key: cord-271485-a1633xxe authors: leaf, rebecca karp; al‐samkari, hanny; brenner, samantha k.; gupta, shruti; leaf, david e. title: abo phenotype and death in critically ill patients with covid‐19 date: 2020-07-01 journal: br j haematol doi: 10.1111/bjh.16984 sha: doc_id: 271485 cord_uid: a1633xxe blood groups are inherited traits that vary across populations, likely due to both founder effects and natural selection. a link between blood groups and susceptibility to infectious disease has been well‐described, with notable examples being h. pylori and plasmodium falciparum infection. blood group antigens may influence disease susceptibility by several mechanisms, including serving as receptors or decoys for infectious organisms and modifying immune response in the form of anti‐abo antibodies. blood groups are inherited traits that vary across populations, likely due to both founder effects and natural selection. 1 a link between blood groups and susceptibility to infectious disease has been welldescribed, with notable examples being h. pylori and plasmodium falciparum infection. 1, 2 blood group antigens may influence disease susceptibility by several mechanisms, including serving as receptors or decoys for infectious organisms and modifying immune response in the form of anti-abo antibodies. 2 data on the relationship between blood group and outcomes in patients with coronavirus disease 2019 (covid-19) are limited. studies from china 3 and europe 4 reported that patients with type o blood may be protected from covid-19 infection, whereas those with type a blood may be at higher risk. data from a related viral outbreak, the severe acute respiratory syndrome coronavirus (sars-cov-1) in 2003, suggested that healthcare workers with type o blood were less likely to contract this disease. 5 in vitro experiments revealed that the interaction between the sars-cov-1 spike protein and angiotensin converting enzyme 2 (ace2)-necessary for viral uptake-may be mitigated by anti-a antibodies. 6 to examine the relationship between blood group and clinical outcomes in patients with covid-19, we studied the distribution and mortality associated with abo phenotype in a large cohort of critically ill patients. we utilized data from the study of the treatment and outcomes in critically ill patients with covid-19 (stop-covid), a multicenter cohort study that enrolled consecutive adults (≥18years-old) with laboratory-confirmed covid-19 admitted to participating intensive care units (icus) at 67 hospitals across the united states. we included patients admitted to icus between march 4 and april 11, 2020. we followed patients until the first of hospital discharge, death, or may 8, 2020the date on which the database for the current analysis was locked. all patients who remained hospitalized at the time of analysis had a minimum of 28 days follow-up. the study was approved with a waiver of informed consent by the institutional review board at each participating site. to examine whether blood type is associated with critical illness in patients with covid-19, we used a chi-square test to compare the observed versus expected distribution of abo phenotypes. we stratified our analyses by race/ethnicity, since race/ethnicity are important determinants of abo phenotype 7 and could also affect hospitalization for covid-19. to improve the reliability of our estimates, we limited our analyses to the three most commonly reported categories of race/ethnicity accepted article in our cohort: white non-hispanic; black non-hispanic; and hispanic. patients missing data on abo phenotype were excluded. we estimated the expected distribution of abo phenotype in each of the above race/ethnicity categories using data from 3.1 million blood donors in the united states. 7 to examine whether abo phenotype is associated with mortality among critically ill patients with covid-19, we used a chi-square test to compare the distribution of observed abo blood phenotypes with 28-day in-hospital mortality, stratified by the above race/ethnicity categories. patients discharged alive from the hospital prior to 28 days were considered to be alive at 28 days. we tested the validity of this assumption in a random subset of 50 patients discharged prior to 28 days, all of whom remained alive at 28 days according to electronic medical records or follow-up by phone. among 3239 critically ill patients with covid-19, 2033 (62.8%) had data available on abo phenotype and were included in the current analysis. the median age was 62 years (interquartile range, 52-71) and 1297 (63.8%) were men. additional characteristics according to abo phenotype are shown in table 1 . the observed and expected frequencies of abo phenotypes in white, black, and hispanic patients are shown in figure 1 . among white patients, the observed distribution of abo phenotypes differed from its expected distribution ( figure 1a) . this difference was primarily driven by patients with blood type a being over-represented (45.1% observed versus 39.8% expected) and patients with blood type o being under-represented (37.8% observed versus 45.2% expected). among black ( figure 1b ) and hispanic patients ( figure 1c ) the observed and expected distributions of abo phenotypes were similar. a total of 799 of the 2033 patients (39.3%) died within 28 days. the mortality rate was similar across abo phenotypes in all race/ethnicity categories (figures 1d-f) . results were qualitatively unchanged when considering rh phenotype. in this large nationwide cohort study of critically ill patients with covid-19, we found significant differences in the observed versus expected distribution of abo phenotypes among white patients, with blood types a and o being over-and under-represented, respectively. we found no difference in the observed versus expected distribution of abo phenotypes among black or hispanic patients, nor did we find an association between abo phenotype and 28-day mortality among any of the three examined categories of race/ethnicity. our finding of a higher than expected frequency of blood type a and a lower than expected frequency of blood type o-at least among white patients-is consistent with other reports. for instance, in a genome wide association study of nearly 2,000 patients in italy and spain, ellinghaus and colleagues recently identified two gene clusters enriched in patients with covid-19. one cluster contained genes relevant to both ace2 functionality and immune response, while the other cluster encoded genes for the abo blood group. in a meta-analysis of two different case-control cohorts, the authors found that type a blood conferred a higher risk of severe covid-19 while type o blood afforded protection. 4 similarly, investigators from both china and the united states reported that patients with type a blood are at increased risk of developing covid-19 whereas those with type o blood have a lower risk. 3 the mechanisms responsible for these observations have yet to be elucidated. one theory is that neutralizing anti-a antibodies protect against viral entry into lung epithelium, as was hypothesized with sars-cov-1; 6 however, if this were the case, we would have expected both type o and type b blood to be under-represented in our cohort. alternatively, individuals with type o blood are known to have lower rates of thrombosis and cardiovascular disease, which is attributed to altered glycosyltransferase activity and increased clearance of von willebrand factor. thus, patients with type o blood may be less likely to develop covid-19-related microvascular thrombosis and endothelial dysfunction. 8 we are unable to provide a discrete biological explanation as to why we found a difference in the incidence of covid-19-associated critical illness only among white patients, though this finding is consistent with prior reports demonstrating a higher risk of acute respiratory distress syndrome in white patients with type a blood but not in black patients. 9 we would also note that there is large variation, subjectivity, and bias in how race/ethnicity is reported in the us, and that these results should be interpreted with caution. 10 this article is protected by copyright. all rights reserved our study has several strengths. we used granular data from a large number of consecutive critically ill patients from 67 geographically-diverse sites across the us. further, whereas prior studies in covid-19 have had limited follow-up, we followed patients until hospital discharge, death, or a minimum of 28 days. we also acknowledge several limitations. as with all observational analyses, we cannot rule out the possibility of residual confounding. additionally, data on abo phenotype were missing in approximately one-third of patients. finally, we were unable to evaluate other blood groups, such as secretor status and lewis antigens, which are also known to affect host immunity. 1, 2 in conclusion, our data suggest that type a blood may be a risk factor for covid-19-related critical illness among white patients, and that type o blood may be protective. future investigations are needed to determine the mechanisms for these findings. this article is protected by copyright. all rights reserved the relationship between blood groups and disease blood groups in infection and host susceptibility relationship between the abo blood group and the covid-19 susceptibility genomewide association study of severe covid-19 with respiratory failure abo blood group and susceptibility to severe acute respiratory syndrome inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies retrovirus epidemiology donor s. abo and rh(d) phenotype frequencies of different racial/ethnic groups in the united states more on 'association between abo blood groups and risk of sars-cov-2 pneumonia' abo blood type a is associated with increased risk of ards in whites following both major trauma and severe sepsis assessing race and ethnicity data quality across cancer registries and emrs in two hospitals accepted article age (yrs) -median (iqr) body mass index -median (iqr) pao 2 :fio 2 , mm hg -median (iqr) a key: cord-252273-mykwzlsu authors: politis, constantina; papadaki, maria; politi, lida; kourti, georgia; richardson, clive; asariotou, marina; tsakris, athanassios; mentis, andreas title: post-donation information and haemovigilance reporting for covid-19 in greece: information supporting the absence of sars-cov-2 possible transmission through blood components date: 2020-10-20 journal: transfus clin biol doi: 10.1016/j.tracli.2020.10.007 sha: doc_id: 252273 cord_uid: mykwzlsu background. although the sars-cov-2 virus is transmitted mainly through the respiratory tract, possible transmission by transfusion from asymptomatic carriers should be explored. as yet there are no reports of transfusion transmission of covid-19. haemovigilance findings within a three-month surveillance period during the new coronavirus pandemic are presented. materials and methods. due to great demand and shortage, blood sessions in outpatient facilities were organized during the high prevalence period of covid-19, alongside a national plan to monitor the evolving public health situation by random molecular screening of high-risk groups of the population. haemovigilance protocols were implemented as well as surveillance for any covid-19 case reported post-transfusion. a 14-day quarantine and follow-up molecular and antibody testing of any covid-19 positive case was obligatory. results. post-donation, post-transfusion information and molecular testing of swab samples collected from three asymptomatic donors at risk for covid-19, revealed the case of an immunosupressed patient who had been transfused with whole blood derived platelets from a donor subsequently diagnosed with covid-19. the recipient exhibited no symptoms of the disease. molecular and antibody testing results were negative. conclusion. haemovigilance provided information supporting the absence of transfusion transmission of covid-19, thus strengthening the hypothesis that, even if it cannot yet be definitively ruled out, covid-19 is not transmitted through blood transfusion. as of early june 2020, a perfect test does not exist, therefore haemovigilance along with the implementation of strict proactive measures is crucial to identify eluding asymptomatic individuals and ensure blood safety during the pandemic. the recent outbreak of the novel coronavirus disease 2019 (covid-19) was officially reported in december 2019 in china and spread quickly around the globe, resulting in its declaration as a pandemic by the world health organization [1] [2] [3] [4] [5] [6] [7] . as with all respiratory viruses, the covid-19 virus is primarily transmitted by the respiratory route [8] [9] [10] [11] [12] . because respiratory viruses have never been reported to be transmitted through blood or blood components, any potential risk of transmission by transfusion of blood collected from asymptomatic individuals remains theoretical 13 . according to the case report described for the first time by cho et al., the transfusion of apheresis platelets to a patient diagnosed with severe aplastic anaemia from a donor who was subsequently diagnosed with covid-19 did not result in the transmission of the disease 14 . any reports indicating possible transmission through transfusion of blood components have not received confirmation and therefore have been discounted. due to the urgent requirement for blood, it remains critically important to know whether the covid-19 virus can be transmitted by blood transfusion, because asymptomatic carriers may donate blood and therefore blood donation could be an unidentified route of transmission. the pandemic has the potential to reduce the supply of blood and blood components by affecting blood system activities, which has led to the publication by authorities worldwide of a variety of statements, precautionary j o u r n a l p r e -p r o o f measures, interim guidelines and risk assessments concerning blood safety and sustainability [15] [16] [17] [18] [19] [20] [21] [22] . following the first confirmed covid-19 case in greece in late february 23 greek cities in safe outpatient facilities. in our study we describe the haemovigilance data over a surveillance period of three months (march to may 2020) when prevalence of the virus was high, focusing on post-donation and post-transfusion information regarding the transfusion to an immunocompromised patient of whole blood derived platelets from a donor who was subsequently diagnosed with covid-19. in greece, a system for monitoring, reporting, investigating and analyzing any adverse events related to donations, processing and transfusion of blood, is constantly in use; however, during the high prevalence period of the disease period. further investigation showed that she had returned from an 11-day trip to bali 33 days before blood donation, travelling via singapore. she did not report contact with suspected or confirmed covid-19 cases during her trip. after diagnosis, she stayed indoors in self-isolation for 14 days. she remained asymptomatic during quarantine. all her close contacts were tested for covid-19 and self-isolated as a precautionary measure. moreover, her colleagues who donated blood in the same session were subjected to molecular testing and 14 days' quarantine. all contacts and colleagues tested negative for covid-19. the blood establishment where the blood session was performed on april 8 th was informed on april 12 th of a confirmed covid-19 case among the donors. haemovigilance protocols were triggered and information about the confirmed covid-19 case was requested. investigations showed that the whole blood derived platelet unit from this donation had been transfused into an immunocompromised male diagnosed with acute myeloid leukemia. red cells and plasma processed from the same blood unit had not been used, and were discarded, according to blood safety protocols. being in the same room as a subsequently confirmed covid-19 case for ten minutes the day before the blood donation. she was advised to remain selfisolated as a precautionary measure and monitor her health closely. the donated whole blood unit was discarded. the result of her molecular testing for covid 19 was negative. the second donor reported close contact with a subsequently confirmed covid-19 person, twelve days before blood donation. haemovigilance protocols revealed that the processed rbcs had already been transfused into an elderly patient admitted to the icu due to a herpetic encephalitis infection. this donor was also advised to self-isolate and monitor his health closely. donor and recipient both tested negative for covid-19. transfusion of platelet rich plasma obtained from an asymptomatic infected individual did not result in disease transmission, even though the platelet recipient was diagnosed with myeloid leukaemia and was taking immunosuppressive drugs. this is the second report described globally, 26--30 . in general, the sicker one becomes due to an infection, the more robust is the immune response that is triggered, and consequently more robust immunity is acquired. serological screening of the donor was performed approximately 4-8 weeks after the assumed date of exposure to the virus and the result was negative. that the donor remained asymptomatic throughout suggests that either she failed to produce antibodies, or antibody secretion was below the detection limit, or she had not yet seroconverted at the time of sampling. there are as yet no data regarding the extent and duration of immunity to the virus and it might be a long time before such information becomes available. we should accept the fact that at that time a perfect test did not exist [28] [29] [30] [31] [32] , and therefore consider the addition of more stringent criteria to the existing ones to avoid the potential transmission of this infection by blood transfusion, such as precautionary deferral from blood donation based on the profession and place of work of candidate donors, as well as the extension of exclusion periods after travelling. asymptomatic transmission of sars-cov-2 remains the achilles' heel of public health strategies for covid-19 pandemic control. symptom-based j o u r n a l p r e -p r o o f screening is useful, but epidemiological evaluation of covid-19 data strongly demonstrates that covid-19 transmission from asymptomatic individuals may play a critical role in this pandemic status. in order to ensure blood safety, most blood transfusion services implement precautionary measures during an outbreak, which involve: a) body in such circumstances of great demand and shortage, along with all the limitations and "lists of unknowns" in serological and molecular testing, the fact remains that haemovigilance is the essential tool for blood safety. in our study (referring to the period from march to may 2020), haemovigilance a novel coronavirus from patients with pneumonia in china clinical features of patients infected with 2019 novel coronavirus in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster report of clustering pneumonia of unknown etiology in wuhan city. wuhan municipal health commission a novel coronavirus genome identified in a cluster of pneumonia cases wuhan world health organization. who timeline-covid-19 covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? epidemiology, genetic recombination, and pathogenesis of coronaviruses origin and evolution of pathogenic coronaviruses coronaviruses and the human airway: a universal system for virus-host interaction studies coronaviruses and the human airway: a universal system for virus-host interaction studies coronavirus disease 2019: coronaviruses and blood safety covid-19 transmission and blood transfusion: a case report american association of blood banks world health organization 2020. maintaining a safe and adequate blood supply during the pandemic outbreak of coronavirus disease (covid-19): interim guidance centers for disease control and prevention 2020. guidance for blood and plasma facilities coronavirus disease 2019 (covid-19) in the eu/eea and the uk: rapid risk assessment the impact of the sars cov-2 outbreak on the safety and availability of blood transfusions in italy blood transfusion during the covid-19 outbreak the greek response to covid-19: a true success story from an ibd perspective primerdesign launches new molecular test for novel coronavirus fda provides emergency use authorization to perkinelmer for serological test to identify covid-19 antibodies 2020 neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications sars-cov-2 serological analysis of covid-19 hospitalized patients, pauci-symptomatic individuals and blood donors understanding antibody testing for covid-19 antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 the time course of the immune response to experimental coronavirus infection of man antibody testing for coronavirus disease 2019: not ready for prime time diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis key: cord-026559-xx52u01h authors: tripathi, siddhartha; agrawal, amit title: blood plasma microfluidic device: aiming for the detection of covid-19 antibodies using an on-chip elisa platform date: 2020-06-10 journal: trans indian natl doi: 10.1007/s41403-020-00123-9 sha: doc_id: 26559 cord_uid: xx52u01h covid-19 is a public health emergency of international concern. detection of sars-cov-2 virus is an important step towards containing the virus spread. although viral detection using molecular diagnostic methods is quite common and efficient, these methods are prone to errors, laborious and time consuming. there is an urgent need for blood-based tests which are simple to use, accurate, less time consuming, portable and cost-effective. human blood plasma contains water, proteins, organic and in-organic substances including bacteria and viruses. blood plasma can be effectively used to detect covid-19 antibodies. the immune system generates antibodies (igm/igg proteins) in response to the virus and identification of these antibodies is related to the presence of the infection in the patient in the past. therefore, detecting and testing the presence of these antibodies will be extremely useful for monitoring and surveillance of the population (petherick, lancet 395:1101–1102, 2020). herein, we describe and propose a microfluidic elisa (enzyme-linked immunosorbent assay) system to detect covid-19 antibodies on a lab-on-chip platform. we propose to first separate plasma from whole human blood using a microfluidic device and subsequently perform the detection of antibodies in the separated plasma using a semi-automated on-chip elisa. the technology presented comprises a microfluidic blood plasma separation device which is capable of effectively separating plasma from whole human blood. human blood constitutes cells and plasma. blood plasma is considered an important source of information pertaining to human health condition; this is due to the presence of important bio-markers. human blood plasma contains water, proteins, organic and in-organic substances including bacteria and viruses. blood plasma is separated from other constituents on a routine basis. use of plasma is preferred over whole blood in several diagnostic tests; this is due to clogging, cell lysis and cell interference issues associated with whole blood testing. we have developed a passive microdevice to separate blood plasma; the device design and other features are shown in fig. 1 . the device is simple, compact and efficient. the principle behind plasma separation revolves around harnessing the bio-physical and geometrical effects of blood flow within a microchannel. experimental results indicate that almost pure plasma (separation efficiency 99.5%, purity) is obtained by injecting whole blood at a flow rate of 0.5 ml/ min. the yield (or amount of plasma obtained to amount of blood infused) of the device is 1% with whole human blood. the plasma separated was found to be hemolysis free and few biomarkers of interest, namely proteins, hcg (human chorionic gonadotropin) hormone, and glucose were successfully recovered from the separated plasma. the device was fabricated in pdms using photolithography and soft lithography techniques (other fabrication materials and techniques can also be employed). the major advantage of such microdevice is its accuracy, ease of operation, use of small sample amount, small size, portability and ease of its integration with a bio-sensing platform. the device has been extensively studied, patented and has been reported in various publications (tripathi et al. 2013 (tripathi et al. , 2015a (tripathi et al. , 2015b (tripathi et al. , 2016 (tripathi et al. , 2018 prabhakar et al. 2015) . recently, this microdevice has been successfully employed by a research group for measuring dopamine from whole blood. researchers have successfully integrated this plasma separation microdevice with an enzyme-free plasmonic neurotransmitter dopamine biosensor to measure dopamine concentration with high detection selectivity (vázquez-guardado et al. 2018) . the reported plasma separation microdevice is not only an alternate to the centrifuge, but it can also be easily integrated with a biosensing platform/detection technology (for example, elisa) and result in a point-of-care device. microdevice ensures separation of high-quality plasma with minimal cell interference enabling selection of an analyte with high specificity and sensitivity. the technology was developed to realize a microdevice to enable blood plasma separation in a lab-on-chip format in an effective way. the study was motivated from the current worldwide effort of developing point-of-care microdevices. there are numerous novel features of this microdevice. the developed microdevice is passive and does not rely on active techniques of separation, the device uses elevated dimensions, so maintaining tight tolerances is not essential; the design is, therefore, easy to fabricate and is cost-effective. the device can work efficiently over a wide range of hematocrit, both whole blood and diluted blood can be used. whole blood is preferred as it ensures sufficiency of bio-markers in the separated plasma. the device can separate plasma in a continuous manner without clogging the microdevice. approximately 10 µl of plasma can be removed using 1 ml of whole human blood in approximately 3 min. the device can easily be integrated with a bio-sensor to enable on-chip detection of a target analyte. the most common tests to detect the sars-cov-2 virus are the rt-pcr test (swab based) and the antibody test (blood based). in addition, field effect transistor (fet)-based sensors for point-of-care testing have been reported. recently, seo et al. (2020) have reported successful detection of sars-cov-2 virus with high sensitivity in swab specimens fig. 1 a blood plasma microdevice design and zoomed view at the junction. symbols: i-blood inlet, o-blood outlet, p-plasma outlet. b experimental photograph showing plasma separation in the microdevice using whole blood at a flow rate of 0.5 ml/min. c exter-nal view of the blood plasma separation taking place in the pdms microdevice. d comparison of the device size with a coin (tripathi et al. 2016) using a fet-based biosensor. the graphene-based device used sars-cov-2 spike antibody to detect the antigen protein. the swab-based tests directly detects the virus and are useful for early detection of the virus whereas blood-based tests are indirect and informs about the infection in the past. the technology of blood plasma separation in a microdevice can be employed for testing of antibodies present in the blood plasma of a covid-19-infected subject, similar to a point-of-care serological test. the immune system generates antibodies (igg/igm proteins) in response to the virus. identification of these antibodies is related to the presence of the infection in the patient in the past. therefore, detecting/ testing the presence of these antibodies will be extremely useful for surveillance of the population and also for plasma transfusion to combat the active virus (petherick 2020). also, blood-based test can allow for the measurement of additional bio-markers of interest such as crp (c-reactive protein). this protein has been found to correlate with the severity of covid-19 infection (vashist 2020) . in the past, various researchers have reported detection of hiv, zika, hepatitis b, dengue, influenza, measles and rubella using microfluidic techniques (yeh et al. 2014 ). immunoassays can be used to measure small amounts of analytes effectively (vashist 2020; yeh et al. 2014; lee and lee 2013; hsu et al. 2014; liu et al. 2017) . herein, we propose the integration of sandwich elisa (enzyme-linked immunoabsorbent assay) with the blood plasma separation microdevice to detect covid-19 antibodies after minor modifications in the design. although the separated plasma from the chip can be analyzed in a conventional elisa, we prefer to propose the on-chip detection of analyte for simplicity and cost-effectiveness. the idea, procedure and steps have been presented in fig. 2 . the whole setup will be similar to an elisa on a microdevice. first, the detection zone area (the plasma reservoir, fig. 2a) is coated with the sars-cov-2 antigen (spike protein) by surface immobilization techniques (direct entrapment of antibodies by spotting and drying methods/physical absorption methods or plasma treatment) on the glass/pdms (heyries et al. 2007; welch et al. 2017) . note that the glass surface is used for bonding purpose. next, the pdms-based microdevice is bonded onto the glass plate such that the plasma reservoir of the device aligns with the antigen-immobilized area. next, bsa (bovine serum albumin) is added to prevent non-specific binding of antibodies. the device is now ready for the injection of whole blood. blood is injected using a syringe pump delivering a constant flow rate of 0.5 ml/min; however, the use of expensive syringe pump can be avoided by devising a spring-loaded syringe capable of delivering the required flow rate. as blood flows into the microchannel, the plasma gets fig. 2 a top: mask of the original blood plasma separation microdevice design with additional inlet for carrying out washing steps and injecting antibodies. bottom: side view of the microsystem. b experi-mental sandwich elisa: showing steps to identify the presence of sars-cov-2 (covid-19) antibodies present in blood plasma separated and flows towards the plasma outlet reservoir. the antibodies, or the target of interest binds to the sars-cov-2 antigens coated on the reservoir; this step involves reaction and incubation time. subsequently, washing step is carried out by injecting pbs (phosphate-buffered saline and 0.1% tween 20) from an additional reservoir connected through a channel near the plasma outlet. this step is essential to remove the unbound molecules. next, labelled secondary antibodies (hrp-horseradish peroxidase conjugated) are injected and added to the reservoir. finally, the substrate addition (tmb-3,3′,5,5′-tetramethylbenzidine + h 2 o 2 ) will result in colorimetric signals for image analysis and determining the concentration of covid-19 antibody (hsu et al. 2014) . though colorimetric methods are simple to employ, the electrochemical methods integrated with smartphone technology can also be employed for detection purposes (lee and lee 2013) . realizing a fully automated testing on a lab-on-chip format is quite challenging, it is expected that the proposed idea will be useful in reducing the sample processing and detection time as compared to conventional elisa technique. in addition, the arrangement of the microdevice is such that other bio-markers of interest can also be measured simultaneously, if desired. the development of the elisa-based microfluidic platform will involve modifications in the current design and fabrication of the microdevice using photolithography and soft lithography techniques, plasma separation and off-chip testing of separated plasma using conventional 96-well elisa, procurement of spike proteins, antibodies, buffers, bio-safety cabinet and spectrophotometer, immobilization of antigens and related experiments, quantification of antibodies (image analysis), and comparison of results obtained from the current microdevice with those obtained using a conventional elisa kit. overall, 12 months will be a reasonable estimate to accomplish the whole task. conflict of interest the authors declare that they have no competing interests. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. developing antibody tests for sars-cov-2 straightforward protein immobilization on sylgard 184 pdms microarray surface based elisa for the detection of autoimmune antibodies in body fluid the case of bullous pemphigoid lab on a chip for in situ diagnosis: from blood to point of care a fully integrated distance readout elisa-chip for point-of-care testing with sample-in-answer-out capability a novel, compact and efficient microchannel arrangement with multiple hydrodynamic effects for blood plasma separation rapid detection of covid-19 causative virus (sars-cov-2) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor blood plasma separation in elevated dimension t-shaped microchannel performance study of microfluidic device for blood plasma separation-a designer's perspective passive blood plasma separation at the microscale: a review of design principles and microdevices microdevice for plasma separation from whole human blood using bio-physical and geometrical effects international patent on microdevice for separating plasma from human blood enzyme-free plasmonic biosensor for direct detection of neurotransmitter dopamine from whole blood in vitro diagnostic assays for covid-19: recent advances and emerging trends point-of-care microdevices for blood plasma analysis in viral infectious diseases orientation and characterization of immobilized antibodies for improved immunoassays key: cord-281003-7pdhxdzc authors: farmakis, dimitrios; giakoumis, anastasios; cannon, lily; angastiniotis, michael; eleftheriou, androulla title: covid‐19 and thalassaemia: a position statement of the thalassaemia international federation date: 2020-07-13 journal: eur j haematol doi: 10.1111/ejh.13476 sha: doc_id: 281003 cord_uid: 7pdhxdzc objectives: many patients with haemoglobinopathies, including thalassaemia and sickle cell disease, are at increased risk of developing severe complications from the coronavirus disease 2019 (covid‐19). although epidemiologic evidence concerning the novel coronavirus (sars‐cov‐2) infection in these patients is currently lacking, the covid‐19 pandemic represents a significant challenge for haemoglobinopathy patients, their families and their attending physicians. methods: the present statement summarizes the key challenges concerning the management of haemoglobinopathies, with particular focus on patients with either transfusion‐dependent or non‐transfusion‐dependent thalassaemia, identifies the gaps in knowledge and suggests measures and strategies to deal with the pandemic, based on available evidence and expert opinions. key areas covered include patients’ risk level, adaptation of haemoglobinopathy care, safety of blood transfusions, blood supply challenges, and lifestyle and nutritional considerations. conclusions: the proposed measures and strategies may be useful as a blueprint for other disorders which require regular hospital visits, as well as for the timely adaptation of patient care during similar future pandemics. the coronavirus disease 2019 (covid-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has affected millions across the world, having caused hundreds of thousands deaths. 1 inherited haemoglobin disorders or haemoglobinopathies, including thalassaemias and sickle cell disease (scd), are the most common monogenic disorders in human, being associated with significant burden, with multisystemic involvement and need for intensive life-long therapy and follow-up. 2, 3 multiple sequelae often complicate the course of the disease, requiring special care. this is particularly true for patients living in developing or low-income countries, where disease-specific management programmes are lacking and access to modern therapy is limited. on the other hand, therapeutic advances of the past decades have resulted in a significant improvement in the once ominous prognosis of thalassaemia patients, and thus, patients with access to modern treatment modalities and well-organized follow-up programmes grow old and face a new spectrum of comorbidities related to ageing. 4 as a result, many haemoglobinopathy pathe information provided herein is relevant to patients with (a) homozygous β-thalassaemia, including thalassaemia major and intermedia; (b) combined forms of thalassaemia, such as β-thalassaemia/ hbe; (c) α-thalassaemia, particularly hbh, the most clinically significant form. at this stage, sickle cell syndromes will not be further discussed, even though patients affected are expected to be at greater risk of covid-19 complications due to the danger of the virus triggering an acute chest syndrome as well as vascular complications. [6] [7] [8] thalassaemia patients stand out in the situation of the pandemic because of their need to frequently visit healthcare facilities for blood transfusion. this makes the need for protection measures imperative as hospital environments may be regarded as "hotspots" for viral transmission. in addition, from tif contact with patient organization across the world, the pandemic has secondary consequences, such as blood shortages, medication shortages reduced access in some situations to expert care, that may have long-term effects on the health status. it should be stressed that as our yet limited knowledge on covid-19 progresses, 5 new evidence becomes continuously available that may challenge some of the information and guidance provided herein. the proposed measures and strategies may be useful as a blueprint for other disorders which require regular hospital visits, as well as for the timely adaptation of patient care during similar future pandemics. the sars-cov-2 infection presents particular challenges and dangers to patients with haemoglobin disorders. the virus affects primarily the respiratory system, with a disease spectrum ranging from nasopharyngeal symptoms to full blown pneumonia. 9 generally, these infections can cause more severe symptoms in people with weakened immune systems, older and/or frail people, and those with long-term conditions like diabetes, cancer and chronic lung disease. 10 most people (about 80%) who become infected experience mild illness and recover, but it can be more severe for others. 9 most deaths are related to respiratory complications requiring intensive care and respiratory support, even though an overexuberant inflammatory response with multi-organ failure may be prevalent in some cases. so far, very little clinical experience of infected patients with haemoglobin disorders has been recorded. 5 any statement on these subjects may be regarded as speculations; cautionary thoughts are however necessary, in view of the rapid spread of the virus and the possible factors which may render these patients vulnerable in front of this infection. tif believes that health services should be alerted to these risks and affected patients warned so that extra precautionary measures can be taken. haemoglobin disorders are not directly associated with respiratory conditions. however, disease-related complications may affect multiple organs including the heart, liver, endocrine glands, lungs and the immune system, thus rendering this patient population at an increased risk to develop serious complications during covid-19. 2, 3, 11 this is especially so in patients who receive suboptimal management and lack access to modern therapy and thalassaemia patients do not have the same risk of pulmonary infections with sickle cell disease patients but, they may have multiple organ complications, often due to iron overload, including cardiac and hepatic, diabetes mellitus and endocrine disease. [2] [3] [4] one particular endocrine complication, adrenal hypofunction, is often not recognized and may render the patient particularly susceptible to severe infections. this condition requires glucocorticoid supplementation; however, corticosteroids have been shown to slow down the clearance of viral rna from respiratory tract in patients with sars-cov or mers-cov infections increasing the complications rate. 12 thalassaemia patients, particularly of the older age groups, have often been splenectomized while scd patients often suffer from functional hyposplenism or asplenia. this renders these patients vulnerable to bacterial infections that may lead to serious illness or life-threatening sepsis. when infected by sars-cov-2, these patients may also develop secondary bacterial infections. patients with thalassaemia can be grossly divided into three risk groups: (a) group a, patients at "moderate risk," (b) group b, patients at "high risk" and (c) group c, patients at "highest risk"; the classification criteria are presented in detail in table 1 . it should be stressed that the proposed risk classification is mainly based on the level of adherence to disease-specific care, since quality of care is the crucial determinant of morbidity in these patients. varying from country to country, and therefore, local and national guidance should be followed. [13] [14] [15] adherence to the instructions and recommendations of the national health committees is of pivotal importance and should be incorporated in the care of patients with haemoglobinopathies. the following are measures that need to be taken in the care of thalassaemia patients. • all patients should comply with the social distancing measures and family practices recommended by local authorities to prevent their exposure to confirmed or possible covid-19 cases among their social contacts, including family members or friends. • in patients with symptoms of cough, fever, fatigue or other symptoms suggestive of an acute respiratory illness, it is required to test for covid-19 along with other respiratory viral pathogens. • if suspicion for covid-19 is high or the test is positive, the treating physician who is fully aware of the individual's care plan should be contacted immediately. • particularly in those patients with comorbidities, a close collaboration of the attending physician team with the patients' treating physician is of utmost importance. tests that may need to be rearranged based on prioritization and individual patient needs. all this must be considered but the need for short-and long-term adhesion to patient treatment programmes and protocols to avoid adverse effects must not be neglected. it is important to stress that all thalassaemia patients should receive proper care regardless of whether they are exposed to or infected by covid-19. this can be accomplished by the adoption of appropriate patient care pathways, as described in detail below. in addition, enrolment of thalassaemia patients into covid-19 trials should be encouraged as it would generate important evidence for both conditions. to will ask the patient a series of questions, as per national guidelines for covid-19) and will check the patient's vitals, including temperature, heart rate, respiratory rate, blood pressure and pulse oximetry along with the haemoglobin level. if any covid-19-like symptoms are present such as high temperature or dry cough, the triage nurse will consult with the treating physicians to decide upon granting admission to the clinic or not. a proposed algorithm for the screening for covid-19 in patients with haemoglobinopathies is presented in figure 3 . in the case that admission is permitted to the patient with covid-19-like symptoms, he/she should receive the required medical care in a designated "isolation room" by doctors and nurses wearing appropriate personal protective equipment. 16 the haemoglobinopathy units and clinics are thus advised to designate a dedicated room within the medical facility that will be used to provide medical care to thalassaemia patients who are suspected or confirmed covid-19 patients. this room should ideally have a separate entrance and exit to the rest of the clinic so as to limit contact with other patients, thus preventing transmission of covid-19. the room should be disinfected regularly. the routine tests that are crucial for the monitoring of thalassae despite the fact that coronavirus rna can be amplified from patients' blood, there is no hitherto evidence of coronavirus transmission through transfusion of blood and blood products. 19 all the routine practices for donor management and infectious disease testing should not be changed. however, in extreme blood shortage, reduction of whole blood donation intervals may be conthe optimization of blood use will help safeguard blood supplies during the pandemic. 21 in this context, adoption of a patient blood management (pbm) system, based on three pillars, optimization of paconversely, immunosuppressed transplant recipients who regularly visit hospital settings may be exposed to the sars-cov-2 in hospital settings with a higher possibility to develop severe illnesses. the influenza a virus subtype h1n1 (h1n1) pandemic had a significant impact on the blood supply due to donors' fear of exposure to the virus at a hospital or a free-standing donor facility. 32 similarly, the covid-19 pandemic has already led to reduced blood supplies due to the cancellation of numerous community-based and mobile blood donation drives, as well as a marked reduction in donors arriving for scheduled appointments. 31 in the us, nearly 4000 american red cross blood drives, that account for more than 80% of its blood collects, have been cancelled across the country, 33 while cancellation of hospital-based collections resulted in 130 000 fewer donations in only a few weeks. 27 besides the cancellation of organized blood donation drives and campaigns and the significant reduction in the number of donors arriving at blood donation facilities due to self-quarantine, social isolation measures and the fear of exposure to covid-19, there is further the inevitable deferral of donors due to the more meticulous donor selection process with additional criteria discussed above. the situation further worsens by the fact that an increasing number of staff members in the blood services become ill or are required to self-isolate, while part of the blood donation and processing procedures may potentially be diverted to the preparation of convalescent plasma from patients who have recovered from covid-19. finally, the blood supply chain can be affected by travel restrictions as well as factory closing that may affect the blood product testing and production. at a later stage of the pandemic, a considerable percentage of the population is expected to be unknowingly infected by sars-cov-2, including the young blood donor population in which asymptomatic cases are more common; this will render the deferral of potential asymptomatic affected donors much more challenging. on the other hand, the demand for blood and components may decrease during the pandemic as elective and non-urgent surgical interventions are deferred and road and other society-related accidents may decrease due to isolation measures. this may compensate to a certain extend the anticipated reduction of blood supplies for chronically transfusion-dependent patients. in addition to the well-known personal hygiene and preventive measures against the new coronavirus, thalassaemia patients can also follow some simple recommendations regarding their lifestyle and nutrition that strengthen their immune system and could better prepare them for the ongoing pandemic. 34, 35 these recommendations are based on the implementation of common sense and moderation, and consist of: 1. sufficient hydration with 2-3 litres of water consumed throughout the day, if health conditions allow it. 2. adequate rest with 7-8 hours of sleep daily. 3. regular but not exhaustive exercise with a half-hour walk, 3 days a week. 4. normal body weight maintenance through a well-balanced diet. especially during the pandemic patients should consume frequent small and light meals, mainly consisting of fruits and vegetables. 5. effective stress management through calm mindfulness. 6. continuous management of chronic comorbidities, especially cardiac and pulmonary conditions, and diabetes mellitus. 7. self-disciplined smoking cessation as even otherwise healthy smokers are considered of high risk for suffering severe complications from covid-19. boosting, sleep regulation, as well as for essential vitamin d production. regarding nutrition in particular, thalassaemia patients are encouraged to consume natural sources containing vitamin c (citrus fruits), vitamin d (sardine, mackerel, dairy products), zinc (legumes, seeds and nuts) and omega-3 fatty acids (sardine, mackerel) as these foods have been found in applied basic research to provide necessary vitamins, minerals and anti-oxidants that boost the immune response. caution is advised if patients turn to dietary supplements as the danger of over-supplementation (especially for vitamin c) is quite consid the clinical impact of covid-19 in haemoglobinopathy patients is not yet defined and thus infected cases should be under meticulous observation with comprehensive reporting of their clinical outcomes. this will contribute to the prompt management of the potential complications that may arise in infected patients, but also to the collection and sharing with other treating physicians of important information and data that will support the better understand world health organization (who) guidelines for the management of transfusion dependent thalassaemia guidelines for the management of non-transfusion dependent thalassaemias the changing epidemiology of the ageing thalassaemia populations: a position statement of the thalassaemia international federation sars-cov-2 infection in beta thalassemia: preliminary data from the italian experience burden of influenza-related hospitalizations among children with sickle cell disease covid-19 pneumonia as a cause of acute chest syndrome in an adult sickle cell patient vasoocclusive crisis and acute chest syndrome in sickle cell disease due to 2019 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implications catio ns-detai l/maint ainin g-a-safe-and-adequ ate-blood -suppl y-durin g-the-pande mic-outbr eak-of-coron aviru s-disea se-(covid -19) protecting the blood supply during infectious disease outbreaks aabb's resources for: fda's updated information for blood establishments regarding the novel coronavirus (covid-19) outbreak updated information for blood establishments regarding the novel coronavirus outbreak european centre for disease prevention and control. coronavirus disease 2019 (covid-19) and supply of substances of human origin in the eu/eea post-donation covid-19 identification in blood donors the essential role of patient blood management in a pandemic: a call for action hydroxyurea for nontransfusion-dependent β-thalassemia: a systematic review and meta-analysis clinical experience with fetal hemoglobin induction therapy in patients with β-thalassemia a phase 3 trial of luspatercept in patients with transfusion-dependent β-thalassemia alternative procedures for blood and blood components during the covid-19 public health emergency: guidance for industry institute of medicine (us) forum on microbial threats. the domestic and international impacts of the 2009-h1n1 influenza a pandemic: global challenges, global solutions: workshop summary coronavirus outbreak: help us continue to deliver our lifesaving mission nationwide due to this public health emergency a useful health & nutrition short guide for the covid-19 pandemic by tif integrative considerations during the covid-19 pandemic herbal antivirals: natural remedies for emerging & resistant viral infections. viral respiratory infections and their treatment. sars and coronaviruses potential antivirals and antiviral strategies against sars coronavirus infections covid-19 and thalassaemia: a position statement of the thalassaemia international federation key: cord-012354-2f5oq9e5 authors: holtkötter, hannah; dias filho, claudemir rodrigues; schwender, kristina; stadler, christian; vennemann, marielle; pacheco, ana claudia; roca, gabriela title: forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault—validating an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and d-dimer date: 2017-10-23 journal: int j legal med doi: 10.1007/s00414-017-1719-y sha: doc_id: 12354 cord_uid: 2f5oq9e5 sexual assault is a serious offense and identification of body fluids originating from sexual activity has been a crucial aspect of forensic investigations for a long time. while reliable tests for the detection of semen and saliva have been successfully implemented into forensic laboratories, the detection of other body fluids, such as vaginal or menstrual fluid, is more challenging. especially, the discrimination between peripheral and menstrual blood can be highly relevant for police investigations because it provides potential evidence regarding the issue of consent. we report the forensic validation of an immunochromatographic test that allows for such discrimination in forensic stains, the seratec pmb test, and its performance on real casework samples. the pmb test is a duplex test combining human hemoglobin and d-dimer detection and was developed for the identification of blood and menstrual fluid, both at the crime scene and in the laboratory. the results of this study showed that the duplex d-dimer/hemoglobin assay reliably detects the presence of human hemoglobin and identifies samples containing menstrual fluid by detecting the presence of d-dimers. the method distinguished between menstrual and peripheral blood in a swab from a historical artifact and in real casework samples of alleged sexual assaults. results show that the development of the new duplex test is a substantial progress towards analyzing and interpreting evidence from sexual assault cases. electronic supplementary material: the online version of this article (10.1007/s00414-017-1719-y) contains supplementary material, which is available to authorized users. identifying the biological source of a crime scene stain is one of the most important components in forensic science practice. this aids police investigations as it possibly provides the investigators with information on the course of the crime. blood is one of the most commonly found body fluids at crime scenes, and accurate differentiation between peripheral blood and menstrual fluid could provide crucial evidence, e.g., regarding the issue of consent in sexual assault cases; while the presence of peripheral blood indicates a traumatic cause, menstrual fluid points towards a natural bleeding cause [1] . some body fluids present in cases of alleged sexual assault, such as semen or saliva [2, 3] , can be detected reliably, but others are more challenging to detect, such as pre-ejaculate, menstrual blood, or vaginal fluid. driven by the importance of forensic body fluid identification, novel methods have been extensively researched over the years including microscopic examination [4, 5] , messengerrna (mrna) [6, 7] , microrna (mirna) [8, 9] , and dna methylation profiling [10] [11] [12] , which have been successfully applied to the identification of blood. however, distinguishing peripheral blood from menstrual fluid with these methods has been shown to be highly challenging, and the application of more complex molecular techniques requires a considerable amount of training and experience. furthermore, results may be influenced by interand intra-individual differences between donors [13] , and a full forensic validation for casework application of these methods is hitherto still outstanding. the most important requirements of newly developed tests are that they do not interfere with or hamper subsequent dna analysis by loss of material, contamination, or inhibition. immunochromatographic assays for body fluid detection have been shown to be highly specific and easy to use. recently, initial work successfully introduced immunochromatographic assays that detect degradation products of fibrinolysis (fdps) as innovative methods for the identification of menstrual fluid in forensic samples; during menstruation fibrinolysis, the endogenous degradation of fibrin after blood coagulation is a crucial step that enables menstrual fluid to easily pour out [1, 15] . the most significant subtype of fdps is d-dimer, a small protein fragment specific to the process of fibrinolysis. the use of d-dimers for menstrual fluid identification has first been described by miyaishi et al. in 1996 [14] who showed that menstrual blood has a mean concentration of d-dimer 200 times greater than that of peripheral blood. with that in mind, baker and colleagues [15] examined four d-dimer assays, examining their specificity and sensitivity in detecting menstrual blood. results showed that clearview® simplify d-dimer (alere, cheshire, uk) was the kit expressing highest sensitivity and specificity, beyond which, its usage was simple and quick, and results were easy to interpret. the kit, originally developed for diagnostic purposes to detect d-dimers in peripheral blood as a marker for peripheral thromboembolism, was validated in 2014 by our group for the detection of menstrual fluid in forensically relevant samples [1] . recently, a novel immunochromatographic assay, the seratec pmb test (seratec gmbh, göttingen, germany), was developed, which combines the detection of human hemoglobin and d-dimers. this is the first ddimer assay that was developed specifically for differentiation between human peripheral blood and human menstrual fluid in forensic samples. to our knowledge, it also represents the first immunochromatographic multiplex assay for forensic use. the immunochromatographic seratec hemdirect test, which detects the presence of human hemoglobin in a forensic sample [16] and therefore confirms the presence of blood, was used as the basis for the development of the novel duplex test. according to seratec's user instruction, the pmb assay detects hemoglobin down to a concentration of 20 ng/ml and d-dimer down to 400 ng/ml, is easy to use directly at crime scenes or in the laboratory, quick, and does not require special training of the analysts. furthermore, the sample material remains suitable for dna extraction and profiling [17] . here, we report the final development and the forensic validation of the seratec pmb test. different antibody quantities as well as buffer amounts and incubation times were tested. moreover, we aimed to evaluate (1) the sensitivity of the pmb test by considering serial dilutions of peripheral blood and menstrual fluid, (2) possible influences of and cross-reactivity with other biological secretions, and blood from various species, (3) possible false-positive results on blood from aged donors and deceased's peripheral blood, (4) the viability of dna extraction and amplification from the residual blood diluted on the remaining buffer, (5) a case example involving a historical ethnological artifact, and (6) the performance of the test on blood samples from ten cases of alleged sexual assault. the hemoglobin/d-dimer assay contains four monoclonal antibodies as active compounds and two polyclonal: two gold-labeled monoclonal for the respective epitopes of human hemoglobin and d-dimer located in the start area and two monoclonal antibodies for the sandwich assay located at the result lines. the two polyclonal antibodies are forming the control line. the cassettes of the assay contain a sample well and a result window with a control line (labeled bc^for control), a result line for hemoglobin (labeled bp^for peripheral blood), as well as a result line for d-dimer (labeled bm^for menstrual fluid). if no line is visible, the test is invalid and should be repeated [17] . initially, five different cut-off levels for d-dimer detection were evaluated during production. preliminary testing revealed that cut-off levels of ≥ 300 and ≥ 400 ng/ml were optimal for reliable detection of menstrual blood and avoidance of false-positive results from peripheral blood (data available on request). two independent research institutions further evaluated these two cut-off levels. for the assessment of both cut-off levels and forensic validation of the assay, menstrual blood was collected on vaginal swabs from 16 healthy female donors (age ranging from 24 to 39 years old) at days 1, 2, and 3 of the menses. informed consent was obtained from all individual participants included in the study. liquid menstrual fluid was collected from one donor using a menstrual cup (mooncup ltd., brighton, uk). peripheral blood samples were collected from 14 healthy donors (six female and eight male). swabs were stored at room temperature (rt) and liquid blood was stored frozen in aliquots, both for 8 months prior to testing. from dried samples, approximately 3 mm 2 of the cotton swabs were used for testing. from liquid samples, 3 μl was used for testing and frozen samples were brought to rt prior to analysis. the sensitivity of the pmb test was assessed by testing various volumes and dilutions of buffer and menstrual fluid: 3 μl of neat menstrual fluid were added to each 500, 1000, 1500, 3000, and 6000 μl of extraction buffer. incubation times of 5, 30, 90, and 120 min were tested. based on the results of the sensitivity, testing samples were prepared using the following protocols: the positive control was prepared with 3 μl menstrual fluid in 1000 μl buffer and extracted for 5 min. fresh liquid samples were extracted in 1500 μl buffer for 5 min. dry samples on cotton swabs were extracted in 300-500 μl buffer for 30 to 90 min. aged-dried samples were extracted in 120 μl buffer for and extended incubation time of 120 min. either 1/3, 1/2, or a full cotton swab was used for extraction, according to the amount of menstrual fluid deposited. after the individual incubation times 120 μl of sample-buffer solution was added to the sample well and results were read after a development period of 10 min. the possibility of obtaining false-positive results was investigated using neat peripheral blood, vaginal fluid, semen, saliva, and urine as well as non-human peripheral blood samples from horse, cat, dog, goat, sheep, and rabbit. mixtures of menstrual fluid were prepared with peripheral blood, vaginal fluid, semen, saliva, and urine (1:1). in addition to the validation samples, postmortem samples were tested for possible false-positive results due to potential postmortem fibrinolysis [14, 18] . for this, peripheral blood from deceased (n = 10) was applied to cotton swabs during medicolegal autopsy and air-dried at rt for 4 weeks prior to testing. as d-dimer levels are known to increase with age [19] , five peripheral blood samples obtained from donors over the age of 60 years were collected with informed consent. the samples were tested by adding 3 μl of blood to 1500 μl of buffer, and additionally by adding 1 μl of blood to a cotton swab that was extracted in 120 μl of buffer after a storage time of 2 weeks. all samples were tested in duplicates and the provided buffer was used as negative control. dna profiles were generated from a mixture of menstrual fluid and semen. dna was extracted from (1) the pad underneath the sample well and (2) the remaining sample-buffer solution. for method 1, the plastic cartridge was opened and the pad was cut out and transferred to a reaction tube. for method 2, 100 μl of sample-buffer solution was transferred to a reaction tube. dna was extracted using the maxwell® instrument (promega, mannheim, germany) and amplified with powerplex® esx 17 pro system (promega) according to manufacturer's recommendations. samples were subsequently analyzed using the 3130 genetic analyzer with the genemapper® id software by thermo fisher scientific. a case study was performed that involved a historical artifact, an african statue used during traditional ceremonies for medicinal purposes and administration of justice in a rural community of gabon. the statue of unknown age was made from wood and was suspected to be covered in human blood and/or menstrual fluid. a swab was taken from the statue and approximately 3 mm 2 was submerged in 500 μl of the provided seratec pmb extraction buffer. after an incubation time of 120 min 120 μl was applied to the sample well and results were read after a development period of 10 min. also, the swab was analyzed using the human-specific rsid-blood test (galantos genetics, mainz, germany) for validation. lastly, blood samples from ten cases of alleged sexual assault were analyzed using the seratec pmb test. blood samples were either collected from the victims' clothing or from vaginal swabs collected during the medical examination. the existence of human blood was additionally tested using the feca-cult one step test (alamar tecno científica ltda), an immunochromatographic test for the qualitative determination of human hemoglobin. all tests were performed in duplicates. the volume of the provided seratec pmb extraction buffer was 300 or 500 μl and the incubation time varied from 30 to 90 min, considering the amount of blood on each sample (see table 1 ). the test dose was 120 μl applied to the sample well and results were read after a development period of 10 min. a brief history of each case and sample is given in the supplementary material, document 1. handling of the test device (see fig. 1 for examples of the cassettes) was easy and straightforward. all replicates showed consistent results and all negative controls gave the expected result (i.e., line bc^present, lines bp^and bm^not). all tests with 300 ng/ml as well as 400 ng/ml as cut-off for the d-dimer antibody worked correctly as shown by the test's c line. all tests reacted positive for hemoglobin presence and negative for d-dimer presence when peripheral blood was tested. furthermore, all of the menstrual blood samples gave a positive result for hemoglobin presence, dried (n = 16) or liquid (n = 1). while fresh liquid menstrual fluid reacted clearly positive for d-dimer presence with both cut-offs, dried menstrual fluid samples showed more varied results (see fig. 2a for details) . results for the cut-off of 300 ng/ml using stored dry menstrual blood (n = 16) from the 48 samples tested results for the cut-off of 400 ng/ml using stored dry menstrual blood (n = 14) all of the 42 samples tested (14 donors, days 1, 2, 3) reacted positive to the presence of d-dimer (100%). the majority gave a strong positive signal (52.4%), 12 from the 42 samples gave a clear positive reaction (28.6%), and only eight of the samples gave a weak positive signal (19%). it was observed that intra-individual differences of the samples result in variances of signal intensity. the day of the menses on which the sample was collected does not seem to influence the results of the test. based on these results, the following tests were performed using the 400 ng/ml cut-off only. the sensitivity testing revealed that menstrual fluid was still detectable down to 120 nl, which is equivalent to 3 μl of menstrual fluid diluted in 3000 μl buffer. here, a clear signal for hemoglobin and an extremely weak signal for d-dimer were observed. a much clearer signal for d-dimer presence was observed when only 1500 μl buffer was used on 3 μl of sample; thus, 240 nl of menstrual fluid. therefore, the detection limit with the pmb test was set to 240 nl of menstrual fluid. best results, with clear, balanced signals for both hemoglobin and d-dimer presence, were received with a concentration of 360 nl of menstrual fluid (3 μl in 1000 μl buffer), fig. 2 heat maps of a signals for d-dimer presence with the 300 and 400 ng/ml cut-off (donor 1 to 16 = dried blood, donor 17 = liquid blood), b the sensitivity study, c the mixtures and cross-reactivity testing, d blood collected from aged donors, and e postmortem blood samples. mb menstrual blood, pb peripheral blood, vf vaginal fluid which is why this was set as optimal for a positive control. it was observed that 3 μl of fresh liquid menstrual fluid in only 500 μl of buffer (720 nl) overloaded the test strips and signals were hard to interpret. hence, when fresh liquid samples are received for analysis, they should either be diluted beforehand or used with the appropriate amount of at least 1000 μl of buffer. no signal was received when 60 nl of menstrual fluid (3 μl in 6000 μl buffer) was analyzed. different incubation times were tested for liquid and dried samples. it was shown that for liquid samples, an incubation time of only 5 min is sufficient. for dried samples, individual decisions on incubation times have to be made by the analyst, depending on the amount of blood deposited on the evidence and the time of deposition. from experience in the application of this test, we suggest the following: a tampon or sanitary pad should generally contain an enormous amount of blood. if it is relatively freshly collected, it is advised that a piece of either is extracted in 500 μl for 30 min. if it has been stored for several weeks or months, the extraction time should be extended to 60 min. for smaller blood stains, i.e., collected on clothing, the amount of buffer should be reduced to 300 μl. if an object is swabbed or only traces of blood are found, the minimal amount of 120 μl buffer should be used. if the swab has been deposited for months or years before analysis, the incubation time should be extended to 90 min. to assess whether the presence of other body fluids influence the test, mixtures and neat body fluids were analyzed. all of the mixtures containing menstrual fluid gave a clear positive result for d-dimer presence and no false positive was observed by peripheral blood, vaginal fluid, semen, saliva, and urine (fig. 2c ). to assess cross-reactivity with other species, animal blood samples were tested. blood samples collected from dog, cat, horse, sheep, goat, and rabbit reacted negative for the presence of human hemoglobin. with the exception of rabbit blood, which reacted very weakly positive for the presence of d-dimer, all other animal blood samples gave a negative result for d-dimer. since rabbit blood reacted negative for hemoglobin presence, and a positive reaction would be expected in case of a menstrual blood sample, this result does not pose a problem for casework samples. positive d-dimer reaction combined with negative hemoglobin reaction might indicate a non-human origin of a suspected blood stain. all samples from donors over the age of 60 (n = 5) tested positive for peripheral blood and negative for menstrual fluid (fig. 2d) . slightly stronger signals were obtained for dried samples than for liquid samples. thus, no false-positive d-dimer results were generated by these samples; hence, samples from elderly people do not seem to influence test results. all of the postmortem samples (n = 10) reacted positive for hemoglobin presence and seven of the samples additionally reacted positive for d-dimer presence. of the seven samples three gave a weak positive signal while four gave such a light signal that was impossible to be captured by camera (fig. 2e) . numerous biochemical reactions take place in dying cells and corpses. the one that is likely to explain the positive reaction to d-dimer presence is a decrease in oxygen levels that leads to a switch to anaerobic metabolism. in turn, this results in the accumulation of lactic acid, initiating a fall in ph postmortem. the plasma ph decrease is noteworthy because it is believed to introduce fibrinolysis, and therefore, the formation of ddimers [18, 20] . consequently, care must be taken when postmortem blood samples need to be assessed. it was possible to generate dna profiles from the samplebuffer solution as well as from the pad beneath the sample well. the two str profiles are given in the supplementary material, document 2. as a relatively high amount of the d-dimer incubation buffer is used for each sample, the possibility to subsequently perform standard methods such as dna profiling from leftover buffer solution is particularly useful. this avoids sample loss for body fluid identification. the possibility to extract dna from the pad is especially important in cases where the entire amount of sample-buffer solution was needed for analysis. no differences in peak height (rfu) were observed between the two extraction methods. using the seratec pmb test, the swab taken from the african figurine tested positive for the presence of hemoglobin and negative for the presence of d-dimer. with the rsid-blood test, the sample tested positive for glycophorin a. as a result, it was concluded that the figurine contained human peripheral blood but no menstrual fluid. the pmb test showed reliable results in a swab taken from a stain that was suspected to contain human blood of unknown age and time of deposition. a clear result was obtained confirming the suitability of this test for forensic settings. all samples except the dress from case #1 reacted positive in the feca-cult test. these results were confirmed by the seratec pmb test. from ten samples, six presented positive results for menstrual blood. the positive detection of d-dimer in case #7 is somewhat surprising and might be explained by residual menstrual fluid in the vagina, i.e., through intermenstrual bleeding [21] . for cases #4, #8, #9, and #10, the incident happened within 3 days after menses, with consistent results. in four cases (#1, #2, #5, #6), the test reacted positive for hemoglobin presence but negative for d-dimer presence indicating blood of a traumatic cause. the test performance seems promising. a summary of the results is shown in table 1 . the seratec pmb test proved to be a helpful tool in interpreting samples containing human menstrual and/or peripheral blood. test cassettes were easy to use, interpretation of results was found to be straightforward and unambiguous. no special training was needed to apply the test successfully. it was shown that tests with a cut-off of 400 ng/ml overall produced clearer results with a stronger signal compared to 300 ng/ml. therefore, this cut-off was chosen for the commercially available product. the results of this study show that the test's detection limit is 240 nl of menstrual fluid, proving the high sensitivity of the assay. highly sensitive tests are especially important for forensic and historical samples, as typically, only limited material is available for testing. slight intra-individual differences were observed in terms of intensity of the test result but no significant differences in line intensities were received when testing menstrual blood from different days of the menses. with an incubation time of 90 min, it was possible to analyze aged-dried samples, which is also crucial for crime scene samples, especially considering stored evidence from socalled cold cases. biological secretions, such as vaginal fluid, semen, saliva, and urine, might be part of a mixed sample on evidence in cases of alleged sexual assault. this enhances the results reported here, since all mixtures containing menstrual blood resulted in a positive signal for d-dimer presence and no false-positive results were obtained for other tested body fluids, proving its high specificity. the test is human-specific and did not react with animal blood. even though rabbit blood yielded a positive signal for d-dimer, no signal for hemoglobin was visible. since menstrual blood contains hemoglobin, both lines need to be visible for a positive test result. in addition, even the blood of elderly people who may present fdp does not appear to influence the test. none of the samples from donors over the age of 60 generated a false-positive result for d-dimer presence. even though a theoretical risk remains to obtain a false-positive result from peripheral blood of an elderly person, the relevance of such a finding is limited in casework; if a blood stain from an alleged sexual assault case does react positive to d-dimer presence but is known to originate from an elderly person, it should be interpreted as a false-positive reaction based on the fact that menstrual fluid cannot be expected to originate from women after menopause. possible limitations need to be taken into consideration when analyzing samples with the seratec pmb test. ddimers are normally not detectable in blood from healthy individuals but d-dimer levels can be elevated in patients suffering from acute peripheral thromboembolisms, pregnancy, previous surgery, or active malignancy, which may lead to a false-positive test result [19, 22] . in unclear cases, a reference sample should therefore be tested to avoid misinterpretation of a positive test result for menstrual fluid. moreover, d-dimer levels have shown to rise in postmortem samples [18] , which was confirmed in our study with 70% of the postmortem blood samples giving a light to strong positive result for d-dimer presence. this should be kept in mind as a possible source of false positivity when postmortem samples need to be assessed. however, this limitation is of only limited relevance because most blood samples observed at crime scenes can be expected to originate from a living (and therefore actively bleeding) individual. aside from these limitations, the test is simple to implement into forensic workflows and it is possible to use the remaining buffer-sample solution or the sample pad for subsequent dna analysis. the performance is not user-dependent as it is standardized and no special training of the analyst is needed. all the above considerations had been useful for the interpretation of the ten sexual assault casework samples. it is worth mentioning that the peripheral blood marker gave consistent results with human blood tests already in use in the forensic field. the seratec pmb test is the first commercially available example for a multiplexed immunochromatographic assay for forensic body fluid identification targeting two different body fluids in a single test. this, in combination with the possibility of performing standard dna profiling from leftover buffer-sample solution, is an important characteristic of this test because it ensures minimal sample loss during body fluid testing. validation of an immunochromatographic d-dimer test to presumptively identify menstrual fluid in forensic exhibits evaluation of semen presumptive tests for use at crime scenes evaluation study about the seratec rapid tests analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene women's gynecologic health rna/dna co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative ednap exercise mrna profiling using a minimum of five mrna markers per body fluid and a novel scoring method for body fluid identification the identification of menstrual blood in forensic samples by logistic regression modeling of mirna expression validation of forensic body fluid identification based on empirically normalized mirna expression data dna methylation-specific multiplex assays for body fluid identification identification of body fluid-specific dna methylation markers for use in forensic science independent validation of body fluid-specific cpg markers and construction of a robust multiplex assay forensic body fluid identification: state of the art identification of menstrual blood by the simultaneous determination of fdp-d dimer and myoglobin contents d-dimer assays for the identification of menstrual blood cross reaction and forensic comparison of blood testing done by private and public sector laboratories seratec pmb user instruction 1-2 usefulness of a latex agglutination assay for fdp d-dimer to demonstrate the presence of postmortem blood age, functional status, and racial differences in plasma d-dimer levels in community-dwelling elderly persons biochemistry changes that occur after death: potential markers for determining post-mortem interval vaginal bleeding between periods d-dimer concentrations in normal pregnancy: new diagnostic thresholds are needed acknowledgments we would like to thank all donors for participating in this project and dr. ivan dieb miziara for authorizing the use of sexrelated crime samples in this study. key: cord-278174-znc99yos authors: ramsey, glenn title: managing recalls and withdrawals of blood components date: 2004-01-31 journal: transfusion medicine reviews doi: 10.1016/j.tmrv.2003.10.005 sha: doc_id: 278174 cord_uid: znc99yos abstract donor centers are issuing a growing number of recalls and market withdrawals to hospital transfusion services about blood components. more than 1 in 2,000 units were recalled in the late 1990s in the united states. the most common reason for these notices from donor centers is postdonation donor information. most of these units had been transfused, and many present a “risk of a risk” (ie, a problem might have been present that might have affected the recipient). a few regulations and standards address recalls in general terms, but transfusion services generally have wide discretion in the management of specific common recall problems. the food and drug administration (fda) is now including posttransfusion evaluations in its guidelines for emerging infectious threats to the blood supply. we suggest that hospital transfusion services should have standard operating procedures for managing recalls and that the hospital transfusion committee and the quality management program should provide local input or oversight. using the fda’s categories of donor center biological product deviations, we provide recommendations to consider for when to notify the recipient’s physician, after postdonation information is received about a previously transfused blood component. more study of this important everyday issue in transfusion medicine is highly desirable. b lood components are regulated as drugs. however, because they are derived directly from humans, they will always be subject to biological variation. their unit-by-unit production, testing, storage, distribution, and record keeping are also more complex than other medications, leading to further opportunities for deviations from the expected. the us food and drug administration's (fda) current good manufacturing practices extend to after manufacturing so that, if problems are found with the finished drug, measures must be taken to correct the product or prevent consequences to patients if possible. over the past 10 to 15 years, the stricter application of these principles to blood components has led to a growing number of recalls and withdrawals by blood suppliers, as well as a concomitant increase in the numbers of notices sent to transfusion services about blood products they have received. furthermore, because platelets and red blood cells have a short shelf life and because hospitals do not keep a large reserve of blood components in storage, most of these notices about nonconforming blood products are often received after the units had been transfused. in retrospect, many of these recalled units presented a "risk of a risk" (ie, that the problem might have affected the patient if it had actually been present but whether it was truly present is often unknown). the lookback requirements for tracing units from donors later found to have human immunodeficiency virus (hiv) and hepatitis c virus (hcv) infections have been formalized by the fda in rules and guidances. 1,2 (a revised rule for hiv and hcv lookbacks was proposed in 2000 but not finalized as of this writing. 3 ) hcv and hiv lookbacks have been discussed elsewhere 4,5 and will not be covered in depth here. in contrast to hiv and hcv lookbacks, other types of notices about blood products have few specific formal rules or guidelines as to how they should be handled by the transfusion service. the purpose of this review is to discuss the management of recalls and withdrawals of blood components. "recall is an effective method of removing or correcting consumer products that are in violation of laws administered by the food and drug administration" (title 21, code of federal regulations (cfr), section 7.40 (21 cfr 7.40)). the fda classifies recalls into 3 categories, from the most serious, class i, to the least serious, class iii ( table 1 ). all recalls under fda jurisdiction are published on line in the weekly fda enforcement report. 6 there is a lag time of weeks to months from the recall to publication, and the enforcement report does not say when the original problem occurred. the fda says recalls are "voluntary" by the manufacturer, although conducting recalls is required, and the fda has the power to initiate recalls if the manufacturer does not act as required. recalls can include public warnings in serious situations (21 cfr 7.42). in 1 blood component recall involving improper donor testing in a large centralized laboratory, several blood centers who had used that laboratory made community media announcements and advertisements notifying transfusion recipients to seek testing if desired. 7 market withdrawals are also defined in table 1 . the fda has stated that withdrawals because of problems beyond the control of the manufacturer may be classified as withdrawals. for example, the us nationwide removal of tylenol (mcneil-ppc, ft washington, pa) from stores because of fatal cyanide tampering in chicago in 1982 was classified as a market withdrawal not a recall. 8 most notices about blood components involving postdonation donor information are now categorized as market withdrawals. market withdrawals are not published by the fda so their magnitude is unknown. ramsey and sherman 9 analyzed recalls of blood components published by the fda from 1990 through 1997. during this 8-year period, recalls were announced for nearly a quarter-million blood components, comprising about 1 in 700 units available to hospitals. seventy-six percent were in class iii recalls, 24% were of class ii, and only 12 units were designated in class i (because of viral or bacterial infection). three fourths of the recalled units involved incorrect or incomplete testing for syphilis or viral infection. the next largest categories were for donors with reactive or previously reactive infectious disease tests, which involved 10% of recalled units. nearly 90% of recalled units were included in a small number (22) of large recalls of over 1,000 units each. by 1998, the final year examined, published blood component recalls had shifted away from incorrect or incomplete infectious-disease testing (down to 20% of units) and toward donors with previously reactive infectious-disease tests (51% of units). 10 also in contrast to 1990-1997, two thirds of the recalled units were included in a growing number of smaller recalls of under 1,000 removal or correction of a marketed product that the fda considers to be in violation of the law it administers and against which the agency would initiate legal action, eg, seizure. recall classification for use of, or exposure to, a violative product: class i: reasonable probability [of] serious adverse health consequences or death. class ii: may cause temporary or medically reversible adverse health consequences or where the probability of serious adverse health consequences is remote. class iii: not likely to cause adverse health consequences. market withdrawal: removal or correction of a distributed product which involves a minor violation that would not be subject to legal action by the fda or which involves no violation, eg, normal stock rotation practices, routine equipment adjustments and repairs. units each. about 10,000 blood components were recalled in the 1998 enforcement reports. when compared with the total annual blood components distributed in the united states, the risk of a unit being recalled after issue was about 1 in 2,000. another gauge of the types of problems found with blood components comes from the recent requirement for reporting biological product deviations (bpds) to the fda. by definition, bpds involve products that had been issued but that were later found to have safety, potency, or labeling problems. bpds should include all problems that lead to recalls or market withdrawals. the fda has summarized the first full fiscal year (fy 2002) of bpds reported from licensed blood facilities. 11 their statistics are presented as the number of reports, not the number of units involved, so the overall frequency of blood components involved is unknown. one bpd for incorrect testing could involve many units, whereas bpds for donor suitability are often reported on a unit-by-unit basis. however, the categories and numbers of bpds offer a useful fda-defined framework for categorizing problems found in blood components currently. nearly 22,000 bpds were reported from licensed facilities (excluding plasma centers) in fy 2002. (reports which actually did not need to be reported were excluded.) seventy-six percent were for postdonation information concerning donor high-risk behavior and history. the most common categories here, in order, were travel to malaria and creutzfeldt-jacob risk areas, cancer, tattoos, disease or surgery, deferring medications, and intravenous drug use. the next largest type of bpd, in 10% of cases, was for quality control and distribution problems, such as clotted or hemolyzed units or segments, inappropriate product release (eg, unacceptable quality control, outdated), incorrect temperature, and failure to quarantine after another problem was found. the rest of the donor-center bpds were in donor screening (6.0%), labeling (4.3%), routine testing (1.2%), component preparation (1.2%), collection (0.7%), miscellaneous (0.6%), donor deferral (0.2%), and viral testing (0.2%). the most common items within each of these areas were as follows: donor screening, deferring history, but not deferred; labeling, incorrect autologous donor tag; routine testing, incorrect rh typing; component preparation, incorrect temperature; collection, bacterial contamination; miscellaneous, hcv lookback deviation; donor deferral, previous deferral for history; and infectious disease testing, incorrect syphilis testing. incorrect infectious disease testing, previously the most common category of recalled units in the 1990s, was the least common category of bpds in fy 2002. this may reflect the current widespread use of large, dedicated centralized testing laboratories for donors. the fda has detailed regulations and support documents for the conduct of recalls by the manufacturer. 21 cfr 7.3 defines recalls, and 21 cfr 7.40-7.59 describe the manufacturer's obligations and the fda's processes for monitoring and assessing recalls. two publications by the fda's office of regulatory affairs provide details for their inspectors about how to inspect recall operations: the investigation operations manual, chapter 8, and the regulatory procedures manual, chapter 7. 12,13 these 2 publications are on line at www.fda.gov/ora. the fda may conduct effectiveness checks, which are follow-up surveys of consignees such as transfusion services, to verify that recalls are carried out appropriately by the manufacturer. in contrast, the fda provides much less general information about the response to recalls. one line in the cfr is addressed to consignees: "responsibility of recipient. consignees that receive a recall communication should immediately carry out the instructions set forth by the recalling firm and, where necessary, extend the recall to its consignees in accordance with . . . this section (21 cfr 7.49 [d])." therefore, if the hospital transfusion service has shipped the recalled component, or part of it, to another facility, it should conduct a recall to the second facility. for example, some hospital transfusion services send source plasma to a manufacturer so the source plasma buyer should be notified if the original unit is recalled. if the transfusion service is notified about an unsuitable product, but then issues it inadvertently, then a bpd report would be required. hiv and hcv lookbacks have been referred to previously (table 2) . [1] [2] [3] [4] [5] donors with reactive tests for hepatitis b virus (hbv) and human t-cell lymphotropic viruses (htlv) i and ii were ad-dressed by the fda in a 1996 recommendation 14 (now listed under blood memos) and a 1997 guidance for htlv. 15 the fda recommended withdrawing in-dated components from donors with hbv and htlv markers but stated that they were not recommending consignee notification for the purpose of recipient notification. in our own practice, we perform lookback on units from donors with confirmed hepatitis b surface antigen (hbsag) or anti-htlv (see management of specific problems), but this is not required by the fda. recent fda guidances about emerging infection issues have included provisions about posttransfusion actions. in the january 2002 guidance 16 on creutzfeldt-jakob disease (cjd), the fda stated that notifications about donors deferred for travel, bovine insulin, united kingdom transfusion, or a single family member with cjd were intended only for product removal, and not for notification of recipients. for other more direct donor connections to cjd, including actual donor cjd subsequent to transfusion, "consignee notification could enable the consignee to inform the physician . . . so that recipient tracing and medically appropriate notification and counseling may be performed at the discretion of health care providers." there is an ongoing study by the us centers for disease control and prevention (cdc) and the national blood data resource center to investigate transfusion recipients of blood from subsequently diagnosed cjd patients. 17 in this approved study, the recipients are not notified, but national deaths are monitored to see whether any of the recipients die of cjd. as of august 2002, 331 transfusion recipients with 1,325 person years of follow-up had been studied. a similar study is being conducted in great britain for recipients of blood from donors with later variant cjd (vcjd). 18 no secondary cases of cjd or vcjd have been reported to date. if a notice were received in the united states about a blood component from a donor with later cjd, the cdc or the national blood data resource center should be contacted. in the may 2003 west nile virus (wnv) guidance, 19 if a donor has a medical diagnosis of wnv, then other units from ϫ14 days before to ϩ28 days after the onset of illness should be traced for consideration of notifying the recipients' physicians. if the donor is the suspected likely source of another wnv transfusion case, then other units from that donor collected from ϫ28 days to ϩ28 days from the infectious donation should also be traced and the recipients' physicians notified. "however, in cases when a donor is potentially associated with a case of transmission of wnv, but the epidemiological investigation has not established the specific donor as a likely source of transmission of wnv, we are not recommending notification of the transfusion service." this is slightly misworded because units from recent donors associated with a transfused wnv patient should be sought for precautionary quarantine and retrieval from the transfusion service, but the implication is that the recipients' physicians need not be notified if the donor is not the likely source. wnv nucleic acid testing (nat) began in the us and canada in the summer of 2003. when a donor tests reactive by wnv nat, the fda has not specified at this writing whether to use a 14-day lookback period (as per donor wnv illness) or a 28-day period (as per donor transmission of wnv) for recent donations. the december 2002 guidance on smallpox vaccination and blood donation 20 addressed postvaccination blood collections. if a donor should have been ineligible, but his/her units already have been transfused, then "we recommend that medical directors consider the need for prompt record tracing and, as appropriate, notification of the treating physicians or notification of prior recipients of the affected blood and blood components previously collected from that donor." the april 2003 fda guidance 21 on severe acute respiratory syndrome (sars) gave recommendations for lookback investigation. if a blood product has been transfused from a donor who should have been ineligible at the time of donation, then "we recommend that the establishments consider notifying the treating physician of those recipients about the post donation information, including whether the donor developed suspected sars." donors are deferred for 28 days after recovering from suspected sars or for 14 days after exposure to a person with sars or travel to sars-risk areas. however, the guidance states that if the donor is symptom free for more than 14 days after exposure, then product retrieval and quarantine (and thus presumably notification of the treating physician) are not necessary. in the june 2003 draft guidance on donor syph-ilis testing, 22 lookback, quarantine, and consignee notification are not recommended for previous units from donors with later syphilis or a confirmed syphilis test. the recent fda draft guidance on xenotransplantation 23 and on anthrax 24 call for withdrawal of blood components inadvertently collected from donors with these unusual exposures. the anthrax guidance has recommendations on when to notify the recipient's physician after transfusion of a suspect unit. in the 22nd edition of the standards of the american association of blood banks (aabb), effective november 2003, chapter 7 is on deviations, nonconformances, and complications. 25 standard 7.0 requires policies, processes, procedures and defined responsibility for detecting, investigating, and reviewing deviations. standard 7.1 and its subsections call for nonconforming products to be evaluated, traced, segregated if still present, and prevented from unintended use. blood banks and transfusion services must have processes for identification, quarantine, retrieval, and recall. nonconforming products that already have been released must be evaluated for quality, and when quality may have been affected, the nonconformance shall be reported to the customer. (the "customer" is defined elsewhere as the receiver of a product or service, either another organization or another department within the same organization. in this context of nonconforming products, the "customer" does not refer to the patient who received the product.) records of product nonconformances and actions about them must be maintained for 5 years (reference standards 6.2a and 6.2c). the transfusion medicine checklist of the laboratory accreditation program of the college of american pathologists (december 2002 edition) 26 has 2 questions touching on some aspects of recalls and notifications. trm.42120 asks if there is a procedure to identify and quarantine all previous components from donors who now test repeatedly reactive in viral marker screening tests. trm.42170 asks for a "procedure consistent with [medicare] and fda regulations/guidelines for notification and counseling of recipients who have been transfused with a potentially infectious blood component." the commentary for this question refers to federal requirements for notifying recipients about subsequent confirmed positive infections in their donors. the main intent of the question is to require hiv and hcv lookback. however, the mention of guidelines in the question may be construed to include other recent fda guidelines as discussed earlier. some hospitals collect blood products, and some blood collection centers have transfusion services. the previously mentioned regulations and standards apply to those facilities as well (ie, notices should be transmitted from the collection arm to the transfusion arm of the same establishment when necessary). within the extant regulations and standards summarized previously, the transfusion service has broad latitude on how to manage recalls and notifications from blood suppliers, other than hiv and hcv lookback. the remainder of this article offers recommendations based on our experience. however, these are only recommendations, and others may choose to use different approaches. given the paucity of literature about this important everyday area of activity in transfusion medicine, we hope the following will be helpful in providing a framework for others to organize their programs as suited for their hospitals. future study, analysis, and discourse on the management and outcomes of recalls and notifications will be very helpful. in particular, the yield of problem investigations and the medical benefit of these notices for transfusion recipients deserve more examination. at a minimum, we would suggest that recall management processes include the following key elements: 1. have a standard operating procedure. however one chooses to manage recalls and notifications, and in however much detail desired, a procedure is a prerequisite for instructing staff in these key elements. 2. act immediately to quarantine and discard, or return, blood components as instructed by the supplier. time is of the essence when a notice is received. immediate action should be taken to quarantine the unit and prevent release. is a recalled unit of plasma being thawed in the waterbath? in case a large number of units is involved, the transfusion service staff should have round-the-clock ac-cess to facsimile or secure electronic mail to facilitate transfer of information and prevent transcription errors from verbal messages. for laboratories with blood bank computer systems, both computer and physical quarantine must be done, although computer quarantine may be done first to expedite prompt blockage of issue. as noted previously, if a unit is erroneously released after a notice is received to quarantine it, then a biological product deviation report must be sent to the fda. 3. review and determine the medical implications of units already transfused. this is further discussed later. 4. keep records of all notices and actions as required (eg, 5 years in aabb standards). 25 telephone instructions should be logged and obeyed, but also documented pending written follow-up. record systems should include the capability to track investigations in progress. transfusion services may want to consider means to search recall records by unit number or patient or the ability to tag the unit or patient laboratory record that a recall has occurred. large hospitals may find a confidential database useful. transfusion services may wish to review their general strategies for oversight, management, and record keeping with the hospital's risk management and/or legal counsel. although most of these problems occur before the hospital receives the units, there could be potential medicolegal implications for the hospital and the physician. for example, it may be advisable to consider these investigations as a subcategory of the hospital's overall incident management program for quality improvement or at least to bring serious problems to this forum. the transfusion committee or its local equivalent may wish to provide general oversight for the recall management process. there are several advantages to performing recall investigations under the aegis of the transfusion committee. this educates key physicians in the range of problems found with blood components after transfusion. it provides the medical staff's representatives a forum to review and approve the general and specific features of the procedure. the transfusion committee also provides a logical venue to bring all or selected recalls into the hospital quality improvement program. some other resources of expertise in the hospital may be helpful in certain problems. the infection control office can provide advice on transfusions, which may have posed a serious infection risk in hindsight. potentially, this consultation could include immediate measures such as baseline patient infectious disease testing (eg, a donor calls back to report recent previously unsuspected exposure to hiv) or antimicrobial therapy (eg, a platelet culture becomes positive after the product was already transfused). for perplexing conundrums in posttransfusion problems, the hospital ethics committee might provide a useful forum for discussion. the hospital public relations office should be informed when a problem could be of concern to the news media and the public, such as a large recall of blood products in the community. many transfusion recipients have died of their underlying illness by the time a notice arrives about one of their blood components. in an hiv lookback, their next of kin must be notified. however, for other problems, no further investigation is needed. table 3 gives suggestions for whether to inform a recipient's physician about a problem transfusion. the list of problems is adapted from the fda's categories of donor-center bpds, with some additions. our general approach is that if a blood component might have posed a tangible infectious or other risk, then the patient's physician should evaluate whether the patient may have been affected. on the other hand, if the problem with the product did not pose a tangible risk to the patient, then the transfusion service physician, with the oversight of the transfusion committee if desired, can exercise informed medical judgment to not notify the recipient's physician. blood suppliers should provide adequate information for the transfusion service to evaluate the problem and counsel the physician if needed. without violating the donor's confidentiality, the transfusion service may seek further information from the blood supplier if the first notice is insufficient for a decision. many physicians are not familiar with the details of when and why blood donors should be deferred. when the patient's physician is informed about a (table 4 and text). hiv or hcv nucleic acid tests (nat) have short seroconversion windows, but postdonation nats have not been incorporated yet into fda rules and guidances on hiv or hcv lookbacks. [1] [2] [3] problem with a nonconforming blood component, some background information is often useful. for recurring notices such as malaria-area travel, a form letter may be convenient. for most routine notices, we have not required follow-up information from the physician. however, for sensitive issues, the transfusion service physician may offer assistance in patient counseling if desired. when there is concern about the possibility of infection risk from the donor, testing of the donor and/or patient may be indicated. when a donor is deferred for a risk factor before donation, no testing is done at that time. from the collection facility's standpoint, this may discourage test seeking by ineligible donors. unfortunately for the transfusion services, which have previously received blood components from that donor, the current infection status of the donor is thus left unknown. if the donor has been tested since the donation in question, the last date of testing should be included in the notification or be sought by the transfusion service. in serious donor risks, such as known hiv exposure, the transfusion service may ask the collection facility to seek donor testing if feasible. seroconversion window information is helpful for counseling and donor testing after exposure or for recipients after transfusion. if the donor has tested negative after the seroconversion period of the test in question has elapsed, then donor infection at the time of the donation can be ruled out. table 4 shows seroconversion window periods for viral tests required in blood donors. the cdc recommendation for hiv antibody testing after needlestick exposure is 6 months, 29 that is more conservative than the table figures. in the 1996 hiv lookback rule and the 2000 proposed look-back rule for hiv and hcv, 1,3 the fda required 12 months before a negative antibody test to rule out the need for lookback in prior donations. nat for hiv and hcv has much shorter window periods than antibody testing. the cdc recommends 4 to 6 weeks of follow-up testing for hcv rna after needlestick exposure. 29 nat has not yet been factored into fda lookback rules and guidances. blood centers have greatly reduced infectious disease testing errors and problems that predominated in recalls of the early and mid-1990s. today's challenge is the donor who does not reveal a deferring risk factor or condition. an anonymous survey of blood donors for risk behaviors revealed that 1.9% had a deferrable risk at the time of their donation (1.7% after testing and confidential unit exclusion). 30 efforts have been made to reshape screening questions to improve their comprehension by donors. 31 computer-assisted interviews may offer donors a more comfortable way to reveal risk factors. 32 because many current donor risk factors are based on international travel, another area for consideration is making information more widely accessible for travelers and their physicians about when not to donate blood. for example, the cdc's key international travel health publication, the "yellow book," does not tell physicians and travelers about when to avoid blood donation, and for how long. 33 likewise, when blood donor deferrals began for sars-area travel, this was not included in cdc information pages for travelers. 34 more publicity about the consequences of travel on blood donation might reduce recall rates for geographic donor risks. in today's regulatory climate, hospital transfusion services receive numerous recalls and market withdrawals from their blood suppliers. hospitals should have procedures for managing the quarantine, medical evaluation, and records of these recalls. the transfusion committee may provide oversight for local preferences about when to inform the recipient's physician. more study of the medical importance of recalls for transfused patients is needed. because the predominant reason for notices about blood products is postdonation information about the donor, this is an important area for quality improvement by blood suppliers. new mea-sures to improve donor understanding and communication about deferring information could help reduce blood component recalls and withdrawals. guidance for industry: current good manufacturing practice for blood and blood components: (1) quarantine and disposition of units from prior collections from donors with repeatedly reactive screening test for antibody to hepatitis c virus (anti-hcv); (2) supplemental testing, and the notification of consignees and blood recipients of donor test results for anti-hcv risk of human immunodeficiency virus (hiv) transmission by blood products before the implementation of hiv antibody screening food and drug administration (weekly) nordenberg t: recalls: fda, industry cooperate to protect consumers blood component recalls in the united states recommendations for the quarantine and disposition of units from prior collections from donors with repeatedly reactive screening tests for hepatitis b virus (hbv) guidance for industry: revised preventive measures to reduce the possible risk of transmission of creutzfeldt-jakob disease (cjd) and variant creutzfeldt-jakob disease (vcjd) by blood and blood products abstr) 19. guidance for industry: revised recommendations for the assessment of donor suitability and blood and blood product safety in cases of known or suspected west nile virus infection draft guidance for industry: precautionary measures to reduce the possible risk of transmission of zoonoses by blood and blood products from xenotransplantation product recipients and their intimate contacts updated us public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis blooddonor perceptions of health history screening with a computerassisted self-administered interview available at: www.cdc.gov/ncidod/sars/travel_advice. htm key: cord-016815-pva22xy7 authors: mannem, hannah c.; donahoe, michael p. title: transfusion and acute respiratory distress syndrome: clinical epidemiology, diagnosis, management, and outcomes date: 2016-06-11 journal: hematologic abnormalities and acute lung syndromes doi: 10.1007/978-3-319-41912-1_11 sha: doc_id: 16815 cord_uid: pva22xy7 transfusion related acute lung injury (trali) is a life-threatening complication of blood product transfusion. it is the leading cause of blood product transfusion related death in the usa. the syndrome is defined by hypoxemic respiratory failure with bilateral infiltrates on chest x-ray in the setting of a blood transfusion and absence of cardiac failure. the exact incidence of trali is unknown, but the incidence is higher in the critically ill patient population. multiple patient and donor related risk factors for trali exist, including critically illness, alcohol use, and receiving transfusions with high plasma volumes. practitioners should have a low index of suspicion for the diagnosis of trali, and blood bank reporting is vital to aid in diagnosis and future prevention. management is primarily supportive care, with supplemental oxygen as the mainstay for therapy. despite the transient course of trali, its morbidity is severe with the majority of patients requiring mechanical ventilation and treatment in the intensive care unit. for patients that survive trali, outcomes are promising without residual pulmonary deficits. prevention strategies over the past 10 years have helped to decrease the incidence of trali and have led to increased awareness of this condition in the medical field. the association was made into a distinct clinical entity with specifi c clinical criteria in 1983 by popovsky and colleagues and redefi ned by the national heart, lung, and blood institute (nhlbi) , as well as the canadian consensus conference (ccc) in 2004 [ 5 -7 ] . two key points were highlighted in the revision of the defi nition. the fi rst was emphasizing an acute and new presentation of respiratory distress. the second focus was to eliminate other temporally associated, alternative risk factors to explain the new lung injury (table 11 .1 ). two other terms were also defi ned-possible trali and delayed trali . possible trali occurs when the acute respiratory distress takes place in the setting of a blood transfusion, as well as other co-existing risk factors for development of acute respiratory distress syndrome (ards) , including: trauma, sepsis, pancreatitis, aspiration, inhalation, drug overdose, or burns. delayed trali is defi ned as trali which occurs after 6 h but within 72 h of a blood transfusion (table 11 .1 ). these distinctions between trali, possible trali, and delayed trali help to further elucidate incidence, pathophysiology, and treatment of this condition by clarifying the disease for future research investigations. two pathophysiologic mechanisms of trali have been recognized, immunemediated trali and non-antibody mediated trali . anywhere from 65 to 90 % of reported cases are found to be immune-mediated trali, which occurs when leukoagglutinating antibodies from the donor blood bind to conjugate recipient antigens [ 8 ] . by defi nition, evidence of antibodies from the blood donor are present; most commonly anti-hla and anti-hna antibodies, with anti-hna3a associated with worse clinical outcomes [ 9 , 10 ] . the second mechanism for development of trali is classifi ed as non-antibody mediated trali and stems from an antibody independent mechanism. approximately 15 % of trali falls into this category, in which no antibodies are found in the donor blood product. silliman and colleagues have described the non-antibody mediated mechanism as a two-hit model. the fi rst hit involves neutrophil priming and sequestration secondary to a preexisting condition in the recipient. in the second hit, biologic modifi ers such as lipids in the blood product activate neutrophils and lead to capillary leak in the lung endothelium [ 11 , 12 ] (see chap. 10 ). risk factors for the development of trali can be broken into two categories, recipient and donor related risks. the recipient of the blood product may have underlying disease states and clinical conditions, which put them at increased risk (table 11 .2 ). also the donor profi le and blood components being transfused may also put the recipient at higher risk for development of trali. multiple recipient related risk factors are noted in the literature. most of these studies are retrospective and small. however, it is evident from clinical data that the critically ill population is at the highest risk for the development of trali [ 13 ] . in one multicenter, prospective trial, history of liver transplant, chronic alcohol use, active tobacco use, shock, increased il-8 levels in serum, increased peak airway pressures of >30 cm h 2 o on the ventilator, and an overall positive fl uid balance were all signifi cant risk factors for trali [ 14 ] . multiple studies reveal sepsis and shock as major risk factors. not only being critically ill, but also being on mechanical ventilation at the time of transfusion may increase risk independently. a prospective cohort study showed 33 % of patients on mechanical ventilation at the time of transfusion developed acute lung injury [ 15 ] . multiple other studies have also shown recipient risk factors such as: major surgery within 72 h of blood transfusion, hematologic malignancy, higher apache ii scores, and active liver disease [ 16 ] . the risk for development of trali also increases with the number of transfusions, as seen commonly in the trauma population where patients are receiving massive transfusions [ 13 ] . not only critically ill patients, but cardiac and orthopedic surgery patients are also at higher risk for trali development [ 17 ] . the time on cardiac bypass appears to be correlated as well, with longer bypass times leading to higher risk of trali development [ 18 ] . despite the multitude of recipient risk factors reported, most of which are seen in the critically ill population, trali is also reported in otherwise healthy individuals at the time of transfusion [ 19 ] . the development of trali in this healthy patient population supports the realization that the risk of trali is not dependent on the recipient alone. all forms of blood products have been reported to cause trali, including: whole blood, packed red blood cells, apheresis platelets, fresh frozen plasma, cryoglobulin, intravenous immunoglobulin, granulocytes, and allogeneic stem cells [ 12 ] . however, blood products with higher plasma volume are at the greatest risk, specifically fresh frozen plasma, apheresis platelets, and whole blood. in the fda reported cases of death due to trali, fresh frozen plasma was the most implicated [ 7 ] . in one retrospective cohort study from 2007, fresh frozen plasma and platelet transfusions led to a higher incidence of trali versus red blood cell transfusion in the icu population [ 20 ] . it remains unknown the exact amount of plasma which must be transfused in order for trali to develop. reports of as little as 10-20 ml of plasma transfused before trali development are in the literature; however, plasma volumes greater than 50-60 ml are thought to be the threshold which puts patients at a higher risk [ 12 ] . another important risk factor is the gender of the donor, and preventive strategies in the past 15 years have focused on gender related donor deferral . female, multiparous donors have allo-immunization from pregnancy. blood from this particular group of donors has a much higher risk of trali development in the recipient secondary to the anti-hla and anti-hna antibodies, which bind to recipient antigens and lead to immune-mediated trali. the prevalence of antibodies in this population increases with parity. a 26 % approximate frequency of anti-hla antibodies exist if a female has had more than three pregnancies [ 21 ] . another potential risk factor where studies have shown controversial data is blood product storage time. experts in the fi eld hypothesize that longer storage times of red blood cells may lead to a higher incidence of trali. experimental models in preclinical trials show a positive correlation between longer blood storage times and trali; however, there remains no overt clinical evidence to support the fi nding [ 22 ] . studies done in the preemie population showed no difference in the incidence of trali based on blood storage time. an ongoing study in the adult intensive care unit population is underway that hopefully will help to clarify the importance of blood storage time as a potential risk factor [ 23 ] . the true incidence of trali is unknown secondary to prior lack of a concise definition, the inconspicuousness of the diagnosis, and lack of a structured reporting system. it occurs in all age groups, including children and the geriatric population. it occurs at the same frequency in women and men. reported trali incidence varies between 0.08 and 15 % of patients transfused and 0.01-1.12 % per product transfused, with the higher incidence in the critically ill patient population [ 24 ] . up to 50-70 % of patients in an intensive care unit receive some form of blood product transfusion, and more independent patient risk factors exist in the critically ill population, which may account for this increase in incidence (see section "risk factors"). even though the overall reported incidence of trali remains low, it is almost certainty an under-recognized and underdiagnosed condition. in the setting of no gold standard for diagnostic testing, a passive reporting system, and an array of mild cases which do not meet the consensus defi nition of the disease, trali remains under-reported [ 25 ] . despite the underestimated incidence of trali, the overall frequency has decreased since the mid-2000s secondary to preventative strategies for plasma and platelet transfusions (see section "prevention"). as stated before, all blood products have been implicated in trali development, and the incidence of trali varies based on blood product components. products with higher plasma volume have higher incidence of trali. reports reveal incidences at approximately 1/432 whole blood products vs. 1/7900 fresh frozen plasma vs. 1/557,000 red blood cells [ 12 , 22 ] . however, the incidence of trali in plasma products has decreased in the past decade secondary to risk mitigation strategies, leaving the incidence of red blood cell transfusions at a higher rate in the more recent years [ 26 ] . trali can present with a large variation in disease severity. by nhlbi and ccc defi nition 100 % of patients with trali have hypoxemic respiratory failure and bilateral pulmonary infi ltrates on chest x-ray. clinically, the most common complaint of patients is dyspnea. however, a large number of patients are critically ill and on mechanical ventilation at the time of blood transfusions leading symptoms to be unhelpful. despite patients being unable to report symptoms, clinical signs of respiratory distress and failure are present, typically within one to 2 h of a blood transfusion in the majority of patients. predominately, patients are tachypneic, and in approximately one-third of patients, fever and/or hypotension may develop. rarely patients may develop new onset hypertension. most notably in the vital signs, spo2 should be decreased compared to before the transfusion. patients on mechanical ventilation may experience a change in pulmonary compliance with an increase in peak and plateau pressures. pink, frothy secretions from the mouth or endotracheal tube occur in roughly half of patients who develop trali. physical exam should help rule out other etiologies of respiratory distress and should be thorough including a complete lung, heart, and skin exam. lung auscultation reveals bilateral crackles. exam fi ndings suggestive of cardiac failure should not be present, such as jugular venous distention and an s3 on cardiac auscultation. it is important to keep in mind that very mild cases of trali do exist, which may not fall into the nhlbi and ccc defi nitions. mild cases may go unrecognized or present with a similar presentation to the underlying disease process, albeit in a less severe form . practitioners should have a high index of suspicion for trali when administering blood products, especially in the critically ill population. diagnosis can be diffi cult as there is no gold standard diagnostic test for trali. any person who develops even the least amount of dyspnea or respiratory distress in temporal association with a blood product transfusion should have further clinical and diagnostic evaluation for trali. patients who meet the 2004 nhlbi and ccc defi nition (table 11 .1 ) including, new hypoxemic respiratory failure with a pao 2 /fio 2 ratio <300 and bilateral pulmonary infi ltrates within a 6 h time frame from blood product transfusion, deserve further workup to confi rm the diagnosis. one of the goals of the diagnostic workup should be to rule out other possible etiologies for the new development of ards, which would then classify the patient as possible trali. no diagnostic lab tests are available that confi rm the diagnosis of trali. an arterial blood gas can be helpful to quantify the degree of hypoxemia. the most common laboratory fi nding is acute and transient leukopenia, which is thought to be secondary to neutrophil sequestration into the pulmonary vasculature and can be seen in 5-35 % of patients [ 27 ] . thrombocytopenia has also been reported in trali. other laboratory tests, although not diagnostic may also be helpful. in other etiologies of ards such as sepsis, a leukocytosis may be present. an elevated brain naturitic peptide can be seen in transfusion associated circulatory overload (taco) and should not be elevated in trali alone. as stated before, a chest x-ray revealing bilateral pulmonary infi ltrates is a ubiquitous fi nding in trali, and should be performed for any patient with suspicion of the diagnosis. historically the pulmonary infi ltrates in trali were described as "white out lungs." this may be the scenario in extreme cases; however, both alveolar and interstitial infi ltrates have been described in a spectrum from bilateral and patchy to diffuse territories of the lung fi elds. despite the fi ndings being nonspecifi c, the presence of bilateral infi ltrates should reach 100 % in this patient population. the chest x-ray is also helpful to eliminate other etiologies of acute respiratory failure, such as pneumothorax . for any suspected trali reaction, it is of vital importance the associated blood bank be contacted. typically a transfusion reaction lab panel is sent, which is directed by the blood bank or transfusion medicine director. the panel includes a complete blood count, haptoglobin, bilirubin, direct coombs test, and most importantly hla and hna antibody testing in the donor blood sample. anti-hla and anti-hna antibodies strongly support the diagnosis of trali but are not essential for diagnosis. 15-25 % of trali reactions are found to be non-antibody mediated [ 21 ] . however, positive antibody results can guide future trali prevention if found in the donor blood product (see section "prevention"). antibody testing may take days to weeks for results, and therefore no acute treatment decisions should be made based on antibody testing alone. in distinguishing trali from other disease states it is important to consider other causes of ali/ards, as well as other transfusion reactions. in 2004, new terminology was instituted as part of the trali defi nition, termed, possible trali . this defi nition takes into account other etiologies of ali/ards, which the patient may be at risk for at the time of blood transfusion (table 11 .1 ). since no gold standard diagnostic test exist for trali, and it occurs most commonly in the critically ill population with multiple other comorbidities, possible trali remains a very relevant diagnosis. if any of these other conditions exist or are suspected, a defi nitive diagnosis of trali cannot be made. further diagnostic workup should be done in order to eliminate the additional etiologies. fever can occur as part of trali; however, pneumonia, pancreatitis, and sepsis should be suspected as well as an etiology of the acute lung injury. cbc, blood cultures, and chest x-ray can all help to further delineate other disease states. other conditions such as inhalation, drowning, cardiac bypass, drug overdose , and trauma may be more obvious from history alone. various other blood transfusion reactions exist, all of which can overlap with aspects of the clinical presentation of trali. each blood transfusion reaction is managed differently, therefore it is vital to establish the correct diagnosis. the transfusion reaction that mimics trali the most is taco (see chap. 12 ). taco may coexist with trali and distinguishing between these two diagnoses may be diffi cult (table 11 .3 ). both conditions present acutely during or after blood product transfusion. also, both lead to acute respiratory distress and hypoxemia with bilateral infi ltrates on chest x-ray. while trali's clinical presentation stems from non-hydrostatic pulmonary edema with capillary leak, taco is secondary to hydrostatic pulmonary edema. the two conditions are both transient but managed differently. diuretics are the mainstay of treatment for taco, but may be detrimental in the treatment of trali (see section "medications"). a positive fl uid balance is a risk factor for development of trali, and if the positive fl uid balance is secondary to compromised cardiac function a higher awareness for taco should exist. while no defi nitive test exists to distinguish between the two, diagnostic tools such as elevated jugular venous pressure, an s3 on cardiac auscultation, a transthoracic echo showing depressed cardiac function, and/or an elevated bnp may suggest taco vs. trali. if the patient has a pulmonary artery catheter in place, an elevated pulmonary capillary wedge pressure and/or central venous pressure also favors the diagnosis of taco. as stated before, chest x-ray is unhelpful in distinguishing between the two diagnoses. other transfusion reactions may also overlap in clinical presentation with trali; however, they are usually more obvious to diagnose. like trali, an anaphylactic reaction from a blood product transfusion may also lead to hypoxia and hypotension. conversely, the clinical presentation of patients undergoing an anaphylactic reaction may demonstrate signs of airway compromise, such as stridor, bronchospasm, laryngeal edema, and/or wheezing, as well as an associated rash, urticaria, and/or diarrhea, all of which are not seen in trali alone. in septicemia from blood product transfusion, which can occur in the setting of contaminated blood products, microbiology is usually positive. patients may also have a leukocytosis , which is very uncommon in trali. platelets are most commonly associated with septicemia from a transfusion. lastly, hemolytic transfusion reactions develop acutely with blood product transfusion, but hypoxia and acute respiratory distress are not the mainstay. fever and hypotension occur in almost all patients with hemolytic reactions and less often in trali. laboratory tests will also reveal a hemolytic pattern, such as a low haptoglobin , elevated unconjugated bilirubin and an elevated lactate dehydrogenase. similar to the diagnosis, the management of trali is also nonspecifi c. no exact therapy for trali exists, and supportive therapy is the mainstay for treatment. if trali is suspected while a blood product is actively being transfused, it should be stopped immediately. all subsequent blood product transfusions should also be held in the acute setting until the diagnosis is made and treatment ensued. as mentioned before, the blood bank or transfusion medicine physician should be notifi ed with any suspicion of trali in order to potentially identify and exclude involved donors if relevant antibodies are present. oxygen supplementation is the primary management in trali. although mild cases are reported where little to no oxygen is necessary, almost all patients require some form of oxygen. studies show up to 70-80 % of patients develop severe enough hypoxemia to require mechanical ventilation [ 28 , 29 ] . there are no specifi c studies looking at mechanical ventilation strategies in trali specifi cally; however, it is reasonable to adopt the ventilation strategies from the ards network trial [ 30 ] . the restrictive tidal volume approach with tidal volumes set at 6 ml/kg of predictive body weight vs. 12 ml/kg has been shown to improve mortality in ards, and therefore should be the mainstay ventilation approach in the trali patient population. maintaining plateau pressures <30 cm h 2 o has also been shown to improve mortality and the incidence of barotrauma in the ards population [ 30 ] . in severe cases where mechanical ventilation fails to support the patient's physiologic demands, the use of extracorporeal membrane oxygenation (ecmo) has been described in case reports [ 31 , 32 ] . however, no randomized control studies exist to support the use of ecmo for trali specifi cally . the volume status of patients who develop suspected trali should be examined carefully, as management decisions are dependent on this judgement. as mentioned above, in the patient who appears to be volume overloaded with depressed cardiac function the diagnosis of taco should be strongly considered, and diuretics should be administered. commonly patients who develop trali are found to be hypovolemic [ 33 ] . trali in the hypovolemic patient may lead to hypotension and shock. intravenous fl uids should be given in this setting, as well as pressors if needed, to support end organ perfusion during the acute episode. while steroids have been studied extensively in ards, no randomized control trials looking at the use of steroids in patients with trali have been completed. the use of steroids in the ards population remains controversial, but data suggest use after 14 days may be harmful [ 34 ] . in patients with trali, case reports with intravenous corticosteroids do exist [ 27 ] . however, in the setting of no true prospective clinical trials, the negative side effects, and the transient clinical course of trali, the use of corticosteroids is not routinely recommended in the treatment of trali. evidence from the factt trial supports the use of a conservative fl uid strategy in the ards population [ 35 ] . however, as stated before patients who develop trali are at risk for hypotension and shock, especially in the setting of hypovolemia. intravenous fl uids are the mainstay of therapy for hemodynamic support early on in trali, especially without evidence of coexisting taco. diuretic therapy should be used judiciously in this patient population, as it may worsen outcomes early on. based on evidence from the ards population, if patients are still requiring high levels of oxygen supplementation once they are hemodynamically stable and volume resuscitated, a role for diuretic use in trali may still exist [ 8 , 36 ] . with no specifi c management strategies for trali exist, prevention measures are of the utmost importance. over the past 10 years policies have been put into place at blood product donation centers in order to guide risk mitigation. the largest risk mitigation strategies so far have focused on plasma donation. no practical risk reduction measures are established for red blood cell transfusion prevention from a donation perspective. some experimental models suggest washing of stored red blood cell products to prevent trali, but it is yet to be determined if this strategy makes a difference and can be feasible in a clinical setting. however, strategies exist to assist in the prevention of all adverse transfusion reactions, most importantly being the use of conservative transfusion practices. an overall judicious approach to blood product transfusion is the simplest and most effective strategy for trali prevention. evidence from a randomized, doubleblinded control trial shows the incidence of ards is decreased with a conservative red blood cell transfusion strategy vs. a liberal one [ 37 ] . other studies suggest ffp is still over utilized at times by physicians with no clear indications for its use [ 24 ] . with electronic medical records in the forefront of today's health care, data suggest that electronic decision support to further guide the ordering of blood product transfusions not only decreased the amount of blood transfusions given but also decreased the incidence of acute lung injury [ 38 ] . blood utilization guidelines and blood conservation programs should be established in health care centers to help minimize unnecessary transfusions. a patient tailored approach should be taken for patients who do need non-emergent blood product transfusions. patient related risk factors for trali should be considered, and an attempt to minimize these risk factors prior to transfusion is an important component of primary trali prevention . as mentioned above, the reporting of any suspected or confi rmed trali episode is vital to secondary prevention. the american association of blood banks (aabb) advocates that implicated donors abstain from any type of blood product donation until leukocyte antibody testing has been complete. in the donors who are found to have leukocyte antibodies which match or are likely to match recipient leukocyte antigens, deferral from at least plasma and platelet apheresis donation is mandatory. if the donor is found to have anti-hna3a antibodies, which have been shown to lead to an increase severity of trali, they are deferred from all types of blood donation [ 39 ] . in the mid-2000s, risk mitigation strategies for trali were instilled in order to exclude "at risk" donors from certain types of blood product donation. an observational study, leukocyte antibody prevalence study (laps) looked at antibody levels in 8000 volunteers for blood donation using fl ow cytometry. only 1-2 % of anti-hla and anti-hna antibodies were present in the male, never-pregnant female, and prior blood product recipient populations compared to multiparous female donors with approximately 24 % of antibodies present [ 40 ] . other studies report higher frequency of antibodies in the multiparous, female population as well, putting patients who receive blood products from this population at an increased risk for immune-mediated trali [ 10 , 41 ] . in 2007 the aabb published the recommendation, "â�¦blood collecting facilities should implement interventions to minimize the preparation of high plasma-volume components from donors known to be leukocyte-allo-immunized or who are at increased risk of leukocyte alloimmunization ." based on this recommendation, the deferral of multiparous, female donors from plasma donations was implicated. the policy to use solely male donors for plasma donation led to a two-thirds decreased incidence in trali [ 24 ] . data also shows since the deferral of multiparous females from plasma donation, the reported cases of deaths to the fda from plasma associated trali decreased from 48 % before 2007 to 27 % from 2008 to 2011 [ 42 ] . the multiparous, female donor deferral strategy also has been used in platelet apheresis donation; however, with the shortage of donors available to meet the demanding needs of platelets, it is not completely feasible to implement complete deferral of high risk donors. another option for primary prevention of trali is the concept of leukocyte reduced blood. reduction of leukocyte antibodies in high volume plasma products has been shown to reduce trali incidence [ 21 ] . however, patients still may be at risk for the non-immune mediated form of trali. pooled solvent detergent plasma was approved by the fda in 2013 as an alternative to ffp. in observational data, there was no reports of trali in ten million units of solvent detergent plasma [ 43 ] . multiple studies from other countries as well have confi rmed the lack of trali in transfusions with solvent detergent products [ 8 , 24 , 44 ] . the pooling and dilution of anti-hla antibodies is thought to play a large role in this decreased incidence. potential risks of pooling high volume plasma products also exist including, exposure to multiple donors and increased transmission of viruses . the majority of patients who develop trali require close monitoring in an intensive care setting. the degree of hypoxemia and lung injury is variable but commonly can be very severe. however, a subset of patients who develop trali will only require minimal supportive care and may even go undiagnosed. no studies have shown clinical severity correlating to the type of blood product or the amount of plasma transfused. worse clinical outcomes have been shown in patients who are positive for hna-3a and hla-a2 antigens [ 9 ] . despite the potential severity of trali, the timeframe is short-lived. studies show that even when patients require mechanical ventilation, the respiratory distress from trali resolves on average within 48 h. in the patient population who is already critically ill, the time course may extend up to 3-10 days [ 14 , 29 ] . one report found that 80 % of trali cases resolved within 48-96 h [ 8 , 45 ] . unlike ards from other etiologies where mortality rates can range from 29 to 70 %, trali has signifi cantly lower rates of death. studies show that mortality rates from trali alone range from 5 to 10 %, with higher percentages quoted from the icu population [ 24 , 46 ] . reports as high as 67 % mortality have been shown in trali patients who were critically ill at the time of trali diagnosis; however, the cohorts utilized in these studies included some " possible trali " cases as well [ 14 , 16 , 32 ] . despite a very similar clinical presentation as ards, trali also differs in the fact that it has minimal to no physical or pulmonary sequelae. in ards, patients are known to have decreased exercise capacity and decreased lung function on pulmonary function tests for up to 5 years after initial pulmonary insult [ 47 ] . in patients who recover from trali there are no residual pulmonary complications. this population of patients returns back to baseline pulmonary function and does not have complications of pulmonary fi brosis. permanent lung damage is rare [ 48 , 49 ] . based on limited evidence, it also appears patients who develop trali are not at increased risk for recurrent episodes of blood transfusion reactions from other donors. caution should be taken with blood transfusions from previously implicated donors; however, overall patients should not be restricted from receiving blood products in the future [ 27 , 50 , 51 ] . current risks of transfusion-transmitted agents: a review fatalities caused by trali giving trali the one-two punch indiscriminate transfusion: a critique of case reports illustrating hypersensitivity reactions toward an understanding of transfusion-related acute lung injury: statement of a consensus panel transfusion-related acute lung injury associated with passive transfer of antileukocyte antibodies proceedings of a consensus conference: towards an understanding of trali transfusion-related acute lung injury: a dangerous and underdiagnosed noncardiogenic pulmonary edema the role of neutrophils in the pathogenesis of transfusion-related acute lung injury the effect of previous pregnancy and transfusion on hla alloimmunization in blood donors: implications for a transfusion-related acute lung injury risk reduction strategy donor antibodies to hna-3a implicated in trali reactions prime neutrophils and cause pmn-mediated damage to human pulmonary microvascular endothelial cells 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prevention and treatment of acute respiratory distress syndrome (ards) in adults: meta-analysis comparison of two fl uid-management strategies in acute lung injury transfusion-related acute lung injury (trali): current clinical and pathophysiologic considerations a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. transfusion requirements in critical care investigators, canadian critical care trials group toward the prevention of acute lung injury: protocol-guided limitation of large tidal volume ventilation and inappropriate transfusion transfusion reactions: newer concepts on the pathophysiology, incidence, treatment, and prevention of transfusion-related acute lung injury prevalence of hla antibodies in remotely transfused or alloexposed volunteer blood donors measures to prevent transfusion-related acute lung injury (trali) cost-effectiveness of solvent/detergenttreated fresh-frozen plasma transfusion-related acute lung injury: reports to the french hemovigilance network transfusion-related acute lung injury: a rare and lifethreatening complication of a common procedure transfusion-related acute lung injury (trali): clinical presentation, treatment, and prognosis functional disability 5 years after acute respiratory distress syndrome case report: transfusion-related acute lung injury (trali)-a clear and present danger transfusion-related acute lung injury transfusion-related acute lung injury recurrent transfusion-related acute lung injury key: cord-003974-cr6omr9l authors: rutter, sara; snyder, edward l. title: how do we … integrate pathogen reduced platelets into our hospital blood bank inventory? date: 2019-03-18 journal: transfusion doi: 10.1111/trf.15241 sha: doc_id: 3974 cord_uid: cr6omr9l for more than 50 years there has been an ongoing effort to combat transfusion‐transmitted infections and provide patients with the safest possible blood. this initiative has driven much of the research within the transfusion community. initial methods included screening donors for travel histories to banned areas and for high‐risk behaviors, but pathogen‐specific assays performed at the collection and manufacturing sites also have become key factors in assuring blood safety. many of these have focused on donor and laboratory‐based screening for transfusion‐transmitted diseases, as evidenced by the hepatitis and human immunodeficiency virus screening in the 1970s, 1980s, and 1990s. more recently, this effort has expanded to develop donor screening assays to identify other blood‐borne pathogens, such as zika and west nile viruses and babesia. bacterial contamination of units of platelets (plts), however, remains a significant concern. in recent years, the food and drug administration has approved rapid tests to identify bacterially contaminated plt units in the blood bank before transfusion. other supplemental methods have been developed, however, that aim to inactivate blood‐borne pathogen(s) present in the blood product, rather than to rely on our ability to identify and interdict contaminated and infected components. pathogen reduction technology, as this is referred to, provides a proactive way to further reduce the risk posed by transfusion‐transmitted infections. for more than 50 years there has been an ongoing effort to combat transfusion-transmitted infections and provide patients with the safest possible blood. this initiative has driven much of the research within the transfusion community. initial methods included screening donors for travel histories to banned areas and for high-risk behaviors, but pathogen-specific assays performed at the collection and manufacturing sites also have become key factors in assuring blood safety. many of these have focused on donor and laboratory-based screening for transfusion-transmitted diseases, as evidenced by the hepatitis and human immunodeficiency virus screening in the 1970s, 1980s, and 1990s. more recently, this effort has expanded to develop donor screening assays to identify other blood-borne pathogens, such as zika and west nile viruses and babesia. bacterial contamination of units of platelets (plts), however, remains a significant concern. in recent years, the food and drug administration has approved rapid tests to identify bacterially contaminated plt units in the blood bank before transfusion. other supplemental methods have been developed, however, that aim to inactivate bloodborne pathogen(s) present in the blood product, rather than to rely on our ability to identify and interdict contaminated and infected components. pathogen reduction technology, as this is referred to, provides a proactive way to further reduce the risk posed by transfusion-transmitted infections. pathogen reduction (pr) of platelet (plt) concentrates using a psoralen as the photoactivating agent is a relatively new food and drug administration (fda)-approved technology that has been deemed suitable by the agency for all patient demographics. 1, 2 the manufacturing process features the addition of a synthetic psoralen compound, amotosalen, to single-donor apheresis plt concentrates, which are then exposed to ultraviolet (uv)-a illumination. psoralens are natural compounds that are found in a number of foods and plants. forms of psoralens have been used in a variety of other therapies including photopheresis. 3 amotosalen intercalates into dna and rna and, once activated by uv-a light, produces an irreversible inter-and intrachain cross-linking of nucleic acids. 4 this cross-linking prevents replication of nucleic acids and, in turn, the pathogen. since intact nucleic acid activity is not needed for plts to function, this approach is believed to diminish the infectious risk posed by a wide variety of pathogens without damaging the hemostatic efficacy of the plts. free photoproducts created by the illumination of amotosalen in this process are adsorbed by a compound adsorption device, minimizing the amount of amotosalen and its byproducts that could be transfused to the patient. [3] [4] [5] [6] any amotosalen that the patient does receive is cleared quickly, with excretion being primarily via the fecal route. 5, 6 the half-life of amotosalen is approximately 6.5 hours, and studies have shown that the peak amotosalen level post-pr plt transfusion is approximately 900 pg/ml. 3, 5, 7 while bacterial contamination remains of primary concern, pr is effective against a wide variety of organisms ranging from viruses to protozoa to bacteria, including those most commonly responsible for plt contamination. some pathogens, however, such as hepatitis a, hepatitis e, parvovirus b19, poliovirus, and prions, the agent of variant creutzfeldt-jacob disease (vcjd), are resistant to the treatment. 2, [8] [9] [10] [11] [12] [13] plt contamination with these latter organisms, however, is uncommon. in addition, due to the intercalation of amotosalen into white blood cell nucleic acid chains, the pr process also prevents t-cell proliferation, and thus it is effective for the prevention of transfusion-associated graft-versushost disease (ta-gvhd), eliminating the need for gamma or x-ray irradiation of these plts. [14] [15] [16] [17] [18] psoralen-based pr techniques have been utilized in europe for many years, and multiple published studies support the benefits and safety of pr products. [19] [20] [21] [22] [23] [24] phase iii clinical trials are also ongoing for riboflavin (miplate) and uv-c light-based pr techniques for plts (capture). 4, 25, 26 pr systems are also currently available for plasma and cryoprecipitate. systems for pr red blood cells are in phase iii clinical trials. [25] [26] [27] [28] [29] impetus and rationale for inventory conversion the plan for conversion to a full pr plt inventory at our academic tertiary care medical facility followed fda product approval and was contemporaneous with the expansion of our cancer hospital. as a result of the growth of our facility and the increasing acuity of our patients, plt usage has increased over the past several years from approximately 7500 units to more than 10,000 units per year. an increase in volume and acuity has led to an increase in pathogenassociated plt transfusion cases. in one incident, a blood bank technologist identified a unit of plts with obvious plt clumping during the sign-out process. as the technologist was aware that this could be indicative of a contaminated product, the unit was quarantined. this unit was one of a non-pr triple-apheresis collection and on testing in the microbiology laboratory was shown to be contaminated with staphylococcus aureus. that unit and the other two contaminated single-donor plt (sdp) cocomponents were interdicted and, fortunately, never reached a patient. the occurrence of this and several other clinical events involving bacterially contaminated plt units at our institution raised serious concerns regarding our organization's ability to fully protect patients from transfusion-transmitted infections. many of our patients are severely immunosuppressed and vulnerable to life-threatening blood-borne infections. such occurrences substantially increased the institutional desire to seek the safest possible plt products. initially we planned a rapid conversion from a conventional plt inventory (pooled random-donor and non-pr sdp) to a full 100% pr inventory. it became clear early in this process, however, that due to our being an early adopter and with the existing supply-side constraints, we were going to have to maintain a dual plt inventory consisting of both pr plts and conventional plts, for several years. during this time, our blood suppliers were developing the infrastructure needed to support our request for a 100% (full) pr plt inventory. in this article, we explain the steps and ramp-up strategies we employed to implement pr plts on an institutionwide basis. we also address what we believe are some key factors others may wish to consider as they investigate the process of converting to an all pr plt inventory. once our transfusion medicine service reached an internal consensus to integrate pr plts into our inventory, we approached our department's chairman for his support and agreement (fig. 1 ). this is an important step, since such a discussion should be held within the department before it is brought to outside administrative areas and service lines. the next approval was sought from the hospital's transfusion committee. once that committee agreed and we had their administrative support to move the project forward, we contacted the institution's chief medical officer with our plan. our guiding philosophy, presented to all levels of the administration, was that patient safety was paramount. concurrence at the c-suite level was an essential early step, as the increased cost of pr plts, compared to that of conventional (non-pr) plts, was substantial. the chief medical officer agreed with the safety-over-cost concept and facilitated communication with other hospitals in our health system. approval from other members of the health system reinforced the importance and timeliness of our goal and the necessity of efforts to foster safer transfusion therapy. it is also worth noting that uniform policies and practices are increasingly being sought among all our health system's member hospitals. we then proceeded to communicate the upcoming policy change to multiple stakeholder groups and service lines (fig. 1 ). our hypothesis was that the more people we included in the decision process, and the sooner we informed them of the plan, the smoother the transition would be once we launched the pr plt program. within the department of laboratory medicine, our blood bank manager and the transfusion safety and compliance officers were key figures assisting with early notification and consultation across multiple service lines (fig. 1) . approval of our institution's transfusion committee was supplemented with consultation and agreement from our hospital's risk management (legal) office as well as the hospital's ethics committee (fig. 1 ). the risk management and ethics committees were approached due to both the medicolegal and the ethical implications of managing a dual plt inventory while we converted our stock from conventional to pr plts. such a shift in transfusion practice has implications regarding standard of care, which are discussed further in the next section. in addition, we consulted our department business office and hospital finance department to develop a business plan that would provide the key financial data needed to inform the decision to migrate the plt inventory to a safer, albeit more expensive, product. it is critically important not to underestimate the value of a well-thoughtout and well-prepared business plan when addressing senior administration. such a plan not only provides necessary fiscal data, but it also conveys the message to the hospital administration that the blood bank director understands the impact that such a move has on the financial health of the institution. this approach lent valuable credibility to our efforts. a key part of this process was the provision of training and education regarding pr plts before the inventory conversion for the blood bank staff involved in plt handling and distribution. the blood bank technical staff was educated on policies regarding pr plt management by the blood bank medical staff using in-service presentations across all shifts. technologists also must be familiar with manufacturing issues and electronic medical record (emr) management, to respond appropriately to clinician questions. we communicated with the information technology (it) department early in the planning process to ensure that once the pr inventory was introduced, component coding, bar code reading, and order entry activities would be seamless in epic, our emr, as well as in soft, our blood bank laboratory information system. inability to easily scan the new plt bar codes into the emr would be very problematic for the nursing staff. the blood bank similarly needed to have the appropriate codes added to the laboratory information system. the lead time for it notification is a key consideration since this department's time and availability to work on new projects often may be quite constrained. failure to consider these logistic issues could result in delayed patient care, patient safety concerns, and delayed system implementation. supply issues must be considered by any hospital planning to shift to a pr plt inventory. extensive discussions were held with our blood suppliers regarding the inventory shift and their ability to meet our anticipated needs (fig. 1) discussions were critical to allow planning at our hospital. as mentioned, frank conversations with our primary blood supplier disclosed that they would not be able to commit to meeting 100% of our demand for pr plts for at least 1 to 2 years after we decided to restructure our inventory. at that time, this was due to a lack of sufficient manufacturing infrastructure, a paucity of trained personnel, an uncertain supply of plt donors with high enough plt counts, difficulty in meeting the requisite manufacturing "guard bands," and the lack of a biologics license application (bla) to allow interstate shipping of plts from out-of-state manufacturing sites. 30 without a bla, interstate shipment of even fda-approved biologics is generally prohibited. moreover, seasonal and holiday plt shortages were certain to further infringe on our ability to obtain pr plts. when all these factors were considered, it became evident to us that a dual plt inventory was unavoidable. 31 even when our secondary blood supplier (in a neighboring state) received their bla 18 months into our program, a sufficient number of pr plts to meet 100% of our demand still could not be obtained (fig. 2 ). as the administrative notifications and discussions progressed, we met with physicians caring for both low-and high-risk/-acuity patients (fig. 1) . the latter represented the patient groups considered the most vulnerable to adverse events of any sort and included stem cell transplant/oncology patients, solid organ transplant candidates/patients, patients admitted to intensive care units (including the neurologic, surgical, medical, and cardiothoracic units), the emergency department, obstetrics, pediatrics (including neonatal and pediatric intensive care units), and geriatric services. nursing staff and business associates (formerly ward clerks) were also involved in these discussions. we launched a major institutional educational campaign to familiarize the nursing staff with the changes that would accompany the conversion of our plt inventory. this included holding educational sessions as well as developing computer screen saver-based announcements and informational articles in our hospital bulletin. for nurse and house staff training, we chose not to use spokespersons from the company producing the pr technology. we also declined company offers to present at grand rounds and other key conferences. at our institution, the optics of such presentations likely would be viewed as more commercial than educational. instead, we chose to have the blood bank medical directors provide key information at such conferences and have the blood bank's transfusion safety officer disseminate information and educational materials to floor and infusion clinic nurses through hospital's nurse educators. the company's offer to give didactic presentations to our hospital staff was declined to avoid the appearance of bias or sponsorship. educational efforts were essential to ensure that the floor staff medical and patient support personnel were familiar with the different appearance of pr plt bags, labels, color of the contents, and so forth. due to the need to remove 65 ml of plasma and replace the volume with a pas-c plt additive solution (as), the contents of pr plts collected on the apheresis device (amicus, fresenius kabi) are much paler than conventional sdp plt products suspended in 100% donor plasma or pr products collected on another apheresis device (trima, terumo corp.) in donor plasma. this educational practice was instrumental in preventing pr plts from unnecessarily being returned to the blood bank as being abnormal when clinical staff encountered these products with a different and unfamiliar appearance. no major change to the blood bank inventory of an academic medical center can be achieved overnight. we anticipated that a goal of 100% pr plts would not be attainable rapidly or consistently, owing to the previously noted logistic issues as well as fluctuating inventory demands and periodic seasonal changes in plt donor supply 30, 32 (fig. 2) . in our discussions with the hospital's ethics committee, a question was raised as to how an incremental introduction of pr plts would affect our institution's standard of care. this was due to concerns regarding which of the plt types in a dual inventory, pr or conventional, would be considered the safer product and which patients should have priority for those products. our solution to the ethical quandary presented by a dual inventory was to enhance the safety profile of our remaining conventional (non-pr) apheresis plt inventory as per the fda draft guidance on bacterial contamination risk control for plts. 2 we chose to use an fda-cleared plt "safety measure" device, pgd (pan genera detection, verax corp.). with administrative, legal, and regulatory approval (and with concurrence from the transfusion committee) we chose to consider conventional plts, including those on storage day 5 that were tested with the pgd assay and pr plts at any storage time, to be the standard of care at our institution. accordingly, this eliminated the need to determine whether a patient received a pr or conventional plt product treated with a safety measure, both of which are fdaapproved products. as mentioned, both were considered to be "standard of care" by our hospital's ethics and risk management committees. no patients were assigned to receive one product over another. thus, after receipt of a request for plts, either a pr unit or a conventional non-pr unit (the latter tested with the pgd assay if stored for 5 days before distribution), would be issued by the blood bank staff as equivalent products without concern for patient acuity or demographics. this added safety measure test was initially instituted only for day 5 conventional plts in our inventory. during the conversion process, while we were maintaining a dual inventory, a septic reaction to day 4 conventional plts occurred. since we only tested conventional plts on day 5 of storage and the contaminated units were at day 4 of storage, they were not tested. after this incident, we thus changed our policy to include safety measure testing of day 4 as well as day 5 conventional products. 2, 30 subsequently, we received requests from some service lines asking that their high-acuity patient groups receive only pr plts. we felt that we could not honor such a request from an ethical perspective and because it was problematic, at a practical level. such a practice would require blood bank staff to check a patient's chart after every plt request to verify that the correct type of plt was being issued based on the patient's diagnosis and classification. this would likely result in delays to patient care and would certainly put significant strain on our staff. from an ethics standpoint, this would require a definition of which patients are "more highly vulnerable". this topic continues to generate discussion among our service lines. there is still some concern over whether infusion of pr plts is associated with an increased incidence of acute respiratory distress syndrome (ards) compared to conventional plts. 7 several publications, however, have shown no association between development of ards and infusion of pr plts. [33] [34] [35] [36] based on these data, our institution decided that the risk of bacterial septic reactions from conventional plts outweighed the potential risk of respiratory complications from pr plts. to further address the risk of ards from pr plt products, an fda-mandated phase iv clinical trial comparing pr versus conventional plts (piper) has been initiated. our institution is one of the enrollment sites for this study. we soon found that performing a safety measure test on all day 4 and day 5 stored conventional products, especially those that arrived as an emergency shipment during the night, was logistically difficult to manage. we chose to address this issue by entering such day 4 or day 5 non-pr units into inventory if circumstances require that these plts be infused emergently before safety measure testing can be completed. these plts are thus released with appropriate documentation in the emr. the next morning, any remaining non-pr day 4 or day 5 stored units in inventory were sent for pgd testing. it should be noted that the fda has only issued a draft guidance in this area and that day 4 or day 5 safety measure testing is not required at this time. 2 this situation has provided an even stronger impetus for us to work with our two blood suppliers to obtain a 100% pr inventory as soon as possible (fig. 2) . the "roll out" for the pr plt inventory was coordinated by the blood bank's medical director, and the transfusion safety officer, in conjunction with senior blood bank technologists. the "go live" date was advertised throughout the hospital to all clinical services, starting approximately 4 weeks in advance. importantly, the blood bank medical staff was continuously available to our clinical colleagues to address issues and concerns. a review of our institution's quality and error tracking system after implementation of the pr ramp-up showed, gratifyingly, that there were no complaints filed regarding the introduction of pr plts. we attribute this positive outcome to our extensive training and educational efforts and the it preparations conducted before and during the roll out. in addition to educating our clinical staff on "what to expect," we also emphasized the importance of ongoing hemovigilance as pr plts made their way to the wards. although there are data in the literature discussing quality monitoring, to monitor our investment in and the performance of these products, we collected and analyzed patient data to assure that pr plts were indeed as safe and efficacious as were conventional apheresis plts. continued reporting of transfusion reactions is critical for monitoring product safety. similarly, data on product usage per patient are necessary to document the efficacy of pr plts. data addressing these points have been published by our group [37] [38] [39] and others [19] [20] [21] [22] [23] [24] [34] [35] [36] 40 providing evidence supporting the concept that diminishing the infectious risk by crosslinking nucleic acids does not negatively impact plt activation or hemostatic efficacy. other studies, however, including the effipap trial have also addressed this issue. 41 this group noted differences regarding the hemostatic efficacy of pr plts and non-pr plts collected in pas-c as, when compared to non-pr plts collected in plasma. 41 successful introduction of pr plts into inventory is just the beginning; continued monitoring must be performed as the program grows and matures. this should be undertaken from a quality assurance perspective. we began our audit process by examining patient groups of particular concern as reported by others. one such group included neonates, as there was an early and ongoing concern that even minimal residual psoralen in pr plts could be harmful to these patients. 42 this concern was especially applicable to those newborns undergoing phototherapy for hyperbilirubinemia, and our neonatologists also had some reservations in this regard, despite fda clearance of pr plts for all patient groups. 1,7 a review of neonates receiving both pr plts and blue light phototherapy at our institution, however, showed no evidence of rash, toxicity, or adverse effects. 37 this was not unexpected since the synthetic psoralen used in the pr process absorbs light in the 320-to 375-nm wavelength range, while phototherapy devices in use in the western world typically emit light in the 400-to 520-nm wavelength range. 43 older pediatric and obstetric patients were also populations of interest. again, we and others have observed no difference in the rate of adverse transfusion events or in plt utilization for patients receiving pr plts compared to conventional plts in these groups. [33] [34] [35] [36] [37] [38] [39] 44 although the small sample size precludes generalization, no cases of ta-gvhd were identified among any of our autologous and allogeneic hematopoietic stem cell transplant patients who received pr plt transfusions that were not irradiated. 39 similar findings regarding the lack of reports of ta-gvhd in comparable recipients of nonirradiated pr plts have been published. [34] [35] [36] among adult nonpregnant patients, no increase in transfusion reactions or plt usage was seen in patients given pr plts compared to those receiving conventional plt products. 38, 39 these data findings were communicated to our hospital service line medical personnel. pathogen-reduced plts are more expensive than conventional sdp. shifting our plt inventory to pr products required an increased expenditure of 50% above the cost of conventional plt products. of this increase, half was attributed to requiring a change to sdps from pooled random plt donor units (pooled units are not approved for use with pr treatment) and the other half from the cost of the pr process itself. blood banks are cost centers, not revenue centers. as they do not generate revenue, there are very few ways to shift costs and save money when implementing pr plts. other seemingly less expensive options, however, such as those aimed at mitigating bacterial contamination risk alone, may be as expensive as pr when space, equipment, training, and staffing requirements are considered. 32, [45] [46] [47] furthermore, as new nonbacterial pathogens, such as viruses and protozoa, threaten the blood supply, new pathogen-specific tests would need to be developed for each agent and paid for by hospitals not using pr products. this scenario has already occurred with the zika virus, as pr plts do not require testing for zika. this does represent a degree of cost saving for pr plt users. the financial impact of paying for additional assays as new nonbacterial pathogens inevitably appear may prove to be substantial. pr treatment of plts, we believe, thus presents an efficient and, in the long run, cost-effective method by which to mitigate the risk of bacterial and nonbacterial pathogens. [45] [46] [47] our institution ultimately decided that a commitment to patient safety superseded cost and chose to pursue pr plts despite the anticipated monetary impact. new technology is typically expensive. currently, only one company has received fda approval for its plt pr system; however, at least two other companies as previously mentioned are working on their pr technologies. 6, 25, 26, 42 other considerations it must be noted that it is theoretically possible that pr plts can become contaminated after the pr process is completed. the actual pr step in the currently fda-approved system occurs when the plts, combined with the added amotosalen, are irradiated under uva light. after the illumination is extinguished, however, the plts are again vulnerable to contamination, such as might occur if there is a leak in the plastic container or if the product is improperly stored. integration of pr plts into an academic medical center blood bank inventory is a daunting task. we have taken an early-adopter role because we believe that based on published data, and regulatory approval by multiple countries, pr represents an efficient and cost-effective technology, capable of mitigating the risk of transmission by bacterial and nonbacterial blood-borne disease pathogens. 32, [45] [46] [47] with thoughtful planning and interdepartmental collaboration, we found that this can be accomplished in an academic tertiary care medical center both effectively and efficiently. clear communication throughout the institution, from nursing units to the c-suite, is vital. training and education across service lines promotes a smooth transition, as does strong involvement of blood bank leadership throughout the "go live" process. a plan for maintaining and managing a dual inventory is critically important, and continued hemovigilance monitoring of pr plts after the rollout ensures a high level of transfusion safety and efficacy. we recommend that hospital financial personnel be involved in the process in the early planning stages. similarly, involvement by it must not be overlooked, as problems with the emr ordering and scanning programs are a major source of clinician end-user dissatisfaction. pr plts are more expensive than conventional plts. we believe, however, that conversion to a full pr plt inventory is prudent, cost-effective, and foresightful and appropriately prioritizes patient safety. cerus corporation. fda approves pathogen reduction system to treat platelets bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion: draft guidance for industry amotosalen interactions with platelet and plasma components: absence of neoantigen formation after photochemical treatment chemical and biological mechanisms of pathogen reduction technologies pharmacokinetic and toxicology assessment of intercept (s-59 and uva treated) platelets summary of safety and effectiveness data: intercept blood system for platelets cerus corporation. intercept blood system for platelets efficacy of intercept treatment for the inactivation of dengue virus in single-donor platelet concentrate: sp266 inactivation of orientia tsutsugamushi using intercept blood system for plasma, as demonstrated in an animal model helinx ® technology inactivates vaccinia virus in human platelet concentrates simultaneous inactivation of co-circulating arboviruses through nucleic acid crosslinking transfusion of platelet components prepared with photochemical pathogen inactivation treatment during a chikungunya virus epidemic in ile de la reunion amotosalen photochemical inactivation of severe acute respiratory syndrome coronavirus in human platelet concentrates inactivation of leukocytes in platelet concentrates by photochemical treatment with psoralen plus uva irradiation of platelet components: inhibition of lymphocyte proliferation assessed by limiting-dilution analysis effect of gamma irradiation of red blood cell units on t-cell inactivation as assessed by limiting-dilution analysis: implications for preventing transfusion associated graft versus host disease pathogen reduction of platelet rich plasma abrogates t cell allo response in mice irradiation of blood components. bethesda (md): aabb; 1992 a prospective, active haemovigilance study with combined cohort analysis of 19 175 transfusions of platelet components prepared with amotosalen-uva photochemical treatment impact of platelet pathogen inactivation on blood component utilization and patient safety in a large austrian regional medical center patient outcomes and amotosalen/uva-treated platelet utilization in massively transfused patients haemovigilance annual report rapport d'activité hémovigilance 2013. france an active hemovigilance program characterizing the safety profile of 7483 transfusions with plasma components prepared with amotosalen and uva photochemical treatment pathogen inactivation technologies for cellular blood components: an update pathogen inactivation of cellular blood products-an additional safety layer in transfusion medicine assessment of safety in neonatal rates for transfusion of red blood cells prepared with the amustaline-gsh pathogen reduction treatment transfer of the amustaline/gsh pathogen reduction system to two us blood research centers abstract presentations from the aabb annual meeting qualification of amustaline-gsh red blood cell pathogen reduction system for a phase 3 clinical trial bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion" draft guidance dual apheresis platelets inventory for a hospital-based donor center and transfusion service platelet availability and economic impact of bacterial risk reduction studies at us hospitals determination of acute lung injury after repeated platelet transfusions haemovigilance annual report the role of hemovigilance and postmarketing studies when introducing innovation into transfusion medicine practice: the amotosalen-ultraviolet a pathogen reduction treatment model blood utilization and transfusion reactions in pediatric patients transfused with conventional or pathogen reduced platelets a retrospective evaluation of the hemostatic efficacy of pathogen reduced (pr) vs conventional (conv) platelets at an academic medical center transfusion reaction rates of pathogen reduced (pr) vs conventional (conv) platelets in adults: a single academic center experience activation status of pathogen reduced platelet components in plasma in comparison with conventional plasma platelet components comparison of the hemostatic efficacy of pathogen-reduced platelets vs untreated platelets in patients with thrombocytopenia and malignant hematologic diseases: a randomized clinical trial pathogen-inactivated blood products for pediatric patients: blood safety, patient safety, or both? fundamentals of phototherapy for neonatal jaundice analysis of pediatric adverse reactions to transfusions economic implications of pathogen reduced and bacterially tested platelet components: a us hospital budget impact model cost implications of implementation of pathogen-inactivated platelets financial impact of alternative approaches to reduce bacterial contamination of platelet transfusions els receives research support from cerus corporation as pi for the piper and recepi studies. he receives no personal remuneration from cerus. sr has disclosed no conflicts of interest. key: cord-102668-1yc38ok1 authors: siddiqui, shoib s.; dhar, chirag; sundaramurthy, venkatasubramaniam; sasmal, aniruddha; yu, hai; bandala-sanchez, esther; li, miaomiao; zhang, xiaoxiao; chen, xi; harrison, leonard c.; xu, ding; varki, ajit title: acidosis, zinc and hmgb1 in sepsis: a common connection involving sialoglycan recognition date: 2020-07-15 journal: biorxiv doi: 10.1101/2020.07.15.198010 sha: doc_id: 102668 cord_uid: 1yc38ok1 blood ph is tightly regulated between 7.35-7.45, with values below 7.3 during sepsis being associated with lactic acidosis, low serum zinc, and release of proinflammatory hmgb1 from activated and/or necrotic cells. using an ex vivo whole blood system to model lactic acidosis, we show that while hmgb1 does not engage leukocyte receptors at physiological ph, lowering ph with lactic acid facilitates binding. at normal ph, micromolar zinc supports plasma sialoglycoprotein binding by hmgb1, which is markedly reduced when ph is adjusted with lactic acid to sepsis levels. glycan array studies confirmed zinc and ph-dependent hmgb1 binding to sialoglycans typical of plasma glycoproteins. thus, proinflammatory effects of hmgb1 are suppressed via plasma sialoglycoproteins until drops in ph and zinc release hmgb1 to trigger downstream immune activation. significance statement hmgb1 sequestered by plasma sialoglycoproteins at physiological ph is released when ph and zinc concentrations fall in sepsis. the ph of body fluids in healthy individuals spans a very broad range in different tissue types and organs, ranging from ph 1.5 (stomach contents), to 8.0 (urine). human cells in tissue culture can also tolerate a wide range of ph values. in contrast, blood ph is tightly regulated between 7. 35-7.45 (1) , and departure out of this range (acidosis or alkalosis) can be very detrimental. for example, in the recent covid-19 pandemic, 30% of non-survivors had acidosis, compared to 1% among survivors (2) . acidosis in sepsis is partly due to lactic acid release from anoxic tissues, which overwhelms the buffering capacity of circulating blood (3) . a "cytokine storm" of proinflammatory mediators in sepsis triggers a cascade of destructive outcomes such as multiple organ failure (4) (5) (6) (7) (8) as currently seen in severe cases of covid-19 infection (9) . the mechanisms underlying lethality associated with low blood ph are not clear, but include low zinc levels and release from apoptotic or necrotic cells of hmgb1, a damageassociated molecular pattern (damp) defined as one of the late mediators of sepsis, further upregulating many other proinflammatory cytokines (10) (11) (12) . importantly, a recent study indicates hmgb1 levels are strongly associated with mortality in patients infected with sars-cov-2 (13) . here we show that sialylated plasma glycoproteins bind hmgb1 to suppress its ability to promote inflammatory responses in a zinc and ph-dependent manner. this finding provides an avenue for developing a new therapeutic strategy for treating sepsis. mimicking lactic acidosis ex vivo in hirudin-anticoagulated whole blood. in vivo studies of acidosis and sepsis involve many complex factors and interactions. on the other hand, ex vivo reconstitution of purified blood components can result in artifacts, e.g., neutrophils get activated when separated away from erythrocytes and plasma (14) . to study the significance of tightly regulated blood ph ex vivo, we sought to create a whole blood system mimicking lactic acidosis. conventional anticoagulation with edta or citrate abrogates divalent cation functions, and heparin has many biological effects independent of anticoagulation. we have previously shown that the leech protein hirudin can be used to obtain whole blood anticoagulation in vitro (15) . when lactic acid was added to freshly collected hirudin-anticoagulated whole blood, the ph first rose until a concentration of about 1 mm lactic acid was reached. further addition then caused a sharp drop in blood ph. such an initial rise in blood ph followed by a subsequent drop is seen in patients with sepsis (16) . to further develop this model, we introduced hmgb1, a damp (17) (18) (19) associated with poor prognosis in late sepsis (20, 21) . which is partially attenuated by an hmgb1-blocking antibody: cd11b expression was determined by flow cytometry after incubating whole blood with/without hmgb1 (1 µg/ml). a) neutrophils are activated when incubated with hmgb1 in whole blood at ph 7.2 (chromatograms: red-control, blue-whole blood at ph 7.5, orange-whole blood at ph 7.2, green-whole blood at ph 7.5 with hmgb1, cyan-whole blood at ph 7.2 with hmgb1). b) activation is partially attenuated with an hmgb1-blocking antibody (50 µg/ml) (chromatograms: red-isotype control, blue-whole blood at ph 7.2, green-whole blood at ph 7.2 with hmgb1, orange-whole blood at ph 7.2 with hmgb1 and an hmgb1-block neutrophils in whole blood are activated by hmgb1 at low ph due to better binding, and activation is attenuated with an hmgb1 blocking-antibody. interaction of hmgb1 with toll-like receptors (tlrs) during sepsis is well documented (22) . the proinflammatory activity of hmgb1 is due to binding to targets such as tlr-2, tlr-4, tlr-9 and rage that are expressed on leukocytes and endothelial cells (23, 24) . we, therefore, introduced exogenous hmgb1 into our whole blood acidosis model and tracked cd11b expression on neutrophils, as a sensitive marker of activation triggered by hmgb1. increased neutrophil activation was noted when hmgb1 was incubated with whole blood at low ph as compared to physiological ph ( figure 1a ). this effect was partially attenuated by adding hmgb1 blocking antibody ( figure 1b) . enhanced activation at low ph coincides with increased hmgb1 binding to neutrophils and monocytes (compare upper and lower panels of figure 2a and b). thus, physiological blood ph limits interaction of hmgb1 with leukocyte receptors, suggesting natural inhibitor(s) of hmgb1 interaction in blood. looking for candidate inhibitors, we noted earlier evidence that hmgb1 can interact with cd24 and cd52, two heavily sialylated proteins (25, 26) in a trimolecular complex with siglec-10, a known sialic acid-binding protein. cd52-fc bound specifically to the proinflammatory box b domain of hmgb1, and this, in turn, promoted binding of the cd52 n-linked glycan sialic acid with . furthermore, sialidase treatment abolished cd52 binding to hmgb1, indicating that it might be a sialic acid-binding lectin. since normal blood plasma contains ~2 mm sialic acid attached to glycans on plasma proteins, (27) , we hypothesized that the unknown natural inhibitor might be the sialome (the total sum of all sialic acids presented on plasma glycoproteins). ability of hmgb1 to bind to different cell types of the blood (erythrocytes, monocytes and neutrophils) was determined by using different concentrations (100 ng/ml, 500 ng/ml and 5 µg/ml) of hmgb1 at physiological conditions. b) different cell types of blood were used for binding with hmgb1 (100 ng/ml) at physiological and lower ph (ph 7.2, adjusted with lactic acid). among divalent cations, only zinc supported the robust binding of hmgb1 with sialylated glycoproteins at physiological ph. the binding buffer used in prior hmgb1 studies included millimolar concentrations of manganese cation (mn 2+ ), a feature likely carried over from the unrelated function of nuclear hmgb1 binding to dna. looking at earlier studies of the interaction of hmgb1 with cd24 and cd52, we noticed that all those experiments were performed in a buffer containing millimolar mn 2+ concentrations (25, (28) (29) (30) . these concentrations were very high in comparison with the physiological levels of mn 2+ in the blood (4-15 µg/l). we predicted that there might be other divalent cation(s) that are better co-factor(s) for hmgb1 and facilitate its binding with sialic acids. indeed, upon testing micromolar concentrations of many divalent cations, we found that only zinc cation (zn 2+ ) supported robust binding with sialylated glycoproteins ( figure 3a ). we tested α1-acid glycoprotein and 3'-sialyllactose as binding partners for hmgb1 in the presence of different cations and again found that only zn 2+ facilitated binding. there was a modest binding of 3'-sialyllactose with hmgb1 in the presence of mn 2+ , but robust binding was only seen with zn 2+ -containing buffer ( figure 3b ). replacing plasma with buffer at physiological ph allows hmgb1 to activate neutrophils, suggesting sequestration by plasma sialoglycoproteins. we next asked which whole blood components were preventing neutrophil activation under physiological conditions. hirudin-anticoagulated whole blood at physiological ph was spun down and plasma either replaced with hepes buffer (ph 7.5) supplemented with zn 2+ or with the same plasma that had been removed. after incubating with hmgb1, neutrophils were in a more activated state when incubated in the buffer as compared to when plasma was added back ( figure 4a ). independent studies have shown that hmgb1 binds to sialic acid on glycoproteins (26, 31) and we posited that the ~2 mm bound sialic acid present on plasma glycoproteins might lead to sequestration of hmgb1 under physiological condition. we also tested the effect of ph on the binding of hmgb1 to α1-acid glycoprotein and found that optimal binding was at physiological ph, with less binding at ph 7.2 with buffer containing zn 2+ ( figure 4b ). replacing plasma with a buffer at physiological ph allows hmgb1 to activate neutrophils a) 1 ml of blood was drawn from a healthy individual and spun down. the plasma was replaced with hepes buffer containing zinc (500 µm of zn 2+ ) or plasma was added back. the cd11b expression as a marker of neutrophil activation was measured. b) the binding of hmgb1 to α1-acid glycoprotein was checked with a binding buffer using different ph ranging from 7.1 to 7.8. sialoglycan array studies of hmgb1 confirm that it is a sialic acid-binding lectin with optimal binding at physiological blood ph in the presence of zinc cations. we previously reported a sialoglycan microarray platform used to identify, characterize, and validate the sia-binding properties of proteins, lectins, and antibodies (32) (33) (34) . after identifying zn 2+ -dependent hmgb1 binding to sialoglycoproteins, we next investigated the ability of hmgb1 to bind with multiple sialoglycans abundantly found in plasma proteins. we performed sialoglycan array studies of hmgb1 under four different conditions: 1) at physiological ph with zn 2+ , 2) at physiological ph without zn 2+ , 3) at ph 7.2 with zn 2+ 4) at ph 7.2 without zn 2+ . these array studies further confirmed the binding of hmgb1 with multiple sialylated glycan sequences that are typically found on plasma glycoproteins, in ph-and zn 2+ -dependent fashion ( figure 5a and 5b respectively). additionally, we checked the binding of hmgb1 to sialic acids in sialoglycan microarray using 0, 15 and 150 µm concentrations of zn 2+ and observed a dose-dependent effect ( figure 5b ). this assay showed the relevance of zn 2+ in this binding phenomenon at a physiological concentration (~15 µm). on resolving the binding of hmgb1 at physiological ph and in the presence of zinc, the binding on the microarray was exclusively to sialylated glycans confirming our findings ( figure 5c ). a heat map representation of all these findings and hmgb1 binding to individual glycosides is provided in supplementary figures 3 and 4 respectively. **** represents p-value < 0.0001) heparin, a previously known anionic glycan binding partner of hmgb1, does not exhibit ph sensitivity, and zn 2+ only partially facilitates binding. hmgb1 is known to bind heparin, a heavily sulfated glycan carrying many negatively charged groups (35, 36) . we checked the binding of hmgb1 with heparin at different ph values and found that unlike binding with sia, it was not ph-sensitive (supplementary figure 1a) . moreover, there was appreciable baseline binding of hmgb1 with heparin that only increased partially with zn 2+ supplementation (supplementary figure 1b) . this data indicates that the binding of heparin and sialic acid are very different. the b-box of hmgb1 that mediates sialic acid binding (26) has three arginine residues (26) that might be involved in sialic acid recognition. we made single mutants of arginine residues at positions 97, 110 and 163. when we checked the sialic acid binding, we could not find any difference between either of the mutants and wt hmgb1 ( supplementary fig 2) . we suspect other positively charged residues and/or multiple arginines to mediate sialic acid binding. here we report one plausible explanation for the tight regulation of blood ph between 7.35-7.45, showing that even a slight reduction to ph 7.2 abolishes the zinc-dependent sequestration of hmgb1 by plasma sialoglycoproteins, releasing it to bind to activating receptors on neutrophils. hmgb1 was originally discovered in the cell nucleus (37) (38) (39) (40) , playing a role in dna bending, replication and transcription (41, 42) . much later, hmgb1 was found to be passively or actively released in conditions like sepsis, leading to inflammation (21, 41, 43) . i.e, it is as a damp (44) . hmgb1 retention inside the nucleus is dictated by conserved lysine residues (45) . inflammatory stimuli trigger acetylation of these lysine residues and trafficking of hmgb1 to the cytosol, and eventually to the extracellular space. the different domains of hmgb1 are box a, box b and an acidic tail. while box a and box b possess many arginine and lysine residues, the acidic tail is enriched with glutamic and aspartic acid residues. box b is proinflammatory whereas box a behaves like an antagonist and mimics an anti-hmgb1 antibody (26, 46) . while tnf-α and il-1β are released early during sepsis, hmgb1 is a late mediator expressed only after about 24 hours and remains at elevated levels before death occurs (47) . many preclinical studies show protection against sepsis upon injection of blocking antibodies of hmgb1 or just injection of box a protein (48) . the proinflammatory activity of hmgb1 is well studied. however, the anti-inflammatory activity of hmgb1 also has been documented in multiple studies (49) (50) (51) . recently, it was shown that hmgb1 binds soluble cd52 and this complex binds with siglec-10 on t-cells leading to shp-1 (phosphatase) recruitment that dephosphorylates lck and zap70, thus activating an anti-inflammatory cascade (26, 52) . in addition, haptoglobin (49) , c1q and tim3 also show anti-inflammatory activity of hmgb1 (50, 51) . in this study, we found that at physiological blood ph, there is no interaction of hmgb1 with its receptors on leukocytes. surprisingly, when we lowered the ph using lactic acid (to mimic lactic acidosis, a characteristic feature of sepsis), the interaction was restored. furthermore, the high concentration of sialic acids in plasma glycoproteins was found to be the likely inhibitor of interactions between hmgb1 and tlrs. we further characterized the role of hmgb1 as a sialic acid-binding lectin and found that zinc is a required co-factor. moreover, we confirmed all our findings with lipopolysaccharide (lps)-free hmgb1 and used a glycan array that detected the binding of hmgb1 with several sialic acid probes (see supplemental table 1 ) in a ph and zincdependent manner. taken together, our findings lead us to propose that under physiological conditions (ph 7.35-7.45) and normal zinc concentrations, there is a potent binding of hmgb1 with plasma sialoglycoproteins ( figure 6a ). under septic conditions, drops in ph and zinc concentration decrease interactions between hmgb1 and plasma sialoglycoproteins leading to the liberation of hmgb1 to bind with tlrs, to enhance inflammation ( figure 6b ). therefore, proinflammatory and anti-inflammatory activities of hmgb1 are the two sides of the same coin and are dependent on the different physiological conditions. while the proinflammatory role of hmgb1 is very well studied, recent studies have reported an anti-inflammatory role for hmgb1 (25, (50) (51) (52) . the exact mechanism that enables hmgb1 to switch from its proinflammatory to anti-inflammatory role and vice-versa is not very well described. one factor known to enable its switch from being proinflammatory to anti-inflammatory is its oxidative state. the disulfide form of hmgb1 is proinflammatory, and the sulfonate form is involved in the resolution of inflammation (53) (54) (55) . in the current study, we have identified another mechanism by which hmgb1 switches from its proinflammatory to anti-inflammatory role in a ph-and zinc-dependent manner. sepsis is characterized by a decrease in ph and zinc concentration of the blood. we hypothesize that under physiological conditions, hmgb1 binds with sialoglycoproteins of blood keeping it in a quiescent state. during sepsis, the drop in ph and zinc concentration of the blood leads to disruption of hmgb1's binding with sialic acid, enabling the free hmgb1 to bind with tlrs and rage present on immune cells and the endothelium. this activates a cascade of the inflammatory response, which if untreated, might lead to multiple organ failure or even death. also consistent with our hypothesis are the findings that survival in mouse models of sepsis can be improved by infusion of soluble cd52 (56) , and that the sialic acid binding feature of hmgb1 is restricted to the disulfide-form of hmgb1 (26) , which is expected to be formed when the cytosolic reduced form is released into the oxidizing environment of the bloodstream. we suggest that the potent proinflammatory effects of hmgb1 are normally kept in check via sequestration by plasma sialoglycoproteins at physiological ph and zinc levels and is triggered when ph and zinc levels fall in the late stages of sepsis. in this regard, it is notable that the acute phase response to inflammation results in high production of hypersialylated molecules such as α1-acid glycoprotein from the liver and endothelium, which may then act as a negative feedback loop (57) (58) (59) . current clinical trials that are independently studying zinc supplementation (clinicaltrials.gov identifier: nct01328509 nct02130388) or ph normalization (nct03530046) may be more successful if these approaches are combined, and perhaps supplemented by infusions of heavily sialylated molecules like cd52. additionally, studies evaluating plasma exchange in subjects with septic shock (example nct03366220) may show superior efficacy if supplemented with zinc infusions and ph correction. pre-clinical studies are presently evaluating a function blocking anti-hmgb1 antibody (60) . we performed our assays with hmgb1 purchased from hmg biotech, also produced it in e. coli and finally confirmed findings using hmgb1 expressed in 293 freestyle cells. in order to recapitulate the characteristics of hmgb1 in septic conditions, we used the disulfide linked form in all our assays. future studies should address whether other post-translational modifications such as acetylation, methylation, phosphorylation or oxidation have any further effect on hmgb1's propensity to bind sialic acids. numerous studies have shown that zinc is protective against sepsis (61-63). additionally, blood zinc levels usually decrease during inflammation because it is sequestered to the nucleus where it is required as a cofactor for expression of proinflammatory genes and proteins (61, 64, 65) . thus, lowering of the zinc level in blood is detrimental. the mechanism of action for the anti-inflammatory effect of zinc is extensively studied. these include effect impact on the microbiome, lowering of nf-κb levels, chemotaxis and phagocytosis by immune cells, anti-oxidative stress and adaptive immune response (61) . in this regard, it is notable that a recent study also shows the role of zinc, ph and ionic strength on the oligomerization of hmgb1 (66). we did not investigate any role of zinc or ph on the structural changes or oligomerization of hmgb1. it seems that at particular ph and zinc concentration, a positively charged residue of hmgb1 is exposed for binding with sialic acid. this residue may not be surface available at lower ph and low zinc concentration. in this study, we could not pinpoint the critical residue that is important for sialic acid binding. hmgb1 has been reported to bind many ligands and some of which are highly negatively charged molecules such as heparin/heparan sulfate (35) . we wanted to determine if the interaction of hmgb1 with sialic acid, which is also negatively charged, is a generic electrostatic charge-based interaction. therefore, we tested the binding of hmgb1 with the acidic glycosaminoglycan, hyaluronan, but could not detect any binding (data not shown). upon testing with heparin, we found that while hmgb1 did bind with heparin, it did not show any ph dependency. moreover, binding was only partially enhanced in the presence of zinc. this shows that a different set of amino acid(s) might be required for binding to heparin and sialic acid. notably, under physiological conditions, sialic acid is present in the blood, but the concentrations of other anionic glycans (heparan sulfate, hyaluronic acid etc.) are low. our findings, if confirmed in randomized clinical trials, have broad implications in the management of sepsis and possibly other types of acidosis. sepsis is a significant cause of mortality, with a recent study implicating it as the cause of twice as many deaths as earlier estimated (67) . these findings are of particular importance in light of the present covid-19 pandemic/survivorship in these patients. acute respiratory distress syndrome (ards), a deadly complication of the sars-cov-2 and sars-cov-1, has been linked with hmgb1 production (68) (69) (70) . recent articles suggest a potential link between hmgb1 and the pathogenesis of covid-19 (71, 72) . a recent study showed that hmgb1 strongly correlates with mortality in covid-19 patients (13) . additionally, another recent study showed 100% of covid-19 non-survivors had sepsis and 30% of these had acidosis (2) . while the surviving sepsis campaign does not suggest the use of convalescent plasma in critically ill patients, (73) , the fda has approved its use as an investigational new drug. a small study of five critically ill covid-19 patients treated with convalescent plasma showed improvements in sepsis related sofa scores (74). a clinicaltrials.gov search for "covid" and "convalescent plasma" on april 6, 2020 yielded 9 results of trials ranging from phase 1 to phase 3. while the circulating antibodies are likely to be beneficial on their own, the hmgb1-sequestering properties of plasma sialoglycoproteins may also contribute to suppressing the "cytokine storm". these effects are likely to be further enhanced if plasmapheresis is supplemented with aggressive ph correction and zinc supplementation. elisa for binding of hmgb1 with α1-acid glycoprotein or 3'-sialyllactose: 500 ng-1µg of hmgb1 recombinant protein (hmg biotech) diluted with the binding buffer (20 mm hepes, 150 mm nacl, 500 µm and the absorbance was acquired at 490 nm with a plate reader. for the elisa with different divalent cations, the binding buffer was prepared using the particular cation containing salt instead of zncl2. each incubation and wash was performed using the respective binding buffer. informed consent was obtained from healthy individuals as per a protocol approved by the ucsd human research protection programs institutional review board and venous blood was collected in hirudin coated tubes (thermofischer catalogue number-nc1054637). hirudin was chosen as the anticoagulant as edta and heparin interferes with normal bioprocesses (chelation by edta and binding to and modulating cell-surface proteins by heparin). the ph of blood, when measured at the start of various assays varied between 7.5 and 7.6 and is referred to as the "physiological" ph. to test for neutrophill activation, 100 µl of whole blood was incubated with 1 µg/ml of hmgb1 for 30 minutes at 37 °c. cd11b expression was measured by flow cytometry as described earlier (14, 75) . blocking with an anti-hmgb1 antibody (clone 3e8, biolegend, catalogue number-651402) was performed with 50 µg/ml antibody as described earlier (60) . for plasma addback studies, whole blood was spun down at 500 x g for 5 minutes and replaced with hepes buffer supplemented with 500 µm zncl2. binding assays were performed with 500 µl of whole blood. the required amount of hmgb1 (0, 100, 500, 5000 ng/ml) was added to 500 µl of blood and incubated at 37 o c for 60 minutes with rotation. after centrifuging at 600 x g for 5 minutes, the cells were washed with 1 ml of pbs and finally resuspended in 100 µl of facs buffer (1% bsa in pbs with ca 2+ /mg 2+ ) with anti-hmgb1 antibody (10µg/ml, biolegend, catalogue number-651402). the cells were incubated at 4 o c for 30 minutes on ice and were washed with 1 ml pbs (containing ca 2+ /mg 2+ ). the cells were subsequently resuspended in 100 µl of facs buffer with a secondary anti-mouse-apc antibody (biolegend, catalogue number-405308). the cells were incubated at 4 °c for 30 minutes on ice and washed with pbs as before. 10 µl was taken from each sample for rbc analysis and the rest of the sample was fixed with 4% paraformaldehyde (pfa) and incubated on ice for 20 min. the sample was then washed with pbs and subsequently treated with ack lysis buffer (gibco, catalogue number-a10492-01) to perform analysis of rbcs. the sample was washed and resuspended in 500 µl of facs buffer. in the forward and side scatter profile, monocytes and neutrophils were gated for the analysis. for gating of monocytes forward and side scatter pattern was used. the surface markers were not used for this gating. chemoenzymatically synthesized sialyl glycans were quantitated utilizing dmb-hplc analysis and were dissolved in 300 mm sodium phosphate buffer (ph 8.4) to a final concentration of 100 µm. arrayit spotbot ® extreme was used for printing the sialoglycans on nhs-functionalized glass slides (polyan 3d-nhs slides from automate scientific; catalogue number-po-10400401). purified mouse anti-hmgb1 antibody (biolegend; catalogue number-651402, lot# b219634) and cy3-conjugated goat anti-mouse igg (jackson immunoresearch; catalogue number-115-165-008) were used. fresh hepes buffer (20mm hepes, 150mm nacl ± 500µm zncl2) was prepared immediately before starting the microarray experiments. method described in (34) was adapted to perform the microarray experiment. each glycan was printed in quadruplets. the temperature (20 °c) and humidity (70%) inside the arrayit ® printing chamber was rigorously maintained during the printing process. the slides were left for drying for an additional 8 h. printed glycan microarray slides were blocked with pre-warmed 0.05 m ethanolamine solution (in 0.1 m tris-hcl, ph 9.0), washed with warm milli-q water, dried, and then fitted in a multi-well microarray hybridization cassette (arrayit, ca) to divide it into 8 subarrays. each subarray well was treated with 400 µl of ovalbumin (1% w/v) dissolved in freshly prepared hepes blocking buffer ± 500 µm of zn 2+ (ph adjusted for individual experiments) for 1h at ambient temperature in a humid chamber with gentle shaking. subsequently, the blocking solution was discarded, and a solution of hmgb1 (40 µg/ml) in the same hepes buffer (± zn 2+ , defined ph) was added to the subarray. after incubating for 2 hours at room temperature with gentle shaking, the slides were extensively washed (first with pbs buffer with 0.1%tween20 and then with only pbs, ph 7.4) to remove any non-specific binding. the subarray was further treated with a 1:500 dilution (in pbs) of cy3-conjugated goat anti-mouse igg (fc specific) secondary antibody and then gently shaken for 1 hour in the dark, humid chamber followed by the same washing cycle described earlier. the developed glycan microarray slides were then dried and scanned with a genepix 4000b (molecular devices corp., union city, ca) microarray scanner (at 532 nm). data analysis was performed using the genepix pro 7.3 analysis software (molecular devices corp., union city, ca). expression and purification of full-length murine his-hmgb1 in e. coli were performed as described before (35) . mutagenesis was performed using a quikchange site-directed mutagenesis kit (agilent). acid-base homeostasis clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study lactic acidosis sepsis-associated hyperlactatemia novel insights for systemic inflammation in sepsis and hemorrhage sepsis: something old, something new, and a systems view harmful molecular mechanisms in sepsis the immunopathology of sepsis and potential therapeutic targets the covid-19 cytokine storm; what we know so far targeting hmgb1 in the treatment of sepsis hmgb1, a potent proinflammatory cytokine in sepsis high mobility group 1 protein (hmg-1) stimulates proinflammatory cytokine synthesis in human monocytes elevated serum levels of s100a8/a9 and hmgb1 at hospital admission are correlated with inferior clinical outcomes in covid-19 patients erythrocyte 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innate immune responses through interactions between the receptor tim-3 and the alarmin hmgb1 t cell regulation mediated by interaction of soluble cd52 with the inhibitory receptor siglec-10 mutually exclusive redox forms of hmgb1 promote cell recruitment or proinflammatory cytokine release regulation of posttranslational modifications of hmgb1 during immune responses high-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair cd52 inhibits toll-like receptor activation of nf-κb and triggers apoptosis to suppress inflammation alpha-1-acid glycoprotein hernàndez pando r. alpha-1-acid glycoprotein, its local production and immunopathological participation in experimental pulmonary tuberculosis detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, o-acetylated sialic acids but not disialyl residues generation of monoclonal antibodies against highly conserved antigens mechanistic insights into the protective impact of zinc on sepsis persistent low serum zinc is associated with recurrent sepsis in critically ill patients -a pilot study low zinc and selenium concentrations in sepsis are associated with oxidative damage and inflammation zinc dyshomeostasis during polymicrobial sepsis in mice involves zinc transporter zip14 and can be overcome by zinc supplementation interleukin-6 regulates the zinc transporter zip14 in liver and contributes to the hypozincemia of the acute-phase response the effect of physicochemical factors on the self-association of hmgb1: a surface plasmon resonance study global, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the global burden of disease study involvement of high mobility group box 1 and the therapeutic effect of recombinant thrombomodulin in a mouse model of severe acute respiratory distress syndrome platelet-derived hmgb1: critical mediator of sars related to transfusion pathogenic role of hmgb1 in sars extracellular hmgb1: a therapeutic target in severe pulmonary inflammation including covid-19 hmgb1: a possible crucial therapeutic target for covid-19 surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) we thank sandra diaz and patrick secrest for their excellent technical help with the work. key: cord-010772-e7kfe87q authors: hospach, ingeborg; goldstein, jacques; harenski, kai; laffey, john g.; pouchoulin, dominique; raible, manuela; votteler, stefanie; storr, markus title: in vitro characterization of prismalung+: a novel ecco(2)r device date: 2020-05-13 journal: intensive care med exp doi: 10.1186/s40635-020-00301-7 sha: doc_id: 10772 cord_uid: e7kfe87q background: invasive mechanical ventilation is lifesaving in the setting of severe acute respiratory failure but can cause ventilation-induced lung injury. advances in extracorporeal co(2) removal (ecco(2)r) technologies may facilitate more protective lung ventilation in acute respiratory distress syndrome, and enable earlier weaning and/or avoid invasive mechanical ventilation entirely in chronic obstructive pulmonary disease exacerbations. we evaluated the in vitro co(2) removal capacity of the novel prismalung+ ecco(2)r device compared with two existing gas exchangers. methods: the in vitro co(2) removal capacity of the prismalung+ (surface area 0.8 m(2), baxter) was compared with the prismalung (surface area 0.35 m(2), baxter) and a.l.one (surface area 1.35 m(2), eurosets) devices, using a closed-loop bovine blood–perfused extracorporeal circuit. the efficacy of each device was measured at varying pco(2) inlet (p(in)co(2)) levels (45, 60, and 80 mmhg) and blood flow rates (q(b)) of 200–450 ml/min; the prismalung+ and a.l.one devices were also tested at a q(b) of 600 ml/min. the amount of co(2) removed by each device was assessed by measurement of the co(2) infused to maintain circuit equilibrium (co(2) infusion method) and compared with measured co(2) concentrations in the inlet and outlet of the co(2) removal device (blood gas analysis method). results: the prismalung+ device performed similarly to the a.l.one device, with both devices demonstrating co(2) removal rates ~ 50% greater than the prismalung device. co(2) removal rates were 73 ± 4.0, 44 ± 2.5, and 72 ± 1.9 ml/min, for prismalung+, prismalung, and a.l.one, respectively, at q(b) 300 ml/min and p(in)co(2) 45 mmhg. a bland–altman plot demonstrated that the co(2) infusion method was comparable to the blood gas analysis method for calculating co(2) removal. the resistance to blood flow across the test device, as measured by pressure drop, varied as a function of blood flow rate, and was greatest for prismalung and lowest for the a.l.one device. conclusions: the newly developed prismalung+ performed more effectively than prismalung, with performance of co(2) removal comparable to a.l.one at the flow rates tested, despite the smaller membrane surface area of prismalung+ versus a.l.one. clinical testing of prismalung+ is warranted to further characterize its performance. patients with severe acute hypoxemic and/or hypercapnic respiratory failure require invasive mechanical ventilation (imv) to facilitate gas exchange and to support breathing. while imv may be lifesaving in this setting, it is associated with significant short-and long-term side effects. consequently, there is considerable interest in developing strategies such as extracorporeal co 2 removal (ecco 2 r), which can facilitate co 2 removal [1] , or extracorporeal membrane oxygenation (ecmo), which, in addition, provides oxygenation in instances of severe hypoxemic respiratory failure [2] . these approaches may enable reductions in the intensity and/or the duration of imv in these patients. in patients with severe hypoxemia, particularly those with acute respiratory distress syndrome (ards), the loss of alveolar ventilation capacity due to alveolar consolidation, edema and/or collapse contributes to the need for ventilatory support [3] . the discovery that high tidal and minute ventilation strategies can cause harm-termed "ventilator-induced lung injury" (vili ) [4] [5] [6] -has led to the use of lung "protective" ventilation (lpv) strategies, where low tidal volumes (4-8 ml/kg of per body weight [pbw ] [7] versus 10-15 ml/kg of pbw in conventional mechanical ventilation [mv ] [6] ) decrease lung stretch, reduce vili [8] , and can potentially improve survival and reduce mortality in patients with acute lung injury and ards [6, 9] . amato et al. showed that lower driving pressure was the physical variable that best correlated with survival in patients with ards [10] ; higher positive end-expiratory pressure (peep), lower peak and plateau pressures, and lower respiratory rate, may also be associated with improved survival [11, 12] . the use of lower tidal and minute volumes with lpv strategies is limited by the resultant respiratory acidosis [13] [14] [15] . the rationale to integrate ecco 2 r into the management of severe ards is to allow more protective ventilation, i.e., providing very low tidal volumes (v t ) (less than 6 ml/kg pbw) with conventional mv, while avoiding extreme levels of respiratory acidosis. arterial co 2 tensions are generally maintained in the range 45-60 mmhg rather than targeting normocapnia with this approach [16] . the potential for use of ecco 2 r in patients with ards has been evidenced in a number of clinical studies [17] [18] [19] , indicating it may be an effective strategy in ards management and a viable option to further reduce tidal and minute volumes in these patients [15, 16] . in patients with acute exacerbations of chronic obstructive pulmonary disease (aecopd), where hypercapnia is predominant, non-invasive positive pressure ventilation (niv) is used as a first-line strategy in order to avoid mv [20] . use of niv has been reported to reduce mortality by approximately 70% [21] ; however, in some patients, additional assistance is required to prevent the need for intubation [22] . niv fails in almost 40% of cases, and patients must undergo endotracheal intubation and imv to restore adequate gas exchange [22] [23] [24] [25] . there is increasing clinical evidence supporting the use of low-flow, partial ecco 2 r for patients experiencing aecopd who are failing support with niv [22] , avoiding the need for imv and/or decreasing the length of time on the ventilator [26] . advances in extracorporeal device technologies have made selective ecco 2 r devices a less invasive and more feasible option than ecmo, with several devices clinically available that utilize blood flow rates between 180 ml/min and 1700 ml/ min [27] . however, these devices were historically designed for use as oxygenators for ecmo treatment in the neonatal or pediatric setting, rather than being optimized for co 2 removal [28] . here, we describe a newly developed ecco 2 r device, the prismalung+ (additional file 1: figure s1 ), created specifically for co 2 removal. we compared the in vitro co 2 removal rates during low blood flow (q b 200-450 ml/min) of three devices: prismalung+ (baxter), prismalung (baxter), and eurosets a.l.one (eurosets) , and during a q b of 600 ml/min for the prisma-lung+ and a.l.one devices [29] [30] [31] . we hypothesized that prismalung+ with a membrane surface area of 0.8 m 2 provides significantly higher co 2 removal rates than prismalung (surface area 0.35 m 2 ), whereas we expected similar performance for prismalung+ and the a.l.one device (surface area 1.35 m 2 ), since with increasing membrane surface area, the low blood flow rates limit co 2 removal. in vitro experimentation to determine co 2 removal rates was performed using three different ecco 2 r devices: prismalung+ (baxter), prismalung (baxter), and eurosets a.l.one (eurosets) ( table 1) . the devices were selected as they had the same membrane composition, i.e., polymethylpentene hollow-fiber mats, in order to remove this potential source of variability from the experiments. five test devices of each type were test media bovine blood parameters were adjusted as listed in table 2 . nacl and nahco 3 solutions were used to adjust the required ranges of blood parameters. the experimental setup was a closed-loop circuit in which a continuous co 2 infusion balanced the co 2 removal from the test gas exchanger to establish a steady-state condition and allowing the co 2 removal rate to be determined (fig. 1 ). the total amount of blood used in the circuit was approximately 600-700 ml. the test setup comprised the following: blood reservoir, 250 ml duran glass bottle (schott ag, germany) with a temperature sensor pt100 (technetics, germany); tubing (promedt, germany) with inserted septum as sample port and valves; 2 × peristaltic blood pumps (made in-house, baxter, germany); datalogger for sensor read-out mikromec® logger (technetics, germany); control loop: gas exchanger for co 2 input, prismalung (baxter, germany) (closed at gas outlet with plugs), a thermax blood warmer bag (baxter, france) inside an in-house made holder, and water bath with thermostat eh (julabo, germany); co 2 gas bottle ≥ 99.5% purity (linde, germany) including pressure regulator and gas tubing; co 2 mass flow regulator gsc-a9ta-bb22 (vögtlin, switzerland); 2 × pressure sensor, pe2 bar (technetics, germany); test loop: sweep gas mass flow regulator gsc-c9ta-bb12 (vögtlin, switzerland); 3 × pressure sensor, pe1 bar (technetics, germany); syringe pump 540270 (tse systems, germany); compressed air as sweep gas (in-house) including pressure regulator and tubing. blood samples were analyzed with an abl 90 blood gas analyzer (radiometer, germany) . before use, an integrity test of the co 2 infusion circuit was performed, after which the whole test setup, including test devices, was primed with saline or dialysate solution (e.g., prismasol 2, baxter) to remove all air. the setup was then filled with bovine blood and all saline or dialysate solution was replaced. the test circuit comprised a central reservoir filled with 200-300 ml of blood as well as two loops. the control loop had a gas exchanger connected to a co 2 supply, which was used to achieve the targeted p in co 2 levels. in addition, a blood warming system, where the blood warmer bag was submerged in a water bath, was used to maintain blood pool temperature at 37 ± 1°c. the control loop was fed through the central reservoir then connected to the test loop in which the test device was attached, and co 2 removal was determined. loss of water due to evaporation through the membrane and into the sweep gas was balanced by infusion of reverse osmosis water. the sodium concentration was kept constant throughout experimentation, as analyzed by the blood gas analyzer, to maintain a constant water flow. when the test was initiated, the blood flow of the control loop was set to 500 ml/ min and the sweep gas flow was set at the targeted rate. the co 2 inlet flow was adjusted in a stepwise fashion to maintain p in co 2 at the targeted value and to reach steady-state conditions (constant values for p in co 2 , co 2 inflow rate, co 2 removal rate). the pco 2 value was measured by blood gas analysis, after samples were taken at the blood inlet of the test device. following an equilibration time of at least 13 minutes, during which co 2 removal from the test circuit was demonstrated to be balanced by co 2 addition to the control loop, co 2 removal rate was determined based on the co 2 inflow rate. if blood samples were taken, a syringe with a volume of 0.5-1 ml was used. on average, no more than 2-3 blood sample measurements were necessary to confirm a steady state, which is below 0.5% of the total circuit blood volume. for each test device, at all requested test parameters (9 settings of varying q b and p in co 2 , table 3 in the appendix), measurements were taken at the inlet and outlet, with samples taken in triplicate. po 2 inlet values (160-183 mmhg) indicated that the blood used in this study was oxygen saturated. all devices were tested at q b 200, 300, and 450 ml/min, with additional testing of the prismalung+ and a.l.one devices at q b 600 ml/min and p in co 2 45 mmhg. test conditions are outlined in table 2 . the primary method utilized for measuring co 2 removal was the infusion method, which was validated using the blood gas analysis method. in the infusion method, normalized co 2 removal rate (jco 2(inf) ) was determined based on the co 2 input flow rate at equilibrium, controlled by sample taking and analysis at the inlet. the blood gas analysis method utilized the same setup as the infusion method, but blood samples were taken additionally after the test device, at the outlet. only samples, where measured pco 2 and/or ph were inside the reportable range (i.e., pco 2 > 12 mmhg, ph < 7.85) were used to validate the data from the infusion method. prior to the series of experiments, the suitability of the analyzer, with respect to its intended use, was verified through the measurement principles of the blood gas analyzer [32, 33] , to ensure that the device was able to measure bovine parameters. furthermore, the same device was used for all replicates. measurements taken included the analysis of cthb (g/dl), ph, pco 2 (mmhg), po 2 (mmhg), and fmethb (%) ( table 3 in the appendix). following experimentation, an integrity test of the circuit was again performed to confirm its co 2 gas integrity. mean values were determined from triplicate measurements. in the infusion method, jco 2(inf) was determined based on co 2 input flow rate, using the following equation: where qco 2 is the co 2 input flow within the control loop, p in co 2(ref) is the target inlet pco 2 of 45, 60, or 80 mmhg, and p in co 2(inlet) is the actual partial pressure of co 2 in the blood reservoir or gas exchanger inlet. p in co 2 was normalized to reduce variability in measurements resulting from small deviations from target p in co 2 values (± 10%). qco 2 values are referred to in ml/min under normal conditions (0°c, 1013 mbar) and are re-calculated where appropriate, applying the ideal gas equation: where q (t) is the volumetric flow at a defined temperature (t, in°c). the temperatures used for calculation ranged from 0 to 37°c. note: the atmospheric pressure is assumed to be constant at 1013 mbar. in addition, using the more commonly utilized blood gas analysis method, the normalized co 2 removal rate (jco 2(bga) ) was determined according to the following equation: where v m is the temperature-dependent molar volume; q b is the blood flow within the test loop; ctco 2 is the total blood concentration of co 2 (given by the blood gas analyzer, derived from ph, pco 2 , saturation of oxygen so 2 , and hemoglobin concentration); p in co 2(ref) is the target inlet pco 2 of 45, 60, or 80 mmhg; and p in co 2(inlet) is the partial pressure of co 2 in blood. co 2 removal rates were additionally calculated in units of mmol/min to remove any dependency of reported values upon pressure and reference temperature. a total of 5 test runs were performed for each device and parameter settings. data are expressed as mean ± sd and the normal distribution of the data sets was assessed using the kolmogrov-smirnov test (α = 0.05). co 2 removal performance results were compared using an anova test with p values of < 0.05 considered as indicating a significant difference. bland-altman analysis was used to compare the two different performance test methods, generated using sigmaplot software [34, 35] . a linear regression comparing both methods was additionally used. a paired t test was used to compare data obtained via the infusion and blood gas analysis methods. the co 2 removal rates of the different devices were analyzed using the infusion method at p in co 2 levels (45, 60, and 80 mmhg) and q b (200, 300, and 450 ml/min), at 37°c (fig. 2 a-c, table 4 in the appendix). the a.l.one and prismalung+ devices provided comparable co 2 removal rates across the range of different test conditions (p > 0.05, not significant). for both devices, removal rates were significantly higher than those observed with the prismalung device (p < 0.05). co 2 removal rates at an increased blood flow rate of 600 ml/min were additionally evaluated for the prismalung+ and a.l.one devices only and were comparable for both devices (p > 0.05) (additional file 2: figure s2 ). at a p in co 2 of 45 mmhg, at 37°c, the mean co 2 removal rates at a blood flow rate of 600 ml/min were 106 ± 3.8 ml/min and 106 ± 5.8 ml/min for the prismalung+ and a.l.one devices, respectively. as the volume flow of gases, i.e., the co 2 removal rate, is temperature-and pressure-dependent, data were calculated at standard reference conditions, 0°c and 25°c (stp as defined by iupac), in addition to the physiological conditions, 37°c, for the prismalung+ device at a p in co2 of 45 mmhg and q b range of 200-450 ml/min (fig. 3a) . results illustrate the dependence of co 2 removal on the chosen reference temperature. by definition, calculation of co 2 removal rates in mmol/min across q b 200-450 ml/min, at a p in co 2 of 45 mmhg, is independent from any reference temperature (fig. 3b) . to examine the blood flow resistance for each device, pressure drop was analyzed at the blood side, for all p in co 2 levels (45, 60, and 80 mmhg). pressure drop was observed to be largest for the prismalung, and lowest for the a. the co 2 removal performances were analyzed using the infusion and blood gas analysis methods. however, at the lower blood flow rates, many of the outlet samples were below the measuring range of pco 2 , among others, using the blood gas analyzer method. a comparison of the two methods using a bland-altman analysis (fig. 5a) and linear regression analysis (fig. 5b) across the different p in co 2 levels (45, 60, and 80 mmhg) and blood flow rates (200, 300, and 450 ml/min), using valid data within the reportable range of the blood gas analyzer used, indicated a linear relationship between the data obtained by the two methods, suggesting comparability. statistical analysis revealed that co 2 removal performance values obtained with the infusion method were, on average, 4.2 ml greater than the values obtained with blood gas analysis (p < 0.05). in addition, the difference was shown to be independent of the test conditions and a constant offset between the two methods. the infusion method was used for the analysis of the full data set as the two methods are similar in terms of validity. in this in vitro study, the co 2 removal performance of the new prismalung+ device was comparable to the a.l.one device, with both devices demonstrating co 2 removal rates~50% greater than the prismalung device. the performance of the three devices was consistent over a range of blood p in co 2 levels and at flow rates from 200 to 450 ml/min, with both the prismalung+ and a.l.one devices also performing comparably at the higher flow rate of 600 ml/min. co 2 removal data obtained with the co 2 infusion method were comparable to those obtained with the blood gas analysis method. the resistance to blood flow across the test device, as measured by pressure drop, varied as a function of blood flow rate, being greatest for the prismalung, intermediate for the prismalung+, and lowest for the a.l.one device, most likely driven by the differences in the surface areas. taken together with prior clinical studies of ecco 2 r devices [17] [18] [19] 22] , these findings suggest that the prismalung+ may be an effective device and further testing in the clinical setting is warranted. rationale for co 2 removal ecco 2 r technologies may have important roles in the management of patients with ards and patients with aecopd. ecco 2 r can help facilitate lung-protective strategies by enabling very low v t (< 6 ml/kg pbw) ventilation [36, 37] . the safety and feasibility of ecco 2 r has been demonstrated in multiple studies [16, 18, 19] of patients with ards, with reduced lung injury and benefits in terms of pulmonary inflammation with low v t ventilation [17] . several studies also support the use of ecco 2 r in patients with aecopd requiring ventilatory support [38] [39] [40] [41] [42] . devices with reduced blood flow requirements will, by design, be less efficient at removing co 2 than higher flow devices, but they do have several advantages. the prismalung+ device in this study has a design tailored to specifically remove co 2 . the lack of a need for a heat exchanger inside the device allows for reduced size and weight, given that a heat exchanger is available for use next to the machine during treatment. the new prismalung+ device has the lowest ratio of blood volume to membrane surface of the tested devices reducing the risks associated with large extracorporeal blood volumes. the removal of the heater also allows for a streamlined design, which should reduce the potential for pooling and low flows of blood within the device. further aspects of the device design, including the fluid path and dimensional parameters, have been developed to enable an intended operating blood flow of 200 to 450 ml/min. namely blood flow velocity distribution was calculated to avoid stagnant areas or areas with very low blood flow velocity and to ensure that channeling of the blood did not occur. the residual volume space is smaller than other devices, minimizing the space for blood to clot. the ratio of co 2 removal rate to blood volume of the prismalung+ device allows for optimized performance at these flow rates. it has been shown that an extracorporeal co 2 removal rate of 51 ± 26 ml/min was associated with an increase in paco 2 from 43 ± 8 to 53 ± 9 mmhg when applying low tidal volume ventilation (v t = 4 ml/kg) in patients with mild-to-moderate ards [43] . whereas a mean co 2 removal of 81 ± 9 ml/min enabled a reduction in v t to 4.29 ± 9 ml/min without an increase in paco 2 of more than 10% [19] . therefore, larger co 2 removal rates are desirable to allow ultraprotective ventilation in ards patients without a significant increase in paco 2 . furthermore, it is assumed that a reduced interaction between blood and foreign material, i.e., a preferably small device, may potentially support biocompatibility [44] . the streamlined design of prismalung+ might require less anticoagulation, which entails a lower risk of bleeding complications in patients, as there is less potential for pooling and low flows of blood within the device. this hypothesis needs to be investigated in future studies. an advantage of lower flow ecco 2 r devices is that smaller bore catheters can be used. a second advantage is that they may be integrated with other organ support strategies familiar to critical care physicians and nurses, such as continuous renal replacement therapy (crrt), making these approaches much more feasible in the busy critical care environment. the potential to integrate ecco 2 r into continuous renal replacement circuits may improve the risk/benefit ratio for hypercapnic patients with acute kidney injury (aki) [45] . if effective, such devices could also be used in patients that do not have aki, given the familiarity of the critical care team with this equipment. a feasibility study demonstrated that the use of a low-flow ecco 2 r device managed with an rrt platform easily and safely enabled very-low-tidal-volume ventilation with moderate increase in paco 2 in patients with mild-to-moderate ards [43] . in this study, the new prismalung+ device performed similarly to the a.l.one device, with both devices demonstrating co 2 removal rates~50% greater than the prismalung device. while the increase in co 2 removal observed with prismalung+ compared with prismalung can, at least in part, be explained by an increase in membrane surface area from 0.35 m 2 to 0.8 m 2 , the similarities observed for pris-malung+ and a.l.one occurred despite an increase in surface area, suggesting a more complex explanation. recent data from karagiannidis and colleagues suggest that the capability of different ecco 2 r devices to eliminate co 2 is dependent upon a dynamic interplay within the device between the surface area available for gas exchange and the blood flow rate [44] . devices with gas exchange membrane surface areas ranging from 0.35 m 2 (e.g., prismalung) up to 1.3 m 2 (e.g., a.l.one) are currently used in clinical practice [19, 22, 26, [46] [47] [48] . furthermore, recent in vitro and in silico studies suggest that co 2 removal rate can increase with increasing blood flow rate [49, 50] , in line with the observations we report here. our study also confirms the findings from karagiannidis and colleagues [44] , as both surface area and blood flow rates govern the rate of co 2 removal; however, an increase in the surface area above a certain threshold has limited impact on co 2 removal when low blood flows are applied, as is the case with the a.l.one device. larger membrane surface areas are thought to result in greater levels of co 2 removal at higher blood flow rates, with a smaller pressure drop across the gas exchanger [44] . in our in vitro study, pressure drop values across the three devices were relatively low, with levels of up to 25 mmhg with prismalung+ (surface area 0.8 m 2 ) at a blood flow rate of 450 ml/min. some challenges do exist for devices with larger membrane surface areas and those that require larger priming volumes, as they may have increased thrombotic potential due to increased interaction with an artificial surface [44] . a lower blood flow rate combined with a larger surface area may lead to more clotting events due to the increased time blood spends passing through the membrane [43, 51] . furthermore, in the clinic, larger priming volumes can negatively affect exposure time and the hemolysis index [51] , potentially resulting in increased blood loss due to clotting events and the device having to be replaced. we did not observe clotting events; however, this was not a focus of the study and would require investigation in the clinic. these findings highlight the potential for lower flow devices with higher surface areas to remove co 2 from blood. the data also demonstrate the restrictions of conventional diffusive co 2 removal determined by blood flow rates. to further enhance co 2 removal at low blood flow rates novel systems, such as approaches involving acidification of bloo d [52] or bicarbonate dialysi s [53] , need to be investigated. we used the infusion analysis rather than the more commonly accepted blood gas analysis method to determine the co 2 removal rates. this was done because of the limitations of the blood gas analysis method, because test conditions utilizing lower blood flow produced co 2 results below the reportable range of the analyzer. the blood gas analysis method was used to determine co 2 removal rates when test results where within the reportable range of the blood gas analyzer. the results obtained with the blood gas analysis method demonstrated the validity of the infusion method. the comparison of the valid data obtained by blood gas analysis versus the infusion method by slope analysis indicated that these two data sets are comparable (fig. 5) . it is important to note that the comparative data indicated that a small amount of gas loss from the test setup was likely, as for example, if the reservoir and/or tubing are not fully gas-tight. this is demonstrated by the data being slightly off the line of identity, the offset of the mean, as shown in the bland-altman diagram (4.2 ml/min) and the paired t test. the general suitability of the blood gas analyzer, with respect to its intended use, was verified through the measurement principles of the analyzer [32, 33] , to ensure that the abl90 device used here could measure bovine blood parameters. in addition, the abl devices from radiometer have been routinely used to perform experiments on blood from different species [54] [55] [56] [57] . we used a flow rate that would be achievable by a monitor that the prismalung+ device is intended to be used on in the clinic, namely 200 to 450 ml/min, allowing us to characterize the device in conditions comparable to mild-to-moderate hypercapnia where the device would be used. the a.l.one device is designed to run at higher blood flow rates than the flow rates used in this study; therefore, conditions perhaps did not favor the co 2 removal rate of the device, despite the high surface area in comparison with prismalung+ [31] . further methodological limitations to consider include the use of bovine blood for experimentation, as it is easier to obtain than human blood and well-accepted for use in in vitro studies. it should be noted that as the blood was obtained from healthy animals, levels of blood components will be different from those for icu patients; however, levels were consistent across experiments. furthermore, a high dose (5 u/ml) of heparin was used that is higher than that routinely used in the clinic; this dose was selected to ensure no clotting occurred during transportation from the slaughterhouse and during the in vitro experiment and is not expected to impact co 2 removal. given the in vitro nature of the data, caution should be exercised when translating these data to the clinical setting, and further studies are needed to explore coagulation in the clinic. here, and in similar studies, the co 2 removal rates are stated in units of ml/ min, with dependency upon pressure and chosen reference temperature. to remove the dependency upon these parameters, units of mmol/min would be more appropriate and comparable when reporting co 2 removal rates. despite this, the units of mmol/min are not standard and are not used in the clinical setting . ecco 2 r uses similar gas-exchange principles as ecmo, but the main goal is to remove co 2 in those with sufficient oxygenation and at lower blood flow rates than ecmo [58] . oxygenation and o 2 transfer rates were not a focus in this study. in the clinical setting, venous so 2 levels are expected to be around 70% and therefore the testing of fully oxygen-saturated blood in this setting may slightly underestimate co 2 removal performance. this will require further experimental confirmation. in summary, at the flow rates tested, prismalung+ performed more effectively than prismalung for co 2 removal, with comparable performance to a.l.one, despite the smaller surface area. the prismalung+ may be an effective device for reducing co 2 levels, and further testing in the clinical setting is warranted. extracorporeal carbon dioxide removal (ecco2r) in respiratory failure: an overview, and where next? blood oxygenation and decarboxylation determinants during venovenous ecmo for respiratory failure in 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validation of a prototype device for measurement of ionized calcium concentrations cow-side against a point-of-care instrument and a benchtop blood-gas analyzer reference method in vitro tests and modelization of bicarbonate kinetics and mass transfers during online hemodiafiltration evaluation of h2o2 and ph in exhaled breath condensate samples: methodical and physiological aspects bench to bedside review: extracorporeal carbon dioxide removal, past present and future editorial support for the development of this manuscript was provided by daniel johnson phd, ailsa bennett phd, ruth brown phd, and siobhán ahern phd, scimentum (nucleus global), with funding provided by baxter. supplementary information accompanies this paper at https://doi.org/10.1186/s40635-020-00301-7.additional file 1: figure s1 . cross section of the prismalung+ device additional file 2: figure s2 . co 2 removal rates for the prismalung+ and a.l.one devices. assessed at 37°c, q b 600 ml, and a p in co 2 of 45 mmhg. p in co 2 , partial pressure of carbon dioxide at the inlet; q b , blood flow rate. data are plotted as mean values ± sd. p > 0. 05 this study was sponsored by baxter. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. ih, jg, ms, kh, dp, mr, sv are full-time employees of baxter international. j.l. has received consulting fees from baxter international. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-269839-jxqs51o5 authors: bitome-essono, paul-yannick; ollomo, benjamin; arnathau, céline; durand, patrick; mokoudoum, nancy diamella; yacka-mouele, lauriane; okouga, alain-prince; boundenga, larson; mve-ondo, bertrand; obame-nkoghe, judicaël; mbehang-nguema, philippe; njiokou, flobert; makanga, boris; wattier, rémi; ayala, diego; ayala, francisco j; renaud, francois; rougeron, virginie; bretagnolle, francois; prugnolle, franck; paupy, christophe title: tracking zoonotic pathogens using blood-sucking flies as 'flying syringes' date: 2017-03-28 journal: elife doi: 10.7554/elife.22069 sha: doc_id: 269839 cord_uid: jxqs51o5 about 60% of emerging infectious diseases in humans are of zoonotic origin. their increasing number requires the development of new methods for early detection and monitoring of infectious agents in wildlife. here, we investigated whether blood meals from hematophagous flies could be used to identify the infectious agents circulating in wild vertebrates. to this aim, 1230 blood-engorged flies were caught in the forests of gabon. identified blood meals (30%) were from 20 vertebrate species including mammals, birds and reptiles. among them, 9% were infected by different extant malaria parasites among which some belonged to known parasite species, others to new parasite species or to parasite lineages for which only the vector was known. this study demonstrates that using hematophagous flies as ‘flying syringes’ constitutes an interesting approach to investigate blood-borne pathogen diversity in wild vertebrates and could be used as an early detection tool of zoonotic pathogens. doi: http://dx.doi.org/10.7554/elife.22069.001 emerging and re-emerging human infectious diseases have increased in recent years. around onefourth of the 1415 pathogens known to infect humans appeared between 1940 and 2004 and their appearance has gradually increased since 1980 (taylor et al., 2001; woolhouse and gaunt, 2007; jones et al., 2008; daszak et al., 2004) . today, seven new pathogens appear every year and this number should reach 15-20 by 2020 (woolhouse et al., 2008) , mostly due to the growth of human activities that increase contact with novel sources of pathogens and favor their spread worldwide (murray et al., 2015) . emerging threats mainly concern viruses, such as hiv (sharp and hahn, 2011) , sars-cov and mers-cov (de wit et al., 2016) , avian flu (alexander, 2007) and more recently ebola (baize et al., 2014) , chikungunya (burt et al., 2012) and zika (wikan and smith, 2016) . however, disease emergence and re-emergence also concern bacteria (e.g. helicobacter pylori, salmonella sp., etc.) and parasites (e.g. plasmodium knowlesi in south-east asia). sixty per cent of diseases emerging in humans are zoonoses and wildlife plays a key role by providing a zoonotic pool from which previously unknown pathogens may emerge (taylor et al., 2001; woolhouse and gaunt, 2007; jones et al., 2008; daszak et al., 2004) . the case of p. knowlesi in south-east asia is a good example. this parasite emerged in the human population after a transfer from asian macaques. it is now considered as the fifth human malaria agent after plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale (singh and daneshvar, 2013) . such emerging diseases constitute a massive public health issue that requires active monitoring for signs of outbreaks and rapid diagnosis of the involved pathogen. therefore, it is crucial to anticipate and prevent potential epidemic and pandemic outbreaks by developing new methods for the early detection and monitoring of infectious agents in wild animal sources (kuiken et al., 2005; wolfe et al., 2005) . however, in many cases, monitoring is limited or impossible due to our poor knowledge about the ecology of these pathogens (i.e. where, when and how these agents circulate in the wildlife). the case of the ebola virus is quite exemplary. indeed, the exact nature of its reservoir(s) remains uncertain, although thousands of animals have been screened during the last 40 years (e.g. [marí saéz et al., 2015] ). nowadays, pathogen circulation in wild animals is screened using mainly two methods: bushmeat analysis or direct trapping of animals for organ and tissue collection. these methods are pertinent in many cases, but present some weaknesses. bushmeat represents only a fraction of the fauna (the one consumed by humans), whereas animal trapping can be difficult or dangerous. moreover, such manipulation may be harmful for threatened and protected species. as a consequence, several methods were developed in the last years to study pathogen diversity from wild fauna without the need of direct contacts with animals, for example, by using fecal, urine or saliva samples (e.g. [santiago et al., 2002; prugnolle et al., 2010; pesapane et al., 2013; taberlet et al., 2012] ). however, the value of these non-invasive methods remains limited because not all pathogens can be detected and not all reservoirs can be explored by these methods (for instance, it is difficult to collect feces or saliva of reptiles without trapping them). therefore, new non-invasive methods are crucially needed to provide new opportunities for screening a larger range of hosts and pathogens. the use of hematophagous flies as 'flying syringes' may constitute a new approach to track and survey blood-borne pathogens in the wild (calvignac-spencer et al., 2013) . nucleic acids (dna or elife digest about 60% of new infectious diseases in humans come from animals. their increasing number and rapid spread are linked to increasing levels of contact between humans and wildlife, as recently highlighted by the epidemics of zika in brazil or ebola in west africa. to anticipate and prevent similar outbreaks in the future, it would be ideal to develop new methods for the early detection and monitoring of infectious diseases in wild animals. currently, three methods are mainly used to screen wild animals for infectious disease, but these all have limitations. analyses of bushmeat and game meat only investigate those animals that are eaten by humans. testing the organs and tissues of trapped animals can be difficult and harmful for both the humans and animals involved. collecting and examining samples of feces, urine or saliva cannot detect all diseases and can be difficult to do for some species. bitome-essono et al. now demonstrate a new method for assessing the diseases carried by wild animals: using blood-sucking flies as 'flying syringes' to collect their blood. during several weeks of sampling in gabon, central africa, bitome-essono et al. trapped thousands of these flies, about a third of which were engorged with blood. analyses of these blood samples revealed that they had come from 20 different species, including birds, mammals and reptiles. different malaria parasites could also be detected in the blood. although the study performed by bitome-essono et al. only focused on malaria parasites, in the future the technique could be extended to analyze a number of disease-causing microbesincluding viruses, bacteria, protozoa and macroparasites -that are found in the blood of wild animals. rna) of vertebrate hosts or of pathogens in arthropod blood meals are preserved and detectable for several days (calvignac-spencer et al., 2013; kent, 2009; muturi et al., 2011; grubaugh et al., 2015; lee et al., 2015) . for example, hiv was detected 8 days and 10 to 14 days after blood ingestion by bugs and by ticks, respectively (webb et al., 1989; humphery-smith et al., 1993) . recently, the h5n1 flu virus was found viable in mosquitoes (barbazan et al., 2008) , although its transmission by these insects is unproven (sawabe et al., 2006) . grubaugh and colleagues (grubaugh et al., 2015) applied such an idea (that they called 'xenosurveillance') using anohpeles mosquitoes to estimate the diversity of viruses infecting human populations in remote areas. nevertheless, bloodengorged mosquitoes are very difficult to collect in forest and often show strong host preferences (in particular for mammals). arthropods with more generalist blood feeding patterns would be more useful to survey pathogens from a large range of vertebrates (including mammals, birds and reptiles) in these highly complex ecosystems. hematophagous flies (tsetse flies, stomoxids and tabanids) could be good candidates for this purpose since they are usually large diptera (length comprised between 3 and 25 mm) and hematophagous in both sexes, with the exception of male tabanids (mullens, 2002) . they are easy to trap and some studies performed on tsetse flies and stomoxids showed that 20 to 40% of trapped flies are engorged with blood (mavoungou et al., 2008; simo et al., 2012) . these flies feed on a large spectrum of vertebrate hosts, including birds, reptiles and mammals (muturi et al., 2011; clausen et al., 1998; muzari et al., 2010) . the omnipresence of hematophagous flies in certain habitats and their opportunistic blood-feeding behaviour (muturi et al., 2011; muzari et al., 2010; späth, 2000) make of them compelling candidates to obtain blood meals from different vertebrate hosts for pathogen detection. in the present study, we investigated the possibility of using hematophagous flies as 'flying syringes' to explore the diversity of extant malaria parasites (haemosporida) infecting wild vertebrates living in the forests of gabon (central africa). a total of 4099 hematophagous flies were caught in four national parks of gabon during dry and rainy seasons over a cumulated sampling period of 16 weeks ( figure 1a ). among them, six tsetse fly species, six stomoxid species and six tabanid species were identified ( table 1) . among the 4099 caught flies, 1230 (30%) were engorged with blood. these were mostly tsetse flies (n = 1218; 99%), particularly glossina palpalis palpalis (n = 662; 54%) and g. fuscipes fuscipes (n = 214; 18%) specimens. the blood meal origin was successfully identified in 33% and 43% of these flies, respectively (table 1) . overall, the blood meal origin was successfully identified in 428 fly samples (35%) using a pcr system amplifying long fragments of cytb (450 bp) or coi genes (330 bp or 660 bp). specifically, blood meals were from 20 vertebrate species, including 12 families and 8 orders (figure 1b and tables 2 and 3). a trial study using a pcr system amplifying a shorter fragment (150 bp of the gene 16s) to deal with potential dna degradation in the blood meal showed a high gain of sensitivity in the determination of the origin of the blood meal. thus, out of 89 previously unidentified blood meals, the host was identified for 76% (n = 68) of them. the list of newly identified hosts is given in figure 2 . this shows a high gain of sensitivity with the new pcr system. extant malaria parasites were detected in 37 (8.7%) of the 428 identified blood meals (figure 1c , red isolates). phylogenetic analyses revealed that 29.7% of these parasites belonged to plasmodium falciparum (n = 11, figure 1c ; group 1), 8.1% to plasmodium adleri (n = 3, figure 1c ; group 2), and 8.1% to a recently described lineage of parasites infecting wild ungulates (n = 4, figure 1c ; group 3) (boundenga et al., 2016) . for all blood meals, the identified host represented the known natural host (or one of the hosts) of such parasites. sequences of unknown parasite lineages or of parasites for which the hosts were not known were also obtained. for instance, one sequence ( figure 1c ; group 4) detected in a blood meal originating from an ungulate was related to parasites previously :"$6(-,$6/%)%3-@05/-+,#) isolated only from anopheles mosquitoes (boundenga et al., 2016) . one sequence detected in a blood meal originating from a bird was related to bat haemosporida (nycteria), (figure 1c ; group 5). finally, 18 sequences ( figure 1c ; group 6) that were amplified from blood meals originating from ungulates formed an independent and never described lineage related to groups 3 and 4. in addition, 100 additional samples for which identification of the blood meal failed were randomly chosen for malarial parasite screening. this analysis showed that 7% were infected with p. falciparum (n = 4, group 1), p. praefalciparum (n = 1, group 7), malaria parasites of antelopes from group 6 (n = 1) and parasites of tortoises (group 8, n = 1) ( figure 1c , green isolates). for the parasite, the use of a shorter pcr system led to less conclusive results than those obtained for the host identification. out of the 91 blood meals that were negative to plasmodium with a pcr system amplifying a long cytb fragment, only one was found positive with the new system. the positive individual corresponded to a tragelaphus spekii and was infected with a parasite belonging to group 3 ( figure 1c ). in this study, we tested whether hematophagous flies could be used as 'flying syringes' to identify blood-borne pathogens circulating in the wild vertebrate fauna of gabon. our results show that the blood meals of the captured engorged flies can be successfully used to analyze the diversity of extant malaria parasites. despite a limited sampling effort (a total of 4 weeks of sampling for each park), we could screen the diversity of haemosporidian parasites from a large range of vertebrate hosts, including mammals, birds and reptiles. parasites were detected in more than 8% of the analyzed samples. these malaria parasites belonged to already known, but also to never previously table 1 . number and proportion of specimens captured per fly species. the number of engorged specimens and blood meals identified in each fly species are also indicated. mammals artiodactyla table 4 continued on next page concerning the method efficiency, 30% of blood meals were obtained from 4099 hematophagous flies. this result is consistent with previous studies (mavoungou et al., 2008; simo et al., 2012) showing that most hematophagous flies caught using traps are often seeking hosts for a blood meal. other methods using a dip net seem to have a better capture efficiency with more than 40% of engorged flies caught on their resting places (gouteux et al., 1984) . however, this method requires spending a lot of time in the field because of difficulties in finding their resting sites and catching the flies. tsetse flies provided 99% of the collected blood meals (54% by glossina palpalis palpalis) and they are an interesting candidate as 'flying syringes'. indeed, differently from stomoxids and tabanids, both sexes are exclusively hematophagous in tsetse flies. in addition, g. p. palpalis is considered to be an opportunistic species concerning its feeding behaviour, thus explaining the large diversity of blood meals (clausen et al., 1998; simo et al., 2008; weitz, 1963) . conversely, stomoxids and tabanids show sex-specific differences in feeding behaviour and this may partly explain the smaller number of blood meals collected in these two families. in stomoxids, both sexes are unknown_host_819 ky631984 doi: 10.7554/elife.22069.008 hematophagous, but males sometimes feed on nectar (wall and shearer, 1997) . moreover, the digestion of stomoxids starts more rapidly than in the other hematophagous flies (moffatt et al., 1995) . male and female tabanids feed on nectar just after their emergence as adults. only after having been fertilized, females start sucking blood (mullens, 2002) . therefore, engorged stomoxid and tabanid flies are more difficult to capture. additionally, the lack of engorged stomoxids and tabanids could be explained by the fact that we sampled flies only at floor level. indeed, some stomoxid species readily feed on arboreal monkeys that are mostly found higher in the tree layer (mavoungou et al., 2008) . the low rate (35%) of blood meal identifications could be explained by the degradation of host dna during digestion in the fly midgut or by a too small blood quantity in the midgut. the stage of digestion might influence dna degradation and the host identification efficiency. nevertheless, the diversity of hosts we successfully identified, mainly in tsetse fly blood meals, was large, including big terrestrial (elephants) and semi-aquatic mammals (hippopotamus) and also reptiles and birds. as previously noted, the diversity of blood meals can be due to the fly high mobility, their opportunistic feeding behaviour and their frequent feeding. in our study, most blood meals were from terrestrial animals (i.e. that live primarily on the ground) and very few from arboreal species. as mentioned above, this result is potentially biased by the trophic preferences of tsetse flies and by the capture method that excluded canopy levels. previous studies have shown that hematophagous flies sampled in canopies mainly feed on arboreal species (mavoungou et al., 2008) . therefore, changes in trap position could broaden the range of host species analysed. we can also notice the absence of small mammals (e.g., rodents or bats) within the diversity of host vertebrates we identified. this may be explained by the trophic preferences of the flies we sampled which could have a preferential taste for large vertebrates as previously documented for tsetse flies (e.g. [muturi et al., 2011; späth, 2000] ). concerning pathogen detection, we detected extant haemosporidian parasites in 8.65% of the 428 blood meals for which the host origin was successfully identified. moreover, we also detected parasites in blood meals of unknown origin, thus increasing the number of detected parasites. together, these results show that blood meals collected from hematophagous flies are suitable for tracking blood-borne pathogens from wild animals. haemosporidian pathogens ingested by hematophagous flies during their blood meal can remain detectable in the fly digestive tract even after partial digestion of the blood meal. we observed congruence between the identified hosts and the detected pathogens. as expected, p. falciparum was detected in human blood and p. adleri in gorilla blood. haemosporidian lineages are often host-specific or restricted to certain classes of vertebrate hosts. therefore, the unknown host could be inferred from the detected haemosporidian species (figure 1c) . for example, the blood meal from unknown host n˚110 could have originated from a kinixys turtle (kinixys sp.). similarly, the blood meals from the unknown hosts n˚649, 520, 665, 512 and 819 could have originated from humans (homo sapiens). the present study demonstrates the possibility to use hematophagous flies as 'flying syringes' to analyze the diversity of pathogens circulating in wildlife. we think that there is now room for improvement of the tool; for instance, by improving the methods used to identify the blood meals and the pathogens. since dna is likely to be degraded in many blood meals (calvignac-spencer et al., 2013; schnell et al., 2012) , the use of pcr systems targeting fragments of shorter size could potentially improve the performance of detection. a trial study based on 89 previously unidentified blood meals using a pcr system amplifying a shorter fragment (<150 bp) (boessenkool et al., 2012) than the one used in the present study allowed the identification of 76% (n = 68) of the hosts (figure 2) . this represents an important gain of sensitivity. however, these primers are still not ideal for our purpose as they were designed for optimal amplification of mammal dna and often fail to properly amplify the dna of other classes of vertebrates. a similar pcr system targeting the entire range of vertebrates still remains to be developed. for plasmodium, our trial for amplifying a shorter fragment of cytb (<200 bp) using a combination of previously published primers did not increase the sensitivity. indeed, out of 91 samples for which the blood meal was successfully identified but in which no haemosporidian infection was detected with our long cytb pcr system, only one was shown to be positive with the short pcr system. however, it is possible that other pcr systems, more optimized, could indeed improve the sensitivity of plasmodium detection. another direction of improvement could be the use of high-throughput sequencing technologies on pools of blood-engorged flies or amplicons to ease the identification of both hosts and parasites (especially in the case of mixed blood meals or mixed infections). finally, another way to improve the tool could be to use high-throughput multiplexed pathogen detection methods for the simultaneous testing of many samples in rapid succession. with such improvements, this approach of 'xenorsurveillance' could usefully complete recently developed methods based on the analysis of other invertebrates (carrion flies (hoffmann et al., 2016) , mosquitoes [grubaugh et al., 2015] ) and become an innovative way for the concomitant surveillance of many enzootic blood-borne pathogens, such as viruses (chikungunya, zika), bacteria, protozoa and macro-parasites. the use of hematophagous flies as 'flying syringes' could indeed improve public health management by allowing the surveillance and early detection of zoonotic pathogens and thus prevent they spread to humans before they cause massive infections. this tool could also help to better understand the circulation in wildlife of other enzootic viruses, such as chikungunya or zika, especially at the interface between natural/sylvan environments and, consequently, improving our knowledge of their natural history. from a broader perspective, this method could also be useful for people interested in wildlife biodiversity and conservation. indeed, it could help monitoring the wildlife diversity within a specific region as demonstrated with other invertebrate systems (calvignac-spencer et al., 2013; lee et al., 2015; schnell et al., 2012; schubert et al., 2015) . more importantly, it could also allow detecting the emergence of new diseases in wild animals that may threaten their long-term survival. despite the significant scientific advances in the medical field, humans are still unable to predict where, when and how epidemics arise. around 60% of emerging diseases in humans are of zoonotic origin. the progressive reduction of wild habitats will increase the contacts between humans and species that are potential reservoirs of diseases. we propose here a new non-invasive tool that can help identifying pathogens that circulate in wildlife before they spread in humans. the fly sampling was carried out in four wildlife reserves in gabon (figure 1a hematophagous flies were sampled during the rainy and dry seasons between 2012 and 2014. in inp and mdnp, sampling was done during two years following a gradient of human activity from primary forest to villages. in the other parks, flies were sampled during a single year. flies were collected by using vavoua and nzi traps (laveissiere and grebaut, 1990; acapovi et al., 2001; mihok, 2002; gilles et al., 2007) . the vavoua trap, initially developed for the capture of tsetse flies was also successfully used for the capture of stomoxids at la ré union island (laveissiere and grebaut, 1990; gilles et al., 2007) . the nzi trap was more adapted to the capture of glossina pallidipes and tabanids in africa (acapovi et al., 2001; mihok, 2002) . in each park, we placed 24 traps (12 vavoua and 12 nzi) during 2 weeks per climatic season. each trap was activated from 7:00 am to 5:00 pm. freshly collected hematophagous flies were identified using a stereo-microscope and taxonomic procedure. the fly species (tsetse, stomoxids and tabanids) was determined following the determination keys of pollock (1982) , brunhes et al., 1998 , zumpt, 1973 , garros et al. (2004 and oldroyd (1973) , on the basis of their morphological characteristics, such as size, color, wing venation structure and proboscis. after species identification, engorged flies were dissected individually in a drop of dulbecco's phosphate buffered saline solution (1x dpbs) to isolate blood meals from midgut. each hematophagous fly was dissected on a slide using one forceps and one scalpel that were changed each time to avoid contaminations. each blood meal was transferred in a 1.5-ml microtube containing 50 ml of rnalater stabilization solution (qiagen: store at rt tissue collection) to stabilize and protect nucleic acids of vertebrate hosts and pathogens contained in the blood meals. samples were kept at ambient temperature during field session and then frozen at à80˚c until dna extraction. samples were centrifuged at 15,000 rpm at 4˚c for 10 min to remove the rnalater solution. pellets were used to extract dna using the dneasy blood and tissue kit (qiagen) according to the manufacturer's instructions. extracted dna was eluted in 100 ml of buffer ae and stored at à20˚c. the origin of blood meals was determined using the extracted dna to amplify a 450 bp fragment of the cytochrome b (cytb) gene using previously published primers (townzen et al., 2008) . pcr amplifications were performed using a geneamp 9700 thermal cycler (applied biosystems, usa) with 50 ml reaction mixtures containing 4 ml template dna, 10 mm tris-hcl (ph = 9), 50 mm kcl, 3 mm mgcl 2 , 20 pmol each primer (5'cccctcagaatgatatttgtcctca3' and 5'ccatccaacatc tcagcatgatgaaa3'), 200 mm dntp and 1 u taq polymerase. the thermal cycling conditions consisted of 3.5 min at 95˚c, 40 cycles of 30s at 95˚c, 50s at 58˚c, and 40s at 72˚c, followed by 5 min at 72˚c. when cytb amplification failed, a 330 bp and/or a 660 bp fragment of the cytochrome oxydase subunit i (coi) gene was amplified using previously described primers and protocols (townzen et al., 2008) . all pcr-amplified products (10 ml) were run on 1.5% agarose gels in tbe buffer, and positive samples were sent to beckman coulter genomics (france) for sequencing in both directions (forward and reverse) after purification. consensus sequences were compared with existent sequences using the ncbi nucleotide blast search (altschul et al., 1990) to determine the host species. hosts were identified when the amplified and reference sequences showed at least 98% similarity. haemosporidian parasite detection was performed in samples with identified blood meal origin and also in 100 randomly chosen samples for which blood meal origin could not be identified. haemosporidian parasites were detected by pcr amplification of a portion of the cytb gene (~790 bp) using a nested pcr protocol, as previously published (ollomo et al., 2009) . pcr products were checked on 1.5% agarose gels before shipment to eurofins mwg (germany) for sequencing in both directions (reverse and forward) after purification. multiple alignments of haemosporidian sequences were done using muscle (edgar, 2004) . a phylogenetic tree with the haemosporidian sequences obtained in our study and a set of reference sequences was built using maximum likelihood (ml) methods and phylogeny.fr (dereeper et al., 2008 ) (see table 4 for accession numbers). the ml model used for construction of the tree was gtr (general time reversible)+g (gamma distribution)+i (invariable site distribution). several measures were taken to avoid contaminations during our manipulations. extraction of dna was performed at the cirmf (gabon) in a laboratory working on mosquitoes. the room in which extraction was performed was away from the rooms in which dna was amplified in this lab. dna extracts were then sent to france at the ird (montpellier). there, blood meal and plasmodium identification was performed. this lab had never worked before on plasmodium from ungulates or reptiles. amplification of host dna was never or very rarely performed in this lab. when the work was performed, no work on plasmodium has been performed in this lab for almost 4 years. in addition, the laboratory is designed to avoid contaminations. clearly defined and separated areas are devoted for each step of the pcr process: one area is devoted to the preparation of reagents (mix pcr). another room is dedicated to the pre-pcr manipulation (loading of native dna). this step is done under a cabinet to avoid contamination of the sample with dna from the operator. finally, an area is devoted to pcr-amplified dna. in this area, cabinets are used to deposit the first pcr product into the reagents of the second pcr (for nested pcrs). all cabinets are equipped with uv lamps and are always decontaminated with dna-free solutions before and after manipulations. gloves and coats are changed when moving between the areas and plugged tips are used at all steps. blank controls were always incorporated at all steps of the experimental procedure and were always negative. several observations confirm the authenticity of our results: (1) >80% of the hosts that were found have never been manipulated in our lab (hosts that are not humans or non-human primates); (2) the parasite always corresponded to the expected host (antelope parasites were always found in antelopes, human parasites in humans and gorilla parasites in gorillas). contaminations by external dna would have lead to random association of hosts and parasites; (3) a new lineage of parasites was discovered. since dna is likely to be degraded in many of our samples, the use of pcr systems targeting fragments of shorter size might improve performance. to determine if this could be the case with our study system, we performed supplementary analyses using (1) a pcr system targeting a shorter fragment of the vertebrate mitochondrial dna to identify the blood meal origin and (2) a pcr system targeting a shorter fragment of the cytb dna to identify the parasite. for the identification of the host, the pcr system used was the one amplifying a fragment of 150 bp of 16s as described in boessenkool et al. (2012) and using the primers 16smam1 and 16smam2. this pcr system was used on blood meals that failed to be identified using our original pcr system (see the paragraph 'blood meal identification'). a total of 89 blood meals were tested for this trial study. for the parasite, we designed new primer sets to amplify a shorter fragment of the cytb gene of the parasite (~177 bp). this new pcr system was applied to blood meals for which the host was identified but that were negative to plasmodium with our long pcr system (~790 bp, see material and methods above). a total of 91 blood samples were tested. for the first round of amplifications, we used 6 ml of dna template in a 25 ml reaction volume, containing: 12.5 ml of mix pcr (qiagen), 2.5 ml solution q (qiagen), and 4 pmol of each primer (cytb1f ctctattaatttagttaaagcacactt and 454r ccwgtwgcytgcatytatct). cycling conditions were 15 min at 95˚c, 30 s at 94˚c, 90 s at 57˚c, 90 s at 72˚c (40 cycles), and 10 min at 72˚c. for the second round of amplification, we used 1.5 ml of the first pcr template in a 25 ml reaction volume, containing 2.5 ml of 10â buffer, 1.25 mm mgcl 2 , 250 mm of each dntp, 10 pmol of each primer (454f2 waattayccatgyccattraa and plas1rc caccatccactccataattctc), and 0.1 unit taq platinum (invitrogen). cycling conditions for the second round were 5 min at 95˚c, 30 s at 94˚c, 30 s at 50˚c, 90 s at 72˚c (35 cycles), and 10 min at 72˚c. the amplified products (5 ml) were run on 1.5% agarose gels in tae buffer. the pcr-amplified products (177 bp) were used as templates for sequencing. dna sequencing was performed by eurofins mwg. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. abondance relative des tabanidé s dans la ré gion des savanes de cô te d'ivoire. revue d'élevage et de médecine vétérinaire des pays tropicaux an overview of the epidemiology of avian influenza basic local alignment search tool emergence of zaire ebola 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zusammenarbeit reading mammal diversity from flies: the persistence period of amplifiable mammal mtdna in blowfly guts (chrysomya megacephala) and a new dna mini-barcode target ecology of stomoxyine fulies (diptera: muscidae) in gabon. ii. blood meals analysis a nd epidemiologic consequences the development of a multipurpose trap (the nzi) for tsetse and other biting flies studies on the synthesis and secretion of trypsin in the midgut of stomoxys calcitrans horse flies and deer flies (tabanidae). in: medical and veterinary entomology global biogeography of human infectious diseases tracking the feeding patterns of tsetse flies (glossina genus) by analysis of bloodmeals using mitochondrial cytochromes genes host preferences of tabanid flies based on identification of blood meals by elisa tabanidae. in: insects and other arthropods of medical importance a new malaria agent in african hominids tracking pathogen transmission at the human-wildlife interface: banded mongoose and escherichia coli tsetse biology, systematics and distribution, techniques african great apes are natural hosts of multiple related malaria species, including plasmodium falciparum investigating the zoonotic origin of the west african ebola epidemic sivcpz in wild chimpanzees detection and isolation of highly pathogenic h5n1 avian influenza a viruses from blow flies collected in the vicinity of an infected poultry farm in kyoto screening mammal biodiversity using dna from leeches targeted detection of mammalian species using carrion fly-derived dna origins of hiv and the aids pandemic tsetse fly host preference from sleeping sickness foci in cameroon: epidemiological implications identification of different trypanosome species in the mid-guts of tsetse flies of the malanga (kimpese) sleeping sickness focus of the democratic republic of congo human infections and detection of plasmodium knowlesi feeding patterns of three sympatric tsetse species (glossina spp.) (diptera: glossinidae) in the preforest zone of cô te d'ivoire risk factors for human disease emergence identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit i and cytochrome b gene sequences veterinary entomology potential for insect transmission of hiv: experimental exposure of cimex hemipterus and toxorhynchites amboinensis to human immunodeficiency virus the feeding habits of glossina zika virus: history of a newly emerging arbovirus bushmeat hunting, deforestation, and prediction of zoonoses emergence ecological origins of novel human pathogens temporal trends in the discovery of human viruses the stomoxynae biting flies of the world authors thank all the reviewers for their constructive and helpfull comments. this study was carried out with a financial support of: 'agence universitaire de la francophonie' (auf), the 'service de cooperation et d'action culturelle' (scac) of french embassy in gabon, the 'institut franç ais' of libreville (if), the 'conseil ré gional de bourgogne' and the 'bonus qualité recherche' (bqr) of université de bourgogne. this work was also funded by institut de recherche pour le dé veloppement (laboratoire mixte international zofac), centre international de recherches mé dicales de franceville (cirmf), as well as the agence nationale de la recherche (anr) programme jeunes chercheuses jeunes chercheurs (jcjc) sciences de la vie, de la santé et des ecosystè mes 7-2012 project origin (anr jcjc svse 7-2012 origin). we thank the agence nationale des parcs nationaux (anpn) and the centre national de la recherche scientifique et technologique (cenarest) of gabon who authorized this study and facilitated the access to national parks. authors also thank eric willaume from the park of la lé ké di for his help. key: cord-294684-wfsdjs1f authors: vesnaver, elisabeth; goldman, mindy; o’brien, sheila; macpherson, paul; butler-foster, terrie; lapierre, don; otis, joanne; devine, dana v.; germain, marc; rosser, andrew; macdonagh, richard; randall, taylor; osbourne-sorrell, william; clement-thorne, broderic; al-bakri, taim bilal; rubini, kyle a.; hill, nolan e.; presseau, justin title: barriers and enablers to source plasma donation by gay, bisexual and other men who have sex with men under revised eligibility criteria: protocol for a multiple stakeholder feasibility study date: 2020-11-02 journal: health res policy syst doi: 10.1186/s12961-020-00643-4 sha: doc_id: 294684 cord_uid: wfsdjs1f background: blood donation policy in canada for gay, bisexual and other men who have had sex with men (gbmsm) has changed progressively in the last decade from indefinite deferral to 3-month deferral from last male-to-male sex. driven by safety data and overseen by the national regulator, more inclusive policies continue to redress the disparity in donation for gbmsm. at the same time, the need for source plasma to prepare fractionated blood products is growing worldwide. the collection and processing of source plasma ensures greater safety compared to whole blood donation with respect to transfusion-transmitted infection. this greater safety offers an opportunity to evolve policies for gbmsm from time-based to behaviour-based deferral using revised eligibility criteria. however, changing policies does not in itself necessarily guarantee that gbmsm will donate or that staff in donor clinics are ready to support them to do so. in anticipation of a move to behaviour-based donation screening for gbmsm in canada, we aim to assess the acceptability of and perceived barriers and enablers to source plasma donation using revised screening criteria for gbmsm among key stakeholders to inform policy implementation strategies. methods: this mixed-methods feasibility study will involve gbmsm and donor centre staff to understand modifiable barriers to implementing more inclusive eligibility criteria. key informant interviews and surveys will be rooted in the theoretical domains framework to identify modifiable factors associated with source plasma donation motives in gbmsm and training needs in donation centre staff. we will use an integrated knowledge translation approach involving a partnership between researchers, the national blood operator and gbmsm, situating knowledge users as key research team members to ensure their perspectives inform all aspects of the research. discussion: our integrated knowledge translation approach will provide a more comprehensive and collaborative understanding of blood operator and gbmsm needs while accelerating the implementation of study findings. given the historical backdrop of the decades of exclusion of sexually active gbmsm from blood donation, this study has the potential not only to inform a process and policy for gbmsm to donate source plasma, a blood product, but also offers opportunities for new relationships between these knowledge users. the demand for plasma proteins (e.g. immunoglobulins) continues to rise globally, outstripping the supply. canada does not collect enough source plasma to meet the needs of its citizens and would benefit from widening the range of possible donors, especially to those who may be interested but limited from doing so, such as gay, bisexual and other men who have sex with men (gbmsm). source plasma is a type of plasma donation that is frozen and then sent to a manufacturer for the production of specific plasma protein products such as intravenous immunoglobulins. the additional processing involves pathogen reduction and additional assurance of supply safety compared to whole blood donation with respect to transfusion-transmitted infection. this greater safety offers an opportunity to widen the range of possible source plasma donors with more inclusive eligibility screening. in the early 1980s, in canada and many other countries, blood donation by gbmsm was banned based on the higher seroprevalence of hiv in this group and because there was no test available to detect hiv infection at the time [1] . donor criteria have moved progressively from a permanent deferral (cannot donate) to a 3-month deferral since the last time of sexual encounter with a goal of moving to behavioural risk screening for gbmsm donors. the risk of hiv infection is not the same for all gbmsm; behavioural risk screening would enable identification based on sexual behaviour rather than time since sexual encounter [2, 3] . currently, canadian blood donor criteria do not permit any sexually active gbmsm to donate whole blood or source plasma-men who have had sex with a man in the past 3 months are deferred. however, canadian blood services, one of canada's two national blood operators, is reviewing its policies regarding source plasma donation in an effort to be more inclusive while maintaining the safety of the blood supply, including some gbmsm that are at low to no risk of hiv infection such as those in monogamous relationships. it is likely that many in the general population, including gbmsm, are not yet as aware of what source plasma donation entails and how it differs from whole blood donation. source plasma donation involves drawing whole blood, separating out the plasma, and then returning red and white blood cells and platelets back to the donor. the source plasma donation process takes approximately 45 min and can be donated as frequently as every week. once collected, source plasma can be frozen and stored for months and the manufacture of plasma protein products includes several pathogen reduction steps to kill any residual infectious agents. conversely, fresh components collected during whole blood donation, such as platelets and red blood cells, have a short storage period of up to 7 or 42 days, respectively, and are currently not subjected to the same pathogen reduction processes as source plasma in canada. both the longer storage time of source plasma before pooling and the pathogen reduction manufacturing processes involved in the production of plasma protein products make it feasible to apply additional safety steps that may allow some sexually active gbmsm to donate source plasma. expanding the eligibility to gbmsm for source plasma donation first provides an opportunity to collect data on gbmsm donors whilst maximising the continued safety of the supply. data on gbmsm donors is critical to support changes to whole blood screening policies; with current deferrals in place and gbmsm unable to donate, no such data can be collected. as of 2019, all male donors are asked 'in the last 3 months, have you had sex with another man?' responding yes to this question results in a 3-month deferral from last sexual contact, which amounts to an indefinite deferral for a donor in an ongoing relationship. revised eligibility criteria could include adding additional behavioural screening questions to those who answer 'yes' to identify gbmsm that are at low to no risk of hiv infection. the additional behavioural questions under consideration include: 1 have you had sex with a new partner in the last 3 months? 2 have you had sex with more than one partner in the last 3 months? 3 are you in a mutually exclusive (monogamous) relationship? these questions are based on those in use in jurisdictions such as france (quarantined plasma programme), italy and spain [4] [5] [6] as well as data generated in the ongoing canadian research studies on gbmsm and blood donation. the use of the draft questions in this study will permit further validation of their clarity and acceptability, which may lead to modifications in the actual questions submitted to health canada for implementation in a source plasma collection site. making donation possible is necessary but likely insufficient for a successful gbmsm plasma donation programme. given the historical exclusion of gbmsm from any blood or blood product donation, any gbmsm donation programme would require general acceptability among gbmsm. in canada, caruso et al. [7] explored the acceptability among gbmsm of a plasma donation programme involving quarantining plasma until donors return 2 months or more after initial plasma donation and are retested for transmissible diseases, thus ensuring the safety of the supply prior to release. the programme was considered by some participants to reinforce the exclusion and discrimination experienced with whole blood deferral policies because gbmsm donors and their plasma are treated differently. while behavioural risk screening is an approach that has previously been supported by gbmsm with respect to whole blood donation [8, 9] , there is also a need to explore how gbmsm understand and would respond to the screening questions. studies of gbmsm that have donated blood despite not being eligible reported that participants donated because they were uninformed about the policies or they assessed their own hiv risk to be low with a mixed understanding of the 'window period' , the period in which new hiv infection is not detectable but potentially infectious [8] [9] [10] . exploring how gbmsm understand the behavioural screening questions prior to implementation can help uncover and address the challenges of adherence to the policies. the ineligibility of sexually active gbmsm from blood and plasma donation has attracted debate worldwide with regards to justice, exclusion, safety, risks and community [11] [12] [13] [14] . time-based deferrals that involve policies specific to gbmsm have been described by some as discriminatory, disproportionate to risk and unnecessarily strain the supply of available blood (and plasma) products [15, 16] . the gbmsm blood donor debate has ignited many gbmsm and allies to equate blood donation with equality and full citizenship and has resulted in lawsuits, boycotts of blood drives and petitions [16, 17] . the effects of group exclusion from blood donation can be long lasting as has been observed in the haitian-canadian community [18] . an extension of existing donation promotion strategies without such considerations is likely to miss the mark. understanding how gbmsm view the historical context and changes of the policies in relation to how they view the proposed behavioural risk screening approach will generate insights for the development of appropriate strategies for communication of the policy and promotion of source plasma by gbmsm. the staff in donation clinics who would be tasked with implementing the revised eligibility criteria for gbmsm play a central role in the safety and success of such a programme. alteration of the eligibility criteria may require additional training and staff may have views and perspectives that, if surfaced early, would help to ensure a feasible and acceptable roll-out of a gbmsm source plasma donation programme. indeed, hughes et al. explored the views of blood collection organisation staff on the transition from indefinite deferral to 12-month deferral in gbmsm in california [19] . they showed that, while some staff voiced reservations, most staff supported this transition. they also showed that staff valued their professionalism and would follow the regulations imposed by the food and drug administration in the united states regardless of their own personal views. furthermore, most staff expressed a desire for further training and materials. findings such as these highlight the importance of understanding the potential barriers and enablers to the implementation of revised eligibility criteria to ensure that such a change could be feasibly implemented. however, the hughes et al. study focused on whole blood donation and the move from indefinite to 12-month donation deferral in gbmsm. there is a need to explore such views in more detail in a canadian setting for source plasma donation and for revised eligibility criteria that moves away from a time-since-sexual-encounter criterion. while this is a promising opportunity for more inclusive donation to enhance the donor pool with a previously excluded segment of the population, it is important to assess the feasibility with key knowledge users prior to implementation. the proposed study is designed to fill this gap and provide information from gbmsm and donor centre staff on whether revised eligibility criteria for gbmsm is viewed to be feasible and acceptable and what factors could be adapted to enhance these features [20] . the implementation of revised eligibility criteria and gbmsm source plasma donation can be described as an intervention designed to support behaviour change in gbmsm. due to the number of different knowledge users that may each require different targeted strategies, the intervention can be described as a 'complex' intervention. the united kingdom medical research council guidance on the development and evaluation of complex interventions highlights the importance of careful intervention development using appropriate theory and feasibility assessment prior to evaluation and implementation [21] . although there are extant theories to draw from to understand first time blood donation (as it would be for most gbmsm), the application of behavioural science to source plasma donation is scant and, in canada, source plasma donors are typically recruited from among whole blood donors [22] . furthermore, the historical backdrop of exclusion of gbmsm from donation may have resulted in drivers that are unique to this population. a systematic approach to intervention development can provide transparency and foster cumulative evidence to ensure that the strategies devised to support gbmsm in donating and staff in screening are fit for purpose. french et al. [23] proposed a four-step approach for designing theory-based implementation interventions that suggests key steps in the systematic development of an intervention to change behaviour as follows: first, identify who needs to do what differently; second, identify what barriers and enablers might be relevant; third, select change strategies and techniques that are fit for purpose to address identified barriers and enablers; and fourth, identify how change can best be measured. this study will focus on completing step two using a comprehensive theoretical framework. to ensure the findings are useful to all knowledge users, this study will be rooted in an integrated knowledge translation (ikt) approach, a form of research coproduction [24] . research co-production is an equitable collaborative approach to research that meaningfully engages knowledge users who are directly impacted by the results of the research [25] . ikt is distinguished from other co-production approaches with its emphasis on collaboration with knowledge users that are in positions of power to create change [26] , the focus of study is identified by knowledge users, and research focuses on "generating real-life solutions to complex problems" [27] . the purpose of this study is to assess the feasibility and acceptability of source plasma donation with revised eligibility criteria for gbmsm in two canadian cities. our specific objectives are to identify the following: 1 views and experiences of gbmsm regarding source plasma donation, current eligibility criteria and revised eligibility criteria. 2 the acceptability of additional behavioural questions during the screening process from the perspective of gbmsm. 3 potential barriers and enablers to source plasma donation from the perspective of gbmsm. 4 the acceptability of additional behavioural questions during the screening process from the perspective of donor centre staff. 5 potential barriers and enablers from the perspective of donor centre staff in the donation clinic to implementing new eligibility criteria for gbmsm to donate and to inform the adaptations needed to centre flow and processes prior to piloting. 6 consistencies and discrepancies between the two canadian cities and implications for tailoring strategies to support gbmsm source plasma donation in each context. consistent with an ikt approach, the initial topic was born out of canadian blood services seeking to better understand the feasibly of a source plasma programme for gbmsm with more inclusive donation eligibility criteria and the broader voice of gbmsm demanding greater equality as it relates to donation. the research questions were collaboratively developed by the research team consisting of scientists and collaborators from research institutions, canada's two national blood operators, and a local lgbt2q+ community organisation to ensure diverse perspectives and expertise. while our team offers both insider and outsider perspectives with respect to the donor centre working environment and identifying as gbmsm, we sought out greater inclusion of gbmsm voices in the design and conduct of the study design. we first consulted with community members through outreach and engagement activities and these activities resulted in the formation of a local advisory group of gbmsm. local advisors are key members of the research team (rather than participants) who provide ongoing input in monthly group meetings and in individual exchanges by email or phone as needed. they provide feedback on study design, contribute to survey and interview guide development, and help to develop and facilitate the recruitment strategies. they will review and provide feedback of summaries of analysis and results, advise on next steps, and help disseminate findings. this mixed-methods feasibility study will explore the views of gbmsm and donor centre staff regarding source plasma donation and eligibility criteria to better understand the modifiable barriers and enablers to implementing revised eligibility criteria. we will use qualitative interviews and an online anonymous survey to identify the barriers and enablers to source plasma donation that may emerge with the implementation of revised screening criteria for source plasma donation by gbmsm. qualitative interviews will be used to elicit potential barriers and enablers to implementing revised eligibility from the perspectives of donor centre staff. this study will be conducted in london (ontario) and calgary (alberta). canadian blood services operate two dedicated source plasma donation centres that are located in london (ontario) and calgary (alberta). these centres are potential locations for a first implementation of a gbmsm source plasma donation programme if approved by health canada. the study was first developed in london (ontario) due to operational feasibility and strong relationships with the local gbmsm community. the research team sought additional funding to expand the project to calgary (alberta). many factors may emerge as barriers and enablers to source plasma donation by gbmsm or to donor centre staff 's implementation of revised eligibility criteria. solutions and strategies for encouraging and supporting donation should be tailored to address these barriers to ensure that the supports developed are fit for purpose [28] . theories of behaviour provide a useful set of factors to consider when investigating barriers and enablers. by providing an understanding of which modifiable factors may be associated to implementing revised criteria, such theories provide a source of factors that could then be directly targeted to develop strategies and materials to support donation. there are many different theories that could be used as a basis for identifying such factors in a systematic way. a group of researchers sought to synthesise key content across 33 predominant theories and the 128 constructs within them. they developed the theoretical domains framework (tdf) [29, 30] , which summarises key factors from theory that are known to be associated with behaviour and behaviour change and is well suited to explore the full breadth of factors that are relevant in this behaviour and population. the tdf identifies 14 different modifiable factors, as follows: knowledge, skills, beliefs about capabilities, optimism, beliefs about consequences, intention, goals, professional/social role and identity, social influences, reinforcement, behavioural regulation, emotion, memory/attention/decision processes, and environmental context and resources. clear guidance has been developed to use the tdf for developing qualitative interview guides [31] and quantitative surveys [32] . the tdf has been used broadly as a basis for understanding barriers and enablers in the healthcare setting and with the public [32] [33] [34] [35] . once identified, this approach specifically suggests particular strategies and behaviour change techniques that best suit addressing the barriers and enablers identified based on expert review and the evidence base [36] . participants and recruitment we will use a combination of purposive and snowball sampling to recruit adult (18+) gbmsm in london (ontario) and calgary (alberta) as well as in surrounding communities for interviews. purposive sampling will be used to recruit gbmsm who represent a breadth of age, cultural backgrounds and geographic locations (rural/urban). we will work with our local advisory groups, local organisations and social groups that provide services to gbmsm to help identify potential participants. in 2020, many countries, including canada, were practicing social distancing to reduce the spread of covid-19. in light of this, we will advertise on social media platforms with assistance from organisations that provide services to gbmsm. we recognise that this method of sampling may not reach those who are not active on social media and, as such, we will supplement this strategy with snowball sampling methods by inviting participants to recommend others for participation. snowball sampling is well suited for the recruitment of traditionally underserved groups and those who experience stigmatisation, including gbmsm. procedure we will conduct up to two semi-structured interviews per participant, by phone, scheduled over a period of 2-6 weeks. informed consent will be obtained prior to the interview. interviews will be approximately 60 min in length and audio-recorded for verbatim transcription. the use of a multiple interview format will promote the development of rapport over time, facilitating the discussion of sensitive and personal topics while allowing time for reflection and elaboration [37] . we will offer the option of one longer interview if participants prefer, to take a participant-centred approach to data collection. field notes will be captured after each interview to assist with analysis and reflection on the impact of the interviewer's positionality on the data generated. interview participants will receive a cad$ 20 gift card at each interview session to thank them for their time, to a maximum of cad$ 40 per person. participants that opt for one longer interview will receive a cad$ 40 gift card. interview guide development the first interview will explore the context of how source plasma donation is perceived by gbmsm using a semi-structured approach to interviewing. key interview questions will be used to help define the areas to be explored but the interview style will remain flexible to allow for the discovery and discussion of topics of importance to the participant [38, 39] that may not have otherwise been thought of as pertinent by the research team [40] . the topic guide includes questions regarding experiences of donation, deferral or exclusion, views on current gbmsm donor deferral criteria, and the acceptability of the three behavioural screening questions suggested for inclusion in revised eligibility criteria. the second interview will build on the first to elaborate on emerging themes and explore participants' views regarding the implementation of revised eligibility criteria, potential impacts of revised eligibility criteria on donation practices, and possible barriers and enablers to source plasma donation. the topic guide for the second interview draws on existing literature [31] and previously developed guides [33] to assess if and how the identified barriers align with tdf domains. interview guides will be reviewed by the study's local advisory groups in each city and piloted prior to broad enrolment. at the end of the interviews, participants will be asked about the interview experience and this feedback will be considered and incorporated as appropriate. sample size our sample size will be determined by the available number of interviewees. we aim to recruit 15-20 men from each region to complete a series of two interviews. this sample size estimate is informed by the scope of the study, the nature of the topic and the use of a multiple interview format [41] . given that data collection and analysis are concurrent, informational and thematic redundancy [42, 43] will be assessed on an ongoing basis and the number of interviews will largely be driven by the quality and richness of data [44] . drawing on the literature, saturation is often reached within 15-20 tdf interviews [33, 34] . data analysis interviews will be audio recorded, transcribed verbatim and de-identified prior to analysis. as an additional step, we will send each participant their written transcript to review for completeness and resonance. we will use nvivo12 to facilitate analysis. data collection and analysis will be concurrent to generate emerging understanding of the research questions, which will inform both the sampling and the questions being asked [39] . we will conduct two phases of qualitative descrip-tive analysis-inductive thematic analysis [45] to identify, analyse and report patterns within the data, and theorydriven, directed content analysis to code barriers and enablers expressed by gbmsm to specific tdf domains [46] . throughout, reflexive journaling will be used to capture the analytic process and any developing insights about the patterns in the data. data from each region will be analysed separately as each context may have unique cultural, societal and political forces that shape the views of gbmsm regarding source plasma donation. we will contrast findings between cities to inform city-specific modifications for implementation. during thematic analysis, we will follow the six analytic steps proposed by braun and clark [45] : becoming familiar with the data, generating initial codes, searching for themes, reviewing themes, defining and naming themes, and producing the report. we will then re-visit the data during directed content analysis and use guidance from the literature [31] to code barriers and enablers to source plasma donation expressed by gbmsm to specific tdf domains [46] . we will develop a code book to enhance the reliability of coding. to enhance the trustworthiness of our findings, we will use a combination of duplicate coding by two researchers trained in both inductive analysis and the use of the tdf, peer debriefing activities and consensus-building measures. participants we will invite adult gbmsm (aged 18+) living in london (ontario) and calgary (alberta) to complete an anonymous questionnaire regarding their views about current and potential future barriers and enablers to donating source plasma. questionnaire items will enable the assessment of respondents' eligibility to donate according to the revised criteria but will not exclude participants to the survey on the basis of these criteria. recruitment and procedure our local advisory groups of gbmsm will facilitate recruitment by providing access to the venues and organisations through which a link to the online survey can be circulated as well as by advising on additional non-traditional venues for recruiting participants. we will offer the opportunity to enter a draw for one of ten pre-paid visa gift cards valued at cad$ 125 each. questionnaire development the questionnaire will be designed for completion by adult gbmsm and will include screening questions to ensure that we involve our targeted respondents. we will use items from a tdf questionnaire previously assessed for its discriminant content validity [32] to assess barriers and enablers to donating source plasma if they were eligible under revised eligibility cri-teria. appropriate language and response options will be informed by the interviews; thus, certain questions may be unique to each city. the questionnaire will be piloted by gbmsm prior to launching. data analysis our analytical approach is modelled on an approach used by presseau et al. [33] . we will conduct descriptive analyses to identify mean scores and standard deviations on each of the 14 tdf domains. as donation represents a hypothetical behaviour for respondents at the moment, we will assess intention to donate as a proxy for actual donation (consistent with other donation studies) [47] . we will assess bi-variate associations between intention to donate source plasma with socio-demographic factors, including self-identified gender, eligibility to donate (based on revised criteria) and responses to each tdf domain. informed by behavioural theory [33, 48] , we will then conduct multiple regression analysis to investigate which variables are associated with gbmsm's intention to donate source plasma. we will investigate whether these associations are moderated by their eligibility as determined by the revised criteria. if the surveys end up very similar between sites, we will analyse the combined sample and investigate whether the associations differ by location. we are powered to conduct independent analyses in each region if needed. sample size for analysis of our survey data, for a regression model comprised of data covering 20 independent variables [i.e. 13 tdf domains (not including intention), as well age, self-identified gender, rural/urban, sex with new partner in last 12 months (yes/no), sex with more than one partner in last 3 months (yes/no), in a monogamous relationship (yes/no), married (yes/no)], we will require a total sample size of 314 participants (157 participants in each region) to detect a medium effect size (r 2 = 0.15). we will aim to continue to recruit until this sample is exceeded or the recruitment period is completed (6 months). we will interview english-speaking donor centre staff involved in applying current eligibility criteria or discussing eligibility with potential donors at the london donor centre. recruitment will be facilitated by our stakeholder contacts at the donor centre. nurses and donor care associates will be invited to participate in a one-on-one semi-structured telephone interview lasting 30-45 min. interviews will focus on the barriers and enablers to using each of the proposed revised screening criteria. our approach to interview guide development, data analysis and sample size is rooted in the tdf framework and will follow the methods outlined for the interviews with gbmsm. the findings of the study may be of interest to diverse audiences and require a multi-pronged dissemination strategy. a key advantage of an ikt approach is that stakeholders are members of the research team and provide real-time guidance on the type of dissemination that is needed at different stages of the project. our first priority is to work with our stakeholders to ensure they have the findings in an appropriate format and to develop a dissemination plan for their respective communities. we will collaborate with our local advisory groups of gbmsm to disseminate among their local communities and beyond, which may involve a website and online events or articles in gbmsm-focused venues. we will also work with our blood operator stakeholders to disseminate appropriately through their organisations and international networks. beyond our stakeholders, dissemination of this work is likely relevant to stakeholders outside the traditional scientific audience and, thus, we will aim to publish in open-access journals or repositories and share these via social media to reach a broader audience. source plasma donation by sexually active gbmsm is not currently permitted. the data generated in this study is based on a hypothetical change in policy. the barriers and enablers will be elicited by asking respondents to answer to a hypothetical scenario, which may be different from their responses to real-life scenarios [49] . if revised eligibility is approved, ongoing investigation is needed alongside any initial implementation. this protocol is based on a grant funded by health canada, administered by canadian blood services, and peerreviewed by experts external to the funder and blood operator. the study has been reviewed and received ethics approval from the ottawa health science network research ethics board (id # 20190287-01h and 20200255-01h). the canadian blood services ethics board has also reviewed and approved the parts of the study involving donor centre staff (id 2019.020). at the time of submission, recruitment and data collection had begun with donor centre staff and gbmsm. the findings from this study will contribute to our understanding of the views of gbmsm regarding source plasma donation and source plasma donation eligibility policies as well as of the perceived barriers and enablers to donating source plasma should they become eligible. findings will help to inform the development of implementation strategies and support donation promotion if policies change to enable some sexually active gbmsm to donate. this study will also generate insights regarding staff anticipated barriers and enablers to implementing revised criteria for source plasma donation that will inform intervention development in preparation for wide-scale implementation if policy changes are approved by health canada. a strength of using the tdf is that the domains have been mapped to specific evidence-based behaviour change techniques [36] . thus, the findings will specify theory-driven and evidence-based strategies to be incorporated into future intervention development to address the barriers and enablers identified, increasing the likelihood that resulting strategies are likely to be effective. although the findings are based on two collection sites, by rooting our approach in behaviour change theory, we will enhance the transferability of findings to other sites. the multiple sources and types of data that will be collected in this study will enable a broader understanding of the needs to be addressed prior to implementation. qualitative methods will help us to explore the full range of factors that may impact implementation by staff and donation among gbmsm. quantitative findings will inform which of these factors identified by gbmsm are likely to have the greatest impact on donation intention, elucidating the areas most amenable to the targeted strategies to support donation. canada is not alone in its consideration of behaviourrisk screening for blood donation [50] [51] [52] . while many countries have followed a similar evolution to blood donation policies for gbmsm, the context in which these policies are made and enacted vary greatly. the contextual findings can help decision-makers and researchers in other jurisdictions to better situate the findings and assess the transferability of our findings to their context [42] . as of 2020, the public discourse related to blood donation by gbmsm was intensifying in response to the repeated calls for blood donation by blood operators internationally due to covid-19-related blood shortages [53, 54] . there was also increasing public interest in plasma donation and the ineligibility of gbmsm due to the clinical trials investigating convalescent plasma as a treatment for covid-19 [55, 56] . convalescent plasma is a type of plasma collected from patients recovered from covid-19; gbmsm who have recovered from covid-19 are not eligible to donate. the iterative and adaptable nature of an ikt approach that engages both the blood operator and gbmsm combined with the flexibility of qualitative methods makes this project uniquely situated to adapt and respond to the shifting social context of the study. due to the decades of exclusion from blood and bloodproduct donation that gbmsm have experienced and the social and political movement that has arisen in response, there may be divisions between blood operators in different countries and gbmsm that will need to be bridged for gbmsm to engage enthusiastically with a source plasma donation programme. research using ikt has the potential to create impact beyond addressing the research objectives. there is potential for the different knowledge user groups involved in the research to experience mutually beneficial learning cycles throughout the project's stages of design, data collection, analysis, reporting and dissemination [25] . as each unique perspective contributes to the work, all knowledge users and researchers learn from these perspectives and may shift their own perspectives. furthermore, in this process, there is the potential to strengthen relationships across knowledge users. partnerships between gbmsm and canadian blood operators can only aid in improving the development of appropriate and sensitive implementation supports and ultimately facilitate bringing gbmsm into the donor base. this research will explore the perceptions and experiences of gbmsm and donor centre staff regarding the feasibility and acceptability of implementing revised eligibility criteria for source plasma donation by gbmsm. the findings from this study will help refine or provide feedback on three proposed behaviour-based screening questions for donation eligibility and provide a basis for developing sensitive materials that could support a change in policy. abbreviations gbmsm: gay, bisexual, and other men who have sex with men; ikt: integrated knowledge translation; tdf: theoretical domains framework. donor criteria for men who have sex with men: a canadian perspective state ment-from-the-minis ter-of-healt h-on-furth er-reduc ing-barri ers-for-blood -donat ion-by-men-who-have-sex-with-men donor deferral policies for men who have sex with men: past, present and future sexual risk behaviour and donor deferral in europe changing blood donor screening criteria from permanent deferral for men who have sex with men to individual sexual risk assessment: no evidence of a significant impact on the human immunodeficiency virus epidemic in italy the evolving blood donor deferral policy for men who have sex with men: impact on the risk of hiv transmission by transfusion in france one step closer": acceptability of a programme of plasma donation for fractionation from men who have sex with men views and experiences of men who have sex with men on the ban on blood donation: a cross sectional survey with qualitative interviews saving lives, maintaining safety, and science-based policy: qualitative interview findings from the blood donation rules opinion study (blood drops) context and social perceptions of blood donation in donors found positive for human immunodeficiency virus in france should men who have ever had sex with men be allowed to give blood? no is there a right to donate blood? patient rights; donor responsibilities should men who have ever had sex with men be allowed to give blood? yes end the gay blood ban. the guardian blood donation, deferral, and discrimination: fda donor deferral policy for men who have sex with men reconsidering the lifetime deferral of blood donation by men who have sex with men negotiating exclusion: msm, identity, and blood policy in the age of aids the paradoxical situation of blood donation in the haitian-quebec community transition to a 1-year deferral for male blood donors who report sexual contact with men: staff perspectives at one blood collection organization what can qualitative research do for randomised controlled trials? a systematic mapping review developing and evaluating complex interventions: the new medical research council guidance how do people become plasma and platelet donors in a vnr context? developing theory-informed behaviour change interventions to implement evidence into practice: a systematic approach using the theoretical domains framework knowledge translation in health care. hoboken: wiley embracing complexity and uncertainty to create impact: exploring the processes and transformative potential of co-produced research through development of a social impact model how does integrated knowledge translation (ikt) compare to other collaborative research approaches to generating and translating knowledge? learning from experts in the field building an integrated knowledge translation (ikt) evidence base: colloquium proceedings and research direction lost in knowledge translation: time for a map? making psychological theory useful for implementing evidence based practice: a consensus approach. qual saf health care validation of the theoretical domains framework for use in behaviour change and implementation research a guide to using the theoretical domains framework of behaviour change to investigate implementation problems discriminant content validity of a theoretical domains framework questionnaire for use in implementation research identifying determinants of medication adherence following myocardial infarction using the theoretical domains framework and the health action process approach • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? choose bmc potential determinants of health-care professionals' use of survivorship care plans: a qualitative study using the theoretical domains framework from theory to intervention: mapping theoretically derived behavioural determinants to behaviour change techniques use of serial qualitative interviews to understand patients' evolving experiences and needs qualitative interviews in medical research the qualitative research interview methods of data collection in qualitative research: interviews and focus groups determining sample size naturalistic inquiry. thousand oaks: sage; 1985 sample size in qualitative interview studies: guided by information power characterising and justifying sample size sufficiency in interview-based studies: systematic analysis of qualitative health research over a 15-year period using thematic analysis in psychology three approaches to qualitative content analysis factors explaining the intention to give blood among the general population modeling health behavior change: how to predict and modify the adoption and maintenance of health behaviors what we say and what we do: the relationship between real and hypothetical moral choices an end to lifetime blood donation ban in israel for msm would be a major step toward a science-based policy that reduces stigma blood donation deferral policies among men who have sex with men in brazil men having sex with men and blood donation: is there a game changer on the horizon? relaxes rules for gay blood donors amid coronavirus-will canada go further? global news pm trudeau urges canadians to donate blood during covid-19 pandemic i recovered from covid-19. but i can't donate my plasma because i'm gay publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. jp and mg conceived the study. all authors participated in the design of the study. jp and ev led the drafting of the manuscript with input from all authors. all authors read and approved the final manuscript. this study received funding from canadian blood services msm research grant program and canadian blood services msm plasma program, funded by the federal government (health canada) and the provincial and territorial ministries of health. the views herein do not necessarily reflect the views of canadian blood services or the federal, provincial, or territorial governments of canada. not applicable. key: cord-018809-3nrvm4jt authors: mcmullin, n. r.; holcomb, j. b.; sondeen, j. title: hemostatic resuscitation date: 2006 journal: yearbook of intensive care and emergency medicine doi: 10.1007/3-540-33396-7_25 sha: doc_id: 18809 cord_uid: 3nrvm4jt component therapy is useful for the majority of patients when blood requirements are minimal and there is no associated coagulopathy. of concern are requirements for massive transfusion and resuscitation that absorb resources and create a short-fall for patients whose injuries are less severe. additionally, the conventional massive transfusion model of packed rbcs, plasma and platelets actually further dilutes the patient compared to the blood he or she has lost and thus is not the ideal fluid for patients who require this massive transfusion of products. fresh whole blood has three vital properties: oxygen carrying capacity, volume, and hemostatic effect. in the austere environment of combat the practice of fresh whole blood transfusion has proven beneficial to patients who are coagulopathic and require massive transfusion. appropriate use following established guidelines can be beneficial and may even be superior to packed rbcs. a fluid containing the vital properties of fresh whole blood would serve as a bridge to allow a patient to be resuscitated without initiating the ‘bloody cycle of death’ that is seen all too often in our current paradigm of massive resuscitation. combat medicine is an ebb and flow of casualties. several days may pass when there are no casualties, followed by an onslaught of wounded. casualties from explosive devices, such as those seen in operation iraqi freedom and civilian terrorist attacks, tend to result in a large number of casualties at one time. this paradigm does not lend itself to component therapy. packed rbcs, plasma, and crystalloid solutions may be in abundance, but a five-day shelf life prohibits the banking of platelets. the casualties who receive massive transfusions (> 10 units packed rbcs) quickly deplete precious stores. ffp bags, not designed to support the temperature (±70 8f) associated with dry ice used during transport, become brittle and break one third of the time during the thawing process. platelets generated by plasmapheresis became available only two years after the start of the war in iraq, require significant and dedicated resources and availability remains limited. limited blood products, manpower shortages, and warming methods increase the vulnerability of wounded soldiers to the onset of a second enemy: hypothermia, acidosis and coagulopathy. in an attempt to resolve this problem combat surgeons often resort to the`walking blood bank'. predetermined donors are mobilized and fresh whole blood is collected, tested, and given to the wounded. fresh whole blood protocols were followed in post-war kosovo and in somalia where over 120 units of fresh whole blood were collected and administered to critically wounded patients [1, 2] . a combat support hospital deployed to baghdad transfused 598 units of fresh whole blood over a 6 month period [3] . reports of the rapid positive response of recipients to fresh whole blood are thought-provoking and suggest profound possibilities for fresh whole blood transfusion. in combat, fresh whole blood for massive transfusion becomes a blood bank multiplier, providing within a single unit, rbcs, volume, coagulation components, and functional platelets in a warm fluid. in 1999, pearce and lyons contrasted blood product usage from world war ii and korea to the practice in vietnam [4] . in world war ii and korea, resuscitation consisted of colloid in the form of concentrated plasma and fresh whole blood. however, the incidence of hepatitis transmission rose to unacceptable levels ± as high as 21% in some units in korea [5] . in vietnam, tested units of packed rbcs and crystalloid solutions (lactated ringer's) were used and the incidence of infectious disease decreased. however, complications of the switch from fresh whole blood to stored rbcs became evident in vietnam as acute respiratory distress syndrome (ards) or`da nang lung' was so prevalent that it became the focus of thousands of studies and book chapters. in comparison, very few cases of ards were described during world war ii and none during korea despite the administration of large volumes of whole blood and colloid [4] . these observations from the front lines of the advantages of fresh whole blood transfusion have been borne out in the civilian literature. in particular, two well designed, prospective studies support the clinical testimony of combat surgeons that fresh whole blood improves coagulopathy and decreases blood loss when compared to component therapy. the first study randomized cardiopulmonary bypass (cpb) patients to receive either one unit of fresh whole blood or 10 units of platelet concentrates after surgery [6] . the patients who received one unit of fresh whole blood increased platelet counts by 34 ô 17´10 9 /l, an increase equivalent to four to six units of platelets. platelet aggregation response to collagen and epinephrine after fresh whole blood transfusion was superior to that achieved by 10 units of platelets. furthermore, bleeding time after administration of one unit of fresh whole blood approximately equaled bleeding time following eight units of platelets. in the second study, the beneficial effects of fresh whole blood on the coagulation cascade were demonstrated in a double-blinded, randomized controlled study comparing the use of fresh whole blood less than 24 hours old (n = 52), fresh whole blood between 24 and 48 hours old (n = 57), and reconstituted blood using packed rbcs, plasma, and platelets (n = 52), in a pediatric population undergoing cpb operations [7] . the 24-hour blood loss was no different between the two whole blood groups. the group that received reconstituted products had an increase in average blood loss that was significantly greater than either of the fresh whole blood groups (p = 0.03). after age stratification, the 24-hour blood loss for children less than 2 years old who received reconstituted blood was 85% greater than those who received fresh whole blood (p = 0.001). when platelet aggregation times were compared among the three groups, the group that received the reconstituted blood had a greater incidence of abnormal studies in the presence of the agonists adp, epinephrine and collagen (p < 0.001, p = 0.02, and p = 0.007, respectively). other recent studies have linked increasingly negative outcomes to each unit of packed rbcs received. fresh whole blood transfusion has also been shown to improve hemodynamic and oxygen delivery parameters in animal studies. one such study comparing transfusion of packed rbcs with whole blood in a canine model of hemorrhagic shock demonstrated that animals resuscitated with packed rbcs had significantly less hemodynamic recovery, demonstrated by lower mean arterial pressure and cardiac output (p < 0.05) [8] . resuscitation with packed rbcs elevated total peripheral resistance when compared with resuscitation with fresh whole blood, suggesting vasoconstriction or obstruction of the peripheral vasculature by noncompliant rbcs [8] . this study corroborated findings of an earlier study that showed improved cardiac output and oxygen delivery in dogs resuscitated with whole blood compared to those resuscitated with crystalloid or colloid [9] . in an attempt to explain these observations of improved oxygen delivery, macroaggregated albumin clearance rates from the lungs of hemorrhaged rats were studied. rats that received fresh whole blood demonstrated improved clearance of the tracer compared to animals resuscitated with either crystalloid or colloid [10] . another rat model of hemorrhage and resuscitation demonstrated that fresh whole blood for resuscitation provided superior end organ perfusion by preventing ultrastructural kidney damage [11] . in a recent severe porcine hemorrhage study, resuscitation with fresh whole blood resulted in 90% 72-hour survival compared with 27% survival in animals receiving standard of care resuscitation fluids (lactated ringer's, hextend, 5% nacl, 7.5% nacl-6% dextran-70 [hsd]) [12] . this was a non-coagulopathic model of controlled hemorrhage (53% bled in 5 minutes), indicating the metabolic benefits of fresh whole blood as a resuscitation solution. fresh whole blood transfusion is not without risk. the emergency conditions of the battlefield permit little time to test the blood for infection prior to administration. aliquots from each unit of transfused fresh whole blood are sent back to the united states for testing. over 1700 units of fresh whole blood have been transfused in the iraqi operating room. in the aliquots of this blood, there were two positive confirmatory tests for hepatitis c (1 : 1000) (riba), and one each indeterminate test (western blot) of human immunodeficiency virus (hiv) and human tcell lymphocytic virus (htlv). compared to the extremely low incidence of transmission of these diseases in component therapy (1 : 493,000 hiv, 1 : 641,000 htlv, 1 : 103,000 hepatitis c, 1 : 63,000 hepatitis b) [13] , the risk of infectious disease transmission is still very low, but the surgeon must weigh the risk of infection against the risk of poor outcome if fresh whole blood is not received. rapid enzyme-linked immunosorbent assay (elisa) for disease can be accomplished in two hours. elisa was successfully utilized at one of the large combat support hospitals and has since seen more widespread use [3] . without elisa fresh whole blood must be administered sparingly and only under extreme circumfig. 1 . this flow diagram is the massive transfusion/fresh whole blood transfusion protocol employed by the 31 st combat support hospital, while serving in baghdad, iraq. this is a two-pronged approach where patients are identified early and the mechanism for obtaining fresh whole blood initiated. patients receive emergency release packed red blood cells (prbcs) and when available are transitioned to fresh whole blood. this transition time coincides with the point where platelet transfusion would likely be indicated. ffp: fresh frozen plasma; cryoppt: cryoprecipitate. from [3] with permission stances and protocols have been established that delineate the clinical guidelines for determining who should receive fresh whole blood. figure 1 illustrates one such protocol employed at a combat support hospital [3] . fresh whole blood was requested when large quantities of blood were required in order to save life and when the onset of coagulopathy and the need for platelets was imminent. this protocol illustrates a two-pronged approach in the event a massive transfusion is required. first, uncrossmatched, type o packed cells are made available as emergency-release blood and the`massive transfusion' protocol is initiated. at the same time, since there are usually no platelets and nominal ffp and cryoprecipitate available on the battlefield, the`walking blood bank' of predetermined donors is activated so that required coagulation factors and components can be provided in the form of fresh whole blood. the first unit of fresh whole blood usually becomes available in about 120 minutes and the surgical team transitions to fresh whole blood as an adjunct to the massive transfusion protocol [3] . many civilian hospitals utilize this template of a`massive transfusion protocol' where, upon activation, predetermined quantities of packed rbcs, ffp, platelets and cryoprecipitate are sent at scheduled time intervals. the main difference is the effort to obtain fresh whole blood and the transition to whole blood transfusion. following a landmark study published in the new england journal of medicine, the recommendations for red blood cell transfusion when serum hemoglobin levels are less than 10 mg/dl was changed to hemoglobin levels of 7 mg/dl in patients with signs of systemic compromise [14] . this randomized controlled trial compared a practice of liberal transfusion (keeping the hematocrit > 30%) with a practice of restrictive transfusion (keeping the hematocrit between 21±27% and only transfusing if the hematocrit fell below 21%). there was no difference in overall mortality. however, less ill patients (apache < 20) and younger patients (age < 55) showed a statistically significantly improved survival with the use of a restrictive transfusion. of clinical significance, the liberal practice group showed more cardiac complications and the restrictive practice population had lower hospital mortality and a lower adjusted multi-organ dysfunction score. a growing body of literature underscores the potential risks of stored rbc transfusion. a retrospective study assessed over 15,000 patients for the independent predictors of mortality, intensive care unit (icu) admission, icu length of stay, and hospital length of stay [15] . after adjustment for injury severity, blood transfusion was shown to be an independent predictor of mortality, icu admission, and hospital length of stay. patients who underwent blood transfusion were nearly three times more likely to die and greater than three times more likely to be admitted to the icu. coagulopathy can be caused by the dilution of coagulation factors by packed rbcs which do not contain any factors. patients who undergo a massive transfusion (more than 10 units of packed rbcs or receipt of twice the normal blood volume) have prolonged prothrombin time, thrombocytopenia and decreased levels of fibrinogen. seventy percent of those who receive more than 20 units of red blood cells become coagulopathic, with thrombocytopenia being the most common abnormality [16] . a single unit of packed rbcs is approximately 335 ml with a hema-tocrit of 55%. a single unit of platelets is 50 ml and contains 5.5´10 10 /l platelets. a single unit of plasma is about 275 ml with only 80% coagulation activity of fresh whole blood. when all of these components are combined, the result is a fluid with a hematocrit of 29%, 8.8´10 7 /l platelets and coagulation activity that is 65% of normal [17] . even when rbcs are administered at a high one-to-one ratio with ffp, dilution of coagulation factors and platelets and iatrogenic anemia is unavoidable. it is well known that the trauma related to surgery results in the activation of the immune system. levels of inflammatory cytokines are elevated to a greater extent in patients with severe blood loss when compared to patients with isolated trauma. additionally, the degree of elevation of pro-inflammatory cytokines correlates with severity of injury [18, 19] . when blood transfusion, which is inherently pro-inflammatory, is combined with the inflammatory response associated with trauma, the result is an exacerbated acute phase response [20] . this exacerbated inflammatory response is associated with increased infectious complications and increased mortality [19] . a biphasic inflammatory response begins, with initial proinflammation followed by immunosupression which begins at about 24 hours [18] . it is during the subsequent phase of immunosuppression that the patient is most susceptible to infection. historically, this immunosuppressive effect was actually used to the advantage of the patient. prior to the use of cyclosporine, blood transfusion was used in transplant patients as an immunosuppressant, improving outcomes [21] . several studies further illustrate the disruption of the immune system by rbc transfusion. a prospective trial demonstrated a significantly increased rate of nosocomial infection of 15.4% in 412 patients who received transfusions compared to a 2.9% rate in 1,301 control patients who did not receive transfusions [22] . there was a dose-related response between the number of units of packed rbcs received and the risk of developing a nosocomial infection. additionally, patients who received transfusions showed increased mortality, length of icu stay, and hospital stay (p < 0.05). this single-center prospective trial corroborated the observations of several basic science studies. detrimental immunomodulation is thought to occur following increased production of both serum and tissue tumor necrosis factor (tnf)-a, interleukin (il)-1b, il-6, il-8, interacting with soluble cytokine receptors [23] . fransen et al. showed that surgical patients who received packed rbcs compared to those who did not had increased levels of il-6 (p < 0.01) and bactericidal permeability increasing protein (bpi) (p < 0.05), which is a marker of neutrophil activation [20] . in addition to these serum markers of inflammation, patients who received transfusions had increased ventilator time (42 ô 12 vs. 22 ô 2 h, p < 0.025) and increased icu days (45 ô 6 vs. 89 ô 21, p < 0.025) [20] . transfusion of blood components has also been associated with impaired natural killer cell function and decreased helper-suppressor cell ratios as well as effects on b lymphocytes [24] . again, there appeared to be a linear relationship between the amount of blood transfused and the degree of impairment [24] . approximately twenty-four hours is required for transfused packed rbcs to recover their full ability to deliver oxygen, but the precise mechanism of this immunomodulation is a mystery. one theory is that immune system derangement is the result of the storage age of blood. gastric mucosal ph can be used as an indicator of oxygenation of the gastric mucosa or the organs of splanchnic distribution and studies have shown that direct measurements of gastric mucosal ph are related to the age of the blood transfused to critically ill patients [25, 26] . two mechanisms that have been proposed to account for the inability of old transfused rbcs to improve systemic oxygen consumption include a left shift in the oxyhemoglobin dissociation curve due to 2,3-diphosphoglycerate (dpg) depletion with storage, and loss of the rbc compliance and the ability to deform, impeding delivery of oxygen to the microcirculation [27] . another emerging theory speculates that due to the high affinity of nitric oxide (no) with free hemoglobin, cell-free ferrous hemoglobin in the plasma is oxidized to methemoglobin and nitrates that rapidly destroy no [28, 29] . the decreased availability of no results in regional and systemic vasoconstriction. the exact mechanisms are uncertain, but it is becoming clearer that the administration of stored rbcs is a clinical decision that should not be taken lightly. the negative repercussions of packed rbc transfusion may become evident in trauma patients, who present in a state of acute inflammation derived from the trauma sustained. the literature supports the theory that the transfusion of packed rbcs can actually result in a deranged inflammatory response and that this may be related to the storage age of the blood. the transfusion of packed rbcs remains the standard of care for restoring rbc concentration and circulating oxygen content during ongoing hemorrhage, but it is not a solution that is devoid of risk. in severe hemorrhage, oxygen delivery to tissues must be restored. hence, the rationale for hemoglobin-based oxygen carriers is the ability to deliver oxygen to vital organs via plasma components. but the development of hemoglobin-based oxygen carriers to replace packed rbcs has led a rocky course over the past twenty-five years. several variants of hemoglobin-based oxygen carriers attempted in the past were either abandoned by the developing company or discontinued in phase iii trials out of concern for patient safety [30] . concerns about hemoglobinbased oxygen carriers are their effects on vasoactivity resulting from binding of no by hemoglobin and subsequent hypertension. currently, however, in the united states a phase iii trial of pyridoxalated hemoglobin (polyheme, northfield laboratories, evanston, il.) is 50% completed. this multicenter pre-hospital trial is examining the administration of hemoglobin based oxygen carriers to trauma patients in severe hemorrhagic shock. hopefully, upon completion of this study, hemoglobin based oxygen carriers will be added to the armamentarium of the caregiver. an effective and safe hemoglobin-based oxygen carrier appeals to both the civilian and military communities. polyheme can be stored for 72 hours at room temperature, virtually eliminates any risk of disease transmission, does not require type and cross-matching and provides a fluid which can enhance oxygen delivery [31] . it does not, however, have any restorative effect on coagulation. for military use, polyheme will require availability at room temperature extended beyond 72 hours. whether it will be carried in the rucksack of a medic for a limited time or in a civilian ambulance, this potentially lifesaving product deserves further exploration. z fresh frozen plasma plasma contains various components including stable coagulation factors, fibrinogen and albumin. lyophilized plasma was the principle resuscitation fluid during both world war ii and the korean war due to its long shelf life without refrigeration. at the end of world war ii and the korean war, the discovery of infectious diseases as the result of plasma obtained from multiple donors, i.e., pooled plasma, halted this practice. consequently, the indications for plasma transfusion today no longer include that of a volume expander. ffp is indicated for the correction of coagulopathy associated with factor deficiency (ii, vii, ix, x), hemorrhage, reversal of warfarin effect, and antithrombin deficiency. during massive transfusion of packed rbcs in a patient with exsanguinating hemorrhage, the onset of coagulopathy is usually described as the result of dilution and consumption of coagulation factors, acidosis and hypothermia. the incidence of coagulopathy increases with injury severity. twenty-one percent of patients with an injury severity score (iss) of 15±29 are coagulopathic, 41% with an iss of 30± 44, 59% with iss of 45±59, and 79% of those with an iss of 60±75 [32] . however, importantly, the most seriously injured patients are coagulopathic upon admission, unrelated to dilution. in a large study of 1,088 trauma patients, 24.4% were coagulopathic on admission [32] . as a general rule, hemostasis is possible as long as coagulation factor activity is kept at 20 to 30% of normal and fibrinogen levels are at least 100 mg/dl. ffp contains plasma clotting factors and fibrinogen; in addition, administration will increase intravascular volume. because the incidence of coagulopathy increases with severity of injury, many institutions include the transfusion of single donor ffp in their massive transfusion protocols. often, the first application of blood products contains six units of type o rbcs and four units of ab negative ffp. subsequent applications include six units of type specific rbcs and ffp, and a six-pack of platelets with a unit of cryoprecipitate in every other application. cryoprecipitate provides fibrinogen and some clotting factors which become depleted in a massive transfusion. volume expansion inherently occurs with the administration of these products and is also augmented with either crystalloid or colloid solutions. the purpose of this protocol is to restore circulating rbcs, volume, clotting factors, and clotting substrate and to sustain the patient while the surgeon stops the source of bleeding. the onset of coagulopathy in the hemorrhaging patient can be rapid, and the time required to obtain plasma can further propel the patient along the`bloody vicious cycle' of coagulopathy, acidosis, and hypothermia. therefore, to prevent a dangerous situation the caregiver must be ready to transfuse blood products based on immediate clinical evaluation rather than delayed laboratory assessment. the current laboratory guidelines for the transfusion of ffp are prothrombin time (pt) more than 1.5 times greater than normal, activated partial thromboplastin time (aptt) ratio more than 1.5 times greater than normal, fibrinogen less than 0.8 g/l, and coagulation factor levels 30% of normal [17] . some authors advocate early ffp transfusion at a 1:1 ratio with packed rbcs after hemorrhage of one blood volume, or pt and aptt greater than 1.5 times normal with ongoing hemorrhage [33] . even with this aggressive practice of plasma transfusion, hemodilution is unavoidable. as stated earlier, when standard blood products are administered in a 1 : 1 ratio the resulting fluid has a hematocrit of 29%, 8.8´10 7 /l platelets, and coagulation activity that is 65% of normal [17] . the primary risks associated with fresh frozen plasma transfusion are infection, transfusion-related acute lung injury (trali), acute aller-gic and anaphylactic reactions, hemolysis due to anti-a and anti-b, and fluid overload [34] . of these, the most common serious complication of ffp transfusion is trali, thought to occur in a graft versus host mechanism as donor antibodies react with host leukocytes [35] . the rate of infectious disease transmission resulting from ffp administration is similar to that of packed rbcs. it is important that clinicians take into consideration the etiology of thrombocytopenia, platelet dysfunction, risk of bleeding, planned invasive procedures, and the presence of concomitant disorders when deciding whether or not to transfuse platelets. patients with severe sepsis, regardless of the presence of hemorrhage, usually receive a transfusion of platelets if their platelet count falls below 5000/mm 3 . platelet transfusion should be considered if the platelet count is between 5000±30,000/ mm 3 , and there are signs of bleeding. if the patient requires surgery or other invasive procedures it is recommended that the platelet count be maintained above 50,000/mm 3 . the conundrum presented by these classic recommendations is the highly variable relationship between platelet counts and platelet efficacy. platelets pose significant logistical problems. since the development of platelet aphaeresis technology, it has been well known that platelets stored in the cold have poor recovery and survival in vivo. they cannot be refrigerated even for short periods of time, thus platelets are stored at 22 o c. studies of chilled platelets have shown that this irreversibly alters their morphology as well as the expression of the gpiba receptor on the platelet, causing rapid clearance of the transfused platelet from the circulation [36] . the changes which occur in the structure and function of stored platelets are known as the`platelet storage lesion' and are poorly understood. there are few data regarding the function of platelets in the bleeding patient. after room temperature storage of up to 5 days, the risk of bacterial contamination becomes significant. the rate of septic reaction to platelet transfusion in the united states is between 1 : 10,000 and 1 : 20,000 [37] . the short shelf-life of platelets combined with an uncertain demand for platelets results in wastage rates as high as 50%. significant efforts are being made in the development of novel platelet products and platelet substitutes. lyophilized platelets are one such product, initially investigated nearly fifty years ago and recently resurfaced [38] . lyophilized platelets have shown encouraging preclinical results in animal models [39] . a second product, synthocyte tm (profibrix, inc., the netherlands), is a fibrinogen-coated albumin microsphere that has shown a reduction in surgical bleeding in a thrombocytopenic animal model [40] . the mechanism of action of synthocytes tm is thought to be due to the cross-linking of fibrinogen which has been directly imbedded into the surface of the microcapsules [41] . other areas of platelet product development include liposome-based hemostatic agents, thromboerythrocytes, and platelet-derived microparticles. z fibrinogen one of the end products of the coagulation system is the production of thrombin which stimulates the sequential proteolytic cleavage of fibrinogen to release fibrinopeptides a and b. the fibrin monomers that result from this process spontaneously polymerize to form an insoluble matrix. this matrix is stabilized by factor xiiia, which is another product of thrombin generation. fibrinogen, or factor i, is essential for hemostasis and it is recommended that fibrinogen be administered in the form of cryoprecipitate once fibrinogen levels fall below 100 mg/dl. this number applies to patients with non-surgical bleeding and not necessarily to those with active blood loss. cryoprecipitate derived from a unit of whole blood contains 10±20 ml of fluid per unit, providing up to 150±250 mg of fibrinogen, 80±100 units of factor viii, and 50±60 mg of fibronectin and in a non-bleeding 70 kg patient one pool of cryoprecipitate will increase the fibrinogen levels 45 mg/dl. cryoprecipitate does not contain all of the necessary coagulation factors and should not be used in place of ffp. fibrinogen substitutes are being actively investigated for fibrinogen supplementation. recombinant fibrinogen, rhfib, (pharming group, denmark) is currently under investigation by the united states army as an adjunct for hemostasis. another group has shown that fibrinogen derived from salmon activates human platelets and may provide a hemostatic function [42] . a third product, haemocomplettan (aventis behring gmbh, marburg, germany), is a lyophilized human fibrinogen concentrate that has been shown to correct thromboelastograph abnormalities and improve mortality as seen in rat models of sepsis-induced disseminated intravascular coagulation (dic) [43] . fibrinogen substitutes would allow more rapid correction of fibrinogen dilution than cryoprecipitate. over the past several years recombinant factor viia (rfviia) (novoseven, novonordisk, malov, denmark) has emerged as a promising therapy for treatment of coagulopathy associated with trauma and massive transfusion. this emergence began as observational experiences [44] , translated into pre-clinical studies in animal models [45, 46] , and progressed to large, multicenter, randomized and controlled studies which clearly show the safety and efficacy of rfviia in severely injured trauma patients. phase ii trials will soon advance to larger phase iii studies and rfviia holds promise as an adjunct to hemostasis in patients who receive massive transfusions for both blunt and penetrating trauma. used as a surgical adjunct rfviia reduces blood loss. in a randomized, doubleblinded prospective study, surgical patients who underwent a retropubic prostatectomy were given 20 lg/kg rfviia, 40 lg/kg rfviia or placebo prior to surgery. the higher dose group showed significant decreases in blood loss (p < 0.01) and surgical time (p < 0.05) and elimination of packed rbc transfusion requirements [47] . on the other hand, studies regarding the proper administration of rfviia have elucidated that rfviia is ineffective as a`last resort' [48] . we recommend that the administration of rfviia follow a clinical protocol according to set guidelines for dose and other parameters in order to optimize success. one early guideline published by dutton et al. outlines the use of clinical ªgatekeepersº who are available to assess the patient and clinical circumstances surrounding the administration of rfviia [49] . criteria include evidence of ongoing active hemorrhage, with clinical evidence of coagulopathy, and ongoing utilization of conventional transfusion and hemostatic therapy which must be judged unlikely to succeed. patients must have received at minimum 10 units of rbcs, eight units of ffp, one unit of platelets, with continued derangement of coagulation studies and ongoing hemorrhage. this approach reserves rfviia for the most critically injured. with its safety demonstrated in a large randomized clinical trial, most clinicians are utilizing rfviia earlier in an attempt to prevent massive bleeding and coagulopathy rather than treating them after the fact. in a protocol in may 2004 from the trauma consultant to the surgeon general of the united states, this change from reacting to coagulopathy to actively preventing coagulopathy with rfviia was made manifest. it is recommended that combat surgeons ªconsider rfviia administration for use in patients that require damage control procedures, have coagulopathic bleeding, or difficult to control bleeding associated with hypothermia or significant operative bleeding.º it is further recommended that the surgeon consider administering two units of fresh whole blood prior to rfviia to ensure the presence of functional platelets and coagulation factors. a significant drawback to the administration of rfviia is cost. on average, an 80 kg man receiving a 120 lg/kg dose, costs about $ 8,000. though this cost appears exorbitant, most studies demonstrate a reduction in blood product requirements, which in turn should translate to lower costs and prevention of subsequent infection and deleterious inflammatory response. efforts are also being taken to decrease cost by making rfviia more efficient. in a rat model of hemophilia, threè super analogs' of rfviia were compared with rfviia [50] . the rfviia groups received 1, 3, 6 or 10 mg/kg, and the super-analog groups 1 or 3 mg/kg. two of the 3 mg/kg groups of the super-analogs and the 10 mg/kg group of rfviia showed significant improvement in bleeding times (p < 0.001) when compared to the hemophilia control group. average blood loss was reduced in the 3, 6 and 10 mg/kg groups of rfviia (p < 0.05) and in all groups of the super-analog, including the 1 mg/kg groups (p < 0.05). a dose response was seen between the 1 and 3 mg/kg groups of all three super-analogs, with decreased blood volumes in the higher dose super analog groups (p < 0.001) [50] . with further research and the development of rfviia analogs with greater potency, we hope to see the cost associated with this drug decrease while effectiveness increases. z where are we heading? the potential hematologic and physiologic benefits of fresh whole blood as a hemostatic and resuscitative fluid are clear. this straightforward approach is akin to the damage control procedures so widely accepted in trauma surgery, although it is temporized by inherent logistical problems and the small but real threat of infectious diseases. what is needed is a fluid which contains the oxygen-carrying capability as well as the volume and hemostatic qualities provided by fresh whole blood. for hemostatic resuscitation on the battlefield we also envision a fluid that is stable at room temperature and has a long shelf life. additionally, a fluid that can transport oxygen, possibly by a hemoglobin-based oxygen carrier could serve as a volume expander and contain active clotting factors and platelets, like those found in freeze-dried plasma, platelets, fibrinogen and recombinant factor viia. being fielded forward to the point of injury, this fluid could prevent the onset of coagulopathy and decrease blood product transfusion requirements, all in hope of improving survival. fascinating and promising products on the horizon may make hemostatic resuscitation a possibility. component therapy is useful for the majority of patients when blood requirements are minimal and there is no associated coagulopathy. of concern are requirements for massive transfusion and resuscitation that absorb resources and create a shortfall for patients whose injuries are less severe. additionally, the conventional massive transfusion model of packed rbcs, plasma and platelets actually further dilutes the patient compared to the blood he or she has lost and thus is not the ideal fluid for patients who require this massive transfusion of products. fresh whole blood has three vital properties: oxygen carrying capacity, volume, and hemostatic effect. in the austere environment of combat the practice of fresh whole blood transfusion has proven beneficial to patients who are coagulopathic and require massive transfusion. appropriate use following established guidelines can be beneficial and may even be superior to packed rbcs. a fluid containing the vital properties of fresh whole blood would serve as a bridge to allow a patient to be resuscitated without initiating the`bloody cycle of death' that is seen all too often in our current paradigm of massive resuscitation. whole blood transfusion for exsanguinating coagulopathy in a us field surgical hospital in postwar kosovo united states army rangers in somalia: an analysis of combat casualties on an urban battlefield the use of fresh whole blood in massive transfusion logistics of parenteral fluids in battlefield resuscitation blood program in world war ii. office of the surgeon general, washington dc the hemostatic effect of transfusing fresh whole blood versus platelet concentrates after cardiac operations comparison of the hemostatic effects of fresh whole blood, stored whole blood, and components after open heart surgery in children a comparison of resuscitation with packed red blood cells and whole blood following hemorrhagic shock in canines regional and systemic oxygen delivery/uptake relations and lactate flux in hyperdynamic, endotoxin-treated dogs lung perfusion in hemorrhagic shock of rats: the effects of resuscitation with whole blood, saline or hes 6% effects of whole blood, crystalloid, and colloid resuscitation of hemorrhagic shock on renal damage in rats: an ultrastructural study fresh whole blood is the best hypotensive resuscitative fluid in a severe hemorrhage pig model the risk of transfusion-transmitted viral infections. the retrovirus epidemiology donor study a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. transfusion requirements in critical care investigators, canadian critical care trials group blood transfusion, independent of shock severity, is associated with worse outcome in trauma blood component supplementation during massive transfusion of as-1 red cells in trauma patients treating coagulopathy in trauma patients clinical review: immunodepression in the surgical patient and increased susceptibility to infection cytokine patterns in patients after major vascular surgery, hemorrhagic shock, and severe blunt trauma. relation with subsequent adult respiratory distress syndrome and multiple organ failure impact of blood transfusions on inflammatory mediator release in patients undergoing cardiac surgery planned random donor blood transfusion in preparation for transplantation. sensitization and graft survival impact of allogenic packed red blood cell transfusion on nosocomial infection rates in the critically ill patient effects on complement activation and cytokine (tnf-alpha and il-8) release of infusion of anti-tnf-antibodies or a xanthine derivative (hwa 138) in septic baboons vascular access and fluid resuscitation in trauma effect of stored-blood transfusion on oxygen delivery in patients with sepsis gastric tonometry in patients with sepsis. effects of dobutamine infusions and packed red blood cell transfusions tissue oxygen delivery and the microcirculation cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease hemoglobin and the paracrine and endocrine functions of nitric oxide the clinical trials of diaspirin cross-linked hemoglobin (dclhb) in severe traumatic hemorrhagic shock: the tale of two continents insights from studies of blood substitutes in trauma acute traumatic coagulopathy are we giving enough coagulation factors during major trauma resuscitation? risks of fresh frozen plasma (ffp) and platelets diagnostic and pathogenetic considerations in transfusionrelated acute lung injury the clearance mechanism of chilled blood platelets bacterial contamination of blood components novel platelet products, substitutes and alternatives preservation of hemostatic and structural properties of rehydrated lyophilised platelets: potential for long-term storage of dried platelets for transfusion fibrinogen-coated albumin microcapsules reduce bleeding in severely thrombocytopenic rabbits interactions of platelets with synthocytes, a novel platelet substitute purification and characterization of atlantic salmon (salmo salar) fibrinogen the effect of fibrinogen concentrate administration on coagulation abnormalities in a rat sepsis model treatment of traumatic bleeding with recombinant factor viia the effect of recombinant factor viia on coagulopathic pigs with grade v liver injuries recombinant factor viia increases the pressure at which rebleeding occurs in porcine uncontrolled aortic hemorrhage model effect of recombinant activated factor vii on perioperative blood loss in patients undergoing retropubic prostatectomy: a doubleblind placebo-controlled randomised trial last-ditch' use of recombinant factor viia in patients with massive haemorrhage is ineffective factor viia for correction of traumatic coagulopathy improved hemostasis with superactive analogs of factor viia in a mouse model of hemophilia a key: cord-280480-djv7pc3m authors: jägers, johannes; wrobeln, anna; ferenz, katja b. title: perfluorocarbon-based oxygen carriers: from physics to physiology date: 2020-11-03 journal: pflugers arch doi: 10.1007/s00424-020-02482-2 sha: doc_id: 280480 cord_uid: djv7pc3m developing biocompatible, synthetic oxygen carriers is a consistently challenging task that researchers have been pursuing for decades. perfluorocarbons (pfc) are fascinating compounds with a huge capacity to dissolve gases, where the respiratory gases are of special interest for current investigations. although largely chemically and biologically inert, pure pfcs are not suitable for injection into the vascular system. extensive research created stable pfc nano-emulsions that avoid (i) fast clearance from the blood and (ii) long organ retention time, which leads to undesired transient side effects. pfc-based oxygen carriers (pfocs) show a variety of application fields, which are worthwhile to investigate. to understand the difficulties that challenge researchers in creating formulations for clinical applications, this review provides the physical background of pfcs’ properties and then illuminates the reasons for instabilities of pfc emulsions. by linking the unique properties of pfcs and pfocs to physiology, it elaborates on the response, processing and dysregulation, which the body experiences through intravascular pfocs. thereby the reader will receive a scientific and easily comprehensible overview why pfocs are precious tools for so many diverse application areas from cancer therapeutics to blood substitutes up to organ preservation and diving disease. humankind has pursued the idea of rejuvenation and blood substitution since ancient times. in 8 ad, ovid versified his thoughts on the magician medea who-with a complex mixture of plants, stones and sand she had collected for 9 nights and 9 days-successfully regenerated the blood of the doter aeson who rejuvenated into a 40-year-old man [45] . the first effective inter-human blood transfusion was performed on september 1st 1818, by james blundell. but as human blood has always been a very precious and finite good, amberson and rhode were among the first researchers mixing isolated red blood cells (rbcs) from cattle, cats, dogs or humans with ringer's solution to simulate human blood in the 1930s [1] . they focused on the main ability of blood: oxygen (o 2 ) transport via rbcs. it took until the 1960s for the first artificial oxygen carriers made of engineered haemoglobin or other synthetic compounds to replace rbcs in blood substitution. this promised the end of the subjection to donated human blood for transfusion [83] . however, the interest in synthetic blood substitutes only increased gradually until the hi-viruscontaminated-donor-blood-crisis arose in the 1980s and vigorously accelerated the developmental research on artificial blood substitutes [8] . the use of perfluorocarbons bears attractive advantages over blood: unlimited availability, no transmittance of diseases and no dependence on blood types [23] . this review intends to introduce the reader to these astonishing materials, which offer so many more chances of application beyond ordinary blood substitution. the work of leland c. clark and frank gollan on fluorinated hydrocarbons (perfluorocarbons, pfc) laid the foundation to the idea to utilize the o 2 carrying and conducting features of these liquids for clinical application. in their famous experiment in 1966, they made mice dive in fluorobutyltetrahydrofuran (fx80) , which was equilibrated with 100% o 2 [9] . within this experiment, they showed that anaesthetized mammals could breathe this fluid and maintain their respiration for 4 h. in the same publication, they describe their second setup, in which they placed mice under a reverse funnel. the wide side of this funnel was placed under the surface of liquid pfc, which was equilibrated with 100% o 2 so that the only o 2 these mice were breathing emerged from the pfc. the mice showed no sign of hypoxia for hours, whereas mice in the same setup with water instead of the pfc died after a few minutes [9] . the latter experiment showed that pfcs are optimal gas transporters as they are not only able to store o 2 but also to release enough of it to maintain mammalian life. these key properties gave rise to the idea of using these kinds of chemicals as artificial oxygen carriers in blood substitutes resulting in the first clinical use of a pfcbased artificial oxygen carrier named fluosol-da in 1980 [38] . pfcs are hydrocarbons whose hydrogen atoms are substituted either completely by fluorine atoms and sometimes additional other halogens (fig. 1a) . this substitution changes the physical properties of these organic compounds radically. fluorine is the halogen of the second period; its atom radius is only twice the one of hydrogen, while its weight is 20 times the one of hydrogen. among all the elements, fluorine is the most eager to suck electrons from other atoms, which tightens its bonds along the whole molecular structure and stiffens the carbon backbone of these fluorinated compounds (fig. 1b ) [88] . the carbon-fluorine bond is very strong (~484 kj/mol) and extremely polar (almost ionic). however, this does not result in water solubility because the intrinsic symmetry annuls the polarity of each c-f bond with the whole pfc molecule becoming nonpolar (fig. 1a) . the tight bonds cause a more bulky shape of the pfc molecule so it needs more space in the water, which increases the hydrating energy. this effect decreases the solubility in water, compared to the corresponding hydrocarbon derivative [14] . on the other hand, this extreme polarity inhibits the formation of induced dipoles, which would lead to van der waals forces, necessary for solubility in lipids. therefore, pfcs are one of those rare species that are both hydrophobic and lipophobic. all pfcs are able to dissolve tremendous amounts of gases. the two most commonly used pfcs are perfluorooctylbromide (pfob) and perfluorodecalin (pfd, table 1 ). these two pfcs can dissolve 527 and 403 ml o2 /l pfc at 1 atm (1 bar, 713 mmhg), respectively. carbon dioxide can be dissolved up to 4 times the amount of o 2 [102] . in comparison, the solubility of o 2 in water is about 9 to 10 ml o2 /l water and in blood around 200 ml o2 /l blood [86, 94] . at this point please be reminded that 1 l of water contains 55 mol, whereas 1 l of pfd contains only 4.2 mol. therefore, the molecular ratio of dissolved o 2 is 1 o2 :200 water in water, but 5 o2 :1 pfd in pfd, resulting in a 1000× increased molecular solubility for pfd compared to water (fig. 1c ). major properties for these calculations are listed in table 1 . the low molecule density of pfcs, mentioned above, explains the high o 2 capacity and conductivity, which clark and gollan tested in their experiment from 1966. let us carry out a thought experiment and consider solvent molecules to be pebbles on a road that you walk on barefoot. you can pass the pebbles, but you need to take your time and energy to carefully step between them. the o 2 passes the "pfd pebbles" substantially faster than the "water pebbles", because the pfd molecules leave substantially more space so that the o 2 can move freely between the solvent molecules and pass them (fig. 1c ). the o 2 capacity obeys henry's law, which means it is theoretically infinite assuming an infinite po 2 . less than a year later than their mouse-diving experiment, clark and gollan kept a rat langendorff-heart beating by perfusing it with pure pfc [32] . in 1967, sloviter annotated in his study: "water and polar substances such as glucose and salts are virtually insoluble (in pfcs)" [87] . he was the first to emulsify (emulsifier: bovine serum albumin (bsa)) pfc in water and successfully perfuse isolated rat brains [87] . this emulsification paved the way to using pfcs as oxygen carriers in physiological systems by developing a way to allow the addition of, e.g., water-soluble nutrients and pharmaceuticals. emulsification is the process of mixing two or more immiscible liquids via building microbubbles. it generates thermodynamically metastable but kinetically stable mixtures. for deeper knowledge about the physics of emulsification, numerous reviews have been published [29, 93] . briefly, the extreme hydrophobicity of pfcs makes the use of emulsifiers such as lipids, fluorinated compounds, tensides or proteins necessary to bypass the immiscibility with aqueous media, e.g. blood. forming a microbubble is a thermodynamic process that is described by a variant of the gibbs-helmholtz-equation: [28] . the cost of energy (enthalpy) of a reaction must be negative to make the reaction proceeds spontaneously. as you can see in the equation, the interfacial area should be small to minimize the cost of energy. the lack of possible hydrogen bonds at the interface of the immiscible liquids causes interfacial tension, which increases the inner energy of the whole system. however, a few millilitres of an emulsion can contain an interfacial area the size of a football field or even larger. emulsifiers adhere to the interface and represent a hydrogen bond acceptor that lowers the interfacial tension. this minimizes the unfavourable rise of energy caused by the increase of the interfacial area. considering the second law of thermodynamics, systems tend to increase the intrinsic disorder. solving an emulsifier in a solvent, e.g. water, decreases the disorder by forcing the water to form hydration spheres around the emulsifier. adsorption at interfacial areas, therefore, increases the disorder of the system [27, 93] . as mentioned above, emulsions are metastable because the emulsion reaches the thermodynamic equilibrium only after a complete phase separation, and two major processes push this decay forward. the first one is coalescence. if you have ever observed raindrops running down a window, you might have seen them approaching each other, and as soon as the droplets' surfaces touch, fusing into one big droplet. this event is coalescence and the driving force is the minimization of the interfacial area. the only effective way to avoid the fusion is to avoid the contact between the droplets. one way to accomplish this is to reduce brownian movement of the droplet via cooling so that the droplets may not move too close to each other. a disadvantage of freezing an emulsion is that it may lead to decay [16, 30] . another way to inhibit coalescence is to use charged emulsifiers like lipids or proteins to generate a high surface charge density, which leads to the repulsion of droplets [48, 77] . to envision this repulsion think of washing your hands with soap. the tensides in the soap give a dense negativity to your skin, which causes the two hands to repulse, leading to reduced friction, which feels slippery [40] . furthermore, the emulsifier's layer thickness, the emulsifier's density at the interface and the droplet size contribute to the steric stabilization of an emulsion [46] . the repulsion between two droplets occurs at distances below twice the layer thickness. the thicker the layer, the earlier the repulsion occurs, which limits the possibility for the attractive forces (van der waals forces) between two droplets to prevail at short distances. additionally, the repulsive forces augment with increasing layer-thickness to droplet-size ratio [93] . therefore, nano-emulsions, which show droplet diameters below 1 μm, are more stable than micro-emulsions, which exhibit droplet diameters larger than 1 μm. when the first food and drug administration (fda)-approved pfc emulsions such as fluosol-da and perftoran came up in the 1980s, they suffered from flocculation, which gave the products weak shelf life [81] . the used emulsifiers were block polymers sometimes mixed with lecithin or phospholipids, which generate too little surface charge density to prevent this process [24] . freezing the emulsion prior to storage was insufficient because of decay during thawing, which made it necessary to sonicate the emulsion prior to use. even though these first-generation pfc-based oxygen carriers (pfocs) showed major physiological side effects, the main reason for stopping the clinical trials in the 1990s was the insufficient shelf life [36, 51, 101] . short time later, new pfc emulsions such as oxycyte and oxygent were developed using egg yolk lecithin as emulsifier and further block polymers (proxanol 268), which allow for the formation of smaller droplets (50-100 nm) with a higher charge density [50, 62] . those emulsions showed a higher stability, were heat sterilizable and could be stored frozen without decaying when thawing [23, 101] . decay of these second-generation emulsions is the result of a second process: ostwald ripening. small droplets vanish in favour of the growth of bigger more stable ones. again, the reduction of the interfacial area is the driving force, but in this case, diminishment by kinetic stabilization is impossible. the pfc inside the droplet evaporates or dissolves in the surrounding solvent until a bigger droplet captures it. crucial factors herein are the solubility of the pfc in water and its vapour pressure, which forces the pfc to evaporate. minimizing these factors by choosing the right pfc can minimize ostwald ripening [43] . recently developed pfc emulsions using pfd, which displays extremely low solubility in water, can reduce ostwald ripening. in addition, the use of charged proteins like albumin as an emulsifier that forms a thick layer with a high surface charge density such as in a-aocs helps to prevent flocculation and coalescence [17, 34] . entering the vascular system, pfc droplets face different obstacles, which they have to overcome to function as appropriate o 2 shuttles. as mentioned above, size is an important factor influencing the stability of emulsions regarding shelf life but also the intravascular half-life [75] . the droplet size should be smaller than 0.5 μm mainly because of two reasons. on the one hand, the beneficial effect of the enhanced oxygen transport capacity decreases with increasing size of the droplet, because of reduced diffusivity, so that droplets with a size bigger than 0.5 μm need a treatment with 100% oxygen to work at their full potential [26] . on the other hand, droplets with a diameter over 0.5 μm may cause microembolism through obstruction of the microvascular system [104] . both cause injury by oxygen toxicity and embolism, of the lung for example [80] . therefore, especially the older formulations (fluosol-da and perftoran) were associated with pneumonia. additionally, the body is capable of discriminating endogenous and exogenous structures efficiently. therefore, the body may confuse the droplets with vicious intruders such as microorganisms or tumour cells and might try to defend itself against this alleged danger. the frontline of this defence is the reticuloendothelial system (res), which consists of several different cell types and proteins. the first step is that complement factors and antibodies recognize the pfc droplets and opsonize them. there are three ways to reduce this process: firstly, through choosing the adequate emulsifiers such as endogenous proteins and lipids. secondly, via the attachment of polyethylene glycol (so-called pegylation) to the outside of the particle, which sterically hinders the general interaction of proteins with the droplet surface [25, 35, 52] . thirdly, by carefully adjusting the droplet size, which influences the circulation of pfc droplets in the blood. in general, the smaller the droplet, the longer it will circulate in the bloodstream, with a minimum size of around 100 nm, below which the droplets will enter the endothelial cells via pinocytosis [15, 44] . by saying that, one has to admit that the res might not be the only factor to explain the elimination from the vascular system. in addition, the fusion of pfcs wrapped in phospholipids with natural lipid vesicles inside the bloodstream may contribute to the particle clearance (fig. 2c) . the rate of this fusion of phospholipid-coated pfc droplets and, e.g., cholesterol vesicles or lipoproteins is proportional to the vapour pressure of the pfc used [53, 99] . once the droplets are opsonized, circulating macrophages and monocytes take them up and transport them directly to the lung, where they can evaporate [95] . in addition, splenocytes and kupffer cells also start phagocytosis and, thereby, store the incorporated pfc droplets in the liver ( fig. 2a ) and the spleen. liver damage parameters (aspartate transaminase and alanine transaminase) increase transiently [6, 59, 68] , although the storage of the inherently inert pfc does not cause organ damage per se, except for the formation of foamy cells from primary kupffer cells ( fig. 2a ) [84, 105] . the increase of plasma transaminase activity seems to be linked to oxidative stress caused by the o 2 release from the pfc droplet that occurs inside the macrophages (fig. 2d ) [4, 56] . furthermore, once pfc has reached the liver, it starts to uncouple cytochrome p-450 (cyp450) enzyme reactions. the pfc and the enzyme form a pseudo-enzyme-substrate complex that leads to the hydrolysis of the coenzyme nadph without substrate conversion. these enzymes are monooxygenases that bind o 2 to potentially poisonous organic compounds to increase their water solubility for renal clearance [96] . the degree of the enzyme-substrate complex formation depends on the lipophilicity of the pfc and impairs the detoxification capacity of the liver [58, 70, 73] . the nadph depletion causes the malic enzyme to regenerate nadph at the expense of malate. subsequently, less malate transforms into oxaloacetate inside the mitochondria, thus producing less nadh+h + . as a result, oxaloacetate and nadh+h + are missing for gluconeogenesis (fig. 2b ) [60] . this forces the liver to deplete glycogen [85] . the liver compensates the detoxification impairment by upregulating the cyp450 biosynthesis [60, 71] . furthermore, pfcs lead to a dysregulated lipid storage and utilization in the liver (lack of lipogenesis as well as the inhibition of the uptake of chylomicrons and lipoproteins), which might also result from their cyp450 uncoupling (fig. 2b) [21, 67, 96] . the organ retention can last up to 130 days, while the pfc is constantly excreted via lipoproteins that transport it to the lung, where it evaporates and leaves the body [18] . in between this long period, the pfc seems to be kept from evaporating. therefore, one might wonder what is happening inside the organs. with increasing lipophilicity the pfcs tend more to interact with lipid membranes [69] . as mentioned above, the pfcs are generally lipophobic, but, e.g., pfob displays enough lipophilicity to intercalate into the centre of the lipid bilayers of cell membranes up to 2 mol% without causing conformational changes in the membrane ( fig. 2a ) [19] . still, this intercalation is suspected to cause the inhibition of the function of macrophages and neutrophil granulocytes leading to attenuated immune response [72] . within the few last years, there has been an important shift of paradigm from large-volume "blood replacement" (several litres) to a small-volume use (mostly 250-500 ml) of artificial oxygen carriers as "oxygen therapeutics" [23] . the major field of application for pfocs is clearly the short-term blood fig. 2 pfcs are inherently inert, but dependent on their lipo-and hydrophilicity and their vapour pressure they interact with the tissue and blood. (a) some of the more lipophilic pfcs such as pfob intercalate into the lipid bilayer of cell and organelle membranes [19] . the major part of pfcs are deposited inside the kupffer cells which makes them appear foamy [84] . (b) pfcs can uncouple cyp450 monooxygenases, which reduces the detoxification capacity of the liver and causes differed glucose and lipid metabolism [21, 58, 71, 73, 85] . nad/nadp: nicotinamideadeninedinucleotide/-phosphate. (c) proportional to the vapour pressure, phospholipid-wrapped pfcs (red) and natural lipid vesicles (yellow) inside the bloodstream may fuse and form hybrid vesicles [53, 99] . (d) pfc droplets are opsonized by complement factors or antibodies to be recognized and phagocytized by macrophages [52] . figure created with biorender.com replacement in cases of blood loss and many reviews already cover this topic [23, 36, 41] . so far, pfocs can be divided into four subclasses considering the main pfc used in the product: (i) pfd-based pfocs such as fluosol-da, perftoran and a-aocs, (ii) pfob-based pfocs such as oxygent, (iii) tertbutylperfluorocyclohexane-based pfocs such as oxycyte and (iv) ddfp-based pfocs. their general properties are listed in table 2 . in the usa, the fda granted the approval for fluosol-da in cases of coronary angioplasty but withdrew their approval in 1994 because of its extremely limited shelf life and mainly because of severe complement activation [55] . to treat severe blood loss, perftoran is approved in russia, kazakhstan, kyrgyzstan and ukraine and was approved in mexico from 2005 to 2010 [61] . oxygent and oxycyte reached human trials, but by now were not approved by the fda or elsewhere. oxycyte successfully completed phase ii trials but was abandoned by the sponsor in 2014 due to lack of patient enrolment and economic reasons. oxygent reached phase iii trials, was transiently abandoned because of safety issues that later were disproved to be product-related and is currently under clinical investigation in china [23] . a closer look at the physiological behaviour of blood in the capillaries in comparison to the properties of pfocs is worthwhile. because of the fåhraeus-lindqvist effect in small vessels, rbcs are located in the middle of the bloodstream, surrounded by a cell-free plasma layer. this effect has two consequences leading to enhanced tissue oxygenation in the presence of pfocs. the first consequence is the so-called plasma skimming, which occurs at bifurcations of the vessels (fig. 3a) . whenever a vessel splits into smaller vessels of unequal size, the major fraction of the rbcs remain in the larger vessel, thereby reducing the amount of rbcs within the microcirculation. subsequently, friction is reduced and the flow in microcapillaries is increased. this is necessary to adjust the o 2 supply to physiological o 2 levels in the tissue [79] . however, pathological obstructions exert a similar influence so that the rbc content of the obstructed vessel decreases disproportionally to the flow. combined with the low haematocrit, this may amplify tissue hypoxia. moreover, during shock, a narrowing of vessels occurs as the body centralizes through excessive vasoconstriction, again causing a barrier for rbcs. such vasoconstriction cannot restrain pfocs from entering and passing partially obstructed vessels, thus maintaining the o 2 supply for the periphery [13, 39] . because of the equal distribution of the nano-sized droplets, plasma skimming does not occur with pfc emulsions (fig. 3b) . they maintain the po 2 in the microcirculatory system by passing partially obstructed vessels. recent preclinical and clinical works on heart attack and stroke showed promising results in minimizing the size of the infarct area and, therefore, extending the window for therapy in acute models [3, 11, 12, 22, 92] . the second consequence of the fåhraeus-lindqvist effect is an increased diffusion distance due to the enlarged cell-free layer surrounding the rbcs [54, 90] . as soon as pfocs enter the vascular system, they contribute to the o 2 capacity of the plasma. the uptake of o 2 into the pfc droplet happens faster than the uptake of o 2 into the rbcs. this is unimpeded by the emulsifier [54, 106] . furthermore, pfocs stay rather close to the endothelia and shorten the diffusion distance between rbcs and the endothelium whilst acting as stepping-stones for o 2 (fig. 3c) [20, 37, 78] . this facilitated diffusion is helpful in cases of blood loss caused by extensive bleeding or haemodilution, e. g. during operations assisted by heartlung machines [103] . however, pfocs present exceptional physical and physiological properties (tables 2 and 3) , which differ from those table 2 main properties of the most promising approaches to artificial blood products [5, 12, 38, 65, 80 [105] of rbcs, which allows them to stand up particularly well in their use in additional pathological conditions. one field of application may be the treatment of malignant tumours. radiotherapy induces the synthesis of reactive oxygen species inside the tumour to cause oxidative stress and finally cell death. some tumours tend to be metabolically very active so a lack of o 2 supply and even anoxia occur in the core of the tumour. this anoxia inhibits the formation of reactive oxygen species and renders the tumour cells resistant to radiation therapy [31, 66] . using pfocs in this context leads to the oxygenation of the whole tumour, including the core, thereby sensitizing the tumour to radiation therapeutics, even if the patients do not breathe pure o 2 or carbogen [33, 89, 107] . in the context of organ transplantation, pfc-based oxygen carriers enable the promising new technology of warm machine perfusion. thereby the actual static cold storage procedures on ice after flushing with crystalline solutions such as university of wisconsin solution or custodiol may be replaced in the future. in the last few years, clinical studies showed the advantage of warm machine perfusion after organ harvest to cold storage, either static or perfused [7, 97] . this technique needs o 2 carriers to maintain the physiological status or to regenerate the tissue. some new attempts show promising results in the ex vivo perfusion of different organs with pfocs in low doses oxygenated with carbogen or pure o 2 , respectively [76, 98, 106] . the abovementioned universal gas dissolubility opens another path off the beaten tracks for pfocs. the inertness towards oxidation permits the transportation of excess carbon monoxide (co) from the blood to the lung, and the acceleration of the recovery of co-venomed haemoglobin [47, 105, 108] . in addition, the washout of nitrogen associated with decompression sickness was proven efficient in terms of avoiding embolism in rabbit, pig and rat models [10, 57, 63, 109] . severe nitrogen embolism in rats that underwent simulated diving with fast surfacing could be prevented by infusing albumin-derived pfd oxygen carriers prior to diving [64] . however, pfcs do not only wash out poisonous gases but also balance, e.g., local dysregulated nitrogen monoxide (no) levels. russian researchers focused on the use of pfc emulsions as a pharmaceutical for directed transport of endogenous bioactive no to regulate cardiovascular complications associated with no imbalance [82] . in the 1990s, another field of application for pfc emulsions-use as a contrast agent-was discovered exploiting their ultrasound absorbing properties. ddfp was chosen for this purpose, because of its low boiling point (28°c), which permits phase transition from the liquid into the gaseous phase, immediately after intravenous injection. the gaseous form results in an extremely short blood half-life of 1.45 ± 0.17 min and a main fig. 3 (a) rbcs are located in the middle of the bloodstream, surrounded by a cell-free plasma layer. at bifurcations, the rbc concentration decreases (plasma skimming) [79] . (b) the nanosized pfc droplets (light grey) are equally distributed in the vessel and do not rely on plasma skimming [39] . (c) the uptake of o 2 into the pfc droplet happens fast, which shortens the diffusion distance between rbcs and endothelium whilst pfc droplets act as stepping-stones for o 2 [20] . effected by plasma skimming not effected by plasma skimming excretion route via the lung, which minimizes organ retention of ddfp and therefore its negative side effects [2, 12] . these properties of ddfp are in extreme contrast to the other pfcs mentioned above, normally aiming at a rather long intravascular halflife, when pfc emulsions are used as blood substitutes [42] . nevertheless, ddfpe qualifies as an artificial oxygen carrier therapeutic as the transition leads to a tremendous expansion of intermolecular cavities resulting in an oxygen capacity superior to all other pfcs (table 1) . one application is the short-term oxygenation of hypoxic tissue after stroke [11] [12] [13] . furthermore, ddfp emulsions (ddfpe) can be used for tissue preconditioning prior to a surgery involving tissue ischaemia (e.g. myocard); a technique used to decrease infarct volume during stroke. here, intravenous administration of ddfpe functions as an oxygen tidal wave resulting in production of reactive oxygen species which is acute but limited in time. these reactive oxygen species subsequently activate the mitochondrial atp-sensitive k + -channel, an effect that exceeds intravascular half-life of ddfpe [11] [12] [13] 91] . the current year 2020 presents a major obstacle for humankind in a form of a pandemic that causes thousands to die and millions to be kept in quarantine. the use of pfocs to treat covid-19 patients has not yet been tested. however, their use as an additional o 2 transporter to enhance the peripheral o 2 supply even in narrowed microcapillaries during severe lung inflammation and their cytoprotective effect on rbcs are already discussed [100] . researchers aiming to develop biocompatible artificial oxygen carriers have to work extremely interdisciplinary. more than 50 years after l. c. clark's first experiments, researchers worldwide still work in different fields to obtain more and more knowledge on pfocs. hopefully, this will lead to safe pfc formulations and allow to exploit these materials to a greater extent as universal gas transporters. the former sprint in the 80s has turned into a marathon because of the complexity of simulating blood. researchers are still developing new applications and promising new formulations, which make the research field of pfocs more topical than ever. acknowledgments open access funding enabled and organized by projekt deal. conflict of interest the authors declare that they have no conflict of interest. ethics approval not applicable. consent to participate not applicable. code availability not applicable. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. mammalian life without red blood corpuscles ddfp) following repeated iv administration in the new zealand white rabbit dodecafluoropentane emulsion (ddfpe) decreases stroke size and improves neurological scores in a permanent occlusion rat stroke model cytotoxicity of a perfluorocarbon blood substitute to macrophages in vitro perfluorocarbon-based oxygen 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langendorff-heart (dagger) perfluorocarbon nanodroplets can reoxygenate hypoxic tumors in vivo without carbogen breathing effect of perfluorochemical (pfc) emulsion on acute carbon monoxide poisoning in rats intravenous perfluorocarbon emulsion increases nitrogen washout after venous gas emboli in rabbits doi publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-016248-dxk0i6t7 authors: papa, joey c.; stolar, charles j. h. title: extracorporeal membrane oxygenation date: 2009 journal: pediatric surgery doi: 10.1007/978-3-540-69560-8_32 sha: doc_id: 16248 cord_uid: dxk0i6t7 extracorporeal membrane oxygenation (ecmo) is a life-saving technology that uses partial heart and lung bypass for extended periods. it is not a therapeutic modality, but rather a supportive tool that provides suf-fi cient gas exchange and perfusion for patients with acute, reversible cardiac or respiratory failure. this affords the patient's cardiopulmonary system time to rest, sparing them from the deleterious effects of traumatic mechanical ventilation and perfusion impairment. the extracorporeal life support organization (elso) was formed in 1989 by a collaboration of physicians, nurses, perfusionists, and scientists with an interest in ecmo. the group provides an international registry that collects data from almost all ecmo centers in the united states and throughout the world. at the end of 2005, elso registered nearly 30,000 neonatal and pediatric patients treated with ecmo for a variety of cardiopulmonary disorders with an overall survival rate of 66%. extracorporeal membrane oxygenation (ecmo) is a life-saving technology that uses partial heart and lung bypass for extended periods. it is not a therapeutic modality, but rather a supportive tool that provides suffi cient gas exchange and perfusion for patients with acute, reversible cardiac or respiratory failure. this affords the patient's cardiopulmonary system time to rest, sparing them from the deleterious effects of traumatic mechanical ventilation and perfusion impairment. the extracorporeal life support organization (elso) was formed in 1989 by a collaboration of physicians, nurses, perfusionists, and scientists with an interest in ecmo. the group provides an international registry that collects data from almost all ecmo centers in the united states and throughout the world. at the end of 2005, elso registered nearly 30,000 neonatal and pediatric patients treated with ecmo for a variety of cardiopulmonary disorders with an overall survival rate of 66%. neonates are the patients who benefi t most from ecmo. cardiopulmonary failure in this population can arise from meconium aspiration syndrome (mas), congenital diaphragmatic hernia (cdh), persistent pulmonary hypertension of the newborn (pphn), as well as several congenital cardiac diseases. for the pediatric population, the most common disorders treated with ecmo are bacterial and viral pneumonia, acute respiratory failure, ards, sepsis, and cardiac disease. the experience with pediatric cardiac ecmo has been increasing over the past few years. its use for treating postcardiotomy patients who are unable to wean from bypass as well as cardiac failure with bridge to transplantation have expanded greatly in the last decade. some indications for ecmo that are not yet established clinically include emergency cardiopulmonary bypass and ecmo during cpr (ecpr) respiratory failure second to mediastinal compression (mass effect), smoke inhalation, severe asthma, or rewarming of hypercoagulopathic and hypothermic trauma patients (see fig. 32 .1). the selection of patients as potential ecmo candidates continues to remain controversial. the selection criteria are based on data from multiple institutions, patient safety, and mechanical limitations related to equipment. the risk of performing an invasive procedure that requires systemic heparinization of a critically ill child must be weighted against the estimated mortality of the patient with conventional therapy alone. a predictive mortality of greater than 80% despite maximal medical management is the criterion most institutions use to select patients for ecmo. ecmo is indicated when a reversible disease process is present, tissue oxygenation requirements are not being met, and ventilator treatment is causing more harm than good. all ecmo centers must develop their own criteria and continually evaluate their patient selection based on ongoing outcomes data. a discussion of generally accepted selection criteria for using ecmo follows. reversible disease process: the underlying principle of ecmo relies on the premise that the patient has a reversible disease process that can be corrected with either therapy or "rest" within a relatively short period of time. exposure to high pressure mechanical ventilation with high concentrations of oxygen will frequently lead to the development of bronchopulmonary dysplasia (bpd) . bpd can result from as little as 4 days of high-level ventilatory support. the pulmonary dysfunction following barotrauma and oxygen toxicity from mechanical ventilation can take weeks to months to resolve. therefore, patients who have received aggressive ventilation for greater than 10-14 days are not considered ecmo candidates due to the high probability of established, irreversible lung injury. gestational age: the gestational age should be at least 34 weeks. signifi cant morbidity and mortality related to intracranial hemorrhage (ich) is associated with infants less than 34 weeks gestational age. in preterm infants, ependymal cells within the brain are not fully developed, thus making them susceptible to hemorrhage. in addition, the systemic heparinization necessary to maintain a thrombus-free ecmo circuit also increases the risk of hemorrhagic complications ( fig. 32 .2). birth weight: technical consideration and limitation of cannula size restrict ecmo candidates to a birth weight of 2,000 g. the smallest single lumen ecmo cannula is 6 french (fr), and fl ow through the tube is related to the radius of the tube by a power of 4. babies that weigh less than 2 kg provide technical challenges in performing cannulation and in maintaining adequate fl ow through small catheters. bleeding complications: babies with ongoing, uncontrollable bleeding or an uncorrectable bleeding diathesis pose a relative contraindication to ecmo. coagulopathy should be corrected before initiation of ecmo as the circuit requires continuous systemic heparinization. intracranial hemorrhage: patients who pose a high risk for ich are those with previous history of seizures, intracranial bleed, cerebral infarction, prematurity, coagulopathy, ischemic central nervous system injury, or sepsis. consideration of these patients for ecmo should be individualized. in general, candidates for ecmo should not have an ich. a preexisting ich may be exacerbated by the use of heparin and the unavoidable alterations in cerebral blood fl ow while on ecmo support. patients with small intraventricular (grade i or ii) or intraparenchymal hemorrhages can be successfully treated on ecmo by maintaining a lower than optimal activated clotting time (act) between 180 and 200 s. these patients should be closely observed for extension of intracranial bleeding with frequent neurologic exams and daily cranial ultrasonography. coexisting anomalies: the patient should have no congenital anomalies that are incompatible with life. however, many lethal pulmonary conditions such as congenital alveolar proteinosis, alveolar capillary dysplasia, and overwhelming pulmonary hypoplasia may present as reversible diseases. every effort should be made to establish a clear diagnosis before the initiation of ecmo as it is not intended to delay an inevitable death. other treatable conditions, such as total anomalous venous line pulmonary venous return and transposition of the great vessels, may initially manifest with respiratory failure but should be diagnosed with preoperative echocardiography. failure of medical management/risk assessment: ecmo candidates are expected to have a reversible cardiopulmonary disease process with a predictive mortality of greater than 80% despite maximal medical management. the pharmacologic agents that comprise part of the medical management include vasoconstrictive, inotropic and chronotropic agents, sedatives, and analgesics. ventilatory management usually begins with conventional support but may also include the administration of surfactant, inhaled nitric oxide, inverse inspiratory-expiratory (i/e) ratios, or high-frequency oscillation. because of the invasive nature of ecmo and the potential life-threatening complications, investigators have worked to develop an objective set of criteria to predict which infants and children have 80% mortality without ecmo. pulmonary insuffi ciency with associated hypoxia, hypercarbia, and acidosis is not an indication for ecmo unless tissue oxygen requirements are not being met, as evidenced by progressive metabolic acidosis, decreased mixed-venous oxygen saturation (svo 2 ), and early evidence of multiple organ failure. the two most commonly used measurements for respiratory failure are the alveolar-arterial oxygen gradient (aado 2 ) and the oxygenation index (oi), which are calculated as follows: aado 2 = (p atm − 47) (fio 2 ) -[(paco 2 )/0.8] -pao 2 where p atm is the atmospheric pressure and fio 2 is the inspired concentration of oxygen. where map is the mean airway pressure. although institutional criteria for ecmo vary, it is generally accepted that for neonates with an aado 2 greater than 625 mmhg for more than 4 h, an aado 2 greater than 600 for 12 h, or an oi greater than 40 establishes both relatively sensitive and specifi c predictors of mortality. other criteria used by many institutions include a preductal pao 2 less than 35-50 mmhg for 2-12 h or a ph of less than 7.25 for at least 2 h with intractable hypotension. these are sustained values measured over a period of time and are not accurate individual predictors of mortality. older infants and children do not have such welldefi ned criteria for high mortality risk. the combination of a ventilation index respiratory ´ rate paco 2 ´ peak inspiratory pressure 1,000 greater than 40 and an oi greater than 40 correlates with a 50-70% mortality risk. a mortality of 60-80% is associated with an aado 2 greater than 580 mmhg and a peak inspiratory pressure (pip) of 40 cm h 2 o. indications for support in patients with cardiac pathology are based on clinical signs of decreased peripheral perfusion, including hypotension, despite the administration of fl uid resuscitation and inotropes, oliguria (urine output < 0.5 ml/kg/h), an elevated arterial lactate, and a decreased svo 2 . special mention should be made of infants with cdh who develop respiratory failure. before ecmo is initiated in an infant with cdh, the infant must fi rst demonstrate some evidence of adequate lung parenchyma. this includes maintaining a preductal oxygen saturation ≥ 90% for a sustained period of at least 1 h and at least one recorded paco 2 of less than 50 mmhg. the goal of ecmo support is to provide oxygen delivery. many different cannula confi gurations are possible, but the three most commonly used clinically include venoarterial (va), venovenous (vv), and double-lumen venovenous (dlvv). veno-venous (vv), including single cannula, dual-lumen vv (dlvv): dlvv is used in neonates, infants, and children less than 15 kg due to limitations of fl ow based on cannula size. the catheter is inserted into the rij, with the tip in the ra. vv ecmo bypass is established by draining the ra via the rij, with reinfusion into a femoral vein. the advantages of vv and dlvv over va ecmo include avoidance of arterial cannulation and permanent ligation of the carotid artery, maintaining pulsatile fl ow to the patient, continued blood fl ow to the lungs, and avoiding arterial emboli. a major limitation of dlvv ecmo is that there is mixing of unsaturated and saturated blood in the ra, because blood is both withdrawn and returned to the right atrium (ra). in addition, a fraction of the reinfused, oxygenated blood reenters the pump, called recirculation. recirculation artifi cially raises the svo 2 measurement on the pump and may limit oxygen delivery at higher fl ow rates. veno-arterial (va): va ecmo offers the ability to replace both cardiac and pulmonary function. venous blood is drained from the ra through the right internal jugular vein (rij) and oxygenated blood is returned via the right common carotid artery (rcca) to the ascending aorta. there are many potential disadvantages associated with va ecmo. a major artery must be cannulated and therefore sacrifi ced. the risk of gas and particulate emboli being introduced into the systemic circulation is substantial. a decrease in the preload and an increase in the afterload may reduce cardiac output, resulting in nonpulsatile fl ow. pulmonary perfusion is reduced and the coronary arteries are largely perfused by hypoxic left ventricular blood. transthoracic cannulation is the preferred mechanism of support for cardiac surgery patients who are unable to wean off bypass postcardiotomy or in cases of cardiac arrest in the immediate to early postoperative period. the venous cannula is placed directly into the right atrial appendage and the arterial cannula in the ascending aorta. the chief disadvantages to open-chest cannulation include signifi cant risk of hemorrhage and infection (mediastinitis). patients with left heart or bi-ventricular failure are at risk of left ventricular distention. left heart decompression is needed to reduce pulmonary edema, prevent pulmonary hemorrhage, and reduce ventricular distention that may aid in recovery of function. this can be avoided with a surgically created atrial septostomy or a cannula placed directly in the left atrium via open chest cannulation; patients with a preexisting atrial septal or ventricular septal defect (asd or vsd) do not need further surgical intervention. at our institution, all patients with cardiac failure receive prophylactic atrial septostomy to prevent left-sided dilation and potential worsening cardiac function. with proper monitoring, cannulation can be performed in the neonatal or pediatric intensive care units under adequate sedation and intravenous anesthesia. the child is positioned supine with the head at the foot of the bed. the head is turned to the left and the neck is hyperextended over a shoulder roll. after local anesthesia is administered over the incision site, a transverse cervical incision is made along the anterior border of the sternocleidomastoid muscle, one fi ngerbreadth above the right clavicle. the platysma muscle is divided, the sternocleidomastoid muscle is retracted laterally, and dissection is carried down to the carotid sheath. the sheath is opened and the internal jugular vein, common carotid artery, and vagus nerve are identifi ed. the vein is dissected fi rst and isolated with vessel loops. the common carotid lies medial and posterior, contains no branches, and is mobilized in a similar fashion. the vagus nerve should be identifi ed only to protect it from injury. once the vessels have been isolated, the patient is given a bolus of 100 u/kg of heparin sulfate, which is allowed to circulate for 2-3 min. an act level should be drawn and should be greater than 300 s. for va bypass, the arterial cannula is placed fi rst. the carotid artery is ligated distally and once proximal control is obtained with a vessel loop a transverse arteriotomy is made near the distal ligature. stay sutures can be placed in the artery to retract and to help prevent intimal dissection. the saline-fi lled cannula is inserted to its premeasured position (tip at the junction of the brachiocephalic artery and the aorta) and secured in the vessel with 2-0 silk ligatures. additionally, a small piece of vessel loop may be placed under the ligature on the anterior aspect of the carotid to protect the vessel from injury during decannulation. the patient must be paralyzed with succinylcholine before venous cannulation to inhibit spontaneous respiration and prevent air emboli. the jugular vein is then ligated and a venotomy is made close to the ligature. the saline-fi lled venous catheter is passed to a measured level of the ra and secured as described above. any bubbles are aspirated from the cannulas, which are then connected to the preprimed ecmo circuit and bypass is initiated. the cannulae should then be secured to the patient's skin above the wound and the skin closed in layers to ensure meticulous hemostasis. for vv and dlvv bypass, the procedure is exactly as described above, including dissection of the artery with the placement of a vessel loop to facilitate conversion to va ecmo should the need arise. the venous catheter tip should be positioned in the mid-right atrium with the arterial portion of the dlvv catheter oriented medially to direct the fl ow of oxygenated blood toward the tricuspid valve. the cannula position is confi rmed by chest radiography and transthoracic echocardiogram and readjusted as needed. venous blood is drained from the infant or child by gravity into a small reservoir or bladder. an in-line oxymetric probe is located between the venous return cannula and the bladder to continuously monitor the svo 2 saturation. the bladder is a 30-to 50-ml reservoir that acts as a safety valve. in the event venous drainage does not keep up with the arterial fl ow from the pump, the bladder volume will be depleted, the pump will be automatically shut off, and an alarm will be sounded. this serves to limit the potential for injury to the ra, rij, or cavitation of air and high negative pressures within the circuit. hypovolemia is one of the common causes of decreased venous infl ow into the circuit, but kinking with occlusion of the venous line should be suspected fi rst. in addition, the height of the patient's bed can be raised to improve venous drainage by gravity. a displacement roller pump pushes blood through the membrane oxygenator. the roller pumps are designed with microprocessors that allow calculation of the blood fl ow based on roller-head speed and tubing diameter of the circuit. in other words, the speed at which the pump is set determines what proportion of the patient's cardiac output will be diverted into the circuit and is adjusted according to how much support the patient requires. the pumps are connected to continuous pressure monitoring throughout the circuit and are servoregulated if pressures within the circuit exceed preset parameters. another safety device, the bubble detector, is interposed between the pump and the membrane oxygenator and will stop fl ow if air is detected within the circuit. the blood enters the membrane lung, after exiting the pump. the oxygenator consists of a long, two-compartment chamber composed of a spiral-wound silicone membrane and a polycarbonate core. this provides a large surface area across which blood and gas come into close contact, with blood fl owing in one direction and gas fl owing in the opposite direction. oxygen diffuses through the membrane into the blood circuit and carbon dioxide and water vapor diffuse from the blood into the sweep gas. the size (surface area) of the oxygenator chosen is based on the patient's weight and size. the blood emerges from the upper end of the oxygenator and passes through the countercurrent heat exchanger returning to the body at physiologic temperature into the ra (via dlvv cannulation) or the aortic arch via the rcca. prime management: the tubing of the ecmo circuit is initially circulated with carbon dioxide gas. this is followed by the addition of crystalloid and 5% albumin solution. the albumin coats the tubing to decrease its reactivity to circulating blood. the carbon dioxide gas dissolves into the fl uid. approximately two units of packed red blood cells are required for initial priming of the pump, which displaces the crystalloid and colloid in the circuit. the initial ph, oxygen content, and carbon dioxide content of the circuit are then measured and adjusted to physiologic parameters. if the prime blood is acidotic, this may exacerbate the infant's condition; or if the primed circuit has low carbon dioxide content, this may cause metabolic problems for the neonate. additionally, a heat exchanger warms the prime to normal body temperature. in sum, the primed circuit must be physiologically compatible with life prior to initiating ecmo to maximize support and prevent initial worsening of the child's condition. pump management: the goal of ecmo is to maintain adequate pump fl ow, which will result in good oxygen delivery to the tissues and organs. oxygen delivery to the infant is dependent on the speed or rotations per minute (rpm) of the roller pump. full bypass support is considered 100 cc/kg/min on vv ecmo and 150 cc/kg/min on va ecmo. to increase a patient's oxygen level on ecmo, one can either increase the fl ow rate (∼cardiac output) or increase oxygen carrying capacity with transfusion of prbc to maintain a hemoglobin level of 15 g/dl (∼oxygen content). with va ecmo, adequate perfusion and oxygen delivery can be monitored by the ph and po 2 of a mixed venous blood sample (pre-oxygenator blood sample). the fl ow of the roller pump should be adjusted to maintain a mixed venous po 2 of 37-40 mmhg and svo 2 of 65-70%. with vv ecmo, the mixed venous sample may not be a reliable indicator of perfusion as recirculation may produce a falsely elevated po 2 . therefore, other indicators of poor perfusion should be followed, such as persistent metabolic acidosis, oliguria, seizures, elevated liver function tests, and hypotension. if oxygen delivery is found to be inadequate, then the rpm of the pump may need to be increased to improve perfusion. roller pumps roll against the tubing to propel the blood towards the oxygenator. this area of contact is at risk of tubing rupture over time. to reduce the risk of rupture, the raceway is advanced every 5-7 days after temporarily stopping the pump flow. tubing rupture is a rare event because of modern materials such as supertygon (norton performance plastics corp., akron, oh), a chemically altered polyvinyl chloride (pvc). the tubing should be inspected daily and all connections secured properly and replaced if defective. when a raceway rupture does occur, the pump must be turned off immediately, the patient must be ventilated and perfused with conventional methods (increased ventilator pressures and fio 2 ), and cpr performed if necessary. the raceway tubing is then replaced or the entire circuit can be changed. oxygenator management: the silicone membrane (envelope) oxygenator (avecor, inc., minneapolis, mn) is critical to the success of ecmo and long-term bypass. the mechanism of gas exchange occurs when blood in the tubing enters a manifold region and is distributed around the envelope of a silicone membrane lung. oxygen, which is mixed with a small amount of carbon dioxide to prevent hypocapnea, fl ows through the inside of the membrane envelope in a countercurrent direction to the fl ow of blood. oxygen diffuses across the silicone membrane into the blood as carbon dioxide is eliminated. the oxygenated blood drains into a manifold and is returned to the infant via a heat exchanger. thrombus may form in the oxygenator over time. as the thrombus extends, the membrane surface area decreases, resulting in decreased oxygenation, increased carbon dioxide retention, and increased resistance to blood fl ow. signs of clot formation can be detected by direct visualization of the top or bottom of the membrane, but the extent of the clot cannot be determined. another sign of clot formation within the oxygenator is progressive consumption of clotting factors such as platelets and fi brinogen. the gaseous portion of the oxygenator may also develop obstructions, which may lead to air emboli. long-term use may wear out the silicone membrane resulting in blood and water in the gas phase causing water condensation. therefore, the oxygenator should be replaced when the postoxygenator po 2 decreases to <200 mmhg or pre-oxygenator circuit pressures increase to over 400 mmhg at fl ow rates required to support the patient. in addition, a larger oxygenator may also be required if the gas and blood fl ow rating of the old oxygenator are exceeded in order to maintain adequate perfusion. volume management: while on ecmo, maintenance fl uids for a term newborn under a radiant warmer are estimated at 100 cc/kg/day. water loss through the oxygenator may approach 2 cc/m 2 /h. for a 3 kg baby, this would be about 13 cc/kg/day. fluid losses from urine, stool, chest tubes, nasogastric tubes, ostomies, mechanical ventilation, radiant fl uid loss, and blood draws should be carefully recorded and repleted. fluid management may become diffi cult in the ecmo baby as fl uid extravasates into the soft tissues during the early ecmo course. therefore, meticulous recordings of the net fl uid balance should be maintained on ecmo. classically, the weight increases in the fi rst 1-3 days as the patient becomes increasingly edematous. starting the third day on ecmo, diuresis of the excess edema fl uid begins, and can be facilitated with the use of furosemide. this diuretic phase is often the harbinger of recovery. in the event of renal failure on ecmo, hemofi ltration or hemodialysis can be added to the ecmo circuit for removal of excess fl uid and electrolyte correction. respiratory management: once the desired fl ow is attained, the ventilator should be promptly weaned to avoid further oxygen toxicity and barotrauma. such "rest settings" have been studied and debated. at our institution, we decrease the fio 2 to 0.4, peep to 5 cm h 2 o, pip to 20-25 cm h 2 o, a rate of 12 breaths/min, and inspiratory time of 0.5 s if the infant's arterial and venous oxygenation are adequate. if the baby remains hypoxic despite maximal pump fl ow, then higher ventilator settings may be temporarily required. alternatively, hypoxic neonates on vv ecmo may need to be converted to va ecmo for full cardio-respiratory support. on occasion, the chest x-ray will worsen in the fi rst 24 h independent of ventilator settings and will improve after diuresis. as the patient improves on ecmo and the pump fl ow is weaned, ventilator settings are then modestly increased to support the baby off ecmo. in addition, during the course of ecmo, pulmonary toilet is essential to respiratory improvement and includes gentle chest percussion and postural drainage. special attention should be paid to the ecmo catheters and to keep the head and body aligned. endotracheal suctioning is also recommended every 4 h and as needed based on the amount of pulmonary secretions present. medical management: after the initiation of ecmo, vasoactive medications should be quickly weaned down if the blood pressure remains stable. low-dose dopamine (5 mcg/kg/min) can be administered for renal protection, although its use is controversial. in the event of seizures, phenobarbital is usually given and maintained to prevent further seizures. in addition, gastrointestinal prophylaxis with an h2-blocker, such as ranitidine, is instituted. fentanyl and midazolam is usually administered for mild sedation; however, the use of paralytics should be avoided as muscle activity is not only important for fl uid mobilization but also to monitor neurologic activity. infectious prophylaxis is provided by the use of ampicillin and gentamicin, which covers most common bacterial infections. with the use of gentamicin, attention should be directed to renal function. for this reason, cefotaxime may be used for gram-negative coverage instead of gentamicin. because of the cannula and manipulation of the circuit at stopcocks, the risk of infection is a constant concern; therefore, strict observance to aseptic technique when handling the ecmo circuit should be maintained. daily routine blood, urine, and tracheal cultures should be obtained to monitor for infection. caloric intake on ecmo should be maximized using standard hyperalimentation. for a newborn, total parenteral nutrition (tpn) should be started at 100 kcal/ kg/day. normally, this should be supplied as 60% carbohydrates (14.6 gm/kg/day) and 40% fat (4.3 gm/kg/ day). intralipid infusions may be used as a fat source, although there is some controversy with its use in the setting of severe lung disease. as a result, the percentage of fat in the hyperalimentation may be lowered. amino acids may be added but must be considered in the setting of poor renal function and increasing bun levels. with normal renal function, approximately 2.5 gm protein/kg/day should be provided in the tpn mixture. electrolytes should be closely monitored with potassium, calcium, and magnesium repleted as necessary. while on ecmo, the patient's hemoglobin is maintained at 15 gm/dl to maximize the oxygen carrying capacity of the blood. platelet destruction during ecmo is anticipated and is secondary to the fl ow through the oxygenator. in order to reduce the risk of bleeding during ecmo, the platelet count should be kept above 100,000/mm. we recommend using "hyperspun" platelets in neonates to avoid the excess administration of fl uid, and thus prevent further problems with volume overload and edema. heparin is initially administered as a bolus (50-100 mg/kg) followed by constant heparin infusion (30-60 mg/kg/h) to maintain a thrombus-free circuit. the level of anticoagulation is monitored hourly by the activated clotting time (act). the heparin infusion is adjusted to maintain an act of 180-220 s. after decannulating, the heparin infusion is stopped and not reversed with protamine sulfate. operative procedures on ecmo: surgical procedures, such as cdh repair, may be safely performed while the child remains on bypass. however, care must be taken to obtain meticulous hemostasis to avoid hemorrhagic complications. before any invasive procedure, platelets should be transfused to a level greater than 150,000/mm 3 and the act level dropped to 180-200 s. the fi brinolysis inhibitor aminocaproic acid is administered as a 100 mg/kg bolus 30 min prior to incision and maintained at a continuous drip at 30 ml/kg/h for 72 h postoperatively. weaning and decannulation: as the patient's underlying process improves, less blood fl ow is required to pass through the ecmo circuit in order to maintain adequate tissue oxygenation. the fl ow rate may be weaned slowly (10-20 ml/h) as long as the patient maintains oxygen saturations with evidence of adequate perfusion. the most important guide to weaning on va ecmo is the svo 2 and for vv ecmo, the sao 2 . when fl ow levels have decreased until they approximate 10% of the patient's cardiac output (∼30-50 cc/kg/min), the patient is usually ready for decannulation. as fl ow levels are decreased, the heparin drip should be increased for an act of 200-220 s to prevent thrombotic complications. ventilator settings can be increased moderately if saturations drop during weaning, but should not revert to pre-ecmo settings. if the child continues to tolerate low fl ow, all medications and fl uids should be switched to vascular access on the patient side and the cannulas can be clamped and fl ushed with heparanized saline (2 u/ml). flow is maintained within the circuit with the bridge open as the possibility that the child may not tolerate clamping and may need to be placed back on bypass remains. once the cannulas are clamped, the child is observed for 2-4 h. if he or she remains hemodynamically stable, with adequate saturations and does not become acidotic during this period, decannulation can safely be accomplished. decannulation is performed in the icu using a near-identical manner as cannulation. this should be done under sterile conditions in the trendelenburg position using a muscle relaxant to prevent air aspiration into the vein. once the cannula is withdrawn, the vessel is ligated, the wound irrigated, and closed over a small drain. complications on ecmo can be divided into technical, mechanical, or pump-related and patient-related. the most common technical complications include vessel injury or dissection during cannulation, cannula malposition and kinking, accidental decannulation, and limb ischemia from occlusion of distal fl ow. most technical complications can be avoided with proper surgical technique and securing of the cannulas. limb ischemia can be avoided with the placement of a distal perfusion catheter when signs of ischemia develop (loss of pulse, cool, mottled, or swollen extremity). mechanical complications include oxygenator failure, tubing rupture in the raceway (both described above), clot formation within the circuit tubing, and the introduction of air into the circuit. if clot is detected on the venous or pre-oxygenator side of the circuit, it can often be observed or segments of tubing can be selectively replaced. clots on the arterial or postoxygenator side of the circuit are cause for concern as they can break off and cause emboli with pulmonary and neurologic complications. when a clot is detected on the arterial side, the entire circuit should be exchanged for a fresh preprimed circuit. the introduction of air into the circuit is possible during the initial cannulation as well as through several connectors, tubing stop-cocks, and the membrane oxygenator. prevention of air embolism is vital; when setting up the circuit, all air must be removed and all connections made tight and thoroughly inspected and the circuit must be continuously monitored. if air is detected on the venous side, it can often be aspirated from one of the ports without coming off bypass. air on the arterial side is an emergency and requires the patient to be taken off bypass immediately until it can be safely aspirated. in the event that an air embolism reaches the patient, ecmo should be stopped, the patient placed in trendelenburg position, and an attempt should be made to aspirate any air out of the arterial cannula. if air enters the coronary circulation, inotropic support may be necessary. the most common patient complications are bleeding (cannula site 6.2-9.4%, surgical site 6.1-15.6%, intracranial 4.9-5.8%, gi 1.7-4.0%, tracheal, urinary) and coagulation disorders (hemolysis 12%, dic 1.4%). contact of blood with the foreign surface of the circuit activates the coagulation cascade. platelets are consumed by the circuit and their function is also affected. constant monitoring for signs of bleeding include observing for tachycardia, hypotension, a decreased hematocrit, or inadequate venous return are signs of hemorrhage. treatment includes replenishing lost blood products, including platelets and coagulation factors, if necessary. patients on ecmo may also have hemodynamic compromise, including hypotension or hypertension. according to the 2005 elso registry, 13.2% of neonates and 43% of pediatric patients treated with ecmo for respiratory failure required the use of inotropes while on bypass. hypotension can be from volume depletion (including blood loss) as well as decreased myocardial function from hypoxia prior to the initiation of ecmo support. inotropes are often easily weaned when hypoxia is reversed, but euvolemia and adequate hct should be maintained. hypertension requiring the use of vasodilators was reported in 12.6% of neonates and 11.8% of pediatric patients. the patient should be assessed for reversible causes of hypertension such as pain, hypercarbia, and hypoxia. hypertension should be aggressively treated due to the increased risk of intracerebral hemorrhage in ecmo patients. neurologic complications including intracerebral hemorrhage (ich), infarct and stroke, and seizures can occur with an overall incidence of 20-25%. seizures are widely reported among ecmo neonates, ranging from 20% to 70%. however, only 2% had a continued diagnosis of epilepsy at 5 years of age. seizures in the neonatal ecmo population are associated with neurologic disease and poorer outcomes, including epilepsy and cerebral palsy. the incidence of ich and infarct is recorded at 14% in neonates and 8% in pediatric patients on ecmo. as stated earlier, the risk is increased in low birth weight infants and premature infants <34 weeks gestation. patients with small interventricular (grade i or ii) or intraparenchymal hemorrhages can be successfully treated on ecmo by maintaining a lower than optimal activated clotting time (act) between 180 and 200 s. these patients should be closely observed for extension of intracranial bleeding with frequent neurologic exams and daily cranial ultrasonography. any progression or change in neurologic status requires cessation of anticoagulation and thus removal from ecmo support. oliguria and a slight rise in creatinine are common in ecmo patients and are often seen during the fi rst 24-48 h. the capillary leak seen after placing a child on ecmo may cause decreased renal perfusion, or it may be due to the nonpulsatile nature of blood fl ow seen in va ecmo. once the patient is adequately volume resuscitated, furosemide can be used to improve urine output. the incidence of acute renal failure was 10% in neonates and 14% in pediatric patients on ecmo for respiratory support, with 10-15% requiring hemofi ltration or dialysis. continuous hemofi ltration can be easily added in-line to the ecmo circuit and provides assistance with fl uid balance, hyperkalemia, and azotemia, which is often not needed after ecmo support is withdrawn. hemofi ltration removes plasma water and dissolved solutes while retaining proteins and cellular components of the intravascular space. the incidence of acquiring a nosocomial infection on ecmo has been reported at 26-30%. associated risk factors include the duration of the ecmo run, the length of hospitalization, type of cannulation (open chest vs neck), and surgical procedures performed before or during ecmo. fungal infections and sepsis carry a signifi cantly higher morbidity and mortality rates. in addition, because of the large volume of blood products transfused into ecmo patients, the risk of developing a bloodborne infectious disease is significant. one study states that approximately 8% of children who were treated with ecmo as neonates were seropositive for antibodies to the hepatitis c virus. there has been a decline in the use of ecmo for neonatal respiratory failure secondary to improved medical management (permissive hypercapnea and spontaneous ventilation, ino, surfactant, and hfov). inhaled nitric oxide (ino), a selective pulmonary vasodilator, improves oxygenation and has signifi cantly contributed to the recent decrease in the need for extracorporeal membrane oxygenation (ecmo) in neonates with respiratory failure. in addition to ino, high-frequency ventilation, the adjunct use of surfactant therapy, and improved cardiovascular support have recently been shown to decrease the need for ecmo in this patient population. according to a 10-year retrospective review of the elso registry data published in 2000, the use of surfactant, high-frequency ventilation, and inhaled nitric oxide in patients with respiratory failure who required ecmo increased from 0% in 1988 to 36%, 46%, and 24%, respectively, in 1997. the proportion of neonates with cdh requiring ecmo increased from 18% to 26%, while the proportion with respiratory distress syndrome decreased from 15% to 4%. in contrast, the number of cardiac cases had steadily increased over 15 years with a peak in 2002; however, there was a notable decline in 2003 and 2004. this could be due to decreased use secondary to the poor overall survival reported, increased organ procurement and transplantation, or use of other methods of support, including the berlin heart excor (berlin heart ® , berlin heart ag, berlin, germany), lvad, and bivad. in a recent study at our institution, we reviewed all transplant-related use of ecmo in patients who were placed on extracorporeal life support as a bridge to cardiac transplantation. the aggregate survival of these patients was 29% (6/21) but those who were successfully bridged to a cardiac transplant (i.e., survived on ecmo until transplanted) had 60% (6/10) survival. overall survival to discharge for neonates and pediatric patients treated with ecmo is dependent again on initial diagnosis. higher survival rates are seen in neonates with respiratory diseases (77%) than cardiac diseases (38%). within the neonatal population, newborns with mas that require ecmo have the highest survival rate at 94%, whereas survival for infants with cdh is 52%. the pediatric population of ecmo patients represents a diverse group with regard to patient age as well as diagnosis. over double the number of cardiac cases have been reported in the pediatric population compared to the respiratory cases (6,135 vs 2,934 at the end of 2005). higher complication rates exist with the pediatric patients, refl ecting the more complicated disease states as well as the longer duration of bypass required for reversal of the respiratory or cardiac failure. common long-term problems in ecmo-treated infants and children include feeding and growth sequelae, respiratory complications, and neurodevelopmental delays. these children are at increased risk for complications both as a consequence of ecmo itself and from antecedent hypoxia, acidosis, and reperfusion injury. approximately, one-third of infants treated with ecmo have feeding problems. the possible causes are numerous and include tachypnea, generalized central nervous system depression, poor hunger drive, postsurgical neck soreness (possibly from compression of the vagus nerve), and poor oral-motor coordination. cdh babies have a higher incidence of feeding diffi culties as compared with infants with mas secondary to foregut dysmotility, which leads to signifi cant gerd and delayed gastric emptying. respiratory compromise and chronic lung disease compound the problem. normal growth is most commonly reported in ecmo-treated patients; yet these children are more likely to experience problems with growth than agematched normal controls. head circumference below the fi fth percentile occurs in 10% of ecmo-treated children. growth problems are most commonly associated with ecmo children who have suffered from cdh or residual lung disease. neonatal ecmo survivors have a relatively high incidence of respiratory abnormalities initially with 15% requiring supplemental oxygen at 28 days of age and 25% having at least one episode of pneumonia by the age of 5 years as compared with controls (13%). these children with pneumonia are more likely to require hospitalization, and pneumonia occurs at a younger age, with over half diagnosed in the fi rst year of life. cdh infants, in particular, have been found to have severe lung disease after ecmo and may require supplemental oxygen therapy at home. probably the most serious post-ecmo morbidity is neuromotor handicap. most studies show approximately 20% (18-25%) ecmo survivors exhibit some type of handicap, with an 8-9% incidence of moderate-to-severe cognitive delay. auditory defects are noted in over one-fourth of ecmo neonates at discharge, with sensorineural hearing loss in ∼5%, speech and language delay in ∼6% with roughly 10-15% requiring speech and language therapy. healthcareassociated infection in pediatric patients on extracorporeal life support: the role of multidisciplinary surveillance international registry report of the extracorporeal life support organization extracorporeal membrane oxygenation neurodevelopmental outcome of infants supported with extracorporeal membrane oxygenation after cardiac surgery ecmo in the newborn pediatric surgery how low can you go? effectiveness and safety of extracorporeal membrane oxygenation in lowbirth-weight neonates key: cord-015941-4fz79wzf authors: hu, yuan title: molecular techniques for blood and blood product screening date: 2018-11-10 journal: advanced techniques in diagnostic microbiology doi: 10.1007/978-3-319-95111-9_2 sha: doc_id: 15941 cord_uid: 4fz79wzf blood product safety is a high priority for manufacturing industries, hospitals, and regulatory agencies. an important step in ensuring safety is the screening of donated blood for infectious diseases. molecular technologies for screening infectious diseases have improved remarkably over the years. molecular biological assay significantly reduced the risk of transfusion-transmitted infections. unlike previous methods, molecular technologies for screening infectious diseases are specific, efficient, easy to use, and economical. a new era in molecular biology is coming to the field of blood safety. direct detection of viral antigens and virus-specific antibodies has been a common tool for the diagnosis of virus infections in the past 40 years. there are some limitations. for direct detection of virus antigens, shortly after virus infection, only a few viruses release antigens in amounts sufficiently detectable in the body by an antibody-mediated assay. for indirect virus detection by virus-specific antibodies [e.g., an immunofluorescence assay or enzyme immunoassay (eia), etc.], there is a problem in that shortly after infection by a pathogenic virus, and there is a window period in which antibody generation is insufficient for detection [4] . to reduce this window period of low detection, direct nucleic acid tests are needed. through the application of molecular biology, biological and biochemical analyses have been revolutionized, and nucleic acid, gene-based techniques have been developed to screen blood and plasma donations for evidence of very recent and earlier viral infections that might otherwise be missed by conventional serologic testing. the nucleic acid tests can also provide evidence for genetic variation in viruses. molecular methods include the use of nucleic acid probes as well as amplification-based and dna sequence-based techniques. an increasing number of molecular diagnostic methods are now available commercially [2] . in comparison to classical methods, molecular biological methods are superior in terms of rapidness, specificity, and sensitivity. the current nucleic acid detection methods in the field may be grouped into two major classes: amplifying techniques such as pcr and non-amplifying techniques such as southern blot hybridization. amplifying techniques are more sensitive than non-amplifying techniques. there are two different types of amplifying methods [5] , target amplification methods and signal amplification methods. target amplifying techniques include pcr, nucleic acid sequence-based amplification (nasba) [6, 7] , self-sustaining sequence amplification (3sr), transcription-based amplification (tas), transcription-mediated amplification (tma), strand displacement amplification (sda), and ligase chain reaction (lcr). signal amplification methods include branched dna signal amplification (bdna) [8] , cleavage-based signal amplification (cycling probe technologies and invader assay), qß replicase, hybrid capture, cycling probe technologies (cpt), and rolling-circle amplification (rca) [9] . to further insure the safety of blood products, it is of importance to further improve these and other types of nucleic acid testing [2] [3] [4] [5] . southern blotting [10] was named after edward m. southern who developed this procedure at edinburgh university in the 1970s. this technique is used to detect specific sequences within mixtures of dna, which is size fractionated by gel electrophoresis and then transferred by capillary action to a suitable membrane. after blocking of non-specific binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with an oligonucleotide probe representing the specific dna sequence of interest. should specific dna be present on the blot, it will combine with the labeled probe and be detectable. in 1983, dr. kary mullis at cetus corporation conceived of polymerase chain reaction [11] . there is not a single technique that has had a greater impact on the practice of molecular biology than pcr. with this technique, we can detect infectious disease agents at an extremely low level. it is based on the ability of sense and antisense dna primers to hybridize to a dna of interest. following extension from the primers on the dna template by dna polymerase, the reaction is heat denatured and allowed to anneal with the primers once again. another round of extension leads to a multiplicative increase in dna products. therefore, a minute amount of dna can be efficiently amplified in an exponential fashion to result in larger amounts of dna that are more easily manipulated. by including critical controls, the technique can be made quantitative. the current level of the sensitivity and detection limit is as low as 10-50 copies per ml blood in hiv testing [1, 12, 13] . important clinical examples of the use of pcr are detection of hiv and hcv [14] [15] [16] . pcr techniques have evolved into different branches. some of them are now widely in use for virus detection in clinical diagnostics. these are real-time pcr by taqman (roche), lightcycler (roche), smartcycler (cepheid), in situ pcr, nested pcr, nested real-time pcr [17] , broad-range pcr, multiplex pcr, rt-pcr, arbitrarily primer pcr, long pcr, and quantitative pcr. real-time sequence technology will be coming soon for more detailed detection. in the past, identification of viral serotypes was restricted to investigative methods using antibody detection and restriction fragment length polymorphism (rflp). with real-time sequence technology, we will be able to detect a virus early as well as to obtain the viral sequence. microarrays were developed at stanford university by schena and co-workers in the early 1990s [18] . for medical applications, a microarray analysis offers a very accurate screening technology. it allows hundreds or thousands of nucleic acid hybridization reaction to be performed on a solid substrate. it promises to be a fast and accurate diagnostic tool in the field of clinical microbiology and virology. applied to infection safety for blood and blood products, it will be able to screen for the presence of viral pathogens by matching genetic sequences. compared with existing technologies, it allows for a wider variety of specific tests to be carried out simultaneously to determine the quality of the blood and will provide consumers with extra safety. with the use of molecular biology protocols, the microarray will permit the detection of lower concentrations of microorganisms in the blood and the accurate identification of many types of pathogenic contaminants. in the near future, progress can be expected in the application of microarray technology for screening of donated blood for infectious agents. it can provide vast information about the identity of blood-borne pathogens as well as their gene expression profiles [19] . to ensure a safe blood supply for those who may need a transfusion, an important step in ensuring safety is the screening of donated blood for infectious agents [20] . all of the above tests are referred to as screening tests and are designed to detect as many infectious agents as possible. because these tests are so sensitive, some donors may have a false-positive result, even when the donor has never been exposed to the particular infection. in order to sort out true infections from such false-positive test results, screening tests that are reactive may be followed up with more specific tests called confirmatory tests. thus, confirmatory tests help determine whether a donor is truly infected. if any one of these tests fails, affected blood products are considered unsuitable for transfusion [20] . nucleic acid testing (nat) employs testing technology that directly detects the genomes of viruses. because nat detects a virus's genetic material instead of waiting for the body's response, the formation of antibodies, as with many current tests, it offers the opportunity to reduce the window period during which an infecting agent is undetectable by traditional tests [21] , thus further improving blood safety. nucleic acid testing is becoming the gold standard because of greater sensitivity compared to antibody tests [2] . since 1999, nat has been approved by the fda and used to detect hiv-1 and hcv; this technology currently is under investigation for detecting other infectious disease agents. we know that for many viral infections, viral rna appears very early in the infection, in 1 to 2 weeks, but the antibody doesn't appear until 10-12 weeks, e.g., hiv and hcv [21] . in order to virtually prevent infection by all the transfusion-associated viruses, we need to detect the viruses in their window period, and a nat or gene-based testing method is needed. nat also provides an opportunity for the viral, e.g., hiv or hcv, infected donor to seek early treatment. on the other hand, nat is not only a sensitive method but also a rapid method, which is suitable for a blood bank laboratory because the turnaround time for maintaining blood donations is extremely critical. the hepatitis b virus (hbv) is a highly infectious and often non-symptomatic virus that is transmitted primarily through blood and blood-derived fluids and is a leading cause of liver infection worldwide [22] . the world health organization (who) estimates that 2 billion people worldwide have been infected with hbv and 350,000,000 people are chronically infected. chronic infection results in a high risk for liver cancer and cirrhosis of the liver, which cause about 1000,000 deaths each year. each year up to 200,000 people become newly infected in the united states alone. since screening for hbv began in 1969, the rate of infection through blood transfusions has greatly decreased. however, as of 2000, hbv is still transmitted through blood transfusions in 1 out of 137,000 units of blood. one reason for this is that currently available blood screening technologies detect core antibodies or surface antigens, which appear up to 8 weeks after infection. serologic tests for hepatitis b virus include hepatitis b surface antigen (hbsag) and hepatitis b core antibody (hbcab). hbv, which mainly infects the liver, has an inner core and an outer envelope (the surface). the hbsag test detects the outer envelope, identifying an individual infected with the hepatitis b virus. this virus can cause inflammation of the liver, and in the earliest stage of the disease, infected people may feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice. fortunately, most patients recover completely and test negative for hbsag within a few months after the illness. a small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. chronically infected people can develop severe liver disease as time passes and need to be followed carefully by an experienced physician. to reduce the occurrence of posttransfusion hepatitis, it is essential to screen all blood donations for hepatitis b surface antigen by the most sensitive and specific assays. blood donations that are found to be reactive in the hbsag test are automatically confirmed by the hbsag confirmatory assay. if the specimen is neutralizable in the confirmatory test, the specimen is considered positive for hbsag. hepatitis b surface antigen testing of donated blood has begun in 1975 (table 1) . currently, all blood donors are screened for hbsag, but occasional transmission of hbv still occurs due to the inclusion of window period donations (i.e., blood from recently infected donors who are antibody negative but still viremic). detection of early hbv infection of blood donors is still a major problem of blood transfusion. the current third-generation licensed hbsag tests (mostly radioimmunoassay and enzyme immunoassays) cannot detect hbv in the window period for hbv infection. this is a strong motivation for introducing molecular detection techniques to the field [23] . there are some commercially available test methods for detecting hbv dna in the market now, such as chiron's quantiplex hbv dna [24] , digene's hybrid capture, abbott's hbv dna assay, and roche's amplicor hbv monitor. using these commercial hybridization or pcr-based assays, hbv dna can be detected 1-3 weeks before the appearance of hbsag [25] . some chronically infected patients who have lost their hbsag remain hbv dna positive but are disqualified as potential blood donors. molecular detection of hbv dna is more sensitive than current methods employed for hbsag screening [22] [23] [24] [25] . determination of anti-hbc (total) is also used to monitor the progress of the hepatitis b viral infection. determination of anti-hbc (igm) is employed to distinguish an acute hepatitis b infection from a chronic infection. the anti-hbc test developed in 1987 detects an antibody to the hepatitis b virus that is produced during and after infection. if an individual has a positive anti-hbc test, but the hbsag test is negative, it may mean that the person once had hepatitis b but has recovered from the infection. of the individuals with a positive test for anti-hbc, many have not been exposed to the hepatitis b virus; thus, there is a frequent problem of false positives. although the individual may be permanently deferred from donating blood, it is unlikely that the person's health will be negatively affected. (note: this antibody is not produced following vaccination against hepatitis b [26] . the hepatitis c virus (hcv) is a member of the flaviviridae family of viruses, which are associated with both human and animal diseases [27] . hepatitis caused by hcv is the most common chronic blood-borne infection in the united states. over 4 million americans are believed to be infected. hcv can also be transmitted through blood transfusion. hcv causes inflammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver inflammation, cirrhosis, cancer, and death. eventually, up to 20% of people with hcv may develop cirrhosis of the liver or other severe liver diseases. as in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. because of the risk of serious illness, people with hcv need to be followed closely by a physician with experience evaluating this infection. since the full-length hcv cdna was first cloned in 1989, significant progress has been made in characterizing its molecular biology [13] . but, the natural history of hcv infection is still evolving, and current treatment options for patients are either limited or expensive [27] . there is no vaccine for hcv, and the current treatment includes a combination of alpha interferon and ribavirin as well as the combination of the nucleotide polymerase inhibitor sofosbuvir and the ns5a inhibitor velpatasvir [28] . although the former is efficacious in only a minority of patients [29] , the latter has been shown to be effective in a broad range of patients [28] . the life cycle of the hcv continues to be poorly understood due to the lack of an efficient cell culture system [30] . there is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hepatitis c virus. we recently established a cell culture system for the replication of hcv by using human t and b leukemia cell lines [31] . this model should represent a valuable tool for the detailed study of the initial steps of the hcv replication cycle and for the evaluation of evolving antiviral molecules. currently, appropriate vaccine strategies for hcv have not been developed. early detection and prevention of hcv infection are most important for blood safety. it is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the hcv genome because of a high mutation rate in this genomic region. however, the untranslated region of about 341 nucleotides contains highly conserved domains which allows for stable primer design for qualitative and quantitative diagnostic tests which have equivalent sensitivity against the known six various genotypes of hcv. in 1990, the first specific test for hepatitis c virus, the major cause of "non-a, non-b" hepatitis, was introduced. now, a third-generation elisa kit is available to detect antibodies to hcv, and screening blood for hcv antibodies is recommended. these assays are based on detection of serum antibody to various hcv antigens because these antibodies are nearly universally present in patients who are chronically infected with hcv [32] . the hcv screening tests are known to have significant limitations, and positive samples should be further tested by hcv confirmatory tests. guidelines provided by the cdc recommend that hcv antibody screening testpositive samples should be confirmed with serologic or nucleic acid supplemental testing. hcv confirmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient's serum. in this way, the response to individual antigens can be recognized, and some false-positive elisa results can be eliminated (e.g., riba, chiron hcv 3.0, and pcr assay) (e.g., roche cobas amplicor hcv test, version 2.0). laboratories can choose to perform this testing on all positive specimens or based on screening test-positive (signal to cutoff) ratios. the positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened. hcv antibodies are not generally detectable for at least 6 weeks and may not appear for several months. acute hcv infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. high-sensitivity detection of hcv during the window period is a long-term technical challenge in the field. tests for hcv rna genome detection based on the pcr or other highly sensitive rna detection systems have been used for the diagnosis of acute hepatitis [26] . sensitive detection of hcv rna based on rt-pcr or other nucleic acid amplification techniques can be readily accomplished with kits that are now available commercially. for example, in 1999 the fdaapproved roche's amplicor hiv-1 monitor ultrasensitive quantitative assay. it can measure hiv levels at as few as 50 copies/ml, and another commercial kit, the lcx hiv rna quantitative assay from abbott laboratories, also has a detection limit at 50 copies/ml. some studies even showed a sensitivity limit at one copy [33] . in fact, a qualitative assay should be much more sensitive than a quantitative assay for hiv/ hcv screening. a sensitive qualitative hcv molecular detection assay will possibly interdict and virtually prevent all transfusion-associated hiv/hcv. the current sensitivity standard for clinical diagnostics is 100 copies per ml, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies per ml. hiv-1 and/or hiv-2 virus cause acquired immunodeficiency syndrome or aids. the test is designed to detect antibodies directed against antigens of the hiv-1 or hiv-2 viruses. hiv-1 is much more common in the united states, whereas hiv-2 is prevalent in western africa. donors are tested for both viruses because both are transmitted by infected blood, and a few cases of hiv-2 have been identified in us residents. in 1985, the first blood screening eia test to detect hiv was licensed and quickly implemented by blood banks to protect the blood supply. in 1992, testing of donor blood for both hiv-1 and hiv-2 antibodies (anti-hiv-1 and anti-hiv-2) was implemented. in 1996, hiv p24 antigen testing of donated blood was mandated. now, the p24 antigen testing is going to be compared with a pcr-based test for their ability to detect hiv in the window period. htlv retroviruses are endemic in japan and the caribbean but relatively uncommon in the united states [34] . they cause adult t-cell leukemia/lymphoma and a neurological disorder similar to multiple sclerosis. the infection can persist for a lifetime but rarely causes major illnesses in most people who are infected. in rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. htlv-ii infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. disease associations with htlv-ii have been hard to confirm, but the virus may cause subtle abnormalities of immunity that lead to frequent infections or rare cases of neurological disease. in 1989, human t-lymphotropic virus antibody testing of donated blood was begun. blood is now routinely screened for antibodies to htlv-i/ii. these test screens for antibodies directed against epitopes of the htlv-i and htlv-ii viruses. several commercial assays based on the enzyme-linked immunosorbent assay (elisa) or particle agglutination formats are used for screening of htlv antibodies, followed by confirmatory assays using western blotting. in some infected individuals, the serologic response to htlv infection is very low. these problems have been solved by the application of pcr amplification of specific sequences in the virus genome. pcr can be used to detect htlv-i/ii proviruses and is now the method of choice for detection of htlv dna directly from blood and many other tissues. commercial pcr kits for htlv are available [34] . the west nile virus (wnv) is a single-stranded rna virus of the flaviviridae family and is one of the most recent emerging infectious disease threats to public health and, potentially, to the safety of our blood supply [35] [36] [37] . in 2002, wnv was identified as transfusion transmissible. it is transmitted by mosquitoes to birds and other animals through a mosquito bite. the virus can infect people, horses, many types of birds, and some other animals. wnv was shown in 2002 to be transmissible by blood [35] , with an estimated mean risk of 2/10,000-5/10,000 in outbreak regions in the united states. the most common symptoms of transfusion-transmitted cases of wnv were fever and headache. detection of wnv includes either a measurement of wnv antibodies or of wnv nucleic acid (detecting genetic material from the virus itself). there are two types of wnv antibody testing: igm and igg. in most individuals, igm antibodies will be present within 8 days after the initial exposure to wnv, followed by igg production several weeks later. but, the antibodies tested to detect wnv are not expedient for donor blood screening. nucleic acid testing involves amplifying and measuring the west nile virus's genetic material to detect the presence of the virus in the blood or tissue. wnv nat will be negative in the blood once clinical illness has occurred. in this situation, both nat and igm antibody testing may be needed. nucleic acid tests to screen blood for wnv are commercially available and in current use. but, the viral yield for wnv infection is much lower than other viruses. consequently, a more sensitive wnv nat system for donor blood screening will be required, which could further reduce the risks of transfusiontransmitted wnv. serum samples from all blood units should be subjected to either the vdrl (venereal disease research laboratory) test or a treponemal test, such as the treponema pallidum hemagglutination (tpha) test before transfusion. any unit found positive should be discarded as per standard safety procedures. this test is done to detect evidence of infection with the spirochete that causes syphilis. blood centers began testing for this shortly after world war ii, when syphilis rates in the general population were much higher. the risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in this country for many years) because the infection is very rare in blood donors and because the spirochete is fragile and unlikely to survive blood storage conditions. sensitivity and specificity of serologic tests vary depending on the type of test performed and the stage of the disease. if the donor has spirochetemia, their serologic tests are usually negative, and if the donors are antibody positive, their blood is not infectious. syphilis serological tests for donors have less clinical significance. a nucleic acid test for accurately detecting syphilis is needed. it can be used to determine whether a blood donor is currently or has recently been infected with the spirochete. in recent years, numerous infectious agents found worldwide have been identified as potential threats to the blood supply, and among these are several newly discovered hepatitis viruses that present unique challenges in assessing possible risks. even if the hepatitis virus test is negative for all known a-e hepatitis agents, there are some unidentified hepatitis viruses, called non-a-e hepatitis viruses that can still be transmitted by blood transfusion. in the future, advances in nat may allow rapid discovery of the unknown hepatitis viruses. hepatitis delta virus (hdv) is a small rna virus that can infect only individuals who have hbv; worldwide more than 15 million people are coinfected [38] . hdv is clinically important because it generally makes hbv infections more damaging to the liver. increased understanding of the molecular virology of hdv will identify novel therapeutic targets for this most severe form of chronic viral hepatitis. pcr and real-time pcr methods are available for hdv rna detection [39] . tt virus (ttv) [40] , named for the patient from whom it was first isolated with non-a-e and g posttransfusion hepatitis in japan in 1997, is a newly discovered transfusion-transmitted, single-stranded and circular dna virus [41] . ttv is nonenveloped, and its entire sequence of ~3.9 kb has been determined. it is also often interpreted as a transfusion-transmitted virus [42] . at least 16 genotypes have been identified, and ttv is now found all over the world. ttv infection was sought by detection of ttv dna in serum by polymerase chain reaction using primers generated from a conserved region of the ttv genome, e.g., the utr region [42] . donor blood and blood product can be screened for ttv dna by using pcr or real-time pcr. the significance of positive findings is still unclear, because high-level ttv carriers in healthy populations are currently found [42] [43] [44] . whether ttv actually causes hepatitis remains to be determined. cytomegalovirus (cmv) is a virus belonging to the herpes group that is rarely transmitted by blood transfusion. donor blood is not routinely tested for cmv, and the prevalence of cmv antibody ranges from 50 to 80% of the population. but, blood contaminated with cmv can cause problems in neonates or immunocompromised patients. it also remains a major pathogen for solid-organ transplant recipients causing febrile syndromes, hepatitis, pneumonitis, retinitis, and colitis. potential problems in selected patient populations can be prevented by transfusing cmv negative blood or frozen, deglycerolized red blood cells. serologic tests for antibody to cmv are useful for determining whether a patient had cmv infection in the past, a determination of great clinical importance for organ and blood donors and in the pretransplant evaluation of prospective transplant recipients [45] . commercial nat kits are available for cmv [5] , and these include the amplicor pcr cmv monitor test and hybrid capture system cmv dna test. chagas disease is caused by the blood-borne parasite, trypanosoma cruzi, which is transmitted to humans through insects. in the united states, chagas disease is considered one of the neglected parasitic infections, a group of five parasitic diseases that have been targeted by cdc for public health action [46] . commercial anti-t. cruzi assay kits are available for the qualitative detection of antibodies, trypanosoma cruzi (t. cruzi), the causative agent of chagas disease in human serum and plasma specimens by abbott diagnostics [47] . sensitive screening tests for malaria are neither commercially available nor officially approved yet. the most effective way of screening donors is to take a proper history of malaria or of fever that could be due to malaria [48] . donor selection criteria should be designed to exclude potentially infectious individuals from donating red blood cells for transfusion. because there are no practical laboratory tests available to test donor blood, donors traveling to high-risk malaria areas are excluded from donating blood for 6 months. however, there is a need to develop suitable screening tests, especially for use in an endemic area. a number of clinical research approaches have been developed for the extraction, amplification, and detection of malaria parasite dna from blood products [49] . variant creutzfeldt-jakob disease [50] (vcjd, a rare but fatal brain infection) was first described in 1996 in the united kingdom. vcjd is strongly linked with exposure to the bovine spongiform encephalopathy (bse) agent. bse is a transmissible spongiform encephalopathy (tse) affecting cattle and was first reported in the united kingdom in 1986. it has different clinical and pathologic characteristics from classic cjd. each disease also has a particular genetic profile of the prion protein gene [51] . in recent years, questions have been raised concerning the potential risk of variant creutzfeldt-jakob disease for recipients of plasma-derived clotting factors, including the united states licensed factor viii (pdfviii), factor ix (pdfix), and other plasma-derived products such as immune globulins and albumin. in the past 10 years, there have been some reported cases of probable variant creutzfeldt-jakob disease (vcjd) transmission by red blood cell transfusions in the united kingdom [52] . prion infections are associated with long and clinically silent incubations [50, 51] . the number of asymptomatic individuals with vcjd prion infection is unknown, posing risk to others through blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. in order to decrease the risk, there is a need to establish a blood-based molecular assay for detection of vcjd prion infection [52] . recently research papers have shown that sensitivity detection methods are available for vcjd prion [53] [54] [55] . however, commercial detection kits are not yet available. the dengue virus (denv) is a member of the virus family flaviviridae and is transmitted to people through the bite of an infected mosquito [56] . the dengue virus has been shown to have four subtypes. these subtypes are different strains of dengue virus that have 60-80% homology between each other. dengue has emerged as a worldwide problem only since the 1950s. with more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. according to cdc, as many as 100 million people are infected yearly [57] . dengue is caused by any one of the four related viruses transmitted by mosquitoes. there are not yet any vaccines to prevent denv infection, and the most effective protective measure is to avoid mosquito bites. there have been healthcare-related transmissions, including transmission by blood products [58] . dengue infection has a viremic phase that lasts 4-8 days, and blood collected during this phase may be infective when transfused into susceptible hosts [58] . there are currently no tests for direct detection of dengue virus, but there are, however, commercial elisa tests to detect antibodies of the dengue virus in blood samples from patients [59] . recently, research papers have shown that pcr detection methods are available for any dengue virus strain [57, 60] . babesia is a protozoan parasite of the blood that causes a hemolytic disease known as babesiosis [61] . babesiosis is a malaria-like parasitic disease [62] , and there are over 100 species of babesia identified [63] . in the united states, babesia microti is the agent most commonly reported to cause human infection. clinical confusion between human babesiosis and malaria is often reported in literature [62] . babesia infection can also be acquired by blood transfusion [64, 65] . in fact, there have been many cases of transfusion-induced babesiosis documented [64, 65] . risk of developing this clinical infection is increased for elderly, asplenic, or immunosuppressed patients. current standards issued by the american association of blood banks (aabb) require the indefinite deferral of a blood donor with a history of babesiosis [65] . there is a need to develop methods for identification babesia microti in order to reduce the risk of transmission of babesiosis by transfusion. diagnosis depends upon finding parasites on blood film examination which can be detected 2-4 weeks after a tick bite. hamster inoculation and serology have also been used for diagnosis. the indirect fluorescent antibody test (ifat) is available for b. microti and is the most useful serological test for early diagnosis [66] . also, the pcr screen tests for babesiosis are technically available in the field [67] . chagas disease is named after the brazilian physician carlos chagas, who discovered the disease in 1909 [68] . chagas disease is spread mainly by blood-sucking insects infected with trypanosoma cruzi. chagas disease can also be spread through blood transfusion, organ transplants, and from a mother to an unborn child. national screening of the blood supply [69] was instituted in early 2007 by fda, and more than 1000 donors with t. cruzi infection have been identified within the past 3 years of testing. "screening for t. cruzi is an important safety measure to help protect our blood supply and to help prevent the spread of chagas disease," says karen midthun, m.d., acting director of the fda's center for biologics evaluation and research. currently, serological elisa tests are available to diagnose chronic chagas disease [70] . pcr test is not a tool for diagnosis of chronic chagas disease in clinical practice yet, although some research results have showed that pcr is a very sensitive parasitological test for chagas disease in active transmission regions [71] . more studies are needed for the development of this molecular method. coronavirus is an rna virus known to be associated with respiratory disease. severe acute respiratory syndrome (sars) is a newly recognized coronavirus whose genome sequence does not belong to any of the known coronavirus groups and which quickly spread all over the world from asia in 2003. there has been no evidence that this infection is transmitted from blood donors to transfusion recipients, but the virus associated with sars is present in the blood of people who are sick, and it is possible that the virus could be present in the blood immediately before a person gets sick, so that an individual with infection but no symptoms possibly could transmit sars through a blood donation. to help determine whether or not an individual might be infected with sars, a blood collection facility will ask a potential donor orally or in writing about any travel to a sars-affected country or a history of sars or possible exposure to sars. enzyme-linked immunoassays for detection of specific igg and igm antibodies and rt-pcr for detection of sars coronavirus-specific rna in the sars patients have been developed. rapid, sensitive, and specific identification of sars and other novel coronaviruses by molecular methods will be very important in the future. ebola virus disease (evd) is a rare and deadly disease caused by infection with one of the ebolavirus species [72] . the recent outbreak in 2014-2015 is the largest ebola outbreak since the ebola virus was first discovered in 1976, first in yambuku, democratic republic of congo, and then in nzara, south sudan. the virus family filoviridae includes three genera: cuevavirus, marburgvirus, and ebolavirus. there are five species that have been identified: zaire, bundibugyo, sudan, reston, and taï forest. the first three, bundibugyo ebolavirus, zaire ebolavirus, and sudan ebolavirus, have been associated with large outbreaks in africa. the virus responsible for causing the 2014 west african outbreak belongs to the zaire species (who) [72] . samples from patients are an extreme biohazard risk. currently, a number of approaches have been developed and are available for diagnoses of ebola virus disease: (1) antibody-capture enzyme-linked immunosorbent assay (elisa), (2) antigen-capture detection tests, (3) serum neutralization test, (4) reverse transcriptase polymerase chain reaction (rt-pcr) assay, (5) electron microscopy, and (6) virus isolation by cell culture tests. the who and fda are working to help expedite the development and availability of medical products -such as treatments, vaccines, diagnostic tests, and personal protective equipment -with the potential to help bring the ebola epidemic in west africa under control as quickly as possible (fda) [73] . based on past history, it is not just a hypothetical risk that many people have been infected with unrecognized viruses, for example, many patients with symptoms of non-a-e, g, and ttv posttransfusion hepatitis. it is still possible that unexplained cases of posttransfusion hepatitis may be caused by a new, undiscovered pathogen. in recent years, numerous new infectious agents found worldwide have been identified through time-consuming procedures. by the time a new virus, such as hcv, hiv, and sars, is found, many people are infected, and there could be a large number of fatalities. there is an urgent need to develop methods for rapid identification and characterization of previously unknown pathogenic viruses. the most recent technologies for detecting and identifying previously unrecognized pathogens are expression library screening, representational difference analysis (rda), and broad-range polymerase chain reaction (br-pcr). but they are all time-consuming approaches. the new unrecognized and uncharacterized viral agents can be rapidly identified by some of the new molecular approaches, e.g., subtraction hybridization [74] and dna microarray. ensuring the safety and efficacy of blood and blood products is a critical regulatory challenge. the high safety level of the blood supply is the result of continued improvements in blood donor screening and testing. it will be achieved by introducing more updated nucleic acid tests to the field of blood safety [2, 3] . nucleic acid testing is a method of testing blood that is more sensitive and specific than conventional tests that require the presence of antibodies to trigger a positive test result. also, nat works by detecting the low levels of viral genetic material present when an infection occurs but before the body develops an immune response to a virus. this improved sensitivity should enable us to significantly decrease the infection window period, allowing for earlier detection of the infection and diminishing the chances for transmission of the agent via transfusion. we are not only to protect the blood supply from known pathogens but also the emergence of new and unrecognized and uncharacterized infectious agents [4, 36, 37, 75] . the nat methods are more sensitive and specific compared with non-nat. in the future, nat technology, such as pcr, may allow routine screening of donors for all the known and unknown pathogens of concern to blood safety. progress in blood supply safety advances in testing technology to ensure transfusion safety -nat and beyond the safety of the blood supply -time to raise the bar emerging infectious disease issues in blood safety in vitro nucleic acid amplification techniques evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (nasba) molecular-based methods for quantifying hiv viral load molecular diagnostics of infectious diseases 50 years of the double helix: from the concept of molecular hybridization to microarrays in vitro nucleic acid amplification techniques. diagnostic molecular microbiology principles and applications. mayo foundation multicenter evaluation of the performance characteristics of the nuclisens hiv-1 qt assay used for quantitation of human immunodeficiency virus type 1 rna serological and molecular biology screening techniques for hcv infection clinical evaluation of an hiv-1 and hcv assay and demonstration of significant reduction of the hcv detection window before seroconversion roche amplicor hiv-1 monitor ultrasensitive quantitative assay quantitative assay from abbott laboratories nested real-time pcr for hepatitis a detection introduction to microarray analysis. microarray analysis development and validation of a diagnostic dna microarray to detect quinolone-resistant escherichia coli among clinical isolates food & drug administration. testing requirements for communicable disease agents detection of hiv-1 and hcv infections among antibody-negative blood donors by nucleic acid-amplification testing update on hepatitis b virus infection challenges in hepatitis b detection among blood donors assessment of hepatitis b virus dna stability in serum by the chiron quantiplex branched-dna assay hepatitis b virus. section two: specific virus families immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infection control practices advisory committee (hicpac) hepatitis c virus: virology, diagnosis and treatment sofosbuvir and velpatasvir for hcb genotype 1, 2, 4, 5, and 6 infection recent advances in prevention and treatment of hepatitis c virus infections the scientific challenge of hepatitis c detection of extrahepatic hcv replication by a novel highly sensitive single tube nested-pcr chapter 34: hepatitis c viruses. section two: specific virus families new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunodeficiency virus type 1 rna in plasma chapter 58: human t-cell leukemia virus types 1 and 2. section two: specific virus families estimated risk of transmission of the west nile virus through blood transfusion in the us transfusion-transmitted emerging infectious diseases: 30 years of challenges and progress the potential treat to blood transfusion safety of emerging infectious disease agents hepatitis delta virus. the lancet, early online publication quantification of hepatitis delta virus rna in serum by consensus real-time pcr indicates different patterns of virological response to interferon therapy in chronically infected patients ttv-a virus searching for a disease a novel unenveloped dna virus (tt virus) associated with acute and chronic non-a to g hepatitis role of transfusion-transmitted virus in acute viral hepatitis and fulminant hepatic failure of unknown etiology a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology chapter 77: cytomegalovirus. section two: specific virus families evaluation of a prototype trypanosoma cruzi antibody assay with recombinant antigens on a fully automated chemiluminescence analyzer for blood donor screening transfusion-transmitted malaria in countries where malaria is endemic: a review of the literature from sub-saharan africa detection and species identification of malaria parasites by isothermal thda amplification directly from human blood without sample preparation 18 years of research and surveillance prions: beyond a single protein crerutzfeldt-jakob disease and blood transfusion: updated results of the uk transfusion medicine epidemiology review study detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay preclinical detection of variant cjd and bse prions in blood quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus threat of dengue to blood safety in dengueendemic countries comparison of a commercial igm capture elisa with dengue antigen focus reduction microneutralization test and the centers for disease control dengue igm capture-elisa pcr detection of nearly any dengue virus strain using a highly sensitive primer cocktail do babesiosis and malaria share a common disease process? babesia: a world emerging babesia infection through blood transfusions: reports received by the us food and drug administration transfusion-transmitted babesia spp.: bull's-eye on babesia microti continuous in vivo culture and indirect fluorescent antibody test for zoonotic protozoa of babesia microti detection of babesia species from infected dog blood by polymerase chain reaction silver spring, m.d. 20993. complete list of donor screening assays for infectious agents and hiv diagnostic assays elisa versus pcr for diagnosis of chronic chagas disease: systematic review and meta-analysis pcr-based diagnosis for chagas' disease in bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis virus disease updated ebola response updates from fda rapid approach to identify an unrecognized viral agent emerging infectious disease agents and their potential threat to transfusion safety key: cord-017948-fqhl1qb4 authors: hu, yuan title: molecular techniques for blood and blood product screening date: 2012-04-05 journal: advanced techniques in diagnostic microbiology doi: 10.1007/978-1-4614-3970-7_28 sha: doc_id: 17948 cord_uid: fqhl1qb4 the food and drug administration (fda) is responsible for ensuring the safety of the more than 15 million units of blood and blood components donated each year in the united states. “blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries,” said david a. kessler, md, former fda commissioner [1]. screening donated blood for infectious diseases that can be transmitted through blood transfusion is a very important step in ensuring safety. the united states has the safest blood supply in the world [1] and the fda is striving to keep it safe by decreasing the risk of infectious disease transmission. the regulatory agency is continuously updating its requirements and standards for collecting and processing blood. as mentioned earlier, an important step in ensuring safety is the screening of donated blood for infectious diseases. in the united states, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (table 28.1). the field of clinical microbiology and virology are now focusing on molecular technology. currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests. it is time for all blood safety procedures to include molecular detection techniques. this approach can significantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. this chapter reviews the current antigen/antibody-based technology, molecular biological technology, and published regulatory policy data for blood safety. the food and drug administration (fda) is responsible for ensuring the safety of the more than 15 million units of blood and blood components donated each year in the united states. "blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries," said david a. kessler, md, former fda commissioner [ 1 ] . screening donated blood for infectious diseases that can be transmitted through blood transfusion is a very important step in ensuring safety. the united states has the safest blood supply in the world [ 1 ] and the fda is striving to keep it safe by decreasing the risk of infectious disease transmission. the regulatory agency is continuously updating its requirements and standards for collecting and processing blood. as mentioned earlier, an important step in ensuring safety is the screening of donated blood for infectious diseases. in the united states, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (table 28 .1 ). the fi eld of clinical microbiology and virology are now focusing on molecular technology. currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests. it is time for all blood safety procedures to include molecular detection techniques. y. hu (*) u.s. food and drug administration, northeast regional laboratory , 158-15 liberty avenue , jamaica , ny 11433 , usa e-mail: yuan.hu@fda.hhs.gov no of fi cial support or endorsement of this article by the food and drug administration is intended or should be inferred. this approach can signi fi cantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. this chapter reviews the current antigen/antibody-based technology, molecular biological technology, and published regulatory policy data for blood safety. direct detection of viral antigens and virus speci fi c antibodies has been a common tool for the diagnosis of virus infections in the past 40 years. there are some limitations. for direct detection of virus antigens, shortly after virus infection, only a few viruses release antigens in amounts suf fi ciently detectable in the body by an antibodymediated assay. for indirect virus detection by virus speci fi c antibodies (e.g., an immuno fl uorescence assay or enzyme immunoassay (eia), etc.), there is a problem in that shortly after infection by a pathogenic virus, there is a window period in which antibody generation is insuf fi cient for detection [ 2 ] . to reduce this window period of low detection, direct nucleic acid tests are needed. through the application of molecular biology, biological and biochemical analyses have been revolutionized, and nucleic acid, gene-based techniques have been developed to screen blood and plasma donations for evidence of very recent and earlier viral infections that might otherwise be missed by conventional serologic testing. the nucleic acid tests can also provide evidence for genetic variation in viruses. molecular methods include the use of nucleic acid probes as well as ampli fi cation-based and dna sequence-based techniques. an increasing number of molecular diagnostic methods are now available commercially. in comparison to classical methods, molecular biological methods are superior in terms of rapidness, speci fi city, and sensitivity. the current nucleic acid detection methods in the fi eld may be grouped into two major classes: amplifying techniques such as pcr and nonamplifying techniques such as southern blot hybridization. amplifying techniques are more sensitive than nonamplifying techniques. there are two different types of amplifying methods [ 3 ] , target ampli fi cation methods and signal ampli fi cation methods. target amplifying techniques include pcr, nucleic acid sequence-based ampli fi cation (nasba) [ 4, 5 ] , self-sustaining sequence ampli fi cation (3sr), transcription-based ampli fi cation (tas), transcription-mediated ampli fi cation (tma), strand displacement ampli fi cation (sda), and ligase chain reaction (lcr). signal ampli fi cation methods include branched dna (bdna) signal ampli fi cation [ 6 ] , cleavage-based signal ampli fi cation (cycling probe technologies and invader assay), qß replicase, hybrid capture, cycling probe technologies (cpt), and rolling-circle ampli fi cation (rca) [ 7 ] . to further insure the safety of blood products, it is of importance to further improve these and other types of nucleic acid testing. southern blotting [ 8 ] was named after edward m. southern who developed this procedure at edinburgh university in the 1970s. this technique is used to detect speci fi c sequences within mixtures of dna, which is size-fractionated by gel electrophoresis and then transferred by capillary action to a suitable membrane. after blocking of nonspeci fi c binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with an oligonucleotide probe representing the speci fi c dna sequence of interest. should speci fi c dna be present on the blot, it will combine with the labeled probe and be detectable. by coelectrophoresing dna fragments of known molecular weight, the size(s) of the hybridizing band(s) can then be determined. southern blotting hybridization technology is one of the major tools that have already helped clinical staffs worldwide interpret genomic information. other competing methodologies include in situ hybridization and solution hybridization. important clinical examples of the use of this technology are dna fi ngerprinting and the ability to detect dna gene rearrangements. in 1983, dr. kary mullis at cetus corporation conceived of polymerase chain reaction [ 9 ] . there is not a single technique that has had a greater impact on the practice of molecular biology than pcr. with this technique, we can detect infectious diseases agents at an extremely low level. it is based on the ability of sense and antisense dna primers to hybridize to a dna of interest. following extension from the primers on the dna template by dna polymerase, the reaction is heat-denatured and allowed to anneal with the primers once again. another round of extension leads to a multiplicative increase in dna products. therefore, a minute amount of dna can be ef fi ciently ampli fi ed in an exponential fashion to result in larger amounts of dna that are more easily manipulated. by including critical controls, the technique can be made quantitative. the current level of the sensitivity and detection limit is as low as 10-50 copies per ml blood in hiv testing [ 1, 10, 11 ] . important clinical examples of the use of pcr are detection of hiv and hcv [12] [13] [14] . pcr techniques have evolved into different branches. some of them are now widely in use for virus detection in clinical diagnostics. these are real-time pcr by taqman (roche), light cycler (roche) and smart cycler (cepheid), and in situ pcr, nested-pcr, nested-real time pcr [ 15 ] , broad-range pcr, multiplex pcr, rt-pcr, arbitrarily primer pcr, long pcr, and quantitative pcr. real-time sequence technology will be coming soon for more detailed detection. in the past, identi fi cation of viral serotypes was restricted to investigative methods using antibody detection and restriction fragment length polymorphism (rflp). with real-time sequences technology, we will be able to detect a virus early as well as to obtain the viral sequence. microarrays were developed at stanford university by schena and coworkers in the early 1990s [ 16 ] . for medical applications, a microarray analysis offers a very accurate screening technology. it allows hundreds or thousands of nucleic acid hybridization reaction to be performed on a solid substrate. it promises to be a fast and accurate diagnostic tool in the fi eld of clinical microbiology and virology. applied to infection safety for blood and blood products, it will be able to screen for the presence of viral pathogens by matching genetic sequences. compared with existing technologies, it allows for a wider variety of speci fi c tests to be carried out simultaneously to determine the quality of the blood and will provide consumers with extra safety. with the use of molecular biology protocols, the microarray will permit the detection of lower concentrations of microorganisms in the blood and the accurate identi fi cation of many types of pathogenic contaminants. in the near future, progress can be expected in the application of microarray technology for screening of donated blood for infectious agents. it can provide vast information about the identity of bloodborne pathogens as well as their gene expression pro fi les [ 17 ] . to ensure a safe blood supply for those who may need a transfusion, an important step in ensuring safety is the screening of donated blood for infectious agents. after donation, each unit of donated blood undergoes a series of tests for bloodborne agents such as human immunode fi ciency virus (hiv)-1, hiv-2, hepatitis b virus (hbv), hepatitis c virus (hcv), human t-cell lymphotropic virus (htlv)-1 and htlv-ii, west nile virus (wnv), and treponema pallidum , the agent of syphilis. all of the above tests are referred to as screening tests, and are designed to detect as many infectious agents as possible. because these tests are so sensitive, some donors may have a false-positive result, even when the donor has never been exposed to the particular infection. in order to sort out true infections from such false-positive test results, screening tests that are reactive may be followed up with more speci fi c tests called con fi rmatory tests. thus, con fi rmatory tests help determine whether a donor is truly infected. if any one of these tests fails, affected blood products are considered unsuitable for transfusion [ 18 ] . nucleic acid testing (nat) employs testing technology that directly detects the genomes of viruses. because nat detects a virus's genetic material instead of waiting for the body's response, the formation of antibodies, as with many current tests, it offers the opportunity to reduce the window period during which an infecting agent is undetectable by traditional tests [ 19 ] , thus further improving blood safety. nat will become the gold standard because of greater sensitivity compared to antibody tests. since 1999, nat has been approved by the fda and used to detect hiv-1 and hcv; this technology currently is under investigation for detecting other infectious disease agents. we know that for many viral infections, viral rna appears very early in the infection, in 1-2 weeks, but the antibody does not appear until 10-12 weeks, e.g., hiv and hcv [ 20 ] . in order to virtually prevent infection by all the transfusion associated viruses, we need to detect the viruses in their window period, and a nat or gene-based testing method is needed. nat also provides an opportunity for the viral, e.g., hiv or hcv, infected donor to seek early treatment. on the other hand, nat is not only a sensitive method, but also a rapid method which is suitable for a blood bank laboratory because the turnaround time for maintaining blood donations is extremely critical. the hbv is a highly infectious and often nonsymptomatic virus that is transmitted primarily through blood and blood-derived fl uids and is a leading cause of liver infection worldwide. the world health organization (who) estimates that two billion people worldwide have been infected with hbv and 350,000,000 people are chronically infected. chronic infection results in a high risk for liver cancer and cirrhosis of the liver, which cause about 1,000,000 deaths each year. each year up to 200,000 people become newly infected in the united states alone. since the beginning of screening for hbv in 1969, the rate of infection through blood transfusions has greatly decreased. however, as of 2000, hbv is still transmitted through blood transfusions in 1 out of 137,000 units of blood. one reason for this is that currently available blood screening technologies detect core antibodies or surface antigens, which appear up to 8 weeks after infection. serologic tests for hbv include hepatitis b surface antigen (hbsag) and hepatitis b core antibody (hbcab). hbv, which mainly infects the liver, has an inner core and an outer envelope (the surface). the hbsag test detects the outer envelope, identifying an individual infected with the hbv. this virus can cause in fl ammation of the liver, and in the earliest stage of the disease, infected people may feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice. fortunately, most patients recover completely and test negative for hbsag within a few months after the illness. a small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. chronically infected people can develop severe liver disease as time passes, and need to be followed carefully by an experienced physician. to reduce the occurrence of posttransfusion hepatitis, it is essential to screen all blood donations for hbsag by the most sensitive and speci fi c assays. blood donations that are found to be reactive in the hbsag test are automatically con fi rmed by the hbsag con fi rmatory assay. if the specimen is neutralizable in the con fi rmatory test, the specimen is considered positive for hbsag. hbsag testing of donated blood has begun in 1975 (table 28.1 ) . currently, all blood donors are screened for hbsag, but occasional transmission of hbv still occurs due to the inclusion of window period donations (i.e., blood from recently infected donors who are antibody negative but still viremic). detection of early hbv infection of blood donors is still a major problem of blood transfusion. the current third-generation licensed hbsag tests (mostly radioimmunoassay and enzyme immunoassays) can not detect hbv in the window period for hbv infection. this is a strong motivation for introducing molecular detection techniques to the fi eld. there are some commercially available test methods for detecting hbv dna in the market now, such as chiron's quantiplex hbv dna [ 21 ] , digene's hybrid capture, abbott's hbv dna assay, and roche's amplicor hbv monitor. using these commercial hybridization or pcr-based assays, hbv dna can be detected 1-3 weeks before the appearance of hbsag [ 22 ] . some chronically infected patients who have lost their hbsag remain hbv dna positive, but are disquali fi ed as potential blood donors. molecular detection of hbv dna is more sensitive than current methods employed for hbsag screening. determination of antibodies to the hepatitis b core antigen (anti-hbc) (total) is also used to monitor the progress of the hepatitis b viral infection. determination of anti-hbc (igm) is employed to distinguish an acute hepatitis b infection from a chronic infection. the anti-hbc test developed in 1987 detects an antibody to the hbv that is produced during and after infection. if an individual has a positive anti-hbc test, but the hbsag test is negative, it may mean that the person once had hepatitis b, but has recovered from the infection. of the individuals with a positive test for anti-hbc, many have not been exposed to the hbv; thus, there is a frequent problem of false positives. although the individual may be permanently deferred from donating blood, it is unlikely that the person's health will be negatively affected. (note: this antibody is not produced following vaccination against hepatitis b). the hcv is a member of the flaviviridae family of viruses, which are associated with both human and animal diseases. hepatitis caused by hcv is the most common chronic bloodborne infection in the united states. over four million americans are believed to be infected. hcv can also be transmitted through blood transfusion. hcv causes in fl ammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver in fl ammation, cirrhosis, cancer, and death. eventually, up to 20% of people with hcv may develop cirrhosis of the liver or other severe liver diseases. as in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. because of the risk of serious illness, people with hcv need to be followed closely by a physician with experience evaluating this infection. since the fi rst cloning of fulllength hcv cdna in 1989, signi fi cant progress has been made in characterizing its molecular biology [ 11 ] . but, the natural history of hcv infection is still largely unclear and current treatment options for patients are limited. there is no vaccine for hcv, and the only available treatment, a combination of alpha interferon and ribavirin, is ef fi cacious in only a minority of patients [ 23 ] . the life cycle of the hcv is poorly understood due to the lack of an ef fi cient cell culture system [ 24 ] . there is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hcv. we have recently established a cell culture system for the replication of hcv by using human t and b leukemia cell lines [ 25 ] . this model should represent a valuable tool for the detailed study of the initial steps of the hcv replication cycle and for the evaluation of antiviral molecules. currently, appropriate vaccine strategies for hcv have not been developed. early detection and prevention of hcv infection are most important for blood safety. it is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the hcv genome because of a high mutation rate in this genomic region. however, the untranslated region of about 341 nucleotides contains highly conserved domains which allows for stable primer design for qualitative and quantitative diagnostic tests which have equivalent sensitivity against the known six various genotypes of hcv. in 1990, the fi rst speci fi c test for hcv, the major cause of "non-a, non-b" hepatitis was introduced. now, a third generation elisa kit is available to detect antibodies to hcv and screening blood for hcv antibodies is recommended. these assays are based on detection of serum antibody to various hcv antigens because these antibodies are nearly universally present in patients who are chronically infected with hcv [ 26 ] . the hcv screening tests are known to have signi fi cant limitations and positive samples should be further tested by hcv con fi rmatory tests. guidelines provided by the cdc recommend that hcv antibody screening test positive samples should be con fi rmed with serologic or nucleic acid supplemental testing. hcv con fi rmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient's serum. in this way, the response to individual antigens can be recognized, and some false-positive elisa results can be eliminated (e.g., riba, chiron hcv 3.0, and pcr assay) (e.g., roche cobas amplicor hcv test, version 2.0). laboratories can choose to perform this testing on all positive specimens or based on screening test positive (signal to cutoff) ratios. the positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened. hcv antibodies are not generally detectable for at least 6 weeks and may not appear for several months. acute hcv infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. high sensitivity detection of hcv during the window period is a longterm technical challenge in the fi eld. tests for hcv rna genome detection based on the pcr or other highly sensitive rna detection systems have been used for the diagnosis of acute hepatitis [ 26 ] . sensitive detection of hcv rna based on rt-pcr or other nucleic acid ampli fi cation techniques can be readily accomplished with kits that are now available commercially. for example, in 1999 the fda approved roche's amplicor hiv-1 monitor ultra sensitive quantitative assay. it can measure hiv levels at as few as 50 copies/ml and another commercial kit, the lcx hiv rna quantitative assay from abbott laboratories, also has a detection limit at 50 copies/ml. some studies even showed a sensitivity limit at 1 copy [ 27 ] . in fact, a qualitative assay should be much more sensitive than a quantitative assay for hiv/hcv screening. a sensitive qualitative hcv molecular detection assay will possibly interdict and virtually prevent all transfusion-associated hiv/hcv. the current sensitivity standard for clinical diagnostics is 100 copies/ml, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies/ml. hiv-1 and/or hiv-2 virus cause acquired immunode fi ciency syndrome, or aids. the test is designed to detect antibodies directed against antigens of the hiv-1 or hiv-2 viruses. hiv-1 is much more common in the united states, whereas hiv-2 is prevalent in western africa. donors are tested for both viruses because both are transmitted by infected blood, and a few cases of hiv-2 have been identi fi ed in us residents. in1985, the fi rst blood-screening eia test to detect hiv was licensed and quickly implemented by blood banks to protect the blood supply. in 1992, testing of donor blood for both hiv-1 and hiv-2 antibodies (anti-hiv-1 and anti-hiv-2) was implemented. in 1996, hiv p24 antigen testing of donated blood was mandated. now, the p24 antigen testing is going to be compared with a pcr-based test for their ability to detect hiv in the window period. htlv retroviruses are endemic in japan and the caribbean but relatively uncommon in the united states. they cause adult t-cell leukemia/lymphoma and a neurological disorder similar to multiple sclerosis. the infection can persist for a lifetime but rarely causes major illnesses in most people who are infected. in rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. htlv-ii infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. disease associations with htlv-ii have been hard to con fi rm, but the virus may cause subtle abnormalities of immunity that lead to frequent infections, or rare cases of neurological disease. in 1989, human-t-lymphotropic-virus-antibody testing of donated blood was begun. blood is now routinely screened for antibodies to htlv-i/ii. these test screens for antibodies directed against epitopes of the htlv-i/ii viruses. several commercial assays based on the enzyme-linked immunosorbent assay (elisa) or particle agglutination formats are used for screening of htlv antibodies, followed by con fi rmatory assays using western blotting. in some infected individuals, the serologic response to htlv infection is very low. these problems have been solved by the application of pcr ampli fi cation of speci fi c sequences in the virus genome. pcr can be used to detect htlv-i/ii proviruses and is now the method of choice for detection of htlv dna directly from blood and many other tissues. commercial pcr kits for htlv are available [ 28 ] . the wnv is a single-stranded rna virus of the flaviviridae family and is the most recent emerging infectious disease threat to public health and, potentially, to the safety of our blood supply. in 2002, wnv was identi fi ed as transfusion transmissible. it is transmitted by mosquitoes to birds and other animals through a mosquito bite. the virus can infect people, horses, many types of birds, and some other animals. wnv was shown in 2002 to be transmissible by blood [ 29 ] , with an estimated mean risk of 2/10,000-5/10,000 in outbreak regions in the united states. the most common symptoms of transfusion-transmitted cases of wnv were fever and headache. detection of wnv includes either a measurement of wnv antibodies or of wnv nucleic acid (detecting genetic material from the virus itself). there are two types of wnv antibody testing: igm and igg. in most individuals, igm antibodies will be present within 8 days after the initial exposure to wnv, followed by igg production several weeks later. but, the antibodies tested to detect wnv are not expedient for donor blood screening. nat involves amplifying and measuring the wnv's genetic material to detect the presence of the virus in blood or tissue. wnv nat will be negative in the blood once clinical illness has occurred. in this situation, both nat and igm antibody testing may be needed. nucleic acid tests to screen blood for wnv are commercially available and in current use. but, the viral yield for wnv infection is much lower than other viruses. consequently, a more sensitive wnv nat system for donor blood screening will be required, which could further reduce the risks of transfusion transmitted wnv. serum samples from all blood units should be subjected to either the venereal disease research laboratory (vdrl) test or a treponemal test, such as the t. pallidum haemagglutination (tpha) test before transfusion. any unit found positive should be discarded as per standard safety procedures. this test is done to detect evidence of infection with the spirochete that causes syphilis. blood centers began testing for this shortly after world war ii, when syphilis rates in the general population were much higher. the risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in this country for many years) because the infection is very rare in blood donors, and because the spirochete is fragile and unlikely to survive blood storage conditions. sensitivity and speci fi city of serologic tests vary depending on the type of test performed and the stage of the disease. if the donor has spirochetemia, their serologic tests are usually negative, and if the donors are antibody positive, their blood is not infectious. syphilis serological tests for donors have less clinical signi fi cance. a nucleic acid test for accurately detecting syphilis is needed. it can be used to determine whether a blood donor is currently or has recently been infected with the spirochete. in recent years, numerous infectious agents found worldwide have been identi fi ed as potential threats to the blood supply and among these are several newly discovered hepatitis viruses that present unique challenges in assessing possible risks. even if the hepatitis virus test is negative for all known a-e hepatitis agents, there are some unidenti fi ed hepatitis viruses, called non a-e hepatitis viruses that can still be transmitted by blood transfusion. in the future, advances in nat may allow rapid discovery of the unknown hepatitis viruses. hepatitis delta virus (hdv) is a small rna virus that can infect only individuals who have hbv; worldwide more than 15 million people are coinfected [ 30 ] . hdv is clinically important because it generally makes hbv infections more damaging to the liver. increased understanding of the molecular virology of hdv will identify novel therapeutic targets for this most severe form of chronic viral hepatitis. pcr and real-time pcr methods are available for hdv rna detection [ 31 ] . tt virus (ttv) [ 32 ] , named for the patient from whom it was fi rst isolated with non-a-e and g posttransfusion hepatitis in japan in 1997, is a newly discovered transfusion transmitted, single-stranded and circular dna virus [ 33 ] . ttv is nonenveloped and its entire sequence of ~3.9 kb has been determined. it is also often interpreted as a transfusion-transmitted virus [ 32 ] . at least 16 genotypes have been identi fi ed, and ttv is now found all over the world. ttv infection was sought by detection of ttv dna in serum by polymerase chain reaction using primers generated from a conserved region of the ttv genome, e.g., the utr region [ 34 ] . donor blood and blood product can be screened for ttv dna by using pcr or real-time pcr. the signi fi cance of positive fi ndings is still unclear, because high level ttv carriers in healthy populations are currently found [ 35, 36 ] . whether ttv actually causes hepatitis remains to be determined. cytomegalovirus (cmv) is a virus belonging to the herpes group that is rarely transmitted by blood transfusion. donor blood is not routinely tested for cmv, and the prevalence of cmv antibody ranges from 50 to 80 % of the population. but, blood contaminated with cmv can cause problems in neonates or immunocompromised patients. it also remains a major pathogen for solid-organ transplant recipients causing febrile syndromes, hepatitis, pneumonitis, retinitis and colitis. potential problems in selected patient populations can be prevented by transfusing cmv negative blood or frozen, deglycerolized red blood cells. serologic tests for antibody to cmv are useful for determining whether a patient had cmv infection in the past, a determination of great clinical importance for organ and blood donors, and in the pretransplant evaluation of prospective transplant recipients [ 37 ] . commercial nat kits are available for cmv [ 3 ] , and these include the amplicor pcr cmv monitor test and hybrid capture system cmv dna test. sensitive screening tests for malaria are neither commercially available nor of fi cially approved yet. the most effective way of screening donors is to take a proper history of malaria or of fever that could be due to malaria. donor selection criteria should be designed to exclude potentially infectious individuals from donating red blood cells for transfusion. because there are no practical laboratory tests available to test donor blood, donors traveling to high risk malaria areas are excluded from donating blood for 6 months. however, there is a need to develop suitable screening tests, especially for use in an endemic area. a number of clinical research approaches have been developed for the extraction, ampli fi cation and detection of malaria parasite dna from blood products [ 37 ] . variant creutzfeldt-jakob disease (vcjd-a rare but fatal brain infection) [ 38 ] was fi rst described in 1996 in the united kingdom. vcjd is strongly linked with exposure to the bovine spongiform encephalopathy (bse) agent. bse is a transmissible spongiform encephalopathy (tse) affecting cattle and was fi rst reported in the uk in 1986. it has different clinical and pathologic characteristics from classic vcjd. each disease also has a particular genetic pro fi le of the prion protein gene. in recent years, questions have been raised concerning the potential risk of vcjd disease for recipients of plasma-derived clotting factors, including united states licensed factor eight (pdfviii), factor nine (pdfix), and other plasma-derived products such as immune globulins and albumin. in the past 10 years, there have been some reported cases of probable vcjd transmission by red blood cell transfusions in the united kingdom. prion infections are associated with long and clinically silent incubations. the number of asymptomatic individuals with vcjd prion infection is unknown, posing risk to others through blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. in order to decrease the risk, there is a need to establish a blood-based molecular assay for detection of vcjd prion infection. recently research papers have shown that sensitivity detection methods are available for vcjd prion [ 39 ] . however, commercial detection kits are not yet available. the dengue virus (denv) is a member of the virus family flaviviridae and is transmitted to people through the bite of an infected mosquito. the dengue virus has been shown to have four subtypes. these subtypes are different strains of dengue virus that have 60-80 % homology between each other. dengue has emerged as a worldwide problem only since the 1950s. with more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. according to cdc, as many as 100 million people are infected yearly. dengue is caused by any one of four related viruses transmitted by mosquitoes. there are not yet any vaccines to prevent denv infection, and the most effective protective measure is to avoid mosquito bites. there have been healthcare-related transmissions, including transmission by blood products [ 40 ] . dengue infection has a viremic phase that lasts 4-8 days, and blood collected during this phase may be infective when transfused into susceptible hosts [ 40 ] . there are currently no tests for direct detection of dengue virus, but there are however, commercial elisa tests to detect antibodies of the dengue virus in blood samples from patients. recently, research papers have shown that pcr detection methods are available for any dengue virus strain [ 41 ] . babesia is a protozoan parasite of the blood that causes a hemolytic disease known as babesiosis. babesiosis is a malaria-like parasitic disease, and there are over 100 species of babesia identi fi ed. in the united states, babesia microti is the agent most commonly reported to cause human infection. clinical confusion between human babesiosis and malaria is often reported in literature [ 42 ] . babesia infection can also be acquired by blood transfusion. in fact, there have been many cases of transfusioninduced babesiosis documented [ 43 ] . risk of developing this clinical infection is increased for elderly, asplenic, or immunosuppressed patients. current standards issued by the american association of blood banks (aabb) require the inde fi nite deferral of a blood donor with a history of babesiosis. [ 44 ] there is a need to develop methods for identi fi cation b. microti in order to reduce the risk of transmission of babesiosis by transfusion. diagnosis depends upon fi nding parasites on blood fi lm examination which can be detected 2-4 weeks after a tick bite. hamster inoculation and serology have also been used for diagnosis. the indirect fluorescent antibody test (ifat) is available for b. microti and is the most useful serological test for early diagnosis. also, the pcr screen tests for babesiosis are technically available in the fi eld [ 45 ] . chagas disease is named after the brazilian physician carlos chagas, who discovered the disease in 1909. chagas disease is spread mainly by blood-sucking insects infected with trypanosoma cruzi . chagas disease can also be spread through blood transfusion, organ transplants, and from a mother to an unborn child. national screening of the blood supply was instituted in early 2007 by fda, and more than 1,000 donors with t. cruzi infection have been identi fi ed within the past 3 years of testing. "screening for t. cruzi is an important safety measure to help protect our blood supply and to help prevent the spread of chagas disease," says karen midthun, m.d., acting director of the fda's center for biologics evaluation and research. currently, serological elisa tests are available for diagnose chronic chagas disease. pcr test is not a tool for diagnosis of chronic chagas disease in clinical practice yet, although some research results have showed that pcr is a very sensitive parasitological test for chagas' disease in active transmission regions [ 46 ] . more studies are needed for the development of this molecular method. coronavirus is an rna virus known to be associated with respiratory disease. severe acute respiratory syndrome (sars) is a newly recognized coronavirus whose genome sequence does not belong to any of the known coronavirus groups and which quickly spread all over the world from asia in 2003. there has been no evidence that this infection is transmitted from blood donors to transfusion recipients, but the virus associated with sars is present in the blood of people who are sick, and it is possible that the virus could be present in blood immediately before a person gets sick, so that an individual with infection but no symptoms possibly could transmit sars through a blood donation. to help determine whether or not an individual might be infected with sars, a blood collection facility will ask a potential donor orally or in writing about any travel to a sars-affected country or a history of sars or possible exposure to sars. enzyme-linked immunoassays for detection of speci fi c igg and igm antibodies and rt-pcr for detection of sars coronavirus speci fi c rna in the sars patients has been developed. rapid, sensitive, and speci fi c identi fi cation of sars and other novel coronaviruses by molecular methods will be very important in the future. based on past history, it is not just a hypothetical risk that many people have been infected with unrecognized viruses, for example, many patients with symptoms of non a-e, g, and ttv posttransfusion hepatitis. it is still possible that unexplained cases of posttransfusion hepatitis may be caused by a new, undiscovered pathogen. in recent years, numerous new infectious agents found worldwide have been identi fi ed through time-consuming procedures. by the time a new virus, such as hcv, hiv and sars, is found, many people are infected and there could be a large number of fatalities. there is an urgent need to develop methods for rapid identi fi cation and characterization of previously unknown pathogenic viruses. the most recent technologies for detecting and identifying previously unrecognized pathogens are expression library screening, representational difference analysis (rda), and broad-range polymerase chain reaction (br-pcr). but they are all time-consuming approaches. the new unrecognized and uncharacterized viral agents can be rapid identi fi ed by some of the new molecular approaches, e.g., subtraction hybridization [ 47 ] and dna microarray. ensuring the safety and ef fi cacy of blood and blood products is a critical regulatory challenge. the high safety level of the blood supply is the result of continued improvements in blood donor screening and testing. it will be achieved by introducing more updated nucleic acid tests to the fi eld of blood safety. nat is a method of testing blood that is more sensitive and speci fi c than conventional tests that require the presence of antibodies to trigger a positive test result. also, nat works by detecting the low levels of viral genetic material present when an infection occurs but before the body develops an immune response to a virus. this improved sensitivity should enable us to signi fi cantly decrease the infection window period, allowing for earlier detection of the infection and diminishing the chances for transmission of the agent via transfusion. we are to protect the blood supply from not only known pathogens but also the emergence of new and unrecognized and uncharacterized infectious agents. the nat methods are more sensitive and speci fi c compared with non-nat. in the future, nat technology, such as pcr, may allow routine screening of donors for all the known and unknown pathogens of concern to blood safety. progress in blood supply safety emerging infectious disease issues in blood safety white tj (eds) molecular microbiology: diagnostic principles and practice evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents detection of piscine nodaviruses by real-time nucleic acid sequence based ampli fi cation (nasba) molecular-based methods for quantifying hiv viral load molecular diagnostics of infectious diseases dna, 50 years of the double helix: from the concept of molecular hybridization to microarrays in vitro nucleic acid ampli fi cation techniques multicenter evaluation of the performance characteristics of the nuclisens hiv-1 qt assay used for quantitation of human immunode fi ciency virus type 1 rna serological and molecular biology screening techniques for hcv infection clinical evaluation of an hiv-1 and hcv assay and demonstration of signi fi cant reduction of the hcv detection window before seroconversion hiv-1 monitor ultrasensitive quantitative assay. roche, nutley 14. lcx hiv rna quantitative assay from nested real-time pcr for hepatitis a detection introduction to microarray analysis development and validation of a diagnostic dna microarray to detect quinolone-resistant escherichia coli among clinical isolates testing requirements for communicable disease agents detection of hiv-1 and hcv infections among antibody-negative blood donors by nucleic acid-ampli fi cation testing immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infection control practices advisory committee (hicpac) assessment of hepatitis b virus dna stability in serum by the chiron quantiplex branched-dna assay section two: speci fi c virus families recent advances in prevention and treatment of hepatitis c virus infections the scienti fi c challenge of hepatitis c hirsh fi eld i (2003) detection of extrahepatic hcv replication by a novel highly sensitive single tube nested-pcr section two: speci fi c virus families new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunode fi ciency virus type 1 rna in plasma human t-cell leukemia virus types 1 and 2 (chap. 58) estimated risk of transmission of the west nile virus through blood transfusion in the us hepatitis delta virus quanti fi cation of hepatitis delta virus rna in serum by consensus real-time pcr indicates different patterns of virological response to interferon therapy in chronically infected patients ttv-a virus searching for a disease a novel unenveloped dna virus (tt virus) associated with acute and chronic non-a to g hepatitis role of transfusion-transmitted virus in acute viral hepatitis and fulminant hepatic failure of unknown etiology a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology tt virus section two: speci fi c virus families emerging infectious disease agents and their potential threat to transfusion safety detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay threat of dengue to blood safety in dengue-endemic countries pcr detection of nearly any dengue virus strain using a highly sensitive primer cocktail do babesiosis and malaria share a common disease process? babesia infection through blood transfusions: reports received by the us food and drug administration transfusion-transmitted babesia spp.: bull's-eye on babesia microti detection of babesia species from infected dog blood by polymerase chain reaction pcr-based diagnosis for chagas' disease in bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis rapid approach to identify an unrecognized viral agent complete list of donor screening assays for infectious agents and hiv diagnostic assays key: cord-261788-f728j3bb authors: sabater gonzález, mikel; calvo carrasco, daniel title: emergencies and critical care of commonly kept fowl date: 2016-03-02 journal: vet clin north am exot anim pract doi: 10.1016/j.cvex.2016.01.007 sha: doc_id: 261788 cord_uid: f728j3bb fowl are birds belonging to one of the 2 biological orders, the game fowl or land fowl (galliformes) and the waterfowl (anseriformes). studies of anatomic and molecular similarities suggest these two groups are close evolutionary relatives. multiple fowl species have a long history of domestication. fowl are considered food-producing animals in most countries and clinicians should follow legislation regarding reportable diseases and antibiotic use, even if they are pets. this article reviews aspects of emergency care for most commonly kept fowl, including triage, patient assessment, diagnostic procedures, supportive care, short-term hospitalization, and common emergency presentations. refill time (crt). normally, when the finger is removed from the vein, refilling cannot be witnessed visually. if it can be witnessed visually the bird is considered approximately 5% dehydrated, and if 1 second can be counted, then the bird is about 10% dehydrated or in shock. 8 in chickens, the comb should be firm and red. a crt can be assessed on the comb. it should refill within 2 seconds. 8 mucous membrane color can be assessed by everting the vent or the eyelid. respiratory monitoring includes auscultation of the upper and lower respiratory tracts, assessing breathing frequency and quality, as well as detection of signs of dyspnea (eg, orthopneic gait or tail bobbing). the levels of brightness, alertness, and response should be evaluated as part of a first neurologic examination. birds showing depression or severe weakness should be placed immediately in a prewarmed incubator with 50% to 80% humidified oxygen and complete physical examination or diagnostic procedures may be delayed until the bird is stable enough to tolerate them. secondary survey includes obtaining a complete medical history, a full physical examination, assessment of the response to initial therapy, and more diagnostic procedures, which may provide a comprehensive diagnostic and therapeutic plan as well as orientate the owner about the potential economic costs and prognosis. 3 a complete anamnesis should include, but is not restricted to, species; breed; age; gender; presenting complaint; source of the bird; diet; number of birds in the household; open or closed flock; acquisition date; date of the last addition to the flock; number and species of animals affected; potential exposure to toxins; length of illness; changes in behavior; history of previous diseases, treatments, and outcomes; reproductive history; and clinical signs, including their duration and progression. physical examination in fowl is similar to that of other avian species. careful observation of the bird before handling is mandatory in order to determine the length and depth of the physical examination and further diagnostics that the patient is likely to tolerate. all equipment and supplies are readied before removing the bird from the holding container or the intensive care unit. if the patient is debilitated, examination can be performed in a stepwise fashion with small breaks given to the bird between handling, examination, diagnostics, and treatments. in general, fowl species may be handled without chemical restraint. precautions should be taken in order to avoid physical injuries to the bird or the handler (bites, scratches [eg, from tarsal spurs], or blows from the wings [larger species]). fowl should be grasped across the back with or without a towel to avoid wing flapping. then, the legs should be firmly grasped placing 1 finger between them to prevent pressure damage. the bird should then be restrained close to the handler's body or against a hard, nonmovable surface. fractious birds may benefit from having their heads covered with a cloth. 9 smaller species of waterfowl may be held singlehandedly by restraining the animal with the wings folded or with fingers of one hand under each wing supporting the proximal humerus and the other hand supporting the bird's abdomen. larger species, such as geese and swans, are typically restrained keeping the wings folded and facing backward under the arm of the handler. large, calm species may also be straddled on the floor 10 (fig. 1) . the position of the bird during physical examination, diagnostic procedures, and therapeutic procedures may affect its cardiorespiratory function. dorsal recumbency in conscious chickens decreases tidal volume by 40% to 50% and increases breathing frequency by 20% to 50%. 11 birds showing signs of respiratory distress should be held upright. respiratory compromise may be worsened in fowl by the inertial resistance of the large pectoral muscle mass to respiratory excursions of the keel. enlarged viscera, excessive intracoelomic fat, or fluid within the coelom may compress the air sacs, reducing their effective volume and potentially leading to hypercapnia, respiratory acidemia, and death. 12 cloacal or body core temperature can be measured. cloacal temperature depends on body temperature and cloacal activity over time. 13 normal range for body temperature in waterfowl is 40 c to 42 c. to read body core temperature, the probe of a thermistor thermometer should be inserted along the esophagus until it passes the thoracic inlet. 6 normal range for core body temperature in chickens is 40.6 c to 43.0 c. 8 the body condition should be assessed and an accurate weight obtained on a gram or appropriately sized scale in order to correctly calculate potential drug dosages or to compare with previous or future weights (fig. 2) . the patient's needs must be prioritized. despite preferring that samples for hematologic and biochemical analysis be obtained before treatment for the best diagnostic ability, fowl in shock must be stabilized before extensive diagnostic sampling. a conservative minimum database includes determination of packed cell volume, total solids, and estimated white blood cell count. there is intraspecies variation in blood volume (67 ae 3 ml/kg for common pheasants and 111 ae 3 ml/kg for redhead and canvasback ducks). 14 in healthy patients, the amount of blood that can be removed without deleterious effects is 3% of body weight in ducks, 2% in chickens, and 1% in pheasants. 14 in compromised patients, this should be reduced to 0.5% of body weight. reference values for multiple avian species can be found in the literature. intravenous or intraosseous fluid administration is essential when treating critical patients. catheters can be placed under general anesthesia if necessary. sites for intravenous catheterization include the medial metatarsal vein, the ulnar vein, and the jugular vein (fig. 3) . intraosseous catheters can be placed in the distal ulna or proximal tibiotarsus. pneumatic bones, such as the femur or humerus, should be avoided. most birds benefit from intravenous or intraosseous administration of warmed crystalloids at 3 ml/100 g body weight. because fluid resuscitation in critically ill birds is difficult, administration of 1 bolus of crystalloids with oxyglobin to hypovolemic birds may be beneficial. 15 different types of colloids may be used as an alternative to oxyglobin. capnography, direct and indirect blood pressure, electrocardiography, and blood gas analysis are additional monitoring techniques that may be useful in assessing unstable patients. deciding the instrumentation to use depends on practicality and procedure length. capnography measures end-tidal carbon dioxide concentrations in expired air and is a useful indicator of arterial carbon dioxide concentrations. the use of capnographs with sidestream sensors is recommended for small avian patients. pulse oximetry has not yet been validated for birds. the characteristics of oxygenated and deoxygenated avian and human hemoglobin are different, resulting in underestimation of hemoglobin saturation. 16 an ultrasonic doppler flow detector is most commonly used for cardiac monitoring but can also be used for indirect blood pressure measurement. indirect blood pressure measurement techniques used in fowl include doppler, photoplethysmographic/photoacoustic probes with a sphygmomanometer, and oscillometric monitors. 17 systolic blood pressure determination via ultrasonic doppler flow detection correlates well with direct blood pressure measurements in ducks (a platyrhynchos). 18 diastolic, and therefore mean, blood pressure cannot be obtained with this method. 18 direct arterial pressure measurement is ideal but not commonly used because of the need for specific technical skill, the invasive nature of the procedure, and the cost of equipment. 18 for medium to large birds (>200 g), the deep radial artery is the preferred site for arterial catheter placement, whereas for smaller birds (<200 g) the superficial ulnar artery is preferred. for waterbirds or long-legged birds, the cranial tibial or dorsal metatarsal arteries are acceptable sites for catheterization. catheterization of the external carotid artery usually requires a cut-down for proper visualization. 19 a study performed on anesthetized galliformes comparing glomerular filtration rate and blood pressure found that galliformes were able to maintain their glomerular filtration rate when mean arterial pressure (map) ranged between 60 and 110 mm hg. when map decreased to less than 50 mm hg, chickens were unable to sustain glomerular filtration and urine output ceased. 19 unlike chickens that have normal systolic, mean, and diastolic arterial blood pressures of 99 ae 13, 84 ae 13, and 69 ae 15 mm hg, respectively, values for normotension are higher in other galliformes (eg, turkeys). 18, 19 if the definition of hypotension in humans (reduction of 30% from the baseline of conscious maps) is extrapolated to birds, the level of blood pressure at which birds are considered hypotensive would have a tendency to be higher than that recorded in mammals, with the exception of some galliformes and anseriformes species. 19 hypovolemia is treated with intraosseous or intravenous bolus administration of crystalloids (10-20 ml/kg) or colloids (5 ml/kg) until systolic pressures are restored. 20, 21 reference blood pressure values have been determined for different species of fowl. 6, 19 electrocardiography can be used to monitor cardiac rate and rhythm. electrocardiographic parameters can be highly variable between fowl species, as shown by electrocardiographic studies published for several species including the chicken, turkey, quail, duck, swans, muscovy ducks, guinea fowl, and rock and chukar partridges. [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] arterial blood gasometry is the gold standard for assessing the acid-base status, ventilation, and tissue perfusion. it provides essential physiologic information for patients with critical illness or respiratory disease and is vital in the correction of any metabolic respiratory disorders. 19 detailed information about blood gases in birds has been published. 19 sedation or anesthesia may minimize stress to fractious or painful patients. it also may aid in minimizing risk of capture myopathy in canada geese or turkeys. 35, 36 midazolam is increasingly used in birds to produce sedation, hypnosis, anxiolysis, anterograde amnesia, centrally mediated muscle relaxation, and anticonvulsion activity. 37 the pharmacokinetics of midazolam hydrochloride following intravenous administration at 5 mg/kg were determined in broiler chickens, turkeys, ring-necked pheasants, and bobwhite quail. 38 several articles regarding fowl anesthesia and analgesia have been published. 5,6,39 inhalation anesthesia with isoflurane or sevoflurane is the most common in-hospital method for anesthetizing fowl. oxygen flow rates of 1 to 2 l/min allow rapid changes in anesthetic concentration if vaporizer setting is altered. induction is typically via a face mask. the apnea and bradycardia that occur when an induction mask is placed over the beak and face are consequences of a stress response caused by stimulation of trigeminal nerve receptors. [40] [41] [42] preoxygenation with 100% oxygen for several minutes reduced this response in dabbling but not diving ducks. 41 the isoflurane vaporizer is set to 3% to 4% for induction. 6 intubation with a noncuffed endotracheal tube is recommended for anesthetic procedures lasting more than 15 minutes. waterfowl females may require an endotracheal tube 0.5 to 1 full size larger than males of the same species. 6 if intubation is not feasible because of the nature of the procedure to be performed or anatomic structures preventing intubation (eg, presence of crista ventralis), ventilation can be achieved via air sac perfusion. 43 airway patency should be regularly checked during waterfowl anesthesia because the thickening of mucus in the trachea or glottis may completely obstruct the endotracheal tube, leading to death of the patient. anticholinergic drugs reduce pharyngeal and tracheal secretions but also increase their viscosity, and so are only recommended for treatment of bradycardia. 6 in chickens and ducks, isoflurane has a minimum anesthetic concentration (mac) of 1.32% and 1.3%, respectively. 5,6 isoflurane produces dose-dependent cardiopulmonary depression in birds and in pekin ducks induces tachycardia and hypotension. 44 in geese, an average paco 2 of 53 mm hg was necessary for spontaneous respiration to occur, and no respiration occurred with a paco 2 less than or equal to 40 mm hg. 45 intermittent positive pressure ventilation may be used in anesthetized birds, even if some spontaneous breathing is present, to ensure adequate oxygenation of the blood. 46 ventilation is assisted manually using the reservoir bag on the breathing system or a mechanical ventilator. a spontaneously breathing bird is given greater than or equal to 2 assisted beats per minute. if an anesthetized bird is apneic, the assisted ventilation rate is greater than or equal to 8 to 15 beats per minute depending on size (large birds require fewer breaths than small birds). analysis of blood gases showed that effective gas exchange is achieved using mechanical ventilation. 5 in chickens, sevoflurane mac is 2.21%. at mac, heart rate did not change significantly and cardiac arrhythmias were not observed at less than or equal to 2 times mac. in another study in chickens, hypotension was observed during both spontaneous and controlled ventilation. however, this effect was only dose dependent during critical care of commonly kept fowl controlled ventilation. tachycardia occurred during spontaneous ventilation, whereas heart rate remained unchanged during controlled ventilation. 5 analgesia species variability occurs because of differences in pain sensitivity, the conscious response to pain, and the physiologic response to analgesic therapy. dosages and effects of opioids and nonsteroidal antiinflammatory drugs in fowl have been reviewed. 5, 6, 39 hospitalization many birds benefit from symptomatic treatment such as oxygen supplementation, nebulization, fluid therapy, broad-spectrum antibiotics, antifungals, and/or nutritional support and observation for 2 to 8 hours in a warmed incubator before diagnostic tests are performed. 15 separate equipment and housing should be used for birds with suspected contagious diseases and all equipment and cages should be thoroughly disinfected after use to minimize the risk of disease transmission. 15 the optimum temperatures for ill birds are 29.4 c to 32.1 c. administration of oral or subcutaneous fluids is reserved for stable fowl that are less than 5% dehydrated. oral fluid administration requires a patient that can maintain an upright body position and has a functional gastrointestinal tract to avoid regurgitation and aspiration of fluids. subcutaneous fluids may be administered in the inguinal web, interscapular area, axillary region, lateral flank, or midback. replacement fluid therapy is critical before nutritional support is instituted. diets for stable hospitalized fowl should ideally be selected according to the natural diet of the species. commercially available feeding formulas, such as a formulated critical care diet lafeber's critical care diet (lafeber company, cornell, il) or hill's a/d diet (a/d canine/feline; hill's pet nutrition, topeka, ks) can be used on a short-term basis. the use of emeraid exotic carnivore diet improves postsurgical recovery and survival of long-tailed ducks 47 (fig. 4) . the most common causes of blood loss in birds include traumatic injury and hemorrhagic lesions of internal organs. the ld50 (lethal dose, 50%) for acute blood loss in mallard ducks was 60% of total blood volume. after chronic blood loss, the ld50 of mallard ducks was reached when 70% of blood volume was removed, compared with an ld50 in pheasants and chickens of 40% to 50% loss of total blood volume. recommendations for fluid resuscitation after severe blood loss in birds have included the administration of crystalloids, colloids, and whole blood. although no statistical difference in mortality was appreciated among the 3 fluid resuscitation groups (crystalloids, hetastarch, or a hemoglobin-based oxygen-carrying solution [hbocs]) in the acute blood loss study, a trend of decreased mortality was observed in the hbocs group. an early regenerative response was apparent following acute blood loss. 48 trauma traumatic injuries occur fairly frequently in fowl kept outdoors, either as a result of predator attack, gunshot, electrocution, or as a result of inappropriate housing. a thorough physical examination is essential to determine the extent of trauma and the best approach for treatment. prioritize therapy (oxygen therapy, fluid therapy, and analgesia), control active hemorrhage, cleanse wounds, and stabilize fractures initially until patient stabilization allows a more specific treatment. any animal that has a bite wound should be provided with antibiotics after a sample has been taken for microbiological culture and sensitivity. clinical signs of head trauma may include, but are not restricted to: anisocoria, head tilt, depression, or other neurologic signs, skull fractures, retinal detachment, or hemorrhage from the nares, oral cavity, ears, and/or anterior chamber of the eye. mentation, pupil symmetry and size, and pupillary light reflex should be constantly monitored. changes in pupil size to dilated and loss of pupillary light reflex along with mentation progression to stupors or coma indicate neurologic deterioration. 7 soft tissue injuries in the head and neck are commonly seen, and may require surgical repair. 49 posttraumatic exposed epibranchial bones, part of the hyoid apparatus, can be surgically excised without significant postsurgical impairment, allowing easier surgical repair of wounds in the crown. 49 fractures of the skull bones (eg, mandible, quadrate, jugal arches, palatine, pterygoid, and maxilla) can also occur. if the animal is able to groom and feed, healing by second intention can create a false joint allowing normal function. 49 surgical repair of the beak and the use of prostheses have been reported. 50, 51 anseriformes are prone to foreign body injuries. ingestion of fishing hooks and lines is common in swans (fig. 5) . lesions can be observed in the rhamphotheca, tongue, skin of the neck, and gastrointestinal tract. management varies depending on the severity of the injuries. endoscopy can be attempted, but sometimes surgery is required. management of a neck injury caused by a nail shot from a pneumatic nail gun in a young muscovy duck (cairina moschata) has been reported. 52 ocular injuries can also be seen after head trauma. if the eye is not visual and is severely damaged, enucleation may be considered. injuries over the coelomic cavity should always be assessed to make sure no penetrating injuries are present. in such cases the prognosis is poor. skin and muscle injuries can be surgically repaired when indicated, or managed medically to allow healing by second intention. fracture repair follows the same principles as in other avian species. in laying hens, pathologic fractures caused by hypocalcemia and metabolic bone disease might occur, and calcium status should be assessed and deficiencies corrected before surgical repair. vitamin c deficiency can also cause secondary fractures. 53 emergency care for bone fractures should align the fracture as fully as possible so further damage of the surrounding tissues and pain are minimized, and weight bearing can occur as soon as possible, avoiding excess stress and load on the unaffected limb. luxations should be reduced as soon as possible to provide the best chance for joint mobility. bandaging includes soft bandage material and splints applied for temporary support or permanent fixation of fractures. bandaging techniques commonly used in other avian species (tape splint, football-type bandage, plastic spica bandage, modified robert jones bandage, schroeder-thomas splint, ehmer-type bandage, and figure-of-eight bandage) can be also used in fowl 54 (figs. 6 and 7) . damage to the cervical air sac can cause emphysema because of the leakage of air into the subcutaneous space. this condition is often self-limiting. a cauterized skin defect can also be created over the swelling to allow the air to escape. the cauterized hole takes longer to heal than the air sac lesion, preventing recurrence. 49 older hens quickly run for shelter when the weather conditions are not desirable. however, juvenile animals may stand on the wet ground, becoming hypothermic rapidly, especially those with thin skulls and crest, such as polands and those with wooly feathering, such as silkies. 55 nevertheless, adequate shelter must be always provided. hypothermic patients may be warmed externally and via infusion of warmed fluids. poultry experience heat distress when high temperatures accompanied by high humidity increase beyond their comfort zone. when the environmental temperatures are between 28 c and 35 c, birds use nonevaporative cooling in 2 ways: (1) increasing the surface area by relaxing the wings and hanging them loosely at their sides, and (2) increasing the peripheral blood flow. as the environmental temperature approaches the body temperature of the bird (41 c), the rate of respiration increases and the bird open-mouth breathes in order to increase evaporative cooling or water evaporation. if panting fails to prevent body temperature from increasing, birds become listless, comatose, and finally die of respiratory, circulatory, or electrolyte imbalances. 56 respiratory disease is a common presentation in avian practice. clinical signs are often unspecific, and hardly ever pathognomonic. respiratory signs are not only seen with primary respiratory disease but also with any organomegaly or distended coelom as a result of the pressure to the air sacs, or secondary to other disorders, such as cardiovascular disease. sinusitis is a common presentation in chickens and waterfowl, and often presents because of swelling of the periocular sinuses. 49 different agents could cause sinusitis, such as mycobacterium spp, pasteurella spp, escherichia coli, pseudomonas spp, and some viral agents, such as avian influenza and newcastle disease (both reportable diseases in the united kingdom, european union [eu], and united states (us); fig. 8 ). however, newcastle disease, avian influenza, and infectious laryngotracheitis are all rare in backyard poultry, and the most common causative agent of sinusitis in fowl in the us is mycoplasma. [57] [58] [59] different agents are often isolated from the nasal cavity, aggravating the clinical presentation. in such cases, the authors recommend performing an initial sinus flush with sterile saline, in order to obtain samples for culture and polymerase chain reaction (pcr) identification. sinus flush should be performed under general anesthesia with an uncuffed endotracheal tube placed, to avoid fluid going into the respiratory tract. once samples have been obtained, f10, enrofloxacin (not to be used in the usa and only to be used on label in egg laying chickens and turkeys in europe), amikacin or gentamicin flush could be performed, and repeat it as necessary. if purulent material is present in the sinus, the author recommends surgical access to remove as much purulent material as possible, because antibiosis alone is unlikely to resolve it. mycoplasmosis is the most common respiratory condition seen in backyard poultry. 57, 60 poultry is mainly affected by 4 species of mycoplasma: mycoplasma gallisepticum, mycoplasma synoviae, mycoplasma meleagridis, and mycoplasma iowae. m gallisepticum is often the pathogen causing respiratory signs, although m synoviae can also cause respiratory signs (sneezing, foamy nasal and ocular discharge, conjunctivitis, sinusitis, and/or purulent aural discharge). mycoplasma spp can be latent within the flock and often causes disease when there is immunosuppression, stressful factors, and concomitant infections. tylosin is the recommended treatment because it is licensed (at least in the united kingdom/eu and united states). antibiotic therapy does not eliminate mycoplasma, but it can resolve clinical signs; equally important is to assess and treat any other stressful factors (ammonia and dust sabater gonzá lez & calvo carrasco levels, densities, overall hygiene, food and water quality). however, if symptoms persist despite treatment, euthanasia should be considered for the well-being of the flock. anseriformes are an important reservoir for avian influenza, often being asymptomatic carriers even from some of the high-pathogenic strains. avian influenza or fowl plague is a rare disease in wild waterfowl, with few records in the wild. 59 despite being uncommon, in particular in mixed flocks or flocks exposed to wild waterfowl, avian influenza should be considered and investigated in cases with compatible clinical signs, such as mucopurulent or caseous sinusitis. 59 important management factors to control this disease, such as hygiene and density levels, should be assessed in captive waterfowl showing clinical signs. newcastle disease or avian paramyxovirus can also present with signs of upper respiratory disease, such as conjunctivitis or tracheitis, and it can also cause central nervous system and gastrointestinal signs. 49, 61 zoonosis can also occur, although this only causes mild conjunctivitis in humans. it is a relevant disease given the high economic losses it can produce in the commercial poultry industry, because there is no effective treatment. vaccinations are available to reduce the likelihood of outbreaks. vaccination against newcastle disease is not currently allowed in the uk, but seems to be standard practice in the us. infectious laryngotracheitis (ilt) is caused by a herpesvirus, as well as marek disease. ilt can affects chickens (mainly meat breeds) as well as pheasants, and is similar in presentation to other respiratory diseases. it is characterized by the formation of a diphtheritic membrane in the trachea that can cause obstruction; animals can present gasping. vaccination can be attempted in an outbreak to reduce morbidity and mortality. early vaccination prevents clinical manifestation, but not latent infection. modified live vaccines are available in the uk, eu, and us. aspergillosis is a common condition affecting waterfowl, although it can also affect gallinaceous birds, such as chickens. as in other avian species, aspergillus fumigatus is the main isolated pathogen, although others species of the genus aspergillus can also cause disease. 61, 62 in chickens, despite most healthy birds coping with a moderate exposure to the aspergillus conidia, infection may occur in immunocompromised birds or when exposed to an overwhelming quantity of spores. common sources of aspergillus are contaminated food and moldy substrates. clinical signs might include dyspnea, but it can present as lethargy, anorexia, and significant weight loss. diagnosis and treatment present similar challenges to those faced in other avian species. treatment is based in antifungal therapy, often an azole drug, together with supportive care. infectious bronchitis is caused by a highly infectious coronavirus and is characterized by having 2 main presentations depending on the age of the infected animals; in young chicks, respiratory disease is the predominant manifestation, whereas salpingitis and the subsequent decrease in egg production is most commonly seen in older laying hens. soft, irregular, or rough-shelled eggs are often seen. in certain animals the lesions caused might impair normal laying for the rest of the animal's life, or even cause secondary problems, such as egg coelomitis. c psittaci is a well-known pathogen among avian practitioners worldwide, not only relevant for its high prevalence but also for its zoonotic potential. 63 more than a 100 species have been shown to be infected, including galliformes. 64 because of its very wide infection range, many different species can act as a reservoir, such as pigeons and waterfowl. 65 a recent study in pigeons showed a prevalence of 15% in adult animals, which was twice as high in juvenile birds. 66 outbreaks in fowl occur only occasionally. in poultry, infection is often systemic, and occasionally fatal. clinical signs, incubation periods, morbidity, and mortality vary widely depending on the virulence of the strain infecting the flock. common clinical signs observed with chlamydiosis are sinusitis, rhinitis, diarrhea, and weakness. postmortem findings in affected birds include splenomegaly, hepatomegaly, airsacculitis, pericarditis, and peritonitis. 67, 68 in turkeys, the disease pattern differs from other species and tends to present as an explosive outbreak. 69 clinical signs can be aggravated by concurrent infections, such as salmonella or pasteurella. ideally, a combination of serology and pcr identification is used to diagnosis chlamydia. however, after adequate therapy, there is no currently available test to ascertain whether affected birds are no longer carriers; therefore, treatment should be carefully considered, because of its zoonotic risk, especially in collections open to the public. 61 chlortetracycline (1000 ppm; ie, 18.2 g/kg food daily for 45 days) has been recommended in turkeys, although doxycycline (25 mg/kg po twice a day or 240 ppm in food daily for 45 days; or 50-100 mg/kg im weekly on 6 occasions) is also used in outbreaks to reduce mortality in turkeys as well as in other species. 64, 70 avian tuberculosis can present as a respiratory emergency when lesions are localized in the pharynx or trachea. certain parasites can also cause respiratory disease in fowl, such as syngamus trachea (commonly known as gapeworm), duck leeches (theromyzon tessulatum), streptocariasis, (streptocara spp), and air sac mites (cytodites nudus). [71] [72] [73] if upper airways are affected, animals present gasping for air or open-mouth breathing, coughing, or retching. diagnosis is based on identification of the parasites (adult forms, ova, or larvae). riemerella anatipestifer can cause a peracute infection in ducklings, which might present with upper respiratory clinical signs, such as dyspnea, or nasal or ocular discharge. 74 this condition evolves quickly and can cause sudden death. samples should be obtained for culture and sensitivity, to allow adequate antibiotic therapy. neurologic disease is common in fowl. clinicians must be vigilant and aware of the reportable diseases that can present with neurologic signs, such as newcastle disease, avian influenza, and chlamydiosis. marek disease is common in unvaccinated chickens, and heavy metal poisoning should always be considered in waterfowl. other possible causes are trauma, nutritional deficiencies, central nervous system ischemia, vascular insult, and other intoxications (fig. 9) . marek disease is caused by gallid herpesvirus 2, and has recently been described as the most common disease diagnosed in backyard poultry. 57 the disease is characterized by the presence of t-cell lymphoma as well as mononuclear infiltration of nerves, organs, reproductive tract, internal viscera, iris, muscle, and skin. 75 the mononuclear infiltration of peripheral nerves, in particular the sciatic nerve, causes paralysis. however, there is no treatment of affected birds and euthanasia should be considered in unvaccinated suspicious cases. early vaccination (within the first 3 days of hatching) does not stop infection (the virus is considered ubiquitous worldwide) but achieves more than 90% protection under commercial conditions. 76 lead poisoning is thought to be one of the most significant causes of neurologic disease in waterfowl. 77 a recent report estimated between 50,000 and 100,000 (approximately 1.5%-3.0% of the wintering population) wildfowl deaths each winter are caused by lead poisoning. that number represents a quarter of all recorded deaths regarding migratory swans. 78 not only waterfowl are affected by this, because other terrestrial game birds and fowl may ingest lead pellets that they mistake for grit or food; lead pellets may be buried in mud, in areas where fishing or hunting has previously taken place. animals can experience chronic intoxication when ingesting small numbers of lead pellets intermittently, but can also present acutely and in the form of an outbreak when reduced water levels or other circumstances expose lead that was previously unavailable. in the united kingdom, the sale and use by fishermen of lead leger weights and split shot weighing less than 28 g has been banned since 1987. 79 since then, the incidence of lead poisoning has reduced significantly. 80 however, the environmental contamination will still have an effect for many years. characteristic clinical signs of lead toxicity include weight loss, weakness, and green faces; weakness of the neck muscles causes a typical posture with the head resting on the bird's dorsum. 81 whole-body radiographs might reveal the presence of metallic objects in recent cases; however, the grinding action and ph of the ventriculus dissolves the lead pellets within a few days. other common findings in chronic cases on radiographs are dilatation and impaction of the proventriculus. 79 a blood sample should always be tested for lead levels to achieve a definitive diagnosis (normal, <0.4 ppm; diagnostic, 0.5-2.0 ppm; severe, >2.0 ppm). moderate anemia (20%-38% hematocrit) can be observed. 82 delta amino levulinic acid dehydrase activity has been suggested as a more sensitive diagnostic indicator for lead intoxication. 83 early treatment of lead toxicosis should include stopping any further lead absorption; lead particles within the gastrointestinal tract can be removed by lavage under general anesthesia with warm fluid via a gastric tube. 82 some investigators recommend repeating gastric lavage within 24 to 48 hours if lead particles are still present in postlavage radiographs, because fragments of lead can be trapped in crevices in the koilin. those particles precipitate when muscle activity has restarted. 84 chelation therapy should be started in all affected animals. sodium calcium edetate (10-40 mg/kg intramuscularly every 24 hours for 10 days, with a 5-day break at day 5) is the recommended treatment of lead and zinc toxicosis. penicillamine can be used as an alternative if sodium calcium edetate (nacaedta) is not available, or at the same time in severe cases. zinc toxicosis is less common in animals housed outdoors, and is similar in diagnosis and treatment to lead intoxication. 85 botulism occurs when animals are kept in water with anaerobic conditions, particularly in warm, dry periods. clostridium botulinum overgrows and produces toxin type c, causing flaccid paralysis. other clinical signs are similar to other heavy metal poisoning, such as weakness. a good anamnesis and water analysis allows a presumptive diagnosis. 86 other intoxications are common in fowl, such as coccidiostats in waterfowl (found in chicken-formulated commercial diets) or pesticides (dimetridazole and organophosphorus pesticides). 87, 88 diarrhea diarrhea can have many different causes; after physical examination, clinicians should perform a direct observation, flotation, and diff-quik examination of a fresh fecal sample. samples should also be taken for viral identification. duck plague or duck viral enteritis is caused by a herpesvirus, and can cause significant losses in waterfowl collections. presentation can be peracute, including sudden death without previous obvious signs. other described clinical signs are cloacal lethargy, diarrhea, hemorrhage, prolapse of the penis, photophobia, ataxia, and tremors. 49, 89 outbreaks are often seasonal (may to june in the united kingdom). it can cause morbidities between 10% and 100% in unvaccinated collections. 90 in affected animals, the prognosis is very poor with no effective treatment. annual vaccination is recommended in endemic areas. avian or fowl cholera, caused by pasteurella multocida, is the most common pasteurellosis in poultry. chickens, duck, geese, and turkeys can be affected. outbreaks in turkeys can cause up to 65% mortality. 91 clinical signs include oral and nasal discharge, dyspnea, diarrhea, and sudden death. this condition seems to be less frequent in the united kingdom than in north america, where annual outbreaks can cause significant mortalities. 92 impaction of the crop, proventriculus, or gizzard has occasionally been reported in poultry and waterfowl. affected birds commonly present showing lethargy, emaciation, and esophageal or crop distension. despite the crop/esophagus, proventriculus, and/or gizzard being full of a solid mass of interwoven fibrous material, the intestines of birds with this condition are frequently empty. 56 poultry have been known to ingest poorly digestible items (eg, grass, newspaper, sawdust shavings/ wood chips, and feathers) out of curiosity or as a response to stress, causing crop impaction. crop impaction is most frequently seen in spring, when chickens ingest long stems of grass that get impacted in the crop. captive waterfowl, especially geese, suddenly exposed to new environments may ingest nondigestible items like newspaper or plant products, like grasses. ingestion of grains that have low moisture content with concurrent exposure to water can lead to grain swelling and result in impaction of the esophagus. 93 gizzard impaction can cause high mortality during the first 3 weeks of life in turkey flocks. 56 although rehydration of the impaction, gentle massage or flushing (only for crop or esophageal impactions), and liquid paraffin may help to resolve the impaction in early cases, surgical intervention might be necessary (fig. 10) . these conditions occur sporadically in domestic fowl. intussusception occurs most frequently in the intestine, but it has also been reported in the proventriculus. in young birds volvulus of the small intestine may be caused by twisting around the yolk sac. intussusception and volvulus have been reported in chickens secondary to enteritis or abnormal peristalsis caused by nematode or coccidial infection. intestinal torsion has also been associated with pedunculated neoplastic stalks. clinical signs are anorexia and progressive weight loss, which may lead to death within a few days. diagnosis may be achieved by ultrasonography, radiography, or endoscopy. if an early diagnosis is made, resection of the affected intestine can be performed. 56 coelomitis is an occasional cause of morbidity and mortality in waterfowl and a common condition in chickens, particularly seen in laying or ex-battery hens. 94 infection of the coelom can become established following infection of the respiratory system, penetrating injuries, neoplasia, heavy parasitism, or reproductive diseases. in chickens, e coli is often responsible of the oviduct infections. salmonella pullorum or infectious bronchitis can also cause lesions in the reproductive tract. 60 diagnosis may be achieved by aspiration of coelomic fluid (ultrasonographically guided if possible). powerful antibiotic therapy is recommended (fig. 11) . egg coelomitis may occur because of an ectopic ovulation, when the follicle or yolk misses the infundibulum, or when the follicle in the oviduct moves back in a retroperistaltic manner. this condition can be caused by an underlying disorder or can occur fig. 11 . ultrasonography in a chicken with distended coelom. critical care of commonly kept fowl after a stressful event while the egg was forming within the oviduct. in both situations the yolk reaches the coelomic cavity causing a coelomitis. secondary bacterial colonization can occur. often this occurs because of pathologic changes in the oviduct, with either infectious or neoplastic causes, or because of oviductal damage in battery hens. a recent study performed in backyard poultry in the united states revealed that the most common condition diagnosed was marek disease. 57 in that study, the most common finding observed in gross postmortem was the presence of tumors affecting internal organs or carcinomatosis, which can affect the ovaries. equally, non-viralinduced reproductive neoplasia, despite having significantly different findings in the 2 different institutions involved in the study, is also considered common. salpingitis was one of the most common presentations in 1 of the institutions, with 7.8% of the presented cases. initial treatment can include coelomoentesis when dyspnea is observed; this technique, although not free of risk, also helps in achieving a diagnosis by analyzing the fluid drained. fluid therapy, antibiosis, analgesia, and assisted feeding are required at initial stabilization. salpingohysterectomy is likely to be required for long-term treatment because this condition is likely to reoccur. stress, age, obesity, and poor nutrition can contribute to the presentation of this condition, and good layers seem to have a higher predisposition. 60 this condition can be seen in animals with egg binding. often animals had experienced trauma of exposed tissue from the other animals in the flock. medical management is often unsuccessful and salpingohysterectomy is the preferred treatment option according to the investigators. alternatively, a gonadotrophin-releasing hormone agonist implant (deslorelin acetate) can be used, once the prolapsed tissue has been repositioned and infection and inflammation controlled. repeated applications are required longterm, and in certain animals the duration of the implant seems to decrease after repeated applications. this condition may result from inflammation of the oviduct, partial paralysis of the muscles of the oviduct, or production of an egg so large that it is physically impossible for it to be laid. young pullets laying an unusually large egg are most prone to the problem. 56 as in other avian species, this condition is often linked to calcium imbalance, caused by a combination of dietary deficiencies, stress, and other husbandry-related problems. treatment includes fluid therapy, calcium, and oxytocin administration. if initial medical management is unsuccessful, ovocentesis (either directly into the egg shell or via the coelomic wall) should be the next step. the egg should not be manually broken or pulled, because iatrogenic damage to the oviduct may occur. if the shell of the egg is not eliminated within 24 hours, salpingohysterectomy is indicated because the remnants of the shell might adhere to the oviduct, inevitably causing further complications in future oviposition. phallus prolapse is occasionally seen in anseriformes associated with mechanical damage, infection (ie, cryptosporidum spp., mycoplasma spp, neisseria spp.), hypersexsuality or immunosuppression. frostbite and bacterial infection may occur as a sequela of phallus prolapse. 56 treatment may include analgesics, local and/or systemic antibiotherapy, and decongestive and lubrifying local therapies which allow reposition of prolapsed healthy tissues. severe cases may require amputation of the phallus. euthanasia might be required in cases with a poor prognosis and when certain infectious diseases have been confirmed. euthanasia should always be performed in a humane manner. the authors' preferred method is intravenous administration of barbiturates, but other methods can be used. 95 fowl are stoic patients that commonly mask signs of illness in the early stages of disease and are not commonly presented as emergencies until the acute or chronic condition is severe. an understanding of intraspecific and interspecific anatomic and physiologic variations is crucial to the successful management of critically ill fowl. stabilization of the patient should be prioritized over diagnostic procedures. clinicians treating fowl should be aware of infectious and noninfectious conditions causing emergencies in fowl. a classification of the living birds of the world base on dna-ddna hybridization studies a phylogenetic supertree of the fowls (galloansera, aves) bsava manual of canine and feline emergency and critical care zoonoses, public health, and the backyard poultry flock free-living waterfowl and shorebirds emergency care of raptors backyard poultry medicine and surgery. a guide for veterinary practitioners the maximum capacities of the lungs and air sacs of gallus domesticus evaluation of three heat sources for their ability to maintain core body temperature in the anesthetized avian patient updates in anesthesia and monitoring avian clinical biochemistry emergency and critical care of pet birds evaluation of pulse oximetry as a monitoring method in avian anesthesia arterial blood pressure monitoring in anesthetized animals determination of indirect blood pressure in the companion bird arterial catheterization, interpretation and treatment of arterial blood pressures and blood gases in birds principles of shock and fluid therapy in special species intraosseous cannulation and drug administration for induction of anesthesia in chickens physiological studies on the electrocardiogram of the chicken i: bipolar leads physiological studies on the electrocardiogram of the chicken iii: on the normal values of the electrocardiogram of laying hens the electrocardiogram of birds (chicken, duck, pigeon) electrocardiographic observation on spontaneously occurring arrhythmias in chickens comparative electrocardiographical studies on the wave form of qrs complex in vertebrates the electrocardiogram of the chicken cardiac muscle mass distribution in the domestic turkey and relationship to electrocardiogram the electrocardiogram of the turkey scalar electrocardiographic measurements in unrestrained young japanese quail hyperpotassemia and electrocardiographic changes in the duck during prolonged diving normal electrocardiogram patterns and values in muscovy ducks (cairina moschata) analysis of electrocardiographic parameters in helmeted guinea fowl (numida meleagris) electrocardiography of rock partridges (alectoris graeca) and chukar partridges (alectoris chukar) capture myopathy capture myopathy in wild turkeys (meleagris gallopavo) following trapping, handling, and transportation in colorado advances in avian clinical therapeutics plasma pharmacokinetics of midazolam in chickens, turkeys, pheasants, and bobwhite quail the exercise response and the 'classical' diving response during natural submersion in birds and mammals forced and voluntary diving in ducks: cardiovascular adjustments and their control metabolism in diving birds: studies in the laboratory and the field anesthesia case of the month effects of halothane and isoflurane on mean arterial blood pressure, heart rate, and respiratory rate in adult pekin ducks halothane effects on ventilatory responses to changes in intrapulmonary co2 in geese capnographic monitoring of anesthetized african grey parrots receiving intermittent positive pressure ventilation the use of emeraid exotic carnivore diet improves postsurgical recovery and survival of long-tailed ducks comparison of fluid types for resuscitation after acute blood loss in mallard ducks (anas platyrhynchos) bsava manual of raptors, pigeons and waterfowl. cheltenham (united kingdom): british small animal veterinary association distraction osteogenesis correction of mandibular ramis fracture malunion in a juvenile mute swan (cygnus olor) bsava manual of farm pets. gloucester (united kingdom): british small animal veterinary association what is your diagnosis? clinical avian medicine volume ii bandaging, endoscopy, and surgery in the emergency avian patient bsava manual of farm pets. gloucester (united kingdom): british small animal veterinary association developmental, metabolic, and other noninfectious disorders postmortem survey of disease conditions in backyard poultry prevalence and differentiation of diseases in maryland backyard flocks avian medicine: principles and application. lake worth (fl): wingers publishing bsava manual of farm pets. gloucester (united kingdom): british small animal veterinary association bsava manual of raptors, pigeons, and waterfowl. cheltenham (united kingdom): british small animal veterinary association aspergillus infections in birds: a review outbreak of psittacosis in a group of women exposed to chlamydia psittaci-infected chickens avian chlamydophilosis (chlamydiosis/psittacosis/ornithosis) chlamydia psittaci in ducks: a hidden health risk for poultry workers epidemiological investigations on the possible risk of distribution of zoonotic bacteria through apparently healthy homing pigeons experimental ornithosis in turkeys respiratory and pericardial lesions in turkeys infected with avian or mammalian strains of chlamydia psittaci chlamydiosis (psittacosis, ornithosis) use of ovotransferrin as an antimicrobial in turkeys naturally infected with chlamydia psittaci, avian metapneumovirus and ornithobacterium rhinotracheale leech parasitism of waterfowl in north america laryngeal streptocariasis causing death from asphyxiation in ducks cytodites nudus infestation of chickens dose response study of enrofloxacin against riemerella anatipestifer septicaemia in muscovy and pekin ducklings marek's disease protective synergism among marek's disease vaccine viruses report of the nature conservancy council working group poisoning of birds and other wildlife from ammunition-derived lead in the uk bsava manual of raptors, pigeons and waterfowl. cheltenham (united kingdom): british small animal veterinary association progress on lead-free shot in the uk pathological study of lead poisoning in whooper swans (cygnus cygnus) in japan lead poisoning in trumpeter swans toxicities in waterfowl treatment of lead poisoning in swans emergency care and managing toxicoses in the exotic animal patient triage of botulism in wild birds toxicity of dimetridazole in waterfowl identification of differentially expressed proteins related to organophosphorus-induced delayed neuropathy in the brains of hens duck, geese, swans, and screamers: infectious diseases the comprehensive diagnosis and prevention of duck plague in northwest shandong province of china fowl cholera in turkeys persistence of pasteurella multocida in wetlands following avian cholera outbreaks backyard poultry medicine and surgery. a veterinary guide for veterinary practitioners bsava manual of raptors, pigeons, and waterfowl. cheltenham (united kingdom): british small animal veterinary association key: cord-306881-wrd2rhjz authors: gehrie, eric; tormey, christopher a; sanford, kimberly w title: transfusion service response to the covid-19 pandemic date: 2020-06-25 journal: am j clin pathol doi: 10.1093/ajcp/aqaa111 sha: doc_id: 306881 cord_uid: wrd2rhjz nan emergency preparedness has been an emphasis for blood banks for the past several years, driven largely by the proliferation of high-profile mass shootings and the recognition that such a disaster could immediately impact blood bank operations. 1, 2 while most hospitals included a pandemic scenario into their emergency plans, for better or worse, lack of recent experience with pandemics made specific preparations difficult to identify and expert opinion on these matters remained highly theoretical. highly detailed plans, such as those published by the aabb and canada, had never been previously operationalized for a pandemic response. 3, 4 as a result, even the most well-prepared blood bankers found themselves working diligently to make frequent changes to operational plans as the coronavirus disease 2019 (covid19) pandemic unfolded in the united states in early 2020. in this article, we highlight "best practices" that have emerged during the pandemic, focusing on management of blood supply and blood bank operations, rapid incorporation of covid-19 convalescent plasma into blood bank inventory, and changes to the approach to the patient requiring therapeutic apheresis. in the united states up to 80% of the blood supply is collected at mobile blood drives that are susceptible to cancellation or underperformance without strong support from the public. 5 when large corporations, schools, and universities began closing in response to the need to promote social distancing, there was an immediate effect on blood donation. the american red cross, which is the largest single blood supplier in the united states, estimated that 4,600 blood drives were almost immediately cancelled, correlating in an estimated loss of 143,600 units of blood from the blood supply (pampee young, md, phd, american red cross email communication, march 18, 2020) . in many areas, the changes to businesses and schools preceded major changes to hospital operations, such as cancellation of nonurgent elective surgery, and the blood banking community found itself stuck in the middle between, on the one hand, a rapidly developing blood supply shortage and, on the other, essentially normal hospital operations (eg, elective surgeries were ongoing, and changes were not yet made to transplant or oncology services). many blood banks responded to this issue by doing everything possible to promote community blood donation at fixed donor sites, encourage blood suppliers to increase collection activities at hospitals, while lobbying with hospital administrators to reduce hospital operations or to at least warn the general hospital community that blood was quickly becoming a very scarce resource. in early late march 2020, the blood supply and demand stabilized, due to widespread cancellation of normal hospital operations, including cancellation of nonemergent surgeries and transplants, reduction in routine sickle cell disease transfusion volumes, and a sharp decline in elective cardiac surgery. 6 as a result of these changes, demand for rbcs was significantly impacted and came largely into line with supply ❚figure 1❚. critically, massive transfusion protocol trauma activations were markedly reduced during the pandemic, also suppressing transfusion utilization, especially of rbcs and platelets ❚table 1❚. an area of ongoing concern is coordination of resumption of normal patient activities with increased collection of blood components (pampee young, md, phd, american red cross email communication, may 1, 2020). mobilization of the donor base will be a key part of any plans to resume normal volumes, as will coordination between blood suppliers and transfusion services, especially given that summer months are difficult on the blood supply even in the absence of a pandemic. as blood donation was encouraged, blood bankers were suddenly inundated with concern that blood transfusions could transmit severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection, and the transfusion medicine community was asked to provide reassurance against this possibility. extrapolation from previous experience with sars-cov, middle eastern respiratory syndrome, and influenza, and with the strong backing of statements by aabb, the centers for disease control and prevention, and the food and drug administration (fda), as well as the preliminary experience of other areas that were afflicted by covid-19 prior to its wide spread in the united states, blood bankers were able to convince most stakeholders that the true risk to the blood supply was not sars-cov-2 itself, but rather social distancing practices resulting in an interruption to the critically needed blood supply. 5, 7, 8 to assist with donor recruitment, the fda eased several donor deferral criteria pertaining to low-risk hiv-associated behaviors, travel-related malaria risk mitigation, and creutzfeldt-jakob or variant creutzfeldt-jakob disease ❚table 2❚. [9] [10] [11] the available evidence and experience with covid-19 continues to confirm that sars-cov-2 is not transmitted by transfusion. an area of secondary concern was that potential blood donors could come into contact with sars-cov-2 while participating in a blood drive. this was immediately anticipated by the blood suppliers, many of whom quickly stopped using small confined areas to collect blood (such as trailers) and began asking potential blood drive sponsors to provide large spaces that could allow donors to maintain at least a 6-ft distance from each other and, as much as possible, from collection staff. in addition, blood suppliers began to publicize the cleaning activities that they used to disinfect donor areas. meanwhile, within the hospital, there began to be concern that units of blood could theoretically promote transmission of sars-cov-2 if they were returned from the immediate area of a covid-19 patient to the blood bank and subsequently reissued to a different patient. in many large centers, it is known that blood may be issued to 3 to 4 patients and returned prior to issue to the patient who is ultimately transfused, but data are not kept on this phenomenon and there are no specific regulatory standards or benchmarks that apply. the bags themselves are made of a soft, permeable plastic that cannot be cleaned without the possibility of introducing the cleaning solution to the blood itself. prior to covid-19, many transfusion services already had best practices in place to prevent avoidable exposure of units of blood to potentially biohazardous areas, such as asking transfusionists to avoid opening coolers in patient areas until the decision to transfuse was final. however, due to the airborne nature of sars-cov-2, as well as the potential for large numbers of cases in various treatment wards, hospitals considered various methods to more definitively address this problem. possibilities included mandating the destruction of any units of blood that came into the direct patient area for a covid-19 patient (which is difficult to tolerate at baseline, and even worse during a blood shortage); issuing all blood units in plastic bags, with instructions to not open the outer plastic bag unless the decision to transfuse was final, and to wipe down the outer bag with an approved hospital disinfectant wipe prior to returning to the blood bank; and changing guidance to clinical personnel that is printed on the outside of validated blood transportation coolers ❚image 1❚. these activities required changes to internal blood bank operations, as well as strong communication to the general hospital audience during a time when information flow was already extremely high. as the extent of illness caused by the virus became clearer, the number of possible treatment modalities under investigation literally exploded. overnight, demand drugs with relatively narrow therapeutic applications-and unproven effect in the treatment or prevention of covid-19-suddenly exploded. 12 in this context, convalescent plasma, which has been employed for treatment of emerging infectious disease outbreaks for over 100 years-and had been shown to be of no benefit for treatment of ebola virus and of promise but uncertain efficacy for treatment of sars and h1n1 influenzasuddenly became of extreme interest to the scientific community and to the public. [13] [14] [15] [16] early experience from china, which featured case reports of small numbers of patients treated without controls, was viewed as promising despite the low quality of the underlying studies. 17 blood bankers, the majority of whom had never previously handled a unit of convalescent plasma, were suddenly viewed as content matter experts for this promising, but ultimately unproven treatment, and multiple clinical trials, as well as other access pathways, were planned in a matter of weeks. 18 at the time of this writing, there remained a lack of consensus on the efficacy of convalescent plasma in the treatment of covid-19, and availability of convalescent plasma was gradually improving but uneven. initial safety data, published in preprint format, appears to support the continued application of convalescent plasma to recent tattoo or piercing 12 mo 3 mo 9 travel to malaria endemic area 12 mo 3 mo 10 theoretical risk of creutzfeldt-jakob disease or variant creutzfeldt-jakob disease due to european travel or military service lifetime eliminated many restrictions that previously applied to military personnel, individuals treated with bovine insulin, and time spent in many european countries 11 patients with severe disease. 19 peer reviewed efficacy data are eagerly anticipated. placebo-controlled clinical trials are enrolling, and there is a national debate as to whether placebo-controlled, randomized clinical trials should be prioritized above assured access to convalescent plasma via expanded access. proponents of the randomized clinical trials identify that this approach is the most likely to determine with scientific certainty whether or not convalescent plasma is effective and preventing severe morbidity or death. advocates of expanded access note that information gathered during clinical trials will only apply to covid-19 and not to future emerging pathogens, limiting the import of the clinical trial findings and creating an imperative to provide compassionate access to any patient who could theoretically benefit. initially when considering the impact to the apheresis and cellular therapy units, the first concern was to minimize the risk of exposing patients with compromised immune systems to sars-cov-2. centers across the country were advised to restrict visitors to the units as well as restrict any staff members with respiratory symptoms and where feasible utilize telemedicine visits in place of in-person visits. patients and health care workers were advised to wear masks as well as health care workers with face-to-face care of the patients. all patients who could safely defer cellular therapies were advised to do so. in the event transplant could not be postponed, patients were advised to self-isolate for up to 14 days prior to receiving any cellular therapies as well as undergo covid-19 testing to ensure the donor candidate is negative. any candidates found to be positive were recommended to defer treatment until asymptomatic and negative by pcr on 2 occasions at least 1 week apart. in the event a patient had close contact with a covid-19-positive person, deferral of cellular therapy is recommended for 14 to 21 days and covid-19 testing of the candidate became negative. 20 donors of stem cells should self-isolate or at least avoid crowded locations for 21 days before donation. if donors have returned from travel of an area with community transmission, the recommendation is to defer collection for up to 4 weeks. 21 to accommodate these requirements, units communicated with each provider to defer routine therapies or to increase the intervals between treatments to accommodate maintaining safe distance between patients. patients undergoing therapeutic plasma exchange (tpe) for neurologic conditions were advised to maintain current treatments but to remain vigilant regarding social distancing and good hand hygiene. 22 photopheresis therapies for cutaneous t-cell lymphoma and stable graft-vshost disease were deferred for treatments or converted from every other week to 1 treatment monthly. importantly, patients with sickle cell anemia (sca) were most impacted as antigen-matched units became scarce as mobile blood drives were cancelled nationwide due to the closure of businesses, universities, and schools. the apheresis unit was tasked to work with the sca teams to review all patients and, where possible, convert patients to simple transfusion. those patients who had a history of cerebrovascular accident required more thoughtful examination, including review of hemoglobin fractionations over time and, where appropriate, increased the intervals between exchanges or partial exchanges were performed. 23 providers also considered starting or increasing hydroxyurea in an effort to maintain lower levels of hemoglobin s. there were initially reports of tpe being done in covid-19 patients with florid infections who developed sepsis, pneumonia, acute respiratory distress syndrome, and multisystem organ failure, most likely the result of cytokine storm with endothelial damage, inflammation, and hypercoagulability. [24] [25] [26] [27] in 1 report, 3 patients underwent tpe in a single center with reported recovery in all 3 patients after tpe. the report does not comment on the number of tpe treatments required and other medications utilized for these patients, but in a follow-up letter to the editor, the 3 patients are explained in more detail. surprisingly, the follow-up letter revealed that only 1 patient who developed an antiphospholipid syndrome during his covid-19 infection was treated with tpe successfully with an improvement in symptoms after 3 treatments. the other 2 patients were in fact treated with continuous renal replacement therapy and not tpe. recently there was another report of a single patient undergoing tpe for covid-19; this patient also received a combination of therapies, including intravenous immunoglobulin and steroids, and therefore the contribution of apheresis to recovery is difficult to determine. 24 based on american society for apheresis guidelines, the use of tpe in multisystem organ failure is listed as a category iii, indicating that this is used in patients who have failed medical therapy and is used as a rescue therapy and is most effective early in the course of treatment. randomized controlled trials for sepsis with multisystem organ failure have yielded mixed results. exchange procedures must be performed with donor plasma, and the average length of time for performing the procedure is up to 14 days. 28 currently, the risk to the apheresis nurse is high considering the length of time to perform the procedure. some institutions have advocated extended tubing for both dialysis and apheresis instruments so the nurse performing the procedure can connect the patient to the instrument and then remain outside of the patient's room for the duration of the procedure. in larger institutions, the il-6 agonist, tocilizumab, has been used to treat the cytokine storm and florid inflammatory process in patients with fulminant covid-19 infections. one theoretical risk of tpe is alteration of the coagulation cascade, which is already perturbed in many advanced cases of covid-19 and is associated with a high degree of morbidity and mortality. this risk should be carefully weighed, especially when considering that the literature contains significant advocacy for extensive plasma exposure to covid-19 patients, some with no actual patient experience to support the proposed practice. 27 although early on in the pandemic it was believed that children were very unlikely to become seriously ill, as the pandemic continued some children were disturbingly found to suffer from a multisystem inflammatory syndrome. this condition is marked by fever, shock, and acute heart failure and has been managed with various combinations of immunosuppressive medical therapy, including intravenous immunoglobulin, steroids, anakinra, and infliximab. [29] [30] [31] the role of transfusion, as well as the role of tpe, remains unclear as of this writing. finally, and of note, there are reports of using blood "purification" filters as a component of apheresis therapy to reduce proinflammatory cytokines such as il-3, il-6, il-10, and other chemical markers of inflammation. experience with such inline filters, which are typically integrated downstream of where plasma is separated from whole blood in the apheresis device, are again largely limited to sepsis with multiorgan failure and have shown mixed, at best, outcomes in reducing mortality. 28, 32 nonetheless, the fda did issue an emergency use authorization (eua) for a blood filtration product, the depuro d2000 adsorption cartridge, for the treatment of complications of covid-19. 33 per the fda eua release, the depuro filtration system should be applied only to patients 18 years of age or older admitted to intensive care units with confirmed covid-19 and definitive or imminent respiratory failure. as of this writing, the device is not commercially available for routine use but is being studied in clinical trials per a communication from the manufacturer received by the authors. overall, the hospital transfusion service and the blood suppliers have responded to the covid-19 health crisis by working with the broader medical center to understand the effect of social distancing on supply, alerting the public of the need to donate blood, and working with recovered patients and regulators to safely collect convalescent plasma. at the same time, efforts have been taken to assure safe, continued access to apheresis treatments for patients who are apheresis-or transfusion-dependent, such as patients with myasthenia gravis and sickle cell disease complicated by previous stroke. the role of tpe for the treatment of covid-19 remains uncertain at this time, and, similar to what is ongoing with convalescent plasma, if this therapy is pursued it should be done as a part of a carefully constructed clinical trial. the las vegas mass shooting: an analysis of blood component administration and blood bank donations effect of a national disaster on blood supply and safety: the september 11 experience disaster operations handbook v 2.0 the national plan for management of shortages of labile blood components balancing supply and demand for blood during the covid-19 pandemic but they're "worried about four weeks from now aabb. update: impact of 2019 novel coronavirus on blood donation. 2020 revised recommendations for reducing the risk of human immunodeficiency virus transmission by blood and blood products united states food and drug administration. recommendations to reduce the possible risk of transmission of creutzfeldt-jakob disease and variant creutzfeldt-jakob disease by blood and blood components: guidance for industry treatment of covid-19: old tricks for new challenges convalescent plasma treatment reduced mortality in patients with severe pandemic 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followed by intravenous immunogloblin in a critically ill patient with 2019 novel coronavirus infection a novel treatment approach to the novel coronavirus: an argument for the use of therapeutic plasma exchange for fulminant covid-19 efficacy of therapeutic plasma exchange in severe covid-19 patients get rid of the bad first: therapeutic plasma exchange with convalescent plasma for severe covid-19 guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the writing committee of the american society for apheresis: the eighth special issue multisystem inflammatory syndrome in children during the covid-19 pandemic: a case series acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov-2 pandemic pediatric crohn's disease and multisystem inflammatory syndrome in children (mis-c) and covid-19 treated with infliximab the effect of a novel extracorporeal cytokine hemoadsorption device on il-6 elimination in septic patients: a randomized controlled trial united states food and drug administration key: cord-006039-vbq9izw3 authors: coban, cevayir; lee, michelle sue jann; ishii, ken j. title: tissue-specific immunopathology during malaria infection date: 2018-01-15 journal: nat rev immunol doi: 10.1038/nri.2017.138 sha: doc_id: 6039 cord_uid: vbq9izw3 systemic inflammation mediated by plasmodium parasites is central to malaria disease and its complications. plasmodium parasites reside in erythrocytes and can theoretically reach all host tissues via the circulation. however, actual interactions between parasitized erythrocytes and host tissues, along with the consequent damage and pathological changes, are limited locally to specific tissue sites. such tissue specificity of the parasite can alter the outcome of malaria disease, determining whether acute or chronic complications occur. here, we give an overview of the recent progress that has been made in understanding tissue-specific immunopathology during plasmodium infection. as knowledge on tissue-specific host–parasite interactions accumulates, better treatment modalities and targets may emerge for intervention in malaria disease. supplementary information: the online version of this article (doi:10.1038/nri.2017.138) contains supplementary material, which is available to authorized users. activation-induced cytidine deaminase (aid) . an enzyme that is required for two crucial b cell events in the germinal centre: somatic hypermutation and class-switch recombination. by incomplete immunity to malaria causes long-term hidden pathologies 20 . one such pathology is burkitt lymphoma, a cancer that develops in both childhood and adulthood in africa and that is closely related to p. falciparum and epstein-barr virus co-endemicicity 21 . the robust and long-lasting expansion of germinal centre b cell populations that occurs during plasmodium infection may induce increased activation-induced cytidine deaminase (aid) expression, eventually leading to chromosomal translocations [22] [23] [24] that predispose to the development of lymphoma in immune tissues. an increasing recognition of the association between malaria infection and physical growth retardation in children in africa, regardless of nutritional status 25, 26 , suggests a detrimental effect of chronic malaria infection on growth, possibly via an effect on the bone tissue environ ment 27 . nevertheless, any plasmodium spp. causing infection in humans result in varying degrees of complications, which can not only be severe and life threatening but can also have a long-term impact on patients' quality of life even after recovery. in this review, we focus on how systemic infection by plasmodium parasites causes local but tissue-specific immunopathologies during the blood stage of infection, mainly through the manipulation of inter-tissue interactions between the blood and other host tissues that often result in dysfunction of certain, but not all, organs. acute systemic immune activation, such as pro-inflammatory cytokine production, lymphocyte activation and vessel congestion, is evident; however, how these pathological events affect each tissue or organ is not understood well. current interventions for malaria are based on the administration of anti malarial drugs aimed at killing parasites and on supportive care to reduce systemic symptoms, such as coma, high fever, severe anaemia and acidosis. in this review, we emphasize the need to focus on host interactions with plasmodium parasites at various tissue levels and the importance of targeting local and specific organ failure and/or pathologies during, as well as long after, infection. these pathologies might be critical for determining diagnostic as well as therapeutic targets for the development of novel adjunct therapies to be used in combination with current antimalarials. we describe new evidence and new ways of targeting infected tissues of a few, but not all, unique organs such as the brain, gut and bones. we initially summarize how the blood tissue plays a central role in the initial pathogenesis caused by plasmodium parasites, following which we explore the interaction of infected blood tissue with specific organs composed of multiple, unique interacting tissue layers. largely owing to limitations of space, topics regarding the sporozoite stages of infection in the liver and those on the innate immune recognition of plasmodium para sites 28, 29 are out of the scope of this review. in fact, plasmodium parasites do possess several ligands used to manipulate host cell invasion that could potentially be exploited as host factor targets for anti-malaria therapy 30, 31 , a notion close to the topic of this review, but we exclude and leave this to an excellent recent article focusing on this topic 32 . pathology within the blood tissue blood is a tissue (box 1) that is formed by the plasma and blood cells (that is, erythrocytes, platelets and leukocytes) and is primarily located in the blood vessels. the infection of erythrocytes by plasmodium parasites results in extensive erythrocyte remodelling and dysfunction followed by cell lysis, which contribute to the pathology of severe anaemia. anaemia. plasmodium parasites (a merozoite form) are released into the bloodstream from liver hepatocytes within specialized vesicles called merosomes 33 and continue a repetitive erythrocyte-invasion cycle in the blood tissue. merozoites released from the liver enter into erythro cytes through a very dynamic and complex process 34, 35 (fig. 1) . once erythrocytic infection is established, continuous destruction of erythrocytes due to schizont rupture contributes to anaemia. a moderate degree of anaemia is caused simply by the naturally occurring life cycle of para sites in erythrocytes. however, studies in both humans and mice have clearly indicated an increased removal of infected and uninfected erythrocytes that plays an essential and major role in severe malarial anaemia [36] [37] [38] . clinical observations and mathematical modelling have suggested that for each infected erythrocyte, approximately 10 uninfected rbcs are removed during malaria 37 . chronic anaemia may also be mediated by the generation of self-reactive anti-phosphatidylserine antibodies 42 . phosphatidylserine on infected erythrocytes is thought to be pathologically exposed to the immune system during malaria, leading to the generation of anti-phosphatidylserine antibodies. under usual circumstances, uninfected erythrocytes, mainly young cells called reticulocytes, express high levels of cd47, a 'do not eat me' signal 43, 44 , which allows them to escape from phagocytosis. however, young erythrocytes generated during malaria expose phosphatidylserine earlier, thus leading anti-phosphatidylserine antibodies to bind to uninfected erythrocytes and facilitate their clearance, contributing to chronic anaemia. one of the outcomes of the destruction of high numbers of erythrocytes during malaria is an increase in intravascular haem release 45, 46 , which is an important factor for neutrophil activation 47 but in turn could be the reason for increased oxidative damage and thus decreased macrophage function and the neutrophil exhaustion observed during malaria. this implies that not only erythrocytes but also blood tissue components including various leukocytes collectively react to the presence of plasmodium parasites (covered previously by seminal review articles 35, 48, 49 ) and their related products in the blood circulation and tissues (fig. 1) . erythrocytes undergo extensive deformation and remodelling after invasion by plasmodium parasites. while remodelling allows nutrient acquisition and parasite growth, it leads to changes in erythrocyte structure, with an increase in rigidity 50 and rosetting 51 . hence, the mature schizont stages easily sequester in the host microvasculature, mostly during p. falciparum and p. knowlesi infection and to a lesser extent during p. vivax infection 50 , whereas immature ring stages freely circulate in the vessels. plasmodium berghei anka infection in mice shows a similar sequestration phenotype [52] [53] [54] [55] , and in this instance, sequestration via cd36 may be beneficial for the survival of the parasite 56 . it is believed that infected erythrocytes, mostly those in the schizont stages of parasite infection, adhere along with activated leukocytes (mainly cd8 + t cells) to the endothelial cells of small blood vessels in the brain via binding to cd36, intercellular adhesion molecule 1 (icam1), endothelial protein c receptor (epcr) and platelet endothelial cell adhesion molecule (pecam1) [57] [58] [59] [60] . however, endothelial cell acti vation can occur without direct adhesion of leukocytes to the endothelial cells, possibly as a result of metabolites produced by leukocytes or parasites 61 . indeed, there are unidentified soluble factors released by irbcs that cause endothelial cell pathology 62 . in line with this, endothelial cells, irbcs, platelets, leukocytes and monocytes were shown to increase their release of extracellular vesicles during plasmodium infection, and this contributed to inflammation and correlated with disease severity 63 . extracellular vesicles are mostly released from irbcs during schizogony and contain rbc components, parasite proteins and various rnas that can activate innate immune cells 64 . in addition, endothelial cells have been shown to act as antigen-presenting cells (apcs) through the phagocytosis of merozoites and the presentation of malarial antigens to cd8 + t cells, which leads to ifnγmediated and perforin-mediated disruption of the blood-brain barrier (bbb) 61 . most of these models of malaria-induced pathology are still under debate, and more evidence from both humans and animal models is clearly needed. overall, while the process of sequestration is not completely understood, it is known to cause obstruction of blood flow in small capillaries and post-capillary venules (pcvs), endothelial cell activation and inflammation and severe pathology in many organs including lung, adipose tissue, spleen and brain 52, 53, 65, 66 (fig. 1) . plasmodium-infected erythrocytes, is transported to organs via blood vessels. between large arteries and veins, various smaller sized vessels such as arterioles, capillaries and pcvs are present and have a role in controlling blood pressure and velocity in organs (fig. 2a) . the speed of blood flow substantially decreases as the blood enters arterioles, drops dramatically again in the capillaries and then slightly increases in the venules and veins (fig. 2a) . depending on the size and tissue environment, blood vessel structures vary. generally, the smaller the diameter of the vessel, the less smooth muscle it contains. capillaries, which play a major role in exchanging materials in blood, are the smallest vessels and are composed of endothelial cells with firm tight junctions and a basement membrane and do not include a smooth muscle layer (fig. 2b) . unlike capillaries, pcvs may have a few thin smooth muscle a tissue operates as an orchestration of large numbers of similar cells for a specific function. mammals have four basic types of tissues: epithelial tissue, muscle tissue, nerve tissue and connective tissue. two or more different tissues form and function as an organ. in general, the cells in their related tissue environment and organ are found in harmony, but during infection and inflammation, this harmony is likely to be disrupted. depending on the organ that the tissue or tissues interact with and support, multiple specific layers are present, which gives tissue specificity to healthy and diseased conditions. connective tissue deserves special attention because it includes various subcategories such as blood tissue, which is a major target of plasmodium parasites. this tissue, which mainly connects and supports various other tissues, is composed of various subtypes in addition to blood tissue, such as adipose tissue, areolar tissue, cartilage and bone tissue, lymphatic tissue and fibrous tissue. the main characteristics of connective tissues are that they contain an extracellular matrix composed of various collagen and/or elastin fibres, they contain interstitial fluid and they are vascularized with blood vessels. importantly, blood vessels are lined by endothelial cells, a component of epithelial tissue, and covered by smooth muscles (a muscle tissue); they carry blood cells, infected erythrocytes and various materials to the tissues and organs of the entire body, suggesting that even at the basic blood vessel level, many complex tissues specifically interact with each other. because tissue specificity occurs within an organization of different interacting tissues, it is reasonable to hypothesize that plasmodium parasites will reach different tissues and organ environments and develop unique and specific interactions with other tissues via blood tissue. also known as virchow-robin spaces. spaces located between brain-penetrating pial vessels and the brain tissue grey matter. (isf). the solution present extracellularly, that fills the spaces between cells and tissues. (csf). the solution surrounding the brain and spinal cord, which mainly serves to protect these two important organs. layers (fig. 2c) . importantly, depending on the organ, capillaries and pcvs interact with additional cells in tissues (fig. 2d) . for instance, brain capillaries and pcvs are additionally surrounded by pericytes, astrocyte end-feet and microglia (fig. 2d) . in particular, pcvs differ from capillaries in that they have leakier tight junctions and thus, may create perivascular spaces that apcs can easily home to 67 and that allow for the infiltration of inflammatory cells during inflammatory conditions 68 . moreover, the capillary beds in various organs are mostly in close proximity to lymphatic capillaries, into which drain interstitial fluid (isf; mainly leukocytes) and materials coming from blood vessels. the isf drained into the lymphatic vessels reaches the lymph nodes and finally returns to the final destination veins (fig. 2a) . therefore, it is reasonable to think that the type and diameter of a blood vessel in a specific organ in addition to the specific connective tissue and smooth muscle that is present to support vessel integrity may determine the degree of sequestration of plasmodium parasites and the outcome of disease. the walls of the smallest capillaries and pcvs are presumably more susceptible to internal changes occurring in the vessels. therefore, in the following sections, we detail the blood vessels within a few organs, such as the brain, gut and bones, and highlight their unique involvement with irbcs during malaria. tissue pathology within the brain the brain is severely affected by p. falciparum infection and to a lesser extent by p. knowlesi and p. vivax infections. this unique brain pathology, known as cerebral malaria, involves convulsions, coma and high fever and develops with the presence of mostly ring-stage infected erythrocytes in the periphery (suggesting a sequestration of late-stage parasites in the organs) [69] [70] [71] . disruption of bbb integrity is an established outcome in cerebral malaria and may cause brain swelling and death in both humans and animals 72, 73 . the activation of blood tissue components and their effect on brain immune cells (microglia, astrocytes) have been addressed 53,74-80 , but how an obligate erythrocyte-resident pathogen can cause bbb disruption is not well understood. although the brain is considered an immune-privileged organ that is protected by the presence of the tight and selective bbb and by the lack of lymphatic drainage (a notion that has been disputed recently 81, 82 ), the loss of bbb integrity due to the direct invasion and inflammation of meninges by various extracellular pathogens is well known 83 . to understand why and how brain tissue is affected during malaria, we should consider recent advances in the neuroscience field, which could help to expand our understanding of cerebral malaria pathogenesis and development. the central nervous system (cns) is composed of the brain and spinal cord, which are protected by the meninges (formed by three layers: dura mater, arachnoid mater and pia mater) and are associated with two types of fluids: cerebrospinal fluid (csf) running through the subarachnoid space and isf in the brain and spinal cord parenchyma (box 2) . csf is derived from plasma but has far fewer proteins, no erythrocytes, very nature reviews | immunology cells neutrophils, monocytes, platelets, t and b cells etc. brain, lung, kidney, intestine, bone etc. following the release of plasmodium merozoites from infected hepatocytes into the bloodstream, repetitive erythrocyte-invasion cycles occur in the blood. this leads to the release of several parasite by-products, such as haemozoin, and the parasites themselves into the bloodstream. the blood-stage cycle of plasmodium infection causes various pathologies, such as anaemia, toxic haem release, immune cell activation (modulation of platelets, neutrophils, monocytes, macrophages, t cells and b cells) and can cause a cytokine and chemokine storm. in blood tissue, infected red blood cells (irbcs) and their products may interact with other infected and uninfected rbcs (causing rosetting), or they may interact with immune cell populations (causing a cytokine storm) or with the endothelial cells of blood vessels (causing rbc sequestration and microhaemorrhage). these cell-cell interactions have organ specificity and thus take place in specific tissue environments, resulting in specific immunopathologies. an organ located on the cribriform plate, which functions in smell. the bulb surface is surrounded by complex olfactory nerve structures that originate from the nasal cavity and project to the brain. small trabecular capillary structures are the main vessel structures inside the bulb. few leukocytes and higher sodium, chloride and magnesium contents. isf fills the basement membranes of brain capillaries, where most of the exchange between blood and the cns occurs. the selective bbb inside the brain blood vessels, especially capillaries, is composed of uniquely specialized endothelial cells with intracellular tight junctions, pericytes, astrocyte end-feet and microglia in addition to the basement membrane, suggesting that blood vessel structures in the brain differ from those in other organs of the body, with a unique involvement of brain cell astrocytes and microglia in disease processes 84 (fig. 2d) . advancing our understanding of brain physiology (box 2) clearly has important implications for neurological diseases, such as alzheimer disease and multiple sclerosis 85, 86 , as well as for cerebral malaria. indeed, recent preclinical studies using cutting-edge imaging technologies, such as ultra-high-field 11.7 t magnetic resonance imaging (mri) and multi photon live imaging microscopy, in experimental cerebral malaria models have expanded our understanding of how cerebral malaria develops. below, we consider the roles of the retina, olfactory bulb and the perivascular spaces of the brain during cerebral malaria. arteries supplying blood to the brain divide into smaller arteries on figure 2 | interaction of plasmodium-infected red blood cells with various blood vessels and lymphatics. a | infected red blood cells (irbcs) circulate in blood vessels, which vary in size from large arteries to veins, with blood flow running from arteries, arterioles, capillaries and post-capillary venules (pcvs) to venules and veins, providing controlled blood pressure and velocity in organs. the speed of blood flow is lowest in capillaries and pcvs. in various organs, these capillary beds are mostly near lymphatic vessels, through which interstitial fluid and materials coming from blood vessels drain the lymph nodes and finally return to veins. irbcs and irbc-mediated immune responses, therefore, may have profound effects on these smaller vascular beds in each organ. b | capillaries have only an endothelial cell wall and no smooth muscle; therefore, they are in close contact with irbcs. c | two capillaries fuse and form pcvs, which may have some smooth muscle and where leukocyte rolling can occur. d | capillaries and pcvs in the brain are additionally surrounded by pericytes, astrocyte end-feet and microglia; irbc-related events in capillaries are sensed immediately. a small, perforated bone structure that lies on top of the nasal cavity and supports the olfactory bulb. olfactory nerves running from the nasal cavity to the olfactory bulb pass thorough cribriform plate. the pial surface and penetrate into the brain parenchyma before further dividing into arterioles. arterioles (and venules) are surrounded by perivascular space which is filled with isf drained from the brain parenchyma (fig. 3a) . a newly defined 'glymphatic system' has been proposed in which the glia limitans, a barrier comprising astrocyte end-feet that surrounds small capillaries and veins, creates perivascular spaces via aquaporin 4 (aqp4) channels. the aqp4 channels facilitate the flux of isf into perivascular spaces to be mixed with csf 87, 88 , and this eventually contributes to the clearance of particles from the csf. interestingly, dilatation at perivascular spaces and astroglia and reduced expression of aqp4 protein have been reported during cerebral malaria in animal models 89, 90 . furthermore, in the absence of aqp4, mice with cerebral malaria succumbed to death more quickly 90 , suggesting aqp4 has protective roles during cerebral malaria, perhaps in controlling liquid exchange, oedema and cell transmigration at the bbb. apcs with phagocytic properties such as dendritic cells and macrophages located in the pial surfaces and perivascular spaces have been shown to present antigens to t cells in various cns diseases 91 . as visualized by electron microscopy and intravital two-photon microscopy, it seems that leukocytes and cd8 + t cells, and probably irbcs or their products, increasingly accumulate in perivascular spaces in very deep and branching vessels during cerebral malaria [92] [93] [94] [95] . these phagocytic apcs and activated endothelial cells in vessels have the capacity to present malarial antigens 96, 97 . furthermore, all these events that are mediated by ifnγ could be reversed by blocking the adhesion molecules box 2 | the steady state brain drainage system owing to the presence of the highly selective blood-brain-barrier (bbb), the brain has long been considered to have no classical lymphatic system and thus be an immune-privileged organ (see the figure) . in homeostatic conditions, the cerebrospinal fluid (csf) is continuously produced by the choroid plexus and drained into the bloodstream via arachnoid granulations in the venous sinuses or nasal lymphatics under the cribriform plate next to either the olfactory nerves or the spinal and cranial nerve sheaths. immune surveillance via selective epithelial cells and homeostatic leukocyte trafficking in csf have been acknowledged 145 ; however, recent studies by louveau et al. 82 and aspelund et al. 81 have challenged previous knowledge by demonstrating the presence of a classical lymphatic drainage system located in the brain meninges. the authors clearly showed that this lymphatic network works alongside the classical known csf drainage system, with the additional capacity to drain other constituents such as toxic macromolecules and immune cells into the deep cervical lymph nodes, providing evidence of a connection between the csf, the brain and the periphery. essentially, these newly identified brain lymphatic vessels lay in the dura mater and collect csf and interstitial fluid (isf) together with macromolecules such as amyloids and immune cells from perivascular spaces (pvss) of the brain parenchyma. similar lymphatics are also present around another immune-privileged organ, the eye (along with the optic nerve) 146 , and over the olfactory bulb, aligned throughout meninges as simple narrow vessels that become wider in the transverse sinuses, ultimately reaching the cerebellum. all lymphatic vessels finally drain into the deep cervical lymph nodes. it is of note that similar to blood vessels, these lymphatic vessels are different in size depending on their anatomical location in the central nervous system, and owing to that, their interaction with local blood vessels or astrocyte end-feet may be different during homeostasis than in diseased conditions. pcv, post-capillary venule. figure 3 | infected red blood cells in close proximity with brain components. a | small arteries on the pial surface of the brain penetrate into the brain parenchyma and further divide into arterioles; these are surrounded by perivascular space (pvs) which is filled with interstitial fluid (isf) that has drained from the brain parenchyma. arterioles become capillaries, venules then veins, which finally drain into dural venous sinuses, and each of these areas potentially contain abundant infected red blood cells (irbcs), as well as lymphocytes such as cd8 + t cells. lymphatic vessels located in the dura carry immune cells and cerebrospinal fluid (csf) into deep cervical lymph nodes. b | the retina is rich in photoreceptors (cones, bipolar cells and ganglions), nerve cells and complex blood vessels, especially capillary structures surrounded by müller cells, a retina-specific astrocyte-like cell. the optic nerve serves as a connector between the retinal nervous tissue and the brain, where meningeal membranes, dura lymphatics and csf cover the optic nerve up to the retina border. this area may contain abundant irbcs. c | the olfactory bulb is surrounded by meninges of brain dura and arachnoid and pial layers. csf similarly runs throughout the bulb and reaches the cribriform plate area. olfactory nerves originate from the nasal mucosa and terminate in the olfactory bulb through the cribriform plate, which is also the route of the blood vessels surrounding the olfactory nerves. these olfactory nerves project any signal for smell to the olfactory bulb and the brain. inside the olfactory bulb, very dense blood capillaries are oriented in different directions (radially and tangentially), with thin astrocyte end-feet surrounding the vessels, and sense irbc-related events and secrete several cytokines and/or chemokines including cc-chemokine ligand 21 (ccl21). blood capillaries easily bleed during cerebral malaria. pcv, post-capillary venule. a mechanism explaining the relationship between neuronal activity and the cerebral blood vessels and the blood flow. very late antigen 4 (vla4) and lymphocyte function-associated antigen 1 (lfa1) (ligands for vascular endothelial adhesion protein 1 (vcam1) and icam1, respectively), which are expressed on activated cd8 + t cells during cerebral malaria 98 . one of the important features of inflammation in perivascular spaces is that the parasite and inflammatory products may directly activate astrocytes 53, 99 , microglia 77 and neurons 100 , and may cause an increase in the levels of protein s100b (a biomarker for astrocytes) and in the levels of the microtubule-associated protein tau (a biomarker for degenerated axons) in csf 80 . nevertheless, future studies are needed to provide direct evidence of this. it would also be interesting to investigate in the future whether there are other types of perivascular spaces found at different locations in the brain 101, 102 , because pia mater also covers the cerebellum and other cns areas, such as the spinal cord. therefore, the interaction of perivascular spaces with small blood vessels may vary depending on the anatomical location and unique architecture of the tissue, for example, in the brain cortex, spinal cord, cerebellum or olfactory bulb. the back of the eye ball is lined by the retina, with the optic nerve in the middle and going through to the brain. the optic nerve serves as a connector between the nervous tissue of the retina and the brain, where brain meninges, dura mater lymphatics and csf cover up the optic nerve up until the retina border. the retina is rich in photo receptors, nerve cells and complex blood vessels, especially capillary structures (fig. 3b) . retinal changes (such as haemorrhages, multiple peripheral and macular patchy whitening areas and papilloedema) have been commonly observed in children and in adults who develop sudden comatose cerebral malaria symptoms after a few days of fever [103] [104] [105] . these retinal changes occur in areas that show high sequestration of parasites accompanied by thrombi (composed of fibrin and platelets) 106, 107 , which cause impaired capillary perfusion 108, 109 and are well correlated with the severity of disease 110 . interestingly, other areas in the eye, such as the optic nerve head, ciliary body and iris, are similarly affected by irbcs 110 , indicating that specific blood vessel similarities may occur within other parts of the brain. retinal homeostasis is maintained through neurovascular coupling involving astrocytes, müller cells (a specific type of glial cell found in the retina) and resident retinal microglia, with müller cells, pericytes and astrocytes mainly ensheathing the retinal blood capillaries 111 . the observation that the accumulation of fluid between processes of müller cells and photoreceptor cells in the macular area produces intra-retinal spaces in human cerebral malaria 107 suggests that these areas represent perivascular-space-like areas in the retina that support immunological events similar to those that occur in the perivascular spaces in deep brain areas. furthermore, high-field mri studies have identified damage to the optic and trigeminal nerves during experimental cerebral malaria even before the development of oedema, which may cause loss of visual acuity 112 . the olfactory bulb and cerebral malaria. the olfactory bulb is situated on the cribriform plate, located on top of the nasal sinus. although the olfactory bulb has several characteristics that distinguish it from the brain, it is covered by similar meningeal structures and by the csf and is thus included in the cns. however, in contrast to other parts of the brain, olfactory nerves initiate from the nasal mucosa and terminate in the olfactory bulb through the cribriform plate, which is also the route of the blood vessels surrounding olfactory nerves. these olfactory nerves project any signals for odour to the olfactory bulb and to the brain. inside the olfactory bulb, very dense blood capillaries are oriented in different directions (radially and tangentially) with thin astrocyte end-feet surrounding the vessels (fig. 3c) . owing to complex structural interactions of nerves through the cribriform plate, capillaries, microglia and astrocyte endfeet in this region, the olfactory organ is considered to be the gateway of the otherwise well-protected brain to the outside world. it is known that several molecules, cells and even pathogens can gain access to the brain parenchyma through this route 113 . by use of 11.7 t mri in mice, it was discovered that the olfactory bulb is the first region during experimental cerebral malaria to show vascular leakage and bleeding 53 , a phenomenon possibly related to the unique small capillary trabecular structure of the olfactory bulb. this was also directly visualized and confirmed by intravital two-photon microscopy during cerebral malaria 53 . these dense and directionally structured olfactory blood capillaries 114 , which are thought to be leakier than those in the brain cortex or pons 115 , are a suitable scaffold for irbcs 53 . sequestration of irbcs in the olfactory bulb capillaries results in astrocyte and microglia activation 53, 76, 98 and in the loss of tight junction integrity, and these parasite-mediated events lead to olfactory dysfunction and loss of smell, which could be a valuable early diagnostic marker for cerebral malaria. moreover, astrocytes surrounding small trabecular capillaries in the olfactory bulb were found to sense changes in the vessels and secrete cytokines and chemokines, such as cc-chemokine ligand 21 (ccl21), which are involved in the accumulation of pathological cxcr3 + cd8 + t cells in the olfactory bulb 53 . expression of cc-chemokine receptor 7 (ccr7), which is the canonical receptor for ccl21 and ccl19, was shown to be essential for antigen cross-presentation by cd8α + dendritic cells and for the activation of cxcr3 + cd8 + t cells in the spleen during cerebral malaria 53 . by contrast, ccl21 secreted from astrocytes played a role in the recruitment of pathological cxcr3 + cd8 + t cells in the olfactory bulb via its non-canonical receptor cxcr3 (ref. 53) . a pathological role for several chemokines and chemokine receptors (including cxcchemokine ligand 4 (cxcl4), cxcl10 and cxcr3) is well recognized in experimental cerebral malaria 116 , but the study discussed above 53 highlights how the specific role for chemokines in cerebral malaria might differ depending on the tissue and organ environment. extended mri studies using sensitive contrast reagents have further confirmed these findings and have shown that micro-haemorrhages originate in the olfactory lacteals small lymphatic capillaries located in the villi of the small intestine. vessels with a structure similar to capillaries but that have larger diameters and porous endothelial cells, which allow for permeability and exchange of materials; they are located in bone marrow, liver and spleen. bulb together with oedema, but that the oedema further spreads along the rostral migratory stream, similar to the migration route of immune cells and neuroblasts 76 , and may reach the brainstem 98 . moreover, the glymphatic system may play a key role in the rapid inflammatory spread of cerebral malaria from the olfactory bulb and rostral migratory stream axis to deeper brain areas such as perivascular spaces 76 . it is also possible that these events that occur in the olfactory bulb during cerebral malaria may block the drainage of csf into nasal lymphatics and aggravate brain oedema (fig. 3c) . clearly, the role of the olfactory bulb in cerebral malaria in humans warrants further research. tissue pathology within the gut gastrointestinal symptoms such as abdominal pain, vomiting and diarrhoea are common symptoms during malaria infection. autopsies of human patients who died from severe malaria have shown that sequestration of irbcs occurs in the gastrointestinal tract (including in the colon, jejunum, ileum and stomach) 13 , suggesting that gastrointestinal bleeding occurs during malaria. the presence of plasmodium dna in faeces additionally supports the occurrence of intestinal bleeding during infection 117 ; however, how parasites interact with other cells in gut tissue is a research area that has only recently gained attention. recent studies have confirmed that pathological changes (including detachment of epithelia and shortening of villi and the colon) occur in the intestinal tract during malaria 118 ; such changes may cause increased intestinal permeability, ruptures and leakage of infected erythrocytes into the lumen, causing dysbiosis of the intestinal microbiota, which may also contribute to disease severity 118 . moreover, there is a close correlation between malaria infection and increased rates of infection with other gut pathogens (for example, nontyphoidal salmonella and helminths), possibly owing to an impairment of intestinal barrier function during malaria [119] [120] [121] . several factors may have a role in this, such as increased histamine release from mast cells in the gut 122 or the modulation of intestinal inflammation and immune responses via an increase in the number of exhausted neutrophils 45 or an increase in the levels of il-10 family cytokines, including il-10 and il-22, in the intestinal tissues during malaria infection, as these factors may ameliorate responses to secondary bacterial infections [123] [124] [125] . however, it should be kept in mind that the composition of the gut microbiota has a clear impact on the severity of malaria disease [126] [127] [128] , suggesting that there is a bidirectional relation between the gut and plasmodium parasites that is not well understood and requires further investigation. nevertheless, a close look at the intestinal tissues may give some clues for understanding gut-plasmodium interactions (fig. 4a) . the blood capillaries carrying irbcs reach the lamina propria of the intestinal mucosa and possibly come into close proximity with lacteals, which are specialized lymphatic vessels of intestinal villi that play a key role in nutrient absorption and in gut immune responses, such as the transport of microbial antigens and apcs to the mesenteric lymph nodes, where antigen presentation by apcs promotes the induction of t cells with gut-homing properties 129 . there is a gut-vascular barrier comprising endothelial cells, enteric pericytes and enteric glial cells that is very similar to the barriers found in other parts of the body (such as the bbb) and that actively blocks bacterial dissemination from the gut epithelium to the blood 130 . therefore, it is reasonable to hypothesize that intestinal bleeding may occur as a result of malaria-mediated immune responses that directly affect lacteals. it is possible that lacteals with disturbed remodelling have impaired survival and villi length 118 , and this may have knock-on effects on immune cell composition and on the microbiota of the gut tissue. tissue pathology within bone bone tissue is a mineralized connective tissue supporting the whole body and it houses the bone marrow, which comprises specialized niches that maintain and regulate haematopoiesis, erythropoiesis and bone remodelling (fig. 4b) . arteries in the bone marrow unite and become specialized vessels called bone marrow sinusoids, which allow cells to pass between the circulation and bone marrow 131 . without any cyto adherence, plasmodium parasites can invade and multiply in nucleated erythroblasts located in extravascular spaces of the bone marrow [132] [133] [134] . the parasites can survive and hide in these erythroid precursors and develop into gametocyte stages [135] [136] [137] , likely assisting in the continuous success of malaria transmission to mosquitoes. however, it is still not known why plasmodium parasites preferentially differentiate into gametocytes in the bone marrow and whether a specific bone marrow niche is required to support the development of gametocytes. unlike haematopoietic niches, which are located adjacent to sinusoids, erythroid niches are found throughout the bone marrow and are generally located away from the sinusoids, although they move closer to the sinusoids as they mature 138 . this may suggest a major role for the erythroid niche in plasmodium gametocyte development. from an immunological point of view, it has recently been shown that cd8 + t cells produce ifnγ in response to infected mhc class i-expressing erythroblasts residing in the bone marrow; by contrast, mature erythrocytes residing in the blood circulation lack any mhc molecules and cannot present antigens to cd8 + t cells 132, 139 . activated cd8 + t cells promote the exposure of phosphatidylserine on infected erythro blasts, and this enhances the susceptibility of the infected cells to phagocytosis by macrophages 132 , suggesting a protective role of cd8 + t cells against malaria. however, whether bone marrow erythroid cells contribute to the expansion of pathological populations of cd8 + t cells that can drive cerebral malaria has not been investigated. despite the growing body of information on how bone marrow niches are manipulated by plasmodium parasites, very little is known about the potential outcomes of interactions between plasmodium parasites and bone tissue. in bone marrow, haematopoietic stem cells give rise to blood cells and bone-resident osteoclasts, whereas mesenchymal stem cells give rise to osteoblasts, adipocytes and stromal cells. a very recent study has suggested that during plasmodium infection, parasites and their products -mainly haemozoin -continuously accumulate in bone marrow and cause acute as well as chronic bone loss 27 . although it is not precisely known how plasmodium products are retained long term in the bone marrow, it is possible that engulfment by macrophages or other phagocytic cells, such as bone-resident osteoclasts, or trapping by extracellular matrix in the bone marrow contributes to this phenomenon 27 . chronic bone loss during malaria infection is mediated by over-activation of osteoclast resorption activity. the osteoclasts are activated by the key osteoclastogenic cytokine rankl (also known as tnfsf11), which is upregulated in osteoblasts through myd88-dependent signalling triggered by the persistence of parasite products in the bone marrow 27 . these findings highlight how, not only during acute malaria infection but also after recovery from infection, plasmodium products continue to interact with tissueresident cells, including bone cells, osteoclasts and osteoblasts, and how they can cause long-term effects in the host, such as bone loss and growth problems. the molecular and immunological pathways underlying the interaction between plasmodium spp. and the niches of various bone marrow cells and the effect of these interactions on the immune system 140, 141 need to be addressed in the future. malaria is a serious disease with acute life-threatening and long-term complications, all of which can be attributed to local but specific organs in which plasmodium figure 4 | infected red blood cells in gut and bone marrow niches. a | the intestinal tract is made up of several tissue layers. the villous mucosa is covered by mucus that is secreted by the goblet cells of the epithelium. it lies on a connective tissue, the lamina propria, which is an environment rich in blood vessels, lymphatics, nerves and immune cells. it also contains peyer's patches and is lined by smooth muscle tissue (muscularis mucosa). blood capillaries that are carrying infected red blood cells (irbcs) reach the lamina propria of the intestinal tract and can come into close proximity with lacteals at this location. b | bone tissue supports the whole body and is home to bone cells, blood vessels, nerves and endothelium, as well as containing the bone marrow niches. the bone marrow is composed of haematopoetic and mesenchymal stem cells that are involved in both haematopoiesis and bone remodelling, such as osteoclasts and osteoblasts. cells and irbcs pass through the circulation and bone marrow via specialized venules called bone marrow sinusoids. plasmodium parasites can invade erythroblasts, the erythrocyte precursors, and preferentially form gametocytes in the bone marrow erythroblastic niche. released plasmodium products including haemozoin can be engulfed by osteoclasts and activate osteoblasts and osteoclast precursors. hsc, haematopoietic stem cell; msc, mesenchymal stem cell; osb, osteoblast. haemozoin a unique by-product of plasmodium parasites that accumulates in several organs, including spleen, liver and bone marrow, even after the infection resolves. parasites and their products or irbcs interact with the tissue, causing an imbalance in the specific tissue environ ment. here, we have attempted to summarize the recent progress of research focusing on the tissue environment in a few example organs, such as the brain, gut and bone, where plasmodium parasites reach the tissue via the blood. however, the lungs 142 , kidneys 143 and placenta 144 represent other organs that are seriously affected during malaria infection, and these tissues should also be examined more closely in future studies. here, our aim has been to show that the influence of malarial parasites reaches beyond erythrocytes and may vary depending on, and be tightly regulated by, the vessel-specific and/or tissue-specific microenvironment. on the basis of the available data, we suggest that the brain pathology caused by plasmodium parasites is not homogeneously controlled by parasite sequestration but is rather controlled by the local, specific and unique anatomical structure of vessels, parenchyma and lymphatics, which closely interact with each other during both homeostasis and infection. this concept is in accordance with the recent understanding that the vascular and epithelial barriers do cooperate in an organ-specific manner in homeostatic and diseased conditions 130 . we additionally emphasize that parasite-derived products can continue to stay in the body, interact with tissues and cause chronic pathologies even long after recovery from infection, particularly in the case of bone tissue. the gut pathology caused by malaria, which has recently been recognized, was summarized, and the possible anatomical interaction between blood tissues and special gut lymphatics has been introduced. there are multiple benefits of understanding malaria-related pathologies in various tissues. for instance, understanding the reaction of microglia and astrocytes to irbcs will be beneficial for generating intervention strategies that block these interactions to prevent cerebral malaria and/or related long-term sequelae. supporting the gut microbiota during and after plasmodium infection might help to protect against and promote recovery from secondary infections. similarly, additional therapies like vitamin d 3 supplementation to support the bone environment affected during and after malaria might help the growth of children or support bone health in elderly people. the search for a full understanding of malaria-related pathologies is important for ensuring that malaria is one day a curable and/or preventable disease. towards this goal, novel approaches should be investigated to analyse unknown or overlooked anatomical -as well as immunological -host-parasite interactions at various tissue levels of each organ, not only during infection but also long after clearance of the initial infection by the parasite. by doing so, we will be able to prevent the disease, diagnose and treat patients, predict prognoses and allow patients to receive appropriate medical care through the use of effective vaccines, diagnostic tools, adjunct therapies and antimalarials in a cost-effective and timely manner in the near future. global, regional, and national incidence and mortality for hiv, tuberculosis, and malaria during 1990-2013: a systematic analysis for the global burden of disease study bad air, amulets and mosquitoes: 2,000 years of changing perspectives on malaria mapping plasmodium falciparum mortality in africa between prioritising infectious disease mapping is plasmodium vivax malaria a severe malaria?: a systematic review and meta-analysis clinical profile of plasmodium falciparum and plasmodium vivax infections in low and unstable 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hematopoietic stem and progenitor cells respiratory manifestations of malaria malaria-induced renal damage: facts and myths the sick placenta -the role of malaria orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates the schlemm's canal is a vegf-c/ vegfr-3-responsive lymphatic-like vessel pathophysiology of severe malaria in children increased gastrointestinal permeability in patients with plasmodium falciparum malaria changes in gastric mucosa in acute malaria plasmodium vivax: clinical spectrum, risk factors and pathogenesis plasmodium ovale: parasite and disease plasmodium ovale: a case of not-so-benign tertian malaria plasmodium malariae: parasite and disease human infections and detection of plasmodium knowlesi the authors are supported by grants-in-aid for scientific research (b grant no. 16h05181 to c.c.) and by the japan agency for medical research and development (amed j-pride 17fm0208021h0001 to c.c.). c.c. wrote the manuscript; all authors contributed to researching data, discussing the content and reviewing and editing the manuscript. the authors declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-016871-1mlamf20 authors: streiff, agathe; cotton, bryan a. title: hemorrhage and transfusions in the surgical patient date: 2012-10-30 journal: common problems in acute care surgery doi: 10.1007/978-1-4614-6123-4_12 sha: doc_id: 16871 cord_uid: 1mlamf20 hemorrhage is the leading cause of intraoperative deaths. many cardiovascular and hepatobiliary procedures result in massive hemorrhage and postpartum hemorrhage events in labor and delivery place the patient at a high risk for mortality. gastrointestinal bleeding from diverticulosis, varices, and ulcer disease can result in significant blood loss requiring massive transfusion and resuscitation from hemorrhagic shock. timely and effective transfusion of blood products is of critical in these scenarios. the frequency in which blood component products are transfused in surgical patients begs for a greater understanding of them. the aim of this chapter is to provide clinicians with a discussion of the current literature on the various blood component products, their indications, and unique hemostatic conditions in the surgical patient. while the majority of data concerning optimal management of acquired coagulopathy and hemorrhagic shock resuscitation is based on trauma patients, many of the principles can and should be applied to the surgical patient (or likely any patient) with profound hemorrhage. hemorrhage is the leading cause of intraoperative deaths. many cardiovascular and hepatobiliary procedures result in massive hemorrhage and postpartum hemorrhage events in labor and delivery place the patient at a high risk for mortality. gastrointestinal bleeding from diverticulosis, varices, and ulcer disease can result in signi fi cant blood loss requiring massive transfusion and resuscitation from hemorrhagic shock. timely and effective transfusion of blood products is of critical in these scenarios. the frequency in which blood component products are transfused in surgical patients begs for a greater understanding of them. the aim of this chapter is to provide clinicians with a discussion of the current literature on the various blood component products, their indications, and unique hemostatic conditions in the surgical patient. while the majority of data concerning optimal management of acquired coagulopathy and hemorrhagic shock resuscitation is based on trauma patients, many of the principles can and should be applied to the surgical patient (or likely any patient) with profound hemorrhage. the concept of the lethal triad-hypothermia, acidosis and coagulation-was fi rst promoted in the trauma population in those undergoing emergency surgery. in an effort to combat its development (or at least attenuate its effects), several authors began advocating for damage control surgery [ 1, 2 ] . however, the principles of damage control have spread through the trauma centers and into the operating theaters and intensive care units [ 3 ] . central to this concept is aggressively and rapidly addressing all three components simultaneously, as each greatly affects the other. hypothermia, a core body temperature of 34-36°c, in the trauma patient primarily results from re fl exive peripheral vasoconstriction in the hypovolemic patient. this phenomenon is further exacerbated by rapid infusion of unwarmed crystalloid fl uid during initial resuscitation. this condition impairs coagulation factor activity and platelet function, such as their ability to produce thromboxane, and must be rapidly reversed [ 4 ] . crystalloid and colloid fl uids also contribute to hemodilution of clotting factors, further promoting ongoing bleeding. hence, in this situation, early plasma therapy and platelets have been shown to improve outcomes [ 5 ] . acidosis has been hypothesized to result from hypoperfusion and excess administration of ionic chloride in normal saline administration. the acidosis disturbs platelet function and morphology, reduces coagulation factor complex activity, and degrades fi brinogen. approximately 25% of trauma patients present with abnormal coagulation parameters, and these have been associated with poorer outcomes in these patients. the three conditions previously mentioned contribute to poor clot formation and aggravated coagulopathy [ 4 ] . evidence exists supporting increased survival upon rapid treatment of initial coagulopathy [ 5, 6 ] . preemptive strategies have been shown to actually reduce coagulopathy and the number of overall transfusions required to treat the patient [ 7, 8 ] . however, challenges to implementation include time limitations of laboratory-guided component therapy since the results of the tests are not immediate. another dif fi culty is that once it has been determined that the patient should receive plasma, it may take another 30-45 min to thaw and deliver the products [ 5 ] . as such, hospitals should have in place a thawed plasma program, keeping adequate numbers of "universal" and type-speci fi c thawed plasma available for immediate release. plasma thawing protocols exist to avoid this issue and are discussed in later sections. in acutely bleeding patients, massive transfusion protocols are often activated in order to ef fi caciously restore blood volume and hemostasis and thawed plasma is critical to their success [ 5, 6 ] . a massive transfusion (mt) is de fi ned as more than ten units of red blood cells (rbc) in 24 h [ 5 ] . a massive transfusion protocol (mtp) is the standardization of the delivery and transfusion of rbc, plasma, and platelets in predetermined and prede fi ned ratios as facilitated by a surgical or medical team. in the patient requiring immediate resuscitation, a typical mtp will call for six to ten units of rbc, with a ratio of rbc to plasma and platelets in 1:1:1 to 1:1:2 fashion. this protocol and release of products will continue based on ongoing bleeding ( fig. 12.1 ). these assessments are generally implemented "blind," with subsequent releases guided by routine coagulation laboratory studies as well as thromboelastography (teg) [ 9 ] . even before the transfusions take place, mtps call for the rapid mobilization of blood components by having ab thawed plasma and group o rbc [ 10 ] . a type and screen should be drawn as soon as possible to allow for the transition from universal products to type-speci fi c ones. the ef fi cacy of an mtp also lies in its early implementation as well as identi fi cation of patients who would bene fi t from such an intervention. criteria for activation include laboratory values, anatomic injuries, and mechanism of injury. several authors have demonstrated that the transfusion of uncross-matched rbcs is an independent predictor of substantial hemorrhage and the transfusion of multiple units of rbc, plasma, and platelets [ 10, 11 ] . as such, when one is requesting uncross-matched product for transfusion, the institution's mtp should be activated. prior to the advent of mtps, resuscitation protocols for severely injured patients began with large volumes of crystalloid followed by rbc transfusions. later on, plasma, platelets, and cryoprecipitate were administered if the patient had survived the operating theater and then only based on laboratory values and the opinion of anesthesiologists and transfusion specialists. these guidelines recommended transfusions at prothrombin time ratio of >1.5, platelet counts of <50 × 10 9 /l, fi brinogen level <1.5-2.0 g/l or after a predetermined volume loss. this approach relied on a reactive strategy where the clinician was constantly "catching up" with values representing an earlier hemodynamic state of the patient [ 12 ] . while this standard resuscitation method is adequate for patients who are not in shock or not bleeding, studies have demonstrated that it does not suf fi ce for the subset of patients who have sustained serious injuries, are coagulopathic or in shock [ 5 ] . one reason is that the coagulopathy is addressed after a time lapse since the original laboratory values were obtained. other reasons for the suboptimal results of this method are due to the ratios of each blood component [ 5 ] product infused. speci fi cally, evidence exists that demonstrates that large volume of crystalloid fl uids is associated with increased hemorrhage and lower survival rates [ 13 ] . it has been hypothesized that this effect is due to insuf fi cient replenishing of hemostasis factors, and the complex coagulopathy of dilution, consumption of factors, and fi brinolysis is not adequately addressed. mtps also offer the advantage of reducing intraoperative crystalloid use and hence, reducing opportunities for hemodilution. damage control resuscitation (dcr) expands on the mtp process and calls for low-volume resuscitation, sparing the patient of resuscitation with fl uids such as crystalloids and colloids that are low in hemostasis factors [ 14 ] . instead, dcr adheres to transfusion of blood products in a ratio of plasma and platelets to red blood cells consistent with that which is being lost to hemorrhage. it also involves more permissive hypertension, and acting preemptively on the hypovolemic, hemorrhaging patient. dcr is also supported by fi ndings from the us army's institute of surgical research, which demonstrated improvement in outcomes in severely bleeding patients who were transfused in ratios of products similar to whole blood. civilian trauma data has also shown that rbc to plasma ratio between 3:2 and 1:1 lead to reduced 30-day mortality and increased odds of survival [ 5 ] . fox et al. found that patients undergoing vascular surgery with dcr had improved revascularization and graft patency. their results demonstrated that recombinant viia, whole blood, fresh frozen plasma (ffp), platelets, cryoprecipitate and minimal crystalloid prevented early graft failures [ 15 ] . while there is a wealth of data in the trauma population, less data is available regarding coagulopathy in the severely bleeding patient in other surgical specialties. it is, however, important to consider the underlying pathology responsible for exsanguination, such as in obstetric patients, as well as related comorbidities, such as uremia, pharmacologic anticoagulation, in assessing for need of blood products [ 5 ] . for instance, kılıç et al.'s review of resuscitation in patients with gastrointestinal bleeding found that 1:1:1 ratios of prbcs, ffps, and platelets reduced dilutional coagulopathy, similarly to trauma patients [ 16 ] . patients undergoing open thoracoabdominal aortic aneurysm repair are also vulnerable to coagulopathy due to systemic heparinization, hypothermia, and left-heart bypass with a centrifugal pump [ 17 ] . as well, several authors have noted its bene fi t in the vascular population [ 15, 18, 19 ] . mell evaluated 168 patients with ruptured abdominal aortic aneurysm who had massive hemorrhage in the perioperative period. their fi ndings showed reduced 30-day mortality in patients who were transfused 1:1 rbc to plasma ratios. these patients also experienced lower rates of colonic ischemia. the value of this study is that the average age of patients was 73 years, much older than the average trauma patient, demonstrating applicability of mtps in different patient age populations [ 19 ] . lastly, evidence on mtps has focused on the acutely bleeding surgical patient, and less is known about patients in other surgical settings. due to the less emergent nature of such settings, it is likely that mtps are activated more reactively, and it may have a different effect on patient outcome [ 5 ] . however, some groups have shown that those patients receiving less than massive transfusion levels may still bene fi t from higher plasma to red blood cell ratios [ 20 ] . wafaisade and colleagues demonstrated decreased mortality rates in such patients. because of the nature of frozen plasma, transfusion delays of 45 min occur as units are thawed and prepared. young and colleagues surveyed members of the university health system consortium, consisting of 107 academic medical centers and 232 af fi liated hospitals and found that only 60% of participating hospitals had thawed plasma suf fi cient for the fi rst cycle of their mtp. this problem delays the critical availability of plasma in the initial phase of resuscitation. reviews of plasma, cryoprecipitate and platelet transfusions alongside massive blood transfusion protocols have demonstrated that earlier use of plasma and platelets in trauma patients have decreased the incidence of coagulopathy [ 21 ] . unfortunately, by the time one or more blood volumes have been lost, plasma may still be unavailable in the absence of a thawed or liquid plasma program. hence, protocols have been established to reduce wastage of products and use them for patients in an ef fi cacious manner [ 22 ] . red blood cells are the component of choice used to restore hemoglobin levels in resuscitation. more than 30% of intensive care unit (icu) patients receive rbc transfusions and more than 40% are transfused during hospitalization [ 23 ] . the cardiovascular health study found that anemia is associated with increased mortality in elderly patients, emphasizing the importance of treatment [ 24 ] . however, correction of anemia in surgical patients has not been readily studied, and its bene fi ts remain controversial. in their review, englesbe et al. note that there is not yet a consensus of in what degree of anemia can rbc transfusions offer a bene fi t [ 25 ] . they discuss the current fi ndings by various studies, which have found that survival was not increased when postoperative patients were transfused to correct a hematocrit of 25%, and similarly, while studies favor transfusion in cardiac patients with a hematocrit of 33% or less, a true bene fi t remains to be seen. hence, they recommend making the decision to transfuse using a host of physiological measures and evaluation of the patient's compensatory ability, not only the hematocrit. they have used a trigger of a hematocrit of 16% for initiating transfusion when the patient has excellent compensatory ability, and 21% when this is not the case [ 25 ] . the 21% trigger should also be employed in stable elderly patients without tachycardia or hypoxia. otherwise, their investigations have not yet shown bene fi ts in strati fi cation of surgical patients by specialty or procedures. one surgical population that has been studied with regards to transfusion is patients undergoing infrarenal abdominal aortic aneurysm surgery. a meta-analysis of randomized controlled trials demonstrated that intraoperative autotransfusion in these patients decreased the allogeneic blood transfusion requirement [ 26 ] . high quality evidence, notably hébert et al.'s, exists to support conservative triggers for rbc transfusion in critically ill patients [ 27 ] . this multicenter randomized, controlled, clinical trial of 838 critically ill patients compared the outcomes of patients who were transfused at hemoglobin levels of less than 7.0 g/dl and those who were transfused at hemoglobin levels below 10.0 g/dl. their study ultimately found that the more restrictive trigger of 7.0 g/dl was superior to the liberal one and patients experienced improved 30-day survival rates. of note, of the various patient populations studied, this improvement was not found to be signi fi cant in patients with acute myocardial infarction and unstable angina [ 27 ] . it is important to be mindful of false triggers for transfusion, such as anemia due to hemodilution, commonly seen in patients receiving fl uids during prolonged hospital stays. a peripheral hematocrit is not enough to determine the patient's red blood cell levels, and calculations of total blood volume, red blood cell volumes, and normalized hematocrit are necessary [ 28 ] . van et al. report that relying on peripheral hematocrit alone resulted in overdiagnosis of anemia in 23.8% of analyses, and this fi nding can lead to unnecessary transfusions. blood volume analyzers are one option that has been shown to separate anemia due to hemodilution compared to other sources such as surgical bleeding [ 28 ] . in patients with prolonged hospital stays and critically ill patients, it is important to keep in mind anemia due to phlebotomy for various laboratory testing and other needs [ 23 ] . between 40 and 240 ml of blood per day is collected from icu patients, with surgical patients generally on the higher end. hence, the conservation of blood and reducing unnecessary blood draws is key to preventing a need for prbc transfusions. because rbc transfusions are associated with certain risks that are discussed in a later section, it is important to also consider possible alternatives or treatments that reduce transfusion requirements, such as epoetin alfa. silver et al.'s randomized, double-blind, placebo-controlled trial investigated the role of epoetin alfa, a recombinant erythropoietin, in reducing the rbc transfusion requirement of long-term acute care patients, thereby reducing risks associated with transfusions [ 29 ] . their fi ndings showed that treatment with epoetin alfa signi fi cantly increased hemoglobin concentration and the odds ratio for receiving an rbc transfusion compared to patients on the placebo arm was 0.28 [ 29 ] . additionally, vincent et al.'s randomized, double-blind, placebo-controlled study demonstrated that a once weekly dose of epoetin alfa augmented the erythropoietin response [ 30 ] . knight et al.'s review found that patients with cancers of various organs who did not have anemia, most due to correction with epoetin alfa, required less transfusions and experienced more quality of life [ 31 ] . however, epoetin alfa is limited by its delayed onset at 5-7 days. as for its effects on mortality, corwin et al. conducted a prospective, randomized, placebo-controlled trial of 1,460 medical, surgical, or trauma patients [ 32 ] . weekly injections of epoetin alfa were shown to decrease mortality at day 29 and day 140, especially in trauma patients compared to placebo. however, epoetin alfa was associated with an increase in thrombotic events, and did not affect the number of patients who received a transfusion of rbcs [ 32 ] . iron sucrose has also been investigated as a possible adjunct to rbc transfusions in order to reduce transfusion requirements. to answer this question in colorectal cancer surgery patients, edwards et al. conducted a randomized prospective blinded placebo-controlled trial of 60 patients [ 33 ] . patient outcomes, which were assessed using change in hemoglobin levels, serum iron markers, transfusion rate, length of hospital stay and perioperative events, were found to be unchanged by the addition of 600 mg of iron sucrose [ 33 ] . plasma is an acellular blood product consisting of clotting factors involved in coagulation and fi brinolysis, as well as proteins involved in immune reactions and maintenance the oncotic balance of blood. plasma can be obtained from separation of whole blood or unique plasma donations from a donor using plasmapheresis. common indications for plasma are reversal of warfarin-induced anticoagulation, massive transfusion in trauma and surgery, procedures with limited bleeding or risk thereof, liver disease with coagulation factor de fi ciencies, single coagulation factor de fi ciency, and thrombotic thrombocytopenic purpura (ttp) [ 34 ] . historically, plasma transfusions have been associated with various side effects including transfusion related acute lung injury (trali) [ 35 ] . however, these complications have been dramatically reduced with blood donation centers transitioning to male only and/or nulliparous female donors [ 36 ] . norda et al. studied two types of plasma: thawed plasma and liquid plasma (never frozen). liquid plasma is an aabb approved product and may be stored at 2-6°c for up to 26 days. both of these types of plasma have been considered clinically equivalent. as for their individual components, liquid plasma has been shown to contain levels of factor v and von willebrand factor at levels 70% or greater. however, studies have noted that c1 esterase inhibitor (c1inh) was consumed by day 14 in 22% of plasma products due to coldinduced contact activation [ 37 ] . in order to avoid this effect that places patients at risk for inadequate perfusion, some institutions have introduced a maximum storage time of 7 days for nonfrozen plasma [ 37 ] . murad et al.'s meta-analysis of 37 studies on adults transfused with plasma compared with nontransfused controls demonstrated that in the setting of massive transfusions in trauma patients, transfusion may be associated with increased survival and decreased multiorgan failure. however, they also noted increased mortality in patients who received plasma not part of a massive transfusion protocol. this fi nding may be due to the unbalanced ratio of transfusion of products, unlike in mass transfusion protocols, which call for 1:1 transfusion of rbcs and plasma. in addition, plasma transfusion was associated with increased risk of developing trali, and by itself did not reduce transfusion requirements [ 34 ] . their fi ndings, in the fi rst comprehensive meta-analysis and systematic review of plasma transfusion outcomes, highlight the need of assessing each patient's indications for plasma. the maturation of this fi eld will be needed to strengthen the fi ndings, which the authors did note were subject to survivor biases in some studies. however, none of these studies involved the use of plasma in patients with hemorrhagic shock. in this population of patients, the incidence of multi-organ failure has been shown to be lower than comparison cohorts (most likely as a result of less overall transfusions in the higher plasma group) [ 13, 14 ] . the purpose of platelet transfusions is to avoid spontaneous hemorrhage, which can occur at very low platelet levels, especially in patients who are already hemorrhaging or have various platelet de fi ciencies and abnormalities of function. along with plasma and fi brinogen, platelets are key in achieving hemostasis in the obstetric patient with post-partum hemorrhage [ 38 ] . approximately 50,000 cells/l of platelets are necessary in order to achieve adequate hemostasis. in addition to the total number of platelets, their quality is also important to overall hemostatic function. a patient's platelets must be ef fi cacious, that is, remaining in circulation and completing its physiological role in clot formation [ 39 ] . this ef fi cacy can be assessed by various modalities, from the traditional laboratory coagulation studies to the more recent thrombelastograms (teg), also known as thromboelastography, and this topic is covered in the last section. cryoprecipitate consists of von willebrand factor/viii complex, factor xiii, and fi brinogen. it is used to supplement plasma transfusions with fi brinogen, especially in patients with fi brinogen levels of less than 100 mg/dl, the level at which hypo fi brinogenemia results in bleeding [ 5 ] . it is named cryoprecipitate because single units of plasma are rapidly frozen to −30°c and are slowly thawed overnight to 4°c, causing many clotting factors such as fi brinogen to precipitate out of the solution [ 35 ] . indications for cryoprecipitate include factor vii de fi ciency, congenital or acquired hypo fi brinogenemia, disseminated intravascular coagulation, and massive transfusion. unlike plasma, virus-inactivated cryoprecipitate is not yet available, and studies on the ef fi cacy of sd ffp and mb ffp have not shown a bene fi t [ 35 ] . the complications of cryoprecipitate are similar to those of plasma, with a slightly lower occurrence of complications associated with higher volumes of plasma, such as trali and hemolysis [ 35 ] . the practice of using whole blood is largely uncommon due to the separation of blood components for targeting speci fi c de fi ciencies currently supported by evidencebased medicine. decision-making for each transfusion requires laboratory testing, and each product must carefully be stored and transported to the site of need. when this is not possible, such as in acute settings with limited resources, whole blood transfusions can adequately resuscitate certain patients. grosso et al. recount a case of collecting whole blood from hospital personnel donors in a us fi eld surgical hospital in kosovo [ 40 ] . this whole blood was used to treat exsanguinating coagulopathy in an acutely bleeding patient. the advantage of whole blood is its ability to increase hemoglobin levels, similarly to red blood cells, and its ability to restore blood volumes, similarly to crystalloids [ 40 ] . because of its physiological ratios of each blood component, it may hold an advantage over individual blood component transfusions, but more work is necessary to substantiate this idea. recombinant activated factor vii (rfviia), originally developed for use in hemophilia a and b patients, has recently been explored in various off-label uses, such as stemming acute bleeding alongside standard replacement therapy. mayo et al. demonstrate the use of a coagulopathy score that they found to be statistically correlated to rfviia response and survival in 13 trauma patients in israel [ 41 ] . this fi nding was a turning point in the understanding of rfviia indications due to its previous contraindication in coagulopathy. other uses for rfviia are factor vii de fi ciency, thrombocytopenia, functional platelet disorders, von willebrand disease, intracranial bleeding, and reversal of warfarin overdose, liver disease, and transplantation. however, little evidence is currently available to support these uses [ 41 ] . before entering the discussion on complications related to transfusions, the dif fi culty of study design to answer such questions must be appreciated. there are ethical obstacles to randomizing patients to transfusion and non-transfusion arms. hence, many trials show patients who received more blood component transfusions fared worse than patients who did not, but this may be entirely because of the condition of the patients that necessitated the transfusions [ 25 ] . khorana et al.'s retrospective cohort study of 504,208 patients hospitalized with cancer demonstrated that rbc and platelet transfusions were associated with increased mortality, as well as venous and arterial thrombotic events [ 42 ] . however, it is unclear if this is a causal relationship. as with large-scale introduction of exogenous elements to the body, immune reactions can develop, a sequela that is notorious in blood products. this complication is particularly devastating in severely ill patients. the most notorious of these immune reactions are hemolytic reactions. in order to prevent this event, it is important to cross-match patient and donor blood whenever possible. the most common cause of hemolytic reactions due to transfusion of an incorrect match is clerical error. hemolytic reactions in blood transfusions occur because each individual carries antibodies against the blood group (a or b) that it does not express endogenously. hence, when products containing anti-a or anti-b antibodies in plasma, such as plasma, are transfused to patients of a, b, or both blood groups, the donor antibodies stage an attack on the patient's red blood cells. allergic reactions are another common immune-mediated complication of transfusions. severely anaphylactic reactions are more common after plasma compared to rbc transfusion [ 35 ] . patients present with wheeze, hypotension, tachycardia, laryngeal edema, and urticarial rash. trali is de fi ned as acute lung injury occurring within 6 h of transfusion with a blood product, with most commonly reported cases occurring due to ffp [ 43 ] . trali is the most common cause of death due to transfusion [ 35 ] . trali is characterized by respiratory insuf fi ciency, not limited to but including tachypnea, cyanosis, dyspnea, and acute hypoxemia [ 43 ] . unfortunately, the occurrence of trali in critically ill patients who received a blood transfusion is estimated to be around 25% and increases with each subsequent transfusion, has a mortality rate of approximately 40%, and it is the most common transfusion-related complication [ 16 ] . eighty-fi ve percent of patients with bleeding varices receive blood transfusions, and the trigger for transfusions is much debated. in patients with gastrointestinal bleeding, trali is further exacerbated by the presence of end-stage liver disease. proposed mechanisms for this phenomenon have included antibody-mediated reactions, but these fi ndings are not de fi nitive and many are subject to selection bias due to no screening in the asymptomatic population [ 43 ] . autopsies and animal models have suggested hyperactive pmn involvement, since mass in fi ltration was noted [ 43 ] . a two-event model has also been proposed, with the fi rst event dictated by the clinical health of the patient and the second event by the quality (affected by storage, donor immunologic components) of the blood product [ 43 ] . the treatment of trali is aggressive respiratory support and ventilation in more severe cases, such as in critically ill patients [ 43 ] . practices to reduce the risk of trali include prestorage leukoreduction as well as avoiding the use of old blood products, de fi ned as older than 14 days for rbcs and older than 2 days for platelet concentrates. another prevention strategy is using only male donors or donors who have never been pregnant due to look back studies showing fewer trali events in blood donations from those populations [ 16 ] . eder et al. demonstrated that preferential distribution of plasma from male donors reduced the reported number of trali cases [ 44 ] . transfusion-associated immunomodulation refers to the immunosuppression resulting from the introduction of foreign antigens via blood products to the host [ 25 ] . the exact mechanism of this effect has not yet been elucidated, but plasma components, white blood cells (wbcs), metabolic products from storage processes are thought to play a role. this effect may be responsible for the immunosuppressive effects of transfusions on severely ill patients. transfusions can cause sensitization to hla antigens, creating a unique problem in potential kidney transplant patients. studies have demonstrated increased sensitization of patients on a kidney transplant waiting list after transfusion, rendering them unsuitable candidates for living donation. their only remaining alternative once this has occurred is to wait for a cadaveric graft, which takes up to four times longer, and may never receive a transplant. hence, non-life-sustaining transfusions should be avoided in potential kidney transplant recipients [ 25 ] . red blood cell transfusion is an independent predictor of systemic in fl ammatory response syndrome (sirs), icu admission, mortality, and length of hospital stay, and the development of multiple organ failure (mof) [ 45 ] . in particular, the age of the blood plays an important role, with increased age of rbcs resulting in increased instances of mof. rbcs are not alone in this adverse event. a multicenter prospective cohort study demonstrated that ffp was independently associated with increased risk of mof and acute respiratory distress syndrome (ards) of 2.1% and 2.5% [ 46 ] . the same study found, however, decreased risk of mof per unit of cryoprecipitate, and platelets were not found to be associated with mof or ards [ 46 ] . in addition to mof, blood transfusions are notorious in lay media for their association with infectious agents. in their review of the current literature, englesbe et al. found that patients who received transfusions compared to those who did not experienced signi fi cant increase nosocomial infection rates, and each additional prbc transfused correlated to increased infection risk [ 25 ] . staphylococcus aureus is the most commonly transmitted bacterial pathogen [ 16 ] . bacterial pathogen in blood products arise mainly from donor skin, and platelets are especially prone to these contaminants [ 35 ] . however, bacterial infections are less common than viral infections in blood transfusions. despite increased screening and testing, each rbc transfusion is associated with a risk for viral infections such as hepatitis [ 29 ] . virus risks in the uk in ffp have been estimated at 1 in 8 million for hiv, 1 in 30 million for hcv and 1 in 900,000 for hbv [ 35 ] . since up to 50% of adult donors are cytomegalovirus (cmv) carriers, there is a risk of transmission of this virus to patients, especially the immunosuppressed, transplant patients and neonates [ 35 ] . compared to viral causes, bacterial, endotoxin and prion contamination rates are more rare [ 35 ] . in order to avoid this deleterious complication, virus-inactivated preparations of plasma exist, such as methylene blue and solventdetergent treated products. while these options may offer increased viral protection, they have been associated with loss of clotting factors [ 35 ] . the most stringent testing protocols and sensitive tests may not ever eradicate the risk of infectious agent transmission due to several reasons. first, new pathogens of unknown methods of spread are constantly emerging and may not actively be screened for in its early emergence, such as human immunode fi ciency virus (hiv) and west nile virus. another obstacle in prevention is the incubation period of pathogens before seroconversion of blood [ 29 ] . prion diseases transmitted by transfusion has been a concern in the uk, following the bovine spongiform encephalopathy (bse) epidemic. unfortunately, no screening test for this condition has been established, and the occurrence of prion diseases in blood products in the uk is largely unknown. in order to avoid transfusions with prion disease, plasma has been imported from the usa since 2002 for pediatric transfusions [ 35 ] . another concerning complication is the loss of ef fi cacy in stored blood, and the adverse effects it causes. these consequences of the storage process are known as a storage lesion. with current technology, the shelf life of red blood cells cannot be extended further than its physiological shelf life of 120 days, and 35 and 42 days is the limit of viability in whole blood and adenine-saline preservation, respectively [ 29 ] . even this length of shelf-life results in counterproductive transfusions. speci fi cally, rbc products older than 2 weeks have been shown to not improve oxygen uptake in septic patients. in fact, rbcs of that age have been associated with higher mortality, increased adverse events, extended hospital stay, and electrolyte imbalances. this reduction in ef fi cacy may be due to decreased ability of the older rbcs to unload oxygen [ 29 ] . another proposed mechanism is that since stored rbcs have depleted nitric oxide, this may have a vasoconstrictive effect, leading to thrombosis and the observed increases in venous and arterial thrombotic events in patients with increased prbc and platelet transfusions [ 42 ] . the question is how realistic it is to maintain strict storage age in a fi nite and scarce resource such as blood. a doubleblind, prospective randomized pilot study demonstrated that controlling the storage age of rbcs in transfusion compared to the current standard of care is feasible and results in decreased exposure to older blood [ 47 ] . more evidence is needed to determine precisely the cut off age of rbcs in their ef fi cacy and availability. in stored platelets, it has been estimated that the recovery rate of 5-day old platelets is about 50%, with many nonviable platelets being sequestered into the spleen [ 21 ] . for these reasons, there is some concern that platelet counts performed immediately after transfusion do not provide an accurate picture of platelet function [ 21 ] . given the complications listed previously, a discussion of known preventative measures is warranted. transfusion with rbcs that have not been leukoreduced has been associated with increased risk of multiple organ failure and degenerating leukocytes may cause rbc toxicity. furthermore, nationwide leukoreduction protocols in canada were shown to lower mortality rates [ 29 ] . currently, in the usa, leukoreduction is not a standard practice despite evidence of bene fi t, and additional work is required to determine effects on outcome in various patient populations, such as icu patients [ 29 ] . hospitalized patients receiving transfusions are already in a vulnerable state of health, and when transfusion-related adverse events occur, it is most regrettable. with institutional triage protocols and transfusion guidelines, such unnecessary harm can be avoided, and cost reduction of a limited and precious resource can be achieved [ 48 ] . protocols and scoring systems, such as the emergency transfusion score (ets), have been successfully shown to triage patients in need of transfusions and those for whom it would be unnecessary [ 49 ] . there are many considerations to address in the management of an anticoagulated surgical patient, such as reversing anticoagulation fully before operation, in order to avoid bleeding complications. in the nonelective setting, such as life-threatening hemorrhage or emergent surgical indications, this process must be sped up, using prothrombin complex concentrate (pcc) [ 50 ] . unlike ffp, pcc can be administered without the need for cross-matching or thawing, has more predictable concentrations of clotting factors, and has been shown to reverse warfarin-related coagulopathy. the clotting factors are also in high concentrations, approximately 25 times that of plasma, decreasing the volume of pcc needed. in addition, the inr is rapidly corrected, taking about 15 min [ 50 ] . anticoagulated patients and patients using antiplatelet agents are especially vulnerable to coagulopathies, which may develop during resuscitation. kılıç et al.'s fi ndings recommend using individualized treatment, providing the de fi cient blood component as per laboratory value de fi ciency [ 16 ] . in addition, patients who are overly anticoagulated with warfarin may also be treated with pcc containing vitamin k dependent factors [ 16 ] . due to the teratogenocity of warfarin, pregnant patients requiring anticoagulation receive heparin as the preferred drug for preventing pulmonary embolism or in thromboprophylaxis in atrial fi brillation [ 51 ] . insertion of a venal caval fi lter is another option. in the surgical patient, it is important to discontinue aspirin and reversible platelet inhibitors such as clopidogrel 10 and 7 days respectively before an operation to avoid bleeding complications [ 50 ] . however, risks of thrombotic events in discontinuation of these agents in cardiovascular surgeries have been noted [ 50 ] . because of these risks with anticoagulated patients and patients receiving antiplatelet agents, it is important to weigh the bene fi ts of the surgery against these risks, among others. obstetric patients are one subpopulation of actively bleeding surgical patients that can easily confuse the provider. their generally young age may lead one to dismiss some vital sign changes or lab values, while alterations of their physiology in response to pregnancy often results in the misinterpretation of critical fi ndings. during pregnancy, blood becomes less viscous in order to increase oxygen carrying capacity while minimizing increased cardiac load as much as possible. intravascular volume, and more speci fi cally, plasma volume increases proportionately more than red cell volume, creating a "physiologic anemia of pregnancy" [ 51 ] . fibrinogen, von willebrand factor and factors vii, viii, ix, x, xii are synthesized more frequently while levels of factors xi and xiii and platelets decrease [ 38 ] . levels of factor ii decrease, yet interestingly, prothrombin time (pt) and partial thromboplastin time (ptt) remain unaffected [ 51 ] . mechanical obstruction of the uterus on the inferior vena cava and other vessels encourage stasis and the formation of thrombi. the summation of these effects result in a net hypercoagulable state [ 51 ] . the utero-placental circulation has increased activity of both coagulation and fi brinolysis, contributing to increased levels of fi brin degradation products such as d -dimer, especially in the third trimester [ 38 ] . this effect may contribute to the hemostatic challenges in obstetric patients. anti fi brinolytics such as tranexamic acid and aminocaproic acid can be used to treat hyper fi brinolysis. in fact, tranexamic acid has been shown to reduce blood loss after elective caesarean section and vaginal delivery [ 38 ] . plasma and cryoprecipitate contain fi brinogen and may be used to replenish fi brinogen in states of hypo fi brinogenemia (<180 mg/dl). post-partum hemorrhage (pph) is a major cause of obstetric mortality that may require peripartum hysterectomy and is the most common cause of maternal mortality worldwide. pph, in general, is not associated with underlying coagulation disorders but rather acute events related to placenta abnormalities, trauma from large births or instrumentation, or uterine atony [ 38 ] . in addition to rapid surgical intervention, hematologic management of pph includes rapid volume replacement and blood transfusions. these patients are likely to bene fi t from management strategies similar to that for acutely injured patients who are in shock from hemorrhage. in obstetrical patients, rfviia has also been found to control and decrease hemorrhage. segal et al.'s observation of three patients with pph, hypovolemic shock, and dic who received massive transfusions suggests that rfviia may be bene fi cial adjunctive therapy after the completion of hysterectomy [ 52 ] . the therapeutic effect of rfviia may be due to its binding of tissue factor at the site of vessel injury and forming a complex, activating platelets and facilitating fi brin clot formation [ 52 ] . however, these fi ndings have not been consistent in the current literature, and especially because of the expense of rfviia, the decision to administer this to the patient must involve a thorough consideration of the bene fi ts, if any [ 38 ] . intensive care unit (icu) patients are another patient population that frequently receives blood transfusions in order to correct their anemia, which has been shown by a large body of work to indicate worse prognosis [ 29 ] . these patients are anemic due to sepsis, occult blood loss, hemorrhage, decreased production and functional iron de fi ciency. icu patients with low hemoglobin levels are more likely to suffer from complications such as sepsis, and they are more likely to experience delayed weaning from ventilator support. the decision to transfuse such patients should weigh the bene fi ts and the risks of blood transfusions, especially given the patients' increased susceptibility to infections, iatrogenic events and increased metabolic demands [ 53 ] . vincent et al.'s multicenter prospective observational study of 1,136 patients demonstrated that icu patients frequently received transfusions, with a transfusion rate of 37% during their stay. the patients who received transfusions also experienced a higher mortality rate, prolonged hospital stay, and decreased organ function [ 53 ] . there is also evidence suggestive of increased transfusions in patients with hemoglobin levels higher than the generally accepted trigger value of 8 g/dl. speci fi cally, vincent et al. found that in under 30% of cases, patients with hemoglobin levels greater than 9 g/dl received blood transfusions [ 53 ] . hence, future work is needed to recommend strict hemoglobin cut offs for transfusion. in the acute trauma setting, conventional coagulation testing (cct), which consists of prothrombin time, international normalized ratio (inr), partial thromboplastin time, and platelet count, is used to assess coagulation status. this approach, however, is limited by slow results, incomplete characterization of the coagulation abnormality, and poor prediction of patient outcome. furthermore, ccts, which are riddled with delays from time to arrival in the laboratory and duration of testing, end up re fl ecting the coagulation state of the patient after 30-45 min of interventions and resuscitation [ 54 ] . since cct only examines plasma factors, the integral role of platelets and their function is ignored. in addition, the cct assesses only the extrinsic pathway, intrinsic pathway, and platelet count, painting an incomplete picture of the pathologies of clotting in the severely exsanguinating patient. these de fi ciencies are addressed by thrombelastography (teg), a test that creates a dynamic, graphical representation of the coagulation characteristics of a blood sample from initial clot formation to fi brinolysis. since speci fi c coagulation components have speci fi c disturbances on teg, this test reveals diagnostic as well as therapeutic information [ 55 ] . the procedure involves obtaining an uncitrated whole blood sample, activation of the specimen with kaolin and spinning the sample in a thrombelastograph machine within 4-5 min in order to avoid clotting [ 55 ] . if this timeframe cannot be achieved, a "reversal" method can be used, where citrate is used to avoid clotting until the sample has arrived at the laboratory, at which point, the citrate will be "reversed" using calcium chloride as per manufacturer instructions. while this method has been shown to affect teg results, it has not been shown to be inferior to the standard method and may be used in centers where 4-5 min from sample collection to running the teg is not realistic [ 55 ] . rapid teg differs from conventional teg in its addition of tissue factor to the blood sample and kaolin, accelerating activation of the clotting cascade. this modi fi cation makes it well suited for the trauma setting since its results are available much earlier, namely under 20 min, compared to kaolin teg and ccts, which can take over 30 min, without sacri fi cing accuracy [ 55 ] . interpreting the results involves analyzing each of the sequential measurements ( fig. 12. 2 ). reaction time, or r-time, in teg is the time until initial clot formation. it is also known as activated clotting time (act) in r-teg in order to denote intentional anticoagulant agents in the sample. factor de fi ciency or severe hemodilution can prolong reaction time or act. next, k -time represents the time needed to reach 20-mm clot strength, and has a normal range of 1-2 min. the -angle, normally between 66 and 82°, represents the rate of clot formation. in platelet de fi ciency or hypo fi brinogenemia, where one of the two key components of clots are missing, the k -time is increased and the -angle is decreased. oshita et al.'s linear regression analysis of 36 samples from healthy individuals reported that ma and k -time were linearly related to platelet count [ 56 ] . the maximal amplitude (ma) of the tracing represents platelet contribution to clot strength (normal range 54-72 mm). it is decreased in states of platelet dysfunction and hypo fi brinogenemia. the g -value represents overall clot strength, including platelet function as well as enzymatic, and is decreased in hypocoagulable states (normal 5.3-12 k dynes/cm 2 ). the ly30 is the percent of amplitude reduction at 30 min after the ma, and is elevated in hyper fi brinolytic states (normal range 0.0-7.5%) [ 55 ] (figs. 12.3 and 12.4 ) . the use of r-teg is further facilitated by advanced software that displays the r-teg tracing as the test is being performed, providing physicians with "real time" results. cotton et al. report that early r-teg parameter tracings (act, k -time and r -value) appeared within 5 min while later values (-angle, ma) were seen within 15 min, compared to cct panels, fig. 12.2 the various sequential and parallel measurements of teg and rotem [ 4 ] which were not available until 48 min [ 55 ] . installation of graphical software in the trauma bay, operating room and shock-trauma intensive care unit computers can further facilitate the rapid access to teg results [ 55 ] . teg data results compare well to the previous standard, ccts. in 2011, cotton et al. conducted a pilot study of 272 patients to investigate the role of rapid thrombelastography (r-teg) in (1) assessing speed of results, (2) correlation with cct fi ndings, and (3) predictability of early transfusions of prbcs, plasma, and platelets [ 55 ] . their fi ndings demonstrated that graphical r-teg is available within minutes, an improvement compared to ccts. they also demonstrated that act, r -value and k -time strongly correlated with pt, inr, and ptt. ma and -angle strongly correlated with platelet count, and act, r -value, -angle and ma were predictive of prbc, plasma and platelet transfusions within the fi rst 2 h of arrival. in fact, an act > 128 predicted massive transfusion in the fi rst 6 h and an act < 105 predicted patients that did not receive transfusions in the fi rst 24 h [ 55 ] . in addition, comparison of teg and cct in cardiopulmonary bypass patients found that teg measures were useful surrogates for cct values [ 57 ] . because of the speed of their availability and predictive ability, integrating teg results in mtps can strengthen decision-making and management of patients and improve patient outcomes. a wide array of evidence exists in surgical patients in support of teg's ability to predict prognosis, and in some instances, guide therapy that improves it. table 12 .1 is an example of teg-guided protocol with such an aim. platelet dysfunction in cardiopulmonary bypass patients has been attributed to microvascular bleeding, and teg has been used in the setting of cardiac surgery as a predictor of worsening patient outcomes due to this mechanism [ 17 ] . solomon et al. demonstrated that fi brinogen clot elasticity assessed by teg correlated to fi brinogen concentration in cardiopulmonary bypass patients [ 58 ] . teg has been found to predict the risk of postoperative bleeding, and has been used to direct desmopressin therapy and ffp transfusion requirement in cardiopulmonary bypass patients [ 17 ] . teg has been shown to be useful in liver surgery, especially in transplantation. unlike other surgeries, liver surgery poses the additional problem of increased risk of coagulation factor de fi ciencies due to hepatic dysfunction and lack of synthesis. teg-guided transfusion algorithms in this area have been shown to reduce the transfusion requirements in such patients [ 17 ] . however, ogawa et al.'s prospective observational study of 26 patients undergoing cardiac surgery did not fi nd a signi fi cant correlation between teg measures and volume of intraoperative and total transfusions. despite these fi ndings, [ 59 ] . they investigated thromboelastography in cardiac surgery patients and found 14 studies that provided the best evidence. their synthesis concluded that teg can guide transfusion therapy algorithms and result in decreased blood component requirements. in orthopedic surgery patients, teg was used in a prospective study to identify disturbed fi brin polymerization as a pathological mechanism in dilutional coagulopathy, and to rescue this state with fi brinogen administration [ 60 ] . however, teg has been found to be less sensitive for certain categories of platelet inhibition. in addition, hemostatis point of care tests such as pfa-100 and teg are affected by nonopiod analgesic drugs. scharbert et al.'s crossover, double-blinded, placebo controlled study demonstrated that in low back pain patients scheduled for invasive pain therapy, cytochalasin d -modi fi ed thromboelastometry had a low sensitivity for detecting platelet inhibition by diclofenac [ 61 ] . there are hemostatic states unique to the surgical patient as a result of medications such as warfarin, perioperative bleeding especially in high bleeding risk surgeries, and emergent surgical indications such as trauma. various mechanisms affect coagulation cascades in these patients, and techniques from the standard coagulation tests to teg are currently available. these have shown mostly success in predicting the course of the patient and guiding therapy. the therapeutic options include various blood product components, ranging from whole blood to concentrations of individual factors. using physiological ratios of prbcs, ffp, and platelets have improved patient survival in the massively hemorrhaging patient. however, like all powerful therapy, they are associated with adverse effects. preventative options, such as decreasing storage lengths and screening for infectious agents have drastically reduced these risks. lastly, administering these products in a rapid and directed fashion would not be feasible without in-house triage and massive transfusion protocols. these algorithms include steps that must be taken to smooth out logistics of urgent transfusions, such as anticipating adequate thawing times of ffps and collaborating with blood banks to crosscheck appropriateness of each order (fig. 12.5 ). damage control': an approach for improved survival in exsanguinating penetrating abdominal injury management of the major coagulopathy with onset during laparotomy on behalf of the dutch peritonitis study group. comparison of on-demand versus planned relaparotomy strategy in patients with severe peritonitis management of major blood loss: an update massive transfusion protocols for patients with substantial hemorrhage prede fi ned massive transfusion protocols are associated with a reduction in organ failure and post-injury complications damage control hematology: the impact of a trauma exsanguination protocol on survival and blood product utilization a massive transfusion protocol to decrease blood component use and costs initial experiences with point-of-care rapid thrombelastography for management of life-threatening postinjury coagulopathy emergency department blood transfusion predicts early massive transfusion and early blood component requirement the impact of uncrossmatched blood transfusion on the need for massive transfusion and mortality: analysis of 5166 uncross-matched units resuscitation of the trauma patient: tell me a trigger for early haemostatic resuscitation please! crit care room for performance improvement: provider-related factors associated with poor outcomes in massive transfusion damage control resuscitation reduces resuscitation volumes and improves survival in 390 damage control laparotomy patients damage control resuscitation for vascular surgery in a combat support hospital resuscitation and monitoring in gastrointestinal bleeding the surgical application of point-of-care haemostasis and platelet function testing proactive administration of platelets and plasma for patients with a ruptured abdominal aortic aneurysm: evaluating a change in transfusion practice effect of early plasma transfusion on mortality in patients with ruptured abdominal aortic aneurysm trauma registry of dgu. high plasma to red blood cell ratios are associated with lower mortality rates in patients receiving multiple transfusion (4 £ red blood cell units < 10) during acute trauma resuscitation indications for early fresh frozen plasma, cryoprecipitate, and platelet transfusion in trauma instituting a thawed plasma procedure: it just makes sense and saves cents blood conservation for critically ill patients a prospective study of anemia status, hemoglobin concentration, and mortality in an elderly cohort transfusions in surgical patients intraoperative autotransfusion in abdominal aortic aneurysm surgery transfusion requirements in critical care investigators for the canadian critical care trials groups. a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care blood volume analysis can distinguish true anemia from hemodilution in critically ill patients anemia in the long-term ventilator-dependent patient with respiratory failure pharmacokinetics and pharmacodynamics of once-weekly subcutaneous epoetin alfa in critically ill patients: results of a randomized, double-blind, placebo-controlled trial prevalence and outcomes of anemia in cancer: a systematic review of the literature for the epo critical care trials group. ef fi cacy and safety of epoetin alfa in critically ill patients randomized clinical trial of preoperative intravenous iron sucrose to reduce blood transfusion in anaemic patients after colorectal cancer surgery the effect of plasma transfusion on morbidity and mortality: a systematic review and meta-analysis risks of fresh frozen plasma and platelets appropriateness of fresh-frozen plasma usage in hospital settings: a meta-analysis of the impact of organizational intervations utilisation de plasma: indications médicales et préparation de plasma en suède [use of plasma: clinical indications and types of plasma components in sweden management of post-partum haemorrhage ef fi cacy evaluation of current and future platelet transfusion products whole blood transfusion for exsanguinating coagulopathy in a u.s. fi eld surgical hospital in postwar kosovo recombinant activated factor vii (novoseven): addition to replacement therapy in acute, uncontrolled and life-threatening bleeding blood transfusions, thrombosis, and mortality in hospitalized patients with cancer transfusion-related acute lung injury (trali): current concepts and misconceptions effective reduction of transfusion-related acute lung injury risk with male-predominant plasma strategy in the american red cross cumulative risks of early red blood cell transfusion in fl ammation and the host response to injury investigators. fresh frozen plasma is independently associated with a higher risk of multiple organ failure and acute respiratory distress syndrome a prospective, double-blind, randomized clinical feasibility trial of controlling the storage age of red blood cells for transfusion in cardiac surgical patients prospective monitoring of plasma and platelet transfusions in a large teaching hospital results in signi fi cant cost reduction emergency transfusion score (ets): a useful instrument for prediction of blood transfusion requirement in severely injured patients management of surgical patients receiving anticoagulation and antiplatelet agents critical care obstetrics and gynecology treatment of obstetric hemorrhage with recombinant activated factor vii (rfviia) peres-bota d, the abc investigators. anemia and blood transfusion in critically ill patients all bleeding stops: how we can help… rapid thromboelastography delivers realtime results that predict transfusion within 1 hour of admission quantitative measurement of thromboelastography as a function of platelet count a comparative evaluation of rotation thromboelastometry and standard coagulation tests in hemodilution-induced coagulation changes after cardiac surgery a comparison of fi brinogen measurement methods with fi brin clot elasticity assessed by thromboelastometry, before and after administration of fi brinogen concentrate in cardiac surgery patients can the use of thromboelastography predict and decrease bleeding and blood and blood product requirements in adult patients undergoing cardiac surgery? hemostatic changes after crystalloid or colloid fl uid administration during major orthopedic surgery: the role of fi brinogen administration point-of-care platelet function tests: detection of platelet inhibition induced by nonopioid analgesic drugs thromboelastography: theory and practice in measuring hemostasis admission rapid thrombelastography (r-teg) can replace conventional coagulation tests in the emergency department: experience with 1974 consecutive trauma patients key: cord-280379-1o9tzwjg authors: touyz, louis z. g. title: liquorice health check, oro-dental implications, and a case report date: 2009-07-08 journal: case rep med doi: 10.1155/2009/170735 sha: doc_id: 280379 cord_uid: 1o9tzwjg liquorice has an active substance, glycyrrhizin which inhibits the conversion of precursor cortisol to cortisone by inhibiting the enzyme 11-betahydroxysteroid dehydrogenase. when imbibed, liquorice acts like hyperaldosteronism which presents with typical symptoms including high blood pressure, low blood potassium, and muscle pain and weakness. this article appraises physiological and pharmacological effects on health of liquorice, critiques products containing liquorice, describes a typical case report of liquorice-induced hypertension, and appraises oral effects from consumption of liquorice products. liquorice, or liquorice, is a uniquely tasting herb derived from glycyrrhiza glabra, and has been used in medicine for thousands of years. liquorice is used as a flavorant in a variety of edibles, medicine, and tobacco, and is often innocently consumed in vast amounts without any regard or only with vague concepts of side effects liquorice may produce. this paper appraises liquorice provides a reality check of its properties (botanical sources, chemical structure, active liquorice ingredient, physiological/pharmacological activity, some common liquorice containing consumables), their systemic impact on health, a typical case report of liquorice-induced hypertension, and effects of consumption of liquorice on oro-dental structures. important clinical management principles for moderating liquorice consumption are suggested. it is the roots (rhizomes) and stolons of glycyrrhiza glabra (a.k.a sweet root, spanish or italian liquorice), which is the commonest variety source of liquorice. g.g lepidota is american wild liquorice, while g.g violacea and g.g glandulifera are persian/turkish and russian varieties, respectively. glycyrrhiza uralensis (a.k.a. manchurian liquorice) is the species favoured for traditional chinese eastern medicines. liquorice flavours are also found in the plants like fennel (foeniculum vulgare), anise seeds (from pimpinella anisum), and other plants [1] . ingredient. the active chemical ingredients imparting the unique liquorice taste are glycyrrhizic acid and its glucoside, glycyrrhizin (c 42 h 62 o 16 ). these molecules are regarded as nearly synonymous, are powerful organoleptic flavorants, and impart characteristic liquorice taste and aroma to mixtures in small concentrations [2] . glycyrrhizin is 50 times sweeter than sucrose. it retains, when sapid, a singular liquorice flavour. the liquorice sweetness has a slower onset than sugar and lingers. unlike artificial sweeteners like aspartame, saccharine, and cyclamates, it contains no sulfur molecule [3] , and retains its sweetness when heated [2] . on hydrolysis glycyrrhizin yields 2 mols of glucuronic acid and 1 of glycyrrhetenic acid, a pentacyclic, tri-terpene which structure partially resembles that common to steroids, with a moiety attached. glycyrrhizin inhibits the conversion of the precursor cortisol to cortisone by inhibiting the enzyme 11betahydroxysteroid dehydrogenase [4] . hydrolysis of slowly absorbing glycyrrhizin into the more rapidly absorbed glycyrrhetenic acid is performed by intestinal microbiota. consequently antibiotics affecting gut flora, adversely affects absorption of liquorice. liquorice boosts cellular formation of endogenous interferon, and has a positive long-term healing effect on hepatitis-c-infected patients [5] [6] [7] [8] [9] . liquorice is marketed in various forms, and because its often sold as the natural grown product, concentrations in the plant varies. solid extract 250-500 mg, three times daily, is suggested for medicinal purposes. dried root is dispensed at 1-4 g, three times daily to a maximum of 12 g [2] . at 75 mg daily glycyrrhetenic acid (derived from 50 g/day liquorice), a raising effect on blood pressure is noted after 2 weeks. more than this, daily dose increases blood pressure proportional to increased liquorice intake [2, 4] . toxicity. this on its own is rare, yet not infrequent when encountered clinically and usually occurs in diuretic medicated patients unwittingly combining consumption of commercial products containing high amounts of liquorice extract like chewing tobacco, laxatives, or confections with concentrated liquorice extracts [9] [10] [11] . consumption of glycyrrhizin is considered safe at 200 mg per day, a dose accepted as recommendation to japanese. the accepted daily intake (adi) for glycyrrhizin at 0.2 mg/kg/day is deemed safe; up to 1200 mg/day liquorice flavonoid oil shows no clinical noteworthy change of hematological or related biochemical parameters [12] [13] [14] . in the united states of america, glycyrrhizin is classified "as generally recognised as safe" as a flavouring agent, although not as a sweetener [2] . commercially liquorice flavoured sweets rarely have any serious medicinal side effects, especially if consumed irregularly, in moderate amounts, of less than 25 g of liquorice per day. liquorice has many positive and negative health modulating physiological effects which explain a variety of its medical effects. the most widely renowned negative action derives from liquorice's association with hypertension. because of the inhibition of 11-beta hydroxysteroid dehydrogenase, by liquorice, cortisol levels are high within the collecting duct of the kidney, and potassium is excreted while sodium is retained [4] . cortisol has high mineralo-corticoid properties, that is, it acts like aldosterone and increases sodium reabsorption from the glomerular filtration in the proximal tubules of the kidney in enac channels [3, 4] . sodium retention leads to higher osmotic intravascular pressure, which in turn retains more water, which increases circulating blood volume with consequent increased blood pressure leading to hypertension [4, 15] . liquorice has a variety of positive healthy effects on the body. for example, it is known that liquorice has antiviral properties and has some inhibitory effects on hiv, encephalitis, and sars-corona viruses [11, 16, 17] . also glycyrrhetenic acid itself has a retardant effect on peptic ulcers, possibly due to the fact that it has antibacterial properties and retards the growth of helicobacter pylori [18] . other medicinal claims are that liquorice is antiulcer (peptic, duodenal, and aspirin) [19, 20] . liquoriceinduced hypokalemic myopathy may explain why git spasms relax, and also why liquorice containing alcohols are said not to induce emesis [21] . with gastric smooth muscle paralysis, the irritating effect of ethanol is reduced and gastric contraction is temporarily weakened. liquorice among others is claimed to also be anti-inflammatory, an immune-stimulant, a demulcent, an expectorant, anticatarrhal, hepato-protective, a git spasmolytic, a mild laxative, and an antioxidant [20] [21] [22] [23] [24] . liquorice used as a flavorant in candies adds much gustatory joy to the variety of living pleasures. the flavour is so positive and pleasant, it is also used for flavouring other foods like ice-cream, biscuits, cakes, and drinks. it is a very popular flavour for sweet treats as "liquorice all sorts" (see figures 1 and 2) , and also in salty liquorice in holland. liquorice is added to baked confectionery, toffees, chocolates, chewing-gum, and sucking sweets. black and red liquorice varieties are made using food dyes which can discolour the tongue. (figure 3 ). liquorice is also part of spice mixes constituting curry, and liquorice is also used in breath fresheners. it is also used in medications, in many syrups, lozenges, capsules, laxatives, cough-lozenges, and mixtures to mask bitterness and foul-flavours of other drugs. liquorice is included in commercial over-the-counter tobacco products like pipe and chewing tobacco and snuffs. liqourice can stain the tongue and teeth (see figures 3, 4, 5 and 6) . liquorice concentrate-flavour is popular in alcoholic drinks too like absinthe (thujole containing) and pernod in france, anise in europe, and ouzo in greece. a 55-year-old women presented with a brown/black tongue for routine dental check and maintenance. (see figure 3) . there was neither history of antibiotic use nor any current tobacco abuse. she had been diagnosed with hypertension two years previously. despite multiple antihypertensive drugs, her blood pressure remained elevated. preoperative dental of vital signs revealed blood pressure (bp) measures of systolic 160 mm hg/diastolic 125 mm hg. although this was attributed to a possible "white-coat hypertension" phenomenon, she was referred to the hypertension clinic for further investigation and management of hypertension. other than prescribed blood pressure-lowering drugs (diuretics, an ace inhibitor and beta-blocker), she was not taking any other medication or herbal products. she exercised regularly, denied excessive alcohol intake and consumed a "healthy" diet. on examination, casual sitting blood pressure was high, despite treatment. routine laboratory investigations revealed hypokalemia. the diuretic was discontinued, k + supplements were given and she was advised to consume a k +rich diet. two weeks after stopping the diuretic, plasma k + was still low and hypertension was uncontrolled. the patient was investigated for hyperaldosteronism. plasma aldosterone and rennin levels were very low. in light of hypokalemia and reduced plasma aldosterone levels, a diagnosis of pseudohyperaldosteronism was made. a detailed dietary history revealed that since the patient stopped smoking 4 years ago, she started eating liquorice regularly every day. this helped her relieve her cravings for tobacco. she always enjoyed eating liquorice, but since quitting smoking she consumed at least one pack of 200-250 g or more of black liquorice daily. the patient was advised to stop eating liquorice and to continue her k + -rich diet and k + supplementation. three months later k + blood level was normal. eighteen months after presentation at the hypertension clinic and after stopping liquorice consumption, her blood biochemistry remained normal and her blood pressure was controlled to within normal limits. much lower doses of drugs than originally used kept her bp stable and she was clinically well. drug-induced local oral reactions are common [25] . a wide variety of drugs may induce one or more oral reactions including allergic reactions, aphthous-like lesions, burning mouth syndrome, glossitis, ulcerations, erythema multiforme, vesiculo-bullous lesions, color changes, oral lichenoid reactions, black hairy tongue, oral mucositis, gingival hyperplasia, salivary gland changes, dental changes, oral motor disorders, oral malodours, oral infections including osteonecrosis of the jaws, angioedema, and cheilitis [25] . liquorice not only has local oral effects but also has systemic effects, as the above case report shows. glycyrrhyzin by itself does not stain teeth, but when combined with dark food dyes, tobacco and/or curries, liquorice is associated with stains. frequently liquorice is mixed with dark caramel and food colorings which leave a surface brownish/black tongue stain (figure 3 ). it contributes to increased tobacco staining, especially when included in aromatic pipe tobaccos; the dental stain is directly proportional to the amount and frequency of the pipe smoking. not only is the palatal and lingual side of teeth prone to accumulating dark tobacco stain but also the mucosa undergoes specific changes. (figure 4 ) combined with chewing tobacco, liquorice additives enhance and prolong the flavour of the chewing tobacco experience, and consequently damage from longer contact time onto the gingiva, seeming to derive more from tobacco contents rather than just liquorice. (figures 5 and 6 ), adjacent recession, cervical dentinal staining, and thickening with hyperkeratosis of mucosa are seen. liquorice effects on the body are not widely acknowledged or understood by the public. this is because it is generally a pleasant experience and safe to eat liquorice flavoured candies, and although it is rare for someone to gormandise on them to reach toxic levels, as the case report indicates, this does occur. figure 5 . there is cervical recession and staining, and keratotic changes to the adjacent mucosa. this is the result of prolonged contact of the tobacco wedge against the teeth and mucosa. hypertension (as high blood pressure) is presumed to obtain when a measure of 130/90 mm mercury (systolic/diastolic pressure) or more is measured, normal being 120/70 mm mercury. hypertension is among the major causes of morbidity and mortality in the world today, and because it is mainly symptomless and painless, hypertension has been labelled "the silent killer." atherosclerosis, myocardial infarction and stroke can all result from chronic undetected hypertension [15, [26] [27] [28] [29] . a linear dose-related rise in blood pressure has been reported with liquorice consumption in various doses of (20-200 g a day for 4-2 weeks), corresponding to a daily intake of 75-540 mg glycerrhetinic acid [14] . this is an inordinate amount of daily liquorice consumption and would be regarded as a "fad-diet" and not to be sustained for health. an acceptable daily intake of 0.015-0.229 mg glycyrrhizin/kg body weight/day has been proposed [30] . most problems derive from people on diuretic medications in combination with other sources of liquorice. hypokalemia partially paralyses smooth muscle contraction, and excess imbibing of liquorice-flavoured alcohols contributes to gastric paralysis, prevents emesis, and indirectly contributes to the development of alcoholism. excess consumption affects blood pressure, kidney function, and gastrointestinal tract [30, 31] . other than the proven aldosterone effect of liquorice, other medical ailments like headache, myalgia, and muscle fatigue also present [32, 33] . liquorice contributes in part positively to all these, but eliminating liquorice may only improve, but not cure or totally prevent severe conditions presenting with these symptoms. some herbal remedies and other common consumables like chewing gum containing liqourice may precipitate hypertension and associated hypokalemic symptoms [34, 35] . liqourice-induced hypertension with hypokalemia must be differentiated from other genetic deficiencies which may present with similar findings: three monogenetic types of mineralo-corticoid hypertension have been identified, liddle's syndrome, glucocorticoid-remediable hypertension, and apparent mineralo-corticoid excess, an autosomal recessive disorder with mutations in the 11β-hsd2 gene [36] [37] [38] [39] . use of chewing and sucking tobacco snuffs and other products containing liquorice-like herbal medications, teas, breath fresheners, chewing gums, alcoholic drinks, and food products, all contribute to chronic habitual frequent swallowing of large quantities of liquorice [34, 35] . excess liquorice consumption contributes not only to deteriorating general health through potassium loss and sodium retention [40] but also to oro-dental compromise. some people stop smoking and then help control tobacco cravings by consuming large quantities of liquorice as a chronic organoleptic stimulus to help quit. tooth staining from black liquorice is known, but the tooth staining derives mainly from added dyes to liquorice confections and from liquorice-flavoured tobacco. accumulation of extracellular polysaccharides from microbial activity contributes to biofilm formation and bacterial plaques. this allows for a tacky gummy surface of muco-polysaccharides to stick to stagnant areas on teeth, and with adherent chromogenic case reports in medicine 5 bacteria, liquorice tobacco products discolour teeth and accelerate adjacent gingival breakdown. quitting the tobacco habit with safe stain removal through scaling and polishing from teeth is feasible. liquorice sweets are generally health promoting, pleasurable to eat, and in moderation on their own rarely stain teeth. health care workers, including all in the dental team, discovering new hypertension patients, or noting a history of taking diuretics, should always enquire about consumption or use of any liquorice containing product [14] . health care workers should update their knowledge about which drugs their patients are using, and follow-up on unwanted or toxic side effects [40, 41] . unduly stained teeth, a stained tongue or other orodental signs of intraoral chewing tobacco abuse combined with elevated blood pressures, should alert dentists to the possibility of morbidity arising from liquorice toxicity or abuse. with regular dental maintenance, regular medical history updates are essential and dental practices should measure blood pressure before any surgery. some patients may even volunteer information about raised blood pressure. health care professionals should check diets of all hypertensive patients and besides giving advice about eschewing liquorice products could refer affected people for early diagnosis and treatment for hypertension. encyclopaedia of herbs and their uses liquorice and its health implications chemical formulations. glycyrrhizin-4515. cyclamate-2770 hypertension and the cortisol-cortisone shuttle glycyrrhiza glabra. monograph a preliminary open trial on interferon stimulator (snmc) derived from glycyrrhiza glabra in the treatment of subacute hepatic failure the long term efficacy of glycyrrhizin in chronic hepatitis c patients intravenous glycyrrhizin for the treatment of chronic hepatitis c: a double-blind, randomized, placebo-controlled phase i/ii trial glycyrrhizin-induced reduction of alt in european patients with chronic hepatitis c licorice-induced hypermineralocorticoidism liquorice risk and safety assessment on the consumption of locorice root (glycyrrhiza sp.) its extract and powder as a food ingredient clinical safety of licorice flavonoid oil (lfo) and pharmacokinetics of glabridin in healthy humans liquorice-induced rise in blood pressure: a linear dose-response relationship fifty years of framingham study contributions to understanding hypertension in vitro antiviral activity of indigenous glycyrrhizin, liquorice and glycyrrhizic acid (sigma) on japanese encephalitis virus glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus preliminary evidence for inhibitory effect of glycyrrhizin on hiv replication in patients with aids inhibitory actions of glycyrrhizic acid on arylamine n-acetyl transferase of helicobacter pylori from peptic ulcer patients effect of deglycyrrhizinated liquorice on gastric mucosal damage by aspirin the plant kingdom as a source of anti-ulcer remedies glycyrrhizin (licorice)-induced hypokalemic myopathy: report of 2 cases and review of the literature herbs of choice: the therapeutic use of phytomedicinals a drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice current opinion on drug-induced oral reactions: a comprehensive review prehypertension and risk of cardiovascular disease prevalence of chronic kidney disease and associated risk factors-united states prevalence, awareness, treatment, and control of hypertension among united states adults hypertension: catching the silent killer licorice-induced hypokalemia liquorice and hypertension low doses of liquorice can induce hypertension encephalopathy an autopsy case of licorice-induced hypokalemic rhabdomyolysis associated with acute renal failure: special reference to profound calcium deposition in skeletal and cardiac muscle a hypertensive urgency induced by the continuous intake of a herbal remedy containing liquorice hypokalaemia and hypertension associated with use of liquorice flavoured chewing gum glucocorticoid-remediable aldosteronism is associated with severe hypertension in early childhood genetic dissection of human blood pressure variation: common pathways from rare phenotypes pseudohyperaldosteronism: pathogenetic mechanisms pseudohyperaldosteronism, liquorice, and hypertension liquorice, hypertension and the dentist antioxidant constituents from liquorice roots: structure elucidation and anti-oxidative capacity towards ldl oxidation key: cord-302690-0v7ne7vi authors: chow, clara k.; atkins, emily r.; billot, laurent; chalmers, john; hillis, graham s.; hay, peter; neal, bruce; nelson, mark; patel, anushka; reid, christopher m.; schlaich, markus; usherwood, tim; webster, ruth; rodgers, anthony title: ultra-low-dose quadruple combination blood pressure lowering therapy in patients with hypertension: the quartet randomized controlled trial protocol() date: 2020-10-02 journal: am heart j doi: 10.1016/j.ahj.2020.09.017 sha: doc_id: 302690 cord_uid: 0v7ne7vi high blood pressure is the leading cause of preventable morbidity and mortality globally. many patients remain on single-drug treatment with poor control although guidelines recognize that most require combination therapy for blood pressure control. our hypothesis is that a single-pill combination of four blood pressurelowering agents each at a quarter dose may provide a simple, safe and effective blood pressure lowering solution which may also improve long term-adherence. the quartet (quadruple ultra-low-dose treatment for hypertension) double-blind, active controlled, randomized clinical trial will examine whether ultra-low-dose quadruple combination therapy is more effective than guideline recommended standard care, in lowering blood pressure. quartet will enroll 650 participants with high blood pressure, either on no treatment or on monotherapy. participants will be randomized 1:1 and allocated to intervention therapy of a single pill (quadpill) containing irbesartan 37.5 mg, amlodipine 1.25 mg, indapamide 0.625 mg and bisoprolol 2.5 mg or to control therapy of a single identical appearing pill containing irbesartan 150 mg. in both arms step up therapy of open-label amlodipine 5 mg will be provided if bp is >140/90 at 6 weeks. the primary outcome is the difference between groups in the change from baseline in mean unattended automated office systolic blood pressure at 12 weeks follow-up. the primary outcome and some secondary outcomes will be assessed at 12 weeks, there is an optional 12 months extension phase to assess longer term efficacy and tolerability. our secondary aims are to assess if this approach is safe, has fewer adverse effects and better tolerability compared to standard care control. quartet will therefore provide evidence for the effectiveness and safety of a new paradigm in the management of high blood pressure. burden of high blood pressure and treatment gaps 84 high blood pressure is the leading cause of preventable morbidity and mortality globally. 1 the benefits of blood 85 pressure lowering in reducing cardiovascular events are unequivocal 2 and there is clear evidence of greater benefits 86 for combination-based therapy compared to monotherapy. 3 furthermore, numerous studies have indicated the 87 benefits of more rapid control of blood pressure, and have shown that this is more likely to occur with use of 88 combination therapy. 4 yet, control of high blood pressure is poor, with only 1 in 3 on treatment achieving blood 89 pressure targets. 5-8 90 previous guidelines typically recommended initiating monotherapy, up-titration of dose, switching drugs if not 91 tolerated, and adding other agents if needed. 7 this often takes multiple visits to achieve target blood pressure -and 92 studies show that most individuals remain on monotherapy and with inadequate blood pressure control. 5 the largest 93 global survey of hypertension practice showed only 34% of those treated for high blood pressure were controlled 94 (sbp<140 and dbp<90mmhg), and 31% of treated patients were receiving combination therapy. 5 the 2017 may 95 measurement month blood pressure screening campaign included a convenience sample of 1.2 million across 34 96 countries, and found 54% of those treated had adequate blood pressure control. a 2013 survey of 31 international 97 hypertension guidelines showed that 27 (87%) now recommend use of combination for initial treatment, but typically 98 only as an option for patients at >20/10mmhg from goal. 9 as 50 to 75% of patients require combination treatment for 99 blood pressure control, there has been increasing interest in the initial use of combination therapy. 10 most recently 100 the esc/esh guidelines recommended initial combination therapy for most people, except those with low 101 cardiovascular risk and sbp<150mmhg and frail older adults. 11 102 there are multiple barriers to blood pressure control that are patient, healthcare system and physician related. 103 patient adherence is a major factor and is worsened by increased number of medications, complexity of dosing 104 regimens and medication side effects. 12, 13 'therapeutic inertia', the reluctance of physicians to treat mild 105 hypertension and up-titrate medications, is also a barrier to blood pressure control. a large study conducted in 106 western europe and the us of more than 20,000 people with hypertension found that blood pressure control rates 107 ranged from 31 to 63%, and only 15 to 38% of instances of elevated blood pressure had up-titration during the visit. 14 108 there is a clear need for improved strategies that will: a) make the treatment of high blood pressure more effective 109 and easier to implement for doctors and patients; b) quickly and safely bring blood pressure under control and; c) 110 increase long term adherence with therapy. we hypothesize that a single-pill combination of four blood pressure 111 lowering agents at quarter dose may achieve these goals. 112 rationale for very low-dose combination therapy 113 dose response data on blood pressure reduction 114 pharmacological dose response curves for blood pressure lowering drugs indicate that a quarter-dose has at least half 115 the blood pressure-lowering effect of a standard dose (usual maintenance dose) but with much fewer side effects. a systematic review of all randomized trials of quarter dose blood pressure lowering identified a total of 42 trials, 38 117 of single quarter-dose comparisons, seven of dual quarter-dose comparisons and two of a quadruple quarter-dose 118 combination. 15 compared to placebo, single quarter dose therapy reduced blood pressure by 5/2 mmhg (p<0.0001) 119 with no increase in adverse events. dual quarter-dose therapy was similarly efficacious as standard-dose 120 monotherapy. two studies of quadruple quarter-dose therapy have been published. one unblinded pilot with four 121 control groups of standard-dose monotherapies of the components showed a reduction of 13/8mmhg compared to 122 the average reduction of all four controls after four weeks. 16 the other, our pilot, a double-blinded placebo-controlled 123 cross-over trial in people with newly diagnosed hypertension showed a reduction of 22/13mmhg after four weeks 124 active treatment versus placebo. 17 125 there is strong evidence that the blood pressure lowering effects of different classes of drugs are independent and 126 fully additive. 15, 18 the effects of adding a second blood pressure lowering agent are closely concordant with those 127 predicted by independent effects, occur across all pairs of medication classes, and are about five times more effective 128 than doubling the dose of the first agent. 18 the additive effects across three classes of low-dose drugs were also 129 j o u r n a l p r e -p r o o f demonstrated in a placebo-controlled, crossover trial of three half-dose blood pressure drugs in 86 participants aged 130 over 50 years without a history of cardiovascular disease. 19 overall a 17.4/9.4 mmhg blood pressure reduction was 131 observed, compared to the anticipated 17.9/9.5 mmhg decline expected from the cumulative effects of the three 132 separate agents. 133 dose response data on adverse effects biochemical changes appear minimal with quarter-dose therapy compared to standard-dose monotherapy. 15 these 149 data suggest that dose-dependent adverse effects will be minimal with this intervention, and idiosyncratic reactions 150 are uncommon with these component medications. 151 objective 152 the primary objective of the quadruple ultra-low-dose treatment for hypertension (quartet) trial is to examine 153 whether ultra-low-dose quadruple combination therapy (quadpill) is more effective than guideline recommended 154 therapy with an arb plus a ccb if required in lowering blood pressure. our secondary aim is to assess if this approach 155 is safe and has fewer adverse effects compared to standard care. 156 trial design 158 this is a 12-week double blind randomized controlled trial of 650 patients with high blood pressure. participants are 159 randomized in a 1:1 allocation ratio using a central computer-based service, to initial therapy with quadpill or to a 160 standard dose of an arb, with a ccb added as required, as per current guideline recommendations ( figure 1 ). the 161 primary outcome is reduction in mean office systolic blood pressure (sbp) measured using omron hem-907 at 12 162 weeks. secondary outcomes include: the proportion of participants with controlled blood pressure (sbp<140mmhg 163 and diastolic blood pressure [dbp] <90mmhg) at 6 weeks and 12 weeks, ambulatory blood pressure (abp) measures at 164 12 weeks, tolerability and the occurrence of adverse events. learnings from the quadpill pilot informed the design and 165 conduct of the present trial. 17 166 extension study 167 an extension study to 12 months follow-up involves two more visits, at 26 and 52 weeks after randomization to 168 examine longer term efficacy and tolerability. 169 setting, locations and recruitment 231 participants are recruited from community general practices and outpatient clinics. current active sites are listed in 232 the appendix. there is a total of 10 sites in 4 of the 8 states and territories of australia (new south wales, victoria, 233 tasmania and western australia), with 3 of these based in primary care and the rest in hospital or university locations. 234 we employ several methods to identify potentially eligible participants. this includes community advertising and 235 awareness campaigns (using print and electronic media advertisements and radio), referral by clinicians aware of the 236 study ( hydrochlorothiazide is included in a number of fixed dose combinations, some recent guidelines 23 and literature 249 recommend indapamide or chlorthalidone, principally on the basis that some data suggest more cardiovascular event 250 reduction with these agents, 24, 25 though a recent paper suggests no difference. 26 the additional blood pressure 251 reduction expected from including a quarter-dose of a different class of drug is about three times as great as would be 252 achieved by doubling the dose of any other component. 15 we chose the 4 th agent to be a bb, due to its long duration 253 of action, relatively minimal side effects at a quarter dose. the choice of a beta-blocker as a 4 th agent of choice is also 254 consistent with a number of international hypertension guidelines which specify beta-blocker use after renin 255 angiotensin system blockers, ccbs and thiazide type diuretics. 22, 27, 28 we chose bisoprolol over atenolol due to its 256 longer duration of action. the other major consideration was use of off-patent components to minimize costs. 257 the control group follows the recommendations of the current australian guidelines, 22, 29 i.e. initiating with an ace-i or 258 arb, and if blood pressure is not controlled adding a ccb. this approach is also consistent with the 2011 nice 259 hypertension guidelines, and among the preferred treatment options in the 2013 jnc-8 guidelines and the 2013 260 esc/esh guidelines, which were current at the inception of this study. 23, 30, 31 we chose irbesartan as it is the most 261 commonly prescribed arb in australia and amlodipine because it is the most commonly prescribed ccb. 262 patients who are on monotherapy at time of recruitment will be asked to stop their treatment while they are taking 263 the study treatment. the drug is provided to both intervention and control arms at no cost to the participant. 264 medications are provided in quantities of 99 tablets at 3 monthly intervals. that is a medication kit is given to patients 265 at baseline, week 12 and at 6 months and 9 months in participants participating in the extension study. each kit 266 consists during the study we will obtain information on self-reported health service utilization, and specifically ask if patients 295 have seen and how frequently they have seen the following health providers -practice nurse, general practitioner, 296 doctor in public hospital emergency department (not admitted), doctor in public outpatients clinic for any reason and 297 doctor in private specialist clinic for any reason. we also request consent to link data to mbs (medical benefits 298 schedule -listing of medicare services subsidized by the australian government) and pbs (pharmaceutical benefits 299 scheme -listing of medicines subsidised by the australian government). 300 information is collected on serious adverse events and adverse events of special interest (see list in appendix). we 301 specifically query participants about adverse events of special interest at each visit (6 week, 12 weeks and additional 302 visits for extension participants 6 months and 12 months). adverse events of special interest include: dizziness, 303 hypotension, pedal oedema, muscle cramps, bradycardia, heart failure, hypersensitivity reactions (skin rashes, itching), 304 gastrointestinal complaints (nausea, vomiting, diarrhea), musculoskeletal complaints, headaches. adverse events are 305 not adjudicated. the euroqol group (eq-5d-3l) quality of life questionnaire is completed by participants at their 306 baseline, 12 week and final visits. 307 308 outcome measures and outcome assessment 309 the primary and secondary outcomes are listed in table 1 . 310 the blood pressure measurements are recorded using an omron hem907. an appropriate cuff size is selected for all 311 bp measurements. first a measure of clinic blood pressure is observed and recorded by research staff. then, 312 automated office blood pressure is measured following the recommendations of the european society of 313 hypertension/european society of cardiology and australian national heart foundation. 22, 30 this requires the 314 research staff to set the automated device to take three separate bp measurements while the researcher steps out of 315 the room (unattended bp measurement). the omron hem907 is programmed to start the first measurement after 316 five minutes of rest, then at one-minute intervals. the primary outcome "mean sbp" will be calculated using the 317 j o u r n a l p r e -p r o o f average of these three unattended measures. in addition, 24-hour abpm is conducted at baseline, 12 and 52 week 318 follow-up visits using a suntech oscar-2 programmed to measure every 30 minutes while participant is awake, and 319 hourly during sleep. 32, 33 320 sample size 321 a sample size of 650 patients provides 90% power at p=0.05 to detect a difference between randomized groups of 4 322 mmhg in the primary outcome, assuming a standard deviation (sd) of 15mmhg. 34 a sample of 650 also has 85% 323 power to detect a 3mmhg difference in average 24hr sbp (sd 12 mmhg) 34 and 85% power to detect a 25% increase in 324 the proportion with controlled blood pressure assuming 50% are controlled in the comparator group. all calculations 325 allow for a 10% dropout or data loss rate. it is assumed that irbesartan 150 mg and up-titration with the addition of 326 amlodipine in 75% of participants in the control group will give an average reduction of 12mmhg from an average 327 baseline sbp of 150mmhg. 35 based on the information presented in the background, quadruple combination therapy 328 will reduce sbp by at least 16mmhg. 16, 20 329 the rate of all adverse events is predicted to be around 15% in the control group, 35 and this study will have 90% power 330 to rule out an increase of 5 percentage points (i.e. a non-inferiority margin of 20%) assuming the true incidence of 331 adverse events in the quadpill group is 10% and a one-sided test with alpha=2.5%. the 10% incidence of adverse 332 events is a conservative estimate from adding up the incidence of side effects from each treatment class at ½ standard 333 dose described in a previous systematic review: bb 5.5%, tz 2.0%, ccb 1.6% and arb 0%. interim analyses, monitoring, and stopping guidelines 335 the trial data safety and monitoring committee (dsmc) monitors safety data on an ongoing basis, with the analyses 336 performed by an independent statistician from the george institute for global health. the dsmc can recommend the 337 steering committee of the quartet study should continue the study unchanged, adjust the duration of follow-up, or 338 terminate the study early if there is clear and substantial evidence of benefit, if the data suggests the risk of adverse 339 events substantially outweighs the potential benefits, or for futility. the first dsmc meeting was held after 25% of 340 participants completed 12 weeks follow up and recommended continuation of the study without modification. 341 the unblinded statistician prepared a computer-generated randomization schedule stratified by site and using 343 permuted blocks of variable size. this was loaded into the web-based data management system (ibm clinical 344 development, morrisville usa). allocation concealment is maintained as only the unblinded statistician and unblinded 345 data manager have access to the randomization list and allocation within the database. 346 participants are enrolled at sites by blinded staff, with participant randomization and study drug allocation conducted 347 through the database with blinding maintained. the study drug kit numbering is separate to the randomization 348 sequence to prevent the kit allocation potentially unblinding site staff. the investigators, project management, site 349 staff, and participants are blinded to the randomization sequence and treatment allocation. 350 the main analyses of study outcomes will be conducted according to the principle of intention-to-treat. the primary 352 analysis of change in sbp at 12 weeks will be performed using an analysis of covariance including the treatment arm 353 and baseline sbp as a covariate. continuous secondary outcomes will be analyzed similarly. additional analyses will 354 include all follow-up measurements in a longitudinal model including treatment arm, visit, and a treatment by visit 355 interaction term as well as the baseline measurement. within-patient correlations will be modelled using generalized 356 estimating equations or random effects. a similar approach will be applied to binary endpoints (e.g. blood pressure 357 control) with log-binomial regression used in place of linear regression. a per-protocol analysis will be performed to 358 provide information on the difference in efficacy between the two study treatments. there will also be pre-defined 359 subgroup analyses, including by baseline blood pressure, gender, age, diabetes, education and by bp lowering 360 treatment at baseline (no treatment versus monotherapy). a detailed analysis plan will be finalized prior to unblinding. cost-effectiveness and cost-utility analysis 364 an incremental cost-effectiveness analysis will be used to compare the costs and outcomes of the treatment arms 365 from a health system perspective. this will consider the cost per mmhg reduction in systolic blood pressure and the 366 cost per quality adjusted life-year gained for quadpill versus monotherapy to facilitate comparison with other 367 interventions. costs will be determined through the collection of resource use during the study period and estimates 368 of commercial costs for the quadpill. information on hospital admissions, doctors' visits and medications is collected at 369 follow-up visits. 370 a semi-quantitative survey and in-depth interviews will be conducted to assess the acceptability of quadpill, and to 372 identify which factors are important to participants and health providers in blood pressure reduction. patient 373 acceptability is a critical component of healthcare innovation. patients and health providers in the study will be invited 374 to answer questions assessing their perceptions, experience and the degree of engagement with the intervention at 375 the completion of the trial. patients and health providers will be invited to participate in semi-structured interviews on 376 perceptions of the utility and acceptability of the intervention program. examples of questions are included in the 377 appendix. interviews will be recorded and transcribed, then coded using nvivo. from the coded data key themes will 378 be identified. 379 trial management, funding, and sponsorship the trial conduct is overseen by a steering committee (list in appendix). the central coordinating center ensures 381 implementation of the study according to the protocol, timelines and recruitment targets. we use an electronic data 382 management system incorporating study checks and omissions. an independent data and safety monitoring 383 committee meets regularly to assess emerging evidence on safety and efficacy. the quartet trial is the first large-scale trial to examine a quadruple, quarter-dose regimen. this approach has many 406 theoretical benefits, including greater efficacy and fewer side effects as well as pragmatic benefits that should 407 improve adherence and decrease costs. if this new intervention achieves its conservative additional 4mmhg of blood 408 j o u r n a l p r e -p r o o f pressure reduction compared to that conferred by optimal guideline-recommended care, such a difference could 409 translate into an additional 15 to 20% reduction in cardiovascular events. 3 there has been increasing acceptance of the role of dual anti-hypertensive combinations in blood pressure 411 management both due to the observation that most patients require more than one agent to achieve bp control, and 412 by trials showing early use of combination is beneficial. the triumph trial evaluated a half-strength triple pill, but with several points of difference, most importantly the 426 comparison against a variety of usual care options in sri lankan outpatient hospital care, with a focus on improving 427 the access and affordability of blood pressure lowering medications in this setting. 44 this study found 70% of 428 participants in the triple pill group achieved their target blood pressure versus 55% in the usual care group, 44 and the 429 triple pill was cost-effective compared to usual care. 45 the quadpill pilot trial was a placebo-controlled pilot study was 430 conducted in treatment-naïve people with newly diagnosed high blood pressure in primary care. 17 the ultra-low dose 431 quadruple combination was very effective at lowering blood pressure in the short-term single center pilot study, 432 hence the current study is needed. a sister trial, quartet usa (clinicaltrials.gov nct03640312), is currently underway 433 in chicago usa and an individual patient data meta-analysis is planned once both trials are completed. 434 if the intervention tested here is proven to be safe and effective, the trial results could be rapidly implemented, with 436 immediate benefits in routine clinical practice. similar therapy could be provided to patients using available 437 medications, including existing dual combinations and the use of dose administration aids. ultimately, most advantage 438 will be gained from single pill formulations. the results of the current trial would stimulate the development of such 439 products if the results were favorable. 440 in summary, ultra-low-dose combination therapy has the potential to have a major impact on current poor rates of 441 blood pressure control globally. the critical next step is direct evidence on effectiveness and safety in a large-scale 442 randomized controlled trial, which the quartet trial aims to provide. 443 authors' contributions 444 ckc wrote the first draft of the quartet protocol that was subsequently funded by nhmrc with critical review from 445 ar as the senior author and all cis of the nhmrc protocol including gh, ms, tu, rw, lb. era has been the 446 postdoctoral fellow on quartet, prepared the first draft of the manuscript and revised the manuscript. all authors 447 have reviewed the final manuscript. we also acknowledge henry krum (deceased) who provided critical review of the 448 quartet protocol submitted to nhmrc. 449 the quartet trial received principal funding through a project grant from the national health and medical research 451 council australia (app1100377). the trail was also supported by funding from nhmrc program grants (app1052555 452 and app1092642). individual investigators also received support to enable their time allocation to the trial: cc was 453 j o u r n a l p r e -p r o o f supported by nhmrc career development fellowship level 2 (app1105447), era was supported by a national j o u r n a l p r e -p r o o f table table 1 difference between groups in change in mean office systolic blood pressure from baseline to 12 weeks secondary outcomes 24-hour ambulatory blood pressure a) difference between groups in mean 24-hour sbp and dbp at 12 and 52 weeks b) difference between groups in mean change in 24-hour sbp and dbp from 0 to 12 weeks, 0 to 52 weeks and 12 to 52 weeks c) difference between groups in mean daytime sbp and dbp at 12 and 52 weeks d) difference between groups in mean night-time sbp and dbp at 12 and 52 weeks e) difference between groups in daytime, night-time, and 24-hour bp load (percentage area under the blood pressure curve above normal day, night, and 24-hour values as per national heart foundation guidelines f) difference between groups in the proportion of non-dippers (night-time bp is not more than 10% lower than average daytime bp as per national heart foundation guidelines) and coefficient of variability of bp 33 other blood pressure measures a) difference between groups in mean automated office systolic (52 weeks) and diastolic blood pressure (12 and 52 weeks). b) difference between groups in standard clinic sbp/ dbp at 12 and 52 weeks c) hypertension control (% with sbp <140 mmhg and dbp <90 mmhg) at 6, 12, 26 and 52 weeks, d) percentage requiring step-up treatment at 6 weeks e) percentage requiring step-up blood pressure lowering treatment over 52 weeks f) percentage with both bp control (as defined above) and no adverse events. g) difference between groups in sbp and dbp variability tolerability a) difference between groups in potentially related side-effects (dizziness, blurred vision, syncope/ collapse/ fall, chest pain/ angina, shortness of breath, cough, wheeze, ankle edema, skin rash, itching, gout, hyperkalemia, hypokalemia, hyponatremia, other) b) difference between groups in mean potassium, uric acid, blood glucose, cholesterol and fractions, alt, ast, uacr (urine albuminto-creatinine ratio) and creatinine levels. c) difference between groups in participant withdrawals from treatment safety percentage with any severe adverse event medication adherence self-reported measures and pill counts the ratio of the difference in costs and outcomes between treatment arms patient and prescriber acceptability end of study feedback questionnaires note: key secondary outcomes have been put in bold j o u r n a l p r e -p r o o f a suspected unexpected serious adverse reaction is any uar that at any dose meets the definition of an sae (refer to section 3). any event that meets the definition of a susar between when the informed consent form is signed and the end of study visit at week 12 or week 52 will be reported to the local hrec/ irb and the relevant regulatory authorities as per local requirements and ich clinical safety data management: definitions and standards for expedited reporting. examples of questions asked of providers and participants to assess acceptability of the quadpill intervention examples of questions asked of participants include: -during the trial, how easy did the participant find it to take the trial medications? -if the ldqt is available to be prescribed by participant's usual doctor, how likely would the participant be to request it? -are there any other comments the participant had about the ldqt? examples of questions addressed to healthcare providers about the quad pill include: -what do you think are the potential benefits of ldqt or your concerns about ldqt? -if ldqt was available, in what circumstances would you prescribe it or what evidence would you require to start prescribing ldqt? -what do you consider to be important factors in patients' decisions to take blood-pressure lowering medications? j o u r n a l p r e -p r o o f 2013 esh/esc 575 guidelines for the management of arterial hypertension: the task force for the management of arterial 576 hypertension of the european society of hypertension (esh) and of the european society of cardiology 580 evidence-based guideline for the management of high blood pressure in adults ambulatory blood pressure monitoring in australia: 2011 consensus position 583 statement ambulatory blood pressure measurement: what is the international 585 consensus? efficacy and tolerability of fixed-dose combination of perindopril/indapamide in type 2 diabetes 587 mellitus: picasso trial i-add study: assessment of efficacy and safety profile of irbesartan/amlodipine 589 fixed-dose combination therapy compared with irbesartan monotherapy in hypertensive patients 590 uncontrolled with irbesartan 150 mg monotherapy: a multicenter, phase iii, prospective, randomized, open-591 label with blinded-end point evaluation study thrift 656 global, regional, and national comparative risk assessment of 84 664 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2016: a 665 systematic analysis for the global burden of disease study for the for the institute for health m, evaluation. global deaths 667 attributable to high systolic blood pressure a simplified approach to the 669 treatment of uncomplicated hypertension: a cluster randomized, controlled trial optimisation of antihypertensive treatment by 672 crossover rotation of four major classes long term monitoring in patients receiving treatment to lower blood 674 pressure: analysis of data from placebo controlled randomised controlled trial compliance, safety, and effectiveness of fixed-dose combinations of 676 antihypertensive agents: a meta-analysis the efficacy and safety of triple vs dual combination of angiotensin ii receptor 678 blocker and calcium channel blocker and diuretic: a systematic review and meta-analysis. the journal of 679 clinical hypertension fixed low-dose triple combination antihypertensive medication vs usual 683 care for blood pressure control in patients with mild to moderate hypertension in sri lanka: a randomized 684 clinical trial fixed-combination, 687 low-dose, triple-pill antihypertensive medication versus usual care in patients with mild-to-moderate 688 hypertension in sri lanka: a within-trial and modelled economic evaluation of the triumph trial. the lancet 689 global health j o u r n a l p r e -p r o o f harms all serious adverse events (saes) and adverse events of special interest (aesi) experienced by a participant after the informed consent document is signed and until the end of the study at week 12 or 52 will be collected and reported to the ccc as per applicable ich gcp and applicable regulatory guidelines. if an sae is unresolved at the conclusion of the study, a clinical assessment will be made by the medical monitor as to whether continued follow up of the sae is warranted. sae criteria, definitions and guidance for reporting are outlined in section 1 to 4 an adverse event is defined as any untoward medical occurrence in a subject or clinical investigation subject administered a pharmaceutical product at any dose that does not necessarily have to have a causal relationship with this treatment. therefore, an ae can be any unfavourable and unintended sign (including an abnormal laboratory finding, for example), symptom, or disease temporally associated with the use of an investigational product, whether or not considered related to the investigational product. this definition includes intercurrent illnesses or injuries and exacerbation of pre-existing conditions. the expected adverse reactions to the bp lowering medications that will be used in quartet are well known (appendix 2). to better assess participants' tolerability to the study medications the following aesi's and whether they are new or ongoing from baseline will be reported to the ccc regardless of severity and seriousness: a serious adverse event (sae) is defined as any untoward medical occurrence that at any dose:  results in death  is life threatening in the opinion of the attending clinician (i.e. the patient was at risk of death at the time of the event; it does not refer to an event that might hypothetically have caused death had it been more severe)  requires inpatient hospitalisation or prolongation of existing hospitalisation (any hospitalisation that was planned prior to randomisation will not meet sae criteria. any hospitalisation that is planned post randomisation will meet the sae criteria)  results in persistent or significant disability or incapacity  results in congenital anomaly or birth defect (note that the females in the study population are likely to be postmenopausal)  is an important medical event in the opinion of the attending clinician that is not immediately life-threatening and does not result in death or hospitalisation but which may jeopardise the patient or may require intervention to prevent one of the other outcomes listed above an adverse event that meets the above categories between when the informed consent form is signed, the end of study visit at week 12 or at 26 and 52 weeks if patient is participating in the study extension and until the 28 days after the study drug discontinued will be reported as an sae. all saes are required to be reported to the sponsor team within 24 hours of the study team first becoming aware of the event. the sae will also be required to be reported to the relevant hrec/ irbs within the timeframe specified in the relevant committee guidelines. if irbesartan or the ldqt is discontinued as a result of an ae, the study team will document all events leading to the discontinuation of treatment. adverse events which do not fall into these categories are defined as non-serious. an unexpected adverse reaction (uar) is an adverse reaction, the nature or severity of which is not consistent with the applicable product information. refer to (quartet protocol, appendix 2) for a list of expected adverse reactions for the interventions used in this protocol.j o u r n a l p r e -p r o o f key: cord-274061-ynqxgyw6 authors: epstein, jay s.; jaffe, harold w.; alter, harvey j.; klein, harvey g. title: blood system changes since recognition of transfusion‐associated aids date: 2013-10-17 journal: transfusion doi: 10.1111/trf.12373 sha: doc_id: 274061 cord_uid: ynqxgyw6 nan the fact that transfusions could transmit infectious diseases, namely, bacterial infections, syphilis, and hepatitis, was recognized before taa with progressive interventions dating back to the dawn of blood banking. donor testing for antibodies to syphilis began in 1938. 7 bacterial infections, a major threat at the time of world war ii, were later decreased by cold storage of whole blood and red blood cells (rbcs) in plastic containers. 5, 7 in the 1970s, transfusion-associated hepatitis (tah) was largely prevented by near elimination of paid donation through product labeling to identify paid collections, concurrent with testing for hepatitis b virus (hbv) infections. 5 however, the medical importance of the residual hepatitis risk, mostly attributed to non-a, non-b hepatitis (nanbh), was recognized slowly. 5 with the acute threat of bacterial infections largely controlled, syphilis effectively prevented, and the full consequences of nanbh transmission unappreciated, the blood community in the late 1970s was more focused on systemic issues of economic competition and supply instabilities than on transmissible disease. then came aids! aids was first reported as a "gay-related immune deficiency" in 1981, but soon was identified in other risk groups including sex workers, haitian entrants to the united states, and injection drug users. 3, 4 evidence for transfusion transmission emerged in 1982 when a few cases of aids were reported in hemophilia patients and later in transfusion recipients. however, despite a number of high-level federal meetings, actions by the national government to contain the aids risk from transfusion were not undertaken until 1983. 8 although transfusion transmission of hiv undoubtedly took place at least 5 years before the recognition of taa due to the very long asymptomatic period of the disease, the delay in a response to taa subsequent to these initial reports of disease in persons with hemophilia and transfusion recipients also contributed to the aids tragedy. rage within the hemophilia community, due both to the fact of transmission of a fatal infection and to the failure of authorities to provide adequate warnings and preventions, was expressed in a demand for a congressional investigation. members of congress instead directed the department of health and human services (hhs) to look into the matter. this was accomplished through a contract with the institute of medicine (iom) to study the evolving hiv-related events impacting blood safety and the decision-making process in this crisis period. in its report, entitled "hiv and the blood supply: an analysis of crisis decision making (1995)," 9 the iom found no wrongdoing by organizations or officials, but identified failed opportunities to better protect public health. these failures to act more rapidly and aggressively in the face of taa were seen to unmask an underlying weakness in the ability of federal agencies to address a new threat in the face of substantial scientific uncertainty. this weakness was attributed to systemic deficiencies, primarily of leadership and coordination. in particular, the iom criticized the federal agencies for lack of top-level leadership needed to overcome inherent bureaucratic inertia; absence of a systematic approach within advisory committees sufficient to maintain their focus; over dependency on the regulated industry as a source of data given the inherent conflict of interest; and failure to engage in forward thinking both with respect to new technologies and emerging safety threats. as a consequence, the risk of taa was severely underestimated; patients and care providers were not suitably warned of the risk; and resistance to a change in the status quo caused delayed intervention. in a set of 14 recommendations directed primarily at federal agencies, the iom called for a more responsive and integrated decision-making process including establishment of a blood safety council reporting to a designated blood safety director within hhs and a standing "expert panel" to assure communication of blood product risks and alternatives to their use both to care providers and to the public. specifically to the food and drug administration (fda), the iom recommended that, "where uncertainties or countervailing public health concerns preclude eliminating potential risks, the fda should encourage, and where necessary require, the blood industry to implement partial solutions that have little risk of causing harm." while not itself a mandate, the iom's admonition that the fda should institute measured precautions in the face of uncertainty has become a dominant factor in blood safety decision making. a more vigilant and proactive fda approach to blood safety unfortunately has had the unintended consequence of dramatically increasing the manufacturing costs and therefore the price of blood. 10 the hhs response to the iom report established a new landscape for federal oversight of the blood system, which continues to the present day. the present structure includes the assistant secretary for health (ash) as the blood safety director; heads of public health service and related agencies as members of a blood, organ and tissue safety executive council (botsec); and an hhs secretary's advisory committee for blood and tissue safety and availability (formerly the advisory committee for blood safety and availability). the ash is the acknowledged national blood safety director with final responsibility and authority for decisions regarding blood safety and availability. an interagency blood, organ and tissue safety working group meets monthly by teleconference, more often when necessary, and the botsec meets approximately quarterly face to face with the ash to provide information and guidance regarding current and emerging issues involving the nation's blood supply. 11 unlike fda's blood products advisory committee, whose function is to provide external scientific advice relevant to regulation, the secretary's advisory committee is empowered to discuss broad legal, ethical, social, and economic issues affecting the blood system. to give voice to patient concerns, both advisory committees seat voting representatives of communities that have been particularly affected by taa. additionally, in response to a series of congressional hearings, reports from the government accountability office, and the iom study, the fda developed and hhs subsequently adopted a comprehensive "blood action plan" 12 designed to address the identified shortcomings, to ensure greater coordination among the department's public health agencies, and to increase the effectiveness of the fda's scientific and regulatory activities. notably, the post-taa era has witnessed an aggressive effort by the fda to improve blood safety through enforcement of cgmp in blood product collection and processing aligned with the model of pharmaceutical manufacturing and a more formal relationship than blood establishments experienced in the past. the fda initiative also involved promotion of automation to reduce human errors, including use of validated blood bank software. an intensive program of field inspections designed to assure universal regulatory compliance of blood collection establishments resulted in a number of court-enforced voluntary injunctions (consent decrees). known and emerging infectious threats to blood safety have continued to demand attention in the post-taa era, repeatedly testing whether the lessons of taa were learned. are we prepared to deal with potential threats from bioterrorism agents? how much effort should be expended to prepare for an outbreak of chikungunya virus that might never happen? what should we do about pandemic influenza and middle east respiratory syndrome coronavirus in the absence of studies to establish the presence or absence of viremia in the course of the infections? does it make sense to screen all blood donations when risks of babesiosis and dengue are seasonal and geographical? what changes to the current paradigm of donor screening and testing can be considered when pathogen reduction becomes available for all blood components? more generally, as we become increasingly proactive in addressing infectious risks, are we misdirecting resources that could be better spent to improve blood safety in other ways? readers of this commentary are encouraged to ask themselves whether the lessons of taa have been optimally incorporated during the decades of challenge and response that followed. a sentinel event in the history of blood safety was the recognition and response to taa. although the etiology remained unknown, the report of aids in three persons with hemophilia a in july 1982 suggested a blood-borne pathogen as the causative agent. 3 these three individuals were reported to be heterosexual, had no other known aids risk factors, and had all received frequent administration of factor viii concentrate. the evidence for transmission of the "aids agent" through blood was further strengthened in december 1982 by the report of a 20-month-old infant in san francisco who had developed unexplained immunodeficiency after transfusion of multiple blood products to treat erythroblastosis fetalis. 4 one of the blood donors was a man who was healthy at the time of donation, but subsequently died of aids. to address the possibility that aids was associated with the receipt of blood and blood products, the centers for disease control and prevention (cdc) convened a meeting on january 4, 1983, with participation by the fda, the national hemophilia foundation, blood banking officials, and patient advocacy groups. from the cdc perspective, the purpose of the meeting was to discuss how to reduce the risk of aids in transfusion recipients and persons with hemophilia in the absence of a test for the etiologic agent. several possible strategies were presented, including deferral of blood donations by persons known to be at increased risk for aids and the use of surrogate tests to identify persons at increased risk of transmission, such as those with detectable antibody to hepatitis b core antigen (anti-hbc) or low cd4/cd8 t-cell ratios. however, the meeting turned into a contentious debate about the existence of aids in transfusion recipients and persons with hemophilia, and no agreement was reached on a risk reduction strategy. on march 4, 1983 , the us public health service published the first recommendations for prevention of aids. 8 among the recommendations was a statement that, "as a temporary measure, members of groups at increased risk for aids should refrain from donating plasma and/or blood." in addition to persons with clinical evidence of aids and their sexual partners, those considered to be at increased risk included "sexually active homosexual or bisexual men with multiple partners; haitian entrants to the united states; present or past abusers of iv drugs; patients with hemophilia; and sexual partners of individuals at increased risk for aids." at the time, these recommendations were controversial. in particular, restricting blood donation by homosexual men was seen as a civil rights issue, and deferring donations by haitian entrants undoubtedly led to discrimination against haitian americans. from a public health perspective, however, these measures were needed to increase blood safety. with the identification of hiv, screening of donated blood and plasma became possible. bulk preparations of the virus, known at the time as htlv-iii, were provided by the national cancer institute to diagnostics companies for the development of antibody detection tests. the first such screening test, developed by abbott laboratories, was approved by the fda in march 1985. because of concerns that persons would donate blood for the purpose of learning their hiv infection status, the cdc funded the first alternative hiv test sites, where individuals could obtain free and confidential testing. blood banks also established the option of confidential unit exclusion to allow persons who had donated blood to confidentially indicate that the blood should not be used for transfusion. a watershed event in blood safety was the statement by the fda commissioner at a september 1994 workshop that nucleic acid technology should be implemented to close the window period for hiv detection by serology. this technology had been considered too costly and cumbersome for practical application in blood banking. the introduction of direct testing for hiv in donor blood, first by p24 antigen assays, which proved largely unproductive, 13 and then by nucleic acid tests (nats) for viral rna, which proved beneficial, put to rest a decade of concern about residual hiv risk from donations in the 3-to 6-week infectious "window period" before seroconversion dependent on the sensitivity of different screening tests. the successful adaptation of nat to donor screening, including testing of specimens in small pools of 16 to 24, established a new era in risk reduction from transfusiontransmitted viral diseases. in addition to increasing the safety of transfused blood, hiv antibody screening of donors led to "lookback" programs in which recipients of previous unscreened donations from infected donors were identified. these recipients were found to be at substantial risk for infection. 14, 15 although no effective treatment was available at the time, infected recipients could be counseled to reduce the risk of hiv transmission to others. another retrovirus, htlv-i, was also found to cause disease, including adult t-cell leukemia or lymphoma and htlv-1 associated myelopathy or tropical spastic paraparesis. the virus can be transmitted by transfusion of cellular blood products, but not plasma fraction or plasma derivatives. 16 in november 1988, the fda issued guidance recommending antibody testing of donated whole blood and cellular components for htlv-i. because of a high degree of sequence homology, the currently approved htlv-i screening assay also detects antibodies to htlv-ii, a virus with transmission routes similar to htlv-i but with less clear disease associations. although not fda approved, western blot and pcr tests can be used to distinguish between the two viruses. world war ii led to recognition of the frequent occurrence of hepatitis among military personnel through the confluence of contaminated water, massive immunizations, and for the first time, blood transfusion. it was during this time that food-and water-borne "infectious hepatitis" was distinguished from parenterally transmitted "serum hepatitis" and these entities were later termed hepatitis a and b, respectively. in 1943, beeson 17 reported seven cases of jaundice occurring 1 to 4 months after transfusion of blood or plasma. a dramatic outbreak of hepatitis involving 50,000 us soldiers was traced to serum-contaminated preparations of yellow fever vaccine, which conclusively documented parenteral transmission. decades later, this outbreak was shown by seeff and colleagues 18 to be due to the hepatitis b virus. the us army extensively studied serum hepatitis during and after the war and characterized both the epidemiology and the resultant disease, but could not identify the causative agent. the etiologic breakthrough began in the early 1960s with the discovery of the australia antigen by blumberg and coworkers at the national institutes of health (nih). 19 this single finding changed the course of hepatitis history when in 1968, the australia antigen was shown by the blumberg group to be associated with viral hepatitis 20 and then by prince and colleagues 21 to be specifically associated with hepatitis b. in england, dane and coworkers 22 showed by immune electron microscopy that the australia antigen represented the envelope protein of hbv and it was renamed the hepatitis b surface antigen (hbsag). the serologic distinctions between hepatitis a and b were further solidified by the controversial, but definitive prospective studies by krugman and colleagues 23 at the willowbrook state school. the us government played a pivotal role in these momentous events, first through the initial discovery of the australia antigen in the intramural program at nih and then through extensive grant support of the blumberg laboratory at the institute for cancer research in philadelphia. in the late 1960s and early 1970s, prospective studies at the nih clinical center revealed several critical elements of tah, including: 1. that the primary risk factor for tah was the use of paid donor blood 24 confirming earlier studies; 25 in 1972, this led to an fda mandate requiring the labeling of paid donor blood, which effectively resulted in the near-universal adoption of blood collection only from unpaid volunteers, one of the most important transfusion-transmitted infectious disease interventions ever implemented. 2. that hbsag testing of blood donors was effective even when using insensitive techniques such as agar gel diffusion and counterelectropherseis; 26 nationwide testing for hbsag was delayed until more practical and confirmable assays were introduced in 1972. volunteerism and first-generation hbsag screening reduced the incidence of tah from 30% to approximately 10% 26 and that this massive reduction was more dependent on the donor source than on blood screening because hbv was shown to account for less than 30% of total tah. feinstone and coworkers at nih, 27 it became evident that hav was not responsible for the residual cases of tah, giving rise to the cumbersome, but nonpresumptive designation nanbh. 28 while intensive efforts to isolate the nanbh agent in the decade from 1975 to 1985 were unsuccessful, studies at the nih and cdc revealed that the agent was small, lipid-enveloped and most similar to the small rna alpha and flaviviruses. [29] [30] [31] despite the absence of a specific test for detecting the nanbh agent, tah incidence declined because of the more judicious use of blood fostered by the recognition that nanbh could result in cirrhosis and death 32 and by the devastating consequences of transfusion-transmitted hiv. 5 further, in the absence of specific nanbh assays, surrogate assays were advocated. the transfusion transmitted virus study, supported by the national heart, lung and blood institute, published a retrospective analysis of a prospective study that showed that alanine aminotransferase (alt) testing of donors might effect a 30% reduction in tah incidence. 33 this was confirmed by a similar analysis of the nih prospective tah study, 34 but implementation of alt donor screening at the nih failed to demonstrate the predicted benefit. 35 similar retrospective testing of the transfusion transmitted virus study 36 and the nih 37 prospective studies suggested that anti-hbc testing might result in a 30% to 40% reduction in tah, and this fostered the voluntary introduction of alt and anti-hbc donor testing in 1986 to 1987; the fda recommended routine donor testing for anti-hbc in 1992. although anti-hbc screening was introduced specifically to detect hbv carriers who were hbsag negative (now termed occult hepatitis b), it also served as a surrogate for nanb carriers and for seronegative hiv carriers because of overlapping transmission routes. had anti-hbc surrogate testing been introduced in the early 1980s it presumably would have prevented some cases of transfusion-transmitted aids and nanbh. this delayed implementation was the basis for extensive litigation, but also served as the driver for the iom recommendation of invoking the "precautionary principle" when weighing new donor screening interventions and this precautionary approach has significantly improved transfusion safety. industry has played a major role in hepatitis prevention, first by developing increasingly sensitive assays for hbsag, by developing nucleic acid detection assays for all the major viruses, and particularly by cloning the nanb agent. 38 the latter was a monumental achievement by chiron corporation in collaboration with dan bradley at the cdc. using the then-novel technique of expression cloning, these investigators identified a single clone among millions tested that reacted with serum from patients with nanbh. houghton and associates at chiron then "walked" the genome, characterized an antigen derived from the nonstructural region of the viral genome, and developed an antibody assay to detect this viral protein. 39 studies at the nih 40 confirmed that the cloned agent, designated hepatitis c virus (hcv), was detected in virtually all nanbh cases and identified an implicated donor in near 90% of these cases. first-generation anti-hcv testing was introduced in 1990 and secondgeneration assays in 1992. prospective studies at the nih clinical center documented the virtual eradication of tah by 1997; 35 mathematical modeling after the introduction of nat screening in 1999 predicts that the current risk of transfusion-related hepatitis c is approximately one case in every 2 million transfusions, approximately the same risk as being hit by lightning. west nile virus (wnv) was first identified in the united states in 1999 after an outbreak of encephalitis in newyork. four cases of unexplained fever and encephalitis in recipients of organ transplants from a common donor proved to be caused by wnv and raised the possibility of transmission through blood transfusion. initial efforts to screen blood donors using signs and symptoms of wnv infection proved ineffective. 41 in 2002, a total of 4156 cases of human illness were reported, and at least 21 people contracted wnv through transfusion, six of whom died. 42 the rapid expansion of wnv across the united states and reported to botsec by the cdc lent urgency to developing a screening test before the next epidemic season. 43 the fda requested that industry develop such a test; the national heart, lung and blood institute provided $3.47 million in research support; the american red cross provided 35,000 archived specimens; and the fda facilitated rapid national test implementation and ultimate approval. although development of blood screening tests usually takes years, the nat assay for wnv was available for the 2003 epidemic season, building on technology platforms already developed for hiv and hcv. west nile virus was the first acute infection with a short asymptomatic viremia and an epidemic spread to warrant routine donor testing and demonstrated a successful collaboration of government, blood collectors, and the diagnostics industry. 44, 45 a footnote to the wnv screening success was the recognition that testing of pooled samples was insufficiently sensitive to detect low-titer viremia in blood donations, including in the infectious preseroconversion donations commonly encountered during epidemic spread. however, universal testing of individual units in nonepidemic areas nationwide was inefficient and costly. this problem was solved through a novel strategy of triggering individual testing based on the yield of pool testing. this approach effectively detected and interdicted approximately 1400 potentially infectious blood donations during 2003 to 2005. 46 the emergence of variant creutzfeldt-jakob disease (vcjd) in the united kingdom and france first reported in 1996 posed what has been arguably the most challenging blood safety problem for decision makers since the beginning of the aids epidemic. like aids, vcjd presented a new disease with unknown transmission dynamics, the potential for transmission through blood transfusion, the recognition of a novel infectious agent (prions), and near invariable fatality. 47 vcjd was linked to bovine spongiform encephalopathy, a disease recognized in the united kingdom since 1986, so the incubation period of the disease was assumed to be lengthy. the scope of the epidemic was and remains unknown. 48, 49 for prions, unlike for bacteria and viruses, no technology for developing diagnostic or screening assays was available. the fda established a transmissible spongiform encephalopathies advisory committee to assure focused, objective, and transparent input to its decision making. based on the available epidemiologic data in 1999, the fda recommended that blood components collected from donors diagnosed with vcjd be withdrawn and developed a mathematical model for indefinite donor deferral based on geographic exposure (donors who resided in the united kingdom for a total of 6 months or more, between 1980 and 1996) that eliminated an estimated 87% of donor exposuredays to bovine spongiform encephalopathy in the united kingdom with a projected loss of approximately 2% of donors, which was considered a difficult balance of safety and supply, necessitating close monitoring of the blood supply. 50 based on continuing surveillance of vcjd, the geographic exclusion was expanded in 2002, providing approximately a 90% reduction in total risk-weighted person-days of donor exposure to bovine spongiform encephalopathy in western europe including the united kingdom with an estimated total donor loss of approximately 7%. the question of blood transmission was answered when the united kingdom reported four cases of vcjd infections associated with blood transfusion that occurred between 2003 and 2007. 51 all four recipients had received transfusions of nonleukoreduced rbcs between 1996 and 1999, which confirmed the long incubation. only time will tell whether the steps taken in the united states will prove both warranted and sufficient, but the policy reflects adoption of a "partial solution" when it appears to reduce risk and an attempt to act expeditiously and responsibly with a benefit-to-risk model to address risk in the face of scientific uncertainty. chagas disease, caused by the protozoa trypanosoma cruzi, affects an estimated 8 million people globally; an estimated 300,000 people in the united states and canada are infected. most infections are found in immigrants from latin america. whereas most new infections are vector borne, transmission by blood transfusion is well recognized. six transmissions had been reported in the united states before the ability to screen blood donors. 52 as early as 1989, the fda blood products advisory committee recognized that while only 20% to 30% of those infected with t. cruzi develop symptomatic disease, the infection is lifelong in the absence of early treatment and can be fatal. 53 in view of increasing immigration to the united states from endemic regions, the blood products advisory committee recommended testing donors when a suitable test became available. donor history screening proved insufficiently sensitive and specific. not until 2006 was a test found suitable for licensure. shortly thereafter, the major blood collectors undertook universal donor screening for antibodies to t. cruzi. in retrospect, an earlier study in los angeles and miami suggested that seropositivity did not equate with infectivity; none of 18 recipients of blood from a subsequently identified seropositive donor had evidence of infection. 54 two years of screening in the united states established that whereas the seroprevalence may be as high as 1 in 13,292 donors in some regions, infections confirmed by lookback studies are rare. 55, 56 reexamination by the fda of its decision to recommend universal donor screening led to a novel policy of once-in-a-lifetime donor testing based on the demonstrated rarity of acute or incident t. cruzi infections in us donors. anthrax: bioterrorism, public concern, and the blood supply anthrax is caused by infection with a spore-forming gram-positive bacterium bacillus anthracis found globally in temperate zones, but uncommon in the united states. 57 only seven cases of cutaneous anthrax had been reported to the cdc between 1980 and 2000 when in 2001 an outbreak of bioterrorism-related anthrax resulted in 22 confirmed or suspected cases including five fatalities. 58 this episode raised public concern about the blood supply during a period of high anxiety regarding threats of bioterrorism. bacteremia is present during fulminant cutaneous and respiratory anthrax; however, bacteremia in asymptomatic individuals has not been described. the period between exposure to b. anthracis and development of clinical anthrax is reported as 1 to 7 days but may be as long as 60 days. little information exists regarding transmission via blood transfusion from an asymptomatic individual who has been exposed to b. anthracis. no such cases have been reported and no licensed diagnostic or blood donor screening test exists. the fda received several inquiries regarding the risk to the blood supply from donors in direct contact with material contaminated with b. anthracis. after consulting with experts at the cdc, the nih, and the us army medical research institute for infectious diseases, the fda issued guidance regarding measures to reduce possible risk for transmission of anthrax from blood. 59 the guidance did not recommend any changes to standard donor screening and blood collection procedures, but emphasized that standard blood collection procedures already in place include deferral of any donor who is not in good health at the time of donation. nevertheless, to address public concerns as well as the dearth of scientific information regarding blood transmission, the fda provided prudent but specific recommendations concerning donors with a confirmed medical diagnosis of anthrax or proven colonization with b. anthracis and provided criteria for product quarantine and retrieval related to reports of postdonation illness. 59 in 2009, the journal science reported that a gamma retrovirus, xenotropic murine leukemia virus-related virus (xmrv) was isolated from blood in two-thirds of patients diagnosed with chronic fatigue syndrome (cfs) and, most alarmingly, in 3.7% of healthy subjects. 60 a second article reported a related retrovirus (pmlv) with an even higher prevalence of 6.8% among blood donors. 61 these reports generated enormous public interest and concern. given the possibility that xmrv could be transmitted by transfusion, immediate calls arose to screen blood donors for signs and symptoms of cfs and to test donations for xmrv. at the same time, intensive efforts were being undertaken worldwide to resolve this potential safety concern. a federal interagency working group met repeatedly by teleconference and electronic communication, and laboratories within the cdc, nih, and fda invested resources into investigating discrepant laboratory results. 62 additionally, representatives of the cdc, the fda, and the intra-and extramural programs at the nih participated in a public-private interorganizational task force assembled within 60 days by the aabb (formerly american association of blood banks). 63 the result was voluntary implementation of an interim aabb recommendation that blood collectors should "actively discourage potential donors who have been diagnosed by a physician with cfs, chronic fatigue and immune dysfunction syndrome, or myalgic encephalomyelitis from donating blood" and ultimately definitive laboratory evidence that xmrv or pmlv bore no association with cfs and posed no threat to the blood supply. 64, 65 ongoing threats and challenges several infectious threats are currently challenging federal decision makers. bacterial contamination of platelets is a clearly identified risk that is being addressed with "partial solutions," culture, and point-of-issue serologic testing. 66 hepatitis e virus is known to be transfusion transmitted, but potential disease burden has not been defined. 67 the geographic and travel exclusions to limit the risk of malaria transmission continue to be refined pending development of screening assays or pathogen reduction technology. surveillance for the coronaviruses responsible for severe acute respiratory syndrome and middle east respiratory syndrome is active and the possibility that these agents as well as pandemic influenza and monkey pox might be transfusion transmitted or disrupt blood donation is unresolved. the possibility of seasonal and geographic-based donor screening with validated tests for dengue and babesiosis has been modeled even as pilot studies of screening assays are ongoing. 68, 69 pathogen reduction technology offers an alternative approach to risk mitigation. such technology would change the riskbenefit paradigm both for the known infectious agents and for those likely to threaten the blood supply in the future. 70 federal decision makers are involved in deter-mining when and how this technology should be applied to the nation's blood and blood components. the federal response to transfusion-transmitted infections has evolved dramatically since the emergence of hiv as a transfusion-transmitted infection. the philosophy of risk management has become more precautionary and patient focused, yet still data driven. regulation of notfor-profit blood collectors has become more formal and stringent. manufacturers of blood components are now held accountable for meeting cgmp standards similar to those that apply to the manufacture of medical devices and pharmaceutical-type drugs. a new and arguably more responsive federal structure for addressing issues of blood safety and availability has been adopted. the decisionmaking structure places a premium on clear lines of authority, internal and public communication, flexibility, and coordination among the federal agencies with major roles in blood safety. federal agencies have encouraged public discourse through workshops, joint initiatives with industry, and participation in public-private partnerships with professional societies and blood collectors. these adjustments have allowed federal agencies to respond with appropriate urgency to the differing situations posed by emerging infectious agents in the era since recognition of taa 30 years ago. 71 hiv's leading men 25 years after hiv discovery: prospects for cure and vaccine epidemiologic notes and reports pneumocystis carinii pneumonia among persons with hemophilia a possible transfusion-associated acquired immune deficiency syndrome (aids)-california the hazards of blood transfusion in historical perspective transfusion-associated infections: 50 years of relentless challenges and remarkable progress syphilis: a disease of direct transfusion hiv and the blood supply: an analysis of crisis decision making. washington dc: institute of medicine national academy press staff costs associated with the implementation of a comprehensive compliance program in a community blood center risk-based decision-making for blood safety: preliminary report of consensus conference us department of health and human services. improving blood safety and supply in the u.s the efficiency of hiv p24 antigen screening of us blood donors: projections versus reality risk of human immunodeficiency virus infection from blood donors who later developed the acquired immunodeficiency syndrome risk of human immunodeficiency virus (hiv) transmission by blood transfusions before the implementation of hiv-1 antibody screening guidelines for counseling persons with human t-lymphotropic virus type i (htlv-i) and type ii (htlv-ii) jaundice occurring one to four months after transfusion of blood or plasma: report of seven cases a serologic follow-up of the 1942 epidemic of post-vaccination hepatitis in the united states army a "new" antigen in leukemia sera australia antigen and acute viral hepatitis immunologic distinction between infectious and serum hepatitis virus-like particles in serum of patients with australia-antigen-associated hepatitis infectious hepatitis: evidence for two distinctive clinical, epidemiological, and immunological types of infection posttransfusion hepatitis after open-heart operations serum hepatitis from transfusions of blood posttransfusion hepatitis after exclusion of the commercial and hepatitis b antigen positive donor hepatitis a: detection by immune electron microscopy of a virus-like antigen associated with acute illness transfusion-associated hepatitis not due to viral hepatitis type a or b posttransfusion non-a, non-b hepatitis: physiochemical properties of two distinct agents inactivation of hepatitis b virus and non-a, non-b virus by chloroform determining the size of non-a, non-b hepatitis virus by filtration the chronic sequelae of non-a, non-b hepatitis serum alanine aminotransferase of donors in relation to the risk of non-a, non-b hepatitis in recipients: the transfusion-transmitted virus study the relationship of donor transaminase (alt) to recipient hepatitis: impact on blood transfusion services hepatitis c virus and eliminating post-transfusion hepatitis hepatitis b virus antibody in blood donors and the occurrence of non-a, non-b hepatitis in transfusion recipients: an analysis of the transfusion-transmitted virus study antibody to hepatitis b core antigen as a paradoxical marker for non-a, non-b hepatitis agents in donated blood isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome an assay for circulating antibodies to a major etiologic virus of non-a, non-b hepatitis detection of antibody to hepatitis c virus in prospectively followed transfusion recipients with acute and chronic non-a, non-b hepatitis and update on west nile virus infections in recipients of blood transfusions west nile virus transmission investigation team. transmission of west nile virus through blood transfusion in the united states in 2002 as west nile virus season heats up, blood safety testing lags behind west nile virus among blood donors in the united states screening the blood supply for west nile virus rna by nucleic acid amplification testing triggers for switching from minipool testing by nucleic acid technology to individual-donation nucleic acid testing for west nile virus: analysis of 2003 data to inform 2004 decision making transmissible spongiform encephalopathies estimation of epidemic size and incubation time based on age characteristics of vcjd in the united kingdom uncertainty due to model choice in variant creutzfeldt-jakob disease projections guidance for industry: revised preventive measures to reduce the possible risk of transmission of creutzfeldt-jakob disease (cjd) and variant creutzfeldt-jakob disease (vcjd) by blood and blood products transfusion transmission of human prion diseases transfusion-associated chagas disease (american trypanosomiasis) in mexico: implications for transfusion medicine in the united states guidance for industry: use of serological tests to reduce the risk of transmission of trypanosoma cruzi infection in whole blood and blood components intended for transfusion trypanosoma cruzi in los angeles and miami blood donors: impact of evolving donor demographics on seroprevalence and implications for transfusion transmission epidemiological and laboratory findings from 3 years of testing united states blood donors for trypanosoma cruzi the united states trypanosoma cruzi infection study: evidence for vector-borne transmission of the parasite that causes chagas disease among united states blood donors anthrax as a biological weapon: medical and public health management. working group on civilian biodefense summary of notifiable diseases-united states detection of an infectious retrovirus, xmrv, in blood cells of patients with chronic fatigue syndrome detection of mlv-related gag gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the united states xenotropic murine leukemia virus-related virus (xmrv) and blood transfusion: report of the aabb interorganizational xmrv task force failure to confirm xmrv/mlvs in the blood of patients with chronic fatigue syndrome: a multi-laboratory study a multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus aabb bacterial contamination task force. survey of methods used to detect bacterial contamination of platelet products in the united states in 2011 seroprevalence and incidence of hepatitis e virus infection in german blood donors dengue viremia in blood donors identified by rna and detection of dengue transfusion transmission during the 2007 dengue outbreak in puerto rico babesia microti real-time polymerase chain reaction testing of connecticut blood donors: potential implications for screening algorithms protecting the blood supply from emerging pathogens: the role of pathogen inactivation emerging infectious agents and the nation's blood supply: responding to potential threats in the 21st century none. key: cord-294241-11abmmyl authors: jersild, c.; hafner, v. title: blood transfusion services date: 2008-08-26 journal: international encyclopedia of public health doi: 10.1016/b978-012373960-5.00520-7 sha: doc_id: 294241 cord_uid: 11abmmyl blood transfusion services are an important part of the health-care system since blood transfusion is required in a number of frequently occurring clinical situations: major surgical procedures, including treatment of trauma patients; obstetric care with major bleeding during child birth; and treatment of several medical diseases, especially hematological diseases. this article outlines the essential safety aspects of blood transfusion from the proper selection of the unpaid blood donor to processing and appropriate testing of the blood unit to safety aspects of transfusion, which include the appropriate clinical use of blood and blood components. the importance of accurate reporting of any adverse reactions to blood transfusion, which form the basis for a hemovigilance system, is reviewed. 'blood' is a fluid tissue, composed from various cells with complex functions (erythrocytes, red cells -oxygen carriers; leucocytes and lymphocytes, white cells -immune response to disease; and thrombocytes -clotting factor), which are carried by plasma. blood is vital as carrying oxygen, nutrients, and other essential elements to the tissues and removing residues of cellular metabolism. it also carries various clotting factors which normally intervene when bleeding occurs. blood donation is a benevolent gesture of offering blood for patients in need. every healthy individual 18 to 65 years of age can donate blood, about 7 ml per kg body weight, up to a maximum of 450 ml whole blood per donation. the interval between two whole blood donations has to be at least 72 days. voluntary nonremunerated blood donation is considered a cornerstone of blood safety and it is advocated and supported by many international bodies, such as world health organization, the international federation of red cross and red crescent societies, and the international federation of blood donor organizations (see relevant websites). blood transfusion is a medical treatment in which blood, blood components, or blood products are collected and prepared from a healthy person (a donor) and administered to the patient being treated. the most appropriate blood transfusion therapy should provide for the missing or reduced element. transfusion of whole blood can be life-saving in situations such as massive blood loss due to trauma or extensive surgery. blood component therapy is used to treat conditions in which the specific element is missing; for example, severe anemia (a reduction in oxygen supply to vital organs) or an abnormal decrease in the number of platelets. people suffering from sickle-cell disease may require frequent transfusion of red cell concentrates. in the case of hemophilia, the administration of the missing clotting factor is required. blood transfusion treatment is usually performed in health-care settings. blood transfusion service is an organization which deals with various aspects of the blood transfusion chain, from the potential donor (information and selection of donors, blood collection, blood testing, blood processing, blood storage, blood transportation) to the potential recipient (selection and distribution of appropriate components for transfusion), and should link to the clinical interface or administration of blood and patient follow-up. this process is usually carried out by organizations or health departments operating at a local, regional, or national level. in some countries blood services are hospital based. the range of responsibilities of the hospital blood banks varies, therefore, according to the organization of the national health-care system. usually, the hospital transfusion department bears the responsibilities of storage, selection (compatibility testing), and distribution of blood components according to medical prescription. a blood component results from separation of collected whole blood through centrifugation at various speeds. components include red cells, white cells, thrombocytes, and plasma. each component has specific characteristics, including a special storage temperature and shelf life (e.g., thrombocytes must be stored at þ21 c; plasma must be frozen at -40 c). blood components can also be collected from the individual donor through apheresis procedures (separation of the required component from collected blood during the donation procedure, with the remaining being returned to the donor at the end of the separation process). the procedure can be fully or partially automated. in separating blood derivatives or components, raw plasma is subject to industrial fractionation procedures. these procedures must follow the pharmaceutical current good manufacturing practices (cgmp) so that resulting products comply with required safety standards. the purified and concentrated coagulation factors (e.g., factor viii, factor ix) and human albumin are usually prepared as 5% or 20% solutions, and immune globulins are the most frequently used blood derivatives. technological progress has been achieved in obtaining recombinant coagulation factors, as well as volume replacement solutions and plasma expanders, in the continuous search to identify responses to an insufficient blood supply which is increasingly exposed to safety threats. being a complex but undeniable life source, blood has been given historically mythical attributes. after unsuccessful transfusions of blood from animals to humans, and sometimes from humans to humans as well, it was only after 1900, when karl landsteiner discovered the abo major blood groups, that blood transfusion therapy became possible. the history of blood transfusion has shown that once the blood group compatibility barrier had been overcome, there are infectious threats to which increased attention must be given. blood-borne pathogens can be transferred with the donor's blood to the recipient patient. such pathogens may be viruses (e.g., hiv/ aids, hepatitis b and c), parasites (e.g., malaria, chagas' disease), bacteria (e.g., syphilis, brucellosis), or prions (e.g., vcjd). in the late 1980s the transmission of the hiv virus by blood, blood components, and by pharmaceutically manufactured blood derivatives raised important concerns. a large number of patients chronically depending on therapy with blood components or derivatives were proven to be infected with hiv and other blood-borne pathogens. these findings led to more firm regulations of the entire blood transfusion area, with patient protection being of central importance. intensive work in the field of quality assurance of blood-banking operations, including viral inactivation of blood derivatives, was initiated. for a more comprehensive overview of the history of blood transfusion, see starr (1998) . other crisis situations, such as the variant creutzfeldt-jakob disease (vcjd) infections (mad cow disease), severe acute respiratory syndrome (sars), or avian flu epidemics, have strongly affected the availability of the blood supply at times. a more complete list of infectious agents which can be transmitted by blood transfusion is presented in table 1 . it is important to note that these agents in their early stage of infection induce a healthy carrier state with few or no symptoms of disease. the safety and availability of and access to adequate blood supplies remain challenges in many parts of the world. according to the who global database for blood safety, 81 million units of whole blood and 20 million liters of plasma were donated annually in the period 2001-2002. analysis of blood donations in relation to the human development index (hdi -united nations indicators) showed that 82% of the world's population, which lives in low and medium hdi countries, has access to only 39% of the total blood supply. this limited access has a dramatic impact on the capacity to respond to various health needs of the population, and the strong links between blood availability and maternal mortality rates have been demonstrated. the differences in blood donation rates reflect not only the degree of awareness of the population with respect to blood transfusion, but also the level of development of health care in which the blood service is embedded. with mounting safety requirements and technological progress, blood transfusion services have become an increasingly expensive part of public health. in countries with restricted resources, the safety of blood transfusion is of particular concern, especially because these countries often have high prevalence rates of hiv, hepatitis b and c, and other particular blood-borne diseases. (see relevant websites for the who global database on blood safety.) an appropriate blood supply -defined as a situation in which no patient will die due to lack of appropriate treatment with blood or blood components -varies from one country to another. important components of blood safety are: . organization and regulatory framework for the blood transfusion services; . safe blood donors -education, recruitment, retention; . testing of all blood units and further processing; table 1 transfusion-transmitted infectious diseases remarks with respect to geographical distribution or generated disease . appropriate use of blood and blood components at the clinical site; . hemovigilance, reporting and follow-up on safety issues and transfusion outcomes. who-integrated strategy for blood safety recommends nationally coordinated blood services with quality systems functional in all areas and a blood supply based on voluntary nonremunerated blood donors from low-risk populations that is appropriately tested (100% for blood-borne pathogens, blood grouping, and compatibility testing), processed, stored, and distributed, as well as adequately used at the hospital site (alternatives to transfusion should be available), including posttransfusion follow-up. the blood transfusion service should be integrated with the health-care system of a country. its appropriateness depends on the strength of public health interventions, the level of health service delivery, and on the health status of the population and existing patterns of disease. the blood transfusion service usually functions under government responsibility. a national blood policy and plan should define the vision and the steps required for strengthening quality, safety, availability, and access to adequate blood supplies. a national blood program for defined periods of time will allow addressing priorities and monitoring progress in the field. constantly updated, dedicated, legal provisions and regulatory frameworks are part of the supportive mechanisms to implementation. appropriate and sustainable resources, including funding mechanisms, are vital. involvement of the various stakeholders in the process is essential, and a dedicated national commission can play an advisory function at a high decision-making level with respect to blood safety issues, as well as enhance dialogue among interested parties, such as blood transfusion specialists, clinicians, and patients. experience has shown that the organization of blood services on a nationally coordinated basis ensures harmonized quality standards along the blood chain and increases consistency in delivery of products, information, and statistics. in addition, such organization has been proven to be more cost effective owing to economies of scale. following the early period of the hiv/aids epidemic, the blood transfusion community began to realize that production of blood should take place in a more controlled environment. in order to accomplish this, two specific initiatives were taken: (1) quality standards for blood components were established, as well as for the raw plasma used in pharmaceutical processing, and (2) quality management systems were implemented to ensure that products meet the established standards. this has generated enhanced work of national and international organizations to support both the development and implementation of quality and safety standards for the blood services and deliverables (e.g., council of europe ''guide to the preparation, use and quality assurance of blood components,'' aabb standard for blood banks). these standards are all grounded in more general standards, such as the iso 9000series standards and the current good manufacturing practice (cgmp) guidelines for medicinal products for human and veterinary use (e.g., eu published by the european commission). these specify basic requirements and principles for quality management, including personnel management and training, premises and equipment layout, production operations and requirements for standard operating procedures, design and methods for ensuring maintenance and documentation for all activities, how to perform quality controls, contract manufacture and analysis, and how to perform and document self-inspection. monitoring the compliance to defined standards is part of a regular process of quality assessment and control. in line with the above, who developed a comprehensive training program (including interactive toolkit and external quality assessment schemes) in quality management principles for the blood service. according to data available from various sources and studies, voluntary nonremunerated regular blood donors recruited from low-risk populations carry a much lower risk of infections. the safe blood donors are the cornerstone of a safe blood supply and therefore an important and central point in who recommendations as well as those emerging from the ifrcrcs, the isbt, and other international organizations concerned. establishing a pool of safe blood donors requires longterm commitments to education, information, and social involvement. efforts in this respect have been particularly enhanced with the world blood donor day, which became a global event after its endorsement by the world health assembly in 2005. co-sponsored by who, the international federation of red cross and red crescent societies (ifrcrcs), international federation of blood donor organizations (ifbdo/fiods) and international society of blood transfusion (isbt), it aims to increase awareness toward blood donation and enhance the different steps in the process of education, recruitment, and retention of low-risk donors and establishment of youth donor programs. safe donor selection criteria -the education to healthy lifestyles -proved to be highly effective in the prevention of sexually transmitted infections, including hiv/aids. the 'club 25' initiative in africa (youth initiative promoting lifestyles and regular blood donation, aiming for about 20 blood donations by the age of 25) led to a dramatic decrease in transfusion-transmitted infections and related risks, to the mutual benefit of donors and recipient patients. collected blood needs to be screened for blood-borne pathogens prior to transfusion. basic screening includes tests for hiv, hepatitis b, hepatitis c, and syphilis. in some parts of the world, additional tests are necessary for local epidemiological threats. it is the responsibility of health authorities to outline a national strategy for screening of all donated blood and specify the most appropriate testing and diagnostic algorithms to be used. in addition, testing for abo and rhd blood groups, as well as screening for irregular antibodies, are performed to avoid an incompatible (hemolytic) transfusion reaction. reliable testing of blood units requires the following: 1. uninterrupted supply of high-quality test systems; this includes procurement, supply, central storage, and distribution of reagents and materials to ensure continuity of testing; 2.. maintenance of a quality assurance system and good laboratory practice, including the use of standard operating procedures (sops) for all aspects of blood screening and processing; 3. continuous training of staff members in all aspects of blood screening and processing of blood units, including storage and transportation of blood products. collected whole blood can be stored for a time depending on the mixture of anticoagulant preservation solution used. the whole blood unit may be separated into major blood components -red cell concentrates, fresh frozen plasma, and platelet concentrate -to increase efficiency of use. processing of donated blood into its different components reduces the occurrence of adverse transfusion reactions and tailors therapeutic response to the particular needs of the patient. since each blood component can be stored according to its specific requirements, effectiveness is increased and shelf-life adjusted. for example, fresh frozen plasma can be stored at -40 c for 24 months, platelet suspended in plasma or suspension media (t-sol) can be stored at þ20-24 c for 3-5 days, and red cells suspended in an additive solution (sag-man or adsol) can be used up to 45 days if stored at þ4 c. blood is a scarce resource and should always be used with care. as with any medical procedure, it carries also a measurable risk to the recipient patient. in evaluating the indication for blood transfusion therapy, the benefits for the patient should outweigh the risks. several international recommendations and guidelines have been developed in this respect, but these are not always available and in many circumstances the process continues to rest on historically based practices and the clinical experience of the attending physicians. the training of clinical users in the adequate use of blood is an important endeavor which requires constant updates not only regarding scientific progress and technologies but also the availability of and access to alternatives to blood (e.g., volume replacement solutions). strengthening the cooperation with other sectors of health care (i.e., primary health care) may help reduce blood usage through (1) early diagnosis and treatment of diseases or conditions which may lead to the need for blood transfusion (obstetric antenatal care, iron substitution); (2) pre-operative blood collection for auto-transfusion; (3) intra-operative normo-volemic hemodilution; (4) per-operative and post-operative blood salvage. blood usage in western europe and north america shows a moderate decrease in number of units used, mostly due to modern surgical techniques, such as endoscopic interventions with minimal tissue injury. to further respond to blood availability challenges, increasing attention is given to the development of blood substitutes and oxygen carriers. the network for advancement of transfusion alternatives also aims to reduce unnecessary blood transfusion and provides additional information. the word 'hemovigilance' is derived from the word 'pharmacovigilance,' which encompasses activities and systems to collect information useful in supervising medicinal products, with particular reference to adverse drug reactions in human beings, and to evaluate such information scientifically. it comprises a set of surveillance procedures covering the whole transfusion chain (from the collection of blood and its components to the follow-up of the transfused patient). the aim of hemovigilance is to detect, collect, and analyze all information of unexpected or undesirable effects resulting from the therapeutic use of blood and blood components in order to correct their cause, prevent recurrence, and improve the safety of blood transfusion. adverse reactions are defined as reactions which are harmful and unintended and which occur at doses normally used for prophylaxis, diagnosis, or treatment of various conditions. hemovigilance concerns blood components (e.g., whole blood, erythrocyte concentrates, platelet concentrates, and fresh frozen plasma). pharmacovigilance in transfusion medicine concerns plasma derivatives (e.g., clotting factor concentrates, immunoglobulins, albumin, and other fractionated products). reporting of serious adverse events related to transfusion therapy appears to be one of the oldest reporting systems in place. however, the development of national hemovigilance systems poses increasing challenges due to the various taxonomies in use, the limited resources for supportive informative technologies, and, mainly, the need for enhanced communication between blood services and hospital services. the increasing role of the patient in this process is expected to strengthen hemovigilance at its operational level. several websites provide data on hemovigilance (national and regional), such as those from the united kingdom, the european haemovigilance network, and the danish haemovigilance network. botulism is a rare, severe, neuroparalytic disease caused by accidental or intentional exposure to seven distinct botulinum toxins (bonts, a-g types) that affect humans and a variety of domestic and wild lower animals. the disease is characterized by symmetrical cranial nerve palsies that may be followed by descending, symmetric flaccid paralysis of voluntary muscles, which may lead to death because of respiratory or heart failure. botulinum toxins are neurotoxins of extreme potency and lethality (they can be lethal at doses as low as 1 mg/kg orally) released by vegetative cell death and lysis. four toxigenic anaerobic gram-positive spore-forming bacteria of the genus clostridium produce the botulinum toxins: the classic c. botulinum that produces type a, b, c, d, e, and f toxins (bont/a-f), c. argentinense that produces type g toxin (bont/g), and two rare strains of c. butyricum and c. baratii that produce types e and f botulinum-like toxins, respectively. blood: an epic history of medicine and commerce further reading brecher me guide to the preparation, use and quality assurance of blood components, 13th edn blood banking and transfusion medicine. basic principles and practice mollison's blood transfusion in clinical medicine, 11th edn global perspectives in transfusion medicine gift or good? paying for blood donations: still a risk safety (2001) the clinical use of blood: handbook -international federation of blood donor organizations -international federation of red cross and red crescent societies advancement of transfusion alternatives world health organization: blood transfusion safety centro nazionale per la qualita' e i rischi alimentari key: cord-026021-b8vtmr9h authors: hohenhaus, ann e. title: blood transfusion and blood substitutes date: 2011-06-22 journal: fluid, electrolyte, and acid-base disorders in small animal practice doi: 10.1016/b978-1-4377-0654-3.00031-7 sha: doc_id: 26021 cord_uid: b8vtmr9h nan ann e. hohenhaus blood transfusions have many things in common with fluid therapy. like crystalloid and colloid solutions, blood products are not used to treat disease; they are supportive therapies given to correct deficiencies in the patient until the underlying disease process can be treated. for example, a red blood cell transfusion is given to replace red blood cells lost as a result of a traumatic laceration. the transfusion of red blood cells increases the oxygen-carrying capacity of the blood, allowing for surgical repair of the laceration; it is not the primary treatment for hemorrhage. likewise, sodium chloride is used to replace sodium, chloride, and water in a dehydrated patient with hypoadrenocorticism until adrenal hormones can be replaced. the use of both blood transfusions and fluid therapy must be carefully assessed before inclusion in a patient's treatment plan, and the veterinarian should evaluate the risk/benefit ratio for each patient. volume overload, electrolyte disturbances, and transmission of infection can occur from administration of pathogen-contaminated blood products or fluids. 31, 66, 130 despite the potential negative effects of transfusion, most veterinarians view it as lifesaving therapy, allowing the transfusion recipient to receive other necessary treatments such as surgery, chemotherapy, or medical care. 56 three major differences exist between the more commonly used fluids and blood products. the differences between crystalloid or colloid solutions and blood products are their immunogenicity, availability, and cost. the immunogenicity of blood products stems from the proteins and cellular material in the blood. because crystalloid solutions lack proteins and cellular material, they are not considered immunogenic; however, certain colloid solutions such as hydroxyethyl starch have been reported to cause acute anaphylaxis in rare instances in humans. 96 the mechanism of this reaction is unknown. limited availability differentiates blood products from crystalloid and colloid solutions. crystalloid and colloid solutions are readily available because they can be manufactured according to market demand. only a living animal can produce blood, and the donor's physiologic capability limits production. the small number of commercial canine and feline blood banks providing a convenient source of blood for the veterinary practitioner further limits availability of blood for transfusion (box 24-1). furthermore, blood products require a more regulated storage environment and have a significantly shorter shelf life than crystalloid or colloid solutions, making blood a less convenient product to store and use in a veterinary hospital. nearly 20 years ago, veterinarians estimated costs associated with transfusions, but an exact analysis of cost is lacking. in 1992, the estimated cost of a 500-ml whole blood transfusion ranged from $25 to more than $300. 56 the cost of 500 ml of lactated ringer's solution is about $1. despite the fact that the first documented transfusion was given to a dog in 1665 by richard lower at oxford university, veterinary transfusion medicine scientifically and technologically lags behind its counterpart in human medicine. 76 information in this chapter is based on veterinary studies whenever possible. when none is available, currently accepted guidelines from human medicine will be applied to the veterinary patient. the purpose of this chapter is to provide the reader with the following: 1. a basic understanding of the theory of blood component therapy 2. information on the technical aspects of obtaining blood for transfusion 3. suggestions for the administration and monitoring of transfusions 4. a description of the clinical applications of a veterinary blood substitute blood is the body's largest connective tissue. when collected from the donor, it contains all the elements of blood: red blood cells, white blood cells, platelets, coagulation factors, immunoglobulins, and albumin. whole blood can be transfused into the recipient as it is collected from the donor, but it is neither a specific therapy nor an economical use of blood. the optimal method of preservation of blood for transfusion is to separate whole blood into its component parts. appropriate use of blood components not only conserves the products but also allows the most specific and safe product to be administered for each patient. when blood components are used instead of whole blood for transfusion, two dogs can benefit from 1 unit of whole blood. a plasma transfusion counteracts the anticoagulant effects of rodenticide intoxication in one dog, and red blood cells from the same donor provides enhanced oxygen-carrying capacity in a second, anemic dog. component transfusions also have been used in cats, but preparation of components is more difficult because of the small volume of blood collected from donor cats. 23, 53, 68, 99 production of components is not feasible for most veterinary practices. most will purchase their blood inventory because they lack the time and equipment to recruit donors, and collect and process whole blood into components. blood components predominate in the inventory of commercial blood banks, requiring veterinarians to become familiar with their usage. the technical aspects of component production are not included in this chapter but can be found elsewhere. 85, 99 a brief summary follows. preparation of blood components from whole blood requires that the blood from the donor be collected into the anticoagulant-containing bag of a multibag plastic blood collection system. the whole blood then is separated into packed red blood cells (prbcs) and plasma by differential centrifugation in a refrigerated blood bank centrifuge, and the plasma is transferred into one or more of the attached satellite bags via the sterile tubing linking the bags. the bags are separated, and prbcs are stored in a refrigerator and plasma is stored in a freezer. blood collected into glass bottles is not amenable to centrifugation and cannot be processed into components. additionally, storage of canine blood in a glass bottle results in lower levels of 2,3-diphosphoglycerate and adenosine triphosphate (atp) than blood stored in plastic bags; consequently, plastic bags are the preferred storage container for blood. 28 the most commonly used blood products, their indications, and suggested dosages are described later. the dosage of a blood product depends on the physical state of the patient and the response of the patient to the treatment: in essence, the treatment is "to effect." whole blood is the blood collected from the donor plus the anticoagulant. in veterinary medicine, no standards have been established for the volume of blood that constitutes 1 unit. when a human blood collection system is used for dogs, 450 ae 45 ml of blood is collected and combined with 63 ml of anticoagulant, and often is designated as 1 unit. whole blood contains red blood cells, clotting factors, proteins, and platelets and is the product most commonly transfused into dogs and cats. 56 once whole blood is refrigerated, the white blood cells and platelets become nonfunctional. as a starting point, the dosage for whole blood is 10 to 22 ml/kg. prbcs are the cells and the small amount of plasma and anticoagulant that remains after the plasma is removed from 1 unit of whole blood. if 450 ml of blood are collected, the volume of prbcs obtained is approximately 200 ml. because the plasma has been removed, the total volume transfused is less than 1 unit of whole blood but contains the same oxygen-carrying capacity as 450 ml of whole blood. in cats, the increase in packed cell volume (pcv) after transfusion of 1 unit of prbcs has been shown to be equivalent to the increase after transfusion of 1 unit of whole blood. 68 prbcs are used only to treat clinically symptomatic anemia because they do not contain platelets or clotting factors. red blood cell transfusions are administered to cats for a variety of reasons. data on 126 cats administered whole blood or prbcs indicated 52% were transfused for blood loss anemia, 38% for erythropoietic failure, and 10% for hemolytic anemia. 68 similar reasons for transfusion of cats have been reported elsewhere. 22, 126 dogs more commonly are transfused for blood loss anemia (70%) with 14% to 22% being transfused for hemolytic anemia and 8% to 14% for erythropoietic failure. 17, 64 the initial dosage of prbcs is 6 to 10 ml/kg, and transfusion is continued until the clinical signs of anemia resolve. fresh frozen plasma is the plasma obtained from whole blood plus the anticoagulant solution, which is frozen within 8 hours of collection. when whole blood is centrifuged to produce plasma and prbcs, the anticoagulant segregates with the plasma fraction. fresh frozen plasma contains all clotting factors, which, if frozen at à30 c in a blood bank freezer, maintains activity for 12 months. 121 fresh frozen plasma maintained in an upright freezer at à20 c maintains clotting factor activity for 6 months. when frozen, the plastic storage bag becomes brittle and if not carefully handled can crack, rendering the plasma unusable. for this reason, plasma is stored in special boxes to protect the plastic bag and must be handled carefully before transfusion. if plasma is thawed, and not transfused, it can be refrozen within 1 hour of thawing without loss of coagulation factor activity. 131 fresh frozen plasma has been used to treat a wide variety of clinical patients. a retrospective analysis of fresh frozen plasma usage in dogs identified multiple indications for administration of fresh frozen plasma, including replacement of coagulation factors, albumin, a 2 -macroglobulin, and immunoglobulin despite the recommendation that fresh frozen plasma should not be used as a source of albumin, for volume expansion, or nutritional support. 75, 87 calculations suggest 45 ml/kg of plasma would need to be given to increase albumin serum concentration by 1 g/dl. 120 in cases of coagulation factor deficiencies, plasma should be given to effect (i.e., until active bleeding ceases). 70 for the treatment of coagulation disorders, 6 to 10 ml/kg is the recommended starting dosage. multiple doses may be required to control bleeding because of the short half-life of clotting factors, especially in patients with disseminated intravascular coagulation. normalization of previously abnormal coagulation tests can be used as a guide for discontinuation of plasma therapy. cryoprecipitate is prepared by thawing fresh frozen plasma at 0 c to 6 c. a white precipitate forms, the liquid plasma is removed after centrifugation, and both aliquots are refrozen. the cryoprecipitate is a concentrated source of von willebrand's factor, fibrinogen, and factors xiii and viii (antihemophilia factor). it is useful in the treatment of deficiencies of these clotting factors and is handled in the same manner as fresh frozen plasma. two studies have shown cryoprecipitate to be the blood product of choice for the treatment of von willebrand's disease because it concentrates the larger, more hemostatically active von willebrand's multimers into a smaller volume than fresh frozen plasma. 23,108 a preliminary study suggests cryoprecipitate corrects the hypocoagulable state associated with disseminated intravascular coagulation when administered at a dose of 5 to 7 ml/kg. 117 cryoprecipitate is equivalent to fresh frozen plasma for the treatment of hemophilia a. the dose is 1 unit per 10 kg body weight. 84 cryo-poor plasma is the supernatant plasma removed from the cryoprecipitate. cryo-poor plasma contains factors ii, vii, ix, and x, which make it useful for the treatment of rodenticide intoxication. storage and handling of cryo-poor plasma is similar to fresh frozen plasma. the initial dose is 1 unit per 10 kg of body weight. platelet-rich plasma is prepared from fresh whole blood by centrifugation at a slower rate than is used for production of prbcs and plasma. 85 the platelets are suspended in a small amount of plasma to facilitate transfusion. storage of fresh platelets is impractical outside a blood bank because of their requirement for storage at 20 c to 24 c in special plastic bags and continuous agitation. 2 transfused platelets are rapidly destroyed in human patients with immune-mediated thrombocytopenia, and because immune-mediated thrombocytopenia is a common cause of profound thrombocytopenia in dogs, most cases of thrombocytopenia-mediated hemorrhage may not be amenable to successful platelet transfusion. if a platelet transfusion is given, the dose is the platelets collected from 1 unit of whole blood per 10 kg of body weight. cryopreserved canine platelets are collected from a single donor via plateletpheresis, and the manufacturer reports one bag contains 1 â 10 11 platelets preserved in dimethyl sulfoxide (dmso). 57 the bag also contains a small amount of fresh frozen plasma. efficacy data on this product have not been published, but the manufacturer recommends this product be used for the treatment of immune-mediated thrombocytopenia. the dose is 1 unit of frozen platelets per 10 kg of body weight. according to the manufacturer, anticipated increase in platelet count is 20,000/ ml 1 to 2 hours posttransfusion. because the product contains dmso, it must be infused slowly to prevent bradycardia. cryopreserved canine platelet concentrate was compared with fresh platelet rich plasma in the laboratory. 48 this study identified decreases in platelet number and function as a result of the freeze-and-thaw process. platelet number decreased 59% compared with the manufacturer's reported platelet count and platelets demonstrated multiple features of activation. the impact of cryopreservation on platelet function and number in vitro has not been studied. the use of serum has been recommended for the treatment of kittens and puppies with failure of passive transfer. kittens treated with 5 ml subcutaneously or intraperitoneally three times in 24 hours achieved immunoglobulin g (igg) concentrations comparable to kittens receiving colostrum. 72 treatment of puppies with 22 ml/kg of serum given orally or subcutaneously at birth did not result in equivalent igg and iga concentrations when serum-treated puppies were compared with nursing puppies. 90 igm was higher in the puppies treated with subcutaneously administered serum. human albumin is a concentrate of albumin derived from pooled human plasma. homology between canine and human albumin is approximately 79%, and human albumin is antigenic in dogs. 30, 80 previous human albumin transfusion does not appear to be required for production of antibodies in dogs. 80 hypoalbuminemia predicts a negative outcome in several canine diseases; consequently, the ability to correct hypoalbuminemia by albumin transfusion would be a medically desirable intervention. 1, 20 because canine albumin was not previously available, human albumin has been used in dogs. two retrospective studies have evaluated transfusion of human albumin to critically ill dogs. 81, 115 one associated improved albumin levels and blood pressure with human albumin administration and did not report serious adverse events. 81 the second concluded the serious nature of the diseases treated with precluded recognition of complications of the transfusion. 115 a recent study performed in normal dogs has identified serious adverse events suggestive of anaphylactic and fatal type iii hypersensitivity reactions. 25 transfusion of dogs with human albumin should be undertaken with great caution especially because lyophilized canine albumin is available. 58 human intravenous immunoglobulin (hivig) is a highly purified preparation of immunoglobulin g, obtained from large pools of donated human plasma. the manufacturer provides the product as a lyophilized powder, which is reconstituted before transfusion. sporadic availability of hivig limits its use, as does its high cost. estimates indicate the cost of the drugs alone may be as high as $3000 to treat a 20 kg dog. 129 reconstituted hivig is infused over 6 hours. most report a single administration of the drug at a dosage of 0.5 to 1.0 g/kg, but in some cases the dose is administered three times on 3 consecutive days. 8, 9, 113, 129 because of its immunomodulatory properties, transfusion of hivig has become more common in veterinary patients. 94 the two major diseases treated with hivig have been immune-mediated hemolytic anemia (imha) and immune-mediated thrombocytopenia (itp), but hivig has also been used to treat some immune-mediated dermatologic disorders as well. 8, 9, 113, 129 randomized, controlled prospective studies of glucocorticoids with and without hivig for the treatment of imha and itp have been published. 8, 129 the itp study demonstrated reduction in platelet recovery time without a concurrent increase in associated charges in the group randomized to receive glucocorticoids and hivig. the imha study did not show an advantage to treatment with hivig and glucocorticoids compared with glucocorticoids alone, but the study was underpowered to distinguish a difference between the two treatment groups. administration of hivig to normal dogs promoted a hypercoagulable state, but in clinically ill dogs causality of thromboembolism is difficult to determine given the complexity of diseases undergoing hivig transfusion. 100, 116 the most convenient source of blood for a veterinary clinic is a commercial blood bank. currently, there are only a limited number of commercial veterinary blood banks in the united states, and they cannot adequately supply all the small animal practices in the country with blood (see box 24-1). veterinary school blood donor programs may serve as an additional source of blood for the practitioner. 56 because of the limited supply of blood from commercial animal blood banks, small animal practitioners typically borrow a donor from an employee or maintain a blood donor on the premises. 56 borrowing a donor from either an employee or a client is a frequently used, if less convenient, option and is less expensive than maintaining an in-hospital donor. maintaining a donor on the premises is advantageous because they are readily available for donation and their health status and disease exposure can be controlled, but the expense associated with feeding, housing, and caring for a blood donor is significant. 54 volunteer blood donor programs have replaced many on-site blood donors. 14, 59 collecting blood from stray animals is unsafe because infectious disease exposure and health status are unknown. identification of donor dogs and cats before blood is needed is essential to allow blood type to be determined and the health status of the donor to be assessed before blood collection, thus ensuring the safety of the blood being transfused. the american college of veterinary internal medicine has published recommendations on infectious disease screening for canine and feline blood donors as a consensus statement. 124 the recommendations have been incorporated into the following sections. for nearly 60 years, the best blood donor was believed to be a large, quiet dog not requiring anesthesia during blood collection. 78 the current recommendation is unchanged. a canine blood donor weighing more than 27 kg can safely donate 450 ml of blood in one donation, allowing collection of blood into commercially manufactured blood collection bags designed to facilitate sterile processing of components. dogs weighing 27 kg or more have been shown to consistently donate 1 unit of blood for 2 years at 3-week intervals. 92 dogs selected as donors also should have an easily accessible jugular vein to facilitate venipuncture. prior pregnancy does not exclude female dogs from donation because pregnancy does not induce alloantibodies. 12 greyhounds have been promoted as ideal blood donors because of their gentle disposition, high hematocrits, and lean body type, which simplifies blood collection. 45 many greyhounds are euthanized because of poor racing performance, and these dogs are available from racetracks, breeders, and rescue organizations. 36 blood banks choosing greyhounds as blood donors should be aware of certain breed idiosyncrasies that will impact on the management of a greyhound donor. the greyhound idiosyncrasy most important in transfusion medicine is the high red blood cell count, pcv and hemoglobin concentration, and low white blood cell counts and platelet count compared with mixed breed dogs. 91, 109 greyhounds in florida have a 46% seroprevalence of babesiosis. 111 because the geographic origin of greyhounds serving as blood donors cannot always be determined, all greyhounds being screened as donors should have serologic testing for babesia canis performed, and dogs with positive titers should be excluded as donors. greyhounds with negative titers against b. canis should have b. canis polymerase chain reaction (pcr) performed, and if the test is positive, the dog should be excluded as a donor. in addition to the tendency of greyhounds to be asymptomatic carriers of b. canis, some other breeds of dogs should be used cautiously as blood donors because they are known to be asymptomatic carriers of infectious organisms transmitted by transfusion. american pit bull terriers and staffordshire bull terriers recently have been recognized as carriers of babesia gibsoni. 10, 77 use of these dogs as blood donors should be restricted to those dogs that are seronegative and pcr-negative for b. gibsoni. leishmaniasis has been identified in american foxhounds and transfusion of leishmania infantum-infected blood from american foxhounds resulted in clinical leishmaniasis in transfusion recipients. 47, 89 all potential foxhound donors should be screened for leishmania sp. determination of blood type is critical to the selection of a blood donor dog. although seven canine blood groups or blood type systems have received international standardization, typing sera are available for only five types (box 24-2). red blood cells can be negative or positive for a given blood type, except for the dog erythrocyte antigen (dea) 1 system, which has three subtypes: dea 1.1, 1.2, and 1.3. canine red blood cells can be negative for all three subtypes (a dea 1-negative blood type) or positive for any one of the three subtypes. naturally occurring alloantibodies occur infrequently and without previous sensitization from transfusion do not appear to cause transfusion incompatibility in the dog 51 (see . a new canine red blood cell antigen, dal, has recently been described. 11 this antigen appears to be common in the general canine population and lacking in dalmatians. transfusion with dal-positive blood, induced an anti-dal antibody producing multiple incompatible crossmatch tests. dogs producing anti-dal antibodies are at risk for hemolytic transfusion reactions. the blood type of the ideal canine blood donor is not uniformly agreed on among transfusion experts. of the five blood groups for which typing sera are available, a transfusion reaction has been attributed to antibodies against dea 1.1 induced by a dea 1.1-positive transfusion in a dea 1.1-negative recipient and to an antibody induced by a dea 4-positive transfusion in a dea 4-negative dog. 40, 83 in theory, red blood cells expressing dea 1.2 can sensitize a dea 1.2-negative transfusion recipient, resulting in an acute hemolytic transfusion reaction if a second transfusion of dea 1.2-positive blood is given. in a laboratory setting, antibodies against dea 1.2 have been reported to cause transfusion reactions, but clinical reports of hemolytic transfusion reactions mediated by anti-dea 1.2 antibodies are lacking. dea 7 is believed to be structurally related to an antigen found in common bacteria. a naturally occurring antibody against dea 7 has previously been described in 20% to 50% of dea 7-negative dogs, but recently revised down to 9.8% of dogs. 51 this antibody may result in accelerated removal of dea 7-positive cells from a dea-negative donor with anti-dea 7 antibodies. 51, 102 based on this information, the recommendation has been made to select donors that are negative for dea 1.1, 1.2, and 7. others suggest the donor dog should also have red blood cells positive for dea 4 to be designated as a universal donor. 50 the recent description of a transfusion reaction attributed to antibodies against dea 4 in a dog with dea 4-negative red blood cells calls into question this recommendation. 119 ninety-eight percent of dogs are dea 4-positive, making it easy to find donors of this blood type. the importance of dea 3 and 5 and dal in blood donor selection remains to be determined. one other feature that should be considered before selection as a blood donor is the dog's plasma von willebrand factor concentration. von willebrand's disease is the most common inherited coagulopathy in dogs and has been reported in many breeds of dogs and in dogs of mixed breeding as well. because of the high frequency of this disease in the canine population, plasma from a canine blood donor will likely be used to transfuse a dog with von willebrand's disease-induced hemorrhage, and a donor with a normal concentration of von willebrand's factor is essential to replace the deficient coagulation factor. the physical requirements for a feline blood donor are similar to those for a canine donor. the ideal feline donor is a large cat, more than 5 kg of body weight, with a pleasant disposition. easily accessible jugular veins facilitate collection of blood, and choosing a shorthair cat decreases the clipping required before phlebotomy. it is essential to determine the blood type of potential donors. one feline blood group system has been identified with three blood types: a, b, and ab (see box 24-2) and recently a new common red blood cell antigen, mik has been identified. 6, 128 unlike dogs, cats have naturally occurring alloantibodies. type a cats have naturally occurring alloantibodies against type b cells and type b cats against type a cells. 38 cats of blood type b have strong hemagglutinating antibodies of the igm type against type a cells, and cats of blood type a have weak hemolysin and hemagglutinating antibodies of the igm and igg type against type b cells. the clinical significance of these alloantibodies is threefold in transfusion medicine. first and most importantly, a cat may have a transfusion reaction without sensitization from a previous transfusion; second, type a kittens born to a type b queen are at risk for neonatal isoerythrolysis 21 ; and third, the antibodies are useful in determining the blood type of a cat. mik appears to be a common red blood cell antigen. only a few cats lacking mik have been identified and they all produce anti-mik alloantibodies. donors of both type a and type b blood must be available because there is no universal donor in cats. incompatible transfusions result in shortened red blood cell survival and potentially death in the transfusion recipient; therefore the serologic compatibility between recipient and donor must be determined before every transfusion in cats. 38 donors of type a blood are easy to find because more than 99% of the domestic cats in the united states are type a. 42 the prevalence of domestic cats with type b blood varies geographically. in the united states, the western states have the highest percentage of type b cats, 4% to 6%. 42 australia has the highest reported percentage of type b cats in their domestic cat population, 73%. 6 in europe, the frequency of blood type b in domestic cats varies from 0% in finland, 14.9% in france, and 24.6% in turkey. 3, 41 some purebred cats have a higher frequency of type b in their population. 39 the british shorthair, devon rex and turkish van have been reported to have the highest proportion of type b individuals, approximately 50% to 60%. 4 the siamese, oriental shorthair, burmese, tonkinese, american shorthair, and norwegian forest cat breeds have not been reported to have any members with type b blood. blood type ab is extremely rare, occurring in 0.14% of cats in the united states and canada. 46 fortunately, a type ab donor is not required to successfully transfuse a type ab cat. blood from a type a cat is adequate. screening blood donors for infectious diseases transmitted by blood transfusion is an integral step in maintaining a safe blood supply. infectious disease screening of canine and feline blood donors varies within the different geographic regions of the united states and with the breed of the blood donor. an american college of veterinary internal medicine consensus statement, developed by a committee consisting of members of the infectious disease study group and the association of veterinary hematology and transfusion medicine should serve as the guideline for donor screening. 124 organisms infectious to dogs and known to be transmitted by blood transfusion include b. canis, b. gibsoni, haemobartonella canis, and leishmania sp. 31 124 dogs should not donate if they are ill or have fever, vomiting, or diarrhea; using donors with these clinical signs has resulted in yersinia enterocolitica contamination of human units of blood. 32 organisms infectious to cats and known to be transmitted by blood transfusion include: feline leukemia virus (felv), feline immunodeficiency virus (fiv) bartonella henselae, anaplasma phagocytophilum, ehrlichia spp. and neorickettsia spp., and the organisms formerly classified as haemobartonella sp. (mycoplasma haemofelis and candidatus mycoplasma haemominutum). 33, 49 potential donor cats should be screened for felv and fiv. because the prepatent period for felv infection can be 3 months, cats being considered as donors should be screened monthly for felv for 3 consecutive months. testing for fiv antibodies can be performed simultaneously. bartonella henselae is an emerging feline infectious disease and has been transmitted to cats by infected blood. 69 the use of cats with positive serology or cultures for b. henselae as blood donors is controversial and eliminating these cats from the donor pool is the safest approach. testing for hemoplasma should include both light microscopy and pcr, and infected cats should be eliminated from the donor pool. 33 screening of donor cats for feline infectious peritonitis (fip) is problematic because there is no reliable test to identify the fip-causing coronavirus. feline blood donors should be screened for infection with cytauxzoon felis and the agents causing feline ehrlichiosis if they reside in or are known to have traveled to endemic locations. a safe blood supply begins with healthy blood donors. all blood donors should undergo a complete physical examination each time they donate blood. complete and differential blood counts, biochemical profile, and fecal examination should be performed annually. donor cats and dogs with exposure to the outdoors or to ectoparasites should be routinely screened for infectious disease. blood donors should be tested for heartworms, treated for ectoparasites, and vaccinated for the diseases on the schedule recommended for pets residing in the geographic region of the blood bank. because the ideal feline blood donor lives in an indoor environment and is not exposed to other cats, the author believes vaccinations against felv, fiv, and fip are unnecessary in donor cats. exposure to the outdoors or to fleas approximately doubles the prevalence of hemoplasma infections in donor cats and restricting access to the outdoors, fleas, and other cats can prevent most infectious diseases in feline blood donors. 49 strict aseptic technique must be used during the blood collection process to prevent contamination of blood with microorganisms. whenever possible, solutions and equipment used for the collection process should be single-use products to prevent inadvertent contamination of blood. 55 after clipping the hair over the venipuncture site, the skin is surgically scrubbed. the ideal skin preparation regimen is yet to be determined in animals; however, in human blood donors, a 30-second, 70% isopropyl scrub followed by a 2% iodine tincture resulted in better skin surface disinfection than alcohol followed by chlorhexidine or green soap. 44 the phlebotomist wears sterile surgical gloves and performs venipuncture without touching the scrubbed area. several different solutions are available to anticoagulate and preserve blood for transfusion and species-specific storage times are listed (table 24-1) . anticoagulants provide no nutrients to preserve red cell metabolism during storage. blood collected in anticoagulants should be transfused immediately. anticoagulant-preservative solutions have been designed to provide nutrients to maintain red blood cell function during storage. one common anticoagulant solution for preservation of canine red blood cells, citrate phosphate dextrose adenine (cpda-1), is found in commercially prepared, multiple-bag systems. maximal storage time for feline blood in cpda-1 has yet to be determined but may be as long as 35 days. 15 acid citrate dextrose or anticoagulant citrate dextrose (acd) formula b can be used to store either canine or feline blood. 29, 79 it can be purchased in 500-ml bags and placed in syringes for collection of blood. cat and dog red blood cells maintain adequate viability following storage in acd for 30 and 21 days, respectively. additive solutions are contained in a multibag system containing citrate phosphate dextrose (cpd) or citrate phosphate dextrose 2 (cpd-2) as the anticoagulant. the additive solution is contained in a bag separate from the main bag and is added to prbcs after the plasma is removed. additive solutions that have been evaluated in dogs are adsol (fenwal laboratories, baxter health care corporation, deerfield, ill.) and nutricel (miles pharmaceutical division, west haven, conn.). 123, 125 additive solutions have not been evaluated for storage of feline blood, but are sometimes used as storage media for feline red blood cells. white blood cells are responsible for some adverse effects of transfusion and donot contribute to transfusion efficacy(see "adverse effects of transfusion"). an integral filter to remove white blood cells from whole blood is incorporated into some blood bag systems. one system has been evaluated using canine blood and effectively removed white blood cells without affecting red blood cell viability. 13 98 the choice is strictly a matter of personal preference and skill. acepromazine is not recommended because it causes hypotension and platelet dysfunction. the flow of blood into the bag can occur by gravity or suction. blood collected by suction does not have a greater rate of hemolysis than that collected by gravity flow, and it can be collected more rapidly. 27 suction collection of blood is facilitated using a vacuum chamber manufactured by the animal blood resources international (stockbridge, mich.). this device requires an external vacuum source during collection of blood. it is unusual to find a feline blood donor that does not require sedation during blood donation. the author prefers a combination of ketamine (10 mg) and diazepam (0.5 mg) intravenously for cats, whereas others recommend using midazolam, and isoflurane or sevoflurane. 98, 114 if the sedative agent is to be given intravenously, a peripheral vein (cephalic or medial saphenous) should be used to preserve the jugular veins for blood collection. no commercially available system is manufactured for the collection of blood from cats because of the small volume of blood that can safely be withdrawn from a cat. typically, anticoagulant can be withdrawn from a blood bag port using a syringe. it is placed in one or two large syringes (25 to 60 ml) depending on the volume of blood to be collected (see table 24 -1). a large (19gauge) butterfly needle is used for jugular venipuncture so that if a second syringe of blood is to be collected, the full syringe can be removed and the second syringe connected without a second venipuncture. by the definition of the american association of blood banks, this is an "open" system, and blood collected in this manner should not be transfused more than 4 hours after collection. 118 alternatively, a standard blood collection bag containing cpda-1 can be used. all cpda-1 is expelled from the bag except for the amount remaining in the tubing. feline blood is collected directly into the bag. 93 a commercially available vacuum system can be used for collecting blood from cats, but some authors find this system less satisfactory than the syringe method. 63, 98 selection and transfusion of compatible blood is one component of the process to provide a safe and efficacious red cell transfusion. with the identification of a new red blood cell antigen in both dogs and cats, recommendations for appropriate compatibility testing before the first transfusion are a currently being revaluated. because each unit of red blood cells is antigenically distinct, the recipient may form antibodies after transfusion of any unit of blood. the immune system will take a minimum of 5 days to make antibodies against transfused red blood cells; therefore, a crossmatch should be performed if more than 4 days elapse between transfusions. performing a crossmatch will not prevent an immune reaction to subsequent transfusions; it can only identify those units of blood with potential to cause acute hemolytic transfusion reactions. because of the lack of clinically significant preformed alloantibodies in the dog, blood typing and crossmatching are not routinely performed before the first transfusion. when dea 1.1-positive blood is transfused, ideally it would be given to a dea 1.1-positive recipient to prevent sensitization of a dea 1.1-negative dog. dea 1.1 status can be determined by using the typing systems described below. crossmatching should be performed before any subsequent transfusion to identify a compatible unit of red blood cells. blood typing or crossmatching is not required before transfusion of canine plasma. previously, blood typing was considered adequate pretransfusion testing before administration of red blood cells or plasma to cats. blood typing prevented administration of type b blood to a type a cat and vice versa. however, transfusion of a-b mismatched blood results in decreased red blood cell survival or a potentially fatal, acute hemolytic transfusion reaction. 38 blood typing will not identify the mik antigen and its naturally occurring alloantibody; however crossmatching will detect the anti-mik antibody and crossmatching may become the preferred compatibility test for all feline transfusions to prevent incompatible transfusions due to anti-a, anti-b, or anti-mik alloantibodies. determining a-b blood type in the cat has been simplified by the availability of in-clinic typing systems (figure 24-1) . a special situation with regard to blood typing and crossmatching exists in cats. when blood typing is unavailable, crossmatching will administration of an incompatible transfusion due to a, b, or mik alloantibodies. when crossmatching is performed with a known type a donor, an incompatible major crossmatch strongly suggests the potential recipient is a type b or a mik negative cat because of the naturally occurring alloantibodies in these cats. if cat plasma is administered, it should be the same blood type as the recipient because plasma will contain anti-a, anti-b, and anti-mik antibodies. crossmatching the donor to the recipient cat will prevent a reaction because of a, b, or mik alloantibodies. the person administering the blood should pay careful attention to the blood bag label before transfusion. the most common reason for an acute hemolytic transfusion reaction in human patients is clerical error-the wrong unit of blood is released from the blood bank or a unit of blood is given to a patient who was not intended to receive a transfusion. 110 in veterinary medicine, it is crucial to confirm that the blood comes from the correct species of blood donor in addition to being typed and matched to the patient requiring a transfusion. the contents of the bag also should be examined for normal color and consistency. bacterially contaminated blood often appears brown or purple because of deoxygenation, hemolysis, and formation of methemoglobin. 55, 65 blood and plasma can be administered using several routes. most commonly, blood is given intravenously. the diameter of the catheter used for transfusion is important in determining the rate of blood flow because blood flows more slowly through a small catheter; however, small diameter catheters have not been associated with increased risk of hemolysis during transfusion. 118 the intraosseous route can be used successfully for administration of blood and plasma. 88 in normal dogs, 93% to 98% of red blood cells administered through an intraosseous catheter are found in the peripheral circulation within 5 minutes. 24 this rapid and simple method is especially useful in animals with vascular collapse and in extremely young puppies and kittens. special intraosseous catheters are available, but a spinal needle, bone marrow aspiration needle, over-the-needle catheter, or even an ordinary hypodermic needle can be used. sites for the placement of the intraosseous catheter include the trochanteric fossa of the femur, the medial tibia, and the iliac crest. blood flows very rapidly through an intraosseous catheter, and rate of administration should be monitored closely. plasma can be administered intraperitoneally in emergency situations, but red blood cells are slowly and poorly absorbed when administered by this route, and it is not recommended for red blood cell transfusions. a blood transfusion administration set is required for administration of red blood cells or plasma to remove blood clots and debris, which form during storage and which could cause embolism. the filter typically used in veterinary medicine is 170 mm in size. for smallvolume transfusions, an 18-mm filter attached to intravenous tubing is useful. an 18-mm filter does not work well for large-volume transfusions because it rapidly becomes obstructed with debris, and transfusion rate slows. the risk of an air embolism is increased when blood is collected into glass bottles. a blood administration set does not remove air from stored blood; accordingly, glass bottles are not recommended for collection and storage of blood. the american association of blood banks explicitly states that medications should not be added to blood or components. 118 in addition, no fluid should be added to blood excep. 0.9% sodium chloride when it is necessary to decrease the viscosity of prbcs. fluids containing calcium such as lactated ringer's solution may overcome the anticoagulant properties of citrate, resulting in coagulation of the blood. solutions such as 5% dextrose in water are hypotonic and may induce hemolysis. the recommended rate of transfusion of red blood cells depends on the status of the recipient. in massive hemorrhage, the transfusion should be given as rapidly as possible. in a normovolemic, stable transfusion recipient, some clinicians recommend a rate of 0.25 ml/ kg for the first 30 minutes, after which the rate is increased if no reaction is seen. 112 in patients with heart disease, a rate of 4 ml/kg/hr should not be exceeded. 45 transfusion rates of 10 ml/kg/hr, 4 ml/kg/hr, and up to 60 ml/kg/hr were used to transfuse red blood cells to cats with normovolemia, cardiovascular dysfunction. and hypovolemic shock, respectively. 126 plasma can be given more rapidly (4 to 6 ml/min). 67 whatever the rate chosen, it should be rapid enough to complete the transfusion within 4 hours of initiation because of the risk of bacterial growth in blood maintained at room temperature for a prolonged period. control of blood product delivery rate can be accomplished by use of infusion pumps to deliver a preset volume over a specific period of time. the use of infusion pumps must be limited to devices approved for use with blood because some infusion pumps can result in hemolysis of red blood cells as a result of excessive pressure. 107 because blood does not contain any antibacterial agents, it must be refrigerated until used to retard bacterial growth and maintain red blood cell viability. if the clinical status of the animal requires that the transfusion be given over a period greater than 4 hours, the blood can be split into smaller units with a transfer bag. one portion of the blood is transfused while the other is returned to the refrigerator until the first half of the transfusion is completed. in patients with cardiac disease at risk for volume overload, the risk can be further minimized by use of prbcs, which require infusion of a lower volume than whole blood. warming of blood before transfusion has been recommended to prevent hypothermia in the transfusion recipient. warming of blood probably is only necessary if a large volume of blood is to be given or if the recipient is a neonate. for adult animals receiving a single unit of blood, the blood can be administered directly from the refrigerator. warming blood has the potential for excessive heating, causing red blood cell membrane damage and hemolysis or promoting bacterial growth if contamination is present. blood warming devices that use dry heat, radio waves, microwaves, or electromagnetic energy are available, but cost often is prohibitive. refrigerated human blood can be warmed quickly by admixing it with warm (45 c to 60 c) 0.9% saline in a ratio of 1:1 without damage to red blood cells. 61 this method has not been tested for dogs or cats. once blood is warmed to 37 c, it deteriorates rapidly and, if not used, should be discarded. fresh frozen plasma must be thawed before transfusion. a method for thawing canine fresh frozen plasma in a microwave oven has been described, but the author has found this unsatisfactory because of uneven heating by household microwave ovens. 60 plasma can be thawed at room temperature, and if the thawing time needs to be shortened, the plasma can be placed into a plastic bag and thawed in a 37 c water bath. the plastic bag is necessary to prevent contamination of the infusion ports in the water bath. if thawed and not used within 1 hour, it maybe refrozen with out loss of anticoagulant activity. 131 plasma should be used within 4 hours of thawing or discarded. transfusion recipients should be monitored during transfusion to allow early detection of a transfusion reaction. rectal temperature, heart rate, and respiratory rate should be recorded every 10 minutes during the first 30 minutes and then every 30 minutes thereafter. the patient should be monitored for vomiting, diarrhea, urticaria, and hemoglobinuria or hemoglobinemia. changes in vital signs or clinical status may indicate a transfusion reaction. patients developing volume overload will become tachypneic or dyspneic, and tachycardic. massive transfusions (1 blood volume in 24 hours) have been reported in both dogs and cats. 16, 62, 97 patients receiving massive transfusions of stored blood may develop specific abnormalities. consequently, patients receiving massive transfusions should be monitored for changes in serum potassium, ionized calcium, and ionized magnesium concentrations, as well as hypothermia and coagulation abnormalities. 16, 62, 97 an adverse effect of transfusion or transfusion reaction consists of the range of immunologic and metabolic changes that occur during or after administration of a blood product. four classes of adverse effects of transfusion have been described (box 24-3). acute transfusion reactions occur during or within a few hours after a transfusion, and delayed transfusion reactions occur after the completion of the transfusion. the delay may be hours to years. reports describing adverse effects of transfusion in dogs and cats are limited to case reports and retrospective series.* acute immunologic transfusion reactions occur because antibodies that elicit an immune response are present in the plasma of either the donor or recipient. the sequelae of an acute immunologic transfusion reaction are rapid, often irreversible, and sometimes fatal. current theories on the pathogenesis of acute hemolytic transfusion reaction in humans propose that hemolysis induces the release of cytokines, such as tumor necrosis factor, interleukin 1 (il-1), il-6, and il-8, complement, endotheliumderived relaxing factor (nitric oxide), and endothelin, resulting in the clinical syndrome of disseminated intravascular coagulation, shock, and acute renal failure. 9 the pathophysiology of acute hemolytic transfusion reaction in dogs and cats must differ in some manner from that described in humans because acute renal failure is not reported to be a feature in dogs and cats. 5, 37, 40, 132 the best example of an acute hemolytic transfusion reaction in veterinary medicine is the administration of type a red blood cells to a type b cat. in the recipient cat, naturally occurring alloantibodies and complement bind to the transfused red blood cells and cause hemolysis. clinical signs described in cats having an acute hemolytic transfusion reaction include fever, vomiting, lethargy, icterus, and death. 5 results of laboratory testing often show a positive coombs test, rapidly declining pcv, and increasing serum bilirubin concentration. dogs experiencing an acute hemolytic transfusion reaction show clinical signs similar but not identical to those observed in cats. most affected dogs exhibit fever, restlessness, salivation, incontinence, and vomiting. some dogs develop shock, and an occasional dog experiences acute death. plasma and urine hemoglobin concentrations increase within minutes of transfusion. incompatible cells are cleared from circulation in less than 2 hours. dogs whose red blood cells lack the dea 1.1 antigen that have previously been sensitized by transfusion of dea 1.1-positive cells are at the greatest risk for an acute hemolytic transfusion reaction. 40 other acute immunologic transfusion reactions reported in dogs and cats include nonhemolytic fever and urticaria. 17, 53, 64, 120 in humans, nonhemolytic fever is a result of antibodies against donor white blood cells, and urticaria occurs as a result of antibodies-against donor plasma proteins. nonhemolytic febrile transfusion reactions do not require treatment, but antipyretics may be used if the patient is uncomfortable (table 24 -2). urticaria is the most common reaction to plasma transfusion in dogs. 120 if urticaria caused by plasma administration is diagnosed, it should be treated with short-acting corticosteroids and antihistamines. the plasma transfusion then may be restarted at a slower rate and the recipient carefully observed. delayed immunologic transfusion reactions are classified as delayed hemolytic, transfusion-induced immunosuppression, posttransfusion purpura, and graft-versus-host disease. these reactions are not preventable by crossmatching or blood typing. delayed hemolytic transfusion reactions invariably occur in persons who have been previously sensitized to allogenic red blood cell antigens by transfusion or pregnancy. even though compatible blood is given to a patient, the recipient may develop antibodies against any one of the hundreds of red blood cell antigens present on the transfused cells. an anamnestic response to the antigens on the transfused red blood cells results in a delayed hemolytic transfusion reaction that occurs 7 to 10 days after a transfusion and is a well-described complication of red cell transfusion in humans. it has not been reported in dogs, but there is no reason it could not occur. fever is the most common sign of a delayed hemolytic transfusion reaction in humans. icterus also may be noticed 4 to 7 days after a transfusion. the only delayed immunologic transfusion reaction that has been reported in veterinary medicine is posttransfusion purpura occurring in a previously transfused dog with hemophilia a. 122 five to eight days after subsequent transfusion, thrombocytopenia and petechiation were evident. blood collected during a thrombocytopenic episode was positive for plateletbound igg, indicating an immune mechanism for platelet destruction. acute immunologic acute nonimmunologic transfusion reactions are caused by physical changes in the red blood cells during collection, storage, or administration. improper collection of blood can result in an adverse reaction to transfusion. collection of blood from an inadequately screened donor can result in transmission of bacteria, spirochetes, or protozoa and eventually clinical signs of the associated disease in the recipient. transfusion of blood contaminated by bacteria can cause shock, which is managed with volume expansion and pressor agents, as well as empirical antibiotic administration based on results of a gram stain. endotoxic shock results from transfusion of blood heavily contaminated with endotoxin-producing bacteria. clinical signs in cats transfused with blood contaminated by bacteria include collapse, vomiting, diarrhea, and acute death, but most cats did not exhibit clinical signs after receiving bacterially contaminated blood. 55 hypotensive shock developed in a dog that received a b. canis-infected transfusion. 18 during storage, the atp content of red blood cells decreases, and some cells undergo hemolysis resulting in leakage of potassium out of the cells into the storage medium. the increase in potassium in the storage medium is a contributing factor in the development of hyperkalemia in patients receiving large volume transfusions of stored blood. a large-volume transfusion of stored blood can cause hyperkalemia, but this is rare unless the patient has renal failure or preexisting hyperkalemia. 62 hyperkalemia in a transfusion recipient is as it would be in any patient with hyperkalemia. the transfusion should be discontinued and 0.9% nacl administered because 0.9% nacl does not contain added potassium and will facilitate renal excretion of potassium. intravenous administration of insulin, followed by administration of 50% dextrose and frequent monitoring of blood glucose and potassium concentrations until serum potassium concentration normalizes, is all that is necessary. physical damage (such as freezing or overheating) to red blood cells during storage causes hemolysis. while being transfused, the patient exhibits hemoglobinuria and hemoglobinemia without evidence of other signs of an acute hemolytic transfusion reaction, such as fever, vomiting, or collapse. during storage of blood, formation of clots or introduction of air into the bag may occur, resulting in embolism during transfusion. a rare adverse event associated with transfusion is an embolism. venous air embolism causes sudden onset pulmonary vascular obstruction, a precordial murmur, hypotension, and death as a result of respiratory failure. administration of large-volume transfusions can result in multiple adverse events. ionized hypocalcemia or ionized hypomagnesemia can result from the citrate used as an anticoagulant complexing with calcium or magnesium, and lead to myocardial dysfunction and potential cardiac arrest and tetany. 66 routine empirical administration of calcium to transfusion recipients cannot be recommended because of the risk of hypercalcemia and increased myocardial irritability, but animals with ionized hypocalcemia resulting from large transfusion should be treated with calcium gluconate or calcium chloride to effect. 26 hypothermia is common after large-volume transfusion in veterinary patients, and use of warming blankets should be instituted whenever possible. dilution of coagulation factors by large-volume transfusion of coagulation factor-depleted stored blood results in prolongation of coagulation times. in dogs receiving large-volume transfusions, prolongation of coagulation times is associated with a poor prognosis. 62 administration of fresh frozen plasma is indicated to correct the coagulation abnormalities. any transfusion can cause circulatory overload. dogs and cats with chronic severe anemia or compromised cardiac and pulmonary systems are at greater risk for circulatory overload and pulmonary edema than are those without cardiopulmonary disease. dogs and cats developing volume overload from transfusion are treated with oxygen supplementation, diuretics, and vasodilators. improvement should be seen within 1 to 2 hours. in humans, human immunodeficiency virus, hepatitis virus, and cytomegalovirus infections are documented as late effects of transfusion. one late complication of transfusion described in veterinary medicine is hemochromatosis. 104 a schnauzer received blood transfusions every 6 to 8 weeks for 3 years to treat chronic anemia. hemochromatosis was confirmed by necropsy when the dog was euthanized because of progressive liver disease. when an acute transfusion reaction is suspected, immediate intervention is critical because of the life-threatening nature of acute transfusion reactions. in all animals suspected of having some form of acute transfusion reaction, the transfusion should be stopped and samples of patient blood and urine obtained for baseline evaluation of biochemical, hematologic, and coagulation values. the unit of blood should be inspected to ensure it is from the appropriate species and is the intended unit based on the crossmatch or blood type. a gram stain and bacterial culture of the blood remaining in the blood bag should be submitted to the laboratory. urine can be visually inspected to determine the presence or absence of hemoglobin. rectal temperature of the recipient should be compared with the pretransfusion value. a transfusionassociated fever is defined as an increase in 1 f over the pretransfusion temperature. 118 the cardiovascular system should be monitored by electrocardiogram and blood pressure measurement. immediate evaluation of serum ionized calcium and potassium concentrations are critical, but certain electrocardiographic changes serve as surrogate markers of hypocalcemia (long qt-interval with a normal heart rate) or hyperkalemia (decreased height of p waves, loss of p waves, or widening of the qrs complex with large t waves) if rapid measurement of serum electrolyte concentrations cannot be obtained. venous access and blood pressure should be maintained by an infusion of a crystalloid solution such as lactated ringer's solution or 0.9% nacl. intravenous administration of short-acting glucocorticoids may suppress some of the mediators of acute hemolytic transfusion reactions and lessen the clinical progression, but their efficacy in transfusion reactions has not been evaluated in veterinary patients. when the evaluation of a patient with a suspected transfusion reaction suggests that an acute hemolytic transfusion reaction is occurring, the blood typing and crossmatching must be repeated to determine whether a laboratory error is responsible for the reaction. in patients with fever, without evidence of hemolysis, the transfusion may be restarted if the gram stain is negative for bacterial contamination. it is important to recognize the late effects of transfusion and not mistake them for another disease process. delayed transfusion reactions usually are managed with supportive care. the only specific treatment for a delayed transfusion reaction consists of treating a transfusionacquired infection appropriately. a special effort is not necessary to prevent transfusion reactions. by simply following the transfusion guidelines discussed here with reference to donor selection, blood typing, blood storage, and administration, most transfusion reactions can be prevented. crossmatching should be included in the guidelines for providing a safe blood transfusion. major and minor crossmatches detect antibodies in the plasma of the donor or recipient capable of causing an acute hemolytic transfusion reaction; however, a transfusion reaction may still occur despite a compatible crossmatch. crossmatching does not prevent sensitization to red blood cell antigens, which may result in a hemolytic reaction during future transfusions because it detects only antibodies that are currently present in the donor or recipient. it should be performed routinely in veterinary clinics either by a commercially available gel tube method (dms laboratories, inc., flemington, n.j.) or by the tube method. a tube crossmatch is described below. performing a crossmatch is an intimidating but simple procedure once all the equipment is assembled (box 24-4) . several descriptions of the procedure have been published, all of which describe the same basic procedure with minor variations. 14, 35, 103 not all protocols recommend the use of phosphate-buffered saline; others have an additional step at the end using species-specific coombs reagent to increase test sensitivity, and some recommend that tubes be incubated at 4 c, 37 c, and 42 c. the following is the protocol the author uses: 1. obtain edta-anticoagulated blood from the recipient and the potential donor or the tube segments of blood from the units being considered for transfusion. 2. centrifuge both donor and recipient blood for 5 minutes at 1000 g. 3. using pipettes, remove the plasma, and save in separate labeled tubes. 4. wash the red blood cells by adding phosphate-buffered saline to the red cells to fill the tube. resuspend the red cells in the saline by tapping the bottom of the tube with a finger. 5. centrifuge the red cells and saline for 5 minutes at 1000 g. pipette off saline, and discard. 6. repeat step. 4 and 5 twice. 7. after the third washing of the red cells in saline, resuspend the red cells to a 3% to 5% solution. it will appear bright cherry red. 8. for each potential donor, mix two drops of recipient plasma and one drop of donor red cell suspension for the major crossmatch. mix gently. 9. for each potential donor, mix two drops of donor plasma and one drop of recipient red cell suspension for the minor crossmatch. mix gently. 10. for the recipient control, mix two drops of recipient plasma and one drop of recipient red cell suspension. mix gently. 11. incubate the tubes at room temperature for 15 minutes. 12. centrifuge the tubes for 15 seconds at 1000 g. 13. observe the plasma for hemolysis. 14. resuspend the centrifuged cells by shaking gently. 15. observe the red blood cells for agglutination. interpretation. hemolysis or agglutination in a crossmatch indicates transfusion incompatibility. the degree of agglutination is graded 0 to 4þ (box 24-5 and figure 24 -2). units of blood that are incompatible should not be used. if all available units are incompatible, the least reactive unit should be chosen. when the recipient control shows hemolysis or agglutination, the crossmatch cannot be interpreted. this is common in patients with hemolytic anemia. multiple methods of blood typing dogs and cats have been described, including tube tests, typing cards, slide tests, immunochromatography, and gel tubes. 43, 106 a reference laboratory can perform blood typing for dea 3, 4, 5, and 7 and the recently described dal and mik of dogs and cats, respectively. in clinical situations in the united states, the commercially available blood typing cards for feline types a, b, and ab are commonly used (dms laboratories, inc., flemington, n.j.). however, when results indicate type ab, the results should be interpreted with caution as the card typing method commonly gives false positive results as type ab when the cat is actually type a. 7 any cat typed as ab should be confirmed by a second typing method. gel tube typing tests, available in europe, but not currently available in the united states, give accurate results when used in cats of type a, b, and ab. a simple immunochromatography method of blood typing has recently become available in the united states. 57 a paper strip impregnated with anti-a and anti-b monoclonal antibodies is placed in a red blood cell solution, allowing the cells to migrate up the strip and bind to the antibodies. results are rapidly available and easily interpreted. when using any blood equipment for performing crossmatch ) is an ultrapurified, polymerized hemoglobin of bovine origin (13 g/dl) in a modified ringer's lactate solution with a physiologic ph (7.8). the hemoglobin polymers range in molecular mass from 65 to 500 kda, with an average of 200 kda. the viscosity is low compared with blood (1.3 and 3.5 centipoise, respectively), and the solution is isosmotic (300 mosm/kg) with blood. the concentration of methemoglobin, the inactive form of hemoglobin, is 10%. oxyglobin can be stored at room temperature or refrigerated (2 c to 30 c) for up to 3 years. its intravascular half-life is dose dependent (18 to 43 hours, at a dosage of 10 to 30 ml/kg), as measured in healthy dogs. it is expected that more than 90% of the administered dose will be eliminated from the body in 5 to 7 days after infusion. the oxygen half-saturation pressure (p-50) of oxyglobin is greater than that of canine blood (38 vs. 30 mm hg, respectively). this increase in p-50 facilitates off-loading of oxygen to the tissues. the hemoglobin is packaged in the deoxygenated state in an overwrap that is impermeable to oxygen. complications of severe anemia result from poor oxygenation of tissues. restoration of adequate tissue oxygenation typically is achieved by administering a blood transfusion. improvement in the clinical signs of anemia results from a corresponding increase in hemoglobin concentration, which in turn increases the arterial oxygen content of the blood. the increased oxygen content of the blood supplied by oxyglobin also relieves the clinical signs of anemia. two prospective randomized trials have evaluated oxyglobin for the treatment of anemia. 95, 133 one was a multicenter clinical trial for dogs with moderate to severe anemia (pcv, 6% to 23%). 95 sixty-four dogs in need of blood transfusion were studied, including those with anemia caused by blood loss (n ¼ 25), hemolysis (n ¼ 30), or ineffective erythropoiesis (n ¼ 9). dogs in both groups were monitored for a decrease in hemoglobin concentration or deterioration in physical condition at which time they received additional oxygen-carrying support. if additional oxygen-carrying support was needed, oxyglobin-treated dogs received prbcs (n ¼ 1), and untreated control dogs received oxyglobin (n ¼ 19). treatment success was defined as the lack of need for additional oxygen-carrying support for 24 hours. the success rate in the 30 treated dogs (95%) was significantly greater than the success rate in the 34 control dogs (32%). this difference between treated and control dogs was significant, regardless of the cause of anemia. the other trial randomized 12 dogs with severe anemia (pcv ¼ 10% to 20%) secondary to babesiosis to receive either 20 ml/kg of oxyglobin, or packed red blood cells. 133 blood gas, acid-base, and blood pressure were objective measures of response to treatment. similar overall improvements were seen in both the oxyglobin and prbc transfusion groups. although oxyglobin is approved only for use in dogs, other species have been infused with the solution. oxyglobin administration to cats has been retrospectively evaluated. 34, 127 the median dosage was approximately 10 to 11 ml/kg/24 hours. oxyglobin has also been administered to other species to increase oxygen carrying capacity: mallard duck, miniature horse, and serval cat. 73, 82, 101 one published dosage for birds is 5 ml/kg iv or interosseously for the treatment of shock and for the treatment of shock in small mammals, 2 ml/kg as a 10 to 15 minute intravenous bolus followed by a continuous rate infusion at 0.2 to 0.4 ml/kg/hr. 74 because it lacks the antigenic red blood cell membrane, oxyglobin is not only useful in multiple species, but it eliminates some of the pretransfusion testing required with red blood cell transfusions. blood typing and crossmatching are not necessary because the red blood cell membrane, which is the major cause of transfusion incompatibility, has been removed during the manufacturing process. repeated dosing of oxyglobin was reported in both feline retrospective studies. 34, 127 no allergic reactions were reported. a laboratory study of repeated dosing in dogs showed antibodies to oxyglobin did form, but those antibodies did not decrease binding of oxygen to oxyglobin and did not result in systemic allergic reactions. 52 adverse effects of treatment with oxyglobin are similar in dogs and cats. after treatment, a transient discoloration (yellow, brown, or red) of the mucous membranes, sclera, urine, and sometimes skin occurs. overexpansion of the vascular volume may occur, especially in normovolemic animals. rates of administration greater than 10 ml/kg/hr in anemic, clinically ill dogs sometimes resulted in increased central venous pressure, with or without pulmonary edema or other respiratory signs of circulatory overload. pleural effusion and pulmonary edema were found commonly in cats given oxyglobin, but evidence was insufficient to directly link either to the administration of oxyglobin. 34, 127 cats recently administered blood transfusions or having underlying cardiac disease appear to be more likely to develop pleural effusion and pulmonary edema. low infusion rates are recommended in these cats (<5 ml/kg/hr). in the canine clinical trial, vomiting occurred in 35% of the treated dogs. diarrhea, fever, and death also were seen in approximately 15% of oxyglobin-treated dogs; however, an association with oxyglobin or the underlying disease could not be determined. these findings were most common in dogs with immune-mediated hemolytic anemia that received oxyglobin. the presence of oxyglobin in serum may cause artifactual changes in the results of serum chemistry tests. interference by oxyglobin depends on the type of analyzers and reagents used but is not typical of hemolysis. 18, 86 blood samples for analysis should be collected before infusion. a list of valid chemistry tests by analyzer is included in the product labeling. results of any clinical chemistry test performed on serum containing oxyglobin should be interpreted with consideration of the validity of the test. in general, all tests using colorimetric techniques are invalid, but other methodologies also show some interference. no interference is seen with hematologic or coagulation parameters except when optical methods are used for measuring prothrombin time and activated partial thromboplastin time. dipstick measurements (ph, glucose, ketones, protein) of urine are inaccurate when gross discoloration of the urine is present. the urine sediment is not affected. chronic enteropathies in dogs: evaluation of risk factors for negative outcome room temperature storage and cryopreservation of canine platelet concentrates blood type a and b frequencies in turkish van and angora cats in turkey frequencies of blood type a, b and ab in non-pedigree domestic cats in turkey blood transfusion reactions in the cat the ab blood group system in cats erythrocytic pyruvate kinase deficiency and ab blood types in australian abyssinian and somali cats a prospective, randomized, double-blinded, placebo-controlled study of human intravenous immunoglobulin for the acute management of presumptive primary immune-mediated thrombocytopenia in dogs treatment of severe immune-mediated thrombocytopenia with human iv immunoglobulin in 5 dogs serosurvey of anti-babesia antibodies in stray dogs and american pit bull terriers and american staffordshire terriers from north carolina canine dal blood type: a red cell antigen lacking in some dalmatians lack of evidence of pregnancy-induced alloantibodies in dogs use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability problems in veterinary medicine. philadelphia: jb lippincott storage of feline and canine whole blood in cpda-1 and determination of the posttransfusion viability massive transfusion and surgical management of iatrogenic aortic laceration associated with cystocentesis in a dog canine red blood cell transfusion practice in vitro effects of a novel hemoglobin-based oxygen carrier on the routine chemistry, therapeutic drug, coagulation, hematology, and blood bank assays acute hemolytic transfusion reaction, a paradigm of the systemic inflammatory response: new insights into pathophysiology and treatment prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs transfer of colostral antibodies from queens to their kittens clinical use of blood products in cats: a retrospective study effect of cryoprecipitate and plasma on plasma von willebrand factor multimeters and bleeding time in doberman pinschers with type-i von willebrand's disease the absorption of red blood cells after parenteral injection at various sites response of healthy dogs to infusions of human serum albumin calcium chloride versus calcium gluconate: comparison of ionization and cardiovascular effects in children and dogs post transfusion viability of stored canine red blood cells after vacuum facilitated collection evaluation of preservatives and containers for storage of canine blood use of biochemical measures to estimate viability of red blood cells in canine blood stored in acid citrate dextrose solution with and without added ascorbic acid adverse reactions suggestive of type iii hypersensitivity in six healthy dogs given human albumin hypotensive shock syndrome associated with acute babesia canis infection in a dog transfusion acquired yersinia enterocolitica survival of mycoplasma haemofelis and "candidatus mycoplasma haemominutum" in blood of cats used for transfusions use of a hemoglobin-based oxygen-carrying solution in cats: 72 cases problems in veterinary medicine. philadelphia: jb lippincott where to get blood donors? acute hemolytic transfusion reaction in an abyssinian cat with blood type b transfusion of type-a and type-b blood to cats frequency and inheritance of a and b blood types in feline breeds of the united states an acute hemolytic transfusion reaction caused by dog erythrocyte antigen 1.1 incompatibility in a previously sensitized dog frequencies of feline a and b blood types in europe geographical variation of the feline blood type frequencies in the united states comparison of various canine blood-typing methods evaluation of donor skin disinfection methods blood transfusion therapy: an updated overview blood type ab in the feline ab blood group system seroprevalence of antibodies against leishmania spp among dogs in the united states assessment of a dimethyl sulfoxide-stabilized frozen canine platelet concentrate anaplasma phagocytophilum and species of bartonella, neorickettsia and ehrlichia in cats used as blood donors in the united states canine blood groups and their importance in veterinary transfusion medicine incidence of canine serum antibody to known dog erythrocyte antigens in potential donor population absence of immunopathology associated with repeated iv administration of bovine hb-based oxygen carrier in dogs feline blood component therapy: retrospective study of 246 transfusions problems in veterinary medicine. philadelphia: jb lippincott; 1992 serratia marcescens contamination of feline whole blood in a hospital blood bank transfusion practices and costs in dogs evaluation of microwave-thawed canine plasma for transfusion blood warming: current applications and techniques massive transfusion in dogs problems in veterinary medicine. philadelphia: jb lippincott packed red blood cell transfusions in dogs: 131 cases (1989) pseudomonas fluorescens contamination of a feline packed red blood cell unit and studies of canine units response of canine plasma-ionized calcium and magnesium to the rapid infusion of acid-citrate-dextrose (acd) solution use of blood and blood components for feline and canine patients red blood cell transfusions in cats: 126 cases (1999) in press relapsing bacteremia after blood transmission of bartonella henselae to cats general principles of small animal blood component administration haemobartonella canis infection following splenectomy and transfusion use of adult cat serum to correct failure of passive transfer in kittens comparison of fluid types for resuscitation after acute blood loss in mallard ducks (anas platyrhynchos) emergency care and managing toxicoses in the exotic animal clinical indications for use of fresh frozen plasma in dogs: 74 dogs a treatise on the heart on the movement and colour of the blood and on the passage of the chyle into the blood babesia gibsoni infection among dogs in the southeastern united states the blood and plasma bank posttransfusion viability of feline erythrocytes stored in acid citrate dextrose solution serum antibodies against human albumin in critically ill and healthy dogs the use of 25% human serum albumin: outcome and efficacy in raising serum albumin and systemic blood pressure in critically ill dogs and cats use of a bovine hemoglobin preparation in the treatment of cyclic ovarian hemorrhage in a miniature horse a hemolytic transfusion reaction due to dea 4 alloantibodies in a dog canine von willebrand's disease; pathobiology, diagnosis and shortterm treatment problems in veterinary medicine. philadelphia: jb lippincott effect of hemopure w on the performance of ektachem and hitachi clinical analyzers national institutes of health consensus conference fresh-frozen plasma: indications and risks intraosseous infusion of fluids and therapeutics transmission of visceral leishmaniasis through blood transfusions from infected english foxhounds to anemic dogs use of adult dog serum as a substitute for colostrums in the neonatal dog hematologic values in mongrel and greyhound dogs being screened for research use effects of collection interval, body weight, and season on the hemograms of canine blood donors a method for collecting and storing feline whole blood effect of human intravenous immunoglobulin on canine monocytes and lymphocytes a clinical trial of a hemoglobin based oxygen carrier (hboc) fluid in the treatment of anemia in dogs incidence and severity of anaphylactoid reactions to colloid volume substitutes ): indications, complications and outcomes blood components: collection, processing and storage principles of blood collection and processing intravenous administration of human immune globulin in dogs with immune-mediated hemolytic anemia successful treatment of a southern pacific rattlesnake (crotalus viridis helleri) bite in a caracal (caracal caracal) transfusion medicine: the challenge of practical use hemochromatosis secondary to repeated blood transfusions in a dog transfusion-associated babesia gibsoni infection in a dog comparison of various blood-typing methods for the feline ab blood group system hemolysis of canine fresh and stored blood associated with peristaltic pump infusion efficacy of fresh frozen plasma and cryoprecipitate in dogs with von willebrand's disease or hemophilia a platelet concentration and hemoglobin function in greyhounds reports of 355 transfusion associated deaths: 1976-1985 seroprevalence of babesiosis in greyhounds in florida blood transfusion in dogs and cats. part ii. administration, adverse effects and component therapy treatment of severe adverse cutaneous drug reaction with human intravenous immunoglobulin in two dogs comparing chemical restraint and anesthetic protocols used for blood donation in cats: one teaching hospital's experience evaluation of use of human albumin in critically ill dogs: 73 cases prothrombotic and inflammatory effects of intravenous administration of human immunoglobulin g in dogs hemostatic effects of cryoprecipitate in dogs with disseminated intravascular coagulation technical manual. 11th ed. bethesda, md: american association of blood banks new red blood cell antigens in dogs and cats -a welcome discovery canine plasma therapy stability of hemostatic proteins in canine fresh frozen plasma units posttransfusion purpura in a dog with hemophilia a evaluation of an additive solution for preservation of canine red blood cells consensus statement on canine and feline blood donor screening for infectious disease evaluation of canine red blood cells stored in a saline, adenine and glucose solution for 35 days whole blood transfusions in 91 cats: a clinical evaluation clinical use of a haemoglobin-based oxygen carrying solution (oxyglobin) in 48 cats a newly recognized blood group in domestic shorthair cats: the mik red cell antigen use of human immunoglobulin in addition to glucocorticoids for the initial treatment of dogs with immune-mediated hemolytic anemia severe cardiomegaly secondary to anemia in a kitten comparative stability of canine hemostatic factors in freeze-thaw-cycled fresh frozen plasma hemolytic reactions produced in dogs by transfusion of incompatible dog blood and plasma a prospective, randomized comparison of oxyglobin (hb-200) and parked red blood cells transfusion for canine babesiosis key: cord-027659-rxbo7b0e authors: bates, imelda; owusu-ofori, shirley title: blood transfusion date: 2020-06-22 journal: manson's tropical diseases doi: 10.1016/b978-1-4160-4470-3.50018-5 sha: doc_id: 27659 cord_uid: rxbo7b0e nan only 39% of the global blood supply is donated in the poorest countries where 82% of the world's population lives. 1 blood transfusion is a vital component of every country's health service (table 14 .1). it can be a life-saving intervention for severe, acute anaemia, but mistakes in the transfusion process can be life-threatening, either immediately or years later through transmission of infectious agents. it is imperative that clinicians have a good understanding of how blood is acquired and prepared for transfusion, and when it should be used, and that governments put in place quality assurance mechanisms to guarantee that blood for transfusion is safe. transfusion medicine is critical to the success of most clinical specialties and should be incorporated into all national health plans and budgets. only 16% of member states meet all the world health organization's (who) recommendations for a national quality blood transfusion system. 1 at the national level the transfusion service should have a director, an advisory committee and clear transfusion policies and strategies (table 14. 2). blood collection, testing and distribution need to be standardized. although centralization of these services may offer the best guarantee of quality, it is often not practical in countries with poorly developed communications and transport infrastructure. in such countries, each hospital organizes its own blood transfusion service and it is then diffi cult to ensure national standardization and quality. hospital-based transfusion services place an enormous burden on laboratory resources and on the families of patients because they are responsible for fi nding suitable blood donors. in a typical district hospital in malawi, the overall cost of the transfusion service, including consumables, proportional amounts for capital equipment, staff time and overheads, was 53% of total laboratory costs and each unit of whole blood cost the laboratory approximately £10 to collect and process. 2 in wealthy countries with nationally or regionally centralized transfusion services, blood donor recruitment, and screening and processing of donated blood, are carried out in purpose-built centres which are separate from the hospitals where the blood is transfused. these centres operate to good manufacturing stan-dards similar to those laid down for the pharmaceutical industry. after donation and exclusion of potentially infected units, the blood is separated into components and fi ltered to remove white cells. computerization enables individual components to be barcoded so they can be tracked back to the original donor. hospitals are profi cient at predicting how much blood they will require and they receive regular consignments through a well-established delivery network. the effi ciency of the system means that one donor centre may provide blood to many hospitals and cover a population of several million. this process is expensive and one unit of blood currently costs over £100. 3 in wealthy countries it is standard practice to optimize the use of each donation of blood by separating it into individual components. these components, which may include plasma, platelets and cryoprecipitate, are prepared by centrifugation using a closed, sterile system. each component has different storage requirements. plasma and cryoprecipitate are kept frozen, red cells are stored at 1-5°c, and platelets at 18-22°c with constant agitation. separation of blood, even into simple components such as cells and plasma, requires equipment and expertise, so in the poorest countries blood components may only be accessible to those living close to a central hospital with blood separation facilities. an unsafe blood supply is costly in both human and economic terms. transfusion of infected blood causes morbidity and mortality in the recipients, and has an economic and emotional impact on their families and communities. those who become infected through blood transfusion are infectious to others and contribute to the spread of disease throughout the wider population. this increases the burden on health services and reduces productive labour. strategies for recruiting blood donors have to balance supply with demand, and yet ensure that the blood is as safe as possible. in general, the safest sources of blood are altruistic voluntary unpaid donors who should be anonymous to the recipient. only 32% of who member states report having at least 90% of their blood supply from voluntary donors, and developing countries have not shown any improvement in recruitment of voluntary donors for several years. 1 in countries without a national transfusion service, each hospital is responsible for fi nding its own donors and processing blood for transfusion. recruiting voluntary donors from the community is expensive and logistically complicated, requiring resources such as a local education programme, dedicated venesection team, vehicles and cold storage. paid donors or 'loan' systems, where family members are responsible for providing blood for their relatives in the hospital, are therefore widespread in poorer countries. cultural taboos and misinformation about donating blood (e.g. 'men will become impotent if they donate blood'; 'hiv can be caught from the blood bag needle') mean that relatives may be reluctant to donate. families are open to exploitation by 'professional donors' who charge a fee to donate in place of a family member. by the time a donor has been found, screened and venesected, and the blood is transfused into the patient, several hours or even days can elapse, especially if blood of a rare group is required. because patients in poorer countries often present late in the course of their disease, severely anaemic patients may die in hospital without ever receiving a blood transfusion. it is unfortunate that in many countries where the majority of transfusions are performed as an emergency and where it is imperative to have a well-stocked blood bank, the 'loan' system, with its inherent delays, predominates. potential 'high-risk' donors, such as commercial sex workers or those having frequent contact with these individuals, intravenous drug abusers, or persons with itinerant or fl uctuating activities such as traders, drivers and military personnel, should be permanently deferred from the donor pool. 4 even in areas where hiv infection rates in the general population are high, donor deferral can be effective in excluding hiv-infected donors. 5 the whole donation process, including tests for hiv and other infections, should be explained to the donor before blood is collected and donors should have the option of knowing the results and receiving counselling. it is imperative that complete confi dentiality is maintained throughout all procedures. infections with organisms that are common in tropical countries, such as hiv-1 and -2, hepatitis a, b, c and d, cytomegalovirus, syphilis, lyme borreliosis, malaria, babesiosis, american trypanosomiasis (chagas' disease) and toxoplasmosis, can all be acquired through blood transfusions. there have also been recent reports of transmission of variant creutzfeldt-jakob disease through blood transfusion and there is a theoretical risk of acquiring severe acute respiratory syndrome (sars) through transfusion of labile blood products. 6, 7 who recommends that all donated blood should be screened for hiv, hepatitis b and syphilis and, where feasible and appropriate, for hepatitis c, malaria and chagas' disease. between 5% and 10% of hiv infections worldwide are thought to have been transmitted through the transfusion of infected blood and blood products. hiv testing of blood donors needs to be highly sensitive, and blood which tests positive should be rejected. before informing the donor of the outcome, all positive results should be confi rmed using a test with a high degree of specifi city. where blood donation is organized locally, the confi rmatory test is often performed at a central laboratory, so there may be delay in informing the donor of the result. malaria can be transmitted by transfusion and has an incubation period of between 7 and 50 days, depending on the species. in areas of low or no malaria transmission, screening for the parasite is important, as recipients are likely to have no immunity. in countries with high malaria transmission, exclusion of parasitaemic donors could result in deferral rates exceeding 30% and consequently would have a major impact on blood supply. 8 it is unclear whether malaria screening is necessary in regions where the disease is common, particularly because most of the blood is given to hospitalized children with malaria who are likely to be receiving antimalarial drugs, or adults who are clinically immune. further research to assess the risks and benefi ts of screening blood for malaria is needed, particularly in relation to pregnant women and patients with hiv infection. screening for hepatitis b surface antigen should be carried out on all donated blood, as hepatitis b-infected blood is almost 100% infectious. fresh blood is potentially infectious for syphilis, but storage at 4°c can inactivate treponema pallidum. globally, the prevalence of hepatitis c, htlv-1 and -2 and chagas' disease is variable and the decision to introduce donor screening for these infections will be based on local assessments of the risks, benefi ts, feasibility and costs. blood should not be separated into components if the residual risk of infection is high, as this will increase the number of potentially infected recipients. in some wealthy countries nucleic acid amplifi cation techniques (nat) have been introduced to improve the safety of blood. although nat may not be cost-effective where infection prevalence is low, it has reduced the residual risk for hiv, hepatitis c virus (hcv) and hepatitis b virus (hbv) infection in germany to 1 in 5 540 000, 1 in 4 400 000 and 1 in 620 000, respectively. 9 blood is usually taken from donors and stored in a blood bank until screening tests for infections have been completed. this system has several drawbacks: potentially infected blood may be mixed up with units that have already been screened, and the whole process of venesection with wastage of blood collection bags is costly. pre-donation screening, by which potential donors are tested for hiv, hepatitis b and possibly hepatitis c at the site of donation before being venesected, may be a more cost-effective way of ensuring safe blood. 10 in wealthy countries the majority of transfusions are planned and carried out electively. by contrast, in poorer countries, and particularly those where the malaria transmission rate is high, most transfusions are given for life-threatening emergencies. in these countries 50-80% of transfusions are administered to children, predominantly for malaria-related anaemia. transfusion can signifi cantly reduce the mortality of children with severe anaemia but it may not have any benefi t unless it is given within the fi rst 2 days of hospital admission. 11 in areas of high hiv prevalence, young children have a relatively low risk of being infected with hiv and potentially have a long life expectancy. however, this is the age group that is predominantly affected by severe malariarelated anaemia and so they are particularly at risk of transfusionacquired hiv infection. 12 pregnant women are the second most common recipients of blood, particularly for haemorrhagic emergencies. 13 other specialities which are signifi cant users of blood are surgery, trauma and general medicine. whether a patient needs a blood transfusion or not is ultimately a clinical decision. emergency transfusions can be life-saving for patients in whom the anaemia has developed too quickly to allow physiological compensation. examples of such emergencies include severe malaria-related anaemia in children, and sudden, severe obstetric bleeding. in contrast, if the anaemia has developed slowly, for example due to hookworm infestation or nutritional defi ciency, patients can generally be managed conservatively by treating the cause of the anaemia and prescribing haematinic replacements. these should be continued for at least 3 months after the haemoglobin has returned to normal, so that body stores can be replenished. it is possible to avoid unnecessary transfusions through the use of clinical transfusion guidelines, and most institutions or organizations have developed guidelines to help clinicians make rational decisions about the use of blood transfusions (table 14. 3). 14 strict enforcement of a transfusion protocol in a malawian hospital reduced the number of transfusions by 75% without any adverse effect on the mortality rate. 15 while the details may vary, the principles underlying most transfusion guidelines are similar and combine a clinical assessment of whether the patient is developing complications of inadequate oxygenation, with measurement of their haemoglobin. the haemoglobin level is used as a surrogate measure for intracellular oxygen concentration. increasingly, transfusion guidelines are making use of evidence which shows that adequate oxygen delivery to the tissues can be achieved at haemoglobin levels that are signifi cantly lower than the normal range. 16 it is easier to develop guidelines than to ensure that they are used in routine practice. implementation of transfusion guidelines is particularly diffi cult if clinicians do not have confi dence in the quality of haemoglobin measurements. it has been shown that when doubtful of the quality of haemoglobin result, clinicians rely entirely on clinical judgement to guide transfusion practice. this may lead to signifi cant numbers of inappropriate transfusions. 17 in a typical district hospital in africa, the cost of providing a unit of blood through the family 'loan' system is approximately 30 times the cost of a quality-assured haemoglobin test. a lack of investment in assuring the quality of a basic but critical test such as haemoglobin measurement can result in a signifi cant waste of resources downstream in the transfusion process, with the additional unnecessary exposure of recipients to the risk of transfusion-related infections. in resource-poor countries, the recommended haemoglobin threshold for transfusions is often well below that which would be accepted in more wealthy countries. for example, american anaesthetists suggest that transfusions are almost always indicated when the haemoglobin level is less than 6 g/dl, 18 whereas in malawi transfusions are recommended for children with haemoglobin levels less than 4 g/dl, provided there are no other clinical complications. 19 complications such as cardiac failure or infection may necessitate transfusion at a higher haemoglobin level. transfusion should be combined with adequate iron and folate replacements and treatment of any underlying conditions that contribute to anaemia, so that a normal haemoglobin count can be achieved during the weeks following transfusion. any transfusion service must be able to guarantee the quality of haemoglobin results. these results are crucial in donor selection and are also used to guide the decision to transfuse patients. although it is the most commonly performed test, accurate haemoglobin estimation is diffi cult to achieve in laboratories without automated blood analysers. 20 the reference technique for haemoglobin measurement is the haemiglobincyanide method. not only does this method need a constant electricity source for the spectrophotometer, but also technicians need arithmetic expertise to calibrate the equipment and automatic pipettes for accurate measurements. in under-resourced countries, district hospitals may use simpler, cheaper and less accurate methods of haemoglobin measurement, many of which are based on visual colour comparisons. while a few individual laboratories in resource-poor countries may be registered with an external system to monitor the quality of laboratory tests, almost no country has a nationwide programme. this means that for many laboratories and their users, the quality of tests, including haemoglobin and those used for screening and determining blood groups, is unknown. complications can occur immediately during transfusion, within a few hours of its completion, or be delayed for many years, as in the case of viral infections. see table 14 .4. transfusion of blood into a recipient who possesses antibodies to the donor's red cells can cause an acute, and occasionally fatal, intravascular haemolysis. this could occur, for example, if group a cells are transfused into a group o recipient who has naturally occurring antibodies to group a cells. the profound haemolysis induces renal vasoconstriction and acute tubular necrosis. treatment involves stopping the transfusion, cardiorespiratory support and inducing a brisk diuresis. in addition to abnormalities indicating renal failure, laboratory fi ndings include haemoglobinuria and haemoglobinaemia. proof of the diagnosis involves rechecking the whole transfusion process including all documentation stages, regrouping the donor and the recipient, and screening for antibodies on red cells with a direct antiglobulin test. these tests are usually available in any hospital laboratory capable of providing a transfusion service. delayed haemolysis has a similar physiological basis to acute intravascular haemolysis but tends to be less severe. the antibody-antigen reaction develops 7-10 days after the transfusion and it is less likely than acute haemolysis to present as a clinical emergency. bacteria can enter the blood bag during venesection or if the bag is perforated at a later stage, perhaps to reduce the volume for a paediatric recipient or during component preparation. gramnegative bacteria, including pseudomonas and yersinia, grow optimally at refrigerator temperatures and infected blood may not necessarily appear abnormal. reactions following infusion of infected blood are often due to endotoxins and may occur several hours after the transfusion has fi nished. although these reactions are rare, they can be severe and fatal. if bacterial contamination is suspected, the transfusion should be stopped and samples from the patient and the blood bag sent to the laboratory for culture. cardiorespiratory support may be needed and broad-spectrum antibiotics should be started immediately and continued until culture results are available. these are episodes of fever (i.e. ≥1°c rise in temperature) and chills for which no other cause can be found. they are due to the recipient's antibodies reacting against antigens present on the donor's white cells or platelets. these reactions are most common in patients who have received multiple transfusions in the past and have therefore been exposed to a broad range of antigens. mild febrile reactions usually respond to simple antipyretics such as paracetamol. more severe reactions may be the fi rst indication of a haemolytic transfusion reaction or bacterial contamination and should be investigated and managed accordingly. these are due to infusion of plasma proteins and manifestations include erythema, rash, pruritus, bronchospasm and anaphylaxis. the transfusion should be stopped and the patient treated with antihistamines. if the reaction is mild and the symptoms and signs completely disappear, the transfusion can be restarted. if this type of mild reaction occurs repeatedly with more than one unit of blood, the red cells can be washed before transfusion. this should only be done if absolutely necessary, as it carries the risk of introducing potentially fatal bacterial infection. severe allergic reactions with evidence of systemic toxicity should be managed as acute anaphylaxis. blood should always be transfused slowly, to avoid overloading the circulation, unless the patient is actively and severely bleeding. overload may be a particular problem when paediatric blood bags are not available, as children may be over-transfused due to miscalculation of the required volume, lack of accurate infusion devices or inadvertent administration of an adult-sized unit of blood. in tropical practice, blood transmission of hepatitis b, hiv-1 and -2, and, in some areas, american trypanosomiasis (chagas' disease) is of particular concern. in general, transfusions are not the major route of transmission of these infections and they may not cause clinical problems until many months or years after the transfusion. four units of blood contain the equivalent of the amount of iron stored in the bone marrow (approximately 1 g). repeated transfusions for chronic haemolytic anaemia, as in thalassaemia major and sickle cell disease, lead to iron deposition in parenchymal cells. eventually, failure of the heart, liver and other organs supersedes. adequate doses of iron chelators, such as injectable desferrioxamine or the newer oral chelator, deferiprone, are able to maintain acceptable iron balance in patients with chronic anaemia receiving regular transfusions. it is not usually necessary to warm blood unless rapid transfusion of large quantities is needed. this may lower the temperature of the sino-atrial node to below 30°c, at which point ventricular fi brillation can occur. if blood needs to be warmed, an electric blood warmer specifi cally designed for the purpose should be used. this keeps the temperature below 38°c, thereby avoiding the haemolysis associated with overheating blood. graft-versus-host disease occurs when donor lymphocytes engraft in an immune-suppressed recipient. the lymphocytes recognize the recipient's bone marrow as foreign and induce aplasia. graftversus-host disease is almost universally fatal and can be prevented by irradiating the donor blood, which inactivates the donor lymphocytes. where blood is in short supply, it is particularly important to ensure that the best anaesthetic and surgical techniques are used, to minimize blood loss during surgery. drugs which improve haemostasis or reduce fi brinolysis, such as aprotinin and cyklokapron, and fi brin sealants, can be effective in reducing perioperative blood loss and hence the need for blood transfusion. cost is a major limiting factor to the use of these therapies in poorer countries, and surgical blood loss is generally not a major contributor to the overall transfusion needs. patients undergoing planned surgery who are likely to require a blood transfusion can have units of their own blood removed and stored prior to surgery for use by themselves only, if signifi cant intraoperative blood loss is anticipated. preoperative autologous donation can reduce the need for allogeneic transfusions by 46-74% 21 but it requires careful organization: the surgeon needs to predict how much blood will be required, the patient has to be fi t enough to withstand removal of one or more units of blood over the weeks preceding the surgery (preoperative haemoglobin will drop by about 1 g/dl), and the surgery must take place within the shelf-life of the blood. as the blood has to be stored in the blood bank there is still a risk that the patient may receive blood which is not his or her own or that the blood may become infected with bacteria during the process. this involves collecting blood lost during the operation and reinfusing it into the patient either during or after surgery. although this technique is practical and safe, and reduces the need for donor blood by 27-53%, 21 it requires specialized equipment and training and may be more expensive than routinely donated blood. 22 normal saline or intravenous replacement fl uids can be used judiciously in acute blood loss, and in certain circumstances may be as effective as whole blood, red cells or plasma. erythropoietin, which stimulates endogenous red cell production, has wellestablished uses in chronic anaemias such as those due to renal failure, cancer and hiv infection. its delayed action makes it unsuitable for use in acute anaemias, the major reason for transfusions in poorer countries. the development of synthetic oxygen carriers, generally perfl uorocarbons, has been fraught with problems and they are not routinely available. 23 in under-resourced countries, especially those with a heavy burden of malaria, the most effective way to avoid the need for transfusions is to reduce the prevalence of anaemia in the community. more studies on the ability and cost of combined interventions such as the provision of bed nets, nutritional supplements and anthelmintic drugs to children to prevent anaemia and reduce transfusion requirements are needed. when resources are very limited, governments may need to make some diffi cult decisions in order to achieve an equitable balance between investing in a transfusion service and public health measures to reduce anaemia. laboratory costs of a hospitalbased blood transfusion service in malawi better blood transfusion safe blood in developing countries excluding blood donors at high risk of hiv infection in a west african city possible transmission of variant creutzfeldt-jakob disease by blood transfusion world health organization. who recommendations on sars and blood safety the risk of malaria transmission by blood transfusion at cotonou human immunodefi ciency virus, hepatitis c and hepatitis b infections among blood donors in germany 2000-2002: risk of virus transmission and the impact of nucleic acid amplifi cation testing predonation screening of blood donors with rapid tests: implementation and effi cacy of a novel approach to blood safety in resource-poor settings effect of blood transfusion on survival among children in a kenyan hospital trends and risk factors of hiv-1 seropositivity among outpatient children anaemia, blood transfusion practices, hiv and mortality among women of reproductive age in western kenya world health organization. blood safety . . . for too few. press release who/25 whd/3 information sheet for clinicians prevention of transfusion-associated hiv transmissions with the use of a transfusion protocol for under 5s electrocardiographic st-segment changes during acute, severe isovolemic hemodilution in humans use of clinical judgement to guide administration of blood transfusions in malawi american society of anesthesiologists task force. practice guidelines for blood component therapy ministry of health and population, malawi. aids control programme. recommended guidelines for the practice of safe blood transfusion in malawi evaluation and costs of different haemoglobin methods for use in district hospitals in malawi autologous transfusion techniques: a systematic review of their effi cacy intra-operative autologous blood management artifi cial o2 carriers: status in 2005 key: cord-022474-xxy83c6u authors: tenorio, grace c.; gupte, snehalata c.; munker, reinhold title: transfusion medicine and immunohematology date: 2007 journal: modern hematology doi: 10.1007/978-1-59745-149-9_22 sha: doc_id: 22474 cord_uid: xxy83c6u blood transfusion is essential and vital in the successful treatment of many malignant and nonmalignant hematological disorders. children with thalassemia, adults with myelodysplastic syndromes, and patients with autoimmune hemolytic anemias, leukemias, or aplastic anemias become chronically dependent on blood transfusions. modern treatment procedures such as high-dose chemotherapy and progenitor cell transplantation require intensive support with blood components and products. the serological basis of blood transfusion, the available blood components and products, and adverse effects of blood transfusion with special emphasis on infectious disease transmission are discussed in this chapter. blood transfusion is essential and vital in the successful treatment of many malignant and nonmalignant hematological disorders. children with thalassemia, adults with myelodysplastic syndromes, and patients with autoimmune hemolytic anemias, leukemias, or aplastic anemias become chronically dependent on blood transfusions. modern treatment procedures such as high-dose chemotherapy and progenitor cell transplantation require intensive support with blood components and products. the serological basis of blood transfusion, the available blood components and products, and adverse effects of blood transfusion with special emphasis on infectious disease transmission are discussed in this chapter. genes for three different blood group systems (abo, hh, and sese) indirectly control the expression of the a, b, and o antigens because antigenic activity is determined by sugars linked to either polypeptides (forming glycoproteins) or lipids (forming glycolipids). each of the a, b, and h genes code for a specific enzyme (glycosyltransferase) that adds a different sugar on a polypeptide or lipid to form the abh antigens. the abo system is the most important system in red cell serology and involves three allelic genes (a, b, and o) in chromosome a. the a and b genes encode glycosyltransferases (enzymes) that produce the a and b antigens, respectively. the o gene is considered to be nonfunctional because it determines no detectable blood group antigen. the expression of the o gene results in loss of production of a functional protein or enzyme; consequently, no product is formed. the red cells of a group o individual lack a and b antigens, but have an abundant amount of h antigen. the precursor h enzyme (fucosyltransferase) specifically adds fucose to a terminal galactose, thus giving h antigenic expression. if the glycosyltransferase adds n-acetyl-d-galactosamine to the terminal d-galactose of an h antigen, then the red cells will have the a antigen on their surface. correspondingly, if the glycosyltransferase adds d-galactose, a b antigen is formed. abo specificity is dependent on both abo and hh genes. table 1 shows the incidence of the four main phenotypes of the abo system. the a blood group has two main subgroups: a 1 and a 2 , which could be distinguished using the dolichos biflorus lectin reagent. a 1 individuals have more a antigen sites than a 2 individuals. the a 2 gene differs from the a 1 gene by one base pair. variability in genes causes variable reactivity as well add after "as well." subgroups of b are very rare and less frequent than a subgroups. in the abo system, naturally occurring antibodies (isoagglutins) are present against a or b antigens. individuals with the blood group a have isoagglutinins against b red cells and vice versa (see table 1 ). these antibodies invariably are immunoglobulin (ig)m that activate complement and cause immediate intravascular hemolysis resulting in severe acute hemolytic transfusion reactions (htr). they are absent at birth and develop within 3-6 mo of age, when the immune system is exposed to abh antigenic determinants present in our environment (i.e., bacteria, plants, dust, and food). antibodies are naturally developed against the abh antigens absent in the individual. generally, the antibody titer increases until the age of 10 yr and progressively falls with increasing age in adults. in acquired immunodeficiency states (e.g., leukemias and lymphomas) the levels may be significantly low. the abh antigens present in red cells have been demonstrated in most tissues of the body, including platelets and leukocytes. the ability to secrete soluble abh antigens is controlled by the secretor (se) gene that is separate from the abh system. about 80% of the population have the dominant secretor (se) gene that controls one's ability to secrete soluble abh antigens. these individuals (secretors) distinctly have soluble abh substances in their plasma and secretions (i.e., saliva, semen, and sweat). the rhesus (rh) system is the second most clinically important and complex blood group system. it consists of some 50 different antigens, but only 5 antigens-d, c, c, e, and e-are inherited in various combinations and account for most of the rh-related problems encountered in practice. the rh antigen with the strongest antigenicity is the rh (d) antigen. as a simple rule, it can be noted that persons whose red cells express the d antigen are rh (d) positive and individuals whose red cells lack the d antigen are rh (d) negative. the different genotypes, their rh status, and the frequency of these genotypes in caucasians are shown in table 2 . about 85% of north american caucasians are rh (d) positive. after the discovery of the rh system in 1940, various theories were postulated to explain the mode of inheritance and different nomenclatures were proposed. the wiener system proposed that the gene product was a single entity with multiple serological specificities. the fisher-race system postulated three sets of closely linked genes and gene products (c and c, d and d, and e and e) . rosenfield proposed a third nomenclature system based on serological reactions, which assigns a number for each rh antigen. the world health organization in genomic studies have revealed the presence of two closely linked genes (rhd and rhce) with considerable homology that refutes both wiener and fisher-race postulates. the rhd gene controls the production of the d antigen and is absent in rh (d)-negative individuals and explains the absence of the "d" antigen. the rhce gene encodes for cc and ee antigens. the d and ce polypeptides differ in only 36 amino acids, the c and c polypeptides differ in four amino acids, and e and e differ in only one amino acid. the approximate molecular weight of a nonglycosylated rh protein is 30 kda. recently, a d protein (a mixture of rh [c] and [e]) has been isolated from rh (d)-negative red cells that differ from the d protein of rh (d)-positive cells. genetic polymorphism may account for the difference. individuals whose red cells give weaker reactions with anti-d reagents are classified as quantitative weak d (red cells that require additional steps to demonstrate d were formerly classified as d variant or d u ). the d antigen has more than 37 epitopes, and if a significant number of epitopes are absent, then the individual is known to have partial d antigen (formerly classified "d mosaic" or "d variant") and can produce an antibody to the portion of the d antigen they lack. partial d phenotypes arise from nucleotide interchange between the rhce and the rhd genes or from single mutations. gene interaction also depends on the position of the genes that ultimately affect the expression of the d antigen. a weak d antigen can also result from the suppressive effect of c in trans position to a d on the opposite chromosome exemplified by cde/cde. a weak d individual because of gene interaction has the entire d antigen and can receive dpositive blood. in contrast, weak d individuals who have partial absence of the d should only be transfused with d-negative blood because they can produce anti-d antibodies. the american association of blood banks (aabb) requires that blood donors be screened for weak expression of the d antigen and to be labeled as rh (d)-positive if the test is positive; however, recipients need not be tested for weak d. on very rare occasions, red cells may lack the expected rh antigens (e.g., d de, cd ). in rh-null individuals, the rh antigens are completely absent. this can arise from the absence of the gene that regulates rh antigen expression or the presence of an amorphic gene at the rh locus. rh-null individuals have a compensated hemolytic anemia and abnormal red cell morphology (stomatocytosis). if transfused, they will produce antibodies against the different rh antigens; therefore, rh null individuals should be transfused only with rh-null cells from the rare donor registry or with autologous red cells. rh antibodies can be acquired during pregnancy or a blood transfusion. the most common rh antibody is anti-d. rhesus immunization during pregnancy or delivery may occur when an rh (d)-negative woman has an rh (d)-positive child. this can be prevented by the prophylactic injection of anti-d igs (rhig). rh antibodies are predominantly igg and react at 37 c. they do not fix complement effectively, but can cause hemolytic disease of newborn (hdn; see chapter 6) and hemolytic transfusion reaction (htr). extravascular hemolysis occurs through the mononuclear phagocyte system. red cells bear antigens of many other blood group systems besides the abo and rh systems (kell, duffy, lewis, i, p, mn, lutheran, kidd, and others). these red cell antigens are not routinely typed and generally are rare causes of hdn. the kell and duffy systems are briefly discussed. for more comprehensive information on the other red cell antigen systems, the reader is referred to reference texts. (see "suggested reading"). the kell blood group system is clinically important, as the k antigen follows the d antigen in immunogenicity and its antibodies can cause hdn and htr. currently, this system includes 24 alloantigens, the most common being the k, k, kp(a), kp(b), js(a), and js(b) antigens. a defective and weak expression of kell antigens (also lack kx) is observed in individuals with the mcleod phenotype. these individuals have a chronic compensated hemolytic anemia and abnormal red cell morphology (acanthocytosis). individuals with mcleod red cells also have neuromuscular and cardiovascular abnormalities (myopathy, areflexia, and cardiomyopathy). rarely, the mcleod phenotype is associated with chronic granulomatous disease and arises from the deletion of the x chromosome that includes both xk and x-cgd loci. the duffy system is unusual in that the antigen frequency varies in different racial groups. the duffy glycoprotein is the receptor for the malarial parasite and serves as an erythrocyte receptor for a number of cytokines, notably interleukin (il)-8. duffy glycoproteins also serve as a sponge for excess chemokines without any adverse effect on the red cell. this system has six antigens; two of these are important and deserve mention. both fy a and fy b antigens have low incidence in africans. in west africa, most probably by natural selection, both antigens are absent in the majority of blacks [fy (a b )]. their red cells exhibit resistance to infection by plasmodium vivax and p. knowlesi. anti-fy a antibody may cause mild hdn and rare but severe htr. infrequently, anti-fy b is associated with either hdn or htr; other antibodies of this system have not been implicated at all. prior to any blood transfusion, the red cell abo and rh(d) blood group (blood type) of the recipient is determined, and the serum is screened for any unexpected red cell antibodies (usually igm and igg antibodies). thereafter, a cross-match is carried out between the donor's red cells and the recipient's serum. blood group antigens or antibodies are determined with agglutination methods. the igm antibodies (i.e., anti-a or anti-b) are usually detected by saline techniques, whereas enzyme, albumin, or antiglobulin methods are employed for the igg antibody detection. low ionic strength solution (liss) is widely used in blood group serology as it shortens the incubation period and is helpful with emergency blood requests. the antiglobulin (coombs') test detects antibodies coated on the red cells. the direct antiglobulin test (dat) detects antibodies that are already bound to red cells in vivo, whereas the indirect antiglobulin test (iat) detects antibodies present in the serum. the direct antiglobulin test may be positive in: (1) autoimmune hemolytic anemias (seen in lymphomas, system lupus erythematosus, cold agglutinin syndrome, and paroxysmal cold hemoglobinuria); (2) alloimmune hemolytic anemias (hdn and htr); and (3) drug-induced hemolytic anemia. figure 22 .1 schematically outlines the determination of the abo blood group with agglutination methods, whereas fig. 22 .2 shows the principles of both dat and iat. human leukocytes bear two types of surface antigens: the human tissue or cell-specific antigens and individual type-specific antigens. the first group is described in the cluster designation (cd) nomenclature. these antigens characterize the lineage, function, or activation state of the individual type of leukocyte (e.g., cd3 for mature t-cells). a list of the most current cd markers is given in appendix 2. the second group, the family of human leukocyte antigens (hlas) (class i, ii, and iii antigens), is encoded by the major histocompatibility complex (mhc) genes on the short arm of chromosome 6. class i and ii antigens are the classic transplantation antigens that define tissue tolerance or rejection and are important for organ transplantation, but their primary role is in immune response regulation. class iii antigens (i.e., complement c2 and c4 or tumor necrosis factor [tnf]-) may be directly or indirectly involved with mhc function. the hla system is expressed on many tissues. the class i antigens (hla-a, -b, and -c molecules) are present on all nucleated cells and platelets. class ii antigens (hla-d molecules) are expressed on b-lymphocytes, antigen-presenting cells (monocytes, macrophages, and dendritic and langerhans cells), and activated t-lymphocytes. hla class i and class ii antigens differ in immunological function. class i antigens interact with cd8 + lymphocytes, which recognize endogenous antigens. class ii molecules on the surface of antigen-presenting cells bring exogenous antigens in contact with cd4 + lymphocytes. class i molecules consist of two glycosylated heavy chains of 44-45 kda and a noncovalently bound 12 kda molecule ( 2 -microglobulin). the class i heavy chain has three extracellular domains, a transmembrane region, and an intracytoplasmic domain. each of the class ii molecules (hla-dr, dq, and dp) consists of two transmembrane noncovalently associated glycosylated polypeptide chains. the -chain has a molecular weight of about 30-34 kda; the -chain has a molecular weight of about 26-29 kda. the inheritance of the hla genes is closely linked, and the entire mhc is inherited as an hla haplotype (half of the genotype) in a mendelian fashion from each parent. for example, a haplotype of a3, b7, cw7, and dr2 may come from the father and the other haplotype of a9, b27, cw1, and dr7 may come from the mother. distances between loci may permit some chance of recombination within the hla system, but this occurs only infrequently (<1%) and usually between dp and dq loci. statistically, siblings have a 25% chance of inheriting the same pairs of hla molecules from the parents (i.e., being hla identical). the hla system is the most polymorphic human antigen system, thereby showing an enormous number of different hla haplotype combinations. the hla antigen pattern varies among different ethnic groups. because of linkage disequilibrium, some haplotypes occur more frequently in certain populations. for example, hla-a1, b8, dr3 is the most common hla haplotype among caucasians with a 5% frequency. the hla complex is divided into three regions indicating the locations of loci. serological and dna techniques differ in the number of alleles that they can identify for each locus. hla-a, hla-c, and hla-b loci have 20, 8, and 30 serologically defined alleles but have 309, 167, and 563 alleles detected by dna analysis. among the class ii antigens, the hla-dr, hla-dp, and hla-dq molecules expressed on the cell membranes are most important with 442, 81, and 127 alleles defined by dna techniques. the allelic variations of dq-a, dp-a, and dp-b can only be defined by dna methods. two hla nomenclatures are currently in use. the older list of specificities is based on detection of epitopes by immunological techniques (serology or mixed leukocyte culture reactions) and the newer molecular nomenclature is based on specific nucleotide sequences of alleles using dna-based methods, now a commonly used technique in hla-typing laboratories. peripheral blood lymphocytes express class i antigens and are used for the serological typing of hla-a, hla-b, and hla-c. class ii antigens are typed using b-lymphocytes. classic serology uses lymphocyte microcytotoxicity tests (by terasaki) utilizing sera from multiparous women who have been immunized against certain hla antigens. clinical molecular techniques have revealed the complexities of both class i and ii antigens. for example, the identities of class ii antigens can be shown by the mixed lymphocyte reaction but hla-d identical pairs remain nonreactive using this method. however, molecular typing is able to distinguish serologically indistinguishable but functionally discrete hla alleles. all current dna-based hla typing assays utilize pcr to amplify the genes of interest. there are three commonly used procedures: sequence-specific primers (pcr-ssp), sequence specific oligonucleotide probes (pcr-ssop), and sequence-based typing (pcr-sbt). dna typing is specific (no batch-to-batch variation in specificity) and flexible (new reagents can be designed as new alleles or new nucleotide sequences are identified). it is highly reproducible (with ssop) and more robust than other techniques because it does not require viable lymphocytes nor is it influenced by the patient's health. dna based methods have the added advantage of hla-typing large numbers of volunteers for donor registries. furthermore, it can detect the full range of hla diversity. hla alleles can specify the hla proteins that are indistinguishable by serology. for example, drb1*0401 and drb1*0412 are allele splits identified by dna typing that belong to the broad specificity dr4 serological type. in addition to the major histocompatibility antigens just described, minor histocompatibility antigens (mhags) have been defined by both class i and ii mhc-restricted t-cells and may affect the outcome of progenitor stem cell and solid organ transplantation. the mhags are immunogenetic peptides bound to class i molecules that stimulate t-cell responses. they are also inherited and the number of minor histocompatibility loci is probably high. to date, the range of polymorphism of these antigens is not well characterized. the disparity of mhags can be associated with graft-vs-host disease (gvhd) in hla-identical transplants (i.e., h-y antigen in a male recipient and a female donor immunized by pregnancy). the frequency of allelic forms, immunogenicity of peptides and tissue-specific expression of proteins will determine the role of mhag disparity in either gvhd or graft rejection. hla testing is used in progenitor stem cell and organ transplantation, disease susceptibility studies, and parentage testing. hla identity is the sine qua non of allogeneic bone marrow transplantation. despite full hla-identical progenitor cell grafts, a substantial number of patients develop graft-vs-host mhag reactivity (details about allogeneic progenitor stem cell and bone marrow transplantation are described in chapter 4). the hla antigens are also important for solid organ transplantation (i.e., kidney or liver). hla-a, hla-b, and hla-dr are considered to be the major transplantation antigens, whereas hla-c, hla-dp, and hla-dq are generally of minor importance. in kidney transplantation, hla matching between donor and recipient is done routinely. hla-matched kidney grafts have better outcomes than unmatched grafts. in contrast to bone marrow transplantation, solid organ transplantation requires abo-compatible grafts. for logistic reasons, other solid organs (heart, liver, lung, and pancreas) are not routinely matched for hla-antigens. hla antibodies play a major role in graft survival and chronic rejection. the presence of cytotoxic hla antibodies in the serum of transplant recipients reactive against a panel of cells (expressed as panel reactive antibodies or [pra]) lowers the graft survival rates and may be a contraindication for kidney transplantation. the pra effect is greater among recipients of a second transplant. multiparous women and patients who receive multiple blood transfusions are frequently alloimmunized to hla antigens. these hla antibodies are broadly reactive. post-transfusion hla alloimmunization is variable and dependent on the patient's diagnosis and therapy. patients with leukemia have lower detectable antibodies (25 to 30%) than patients with aplastic anemia (80%) because they are usually immunosuppressed from intensive chemotherapy when the transfusions are given. leukocyte reduction of blood components to less than 5 10 6 has significantly reduced the development of primary hla alloimmunization. this can be achieved by the use of third-generation leukocyte filters for red blood cells (rbcs) and platelets or by inline leukoreduction systems of blood cell separators used to collect pheresis blood components. as will be discussed later in the chapter, hla antibodies have been implicated in febrile nonhemolytic transfusion reactions and transfusion-related acute lung injury. immunological refractoriness to platelet transfusions results from immune destruction of transfused platelets more often by hla antibodies (class i antigens are expressed on platelets) than by platelet-specific antibodies. nonimmune causes of platelet refractoriness need to be ruled out such as splenomegaly, disseminated intravascular coagulation (dic), bleeding, infection, marrow transplantation, and antibiotics (amphotericin b, vancomycin, ciprofloxacin). detection of hla-or platelet-specific antibodies is usually done by solid phase red cell adherence assay (sprca), flow cytometry, elisa, monoclonal antibody specific immobilization of platelet antigen assay, or mixed passive hemagglutination assay. once immune refractoriness is established, special platelet products are indicated. these patients can receive hla-matched platelets, or platelet crossmatching can be done using sprca or flow cytometry. both methods of crossmatching will detect platelet antibodies against class i and platelet-specific antigens. the efficacy of crossmatched platelets may be as good as hla-matched platelets. platelet-crossmatched units have the additional advantage of being readily available for transfusion. crossmatching is not always practical because alloimmunized patients may have hla antibodies that react to more than 90% of the random population. these patients may also have few broadly reactive antibodies against public epitopes of class i molecules, which will make it harder for a blood center to provide a product because the best hla-matched platelet unit can still have some incompatibility. numerous diseases have a more or less strong association with certain hla antigens. well-known examples are ankylosing spondylitis associated with hla-b27, narcolepsy associated with hla-dr2, hemochromatosis associated with hla-a3, celiac disease with hla-dqb1*02, and type i diabetes mellitus with dr-3 and -4 heterozygotes. the hla-a1, b8, dr3 haplotype is frequently involved in autoimmune disorders. these disease associations indicate the central role of the major histocompatibility complex in determining the susceptibility to disease and immune responsiveness. the hla system is used in parentage testing because of its polymorphism with a low recombination rate and mendelian inheritance. there is a decreasing use of hla typing because it does not provide a high exclusion probability when a case involves a paternal haplotype common in one particular ethnic group. thus, molecular techniques using non-hla genetic systems are widely favored. the use of whole blood is limited and is indicated in massive blood loss to replace the loss of both rbc mass and blood volume. many trauma centers have abandoned the transfusion of whole blood in favor of intravenous solutions in conjunction with rbcs and other blood components. currently, whole blood units serve as source material for blood components and plasma products. rbcs are indicated for replacement of red cell mass in patients who require increased oxygen-carrying capacity to prevent tissue hypoxia. the hemoglobin or hematocrit value, at which a transfusion is given, depends on the clinical circumstances. current information supports a "restrictive strategy." transfusions are indicated when hemoglobin concentration falls below 7 g/dl. the hemoglobin concentration should be maintained between 7 and 9 g/dl. note: in younger patients, it may be necessary to transfuse if the hemoglobin concentration drops below 6 g/dl. in elderly anemic patients with cardiovascular disease (acute myocardial infarction and unstable ischemic syndromes), the threshold for transfusion may be 9 or 10 g/dl. to avoid volume overload, transfusions should be given slowly. in recent years, the recommended hemoglobin value for transfusion during surgery has been lowered from 10 to 7 g/dl. in a typical 70-kg (155-lb) patient, each unit of transfused rbcs is expected to raise the hemoglobin 1 to 1.5 g/dl and the hematocrit by 3 to 5%. two types of platelet components are available for transfusion: "pheresed platelets," derived from single donors using an automated cell separator, and "pooled platelets," derived from whole blood donation and multiple donors. automated cell separators effectively collect platelets (3 to 4 10 11 /u) from donors. the major goal of prescribing platelet transfusions is to effectively and safely prevent and/or treat bleeding in thrombocytopenic patients. platelets may be given prophylactically in severely thrombocytopenic patients who have a hemorrhagic tendency or to patients on intensive myelosuppressive chemotherapy to keep the platelet count above 10 10 9 /l. the success of modern chemotherapy in patients with hematological disorders (i.e., acute leukemias and myelodysplastic syndromes) and progenitor cell transplantation (bone marrow or stem cell transplantation) is largely dependent on effective platelet transfusions. platelet counts for those at risk for spontaneous bleeding (patients with fever, infection, impaired platelet function from drugs, or hepatic or renal failure) should be kept above 15 to 20 10 9 /l. platelets are also indicated in other thrombocytopenic states: consumption coagulopathy, massive transfusion, gvhd, von willebrand disease, and congenital and acquired platelet defects. invasive procedures (i.e., lumbar puncture and liver biopsy) can be performed safely when the platelet count is at least 50 10 9 /l. counts of 100 10 9 /l should be maintained if excessive bleeding cannot be tolerated (i.e., cns or retinal procedures). in autoimmune thrombocytopenia, hypertransfusions of platelets are only indicated in cases of major hemorrhage. platelets are not useful in most other instances, as the autoantibody shortens the survival of both transfused and patient's own platelets. to investigate the mechanism of a poor response to a platelet transfusion, a platelet count is usually obtained within 1 h of the transfusion and the corrected count increment (cci) or percent recovery is calculated. patients who are refractory to platelet transfusions (cci < 7.5 10 9 /l or percent recovery of <15 or 20%) may have a better response if transfused with a sufficient dose of apheresis platelets. for those who become immunologically refractory, hla-matched or crossmatched compatible platelets may offer satisfactory results. however, patients refractory to all available platelet concentrates may benefit from intravenous immunoglobulin (ivig), plasma exchange, massive abo-identical platelet transfusions, and acid-treated platelets (stripped of hla antigens). platelets are stored at 20-22 c (room temperature) with agitation for no more than 5 d. more recently (april 2005), the food and drug administration (fda) extended the shelf-life of apheresis platelets to 7 d only if collected by the trima â® cobe spectraâ�¢ blood separator in conjunction with 100% testing by bacterial culture system, biomerieux bactt/alert microbial detection system release test â® , to monitor for any bacterial contamination. current blood separators can collect granulocytes at high yields (20 to 30 10 9 granulocytes) from donors stimulated with recombinant granulocyte colonystimulating factor (g-csf) and steroids; however, granulocyte transfusions lost popularity between 1985 and 1995 because of reported adverse pulmonary reactions and marginal clinical results. the unimpressive clinical efficacy may be attributed to rapid postcollection neutrophil apoptosis and inadequate doses (because previous donors did not undergo any stimulation/mobilization). renewed interest in granulocyte transfusion stems from the availability of g-csf that not only increases the yield of granulocyte collections but also inhibits neutrophil apoptosis. currently, granulocyte transfusion has limited indications that include refractory fungal or bacterial infections in neutropenic patients and those with qualitative neutrophil defects. granulocyte transfusions may also be beneficial to newborns with sepsis, neutrophil counts of less than 3000/ l or a defective marrow response. granulocytes have no defined regulatory specifications because the fda does not license them. however, the american association of blood banks (aabb) requires that each unit contain at least 1.0 10 10 granulocytes (note: value intended as a goal for adequate collection but not as an adequate clinical dose). at least four consecutive transfusions of 1.0 10 10 granulocytes are recommended. the use of additional transfusions is based on the patient's response or clinical course and the clinician's judgment. granulocyte units are suspended in 200 to 400 ml plasma and contain significant numbers of platelets (1 to 6 10 11 platelets, equivalent to an apheresis platelet unit), rbcs, and viable lymphocytes. thus, they must be abo and rh compatible with the recipient and irradiated to prevent transfusion-associated (ta)-gvhd. granulocytes have to be transfused as soon as possible and, if necessary, stored without agitation at 20-24 c for no more than 24 h. fresh-frozen plasma (ffp) is a single-donor unit of plasma that has been separated from one unit of whole blood or collected by apheresis and frozen at -18 c or lower within 6 to 8 h of collection. it contains coagulation factor levels at 1 u/ml (or 100% activity) and is indicated to treat global or multiple coagulation factor deficiencies (i.e., liver failure and dilutional coagulopathy in massively transfused patients). other uses for ffp include emergent reversal of warfarin therapy (when time does not allow the use of vitamin k) and plasma exchanges in thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. the timing and dose are important. correction of a markedly abnormal prothrombin time and activated partial thromboplastin time requires ffp transfusions immediately before surgery because several of the coagulation factors have very short half-lives (particularly factor vii with a biological half-life of 3 to 6 h). the dose is based on the patient's weight at 10 to 20 ml/kg. ffp contains abo antibodies and therefore must be compatible with the recipient's red cells. cryoprecipitated antihemophilic factor (cryo) is the cold insoluble portion of plasma processed from ffp by cold precipitation. the precipitate is formed as ffp is thawed at 1 to 6 c. the stability of the coagulation factors is maintained for up to 1 yr at -18 c storage. it is a plasma-derived product that contains the highest concentration of fibrinogen (250 mg, primarily used in acquired hypofibrinogenemia of dic), factor viii (80-120 u/concentrate), and a variable percentage of the original plasma concentration of von willebrand factor (40-70%) and factor xiii (30%). it is indicated for the treatment of hemorrhagic disorders resulting from either quantitative or qualitative defects of these factors. however, the availability of factor concentrates and recombinant factors have contributed to its diminished usage. cryo is also used as a source of fibrinogen to form a fibrin glue or sealant (when added to thrombin) and is also indicated to ameliorate the platelet dysfunction in uremia. like ffp, it contains abo antibodies, requiring consideration of recipient red cell compatibility. parenteral igs are manufactured from pooled human plasma and are used in primary or acquired hypogammaglobulinemia to protect against viral and bacterial infections. they consist of all subclasses and allotypes of igg with only trace amounts of igm and iga. polyvalent/multispecific immunoglobulins offer protection against many infections, whereas hyperimmune/specific immunoglobulins are obtained from actively immunized donors and contain high titers of disease-specific antibodies (i.e., hepatitis b virus [hbv]), varicella zoster, rabies, or cell-specific [rhig] ). rigorous donor screening and modified cohn fractionation have made immunoglobulin preparations relatively safe. immunoglobulins can either be administered intravenously (ivig) or intramuscularly (immune serum globulins) and are generally well tolerated. adverse effects are rare (<1%) and include headache, nausea, vomiting, chills, volume overload, and allergic and pulmonary reactions that can be prevented by slow infusion and pretreating with diphenhydramine and/or hydrocortisone. true anaphylactic reactions are very rare and are seen in common variable immune deficiency and iga deficiency. iga deficient products are recommended in these cases. polyvalent immunoglobulins are indicated in hypogammaglobulinemias (congenital or acquired) and for immunomodulation in certain disorders. note: the plasma half-life of igg (the major human immune globulin) is 21-23 d, but longer in hypogammaglobulinemia. in hypogammaglobulinemia, a dose of 0.4-0.6 g/kg body weight is given every month to maintain serum igg levels. in acquired hypogammaglobulinemia (e.g., multiple myeloma), immunoglobulins are recommended in patients with frequent infections. established indications for immunoglobulin replacement or prophylaxis are parvovirus b19 infections (among immunosuppressed patients) and recurrent life-threatening infections (among hiv-infected children). the use of prophylactic ivig in adults with advanced hiv infection has shown equivocal results. high-dose polyvalent immunoglobulins achieve immunomodulatory effects through multiple mechanisms: (1) by suppressing antibody production directly through its effect on b-lymphocytes, (2) by interfering in the interaction between autoantibodies and cellular targets through its anti-idiotypic antibodies, and (3) by blocking the fc-receptors on macrophages. higher doses of ivig (e.g., 0.4 g/ kg 5 d) are recommended for immunomodulation in kawasaki syndrome (a mucocutaneous lymph node syndrome in children) and idiopathic thrombocytopenic purpura. acute hemolytic transfusion reactions (ahtr) involves rapid destruction of blood cells immediately or within 24 h of a transfusion, commonly of rbcs or whole blood. it has a low rate of occurrence but is the most dangerous complication with a high mortality. mortality depends on the amount of incompatible blood infused (25 to 44 % with infusion of less than or more than 1000 ml, respectively). abo incompatibility is the most common cause of immune ahtr and accounts for 74% of all fatal reactions. ahtr is due to preformed antibodies, often igm (commonly anti-a), that bind complement and cause red cell lysis. nonimmune causes of hemolysis mimic immune mediated hemolysis and have to be excluded. chemical, mechanical, thermal, and physical damage of red cells may result from bacterial contamination, mechanical trauma during infusion, thermal hemolysis (from overheating or freezing a unit), and osmotic hemolysis (use of hypotonic reconstituting solutions and co-administration of drugs during transfusions) the patients commonly experience fever and chills, hypotension, and chest pain. other signs and symptoms include low back pain, flushing, dyspnea, hemoglobinuria, gastrointestinal symptoms (abdominal pain, diarrhea, and vomiting), and unexpected bleeding from dic. in patients under anesthesia, no immediate reactions may be recognized. changes in blood pressure, diffuse bleeding, and hemoglobinuria may be the only signs. ahtr may cause oliguria and acute renal failure. the classic laboratory changes include a drop in hemoglobin and hematocrit, hemoglobinemia (free hemoglobin can be demonstrated in serum or plasma), hemoglobinuria, reduced serum haptoglobin, and positive dat. elevations of unconjugated bilirubin, methemalbumin, and lactate dehydrogenase, and an unexpected red cell antibody may be present. the signs and symptoms of an igm-type acute hemolysis result from complement-mediated lysis of red cells and the release of cytokines. binding of complement to the red cell surface is a major factor in cytokine production. the symptoms of igg-type hemolysis also involve several cytokines (il-1, il-6, il-8, and tnf). coagulopathy, frequently seen in hemolytic transfusion reactions, especially due to igm antibodies, results from several mechanisms. the coagulation cascade is activated by (1) antigen-antibody complexes (activates hageman factor); (2) thromboplastic substances of the red cell stroma; (3) platelet factor 3 released by activated platelets; and (4) tissue factor release secondary to hypotension. in order to minimize ahtrs, all transfusions should have clearly defined indications. if an ahtr is suspected, the transfusion must be stopped and immediate steps taken to confirm or exclude this possibility. management of ahtr is dependent on the clinical status of the patient and may include cardiopulmonary support, prevention of renal failure (i.e., fluid resuscitation, vasopressors, and diuretics), and treatment of dic. severe bleeding in dic requires platelets, replacement of procoagulant factors and fibrinogen (ffp and cryo transfu-sions) , and administration of low-dose heparin. heparin limits both hemolysis (by its direct anticomplement activity) and inappropriate activation of procoagulant activity (by enhancing antithrombin iii-neutralizing serine proteases). delayed hemolytic transfusion reaction (dhtr) represents an anamnestic response to a red cell antigen to which the patient has been previously sensitized by pregnancy or previous transfusions. it occurs in patients without identifiable antibody detected in the pretransfusion compatibility testing who experience accelerated red cell destruction of the transfused red cells after an interval of 3 to 10 d from transfusion. such reactions are due to weaker antigen-antibody reactions and may develop over days. because of the low titer of reactivity, the implicated antibody is not detectable at the time of screening or compatibility testing. the sensitivity of the antibody-screening test is important in preventing dhtrs because insensitive tests will miss weak-reacting antibodies. note: these weak-reacting antibodies commonly include rh antibodies (against cece) and other antibodies in the kell (anti-k), kidd (anti-jk a ), and duffy (anti-fy a ) blood group systems. dhtrs often remain asymptomatic and have milder symptoms than ahtrs, including unexplained anemia, jaundice, and fever. in patients with sickle cell disease, dhtrs may precipitate a sickle cell crisis. laboratory findings are similar to those of ahtr, except for the identification of a new alloantibody in the patient's rbc eluate or serum or both. the degree of hyperbilirubinemia will depend on the rate and amount of hemolysis and the patient's liver function. hemolysis is usually extravascular but intravascular hemolysis can occur. both ahtrs and dhtrs can demonstrate a positive, mixed-field, or negative dat. a mixed-field reaction will show a mixture of agglutinated transfused donor cells along with unagglutinated patient's cells. the dat can be negative if all the incompatible transfused donor cells are immediately destroyed. there is a need to differentiate dhtr from delayed serological transfusion reactions (dstrs), in which only serological incompatibility is evident without clinical evidence of hemolysis. dstrs are more common than dhtrs in the multiply transfused patient as more sensitive screening methods are employed and the length of stay for in-patients increases. dhtrs are tolerated well by many patients and may only require close monitoring. typically, fluid loading and diuresis are not indicated unless active intravascular hemolysis is present. complications, such as renal failure and sickle cell crisis, should be treated accordingly. a red cell exchange is indicated if there is a large burden of antigen-positive cells. ivig may be useful because extravascular hemolysis is similar to acute immune hemolytic anemia. transfusion should be avoided until the causative antibody is identified and antigen-negative units are available. withholding transfusions because of the lack of serologically compatible blood in patients with severe anemia is associated with significant morbidity. this can be avoided by good communication between the clinician and transfusion service. febrile nonhemolytic transfusion reactions (fnhtr) are defined as a temperature rise of at least 1 c in association with a transfusion or up to 4 h after that may be accompanied by chills or rigors. such reactions are due to acquired antibodies to donor leukocyte antigens or pyrogenic cytokines (il-1, il-6, il-8, and tnf-) elaborated by leukocytes present in the blood components or products. fnhtrs are less frequent with prestorage leukocyte-depleted blood components. they occur in 0.5-1.4% of all transfusions. the fever is self-limited and resolves after 4-6 h. antipyretics are effective as symptomatic treatment. meperidine can be useful for treating rigors. among patients who have experienced a fnhtr, there is a 15% recurrence rate. allergic reactions are probably the most frequent, occurring in 1-2% of all transfusion reactions. the symptoms range from local or diffuse pruritus, urticaria, erythema, and cutaneous flushing to anaphylactic allergic reactions occurring within minutes of the transfusion. anaphylactoid reactions fall in between the two ends of the spectrum. allergic disorders afflict 30% of donors, and passive transfer of ige antibodies may be involved. uncomplicated allergic reactions are associated with increased histamine (increased during storage), cytokines, mast cell activators (i.e., leukotrienes), and other vasoactive substances (c3 a and c5 a ) produced by donor leukocytes during storage. some allergic reactions only have pulmonary signs and symptoms without cutaneous involvement (10%). severe anaphylactic reactions may occur after infusion of a very small volume (<10 ml) in patients with iga deficiency and are due to preformed, class specific, recipient anti-iga antibodies against infused donor iga proteins. additionally, they may be due to antibodies against complement c 4 and haptoglobin. patients with chido (ch) and rogers (rg) antibodies (against the ch/rg blood group antigens carried by complement c4d of the classic complement pathway) also exhibit anaphylactoid reactions following plasma product transfusions. haptoglobin deficiency is rare in north american populations but more common than iga deficiency among japanese patients experiencing anaphylactic reactions. anaphylactoid reactions are typically associated with subclass, allotypic, or specific anti-iga in patients with normal or demonstrable levels of iga. these reactions may be seen in other transfused products, such as peanut allergen transfused to patients with peanut allergy. mild uncomplicated allergic reactions involving hives (without other symptoms) respond to antihistamines (such as diphenhydramine). the transfusion may be restarted after treatment if there is no recurrence or progression of symptoms. restarting a transfusion is not advisable with more serious reactions; in particular, pulmonary symptoms with airway involvement. treatment of severe anaphylactic reactions is the same as for any anaphylactic reaction and requires immediate cessation of the transfusion, epinephrine, and other supportive care. prevention of anaphylaxis in iga-deficient individuals requires avoidance of all plasma containing products unless collected from a known iga-deficient donor and washing of all red cell and platelet products. the major concern is to make all blood transfusions as safe and effective as possible. several infectious agents, viruses, bacteria, and parasites are transmissible by transfusion of whole blood, blood components, or products. attention will focus on those transfusion-transmissible diseases (ttd) that present risks of chronic infection in the recipient. major strategies available to reduce transmission include: stringent donor selection and laboratory testing; use of autologous blood, pharmacological substitutes, or new transfusion strategies; inactivation of residual infectious agents in the units to be transfused, and limiting the number of donor exposures and allogeneic transfusions. blood donor screening is one of the more important steps in protecting the safety of the blood supply. nonremunerated, voluntary donors with low infectious risks are encouraged and recruited to become blood donors. the scarcity of the blood supply in many countries has spurred the use of paid donors. direct interviews, miniphysical examinations and laboratory testing of all blood collections help to exclude donors who have exposure to transmissible infections or who have the risk factors for ttd. the blood supply is screened or tested for bacterial and viral infections (including tick borne infections), parasites (i.e., malaria, babesia, chagas' disease, and microfilariasis). other infectious agents can be tested or excluded based on the epidemiology of the infectious agent, geographic area, and intended use of the blood product. in countries with endemic malaria, testing for malaria parasites is mandatory. the general safety of the blood supply will be enhanced if these concerns are addressed; however, minimal risks still remain because very recent infections are not detected by recommended/standard laboratory tests (see table 3 ). bacterial contamination resulting in transfusion-associated bacterial sepsis is now believed to be the most common infectious source of morbidity and the most frequently reported cause of transfusion-related fatalities in the united states (accounting for 16% of transfusion fatalities). bacteria are believed to originate from the donor either from the venipuncture site or unsuspected bacteremia. excluding donors with chronic diseases or recent febrile infections and scrupulous disinfection of the skin reduces the risk of bacterial transmission. both gram-positive and -negative bacteria have been implicated. bacterial multiplication is more likely in components stored at room temperature (i.e., platelets) than refrigerated components (i.e., rbcs) or frozen components (ffp and cryo). bacterial contamination of platelet transfusion is one of the most common infectious risks of transfusion, causing life-threatening sepsis in 1 in 100,000 recipients and immediate fatal outcome in 1 in 500,000 recipients. despite limiting platelet storage to 5 d, various pathogens have been implicated (staphylococcus, enterobacteriaceae, streptococcus, bacillus, and pseudomonas) . the low incidence of platelet-associated sepsis may be due to underreporting because (1) they typically occur several hours after transfusion and are not as catastrophic as rbc-associated sepsis; and (2) sepsis is attributed to other causes because platelets are commonly transfused to immunocompromised patients with other complex problems. psychrophilic gram-negative bacteria can multiply in refrigerated blood and components. rbc contamination is primarily from yersinia enterocolitica and serratia liquifaciens. y. enterocolitica proliferates and produces an endotoxin in refrigerated anticoagulated blood because it can grow at temperatures below 37 c in a calcium-free medium even after a long lag period. any bacterial contamination of blood products is potentially serious. p. cepacia and p. aeruginosa are environmental organisms that grow optimally at 30 c and have been isolated from cryoprecipitate and plasma thawed in contaminated water baths. syphilis transmission by blood transfusion is possible but its occurrence is extremely rare because the phase of spirochetemia is short and the infective organism, treponema pallidum, does not survive refrigerated storage for more than 96-120 h. seroconversion occurs after the phase of spirochetemia so testing donor blood by standard sts does not effectively prevent transmission. however, most positive sts results are demonstrated by donors with inadequately treated noninfectious syphilis, or are biological false positives (may be positive for hepatitis, mononucleosis, measles, chickenpox, immunizations, rheumatoid arthritis, and pregnancy). there have only been two cases of transfusion-transmitted syphilis reported in the english literature. another spirochete, borrelia burgdorferi, causing lyme disease (transmitted by the ixodes deer tick) can survive routine storage of rbcs and ffp. however, transfusion-related transmission has not been reported at this time. the phase of spirochetemia is associated with clinical symptoms that would render potential donors ineligible for donation. donors diagnosed with lyme disease are accepted provided they have completed antibiotic therapy and are completely asymptomatic. before the 1980s, the transmission of hepatitis was the major transfusionrelated viral infection. the absolute number of hepatitis infections post-transfusion has decreased significantly, because reliable tests for hbv and hcv were introduced. in the past, donors having elevated alanine aminotransferases were rejected. this surrogate marker is no longer used with the availability of more specific tests for anti-hcv and hcv rna. anti-hbc testing has been retained because it may still detect a few donors with infectious hbv who are negative for hbsag. hepatitis a (hav), an rna virus, is generally transmitted by the oral-fecal route and very rarely transmitted by transfusion. the main concern of hav (and parvovirus b19 as well) is transmission by plasma derivatives (particularly human source factor viii concentrates) because it does not have a lipid envelope and is not inactivated during the manufacturing process. nucleic acid testing (nat) is available and is done only for source plasma (plasma intended for manufacture of blood derivatives/products). nat testing for this virus is currently considered an "in-process control" by the fda so donor notification (for positive tests) is not required. in contrast to persons infected with hbv, persons who are exposed to hav do not develop a chronic carrier state. blood donors are not routinely screened for hav because of the rarity of transfusion transmissible hav and the absence of hav antibody at the time of viremia. the risk for hav is estimated at 1 per 10 million units. hbv, a dna virus, is primarily transmitted by the parenteral route. it can be transmitted within the first 100 d after infection when viremia is present and no protective immunity has yet developed, or in the chronic carrier state. donor screening to detect hbv infection includes assays for the hbsag and for anti-hbc. most infections are asymptomatic and hbsag positivity occurs 2 to 6 wk before the onset of symptoms; thus, an apparently healthy but infectious donor will be eligible for donation. a "window period" for any transfusion-transmissible agent is defined as the period of time that an individual is potentially infectious but demonstrates negative serological tests (without detectable antibodies). a licensed nat test is not available for hbv because of low levels of viremia during the "window phase" resulting from a slow viral doubling time. however, a recent publication reported that the "window period" can be reduced by 25-36 d using single donor nat (sdnat), further reduced by 9-11 d using minipool nat (mpnat) and reduced by 2-9 d using a new and more sensitive hbsag assay. disagreement exists as to whether hbv-nat will be cost effective to further reduce the risk of transfusion-transmissible hbv. hcv, an rna virus, was discovered in 1989 and was linked with most cases of non-a, non-b hepatitis in the past. hcv infections are mild and generally (80%) asymptomatic. the long-term effects are far more serious in that the majority of hcv infected patients develop chronic liver disease with 20% developing cirrhosis. in addition, those with hcv cirrhosis develop hepatocellular carcinoma. it was estimated that the elimination of hepatitis c by anti-hcv antibody testing prevented 40,000-50,000 new cases of post-transfusion hepatitis per year (an additional 10,000-13,000 cases may have been prevented by newer versions of the test). the risks of transfusion-transmissible hcv has further been reduced by hcv-nat. prior to hcv-nat, the window period because of a slower doubling time was 70 d. the newly developed hcv-nat (mpnat/sdnat) has further closed the window phase to 10 d, so that the transfusion-transmissible risk for hcv is reduced to 1:1,935,000 per unit transfused from 1:276,000 in the past (see table 3 ). hepatitis d (or ), another rna virus, exists as a co-infection in patients chronically infected with hbv because the "delta agent" cannot multiply in the absence of hbv. testing for hbv markers eliminates hepatitis d-positive donors. the hepatitis g virus (hgv, also known as gbv-c), a newly discovered rna virus, is potentially transmitted by blood products. among normal donors, the reactivity is 1-4% by pcr techniques. currently, hgv has not been associated with a specific disease entity and its designation as a "hepatitis" virus may have been premature. thus, no routine donor testing is performed. two other hepatitis viruses, hepatitis e virus (hev) and sen virus, are apparently transmitted by transfusion. hev does not occur in the united states but is endemic in other parts of the world, although its true incidence remains unknown. sen virus is the latest virus postulated to cause the remaining non a-e transfusion-transmitted hepatitis. at present, the biological role of sen virus has not been clearly defined. lastly, tt virus, a dna virus, first described by a japanese group was originally postulated to cause non-a-e hepatitis. this virus is prevalent in many countries and is currently not associated with hepatitis. retroviral infections transmitted by blood transfusion include hiv-1 and hiv-2 and human lymphotropic virus (htlv-i and htlv-ii). hiv is transmitted by both cellular blood components and plasma; however, both types of htlv are highly cell-associated and require viable lymphocytes for transfusion transmission. hiv is a cytopathic retrovirus that preferentially infects cd4-positive tlymphocytes. the infection begins as a viremia of cell-free virions that may be clinically manifested by an acute nonspecific flu-like illness. viremia is detectable in the plasma 10 d to 3 wk after infection. as hiv antibodies appear, the disease enters a clinically latent phase. viral replication and dissemination continues and the virus can be transmitted by blood or genital secretions. hiv-2 causes endemic infection in west africa and has apparently spread with population movements. it is indistinguishable from hiv-1 in disease spectrum although hiv-2 has a longer incubation period and is less efficiently transmitted than hiv-1. the aids epidemic in the early 1980s had a catastrophic impact on the safety of blood transfusions but triggered a major impetus for improvement in blood safety. the risk of hiv transmission through blood components/products dramatically decreased with more stringent donor history screening and improvement of donor testing (see table 3 ). combination hiv-1/hiv-2 tests implemented in the united states in 1992 have identified to date three hiv-2 infected donors, none appear to have been infected in the united states. prior to 1992, the window period for hiv averaged 45 d. more sensitive hivantibody screening tests closed this window to 22 d. in 1996, hiv antigen (p24 antigen) testing reduced this infectious window by an estimated 6 d; such that circulating cell-free virions could be detected as early as 16 d following infection. with the newly developed pcr-based nat, this window period was further reduced to about 10 d. although sdnat has a detection sensitivity of less than 50 viral copies/ml, its higher cost prompted many blood centers into using mpnat with 14-16 donors per pool. an estimate of residual risk of hiv infection from repeat donors after nat is 1 per 2,135,000 (per unit transfused). currently, many blood centers have implemented nat and discontinued p24 antigen testing following the fda's licensure of the hiv-nat assay. htlv-i and ii are human retroviruses that can be transmitted by blood, sexual contact (predominantly male-to-female), and through breast milk. it circulates as a provirus that is incorporated into the dna of lymphocytes. no cases of transfusion-transmitted htlv have been reported with noncellular blood components. prolonged storage for more than 10 to 14 d (refrigeration inactivates the lymphocytes) also reduces transmission risk. compared to other viruses, htlv-i and -ii transfusion transmission is less efficient because exposure invariably does not result in infection. look-back studies have shown that one in three htlv-contaminated units transmitted the virus. risk factors for htlv-1 infection are birth or sexual contact in areas where the disease is endemic (japan and certain pacific islands, caribbean basin, sub-saharan africa, central and south america). the natural epidemiology of htlv-ii is not fully known, although a high prevalence is seen in some native american populations. htlv-ii is presumably associated with parenteral transmission with risk factors of intravenous drug use (1 to 20% seroprevalence) and sexual contact with an iv drug user. an excess of infectious syndromes (i.e., bronchitis, pneumonia, and urinary infections) is seen among blood donors infected with htlv-i or -ii. however, most htlv-1-positive individuals remain asymptomatic donors with an extremely long latency period (decades) and a 2-5% life-long risk of developing adult t-cell lymphoma/leukemia or htlv-associated myelopathy/tropical spastic paresis in areas of high endemicity. enzyme immunoassay (eia) for both htlv-i and -ii is used to screen donors, and eia-reactive donors are indefinitely deferred regardless of investigational supplemental tests since there is no licensed confirmatory test. the risk of transfusion-transmissible htlv with the same "51-d window period" has decreased from 1: 514,000 (in 1998-1999) to 1:2,993,000 (in 2000-2001) . such a dramatic risk reduction may be partly attributed to the implementation of universal leukocyte reduction. viral load reduction by removal of infected leukocytes remains controversial. leukocytotropic human herpesvirus (hhv) may contaminate blood components. cytomegalovirus (cmv or hhv-5) and epstein-barr virus (ebv or hhv-4) have the greatest clinical relevance to transfusion medicine. cmv is transmitted by transfusion in the latent, noninfectious state in the genome of leukocytes present in cellular blood components. seropositivity in the general population, united states ranges from 20 to 80%, but only a small fraction of these individuals have circulating virion. exposure and host factors determine symptomatology. among immunocompetent individuals, cmv causes a mononucleosis-like syndrome or an asymptomatic infection and remains latent in tissues and leukocytes. symptomatic cmv infections develop in immunosuppressed, seronegative hosts. human progenitor cell transplant (hpct) recipients (develop cmv pneumonitis), low birth weight neonates (<1500 g) of seronegative/seropositive mothers and hiv-infected patients (develop cmv chorioretinitis, encephalitis, and enteritis) are particularly at risk. among seropositive hpct recipients, viral reactivation is the most common cause of cmv infection (up to 69% in one study). generally, the donor organ is the source among transplant recipients. additionally, the risk of transfusion-transmitted cmv is high in heavily transfused recipients (liver, heart-lung, and pancreas transplants). "cmv-reduced-risk blood components" are recommended to reduce cmv transmission. leukoreduced cellular blood components are comparable to seronegative cellular blood components. nonetheless, there remains a small risk of transmission with either type of component. most centers in the united states, canada, australia, and europe recommend transfusion of cmv-negative blood components to cmv-negative pregnant women and for intrauterine transfusions (to prevent transplacental infection), and to cmv-negative immunosuppressed individuals (i.e., hpct recipients, hiv-positive with aids, and other immunosuppressed individuals). it is also prudent to extend this consideration to cmvnegative candidates for hpct. despite transfusions of "cmv-reduced-risk blood components," a few marrow transplant patients (1 to 4%) still develop primary cmv infection. ebv targets b-lymphocytes causing polyclonal proliferation with t-lymphocyte response and demonstration of "atypical lymphocytes." it causes infectious mononucleosis, the endemic form of burkitt's lymphoma in africa and nasopharyngeal carcinoma. ebv can be transmitted by blood transfusion, but is a rare cause of significant disease in immunocompetent individuals. transfusion-transmitted ebv is usually asymptomatic. rarely, ebv causes posttransfusion hepatitis and "postperfusion syndrome." the latter is characterized as a viral-like illness following massive transfusion of fresh blood during cardiac surgery. ebv contributes to the development of lymphoproliferative disorders among immunosuppressed hpct and organ transplant recipients from a reactivation of a latent infection. the high seropositivity rate (90%) among blood donors and the low risk of acquiring clinical disease among immunocompetent recipients make blood donor screening and laboratory testing less beneficial. parvovirus b19 is the etiological agent of erythema infectiosum ("fifth disease") in children, arthritis in adults, and hypoplastic anemias in hiv-infected individuals. more ominously, it causes an aplastic crisis among patients with chronic hemolytic anemias who rely on active erythropoiesis to offset the shortened red cell survival. the red cell p antigen is the cellular receptor for parvovirus b19 so those who do not possess the antigen are naturally resistant to infection. the presence of parvovirus b19 antibodies (30 to 60% prevalence) and the brief viremia in blood donors make viral transmission uncommon (ranging from 1 in 3300 to 1 in 50,000). the rarity of clinically significant disease or viral transmission has not made donor screening and testing imperative. there are reports of parvovirus b19 transmission by solvent-detergent plasma (solvent detergent viral inactivation is ineffective because the virus lacks a lipid envelop), cellular blood components, and clotting factor concentrates. there are no reports of transmission from ivig and albumin. nat screening has been implemented only for in-process manufacturing control of plasma derivatives. west nile virus (wnv), a flavivirus, is transmitted through mosquito bites (an arthropod-borne-virus), blood transfusions (first reported during the 2002 epidemic), and organ transplants to humans causing encephalitis, meningitis, and very rarely asymmetrical flaccid paralysis. immunocompromised and elderly individuals are at risk of developing severe disease. viremia occurs 1 to 3 d following an infecting mosquito bite and lasts from 1 to 11 d. wnv transfusion-transmission risk (per 10,000 donations) ranges from 1.46 to 12.33 for selected metropolitan areas and from 2.12 to 4.76 for six highincidence states. serological tests are ineffective in donor screening as viremia disappears by the time igm antibodies are detected by elisa tests. the seasonal increase in 2003 prompted mpnat under a clinical protocol in the united states. likewise, donor history questionnaires have been implemented to reduce the risk of wnv transmission. during periods of high endemicity, frozen products have been withdrawn voluntarily from the supply, as cessation of donor collection is not feasible. other blood derivatives do not appear to be at risk for transmission as the wnv is inactivated by heat or solvent-detergent treatment. the severe acute respiratory syndrome (sars) virus that first appeared in guangdong, china is spread by close person-to-person contact and is believed caused by a corona virus that causes the common cold and/or probably a paramyxovirus. its spread to other countries was linked to airline travel, often by health care workers in contact with sars patients. the virus has been isolated from the blood of an infected individual but its risk of transmission through blood transfusion remains unknown. however, because of its highly contagious nature, transfusion transmission is possible if blood collection coincides with the viremic phase of the disease. the fda recommends deferral of at-risk donors for 14 d after a possible exposure and at least a 28-d deferral after resolution of symptoms. likewise, deferral is extended to donors with a history of travel or residence in sars-affected areas. transmissible spongiform encephalopathies (tses) are rare, fatal degenerative neurological disorders caused by infectious agents classified as prions or proteinaceous infectious particles that lack nucleic acid. a prion is an abnormal isoform (prp sc ) of a normal cellular protein (prp c ) that is resistant to inactivation by alcohol, formalin, ionizing radiation, proteases, and nucleases; but is disrupted by autoclaving, phenols, detergents, and extremes in ph. tses have long incubation periods (years to decades). two such tses, classic creutzfeld-jakob disease (cjd) and variant creutzfeld-jakob disease (vcjd) are important from the transfusion medicine perspective. unlike classic cjd, which presents in older patients, vcjd is observed in young adults and has an acute course with rapid progression to death in 2 yr. the majority of classic cjd is sporadic (80%). familial cases (10-15%) are caused by mutations and the rest (10%) arise from iatrogenic transmission (administration of growth hormone and gonadotropic hormone derived from pooled human pituitary tissue, allografts of dura mater and cornea, and reuse of intracerebral electroencephalographic electrodes from such patients). to date, transfusion transmission of cjd has been reported in experimental rodent models, but not in humans. although theoretically possible, there is growing consensus that cjd transmission by blood or its components is unlikely. variant cjd (vcjd), first reported in the united kingdom in 1996, is caused by the same prion responsible for bovine spongiform encephalopathy (bse) but might be entirely different. its potential for transmission by blood and blood components is heightened by the fact that vcjd can spread from cattle to humans (presumably by ingestion) and from human to humans (a recipient developed symptoms 6.5 yr after a red cell transfusion). in december of 2003, a potential case of vcjd associated with blood transfusion was reported in the united kingdom. its presence in lymphoreticular tissue of vcjd patients, determined by animal studies and its association with b-lymphocytes, suggests possible transmission through blood transfusions. as a safety measure, several european countries and canada implemented universal leukocyte reduction for all blood products to prevent lymphocytes from transmitting vcjd. however, the efficacy of such intervention remains uncertain. expanded donor deferral criteria have been implemented to reduce the risk of transmission of both tses. this includes deferral of donors at risk of exposure due to travel or residency in areas with bse epidemics, deferral of donors who received bovine insulin and pituitary-derived human growth hormone or dura transplants since 1980, and deferral of donors with blood relatives diagnosed with cjd. malaria is caused by several species of the intraerythrocytic protozoan plasmodium and can be transmitted by transfusion of parasitemic blood. malarial parasites survive for at least 1 wk in blood components stored at room temperature (i.e., platelets) or at 4 c. they can survive cryopreservation with glycerol. any blood component that contains red cells can transmit infection via the asexual form of the parasite. it is frequently transmitted by red cell transfusions, rarely by platelet transfusions, and is absent in plasma products. it is recognized as a global health problem, but is very rare in the united states. however, it is the most commonly recognized parasitic complication of transfusion and occurs as at an estimated rate of 0.25 cases per 1 million blood units collected. asymptomatic carriers are the general source of transfusion acquired infections. at present, there are no practical serological tests to screen asymptomatic carriers. prevention of malaria transmission is possible by deferral of prospective donors with increased risk of infectivity based on medical and travel history. in western europe and the united states, blood donors are deferred for 12 mo after travel to malaria-endemic areas. individuals born in areas endemic for malaria generally are excluded from blood donation for 3 yr after leaving the area. chagas' disease (american trypanosomiasis) endemic in south and central america is caused by another protozoan parasite, trypanosoma cruzi, transmitted by reduviid bugs (cone-nosed or "kissing" bugs). infection occurs from fecal contamination of the reduviid bug bite wound by the infectious trypomastigote. acute infections are mild to asymptomatic and 20-40% of infected individuals enter a chronic phase of intermittent parasitemia manifested by "megasyndromes" (cardiomegaly, megaesophagus, and megacolon). blood transfusion is a major source of infection in south america, where parasite reduction using chemicals (like gentian violet) on donated blood pose additional risks. six cases of transfusion-transmitted chagas' disease have been reported in the united states (new york, los angeles, texas, and florida) and canada, involving platelet concentrates in at least four cases. currently, there is a 0.1 to 0.2% seroprevalence in areas with high immigrant populations from central and south america. to date, no tests are licensed by the fda for screening, but highly specific confirmatory tests (i.e., western blot assays) are available. close monitoring is needed to define the risk of transfusion transmitted chagas' disease. patients who are transfusion-dependent for aplastic anemias or chronic hemolytic anemias (sickle cell anemia and thalassemias) such as genetic hemochromatosis may develop iron overload (hemosiderosis), which may result in organ failure, primarily of the heart, liver, and pancreas. every milliliter of transfused rbcs contains approx 1 mg of elemental iron. signs of clinical toxicity become evident when total body iron reaches 400 to 1000 mg/kg of body weight. patients who develop iron overload on chronic transfusion are candidates for chelation therapy with parenteral deferoxamine or oral iron chelators (see chapter 23). transfusion of rbc units enriched with a younger cell population or "neocytes" (reticulocytes), advocated by some investigators, potentially increases transfusion intervals and intravascular survival of rbcs (41%). however, its value in reducing hemosiderosis has not yet been established. a transfusion recipient's ability to mount an immune response and inability to reject transfused (donor) t-lymphocytes in cellular blood components is fundamental to the pathogenesis of ta-gvhd. only whole blood and its cellular components (rbcs, granulocytes, and platelets) containing sufficient, viable, cytotoxic t-lymphocytes can meditate ta-gvhd. no cases have been reported following ffp transfusions. this disease has been reported in immunosuppressed patients with hematological and solid malignancies. rarely, it occurs after organ and hpct and in patients receiving myeloablative therapy. immunocompetent transfusion recipients at risk of ta-gvhd share a haplotype with related or unrelated hla homozygous donors (hla haploidentical). ta-gvhd typically appears 2 to 50 d after transfusion. the development of marrow aplasia and progressive pancytopenia distinguishes ta-gvhd from gvhd following hpct. these patients may develop a skin rash, diarrhea, fever, and abnormal liver function accompanied by extensive hepatocellular damage. most cases (>90%) of ta-gvhd are fatal. treatment with immunosuppressive regimens (steroids, cytoxan, and antithymocyte globulin, including okt3) has been ineffective. as a consequence, immunosuppressed patients (with congenital immunodeficiency syndromes, hematological malignancies undergoing myeloablative therapy, allogeneic or autologous hpct) premature neonates (<1200 g), intrauterine transfusions, recipients of blood from biological relatives and hla-matched platelets should receive only irradiated blood. solid organ transplant recipients receiving immunosuppressive therapy or those undergoing chemotherapy/radiation therapy for solid tumors do not require irradiated blood componenets. interestingly, ta-gvhd has not been reported in patients with aids. gamma irradiation with 25-30 gy inactivates all immunoreactive lymphoid cells and prevents ta-gvhd. leukocyte reduction is insufficient to prevent ta-gvhd. the transfusion-related acute lung injury (trali) reaction is rare (1:2000 to 1:5000 units) and occurs during or within the 4-6 h after a transfusion. patients suddenly develop shortness of breath with severe hypoxemia, chills, fever, cough, and tachycardia. noncardiogenic pulmonary edema recognized on chest x-ray as bilateral diffuse interstitial infiltrates results from leukocytic activation and aggregation in the pulmonary bed causing "capillary leak." the pathophysiology of this reaction is thought to be immune-mediated, typically resulting from (1) donor antibodies (granulocytic or lymphocytotoxic antibodies) directed against white blood cell antigens, (2) hla class i and ii antibodies, and (3) lipid activators of neutrophils in donor plasma. in very rare cases, these antibodies may be present in the recipient. most donors associated with trali are multiparous females or donors with multiple exposures to varying hla types. treatment is supportive; however, ventilatory and pressor support may be necessary. resolution occurs within 3 to 7 d in 81% of cases but is fatal in a small proportion of cases. trali is the third leading cause of transfusion-related deaths, accounting for 13% of all transfusion fatalities. an autologous donor donates blood for his or her own future use. for practical reasons, the transfusion of autologous rbcs is an attractive option for patients undergoing elective surgery. autologous blood can be collected from a patient in advance of anticipated need (preoperative collection) or at the start of surgery (acute normovolemic hemodilution); additionally, shed blood can be recovered for reinfusion during surgery (intraoperative collection) or during the postoperative period from drainage devices (postoperative collection). clinicians should be aware that recovered blood does not provide platelets or coagulation factors. autologous blood transfusion avoids the small risk of transfusion-transmitted infectious agents, red cell alloimmunization, adverse reactions resulting from antibody-mediated hemolysis, and leukocyte-associated febrile reactions. in a larger perspective, autologous transfusion supplements and preserves the blood supply for other patients who acutely or chronically need allogeneic transfusions. autologous donors undergoing preoperative collection should be in satisfactory or good health without major cardiac problems or anemia. the predonation hemoglobin requirement is lower (11 g/dl) for autologous donation than for allogeneic donation (12.5 g/dl). autologous donors can donate as often as every 72 h before the scheduled surgery. ideally, supplemental iron is prescribed before the first collection because iron-restricted erythropoiesis is one of the limiting factors in collecting multiple units of blood over a short period of time. oral or parenteral iron supplement enhances recovery of hematopoiesis. rarely, recombinant human erythropoietin must be administered to donors who have insufficient erythropoietic response. in order to justify the cost effectiveness of autologous collections, there should be a high likelihood that at least two units of blood will be used during surgery. hospitals must establish guidelines regarding indications for autologous blood. during a 4-5 wk collection period, 2-4 units of rbcs can be collected. the storage time of autologous blood is comparable to blood from other donors (up to 42 d with additive solutions). unused autologous blood may be discarded or may be used for allogeneic transfusions in facilities that allow "crossover" only after infectious disease testing. only 30% of collections are typically eligible for allogeneic use. many institutions choose not to "crossover" autologous units because of the cost, complexity, and error risks associated with the process. freezing and long-term storage of rbcs is indicated for patients with rare blood group antibodies, making it difficult to find compatible blood (see p.439). scientific basis of transfusion medicine estimated risk of transmission of the west nile virus through blood transfusion in the us american association of blood banks current and emerging infectious risks of blood transfusions current prevalence and incidence of infectious disease markers and estimated window-period risk in the american red cross blood donor population modern blood banking and transfusion medicine blood banking and transfusion medicine standards for blood banks and transfusion services transfusion medicine transfusion therapy: clinical principles and practice the hla system: basic biology and clinical applications clinical practice of transfusion medicine risks of transfusion-transmitted infection rossi's principles of transfusion medicine principles of transfusion medicine request for an exception under 21 cfr (code of federal regulation) 640.120, alternative procedures, to 610.53 (d), dating periods, to use the gambro 7-day elp platelet storage system using apheresis platelets collected with the cobe spectra apheresis system and gambro trima automated blood component collection system us food and drug administration approves gambro bct's 7-day platelet post-market surveillance plan. gambro.bct march 2005 press release key: cord-278032-27ikx97x authors: göker, hakan; aladağ-karakulak, elifcan; demi̇roğlu, haluk; ayaz, cağlayan merve; büyükaşik, yahya; i̇nkaya, ahmet cağkan; aksu, salih; sayinalp, nilgün; c. haznedaroğlu, i̇brahim; uzun, ömrüm; akova, murat; özcebe, osman; ünal, serhat title: the effects of blood group types on the risk of covid-19 infection and its clinical outcome date: 2020-06-23 journal: turk j med sci doi: 10.3906/sag-2005-395 sha: doc_id: 278032 cord_uid: 27ikx97x background/aim: covid-19 (coronavirus disease of 2019) is an infectious disease outbreak later on declared as a pandemic, caused by the sars-cov-2 (severe acute respiratory syndrome coronavirus-2). it spreads very rapidly and can result in severe acute respiratory failure. the clinical studies have shown that advanced age and chronic diseases increase the risk of infection. however, influence of the blood groups on covid-19 infection and its outcome remains to be confirmed. the aim of this study is to investigate whether there exists a relationship between the blood groups of the patients and risk of sars-cov-2 infection and the clinical outcomes in covid-19 patients. material and method: 186 patients with pcr confirmed diagnosis of covid-19 were included in this study. age, sex, blood groups, comorbidities, need for intubation and intensive care unit follow up and mortalities of the patients were analyzed retrospectively. 1881 healthy individuals, who presented to the hacettepe university blood bank served as the controls. results: the most frequently detected blood group was blood group a (57%) amongst the covid-19 patients. this was followed by blood group o (24.8%). the blood group types did not affect the clinical outcomes. the blood group a was statistically significantly more frequent among those infected with covid-19 compared to controls (57% vs. 38%, p < 0.001; or: 2.1). on the other hand, the frequency of blood group o was significantly lower in the covid-19 patients, compared to the control group (24.8% vs. 37.2%, p: 0.001; or: 1.8). conclusions: the results of the present study suggest that while the blood group a might have a role in increased susceptibility to the covid-19 infection, the blood group o might be somewhat protective. however, once infected, blood group type does not seem to influence clinical outcome. covid-19 was first described as a serious infection leading to significant morbidity and mortality in wuhan, china in january 2020 [1] . world health organization (who) declared covid-19 infection caused by sars-cov-2 virus as a pandemic on march 2020. sars-cov-2 is beta coronavirus that is closely related to sars-cov, both viruses use the angiotensin-converting enzyme-related carboxypeptidase (ace2) in order to enter the cell [1, 2] . covid-19 disease has been observed over 5,5 million people worldwide as of may 25, 2020; and it has caused the death over 350,000 of these patients. many studies have shown that advanced age, male sex, and comorbidities of the person increase the risk and severity of the infection [2] . to date, there is no specific biological marker that has been demonstrated to predict the disease. many previous studies indicated that the connection between hepatitis b and the norwalk virus infection with the blood groups [3, 4] . few studies on sars-cov-1 demonstrated that there exists a relationship between infection risk and the blood types and that the blood group o was somewhat protective against the sars-cov-1 [5, 6] . there are only few clinical studies examining the relationship between sars-cov-2 and the blood groups. in these studies, it was demonstrated that the blood group o had a negative predictive effect and the blood group a was more frequent in patients who presented with severe pulmonary damage [7] [8] [9] . the aim of this study is to investigate the distribution and relationship between the blood groups amongst the covid-19 patients and their clinical outcomes at a referral university hospital. the present study included 207 patients who were followed at hacettepe university school of medicine hospitals between 10/03/2020 and 05/05/2020 with the covid-19 infection who were positive for the sars-cov-2 rna test through pcr from the nasopharyngeal swab, and who were approached in accordance with the treatment guidelines of the turkish ministry of health. the medical records of patients were retrospectively analysed. the patients, whose blood group analysis that were not done at our hospital, were later on contacted, and their blood groups were also obtained. a total of 186 patients were evaluable for the final statistical analysis. the remaining 21 patients were excluded from the study because their blood group information was not reliable or the patients could not be reached. clinical data including age, sex, comorbidies, intubation needs, intensive care admissions and outcome of the patients were obtained from medical records. due to the existing protocols of the hospitals of hacettepe university faculty of medicine, all of the ethical considerations were strictly followed. as a standard care/action of the hospitals of the hacettepe university faculty of medicine, it has been recognized from the patient records that all of the studied patients gave informed consent at the time of admission to the hospital for diagnostic/therapeutic procedures as standards of care. local ethical committee approval was obtained from hacettepe university numbered go 20/434 and additionally, turkish health ministry approval was also obtained on may 4th, 2020 for this study as required. as for the comorbidities, histories of cardiovascular disease, respiratory disease, obesity, diabetes mellitus, and malignancy were screened. hypertension, coronary artery disease, congestive heart failure, and arrhythmias were classified as cardiovascular diseases; copd and asthma were classified as respiratory diseases. clinical outcomes were determined as the need for intubation, the need for icu, and death. in order to determine the normal distribution of the blood groups, 1881 healthy individuals, who applied to the hacettepe university blood bank affiliated with our center between 01/03/2020 and 01/05/2020, were included as the control group. the blood group distributions, age and sex of these patients were recorded. analyses were made using spss version 25.0 (ibm corp., armonk, ny, usa). the distribution between the blood groups of the patient and control groups was evaluated using chi-square and fisher exact tests. the impacts of blood groups and comorbidities on the clinical outcomes were evaluated using logistic regression analysis. hosmer-lemeshow test was used for goodness-of-fit. the cases with the p-value below 0.05 were accepted as statistically significant. the clinical characteristics and outcomes of 186 patients included in the study according to their blood groups are presented in tables 1 and 2. the median age of patients was 42 (19-92) and the percentage of female patients was 46.2%. the most frequently detected blood group was blood group a with 57% amongst the covid-19 patients. this was followed by blood group o with 24.8%. there was a history the distribution of the patient and control groups according to their blood groups is presented in table 3 . when the healthy control group was compared to the covid-19 patient group, it was observed that the covid-19 infection rate was statistically significantly higher in those with blood group a (57% vs. 38%, p <0.001; or: 2.1). though, covid-19 was significantly more seen with the blood group a, on the other hand, rh factor did not make any significant difference (p > 0.05) it was observed that the blood group o was significantly lower in the covid-19 patient group in comparison to the controls (24.8% vs. 37.2%, p: 0.001; or: 1.8). there was no difference between blood groups of b and ab. the effect of blood groups on clinical outcomes of covid-19 patients are presented in table 4 . no significant effect of abo and rh systems were demonstrated on the clinical outcomes. a total of 186 covid-19 patients, who were followed at the hacettepe university, were included in the present study. it was observed that blood group a was more frequent and the blood group o was less frequent in covid-19 patients compared to the control group. the blood group a was significantly more frequent and the blood group o was less frequent amongst the covid-19 patients when compared to the controls (p < 0.001 and p: 0.001, respectively) when we looked at the effect of blood groups and rh type on the clinical outcomes, it was demonstrated that the blood groups did not have significant predictive effects on the need for intubation, need for icu hospitalization and mortality; and that the most significant factors affecting the few studies evaluated were evaluated in blood groups in terms of the needs for intubation, icu hospitalization and clinical outcomes [7, 8] . there have been studies indicating the predictive effect of abo blood groups on the helicobacter pylori, norwalk virus, and sars-cov [6] . in a previous study, conducted with 42 healthcare professionals in hong kong during the sars epidemic period, it was presented that those with blood group o had a lower incidence of sars-cov infection than the non-o (or, 0.18; 95% confidence interval, 0.04-0.81). the patients with blood group b also demonstrated a tendency to the disease; however, there was no statistical significance (or: 1.46) [5] . in an animal model, which was carried out to present the mechanism of this result, it was found that the anti-a antibodies found in individuals with blood group o inhibited the interaction of sars-cov-1 virus s spike protein and the ace-2 receptor [10] . a number of studies have been carried out on this subject since the beginning of the sars-cov-2 pandemic. first of all, in a study on 265 covid-19 patients, it was presented that the blood group o was less frequent in severe covid -19 patients who required long hospitalization (p < 0.01), and the blood group a was more frequent in patients with severe covid-19 infections compared to the normal population (0.017) [10] . two different studies have also demonstrated the possible protective effect of the blood group o [7,9,] . the relationship between abo blood groups and cardiovascular diseases was well established previously [11] . it is known that thrombotic risks decrease significantly in blood group o compared to the non-o [11, 12] . studies have shown that the micro thrombosis developing in the covid-19 infection in pulmonary vascular bed lead to a serious contribution in the acute respiratory syndrome; therefore, the use of prophylactic anticoagulants was included in the guidelines, as well [13, 14] . there are opinions arguing that the protective effect demonstrated in blood group o is based on this phenomenon [15] . the present study, there was no significant relationship between the clinical outcomes and the blood groups. however, this is an important study amongst turkish covid-19 patients that demonstrate the blood group a might be susceptible to covid-19 infection and the blood group o might be somewhat protective which should be better elucidated in prospective much larger studies, as well. the limitation of this study is the somewhat small number of patients with the clinical outcomes may have caused the failure in demonstrating the effect of blood groups on clinical outcomes in statistical terms. in conclusion, the present study demonstrate that the blood group o might be protective while the blood group a might have increased susceptibility to the disease, and guidelines set forth by the centers for disease control and prevention (cdc), who and healthy authorities, such as social distancing, hand hygiene, mask use strictly should be followed. one might conclude that stronger, stringent measures should be taken when taking care of individuals with the blood group a for possible covid-19, i.e. sars-cov-2 infection susceptibility. there is a need for further molecular studies to elucidate the relationship between the blood groups and the disease. prospective larger multicentre studies may be needed in order to further elucidate the role of possible somewhat protective role of the blood group o. a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study human susceptibility and resistance to norwalk virus infection abo blood groups and hepatitis b virus infection: a systematic review and meta-analysis abo blood group and susceptibility to severe acute respiratory syndrome blood groups in infection and host susceptibility relationship between the abo blood group and the covid-19 susceptibility association between abo blood groups and risk of sars-cov-2 pneumonia testing the association between blood type and covid-19 infection, intubation, and death inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies abo(h) blood groups and vascular disease: a systematic review and metaanalysis abo blood group determines plasma von willebrand factor levels: a biologic function after all? abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia more on "association between abo blood groups and risk of sars-cov-2 pneumonia key: cord-022035-annn4qn1 authors: menitove, jay e.; tegtmeier, gary e. title: other viral, bacterial, parasitic and prion-based infectious complications date: 2009-05-15 journal: blood banking and transfusion medicine doi: 10.1016/b978-0-443-06981-9.50053-3 sha: doc_id: 22035 cord_uid: annn4qn1 nan during the last decade of the 20th century, diagnostic advancements dramatically reduced the transmission of human immunodeficiency virus (hiv), hepatitis c virus (hcv), and hepatitis b virus (hbv) by transfusion. however, simultaneously, the emergence of additional pathogens as potential blood contaminants gained attention. some of these agents represented newly discovered entities (e.g., severe acute respiratory syndrome [sars-coa]). others were known sources of transfusion complications that expanded into the united states (e.g., chagas disease). additionally, some agents demonstrated species jumping from animal hosts to humans (e.g., variant creutzfeldt-jakob disease [vcjd] , asian influenza, and west nile virus). increasing globalization through commerce, travel, and social interaction requires that many infectious agents, once thought exotic or of remote significance, must be considered as potential blood-component contaminants. 1, 2 alternatively, new information may reduce some concerns. for example, porcine endogenous retrovirus (perv), previously linked to humans undergoing xenotransplantation, currently appears less threatening. 3 this chapter addresses agents endemic to the united states and those emerging in other parts of the world that have been transmitted or are theoretically capable of transmission by transfusion, and approaches to reduce the associated risks. babesiosis, a zoonosis caused by the rodent-borne piroplasm protozoan, babesia microti, is transmitted by ixodes scapularis, the deer or black-legged tick. i. scapularis also transmits the agents of lyme disease and human granulocyte ehrlichiosis (discussed later). [4] [5] [6] [7] [8] [9] [10] the white-footed mouse (peromyscus leucopus) is the natural reservoir for b. microti; once infected, a mouse remains parasitemic indefinitely. i. scapularis transmits the piroplasm most frequently during the nymphal stage when the tick is 1.5 mm long. tick bites, at this stage, often go unnoticed despite the 48-to 72-hour feeding time during which infection occurs. 11 b. microti is the agent most frequently associated with clinical illness; mo1-type, wa1-type, and ca1-type also cause clinical disease. 12, 13 endemic areas include coastal and island areas of new england and new york as well as parts of california, washington, missouri, wisconsin, and minnesota. 6, 7, 9, 12, 13 ticks coinfected with b. microti and borrelia burgdorferi (the agent of lyme disease) transmit b. microti less frequently than b. burgdorferi because the tick is a less competent host for b. microti. the intraerythrocytic localization of b. microti, however, favors transfusion transmission of this agent over that of b. burgdorferi. 6 in humans, circulating b. microti dna persists, on average, for 82 days in asymptomatic patients and in those not given specific treatment. co-infection with lyme disease does not alter the duration of parasitemia. parasites circulate for only 16 days in persons who are treated with clindamycin and quinine; alternative antibiotic regimens include atovaquone and azithromycin. 13 silent babesia infections occur commonly. some infected individuals develop a chronic carrier state lasing months to years. in others, recrudescence occurs spontaneously or after splenectomy or immunosuppression. 11, 14 the parasite retains infectivity in red blood cell (rbc) components at refrigerated or frozen temperatures and in the residual rbcs contained in platelet concentrates stored at room temperature. 5, 8 to date, more than 50 post-transfusion cases involving b. microti and other babesia species have been reported. 5, 6, 9, 12, 15 several reports involve donors who transmitted infections through multiple donations given up to 6 months apart. 11, 16 the overall risk of acquiring transfusion-associated babesiosis is low, but varies regionally. in connecticut, 1.9% of seronegative donors became seropositive on a subsequent donation. in another study, 0.9% of donors in endemic and nonendemic areas of connecticut had confirmatory indirect immunofluorescence assay (ifa)-positive test results for babesia infection; the prevalence rates peaked in july when 1.2% of donors were seropositive. 15 this represents a relatively high potential threat in an endemic area because 8 of 51 recipients became seropositive after receiving blood from ifa-positive blood donors. 17 asplenia, older age, immunodeficiency, organ transplantation, and liver disease increase the risk of severe babesia illness. in acute symptomatic cases, fatigue, malaise, weakness, and fever occur in more than 90% of the patients. shaking chills, diaphoresis, nausea, anorexia, headaches, and myalgia occur frequently. heart murmurs, hepatomegaly, and splenomegaly are found in 10% to 20% of patients; jaundice occurs less frequently. renal failure, disseminated intravascular coagulation, and adult respiratory distress syndrome have been reported. 13 the average hemoglobin concentration was 11.3 g/dl in a review of hospitalized patients with community-acquired babesiosis. 4 examination of blood smears for intraerythrocytic ring forms and maltese cross-like tetrads (including more than two parasites per cell, contorted shapes, vacuoles, and budding), 18 antibabesial antibody assays, and polymerase chain reaction (pcr) assays for babesial dna provide laboratory evidence of infection. 4, most transfusion-associated babesia cases involve rbc transfusions, although frozen-deglycerolized rbc and platelet units have been implemented. 5, 8, 13 transfusion-acquired cases have an incubation period of 2 to 6.5 weeks. 4, 6, 8, 13, 15 blood-collection agencies ask all prospective donors whether they have ever had babesiosis. those answering affirmatively are deferred. however, donors are not asked about a recent history of tick bites or geographic residence because of the low predictive value associated with these questions. for example, 0.4% of donors in connecticut reporting tick bites were seropositive for babesiosis antibodies compared with 0.3% in those not reporting tick bites. 9 serologic or pcr testing is impractical at this time. the absence of specific interventions to interdict donors capable of transmitting babesia infections relegates clinical awareness and prompt antibiotic therapy as the primary modality for treating this infrequent complication of transfusion therapy. the borrelia burgdorferi spirochete causes lyme disease, a tick-borne zoonosis present in mice, squirrels, and other small animals. more than 20,000 human lyme disease cases occur annually in the united states, although none has been associated with transfusion. endemic areas include the northeastern, mid-atlantic, and upper north-central regions of the united states. 10, 19 ixodes scapularis, the black legged deer tick, transmits b. burgdorferi in the northeastern and north-central parts of the united states. i. pacificus, the western black-legged tick, transmits the infection along the pacific coast. the ticks feed predominantly in the late spring and early summer during their nymphal stage, and lyme disease usually results from bites of infected nymphs. deer do not become infected but rather transport and maintain the ticks. 10 patients with lyme disease typically present with a characteristic erythema migrans rash accompanied by fever, malaise, headaches, myalgia, arthralgia, or bell's palsy. the rash occurs 3 to 30 days after a tick bite. b. burgdorferi spirochetes disseminate from the entry site via cutaneous, lymphatic, and blood-borne routes. in one study, spirochetes were isolated from the blood of 44% of patients with symptomatic lyme disease. 20 b. burgdorferi has also been isolated from erythema migrans lesions. the diagnosis of lyme disease is based primarily on characteristic symptoms, physical examination findings, and a history of possible tick exposure. 10, [20] [21] [22] [23] serologic tests, including enzyme-linked immunoassays and ifa tests, become positive 4 to 6 weeks after infection. western blot testing is used to confirm the results of reactive screening tests. treatment with antibiotics clears the infection, but additional treatment to relieve symptoms is prescribed when arthritis persists after two antibiotic courses and for post-lyme disease syndrome. [20] [21] [22] [23] despite documentation that the spirochete survives routine rbc and frozen plasma storage, testing blood donors is not under consideration because no reports exist of transfusion-associated lyme disease. 10 of note, transfusion of rbcs or platelets collected during peak deer tick activity to 155 patients undergoing cardiothoracic surgery resulted in no serologic or clinical evidence of lyme disease. 5 individuals with a history of lyme disease are accepted as blood donors provided they have been treated and are asymptomatic 12 months after the last dose of antibiotics. the rickettsial agents human monocytic ehrlichiosis (hme) and human granulocytic ehrlichiosis (hge) are intracellular organisms that survive in stored blood and cause mild to severe illnesses. 10 ehrlichia chaffeensis causes hme and is transmitted to humans through the bite of the lone star tick (amblyomma americanum) previously infected by contact with deer or possibly dogs. 9,10 most of the reported cases have occurred in the south-central and southeastern united states. anaplasma phagocytophila causes hge and is related closely to species infecting horses (ehrlichia equi) or ruminants (ehrlichia phagocytophila). this illness occurs predominantly in the northeastern, upper midwestern, and northwestern areas of the united states and is transmitted to humans by i. scapularis or i. pacificus ticks. 5, 9, [24] [25] [26] fifty percent of ticks examined in one study in connecticut were infected with the hge agent, but none was infected with e. chaffeensis. 27 patients with hme and hge present similarly, with fever, headache, myalgia, thrombocytopenia, leukopenia, and elevated liver enzyme concentrations. a rash occurs in one third of patients with hme but in fewer patients with hge. membrane-bound intracytoplasmic ehrlichia aggregates, or morulae, are present in monocytes. complications include respiratory distress, renal failure, neurologic disorders, and disseminated intravascular coagulation. septicemia, vasculitis, and thrombotic thrombocytopenic purpura should be considered in the differential diagnosis. 5, 10, 24, 25 doxycycline is the treatment of choice. because ehrlichia are present in blood, transfusion transmission must be considered. one case of transfusionassociated hge occurred 9 days after an rbc transfusion donated by an asymptomatic donor who had been exposed to extensive deer ticks 2 months previously. the infected rbcs were stored for 30 days before transfusion. 28 an in vitro study suggested that leukocyte reduction may not be completely effective at preventing e. chaffeensis transmission because some pathogens are found in the cell free plasma fraction. 29 an extensive epidemiologic study in arkansas involving military trainee blood donors who had been exposed to tick bites and unknowingly infected with the agents of ehrlichiosis and rocky mountain spotted fever (rmsf) found no clinical illness among the recipients of rbcs and platelets donated by these soldiers. however, possible seroconversion to rmsf occurred in one of the recipients. 30 a single case report has been published of clinical illness associated with transfusion-transmitted rmsf infection. the donor developed symptoms of rmsf 3 days after donation and died 6 days later. the recipient, who developed fever and headache 6 days after receiving the implicated rickettsia rickettsii-infected transfusion, was notified about the donor's illness and was treated effectively. 10 other tick-borne agents implicated in transfusionassociated cases include colorado tick fever virus and tick-borne encephalitis virus. 10 although the risk of transfusion transmission of these agents is low, clinical suspicion is important as a mechanism for determining infection by these organisms. malaria is a protozoan disease caused by four species of the genus plasmodium: p. falciparum, p. vivax, p. ovale, and p. malariae (table 48-1) . these protozoa are transmitted to humans by the bite of an infected female mosquito of the genus anopheles. infection of the human host, absent treatment, results in a chronic intraerythrocytic infection that can be transmitted by blood transfusion. the two-host life cycle of the malaria parasite is diagrammed in figure 48 although the signs and symptoms of malaria are variable, most patients are febrile, and many also manifest headache, chills, sweating, nausea, vomiting, diarrhea, back pain, myal-gia, and cough. a diagnosis of malaria should be considered for any patient with these symptoms who has a history of travel to a malaria-endemic area or recent blood transfusion. given the periodic reports of local mosquito-borne transmission, malaria should also be considered in the differential diagnosis of patients who have fever of unknown origin regardless of their travel history. malaria is diagnosed microscopically by finding intraerythrocytic parasites on giemsa-stained peripheral blood smears. properly prepared thick and thin smears must be examined by trained laboratory personnel to make an accurate laboratory diagnosis. patients with negative smears suspected of having malaria should have additional smears examined daily for 3 days. pcr can be a useful adjunct in cases in which serial testing of smears yields negative results. malaria is a huge global public health problem with an estimated annual incidence of 300 to 500 million cases and 3 million deaths per year. 31 malaria-endemic areas include parts of africa, asia, central america, hispaniola, north america, oceania, and south america. during the early part of the 20th century, specifically 1914, an estimated 600,000 cases of malaria occurred in the continental united states, but since the 1940s, improved socioeconomic conditions, water management, vector control, and case management have prevented endemic malaria transmission. 32 ongoing malaria surveillance in the united states by the centers for disease control and prevention (cdc) continues to identify cases in immigrants and in residents and travelers to areas of the world where malaria transmission still occurs. additionally, each year, a few cases are reported that might represent local mosquito-borne transmission. 33 for example, seven cases of locally acquired, mosquitotransmitted p. vivax malaria were reported in palm beach county, florida. multilocus genotyping of the ribosomal rna of the isolates from the seven patients revealed that they were infected by the same strain. 34 congenital infections and transfusion-acquired infections round out the sources of malaria cases diagnosed each year in the united states. of 1337 cases of malaria in the united states with onset of symptoms in 2002, one was due to transmission of p. malariae by blood transfusion. 35 of 1278 cases reported in 2003, one was due to transmission of p. falciparum after a blood transfusion. 36 the overwhelming majority of reported cases in both years were imported (i.e., acquired outside the united states). data from 1979 through 1986 showed that cases were more frequently identified in foreign civilians than in u.s. civilians. however, since 1997, the situation has reversed. cases in united states civilians are now reported at 2.5 to 3 times the number in foreign civilians, most likely due to increased travel by u.s. civilians to endemic areas and decreased immigration since 2001. 36 from 75% to 90% of the cases among u.s. civilian travelers occurs in persons who failed to take prophylactic drugs, had not taken cdc-recommended drugs, or were noncompliant with a recommended drug. mosquitoes of the genus anopheles, with few exceptions, feed between dusk and dawn. the exceptions are daytime feedings in densely shaded woodlands or dark interiors of houses or shelters. therefore, travelers who visit malarial areas during bright daylight hours are at little or no risk for acquiring malaria if they return to a nonmalarial area before dusk. transfusion-transmitted malaria occurs at an estimated rate of 0.25 cases per 1 million blood units collected. 37 because of this low incidence and the lack of a laboratory test approved by the u.s. food and drug administration (fda), prevention of transfusion-transmitted malaria continues to depend solely on the donor-deferral guidelines established by the fda and most recently updated in 1994. 38 currently, prospective donors who are residents of countries where malaria is not endemic but who have traveled to a malaria-endemic area are temporarily deferred until 1 year after their departure from the endemic area if they have remained free of symptoms suggestive of malaria. immigrants, refugees, citizens, and residents of malaria-endemic areas are deferred for 3 years after can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.) after this initial replication in the liver (exo-erythrocytic schizogony), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony). merozoites infect red blood cells. the ring-stage trophozoites mature into schizonts, which rupture, releasing merozoites. some parasites differentiate into sexual erythrocytic stages (gametocytes). blood-stage parasites are responsible for the clinical manifestations of the disease. the gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an anopheles mosquito during a blood meal. the parasites' multiplication in the mosquito is known as the sporogonic cycle. while in the mosquito's stomach, the microgametes penetrate the macrogametes, generating zygotes. the zygotes in turn become motile and elongated (ookinetes) and invade the midgut wall of the mosquito where they develop into oocysts. the oocysts grow, rupture, and release sporozoites, which make their way to the mosquito's salivary glands. inoculation of the sporozoites into a new human host perpetuates the malaria life cycle. ( their departure from the endemic area if they have remained free of symptoms suggestive of malaria. prospective donors who were diagnosed and treated for malaria are deferred for 3 years after becoming asymptomatic. between 1996 and 1998, three cases of post-transfusion malaria due to p. falciparum, two of which were fatal, 39 were diagnosed in the united states, prompting a review by the cdc of all cases of transfusion-transmitted malaria reported between 1963 and 1999 40 (see table 48 -1, which has been updated to include reported cases in 2002 and 2003 35, 36 ). in total, 95 cases (2.5 per year) were reported through 2003. thirty-four (36%) cases were caused by p. falciparum, 25 (27%) by p. vivax, 26 (28%) by p. malariae, 5 (5%) by p. ovale, 3 (3%) by mixed species, and 2 (2%) by an undetermined species. p. falciparum cases increased in frequency over the period 1990 to 2003, accounting for 11 (73%) of 15 cases during that interval, compared with 15 (24%) of 62 cases reported between 1970 and 1989. of 10 (11%) fatal cases overall, 6 were associated with p. falciparum, 2 with p. vivax, and 2 with p. malariae. the incubation period in these cases ranged from 8 to 90 days, with p. falciparum having the shortest time (mean, 17 days; range, 8 to 36 days) and p. malariae having the longest (mean, 51 days; range, 8 to 90 days). the period between onset of symptoms and the time of diagnosis ranged from 1 to 180 days, with a median of 10 days. ninety-four percent of the cases were associated with transfusion of whole blood or rbcs; 6% were platelet-associated. implicated donors were defined as having met one or more of the following criteria (1) a blood smear that demonstrated malaria parasites, (2) a positive result on malaria serology, and (3) being the only donor. ninety-three donors were implicated in the 95 cases. the median number of donors per case was seven (range, 1 to 192). donors were overwhelmingly male (90%) and ranged in age from 19 to 59 years (median, 27 years). foreign-born donors accounted for 60% (64% of those from africa); 40% were born in the united states. of 60 donors implicated in the cases for which epidemiologic follow-up was complete, serology was the most effective tool for identifying transmitting donors (73%); only 10% were identified by a positive blood smear. serology and blood smear were both positive in 15%, and 3% were implicated as the only donor to a case. analysis of all cases using current donor deferral guidelines revealed that 23 (24%) cases occurred despite proper application of the guidelines. when reviewed against the guidelines in place at the time they occurred, 3 cases could not be evaluated because their dates of onset were before 1970 when guidelines were vague; 18 of the remaining 20 cases would still have occurred, but 2 would have been prevented if then-current guidelines had been applied properly. not surprisingly, most (65%) of the cases that occurred despite following guidelines were caused by p. malariae. the continued occurrence of cases in the face of current history questions highlights the reality that malaria risk from transfusion, although low, cannot be fully prevented by questioning of donors. although the deferral guidelines currently in place are based on the biology of the four species of plasmodia that cause malaria, they represent a balance struck between maximizing safety and minimizing donor loss. p. vivax and p. ovale, species that give rise to relapsing infections, rarely persist longer than 3 years. 42 however, some infections do persist, and individuals with these prolonged infections will transmit malaria if their blood is transfused. likewise, disease caused by p. falciparum, a nonrelapsing species, manifests within 1 year after departure from a malarious area 99% of the time, but a report of falciparum malaria occurring 13 years after departure from a malarious area has been published. 41 the well-known ability of p. malariae to persist asymptomatically for decades in some individuals further highlights the difficulty of eradicating the risk of post-transfusion malaria through questioning of donors. 42 the aabb has advocated the use of uniform donor screening questions to elicit malaria risk from prospective donors, including questions that inquire about a history of malaria and about the prospective donor's travel history within the past 3 years. a "yes" answer to travel outside the united states and canada triggers further inquiry to pinpoint travel destinations in malarious areas. the fda is in the process of revising its guidelines for deferral of blood donors because of risk of malaria. however, it is unclear when the agency will issue the new guidelines. the proposed guidelines were discussed at the fda's blood products advisory committee meeting in june 1999. 46 in addition to retaining the provisions for donor deferral outlined in the fda memo of july 26, 1994, 38 the revised guidelines recommend adding the following question sequence to the donor history form: (1) "were you born in the united states?" if yes, ask: (2) "in the past 3 years, have you been outside the united states or canada?" if the answer to (1) is no, ask: (3) "when did you arrive in the united states, and, since your arrival, have you traveled outside the united states or canada?" if the answer to question (2) or the second question in (3) is yes, follow-up questions will be asked of the donor to determine when and which country or countries were visited. the impetus for revision of the guidelines includes the increased number of imported malaria cases in the united states, the large number of postdonation events related to malaria reported to the fda, and the recognition that eliciting an accurate donor history is the only currently available defense against transfusion-transmitted malaria. from time to time, proposals to test donors for evidence of malaria have been advanced, but no fda-approved tests or policies for screening donors are currently in place. selective screening of high-risk donors has been suggested as an alternative to universal screening. 43 blood-smear diagnosis is both impractical and insensitive as a donor-screening technique. the ifa test is useful diagnostically but is unsuitable for large-scale donor screening, although it could be used to test high-risk donors and to determine their suitability. 43 although antibody assays detect most individuals with parasitemia, they also are positive in treated persons who are no longer parasitemic. 44 hence, noninfectious donors would also be deferred if selective antibody screening were implemented. pcr is a promising approach that may have the required sensitivity and specificity, but it is currently not standardized and not available outside research laboratories. 45 american trypanosomiasis, or chagas disease, is a zoonosis caused by the hemoflagellate protozoan parasite trypanosoma cruzi. the life cycle of t. cruzi involves transmission from insect vectors to mammalian hosts including humans. t. cruzi infects humans when triatomid (reduviid) or kissing bugs ingest a blood meal from the host and deposit infected feces into the wound or when contaminated feces contact the mucosal surface of the eye or mouth. hematogenous spread occurs subsequently. in addition, t. cruzi crosses the placenta and can cause congenital disease [47] [48] [49] [50] [51] (fig. 48-3 ). acute chagas disease is associated with fever, facial edema, generalized lymphadenopathy, and hepatosplenomegaly. symptomatic myocarditis and meningioencephalitis can occur, and fulminant illness can develop in immunologically immature children or immunocompromised adults. however, in more than 95% of patients, the illness is mild and symptoms resolve in 4 to 6 weeks. if untreated, hosts then enter an indeterminate phase. ten percent to 30% of patients progress from the indeterminate asymptomatic phase to a chronic symptomatic phase associated with cardiac enlargement, apical aneurysms, mural thrombi, megaesophagus, or megacolon, appearing years to decades after infection. [48] [49] [50] [51] epidemiology an estimated 16 to 18 million people are infected in south america, central america, and mexico, where chagas disease is endemic and, historically, triatomid insects reside in cracks of rural and suburban houses with adobe walls. in the united states, an estimated 25,000 to 100,000 persons and 1 in 25,000 blood donors may be infected with t. cruzi. 48 almost all are immigrants from central and south america. chagas disease is responsible for 50,000 deaths worldwide annually. 53 lifelong low-grade parasitemia persists in approximately 50% of those infected, and up to 63% of seropositive blood donors have parasitemia. 52 this presents a risk of transfusion transmission and of vertical transmission to infants. between 12% and 48% of recipients of parasitemic blood become infected. estimates of risk for transfusion-associated chagas disease are related to immigration patterns from endemic regions. during the mid-1980s, 4.9% of 205 nicaraguan and salvadorian immigrants living in washington, d.c., had serologic evidence of t. cruzi infection. parasites were isolated from half. 54 in the early 1990s, 0.11% of a selected blood-donor population in california and the u.s. southwest was seropositive for t. cruzi antibodies. at least 50% of these donors were of hispanic origin. 55 during the mid-1990s, 39.5% of donors at a hospital in los angeles responded affirmatively to questions inquiring about birth in chagas disease-endemic areas or residing in dwellings constructed of palm leaf-thatched roofs or walls made of mud, 56 and 0.5% tested positive for t. cruzi antibodies. in a study conducted in the mid-to-late 1990s involving more than 1.1 million blood donors, 1 in 7500 in los angeles and 1 in 9000 in miami were t. cruzi seropositive. 52 although a correlation exists between the percentage of immigrants from endemic areas and the percentage of blood donors with serologic evidence of t. cruzi infection, investigators have also identified seropositive blood donors who were born in the united states. 57 congenital transmission may explain infection in these individuals. in addition, autochthonous transmission has been reported in the united states, 58 and an infestation of triatomines has been reported in texas. 59 since 1989, seven cases of transfusion-associated chagas disease have occurred in the united states and canada. [61] [62] [63] [64] [65] symptoms developed approximately 2 to 3 months after transfusion. in at least six of the cases, platelets were the implicated blood component; however, in the seventh case, the implicated unit was not identified. 63 centrifugation may sediment t. cruzi into the platelet layer during component preparation, accounting for the association with platelet transfusions. whereas room-temperature storage of platelets may favor parasite survival, t. cruzi has been shown to survive in refrigerated rbcs and whole blood for at least 18 to 21 days. 51 in six of the north american transfusion-associated cases, a donor emigrating from a t. cruzi-endemic region (bolivia, mexico, paraguay, chile) was identified. four of the donors emigrated between 16 and 33 years before the implicated donation. given this small number of cases, transfusion transmission of chagas disease may be inefficient. in a study of 18 patients receiving blood from blood donors subsequently found to be t. cruzi seropositive, none of the recipients became seropositive after transfusion. however, only two received platelet transfusions. 52 a report of chagas disease also has been reported after transplantation involving an organ donor who emigrated from central america appeared in 2002. 60 the recipient of a kidney and pancreas died of acute chagas myocarditis 5 months after transplant. the recipients of the other kidney and the liver were both also infected with t. cruzi. interventions to reduce the risk of transfusion-transmitted chagas disease include questioning donors about geographic location of birth, extended stay or transfusion in areas endemic for chagas disease, and serologic testing. 51, 66, 67 donor history questions may be only 75% effective. 51 at least one candidate serologic screening assay has undergone clinical trials in the united states and is currently under review at the fda. the u.s. fda has indicated that it will require testing for chagas disease if an appropriate screening assay achieves licensure. this decision reflects the reported transfusion-and organ transplant-associated cases and the concern that up to 600 transmissions may occur annually in the united states. 52 leukocyte reduction by filtration is modestly effective, reducing t. cruzii transmission by 50% to 70% in a mouse transfusion model. 68 serologic testing of blood donations for syphilis was instituted in 1938 and required by regulation in 1958. no cases of transfusion-associated syphilis have occurred in the united states since 1966. 69 multiple factors-improved donor selection, uniform serologic testing, lack of spirochete viability in blood stored at refrigerated temperatures, and widespread antibiotic use-apparently contribute to the current absence of transfusion-transmitted syphilis cases. [69] [70] [71] in 1978, the aabb standards committee deleted the requirement for syphilis testing, and an fda advisory panel proposed eliminating the requirement for serologic syphilis testing in 1985. however, these changes were not made because of the belief that such testing might identify those at risk of transmitting the hiv. subsequently, observational data did not support this assumption. nonetheless, a national institutes of health consensus statement, issued in january 1995, recommended continuation of syphilis testing because its role in preventing transfusion-transmitted syphilis was not "understood." 70 a lack of complete laboratory data also supports test retention. although spirochetes survive 96 to 120 hours at refrigerated temperatures, 72,73 viability at room temperature (e.g., in platelet concentrates) has not been studied. furthermore, loss of viability during storage is an incomplete protection mechanism. no single optimal laboratory test exists for syphilis. the infectious agent, treponema pallidum, is an anaerobic organism that cannot be cultured in vitro. 73 during treponema infection, nontreponemal and treponemal antibodies are produced. the nontreponemal antibodies (reagin antibodies) react against phospholipid isolated from beef heart or cardiolipin. these antibodies are detected by the venereal disease research laboratory (vdrl), rapid plasma reagin (rpr), and other tests in response to the interaction of infected host tissue with t. pallidum. they parallel the pathologic course but have no relation to immunity. treponema-specific antibodies have a higher serologic sensitivity in the early stages of syphilis but are less effective indicators of disease activity. during the first 3 weeks after primary infection, the vdrl is positive in 30% of cases, and the fluorescent treponemal antibodyabsorption (fta-abs) test is positive in 50%. other treponemal antibody tests, often used to confirm nontreponemal tests, include t. pallidum particle aggregation (tp-pa) and recombinant antigen tests. an automated test for treponemal antibodies, pk (tm) treponema pallidum (pk-tp), performed on the olympus pk 7200, is widely used. [74] [75] [76] reaction patterns characterized by positive rpr or pk-tp tests and negative fta-abs reactions (socalled false-positive reactions) may be caused by hepatitis, mononucleosis, viral pneumonia, chickenpox, measles, immunizations, pregnancy, or laboratory error. persistent false-positive reactions have been reported in patients with rheumatoid arthritis, cirrhosis, ulcerative colitis, vasculitis, and older age. [74] [75] [76] among pk-tp-and fta-abs-positive blood donors, approximately half give a prior history of a treated syphilis infection. 75 a history of lupus, rheumatoid arthritis, and diabetes did not provide an explanation for nonconfirmed pk-tp results. the typical first sign of syphilis, a chancre, appears 3 to 90 days (average, 21 days) after exposure. the exact timing of spirochetemia and t. pallidum dissemination from the chancre and of seroconversion is not known. secondary syphilis, characterized by a disseminated rash and spirochetemia, occurs 6 to 8 weeks after infection. serologic tests are almost universally positive. if patients remain untreated, recurrent fulminant secondary syphilis recurs within 2 years in approximately 20%. subsequently, patients become immune to reinfection and become noninfectious. vdrl titers decrease over time. unless patients are treated in the primary stage, treponemal antibodies persist in both treated and untreated patients. tertiary syphilis develops after a variable length of time. reactivation is clinically and serologically noticeable via anticardiolipin and treponemal antibody detection. 72, 74, 76 currently, donations with reactive syphilis screening tests are unsuitable unless nonreactive in a confirmatory test. if the confirmatory test is positive, donors are deferred for 1 year; they are then allowed to donate again, provided that they have undergone adequate treatment for syphilis, and a nontreponemal assay is negative. 76 human parvovirus b19 was discovered serendipitously in human plasma during blood-donor screening for hepatitis b surface antigen in 1975. initially, parvovirus was linked causally with transient aplastic crises in patients with sickle cell anemia and subsequently in patients with other inherited hemolytic diseases, as a result of severe reticulocytopenia and anemia. b19 was later found to be the etiologic agent of fifth disease or erythema infectiosum, a common childhood illness that manifests as an erythematous rash. 77, 78 the rash occurs less often in infected adults than children. fever and nonspecific symptoms precede the rash and arthralgia, both of which probably result from immune complex deposition in the skin and other organs. hepatitis, myocarditis, vasculitis, and the gloves-and-socks syndrome have also been linked to b19 infection (fig. 48-4) . b19 infects only humans, and transmission occurs most commonly via the respiratory route. in addition, transplacental transmission of parvovirus b19 occurs in 30% of women infected during pregnancy. in women infected during weeks 9 to 20 of pregnancy, hydrops fetalis and fetal death occur in approximately 11%. 79 the virus is highly tropic for erythroid progenitor cells, gaining access to cells through the blood group p antigen, or globoside, which has been identified as the virus receptor. 77, 78, 80 the viral genome consists of single-stranded dna that codes for three proteins. the nonstructural protein, ns1, is cytopathic to host cells. viral protein 1 and viral protein 2 code for î±-helical loops that appear on the capsid surface. neutralizing antibodies recognize vp1. the nonenveloped virus consists of symmetric particles 25 mm in diameter. b19 infection is ubiquitous in human populations and is already prevalent in pediatric age groups. seroprevalence studies show antibody frequencies of 50% in high schoolage children and up to 90% in older adults. 77, 78 epidemics and sporadic infections may occur at any time of year, with major outbreaks of erythema infectiosum occurring every 3 to 6 years. persistent parvovirus infection, including pure rbc aplasia, occurs in those not developing neutralizing antibodies to vp1. the virus circulates at high titer, greater than 10 12 genome copies per milliliter. 77, 78 patients receiving cytotoxic chemotherapy, immunosuppressive drugs, organ transplant recipients, and patients with immunodeficiency and the acquired immunodeficiency syndrome (aids) are at higher risk of developing chronic infections. the therapeutic approach for persistent parvovirus infection involves discontinuing immunosuppressive therapy, administering intravenous immunoglobulin (ivig) preparations, instituting antiviral therapy for aids patients, and giving repeated courses of ivig as needed. 77, 78 the transient 1-to 2-week, high-titer viremia accompanying acute asymptomatic b19 infection allows virus transmission by blood, blood derivatives, and organ transplantation. 77, 78, 81 the infrequent recognition of transfusion-associated cases reflects the short viremic phase and the high frequency of immunity among transfusion recipients. in contrast to recipients of blood transfusions, almost all recipients of plasma-derived factor viii and ix concentrate are at risk for b19. parvovirus circulates in the blood of approximately 1 in 800 plasma donors. 90 fourteen percent had titers between 10 4 and 10 7 genome equivalents per milliliter, and 1 in 13,000 had greater than 10 7 genome equivalent per milliliter. not surprisingly, in plasma derivatives prepared from large-scale plasma pools, pcr testing detects parvovirus b19 in most lots. in observational studies, recipients of solvent/detergent-treated plasma seroconverted after infusion of products with high-titer parvovirus dna, 10 7.5 to 10 8.5 genome copies per milliliter, suggesting that the presence of anti-b19 antibodies was not protective against large viral loads. seroconversion did not occur among recipients of lots with viral titers between 10 0.5 and 10 3.5 genome copies per milliliter. 78 the virus is resistant to viral-inactivation steps such as solvent/detergent treatment and heat after lyophilization or in the vapor stage. heat may reduce infectivity if applied in the liquid state. children receiving plasma-derived factor viii concentrates were at least 1.9-to 7.6-fold more likely to be b19 seropositive than were those receiving no product or recombinant-derived anti-hemophilic factor. 87 parvovirus seemingly becomes concentrated in the plasma fraction used in factor viii preparations. despite the high frequency of parvovirus exposure, long-term sequelae appear subtle. 82, 87 for example, parvovirus b19-seropositive hemophilic children had an 8-degree loss in joint range of motion, a 0.48% difference, compared with seronegative children. unlike factors viii and ix, albumin has not transmitted parvovirus b19. [82] [83] [84] [85] [86] [87] [88] one report implicated parvovirus transmission by ivig based on detection of viral dna by pcr. 89 however, no documentation showed the same viral genotype in the recipient and the immunoglobulin preparation. in light of these data, in 1999, regulatory agencies and manufacturers of plasma derivatives sought to reduce b19 dna levels below 10 4 genome copies per milliliter in plasma pools containing 1000 to 3300 plasma donations. however, subsequent reports showed that parvovirus transmission occurred in a recipient of solvent/detergent-treated antihemophilic factor containing 1.3 ã� 10 3 genome equivalents per milliliter 83 and a recipient of a dry-heat-treated factor viii product containing 4 ã� 10 3 genome equivalents per milliliter. 85 in these cases, smaller plasma batches with high viral loads were combined to form larger pools used in manufacturing the antihemophilic factor concentrates. currently, manufacturers conduct "in-process" testing to eliminate plasma donors with high-titer b19 levels. for example, "recovered plasma" (plasma obtained from wholeblood donations) that is intended for fractionation into plasma derivatives undergoes b19 dna testing via nucleic acid amplification testing (nat) assays on aliquots from pooled samples. subpool analyses are performed to determine which of the samples contained the high-titer donor. these high-titer units, approximately 1 per 10,000 donations, are withheld from product manufacture. because the infection is transient and a carrier states does not exist, the infected donor is not identified specifically or permanently deferred. because whole-blood donations rarely transmit parvovirus infections, testing of single unit rbcs, platelets, or plasma for parvovirus b19 is not under consideration in the united states. transmissible spongiform encephalopathies (tses) occurring in humans include kuru, cjd, gerstmann-strã¤ussler-scheinker disease (a phenotypic variant of cjd), fatal familial insomnia, and variant cjd (vcjd). tses occurring in animals include scrapie (sheep and goats), wasting disease of deer and elk, transmissible mink encephalopathy, and bovine spongiform encephalopathy. [91] [92] [93] the tse infectious agents are classified as prions, or proteinaceous infectious particles that lack nucleic acid. tses resist inactivating agents such as alcohol, formalin, ionizing and ultraviolet irradiation, proteases, and nucleases but are disrupted by autoclaving, phenols, detergents, and extremes in ph that affect proteins. the normal host membrane prion protein prp (designated prp c ), whose function is unknown, is protease sensitive, soluble, and has a high î±-helix content. all prion diseases appear to involve conformational modification of prp c to a protease-resistant altered isoform that forms amyloid fibrils (designated prp sc ). the conversion of prp c to prp sc results in refolding of a portion of the î±-helical and coil structure of prp c into î²-sheets. neuronal loss and vacuolization leads to a spongioform appearance in the brain cortex and deep nuclei. cjd occurs at an incidence of 0.5 to 1.5 cases per 1 million population worldwide. this rate has increased slightly over the past decade presumably on the basis of improved diagnostic accuracy and greater numbers of older individuals. 92, 94 fewer than 300 cases per year are reported in the united states. sporadic cjd, causing approximately 85% of cjd cases, occurs in persons 40 to 80 years of age (average age at onset is 60 years) and is manifested by disordered sleep and decreased appetite, behavioral or cognitive changes or focal signs such as visual loss, cerebellar ataxia, asplasia, and motor deficits. 91 the mean survival time is 5 months. the mode of infection for sporadic cjd is uncertain. approximately 10% to 15% of cjd cases occur in patients with a family history of cjd, suggesting an autosomal dominant inheritance pattern and mutations in the prmp gene that codes for the prion protein. more than 50 mutations in this gene, located on the short arm of chromosome 20, have been identified, but 4 point mutations at codons 102, 178, 200, and 210 occur in 95% of familial cases. 91 approximately 1% of cjd cases involve iatrogenic transmission. for example, cjd was transmitted by a corneal transplant from a patient with undiagnosed cjd, whereas stereotactic electroencephalographic silver electrodes previously implanted in a patient with cjd subsequently resulted in two iatrogenic cjd cases. 91, 92 more than 130 young adults have died 5 to 30 years after receiving intramuscular human growth hormone injections prepared from cadaveric pituitary glands from donors with unsuspected cjd. 91, 95 cadaveric dura mater grafting with a commercial product prepared by batch processing resulted in at least 100 cjd cases worldwide, some occurring 18 years after graft placement. 91, 96 in sporadic and iatrogenic cases of cjd, a polymorphism involving codon 129 in the prp gene appears to affect susceptibility. normally 37% of the population are methionine/methionine homozygous, and 11% are valine/valine homozygous at codon 129. the remaining 52% are heterozygous. homozygous individuals represent almost 90% of sporadic and iatrogenic cjd cases. 96, 100 those homozygous for methionine are at risk for fatal familial insomnia, whereas those homozygous for valine are at risk of clinical cjd. 91 in experiments involving mice infected with a strain of gerstmann-strã¤ussler-scheinker disease, blood-component infection was demonstrated. 97 in contrast, no evidence of transfusion-associated cjd was documented in case-control studies involving more than 600 patients with cjd or in recipients of blood from persons who subsequently developed cjd. [98] [99] [100] examination of brain tissue from deceased hemophilia patients showed no evidence of cjd. 91, 101, 102 no transfusion-associated cjd cases have been reported to date. nonetheless, the occurrence of iatrogenic cases and the theoretical risk of cjd transmission by blood led the fda to issue a recommendation to defer donors if they have one or more blood relatives with cjd or if they have received human pituitary-derived growth hormone injections or a dura mater transplant. all in-date products from donors with these risk factors must be quarantined and destroyed, and the previous recipients of blood from implicated donors, with the exception of those who have only one family member with cjd, must be notified. 92 in the spring of 1985, several dairy cows in the united kingdom displayed aggressive behavior, ataxia, and falling. these "mad cows" were found to have spongiform lesions in brain tissue resembling scrapie that was subsequently termed bovine spongiform encephalopathy (bse). more than 180,000 cattle succumbed to bse, but almost 1 million may have been infected. because the mean incubation period for bse is 5 years and most cows were slaughtered between 2 and 3 years of age, most cattle did not manifest disease. [91] [92] [93] approximately 50,000 bse-infected cattle entered the food chain before the first bse case was recognized in 1986. subsequently, the onset of the bse epidemic was traced to a meat-and-bone cattle feed made from sheep, cattle, and pig offal. the rendering process presumably resulted in the feeding of scrapie-infected material to cows. use of sheep offal or other tissues from ruminant animals as feed for other ruminant animals was banned in 1989. the annual incidence of clinical cases in cattle peaked in 1992. after march 1996, only animals younger than 30 months were eligible for food preparation. 93 surveillance for human cjd cases heightened in the united kingdom after recognition of the bse epidemic. ten of 207 cjd patients in 1994 and 1995 had unusual neuropathologic changes. 103, 104 they had predominantly psychiatric and sensory symptoms, ataxia, dementia, and myoclonus. all were younger than 45 years, a distinctly unusual characteristic for cjd. electroencephalographic features were not typical of cjd, and florid prp plaques were seen on neuropathologic examination. median survival time was 14 months, in contrast to 4 months for cjd. these cases were considered a new variant of cjd (vcjd). the median incubation period for food-borne vcjd is 13 years. 105 extensive investigations using animal models provided evidence that the same prion strain causes bse and vcjd. 106 ingestion of british beef, therefore, was identified as a risk factor for bse. 107 as of june 2006, 159 cases of vcjd have been reported in the united kingdom, 17 in france, 4 in ireland, 1 each in portugal, spain, italy, the netherlands, saudi arabia, japan, and canada, and 2 in the united states. the latter three plus one irish patient were thought to result from exposure in the united kingdom. the japanese patient spent only 24 days in the united kingdom. all patients tested were homozygous for methionine at prp codon 129. by 2000, the incidence of human vcjd cases peaked, suggesting that clinical manifestations among methionine/methionine homozygotes may be less than anticipated after extensive exposure to cattle with subclinical disease. 105, 108, 109 concern about transfusion transmission of vcjd increased because prp sc is found consistently in the lymphoreticular system of vcjd patients, the possibility that circulating prions transfer the infection from the gut to the brain, and eventually because of animal studies. 105, 108, 109 in animal model experiments, sheep were fed aliquots of brain obtained from bse-infected cattle. subsequently, the sheep underwent phlebotomy at periodic intervals. among 24 sheep receiving blood from iatrogenically infected donor sheep, 2 given blood from donors in the preclinical bse phase developed bse, and 2 receiving blood from clinically affected sheep showed clinical signs of bse. among 21 sheep transfused with blood from natural scrapie-infected animals, 4 demonstrated clinical signs of scrapie. 110, 111 an active investigation to determine whether transfusion associated-vcjd transmission occurs in humans began in the united kingdom in 1997 by identifying vcjd patients who donated blood before illness. eventually, 48 recipients of blood from 15 donors with vcjd were identified. three of the 48 recipients, to date, have evidence of vcjd. one, at age 62 years, received non-leukocyte-reduced rbcs from a 24year-old donor who developed vcjd 3.5 years after the blood donation. the transfusion recipient developed vcjd 6.5 years after transfusion. 112 the second patient received a transfusion of non-leukocyte-reduced rbcs in 1999. 113 the donor developed vcjd 18 months later. the asymptomatic recipient died of a ruptured abdominal aortic aneurysm 5 years after transfusion. at autopsy, protease-resistant prions were present in the spleen and cervical lymph nodes. prions were not detected in the brain. the recipient, found to be heterozygous (methionine/valine at codon 129), did not have clinical vcjd, raising concern that the incubation period may be longer in codon 129 heterozygotes. in animal studies, a primary challenge with vcjd prions resulted in a significantly reduced transmission rate in mice with valine at codon 129 compared with that in animals homozygous for methionine. 114 additional data are needed to confirm whether the incubation period varies among methionine homozygous and heterozygous individuals. the third case developed vcjd approximately 8 years after receiving non-leukocyte-reduced red cells from a person who developed vcjd 20 months postdonation. 114a the u.k. national blood service also determined that approximately 100 people donated blood to four patients who subsequently showed clinical signs of vcjd. these donors were notified that they may be at higher risk of developing vcjd despite the uncertainty of whether the patients contracted vcjd through food or blood transfusion. in addition, uk authorities notified recipients of factor xi concentrates that donors of these components developed vcjd after donation on the ethical tenet of transparency. the united kingdom currently imports plasma from the united states for patients younger than 16 years and uses apheresis-derived platelets in these patients to reduce donor exposures. 94, 116 the identification of presumed transfusion-associated vcjd cases appears to validate the steps taken in response to the precautionary principle to decrease the risk of transmitting vcjd by transfusion. 92 donors who visited or resided in the united kingdom for a cumulative period of 3 months or longer between 1980 and 1996 are deferred indefinitely. donors who spent 5 years or more in europe before 1980 and the present are also deferred. in addition, donors are indefinitely deferred if they injected bovine insulin after 1980, received transfusions in the united kingdom and france between 1980 and the present, or served in the military on bases in europe for 6 months or more between 1980 and 1996. this geography-based donor-deferral protocol evolved in various phases beginning in 1999. approximately 3.5% of potential donors in the united states have been deferred as a result of this policy. 117 the impact was higher in canada. 118 in-date blood components and plasma intended for derivative production from these donors must be recalled, quarantined, and destroyed. ongoing surveillance of vcjd cases, which increased after identification of a texas cow with bse, is currently being conducted, including a recommendation to notify the cdc about all patients younger than 55 years who are diagnosed with cjd. 115 in addition to geographic exclusion policies, other strategies for preventing vcjd transmission include removal of the infectious agent and testing. in the united kingdom, all blood components undergo leukocyte reduction by filtration, based on observations that prions associate with leukocytes. leukocyte reduction, however, is only partially effective, removing only 42% of total prion infectivity. 120 filters that specifically remove prions and laboratory tests that detect infectious prions are currently being developed and evaluated. the latter, if implemented, will be accompanied by significant ethical concerns. 119 visceral forms of leishmaniasis result from infection with leishmania donovani or leishmania infantum. cutaneous lesions occur in persons infected with leishmania braziliensis or leishmania tropica, the cause of old world cutaneous leishmaniasis. however, at least eight soldiers returning from eastern saudi arabia after operation desert storm developed visceral leishmaniasis that was attributed to l. tropica. 121, 122 the leishmania organisms, transmitted primarily by bites from infected sand flies, are endemic in the tropical and subtropical regions of the sudan, eastern india, bangladesh, nepal, brazil, and the mediterranean. 123 after transmission by sand fly bite, parasites reside intracellularly in monocytes, which circulate before taking up residence in internal organs. in the most severe manifestation of visceral leishmaniasis, kala-azar, patients have marked hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and cachexia. the incubation period is approximately 6 months. 123 anti-l. donovani antibodies form shortly after infection. in studies conducted in brazil, 21 seropositive asymptomatic blood donors were found to have positive pcr results for l. donovani, demonstrating the ongoing potential of transfusion transmission in endemic areas. 124 at least 10 transfusion-associated cases of leishmaniasis attributed to l. donovani have been reported in endemic regions. most of those infected were young children or neonates. a probable case of platelet transfusion-transmitted leishmania was reported recently. 125 transfusion-transmission also appears to occur in dogs receiving transfusions of rbcs from seropositive dog-blood donors. 126 veterans of operation desert storm who served in the persian gulf region between august 1990 and december 1992 were deferred from blood donation for 1 year, after the report of l. tropica-related viscerotropic leishmaniasis. the patients had nonspecific clinical manifestations, including prolonged fever, malaise, abdominal pain, and intermittent diarrhea, which occurred up to 7 months after they returned to the united states. 121 l. tropica was found in the bone marrow of seven patients and in a lymph node in one patient. intracellular amastigotes were seen in the peripheral blood of the one patient in whom this was studied. 127 after reports of hundreds of cases of cutaneous leishmaniasis and two cases of visceral leishmaniasis in troops involved in the iraq war, a similar 1-year deferral after departure from iraq was instituted in october 2003. 127 l. tropica within human monocytes survives in blood stored at 1â° c to 6â° c, in frozen rbcs, and in platelet concentrates stored at room temperature. however, l. tropica has not been detected in relatively cell-free fresh frozen plasma. animal studies demonstrate transmission by contaminated blood. 126 no cases of transfusion-transmitted leishmaniasis have been reported in the united states to date. for this reason, surveillance and targeted donor deferral appear to be appropriate. use of leukocyte filters to reduce leishmania transmission is under investigation. 128 toxoplasma gondii is a ubiquitous parasite whose usual host is the domestic cat. infection sometimes results in lymphadenopathy, malaise, fever, headache, sore throat, splenomegaly, hepatomegaly, and rash. retinopathy and lethal infections occur in immunocompromised hosts. transfusion transmission was reported in 1971. however, the cases occurred among leukemia patients given granulocyte transfusions obtained from other leukemic patients. another case report suggested that a patient undergoing chemotherapy for a leukemic relapse 3 years after receiving an allogeneic marrow transplant developed toxoplasma pneumonitis. a person with serologic evidence of recent toxoplasma infection donated one of the units of blood transfused to the patient. 130 in addition, a 52-year-old woman with drug-induced thrombocytopenia developed toxoplasma retinochoroiditis, presumably related to a platelet transfusion. 130 a case further emphasizing the importance of nontraditional routes of infection in immunocompromised patients involved a renal transplant recipient who developed toxoplasmosis. 131 the infection was presumably transmitted by a kidney obtained from a seropositive organ donor. dengue, transmitted by aedes mosquitoes, has infected at least 77 u.s. travelers to caribbean islands (including puerto rico and the u.s. virgin islands), pacific islands, asia, central america, africa, and hawaii between 2001 and 2004. 132, 133 the incubation period is 3 to 14 days. infections cause either no symptoms, mild illness, or severe disease including hemorrhagic manifestations and shock. 134 transmission by bone marrow transplantation and several reports of transmission after needle-stick injuries involving symptomatic patients raise the possibility of transfusion transmission by asymptomatic travelers returning from endemic areas. [135] [136] [137] more than 70% of nonhuman primates in zoos or in animal research facilities are infected with simian foamy virus (sfv), an endogenous, cell-associated retrovirus found in new and old world primates. 138 surveillance studies indicate that approximately 5% of zoo and biomedical research personnel working with chimpanzees and baboons are infected with sfv. evaluation of archival samples documented infection for 8 to 26 years (median, 22 years). all subjects remained healthy, and each of three spouses undergoing testing for sfv were nonreactive. in addition, 1% of bush hunters in central africa and 1% of those exposed to free-ranging nonhuman primates in asia tested positive for sfv. 139, 140 presumably, those infected were inoculated through exposure to saliva from bites or close contact through exposure to body fluids. only limited information is available about transfusion transmission. one occupationally exposed sfv-infected individual donated blood 6 times during an interval when sfv test results, conducted retrospectively, were positive. none of the tested recipients of rbcs, leukocyte-reduced rbcs, or platelets was sfv positive. three of these blood components were stored for less than 8 days. 141 infections with lymphocytic choriomenigitis virus (lcmv), a rodent-borne arenavirus, usually cause mild, self-limited illness or aseptic meningitis in nonimmunosuppressed patients. human infections typically follow exposures to body fluids or infected animal excretions. vertical transmission occurs, but lcmv is not considered to be communicable from person to person. lcmv has been transmitted to four organ transplant recipients via an asymptomatic organ donor who had a cerebrovascular accident and subsequent brain death. 142 the donor apparently became infected by exposure to a pet hamster. within 3 weeks of transplantation, the recipients of the liver, lungs, and two kidneys developed fever, rash, or diarrhea; three of the four recipients died. a previous case also involving four transplant recipients was unrecognized until this case was reported. transfusion transmission has not been reported but is a possibility, given transmission by solid-organ transplantation. the avian influenza a/h5n1 virus has spread epidemically among birds and poultry since emerging in hong kong in 1997. 143 since that time, more than 100 million birds and poultry have died or been culled to prevent epidemic progression via bird migration in cambodia, china, indonesia, japan, laos, south korea, thailand, vietnam, malaysia, turkey, romania, and russia. transmission to humans via contact with infected poultry or contaminated surfaces has resulted in more than 60 deaths. to date, human-to-human transmission has occurred infrequently. 144 however, concern exists that mutations, reassortments, or recombinant rearrangements of the virus with pathogenic human influenza viruses could produce a virus capable of jumping the species barrier and causing a worldwide pandemic. influenza viremia is infrequent, although highly pathogenic avian influenza can be transmitted via blood. 145 although transfusion transmission is a theoretical risk, a more likely impact would be large-scale donor illness and blood shortages. sars, caused by a novel enveloped rna coronavirus, infected more than 1800 patients in 17 countries in february and march 2003. 146 this highly contagious illness dominated worldwide public health attention, resulting in rapid identification, travel advisories, patient quarantine, and eventual eradication of the epidemic. 147 the 2-week asymptomatic incubation period fostered spread of the virus through close person-to-person contact and raised the possibility of blood-borne infection. for this reason, the u.s. fda issued a guidance document in april 2003 requiring blood-collection agencies to defer anyone from donating blood for at least 14 days after possible exposure to sars. 148 those with a suspected sars illness were deferred for at least 28 days after recovery. notices were posted in blood centers apprising donors about sars-affected areas, and donors were asked about recent travel history. those traveling to affected areas, including transit in an airport at these locations, were deferred from blood donation. the epidemic subsided within months. no reports of transfusion-associated sars exist. west nile virus (wnv) is a mosquito-borne, lipid-enveloped, rna virus in the japanese encephalitis flaviridae complex. the viral genome codes for capsid, membrane, envelope, and nonstructural proteins. 149 the virus, transmitted from bird to bird by mosquito vectors, infects humans as incidental hosts. the virus was identified in the west nile district of uganda in 1937. outbreaks occurred subsequently in the mid-east, south africa, and europe. the first north american cases were recorded in new york city in 1999. 150 154 in addition, 372 viremic blood donors were identified. wnv activity in birds and mosquitos occurs throughout the year, especially in warmer regions. the virus becomes detectable in blood 1 to 3 days after a mosquito bite, followed by an increase in viral loads. however, peak titers are relatively low (median of 3500 copies per milliliter) compared with hiv and hcv (10 5 to 10 7 per milliliter). rna levels decrease markedly 7 to 10 days after infection when immunoglobulin m (igm) antibodies, and subsequently igg anitibodies, appear. igm antibodies persist for more than 398 days in approximately two thirds of those infected. 155 the mean duration of viremia is 6 days. however, wnv rna was detected up to 104 days after infection in one blood donor. [157] [158] [159] [160] [161] [162] approximately 80% of persons infected with wnv remain asymptomatic. the 20% with symptomatic infections report abdominal pain, chills, fever, generalized weakness, headache, joint pain, muscle weakness and pain, new macular rash on the trunk and extremities, new difficulty thinking, painful eyes, and swollen glands. 163 one in 150 infected persons develops meningitis, encephalitis, or asymmetric flaccid paralysis. fatal outcomes occur in 4% to 14% of those with severe disease. wnv transmission in four recipients of organ donations was reported in 2002. 163 the organ donor, in turn, received blood transfusions from 63 donors, one of whom subsequently was found to be wnv infected. a sample from the organ donor subsequently tested wnv rna positive, but wnv igm negative. initial reports of transmission by blood transfusion in 2002 eventually resulted in confirmation of 23 cases of transfusion-associated wnv infection. 162 the interval between transfusion and symptom onset was 10 days (median interval range, 2 to 21 days). nine of 14 implicated blood donors reported wnv-associated symptoms before donation. after intense collaboration among u.s. public health authorities, test manufacturers, and blood-collection agencies, nat for wnv rna was implemented before the 2003 wnv season. as a direct result of testing, more than 1000 donors were found to be wnv rna positive in 2003, preventing wnv transmission to approximately 1500 recipients of rbcs and components prepared from these donations. 156, 157, 159 during the summer months, approximately 1 per 7000 units was wnv rna positive. in high wnv endemic areas, 1 in approximately 150 donors was wnv viremic. in 2003, six transfusion-associated wnv cases were reported. 158 all of the implicated donors had extremely low-level viremia that escaped detection by routine testing in minipools containing aliquots from 6 to 16 donations. testing of individual samples in high-incidence areas increases test sensitivity by approximately 7% and was introduced in 2004 when incident cases exceeded preestablished thresholds (approximately 1 wnv-positive donor per 1000 donations). only one confirmed transmission occurred in 2004. among the 30 confirmed transfusion cases, all implicated donations were wnv igm antibody negative. 164, 165 a second transplant-associated incident involving three of four organ recipients who developed wnv infection after transplant was reported in 2005. 166 of note, the organ donor (infected through mosquito bites) was wnv rna positive and igm antibody positive. this report raises concern that organ-transplant recipients and other heavily immunosuppressed patients are at extremely high risk for severe wnv complications and that the virus remains viable in organ/ tissue reservoirs despite a humoral immune response. overall, the rapid implementation of wnv testing within months of the initial transplant and transfusion-associated cases resulted in dramatic reduction of further transfusiontransmitted cases. assuming that rna-positive, igm antibody-positive donors do not transmit wnv through blood transfusion, the residual risk of transfusion-associated wnv after implementation of nat is approximately 1 per 350,000 donations. 164, 165 in june 2004, the cdc confirmed the diagnosis of rabies in recipients of a liver and two kidneys from an organ donor subsequently found infected with rabies via a bat bite. the recipients developed tremors, myoclonic jerks, altered mental status, or anorexia 21 to 27 days after transplant. all died. it is unlikely that these transplant-related cases portend a risk for transfusion-transmission, in that exposure to infected neuronal tissue appears to be the vector in these cases. the rabies virus is not transmitted hematologically, and contact with blood, urine or feces is not considered an exposure risk. 167 days post infectious mosquito bite wnv rna (geq per ml) emerging infectious diseases: a clear and 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[two cases of transfusional malaria parasitic infections and their impact on blood donor selection and testing le paludisme transfusionnel: risque, prã©vention et coã»t the indirect fluorescent antibody test for the detection of occult malaria in blood donors maxcy-rosenau-last textbook of public health and preventive medicine department of health and human services, food and drug administration emerging chagas disease: trophic network and cycle of transmission of trypanosoma cruzi from palm trees in the amazon threats to blood safety posed by emerging protozoan pathogens american trypanosomiasis (chagas' disease): a tropical disease now in the united states strategies for prevention of transfusionassociated chagas' disease intervention strategies to reduce the risk of transfusiontransmitted trypanosoma cruzi infection in the united states trypanosoma cruzi in los angeles and miami blood donors: impact of evolving donor demographics on seroprevalence and implications for transfusion 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epidemic analytical and clinical sensitivity of west nile virus rna screening and supplemental assays available in 2003 transmission of west nile virus through blood transfusion in the united states in 2002 and investigations of west nile virus infections in recipients of blood transfusion and organ tranplantation insights on donor screening for west nile virus problem solved? west nile virus and transfusion safety nile virus infections in organ transplant recipients: new york and pennsylvania cdc. investigation of rabies infections in organ and transplant recipients key: cord-308614-gsgntf4c authors: eshar, david; weinberg, maya title: venipuncture in bats date: 2010 journal: lab anim (ny) doi: 10.1038/laban0610-175 sha: doc_id: 308614 cord_uid: gsgntf4c though not as common as small rodents in laboratory settings, bats are being increasingly used in research studies. knowledge of proper blood sampling techniques is essential for care and management of bats. to minimize handling and to avoid sample failure, all needed equipment should be prearranged and organized in advance. equipment needed includes a 1-to 3-ml syringe or 0.5-ml insulin syringe; 25-or 27-gauge needles; microtainer collection tubes with heparin and calcium-edta; microhematocrit capillary tubes and sealing clay; glass slides; chlorhexidine-based scrub equivalent to 1% of the total body weight at each draw. several bat species can act as reservoir hosts of zoonotic pathogens including lyssaviruses, severe acute respiratory syndrome (sars) coronavirus, ebola virus, henipaviruses, west nile virus, st. louis encephalitis virus and leptospira spp. 2, 5 . all personnel working with bats should be vaccinated against rabies 2 . coats and gloves should be used to minimize contact with body secretions, such as blood or saliva, that may contain infectious organisms 2 . manual restraint is usually not a problem in bats, but healthy bats can wriggle excessively unless properly restrained. all restrained bats can deliver a defensive bite, and the digits and associated claws of large bats can scratch an unprotected handler or pull fingers in for a bite 2 . bats should be restrained using either protective thick gloves or a towel to hold the head and the limbs (fig. 3) . the duration of manual restraint should be minimized to reduce stress and prevent hyperthermia and marked elevations in cortisol and glucose 6 . stressed bats may require chemical restraint, either by inhalation or by injection, although chemical restraint may have complicating side effects. bats should be anesthetized for procedures requiring close contact to reduce risk of biting and transmission of secretions 2 . bats are homeotherms, maintaining their body temperature between 35 °c and 39 °c (ref. 2). additionally, large lungs and naked flight membranes result in greater heat loss (6 times greater) and thermal conductance in recent years, interest in research using bats has grown. these small mammals have many unique features that attract great attention and can now be more commonly found in many research centers 1 . second to rodents, bats are the most abundant and diverse mammalian group in nature. the order chiroptera is divided into suborders microchiroptera and megachiroptera, with further subdivision into 18 families and 1,116 recognized species. bats used in studies are either wildcaught or originate from captive breeding colonies 1 . blood testing in bats may be done as part of a clinical evaluation or for hematological research. sites for blood collection from bats include the median (brachial) vein, cranial vena cava, jugular vein, orbital sinus and heart [2] [3] [4] . venipuncture of the cephalic and the saphenous (interfemoral) veins are two of the best techniques for quick and safe collection of blood in bats. these nonterminal techniques can be used to collect small to medium volumes of blood with good visualization of the blood vessels and can be done on anesthetized or manually restrained, unsedated bats. the cephalic vein is located along the leading edge of the patagium or the antebrachial wing membrane (fig. 1) . the saphenous (interfemoral) vein is found in the uropatagium (tail membrane) parallel to the femur (fig. 2) . the blood volume of bats comprises ~10% of the total body weight (9.0-11.0 ml per 100 g) 2 . as in other species, it is considered safe to remove a blood sample david eshar, dvm 1 though not as common as small rodents in laboratory settings, bats are being increasingly used in research studies. knowledge of proper blood sampling techniques is essential for care and management of bats. can also be centrifuged so that plasma can be removed and frozen until later analysis 7, 8 . cephalic and saphenous (interfemoral) venipuncture is a non-terminal technique that allows simple, safe and efficient blood sampling in bats of various sizes. be used for the venipuncture using a chlorhexidine-based scrub solution. for most cases, a 25-gauge needle with a 1-ml syringe or a 0.5-ml insulin syringe with attached 27-gauge needle is used (fig. 2) . once the needle is inserted into the vessel and blood is identified in the needle hub, a gentle negative pressure (suction) is intermittently applied until the desired sample volume is obtained. care should be taken not to use excessive pressures that can cause collapse of the vein or hemolysis of the blood sample. alternatively, microhematocrit capillary tubes can be used for sample collection. a 25-gauge needle can be used to puncture the vessel in small bats, and the tube can be inserted into the hub of the needle to collect the sample, or if a vein is accidently punctured, blood can be collected into a microhematocrit capillary tube directly from the incision site 2 (fig. 1) . bats' wing membranes are very thin and lack subcutaneous fat to aid in hemostasis of the punctured blood vessels. the venipuncture site should be gently pressed using a piece of cotton gauze until bleeding completely stops. special attention should be given to small bats because large hematomas and ongoing bleeding can result in severe and even life-threatening blood loss. once back in its cage, the bat should be closely monitored for renewed bleeding resulting from wing flapping. bat blood can altered by inappropriate collection, handling and storage 2 . moderate hemolysis during sample collection can cause test results to show hyperkalemia, hyponatremia, hypochloremia and elevated levels of aspartate transaminases 8 . the sample should be processed within 6 hours of sampling, as prolonged contact between the resting plasma and the red cells causes elevation in phosphorus and decreases in chloride and glucose concentrations 8 . blood samples treated with heparin can be analyzed immediately after collection and solution; leather and non-sterile gloves; and chemical restraint agents (as needed). some bat species weigh less than 100 g, and in many cases, only a small sample is obtained. hence, we prefer to use blood analyzers (such as the abaxis vetscan chemistry analyzer) that require only small blood sample volumes (0.l ml of whole blood or plasma). to avoid clotting and to reduce sample collection time, syringes should be prepared with heparin in advance 7 . venipuncture of the cephalic and saphenous veins without anesthesia usually requires one phlebotomist and one or two individuals to restraint the bat. one assistant will hold the bat's head and body while a second assistant gently spreads the wing and holds the vein at its most proximal end, to allow the vessel to engorge with blood and dilate. for a right-handed phlebotomist, the right wing of the bat is extended while the left wing is securely held by the first assistant. the left hand of the phlebotomist is used to either compress the vein or add stability while the right hand holds the syringe. the venipuncture site can be warmpacked prior to the procedure to facilitate blood flow. aseptic techniques should figure 2 | venipuncture of the right saphenous (interfemoral) vein in the uropatagium of a male egyptian fruit bat (rousettus aegyptiacus) using a 1-ml syringe with a 27-gauge needle. the restrainer manually holds the bat's neck and feet using thick gloves to protect against bites. science and the conservation of bats chiropterans (bats). in zoo animal and wildlife immobilization and anesthesia a method of bleeding small bats a new method of bleeding small and infant bats public health awareness of emerging zoonotic viruses of bats: a european perspective basal, diurnal, and stress-induced levels of glucose and glucocorticoids in captive bats comparison of serum and plasma for determination of blood biochemical values in malaysian flying foxes (pteropus vampyrus) the effect of time at which plasma separation occurs on biochemical values in small island flying foxes (pteropus hypomelanus) key: cord-217663-3g2j9tnk authors: li, na; chiang, fei; down, douglas g.; heddle, nancy m. title: a decision integration strategy for short-term demand forecasting and ordering for red blood cell components date: 2020-08-17 journal: nan doi: nan sha: doc_id: 217663 cord_uid: 3g2j9tnk blood transfusion is one of the most crucial and commonly administered therapeutics worldwide. the need for more accurate and efficient ways to manage blood demand and supply is an increasing concern. building a technology-based, robust blood demand and supply chain that can achieve the goals of reducing ordering frequency, inventory level, wastage and shortage, while maintaining the safety of blood usage, is essential in modern healthcare systems. in this study, we summarize the key challenges in current demand and supply management for red blood cells (rbcs). we combine ideas from statistical time series modeling, machine learning, and operations research in developing an ordering decision strategy for rbcs, through integrating a hybrid demand forecasting model using clinical predictors and a data-driven multi-period inventory problem considering inventory and reorder constraints. we have applied the integrated ordering strategy to the blood inventory management system in hamilton, ontario using a large clinical database from 2008 to 2018. the proposed hybrid demand forecasting model provides robust and accurate predictions, and identifies important clinical predictors for short-term rbc demand forecasting. compared with the actual historical data, our integrated ordering strategy reduces the inventory level by 40% and decreases the ordering frequency by 60%, with low incidence of shortages and wastage due to expiration. if implemented successfully, our proposed strategy can achieve significant cost savings for healthcare systems and blood suppliers. the proposed ordering strategy is generalizable to other blood products or even other perishable products. blood transfusion is one of the most crucial and commonly administered therapeutics worldwide. the need for more accurate and efficient ways to manage blood demand and supply is an increasing concern in many countries, including canada. building a technology-based, robust blood product demand and supply system that can achieve the goals of reducing wastage and shortage, while maintaining the safety of the blood system, is essential in modern health care systems. canadian blood services (cbs) is the national blood supplier in canada (excluding québec 1 ). as shown in figure 1 , the current blood supply chain network in canada is a centralized regional network consisting of two levels: regional cbs distribution centres and hospital blood banks. for example, there is one cbs regional blood distribution centre in brampton, ontario that covers the demands from the majority of hospitals in ontario. there are nine regional cbs blood distribution centres across canada [1] . each regional centre sets priorities to meet the demands of its own network; if there is excess supply, cbs decides centrally where the products are to be reallocated. cross-matching, placing orders to cbs, receiving products from cbs, issuing products to patients, managing inventory • supply: blood from regional cbs distribution centre • demand: physician orders two-level supply chain with one regional blood centre and multiple hospitals currently, cbs has no information on recipient demographics and clinical utilization of the blood products distributed to hospitals. as a result, it has been very challenging for cbs to predict future demand and plan donor collection. for example, through this collaboration we discovered that decision makers at cbs have noticed a significant reduction in red blood cell (rbc) transfusions over recent years. they hypothesize this reduction could be caused by changes occurring in local hospitals, such as increased engagement on rbc clinical transfusion guidelines or improved surgical techniques. however, without clinical data, any such hypothesis cannot be investigated. in addition, the impact of clinical changes on the rbc demand cannot be evaluated. it is essential to maintain robust blood demand and supply management not only at the regional blood centre and individual hospital levels, but also as a complete supply chain. increasing the transparency of blood utilization between the national blood supplier and the hospitals is important to promote efficient blood supply chain management (bscm). at the level of hospital blood banks, current inventory management practices rely heavily on human decisions. there are three key limitations at hospital blood banks: 1) the blood product ordering decisions are made from human experience. there is little quantitative evidence of the fluctuations in rbc demands to support decision making. 2) since the electronic data systems in hospitals are primarily designed for clinical data collection, the systems do not have functions such as inventory reporting, forecasting or planning. 3) they are facing heterogeneous demand processes. hospital demands are made by physicians based on individual patient diagnoses with different requirements, such as multiple units issued at the same time and units with specific dose or brand requirements. these limitations increase the complexity of inventory management. as a result, hospital blood banks tend to cope with the variation in demand by holding excess inventory. while holding excess inventory can help hospital blood banks decrease the risk of shortages, it increases the holding costs for storing blood and the risk of wastage 2 . it may also lead to extra costs for reallocating units close to expiry. moreover, studies [2] [3] [4] [5] have shown that duration of rbc storage can affect functional integrity and quality standards which could impact patient outcomes. results of randomized trials suggest that there is no benefit to transfusing very fresh blood compared to blood stored for a longer duration (i.e. less than 10 days since blood donation compared with 24 days or more [6] ); however, it has been suggested that the clinical impact of blood storage over its 42-day shelf life may not be linear. more specifically, the risk of a specific outcome (i.e. mortality) by days of storage could be a convex curve [7] . through controlling inventory levels in hospital blood banks, it is possible to restrict the age of transfused blood into a reasonable range that may correlate with better patient outcomes. furthermore, from the national blood supplier's point of view, when hospital blood banks hold a large amount of inventory, it prevents cbs from understanding the real demand and restricts the ability of cbs to adjust for demand variability. as a result, both hospital blood banks and cbs cannot operate the demand and supply chain efficiently. with the availability of a large amount of clinical data and advanced analytical methodologies, there are opportunities to increase the accuracy of blood demand forecasting and improve the efficiency of the entire demand and supply chain. fresh blood components include rbc, platelets, and plasma. the rbc component experiences the highest demand among all the fresh components. it is prepared by removing plasma from a whole blood donation and is used to treat hemorrhages and to restore tissue oxygenation [8] . the demand for rbc components determines the plan for blood collection and production. in this study, we aim to tackle the demand forecasting and inventory management challenges for rbc components. we study a large clinical database with over 1.2 million blood transfusions for nearly 100,000 patients in hamilton, ontario from 2008 to 2018. we perform a thorough investigation of rbc utilization with associated trends, and formulate a hybrid model for short-term rbc demand forecasting using clinical indicators. the demand forecasting model is then used to develop an integrated ordering strategy. the proposed integrated methodology achieves three goals: i) a more accurate forecasting method that reflects the actual rbc demand at hospital blood banks, which increases the transparency between cbs and hospital blood banks; ii) a leaner and fresher inventory at hospital blood banks, which may correlate with better patient outcomes; iii) a simpler ordering strategy that requires less frequent orders on scheduled 2 throughout this paper, the terms "wastage" and "waste" are all referring to the blood products wasted due to expiration. days, which reduces human resources and costs both at hospitals blood banks and cbs. the main contributions of this study are as follows: 1. we summarize the key challenges in hospital blood banks and provide quantitative evidence to justify the need for a comprehensive methodology to resolve these issues. in section 2, we describe the inventory management process at hospital blood banks, and existing challenges. in section 3, we provide a literature review of demand forecasting models for blood products, methods for blood demand and supply chain management, and implementations for blood product management in healthcare systems. section 4 provides the data description, model background, model development, model training, model evaluation, and the proposed integrated ordering strategy for rbc inventory management. section 5 presents the results of the hybrid demand forecasting models for daily and semiweekly demand predictions, and the comparisons between the proposed ordering strategy and the existing strategy used in current hospital blood banks. section 6 discusses limitations and future opportunities of this study. this section describes the daily routine of blood product ordering and inventory management in a typical hospital blood bank. there are four hospital blood banks in hamilton, ontario operated by one all the blood products and fresh blood components in the hamilton region, as well as provide expert diagnostic, consultative and educational services for patients, physicians, and allied health care providers. hospital blood bank daily routine : the tm labs are responsible for ordering, storing, managing and issuing all fresh blood components including rbcs, platelet components, frozen plasma components, frozen cryoprecipitate and other experimental blood components such as covid-19 convalescent plasma. at mumc, every monday to saturday at approximately 4 pm, a technical specialist in the lab performs a routine physical count for each product in their inventory, compares the physical counts with the pre-defined inventory targets and sends an order to cbs (brampton) through fax. the order delivery from the cbs distribution centre usually arrives between 8:30 am and 10 am on the next day. cbs provides a paper-copy summary list of the delivered products with the shipment. the technical specialist compares this summary list with the order form they faxed to ensure they received all the products they requested. (feedback from the hospital blood bank indicates that only occasionally their orders are replaced, cancelled or delayed. however, due to the fact that there are no electronic data entries to trace the orders, a statistical summary of such information is not possible.) on the cbs side, a specific person manually transfers ordering information from faxed paper copies to an electronic spreadsheet 4 . the data are collected for cbs administration and operation purposes and are not shared with hospital blood banks. after a hospital blood bank receives blood products from cbs, the technical specialist enters the information into an electronic health system named meditech 5 . during the day, physicians may make prescription orders at any time through meditech or by fax to the hospital blood banks. usually, physicians make daily orders per patient, and the ordered products are available for pick up at different scheduled times. for every physician order, the technical specialist reviews the patient's historical clinical information which in this case is a custom meditech report. the report includes the most recent lab test results and antibody screening results. these are used to determine whether special transfusion requirements are needed. finally, a compatible product will be assigned to the patient. current ordering strategy: using their experience, the technical specialists at each hamilton hospital determine fixed inventory targets for different products. orders are made to raise inventory up to these targets. we find the targets set by hospital blood banks are significantly higher than the actual demands. for example, table 1 shows the inventory targets for rbc units by blood group at mumc. the total inventory target in the table is eight times larger than the mean daily demand at mumc, which has the smallest number of days of inventory on hand among all hospital blood banks in hamilton, ontario. more details are described in challenge 2 below. it is natural that hospital blood banks are most concerned about having sufficient inventory. however, without the quantitative evidence as we provide in this study, the consequence is that the order quantities cannot reflect fluctuations in the actual demand, resulting in excess inventory (prolonged days of inventory on hand), increased risk of wastage, and overly frequent same-day urgent orders. challenge 1: excess inventory level. the mean and standard deviation (sd) statistics in hamilton, ontario for the days of inventory on hand, age (days) of blood, and daily number of units in-stock are shown in table 2 table 2 . mean (sd) of days of inventory on hand (doh), age (days) of blood prior to transfusion, and daily number of units in-stock, as well as wastage rate by year in hamilton hospital blood banks challenge 2: large variation of the differences between ordered quantity and actual demand. the mean and sd statistics for the daily number of units received, transfused and their difference are shown in table 3 . as shown in the table, although the average difference between the number of units received (as a proxy of the order quantity by hospital blood banks on the previous day) and units transfused (actual demand) was close to zero, the standard deviation of the differences was very large. figure 2 (a) presents the boxplots of the differences between ordered quantity and actual demand by year from 2008 to 2018, which shows the ranges of the differences were wide for all the years and there was no observed trend across the years. in figure 2 weekday difference of units between received and transfused q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q challenge 3: frequent same-day urgent orders. we have described the process for routine ordering requests between hospital blood banks and cbs above. in urgent situations, non-scheduled orders that require same-day delivery can be placed. there are two types of urgent orders: "as soon as possible" (asap) orders and "stat" orders. asap orders are usually dispatched by parcel express, and stat orders are typically required in an emergency situation for bleeding patients, thus faster transportation methods such as taxis are used (the lead time for delivery of such orders is 2 to 3 hours to hamilton, ontario there was a small number of days with same-day urgent orders but without routine orders. of these orders, 37 (90.2%) occurred on weekends; 2) the same-day urgent orders were mostly made on different days for different hospitals. although the rate of same-day urgent orders may not be too concerning for individual blood banks, the pooled rate for all blood banks was significantly higher than the rate stratified by hospitals. this reflects the need for optimizing the inventory management as a network. table 5 presents the difference in means of demand patterns between dates with same-day urgent orders and dates with routine orders. the numbers of units received and transfused were much higher on dates with urgent orders, and there were significant differences of units transfused to trauma patients (doubled) and patients with abnormal laboratory tests (defined in table 6 ). table 4 . summary of rbc orders in hamilton hospital blood banks from april 1st, 2012 to may 31st, 2015 * hospitals a, b, c, and d represent the four hospital blood banks in hamilton, ontario. ‡ the denominators for the percentages of days with routine orders, stat orders, and asap orders are the number of days with any order in the last column. † the denominator for the percentages of days with any order in the last column is 1,156 calendar days during the period. it is interesting to observe the issues of excess inventory levels and over-frequent same-day urgent orders simultaneously exist. the data shows hospital blood banks made same-day urgent orders when more patients were in severe condition even when there were units available in inventory, revealing a potential cognitive bias for overestimating shortage risks. these biases can be controlled using mathematical models for quantitative analysis. the integrated data-driven demand forecasting and inventory management model we propose can produce accurate demand predictions and generate robust ordering decisions based on historical data. it can significantly reduce the occurrence of asap and stat orders while reducing inventory levels, resulting in significant savings with respect to both costs and resources. simulation models have been developed for bscm, including blood collection, production, inventory and distribution. mansur et al. [15] reviewed articles on bscm from 1960 to 2017, and provided a concise summary of the articles based on four categories: blood product type, performance measurement, coordination hierarchy level, and blood inventory model. they point out that the solutions offered are not comprehensive and sometimes are difficult, if not impossible, to implement. they then used the blood management system in indonesia as an example to suggest the need for a reliable inventory management system adaptive to demand fluctuation and blood supply pattern. beyond the articles surveyed in [15] , a number of additional references are pertinent for our proposed approach. sirelson and brodheim [16] built a predictive model using simulation and linear regression that determines the outdate rate and the shortage rate as a function of the fixed base stock level and the mean daily demand, for blood banks with scheduled daily deliveries of platelet components from a regional blood centre. they showed that for blood banks with moderate to large mean demands there exist optimal base stock levels that can effectively keep the outdate rate and the shortage rate within favorable ranges. they also extended the model to distinguish the platelet demand by blood groups. haijema et al. [17] presented a combined markov decision process and simulation approach with an application in a dutch blood bank. hemmelmayr et al. [18] established a two-stage stochastic optimization problem, which relies on sampling-based approaches involving integer programming to handle the stochastic demand and variable neighborhood search to improve computational efficiency. zhou et al. [19] analyzed a periodic review inventory system for platelet components under two replenishment modes: regular orders placed at the beginning of a cycle, and expedited orders within the cycle characterized by an order-up-to level policy. they started with a single-item periodic review inventory system and then expanded their work to a multi-period inventory problem. they provided a numerical illustration and a sensitivity analysis using historical data, and showed that the optimal cost is significantly affected by demand uncertainty, lead times, seasonality, and age of expedited orders. although there have been many studies in the field of blood demand and supply management, the methods are typically developed based on various assumptions and are difficult to implement. furthermore, to our knowledge, no study has considered integrating demand forecasting models into inventory management strategies for blood products. in this study, we investigate a multi-period inventory problem that mitigates the effects of forecasting errors from a data-driven demand forecasting model. the proposed integration strategy can help resolve practical challenges for rbc demand and supply chain management. there have been multiple inventory management approaches implemented in healthcare systems. heitmiller et al. [20] was the first major study focusing on reducing blood wastage from the hospital side. they used the five-part lean sigma process, i.e., define, measure, analyze, improve, and control, to reduce rbc wastage with an emphasis on container wastage, where a control plan and a list of interventions by lean sigma were developed. they demonstrated there could be a 60% reduction in rbc wastage with savings of more than $800,000 over four years. kort et al. [21] showed significant improvements, including a reduction of the median weekly outdating rate and a gain in the time until outdating, after implementing a combined approach of stochastic dynamic programming and simulation techniques. they stated the results brought confidence to personnel to apply and adopt the mathematical approach and the thrombocyte inventory management optimizer software tool. collins et al. [22] evaluated the effectiveness of multiple low-cost interventions that were implemented in january 2013 in the u.s., including educational outreach, print and digital messaging, and improved transportation and component identification modalities. they compared the rbc, platelet, and plasma wastage rates in the 16 months after these interventions with the rates prior to the interventions. they found significant decreases in the rbc and platelet wastage rates, however, there was an increase in the plasma wastage rate. the overall net cost savings of the reduced waste was estimated at $131,520, excluding the intervention costs. quinn et al. [23] designed and implemented a blood ordering algorithm, using a mathematical model based on the probability of rbc transfusion within 48 hours given certain hemoglobin levels, to provide a more accurate measure of rbc utilization. after implementation, they observed a significant reduction of the mean daily total rbc inventory level and the monthly rbc outdated units. these applications showed significant cost savings could be achieved by applying mathematical modeling in bscm. our proposed integrated methodology framework, being data-driven produces more robust results, is generally applicable to a range of decision problems in bscm, e.g., for different blood products. moreover, to support accessibility and knowledge translation, we plan to develop a prototyping tool with a user-friendly interface using the proposed methods. our study dataset is constructed by processing the trust ("transfusion research for utilization, the data for analysis is processed in two steps: 1) the dataset consists of all the transfused rbc units, and each row contains a unique rbc unit with product-related information and the transfusion recipient's characteristics as specified above. 2) a daily aggregated dataset is then constructed for demand forecasting. the dataset is organized by date, and each row contains the daily product and patient-related information. there are over 200 processed variables in the daily aggregated dataset using straightforward statistical transformations (e.g. mean, min, max, sum). the residuals forecast from the xgboost model are denoted byê i , given the input predictors x i . the the structure of each tree that maps the input predictors to the corresponding leaf index is represented by q : r d → {1, . . . , θ }, where θ is the number of leaves in the tree, and w ∈ r θ represents the weight of each leaf. let f k correspond to an independent tree structure q and leaf weights w. letê i be the prediction of the i-th instance at the k-th iteration. the objective function at the k-th iteration is where the function l is a differentiable convex loss function that measures the difference between the target e i and the predictionê for a fixed structure q(x), the optimal weight w * j of leaf j is given by and substituting (4) into (3),l this is the optimal loss for a fixed tree structure, however there might be thousands of possible trees. instead of searching all possible tree structures, xgboost uses a greedy algorithm to build a tree structure where the split is chosen with the maximum gain in the loss reduction. let i l and i r be the instance sets for the left and right nodes, respectively. the gain in the loss reduction is calculated by there are a number of hyperparameters for an xgboost model, including learning rate, the maximum depth of a tree, the minimum sum of weights of all observations in a leaf, the fraction of observations to be randomly sampled for each tree, the fraction of columns to be randomly sampled for each tree, and the regularization term on weights. in addition, s.window and t.window for the stl model are also considered as hyperparameters in the hybrid model. the hyperparamters are tuned using grid search based on a pre-defined parameter space. the tuning process is measured by cross-validation on the training data and evaluated with the root mean squared error (rmse). the optimal hyperparameters are selected with the minimum rmse using 5-fold cross-validation. variable selection proceeds in an iterative manner based on the variable importance scores calculated from the xgboost models. variable importance is the relative improvement in the performance measure contributed by a variable weighted by the number of observations for each decision tree, then averaged across all the decision trees within the model [31] . we initialize the iterative process with a model using all variables on the training dataset and evaluate the model performance measure on the test dataset, then select a subset of variables based on a pre-defined variable importance threshold for the next iteration. we repeat the process until there is no improvement observed in the performance measure, then the subset with the most important variables is finalized. the variables reported in the result section are the final set of variables selected through this process. the prediction target for the demand forecasting model is the daily rbc demand. in order to reduce costs, a second target, semiweekly demand, is considered. the semiweekly demand is defined as the three-day demand for tuesday, wednesday and thursday, and the four-day demand for friday, saturday, sunday and monday. the semiweekly demand is calculated from the daily demand prediction. we evaluate the model performance using the following two accuracy measures: mean absolute percent we consider a multi-period inventory problem for rbc units with fixed shelf life in a rolling horizon framework. the model assumptions are as follows: i) based on our data, we assume a fixed duration of 10 days from blood collection date to received date resulting in a shelf life of 32 days for units arriving at hospital blood banks. usually, the first two days after collection are spent on testing in blood production sites at cbs and the units are available for distribution on the third day, however, this could vary due to many reasons. ii) the rbc issuing policy in hospital blood banks is assumed to be a first-in first-out (fifo) withdrawal policy. iii) we assume an infinite supply of rbc units at cbs, so that all orders made from hospital blood banks to cbs can be fulfilled 8 . the order of events occurring in each period is given as follows: i) based on the inventory policy used, an order is placed (if necessary) at the end of each period. a routine delivery cost is charged for every order. ii) fresh units arrive at the beginning of each period according to the quantity ordered at the end of the previous period. the inventory level of each age is then updated. iii) the demand is observed and satisfied as much as possible. if there is a shortage, an urgent delivery order for the unmet demand is requested and the unmet demand is satisfied during the same period. iv) at the end of each period, the remaining inventory is carried over to the next period. expired units are discarded. wastage costs are charged for expired units and, if applicable, same-day urgent delivery costs are charged. usually, an optimization problem to determine the ordering strategy is set up with prior assumptions on the demand and supply distributions [33] . recently, bertsimas and kallus [34] considered a conditional stochastic optimization problem given imperfect observations, and developed a framework to prescribe optimal decisions using observed explanatory variables. in this study, since our goal is not only to propose an ordering decision but also to develop an accurate model that identifies clinical predictors for rbc demands, we do not formulate the optimization problem using observed clinical indicators directly, as in bertsimas and kallus [34] . instead, we proceed with the demand estimation and ordering strategy optimization in two separate steps. the structure of this data-driven inventory management problem is consistent with the structure for a newsvendor policy proposed in huber et al. [35] . as described in section 4.2, we have developed a demand forecasting model to predict future rbc demand using clinical predictors. this provides important information to hospital blood banks and blood suppliers for model generalisation and knowledge translation. multiple techniques have been applied to improve model accuracy, however, the existence of forecasting errors cannot be avoided. in the inventory optimization step for ordering decisions, we propose an ordering strategy considering two extra decision variables to control the cumulative loss due to the forecasting errors from the demand predictions of the hybrid model for this mutli-period inventory problem: inventory target (s) and reorder level (s). the proposed ordering strategy is a modified version of classical (s, s) policies [36] . the inventory target, s, defines an upper limit on the inventory level to avoid excessive inventory levels that may arise from demand over-estimation. the reorder level, s, sets a lower limit on the inventory level to avoid the need for urgent deliveries that may arise from demand under-estimation. under this policy, the order quantity is driven by the demand prediction from the hybrid model controlled by the following criteria: when the inventory level is below the reorder level, the order quantity is at least the number of units to bring the inventory level back to the reorder level, but cannot make the inventory level greater than the inventory target; if the inventory level is above the reorder level, no order is required. thus, the final ordering strategy is a function of the predictions from the hybrid demand forecasting model, the inventory target, and the reorder level. the age of an rbc unit is denoted by m ∈ {1, . . . , m}. let a, h, u, w be the routine delivery cost (per order), unit inventory holding cost, unit urgent delivery cost, and unit wastage cost for each expired unit, respectively. let i m i denote the inventory level of units of age m at the end of period i ∈ 1, . . . , t . the remaining demand after withdrawing all products having ages from m to m using the fifo withdrawal policy is denoted by r m i . the order quantity is denoted as z i in each period i ∈ 1, . . . , t , and i(z i > 0), is the indicator that an order occurs in period i. at the end of period i, the actual demand y i is updated. then, the inventory level i m i for each age m, units required for urgent delivery b i , and wasted items due to expiration i m i are calculated. the proposed cost function is given as follows: where the cost function in equation (7) includes four types of costs: routine delivery, inventory holding, urgent delivery and wastage cost over the planning horizon of t periods. equation (8) defines r m i according to the fifo withdrawal policy. equations (9) and (10) define the inventory dynamics. equation (11) calculates the number of units requiring urgent delivery. in the model, all variables are non-negative. the average cost can be written as e[c(z)] = 1 t ∑ t i=1 c(z i ). we propose an integrated ordering strategy where the order quantity is the predicted demand,ŷ i , from the hybrid model in section 4.2 controlled by an optimal inventory target, s * , and an optimal reorder level, s * . the optimal inventory target and reorder level values are learned through the training data by minimizing the difference between the average costs under the predicted demands and the actual demands as a prior [37] , rather than using the classical ordering structure based on estimated demand distribution [36, 38] . we set the initial inventory, i 0 , to be the mean inventory level according to the first three months of data. we assume this is an inventory level that the decision makers at hospital blood banks can accept, but it can be adjusted if needed (in particular the effect of lowering the initial inventory could be explored). let i i denote the aggregate inventory level of all non-expired units for period i, such that, i i = ∑ m−1 j=1 i j i for i = 1, . . . , t . let s * be the optimal inventory target and s * be the optimal reorder level. the procedure to generate the ordering strategy is described as follows: 1. determine the optimal inventory target, s * : first, we calculate the cost for each period using the actual demand, y i , as the ordering decision, z i , and denote the average cost as e[c(y)] = 1 t ∑ t i=1 c(y i ) over the planning horizon of t periods. when the order quantity is equal to the actual demand, the inventory level is always the same as the initial inventory level for all time periods. in reality, it is not possible to know the actual demand in advance. ordering according to the actual demand at a given initial inventory level is used as a gold standard for obtaining the optimal inventory target. second, we calculate the cost of each period using the predicted demand bounded by the inventory target, defined as min(ŷ i , s − i i−1 ), as the ordering decision. we denote the average cost where ξ is the feasible set of s. the optimal inventory target, s * , is determined by minimizing the absolute difference between the two average costs, such that, s * = arg min s∈ξ e[c(y)] − e[c(ŷ, s)] . seek the optimal reorder level, s * : using the optimal inventory target as an input variable, we consider the decision variable as the predicted demand bounded by the optimal inventory target, s * , and the reorder level, s ∈ ξ , where ξ is the feasible set of s. if s > i i−1 , the order quantity is the 3. generate the proposed order quantity, z * i : the proposed ordering decision integrating the prediction generated by the hybrid demand forecasting model, the optimal inventory target, and the optimal reorder level can be calculated as follows: for i = 1, . . . , t , the procedure is applicable to both the daily and the semiweekly demand predictions. the optimal inventory target is determined by errors in the daily demand predictions. under a given optimal inventory target, since the reorder level is used to control the inventory variations due to forecasting errors, the calculation of the optimal reorder levels for the daily and semiweekly predictions are performed separately. a statistical summary of selected variables is presented in table 7 . after time series decomposition using stl, we find significantly higher correlations between the stl residuals and selected clinical predictors than the correlations between the stl trend + seasonality and those predictors, as shown in figure 4 . moreover, figure 4 shows the correlations were high among the abnormal laboratory test results. as described in section 4. the number of orders, inventory level, number of units requiring same-day urgent delivery, wastage, average cost, and total cost are calculated using equations (7) to (12) , assuming the routine delivery cost per order a = 100, unit holding cost h = 1, unit urgent delivery cost u = 300, and unit wastage cost w = 50. using the procedure described in section 4.3, the optimal inventory target is 1040 units, the optimal reorder level for daily orders is 830 units, and the optimal reorder level for semiweekly orders is 770 units. comparisons are made during the time period of the test dataset (365 days) for four ordering strategies: current practice (baseline), ordering according to actual demand (gold standard), the proposed daily ordering strategy, and the proposed semiweekly ordering strategy. ordering as current practice refers to the current ordering strategy used in hospital blood banks that reflects the actual order quantity, inventory level, and wastage. since the actual number of units requiring urgent delivery was not captured in the database, the total cost calculated for the current ordering practice assumes that the urgent delivery cost is zero, and thus underestimates the actual cost. it is considered as the baseline strategy, whereas the results for ordering according to actual demand is considered as a gold standard. table 10 shows the results of the four ordering strategies. a summary of findings include: 1. there is a 60.55% reduction in the percentage of days with orders between the proposed semiweekly ordering strategy and baseline, whereas the proposed daily ordering strategy results in a 4% reduction. 2. the average order quantities for the proposed daily and semiweekly strategies over the entire period are less than the actual order quantities under current practice, and better reflect the actual demands. the mean order quantity for the semiweekly strategy on the 141 days with orders doubles the order quantity under current practice, which may raise operational problems such as requiring more packing boxes per order. our collaborators indicate this is not of great concern for cbs. 3 . the average inventory level for the proposed daily strategy is significantly lower, a reduction of 41%, as compared to the actual inventory level in hospital blood banks. similarly, the proposed semiweekly ordering strategy results in a 39% reduction of the inventory level. both the means of the inventory levels for the daily and semiweekly strategies are close to the inventory level of the gold standard, but the semiweekly ordering strategy is associated with a larger variance since over 60% of days have no orders. a leaner inventory leads to fresher rbc transfusions for patients. the doh is reduced to 8.6 days for the proposed daily and semiweekly strategies from 12.47 days for current practice. 4. there are no same-day urgent deliveries or wastage observed for the proposed daily and semiweekly strategies. however, there is no guarantee that this will always happen. 5. the proposed daily and semiweekly ordering strategies achieve remarkable cost savings. figure 7 illustrates the daily costs of the proposed daily strategy (red line), the semiweekly strategy (grey line), the actual costs under current practice (black line), and the constant cost of the gold standard (blue dashed line). notably, the cost savings are driven by the significant decreases in the inventory level and routine order delivery costs. the proposed semiweekly strategy has the lowest average cost since it not only leads to a lower inventory level but also requires less frequent deliveries. overall, both of the proposed daily and semiweekly ordering strategies result in a leaner inventory level, fresher blood, and lower costs, while no shortages (units requiring urgent delivery) or wastage are observed. particularly, the semiweekly ordering strategy creates a "win-win" situation, since it also provides a reduced, fixed delivery schedule that reduces costs and can free human resources both at cbs and hospital blood banks. improving the demand forecasting accuracy and building an efficient inventory management strategy for blood products are important to modern healthcare systems. it not only impacts the blood demand and supply management for blood suppliers and hospital blood banks, but may exert positive influence on patient outcomes. among all fresh blood components, rbc is the most commonly administered therapeutic. it accounts for the largest portion of blood inventory, and determines the plan for blood collection and production. in canada, due to the uncertain daily demand and the lack of quantitative evidence to support decision making, the rbc supply chain faces multiple challenges in hospital blood banks including excess inventory, highly variable ordering decisions, and over-frequent urgent orders. as a result, it has been very challenging for cbs to predict future demand and plan blood production. in this study, we develop an stl + xgboost hybrid algorithm for demand forecasting. it has the same level of prediction performance as a more complex lstm model. it handles changing trend and seasonality, nonlinear patterns in residuals, and correlations among predictors in an efficient and accurate manner. we then construct a data-driven multi-period inventory problem for rbc ordering. the procedure to generate the data-driven ordering strategy involves solving for an optimal inventory target and an optimal reorder level through minimizing the absolute difference between the average costs using the predicted demands from the hybrid model and the actual demands. the proposed ordering strategy is a modified version of the classical (s, s) policies considering a specific cost function. shi et al. [37] studied a nonparametric data-driven algorithm for the management of a stochastic periodic review inventory system with a constrained inventory target. there are two major differences between their optimization problem and ours: 1) the demand distribution is not stationary in our problem; 2) the cost function they considered does not include delivery costs, consequently there is no need to consider a reorder level to control the frequency of orders. they proved the asymptotical optimality of their algorithm under some technical assumptions and regularity conditions on the demand distribution. as a follow-up work, we plan to explore the optimality of our proposed ordering strategy in the setting for non-stationary demand. we expect that this will involve an appropriate asymptotic approach. this study considers the aggregated rbc demand of all hospitals in hamilton, ontario for the development of the demand forecasting model rather than the demand of each hospital stratified by abo blood groups. we chose to forecast the aggregate demand at the city level for the following reasons: i) all hamilton hospital blood banks are managed by one transfusion medicine laboratory team. this is a common hospital blood bank management structure for canadian cities, such as toronto and ottawa, ontario. there are existing transaction networks available that allow blood delivery within cities at low costs. the central management structure enables the pooling of demand and inventory that may yield operational improvements. ii) the model accuracy is increased as the demand variability is decreased due to pooling. in other words, considering the aggregate demand can reduce the level of demand uncertainty. iii) the model provides important clinical predictors for the aggregated rbc demand of a diverse patient population in hamilton, ontario that could be representative of the overall canadian rbc demand. when considering abo blood groups, there is no significant trend observed for each abo blood group of transfused patients over the years. the proportions of abo blood groups are consistent with the blood group distribution for canadian population with very low variation. our proposed methodology can be adapted to ensure sufficient inventory for each abo blood group. moreover, withdrawal policies to prioritize abo identical rbc transfusion will be investigated in future studies. when defining the rbc inventory optimization problem, the assumption of a fixed storage duration at cbs from the blood collection date to received date at hospital blood banks is a limitation of this work. seasonality and nonlinear trend patterns were observed in the blood storage duration data from 2008 to 2018, reflecting the changes of the blood donation process at cbs. we found a significant increasing trend of the storage duration after 2017. this could be associated with new tools, such as chat bots and online appointment booking systems, launched at cbs in 2017 which increased the number of blood donations. since the increase in blood supply exceeded the demand, a longer storage durations resulted at the cbs distribution centres. both the storage duration at cbs and the doh at hospital blood banks increased, consequently, the age of blood for transfusions has been increasing in the past couple of years. this also addresses the need of a better inventory management strategy that can help control the impacts of such changes at hospital blood banks, and share more accurate blood utilization information with cbs for blood collection planning. to conclude, we propose a decision integration strategy for short-term demand forecasting and ordering for rbc blood components. it incorporates a robust and accurate hybrid model and a multiperiod inventory optimization problem for ordering decisions. this leads to a significantly lower inventory level under a policy that has easy-to-compute order quantities and allows for a less frequent delivery schedule. it can potentially reduce the inventory by 40% and decrease the number of deliveries by 60%. the proposed ordering strategy can resolve the challenges faced by hospitals and cbs, and increase the transparency of blood utilization between blood suppliers and hospitals to promote an efficient blood supply chain, which may lead to better patient outcomes. furthermore, the proposed data-driven ordering strategy is generalizable to other blood products or even other perishable products. we have initiated work to apply the proposed strategy to the inventory management of platelet components. we plan to develop a software application to implement the proposed methodology at hospital blood banks in hamilton, ontario in the near future, and expand to other hospital blood banks across canada as a long-term plan. this study was funded by mitacs through the accelerate industrial postdoc program (grant number: it3639) in collaboration with canadian blood services. the funding support from canadian blood services was through the blood efficiency accelerator program, funded by the federal government (health canada) and the provincial and territorial ministries of health. the views herein do not necessarily reflect the views of canadian blood services or the federal, provincial, or territorial governments of red blood cell storage duration and trauma prolonged red cell storage before transfusion increases extravascular hemolysis duration of red blood cell storage and inflammatory marker generation storage time of red blood cells and mortality of transfusion recipients can we be certain that storage duration of transfused red blood cells does not affect patient outcomes? red blood cell transfusion in clinical practice. the lancet a decision-making tool for demand forecasting of blood components demand forecasting for blood components distribution of a blood supply chain an efficient inventory model to reduce the wastage of blood in the national blood transfusion service artificial neural network based approach for blood demand forecasting: fez transfusion blood center case study big data modeling to predict platelet usage and minimize wastage in a tertiary care system forecasting demand in blood supply chain (case study on blood transfusion unit) challenge and opportunity research in blood supply chain management: a literature review computer planning model for blood platelet production and distribution blood platelet production: optimization by dynamic programming and simulation. computers & operations research vendor managed inventory for environments with stochastic product usage inventory management of platelets in hospitals: optimal inventory policy for perishable products with regular and optional expedited replenishments blood wastage reduction using lean sigma methodology platelet pool inventory management: theory meets practice effectiveness of multiple initiatives to reduce blood component wastage the successful implementation of an automated institution-wide assessment of hemoglobin and abo typing to dynamically estimate red blood cell inventory requirements r: a language and environment for statistical computing why giving two when one will do? a toolkit for reducing unnecessary red blood cell transfusions in hospitals stl: a seasonal-trend decomposition procedure based on loess (with discussion) xgboost: a scalable tree boosting system acm sigkdd international conference on knowledge discovery and data mining extreme gradient boosting contributors a new hybrid method for china's energy supply security forecasting based on combining deep neural networks and classical time series regression models for forecasting patient flows in hong kong the elements of statistical learning: data mining, inference, and prediction long short-term memory recurrent neural network architectures for large scale acoustic modeling introduction to stochastic programming from predictive to prescriptive analytics a data-driven newsvendor problem: from data to decision the optimality of (s, s) policies in the dynamic inventory problem nonparametric data-driven algorithms for multiproduct inventory systems with censored demand confidence intervals for data-driven inventory policies with demand censoring leveraging cloud data to mitigate user experience from 'breaking bad canada. the authors thank dr. john blake for his expertise in blood supply chain management. the authors thank dr. donald arnold for providing valuable comments on the manuscript. the authors thank tom courtney, rick trifunov, marianne waito, and masoud nasari for arranging partner interaction activities, and providing information about blood collection, blood processing, and blood distribution at canadian blood services. all final decisions regarding manuscript content were made by the authors. key: cord-281990-x5nql0cw authors: liu, y.; steinacker, j. m.; haeussinger, l.; dinse-lambracht, a. title: association between epidemic dynamics of covid-19 infection and abo blood group types date: 2020-07-15 journal: nan doi: 10.1101/2020.07.12.20152074 sha: doc_id: 281990 cord_uid: x5nql0cw background: covid-19 pandemic is the most critical challenge nowadays for the manhood, and the infection and death cases are still speedily increasing. since there are no available vaccine and specifically effective treatment, to break the infectious way of the pandemic remains the unique measure to efficiently combat covid-19 infection. understanding factors that affect the covid-19 infection can help make better balance between activity restriction and infection dynamics. this study sought to investigate association between covid-19 infection and blood type distribution. methods: the big data provided by world health organization and johns hopkins university were taken to assess epidemic dynamics of covid-19 infection. growth rate and doubling time of infection and death cases, reproductive number, infection and death cases in the mid-exponential phase were analyzed in relation to blood type distribution. results: growth rate of infection and death cases correlated significantly to blood type a proportion of the population positively while to blood type b proportion negatively. in comparison with lower blood type a population (< 30%) people with higher blood type a ([≥] 30%) had more infection and death cases in the early exponential phase, higher growth rates, and shorter case doubling time for infection and death. discussion: covid-19 infection is significantly associated with blood type distribution and people with blood type a are more susceptible to covid-19 infection and have higher epidemic dynamics and higher case fatality rate. the results of this study provide important and useful information for fighting covid-19 pandemic. the pandemic of covid-19 infection still keeps rapidly increasing worldwide, which threatens very much the public health and causes dramatic knockdown of the global economics and social life. according to the data derived from world health organization (who) database, up to date there was over 10 million confirmed cases of covid-19 infection (who situation reports on june 29, 2020), and the daily new infection cases maintain at very high level. to control this pandemic outbreak is difficult because factors affecting the covid19 infection have yet not been thoroughly understood, and specific vaccine and treatments are still unavailable. if one looks the covid-19 infection map provided by johns hopkins university (jhu) (for example the map picture on may15, 2020), one would immediately recognize that there is quite a difference among the geographic districts. in fact, after the initial outbreak in china, the covid-19 pandemic speedily advanced to europe and simultaneously to new york city, and afterwards this pandemic spread to south america and east mediterranean zones. trying to understand the factors that have profound impact on the pandemic is crucial for bringing the pandemic under control since factors associated with the pandemic must be considered to make public policy and medical decisions. abo blood types are attributed to diverse infectious diseases like malaria, hiv and influenza (1) (2) (3) (4) . it has been reported that among the confirmed covid-19 infection cases that were treated in the hospitals the proportion of blood type a was significantly higher than that of blood type b (5), and furthermore it has been reported that the severity and clinical outcome of the covid-19 infection disease were associated with blood types (6) . however, in these previous studies the study subjects were in a relatively small number and/or limited locality so that the data cover only a regional geographic zone and do not reveal the worldwide geographically uneven distribution of covid-19 infection. furthermore, the dynamics of covid-19 had not been dealt with worldwide. we therefore conducted this big-data-analysis on association between dynamics of covid-19 pandemic and abo blood types. the big data are derived from the official database presented by world health organization (who). abo blood type distribution serves as a typical genetic marker for geographic distribution over the globe for diverse diseases as well as public health issues. finding out any factors that are attributed to covid-19 infection might be thus important in fighting covid-19 pandemics. for an epidemic of an infectious disease the dynamic development of the infection is determinant, and this can be assessed by several parameters classically used, that are among others infection case growth rate (icgr), infection case doubling time (ic-dt) reproductive number (rn), death case growth rate (dcgr) ,and death case doubling time (dc-dt). the difficulty to determine these parameters is that the current high dynamic in the infection development worldwide so that an endpoint of total infection number remains yet unreached. instead, we tried to assess on the mid-way of the exponential phase of infection the infection cases and death cases, which was performed based on the epidemical curves of the involved countries displayed by jhu. this study sought to investigate the relationship between the distribution of blood group types and the epidemic dynamics of covid-19 infection based on analyses of big data that cover worldwide population majority. for analysis of the pandemic of covid-19 infection the population of six geographic regions divided according to who is included: africa, america, east mediterranean, europe, south east asia and western pacific. within in each geographic zone six countries are randomized selected for the analysis (table 1 supplement material). however, at the time point of the analysis two countries, i.e. botswana and papua new guinea, got only a few covid-19 infection cases that did not show an epidemic and therefore were excluded from the analysis. finally, the total population of the 34 countries was about 5391149 thousand (2016 data of who). data of blood type distribution of each country are derived from original research studies with respect to each corresponding countries (table 1 of supplement material). since the blood type a seemed more relevant through prior correlation analysis (figure 2a), and mean blood type a proportion of all 34 countries was 30%, the population of all included countries was divided in to higher (≥ 30%) and lower (<30%) blood type a groups for advanced analysis (figure 3). the confirmed infection cases as well as death cases of each country were taken from the daily situation reports of who. to calculate icgr the start point (1 st day) was set on the day when the infection cases began to increase exponentially. this point could be identified on the curve of jhu by two independent coauthors. taking germany as an example, the day when the infection cases began to increase was march 12, 2002 with 1567 infection cases. the afterward-consecutive 14 daily cases were input into spss ® . icgr was calculated according to the following formula in the spss software: icgr = exp (((ln (infection cases on 15 th day)ln (infection cases on 1 st day)) ÷ 14). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 15, 2020. . https://doi.org/10.1101/2020.07.12.20152074 doi: medrxiv preprint the calculation of rn was referred to the methods used by robert-koch-institute (cologne. germany) (6) (7) (8) . the calculation was based on the assumption of the viral generation time with 4 days and the incubation time 5 days. according to the following formula, the rn was calculated: the death cases caused by or with covid-19 infection were taken from the daily situation report of who. based on the clinical consideration that a death case took place about 15 days after infection, the death cases on the 16 th day and the following consecutive 14 days were input into spss ® to calculate the death case growth rate (dcgr) that was calculated analogously to that of icgr as following: dcgr = exp (((ln (mortal cases on 30 th day)ln (mortal cases on 16 th day)) ÷ 14). furthermore, the doubling time was calculated for infection cases (ic-dt) and for death cases (dc-dt), respectively as follows: from the epidemical curve provided by jhu, a plateau phase for the infection cases could be considered when the increase of new infection cases was no longer exponential, which could be assessed for a part of the countries. the time interval from the start point of exponential phase to the begin of the plateau phase could be determined (days to begin of the plateau phase, dpp). furthermore, the cases on the day when the plateau phase was reached (nine countries up to june 30, 2020) were taken for calculation of covid-19 infection of the corresponding country. the mean dpp was 51.2 days among these nine countries and thus dpp(1/2) was set at 26 days after begin of the exponential phase. since most of the analyzed countries did not reach their plateau phase, dpp(1/2) was taken for all analyzed countries to count the infection and death cases (icdpp(1/2) and dc dss(1/2), respectively). all these methodological measures are depicted in figure 1 . in addition, a comparison of the epidemic dynamics was performed by dividing the population into higher and lower groups of blood type a proportion either with nonparametric (mann whitney) (figure 3a) or anova (figure 3b) accordingly. because the mean proportion of blood type a was 30%, the higher and lower blood type a groups were defined respectively by ≥ 30% or < 30%. the mathematic and statistical procedures were performed with spss ® (ibm. 25 th edition. usa). a difference was assumed to be significant by p<0.05. in table 2 are summarized the parameters describing the covid-19 epidemic dynamics, an overall difference among the selected countries was found for each parameter through anova analyses (p<0.01). icgr correlates positively with blood type a (r² = 0.328, p<0.05. figure 2a) and negatively with type b (r² = 0.210, p<0.05. figure 2b ), but not with blood type o or ab. the relationship between dcgr and blood types is shown in fig.3 . dcgr correlated positively with blood type a (p < 0.01, figure 2c ) and negatively with blood type b (p < 0.05, figure 2d ), but not with blood type o or ab. at begin of exponential phase of covid-19 infection, the infection cases were comparable between the higher and lower blood type a groups (p > 0.05, figure 3a ), but at dpp(1/2) the infection cases were distinctly higher in group with higher blood type a than that with lower blood type a (p < 0.05), which was also the case for to dcdpp(1/2) (p < 0.05). during the first 15 days of exponential growth phase icgr was significantly higher in the group with higher blood type a than that in the group with lower blood type a (p < 0.05, figure. 3b ). ic-dt was reciprocally shorter in the higher blood group a than that of the lower blood type group a (p < 0.05, figure 3c ). though the reproductive number showed slightly higher in the group with higher blood type a than that in the lower blood group a, the difference was statistically not significant (p > 0.05, figure 3b ). the dynamic changes in death cases indicated by dcgr showed significant difference between both groups, i.e. higher in the group with higher blood type a proportion (p < 0.01, figure 3b) . accordingly, the death case doubling time was significantly shorter in this group (p < 0.05, figure 3c ). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 15, 2020. . https://doi.org/10.1101/2020.07.12.20152074 doi: medrxiv preprint the worldwide pandemic of covid-19 infection is dramatically challenging the global health care, social life and economy, and is spreading with still speedy increase in new infection and death cases. to control and conquer this pandemic is of critical interest for the manhood and global society. classically, to control an epidemic disease, four major measures are essential, they are controlling disease origin, cutting the infectious way, effectively treating patients and improving herd immunity. however, up to date, the origin of covid-19 epidemic remains uncertain; a clinically proven vaccine to improve immunity against covid-19 virus is still unavailable; an effective therapy for covid-19 infected patients has yet not established. therefore, the unique certain effective measure at present is to break the way of wide spreading, i.e., activity restriction and community lockdown. unfortunately, lockdown causes a series of adverse effects, e.g., down of community economy, reduction of productivity, restriction of people freedom, and hamper of the public health care. therefore, to balance rationally the epidemic dynamics and lockdown is of critical social and health interest, which bases on the observation and analysis of covid-19 infection dynamics. to determine finally the dynamics of covid-19 infection is by no means easy because a number of factors have profound impacts on this dynamics. it is hardly possible to get the exact data of infection and death cases due to limited manpower and health care resource in testing the population, not to mention limited test certainty in terms of sensitivity as well as specificity, variant incubation time, different social environments and activities, etc. therefore, analysis based on big data seems a reasonable way to assess the dynamics of covid-19 infection globally. at present, who and jhu are monitoring this dynamics and providing useful information, we therefore take these useful tools and go further with analysis with specific interest on genetic factor. in fact, genetic factor plays important role in many diseases like cardiovascular diseases (9), diabetes mellitus (10), cancers (11), , and infectious diseases including malaria (12) , hiv and influenza (13;14) . as a genetic factor, abo blood type distribution has been extensively investigated in the field of infectious diseases (13) (14) (15) . meanwhile, investigation interest on relationship between abo blood system and covid-19 infection is increasingly (6;16;17) . the first study suggested that among the infected individuals the proportion of blood type a patients was higher (17) . in this study, total 2173 confirmed covid-19 infection cases treated in hospitals were analyzed, and of these patients a higher population with blood group a than that of comparable population without covid-19 infection was found. since the study subjects were in a small number and from the same locality, the data were quite limited. a larger multicenter study has been recently reported by ellinghaus et al, and a distinct association between abo blood types and severity of covid-19 infection disease could be demonstrated (6;16;17) . unfortunately, in their study the dynamics of the covid-19 infection was not investigated. we thus conducted this study based on the worldwide available big data trying to ascertain an association between the distribution of blood types and covid-19 infection dynamics. the results of our analyses show that there was a positive correlation between proportion of blood group a in the worldwide population and the dynamics of covid-19 infection reflected by the infection case growth rate, and a negative correlation was found between blood group b and the infection dynamics ( figure 2) . furthermore, similar results could be also elucidated for the relationship between blood group types and death case growth rate ( figure 2 ). in the context, our results are in a good accordance to these aforementioned studies, and strongly demonstrate the association between the distribution of blood group types and covid-19 infection. based on the above described results we divided the worldwide population into two groups according to the proportion of blood type a, i.e., higher blood type a group (≥30%) and lower group (<30%), and compared the differences in terms of the epidemic dynamic parameters between both the groups. it is shown in figure 3 that in the higher blood type a group except for the infection cases at begin of exponential phase and the reproductive number, all other parameters were significantly higher than those in the lower blood type a group. these results demonstrate further that an association between blood type distribution and the epidemic dynamics of covid-19 infection. we have also analyzed a relationship between the population life expectance as well as health care expense and covid-19 infection cases (data not shown) based on data provided by who. that with increase in life expectance the covid-19 infection cases per 100,000 people increased, suggesting that the older people have a higher covid-19 infection susceptibility, is already well-known. there was no significant correlation between health care expense and covid-19 infection cases in the analyzed countries. all these results mentioned above strongly demonstrate that the epidemic dynamics of covir-19 infection is associated with the distribution of blood types worldwide, not only infection cases but also death cases. people with blood group a are therefore more susceptible to covid-19 infection and their case fatality rate though covid-19 infection is higher. mechanisms responsible for this association are yet to be explored. nowadays, it is believed that covid-19 infection begins with the docking of its spiking protein to acer2 (18) (19) (20) . whether acer2 expression level is different among the blood types, and whether people with blood type a have higher expression level of acer2, remain unclear. certainly, further studies are necessary to explore the mechanisms. bearing the association between covid-19 infection and blood type distribution in mind may important and meaningful in order to make reasonable decision in combating the covid-19 infection among different people with their blood type distribution. in general, countries with lower proportion of blood type a have lower economic income and poorer health care resource, which might be exaggerated by community lockdown. it might reasonable for these countries to make slower or later strict measures owing to the lower epidemic dynamics of covid-19 infection, whereas for those countries where the people with higher proportion of blood type a the strict measure to combat the covid-19 infection ought to be more active and earlier. illustration of mathematical analyses of the study parameters involving covid-19 infection. as an example the development of infection cases in germany over 140 days is displayed to determine the points taken for calculations. from the course of cumulative infection cases a curve with different phases can be identified. the 1 st day was set on the day when the infection cases began to increase exponentially. in the early exponential phase the infection growth rate (icgr) was calculated. the death cases were taken from the period day14 days after the start point through further 14 days. the plateau phase was reached when the course of cumulative infection cases did no longer show an exponential increase. the time interval (days) between start point and begin of plateau phase (dpp) was calculated. for in most of the countries a plateau phase was yet not reached, dpp(1/2) was set as half mean dpp and calculated for all the countries. correlation between blood type distribution and covid-19 infection case growth rate per day and death case growth rate per day. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 15, 2020. . https://doi.org/10.1101/2020.07.12.20152074 doi: medrxiv preprint figure 3 comparison of the covid-19 infection epidemic dynamics between higher and lower blood type a population. in figure 3a : ic-begin, the first day when infection case began to increase exponentially; dcdpp(1/2), death cases on 26 th day after ic-begin; icdpp(1/2), infection cases on 26 th day after ic-begin. in figure 3b : icgr, infection case growth rate per day; rn, reproductive number; dcgr, death case growth rate per day. in figure 3c: ic-dt, infection case doubling time in day; dc-dt, death case doubling time in day. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 15, 2020. . https://doi.org/10.1101/2020.07.12.20152074 doi: medrxiv preprint genome-wide association studies of severe p. falciparum malaria susceptibility: progress, pitfalls and prospects effect of the abo blood group on susceptibility to severe malaria: a systematic review and meta-analysis abo/rh blood groups and risk of hiv infection and hepatitis b among blood donors of abidjan the role of host genetics in susceptibility to influenza: a systematic review relationship between abo blood group distribution and clinical characteristics in patients with covid-19 genomewide association study of severe covid-19 with respiratory failure schätzung der aktuellen entwicklung der sars-cor-2-epidemie in deutschland -nowcasting testing the association between blood type and covid-19 infection, intubation, and death the relationship between abo blood group and cardiovascular disease: results from the cardiorisk program genetic polymorphisms in diabetics and non-diabetics pancreatic cancer risk is modulated by inflammatory potential of diet and abo genotype: a consortia-based evaluation and replication study assessing abo/rh blood group frequency and association with asymptomatic malaria among blood donors attending arba minch blood bank, south ethiopia relation between blood groups and resistance to infection with influenza and spome picornaviruses do blood group antigens and the red cell membrane influence human immunodeficiency virus infection? abo blood groups and viral diseases covid-19 and the abo blood group connection relationship between the abo blood groups and the covid-19 susceptibility inhibition of the interaction between ths sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor key: cord-335733-u1g03s2y authors: lakshmanan, hari hara sudhan; pore, adity a.; kohs, tia c. l.; yazar, feyza; thompson, rachel m.; jurney, patrick l.; maddala, jeevan; olson, sven r.; shatzel, joseph j.; vanapalli, siva a.; mccarty, owen j. t. title: design of a microfluidic bleeding chip to evaluate antithrombotic agents for use in covid-19 patients date: 2020-08-06 journal: cell mol bioeng doi: 10.1007/s12195-020-00644-x sha: doc_id: 335733 cord_uid: u1g03s2y introduction: interventions that could prevent thrombosis, clinical decompensation, and respiratory compromise in patients with novel coronavirus disease (covid-19) are key to decrease mortality rate. studies show that profound cytokine release and excessive activation of blood coagulation appear to be key drivers of covid-19 associated mortality. since limited in vitro methods exist for assessing the effects of anticoagulants on hemostasis, the development of novel therapies to safely prevent thrombosis in covid-19 patients relies on preclinical animal models and early phase human trials. herein we present the design of a microfluidic “bleeding chip” to evaluate the effects of antithrombotic therapies on hemostatic plug formation in vitro. methods: the design of the microfluidic device consists of two orthogonal channels: an inlet that serves as a model blood vessel, and a bleeding channel to model hemostatic plug formation at sites of compromised endothelial barrier function. this is achieved by placing a series of 3 pillars spaced 10 μm apart at the intersection of the two channels. the pillars and bleeding channel are coated with the extracellular matrix protein collagen. results: perfusion of human whole blood through the microfluidic bleeding chip led to initial platelet adhesion and aggregation at the pillars followed by hemostatic plug formation and occlusion of the bleeding channel. conclusions: safe and effective mitigating agents are needed for treatment and prevention of thrombotic complications in covid-19 patients. this simple microfluidic device holds potential to be developed into a tool for assessing the effects of anticoagulant therapy on hemostasis. coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection is a global pandemic, having infected nearly 13 million around the world by the time of this writing. primarily a respiratory disease, cov-id-19 patients with severe infection suffer from inflammatory and thrombotic complications. 6, 34 a retrospective analysis of covid-19 patients revealed that 71.4% of patients who died developed disseminated intravascular coagulation (dic) compared to only 0.6% of survivors. 30 critically ill covid-19 patients have been found to frequently develop thrombotic manifestations including microvascular thrombosis, venous thromboembolism, and acute arterial thrombosis. despite standard prophylactic anticoagulant administration the rate of thrombosis approach 20% in hospitalized patients. 2, 5, 8, 19, 32 while heparin is a convenient and biologically plausible attractive anticoagulant for covid-19 due to its potential anti-inflammatory, anti-complement, and even direct antiviral effects, the persistently high rate of thromboembolic disease despite routine heparin use implies an urgent unmet need for alternative agents. 11, 22, 25 several direct and indirect mechanisms have been proposed by which sars-cov-2 infection induces pathologic upregulation of inflammation and coagulation, as illustrated in fig. 1 . this has led to significant enthusiasm for mobilizing clinical trials targeting either pathway, balancing efficacy with safety. with regards to developing novel anticoagulants for treatment of covid-19 patients, this demands that these strategies ensure hemostatic safety, i.e. no abnormal bleeding. while the most clinically relevant conclusions regarding the safety of anticoagulants will ultimately be learned through clinical trials, in vitro models hold potential usefulness in revealing early safety and efficacy signals to guide future anticoagulant development of agents to prevent thrombosis in cov-id-19 patients without compromising hemostasis. the key mechanisms that drive intravascular thrombus formation in diseased vascular beds were elucidated in part due to the creation and use of in silico, in vitro, ex vivo and in vivo models of thrombus formation under shear flow. 7, 13, 16 building upon this knowledge, a number of antithrombotic agents targeting either platelets or the coagulation cascade have been brought to market for use in the prevention or treatment of cardiovascular diseases including heart attack and stroke. yet, despite advances in identifying targets to improve the efficacy of antithrombotic therapy, in vitro models to predict clinical bleeding are limited. this is particularly relevant in covid-19, as new antithrombotic agents including inhibitors of platelet function, coagulation factor (f) x, or the figure 1. hypothesized procoagulant mechanisms of sars-cov-2 infection including endothelialitis, leukocyte recruitment, inflammatory cytokine release and direct activation of coagulation enzymes. use of anticoagulants to block the development of covid-related thrombosis must take into account their potential to incite pathologic bleeding. contact activation system will likely have to be used in combination with heparin as a standard-of-care therapy. 28 an understanding of any potentially deleterious effects of such drug combinations on hemostasis will have to await the results of ongoing or proposed early phase clinical trials. this is due in part to the fact that the physical biology and rheology underlying hemostatic plug formation are ill-defined relative to our understanding of thrombosis. 9 the events that support hemostasis (extravascular) vs. thrombosis (intravascular) are distinct in part due to the rheology of blood flow that differentially distributes blood constituents inside and outside blood vessels. several elegant microfluidic models of the hemostatic response to a mechanical injury of the microvasculature have recently been developed. 26, 27 here we extend these models based on the percolation theory of fluid dynamics to develop a model of blood transport into the tissue space at sites of compromised endothelial cell barrier function, such as in inflamed tissue beds and other forms of microangiopathy. 3 to achieve this, we present the design of a microfluidic device with two orthogonal channels: a main channel that serves as a model blood vessel, and a bleeding channel to model hemostatic plug formation. we propose this model could be utilized to identify potential anticoagulant and antithrombotic targets and test agents to mitigate the thrombotic complications associated with covid-19 whilst preserving hemostasis. the design consists of two orthogonal channels: a main channel of width 150 lm, length 10 mm and a side channel of width 100 lm, length 3 mm as shown in fig. 2a . we designed 3 circular pillars of diameter 20 lm, placed 10 lm apart at the intersection of two channels. all features have a uniform height of 50 lm. one side of the main channel serves as the inlet for blood, referred to as the inlet channel and the side channel with the pillars is termed the bleeding channel as indicated in fig. 2a . only the bleeding channel is functionalized with fibrillar collagen to represent the subendothelial extracellular matrix. this method can be modified to include coating the bleeding channel with tissue factor alone or in combination with extracellular matrix proteins including collagen, nidogen, or laminin. 15, 37 the gaps in the pillars represent compromised endothelial barrier function as a result of inflammation or vascular injury. the physical parameters of the pillars including number, radius, and spacing could be modified to study the effects of the physical biology of barrier function on the kinetics of hemostatic plug formation. the design for the bleeding chip was drawn in au-tocad and was printed on photomask (cad/art services inc., oregon, usa). standard soft-lithography techniques were used for making the bleed chip. 23, 35 the master mold was a single layer design, fabricated using a negative photoresist su-8 2050 (kayaku advanced materials, usa) with feature height of 50 lm. microfluidic devices were made by mixing polydimethylsiloxane (pdms) (sylgard 184, dow corning, usa) monomer and the curing agent in a 10:1 ratio followed by degassing of the solution, pouring it over the master mold and curing it at 70°c for 2 h. after curing, the pdms replicas were cut and peeled from the su-8 mold. fluid inlets and outlets were punched using a 1 mm biopsy punch (miltex, japan). the pdms replicas were bonded to a cover glass (thermo scientific, usa) of thickness 170 lm, after plasma treating (harrick plasma, ithaca, new york) the surfaces for 90 s. this assembly was baked at 70°c for 4 mins to strengthen the bonding. to coat the bleeding channel with fibrillar collagen (100 lg ml à1 , chronolog), a collagen droplet of 2 ll volume was introduced by pipette into the bleeding channel; the leading edge of the droplet meniscus was advanced until the collagen solution reached the pillars as observed with a 109 microscope objective. capillary forces were sufficient to restrict the collagen solution from entering the orthogonal vessel channel, thus allowing selective coating of the bleeding channel and pillars with collagen as shown in fig. 2b . after an hour of incubation at room temperature, both channels were washed with phosphate buffer saline (pbs) and blocked with 5 mg ml à1 denatured bovine serum albumin (bsa) for 1 h followed by washing with pbs. relevant controls would include coating the bleeding channel with bsa. human venous blood was collected from healthy adult volunteers into a syringe filled with 3.8% sodium citrate (1/10th blood volume) in accordance with the oregon health & science university institutional review board. citrated blood was recalcified with the addition of (1:9 recalcification buffer:blood) recalcification buffer (75 mmol l à1 cacl 2 and 37.5 mmol l à1 mgcl 2 ), to allow for coagulation. this method could be modified for use with reconstituted blood (purified red blood cells, platelets, and plasma) to allow for parameters such as platelet count to be adjusted. recalcified blood was loaded into a 1 ml syringe and assembled onto a harvard 2000 syringe pump as shown in fig. 3 . the syringe was connected to the device inlet with a 0.5 mm polyethylene tubing (braintree scientific, usa) and the blood was perfused at a constant flow rate of 10 ll min à1 , which corresponds to an arterial shear rate of 1000 s à1 in the inlet channel. the outlet of the bleeding channel was con-nected with polyethylene tubing (20 cm length) graduated every 0.5 cm to record the velocity of blood flow in the bleeding channel. the main channel outlet was also connected to polyethylene tubing of similar length for waste collection. platelet adhesion, aggregation and thrombus formation in the device near the pillars during blood perfusion was recorded using kohler illuminated nomarski differential interference contrast (dic) optics with a zeiss 109 lens on a zeiss axio imager m2 microscope (carl zeiss microimaging gmbh, germany). graduations in the polyethylene tubing connected to the bleeding channel enabled manual monitoring and recording of blood velocity and volume in the bleeding channel. we performed computational fluid dynamic simulations using a 2d model of the bleeding chip created in comsol to predict the hemodynamics at and near the interface created by the pillars. our model included biomedical engineering society . experimental setup for the use of bleed chip with blood sample. device inlet is connected to a syringe pump with a syringe filled with recalcified whole human blood. the bleeding channel is connected to a 0.5 mm polyethylene tube with graduations every 0.5 cm to record the blood flow rate through the bleeding channel. an equal length of polyethylene tubing is connected to the second outlet leading to waste collection. real time dynamics of blood flow inside the bleed chip is captured with the 103 objective of a dic zeiss axio imager 2 microscope. arrows denote the direction of blood flow. the constant flow rate of 10 ll min à1 at the inlet, a constant atmospheric pressure at the main channel outlet and a constant flow rate of 2 ll min à1 at the bleeding channel outlet. blood was modeled as a non-newtonian fluid using power-law as described in the literature. 36 our initial model was based on the average physical parameters of density and viscosity of human whole blood and did not take into account changes in these parameters due to the addition of anticoagulants, recalcification buffer, or differences in donor hematocrit. based on the inlet flow rate, we estimated a reynold's number (re) % 0.5 for flow in the device resulting in an assumption of creeping flow inside the device. although the low re flow in the model may not capture the physiology of blood flow, this is a limitation associated with the field of microfluidics in general and there remains a need for scaling approaches that better relate the rheology observed in microfluidic models to the biophysical parameters of veins, capillaries and arteries. the navier-stokes equation for creeping flow was solved using comsol under steady state conditions to create velocity and shear rate profiles as shown in figs. 4a and 4b. velocity profiles derived from the microfluidic device indicated a series of stagnation points along the pillar surface and the corners at the channel intersection (fig. 4a) . platelet aggregation at stagnation points has been shown to initiate a core and shell thrombus wherein the core consists of procoagulant platelets which facilitates localized thrombin generation whereas the shell is comprised of weakly activated platelets which acts as a rheological shield. 12, 29 for our bleeding chip model, the shear rate profile shows a change in shear rate from 1000 s à1 in the inlet channel to 9000 s à1 in the spaces between the pillars of the bleeding channel, thus introducing a shear gradient. this region of increasing shear rate was followed by decreasing shear rates in the bleeding channel downstream of the pillars as shown in fig. 4b . rapid transition of shear rate over short distances has been shown to promote platelet activation and aggregation due to platelet gpib-von willebrand factor (vwf) interaction. 21 the peclet number (pe) >> 1 for the transport of zymogens from bulk flow to the pillar surface at steady state conditions implies a significant dependence on advection as the primary transport mechanism. 4, 17 we experimentally observed the initiation and propagation of hemostatic plug formation at the sites predicted by our computational model as ''locations of shear gradients''. the dynamics of hemostatic plug formation were imaged by dic microscopy. figure 5a shows the progression of platelet aggregation and coagulation around the pillars at the entrance of bleeding channel at 0, 5, and 10 min perfusion times. hemostatic plug formation was observed to initiate at and around the pillars as indicated by the dotted circle in fig. 5a . the growth of the hemostatic plug continued in the direction of flow inside the bleeding channel over the next 5 min. by 10 min a patent hemostatic plug was formed and blood flow ceased. in contrast, in the absence of coagulation using sodium citrate-anticoagulated whole blood, hemostatic plug formation was incapable of stopping blood flow in the bleeding channel. at present the time to occlusion is a laboratory-based experimental parameter, akin to a clotting time in an aptt assay; future studies are required to relate this parameter to the kinetics of hemostatic plug formation at sites of vascular injury or compromised barrier function and relevant scaling of the model to vascular beds, veins and arteries. the velocity of blood in the bleeding channel was recorded by quantifying the rate of blood flow within a graduated polyethylene tubing connected to the distal end of bleeding channel. we report the scaled velocity ð uþ ¼ u i u 0 in fig. 5b where u i is the velocity of blood measured at a given time point and u 0 is the initial velocity of blood measured in the tubing connected to the bleeding channel. our pilot data show a reduction in blood flow velocity as a function of time. of note, we observed fluctuations in scaled velocity due to 'rebleeding' wherein a portion of the hemostatic plug would become unstable and erode in a similar manner as has been observed in animal models of hemostatic plug formation. 31, 33 our current technique to measure velocity of blood in the bleeding channel is limited to the granularity of measuring discrete time intervals associated with 1.3 ll gradations in blood volume; continuous monitoring of the blood volume and velocity with techniques including gravimetric or doppler measurements of volumetric flow rate would improve the precision of this platform, albeit at an increased cost of design. this method can be developed to study hemostasis in vitro under physiologically relevant flow conditions. the effect of inhibitors of the intrinsic, extrinsic and common pathways of coagulation alone or in combination with anti-platelets inhibitors including par4 and p2y12 antagonists on hemostatic plug formation can readily be evaluated in this model. 20, 24 the sensitivity of this model to changes in platelet count and hematocrit remain to be validated. the primary cause of death in covid-19 is progressive respiratory failure, which has been increasingly associated with the presence and frequency of microangiopathy thrombi in the pulmonary vessels of covid-19 patients. 10, 18 for instance, reports have shown the rate of alveolar capillary microthrombi were nearly 9-fold higher in covid-19 patients as compared to patients with influenza. 1 in the intensive care unit, cumulative incidence rates nearing 50% have been reported for arterial and venous thrombosis in covid-19 patients despite the widespread adoption of low molecular weight heparin (lmwh) as primary thromboprophylaxis. 14, 19 these observations highlight both the need for novel safe and effective antithrombotic strategies to combat thrombosis in covid-19 patients, as well as our lack of understanding of the pathology of thrombosis in infectious diseases including covid-19. novel anticoagulant strategies such as inhibitors of the contact activation system (cas) of coagulation and stimulators of the protein c pathway will likely see introduction into clinical trials in combination with heparin products. 28 as lmwh is known to compromise hemostasis, combinations of novel anticoagulants with lmwh will require careful study to ensure no compounding effects on hemostasis. the development of in vitro or ex vivo models of hemostasis may be useful in predicting the potential for adverse major bleeding events for the use of novel anticoagulants as monotherapy or in combination with lmwh prior to evaluation in clinical trials. herein we describe the development of a simple microfluidic-based assay to model hemostasis. we aim to use this model for evaluation and dose selection of cas inhibitors for use in clinical trials to evaluate their safety and efficacy in mitigating thrombosis in covid-19. pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 covid-19 and thrombotic or thromboembolic disease: implications for prevention transport phenomena transport physics and biorheology in the setting of hemostasis and thrombosis id-19? is the recommendation to use high-dose heparin for thromboprophylaxis justified? covid-19 and its implications for thrombosis and anticoagulation systems analysis of thrombus formation coronavirus disease 2019 and stroke: clinical manifestations and pathophysiological insights fluid mechanics of blood clot formation severe covid-19 infection and thrombotic microangiopathy: success does not come easily heparin: an essential drug for modern medicine thrombi produced in stagnation point flows have a core-shell structure using microfluidic devices to study thrombosis in pathological blood flows endeman. incidence of thrombotic complications in critically ill icu patients with covid-19 the basement membrane protein nidogen-1 supports platelet adhesion and activation factor xii inhibition reduces thrombus formation in a primate thrombosis model dimensional analysis and scaling relevant to flow models of thrombus formation: communication from the ssc of the isth immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia incidence of venous thromboembolism in hospitalized patients with covid-19 potentiation of trap-6-induced platelet dense granule release by blockade of p2y12 signaling with mrs2395 a shear gradient-dependent platelet aggregation mechanism drives thrombus formation heparin and anticoagulation soft lithography for micro-and nanoscale patterning protease-activated receptor 4 activity promotes platelet granule release and plateletleukocyte interactions identification of multiple sites of interaction between heparin and the complement system a microengineered vascularized bleeding model that integrates the principal components of hemostasis a microfluidic model of hemostasis sensitive to platelet function and coagulation the contact activation system as a potential therapeutic target in patients with covid-19 hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia interrelationships between structure and function during the hemostatic response to injury covid-19 associated pulmonary thrombosis hierarchical organization of the hemostatic response to penetrating injuries in the mouse macrovasculature the pathogenesis and treatment of the 'cytokine storm' in covid-19 soft lithography dynamics of blood flow and thrombus formation in a multi-bypass microfluidic ladder network design and utility of a point-of-care microfluidic platform to assess hematocrit and blood coagulation publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this work was supported by grants from the national institutes of health (r01hl101972 and r01hl144113) and the american heart association (18ufel33960363). r.m. thompson is an aha undergraduate research fellow. all human subjects research was carried out in accordance with institutional guidelines approved by the oregon health & science university institutional review board. no animal studies were carried out by the authors for this article. key: cord-004133-32w6g7qk authors: walker, faye m.; hsieh, kuangwen title: advances in directly amplifying nucleic acids from complex samples date: 2019-09-30 journal: biosensors (basel) doi: 10.3390/bios9040117 sha: doc_id: 4133 cord_uid: 32w6g7qk advances in nucleic acid amplification technologies have revolutionized diagnostics for systemic, inherited, and infectious diseases. current assays and platforms, however, often require lengthy experimental procedures and multiple instruments to remove contaminants and inhibitors from clinically-relevant, complex samples. this requirement of sample preparation has been a bottleneck for using nucleic acid amplification tests (naats) at the point of care (poc), though advances in “lab-on-chip” platforms that integrate sample preparation and naats have made great strides in this space. alternatively, direct naats—techniques that minimize or even bypass sample preparation—present promising strategies for developing poc diagnostic tools for analyzing real-world samples. in this review, we discuss the current status of direct naats. specifically, we surveyed potential testing systems published from 1989 to 2017, and analyzed their performances in terms of robustness, sensitivity, clinical relevance, and suitability for poc diagnostics. we introduce bubble plots to facilitate our analysis, as bubble plots enable effective visualization of the performances of these direct naats. through our review, we hope to initiate an in-depth examination of direct naats and their potential for realizing poc diagnostics, and ultimately transformative technologies that can further enhance healthcare. nucleic acid amplification tests (naats) have become indispensable tools in biology and medicine. for example, for infectious diseases diagnostics, naats are generally faster, more sensitive, and more specific than the current gold standard of culture-based techniques. in fact, a number of dna-and rna-based diagnostics are now recommended by the us food and drug administration (fda) for infectious diseases such as human immunodeficiency virus (hiv) [1, 2] . bringing naats to the point of care (poc), and particularly to resource-poor settings, is envisioned to revolutionize healthcare. unfortunately, many naats require access to expensive, specialized equipment and a degree of expertise that is highly unlikely to be found in decentralized laboratories. as an additional challenge, these tests typically require an extraction step to isolate dna or rna from blood, urine or sputum, and a purification step to eliminate contaminants from the sample matrix that can confound the actual detection procedure (figure 1 ). these procedures necessitate expensive instrumentation and can add up to several hours to sample-to-answer results, which further restricts the use of naats within centralized laboratories. direct nucleic acid testing is much more convenient and streamlined than the three-step method with preparatory techniques. in a typical extraction experiment, buffer with lytic agents is added to dilute the sample and homogenized with a mixer. sonication creates pressure waves that burst the cells in mechanical lysis. lysozyme enzymatically destroys cells, and is removed from the reaction with vortexing and centrifugation in a phenol/chloroform phase separation. the dna is precipitated in fresh ethanol and the resulting mixture is washed to remove excess contaminants. excess liquid is removed so that the dna can be resuspended in an appropriate buffer. many groups have attempted to develop portable, integrated, microfluidics-based platforms to increase the functionality of diagnostic sensing and analysis [3] [4] [5] , and some of these have even been commercialized (e.g., biomerieux's nuclisens easyq tests, twistdx's twistamp kits, and enigma diagnostic's minilab). these platforms present breakthrough technologies for rapid, cost-effective, and user-friendly diagnostics. while it remains to be seen whether these systems are simple and error-free enough for developed and developing settings, they demonstrate the feasibility of implementing existing nucleic acid amplification methods for poc use [6] [7] [8] [9] . an alternative approach to time-consuming and cumbersome sample preparation is performing naats directly from complex samples ( figure 1 ). the advantage of traditional amplification technologies, such as pcr with real-time spectroscopic or mass spectrometry detection, is that the results are highly specific and quantitative. however, these sensing platforms are expensive and require prior extraction of genetic material from the sample. direct naats are advantageous when complicated, costly laboratory apparatuses are not available. they not only reduce the time, labor, and technical constraints of molecular testing, but also bring the additional benefit of standardizing results [10] . indeed, a growing number of groups are developing such "direct" naats. most notably, the alere i influenza a&b assay became the first fda clinical laboratory improvement amendments (clia)-waived nucleic acid-based test [11] in january 2015. as the alere i system requires no front-end nucleic acid extraction, and can be used outside of traditional laboratory sites [12] [13] [14] [15] [16] , its development and clia-waived status provide strong support for further development of direct assays that can minimize or even bypass sample preparation. thus motivated, we present the current state of direct assays and platforms that achieve nucleic acids detection and analysis from clinically-relevant, complex samples but with either minimal or biosensors 2019, 9, 117 3 of 29 even no sample preparation procedures. we surveyed the literature from 1989-2017 and came across a significant number of works that reported naats from bodily samples (e.g., blood-based liquids, oral samples, swabs) without the complex steps generally involved in sample preparation. this meant discarding the works that depended on sophisticated instruments and operations that are labor-, time-, and cost-intensive, such as enzymatic (proteinases), chemical (acids, detergents), or physical (temperature shock, mechanical disruptions) treatments. then, we describe examples whereby data visualization can be used to reveal the connections between the robustness, sensitivity, and efficacy of technologies developed for direct dna-and rna-based tests. it is our hope that in reviewing technologies such as these, and presenting these promising early findings in an information-rich and accessible fashion, we can help to accelerate the development of approaches that make poc nucleic acid testing rapid, accurate, simple, and affordable. in order to find relevant articles with data on naat parameters, we performed literature searches from december 2014 to february 2018. we searched google scholar with a combination of search terms. these followed a formula of combining a descriptor (e.g., "point-of-care"), an amplification technology (e.g., "lamp" or "loop-mediated isothermal amplification") and a sample matrix (e.g., "blood"). references of previously published reviews, as well as those included in original studies, were checked for possible candidate articles. articles were initially screened on the title, and secondly on the abstract. any articles that relied on microfluidic platforms or commercialized extraction devices were excluded. publications that required complex pre-processing with enzymatic treatment or chemical purification were not selected. studies were included if they involved direct amplification and detection of genetic material from one of six representative sample types: blood, dried blood spot, serum and plasma, saliva and sputum, swabs, urine, and stool. the full text of appropriate articles was read to extract the necessary information. from each of the 174 published works surveyed, we extracted and recorded data that corresponded to test performance. there are many parameters that cannot be ignored when considering naats: accuracy, specificity, user-friendliness, training requirements, and so on. as such, we provide an extensive examination of nucleic acid template specificity (including single or multiplexed reactions), amplification methodologies (enzymes, operating temperatures, and amplification technology), and user-friendliness (storage considerations, pretreatment requirements, and physical involvement) in supplementary table s1 . in addition, we have classified assays that can feasibly be completed without extensive training or high-end instrumentation as "direct," whereas those with greater labor or equipment demands (e.g., freezers, high-speed centrifugation, or incubation for multiple hours) are deemed "semi-direct." specifically, the "semi-direct" assays have the following exceptions to a simple laboratory setup: alternating between two or more incubation temperatures (other than room temperature), relying on enzymatic activity, or requiring more than a brief, low-speed (<100× g) centrifugation. methods categorized as "semi-direct" face some hurdles to implementation as an on-site service for patient care. what these tests do offer is a way to deliver actionable results that can link diagnosis to treatment. with appropriate conversion from requirements for highly trained staff and sophisticated tools to easy-to-use methods, "semi-direct" procedures will meet the requirements for poc diagnostic devices. we found certain parameters could be distilled into numerical data, yielding particularly useful insights when examining different tests. we have devised three major criteria that are indicative of each platform's robustness, sensitivity, and clinical efficacy: tolerance to the sample of interest-ideally, the assay should be able to detect its target against a high concentration of background contaminants. we note that, although sample dilution sometimes provides a convenient way of permitting amplification, doing so inevitably reduces the limit of detection (lod). most naats analyze only a fraction of the sample volume. sample dilution therefore increases the likelihood of false negative results, especially when the samples already have low target concentrations. 2. lod-by foregoing sample preparation, one generally sacrifices the opportunity to concentrate bulk samples, reducing the limit of detection and making sensitivity an important consideration. clinical evaluation-recognizing assays that have been validated with clinical samples. finally, we sought to devise a visual strategy that would clearly and quickly communicate the importance of our criteria, compare the wide range of assays, discover trends in the data, and reveal patterns in a single glance. specifically, the essential information of the 174 reviewed publications is presented quantitatively in a single plot. relevant values are standardized and communicated in terms of visual attributes of position, size, shape, and color. we have found it particularly useful to visualize the data as "bubble plots." in a bubble plot, numerical values from three parameters are simultaneously visualized via the two axes and the size of the circular marker. different categories can also be grouped according to the color of the markers. in our case, we can readily display the essential information (e.g., sample tolerance, lod, and instances of clinical testing) of related procedures to discern those that enhance test performance. in our survey, we came across eight dna-and rna-based testing techniques. as expected, pcr (and reverse transcription pcr, or rt-pcr) has been the predominant technique. notably, a number of isothermal amplification techniques have also been used to develop direct naats. herein, we provide brief overviews of these lesser known isothermal amplification techniques. while pcr is the most commonly reported method of amplification, there is an increasing number of isothermal amplification technologies that can be truly used at the poc. the single reaction temperature enables the use of less costly, complicated instruments than for thermal cycling tests. loop-mediated isothermal amplification (lamp) is one such widely researched, developed, and characterized method [17] . amplification employs a strand-displacing polymerase and two or three pairs of primers: one that is sacrificed to linearize the template, and one or two others that prime the dna synthesis to produce concatenated, cauliflower-like products [18] . as with pcr, lamp has been modified to target rna as reverse-transcription (rt)-lamp [19] . lamp has been compared to pcr in other ways as well, including applications with bacterial, viral, fungal, and parasitic assays. not only has the specificity and sensitivity been equivalent to that of pcr, the robustness of lamp to certain preparations of serum, swabs, and blood has shown it to be more tolerant to inhibitors than pcr [8] . the nucleic acid sequence-based amplification (nasba) method is unique in its ability to amplify single-stranded rna directly [20] . this is most desirable for targeting rna viruses and for transcriptome analysis [8] . the continuous, homogeneous, isothermal process relies on rna polymerase, rnase, and reverse transcriptase. first, the reverse transcriptase creates a double stranded rna:dna hybrid from the rna template; next, the original rna is destroyed; a dna duplex is synthesized; then, the polymerase can transcribe rna from the dna. each new rna molecule can repeat the cycle for exponential amplification. nasba has been applied to a wide-ranging set of research problems, including hiv diagnosis during the aids epidemic of the 1990s and automated, real-time, clinical tests in blood with the modern nuclisens (biomerieux, inc., durham, nc, usa) or in urine with the aptima assay (hologic, san diego, ca, usa). nasba is also used outside of the commercial sector with systems to monitor viruses in serum [21] . the strand displacement amplification (sda) technique is based upon the abilities of a restriction enzyme and a dna polymerase. a primer containing a recognition sequence for the restriction enzyme binds to its complementary, single stranded dna target. after extension by the polymerase, the restriction enzyme nicks the unmodified strand of the double-stranded hemiphosphorothioate recognition site. dna polymerase then extends the 3 end of the nick, displacing the downstream strand. the end result is exponential target amplification from the displaced strands, which serve as targets for new reactions. sda is not complex, but it does suffer from sensitivity issues in the presence of background dna. the best way to overcome off-target amplification, and hence reduce false-positives, is to use simple pretreatment procedures like those that have been developed for detection with the bdprobe-tec (becton dickinson microbiology systems, sparks, md, usa) and in-house systems for urine [22] . recombinase polymerase amplification (rpa) avoids thermal cycling by using three core proteins that operate optimally between 37-40 • c [23] . the first protein, recombinase, binds to primers that recombine with a duplex target for strand displacement. the second, a single-stranded dna binding protein, attaches to the displaced strand before a strand-displacing polymerase copies the dna from the primer onwards for exponential amplification. one of the requirements for rpa technology is sequence-specific detection. this avoids the problem of primer artifacts that add to background fluorescence with nonspecific, intercalating dyes. with its specific readout and rapidity (<20 min to results) as two main features, rpa provides an alternative to the time-consuming processes of culturing and bacterial genotyping when testing for pathogens [7] . strand invasion based amplification (siba) is another amplification process that relies on recombinase activity. in siba, there is a separate recombinase substrate that is inserted between two primer-binding sites. the duplex peripheral to this insertion site is separated, enabling the primers to bind. dna polymerase can then extend the template from the bound primers. this use of an invading substrate, one that is neither consumed nor included in the extension of dna, is advantageous because it abolishes primer artifacts. siba can therefore be used to reliably detect low copy numbers of pathogens-other isothermal methods generate non-specific amplification products in the absence of target dna [24] . going further, the specificity of siba enables multiplexing for the detection of templates that differ by as little as two bases [25] . multiple displacement amplification (mda) is a technique that exploits the strand displacement, proofreading, and polymerase activity of the φ29 bacteriophage dna polymerase [26] . the highly processive polymerase uses random primers to amplify an entire genome. mda is therefore well-suited for whole genome amplification from crude biological samples, which can be followed by single nucleotide polymorphism (snp) testing and genotyping [8] . the concept of hybridization chain reaction (hcr) [27] -an enzyme-free, room-temperature method-relies on a dna trigger to initiate amplification. the initiator interacts with two stable dna hairpins to create nicked double helices. amplification of this initiation event continues until the hairpins are depleted. hcr is a useful assay for detecting short dnas, such as human immunodeficiency virus type 1 (hiv-1) in serum [28] . time-series plots offer an effective means for showing the growing prevalence of direct naats. specifically, within each sample type, we plotted the number of clinical samples that have been analyzed by direct naats from 1989 to 2017 ( figure 2 ). here, we also divided the data into two cohorts based on whether samples were subjected to pcr (figure 2 , black) or isothermal amplification techniques ( figure 2 , red). as expected, we saw an overall rise in the number of clinical samples analyzed via naats with minimal or no sample preparation over the analyzed period. across all six sample types, we observed sharp spikes, which indicate studies of high numbers of clinical samples. based on the sample type, swab samples had been most analyzed, while urine and stool samples had been least analyzed. within each sample type, after an initial lag, we saw a notable rise in the use of isothermal amplification techniques. this first became apparent as early as 2003, several years after the advent of lamp in 2000, and twelve years after the introduction of nasba [20] . pcr-based systems emerged within five years of the technique's inception in 1986 [29] , and pcr largely continues to dominate the realm of nucleic acid testing. the one notable exception is seen in our time-course of blood testing, where isothermal techniques have surpassed pcr and rt-pcr in terms of the number of assays performed on whole blood samples. the concept of hybridization chain reaction (hcr) [27] -an enzyme-free, room-temperature method-relies on a dna trigger to initiate amplification. the initiator interacts with two stable dna hairpins to create nicked double helices. amplification of this initiation event continues until the hairpins are depleted. hcr is a useful assay for detecting short dnas, such as human immunodeficiency virus type 1 (hiv-1) in serum [28] . time-series plots offer an effective means for showing the growing prevalence of direct naats. specifically, within each sample type, we plotted the number of clinical samples that have been analyzed by direct naats from 1989 to 2017 ( figure 2 ). here, we also divided the data into two cohorts based on whether samples were subjected to pcr (figure 2 , black) or isothermal amplification techniques (figure 2 , red). as expected, we saw an overall rise in the number of clinical samples analyzed via naats with minimal or no sample preparation over the analyzed period. across all six sample types, we observed sharp spikes, which indicate studies of high numbers of clinical samples. based on the sample type, swab samples had been most analyzed, while urine and stool samples had been least analyzed. within each sample type, after an initial lag, we saw a notable rise in the use of isothermal amplification techniques. this first became apparent as early as 2003, several years after the advent of lamp in 2000, and twelve years after the introduction of nasba [20] . pcr-based systems emerged within five years of the technique's inception in 1986 [29] , and pcr largely continues to dominate the realm of nucleic acid testing. the one notable exception is seen in our time-course of blood testing, where isothermal techniques have surpassed pcr and rt-pcr in terms of the number of assays performed on whole blood samples. because blood contains circulating nucleic acids, cells, and over 20,000 different proteins, it offers an abundance of biomarkers for disease detection. molecular diagnostics in blood are useful for detecting specific dna or rna sequences from a range of bacterial, toxic, and viral infectious agents. platforms for hepatitis and human immunodeficiency virus [30] , staphylococcus aureus [31] , and plasmodium species are just a few of the most-used systems enabling rapid diagnostics in whole blood. blood-based testing generally demands sophisticated detection instruments or extensive preparation to recover inhibitor-free and high-purity dna. not all inhibitory blood components are known [32] , but heme compounds, anticoagulants, and immunoglobulin g (igg) can all interfere with amplification reactions by inhibiting dna polymerase activity [33] or chelating necessary cofactors [34, 35] . although a wide range of bloodborne viruses, bacteria, and parasites can in principle be detected with nucleic acid testing, extraction-and purification-free means of detecting these pathogens are not currently commercially available. we have visualized the general trends of direct and semi-direct nucleic acid testing in blood as a function of the lods ( figure 3 ). the % (v/v) of blood tolerated in a reaction is plotted against the lod in g of template, with the number of clinical samples encoded as the area of the bubble. we have also assigned colors to indicate the type of amplification technology. it is evident that many studies have achieved high sensitivity in detecting their target in a low concentration of blood. this shows that nucleic acid testing has great potential for blood-based tests in poc situations where collection volumes are small (e.g., finger pricks) and parasite loads may be low. those examples from the literature that were not demonstrated on patient samples are considered separately in the plot, and represented by xs rather than bubble markers. some of these are purported to have very low lods that reach below the fg level (table s1 )-it remains to be seen whether such tests will perform with the same extreme sensitivity in a clinical context. because blood contains circulating nucleic acids, cells, and over 20,000 different proteins, it offers an abundance of biomarkers for disease detection. molecular diagnostics in blood are useful for detecting specific dna or rna sequences from a range of bacterial, toxic, and viral infectious agents. platforms for hepatitis and human immunodeficiency virus [30] , staphylococcus aureus [31] , and plasmodium species are just a few of the most-used systems enabling rapid diagnostics in whole blood. blood-based testing generally demands sophisticated detection instruments or extensive preparation to recover inhibitor-free and high-purity dna. not all inhibitory blood components are known [32] , but heme compounds, anticoagulants, and immunoglobulin g (igg) can all interfere with amplification reactions by inhibiting dna polymerase activity [33] or chelating necessary cofactors [34, 35] . although a wide range of bloodborne viruses, bacteria, and parasites can in principle be detected with nucleic acid testing, extraction-and purification-free means of detecting these pathogens are not currently commercially available. we have visualized the general trends of direct and semi-direct nucleic acid testing in blood as a function of the lods ( figure 3 ). the % (v/v) of blood tolerated in a reaction is plotted against the lod in g of template, with the number of clinical samples encoded as the area of the bubble. we have also assigned colors to indicate the type of amplification technology. it is evident that many studies have achieved high sensitivity in detecting their target in a low concentration of blood. this shows that nucleic acid testing has great potential for blood-based tests in poc situations where collection volumes are small (e.g., finger pricks) and parasite loads may be low. those examples from the literature that were not demonstrated on patient samples are considered separately in the plot, and represented by xs rather than bubble markers. some of these are purported to have very low lods that reach below the fg level (table s1 )-it remains to be seen whether such tests will perform with the same extreme sensitivity in a clinical context. bubble plot of nucleic acid diagnostics performed in whole blood. percent concentration (v/v) of blood per reaction in a given procedure is displayed as a function of the limit of detection (lod) in g of template. the number of patient samples tested is proportional to the log of the marker area, as shown at top, and the testing methodology is indicated by marker color. cases shown with × instead of bubble markers illustrate that patient testing was not reported. by assessing the pcr-and isothermal-based data, we could obtain some insight into how to optimize these techniques to better tolerate blood as a sample matrix. several of the semi-direct works with pcr have employed over 50% blood in a reaction after heat-cold shock [36] . more noteworthy is a truly direct example that relied on the specificity and efficacy of the phusion polymerase (new england biolab, ipswich, ma, usa) to perform pcr in 40% blood [37] . pcr typically employs the taq polymerase from thermus aquaticus. chemical additives, whether commercially-available cocktails [38, 39] or in-house buffers [39-52], allow the taq family of polymerases to amplify dna from whole blood. pcr can likewise be optimized through the use of more unconventional polymerases [53-58] and physical heating steps [53, [59] [60] [61] [62] [63] [64] to reduce the inhibitory effect of blood components. these referenced works offer expedited methods to obtain amplifiable templates with similar sensitivities to chemical-based extraction kits [59] . though several authors include the use of a centrifuge in the extraction process, these semi-direct methods of template preparation could likely be completed by relying on careful pipette-based transfer of supernatants rather than high-speed centrifugation [59, 63] . most isothermal amplification-based diagnostics in blood make use of lamp [17] , which offers a highly tolerant means of amplification [8] . simple treatments with heat [59, [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] or chemicals [73, [77] [78] [79] [80] [81] can increase the sensitivity of the lamp or rt-lamp reaction. most impressive are the examples of direct amplification of dna in blood with lamp-based technologies [82, 83] and other isothermal amplification methods like mda [84] . some of these assays employ a post-heating centrifugation step, but since poon et al. have demonstrated that lamp can be performed directly on heat-treated blood without a spin-down process, this step could likely be avoided in semi-direct processes [68] . even though these successful examples of simple, direct nucleic acid testing methods highlight the promise of dna amplification in whole blood, there is an ongoing need for further improvements. no assay has come close to reaching the capacity of burckhardt et al.'s pcr amplification with taq polymerase in nearly 80% whole blood, as demonstrated over 20 years ago in 1994 [36] . the associated treatment method is one of the more technically-involved and time-intensive, demanding 20 cycles of heating and cooling. it remains to be seen whether an isothermal amplification method could equal this tolerance. perhaps these techniques will make up for their decreased level of tolerance in their ease of use, as evidenced by suzuki et al. achieving 20% incorporation of whole blood in lamp with only a five-minute heating [76] . dried blood spots offer a convenient alternative for screening for genetic disorders, testing for infectious diseases, and profiling drug metabolism in settings with limited laboratory or storage capabilities. such samples are typically prepared by spotting whole blood, either from venous blood or a finger prick, onto filter paper [85] . sampling time is quick, temperature-controlled storage is unnecessary, and biohazard risks are minimized for health care workers [86] . the downside of such samples is that the dna in the dried blood must be eluted from the paper-based cellular components before it can be amplifiable. filter paper has been used as medium to test blood for infectious diseases since the 1940s [87] . from syphilis diagnosis during world war ii [88] , to infant screening in the 1960s [89] , to hiv detection and monitoring in the modern day [85, 90] , there are important assays with dried blood spots in naats. commercial technologies are even becoming widely available to map, monitor, and survey blood spots from patients infected with malaria or other neglected tropical diseases [74, 91] . in a similar manner, the preparation and processing techniques for dried blood samples presented below could open new avenues for disease control and elimination when combined with well-standardized assays for detecting bloodborne pathogens. as shown in figure 4 , all of the tests we surveyed have been validated with actual dried blood spots. in the most-heavily tested example (720 clinical samples) by raskin et al., pretreatment with heating-cooling cycles and addition of spermidine to the reaction helped boost the efficiency and yield of the amplification [92] . blood spot-containing filter paper can typically be directly added into a mixture of reagents, though it necessitates overcoming the high background interference of the filter paper and detecting small amounts of dried blood. biosensors 2019, 9, x for peer review 9 of 28 as shown in figure 4 , all of the tests we surveyed have been validated with actual dried blood spots. in the most-heavily tested example (720 clinical samples) by raskin et al., pretreatment with heating-cooling cycles and addition of spermidine to the reaction helped boost the efficiency and yield of the amplification [92] . blood spot-containing filter paper can typically be directly added into a mixture of reagents, though it necessitates overcoming the high background interference of the filter paper and detecting small amounts of dried blood. to overcome the background interference from filter paper in directly amplifying dried blood spots via pcr, researchers have bolstered the enzyme's resistance to inhibitors and included various buffer components [47, 93] . pretreatments, such as fixing [92, [94] [95] [96] [97] or heating [43, [98] [99] [100] , also aid in improving sensitivity and specificity. even if the procedures are reported as being too lengthy for poc, there are appropriate ways to scale down the waiting period: for instance, an overnight drying period with methanol while under vacuum [98] can be streamlined into a five-minute methanol fix [100] . most of the approaches to amplify dna directly in blood spots use eluants-either from commercial kits [51, 101] , in-house buffers [44,59,102-105], or water [106, 107] -to overcome the various difficulties that impede pcr reactions with paper matrices. buffer-based eluants, in addition to being cost-effective, can achieve even higher sensitivities than standard extraction protocols [105] . similar preparatory approaches are used for lamp, wherein heating in water [72, 108] , phosphate buffered saline (pbs) [59] , or sodium dodecyl sulfate (sds) buffer [74] enables a fast and easy nucleic acid elution with amplification results that are comparable to the conventional gold standard of microscopy [72, 74, 108] . one of the difficulties in making blood spots suitable for lamp and pcr is the need to resuspend the spots in liquid, then filter out the species of interest. the easiest way to address the former problem is through long elution times; the latter, through centrifugation. this makes taylor et al.'s amplification of plasmodium spp. dna directly from clinical filter paper samples such a remarkable achievement for low-resource settings. the combination of an inhibitor-resistant taq mutant and an enhancer cocktail resulted in a specificity and sensitivity of 100% for 48 patient to overcome the background interference from filter paper in directly amplifying dried blood spots via pcr, researchers have bolstered the enzyme's resistance to inhibitors and included various buffer components [47, 93] . pretreatments, such as fixing [92, [94] [95] [96] [97] or heating [43, [98] [99] [100] , also aid in improving sensitivity and specificity. even if the procedures are reported as being too lengthy for poc, there are appropriate ways to scale down the waiting period: for instance, an overnight drying period with methanol while under vacuum [98] can be streamlined into a five-minute methanol fix [100] . most of the approaches to amplify dna directly in blood spots use eluants-either from commercial kits [51, 101] , in-house buffers [44,59,102-105], or water [106, 107] -to overcome the various difficulties that impede pcr reactions with paper matrices. buffer-based eluants, in addition to being cost-effective, can achieve even higher sensitivities than standard extraction protocols [105] . similar preparatory approaches are used for lamp, wherein heating in water [72, 108] , phosphate buffered saline (pbs) [59] , or sodium dodecyl sulfate (sds) buffer [74] enables a fast and easy nucleic acid elution with amplification results that are comparable to the conventional gold standard of microscopy [72, 74, 108] . one of the difficulties in making blood spots suitable for lamp and pcr is the need to re-suspend the spots in liquid, then filter out the species of interest. the easiest way to address the former problem is through long elution times; the latter, through centrifugation. this makes taylor et al.'s amplification of plasmodium spp. dna directly from clinical filter paper samples such a remarkable achievement for low-resource settings. the combination of an inhibitor-resistant taq mutant and an enhancer cocktail resulted in a specificity and sensitivity of 100% for 48 patient samples [47] . all the approaches have interesting characteristics that make them special, but none achieve the ease in use of this assay for malaria. blood plasma and serum are widely used for quantitative molecular diagnostics in the areas of clinical decision-making and therapeutic management [109] . plasma is the pale yellowish fluid that normally holds the blood cells of whole blood in suspension, whereas serum is the remnants of blood plasma after the removal of clotting factors [110] . circulating dna in serum and plasma is a biomarker for a diverse array of systemic, infectious, and genetic diseases. these include particular disorders such as diabetes [109] and hepatitis b virus [111] . refining blood into serum or plasma historically requires expensive equipment for centrifugation or sedimentation. recovering dna or rna from blood-based proteins, nutrients, electrolytes, antibodies (particularly igg), antigens, hormones, and exogenous substances becomes even more challenging when considering the low relative levels of cell-free or cell-bound nucleic acids [112] [113] [114] . more recently, however, paper-or card-based devices [115, 116] , membrane-based sedimentation [117] , and microscale devices for cell differentiation and filtration [118] have made blood separation a single step process at the poc. as such, we include these sample types here. in assessing nucleic acid testing with plasma or serum, we see that most reactions are performed at sample concentrations in the 20% range ( figure 5 ). however, it is important to note that the sensitivity does not necessarily suffer in much more concentrated samples-in liu et al.'s highly robust two-step amplification process with direct hairpin assembly and hcr-based detection of snp dna sequences in 50% (v/v) serum, they achieved a very low lod of 100 pg [119] . these plasma/serum-based tests are especially promising for use in real-world contexts, because their clinical relevance is well-documented-18 out of the 24 cases examined here included testing with patient samples. samples [47] . all the approaches have interesting characteristics that make them special, but none achieve the ease in use of this assay for malaria. blood plasma and serum are widely used for quantitative molecular diagnostics in the areas of clinical decision-making and therapeutic management [109] . plasma is the pale yellowish fluid that normally holds the blood cells of whole blood in suspension, whereas serum is the remnants of blood plasma after the removal of clotting factors [110] . circulating dna in serum and plasma is a biomarker for a diverse array of systemic, infectious, and genetic diseases. these include particular disorders such as diabetes [109] and hepatitis b virus [111] . refining blood into serum or plasma historically requires expensive equipment for centrifugation or sedimentation. recovering dna or rna from blood-based proteins, nutrients, electrolytes, antibodies (particularly igg), antigens, hormones, and exogenous substances becomes even more challenging when considering the low relative levels of cell-free or cell-bound nucleic acids [112] [113] [114] . more recently, however, paper-or card-based devices [115, 116] , membrane-based sedimentation [117] , and microscale devices for cell differentiation and filtration [118] have made blood separation a single step process at the poc. as such, we include these sample types here. in assessing nucleic acid testing with plasma or serum, we see that most reactions are performed at sample concentrations in the 20% range ( figure 5 ). however, it is important to note that the sensitivity does not necessarily suffer in much more concentrated samples-in liu et al.'s highly robust two-step amplification process with direct hairpin assembly and hcr-based detection of snp dna sequences in 50% (v/v) serum, they achieved a very low lod of 100 pg [119] . these plasma/serum-based tests are especially promising for use in real-world contexts, because their clinical relevance is well-documented-18 out of the 24 cases examined here included testing with patient samples. researchers have developed convenient pcr assays for both direct and semi-direct testing in blood-based fluids. by using enhanced enzymes, dna [49,50,67,120] and rna [121, 122] targets have been successfully amplified in plasma and serum. additionally, heat-based pretreatment can be used to release nucleic acids prior to carrying out amplification [36, 60, [123] [124] [125] [126] . the effect of preheating can be seen in lamp as well. lamp is generally tolerant to the serum or plasma environment [120, 127] , but preheating the input sample has been found to have a favorable effect [128] [129] [130] [131] that produces up to a 100-fold improvement in sensitivity [132] . this heating also enabled pardee et al. to detect zika virus rna in serum with high sensitivity using nasba [21] . hcr performs especially well in serum without any pretreatment [28, 119, [133] [134] [135] , presumably because the reaction relies on cascaded hybridization events instead of polymerases. because plasma and serum contain very low-abundance analytes, nucleic acid tests need to operate with high sensitivity. fortunately, lamp-based applications are achieving increasingly low limits of detection. nijru et al., for instance, demonstrated that their lod of 1 trypanozoan parasite/l serum in hat diagnosis was 100-fold more sensitive than pcr testing. such methods could still benefit from user-friendly techniques for large-scale processing. some semi-direct examples presented above include a centrifugation step to collect condensate formed after heating, but could just as easily rely on pipette collection to obviate the need for a high-speed centrifuge. others might benefit from certain stand-alone modules for plasma and serum separation that could be integrated into a poc workflow [117, 136] . saliva and sputum are abundant and easy to obtain, and are thus attractive samples for diagnostics. saliva flows into the oral cavities through salivary glands, where blood vessels secrete the same protein and nucleic acid biomarkers as in peripheral blood. in contrast with blood-based samples, saliva sampling does not require trained technicians, presents fewer antigen-associated risks, and can be more easily purified (saliva is 95% water) [137] . sputum, a necessary sample for respiratory infections, is mucus from the lower airways. unfortunately, saliva and sputum are very heterogeneous with respect to the distribution of organisms, chemical composition, and the presence of outside contaminants such as toothpaste, cigarette smoke, coffee, or mouthwash. technical extraction kits such as rnaqueous and magmax (life technologies, grand island, ny, usa) are often used to eliminate inhibitors and nucleases from oral samples. the viscosity of sputum requires particularly cumbersome protocols for sample preparation: full processing begins with mucolytic agents such as n-acetyl-l-cysteine (nalc) and dithiothreitol (dtt), disruption of mycobacteria by detergents and proteolytic enzymes, then isolation of target dna by organic solvents or capture reagents [138, 139] . the human salivary microbiome has importance as a diagnostic indicator of oral cancer, oral diseases such as periodontitis, and systemic diseases such as pneumonia [140] . as for sputum, it has become the specimen of choice for detecting tuberculosis [141, 142] . naats for mycobacterium tuberculosis have been endorsed by the who (world health organization) and the fda for their high accuracy [143, 144] . the systems introduced below extend the practical usage of sputum for poc testing by reducing the requirements for sputum manipulation. most reported nucleic acid testing methods for saliva and sputum show fairly high numbers for patient samples tested, with only two of 30 cases that did not examine clinical specimens ( figure 6 ). the approaches we examined generally employ dilutions of 20% or less, and achieve low detection limits. this is critical for avoiding false positives, as the target concentrations in sputum and saliva are small. the lowest lod achieved-2 fg of acinetobacter baumannii bacterial gdna in sputum-required pretreatment with sputazyme (kyokuto, tokyo, japan) and heat before lamp analysis [145] . in contrast with blood, the high water content of saliva should make it relatively easy to augment the concentration of matrix that can be employed in an amplification reaction. there are several examples of amplification directly on dried sputum collected via filter cards, which is an especially promising direction for direct testing at the poc if samples need to be stored, handled by multiple clinicians, or reevaluated at later dates [87, 146, 147] . amongst the collection of approaches for direct pcr amplification on saliva samples, those that begin with dried saliva swabs fully circumvent dna extraction, purification, and quantification [93] . hall et al. added punches of saliva stains directly to the reaction mixture in order to perform str analysis. genetic testing in saliva or sputum is often performed directly in liquid samples, and dilution into the reagents is typically sufficient to negate the effect of any inhibitors [148] [149] [150] , although heat [151] or additives such as polyethylene glycol (peg), hydroxides, or dithiothreitol (dtt) may be added to further process samples [42, 43, 56, 145] . with the broad range of bacterial species present in the mouth (over 600 inventoried), differing in terms of their contributions to health and disease, pcr is very useful for profiling large numbers of bacteria. some species are recalcitrant to lysing, like streptococcus. so, instead of relying on bead-beating, phenol treatments, or other steps, aas et al. applied proteinase k lysates directly to pcr reagents [140] . several other groups have followed suit in detecting bacterial taxa in healthy [152, 153] and diseased saliva samples [154] . direct amplification is also possible in the field of lamp-based diagnostics, as evidenced in testing for zika [82] and malaria [65] . du et al. went further than simplifying the sample treatment, a ten-minute heating of zaire ebolavirus dna in saliva, by actually providing a lamp-to-glucose transduction that can be read out on a handheld glucometer [155] . designing direct tests for sputum is inherently difficult because many nucleic acid-based methods process samples analogously to culture-based protocols. in this n-acetyl-l-cysteine (nalc)-naoh method [156] , the viscous sputum matrix is liquefied through several buffer exchanges and high-speed centrifugation into a more manipulative sample for testing. an additional concern is decontamination. this is necessary for culture, but also useful to protect the operator from biosafety hazards in molecular testing [157] . several semi-direct examples based on lamp [158] [159] [160] , recombinase polymerase amplification (rpa) [23, 161] , or pcr [162] [163] [164] [165] [166] have adopted this practice. however, side-by-side comparisons of nucleic acid testing on sputum samples with or without naoh-nalc treatment have indicated that naoh-nalc processing could be removed to enable truly direct protocols for sputum. mitarai et al. reported that the addition of nalc to a sputum specimen prior to extraction had no effect on testing for tb [167] . furthermore, tarhan et al. showed amongst the collection of approaches for direct pcr amplification on saliva samples, those that begin with dried saliva swabs fully circumvent dna extraction, purification, and quantification [93] . hall et al. added punches of saliva stains directly to the reaction mixture in order to perform str analysis. genetic testing in saliva or sputum is often performed directly in liquid samples, and dilution into the reagents is typically sufficient to negate the effect of any inhibitors [148] [149] [150] , although heat [151] or additives such as polyethylene glycol (peg), hydroxides, or dithiothreitol (dtt) may be added to further process samples [42, 43, 56, 145] . with the broad range of bacterial species present in the mouth (over 600 inventoried), differing in terms of their contributions to health and disease, pcr is very useful for profiling large numbers of bacteria. some species are recalcitrant to lysing, like streptococcus. so, instead of relying on bead-beating, phenol treatments, or other steps, aas et al. applied proteinase k lysates directly to pcr reagents [140] . several other groups have followed suit in detecting bacterial taxa in healthy [152, 153] and diseased saliva samples [154] . direct amplification is also possible in the field of lamp-based diagnostics, as evidenced in testing for zika [82] and malaria [65] . du et al. went further than simplifying the sample treatment, a ten-minute heating of zaire ebolavirus dna in saliva, by actually providing a lamp-to-glucose transduction that can be read out on a handheld glucometer [155] . designing direct tests for sputum is inherently difficult because many nucleic acid-based methods process samples analogously to culture-based protocols. in this n-acetyl-l-cysteine (nalc)-naoh method [156] , the viscous sputum matrix is liquefied through several buffer exchanges and high-speed centrifugation into a more manipulative sample for testing. an additional concern is decontamination. this is necessary for culture, but also useful to protect the operator from biosafety hazards in molecular testing [157] . several semi-direct examples based on lamp [158] [159] [160] , recombinase polymerase amplification (rpa) [23, 161] , or pcr [162] [163] [164] [165] [166] have adopted this practice. however, side-by-side comparisons of nucleic acid testing on sputum samples with or without naoh-nalc treatment have indicated that naoh-nalc processing could be removed to enable truly direct protocols for sputum. mitarai et al. reported that the addition of nalc to a sputum specimen prior to extraction had no effect on testing for tb [167] . furthermore, tarhan et al. showed that the sensitivity in tb testing was better for sputum samples that were measured directly, rather than after extraction with naoh-nalc [168] . in pursuing alternatives to the lengthy naoh-nalc method, sputum has been used in pcr after adding mucolytic agents [56, 169] , diluting in buffer [148] , or bead-beating [157] to reduce viscosity. a more aggressive pretreatment, relying on chemical, thermal, and mechanical means of disruption, used heating and centrifugation to detect tb in 548 sputum samples with comparable performance to more expensive molecular-based systems [170] . one particularly noteworthy example of direct testing used lamp to diagnose tuberculosis at three peripheral laboratories [147] . in the study, lamp had a sensitivity of 97.9%, detecting 173 out of 177 smear-negative, culture-positive sputum samples. the authors canvassed the laboratory personnel after they implemented the heating, washing, and filter-tip capture steps before direct amplification to verify that the assay had significant potential to be adopted for routine use. several early examples of pcr on sputum samples with mycobacterium spp. reported sensitivities in the single-digit copy number range. however, the pretreatment methods were quite divergent. sjobring et al. used long centrifugation and sonication steps, in addition to boiling, to detect down to eight organisms [171] . sritharan et al. was able to cut down the steps to a 30 min boiling period, with a resulting lod of 1 organism [172] . since these examples in the 1990s, only one technique with a pre-amplification wash and an rpa reaction has been able to match this performance in detecting a single mycobacterium [161] . as far as combining specificity and sensitivity without adding technical difficulty, priye et al.'s recent multiplexed rt-lamp detection system for zika, dengue, and chikungunya achieved lods of 44 copies/reaction with no need for lysis or extraction [82] . these impressive outcomes from isothermal technologies like rpa and lamp illustrate that fancy hardware is not necessary for testing modalities to achieve high specificity directly in human samples. swabs have become a mainstay in testing for viral pathogens. molecular systems that identify respiratory tract infections in nasal swabs [13] or stis (sexually transmitted infections) in dermal, genital, and conjunctival swabs [173] are used for rapid, accurate patient diagnosis. dna collection from swabs is attractive because it is simple, minimally invasive, and even enables self-sampling. however, swab-collected specimens are likely to contain polymerase inhibitors such as secreted minerals, electrolytes, hormones, enzymes, immunoglobulins, and cytokines, as well as topical medications [137, [174] [175] [176] [177] . as a result, many swab tests now on the market remove these inhibitors via extraction methods that are too involved and complex to be suitable for the poc [178] . all of the swab sample studies we examined employed at least one patient sample, and most achieved high sensitivity at a reasonable level of dilution in the buffer used for dna elution from the solid swab (figure 7) . one remarkable study examined 4518 patient swabs in direct pcr for str (short tandem repeat) analysis [179] -unfortunately, the authors did not report the yields of dna obtained or the lowest amounts detected. this is a particularly common problem amongst these references-when the lods are not reported, it is especially difficult to replicate these procedures or compare the manipulations used in sample storage, dna replication, and detection [180, 181] . special attention should therefore be paid to reproducibility in future efforts at direct amplification of swab samples. typically, elution either at room temperature [51, 176, [182] [183] [184] [185] [186] or with heating [43,44, [187] [188] [189] [190] [191] is sufficient to generate pcr-amplifiable template from swabs in solution. these methods of dilution or heating can save over thirty minutes of processing time, as with nihonyanagi et al.'s heating protocol to release mrsa in celleaseii (biocosm inc., hyogo, japan) diluents [169] . lamp-based testing of swab samples can also be performed at room-temperature [192] [193] [194] [195] [196] or while heated [145, [197] [198] [199] [200] [201] [202] , as can mda [84] . in particular, mahony et al.'s lamp-based test for influenza a and b achieved an analytical sensitivity of one genome equivalent, operating via a novel swab preparation procedure of vortexing and heating [198] . moving towards instrument-free molecular diagnostics systems, a lateral-flow stranddisplacement amplification (sda) [203] assay could directly detect mrsa from nasal swabs with a sensitivity of 600 copies/reaction [204] . lateral flow eliminates the need for expensive detectors. rogdriguez et al. went one step further by combining paper-based extraction and in situ amplification with lateral flow to develop an rt-lamp assay for h1n1 in patient nasopharyngeal specimens. their sensitivity of 500 copies/reaction was well below the mean viral load for h1n1 patients [205] . pushing to even lower limits of detection, an hda-based assay with a vertical-flow dna strip readout [206] could directly test clinical genital swabs in transport medium for hsv types 1 and 2 [207] . the nucleic acid assays had lods of 5.5 and 34.1 copies/reaction for hsv-1 and hsv-2, respectively, and were able to detect low-viral loads below the sensitivity of culture tests. the most sensitive tests on swabs rely on lamp reactions: for example, fewer than 10 copies/reaction of viral targets were seen after brief heating steps in hank's buffer (whittaker bioproducts, boston, ma, usa) [197] , m-swab diluent (copan diagnostics inc., murrieta, ca) [198] , or water [202] . it is noteworthy is that entire swab samples can be used in amplification reactions, eliminating the loss of starting material that accompanies liquid and hardware transfers. this approach has also been successful in several instances of str testing [43, 179] . rodriguez et al. used a similar approach in detecting clinical levels of h1n1 by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [205] . future developments could focus on direct reactions with swabs that integrate extraction, figure 7 . swab-based procedures for nucleic acid testing, with data presented as in figure 3 . typically, elution either at room temperature [51, 176, [182] [183] [184] [185] [186] or with heating [43, 44, [187] [188] [189] [190] [191] is sufficient to generate pcr-amplifiable template from swabs in solution. these methods of dilution or heating can save over thirty minutes of processing time, as with nihonyanagi et al.'s heating protocol to release mrsa in celleaseii (biocosm inc., hyogo, japan) diluents [169] . lamp-based testing of swab samples can also be performed at room-temperature [192] [193] [194] [195] [196] or while heated [145, [197] [198] [199] [200] [201] [202] , as can mda [84] . in particular, mahony et al.'s lamp-based test for influenza a and b achieved an analytical sensitivity of one genome equivalent, operating via a novel swab preparation procedure of vortexing and heating [198] . moving towards instrument-free molecular diagnostics systems, a lateral-flow strand-displacement amplification (sda) [203] assay could directly detect mrsa from nasal swabs with a sensitivity of 600 copies/reaction [204] . lateral flow eliminates the need for expensive detectors. rogdriguez et al. went one step further by combining paper-based extraction and in situ amplification with lateral flow to develop an rt-lamp assay for h1n1 in patient nasopharyngeal specimens. their sensitivity of 500 copies/reaction was well below the mean viral load for h1n1 patients [205] . pushing to even lower limits of detection, an hda-based assay with a vertical-flow dna strip readout [206] could directly test clinical genital swabs in transport medium for hsv types 1 and 2 [207] . the nucleic acid assays had lods of 5.5 and 34.1 copies/reaction for hsv-1 and hsv-2, respectively, and were able to detect low-viral loads below the sensitivity of culture tests. the most sensitive tests on swabs rely on lamp reactions: for example, fewer than 10 copies/reaction of viral targets were seen after brief heating steps in hank's buffer (whittaker bioproducts, boston, ma, usa) [197] , m-swab diluent (copan diagnostics inc., murrieta, ca, usa) [198] , or water [202] . it is noteworthy is that entire swab samples can be used in amplification reactions, eliminating the loss of starting material that accompanies liquid and hardware transfers. this approach has also been successful in several instances of str testing [43, 179] . rodriguez et al. used a similar approach in detecting clinical levels of h1n1 by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [205] . future developments could focus on direct reactions with swabs that integrate extraction, amplification, and detection in a single tube for poc usage. this would give low-resource settings alternatives to instrument-dependent assays like the alere i platform for detecting influenza a & b from nasal swabs. diseases of the kidney or genitourinary tract can often be detected from stool or urine. urea destabilizes interactions between primers, template and polymerase, and since urea is typically present in adult urine at concentrations six-fold greater than can be tolerated in pcr reactions, ultracentrifugation, or related procedures are typically used to prepare such samples for nucleic acid testing [208] [209] [210] . molecular tests for the identification of pathogens in stool rely on extraction methods to remove the proteinases, bile salts, polyphenols, and acids that directly inhibit the activity of dna polymerases [211, 212] . methods for testing external specimens have been integrated into diagnostic screens for pathogens such as chlamydia trachomatis, neisseria gonorrhoeae, and clostridium difficile with high sensitivity and specificity [213] [214] [215] [216] [217] . in surveying direct nucleic acid testing methods (figure 8 ), we noted that the lods are generally lower for urine-based tests than for feces-based. however, there is one example of direct pcr in stool samples, taking advantage of buffer additives and enhanced phire polymerase (new england biolabs, ipswich, ma, usa), which achieved a lod of 0.002 pg of bacterial dna. however, there is a clear need for increased testing with patient samples, as nearly half of the referenced works do not include any clinical validation. this issue is especially notable with fecal testing, where patient testing is only reported in one study. diseases of the kidney or genitourinary tract can often be detected from stool or urine. urea destabilizes interactions between primers, template and polymerase, and since urea is typically present in adult urine at concentrations six-fold greater than can be tolerated in pcr reactions, ultracentrifugation, or related procedures are typically used to prepare such samples for nucleic acid testing [208] [209] [210] . molecular tests for the identification of pathogens in stool rely on extraction methods to remove the proteinases, bile salts, polyphenols, and acids that directly inhibit the activity of dna polymerases [211, 212] . methods for testing external specimens have been integrated into diagnostic screens for pathogens such as chlamydia trachomatis, neisseria gonorrhoeae, and clostridium difficile with high sensitivity and specificity [213] [214] [215] [216] [217] . in surveying direct nucleic acid testing methods (figure 8 ), we noted that the lods are generally lower for urine-based tests than for feces-based. however, there is one example of direct pcr in stool samples, taking advantage of buffer additives and enhanced phire polymerase (new england biolabs, ipswich, ma, usa), which achieved a lod of 0.002 pg of bacterial dna. however, there is a clear need for increased testing with patient samples, as nearly half of the referenced works do not include any clinical validation. this issue is especially notable with fecal testing, where patient testing is only reported in one study. though complex extraction methods are recommended prior to pcr in order to remove inhibitory components of urine and feces, changes to the reaction chemistry could effectively relieve the negative effects of the sample matrix. with urine, this alteration takes the form of a hydrogelencased reaction [183] . the authors developed a pre-assembled, desiccated, gel-based cassette that is rehydrated by the liquid in raw urine samples. the polyacrylamide gel matrix effectively filters though complex extraction methods are recommended prior to pcr in order to remove inhibitory components of urine and feces, changes to the reaction chemistry could effectively relieve the negative effects of the sample matrix. with urine, this alteration takes the form of a hydrogel-encased reaction [183] . the authors developed a pre-assembled, desiccated, gel-based cassette that is rehydrated biosensors 2019, 9, 117 16 of 29 by the liquid in raw urine samples. the polyacrylamide gel matrix effectively filters biological inhibitors out of the amplification reaction, enabling detection of mycoplasma homonis and ureaplasma urealyticum. for feces, the options are to employ inhibitor-resistant polymerases [57] and buffer additives [212] . in the case of one large-scale characterization by hall et al., the combination of both phire polymerase (new england biolabs, ipswich, ma, usa) and ampdirect (biomatrica, san diego, ca) gave an lod of nearly one copy/reaction in pcr for francisella tularensis in 0.5% stool [56] . one can also lyse bacteria in urine [218] or stool [219, 220] through heating to release an amplifiable amount of target dna with minimal levels of inhibitors. moore et al. managed to detect human norovirus repeatedly in 11 out of 12 outbreak stool samples after boiling the diluted feces in pbs [221] . although centrifugation is employed in some semi-direct methods to create a supernatant from the collected stool, we believe a dilution step could accomplish the same feat by allowing solids to settle at the base of a highly aqueous, non-viscous sample. isothermal amplification techniques like lamp and rpa generally demonstrate a higher tolerance than pcr for urine, as they can be carried out directly. as such, lamp-based assays without any pre-processing steps or chemical enhancements have detected the causative agents of viral infections [82, 127, 131] or stis [222, 223] , and pathogenic bacteria such as escherichia coli [224] . the developers of the recently established isothermal method siba showed the utility of this amplification technique by detecting chlamydia trachomatis and neisseria gonorrhoeae in a low-copy urine sample [25] . the landscape of molecular diagnostics is constantly advancing, as is the current paradigm of healthcare. the advent of mobile health and telemedicine has decentralized patient care. it has also put a new emphasis on usability and non-invasiveness in disease testing. nucleic acid diagnostics that reduce the difficulties and expenditures of standard multi-step procedures by direct amplification can expedite patient testing in poc, hospital, and laboratory situations [225] . in this review, we are thus motivated to discuss the current state of the art for direct naats: assays and platforms that require minimal or no sample preparation procedures. we search the literature from 1989 to 2017 and find 174 published works that we consider direct naats. we first categorize these works based on the type of complex samples. we subsequently employ bubble plots to facilitate the comparison of the amplification method, robustness in complex media, sensitivity to target, and clinical usage. our findings indicate that the majority of direct naats exhibit a tolerance of less than 17% for their sample of interest, and fewer than 30 patient-based evaluations. still, there are diagnostic procedures that far surpass these averages. sim et al.'s study of direct pcr on 4518 buccal swabs [179] , for instance, is robust and carried out directly on the entire sample for maximal ease. improvements must continue to be made for all sample types in terms of facilitating this level of evaluation with clinical samples. despite significant developments to date, there remain several challenges for realizing direct naats with user-friendliness, consistency, and generalizability. in furthering the development of direct testing, it is important to take a holistic approach and consider the type of sample to be analyzed, the method of sample acquisition, the throughput and volume, and any chemical or mechanical requirements, and the amplification technique. another key point to note is that different matrices will function best in different environments. an improved understanding of the mechanisms behind emerging nucleic acid amplification reactions and mutant polymerases will enable the rationalization of how inhibitory compounds can make or break an amplification system. furthermore, molecular assays have much to learn from diagnostics that are continuously being developed in the commercial pipeline. proprietary technology will always hold knowledge at a cost to the user, but the implementation of new ideas can lead researchers towards better and more successful ways in which to modernize the ever-changing field of disease testing. as we enter the age of electronic, mobile, and personalized medicine, there remains much room for creativity and innovation in the design of naats and poc diagnostics. molecular diagnostics, as the highest-growing segment of all in vitro diagnostic products [226] , truly have great potential for both developed and low-resource areas. the diagnostic community continues to strive for tests that are reliable against variable electrical resources, water quality, trained staff, or harsh environmental conditions [227, 228] . researchers continue to seek approvals such as the fda's clia waivers or the who's assured (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and delivered to end-users) criteria. in this regard, direct naats present a promising approach. through this review, it is our hope to stimulate the discussion on direct naats and their potential as poc diagnostics. ultimately, we seek to help accelerate the development of poc diagnostics that can be clia waived and/or meet the who assured criteria, thereby ushering in the next revolution in healthcare. the following are available online at http://www.mdpi.com/2079-6374/9/4/117/s1, table s1 : detailed properties of amplification assays. point of care diagnostics for sexually transmitted infections: perspectives and advances advances in developing hiv-1 viral load assays for resource-limited settings commercialization of microfluidic point-of-care diagnostic devices micro total analysis systems for cell biology and biochemical assays microfluidic dna amplification-a review nucleic acid isothermal amplification technologies: a review point-of-care nucleic acid testing for infectious diseases isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings fully automated quantification of cytomegalovirus (cmv) in whole blood with the new sensitive abbott realtime cmv assay in the era of the cmv international standard fda grants first clia waiver for nucleic acid-based flu diagnostic test; food and drug administration focus diagnostics receives fda clearance for moderate complexity simplexa hsv 1 & 2 direct molecular test for aiding the diagnosis of encephalitis performance of the molecular alere i influenza a&b test compared to that of the xpert flu a/b assay detection of influenza a and b with the alere tm i influenza a&b: a novel isothermal nucleic acid amplification assay evaluation of the alere i influenza a&b nucleic acid amplification test by use of respiratory specimens collected in viral transport medium evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus nucleic acid sequence-based amplification low-cost detection of zika virus using programmable biomolecular components reliability of nucleic acid amplification methods for detection of chlamydia trachomatis in urine: results of the first international collaborative quality control study among 96 laboratories reliability of nucleic acid amplification methods for detectio dna detection using recombination proteins invasion based amplification (siba): a novel isothermal dna amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte multiplex strand invasion based amplification (msiba) assay for detection of chlamydia trachomatis and neisseria gonorrhoeae comprehensive human genome amplification using multiple displacement amplification triggered amplification by hybridization chain reaction highly sensitive electrogenerated chemiluminescence biosensor based on hybridization chain reaction and amplification of gold nanoparticles for dna detection specific enzymatic amplification of dna in vitro: the polymerase chain reaction. cold spring harb standardization of nucleic acid tests for clinical measurements of bacteria and viruses highly sensitive detection of staphylococcus aureus directly from patient blood a potent inhibitor of taq polymerase copurifies with human genomic dna identification of the heme compound copurified with deoxyribonucleic acid (dna) from bloodstains, a major inhibitor of polymerase chain reaction (pcr) amplification capacity of nine thermostable dna polymerases to mediate dna amplification in the presence of pcr-inhibiting samples pcr based diagnosis in the presence of 8% (v/v) blood a reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification detection of toxoplasma gondii dna by pcr following microwave treatment of serum and whole blood comparison between toxoplasma gondii dna and specific immunoglobulins during pregnancy. la rev whole-blood polymerase chain reaction and restriction fragment length polymorphism: a simplified method by microwave irradiation improved method for direct pcr amplification from whole blood direct pcr from whole blood, without dna extraction rapid point-of-care isothermal amplification assay for the detection of malaria without nucleic acid purification rapid and sensitive detection of orientia tsutsugamushi by loop-isothermal dna amplification african trypanosomiasis: sensitive and rapid detection of the sub-genus trypanozoon by loop-mediated isothermal amplification (lamp) of parasite dna sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification a simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (htlamp) assay for malaria elimination highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in uganda clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria short report: evaluation of loop-mediated isothermal amplification (lamp) for malaria diagnosis in a field setting rapid detection of haptoglobin gene deletion in alkaline-denatured blood by loop-mediated isothermal amplification reaction mitochondrial dna targets increase sensitivity of malaria detection using loop-mediated isothermal amplification detection of plasmodium vivax infection in the republic of korea by loop-mediated isothermal amplification (lamp) heat denaturation increases the sensitivity of the cytomegalovirus loop-mediated isothermal amplification method molecular diagnostic 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circulating subtypes by use of nested three-monoplex dna pcr and dried blood spots review article: an overview of the clinical use of filter paper in the diagnosis of tropical diseases the diagnosis of syphilis on a dessicated and defibrinated blood drop a simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants dried blood spots for hiv-1 drug resistance and viral load testing: a review of current knowledge and who efforts for global hiv drug resistance surveillance a diagnostics platform for the integrated mapping, monitoring, and surveillance of neglected tropical diseases: rationale and target product profiles cystic fibrosis genotyping by direct pcr analysis of guthrle blood spots an evaluation of direct pcr amplification enhanced direct amplification of guthrie card dna following selective elution of pcr inhibitors dna analysis of cystic fibrosis in brazil by direct pcr amplification from guthrie cards screening for cystic fibrosis: feasibility of molecular genetic analysis of dried blood specimens polymerase chain reaction amplification from dried blood spots on guthrie cards rapid, efficient method for multiplex amplification from filter paper gene amplification directly from guthrie blood spots molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening dna extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction validation of a fast and low-cost alkaline lysis method for gdna extraction in a pharmacogenetic context a reliable and effective method of dna isolation from old human blood paper cards comparison of six commercially-available dna polymerases for direct pcr short report: rapid dna extraction from archive blood spots on filter paper for genotyping of plasmodium falciparum polymerase chain reaction amplification of dna from aged blood stains: quantitative evaluation of the "suitability for purpose" of four filter papers as archival media improved procedure for eluting dna from dried blood spots adaptation of a visualized loop-mediated isothermal amplification technique for field detection of plasmodium vivax infection point-of-care technologies for molecular diagnostics using a drop of blood multilaboratory evaluation of real-time pcr tests for hepatitis b virus dna quantification cell-free nucleic acids as biomarkers in cancer patients characterization of circulating dna in healthy human plasma isolation and characterization of dna from the plasma of cancer patients blood separation on microfluidic paper-based analytical devices simple, miniaturized blood plasma extraction method membrane-based, sedimentation-assisted plasma separator for point-of-care applications micro-scale blood plasma separation: from acoustophoresis to egg-beaters enzyme-free and ultrasensitive electrochemical detection of nucleic acids by target catalyzed hairpin assembly followed with hybridization chain reaction comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus direct serum assay for cell-free bmi-1 mrna and its potential diagnostic and prognostic value for colorectal cancer direct serum assay for microrna-21 concentrations in early and advanced breast cancer circulating dna of hotair in serum is a novel biomarker for breast cancer simple and rapid identification of low level hepatitis b virus dna by the nested polymerase chain reaction microwave treatment of serum facilitates detection of hepatitis b virus dna by the polymerase chain reaction. results of a study in anti-hbe positive chronic hepatitis b improved detection of hbv dna by pcr after microwave treatment of serum tolerance of loop-mediated isothermal amplification to a culture medium and biological substances direct detection of human herpesvirus 6b by the lamp method using newly developed dry-reagents direct detection of human herpesvirus 6 dna in serum by variant specific loop-mediated isothermal amplification in hematopoietic stem cell transplant recipients rapid detection of hiv-1 by reverse-transcription, loop-mediated isothermal amplification (rt-lamp) development of a loop-mediated isothermal amplification assay for rapid detection of bk virus direct detection of human herpesvirus 6 dna in serum by the loop-mediated isothermal amplification method enzyme-free and label-free ultrasensitive electrochemical detection of dna and adenosine triphosphate by dendritic dna concatamer-based signal amplification g-quadruplex based two-stage isothermal exponential amplification reaction for label-free dna colorimetric detection pyrene-excimer probes based on the hybridization chain reaction for the detection of nucleic acids in complex biological fluids advances in addressing technical challenges of point-of-care 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influenza a&b isothermal nucleic acid amplification test collection of buccal cell dna using treated cards a miniaturized and integrated gel post platform for multiparameter pcr detection of herpes simplex viruses from raw genital swabs an enclosed in-gel pcr amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics quick detection of herpes viruses from skin vesicles and exudates without nucleic acid extraction using multiplex pcr real-time pcr for detection of herpes simplex virus without nucleic acid extraction detection of chlamydia trachomatis in endocervical specimens by polymerase chain reaction genotyping single nucleotide polymorphisms in human genomic dna with an automated and self-contained pcr cassette direct pcr amplification of the hvsi region in mitochondrial dna from buccal cell swabs evaluation of pcr for diagnosis of bordetella pertussis and bordetella parapertussis infections these include: evaluation of pcr for diagnosis of bordetella pertussis and bordetella parapertussis infections use of polymerase chain amplification reaction for the detection of adenoviruses in ocular swab specimens comparison of polymerase chain reaction and culture techniques for detection of chlamydia trachomatis rapid detection of diagnostic targets using isothermal amplification and hybeacon probes-a homogenous system for sequence-specific detection clinical utility of loop-mediated isothermal amplification assay for the diagnosis of common alpha herpesvirus skin infections rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplification method rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (lamp) method. curr four dna extraction methods used in loop-mediated isothermal amplification for rapid adenovirus detection development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 min multiplex loop-mediated isothermal amplification (m-lamp) assay for the detection of influenza a/h1, a/h3 and influenza b can provide a specimen-to-result diagnosis in 40min with single genome copy sensitivity evaluation of a direct reverse transcription loop-mediated isothermal amplification method without rna extraction for the detection of human enterovirus 71 subgenotype c4 in nasopharyngeal swab specimens detection of mycobacterium ulcerans by the loop mediated isothermal amplification method rapid detection of epstein-barr virus dna by loop-mediated isothermal amplification method isothermal in vitro amplification of dna by a restriction enzyme/dna polymerase system a rapid, instrument-free, sample-to-result nucleic acid amplification test paper-based rna extraction, in situ isothermal amplification, and lateral flow detection for low-cost, 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acknowledge the support of h.t. soh in the initial stages of manuscript preparation. the authors declare no conflict of interest. key: cord-017946-fa4ehlb0 authors: lawless, ryan a.; cotton, bryan a. title: adjuncts to resuscitation date: 2018-05-26 journal: damage control in trauma care doi: 10.1007/978-3-319-72607-6_20 sha: doc_id: 17946 cord_uid: fa4ehlb0 damage control resuscitation has been increasingly adopted and practiced over the last decade. the concepts used are not new to this era of medicine but are novel in combination. this chapter will focus on adjuncts to damage control resuscitation (dcr) including massive transfusion protocols, the “other” tenets of damage control resuscitation, hypertonic saline, tranexamic acid, pharmacologic resuscitation, factor viia, and prothrombin complex, and viscoelastic testing. damage control resuscitation (dcr) is a treatment strategy targeting the conditions that potentiate hemorrhage in the traumatically injured patient [1] . the term originates from the us navy in reference to the techniques used to salvage a damaged ship during conflict [2] . in the management of the hemorrhaging patient, dcr refers to an approach to resuscitation initially adopted to improve outcomes of patients undergoing an abbreviated laparotomy or other procedure due to grossly disturbed physiology. however, its early implementation and even adoption in the prehospital setting have resulted in many of these patients now undergoing definitive procedures as the initial operation. the three basic tenets of dcr include permissive hypotension, blood product resuscitation approximating whole blood, and minimizing use of crystalloid prior to surgical control of bleeding [3] . ideally, this process begins in the prehospital setting and continues through the emergency room (er) and operating room, and into the icu, as needed. looking at the incorporation of the other two principles (permissive hypotension and minimizing crystalloids) into a mature trauma center already incorporating a transfusion strategy approaching whole blood, investigators found an improvement in survival among emergent laparotomy patients [4] . cotton and colleagues evaluated 390 patients who underwent damage control laparotomy and were managed with a red blood cell:plasma:platelet ratio of 1:1:1. the investigators found that after adoption of permissive hypotension and minimal crystalloids in the ed and operating room, blood transfusions were reduced, patients arrived to the icu with less coagulopathy and acidosis, and survival was increased 2.5-fold. duke et al. investigated the combination of a restrictive fluid resuscitation strategy and dcr. they reported a significant reduction in preoperative and intraoperative crystalloid administration with improvements in hospital and icu length of stay, a reduction in operative room mortality, and a resultant decrease in overall mortality [5] . the concept of permissive hypotension has been debated for years. maintaining the patient's blood pressure high enough for adequate organ perfusion yet low enough as to avoid exsanguination is the goal. in 1918, walter cannon and john fraser warned of the blood loss that could occur by elevating a patient's blood pressure before a surgeon was available [6] . the endpoint for resuscitation prior to the availability of a surgeon in their study was between 70 and 80 mmhg systolic blood pressure. in his document entitled "surgery in world war ii, general surgery", dr. beecher stated that "when the patient must wait for a considerable period, elevation of his systolic blood pressure to ~85 mmhg is all that is necessary" and that "…one should consider himself lucky if a systolic pressure of 80 mmhg to 85 mmhg can be achieved and then surgery undertaken" [7] . however, human studies validated the data produced by animal studies have been few [8, 9] . in 2002, dutton and colleagues conducted a randomized trial of hypotensive resuscitation [9] . a total of 110 patients were randomized on arrival to a resuscitation targeting a systolic blood pressure of 70 mmhg or a systolic of 100 mmhg. each resuscitation strategy was continued until definitive hemostasis was achieved. while no survival difference was noted, the authors found that aiming for sbp of 70 mmhg (vs. >100 mmhg) was safe in patients arriving with evidence of hemorrhage. carrick et al. recently evaluated a similar strategy in trauma patients undergoing a thoracotomy or laparotomy [10] . aiming for mean arterial pressure of 50 or 65 mmhg, the investigators continued this goal throughout the operating room course. the authors found that while blood loss and transfusions were less in the hypotensive group (50 mmhg goal), this did not translate into an improvement in 30-day mortality. both studies, however, were quite small and their results may reflect a type-ii error. as well, the benefits of reduced hemorrhage outweigh the possible detrimental effects of organ ischemia and reperfusion injury [11] . historically, large quantities of crystalloid and blood were advocated to replace the intravascular and extravascular fluid loss from hemorrhage. this strategy arose from studies in the 1950s and 1960s and was, until recently, endorsed by the american college of surgeons advanced trauma life support (atls) course with the recommendation of 1-2 l of crystalloid during the initial management of trauma patients [12] [13] [14] . however, multiple studies have shown the detriments of aggressive crystalloid resuscitation including cardiac dysfunction, ards, multi-organ failure, and increases in mortality [15] [16] [17] [18] [19] [20] [21] . moreover, the infusion of room temperature, high chloride containing fluid worsens hypothermia, acidosis, and coagulopathy [22] . resuscitation to a normal blood pressure increases hemorrhage through the dilution of coagulation factors, displacement of tenuous clots, and decreases in blood viscosity leading to increased mortality [23] [24] [25] [26] [27] [28] . evaluating the impact of minimizing crystalloids in the clinical arena, bickell and colleagues built on this extensive preclinical data by conducting a randomized trial of standard atls resuscitation (crystalloids) versus no fluid [27] . patients presenting with hypotension (systolic ≤ 90 mmhg) and who had sustained penetrating torso injuries were randomized to one arm or the other beginning in the prehospital setting and the randomization resuscitation strategies were continued until the patient entered the operating room. patients who received no fluid (delayed resuscitation) had lower mortality compared to those who received immediate fluid resuscitation. two decades later, the resuscitation outcomes consortium further investigated these two damage control resuscitation principles [29] . the investigators randomized study patients beginning in the prehospital setting to a systolic pressure of 70 mmhg and small boluses (250 ml) to maintain blood pressure goal, while the control group was randomized to 110 mmhg, received two liters initially, and additional fluid to maintain blood pressure target. each protocol continued until hemorrhage control or 2 h after hospital arrival. the 24-h mortality for blunt trauma was significantly lower in the study arm compared to that observed in the control group (3% vs. 18%). the 1970s brought about the first discussions of massive transfusions (mt) and the associated 90% mortality rate [30] . however, the development of standardized delivery processes and protocolization of these mt processes would take another 30 years to arrive. with the implementation and maturation of these mt protocols, times to delivery of initial blood products, overall product utilization, and mortality were all significantly reduced [31, 32] . to put this in perspective, early improvements in hemorrhagic shock resuscitation (2000-2005) had reported mortalities for patients receiving a mt were 55-65% [33] . however, with increased adoption of mt protocols, mortality soon dropped to 45-50% by 2009. with further maturation and adoption of dcr tenets, mt mortalities continued to decrease to the current rates of 22-26% [34] . the layers of delay for blood product administration include the placement of the individual product orders, communication among providers, decisions regarding the products to transfuse, transportation of blood samples to the lab, and receipt and review of the lab values [35] . mt protocolization allows members of the trauma team to focus on managing the patient and their injuries and be less worried about choosing, obtaining, and transfusion of blood products. that said, establishing a massive transfusion protocol is not an easy task (figs. 20.1, 20.2, 20.3 and 20.4 ). this is a multidisciplinary process involving the emergency medicine physicians, trauma surgeons, anesthesiologists, hematologists, and blood bank. in 2008, cotton et al. first demonstrated an improvement in mortality with the simple implementation of the mt process at their hospital [32] . in 2009, riskin and colleagues also noted an improvement in mortality, which they attributed to improved communication with the blood bank and time to blood product availability [36] . in order for mt protocol to be effective, a center must have thawed or liquid plasma available for immediate delivery and transfusion [37] [38] [39] . determining the content of the mtp cooler is highly debated in the literature to this day. in a recent randomized controlled trial, resuscitation of red blood cells:plasma:platelets in a ratio of 1:1:1 demonstrated a reduction in bleedingrelated mortality [34] . however, overall mortality was not improved. no matter the take on the current literature, delivery of a standardized mt protocol and compliance with that protocol has been shown to improve outcomes in these patients [40, 41] . once in place, knowing which patients will benefit from activation of the mt protocol can be difficult. multiple scoring systems have been developed to identify patients that may benefit from a mt protocol [33] . while the predictive value of each of these scores is good, many of these include laboratory values that are not readily available upon the patient's arrival to the hospital making these scoring systems less useful. to address this, the assessment of blood consumption (abc) score was developed in 2009. it utilizes data readily available upon a patient's arrival to the hospital. the four components of the score are: ed systolic blood pressure ≤90 mmhg, ed heart rate ≥120 beats per minutes, penetrating mechanism, positive fluid on abdominal ultrasound evaluation. each criterion consists of one point. scores ≥2 indicate activation of the mt protocol ( fig. 20.1 ). while the abc score overestimates the need for mt protocol with a positive predictive value of 50-55%, products can be returned and restocked. more importantly, the negative predic[39, 42, 43] . the abc score is but one of many tools available to practitioners managing severely injured patients and should not replace a physician's discretion. other clues to encourage activation of the mt protocol include persistent hemodynamic instability, active hemorrhage requiring surgical intervention, including interventional radiologic procedures, and the transfusion of non-crossmatched blood in the resuscitation bay [39] . a delay in identifying patients that require massive transfusion and delay in the initial cooler arrival, prolong the time to hemostasis and increase mortality [44, 45] . once activated, the blood bank should immediately release mt coolers for transfusion approximately every 10-15 min. one simple rule: remain one cooler ahead of the current transfusion. once the hemorrhage has been controlled and the patient is no longer hemodynami-cally unstable, the mt protocol can be discontinued. laboratory indicators can be helpful in these situations and include hemoglobin levels, traditional coagulation tests, viscoelastic testing, platelet counts, and fibrinogen levels. the decision to terminate the mtp should be determined by the anesthesia and surgery teams jointly [39] . compliance with the protocol is also essential for providing optimal care. knowing when to activate and when to terminate the protocol can be guided by scoring tools and laboratory values. these values should assist, not replace, the judgment of the providers. the east guideline for damage control resuscitation recommends the development and implementation of a massive transfusion/dcr protocol in a multidisciplinary fashion with current literature and target blood product ratios. furthermore, the same group endorses a high ratio mt/dcr strategy, if not whole blood [37] . the anti-fibrinolytic agent tranexamic acid (txa) works by inhibiting the conversion of plasminogen to plasmin. in retrospective studies, administration of txa to hemorrhaging patients has been associated with decreased mortality in both the civilian and military populations [46, 47] . it has also been shown to be a cost-effective adjunct for health care systems in poor and wealthy nations [48] . the identified window of benefit for the administration of txa is within 3 h of injury [49] . however, txa has failed to gain widespread acceptance and incorporation in the usa for multiple reasons. first, the large, multicenter, randomized trial of txa in injured patients (crash-2) has several major methodological flaws. also, whether txa has any impact on trauma outcomes in a region already practicing dcr or a hospital with a mtp in place is unknown. the majority of patients enrolled in crash-2 were in low-income and middle-income countries where blood products, mature trauma centers, and experienced trauma teams are not readily available. finally, how sick or injured (or uninjured) patients were in crash-2 is also a concern; less than half of patients (in either txa or placebo groups) underwent an operation and only half received even one unit of blood. in 2013, napolitano et al. recommended txa be used as part of a mtp in the following situations: (1) hyperfibrinolysis demonstrated on viscoelastic testing or (2) in patients with severe hemorrhagic shock (sbp ≤75 mmhg and a base deficit >5). furthermore, given the increase in mortality seen with administration after 3 h, txa should not be given when the time from injury is known or suspected to be greater than 3 h [50] . the current east guideline conditionally recommends txa use as a hemostatic adjunct in the severely injured patient in the hospital setting only [37] . the case for the addition of fibrinogen, through either cryoprecipitate or fibrinogen concentrate, remains controversial. while in theory an argument can be easily made for its use in the bleeding patient, data in trauma and emergent cases is extremely lacking. the majority of data supporting its use is in the cardiac setting, and especially in those scenarios where intravascular support includes hydroxyethyl starches [51] . hydroxyethyl starches reduce maximal clot firmness through a reduction in fibrinogen activity (as well as that of factors ii, x, and xiii) [52] . one randomized trial of patients undergoing major aortic replacement surgery found a reduction in both red blood cell and plasma transfusions in patients receiving fibrinogen concentrate (compared to placebo) [53] . in the setting of trauma, schochl et al. evaluated 131 patients who received fibrinogen concentrate guided by viscoelastic tests in the setting of bleeding [54] . the authors noted a significantly improved mortality compared to that predicted by the trauma related injury severity score (triss) in patients who received fibrinogen concentrate during their initial resuscitation. as well, german investigators found that exsanguinating patients who received fibrinogen concentrate as part of their resuscitation had improved 6-h mortality (corresponding to the time of bleeding-related deaths [55] ). the matters ii study, which was a retrospective study of uk and us military experience with combat injuries, evaluated the role of cryoprecipitate and tranexamic acid [56] . investigators found that when patients received both cryoprecipitate and tranexamic acid as part of their care, mortality was half that of those who received neither (11.6 versus 23.6%). however, the prommtt investigators evaluated the impact of cryoprecipitate on bleeding patients from ten us level-1 trauma centers and noted that its use varied greatly in their timing and use of cryoprecipitate in severely injured trauma patients [57] . however, the authors could not identify any association of cryoprecipitate use with in-hospital mortality. in addition, some have argued that routine replacement even in exsanguinating patients is unwarranted [58] . opponents note that with early and more aggressive use of plasma, hypofibrinogenemia and/or fibrinogen dysfunction is uncommon. given this, most would agree that randomized controlled studies in trauma are needed to determine if fibrinogen replacement is necessary. to that end, a randomized trial of fibrinogen replacement beginning in the prehospital setting, cryostat-2, will begin enrollment in 2017. prothrombin complex concentrate (pcc) is a concentrated vitamin k-dependent coagulation factor product. it was originally approved for the treatment of hemophilia b and has been expanded to the reversal of vitamin k antagonist (vka) effects. pccs offer potential advantages over plasma including faster inr normalization in patients, delivery of smaller infusion volume, rapid administration, and no requirement for abo blood-type matching. all derived from pooled plasma and carry the possible risk of virus transmission. the 4-factor concentrates contain factors ii, vii, ix, and x, while the 3-factor concentrates contain ii, ix, and x. sarode and colleagues conducted a multicenter, randomized trial in non-trauma, noncritical patients who were on warfarin and had need for urgent inr reversal [59] . the authors demonstrated that pccs more rapidly reversed inr compared to plasma reversal, and did so with a similar adverse event profile. studies in trauma are small and few [60] . however, some of these have noted effectiveness of pcc in the rapid reversal of vka-related coagulopathy in trauma patients. quick et al. demonstrated the safety and efficacy of pcc in the reversal of vka-related coagulopathy in geriatric trauma patients [61] . huynh et al. noted a more rapid correction of inr in patients on warfarin who sustained traumatic brain injury, with no differences in outcome. the utility of pcc incorporation into mt protocols or as a replacement for plasma in early resuscitation remains to be answered. plasma provides volume expansion, acid-base buffering capacity, and high oncotic properties, all of which are beneficial in the patient with hemorrhagic shock, and none of which are present with pccs [58] . in addition, plasma is effective in maintaining vascular endothelium integrity and clot stability, which has not yet been established for pccs [62] . recombinant factor viia (rfviia) is currently approved for the treatment of hemophilia and has previously been advocated for the use in hemorrhaging trauma patients. it has been adopted and incorporated into many massive transfusion algorithms [63] . the rfviia is thought to activate the common coagulation pathway at pharmacologic doses [64] . several retrospective studies show possible benefit in the use of rfviia as an adjunct to dcr by decreasing the overall need for blood transfusions, improvement in traditional coagulation tests, and hastening the resolution of bleeding [65] [66] [67] . however, randomized trials were less encouraging. bouffard et al. published a randomized, placebo-controlled trial showing that patients suffering from blunt traumatic injury received less blood products with rfviia, but no benefit was seen in penetrating trauma [68] . raobaikady et al. reported their randomized trial on the use of rfviia during pelvic reconstruction and noted no difference in transfusion requirements [69] . in 2010, the control trial evaluated the safety and efficacy of rfviia in a multinational randomized trial. there was no difference in survival between patients receiving rfviia and placebo, regardless of the mechanism of trauma. similar to previous studies, rfviia decreased red blood cells and plasma transfusions and a trend toward less multiorgan failure was noted [70] . there was much early enthusiasm for the use of rfviia in the management of the bleeding patient. however, the popularity of dcr, with less crystalloid volume and earlier plasma and platelet transfusions, increased during the study periods for rviia. the current east guideline does not recommend for or against the use of rfviia as it does not appear to improve all-cause mortality and the only benefit is a possible reduction in the need for massive transfusion. they recommend further study to identify the optimal dose and timing for rfviia delivery [37] . arginine vasopressin (avp) is an endogenous neurohypophyseal hormone released in response to changes in plasma osmolality and blood pressure [71] . hypotension can stimulate the release of vasopressin as much as 40 times physiologic concentrations. also, avp clearance is prolonged leading to higher concentrations for a longer duration [72] . its release is suppressed by elevations of norepinephrine and nitric oxide, which can be seen in hemorrhagic shock. primarily, avp works on the extracerebral arteriole v1 receptors leading to vasoconstriction. this causes an increase in systemic vascular resistance and a redistribution of blood flow from capacitance vessels in the periphery toward heart, brain, and kidneys [73] . however, this vasoconstriction is not as great in the coronary and renal system. avp potentially has a vasodilatory effect on cerebral and renal vessels leading to improved blood flow [74] . the compensatory increase in endogenous vasopressin levels has been described in septic and hemorrhagic shock. patients suffering from septic or hemorrhagic shock with a depressed endogenous avp response show a significant increase in blood pressure with low-dose administration of avp, while patients with a normal compensatory response did not show the same response to low-dose avp administration [75, 76] . animal models of liver injury associated hemorrhagic shock have shown decreased blood loss, increased mean arterial pressure, and significantly higher hemoglobin levels with vasopressin administration compared to standard crystalloid resuscitation [74] . avp caused a shift in blood flow from the intra-abdominal injury leading to decreased blood loss. a temporary shunting of blood flow from the mesenteric vessels and profound peripheral vasoconstriction lead to restored perfusion of the liver and kidneys [74, 77] . similar studies showed an increase in cardiac and cerebral blood flow following vasopressin administration secondary to cutaneous, muscular, adipose, gut vasoconstriction. the cardiovascular collapse and blood loss seen with crystalloid infusion was not demonstrated following avp injection [78] . a randomized, placebo-controlled study (avert shock) is currently underway to investigate the potential benefit of vasopressin administration during the early resuscitation of bleeding trauma patients [79]. valproic acid (vpa) is a gaba-ergic medication used in the treatment of epilepsy, bipolar disorder, migraines, and neuropathic pain. there are multiple mechanisms of action that contribute to the biologic activity of vpa including the alteration of gene expression, the downregulation of enzymatic pathways, the enhancement of neurotransmission, and the stabilization of neuronal membranes [80] [81] [82] [83] . histone deacetylase has been implicated in the modulation of the lifespan of certain organisms through its effect on gene expression [84] . hemorrhage and the resuscitation of hemorrhage have been shown to be associated with an imbalance in histone deacetylase (hdac) and histone acetyltransferase (hat) activity. the presence of shock induces changes in histone deacetylation which can be reversed with the infusion of vpa, a hdac inhibitor. animal models have shown that the cytoprotec-tive and restorative effects occur with pretreatment and post-injury infusion of vpa [85] [86] [87] . this leads to improvement in early survival from near lethal hemorrhage. the survival advantage is likely due to better tolerance to the shock state by cells [88] . further research needs to be performed in this area to investigate the utility of vpa in human trauma resuscitation. the infusion of hypertonic saline has been described at multiple points during the management of the injured patient; from the point of injury, to specific injury management, to damage control resuscitation. hypertonic saline infusion increases serum osmolality causing a shift of fluid volume from the intracellular to the extracellular space. this volume shift effectively increases preload, cardiac output, and mean arterial pressure [89] . mazzoni et al. described an improvement in capillary endothelial swelling leading to an improvement in microcirculation with the infusion of hypertonic saline [90] . as described above, volume overload is detrimental to patients requiring management in damage control situations. it leads to the development of acute lung injury and acute respiratory distress syndrome, as well as to multisystem organ failure secondary to the cascade of inflammatory and immune responses associated with volume overload [91] . hypertonic saline infusion allows for the rapid restoration of preload with less volume. furthermore, the edema associated with resuscitation is attenuated leading to improved end organ perfusion [1] . animal studies have shown that hypertonic saline infusion attenuates the proliferation of white blood cells, endothelial adhesion, and the expression of inflammatory markers in the lung and gut [92] [93] [94] . rizoli and colleagues corroborated these findings by demonstrating a decreased neutrophil activation, decreased serum tnf-α levels, increased level of anti-inflammatory cytokines il-1ra and il-10, and attenuated norepinephrine surge associated with shock. this was demonstrated up to 24 h following injury [95] . other animal studies have shown that hypertonic saline not only prevents, but reverses resuscitationinduced intestinal edema [96] [97] [98] . duchesne et al. performed a retrospective comparison of low volume resuscitation with hypertonic saline and isotonic crystalloid infusion during the icu phase of damage control resuscitation. they found a decrease in the volume infused, decreased icu length of stay, lower prevalence of ards and multisystem organ failure, and a trend toward renal failure in the hypertonic saline group [91] . in 2013, harvin and colleagues described a protocol of hypertonic saline (3% saline) infusion following damage control laparotomy with respect to primary fascial closure. they found that the isotonic crystalloid group received significantly more fluid compared to the hypertonic saline group; however, transfusions of blood products were similar. there were also similar rates of renal failure between the group. the investigators found that 100% of patients in the hypertonic saline group achieved primary fascial closure by post-damage control day 7 and a decreased time to fascial closure compared to 76% in the isotonic crystalloid group [99] . approximately 25% of severely injured patients have an established coagulopathy upon arrival to the emergency department [100, 101] . prompt recognition and treatment of this cohort of patients is crucial. most centers monitor five convention coagulation tests: prothrombin time (pt), international normalized ratio (inr), activated partial thromboplastin time (aptt), platelet count, and fibrinogen levels. these tests are representative of a portion of the coagulation system. utilization of these tests is limited by slow result times, poor association with clinical outcomes, and incomplete characterization of the complete coagulation system. viscoelastic tests characterize the lifespan of a clot from the time to initial fibrin crosslinking to breakdown by fibrinolysis in a single assay [102] . the viscoelastic assays available are thromboelastography (teg) (haemonetics corp, niles, in) and thromboelastometry (rotem) (tem international, gmbh, munich, germany). the rapid teg (r-teg) assay has been shown to be readily available within minutes of being drawn, correlates well with conventional coagulation tests, and is predictive of early transfusion necessity [103] . in 2012, holcomb et al. found that r-teg data was clinically superior to the five conventional coagulation assays and identified patients with an increased risk of early prbc, plasma and platelet transfusions, and fibrinolysis. the authors suggested that admission conventional coagulation tests could be replaced with r-teg [104] . further investigation into specific teg values found that the parameter of clot strength (g) provided a consistent, independent prediction of massive transfusion and coagulation-related mortality early in the resuscitation [105] . most recently, gonzalez et al. compared mtp goaldirected by teg versus conventional coagulation tests. they found that using a goal directed, tegguided mtp improved survival and used less plasma and platelet transfusions during the early phase of resuscitation compared with conventional coagulation test directed mtp [106] . damage control resuscitation has become increasingly adopted and practiced over the last 10 years. while many of the concepts used are not new, their application to early trauma resuscitation and their combination unique "cocktails" is novel, and the resultant improved outcomes applauded. this chapter evaluated many of these adjuncts to dcr including massive transfusion protocols, the less known and investigated dcr tenets, viscoelastic testing, hypertonic saline, tranexamic acid, factor viia, and prothrombin complex. in conjunction with early use of blood products (in ratios resembling whole blood), permissive hypotension and limited crystalloid administration are associated with reduced bleeding, less transfusions, and improved survival. protocolization of the massive transfusion process, independent of ratios and products, is associated with improved survival. use of viscoelastic testing to guide the resuscitation or bleeding patients appears to improve survival and reduce overall transfusion rates. while viia and pcc appear to be of little use in the acute resuscitation of hemorrhage, txa appears to improve survival in patients with penetrating mechanism, who are in profound shock, and receive the drug early after injury. both hypertonic saline 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starchinduced dilutional coagulopathy effects of fibrinogen concentrate as first-line therapy during major aortic replacement surgery goal-directed coagulation management of major trauma patients using thromboelastometry (rotem)-guided administration of fibrinogen concentrate and prothrombin complex concentrate administration of fibrinogen concentrate in exsanguinating trauma patients is associated with improved survival at 6 hours but not at discharge association of cryoprecipitate and tranexamic acid with improved survival following wartime injury cryoprecipitate use in the prommtt study a window of opportunity: the aggressive use of plasma in early resuscitation efficacy and safety of a 4-factor prothrombin complex concentrate in patients on vitamin antagonists presenting with major bleeding: a randomized, plasma-controlled, phase iiib study prothrombin complex concentrate in trauma patients experience with prothrombin complex for the emergent reversal of anticoagulation in rural 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trial results of the control trial: efficacy and safety of recombinant activated factor vii in the management of refractory traumatic hemorrhage vasopressin in hemorrhagic shock: a systematic review and meta-analysis of randomized animal trials vasopressin clearance and secretion during haemorrhage in normal dogs and in dogs with experimental diabetes insipidus vasopressin and shock but not fluid resuscitation, enhances survival in a liver trauma model with uncontrolled and otherwise lethal hemorrhagic shock in pigs potential benefit of vasopressin in resuscitation of hemorrhagic shock vasopressin deficiency in the syndrome of irreversible shock arginine vasopressin, but not epinephrine, improves survival in uncontrolled hemorrhagic shock after liver trauma in pigs vasopressin improves survival in a porcine model of abdominal vascular injury anti-tumor mechanisms if valproate: a novel role for an old drug acetylation: a regulatory modification to rival phosphorylation? histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen gene activation by histone and factor acetyltransferase regulation of lifespan by histone deacetylase cardiac histones are substrates of histone deacetylase activity in hemorrhagic shock and resuscitation valproic acid prevents hemorrhage-associated lethality and affects the acetylation patterns of cardiac histones surviving blood loss without fluid resuscitation surviving blood loss without blood transfusion in a swine poly-trauma model a rational approach to perioperative fluid management dynamic fluid redistribution in hyperosmolar resuscitation of hypovolemic hemorrhage damage control immunoregulation: is there a role for low-volume hypertonic saline resuscitation in patients managed with damage control surgery? hypertonic saline and the microcirculation hypertonic saline and pentoxifylline attenuates gut injury after hemorrhagic shock hypertonic saline and pentoxifylline reduces hemorrhagic shock resuscitation-induced pulmonary inflammation through attenuation of neutrophil degranulation and proinflammatory mediator synthesis the immunomodulatory effects of hypertonic saline resuscitation in patients sustaining traumatic hemorrhagic shock: a randomized, controlled, double-blinded trial hypertonic saline prevents inflammation, injury, and impaired intestinal transit after gut ischemia/reperfusion by inducing heme oxygenase-1 enzyme heme oxygenase-1 induction by hemin protects against gut ischemia/reperfusion injury conventional dose hypertonic saline provides optimal gut protection and limits remote organ injury after gut ischemia reperfusion chasing 100%: the use of hypertonic saline to improve early primary fascial closure after damage control laparotomy early coagulopathy predicts mortality in trauma human polymerized hemoglobin for the treatment of hemorrhagic shock when blood is unavailable: the usa multicenter trial coagulation abnormalities in the trauma patient: the role of point-of-care thromboelastography rapid thromboelastography delivers real-time results that predict transfusion within 1 hour of admission admission rapid thromboelastography can replace conventional coagulation tests in the emergency department viscoelastic clot strength predicts coagulation-related mortality within 15 minutes goal-directed hemostatic resuscitation of trauma induced coagulopathy: a pragmatic randomized clinical trial comparing a viscoelastic assay to conventional coagulation assays key: cord-304437-ezqghyid authors: palmieri, tina l. title: children are not little adults: blood transfusion in children with burn injury date: 2017-08-15 journal: burns trauma doi: 10.1186/s41038-017-0090-z sha: doc_id: 304437 cord_uid: ezqghyid blood transfusion in burns larger than 20% total body surface area (tbsa) are frequent due to operative procedures, blood sampling, and physiologic response to burn injury. optimizing the use of blood transfusions requires an understanding of the physiology of burn injury, the risks and benefits of blood transfusion, and the indications for transfusion. age also plays a role in determining blood transfusion requirements. children in particular have a different physiology than adults, which needs to be considered prior to transfusing blood and blood products. this article describes the physiologic differences between children and adults in general and after burn injury and describes how these differences impact blood transfusion practices in children. children and adults have different physiologic and hematologic systems, which impacts therapeutic interventions and their efficacy. in addition, children of different ages have different physiology and anatomy, which further complicates treatment. for example, an infant has a higher metabolic rate than an 8-year-old, a larger body surface area to mass ratio, and a markedly smaller blood volume. hence, different strategies need to be employed when treating children of different ages. these differences are accentuated in burn injury, which further alters metabolism, anatomy, and physiology. understanding the differences among children of different age groups is essential to optimize the use of blood transfusion in children. this article will discuss how differences in the physiologic, hematologic, metabolic, and immunologic systems in burned children impact blood transfusion requirements. although this article describes how children differ from adults in terms of factors with impact on blood transfusion, the unique primary aim of this article is to understand how burned children are impacted by blood transfusion and describe optimal transfusion practices in burned children (table 1) . children and adults have differences in hematologic and physiologic characteristics children clearly have a smaller stature than adults, yet their requirements may actually exceed those for adults on a kilo per kilo basis. for example, young children have a greater body surface area per mass than an adult, and the distribution of that mass is different than in adults. this impacts burn size determination, intravenous fluid requirements, and blood transfusion requirements. even the most essential body systems are impacted by the differences between children and adults. heart rate measurement is simple, yet there are important differences between children and adults that should be considered when instituting burn treatment. the baseline heart rate in a child is higher than that in an adult and varies with age [1] . burned children have a higher cardiac output and heart rate than unburned children, which can predispose them to heart failure. cardiac function also differs with age. as a baseline, a newborn child's myocardium is at near maximum function; hence, the newborn may not be able to compensate for decreased oxygen carrying capacity by increasing cardiac output after injury [2] . in other words, an infant increases heart rate rather than contractility to increase cardiac output. in the burned child, whose hypermetabolic rate adds further demand to an already stressed system, tachycardia is increased. hence, burned infants are at particular risk for heart failure after injury. beta blockade will be problematic, as lowering the heart rate will also lower cardiac output. finally, myocardial ischemia could occur due to decreased oxygen delivery capacity in the newborn or very young infant, which may in part contribute to the increased mortality of burned children less than 2 years. a second difference between adults and children is in blood volume. a child's mean blood volume approximates 70 ml/kg, which exceeds adult blood volume/ weight calculation. this increased blood volume/unit mass impacts a variety of body functions. as indicated above, oxygen consumption in children is higher; in addition, cardiac output to blood volume ratio is also higher in children than in adults [3, 4] . normal hemoglobin levels in children are agedependent and also differ from adults. children are born with a hemoglobin level approximating 19 g/dl and have a nadir of 11.2 g/dl at approximately 2-3 months of age. eventually, a child's hemoglobin stabilizes at approximately 13 g/dl [5] . in infants, fetal hemoglobin can play a role in oxygen delivery, thus decreasing the efficacy of the at-birth oxygen delivery. at birth, fetal hemoglobin constitutes 70% of the child's hemoglobin. at 6 months of age, however, only a trace of fetal hemoglobin remains [6, 7] . in fetal hemoglobin, red blood cell life span is decreased by 30 days (from 120 to 90), causing the oxygen-hemoglobin dissociation curve to be shifted to the left, which may impact tissue ischemia in the face of inadequate erythropoiesis. clearly, the presence of fetal hemoglobin should be considered in children less than 1-2 months of age who sustain burn injury, as younger infants (<6 months) thus have lower oxygen carrying capacity. this is exacerbated by a decreased production of erythropoietin in response to hypoxia or anemia in critically ill infants with sepsis or polytrauma [8] . burned children clearly fall into this category. children who sustain severe burns at birth or shortly after birth due to bathing rituals are at particular risk. the higher blood transfusion/unit volume ratio in children increases their risk for metabolic perturbation with blood transfusion. both the red blood cells themselves and the substances used to help preserve red blood cells contribute to these effects. risks related to transfusion include hyperkalemia, hypomagnesemia, hypothermia, acidosis, and hypothermia. hyperkalemia associated with blood transfusion poses a significant risk in children, and potassium levels should be monitored in children receiving >20 ml/kg transfusion volume (or lower if the patient has renal dysfunction or hyperkalemia at the onset of transfusion). hyperkalemia has been associated with cardiac arrest during large blood volume transfusions intraoperatively in children and infants receiving exchange transfusions [9, 10] . children with small blood volumes are at particularly high risk of hyperkalemia due to both volume/ size considerations and the developing renal function of infants and small children. potassium levels differ among blood products. whole blood, irradiated units, and units nearing the expiration date (i.e., "old blood") contain the largest amounts of potassium [11, 12] . practices that decrease hyperkalemic cardiac arrest risk include using "young" blood (packed red blood cell (prbc) <7 days in age), washing erythrocytes prior to transfusion, and avoiding whole blood transfusion in small infants. the life-threatening arrhythmias associated with rapid large volume can be ameliorated by administering calcium [9, 12] . administration of calcium treats hyperkalemic arrhythmias by opposing the effects of hyperkalemia on the heart's electrical conduction system. additional measures, such as intravenous glucose, insulin, albuterol, and kayexelate, may be needed to resolve the hyperkalemia. in addition to ameliorating hyperkalemia, ionized calcium is an important cofactor in infant coagulation and myocardial contractility [13] . citrate, used in blood storage to prevent clotting, prevents clot formation by chelating calcium. as such, transfusion may induce hypocalcemia. the type of blood product transfused, the rate of the transfusion, and the patient hepatic function all influence the extent of hypocalcemia [5, 14] . whole blood and fresh frozen plasma (ffp) contain the highest concentration of citrate/unit volume of product; hence, they have the highest hypocalcemia risk. hypocalcemia has been reported after the transfusion of ffp [15] . the neonate is at a particular risk of cardiac dysfunction with hypocalcemia due to the relative lack of neonatal cardiac sarcoplasmic reticulum. this reduction makes the table 1 summary of transfusion considerations in burned children 1 . despite a smaller stature, burned children have a greater body surface area per mass than adults. 2. cardiac function, mean blood volume, and normal hemoglobin levels are age-dependent in children; hence, children have a higher blood transfusion/unit volume ratio. 3. the optimal hemoglobin threshold for initiating a blood transfusion in burned children has not yet been defined. 4. hyperkalemia associated with blood transfusion poses a significant risk in children, and potassium levels should be monitored in children receiving >20 ml/kg transfusion volume. 5. the maximal allowable blood loss (mabl), i.e., the volume of blood that can be lost in an operation without transfusing blood, can be calculated from the following (hct = hematocrit, ebv = estimated blood volume): mabl = [(hct start − hct target )/hct start ] × ebv. neonate myocardium dependent on ionized calcium for both normal contraction and relaxation. transfusing blood at a rate of less than 1 ml/kg/min may ameliorate the hypocalcemic effect of the blood. correction of hypocalcemia can be accomplished with administration of either calcium chloride (5-10 mg/ kg) or calcium gluconate (15-30 mg/kg) intravenously. in general, the dose of calcium gluconate required to achieve the same effect is three times that of calcium chloride. because calcium may result in clot formation when in contact with blood, calcium should never be administered in a blood line. magnesium, which is often altered in association with calcium, must also be considered. hypomagnesemia may also occur after massive transfusion, and if a patient is hypocalcemic, magnesium levels should be obtained. magnesium stabilizes resting membrane potential; hence, hypomagnesemia may cause lifethreatening arrhythmias. if ventricular fibrillation or ventricular tachycardia develops after transfusion and does not respond to calcium administration, intravenous magnesium sulfate, in a dose of 25-50 mg/ kg, may be helpful. environmental issues also impact transfusion effects. hypothermia in burned children, in particular, requires special consideration. children, due to their large surface area to volume ratio, are at increased risk for hypothermia. not only do children with burn injury lose skin integrity, and hence the key temperature regulating mechanism, they also actively lose heat through convection and conduction through wet wounds and exposed tissue. hypothermia will increase oxygen consumption and exacerbate coagulopathy and is associated with increased mortality [16, 17] . hypothermia may be exacerbated during periods of rapid transfusion utilizing cold blood products, especially during episodes of massive transfusion in the operating theater. hypothermia may be ameliorated using several different methods, including the use of blood warmers during transfusion, increased ambient room temperature, external warming devices, and potentially warming central venous catheters. hypothermia frequently accompanies another significant complication of transfusion in children: acidosis. hypovolemia in the operating room during massive excision is of particular concern with respect to the development of acidosis. as such, life-threatening acidosis may occur during rapid transfusion for massive blood loss in a hypovolemic patient. because stored blood cells continue to metabolize, lactic acid increases in stored blood, making acidosis more likely. it is also notable that metabolic alkalosis may occur several days after massive transfusion from the metabolism of the citrate in the blood products administered. although transmission of infectious diseases due to blood transfusion has decreased over time, the transmission of infectious diseases remains an important problem in children requiring blood transfusion [2] . parents, understandably, are worried about hepatitis and human immunodeficiency virus from blood transfusion. blood products in different countries differ in the frequency of transmission of infectious organisms. current blood screening tests include hepatitis b surface and core antigen, hepatitis c virus antibody, hiv-1 and hiv-2 antibody, htlv-i and htlv-ii antibody, nucleic acid amplification testing for hiv-1 and hcv, syphilis, and west nile virus [18] . in addition to these commonly measured viral infections, bacteria can also infect blood products. the incidence of bacterial contamination is highest for platelets [19] [20] [21] . other potential infections that could be transmitted via transfusion that are not tested for include htlv, west nile virus, babesiosis, chagas disease, lyme disease, malaria, creutzfeldt-jakob disease, and severe acute respiratory syndrome (sars). screening for zika and ebola virus has recently been released by the food and drug administration [22] . hemolytic transfusion reactions continue to occur despite the careful application of compatibility testing. blood mismatch transfusions is due primarily to clerical error. particularly important is the verification of blood products prior to transfusion by physician and nurse with the patient's identification to make sure that the unit is truly intended for that patient. this simple, inexpensive procedure can prevent a life-threatening transfusion reaction. strict adherence to transfusion protocols is important to avoid this iatrogenic complication. acute hemolytic reactions generally occur due to abo incompatibility and causes immunologic destruction of red cells. however, this complication can also occur due to minor antigens not detected by current screening techniques [23, 24] . anaphylactic reactions rarely occur. transfusion-related graft-versus-host reaction, in which the lymphocytes in the transfused blood cause host cell destruction, occurs primarily in immunocompromised patients and has been reported in neonates and immunocompromised children [25] [26] [27] [28] . this condition occurs primarily in premature infants or children with rapid acute blood loss, cardiopulmonary bypass, cancer, or severe systemic illness [29] . burned children are immunosuppressed and require massive transfusions in the operating room, thus putting them at risk for this complication. transfusion-related graft-versus-host disease can be reduced by using irradiated units, which effectively decrease the lymphocyte count. however, since irradiated blood has a higher potassium content than nonradiated blood, potassium levels must be monitored closely. the child's blood volume varies with age and weight; hence, the amount of blood required in times of acute blood loss varies markedly among children of different ages. the highest blood volume per unit weight is for a premature infant (90-100 ml/kg), while the lowest is for a very obese child (65 ml/kg). a term infant has an 80-90 ml/kg blood volume until age of 3 months, after which the total blood volume drops to 70 ml/kg [2] . the difference in total blood volume in an infant compared to that in an adult is an important consideration in determining how much blood to transfuse in a child. as such, formulas have been developed to guide clinicians during massive blood loss (blood loss greater than 1 blood volume) in a child without preexisting anemia. the blood loss at which transfusion should be considered in a child (or an adult) without preexisting anemia (maximal allowable blood loss (mabl)) can be estimated from the following formula [30] : theoretically, blood loss amounting to the mabl can be replenished by crystalloid or colloid, with blood transfusion reserved for higher blood losses. in general, the hematocrit in prbc approaches 70%; hence, approximately 0.5 ml of packed rbcs should be transfused for each milliliter of blood loss beyond the mabl. although this formula provides a framework for blood transfusion, it is merely an estimate. ultimately, blood transfusion requires careful consideration of patient condition, local resources, and severity of illness. a burned child poses a particular challenge, due to the increased red cell destruction and decreased red cell production that accompanies major burn injury. surgical excision of the burn wound results in major blood loss; a child loses 5% of a blood volume per percent face burn excised and 2% of a blood volume per percent burn excised on other areas [31] . thus, an infant having burn excision of the entire head could potentially lose 90% of total blood volume (body surface area of the head of 18 percent × 5 percent blood volume lost per percent excision of the head). sufficient units of blood products should be ready prior to the onset of surgery. the optimal transfusion threshold for critically ill children has been evaluated in a multicenter trial in pediatric intensive care units [32] . this study reported that a restrictive transfusion strategy, which transfused at a hemoglobin <7 g/dl, had mortality and outcomes comparable to a liberal strategy (maintain hemoglobin >10 g/dl). this study evaluated stable, critically ill children without acute blood loss; hence, its applicability to burn patients is limited. a recently completed randomized prospective trial in adult burn patients with burn size >20% tbsa demonstrated no outcome difference between different transfusion strategies (palmieri, in press). massive blood transfusion may result in the lethal triad: hypothermia, acidosis, and coagulopathy. hypothermia in the operating room, as discussed above, is more prevalent in the infant due to the larger surface area per unit mass. hypothermia is further exacerbated by exposure to the cold operating room suite and anesthetic agents which decrease shivering. acidosis due to hypovolemia and hypothermia develops if patients are under-resuscitated. coagulopathy, the final link in the triad, occurs during massive blood transfusion as a result of depletion of clotting factors. currently, prbcs are the predominant form of red cell transfusion. since 80% of coagulation factors are separated from prbcs during processing, clotting factor deficiency generally occurs at approximately 1 blood volume [33] . however, if whole blood is used, all clotting factors except for labile factors v and viii will be transfused at normal levels. thus, coagulation abnormalities tend to occur later (>3 blood volumes) when using whole blood [34] . however, whole blood carries substantial risks as well, including hyperkalemia, transfusion reactions, and transfusion-related circulatory overload. thrombocytopenia may be caused by dilution of platelets during transfusion. in general, a patient will lose 40% of the starting platelet count in the first blood volume loss, with loss of an additional 20% of the starting count at a second blood volume [33] . it is thus important to record the platelet count prior to an anticipated massive blood loss, such as happens with major burn excision. a child with sepsis and a low starting platelet count is far more likely to require platelet transfusion than a child with a high or normal platelet count. the optimal ratio of fresh frozen plasma to packed red blood cells in massive bleeding associated with extensive surgical burn excision has not been definitively defined; however, a prospective trial in burned children suggests that a 1:1 ffp/prbc strategy may improve outcomes. the use of prbc and other transfusion products also predisposes patients to other potential complications, including transfusion-related immunomodulation (trim), transfusion-related acute lung injury (trali), and transfusion-related circulatory overload (taco). as blood is stored, it releases a variety of agents, including toxic oxygen radicals, cytokines, soluble hla class i antigens, histamine, plasminogen activator inhibitor-1, and leukocyte elastase [35] . older blood may increase infection risk in multiple different patient populations [36] . blood transfusion in general impact the immune system by increasing suppressor t lymphocyte and natural killer cell function, depressing monocyte and macrophage phagocytic activity, inducing immune cell anergy and clonal deletion, decreasing macrophage antigen presentation, suppressing lymphocyte blastogenesis, decreasing delayed-type hypersensitivity, and suppressing mitogen-stimulated human t cell proliferation [37] . trim involves both immune activation (such as transfusion reactions, trali, alloimmunization, autoimmune diseases, and transfusion-associated graft-versus-host disease) as well as immune tolerance and immunosuppression (infection, cancer recurrence, microchimerism, enhanced allograft survival). trali, first described in 1983, is characterized by respiratory distress, hypoxemia, pulmonary edema, hypotension, and fever after receiving blood transfusion. a recent study in canada estimated that the incidence of trali in children is 1.8/100,000 population, much less than in adults [38] . the incidence of trali in burn injury is unknown. taco consists of pulmonary edema that develops within 6 h of transfusion due to increases in hydrostatic pressure. the incidence of taco is <11% in adults and carries a 5-15% mortality [39] . the incidence of taco in burned children and adults has not been determined. multiple strategies can be employed to decrease the immunologic and storage-related impact of blood. the first strategy is to decrease the amount of blood lost due to testing and surgery. for example, reducing volume and frequency of blood draws, adopting a restrictive transfusion policy, and utilizing tourniquets and tumescence during surgical burn excision will all decrease the volume of blood removed from the patient. the second strategy is to minimize the volume of blood administered. this entails using leukoreduced blood, transfusing blood one unit at a time, and investigating alternatives to transfusion. the fewer units of blood the patient receives, the less likely the patient is to have a transfusionrelated complication. children, due to their age-dependent physiology, alterations in body mass ratio, and immature cardiac and immunological status, have variable and complex transfusion needs after burn injury. optimizing the treatment of burn-injured children requires knowledge of these issues and careful consideration of the impact of transfusion on patient outcomes. fastidious attention to the sometimes subtle differences between children and adults is needed to optimize blood utilization in children with major burn injury. normal ranges of heart rate and respiratory rate in children from birth to 18 years: a systematic review of observational studies intraoperative pediatric blood transfusion therapy: a review of common issues the gaseous metabolism of the newborn infant breathing 15% oxygen oxygen consumption in normal newborn infants during moderate hypoxia in warm and cool environments pediatric resuscitation in the operating room physiologic anemia of infancy: normal red-cell values and physiology of neonatal erythropoiesis nathan 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platelet count, apheresis platelet yield, and platelet transfusion dose revised recommendations for reducing the risk of zika virus transmission by blood and blood components unexpected red blood cell antibody distributions in chinese people by a systematic literature review the kidd (jk) blood graoup system transfusion practices in infants receiving assisted ventilation dna polymorphism analysis in transfusion-associated graft-versus-host disease transfusion-associated graft versus host disease (tagvhd)-with reference to neonatal period nonlethal, attenuated, transfusionassociated graft-versus-host disease in an immunocompromised child: case report and review of the literature changes in lymphocyte subpopulations as a result of cardiopulmonary bypass. the effect of blood transfusion intraoperative pediatric blood transfusion therapy: a review of common issues. part ii: transfusion therapy, special considerations, and reduction of allogenic blood transfusions a prospective study of blood loss with excisional therapy in pediatric burn patients transfusion strategies for patients in pediatric intensive care units changes in serial platelet counts following massive blood transfusion in pediatric patients complications of massive blood transfusions red cell changes during storage addressing the question of the effect of rbc storage on clinical outcomes: the red cell storage duration study (recess) duration of red cell storage influences mortality after trauma transfusion-related acute lung injury in the canadian paediatric population incidence and transfusion risk factors for transfusion-associated circulatory overload among medical intensive care unit patients none. the authors received no funding for this work.availability of data and materials not applicable. ethics approval and consent to participate not applicable. not applicable. the author declares that she has no competing interests. submit your next manuscript to biomed central and we will help you at every step: key: cord-349803-tsjgypy5 authors: rouka, erasmia title: the effect of the covid-19 pandemic on the adequacy of blood supply: specialists in transfusion medicine need to establish models of preparedness date: 2020-09-28 journal: transfus apher sci doi: 10.1016/j.transci.2020.102960 sha: doc_id: 349803 cord_uid: tsjgypy5 nan on 11 march 2020, coronavirus disease 2019 (covid-19) was declared a pandemic by the world health organization. the disease, caused by an emerging pathogen, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) continues to spread rapidly at a global scale with severe impacts on public health and economy. among the major challenges faced by the national health systems is the maintenance of blood availability. at this time point, national blood centers are fighting to maintain blood adequacy through concerted actions which include the public's awareness of the situation, the encouragement of healthy individuals to donate blood and the planning of outdoors blood donations. however, as the pandemic continues to evolve the question that logically arises is whether current efforts are enough to address the crisis in the field of transfusion. the management of transfusion services in disasters like the pandemic is of critical importance for any blood bank [1] . a study by zimrin and hess (2007) on the effect of a modern pandemic influenza on blood supply in the united states (us) predicted that transfusion services are likely to face significant losses of blood donors, personnel, supplies and reagents [2] . on the other hand, there may be some mitigation owing to the reduced demand for blood products which results from the restriction in hospital admissions [3] and the postponing of scheduled, non-urgent surgeries. simulation models for the blood supply system in threats like the pandemics have been developed in the us as reported by two independent studies [4] [5] . both reports highlighted the importance of strategic planning towards the apt timing and duration of recruitment efforts [4] and the minimization of blood donation j o u r n a l p r e -p r o o f disruptions [5] . a european simulation study conducted in germany, has reported that the estimation of the expected blood deficit during a severe pandemic largely depends on the detailed information regarding the fraction of transfusions that can't be postponed [6] . the use of computer simulation tools for increasing the efficiency in the blood supply chain was previously reported by a finnish group [7] . the authors concluded that different scenarios should be adapted in particular settings considering the variability of transfusion services policies both at the transnational and national level. today's progress in the field of bioinformatics and computer science enables us to develop complex models that are capable of integrating numerous variables. the covid-19 pandemic will eventually resolve but contagious diseases will continue to perturb human populations [8] . thus, future modelling should incorporate past and existing knowledge on human epidemics along with patterns of blood donation and transfusion in different countries and regions. in addition, new models should include variables related to the consumables and reagents management policy as well as social distancing rules. last but not least, blood transfusion organizations should focus on designing crisis management training programs for scientists working on the field. the author declares no conflict of interest regarding the content of this letter to the editor. management of blood system in disasters planning for pandemic influenza: effect of a pandemic on the supply and demand for blood products in the united states potential impact of pandemic influenza on blood safety and availability a stochastic simulator of a blood product donation environment with demand spikes and supply shocks an interregional us blood supply simulation model to evaluate blood availability to support planning for emergency preparedness and medical countermeasures management of blood supplies during an influenza pandemic using simulation to increase efficiency in blood supply chains history in a crisis -lessons for covid-19 this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-317383-uqg0xwdw authors: weiskopf, richard b.; glassberg, elon; guinn, nicole; james, michael f.m.; ness, paul m.; pusateri, anthony e. title: the need for an artificial oxygen carrier for disasters and pandemics, including covid‐19 date: 2020-09-26 journal: transfusion doi: 10.1111/trf.16122 sha: doc_id: 317383 cord_uid: uqg0xwdw nan transfusion of red cells has saved countless lives owing to (a) the ability to perform far more extensive surgery; (b) treatment of acute hemorrhagic anemia, both civilian and military; and (c) treatment of illnesses of red cell destruction or impaired (including, but not limited to chemotherapy-induced suppression of hemopoiesis) production. there are extensive, largely efficient, blood collection and banking systems in developed countries that ordinarily provide stored red cells (at 2-6 c) for these functions, despite relatively brief periods of local or national shortages. blood banking systems require operational collection and processing facilities, sufficient number of healthy donors with access to a blood collection center, together with functional blood processing and transportation systems to provide the needed products in appropriate quantities. depending on the circumstances, any or all of these are likely to be severely degraded or completely non-operational in some civilian mass casualty events or on a military battlefield. these systems are also stressed at times of pathogen dissemination, owing to a decreased availability of healthy, acceptable donors, and healthy, non-infected health care personnel. for example, considering only the donation issue, with the onset of the current coronavirus pandemic the american red cross "faced a severe blood shortage" 1 with the cancelation of thousands of blood drives, and a "precipitous decrease" in blood donations in seattle was reported. 2 earlier viral epidemics noted a 21% donation decrease in a prefecture in japan, 3 and a greater than 90% decrease in beijing; 4 furthermore, covid-19 has been detected in asymptomatic donors whose donations had entered the blood supply in china. 5 nevertheless, the u.s. blood banking community points to their past capability in domestic cases of mass casualty, such as the twin towers destruction on 9/11, focusing on blood supply and donations [6] [7] [8] and that the above functions remained preserved throughout the 9/11 crisis, but has paid far less attention the isolated nature of this disaster 9 . only 224 units of packed red blood cells (prbcs) were required to satisfy the needs 6, 7 accepted article (less than 300 units were needed for other u.s. disasters 10, 11 ) a . in addition, the conditions are expected to be very different for other disasters (such as detonation of a thermonuclear device [12] [13] [14] , when the number of injuries will be far greater and capabilities for donation, testing, and transportation will be degraded. while there have not been reports of an overwhelming strain on the blood supply following a nuclear event such as that at chernobyl, b a nuclear detonation would be expected to result in a far larger mass casualty (including combined injuries) surge situation. 14 it has been estimated that a 10 kiloton nuclear detonation in a major city could require hundreds of thousands of units of plasma, and presumably a commensurate need for red cells. [19] [20] [21] the financial 22 and logistic 4 stresses on civilian blood collection and processing facilities add to the problem. c early on in the covid-19 pandemic, volunteer blood donations at fixed sites and reduced demand with the cancellation of elective procedures enabled the blood supply to be successfully maintained in its early phases, but the effect on the blood supply if the pandemic intensifies or become more chronic could be problematic. paradoxically, at least in the early phases of covid-19, societal lockdown may have unexpected results. in south africa there was a marked initial decrease in trauma admissions and a reduced demand for blood. however, this does not rule out a later upsurge in blood demand coupled with a delayed lack of availability through inadequate donation and reduced transfusion service staffing. when elective surgery was restored at some institutions in the u.s., shortages did occur as some blood banks/ centers were not prepared for the sudden demand increase and collection difficulties caused by new rules. due to their unique operational challenges, a the las vegas shooting injuries, perpetrated by only a single person, required 278 rbc units transfused, 67% in the first 24 hours, met by fully stocked in-hospital and local blood centers supplies. 11 b the nuclear disaster at chernobyl [15] [16] [17] [18] was an operator-and design caused steam explosion followed by a fire and a nine-to-ten day release of a substantial quantity of radioactive material with resultant many long-term medical issues including bone marrow suppression, but with relatively lesser immediate transfusion need, with two deaths in the first 5 hours, and apparently 31 in the first three days, predominantly from burns. c an hhs-commissioned rand study 23 concluded that disruption of the u.s. blood supply was unlikely, but, as pointed out by klein et al. 22 that was shown to be off the mark when the system could not function properly during the zika virus outbreak without a major financial infusion by the u.s. government. military organizations appear to be far more cognizant of these challenges than are their civilian counterparts. nevertheless, these systems are unprepared to fulfil the needs should the systems become severely degraded, as has been recognized in coordinated us government multi-agency (nih, fda, barda, dod) efforts to develop alternative products as countermeasures in these scenarios. 14 limited shelf-life and cold-chain constraints make the systems dependent on continuous donor availability and uninterrupted laboratory operations. blood collection, processing, and distribution is highly regulated. there are no products approved in the u.s. and most other countries that provide the primary physiologic functions of plasma or red cells that do not require continuous cold storage and a multitude of regulatory constraints and checks. however, there are products approved in some countries that fulfil these functions, have a prolonged shelf-life at 22c, and do not require recipient blood typing and cross-matching. dried plasma is produced in germany, france, and south africa, with limited availability of these products outside these countries; a hemoglobin-based oxygen carrier (hboc) is approved for use in south africa and russia. the need and rationale for a dried plasma has been published recently. 24 here, we address the need and rationale for a non-red cell oxygen carrier. the quest for an efficacious non-red cell oxygen carrier is many decades old. [25] [26] [27] research and development intensified when it became apparent that hepatitis c [28] [29] [30] and hiv 31 could be transmitted by transfusion, with the intent that an artificial oxygen carrier could replace rbc transfusion. however, donor screening and testing has reduced greatly the risk of transmission of these pathogens. 32 that coupled with the perception of a slightly increased risk of infusion of an hboc in comparison to red cell transfusion, 33, 34 resulted in a substantial decrease of research and development and in the number of companies in this field despite the latter meta-analysis having been widely criticized. [35] [36] [37] [38] [39] [40] subsequently, attention has turned to an alternative potential purpose for the use of such biologics, viz when rbc transfusion is not an option. for this indication, the appropriate comparator for the evaluation of product safety is untransfused severe this article is protected by copyright. all rights reserved. 44 the data for the hboc administration for "all-comer" hospitalized patients are less robust and come from "expanded access" use. 53 this article is protected by copyright. all rights reserved. in untransfused anemic patients that myocardial infarction, as defined by troponin elevation, increased with decreasing nadir hb below 8 g/dl, with an overall rate of 10.5%. 57 the hboc orthopedic trial 52 did not analyze the adverse event data according to nadir native hb, e and the data provided by clinicians who treated patients with hboc in the expanded access program is inadequate for analysis. 58 the recent report of guinn et al. has added an additional important novel piece of information to our knowledge of the mortality associated with severe anemia: the time to death as a function of hb. 55 the analyses above have documented well that for hb concentrations less than 8 g/dl, decreasing hb is associated with increased mortality. however, those analyses do not provide information regarding the window for therapy, should any be available. guinn et al. found that decreasing hb was not only associated with increased mortality, but also with decreased time to death. with nadir hb  5 g/dl median time to death from that hb value was 2 days; at nadir hb 5-8 g/dl, median time to death was 4-6 days. 55 these times are likely maximal for the period available for therapy, should any be available, as it is probable that the anemia-induced damage became irreversible with death inevitable some time prior to death. the above analyses and considerations regarding efficacy and safety of use of the hboc for severe anemia when rbc transfusion is not an option (mortality is the ultimate expression of a severe adverse event) is supported by real-world experience emanating from south africa, where hemopure® is approved by the national regulatory authority for use in severe anemia when blood transfusion is not an option. levien reviewed the hemovigilance program of 336 patients who received hemopure for acute surgical anemia owing to when blood was not an option, when blood avoidance was medically desirable, or physician or patient e there was a greater sae rate in the hboc group compared with prbcs, with most attributed to fluid overload or undertreatment. preference, finding no pattern of significant adverse events attributable to the hboc. 59 no deaths were attributable to the hboc. 59 oxidation. 65 when infused into patients with proven clinically significant myocardial vascular disease, hemopure® appears not to be harmful, but rather protective. 66, 67 a small incomplete safety trial in hemorrhagic shock in trauma, in south africa, found no mortality difference between those randomized to receive hemopure (4/10) and those treated with standard of care (5/9; p>0.99), but a 40% lesser need for crystalloid (11.5 l / patient v 19.3 l/ patient, respectively; p<0.0001). f the large crystalloid volume difference is of substantial importance, as increased crystalloid administration in trauma is associated with increased mortality. 69 the data presented and discussed suggest that hbocs could fill the critical underdosing, and volume overloading as may occur with the administration of any intravenous fluid, including rbcs or plasma. nevertheless, our assessment is that for the described potential use, when red cell transfusion is not an option, the benefit:risk profile for the treatment of severe anemia by administration of the hboc discussed is favorable, in comparison to that of untreated severe anemia. in conclusion, there are international needs for ensuring the large-scale availability of an artificial oxygen carrier for use when the civilian or military blood collection, processing, or delivery systems are degraded. the covid-19 pandemic has shown how vulnerable health care systems are to major disasters in the absence of proper preparation. freeze-dried plasma is approved in some countries, but hbocs are approved only in two: south africa and russia. there are products (fdp and hboc) not approved in most countries that can be so positioned now, for such emergency purposes. it is our opinion that the evidence supports that the relatively small risk of doing so with these products even where unapproved, for emergency use, greatly outweighs the risk of untreated severe anemia or decrease in coagulation factors. key message • world-wide blood collection and processing facilities and systems are illprepared to provide adequate amounts of red blood cells when transfusion is not an option, such as for major disasters: civilian mass casualty, pandemics, and extensive military operations. • at least one hemoglobin-based oxygen carrier under such circumstances has a favorable benefit-risk profile, and several year shelf-life at 22c, and should be considered for advance deployment to meet the needs in the above circumstances. accepted article prepare to adapt: blood supply and transfusion support during the first 2 weeks of the 2019 novel coronavirus (covid-19) pandemic affecting washington state the impact of h1n1 influenza a virus pandemic on the blood donations in hyogo prefecture viral attacks on the blood supply: the impact of severe acute respiratory syndrome in beijing severe acute respiratory syndrome coronavirus 2 rna detected in blood donations the september 11, 2001 disaster and the new york blood supply effect of a national disaster on blood supply and safety: the september 11 experience an interregional us blood supply simulation model to evaluate blood availability to support planning for emergency preparedness and medical countermeasures blood and disaster--supply and demand the las vegas mass shooting: an analysis of blood component administration and blood bank donations blood and disaster comprehensive us government program for dried plasma development the interagency strategic plan for research and development of blood products and related technologies for trauma care and emergency preparedness 2015-2020 immediate medical consequences of nuclear accidents. lessons from chernobyl the global impact of the chernobyl reactor accident chernobyl in retrospect united nations scientific committee on the effects of atomic radiation national security staff interagency policy coordination subcommittee for preparedness & response to radiological and nuclear threats toward a sustainable blood supply in the united states. an analysis of the current system and alternatives for the future the need for dried plasma -a national issue the large-scale production of crystalline human hemoglobin: with preliminary observations on the effect of its injection in man, in blood substitutes and blood transfusion clinical experience with hemoglobinsaline solutions survival of mammals breathing organic liquids equilibrated with oxygen a atmospheric pressure serum alanine aminotransferase of donors in relation to the risk of non-a,non-b hepatitis in recipients: the transfusiontransmitted viruses study non-a, non-b posttransfusion hepatitis in the united states non-a, non-b hepatitis following blood transfusion: risk factors associated with donor characteristics acquired immunodeficiency syndrome (aids) associated with transfusions a new strategy for estimating risks of transfusion-transmittted viral infections based on rates of detection of recently infected donors for the planning committee and speakers. hemoglobin based oxygen carriers: current status and future directions cell-free hemoglobinbased blood substitutes and risk of myocardial infarction and death: a metaanalysis hemoglobin-based blood substitutes and risk of myocardial infarction and death hemoglobin-based blood substitutes and risk of myocardial infarction and death hemoglobin-based blood substitutes and risk of myocardial infarction and death hemoglobin-based blood substitutes and risk of myocardial infarction and death hemoglobin-based blood substitutes and risk of myocardial infarction and death users guide to pitfalls and lessons learned about hboc-201 during clinical trials, expanded access, and clinical use in 1,701 patients the efficacy and safety of liquid stored blood and storage duration: a confused subject; are patients confused? hemoglobin-based oxygen carriers (hboc)-what the next generation holds: when red blood cells are not an option addressing the unmet need of life-threatening anemia with hemoglobin-based oxygen carriers acute severe isovolemic anemia impairs cognitive function and memory in humans oxygen reverses deficits of cognitive function and memory and increased heart rate induced by acute severe isovolemic anemia fresh blood and aged stored blood are equally efficacious in immediately reversing anemia-induced brain oxygenation deficits in humans human cardiovascular and metabolic response to acute, severe isovolemic anemia mortality and morbidity in patients with very low postoperative hb levels who decline blood transfusion an update on mortality and morbidity in patients with very low postoperative hemoglobin levels who decline blood transfusion (cme) clinical benefits and cost-effectiveness of allogeneic red-blood-cell transfusion in severe symptomatic anaemia hboc-201 as an alternative to blood transfusion: efficacy and safety evaluation in a multicenter phase iii trial in elective orthopedic surgery food and drug administration. 21 cfr parts 312 and 316: expanded access to investigational drugs for treatment use guidance for industry: individual patient expanded access applications: form fda 392 lower hemoglobin concentration decreases time to death in severely anemic patients for whom blood transfusion is not an option mortality risk stratification in severely anaemic jehovah's witness patients severe anemia associated with increased risk of death and myocardial ischemia in patients declining blood transfusion south africa: clinical experience with hemopure the use of hemopure(r) at groote schuur hospital, cape town: 4 cases south africa: consensus usage guidelines from clinician experts who have treated patients a case study of 10 patients administered hboc-201 in high doses over a prolonged period: outcomes during severe anemia when transfusion is not an option the rate of uptake of carbon monoxide and of nitric oxide by normal human erythrocytes and experimentally produced spherocytes recombinant human hemoglobin does not affect renal function in humans: analysis of safety and pharmacokinetics redox reactions of hemoglobin: mechanisms of toxicity and control proof-ofconcept trial to evaluate haemoglobin based oxygen therapeutics in elective percutaneous coronary revascularisation. rationale, protocol design and haemodynamic results haemodynamic effects, safety, and tolerability of haemoglobin-based oxygen carrier-201 in patients undergoing pci for cad briefing book for the 14 crystalloid resuscitation in trauma patients: deleterious effect of 5l or more in the first 24h. bmc surg 2018whom blood transfusion was not an option. this relationship from the new zealand database 44,51 () and from duke university all whom blood transfusion was not an option, comparing all hospitalized patients in the databases of beliaev et al. 51 plus guinn et al. 55 () with patients treated with an hboc, hemopure®, in an expanded access program all authors participated in the writing of this manuscript. the corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. no others contributed to the writing in any manner.no medical writers or editors have been involved in any way. rbw produced the figures. nobody other than the authors have influenced the writing of this manuscript.no funding or compensation of any kind (including from nih, wellcome trust, or hhmi) was received by any of the authors for this manuscript.the corresponding author confirms that he has had full access to all data and has had final responsibility for the decision to submit this manuscript for publication. this manuscript has not been published elsewhere. r.b. weiskopf has consulted for sponsors of hemoglobin-based oxygen carriers in the past, but has not received any compensation from these entities in the past 3 years.m.f.m. james has consulted with sponsors of hemoglobin-based oxygen carriers in the past but has received no financial compensation for these activities at any time.p. ness has consulted for sponsors of hemoglobin-based oxygen carriers in the past, but has not received any compensation from these entities in the past 3 years. e. glassberg, n. guinn, and a.e. pusateri report no competing interests. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. key: cord-294585-dl5v9p50 authors: klein, h. g.; bryant, b. j. title: pathogen‐reduction methods: advantages and limits date: 2009-02-13 journal: isbt sci ser doi: 10.1111/j.1751-2824.2009.01224.x sha: doc_id: 294585 cord_uid: dl5v9p50 pathogen‐reduction (inactivation) provides a proactive approach to reducing transfusion‐transmitted infection. pathogen‐reduction technologies have been successfully implemented by plasma fractionators resulting in no transmission of human immunodeficiency, hepatitis c, or hepatitis b viruses by us‐licensed plasma derivatives since 1987. fractionation technologies cannot be used to treat cellular blood components. although blood donor screening, deferral and disease testing have drastically reduced the incidence of transfusion‐transmitted diseases, the threat of new or re‐emerging pathogens remains. of particular concern is the silent emergence of a new agent with a prolonged latent period in which asymptomatic infected carriers would donate and spread infection. the ultimate goal of pathogen‐inactivation is to reduce transmission of potential pathogens without significantly compromising the therapeutic efficacy of the cellular and protein constituents of blood. the acceptable technology must not introduce toxicities into the blood supply nor result in neoantigen formation and subsequent antibody production. several promising pathogen‐inactivation technologies are being developed and tested, and others are currently in use, but all of them have limits. pathogen‐reduction promises an additional ‘layer of protection’ from infectious agents and has the potential to impact the safety of blood transfusions worldwide. the first decade of the 21st century remains an age of emerging and re-emerging pathogens that threaten the blood supply. blood collectors appreciate the dramatic reduction in risk of transfusion-transmissible infections, even as they are sobered by the failures of the 20th century safeguards to prevent widespread transmission of human immunodeficiency virus (hiv) and hepatitis viruses [1] . the specter of a new, as yet undiscovered agent with an extended latent phase raises the concern that the current system of overlapping safeguards that protects patients from infectious blood components is still disturbingly vulnerable. until recently, the approach to blood safety depended upon a combination of donor education, screening, testing for selected agents and discarding components in inventory if donor exposure or illness was reported post-donation. this strategy, while effective, is reactive. pathogen reduction of blood components represents a proactive approach to blood safety [2] . inactivation technologies promise an additional layer of protection both from infectious agents that are known as well as from those not yet recognized as threats to the blood supply (table 1) . a method with broad antimicrobial activity could eliminate emerging agents before they become recognized as transfusion-transmitted pathogens. however, because blood contains numerous labile proteins and fragile cells, and because there is a wide array of potentially infectious agents, no single method of pathogen-inactivation will likely preserve all blood components, yet effectively remove all viruses, bacteria, spores, protozoa and prions. furthermore, any chemical or physical process applied to blood must be 'safe' or at least less toxic to recipients than the infectious risk of blood. many pathogens that have the potential to invade the blood supply are not yet screened by testing because of low prevalence in the general population, unknown transmission rate of infection through transfusion or the lack of a readily available test for the agent (table 1) . agents with the potential to infect the blood supply are numerous and include the known viral pathogens -more than 35 arboviruses such as the flaviviruses den-1 through den-4 and st louis encephalitis virus; the togaviruses western and eastern equine encephalitis and chikungunya; the coronavirus severe acute respiratory virus; the circovirus tt and its variant sen; and the deltavirus hepatitis d. other blood-borne viruses include the herpes viruses such as the epstein-barr virus and human herpes viruses-6, -7 and -8, as well as the human parvovirus b19 virus. numerous animal blood-borne agents (zoonotics) have been able to cause infection across species barriers; most do not cause disease, but have the potential to do so. of particular concern are the simian viruses such as the foamy viruses and sv 40 that have been reported in animal handlers and in some vaccine recipients [3] . protozoa that threaten the blood supply include the four malarial, babesia microti , found primarily in the northeastern usa, toxoplasma gondii , the causative agent of toxoplasmosis, leishmania donovani , and numerous other subspecies that result in a high disease burden in the developing world [4] . borrelia burgdorferi , the cause of tick-borne lyme disease, has the potential for blood transmission and another tick-borne illness, human granulocytic ehrlichiosis has been reported from transfusion-transmission of the bacterial pathogen anaplasma phagocytophilum [5] . four instances of transfusion transmission of the infectious protein or prion pr sc have been reported in the uk, at least three of which almost certainly caused the human equivalent of 'mad cow disease', variant creutzfeldt-jakob disease [6] . there is no blood screening test for the prion diseases. most of the world does not have access to safe blood [7] . most developing countries do not screen donor units for all viral markers, because the technology is sophisticated, the cost prohibitive and/or the incidence of infected individuals so high that little blood would be available if all markerpositive donors were excluded. approximately 50% of blood donations in developing nations are from family members or paid donors [8] . each year, unsafe blood transfusions in third world countries result in an estimated 8-16 millions hepatitis b virus (hbv) infections, 2·3-4·7 million hepatitis c virus (hcv) infections and 80 000 to 160 000 hiv infections. pathogen-reduced blood could have a dramatic impact on blood safety in these circumstances. in 1946, edwin cohn introduced what has become the most widely used commercial fractionation method for plasma proteins involving multiple steps of precipitation and physical separation by centrifugation or filtration of the precipitant and effluent using changes in ph, temperature ionic strength and ethanol concentration gradients. as pathogen-reduction occurrs after different steps of precipitation and filtration in the fractionation process, proteins isolated later in the schematic were generally safer from infectious agents [9] . the albumin and globulin fractions proved extremely safe, particularly regarding transmission of hepatitis viruses and hiv. however, the process was not infallible and deviations from proper processing have resulted in disease transmission. some of the most important plasma protein derivatives such as factors viii and ix are separated early in the fractionation process and do not reap the benefits of added layers of fractionation and processing. pasteurization has been used effectively to inactivate viruses in albumin fractions stabilized with small chain fatty acids from as early as 1948 [10] . other proteins, especially clotting factors, denature during the pasteurization process unless additional stabilizers are added. terminal heat inactivation of lyophilized clotting factors has brought varied results depending upon temperature and processing time. dry heat treatment of lyophilized clotting factors at 60-68 ° c does not prevent transmission of hbv, hcv and hiv. increasing the dry heat temperature to 80 ° c for 72 h destroys hiv, hbv and hcv, although the non-enveloped viruses, especially hav and human parvo b19, may not be completely inactivated. adding humidity (vapor heating) improves viral kill. heat inactivation at 100 ° c for as little as 1 h has resulted in viral inactivation of both lipid enveloped and non-enveloped viruses. when this process is applied to intravenous immunoglobulin concentrates, little protein is lost. exposure to low ph inactivates many enveloped viruses, however among commercial proteins, only the immunoglobulins are stable under the necessary acid conditions (ph 4·0). use of organic solvents and detergents in the processing of coagulation factors inactivates the lipid-enveloped viruses, but not the non-lipid enveloped viruses [11] . the solvent and detergent combination disrupts the lipid envelope and prevents the virus from binding to cells and replicating. incorporation of a virucidal detergent and solvent (solvent 1% tri-n-butyl phosphate and the detergent 1% triton-x-100 for 4 h at 30 ° c) into the processing of plasma from pools of approximately 12 000 donors produces a product known as solvent-detergent plasma [12] . the tri-n-butyl phosphate is removed by oil extraction, and triton-x-100 is removed by chromatographic adsorption. plasma protein concentrates have been made from solvent-detergent-treated plasma, and fresh-frozen plasma (ffp) equivalent has been licensed in the usa and europe. as with the heat-inactivated coagulation factors, solvent-detergent technology does not inactivate the nonenveloped viruses. solvent-detergent treatment also results in some loss of integral plasma proteins such as α -2 antiplasmin and protein s [12, 13] . alpha-2 antiplasmin is crucial in maintaining haemostasis especially in patients with liver dysfunction. decreased levels of the natural anticoagulant protein s can lead to a hypercoaguable state especially when massive solvent-detergent plasma transfusions are used as in massive traumas or therapeutic plasma exchanges. these concerns, along with economic factors, resulted in the removal of solvent-detergent plasma from the us market. solvent-detergent plasma is still used widely in europe. recently, two new solvent-detergent treatment procedures have been developed for single unit or mini-pools of 10-12 units of plasma that yield > 90% mean recovery of coagulation factors, anticoagulants (including protein s), protease inhibitors (including α -2 antiplasmin), total protein, albumin and immunoglobulins. single unit and mini-pool solventdetergent treatment technologies show promise and have the potential to overcome some of the drawbacks of the original industrial solvent-detergent treatment processes [14] . nanofiltration has proven effective in removing a wide range of viruses including the non-enveloped viruses, and may even remove viruses smaller than the filter pore size [15] . many currently licensed plasma derived coagulation factors and immunoglobulins that are subjected to heat, pasteurization and/or solvent-detergent treatment are also nanofiltered. all classes of plasma protein fractions such as antithrombin, c-1 inhibitor, protein c, fibrinogen and ceruloplasmin have been nanofiltered without apparent change in the protein characteristics. methylene blue (mb) is a photoactive phenothiazine dye that has been used in europe for more than 15 years for the pathogen-inactivation of single units of plasma. mb has an affinity for nucleic acids and the surfaces of viruses [16] . when mb-treated plasma is exposed to ultraviolet light, most enveloped viruses are easily inactivated; however nonenveloped viruses are more resistant. intracellular viruses are not inactivated by mb/ultraviolet light, but freezing and thawing plasma often disrupts the cell membranes of leucocytes, thus liberating the viral particles and leaving them susceptible to mb pathogen-inactivation. residual intact white blood cells containing viruses are removed by a micropore filter. neither protozoa nor bacteria are inactivated by mb treatment. plasma proteins are moderately affected; fibrinogen and fviii activity is reduced by up to 30% [17] . mb treatment can be used for pathogen-inactivation of single units of plasma, thus eliminating the risk of large plasma pools currently used to manufacture solventdetergent plasma. macopharma uses an in-line system consisting of a membrane filter, mb dye, illumination bag, elimination filter and storage bag. the 0·65 μ m membrane filter removes platelets, leucocytes and debris. the plasma then passes through tubing containing an mb pill that dissolves as the plasma flows through the tubing into the illumination bag. the mb-containing plasma is subjected to double-sided illumination by sodium high-intensity low-pressure lamps emitting yellow light at a wavelength of 590 nm for 15-20 min. plasma is then passed through an mb elimination filter that removes greater than 95% of the residual dye and photoderivative by-products [18] . millions of mb single unit of plasma have been transfused in europe without unexpected adverse outcomes. until recently, attempts at pathogen-reduction for cellular blood components have achieved little success. leucoreduction of blood has reportedly decreased the risks of transfusion transmitted cell-associated viruses, such as cytomegalovirus, human t-lymphotropic virus i/ii and probably epstein-barr virus and human herpesvirus-8 (hhv-8), as leucocytes are the principal reservoir for these infectious agents. psoralens are small, planar molecules that cross cell membranes and viral capsids and intercalate between the bases of the nucleic acids. upon illumination with ultraviolet a (320-400 nm), the psoralens react with the dna or rna pyrimidine bases to form covalently bonded intranucleic and internucleic acid cross-links. this cross-linking prevents replication and transcription of the rna or dna [19] . psoralen treatment with ultraviolet a light results in the reduction of a broad array of viruses, bacteria and protozoa to a level unlikely to transmit infection. aminomethyl-trimethyl psoralen, a three-ringed synthetic psoralen known as amotosalen hydrochloride or s-59, has been extensively tested in the pathogen-reduction of platelets and plasma. amotosalen and photochemical treatment have demonstrated an acceptable safety profile through extensive toxicological studies for acute toxicity, repeat dose toxicity, reproductive toxicity, phototoxicity, and mutagenic and carcinogenic potential. in order to pathogen-inactivate with psoralens, the platelet concentrate must be volume reduced and re-suspended in 30-45% plasma and 70-55% platelet additive solution. the amotosalen is added to the platelet and incubated for 3-5 min [20] . the product is then exposed to ultraviolet a light after which approximately 80% of the psoralen have been photodegraded to by-products. the remaining psoralen and by-products are removed by a 'compound absorption disc' . intercept, an s-59 amotosalen system, has been evaluated in three clinical trials in europe (eurosprite) involving 166 thrombocytopenic patients. two of these trials evaluated whole blood-derived buffy-coat platelets, and one assessed single donor apheresis platelets. these studies demonstrated that when equal platelet doses were transfused, the inter-cept and conventional platelet transfusions resulted in comparable post-transfusion platelet count increments without significant differences in adverse reactions. in the usa, the sprint trial evaluated the haemostatic efficacy and safety in 645 thrombocytopenic oncology patients receiving intercept single donor apheresis platelets collected on the amicus separator [20] . a total of 4719 platelet transfusions were given; 2678 intercept platelets and 2041 conventional platelets. the incidence of world health organization grade 2 bleeding between the groups was comparable, and the incidence between the groups of world health organization grade 3 or 4 bleeding was equivalent. patients receiving the intercept platelets had lower post-transfusion platelet count increments, required more platelet transfusions and had a shorter interval between transfusions. explanations for the differences in post-transfusion platelet count increments in the photochemical-treated (pct) platelets were partly explained by the lower mean platelet dose and disproportionate number of transfusions containing platelet doses less that 3·0 × 10 11 cells. transfusion reactions were fewer with pct platelets most likely attributed to the reduced volume of plasma in the pct units as well as increased leucocyte inactivation resulting in less cytokine production during storage [21] . however a trend towards a higher frequency of respiratory complications has been noted and is of great concern to the us food and drug administration, but apparently less so for other national regulatory bodies. amotosalen and ultraviolet a light has been used to pathogeninactivate plasma in a system much like that used for platelets [22] . post-thaw coagulation studies of the pct plasma demonstrate that most coagulation factors are well-preserved in the range of 73-98% of control thawed plasma. factor viii levels, while decreased to 73%, are still sufficient for therapeutic use. there were no significant differences in the quantity and activity of the von willebrand factor, the pattern and distribution of the von willebrand multimers, or the activity of the adamts-13. fibrinogen maintained functional activity of approximately 87%. protein c and s as well as antithrombin were maintained at > 95% pre-treatment activity levels. there was no evidence of coagulation factor activation as a result of treatment. the factor vii kinetics of post-transfusion pct plasma was compared to standard ffp in a crossover study [23] . in a study of 34 patients with congenital coagulation factor deficiencies, coagulation factor kinetics and therapeutic efficacy of ffp treated with amotosalen and ultraviolet a light has been shown to be consistent with that of conventional ffp [24] . randomized controlled trials of pct-ffp supported haemostasis for the treatment of acquired co-agulopathy of liver disease and liver transplantation have revealed outcomes similar to that of conventional ffp [25] . additional studies utilizing pct-ffp for plasma exchanges in thrombotic thrombocytopenia purpura demonstrated similar results as those with conventional plasma. cryoprecipitate can also be produced from amotosalen and ultraviolet a-treated plasma. preliminary studies indicate that pct cryoprecipitate coagulation factor levels are acceptable. riboflavin, vitamin b2 a naturally occurring essential nutrient, has been used as a pathogen-inactivating agent for platelets and plasma. riboflavin is a 3-ringed planar structure that binds to nucleic acids and intercalated between dna and rna bases. upon activation of cross-linked riboflavin with ultraviolet or visible light, guanosine bases are oxidized resulting in single strand breaks in the nucleic acids. the damaged and disrupted nucleic acids are incapable of repair and replication. toxicities of riboflavin and its photoderivative by-products do not appear to cause concern, because riboflavin and its breakdown products are present in many food and natural products. removal of the spent riboflavin and products post-illumination may not be necessary in a pathogen-inactivation system using riboflavin. the us food and drug administration has classified riboflavin as a 'generally regarded as safe' compound. the mirasol prt system contains 30 ml of riboflavin (500 μ m) in a light protective pouch and a pathogen-reduction illumination/storage bag. platelets or plasma are sterilely connected to the system, and 250 ml of plasma or platelet product is transferred to the bag containing the riboflavin diluting it to a final concentration of 50 μ m. the riboflavin-treated product is subjected to double-sided ultraviolet illumination [27] . riboflavin/ultraviolet light treatment has been evaluated in preclinical studies and found to result in reduction of infectivity by many pathogens including west nile virus, intracellular hiv, bacteria and protozoa. the mirasol system demonstrated successful pathogen reduction of selected pathogen-spiked platelet units after treatment and storage for 5 days. the viral reduction of platelets was sufficient to close the window period of transmission of hiv and chronic phase of parvo b19, eliminate the viraemic period of west nile virus, prevent infection due to staphylcoccus epidermidis and escherichia coli , and result in a 5-6 log reduction of leishmania donovani infantum [27] . additionally, leishmaniaspiked plasma units treated with riboflavin/ultraviolet light demonstrated a 5-7 log reduction in parasites. studies have demonstrated significant differences in control and treated platelets after 5 days of storage in regards to accelerated changes in platelet morphology, increased platelet activation and induced partial platelet aggregation. riboflavin/ultraviolet light-treated plasma has been shown to retain acceptable levels of clotting factors without evidence of increased compliment activation. pathogen-inactivation of components containing red blood cells presents a particularly challenging dilemma. methods utilizing photoactivation must do so in the red wavelength region of the light spectrum above that of haemoglobin in order to avoid absorption or scattering of the light by the red blood cell. many potential methods of pathogen-inactivation easily alter or disrupt the red blood cell membrane resulting in decreased red cell survival, haemolysis or immunogenicity. s303 (helinx), a small molecule designed for pathogeninactivation treatment of red blood cells, is an alkylating agent derived from a quinacrine mustard that belongs to a class of 'frangible anchor linker effectors' (frale) compounds. frale compounds contain an intercalator group that inserts into the helical region of dna and rna, an effector group that permits covalent attachment of nucleic acids and a central frangible bond that orchestrates the degradation of the compound [28] . s-303 is a positively charged planar structure that easily intercalates into the helical regions of the negatively charged nucleic acids. the process does not depend on light for activation. frale compounds are activated by a shift from lower ph storage environment to the higher neutral ph of red blood cells causing hydrolysis and generating s-300 the primary degradation product; cross-linking of the dna and rna ensues. s-300 is rapidly metabolized and excreted leaving no detectable parent compound. the remaining free degradation products are absorbed and removed by a compound removal step. s-303 binds to other proteins and cell membranes as well as nucleic acids, and up to 20% can potentially remain bound to the surface or contained within the red blood cells. s-303 has demonstrated pathogen-inactivation of a wide range of viruses, bacteria and protozoa. no unexpected toxicities have been described. assays for red blood cells storage lesions (extracellular potassium leakage, plasma-free haemoglobin, adenosine diphosphate, 2,3-diphosphoglycerate, glucose and lactate) are comparable to control red blood cells. the red blood cell function appears to be normal, and in vivo 51 cr-labelled survival studies exceed the standard of 75% at 24 h. two randomized, controlled trials involving patients either undergoing first-time cardiovascular surgery or with haemoglobinopathies were in progress when antibodies to residual red blood cell bound s-303 were discovered in two subjects [29] . the trials were suspended as a consequence of these findings. additional studies have revealed that 1% of patients and healthy donors that had never been exposed to s-303 had naturally occurring antibodies that reacted with s-303 treated red blood cells. modifications have been made to the s-303 treatment process to reduce the amount of red blood cell bound s-303 in attempts to eliminate immunoreactivity and immunogenicity. preliminary finding indicates that red blood cells from the modified s-303 treatment process were cross-match-compatible with the anti-s-303 antibodies formed after exposure to the original s-303 formulation as well as the anti-s-303 antibodies found in the patients and donors never exposed to s-303. the antibodies do not appear to impair transfusion or to pose any clinical problem. new clinical trials have been initiated. riboflavin-based pathogen-inactivation systems for red blood cells are currently under development if found to be a successful means of pathogen-inactivation of red blood cells, riboflavin may serve as the one material to inactivate pathogens in three blood components (red cells, platelets and plasma). current leucoreduction filters are effective in removing cell-associated viruses, but remove only 42% of total prion infectivity in endogenoussly infected blood. a leucoreduction filter under development removes prions in exogenously and endogenously infected blood more effectively [30] . exogenous infectivity studies were conducted using 270 ml of red blood cells and 30 ml of 10% (wt/vol) with high titre brain homogenates from hamsters infected with scrapie for a final 1% homogenate concentration. endogenous infectivity studies utilized red blood cells processed from 500 ml of whole blood collected from 120 scrapie-infected hamsters. the new prototype leucoreduction, prion reduction filter was effective in removing 3·7 logs of scrapie infectivity from exogenously infected red blood cells and all of the detectable prp sc from the endogenously infected hamsters. in the endogenous infectivity study, the pre-filtered-infected red blood cells transmitted disease to six of 43 animals, and the post-filtration red blood cells did not transmit disease to any of 35 animals. allogeneic blood is a critically important therapeutic, but also an inherently risky biologic source material. among the dangers is transmission of a wide range of pathogens. donor selection and blood testing have reduced this risk dramatically and will remain the cornerstone of blood safety programmes. nevertheless, infectious units still elude screening and testing; testing errors and product release errors are probably impossible to eliminate. the largest current infectious risks involve pathogens for which we do not test, for which we have no test, or for which demographic screening and testing are inadequate. the greatest fear concerns the emergence of a 'new' transmissible agent, particularly one that has not previously been associated with human disease, has a long silent period, can infect others by secondary spread and is highly lethal -as was the case with hiv. ideally, pathogeninactivation techniques would provide an additional safeguard. that has been the experience in the plasma fractionation industry. no single pathogen-reduction method will likely be effective for every class of agent and for every blood component. some combination of techniques that remove and inactivate infectious agents will probably be needed. these technologies are generally expensive and have the potential to profoundly escalate the cost of blood, but this cost may be partially offset by the elimination of some testing markers. however, if potent pathogen-inactivation techniques that preserve blood function and do not evidence some new toxic risk can be created, the developed world, which embraces the myth of zero-risk transfusion, will likely adopt them almost regardless of cost. for the developing world, in which low-risk blood donors are at a premium and elegant testing methods often not feasible, a good but not perfect pathogen reduction method, especially if relatively inexpensive and easy to implement, could save millions of lives. meeting transfusion safety expectations pathogen inactivation: making decisions about new technologies. report of a consensus conference frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates protecting the blood supply from emerging pathogens: the role of pathogen inactivation risk and prevention of transfusion transmitted babesiosis and other tick-borne diseases preclinical vcjd after blood transfusion in a prnp codon 129 heterozygous patient who: fact sheets: blood safety and volunteer donations blood supply and demand clearance of prions during plasma protein manufacture an adventure in biotechnology: the development of haemophilia a therapeutics -from whole blood transfusion to recombinant dna to gene therapy sterilization of hepatitis and htlv-iii viruses by exposure to tri (n-butyl) phosphate and sodium cholate current status of solvent/detergenttreated frozen plasma venous thromboembolism associated with the management of acute thrombotic thrombocytopenic purpura a process for solvent/detergent treatment of plasma for transfusion at blood centers using a disposable bag system removal of small non-enveloped viruses by nanofiltration virus inactivation in blood components by photoactive phenothiazine dyes methylene blue treated fresh-frozen plasma: what is its contribution to blood safety? methylene blue and thionine in pathogen inactivation of plasma and platelet concentrates photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light therapeutic efficacy and safety of platelets treated with a photochemical process for pathogen inactivation: the sprint trial clinical safety of platelets photochemically treated with amotosalen hcl and ultraviolet a light for pathogen inactivation: the sprint trial preclinical safety profile of plasma prepared using the intercept blood system pharmacokinetic study of ffp photochemically treated with amotosalen (s-59) and uv light compared to ffp in healthy volunteers anticoagulated with warfarin fresh frozen plasma prepared with amotosalen hcl (s-59) photochemical pathogen inactivation: transfusion of patients with congenital coagulation factor deficiencies photochemically treated fresh frozen plasma for transfusion of patients with acquired coagulopathy of liver disease photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light the use of riboflavin for the inactivation of pathogens in blood products helinx technology for inactivation of infectious pathogens and leukocytes in labile blood components: from theory to clinical application therapeutic efficacy and safety of red blood cells treated with a chemical process (s-303) for pathogen inactivation: a phase iii clinical trial in cardiac surgery patients removal of exogenous (spiked) and endogenous prion infectivity from red cells with a new prototype of leukoreduction filter key: cord-277535-u283k70i authors: vaja, rakesh; rana, meenal title: drugs and the liver date: 2020-09-22 journal: nan doi: 10.1016/j.mpaic.2020.07.001 sha: doc_id: 277535 cord_uid: u283k70i the liver is a major organ with multiple functions. a number of drugs are metabolized by the liver during phase 1 and 2 reactions which include complex processes involving cytochrome p450 enzymes. genetic and acquired variability in cytochrome p450 activity may have profound effects on pharmacokinetics. additionally, drugs can also modify how the liver functions and cause dysfunction or even failure of the organ both by a direct effect on the liver or by alteration in liver blood flow. it is important to recognize the signs and symptoms of liver failure in patients and identify possible causes including drug interactions. furthermore, once a patient has been recognized to be suffering with liver dysfunction or failure drug choice and dosing regime will need to be rationalized. paracetamol overdose can have severe and life threatening consequences for patients due to its effect on liver function. it is the leading cause of acute liver failure in the uk, 1 correct and early management is crucial and will be discussed within this article. the liver receives approximately 30% of cardiac output. uniquely it receives both arterial blood from the hepatic artery and venous blood from the portal veins. the portal vein supplies 70e75% of hepatic blood flow but only 50% of oxygen supply, the remaining blood flow and oxygen supply being from the hepatic artery. anatomically the liver is divided into two lobes and further into functional lobules based around a central vein, which contains blood from the hepatic arterial and portal venous circulations. blood arriving to the liver flows into the sinusoids which are spaces lined by hepactocytes. blood then drains towards the centre of the lobule and the central vein then hepatic vein to return blood back to the heart via the inferior vena cava. it is the portal veins taking blood directly from the gut to the liver which allows for first pass metabolism, making the liver susceptible to ingested drugs as they are absorbed from the gastrointestinal tract and transported to the liver. the liver has a broad range of functions categorized in table 1 . the liver metabolises a wide range of drugs the end result being to produce water soluble compounds which can be excreted in the bile. this results from phase 1 reactions mediated by cytochrome p450 including oxidation, reduction and hydrolysis reactions. this is followed by phase 2 reactions which are conjugative. the cytochrome p450 family are a group of enzymes found mainly in the liver, which perform oxidation and reduction reactions (phase 1) using iron to enhance the water solubility of drugs to aid excretion. cyp450 enzymes are so named as they are bound to membranes within the cell and contain a haem pigment that absorbs light at a wavelength of 450nm when exposed to carbon monoxide. after reading this article you should: c understand the mechanisms of drug metabolism by the liver c have an appreciation of alterations to drug choice and dosing regimens in patients with liver disease due to their altered pharmacokinetics c know the management of a patient with paracetamol overdose there are many different isoforms of cyp450, classified according to their amino acid sequencing into families, subfamilies and individual genes. their importance can be seen in certain subgroups that lack particular genes. an example pertinent to anaesthesia is deficiency in cyp2d6 which metabolises codeine to morphine, these patients therefore find codeine ineffective. conversely there is a small subgroup of people of saudi arabian and ethiopian decent with very high expression of 2d6 who metabolize codeine into vast amounts of morphine. refer to table 2 for more details. an individual more detailed breakdown of cyp450 genes is beyond the scope of this article. some drugs can induce or inhibit cyp450 enzymes which have the sequential effect on the metabolism of other drugs, either increasing or reducing it respectively. possibly the most important example is cyp3a4 which metabolises many substrates and is induced by rifampicin, carbamazepine, phenytonin and dexamethasone. of interest to anaesthesia this will increase metabolism of opioids, benzodiazepines and local anaesthetics. another well cited example is the increased metabolism of the oral contraceptive pill and its reduction in efficacy. for a more exhaustive list of substrates, inducers and inhibitors see table 3 (see table 4 ). a number of non-cytochrome p450 dependent reactions occur in the liver eg oxidation of dopamine and alcohol and hydrolysis of amides and esters (eg lignocaine and pethidine respectively). a predominant rise in aspartate aminotransferase (ast) and alanine aminotransferase (alt) signals hepatocellular injury or death. this can be caused by drug reactions or toxicity (e.g. paracetamol), viral hepatitis, autoimmune hepatitis, alcoholic hepatitis, ischaemic hepatitis secondary to profound hypotension, and rare causes such as wilson's disease. an obstructive pattern has a rise predominantly in alkaline phosphatase (alp) and gamma-glutamyltransferase (ggt), these are canalicular enzymes and suggest cholestasis. this is caused by obstruction, either calculi or tumour (primary biliary, pancreatic or metastases), and liver disease such as primary biliary cirrhosis. pharmacological causes include antibiotics, anabolic steroids and oral contraceptives. a mixed pattern can be seen in sepsis, some drug reactions, cholangitis, congestive cardiac failure and alcoholic liver disease. halothane hepatitis can cause raised liver enzyme assays, raised bilirubin and jaundice. an isolated rise in unconjugated bilirubin may be attributed to gilbert's syndrome or haemolysis. the sars-cov-2 virus uses angiotensin-converting enzyme 2(ace-2) receptors to gain entry into cells. 3 liver particularly in the ductal region has abundance of these receptors, hence may be susceptible to sars-cov-2 3 elevated lft have been reported widely in hospitalized patients with covid-19, the range of elevation is highly variable from 14 to 58%. 4 surprisingly, the pattern of elevation mimics hepatocyte damage (ast/alt higher than bilirubin or alp) rather than cholangiocytic damage, as would have been expected given the density of ace-2 receptors is higher in the ductal system. 4 additionally low albumin has been seen and is a marker of severe disease. 5 one study reported longer hospital stay in patients with elevated lft. 6 an international registry has suggested that has many as 25% of patients with covid-19 may present with hepatic decompensation in absence of respiratory symptoms. 7 the above findings hold important implications for anaesthetists, especially in preop assessments. most drugs given in anaesthesia and intensive care are given intravenously, thus having a bioavailability of 1. however some maybe given orally or nasogastrically and absorbed enterally. the absorption will be affected by delayed gastric emptying or reduced by diarrhoea and increased gastric transit time seen in liver failure. additionally if vasopressors are used there maybe splanchnic vasoconstriction with associated reduced absorption. volume of distribution is a theoretical calculated volume within which a dose of a drug is dissolved. hepatic dysfunction can cause fluid retention and will increase the volume within which drugs are present, particularly those which usually remain in the plasma, thus increasing their volume of distribution and reducing their plasma concentration. 2 in liver disease, protein synthesis may be reduced. these proteins are important as binding sites for drugs and as such alter the amount of free drug available, volume of distribution, half life and duration of action. an important example is albumin. hypo-albuminaemia will increase the proportion of free drug which is active, therefore doses of highly protein bound drugs may need to be reduced, for example phenytoin and benzodiazepines, aspirin and warfarin. 8 another protein produced by the liver, a 1 acid glycoprotein binds basic drugs such as carbamazepine, propanolol, alprenolol and imipramine as well as steroids. bilirubin can also compete for protein binding sites, so raised levels can increase amount of free drugs, the effect however is less in vivo than in vitro. 1 problems with absorption of enterally delivered drugs have been described. once absorbed these drugs undergo the "first pass effect" by the liver before reaching the systemic circulation. in liver failure the degree of metabolism will be reduced, therefore the extraction ratio will also be reduced and more drug will reach the systemic circulation, thus increasing bioavailability. prevalence of ultrarapid metabolisers metabolism of drugs in liver disease depends on liver blood flow. this can be reduced in a cirrhotic liver as portovenous shunting in the form of varices which are created and blood is diverted directly into the systemic circulation by-passing the liver. thus first pass metabolism is reduced. drug metabolism by the liver may also be reduced by the use of vasopressors on intensive care which reduce liver blood flow due to varying degrees of splanchnic vasoconstriction. the phase 1 and 2 reactions performed by the liver are affected and metabolism and thus extraction ratios are reduced. drugs can be divided into those with high extraction ratios >0.7, for example fentanyl and morphine and low extraction ratios <0.3 such as lorazepam, diazepam and methadone. most drugs have low extraction ratios <0.3 that is they have poor permeability and are metabolized by the liver but poorly extracted, therefore clearance is limited by reduced metabolism not by blood flow. those with high extraction ratios >0.7 are highly permeable and clearance is dependent on blood flow. 9 hepatic dysfunction is not uncommon within the intensive care setting affecting 11e54% of critically ill patients depending on definitions used. 2 there is currently no tool akin to renal clearance to indicate degree of liver dysfunction. 9 therefore clinicians use liver function blood tests, international normalized ratio (inr), serum albumin and clinical scores such as the child pugh score act as a surrogate for function. more recently the model for end stage liver disease (meld score) and the meld-na have been used to more accurately predict the severity of liver dysfunction. 10, 11 although their correlation with pharmacokinetic function not well understood. due to the alterations discussed in pharmacokinetics in liver dysfunction drug choices, dosages and frequency may need to be rationalized and altered accordingly. for example the induction dose and maintenance dose, for either anaesthesia or sedation, needs to be reduced. historically inhalational agents, particularly halothane have been implicated in causing hepatitis. the risk is related to the generation of trifluroacteyl chloride (tfa) by metabolism of agents, which is implicated in toxicity 12 around 20% of administered dose of halothane is metabolized by the liver, more specifically by cytochrome p450. this is a relatively high percentage when compared to more modern inhalational agents, for example, 0.2% isoflurane, 0.02% desflurane and 3% sevoflurane. even though sevoflurane undergoes 3% metabolism it does not generate tfa and hence is not linked to immune mediated injury 13 sevoflurane metabolism produces fluoride which is not linked to hepatotoxicity. 14 inhalational agents themselves cause a dose dependent reduction in hepatic blood flow (hbf). isoflurane and sevoflurane result in relatively lower reduction in hbf at 1 mac as compared to desflurane. 15 as long as hypotension is avoided and above effects are kept in mind desflurane is probably the safest choice of inhalational agent due to its' low rate of metabolism and rapid and predictable emergence from anaesthesia. 13 there are two types of halothane hepatitis. type 1 which is mild, transient and has a relatively high incidence (25e30%). type 2 caused by oxidative metabolism of halothane in the liver leading to fever, jaundice, and dramatically elevated serum transaminases. the compounds synthesized by oxidation then bind to trifluoroacetylate proteins in the hepatic endoplasmic reticulum causing cellular dysfunction, it is thought to occur in genetically predisposed individuals. the committee on safety of medicines in 1986 recommended the avoidance of halothane in patients with a history of previous adverse reactions, those who had received halothane within 3 months unless clinically necessary, and those with a history of unexplained jaundice or pyrexia following previous halothane anaesthesia. iv anaesthetics: the induction agents have a marked effect on haemodynamics and may cause sudden precipitious fall in blood pressure. in clinical practice a standard induction dose need not be altered. however they should be titrated slowly to effect. there are no current recommendations on the use of tiva (total intravenous anaesthesia) in patients with liver disease. research is sparse and conflicting. some earlier reports suggested that inhalational anaesthesia results in smaller elevation of liver enzymes than tiva with propofol-fentanyl. 16 a more recent study however suggested slightly lower rate of elevation in lft after using tiva. 17 opiates morphine is metabolized by the liver to active metabolite morphine-6-glucoronide which has potent analgesic properties, and morphine-3-glucuronide, which has no analgesic properties but has adverse neurotoxic side effects such as confusion and respiratory depression. as both metabolites are excreted renally, they accumulate in renal failure. in liver failure morphine itself may accumulate as extraction ratio is reduced thereby enhancing further the effect of morphine. 13 therefore dose of morphine should be reduced. the same is true of fentanyl and alfentanil dose, as although there is no active metabolite they also rely on hepatic metabolism. remifentanil may be good choice as its metabolism is by plasma esterases and it has no active metabolites. a review of pain management in patients with liver disease by the american association for study of liver diseases (aalsd) in 2018 states in general most opioids have prolonged half life. their recommendations include increase the dosing intervals (6e12hr) and using immediate release preparations over extended release 18 in july 2013 the medicine and healthcare products regulatory agency (mhra) produced a drug safety update that restricted the use of codeine in children. 19 this was prompted by case reports of four children suffered serious harm following the administration of codeine in the immediate post-operative period. codeine should only be used to relieve acute moderate pain in children older than 12 years and only if it cannot be relieved by other painkillers such as paracetamol or ibuprofen alone. a significant risk of serious and life-threatening adverse reactions has been identified in children with obstructive sleep apnoea who received codeine after tonsillectomy or adenoidectomy (or both). codeine is now contraindicated in all children younger than 18 years who undergo these procedures for obstructive sleep apnoea. codeine is converted to morphine in the liver by the cyp2d6 enzyme. the extent of conversion of codeine to morphine depends on genetic variations of cyp2d6. people can be classified as: poor; intermediate; extensive; or ultra-rapid metabolisers. poor metabolisers convert very little codeine into morphine and therefore have little or no pain relief, ultra-rapid metabolisers or extensive metabolisers have an excessive amount of morphine in their blood following ingestion of codeine. ethnic origin is an important factor in genetic variability. up to 10% of caucasians are poor metabolisers whereas up to 29% of patients of african origin may be ultra-rapid metabolisers. (table 2) . this genetic variability leads to different plasma morphine concentrations in patients leading to different analgesic effects as well as side effects including respiratory depression. codeine is contraindicated in all patients of any age known to be cyp2d6 ultra-rapid metabolisers and should not be used by breastfeeding mothers because it can pass to the baby through breast milk and potentially cause harm. nsaids are contraindicated for systemic use in most liver disease patients, because of increased bioavalibilty, the high risk of precipitating gastrointestinal bleeding and renal failure. 20 pregabalin and gabapentin are not metabolized in the liver and can be considered for use. these drugs are renally excreted therefore patients with hepatorenal syndrome warrant cautious adminstration. 18, 20 gabapentin is considered as first line non opiod drug for analgesia. among the tricyclic antidepressants nortryptilline appear safer than amitriptyline and imipramine. 20 an increased dose of non-depolarising neuromuscular blockers (nmb) may be required in liver disease possibly due to altered pharmacology anaesthesia and intensive care medicine xxx:xxx protein binding and increased volume of distribution. however, those which are metabolized by the liver have a prolonged duration of action, atracurium as metabolized in the plasma has a more predictable duration of action. however, it is worth noting that in prolonged usage concentrations laudanosine from hoffmann degradation may accumulate with the potential to provoke epileptiform activity on electro encephalography (eeg). 13 this is of greater concern if the patient has concomitant renal failure or impaired blood brain barrier. the metabolism of succinylcholine may be prolonged due to reductions in pseudocholinesterase concentrations, but clinically this is of little significance. suggamadex is a unique reversal agent for amniosteriodal nmb which acts by chelating the nmb. it is not metabolized and is excreted almost exclusively unchanged by the kidneys within 24 h. data regarding use of sugammadex, in patients with liver dysfunction is limited. however as sugammadex is almost entirely excreted renally, no dose reduction is required in patients with mild to moderate liver dysfunction. in patients undergoing liver transplant sugammadex is able to reverse neuromuscular block maintained by rocuronium continuous infusion. 21 however it is important to note that the sugammadex recovery time in this population was found to be considerably longer than in other surgical settings, and should be considered in clinical practice. dexmedetomidine is a highly selective alpha-2 receptor agonist, with analgesic, anxiolytic and sedative. it is primarily metabolized in the liver and may have a prolonged half-life in patients with liver disease. dexmedetomidine has potential protective effects on the liver and intestine during hepatectomy and intraoperative use of dexmedetomidine during liver surgery is subject to ongoing research. 22 additionally patients with elevated bilirubin and bile salts secondary to jaundice may show bradycardias limiting its use. 23 paracetamol overdose paracetamol is the commonest drug taken in overdose in the uk to date, it can result in liver failure and in some cases fatal. 24 hepatocelluar necrosis can occur if as little as 7.5g of paracetamol is ingested. normal pathways of metabolism are saturated and hepatic glutathione stores are exhausted. patients are often initially asymptomatic for the first 24 h before reporting nausea, vomiting, right upper quadrant pain with progressive derangement of liver function tests (lfts) after 18 h. the mhra produced new guidelines for treatment of paracetamol overdose in 2012; the changes included an updated nomogram and a simplified treatment schedule. 25 the administration of acetylcysteine has previously been based on the rumack-matthew nomogram, which divided treatment groups into high and low risk. the dose was then calculated on a weight-based table, which increased the risk of drug error. the updated nomogram has a single treatment line (figure 1) 25 ; thereby eliminating the need for assessing whether the patient falls into the high-risk category. it also advises that in cases of staggered overdoses there should be no delay in the administration of acetylcysteine. another cause for concern was the adverse events that had been reported following the bolus dose of acetylcysteine, this has been addressed by increasing the duration over which it is infused, from 15 min to 60 min. acetylcysteine should be administered when the plasma paracetamol level is on or above a single treatment line joining points of 100mg/l at 4 h and 15mg/l at 15 h after ingestion. a baseline full blood count (fbc), urea and electrolytes (u&es), lfts and coagulation screen along with an arterial blood gas sample should also be done at the earliest opportunity. intravenous preparations of paracetamol were licensed in the uk in 2004 and it is routinely used in anaesthetic practice. however, there have been some concerns regarding the dosage and administration; especially in adults and children under 50kg, patients with pre-existing hepatic dysfunction and the elderly. children and infants weighing less than 10kg should receive the reduced dose of 7.5mg/kg not exceeding the daily dose of 30mg/ kg, whilst those >10kg can be prescribed up to 15mg/kg not exceeding the daily dose of 60mg/kg. both the mhra and the npsa have issued alerts, regarding the correct dose prescription as there have been reported cases of accidental overdose due to ml to mg conversion and the administration of 1g in adults weighing less than 50kg. the key issue is that with intravenous paracetamol plasma levels will peak immediately after administration. the traditional nomograms used to predict plasma levels after overdose refer to oral ingestion where the levels peak some hours after. indicators of severe paracetamol poisoning which is likely to require referral to a specialist liver centre include: inr of >2.0 at 24 h, >4 at 48 h or >6 at 72 hours; renal impairment (creatinine >200 micromol/l); hypoglycaemia; metabolic acidosis despite rehydration; hypotension despite resuscitation or encephalopathy. 26 the only other treatment in fulminant liver failure is transplantation. paracetamol as analgesic in chronic liver disease given the potential of paracetamol to cause liver injury, there is a common misconception that these patients should never take paracetamol. however, various studies have shown that if taken in appropriate doses, paracetamol is one of the safest analgesics for patients with cirrhosis. limiting the total daily dosage to 2e3 g/day with thrice daily dosing is generally recommended. 27 patients should be educated about over-the-counter and prescription medications that may also contain acetaminophen to avoid overdose. remdesvir: elevated liver enzymes are commonly observed in clinical trials of patients with remedevir. 28 the elevated values rarely warrant treatment discontinuation. current recommendations suggest that if the enzymes are elevated to 5 times or more above baseline, to discontinue the drug. tocilizumab: alt elevations are frequent but fulminant hepatitis is rare. the risk of re activation of hbv should be kept in mind if patient had chronic liver disease secondary to viral etiology. 28 steriods: low dose dexamethasone is probably safe in patients with chronic stable liver diease. however use of methylprednisolone in high doses may reactivate hbv and increase risk of spontaneous bacterial peritonitis (sbp) in severe cases. 28 hydoxychloroquine: data is limited but generally has not been associated with elevations in alt levels and is an extremely rare cause of drug induced liver injury. 28e31 changing patterns of causation and the use of transplantation in the united kingdom drug dosing considerations for the critically ill patient with liver disease sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor liver injury in covid-19: the current evidence hypoalbuminemia predicts the outcome of covid-19 independent of age and co-morbidity liver injury during highly pathogenic human coronavirus infections high mortality rates for sars-cov-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry differentiated effects of liver cirrohosis on albumin binding sites for diazepam, salicylic acid and warfarin anaesthesia for the patient with liver disease a model to predict poor survival in patients undergoing transjugular intrahepatic portosystemic shunts meld-na (the new meld) and peri-operative outcomes in emergency surgery buggy donal j. pharmacology of anaesthetic agents ii: inhalation anaesthetic agents anaesthesia for patients with liver disease the effects of prolonged lowflow sevoflurane anesthesia on renal and hepatic function blood supply to the liver in the human after 1 mac desflurane in comparison with isoflurane and halothane the effect of isoflurane or propofol anaesthesia on liver injury after partial hepatectomy in cirrhotic patients comparison of the postoperative liver function between total intravenous anesthesia and inhalation anesthesia in patients with preoperatively elevated liver transaminase levels: a retrospective cohort study pain management in patients with cirrhosis analgesics in patients with hepatic impairment: pharmacology and clinical implications sugammadex versus neostigmine after rocuronium continuous infusion in patients undergoing liver transplantation dexmedetomidine reduces intestinal and hepatic injury after hepatectomy with inflow occlusion under general anaesthesia: a randomized controlled trial bile acids induce arrhythmias: old metabolite office for national statistics) paracetamol overdose: an evidence based flowchart to guide management the therapeutic use of analgesics in patients with liver cirrhosis: a literature review and evidence-based recommendations care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper liver injury in covid-19: management and challenges covid-19: abnormal liver function tests pharmacokinetics in disease states modifying hepatic and metabolic function key: cord-291644-5y0ioety authors: akiyama, tomohiro; hirata, takamichi; fujimoto, takahiro; hatakeyama, shinnosuke; yamazaki, ryuhei; nomura, tomohiro title: the natural-mineral-based novel nanomaterial ifmc increases intravascular nitric oxide without its intake: implications for covid-19 and beyond date: 2020-08-29 journal: nanomaterials (basel) doi: 10.3390/nano10091699 sha: doc_id: 291644 cord_uid: 5y0ioety there are currently no promising therapy strategies for either the treatment or prevention of novel coronavirus disease 2019 (covid-19), despite the urgent need. in addition to respiratory diseases, vascular complications are rapidly emerging as a key threat of covid-19. existing nitric oxide (no) therapies have been shown to improve the vascular system; however, they have different limitations in terms of safety, usability and availability. in light of this, we hypothesise that a natural-mineral-based novel nanomaterial, which was developed based on no therapy, might be a viable strategy for the treatment and prevention of covid-19. the present study examined if it could induce an increase of intravascular no, vasodilation and the consequent increase of blood flow rate and temperature in a living body. the intravascular no concentration in the hepatic portal of rats was increased by 0.17 nm over 35.2 s on average after its application. an ultrasonic doppler flow meter showed significant increases in the blood flow rate and vessel diameter, but no difference in the blood flow velocity. these were corroborated by measurements of human hand surface temperature. to our knowledge, this result is the first evidence where an increase of intravascular no and vasodilation were induced by bringing a natural-mineral-based nanomaterial into contact with or close to a living body. the precise mechanisms remain a matter for further investigation; however, we may assume that endothelial no synthase, haemoglobin and endothelium-derived hyperpolarising factor are deeply involved in the increase of intravascular no. humanity is facing a great turning point in terms of its evolution. the novel coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (sars-cov-2) has become a global health emergency. according to the covid-19 situation report published by the world health organization, on 30 may 2020, more than >5.8 million confirmed cases and 362,705 deaths across the world had been attributed to covid-19 [1] . the explosive rise in the there are currently no promising therapeutic strategies for either the treatment or prevention of covid-19, despite the urgent need. while trials on sars-cov-2 genome-based specific vaccines (e.g., messenger ribonucleic acid (mrna)-1273 by moderna, inc.; an adenovirus type-5 vector vaccine called ad5-ncov by cansino biologics, inc.; a dna-based vaccine called ino-4800 by inovio pharmaceuticals, inc. and beijing advaccine biotechnology co.; an adenoviral vector-based vaccine called chadox1 ncov-19 or azd1222 by astrazeneca plc., the university of oxford and its spinout vaccitech ltd.; a lentiviral minigene vaccine called lv-smenp-dc and lentiviral vector modified artificial antigen presenting cells (aapcs) by shenzhen geno-immune medical institute) [29] and therapeutic antibodies are currently being tested, these solutions are more long-term as they require thorough safety testing (e.g., the estimated study completion date of mrna-1273 is 1 june 2021). repurposing existing drugs previously designed for other virus infections and pathologies is also being examined as a rapid response measure to the emergent pandemic as the safety of most of these agents has already been tested. the agents can be divided into two categories depending on their target [14] . one acts directly on the coronavirus, either by inhibiting the crucial viral enzyme responsible for genome replication (rna-dependent rna polymerase inhibitors such as remdesivir, favipiravir, ribavirin, sofosbuvir, galidesivir and tenofovir and viral protease inhibitors such as lopinavir/ritonavir, azithromycin, ivermectin and nelfinavir) or by blocking viral entry to human cells (virus-cell membrane fusion inhibitors such as apn01 (rhace2), chloroquine/hydroxychloroquine, teicoplanin and umifenovir). however, hydroxychloroquine and azithromycin combination could be lethal to covid-19 patients [30] . the other is designed to modulate the human immune system, either by boosting the innate antiviral immune response (immunotherapies using natural killer cells and recombinant interferon) or by alleviating damage induced by dysregulated inflammatory responses (anti-inflammatory therapies using mesenchymal stem cells, intravenous immunoglobulin, anti-c5 monoclonal antibodies (e.g., eculizumab, siltuximab, tzls-501, sarilumab and tocilizumab), anti-vegf-a monoclonal antibody (bevacizumab), anti-tnf monoclonal antibody (adalimumab), sars-cov-2-specific neutralising antibodies, thalidomide, methylprednisolone and fingolimod). passive immunotherapy, using recovered patients' plasma, has been recommended for severe and critical cases of covid-19, based on the presence of neutralising antibodies against sars-cov-2; however, concerns have been raised related to the potential risk for transfusion-transmitted infection and potential risk for severe disease due to antibody-dependent enhancement [31, 32] . various other treatments and prevention strategies are also currently under investigation, including oxygen therapies such as hyperbaric oxygen, nitric oxide (no) and extracorporeal membrane oxygenation, convalescent plasma therapy, anticoagulant therapy, low-dose corticosteroid therapy, ace inhibitors, angiotensin ii type i receptor blockers, statins, antibiotics, polyclonal antibodies and other pharmacological agents. natural compounds including traditional herbal medicines [33, 34] and nutritional interventions such as vitamins, omega-3 fatty acids, selenium, zinc and iron [35] could also be considered for enhancing host immunity against covid-19 infection. the application of big data analysis is also emerging to systematically identify the potential agents for drug repurposing against sars-cov-2 [36] . both agent efficacy and agent safety, including in terms of adverse drug reactions and drug-drug interactions, await further clinical confirmation. previous studies have proven the potential of several countermeasures, yet large-scale trials with a broader perspective remain necessary to respond to covid-19. the present study proposes an additional strategy for the treatment and prevention of covid-19 by applying no. in the vascular system, no is produced by the endothelium, a single layer of cells that forms the inner lining of all blood vessels [37] . endothelium-derived no has several different functions, one of which is vascular smooth muscle relaxation, resulting in vasodilation and a consequent decrease in blood pressure and increase in local blood flow [37] ; these discoveries were awarded the nobel prize in 1998 [38] [39] [40] . previous studies treating sars-cov-infected patients with inhaled no have shown pharmacological and antiviral effects of no [41] [42] [43] , suggesting that no inhalation might be effective for covid-19 patients [37, 44] . indeed, two clinical trials of inhaled no are being conducted in covid-19 patients [45, 46] . meanwhile, balneotherapy (hot spring therapy), sauna therapy and thermal therapy (hyperthermia) also reportedly increase endothelial no synthase (enos), thus inducing vascular endothelial no production [47] [48] [49] [50] [51] [52] . however, hyperthermia may cause health issues such as heat stroke and cardiac morbidity. for example, there are 14,000 deaths in japan per annum during bathing, which accounts for >15% of all sudden out-of-hospital deaths [53] , while there are countries in which neither such therapies nor no inhalation are accessible. one potential breakthrough in terms of overcoming these existing challenges may be nanomedicine or nanoscience, which have been advancing since the 1990s. different classes of nanomaterials such as polymeric nanoparticles or nanocapsules, liposomes, metallic nanoparticles, metal oxide nanoparticles, silica-based nanoparticles, quantum dots, peptide or carbon nanotubes and up-conversion nanoparticles have been used for controlled no delivery in medical applications [54] . however, this requires introducing no-releasing nanomaterials into the human body, and the use of nanomaterials has become controversial due to health risks associated with their applications [55] . meanwhile, a natural-mineral-based novel nanomaterial that is expected to have an effect similar to that of no therapy without its intake into the body has been available since 2018. it was developed from japanese hot springs and named integrated functional mineral crystal (ifmc) by teikoku pharmaceutical co., ltd., japan [56] . if it were able to sufficiently increase intravascular no, it might present a supportive treatment and prevention option for covid-19 since it is inexpensive and easy to use (just putting it or spraying it in a solution on the body). here, we demonstrate how the natural-mineral-based novel nanomaterial can induce an increase of intravascular no, vasodilation and the consequent increase in blood flow. the experimental object of this research is the natural-mineral-based novel nanomaterial ifmc by teikoku pharmaceutical co., ltd., osaka, japan [56] . a solution of ifmc consists of haematite (fe 2 o 3 ), olivine (mg 2 sio 4 and fe 2 sio 4 ), rhodolite (mnco 3 ), zincite (znco 3 ) and additives such as deionized water, ethanol, methylparaben and sodium metabisulphite. the physical characteristics of ifmc were first investigated using a zeta-potential and particle size analyser, as well as scanning electron microscopy (sem) coupled with an energy-dispersive x-ray spectrometer (eds). then, the intravascular no concentration was measured continuously using a no sensor in the hepatic portal of rats to test whether ifmc could induce an increase of intravascular no. since the long-term continuous measurement of intravascular no was impossible, complementary tests were conducted to determine whether ifmc could increase the surface temperature, blood flow rate, velocity and vessel diameter in the human body. all experimental human and animal protocols were respectively reviewed and approved by the medical research ethics committee (approval no. 413) and animal experimentation committee (approval no. 956) of tokyo city university, japan. all participants provided voluntary written informed consent prior to participation. we conducted a monitoring test prior to the present study. no problem was found in the monitoring results when spraying ifmc solution on the body or when wearing clothes impregnated with ifmc over several months. since the naturally occurring substances that make up ifmc are hot-spring ingredients, which are completely different from chemical substances that may have mutagenic properties, we believe there would be no side effects for small animals or humans, even if they were taken directly. the particle size distribution in the ifmc solution was measured using a zeta-potential and particle size analyser (els z-2, otsuka electronics co., ltd., osaka, japan). dynamic light scattering (photon correlation method) and the contin method were used for the measurement. the measured temperature was 22.5 • c when the scattering angle was 165 o . the result showed no scattering intensity. the possible reasons for this include: (1) there were no particles; (2) the particles were too small to measure (els z-2 cannot measure particles smaller than 0.5 nm); (3) substances were present in the solution as ions and were therefore not present as minute particles. we think that mineral components exist as ions in ifmc solution since crystals were obtained when it dried up. sem images of ifmc were taken using an ultra-high-resolution cold-field emission scanning electron microscope fe-sem (su8230, hitachi high-tech co., tokyo, japan), and eds spectra were obtained using an energy-dispersive x-ray spectroscope (aztecenergy x-max150, oxford instruments, oxfordshire, u.k.). ifmc was dropped onto a microscope slide with a pipette and desiccated using a hot plate. the dried deposit was then secured to an fe-sem carbon tape. the measurement conditions of fe-sem were twofold: (1) magnification, ×100-5000; accelerating voltage, 1 kv; (2) magnification, ×100,000-200,000; accelerating voltage, 10 kv. a silicon-drift detector was used to collect fluorescence spectra. a catheter-type no sensor (inc-020, inter medical co., ltd., aichi, japan) was used with an integrated working electrode and a reference electrode (diameter, 0.5 mm; sensitivity, 500 pa µm −1 ). the electric current generated by an electrochemical reaction of no was fed into a head amplifier (haec-a, b ×1/×10 or ×100/×1000, inter medical co., ltd., aichi, japan) and measured using an electrochemical amplifier (imec-601, inter medical co., ltd., aichi, japan) with the voltammetry method. imec-601/601a is currently the most improved measurement system for no, oxygen, dopamine, glutamine acid and vitamin c both in vivo and in vitro [57] [58] [59] [60] [61] . female slc-wistar rats (8-10 weeks) were obtained from japan slc, inc. all rats were housed individually and maintained on an alternating 12 h light/dark cycle at 23 ±1 • c. after a five day acclimatisation period, the rats were randomly divided into positive (use of ifmc solution; n = 2 rats for three samples) and negative control (placebo, i.e., with normal saline solution; n = 2 rats for six samples), with ad libitum access to drinking water. the small number of samples was due to the necessity of special surgical techniques; if this failed, the rats would quickly die due to heavy bleeding. therefore, the present study limited the number of samples to secure the experiment from potential failures. all rats were anaesthetised using a cocktail of butorphanol tartrate (2.5 ml), midazolam (2.0 ml), medetomidine hydrochloride (1.875 ml) and physiological saline solution (3.625 ml) injected intraperitoneally at 2 ml kg −1 body weight and were maintained in their anaesthesia-induced unconscious state throughout the experiment. the no sensor was inserted into the hepatic portal where blood vessels enter and leave the liver (portal vein, hepatic artery), bile ducts, lymph ducts and nerves all pass. this site was chosen because changes in intravascular no concentration can be measured directly and stable measurement of arterial blood flow can also be made without damaging the artery itself. the chests of the rats in both groups were covered by kimwipes r with 5 ml ifmc solution or normal saline solution to test whether ifmc induces an increase of intravascular no. statistical significance was estimated based on the increase of intravascular no concentration. an ultrasonic doppler blood flow meter (qfm-21, hadeco inc., kanagawa, japan) was used to measure the blood flow rate, velocity and vessel diameter of the human brachial artery non-invasively. the measurement conditions were as follows: ultrasonic frequency, 5.0 mhz (blood flow velocity measurement) and 7.5 mhz (blood vessel diameter measurement); ultrasonic output, ≤1500 mw cm −2 (peak time average output), ≤350 w cm −2 (peak pulse output), and ≤550 w cm −2 (maximum output); blood flow range, 0.15-377 ml s −1 ; blood flow rate, 5-120 cm s −1 ; blood vessel diameter, 2-20 mm; depth, 4-35 mm; blood flow rate measurement accuracy, ±10%. the research participants were four males aged 21-25 years. the measurements were conducted 22 times in total. the upper arm of each participant was covered by ifmc-added or ifmc-free textiles (100% polyester) to test whether ifmc could increase the blood flow rate, velocity and vessel diameter of the human body. statistical significance was determined using a two-sided paired t-test. the research participant was a 22-year-old female who felt coldness of the hands and feet on a regular basis. the measurement was started right after the solution of ifmc was sprayed on the surface of the participant's hand. changes in the hand surface temperature were measured using an auto-range thermo tracer (th9100mr, nippon avionics co., ltd., tokyo, japan) at 1 min intervals during a total measurement period of 10 min. statistical significance was determined based on pixel values using a two-sided paired t-test. and then gradually decreased. the peak value and duration of the increase were comparable to those of other reports, which used acetylcholine [62] [63] [64] [65] . the lower peak value and shorter duration of the increase in this study were probably due to the small size of the rats. the minor spikes right before the increase were probably because of the relaxation of the vascular smooth muscle. indeed, a similar spike was observed when the sensor was moved slightly. the result is, to our knowledge, the first evidence of inducing an increase of intravascular no by bringing the natural-mineral-based nanomaterial into contact with or close to a living body without pharmacological intervention or physical intervention (e.g., acupuncture, massage therapy or balneotherapy). figure 4 shows changes in the blood flow rate, velocity and vessel diameter in the brachial artery. the ranges of the systolic and diastolic peaks of each parameter were comparable to the values of another report [66] . compared with ifmc and placebo applications, significant increases were observed in blood flow rate and vessel diameter, but there was no significant difference in blood flow velocity. statistical significance values of blood flow rate, velocity and vessel diameter between placebo and ifmc applications were 0.0188, 0.1911 and 0.0387, respectively. the significant increases in blood flow rate and vessel diameter were due to vasodilation derived from the increase in intravascular no. the minor change in blood flow velocity was due to the improvement of vascular elasticity followed by relaxation of vascular smooth muscle. the result (i.e., the increase of the blood flow rate due to vasodilation without blood flow velocity change) indicates improvement of the neuroimmunoendocrine system [67] [68] [69] . figure 5 shows changes in the surface temperature of a hand (back) observed by thermography. before spraying the ifmc solution, the mean surface temperature of the entire hand (back) was 30.5 • c, and that of the fingers was 30.5 • c, indicating that the peripheral circulation was poor. after 1 min, the surface temperature decreased due to vaporisation. however, 2 min later, some of the relatively thick blood vessels in the hand began to swell, and after 3 min, almost all of the thick blood vessels on the back of the hand stood out. meanwhile, after 3 min, the temperature of the peripheral blood vessels in the fingertips increased. the surface temperature of the area close to the blood vessels rose. the temperature of the entire hand had increased at 5 min. the clear behavioural transition from thick to thin blood vessels was obviously due to vasodilation. the mean surface temperature of the entire hand (back) was 30.9 • c and that of the fingers 31.2 • c at 10 min. the differences in the surface temperature of the entire hand and fingers before and 10 min after ifmc application were significant. the increase in the fingers (+0.9 • c) was larger than that in the entire hand (+0.4 • c). the difference is evidence of vasodilatation and the increase of blood flow rate in the peripheral blood vessels. the present study confirmed that the natural-mineral-based novel nanomaterial ifmc, with a size of tens of nanometres (figure 1 ), could induce an increase of intravascular no (figure 3) , vasodilation (vessel diameter) and blood flow rate in a living body (figure 4) , as well as an increase of the surface temperature of a hand including fingers ( figure 5 ). despite the lower peak value and shorter duration of the increase in intravascular no concentration (figure 3) due to the small size of the rats, which were comparable to those of other reports [62] [63] [64] [65] , the increase of human hand surface temperature lasted at least 10 min (figure 5) , suggesting that the effect of ifmc was significant. further, the fact that the increase of the mean surface temperature of the fingers (+0.9 • c) was larger than that of the entire hand (+0.4 • c) at 10 min after ifmc application ( figure 5 ) suggested that the vasodilatation and increase of blood flow rate in the peripheral blood vessels were caused by no as an endothelium-derived relaxing factor (edrf). this is corroborated by the increase in the diameter of blood vessels in the brachial artery and the increase in blood flow ( figure 4) . the results are, to our knowledge, the first evidence of inducing an increase of intravascular no and vasodilation by bringing the natural-mineral-based nanomaterial into contact with or close to a living body, without pharmacological intervention or physical intervention (e.g., acupuncture, massage therapy or balneotherapy). the precise mechanism of the induction of intravascular no remains uncertain; however, there are two possibilities. the primary possibility is no synthesis. it is well known that the activity of enos followed by no production in the vascular wall is regulated by receptor-mediated signalling, such as vascular endothelial growth factor receptor 2 (vegfr2) and acetylcholine (ach), as well as protein partners, including heat shock protein 90 (hsp90), calmodulin (cam) and caveolin-1 (cav-1) [70] . the receptor-mediated biological response includes a time lag between actions of chemical mediators (messenger substances) and receptor reaction. in contrast, ifmc caused its effect within 1 min (figures 2 and 5) . the quick response indicates the possibility that ifmc principally involves another mechanism, not receptor-mediated action. therefore, we assume haemoglobin (hb) is deeply involved in the phenomenon. it is known that hypoxic vasodilation does not require the synthesis of edrf/no [71] . hb and no independently fulfil diverse and complex physiological roles, while together, they subtly modulate microvascular perfusion in response to second-by-second changes in local metabolic demand, contributing to hypoxic vasodilation [72] . among the candidate molecular mechanisms, only s-nitrosohemoglobin (sno-hb) directly fulfils the physiological requirements [72] . thus, no is transported by red blood cells to microvascular sites of action in protected form as an s-nitrosothiol on the highly conserved hb β-93 cys residue, invariant in birds and mammals [72] . meanwhile, no as a vascular edrf is a gaseous signal substance and considered to be freely diffused between vascular endothelial cells and vascular smooth muscle cells. however, no does not freely diffuse between the endothelial cells and smooth muscle cells of blood vessels [70, 73] . the oxidation state of hb is harnessed to control how much no reaches the smooth muscle [74] . hb has the structure of haem iron with four iron porphyrin units combined with a globin protein. hb has the ability to combine with oxygen molecules (o). therefore, hb not only plays a role of transporting oxygen (o 2 ), but also is deeply involved in the control of no transport. it is well known that the transport of o 2 and no in blood is facilitated by the spin transition of iron atoms in hb. the ferrous ions in haem iron are five-coordinated high-spin complexes; however, they become six-coordinated low-spin complexes when oxygen molecules are added. ferrous ions move to the centre of the porphyrin ring by changing the spin state of d-electrons from high to low spin. this movement at the molecular level changes the structure of hb to enhance the affinity or adsorbability of the other three haem-iron complexes to oxygen molecules. in other words, the electronic structure of d-electrons controls the uptake and release of oxygen molecules. therefore, we may form the following hypothesis: the affinity of hb for no might be reduced by bringing ifmc into contact with or close to a living body. in light of this, magnetic evaluation using a magnetic probe such as a positive muon may clarify the mechanism of action by ifmc on hb. indeed, the mixture of hb as an organic compound and ifmc as an inorganic nanomaterial, which contains transition metal compounds exhibiting antiferromagnetism and weak ferromagnetism, may trigger several different types of interactions. one of the potential methods of clarifying the mechanism is to estimate the magnitude of the internal magnetic field that the muon experiences when ifmc is irradiated with muon beams. specifically, we would be able to examine the relation between the spin properties of ifmc through muon experiments using a high-energy accelerator and the spin transition changes of iron atoms involved in the changes in the affinity or adsorbability to the o/no molecule of haem iron in hb. through the experiments, it would be possible to examine ifmc's remote action or action over a distance between atoms and atoms or between atoms and molecules. in the fields of semiconductor engineering and spintronics, there are previous studies claiming that simple antiferromagnetic insulators may transmit spin information over a distance of several tens of micrometres [75] . the proposed examination might be the world's first experiment to uncover the spin transmission over a much longer distance. meanwhile, the potential benefits of ifmc are probably not limited to the induction of intravascular no. it is known that there are three different endothelium-derived vasorelaxation factors involved in maintaining vascular homoeostasis: prostacyclin (pgi2), no [38] [39] [40] 76] and endothelium-derived hyperpolarising factor (edhf) [77] . they are all derived from vascular endothelium, whereas they differ in their production processes: no is produced from arginine by enos; edhf is synthesised by a variety of stimulants, such as agonists, shear stress and acetylcholine in a ca 2+ /calmodulin-dependent manner [78] ; and prostacyclin is produced by cyclo-oxygenase and prostacyclin synthase enzymes [79] . it has been shown that no and edhf fulfil complementary roles. no mediates vascular relaxation of relatively large conduit arteries (e.g., aorta and epicardial coronary arteries), whereas edhf plays an important role in modulating vascular tone in small, resistance arteries (e.g., small mesenteric arteries and coronary microvessels) [80] . the vascular tone can be regulated by an edhf-like mechanism in the absence of no [81] . the clear behavioural transition from thick to thin blood vessels (figure 4) suggests that ifmc influences not only the induction of intravascular no, but also the production of edhf. in addition, the effect of ifmc on neuroimmunoendocrine system improvement, indicated by the increase of blood flow rate due to vasodilation without blood flow velocity change ( figure 5) , is also worthy of further examination. thus, the precise mechanism and other potential benefits of ifmc are possible future research topics. however, the limited findings of the present study provide insight into the treatment and prevention of covid-19. the increase of intravascular no (figure 3) can be expected to have both pharmacological and antiviral effects. the pharmacological actions of no are twofold: vascular and nonvascular smooth muscle relaxation and regulation of airway function and the pathophysiology of inflammatory airway disease [82] . no has also been shown to have antiviral actions, such as an inhibitory effect on the synthesis of viral protein and rna, including the replication cycle of sars-cov [41] [42] [43] . in general, the action mechanism of inhaled no has been thought to be pulmonary vasodilation and the consequent improved oxygenation in the blood of the lungs, which kills the virus, as it does not do well in a high-oxygen environment. ignarro further added that no also interacts directly with the virus to kill it and/or inhibit its replication, and no inhalation is more effective as an antiviral agent than simply as a vasodilator in pulmonary circulation [37] . the proposed natural-mineral-based novel nanomaterial might be added as an supportive treatment and prevention option for covid-19, since it is inexpensive and easy to use (just putting it or spraying it in a solution on the body). given its advantages, it has the potential to overcome the limitations of inhaled no therapy, such as the cost and availability of devices and skilled medical staff. the health risks of the existing no-releasing medical nanomaterials might no longer be a matter of concern, since ifmc does not require intake into the human body. it is probable that the increase of intravascular no concentration ( figure 3) is not abnormal; however, quantitative examination of the increase and its persistence in the human body is necessary. nevertheless, there is still an urgent need for promising therapy strategies for both the treatment and prevention of covid-19. to summarise, our inter-and trans-disciplinary approach revealed that the natural-mineral-based novel nanomaterial ifmc can induce an increase of intravascular no, vasodilation and blood flow rate, as well as an increase of hand surface temperature in a living body. therefore, it might become a supportive treatment and prevention option for covid-19. the precise mechanisms remain a matter for further investigation; however, we may assume that enos, hb and edhf are deeply involved in the increase of intravascular no. the remaining research agendas are to (1) examine if ifmc can induce an increase of intravascular no and improve long-term blood circulation in humans, (2) clarify how intravascular no is induced by bringing ifmc into contact with or close to a living body, (3) examine ifmc's remote action or action over a distance between atoms and atoms or between atoms and molecules, (4) explore other potential benefits of ifmc, (5) explore the possibility of other natural-mineral-based nanomaterials, (6) clinically examine whether ifmc is effective in the treatment and prevention of covid-19 and (7) explore urgently and collectively a series of promising therapy strategies for both the treatment and prevention of covid-19 based on larger scale trials from a broader 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crystalline antiferromagnetic iron oxide no news is good news acetylcholine releases endothelium-derived hyperpolarizing factor and edrf from rat blood vessels important role of endothelium-derived hyperpolarizing factor in shear stress-induced endothelium-dependent relaxations in the rat mesenteric artery milrinone enhances relaxation to prostacyclin and iloprost in pulmonary arteries isolated from lambs with persistent pulmonary hypertension of the newborn crucial role of nitric oxide synthases system in endothelium-dependent hyperpolarization in mice endothelium-derived hyperpolarizing factor and vascular function acknowledgments: microanalyses (sem and eds) of ifmc were conducted by dr. a. yoshida, ms. e. shindo and mr. n. hamamura (interdisciplinary research center for nano science and technology, tokyo city university, japan). other measurements, including intravascular no concentration of rats, surface temperature, blood flow rate, velocity and vessel diameter of the human body, were carried out by prof. a. mori (graduate school of integrative science and engineering, electrical engineering and chemistry tokyo city university, japan). the authors declare no competing interests. the following abbreviations are used in this manuscript: key: cord-323906-ro078y52 authors: sardu, celestino; marfella, raffaele; maggi, paolo; messina, vincenzo; cirillo, paolo; codella, vinicio; gambardella, jessica; sardu, antonio; gatta, gianluca; santulli, gaetano; paolisso, giuseppe title: implications of ab0 blood group in hypertensive patients with covid-19 date: 2020-08-14 journal: bmc cardiovasc disord doi: 10.1186/s12872-020-01658-z sha: doc_id: 323906 cord_uid: ro078y52 background: hypertension is the most frequent co-morbidity in patients with covid-19 infection, and we might speculate that a specific blood group could play a key role in the clinical outcome of hypertensive patients with covid-19. methods: in this prospective study, we compared 0 vs. non-0 blood group in hypertensive patients with covid-19 infection. in these patients, we evaluated inflammatory and thrombotic status, cardiac injury, and death events. results: patients in non-0 (n = 92) vs. 0 blood group (n = 72) had significantly different values of activated pro-thrombin time, d-dimer, and thrombotic indexes as von willebrand factor and factor viii (p < 0.05). furthermore, patients in non-0 vs. 0 blood group had higher rate of cardiac injury (10 (13.9%) vs. 27 (29.3%)) and death, (6 (8.3%) vs. 18 (19.6%)), (p < 0.05). at the multivariate analysis, interleukin-6 (1.118, ci 95% 1.067–1.171) and non-0 blood group (2.574, ci 95% 1.207–5.490) were independent predictors of cardiac injury in hypertensive patients with covid-19. d-dimer (1.082, ci 95% 1.027–1.140), interleukin-6 (1.216, ci 95% 1.082–1.367) and non-0 blood group (3.706, ci 95% 1.223–11.235) were independent predictors of deaths events in hypertensive patients with covid-19. conclusions: taken together, our data indicate that non-0 covid-19 hypertensive patients have significantly higher values of pro-thrombotic indexes, as well as higher rate of cardiac injury and deaths compared to 0 patients. moreover, ab0 blood type influences worse prognosis in hypertensive patients with covid-19 infection. hypertension is the most common co-morbidity and cause of death in patients with covid-19 infection [1] . such a negative correlation between hypertension and clinical prognosis in covid-19 patients has been deeply investigated in recent trials [1, 2] . angiotensin converting enzyme 2 (ace2), known to be involved in the molecular pathways underlying hypertension, is a crucial co-factor mediating sars-cov-2 entry into host cells [2] . indeed, the spike proteins of sars-cov-2 have a high binding affinity for ace2, which are mainly expressed in endothelial cells of the lung and the upper airways [3] . moreover, angiotensin converting enzyme inhibitors (acei) and angiotensin receptor blockers (arb) have been shown to up-regulate ace2 levels, which partially mediates their cardiovascular protective effects [4] . nevertheless, according to the recent evidence, acei/arb therapy does not seem to increase the risk of covid-19 infection in hypertensive patients [4] . secondly, acei/arb therapy discontinuation is not recommended, because it may lead to endothelial dysfunction [4] . actually, endothelial dysfunction itself, mirrored by hyper-inflammation could cause alterations of the coagulation thereby aggravating the prognosis of the disease [2, 3] . the ab0 blood group has been previously shown to play a functional role in viral infections [5, 6] . intriguingly, patients with non-0 blood group have higher risk for covid-19 infection when compared to 0 blood groups [7] , and the ab0 blood group could influence the coagulation processes [8, 9] . however, the pathogenic mechanisms underlying these events have not been fully investigated, and could be of great interest to the scientific community and for clinical applications. thus, we hypothesized that non-0 blood group could be a trigger of endothelial dysfunction, via overinflammation and promoting a pro-thrombotic status in hypertensive patients with covid-19. actually, although hypertension is known to trigger endothelial dysfunction and a pro-thrombotic status [9] , no data are currently available exploring the association of ab0 group with inflammatory/thrombotic status in hypertensive patients with diagnosis of covid-19.therefore, in this study we investigated the inflammatory/thrombotic status and clinical outcomes as cardiac injury and death in hypertensive patients with covid-19,comparing 0 vs. non-0 blood groups. in this prospective study, we analyzed covid-19 hypertensive patients consecutively admitted to the department of infectious disease at university of campania "luigi vanvitelli", naples, italy between february 10, 2020 and april 20, 2020. covid-19 infection was categorized as follow: a) mild, patients with fever and no pneumonia evidence in imaging; b) moderate, patients with fever, respiratory tract symptoms, pneumonia confirmed at imaging without the need for invasive ventilation; c) critical, occurrence of respiratory failure requiring mechanical ventilation, presence of shock, other organ failure requiring monitoring and treatment in intensive care unit [1] . patients with previous inflammatory disorders, malignancy, renal diseases; unavailability of a written informed consent; patients without cardiac biomarkers evaluation, including values of high-sensitivity troponin i (hs-tni) and creatinine kinase-myocardial band (ck-mb). the diagnosis of hypertension was made following the international guidelines [10] , and/or by known history of hypertension and current anti-hypertensive therapy. all enrolled patients were treated with the same standard protocol: non-invasive oxygen therapy; hydroxychloroquine (400 mg/daily) and lopinavir/ritonavir (200/ 50 mg daily). according to ab0 blood group, patients were then categorized as "0 and non-0 blood group", [6, 7] . established cardiac biomarkers, including hs-tni, ck-mb, and myo-hemoglobin, were collected for every participant at hospital admission by 2 investigators (v.m. and c.s.).the investigation conforms to the principles outlined in the declaration of helsinki for the study of human subjects or tissues. the institutional ethics committee of the university of campania "luigi vanvitelli" approved the study protocol. written informed consent was obtained from all participating patients. in this study, we investigated the inflammatory and coagulative status, and the cardiac injury and deaths in hypertensive patients with covid-19, with the aim to compare 0 vs. non-0 blood group. cardiac injury and death were reported in a previous study for patients with covid-19 [11] . cardiac injury was defined as blood levels of cardiac biomarkers (hs-tni) above the 99thpercentile upper reference limit (11) . data on cardiac injury and death were collected by two independent physicians (p.m; r.m) during clinical examination, laboratory and imaging tests in hospitalized patients, and by examination of hospital discharge schedules [11] . laboratory and imaging evaluations -real-time reverse transcription (rt-pcr assay for sars-cov-2 respiratory specimens were collected from each patient and then shipped to specialized laboratories designated by the italian government for confirming covid-19 infection. the presence of sars-cov-2 in respiratory specimens was detected by established rt-pcr methods. laboratory analyses were obtained on admission before starting covid-19 medical therapy and during hospitalization. we tested respiratory specimens, including nasal and pharyngeal swabs or sputum, to exclude evidence of other viral infections, including influenza, respiratory syncytial virus, avian influenza, para-influenza, and adenovirus. we also performed routine bacterial and fungal examinations. laboratory assessments consisted of a complete blood count, blood chemical analysis, coagulation testing, evaluation of liver and renal function, and measures of electrolytes, c-reactive protein, procalcitonin, lactate dehydrogenase, and creatine kinase. venous blood for il-6 (human elisa kit, rd system) and d-dimer (human elisa kit, invitrogen) levels was collected in edta-coated tubes immediately after patients arrived at the department and weekly during hospitalization. the ab0 phenotypes were ascertained by genotyping for four single nucleotide polymorphisms of the ab0 gene: g261del, a297 g, g703a and c526g, as described [9] . briefly, we used single nucleotide polymorphisms of the c526g to decipher the o303 allele, which, unlike other o alleles, does not have a deletion at nucleotide position 261 [9] . we determined genotyping by using the multiplexing capability of the massarray homogenous massextend assay of the sequenom system (san diego, ca, usa). therefore, the dna fragments surrounding the single nucleotide polymorphisms sites were amplified by pcr, treated with shrimp alkaline phosphatase to dephosphorylate unincorporated dntps, followed by the extension primers that form allelespecific extension products. however, each extension product had a unique mass, measured using maldi-tof. genotypes were automatically assigned to each sample using the mass array rt software. the presence or absence of fv leiden (a1691 g, r506q) and the prothrombin g20210a polymorphism was assessed by standard methods [9] . all patients underwent ecg at hospital admission, and in case of elevation of cardiac biomarkers during hospitalization; findings compatible with myocardial ischemia included t-wave depression and inversion, st-segment depression, and q waves. two blinded physician (c.s, r.m) reviewed and analyzed ecg patterns. radiologic assessments included chest radiography and/or computed tomography (ct) at admission and weekly during hospitalization, and all laboratory testing was performed according to the clinical care needs of each patient. we determined the presence of radiologic abnormalities on the basis of the documentation or description in medical charts; if imaging scans were available, they were reviewed by attending physicians in respiratory medicine who extracted the data. two blinded physician experienced in lung imaging (g.g, v.c.) reviewed and analyzed chest radiography and ct patterns. major disagreement between two reviewers was resolved by consultation with a third reviewer. continuous variables were expressed as medians and interquartile ranges or simple ranges, as appropriate. categorical variables were summarized as counts and percentages. we performed only descriptive statistics, because the cohort of patients in our study was not derived from random selection. we performed a risk adjusted cox-regression analysis to assess survival from cardiac injury and deaths through days of hospitalization; cox models were adjusted for; age, gender, body mass index, heart rate, cholesterol, high density lipoproteincholesterol, low density lipoprotein-cholesterol, triglycerides levels, heart diseases, dyslipidemia, diabetes, current smoking, beta-blockers, ace-inhibitors, calcium inhibitors, thiazide diuretics, aspirin. only variables presenting a p value ≤0.25 at the univariate analysis were included in the model. we used a stepwise method with backward elimination, and we calculated odds ratios (or) with 95% confidence intervals. the model was evaluated with a hosmer and lemeshow test. kaplan-meier survival analysis was performed for cardiac injury events and deaths in patients divided in: 0 vs. non-0 blood group. a p value < 0.05 was considered statistically significant. all calculations were performed using the software spss23. we enrolled 164 hypertensive covid-19 patients; the study population was then divided according to the ab0 blood group in0 (n = 72) vs. non-0 (n = 92). the main clinical characteristics of our population are shown in table 1 . comparing 0 vs. non-0 blood group, we found significantly different values of activated pro-thrombin time, d-dimer, and thrombotic indexes including activated pro-thrombin time, von willebrand factor (vwf) and factor viii (p < 0.05). patients in non-0 vs. 0 blood group had higher rate of cardiac injury [10 (13.9%) vs. 27 (29.3%)] and deaths [6 (8.3%) vs. 18 (19.6%)], (p < 0.05) as shown in table 1 . cardiac injury was diagnosed in 13 (54%) of patients. then, we performed a multivariate analysis, which revealed that interleukin-6 (il-6, 1.118, ci 95% 1.067-1.171) and non-0 blood group (2.574, ci 95% 1.207-5.490) were identified as independent predictors of cardiac injury in hypertensive patients with covid-19 (table 2) . moreover, at multivariate analysis, d-dimer (1.082, ci 95% 1.027-1.140), il-6 (1.216, ci 95% 1.082-1.367) and non-0 group (3.706, ci 95% 1.223-11.235) were identified as independent predictors of death in hypertensive patients with covid-19 (table 3) . finally, we analyzed kaplan curves of survival ( fig. 1) , observing a significant (p < 0.05) difference between o vs. non-0 hypertensive patients with covid-19 in terms of cardiac injury (upper panel) and death (lower panel). from the analysis of hypertensive patients with covid-19 infection, the main study results are: i) non-0 vs. 0 patients have significant higher values of pro-thrombotic indexes; ii) non-0 vs. 0 patients have higher rate of cardiac injury; iii) non-0 vs. 0 patients have increased rate of deaths (p < 0.05); iv) il-6 level is an independent predictor of cardiac injury and death; v) d-dimer is an independent predictor of death; vi) non-0 group is an independent predictor of both cardiac injury and deaths in hypertensive patients with covid-19. in our study, non-0 patients had higher values of prothrombotic indexes, and higher rate of cardiac injury and death. of interest, the ab0 blood type has been previously shown to influence the hemostasis by increasing vwf and fviii blood levels, as well as by genetic variations and over-inflammation, that can lead to thrombosis independently of factor viii [12] . notably, non-0 blood group can influence the traditional risk factors for arterial or venous thrombotic events [12, 13] ; besides, in patients with sepsis the non-0 blood group increases the risk for disseminated intravascular coagulopathy (dic) independently from disease severity [12] . equally important, endothelial dysfunction in hypertensive patients can cause cardiac injury and stroke via thromboembolism [13] . therefore, these events could underlie the worse prognosis in patients with hypertension and covid-19. indeed, we have shown that covid-19 infection could affect endothelial cell function leading to thrombotic complications [3] . according to the data presented here, the extent of endothelial dysfunction present in this class of patients could be further enhanced by a pro-thrombotic status in non-0patients [7] . hence, hypertension could confer a pro-thrombotic state and over inflammation in covid-19 patients, that could be , and myohemoglobin (μg/l), ast (mg/dl), alt (mg/dl), ck-mb (mg/dl) and ldh (mg/dl) as means ± standard deviations. the means for continuous variables were compared using independent group t tests when the data were normally distributed (normal distribution verified applying the kolmogrov-smirnov test), otherwise, the mann-whitney test was used. the pearson correlation coefficient and spearman rank correlation coefficient were used for liner correlation analysis. proportions for categorical variables were compared using the χ 2 test, whereas the fisher exact test was used when data were limited. wilcoxon rank sum matched-pair tests were used to assess differences among the admission, hospitalization, and impending death. a 2-sided p < .05 was considered statistically significant. analysis began february 29, 2020 ami acute myocardial infarction, cabg coronary artery bypass grafting, ptca percutaneous coronary angioplasty, pt pro-thrombin time, aptt activated prothrombin time, ast aspartate amino transferase, alt alanine amino transferase, ck-mb creatinine kinase-myocardial band, ldh lactate dehydrogenase, bnp b type natriuretic peptide, hb1ac glycated hemoglobin, pao2/fio2 pressure of arterial oxygen to fractional inspired oxygen concentration, hs high specificity, lvtdd left ventricle end-diastolic diameter, lvtsd left ventricle end-systolic diameter, lvef left ventricle ejection fraction, * is for statistical significant (p < 0.05) then increased in non-0 blood [2] [3] [4] [5] [6] . in line with this view, il-6, a widely recognized marker of inflammation, is up regulated in non-0 vs. 0 covid-19 patients, and independently predicts cardiac injury and death. indeed, il-6 plays a crucial role in the cytokine release syndrome [14] . thus, the increased il-6 levels detected in covid-19 patients could result in worse prognosis and death [14] . therefore, the therapeutic block of il-6 mediated signal transduction pathway by tocilizumab, has been proposed as an effective rescue treatment in severely ill covid-19 patients [14, 15] . hence forth, il-6 serum levels could be used as marker of disease severity, such as predictor of worse prognosis for non-0 vs. 0 blood group of hypertensive patients with covid-19. in addition, assaying for the d-dimer could be used to add other predictive information on the risk of death in non-0 vs. 0 hypertensive patients with covid-19. consistent with our findings, a d-dimer greater than 1 μg/ml has been proposed to help clinicians in identifying patients with poor prognosis at an early stage of covid-19 disease [16] . indeed, augmented d-dimer level is a marker of enhanced thrombosis in patients with covid-19, [16, 17] . thus, the functional association linking d-dimer, thrombosis, fibrinolysis and poor prognosis could be extended to hypertensive patients with non-0 blood group, identifying these patients as individuals at high risk of a severe outcome following covid-19 infection. finally, in hypertensive patients all these adverse events could be seen as complications of an increased inflammation, thrombosis, and fibrinolysis [18] , all phenomena that are particularly enhanced in patients with non-0 blood group [19] . indeed, ab0 blood group could cause a higher susceptibility to severe acute respiratory syndrome [19] , leading to the development of neutralizing antibodies against protein-linked n-glycans [20] and acting on the stabilization of vwf [21] . intriguingly, in a recent genomic study, authors identified a 3p21.31 gene cluster as a genetic susceptibility locus in covid-19 patients with respiratory failure [21] . moreover, the ab0 blood-group showed a potential involvement in covid-19 disease, with a protective effect for blood group 0 as compared with the other blood groups [22] . consequently, in the non-0 group all these adverse events could cause an increased rate of cardiac injury and death in hypertensive patients. in this regard, including elevations in serum biomarkers of cardiac damage, the standard ecg may represent a crucial test in the diagnosis of myocardial injury or heart rhythm disturbances in patients with covid-19 [23] . indeed, ecg abnormalities, independently from the severity of pulmonary tract infection, could reflect a wide spectrum of cardiovascular complications and frequently occur after negative nasopharyngeal swabs [23] . however, ecg abnormalities of covid-19 are still undefined, particularly during the acute phase of the disease [23] . in this sense, it is critical to note that we found that the non-0 blood group results in 2.6-fold and 3.7-fold increased risk to develop cardiac injury and death in hypertensive patients with covid-19. our study is not exempt from limitations. for instance, we did not report data on magnetic resonance imaging or echocardiography to determine the features of myocardial injury. however, we diagnosed cardiac injury by evaluation of hs-tni serum increase and ecg findings. thus, we cannot have definitive data and evidence about the mechanisms of covid-19 directly heart injury. thereby, this aspect requires further studies in order to be confirmed. again, we did not evaluate effects of 0 vs. non-0 blood group in non-hypertensive covid-19 patients, that could be seen as control group and could limit the generalization of the present study results in overall population. taken together, our data indicate that covid-19 associated coagulopathy should be carefully managed in hypertensive patients with non-0 group as critically ill patient because such association could result in an increased risk of unfavorable outcomes as cardiac injury and death via inflammatory and hyper-coagulative mechanisms. therefore, we speculate that targeted anticoagulant therapies have to be introduced early in these high-risk covid-19 patients, namely hypertensive individuals with non-0 blood group, in order to reduce cardiac injury and death. further studies conducted on larger populations are needed to confirm these results. clinical characteristics of coronavirus disease2019 in china hypertension, thrombosis, kidney failure, and diabetes: is covid-19 an endothelial disease? a comprehensive evaluation of clinical and basic evidence negative impact of hyperglycaemia on tocilizumab therapy in covid-19 patients could anti-hypertensive drug therapy affect the clinical prognosis of hypertensive patients with covid-19 infection? data from centers of southern italy human susceptibility and resistance to norwalk virus infection abo blood group and susceptibility to severe acute respiratory syndrome relationship between the abo blood group and the covid-19 susceptibility. medvis preprint abo blood group, other risk factors and incidence of venous thromboembolism: the longitudinal investigation of thromboembolism etiology (lite) the abo blood group system revisited: a review and update acc/aha/aapa/abc/acpm/ags/ apha/ash/ aspc/nma/pcna guidelines for the prevention, detection, evaluation, and management of high blood pressure in adults: a report of the american college of cardiology/american heart association task force on clinical practice guidelines association of cardiac injury with mortality in hospitalized patients with covid-19 in wuhan, china advance in the management of sepsis-induced coagulopathy and disseminated intravascular coagulation hypertension and the prothrombotic state the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality negative impact of hyperglycaemia on tocilizumab therapy in covid-19 patients clinical course and risk factors for mortality of adult in patients with covid-19 in wuhan, china: a retrospective cohort study covid-19 and its implications for thrombosis and anticoagulation outcomes in patients with hyperglycemia affected by covid-19: can we do more on glycemic control? diabetes care abo blood group and susceptibility to severe acute respiratory syndrome harnessing the natural anti-glycan immune response to limit the transmission of enveloped viruses such as sars-cov-2 abo blood group is a determinant of von willebrand factor protein levels in human pulmonary endothelial cells genomewide association study of severe covid-19 with respiratory failure electrocardiographic features of patients with covid-19 pneumonia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we thank all physicians those who have helped in carrying out this research.authors' contributions c.s: study design, data interpretation, major contributor in writing the manuscript and revision; r. m and g.p.: study editing and revision; p. m, v. m, p. c, v. c, j. g, a. s, g.g: data collection; g. s: study revision. all authors read and approved the final manuscript. the santulli's lab is supported in part by the nih (r01-dk123259, r01-hl146691,r01-dk033823, and r00-dk107895 to g.s.) and by the american heart association (aha-20post35211151 to j.g.). the funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.availability of data and materials the datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.ethics approval and consent to participate the institutional ethics committee of the university of campania "luigi vanvitelli" approved the study protocol. written informed consent was obtained from all participating patients. competing interests none to declare. received: 9 may 2020 accepted: 6 august 2020 key: cord-273388-615acz0l authors: he, miao; wang, jingxing; chen, limin; liu, jing; zeng, peibin title: the impact of emerging infectious diseases on chinese blood safety() date: 2016-11-04 journal: transfus med rev doi: 10.1016/j.tmrv.2016.10.002 sha: doc_id: 273388 cord_uid: 615acz0l emerging infectious diseases (eids) have always been one of the major threats to public health. although the implementation of mandatory testing for 4 classical transfusion-transmitted infectious—human immunodeficiency virus, hepatitis b virus, hepatitis c virus, and syphilis—has reduced the transfusion risk of these pathogens, the potential threat of various eid agents and their constantly evolving variants to blood safety in china is not fully understood. this review presents 9 representative eid agents that are autochthonous and epidemic nationally or regionally in china. the epidemiologic status and distribution of these eid agents among donors and/or healthy populations are summarized. the potential risks of these eid agents to blood safety are discussed. the review also explores strategies to strengthen hemovigilance systems and studies to further evaluate the impact of eid agents on blood safety. emerging infectious diseases (eid) agents are considered as major threats to transfusion safety. the most notorious eid agent that sabotaged blood safety during 1980s was human immunodeficiency virus (hiv). it raised global concerns of eids and triggered organized activity to systematically prevent eid agents from threatening transfusion safety. nowadays, blood donations are screened for various infectious agents in developed countries or regions where eid agents have been well studied. in the united states, the blood supply is routinely screened for human t-lymphocyte virus (htlv). west nile virus (wnv); trypanosoma cruzi [1] [2] [3] and babesia spp. [4] [5] [6] [7] have been systematically scrutinized to evaluate the value of donor screening [8] ; and zika virus has recently emerged as an eid threat [9] . in china, however, many of the eid agents are rarely investigated among blood donor and evidence for their impact on blood safety is absent. in 2009, the american association of blood banks published a catalog of pathogens relevant to blood transfusion medicine reviews j o u r n a l h o m e p a g e : w w w . t m r e v i e w s . c o m safety in which 68 eid agents were listed as confirmed or suspected to be associated with transfusion transmissible infection (tti) [10] . the threat of these eid agents to blood safety varies, due to different spreading patterns, transmission routes, epidemiologic characteristics, and endemic status; therefore, they need to be specifically evaluated in each country or area. in china, blood donors are routinely tested for only 4 pathogens: hiv-1/2, hepatitis b virus (hbv), hepatitis c virus (hcv), and syphilis [11] . with the broad implementation of nucleic acid testing (nat), the improvement in performance of enzyme immunoassay assays, and the more rigorous policies for donor recruitment and testing, the transfusion risks of the conventional ttis have been largely reduced in china [11, 12] . however, many of the diversely distributed eid agents still pose potential threats to blood safety, yet their risks have never been fully evaluated. nevertheless, china has witnessed recurrent outbreaks of highly virulent eids including severe acute respiratory syndrome (sars), highly pathogenic avian influenza (h5n1) and streptococcus suis, a zoonotic bacterium mostly carried by pigs or pork products which causes syndromes of streptococcal toxic shock, sepsis and meningitis. [13] . for example, outbreaks of human streptococcus suis infection in china were reported in sichuan province, resulting in 66 laboratory confirmed cases and 39 deaths from mid-july to the end of august 2005 [14] . some eid agents with asymptomatic distribution among chinese general population, such as malaria, hepatitis e virus, and dengue virus may emerge as greater threats to chinese blood safety and require interventions. in this review, we highlight 9 representative, autochthonous eids agents that are nationwide or regionally epidemic. the epidemiology of these eid agents and the investigations among blood donors and/or the general population are reviewed. the geographic distribution for eid agents that are regionally epidemic is summarized, and the prevalence of these eid agents among chinese donors is summarized. human parvovirus b19 (b19v) is a small, non-enveloped, singlestrand dna virus [15] , causing various clinical manifestations such as chronic anemia, aplastic crises and arthropathies, and a variety of other syndromes among immune-compromised or immunosuppressed patients [16] [17] [18] [19] . b19v has been confirmed to be one of the ttis transmitted through blood or blood products [20] . the us food and drug administration (fda) and european pharmacopeia have proposed a limit of 1 × 10 4 geq/ml for pooled-plasma in order to reduce the potential risk of transmission [21, 22] . there is limited data on b19v prevalence among blood donors or the general population in china. according to several reports, the estimated prevalence rate could be as high as 3.5% in the general chinese population [23] and 4.5% in hiv co-infected individuals [24] . in the tibetan area, the b19v dna positivity rate was 4.8% among the general population [23] . among chinese blood donors, the b19v dna prevalence rate was 0.58%, which is much lower than that in the general population [25] . the same study also reported that different geographic locations demonstrated different prevalence rates for b19v, and that the dna sequences in xinjiang province showed a different genetic lineage than in other places of china [25] . in another study, b19v dna was detected in 54.2% (77/142) of plasma pools from 2 chinese blood product manufacturers of intravenous immunoglobulin (ivig), factor viii, fibrinogen and prothrombin complex concentrates, with levels of b19v-dna varying from 1 × 10 2 geq/ml to 1 × 10 7 geq/ml [26] . the viral load in one donation sample was 1.09 × 10 10 geq/ml, which was significantly higher than the threshold recommended by the us fda and european pharmacopeia (1 × 10 4 geq/ml). while further investigation is necessary to determine whether b19v nat screening should be implemented as a routine in chinese blood centers, the current b19v contamination in plasma products is a serious concern. malaria malaria is caused by infection with the parasites of plasmodia spp. and the current distribution covers the tropics and large parts of the subtropics [27] . infection with plasmodia spp. often resembles a common viral infection which may lead to a delay in diagnosis [28] . malaria is considered a transfusion risk since asymptomatic immigrants who reside in and travelers who visit endemic areas might import malaria to non-endemic areas [10] . therefore, us fda has initiated a donor deferral policy that temporarily defers donors who travel to endemic areas. as a result, about 1% of us donors are deferred for that reason [29] . malaria was once highly endemic in china with an estimated 30 million cases per year [30] . the chinese government began a tremendous effort to eliminate malaria in 1955 when the national malaria control program was launched [31] . the main species that causes malaria in china is p vivax and has been found in many regions; however, other species of plasmodia have also been reported [32] . although malaria has been well controlled in the last 2 decades, sporadic outbreaks are frequently reported [31] , and malaria remains a reportable infectious disease in china [13] . several cases of malaria transmission by transfusion were recently reported ( table 1 ) [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . meanwhile, the dna of p knowlesi was also found in pooled plasma from a manufacturer in guizhou [50] . however, currently chinese blood centers do not have a policy to defer donors who have traveled to malaria endemic areas or malaria infected countries during epidemic seasons. the actual prevalence of malaria and its residual risks among voluntary blood donors is unknown. further studies on malaria infected blood donors in china, such as a survey on the risks of infection and demographic characteristics of plasmodium infected donors, are crucial to better understand the transfusion risks of malaria in china. hepatitis e virus (hev) is an enterically transmitted, positive-sense, single-stranded non-enveloped rna icosahedral virus [51] . it usually causes an acute and self-limiting infection. hev has a worldwide distribution and substantial morbidity and mortality in some developing countries [52] . many cases of hev transmission by blood transfusion have been documented all over the world [53] [54] [55] [56] [57] [58] [59] . routine screening of donors for hev rna was suggested by some studies [60] . in china, the anti-hev seroprevalence is about 40% in the general population and increases with age by 1% per year [61] . approximately 2.7% of individuals are igm positive (indicating acute infection) and 0.3% are asymptomatic with viremia [62] . in the early 1990s, due to the frequent occurrence of illegal blood donation in central china, the anti-hev igg prevalence spiked to 22.7% and anti-hev igm prevalence was 1.8% among illegal blood donors [63] . in a recent study using test results from routine donations collected at 6 urban blood centers, investigators reported a prevalence of 32.6% for anti-hev igg, 0.94% for anti-hev igm, and 0.07% for hev rna among 44 816 donations. in addition, they found that prevalence rates varied by blood center locations [64] . in a comparative study [63] , where samples from both qualified blood donors and donors deferred due to elevation of alanine aminotransferase (alt) were examined, the prevalence rates of anti-hev igm and anti-hev igg in alt-elevated donors (2.76% and 40.02%, respectively) were significantly higher than those in qualified donors (1.02% and 27.42%, respectively). meanwhile, the prevalence of hev antigen among the alt-elevated donors (0.25%) was also higher than that among qualified donors (0.06%), but not at a statistically significant level. these data suggest that routine pre-screening and post-donation alt tests can reduce, but not eliminate the potential risks of hev infection from otherwise qualified donations in china. dengue viruses (denv) denv causes dengue fever, dengue hemorrhagic fever (dhf), and dengue shock syndrome. denv is a life-threatening mosquito-borne disease with global spread [65] . denv was shown to be capable of transmission by transfusion [66] . transfusion of red blood cells which tested denv seronegative but were rna positive with high viral load (n10 7 copies/ml) can lead to denv infections in transfusion recipients [67] . several countries and regions with a high prevalence of denv have investigated denv among blood donors and recipients to evaluate its impact on blood safety, as well as to develop a proper donor enrollment strategy to reduce the transfusion-transmission risk [68] [69] [70] . in china, early outbreaks of dhf in 1980s were reported mostly in guangdong, guangxi, yunnan, hainan, fujian, and zhejiang provinces located in the southeast coastal regions or border areas next to southeast asia [71] . more than 80% of the documented dhf cases were from guangdong province, one of the most developed provinces located in the south coastal area [72] . since dengue fever first reemerged in guangdong province in 1978 [73] , the epidemic of denv spread to 26 chinese provinces by 2014 [74] . from 1990 to 2013, the epidemic status of dhf gradually changed from sporadic imported to autochthonous endemic cases [75] . in 2013, there was a large dhf outbreak from july to november in foshan city, guangdong province with 5173 suspected febrile cases [76] . among them, 641 denv infections were confirmed by laboratory testing (436 rna positive and 205 igm positive) [76] . the following year, the third historically largest dhf outbreak spread throughout guangdong province (20 of 21 cities with reported cases) with 45 236 febrile cases (6024 denv infections confirmed) resulting in 6 deaths [77] . partly in response to the outbreaks in 2013 and 2014, the chinese center for disease control and prevention (cdc) amended the guidelines for dhf prevention and control to strengthen the implementation of mosquito control [78] . in 2015, the dhf cases in guangdong province dropped sharply to 739 cases by the end of september [79] , reflecting the effectiveness of denv control measures in this area. denv sentinel monitoring by sero-surveillance has been in operation in dhf epidemic regions in china since 1990s. the denv seroprevalence rate varied (igg: 0.25%-14.49%; igm: 0.56%-5.2%) in the denv endemic areas in guangdong [80, 81] , guangxi [82] , yunnan [83, 84] , hainan [85] , and fujian [86] provinces. a pilot study of denv serology and viremia among asymptomatic donors in guangzhou city (guangdong province) in september and october 2014 found that the denv igm prevalence rate was 2.4% (n = 3000). the study also identified one denv rna positive donor with viral load of 944 copies/ml [87] . another post-outbreak serological investigation among healthy populations in foshan city (next to guangzhou city) found significantly higher denv seroprevalence rates in the 4 towns that experienced dhf outbreaks in 2013 (igg rate: 2.7%, n = 817) compared with the 2 towns without autochthonous cases (igg rate: 0.6%) [76] . a blood product with denv rna viremia can potentially lead to transfusion-transmitted infection. however, to date, there are no published studies on denv transmission via transfusion in china. human brucellosis caused by brucella spp. is one of the most severe zoonotic diseases worldwide [88] . transfusion-transmitted brucellosis cases have been reported since the 1950s [89] [90] [91] [92] , and brucella spp. are considered a potential risk for blood safety [10] . human brucellosis is one of the leading threats to public health in farming areas with livestock in china [93] . more than 90% of brucellosis cases in china were found in north china and were concentrated in inner mongolia, shaanxi, heilongjiang, jilin, and hebei provinces [94] . a total of 141 604 brucellosis cases were confirmed in these areas. in addition, a rapidly increasing trend was observed in brucellosis incidence, from 0.92 cases per 100 000 people in 2004 to 2.62 clinical cases per 100 000 people in 2010 [94, 95] . an epidemiological investigation in inner mongolia reported the incidence rate as high as 12.94% among people who had close contact with livestock [96] . in china, most of brucella surveillance has focused on risk factors for infections [97, 98] , prevalence in dairy cattle [99] and local animals [100, 101] with the goal of providing information for brucellosis prevention and control. the dna prevalence rate of brucella in raw whole milk samples was found to be 1.07% in 15 provinces [102] . the low-risk population in urban areas is at increasing risk of brucellosis from contaminated milk or raw meat [103] . limited data showed the seroprevalence rate of brucella among the healthy human population to be 9.82% in endemic areas [104] . recently, a brucella spp. investigation based on enzyme immunoassay testing among blood donors in an endemic area (kashi, xinjiang province) showed that the reactive rate was 1% (39/3896), in which 0.64% (25/3896) samples were further confirmed by western blot (wb) testing, and the brucella dna prevalence rate was 0.39% (15/3896) [105] . although no infection cases transmitted by transfusion have been reported, the existence of brucella dna in donors' plasma samples indicates potential risk of transfusion-transmitted brucellosis in endemic areas and warrants future investigation. as the first retrovirus discovered in humans [106] , htlv is one of the important ttis that may lead to various human diseases such as adult t-cell leukemia/lymphoma, myelopathy/tropical spastic paraparesis, opportunistic infections, and inflammatory disorders [107] . the implementation of mandatory testing on donors has been in effect since mid-1980s in many countries to reduce the risk of transfusion transmission. [43] in china, sero-surveillance of htlv among the general population started in 1980s when an early study in beijing and other 28 provinces found a 0.08% positive rate in 9303 samples-all connected to the endemic country japan [108] . in 2005 and 2012, 2 nationwide investigations reported the prevalence of htlv among blood donors to be 0.05% (n = 145 293) [109] and 0.03% (n = 122 468) [110] respectively. a meta-analysis of 40 studies among 458 525 donors in 21 provinces and regions found that the pooled estimates of htlv-1 prevalence in fujian and guangdong were 9.9/10 000 (95% ci, 4.4/10 000-22.2/10 000) and 2.9/10 000 (95% ci: 1.7/10 000-4.8/10 000), respectively. most isolates belonged to the transcontinental subgroup a whereas only 2 cases of htlv-1 infection were found among 204 763 donors in other provinces and regions [111] . data from these studies indicate that while htlv prevalence is low in china, the infection has expanded from concentrated coastal regions in fujian and guangdong to the neighboring provinces where seroprevalence remains low (ranging from 0.02% in shanghai and 0.12% in jiangxi) [108, 110] . although no transfusion-transmitted htlv infections have been reported, the surveillance of htlv among blood donors and the evaluation of its impact on blood safety continue to be studied in china. severe fever with thrombocytopenia syndrome virus (sftsv), a novel tick-borne bunyavirus, was first identified in china to be the etiologic agent of severe fever with thrombocytopenia syndromes (sfts) with initial fatality rates between 12% and 30% in 2009 [112] . sftsv is concentrated in the mountainous rural areas in central and eastern china [112] [113] [114] . the epidemic seasons of sfts are mainly from spring to autumn and peak in may to july [113] . farmers are at high risk for sfts due to more exposure to ticks [115] . the molecular characteristics, epidemiologic distribution, risk factors and clinical symptoms have been well summarized in several reviews [116, 117] . by the end of 2013, sftsv had spread from 6 provinces in 2009 [113] to 14 provinces with a growing incidence but a decreasing fatality rate [118] . the increasing epidemic of sfts was reported and annual incidence was estimated to be 3 cases per 100 000 populations in autochthonous endemic area [118] . meanwhile, increasing numbers of sfts cases were reported in japan [119] [120] [121] and south korea [122, 123] . migratory birds are suspected to have played an important role in promoting the spread of sftsv in east asia [124] . although there is no recorded transfusion-transmitted sfts case at present, sftsv has the potential to become a transfusion-transmitted infectious agent due to several considerations: (1) nosocomial transmissions caused by direct exposure to sfts patients' blood have demonstrated that sftsv is a blood-borne pathogen [125] [126] [127] [128] . (2) the incubation period from infection to onset of the disease is one or 2 weeks on average [129] , and in some cases up to thirty days [130] . viremic asymptomatic blood donors within the incubation period may therefore potentially transmit sftsv to recipients leading to sfts. in a study launched by the national heart, lung, and blood institute, johns hopkins university, the chinese institute of blood transfusion, and 3 chinese blood centers (one located in an endemic and 2 in nonendemic regions), antibody screening and follow-up sftsv rna detections were performed on 17 208 blood donor plasma samples collected between april and october 2012. the seroprevalence rates were 0.54% (80/14 752), 0.27% (3/1130) and 0.28% (3/1326) in an endemic area (xinyang) and non-endemic regions (mianyang and luoyang) respectively, with no significant difference observed (p n .1). among 9964 donors screened by 4-sample minipool sftsv rna testing, 2 suspected viremic samples were detected each with a viral load less than 20 plaque-forming units (pfu)/ml [131] . other regional sftsv among the healthy individuals reported seroprevalence rates varying between 0.44% and 7.2% [132] [133] [134] in epidemic areas, with finding asymptomatic viremic cases. continued investigation is needed to evaluate whether sftsv presents a risk to transfusion recipients in china. leishmania infection is responsible for cutaneous and visceral leishmaniasis (kala-azar). leishmania spp. are usually transmitted to people through the phlebotomine sandfly [135] . the infected individual may harbor a persistent infection up to 30 years before recovery [136] . the transmissibility of leishmania infection via blood has been demonstrated in animals [137, 138] as well as human beings [139] [140] [141] [142] . leishmania in human red blood cells (rbcs) can survive for as long as 15 days under blood bank storage conditions [143] . it has been reported to cause cutaneous or visceral leishmaniasis in infants and immunocompromised patients [139] [140] [141] [142] . asymptomatic infections are usually found in healthy blood donors from endemic areas [144] [145] [146] [147] [148] . us military blood banks enforce permanent deferral for individuals with any history of leishmaniasis, but there is no existing regulation or standard for leishmania testing in civilian donor screening [10] . during the 1950s, there were more than 500 000 documented kalaazar cases in china, mainly in rural areas in the north [149] . with the efforts of a national control program, kala-azar was almost eliminated in 1960s [150] . currently, more than 300 cases of kala-azar are reported each year, mainly from xinjiang province and other provinces in western china [150] . recently, a retrospective study of visceral leishmaniasis by the chinese cdc reported an increase in the number of visceral leishmaniasis cases in endemic areas between 2005 and 2010 [149] , indicating that prevention and control strategies must be taken to restrain the increasing incidence and spread of leishmaniasis. otherwise, frequent population migration, tourism, and a rapidly growing public transportation may lead to further spread of the infection and push it up on the list of transfusion-transmitted infectious diseases in china. to date, there are still no survey studies of infection among donors and no documented transfusion-transmitted cases for leishmania infection in china. q fever is a zoonosis due to coxiella burnetii infection. c burnetii usually causes asymptomatic clinical manifestations such as a flu-like disease or atypical pneumonia in humans [151] ; however, acute [152, 153] and fatal chronic infections [154] [155] [156] [157] have been reported. in 2007, there was a large outbreak of q fever in the netherlands [158] . hogema et al initiated a surveillance of c burnetii dna and antibodies in local blood donations and found that the c burnetii dna positive rate was 0.3%, while the igg seropositive rate was 12.2% [159] . furthermore, 10 seroconversions were detected in donors with an incidence rate of 5.7% per year during the outbreaks from 2007 to 2009 [159] . after the great outbreaks of q fever, slot et al. started to investigate if chronically infected donors posed a threat to blood safety in the netherlands. the serological results from 2490 serum samples collected in the most affected area during august 2012 to january 2013 showed that chronic c burnetii infection was absent in the epidemic blood donors, which led to the donor re-entry policy that had already been initiated elsewhere in europe [160] . in china, most c burnetii infections have been observed in tibet, yunnan, xinjiang and inner mongolia [161] . the c burnetii dna positivity rate was about 10% in ticks and 7% in humans in western china [162] . as q fever is mainly an airborne disease, the individuals with exposure to livestock bear a higher risk. some epidemiological studies show that more than 50% of rural farmers had antibodies to c burnetii [163, 164] . although blood donor screening of c burnetii has not been initiated in china, the dna of c burnetii was found in pooled plasma from a manufacturer in guizhou [50] . a cross-sectional study of the seroprevalence and viremia status of c burnetii among blood donors, especially in nomadic herding and livestock regions, would provide a more comprehensive assessment of its impact on blood safety in china. due to its biological and geographic diversity, china will always be at high risk for various eid agents. as a populous country, a dramatic ramp-up in the number of blood donations was reported recently, with more than 21 million donations collected in 2012 at the donation index of 8.5 donations per 1000 people [165] . however, monitoring of the classical ttis such as hiv, hbv, hcv and syphilis, as well as eid agents has only been implemented by the chinese cdc and mostly based on sentinel investigations and clinical case report systems. the monitoring systems have demonstrated effectiveness in establishing proper guidelines for disease prevention and control based on epidemiological analysis of transmission routes and infection risk factors. the fatality rates and clinical symptoms from the documented cases are periodically summarized and analyzed to help with diagnosis and therapy of these pathogens, as well as with the development and improvement of laboratory diagnostic assays. effort towards disease control has benefitted from the system in the face of several outbreaks of eid epidemics, such as: sars [166] , h5n1 [167] , sftsv [113] , and denv [76] . the regional spread of infection is an important aspect to consider when evaluating the transfusion risk for eid agents. among the 9 eid agents listed above, all of them have been proven to result in pathological and autochthonous epidemics in china. b19v, hev and plasmodium spp. have nationwide distribution. three insect-borne eid agents (denv, sftsv, and leishmania spp.) and 2 zoonoses (brucella spp. and c burnetii) display significant geographic diversity due to their patterns of transmission. htlv is clustered in one coastal region. the regional distribution of 6 eid agents is shown in fig. 1 . not surprisingly, the regional distribution of insect-borne pathogens matches that of disease vectors (mosquitos, sand-flies and ticks). denv, a well-described mosquito-borne virus, has a global distribution in tropical and subtropical areas, the location of recent outbreaks of dhf. the infections of sftsv, the novel tick-borne bunyavirus, were identified from sfts patients in rural mountainous areas in central and eastern china, mainly because of the higher risk of tick exposure. in a similar way, most of the infections of zoonotic diseases (q-fever and brucellosis) were found in rural livestock farming areas in northern and western china. by compiling the distribution of reported clinical cases and the prevalence of eid agents in donors and/or general population, we provide estimates of the frequency of eid agents among chinese blood donor populations. see fig. 2 . hev and b19v are estimated to have national distribution among donors and general population in china based on previous cross-sectional studies. spread of brucella spp. depends on transmission routes from contaminated milk and raw meat produced from endemic regions. considering the current dna prevalence rate (0.39%) of brucella spp. among blood donors in endemic regions and its potential high frequency in the general donor population, this eid is a matter for concern. in the shadows of recent global outbreaks and the fast expanding trends of dhf, denv should also be highlighted for its potential risks in endemic areas in china. the other 3 regionally concentrated eid agents: qfever, leishmania spp., and sftsv are estimated to have moderate or low frequency among the blood donor population. however, ongoing surveillance on these eid agents in donors is prudent in order to provide sound evidence regarding their impact on blood safety in china. in order to evaluate the impact of eids on blood safety, a practical and feasible approach is to monitor the existence of eid agents among asymptomatic blood donors and obtain direct evidence from transfusion-transmitted cases confirmed by molecular analysis. firstly, the surveillance among blood donors through sero-markers and nucleic acid of pathogens provides data for estimating transfusion risk. among the 9 eids discussed in this review, only htlv has been continuously investigated at blood centers since 1980s. there are a few investigations and case reports on b19v, hev and plasmodium spp. among blood donors, but no systematic and continuous investigation on these eids. data on sftsv, denv and brucella spp. in china are very limited with only one documented donor surveillance study available for each agent. c burnetii (q fever) and leishmania spp. have not been investigated. in addition, well-organized and appropriately designed donorrecipient linkage studies may directly confirm transmission by transfusion, facilitate further evaluation of the post-transfusion outcomes, and yield more convincing evidence to support strategies for screening donors for eid agents. although china has not performed such linkage studies, brazil examined donor-recipient linkage for dengue virus using a large, nationally representative database [168] . this study may serve as an example to follow in order to evaluate transfusion risk of eids in china. collaborative efforts from global surveillance programs may prove useful to mitigate the transfusion risk of eids. although most eid agents in china were historically imported, some agents, such as sars and sftsv, originated in china and are expanding to other adjacent countries. also, non-endemic eid agents such as west nile virus (wnv), chikungunya virus, variant creutzfeldt-jacob disease (vcjd) and zika virus (zikv) should be immediately addressed due to their worldwide distribution and severity of clinical outcomes. recently, transfusiontransmitted zikv has been documented [169] [170] . several cases of zikv infection among travelers from epidemic areas have been reported in china [171] [172] . currently immigration authorities recommend oral fig. 1 . the distribution of 6 major regional eid agents in china. declaration of infection at entry-exit inspection and quarantine of symptomatic travelers from zikv epidemic areas upon entry into china. travelers from countries epidemic for vcjd are deferred from donation by health questionnaire in chinese blood centers. however, whether wnv, chikungunya virus and zikv should also be added to the deferral list remains a matter of controversy that needs evidence from further investigation. pathogen reduction of blood products is an alternative defense against eid agents. several studies described effective inactivation of eid agents such as denv [173] and wnv [174] . currently the only pathogen 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endemicity in southern france current epidemiological profile and features of visceral leishmaniasis in people's republic of china major trends in human parasitic diseases in china q fever as a biological weapon q fever: a biological weapon in your backyard chronic q fever: diagnosis and follow-up host factors in the severity of q fever q fever and hiv infection postinfection fatigue syndrome following q fever q fever in the netherlands: an update on the epidemiology and control measures coxiella burnetii infection among blood donors during the 2009 q-fever outbreak in the netherlands screening of blood donors for chronic coxiella burnetii infection after large q fever outbreaks progress of q-fever epidemiological research in china molecular epidemiology investigation for q-fever in northeast of china sero-investigation of q-fever among different population and livestock in anhui province q-fever detection on antibody of one causal fever blood donation in china: sustaining efforts and challenges in achieving safety and availability epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china probable limited person-to-person transmission of highly pathogenic avian influenza a (h5n1) virus in china transfusion-transmitted dengue and associated clinical symptoms during the 2012 epidemic in brazil evidence for transmission of zika virus by platelet transfusion survey of blood collection centers and implementation of guidance for prevention of transfusion-transmitted zika virus infection-puerto rico first case of laboratoryconfirmed zika virus infection imported into china early detection of zika virus infection among travellers from areas of ongoing transmission in china clearance of dengue virus in the plasma-derived therapeutic proteins nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data quantitative evaluation of plasma after methylene blue and white light treatment in four chinese blood centers the authors acknowledge dr hua shan and dr ling shi for their careful review of the paper. key: cord-345679-ydwcp75s authors: younas, amber; waheed, samra; khawaja, shabnum; imam, mehjabeen; borhany, munira; shamsi, tahir title: seroprevalence of sars-cov-2 antibodies among healthy blood donors in karachi, pakistan date: 2020-08-24 journal: transfus apher sci doi: 10.1016/j.transci.2020.102923 sha: doc_id: 345679 cord_uid: ydwcp75s background: covid-19 spread through blood transfusion has not yet been reported. despite the prevailing pandemic, there are no recommendations available as yet for testing sars-cov-2 antibodies as part of blood screening. objective: to determine the seroprevalence of sar-cov-2 antibodies, its clinical significance and to identify if total antibodies(iga, igm, igg) should be tested or just the specific igg antibodies only. method: consecutive blood donors donated were screened for standard serological panel of hbsag, anti-hcv, anti-hiv and syphilis using cobas-411 analyser and malaria. all seronegative donors were then screened for covid serology using the same instrument. these results were compared with the blood donors’ seroprevalence checked in a cohort in the first week of june 2020. pre-covid-19 period (october 2019) blood donors’ archived samples were also compared. donors who were positive on eclia were then tested for specific antibodies (igm or igg) by elisa. results: a total of 380 healthy blood donors were included. all were males with the mean age being 30.6 ± 6.3 years. ten pre-pandemic samples did not show covid-19 antibodies, whereas out of 70 samples in the3(rd) week of june, only 15 (21.4%)were positive. however, in july out of the 300 blood donors, 113 (37.7%) were found to be reactive. to reconfirm our findings, these 113 donors were then tested on elisa for presence of igg specifically. out of these 128 samples, 81 were igg positive, 23 were borderline positive and 24 were negative. conclusion: almost 40% of blood donors are now seroconverted for covid-19. this is a reflection of widespread seroprevalence in the adult male population. corona virusfirst emerged in wuhan, china in december 2019and now due to its rapid transmission throughout the world, it is regarded a pandemic. (1) sars-cov-2,is a betacorona j o u r n a l p r e -p r o o f virus, which is an enveloped, positive sense-single stranded rna virus containing four major structural proteins: spike(s), membrane (m), envelope (e) and the nucleocaspid (n). (2) virus invades the host cells by interaction of its spike protein (s) with specific receptor on host's cell membrane (ace-2 receptor) and then gains entry into cell via the process of endocytosis and uses its own rna and host machinery for replication. (3) coronavirus has a range of presentation varying from no or mild symptoms like fever, cough cold, body aches, abdominal pain, diarrhea to severe acute respiratory symptoms including acute respiratory distress syndrome that could lead to death. (4,5,6,) . more than 20 million covid-19 cases are recorded throughout the world causing around 0.7 million deaths and around 14 million recoveries. (7) in pakistan, thefirst 2 cases of were reported in february 2020 (8) in individuals who travelled from iran, with all the appropriate measures immediately taken to prevent its spread. pakistan has reported around 0.28 million cases till date, with more than 6000 deaths. (9) this data revealed better control of covid-19 in our population and decreased mortality as compared to some other countries; however, these are the cases that had been symptomatic or tested because of contact tracing and were c-ovid-19 pcr positive. the testing rate of pakistan is much lower compareto the rest of the world and south east asia due to many reasons. one is the fear of community to the covid-19 infection, the other and very important being the limited socioeconomic resources. as covid-19 has become a global threat, it was really important to force the health care systems around the world to take necessary steps in early recognition and prevention of spread of the virus. nucleic acid testing for sars-cov-2 by rt-pcr (real time polymerase chain reaction) is the gold standard and helps in early recognition of confirmed covid cases. (10)however rt-pcr sensitivity can be influenced by many factors like biological sampling, inadequate sample collection, time between sample collection and onset of symptoms and fluctuation in viral load, giving false negative results. (11) . moreover, because of a major proportion of patients being asymptomatic, an easy, sensitive and inexpensive investigation is required to know the actual frequency and seroprevalence of the virus. this could be achieved by performing specific antibody screening by validated serological assays in order to promptly identify the individuals who have been infected with covid 19in order to facilitate the control of transmission of the disease and ensure timely public health management. (12) j o u r n a l p r e -p r o o f evaluatingthe prevalence of covid-19 infection among healthy blood donors is important. who has currently provided no recommendations about screening the donors for sars-cov-2 by rt-pcr or immunoassays, however, it recommends temporary deferral for 28 days ifany symptoms (cough, fever, flu) are present, or if they are exposed to a confirmed covid-19 patient or have travelled to an epidemic area. who also recommends that the potential donors also have to inform the blood bank if they develop symptoms within 28 days of donation. (14) however, covid-19 virus does not transmit through blood donations and is not a blood borne disease but identification of seroprevalence among the blood donors can give an estimate of circulation of the virus among healthy individuals, providing actual disease burden and real case fatality rate in a population. information regarding antibody response against sars-cov-2 in asymptomatic individuals is lacking in our part of the subcontinent. in our study, we conducted specific serological testing (total antibodies) to identify prevalence of sars-2-cov antibodies among the healthy blood donors who visited blood bank at our institute.their results were compared with specific serologic results of blood donors that came before the onset of pandemic(october, 2019). with this aim, we elucidate the seroprevalence of blood donors and association of any blood groups with the seroconversion along with association with the certain age group. a cohort of voluntary/exchange blood donors who came atthe national institute of blood diseases and bone marrow transplantation,karachi, during the pandemic in may, june and july were enrolled in the study. retrospectively, healthy blood donors who visited our blood bank in october 2019 were also tested by recovering their previous samples. blood donors meeting the aabb 18 th edition donor acceptance and deferral criteria were included or excluded accordingly. moreover, history was thoroughly taken for presence of fever and any respiratory symptoms for at least 28 days;donors, who had a history of covid-19 infection, were excluded from the study. any donors having close contact with other covid-19 patients were also excluded. furthermore, donors having positive screening for hepatitis b, hepatitis c, hiv, syphilis or malaria were also excluded. demographic data of donors including age and gender was noted. for categorical variables, frequency with percentage was calculated whereas mean and standard deviation was calculated for quantitative variables. anti-sars-cov-2(total) is qualitative assay; mean was calculated by using numerical cut of index (coi) value. t-test was used to compare the mean and p value of less than 0.05 was taken as statistically significant. all analysis was done on statistical package for social science spss (version 23). a total of 380 healthy blood donors were included in the study. all were males and their mean age was 30.6±6.3years. ten samples from october 2019 were checked for anti-sars-cov antibodies by eclia, and none of them was found to be positive. in 3 rd week of june, 70 donors were tested for presence or absence of anti sars-cov antibodies and 15 were tested positive (21.4%). in july 2020, we tested 300 healthy blood donors, 113 donors (37.7%) were found to be reactive for anti-sars-cov-2antibodies. to reconfirm our findings, these 128 donors were then tested on elisa for presence of igg specifically. out of these 128 samples, 81 were igg positive, 24 were negative and 23 were found to be borderline positive. to further assess our findings, 24 negative igg samples were tested for igm elisa and out of these 24, 22 were found to be negative for igm whereas 2 were borderline positive as shown in figure 01. analyzing the blood groups of these healthy donors, we found out that 109 blood donors were o these findings elaborated upon some important concerns. the first being the actual number of covid cases in pakistan because only symptomatic individuals were tested for covid by pcr. secondly, the increasing number of sars cov-2 antibodies in healthy population is raising herd immunity. lastly, the samples that were positive for sars cov-2 antibodies by eclia but were negative for elisa igg and igm, either that were false positive or some other category of antibody was present in those donors.we rechecked the samples of eclia and the result was found to be same. considering the high specificity of eclia, we preclude the idea of false positivity. hypothetically, we can assume those antibodies to be iga but we could not confirm due to the lack of testing availability. a study in china was published regarding antibodies in healthy blood donors. they demonstrated overall covid-19 antibody prevalence as 2.29%, 0.029% and 0.0074% in wuhan, shenzhen and shijiazhuang respectively from january 2020 to april 2020.(1) these results contradict our findings probably because of the strict social restrictions followed in china as compared to our country. in another study in china, association of gender was also demonstrated with a gender ratio was 0.99(male/female). (14) as all of our blood donors were males, we could not make any association with the gender. again, since the majority of our blood donors were young adults, any association with age could not be established. however, j o u r n a l p r e -p r o o f compared to rest of the world, pakistan has a higher ratio of young adults who were covid-19 positive;althoughthe mortality ratio was lower among them. it could be due to the fact that our country has a much lower average age as compared to the rest of the world. association of blood group with covid-19 infection has also been described in recent studies. individuals with blood group a have been found to be more at risk as compared to those with blood group o. (15, 16) the exact reason for this is unknown, but partly may be due to the protective mechanism of circulating anti-a antibodies which inhibit the interaction of virus to its specific host ace2 receptor. (17) . however, in our study we did not observe any significant difference between different blood groups, which might be because none of the donors were symptomatic during disease period. different studies throughout the world were conducted in the general population for checking antibodies status; one such study in netherlands was reported in april 2020 which showed 2.7%of population being reactive for covid antibodies (18), however they did not exclude previously symptomatic cases. another study in northern france reported 25.8% of population positive for covid-19 antibody(19)but they also did not exclude previously symptomatic cases. in april 2020, italy reported around 9.4% of healthy blood donors to be seropositive for covid-19(20). our study showed the largest number of seroconversion partly because of the time period selected for testing and because of implementation of social restrictions stringently in other parts of the world. a study has demonstrated that if around 60% of population(21) develops antibodies, the prevention of disease can be strengthened. however, whether these antibody responses are long lasting or not needs to be studied and investigated. as rna viruses have a tendency to mutate, it is still not clearthat if the virus mutates, will these antibodies be protective against the disease or not. antibody testing of general population can help in detecting the actual number of asymptomatic carriers; however, it cannot be used for the diagnosis and pcr remains the choice of investigation for diagnosis.talking about limitations, this is a single centre study but the first local study of its type to our knowledge to elucidate the seroprevalence of blood donors, its j o u r n a l p r e -p r o o f association with any blood groups and age group. we need more local studies to authenticate these findings. moreover, there were limited pre-covid samples available to study from. to conclude, seroprevalence of sars-cov-2 antibodies has increased in pakistan over a period of time and could help in recognizing the actual number of covid-19 cases. ethical review board: the study was approved by the ethical review committee of national institute of blood disease and bone marrow transplantation. ay and sw wrote the manuscript. swk and mi did data collection and analysis. mb, tss conceived main idea andcritically evaluated the paper. the prevalence of antibodies to sars-cov-2 among blood donors in china.medrxiv properties of coronavirus and sars-cov-2.the malaysian journal of pathology ace2: the molecule that helps coronavirus invade your cells pathological findings of covid-19 associated with acute respiratory distress syndrome. the lancet respiratory medicine clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet world health organization, covid 19 pandemic emergency information pandemic & pakistan; limitations and gaps. global biosecurity government of pakistan, covid-19 updates the sars-cov-2 outbreak: diagnosis, infection prevention, and public perception detection of sars-cov-2 in different types of clinical specimens the role of antibody testing for sars-cov-2: is there one? world health organization. maintaining a safe and adequate blood supply during the pandemic outbreak of coronavirus disease (covid-19).interim guidance the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china.zhonghualiuxingbingxuezazhi= zhonghualiuxingbingxuezazhi clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet relationship between the abo blood group and the covid-19 susceptibility.medrxiv association between abo blood groups and risk of sars-cov-2 pneumonia we thank all the staffs and technologists of the blood bank, laboratory and donor centre for providing endless support during the pandemic. key: cord-009417-458rrhcm authors: luce, judith a. title: use of blood components in the intensive care unit date: 2009-05-15 journal: critical care medicine doi: 10.1016/b978-032304841-5.50082-0 sha: doc_id: 9417 cord_uid: 458rrhcm nan most patients admitted to an intensive care unit (icu) require the administration of one or more blood components during their stay. such patients exhibit great diversity in conditions necessitating care in the icu, age, underlying medical problems, and integrity of physiologic compensatory mechanisms. all these patients, however, share the need for optimized oxygen-carrying capacity and tissue perfusion. ongoing blood loss resulting from injuries, surgical wounds, invasive monitoring equipment, and blood sampling requirements, coupled with inadequate marrow function and, in some, red cell destruction, makes red cell transfusion a necessity for many icu patients. additionally, many patients are susceptible to the development of hemostatic disorders requiring the administration of such blood components as plasma, cryoprecipitate, or platelet concentrates. blood components should be considered drugs because they exert potent therapeutic responses yet are also capable of causing signifi cant adverse effects. the food and drug administration (fda) regulates blood component preparation, testing, and administration. 1 unlike pharmaceutical agents, however, blood components have fewer objective indications for use and no therapeutic index relating dose to safety. it is not as simple to monitor the effi cacy and continuing need for a blood component as it is to determine the blood level of a drug. in addition, the risks associated with transfusion cannot be known in advance and may be lethal; such risks include medical errors, as well as infectious and immunologic hazards. unlike pharmaceutical agents, these prescribed products require documentation of patient consent and indication for use. although the american blood supply is now safer than ever before, zero-risk transfusion is not achievable, even if blood components could be sterilized. the process of donor selection and screening has become increasingly stringent, an evolution that began in response to the welldefi ned risks of transfusion-transmitted hepatitis and human immunodefi ciency virus (hiv) infection. although the value of maximizing recipient safety is unarguable, increasing donor selectivity has its price. as more tests are added and more conditions placed on the donor, the number of usable donations has declined. this trend has led to occasional regional and seasonal blood shortages and, rarely, outright inability to provide certain blood components. clinicians who prescribe blood components must be aware of these uncertainties in availability and contribute by using blood products appropriately while the national blood banking system seeks strategies to ensure an adequate, safe blood supply. donor screening strategies to ensure recipient safety take several forms. 1, 2 american blood donors are voluntary donors; cash payment was eliminated in the 1970s after studies linked professional donors with transmission of hepatitis. confi dential questionnaires were initiated to limit transmission of hiv and hepatitis and to allow voluntary self-exclusion and involuntary exclusion of donors who pose an increased risk of transmitting infectious agents. multiple specifi c serologic and biochemical tests are performed to detect the potential for transmission of hiv and other retroviruses, hepatitis, and syphilis. any donor who indicates high-risk behavior or who tests repeatedly positive is placed on a permanent deferral list. some patients may insist on blood obtained from relatives or friends. this practice is termed directed or designated donation. these selected donors must undergo the same rigorous questioning and testing as volunteer donors. some studies have found an increased frequency of hepatitis markers in the blood of directed donors when compared with blood drawn from unselected volunteers, but others suggest that designated donors may be no different from new volunteers. 3, 4 there continues to be no consensus about whether directed donors are, as a group, as safe as volunteer donors. 5, 6 institutional policies about the acceptability and processing of directed donations vary widely. in any case, supporting icu patients who require large-volume transfusion with directed donations is unlikely to be advantageous or practical. the basic principle of blood component therapy is prescription of the specifi c blood product needed to meet the patient's requirement. a single whole blood (wb) donation can be separated into its composite parts, or components, which can be distributed to several recipients with differing physiologic needs. component therapy thus meets the clinical requirements of increased safety, efficacy, and conservation of limited resources. as the variety of blood product components increases, however, the complexity of transfusion medicine also increases. a wb donation is typically separated into red blood cells (rbcs), a platelet concentrate, and fresh frozen plasma (ffp) within hours of its collection. the plasma may be further processed into cryoprecipitate and supernatant (cryopoor) plasma. one unit of wb measures approximately 500 ml, including 63 ml of citrate anticoagulant/preservative solution. each unit of wb supplies about 200 ml of rbcs and 300 ml of plasma for volume replacement. wb is refrigerated for 21 to 35 days, depending on the preservative used. after less than 24 hours of refrigerated storage in this preservative and bag system, platelet and granulocyte function is lost. with further storage, levels of the "labile" coagulation factors v and viii decrease. 7 some blood centers offer modifi ed wb, which is produced by removal of the platelet or cryoprecipitate fraction and return of the supernatant plasma to the red cells. this permits provision of the more labile components to patients with specifi c needs, with the remainder forming a product having a composition essentially the same as cold-stored wb. however, the growing need for specialized blood components has resulted in processing the majority of blood donations into components, thus limiting the availability of wb and modifi ed wb. rbcs, or in common usage, "packed" red cells (prbcs), are the blood component most commonly transfused to increase red cell mass. prbcs are derived from the centrifugation or sedimentation of wb and removal of most of the plasma/anticoagulant solution. if collected into citrate-phosphate-dextrose-adenine solution, the volume is approximately 250 ml, the hematocrit (hct) is 70% to 80%, and the storage life is 35 days. extended additive solutions permit storage up to 42 days but increase the volume to 300 ml and decrease the hct to 60%. these extended storage units are commonly used and easier to transfuse because of lower viscosity, but they may pose a problem because of their larger volume. the transfusion of leukocyte-reduced rbcs may benefi t certain patients. transfusion of blood components containing leukocytes may lead to febrile reactions, a greater propensity for alloimmunization, platelet alloimmunization, and transmission of pathogens carried by leukocytes, such as cytomegalovirus (cmv). leukocyte reduction, as defi ned by the fda, requires fi ltration of the blood component by a special fi lter. 1 filtration may be performed either at the time of blood donation and processing or later at the time of transfusion ("bedside fi ltration"). filtration before storage conveys the benefi t of removing white blood cells (wbcs) before they can deteriorate and elaborate cytokines and other unwanted substances during storage. 8 because of proven and theoretical benefi ts of leukocyte reduction of blood components (discussed later in the section covering the adverse effects of transfusion), many european countries and canada require that all transfusions be leukocyte reduced, a process called universal leukoreduction (ulr). some institutions in the united states have also made that decision, but either method of leukocyte reduction adds signifi cantly to the cost of each transfusion ($25 to $30), and the benefi ts of this measure when applied globally have yet to be quantifi ed. 9 washing prbcs involves recentrifuging to remove the plasma/preservative solution from the unit. however, washing may take an hour or more, limits subsequent storage time, and causes some loss of rbcs. washing is also not an effective method of leukoreduction. there are very few indications for the use of washed rbcs, although some recipients with plasma reactions may benefi t. prbcs can be frozen in cryoprotective solution and stored for extended periods. frozen rbcs are generally limited to units of special value, such as those with a rare rbc antigen profi le or autologous blood donations that need to be stored for future use. a rare-donor registry of frozen prbcs exists to assist in providing blood to patients with complex or multiple alloantibodies to red cell antigens. signifi cant advanced planning is necessary to acquire and thaw frozen prbcs for transfusion, thus limiting their use in acute situations. wb and prbcs suffer some cell loss during storage. the current technology of bag and preservative solutions attempts to optimize cell quality and quantity by using strict criteria to determine the length of allowable storage time. nonetheless, as red cell metabolism decreases progressively, a "storage lesion" results, 10 with accumulation of a variety of undesirable substances and loss of cellular function. over time in storage, a slow rise in the concentration of potassium, lactate, aspartate aminotransferase, lactate dehydrogenase, ammonia, phosphate, and free hemoglobin and a slow decrease in ph and bicarbonate concentration occur. cytokines and infl ammatory mediators such as interleukin-1, interleukin-6 and tumor necrosis factor also accumulate. the ph of freshly stored blood in citrate solution is 7.16, which declines to approximately 6.73 at the end of the unit's shelf life. as potassium leaks from red cells during storage, levels as high as 25 meq/l may result. however, each unit transfused supplies at most 7 meq of potassium, which is well tolerated under most circumstances. during the storage period there is also a progressive decrease in rbc-associated 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp). 10 a decrease in 2,3-dpg increases the affi nity of hemoglobin for oxygen, which shifts the oxygen dissociation curve to the left and decreases oxygen delivery to tissues. there is little evidence, however, that this transient increase in oxygen affi nity has clinical importance. after infusion, 2,3-dpg gradually increases as the transfused red cells circulate, with 25% recovery in 8 hours and full replacement by 24 hours. 11 decreased atp during storage diminishes the viability of red cells after transfusion and is one of the chief factors limiting storage time. there is no currently available storage or rejuvenation solution that optimizes these cellular constituents. the majority of blood transfusions are in the form of prbcs, the component indicated for normovolemic patients or those for whom intravascular volume constraints are necessary. the use of wb may be desirable for patients who require both increased oxygen-carrying capacity and volume resuscitation because of a large and ongoing hemorrhage; however, the availability of wb is generally limited. resuscitation is effectively achieved with the use of prbcs and crystalloid solutions. each unit of prbcs or wb is expected to raise the hemoglobin level by 1 g/dl and the hct by 3% in stable, nonbleeding, average-sized adults. although some studies have demonstrated a slight superiority of fresh wb over components when used during cardiac surgery in selected patients, 12 the benefi ts of fresh blood remain controversial, and current testing and processing requirements limit general availability. despite a long tradition of transfusion of rbcs in critically ill patients, the precise indications for transfusion remain a source of controversy, and specifi c transfusion practices may vary widely among clinicians. before the major randomized studies of rbc transfusion policies, a survey of transfusion practice showed that about half of icu patients were receiving red cell transfusions, 13 and another showed that if the icu stay was longer than a week, the rate of transfusion was 85%. 14 the total number of transfusions was high, and icu practice was characterized by high rates of transfusion. 15 the reasons for the controversies are clear: rbcs should be transfused only to enhance tissue oxygen delivery, but the underlying physiology of anemia, the complex adaptations to anemia, and the potential advantages and disadvantages to particular groups of patients are not as well understood. compensatory mechanisms for acute and chronic anemia are diverse and complex. 16, 17 all work in concert to maintain oxygenation within the microcirculation. cardiovascular adjustments leading to increased cardiac output include decreased afterload and increased preload resulting from changes in vascular tone, increased myocardial contractility, and elevated heart rate. lowered blood viscosity permits improved fl ow of erythrocytes within capillaries. blood fl ow is redistributed to favor critical organs with higher oxygen extraction. pulmonary mechanisms, though contributing relatively little to shortterm oxygenation demands, exert potent effects on related metabolic variables. finally, the hemoglobin molecule can undergo biochemical and conformational changes to enhance unloading of oxygen at the capillary level. all these mechanisms contribute to an "oxygen reserve" capacity that exceeds baseline requirements by approximately fourfold. 16 no experimental model exists that encompasses the diversity of physiologic compensations for hypoxia. experiments carried out in animals and case reports in patients refusing transfusion indicate that an extremely low hct is tolerated if tissue perfusion is adequate. [18] [19] [20] certain objective, though indirect, measurements of tissue oxygenation exist and are available to clinicians caring for patients monitored invasively in the icu. mixed venous oxygen content (pv o 2 ) and cardiac output can be measured in patients undergoing pulmonary artery catheterization; arterial oxygen content can also be measured directly. the oxygen extraction ratio (er) can be calculated directly, and in the presence of normal or high cardiac output it is a measure of tissue oxygen extraction and, indirectly, the adequacy of tissue oxygen delivery. the total body er at baseline is about 25%. a falling pv o 2 and an er increasing to greater than 50% have been proposed as indicators of the need for red cell transfusion. 21 there have been only 10 randomized trials of transfusion policy in the icu, and only 1 of them was large enough to draw specifi c, statistically signifi cant conclusions. 22 the canadian critical care trials group compared a liberal (target hemoglobin, 10 to 12 g/dl) with a restrictive (target hemoglobin, 7 to 9 g/dl) red cell transfusion policy in patients stratifi ed for disease severity. at 30 days from randomization, the restrictive strategy was at least as good as, if not better than (p = .11) the liberal strategy, and overall hospital mortality was signifi cantly lower in the restrictive strategy group (p = .05). for patients younger than 55 years and for patients with lower (<20) apache (acute physiology, age, and chronic health evaluation) ii scores, the restrictive strategy was clearly superior. in addition, liberal transfusion was not associated with shorter icu stays, less organ failure, or shorter hospital stays; longer mechanical ventilation times and cardiac events were more frequent in the liberal strategy group. a later subgroup analysis of patients with cardiovascular disease, though small enough to have statistical doubt, suggested that a more liberal transfusion strategy was probably appropriate for patients with severe ischemic coronary disease. 23 this observation has some support in experimental studies of the effects of anemia in laboratory animals with coronary occlusion. 24 the canadian study has highlighted the many and complex issues involved in transfusion decision making in the icu. since publication of the canadian study, several large reports have examined the use of red cell transfusions in critical care units. vincent and colleagues 25 surveyed european icus and found that the transfusion rate in 3534 patients was 37% during the icu stay and 12.7% after the stay. the mean pretransfusion hemoglobin level was 8.4 g/ dl. corwin and colleagues 26 studied 284 icus in the united states a year later and found great similarity: nearly 50% of patients received transfusions, and the mean threshold hemoglobin level was 8.6 g/dl. a single large scottish teaching hospital reported a more parsimonious practice: the rate of transfusion was still 52% in its icu patients, but the total volume of blood used was slightly smaller and the mean pretransfusion hemoglobin level was only 7.8 g/dl. 27 all these authors have concluded that icu practice has not fully embraced the guidelines of the canadian clinical trial. in contrast, 18 hospitals in australia and new zealand have reported on transfusion in 1808 consecutive icu admissions, and although the authors found a median pretransfusion hemoglobin concentration of 8.2 g/dl, the rate of transfusion was lower, at only 19.7% of patients, 60% of whom were bleeding. 28 the "inappropriate" transfusion rate was 3%. the authors speculate that the practitioners may have been infl uenced by publication of the canadian study and their own regional survey of transfusion practices. nonetheless, they agree that full implementation of the canadian guidelines in their clinical setting might be controversial. the literature on rbc transfusion in the setting of surgery, particularly surgery with the use of blood products, is growing. a mounting body of data illustrate the human tolerance of a low hct during and after surgery. a recent randomized trial of rbc transfusion strategy in orthopedic surgery demonstrated no signifi cant differences in outcome between a restrictive (8 g/dl) and a liberal (10 g/dl) transfusion threshold and included monitoring for silent myocardial ischemia preoperatively and postoperatively. 29 provided that adequate perfusion of the microcirculation is maintained, purposeful maintenance of a low hct during surgery, a technique called normovolemic hemodilution, 30 can be a powerful tool in minimizing blood loss and the attendant need for red cell transfusion. table 80 -1 summarizes guidelines proposed by the national institutes of health, 31 the american society of anesthesiologists, 32 and the american college of physicians 33 relative to the transfusion of rbcs. these guidelines have been provided with the intent of establishing parameters, not with the intent of substituting for the individual clinician's judgment. the art of medical decision making in transfusion, as in other areas of medicine, lies in determination of the appropriate treatment for the individual patient. a platelet concentrate (random-donor platelets) is obtained by centrifugation from a unit of donated wb. each unit contains a minimum of 55 ã� 10 10 platelets suspended in about 50 ml of plasma. platelets are stored at room temperature to avoid loss of function from refrigeration and are constantly agitated to maximize gas exchange. the length of storage varies with the container used, but most systems permit 5-day storage. because of this limited storage time and the increasing demand for this component, platelets are often subject to supply shortages. some loss of viability and platelet numbers occurs during storage, but 5-day-old platelets still effect hemostasis. once the bags are entered for pooling before transfusion, the platelets must be administered within 4 hours. each unit of platelets is expected to increase the platelet count by 10 ã� 10 9 /l in a typical 70-kg adult. the usual dose is 6 units, or 1 u/10 kg of body weight. a 1-hour post-transfusion platelet count should be obtained to determine the adequacy of response. the following equation, which relates platelet number and body size to the post-transfusion increment, can be used to assess the effectiveness of the transfusion: abo-compatible platelets are desirable but not essential. when abo-mismatched platelets are given, removal of some of the incompatible plasma can be carried out at the time of pooling for transfusion. likewise, volume reduction may be necessary for patients at risk for fl uid overload from the 300 to 500 ml of plasma present in 6 to 10 units of platelets. nonetheless, the remaining plasma is a good source of stable coagulation factors and contains diminished but still potentially benefi cial amounts of factors v and viii. there is no contraindication to the use of rh-positive platelets in rh-negative patients; if given to women with future childbearing potential, rh immune globulin (rhig) may be used prophylactically against the small risk of rh alloimmunization from red cells that may be contained in the platelet concentrate. plateletpheresis (common terms: single-donor platelets, apheresis platelets) involves separating and removing platelets from one donor by cytapheresis during a 1 1 / 2 -to 2-hour procedure on an automated device and then retransfusing the remainder of the blood back into the donor. each collection contains an equivalent of 6 to 10 units of platelet concentrates. single-donor platelets are suspended in about 300 ml of plasma, so the same abo and volume considerations discussed earlier pertain. single-donor platelets offer the clear benefi t of reducing the risk of multiple-donor exposure to the recipient. single-donor platelets may also be the only available alternative for recipients who have been alloimmunized by previous platelet transfusions because they may be human leukocyte antigen (hla) or platelet antigen matched to the recipient. the use of apheresis platelets now exceeds the use of pooled random-donor platelets; however, use of this product in emergency situations is limited by the availability of volunteer donors. 35 platelet transfusions are indicated for patients bleeding because of thrombocytopenia or functional platelet defects. 36 guidelines for transfusion continue to evolve, and the current guidelines merely provide a desirable range for platelet counts, assuming normal platelet func-tion (table 80 -2). there is ample evidence that bleeding medical or surgical patients with platelet counts of 50 ã� 10 9 /l or above will not benefi t from transfusion if thrombocytopenia is the only abnormality. for critical invasive procedures in which even a small amount of bleeding could lead to loss of vital organ function or death, maintaining the platelet count at 50 ã� 10 9 /l or greater is typically preferred. the presence of other factors that diminish platelet function, such as certain drugs, foreign intravascular devices (e.g., intra-aortic balloon pump or membrane oxygenator), infection, or uremia, may alter this requirement upward. patients at risk for small but strategically important hemorrhage, such as neurosurgical patients, may need to be maintained at counts of 80 to 100 ã� 10 9 /l. patients without hemorrhage who have platelet counts of 5 ã� 10 9 /l or lower appear to be at increased risk for signifi cant hemorrhage. indications for transfusion to patients with counts above 10 ã� 10 9 /l are less well established; thus, the majority of guidelines propose prophylactic platelet transfusion to prevent hemorrhage at a threshold of 10 ã� 10 9 /l. the bleeding time is not a useful procedure in this situation because it is usually prolonged at counts below 80 ã� 10 9 /l, may be insuffi ciently reproducible, and correlates poorly with the risk for bleeding. 37 patients undergoing cardiac bypass surgery experience a drop in platelet count and often acquire a transient platelet functional defect from damage associated with the bypass apparatus. 38 most patients do not experience platelet-associated bleeding, however, so prophylactic transfusion in the absence of bleeding is not warranted. in a patient who continues to bleed postoperatively, more likely causes are a localized, surgically correctable lesion or failure to reverse heparinization. if these conditions are excluded, empiric transfusion of platelets may be justifi ed. patients thrombocytopenic by virtue of immunologic destructive processes such as idiopathic thrombocytopenic purpura (itp) receive little benefi t from platelet transfusions because the transfused platelets are rapidly removed from the circulation. in the event of life-threatening hemorrhage or an extensive surgical procedure, transfusion may prove benefi cial for its short-term effect. transfusion may be accomplished effectively by pretreatment with high-dose immunoglobulin or high-dose anti-d antiserum (rhig). 39, 40 platelet transfusion has been reported to be deleterious in thrombotic thrombocytopenic purpura (ttp), 41 in the related hemolytic-uremic syndrome, and in heparin-induced thrombocytopenia. cautious administration, in cases of life-threatening thrombocytopenic bleeding only, is prudent. prophylactic platelet transfusion for thrombocytopenia secondary to underproduction remains controversial. the common practice of transfusion to maintain the platelet count above 20 ã� 10 9 /l derives from data published in 1962, which demonstrated an increase in spontaneous bleeding in leukemic patients at that level. 42 however, critical evaluation of the data reveals that serious hemorrhage was not greatly increased until counts fell to 5 ã� 10 9 /l or lower and that these patients received aspirin for fever, which might have compromised platelet function and enhanced the bleeding. a somewhat more recent study quantitating stool blood loss in aplastic anemia patients defi ned a bleeding threshold at platelet counts of 5 to 10 ã� 10 9 /l. 43 a prospective study of a more conservative transfusion protocol found that major bleeding episodes occurred on 1.9% of days with counts of less than 10 ã� 10 9 /l and on only 0.07% of days with counts of 10 to 20 ã� 10 9 /l. 44 the trigger for prophylactic platelet transfusion in the 5 to 10 ã� 10 9 /l range, however, applies primarily to stable thrombocytopenic patients. factors such as fever, use of anticoagulant or antiplatelet drugs, and invasive procedures must be considered when generating a treatment plan for individual patients. patients experiencing rapid drops in platelet count may be at greater risk than those at steady state and thus may benefi t from transfusion at higher counts. benefi ts to the patient with more judicious use of platelet transfusion include decreased donor exposure, which lessens the risk of transfusion-transmitted disease; fewer febrile and allergic reactions that may complicate the hospital course; and the potential delay or prevention of alloimmunization to hla and platelet antigens. 45 the development of refractoriness to platelet transfusions is a serious event heralded by a falling cci. poor response to platelet transfusions can be seen in patients with other reasons for platelet consumption, including splenomegaly, fever, trauma and crush injury, burns, disseminated intravascular coagulation (dic), concomitant drugs, or transfusion of platelets of substandard quality. 46 these factors should be sought and corrected if possible. alloimmunization is characterized by the development of anti-hla or platelet-specifi c antibodies, with resultant immune platelet destruction. as many as 70% of patients receiving multiple red cell or platelet transfusions become immunized. 45 leukocyte depletion of transfused components can prevent or delay this phenomenon, but it is important to use leukoreduced components early in the course of transfusion therapy. 45, 47 when patients fail to achieve expected increments after platelet transfusion, provision of abo-specifi c platelet concentrates that are less than 48 hours old may improve the response. if no improvement is seen and the aforementioned medical conditions are excluded, the patient should be screened for hla antibodies or be hla typed and provided with hla-compatible single-donor platelets. alternatively, platelet crossmatching with the patient's serum can be carried out. there is no advantage to unmatched singledonor platelets in this situation. standard ffp is prepared by centrifugation of wb and is frozen within 8 hours of blood donation. 1,2 ffp may be stored frozen for 1 year. the usual volume is about 250 ml, depending on the donor's hct. the most common method of thawing before transfusion is soaking in a 37â° c water bath, which requires about 30 to 45 minutes. once thawed, ffp can be stored refrigerated for a maximum of 24 hours. when prepared and stored in this manner, ffp supplies all the constituents in the amounts normally present in circulating plasma, including stable and labile coagulation factors, complement, albumin, and globulins. by convention, the coagulation factors are present in concentrations of 1 u/ml. crossmatching to the recipient is not performed, but ffp must be abo compatible. standard ffp is as likely to transmit hepatitis, hiv, and most other transfusion-related infections as cellular components are. new ffp products have recently been introduced in response to concern about the transmission of infectious diseases. one such product is solventdetergent-treated ffp. 48 solvent-detergent treatment is a means of viral inactivation that removes the infectivity of lipid-enveloped viruses, such as hepatitis b and c and hiv. because the product is derived from pooled plasma, with as many as 2500 donors in each lot, it has the potential to actually increase recipient exposure to pathogens not inactivated by the solvent-detergent method, such as hepatitis a and parvovirus b19, and be more vulnerable to any newly emerging non-lipid-enveloped agent. a variety of other techniques for reducing pathogen exposure in ffp have been developed, including exposure to low ph or vapor heating and treatment with ultraviolet irradiation, gamma irradiation, or psoralens and light to inactivate pathogens by inducing dna damage. 49 because none of the ffp products is entirely free from the risk of disease transmission or other adverse effects and because infection-reducing modifi cations add significantly to the cost of the components, ffp should be used judiciously. 50 it should be administered only to provide coagulation factors or plasma proteins that cannot be obtained from safer sources. ffp is commonly used to treat bleeding patients with acquired defi ciency of multiple coagulation factors, as in liver disease, dic, or dilutional coagulopathy, or to treat patients with congenital defi ciency of a coagulation factor or other protein for which concentrates or safer sources do not exist. ffp may be indicated for emergency reversal of the coagulopathy induced by warfarin anticoagulants when more concentrated products are not available or for the provision of protein c or s in patients who are defi cient and suffering acute thrombosis. ffp should be administered as boluses as rapidly as feasible so that the resulting factor levels allow hemostasis. the use of ffp infusions without adequate bolus administration is not helpful. ffp should not be used for volume expansion or wound healing or as a nutritional source of protein. ffp does not reverse anticoagulation induced by heparin and in theory might exacerbate bleeding by supplying more antithrombin, heparin's cofactor. prophylactic administration of ffp does not improve patient outcome in the setting of massive transfusion or cardiac surgery unless there is bleeding with an associated documented coagulation abnormality. 51, 52 patients do not usually bleed as a result of coagulation factor insuffi ciency when the international normalized ratio (inr) is less than about 2.0, and even then the results are not always predictable. 53 the partial thromboplastin time (ptt) is not useful in predicting procedural bleeding risk. 54 ffp is often requested prophylactically before an invasive procedure when the patient exhibits mild prolongation in coagulation studies. most of these procedures may be carried out safely without transfusing ffp. 53, 55 ffp is probably the most misused blood component, as illustrated by retrospective surveys. 56 coagulation factors are normally present in the blood far in excess of the minimum levels required for hemostasis. as little as 10% of the normal plasma concentration of several factors will effect hemostasis. conversely, ffp treatment of acquired multiple defi ciencies, as in hepatic failure, is often ineffective because many patients cannot tolerate the infusion volumes required to achieve hemostatic levels of coagulation factors, even transiently. 57 the plasma half-life of transfused factor vii is only 2 to 6 hours. it may be impossible to administer suffi cient ffp every few hours without encountering intravascular volume overload. finally, in some instances, transfusion of seemingly adequate volumes may still fail to correct the coagulopathy. 58 careful documentation of both the need for ffp and the adequacy and outcomes of therapy is essential. 59 cryoprecipitate is manufactured by thawing and centrifuging ffp below 6⺠c and resuspending the precipitated proteins in about 15 ml of supernatant plasma. 1,2 each bag is a concentrated source of factor viii (80 to 120 units), von willebrand factor (vwf) (50% of original plasma content), fi brinogen (250 mg), factor xiii (30% of original plasma content), and fi bronectin. cryoprecipitate offers the advantage of transfusing more specifi c protein and less total volume than the equivalent dose of ffp does. it has been used to treat patients with inherited coagulopathies, such as hemophilia a, von willebrand disease, or factor xiii defi ciency. in the critical care setting, it is more commonly used to replenish fi brinogen, especially in bleeding patients with hypofi brinogenemia caused by dilutional or consumptive coagulopathy. cryoprecipitate also reportedly improves hemostasis in uremic patients, presumably by reversing the functional platelet defect, 60 but desmopressin acetate (ddavp) 61 or conjugated estrogens exert similar effects and should be used preferentially to avoid potential transfusion-transmitted disease. the usual dose of cryoprecipitate to treat hypofi brinogenemia is 10 bags/units to start, then 6 to 10 bags/units every 8 hours or as necessary to keep the fi brinogen level above 100 mg/dl. each bag/unit of cryoprecipitate carries a risk of disease transmission equivalent to that of 1 unit of blood. for this reason, commercial factor viii concentrates, recombinant or treated to inactivate viruses, are preferred over cryoprecipitate for treating hemophilia a patients. immune serum globulin (ig), rhig, and hyperimmune globulins for diseases such as hepatitis b and varicella zoster are obtained by fractionation of pooled plasma, followed by chromatography, delipidation, and other steps to remove aggregates and infectious agents. intravenous ig (ivig) is available in solution or lyophilized form, with protein content varying by mode of preparation. the available products vary slightly in the amounts of iga and igm contained in them, which are mostly present in only trace quantities. ig preparations can be used to provide passive antibody prophylaxis or to supply ig in certain immunodeficiency states. hyperimmune globulins may be used to treat active infections in immunosuppressed hosts. recent applications have exploited ig's immunomodulatory effects in treating a wide variety of disorders with an immune basis. the specifi c mechanism of action of ivig in such conditions has not yet been identifi ed, but possibilities include interference with macrophage fc receptor function, neutralization of anti-idiotypic antibodies, and interference with the incorporation of activated complement fragments into immune complexes. a recent review more completely discusses the effects of ivig on the immune system and its potential uses. 62 rhig is prepared from pools of plasma obtained from donors sensitized to the red cell antigen d from the rh group. the standard-dose vial contains primarily igg anti-d, with a protein content of 300 âµg in 1 ml. this dose will protect against 15 ml of d + red cells or 30 ml of wb. 63 rhig carries no risk of virus transmission. although rhig is used primarily in obstetrics, it may also be indicated to prevent alloimmunization in rh-negative patients receiving small amounts of rh-positive red cells, as in platelet concentrates. routine prophylaxis against large numbers of red cells, as in a unit of rh-positive wb or prbcs given by accident to an rh-negative recipient, is not reliable and usually involves the administration of large amounts, but instances of its effective use in these circumstances have been reported. higher doses of intravenous rhig have been used in the treatment of itp. plasma-derived colloids include human serum albumin (hsa), available in 5% and 25% solutions, and plasma protein fraction (ppf), available in a 5% solution. both are derived from pooled donor plasma but are essentially pathogen-free. hsa is composed of at least 96% albumin, whereas ppf is subjected to fewer purifi cation steps and contains at least 83% albumin, with correspondingly more globulins. the 5% solutions are iso-oncotic, whereas the 25% solution of hsa is hyperoncotic and requires infusion with crystalloid solutions. potential clinical indications for colloid solutions include hypovolemic shock, hypotension associated with hypoproteinemia in patients with liver failure or protein-losing conditions, as a replacement solution in plasma exchange or exchange transfusion, and to facilitate diuresis in fl uidoverloaded hypoproteinemic patients. albumin solutions are not indicated as a nutritional source to raise serum albumin. their use in some indications, particularly for resuscitation, has become controversial, and pulmonary edema has been reported in association with their infusion. 64 although albumin solutions are reasonably safe products to administer, expense and limited availability restrict their use. anaphylactic reactions have been reported in less than 0.1% of recipients. the use of ppf has been associated with severe hypotensive episodes, with hageman factor fragments or prekallikrein activator being demonstrated, 65 thus making ppf a less desirable resuscitation fl uid and contraindicated in cardiac surgery. granulocyte concentrates for transfusion are obtained from a single donor by cytapheresis methods, which generally involve the administration of hydroxyethyl starch and corticosteroids to the donor to improve granulocyte yield. granulocyte colony-stimulating factor (g-csf) has been added to some collection regimens and increases both cell counts and granulocyte survival substantially. each collection should contain at least 10 10 granulocytes 1,2 and is suspended in approximately 200 ml of plasma. a signifi cant number of red cells are present, so crossmatching for the recipient is required. because of the potential risk for graft-versus-host disease (gvhd), granulocytes are usually collected from hla-matched donors. granulocytes are stored at room temperature and must be transfused within 24 hours of collection, although sooner is better because of rapid deterioration of the cells. patients who may benefi t from granulocyte transfusions include those who are neutropenic (absolute neutrophil count of less than 0.5 ã� 10 9 /l) and those who are unresponsive to appropriate antibiotic treatment but in whom bone marrow recovery is expected to occur. a course of therapy generally involves daily infusion for 4 to 7 days. granulocytes have been used for progressive fungal infections in immunosuppressed granulocytopenic patients, in patients with defective leukocytes (e.g., chronic granulomatous disease), and in the neonatal icu for neonatal sepsis. randomized trials had suggested that granulocyte transfusions under these circumstances can reduce mortality, but such trials have not been conducted for more than 2 decades. 66 effective antibiotic regimens and the signifi cant adverse effects associated with the use of granulocyte concentrates, including pulmonary insuffi ciency related to alloimmunization and cmv infection, have limited their use in recent years. the decision to transfuse blood components, like any therapeutic maneuver, must be made with full awareness of the potential risk to the recipient, as well as the expected benefi ts. public expectations of a zero-risk blood supply help raise the acuity of physicians' decisions. for some patients, the benefi t from transfusion is so obvious that the associated risks pale in comparison to the consequences of withholding transfusion. however, the clinician's knowledge of the incidence and management of adverse reactions to transfusion is vital, not only to ensure the best patient care but also to provide appropriate patient education and true informed consent. almost every patient who receives an allogeneic blood transfusion will experience some adverse reaction if such universal effects as immunomodulation and bone marrow suppression are considered. measurable reactions to transfusion occur in about 20% of patients; more serious adverse responses may be expected in only 1% to 2% of transfusions. 67 the nature of these adverse reactions ranges from those that are common but clinically unimportant to those that may cause signifi cant morbidity or death (table 80-3) . transfusion in the icu is a common and often lightly regarded event. however, because the signs and symptoms of severe, life-threatening reactions are frequently indistinguishable from those of troublesome, but less signifi cant reactions, every transfused patient who experiences a signifi cant change in condition, such as an elevation in temperature, change in pulse or blood pressure, dyspnea, or pain, must be promptly and fully evaluated to identify the cause of the reaction and to institute treatment when necessary. the basic approach to all acute reactions should be to maintain a high index of suspicion for acute hemolytic reactions by stopping the transfusion immediately, maintaining venous access with intravenous fl uids, and informing the blood bank laboratory immediately so that the appropriate transfusion reaction protocol can be in stituted and post-transfusion specimens obtained. early recognition of severe transfusion reactions may be lifesaving. the most feared reaction to blood transfusion is intravascular hemolysis, caused by the recipient's complementfi xing antibodies attaching to donor rbcs with resultant rbc lysis. abo incompatibility is most often implicated in these incidents. intravascular hemolysis is still the single most common acute cause of fatalities associated with the transfusion episode. 68 in addition to hemolysis, complement activation stimulates the release of infl ammatory mediators and cytokines and thereby leads to hypotension and vascular collapse. activation of the coagulation system may result in dic. acute renal failure may also occur, presumably on the basis of immune complex interactions. morbidity and mortality are directly related to the quantity of incompatible blood transfused, which is why prompt recognition and cessation of transfusion cannot be overemphasized. misidentifi cation of the patient, or "clerical error," at any time beginning with the process of specimen acquisition through release of the unit and initiation of infusion is the major cause of acute intravascular hemolysis. 69, 70 this reaction is more likely to occur in critical care settings, such as the icu, operating room, and emergency department, than anywhere else in the hospital. it is far preferable to transfuse uncrossmatched group o red cells than to chance abo incompatibility caused by improper patient and specimen identifi cation procedures. the most common clinical sign of hemolysis is fever, with or without chills. 71 other common signs and symptoms include back or fl ank pain, anxiety, nausea, lightheadedness, dyspnea, and hemodynamic instability. in a comatose or anesthetized patient, many of these symptoms will not be evident; therefore, signs such as hypotension, hemoglobinuria, and diffuse oozing from puncture sites or incisions may be the only notable features. immediate management of hemolytic transfusion reactions must include cessation of the transfusion; the remainder of care is supportive. rapid verifi cation of patient and unit identifi cation must be made, not only to confi rm the suspected reaction but also to prevent a second patient from receiving a reciprocally incompatible unit if a clerical error has been made. desired end points of supportive care include maintenance of blood pressure, high urine output, and support of coagulopathy or further blood loss. steroids, heparin, or other specifi c pharmacologic interventions have no role in treatment. anaphylactic reactions to blood transfusions are fortunately rare but may be life-threatening. the usual cause is recipient antibody to a component of plasma that the patient lacks, most commonly antibody to iga in igadefi cient individuals. 72 signs and symptoms include severe malaise and anxiety, fl ushing, dizziness, dyspnea, bronchospasm, abdominal pain, vomiting, diarrhea, hypotension, and eventually shock. fever and hemolysis do not occur. management includes immediate cessation of transfusion and standard therapy for anaphylaxis. if anti-iga antibodies are determined to be the cause of this reaction, the patient must receive blood components donated by iga-defi cient individuals or, if unavailable, specially prepared washed rbcs and platelet concentrates. plasmaderived preparations, such as albumin, and ig contain varying amounts of iga and pose a substantial risk in these patients. febrile nonhemolytic reactions (fnhrs) are the most commonly occurring immediate transfusion reaction. these reactions are annoying to the clinician, patient, and transfusion service alike in that they can cause signifi cant discomfort and, because they share certain manifestations with acute hemolytic reactions, must be investigated in every instance. fnhrs occur in approximately 0.5% to 1.0% of transfusion episodes. 73 the etiologic factors are probably complex and multiple, but many reactions are caused by the release of cytokines and pyrogens, either within the transfused unit of blood or as a result of recipient antibodies to donor leukocytes. clinical signs include fever, with or without chills, usually beginning 1 to 2 hours after the start of the transfusion but occasionally delayed up to 4 to 6 hours. multiparous women and patients who are multiply transfused are particularly prone to fnhrs. the transfusion must be stopped and the appropriate transfusion reaction evaluation instituted. antipyretics such as acetaminophen may be administered. though commonly used, antihistamines such as diphenhydramine are neither preventive nor therapeutic. once acute hemolysis is excluded, transfusion of a new unit may be instituted. most patients will not experience a second such reaction. 73 if repeated reactions become problematic, leukocyte-depleted blood components may be supplied. the implementation of ulr results in a reduction in the frequency of all fevers seen after transfusion by only about 12%. 74 hives and pruritus are relatively common adverse effects of transfusion. 68 they are a hypersensitivity reaction localized to the skin, and their cause is unknown but may include both donor and recipient characteristics. these reactions consist of localized or generalized urticaria beginning shortly after the start of transfusion without other signs or symptoms of anaphylaxis or hemolysis. the transfusion should be temporarily stopped, and antihistamines may be administered. if the hives resolve in a short time, the same unit of blood may be cautiously restarted. if repeated urticarial reactions occur, premedication with antihistamines may be effective, or blood components washed to remove plasma may be required. intravascular volume overexpansion is particularly likely to occur in critical care patients with limited cardiac reserve. aside from the inherent volume of the blood components, the intravenous normal saline concurrently administered adds to the volume load. unfortunately, normal saline solution is the only intravenous fl uid that may be administered with blood components. with careful attention to transfusion requirements and the use of volume reduction maneuvers available to the transfusion service, volume overload can be minimized in most instances. the frequency of this complication of transfusion is not reported. delayed hemolysis is an uncommon but probably underrecognized reaction to transfusion that results from the stimulation of a primary or secondary (anamnestic) recipient antibody response to foreign rbc antigens. these antibodies are undetected at the time of transfusion but increase after transfusion in a manner analogous to the vaccination "booster" effect. these reactions typically occur 3 to 14 days after transfusion but are unrecognized because of the lack of a clear temporal association with transfusion. fever, chills, and an unexplained decline in hct are the usual signs. 75 transient elevation in bilirubin and lactate dehydrogenase may also occur. the diagnosis is established by a positive direct antiglobulin (coombs) test resulting from recipient antibody coating donor rbcs. the antibody may be identifi ed by eluting it from the rbcs or by demonstrating it within the recipient's serum. the specifi city of the antibody is often against such rbc antigens as the rh family, kidd, duffy, or kell systems. hemolysis may not occur, but if it does, it is likely to be extravascular and only rarely causes renal failure or dic. prevention of these reactions is diffi cult. alloimmunization to foreign rbc antigens occurs in approximately 1% of transfusions. 67 detection of delayed antibodies is the purpose for requiring a new blood bank specimen every 72 hours if the patient has recently been transfused. permanent transfusion records should record the occurrence of delayed antibodies, even though they may not be apparent at a later crossmatch. access to transfusion databases is critical for the care of patients with a past history of transfusion. transfusion-related acute lung injury (trali) is an uncommon (0.02%) 76 but serious adverse effect of transfusion that has only recently been gaining recognition. similar reactions have been called pulmonary leukoagglutinin reaction or noncardiogenic pulmonary edema. these reactions consist of acute respiratory distress syndrome (ards), which develops 1 to 6 hours after transfusion. signs and symptoms include bilateral pulmonary infi ltrates, hypoxemia, fever, and occasionally hypotension. monitored patients are found to have normal or low pulmonary wedge pressure and central venous pressure, as contrasted with patients experiencing volume overload. if adequate respiratory support and oxygenation are established promptly, spontaneous resolution generally occurs within 1 to 4 days. deaths have nonetheless occurred, particularly with a delay in diagnosis. 77, 78 episodes of trali appear to have several possible causative mechanisms. some cases may be caused by donor antibodies reacting with recipient neutrophil or hla antigens. 79 plasma factors related to blood storage have also been implicated, such as lipid substances from deterioration of donor cell membranes that prime recipient neu-trophils, which then damage the pulmonary vasculature and lead to increased capillary permeability and an ardslike syndrome. 80 other clinical factors may contribute to increased risk, such as cardiac bypass surgery or other procedures. in the antibody model at least, the implicated antibody is unique to the donor and the affl icted recipient will probably not experience another such reaction, provided that the recipient is not exposed to the same donor. trali is undoubtedly under-recognized in the critical care setting and may frequently be confused with fl uid overload or cardiogenic pulmonary edema. transfusion-associated gvhd (ta-gvhd) is a welldocumented, but probably under-recognized, highly lethal immunologic complication of blood transfusion. 81 immunocompromised patients infused with blood components containing viable donor lymphocytes are at risk for engraftment of the allogeneic lymphocytes and ensuing rejection of recipient (host) tissues. transfusion recipients who are at highest risk include neonates, especially the very premature, bone marrow and organ transplant recipients, and leukemia and lymphoma patients. ta-gvhd has also been reported in patients after cardiac surgery who received designated donor blood from relatives; presumably, the hla antigenic differences between donor and recipient were insuffi cient to stimulate a recipient immune response but suffi cient to elicit a donor immune response. 82 the onset of ta-gvhd is usually within 8 to 30 days after transfusion, and it is manifested as fever and rash, followed by diarrhea and evidence of liver and bone marrow injury. ta-gvhd differs from that seen in bone marrow transplantation (bmt) by its involvement of the marrow and by far greater mortality. treatment is largely ineffective, and mortality exceeds 90%. irradiation of blood components at 25 gy prevents ta-gvhd by eliminating the donor lymphocyte mitogenic response. all cellular blood components should be irradiated before transfusion to high-risk patients. the functions of the cellular components of blood are unaffected, although damage to rbc membranes limits postirradiation storage of prbcs. 83 blood donated by a relative for any patient should be irradiated, as should hla-matched or crossmatched platelet products. allogeneic blood transfusion has been shown to modulate and suppress the recipient's immune response, an effect fi rst noted with kidney transplantation. 84 immunosuppression in a critical care setting is generally undesirable, but whether transfusion has a signifi cant impact is debated. ongoing clinical issues center around two areas of controversy: the putative association between blood transfusion and increased numbers of postoperative infections and increased and more rapid rates of tumor recurrence in surgical oncology patients with certain malignancies. there has been no resolution of either issue despite a few prospective trials having been performed. the largest pro-spective trial of colorectal cancer resection, for example, is negative, 85 but a meta-analysis of the extant data suggests that an adverse effect on recurrence does exist. 86 similarly, most of the randomized trials of postoperative or critical care unit infections are too small to indicate an effect of transfusion, but all point in the direction of an adverse effect. 87, 88 controversy will continue until larger randomized trials are conducted. the precise mechanism of the immunosuppression induced by allogeneic transfusion has not yet been delineated, and several mechanisms may be involved. 89 alterations identifi ed in laboratory and clinical transfusion recipients have included depression of the t-helper/tsuppressor lymphocyte ratio, decreased natural killer cell activity, diminished interleukin-2 generation, formation of anti-idiotype antibodies, impairment of phagocytic cell function, and chronic persistence of donor lymphocytes (microchimerism), suggestive of low-level gvhd. difficulties in analysis of human data arise because patients requiring blood transfusions have conditions that themselves induce immune changes. there is some evidence, bolstered by the results of two large clinical trials, to suggest that leukocyte reduction of blood components reduces or eliminates this immunosuppressive effect. 90 proponents of this viewpoint argue that for this reason, ulr would benefi t most patients receiving blood transfusions and lead to fewer infections, tumor recurrences, and other related putative risks of transfusion, all potentially resulting in saving lives and cost. prospective trials will be extremely important. 91 public awareness of transfusion-associated acquired immunodefi ciency syndrome (aids) has done more to revolutionize transfusion practice than any other transfusion risk by resulting in more conservative blood use, more stringent donor selection criteria, and improved screening tests. the result is that viral transmission rates are now diffi cult to measure, and the risk of transfusionrelated infectious diseases is lower than ever. 92 the current best estimate is that 3 to 4 units per 10,000 will transmit some kind of infection 93 if agents such as cmv or epstein-barr virus are included. bacterial infection has become the most common infectious risk thanks to increasingly sensitive donor screening tests, including nucleic acid testing (nat) to detect viral dna or rna, which has shortened the infectious period and reduced the risk for post-transfusion hepatitis (pth) and other viral infections. several fatalities are reported yearly from the transfusion of blood components contaminated with viable, proliferating bacteria, with or without the accumulation of endotoxin. 94 platelet concentrates, because they must be stored at room temperature, are particularly prone to bacterial growth, with a reported incidence of 6 in 10,000 transfusions. 95 organisms isolated from platelets and implicated in fatal transfusion reactions include staphylococcus and streptococcus species and gram-negative bacilli. fatalities resulting from bacterial contamination of refrigerated rbcs have occurred as well and more often involve cryophilic bacteria. rbc transfusions contaminated by yersinia enterocolitica have been consistently reported for a decade. 96 transfusion reactions caused by bacterial or endotoxin contamination are fortunately quite rare, but mortality exceeds 60%. signs and symptoms of reactions caused by microbial contamination overlap those of hemolytic transfusion reactions and consist primarily of fever and hypotension, along with other signs of endotoxic shock. if recognized promptly, a gram stain of the implicated unit can be prepared immediately and, if positive, appropriate antibiotic and supportive therapy instituted. autologous blood components may also be contaminated at the time of collection; therefore, reactions occurring in patients who are receiving their own blood should not be dismissed but instead should be evaluated as fully as though the patients had received allogeneic blood. the success of viral screening measures is most clearly illustrated by the fall in the risk for pth over the past 2 decades. although pth continues to be a signifi cant cause of morbidity and mortality, the nature of pth has changed through the years with the stepwise institution of various donor screening measures. the elimination of paid donors in 1972 and the successive introduction of immunologic tests for hepatitis b have resulted in a steady reduction in the rates of pth caused by hepatitis b virus (hbv) to approximately 17 per million units of transfused blood products. although about 30% to 40% of hbv transmissions will result in acute hepatitis, chronic hbv infection develops in less than 10% of such patients. in contrast, the risk for chronic hepatitis c virus (hcv) infection after transfusion is higher, nearly 50%, and the long-term risk for cirrhosis-or hepatocellular carcinoma-related mortality is about 15% over more than 20 years after pth secondary to hcv. 97, 98 the clinical course of hepatitis a is generally milder, and the lack of a chronic carrier state means that with donor screening for symptoms of the acute illness, the risk of transmission is much lower, estimated at less than one in a million units. 99 the prevalence of hepatitis b surface antigenemia among fi rst-time blood donors is 0.7%, and the prevalence of hepatitis c antibodies in donors is approximately 0.1% to 0.5%. at this time, given the sensitivity of current screening assays, including the latest generation of enzyme immunoassays (eias) and nat, the current risk of pth resulting from hcv is believed to be about 1 in 150,000 or less. 100 although hbv is still implicated in pth (attributable to the seronegative "window" period in newly infected donors), the risk of transfusion-associated hepatitis b is about 1 in 200,000 units. 100 retroviruses, rna-based viruses characterized by their reverse transcriptase and integration into the host genome, and lentiviruses, a subset of retroviruses, are ubiquitous in animals and were initially identifi ed in humans in the early 1980s. those known to be capable of transmission by transfusion are hiv-1, hiv-2, and human t-cell leukemia/lymphoma virus (htlv) i and ii. transfusion-associated aids was initially reported in late 1982. 101 the fi rst report of an associated viral agent did not appear until late in 1983, and in march 1985 the screening enzyme-linked immunosorbent assay (elisa) to detect antibody to hiv-1 was licensed and immediately incorporated into the blood-screening process. improved confi dential donor screening appeared to decrease the risk of infectious units appearing in the donor pool. 102, 103 the discovery that heat treatment reduced transmission resulted in a reduction in transmission by plasma products, especially to persons with hemophilia. clinical aids developed in more than 90% of recipients of infected blood products, and the vast majority succumbed to the disease. removal of donor units with seropositivity by elisa was insuffi cient to prevent transmission of hiv-1; several hundred cases were reported annually after introduction of the elisa test. subsequent development of an assay for the p24 antigen and then nat has lowered the risk of transfusion-associated hiv-1 infection to less than one in a million (see table 80 -3). despite donor screening and sensitive assays, including eia, nat, and p24 antigen, an extremely small, but fi nite risk of hiv-1 transmission by screened blood transfusions remains. this risk is largely due to the seronegative "window" period experienced by newly infected donors, which is estimated to be an average of 16 days. 100 a second retrovirus, hiv-2, fi rst described in residents of countries in west africa and subsequently detected in migrants to western europe, causes an immunodeficiency syndrome similar to that caused by hiv-1. although very few cases of hiv-2 have been reported in the united states 104, 105 and there have been no reported transfusion-transmitted cases, experience with other retroviruses suggests that screening may prevent the majority of potential transmission. therefore, donated blood is now screened by an assay for the presence of antibody to hiv-2. the retrovirus htlv-i is the causative agent of adult t-cell leukemia (atl) and is strongly implicated in the chronic, progressive neurologic disorder termed tropical spastic paraparesis or htlv-i-associated myelopathy (tsp/ham). htlv-ii has been linked to hairy cell leukemia, but no transfusion-transmitted cases have been reported. the virus exhibits strong serologic crossreactivity with htlv-i such that screening assays fail to distinguish between antibodies to either virus. transfusion-transmitted htlv-i has been demonstrated. 106 tsp/ham has developed in a small percentage of infected transfusion recipients, but no transfusionassociated cases of atl have been seen. approximately 0.025% of donors in the united states are seropositive for htlv-i and htlv-ii 107 ; further testing reveals the majority of them to be htlv-ii. donated blood is currently screened for antibodies to htlv-i and htlv-ii. the estimated risk of htlv transmission by screened negative blood is believed to be 1 in 250,000 to 2 million. cmv is a human herpesvirus that establishes latent infection in the host's tissues, particularly leukocytes, and is transmitted by all cellular blood components. 108 seropositivity, or the presence of antibody, denotes previous exposure to the virus but does not confer protective immunity. secondary reinfection or reactivation of latent infection can occur. antibodies to cmv persist for life and serve as a marker indicating the potential for transmission of live virus. immunocompetent recipients of transfused cmvpositive blood experience minimal morbidity and mortality. the majority are asymptomatic, whereas a heterophile-negative mononucleosis syndrome may develop in a few. immunocompromised patients, however, may suffer life-threatening manifestations such as severe interstitial pneumonitis, gastroenteritis, hepatitis, or disseminated disease. several groups of patients are at particular risk (box 80-1), 109 and these patients should receive blood incapable of transmitting the virus. other patients may benefi t from cmv-negative blood as well, such as seronegative solid organ transplant recipients or autologous bmt patients. screening of donated blood for cmv is not routinely done but can be performed quickly if necessary. because the prevalence of donor seropositivity is quite high in some regions (50% to 70%), cmvseronegative blood may not be readily available. blood that is leukocyte depleted ("cmv safe") may be as effective as seronegative blood in the prevention of cmv transmission, although a recent meta-analysis of clinical trials comparing the two methods suggests that cmvnegative blood products might have a slight advantage over leukocyte-depleted products. 110 many blood-borne parasites may be transmitted by transfusion, although this is a rare occurrence in the united states because of donor screening questions and the low endemicity of implicated agents. 111 changing immigration patterns and worldwide travel, however, make transfusion-transmitted parasites an increasing concern. on a worldwide basis, malaria is the most important transfusion-transmitted infective organism, although only about three cases occur in the united states each year. such infections are manifested by delayed fever, chills, seronegative pregnant women seronegative premature infants weighing less than 1200 g seronegative allogeneic or autologous bone marrow transplant recipients seronegative transplant recipients of seronegative organs diaphoresis, and hemolysis, often masked by underlying medical conditions. fatalities have occurred. babesiosis, a tick-borne disease, is endemic in regions of the united states, especially the northeast, with a seroprevalence of about 4%. transfusion-transmitted cases have been reported, with asplenic or immunocompromised patients being particularly susceptible. with increases in the number of latin american immigrants to the united states, american trypanosomiasis (chagas' disease), which is endemic in latin american countries, has emerged as a potential pathogen. other parasitic diseases that have been transmitted by transfusion include toxoplasmosis, leishmaniasis, and lyme disease. parvovirus b19 has now been recognized as a pathogen capable of transmission by transfusion, with typical clinical fi ndings and the potential for severe hematologic complications. cases of epstein-barr virus infection with a typical mononucleosis-like illness have been reported after transfusion. west nile virus has also been transmitted by transfusion. h2n1 infl uenza, severe acute respiratory syndrome (sars), and other new viral infections should be capable of transmission by transfusion, although cases have not been reported and the prevalence of asymptomatic disease is unknown. a rising area of concern is the transmission of prion disease, either jacob-creutzfeldt disease or bovine spongiform encephalopathy (bse). donor referral criteria were implemented in 1987 for these diseases, and transmission of bse has been reported in the united kingdom. massive transfusion is defi ned as the administration of blood components in excess of one blood volume within a 24-hour period. in an average adult (70 kg), this represents approximately 10 units of wb or equivalent prbcs, crystalloid solution, and other components. massive transfusion, especially in the range of 20 or more units of blood products, causes complications not generally seen in usual transfusion practice: accumulation of undesirable substances present within banked blood and dilutional depletion of normal blood constituents that are lacking in stored units. trauma victims, surgical patients undergoing extensive procedures, and patients with vascular or coagulation disorders may be massively transfused in the critical care setting. survival of the massive transfusion episode is determined more by the nature and degree of the patient's injuries or medical conditions than by the transfusions themselves, but the presence of adverse effects of massive transfusion can complicate patients' courses in the icu. transfusion of large quantities of stored blood defi cient in functional platelets often results in hemostatic defects or outright thrombocytopenia. circulating platelets consistently decrease in inverse proportion to the amount of blood administered, with the hemostatically signifi cant level of 50 ã� 10 9 /l reached after 20 u. 112, 113 functional defects have also been noted, and the bleeding time is prolonged. 114 despite these laboratory changes, severe diffuse bleeding develops in less than 20% of massively transfused patients, and no laboratory studies predict those who will. prophylactic platelet transfusion has not been shown to be of benefi t. 115 platelet counts may return to hemostatically effective levels quickly in patients with normal marrow function. currently, resuscitation of massively bleeding patients is most often accomplished with prbcs in combination with crystalloid solution. this should result in hemodilution to about 60% of normal plasma factor levels after the transfusion of about 10 units; this factor level can effect normal hemostasis. in reality, however, crystalloids may be given in excess of prbcs, so after 10 units is transfused, less plasma protein may remain. bleeding is unlikely until prothrombin time (pt)/inr and ptt prolongations exceed 1.5 to 1.8 times the midpoint normal range, the equivalent of an inr approaching 2.0. 113 as with platelets, prophylactic administration of ffp has not proved effective in preventing diffuse bleeding. 116 thus, the decision to transfuse should be made on an individual basis, as determined by the presence of bleeding or unacceptable risk in patients with documented abnormalities in coagulation. one new area of controversy in the treatment of patients with massive hemorrhage is the use of recombinant activated factor vii. this new agent was created for the treatment of hemophiliac patients with high titers of antibodies to factor viii, which makes them unable to benefi t from transfusion of recombinant factor viii. activated factor vii bypasses that problem by binding to tissue factor and directly activating thrombin and hence generating fi brin. 117 it is extremely expensive, has a short half-life, and carries a risk of inducing pathologic thrombosis, with potentially grave consequences. nevertheless, in numerous case reports, this new agent appears to potentially be benefi cial if used early in the resuscitation of massively injured patients. unfortunately, its unsupervised use has also resulted in thrombotic complications and relative lack of success, both of which suggest that carefully controlled clinical trials are appropriate. 118 blood preservative solutions contain excess citrate, which anticoagulates stored blood by binding ionized calcium. wb contains approximately 1.8 g of citrate/citric acid per unit in the plasma fraction. patients with normal liver function can metabolize the citrate load in 1 unit of wb in 5 minutes, but hepatic impairment may extend removal to 15 minutes or longer. toxicity may result when citrate is administered in excess of the metabolic rate, thereby causing a decrease in ionized calcium levels. 119 although paresthesias, cramps, and myoclonus accompany citrate excess, the chief danger of hypocalcemia is depression of myocardial contractility and potential prolongation of the qt interval. because the effects of citrate are transient and the use of prbcs containing little residual citrated plasma is far more common than massive transfusion with wb, routine administration of calcium is not indicated; clinically signifi cant rebound hypercalcemia may result. calcium infusion should be limited to hypoperfused patients with hepatic or cardiac failure who manifest citrate toxicity, and careful monitoring is essential. as potassium leaks from rbcs during storage, up to 7 meq of extracellular potassium may accumulate in each unit. however, dangerous levels of potassium rarely develop in adults from stored blood; the potassium level is more likely to be determined by the patient's acid-base status. 117 studies of massively transfused patients have demonstrated a wide range of potassium levels, with hypokalemia seen as frequently as hyperkalemia. because of the many physiologic mechanisms altered during resuscitation, including those of the respiratory, renal, cardiac, and hepatic systems, it is impossible to predict the net effect of massive transfusion on serum potassium levels. the ph of banked blood drops during storage, from 7.16 at the time of collection to as low as 6.73 after several weeks of storage. administration of large quantities of acidic blood, together with the metabolic acidosis common in these patients before resuscitation, would lead one to expect worsening acidosis as the outcome of massive transfusion. however, patients are more likely to exhibit metabolic alkalosis at the end of the transfusion episode, 120,121 partly because of improved tissue perfusion and the metabolism of citrate and lactate to bicarbonate. patients in renal failure may be unable to handle the bicarbonate load and require dialysis. acidosis persisting after transfusion suggests inadequate tissue perfusion. 119 empiric administration of bicarbonate to counter the acid load is not warranted and may contribute to the deleterious effects of hypercapnia in patients with impaired ventilation. as discussed previously, the level of rbc-associated 2,3-dpg in banked blood declines during storage, which increases the affi nity of hemoglobin for oxygen and thereby results in decreased oxygen off-loaded to tissues. even in massively transfused patients, it has been diffi cult to document a clinical impact of this shift, and no reliable method for restoring red cell 2,3-dpg has been developed. wb and prbcs are stored at approximately 4⺠c and require 30 to 45 minutes to warm to room temperature. elective transfusions at standard fl ow rates are tolerated without the need to warm the blood; however, core body temperature, measured by esophageal probe, can fall to 30⺠c or lower with the administration of large volumes of cold blood over a period of 1 to 2 hours. 122 adverse effects of hypothermia include a decreased heart rate and myocardial contractility, cardiac arrhythmias, increased affi nity of hemoglobin for oxygen resulting in decreased tissue oxygen delivery, dic, and impaired ability to metabolize the citrate load of stored blood. both blood warmers and patient warming may be instituted during massive transfusion, and patient core temperature should be monitored during such resuscitative efforts. whether massive transfusion in and of itself is a cause of ards is another source of controversy. there are certainly theoretical reasons why massive transfusion might precipitate ards: all cellular transfusions contain damaged or activated wbcs, cell membranes, aggregated platelets, and microthrombi, all of which are capable of lodging in and damaging pulmonary capillaries. despite this possibility, neither microfi ltration of transfusions nor routine leukocyte depletion has shown a signifi cant impact on the incidence of ards in massively transfused patients. 123 certainly, other causes of ards exist in patients who undergo massive transfusion, and the possibility of volume overload and trali should be considered in the evaluation of patients with hypoxia and diffuse pulmonary infi ltrates after massive transfusion. management of such patients is supportive, consistent with the overall management of massive transfusion. 124, 125 autoimmune hemolytic anemia patients with autoimmune hemolytic anemia (aiha) have an autoantibody, usually of broad specifi city, that fi xes itself to their rbcs and triggers extravascular immune-mediated destruction. patients with aiha have a positive direct antiglobulin test 126 (dat, commonly known as the coombs test) and varying degrees of he molysis, and their autoantibodies cause agglutination of rbcs from all donors during crossmatching. if the hemolysis is brisk, patients may require red cell transfusion to support oxygen needs before medical management of the aiha is effective. hence, transfusion is diffi cult because agglutination during crossmatching interferes with proper defi nition of compatible units of rbcs and because the transfused rbcs are themselves subject to the same immune hemolysis as the host rbcs. many blood banks have methods for depletion of autoantibodies from the recipient's plasma and elution of antibodies from rbcs to arrive at a proper crossmatch. 127 although such crossmatches are time consuming and not generally available on an emergency basis, they can be lifesaving. criteria for transfusion should remain the same as for other recipients. rbcs are crossmatched for red cell antigens in the abo and rh 0 (d) group and for other red cell antigens when antibodies are present. however, there are several hundred other red cell antigens in the human family, and with repeated transfusion recipients may become alloimmunized to other antigens. generally, alloimmunization occurs in approximately 1% of transfusions, 68 but the prevalence of alloantibodies is higher in chronically transfused, relatively immunocompetent patients, especially african americans, whose distribution of red cell antigens has signifi cant variation from the white population. alloimmunization rates of 30% or higher may be found in chronically transfused patients with hemoglobinopathies who have not received rbcs matched to potent minor antigens such as kell, duffy, and lewis. alloimmunization may present diffi culties in crossmatching of blood, to the point that compatible blood must be obtained from raredonor registries, if at all. other patients present unresolved serologic problems in that the alloantibody is never precisely identifi ed yet the majority of blood available for transfusion is incompatible. the delay engendered by working with multiple or unidentifi ed antibodies may be unacceptable in some critical care situations in which the need for oxygen-carrying capacity leaves no choice but to transfuse incompatible blood. the behavior of these antibodies in the laboratory may assist in predicting the clinical outcome of the incompatible transfusion. 128 special procedures such as clearance studies, 129 fl ow cytometry 130 and in vivo crossmatching (cautious administration of a small aliquot of blood, with subsequent observation of serum and urine for evidence of hemolysis) are useful if time permits. emergency transfusion of type o, rh-negative uncrossmatched blood is generally reserved for the resuscitation of trauma patients, for whom the delay in crossmatching may be life-threatening. the risks of alloimmunization are generally accepted as low. even rh-positive type o rbcs may be used because rates of alloimmunization to rh 0 (d) are low under the circumstances of emergency transfusion. 128, 131 dic can present the clinician with diffi cult therapeutic choices. this common disorder in critically ill patients may be manifested as severe hemorrhage or thrombosis. therapy is primarily directed at alleviating the cause and supporting the patient. supportive therapy includes the transfusion of components needed to correct the bleeding diathesis caused by the consumption of platelets and fi brinogen, in addition to prbcs to restore oxygencarrying capacity. platelets and fi brinogen (as cryoprecipitate) are the most useful components needed to repair the coagulopathy, but their use risks merely "fueling the fi re" and increasing the microthrombosis of dic. heparin anticoagulation is controversial 132, 133 and may increase the risk of bleeding, especially if depleted factors are not replenished. no defi nitive clinical trials have endorsed the routine use of heparin, and randomized trials of other components and coagulation inhibitors have uniformly been negative. in general, the use of heparin and antifi brinolytic agents has been confi ned to the most severe and protracted cases of dic. 134 cirrhotic patients or those with fulminant hepatic failure have a variety of hemostatic disorders that complicate transfusion management of a bleeding patient. 135 hepatic synthesis of coagulation factors may be markedly diminished, thereby necessitating replacement by ffp or cryoprecipitate. patterns of factor diminution may vary between acute hepatic necrosis and chronic cirrhosis. 136 associated hemodynamic alterations may make it impossible to administer the volumes required for effective hemostasis, however, and any effect is transient. the use of factor concentrates or antifi brinolytic agents may precipitate thrombosis. activation of fi brinolysis and decreased clearance of activated factors may produce or mimic chronic dic, thus further exacerbating the factor defi ciencies and impairing coagulation. abnormal platelet function and thrombocytopenia may contribute to the coagulopathy of liver disease, with concomitant splenomegaly reducing the effectiveness of platelet transfusions. bleeding in uremic patients is exacerbated by an acquired platelet defect, in part secondary to dialyzable circulating molecules soluble in platelet membranes. plateletassociated vwf and plasma high-molecular-weight vwf multimers have also been shown to be decreased, 137 which may explain the benefi t shown by ddavp 138 and cryoprecipitate in shortening the bleeding time and improving hemostasis in some uremic patients. raising the hct by red cell transfusion in anemic patients has also been shown to shorten the bleeding time, presumably as a result of blood vessel wall-laminar blood fl ow interaction. transfusion of platelets in the absence of thrombocytopenia is unlikely to be of benefi t because the transfused platelets rapidly become dysfunctional. more aggressive hemodialysis is the most widely accepted method of reducing platelet dysfunction. bmt patients are vulnerable to the severe infectious and toxic side effects of ablative treatment and hence may be cared for in critical care units. these patients may have intensive red cell and platelet transfusion requirements and need specialized products such as cmv-negative and irradiated blood components. a blood bank problem uniquely encountered in bmt is the need to switch the patient's abo group because of an abo-mismatched transplant, thus necessitating an exchange transfusion of red cells and plasma-containing products (i.e., platelet concentrates) of differing abo type to avoid hemolysis of donor and recipient cells. bmt patients may also manifest an increased rate of delayed hemolytic reactions 139 as donor "passenger" lymphocytes recognize recipient or transfused red cell antigens. patients should be monitored particularly closely between days 10 and 20 after a minormismatched allogeneic transplant, and aggressive transfusion should be undertaken if the hemoglobin level falls and the dat result becomes positive. the safest transfusion is one that is not given. therefore, alternatives to blood component therapy continue to be sought and are valuable adjuncts in some instances. it is possible to limit homologous blood exposure by the appropriate use of pharmacologic agents that promote hemostasis and the administration of recombinant hematopoietic growth factors or biologic growth modifi ers to stimulate marrow hematopoiesis. only one substitute for rbc transfusions has been approved in the united states, a polyfl uorocarbon oxygen carrier with signifi cant limitations as a blood substitute. 140 other preparations that have been explored in clinical trials are cell-free hemoglobin solutions cross-linked or polymerized by chemical manipulation to prevent rapid clearance from the circulation. they are intended to provide short-term oxygen-carrying capacity for acutely ill patients and have the advantage of not requiring crossmatching or infection control. although these proposed products may have a longer shelf-life and are easier to transport, their drawbacks are many. most have a circulatory half-life of only about 24 hours. the oxygen dissociation curve for these substitutes is also frequently not favorable: either a high fio 2 is required to "load" these molecules or they are less likely to deliver oxygen efficiently at lower po 2 levels. 141 because the hemoglobin source is reclaimed bovine or human red cells, it is unlikely that patients who do not accept blood components because of their religious beliefs (jehovah's witnesses) will accept these types of hemoglobin solutions. one product in development uses recombinant technology to generate hemoglobin, and it is hoped that this solution may be acceptable to these patients. the licensed perfl uorocarbon solutions have failed to demonstrate any utility as intravascular oxygen carriers because of their unfavorable p-50 (oxygen half-saturation pressure) and oxygen off-loading characteristics. they are fi nding limited application in regional oxygenation during angioplasty or stent placement procedures and a more novel use in "liquid ventilation." this involves the ventilation of intubated patients experiencing severe pulmonary compromise with superoxygenated perfl uorocarbon solutions in place of oxygen-enriched air. 142 the synthetic vasopressin analogue ddavp increases plasma factor viii : c and promotes the release of vwf from endothelial stores. 143 ddavp has provided effective hemostasis in bleeding patients with mild hemophilia a and type i von willebrand's disease and has been used as prophylaxis for patients undergoing surgery. ddavp reportedly improves platelet function in some patients with qualitative platelet disorders associated with uremia, 136 cirrhosis, and aspirin ingestion. studies of its effi cacy in cardiopulmonary bypass procedures are confl icting, but a subset of these patients may benefi t. the chief drawback to its use is tachyphylaxis, which develops in essentially all cases after short-term repeated administration. the lysine analogues îµ-aminocaproic acid and tranexamic acid inhibit fi brinolysis by blocking the binding of plasminogen and plasmin to fi brin. these antifi brinolytic agents may decrease bleeding and thus the need for homologous blood components in patients with hemophilia, thrombocytopenia, and systemic fi brinolysis. a novel and effective use of tranexamic acid involves administration as a mouthwash in preparation for oral surgery in patients with hemophilia or those receiving oral anticoagulant therapy. 144 the most serious side effect of these agents when systemically administered is thrombosis; thus, it is important to use them appropriately and monitor the patient carefully during their use. aprotinin is a naturally occurring bovine serine protease inhibitor that acts on plasma serine proteases such as plasmin, kallikrein, trypsin, and some coagulation proteins. aprotinin has been shown to reduce blood loss in patients undergoing cardiopulmonary bypass surgery 145 by inhibiting fi brinolysis and preventing platelet damage. however, more recent reports of renal injury and longterm mortality may mean an end to its use. 146 aprotinin has been used extensively in liver transplantation, which involves high blood loss. repeated administration poses the risk of anaphylaxis and renal dysfunction. when time permits, vitamin k is the preferred agent to reverse the coagulopathy induced by oral anticoagulants. normalization of the pt can be seen in as few as 6 to 12 hours. additionally, selected cirrhotic patients may exhibit improvement in the pt when treated with therapeutic doses of vitamin k. many patients in critical care units exhibit a prolonged pt, especially if dietary supplements are limited and broad-spectrum antibiotic therapy is given. vitamin k is a safe and effective agent for reversing this effect. recombinant erythropoietin (epo) has dramatically reduced the red cell transfusion requirements of patients in chronic renal failure. epo also has applications in the adjunctive treatment of the anemia of premature infants and the anemia of chronic disease, especially rheumatoid arthritis, cancer, and aids. studies of its effi cacy in reducing perioperative red cell transfusion requirements by increasing the yield of predeposited autologous blood or stimulating bone marrow synthesis after surgery have shown benefi t in reducing blood transfusion, although preoperative planning and autologous deposits are required. 147 in contrast and probably because the impact of epo is not immediate, the effi cacy of epo in the icu is unproven and awaits the results of large clinical trials. recombinant growth factors such as granulocytemacrophage colony-stimulating factor (gm-csf) and g-csf stimulate marrow production of leukocytes by enhancing several different granulocyte and macrophage functions. these agents are fi nding application in reducing the neutropenic period in bmt and cancer chemotherapy by increasing the leukocyte count in hypoproliferative marrow conditions. these myeloid growth factors are replacing granulocyte transfusions for their few remaining indications. cell salvage equipment has been in clinical use for several decades, and although cell salvage is clearly capable of rescuing otherwise "lost" red cells, its full impact on transfusions has been poorly documented. cell salvage generally consists of collection of shed blood from a clean, uncontaminated operating fi eld, followed by removal of the cellular elements and retransfusion into the patient. cell salvage has been used both intraoperatively and postoperatively, especially in cardiac surgery. although the clinical studies of cell salvage have many fl aws, the overall success of this therapy in reducing transfusion has resulted in its wide application. 148 risks include bacterial contamination, febrile reactions, triggering of dic, and coagulopathy as a result of dilution. when combined with acute intraoperative hemodilution, this technology is also potentially cost saving. 149 the word apheresis is derived from the greek aphairein, "to take away"; thus, therapeutic hemapheresis is performed to remove unwanted plasma constituents (plasmapheresis) or blood cells (cytapheresis). automated cell separators use centrifugation or membrane fi ltration to remove and concentrate the selected blood element. many of the same devices used to prepare apheresis blood components for transfusion are used to perform patient procedures, so therapeutic apheresis is often administered under the auspices of the transfusion medicine service. rapid removal of plasma or cells may fi nd several applications in intensive care practice (box 80-2). the goal of plasmapheresis, or plasma exchange (pe), is to remove or reduce the levels of an undesirable plasma constituent or, alternatively, by means of plasma replacement, to supply a missing substance. the agent to be removed by pe is thought to be an autoantibody in some of the neurologic, renal, or hematologic conditions treated in this manner. 150 immunomodulation by pe is another explanation for its effect, a theory indirectly supported by the equivalent effi cacy of ivig therapy for several of these disorders. 151 pe for the amelioration of hyperviscosity from either excess igm in waldenstrã¶m's macroglobulinemia or excess ig in multiple myeloma is an effective temporizing measure in the treatment of these conditions. 152 plasmapheresis with pe is the standard therapy for ttp. 153 unfortunately, few controlled trials of pe exist, although anecdotal reports abound. pe is seldom the defi nitive treatment of most of these conditions and is used most appropriately as a short-term adjunct to other medical modalities. the kinetics of pe predicts that a one-volume exchange removes 65% of a given plasma constituent if the blood volume does not change or additional synthesis or mobilization of the substance does not occur. two or three volume exchanges remove 87% and 95%, respectively. highly protein-bound, intravascularly concentrated substances are most effi ciently removed, whereas substances with a large volume of distribution such as igg, active synthesis, or large extravascular stores are removed at less than predicted rates. the usual short-term intense course of pe schedules fi ve one-volume exchanges (approximately 3 l in normal-sized adults) over a 7-day period. the appropriate replacement fl uid in most conditions is an albumin-saline mixture, which provides oncotic support without the risk of disease transmission borne by ffp. pe in patients with ttp uses replacement with ffp to supply the plasma protease that is consumed during the disease. side effects of pe are relatively common (10% to 30% of procedures) but generally minor and are related to vascular access, temporary discomfort, or vasomotor symptoms. 154 patient death is rarely due to the procedure itself but is largely of cardiopulmonary causes. plasma proteins such as coagulation factors, immunoglobulins, and complement will be removed by pe, and laboratory test results of coagulation and electrolytes may be deranged in the hours after pe. clinical bleeding is rarely observed. most coagulation factors do not fall below hemostatic levels and recover within hours, with the exception of fi brinogen, which may require several days for complete replenishment. leukapheresis may be required to urgently reduce the wbc count in patients with acute myeloid or lymphoblastic leukemia or chronic myelogenous leukemia with peripheral counts of 100 ã� 10 9 /l or greater. each procedure is expected to drop the count by a third, but the effect is short lived. leukapheresis should be reserved for use only as an adjunct to chemotherapy in patients with pulmonary or cerebral leukostasis or for cytoreduction before chemotherapy in patients at risk for severe tumor lysis syndrome. plateletpheresis may be benefi cial as short-term therapy in patients with symptomatic thrombocythemia manifested as cerebral or myocardial ischemia, pulmonary emboli, or gastrointestinal bleeding. each procedure should effect a 50% reduction in the platelet count. cytotoxic therapy should be started concomitantly as the defi nitive treatment. litigation related to blood transfusion has become prominent, particularly after the epidemic of transfusionassociated aids. 155 most states regulate blood banking and medical practice, but blood products are regarded as symptomatic hyperviscosity thrombotic thrombocytopenic purpura neurologic diseases: myasthenia gravis, guillain-barrã© syndrome uncontrolled systemic vasculitis with critical end-organ injury symptomatic leukocytosis symptomatic thrombocythemia sickle cell anemia crisis (pulmonary or central nervous system manifestations) a service, not as a commodity, so standard product liability does not pertain to blood components. 156 however, negligence in the course of preparing, testing, transferring, crossmatching, or administering blood products is still a potential cause for legal action. every clinician who orders transfusions must be aware that blood components, like drugs, are approved for specifi c uses and that the indications should be clearly documented in the medical record. the informed consent of the patient is an important area of potential liability. the joint commission on accreditation of healthcare organizations (jcaho) has required written patient consent for blood transfusions since 1996. what constitutes adequate informed consent and who is responsible for advising the patient are still in contention. elements of informed consent include an understanding of the need for transfusion, its risks and benefi ts, and the alternatives, including the risk of not undergoing transfusion, as well as the opportunity to ask questions. whether the clinician documents informed consent with an individual progress note in the patient record or with a standardized form is generally established as institutional policy. similarly, institutions vary with respect to policies for consenting adults who are temporarily incompetent, such as sedated patients in the icu. a competent adult patient may refuse blood transfusion, and jehovah's witnesses commonly do so for religious reasons. case law is clear in upholding this right of the patient, 157 which extends to care given at such time as the patient may become incompetent (i.e., comatose) after such refusal was expressed before becoming incompetent. courts will usually order a lifesaving transfusion for minors. exceptions have been made in the case of some "emancipated minors" who are at the age of reason. most states have evoked a "special interest" in the welfare of a fetus in ordering transfusions to pregnant women. the advent of sentinel event reviews and other quality management procedures for patient safety has had an impact on transfusion practice as well. procedures for patient identifi cation before surgical procedures, including devices such as bar code readers, have also been applied to transfusion practice. however, annual sentinel event reviews reporting transfusion errors have remained constant according to jcaho records. 158 â�  blood components should be prescribed like drugs. appropriate blood component therapy requires that the specifi c blood product needed for a clear indication be prescribed, with avoidance of a formulaic approach. â�  red blood cells should be transfused only to increase oxygen-carrying capacity. transfusion decisions should be based on individual patient physiology. the majority of patients with hemoglobin levels greater than 60 or 70 g/l will not require transfusion unless they have limited cardiopulmonary reserve or active bleeding. â�  platelet transfusions are indicated for patients who are bleeding because of thrombocytopenia or functional platelet defects. guidelines for platelet transfusion are also conservative. prophylactic platelet transfusion remains controversial and is not warranted in many situations. â�  fresh frozen plasma is indicated for the repletion of coagulation factors in bleeding patients defi cient in those factors or to provide specifi c plasma proteins that cannot be obtained from safer sources. â�  cryoprecipitate is a concentrated source of fi brinogen and selected coagulation factors. cryoprecipitate may be more helpful in correcting the hypofi brinogenemia of dilutional or consumptive coagulopathy than fresh frozen plasma. â�  adverse reactions to blood components occur in 1% to 2% of transfusion episodes. adherence to routine protocols for the evaluation of transfusion reactions may save lives. â�  acute hemolytic reactions are the leading cause of immediate transfusion fatalities. prevention of these reactions requires strict adherence to transfusion and patient identifi cation procedures. â�  transmission of infectious agents by transfusion has been markedly reduced, and bacterial infection is now the most common infectious complication of transfusion. â�  adverse effects unique to massive transfusion are likely to occur in the icu and complicate the management of critically ill or severely injured patients. component therapy for such patients should remain conservative. the emerging role of activated factor vii in the treatment of these patients requires further evaluation. â�  informed consent for blood transfusion is a standard of practice. a competent adult has the legal right to refuse blood transfusion. consent in critically ill patients remains subject to individual institution policies. department of health and human services, food and drug administration: the code of federal regulations, 21 cfr parts 600, 606, 640 standards for blood banks and transfusion services markers for transfusiontransmitted disease in different groups of blood donors comparative safety of units donated by autologous, designated and allogeneic (homologous) donors directed blood donations: con goldfi nger d: directed blood donations: pro shelf-life of bank blood and stored plasma with special reference to coagulation factors generation of cytokines in red cell concentrates during storage is prevented by prestorage white cell reduction universal wbc reduction: the case for and against chemical and hematological changes in stored cpda-1 blood restoration in vitro of erythrocyte adenosine triphosphate, 2,3-diphosphoglycerate, potassium ion, and sodium ion concentrations following the transfusion of acid-citrate-dextrose 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extraction ratio: a valid indicator of transfusion need in limited coronary vascular reserve? for the abc investigators: anemia and blood transfusion in critically ill patients the crit study: anemia and blood transfusion in the critically ill-current clinical practice in the united states red cell transfusion practice following the transfusion requirements in critical care (tricc) study: prospective observational cohort study in a large uk intensive care unit appropriateness of red blood cell transfusion in australasian intensive care practice silent myocardial ischaemia and haemoglobin concentration: a randomized controlled trial of transfusion strategy in lower limb arthroplasty mathematical analysis of isovolemic hemodilution indicates that it can decrease the need for allogeneic blood transfusion guidelines for perioperative red blood cell transfusions american society of anesthesiologists task force: practice guidelines for blood component therapy prudent strategies for elective 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survey of transfusion-associated graft-versus-host disease in immunocompetent recipients the effect of prestorage irradiation on post-transfusion red cell survival improvement of kidney-graft survival with increased numbers of blood transfusion blood transfusion-modulated tumor recurrence: first results of a randomized study of autologous versus allogeneic blood transfusion in colorectal cancer surgery transfusion-associated cancer recurrence and postoperative infection: meta-analysis of randomized, controlled clinical trials transfusion practice and nosocomial infection: assessing the evidence transfusion increases the risk of postoperative infection after cardiovascular surgery immunosuppressive effects of blood transfusion transfusion of leukoreduced red blood cells may decrease postoperative infections: two meta-analyses of randomized controlled trials transfusion immunomodulation or trim: what does it mean clinically? risks of blood transfusion transfusiontransmitted cytomegalovirus and epstein-barr virus diseases current status of microbial contamination of blood components: summary of a conference septic reactions to platelet transfusions: a persistent problem red blood cell transfusions contaminated with yersinia enterocolitica-united states, 1991-1997, and initiation of a national study to detect bacteria-associated transfusion reactions routes of infection, viremia, and liver disease in blood donors found to have hepatitis c infection clinical outcomes after transfusionassociated hepatitis c adverse consequences of blood transfusion: quantitative risk estimates stramer sl: current prevalence and incidence of infectious disease markers and estimated window-period risk in the american red cross blood donor population possible transfusionassociated acquired immune defi ciency syndrome (aids): california impact of explicit questions about high-risk activities on donor attitudes and donor referral patterns. results in two community blood centers the effectiveness of the confi dential unit exclusion option human immunodefi ciency virus type 2 infection in the united states: epidemiology, diagnosis, and public health implications update: hiv-2 infection among blood and plasma donors-united states transmission of human tlymphotropic virus types i and ii by blood transfusion a prospective study of transmission by transfusion of htlv-i and risk factors associated with seroconversion post-transfusion cytomegalovirus infections reducing the risk for transfusion-transmitted cytomegalovirus infection is white blood cell reduction equivalent to antibody screening in preventing transmission of cytomegalovirus by transfusion? a review of the literature and meta-analysis transmission of parasitic infections by blood transfusion hemostasis in massively transfused trauma patients laboratory hemostatic abnormalities in massively transfused patients given red blood cells and crystalloid serial changes in primary hemostasis after massive transfusion prophylactic platelet administration during massive transfusion clotting factor levels and the risk of diffuse rnicrovascular bleeding in the massively transfused patient potential role of recombinant factor viia as a hemostatic agent recombinant factor viia: unregulated continuous use in patients with bleeding and coagulopathy dues not alter mortality and outcome massive blood replacement: correlation of ionized calcium, citrate, and hydrogen ion concentration potassium levels, acid-base balance and massive blood replacement acid-base status of seriously wounded combat casualties: resuscitation with stored blood blood temperature: a critical factor in massive transfusion an in vivo evaluation of microaggregate blood fi ltration during total hip replacement massive transfusion as a risk factor for acute lung injury: association or causation? guidelines on the management of massive blood loss autoimmune hemolytic anemia approaches to selecting blood for transfusion to patients with autoimmune hemolytic anemia the clinical implications of platelet transfusions associated with abo or rh(d) incompatibility survival curves of incompatible red cells: an analytical review isotype-specifi c detection of abo blood group antibodies using a novel fl ow cytometric method use of rh positive blood in emergency situations pharmacologic agents in the management of bleeding disorders disseminated intravascular coagulation. approach to treatment the pathogenesis and management of disseminated intravascular coagulation coagulation disorders in liver disease new insights into haemostasis in liver failure plasma and platelet von willebrand factor defects in uremia deamino-8-d-arginine vasopressin shortens the bleeding time in uremia donor-derived red blood cell antibodies and immune hemolysis after allogeneic bone marrow transplantation fluosol-da as a red-cell substitute in acute anemia the prospect for red cell substitutes low-dose perfl uorocarbon: a revival for partial liquid ventilation? response of factor viii/von willebrand factor to ddavp in healthy subjects and patients with haemophilia a and von willebrand's disease management of oral bleeding in haemophiliac patients amelioration of the bleeding tendency of preoperative aspirin after aortocoronary bypass grafting for investigators of the multicenter study of perioperative ischemia research group: mortality associated with aprotinin during 5 years following coronary artery bypass graft surgery does the use of erythropoietin reduce the risk of exposure to allogeneic blood transfusion in cardiac surgery? a systematic review and meta-analysis cell salvage for minimizing perioperative allogeneic blood transfusion cost-effectiveness of cell salvage and alternative methods of minimizing perioperative allogeneic blood transfusion: a systematic review and economic model plasmapheresis in nephrology: an update national institutes of health consensus conference: the utility of therapeutic plasmapheresis for neurological disorders correction of hyperviscosity by apheresis improved survival in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome therapeutic plasma exchange as a nephrological procedure: a singlecenter experience a review of transfusion-associated aids litigation: 1984 through 1993 legal, fi nancial, and public health consequences of hiv contamination of blood and blood products in the 1980s and 1990s legal aspects of transfusion of jehovah's witnesses joint commission on accreditation of hospitals and healthcare organizations: sentinel event statistics: available at key: cord-329176-av4qhu4f authors: liu, nanyang; zhang, tingting; ma, lina; wang, huican; li, hao title: association between abo blood groups and risk of coronavirus disease 2019: a protocol for systematic review and meta-analysis date: 2020-08-14 journal: medicine (baltimore) doi: 10.1097/md.0000000000021709 sha: doc_id: 329176 cord_uid: av4qhu4f background: the 2019 coronavirus disease (covid-19) pandemic has threatened millions of people worldwide. growing evidence suggests that the abo blood type contributed to the susceptibility of covid-19, but the results are controversial. the major objective of this systematic review and meta-analysis study is to investigate the impact of abo blood group on covid-19 pneumonia. methods: two independent reviewers searches the databases of the china biology medicine disc, china national knowledge infrastructure, china science and technology periodical database, wanfang database, pubmed, embase, and web of science from the date of conception to june 30, 2020. we will manually search for gray literature, such as meeting records and dissertations. two independent reviewers will screen studies that meet the criteria, extract data, statistical data, and assess the risk of bias. the dichotomous variable will calculate the odds ratio and the corresponding 95% confidence interval. heterogeneity between included studies will be assessed by heterogeneity χ(2) tests and i(2) index. the forest plots will be used to describe the pooled results. the begg rank correlation test or egger linear regression test will be performed to quantize the publication bias. discussion: this study will provide high-quality evidence to evaluate the contribution of the abo blood group in covid-19 pneumonia infection. prospero registration number: crd42020195615 since the outbreak of the 2019 coronavirus disease (covid19) in wuhan, china, at the end of december 2019, the virus has spread rapidly throughout the country. [1] to date, more than 200 countries/regions in the world reported covid-19 cases which have seriously threatened human life. [2] due to its widespread epidemic and high fatality rate, the who declared it an international public health emergency. studies have reported that the current covid-19 pandemic caused the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is genetically similar to the sars-cov that caused the severe acute respiratory syndrome (sars) outbreak in 2003, and both through ace2 as a carrier to enter the cell. recently, the next-generation sequencing of sars-cov-2 showed a 99.98% sequence identity in 9 patients. epidemiology reports on risk factors for sars-cov-2 susceptibility, including age, gender, and chronic disease. [3, 4] researchers from all over the world are actively engaged in searching biomarkers that can predict this disease. several studies are extremely interested in the abo blood type. it is established knowledge nowadays that the importance of abo blood type in blood transfusion and clinical transplantation. multiple studies demonstrate that the abo blood group is a pivotal independent risk factor for cardiovascular disease and venous thromboembolism. [5] in particular, the risk of thrombosis in blood group o is significantly reduced compared to non-o blood groups. recent data have defined the biological mechanism by which abo regulates the risk of thrombosis. [6] [7] [8] given the increasing evidence that covid-19 is associated with severe coagulopathy [9] and microthrombi distributed through the pulmonary vascular system from acute respiratory distress ethical approval is not required due to all the data used in this systematic review and meta-analysis has been published. funding was supported by the china national science and technology major project for "essential new drug research and development" (no.2018zx09301038-003). syndrome, [10] the abo blood group is associated with the susceptibility of covid-19 is of particular concern. a recent study in china compared covid-19 patients with the general population found an association between the abo blood group and sars-cov-2 infection status. [11] however, another study is not supported by this evidence. [12] this study will systematically review the current evidence, aiming to provide clarity surrounding the role of the abo blood type in patients with covid-19. the systematic review and meta-analysis have been registered in prospero (registration number: crd42020195615). we carry out the protocol according to the preferred reporting item for systematic review and meta-analysis protocol (prisma-p) statement [13] supplemental digital content (additional file 1, http://links.lww.com/md/e709). if there get any amendments to the protocol, the registration information will be updated. (1) participants who were documented to have sars-cov-2 infection, and for whom an abo typing. (2) health control participants were recorded with abo blood type. there are not any restrictions on gender, age, race, or any comorbidities. 2.1.2. type of exposure. abo blood type distribution in all participants that regardless of testing time and methods. 2.1.3. type of outcome measurements. the main outcome is to investigate the relationship between abo blood type and covid-19 susceptibility. the secondary outcome is to evaluate the prognosis of covid patients under the abo blood group. observational studies will be included. the sample size, blood type detection method, follow-up time, publication language or publication status will not be limited. (1) results that cannot be pooled through calculation. (2) case reports, case series, duplicate reports. (3) the full text of the study could not be available. two independent reviewers search the databases of the china biology medicine disc, china national knowledge infrastructure, china science and technology periodical database, wanfang database, pubmed, embase, and cochrane library from the date of conception to june 30, 2020. search terms are: "blood type", "blood groups", "abo", "novel coronavirus infected pneumonia", "covid-19", "corona virus disease 2019", "ncp", "2019-ncov". the search words in the chinese databases are translations of the above words. we will search for the latest research references to obtain potentially relevant articles. 2.2. data collection and analysis 2.2.1. study selection. we will export the records retrieved from the database to endnote x9 software to detect duplicate research. after removing the duplicates, the 2 reviewers will independently check them by reading the title and abstract according to the eligibility criteria. if a study may qualify, the full text will be generated and independently reviewed by 2 reviewers. as for the unverifiable literature, it will be supported by the discussion of the 2 reviewers. if they cannot achieve an agreement, a third reviewer will help. if the full text or user data is not available, the author will be contacted. the prisma flow chart is shown in figure 1 . to ensure data integrity and consistency, 2 independent reviewers will use pre-designed tables to extract data from eligible studies. the table includes the following items: (1) general information: first author, corresponding author, contact information, journal, year of publication, country/ region, funding source, research design; (2) characteristics of participants: age, gender, race, education level, disease stage, and severity; (3) research characteristics: sample size, random sequence generation, allocation concealment, blindness, follow-up time; (4) exposure: abo blood group classification (5) outcomes: the main outcome is to investigate the relationship between abo blood type and covid-19 susceptibility. the secondary outcome is to evaluate the prognosis of covid patients under the abo blood group. this information will be cross-checked by 2 reviewers. any differences will be discussed and resolved with the third reviewer. scale recommended by the cochrane collaboration was used to evaluate the methodological quality of the included studies. [14] the evaluation content includes 3 parts: selection, comparability, and exposure/outcome. there are 8 items in this scale, with a total score of 9. quality assessments were conducted by 2 researchers, any discrepancies existed 2 authors were solved by discussion or consensus. review manager software (version 5.3.5) will be used for statistical analysis of the results. the dichotomous variable will calculate the odds ratio and the corresponding 95% confidence interval. heterogeneity between included studies will be assessed by heterogeneity x2 tests and i 2 index. a rough explanation guide is as follows: 0% to 40% represents mild heterogeneity; 30% to 60% represents moderate heterogeneity; 50% to 90% represents significant heterogeneity, while 75% to 100% represents significant heterogeneity. when heterogeneity cannot be explained, 1 method of analysis is to pool it into a random-effects model to pooled the results. otherwise, a fixed-effect model will be used. if quantitative synthesis is not appropriate, we will describe the included studies. the forest plots will be used to describe the pooled results. we pre-specified the following variables for subgroup analysis: country/region, ethnicity, sample size, and study type. furthermore, we will consider other subgroup analyses in the study. 2.2.6. sensitivity analysis. if there is the heterogeneity, we will conduct a sensitivity analysis to test the robustness of the pooled results. if more than 10 studies met the eligibility criteria, the begg rank correlation test or egge linear regression test will be performed to quantize the publication bias. 2.2.8. quality of evidence. the grading of recommendations assessment, development, and evaluation system will be used to estimate the quality of evidence for each result. [15] each result will be evaluated according to the following 5 aspects: limitations, inconsistency, indirectness, inaccuracy, and publication bias. the grade of each evidence will be defined as high, moderate, low, or very low. recent studies suggest that age, gender, and blood type distribution is related to covid-19 disease susceptibility. among them, elderly and male patients with covid-19 are most likely to progress to severe disease. [1, 16] besides, it was also noted that people with blood type a had high susceptibility, while people with blood type o had low susceptibility. [17] it remains unclear whether these causal relationships are established or whether the association is trivial. however, previous studies confirmed that the association between blood type and other coronavirus infections cannot be ignored. [18] as accumulating evidence data floods in every day, blood types should be registered for each infected individual to correlate with larger data sets in the future. moreover, it should be pointed out that the abo blood type varies by race, [12] so when comparing infected and uninfected people, this may affect the observed outcome. to our knowledge, this is the first review to examine the contribution of the abo blood group to infection with covid-19 pneumonia. we rate the strengths and limitations of existing evidence through a systematic review and meta-analysis. the protocol will be guided by the prisma statement [19] to achieve the highest possible quality in reporting and methodology. the development of this protocol based on the current research design may have limitations, if there get any amendments, the registration information will be updated at prospero. in conclusion, this systematic review and meta-analysis will serve to explore the susceptibility of the abo blood group to covid-19 pneumonia and will help us prepare for future epidemics. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study abo blood group determines plasma von willebrand factor levels: a biologic function after all? blood group alters platelet binding kinetics to von willebrand factor and consequently platelet function an influence of abo blood group on the rate of proteolysis of von willebrand factor by adamts13 the effect of abo blood group on the diagnosis of von willebrand disease more on covid-19 coagulopathy in caucasian patients why the immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia are distinct from macrophage activation syndrome with disseminated intravascular coagulation relationship between abo blood group distribution and clinical characteristics in patients with covid-19 covid-19 and abo blood groups preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015: elaboration and explanation critical evaluation of the newcastle-ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses grade: an emerging consensus on rating quality of evidence and strength of recommendations abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia testing the association between blood type and covid-19 infection, intubation, and death abo blood group and susceptibility to severe acute respiratory syndrome prisma group preferred reporting items for systematic reviews and meta-analyses: the prisma statement the authors have no conflicts of interest to disclose.supplemental digital content is available for this article.all data generated or analyzed during this study are included in this published article and its supplementary information files. key: cord-315077-i1xjcuae authors: branda, john a.; lewandrowski, kent title: utilization management in microbiology date: 2014-01-01 journal: clin chim acta doi: 10.1016/j.cca.2013.09.031 sha: doc_id: 315077 cord_uid: i1xjcuae the available literature concerning utilization management in the clinical microbiology laboratory is relatively limited compared with that for high-volume, automated testing in the central core laboratory. however, the same strategies employed elsewhere in the clinical laboratory operation can be applied to utilization management challenges in microbiology, including decision support systems, application of evidence-based medicine, screening algorithms and gatekeeper functions. the results of testing in the microbiology laboratory have significant effects on the cost of clinical care, especially costs related to antimicrobial agents and infection control practices. consequently many of the successful utilization management interventions described in clinical microbiology have targeted not just the volume of tests performed in the laboratory, but also the downstream costs of care. this article will review utilization management strategies in clinical microbiology, including specific examples from our institution and other healthcare organizations. clinical microbiology includes bacteriology and antimicrobial susceptibility testing, virology, parasitology, mycobacteriology, mycology, serology and molecular microbiology. unlike the core laboratory (chemistry/hematology), where the majority of testing is performed on highly automated analyzers, most testing in microbiology is performed manually or on semi-automated platforms. many microbiology tests also require interpretation by a skilled microbiology technologist, including visual interpretation of culture results and microscopic examinations. for these reasons the unit cost of microbiology testing is usually greater than that for routine automated testing. the results of microbiology tests have a significant impact on the overall cost of clinical care, most notably in the use and selection of antimicrobial therapy. therefore, when approaching utilization management in microbiology, it is important to consider not only the cost of testing within the microbiology laboratory but also the downstream costs resulting from clinical decisions based on the test results. the published literature on utilization management in microbiology is relatively limited when compared to reports on managing utilization of routine automated testing in the chemistry and hematology laboratories. this article will outline a number of utilization management interventions in microbiology that have been reported in the literature. we will also describe several unpublished initiatives that have proven successful in our institution. the specific interventions to be discussed are outlined in table 1 , and will be described in more detail in the text that follows. in a number of cases, the initiative's success arose not only from a reduction in laboratory testing per se, but rather also from its impact in the clinical care arena (for example, a reduction in antibiotic use or hospital length of length-of-stay). this observation highlights the importance of the clinical microbiology director in forming collaborative, interdepartmental teams to improve quality and reduce the cost of medical care. tests for cytomegalovirus (cmv) include antigenemia testing, viral load testing by quantitative polymerase chain reaction (qpcr), viral genotyping, shell vial culture, or serologic tests for the detection of a host immunologic response (cmv igm and igg antibody tests and antibody avidity tests). for an individual patient, the most appropriate test depends on the clinical indication. it is difficult for clinicians to keep upto-date with esoteric tests in rapidly evolving specialties, especially when there are numerous tests that can be ordered. in these situations, the use of a decision support tool is an effective mechanism to assist physicians in proper test selection, potentially avoiding inappropriate test selection. as one example, fig. 1 shows a screen display from the on-line laboratory handbook at the massachusetts general hospital. when the clinician types "cytomegalovirus" or "cmv" into the handbook's search function, the available tests and their appropriate indication are presented. in addition, the same decision-support information is provided in the electronic physician order entry (poe) system when a clinician views cmv-related test options. an advantageous feature of this approach is that when new tests become available, or outdated ones are removed from the test menu, the decision-support function can be updated accordingly. for example, the mgh microbiology laboratory recently discontinued the cmv antigenemia assay in favor of the cmv qpcr test. the information provided in the on-line handbook makes it clear that the preferred test has changed. this approach can be applied to many other tests in microbiology, particularly in areas such as molecular microbiology where new assays are supplanting more traditional assays at a rapid rate. the topic of decision support is covered in more detail in another chapter of this special edition. the problem of contamination of blood cultures from improper or poor technique is well known. it has been estimated that up to 5% of positive blood cultures may represent contaminants [1] , resulting in significant increases in resource utilization. consequently many hospitals have engaged in ongoing efforts to reduce blood culture contamination by improving staff training, or designating specific types of employees to collect the blood culture specimens. for example, blood cultures collected by medical house officers are more likely to be contaminated than those collected by phlebotomists [2] . in a retrospective study, bates et al. studied the impact of contaminated blood cultures on hospital length-of-stay and hospital charges [3] . in patients with falsely positive blood cultures, there was a 4.5-day increase in the median length-ofstay and an increase in hospital charges of 33.4%. false-positive episodes were associated with increased pharmacy charges for intravenous antibiotics (39% increase) and laboratory charges (20% increase). in another study, segal and chamberlain assessed the impact of false-positive blood cultures in a pediatric emergency department [4] . the authors reported an increase in phone calls, return visits to the emergency department, unnecessary laboratory tests, inappropriate antibiotic administration and hospital admissions. finally, tabriz et al. evaluated the practice of repeating blood cultures serially [5] . blood cultures were repeated in 31.6% of cases and amounted to approximately one-third of all blood cultures handled in the laboratory. the results of the repeated cultures showed no growth in 83.4% of cases, the same pathogen in 9.1% of cases, and a new pathogen or contaminant in 2.5% and 5.0% of cases respectively. the authors concluded that repeating blood cultures provides little additional yield and that guidelines for when to repeat blood cultures might decrease utilization. laboratory reports that are not optimally designed can lead to confusion among clinicians, with the potential for misdiagnosis or unnecessary requests for additional testing. ackerman et al. evaluated the interpretation of 5 typical microbiology reports by physicians in a teaching hospital [6] . the investigators found that reports were often misinterpreted. for example, one report of "isolation of a gram negative rod from sputum was misinterpreted by 4 out of 5 physicians." the reasons for misinterpretation were reported to be the use of jargon, unfamiliar names of bacterial species, or ill-defined reporting conventions, and the omission of a clear-cut conclusion in many reports. the misunderstandings resulted in both inappropriate use of antibiotics and orders for unnecessary testing in the laboratory. this study highlights the importance of developing clear, concise, standardized reporting formats in microbiology, and the need for the laboratory to work closely with physicians in designing and communicating microbiology reports. 2.4. rapid identification of bacteria using matrix-assisted laser desorptionionization time of flight mass spectroscopy (maldi-tof ms) to improve clinical decision making and guide antibiotic use it has long been known that rapid bacterial identification and susceptibility testing lead to more appropriate use of antibiotics and a reduction in antimicrobial utilization [7] . in the past, rapid identification and susceptibility testing were mainly accomplished using automated instrumentation to perform conventional tests. more recently, manufacturers have developed maldi-tof ms for rapid organism identification, a method that has been demonstrated to reduce turnaround time for the identification of bacteria and yeasts by 1.45 days compared with conventional methods [8] . another advantage of maldi-tof ms is its simplicity and the relatively low cost of consumables [9] . for table 1 examples of utilization management initiatives in clinical microbiology (see text for details). 1. decision support: test selection for cytomegalovirus testing 2. reducing blood culture contamination 3. proper formatting of microbiology reports to avoid misinterpretation 4. use of maldi-tof mass spectroscopy for rapid identification of pathogens 5. antimicrobial stewardship of carbapenems and other expensive antimicrobial agents 6. rapid point-of-care testing for influenza a and group a streptococcus: impact on test ordering and antibiotic utilization 7. rapid molecular diagnostic testing for patients previously colonized with methicillin resistant staphylococcus aureus (mrsa) 8. use of screening methods to reduce low-yield urine cultures 9. restricting stool examinations in hospital acquired diarrhea 10. rapid testing for respiratory viruses: impact on inpatient bed management 11. application of evidence based medicine: discontinuation of fungal blood cultures 12. selection and oversight of molecular diagnostics in microbiology example, one study performed at a large, academic medical center demonstrated potential cost savings to the laboratory (for reagents and labor) of $100,000 per year [8] . although maldi-tof ms does not provide antimicrobial susceptibility data, rapid organism identification may help clinicians earlier to select an effective empirical antimicrobial strategy [10] . carbapenems including imipenem, meropenem, ertapenem and doripenem are broad spectrum antimicrobial agents active against many gram-positive, gram-negative and anaerobic organisms. they are highly effective when used appropriately but are also very expensive relative to potential alternative agents. indiscriminant use of carbapenems will also contribute to antimicrobial resistance, decreasing the effectiveness of the drug class. many hospitals have established antimicrobial formularies in the pharmacy to assist in management of expensive antibiotic drugs. in our institution some antibiotics are restricted and can only be obtained after approval from the division of infectious diseases. however, once approved there was no requirement or formal mechanism in place to re-evaluate the ongoing need for a restricted antibiotic once the results of microbiology culture and antimicrobial susceptibility testing became available. the clinical pathology staff in our microbiology laboratory recently began an antimicrobial stewardship program related to carbapenems. each day, a microbiology fellow and laboratory director review the clinical history, culture results and susceptibility test results for all patients newly started on a carbapenem, to determine appropriate versus inappropriate use of the drugs. if objective data indicate that a patient's infection can be treated using a non-restricted agent, an email is sent to the clinician as shown in the example below. "dear dr.________ your patient ________ is currently receiving a restricted antibiotic: ertapenem. the use of this restricted drug is being monitored by the mgh antimicrobial stewardship program. recent culture and antimicrobial susceptibility data from your patient reveal that the organism(s) is/are susceptible to other, nonrestricted antibiotics (see sensitivity report below). given these data, if clinically appropriate, please consider discontinuing the restricted carbapenem and/or changing to a non-restricted antimicrobial option. this may help reduce both the development of future resistance to these broad spectrum drugs and costs of therapy. if you have not already done so, you may request an infectious disease consult in order to obtain assistance on the choice of antimicrobial agents". after implementation of this stewardship effort, a decrease in carbapenem use was observed, and carbapenems have been removed from the "top 10" list of money spent on antimicrobials. the microbiology group is now extending the program to include other high cost antimicrobials. point-of-care testing (poct) performed at the patient's bedside provides rapid, real-time results. in some cases this facilitates clinical decision making and improves the efficiency of clinical operations [11] . a number of rapid point-of-care tests are available for the diagnosis of infectious diseases [12] . among these are single use, visually read, lateral flow tests for influenza a and b. in a randomized, prospective controlled study by bonner et al., the use of a rapid poc influenza test in a pediatric emergency department was associated with a significant reduction in laboratory tests ordered (complete blood count, blood cultures, urinalysis and urine cultures), a decrease in chest radiographs performed and a reduction in emergency department length-of-stay [13] . in another study of pediatric patients presenting to the emergency department with acute pharyngitis, the authors compared antibiotic use in patients who received a rapid streptococcus a test to those who were managed by conventional throat culture alone. they reported a decrease in antibiotic use of 50% when the rapid test was employed (22.45% versus 41.38%) and concluded that the rapid test significantly reduced unnecessary prescription of antibiotics [14] . patients who are colonized with mrsa require contact precautions when admitted to the hospital. this entails placing them in a private room or cohorting the patient in a semi-private room with another colonized patient. in addition, hospital staff must wear gloves and gowns, and use dedicated equipment when interacting with the patient. some of these patients also have a longer hospital length of stay due to delays in discharge to other health care facilities. collectively, these features of mrsa colonization result in greater use of hospital resources compared with non-mrsa colonized patients. because mrsa colonization can be transient, protocols have been put in place to identify previously mrsa colonized patients who are no longer colonized, and can be managed without contact precautions. in our institution, patients with a history of mrsa are eligible for discontinuation of contact precautions if for either screening method, the patient cannot have been received antibiotics active against mrsa during the 48 h prior to screening. discontinuation of contact precautions on the basis of a single negative mrsa pcr is faster than screening using the culture-based method, and could result in an increase in discontinuation of contact precautions because of a reduction in the number of samples needed [15] . uncomplicated urinary tract infection is very common, especially among women. various strategies have been employed to make (or rule out) a diagnosis of community acquired uti in adult women without the need for urine culture. some sources suggest that uncomplicated uti in outpatients can be diagnosed and managed without culture, unless the patient fails treatment or has had recurrent utis [16, 17] . others have even suggested that suspected uti can be managed over the telephone in women with typical symptoms of cystitis and without vaginal symptoms or major co-morbidities [18] . this approach eliminates both office visit and any subsequent laboratory testing. another approach is to limit urine culture utilization by pre-screening urine using various methods. for example, dipstick urinalysis for leukocyte esterase and nitrites has a high negative predictive value, and may be used to exclude bacteriuria without a culture step when the results are negative. studies have also demonstrated that urine can be screened for significant bacteriuria prior to culture using automated urine sediment examination [19] or flow cytometry [20] . diarrhea is a common complaint among hospitalized patients. although it is common for clinicians to request a routine stool culture and ova and parasites (o&p) examination for patients with diarrhea, these tests are designed to detect agents of community-acquired rather than hospital-acquired infection. a number of studies have indicated that routine stool culture or stool o&p examination is usually not warranted in adult patients who develop diarrhea more than three days after admission to the hospital [21] [22] [23] . in contrast, testing for clostridium difficile should be considered, as this is a major cause of nosocomial diarrheal illness. new molecular diagnostic tests offer the promise of providing rapid and reliable testing for c. difficile. highly sensitive molecular testing could potentially permit rapid ruleout of c. difficile, obviating the need for unnecessary antibiotics and contact precautions in many patients. upper respiratory viral infections in the united states are frequently caused by rhinoviruses, coronaviruses, influenza a and b, parainfluenza, respiratory syncytial virus, adenovirus or metapneumovirus. while many of these illnesses can be managed on an outpatient basis, patients with severe illness or major comorbidities are often admitted to the hospital. usually they present first to the emergency department, where the initial challenge is to confirm the presence of a respiratory viral infection and then to identify the specific offending virus in order to direct specific therapy (if available) and aid in hospital bed assignment. often, respiratory viral infections occur in seasonal epidemics (especially influenza a and b), resulting in hospital overcrowding and a shortage of hospital beds. when this occurs, managing the availability of hospital beds becomes a priority. in cases where the offending virus has been identified, contact and/or droplet precautions must be instituted to prevent transmission between patients as shown in table 2 . patients on contact or droplet precautions must be placed in a private room. alternatively, if two patients are infected with the same respiratory virus, they can be cohorted together in one hospital room. in our hospital, we offer rapid molecular diagnostic testing for influenza a and b for patients who require hospital admission for a flu-like illness. testing is performed 24 h per day, 7 days per week, in order to facilitate bed management and timely initiation of anti-viral therapy. we also offer a respiratory viral panel by direct immunofluorescence for the many viruses listed above, 7 days per week during peak respiratory virus season. the ability to identify the specific virus causing the infection greatly assists in managing our inpatient beds and maintaining effective infection control measures. the savings to the hospital from improved bed management during epidemics greatly exceeds the cost of the testing in the laboratory. many clinical laboratories offer "fungal blood cultures" that employ specialized media designed to enhance the detection of yeast or mold fungemia. however, it is well-established that specialized fungal blood culture media are not superior to routine blood culture media for the detection of candida fungemia (candidemia) [24, 25] . thus, the main rationale for the use of fungal blood cultures is to improve detection of cryptococcal yeast, endemic fungi (histoplasma capsulatum, coccidioides spp., etc) and filamentous fungi (e.g., aspergillus spp.) in the blood. until recently, our institution offered fungal blood cultures using myco/ f lytic bottles (becton dickinson) designed for use with the bactec automated culture monitoring system. this approach was costly, not only in terms of reagent costs but also in terms of technical labor. the highly-enriched and non-selective culture medium needed to be incubated for up to 30 days before a negative result could be obtained, and this frequently promoted the growth and eventual detection of skin contaminants that would not have been detected using routine blood culture bottles and a 5 to 7 day incubation period. thus, we reviewed our experience with this approach to understand whether or not it provided a significant clinical benefit. we reviewed the results of all fungal blood cultures over a 44 month period. during this time period, 5544 myco/f lytic fungal blood cultures were performed. our review revealed the following: â�¢ no dimorphic fungi were recovered by fungal blood culture. â�¢ mold (fusarium sp.) was recovered twice from a single patient using fungal blood culture. however, in this case fusarium was also recovered from 2 sets of routine blood cultures (2 out of 4 bottles), several days prior to recovery from fungal blood culture. â�¢ cryptococcus neoformans was recovered from 3 patients by fungal blood culture. two of the patients had cryptococcal meningitis, and in both cases the organism was detected in numerous other ways (csf and blood cryptococcal antigen tests, csf gram stain, routine and fungal csf cultures, and routine blood cultures). the third patient had cryptococcal fungemia (but not meningitis); a cryptococcal antigen test on blood was positive, and the organism was recovered twice from routine blood cultures. based on these findings, we concluded that fungal blood cultures had failed to detect any dimorphic fungi, molds or cryptococcal yeast that were not otherwise detected by routine blood culture. furthermore, blood culture is not a useful method for the detection of invasive infection caused by molds or dimorphic fungi. therefore, blood culture specifically designed for the detection of fungi was discontinued at our institution. many microbiology tests suffer from drawbacks that limit their clinical utility. for example, results from traditional culture methods may take several days to become available, and antibiotic treatment prior to specimen collection may degrade sensitivity. serologic studies often cannot distinguish current from past infection unless acute and convalescent specimens are available. techniques for specialized cultures such as for viruses are beyond the capability of most hospital laboratories. finally many microbiology tests do not yield quantitative results which may be desirable in some situations. recent advances in molecular diagnostic techniques for microbiology offer promise to resolve some of these issues. some molecular tests, such as those for influenza a and b, hepatitis b virus dna and hiv viral load are requested in sufficient volume that many hospital laboratories are able to offer the tests in-house. in these examples, the molecular diagnostic test is either superior to alternative testing methods or provides unique information of clinical importance (e.g. viral load). however, in many cases the volume of requests for molecular microbiology tests is too low to warrant performing the test in the hospital laboratory. typically these tests are sent to outside reference laboratories for analysis which, over time, can prove very expensive. intuitively a molecular diagnostic test is expected to be highly sensitive and specific, but this is not always the case. in some situations, conventional microbiology tests are more appropriate [26] . for this reason, it is important for the clinical microbiology director to work with infectious disease specialists to scrutinize the send out budget for potential inappropriate test ordering. further, such analysis may reveal opportunities for in-sourcing the testing at significant savings. for example, our microbiology laboratory recently insourced nucleic-acid testing for epstein bar virus (ebv) saving over $100,000 per year and providing superior turnaround time. tests performed in the clinical microbiology laboratory are ripe for utilization management efforts. clinicians are frequently confused about which test to order, and appropriate test selection can be guided through decision support mechanisms and gatekeeper functions. the diagnostic yield of routine cultures can be improved by facilitating proper specimen collection and transport, or screening specimens prior to culture. clinical decision making, particularly in selecting appropriate antibiotics and implementing (or discontinuing) infection control measures, can be aided by efforts to reduce turnaround time and by antimicrobial stewardship efforts directed by microbiologists. finally, errors can be prevented by carefully formatting reports to avoid miscommunication. analysis of strategies to improve cost effectiveness of blood cultures pathology tests: is the time for demand management ripe at last contaminant blood cultures and resource utilization: the true consequences of false positive results resource utilization and contaminated blood cultures in children at risk for occult bacteremia repeating blood cultures during hospital stay: practice pattern at a teaching hospital and a proposal for guidelines consumer survey on microbiology reports rapid identification and antimicrobial susceptibility testing reduce antibiotic use and accelerate pathogen-directed antibiotic use prospective evaluation of a matrix-assisted laser desorption ionization-time of flight mass spectrometry system in a hospital clinical microbiology laboratory for identification of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identification and costeffectiveness maldi-tof mass spectroscopy: transformative proteomics for clinical microbiology impact of matrixassisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with gram-negative bacteremia: a prospective observational study implementing point-of-care testing to improve outcomes infectious disease testing at the point-of-care impact of the rapid diagnosis of influenza on physician decision making and patient management in the pediatric emergency: results of a randomized, prospective controlled study impact of rapid streptococcal test on antibiotic use in a pediatric emergency department discontinuation of contact precautions for methicillin-resistant staphylococcus aureus: a randomized controlled trial comparing passive and active screening with culture and polymerase chain reaction management of urinary tract infections in adults laboratory diagnosis of urinary tract infections in adult patients urine dipstick for diagnosing urinary tract infection bacteriuria screening by automated whole-field-image-based microscopy reduces the number of necessary urine culture evaluation of the sysmex uf-100 urine cell analyzer as a screening test to reduce the need for cultures for community-acquired urinary tract infection role of the microbiology laboratory in the diagnosis of nosocomial diarrhea rational testing for faeces in the investigation of sporadic hospital-acquired diarrhoea when should a stool culture be done in adults with nosocomial infections optimal use of myco/f lytic and standard bactec blood culture bottles for detection of yeast and mycobacteria principles and procedures for blood cultures; approved guideline. clsi document m47-a. wayne, pa: clinical and laboratory standards institute introducing a molecular test into the clinical laboratory: development, evaluation, and validation key: cord-299422-s5evsj96 authors: abdollahi, alireza; mahmoudi-aliabadi, maedeh; mehrtash, vahid; jafarzadeh, bita; salehi, mohammadreza title: the novel coronavirus sars-cov-2 vulnerability association with abo/rh blood types date: 2020-05-23 journal: iran j pathol doi: 10.30699/ijp.2020.125135.2367 sha: doc_id: 299422 cord_uid: s5evsj96 background & objective: coronavirus disease 2019 (covid-19) is the most recent emerging viral disease. defining the epidemiological aspects and factors influencing the susceptibility of the patients to covid-19 has been an ongoing struggle. in the present study, we have investigated the connection between abo histo-blood group phenotypes and the covid-19. methods: this study was conducted on 397 patients with confirmed diagnoses of covid-19 admitted to our center. also, 500 individuals were selected to form the controls, all of whom had been disclosed to the same medical center in june 2019, before the onset of the outbreak. results: our results demonstrated abo histo-blood phenotypes are correlated with patients’ susceptibility to the infection. a higher rate of infection was observed among patients with the ab histo-blood group, while patients with the o histo-blood group have shown a lower rate of infection. the rh blood group phenotype was not statistically significant in determining a patient’s vulnerability. conclusion: similar to several previous studies about other viral diseases’ association with abo histo-blood groups, we have concluded that an individual’s abo histo-blood group phenotype and his/her susceptibility to covid-19 are indeed connected. so far, only one research has been conducted about this association. interestingly, while we observed a decreased vulnerability to the disease among patients with an o histo-blood group, we have reached discordant results regarding the increased susceptibility among individuals with an ab histo-blood group, unlike a histo-blood group in the previous study. coronaviruses have been the cause of three recent global outbreaks, severe acute respiratory syndrome (sars, 2002 (sars, to 2003 , middle east respiratory syndrome (mers, since 2012), and most recently, the coronavirus disease (covid19) posing an extensive crisis in late 2019 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), formerly recognized as 2019-ncov (1) (2) (3) . although the aforementioned coronaviruses, have caused fatal diseases in humans, other known coronaviruses have only induced mild flu-like symptoms in healthy individuals (4) . covid-19 patients mostly present with fever, dry cough, and dyspnea, which can progress to acute respiratory distress syndrome and multiple organ failures (5) . while most patients have been considered to have a good prognosis, multiple studies demonstrated that older male adults and patients with pre-existing medical conditions such as underlying respiratory, cardiovascular, and immunodeficiency diseases are more susceptible to poor clinical outcomes due to their reduced immune system function (6, 7) . other innate and adaptive factors can affect viral infection susceptibility or resistance. previous researches have proved the potential role of abo blood groups on a host's genetic susceptibility to various viral diseases such as influenza, ebola, enteric viruses, and sars-cov infections (8) (9) (10) (11) (12) . this association could be on account of aboantibodies, which act as a part of innate immunity iranian journal of pathology against some bacterial and viral agents that bear aboantigens (13) . we conducted this study to further investigate the susceptibility variabilities amongst patients with different abo histo-blood groups. knowledge of innate susceptibilities to covid-19 disease could lead to a better understanding of virus pathogenesis, disease management, and patient survival. in this cross-sectional survey, individuals with diagnoses of covid-19 admitted to imam khomeini hospital complex, tehran, iran, during march 2020 were enrolled in the study. we included 397 patients with positive real-time polymerase chain reaction (rt-pcr) test results performed on swabs with synthetic fibers from both nasopharyngeal and oropharyngeal samples. the diagnoses were established by utilizing novel coronavirus (2019-ncov) real-time multiplex rt-pcr kit (daan gene co. ltd.) and novel wuhan cov e-gene kit (tib molbiol, germany) according to manufacturer's instructions on cfx96™ real-time pcr detection system (bio-rad laboratories, inc., usa). demographic characteristics were collected from each patient's file. subjects' abo and rh blood group determination was conducted using the tube method forward type and back type grouping techniques. the forward grouping was performed on a 3%red blood cell (rbc) suspension using commercially prepared monoclonal blood group antisera to check antigens on the surface of rbc provided by ce-immundiagnostika gmbh, germany. in reverse grouping, the patients' plasma was tested against commercially prepared reagent cells of known a1 and b phenotype to determine the anticipated abo antibodies in the patients' serum using red cell reagents provided by diamed gmbh, switzerland. abo and rh blood grouping data retrieved from the imam khomeini hospital's blood bank database of patients referred in june 2019 to our lab, before the onset of the outbreak, were used as subjects uninfected by covid-19 (control). covid-19 negative blood samples referred from outpatient and inpatient services constitute 136 and 364 of specimens, respectively. outpatient samples referred from specialist clinic of the hospital (dr yalda clinic, tehran, iran) and inpatient samples referred from accident and emergency department (n=103), liver transplant unit (n=54), plastic surgery unit (n=53), otolaryngology unit (n=46), neurosurgery unit (n=39), urology unit (n=35), and orthopedic unit (n=34). the research protocol was confirmed by the ethics committee of tehran university of medical sciences (code: ir.tums.vcr.rec.1399.208). all data were entered and analyzed using ibm spss 26 (spss inc., chicago, il, usa). comparisons of proportions of blood group antigens between covid-19 patients and controls were conducted using pearson's chi-square test (χ2) or fisher's exact test where appropriate. we explored the combined effect of various blood groups on covid-19 susceptibility using binary logistic regression analysis. only histo-blood groups ab and o with a statistically significant association with covid-19 status in bivariate analyses were included in the model (multivariate analysis for adjustment of age and gender variables), and the results were reported as odds ratio with 95% confidence interval. we used an independent-sample t-test to compare the means. p-values of less than or equal to 0.05 were considered statistically significant. in the present study, 397 covid-19 patients and 500 normal controls were analyzed to evaluate the association of the abo histo-blood group phenotypes with covid-19 disease in the iranian population. patients' ages ranged from 15 to 95 years old versus controls' ages reached 4 to 93 years old. patients and controls had a mean (sd) age of 58.81 (15.4) and 48.53 (17.9) years, respectively, which showed that the older population is at a higher risk of infection with sars-cov-2 (p<0.001). mean (sd) age of patients admitted to the intensive care unit (icu) and general wards were 62.64 (13.9) and 57 (15.8), respectively, which showed that older patients are more prone to the more severe complications and requiring icu admission (p<0.001). our study demonstrated that men were infected more than women by coronavirus so that among patients and controls, male-to-female ratios were 1.74:1 (252 males and 145 females) and 0.86:1 (231 males and 269 females), respectively (p<0.001). among the patients, 270 (68%) individuals were admitted to the icu, among whom 86 (67.7%) and 41 (32.3%) were males and females, respectively. furthermore, 127 (32%) individuals were admitted to the general wards, among whom 166 (61.5%) and 104 (38.5%) were males and females, respectively. there was no statistically significant difference in the severity of disease with respect to genders (p=0.23). the percentages of a, b, ab, and o histo-blood groups in the patients were 40.3%, 22.4%, 9.3%, and 28%, whereas, in the controls, these values were 36%, 21%, 5%, and 38%, respectively. when the blood groups of the covid-19 patients were compared with the controls, patients with ab histo-blood group (or=2.02; 95%ci: 1.17-3.51) and o histo-blood group (or=0.68; 95%ci: 0.5-0.92) were found to have significantly higher and lower proportions than controls, respectively in univariate and multivariate logistic regressions (table 1) . our study has suggested that o histo-blood group makes individuals less susceptible to sars-cov-2 virus infection, unlike the ab histo-blood group that has the opposite effect. as shown in table 1 , there was no statistically significant association between covid-19 positivity and rh blood group (p=0.66). the percentage of histo-blood groups a, b, ab, and o in the icu admitted patients were 40.2%, 22%, 7.9%, and 29.9%, whereas, in the mild patients, these values were 40.4%, 22.6%, 10%, and 27%, respectively ( table 2 ). as shown in table 2 , there was no association between the severity of covid-19 and abo histo-blood group phenotypes and rh blood groups (p=0.88, p=0.32, respectively). viral diseases are considered massive sources of worldwide mortality and morbidity and have significant potential impacts on the global economy and human health. they are regarded as the cause of 60% and 75% of currently known and newly-appearing infectious diseases, respectively (14, 15) . the covid-19 is the latest fatal zoonotic disease, and since its advent, it has affected thousands of people throughout the world. as yet, multiple risk factors, including older age, male gender, and presence of chronic underlying comorbidities, have been established to be attributed to the higher probability of getting infected with sarscov-2 (6,7) . concordantly, we found that the covid-19 affects older individuals to a greater extent so that the mean age of patients was about ten years higher than uninfected individuals. also, according to our survey, the male gender demonstrated as a risk factor in cases of sars-cov-2 infection, too, with the male-to-female ratios of 1.74:1 among patients and 0.86:1 among controls which demonstrated a statistically significant difference. numerous efforts have been made to identify factors that have an impact on the susceptibility of infected patients. while a connection between abo/rh blood groups and other viral disease susceptibility has been demonstrated previously, there is not much information about this connection regarding sars-cov-2. abo histo-blood group system is composed of three antigens-a, b and h. sequential addition of carbohydrate units on precursor oligosaccharide backbone leads to four phenotypes a, b, ab and o; h antigen is built by the addition of fucose to the oligosaccharide backbone; moreover, a and b antigens are made up of addition of n-acetylgalactosamine (a) and d-galactose (b) to the core h antigen, respectively (13, (16) (17) (18) . rh blood grouping categorizes in two phenotypes d+ and d-depending on the presence or absence of d protein epitopes (16) (17) (18) . abo histo-blood group antigens have been found in rbc's surface lymphocytes, as well as many tissue organs, mucosal surfaces, and exocrine secretions (13, (19) (20) (21) (22) (23) . the human body produces antibodies against the missing abo histo-blood group antigens resulting in the production of anti-a and/or anti-b antibodies (13, 24) . abo histo-blood groups are genetically inherited traits that are distributed variably among different populations, o and ab blood type have the most and least prevalence in many populations (16) (17) (18) (19) . in our study, the percentages of a, b, ab, and o histo-blood groups in uninfected individuals who were employed as controls were 36%, 21%, 5%, and 38%, respectively. our study found that the vulnerability to the covid-19 infection was higher among iranians with an ab histo-blood group and lower in those with an o histoblood group. a study conducted by jiao zhao et al. in china has similarly demonstrated that chinese patients with o histo-blood group are less likely to suffer from a covid-19 infection. on the other hand, according to the chinese study, individuals with a histo-blood group were identified as high-risk. different patterns of the outbreak in iran and china might be due to this disparity given the fact that ab is the least prevalent histo-blood group among populations and the number of individuals with an a histo-blood group is generally higher; as a result, it could be suggested that less percentage of iranians are susceptible to covid -19 comparing to china. however, limitations of these studies and the biologic difference between these two populations should not be overlooked (25) . previous studies propose a mechanism through which abo histo-blood groups interact with viruses. abo histo-blood antigens have an impact on the immune system and affect pathogens spread by means of the host's natural antibodies and complement systems (13, 19, 20) . multiple studies have been conducted about the relationship between various viruses' biological functions and abo histo-blood groups leading to human host viral disease vulnerability or resistance. it has been suggested that some viruses perform their role by binding to abo histo-blood antigens. norwalk-like viruses and bat caliciviruses spread through interaction with abo histo-blood group antigens (1,2). the host's histo-blood group antigens affected human rotavirus susceptibility and reduced vaccine efficacy (23) . the role of abo histo-blood group phenotype on the probability of getting infected with sars-cov, the causative agent of the severe acute respiratory syndrome (sars), is presumed. sars-cov invades respiratory and gi tract mucosal epithelium where the epithelial cells express abo histo-blood group antigens, through interaction between virus spike proteins and receptor angiotensin-converting enzyme 2 (ace2). in one study, yufeng cheng et al. investigated the prevalence of sars disease among forty-five health-care workers who had unprotected exposure to infected patients, finding that individuals with histo-blood group o had a lower likelihood of infection, allegedly because of sars-cov varying binding capacity in different blood group types (10) . patrice guillon et al. used a mathematical cellular viral transmission model. they claimed that this association could be attributed to the protective role of anti-histo-blood group antibodies preventing the virus from adhesion to its receptor on the host cells (26) . considering that the sars-cov and sars-cov-2 viruses are genetically related to each other, and the protective pattern of the o histo-blood group in both viruses is similar, the abovementioned rationalization could be extended to sars-cov-2 as well. further studies are required to determine the exact mechanism through which abo blood group influences covid-19 susceptibility, which could be helpful in patient management and disease control. as demonstrated by previous chinese research on covid-19 and our current study, the statistically significant association of abo histo-blood group with covid-19 susceptibility is clear. however, our results were discordant regarding the abo histo-blood antigens which make people susceptible to covid-19 (ab versus a histo-blood group phenotype in iran and china, respectively). we assume that since the ab histo-blood group constitutes the least prevalent proportion, and a histo-blood group is the second most prevalent blood group among the general population, the observed discrepancy between the findings of these two studies may help explain different epidemiological patterns among various ethnicities. more studies are needed to further investigate precise viral pathogeneses to explain such differences. a pneumonia outbreak associated with a new coronavirus of probable bat origin characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention world health organization. who director-general's opening remarks at the media briefing on covid-19-11 origin and evolution of pathogenic coronaviruses review of the clinical characteristics of coronavirus disease 2019 (covid-19) epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study risk factors 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histo-blood group family applied blood group serology expression of abh and x (lex) antigens on platelets and lymphocytes binding of norwalk virus-like particles to abh histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice bat caliciviruses and human noroviruses are antigenically similar and have overlapping histo-blood group antigen binding profiles histo-blood group antigen phenotype determines susceptibility to genotype-specific rotavirus infections and impacts measures of rotavirus vaccine efficacy abo and rh (d) phenotype frequencies of different racial/ethnic groups in the united states relationship between the abo blood group and the covid-19 susceptibility. medrxiv inhibition of the interaction between the sars-cov spike protein and its cellular receptor by antihisto-blood group antibodies the novel coronavirus sars-cov-2 vulnerability association with abo/rh blood types how to cite this article the authors would like to thank the personnel of the vali-e asr laboratory of imam khomeini hospital complex for their help. the authors confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome. key: cord-327139-u5rzp2h4 authors: barrett, claire l. title: primary healthcare practitioners and patient blood management in africa in the time of coronavirus disease 2019: safeguarding the blood supply date: 2020-05-21 journal: afr j prim health care fam med doi: 10.4102/phcfm.v12i1.2457 sha: doc_id: 327139 cord_uid: u5rzp2h4 the coronavirus disease 2019 (covid-19) pandemic has highlighted various weaknesses in global healthcare services. the blood supply in africa is a critical element of the healthcare service that may be significantly affected by the pandemic. by implementing principles of patient blood management, primary healthcare practitioners may play an important role in the resilience of the blood supply during the covid-19 pandemic. tomorrow belongs to the people who prepare for it today. (african proverb) the world health organization (who) defines resilience in the context of the provision of essential health and health-related services as 'the inbuilt capacity of the system to sustain provision of essential health and health-related services even when challenged by outbreaks, disasters or other shocks'. 1 the coronavirus disease 2019 (covid-19) pandemic will undoubtedly test the global resilience of the blood supply, and significantly so in africa. the primary healthcare practitioner plays a crucial role in reducing the burden of anaemia, thus safeguarding the blood supply in africa. the covid-19 pandemic showcases weaknesses, shortcomings and lack of resilience in global healthcare services. whilst commendable work has been performed in health disaster risk management in africa, 2 and recommendations made on how to maintain the blood supply during infectious outbreaks and the covid-19 pandemic, 3, 4 no recommendation can entirely safeguard the blood supply. many countries have well-established healthcare systems and access safe blood, yet this is not true for most of africa. although the demand for blood is high, blood donation rates are very low, especially in low and lower middle-income countries in africa. 1 a third of maternal deaths in sub-saharan africa are because of maternal haemorrhage, and this region has the highest maternal mortality in the world. access to blood may have prevented up to a quarter of these deaths. 5, 6 in spite of this, many countries in africa collect less than 10 donations per 1000 population, the target recommended by the who. 7 twenty-two african countries depend on family, replacement or paid donors, and these donations account for more than 50% of the blood supply. 8 these challenges, although important, are unlikely to be resolved in the midst of the pandemic. blood donation may well be further reduced because of donor illness, countries imposing travel restrictions and donor fear of contracting the virus by visiting donor centres. the who has published guidelines to ensure the safety of blood donors and staff during the pandemic, 4 and donors should be reassured that they are unlikely to contract the novel coronavirus by donating blood when correct procedures are followed. road safety has improved as a result of covid-19 because of enforced travel restrictions, which has translated to fewer road traffic accidents (rtas) in south africa 9,10 and abroad. 11 although little data are available on the indications for the use of blood and blood products in africa, infectious diseases, obstetric haemorrhage, sickle cell disease and the broad term anaemia are the most common indications for transfusion. 12 in south africa, relatively few blood products are issued for general surgery and trauma, (11.3% and 2.8%, respectively), and the bulk of blood products are issued to medical patients, obstetrics and gynaecology and intensive care units the coronavirus disease 2019 (covid-19) pandemic has highlighted various weaknesses in global healthcare services. the blood supply in africa is a critical element of the healthcare service that may be significantly affected by the pandemic. by implementing principles of patient blood management, primary healthcare practitioners may play an important role in the resilience of the blood supply during the covid-19 pandemic. keywords: blood supply; patient blood management; africa; covid-19; resilience; transfusion. read online: scan this qr code with your smart phone or mobile device to read online. note: special collection: covid-19. (28.9%, 16.9% and 16.7%, respectively), 13 which is in agreement with previous observation. 12 these data suggest that even though lockdown may reduce the number of rtas, the need for blood will persist. this emphasises the need for a sustained blood supply that relies on uninterrupted blood donation, component production, appropriate clinical use of blood and blood products, as well as the implementation of patient blood management (pbm) programmes to alleviate anticipated shortages of donor blood. 14 implementation of the principles of pbm may prove to be a vital step in maintaining a sustainable blood supply in africa in the face of the pandemic. patient blood management is defined by the who as 'a patientfocussed, evidence-based and systematic approach to optimise the management of patient and transfusion of blood products for quality and effective patient care'. 15 in addition, the who emphasises that pbm should minimise unnecessary exposure to blood products and, through health promotion and screening, prevent conditions that may result in the need for transfusion. 15 patient blood management is built on three pillars: optimisation of erythropoiesis, minimisation of blood loss, and bleeding and harnessing and optimising physiological reserve of anaemia. 16 whilst pbm has been shown to be reduce the need for transfusion, reduce costs and improve patient safety and clinical outcomes, the implementation thereof has lagged behind. 14, 17, 18 for the most part, where pbm has been adopted, it has been incorporated into the practice of anaesthetists, surgeons, physicians, intensivists, and obstetricians and gynaecologists. in these disciplines, the focus has been on the identification and management of anaemia and bleeding risk, appropriate and conservative use of blood products and alternatives to transfusion. these are the key elements of pbm; however, conservative transfusion triggers are the norm in most of africa, and the traditional approach to pbm may only aid a minority of patients who have access to hospital and specialist care. in spite of efforts by the who, the burden of anaemia is high in africa, particularly east sub-saharan africa. iron deficiency, malaria, schistosomiasis, hookworm, sickle cell disease and thalassemia are the main causes of anaemia in africa. 19 many of these conditions are managed by the primary healthcare practitioner, who should thus play a pivotal part in the implementation of pbm in the outpatient setting. table 1 is adapted from the three-pillar approach from isbister and spahn, 16, 20 reworked for use by primary healthcare practitioners in the outpatient setting in africa. these recommendations are contextualised in light of the pandemic and can be applied to all patients who access who-compliant priority services, including the care of pregnant women, and patients who access care for emergency conditions, vaccination and the acceptable auxiliary services. 22 whilst it is acknowledged that transfusion cannot be avoided in patients with certain conditions and that many patients will still require transfusion as part of their standard care, the application of these principles may reduce the number of transfusions a potential transfusion recipient would need, and may thereby safeguard the blood supply for those who need it most. the 'common-sense' principles that underpin pbm have been recommended to address regional and national shortages of blood during the pandemic. 14 primary healthcare practitioners may play an important role in the resilience of the blood supply during the covid-19 pandemic. the principles of pbm outlined in this article are inexpensive and relatively easy to implement. if these principles are applied to all patients who receive primary healthcare during the pandemic, the blood supply may be safeguarded for those who need it most. world health organization. the state of health in the who african region: an analysis of the status of health, health services and health systems in the context of the sustainable development goals strengthening health disaster risk management in africa: multi-sectoral and people-centred approaches are required in the post-hyogo framework of action era protecting the blood supply during infectious disease outbreaks world health organization. maintaining a safe and adequate blood supply during the pandemic outbreak of coronavirus disease (covid-19): interim guidance world health organization and international federation of red cross and red crescent societies. towards 100% voluntary blood donation: a global framework for action van look pf. who analysis of causes of maternal death: a systematic review trends and gaps in national blood transfusion services -14 sub-saharan african countries geneva: who sa records 26 road crashes with 28 fatalities over easter weekend. the citizen fikile mbalula reports easter road death toll plunges 82% california covid-19 traffic report finds silver lining | uc davis. sci tech [serial online blood transfusion in sub-saharan africa: understanding the missing gap and responding to present and future challenges towards the future of blood transfusion -the south african national blood service's perspectives on cellular therapeutic services and products the essential role of patient blood management in a pandemic: a call for action anesth analg global forum for blood safety: patient blood management (pbm) structured observations the three-pillar matrix of patient blood management -an overview patient blood management -a new paradigm for transfusion medicine? multimodal patient blood management program based on a three-pillar strategy: a systematic review and meta-analysis a systematic analysis of global anemia burden from 1990 to 2010 alternatives to blood transfusion. lancet [serial online the isth bleeding assessment tool and the risk of future bleeding operational guidance for maintaining essential health services during an outbreak prof. colleen aldous for encouraging me to write the article. the author has declared that no competing interest exists. i declare that i am the sole author of this research article. this article used data and information from the public domain and primary authors are cited where appropriate. this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data sharing is not applicable to this article as no new data were created or analysed in this study. the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. key: cord-310254-ko1sptzm authors: attri, bhawna; goyal, alpesh; gupta, yashdeep; tandon, nikhil title: basal-bolus insulin regimen for hospitalised patients with covid-19 and diabetes mellitus: a practical approach date: 2020-07-18 journal: diabetes ther doi: 10.1007/s13300-020-00873-3 sha: doc_id: 310254 cord_uid: ko1sptzm background and aim: the coronavirus disease 2019 (covid-19) outbreak has rapidly crossed international boundaries and placed increasing demands on healthcare facilities worldwide. patients with diabetes and uncontrolled blood glucose levels are at increased risk for poor clinical outcomes and in-hospital mortality related to covid-19. therefore, achieving good glycaemic control is of paramount importance among hospitalised patients with covid-19. basal-bolus insulin therapy is a safe and effective intervention for the management of hyperglycaemia in hospitalised patients. the aim of this article is to provide a practical guidance for the use of the basal-bolus insulin regimen in hospitalised patients with covid-19 and diabetes mellitus. methods: this guidance document was formulated based on the review of available literature and the combined personal experiences of the authors. we provide a comprehensive review on the use of the basal-bolus insulin regimen, including its principles, rationale, indications, prerequisites, initiation, and dose titration, and also suggest targets for blood glucose control and different levels of capillary blood glucose monitoring. various case scenarios are used to illustrate how optimal glucose control can be achieved, such as through adjustments in doses of prandial and basal insulin, the use of correctional insulin dosing and changes in the timing and content of major and minor meals. conclusion: the practical guidance for the use of the basal-bolus insulin regimen in hospitalised patients with covid-19 and diabetes mellitus presented here can be used for patients admitted to hospital for indications other than covid-19 and for those in ambulatory care. electronic supplementary material: the online version of this article (10.1007/s13300-020-00873-3) contains supplementary material, which is available to authorized users. the coronavirus disease 2019 (covid-19) pandemic has affected 213 countries worldwide, with more than 5.4 million confirmed cases and more than 340,000 deaths at the time of writing this paper [1] . the outbreak of this disease has resulted in an unparalleled increase in admissions to dedicated covid-19 facilities, with diabetes, hypertension and cardiovascular disease reported to be major comorbidities among patients with covid-19 [2] . the pooled prevalence of diabetes in a meta-analysis of nine observational studies of patients hospitalised with covid-19 was reported to be 9% (95% confidence interval 6-12%) [3] . a high insulin requirement has been reported for the management of hyperglycaemia in patients with covid-19, especially in those with severe disease, which has been attributed to a combination of severe insulin resistance and decreased insulin secretion due to beta-cell dysfunction [4] . among patients with covid-19, diabetes is associated with an increased severity of infection, rapid disease progression, poor clinical outcomes and mortality [5] , but this association is mainly driven by the severity of hyperglycaemia. in a retrospective study of [ 7000 patients with covid-19 and pre-existing type 2 diabetes mellitus (t2dm), the in-hospital mortality rate was tenfold lower (1.1 vs. 11%) in patients with well-controlled blood glucose levels (upper limit b 10 mmol/l or 180 mg/dl) than in those with poor blood glucose control (upper limit [ 10 mmol/l or 180 mg/dl) during the course of hospitalisation [6] . thus, it is of paramount importance that hospitalised patients with covid-19 achieve good glycaemic control. intensive insulin regimens, such as basalbolus insulin therapy and continuous intravenous insulin infusion, are known to be safe and effective therapies for the management of hospitalised patients with hyperglycaemia [7] [8] [9] . however, given the rising burden of patients requiring hospital admission for covid-19 and a possible deployment of healthcare personnel without required expertise in the management of hyperglycaemia in these patients, the implementation of an effective insulin therapy may be challenging. here, we discuss a practical and simplified approach for the management of hyperglycaemia in hospitalised patients using the basal-bolus insulin regimen. this practical guidance is formulated based upon the available evidence and the combined clinical experiences of the authors, with the aim to help clinicians better manage the patients under their care. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. rationale basal-bolus insulin regimen implies the use of one or two doses of intermediate-or long-acting (basal) insulin and three, or rarely, four doses of short-acting (bolus or prandial) insulin. the basal insulin regulates the rise in blood glucose due to endogenous glucose production through the processes of glycogenolysis and gluconeogenesis, and the bolus insulin prevents the meal-related rise in blood glucose levels. pharmacokinetics of different insulin preparations basal insulin can be classified into: (1) conventional preparations, such as neutral protamine hagedorn (nph) insulin, which is intermediate acting (duration of action 12-16 h), and (2) insulin analogues, such as insulin detemir, insulin glargine and insulin degludec, which are intermediate or long acting (duration of action 12 to [ 24 h) (table 1) . similarly, bolus insulin can be classified into: (1) conventional preparations, such as regular insulin, which are short acting (onset and duration of action 30-60 min and 6-8 h, respectively); (2) rapid-acting insulin analogs, such as insulin lispro, insulin aspart and insulin glulisine (onset and duration of action 5-15 min and 3-5 h, respectively) ( table 1) ; and (3) ultra-rapid-acting insulin analogues, such as fast-acting insulin aspart (onset of action is twofold faster than that of insulin aspart) [10] [11] [12] [13] . thus, while regular insulin provides coverage during the post-absorptive period (beyond 4 h following a major meal), implying some degree of basal glucose regulation, the action of rapid-acting insulin is mainly limited the following factors should be kept in mind before initiating a basal-bolus insulin regimen (esm table 1 ): 1. the patient should receive enteral nutrition per oral administration or via a feeding tube. the latter may be relevant for a patient emerging from or currently on ventilatory support. a regular diet plan should be followed, with a fixed number of calories and a fixed carbohydrate proportion for each major and minor meal (or major and minor feed). in the hospital setting, especially in the current covid-19 scenario, very little support is likely to be available to assess the adequacy of teaching programmes aimed at educating the patient on carbohydrate counting; therefore, a meal with a fixed carbohydrate proportion should be the preferred option. 2. the patient should consume three major and three minor meals (or feeds) or three major and four minor meals (or feeds) (the latter option is preferred if the interval between lunch and dinner [or the second and third major feed] is large). 3. there should be a gap of at least 2 h between a major meal (or feed) and minor meal (or feed). this gap ensures that the release of prandial insulin is appropriate to match the post-meal rise in blood glucose and that there is no second unregulated wave of glucose release during this period. 4. meal (or feed) timings (both major and minor) should be relatively fixed. glycaemic targets for the management of hospitalised patients with hyperglycaemia are based upon capillary blood glucose monitoring. although blood glucose meters use capillary whole blood for the measurement of glucose, most devices currently used report a plasma equivalent value (to facilitate comparison with the laboratory value) [14] . it should be noted that capillary and venous plasma glucose values better approximate in the fasting state than the postprandial state, with capillary values in the latter state possibly being higher by 20-25% [15] . glycaemic targets for hospitalised patients with covid-19 should be decided based on age, comorbidities and presence or absence of diabetes-related complications. for individuals who are young, have no comorbidities or diabetes-related complications and tight blood glucose control can be achieved without causing undue hypoglycaemia, fasting blood glucose (fbg) of b 120 mg/dl (6.7 mmol/l) and 2-h postprandial blood glucose (ppbg) of b 160 mg/dl (8.9 mmol/l) should be targeted. for older individuals or those with comorbidities and diabetes-related complications for whom the risk of hypoglycaemia is higher, blood glucose targets should be relaxed to fbg b 140 mg/dl (7.8 mmol/l) and ppbg b 180 mg/dl (10 mmol/l). the upper limit for ppbg can be further relaxed to 220 mg/ dl (12.2 mmol/l) if the efforts to achieve target value b 180 mg/dl (10 mmol/l) are met with increased frequency of hypoglycaemia (table 2) . patients who were already on a basal-bolus insulin regimen prior to admission and have good blood glucose control can be continued on the same doses of insulin. further adjustments can be made based on capillary blood glucose monitoring as detailed in the following sections of this review. for patients who are insulin naïve, insulin therapy can be initiated at an empirical total daily dose (tdd) of 0.4 units/ kg/day [8] . the tdd should be reduced to 0.2 units/kg/day in elderly patients ([ 65 years) and in those with renal dysfunction, liver disease, cardiac disease, autonomic neuropathy or hypoglycaemia unawareness due to the heightened risk of hypoglycaemia and its associated complications in this patient subgroup. in the scenario in which regular insulin is used for prandial coverage, the calculated tdd is divided equally into four portions, with the implication of one portion for the basal insulin injection and one portion each for the three prandial insulin injections (25% of tdd in each insulin injection). however, when an insulin analogue is used for prandial coverage, the tdd should be divided into 50% for basal insulin and 50% for prandial insulin (further divided into three equal portions for each meal) [8] . transition from intravenous insulin infusion to the subcutaneous basal-bolus insulin regimen should ideally occur when the patient is consuming meals regularly, blood glucose levels are controlled and stable and any underlying illness has improved significantly. typically, the tdd is calculated as 80% of the total daily insulin requirement on intravenous infusion in the last 24-48 h [9] . the 20% decrement is accounted for by increased ambulation, improvement in glucotoxicity and decreased stress-related hyperglycaemia when patient is switched to a subcutaneous insulin regimen. the tdd can be proportionately divided into basal and prandial components as described above. however, when a marked difference in insulin requirement is evident across the different time segments (breakfast to lunch, lunch to dinner, dinner to midnight and midnight to before breakfast), the insulin dose for each segment should be calculated separately (esm table 2 ). pre-analytical factors, such as blood ph, hypoxia, hypotension and extreme hematocrit concentrations, can affect the performance of capillary blood glucose monitoring and should be given due consideration in the current scenario [16] . while most blood glucose meter test strips use the glucose oxidase method, which is specific for glucose, it is worth mentioning that the glucose dehydrogenase-pyrroloquinolinequinone (gdh-pqq) method is not glucose specific. consequently, gdh-pqq can react with other sugars, such as maltose, galactose and xylose, resulting in false high blood glucose readings in the presence of these molecules [17] . maltose is product of icodextrin metabolism (used in peritoneal dialysis solutions) and also a constituent of intravenous immunoglobulin, which is an experimental therapy for patients with severe covid-19 pneumonia [18] . erroneously high blood glucose readings leading to fatal insulin dose calculation errors may occur in such scenarios when using a gdh-pqq-based glucometer [17, [19] [20] [21] . for this reason, gdh-pqq-based blood glucose meters are not recommended for use in the hospital setting by the us food and drug administration [22] . a mix of preprandial and postprandial blood glucose values should be monitored along with the blood glucose values at 3 a.m., when needed. paired values around a meal are more informative for prandial insulin adjustments, as discussed subsequently in following sections. since intensive blood glucose control is needed in patients with active infection, the monitoring frequency should be higher, especially when blood glucose levels are out of the target range. the frequency of monitoring can be reduced once blood glucose levels are stable and in the target range. we suggest the following three levels of monitoring. level 1 monitoring level 1 monitoring consists of making one paired measurement (pre-and 2-h post-meal) each day. the meal around which the paired measurement is performed should be rotated on a daily basis (e.g. breakfast on day 1, lunch on day 2, dinner on day 3 and so on). this strategy should be used when [ 75% of the values are in target range without any episode of hypoglycaemia (table 3) . level 2 monitoring level 2 monitoring consists of making two paired measurements each day. as in level 1 monitoring, the meals around which the paired measurements are performed should be rotated on a daily basis (e.g. breakfast and lunch on day 1, lunch and dinner on day 2, dinner and breakfast on day 3 and so on). in a scenario where values around a specific meal are predominantly affected (e.g. lunch), one pair may be fixed for that meal and other rotated on a daily basis between other two meals (e.g. breakfast and dinner). this strategy should be used when 50-75% blood glucose values are in the target range (table 4) . level 3 monitoring consists of performing blood glucose tests before all meals (pre-breakfast, pre-lunch and pre-dinner) and after all meals (2-h post-breakfast, postlunch and post-dinner) on each day (6-point monitoring). this strategy should be used when \ 50% of the blood glucose values are in the target range. blood glucose testing at 3 a.m. is required in a scenario where fasting blood glucose is persistently elevated, as discussed in following sections ( table 5) . a continuous glucose monitoring system (cgms) device measures the glucose level in the interstitial fluid instead of the blood. the components of a cgms device are: (1) a sensor, which is inserted subcutaneously into the patient's abdomen or forearm; (2) a transmitter, which is attached to the sensor; and (3) a receiver which displays and stores glucose data. the sensor reports the interstitial glucose level every 5-15 min and can be worn for a duration of 6-14 days (varies according to the type of sensor). unlike conventional monitoring, which provides snapshots of blood glucose values, cgms provides information on glucose trends and fluctuations [23, 24] . cgms devices can be: (1) [25] . while the former devices allow real-time assessment of glucose levels (at a site remote from the patient), in the latter devices, data can only be reviewed retrospectively after the device has been worn for a certain period of time. thus, a real-time cgms not only provides an opportunity for stringent glucose monitoring but also reduces the need for repeated contact between the healthcare worker and the patient, which is very relevant in the current covid-19 scenario. since a lag time of 7-15 min exists between interstitial fluid glucose level and blood glucose level, the sensor value may be less accurate when glucose values are changing rapidly, such level 2 capillary capillary blood glucose monitoring is advised when 50-75% of blood glucose values are in the target range a in case values around a specific meal are predominantly affected (e.g. lunch), one pair may be fixed for that meal and other rotated on daily basis between the other two meals (e.g. breakfast and dinner) b blood glucose should be monitored at 3 a.m. when the fasting blood glucose is persistently out of the target range level 3 capillary capillary blood glucose monitoring is advised when \ 50% of the blood glucose values are in the target range a blood glucose should be monitored at 3 a.m. when the fasting blood glucose is persistently out of the target range as after a meal and during or before a hypoglycaemic episode. once insulin therapy has been initiated, the doses of prandial and basal insulin will need titration based on blood glucose monitoring. the titration of insulin dose is based on two main principles: (1) having an understanding of the segment of the day when insulin works; (2) having an understanding of the type of adjustment needed. each insulin dose in the basal-bolus regimen works during a particular segment of the day. prandial insulin administered before breakfast predominantly acts from breakfast to lunch (around 8 a.m. to 1 p.m.); prandial insulin given before lunch predominant acts from lunch to dinner (around 1 p.m. to 7-8 p.m.); prandial insulin given before dinner predominantly acts from dinner to midnight (8 p.m. to 12 a.m.); and basal insulin given at bedtime (especially nph) predominantly acts from midnight to breakfast (12 a.m. to 8 a.m.). although there may be some overlap in action, this broad concept helps the clinician to determine the appropriate dose adjustment based upon the demarcation of the segment(s) during which blood glucose targets are not achieved. there are four main types of adjustments that can be made to achieve optimal blood glucose control; these are: (1) prandial insulin adjustments; (2) correctional insulin adjustments, (3) basal insulin adjustments; and (4) meals and snacks adjustments. these are described in detail in the following sections. the postprandial rise in blood glucose is physiological and to a certain degree desirable. an idea of the degree of postprandial elevation that can be considered to be normal can be derived from an individual's glycaemic targets. for example, if the optimal preprandial target for an individual is b 120 mg/dl (6.7 mmol/l) and the optimal postprandial target is b 160 mg/dl (8.9 mmol/l), the difference between the two, that is, 40 mg/dl (2.2 mmol/l), can be taken as the upper limit of the allowed postprandial excursion. if the difference is consistently [ 40 mg/dl (2.2 mmol/l), and the postprandial glucose is also out of range ([ 160 mg/dl or 8.9 mmol/l) on two or more consecutive occasions for a given meal, this difference needs to be addressed. the healthcare professional should ensure that the insulin injection technique is correct, that there is an adequate time gap between the injection of prandial insulin and the meal (30 min for regular insulin) and that the quality and quantity of carbohydrate in the meal is appropriate and relatively fixed. in a scenario where these issues are not relevant to the problem or postprandial excursion persists despite these issues being addressed, the dose of prandial insulin should be increased to reduce the postprandial excursion to \ 40 mg/dl (2.2 mmol/l). if the postprandial excursion is \ 40 mg/dl (2.2 mmol/l), but the postprandial value is out of the target range due to a high preprandial value ([ 120 mg/dl or 6.7 mmol/l), measures to control preprandial blood glucose should be adopted (discussed later in review). it is also important to understand that the postprandial blood glucose value should not be lower than the preprandial blood glucose value as this situation increases the chances of hypoglycaemia. in such a scenario, if the meal intake was adequate and there were no other factors which could explain the fall in blood glucose (such as exercise, vomiting or diarrhea), the dose of prandial insulin should be reduced. a dose reduction should be strongly considered if postprandial blood glucose is \ 100 to 120 mg/ dl (5.6-6.7 mmol/l) or hypoglyceamia occurs in the postprandial period. as a general strategy, if postprandial blood glucose is less than preprandial blood glucose by b 20 mg/dl table 6 . when the elevation in preprandial blood glucose is very remarkable ([ 200 to 250 mg/dl or 11.1 to13.9 mmol/l), the treating physician may need to administer an additional correctional dose of short-acting insulin in addition to the usual prandial dose [26] . this correctional dose is given with the aim to rapidly reduce the elevated blood glucose levels, and the dose is determined based on a correction factor. the correction factor indicates the decrease in blood glucose (mg/dl) expected with 1 unit of shortacting insulin and depends upon the insulin sensitivity of the person being treated. as a rule, the higher the tdd (units/kg body weight), the lower the insulin sensitivity and correction factor. correction factors based on tdd are shown in table 7 . the practical use of a correctional dose of insulin can be better explained with an example, as follows. a 46-year-old man with t2dm for 3 years, a body weight of 72 kg and no comorbidities or diabetes-related complications is being treated with a basal-bolus insulin regimen. his tdd of insulin is 45 units (0.63 unit/ kg body weight), injected as 10 units of regular insulin before each meal and 15 units of insulin glargine at bedtime. his correction factor (table 7) is 40, that is, 1 unit of regular insulin is expected to decrease the blood glucose level by 40 mg/dl (2.2 mmol/l). in a scenario in which his before-lunch blood glucose is 280 mg/dl (higher than the target blood glucose of 120 mg/dl since insulin sensitivity is dynamic and varies depending on multiple factors, such as time of the day (higher at night), severity of underlying illness and use of concomitant drugs, the correction factor should be adjusted based on the changes in blood glucose observed with correctional insulin doses over the previous 2-3 days. adjustments in bedtime basal insulin may be done based upon the fasting or pre-breakfast blood glucose value. the ideal decrease in blood glucose expected with basal insulin is determined by the difference in the upper limit of targets for post-dinner (160 mg/dl or 8.9 mmol/ l) and pre-breakfast blood glucose (120 mg/dl or 6.7 mmol/l) values. therefore, the action of basal insulin can be considered to be optimal if the decline in blood glucose is around 40 mg/dl (2.2 mmol/l). in case the fasting or pre-breakfast blood glucose is above target despite a decline of c 40 mg/dl (2.2 mmol/l), the elevated post-dinner blood glucose value may need to be addressed with the principles delineated above. if the fasting or pre-breakfast blood glucose is out of the target range and the overnight in refractory cases, addition of short-or rapid-acting insulin before evening snack may be warranted 12 and 8 a.m.) . blood glucose should be checked at 3 a.m. if two or more consecutive fasting or pre-breakfast blood glucose values are above the target. a 3 a.m. blood glucose value of \ 70 mg/dl (3.9 mmol/l) suggests that late night hypoglycaemia is the possible cause of the raised morning blood glucose level. this situation will require a decrease in the dose of basal insulin and/or the addition of or increase in bedtime snacks. if the 3 a.m. blood glucose blood is c 100 mg/dl (5.6 mmol/l), the dose of basal insulin should be increased after excluding early morning tea or snacks and a faulty insulin injection technique. if the fasting or pre-breakfast blood glucose is 70-90 mg/dl (3.9-5.0 mmol/l), the dose of basal insulin should be decreased by 1 unit (10% if the dose of basal insulin is [ 10 units). if the fasting or pre-breakfast blood glucose is \ 70 mg/dl (3.9 mmol/l) or the patient experiences nocturnal hypoglycaemia, the basal insulin dose should be decreased by 2 units (10-20% if the dose of basal insulin is [ 10 units) and the bedtime snack(s) policy reviewed. in a scenario where twice-daily basal insulin is used, the adjustments may be more complex. it should be understood that the primary action of basal insulin is that of regulating pre-meal blood glucose values. accordingly, the morning dose of basal insulin should be adjusted based upon the pre-dinner blood glucose value and less commonly the pre-lunch blood glucose value. the increase in the dose of morning basal insulin is only justified if the upward drift of blood glucose starts after the peak of lunch time prandial insulin is over, implying that the postlunch blood glucose is in target but the predinner and evening blood glucose values are elevated. however, when both the pre-dinner blood glucose and post-lunch blood glucose levels are elevated, it is the dose of prandial (before lunch) insulin that should primarily be adjusted, and not that of basal insulin. similarly, in the case of preprandial hypoglycaemia (during the pre-lunch or predinner period), the morning basal insulin dose should be decreased, provided the hypoglycaemia is not explained by missed or inadequate snacks or other factors (such as exercise, vomiting and/or diarrhea). basal insulin adjustment is explained in the context of various case scenarios in table 8 . medical nutrition therapy (mnt) is the cornerstone of effective blood glucose management in patients with diabetes [27, 28] . the broad aim of mnt is to provide an individual with adequate calories to meet his/her nutrition demands. total calories are split in three major meals (breakfast, lunch and dinner; or major feed 1, major feed 2 and major feed 3) and three to four minor meals (snacks) or feeds (mid-day [10-11 a.m.], early evening [4-5 p.m.] , late evening [6-7 p.m.] and bedtime [10-11 p.m.] ) to provide nutrition throughout the day, regulate the rise in blood glucose after a meal and avoid hypoglycaemia. a fixed meal pattern in terms of timing and content is desirable in the hospital setting (as discussed above). however, even with relatively fixed diet patterns, individual adjustments may be needed. in a scenario in which postprandial blood glucose is in target, but the subsequent preprandial value is elevated (for instance, prebreakfast blood glucose of 115 mg/dl (6.4 mmol/l), post-breakfast blood glucose of 145 mg/dl (8.1 mmol/l) and pre-lunch blood glucose of 140 mg/dl (7.8 mmol/l), the carbohydrate quality (glycaemic index) and quantity (calories) of the intervening snack (mid-morning snack in this case) should be checked. similarly, if the post-prandial blood glucose is within normal range, but the subsequent preprandial value is low (\ 80 mg/dl or 4.4 mmol/ l) or patient experiences hypoglycaemic symptoms at this time [for instance, pre-breakfast blood glucose of 100 mg/dl (5.6 mmol/l), postbreakfast blood glucose of 120 mg/dl (6.7 mmol/l) and pre-lunch blood glucose of 72 mg/dl (4.0 mmol/l)], it should be checked if the intervening snack (mid-morning snack in this case) was missed or was inadequate in terms of calories. meals and snacks adjustment has been explained with various case scenarios in table 9 . discrepancies in patient's diet pattern can result in glycaemic variability. therefore, the patient (or attending healthcare provider) should be advised to note diet content, meal timings and any variance from the previous days in the remarks column of the blood glucose log or in a separate diary for review by the treating physician. type 1 diabetes mellitus and diabetic ketoacidosis one study reported that a large number of patients with covid-19 developed diabetic ketoacidosis (dka), hyperosmolar hyperglycaemic state (hhs) or a combination of both [29] . it has also been found that covid-19 may mask the symptoms of dka [30] . in a study of 64 patients with type 1 diabetes mellitus (t1dm) who tested positive for covid-19 or covid-19-like disease, a significant proportion had hyperglycaemia (n = 32, 50%) or dka (n = 19, 30%) [31] . these results strongly suggest that all patients with covid-19 should be screened for hyperglycaemia and that blood or urine ketone testing should be considered in patients with a blood glucose value [250 mg/dl (13.9 mmol/l) [32, 33] . for patients with t1dm who are already on a basal-bolus insulin regimen, the same regimen can be continued, with dose titration performed according to the results of blood glucose monitoring. however, in cases where ketosis or ketoacidosis is present, intravenous insulin infusion should be the initial preferred strategy. on the other hand, in the current scenario of covid-19, switching patients on continuous subcutaneous insulin infusion or insulin pump to the basal-bolus insulin regimen should be strongly considered-unless expert guidance is available to manage the former. use of glucocorticoids selected patients with moderate to severe covid-19 may require glucocorticoids, usually administered as intravenous methylprednisolone 0.5-1.0 mg/kg/day in two divided doses [34] . the use of glucocorticoids may worsen hyperglycaemia and necessitate adjustments in insulin therapy. a detailed discussion of the management of glucocorticoid-induced hyperglycaemia is beyond the scope of this article and the reader is directed to excellent reviews on this topic [35] [36] [37] . acute kidney injury acute kidney injury (aki) has been reported as a complication of severe covid-19. in a multi-hospital study involving [ 5400 patients admitted with covid-19, aki developed in 36.6% patients, of whom 14.3% required dialysis [38] . the development of renal dysfunction is associated with a reduced clearance of insulin and hence necessitates a reduction in the dose of exogenous insulin to prevent hypoglycaemia [39] . in addition, the requirement of insulin may vary on pre-and post-dialysis days. initiation of dialysis is associated with an improvement in peripheral insulin resistance and hence the dose requirement may decrease further following dialysis [40, 41] . effect on blood glucose patients with covid-19 may receive oral hydroxychloroquine (hcq), which is also an effective anti-hyperglycaemic agent [42] . in such cases, the patient should be kept under close watch for hypoglycaemia, and an adjustment in insulin dose should be considered. the use of oral dipeptidyl peptidase-4 (dpp-4) inhibitors has also been reported to be a safe and effective treatment in patients with mild to moderate hyperglycaemia and in stable clinical condition, and a reduction of insulin dose may be needed in situations where these agents are co-administered with insulin [43] . the use or continuation of glucoselowering oral drugs other than dpp-4 inhibitors is generally not recommended for acutely ill patients with covid-19 due to the potential for abrupt deterioration in clinical status [44] . the risk for euglycaemic dka and volume depletion contraindicates the use of sodium-glucose cotransporter-2 (sglt2) inhibitors in any hospitalised patient at this time [44] . we have presented a practical guidance for the management of patients on basal-bolus insulin therapy. this guidance can also be used for patients hospitalised for indications other than covid-19. some patients are primarily admitted for glycaemic control and are often encouraged to perform some physical activity (such as walking) in the hospital premises, when possible. in such a scenario, one should ensure that the timing and quantum of physical activity is relatively fixed. preferably, physical activity should be avoided late in evening or after dinner to prevent nocturnal hypoglycaemia. additionally, brisk walking after an insulin injection in the thigh should be avoided as it can result in rapid absorption of insulin and subsequent hypoglycaemia [45] . a close watch for hypoglycaemia should be maintained in every hospitalised patient receiving insulin therapy. during the course of the hospital stay, the patient should be educated on recognising the symptoms of hypoglycaemia, as well as on its acute management and prevention of future episodes. in ambulatory patients, the adjustments in insulin doses should not be too frequent and should be done preferably after the patient's blood glucose log has been reviewed for at least 3-4 days. the patient should be educated on principles of insulin dose self-adjustment highlighted in the previous section. the unsupervised use of correctional insulin could be risky and should be avoided. the proposed guidance is based on a review of available literature and the combined extensive clinical experiences of the authors. it provides a comprehensive review on the use of the basalbolus insulin regimen in routine clinical practice with the help of different case scenarios. the principles and concepts highlighted can be easily understood by healthcare personnel with lesser training and experience than skilled physicians in the management of hyperglycaemia in hospitalised patients. the guidance may be used for settings beyond covid-19 and for ambulatory patient care. a limitation of this practical approach is that it has not been tested in a formal manner. this review provides a practical guidance on the use of the basal-bolus insulin regimen in patients with diabetes mellitus hospitalised with covid-19. we have used various case scenarios to illustrate adjustments in doses of prandial and basal insulin, the use of correctional insulin and changes in timing and content of major and minor meals for achieving optimal blood glucose control. this guidance can be used in the future for patients admitted for indications other than covid-19 and for those in ambulatory care. author contributions. the concept for this paper was conceived by ag, yg and nt. the first draft was prepared by ba and ag, which was read and edited by yg and nt. all authors approved the final version of this manuscript. disclosures. bhawna attri, alpesh goyal; yashdeep gupta and nikhil tandon have nothing to disclose. compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. data availability. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. open access. this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/bync/4.0/. who coronavirus disease (covid-19) dashboard diabetes in covid-19: prevalence, pathophysiology, prognosis and practical considerations prevalence of diabetes mellitus in 2019 novel coronavirus: a metaanalysis practical recommendations for the management of diabetes in patients with covid-19 diabetes mellitus is associated with increased mortality and severity of disease in covid-19 pneumonia-a systematic review, meta-analysis, and meta-regression. diabetes metab syndr association of blood glucose control and outcomes in patients with covid-19 and pre-existing type 2 diabetes insulin therapy for the management of hyperglycemia in hospitalized patients 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publication of this article.authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. key: cord-018414-6ffhm895 authors: kang, yoogoo; elia, elia title: anesthesia management of liver transplantation date: 2016-07-22 journal: contemporary liver transplantation doi: 10.1007/978-3-319-07209-8_9 sha: doc_id: 18414 cord_uid: 6ffhm895 anesthesia for liver transplantation pertains to a continuum of critical care of patients with end-stage liver disease. hence, anesthesiologists, armed with a comprehensive understanding of pathophysiology and physiologic effects of liver transplantation on recipients, are expected to maintain homeostasis of all organ function. specifically, patients with fulminant hepatic failure develop significant changes in cerebral function, and cerebral perfusion is maintained by monitoring cerebral blood flow and cerebral metabolic rate of oxygen, and intracranial pressure. hyperdynamic circulation is challenged by the postreperfusion syndrome, which may lead to cardiovascular collapse. the goal of circulatory support is to maintain tissue perfusion via optimal preload, contractility, and heart rate using the guidance of right-heart catheterization and transesophageal echocardiography. portopulmonary hypertension and hepatopulmonary syndrome have high morbidity and mortality, and they should be properly evaluated preoperatively. major bleeding is a common occurrence, and euvolemia is maintained using a rapid infusion device. pre-existing coagulopathy is compounded by dilution, fibrinolysis, heparin effect, and excessive activation. it is treated using selective component or pharmacologic therapy based on the viscoelastic properties of whole blood. hypocalcemia and hyperkalemia from massive transfusion, lack of hepatic function, and the postreperfusion syndrome should be aggressively treated. close communication between all parties involved in liver transplantation is also equally valuable in achieving a successful outcome. dr. thomas starzl of denver, colorado, usa, who believed that "liver transplantation is an effective treatment providing exactly what is needed for patients with end-stage liver disease (esld)," performed the first successful orthotopic liver transplantation (olt) in a 3-year-old boy with biliary atresia in 1963 (starzl et al. 1963) . during the first two decades of the procedure's history, liver transplantation led by starzl and sir roy calne of cambridge encountered almost insurmountable challenges, including complexity of surgical technique, primitive anesthesia and intensive care, less-than-adequate immunosuppression and organ preservation, and devastating infection. the number of procedures performed was relatively few, and the success rate was low. however, their keen observations on these early clinical experiences laid the foundation of modern liver transplantation (starzl and putnam 1969; calne 1983) . breakthroughs were made in each decade following the first transplantation. in the 1980s, venovenous bypass was introduced to maintain better hemodynamic stability (shaw et al. 1984) , cyclosporine was found to be a superior immunosuppressant to azathioprine, and anesthesiologists answered important clinical questions, including those relating to the monitoring and treatment of coagulopathy, hemodynamic changes, and the role of the electrolyte imbalance. in the 1990s, fk506 (tacrolimus) became the immunosuppressant of choice ), university of wisconsin solution was introduced to extend the safe cold ischemia time to 24 h (kalayoglu et al. 1988) , and the piggyback technique simplified surgery in select patients (tzakis et al. 1989 ). in the past 15 years, liver transplantation has been performed in most major medical centers with a 1-year survival rate of greater than 85 %, and living donor liver transplantation has become a valuable alternative. liver transplantation requires a true multidisciplinary approach, and anesthesiologists and intensivists have played a major role in the successful outcome of liver transplantation. in support of the important role of anesthesiologists in liver transplantation, the american society of anesthesiologists (asa) developed the guidelines for director of liver transplant anesthesia in 2001. the guidelines specified that the director should have fellowship training in critical care medicine, cardiac anesthesiology, or transplantation anesthesiology that includes the perioperative care of at least ten liver transplant recipients or experience in the perioperative care of at least 20 liver transplant recipients in the operating room. in addition, the director is expected to obtain a minimum of 8 h of accreditation council for continuing medical education (accme) category i continuing medical education (cme) credit in transplantation-related educational activities within the most recent 3-year period. in this chapter, physiology and pathophysiology of liver disease and anesthesia care of liver transplantation are described based on clinical experience at the university of pittsburgh (pittsburgh, pa, usa) and thomas jefferson university (philadelphia, pa, usa). the liver, which weighs 1200-1500 g in adults, is traditionally divided into the right and left lobe in reference to the location of the falciform ligament. couinaud, however, divided the liver into the right and left hemiliver using the cantlie's line, which extends from the inferior vena cava (ivc) to the gall bladder, and each hemiliver is further divided into four segments (couinaud 1954) . the left hemiliver is composed of the traditional left lobe along with the caudate and quadrate lobe. liver resections based on these segmental definitions are right hepatectomy (segments 5-8), right lobectomy (segments 4-8), left hepatectomy (segments 1-4), and left lobectomy (segments 1-3) ( fig. 1) (bismuth 1982) . the liver has a unique dual blood supply: arterial supply from the hepatic artery, a branch of the celiac axis, and venous supply from the portal vein formed by the union of the splenic and superior mesenteric vein. despite liver mass constituting only 2.5 % of the total body weight, the total hepatic blood flow is approximately 100 ml/ 100 g/min, or 25 % of cardiac output. the hepatic artery supplies approximately 25-30 % of hepatic blood flow and 45-50 % of the oxygen requirement, while the portal vein supplies 70-75 % of hepatic blood flow and 50-55 % of oxygen. the venous drainage is through the right, middle, and left hepatic veins, which merge into the ivc. the valveless portal vein is a low pressure/low resistance circuit, while the hepatic artery is a high pressure/high resistance system. hepatic 553-565, copyright (2000) , with permission from elsevier) blood flow is primarily regulated by local metabolic demand with an inverse relationship between portal venous and hepatic arterial flow: an increase in the hepatic adenosine level triggered by a reduced portal venous flow increases hepatic arterial blood flow (gelman and ernst 1977; lautt et al. 1985) . the hepatic artery buffer response appears to be functional even after liver transplantation (payen et al. 1990) , and this response may be responsible for the development of the small-for-size syndrome after living donor or split liver transplantation (kiuchi et al. 1999 ). small-for-size syndrome develops in a patient who received a donor graft that was less than 1 % of the recipient's body weight and is caused by decreased hepatic arterial flow in response to increased portal venous flow and pressure. subsequently, a prolonged postoperative reduction in hepatic arterial flow can lead to centrilobular tissue necrosis, biliary ischemia, and hepatic arterial thrombosis (smyrniotis et al. 2002) . there is no buffer response in the portal system because the portal vein cannot regulate its blood flow. therefore, alterations in the hepatic arterial blood flow do not induce compensatory changes in the portal blood flow (lautt 1983) . the mean pressure in the hepatic artery is similar to that in the aorta, while portal vein pressure ranges between 6 and 10 mmhg. the portal pressure depends primarily on the degree of constriction or dilatation of the splanchnic arterioles and on intrahepatic resistance. both afferent systems merge at the sinusoidal bed, where the pressure is estimated to be 2-4 mmhg higher than that in the ivc. the liver serves as a blood reservoir, and it replenishes blood volume of up to 25 % rapidly in the case of an acute bleeding episode (lautt 2007) . hepatic blood volume may expand considerably in cardiac failure by venous congestion. the liver is innervated by the left and right vagi, the right phrenic nerve, and fibers from the t7-t10 sympathetic ganglia. the hepatic artery is innervated mainly by sympathetic fibers, and hepatocytes, by the unmyelinated sympathetic fibers. the bile ducts are innervated by both sympathetic and parasympathetic fibers. the role of hepatic innervation is unclear, as denervation of the transplanted liver does not affect its function (kjaer et al. 1994) . bile flow begins from the bile canaliculi to the common bile duct. hepatic lymph forms in the space between the sinusoid and the hepatocyte (space of disse) and flows to lymph nodes in the hilum and ivc. the transdiaphragmatic lymphatic flow is the cause of pleural effusions in the presence of large ascites. the liver is made of parenchymal cells (hepatocytes) and non-parenchymal cells (sinusoidal endothelial, kupffer, stellate, dendritic, and lymphocyte) . hepatocytes make up 60-80 % of liver cells and carry out hepatic metabolic, synthetic, and detoxification functions. polyhedral hepatocytes are arranged in one-cell thick plates with endothelium-lined sinusoids on both sides. each hepatocyte cell membrane has three distinct membrane domains. the sinusoidal membrane is adjacent to the sinusoidal endothelium and has numerous microvilli abutting into the space of disse. fenestrae within the sinusoidal endothelium without the basement membrane permit intimate contact between sinusoidal blood and the hepatocytes to allow the passage of big molecules, including lipoproteins. liver sinusoidal endothelial cells make up 15-20 % of liver cells and release nitric oxide to regulate vascular resistance. they are, along with dendritic cells and lymphocytes, part of the innate immune system. the space of disse contains phagocytic kupffer cells that participate in the hepatic inflammatory process. the ito cells, also known as stellate cells, are the major site of vitamin a storage, and their activation results in hepatic fibrosis and cirrhosis. reticulin fibers in the space of disse support the sinusoidal framework, and weakening of these supporting fibers results in rupture of sinusoidal walls and formation of blood-filled cysts known as peliosis hepatis, a forerunner of cirrhosis. the apical membrane circumscribes the canaliculus, the earliest component of the biliary system. the lateral hepatic membrane is found between adjacent hepatocytes. the functional unit of the liver is the acinus. terminal portal veins communicate with terminal hepatic venules, with sinusoids bridging the gap between the two vessels ( fig. 2) . each sinus contains three zones with equal blood pressure and oxygen content. the periportal zone (zone 1) receives blood highest in oxygen content and the pericentral or perivenular zone (zone 3) receives blood lowest in oxygen content. as a result, the hepatocytes in the perivenular zone (zone 3) are more vulnerable to ischemic damage and nutrient depletion. oxidative and reductive functions are predominantly performed by hepatocytes at the periportal zone and glucuronidation is performed by those at the perivenular zone, although hepatocytes of the two different zones are functionally integrated (lamers et al. 1989 ). the unique structure of the liver acinus is well-suited for bidirectional transfer of nutrients. the low pressure in the portal venous system allows blood to flow slowly through the sinusoids. hepatic arterial blood flows mainly to the terminal bile canaliculi, although it augments sinusoidal flow to give a gentle pulsatility. in patients with liver cirrhosis, the sinusoids acquire features of systemic capillaries: the space of disse widens with collagen deposits at the basement membrane, endothelial fenestrations become smaller and fewer, and hepatic microvilli efface. all of these changes reduce transport across the sinusoidal walls and result in hepatic dysfunction. furthermore, widespread fibrosis and scarring reduce the number and size of the small portal and hepatic veins and increase intrahepatic vascular resistance to the development of portal hypertension (popper 1977) . the sluggish blood flow in the altered vascular architecture promotes thrombosis, causing further cell necrosis and fibrosis (wanless et al. 1995) . the liver undergoes rapid regeneration through proliferation of hepatocytes to maintain the critical mass necessary for normal liver function. for example, the newly transplanted hemiliver from a living related donor regenerates to about 85 % of its original whole liver size in 7-14 days. the major hepatic growth factors are epidermal growth factor and hepatocyte growth factor (michalopoulos 1990) . administration of the epidermal or hepatocyte growth factor to normal rats, however, does not cause hepatocyte replication. this negative response suggests that liver regeneration involves a two-step process: the initial signal generated by an acute increase in metabolic demand associated with the loss of hepatocytes triggers a set of early response genes that prime hepatocytes to respond to various growth factors. in apoptosis or programmed cell death, aging hepatocytes are removed and new cells are produced in a continuous manner (ellis et al. 1991 ). the liver has three major functions: metabolism, bile production and secretion, and filtration of harmful substances. the principal role of the liver is to provide the body with normal glucose levels, which are regulated by insulin, glucagon, growth hormone, and catecholamines (pilkis and granner 1992) . the liver converts glucose into glycogen (glycogenesis) and utilizes glucose for the synthesis of fatty acids. cirrhotic patients are frequently hyperglycemic although their insulin level is elevated (petrides and defronzo 1989) . this insulin resistance is caused by multiple mechanisms. cirrhotic patients have an increased basal metabolic rate and use preferentially fatty acids as an energy source. reduced glucose uptake and limited glucose storage in the liver and muscle lead to hyperglycemia. other contributing factors are increased serum fatty acids, which inhibit glucose uptake by muscle; altered second messenger activity after insulin binding to its receptors; an increased concentration of serum cytokines associated with elevated levels of endotoxins; and increased levels of glucagon and catecholamines. the liver is the major organ for protein synthesis, and albumin is the most important protein product. albumin is the major contributor to plasma oncotic pressure and binds and transports bilirubin, hormones, fatty acids, and other substances. hypoalbuminemia is commonly caused by decreased hepatic synthetic function, although it can be secondary to an enlarged volume of distribution, reduced level of amino acid precursors, and losses into the urine, peritoneum and pleural cavity, and leads to peripheral edema, ascites, and pleural effusions. the low serum oncotic pressure stimulates the hepatic albumin synthesis in healthy subjects, but this is impaired in patients with cirrhosis (pierrangelo et al. 1992) . the liver synthesizes all coagulation factors (except von willebrand factor) and protein c and s. factors ii, vii, ix, and x undergo a posttranslational vitamin k-dependent modification involving γ-carboxylation of specific glutamic acid residues in the liver. the liver is the primary site of interconversion of amino acids. anabolic processes synthesize proteins from amino acids, while catabolic processes convert amino acids either to keto acids by transamination or ammonia by oxidative deamination. ammonia, in turn, is converted to urea by the krebs-henseleit cycle. in patients with liver disease, derangement of both anabolic and catabolic processes results in decreased production of blood urea nitrogen (bun) and accumulation of ammonia, a contributing factor in the development of hepatic encephalopathy. the liver produces acute-phase reactants, such as α-fetoprotein, ceruloplasmin, fibrinogen, transferrin, complement, and ferritin. they are expressed during acute and chronic systemic inflammation, and their activation is mediated by interleukin-6, tumor necrosis factor, interferon-γ, and glucocorticoids. the liver takes up fatty acids and cholesterol from diet and peripheral tissues to produce and release lipoprotein complexes into circulation. fatty acids released from adipocytes are bound to serum albumin and transported to the liver for the synthesis of phospholipids and triglycerides. the liver produces fatty acids from small molecular weight precursors, and cholesterol synthesis is regulated by the ratelimiting enzyme 3-hydroxyl-3 methylglutaryl coenzyme a reductase (hmg-coa reductase). lipids are exported out of the liver by very low-density lipoprotein (vldl) particles, which are the major carriers of plasma triglycerides during non-absorptive states. lipids are temporarily stored in the liver as fat droplets, or as cholesteryl esters in the case of cholesterol, and are directly excreted into bile or metabolized into bile acids. the liver is the major site for sterol excretion and production of bile acids. various abnormalities in lipid metabolism are common in liver disease. hypertriglyceridemia (250-500 mg/dl) is the most common presentation and may be caused by decreased synthesis of lipoproteins, decreased hepatic clearance of lipoprotein complexes, or re-entry of biliary content into the serum. alcoholic liver injury results in increased fatty acid synthesis and steatosis (lieber 1993) . paradoxically, an increased high-density lipoprotein (hdl) 3 level has been noted with moderate alcohol consumption, which may explain the reduced risk of atherosclerosis in these patients (chait and brunzell 1990) . patients with cholestatic liver diseases have elevated total serum cholesterol and triglycerides because the bile is rich in cholesterol, phospholipids, and lecithin. the liver eliminates drugs through two types of reactions. the phase 1 reactions include oxidation, reduction, hydroxylation, sulfoxidation, deamination, dealkylation, and methylation of reactive substances. these reactions involve systems such as cytochrome p450 and typically occur in the periportal area of the liver. the phase 2 reactions, which transform lipophilic agents into more water-soluble compounds, take place in the pericentral area. in patients with liver disease, hepatic drug clearance is usually reduced due to the enlarged volume of distribution and decreased hepatic metabolism. as a result, a large initial dose of medications followed by small, titrated maintenance doses are required to achieve the desired pharmacologic effects. several hormones are deactivated or altered in the liver. the deactivated hormones are insulin, glucagon, steroid hormones, aldosterone, thyroxine, and triiodothyronine. the liver converts testosterone into androsterone and estrogen into estrone and estriol. abnormal levels of estrogen and testosterone in patients with liver disease lead to testicular atrophy, loss of pubic and axillary hair, spider angioma, and gynecomastia. the liver removes various substances from the body, and bile formation is one of the most important excretory functions. when membranes of old erythrocytes rupture, the released hemoglobin is taken up by the reticuloendothelial cells and is split into heme and globin. heme converts to biliverdin, which, in turn, is reduced to free bilirubin and released into the plasma. the free bilirubin-albumin complex is taken up by the hepatocytes. bilirubin conjugates primarily with glucuronic acid and is actively transported into the bile. a small portion of conjugated bilirubin returns to the plasma directly from the sinusoids or indirectly by absorption from the bile ducts and lymphatics. bilirubin is converted into urobilinogen by the intestinal bacterial flora. some urobilinogen is reabsorbed through the intestinal mucosa and is re-excreted into the intestine. bile acids, which enhance absorption of vitamin k, are also excreted into the bile by the liver. the liver, located between the splanchnic and systemic venous system, acts as a vascular filter. kupffer cells phagocytose immune complexes, endotoxins, and bacteria in the portal venous blood and process antigens for presentation to immunocompetent cells. the liver also removes activated coagulation elements from circulation to prevent excessive coagulation or fibrinolysis. liver cirrhosis is defined as progressive fibrosis and the formation of regenerative nodules, and is the final common pathway in which hepatocytes are replaced by connective tissue after various, repetitive insults. the amount of remaining functional hepatic mass and the degree of architectural distortion determine the functional state of the liver. portal hypertension is inevitable in advanced cirrhosis and leads to ascites, variceal bleeding, and encephalopathy. the severity of cirrhosis is frequently classified using the child-pugh score (table 1) , and a score of >6 suggests a short life expectancy. hepatic encephalopathy is a reversible neuropsychiatric condition in both acute and chronic liver failure. in chronic liver disease, hepatic encephalopathy develops in 28 % of patients within 10 years of compensated cirrhosis and is associated with spontaneously developed or surgically created portosystemic shunting (butterworth 2001) . the degree of encephalopathy is stratified by a coma scale: grade 1, subtle confusion; grade 2, somnolence; grade 3, unconsciousness with response to pain stimulation; and grade 4, deep coma. clinically, asterixis, flapping tremor, and fetor hepaticus (musty, sweet breath odor) are confirmatory of hepatic encephalopathy. the main cause of hepatic encephalopathy is the altered expression of several genes for various neurotransmitter proteins in the brain (butterworth 2001) . decreased expression of the glutamate transporter (glt-1) increases extracellular brain glutamate. an increased expression occurs in some receptors: monoamine oxidase increases degradation of monoamine transmitters, the peripheral-type benzodiazepine receptor increases inhibitory neurosteroids, and neuronal nitric oxide synthase increases nitric oxide production. although its plasma level is not closely related to the severity of encephalopathy, ammonia is still considered to be a major contributing factor. ammonia and manganese are known to alter the expression of the peripheraltype benzodiazepine receptor and neuronal nitric oxide synthase in exposed cells (warskulat et al. 2001) . magnetic resonance spectroscopy reveals brain edema and increased brain glutamine/glutamate in the frontal and parietal lobes; histologic findings are swelling and glycogen deposition in astrocytes. these changes in the brain coincide with impairment in the visuopractic capacity, visual scanning, and perceptual-motor speed on neuropsychiatric testing (tarter et al. 1984) . subclinical hepatic encephalopathy can be detected by having patients perform a simple timed connect-the-numbers test. treatment of hepatic encephalopathy is based on the ammonia-lowering strategy, such as protein restriction, oral non-absorbable antibiotics, and lactulose. rifaximin reduces the plasma ammonia level by destroying intestinal bacteria that produce urease. metronidazole (800 mg/day) is another antibiotic, although its adverse effects limit its use to 1 week at a time. lactulose is a substrate for gut bacteria and reduces the formation of ammonia by lowering intestinal ph. rifaximin class a = 5-6 points, b = 7-9 points, and c = 10-15 points inr international normalized ratio and lactulose are commonly used together, and the antibiotic is discontinued once eradication of disaccharide-metabolizing intestinal bacteria is indicated by an increase in stool ph. oral or parenteral ornithine aspartate, a substrate for the conversion of ammonia to urea and glutamine, has effects similar to those of lactulose, but with fewer adverse effects. in patients with severe encephalopathy, the molecular-absorbent recycling system (mars) may be utilized to remove small and middle molecular weight water-soluble substances (sorkine et al. 2001) . the system appears to increase blood pressure and systemic vascular resistance, possibly by removing nitric oxide. in fulminant hepatic failure, progressive hepatic coma is accompanied by a gradual increase in cerebral blood flow and intracranial pressure (icp) (see ▶ chap. 12, "fulminant hepatic failure: diagnosis and management"). subsequently, vasogenic cerebral edema and severe intracranial hypertension develop and approximately 30-50 % of patients die of brain herniation. monitoring of icp using a ladd epidural sensor is useful in detecting intracranial hypertension, monitoring the therapeutic effects, and identifying patients who would survive after transplantation without neurologic damage (lidorsky et al. 1992) . non-invasive neurologic assessment includes transcranial doppler (tcd) to measure cerebral bloodflow velocity, determination of the cerebral metabolic rate for oxygen by calculating the oxygen content difference between arterial and jugular bulb venous blood, evoked potentials, and serial computed tomography (ct) scans (aggarwal et al. 1994) . treatment includes osmotic and loop diuretics, barbiturateinduced coma, and hypothermia. the definitive treatment is usually transplantation. the presence of hyperdynamic circulation with a markedly increased cardiac output and decreased systemic vascular resistance was first described by kowalski and abelmann in the early 1950s (kowalski and abelmann 1953) . several hypotheses have been proposed to explain this phenomenon, including an overactive sympathetic nervous system, inadequate clearance of vasoactive substances by the diseased liver, the presence of arteriovenous shunts, nitric oxide-induced vasodilation, and relative hypoxia in peripheral tissues (benoit et al. 1984; yokoyama et al. 1989; kalb et al. 1993; d'souza et al. 1993) . although cardiac output is frequently two to three times normal, impaired systolic and diastolic function together with attenuated cardiac responsiveness to stimuli suggests that cardiomyopathy is present in cirrhotics (cirrhotic cardiomyopathy) (lee 1989) . caramelo et al. noted a 50 % decrease in cardiac output with volume expansion in a ccl 4 -induced cirrhotic rat model (caramelo et al. 1986) . in another rat model, the chronotropic response to isoproterenol was attenuated compared with that in control animals (lee et al. 1990 ). cardiac response to physical exercise is blunted in patients with cirrhosis, indicated by alterations in the pre-ejection period, isometric contraction time, and ratio of the pre-ejection period to left ventricular ejection time. in addition, abnormalities in myocardial diastolic indices suggest non-compliant ventricles. histologically, myocardial fibrosis, mild subendocardial edema, and vacuolation of myocyte nucleus and cytoplasms are observed. the development of cirrhotic cardiomyopathy is multifactorial. it appears that the β-receptor system, the main stimulant of the ventricle, is dysfunctional. in humans, lymphocyte β-receptor density, which reflects cardiac β-receptor status, is reduced in patients with severe ascites (gerbes et al. 1986) , and β-receptor density of the cardiomyocyte sarcolemmal plasma membrane is reduced in cirrhotic rats (liu and lee 1999) . further, the β-receptor signal transduction pathway is impaired at several levels (ma et al. 1996) . although cardiac contractile impairment may result from overactivity of the muscarinic m 2 receptor, the receptor density and binding affinity are unchanged, suggesting normal parasympathetic function (jaue et al. 1997) . high serum catecholamine levels, a result of desensitization and down-regulation of β-receptors, may lead to myocardial dysfunction in the presence of α-mediated coronary vasoconstriction. additionally, overproduction of nitric oxide inhibits β-receptor-stimulated cyclic adenosine monophosphate (camp) release, causing myocardial dysfunction and vasodilation (hare and colucci 1995) . coronary artery disease (cad) was previously believed to be relatively uncommon in patients with cirrhosis as a result of generalized vasodilation and elevated levels of hdl and estrogen. in addition, autopsy findings showed relatively fewer atherosclerotic changes and myocardial infarction. however, studies have shown that cad is not uncommon, and moderate-to-severe cad was found in approximately 27 % of patients who underwent coronary artery catheterization as a part of liver transplantation workup (carey et al. 1995) . in another study of 161 liver transplantation candidates who were at risk for cad and referred for coronary angiography, 25 % of patients had at least one moderate or severe (>50 %) coronary stenosis (tiukinhoy-laing et al. 2006) . endocarditis is three times more common in patients with liver disease (snyder et al. 1977) . this is attributed to translocation of intestinal bacteria through the intestinal wall and portosystemic collaterals, and reduced immune response. the incidence of pericardial effusion in cirrhotic patients is approximately 32-63 % and correlates with the degree of liver failure (shah and variyam 1988) . the effusion is usually small and may require drainage if it affects cardiac function. patients with liver disease exhibit three common cardiac electrophysiological disturbances: electromechanical dissociation, prolongation of ventricular repolarization (the q-t interval), and chronotropic incompetence (milani et al. 2007) . pulmonary hypertension associated with portal hypertension was first described in 1951 (mantz and craige 1951) . pulmonary hypertension defined as a mean pulmonary artery pressure of >25 mmhg and pulmonary vascular resistance of >240 dyn/s/cm à5 (3 wood units) is more common in patients with liver disease, with a prevalence of 0.25-0.73 % (lebrec and capron 1979; mcdonnell et al. 1983) . pulmonary artery pressure is a function of pulmonary venous pressure, pulmonary vascular resistance, and cardiac output [(pulmonary artery pressure = pulmonary venous pressure + (pulmonary vascular resistance â cardiac output)]. therefore, pulmonary hypertension is not uncommon in patients with liver disease because of their poor left ventricular compliance, increased pulmonary vascular resistance, and increased pulmonary blood flow from portosystemic shunting. the pathophysiology and management of pulmonary hypertension are well-described in ▶ chap. 10, "hepatopulmonary syndrome and portopulmonary hypertension". portal hypertension is caused by an increased intrahepatic vascular resistance and increased splanchnic blood flow. endothelin-1, a powerful vasoconstrictor produced by the sinusoidal endothelial cells, is known to increase intrahepatic vascular resistance and activates stellate cells, and its level increases as cirrhosis progresses (kojima et al. 2002; gandhi et al. 1996) . normally, vasodilatory compounds, such as nitric oxide, counterbalance the increased intrahepatic vascular resistance induced by endothelin. in liver cirrhosis, however, nitric oxide production is inhibited by caveolin-1, a hepatic membrane protein that binds with endothelial nitric oxide synthase. hypoxemia of varying severity is present in 45-69 % of patients with significant liver disease (krowka and cortese 1985) . the common causes are pleural effusions, impaired diffusion capacity, arteriovenous shunting, atelectasis caused by ascites or diaphragmatic dysfunction, aspiration secondary to encephalopathy, and deconditioning (hourani et al. 1991) . ventilation-perfusion mismatch, pulmonary vasodilation, and infection also contribute to hypoxemia. mild forms of hypoxemia are most common, although moderate-tosevere hypoxemia may be found in patients with advanced liver disease complicated by adult respiratory distress syndrome (ards), infection, and multiple organ failure. hepatopulmonary syndrome, first described by fluckiger in 1884 (fluckiger 1884) , may cause severe hypoxemia in a subset of patients with liver disease. the syndrome consists of a triad of liver dysfunction, severe hypoxemia (pao 2 < 70 mmhg in room air), and pulmonary vasodilation, and is characterized by dyspnea, cyanosis, clubbing of the digits, exercise desaturation, and orthodeoxia (hypoxemia in upright position). other concomitant clinical signs are a markedly increased alveolar-arterial oxygen gradient, portal hypertension, and vascular abnormality such as spider angioma and pulmonary vasodilation. the pulmonary vascular dilation (from 8-15 μ to 15-100 μ) at the precapillary level is believed to be the main pathology of the hepatopulmonary syndrome, which is caused by decreasing erythrocyte transit time and impairing diffusion of oxygen to the erythrocytes at the center of the bloodstream (genovesi et al. 1976 ). in contrast with other pulmonary diseases, oxygenation improves dramatically with a high inspired oxygen concentration (fio 2 ), because a high alveolar concentration of oxygen overcomes the diffusion barrier and oxygenates the erythrocytes in the center of the bloodstream. the pathophysiology and management of hepatopulmonary syndrome are described in ▶ chap. 10, "hepatopulmonary syndrome and portopulmonary hypertension". non-cardiogenic pulmonary edema occurs in 37-79 % of patients with advanced liver disease, particularly in those with fulminant hepatic failure, and appears to be associated with sepsis and a neurogenic mechanism. the presence of this complication is ominous: matuschak and shaw reported that all 29 patients who developed non-cardiogenic pulmonary edema died before liver transplantation (matuschak and shaw 1987) . in contrast, a rapid reversal of ards after liver transplantation has been reported (doyle et al. 1993) . pulmonary edema caused by fluid overload responds to diuretics and has a relatively benign course. pleural effusions are found on chest x-rays in about 10 % of patients. these are caused by the unidirectional passage of ascites via diaphragmatic defects into the pleural space. diagnostic thoracentesis is necessary to confirm the transudative nature and to exclude infection, malignancy, or embolic disease. optimal control of ascites may prevent symptomatic pleural effusions, and transjugular intrahepatic portosystemic shunt (tips) is effective in treating refractory hydrothorax in 84 % of patients (siegerstetter et al. 2001 ). approximately 10 % of hospitalized cirrhotic patients with ascites develop the hepatorenal syndrome, which is a form of acute pre-renal kidney injury caused by circulatory dysfunction secondary to an imbalance between circulating vasodilatory and vasoconstrictive substances. the primary contributing factor for the hepatorenal syndrome is nitric oxide-induced vasodilation of the splanchnic vascular bed causing systemic arterial underfilling and relative hypovolemia (arroyo et al. 1996) . this relative hypovolemia activates baroreceptor-mediated sympathetic and the renin-angiotensin system to constrict all vascular beds including the renal vasculature (guevara et al. 1998 ). the initial prostaglandin-mediated compensatory renal vasodilation is followed by renal arterial vasoconstriction and renal hypoperfusion. a striking feature of the hepatorenal syndrome is the lack of any histologic change and its reversibility: the affected kidneys resume their function after successful liver transplantation. the renal failure may be rapid (type 1) or insidious (type 2) and results in sodium and water retention and dilutional hyponatremia. since the hepatorenal syndrome is a functional renal failure, the urine is similar to that found in pre-renal azotemia: oliguria, low urinary sodium, and an increased urine osmolality and urine to plasma osmolality ratio. the major criteria for the diagnosis of the hepatorenal syndrome are as follows: (1) advanced hepatic disease and portal hypertension; (2) low glomerular filtration rate (serum creatinine >1.5 mg/dl or creatinine clearance <40 ml/ min); (3) absence of nephrotoxic drug use, shock, systemic infection, or recent fluid losses; (4) lack of sustained improvement after diuretic withdrawal and volume resuscitation with 1.5 l of normal saline; (5) proteinuria (<500 mg/dl); and (6) no ultrasound evidence of urinary obstruction or parenchymal disease. minor criteria include oliguria (<500 ml/day), urinary sodium <10 meq/l, urinary osmolality greater than plasma osmolality, urinary red blood cells (rbcs) <50/hpf, and serum sodium <130 meq/l. it is noteworthy that conventional renal function tests, such as bun and creatinine levels, overestimate renal function in patients with liver failure because malnutrition and muscle wasting contribute to a low creatinine level and liver dysfunction impairs urea synthesis. the hepatorenal syndrome is treated with the administration of vasopressin-1 agonists (i.e., terlipressin), tips, and, most reliably, liver transplantation. one uncontrolled trial using terlipressin with albumin for a median duration of 26 days (range 8-68 days) showed improvement in serum sodium as well as a decrease in the creatinine level below 2 mg/dl (mulkay et al. 2001) . hemodialysis is a temporary measure and its efficacy is not reliable. the only primary preventive measure showing some promise is the administration of albumin along with antibiotics as soon as the presence of spontaneous bacterial peritonitis is diagnosed; this possibly works by preventing hypovolemia and subsequent activation of vasoconstrictor systems. all phases of hemostasis are impaired in patients with liver disease, including clot formation, fibrinolysis, and their inhibitory processes. thrombocytopenia is found in 30-64 % of cirrhotic patients, and platelet count is commonly below 75,000/mm 3 . thrombocytopenia is primarily caused by splenomegaly associated with portal hypertension, which pools up to 90 % of platelets in the spleen. however, the degree of thrombocytopenia does not closely correlate with the size of the spleen. impaired hepatic synthesis of thrombopoietin also leads to thrombocytopenia. thrombopoietin is involved in the maturation and formation of platelets, and its return to a normal level coincides with a gradual increase in platelet count by the fifth day after liver transplantation (kawasaki et al. 1999) . other contributing factors are increased destruction of platelets by immune mechanisms, excessive activation of coagulation, and direct bone marrow suppression by toxins such as ethanol and folate deficiency. additionally, platelet dysfunction is common, as demonstrated by impaired platelet aggregation to adenosine diphosphate (adp), collagen, and thrombin (rubin et al. 1979) . the liver produces all coagulation factors except for von willebrand factor. therefore, plasma levels of clotting factors are directly related to the severity of liver disease, and prothrombin time (pt) is considered to be one of the most sensitive hepatic synthetic function tests. the plasma fibrinogen level, being an acutephase reactant, typically is normal or increased in chronic liver disease. a reduction in the fibrinogen level may indicate either a greatly reduced hepatic reserve or significant extravascular loss to ascites. markedly prolonged thrombin time indicates the presence of dysfibrinogenemia in some patients. dysfibrinogenemia is characterized by an excessive number of sialic acid residues in the fibrinogen molecule and abnormal polymerization of fibrin monomers. its clinical significance is unclear. patients with liver disease have a tendency to develop fibrinolysis due to decreased hepatic clearance of plasminogen activators, especially tissue plasminogen activator (tpa), and reduced production of α 2 -antiplasmin and thrombin activatable fibrinolysis inhibitors (van thiel et al. 2001) . elevated levels of d-dimers, fibrin degradation products, and plasminogen are present in ascitic fluid, indicating that absorption of ascitic fluid may contribute to the hyperfibrinolysis. on the other hand, excessive activation of coagulation is common in liver disease because of inadequate hepatic clearance of activated coagulation factors, reduced level of coagulation inhibitors, and enlarged vascular beds. the hypercoagulable state may lead to localized or disseminated intravascular coagulation (dic), particularly in the presence of sepsis, trauma, or major surgery. the diagnosis of excessive activation of coagulation is based on the presence of a known triggering factor and the progressively worsening of coagulation with thrombocytopenia. an anesthesia consultation is performed once a patient with esld is referred to the liver transplantation center. the type of liver disease is identified because patients with hepatocellular disease may have more pronounced hepatic dysfunction than those with cholestatic disease or hepatocellular cancer, and certain types of liver disease may affect other vital organ function (i.e., hemochromatosis, familiar amyloidosis, etc.). the anesthesia consultation is focused on evaluation of the functional reserve of extrahepatic organs, and various tests or specific consultations may be requested (table 2) . cardiovascular assessment is performed to determine two things: (1) whether a patient can be expected to survive the operation and immediate postoperative period; and (2) whether transplantation in patients with severe cardiopulmonary disease would be futile and an inappropriate use of a scarce donor organ (lentine et al. 2012) . a suggested strategy for cardiac assessment is shown in fig. 3 (raval et al. 2011) . overall cardiac performance is evaluated by transthoracic echocardiography to assess myocardial contractility, abnormality in cardiac anatomy, intracardiac or intrapulmonary shunting, and pulmonary artery pressure. most patients over age 50 years undergo non-invasive stress testing because they may have multiple cad risk factors (i.e., diabetes, hypertension, hyperlipidemia, and pre-existing cardiovascular disease), and limited physical activity masks underlying ischemic heart disease. an exercise stress test may not be feasible in many patients with advanced liver disease, and dobutamine stress echocardiography (dse) is commonly used, although adenosine or dipyridamole may be used when dobutamineinduced tachycardia is not desirable. dse, with its high sensitivity and specificity, appears to be the most reliable screening test (plotkin et al. 1998) , and dobutamine-induced tachycardia may mimic intraoperative stress on the cardiovascular system. on the contrary, dse has been reported to have poor sensitivity (as low as 13 %) and negative predictive value (as low as 75 %) (harinstein et al. 2008) , and its results may not correlate with adverse cardiac events within 30 days after transplantation (safadi et al. 2009 ). cardiac ct scan is a non-invasive technique measuring calcium deposits within the coronary vasculature. the total amount of calcium, adjusted to the age and gender of the patient, is reported as a calcium score. high scores suggest a greater potential for coronary artery stenosis (shaw et al. 2003; o'rourke et al. 2000) , and a calcium score of >400 has a predictive value of cardiac complications within 1 month after transplantation (kemmer et al. 2014 ). this test, however, may have limited predictive value as a single screening study for cad. cardiac ct angiography is an alternative to invasive coronary angiography. it does appear to have negative predicting value of 100 % for clinical coronary events in patients undergoing liver transplantation (cassagneau et al. 2012 ) but may not be suitable for the diagnosis of obstructive lesions at this time. because of the difficulty in diagnosing cad using non-invasive testing methods, coronary angiography is recommended for patients with a positive dse or multiple high-risk factors to identify the degree and type of obstruction. in addition, coronary angiography should be able to detect non-obstructive lesions (coronary artery stenosis <50 %), which are unlikely to be detected by stress tests but can be responsible for acute coronary syndromes (unstable angina, myocardial infarction, or sudden cardiac death) (rubin et al. 1994; gulati et al. 2009 ). cardiac catheterization, however, can be difficult in patients with severe liver disease due to bleeding complications and the increased risk of contrast-induced nephropathy (sharma et al. 2009 ). if significant coronary artery stenosis (>70 % stenosis) is detected, revascularization may be table 2 liver transplantation evaluation at the thomas jefferson university hospital attempted before liver transplantation. bare metal stents are favored over drug-eluting stents to avoid the need for long-term antiplatelet therapy (6 weeks vs. 1 year). when angioplasty is not amenable, coronary artery bypass grafting (cabg) is performed. it is clear that 1-year survival after cabg is greater in patients with child-pugh class a (80 %) than with child-pugh class b (45 %) and c (16 %) (filsoufi et al. 2007 ). therefore, patients with child-pugh class a can undergo cabg relatively safely while waiting for liver transplantation. on the other hand, patients with child-pugh class b and c may require simultaneous cabg and liver transplantation. patients with mild-to-moderate valvular disease undergo liver transplantation without excessive complications. similar to that of cabg, mortality after corrective valvular surgery depends on the severity of liver disease. therefore, child-pugh class c patients with severe aortic or mitral valve stenosis may undergo percutaneous balloon valvuloplasty or simultaneous valve replacement with cardiopulmonary bypass and liver transplantation. myocardial disease is commonly detected by transesophageal echocardiography (tee). patients with chronic cardiomyopathy may have attenuated systolic contraction and diastolic relaxation, altered repolarization, and reduced cardiac response to β stimulation (liu et al. 2002 for pulmonary evaluation, results of chest x-ray, arterial blood oxygen tension in 100 % oxygen, and spirometry are reviewed to identify the degree of pulmonary shunting, obstructive, or restrictive disease. when the hepatopulmonary syndrome is suspected, contrast tee or tc-99 m macro aggregated albumin scintigraphy may be performed for its definitive diagnosis (krowka et al. 2000) . for evaluation of renal function, results of bun, creatinine, glomerular filtration rate, levels of serum and urine electrolytes, urine output, and renal ultrasound are reviewed. the diagnosis criteria of the hepatorenal syndrome have been described earlier. in patients with chronic renal failure, simultaneous liver and kidney transplantation is performed, the criteria for which are end-stage renal disease with dialysis, no dialysis but a glomerular filtration rate <30 ml/min and proteinuria >3 g/day with a 24-h urine protein/creatinine ratio >3, and acute kidney injury requiring dialysis at least twice per week for more than 6 weeks (charlton et al. 2009 ). in patients with fulminant hepatic failure, reversibility of the neurologic function should be investigated using clinical signs, eeg, brain ct scan, the cerebral metabolic rate of oxygen, and tcd. in addition, icp monitoring is recommended when a high icp (>25 mmhg) is suspected, although its benefit should be weighed against potential complications (vaquero et al. 2005) . poor prognostic indicators of fulminant hepatic failure are progressive hepatic failure for 7-14 days, grade 3-4 encephalopathy, intracranial hypertension, cerebral swelling, severe coagulopathy, rapid shrinkage of the liver, metabolic acidosis, hemodynamic instability, and sepsis. for the coagulation system, pt, activated partial thromboplastin time (aptt), and platelet count are reviewed. in general, no specific coagulation therapy is requested because of the potential long waiting period and fluid overloading. abdominal magnetic resonance imaging (mri) is reviewed to assess the degree of portosystemic shunting and anatomy of hepatic vasculature. additional consultation may be requested from various specialists to identify the type and severity of the specific organ dysfunction. after the evaluation, all information of the potential recipient is compiled to stratify whether the patient's condition can be optimized or meet the criteria of contraindications. contraindications are diseases or conditions patients could have that may not improve survival after liver transplantation. they include malignancy with poor prognosis, active bacterial and viral infection, severe cardiopulmonary dysfunction, and technical difficulties. active alcoholism is a contraindication, although demonstrable abstinence for 6 months is considered acceptable. the presence of multiple organ dysfunction is a relative contraindication for liver transplantation as the 2-year survival is approximately 25 %. indications and contraindications of liver transplantation, however, have evolved over the past 50 years, and further modifications are expected to occur. anesthesiologists participate in the transplantation candidate selection committee for discussion of the hepatic disease, its complications, and the extrahepatic organ function of each patient. once the patient is placed on the active candidate list, the united network for organ sharing (unos) is notified and the patient is given a meld (model for end-stage liver disease) or peld (pediatric end-stage liver disease) score for fair distribution of donor livers. although surgical techniques are fully described elsewhere, a brief description of their physiologic effects is warranted here. in olt, after removal of the diseased liver, the donor liver is placed anatomically in the right upper quadrant. for the convenience of description, the procedure is divided into three stages: stage 1 (dissection stage), stage 2 (anhepatic stage), and stage 3 (neohepatic stage). the dissection stage begins with an inverted y-shaped bilateral subcostal skin incision and ends with the skeletonization of the diseased liver. the anhepatic stage begins with the occlusion of the hepatic artery, portal vein, and ivc for hepatectomy. however, the patient is virtually anhepatic once the hepatic artery or portal vein is occluded. three surgical techniques are used for hepatectomy and vascular reconstruction during the anhepatic stage: olt with simple venous crossclamping, olt with venovenous bypass, and the piggyback technique. in olt with simple venous cross-clamping, the diseased liver is removed together with the retrohepatic portion of the ivc after crossclamping of the suprahepatic and infrahepatic ivc, hepatic artery, and portal vein (fig. 4) . after surgical hemostasis of the hepatic bed, the donor liver is placed in the right upper quadrant, and sequential anastomoses of the suprahepatic ivcs, infrahepatic ivcs, portal veins, and hepatic arteries are performed. during the infrahepatic ivc anastomosis, the liver allograft is flushed with 1000 ml of cold lactated ringer's solution or 5 % albumin solution through a cannula in the portal vein. this flush technique allows preservation solution, metabolites, and air in the donor liver to escape through the incompletely anastomosed infrahepatic ivc. a second flush may be used by allowing 300-500 ml of blood to escape through the incompletely anastomosed portal vein by unclamping the infrahepatic ivc (back-bleeding technique). when the portal vein of the recipient is less than optimal, the superior mesenteric vein, collateral vein, or venous graft may be used for portal blood supply. the hepatic artery is reconstructed by end-to-end hepatic arterial anastomosis. however, an arterial graft is placed between the graft hepatic artery and the infrarenal aorta of the recipient with a side clamp on the aorta when the size or anatomy of the recipient hepatic artery is less than optimal. the liver is reperfused by the sequential unclamping of the infrahepatic ivc, portal vein, suprahepatic ivc, and hepatic artery. after hemostasis, choledochocholedochostomy is performed frequently with a t-tube. choledochojejunostomy using a roux-en-y loop is performed when the bile ducts are diseased or mismatched in size. the abdomen is closed once the absence of foreign bodies in the peritoneal cavity is confirmed. in patients with a large graft or swollen intestine, the abdomen may require secondary closure. olt with venovenous bypass was developed in 1983 to minimize reduction of venous return associated with the cross-clamping of the ivc and portal vein by diverting blood from the ivc and portal vein to the axillary vein using a centripetal magnetic pump (shaw et al. 1984) . once the hepatic hilum is dissected, cannulas are inserted into the left superficial femoral vein (7 mm) and portal vein (9 mm) for outflow from the patient and into the left axillary vein (7 mm) for venous inflow. the cannula site and size may vary depending on the preference of the surgical team or anatomic variations. the cannulas and heparin-bonded tubings are flushed with heparin solution (2000 u/l) to avoid thrombosis during preparation. systemic heparinization is not used because of the presence of pre-existing coagulopathy and the use of heparin-bonded tubings. the bypass run begins by unclamping all cannulas while the pump speed is gradually increased to achieve the maximal flow rate. hepatectomy and anastomoses of the suprahepatic and infrahepatic ivc are performed once full bypass is achieved. the removal of the portal cannula for portal venous anastomosis leads to a partial bypass, which reduces venous return. bypass is terminated after the engrafted liver is reperfused, and cannulas are removed. the advantages of venovenous bypass are (1) well-preserved cardiac output by uninterrupted venous return from the viscera and lower extremities; (2) effective decompression of the portal venous system, which decreases bleeding and intestinal congestion; (3) avoidance of renal congestion, oliguria, and hematuria; and (4) simplified anhepatic stage allowing meticulous hepatectomy and vascular anastomoses. long-term complications are neurovascular injury, thrombosis, infection, lymphocele, and seroma at the cannulation sites. as an alternative to the traditional venovenous bypass technique, percutaneous cannulation was introduced. in this technique, inflow to the patient is achieved by percutaneous cannulation of the right internal jugular vein (16-20 french) performed by the anesthesia team using a seldinger technique, and outflow from the patient by percutaneous cannulation of the left femoral vein (16-20 french) and a portal cannula by the surgical team. this technique is generally safe, but the inadvertent extravascular placement of an inflow cannula may cause a massive hemothorax (sakai et al. 2007 ). the piggyback technique was originally designed for patients with significant cardiovascular disease, portacaval shunt, superior vena caval syndrome, or small donor livers (tzakis et al. 1989) . in this technique, the diseased liver is removed without the retrohepatic portion of the ivc by peeling the diseased liver off the ivc after transaction of the hepatic veins, hepatic artery, and portal vein. therefore, systemic venous return can be relatively well preserved via the intact ivc during the anhepatic stage. vascular anastomoses are made between the reconstructed ostia of the recipient by combining hepatic veins and the suprahepatic ivc of the graft for the drainage of the hepatic venous blood. the portal vein of the recipient and the graft are anastomosed for portal blood supply, and the infrahepatic ivc of the graft is ligated. the neohepatic stage begins with reperfusion of the grafted liver by sequential unclamping of the infrahepatic ivc, portal vein, suprahepatic ivc, and hepatic artery, although the sequence of unclamping may vary depending on the surgical technique. reperfusion is followed by hepatic arterial anastomosis (if it has not been performed already), biliary reconstruction, and closure of the abdomen. immediate preoperative consultation is made when a donor organ is identified. the patient is re-evaluated to identify any interval changes during the waiting period. anesthetic and postoperative management and their risks are explained to the patient one more time. in general, pre-medication is withheld in most cases because of potential encephalopathy and hypovolemia, and narcotics (e.g., fentanyl 1-5 μg/kg) are commonly administered intravenously in the operating room. necessary medications and anesthesia equipment are listed in table 3 . a device that delivers fluids and blood rapidly on demand is considered standard equipment (i.e., fms2000 ® fluid warming system, belmont instrument corp., billerica, ma, usa) (elia and kang 2002 ). an autotransfusion system is helpful in minimizing the need for bank blood (dzik and jenkins 1985; kang et al. 1991) . a system that monitors coagulation, either a conventional coagulation profile, thromboelastography r with circle (teg; haemonetics, braintree, ma, usa), or (kang 1986 (kang , 1997 . teg and rotem provide similar physical properties of blood coagulation, although teg monitors shear elasticity and rotem monitors viscoelasticity. in general, 20 units each of cross-matched packed rbcs (prbcs) and fresh frozen plasma (ffp) are available at all times, and 10 units of each are prepared in the operating room. platelets (10-20 units) should be available on demand. two large-bore intravenous (iv) catheters (up to 8.5 or 9 french) are secured, typically in the right antecubital and right or left internal jugular vein. when the antecubital vein is unavailable, two catheters may be placed in the same internal jugular vein. catheter patency is confirmed by noting the line infusion pressure of <300 mmhg during fluid infusion at 500 ml/ min. sterile technique should be followed during catheterization, and antiseptic ointment or antiseptic patch is applied at the skin puncture site. a nasogastric tube is placed with copious lubrication and topical vasoconstrictor to avoid nasal or esophageal variceal bleeding. proper monitoring is prerequisite to a successful outcome because patients undergoing liver transplantation develop clinically significant hemodynamic, hematologic, metabolic, and other homeostatic abnormalities. non-invasive monitoring is similar to that for patients undergoing any major surgery. for invasive monitoring, two intra-arterial catheters (20 gauge in the left radial artery and 16-18 gauge in the right femoral artery) are used at the thomas jefferson university hospital. femoral arterial pressure monitoring is preferred because it reflects central arterial blood pressure more accurately in the presence of low systemic vascular resistance, particularly after reperfusion (lee et al. 2015) . radial arterial pressure monitoring is useful for blood sampling and backup pressure monitoring when the aorta is partially or completely clamped during aorta-tohepatic artery anastomosis. a pulmonary artery catheter (pa catheter) is inserted via the right internal jugular vein to monitor cardiac output, intracardiac pressures, and core temperature. carotid artery puncture should be assiduously avoided because of the presence of coagulopathy. an oximetric-type pa catheter provides additional information on mixed venous hemoglobin oxygen saturation (svo 2 ). the right ventricular ejection fraction-type pa catheter monitors the right ventricular ejection fraction and right ventricular end-diastolic volume. it has been shown that central venous pressure (cvp) and pulmonary capillary wedge pressure (pcwp) are not as sensitive as right ventricular end-diastolic volume in estimating preload, particularly during the anhepatic stage (dewolf et al. 1993b ). recently, non-invasive, continuous cardiac output monitoring was introduced; however, the technique is not reliable in monitoring cardiac output in hyperdynamic patients. in some centers, a cvp catheter is used instead of a pa catheter. this, of course, is justified if hemodynamic derangement is kept minimal during the entire surgical procedure. however, most centers use a pa catheter for three reasons: (1) hemodynamic instability can be unpredictable during liver transplantation; (2) determination of cardiac output and preload is more clinically significant than cvp monitoring; and (3) it is an important educational tool for trainees. tee is used in all patients at the thomas jefferson university hospital to monitor myocardial contractility, ventricular end-diastolic volume, wall motion abnormality, air or thromboembolism, intrapulmonary shunting, and patency of the reconstructed major veins. a tee probe may cause esophageal variceal bleeding (burger-klepp et al. 2012) and it should therefore be placed gently. various laboratory tests are performed, including arterial blood gas tension and acid-base state, and serum level of electrolytes, ionized calcium, glucose, lactate, and ionized magnesium if available. typical test times are before and after induction of anesthesia, every hour during the dissection stage, 5 min after the onset of the anhepatic stage, every 30 min during the anhepatic stage, 15 min before reperfusion, 5 and 30 min after reperfusion, and every hour thereafter. coagulation is monitored by conventional coagulation profile (pt, aptt, fibrinogen level, and platelet count) and teg or rotem at the following times: before induction of anesthesia, every hour during the dissection stage, 15 and 60 min after onset of the anhepatic stage, 15 min before reperfusion, 5 and 30 min after reperfusion, and every hour thereafter. monitoring of teg or rotem and the platelet count is preferable as a conventional coagulation profile has several drawbacks when used during liver transplantation (kang 1995) . pt is a very sensitive hepatic function test and is prolonged in most patients undergoing liver transplantation. administration of ffp to correct the pt may not be possible or desirable in the course of surgery. aptt follows a similar time course to pt, and its correction may not be practical. it is a sensitive test for the heparin effect, and its prolongation indicates the presence of heparin released from the bypass circuit or grafted liver. the fibrinogen level is frequently maintained within the acceptable range, although severe hypofibrinogenemia may indicate either active fibrinolysis or excessive activation of coagulation. the level of fibrin(ogen) degradation products is usually elevated in most patients due to excessive activation of coagulation and reabsorption of defibrinated blood from the abdominal cavity and does not have any immediate clinical significance. further, coagulation profile results may not be available in a timely manner. teg/rotem has several advantages over a conventional coagulation profile and has been accepted as a standard coagulation monitoring tool by the asa (american society of anesthesiologists 2015). it rapidly and reliably measures blood coagulability (quality) instead of the quantity of each coagulation component. an accurate differential diagnosis can be made for replacement therapy and pharmacologic therapy by comparing teg/rotem of untreated blood with that of blood treated with various blood components (ffp, platelets, cryoprecipitate) or pharmacologic agents (protamine sulfate, heparinase, ε-aminocaproic acid [eaca], aprotinin) (fig. 5 ) . lastly, circumferential identification tags around the wrists or ankles are removed to avoid the compartment syndrome. both arms are placed on padded arm boards in an abducted position, and excessive abduction should be avoided to prevent a plexus stretch injury. the extremities are protected with foam padding to avoid pressure injuries. a rapid-sequence induction is preferred because of uncertain gastric emptying. anesthesia is commonly induced with propofol (2-3 mg/kg) or etomidate (0.3 mg/kg), and fentanyl (2-5 μg/kg) is frequently added. succinylcholine (1-2 mg/kg) or rocuronium bromide (1.2 mg/kg) is used to facilitate intratracheal intubation. anesthesia is maintained using volatile inhalation agents and narcotics. isoflurane is the preferred inhalation agent because its effect includes less myocardial depression and biotransformation. nitrous oxide is avoided because it distends the bowel and increases the size of any entrained air. midazolam (1-4 mg) may be added for amnesia. for muscle relaxation, protamine-treated blood untreated blood 5 min after reperfusion fig. 5 effects of pharmacologic agents on pathologic coagulation immediately after reperfusion (from kang yg (1986) monitoring and treatment of coagulation. in: winter pm, kang yg (ed) hepatic transplantation, anesthetic and perioperative management. prager, new york, with the permission of the publisher) rocuronium bromide, vecuronium bromide, or cisatracurium besilate are commonly used. antibiotics and immunosuppressants administered during surgery may vary from center to center. at the thomas jefferson university hospital, unasyn ® (ampicillin/sulbactam 3 g) is given before incision and every 4 h thereafter. for patients allergic to cephalosporin or penicillin, vancomycin is administered within 2 h before skin incision (1 g for patients <80 kg and 1.5 g for those >80 kg). for immunosuppression, methylprednisolone (500 mg iv) and basiliximab (20 mg iv) are given during the anhepatic stage and tacrolimus is given in the postoperative period. liver transplantation imposes a great deal of physiologic stress on patients, and maintenance of physiologic homeostasis is essential to a successful outcome. the goal of hemodynamic management is to optimize tissue perfusion by maintaining the hyperdynamic state characteristic of esld. in general, there are two schools of thought about maintaining hemodynamic stability. the first endorses maintaining the hyperdynamic state to optimize cardiac output and tissue perfusion. patients with esld have generalized vasodilation and are known to have oxygen debt at the tissue level. therefore, maintaining the hyperdynamic state, instead of 'normal blood pressure,' ensures ample oxygen delivery to tissues and avoids tissue acidosis. this, in turn, optimizes tissue metabolism and hepatic blood flow. the second school of thought endorses maintaining arterial blood pressure within the normal range. this may include the use of various vasopressors (phenylephrine, norepinephrine, vasopressin, etc.) with or without hypovolemia. in extreme cases, blood is removed from the patient to induce hypovolemia, and blood pressure is supported by vasopressors (massicotte et al. 2010) ; it has been claimed that blood loss is minimal without increasing perioperative complications, although fluid restriction may lead to tissue ischemia, renal failure, and air embolism (melendez et al. 1998; schroeder et al. 2004 ). further, α-vasopressors (norepinephrine and phenylephrine) decrease hepatic blood flow by reducing portal venous flow dramatically in the presence of a limited hepatic arterial buffer response (mehrabi et al. 2005 ). hemodynamic instability represented by reduced cardiac output and hypotension is typically caused by hypovolemia associated with drainage of ascites, rapid third-space fluid loss, surgical bleeding, and inadvertent compression of major vessels (ivc, portal vein, hepatic veins, and aorta). intravascular volume is usually replenished by administration of a mixture of prbcs and ffp (typically, prbc:ffp: plasmalyte-a ® = 200:300:250 ml) using a rapid-infusion device. this mixture yields hematocrit of 26-28 vol.% and coagulation factor levels of 30-50 % of normal. a low hematocrit is chosen to optimize microcirculation and minimize the rbc wastage. calcium-containing fluid (i.e., lactated ringer's solution) should not be used to prevent clot formation in the reservoir of the rapid-infusion device. continuous administration of ffp is necessary to compensate for the loss of coagulation elements (procoagulants, prolysins, and their inhibitors) by surgical bleeding and excessive activation of coagulation. in patients with minimal blood loss, colloids (albumin or ffp) and crystalloids may be required to compensate for the third-space fluid loss and continuous production of ascites. close communication with the surgical team is essential to identify the cause of hemodynamic instability, as is communication with the blood bank to facilitate adequate supply of blood products. intraoperative autotransfusion has been shown to be effective and safe during liver transplantation, and its use may be considered when the prbc requirement is >5 units. its use is not recommended for patients with peritoneal infection or malignancy (liang et al. 2008 ). when lower cardiac output and/or hypotension persists even with adequate preload, dopamine or epinephrine may be infused for patients with hypotension, while dobutamine can be used when patients are normotensive. high venous pressures (cvp and pcwp) may be seen in patients with volume overload, large ascites, and pleural or pericardial effusion. drainage of ascites and effusion may decrease intrathoracic pressure and central venous pressures and improve cardiac performance. thoracentesis and pericardiocentesis can be performed after the abdomen is opened to minimize the risk of injury to the thoracoabdominal organs. unexpected pulmonary hypertension may be observed in some patients. because of the high perioperative mortality in patients with pulmonary hypertension, it deserves a thorough intraoperative investigation. the pa catheter should be able to differentiate between pulmonary hypertension with high pulmonary vascular resistance and pulmonary hypertension with fluid overloading. pulmonary hypertension caused by fluid overloading may dissipate gradually by intraoperative fluid loss, and phlebotomy may be required in severe hypervolemia. in portopulmonary hypertension with increased pulmonary vascular resistance, the presence of right ventricular function is investigated. a low cardiac output with a high cvp suggests the presence of right ventricular dysfunction. tee findings of right ventricle dysfunction are low fractional area change (fac), tricuspid annular plane excursion (tapse) of <16 mm, flattening of the ventricular septum, apicalization of the right ventricle, and right ventricular dilation. additionally, the pulmonary vascular response to various vasodilators (i.e., diltiazem, nitroglycerin, epoprostenol, and nitric oxide) may be evaluated. liver transplantation may continue when pulmonary hypertension is mild to moderate with normal right ventricular function. in such cases, right ventricular function is supported by maintaining optimal preload and improving myocardial contractility by inotropes. complications of massive blood transfusion (ionic hypocalcemia, ionic hypomagnesemia, hyperkalemia, and acidosis) may develop at this stage and should be treated aggressively. normothermia can be well-maintained even during massive transfusion when a rapid-infusion device is used. hemodynamic changes that occur during the anhepatic stage are caused primarily by interruption of venous return from the ivc and portal vein. in the simple cross-clamping technique, clamping of the ivc and portal vein reduces venous return by up to 40 %, leading to low cardiac output, hypotension, and compensatory tachycardia (pappas et al. 1971) . calculated systemic vascular resistance is frequently elevated, although this is a reflection of the exclusion of the vascular tree of the lower extremities and splanchnic bed. it is noteworthy that cross-clamping of the ivc and the portal vein decreases the central blood volume and pressure (cvp and pcwp) but progressively increases total intravascular blood volume as blood is sequestered in the vascular bed of the gastrointestinal and pelvic organs, kidneys, and lower extremities. a prolonged low output state, portal hypertension, and renal venous congestion may lead to acidosis, intestinal swelling, and hematuria. we, at thomas jefferson university hospital, prefer to treat the low output state by administration of fluid and/or inotropes (dopamine or dobutamine 2-5 μg/kg/min). venovenous bypass is more physiologic technique than a simple cross-clamping technique as it returns venous blood from the portal and ivc system (shaw et al. 1984) . hence, hemodynamic changes that occur during the anhepatic stage with venovenous bypass are minimal when the bypass flow rate is greater than 25 % of the baseline cardiac output and, therefore, the bypass flow should be monitored and adjusted as needed. improper positioning of the cannula tip in the femoral or portal vein, or a kinked bypass circuit, may not drain blood adequately and the surgical team should correct their positions. a low pump speed reduces venous return, while a high pump speed collapses the outflow venous wall and decreases the bypass flow. the perfusionist, therefore, should adjust the pump speed to maximize the bypass flow. in addition, hypovolemia decreases the bypass flow, and it should be corrected by the anesthesia team. the anesthesia and surgical teams should be prepared for potential acute complications of venovenous bypass. bleeding or air entry may result from venous laceration during cannulation or improperly secured cannulas. entry of a small volume of air (up to 50 ml) into the bypass pump may not cause immediate systemic air embolism because it is trapped in the cone-shaped pump head by centripetal force. thromboembolism may be caused by the migration of pre-existing thrombi or those developed during a low bypass flow rate (<1000 ml/min), particularly in hypercoagulable conditions (i.e., budd-chiari syndrome, neoplasms, and congenital protein c deficiency). most importantly, the bypass may have to be terminated unexpectedly when serious complications occur. therefore, the anesthesia team should be prepared for unexpected cross-clamping of the ivc and portal vein at all times. after completion of the ivc anastomosis, the portal cannula is removed to facilitate the portal venous anastomosis, resulting in partial bypass. low bypass flow and low cardiac output during this period can be improved by the administration of fluids or dopamine, but full correction of central hypovolemia, as reflected on cvp, pcwp, or tee, is avoided to prevent fluid overload on reperfusion. in the original description of the piggyback technique, adequate venous return is maintained through the intact ivc and portal vein using portoaxillary venovenous bypass (tzakis et al. 1989 ). currently, many transplantation centers do not incorporate the portoaxillary venovenous bypass in the piggyback technique, which makes patients vulnerable to significant hypovolemia. hepatectomy in the presence of portal hypertension can be difficult, and hypovolemia is not uncommon as a consequence of inadvertent compression of the ivc and portal vein, partial side-clamping of the ivc, and cross-clamping of the portal vein during portal anastomosis. hence, temporary portacaval shunt or portal-axillary venovenous bypass may be instituted in surgically challenging patients to maintain preload. as described earlier, 1000 ml of cold lactated ringer's solution or albumin (5 %) is flushed through the portal vein and drained via the incompletely anastomosed ivc to remove preservative solution, metabolites, and air from the allograft. additionally, approximately 300-500 ml of blood may be allowed to escape through the incompletely anastomosed portal vein to enhance the washout by partial unclamping of the infrahepatic ivc (back-bleeding technique) immediately before reperfusion of the grafted liver. in this case, blood should be administered simultaneously to avoid hypovolemia. other factors that affect circulation during the anhepatic stage are similar to those of the dissection stage, although lactic acidosis, citrate intoxication, hypomagnesemia, hyperkalemia, and coagulopathy are more pronounced. hyperkalemia is treated by dextrose (5-10 g) and insulin (5-10 units) to move potassium intracellularly (dewolf et al. 1993a ). in severe hyperkalemia, prbc or phlebotomized blood can be washed using an autotransfusion system to remove potassium before transfusion (ellis et al. 1987) . at the end of the anhepatic stage, all biochemical variables are normalized to prepare for reperfusion. significant hemodynamic changes occur on reperfusion of the grafted liver (fig. 6) . unclamping of the infrahepatic ivc and portal vein results in transient hypovolemia and hypotension due to acute sequestration of the blood in the engrafted liver. unclamping of the suprahepatic ivc increases preload by mobilizing blood from the low extremities and splanchnic circulation. this is followed by severe hemodynamic changes, the so-called postreperfusion syndrome (aggarwal et al. 1993) . the postreperfusion syndrome, which occurs in approximately 30 % of patients, is defined by abrupt hypotension (below 70 % of the baseline value) that develops within 5 min of reperfusion and lasts for more than 1 min. other associated hemodynamic changes are bradycardia, high cvp and pcwp, low systemic vascular resistance, and conduction defects. acute reduction in myocardial contractility is observed in tee. the postreperfusion syndrome appears to be caused by a combination of several factors. for example, an acute increase in preload may result in right ventricular strain and an acute decrease in blood temperature (2-3 c) by the systemic entry of the cold preservation solution may decrease cardiac conduction and contractility. other physical factors are air embolism and thromboembolism, which may cause right ventricular strain or right ventricular outflow tract obstruction (ellis et al. 1989; suriani et al. 1996) . chemical factors involved are acute hyperkalemia and acidosis. systemic entry of hyperkalemic preservation solution increases serum potassium level to a very high level (up to 12 mmol/l), causing severe bradycardia and conduction defects (martin 1986 ). return of the acidic blood from the viscera and lower extremities increases the base deficit by 5-10 mmol/l. in addition, unknown endogenous vasodilators or myocardial depressants (i.e., vasoactive intestinal polypeptide, nitric oxide, and eicosanoid) released from the allograft or congested viscera may decrease systemic vascular resistance and impair myocardial function. several measures may be taken to prevent the postreperfusion syndrome, although they are not always successful. at the end of the anhepatic stage, blood volume is adjusted to avoid fluid overloading on reperfusion, and ionic hypocalcemia, hyperkalemia, and metabolic acidosis are corrected. prophylactic administration of cac1 2 (15 mg/kg), nahco 3 (0.5-1 mmol/kg), regular insulin (10 units), 50 % dextrose (1 ml/kg), and epinephrine (5-10 μg) are recommended by some centers (ellis et al. 1989) . once the postreperfusion syndrome develops, severe hypotension and bradycardia are treated with small doses of epinephrine (5 μg increments) to support contractility, heart rate, and vasomotor tone, followed by a dopamine or epinephrine infusion, if necessary. symptomatic hyperkalemia (tall, peaked-t wave, and widening qrs complex with bradycardia) is treated by administration of cac1 2 (15 mg/kg) and nahco 3 (0.5-1 mmol/kg). arrhythmias are treated in the standard fashion. when pulmonary edema develops, positive end-expiratory pressure (peep) is applied and inotropes may be given. patients who develop intracardiac or pulmonary embolism are supported by inotropes. when severe fluid overloading is a concern, phlebotomy may be considered. the postreperfusion syndrome dissipates gradually over the next 5-15 min, although low (1987)) systemic vascular resistance and hypotension with a high cardiac output may persist for several hours. when hypotension is suspected to cause tissue and myocardial ischemia, it may be treated with ephedrine, dopamine, or epinephrine. overzealous administration of fluids may result in hepatic congestion, while norepinephrine may interfere with hepatic blood flow by decreasing portal venous flow. octreotide and vasopressin may increase arterial blood pressure by decreasing portal pressure and flow, although its effects on hepatic circulation and metabolism are unclear (fayed et al. 2013; wagener et al. 2008) . hemodynamic changes that occur during hepatic arterial and biliary reconstruction are relatively minor, except for intermittent fluctuation of the preload associated with continuous third-space fluid loss and compression of the liver and great vessels. gas exchange is maintained satisfactorily in most patients. minute volume is gradually decreased during the anhepatic stage to match the reduced oxygen consumption and carbon dioxide production, and is increased during the neohepatic stage. alveolar recruitment maneuvers are performed intermittently to avoid atelectasis caused by pleural effusions, cephalad traction of the rib cage, and compression of diaphragm. intermittent endotracheal suctioning, using a suction catheter or bronchoscope, may be required to remove secretions. drainage of pleural effusions and ascites decreases intrathoracic pressure and improves oxygenation within 2 h. patients with preoperative ards may require a high fio 2 and a high level of peep to ensure adequate gas exchange, and a volume ventilator may be necessary to overcome the high airway pressure. frank pulmonary edema can develop, particularly after reperfusion, from the increased pulmonary capillary permeability or fluid overload. in such cases, patients are ventilated with a high level of fio 2 and peep, while the underlying cause is treated. closure of the abdominal cavity may interfere with ventilation by increasing intrathoracic and airway pressures. primary closure with mesh or secondary closure may be necessary. in patients with fulminant hepatic failure, preoperative cerebral monitoring is continued as cerebral hyperemia and intracranial hypertension persist during surgery, although they are somewhat attenuated by general anesthetics. however, a sudden increase in preload may dramatically exacerbate intracranial hypertension on reperfusion of the grafted liver. hence, optimal preload should be maintained during the entire procedure. after reperfusion, cerebral hyperemia may gradually decrease as the liver begins to function. intraoperative changes in coagulation are summarized in table 4 . surgical bleeding is common due to numerous collateral vessels associated with portal hypertension, difficulty in dissection of the diseased liver, pre-existing coagulopathy, and pathologic changes in coagulation. the average blood loss in adults is 5-15 units each of prbc and ffp, although blood loss may reach more than 100 units each. during the dissection stage, dilutional coagulopathy develops as bleeding reduces the levels of coagulation factors and platelets (fig. 7) . fibrinolysis may develop, particularly in patients with hepatocellular disease, as a result of a low level of inhibitors of fibrinolysis and impaired hepatic clearance of tpa (lewis et al. 1989a) . excessive activation of coagulation, evidenced by a gradual increase in thrombin-antithrombin complex, develops at the end of the dissection stage (kratzer et al. 1991) . management of coagulation begins with normalization of physiologic variables, such as ionic hypocalcemia, hypothermia, and acidosis impair coagulation (rohrer and natale 1992) . this is followed by continuous infusion of coagulation factor-rich blood (rbc:ffp: plasmalyte-a ® or normal saline = 1 unit:1 unit:250 ml) to maintain coagulation factor levels above the critical level (30-50 % of normal). specific blood components may be administered based on teg/rotem. in general, platelets (5-10 units) are administered for a small maximum amplitude (ma) (<40 mm). platelet administration, in addition to increasing ma, improves reaction time (r) and clot formation rate (α), because the coagulation cascade leading to fibrin formation occurs on the surface of platelets. its administration, however, is withheld during the anhepatic stage to avoid potential thrombosis and during massive blood transfusion (>150 ml/min) to minimize wastage. two units of ffp may be administered when the reaction time is prolonged (r> 12 min) even after platelet administration cryoprecipitate (6 units) containing factors i and viii are rarely required unless severe fibrinolysis is left untreated because plasmin selectively destroys factors i, v, and viii. however, cryoprecipitate may be used for patients with severe hypofibrinogenemia (<100 mg/dl). pathologic coagulation superimposes on dilutional coagulopathy during the anhepatic stage. the heparin effect is seen as a prolonged aptt and reaction time on teg/rotem at the onset of the venovenous bypass as a small dose of heparin (2000-5000 units) in the bypass circuit enters systemic circulation. this heparin effect dissipates over the next 30-60 min. the effects of the absence of the hepatic synthetic and clearance function begin to develop during this stage. the absence of hepatic clearance of tpa promotes fibrinolysis in approximately 30 % of patients . similarly, the absence of hepatic clearance of activated coagulation factors results in excessive activation of coagulation evidenced by a progressive increase in thrombin-antithrombin complex and fibrin (ogen) degradation products. severe fibrinolysis (fibrinolysis time <60 min) may be treated by the administration of a single, small dose of eaca (250-500 mg) ). administration of a large or repeated dose of eaca is not recommended in order to avoid potential thromboembolism (gologorsky et al. 2001) . the postreperfusion syndrome occurs in coagulation at the onset of the neohepatic stage. a typical coagulation profile shows prolonged pt, aptt, reptilase time, and thrombin time. a generalized decrease in coagulation factors (i, v, vii, and viii) and platelets is accompanied by a sharp increase in the tpa level, a shortened euglobulin lysis time, and a moderate increase in fibrin(ogen) degradation products and thrombin-antithrombin complex. fibrinolysis is observed in up to 80 % of patients and is severe in about 40 % . fibrinolysis is caused by a 20-fold increase in tpa being released from the allograft and congested viscera, which overwhelms the activity of the plasminogen activator inhibitor (virji et al. 1989; porte et al. 1989 ). there are ample data to support the finding that fibrinolysis is primary in origin: a relatively steady antithrombin level, only moderate levels of fibrin(ogen) degradation products and d-dimers, selective decreases in factors i, v, and viii, and no known microthrombi formation (lewis et al. 1989a, b) . fibrinolysis resolves over the 120 min following reperfusion. the heparin effect occurs in approximately 30 % of patients, as heparin is released from the allograft and dissipates over the next 60-90 min. to identify the presence of fibrinolysis and the heparin effect, teg/rotems of untreated blood (native), blood treated with antifibrinolytic agent (eaca or aprotinin), and blood treated with an agent neutralizing heparin (protamine sulfate or heparinase) are compared 5 min after reperfusion. when fibrinolysis is present, early treatment using a single, small dose of eaca (250-500 mg) is recommended in order to reduce delayed oozing and to minimize the loss of factors i, v, and viii . prophylactic administration of eaca or tranexamic acid (amca) is a common practice in many centers, but has not shown scientific efficacy. fibrinolysis prophylaxis is not recommended by the authors, because the presence of fibrinolysis can easily be detected by teg/rotem and can be treated effectively with a small dose of eaca in most patients. when the heparin effect is present, a small dose of protamine sulfate (25-50 mg) may be given in severe cases. in addition, blood coagulability can be impaired by reperfusion hypothermia, acidosis, and ionic hypocalcemia. in contrast, excessive activation of coagulation leading to fatal intracardiac or pulmonary embolism may occur in some patients (gologorsky et al. 2001; warnaar et al. 2008 ). this complication appears to be associated with a massive transfusion, release of a large quantity of tissue thromboplastin from the less than optimal allograft, impaired tissue perfusion, and possibly antifibrinolytic therapy. intracardiac thrombosis can be treated by infusion of tpa (40-100 mg over 2 h) while observing resolution of thrombi using tee (boone et al. 2011; jackson et al. 2006) . coagulopathy improves gradually after reperfusion. generalized oozing, however, may occur even in the presence of acceptable coagulation profiles and teg/rotem, possibly due to delayed bleeding caused by the loss of a poorly formed clot or by the residual effects of reperfusion fibrinolysis. several other pharmacologic agents are reported to improve coagulation. aprotinin (2,000,000 kiu followed by 500,000 kiu/h), a non-specific inhibitor of plasminogen and serine protease, may reduce blood loss by inhibiting fibrinolysis and excessive activation of coagulation (neuhaus et al. 1989; cottam et al. 1991 ). however, clinical use of aprotinin declined even before the drug was withdrawn by the manufacturer: clinical reports did not show a significant reduction in blood loss (ickx et al. 1993; groh et al. 1993) , and fibrinolysis can be treated with eaca or amca more efficiently with negligible side effects boylan et al. 1996) . recombinant factor viia (rfviia) has been suggested to improve coagulation and reduces bleeding by actively enhancing coagulation and stimulating fibrin formation in the presence of tissue factor. its beneficial effects have been shown in patients with fulminant hepatic failure, "critical bleeding," and a ruptured liver (meadows et al. 2011; merchant et al. 2004; yamaguchi et al. 2015) . however, results of clinical trials are controversial (planinsic et al. 2005; gasperi and baudo 2006; niemann et al. 2006 ) and a european consensus concluded that a paucity of data from clinical trials with rfviia limits both the strength and the scope of clinical recommendations (vincent et al. 2006) . recently, the use of prothrombin complex concentrate has been assessed in a limited number of centers. prothrombin complex concentrate is prepared from ffp and contains clotting factors ii, vii, ix, and x, protein c, and protein s, and its use may improve coagulation without increasing preload (arshad et al. 2013 ). however, it has limited components of coagulation and its clinical advantage requires further investigation. desmopressin acetate (ddavp), a synthetic analog of 8-arginine vasopressin, increases the endothelial release of factor viii, von willebrand factor, and plasminogen. its beneficial effects have been demonstrated in vitro and in patients with liver disease, and it may be used to improve coagulation (0.3 μg/kg) . conjugated estro-gen has been reported to improve coagulation and reduce blood loss (frenette et al. 1998) , although its use has not been accepted widely. calcium metabolism patients with hepatic dysfunction invariably develop ionic hypocalcemia during massive blood transfusion, which is caused by chelation of serum calcium with citrate in the banked blood. ionic hypocalcemia begins to appear during the dissection stage (marquez et al. 1986 ) and becomes severe during the anhepatic stage. the serumionized calcium level is inversely related to the serum citrate level, as the absence of hepatic metabolism of citrate increases the serum citrate level close to that in the banked blood (fig. 8) . significant hypocalcemia (ca 2+ <0.55 mmol/l) is associated with a prolonged q-t interval and decreases in the cardiac index, stroke-work index, and blood citrate level ca ++ level mg ++ level fig. 8 intraoperative changes in serum calcium, magnesium, and citrate level (results of two studies (marquez et al. 1986 and scott et al. 1996) are superimposed, with the permission of the publisher) pressure. therefore, the ionized calcium concentration is monitored hourly or more frequently, and cac1 2 (15 mg/kg) or calcium gluconate (30 mg/kg) is administered to maintain a normal level (martin et al. 1990 ). ionic hypocalcemia improves gradually as the engrafted liver begins to metabolize citrate, unless the speed of the transfusion exceeds the metabolic function of the liver. hypokalemia is not uncommon in patients with liver disease due to poor dietary intake of potassium and its loss from chronic diuretic therapy and diarrhea. severe hypokalemia (<2.5 mmol/l) is treated with potassium chloride to increase its level to 3.0-4.0 mmol/l. moderate hypokalemia (<3.5 mmol/l) is not treated because it is welltolerated by patients and self-corrected by blood transfusion. hyperkalemia is a serious concern because it interferes with myocardial conduction and contractility, particularly in the presence of acidosis and hypocalcemia. progressive hyperkalemia (up to 6-7 mmol/l) may occur in patients with renal dysfunction or those requiring massive blood transfusion. mild hyperkalemia (up to 5.5 mmol/l) is treated with insulin (10 units) and glucose (12.5 g). it has been shown that glucose and insulin therapy is effective in lowering the serum potassium level even in the absence of hepatic function (dewolf et al. 1993a) . for moderate-to-severe hyperkalemia (>5.5 mmol/l), in addition to insulin therapy, prbc or phlebotomized blood can be washed to remove potassium using an autotransfusion system before transfusion (ellis et al. 1987) . reperfusion hyperkalemia is caused by potassium influx from the preservation solution and hepatocytes, and its systemic effects and treatment have been described previously. acute hyperkalemia returns to a normal range within 5-10 min as a result of redistribution. the potassium level gradually returns to the baseline value as the rbcs and the engrafted liver take up excess potassium. hypokalemia (<3.5 mmol/l), which occurs toward the end of procedure, is treated using a kcl infusion (20 mmol increments). hyponatremia (<130 mmol/l) is a common occurrence in patients with liver disease, particularly those with fluid retention, ascites, diuretic therapy, and restricted sodium diet. the serum sodium level gradually increases towards normal during surgery via administration of blood products and a balanced salt solution. a rapid rise in the serum sodium level (>10 mmol/l) is a clinical concern because it may contribute to the development of central pontine myelinolysis, a serious neurological injury caused by the destruction of the myelin sheath in the pons (videira et al. 1991) . therefore, the preoperative serum sodium level should be raised to >130 mmol/l, if possible, and a rapid increase in sodium should be prevented by administration of low sodium-containing crystalloids during surgery. in addition, tromethamine (tham) is the preferred drug for treatment of metabolic acidosis as it does not contain sodium. hypernatremia may be seen in some patients who receive a large dose of nahco 3 preoperatively. this hypernatremia is gradually normalized by administration of blood products and a balanced electrolyte solution. a clinical investigation showed that the serum ionized magnesium level, similar to the ionized calcium level, has an inverse relationship with the serum citrate level as magnesium ion chelates with citrate in banked blood (scott et al. 1996) . although the clinical significance of ionic hypomagnesemia during liver transplantation is unclear, mgso 4 (1-4 g) can be administered to minimize potential cardiac irritability and myocardial depression. metabolic acidosis begins to appear during the dissection and anhepatic stages because of impaired hepatic metabolism of the acid load from the banked blood and the peripheral tissues. the base deficit and lactate level increase further (approximately 5 mmol/l) on reperfusion due to the acid load from the graft and congested viscera and lower extremities. it gradually improves as hepatic function is restored and tissue perfusion improves during the neohepatic stage. persistent lactic acidosis (>15 mmol/l) appears to be associated with graft dysfunction (begliomini et al. 1989) . metabolic acidosis is aggressively corrected by administration of nahco 3 to maintain base deficit levels <5 mmol/l because acidosis is frequently progressive and leads to myocardial depression, inadequate cellular respiration, and decreased sensitivity to catecholamines. as described earlier, tham is preferred in hypo-or hypernatremic conditions to minimize fluctuation of the serum sodium level: 150 ml of 0.3 m tham is equivalent to 50 mmol of nahco 3 . alternatively, dichloroacetate (40 mg/kg every 4 h) appears to reduce lactate production by stimulating pyruvate oxidation (shangraw and robinson 1997) . metabolic alkalosis may develop during the neohepatic stage, and this was believed to be associated with nahco 3 -administered and citrate metabolism-generating bicarbonate. however, it has been shown that the degree of metabolic alkalosis is unrelated to the citrate and nahco 3 load (fortunato et al. 1987) and may be associated with residual hyperaldosteronism. body temperature may gradually decrease to 34 c during the dissection stage as a result of the exposure of the abdominal contents to the cold environment, vasodilatation, and lack of shivering. hypothermia continues during the anhepatic stage as energy production decreases further. an abrupt decrease in core temperature (2-3 c) occurs on reperfusion as cold preservation solution enters systemic circulation. the temperature increases during the neohepatic stage, and the surgery ends with a body temperature of approximately 35-36 c. hypothermia is difficult to avoid, although raising the room temperature, application of forced warm air devices, use of a warming blanket, and a heat exchanger in the venovenous bypass system may be beneficial. the blood glucose level is relatively wellmaintained (100-200 mg/dl) with blood transfusion, as the banked blood contains glucose (approximately 200 mg/dl). a gradual decrease in glycogenolysis reduces the blood glucose level during the dissection and anhepatic stages. in patients with fulminant hepatic failure or severe hepatocellular disease, the blood glucose level may decrease precipitously, making glucose supplementation necessary. hyperglycemia (up to 300 mg/dl) occurs on reperfusion as glucose is released from the engrafted liver (dewolf et al. 1987 ). insulin does not appear to be effective in treating reperfusion hyperglycemia because glucose reuptake requires restoration of hepatic function. the insulin level is relatively steady during surgery, and the glucagon level increases after reperfusion. the blood glucose level usually returns to normal within 12-24 h. persistent hyperglycemia caused by impaired hepatic glucose reuptake and hormonal imbalance is an early sign of poor graft function (mallett et al. 1989 ). urine output is well-preserved in most patients once the intravascular volume is optimized. oliguria or anuria, however, may persist in patients with the hepatorenal syndrome or underlying renal disease. the presence of oliguria and hematuria during the anhepatic stage of the simple cross-clamping technique has been described earlier. urine output increases during the neohepatic stage as a result of the restoration of renal function and circulation. various agents have been tried to protect or improve renal function: the role of dopamine is controversial, dopexamine appears to be beneficial, and triple-drug therapy (dopamine [2-3 μg/kg/min], mannitol [250 mg/kg], and furosemide) improves urine output but not renal function (gray et al. 1991; planinsic et al. 1997 ). when fluid overload or severe electrolyte imbalance is a concern, intraoperative venovenous ultrafiltration or hemodialysis may be utilized. the restoration of hepatic function is evident about 2 h after reperfusion: levels of citrate and lactate decrease, the glucose level returns toward normal, coagulopathy improves, and bile production begins. persistent citrate intoxication, acidosis, hyperglycemia, coagulopathy, and pale-colored bile are poor prognostic signs. recently, tracheal extubation in the operating room has been successful in several centers when the patient meets the liver transplantationspecific extubation criteria, including the severity of pre-existing liver disease, blood loss, and hemodynamic stability (mandell et al. 2002; biancofiore et al. 2005; glanemann et al. 2007 ). however, most patients are still transported to the intensive care unit (icu) while receiving invasive monitoring and ventilatory support. upon arrival to the icu, the ventilator setting is reported to the respiratory therapist, the lungs are auscultated, and vital signs are displayed on the icu monitor. detailed intraoperative information is reported to the icu physician and nursing staff. primary non-function primary non-function is defined as graft failure occurring within 90 days after liver transplantation in the absence of either rejection or technical factors such as hepatic arterial thrombosis (bzeizi et al. 1997) . this complication occurs in up to 10 % of patients and is frequently caused by hepatic dysfunction of the donor liver or prolonged cold ischemia (>18 h). the patient develops progressive multi-organ failure including encephalopathy, coagulopathy, minimal bile production, and oliguria. supportive therapy may be helpful until the liver resumes its function, although urgent retransplantation is the only solution in many patients. worsening liver function without technical complications in the second week after liver transplantation suggests acute cellular rejection. biopsy findings are inflammation of the intrahepatic endothelium and bile duct and a mononuclear cell infiltration with eosinophilia (wiesner 1996) . hepatic arterial stenosis occurs in approximately 5 % of patients, and is four times more common in children. common clinical signs are biliary tract breakdown, recurrent bacteremia, hepatic abscess, and occasionally massive hepatic necrosis (tzakis et al. 1985) . hepatic arterial stenosis is suspected when an ultrasound examination reveals increased focal arterial flow velocities and is confirmed by angiography. in the immediate postoperative period, direct repair or reconstruction using an infrarenal arterial conduit is usually successful. stenosis occurring several weeks after transplantation is treated with percutaneous hepatic arterial angioplasty, which has a success rate of more than 90 % in achieving long-term patency. vena caval stenosis and thrombosis occur in 1-2 % of patients. in traditional liver transplantation, outflow obstruction is managed by balloon angioplasty with or without a metallic stent placement (simo et al. 1993) . in the piggyback technique, it is treated with end-to-side anastomoses between the donor infrahepatic ivc and the recipient retrohepatic ivc (stieber et al. 1997) . portal venous stenosis and thrombosis are relatively uncommon in the adult population and present with graft dysfunction, massive ascites formation, and hemodynamic instability. this complication is corrected by an urgent reconstruction of the portal vein or construction of a superior mesenteric venous graft to the liver, together with a ligation of large collaterals that may reduce the portal flow. biliary complications are more common in children and have an overall incidence of 8-15 %. early recognition is difficult, leading to high morbidity and mortality. bile leaks usually occur at the anastomotic site, although they may be found at the t-tube site or aberrant ducts. most biliary complications occur within the first 3 months and are diagnosed by liver function tests (serum bilirubin, γ-glutamyltransferase, and alkaline phosphatase) and imaging techniques. these complications are treated by percutaneous or endoscopic drainage of bile collections. in cases of roux-en-y choledochojejunostomy, surgical reconstruction is required. intra-abdominal bleeding occurs in about 7-15 % of patients and requires exploration in about half of these cases (ozaki et al. 1994) . gastrointestinal bleeding may develop from ulcers, viral enteritis, varices, and an afferent roux-en-y loop. variceal bleeding is usually associated with portal vein thrombosis and requires an urgent ultrasound or angiographic evaluation. bleeding from the roux-en-y limb occurs 1 week after surgery and is usually self-limited. additionally, bleeding can be caused by persistent thrombocytopenia associated with splenic sequestration, drug toxicity, heparin-induced thrombocytopenia, and immunologic reactions. intestinal perforation is caused by serosal injury to the intestines and usually occurs in patients who have had a technically difficult hepatectomy, prolonged portal venous clamping, or a massive blood transfusion. intestinal perforation or leakage is treated by urgent surgery and antifungal therapy. cardiac complications any type of cardiac complication can develop in the postoperative period. hypotension can occur due to hypovolemia, either from underresuscitation or ongoing bleeding, decrease in contractility secondary to the pre-existing myocardial disease, or new onset of dilated or ischemic cardiomyopathy. other potential causes are acidosis, hypocalcemia, or vasodilation from sepsis or graft failure. management of cardiac complications is based on the underlying cause. hypertension occurs in patients with pre-existing hypertension, inadequate pain control, hypoglycemia, and cerebral edema. restoration of normal liver function may increase systemic vascular resistance, and calcineurin inhibition can increase systemic blood pressure. calcium channel blockers (i.e., diltiazem and verapamil) are avoided because they can increase the levels of the calcineurin inhibitors. myocardial infarction is relatively rare due to thorough preoperative evaluation being undertaken to detect cad. however, when it does develop, a cardiologist should be consulted for possible emergent cardiac catheterization during surgery and revascularization. pulmonary edema is commonly seen postoperatively and may be caused by significant transfusion requirements, increased capillary permeability, prolonged intubation, and reversible dilated cardiomyopathy. reversible dilated cardiomyopathy with pulmonary edema may develop in the first 5 days after transplantation. sampathkumar et al. reported that 1 % of patients who did not have ventricular dysfunction developed dilated cardiomyopathy postoperatively, most of whom recovered completely without any long-term complications (sampathkumar et al. 1998) . the cause of this condition is unknown, although it may be a form of stress-induced cardiomyopathy. atrial fibrillation, ventricular tachycardia, and other arrhythmias may develop as a result of electrolyte abnormalities (i.e., hypomagnesemia, hyperkalemia, and hypocalcemia), cardiac ischemia, or irritation from cvp or pa catheters. in a study by xia et al., atrial fibrillation was observed in 7.4 % of patients and was associated with increased mortality, graft failure, and acute kidney injury (xia et al. 2015) . all arrhythmias are treated following the standard guidelines. thromboembolism is a common cause of sudden postoperative death. deep vein thrombosis should be prevented by early extubation and mobilization, use of compressive stockings, and administration of heparin (subcutaneous or low molecular weight). most patients require mechanical ventilation for only a few hours or days after transplantation. however, prolonged ventilatory support is required in some patients with atelectasis, pleural effusions, and central nervous system (cns) depression. intraoperative cross-clamping of the ivc occasionally results in right phrenic nerve crush injury and diaphragmatic paralysis in the immediate postoperative period (mcalister et al. 1993 ). ards may develop in patients with intra-abdominal infection, pancreatitis, hepatic necrosis, acute cellular rejection, and occasionally with muromonab-cd3 (okt3) treatment. bronchoalveolar lavage and bacterial culture are frequently performed to rule out pulmonary infection from any other pulmonary pathology. pre-existing pulmonary hypertension may persist postoperatively and is controlled by epoprostenol or nitroglycerin. neurological complications occur in 12-20 % of patients, mostly in the first week of transplantation (singh et al. 1994 ). these are more common in adults and present as mental status changes ranging from dysphasia to frank coma. dysfunction of the cns is commonly caused by medications, such as cyclosporine, tacrolimus, histamine h 2 -blockers, acyclovir, and antibiotics such as imipenem. non-convulsive seizures may occur, and an eeg is performed for patients with unexplained mentation changes. intracranial hemorrhage and watershed infarcts are ruled out using ct scans. hyponatremia and hypomagnesemia can also delay awakening. central pontine myelinolysis may develop several days after transplantation, and recovery is often slow and incomplete (winnock et al. 1993) . hepatic encephalopathy may be present for several days after transplantation in patients with persistent portosystemic shunting. meningitis should be ruled out when the mental status change is accompanied by fever. disseminated aspergillosis is a devastating complication in a patient with multiple brain infarcts and fever. peripheral neuropathy presenting as weakness is usually myopathic in nature and is more common in patients with preoperative severe liver disease, poor graft function, high steroid doses, and uremia, and are confirmed by electromyography and muscle biopsy. in patients with fulminant hepatic failure, cerebral hyperemia and hypertension usually decrease gradually, and the patient regains consciousness as the liver begins to function. renal dysfunction is usually transient and is commonly associated with intraoperative hypovolemia and hypotension, allograft dysfunction, and nephrotoxicity of cyclosporine and tacrolimus. oliguria is an early sign of renal dysfunction and is managed by restoring intravascular volume and renal perfusion. the hepatorenal syndrome may persist after transplantation, and its recovery depends on its preoperative severity and allograft function. in some patients, addition of vasoconstrictive immunosuppressants (cyclosporine and tacrolimus) may lead to acute tubular necrosis. in general, renal function returns to the normal range in most patients, and approximately 10 % of patients require temporary dialysis (mccaulley et al. 1990 ). long-term prognosis is fair, although hypertension, diabetes, and chronic nephropathy induced by steroids and the calcineurin inhibitors may result in chronic renal failure. more than half of the postoperative infections following liver transplantation are bacterial in origin. these infections typically occur in the first 2 weeks, when blood levels of immunosuppressants are high. the most common sites of infection are the liver, biliary tract, peritoneal cavity, and pulmonary system. common organisms in the abdomen are aerobic gram-positive organisms (streptococci and staphylococci) and gram-negative bacilli (escherichia coli, enterobacter species, and pseudomonas), while pseudomonas infection is most common in the lungs. approximately 20 % of infections are caused by fungus, with candida species accounting for more than 80 % of all fungal infections. the risk factors are a high steroid dosage, usage of broad-spectrum antibiotics, and prolonged surgical time. candida infection is treated with amphotericin or fluconazole. aspergillus infection accounts for 15 % of all fungal infections and is associated with a very high mortality; high-dose liposomal amphotericin b followed by prolonged itraconazole is the treatment of choice. viral infections are seen 2-3 months after transplantation, with cytomegalovirus and herpes simplex accounting for the bulk of these infections. epstein-barr virus is not usually seen until approximately 6 months after transplantation, but is an important cause of lymphoproliferative disease. pneumocystis pneumonia, an opportunistic infection, responds to trimethoprim-sulfamethoxazole. late metabolic complications following liver transplantation include diabetes, hyperlipidemia, weight gain, and hypertension. diabetes is induced by steroids, cyclosporine, and tacrolimus and may respond to oral hypoglycemic agents or insulin. hyperlipidemia is associated with diabetes, obesity, steroids, and immunosuppressive drugs and is treated by diet and exercise. hypertension is seen in as many as 85 % of patients after transplantation; the use of steroids or tacrolimus is the most likely cause. hypomagnesemia has been implicated as the cause of the hypertension in some cases. approximately 15 % of all patients require retransplantation of the liver. early retransplantation is performed within several days after the primary transplantation to rescue patients from primary non-function (graft factor), acute rejection, and technical failure (vascular thrombosis), or secondary non-function (host factor) associated with poor hepatic perfusion. hepatic necrosis is the common pathway of graft non-function and results in progressive, severe encephalopathy, ards, lactic acidosis, coagulopathy, hypoglycemia, and significant circulatory instability. although infrequent, hepatectomy with a portacaval shunt may be performed to protect the patient from the ill effects of the necrotizing liver on extrahepatic organ functions. in such a case, retransplantation should be performed as soon as the donor organ is available. the surgical procedure itself is relatively simple because surgical dissection has already been made and adhesions have not yet formed. anesthetic management of these patients is similar to that of patients undergoing primary transplantation. late retransplantation is performed in patients with chronic rejection, vascular complications, and recurrence of the original disease. the physical condition of the patient may have improved, but complications of immunosuppression (i.e., hypertension, renal insufficiency) may be present. adhesions and the steroid-induced fragile tissues frequently complicate late retransplantation. anesthetic management is similar to that of primary liver transplantation, but a large amount of blood loss is anticipated. in pediatric liver transplantation, rapid-sequence iv induction is preferred, although mask induction is chosen in patients in whom there is difficulty obtaining iv access (borland et al. 1985) . large-bore iv catheters are placed in the upper extremities after induction of anesthesia. a central venous catheter with cvp monitoring is the usual procedure, and pulmonary arterial catheterization is rarely indicated. blood pressure is monitored using a femoral intra-arterial catheter. it appears that children tolerate cross-clamping of the ivc and portal vein reasonably well without significant hemodynamic changes, possibly by compliant vasomotor tone. therefore, venovenous bypass is rarely used in children under 20 kg. coagulation changes that occur during liver transplantation are not as severe as those of adults, and this may be associated with more prevalent cholestatic diseases in children ). blood loss in children with biliary atresia can be large due to the technical difficulty associated with previous biliary surgery (i.e., ksai procedure). maintenance of body temperature is difficult, as the large surface area promotes heat loss. live-donor hepatectomy is usually a challenging procedure. the young and healthy donors (asa physical status [ps] 1 or 2) undergo a complete evaluation by hepatologists, surgeons, anesthesiologists, and psychologists. the anesthetic goals are minimizing surgical blood loss and allogeneic blood transfusion, maintaining liver blood flow, facilitating early extubation, preventing deep venous thrombosis and infection, and providing adequate postoperative pain control. preoperatively, donors may be given erythropoietin to boost rbc production and they can donate 2 units of autologous whole blood 2-3 weeks before surgery. on the day of surgery, donors may be given heparin (5000 units, subcutaneous injection) to prevent deep venous thrombosis, and 6 units of typed and cross-matched prbcs are prepared. in the holding area, a peripheral iv catheter is secured, anxiolytics are administered, and donors may elect to receive thoracic epidural anesthesia for postoperative analgesia. the need for epidural local anesthetics with or without narcotics is determined by the attending anesthesiologist and pain service. in the operating room, unasyn ® (3 g iv) or vancomycin (if allergic to penicillin) is administered to prevent infection, and the patient is positioned with minimal stress to the brachial plexus to avoid neurologic injury (dulitz et al. 2005) . induction and maintenance of anesthesia follows the standard guidelines of any major surgical procedure. ultra-short-acting narcotics such as remifentanil may be beneficial for early extubation after surgery as it is rapidly metabolized by plasma esterase and does not have a prolonged effect in the presence of hepatic and renal dysfunction. intraoperative monitoring is similar to that of patients undergoing major surgery, and a radial arterial catheter and cvp are placed for hemodynamic monitoring. additional ivaccess is secured to prepare for the potential need for rapid infusion of fluids using a rapid-infusion system. immediately after induction of anesthesia, isovolemic hemodilution may be performed: 2 units of the patient's whole blood is collected in cpda (citrate phosphate dextrose adenine) blood collection bags, agitated to prevent clot formation, stored at room temperature, and returned to the patient within 8 h. intraoperatively, physiologic condition should be maintained at all times to ensure adequate perfusion of all tissues including the liver by monitoring the cardiopulmonary system and stat laboratory. metabolic acidosis should be avoided, and use of a balanced salt solution (lactated ringer's solution or plasmalyte-a ® ) is the preferred choice in order to avoid the acid load from normal saline (waters et al. 2001 ). blood loss is not excessive and pre-and intraoperatively donated autologous blood and intraoperative autotransfusion are sufficient in most patients. the relationship between the cvp level and surgical blood loss is controversial: chhibber et al. reported that intraoperative blood loss did not correlate with cvp (<5 mmhg) in their study (chhibber et al. 2007 ), while jones et al. demonstrated a significant reduction in blood loss with low cvp (jones et al. 1998 ). the authors recommend euvolemia to maintain hepatic blood flow during dissection of the liver. however, fluid overloading should be avoided after hepatectomy because relatively high portal venous flow to the reduced liver mass may lead to liver congestion and small-for-size syndrome (dahm et al. 2005) . at the conclusion of surgery, the patient can be extubated safely in the operating room and transported to the icu. all types of surgical procedures may be necessary in the early postoperative period. within the first 2 months after transplantation, surgical procedures are performed to treat complications of transplantation, such as exploratory laparotomy for abdominal bleeding or reconstruction of the biliary system. some degree of hepatic dysfunction may still be present, and ventilatory and circulatory support and invasive monitoring may be required. regional anesthesia is not recommended because of potential bleeding and infectious complications. anesthesia care of these patients is similar to that of other urgent abdominal procedures. patients may return to the operating room at any time for biliary reconstruction, replacement of a hip joint, or almost any other procedure. liver function and drug metabolism are usually within the normal range, and anesthetic management differs little from that of other patients. side effects of immunosuppressants (hypertension and renal insufficiency) and drug interactions should be considered. the main goal of organ procurement is the maintenance of optimal conditions for all organ systems to promote as normal as possible an environment for the organs prior to harvesting. specifically, integrity of organs should be maintained by optimizing organ perfusion and preventing further damage associated with pre-existing illness or trauma. therefore, donor care during procurement is a continuum of the intensive care provided before brain death. the donor is reviewed and examined by the anesthesia team to evaluate their medical history and vital organ function. the equipment and medications necessary for multiple organ procurement are shown in table 5 . a multiple-channel vital-sign monitor is an essential piece of equipment because of the unavoidable hemodynamic changes associated with the absence of brain stem function, surgical manipulation, and fluid shift. a volume ventilator may be required for donors requiring high levels of peep or airway pressure. a large volume of crystalloids and colloid solutions is prepared, and 5 units of prbcs are frequently required. the transit from the icu to the operating room is a crucial period; the anesthesia care team directs the transportation while the donor is continuously monitored, ventilated, and treated. intraoperatively, blood pressure is monitored by an indwelling radial or brachial arterial catheter as abrupt changes in blood pressure are anticipated. cvp monitoring is essential, and a pa catheter may be used in unstable donors. general anesthesia is provided as donors respond to surgical stimulation by dramatic hemodynamic changes such as tachycardia, hypertension, perspiration, and involuntary movement (wetzel et al. 1985) . this so-called mass reflex is caused by the neurogenic vasoconstriction and stimulation of adrenal medulla by reflex spinal arc. isoflurane is the most commonly used agent, because its myocardial depression is relatively benign, and short-acting narcotics (i.e., fentanyl, up to 5 μg/kg/min) may be used in unstable donors. rocuronium bromide or vecuronium bromide is administered for muscle relaxation. the specific goals of ventilatory care are to maintain normal pao 2 (70-100 mmhg), arterial hemoglobin oxygen saturation (>95 %), and paco 2 (35-45 mmhg) as well as to avoid pulmonary complications. this goal is frequently achieved by ventilating with a tidal volume of 10-15 ml/kg, fio 2 of 30-40 %, respiratory rate of <20/min, and a low level of peep (<5 cmh 2 o). however, in donors with pulmonary complications, adjustments are made in tidal volume (up to 20 ml/kg), respiratory rate (up to 20/min), and peep (up to 10 cmh 2 o). aggressive circulatory care is essential because hemodynamic instability may impair organ perfusion. specifically, hypotension (systolic blood pressure <80 mmhg or mean arterial pressure <40 mmhg) is associated with a high incidence of acute tubular necrosis, non-function of the graft kidneys, and poor hepatic function. it is generally agreed that systolic blood pressure should be within the normal range (100-120 mmhg) and cvp should be <10 cmh 2 o with minimal vasopressor support. maintaining circulatory homeostasis, however, can be challenging. preload is frequently decreased because of blood loss, vasomotor paralysis, diuretic therapy, and diabetes inspidus, although fluid resuscitation may result in overload. the heart rate may vary depending on the degree of brain injury, ranging from tachycardia to bradycardia. arrhythmia is not uncommon, and myocardial contractility is frequently impaired by myocytolysis, myocardial necrosis, coronary spasm, and reduction of myocardial energy storage (novitzky et al. 1988 ). afterload may be high, from excessive sympathetic tone, or low, from vasomotor paralysis. volume deficit is usually corrected with lactated ringer's or colloid solution, and transfusion of prbcs (1-3 units) may be necessary to maintain hematocrit between 25 and 35 % (hardesty and griffith 1986) . once the fluid deficit is corrected, a glucose-containing hypotonic solution (5 % dextrose in 0.45 % nacl 1 ml/kg/h) is administered to replace urine output and insensible loss, guided by cvp and urine output. excessive urine output (>200-250 ml/h) is replaced using a hypotonic electrolyte solution with supplementation of kcl (20 meq/l). tachycardia with hypertension should be avoided as it may cause pulmonary edema, decrease organ perfusion, and increase myocardial oxygen consumption. a β-antagonist (i.e., labetalol hydrochloride or esmolol hydrochloride) or a calcium channel blocker (verapamil hydrochloride) is used to treat tachycardia and arrhythmia (novitzky et al. 1984) . for bradycardia, isoproterenol or epinephrine is used for positive chronotropic effects because donors are unresponsive to centrally acting chronotropic drugs (i.e., atropine). supraventricular or ventricular arrythmia is treated using antiarrhythmic drugs. low afterload is compensated for by increasing preload because α-vasopressors increase the myocardial work load and decrease splanchnic and coronary blood flow. in severely hypertensive donors, an α-blocker (hydralazine or sodium nitroprusside) may be given to reduce the afterload. when cardiac output and organ perfusion are impaired, inotropes (dopamine hydrochloride, dobutamine hydrochloride, and isoproterenol hydrochloride) are recommended to improve cardiac contractility. in brain-dead animal models, serum levels of triiodothyronine, insulin, and cortisol have been found to be low, and the administration of triiodothyronine may improve hemodynamic stability by maintaining myocardial high-energy stores and glycogen (james et al. 2010) . circulatory arrest, which occurs in 10 % of potential donors (emery et al. 1986) , is managed in the standard fashion, except atropine is not effective. adequate diuresis (>0.5 ml/kg/h, preferably 1-1.5 ml/kg/h) is recommended as urine output (>100 ml/h) is the most significant factor that determines the outcome of the kidney and liver graft. oliguria is generally caused by hypovolemia and hypotension and frequently responds to fluid administration. diabetes insipidus leads to polyuria, hypovolemia, and electrolyte imbalance. in addition to the fluid replacement, ddavp (0.5-1 units/h) may be administered (richardson and robinson 1985) , although an excessive dose of ddavp may increase the risk of acute tubular necrosis and reduce hepatic blood flow (burggraaf et al. 1994) . donors are poikilothermic, and hypothermia plays a major role in hemodynamic instability. body temperature should be kept above 35 c by raising the operating room temperature, infusing all fluids through a blood warmer, and using heating lamps, a warming blanket, and a heated humidifier in the ventilation circuit. metabolic acidosis, caused by inadequate tissue perfusion, is corrected by administration of nahco 3 or tham. commonly seen electrolyte imbalances are hypernatremia, hypokalemia, hypocalcemia, hypophosphatemia, and hypomagnesemia, and they are treated in a standard fashion. glucose metabolism is relatively well-maintained, and any abnormality in glucose metabolism is corrected by administration of insulin or glucose on the basis of the serum glucose level. dilutional coagulopathy is common, and consumption coagulopathy may develop secondary to the release of tissue thromboplastin from injured tissues and the ischemic organs (kaufman et al. 1984) . fibrinolysis is not uncommon in donors, possibly as a result of the release of tpa from the necrotic brain. replacement of coagulation factors and platelets or any pharmacologic therapy is rarely indicated as donors are fully heparinized when the aorta is cannulated. once cardiac arrest is induced by cardioplegia, no further supportive care is necessary. liver transplantation is one of the most stressful procedures for patients with multiple organ dysfunction and it is a challenge for anesthesiologists. it is remarkable that anesthesiologists have played a major role in the progress of liver 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rats agreement between radial and femoral arterial blood pressure measurements during orthotopic liver transplantation cardiac disease evaluation and management among transplantation candidates: a scientific statement from the american heart association and the american college of cardiology foundation liver transplantation: intraoperative changes in coagulation factors in 100 first transplants antithrombin iii during liver transplantation intraoperative blood salvage during liver transplantation in patients with hepatocellular carcinoma: efficiency of leukocyte depletion filters in the removal of tumor cells intracranial pressure monitoring in liver transplantation for fulminant hepatic failure biochemical factors in alcoholic liver disease cardiopulmonary dysfunction in cirrhosis cirrhotic cardiomyopathy role of altered beta adrenoceptor signal transduction in the pathogenesis of cirrhotic cardiomyopathy in rats prognostic significance of reperfusion hyperglycemia during liver 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to chronic liver disease dobutamine stress echocardiography for preoperative cardiac risk stratification in patients undergoing orthotopic liver transplantation pathologic aspects of cirrhosis systemic effects of tissue plasminogen activator-associated fibrinolysis and its relation to thrombin generation in orthotopic liver transplantation cardiovascular risk assessment of the liver transplant candidate effect of hypothermia on the coagulation cascade platelet function in chronic liver disease; relationship to disease severity myocardial ischemia after orthotopic liver transplantation perioperative risk predictors of cardiac outcomes in patients undergoing liver transplantation surgery complications associated with percutaneous placement of venous return cannula for venovenous bypass in adult orthotopic liver transplantation postliver transplantation myocardial dysfunction intraoperative fluid management during orthotopic liver transplantation ionized hypomagnesemia in patients 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transplantation increased concurrence of cirrhosis and bacterial endocarditis role of the molecular adsorbent recycling system (mars) in the treatment of patients with acute exacerbation of chronic liver failure experience in hepatic transplantation. saunders, philadelphia starzl te, marchioro tl, von kaulla kn et al (1963) homotransplantation of the liver in humans fk 506 for liver, kidney, and pancreas transplantation a simple solution to a technical complication in piggy back liver transplantation intraoperative transesophageal echocardiography during liver transplantation non alcoholic cirrhosis associated with neuropsychological dysfunction in the absence of overt evidence of hepatic encephalopathy cardiac hemodynamic and coronary angiographic characteristics of patients being evaluated for liver transplantation clinical presentation of hepatic artery thrombosis after liver transplantation in the cyclosporine era orthotopic liver transplantation with preservation of the inferior vena cava low levels of thrombin activatable fibrinolysis inhibitor (tafi) in patients with chronic liver disease complications and use of intracranial pressure monitoring in patients with acute liver failure and severe encephalopathy a rapid increase in sodium is associated with cpm after liver transplantation recommendations on the use of recombinant activated factor vii as an adjunctive treatment for massive bleeding-a european perspective alterations in plasminogen activator and plasminogen activator inhibitor levels during liver transplantation vasopressin decreases portal vein pressure and flow in the native liver during liver transplantation hepatic and portal vein thrombosis in cirrhosis: possible role in development of parenchymal extinction and portal hypertension intraoperative pulmonary embolism and intracardiac thrombosis complicating liver transplantation: a systematic review identification of osmosensitive and ammonia-regulated genes in rat astrocytes by northern blotting and differential display reverse transcriptase-polymerase chain reaction normal saline versus lactated ringer's solution for intraoperative fluid management in patients undergoing abdominal aortic aneurysm repair: an outcome study hemodynamic responses in brain dead organ donor patients is hepatic histology the true gold standard in diagnosing acute hepatic allograft rejection pontine myelinolysis following liver transplantation: a report of two cases postoperative atrial fibrillation in liver transplantation successful anesthetic management of a patient with critical bleeding during hepatectomy using recombinant activated factor vii and intraoperative blood salvage endotoxemia and human liver transplantation key: cord-350224-dt3li3bk authors: ye, qingsong; wang, hua; xia, xia; zhou, chenliang; liu, zhiming; xia, zun-en; zhang, zhan; zhao, yang; yehenala, jun; wang, si; zhou, gangqiao; hu, ke; wu, bin; wu, chu-tse; wang, songling; he, yan title: safety and efficacy assessment of allogeneic human dental pulp stem cells to treat patients with severe covid-19: structured summary of a study protocol for a randomized controlled trial (phase i / ii) date: 2020-06-12 journal: trials doi: 10.1186/s13063-020-04380-5 sha: doc_id: 350224 cord_uid: dt3li3bk objectives: to assess the safety and therapeutic effects of allogeneic human dental pulp stem cells (dpscs) in treating severe pneumonia caused by covid-19. trial design: this is a single centre, two arm ratio 1:1, triple blinded, randomized, placebo-controlled, parallel group, clinical trial. participants: 1. adults aged 18-65 years; 2. voluntarily participate in this clinical trial and sign the “informed consent form” or have consent from a legal representative. 3. diagnosed with severe pneumonia of covid-19: nucleic acid test sars-cov-2 positive; respiratory distress (respiratory rate > 30 times / min); hypoxia (resting oxygen saturation < 93% or arterial partial pressure of oxygen / oxygen concentration < 300 mmhg). 4. covid-19 featured lung lesions in chest x-ray image. 1. patients have received other experimental treatment for covid-19 within the last 30 days; 2. patients have severe liver condition (e.g., child pugh score >=c or ast> 5 times of the upper limit); 3. patients with severe renal insufficiency (estimated glomerular filtration rate <=30ml / min/1.73 m(2)) or patients receiving continuous renal replacement therapy, hemodialysis, peritoneal dialysis; 4. patients who are co-infected with hiv, hepatitis b, tuberculosis, influenza virus, adenovirus or other respiratory infection viruses; 5. female patients who have no sexual protection in the last 30 days prior to the screening assessment; 6. pregnant or lactating women or women using estrogen contraception; 7. patients who are planning to become pregnant during the study period or within 6 months after the end of the study period; 8. other conditions that the researchers consider not suitable for participating in this clinical trial. intervention and comparator: there will be two study groups: experimental and control. both will receive all necessary routine treatment for covid-19. the experimental group will receive an intravenous injection of dental pulp stem cells suspension (3.0x10(7) human dpscs in 30ml saline solution) on day 1, 4 and 7; the control group will receive an equal amount of saline (placebo) on the same days. clinical and laboratory observations will be performed for analysis during a period of 28 days for each case since the commencement of the study. main outcomes: 1. primary outcome the primary outcome is time to clinical improvement (ttci). by definition, ttci is the time (days) it takes to downgrade two levels from the following six ordered grades [(grade 1) discharge to (grade 6) death] in the clinical state of admission to the start of study treatments (hdpscs or placebo). six grades of ordered variables: 2. secondary outcomes 2.1 vital signs: heart rate, blood pressure (systolic blood pressure, diastolic blood pressure). during the screening period, hospitalization every day (additional time points of d1, d4, d7 30min before injection, 2h ± 30min, 24h ± 30min after the injection) and follow-up period d90 ± 3 days. 2.2 laboratory examinations: during the screening period, 30 minutes before d1, d4, d7 infusion, 2h ± 30min, 24h ± 30min after the end of infusion, d10, d14, d28 during hospitalization or discharge day and follow-up period d90 ± 3 days. 2.3 blood routine: white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, neutrophils, lymphocytes, monocytes, eosinophils acidic granulocyte count, basophil count, red blood cell, hemoglobin, hematocrit, average volume of red blood cells, average red blood cell hb content, average red blood cell hb concentration, rdw standard deviation, rdw coefficient of variation, platelet count, platelet specific platelet average volume, platelet distribution width,% of large platelets; 2.4 liver and kidney function tests: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, prealbumin, total protein, albumin, globulin, white / globule ratio , total bilirubin, direct bilirubin, cholinesterase, urea, creatinine, total carbon dioxide, uric acid glucose, potassium, sodium, chlorine, calcium, corrected calcium, magnesium, phosphorus, calcium and phosphorus product, anion gap, penetration pressure, total cholesterol, triacylglycerol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, lipoprotein a, creatine kinase, lactate dehydrogenase, estimated glomerular filtration rate. 2.5 inflammation indicators: hypersensitive c-reactive protein, serum amyloid (saa); 2.6 infectious disease testing: hepatitis b (hbsag, hbsab, hbeag, hbeab, hbcab), hepatitis c (anti-hcv), aids (hivcombin), syphilis (anti-tp), cytomegalovirus cmv-igm, cytomegalovirus cmv-igg; only during the screening period and follow-up period d90 ± 3. 2.7 immunological testing: collect peripheral blood to detect the phenotype of t lymphocyte, b lymphocyte, natural killer cell, macrophage and neutrophil by using flow cytometry. collect peripheral blood to detect the gene profile of mononuclear cells by using single-cell analyses. collect peripheral blood serum to detect various immunoglobulin changes: iga, igg, igm, total ige; collect peripheral blood serum to explore the changes of cytokines, th1 cytokines (il-1 β, il-2, tnf-a, itn-γ), th2 cytokines (il-4, il-6, il -10). 2.8 pregnancy test: blood β-hcg, female subjects before menopause are examined during the screening period and follow-up period d90 ± 3. 2.9 urine routine: color, clarity, urine sugar, bilirubin, ketone bodies, specific gravity, ph, urobilinogen, nitrite, protein, occult blood, leukocyte enzymes, red blood cells, white blood cells, epithelial cells, non-squamous epithelial cells , transparent cast, pathological cast, crystal, fungus; 2.10 stool routine: color, traits, white blood cells, red blood cells, fat globules, eggs of parasites, fungi, occult blood (chemical method), occult blood (immune method), transferrin (2h ± 30min after the injection and not detected after discharge). randomization: block randomization method will be applied by computer to allocate the participants into experimental and control groups. the random ratio is 1:1. blinding (masking): participants, outcomes assessors and investigators (including personnel in laboratory and imaging department who issue the sample report or image observations) will be blinded. injections of cell suspension and saline will be coded in accordance with the patient’s randomisation group. the blind strategy is kept by an investigator who does not deliver the medical care or assess primary outcome results. numbers to be randomized (sample size): twenty participants will be randomized to the experimental and control groups (10 per group). trial status: protocol version number, hdpsc-covid-2019-02-2020 version 2.0, march 13, 2020. patients screening commenced on 16(th) april and an estimated date of the recruitment of the final participants will be around end of july. . trial registration: registration: world health organization trial registry: chictr2000031319; march 27,2020. clinicaltrials.gov identifier: nct04336254; april 7, 2020 other study id numbers: hdpsc-covid-2019-02-2020 full protocol: the full protocol is attached as an additional file, accessible from the trials website (additional file 1). in the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol. 1. patients have received other experimental treatment for covid-19 within the last 30 days; 2. patients have severe liver condition (e.g., child pugh score >=c or ast> 5 times of the upper limit); 3. patients with severe renal insufficiency (estimated glomerular filtration rate <=30ml / min/1.73 m 2 ) or patients receiving continuous renal replacement therapy, hemodialysis, peritoneal dialysis; 4. patients who are co-infected with hiv, hepatitis b, tuberculosis, influenza virus, adenovirus or other respiratory infection viruses; 5. female patients who have no sexual protection in the last 30 days prior to the screening assessment; 6. pregnant or lactating women or women using estrogen contraception; 7. patients who are planning to become pregnant during the study period or within 6 months after the end of the study period; 8. other conditions that the researchers consider not suitable for participating in this clinical trial. intervention and comparator: there will be two study groups: experimental and control. both will receive all necessary routine treatment for covid-19. the experimental group will receive an intravenous injection of dental pulp stem cells suspension (3.0x10 7 human dpscs in 30ml saline solution) on day 1, 4 and 7; the control group will receive an equal amount of saline (placebo) on the same days. clinical and laboratory observations will be performed for analysis during a period of 28 days for each case since the commencement of the study. main outcomes: 1. primary outcome the primary outcome is time to clinical improvement (ttci). by definition, ttci is the time (days) it takes to downgrade two levels from the following six ordered grades [(grade 1) discharge to (grade 6) death] in the clinical state of admission to the start of study treatments (hdpscs or placebo). six grades of ordered variables: 2. secondary outcomes 2.1 vital signs: heart rate, blood pressure (systolic blood pressure, diastolic blood pressure). during the screening period, hospitalization every day (additional time points of d1, d4, d7 30min before injection, 2h ± 30min, 24h ± 30min after the injection) and follow-up period d90 ± 3 days. 2.2 laboratory examinations: during the screening period, 30 minutes before d1, d4, d7 infusion, 2h ± 30min, 24h ± 30min after the end of infusion, d10, d14, d28 during hospitalization or discharge day and follow-up period d90 ± 3 days. 2.3 blood routine: white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, neutrophils, lymphocytes, monocytes, eosinophils acidic granulocyte count, basophil count, red blood cell, hemoglobin, hematocrit, average volume of red blood cells, average red blood cell hb content, average red blood cell hb concentration, rdw standard deviation, rdw coefficient of variation, platelet count, platelet specific platelet average volume, platelet distribution width,% of large platelets; 2.4 liver and kidney function tests: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, prealbumin, total protein, albumin, globulin, white / globule ratio , total bilirubin, direct bilirubin, cholinesterase, urea, creatinine, total carbon dioxide, uric acid glucose, potassium, sodium, chlorine, (continued on next page) grade 1: discharged of patient; grade 2: hospitalized without oxygen supplement; grade 3: hospitalized, oxygen supplement is required, but niv / hfnc is not required; grade 4: hospitalized in intensive care unit, and niv / hfnc treatment is required; grade 5: hospitalized in intensive care unit, requiring ecmo and/or imv; grade 6: death. collect peripheral blood to detect the gene profile of mononuclear cells by using single-cell analyses. collect peripheral blood serum to detect various immunoglobulin changes: iga, igg, igm, total ige; collect peripheral blood serum to explore the changes of cytokines, th1 cytokines (il-1 β, il-2, tnf-a, itn-γ), th2 cytokines (il-4, il-6, il -10). 2.8 pregnancy test: blood β-hcg, female subjects before menopause are examined during the screening period and follow-up period d90 ± 3. 2.9 urine routine: color, clarity, urine sugar, bilirubin, ketone bodies, specific gravity, ph, urobilinogen, nitrite, protein, occult blood, leukocyte enzymes, red blood cells, white blood cells, epithelial cells, non-squamous epithelial cells , transparent cast, pathological cast, crystal, fungus; 2.10 stool routine: color, traits, white blood cells, red blood cells, fat globules, eggs of parasites, fungi, occult blood (chemical method), occult blood (immune method), transferrin (2h ± 30min after the injection and not detected after discharge). randomization: block randomization method will be applied by computer to allocate the participants into experimental and control groups. the random ratio is 1:1. blinding (masking): participants, outcomes assessors and investigators (including personnel in laboratory and imaging department who issue the sample report or image observations) will be blinded. injections of cell suspension and saline will be coded in accordance with the patient's randomisation group. the blind strategy is kept by an investigator who does not deliver the medical care or assess primary outcome results. numbers to be randomized (sample size): twenty participants will be randomized to the experimental and control groups (10 per group). full protocol: the full protocol is attached as an additional file, accessible from the trials website (additional file 1). in the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol. keywords: covid-19; randomised controlled trial; protocol; human dental pulp stem cells; dental stem cell banking beijing institute of radiation medicine, beijing 100850, china. 3 beijing sh biotechnology co., ltd publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors acknowledge the chu-tse wu foundation for science & technology development for sponsoring this study. beijing sh biotechnology co., ltd. and utooth biological technology co., ltd. are acknowledged for providing the dpscs for this study. we would like to extend our appreciation to our collaborators and the nurses, clinicians and lab technicians at the renmin hospital and center of regenerative medicine, wuhan university. supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-020-04380-5.additional file 1. full study protocol.authors' contributions qy, zw and slw conceived the research idea; qy, hw, xx, yh and gz designed the study protocol and developed the research plan; cz and qy obtained the ethics approval; qy and zl coordinated the tasks among different investigators; cz, zz, zl and qy yz and kh recruited the participants and collected data. jy and sw will deliver the treatment to the participants; zx and bw will be the quality controller for the dpscs intervention; xx manages the blind strategy. qy and yh will analyse the data and interpret the result. yh drafted the manuscript; qy, hw, xx revised and finalised the manuscript; qy, zw and slw proofread the manuscript prior to submission. the author(s) read and approved the final manuscript. the trial has been financially funded by the chu-tse wu foundation for science & technology development, a non-profit organization. the design of the study, as well as the collection, analysis and interpretation of data will not be influenced by the funding body. the writing of the protocol and the decision to submit for publication are compiled independent from the funding body. the data will be stored in a repository managed by a third party called clinflash, a professional data management service for clinical studies (weblink: https://edc.clinflash.net/shbio). member investigators of the trial will have access to the final trial dataset, depending on level of need-to-know. the study was approved by the committee of ethics for clinical research, renmin hospital of wuhan university, on 03/14/2020. the ethical approval number is wdry2020-k106. recruitment has been ongoing by the investigators. once informed consent is signed off by both parties, the investigator and participating patient/ legal representative, the participant will be enrolled in the study. the consent form and materials are available from the corresponding author on request. the authors declare that they have no competing interests.author details 1 key: cord-318598-pzlf2zpc authors: roberts, brian k. title: basic shock physiology and critical care date: 2016-04-28 journal: vet clin north am exot anim pract doi: 10.1016/j.cvex.2016.01.010 sha: doc_id: 318598 cord_uid: pzlf2zpc veterinarians practicing emergency medicine and/or working with exotic animals must be well versed in the pathophysiology of shock because many exotic pets present with an acute crisis or an acute manifestation of a chronic process causing poor organ perfusion. this article discusses the pathophysiology of shock and the systemic inflammatory response syndrome, which may lead to organ dysfunction, organ failure, sepsis, and death. the physiology of perfusion, perfusion measurements, categories of shock, and altered function of the immune system, gastrointestinal barrier, and coagulation system are discussed. veterinarians providing emergency care to patients with shock must also be aware of comorbidities. volume) and cao 2 is the oxygen carrying capacity of arterial blood, which is determined by the saturation of oxygen bound to hemoglobin, the amount of hemoglobin, and the small amount of dissolved oxygen in the serum (fig. 1) . 3 . oxygen consumption (vo 2 ) is the amount of oxygen used by the tissues after being delivered and off-loaded from hemoglobin. 3 it can be determined by using the equation co â (c a o 2 -c v o 2 ) which is cardiac output multiplied by the difference between arterial oxygen content (c a o 2 ) and venous oxygen content (c v o 2 ). 4. systemic inflammatory response syndrome (sirs) is a condition characterized by changes in heart rate, body temperature, and respiration rate with either leukocytosis or leukopenia. 4 sirs can be caused by trauma; organ inflammation, such as pancreatitis; infection; and environmental stressors, like heat stroke. 5 criteria for sirs have been described for humans and extrapolated for small animal veterinary medicine. 6 sirs has been described experimentally using similar criteria in rabbits 7 and in rodents. 8 sirs in reptiles, amphibians, and other exotic species has not been characterized to the author's knowledge. 5. sepsis is defined as sirs secondary to infection. an infection is a pathologic process caused by the invasion of microorganisms that cause disease of normally sterile tissue or fluid. 9 the term has been used since hippocrates' time to describe putrefaction and decomposition. 10 many clinicians use terminology such as sepsis syndrome, bacteremia, and sirs interchangeably with sepsis. note that sirs can occur without infection. 6. multiple organ dysfunction syndrome is a condition whereby disease, injury, lack of perfusion, and so forth cause at least 1 vital organ (heart, lung, kidney, nervous system) to no longer function normally. failure of multiple organs (>1) is multiple organ failure (mof). 11 to provide oxygen to tissues and their cells, there are 3 basic requirements: functional cardiovascular system comprising a pump (heart) and responsive vasculature, the ability of the fluid pumped to carry oxygen (red blood cells, normally working hemoglobin, and saturation of the hemoglobin), and a way for the oxygen to become bound to hemoglobin through a normally working respiratory system. oxygen delivery (do 2 ) is the amount of oxygen carried to the tissues in blood by hemoglobin. it is determined by cardiac output and oxygen carrying capacity (see fig. 1 ). in rats, normal do 2 is approximately 1.8 ae 0.2 ml o 2 /min/kg. 12 in rabbits, the normal do 2 reference range is 27.1 ae 2.7 ml/kg/min. 13 determination of oxygen delivery is more difficult in reptiles because of the various species and slow heart rates. instead of do 2 , researchers sometimes measure oxygen pulse (op), which was used in several tissue oxygenation studies summarized by gleeson and bennett. 14 op is the amount of oxygen delivered per heartbeat, which differs among turtles, snakes, and lizards according to their body temperature, with values ranging between 1 and 4 ml o 2 â 10 à5 /g/beat. in turtles, as body temperature increased, so did op, 15 whereas in lizards 16 and snakes 17 op decreased with increasing body temperature. therefore, species differences and habitat affect emergency treatment such as warming or not warming a reptile in shock. oxygen use by the tissues, termed vo 2 , has a direct association with the amount of oxygen being delivered and extracted by cells. the ratio of arterial oxygen delivery to oxygen consumption is the oxygen extraction ratio (o 2 er) (see fig. 1 ). approximately 20% to 30% of the oxygen delivered to tissues is extracted in mammals under normal conditions. 18 during periods of increased oxygen need caused by changes in metabolism, such as fever, the tissues can extract more oxygen. this ability also occurs during times when there is diminished oxygen delivery caused by hypovolemia, bradycardia, dyshemoglobinemia, pulmonary disease, and so forth. therefore, the cells, and specifically the mitochondria of cells, can still produce enough atp despite diminished hemoglobin saturation and lowered cardiac output by extracting more oxygen. during exercise, acute hypoxia, and acute anemia, do 2 is maintained by increasing cardiac output. 19 however, once a critical point is reached, the tissues can no longer extract enough oxygen unless more is delivered. for example, you are stocking up on water because of an impending hurricane. you fill up a whole shopping cart with bottles of water. because you have a big family, you need more than 1 cart-full, so you fill another cart. soon, the bottled water shelves become sparse. the only way you can extract more water (ie, put more in the cart) is if more is delivered and stocked on the shelves. during states in which the tissues and cells cannot extract enough oxygen and more must be delivered, anaerobic metabolism ensues. this critical point is termed oxygen supply dependency. 20 it is at this point that symptoms of shock are seen; a response that tries to increase oxygen delivery. physiologic changes that occur include tachycardia, tachypnea, and vasoconstriction of peripheral and splanchnic vessels. this condition is shown in fig. 2 ; once the do 2 cannot be maintained at greater than or equal to twice the level of vo 2 , biochemical changes associated with shock, such as hyperlactatemia and acidemia, ensue. not only does inadequate do 2 result from hypovolemia, pulmonary disease, and dyshemoglobinemia but it can also be caused by altered cellular function, as seen with sepsis and mitochondrial dysfunction in the critically ill. during such insults, the cell's machinery, cell membrane, protein receptors, and genes become dysfunctional, leading to apoptosis and loss of integrity. 21 nonmammalian species such as reptiles and birds have different values for do 2 , vo 2 , and o 2 er because of slower or faster heart rates, hemoglobin concentration, and saturation of hemoglobin. reptiles, depending on the species, balance oxygen delivery during exercise differently. iguanas maintain do 2 primarily by increasing heart rate because stroke volume decreases by 20% from a resting state during exercise. however, varanus spp such as monitor lizards increase stroke volume and heart rate to normalize oxygen delivery during exercise. 22 reptiles also tend to have lower oxygen saturation of arterial blood. most reptiles have sao 2 of 70% to 90%, with turtles at the higher end of the range and lizards at the lower end. 23 reptiles differ from birds and mammals in their higher reliance on anaerobic metabolism, which can make evaluation of the importance of oxygenation and perfusion more challenging. another feature that affects reptile oxygen dynamics is blood shunting. reptilian hearts (other than those of the crocodylia) have an incomplete interventricular septum. in these animals, both left-to-right shunting and right-to-left shunting can occur. with intracardiac left-to-right shunting, areas of the myocardium that are normally oxygen poor receive more oxygen. 24 reduction of right-to-left shunting of deoxygenated blood leads to an increase in oxygen carrying capacity. it is thought that alteration of the shunt fraction is controlled by changes in physiologic conditions. this change, along with a right-shift to the oxygen dissociation curve, maintains oxygen delivery in reptiles that have been studied. 25 studies in turtles have shown right-to-left shunting facilitates warming while left-to-right shunting improves systemic oxygen transport. left-to-right shunting is partly responsible for increasing myocardial oxygenation, which is needed for the tachycardic response to hemorrhage. 26 during episodes of decreased tissue oxygenation, or shock, cells try to maintain homeostasis by altering their metabolism to create atp anaerobically via glycolysis. it is thought that, in mammals, the stimulus for anaerobic glycogenolysis is not oxygen deficiency but stimulation of sarcolemmal na 1 /k 1 /atpase secondary to stress hormone release (ie, epinephrine). 27 stimulation of the sympathetic nervous system (sns) occurs through baroreceptor-mediated response to poor cardiac output and decreased tissue oxygen delivery. 28 the increase of sympathetic activity results in tachycardia, increased contractility, and vasoconstriction. fluid shifts from the interstitial and extravascular space to the intravascular space occur following the starling law, especially during episodes of diminished hydrostatic pressure. 28 activation of the rennin-angiotensin-aldosterone system (raas) also occurs because of reduced renal perfusion and also sympathetic system-induced vasoconstriction of the splanchnic vasculature. raas activation results in sodium and water retention, vasoconstriction, more upregulation of the sympathetic response, and release of antidiuretic hormone (adh). 29 the purpose of raas activation is to increase effective circulating volume, increase cardiac output, and shunt blood to the heart and brain. a summary of compensatory mechanisms is outlined in fig. 2 . the stimulation of the sns and release of raas and adh result in classic clinical signs of dull mentation, pallor, and tachycardia. during this stage, capillary refill time may be normal and blood pressure is preserved. 30 if perfusion, effective circulating volume, cardiac output, and/or oxygen delivery do not improve from compensatory mechanisms, additional symptoms of prolonged capillary refill, poor pulse quality, and hypotension occur. as poor perfusion persists, the eventual result of this phase is unresponsive hypotension. 31 this final phase of decompensatory shock results from hemodynamic collapse of the autoregulatory escape mechanism. as organs are starved of oxygen, fluid, and nutrients, arterioles dilate to increase flow. this process is a global phenomenon that leads to systemic hemodynamic collapse and death. 32 intervention (fluid therapy, oxygen supplementation, pressor drugs, transfusion, and so forth) does not reverse this reflexive mechanism. as previously defined, sirs is a state in which there is clinical and clinicopathologic evidence of systemic inflammation, characterized by tachycardia, tachypnea, hypothermia or hyperthermia, and leukocytosis or leukopenia. insults causing sirs, such as trauma, neoplasia, immune-mediated disease, can progress to cause shock, multiple organ dysfunction, and mof. 33 the underlying cause of sirs is the immune system's lack of balance between the activators of inflammation and inhibitors of inflammation. if the balance is tipped in either direction, organ damage occurs. injury to organs occurs from activated leukocytes' release of damaging cytoplasmic granules, microcirculatory ischemia from leukocyte obstruction, altered apoptosis, and production of reactive oxygen species. 34 infection causing sepsis is the classic example of sirs and results in the production of inflammatory mediators, called cytokines, such as tumor necrosis factor-alpha, interleukin-1, and interleukin-6. cytokines are produced by immunocytes, especially b lymphocytes, t lymphocytes, and macrophages. 35 there are more than 20 different cytokines and many more non-immunocyte-produced inflammatory proteins, such as platelet activating factor, interferons, and products of the arachidonic acid cascade. 36 these inflammatory mediators bind to receptors of endothelial cells, other immunocytes such as neutrophils, and basic shock physiology and critical care mast cells. effects of cytokine binding include alteration of vascular permeability, increased chemotaxis of immunocytes, and activation of the coagulation cascade. to limit this robust inflammatory response, many of the same cells that produce inflammatory cytokines also produce antiinflammatory molecules known as the compensatory antiinflammatory response syndrome (cars). antiinflammatory mediators include interleukin 10, transforming growth factor-beta, and interleukin-13. 37 sirs and cars are balanced to produce enough inflammation to respond to an insult, such as an infection or a neoplasm, whereby the inflammatory mediators attack the infectious agent/neoplastic cell and activate the complement cascade while sparing noninfected/nonneoplastic tissue. if the inflammatory response overwhelms the counteractive antiinflammatory response, sirs progresses, causing organ damage, cellular apoptosis, and a hypermetabolic state. in contrast, if the antiinflammatory response is too forceful, a syndrome of immunoparalysis develops, increasing the likelihood of dying of something like a simple infection. 38 pathogen recognition through receptor activation is a key factor in how the immune system responds to infection and targets the organism. receptors against pathogens are expressed by immunocytes and other cells, such as the endothelium. targets of pathogen recognition are called pathogen-associated molecular patterns (pamps). these signaling molecules are produced by microbial pathogens and are structures that give the pathogen the ability to survive and cause disease. the classic example of a pamp is lipopolysaccharide from gram-negative bacteria. pamps bind to many different receptors, the largest class being toll-like receptors, of which there are more than 10 described in the literature. 39 once bound to a pattern recognition receptor, internal signaling pathways result in a change of cellular metabolism, protein production, or other cell activation, such as chemotaxis. pamp production is influenced by genetic factors and aging. in dogs, one study noted that the cytokine response to pamps alters with age and is likely a contributing factor to morbidity in geriatric dogs with gram-negative sepsis. 40 danger-associated molecular patterns (damps) are similar to pamps. these molecules also can initiate and perpetuate the inflammatory response by binding to many of the same receptors of pamps found on leukocytes. the main difference is that damps are proteins and molecules from host cells, not the pathogen. damps constitute molecules from the plasma membrane, endoplasmic reticulum, nucleus, and cytosol from apoptotic cells and damaged cells. 41 for this reason, noninfectious sources of injury, such as trauma, pancreatitis, and heat stroke, can lead to sirs. another organ system heavily involved in the pathophysiology of sirs is the endothelial system. activation of endothelial cells from trauma, infection, or inflammation results in expression of receptors (p and l selectins, endothelial cell immunoglobulin adhesion molecules). 42 leukocytes, namely neutrophils, bind to expressed endothelial receptors, adhering to the wall of the vessel lumen. as this process continues with more neutrophils binding and rolling, microvascular flow can be disrupted, causing changes in capillary integrity and vascular tone. 43 in addition, cytokines cause changes in vascular tone, resulting in contraction of arteries but not venules. this process, mediated by tumor necrosis factor-alpha, interleukin-6, and other cytokines, modifies the blood supply to ischemic and inflamed tissues. 44 interstitial edema, especially that of the pulmonary parenchyma, occurs as the result of cytokines and inducible nitric oxide which is a potent vasodilator. several studies have found that acute lung inflammation, known as acute respiratory distress syndrome, is in part caused by activation of immunocytes, their production of cytokines, and the response by the pulmonary microvascular system. 45, 46 this type of edema, termed capillary leak syndrome, not only affects the lungs but also other organ systems, causing interstitial edema, which makes it more difficult to deliver oxygen from the capillaries to their target cells. as this process progresses, multiple organ system failure ensues. 47 the coagulation system is also altered during the sirs cascade. tissue factor expression by monocytes, and its expression from activated platelets and endothelial cell microparticles, results in thrombin formation. thrombin formation leads to additional thrombin activation and the final formation of cross-linked fibrin. 48 fibrin and activated platelets form thrombi in the microvasculature, disrupting blood flow and oxygen delivery. protection against thrombin formation is overwhelmed during states of sirs, exhausting stores of endogenous anticoagulants such as activated protein c, antithrombin, and protein s. as this process becomes more systemic, no longer isolated to the site of inflammation or infection, disseminated intravascular coagulopathy (dic) occurs. dic is an independent predictor of mortality and its diagnosis is often a poor prognostic sign. 49 an overview of some of the pathways of sirs, with resulting effects on target organs, is provided in fig. 3 . clinically, 2 common effects of poor perfusion and the subsequent sympathetic response are alteration of the gastrointestinal tract and the renal system. when there is significant hypovolemia, trauma, or sepsis with resultant sympathetic-induced vasoconstriction, the splanchnic vasculature is affected, 50 leading to ischemic injury and loss of the protective gastrointestinal barrier, which is primarily sustained because of normal capillary mucosal blood flow. once the barrier is compromised, commensal pathogenic bacteria have the ability to increase in number and translocate. primarily, translocation occurs via lymphatic vessels but can also occur through the blood supply via the portal system. 51 this process is one reason whereby patients who present for trauma or hypovolemic shock from an inflammatory, noninfectious process can become septic without being inoculated with bacteria. the main effect of poor perfusion on the renal system is a decrease in glomerular filtration rate (gfr). this phenomenon is now termed acute kidney injury, in contrast with the older terminology of acute renal failure. 52 however, traditional biochemical methods of estimating gfr using blood urea nitrogen and creatinine concentrations do not alert clinicians to the injury until 75% of functioning nephrons are no longer working. in humans, sepsis accounts for 50% of acute kidney injury and some degree of acute kidney injury is documented in up to 20% of persons hospitalized in intensive care units. 53 acute kidney injury has been described in birds with both viral and bacterial infections. 54 small mammals, mainly rodents, have served as models of acute kidney injury secondary to sepsis for some time. it is well recognized that sepsis and septic shock induced through cecal ligation result in renal injury. 55 although chronic renal failure is more commonly reported in reptiles, acute renal injury has been reported and is more likely to occur because of injected medications to the hind limbs. the reptile renal-portal system allows circulation from the hind limbs and tail to traverse directly to the kidneys. overall, there is a paucity of clinical data regarding shock and sirs in the clinical setting. much of the shock research on pocket pets, rabbits, and rodents are controlled experiments. rodents and rabbits have served as research animals in this field of study for many decades, and there are numerous reports inducing shock, sepsis, and sirs in rats, mice, and rabbits. [56] [57] [58] in rats, typical increases in heart rate basic shock physiology and critical care and sympathetic response occur during sepsis and shock, characterized by tachycardia and decreased mean arterial pressure. 59 rats also develop hypothermia associated with shock, similar to dogs, cats, and humans. 60 hamsters have been used to study sepsis and, similar to rats in shock, develop hypotension and leukocyte activation with vessel margination. 61 similarly, in a ferret model of sars -coronavirus, hyperthermia associated with viremic sepsis progressing to hypothermia associated with decompensatory shock was described. that same study also showed leukopenia in moribund ferrets on day 2 after infection. 62 one study of rabbits that underwent experimental hemorrhagic shock by removing 26% of blood volume noted tachycardia, hypotension, and significantly decreased cardiac output versus controls. 63 that same study, by chalmers and colleagues, 63 which compared normal rabbits with adrenalectomized rabbits, noted that the sympathetic response in normal animals minimized reductions in co and blood pressure. contrasting this research, clinical reports note the difficulty in resuscitating hypotensive rabbits and small mammals because of vagal stimulation, which becomes active simultaneously with sympathetic activity. 64 rabbits in an uncontrolled hemorrhage shock study required 123 to 133 ml/kg of crystalloid fluid in attempts to control hypotension. 65 the aforementioned dose is significantly higher than crystalloid shock dose recommendations for lagomorphs in the literature, which are 15 ml/kg or initial boluses of 3 to 5 ml/kg. 66 there is limited experimental research on hypovolemia and its effects on cardiovascular measurements in reptiles. reptiles have vascular adaptations allowing blood shunting, known as the renal-portal system. during periods of dehydration and prolonged hypovolemia, blood from their hind limbs and tail traverses to the afferent renal portal veins, preserving renal perfusion. 67 one study noted that, during acute hemorrhage, snakes were able to maintain their blood volume by restoring up to 90% of deficit within 2 hours of hemorrhage by shifting extravascular fluid to the intravascular space. 68 although snakes are able to restore their circulating volume after hemorrhage, their autonomic response is minor and does not allow compensation of hypotension. 69 because of their body configuration, environmental temperature effects on metabolism, and cardiovascular anatomy (3-chambered heart), snakes have highly variable blood pressure. depending on their habitat (arboreal vs terrestrial vs aquatic), systemic blood pressures in snakes correspond with gravitational stress. arboreal snakes have higher arterial pressures than aquatic snakes. blood pressure in these species also depends on body mass, with larger snakes having higher blood pressure. 70 turtles also have temperature and habitat influences on their cardiovascular system. turtles that underwent controlled hemorrhage had significantly increased heart rates and hypotension. in that same study, turtles were able to restore prehemorrhage blood volume 2 hours after hemorrhage, which is similar to snakes. 71 the ability of turtles to recover from hemorrhage is not highly dependent on vasoconstriction but depends on increased heart rate. baroreceptor response occurs in these species, resulting in tachycardia and increased contractility. catecholamine effects on their vasculature causes accelerated resorption of interstitial fluid versus increased pressure. 72 lizards, specifically the green iguana, have undergone similar controlled hemorrhage studies to determine cardiovascular responses. progressive hemorrhage resulted in tachycardia, decreased femoral blood flow, and hypotension. changes in body position affect arterial pressure, and passive, head-up tilting induce reflexive cardiovascular changes to regulate blood pressure. 73 hemorrhage and subsequent clot formation takes significantly longer in iguanas than in mammals. higher temperatures have been shown to improve blood clotting in iguanas. 74 much of the scientific literature discussing hypotension and shock in avian species uses poultry as models. one article compared fluid types for resuscitation in leghorn chickens. that study only used colloidal-type fluids (hetastarch) and a hemoglobinbased oxygen carrier (hboc) (hemospan) to resuscitate chickens that had undergone experimentally induced hemorrhage of 50% of their blood volume. surprisingly, there were no significant differences found between those two fluids and autotransfusion with respect to heart rate, respiratory rate, and systolic blood pressure. 75 in a similar study by lichtenberger and colleagues, 76 mallard ducks underwent controlled hemorrhage and fluid resuscitation with either crystalloid (plasmalyte a), colloid (hetastarch), or an hboc (oxyglobin). tachycardia was not noted until 25% to 45% of blood volume was lost. there was no statistical difference in mortality among the different types of fluid. however, oxyglobin is no longer commercially available. birds tolerate hypovolemia secondary to hemorrhage very well. clinical signs of anemia and shock are not seen until 50% to 60% of blood volume is lost. in pigeons, loss of 60% of blood volume resulted in no clinical signs and their hematocrits returned to normal within 7 days. the ability of birds to tolerate such large volumes of blood loss is thought to be caused by 3 adaptations. there is rapid extravascular fluid resorption from muscles that have a large capillary surface area, which then replenishes the intravascular deficit. in addition, birds have blunted autonomic response, resulting in fewer clinical signs of tachycardia, tachypnea, and hypothermia. the ability to mobilize large numbers of immature red blood cells is a third adaptation to hemorrhagic shock that birds possess. 77 this unique response to hemorrhage was also noted in a study that compared the hemodynamics of hemorrhage in chicks with that of rats. controlled hemorrhage caused significant reduction of mean arterial pressure and cardiac output in rats, by 23% and 43% respectively. in the chicks, mean arterial pressure was only reduced by 15% and cardiac output reduced by 4% because of their ability to maintain stroke volume. the investigators concluded that chicks were able to maintain mean arterial pressure independently of changes in peripheral vascular tone. 78 in conclusion, most of the literature discussing shock, systemic inflammation, and sepsis relates to experimental studies using small mammals such as rodents and rabbits. there is a paucity of research of these syndromes in other species such as ferrets, birds, and reptiles. although small mammals, experimentally, have a similar autonomic response to dogs during induced hemorrhage and shock states, that response is not noted in the clinical setting, in which decompensated shock is more commonly reported. poor perfusion affects every major organ system, but particular attention should be given to the immune system, coagulation system, cardiovascular system, gastrointestinal tract, and kidneys. knowing how these systems are affected helps to explain to clinicians why patients with shock can later become septic or coagulopathic. to ensure the best outcomes in patients who have shock or sirs, early recognition and intervention by provision of supplemental oxygen and fluid resuscitation is essential. treatment to combat shock in exotic species is discussed elsewhere in this issue. tissue oxygenation small animal critical care medicine inflammation and infection in the icu the natural history of the systemic inflammatory response syndrome: a prospective study principles of fluid therapy in sepsis & sirs in dogs & cats a comparative study of the morphology of the systemic anaphylactic reaction (sar) and the shock reaction induced by antigen-antibody complexes in rabbits morphology of haemorrhagic shock, systemic immunocomplex reaction and systemic anaphylactic reaction. a comparative study of critical care medicine consensus conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis historical perspective of the word "sepsis multiple organ dysfunction syndrome in humans and animals vasodilatation, oxygen delivery and oxygen consumption in rat hindlimb during systemic hypoxia: roles of nitric oxide vasodilating prostaglandin e1 does not reproduce interleukin-1b-induced oxygen metabolism abnormalities in rabbits circulation, respiration, and metabolism: current comparative approaches effects of temperature and activity on aerobic and anaerobic metabolism and heart rate in the turtles pseudemys scripta and terrapene ornate blood physiology and oxygen transport during activity in two lizards, varanus gouldii and sauromalus hispidus thermal behavior, heat exchange and metabolism in the desert snake spalerosophis cliffordi oxygen delivery and haemoglobin in: critical care physiology. little, brown and co the oxygen supply dependency phenomenon is associated with increased blood lactate levels current concepts on hemodynamic support and therapy in septic shock metabolic recovery from exhaustive activity by a large lizard summary of oxygen transport characteristics of reptilian blood. smithson the intracardiac shunt as a source of myocardial oxygen in the turtle, trachemys scripta effect of o2 affinity on arterial po2 in animals with central vascular shunts the intracardiac shunt as a source of myocardial oxygen in a turtle, trachemys scripta lactate and shock state: the metabolic view shock syndromes. in: dibartola, editor. fluid, electrolyte and acid-base disorders in small animal practice small animal critical care medicine understanding hypovolaemic, cardiogenic and septic shock clinical pathology of the shock syndromes hemorrhage and hypovolemia systemic inflammatory response syndrome s1otwi nski r. current views on the mechanisms of immune responses to trauma and infection cytokine-producing b lymphocytes -key regulators of immunity compensatory anti-inflammatory response syndrome the compensatory anti-inflammatory response syndrome (cars) in critically ill patients the immunopathology of sepsis: pathogen recognition, systemic inflammation, the compensatory anti-inflammatory response, and regulatory t cells age-associated changes to pathogenassociated molecular pattern-induced inflammatory mediator production in dogs emerging role of damage-associated molecular patterns derived from mitochondria in inflammation leukocyte-endothelial cell adhesion alert cell strategy: mechanisms of inflammatory response and organ protection human cytokines modulate arterial vascular tone via endothelial receptors cutting edge: hmg-1 as a mediator of acute lung inflammation phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice the current opinions of capillary leak syndrome the relationship between inflammation and the coagulation system disseminated intravascular coagulation is a frequent complication of systemic inflammatory response syndrome gastrointestinal mucosal injury in experimental models shock, trauma, and sepsis gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock available at: www.iris-kidney.com pathophysiology of septic acute kidney injury: what do we really know acute renal failure due to combined infection with psittacosis and salmonellosis effects of fluid resuscitation with 0.9% saline versus a balanced electrolyte solution on acute kidney injury in a rat model of sepsis a standardized method for the production of hemorrhagic shock in the rat traumatic shock in mice: comparison of survival rates following therapy the role of the reticulo-endothelial system in hemorrhagic shock changes in renal sympathetic nerve activity during experimental septic and endotoxin shock in conscious rats studies on the mechanism of shock. the effect of catecholamines on the temperature response to injury in the rat fluid resuscitation therapy in endotoxemic hamsters improves survival and attenuates capillary perfusion deficits and inflammatory responses by a mechanism related to nitric oxide the sars-cov ferret model in an infection-challenge study the effects of haemorrhage in the unanaesthetized rabbit monitoring and treatment of hypovolemic shock in small mammals rabbit model of uncontrolled hemorrhagic shock and hypotensive resuscitation emergency and critical care in exotic companion mammals the reptilian renal portal system -a review maintenance of blood volume in snakes: transcapillary shifts of extravascular fluids during acute hemorrhage control of arterial pressure in aquatic sea snakes anatomic and physiologic considerations for reptile anesthesia partitioning of body fluids and cardiovascular responses to circulatory hypovolaemia in the turtle, pseudemys scripta elegans baroreflex sensitivity in an amphibian, rana catesbeiana, and a reptilian, pseudemys scripta elegans regulation of arterial blood pressure in the common green iguana investigations on blood coagulation in the green iguana (iguana iguana) comparison of fluid types for resuscitation in acute hemorrhagic shock and evaluation of gastric luminal and transcutaneous pco2 in leghorn chickens comparison of fluid types for resuscitation after acute blood loss in mallard ducks (anas platyrhynchos) practical hematology and transfusion medicine in birds hemodynamics of hemorrhage in the conscious rat and chicken key: cord-318792-psw8bs17 authors: lee, jaewon; lee, sang-hoon title: lab on a chip for in situ diagnosis: from blood to point of care date: 2013-08-01 journal: biomed eng lett doi: 10.1007/s13534-013-0094-y sha: doc_id: 318792 cord_uid: psw8bs17 as the point of care diagnosis devices are becoming ever more popular, this paper suggest a miniaturized testing device from a drop of blood to diagnosis of disease for the global healthcare. the minimal requirements for the poc blood-testing device are blood microsampling, blood separation, immunoassay, and detection and communication of the signals. the microsampling of the blood can be achieved by specialized needle, which can be connected to the microchip or analytical devices. the sampled blood is then separated using either a filter (weir or pillar type), or by the phenomena unique to microfluidic system. the separated blood should then go through sandwich, homogeneous non-competitive, or competitive immunoassay, which can effectively diagnose diverse diseases. lastly, the device should detect and translate the immune-signals to readable, and clinically significant signals. the development of such device will play a great role for improving healthcare technology. despite recent remarkable progress in medical technology, the challenge of overcoming diverse critical diseases−including cancer, cardiovascular disease (cvd), and alzheimer's−still remains. even though the complete treatment of these diseases is difficult, early diagnosis might be one of the crucial clues for the patient survival and successful prognosis of the disease. to date, several techniques have been developed for the early detection of such diseases, but most of these techniques are lab-based, complex, slow, and also require experienced personnel to conduct the analysis. moreover, the patients need to visit medical service centers, such as hospitals, for the diagnosis. however, the contemporary generation simply does not have time to spare on regular medical checkups, which prevents the early detection of disease. to minimize the patients' inconvenience, the analysis of blood sample, saliva, urine and body fluid at home or office and the direct transfer of data through networks are highly required. therefore, there has been an ever-increasing need for novel, efficient, easy and personal diagnostic tools for the early detection of diseases, especially at the point-of-care (poc) [1, 2] . however, the technical limits (size, sensitivity, etc.) have hindered the realization of such poc devices. recent rapid progress in microfluidic technologies has played a pivotal role in handling and processing quite a small amount of liquid, and the integration of biosensors in the microfluidic chips has extended the application of 'lab on a chip (loc)' to biomedical areas [3] [4] [5] [6] [7] [8] . more importantly, size, compact integration of the components, low sample volume consumption, and fast analysis and response time due to short diffusion distances may enhance the portability of device as well as shortening the diagnosis time and enabling the integration of loc to electronic devices such as smartphone, and personal computers (pc). supported by loc technology, the poc device for the ubiquitous diagnosis could be achieved. in this mini-review, we suggest an overview of locbased diagnostic technology, which is currently being actively developed and researched, as well as being readily available for in situ diagnosis. to narrow the focus, we concentrated on the diagnosis from a small amount of blood. for this purpose, blood microscale sampling, blood separation, immunochemical assay, and detection (optical and electrical), and communication to personal device (such as smart phones or pc) are the essentially required processes (fig. 1) . especially, the data stored in the pc or smartphone can be transmitted to doctors and nurses for analysis and earliest possible diagnosis. here, we have described the technical status and trends about these four processes and considered a future prospects. the described loc technologies for in situ diagnosis can be extensively used even for diagnosis of the extremely contagious and infectious diseases such as severe acute respiratory syndrome (sars) and avian influenza. the early detection of such contagious diseases will greatly contribute to prevent the rapid spread throughout the global world. human blood is an important key to point of care diagnosis, as they are easily accessible, as well as containing a lot of important information about our bodies. therefore, a small sample of blood can reveal a lot about a patients' health status. the blood contains white blood cells (that can be counted for the immune-responses), red blood cells (anemia, sickle cell disease), and platelets (blood clotting activity), as well as plasma that contain the possible bacteria and virus that can affect the bodily functions. thus, blood sampling in a microscale amount, and loading the sample to diagnostic device is important. traditional sampling method is a simple collection of blood using the syringe. a rather large amount of blood was drawn using the needle of the syringe, and then discharged into the prepared test tubes containing the additives such as ethylenediaminetetraacetic acid (edta) and citrate. sometimes, the blood had to be collected multiple times if a patient needs to be tested for multiple diseases. this method of blood sampling had many shortcomings, as the whole process was time consuming as well as providing a lot of room for error. sampling of blood and body fluid using microfluidic technology has emerged in recent few decades, and such methods allowed for diagnosis with small amount of sample, and with more accuracy. li et al. have developed a system that can sample blood from a laboratory mouse in the ~ 1 l range by eliminating the large dead/ transfer volumes of conventional approaches (fig. 2) [9]. the system draws blood sample through the needle and into a biocompatible steel reservoir that could be interchanged for a microanalytical chip. in the microfluidic sampling system, the needle must at least penetrate through the stratum corneum layer of the skin into the dermis layer, which indicates the importance of the needle [10] . several blood or body fluid sampling systems that are simple, biocompatible and stable have been developed. s. aoyagi proposes a biodegradable polymer (poly lactic acid (pla)) needle with a trench for collecting blood [11] . because hollow needles are difficult to fabricate, etching a trench on the needle surface has been proposed as an alternative. e.v. mukerjee et al. have developed micro-needle array for the effective puncturing of human skin and extracting and delivering interstitial fluid (isf) to the microchannels [12] . besides, diverse micro-pumping and valve systems can be combined with microneedles to enable delivery and extraction of blood and body fluids of accurate amount. second step in on chip diagnosis using blood is separating the collected blood sample into cells including rbc and wbc, platelet and plasma. the cells can be separated from the blood based on their sizes, and weights. the most widely used methods of today are physical filtration, fluorescenceactivated cell sorting (facs), and magnetically actuated cell sorting (macs). conventionally, the physical membranebased filtration has been widely used because it is simple and relatively efficient. however, the high cellular fractions lead to membrane clogging and compromise the separation efficiency. the facs and macs are methods that sort cells with high precision. but the technical requirements for these methods are too expensive and complicated to be used for a point of care diagnosis, and the machines required for these are not mobile at all, being bulky and heavy. in addition, these macro-scale cell-sorting methods have their limitations in complicated sample preparation procedures, target cell loss, requirement of skilled technician and introduction of artifacts, which are highly undesirable for the point of care diagnosis. recent progress in microfluidic technologies has enabled the blood separation to overcome aforementioned problems. due to the microscale effect [13] , microfluidic chip has demonstrated many appealing characteristics compared to the larger scale blood separation systems. in addition, the small size and simple sampling procedure of microfluidic system reduce the introduction of the unwanted artifacts, as well as allowing for dramatic reduction of target-cell loss, which enables even the detection of wanted rare cells such as circulating tumor cells (ctcs) [14] . blood separation method using microfluidic chip can be categorized as follows: separation by size and weight, and by hydrodynamic mechanisms. the separation using filter or mesh is most common, and integration of such small filter in the microfluidic chip is one of popular methods in blood separation. in a microfluidic chip, diverse shapes of filters are used for blood separation, and among them, the comb, pillar, weir-type and microparticle based filters are popular ( fig. 3a and fig. 3b ). vandelinder and groisman devised weirs of gap size 0.5 m that can weed out the wbcs, rbcs, and platelets while letting the plasma pass through [15] . the device can operate continuously for an hour extracting up to 8% of plasma at the flow speed of 0.65 l/min and with less than 0.1% hemolysis. moorthy and beebe came up with a microporous filter within the microchannel using the emulsion photo polymerization to filter through the whole blood that can be utilized to extract the plasma from the sample [16] . crowley and pizziconi also utilized the microfilters of planar shape to separate the plasma from the whole blood. they demonstrated the design and operation of passive microfilter devices applicable to the separation of plasma from whole blood in miniaturized clinical diagnostic devices [17] . chen suggested pillar-and weir-type filtration microchips, and optimized cell concentration and length of separation channels to enhance the separation efficiency, and 95% rbc can be removed from the initial whole blood, while 27.4% wbc can be obtained [18] . yang et al. has suggested plasma separation by designing a simple microfluidic network based on the zweifach-fung effect (fig. 3c ). the effect states that at the branching points of capillaries, or microchannels, the particles will always enter the channel with higher flow-rate [19] . the microfluidic network was designed using an analogous electrical circuit and the experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% [20] . some researchers also developed a microfluidic device capable of separating wbcs from the whole blood without any pretreatment and lysing the separated wbcs in a continuous and near real-time fashion. choi et al. have developed isolation method of wbcs using a microfluidic device composed of slanted obstacles and filtration obstacles, and the device isolated wbcs with 210-fold enrichment within a short filtration time of ~ 0.3 s [21] . this proved that the traditional centrifuge-based blood separation method could be replaced by microfluidic technology. after blood separation, immunoassay should be performed, and perhaps this is the most important step in blood-based diagnosis. the term "immunoassay" refers to a quantitative measurement, or a detection that depends on recognizing an analyte through the use of antibodies or highly purified antigens. the significance of immunoassay lies in detection of very small amount of analyte in any fluid, as immunoassays are highly sensitive and specific. through immunoassay, not only the measurement of drugs, specific hormones, tumor markers, and many more analytes of extremely minute volume are possible, but also the qualitative detection of viral hepatitis, hiv and lyme disease is possible. the effectiveness of the immunoassays is maximized when done in micro-scale. the most distinguished aspects of the micro-scale immunoassays are its simplicity, efficiency, and the speed. first of all, the on-chip immunoassay allows for extreme simplification of the whole procedure. the simplicity comes from the integration of all immunoassay steps on one chip. the traditional immunoassay procedures require sampling of the whole blood, separation of the cells, and many other steps before the actual immunoassay takes place. also, data needs to be collected in a separate apparatus, and analyzed before the results can be presented. however, the on-chip immunoassay integrates all those preceding steps, detection, and analysis into one, which increases the mobility of it. this makes the micro-scale immunoassay highly desirable for poc, or single use assays that can be widely distributed to the public. the on-chip immunoassay allows the increase of precision by decreasing the volume of the analytes. the small size of it not only increases the mobility and precision of this diagnostic tool, but also allows multiple assays to be done in simultaneously: the micro-scale immunoassay widens the scope of the analysis [22] . lastly, the on-chip immunoassay is kinetically efficient. by reducing the distance the molecules need to travel, the assay can be done in a speedier manner without affecting its results [23] . although the on-chip micro-scale immunoassays possess these obvious advantages as opposed to the macro-scale immunoassays, they are still in the development stage. many studies are being actively conducted as we speak. here, three types of on-chip immunoassay methods will be discussed. immobilization (the "sandwich type" immunoassays) the first and the most common type of the immunoassays is the "sandwich type"−also known as, noncompetitive heterogeneous−immunoassay. this assay utilizes primary−or capture−antibody, a target antigen, and a labeled secondary antibody. this assay provides the highest sensitivity and specificity. the "immobilized" antibodies are coated onto a surface, then the antigens are bound to the immobilized antibodies, and the labeled secondary binds to the antigens, making a "sandwich" [24] . one method of immobilization is surface based immobilization. in 2011, beck and his coworkers devised an on-chip cluster of differentiation 4 (cd4) counting kit using the timed release of antibodies [25] . they first pre-coated the microfluidic chamber of 25m height with the antibodies and other reagents, and let the coating dry. then, the whole blood was sampled and incubated in the chamber for 10-30 minutes. finally, two images of the chamber−one with red excitation light, and the other with blue−were taken. the cd4, or the target cell was counted after digitally summing up the two images. the immobilization in this case, has been done on the hydrogel, and the activation was achieved through diffusion, making the timed release of the antibodies possible. while diffusion may take long in the case of non-microscalse chambers (as long as a month), the microchamber of miniscule height made the assay possible in this case. darain et al. also successfully immobilized primary antibodies onto the polystyrene (ps) surface based on the covalent bonding through a self-assembled monolayer of the thiol [26] . apart from surface based immobilization, the bead-based immobilization is also common. this allows for maximum surface area, thus improving the performance of the immunoassays. ko et al. developed a microfluidic electroimmunosensing chip for simultaneous determination of cancer biomarkers. they used the combination of antibodyconjugated ps beads for immobilization, gold nanoparticles for labeling, and silver enhancer for signal amplification. ps beads were coated with streptavidin for conjugation with biotinylated capture antibodies. these antibody-conjugated ps beads were trapped in the reaction area with the pdms pillars, then the sample fluid containing the cancer markers were introduced, followed by the gold nanoparticles, and the silver enhancers. the whole assay was done in 55 minutes [27] . one site-noncompetitive assay (microfluidic reactor) another type of immunoassay is one site-noncompetitive assay. unlike the "sandwich" type immunoassay, the antigens of interest each only bind to one labeled antibody. then, the unbound labeled antibodies are washed out, leaving the bound labeled antibodies for measurements and analysis. this type of immunoassays is usually done on electrophoresis-based microchips, and requires many concentration strategies to stack the target analytes for detection and measurements [28] . meagher and the coworkers developed a microfluidic apparatus with a photo-polymerized membrane and polyacrylamide gel-filled microchannels. the former is for pre-concentration and the latter for sample loading and separation of left over antibodies from the complexes. first, the sample was loaded onto the chip and went through the membrane to trap the preconcentration. then, the dyelabeled antibodies were loaded and were allowed to mix. after mixing and conjugating, the remaining antibodies and the complexes were separated in the channels. finally, measuring the ratio of unbound and bound immune-complex using sensitive laser-induced fluorescence detection provides quantitation of analyte in the sample [29] . while this assay was relatively simple, it required separation channels. reichmund et al. developed a microfluidic chipbased immunoassay without the separation channels. this apparatus only needed a chamber and a polyacrylamide membrane. whereas meagher et al. utilized the membrane only for pre-concentration, reichmund and the coworkers utilized it for both concentrating the viral particles, and separating the labeled complexes from the unbound antibodies. the assay was done in following steps: first, the sample was mixed with the fluorescently labeled antibodies. then, the antibody-virus complexes were concentrated on the membrane. the excess antibodies were removed by electrophoresis through the membrane, and the complex was then detected downstream. the total assay time was 6 minutes, while successfully detecting the inactivated influenza virus with less than 50m sample volume [30] . the last type of immunoassay technique is the competitive assay. there are two types of competitive immunoassay− homogeneous competitive assay, and heterogeneous competitive assay. in both cases, the labeled analytes compete with the unlabeled analytes in the sample to bind to the antibody. the homogeneous competitive assay measures the labeled unbound analytes, which would be proportional to the unlabeled analyte of interest, whereas the heterogeneous competitive assay measures the labeled bound analytes. lee et al. used the competitive immunoassay to measure the amount of hippuric acid (ha) in human urine. the system put ha and the ferrocene-hippuric acid complex (fc-lys-ha) in competition to bind to ha antibody coated onto polybeads which generated electric signals proportional to the ha concentration. the total assay only took 1 minute, which is much faster than the traditional electrochemical ha immunoassay (20 minutes) [31] . depending on the label (or label-free), several different detection methods have been developed. for detection, transducers play an important role in signal transduction of the immune-recognition event to electrical signals. the electrochemical (amperometry, potentiometry, conductimetry/ impedimetry), optical (colorimetric, fluorescence, luminescence, interferometry), calorimetric (thermistor), mass change (piezoelectric/acoustic wave) or magnetic events are the major immune-recognition events. however, detection with high sensitivity still remains a challenge. in addition, a detection strategy with highly effective signal transduction and multiplexed analysis is another important factor for high quality immune-sensor. the detection methods can be categorized into 3 groups: optical, electrical and mechanical detection. recent microtechnologies enable these 3 categorized detection methods with high accuracy and fast processing time. optical immune-sensing is the simplest and highly popular method among the immunoassay methods, and several labels (e.g., a fluorescent label, enzyme, or metallic particle) facilitate optical signal enhancement and increase detection sensitivity. recently, lots of optical microfluidic immune-sensing systems have been reported, and they have extensive potential applications in clinical diagnostics such as poc due to their versatility in functions. optical detection methods can be categorized into five types based on the detection signals: fluorescence, luminescence, absorbance (colorimetry), surface plasmon resonance (spr), and surface-enhanced raman scattering (sers). fluorescence detection is most popular method and suitable labels conjugated with the antibodies or antigens are excited by a laser or light-emitting diode light source (fig. 5 (left) ). the emitted light from the labeled molecules is detected by the photodetector. hu et al. utilized the aqueousphase-synthesized quantum dots (aqqds) as fluorescent markers to develop highly sensitive (femtomolar sensitivity) microfluidic platform for detecting the cancer biomarkers [32] . luminescence detection, on the other hand, measures the spontaneously generated luminescence intensity without the excitation light source because an enzymatic reaction between luminescent substrates and enzyme reagents causes photochemical emission. in 2007, battacharyya et al. developed a microfluidic chip for detection of c-reactive protein (crp) using chemiluminescence-based immunoassay. the assay results were presented on an on-chip instant photographic film, which was then read by an imager that can detect the chemiluminescent signals [33] . in colorimetry, colored stains resulting from the immunoassay is measured by a spectrophotometer. despite the poor sensitivity of colorimetry compared to that of other optical methods, the simplicity and potential for miniaturization of colorimetry provide several advantages. yu et al. developed a dextran-functionalized microfluidic immunosensor that can demonstrate colorimetric detection of several biomarkers (il-5, hepatitis b surface antigen, and immunoglobulin g) in sera through spectrophotometer or naked eyes. the dextran modification on the microfluidic channel surface enhanced the hydrophilicity and the efficiency in protein immobilization, distinguishing this particular platform from the other colorimetric platforms with a high sensitivity (detection limit of up to 100pg/ ml) [34] . spr is one of the useful biosensing systems to investigate biomolecular interactions and has diverse features including label-free, real-time, high-throughput, and sensitive analysis. spr is also the most common optical evanescent wave biosensor. this type of detection uses the variation of the reflectivity on a metallic layer, which when closely in contact with a dielectric media, reflects the concentration of bound target (fig. 5 (right) ). kurita et al. successfully detected a cardiac marker b-type natriuretic peptide (bnp) with the detection level of 5 pg/ml -100 ng/ml. this was achieved by monitoring the real-time spr angle shift while accumulating the thiol compound (generated by the enzymatic reaction during the immunoreaction) on a gold thin film on the microfluidic platform [35] . sers was first introduced in order to enhance the sensitivity of raman spectroscopy. the method incorporates metallic nanostructures or surfaces to improve the sensitivity from 10 6 to 10 14 . sers provides molecular information and its unique features are non-destructive, non-invasive, works in-situ and in-vitro for biological samples. in addition, sers can operate irrespective of temperature and pressure. the molecules adsorbed on rough metal surfaces enhance raman scattering and enhancement factors can be over 10 10 , which are sufficient to allow even single molecule detection. chon et al. used sers of hollow gold nanospheres on a microfluidic chip to successfully detect the target marker. the limit of detection on the specific target marker they used (rabbit immunoglobin, igg) was estimated to be 1 -10 ng/ml [36] . electrochemical immune-sensing is another commonly used analytical technique for the detection of biomarkers in a sample, followed by optical detection. this method of detection is highly sensitive as well as rapid and selective in determining the analytes, and they can be easily incorporated into the miniaturized poc devices. the electrochemical immunosensors detect the electric signals that arise from the immunoreactions that occur near the surface of the electrodes. the two main types of electrochemical reactions-redox, and non-redox−that can be detected by the immunosensors are discussed here. most redox-based detection relies on the electrochemical signals that the conjugated enzyme generates in enzymelabeled assays. commonly, the redox species such as prussian blue (pb) are added to monitor the electron transfer in the enzymatic reaction [37] . rossier and girault presented elisa with electrochemical detection on a microchip with the horseradish peroxidase-secondary antibody conjugate (hrp-conjugate) as the enzymatic mediator (to catalyze electron transfer reaction). they incorporated the electrode inside a 40 nl microchannel that can detect the redox active enzyme substrate directly in the microchannel. the apparatus detected 0.1 -100 nm of d-dimer with the sandwich immunoassay, which was the relevant concentration level for clinical settings [38] . meanwhile, enzymatic reactions that convert an electrochemically inactive substrate into active product can be another signal that can be electrochemically detected. jang et al. used the alkaline phosphate (ap) labeled secondary antibody to convert the electrochemically inactive substrate into active p-aminophenol, which was measured by the oxidation peak current during electro-oxidation into pquinoneimine (pqi) at the working electrode. using this method, jang and her coworkers were able to detect with the limit as low as 485 pg/ml with 95% accuracy [39] . the electrochemical signals can also be generated by the nonredox reactions. zhao et al. reported a novel non-redox based electrochemical immunoassay. they used the cadmium-sulfur (cds) quantum dots (qds) on the electrode surface, allowing for the photoelectrochemical sensing. the hrp conjugated with the antibody allowed for the precipitation on the cds-qd electrodes, which was directly proportional to the target concentration [40] . detecting for mechanical signals is another way of immunosensing. mechanical detection provides highly sensitive and multiplexed analysis in a short period of time. this method usually involves immobilized antibodies on a sensing surface, and the binding of the target antigens onto these antibodies, which causes physical disruption of the equilibrium state, and ultimately, a mass change. this change in mass can be detected by the use of microcantilevers (mcs) or acoustic wave sensors. these offer highly sensitive detection of the mass changes, and do not require labels, thus deeming suitable for the poc diagnostic devices. the use of microcantilevers in immunosensors provides sensitive and label-free detection. the deflection mode of detection is based on the change in the mechanical deflection of the mc that occurs as a result of antigen binding. in 2005, backmann et al. reported a mcbased immunosensor that operates on a deflection mode with a sensitivity level comparable to the spr. using the single chain fv antibody fragments as the receptor molecules, backmann and the coworkers were able to attain the sensitivity of ~1 nm [41] . mcs working in the resonance mode are generally used to measure the change in resonance frequency resulting from the target binding. these are generally called the "piezoelectric" microcantilever (pemc). hwang et al. utilized the pemc in a microfluidic immunosensor. the device allowed for dynamic detection and measurement of prostate-specific antigen (psa) without using the labels [42] . for the one-step assay from small amount of blood sample to in-situ analysis, four procedures including blood microscale sampling, blood separation, immunochemical assay and detection should be performed on a single platform. in this review, we overviewed the microfluidic technologies that are required per procedure and are currently being actively developed and researched. the developed microfluidic technologies surpass the limit of conventional immunosensing systems by enabling the reduced consumption of reagents and samples, and reducing the assay time, cost, power consumption, and size. in addition, the versatile technologies facilitate the sensitive multiplexed immunosensing and increased realistic clinical applications for poc diagnostics. we expect that the microfluidic immunosensing system on a single microfluidic platform is relevant for use by primary care physicians and outpatient clinics even in developed countries, because in-situ diagnosis can lead to higher rates of correct treatment and lower rates of unnecessary overtreatment [43] . furthermore, in developing countries or on-going battlefields, the proposed microfluidic immunosensing system will play a great role to save lives and prevent the disease breakouts, such as highly infectious bird flu. microfluidics closes in on point-of-care assays returning to diagnostic basics a biomems review: mems technology for physiologically integrated devices applications of micromixing technology microfluidic platforms for the study of cancer metastasis regulating microenvironmental stimuli for stem cells and cancer cells using microsystems from micro-to nanofabrication with soft materials soft lithography in biology and biochemistry a blood sampling microsystem for pharmacokinetic applications: design, fabrication, and initial results quantitative analysis of applied force on biopsy needle 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flow-through functionalized pdms microfluidic channels with dextran derivative for elisas on-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system on-chip immunoassay using surface-enhanced raman scattering of hollow gold nanospheres prussian blue modified amperometric fia biosensor: one-step immunoassay forfetoprotein enzyme linked immunosorbent assay on a microchip with electrochemical detection in situ electrochemical enzyme immunoassay on a microchip with surface-functionalized poly (dimethylsiloxane) channel highly sensitive photoelectrochemical immunoassay with enhanced amplification using horseradish peroxidase induced biocatalytic precipitation on a cds quantum dots multilayer electrode a label-free immunosensor array using single-chain antibody fragments in-situ quantitative analysis of a prostate-specific antigen (psa) using a nanomechanical pzt cantilever point of care diagnostics: status and future acknowledgement this study was supported by the grant of public welfare and safety research program through the national research foundation of korea (nrf), funded by the ministry of education, science, and technology (20120006510). lee j declares that s/he has no conflict of interest in relation to the work in this article. lee sh declares that s/he has no conflict of interest in relation to the work in this article. key: cord-347890-kx5vka0o authors: fan, qian; zhang, wei; li, bo; li, de-jia; zhang, jian; zhao, fang title: association between abo blood group system and covid-19 susceptibility in wuhan date: 2020-07-21 journal: front cell infect microbiol doi: 10.3389/fcimb.2020.00404 sha: doc_id: 347890 cord_uid: kx5vka0o background: the abo blood group system has been associated with multiple infectious diseases, including hepatitis b, dengue haemorrhagic fever and so on. coronavirus disease 2019 (covid-19) is a new respiratory infectious disease and the relationship between covid-19 and abo blood group system needs to be explored urgently. methods: a hospital-based case-control study was conducted at zhongnan hospital of wuhan university from 1 january 2020 to 5 march 2020. a total of 105 covid-19 cases and 103 controls were included. the blood group frequency was tested with the chi-square statistic, and odds ratios (ors) with 95% confidence intervals (cis) were calculated between cases and controls. in addition, according to gender, the studied population was divided into two subgroups, and we assessed the association between cases and controls by gender. finally, considering lymphopenia as a feature of covid-19, the relationship between the abo blood group and the lymphocyte count was determined in case samples. results: the frequencies of blood types a, b, ab, and o were 42.8, 26.7, 8.57, and 21.9%, respectively, in the case group. association analysis between the abo blood group and covid-19 indicated that there was a statistically significant difference for blood type a (p = 0.04, or = 1.33, 95% ci = 1.02–1.73) but not for blood types b, ab or o (p = 0.48, or = 0.90, 95% ci = 0.66–1.23; p = 0.61, or = 0.88, 95% ci = 0.53–1.46; and p = 0.23, or = 0.82, 95% ci = 0.58–1.15, respectively). an analysis stratified by gender revealed that the association was highly significant between blood type a in the female subgroup (p = 0.02, or = 1.56, 95% ci = 1.08–2.27) but not in the male subgroup (p = 0.51, or = 1.14, 95% ci = 0.78–1.67). the average level of lymphocyte count was the lowest with blood type a in patients, however, compared with other blood types, there was still no significant statistical difference. conclusions: our findings provide epidemiological evidence that females with blood type a are susceptible to covid-19. however, these research results need to be validated in future studies. coronavirus disease 2019 , also named novel coronavirus pneumonia (ncp), was first reported in wuhan in december 2019 and then gradually spread throughout the country. by early march 2020, more than 80,000 people were infected, nearly 3,200 of whom died in china. the pneumonia outbreak has become a serious public health event. covid-19 is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2), which is a new member of the coronavirus family. there are currently 7 known coronaviruses that can infect humans, such as severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome (mers) coronavirus. based on current epidemiological investigations, the incubation period is 1-14 days and typically 3-7 days, but there are also cases in which an incubation period of over 14 days is reported wu and mcgoogan, 2020) . individuals are contagious during the incubation period, and asymptomatic infection may also become the source of infection. respiratory droplets and close contact are the major transmission routes. covid-19 is clinically characterized by fever, fatigue, and dry cough. in severe cases, affected individuals can undergo acute respiratory distress syndrome, septic shock, and even death (chan et al., 2020; huang et al., 2020) . the abo blood group is the most important blood group system in humans and includes 4 blood types, namely, a, ab, b, and o. the human abo blood group is located on chromosome 9 (9q34.2) (melzer et al., 2008; wiggins et al., 2009 ). many studies have found that the abo blood group plays an important role in various human diseases, such as cardiovascular, oncological, and some infectious and non-infectious diseases (wolpin et al., 2010; chen et al., 2016) . meanwhile, the system can play a direct role in infection by serving as receptors or coreceptors for microorganisms, parasites, and viruses. blood group antigens, also named human histo-blood group antigens (hbgas), are one of the main antigens on the surface of human red blood cells. they represent polymorphic traits inherited among individuals and populations. differences in blood group antigen expression can increase or decrease host susceptibility to many infections. in addition, many blood group antigens facilitate intracellular uptake, signal transduction, or adhesion through the organization of membrane microdomains and modify the innate immune response to infection (behal et al., 2010; singh et al., 2016; chakrani et al., 2018; liu et al., 2018) . the abo blood group has been previously found to contribute to the risk of multiple infectious diseases in a series of studies. mohammadali et al. reported that the presence of blood group o might significantly decrease the risk of hepatitis b, and the distribution of rh in hbv-infected individuals was higher between rh-positive donors (mohammadali and pourfathollah, 2014) . elnady et al. found that rota-positive status for rotavirus gastroenteritis was significantly more prevalent among those with blood type a and significantly less prevalent among those with blood type b (elnady et al., 2017) . another recent study carried out by degarege et al. reported that malaria patients with blood group a had a higher risk of anemia than did those with o and non-a phenotypes (degarege et al., 2012) . among patients infected with dengue virus, murugananthan et al. found that patients with ab blood had a risk that was more than 2.5 times higher of developing dengue haemorrhagic fever than did those with other blood types (murugananthan et al., 2018) . in addition, a meta-analysis suggested that blood types a, b, and ab might not affect susceptibility to norovirus infection. however, those with blood type o appeared to be more susceptible to this infection (liao et al., 2020) . because sars-cov-2 is a completely new virus, it is unclear whether the abo blood groups affect individuals' susceptibility to covid-19. hence, we performed a case-control study to explore the relationship between the abo blood group and covid-19 in wuhan and further classified the populations according to gender. additionally, lymphopenia is a common feature of patients with covid-19 and might be a critical factor associated with the severity and mortality of the disease (xu z. et al., 2020) . the association between abo blood type and the count of lymphocyte was also investigated in cases. a retrospective case-control association study was performed during the period from 1 january 2020 to 5 march 2020, with a total of 208 subjects (105 cases vs. 103 controls). all subjects were enrolled from zhongnan hospital of wuhan university, which is a hospital designated for the treatment of patients with covid-19. all study individuals were subjected to demographics, clinical features, laboratory findings, reports, and chest ct scans. demographics included age, gender, hypertension, diabetes, and heart disease. clinical features involved disease manifestations such as fever, cough, dyspnoea, chest tightness, and diarrhea. laboratory findings included white blood cell count, lymphocyte count, neutrophil ratio, lymphocyte ratio, blood type, and throat swab nucleic acid test results. all information was obtained and analyzed with the standard excel program. two doctors independently extracted the data of the eligible individuals, and the results were reviewed by a third investigator. this study was reviewed and approved by the medical ethical committee of zhongnan hospital of wuhan university. oral consent was obtained from patients. the criterion for enrolment as a case was defined according to the diagnosis and treatment scheme for new coronavirus pneumonia (trial version 5, trial version 6) issued by the general office of national health commission of the people's republic of china and the office of state administration of traditional chinese medicine. covid-19 cases were diagnosed as "clinically diagnosed cases" or "confirmed cases" according to the above criteria. the specific diagnostic criteria for clinically diagnosed cases are as follows: (a) history of epidemiology: i travel history or residence history in wuhan and surrounding areas within 14 days prior to onset of the disease, or other cases reported in the community; ii contact with patients from wuhan and surrounding areas, or with fever or respiratory symptoms from the community prior to the onset of the disease, within 14 days prior to onset of the disease; iii cluster disease; and iv. contact with a new type of coronavirus infection; (b) clinical manifestations: i fever and/or respiratory symptoms; ii imaging features of the above pneumonia; and iii normal or decreased total white blood cell count or decreased lymphocyte count at the early stage of onset; and (c) comprehensive evaluation by three covid-19 consultation experts in the hospital. the specific diagnostic criterion for confirmed cases is as follows: covid-19 nuclear acid test positive for viral nucleic acid by reverse transcription polymerase chain reaction real-time (rt-pcr) detection with specimens from the respiratory tract or blood samples. the eligible control subjects were selected from individuals with the following characteristics: (1) gender-and age-matched; (2) no other history of respiratory infections, such as bacterial pneumonia, mycoplasma pneumonia, tuberculosis and other types of pneumonia; (3) no other infectious diseases, such as hepatitis b and aids; and (4) no severe liver and kidney dysfunction. the association between different blood types and covid-19 was performed in the selected population. according to gender, subgroups were stratified to assess whether there was a significant difference between blood type and the incidence of covid-19. in addition, because lymphocyte decline was related to the severity of covid-19, we performed a correlation analysis between blood group and lymphocyte count in the covid-19 patients . statistical analysis was carried out using the statistical package for social sciences (spss) version 21.0. independent sample ttests were used for age, white blood cell count, lymphocyte count, neutrophil ratio, and lymphocyte ratio. a chi-square test was used for hypertension, diabetes, heart disease, tumor, liver disease, and kidney disease. the abo blood group frequency in all populations and different gender subgroups was tested using chi-square tests and odds ratios (ors) with 95% confidence intervals (cis). analysis of the association between the abo blood group and the lymphocyte count was performed with analysis of variance (anova) and a linear regression model. a p < 0.05 was considered significant. 14.4 ± 10.5 27.6 ± 9.37 < 0.001 the data are presented as the mean ± standard deviation or a percentage. as shown in table 2 , we performed a combined association analysis between abo blood group and covid-19, which showed a statistically significant difference in covid-19 infection among those with blood type a (p = 0.04, or = 1.33, 95% ci = 1.02-1.73) but not blood types b, ab or o (p = 0.48, or = 0.90, 95% ci = 0.66-1.23; p=0.61, or = 0.88, 95% ci = 0.53-1.46; and p = 0.23, or = 0.82, 95% ci = 0.58-1.15, respectively). an additional statistical analysis was performed by dividing the entire population into two subgroups by gender, as shown in table 3 . the male group comprises 111 subjects, and the female group includes 97 individuals. the association analysis revealed a significant relation between blood type a and covid-19 in the female subgroup (p = 0.02, or = 1.56, 95% ci = 1.08-2.27) but not in the male subgroup (p = 0.51, or = 1.14, 95% ci = 0.78-1.67). in addition, blood types b, ab, and o were not significantly associated in either male or female subgroups (p > 0.05). as illustrated in table 4 and figure 1 , the average lymphocyte count levels of individuals with blood type a were lower than those of individuals with blood types b, ab, and o in the case group (0.76 * 10 9 /l, 0.85 * 10 9 /l, 0.83 * 10 9 /l and 0.85 * 10 9 /l, respectively). unfortunately, statistical analysis showed that blood type a was not significantly associated with lymphocyte count levels in case subjects (p = 0.83, f = 0.30). of the human blood group systems, the abo blood group is widely used in clinical practice. as some of the important antigens, hbgas are complex carbohydrate molecules with specific oligosaccharide sequences expressed on the surface of red blood cell membranes. these antigens are also highly expressed on a large number of human cells and tissues, including epithelia, platelets, vascular endothelia and neurons (storry and olsson, 2009; liumbruno and franchini, 2013; heggelund et al., 2017; kazi et al., 2017) . hbgas have been postulated to modify the spread of pathogens through the action of natural antibodies and complements (neil et al., 2005; ewald and sumner, 2018) . abo antibodies are part of the innate immune system against some parasites, bacteria and enveloped viruses, and hbgas are important as receptors for immune and inflammatory responses (cooling, 2015; jing et al., 2020) . meanwhile, this system is often used as a genetic marker in the human genome, generated by a polymorphic glycosyl-transferase encoded by 2 dominant active and a recessive inactive alleles. the association between abo blood groups and infectious and non-infectious diseases has been widely explored (groot et al., 2020 ). in the current study, we aimed to evaluate the contribution of the abo blood group to covid-19 susceptibility in wuhan by employing a case-control association analysis. our present results demonstrated that there was a significant association between the a blood group and covid-19, such that females (but not males) with blood type a were more susceptible to covid-19 infection. compared with other patients, female patients with blood type a had a relative risk of 1.33 for coronavirus infection. xiong et al. recently also found that women show different characteristics from men in the transmission of covid-19 (xiong et al., 2020) . we speculate that this result may be related to the different anatomic structures, estrogen levels, immune systems and genetic backgrounds of men and women. we further investigated the possible association between abo blood group and lymphocyte count, the latter was considered as one of the index to evaluate the severity of covid-19. although statistical analysis showed no significant difference in abo blood group and lymphocyte counts, our study found that the decreased lymphocyte counts in patients with blood type a were lower than those in patients with other blood types. the possible explanation for this finding may be related to the small sample size. in fact, a number of epidemiological studies had also been conducted. for instance, the study of li et al. reported that the proportion of blood type a in patients infected with sars-cov-2 was significantly higher than that in healthy controls (0.38 vs. 0.32%, p < 0.001), while the proportion of blood type o in sars-cov-2 infected patients was significantly lower than in healthy controls (0.26 vs. 0.34%, p < 0.001) . in another study, zhao et al. also showed that blood type a was associated with an increased risk of sars-cov-2 infection, whereas blood type o was associated with a decreased risk (gerard et al., 2020; zhao et al., 2020) . the main finding of our study was consistent with the above analysis by li et al. and zhao et al., but slightly different. in our cases, the relationship between abo blood type and the count of lymphocyte was further investigated, due to the importance of lymphocyte count in the evaluation of severity of covid-19. as with covid-19, sars is also a serious respiratory infectious disease. nevertheless, abo blood group-associated susceptibility to sars is different from the corresponding susceptibility to covid-19. in 2005, cheng et al. found that individuals with blood type o had a reduced susceptibility to sars infection in the hong kong population. variable binding affinity to differing abh substances present in gut epithelial cells may be the cause of the above phenomenon (cheng et al., 2005) . sars-cov-2 belongs to lineage b betacoronavirus and shares high sequence identity with that of bat or human severe acute respiratory syndrome coronavirus-related coronavirus (sarsr-cov) (tian et al., 2020) . the structural analysis of sars-cov-2 contains two important viral proteins, the nucleocapsid and the spike (s) proteins. s proteins of coronaviruses are large transmembrane heavily n-glycosylated proteins that mediate association with a cell surface receptor. sars-cov-2 makes use of the s protein to gain entry into the host (li et al., 2006; wrapp et al., 2020) . angiotensin-converting enzyme 2 (ace2) is the main host cell receptor of sars-cov-2 and plays a crucial role in the entry of the virus into the cell to cause the final infection (cao et al., 2020; wu, 2020; xu h. et al., 2020) . the relationship between natural antibodies of the abo blood system and the ace2 interaction has been experimentally investigated. in 2008, guillon et al. observed that s protein/ace2-dependent adhesion of special chinese hamster ovary cells to an ace2-expressing cell line could be specifically inhibited by either monoclonal or human natural anti-a antibodies. their findings indicated that anti-a antibodies may block the interaction between the sars coronavirus and its receptor-ace2, thereby providing protection (guillon et al., 2008) . this is consistent with our findings, suggesting that those with blood type a may be more susceptible to viral infection. meanwhile, several drawbacks existed in our study. first, due to the limited sample size of covid-19 in the early stages, the sample size included in our research is not very large. second, regional selection bias needs to be considered. third, other potential diseases may affect the research results. finally, some of the control individuals might develop covid-19 in the future. in conclusion, female patients with blood type a are susceptible to covid-19 in wuhan after gender stratification. however, more studies are necessary to confirm these findings in a larger sample and among individuals of different ethnicities. the underlying mechanism between the abo blood groups and ace2 needs to be further explored. all datasets generated for this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by medical ethics committee, zhongnan hospital of wuhan university. the patients/participants provided their written informed consent to participate in this study. written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. fz and qf had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. qf, d-jl, and jz performed statistical analysis. all authors contributed to the article and approved the submitted version. this 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coronavirus-infected pneumonia in wuhan, china abo genotype and risk of thrombotic events and hemorrhagic stroke pancreatic cancer risk and abo blood group alleles: results from the pancreatic cancer cohort consortium cryo-em structure of the 2019-ncov spike in the prefusion conformation compensation of ace2 function for possible clinical management of 2019-ncov-induced acute lung injury characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention women may play a more important role in the transmission of the corona virus disease (covid-19) than men high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa pathological findings of covid-19 associated with acute respiratory distress syndrome relationship between the abo blood group and the covid-19 susceptibility. medrxiv the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 fan, zhang, li, li, zhang and zhao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-354640-5p79qpok authors: taylor, kirk a.; machlus, kellie r. title: blood and bone: the quarantine chronicles date: 2020-06-28 journal: res pract thromb haemost doi: 10.1002/rth2.12394 sha: doc_id: 354640 cord_uid: 5p79qpok in the midst of the chaos of the global pandemic, the online daily webinar series blood and bone was created. the series started with a blank schedule on a google doc and, with enthusiasm and participation from the hematopoiesis and hemostasis/thrombosis communities, was quickly filled through september 2020. in the absence of any editing of the speaker list, a diverse, well‐balanced, and scientifically exciting program emerged. the seminar is hosted on zoom and live‐streamed on youtube daily, and can accommodate up to 1000 attendees. attendance has topped over 600 and averages 200 to 300 people daily; this has been sustained for 10 weeks. in addition, there is a weekly thursday trainee series that hosts three 20‐minute seminars. in this forum, we reflect on a series that allowed global scientists to come together to help shape chaos into an opportunity for community and growth. recognizing chaos is a much different skill than embracing it. in the lab, i regularly look into a microscope and marvel at the beauty of megakaryocytes extending long, delicate proplatelets, a process that makes platelets, cells essential for life. platelet birth and megakaryocyte death, whose mysterious patterns invite us toward them as something waiting to be unraveled. as scientists, if nothing else, we are drawn to the idea that we can turn chaos into, well, less chaos. but i dare you to tell that to a precocious 5-going-on-15-year-old, an aging and very territorial dog, and a rambunctious puppy who have been confined to share the same limited space for the foreseeable future. every morning we wake up to the macroscopic chaos of sickness, schools being closed, conferences being canceled, and trips being postponed indefinitely. we were all struggling to rearrange our lives and try to figure out what this new normal means as parents, students, teachers, and scientists. having been separated from my peers, my lab, and my profession, the cancellation of a much-anticipated keystone meeting was a particularly hard blow for me. the literal isolation of quarantine destroys more than routine-it threatens the progress that is gained only through solidarity and symbiosis. one evening in mid-march, over a glass of wine, i thought about my friends and colleagues around the world who were in the exact same situation. those meticulously prepared talks that were waiting to be shared with peers; those talks that no longer had an audience. the blood and bone seminar was my small attempt to refute stagnation and isolation. i had no grandiose ideas at the time, but rather was looking for a way to offer new content to my lab members and perhaps connect with a few friends. i began by reaching out to about 50 friends and colleagues and explaining my idea. there was something cathartic about this simple act of checking in and asking for help. i created a blank google doc and invited people to sign up for time slots to give talks. then i went to bed, expecting that the next day would be entirely devoted to pleading with friends to sign up for enough slots to sustain the series for a few weeks. as is the true nature of entropy, when i awoke, i was stunned to see that same google doc full through may. by the end of the next day, the blood and bone seminar was fully booked until september 2020, over 160 sign-ups. the skill set of a lab scientist does not exactly prepare one to become a virtual scientist. between organizing, planning, advertising, moving a children's ikea desk into my spare bedroom, and hanging an old pink blanket up as a shabby but functional backdrop, the first few days were completely overwhelming. i wasted no time in consulting dr taylor, a friend and colleague who i knew was skilled in public outreach. i felt lucky to have dr taylor as a team member, helping me to disseminate information and brainstorm. with our first seminar topping out at over 500 attendees, we had no choice but to rapidly acclimate to zoom and youtube and adapt to their many issues as they arose. using this technology of live information sharing and streaming talks via zoom and youtube meant that we were able to reach an even wider audience than a traditional conference; this came with its own particular set of advantages and drawbacks. the challenge of scheduling across multiple, if not all, time zones was incredibly stressful and ultimately proved impossible. therefore, we implemented supplemental social hours to accommodate our friends in australia and asia. on the other hand, the amount of co 2 emissions spared by reducing conference travel was notable and sets an important precedent for our responsibility, as scientists, to the planet. "reading" a virtual room can be challenging, but not having audience videos meant that the system was capable of handling large viewing figures (up to 1000), and most talks attracted more attendees and engagement than a traditional conference presentation. although the level of interaction will never compare to an in-person conference, the undoubtable pros such as accessibility, cost, and the like made this online seminar, and future ones like it, an important resource and something we should consider incorporating more as a complement to physical meetings. quite differently from traditional conferences, those who signed up were automatically given time to speak; we did not make any cuts or assign any type of speaker hierarchy. with a self-selecting speaker list, the topics have varied wildly from one day to the next. some chose to stick to their specific topics (eg, coagulation, hematopoiesis, platelet disorders, or myeloproliferative neoplasms) while other researchers looked more broadly at the research on offer and thought laterally about their skill set. this was also reflected in the structure and content of people's talks, where complex and fascinating science was presented in accessible and creative formats to reach a broad audience. a particularly fun example from dr shavit was a detour about how zebrafish mutants are named, during which we found out through 2017, and found the proportion of female speakers to be approximately 34%, 2 suggesting that blood and bone surpassed the reported average. a follow-up study in 2020 presented evidence that participation of women in conferences is influenced by the proportion of women on planning committees; committee composition was statistically significantly associated with speaker composition. 3 therefore, it is interesting to speculate that the encouraging number of women participants may have been influenced by the fact that the series was organized by a young, female faculty member. the blood and bone seminars not only featured a promising balance regarding the sex of the participants but also in the quality of talks and diversity of topics. while this is encouraging, further work in the community is needed to increase the representation of our black, asian, minority ethnic, lesbian, gay, bisexual, transgender, queer, and related communities (lgbtq+), and disabled colleagues. for example, our lgbtq+ colleagues may face additional barriers (eg, cultural and structural) to being "out" at work. 4 this highlights why strong allyship is required to create open platforms where people can not only present their science but also bring their whole selves to work. likewise, in addition to structural racism, geographic diversity is always a challenge. figure 1 shows the current geographic locations represented by all of the speakers. given the challenges of different time zones, it is perhaps unsurprising that we have such a high representation from the united states and europe and none from asia. however, the lack of participants from south america and africa is well noted and cannot be explained simply as a result of scheduling. the causes that disenfranchise particular geographic communities is something that must be addressed in the future. apart from the practicalities of time zones, to increase diversity of both speakers and attendees, conferences must be safe spaces that confront discrimination. an example of this obstacle can easily be seen in zoom security, which was an issue in the early days and continues to be a challenge. the harassment received from trolls who would log into seminars and comment about the appearance of female speakers or say lewd things via the private chat made some seminar participants feel uncomfortable in what was supposed to be a safe space. this highlights the importance of working to create science spaces that are safe and inviting for everyone. we know that a more diverse science community results in more and better information. besides shifting attendee statistics through approaching specific people and groups, we envision conferences in the future where there is a designated space for people to report discrimination and/or discomfort. conferences would also benefit from being able to provide closed captions for audiences who have hearing and/or vision disabilities. throughout the seminar, we reached out to our community to get feedback. presenters reported receiving congratulatory messages and collaboration requests from both academic and industrial partners, which may not have occurred outside of this series. the most common feedback on format related to how strange it was to not be able to see the audience and associated visual cues while presenting. according to speakers, they still experienced levels of anxiety, like stage fright, when putting together the talks and adapting to the virtual presentation format, but this adrenaline was also said to be a motivating factor. speakers also described the process of putting talks together and presenting as an island of normality within the sea of anxiety and chaos. people reported that even if they were having an "unproductive" day, the seminar meant they would get in at least 1 hour of science and the associated sense of achievement. consistent among participant feedback was also the sense of community and routine as being essential for mental well-being and was even described as an act of self-care. it is miraculous, but not surprising, that global scientists coming together could shape chaos into an opportunity for community and growth. dr machlus may have had the idea and sent that first email, but it was the hematopoiesis and hemostasis/thrombosis communities that brought the blood and bone seminar to life. we have learned through this experience that global cooperation is not just absolutely possible, but it is what so many of us crave. although we look forward to the day we finally get to see everyone face to face, we will do so with an entirely new appreciation of our combined capabilities to both shape and be shaped by the future. blood and bone website: https://bloodandboneseminar.com link to blood and bone google doc: https://docs.google.com/ sprea dshee ts/d/1qu_e94d5 stscx b-0gpn7 9r4nr e9qvo 3vzix id8rr zag/edit#gid=0 blood and bone slack: https://join.slack.com/t/blood bonew ebina r/shared_invit e/zt-evoce u8h-v1e5i 4woe3 ak4ex 1ymojha blood and bone youtube: https://www.youtu be.com/chann el/ uc7pj wzylh aaadn msacm lpsg we acknowledge and thank shaina joy machlus for her help in the conceptualization and editing of this manuscript. we thank dr kirill butov for his help in creating the figure. the authors (krm and kat) declare that they have no conflicts of interest. #womeninmedicine: progress in gender equity at research and practice in thrombosis and haemostasis trends in the proportion of female speakers at medical conferences in the united states and in canada association between the proportion of women on a conference planning committee and the proportion of women speakers at medical conferences navigating lgbtq+ discrimination in academia: where do we go from here? the biochemist blood and bone: the quarantine chronicles key: cord-331289-02411gfv authors: di minno, giovanni; perno, carlo federico; tiede, andreas; navarro, david; canaro, mariana; güertler, lutz; ironside, james w. title: current concepts in the prevention of pathogen transmission via blood/plasma-derived products for bleeding disorders() date: 2015-07-20 journal: blood rev doi: 10.1016/j.blre.2015.07.004 sha: doc_id: 331289 cord_uid: 02411gfv the pathogen safety of blood/plasma-derived products has historically been a subject of significant concern to the medical community. measures such as donor selection and blood screening have contributed to increase the safety of these products, but pathogen transmission does still occur. reasons for this include lack of sensitivity/specificity of current screening methods, lack of reliable screening tests for some pathogens (e.g. prions) and the fact that many potentially harmful infectious agents are not routinely screened for. methods for the purification/inactivation of blood/plasma-derived products have been developed in order to further reduce the residual risk, but low concentrations of pathogens do not necessarily imply a low level of risk for the patient and so the overall challenge of minimising risk remains. this review aims to discuss the variable level of pathogenic risk and describes the current screening methods used to prevent/detect the presence of pathogens in blood/plasma-derived products. acute bleeding episodes can arise either because of inherited bleeding disorders (e.g. haemophilia, von willebrand disease), acquired deficiency of haemostatic components (e.g. due to infection, malignancy or autoimmune disease), trauma, surgery or as a result of infection with an organism that causes haemorrhagic disease (e.g. ebola or marburg virus) [1] . various treatment options exist for preventing or treating acute bleeding episodes, including fresh-frozen plasma/cryoprecipitate, platelets and plasma-derived/recombinant clotting factor concentrates [2, 3] . the use of blood-derived and recombinant haemostatic products has increased markedly over recent years, as exemplified by the global use of factor viii products ( fig. 1) [4] . this increased use has been driven by improved availability of clotting factors, increased life expectancy of people with bleeding disorders [5, 6] , increased use of prophylaxis for severe bleeding disorders [7, 8] and decreased risk of transmission of infectious agents. historically, the risk of transmission of infectious agents via blood/ plasma-derived products has been of great concern to the medical community. this risk has reduced dramatically since the implementation of stricter donation screening/donor selection procedures and improved purification procedures, but cannot be fully eradicated. furthermore, the implementation of pathogen inactivation technology for blood/plasma-derived products has further reduced the risk of transmission of both known and emerging pathogens, although results can be variable according to the methods used [9, 10] . however, it is important to note that patient risk is highly dependent on the circumstances under which blood products are collected, handled and used. in general, clinicians assess the level of risk associated with the use of blood/ plasma-derived products by evaluating factors such as patient characteristics (e.g. age, immune status, geographical location, lifestyle) and the nature of the pathogen (e.g. physical characteristics, level of virulence, chronicity of infection, prevalence). the presence of a particular pathogen within blood/plasma-derived products may pose a significant threat to specific patient groups (e.g. the elderly or immunocompromised), while being of low risk to the general population (e.g. hev). while the clinical assessment of risk is based on a variety of factors, the virological assessment of risk is based solely on the presence or absence of pathogens. the presence of pathogens implies the possibility of infection, so only pathogen-free products can be described as entirely risk-free. adopting the virological approach (i.e. discarding all products a large number of pathogenic agents (including viruses, protozoan parasites and prions) can be transmitted via blood/plasma-derived products and are capable of causing disease in humans ( table 1) . the presence of viruses in plasma-derived products became a concern in the 1980s, when 60-70% of patients with severe haemophilia became infected with human immunodeficiency virus (hiv-1) [6] . this concern continued with the discovery that 80% of patients treated with plasmaderived products prior to 1992 had become infected with hepatitis c virus (hcv) [5] . current donor selection and screening practices have improved our ability to detect or reduce the presence of pathogens in blood/plasma-derived products; for example, the residual risk of transfusion-transmitted infection (tti) with hiv/hbv/hcv has fallen to near or less than 1 per million transfused units [14, 15] . despite this success, however, a residual risk still remains. the pathogenic agents shown in table 1 (and the supplementary appendix) do not form an exhaustive list. many microorganisms that are normally non-pathogenic have the potential to cause disease when responding to changes in the biological environment, or when transfused to an immunosuppressed patient. in addition, there is still a risk that new and emerging pathogens may enter the blood supply ( table 2) . the standard assays commonly used for blood screening are nucleic acid amplification technology (nat) and immunoassays for detection of antibody and/or antigen. immunoassays are frequently used for screening purposes as multiple samples can be processed together and they may yield semi-quantitative results. nat assays allow earlier pathogen detection than with immunoassays, but they are also more costly and complex. assay selection is generally determined by the level of accuracy/speed required, but factors such as the resources available (e.g. staff, infrastructure), assay complexity and cost considerations (e.g. consumables) must also be considered. most assays for blood donation screening are mandatory (particularly in europe and north america) and the world health organization (who) recommends that all whole blood (and blood which has been processed by apheresis) should undergo pathogen screening before it is used for clinical or manufacturing purposes. screening for hiv-1, hiv-2, hbv, hcv and treponema pallidum subspecies pallidum (t. pallidum; the causative agent of syphilis) is strongly advised. the who and world federation of hemophilia (wfh) suggest that countries should carry out individual routine screening for further pathogens based on epidemiological information for their region e.g. htlv-1 and trypanosoma cruzi [16] . the wfh also acknowledges the positive impact of hiv, hbv and hcv screening on global blood safety and recommends that these screening tests be implemented whenever possible [2] . details of the serological tests carried out on individual donor plasma and nat testing of plasma mini-pools are shown in tables 3 and 4 [17] . it is recommended that the minimum evaluated sensitivity/specificity level of any assay used for blood donation screening should be 99.5% or higher [16] . however, not all assays fulfil these criteria as sensitivity/ [196] . specificity levels vary according to assay type and type of microorganism being tested for (see table 1 for details). therefore, the general advice is to screen donated blood to as high a standard as possible [16] . in the early stages of infection with hiv, hbv or hcv, viraemia occurs in the host's bloodstream at variable levels. viral antigens may appear at the same time as dna/rna, but more often become detectable several weeks later. specific antibodies are measurable 2 to 6 weeks after infection; the time between initial infection and the appearance of detectable parameters of infection (e.g. viral nucleic acid/antigens/ antibodies) is known as the 'window period' [18] [19] [20] . 4.1.1.1. hiv. when screening blood for the presence of hiv-1/hiv-2, the use of a combined antigen/antibody assay is advised (combined hiv p24 ag and anti-hiv-1 + anti-hiv-2 antibodies) as it allows earlier detection of infection. the performance of nat testing is mandatory in many countries and further reduces the window period (from around 20 to 11 days) [20] [21] [22] . 4.1.1.2. hbv. the majority of diagnostic laboratories focus on the detection of hbsag, which is the first detectable serological marker of infection. however, there is a risk that the hbsag concentration may decline to undetectable levels during the course of infection, yielding a false negative result [23] . screening for antibodies to hbc is the most conservative approach for identifying potentially exposed donors, as this identifies all individuals who have ever immunologically experienced any type of hbv infection (either current, chronic or resolved) and who may experience viral reactivation during their lifetime (particularly under conditions of immunosuppression). however, assays to measure hbc antibodies are relatively non-specific and do not always correlate with the presence of hbv virus in plasma [18] . also, we cannot exclude the possibility that donors who test positive for anti-hbc do not have pulsed recurrences of virus replication, resulting in the presence of low levels of hbv-dna in plasma. for these reasons, national transfusion services do not always routinely screen donations for anti-hbc. nat assays can be carried out, but their use is restricted by potentially low levels of viral dna [16, 18] . the combined use of hbsag screening tests and nat assays has reduced the window period for detection of hbv infection from approximately 60 to 35.5 days [20, 24] . mutant hbv strains (escape variants) should also be considered, as they may occasionally escape serological detection (although most can be detected by nat assays) [24, 25] . these hbv-variants are rare, but therefore more likely to enter and contaminate the blood supply as they are more difficult to detect [25] . in summary, the screening of blood supplies for the presence of hbv is effective, but an optimal screening system has not yet been defined. 4.1.1.3. hcv. hcv (both recent and chronic infection) can be detected by screening blood for the presence of both hcv antigen and hcv antibody (anti-hcv). seroconversion occurs at approximately 6-8 weeks postinfection; however, steady improvements in screening technology (including the adoption of nat assays) have reduced the window period to approximately 1-3 weeks [20, 26] . as with hiv, nat assays are more useful for detecting early infection, although the issue of low viral rna concentration persists [27, 28] . htlv-1 and htlv-2 are endemic in some regions, but very rare in others, and therefore screenings are conducted on a geographical or at-risk basis. the presence of virus is mostly inferred by the detection of virus-specific antibodies, using sensitive immunoassays [16] . in this instance, screening involves detection of non-specific, nontreponemal or specific treponemal antibodies. nat assays are generally not used [29] . specific antibody tests identify all individuals who have ever been exposed to this bacterium (and may continue to yield positive results for more than ten years following initial infection), while the non-specific tests (e.g. vdrl or cardiolipin tests) are primarily of use in identifying donors who may have an active infection. since t. pallidum is heat-sensitive and cannot readily withstand extended storage at low temperatures, storage at 4°c for more than three days is sufficient to render the pathogen non-infectious. however, blood components (e.g. platelets) that are stored at temperatures of around 20°c do present a risk of t. pallidum persistence. therefore screening for antibodies of this organism is recommended [16] . blood can be screened for further pathogens as appropriate, according to geographical location, seasonal activity of the vector and also patient risk factors. a current viral pathogen of interest is the mosquitoborne flavivirus west nile virus (wnv), which was confirmed to have been transmitted via transfusion in 2002 [30] . an immediate screening policy was put in place in the usa in order to reduce the risk of further transmission. this policy included deferral of any individual displaying symptoms of infection, quarantine of plasma collected during periods of high mosquito activity (when wnv is most prevalent) and the rapid development/use of wnv-specific nat and serological assays. these measures were highly effective and caused a significant reduction in the number of confirmed cases of wnv transfusion-related transmission. however, wnv outbreaks still occur within the americas, indicating a potential need for seasonal blood screening for wnv [31] . wnv outbreaks have also occurred in europe (including italy and greece), prompting the implementation of seasonal blood screening procedures in the affected regions of those countries [32] . this is another mosquito-borne pathogen that could potentially pose a risk to transfusion safety, although to date reports of transfusionrelated transmission of this virus are rare [33] . a mutated form of the chikungunya virus has been responsible for several epidemics in the past decade, spreading to the reunion islands in the indian ocean (2005), italy (2007) and the caribbean area (2012/2013) [34] [35] [36] . the virus may be detected in blood donors by nat, which will help to reduce the level of transmission risk [35] . there is also concern about the possibility of parvovirus b19 in the blood supply. b19 is prevalent worldwide, with seroprevalence in blood donors varying from between 0.2-1.3% in the usa, europe and africa and 25-40% in asia [37] . the risk of parvovirus transmission is higher when units of blood are pooled (e.g. to create batches of clotting factor concentrates, albumin etc.) and so individuals with bleeding disorders are at a higher risk of infection. b19 dna was detected in 26% of clotting factor concentrates in a recent german study [38] , and another study found that populations receiving blood-derived products were 1.7 times more likely to display antibodies to b19 than populations who had not received blood products [39] . b19 lacks a lipid envelope, which renders it highly resistant to some methods of pathogen inactivation [40] . screening of blood donations for b19 dna is not currently routine, but many manufacturers only process plasma that has been screened for the absence of b19 dna in order to reduce the risk of transmission [41] . given the prevalence of b19 in different populations, it is difficult to define the residual tti risk of this virus. however, it is clear that a transmission risk does exist. trypanosoma brucei gambiense/rhodiense africa, 10% at risk [160] direct microscopic visualisation/antibody/nat nat: b100 trypanosomes/ml [161] nat: 1-10 trypanosomes/ml [162] antibody: catt -87-98% [161] nat: 88% [163] antibody: catt -95% [161] nat: 99.2% [163] (continued on next page) the potential presence of hepatitis e virus (hev) in blood or bloodderived products is relevant. currently there seems to be a discrepancy between the number of hev-rna positive blood donations in europe (ranging from 1 in 1240 in germany to 1 in 1761 donations in the netherlands) [42] , and the low number of confirmed cases of hepatitis e in blood transfusion recipients (one confirmed case in the uk [2006] , one in france [2007] and two in germany [2014]) [43] [44] [45] . this indicates that the subject of hev infectivity and pathogenicity needs to be investigated further [46] [47] [48] [49] . there is also a debate over the necessity of introducing screening blood for the presence of hev; the virus is not currently screened for in the uk and other european countries, although one study of english donors found hev to be widespread (1 in 2848 donations) within the donor population [50] . since infection with this virus can be harmful to immunocompromised individuals, the potential need for introducing hev screening should be considered [42] . the residual risk for tti of hbv, hcv, hiv and hev in selected countries is given in table 5 . despite recent advances in methods for the detection of prions, no single method has been developed as a screening test for blood, although several methods in animal models show great promise [51] [52] [53] . in humans, a protocol for the evaluation of a blood-based test for its suitability in the diagnosis of variant creutzfeldt-jakob disease (vcjd) has been established, but no test yet appears to satisfy the requirements of sensitivity and specificity [54] . the only report so far of a blood-based diagnostic test for vcjd claimed an assay sensitivity of 71.4% and a specificity of 100% in symptomatic patients and its potential applicability as a screening test to detect asymptomatic vcjd infection has recently been investigated [55] . there is currently no strategy for confirming a positive screening result, although the protein misfolding cyclical amplification technique has recently been shown to yield positive results in buffy coat/white blood cell samples from a small number of patients with vcjd [56] . extensive use of blood donor selection and testing does not always guarantee a safe product. if the test is insufficiently sensitive, then false negatives may occur. alternatively, tests which are not sufficiently specific (e.g. anti-hbc assay for hbv) may cause false positive results, leading to an unnecessary decrease in the number of clotting products available [16] . torque tenovirus (ttv) is an example where testing specificity has been an issue, as this virus exists in various divergent forms (23 distinct genotypes have been identified thus far) [57] . since ttv is often detected in healthy individuals and is not associated with any particular disease, routine screening for this virus is not considered to be necessary; even a test with excellent sensitivity/specificity would not contribute to increase the level of safety of blood/plasma-derived products with regard to ttv. insufficient assay sensitivity remains a rare but potential problem when blood donations are screened during the window period of initial hbv, hcv or hiv infection. an increase in testing sensitivity threshold is needed to prevent hbv transmission via blood/plasma-derived products and by blood transfusion, as extremely low concentrations of hbv (e.g. 1.6 copies/ml) is capable of viral transmission (see above section on anti-hbc positive patients) [58] . in the case of hiv and hcv, even nat testing may not always be sufficient to ensure sufficiently high levels of safety as virus transmission has previously occurred after transfusion of blood with undetectable levels of viraemia [59] . recent reports have highlighted concerns about the inability of nat assays to detect different variants of hiv. there have been at least four cases in which the presence of hiv-1 rna was undetected by nat assay screening, potentially putting transfusion recipients at risk [21] . two of these false-negative results occurred due to genetic mutation in the viral rna regions targeted by nat assay primers (although in in cases where no overall figures are available, specificity/sensitivity has been described as low, medium or high (as appropriate). catt, card agglutination test for trypanosomiasis; id-nat, individual donation nat; mp-nat, mini-pool nat; nat, nucleic acid testing; pcr, polymerase chain reaction; pfu, plaqueforming unit. the majority of cases serology yielded a positive result) [60, 61] . falsenegative results can be avoided by designing nat assays that target a minimum of two amplification regions; such testing will be mandatory in germany from 2015 [21, 62] . it is hoped that as the sensitivity and specificity of nat tests continue to improve, cases of undetected infection may become less of an issue in the future. a recent report suggests that it is not necessary to carry out serological screening for multiple hbv markers, and that nat based screening is preferred [58] . however, there is a risk that the practice of relying upon a single method of screening may lead to a higher incidence of false-negative results. in one case of transfusion-associated parvovirus b19 transmission, donated blood was screened for the presence of b19 antigen and deemed to be safe since no antigen was detected. since the recipient subsequently developed b19 infection, the donation was re-analysed and found to contain b19 dna [63] . reports such as this support the argument for multiple parameter testing. the localisation of pathogens within blood may also influence ease of detection. for example, wnv is present at 10-fold higher levels in whole blood than in plasma in viraemic seropositive donors. the situation is reversed in viraemic seronegative donors, who display higher wnv levels (4-fold) in plasma than in whole blood [64] . also, cellassociated viruses such as cmv and htlv-1 are less frequently transmitted with the use of leukoreduced products [65, 66] , indicating that these viruses are less likely to be found in blood/plasma-derived products. the minimum infective dose (mid: the lowest number of pathogenic particles required to successfully infect a host) of a particular pathogen is more likely to be reached in whole blood or blood-derived components, making the use of plasma-derived or recombinant clotting factors the safest option. to date, studies attempting to measure human mid values have generally determined the viral concentration needed to infect a particular percentage of the exposed population (e.g. 50%). this value (the human infective dose for 50% of the population) is referred to as hid 50 and is often described as the human mid [67] . the hid 50 value varies greatly between pathogens (even if they are physically similar) [68] and also varies depending upon the immune status of the recipient, as immunocompromised individuals, neonates and the elderly are at greater risk of infection than healthy individuals [67] . when this finding and the prevalence of immunocompromised patients receiving blood products are both taken into account, it implies a need for screening tests to have the highest sensitivity possible. the impact of transmission on morbidity and mortality is dependent on patient characteristics. for example, although the vast majority of cases of cmv infection are not clinically important, influencing factors such as genetic predisposition, malnutrition and pre-existing infection can lead to the development of severe disease [69] . national screening programmes do not currently screen for cmv as standard, but nat assays exist and may be carried out if required, e.g. if blood is specifically intended for vulnerable recipients, such as pregnant women or transplant patients [70] . it is the opinion of the authors that both serological assays and nat tests should be used in order to reach the highest level of safety possible, particularly when in the case of immunosuppressed patients. as well as blood donation testing, a range of other measures are used to increase the pathogen safety of blood-and plasma-derived products. these include donor selection and screening, recipient vaccination and the use of blood product purification/inactivation methods. the choice of inactivation method also impacts upon the level of risk. questionnaires are often used to attempt to assess donors' health status and their potential exposure to various risks. donors can be accepted or rejected on the basis of these answered questionnaires, or alternatively their blood may be put through additional screening tests as appropriate [16, 31] . patients with bleeding disorders should be vaccinated against hav and hbv. european studies have reported that universal hbv vaccination of blood donors could be cost-effective as this measure would reduce the risk of hbv transmission in general and might even remove the necessity for general hbv nat testing; however, this would not reduce the risk posed by hbv escape variants (as described earlier) [71, 72] . blood/plasma-derived products typically undergo various procedures designed to reduce the pathogen level, although these procedures are not effective against all pathogens. plasma donations undergo quarantine (approximately 4-6 months) prior to fractionation and when the donor is again screened negative ffp can then be subjected to chromatographic fractionation, solvent-detergent treatment, nanofiltration and/or heat inactivation [73, 74] . prolongation of product storage time can be effective in reducing the infectivity of temperature-sensitive pathogens (such as t. pallidum). production of recombinant products also follows strict protocols to remove and inactivate any viruses that might be present, even though the risk of viral presence is minimal. although current purification/inactivation techniques (such as solvent-detergent treatment, nanofiltration and heat activation) do reduce the risk of pathogen transmission, they are not always sufficient to render blood/plasma-derived products safe [75] . small nonenveloped viruses (e.g. hav, b19 and picornavirus) are often highly resistant to inactivation procedures and may still be infectious in some plasma-derived concentrates [75, 76] . the presence of prions is also a concern. attempts to remove prions from plasma-derived products have involved several techniques, including ion-exchange chromatography and nanofiltration [77, 78] . several problems exist with these approaches, in particular the use of exogenous "spikes" derived from prion-infected brain homogenates to measure prion clearance, which may result in an overestimation of the amount of prion removal, and the methods used for the estimation of the reduction in prion load, which ideally should involve bioassay to measure infectivity [79] . further developments in this field are required to address these issues. a recently proposed approach for the inactivation of infectious agents in blood is whole-blood treatment with ultraviolet (uv) light in combination with a photosensitiser such as riboflavin or amotosalen [80, 81] . the main disadvantage of this approach is that uv treatment has been linked to the formation of neoantigens, which may be generated via modification of the surface antigens of platelets. the presence of neoantigens may provoke a recipient immune response during transfusion of uv-treated platelets, causing them to be rapidly cleared from the circulation [81] . while this approach to pathogen inactivation is currently used for platelets and is effective, it needs further refinement for the inactivation of whole blood [81, 82] . the wfh strongly recommends the use of plasma-derived or recombinant products in preference to cryoprecipitate or fresh frozen plasma, as the infectious load of any infectious human pathogen is lower in plasma-derived products than in cryoprecipitate, and even lower in recombinant products [2] .in some european countries, recombinant products have almost completely replaced plasmaderived products [12] . the use of recombinant products, which have been manufactured and formulated with minimal addition of human/animal-derived materials, greatly reduces the risk of recipient exposure to plasma-derived infectious agents and after they have undergone virus removal/inactivation processes, recombinant products can be considered to be as safe as currently possible [83] . despite these benefits, the use of recombinant products may be limited by higher costs and perceived problems of inhibitor formation [83] . for indications where no recombinant factor concentrates are available, the use of inactivated plasma-derived concentrates is safer than fresh frozen plasma and will reduce the risk of other adverse effects such as hypervolemia, transfusion-related lung injury (trali) and hypersensitivity [84] . a minimal risk approach would ensure that patients receive effective treatment with the lowest possible risk, but this is difficult to achieve in practical terms. regulatory needs in different european countries are usually based on recommendations from the medical community, so in order to achieve minimal risk, it would be ideal for these regulations to be standardised and mandatory in all countries. directives issued by the european union commission describe the regulatory requirements for the safety of whole blood and plasma, stating that "all precautionary measures during their collection, processing, distribution and use need to be taken making appropriate use of scientific progress in the detection and inactivation and elimination of transfusion transmissible pathogenic agents" [85] . however, the relative safety of different screening tests, products and processing methods is not discussed and so individual countries may adopt different approaches towards minimising risk. although recombinant products are associated with the highest level of pathogen safety, higher costs for development and production may make them too expensive for some healthcare systems [86] . inhibitor formation also remains an important element of concern with both plasma-derived and recombinant products, particularly with regard to fviii in haemophilia a [87] . the risk of inhibitor formation was shown to be greater with recombinant versus plasma-derived factor viii concentrates in some cohort studies [88] , but similar in others [89] . in light of these considerations (availability, adverse reactions and cost), it appears that the issue of pathogen safety of blood/plasmaderived products is highly important but may not be the limiting factor with respect to overall patient safety. the benefits of treatment with a hypothetically 'unsafe' plasma-derived product may outweigh the risk of a negative outcome (e.g. bleeding, inhibitor formation), although we suggest that it may be clinically simpler to deal with inhibitor formation than to combat an infection from an unknown or untreatable pathogen. there are still significant knowledge gaps and areas of unmet need with respect to the pathogen safety of blood/plasma-derived and recombinant products. the incidence of hbv, hcv and hiv tti has fallen to near or below 1 per million transfused units in the industrialised world, indicating that current donor selection and blood screening strategies have had a positive impact on blood safety [14] . however, it is clear that screening both donors and donated blood cannot exclude all known pathogens or eliminate all risks from emerging pathogens [63, 90] . historical precedent indicates that the blood supply is always vulnerable to contamination by hitherto non-prevalent/unknown pathogens, and that this risk cannot be accurately gauged [91] . as we identify new infectious agents of concern and develop new tests for their detection, it will also be necessary to clearly define the infectious dose range for each agent and use appropriately sensitive tests for their identification. for example, in suspected cases of vcjd infection, considerable challenges remain in the development of screening and confirmatory tests that have sufficient sensitivity and specificity to be of use in both a clinical setting and within blood banks [92] . surveillance of people with haemophilia is required to monitor pathogen safety issues related to blood and plasma products. the european haemophilia safety surveillance system (euhass), which began in 2008, is a pharmacovigilance programme which spans 25 european countries and is designed to detect, monitor and investigate adverse drug reactions. reports of adverse events (such as acute/allergic events, ttis and inhibitors) are submitted to euhass by participating centres and cumulative patient and clotting factor data are recorded annually [93] . a formal coordinated risk-assessment and management action plan, in addition to a task force, should be developed to respond quickly to any emerging infections. such a plan should include long-term storage of samples from produced batches (for retesting in the case of outbreaks with known or recently emerged infectious agents) and guidance on responsibility for developing/performing tests for emerging pathogens (industry vs. regulatory agencies). guidance on approaching patients who have potentially been infected and surveillance strategies for patients at high risk would also be beneficial. the majority of evidence indicates that the concept of clinical safety of blood/plasma derivatives does not necessarily correspond to the concept of pathogen safety; blood can only be classed as microbially safe in reference to the infectious agents that are known and have been screened for. establishing whether the presence of undetected microbes in the blood is clinically relevant will require further long-term, detailed studies. it should also be noted that even though the risk of transmission of key detectable viruses (such as hiv, hbv and hcv) via transfusion has fallen significantly, transmission does still occur. in general, balancing safety, efficacy and practicalities is a difficult goal to achievepatient safety is typically the key driver, but striving for near-complete safety at the expense of the patient's health or quality of life may not be the best course of action for patients or clinicians. the lack of a cohesive international strategy for blood donation and screening is a pressing concern that needs to be addressed. furthermore, a formal coordinated continuous risk-assessment and management action plan needs to be developed to deal with the constant potential risk of emerging infections. establishing an international registry (or harmonising data collection in national registries) and dedicated task force may help to identify newly emerging pathogens more rapidly than in the past and to further improve pathogen safety of blood/plasma-derived products and the blood supply in general. • the use of blood/plasma-derived products for the treatment of bleeding disorders carries a risk of pathogen transmission. • blood donations are screened for key pathogens such as hbv, hcv, hiv and the causative agent of syphilis, but other screening tests should be conducted as required according to geographical location and patient risk factors. • screening tests for pathogens may lack sensitivity/specificity and so false negatives may occur, resulting in a residual pathogen risk to patients. • in terms of pathogen 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sweden, germany and the united states world federation of hemophilia (wfh) annual global surveys we wish to thank professor hermann eichler for his appraisal of this manuscript and helpful suggestions. medical writing assistance was provided by hanna mourad-agha of inscience communications, springer healthcare. this assistance was funded by pfizer. a.t. has received grants and personal fees from bayer, grants and personal fees from baxter, grants and personal fees from biotest, grants and personal fees from csl behring, grants and personal fees from novo nordisk, grants and personal fees from pfizer, and grants and personal fees from octapharma, during the conduct of the study.c.f.p. has received grants as bureau speaker, consultant, or advisor, from gilead, merck sharp and dohme, roche, pfizer, abbott, bristol-myers squibb, viiv, and boehringer-ingelheim. none of these personal activities is in conflict with the opinions he expressed in this manuscript. d.n. has received honoraria for conferences from pfizer, roche pharma, roche diagnostics, abbott, msd, and astellas.g.d.m. has disclosed the following financial relationshipsspeaker or a member of a speaker bureau for: boehringer-ingelheim, sanofi-aventis, bayer, novo nordisk, pfizer, biotest, and grifols. consultant or ad hoc speaker/consultant for: boehringer-ingelheim, eli-lilly, sanofi-aventis, bayer, csl behring, novo nordisk, pfizer, biotest, and grifols.j.w.i. has received personal fees from piramal and grants from the department of health, uk, outside the submitted work.l.g. reports no potential conflicts of interest. m.c. has received research grants, lecture fees, and honoraria for consultancy from baxter, bayer, and pfizer. funding for medical writing assistance and two author meetings were provided by pfizer. pfizer had no editorial influence regarding the contents of this manuscript and did not comment on the outline or any drafts prior to journal submission. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.blre.2015.07.004. key: cord-312418-e4g5u1nz authors: melillo, alessandro title: rabbit clinical pathology date: 2007-09-18 journal: j exot pet med doi: 10.1053/j.jepm.2007.06.002 sha: doc_id: 312418 cord_uid: e4g5u1nz with rabbit patients, as in other species, analyzing blood and urine samples can be useful and informative, although interpretation of the results is sometimes challenging. this article summarizes the interpretation of laboratory results from rabbits. hematological parameters can yield information about the red blood cell population and leukocyte response to stress and pathogens. biochemistry evaluation can be used to investigate liver, kidney, and other organ function, and urinalysis results may yield additional information about kidney function and electrolyte imbalances. serological tests are available for several pathogens of rabbits, including encephalitozoon cuniculi, although the significance of positive results and antibody titers is not clear. serum protein electrophoresis aids the understanding of protein disorders and the immune response to acute and chronic inflammation. r abbits can mask signs of illness or show few or confusing clinical signs. additional information may be gained from laboratory tests, and in-house analyzers can provide a complete profile with a small volume of the patient's blood. unfortunately, most of the published data on rabbit hematology and biochemistry values are descriptions of the effects of toxins on hematological and biochemical parameters of laboratory rabbits. there is little information available that describes the effect of clinical disease on the blood parameters of companion rabbits, or on the use of blood tests as diagnostic and prognostic indicators. the lack of biochemical data for pet rabbits is changing as practitioners collect information and researchers are more cognizant of diagnostic and prognostic hematologic indicators. the blood volume of a healthy rabbit is approximately 55 to 65 ml/kg, and 6% to 10% of the blood volume may be safely collected. many sites are described for blood collection in rabbits. cardiac punc-ture is used in laboratory rabbits but is not recommended for pet animals. both the marginal ear vein and central ear artery are easily accessible, but they may be difficult to access in some patients. collecting a sufficient volume of blood from these sites to perform all of the desired clinical tests may also be difficult, especially from dwarf breeds with small ears. sampling from ear vessels can occasionally result in thrombosis and subsequent avascular necrosis of parts of affected pinna tissue. blood may be collected from the cephalic vein, which is straight and easily accessible, but, because of the short antebrachium, occlusion of the vessel by encircling the limb at the elbow is difficult. the cephalic vein is also small and easily collapses. the jugular veins are large and allow for ample-sized blood volumes to be collected, but jugular phlebotomy can be stressful for rabbits and may require chemical restraint. the dewlap may interfere with jugular vein visualization, especially in obese does. an accessible and efficient site for blood sampling in rabbits is the lateral saphenous vein (fig 1) . most biochemical parameters of rabbits can be measured from serum or plasma. rabbit blood clots easily at room temperature and will coagulate quickly if not mixed with anticoagulant during collection. heparin is a suitable anticoagulant because it does not alter biochemical parameters, even though the anticoagulant/blood ratio is not always optimal. hemolysis can be prevented by letting the blood drop from the needle into the tube, but often, only a few drops can be collected before the blood clots. the technique can be improved by heparinizing syringes and needles through aspiration of a few drops of heparin into the syringe, then removing the excess with injection pressure. the small amount of heparin remaining in the needle prevents clotting without significant alterations in biochemical parameters. it is important to make several air-dried blood smears at the time of venipuncture before the anticoagulant in the tube and transport can modify red and white cell morphology. most of the standard blood stains work well. automated flow cytometry is reliable for analyzing most hematological parameters for rabbit patients. age, sex, breed, and circadian rhythms all affect hematological and biochemical parameters in rabbits. rabbits under 12 weeks of age have lower red blood cell (rbc) and white blood cell (wbc) counts. the total wbc and lymphocyte counts are lowest in the late afternoon and evening, when the heterophils and eosinophil counts rise. urea and cholesterol levels tend to increase at the end of the day. stress can alter many different hematological parameters (e.g., blood glucose). prolonged stress, such as transportation, unfamiliar noises, smell, chronic pain, and poor environment, can induce heterophilia, lymphopenia, and leukocytosis. simple handling does not induce this response, but several muscle enzymes including lactate dehydrogenase (ldh), aspartate aminotransferase (ast), and creatine kinase elevate after physical restraint of the rabbits, especially if they are fractious or unfamiliar with handling. sedation or general anesthesia can be helpful. isoflurane anesthesia does not appear to affect blood parameters in rabbits. hemolysis can induce several artifacts, such as decreased rbc and amylase values and increased ldh, ast, creatine kinase, total protein, and potassium levels. in-house analyzers can be very sensitive to hemolysis, thereby altering the true blood parameters of the patient. hematology results of rabbits can be difficult to interpret. most reference ranges are from experimental laboratory studies that are run on homogeneous groups of rabbits belonging to the same breed, strain, age, and environmental conditions. this can be very different from the clinician's situation of dealing with a heterogeneous population of pet rabbits. many texts amalgamate reference ranges from different sources to create ranges so wide that they include almost any result. another problem is that healthy pet rabbits are very hard to find, so samples from rabbits with an apparently acute condition may also show changes due to an underlying chronic problem, such as malnutrition, improper husbandry, or subclinical disease. for example, harcourt-brown and baker 1 showed that rabbits that were caged, fed on commercial mixes, and suffered from dental disease had consistently lower packed cell volumes (pcv), rbc counts, hemoglobin values, and lymphocyte counts in comparison with rabbits kept outside with a more natural diet and exercise. rabbit erythrocytes are typical mammalian anucleate biconcave discs with an average diameter of 6.8 m, which is midway between cats and dogs. 2 erythrocyte size varies between 5.0 and 7.8 m, which is often reported as a marked anisocytosis (fig 2) . the short life span (57 days) and high turnover of erythrocytes is reflected as polychromasia, which is not clinically significant. the presence of a few nucleated rbcs (1-2 ϫ 100 leukocytes) and the occasional howell-jolly bodies (fig 2) should be considered within the normal reference range for rabbits and not an indicator of cellular regeneration. the published reference range for pcv in the rabbit is 30% to 50%, but pet rabbits often have lower values of 30% to 40%. 2 values higher than 45% may indicate dehydration, which is usually linked to gastrointestinal (gi) stasis. combined pcv and total protein (tp) is useful to differentiate acute conditions from subclinical chronic diseases that have suddenly deteriorated. a pcv of less than 30% indicates anemia, especially if the rbc and hemoglobin levels are low as well. nonregenerative anemia associated with chronic disease is common in pet rabbits. otitis media, dental disease with or without abscesses, pneumonia, pododermatitis, mastitis, endometritis and pyometra, renal disease, and osteomyelitis are all examples of chronic infections that can be associated with nonregenerative anemia in pet rabbits. regenerative anemia is manifested by significant and rapid reticulocyte production and usually indicates blood loss. causes of external hemorrhage in rabbits include trauma and severe flea infestation. common internal causes include hematuria due to kidney or bladder stones or bleeding uterine adenocarcinomas or endometrial aneurysms in does. intravascular hemolysis is an unusual cause of regenerative anemia after ingestion of leaves and stems of potato plants and possibly other solanaceae. alliums (onion, garlic, and chives) may also cause heinz body anemia. 3 autoimmune hemolytic anemia has been reported in laboratory rabbits in association with lymphosarcoma, and isolated cases have been treated in private practice (harcourt-brown, personal communication, november, 2006). 4 lead toxicosis is a cause of regenerative anemia, characterized by many nucleated erythrocytes, hypochromasia, poikilocytosis, and basophilic cytoplasmatic stippling. 2 nucleated red cells of more than 1% to 2% of the rbc can be linked with the acute, septicemic phase of an infectious disease, although this is unusual because of the presence of a nonregenerative anemia from underlying chronic disease. a well-prepared air-dried smear is required for an accurate differential white cell count and evaluation of the cytological appearance of each cell type (figs 2-6). differential white cell counts and cell morphology can be used to develop a differential diagnoses list and to determine the general condition of the patient. interpretation of wbc data from rabbits is different from other domestic species including dogs, cats, and birds, in which a leukocytosis is the response to inflammation. with rabbits, although leukocytosis can be identified in cases that have been diagnosed with lymphosarcoma, it is not the usual response to inflammation. laboratory investigations have shown no increase in the total number of circulating leukocytes in rabbits injected with bacteria or yeast, in domestic carnivores, anisocytosis usually reflects the presence of reticulocytes and indicates regenerative anemia. this is not true in rabbits in which 1% to 4% of the circulating erythrocytes may be reticulocytes. the occasional howell-jolly body is not clinically significant either. rabbit clinical pathology although fever, increased plasma cortisol concentrations, neutrophilia, and lymphopenia were observed. 5, 6 in clinical practice, rabbits with sepsis can show a variety of responses including a neutropenia, normal neutrophilic count, or a mature neutrophilia, accounting for more than 90% of wbcs. band neutrophils appear to be a rare finding in clinical infection, and the absence of a left shift does not rule out an infectious problem. an alteration of the neutrophil/lymphocyte ratio showing a relative neutrophilia coupled with lymphopenia may indicate a response to infection. this ratio is approximately 1:1 in an adult healthy rabbit, but stress can alter the neutrophil/lymphocyte ratio. transport, waiting in a room full of unfamiliar sounds and smells, or even restraint for clinical examination can change the ratio. gentle clinical examination and blood collection do not seem to affect the differential white cell count, whereas more prolonged stress like travel or exposure to barking dogs in the waiting room can induce a lymphopenia and relative neutrophilia, that may persist for up to 24 to 48 hours. 7 the total wbc can be used to further characterize acute stress from chronic stress (e.g., malnutrition, improper husbandry, prolonged social stress, dental disease), as both a leukopenia and lymphopenia are eosinophil. rabbit eosinophils measure 10 to 16 m in diameter and have a purple bilobed or horseshoe-shaped nucleus. the cytoplasm is obscured by so many granules that the cell looks orange-pink and foamy. the abundance of granules is the main difference between the eosinophils and the neutrophils. removal of histamine and histaminelike toxins is the most important function of the eosinophils, suggesting that they therefore play an important role in controlling allergic reactions. more common with chronic stress. chronic stress is often reported as general leukopenia and lymphopenia in a rabbit's differential wbc evaluation. the primary role of the lymphocytes is to respond to those activities that stimulate the immune system. in rabbits, lymphocytes are primarily found in the blood, spleen, bone marrow, lymph nodes and the lymphatic tissues in the gi tract. the number of circulating lymphocytes is a balance between the cells entering and leaving the bloodstream and does not necessarily reflect a change in lymphopoiesis. in rabbits, it has been shown that an increase in adrenaline levels (acute stress) induces lymphocytosis, whereas raised cortisol levels (chronic stress) leads to lymphopenia. viral diseases may result in a normal or higher lymphocyte count. other causes of lymphocytosis are lymphoma and lead poisoning. 8 eosinophilia in rabbits can occur when tissues rich in mast cells, such as the skin, lungs, gi tract, or uterus, are involved in disease. eosinophilia can indicate the presence of an abscess and may be found during wound healing. in other species, eosinophilia is linked to parasitic diseases, especially when larvae are moving through tissues, but this is rare in domestic rabbits. encephalitozoon cuniculi does not stimulate an eosinophilic response. in clinically healthy rabbits, a very low eosinophilic count, even 0, is a common finding. high levels of cortisol (chronic stress) can induce eosinopenia. monocytosis is linked with chronic inflammation (e.g., abscesses, mastitis, tympanic bulla empyema). however, the absence of a monocytosis does not rule out inflammation. monocyte counts within the normal range are a common finding in rabbits with osteomyelitis due to dental disease. the diameter of rabbit basophil measures 8 to 12 m. its nucleus is less segmented than the eosinophil or heterophil and difficult to see because of the many deep purple granules that obscure the light gray cytoplasm. as in other species, the function of the rabbit basophil is not fully understood, but these cells are often present in large numbers of rabbit blood smears. basophilia with concurrent eosinophilia has been described in rabbits with chronic skin problems (e.g., atopy, pyoderma). 3 in rabbits, ast is widely distributed in many tissues. it is present in cardiac tissue and muscle, as well as the liver, and has a short half-life (5 hours). although higher ast levels may be found in patients diagnosed with liver damage, struggling during collection or hemolysis of the sample also raises ast levels. creatine phosphokinase levels also increase after restraint and are purely muscular in origin. ldh is produced by muscle and liver cells in rabbits and therefore is not beneficial as a diagnostic tool for many disease evaluations. reference levels for ast, ck, and ldh can be found in table 1 . in many mammalian species, alanine aminotransferase (alt) is a useful indicator of hepatocyte damage because of its specificity for liver tissue and its long half-life (45-60 hours in dogs). in rabbits, alt is not as useful as an indicator of liver damage as with other species because, like other herbivores (e.g., horses, cattle, guinea pigs), alt is not liver specific and has a shorter half-life (around 5 hours). however, alt concentrations are not affected by restraint and therefore can be used as a diagnostic tool. slightly increased alt levels are a common finding in apparently healthy rabbits. mildly increased alt levels in healthy rabbits have been attributed to exposure to low concentration of toxic substances, such as resins in wood-based litter or aflatoxins in food. 9 raised alt levels (with alkaline phosphatase (alp), bilirubin and glutamyltransferase [ggt]) can be associated with hepatic lipidosis or may be found in patients with hepatic coccidiosis (eimeria steidae) or torsion of a liver lobe. alp is a widely distributed enzyme; liver and bone contain the highest concentrations, but it is also found in bowel epithelium, kidney tubules, and placenta. a physiological cause of high-serum alp concentrations is osteoblastic activity in growing animals. animals with bone lesions will show raised alp levels. as a liver enzyme, alp does not increase because of hepatocellular damage but is indicative of bile stasis (e.g., hepatic coccidiosis, liver abscesses, neoplasia, lipidosis). extrahepatic causes, such as abscesses or neoplasia, can cause bile stasis by occluding the bile ducts. rabbits produce 2 alp isoenzymes in the liver. an intestinal isoenzyme is quite abundant, so serum alp concentrations are actually the sum of these 3 isoenzymes, which may explain why many reference ranges are vague and wide and why raised alp levels in clinically healthy animals are a common finding. alp does have a diagnostic value because it is not altered by restraint and thus is considered a good indicator of real tissue damage. reference levels for alt and alp can be found in table 1 . ggt is a useful indicator of chronic liver disease with bile stasis in horses, cattle, and domestic carnivores; however, in rabbits, the activity of ggt is low. activity of this enzyme is high in the kidney, yet renal ggt does not reach the circulation because it is eliminated with the urine. therefore, elevated ggt levels in the rabbit are most often linked to obstructive lesions of the bile ducts, but with a lower sensitivity than that found in other species. reference levels for ggt can be found in table 1 . bile pigments are produced during the breakdown of the heme molecule by hepatocytes and are excreted into the bowel. bilirubin levels reflect either hepatocyte or bile tract function. rabbits produce a large amount of bile for their weight, and the main compound is biliverdin, for which there is no commercial laboratory test. about 30% of biliverdin is converted to bilirubin, which is found in the blood in measurable amounts. the main cause of hyperbilirubinemia is bile flow obstruction. in young rabbits, hepatic coccidiosis is the most common cause of biliary obstruction, while in adult rabbits it is biliary neoplasia. cellular causes of hyperbilirubinemia, and rarely icterus, are aflatoxicosis (e.g., eating moldy food), which induces hepatic fibrosis (alt is usually raised too), and viral hemorrhagic disease, which causes acute hepatic necrosis with concurrent high levels of all hepatocellular enzymes. if the rabbit survives long enough, icterus may be seen. bilirubin may also be increased in diseases that cause hemolysis (e.g., immune-mediated hemolytic anemia). in other species, a comparison of preprandial and postprandial serum bile acid concentrations is used as an indicator of liver function. in rabbits, cecotrophy makes it almost impossible to fast a rabbit for the preprandial sample, so bile acid measurement is not a routine procedure in clinical practice, although persistently raised bile acids have been reported in association with hepatic disease. 10 cholesterol is synthesized in the liver or obtained from the diet and is a precursor of steroids. it is metabolized by the liver and excreted in bile. in carnivores, hypercholesterolemia is linked with several metabolic diseases, such as hypothyroidism, hyperadrenocorticism, diabetes, and hepatopathies. hypocholesterolemia indicates liver failure. cholesterol and triglycerides levels peak after a meal and fasting is needed for accurate measurement, which limits their diagnostic value in rabbits because of cecotrophy. abnormal levels of cholesterol and tri-glycerides can be related to a diet rich in fats, obese patients, or hepatic disease. in anorexic patients, hypercholesterolemia carries a poor prognosis because it indicates end-stage hepatic lipidosis. hypercholesterolemia has also been linked with pancreatitis, diabetes mellitus, nephrotic syndrome, and chronic renal failure. 3 decreased cholesterol levels in rabbits might be found in cases of liver failure, chronic malnutrition, and even pregnancy (up to 30% below the range). unlike other species, amylase is an almost pure pancreatic enzyme in rabbits, with little or no content in salivary glands, intestinal tissue, or liver. therefore, raised amylase levels in rabbits reflects pancreatic damage from pancreatitis, pancreatic duct obstruction, peritonitis, or abdominal trauma. renal failure can also cause hyperamylasemia because this enzyme is cleared by renal filtration. corticosteroids (exogenous or endogenous) can raise amylase values in serum, whereas hemolysis lowers it. there is little information on the function and diagnostic value of lipase in rabbits. increased lipase values may indicate cellular damage to the pancreas as it does in other species. as with amylase, lipase is artifactually elevated by corticosteroids. urea is a by-product of protein catabolism and is excreted by the kidneys into the urine. urea levels in rabbits depend on the circadian rhythm (peak in late afternoon and early evening), quantity and quality of proteins in the diet, nutritional status, liver function, intestinal absorption, urease activity of the caecal flora, and hydration status. often, small changes in urea levels are difficult to interpret. reference ranges have been determined from laboratory rabbits fed on a standardized diet and bled at the same time of the day, whereas clinicians see pet rabbits fed on a variety of foods and samples are taken at random. slight elevations in blood urea are a common finding. reference levels for urea can be found in table 1 . creatinine is a protein catabolite that is produced from the muscle creatine and excreted by glomerular filtration at a constant rate. creatinine is a more reliable test of renal function than blood urea. in rabbits, prerenal azotemia can be caused by dehydration because rabbits have a limited ability to concentrate urine. only a few hours without drinking or losing fluids, as in cases of ileus or diarrhea, may cause an increase of urea and creatinine to levels compatible with renal failure. urea and creatinine levels rapidly return to normal once the dehydration deficit is corrected. stress can also induce shock and cardiac disease, classified as prerenal disease, causing a decrease in renal perfusion. another potential cause of prerenal azotemia is gi hemorrhage, which results in increased protein digestion. azotemia is also indicative of renal disease, usually affecting the rabbit patient in association with hyperkalemia or hypokalemia, hypercalcemia and coexisting hyperphosphatemia, nonregenerative anemia, and isostenuric urine. the most common cause of renal failure in rabbit patients is e. cuniculi, which causes granulomatous and then fibrotic lesions in the renal parenchyma. other possible causes of renal failure are chronic interstitial nephritis, glomerulonephritis, pyelonephritis, nephrolithiasis, renal cysts, and lymphosarcoma. postrenal azotemia can occur because of obstruction to urine flow as a complication of bladder sludge or urolithiasis. an abdominal radiograph is mandatory in any azotemic rabbit. blood urea levels below the reference range indicate hepatic insufficiency or muscle mass loss (e.g., dental disease). reference levels for creatinine can be found in table 1 . glucose metabolism in rabbits is different from dogs or cats. not only do rabbits eat continuously during the day, but they also use volatile fatty acids produced by cecal flora as a primary energy source. a fasting blood sample is impossible to obtain because rabbits ingest fecal pellets. a rabbit that is not given food can continue to ingest cecotrophs. it has been shown that 4 days of starvation does not reduce blood glucose levels in rabbits. 11 diabetes mellitus is rare in rabbits, although hyperglycemia is a common finding and may be associated with glucosuria. reports of confirmed diabetes mellitus are from laboratory strains bred as a model for human diabetes. clinical signs commonly observed in pet rabbits with diabetes mellitus are polyphagia, polyuria, polydipsia, very high blood glucose levels (ͼ500 mg/dl), and glycosuria with significantly elevated glycosylated hemoglobin and raised triglycerides; however, obesity and ketoacidosis are not observed. 12 in clinical practice, most cases of hyperglycemia are due to stress (e.g., transport, handling, venipuncture, underlying disease). a marked hyperglycemia (around 350 mg/dl) is reported in cases of acute intestinal blockage by a foreign body. 10 early mucoid enteropathy may be associated with hyperglycemia. in rabbits with gi stasis, hyperglycemia carries a bad prognosis because it may indicate hepatic lipidosis. other causes of raised serum glucose levels are traumatic or hypovolemic shock and hyperthermia. acute pancreatitis could cause blood glucose abnormalities, even though the role of the pancreas in glucose metabolism in rabbits is less important than in other species. glucocorticoids and other drugs can raise blood glucose. hyperadrenocorticism has not been described in rabbits. hypoglycemia is an important finding. in anorexic patients, it indicates that the rabbit is using adipose tissue and is at risk of developing hepatic lipidosis. hypoglycemia may occur in terminal mucoid enteropathy, liver failure, or other chronic diseases. rabbits with acute sepsis may be hypoglycemic too. 3 insulinoma has not been described in rabbits. reference levels for glucose can be found in table 1 . the complex gi physiology and the limited ability of the kidneys to correct acid-base alterations make rabbits susceptible to electrolyte imbalances. any disturbance of serum electrolytes should alarm the clinician to possible pathology of the digestive or excretory system. anorexia can rapidly lead to metabolic acidosis. the diagnostic value of sodium for rabbit patients is low. hypernatremia can be due to dehydration or loss of fluids (e.g., diarrhea, peritonitis, burns, myiasis). hyponatremia is usually associated with polyuric renal failure (acute or more commonly chronic), when urine flow in the renal tubules is too fast to impede the sodium-potassium exchange. lipemia or hyperproteinemia can artifactually decrease sodium levels in serum. reference levels for sodium can be found in table 1 . potassium is homeostatically important because it is essential for maintenance of membrane potential. changes in membrane potential can be lethal (e.g., impaired contractility of myocardial cells due to hyperkalemia can cause arrhythmias and cardiac arrest). intracellular and extracellular potassium levels are maintained by a complex mechanism of exchange between cells and microenvironment, and regulated by several hormones such as aldosterone, insulin, and catecholamines. hypoadrenocorticism has not been described in rabbits. instead, raised potassium levels are more often due to acute renal failure or urine flow obstruction. severe tissue damage can also cause hyperkalemia by dispersing potassium into the extracellular space. for the same reason, hemolysis (e.g., intravascular, bad sampling technique, letting the sample wait too long before separating the serum) can artifactually raise potassium levels. another indirect cause of higher potassium levels in serum is metabolic acidosis, which increases the exchange of potassium ions across the cell membrane. reference levels for potassium can be found in table 1 . causes of hypokalemia in rabbits include dietary insufficiency and loss of fluids from the gi system (e.g., saliva, mucoid diarrhea) or the kidneys (e.g., renal failure, diuretic drugs). stress-induced increases in catecolamine levels can also cause hypokalemia. alkalosis, although rare in rabbits, decreases the blood potassium concentrations by stimulating the cellular uptake of potassium ions. hyperproteinemia and lipemia can artifactually decrease blood potassium levels that may present clinically as sensory depression and muscle weakness. a correlation between low potassium levels in blood and "floppy rabbit syndrome" has been reported. 10 in other herbivorous species (e.g., horses), blood potassium levels fluctuate widely, depending on physical activity or even on the quantity of saliva produced during the meals. 13 these examples of serum potassium level fluctuation are purely physiological and may occur in rabbits. calcium is found in blood either bound to serum proteins or ionized free in the serum. most laboratories report a total serum calcium value, which is the sum of bound and ionized calcium. ionized calcium is a more precise measurement but is a more difficult and expensive parameter to test. total serum calcium is influenced by dietary intake, serum protein levels, and other metabolic conditions. calcium metabolism in rabbits is different from that in other animals. blood calcium levels are influenced more by the calcium content of the diet than in the dog or the cat. rabbits absorb calcium in proportion to the concentration of the ion in the gut, and the kidney eliminates the excess. vitamin d is not important in calcium absorption if dietary levels are high, yet it does play an important role if dietary levels are low. vitamin d is also important in calcium distribution within the body. as with other species, parathyroid hormone regulates blood calcium levels, but the level at which calcium is moved from the blood to the bones is high in rabbits. consequently, blood calcium levels are higher, and the normal range is broader than that in other species. growing youngsters and pregnant does use more calcium, resulting in lower blood calcium concentrations. in these rabbits blood calcium concentrations rarely rise above 14 mg/dl, even when fed calcium-rich diets, whereas adult rabbits on a varied diet can show calcium levels up to 16 to 17 mg/dl ( table 1) . the urinary excretion rate of calcium is around 45% to 60% for rabbits, whereas most mammals excrete no more than 2% of their calcium through renal filtration. this predisposes rabbits to the formation of sludge and stones in the rabbit urinary system. although the constant excretion of calcium could be a cause of renal failure in rabbits fed unbalanced diets, hypercalcemia is also a consequence of renal disease in rabbits because of the inability of the kidney to eliminate excess calcium. measurement of blood calcium is essential to diagnose and treat renal disease in rabbits. hypocalcemia is rare but is reported in rabbits. the most frequent cause of low blood calcium levels is hypoalbuminemia due to poor nutrition. hypocalcemic seizures have been described in late-pregnant and lactating does. 13 phosphorus is involved in many enzymatic systems in rabbits, but its main function is contributing to the proper formation of bones and teeth. because phosphorus is present inside cells, blood phosphate concentrations are easily increased by hemolysis (e.g., spontaneous or sampling problems). blood phosphate values should always be evaluated with blood calcium levels to determine the mineral balance in patients diagnosed with urinary tract stones, dental disease, or other signs of nutritional secondary hyperparathyroidism. because the kidney is the main organ involved in phosphorus balance by regulation of glomerular filtration and tubular reabsorption, blood phosphate levels can be an indirect measurement of kidney function and, in general, parallel azotemia. serum phosphorus levels can be elevated as a result of prerenal, renal, and postrenal effects. hyperphosphatemia usually indicates chronic kidney failure (a loss of more than 80% of nephrons) given that serum phosphorus levels are normalized by compensatory mechanisms in early-onset renal disease. hyperphosphatemia may also be an indicator of soft tissue trauma. reference levels for phosphorus can be found in table 1 . hypophosphatemia is not rare, but its clinical significance is unknown at this time, with dietary deficiencies or reduced intestinal absorption possibly involved. total protein, consisting of the sum of albumin and globulin, is an important parameter in any species of animal. many factors (e.g., age of the animal, reproductive status, pregnancy) can affect tp levels. total serum protein levels can be artifactually raised by hemostasis at the sample site through fluid loss because of digital pressure on the vessel from which the blood is collected. reference levels for tp, albumin and globulins can be found in table 1 . the main cause of hyperproteinemia in rabbits is dehydration. raised tp levels may also indicate a chronic infectious or metabolic process. measuring albumin and globulin fractions helps to differentiate the causes of hyperproteinemia. hypoproteinemia is usually due to chronic malnutrition or protein loss. if both albumin and globulin are low, hemorrhage or protein loss through exudative skin lesions such as burns or flystrike should be considered. other possible causes must be examined in cases of hypoalbuminemia with normal or raised globulins. because the liver is the only site of albumin synthesis, a lowered albumin level may indicate an advanced hepatic disease, such as hepatic coccidiosis (e stiedae) or scarring and necrosis due to the migrations of cysticercus (taenia) pisiformis larvae. protein-losing nephropathies (glomerulonephropathy) and enteropathies are rarely diagnosed in rabbits. a common cause of hypoalbuminemia in pet rabbits is chronic malnutrition either from poor diet or advanced dental disease. all causes of reduced cecotrophy (e.g., dental disease, obesity, back pain) can be reflected as low protein, especially albumin levels. a method of evaluating serum proteins is by electrophoresis (eph), which divides the globulins into distinct fractions. in acute disease, alphaglobulins are elevated; consequently, a rabbit with an alpha-globulin peak may have a bacterial infection or a developing abscess, and/or present febrile. the beta portion of globulins consists of several proteins classified as "acute-phase" proteins including fibrinogen. fibrinogen correlates with inflammation in rabbits, although the correlation is not as evident as in other species. plasma is preferable to serum for eph because it does include fibrinogen. gamma-globulins are mainly antibodies, and a peak of this fraction indicates a subacute to chronic inflammation, especially when associated with a bacterial infection. coronavirus infections lead to an impressive increase of rabbit globulins as feline coronavirus does in cats, but this disease is probably limited to laboratory set-tings. the correlation between eph curves and different rabbit pathologies is unknown and currently under investigation. there are serological tests for antibodies against e cuniculi, toxoplasma gondii, treponema cuniculi, myxomatosis, viral hemorragic disease, and pasteurella multocida, although their availability varies in different parts of the world. the most common serological assay used in private practice is for the antibodies to e cuniculi. laboratory studies have shown that infected rabbits start to develop a measurable serological immune response 4 weeks after infection, 2 weeks before e cuniculi is found in the kidney or in the urine, and at least 8 weeks before any brain lesion. 14, 15 if a serologic test for e cuniculi is negative for a neurological rabbit patient, one could assume, with confidence, that this patient does not have the disease. if the test is positive, neurological signs may be due to e cuniculi or a different disease process. the seropositive result may be due to past exposure to the pathogen, which is common within most rabbit populations. further laboratory tests may be helpful to determine a definitive diagnosis. a correlation between antibody titers and neurological signs is hard to prove. a rising titer after 2 weeks is often considered diagnostic in suspect cases. the kidney is a target organ for e cuniculi, so evaluation of renal function and structure by biochemistry, urinalysis, and ultrasound may aid one's diagnostic overview. inflammatory changes in the cerebrospinal fluid are suggestive of e cuniculi but are nonspecific. at the present time, the definitive diagnosis of rabbit encephalitozoonosis requires histopathology or isolation of the spores from the urine by microscopy or polymerase chain reaction assay. although serology for t cuniculi can be used to screen a breeding facility, it has little value in clinical practice. at least 3 months are needed for antibody levels to become measurable in the blood even with clinically evident dermatological lesions. this substantial lag in the development of antibody titers can lead to false-negative test results. positive titers without skin lesions can indicate subclinical disease or previous infection in which the skin lesions are missed. antibody titers fall rapidly once the rabbit is treated for t cunicoli. 2 serological testing for p multocida is available in some countries, but its use in clinical practice is limited. a single positive response has no clinical significance because many rabbits harbor the bacteria in the nasal cavities without illness. a positive titer can also indicate previous exposure to the bacteria rather than clinical infection. repeat testing after 2 weeks, while looking for a rising titer, can be helpful in obtaining a correct diagnostic evaluation. results from rabbits younger than 2 months can be difficult to interpret because maternal antibodies are still present. a high antibody titer is not protective and may indicate subclinical disease. urine samples can be collected from spontaneous micturition, bladder expression, catheterization (quite difficult because sedation/anesthesia is required), or cystocentesis, with the latter being the preferred method for obtaining a sample for bacterial culture. care should be taken when performing a cystocentsis, because microtrauma to the bladder wall can stimulate local mineralization and it is possible to puncture the cecum, an enlarged uterus, or even an abscess. if bacteriology is not required, free catch from a clean litter tray is the easiest way to obtain a urine sample. manual expression of the bladder should be done carefully because the wall is thin and ruptures easily, especially if an obstruction is present or the rabbit struggles. in cases of chronic cystitis, the risk for bladder rupture is lower because the bladder is thickened. it is preferable to collect the first urine of the morning and to run the analysis as soon as possible. normal rabbit urine is dense and rich in minerals, so it should be centrifuged or filtered for biochemical analysis or examinations. clear urine indicates low calcium excretion, which may be pathological because of renal failure or physiological in the case of growing or lactating rabbits. the color may range from light yellow to reddish brown. in most cases, dark urine is caused by dietary pigments; however, the urine should be checked for hematuria, which may be caused by uroliths, urinary tract inflammation/infection, uterine problems, or anticoagulants. test dipsticks work well to evaluate blood, glucose, ketone, and ph levels in rabbit urine but are not accurate for other parameters. glucose may be found as a consequence of stress hyperglycemia; checking urine collected at home can help to rule out stress-related problems. true glucosuria indicates altered energy metabolism, such as hepatic lipidosis, or, very rarely, diabetes mellitus. ketones are always abnormal and indicate anorexia (even short duration), hepatic lipidosis, pregnancy toxemia, or diabetes. the ph of rabbit urine tends to be high (7.5 to 9) in rabbits fed a correct diet. acidic urine indicates acidosis due to anorexia, fever, pregnancy toxemia, or hepatic lipidosis. it is possible that, because a normal kidney would eliminate acidic urine in cases of acidosis, the finding of alkaline urine in an anorexic rabbit could indicate compromised renal function. specific gravity (sg) indicates the ability to concentrate urine. a refractometer is a more reliable tool to measure sg than dipsticks. most normal rabbit urine is quite dilute, with an average sg of 1.015 (range, 1.003-1.036). prerenal azotemia is associated with raised sg (ͼ1.030), whereas true azotemia linked to renal failure is associated with dilute urine (sg ͻ1.013). urine-specific gravity is useful if it is examined alongside urine protein concentrations. protein traces in clinically normal rabbits, especially the young, are not significant, whereas a dilute urine (ͻ1.020) with proteins is very significant. proteinuria appears earlier than biochemical changes in renal disease, making this test useful in clinical practice. measuring urine proteins/urine creatinine ratio (ͻ0.6 is suggested as normal) may further improve the sensitivity of urine-specific gravity testing. sediment examination can differentiate normal urine that is rich in crystals from sludge. after centrifugation, normal crystals should resuspend when shaken, while sludge remains as a solid mass. cytology is not very different from that of other mammals; a small number of leukocytes are considered normal in rabbits. gram or trichrome stains can reveal e cuniculi spores. 3 parathyroid hormone, hematological and biochemical parameters in relation to dental disease and husbandry in pet rabbits laboratory medicine: avian and exotic pets notes on rabbit internal medicine the biology of laboratory rabbit alteration of sleep in rabbits by staphylococcus aureus infection hematological effects of exposure to three infective agents in rabbits physiological stabilization of rabbits after shipping rabbit surgical pathology effects of t-2 toxin on ovarian activity and some metabolic variables of rabbits textbook of rabbit medicine the anatomy, physiology and the biochemistry of the rabbit spontaneous diabetes mellitus in the new zealand white rabbit veterinary laboratory medicine serological and histological studies on adult rabbits with recent naturally acquired encephalitozoonosis ferrets, rabbits and rodents clinical medicine and surgery key: cord-284893-qi6dkcb3 authors: wilson, kumanan; graham, ian; ricketts, maura; dornan, christopher; laupacis, andreas; hebert, paul title: variant creutzfeldt–jakob disease and the canadian blood system after the tainted blood tragedy date: 2006-10-02 journal: soc sci med doi: 10.1016/j.socscimed.2006.08.023 sha: doc_id: 284893 cord_uid: qi6dkcb3 the transfusion transmission of hepatitis c and hiv to thousands of canadian blood recipients was one of this country's largest public health catastrophes. in response to this crisis, and in an effort to prevent such a tragedy from occurring again, the canadian blood system has undergone substantial reform. variant creutzfeldt–jakob (vcjd) disease was the first infectious threat faced by the blood system since undergoing reform. the response at the time to this risk provides insights into the canadian blood system's new approach to infectious threats. our analysis of the decision-making concerning vcjd identifies two dominant themes characterizing the new blood system's approach to safety: (1).. the adoption of a precautionary approach to new risks which involves taking action in advance of definitive evidence, and (2).. risk aversion amongst policy makers, which has contributed to the adoption of safety measures with comparatively high cost-effectiveness ratios. overall the principles governing the new blood system have contributed to the system both providing protection against emerging infectious risks and regaining the confidence of the public and recipients. however, the current set of policy factors will likely contribute to increasingly risk-averse policy making that will contribute to continued increases in the cost of the blood system. the challenge the blood system now faces is to find the appropriate balance between maximizing safety and ensuring the system remains affordable. the transfusion of individuals with products infected with hiv and hepatitis c was, arguably, the largest public health catastrophe in canada's history. estimates suggest that infected transfusions led to more than one thousand individuals acquiring hiv and up to 30 000 individuals acquiring hepatitis c (krever, 1997a) . a national inquiry into the functioning of the blood system and how it could have led to the tragedy, headed by justice horace krever, precipitated a transformation of the delivery of transfusion services in this country. this transformation involved the replacement of the canadian red cross as the operator of the blood system and the creation of new financial arrangements and operating principles (krever, 1997b) . it has now been 8 years since canada's blood system has undergone reform and the opportunity exists to reflect on how successful the transition to a new system has been. from the perspective of managing infectious threats, the new blood system would have to be viewed as a major success. canada has acted aggressively to protect the blood supply from real and theoretical risks such as variant cjd, west nile virus and severe acute respiratory syndrome (canadian blood services, 2004) . in this regard, the post-krever blood system has received high marks, including from hemophiliacs who were one of the primary groups affected by the tainted blood tragedy (canadian hemophilia society, 2005) . however, the blood system has not been without criticism, which has primarily been directed at the introduction of safety measures with prohibitive cost-effectiveness ratios that have contributed to escalating blood costs. there are several opportunities to learn from the canadian blood system's reform efforts and in many ways the blood system provides an important model for other public health sectors which are addressing new and emerging risks in an increasingly risk averse environment. to better understand how decisionmaking has been transformed in the post-krever era we provide a detailed examination of the canadian blood system's management of variant creutzfeldt--jakob disease, its first infectious threat since undergoing transition. while many faults could be found with how blood officials managed affairs leading to the tainted blood tragedy, one of the areas of particular concern was how scientific evidence was utilized to formulate policy. our analysis is therefore assisted by the use of a descriptive policy analysis framework that focuses on the role of information in the policy process. we review our findings in the context of subsequent blood policy decisions to arrive at general principles governing policy making in the post-krever environment. in the late 1990s, the canadian blood system and health officials were confronted with a true policy dilemma: how to manage the theoretical risk of blood transmission of cjd. this challenge emerged just as justice krever was releasing his final report and federal/provincial/territorial officials were creating a new blood system in line with many of the recommendations of this report. the challenge also presented itself as the united kingdom was addressing the outbreak of bovine spongiform encephalopathy, which eventually led to the emergence of vcjd in humans, and dealing with criticism of their response to this crisis. thus, the potential transfusion transmission of cjd combined aspects of two of the highest profile public health controversies at the time. further contributing to the challenge of managing the infections risk posed by cjd to the blood supply was the unusual nature of the disease itself. cjd is a rare infectious condition that is believed to be caused by a new form of infectious agent, an infectious protein, known as a prion (prusiner, 1982) . the infection is devastating and affected patients suffer progressive neurological deterioration and dementia due to spongiform changes in the brains of the victims. patients inevitably die from the infection, usually within a year of the diagnosis. while extremely rare (o1% of all cases), there have also been several documented cases of iatrogenic transmission of cjd. the documented iatrogenic transmission of cjd via human growth hormone, in particular, raised concerns in the united states and canada about whether the condition may also be transmissible via blood products. this concern arose in the canadian blood system, as it was recovering from the tragedy of hepatitis c and hiv blood transmission. as a consequence, the potential transfusion transmission of cjd was viewed as the first major test for the blood system after these infectious threats (vaughan, 1996) . the challenge of managing the potential transmission of cjd via blood products was further complicated by the discovery, in 1996, of a variant form of cjd in the united kingdom . the condition was believed to have arisen from bovine spongiform encephalopathy (bse). while there was no epidemiological evidence of blood transmission of vcjd, the theoretical risk was considered higher than classical cjd for a variety of reasons (cashman, 1999) . policy makers in the united kingdom decided to reject donations from their citizens and import their plasma requirements due to concerns over the potential blood transmission of vcjd. this prompted decisionmakers in the united states and canada to evaluate whether they should accept donations from individuals who had traveled to the united kingdom. however, this would potentially reduce the blood supply and cause shortages. ultimately canadian policy makers decided to defer donations from individuals who had spent 6 months in the united kingdom between the years 1980 and 1996 (the peak period of the bse epidemic). it was anticipated that this policy would reduce the risk of vcjd to the blood supply and ensure that the blood supply remained adequate (wilson et al., 2001) . these policies have been modified as further evidence has accumulated of the prevalence of bse and donor deferral policies have been instituted for individuals who have resided in france and all of western europe. most significantly, scientific evidence has accumulated which validates the institution of the precautionary policies, both from animal models demonstrating transfusion transmission and from case reports of transfusion transmission in humans (wilson & ricketts, 2004) . a framework for understanding decision-making on health risks to understand decision-making on issues of risk pertaining to the blood system, analytical tools that provide a comprehensive understanding of the multiplicity of factors that influence policy are required. descriptive policy analyses are well suited to analyzing policy making concerning risks by identifying the critical factors influencing decisionmaking as well as providing insights on where decision-making breaks down and, equally important, where it works effectively (hogwood & gunn, 1984) . descriptive policy analyses in health, however, have often been based on the assumption that rational decisions are made on the basis of complete information (pal, 1992) . given the uncertainty of scientific information on potential health risks, these information-based models may not be adequate. alternative models to evaluate decision-making have focused on the role of value systems or institutions. however, to be truly comprehensive, approaches to policy analysis integrating the impact of information, values and institutions are needed to ensure that both the policy makers and the public understand how decisions are made concerning health risks. paul sabatier has compiled some of the available frameworks for conducting descriptive policy analyses (sabatier, 1993a) . one of the criteria used for selecting frameworks is that they address the roles of conflicting values and interests, information flows, institutional arrangements and variations in socioeconomic environment on the policy process. the following frameworks were identified as useful for describing decision-making within a given political system or set of institutional arrangements: the stages heuristic, institutional rational choice, multiple-streams framework, punctuated equilibrium framework and the advocacy coalition framework. the last framework, the advocacy coalition framework (acf), was proposed by sabatier and suggests that decision making occurs in a policy subsystem involving individuals from a variety of public and private organizations who are actively concerned with a policy problem. within this subsystem, individuals aggregate into advocacy coalitions based on shared ideologies and beliefs. the success of the coalitions in translating their beliefs into policy is dependent upon their resources (money, expertise, legal authority and size). policy brokers attempt to mediate the conflict between the various advocacy coalitions. this process of conflict and mediation eventually results in policy outputs by the governing structures in the subsystem (sabatier, 1987; sabatier, 1993b ). the acf model has been identified as being effective in describing decision-making in a variety of policy sectors (jenkins-smith & sabatier, 1994) . a modification of the acf model has been put forward by jonathan lomas (fig. 1) (lomas, 2000) . in this model individuals make policy in an ''institutional structure for decision-making''. this structure consists of ''formal'' actors, who actually participate directly in the decision-making process, and ''informal'' actors, who influence decisionmaking through other means. values in this model are divided into ideologies (views on how things ought to be), beliefs (causal assumptions on how things are), and interests (responses to incentives and rewards). in addition, this model pays greater attention to the role of information producers and purveyors. it specifically examines how information is produced and spread and how the value systems of those involved in the policy subsystem influence the interpretation and use of this information. our choice of the lomas framework was based on face and content validity parameters; i.e. the domains we felt would be important are appropriately captured in the framework. the vcjd decision was primarily characterized by how policy makers utilized scientific information. in this sense, the lomas framework is well suited to conducting the analysis given its focus on knowledge development and knowledge transfer. the framework's hypothesis that policy makers resolve conflicts between their various value systems through a process of cognitive dissonance reduction is also well suited to analyzing a policy problem where information is uncertain and value systems would necessarily play a particularly prominent role. the objective of our overall study was to understand and compare the decision-making processes concerning two creutzfeldt-jakob disease-related decisions: a 1995 withdrawal of blood products from a classical cjd donor and a 1999 decision to defer donations from individuals who had traveled to the united kingdom for 6 months during the peak of the bse outbreak (1980) (1981) (1982) (1983) (1984) (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) . we present, here, the full analysis of the second decision, with the first decision acting as important historical context. our study consisted of a literature review and semi-structured interviews. the literature review included a systematic review of the risk of transmission of cjd via blood products, a content analysis of newspaper reporting as well as a review of important policy documents. in total we conducted 31 semi-structured interviews with key informants of all major decision-making organizations. individuals were asked to describe decision-making leading to the 1995 and 1999 decisions as well as other relevant decisions. individuals were also asked about the role of information, information purveyors, decision-making organizations and external factors in the decision-making process. all information from the interviews was coded using qsr nudist, a qualitative software program. findings of the study were fed back to decision-makers through member check sessions with key stakeholder organizations. our findings have been presented in a series of papers, initially focusing on information (wilson, code, & ricketts, 2000) and information purveyors , then institutions (wilson et al., 2001; wilson, mccrea-logie, & lazar, 2004) and finally the role of values and changes in ideology (wilson, wilson, hebert, & graham, 2003) . we synthesized the results of these studies using the lomas framework. based on this synthesis we isolate the key policy factors that are driving current decision-making in the canadian blood system. we then propose mechanisms by which acting on these policy factors could influence future decisions. factors influencing decision-making (table 1 ) in canada three pieces of information played an important role in influencing the decision-making process leading to the donor deferral decision: (1) the risk of transmission of vcjd via blood products, (2) the impact of donor deferral on the blood supply, and (3) the degree of reduction in the blood supply the blood system could sustain. all of these pieces of information had substantial levels of uncertainty associated with them. there were no epidemiological studies on risk of blood transmission of vcjd and an estimation of the risk was based primarily on biological models. based on these models the theoretical risk possibility of blood transmission of vcjd was considered for the following reasons: vcjd had been demonstrated to be transmissible through a peripheral route (gi tract) and thus peripheral transmission via blood products was believed to be possible, the prion concentration in affected tissues was high in vcjd and prions are found in the lymphoreticular system in vcjd which is intimately linked to the blood supply (cashman, 1999) . scientists argued that this evidence of risk based on biological models was substantial enough to warrant government action to protect the blood supply. given the uncertainty of information on risk of blood transmission of vcjd the impact of a policy on the blood supply needed to be considered before proceeding. this information was obtained from a survey that suggested that a 3% reduction in the blood supply would occur with a 6-month deferral policy. previous experience in the blood system indicated that a 3% reduction was sustainable, although there was considerable uncertainty over this estimate. in an attempt to integrate all of this information, as well as the likelihood of developing vcjd over different residency periods in the united kingdom, a risk modeling exercise was performed. this analysis also suggested that the risk of contracting vcjd increased after 6 months residence in the united kingdom. however, this model was based on infectivity rates of bse that, to this date, remain unclear (elsaadany & giulivi, 2000) . three major information purveyors influenced the policy process leading to the donor deferral decision: scientists, the media and think tanks. the media's primary role was to disseminate the release of a report by a think tank advisory committee on bioethics, which advocated that the canadian blood (bayer advisory council on bioethics, 1998) . in combination, this report and the media coverage it received played an important agenda setting function by bringing the emerging issue to the public's attention . scientists also played an important role in communicating information on risk to policy makers. in particular, due to the lack of scientific expertise on the subject, one scientist came to play a prominent role in the decisionmaking process. this individual was a consultant on three separate sources of information that were supplied to the decision-makers. all of these recommended that the government take action to protect the blood supply from vcjd (bayer advisory council on bioethics, 1998; cashman, 1999 ; expert advisory committee on blood regulation, 1998) . we had a unique opportunity to assess the impact of institutions on decision-making by being able to compare the vcjd donor deferral decision to another cjd related decision that took place prior to institutional change in the blood system; the 1995 recall of blood from a classical cjd donor. the major structural changes to the blood system that took place over this time period was the replacement of the canadian red cross as sole operator of the blood system by two separate operators, he´ma-que´bec in the province of quebec, and canadian blood services in the rest of canada. we observed that the movement to a two-operator system had an important impact on the decision-making process. the presence of a second operator introduced a form of ''check and balance'' on the decisionmaking processes of the larger operator by proposing competing policy options to address the vcjd problem. however, it also increased the complexity of the decision-making process and initially produced some inter-institutional conflict (wilson et al., 2001) . distribution of regulatory authority and funding also played an important role in the decisionmaking process, specifically the separation of these two functions. in canada regulatory authority for blood products exists at a federal level. financing of the blood system is the responsibility of the provinces. in this system, the incentive exists for the regulator to introduce policies that maximize the safety of the blood supply, and the financial considerations of the decision play a secondary role (wilson, mccrea-logie et al., 2004) . other institutional effects include decisions made by institutions in parallel subsystems, in particular other countries' blood systems. the uk decision to ban plasma donation from its own citizens played a large role in initiating the decision-making process in both canada and the united states as to how to handle donations from individuals who had traveled to the united kingdom. of particular importance, canadian policy makers attempted to coordinate canada's donor deferral policy with that of the united states. this was largely a consequence of the fact that canada imports a substantial portion of its plasma requirements from the united states. in general, canadian policy makers are expected to meet international standards in protecting the blood supply (canadian blood services, 2003) . we observed that most individuals shared a common belief, defined as a ''causal assumption of what is'', on the risk of transmission of cjd; that transfusion transmission was a theoretical risk with no known documented cases of transmission. however, while the risk was viewed as theoretical, the type of risk which was presented is one to which the public and policy makers would be particularly averse. the factors contributing to this perception of risk include the potentially catastrophic, involuntary nature of the risk, the lack of knowledge of the risk and the lack of trust in the system's ability to manage the risk (slovic, 1987) . ideologies, defined as ''causal assumptions of how things ought to be'', played an important role in determining how information was interpreted and utilized to develop policy. two dominant ideologies on how decision-making should take place on issues of risk were at play: evidence-based decision-making and the precautionary principle. the precautionary principle came to play a particularly prominent role largely as a consequence of the krever inquiry into the blood system use. however, we observed a clear tension in which decision-makers struggled with the idea of introducing a policy that could create a health risk (blood shortages) to protect against a risk for which no epidemiological evidence existed . at the institutional level, we found competing interest systems at work. the regulator's primary responsibility was to protect the safety of the blood system. the operators, in contrast, were interested in balancing safety with adequacy of supply. however, all players in the blood system recognized the crucial need to reestablish public confidence in the blood system and their responsibility to protect the blood supply on behalf of the public. at the level of the policy-maker, the lasting effects of the krever inquiry played an important role in influencing individuals' actions. the spotlight that was placed on previous decision-makers in the blood system and the legal consequences of the subsequent criminal probe created a climate that encouraged implementation of a risk-averse policy (picard, 2002) . reconstructing the policy process from the factors we have analyzed presents us with the following explanation of why events unfolded as they did. agenda setting in canada primarily occurred due to policy decisions made in other countries (e.g. the uk blood system). public awareness was raised by the release of a think-tank report and the dissemination of the information from this report via the print media. the decision to introduce a partial measure to protect against the theoretical risk, in the absence of definitive evidence of the risk, was largely influenced by the knowledge that the united states would proceed with a similar decision. the canadian decision-making process was also shaped by the emergence of the precautionary principle as a dominant ideology in public health. however, perhaps the most important driving factors in the decision-making process were the past experience of the blood supply with hepatitis c and hiv, the general shadow cast by the recent krever inquiry, policy maker fiduciary duty to the pubic and their need to re-establish public trust by being seen to be acting pro-actively. the vcjd policy decision deserves closer examination for several reasons. it demonstrated how policy was made to address an, at the time, theoretical risk. the canadian vcjd donor deferral decision was also emblematic of how other nations addressed the vcjd threat. in addition to withdrawing blood products derived from individuals subsequently diagnosed with vcjd and importing fractionated products from abroad, the uk has recently decided to ban donations from individuals who had previously received a transfusion. france also has instituted precautionary policies including the introduction of leukoreduction, which theoretically would remove infectious material from donated blood (lee, 2001) . the us policy regarding vcjd was similar to canada's, choosing to introduce donor deferral policies for individuals who had traveled to countries in which bse was endemic (fda, january 2002) . the vcjd decision-making process in all of these countries reflected a paradigm shift in how to manage emerging risks. this new paradigm involved the institution of protective measures at an early stage of the risk identification process and reflected a conscious decision by policy makers to act in advance of complete scientific information. important lessons can be learned from the canadian vcjd policy-making process and the decision-making process stands in stark contrast to the decisions concerning hepatitis c and hiv in the pre-krever blood system. in particular, two key themes that have come to dominate decisionmaking in the post krever era deserve further analysis-the application of the precautionary principle to blood policy and the challenge of rising costs in a risk-averse blood system. blood policy makers, in addressing the potential threat of vcjd, ultimately embarked upon a strategy that they believed balanced the reduction in blood supply with reducing the risk of exposure of canadians to potentially infected blood products. this decision explicitly acknowledged the possibility of risk in the absence of epidemiological studies and represented a critical shift from the previous mechanism of policy making. many criticisms exist of blood policy making in several countries leading to the transfusion transmission of hepatitis c and hiv. particularly, in the instance of hiv, the criticisms surround unacceptable delays implementing policies recognized as providing some protection to blood recipients (gilmore & somerville, 1999; picard, 1998; weinberg et al., 2002) . however, one of the primary limitations of pre-krever decisionmaking in canada was the manner in which scientific information was utilized in the formulation of policy. this was perhaps most glaringly demonstrated when considering the decision-making process concerning the adoption of surrogate testing for hepatitis c. the details of the canadian decision regarding surrogate testing has been well described elsewhere (krever, 1997c) . in summary, blood officials in the 1980s were confronted with the threat of a new form of hepatitis referred to as nona-nonb hepatitis, later discovered to be hepatitis c. this form of hepatitis was known to be transfusion transmissible, however, the virus had not been identified and thus no specific test existed to identify contaminated blood. consideration was therefore given to the use of surrogate tests which could not only identify some infected donations but also would result in the discarding of some donations that were not infected (aach et al., 1981; alter, purcell, holland, alling, & koziol, 1981) . canadian officials awaited the results of a prospective trial that compared the rates of post transfusion hepatitis from individuals who received blood from donors who had surrogate testing compared to those who received blood from donors who had not undergone surrogate testing. unfortunately, by the time evidence demonstrating the efficacy of the surrogate testing strategy become available, thousands of individuals had become infected by hepatitis c through blood transfusions, many of which could potentially have been prevented (blajchman, bull, & feinman, 1995) . on reflection, it becomes apparent that a fundamental failing of the canadian blood system's management of hepatitis c was the adoption or perhaps misapplication of the evidence-based paradigm when developing policy concerning safety. the evidence-based paradigm is dominated with the belief in a hierarchy of evidence that asserts that randomized trials are the highest level of evidence (upshur, 2003) . in the tainted blood tragedy such an approach was found to be wanting in many respects, primarily related to the consequences of waiting for high quality evidence when the health of populations, as opposed to the health of individuals, is at risk. reflecting this recognition, the ''precautionary principle'' has emerged as a new paradigm governing the use of scientific information. the precautionary principle essentially states that complete evidence of risk does not have to exist before action is taken to protect against the risk, particularly when the risk is potentially catastrophic (wingspread conference participants, 1998). although there are numerous interpretations of the principle, applications generally advocate an-ticipatory action to protect against harm, prioritize protection of public health and the environment and promote public participation in decision-making (stoto, 2002) . while the principle has become highly influential in risk decision-making in the environment and in health, it also has been heavily criticized. opponents of the principle point to its lack of clarity, potential to create unnecessary fear and potentially denying the public the benefits of new technology (morris, 2000) . in addressing the theoretical risk of vcjd, the canadian blood system, and blood systems around the world, was guided by the precautionary principle. however, at the same time they also integrated components of evidence-based policy making in an attempt to find a middle ground between these potentially conflicting paradigms. specifically, they chose to introduce a measured response that would not cripple the blood supply. this response was then calibrated as new evidence emerged on risk of transmission. in doing so they succeeded in accomplishing several policy objectives including reestablishing confidence in the blood system and demonstrating to the public that policy makers were acting proactively to protect the public. most importantly, as evidence accumulated to demonstrate the probable transfusion transmissibility of vcjd, the policy decisions made by canada and other countries appear to have been warranted and likely prevented further spread of vcjd through transfusion (llewelyn et al., 2004; peden, head, ritchie, bell, & ironside, 2004) . in hindsight, the integration of precautionary policy making in the new blood system would have to be considered a major success (wilson & ricketts, 2004) . while the new blood system's precautionary approach to blood safety has received praise it also has not been without some criticism. in canada, over a three-year period since the blood system underwent structural reform, expenditures in the blood system have increased by 50% . these rising blood system costs have been attributed to several factors including the increase in use of blood products, and the increase in cost of specific blood products such as intravenous immunoglobulin (wilson, macdougall et al., 2003) . however, attention has particularly been focused on the introduction of new safety measures that have only marginally improved the safety of the blood supply. some transfusion policy analysts have described the introduction of these safety measures as ''irrational''. these individuals point to the normally prohibitive cost-effectiveness ratios of many of theses measures (bayer & feldman, 1999) . the cost-effectiveness ratios associated with several of the post-krever safety measures has far exceeded the generally accepted cost-effectiveness ratios of $50 000 to $100 000 per qaly (laupacis, feeny, detsky, & tugwell, 1992) . for example, the cost/quality adjusted life year of nucleic acid amplification testing for hepatitis c is $4 million/ qaly and for solvent detergent plasma $8 million/ qaly (aubuchon & petz, 2001) . these tests have also impacted upon the cost of blood. nucleic acid amplification testing for both hepatitis c and hiv has been estimated to contribute 13-20% of the cost of a unit of blood in the united states (weinberg et al., 2002) . blood systems are also being confronted with the decision of adopting several expensive new safety measures, such as pathogen inactivation technologies (council of europe, 2001) . in the united states (us) the medicare payment advisory committee identified that blood-related costs have been increasing more rapidly than other hospital costs placing strains on the current diagnosis related group (drg) payment system (medicare payment advisory committee, 2001) . in canada, provinces, which are responsible for funding the blood system, have expressed unease about rising blood costs and asked for a reconsideration of how policy decisions concerning the introduction of safety measures are being made (ibm consulting, 2002) . the decision to introduce highly risk averse policies (i.e. the choice of policies with a high certainty of eliminating remote risks) does not appear to be driven by ''public hysteria'' but rather by incentive systems that act directly on the policymakers. evidence for the lack of public demand driving the policy process is provided by the introduction of other similar protective policies. for example leukoreduction, a process by which white blood cells are removed, was introduced to protect against transfusion reactions and potentially other immune mediated effects. the policy met some controversy over its necessity and could not be expected to have been a high agenda issue for the public who would have little understanding of the process and for whom transfusion reactions would not be a major health concern (goodnough, 2000) . the other explanation for the lack of public influence on blood policy is the absence of welldefined advocacy coalitions representing the public and the interests of consumers of blood products (orsini, 2002) . the majority of recipients of blood products are members of the general population who cannot necessarily be identified in advance. the canadian blood system has also explicitly involved representatives of various consumer groups in their policy making process which has reduced the need for public lobbying by these individuals. in contrast to the relative lack of risk aversion amongst the public, our analyses suggest that risk aversion on the part of policy makers is likely responsible for the introduction of several of the safety policies. canadian officials are eminently aware of the public health consequences of the transfusion transmission of hepatitis c and hiv. they also cannot help but be aware of the legal consequences of those who were involved in the decision-making processes at the time. the current incentive structure does little to protect against liability because the recommendation of the justice krever to introduce a no-fault compensation system for transfusion related injury was not implemented. such systems have been found to be effective in controlling litigation in pediatric vaccination, an analogous policy area (plotkin, 2001) . further contributing to risk-averse policy making is the separation of funding from decision-making in the blood system, with the federal government having the authority of introducing safety measures but not the responsibility for paying for them. the impact of this structural factor on blood system costs could have been mitigated if justice krever's recommendation to have hospitals pay for blood products had been introduced since the budget restrictions of the hospitals would have limited their ability to pay for expensive products. despite the growing cost pressures on the blood system, we would expect the canadian blood system's proactive response to threats to blood safety to continue, given the current set of operating principles and policy factors at play. consequently, so will the trend towards the adoption of risk-averse safety measures with marginal cost-effectiveness ratios. if individuals in the blood system are interested in continuing the current practice of ensuring a safe blood supply with cost being a secondary concern, little change needs to occur in the decision-making process. on the other hand, if blood system decisionmakers or provincial officials responsible for funding the blood system desire a change to this approach, it is unlikely that additional studies demonstrating the comparatively poor cost-effectiveness of safety measures alone will have much impact. the current set of institutional arrangements, in which the regulator can introduce safety regulations and not be held directly responsible for the costs of these regulations, will continue to encourage the implementation of safety measures to ensure a high level of blood safety. this is also encouraged by the arms length relationship between the blood system operator and the provincial funders that permits the operator to independently introduce safety measures. to combat the impact of these factors provincial governments will have to make efforts to regain control of the policy making process in the blood system, or perhaps require federal regulators to pay a component of the costs associated with their safety regulations. the scenario of such federal unfunded mandates imposing cost burdens on other orders of government has been observed in the us where it was partially addressed through legislative means (conlan, riggle, & schwartz, 1995) . a risk-averse approach to blood safety is further encouraged as long as decisionmakers are aware of their potential legal and public accountability and a no-fault compensation program for transfusion injured recipients that limits legal liability may help address this issue. our analysis also suggests that as long as the canadian blood supply is reliant upon importing plasma from the united states, canadian blood policy will be heavily influenced by us blood policy. the continued goal of the canadian blood system to achieve selfsufficiency may address this concern. nevertheless, in an increasingly integrated world, decisions made in parallel policy subsystems of other nations' will play a crucial role in determining policy. standard of care may be defined as the international response to a threat and a decision-maker who disagrees with this response may still be left with little option but to meet the international standard. despite the presence of these factors, the adoption of highly risk averse policies cannot continue endlessly and the opportunity costs of these policies will become increasingly evident. eventually, policymakers will have to decide at what level of uncertainty of risk or at what level of cost/qaly safety measures will not be introduced. the decision of whether to adopt pathogen inactivation technologies, which offer the promise to remove both known and unknown pathogens from transfusions although at a substantial cost, will present an interesting challenge to the continued adoption of new safety measures. in many ways, the canadian blood system serves as a model for a transformed system emerging from a crisis of confidence. vcjd represents the first infectious threat to this transformed system and important lessons can be learned from how this threat was managed. decision-making 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latest unknown in struggle to restore faith in blood supply legal, financial, and public health consequences of hiv contamination of blood and blood products in the 1980s and 1990s a new variant of creutzfeldt-jakob disease in the uk the reporting of theoretical health risks by the media: canadian newspaper reporting of potential blood transmission of creutzfeldt-jakob disease risk of acquiring creutzfeldt-jakob disease from blood transfusions: systematic review of case-control studies the challenge of an increasingly expensive blood system a policy analysis of major decisions relating to creutzfeldt-jakob disease and the blood supply how should canada fund the blood system? an evaluation of the chargeback proposal understanding the impact of intergovernmental relations on public health: lessons form reform initiatives in the blood system and health surveillance the success of precaution? managing the risk of transfusion transmission of variant creutzfeldt-jakob disease the application of the precautionary principle to the blood system: the canadian blood system's vcjd donor deferral policy wingspread statement on the precautionary principle this study was supported by a grant from the canadian institutes of health research. thanks also to cathy code and nadya ahmad. key: cord-315293-kng4z4kf authors: quesenberry, katherine e.; de matos, ricardo title: basic approach to veterinary care of ferrets date: 2020-05-29 journal: ferrets, rabbits, and rodents doi: 10.1016/b978-0-323-48435-0.00002-2 sha: doc_id: 315293 cord_uid: kng4z4kf the approach to preventive medicine and basic veterinary care in ferrets is very similar to that used in dogs and cats. special equipment needs are minimal, and pet ferrets can be easily incorporated into a general small animal practice. this chapter describes the unique aspects of handling, restraint, and clinical and treatment techniques used in ferrets. the approach to preventative medicine and basic veterinary care in ferrets is very similar to that used in dogs and cats. special equipment needs are minimal, and pet ferrets can be easily incorporated into a general small animal practice. however, there are unique aspects of handling, restraint, and clinical and treatment techniques used in ferrets. for procedures not discussed here, modify techniques used in other small animals by using instrumentation appropriate for the ferret's small size and choosing appropriate sedation or anesthesia to facilitate the procedure while minimizing stress or discomfort in the ferret. most ferrets are docile and can be easily examined without assistance. however, an assistant is usually needed when taking the rectal temperature, when administering injections or oral medications, or if an animal tends to bite. young ferrets often nip, and nursing females and ferrets that are handled infrequently may bite. ferrets often bite without warning. therefore always ask the owner if the ferret bites before handling it and take precautions accordingly. obtain the rabies vaccination history before physical examination. be aware that rabies protocols for animal bites from vaccinated and unvaccinated ferrets differ by locale. ferrets that are prone to biting and are not currently vaccinated for rabies may need to be sedated for procedures requiring restraint. depending on the ferret's disposition, several basic manual restraint methods can be used for examination. for tractable animals, lightly restrain the ferret on the examination table. examine the mucous membranes, oral cavity, head, and skin. then pick the ferret up and support its body with one hand while using the other hand to auscultate the thorax and palpate the abdomen. for an active animal or one that bites, scruff the ferret and suspend it with all four legs off the table (fig. 2.1 ). most ferrets become relaxed with this hold, and the veterinarian can examine the oral cavity, head, and body; palpate the abdomen; vaccinate; and clean the ears. however, even scruffing may not work for fractious animals. to restrain a ferret for a procedure, hold it firmly by the scruff of its neck and around the hips without pulling the legs back. most ferrets struggle if their legs are extended by pulling on the feet. some animals can be distracted during a procedure by feeding a meat-based canned food or a small amount of a supplement such as ferretone (8-in-1 pet products, islandia, ny) by syringe. for very fractious or anxious animals or for procedures requiring lengthy restraint, light sedation or anesthesia may be indicated (see chapter 37) . flexible digital thermometer that is well lubricated. the normal rectal temperature of a ferret is 100.5°f to 102.5°f (38.0°c to 39.2°c); however, a mean of 102°f (38.8°c), with a wider range of 100°f to 104°f (37.8°c to 40.0°c), is also reported. 22 physical examination of a ferret can be performed quickly and efficiently if a few simple guidelines are followed. observe the attitude of the animal. ferrets may sleep in the carrier in the veterinary office; however, once awakened for the examination, a ferret should be alert and active. assess hydration by observing the skin turgor of the eyelids, tenting of the skin at the back of the neck, and moistness of the oral mucous membranes. note that skin turgor can be difficult to evaluate in a cachectic animal. estimate the capillary refill time by digitally pressing on the gingiva. examine the eyes, nose, ears, and facial symmetry. cataracts can develop in both juvenile and adult animals. retinal degeneration occurs in ferrets and may be indicated by abnormal pupil dilation. inspect for nasal discharge, and ask the owner about any history of sneezing or coughing. the ears may have a brown waxy discharge, but excessive brown exudate may indicate infestation with ear mites. observe the facial symmetry. although uncommon, salivary mucoceles occur in ferrets and present as a unilateral swelling on the side of the face, usually in the cheek or temporal area (see chapter 3) . the teeth should be clean and the gingiva pink. dental tartar is common in pet ferrets, possibly related to feeding kibble instead of natural prey. 13 plaque buildup may be exacerbated by feeding a diet with a high mineral content. 26 remove excess dental tartar by prophylactic techniques used in dogs and cats, and recommend measures to prevent tartar buildup. a pet toothpaste can be used to decrease the rate of calculus formation. 26, 28, 38 gingivitis is a common sequela of excessive dental tartar. ferrets often break off the tip of one or both canine teeth, and the broken tooth may appear dark. however, ferrets rarely exhibit sensitivity associated with a fractured canine. if the ferret exhibits sensitivity when the tip of the canine is probed, recommend a root canal or extraction, depending on the degree of tooth damage (see chapter 36) . bruxism often indicates gastrointestinal discomfort. palpate the submandibular, axillary, popliteal, and inguinal lymph nodes. nodes should be soft and may sometimes feel enlarged in overweight animals because of surrounding fat. any firmness or asymmetry warrants fine-needle aspiration or biopsy. if two or more nodes are enlarged and firm, a diagnostic workup is indicated. auscultate the heart and lungs in a quiet room. ferrets have a rapid heart rate (180 to 250 beats/min) and often a pronounced sinus arrhythmia. if a ferret is excited and has a very rapid heart rate, subtle murmurs may be missed. valvular disease, cardiomyopathy, and congestive heart failure are seen in ferrets, and any murmur or abnormal heart rhythm should be investigated further (see chapter 5) . the ferret's normal respiratory rate is 33 to 36 breaths/min (see chapter 6) . palpate the abdomen while either scruffing the ferret or supporting it around the thorax with one hand. this allows the abdominal organs to displace downward, facilitating palpation. if the history is consistent with an intestinal foreign body or urinary blockage, palpate gently to avoid causing iatrogenic injury, such as a ruptured bladder. palpate the cranial abdomen, paying attention to the presence of gas or any firm, irregularly shaped material in the stomach area, especially in ferrets with a history of vomiting, melena, or chronic weight loss. the spleen is often enlarged, which may or may not be significant, depending on other clinical findings (see chapter 5) . a very enlarged spleen may indicate systemic disease or, very rarely, idiopathic hypersplenism, and further diagnostic workup is warranted. examine the genital area, observing the size of the vulva in females. vulvar enlargement in a spayed female is consistent with either adrenal disease or an ovarian remnant; the latter is rare. examine the preputial area and size of the testicles of male ferrets; preputial and testicular tumors are sometimes seen. check the fur for evidence of alopecia. tail tip alopecia is common and may be an early sign of adrenal disease. symmetric, bilateral alopecia or thinning of the fur that begins at the tail base and progresses cranially is a common finding in ferrets with adrenal disease. examine the skin on the back and neck for evidence of scratching. pruritus is common with adrenal disease and also may indicate ectoparasites (e.g., fleas or sarcoptes scabiei). palpate and visually examine the skin thoroughly for masses. mast cell tumors are common and are variable in size. often, the fur around a mast cell tumor is matted with dried blood from the animal's scratching. other types of skin tumors, such as sebaceous adenomas and basal cell tumors, are also common (see chapter 9) . perform an excisional biopsy of any lump found on the skin. young, recently purchased ferrets need serial canine distemper vaccinations until they are 14 weeks of age. 3 rabies vaccines should be given annually beginning at 3 months of age. 14 ferrets should be examined annually until they are 4 to 5 years of age; then, older animals may need examinations twice yearly because of the high incidence of metabolic disease and neoplasia. annual blood tests are recommended for older animals. measure the blood glucose concentration twice yearly in healthy middle-aged and older ferrets; more-frequent monitoring is needed in ferrets with insulinoma. abdominal ultrasound scanning or an endocrine panel is indicated in ferrets with thinning fur on the tail or other clinical signs suggestive of adrenal disease (see chapter 7) . testing for infectious diseases may be warranted, especially in new or young ferrets that will be introduced into a multi-ferret household or those that are taken to ferret shows. ferrets can be tested for aleutian disease virus and ferret enteric coronavirus by polymerase chain reaction testing (michigan state university, diagnostic center for population and animal health, www.animalhealth.msu.edu; veterinary molecular diagnostics, www.vmdlabs.com; zoologix, www.zoologix.com). serologic tests for aleutian disease by enzyme-linked immunosorbent assay (elisa) and counterimmunoelectrophoresis are also available (see chapter 5). ferrets must be vaccinated against canine distemper virus (cdv). currently, one vaccine is approved by the u.s. department of agriculture for use in ferrets: purevax ferret (boehringer ingelheim animal health, duluth, ga). purevax is a canarypox-vectored recombinant vaccine that does not contain complete cdv or adjuvants; thus, post-vaccination risks are reduced. this product has a wide safety margin and has proved effective in protecting ferrets against cdv. 69 although supply from the manufacturer has been intermittently problematic in the united states, the vaccine is available. canine distemper vaccines that were previously used in ferrets but are now discontinued include fervac-d (united vaccines, inc, fitchberg, wi), a modified-live virus vaccine propagated in avian cell lines, and galaxy d (schering-plough animal health/merck), a modified-live virus vaccine derived from the onderstepoort canine distemper strain and attenuated in a primate cell line. galaxy vaccines are now marketed under the nobivac (merck animal health, madison, nj) trade name. in a safety and efficacy study, galaxy d proved effective in preventing canine distemper in young ferrets challenged after serial vaccination. 74 other canine distemper vaccines have been used off-label in ferrets in countries other than the united states or when purevax has been unavailable. recombitek cdv (boehringer ingelheim animal health) is also a recombinant canarypox vaccine approved for use in dogs that has been used in ferrets. this cdv vaccine is marketed in several multivalent combinations including cdv with parvovirus: a monovalent product is not available in the united states. nobivac puppy-dpv (merck animal health) is a modified live virus canine distemper vaccine combined with parvovirus vaccine, a virus that does not affect ferrets. although these vaccines have been used clinically in ferrets, their safety and efficacy in ferrets have not been studied. a cdv vaccine approved for use in mink (distemink; united vaccines inc, fitchberg, wi) is available in 250-dose vials only. because of the possibility of vaccine-induced disease, especially in immunosuppressed or sick ferrets, avoid using multivalent canine vaccines and do not use modified live cdv vaccines of ferret-cell or low-passage canine-cell origin. standard vaccination protocols for canine distemper in ferrets have been based on serial vaccinations of young ferrets at 6, 10, and 14 weeks, 3 with annual boosters. however, recent data suggest a modified vaccine schedule consisting of two initial vaccines with less-frequent boosters is effective. in an efficacy study of 150 ferrets, 90% of ferrets that were initially vaccinated at 9 weeks and given a booster vaccine between 14 and 16 weeks of age with one of three commercial vaccines (purevax ferret, fervac-d, or galaxy d) maintained protective antibody titers of >1:50 for at least 3 years. 72 the three commercial vaccines did not differ in efficacy of eliciting protective titers. therefore an initial vaccination protocol of two vaccinations, starting at 8 to 9 weeks of age and separated by 4 weeks, followed by a booster every 3 years, should suffice in most cases (d. perpiñan, personal communication, may 2018). for ferrets at high risk of contracting canine distemper or when highly pathogenic strains of cdv are circulating, consider more-intensive vaccination protocols. if the ferret is first vaccinated after 3 to 4 months of age, a series of two vaccinations separated by 4 weeks is sufficiently protective (d. perpiñan, personal communication, may 2018). 29 all ferrets should be vaccinated against rabies. 14 two inactivated (killed) rabies vaccines are approved for use in ferrets in the united states: imrab-3 or imrab-3 tf (boehringer ingelheim animal health) and defensor 1 or defensor 3 (zoetis, parsippany, nj). inactivated vaccines are effective at producing immunity for at least 1 year. 63 current recommendations are to vaccinate healthy ferrets at 3 months of age at a dose of 1 ml administered subcutaneously (sc). give booster vaccinations annually. titers develop within 30 days of rabies vaccination. 63 clinical signs of rabies in ferrets can vary. in studies of experimentally induced rabies in ferrets, clinical signs range from restlessness, apathy, and paresis to ascending paralysis, ataxia, cachexia, bladder atony, fever, hyperactivity, tremors, and paresthesia. 8, 48 mean incubation period in experimental studies varies from 28 to 33 days. 48, 49 virus is present in the brain tissue and salivary glands of inoculated ferrets, and virus is shed in the saliva 2 to 6 days after onset of illness. 48, 49 ferrets are at least 25 times less susceptible than skunks to rabies infection when fed mice carcasses infected with rabies virus. 5 survival and clearance of rabies virus infection was reported in one ferret experimentally infected with rabies virus of skunk origin. the ferret initially exhibited hindlimb paralysis that resolved to paraparesis. no virus antigen was found at necropsy 6 months after inoculation. 24 ferrets are considered currently immunized 28 days after the initial rabies vaccination and immediately after a booster vaccination. 14 if a healthy pet ferret bites a person, current recommendations of the compendium of animal rabies prevention and control are to confine and observe the animal for 10 days, during which the ferret should not be vaccinated. 14 any illness that develops during observation should be reported immediately to the local health department. if signs suggest rabies, the ferret must be euthanized, and protocols for rabies evaluation followed. for a ferret with a current vaccine status exposed to a possible rabid animal, recommendations are to revaccinate the ferret within 96 hours of exposure and then keep the ferret under the owner's observation and care for 45 days. exposed ferrets that are overdue for a booster rabies vaccination should be evaluated on a case-by-case basis by the health department. an unvaccinated animal that is exposed to a rabid animal or a stray ferret that bites a person should be euthanized immediately and submitted for rabies testing. see the website of the centers for disease control and prevention (https://www.cdc.gov/ rabie s/specific_groups/veterinarians/) or the national association of public health veterinarians (http://www.nasphv.org/ documentscompendia.html) for specific guidelines. in ferrets, adverse events associated with vaccination are primarily type i hypersensitivity reactions or anaphylaxis. 43 ferrets with mild reactions may exhibit pruritus and skin erythema. more severe reactions are typified by vomiting, diarrhea, piloerection, hyperthermia, cardiovascular collapse, or death. vaccine reactions are most common after canine distemper vaccination but may also occur after rabies vaccination. in a study of vaccine-associated adverse events in 3857 ferrets in the united states, the incidence of adverse events associated with rabies vaccine alone, canine distemper vaccine alone, and rabies and canine distemper vaccines together were 0.51%, 1.0%, and 0.85%, respectively, with no significant difference among groups. 45 however, occurrence of a vaccine-associated adverse event was significantly associated with the cumulative number of canine distemper vaccinations, with an 80% increase in risk of an adverse event with each additional distemper vaccine. the canine distemper vaccines used in this cohort of ferrets were purevax ferret and fervac-d; the two vaccines were grouped collectively in the analysis, and the incidence of adverse events associated with individual vaccines was not reported. all reactions occurred immediately after vaccination and most commonly consisted of vomiting and diarrhea. in another study of 143 ferrets, the incidence of adverse events after administering either canine distemper (5.9%) (fervac d), rabies (5.6%) (imrab-3), or both vaccines (5.6%) did not differ significantly between groups. 23 in a 2001 report of 83 vaccine reactions in ferrets reported to the u.s. pharmacopeia veterinary practitioners' reporting program, 65% involved administration of fervac d, 24% involved concomitant administration of fervac d and imrab 3, and 11% involved administration of imrab alone (purevax was not approved for use at the time these data were collected). 43 according to merial's (now boehringer ingelheim) product information, the incidence of vaccine reactions with purevax is 0.3%. no adverse events were reported in an efficacy study of vaccination of 150 ferrets with either purevax ferret, fervac-d, or galaxy d. 72 surveillance of vaccine-associated adverse events relies on voluntary reporting by practitioners. 43 vaccine-associated adverse events can be reported to the center for biologics, u.s. department of agriculture (https:// www.aphis.usda.gov/aphis/ourfocus/animalhealth/veterinarybiologics/adverse-event-reporting). always follow the manufacturer's instructions for vaccine administration, and inform the owner of the possibility of an adverse reaction before vaccinating. because most reactions occur almost immediately after vaccination, have the owner monitor the ferret in the waiting area for 30 minutes or more after vaccination with any product. if a ferret has an adverse reaction, administer an antihistamine (e.g., diphenhydramine hydrochloride, 0.5 to 2.0 mg/kg intravenously [iv] or intramuscularly [im]), epinephrine (0.01 mg/kg iv, im or intratracheally), or a short-acting corticosteroid (e.g., dexamethasone sodium phosphate, 1 to 2 mg/kg iv or im), and give supportive care. for any biologic product, veterinarians must assess risk versus benefit of vaccination. the treatment options for ferrets that have had a vaccine reaction are to administer diphenhydramine (2 mg/kg orally [po] or sc) at least 15 minutes before vaccination or not to vaccinate if exposure risk is minimal. vaccine injection-site sarcomas have been described in ferrets. 46, 47 in one report, 7 of 10 fibrosarcomas in ferrets were from locations used for vaccination. 46 fibrosarcomas had similar histologic, immunohistochemical, and ultrastructural features as those reported for feline vaccine-associated sarcomas. in cats, adjuvanted vaccines are most likely to be involved in tumor development; however, no definitive association was made between the fibrosarcoma and the type of vaccine in ferrets. ferrets appear less prone than cats to development of vaccine-induced tumors. gastrointestinal parasitism is most common in young or recently purchased pet ferrets and is relatively uncommon in mature ferrets in the united states. in a survey of ferrets that were either privately owned or in pet shops in italy, 14 of 50 (28%) were positive for ancylostomids (hookworms) and one (2%) was positive for sarcocystis. 17 ferrets can be intermediate hosts or vectors of parasites from other natural hosts. protozoan parasites are occasionally seen. therefore perform routine fecal flotations and direct fecal smears for all young or recently acquired ferrets. coccidiosis (isospora species) usually is seen in young ferrets, which shed oocysts between 6 and 16 weeks of age. 4 infection is often subclinical, although ferrets occasionally may have loose stool or bloody diarrhea. coccidiostats, such as sulfadimethoxine and amprolium, are effective and safe, and treatment should be continued for at least 2 weeks. coccidia in ferrets may cross-infect dogs and cats; therefore check other pets in the household for coccidia and treat as needed. giardiasis is occasionally seen in ferrets. results of studies on molecular characterization and host specificity of giardia duodenalis isolates from pet ferrets vary. in one study, genetic sequences of giardia isolates from ferrets were similar to those of giardia associated with human infections. 55 results of another study showed genetic sequences of giardia differed in ferrets and people and other mammals, suggesting that giardia isolates from ferrets may be host specific. 2 giardia can be detected by identifying cysts or trophozoites in a fresh fecal smear or zinc sulfate flotation, or by fecal elisa. treat ferrets with giardiasis with metronidazole (20 mg/kg po every 12 hours for 5 to 10 days) or fenbendazole (50 mg/kg po every 24 hours for 3 to 5 days). cryptosporidiosis is described primarily in young ferrets. 60 infection is associated with the ferret genotype of cryptosporidium parvum and therefore is an unlikely source of human infection. 1 infection is usually subclinical and, although most ferrets recover within 2 to 3 weeks, can persist for months in immunosuppressed animals. oocysts of cryptosporidium are small (3-5 μm) and difficult to detect but can be found in fresh fecal samples examined immediately after acid-fast staining. 4, 60 various drugs, including azithromycin, tylosin, and nitazoxanide, are used for treatment in dogs and cats, but efficacy in ferrets is not known. 64 heartworms (dirofilaria immitis) can cause disease in ferrets. ferrets that are housed outdoors in heartworm-endemic areas are most susceptible to infection; however, all ferrets in endemic areas should be treated with heartworm preventive (see chapter 5 and appendix). ear mites (otodectes cynotis) are common in ferrets, but affected animals may not exhibit pruritus or irritation. this mite species also infects dogs and cats, and animals in households with multiple pets can transmit mites to other animals. a red-brown, thick, waxy discharge in the ear canal and pinna characterizes infection. a direct smear of the exudate reveals adult mites or eggs. because ferrets normally have brown ear wax, the color or appearance of debris in the ear canal is not pathognomonic for mites. at the initial examination, check for ear mites and do follow-up checks at the annual examination in ferrets kept in multiple-pet households. several products, including selamectin, are effective in treatment (see chapter 9) . flea infestation (ctenocephalides species) is most common in ferrets kept in households with dogs or cats. ferrets with chronic infestations can become severely anemic. check ferrets during the physical examination for signs of fleas or flea dirt. treat infested animals with products safe for use in cats, and institute flea control measures (see chapter 9) . ticks are rarely seen on domestic ferrets, and lyme disease in ferrets has not been reported. ferrets can be hospitalized in standard hospital cages with some adaptations. ferrets are agile escape artists and often can squeeze through vertical cage bar openings on standard hospital cages. for housing ferrets, use only commercial cages with small spacing between bars or use cages with attached plexiglass fronts at least half the height of the cage door or higher to prevent escape. small animal intensive care cages or incubators also can be used to house ferrets and are especially useful for animals that need supplemental heat or oxygen. closely monitor the temperature in these cages when using supplemental heat to prevent hyperthermia or hypothermia. the cage should be large enough to accommodate a sleeping area and an area for defecation and urination. ferrets typically do not soil their sleeping area, even when very sick. all ferrets like to burrow and should be given opportunity to do so while hospitalized. clean towels or a mound of shredded paper make excellent burrowing material. take care with plastic-backed underpads, which ferrets may chew and ingest. small padded pet beds and fleece pet "pockets" work well as sleeping areas. provide water in either water bottles or small weighted bowls. before hospitalization, ask the owner which type of watering system the ferret is accustomed to. ferrets can be finicky eaters and should be fed their regular diet while hospitalized, if possible. otherwise, feed a very palatable ferret food or a premium high-protein cat/kitten kibble. for animals that are anorectic, force-feed a high-calorie semisolid food or supplement until the animal is eating on its own (see later discussion). most veterinary laboratories offer small mammal hematologic and biochemical panels that require 1.0 ml or less of blood. in-clinic, point-of-care analyzers require very small sample sizes (usually 100 μl). the blood volume of healthy ferrets is approximately 40 ml in average-sized females weighing 750 g and 60 ml in males weighing 1 kg. 22 up to 10% of the blood volume can be safely withdrawn at one time in a normal ferret; however, collect only the minimum amount needed for analysis. repeated blood drawing can contribute to anemia in sick animals hospitalized for long periods. obtaining a blood sample from a ferret is relatively easy. venipuncture usually can be done with manual restraint, but some veterinarians prefer sedating or anesthetizing especially active animals. several venipuncture sites are readily accessible; the technique and site chosen depend on how much blood is needed and the availability of assistants for restraint. anesthesia or sedation can be used if assistants are unavailable. if needed, ferrets often can be distracted during restraint for venipuncture by offering semisolid food or a product such as ferretone (8-in-1 pet products) by syringe. avoid using supplements with corn syrup or other sugars, because this will affect blood glucose levels, and collect blood for glucose determination or other fasting samples before offering food. the blood collection technique can affect hematologic test results. isoflurane anesthesia can cause decreases in all hematologic values beginning at induction of anesthesia and reaching maximal effects at 15 minutes after induction. 41 both isoflurane and sevoflurane can cause a decrease in packed cell volume. 32 therefore hematologic results of blood samples collected while a ferret is anesthetized must be interpreted carefully (see chapter 39) . 41 two sites are commonly accessed to obtain large blood volumes in ferrets. jugular venipuncture can be accomplished by extending the ferret's forelegs over the edge of a table and the neck up, or by restraining the ferret in lateral recumbency (fig. 2.2) . have a second assistant restrain the ferret's hind end on the table to prevent twisting, or wrap the lower body in a towel. use a 25-gauge needle bent slightly to a 20-degree angle with a 1-to 3-ml syringe for venipuncture in most ferrets; a 22-gauge needle can be used in large males. shave the neck at the venipuncture site to enhance visibility of the jugular vein. the vein is relatively superficial and is located more laterally in the neck than it is in dogs or cats. once the needle is inserted, the blood should flow easily into the syringe; if the neck is overextended and the head is arched back, the blood may not flow readily from the vein. relax the hold on the head or gently "pump" the vein by moving the head slowly up and down to enhance blood flow into the syringe. with ferrets that resist limb extension, a towel-wrap technique can be used. scruff the ferret with its front legs extended caudally against the ventral thorax and wrap the animal's body firmly with a towel from the base of the neck down. have an assistant restrain the toweled ferret in dorsal recumbency while scruffing the neck or holding the head. however, with very fractious animals, even this technique may be difficult. the second venipuncture site from which to obtain large blood samples is the cranial vena cava. the site of venipuncture is the anterior vena cava or the right or left brachiocephalic trunk, depending on the point of entry and the depth of needle penetration (fig. 2.3, a) . this technique is relatively safe in ferrets because of the long anterior vena cava and the caudal location of the heart in the thoracic cavity, which is approximately 3 cm from the thoracic inlet. however, rare instances of hemorrhage into the anterior thoracic cavity can occur. for this technique, restrain the ferret on its back with the forelegs pulled caudally and the head and neck extended ( fig. 2.3, b) . with manual restraint, two assistants are usually needed, one to restrain the forelegs and head and the other to restrain the rear just cranial to the pelvis. use a 25-gauge needle with an attached 1-ml or 3-ml syringe; insert it into the thoracic cavity between the first rib and the manubrium at an angle 30 to 45 degrees to the body. direct the needle toward the opposite rear leg or most caudal rib and insert it almost to the hub. pull back on the plunger as the needle is slowly withdrawn until blood begins to fill the syringe. if the ferret struggles, quickly withdraw the needle and wait until the ferret is quiet before making a second attempt. in very fractious or active ferrets, jugular venipuncture or use of tranquilization are safer choices to avoid lacerating the vessels. the lateral saphenous or cephalic vein can be used if only a small amount of blood is needed to measure a packed cell volume or blood glucose level. to prevent collapse of the vein during venipuncture, use an insulin syringe with an attached 27-or 28-gauge needle. the saphenous vein lies just proximal to the tarsus on the lateral surface of the leg; the cephalic vein is in the same anatomic location as in a dog. before venipuncture, shave the fur from the area to enhance visibility of the vein. venipuncture of the tail artery is possible but rarely used in pet ferrets. 9 venipuncture at this site is painful and requires anesthesia. insert a syringe with a 21-gauge needle into the ventral midline of the tail directed toward the body. once the artery is entered 2 to 3 mm deep into the skin, slowly withdraw the plunger until blood fills the syringe. apply pressure to the venipuncture site for 2 to 3 minutes after withdrawing the needle. most commercial veterinary diagnostic laboratories provide laboratory-specific reference intervals for ferret hematologic and biochemical values (see chapter 39) . published sources of reference intervals for both laboratory and pet ferrets are available. 22 contains blood coagulation values determined in normal ferrets that can be used as guidelines in the absence of reference intervals specific to a laboratory or to coagulation analyzer equipment. indwelling intravenous catheters can be placed in the lateral saphenous or cephalic vein (fig. 2.4) in ferrets that are collapsed with poor blood pressure or in young or very small ferrets, attempts to place an intravenous catheter may be unsuccessful. an intraosseous catheter can be placed in these animals. the proximal tibia is the most common site for intraosseous catheter placement in small mammals, 34 but the proximal femur can also be used and allows for a greater range of movement. unless the ferret is very depressed, anesthesia is required to place the catheter. sterilely prepare the insertion site and infiltrate the area with 1% to 2% lidocaine (maximum dose, 1 mg/kg). 34 insert a 20-or 22-gauge, 1.5-inch spinal needle into the marrow cavity. alternatively, use a 20-or 22-gauge hypodermic needle with a surgical steel wire inserted into the lumen to prevent a bone plug from occluding the needle during insertion. 52 the intraosseous catheter should occupy approximately 30% to 70% of the marrow cavity at the narrowest portion. 34 administer drugs through intraosseous catheters in small volumes with minimal pressure to prevent leakage from the insertion site. if possible, change to an intravenous catheter as soon as the animal is rehydrated or blood pressure improves. intraosseous catheters should not be left in place for more than 72 hours. 34 vascular access ports can be placed when repeated vascular access is required for any reason, such as for chemotherapy. 59 the technique used to place the catheter and port has been described and illustrated. 53 hospitalized ferrets usually require fluid therapy to maintain hydration and correct dehydration. daily fluid requirements of ferrets have not been determined; however, calculating fluid requirements at a rate of 60 to 70 ml/kg per day appears to be adequate for maintenance. one source estimates daily water consumption of adult ferrets as 75 to 100 ml/day. 44 provide additional fluids to compensate for ongoing fluid loss and to correct dehydration calculated as a percentage of the body weight. intravenous fluids are preferred in ill animals. if possible, administer intravenous fluids by continuous-rate infusion. alternatively, divide the calculated daily fluid volume into two or three doses and administer by a buretrol (baxter healthcare, glendale, ca) or syringe pump. depending on the clinical condition of the ferret, supplements and drugs can be added to fluids by using the same criteria and calculations as for dogs and cats. although subcutaneous fluids can be given, ferrets react painfully to subcutaneous administration, and adequate restraint is needed. administer subcutaneous fluids in the loose skin along the back and dorsal cervical area, dividing the calculated daily fluid volume into doses given two or three times daily. colloids are effective in improving intravascular fluid volume and oncotic pressure in ferrets that are hypoproteinemic or in shock. dosage and administration are like those used in other small animals (see chapter 41). antibiotics and other therapeutic agents are usually given at dosages like those in cats per kg of body weight (see appendix). in hospitalized animals with an indwelling catheter, give medications intravenously if possible. however, monitor the leg where the catheter is placed carefully for phlebitis and cellulitis, particularly when caustic medications are administered. antibiotics can be administered im; however, if treatment continues over several days, subcutaneous administration is preferred in cachectic animals because of the limited muscle mass. pills are very difficult to administer to ferrets; therefore oral medications are most easily given compounded into liquid form. ferrets generally accept chicken and beef flavors of compounded medications but do not like the taste of fish flavors. pain management is important in the postoperative period, with diseases that create significant discomfort (such as gastrointestinal ulceration), and for traumatic injuries (see chapter 37) . ferrets in pain may exhibit tachypnea, a stiff gait, a strained facial expression, teeth grinding, shivering, half-closed eyelids, aggression, focal muscle fasciculation, hiding, general malaise, bristling of tail fur, and appear "tucked" in the abdomen. 42, 71 many analgesic agents are used effectively in ferrets, including opioids, α-2 agonists, nonsteroidal antiinflammatory drugs (nsaids), and local anesthetics. 30 clinically, buprenorphine, butorphanol, or meloxicam or combinations thereof are most commonly used for hospitalized animals and outpatients (see chapter 37 and appendix). although no studies have evaluated efficacy of meloxicam for analgesia in ferrets, a meloxicam dose of 0.2 mg/kg in ferrets achieves plasma concentrations considered effective in other companion animals. 12 ferrets are sensitive to acetaminophen toxicity. 15 the activity of uridine 5′-diphospho (udp)-glucuronosyltransferase in their livers compares with that of cats. acetaminophen glucuronidation is slower in ferrets than in other nonfelid species. unlike cats, however, no genetic mutations are associated with this slow metabolism, and the exact cause is unknown. ibuprofen can be toxic in ferrets at high doses, 11 and accidental ibuprofen ingestion of 220 mg/kg can cause death. 62 clinical signs of toxicosis are depression, coma, ataxia, recumbency, tremors, weakness, and death. 62 therefore avoid acetaminophen in ferrets and use nsaids with caution. do not use nsaids in ferrets being treated with a corticosteroid for insulinoma or other disease. epidural administration of analgesics before surgery appears to be effective in controlling postoperative pain in ferrets undergoing procedures involving the abdomen, spine, pelvis, hind legs, tail, and perineum (see chapter 37) . 18, 66 the technique has been well described in ferrets and is similar to that used in other small animals. 18, 25 epidural injection is contraindicated in cases of coagulopathy, sepsis, hypovolemia, skin infection, and local fractures. 18 be careful in the immediate postoperative period when opioids, such as butorphanol or buprenorphine, are used. in cats, as well as in other species, opioids including hydromorphone, butorphanol, and buprenorphine are associated with an increase in body temperature. 58 opioid analgesics also can cause pronounced sedation, and ferrets can remain very lethargic and immobile for long periods. because of these two factors, immobile ferrets can overheat quickly if a heating lamp or other focused heat source is used during anesthetic recovery. therefore closely monitor the body temperature of any immobile or lethargic ferret given opioids or sedatives to prevent overheating when using heat lamps or forced-air warming devices. many sick ferrets are either cachectic or hypoglycemic or have minimal body fat and require nutritional support. products formulated as recovery diets for carnivores are available and readily accepted by most ferrets (carnivore care, oxbow animal health, murdock, ne; emeraid carnivore, emeraid, cornell, il). ferrets can be syringe-fed meat-based soft foods marketed for hospitalized dogs and cats (maximum-calorie plus, the iams company, mason, oh; canine/feline a/d, hill's pet nutrition, topeka, ks). do not offer supplement gels that contain corn syrup because of the high sugar content. force-feed anorectic ferrets as much as they will accept comfortably, usually 8 to 12 ml syringe fed three or four times daily. ferrets that develop a taste for the food may eat it directly from a bowl. ferret owners often prepare homemade diets of "duck soup" or "chicken gravy" to nurse their pets at home. many different recipe variations are available, and most are based on whole chicken with additives ranging from beef fat, dog kibble, nutritional supplement gels, or brewer's yeast to echinacea capsules. the "duck soup" variations all provide a soft, porridge-consistency food that is usually readily eaten by sick and convalescing ferrets. some recipes are very high in fat and carbohydrates. unless the homemade diets are based on or used with a commercial ferret diet, they should not be used long term because of possible nutritional imbalances and deficiencies. although seldom used clinically, esophagostomy feeding tubes can be placed in ferrets to manage debilitated animals over the long term. the technique is like that used in cats. 21 gastric feeding tubes have been placed in ferrets both experimentally and clinically. 6, 10 in a study of 14 ferrets, gastrostomy tubes were placed percutaneously by a nonendoscopic technique. a gastrostomy tube was placed and maintained successfully in a ferret after surgical repair of an esophageal perforation caused by an esophageal foreign body. 10 total nutrient admixtures have been formulated to provide partial parenteral nutrition to ferrets. 52, 61 depending on the osmolarity of the solution, parenteral nutrition solutions can be delivered via a central, peripheral, intraosseous, or intraperitoneal catheter; however, the relatively high protein requirements of ferrets usually result in a solution of 600 to 800 mosm/l, which should be administered into a large (central) vein. 61 urine samples can be collected by cystocentesis or by free catch after natural voiding or gentle manual expression of the bladder. the techniques for manually expressing the bladder and cystocentesis are the same as those used in dogs and cats. anesthetize fractious ferrets to avoid trauma to the thin bladder wall. use a 25-gauge needle for cystocentesis. reference values for urinalysis are listed in table 2 .5. in one study, the reference range for urine ph in ferrets was reported as 6.5 to 7.5; however, urine ph can vary according to the diet. the normal urine ph in ferrets fed a high-quality, meat-based diet is approximately 6.0. urinary catheterization is commonly indicated in male ferrets, but the procedure can be challenging. although techniques have been described for both sexes, 40 in male ferrets, the urethral opening is very small and is located on the ventral surface of the penis, proximal to the j-hook in the end of the os penis. use a 3.0-or 3.5-fr polytetrafluoroethylene, silicone, 35 or polyurethane urinary catheter (slippery sam tomcat urethral catheter, surgivet, smiths medical, dublin, oh; tomcat urethral catheter, surgivet; tomcat/small animal urinary catheter, mila international inc., florence, ky). alternatively, use a 3.5-fr rubber feeding catheter; estimate the length of the catheter that must be inserted to reach the bladder before placing (see also chapter 4). if using a long rubber catheter, use a stylet or flexible wire to stiffen the catheter if needed; the stylet can be retracted slightly while passing the catheter, but be very careful when rounding the pelvic flexure to avoid perforating the urethra. another option is to use a 20-gauge or 22-gauge, 8-inch jugular catheter with the stylet removed. 52 to pass the urinary catheter, aseptically prepare the preputial area, locate the urethral opening, and introduce the catheter tip (fig 2.5, a) . if the urethral opening is difficult to see, dilate the opening by passing a 24-gauge intravenous catheter just inside the tip of the urethra and flushing gently with saline solution (fig 2.5, b) . slip the tip of the lubricated urinary catheter gently into the dilated urethral opening alongside the intravenous catheter and, while gently flushing with saline solution, pass the catheter into the bladder. often resistance is met at the pelvic flexure; if this occurs, try repeated gentle flushing and relubricating the catheter until it passes. once in place, if using a red rubber catheter, place butterfly tape strips around the catheter just as it enters the urethra and at another point 3 to 5 cm distal, and suture these to the skin (fig 2.5, c) . if using a urethral catheter, suture the catheter hub to the prepuce (fig 2.5, d) . attach a sterile urinary collection device and tape the tubing to the tail to further prevent tension. if needed, bandage the ferret's abdomen to minimize rotation of 70 8-48 70 28 70 8-140 70 24.9 ± 14.3 20 ph 6.2 ± 0.1 22 6.5-7.5 70 6.0-6.5 22 6.3 ± 0.3 22 6.5-7.5 70 the catheter and to restrict the ferret from traumatizing it. soft elizabethan collars are needed in some ferrets to prevent chewing at the catheter. maintain sterility of the collection system, and drain the bag by needle and syringe rather than opening the system (see also chapter 4) . temporary tube cystostomy has been used successfully to manage male ferrets with urinary obstruction caused by adrenal disease. in four ferrets treated surgically, a 5-or 8-fr foley catheter was placed in the bladder at the time of adrenalectomy and left in place for 5 to 14 days. 50 in these ferrets, immediate treatment of urinary blockage was by cystocentesis. a cystostomy catheter also can be placed by using interventional radiographic techniques. this is an option in obstructed ferrets in which a urethral catheter cannot be passed and surgery is considered high risk. a cystostomy catheter allows azotemic ferrets to be managed with diuresis before surgery; alternatively, in cases in which surgery is not an option, the catheter can remain in place pending response to medical management with leuprolide acetate. indirect blood pressure monitoring techniques, using both doppler ultrasonography and oscillometric methods, have been described in ferrets. 36, 51, 65, 73 however, both methods poorly approximate direct arterial blood pressure measurements. indirect blood pressure readings may be inaccurate because the neonatal blood pressure cuff does not fit securely on the short legs of ferrets (doppler method), and pressure changes of the tail artery may not be sufficiently high to be detected by the oscillometric system. 51 in one study, the indirect systolic blood pressure values measured by doppler on the tail of ferrets measured 28 mmhg less than the direct arterial systolic blood pressure values. 51 in a study using 14 male ferrets that compared indirect blood pressure measurements on the tail, forelimb, and hindlimb using an oscillometric sphygmomanometer and a high-definition oscillometric monitor with direct arterial blood pressure measurements, measurements using the tail were considered most reliable. 65 however, the oscillometric sphygmomanometer consistently overestimated the systolic arterial pressure. indirect measurements of systolic, mean, and diastolic arterial pressures were consistently higher during hypotensive states but substantially lower in hypertensive states than corresponding direct blood pressure measurements. in a study of 63 healthy ferrets, sedation with butorphanol (0.2 mg/kg im) and midazolam (0.2 mg/kg im) was found optimal to allow indirect blood pressure measurement. 73 reference values for indirect blood pressure measured with the cuff placed at the base of the tail were 94 to 155 mmhg systolic, 69 to109 mmhg mean, and 51 to 87 mmhg diastolic arterial pressure. be aware of the above-described inconsistencies of indirect compared with direct blood pressure measurements and interpret clinical results carefully. in clinical situations, indirect blood pressure is most useful for evaluating changes in blood pressure from a known baseline or for surgical monitoring of general trends rather than for exact blood pressure measurement. the doppler method of indirect blood pressure measurement is most commonly used in ferrets. shave the hair on the ventral tail and place a no. 1 (3 to 6 cm) cuff on the most proximal part of the tail and attach it to a sphygmomanometer. place the doppler transducer probe crystal with ultrasound gel on the shaved skin approximately 1 cm distal to the cuff and tape or hold in place. alternatively, place the cuff just above the carpus or tarsus (overlying the radial artery on the front leg or the digital branch of the tibial artery on the rear leg, alternatively) or on the distal humerus. 36 evaluating a bone marrow sample is a valuable diagnostic tool for many disease conditions, including anemia, thrombocytopenia, pancytopenia, proliferative abnormalities, and suspected hematopoietic malignancies. although the proximal femur is usually the most readily accessible site (fig. 2.6) , the iliac crest, tibial crest, and humerus can also be used to collect bone marrow samples. anesthesia is necessary to aspirate the bone marrow or perform a core biopsy. with the ferret in lateral recumbency, shave and aseptically prepare the area around the collection site. for the proximal femur, 54 make a small incision over the greater trochanter. stabilize the femur with one hand while inserting a 20-gauge, 1.5-inch spinal needle into the bone, medial to the greater trochanter. use steady pressure and an alternating rotating motion to advance the needle into the marrow cavity. withdraw the stylet and attach a 6-to 12-ml syringe to the needle to aspirate the marrow, stopping suction as soon as the sample is visible (to prevent blood contamination). to collect a core biopsy sample, use the same technique, but use a 1.5-inch, 18-gauge needle in place of the spinal needle (see also chapter 8) . collect samples from alternate sites by using the same basic technique. blood transfusions may be needed in ferrets that are anemic from chronic disease, blood loss, or estrogen toxicosis, or in ferrets that are thrombocytopenic. as in other species, evaluate the need for a transfusion based on the packed cell volume or platelet count and clinical status of the ferret. consider a transfusion if the packed cell volume is 25% or less in a ferret that exhibits clinical signs of anemia or requires surgery, or if a ferret is thrombocytopenic and exhibits ecchymosis, petechiation, or bleeding. ferrets lack detectable blood groups, and risk of transfusion reaction is minimal, even without cross-matching. 39 because of a greater blood volume, large male ferrets are preferred over females as blood donors. depending on the size of the donor ferret, 6 to 12 ml of blood can be safely collected for transfusion. collect blood into an anticoagulant such as acid-citrate-dextrose or citrate-phosphate-dextrose-adenine (cpda) at a ratio of 1 ml of anticoagulant to 6 to 9 ml of donor blood. 31 ferret blood collected into cpda at a 6:1 ratio has a shelf life of 7 days when stored at 4° c; after 7 days, deterioration of red blood cells (rbcs) causes a decrease in blood ph, glucose, and sodium and an increase in lactate and potassium. 56 whenever possible, collect blood from donor ferrets shortly before transfusion. always use a filter when transfusing whole blood, and use at least a 22-gauge catheter to prevent cell lysis during the transfusion. intraosseous blood transfusions can be given to ferrets if an intravenous catheter cannot be placed. splenic aspiration is a common diagnostic technique in ferrets with enlarged spleens (see chapter 5) . the technique is simple and can be done in unanesthetized ferrets; however, if a ferret is fractious, use an injectable sedative. an ultrasound-guided aspirate is preferred, especially if a splenic mass is present. alternatively, palpate the abdomen and immobilize the enlarged spleen next to the body wall and aspirate directly. a positive aspirate appears bloody. the most common findings on cytologic examination of a splenic aspirate are extramedullary hematopoiesis and lymphoma. analysis of cerebrospinal fluid (csf) as a diagnostic tool is occasionally indicated in ferrets that present with neurologic signs. the volume of csf fluid that can be removed safely is 0.26 ml/kg. 57 for a csf tap, place the anesthetized ferret in lateral recumbency. using a 25-gauge, 1.6-cm-long hypodermic needle, puncture the cerebromedullary cistern and allow free flow of the csf fluid. in clinically normal ferrets, reference values for csf tap are mean white blood cells (wbcs), 1.6 cells/μl (range 0-8 cells/μl); mean rbcs, 1041 cells/μl (range 0-11,560 cells/μl; and mean protein concentration, 31.4 mg/dl (range 28-68 mg/dl). 57 in a study of 42 ferrets in which a csf tap was performed, rbcs of <100 cells/μl were obtained in 64% of cases, and the number of wbcs was significantly affected by blood contamination, but protein concentration was not. 57 identification of genotypes of cryptosporidium parvum isolates from ferrets in japan molecular characterization of giardia duodenalis isolates from domestic ferrets antibody titers in domestic ferret jills and their kits to canine distemper virus vaccine parasites of domesticated pet ferrets susceptibility of carnivore to rabies virus administered orally percutaneous placement of a gastric feeding tube in the ferret coagulation values in normal ferrets (mustela putorius furo) using selected methods and reagents experimental rabies in the ferret (mustela [putorius] furo): susceptibility-symptoms-excretion of the virus simple technique for bleeding ferrets (mustela putorius furo) medical and surgical management of esophageal foreign body in a ferret acute ibuprofen toxicosis in a ferret meloxicam pharmacokinetics using nonlinear mixed-effects modeling in ferrets after single subcutaneous administration impact of diet on the dentition of the domesticated ferret compendium of animal rabies prevention and control. national association of state public health veterinarians acetaminophen udp-glucuronosyltransferase in ferrets: species and gender differences, and sequence analysis of ferret ugt1a6 rabbit and ferret hemostasis first survey of endoparasites in pet ferrets in italy epidural anesthesia and analgesia in ferrets urine specific gravity values in clinically healthy young pet ferrets (mustelo furo) estimation of glomerular filtration rate and evaluation of renal function in ferrets (mustela putorius furo) esophagotomy feeding tube placement in the ferret normal clinical and biologic parameters incidence of adverse events in ferrets vaccinated with distemper or rabies vaccine: 143 cases recovery from and clearance of rabies virus in a domestic ferret epidural analgesia in ferrets measurement of dietary and dentrifice effects upon calculus accumulation rates in the domestic ferret reference ranges for laboratory parameters in ferrets diagnosis and treatment of dental disease in ferrets viral diseases of ferrets anesthesia and analgesia in ferrets principles of transfusion medicine in small animals comparison of sevoflurane and isoflurane in domestic ferrets (mustela putorius furo) haematological and serum chemistry profiles of ferrets (mustela putorius furo) intraosseous catheterization of exotic animals a new type of urinary catheter for catheterization of the male ferret critical care monitoring comparative hemostasis in vertebrates reduction of calculus accumulation in domestic ferrets with two dentifrices containing pyrophosphate lack of detectable blood groups in domestic ferrets: implications for transfusion a technique for catheterization of the urinary bladder in the ferret effect of isoflurane on hematologic variables in ferrets use of behavior analysis to recognize pain in small mammals vaccine-associated adverse events laboratory management of the ferret for biomedical research incidence of and risk factors for adverse events associated with distemper and rabies vaccine administration in ferrets histology and immunochemistry of seven ferret vaccination-site fibrosarcomas vaccine injection-site sarcoma in a ferret pathogenesis of experimentally induced rabies in domestic ferrets viral excretion in domestic ferrets (mustela putorius furo) inoculated with a raccoon rabies isolate temporary tube cystostomy as a treatment for urinary obstruction secondary to adrenal disease in four ferrets evaluation of noninvasive monitoring techniques in domestic ferrets (mustela putorius furo) emergency and critical care of ferrets use of vascular access ports in exotic animals a technique for femoral bone marrow collection in the ferret occurrence and molecular typing of giardia isolates in pet rabbits, chinchillas, guinea pigs and ferrets collected in europe during assessment of a blood preservation protocol for use in ferrets before transfusion composition of cerebrospinal fluid in clinically normal adult ferrets effects of opioids and anesthetic drugs on body temperature in cats use of a vascular access system for administration of chemotherapeutic agents to a ferret with lymphoma cryptosporidiosis in ferrets parenteral nutrition support in rabbits and ferrets ibuprofen ingestion in ferrets: 43 cases evaluation of an inactivated rabies vaccine in domestic ferrets update on the diagnosis and management of cryptosporidium spp infections in dogs and cats intra-arterial blood pressure in ferrets compared to peripheral blood pressure evaluation of epidural morphine for postoperative analgesia in ferrets (mustela putorius furo) hematology of the domestic ferret (mustela putorius furo) comparison of the blood coagulation profiles of ferrets and rats minimum protective dose (mpd) and efficacy determination of a recombinant canine distemper virus vaccine for ferrets the ferret, mustela putorius furo, as a new species in toxicology pain management in ferrets serum-neutralizing antibody responses to canine distemper virus vaccines in domestic ferrets (mustela putorius furo) non-invasive blood pressure measurement in sedated ferrets (mustela putorius furo): a study to find the optimal dosing regimen and reference ranges. faculty of veterinary medicine theses serologic evaluation, efficacy, and safety of a commercial modified-live canine distemper vaccine in domestic ferrets key: cord-006226-fn7zlutj authors: nan title: abstracts of the 4th annual meeting of the german society of clinical pharmacology and therapy: hannover, 14–17 september 1994 date: 1994 journal: eur j clin pharmacol doi: 10.1007/bf00193489 sha: doc_id: 6226 cord_uid: fn7zlutj nan grapefruit juice may considerably increase the systemic bioavailability of drugs as felodipine and nifedipine. this food-drug interaction has potential practical importance because citrus juices are often consumed at breakfasttime when drugs are often taken. it is likely that a plant flavonoid in grapefruit juice, naringenin, is responsible for this effect (inhibition of cytochrome p-450 enzymes in the liver or in the small intestinal wall). ethinylestradiol (ee2), the estrogen of oral contraceptive steroides, shows a high first-pass-metabolism in vivo. therefore, the purpose of this study is to test the interaction between grapefi-uite juice and ee 2, the area under the serum concentration-time curve (auc0_24h) ofee 2 was determined in a group of young healthy women (n = 13) on day 4 + 1 ofmenstruale cycle. to compare intraindividually, the volunteers were randomly allocated to two test days. the female volunteers took 50 lag ee 2 together with either 200 ml of herb tea or with the same amount of grapefruit juice (content of naringenin 887 mg/1). furthermore, on the day of testing the women drank 4 times 200 ml of the corresponding fluid every three hours up to four times. the auc0.24 h of ee 2 amounts to 1110.5 + 637.7 pg x mi-1 x h after the administration of the drug with grapefruit juice; that means 28 % higher in comparison to 868 0 + 490.0 pg x m1-1 x h after concomitant intake of tea. also, the mean cmax-value increases to 37 %, p _< 0.01 (117.5 + 53.2 pg x m1-1 and 85.5 + 32.9 pg x m1-1, respectively). this result shows that the systemic bioavailability ofee 2 increases after intake of the drug with grapefruit juice. the extent of this effect is lower than the extent of known interindividual variability. procarbazine is a tumourstafic agent widely used m hodgin's disease, non-hodgldn's lymphomas and mmours of brain and lung. procarbazine is an inactive prodrug which is converted by a cytochrome p 450 mediated reaction to its active metabolites, in the first step to azoprocarbazine. the kinetics of both procarbazine and azoprocarbazine is not described in humans up to now. on 10 turnout patients we have investigated the plasma kinetics of both procarbazine and azoprocarbazine after oral adminislxation of 300 mg procarbazine in form of capsules and drink solution, respectively. a hplc method with uv-detection (254 nrn) and detection limits of 50 and 150 ng/ml was developed for procarbazine and azoprocarbazine respectively. after both the capsules and drink solution the parent drug could be detected in plasma only for 1 h. in contrast the tl/2 of terminal elimination of azoprocarbazine was estimated in the range of 0,9 to 6,5 h with a mean of 2,96 h -2 + 1,46 h. the auc of procarbazine was less than 5 % of that of azoprocarbazine. cma x values of azoprocarbazine were determined in the range of 1,3 to 6,l gg/ml. in comparison to the drink solution we determined on the basis of the plasma levels of azoprocarbazine a bioavailability of the therapeutic used procarbazine capsules of 93,1 + 26,3 %. prostaglandin e1 (pge1) is used for the treatment of patients with peripheral arterial disease, and probably effective due to its vasodilator and antiplatelet effects. l-arginine is the precursor of endogenously synthesized nitric oxide (no). in healthy human subjects, larginine also induces peripheral vasodi]ation and inhibits platelet aggregation due to an increased no production. in the present study the influence of a single intravenous dose of l-arginine (30g, 60 min) or pge1 (40p.g, 60 min) on blood pressure, peripheral hemodynamics (femoral artery duplex sonography), and urinary no3-and cgmp excretion rates was assessed in ten patients with peripheral arterial disease (fontaine iii -iv). blood flow in the femoral artery was significantly increased by l-arginine by 68% (p < 0.01), and by pge1 by 25% (p < 0.05). l-arginine more strongly decreased systolic and diastolic blood pressure than pge1. plasma arginine concentration was increased 4-fold by l-arginine, but unaffected by pge1. urinary excretion of no3-increased by 118% after l-arginine (p < 0.05), and by 78% after pge1 (p = n.s.). urinary cgmp excretion increased by 76% after l-arginine and by 43% after pgei (each p = n.s.). we conclude that intravenous l-arginine decreases peripheral arterial resistance, resulting in enhanced blood flow and decreased blood pressure in patients with peripheral arterial disease. these effects were paralleled by increased urinary no3-excretion, indicating that systemic no production was enhanced by the infusion. increased no3-excretion may be a sum effect of no synthase substrate provision (l-arginine) and increased shear stress (pge1 and l-arginine). it is weli established that the endothelial edrf/no-mediated relaxing mechanism is impaired in atherosclerotic and in hypertensive arteries. recently it was suggested that primary pulmonary hypertension might be another disease in which the endothelial edrf/no pathway is disturbed. we tested the hypothesis that intravenous administration of l-arginine (l-arg), the physiological precursor of edrf/no, stimulates the production of no, subsequently increasing plasma cgmp levels and reducing systemic and / or pulmonary vasular resistance, in patients with coronary heart disease (chd; n = 15) and with primary pulmonary hypertension (pph; n = 5). l-arg (30g, 15 min) or placebo (nac1) was infused in chd patients, and l-arg was infused in pph patients undergoing cardiac catheterization. mean aortic (pao) and pulmonary (ppul) arterial pressures were continuously monitored. cardiac output (co; by thermodilution), and total peripheral resistance (tpr) were measured before and during the infusions. plasma cgmp was determined by ria. in chd patients, pao decreased from 87.2 + 4.9 to 81.8 + 5. mm hg during l-arg (p<0.05), whereas ppul was unchanged. tpr decreased from 1008.9 -+ 87.9 to 845.0 +81.7 dyne sec cm -5 during l-arg administration (p<0.01). co significantly increased during l-arg (from 7.3 + 2.8 to 8.1 + 0.9 1/min, p<0.05). placebo did not significantiy influence any of the haemodynamic parameters, cgmp slightly increased by 12.2 + 9.6% during l-arg, but slightly decreased during placebo (-12.3 +9.2 %)(p < 0.05 for l-arg vs. placebo). in pph patients, l-arg induced no significant change in pao, tpr, and co. mean ppul was 63.4 + 8.8 mm hg at the beginning of the study, but was only slightly reduced by l-arg to 55.0 + 12,3 mm hg (p = n.s.). plasma cgmp was not affected by l-arg in these patients. we conclude that l-arg stimulates no production and induces vasorelaxation in chd patients, but not in patients with primary pulmonary hypertension. thus, the molecular defects underlying the impaired no foimation may be different m both diseases. institutes of clinical pharmacology, *cardiology, and **pneumology, medical school, hannover, germany. the influence of submaximal exercise on the urinary excretion of 2,3-dinor-pgflc, (the major urinary prostacyclin metabolite), 2,3dinor-txb2 (the major urinary thromboxane a2 metabolite), and pge2(originating from the kidney), and on platelet aggregation was assessed in 6 untrained and 10 endurance-trained male subjects before and after 7 days of 50 rag/day of aspirin. urinary 2,3-dinor-txb2 excretion was significantly higher in the athletes at rest (p < 0.05). submaximal exercise increased urinary 2,3-dinor-6-keto-pgfl~ excretion without affecting 2,3-dinor-txb2 or pge2 excretion or platelet aggregation. aspirin treatment induced an -80% inhibition of platelet aggregation and 2,3-dinor-txb2 excretion in both groups. however, urinary 2,3-dinor-6-keto-pgfl~ was inhibited by only 24% in the untrained, but by 51% in the trained group (p <0.05). urinary pge2 was unaffected by aspirin in both groups, indicating that cyclooxygenase activity was not impaired by a systemic aspirin effect. after low dose aspirin administration, the same selective stimulatory effect of submaximal exercise on urinary 2,3-dinor-6-keto-pgfl~ excretion was noted in both groups as before. the ratio of 2,3-dinor-6-keto-pgfld2,3-dinor-txb2 was increased by exercise; this effect was potentiated by aspirin (p < 0.05). our results suggest that the stimulatory effect of submaximal exercise on prostacyclin production is not due to an enhanced prostacyclin endoperoxide shift from activated platelets to the endothelium, but rather the result of endothelial prostacyclin synthesis activation from endogenous precursors. 50 mg/day of aspirin potentiates the favorable effect of submaximal exercise on endothelial prostacyclin production by selectively blocking platelet cyclooxygenase activity. institute of clinical pharmacology, medical school, hannover, germany. soluble guanylyl cyclases (gc-s) are heterodimeric hemeproteins consisting of two protein subunits (70 kda, 82 kda). the enzyme is activated by nitric oxide (no) and catalyzes the formation of the signal molecule "cgmp" (cyclic guanosine-3's'-monophosphate) from gtp. numerous physiological effects of cgmp are already very well characterized. however, detailed insights in the no-activation mechanism of this enzyme have been described to date only in a hypothetical model (1). recently, this concept was supported by experimental data using sitedirected mutagenesis to create a no-insensitive soluble guanylyl cyclase mutant (2). it is generally accepted that the prostethic heine-group plays a crucial role in the activation mechanism of this protein. nonetheless, some interesting questions with regard to structure and regulation of soluble guanylyl cyclases still need to be uncovered (e.g. activation with other free radicals, such as carbon monoxide). since this kind of studies is limited so far by isolating large quantities of a biologically active enzyme with conventional purification techniques, the recombinant protein was expressed in the baculovirus / insect cell system. we describe here the construction and characterization of recombinant baculoviruses, harboring the genes that encode both protein subunits of the soluble guanylyl cyclase. insect cells infected with these recombinant baculoviruses produce between 20-30% (as related to total cell protein) of functional soluble guanylyl cyclase. positive infection was monitored as a change in morphology of the cells and by production of the respective recombinant viruses detected by polymerase-chain-reaction (pcr). so far examined, the recombinant enzyme exhibits similar physicochemical characteristics as the "natural" protein. exogenous addition of several heme analogues to the infected cells is able to either stimulate or inhibit the enzymatic activity of gc-s. we are confident to purify milligram amounts of the recombinant protein in the near future. pet studies of myocardial pharmacology have principally concerned the sympathetic nervous system and u'acers have been developed to probe the integrity of both pre-and post-synaptic sites. the sympathetic nervous system plays a crucial role in the control of heart rate and myocardial contractility as well in the conlrol of the coronary circulation. alterations of this system have been implicated in the pathophysiology of a number of .cardiac disorders, in particular, heart failure, ventricular arrhythmogenesis, coronary artery disease, idiopathic dilated and hypertrophic cardiomyopathy. several beta blockers have been labelled with carbon-ll for imaging by pet. the most promising of these is cgp 12177 which is a non-selective beta adrenoceptor anatagonist particularly suited for pet studies due to its high affinity and low lipophilicity, thus enabling the functional receptor pool on the cell surface to be studied. studies in our institution in a group of young healthy subjects have yielded bmax values of 10.4_+1.7 pmol/g myocardium. these data are consistent with literature values of bmax for beta adrenoceptors in human ventricular myocardium determined by a variety of in vitro assays. a recent study in patients with hypertrophic cardiomyopathy has shown that myocardial beta adrenoceptor density is decreased by approximately 25-30% relative to values in normal subjects. the decrease in receptor density occurs in both hypertrophied and nonhypertrophied portions of the left ventricle. these data are consistent with the hypothesis that sympathetic overdrive might be involved in the phenotypic expression of hypertrophic cardiomyopathy. a further decrease of myocardial beta adrenoceptor density (to levels well below 76_5-7.7 pmol/g) has been observed in those patients with hypertrophic cardiomyopathy who procede to ventricular dilatation and heart failure. cyp1a1 hydroxylates polycyclic aromatic hydrocarbons such as benzo(a)pyrene occurring e.g. in cigarette smoke. two hereditary mutations are discovered: ml, a t to c transition 1,194 bp downstream of exon 7; m2, located at position 4,889 in exon 7 representing an a to g transition resulting an isoleucine to valine substitution in the heme-binding region. recently we could demonstrate in caucasians that carriers of the m2-mutation possess an increased risk towards lung cancer (drakoulis et al clin.lnvestig. 72:240,1994) , whereas the ml-mutation shows no such association. the phasg-ii enzyme gstm1 catalyses the conjugation of glutathione to electrophilic compounds such as products of cyp1ai. gstm1 is absent in 50.9% of the caucasian population due to base deletions in exon 4 and 5 of the gene. we found no contrariety in the gstm1 distribution, including frequencies of type a (p.) and type b (v) among lung cancer patients (odds ratio = 1.01, n = 117; cancer res. 53:1004 res. 53: ,1993 . 149 lung cancer patients and 408 reference patients were investigated for mutations of cypia1 and gstm1 by allele-specific pcr and rflp. a statistically significant higher risk for lung cancer among carriers of the m2trait was found (odds ratio = 2.63, p = 0.001). interestingly, amid lung cancer, m2-alleles were less often linked to ml than in controls (odds ratio = 9.50, 95%-confidence limits = 1.50 -99.7, p = 0.0054). however, the frequency of cypia1 mutations did not differ among active and defective gstm1 types. consequently, we could not confirm in the caucasian population the synergistic effects of cypia1 mutations (especially m2) and deficient gstm1 as combined susceptibility factors for lung cancer as described among the japanese (cancer res. 55:2994 ,1993 in healthy subjects the effect of gastrointestinal hormones like somatostatin and glucagon on splanchnic hemodynamics is not well defined due to the invasiveness of the direct measurement of e.g. portal vein (pv) wedged pressure. methods : now, we applied duplex sonography (3.5 m~z) and color coded flow mapping to compare the effects of ocreotide (i00 ~g sc), a long acting somatostatin agonist, and glucagon (i mg iv) on the hemodynamics of the pv, superior mesenteric artery (sma) and common hepatic artery (ha) in 14 healthy volunteers (13 g,i q; 25 ± 2y; x ± sem). basal values of pv flow (12.9 ± 0.8 cm/s), pv flow volume (549 ± 50 ml/min), sma systolic (sf: 195 ± 13 cm/s) and diastolic flow (df: 28 ± 4 cm/s), sma pourcelot index (pi) (0.86 ± 0.01), ha sf (80 ± 8 cm/s) and df (19 ± 1 cm/s) and ha pi (0.75 ± 0.01) well agreed with previously reported results. within 15 min ocreotide resulted in a decrease of sma sf (-32 ± 4%) sma df (-31 ± 4%), ha sf (-18 ± 5%) and ha df (-24 ± 7 %). maximum drop of pv flow (-33 ± 3%) and flow volume (-34 ± 7%) occurred at 30 min. all effects diminished at 60 min. no significant change of vessel diameter and pi was seen. 5 min following its application glucagon caused a highly variable, only short lasting increase of pv flow volume (+51 ± 18%) and sma df (+49 ± 17%). ha fd (+14 ± 4%) showed a tendency to rise (ns). we conclude that in clinical pharmacology duplex sonography is a valuable aid for measuring effects of hormones and drugs on splanchnic hemodynamics. pectanginal pain and signs of silent myocardial ischemia frequently occur in hypertensives, even in the absence of coronary artery disease (cad) and/or left ventricular hypertrophy, probably due to a reduced coronary flow reserve. since the oxygen extraction of the heart is nearly maximal during rest, increases of oxygen demand cannot be balanced by increases of myocardial perfusion: to assess the frequency of ischemic type st-segment depressions in this patients and to determine the influence of heart rate (hr) and blood pressure (bp), simultaneous 24 h hoher-and 24 h ambulatory bp monitoring were performed in 18 hypertensives (age 43 -71 years, 9 f, 9 m) without cad before and after four weeks on therapy with the 13 -blocker betaxolol. 25 episodes of significant st-segment depressions (>0.1 mv,> 1 min) of a total length of 470 min could be demonsu'ated in 9/18 patients (50%) without antihypertensive therapy_ systolic bp significantly increased from 139 + 13.9 mmhg (mean + sd, p < 0.05) 60 min before to a maximum of 191 + 44.5 mmhg during the ischemic episodes, hr and rate-pressure product (rpp) increased from 76 + 6.3 min -t and 100.6 + 20.9 mmhg x rain -t x 102 to 138 _+ 16.8 min-: and 230.4 + 88.9 mmhg x min -1 x 102 (p < 0.05). the extent of st-segment depressions significantly correlated with hr and rpp (p < 0.05). drug therapy with 10 -20 mg/d betaxolol for 4 weeks significantly decreased mean hr, systolic' and diastolic bp (p < 0.05). 6 ischemic episodes of a total length of 38 min were recorded only in 4 of 15 hypertensives (26.7 %; p < 0.05; x2-test). in conclusion, increases of hr and systolic bp seem to be the most important factors which induce myocardial ischemia in hypertensives without cad. as silent ischemia is a independent risk factor for sudden cardiac death and other cardiac events, specific antihypertensive therapy should not only be aimed to normalize blood pressure, but should also address reduction of ischemic episodes as demonstrated here. phosphodiesterase inhibitors exert their positive inotropic effects by inhibiting camp degradation and increasing the intracellular calcium concentration in cardiomyocytes. an identical phosphodiesterase type 1i[ has been demonstrated in platelets and vascular smooth muscle cells. we studied the influence ofpiroximone on platelet function in vitro and ex vivo and the hemodynaimc effects of a bolus application of piroximone in patients with severe heart failure (nyha iii-iv) using a swan -ganz-catheter. in order to study the influence ofpiroximone on platelet function in vitro, platelet rich plasma from healthy volunteers was incubated with piroximone (10-100 ~tmol/l) from 1 minute to 2 hottrs and aggregation was induced by addition of adp. for the ex vivo experiments platelet rich plasma was obtained from patients, who received piroximone in doses of 0.25, 0.5, 1.0 or 2.0 mg/kg bw. blood samples were drawn immediately before and 30, 60, 90, 120 and 240 minutes after bolus application. the adp-induced platelet aggregation was inhibited time-and dosedependently. the ic50 value for piroximoue in vitro amounted to 67 +14 omol/1. in the ex vivo experiments the maximal inhibition of adp-induced aggregation was obtained in prp from patients who had received 2 mg/kg bw piroximune 60 minutes before. the admitdstration ofpiroximone resulted in a marked hemodynamic improvement with a dose-dependent increase in cardiac index and decreases in pulmonary artery pressure and resistance. to treat conditions associated with acute and chronic multiorgan dysfunction. studies indicate patients receive approximately ten drugs, on average during their icu stay, from several drug classes. commonly prescribed drugs include narcotics, sedatives, antibiotics, antiarrhythmics, antihypertensives, drugs for stress ulcer prophylaxis, diuretics, vasopressors, and inotropes. reports suggest surgical icu patients cost the hospital an average of $18,000/patient in un-reimbursed costs under fixed-price reimbursement. furthermore, patients with the greatest drain in revenue received catecholamines, triple antibiotics, or antifungal agents. thrombolytics, antibiotics, plasma expanders, and benzodiazepines account for nearly twothirds of the cost of drugs prescribed in medical and surgical icus. agents with considerable economic impact include biotechnology drugs for sepsis. pharmacoeconomic data in icu patients suggest increased attention should be directed towards several areas, including patients with pneumonia, intraabdominal sepsis, nosocomial bloodstream infections, optimizing sedation and analgesic therapy, preventing persistent paralysis from neuromuscular blockers, preventing stress ulcers, treating hypotension, and providing optimal nutritional support. studies are needed to assess the impact of strategies to improve icu drug prescribing on length of stay and quality of life. if expensive drugs are shown to decrease the length of icu stay, then their added costs can have positive economic benefits to the health care system. the responses to 10 min iv. infusions of the 131-and 132-adrenoceptor agonist isoprenalin (iso) and the 132-(and c~-) adrenoceptor agonist adrenalin (adr) at constant rates of 1 ijg/min were evaluated noninvasively after pretreatment (pre-tr) with placebo (pl), 100 mg of the 131-selective adrenoceptor antagonist talinolol (tal) and 80 mg of the non-selective 13antagonist propranolol (pro) in 12 healthy subjects. the following were analysed: heart rate (hr, bpm), pre-ejection time (pep, ms), ejection time (vet, ms), hr-corrected electromechanical systole (qs2c, ms), impedance-cardiographic estimates of stroke volume (sv, ml), cardiac output (co, i/min) and peripheral resistance (tpr, dyn.s.cm -5) calculated from co and mean blood pressure (sbp and dbp according to auscultatory korotkoff-i and -iv sounds this indicates that 1) about half the rise of hr and co and half the shortening of pep is 131-respectively 1~2-determined, 2) that predominant 132-adrenergic responses, whilst not affecting vet, take optimal benefit from the inodilatory enhancement of pump performance, 3) that an additional 131-adrenergic stimulation is proportionally less efficient, as vet is dramatically shortened, thus blunting the gain in sv so that the rise in co relies substantially on the amplified increase of hr and 4), vet is more sensitive than qs2c in expressing additional 131-adrenoceptor agonism and 5) prime systolic time intervals provide a less speculative and physiologically more meaningful represenation of cardiac pump dynamics than hr-corrected ones. zentrum flit kardiovaskul~re pharmakologie, mathildenstral3e 8, 55116 mainz, brd a20 regression between blunting of ergometric rise of heart rate and l~ladrenoceptor occupancies in healthy man c. de mey, d. palm, k. breithaupt-grsgler, g.g. belz the hr-responses to supine bicycle ergometry (4 min at appr. 200 watt) were investigated at several time points after the administration of propranolol (pro: 40, 80, 160 mg), carvedilol (car: 12.5, 25, 50, 100 mg), talinolol (tal: 25, 50, 100, 400 mg), metoprolol (met: 600 mg) and celipro-iol (cel: 1200 mg) to healthy man. the effects of the agents (= difference of the ergometric response for active drug and placebo) were analysed for both the end values (end) and the increments (inc) from resting values immediately before ergometry up to end. the effects were correlated with the %-~l-adrenoceptor occupancies estimated using a standard emax-model (sigmoidicity=l) from the concentrations of active substrate in plasma determined by i~l-adrenoceptor specific radioreceptor assay. the respective intercepts (i), slopes (s) and correlation coefficients (r) are detailed here below : inhibition of leukotrienes is a promising approach to the treatmer~t of several diseases because excess formation of these lipid mediators has been shown to play an important role in a wide range of pathophysiological conditions. since until recently we were not able to obtain specific drugs suppressing leukotriene biosynthesis or action for clinical practice, we started investigating the effects of putative natural modulators of leukotriene biosynthesis such as fish oil. 10 healthy male volunteers were supplemented for 7 days with fish oil providing 40 mg eicosapentaenoic and docosahexaenoic acid per kg body weight and day. the urinary concentration of leukotriene e4 plus n-acetyl leukotriene e4 served as a measure for the endogenous leukotriene production, treatment resulted in a significant increase in the eicosapentaenoate concentration in red blood cell membranes. fish oil reduced the endogenous leukotriene generation in 8 of the 10 volunteers. the effect was associated with a decrease in urinary prostaglandin metabolites, determined as tetranorprostanedioic acid. in contrast to what was expected from published in vitro and ex vivo experiments, no endogenously generated cysteinyl leukotrienes of the 5 series could be identified. the inhibitory effect of fish oil on the endogenous leukotriene generation was not synergistic to the effect of vitamin e, which also exhibited some suppressive activity. early clinical data on the effects of fish oil on teukotriene production in patients with allergy or rheumatoid arthritis are not yet conclusive. we conclude that fish oil exhibits some inhibitory activity on leukotriene production in vivo. the effectivity of fish oil may be attenuated by concomitant modulation of other mediator systems e.g. up-regulation of tumor necrosis factor production. • the number and affinity of platelet thromboxane (txa2) and prostacyclin (pgi2)-receptors are regulated by several factors. we studied the influence of oral intake of acetylsalieylic acid (asa) on ex-vivo binding studies with human platelet membranes on the binding of the specific thromboxane a 2 antagonist 3h-sq-29548 and the pgi 2 agunist 3h-l]oprost. the number of receptors (bmm) and the binding affinity (kd) were calculated using scatchard's plot analysis. in healthy male volunteers 11o significant difference was seen following intake of 500 mg/d of asa for 14 days (mean -+ sem): the potency of meloxicam (mel), a new anti-inflammatory drug (nsaid), in the rat is higher than that of well-known nsaids, in adjuvant arthrtitis rats, mel is a potent inhibitor of the local and the systemic signs of the disease. mel is also a potent inhibitor of pg-biosynthesis by leukocytes found in pleuritic exudate in rats. conversely, the effect of mel on pg-biosynthesis in isotated enzyme preparations from bull seminal vesicle in vitro, the effect on intragastric and intrarenal pg-biosynthesis and the influence on the txb2-1evel in rat serum is weak. in spite of the high antiinflammatory potency in the rat, mel shows a low gastrointestinal toxicity and nephrotoxicity in rats. -cyclooxygenase-2 (cox-2) has been recently identified as a isoenzyme of cyclooxygenase. nsaids are anti-inflammatory through inhibition of pg-biosynthesis by inducible cox-2 and are ulcerogenic and nephrotoxic through inhibition of the constitutive cox-1. we have investigated the effects of mel and other nsaids on cox-1 of non stimulated and on cox-2 of lps-stimulated guine pig peritoneal macrophages. cells were cultured with and without lps for 6 hrs together with the nsaid. arachidonic acid was then added for further 20 mins, the medium removed and pge 2 measured by ria. bimakalim, emd 52692, is a new investigational k+-channel activator with vasod[lating properties. single pereral doses of 0.2 mg bimakalim, 60 mg diltlazem, either alone or in combination, were investigated in 13 healthy male supine volunteers (20 to 28 years of age) [n a placebo-controlled, periodbalanced, randemised, double-blind, 4way cross-over design. point estimates of the global effects of bimakalim [k] , di]tiazem [d] and their interaction [kxd, =0 in case of mere additivity] incl. 95% confidence intervals (ci) were analysed for systolic and diastolic blood pressure (sbp, dbp; mmhg), heart rate (hr; bpm), pq (ms), systolic time intervals (pep, qs2c, lvetc; ms), cardiac output (co; i.min-1), total peripheral resistance (tpr; dyn.s.cm-5), heather index (hi; q.s-2); 1,5 h after dosing, *statistically significant at a=0.05: -7 to -1 -4 to 3 -8 to 0 -3 to 5 0.5 to 5.9 -3.4 to 1,9 4.9 to 11.8 * -3.9 to 3,1 -8.2 to -1.2 * -5.3tol,6 -5.1 to 2,0 -4.8 to 2.3 -2.9 to 6.6 -3.9 to 5.6 -0.25 to 0.75 -0.66 to 0.35 -161 to 3 -74 to 90 -&07 to 1.14 -0.50 to 0.71 afterload reduction and a drop in dbp occurred with bimakalim associated with a rise in hr and mild increase in cardiac performance, diltiazem (slightly) decreased afterload and bp with little (reflectory) accompanying changes and had a negative dromotropic effect. the combination caused additive effects. center for cardiovascular pharmacology, zekapha gmbh, mathildenstr. 8, 55116 mainz, germany. rheumatoid arthritis (ra) is characterized by an immunological mediated inflammatory reaction in affected joints. infiltration of granulocytes and monocytes is the pathophysiological hallmark within the initial phase of inflammation. these cells are able to synthesize leukotrienes. ltb 4 is a potent chemotactic factor and therefore could be responsible for the influx of granulocytes from the circulation. cysteinyl leukotrienes ltc4, d 4 and e 4 augment vascular permeability and are potent vasoconstrictors. ltb 4 and cysteinyl leukotrienes have been detected in synovial fluid of patients with ra. however, these results are difficult to interprete, because the procedure is invasive and artificial synthesis cannot be excluded. we used a different, noninvasive approach by assessing the excretion of lte 4 into urine. studies with 3hltc4 have demonstrated that lte 4 is unchanged excreted into urine and is the major udnary metabolite of cysteinyl leukotrienes in man. udnary lte 4 was isolated from an aliquot of a 24 hour urine collection by solid phase extraction followed by hplc and quantitated by ria. nine patients were enrolled in the present study. all met the american college of rheumatology criteria for ra. patients were treated with nonsteroidal inflammatory drugs and disease modifying drugs. therapy with prednisolon was started after collection of the initial 24 hour urine sample. disease activity was assessed by crp (mean 59+22 mg/l) and esr (mean 57_+37mm/hour platelet aggregation is mediated by the binding of an adhesive protein, fibrinogen, to a surface receptor, the platelet glycoprotein lib/ilia. gpiib/llla is one of a family of adhesion receptors, integrins, which consist of a ca++-dependent complex of two distinct protein subunits. under resting conditions, gpiib/llla has a low affinity for fibrinogen in solution. however, activation of platelets by most agonists, including thrombin, adp and thromboxane results in a conformational change in the receptor and the expression of a high affinity site for fibrinogen. binding of fibrinogen to platelets is a common end-point for all agonists and therefore is a potential target for the development of antiplatelet drugs. these have included chimeric, partially humanised antibodies (7e3), peptides and peptidomimetics that bind to the receptor and prevent fibrinogen binding. the peptides often include the sequence rgd, a sequence that is present in fibrinogen and is one of the ligand's binding sites. when administered in vivo, antagonists of gpiib/llla markedly suppress platelet aggregation in response to all known agonists, without altering platelet shape change, a marker of platelet activation. they also prolong the bleeding time in a dose and perhaps drug dependent manner, often to more than 30 rain. in experimental models of arterial thrombosis, gpllb/llla antagonists have proved highly effective and are more potent than aspirin. studies in man have focused on coronary angioplasty, unstable angina and coronary thrombolysis and have given promising results. 7e3 given as a bolus and infusion combined with aspirin and heparin reduced the need for urgent revascularisation in patients undergoing high-risk angioplasty, although bleeding was more common. some compounds have shown oral bioavailability raising the possibility that these agents could be administered chronically. antagonists of the platelet gpiib/llla provide a novel and potent approach to antithrombotic therapy. drug databases on computers are commonly textfiles or consist of tables of generic-names or prices for example. until now pharmacokinetic data are not easily available for regular use, because searching parameters in a textfile is time consuming and personal intensive. on the other hand these pharmacokinetic data are the fundamental background of every dosage regimen and individual dosage adjustment. for many drugs elimination is dependent on the patients renal function. renal failure leads to accumulation, possibly up to toxic plasma concentrations. therefore, the decision was to build up a pharmacokinetic database. the aim is to achieve simplicity and effectiveness by using the basic rules. only three parameters are needed to describe the pharmacokinetics: clearance (ci), volume of distribution (vd) and half-life (t~). moreover, with two parameters the third can be calculated ancl'controlled by the equation: cl = vd * 0,693 / t½ according to the dettli-equation and the bayes' theorem estimation of individual pharmacokinetic parameters will be done by a computer program. the advantage is that the impact of therapeutic drug monitoring can be increased. using the population data and the bayesian approach, only one measurement of serum drug concentrations might be enough to achieve an individual dosage regimens (el desoky et al., ther drug monitor 1993, 15: 281) higher therapeutic security for the patient can be achieved. there is also a major pharmacoeconomic aspect: adapting drug dosage reduces costs (susanka et al., am j hosp pharm 1993, 50:909) the basic database for future pharmacokinetic clinical desicions is going to be built up. the pharmacokinetic interactions with grape#uit juice reported for many drugs are attributed to the inhibition of cytochrome p450 enzymes by nanngenin, which is the aglycene of the bitter juice component nadngin. however, only circumstantial evidence exist that naringenin is indeed formed when grapefruit juice is ingested, and the lack of drug interaction when naringin solution is given instead of the juice is still unexplained. we investigated the pharmacokinetics of naringin, naringenin and its conjugated metabolites following ingestion of 20 ml grapefruit juice per kg body weight, containing 621 ijm naringin, in 3 male and 3 female healthy adults. urine was collected 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-16 and 16-24 hours alter juice intake. naringin and naringenin concentrations were measured by reversed phase hplc following extraction using ethyl acetate, with a limit of quantitation of 300 nm. conjugated metabolites in urine were transformed by incubation with glucuronidase (28000 u/ml) / sulfatase (733 u/ml) from abalone entrails for 4 h at ph 3.8 and determined as parent compounds. additionally, naringin and naringenin concentrations were measured in plasma samples from grapefl'uit juice interaction studies conducted previously. neither naringin nor its conjugated products were detected in any of the samples. naringenin was not found in plasma. small amounts of nanngenin appeared in urine alter a median lag time of 2 hours and reached up to 0.365 % of the dose (measured as nanngin). after treatment with glucuronidase / sulfatase, up to 57 % of the dose was recovered in urine: the absence of naringin and its conjugates and the lag time observed for naringenin to appear in urine suggests that cleavage of the sugar moeity may be required before the flavonoid can be absorbed as the aglycone. naringenin itself undergoes rapid phase ii metabolism. whether the conjugated metabolite is a potent cytochrome p450 inhibitor is unknown but not probable. the pronounced variability of naringenin excretion provides a possible explanation for apparently contradictory results in grapefruit and/or naringin interaction studies. grapefruit juice increases the oral bioavailablity of almost any dihydropyridine tested, presumably due to inhibition of first-pass metabolism mediated by the cytochrome p450 isoform cyp3a3/4. the mean extent of increase was up to threefold, observed for felodipine, and more pronounced drug effects were also reported. thus, a such interaction may be of considerable clinical relevance. no data are yet available for nimodipine. we conducted a randomized cross-over interaction study on the effects of concomitant intake of grapefruit juice on the pharmacokinetics of nimodipine and its metabolites m11 (pyridine analogue), m10 (demethylated) and m8 (pyridine analogue, demethylated). 8 healthy young men (4 smokers / 4 nonsmokers) were included into the investigation. nimodipine was given as a single 30 mg tablet (nimotop e) with either 250 ml of water or 250 ml of grapefruit juice (d~hler gmbh, darmstadt, 751 mg/i naringin). concentrations ef nimodipine and its metabolites in plasma withdrawn up to 24 hours p.ostdose were measured by gc-ecd, and model independent pharmacokinetic parameters were estimated. the study was handled as an equivalence problem, and anova based 90 % confidence intervals were calculated for the test (=grapefruit period) to reference (= water period) ratios. the absence of a relevant interaction was assumed if the ci were within the 0.67 to 1.50 range: grapefruit juice was reported to inhibit the metabolism of a variety of drugs, including dihydropyridines, verapamil, terfenadine, cyclosporine, and caffeine. these drugs are metabolized mainly by the cytochrome p450 isoforms cyp1a2 (caffeine and, in part, verapamil) and cyp3a (others). theophylline has a therapeutic range of 5-20 mg/i and is also in part metabolized by cyp1a2. therefore, we conducted a randomized changeover interaction study on the effects of concomitant intake of grapefruit juice on the pharmacokinetics of theophylline. 12 healthy young male nonsmokers were included. theophylline was given as a single dose of 200 mg in solution (euphyllin e 200), diluted by either 200 ml of water or 200 ml of grapefruit juice (d0hler gmbhi darmstadt, 751 mg/i nadngin). subsequently, additional fractionated 0.8 i of either juice or water were administered until 16 hours postdose. theophylline concentrations in plasma withdrawn up to 24 hours postdose were measured by hplc, and pharmacokinetics were estimated using compartment model independent methods. the study was handeled as an equivalence problem, and anova based 90 % confidence intervals were calculated for the test (=grapefruit period) to reference (= water period) ratios (trnax: differences thus, no inhibitory effect of grapefruit juice on theophylline pharmacokinetics was observed. the lower contribution of cyp1a2 to primary theophylline metabolism or differences in naringin and/or naringenin kinetics are possible explanations for the apparent contradiction between the effects of grapefruit juice on caffeine and on theophylline metabolism. the physical stability of erythromycin stearate film tablets was studied according to a 23 factorial design with experimental variables temperature, relative humidity, and storage time. after one half year of storage at 40oc and 20% relative humidity, the fraction of dose released within 30 min in a usp xxl paddle apparatus under standard conditions decreased from 93% for the reference stored at ambient temperature in intact blister packages to 13% for the stress-tested specimens. chemical degradation of the active ingredient did not become apparent before 12 months of storage. under all other storage conditions, no effects of physical aging upon drug release were found. the bioequivalence of reference and stress-tested samples was studied in six healthy volunteers. the extent of relative bioavailability of the test product was markedly reduced (mean: 8.5%, range: 6-12 %), mean absorption times of the test product were significantly prolonged. the results indicate that the product tested can undergo physical alterations upon storage under unfavourable conditions, and lose its therapeutic efficacy. it can be expected that this phenomenon is reduced by suitable packaging, but the magnitude of deterioration may cause concern. on the other hand, incomplete drug release is in this case easily detected by dissolution testing. whether similar correlations exist for other erythromycin formulations remains to be demonstrated. the efficacy of a drug therapy is considerably influenced by patient compliance. within clinical trials the effects of poor compliance on the interpretation of study results frequently leads to underestimating the efficacy of the treatment. in the evaluation of the "lipid research clinics primary coronary prevention trial" and the "helsinki heart study" special attention was focused on compliance with medication. the strong influence of compliance on clinical outcome and the dilutional effect of poor compliance on the efficacy of the respective drugs occured in both these trials. there are indirect (e.g. pill-count, patient interview) and direct methods (e.g. measurement of drugs, metabolites or chemical markers in body fluids) used to assess compliance with drug therapy. the indirect methods mentioned are commonly considered as unreliable. the direct methods can prove dose ingestion a short time before the sample is taken, however, they cannot show the time history of the drug use. an advanced method of measuring compliance is to use electronic devices. the integration of time/date-recording microcirculty into pharmaceutical packaging, so as to compile a time history of package use, provides real-time data as indicative of the time when dosing occurred. this method supports a precise, quantitative definition of "patient compliance" as: the extent to which the actual time history of dosing corresponds to the prescribed drug regimen. by taking real-time compliance data into account the results from clinical trials show not only clearer evaluations of drug efficacy and dese-reponse-relationship but also a better understanding of dose dependant adverse drug reactions. in the present study, we examined the usefulness of eroderm-1 and eroderm-2. seventy five impotent men, 24 to 65 years old, participated in the present trial. the patients were classified into 3 groups, 25 patients each. the first group was treated by cream containing only co-dergocrine mesilate (eroderm-1), the second received a cream containing isosorbide dinitrate, isoxsuprine hcl and co-dergocrine mesilate (eroderm-2), while the third used a cream containing placebo. the cream was applied to penile shaft and gland 1/2-1 hr before sexual stimulation and intercourse. the patients were asked to report their experience via questi'onnaire after one week. the results of treatment are as follows: seven patients (28%) who applied eroderm-1 indicated a full erection and successful intercourse. the use of eroderm-2 restored potency in 14 patients (56%) of the second group. three men (12%) of psychogenic type reported overall satisfaction with placebo cream. treatment of impotence with eroderm cream was most successful in patients with psychogenic disorders which are often coincident with minor vascular or neurological disorders. fair results were reported by patients afflicted by moderate neurological disorders. except for one case of drug allergy following the use of eroderm-2, no side effects were reported. we believe that eroderm cream has obvious advantages and may be a suitable treatment before the use of non-safe method as intracavernous medication. a new type of topically applied drugs (eroderm creams) for impotence is presented. eroderm creams contain vasoactive drugs. these drugs have ability to penetrate the penile cutaneous tissue and facilitate erection. in the present study, we examine the usefulness of eroderm-3 in the treatment of erectile dysfunction. eroderm-3 contains tiemonium methylsulfate, a.f. piperazine and jsosorbide dinitrate. a randomized, double blinded control trial on 36 patients was performed. the etiology of impotence was investigated. all patients received eroderm-3 and placebo cream. the patients randomized into 2 groups of 18. the first group received eroderm-3 on day 1 and placebo cream on day 2, however, group two received placebo on day 1. the patients were advised to apply the cream on the penile shaft 1/2 -1 hr, before sexual stimulation and intercourse. the patients reported their experience via questionnaire. overall 70 percent of patients demonstrated a response with eroderm-3. the other responders reported a partial erection and tumescenous. three men (8%) reported a full crection and satisfied intercourse with either cream. these patients were psychogenic impotence. neither eroderm-3 nor placebo cream produced marked response in 11 patients. four patients were venous leakage which were advised to use tourniquet at the base of penis after 1/2 hr. of cream application. only one of them indicated a good response. the highest activity proved to occur in psychogenic impotence. less rate of success was observed in patients with minor to moderate neurological and/or arterial disorders. no marked side effects were recorded. for these reasons eroderm-3 may be proposed as first line therapy of erectile dysfunction. control of cell proliferation is a basic homeostatic function in multicellular organisms. we studied the effects of some prostaglandins and leukotrienes and of their pharmacological inhibitors on cell proliferation in murine mast cells and mast cell lines, in a human promyelocytic cell line (hl-60 cells) and in burkitt's lymphoma cell lines. in addition, prostaglandin and leukotriene production was investigated in mast cells, representing putative endogenous sources of these lipid mediators. murine mast cells were derived from bone marrow of balb/c mice. proliferation of cells was estimated using a colorimetric assay (mtt-test). production of prostaglandin d2 (pgd2), pgj2, delta-12-pgj2, leukotriene c4 (ltc4) and ltb4 by mast cells was determined by combined use of high performanceliquid chromatography and radioimmunoassay. pgd2 and its metabolites pgj2 and delta-12-pgj2 exhibited significant antiproliferative effects in the micromolar range in mast cells, mast cell lines, hl-60 and burkitt's lymphoma cell lines whereas inhibition of cyclooxygenase by indomethacin was without major effects. ltc4 and ltb4 had a small stimulatory effect on cell proliferation in hl-60 cells. degradation and possibly induction of cell differentiation may have attenuated the actions of leukotrienes. the leukotriene biosynthesis inhibitors aa-861 and mk-886 reduced proliferation of hl-60 and lymphoma cells significantly but had no major effects on mast cell growth. on the other hand, mast cells stimulated with calcium ionophore produced pgd2 and its metabolites, as well as ltb4 and ltc4 in significant amounts. from our data we conclude that prostaglandins and leukotrienes may play an important role in the control of cell proliferation. we compared the pattern of drug expenditures of several hospitals in 1993 (size: 1000 to 1400 beds). a, b are university hospitals in the ,,old"and c,d,e are university hospitals in the ,,new" german countries, f is a community based institution in an ,,old" german country. main data source were lists comprising all drags according to their expenditures in a rank order up to 100%. items were classified into i) pharmaceutical products including immunoglobulines, ii) blood and -derived products (cell concentrates, human albumin, clotting factors) and iii) contrast media (x-ray). with regard to group i) the highest expenditures nccured in hospitals a and b whereas drug costs in c -e were 1/3 less and came to only 20% in hospital f. the main groups of drugs which together account for > 50% of these expenditures are shown in the table. ) products were about 20% up to 40% of group i and highest in hospitals a, b and e, but about 1/3 lower in hospitals c and d. these results suggest meaningful differences in the drug utilization between the old and new countries as well as betv,,een university institutions and community based hospitals. however, although all hospitals provide oncology and traumatology services and all university hospitals offer ntx, differences in other subspecialities e.g bone marrow and liver transplantation and treatment of patients with haemophilia must be considered, too. dr.medsebastian harder, dept c]inicai pharmacology, university hospital frankfurt, theodor stern kai 7, 60590 frankfurt/main frg m. hgnicka, r. spahr, m. feelisch, and r. gerzer organic nitrates like glyceryl trinitrate (gtn) act as prodrugs and release nitric oxide (no), which corresponds to the endogenously produced endothelium-derived relaxing factor. in the vascular tissue, no induces relaxation of smooth muscle cells, whereas in platelets it shows an antiaggregatory effect. both activities are mainly mediated via stimulation of soluble guanylyl cyclase (sgc) by no. in contrast to compounds which release no spontaneously, a membrane-associated biotransformation step is thought to be required for no release from organic nitrates. glutathione-s-transferasea and cytochrome p-450 enzymes have been shown to metabolize organic nitrates in the liver, but little is known as to whether these enzymes are involved in the metabolic conversion of organic nitrates in the vasculature. furthermore, it is still unclear whether or not platelets are capable of metabolizing organic nitrates to no. we isolated the microsomal fraction of bovine aorta in order to characterize the activities towards organic nitrates using the guanylyl cyclase reaction as an indirect and the oxyhemoglobin-technique as a direct measure for no liberation. gtn was metabolized to no by the microsomal fraction under aerobic conditions already in the absence of added cofactors. this activity was not influenced by the cytochrome p-450 inhibitors cimetidine and metyrapone. in contrast, the glutathione s-transferase substrate 1-chloro-2,4-dinitrobenzene and the glutathione s-transferase inhibitors sulfobromophthalein and ethacrynic acid did not affect no release, but potently inhibited sgc activity. blocking of microsomal thiol-groups resulted in a decreased no release from gtn. homogenates of human plateles isolated by thrombapheresis and stabilized by addition of 5 mm n-acetylcysteine did not show no-release from gtn as determined by the stimulation of the platelet sgc even after addition of the possible cosubstrates glutathione and nadph. these data demonstrate (1) that bovine aortic microsomes exhibit an organic nitrate metabolizing and no-releasing activity whose properties are clearly different from classical cytochrome p-450 enzymes and from glutathione s-transferases, and (2) that human platelets itself are not capable of bioactivating organic nitrates and therefore require organic nitrate metabolism in the vessel wall for antiaggregation to occur. bioavailability of acesal ®, acesal ® extra, micristin ® (all 500 mg acetylsalicylic acid -asa), and miniasal ® (30 mg asa), opw oranienbufg, relative to respective listed references was studied in female and male healthy volunteers (age 18-35 y, weight 48-90 kg, height 161-198 cm). asa and salicylic acid (sa) were measured using an hplc method validated from 50 ng/ml to 60 pg/ml. extent of absorption was assessed by auc (bioequivalence range 0.8-1.25), rate by cr~/auc (bioequivalence range 0.7-1.43). geometric means and 90%-confidence limits of the ratios test/reference (multiplicative model) are shown in the acesal ® and micdstin ® were bioequivalent in rate and extent of absorption with the reference formulations. the fast liberating acesal ® extra was bioequivalent with respect to extent only. asa from miniasal ® was absorbed more slowly than from an asa solution (cm= (68%-range): 264-536 ng/ml and 387-726 ng/ml; t~ (min-max): 0.33-2.0 h and 0.17-0.5 h). asa from micdstin ® and the corresponding reference was absorbed more slowly than from acesal ® and acesal ® extra. this was accompanied by decreased aucasa (increase of first pass metabolism) and increased apparent trrz (absorption being rate limiting). all ratios of aucsa/aucasa after administration of 500 mg asa were markedly higher than after 30 mg asa. thus, the formation of salicyludc acid from sa might be capacity limited at doses of 500 mg asa. in the study >>physicians' assessment of internal practice-conditions and regional health-services-conditions in accordance with ambulatory patient-management<< a sampie of 130 primary care physicians -comprising gps and internists -provide data for continuons analyses of arnbulatory health care quality and structure. focussing on the physicians' drug prescription, the impacts of 1993 reform law (gesundheitsstralcturgesctz, gsg) upon primary care providers and their therapeutic decisions were examined in 1993. four different surveys were carded out during the year, dealing with frequent patients' reasons for encounter in gps' offices. after a pretest was carried out, physicians reported on 3728 patient-physician-encounters, basing on mailed questionnaires. for every therapeutic change patients received, the reasons for the change were recorded (e.g, reform law, medical indication) and above the physicians' expectations towards three criteria to measure the quality: 1) physicians' assessment of the patients' satisfaction, 2) adverse drug effects, 3) therapeutic benefit. according to therapeutic changes due to 1993 reform law (drag budgets, blacklist) it can be stated: 1) therapeutic changes due to reform law were carried out with relevant frequency. 2) the reform law was of different concern regarding the different reasons for encounter we investigate& 3) the impacts' strangth of the legal control mechanisms differed among several groups of physicians: those who already have been liable to recourse before 1993 more often carried out therapeutic changes according to fixed drug budget. different multivariate logistic regression-models yield an estimation of the odds-ratio of about 3. 4) therapeutic changes in accordance with the 1993 reform law having been carried out at the beginning of the year more often suffered from negative expectations towards the therapeutic quality then changes during the actual encounter, e.g. >>joint pains<c 26% decrease of the therapeutic benefit in contrast to 7%. during the year a decrease of negative expectations is noticable. 5) patients overestimating the severity of their own health problem in comparison to the physician more often suffered from negative expectations towards the therapeutic change than other patients, e.g. concerning the therapeutic benefit (29% in contrast to 3%). generally speaking, firstly the reform law led to insecurities -particularly in the introduction phase -among susceptible subgroups of physicians and patients, regarding the four investigated reasons for encounter. secondly, there is evidence of a loss of quality in ambulatory health care -predominandy in terms of subjective criteria. these impacts considerably subsided within the field phase duration from may to november, 1993. the presented analyses give evidence of the practicability of studies assessing effects of legally-initiated mechanisms to control health costs upon primary health care quality. postansehrifi: iseg, bissendorfer sm 9, d-30625 hannover the cellular and biochemical mechanisms that lead to the longerm complicatiorts of diabetes mellitus are poorly understood. glycation is a multireaetion beginning with a condensation reaction between reducing sugars and reactive amino groups of proteins. the accumulation of the glycated products of various proteins may contribute to the development of the complications of diabetes and aging. in order to find suitable non-toxic glycation inhibitors we used sulfur deriva-tives (penicillamine, eaptopril and alpha-lipoic acid) and amino derivatives (aminoguani-dine and aminoguanidine propionic acid) and postulated the mechanisms involved.the inhibitou effect of the drags on the early (amadori product) and advanced glycation products (age). determined by affinity chromatography, deglycation method. i-iplc and fluorescence spectroscopy, differs markedly depending on the drag used and is greatest (up to 80 %) with aminoguauidine and penicillamine (20 mm) and smallest with alpha-lipoic acid. aminoguanidine propionic acid had no inhibitory effect on the glycation process. the ability of penicillamine to inhibit both the formation of amadori products and age was demonstrated by isolation of the adducts of penicillamine with ribose, glucose, glucosa-mine and p-tolylglucosamine, a standard for glycated protein. their structures were characterized using ir-and 1h-nmr and 13c-nmr spectroscopy. the oxidative deg-radation of glycated human serum albun{in in the presence of copper (ii), forming the carbox3jmethyllysine, was inltibited by the sulfur derivatives. the in vitro formation of pentosidine, a crossliuking product was influenced by all drugs used however captopril, alpha-lipoic acid and aminoguanidine propionic acid do not condense with sugar derivatives.the mechanism by which captopril and alpha-lipoic acid inhibit the formation of age may be involve radical formation, where a spontaneus thiol/disul-fide interchange between drug and protein takes place. complications can appear at any time in diabetic patients.the cause of these complica-lions in some cases may be due glycation and/or oxidation. the clinical applications of substances which are able to prevent these events may have advantages. these results and the mechanism proposed show that the amino and sulfl~ydryl groups of drags reduce glycation and postglycation of proteins and thus have potential value in this regard. non-enzymatic glycation starts by covalent binding of single sugars to o~ or e amino groups of proteins. in subsequent reactions a number of early glycation products like schiff bases or amadori products, and of advanced glycation endproducts (ages) is formed. glycation is increased in a variety of proteins in diabetic patients [schleicher e (1992) diabetes und stoffwechsel 1: 274] including human serum albumin (hsa), the red cell membrane, hemoglobin, collagen, laminin, immunoglobulin g, low-and high-density lipoproteins. the process is supposed to be a key mechanism in the pathogenesis of diabetic complications and in tisstie ageing [vlassara het al. (1994) lab invest 70: 138] . inhibition of glycation may therefore be important for the prevention of late diabetic complications. we investigated the in vitro glycation of human serum albumin (hsa) and its inhibition by aminoguanidine, penicillamine, captopril and c~-lipoic acid (20 ram). hsa (40 g/l) was glycated by incubation with 20 mm glucose in 0.1 m phosphate buffered saline, ph 7.4, at 37 °c for 28 days under sterile conditions in the presence of 0.1% natrium azide. the glycation rate was determined by two different methods: deglyeation -measures amadori products based on the colorimetry of 2-ketoglucose which is released from the glycated protein (ketoamine) the effect of aniseylated #asminogen streptokinase activator complex (apsac) on left ventricuiar (ll r) function ;,.,as investigated in ! 5 patien~ with acute myocardial infarction (am!). a dose of 3q units apsac was given intraveneusiy to patien~ with ami within 4 hours after onset of pain, patients underwent cardiac angiegraphy at 90 minutes and at day 28. wail motion ,.~s measured by the global ejection fraction and by the centefline method in :'=he infarct and neninfar~ regions and expressed in units of sf~ndard deviations beiew normai, as defined in 50 normai patient. group a with successfui ',th, rombo!ysis (timi 2 and 3) was compared with group b without successfui thrombolysis (timi 0 and !). at 90 minutes there vras no significant difference in giebal ejection fraction between both groups, by using the regiona! wa[i motion by the centerline method group a showed a significant improvement of lv function (p ~ 0.05). the benefit of successeli thrombeiysis was more marked in the peripheral infarct region than in the centrai infarct region. thus early repeffusion is an important factor in maintaining good lv function after ami. the re=gionai wai! motion in the infarct region seems to be more sensmve man me g!oba! eiection fraction to show ear!y differences in lv function after ami. the quinoline derivative bay x 1005 ((r)-2-[4-(quinoliu-2-yl-metho.,qc)phenyl]-2cyclopentyl acetic acid) is a selective inhibitor of ltb4-biosynthesis in various in vitro and in vivo models. it acts via binding to the integral nuclear membrane protein five-lipoxygenase activating protein (flap), which has been shown to be an essential component of the cellular leukotriene synthesis machinary. the function of flap is only poorly understood, but recently it has been shown that a radiolabeled photoaffinity-analog of arachidonic acid binds specifically to flap. consequently, a role of flap in the substrate transfer to 5-lipoxygenase (5-lox) has been suggested. using intact and fractionated human polymorphonuclear leukocytes (pmnl), we present data showin~ that exogenously added arachidonie acid diminishes dose-dependently binding of [14c]-bay x 1005 to its target. this effect correlates with the reduction of bay x 1005 mediated inhibition of ltb 4 synthesis after stimulation of intact pmnl with tile calcium ionophore a23187 in the presence of increasing extracellular arachidonic acid concentrations in addition, enhancing the release of intracellular arachidonic acid from phospholipids after activation of phospholipase a 2 (pla2) with increasing a23187 concentrations or after prolonged incubation of the cells with the ionophore results in a loss of bay x 1005 potency. to further study" the effect of arachidonic acid on the bay x 1005 mediated ltb 4 synthesis inhibition, human pmnl were stimulated with paf, c5a or fmlp in the presence of exogenous arachidouic acid. under such conditions, ltb 4 synthesis is significantly enhanced in response to paf, c5a or fmlp. which are only weak activators of cell-associated pea 2 the effect of extracellular arachidonic acid is dosedependently inhibited by bay x 1005. coincubation of the cells with arachidonic acid and paf, c5a or fmlp, respectively, enhances synergistically the translocation of 5-lox from the cnosol to the membrane fraction. again, this effect is inhibited by bay x 1005. in summary, using bay x 1005, we provide evidence, that flap is involved in the utilization of the 5-lox substrate araehidouic acid from exogenous or endogenous sources for the agonist-induced leukotrieue biosynthesis. switzerland, but their effect on the expression and function of the multiple drug resistance gene product has not been studied in detail. we have therefore examined the effect of gm-csf and g-csf on the uptake and toxicty of vincristine with and without the chemosensitizers r-verapamil and norverapamil in the pglycoprotein-containing vincristine-resistant cell-line, hl-60/vi,o. following a 48h pre-incubation with 10 ng/ml g-csf or gm-csf, cells were washed and reincubated in rpmi medium with3hvincristine and the modulators for 4 hours. the uptake of3hvincristine was increased up to 2-fold and this effect was equally marked when the vincristine-uptake was stimulated with the chemosensitizers in the range of 2.5 pg/ml to 10pg/ml. although cytokine-pretreatment increased the accumulation of vincristine, we could not show an increase in vincristine toxicity over 72h with the same concentrations of vincristine and chemosensitizers. it is concluded that the effects of cytokines on vincristine accumulation are compensated by a positive effect on cell-survival or cell-growth. department of clinical pharmacology, university clinic, frankfurt, theodor stern kai 7, 60590 frankfurt, germany. from the economic point of view people often tend only to regard the price of a product. savings for the health care system as a whole which could be gained by use of these drugs and which could more than compensate the high costs of medication are not considered. the cost-benefit-analysis enables a comprehensive analysis of the economic effectiveness of a pharmaceutical. the relevant cost factors such as the costs for in-and out-patient treatment depending on the therapeutic success and the inability to work are compared to the costs of medications. using the example of stroke prevention, it was possible to show that prophylaxis with an expensive but effective pharmaceutical is economically better than standard medication. according to the tass study ticlopidine can prevent at least 3 more strokes per i00 treated patients than ass. on the basis of this study cost savings of up to dm 92,000 per i00 treated patients can be reached including higher costs of ticlopidine. projected onto i00,000 patients up to dm 92,000 per year can be saved for the german health care system by the use of ticlopidine in secondary prophylaxis after tia. the naphthodianthrones hypericin (h) and pseudohypericin (ph) were proved to be elfectire in mild depressive disorders. recently they were discovered to exhibit high in vitro activity against enveloped viruses fostering clinical trials in infections with hiv or herpes viruses, for studying the pharmacokinetics of h and ph a solidified extract from st. john's wort (hypericum pefforatum l) was orally administered to 12 healthy male volunteers in three single doses of 250, 750, and 1500 ~g of h and 526, 1578, and 3156 p.g of ph, followed by a 14-days course of multiple dosing of 250 p.g h and 526 pg of ph tds. plasma concentrations were determined by hplc, the quantification limit was 0.2 #g/i (staffeldl et al., nervenheilkunde 12(1993) : 331). single,dose kinetics, h showed a median lagtime of 1.9 (range: 1,4-2.3) h, which was significantly longer compared to 0,38 (range: 0.5-1,0) h of ph. h was eliminated with a median half-life of 36 (range: 12 -56) h, and in case of ph the hail-life ranged between 5.0 and 36 (median: 16,5) h. median crux levels were 1.3, 7.2, 16.6 ~g/i for h and 3.4, 12.1, 29.7 pg/i for ph for the three single-dose levels given above, respectively. lowest dose crre, x and auc of h was lower then expected from medium and highesl dose, which was statistically significantly for crnax (p=0.049, anova; p=0.0156, 8onferroni/dunn), indicating nonproportionality of pharmacokinetic response may exist in the lower dosing range. mul!iple-dose kinetics. during long-term dosing steady-state trough levels (ctreugh) of 7.9 (range: 3.4 -13.6) ~g/i for h and 4.9 (range: 12 -to.9) ~g/i for ph were reached after 4 days for both. cmax after 14 days of treatment were 8.8 (range: 5. c. kori-lindner, r. eberhardt the healthcare restructure act (1993) changed the evaluation of drugs from efficacy to cost effectiveness: therapeutic necessity, utility, purpose; medical progress and economic utility have to be considered. criterias and suroundings of pharmacooeconomic evaluations and studies according to the bealthcare restructure act are demonstrated by an example: "costs of nsaid-induced gastro-duodenal ulcers and prevention with miso~rostol". methods: cost-utility, cost-benefit, legal requirements, decision-model and decision -tree, basic calculation and the transfer to the socialsystem of the frg. premissions: rate of ulcers without prevention 21,7%, with prevention 5,6%; rate of compliance 70%, rate of hospitalisation 20% and duration of hospitalisation 16,6 days. rheumatic disorders have become a frequent reason for services m ambulatory care. therefore the use of drugs which are administered in rheumatic complaints is widespread. data of the sientific institute of statutory health insurances (arzneimittelindex 1992) demonstrate that the group of antirheumatics/analgetics held the first rank due to the number of prescriptions. with regard to the costs thts drug class were amongst the top three. apart from anurheumatics and analgetics different other drug classes are used for the treatment of rheumatic disorders for example corticoids. to estimate the total expenditure for drug utilization in out-patients suffer from musculoskeletal disorders data of the statutory health insurance dortmund (aok dortmund) were investigated. a 5% random sample of insurees (n=6478) was analysed for diagnoses and drug therapy. the data were collected in a individual-related way for an observation period of one year (1988). the results demonstrate the high frequency of rheumatic disorders among out-patients: the 12-month prevalence of musculoskeletal disorders was about 40% in the studied population of whom one half suffered from back pain. approximately 80% of the patients were prescribed any drugs for treatment of rheumatic disorders. as might be expected nonsteroidal antiinflammatory drugs were used most frequently. the extensive use of topical products was a considerable factor of costs. more than one half of the patients received drugs for topical application. further details will be presented. diachronous data of statutory health insurences that have been gathered in a person-related way permit the estimation of drug expenditure in outpatients with regard to the several musculoskeletal disorders. misleading results of endocrine tests in patients suffering from liver diseases promted us to investigate the influence of liver function on the pharmacokinetics of dexamethasone (dex). it was the aim of this study to find out whether routine parameters of liver function may be usefull in roughly predicting the clearance of dexamethasone. methods : in 50 patients liver function was rated on the basis of their plasma cholinesterase activity (che) and prothrombin time (pt). blood samples were withdrawn 7, 15, 30, 60, 120, 240, 360, 480 and 960 min following an iv bolus injection of 2 mg of dexamethasone-21-dihydrogenphosphate corresponding to 1,66 mg of dex for the determination of dex pharmacokinetics by ria. results : computer assisted best fit analysis (2 compartment model) of the plasma dex decay did not show any influence of liver function on the distribution half-life (t½-a) and on the initial volume of distribution of dex, but a 56 % increase in the terminal phase, "metabolic" half-life (t½-b) with decreasing liver function. while the t½-b increased from 167 ±12 min in patients with a che activity >5.0 ku/l to 253 ± 14 min in those with a che s 1.0 ku/l, the metabolic clearance rate (mcr) decreased from 226 ±29 ml/min to iii ±180 ml/min. in patients on phenytoin the t½-b was reduced to <i00 min. che activity (r=-0.454) and pt (r=-0.447) were significantly (p<0.001) correlated with the t½-b of dex. we conclude that the dosage of dexamethasone given for diagnostic or therapeutic purpose needs to be adjusted in patients on phenytoin therapy and in patients with liver disease causing a che s 2 ku/l. etofenamate is one of the most frequently applied topical nsaids in germany. it can be regarded as a lipophilic prodrug of flufenamic acid, which is liberated after transdermal absorption and cleavage of the ester. we compared several topical nsaids in patients suffering from osteoarthrosis or rheumatoid arthritis with concomitant knee joint effusions. the products contained between 5 and 10% of etofenamate and were administered according to the recommended dosage schedule. following multiple applications of defined doses we obtained synovial fluid and plasma specimen in order to measure the concentrations and to compare the bioavailabilily of several products. the analysis of synovial fluid and plasma samples was achieved by hplc. following the addition of other n-phenylanthranilic acid derivatives as internal standards the samples were extracted twice with a 5-fold volume of acetonitrile/methanol (1:1). the diluted extracts were concentrated with the use of preconditioned ods-columns and subsequently eluted with methanol. the separation of the products was perlormed on a reversed phase column with an elution medium of methanol/water/acetic acid (72:27.9:0.1). the highest synovial fluid and plasma concentrations were measured after 3 hours following the final administration of etofenamate. the concentrations reached average maximum values of 60.5 ng/ml of flufenanamic acid in plasma and 45.1 ng/ml in synovial fluid. the bioavailability could be estimated from the area under the concentration time interval between 0.5 and 3 hours, during which samples were obtained. they served for the comparison of the topical products. from in vitro data of cyclooxygenase or lipoxygenase inhibition with n-phenylanthranilic acid derivatives it can be concluded, that the synovial fluid levels of etofenamate and its metabolites are unsufficient to produce effects relying on this mechanism. it can not be excluded, however, that other effects contribute to the clinical improvements observed by other investigators. in animal studies it has been shown to have excellent anti-arthritic efficacy and an improved tolerability profile over other nsaids. early preclinical studies have shown that mel preferentially inhibits cyclooxygenase-2 (cox-2). the aims of this three-week, parallel, multicentre trial were to assess the efficacy anct safety of two different doses of mel in comparison to placebo (pla) in patients with active ra. the specific question was, whether cox-2 inhibition relates to therapeutic efficacy and safety in the clinical'setting. methods: 468 patients (337 f, 131 m, mean age 55 years) were randomised (mel 7.5 mg=159, mel 15 mg =162, pla=147) and analysed by an intention-to-treat analysis. all trial drugs were given orally once daily. results: 31 patients (21%) in the pla group, 11 patients (7%) in the 7.5 mg mel group and 16 patients (10%) in the 15 mg mel group discontinued the trial prematurely due to lack of efficacy or adverse events. 15 mg mel was significantly superior (p<0.05) to pla in 3 of the 4 primary endpoints: disease activity assessed by the investigator and by the patient and reduction of the tender/painful joint count. the difference between 7.5 mg mel and pla reached statistical significance in 2 of the 4 primary endpoints: disease activity assessed by the patient and reduction of the tender/painful joint count. in none of the primary endpoints a statistically significant difference between 15 mg and 7.5 mg mel occured. global tolerance assessed by the patient and investigator at the last trial visit was similar in all three treatment groups. adverse events were slightly more frequent with meloxicam than with placebo. in none of the patients gastric or duodenal ulcers, perforations or bleeds were observed. conclusion: the efficacy of meloxicam 7.5 mg and 15 mg per day was significantly superior to placebo in the treatment of patients with ra. there were no relevant differences regarding safety parameters between the 2 meloxicam doses and placebo. the preferential cox-2 inhibition may be the reason for the favorable efficacy and safety profile of meloxicam. the major metabolites of morphine are morphine 3-0-b-d-glucuronide (m3g) and morphine 6-0-13-d-glucuronide (m6g). in humans plasma aucs of m3g and m6g exceed those of morphine depending on form and route of application. m3g and m6g penetrate the blood-brain-barrier due to an unexpectedly high lipophilicity, m3g is an antagonist able to provoke with&awl symptoms even in opioid naive animals. m6g is an agonist markedly more potent than morphine. methods: pig brains were homogenized and the homogenate centrifuged to give a microsomal preparation free of nuclei and coarse debris. aliquots were incubated at 37°c in buffer (ph 7.4) containing 0.5 nm [3h]damgo. results: the ic50 for morphine, m3g and m6g were 3.7 nm, 24 nm and 1.2 pm, respectively. m6g and m3g, respectively, shifted the ic50 for morphine markedly to the right. discussion: our results on the single substances confirm data from the literature indicating that m3g and m6g inhibit binding to the ~t-or less potent than morphine. from the fact that m3g and m6g shift the ic50 for morphine to the right we infer binding of m3g and m6g to the morphine binding site at the ~t-or. bypassing activity and producing cost of dm 876.300,-was not included in the above shown results. • blood products, coagulation factors and anttmierobial agents are the most expensive substances, however their use could be more restricted. • chemotherapy represents also a high-cost factor, but alternatives do not exist. [ntensiv care units are the most expensive wards consuming 15-20% of the hospital budget, drug use reviews on sicu's of the university hospitals frankfurt (22 beds) and jena (25 beds) were compared after recording the indication related administration of pharmaceuticals and blood products over a 4 months investigation period using notebook-pc's. all substances were included, wich produced 60% and 80% of total pharmacentical cost in the year before, respectively. in frankfurt and jena 207 and 465 patients were registered, causing total cost of dm 938.000,-and dm 1.145.000,-and causing cost per patient and day of average dm 796,and dm 513,-, respectively. in jena most expensive diagnoses were severe trauma (38 8% of total cost; n=66), gastre-intestinal bleeding (14.9%; n=16) and malignant growth (14.3%; n=100). in frankfiart nigh cost diagnoses were liver transplantation (27% of total cost; n=2l), severe trauma (10,5%; n=22) and coronary bypass craring (9.3%" n=73). although the patient populations were different, the percentage cost of the substance groups on total drug cost were the same in both sicu's: blced-products 57±1%, antibiotic and chemotherapeutics 19io,5%, immuna-ghibulines 10,5:t2,5% anyway, inside these groups drug usage patterns differed distinctly. for example, in jena the nigh-cost antibiotics were tobramycin, cefuroxim, amoxicillin and ambisome ® causing 16.2 %, 12.3 %, 8.1% and 8% of total antibiotic cost, respectively. corresponding data for franlffurt are ambisome* 39.8%, imipedem 14.3%, cefotaxim 7,4% and cefamandol 6.5%. severe sepsis was the most abundant complication occuring in beth sicu's in 7 patients and producing cost of dm 208.695,-in frankfurt and dm 173.676,-in jena. in sepsis blood products were the most expensive substances with 52.9% (frankfurt) and 38.9% (jena) followed by antibiotics with 26.2% and 14.3% and immuneghibulines with 17.1% and 36.8%, respectively conclusion: * recording the indication related drug administration drug usage patterns may be identified and targets for economical intervention may be found. • blood and blood-products must be included because they produce about 50% of total cost. • complications, especially sepsis, produce nigh cost, which should be included in the discussion with the health assurances concerning the payment system using diagnosis related groups. in 1992 a drug-utilisafion-review-group (durg) was founded by clinical pharmacologists. the task of this group is to identify drug usage patterns, to record indication related drug administration including cost and to analyse these data. as a result recommendations concerning rational and economical use of pharmaceuticals and blood-products were given (e.g. 5ht3-receptor-antagonists, human albumin, g-csf the most expensive substances are blood-products (27%), antimicrobial agents (27%) and immunoglobulines (21%). in conclusion, at the universitiy hospital frankfurvmain the activities of the drug-utilisation-review-group seem to be successful and are at least inpart responsible for the reduction of drug-cost of more than dm 10 mio last year. the aim of this study was to determine the extent and the time course of fenoterol induced tachycardia and hypokalemia during and after a continuous intravenous infusion with three different rates within the therapeutic range for tocolysis. furthermore we compared the point estimators and precision of parameters obtained with a standard two step approach and a population model. according to a randomized double blind multiple cross over design, 6 healthy females received an intravenous infusion of either placebo or fenoterol (0.5 ijg/min, 1 ijg/min, 2 ljg/min) over 3 hours. the plasma concentration of fenoterol, heart rate, blood pressure, plasma potassium and plasma glucose were monitored during and up to 2 hours after the infusion. the plasma concentrations of fenoterol and potassium and the heart rate were fitted to an integrated pkpd model. both individual parameter estimates and population estimates were obtained with nonmem. both effects correlated best with the concentration in the central compartment. the relationship between heart rate and concentration was adequately described by an emax model. modelling of the dose dependent hypokalemic effect of fenoterol required an negative sigmoid emax model with an additional term allowing for passive diffusion of potassium from the intra-to the extracellular compartment. during the observation period, the concentration response relationship remained stable for both effects. as to be expected, the estimates of the population parameters were much more stable than those of the individual patients. it is concluded that the population method might be superior to the sts approach even in a data rich situation. 18 volunteers received 3 different controlled release formulations (a, b, c, containing 60 mg is-5-m2",1) in a randomised cross-over design. plasma samples were taken from 0 to 48 hours after application. one week wash-out phase was kept between investigations. is-5-mn were determined in plasma using a gc-method. in vitro dissolution of a, ]3, c was investigated by using the rotating bottle method according nf xiv. mean absorption time (mat) was calculated as the difference of mrt and ke1-1, the mean dissolution time (mtdiss) was calculated as proposed by brockmeier (1). additionally half value duration (hvd) and plateau time opt) are reported. the difference of the dissolution profiles between the product is not reflected in the parameters mat, hvd and pt. although preparation c has the longest mtdiss it has the largest cmax value and shortest hvd. the origin of this apparent discrepancy between in vivo/in vitro profiles deserves further elucidation (1) acta pharm. technol. 32 (4) calciumantagonists (ca) are lipophilic molecules which, however, have no common structural similarities. nevertheless, ca are characterized by the ability to attach to various receptors and binding places in cardiac and smooth muscle preventing calcium overload, necrosis and cellular death. in order to study the physiological effects of ca on cerebral cortex of normal and vit d3 calcified rats, i.v. infusion of ca have been applied. cerebral blood flow (via hydrogen clearance) and oxygen supply (via surface oxygen tension) have been measured in normal and sclerotic albino rats in order to differentiate cerebral and systemic effects. miniaturized surface oxygen and hydrogen electrodes were placed on top of the exposed cerebral cortex of ketamine~xylazine anesthetized rats. nifedipen, vcrapamil, diltiazem and flunarizine induced hypotension in normal and monckebcrg type sclerotic rats. nifedipen and verapamil showed comparable improvements in cbf and surface po2, the initial hypotension recovered completely under nifedipen but not under verapamil. compared with vcrapamil, the hypotension was even more pronounced under flunarizine (33:18%). nevertheless, in response to both ca cbf was improved to about 60%. however, diltiazem did not increase cbf due to hypotension. sclerotic rats showed a map-decrease from 148 to 91 mm hg, the po2 histogram was left-shifted. diltiazem did not improve the cortical supply of sclerotic animals. the influence of intracoronary enoximone at a dose of 0.075 mg/kg/lo min on global and regional wall motion, myocardial perfusion (contrast echo) and diastolic lv function during pacing-induced ischemia was investigated in 18 pts. with significant lad stenoses. results: enoximone did not change either heart rate at "rest (79±9 vs 80±9 min -i) or blood pressure (lvsp: 159±7 vs 162±5 mmhg) . in the postpacing ischemic period, lvedp fell from a mean of 28.9 to 18.4 mmhg after enoximone (p<0.001), dp/dtmax increased from 1050 to 1369 mmhg/s (p<0.001) and regional ef from 47% to 58% (p<0.01) with global ef remaining unchanged (45% vs 47%). st segment depression was reduced significantly from 2.3 to 1.5 mm (p<0.01). enoximone induced an increase in myocardial perfusion by 129% (p<0.001) in the stenosis-depending myocardial areas with shortening of the wash-out half-time of the echo contrast medium from a mean of 14 to 5 s (p<0.001) . the isovolumetric relaxation (echo) was shortened by 13% (p<0.05), the e wave by 5%, and dp/dtmin was increased by 17% (p<0.01 treated with pivaloyl-pivaloyl-substituted 8-1actamantibiotics belong to a new group of prodrugs with an improved bioavailability following oral administration. after metabolic cleavage the pivalic acid and the free 13-1actam are eliminated separately. pivalic acid is subjected to enzymatic coupling with endogenous camitine leading to carnitinepivaloylesler, which can be found in large amounts in the urine. free camitine is reduced under the treatment which may lead to neurological side effects in patients with metabolic predisposition. we showed, that the pattern of other acylcarnitineesters excreted with the urine was not changed significantly after administration of a pivaloyl.substituted cephalosporine in healthy subjects. in order to compare the pattern of excreted carnitineesters we analyzed urine samples of pediatric patients treated with cefetamet-pivoxil. the analysis of carnitineesters in the urine was achieved by hplc following precolumn derivatization. as an internal standrad we used undecanoyl-carnitine aster, which does not occur in human plasma or urine. after the extraction using ion exchange columns the samples were incubated with p-bromo-phenacyl reagent. the mixture of derivatives was separated on a reversed phase column applying a gradient of different buffers. with this method the whole spectrum of carnitineesters can be quantitatively analyzed. the percentage of the pivalic acid ester eliminated with the urine in pediatric patients did not differ from that observed in healthy subjects, therefore an increased risk in this patient group cannot be inferred. institut for klinische pharmakologie, universit~.tsklinikum charit& humboldt-universit&t zu berlin, schumannstral~e 20, d-10098 berlin; pharmacokinetics and bioavailability of bupranolol-l"rs and oral bupranolol in patients with impaired liver function compared to healthy volunteers k.-h. molz*, w. cawello**, g. haug-pihale*", r. bonn** w. hammes**, a. weil*** and g. popescu*** bupranolol is nearly completely absorbed after oral administration, but more than 90% of the parent compound are metabolized by first pass through the liver. it can be expected that transdermal application of the substance will bypass the high first pass metabolism resulting in higher plasma concentrations or lower therapeutic doses, respectively. a reduced capacity of metabolizing liver enzymes might mimic this effect. the study was performed to investigate if metabolisation by hepatic enzyme systems results in liver function dependent pharmacokinetics. special interest was to be given to possible differences between oral and transdermal application. in an open sequential single dose-study including 10 patients with impaired liver function and 10 age/sex/weight-matched healthy volunteers a bupranolol transdermal therapeutic system (containing 30 mg/24h) and bupranolol tablets (100 mg) were administered to each participant separated by a washout period of a week. phenotyping for mephenytoin and sparteine was done to exclude poor metabolizers. blood samples for the quantification of bupranolol plasma concentrations were drawn before and 1,2,3, 6,9,12,16,23,24 (before patch removal), 25,27,30,36,48,60 and 72 hours after patch application as well as before tablet administration and 0.5, 1,1.5,2,3,4,6,8, 10,12,16, 24,30,36,48 and 60 hours post dose. urine samples were collected in 5 ('its) and 6 (tablets) intervals after dosing. blood pressure and heart rate were measured before and 1,3,6,12 and 24 hours after dosing, ecg's were recorded before and 3,6 and 24 hours after dosing. the pharmacokinetic results indicate only minor differences between patients and healthy controls after patch applications. for example the main parameter auc(0-~) were 65.8±26.3 ng/ml.h (mean±sd) in healthy controls and 57.8±18.1 ng/ml.h in patients. completely other situation was given in comparison of data from oral administration: auc(0-=)decreased dramatically to 5.7±3.7ng/ml-h in healthy controls and increased to 473.3±611.6 ng/ml.h in patients. vascular complications are a common fmding in patients with diabetes mellitus. it has been reported that elevated glucose impairs acetylcholine-but not nitruprusside-mediated relaxation of rabbit aorta in vitro suggesting an abnormal endothelium-mediated vasodilator mechanism (cohen, circulation 1993; 87 (suppl. v): 67). the purpose of the study was to investigate whether this phenomenon 1. implies general impairment of the nitric oxidemediated relaxation (or only specific interference with muscarinic receptors), and 2. can be also demonstrated for coronary arteries. porcine coronary arteries were cut in rings and were set up in organ baths for isometric tension recordings. rings were randomized and incubated with different glucose concentrations (5.5, 22,0, or 44.0 mm) for 6 hours according to cohen and coworkers. rings were preconstricted with prostaglandin f2 ~t (0.5 -3 ~m); the nitric oxidemediated relaxation was induced by either substance p (0.01 -10 nm), bradykinin (0.1 -100 nm), or calciumionophor a23187 (1 -1000 nm), the endothelium-independent relaxation by nitroglycerin (1 -3000 nm). in addition, the effects of glucose were studied on acetylcholine-(0.01-10 ~tm) and serotonin-(0.001 -10 ~m) induced vascular responses. results: surprisingly, elevated glucose concentrations (22 and 44 mm) had neither an effect on the potency nor the efficacy of any of the agonists tested. it is concluded that this previously reported impairment of endothelium-mediated vasodilation of rabbit aorta by elevated glucose concentrations may not be important in other species or types of arteries. nitric oxide production may be induced by cytokine-mediated immune responses. we tested the hypothesis that graft rejection in cardiac transplanted patients is associated with an increased production of nitric oxide. the study group consisted of 83 patients in whom one or several myocardial biopsies (total 194 biopsies) were obtained to assess for graft rejection. at the day of biopsy, urine was collected for a time period of at least 4 hours (10% isopropanol was added for antimicrobial activity). nitrate was measuerd in urine samples by gaschromatography. nitrate excretion was expressed as the quotient of nitrate and creatinine content (gmol nitrate/mmol creatinine). since nitric oxide is oxidized in biologic systems to nitrite and finally nitrate, it was assumed that nitrate excretion reflects total nitric oxide release. graft rejection was classified according to the hannover classification system (a0/i= no/minor rejection, a2-a4=moderate/severe rejection). results: nitrate excretion showed a large interindividual variation (9 to 502 i.tmol nitrate/mmol creatinine). /£1tough not significant, the nitrate excretion tended to increase with the degree of rejection (a0: 99+ 9; a 1: 128 + 12; a2:131 + 15; a3/4:128 + 15; mean + se). in a subgroup of patients (n=32) who underwent several biopsies with and without rejection, moderate/severe rejection was significantly associated with increased nitrate excretion (a0/ai: 111 + 13 vs. a2-a4:161 + 30, p<0.05). conclusion: the urinary nitrate excretion is increased in patients with moderate/severe graft rejection. because of large variation in excretion, this marker may be not suitable for rejection diagnosis. the combination of thremboxane a~/prostaglandin endoperoxide (epo) receptor antagonism with thromboxane synthetase inhibition (trasi) is designed to both abolish the epos' prothrombotic role and direct epos from activated platelets to adjacent cells for the generation of antithrombotic prostaglandins. thus, we have investigated the effect of the potent trasi dt-tx 30 on thrombus formation in models of vascular injury (of the media versus selective deendothelialization) in man ex vivo. healthy volunteers were treated with an oral dose of vehicle (placebo; n=10) or 50, 100, 200 , 400 mg; n=6 per dose). blood was taken before and 1, 2, 4 and 24 h after the ingestion, anticoagulated and allowed to flow over a thrombogenic cell-free subendothelial matrix (cfm) or a layer of human venous smooth muscle cells (smc) for 10 min. the "en face" area of platelets/thrombi deposited was determined by microscopic morphometry. in the absence of cells of the vessel wall (cfm) the thrombus formation was inhibited by 32+15% (+sd) after 50mg, 43+_28% after 100mg, 31+1% after 200mg and 57+p9% after 400mg dt-tx 30 versus 5+21% after placebo. the subclass of the large thrombi (=7% of all platelets/thrombi but incorporating >90% of the platelet mass) was much stronger affected by the dt-tx 30 treatment: the mean area was reduced by 61+p7% after 25rag, 69+20% after 50mg, 78_+9% after 100mg, 53+25% after 200mg and 71_+8% after 400mg dt-tx 30 versus -16+40% after placebo. in the presence of cells of the vessel wall (smc) the overall thrombus formation was reduced by up to 42+21% after only 25 mg, 36+31% after 50mg, 59+_20% after 100mg, 72_+5% after 200rag and 81-+6% after 400 mg dt-tx 30 versus 2+_12% after placebo. dt-tx 30, a molecule combining potent and specific th romboxane synthetase inhibition with prostaglandin endoperoxide/thromboxane a 2 receptor antagonism, has been examined in healthy male subjects. collagen-induced platelet aggregation in platelet rich plasma prepared from venous blood was measu red photometrically before and up to 24 hours after a single oral dose of 25, 50,100, 200 or 400 mg dt-tx 30 in a placebo-controlled, double-blind study. platelet aggregation was induced in the ex vivo samples by collagen in concentrations between 0.5 and 10 p.g/ml to evaluate platelet aggregation in relation to the strength of the proaggregatory stimulus. the ecs0, i.e. the concentration of collagen required for a half-maximal aggregatory response (defined as the maximal change of the optical density), was determined. in the placebo-treated control group, the mean ecso was 365+55 ng/ml collagen (+ se; n=10) before treatment. it then varied between 362+41 and 417+_83 ng/ml collagen after treatment. the'ratio of the post-to the individual pre-treatment ecso values was 1.08 _+0.10 (n=10) at 0.5 h, 1.05_+0.11 at lh, 1.13_+0.14 at 2 h, 1.14-0.20 at 4 h, 1.07+ 0.07 at 8 h and 1.02+0.06 at 24 h. this indicates that the sensitivity of the platelets to collagen was not affected by the placebo treatment. oral treatment with dt-tx 30, however, strongly inhibited the aggregatory response of the platelets to collagen stimulation. the ecs0-ratio was increased to a maximum of 4. the detection of endogenous opioids suggested the opinion that in case of the presence in the organism of a receptor for an exogenous substance there is probably a similar endogenous substance.the occurrence in the blood of persons, who were not treated with cardiac glycosides, of endogenous digoxin-like or ouabain-like [actors confirms that opinion. in our study we took up the research of other drug-like [actors in the blood serum of healthy people. in two hundered and twenty-five healthy volunteers (llom,ll5f) non-smokers not receiving any treatment before or during the test and aged between ib and49 y(mean age 36y) the occurrence of drug-like [actors in blood serum was studied.the examinations were carried out with the use of the fluorescence-polarization-immunoassay (fpia)-tdabbott. th e presence of the following endogenous drug-like foctors in the blood serum was evaluated: quinidine,phenytoin, earbamazepine,theophylline, cyclosporineand gentamicin. the presence of endogenous phenytoin-like, theophyllinelike and cyclosporine-like [actors has been demonstrated. the drug-like [actors were not found in the case of quinidine ,carbamazepine and gentamicin. the phenytoin-like factor was found in 91,1~, theophylline-like [actor 95,1~ and cyclosporine-like [actor in 96,9~ of examined volunteers.the mean value of the drug-like [actors were as follow : phenytoin 0,18~ 0,05 pg/ml,theophylline 0,16~ o,ll pg/ml and cyclosporine 12,41z 4,24 ng/ml. the supposition may be proponued that organism produces drug-like substances according to its needs. the acetylation and oxidation phenotypes were studied in 448 healthy volunteers (235m, 213[) aged between ib and 46 years (mean 36y) in the wielkopolska region in poland. the acetylation phenotype was studied with the use of sulphadimidine which was given in a dose of 44 mg/kg b.w. per os.sulphadimidine was determined by a spectrophotometric method.the border value of m.r. was 75~ in urine. the oxidation phenotype was studied with the use of sparteine which was given in a dose of 1,5 mg/kg b.w.per de. sparteine was determined by the gas chromatographic method in urine. if m~ was 20 <hr4340 these are values for intensive metabolism, if mr was 0,22<mr(12 these are values for poor metabolism. the determinated phenotypes were bimodally distributed. our studies have short that 259 healthy volunteers (57.8~) were slow and 189 (42,2~) were fast metabolizers. intensive oxidation phenotype could be ascertained in 423 volunteers (94,4~) and poor oxidation phenotype in 25 volunteers (5,6~). these findings are comparable with the values typical for the caucasian population. the genes coding for rapid acety!ation were present in 37.5 % of the cases, genes coding for slow acetylation were detected in 62.5 %. the frequency of specific nat2 alleles were: r1, 20.7 %; r2, 1.2 %; sla, 30.4 %; slb, 3.1 %; slc, 6.3 %; s2, 34.9 %; s3, 1.4 %; and s4, 2.0 %. there was no mutation found at 191 nt. in 166 cases (94.3 %) we have stated nat2 genoand phenotype concordance, but in 10 cases (5.7 %), genotype and phenotype deviated from each other. seven individuals were genotyped as slas2, one as slasle, and one as slas4 but were phenotypically rapid acetylators. one child was genotyped as r1 s 1 a but proved a slow acetylator. we can confirm that in about 94 % nat2 genotyping allows to properly predict nat2 phenotype. discrepancies may derive, i. a., from yet unknown mutations which we try to detect by dna-sequencing. several studies demonstrated that cardiac glycosides reduce diameter of epicardial coronary arteries. in patients (ffs) with coronary artery disease (cad) this vasoconstrictive side effect of glycosides may cause ischemic complications in the presence ot high-grade stenoses. this study evaluated quantitatively the effect of an i.v. injection of digoxin on diameter of epicardial coronary arteries in 20 pts with cad. eleven grs (group 1) were treated with aspirin 100mg/d, nine frs (group 2) were treated with aspirin and 10rag nisoldipin 2 hours prior to angiography. coronary angiograms were taken in identical projections before (0) and 5,10,15 and 30 minutes after i.v. injection of 0.8mg digoxin, at 35 rain 0.ling of nitroglycerin (ntg) was given. the mean diameters of normal segments and the minimum diameters of stenoses were analysed with cms (medis, leiden). a total of 149 normal and 28 stenotic segments were analysed. figure shows the change of coronary diameter (% predrug-vahie; mean + se) for group i (left) and ii (right): (1 -(4-cyclohexylureido-phenoxy)-2-hydroxy-3 -tert.-butylaminopropan; awd, dresden) is frequently used as a cardioselective g-adrenoceptor antagonist. talinolol metabolites in urine were detected by hplc and gc/ms with previously characterized reference compounds and were quantified by hplc with a normal phase silica column (oertel et al., br. j. clin. pharmacol. 37 (1994) 496) . less than one per cent of administered dose was recovered in urine as bydroxylated talinolol. other metabolites could be excluded. in serum after therapeutic doses talinolol metabolites were not traceable. however, in a suicidal patient with a parent compound serum concentration of 1400 ng/ml (manifold higher as after usual therapeutic dose) two hydroxy metabolites at concentrations near the detection limit (10 ng/ml) were found. obviously only a small amount of the talinolol dose is metabolized in human. in this study the relation between structure, polarity and urinary recovery of the metabolites was examined. four different isomers of mono-hydroxylated talinolol were identified in urine of volunteers after a single dose of talinolol. eight isomers are possible: six hydroxy-cyclohexyl-compounds and two hydroxy-phenoxy-compounds. there was a low degree of specificity of biotransformation seen with respect to the point of attack on the cyelohexyl ring. hydroxylation of the phenyl ring could be excluded in human. using hplc methods very small quantities of reference compounds are sufficient to estimate and compare the polarity and hydrophily of similar compounds. long retention times in a normal phase system and short retention times in a reversed phase (rp) system are found for polar and hydropbilic compounds. the shortest retention times of all hydroxylated talinolol compounds in the normal phase system as well as the longest retention times in the rp system were found for the main metabolites 4-trans-and 3-cis-hydroxycyclohexyl-talinolol. accordingly, the main metabolites are the most polar and hydrophilic mono-hydroxylated talinolol isomers. lysine clonixinate is a non-steroidal antiinflarnmatory drug with marked analgesic effects. it is used for treatment of acute pain such as postoperative, dental or menstrual pain. we have determined the biosynthesis of thromboxane (tx)b z and prostaglandin (pg)f2~ in clotting whole blood ex vivo as well as collagen-induced platelet aggregation before and 0.25, 0.5 (only txb 2 and pgf2,~ biosynthesis), 1, 2.5, 6, 24 and 48 hours after oral intake of 125 mg lysine clonixinate. the results were compared with data obtained after oral intake of 500 mg acetylsalicylic acid (asa). while both txb 2 and pgf2,~ biosynthesis measured radioimmunologically were inhibited significantly (p<0,05 and p<0.01, respectively) 2.5 hours, but not 6 hours after lysine clonixinate, inhibition by asa was much greater and still highly significant (p<0,01) after 48 hours. the mean concentrations of lysine clonixinate and of asa inhibiting txb 2 biosynthesis in vitro by 50% (ecs0) were 3.2 + 0.5 ~tm and 6.2 _+ 0.9 gm, respectively. aggregation of 0.5 ml aliquots of platelet-rich plasma induced by 1 gg/ml collagen was inhibited by lysine clonixinate significantly (p<0.01), but not completely for up to 24 hours, while aggregation induced by 3 gg/ml collagen was inhibited for up to 6 hours only (p<0.01). platelet aggregation induced by both collagen concentrations was inhibited by asa almost completely (p<0.01) for at least 48 hours. the ecs0 for in vitro inhibition of submaximal collageff-induced platelet aggregation was 59 + 16 btm for lysine clonixinate and 97 + 21 lam for asa. it remains to be investigated, whether other mechanisms than inhibition of cyclooxygenase contribute to the analgesic activity of lysine clonixinate. the profile of side effects including only moderate inhibition of platelet function by lysine clonixinate as compared to asa might be an advantage with regard to its use as an analgesic. increased cell proliferation induced by ras-oncogenes is mediated by growth factors. growth factors bind to cell membrane surface receptors and activate tyrosine kinases leading to protein phosphorylation. this triggers posttranslational modification of the function of low molecular mass gtp binding ras proteins, e.g. p21ras, including heterotrimetric g proteins. th~se proteins carry a c-terminal amino acid sequence, caax where c is cysteine, which is the target of isoprenylation by famesyl protein transferases (fpptases) and geranyl-geranyl protein transferases (ggptases). the amino acids of this sequence determine whether the ras protein is substrate for fptases or ggptases. this biochemical pathway is related to mevalonate metabolism, therefore, cholesterol biosynthesis shares several steps with isoprenylation. the active compounds include a series of isoprenoid inter-mediates, such as geranyl pyrophosphate (c-10), farnesyl pyrophosphate (fpp, c-15) and geranyl-geranyl pyrophosphate (ggpp, which are critical for cell proliferation. inhibitors of isoprenylation may (i) compete with fpp to its binding site on the alpha subunit of the enzyme, (ii) inhibit the zn protease of fptase beta subunit, and (iii) interfere with the binding of the caax peptide to its binding site on the beta subunit. the inhibitors include microbial products like manumycin, gliotoxin, acetylgliotoxin and pepticin-namins, fpp analogues, tetrapeptides and related peptidomimetics that may compete for the caax sequence, products of plant origin like limonene as well as synthetic compounds. all these compounds have been known to retard cell proliferation, and their effect can be abolished by mevalonic acid. inhibitors of hmg-coa reductase, such as lovastatin, which have been developed as antihypeflipidemic drugs, may also effective as anticancer agents. the recognition of the importance of mevalonate metabolism in cancerogenesis provides possibility to test simple chemical comp6unds against tumor growth, and gives promise for pharmacological exploitation of inhibitors of isoprenylation and hmg-coa reductase inhibitors for cancer chemotherapy. dopamine (da) and epinine (epi, the active metabolite of ibopamine) are da-and ~-and ~-adrenergic receptor (r) agonists. to study whether da-r mediated effects can be separated from ~-and ~-ar effects we assessed in 10 male volunteers effects of i.v. da and epi (0.5; 1.0; 2.0 and 4.0 ~g/kg/min for 15 min each) on changes in serum prolactin (prl, da-r mediated), blood pressure (p) and heart rate (hr, ~-and ~-ar mediated), natriuresis (urinary excretion of sodium), diuresis (urine flow rate) and plasma noradrenaline (na) levels. in the 0.5 and 1.0 ~g doses da and epi did not affect psyst, pdiast and hr, but significantly decreased prl (da: from 4.2±0.5 to 2.7±0.4 ng/ml; epi: from 6.3±i.i to 4.4±0.8 ng/ml, p<o.01). in the 4~g dose da and epi increased psyst by 20mmhg, pdiast by 4 and 8mmhg, and hr by 8 and 11 beats/min but further decreased prl. in addition, both da and epi significantiy increased diuresis and natriuresis; in contrast, only da, but not epi, dose-dependently increased plasma na ievels, an effect that was not inhibited by ~-ar antagonists. we conclude that in 0.5 and 1.0 ~g doses (plasma levels of 20-70 nmol/1) oa and epl act only at da-r. in higher doses, however, da and epi can activate ~-and ~-ar whereby epi has a stronger ~-ar component than da. moreover, part of da-effects are indirect via release of endogenous na whereas epl-effects do obviously not include an indirect component. j. raderrnacher, r.mliller-bartels, f. barrels, h. baurngarten, k. thon, d. tsikas and j.c. fr61ich, 20 patients with essential hypertension were ireated with cicletanin (100 rag/d) for 12 weeks. compliance was verified by measuring urinary cicletanin excretion. before entering the study anti_hypertensive medication had been withdrawn for at least 2 weeks (wash out phase). the study was preceeded by a placabo phase of 1 week duration. blood pressure measurements and urine collections for measurements of 2,3dinor-6-keto-prostaglandin f1 a (a stable prostacyclin metabolite) were performed at the start and end of placebo phase, atter 1 week of treatment with cicletanin and in monthly intervalls thereafter. cicletanin decreased systolic and diastolic blood pressure, the effect becoming significant at 4 weeks. urinary excretion of the prostacyclin metabolite 2,3dinor-6 keto pgfla.increased by 21%. hypertensive patients and control subjects (n=13; 2,3dinor-6-keto-pgfla: 120+19 pg/rnl] did not differ significantly in urinary excretion of 2,3dinor-6 keto pgf1 ct. neither systolic nor diastolic blood pressure showed a significant correlation with 2,3dinor-6 keto-pgf1 alpha excretion (systolic bp: r--0,12, p--0,2l; diastolic bp: r=0,0007, p=l,0). these data show that cicletanin is effective in reducing blood pressure. increased prostacyclin production is probably not responsible for the observed reduction of blood pressure. in the past the realization of trials on the pharmacokinetics of nicergoline in human beings break down according to analytical difficulties. from animal studies with labelled compounds it is known, that nicergoline is rapidly absorbed from the gastrointestinal tract and two main metabolites (mdl, i-mmdl) can be detected.the mean elimination half-life for nicergoline is given with 2,5 h, for mdl 12-17 h and for i-mmdl with 4-6 h. the compound is excreted mainly by the kidney. due to known problems of the application of labelled drugs in human beings there are two approaches for the determination of these compounds: first the detection by monoclonal antibodies by using a ria-method, secondly a hplc-analysis. although there are promising in-vitro results of the immunological method, quantitative analyses of serum samples show deceptive results. the hplcassay reveales that in human serum samples after oral administration of nicergoline only the metabolites mdl and i-mmdl can be detected. further analytical development lower the limit of detection which should be i/i0 of c~x. with an improved hplc-method it will be demonstrated that only the measurement of the metabolite mdl is suitable to give sufficient results in pharmacokinetic trials and in studies on bioavailability with formulations of nicergoline. antiplatelet treatment with aspirin is well established as secondary prophylaxis after a transient ischemic attack or minor ischemic stroke, but the effect of aspirin treatment on the course of carotid atheroselerosis is unknown. we tried to investigate the effect of aspirin on the initial stages of carotid atherosc}erosis. patients were recruited from a prospective, randomized, double blind clinical trial to compare two doses of aspirin (900 mg vs 50 mg claity) with respect to restenoses after lower limb angjoplasty. of the 383 patients admitted to the angioplasty trial, 27 patients showing 104 small carotid atheroma (<50% lumen narrowing) were examined at entry and after 1 year of aspirin treatment using a high-resolution ultrasound duplex system. disease progression and regression were defined by a change of maximal plaque area (as measured by longitudinal ultrasound sections) of more than 2 standard deviations of the method. the change in plaque area was significantly different for the treatment groups: average plaque size remained'unchanged after treatment with 900 mg aspirin daily but increased markedly after treatment with 50 mg aspirin daily (p = 0.011). there were significantly more lesions in the 50-mg group showing progression than in the 900-mg group (23 plaques [47%] vs 13 plaques [24%], p = 0.025). ultrasonic disappearance of a lesion was observed only in the 900-mg group in 9 cases (7 soft plaques, 2 ulcerative plaques, p = 0.018). the 6 patients on 50 mg aspirin who continued smoking during the study showed significantly more progression as compared to the 7 non-smokers in the 50-mg group (17 plaques [59%] vs 6 plaques [30%], p = 0.038). the results of our study indicate that aspirin treatment slows carotid plaque growth in a dose-dependent fashion, with a dose of 900 mg daily more efficient than 50 mg a day. gentamicin is frequently applied in neonatal patients for the treatment of severe infections. the use is associated with high incidences of oto-and nephrotoxicity. ototoxicity occurs in 1-1.5 % of the treated patients. it can be avoided, if the trough levels remain under 2 p.g/ml between the administered doses. if the nephrotoxic effect, which occurs in 2-10 % of the treated patients, can also be reduced under these circumstances is not yet clear. recently it has been suggested to administer gentamicin only once daily in order reach the desired trough levels. this applies especially to children, in which the terminal half-life can be prolonged to approximately 9 hours. this dosage regimen assures a sufficient effectivity due to the marked postantibiotic effect, but should reduce the incidence of the toxic effects. in order to test this hypothesis we perform a case-control study with neonatal children treated with gentamicin. the cases of nephrotoxicity during the previous 12 months are identified from the hospital records. for these patients the plasma level time curves are obtained with the use of a population based pharmacokinetics programme, which considers the measured plasma concentrations and the administered doses. plasma concentrations were measured routinely in the process of therapeutic drug monitoring. from these curves the duration of times with concentrations lower than 2 p.g/ml during the last 3 days prior to the diagnosis are estimated. for the cases matched controls were identified according to age, age of gestation and other factors affecting the renal function e.g. comedication. different analyses are carried out according to the definition of exposure. the results wiij be discussed with regards to the modification of the dosage schedule for gentamicin and the application of population kinetics in order to reduce the adverse effects. economic evaluation of drug therapy is an important task of clinical pharmacology. drug costs amount to a relatively small percentage of total health care costs. some drugs may help to avoid more expensive therapeutic procedures. drugs with small therapeutic ratio need e therapeutic drug monitoring (tdm) to optimize therapeutic results and to avoid side effects. tdm may enhance the costs of drug therapy and the question arises, whether the additional costs are justified from the economic point of view. the aim of the study was to investigate the effectiveness of theophylline tdm in chronic obstructive pulmonary disease. methods: 30 outpatients suffering from chronic obstructive pu[monary disease and receiving theophylline as a basic medication were included in this study. during the one-year observation period the parameters of pulmonary function were examined four times (1 st, 3rd, 6th and 12th month) by the same physician and theophylline trough levels were estimated to individualize the dosage. the clinical outcome was assessed by intraindividual comparison. results: impaired parameters of pulmonary function were improved. hospital admissions were reduced. during the one-year observation period 4 out of 30 patients had 6 hospital admissions with totally 64 days. under the conditions of theophylline therapy without tdm during the year before 7 of them had 13 hospital admissions with 197 days of stay in hospital. discussion: the results agree with a previous study on inpatients. there it was observed, that parameters of pulmonary function significantly were improved due to tdm-supported theophy[line therapy in comparison to a group receiving theophylline without tdm. despite the additional costs thaopylline tdm contributes not only to improved clinical outcome but also to decreased costs for the care of patients with chronic obstructive pulmonary disease. in cancer treatment arterial blood flow reduction by embolisation followed by intra-artedal chemotherapy may be advantageous by achieving high and prolonged drug concentrations in the tumor and lower systemic drug exposure. pharmacokinetics of mitomycin-c (mmc) were investigated in 4 colorectal cancer patients (body weight: 55-92 kg) with liver metastases undergoing chemoembolisation. after hepatic artery branch catheterization and microspheres injection (polyvinylalcobel, 20 mg mmc dissolved in 20 ml physiological naci solution were infused in 5-8 rain, followed by ethiblock application. serum mmc-concentrations were determined from peripheral venous blood samples by reverse-phase hplcsystem with ultraviolet detection (c18-column, elution: phosphate-buffer (0.01m, ph:6.5)) : methanol, v:v=70:30, 365 nm). pharmacokinetic parameters were computed using non-compartmental or two-compartment model analysis assuming linear kinetics (top fit 2.0 software). results: on the average, maximum serum concentrations were observed at tmax = 7.7 rain following the beginning of mmc-infusion and amounted to cmax = 1095 ng/ml (range: 562-1790 ng/ml). mean steady state distribution volume was 0.56 i/kg with a central compartment volume of 0.22 i/kg. concentration-time curves could be descnbed by a distributional phase (t1/2 = 8.5 min) followed by a terminal elimination phase (]'1/2 = 72.4 min). mean total clearance amounted to 8.6 ml/min.kg. the area under the concentration time curve was 33 pg.m1-1 .min. conclusion: pharmacokinetic parameters for mmc-disposition in this study do not substantially differ from those reported for similar mmc-amounts administered as intravenous bolus. systemic mmc exposure does not seem to be reduced following the embolisation procedure used compared to intravenous application. pseudocholinesterase-activity (pche) is an important determinant of the elimination kinetics of drugs like mivacudum. therefore, the time course of pche was investigated in 16 patients undergoing cardiosurgery with cardiopulmonary bypass (cpb) in normo-or hypothermia. anesthesia was induced and maintained with midazolam, propofol and fentanyl using mivacurium as a muscle relaxant. by starting cpb, 2 i of pdming fluid were added to circulation volume. from collected blood samples, pche (u/i) was determined photometrically according to eiiman (1961)(butyrylthiocholine, dithiobisnitrobenzoate, 405 nm, ph=7.0, 37oc) . total plasma protein concentration (g%) was measured according to lowry, and the specific pche was calculated as the quotient of pche and protein concentration (u/g). serum hepadn-activity was determined in 4 patients (berichreme heparin test kit). moreover, the influence of hepadn and aprotinin, both contained in the cpb pdming solution, on pche in vitro was examined using fresh serum from healthy volunteers (n=3). results: the induction of anesthesia had no significant influence on pche or protein concentration (p>0.05). cpb induced a significant decrease of pche (-37%)(p<0.05) and protein concentration (-24%)(p<0.05) and a less pronounced numedcal reduction the specific pche (-15%)(p>0.05). the reduction of pche and protein concentration was not significantly affected by ending cpb (p>0.05), and the values remained low over the remaining operation time. there was no significant difference in pche, measured at 37°c in vitro, or protein concentration between the normothermic and hypothermic group (p>0.05). furthermore, there was no correlation between serum hepadn-activity and pche reduction. pche in the plasma of healthy volunteers was not significantly affected by either hepadn up to 100 u/ml or apretinin up to 10 000 u/ml (p>0.05). conclusion: (1) the concentration of the antitumor antibiotic mitomycin c (mmc), used in ophtalmic surgery for its antiproliferative effects, was measured in the aqueous humor of 7 glaucoma patients undergoing trabeculectomy. sponges soaked with mmc-solution (100 ul of mmc-solution 0.2 mg/ml: 20 rag) were applied intraoperatively under the scleral flap for 5 rain. 100 to 200 ul of aqueous humor were drawn with a needle 10 min following the end of topical mmc-treatment. samples were assayed for mmc using a reverse-phase hplc-system with ultraviolet detection (c18-column, elution: phosphate-buffer (0.01m, ph:6.5):methanol, v:v = 70:30 , 365 nm). swabs were extracted in phosphatebuffer (0.1m, ph:7.0) before hplc-analysis. external calibration was used for mmc quantitetion. quantitation limit was 10 ng/ml. in all aqueous humor samples mmc-concentration was below 10 ng/ml. mmc in the swabs amounted to 37 % of the mmc amount applied. conclusion: after intraoperetive topical application, mmc concentration in the aqueous humor of patients is very low. the substantial loss of mmc from the swabs used for the topical mmc-treatment suggests (1) rapid systemic absorption of mmc and/or (2) a loss through irngation of the operative field following topical mmc-application. institut fur pharmakologie und * klinik for augenheilkunde, universitcit k61n, gleuelerstrasse 24, 5093 k61n al14 a 85 due to runaway costs of the national health service which are reflected as well in growing expenditures for drugs at the university hospital of jena investigation of indication related drug administration patterns becomes more and more interesting. this holds especially true for intensive care units (itu's) which are determined by similar high costs for technical equipment as for drugs (1) although any economical considerations seem to be questionable due to ethical reasons (2). over a 4 month period indication related drug administrations of 2 surgical itu's of the university hospital jena have been recorded and analyzed by using a pc-notebook. total expenditures for all 465 included patients add up to dm 1.144.773 regarding these drugs and blood products which caused 80% of total costs in 1993. the 10 leading substances ( antithrombin ill, human albumin 20 %, prothrembine complex, ...) represent 67 % of total costs including blood products, antibiotics and ig m endched intravenous immunglobine. therefore the indication of particulary these drugs became mere interesting for further investigations. already during the study actual discussions with the treating medical staff have been made leading to new developed therapy recommendations. providing same high standard of medical treatment a remarkable cost saving of some drugs by more cdtical and purposeful use could already be achieved as a first result. however, the results of the study underline impressivly the benefit of such investigations for improvement of drug treatment. the simple replacement of expensive drugs ( e.g. prothrembine complex ) by higher quantities of cheaper ones of the same indication group ( e.g. fresh frozen plasma (3)) does not necessarily mean less expenditures in all cases but may cause unsiderable side effects. ( ketokonazole is known to decrease pituitary acth secretion in vitro and inhibits adrenal ll-hydroxylase activity. to work out the clinical significance of both effects analysis of episodic secretion of acth, cortisol (f) and ll-deoxycortisol (df) was performed in patients with cushing's syndrome (cs) requiring adrenostatic therapy. methods : ketokonazole was started in ii patients with cs (9 acth-secreting pituitary adenomas [cd], 2 adrenal adenoma [aa] ). in 9 of them (8 cd, 1 aa) blood samples were obtained for 24 hours at i0 min intervals (144 samples/patient) before and again under treatment (mean dose i000 mg/d, >6 weeks). hormone levels were measured by ria and secretion patterns analysed by means of pulsar, cluster and desade. 2 patients were investigated only once because treatment was stopped due to side effects. results : the we conclude that the observed 34 % increase of plasma acth and the 58 % decrease of f/df ratio demonstrate that inhibition of adrenal li6-hydroxylase activity is the primary mode of action of ketoconzole in vivo. even at high doses acth and f secretion patterns could not be normalized. the improvement of pain and swelling conditions by means of drugs is an important method of achieving an enhanced perioperative quality of life in cases of dentoalveolar surgery. in 5 prospective, randomised, double-blind studies the influence of various concentrations of local anaesthetics and accompanying analgesic and antioedematons drugs was investigated in the case of osteotoimes. all of the studies were carded out according to a standardised study procedure. a comparison of the local anaesthetics articaine 4 % mad articaine 2 % (study 1) demonstrated the superior effect of articaiue 4% with respect to onset relief on pain, period of effectiveness and ischaemia. recordings of the cheek swelling in the remaining studies were made both sonographically and with tape measurement, while the documentation of the pain was carried out by means of visual analogue scales on the day of operation and on the first and third post-operative days. tile perioperative, exculsive administration of 2x6 mg dexamethasone (study 2) resulted in a significant reduction in the swelling (58 %) while the exclusive administration of 3x400 mg lbuprofen (study 3) was accompained by a marked decrease in pain (64%) but no significant reduction of swelling in comparison to the placebo group. the combination of 3x400 mg ibuprofen und 32 mg methylprednisolone (study 4) yielded a decrease in pain of 67.7% and a reduction in swelling of 58%. a cdmparison between a mono-drug ibuprofen and a combination drug ass/paracetamol (study 5) resulted in no significant difference in the reduction of swelling and pain and therefore highlighted no advantages for the combined drug. a mono-drug should therefore be given priority as an analgesic. the combinatton of ibuprofen und methylprednisolone offers the greatest reduction in pain and swelling. using the results of the randomised studies, a phased plan for a patietu-orietued, anti-inflammatory therapy to accompany dento-alveolar surgery is presented. in a placebo controlled study 20 patients with congestive heart failure (nyka class ii) were treated orally for seven days with i00 mg ibopamine t.i.d, i0 subjects had a normal renal function (mean inulin clearance (gfr) 91 ± 3,4 ml/min), i0 patients suffered from chronic renal insufficiency (gfr 36 ± 3,9 ml/min; x ± sem). pharmacokinetic parameters of epinine, the maximum plasma concentration, the time to reach maximum plasma concentration and the area under the curve from 0 to 6 hours were unaltered in impaired renal function when measured on the first or on the seventh treatment day. however plasma concentrations in both groups were significantly higher on the first treatment day than after one week of ibopamine administration. in this context antipyrine clearance as a parameter of oxidative liver metabolism which might have been induced by ibopamine revealed no differences between placebo and ibopamine values. in conclusion kinetic and dynamic behaviour of ibopamine was not altered by impaired renal function. human protein c (hpc) is a vitamin k-dependent in the liver produced glycoprotein with anticoagulant properties. when active protein c splits the coagulation factors va and vuia by means of limited proteolysis (kisiel et al 1977) . its concentration in normal plasma is 2-6 lag/m[ i-ipc's biological importance became evident when a congenital protein c deficiency, which results in difficult recurrent thromboembolic diseases was discovered (griffin eta/1981) . the recognition of a congenital hpc deficiency, as wall as the connection between acquired protein c deficiency and the appearance of thromboembolic complications by means of highly accurate and sensitive ascertained methods is therefore of great practical importance for the clinic. murine monoclonal antibodies (moabs) against hpc were formed. antibody producing hybridomas were tested by an ,,indirect elisa" against soluble antigens. the plates were coated with purified hpc up to 50 ng/1001al. the peroxydase-system was used to identify antibodies the antibodies were tested with the remaining vitamin k-dependent proteins for cross-reactivity, as well as with hpc deficiency plasma for disturbances by other plasma proteins. the above described experiment represents a sensitive and specific method for measuring the hpc concentration with moabs. assessment of local drug absorption differences ("absorption window") in the human gastrointestinal tract is relevant for the development of prolonged release preparations and for the prediction of possible absorption changes by modification of gastrointestinal motility. current methods are either invasive and expensive (catheterization of the intestinum, hf-capsule method) or do not deliver the drug to a precisely defined localization. we evaluated the delay of drug release from tablets coated with methacrylic acid copolymer dissolving at different ph values as an alternative method. three coated preparations of caffeine tablets (onset of drug release in in vitro tests at ph 5.5, 6.0 and 7.0) and an uncoated tablet (control) were given to six healthy male volunteers in a randomized order. caffeine was used because of its rapid and complete absorption and good tolerability. blood samples were drawn up to 24 h postdose (coating ph 7.0 up to 30 h postdose), and caffeine concentrations were measured by hplc. auc, time to reach measurable caffeine concentrations (tia~), tr, ax, cmax and mean absorption time (mat) values for coated preparations were compared to the reference tablet (mean + sd of n=6): the relative bioavailibility for the coated preparations did not differ from the reference, suggesting complete release of caffeine. all coatings delayed caffeine absorption onset. the tlag for the ph 5.5 preparation suggests that release started immediately after the tablet had left the stomach. the mean delay of 2.1 h for the ph 6.0 coating was highly reproducible and should reflect small intestine release. the ph 7.0 coating delayed absorption to the highest extent, however the drug was probably released before the colon was reached. there is evidence that nitric oxide (no) plays a role in cardiovascular disease like hypertension, myocardial ischemia and septic cardiomyopath.y. no stimulates the guanylyl cyclase leading to an increase m cgmp content we investigated by immunoblotting the expression of the inducible nitric oxide synthase (inos) in left ventricular myocardium from failing human hearts due to idiopathic dilative cardiomyopathy (idc, n=10), ischemic cardiomyopathy (icm, n=7), beeker muscular dystrophy (n=2) and sepsis (sh, n=3) compared to non-failing human hearts (nf, n=4). cytokine-stimulated mouse macrophages were used as positive controls sds-polyacrylamide gel electrophoresis (7.5 %) was perfomed with homogenates of left ventricular myocardium and mouse macrophages respectively. proteins were detected by enhanced chemiluminescence using a mouse monoclnal antibody raised against inos. furthermore, we measured the cgmp content in these hearts by radioimmunoassy. a band at about 130 kda was observed in two out of three hearts from patients with sepsis and in stimulated mouse macrophage~ no inos-protein expression was detected in either non-failing human hearts (n=4) or failing human hearts due to idc, ihd or bmd. in ventricular tissue from patients with sepsis cgmp content was increased to 230% (72 + 17 fmol/mg ww, n=3) compared to non-failing hearts (100% or 31 + 6.9 fmol/mg ww, n=4). in left ventricular tissue tissue from patients with heart failure due to idc, ihd and bmd cgmp content did not differ from that in non-failing hearts. it is concluded that an enhanced inos protein expression may play a role in endotoxin shock, but is unlikely to be involved in the pathophysiology of end-stage heart failure due to idc, ihd and bmd. (supported by the dfg.) nitric oxide (no) has been shown to be a major messenger molecule regulating blood vessel dilatation, platelet aggregation and serving as central and peripheral neurotransmitter; furthermore no is a crucial mediator of macrophage cytotoxicity. no production can be assessed reliably by determination of its main metabolites nitrite and nitrate in serum, reflecting no synthesis at the time of sampling, or in 24 h urine, reflecting daily no synthesis. farrell et ai. (ann rheum dis 1992; 51:1219) recently reported elevated serum levels of nitrite in patients with rheumatoid arthritis (ra). we report here total body nitrate production and the effect of prednisolone in patients with ra. nitrate excretion in 24 h urines of 10 patients with ra as defined by the 1987 revised criteria of the american rheumatism association was measured by gas chromatography at 2 times: first before start of a antiinflammatory therapy with prednisolone, when the patients had high inflammatory activity as indicated by mean crp serum concentrations of 71 + sd 61 mg/i and elevated esr with a mean of 62 ]:28 after 1 hour. secondly 2-4 weeks after start of prednisolone therapy in a dosage of 0.5 mg/kg body weight, when the patients showed clinical and biochemical improvement (crp 6 + 5 mg/i, p<0.05, esg 32 + 17, p<0.001, two-tailed, paired t-test). for comparison 24 h urines from 18 healthy volunteers were obtained. before start of predniselone therapy the urinary nitrate excretion in patients with ra (mean 223 + sd 126 p.mol/mmol creatinine) was more than twofold higher (p<0.001, twoaailed unpaired t-test) than in healthy volunteers (83 + 63 ~tmol/mmol creatinine). the urinary nitrate excretion decreased significantly (p<0.001, two-tailed, paired t-test) to 162 + 83 i.tmol/mmol creatinine under therapy with prednisolone, when inflammatory activity was reduced considerably. despite the decrease the urinary nitrate excretion was still twc, fold higher (p<0.05, two-tailed, unpaired t-test) in patients with ra than in the control group. our data suggest that the endogenous no production is enhanced in patients with ra. furthermore the results indicate that this elevated no synthesis could be reduced in accordance with suppression of systemic inflammation by prednisolone therapy. but now as ever the physicians are entitled to prescribe drugs which have to prepare in a pharmacy for a particular patient. little information is available on the frequency and patterns of these prescriptions. we had occasion to analyse the prescriptions of drugs which were prepared in 6 pharmacies in north thuringia (east germany) from october to december 1993 at the expense of a large health insurance company (allgemeine ortskrankenkasse). the selected pharmacies are loealised in 6 cities. we found 2172 prescriptions of drugs made up in pharmacies among a total number of 58472 reviewed drug prescriptions. this is 3.7 % of the total. most of these prescriptions were performed by dermatologists (56.6 %), general practitioners (21.9 %), paediatrists (7.9 %) and otolaryngologists (4. 1%). according to this, the most frequently prescribed groups of drugs were dermatics enteric eoated tablets with 100 nag and 300 nag acetylsalicylic acid (asa) have been developed wluch should avoid the known gastrointestinal adverse events by a controlled drug release mainly in the duodenum after having passed the stomach. a 4-way cross-over study in 24 healthy male subjects, aged from 19-39 years, was conducted to investigate the pharmacokinetics, bioavailability, safety, and tolerance of asa and its metabolites salicylic acid and salicylurie acid following enteric coated tablets in comparison with plain tablets. asa and its metabolites were determined by a sensitive, specific, and validated hplc method. pharmacokinetic parameters were determined by non-compartreental analysis. bioequivalence was assessed by 90% confidence intervals. following the admimstration of enteric coated tablets, a delayed absorption can be observed for both the 100mg dose and the 300rag dose. this is likely due to a delayed release of the active substance from the enteric-coated tablets in the small intestine arer gastric passage. considering the mean residence times (mrt), there is a difference of at least 2.8 h following the enteric coated tablets compared to the plain tablets for asa and the two metabolites measured• this difference represents the sum of residence time in the stomach plus the time needed to destroy the coating of the tablet when it left the stomach• in general, the maximum observed concentrations of both enteric coated formulations occurred 3-6 h post dose. the pharmacokinetics of a novel immunoglobulin g (lgg) preparation (bt507, biotest, dreieich, frg) have been determined in 12 healthy, male anti-hbs-negative volunteers. for this preparation only plasma from hiv-, hbv-and hcv-negative donors was used, the quality control for the product was in accordance with the ec-guideline for virus removal and inactivation procedures. each volunteer received a single, intravenous infusion of 100ml bt507 containing 5g igg and anti-hbs > 5,000 iu. anti-hbs was used as a simply measurable and representative marker for the igg. blood samples for determination of anti-hbs (ausab eia, abbott, frg) were drawn before and directly after the infusion, after 1,3,6,12 and 24 hours, on day 3, 5,8,15,22, 29,43,57,71,85 and 99 . additionally, total protein, igg, iga, igm and c3/c4 complement were measured and blood hematology and clinical chemistry parameters determined. the phar~gacokinetic parameters of anti-hbs were calculated using the topfit ~" pc program assuming a 2-compartment model. pharmacoeconomic evaluations (pe) describe the relationship between a certain health care input (costs) for a defined treatment and the clinical outcome of patients measured in common natural units (e.g. blood pressure reduction in mmhg), quality of life (qol) gained, lifes saved or even in money saved due to the improvement in patients functional status. this implies that the efficacy of a treatment has been measured and proven in clinical trials. in addition, in order to transfer data obtained in clinical trials to the clinical setting, an epidemiological database for diseases and eventually drug utiiization may be required. the evaluation of the efficacy depends on the disease to be treated or prevented and the mode of treatment. for acute, e.g. infectious diseases, the endpoint can be defined easily by the cure rate, but for pe the time (length of hospital stay) and other factors (e.g. no. of dally drug administrations) have to be considered. in the case of chronic diseases, e.g. hypertension or hypercholesterolaemia, surrogate endpoints (blood pressure or serum cholesterol reduction) and information on side effects may be acceptable for the approval, but cannot be used for a meaningful pe. the latter should include the endpoints of the disease, i.e. cardiovascular events (requiring hospitalisation and additional treatment) and mortality. furthermore, the qol has to be measured and considered for chronic treatment. several questionaires have been developed to measure the overall qol or the health related qol. especially the latter may be a more useful tool to detect mad quantify the impact of a treatment on qol. combining the clinical endpoint mortality and qol by using qalys (quality-adjusted lifeyears) may be a useful tool to determine the value and costs of a given drug treatment but cannot be applied to all treatments under all circumstances. sorbitol was used as a model substance to investigate the dynamics of the initial distribution process following bolus intravenous injection of drugs. to avoid a priori assumptions on the existence of well-mixed compartments data analysis was based upon the concept of residence time density in a recirculatory system regarding the pulmonary and systemic circulation as subsystems. the inverse gaussian distribution was used as an empirical model for the transit time distribution of sorbitol across the subsystems, distribution kinetics was evaluated by the relative dispersion of transit (circulation) times. the distribution volumes calculated from the mean transit times were compared with the modelindependent estimate of the steady-state volume of distribution. kinetic data and estimates of cardiac output were obtained from 10 patients after percutaneous transluminal coronary angioplasty. each received a single 0.8 g iv bolus dose of sorbitol. arterial blood samples were collected over 2 hours. while the disposition curve could be well fitted by a tri-exponential function the results indicate that distribution kinetics is also influenced by the transit time through the lungs, in contrast to the assumption of a wellmixed plasma pool underlying compartmental modelling. a karit@ "bu£ter" is used traditionally in west afr%can manding colture as a cosmetic to protect the skin against the.sun. gas chromatography was used to analyze the ingredients of karit@ butter from guinea. we found 3% palmitic acid, 42% stearic acid, 42% oleic acid and 8% linoleic acid and 0.1% of other fatty acids with higher chain lengths like arachidonio acid. some of these are essential fatty aclds (vitamine f). furthermore karit@ contains vitamine a and d as well as triterpene alcohols and phytosterines. an original extract was used to prepare a skin cream. this preparation was tested in 25 volunteers (18 women, 7 men; age 20-55 y.). the cream contained at least 50% karit@, glycerol, emulsifiers and no preservative agent except for sorbic acid. 24 of the volunteers very well tolerated the cream and thought it effective. the skin became more tender and elastic. good results were obtained when the volunteers suffered from very dry skin. two of them who were known to be allergic against the most available skin creams had no problems in using our karit8 cream. pure karit@ butter was used for four months to treat an african infant with neurodermitis. after this time the symptoms had markedly improved whereas previous therapy trials with other usual topical medicaments had been unsuccessful. these pre-studies had shown that dermatologic preparations containing karit# may be a good alternative in the treatment of therapyreslstent skin diseases and may in some cases be able to replace eorticoid treatment. ) and a low molecular weight heparin preparation (fragmin ~, 75 iu/kg bodyweight s.c.) on coagulation and platelet activation in vivo by measuring specific coagulation activation peptides [prothrombin fragment 1+2 (f1+2), thrombin antithrombin iii complex (tat), 13-thromboglobulin (~-tg)] in bleeding time blood (activated state) and in venous blood (basal state). in bleeding time blood, r-hirudin and the heparin preparations significantly inhibited the formation of both tat and f1+2. however, the inhibitory effect of r-hirudin on f1+2 generation was short-lived and weaker compared to ufh and lmwh and the tat/f1+2 ratio was significantly lower after r-hirudin than both ufh and lmwh. thus, in vivo when the coagulation system is in an activated state r-hirudin exerts its anticoagulant effects predominantly by inhibiting thrombin (lla), whereas ufh and lmwh are directed against both xa and ila. a different mode of action of ufh and lmwh was not detectable. in venous blood, r-hirudin caused a moderate reduction of tat formation and an increase (at 1 hour) rather than decrease of f1+2 generation. formation of tat and f1+2 was suppressed at various time points following both ufh and lmwh. there was no difference in the tat/f1+2 ratio after r-h[rudin and heparin. thus, a predominant effect of rhirudin on ila (as found in bleeding time blood) was not detectable in venous blood. in bleeding time blood, r-hirudin (but neither ufh nor lmwh) significantly inhibited ~-tg release. in contrast, both ufh and lmwh caused an increase of ~-tg 10 hours after hepadn application. our observation of reduction of platelet function after r-hirudin compared to delayed platelet activation following ufh and lmwh suggests an advantage of r-h[rudin over heparin, especially in those clinical situations (such as arterial thromboembolism) where enhanced platelet activity has been shown to be of particular importance. the human cytochrome p450 isoform cyp1a2 determines the level of a variety of drugs metabolized by the enzyme, including caffeine (ca) and theophylline (th). more than 50 compounds are potential or proven inhibitors of this enzyme. some of them were reported to be substrates or inhibitors to cyp1a2 in vitro, ethers caused pharmacokinetic interactions with drugs metabolised by cyp1a2. we characterized a series of these compounds with.respect to their effect on cyp1a2 in human liver microsomes in relation to-published pharmacokinetic interactions in vivo. cyp1a2 activity in vitro was measured as ca 3-demethylation at the high affinity site in human liver microsomes, using 15 rain incubation at 37 °c with 125 -20001jm caffeine, an nadph generating system, and inhibitor concentrations covering 1.5 orders of magnitude. apparent kr values were estimated using nonlinear regression analysis. for inhibitory effects on cyp1a2 activity in vivo, the absorbed oral dose causing 50 % reduction in ca or th clearance (edso) was estimated from all published interaction studies using the emax model. %)i followed by disinfectants (14.1%)r ointments (54.8 %) and solutions (17.8%) were the most frequent drug forms 6 %) or german (17.4 %). our results show that even now drugs prepared trend analysis of the expenses at the various departments may be a basis for a ratio-hal and economic use of the drug budget. total drug expenses amounted to 30 mill. dm in 1993. s0 milldm (33%) were used in surgical departments with intensive care units (icu) (general surgery, kardiovascular surgery, neurosurgery, gynecology, anaesthesiology) of wtfich 40 % are needed by the icu and 25 % in the operating rooms. surgical departments without scu but similar patient numbers (ophthalmology, ent, orthopedics and urology) get only 10 % of the budget (30 % needed for the operating rooms). the medical departments spent s0 mill.dm of which icu needs only 10 % whereas the oncology (oncu) and antiinfective units uses more than 50 %• similar relation could be seen in the child hospital (2.4 milldm, 8 %) where 25 % were spent for icu and 40 % for oncu. the departments of dermatology and neurology get 10 %, the depart-merits of radiology, nuclear medicine and radiation therapy only 8 % of the budget. antiinfective drugs (antibiotics, antimycotics, virustatics) are most expensive (21% of budget) followed by drugs used for radiological procedures (8%) sncreasing the knowledge about the costs of medical items and the rational and economical use may stop the overproportional increase of the drug budget the mostly used :20) and a 100-fold higher efficiency than the r-form the elimination of the talinolol enantiomers was studied in 12 healthy volunteers (age: 23 -32 years, body weight: 55 -84 kg) given a single oral dose (50 mg) or an intravenous infusion (30 rag) of the racemi c drug. three volunteers were phenotypically poor metabolisers and nine were extensive metabolisers of the debrisoquine-type of hydroxylation. the r-and senantiomers of talinolol were analysed in urine by a hplc method after enantioselective derivatisation. the concentrations of the enantiomers within every sampling period as well as the amounts of s-and r-enantiomer this corresponds to a s/r-ratio of 1,00 + 0,02. the mean total amount (= s-+ r-enantiomer) eliminated was on average 50 % &the administered dose. after oral administration 26 _+ 7 % of the dose were eliminated within 36 h. the amounts of talinolol enantiomers recovered were equally (senantiomer: 6416 _+ 1624 gg the ratios of s-to r-concentrations at every sampling interval and of every volunteer were assessed between 0,82 and 1,11 (mean: 1,00 after infusion and 1,01 after oral administration, respectively) medizinische fakult~t carl gustav cams, teelmische university, t, fiedlerstr nitric oxide (no), synthesized by the inducib]e form of no synthase, has been implicated as an important mediator of-specific and non-specific immune response, little is known about the in vivo synthesis or no in inflammatory joint diseases. therefore we have studied the excretion of the major urinary metabolite of no, nitrate, in rats with adjuvant arthritis, a well established model of polyarthritis in addition we assessed the urinary excretion of cyclic gmp, which is known to serve as second messenger for the vascular effects of no, synthesized by the constitutive form of no synthase, affecting blood vessels, plate]et aggregation and neurotransmission, in 24 h urines of 12 male sprague daw]ey rats at day 20 after induction of adjuvant arthritis we measured nitrate excretion by gas chromatography and cyclic gmp by radioimmunoassay. for contro] we determined the same parameters in 24 h urines of non-arthritic rats of the same strain and age, we found a significant (p <0 001, two-tailed, unpaired t-test), more than 3-fo]d increase of urinary nitrate excretion in arthritic rats (mean 541 ± sd 273 pmo]/mmol creatinine) as compared to non arthritic rats (169 _+ 39 izmot/mmo] creatinine). urinary cyclic gmp excretion was slightly, but not significant]y lower in arthritic rats (510 ± 44 nmol/mmol creatinine) than in controls (747 ± 33 nmo]/mmo] creatinine).there were no major differences in food or water intake which cou]d account for these results. the increased urinary nitrate excretion accompanied by normal cyclic gmp excretion suggests that no production by the inducible form of no synthase is enhanced in rats with adjuvant arthritis institute of c]inica] pharmacology~ hannover medical school, d-30623 hannover, germany and *research center gr@nentha] gmbh, zieg]erstr 6, d-52078 aachen, germany background: pge1 has been shown to be efficacious in the treatment of critical leg ischemia. despite of an almost complete first pass metabolism in the lung the clinical effects of intraarterial and intravenous pge1 do not differ significantly. in addition, it is not fully understood which of the various pharmacological actions of pge1 is the main factor; by most authors, however, it is thought to be the increase of cutaneous and muscular blood flow. by means of [15-0]-h20-pet, we studied muscular blood flow (mbf) of the leg in patients with peripheral arterial disease comparing intraarterial and intravenous pge1. patients and methods: 8 patients (3 f, 5 m; mean age 59 y) with pad were studied, (5 atherosclerosis, 3 thromboangiitis obliterans). at the first day, 5pg pge1 were infused intraarterially within 50 minutes; pet scanning of the lower leg was performed at minutes 0, 25 und 50. at the following day, 40pg pge1 were infused intravenously within 2 hours; pet scanning was performed at minutes 0, 30, 60 and 120. results: in the infused leg the increase of mbf caused by intraarterial pge1 averaged 79+59% at minute 25 and 1004_85% at minute 50; in the not infused leg there was no effect. the increase rate in the infused leg was highly variable but did not correlate with sex, age, disease or clinical outcome. for intravenous pge1 the change of mbf at any time averaged almost 0%. conclusion: unlike intraarterial pge1, intravenous pge1 does not increase the muscular blood flow of the leg. a comparable clinical effect provided, increase of muscular blood flow may not be considered the main way of action of pge1 in critical leg ischemia. eslrogen(er) and progesterone(pr) receptor status as well as lymph node involvement are important factors in predicting prognosis and sensitivity to hormone and chemotherapy in patients with breast cancer. prognostic relevance of ps2-protein, egfr and cathepsin d is currently under debate. especially ps2 and egfr expression appears to provide additional information regarding the responsiveness of the tumour tissue to tamoxifen. the aim of the present study was to investigate the relationships between these parameters and established prognostic factors in breast cancer. in a prospective study ps2 and cathepsin d were assayed immunoradiometricauy in the tumour cytosol of 122 patients, egfr was measured by elisa. relating the level of these factors to the lymph node involvement, menopausal status as well as turnout size, no significant association could be established. jn our findings er and pr are significantly correlated with the expression of ps2 but none is correlated with the cathepsin d status. egfr was shown to be inversely correlated with the content of er. a significant association between cathepsin d and ps2 could be established in patients with early recurrence. at a median follow-up of 15-24 months, recurrence was more common in patients with tumours having negative status for ps2, independent of receptor status. in conclusion, because of the relative independence on the er and pr status and other prognostic factors and the influence on the recurrence behaviour, demonslrated here, and their role in promoting tumour dissemination and changing hormone therapy sensitivity, all three factors represent markers of prognostic relevance.deparlancnts of clinical pharmacology l, nuclear medicine 2 and surgery 3,pharmacoeconomic studies, conducted either separately from or together with clinical trials are increasing in both number and meaning. in a period of limited health care budgets, political and medical decision makers alike run the risk of accepting the results of such studies without critical reflection. careful evaluation of those studies by state-of-the-art methods is one way out of the trap. another could be to refer to ethical considerations. the problem in this context is, that the discussion concerning ethical aspects of pharmacoeconomic research, at least in europe, is just in its beginning. therefore, no widely accepted standards are available. but they are essential to answer four main questions: 1. who should perfom a pharmacoeconomic study? 2. which objectives should be considered? 3. what kind of study should be performed (e. g. cost-effectiveness, cost-utility, cost-benefit analysis)? 4. which consequences will be drawn from the results?based on the case study-orientated "moral cost-benefit model" (r. wilson, sci. tech. human values 9: 11-22, 1984) , a three-step decision and evaluation model is proposed to handle bioethical problems in pharmacoeconomic studies: 1. moral risk analysis 2. moral risk assessment 3. moral risk management. possible practical consequences for decision making in research policy, study design and assessment of results are discussed. hirudin is the most potent known natural inhibitor of thrombin and is presently gaining popularity as an anticoagulant since recombinant forms have become available. the aim of the present study was to compare platelet aggregation, sensitivity to prostaglandin e1 (pge1) and thromboxane a2 (txa2) release in r-hirudinized and heparinized blood. platelet aggregation was measured turbidimetrically using a dual channel aggregometer (labor, germany) in blood samples of healthy volunteers anticoagulated with r-hirndin w015 (behring) and hepatin (20 gg/mi blood each). aggregation was induced by arachidonic acid (aa; 0.5, 1.0 and 2.0 ram) and adp (1.0 lam). pge1 in concentrations 10, 20 and 40 ng/ml was used. plasma txb2 content was measured by gas chromatography/mass spectrometry. this study showed a significantly lower a.a-induced platelet aggregation in r-hirudinized plasma. three minutes after the aggregation induction by 0.5 mm aa the plasma txb2 concentration was 23 0 ng/ml in blood anticoagulated with rhimdin and 108.4 ng/ml in heparin-anticoagulated blood. the extent of the adp-induced aggregation was nearly the same in rhimdinized and heparinized plasma. platelet sensitivity to pge1 was significantly higher in r-hirudinized blood. thus, aa-induced platelet aggregation is significantly lower and sensitivity to pgei higher in r-himdin-anticoagulated blood in comparison with beparin-anticoagulated blood.university of tartu, puusepa str. 8, tartu ee 2400, estonia anaemia has been reported in renal transplant (ntx) recipients treated with azathioprine (aza) and angiotensin converting enzyme-inhibitors (ace-i). an abnormal aza metabolism with increased 6-thioguanine nucleotide (tgn) levels in erythrocytes is a possible cause of severe megaloblastic anaemia (lennard et al, br j clin pharmaco11984). methods: 15 ntx patients receiving aza (1,9_+0,54 mg/kg/d), prednisolone (0,12+0,06 mg/kg/d) and enalapril (ena) (0,16+0,07 mg/kg/d) for more than 6 months were studied prospectively. blood samples were taken before and 3h after administration of aza on 2 visits during ena treatment and 10 weeks after ena had been replaced by other antihypertensives (x). tgn in erythrocytes, 6-mercaptopurin (mp) and 6-thiouric acid (tua) in 3h post dose plasma (p.) und 24h urine (u.) samples were analyzed by hplc using a mercurial cellulose resin for selective absorption of the thiol compounds. pharmacodynamic variables were hemoglobin (hb), erythropoietin (epo) and creatinine clearance (ci ace~,lcholine plays an important role in regulating various functions in the airway's. in human lung less is known about regional differences in cholinergic innervation and about receptor-mediated r%m.flation of acetylcholine release. in the present study the tissue content of endogenous acetylcholine and the release of newly-synthesized [~h]acetylcholine were measured in human lung human tissue was obtained at thoracotomy from patients with lung cancer moreover, in isolated rat tracheae with intact extrinsic vagal innervation possible effects of g__-adrenoceptor agonists on evoked ph]acctylcholine release were studied. endogenous acetylcholine was measured by hplc with ec-detection; evoked ph]acetylcholme release was measured after a preceding incubation of the tissue with [~h]choline. huma n large (main bronchi) and small (subsegmental bronchi) airways contained similar amounts of acetylcholine (300 pmol/100 mg), whereas significantly less acetylcholine was found in lung parenchym (60 pmol/100 mg). release of [3h]acetylcholine ,,,,'as evoked in human bronchi by transmural electrical stimulation (four 20 s trains at 15 hz). oxotremorine, an agonist at muscarine receptors, inhibited evoked [~hiacetylcholine release indicating the existence of neuronal inhibitor 3' receptors on pulmona~ parasympathetic neurones. scopolamine shifted the oxotremorine curve to the right suggesting a competitive interaction (pa 2 value: 88: slope &the schild plot not different from unity) however, a rather sluggish schdd plot was obtained for pirenzepine. scopolamine but not pirenzepine enhanced evoked [3h]acetylcholine release. the present experiments indicate a dense cholinergic innervation in human bronchi; release of aceu, lcholine appears to be controlled by facilitatory and inhibitou' nmscarinc receptors. in isolated, mucosa-intact rat tracheae isoprenaline (100 nm) inhibited [~h]acetylcholine release evoked by preganglionic nerve stimulation isoprenaline was ineffective in mucosa-denuded tracheae or in the presence of indomethacin thus, 13adrenoceptor agonists appear to inhibit acetylcholine release in the airways by the liberation of inhibitoiy prostanoids from the mucosa. the occurrence of the non-enzymatic reactions between glucose and structural proteins is well known (vlassara h et al. (1994) lab invest 70: 138-151) . the reaction between proteins and fructose (i.e. fmctation), however, can also occur. like glucose-protein adducts the fructose analognes are able to form so-called advanced glycation endproducts (age). the inhibition of early and advanced products of fmctation may be ilnportant for the prevention of diabetic late complications (mcpherson jd et al. (1988 ) biochemistry 27: 1901 -1907 . we investigated the in vitro fmctation of human serum albumin (hsa) and its inhibition by selected drugs. hsa was fmctated by incubation with 20 mmol/1 fructose in 0. i mol/l phosphate buffer, ph=7.4.,at 37 ° c for 21 days. the rate of fmctation was measured by the following methods: -a colorimetric method based on deglycatien of glycated, proteins by hydrazine (kobayashi k et ai.(1994) bioi pharm bull 17: 365-369), -affinity chromatography with aminophenyl-boronate-agarose, -fluorescence measurement for the delermination of age we used aminoguanidine, pcnicillamine, captopril and alpha-lipoic acid(20 mmol/1) to study the inhibition of hsa fmctation. after three weeks incubation the formation of early glycation products was inhibited by aminogalanidine (15%) and captopril (3%) whereas penicillamine and alpha-lipoic acid showed minimal inhibition. aminognanidine inhibited the formation of age by 42%, penicillamine by 37%, alpha-lipoic acid by 11% and captopril by 37%. these results may suggest a potential use of the investigated drags in the prevention of the formation of protein-fructose addncts. key: cord-312580-r57rkrya authors: harcourt-brown, frances title: chapter 6 clinical pathology date: 2002-12-31 journal: textbook of rabbit medicine doi: 10.1016/b978-075064002-2.50009-6 sha: doc_id: 312580 cord_uid: r57rkrya nan as rabbits are used extensively for toxicological and physiological studies, there are many scientific papers about the effects of experimental infections, drugs and toxic substances on haematological and biochemical parameters. there is also information about diseases of commercial rabbits, which are mainly investigated by post-mortem examination. in contrast, there is a dearth of literature about the effects of clinical diseases on the blood picture of rabbits or the use of blood tests as diagnostic or prognostic indicators. it is not always possible to extrapolate from other species, especially carnivores such as dogs and cats to the herbivorous rabbit with its specialized physiology. at the present time, much of the information that is available on the haematology and biochemistry of pet rabbits is anecdotal, although it can be helpful and is better than no information at all. the collection of blood and urine samples is covered in section 3.12. parameters such as glucose, creatine kinase and aspartate aminotransferase (ast) can be altered by stress associated with handling and restraint, or tissue damage that has occurred during sample collection. for example, potassium results appear less reliable in samples taken with a plastic cannula as opposed to a hypodermic needle . rabbit blood haemolyses easily and clots quickly (perry-clark and meunier, 1991). small clots affect haematology results and haemolysis affects certain biochemistry results, especially potassium and serum inorganic phosphorus that are released from erythrocytes. rapid clotting can affect the performance of some analysers and heparinized syringes and needles are required. it is not possible to take a guaranteed fasting sample from a rabbit because they ingest caecotrophs. parameters such as blood glucose are affected by digestion. some parameters such as bile acids, cholesterol and urea follow a diurnal rhythm that also affects the total and differential white cell count (fox and laird, 1970; fekete, 1989; loeb and quimby, 1989) . stress associated with car journeys or a period in unfamiliar surroundings will increase blood glucose and alter haematological parameters such as the distribution of neutrophils and lymphocytes. pregnancy affects parameters such as protein, haematocrit, cholesterol, alkaline phosphatase, triglycerides (viard-drouet et al., 1984) , glucose, sodium, calcium, phosphate and red cell indices (palm, 1997) . serum cholesterol is the parameter that is most affected and can be up to 30% lower in pregnant than in nonpregnant animals (palm, 1997) . anaesthesia affects some blood parameters such as potassium . the effect of anaesthesia on biochemical parameters is minimized by taking samples within 5 minutes of induction. intravenous or intraosseous fluids will also affect haema-clinical pathology tological findings (ros et al., 1991) and blood samples should be taken prior to treatment. there are a number of published reference ranges for haematological and biochemical parameters in rabbits. conversion factors for the variety of units that are used in reference ranges are given in table 6 .1. differences in analytical techniques between laboratories can lead to disparity in results. laboratory data are often derived from populations of rabbits of the same breed, sex and age that are not genetically diverse. in contrast, the pet rabbit population is made up of a variety of breeds, cross breeds and is composed of rabbits of all ages. significant breed and sex differences have been noted for some parameters in laboratory rabbits (kozma et al., 1974) . published reference ranges for pet rabbits are either derived from sets of data from laboratory rabbits or from data collected by the author. some reference ranges are an amalgamation of other references ranges and result in a range so wide that almost any result will fall within it. for example, the reference range for serum albumin concentra-tions of pet rabbits is given as 27-46 g/l (malley, 1996) based on four published sets of data. gillett (1994) gives an even wider range of 27-50 g/l for laboratory rabbits based on five sets of data. for some parameters there are big differences between published reference ranges. an example is blood calcium. gillett (1994) gives a range of 1.5-3.4 mmol/l in comparison with 3.4-4.0 mmol/l by harkness and wagner (1995) . differences in calcium content of the diet between the groups of rabbits or differences in analytical technique could explain these discrepancies. different laboratory methods can result in large differences between reference ranges. an example is alkaline phosphatase. collins (1988) gives a reference range of 4.1-16.2 iu/l in comparison with 10-70 iu/l (gillett, 1994) or 112-350 iu/l (harkness and wagner, 1995) . automated flow cytometers are designed to measure numbers of human blood cells. rabbit erythrocytes are smaller in diameter than human erythrocytes and also vary in diameter. these differences cause problems with some automated analysers. automated differential white cells counts cannot be relied upon for rabbit blood and accurate results can only be obtained using manual counting methods (kabata et al., 1991) . clinical pathology some morphological characteristics of rabbit blood cells are different from other species. the red blood cells vary in diameter within a range of 5.0-7.8 µm (sanderson and philips, 1981) . this variation in diameter (anisocytosis) is a feature of blood smears from rabbits and is not a significant finding (see plate 2). the red cell distribution width (rdw) is a measurement of the variation in size of erythrocytes and is higher in rabbits (11-15, idexx reference range) than in dogs and cats (8-10; bush, 1981) . in dogs and cats, anisocytosis is indicative of the presence of reticulocytes and a regenerative anaemia. in rabbits, 1-4% of circulating erythrocytes may be reticulotytes. polychromasia and reticulocytes in rabbit blood smears have been attributed to the short life span and high turnover of erythrocytes (kraus et al., 1984) . nucleated red cells and howell-jolly bodies can also be found occasionally (mclaughlin and fish, 1994) . rabbit neutrophils have an almost colourless cytoplasm and contain two types of granules. the smaller granules stain pink giving a pinkish colour to the cytoplasm. larger granules stain a deeper pinkish-red. the overall colour of the neutrophils varies according to the proportion of large and small granules. the granular appearance of the cytoplasm has led to different nomenclature. rabbit neutrophils may be called heterophils, pseudoeosinophils, acidophils or amphophils depending on the text (sanderson and philips, 1981; benson and paul-murphy, 1999) . neutrophils measure 10-15 µm in comparison with eosinophils that measure 12-16 µm (sanderson and phillips, 1981) . small lymphocytes are seen more commonly than large lymphocytes. the average cell diameter for small lymphocytes is 7-10 µm (cooke, 2000) . the lymphocytes are round cells with the typical morphology described for other species. an occasional large lymphocyte may have a few azurophilic granules in the cytoplasm (jain, 1986) . monocytes are large nucleated cells measuring 15-18 µm (cooke, 2000) . the nucleus has a diffuse lacey chromatin pattern that lightly stains a purple blue. the vacuolar cytoplasm stains light blue. eosinophils can be distinguished from neutrophils by their greater size and large acidophilic granules. in contrast to other laboratory species, basophils are frequently found in the circulation of rabbits in small to modest numbers (jain, 1986). a reference range for haematological parameters is given in table 6 .2. the haematological picture gives an indication of the general health status of a rabbit. stress and a range of diseases will alter haematological parameters. hinton et al. (1982) analysed the haematolog-• rabbit blood clots quickly and haemolyses easily • food deprivation does not guarantee a fasting blood sample as rabbits ingest caecotrophs • stress associated with transport or handling can affect parameters such as blood glucose and the distribution of neutrophils and lymphocytes • pregnancy, anaesthesia, blood collection techniques and intravenous fluid therapy will influence some blood results • time of day can influence blood results as many parameters follow a duirnal rhythm in common with many physiological processes in rabbits • laboratory reference ranges are often derived from animals of the same breed and strain. pet rabbits come from a more genetically diverse population • reference ranges for pet rabbits are often an amalgamation of laboratory reference ranges that can be so wide that almost any result will fall within them • different analytical techniques can result in disparity between laboratory reference ranges • automated flow cytometry is not suitable for differential white cell counts in rabbits. accurate results can only be obtained by manual counting methods. ical findings in 117 healthy and diseased rabbits and found that blood cellularity was a good indicator of disease especially with regard to erythrocyte and lymphocyte counts. these findings are in agreement with studies of experimental infections in rabbits krueger, 1988, 1989) and in a clinical study by harcourt-brown and baker (2001) . in this study, significantly higher red cell counts, haemoglobin values, haematocrits and lymphocyte counts were found in rabbits kept outside with unlimited access to grazing and exercise. a comparison was made with rabbits kept in hutches and those suffering from dental disease (see figure 6 .1). reference ranges for packed cell volumes (pcv) vary between sources with values between 30% and 50% (malley, 1996) . pet rabbits tend to have pcv values at the lower end of the range, typically between 30 and 40% (harcourt-brown and baker, 2001) . values greater than 45% are indicative of dehydration, especially in rabbits suffering from gut motility problems. values of less than 30% indicate anaemia that can be classi-fied into non-regenerative and regenerative in a similar manner to other species. a regenerative anaemia is associated with chronic blood loss, e.g. due to heavy flea infestation or from a site that intermittently bleeds such as a uterine endometrial aneurysm. uterine adenocarcinomas are a common finding in middle-aged does. lead poisoning can result in a regenerative anaemia with the presence of nucleated erythrocytes, hypochromasia, poikilocytosis and cytoplasmic basophilic stippling (fudge, 2000) . a nonregenerative anaemia is caused by diseases such as lymphoma or chronic renal failure. autoimmune haemolytic disease has not been described as a clinical phenomenon in pet rabbits although there are reports that it occurs. autoimmune haemolytic anaemia has been reported in laboratory rabbits in association with lymphosarcoma (weisbroth, 1994) . chronic debilitating disease such as dental disorders or abscesses often cause a mild anaemia in pet rabbits (harcourt-brown and baker, 2001 ) (see figure 6 .1). nucleated red blood cells can be associated with acute infectious processes although a few may be present in normal blood films. experimentally, infections with escherichia coli and staphylococcus aureus cause an increase in 143 clinical pathology number of circulating nucleated red blood cells during the septicaemic phase of the disease krueger, 1988, 1989 ). there is a natural diurnal variation in total white cell count with lowest counts occurring during the late afternoon and evening (fox and laird, 1970) . the white cell count also varies with age (jain, 1986) . it is higher in young rabbits of approximately 3 months of age and in adults over 1 year old. the first peak in leucocyte count is due to an increase in the number of lymphocytes. the second peak is due to an increase in the number of neutrophils. in other species, an increased white blood cell count is seen in response to bacterial infection or in response to endogenous or exogenous corticosteroids. rabbits do not develop marked leucocytosis after either acute infectious challenge or the intramuscular injection of cortisone acetate (toth and january, 1990) . in two studies by krueger (1988, 1989) controlled experimental infections with staphylococcus aureus, streptococcus pyogenes, escherichia coli and candida albicans resulted in fever, increased plasma cortisol concentrations, neutrophilia and lymphopaenia but no significant increase in total white blood cell count. high white cell counts can be found in rabbits with suffering from lymphosarcoma (mclaughlin and fish, 1994) . low white blood cell counts can be found in association with chronic disease (hinton et al., 1982) . clinical pathology figure 6.1. comparison of some blood parameters of pet rabbits. as part of an investigation into the relationship of metabolic bone disease with dental disease, blood samples were taken from pet rabbits presented for veterinary treatment or for health checks. at the time of sampling the rabbits were assigned to one of four groups: add (advanced dental disease), edd (early dental disease), h (healthy rabbits kept in hutches), fr (rabbits kept in free-range conditions in a large enclosure with unlimited access to exercise and natural daylight all year round). no rabbits in the free-range group (fr) showed signs of dental disease; all the rabbits in the add, edd and h groups were kept in hutches. blood results from rabbits that were found to be suffering from other conditions, such as renal disease or neoplasia, were not included in the statistical analysis. the add group consisted of rabbits that were presented for veterinary treatment for dental disorders such as acquired malocclusion or abscesses. all the other rabbits in the edd, h and fr groups were presented for neutering, health checks, vaccination or euthanasia. rabbits in the edd group had signs of early dental disease, such as horizontal ribs on the incisors, swellings along the ventral border or the mandible or epiphora related to elongation of the roots of the upper primary incisors. the investigation was conducted to compare blood parameters related to calcium metabolism in rabbits with or without dental disease. the healthy free-range group was used as a control. during the course of the study, differences in haematological pictures and albumin values emerged among rabbits kept under the different husbandry regimens. complete blood counts from free-range rabbits were comparable with laboratory reference ranges whereas there were significantly lower red cell and lymphocyte counts in rabbits suffering from advanced dental disease. the low lymphocyte counts of rabbits with dental disease suggest they suffered from chronic stress. serum albumin values were significantly higher in rabbits kept in free-range conditions than in those suffering from advanced dental disease or those unaffected by dental disease but kept in hutches. total serum calcium concentrations were highly correlated with serum albumin levels. rabbits kept in hutches showed trends towards anaemia and lymphopaenia. plasma parathyroid hormone (pth) concentrations were higher and total serum calcium concentrations were lower in hutch-kept rabbits with advanced dental disease in comparison with rabbits kept in free-range conditions. these results indicated that acquired dental disease of pet rabbits is related to husbandry and is associated with alterations in calcium metabolism. reprinted from harcourt-brown and baker (2001) with kind permission from the journal of small animal practice. although the total white cell count of rabbits seldom alters in diseased rabbits, the differential white cell count may show a number of changes due to the redistribution of white blood cells. a feature of many diseases in rabbits is an alteration in the ratio of neutrophils to lymphocytes and a reduction in blood cellularity (hinton et al., 1982) . the neutrophil: lymphocyte ratio has been suggested as a method of predicting whether a rabbit is normal or abnormal (mclaughlin and fish, 1994) . jain (1986) described a physiological variation in the neutrophil: lymphocyte ratio according to the age of the rabbit. the ratio changes from 33:60 in the second month of life to 45:45 in rabbits over 1 year of age. stress and increased cortisol levels can affect this ratio as well as disease. 6.2.4.3 effect of stress on the differential white cell count stress alters the differential white cell count in any species. rabbits are particularly susceptible to the effects of stress. a car journey to the surgery, a period in the waiting room next to a barking dog or the excitement of handling can be reflected in the blood picture. adrenaline and cortisol affect the distribution of lymphocytes throughout the body. administration of exogenous adrenaline to rabbits results in redistribution of lymphocytes from spleen and bone marrow to peripheral blood, lungs and liver (toft et al., 1992a) . conversely, exogenous corticosteroid administration results in a redistribution of lymphocytes from the peripheral blood, bone marrow and spleen to the lymphatic tissue in rabbits (toft et al., 1992b) . prolonged periods of stress cause neutrophilia and lymphopaenia. marked changes in white cell distribution with a relative neutrophilia and lymphopaenia were found in a study of the cortisol levels and haemograms of rabbits after transport, either by air or by lorry. the changes in white cell distribution lasted for 24-48 h and were correlated with increased cortisol levels (toth and january, 1990) . disease is stressful as well as having a direct effect on the production and distribution of white cells. rabbits with experimental infections exhibit a neutrophilia and lymphopaenia in comparison with control rabbits handled and sampled in exactly the same manner but inoculated with heat killed cultures (toth and krueger, 1989) . rabbits inoculated with a heat-killed culture do not experience the same rise in plasma cortisol concentrations as those inoculated with a live culture, indicating that the stress response is initiated by disease rather than by handling. therefore the stressful effects of a long car journey to the surgery or a morning spent in a kennel next to a barking dog is more likely to affect the neutrophil:lymphocyte ratio than the excitement response of taking blood. neutrophils function primarily as phagocytes and are important in infectious conditions and in inflammation. in other species, a neutrophilia occurs in response to inflammation, especially bacterial infection. an increase in the number of circulating neutrophils causes a rise in total white blood cell count. this response is not marked in rabbits. however, a change in the distribution of white cells can occur in response to infection with a relative neutrophilia and lymphopaenia but no alteration in total white cell count (toth and krueger, 1989) . a mature neutrophilia accompanied by an increase in plasma cortisol can also be associated with stress (toth and january, 1990 ). lymphocytes are involved in immunological responses and are distributed throughout the body in various tissues including blood, bone marrow, lymph nodes, spleen and gut-associated lymphoid tissue. the number of lymphocytes in the blood reflects a balance between cells leaving and entering the circulation and does not necessarily reflect a change in lymphopoiesis. increased cortisol levels cause a lymphopaenia and increased adrenaline levels cause lymphocytosis (toft et al., 1992a, b) . in rabbits, lymphopaenia is a feature of a variety of clinical diseases (hinton et al, 1982) . marked lymphopaenia has been reported as a feature of differential white cell counts of pet rabbits especially those suffering from dental disease (see figure 6 .1) (harcourt-brown and baker, 2001) . lymphoma is a relatively common tumour of rabbits and atypical lymphocytes can be found in the peripheral blood of these patients. the main function of eosinophils is detoxification either by inactivation of histamine, or histamine-like toxic materials. eosinophils are important in the allergic response and are capable of phagocytosis (kerr, 1989) . chronic eosinophilia can be seen in diseases of tissues that contain large numbers of mast cells such as the skin, lungs, gastrointestinal tract and uterus. eosinophilia can be associated with parasitism, especially when parasites are migrating through tissue. mild eosinophilia has been associated with experimentally induced chronic ascarid parasitism in rabbits (gupta and trivedi, 1981) . however, heavy worm burdens are rare in pet rabbits. encephalitozoonosis does not appear to cause an eosinophilia. slight to moderate elevations in eosinophil counts can be observed after traumatic wound repair in rabbits (fudge, 2000) . although eosinopaenia can be a significant finding in other species, low eosinophil counts or a zero count are not unusual in rabbits. basophils are similar to neutrophils but have dark blue cytoplasmic granules. although basophils are rare in blood films from species such as the dog, they may be seen commonly in rabbit blood (kerr, 1989) . basophil counts as high as 30% have been reported in clinically normal animals (benson and paul-murphy, 1999 ). in other species, monocytosis is associated with chronic disease, particularly chronic inflammatory conditions. in rabbits, increased monocyte counts can be associated with chronic bacterial infection. hinton et al. (1982) noted increased monocyte counts in rabbits with subcutaneous abscesses, mastitis and 'labyrinthitis'. however, monocyte counts within the laboratory reference do not signify the absence of chronic infection. rabbits with chronic osteomyelitis due to dental disease can have monocyte counts within the laboratory reference range (harcourt-brown, unpublished data). a reference range for biochemical parameters is given in table 6 .3. clinical pathology key points 6.2 • rabbit erythrocytes vary in diameter and anisocytosis can be a normal finding in rabbit blood films • polychromasia and small numbers of reticulocytes and nucleated red cells may be seen on normal blood films • rabbit neutrophils have a granular cytoplasm and may be mistaken for eosinophils • there are several different terms for the rabbit neutrophil. some authors use terms such as heterophil, pseudoeosinophil, acidophil or amphophil instead of neutrophil • basophils are frequently found on blood films from rabbits • low blood cellularity, i.e. anaemia and lymphopaenia, is a non-specific feature of disease in rabbits • high numbers of nucleated red blood cells may be associated with infectious disease • the neutrophil:lymphocyte ratio should be approximately 1:1 in adult rabbits. alterations in the ratio can be associated with stress or disease • adrenaline causes a shift of lymphocytes from the spleen and bone marrow to blood. cortisol causes a shift of lymphocytes away from the bloodstream to the spleen and lymphatic tissue • an increase in total white cell count is unusual in rabbits even in the presence of infection • a neutrophilia with a left shift occurs in response to infection • monocytosis can be seen in association with chronic infection. herbivores, such as rabbits, differ from carnivores in their carbohydrate metabolism. carnivores eat periodically and have sudden large intakes of nutrients that must be stored for utilization during the fast between meals. herbivores graze for long periods of the day and are continually absorbing nutrients from the digestive tract. in rabbits, volatile fatty acids are produced from bacterial fermentation in the caecum and are continually absorbed as an energy source. a fasting sample is difficult to obtain from a rabbit. withholding food does not prevent caecotrophy and the digestion of caecotrophs provides a source of glucose. blood samples taken after 96 h of food deprivation may show no alteration in blood glucose levels (kozma et al., 1974) . hyperglycaemia is a relatively common finding in rabbits and can be accompanied by glycosuria. handling alone can cause an increase in blood glucose to the order of 8.5 mmol/l experimentally (knudtzon, 1988) and 15 mmol/l or more anecdotally. diabetes mellitus has not been described in pet rabbits and there is some difference of opinion about its importance as a clinical disease (hoefer, 2000; jenkins, 2000; rosenthal, 2000) . herbivorous animals withstand the absence of insulin more readily than carnivorous ones (bentley, 1998) and are therefore not so susceptible to diabetes mellitus. diabetes mellitus has been induced in laboratory rabbits by the administration of alloxan. it has also been described as total protein 54-75 g/l e urea 6.14-8.38 mmol/l b vitamin a (plasma) 30-80 µg/ml ± < 10 µg/ml indicates deficiency (liver levels of < 10 µg/g liver denote deficiency) b vitamin e (plasma -tocopherol) > 1 µg/ml (< 0.5 µg/ml indicates deficiency) a hereditary disease in rabbits. a laboratory strain was selectively bred as an animal model of human diabetes mellitus. (roth and conaway, 1982) . affected animals were polydipsic, polyuric and polyphagic with severely impaired insulin release. elevated glycosylated haemoglobin values of 12.2% were observed in the overtly diabetic animals in comparison with 3.9% in normal animals. increased glycosylated haemoglobin levels did not correlate with plasma glucose concentrations (cannon and conaway, 1981) . histologically there was hypergranulation of ␤-cells of the islets of langerhans. obesity and ketoacidosis were not features of diabetes mellitus in the laboratory rabbits. the hyperglycaemia was in the region of 540-590 mg/dl (30-33.4 mmol/l) and there was marked glycosuria. roth and conaway (1982) described the maintenance of one diabetic individual on insulin at a dose of up to 8 units per day for 3 years. ketonuria was not observed. in pet rabbits, a diagnosis of diabetes mellitus cannot be made on a single blood sample and requires serial blood and urine sampling to confirm the diagnosis. in view of the physiological factors that can increase blood glucose levels it is advisable to take repeat blood samples from hyperglycaemic rabbits with time of day, phase of digestion, anaesthesia, influence of handling or car journeys in mind. mild glycosuria is not a significant finding. hyperglycaemia can be seen in the terminal stages of gut stasis and is a poor prognostic sign (harcourt-brown, personal observation). it is associated with fatty degeneration of the liver at post-mortem examination. marked hyperglycaemia is also seen in association with painful conditions such as acute intestinal obstruction. blood glucose levels can rise to 20-25 mmol/l and return to normal once the condition is resolved (harcourt-brown, unpublished observation). experimental haemorrhagic or traumatic shock results in hyperglycaemia proportional to the severity of the condition. hyperthermia also results in hyperglycaemia (mclaughlin and fish, 1994) . diseases that elevate serum glucose levels in other species, such as hyperadrenocorticism or acute pancreatititis have not been reported in pet rabbits, although they could occur. hypoglycaemia is a significant finding in rabbits and is associated with anorexia, starvation or disturbances in the digestion and absorption of carbohydrates. it can be a sign of hepatic dysfunction. a drop in blood glucose leads to mobilization of free fatty acids from adipose tissue and contributes to the development of ketoacidosis and fatty degeneration of the liver. measurement of serum glucose is of value in moribund rabbits as a basis for the selection of appropriate fluid therapy. other causes of hypoglycaemia such as addison's disease or insulinomas have not been reported in pet rabbits although such conditions could occur. interpretation of total protein concentrations is similar to other mammals. artefactual increases in protein concentrations can result from excessive venous stasis during blood collection. fluid and small molecules leave the plasma, resulting in a relative increase in proteins. this situation can occur in rabbits, especially in miniature breeds with small veins. an increase in total protein indicates dehydration, chronic and immune-mediated disease. in rabbits dehydration due to water deprivation or gastrointestinal disturbances commonly occurs. examination of the haematocrit and albumin and globulin fractions can assist differential diagnosis. liver disease, chronic enteropathy, starvation or malnutrition may result in reduced protein levels. glomerulonephropathy or protein-losing enteropathy are uncommon conditions that could cause low total protein levels in rabbits. a decrease in both albumin and globulin may be associated with haemorrhage or exudative skin lesions such as fly strike. the liver is the sole site of albumin synthesis and hypoalbuminaemia is feature of advanced liver disease in all species. in rabbits, heavy parasitism is a cause of liver disease. eimeria steidae causes hepatic coccidiosis (see section 10.10.1.2). cysticercus pisiformis, the larval stage of taenia pisiformis, migrates through the liver and results in the development of fibrous tracks and necrotic foci (see section 16.3.2). severe infestations can result in low albumin levels. non-hepatic causes of low serum albumin include glomerulonephropathy, protein-losing enteropathy, malabsorption and cardiogenic ascites. laboratory reference ranges for serum albumin levels in rabbits can be wide and vary between sources. sex differences have been reported in laboratory rabbits. one study showed that female new zealand white rabbits had higher serum albumin levels than males, although other studies have found no sex differences (kozma et al., 1974) . in rabbits, hypoalbuminaemia is most likely to be associated with nutritional factors such as abnormal caecotrophy, incorrect diet, starvation or malnutrition associated with dental disease. primary or secondary hepatic neoplasia occasionally occurs in pet rabbits. hepatic coccidiosis is a cause of low serum albumin levels, especially in young rabbits that have been kept in colonies. a high serum albumin concentration is not a feature of any specific disease, although an increased albumin level in conjunction with a raised pcv is indicative of dehydration. in a study by harcourt-brown and baker (2001), pet rabbits kept in free-range conditions had significantly higher serum albumin concentrations than rabbits that were suffering from advanced dental disease. they were also significantly higher than rabbits kept in hutches that were not suffering from dental problems (see figure 6 .1). the difference in albumin levels was attributed to differences in diet and husbandry. caecotrophs are a source of amino acids for rabbits and normal caecotrophy is an important element of their protein metabolism. low fibre diets, obesity, dental disease or skeletal abnormalities can prevent rabbits ingesting caecotrophs from the anus and reduce the available amino acids for protein synthesis. rabbits that are kept in hutches are more likely to be eating a low fibre diet and to suffer from obesity and skeletal problems than rabbits living outside with unrestricted access to natural vegetation and exercise. plasma globulins are made up of a range of proteins including carrier proteins and immunoglobulins or antibodies. the types of globulin can be separated into five fractions by electrophoresis. the ␥-globulin fraction is almost entirely composed of immunoglobulins. some globulins can be synthesized in the liver but immunoglobulins are synthesized exclusively in lymphoid tissue. acute inflammation, chronic disease or immune-mediated disease can cause an increase in globulin levels. myeloproliferative disease results in abnormal levels of immunoglobulin production. there are few published data on the significance of globulin concentrations in rabbits. lipaemia can artefactually elevate protein levels with some analytical methods. experimental infections with rabbit coronavirus result in hypergammaglobulinaemia. analogies have been made between coronavirus infection in rabbits and feline infectious peritonitis in cats (digiacomo and mare, 1994) . coronavirus occurs in laboratory rabbits but is an unlikely diagnosis in the pet rabbit. cholesterol is synthesized in the liver or absorbed from the diet. it is a metabolic precursor of steroid hormones. cholesterol is broken down in the liver and excreted in bile. in other species, elevated cholesterol levels are indicative of a variety of metabolic disorders such as hypothyroidism, hepatopathy, diabetes mellitus, and hyperadrenocorticism. low levels can occur in association with impaired hepatic function. changes in serum triglyceride levels reflect a similar range of diseases. blood levels of triglycerides increase after a meal, especially if it is a fatty meal. in rabbits, there are some physiological factors that affect cholesterol levels. male rabbits have lower cholesterol levels than females and there is a diurnal variation with higher levels occurring during the late afternoon (loeb and quimby, 1989) . large variations in blood cholesterol and triglyceride values can occur between individual rabbits (yu et al., 1979) . a fasting blood sample is required for cholesterol and triglyceride assay. in rabbits, it is difficult to obtain a fasting sample because of caecotrophy. abnormal cholesterol or triglyceride levels are most likely to be associated with dietary factors or hepatic impairment. in anorexic rabbits, especially obese ones, a lipaemic sample is a poor prognostic indicator as it signifies impaired fat metabolism and the presence of hepatic lipidosis (see section 10.3). a rise in triglyceride levels has been found in association with experimentally induced chronic renal failure in rabbits (tvedegaard, 1987) . in other species, amylase is found in the pancreas and to a lesser extent in the salivary glands, liver and small intestinal mucosa. amylase has a short half-life and is rapidly removed from the circulation. it is excreted by the kidney. elevated levels indicate pancreatic disease or renal insufficiency. in rabbits, amylase is present in pancreatic tissue in high concentrations. low concentrations are found in the salivary glands and none is produced by the liver (jenkins, 2000) . amylase is also produced by caecal microorganisms and is present in caecotrophs aiding conversion of glucose to lactic acid during digestion in the stomach and small intestine. serum amylase levels are lower in rabbits than other species (mclaughlin and fish, 1994) . pancreatic duct obstruction or pancreatic disease can result in a rise in blood amylase values. rabbits can survive experimental ligation of the pancreatic duct (brewer and cruise, 1994) . the rabbit secretes a large amount of bile, approximately seven times as much as a dog on a weight basis (brewer and cruise, 1994) . the rabbit also differs from other species in the excretion of breakdown products of haemoglobin. the rabbit has low biliverdin reductase activity (fekete, 1989) and only 30% of biliverdin is converted to bilirubin. bilirubin values can be affected by fasting. glucose administration to rabbits lowers serum bilirubin concentrations by modifying hepatic conjugation and increasing biliary secretion (mclaughlin and fish, 1994) . in rabbits, biliary obstruction results in jaundice and raised serum bilirubin values. in young rabbits, hepatic coccidiosis is the most usual cause of jaundice. in older rabbits, bile duct obstruction from neoplasia is more likely. aflatoxicosis from the ingestion of mouldy feed can result in hepatic fibrosis and jaundice (krishna et al., 1991) . viral haemorrhagic disease (vhd) causes acute hepatic necrosis with elevated bilirubin concentrations in association with dramatic increases in ast and alt concentrations. vhd is invariably fatal, although some rabbits may survive long enough to develop jaundice before they die. there is little on information on haemolytic disease as a cause of jaundice in pet rabbits. haemolytic anaemia has been reported in association with lymphosarcoma in laboratory rabbits (weisbroth, 1994 ). in other species, alt is used as an indicator of hepatocellular damage, especially in dogs and cats. alt is also in found in other tissues such as muscle and red blood cells. an increase in alt signifies cell damage, although the degree of the increase does not correlate with the severity of hepatic disease and is not a prognostic indicator (willard et al., 1999) . in rabbits, liver alt activity is lower than in other species and there is less organ specificity (rosenthal, 1997) . alt is also present in cardiac muscle. the half-life of alt in the rabbit is approximately 5 h. in the dog the half-life is 45-60 hours (jenkins, 2000) . hepatic coccidiosis due to eimeria steidae is a cause of increased blood alt concentrations especially in conjunction with an increase in alkaline phosphatase, bilirubin and gamma gt. elevated alt values have been found in asymptomatic house rabbits and have been attributed to the effects of organic solvents in wood shavings used as litter material. other liver diseases such as neoplasia can cause a rise in alt but sometimes not until the condition is advanced (mclaughlin and fish, 1994) . low doses of aflatoxin caused a significant rise in alt concentrations in a group of laboratory rabbits (fekete and huszenicza, 1993) . hepatic lipidosis will elevate alt levels. in other species ast is widely distributed throughout the body. in particular, it is found in skeletal muscle, cardiac muscle, liver and erythrocytes. like alt, ast is an indicator of tissue damage. it is sometimes used as an indicator of liver disease, especially in horses in which alt is not liver specific. in rabbits, ast is found in liver, heart, skeletal muscle, kidney and pancreas with the highest activity in the liver and skeletal muscle (benson and paul-murphy, 1999) . physical exertion or tissue damage during blood collection can elevate results. raised ast levels can be found in association with liver disease. ggt is found in liver and kidney tissue. in other species, ggt is used as an indicator of hepatobiliary disease especially in horses and ruminants where it is associated with longterm liver damage. although ggt is found in high concentrations in renal tubular cells, kidney disease does not lead to elevated blood levels, probably because the enzyme is excreted in the urine (bush, 1991) . in the rabbit ggt is located predominantly in the renal epithelium with low activity in the liver. liver ggt is present primarily in bile duct epithelial cells and is therefore an indicator of hepatobiliary disease rather than hepatocellular damage (mclaughlin and fish, 1994) . in cases where there is renal tissue damage, urine ggt may be increased in addition to plasma concentrations. alkaline phosphatase consists of a group of several isoenzymes that hydrolyse phosphates at an alkaline ph (kerr, 1989) . it is one of the most widely distributed enzymes in the body. alkaline phosphatase is found particularly in bone, liver and intestinal wall. different isoenzymes are produced from each site. increases in plasma activity are usually due to the isoenzymes derived from liver and bone. higher concentrations are found in young animals with high osteoblastic activity. in rabbits, alkaline phosphatase is present in nearly all tissues. it is found in association with cell membranes and especially in intestinal epithelium, renal tubules, osteoblasts, liver and placenta. the rabbit has three ap isoenzymes -the intestinal form as well as two isoenzymes present in both liver and kidney (mclaughlin and fish, 1994) . there is a wide variation between laboratory reference ranges for ap values for rabbits. examples include: 4.1-16.2 iu/l (collins, 1988) ; 10-70 iu/l (gillett, 1994) ; 112-350 iu/l (harkness and wagner, 1995) . different analytical techniques could account for these variations. in a survey of blood parameters relating to calcium metabolism in pet rabbits, serum alkaline phosphatase values varied widely even in apparently healthy individuals (harcourt-brown and baker, 2001) . increased levels of alkaline phosphatase are seen in biliary obstruction, e.g. neoplasia or hepatic coccidiosis. experimental ligation of the common bile duct results in increased levels of alkaline phosphatase up to 600 iu/l (mclaughlin and fish, 1994) . enteric disease can also elevate alkaline phosphatase values (jenkins, 2000). bile acids are derived from cholesterol and are secreted into the intestine to aid fat digestion. from the gut, they are reabsorbed into the circulation and transported to the liver to be resecreted in the bile. impaired hepatic function results in increased concentrations of bile acids in peripheral blood. there are physiological variations in circulatory bile acid concentrations in association with the digestion of food and stimulation of the gall bladder to release bile into the small intestine. in most species, a fasting sample should have a low concentration of bile acids of less than 15 µmol/l (kerr, 1989) . impaired hepatic function can result in marked rises in fasting serum bile acid concentrations. in rabbits, the production of bile acids shows a circadian rhythm (fekete, 1989) . there is a problem in obtaining a fasting sample from rabbits due to the ingestion of caecotrophs. bile acids are not included in published reference ranges for rabbits at the present time. bile acid levels in excess of 100 µmol/l have been found in association with hepatic coccidiosis in comparison with levels that are generally less than 40 µmol/l (harcourt-brown, unpublished data). urea is a nitrogenous waste product that is formed in the liver as the end product of deamination of amino acids. it is transported in the blood to the kidney where it is excreted in the urine. in other species, high blood urea concentrations are indicative of impaired renal function that may be due to renal disease or poor perfusion due to circulatory disorders or cardiac disease. low blood urea levels can reflect hepatic dysfunction. in rabbits, many physiological factors influence the concentration of urea in the blood. dietary protein concentrations and quality, withholding food and natural diurnal rhythms can all affect blood urea concentrations. higher levels occur in the late evening (loeb and quimby, 1989) . the rabbit's urea metabolism is further complicated by urea utilization by caecal microflora during catabolism or during periods of dietary excess. therefore small fluctuations in serum urea concentrations are difficult to interpret. laboratory reference ranges apply to animals that are fed a standard diet and have usually been bled at a specific time of day. pet rabbits are subject to greater fluctuations in blood urea values due to the variation in diet and other factors, and can have values slightly higher than laboratory reference ranges. prerenal azotaemia associated with poor renal perfusion occurs during periods of dehydration. the rabbit has a limited capacity to concentrate urea and a greater volume of urine is required when urea load increases (brewer and cruise, 1994) . increased blood urea values were recorded in a study by licois et al. (1978) of young rabbits with diarrhoea experimentally induced with coccidiosis. the authors suggested that the blood urea values rose as a result of intense nitrogen catabolism during weight loss associated with the disease. water deprivation can lead to high blood urea values as high as 40 mmol/l in association with creatinine values in excess of 200 µmol/l (harcourt-brown, unpublished data). water deprivation can be due to a lack of available drinking water, caused either by an oversight by the owner or by a faulty mechanism on the drinking bottle. in rabbits, dehydration can cause urea and creatinine values that would signify renal disease in the dog and cat. high levels usually return to normal once the animal is rehydrated. therefore, urea and creatinine values should be checked before making an absolute diagnosis of renal failure. as in other species, elevated blood urea values in rabbits are associated with renal insufficiency. nephrolithiasis is a cause of kidney disease in the rabbit (see section 14.5). abdominal radiography is indicated in rabbits with raised urea and creatinine levels. encephalitozoon cuniculi can cause low-grade kidney disease in rabbits with mild elevations in blood urea. most cases are subclinical. e. cuniculi infection causes granulomatous lesions in the kidneys that become pitted and scarred with fibrotic areas. the parasite has been associated with chronic renal failure with blood urea values in the region of 152.7 mg/dl (25.45 mmol/l) and creatinine of 5.8 mg/dl (512.72 µmol/l) in a study by ewringmann and göbel (1999) . affected rabbits were anaemic with low haemoglobin and red cell counts and had elevated serum potassium concentrations. neoplasia, interstitial nephritis, nephrotoxicity also occur in rabbits and cause renal disease. low blood urea values in association with impaired hepatic function and the use of anabolic steroids have been described (benson and paul-murphy, 1999 ). creatinine is a nitrogenous waste product that, like urea, is transported in the blood to the kidney where it is excreted in the urine. creatinine is not the product of amino acid breakdown but of creatine which is a substance present in the muscle and is involved in high energy metabolism (kerr, 1989) . the slow catabolism of creatine results in a slow inflow of creatinine to the plasma at a rate which is directly proportional to the individual's muscle mass but is unaffected by any change in muscular activity or muscle damage. any changes in blood creatinine concentrations are due to changes in excretion and are a reflection of renal function. concentrations rise quickly at the outset of renal disease and decrease when an improvement of renal function takes place. creatinine deteriorates in plasma and readings from old samples (>24 h) cannot be relied upon. there is interference from a variety of other substances such as bilirubin (which decreases creatinine) or cephalosporins (which increase creatinine). the rabbit's complex digestive physiology and the compromised renal capability of correcting acid-base disorders make the rabbit a prime candidate for electrolyte imbalances (see section 1.6). dietary deficiency of electrolytes such as sodium and potassium is unlikely in the herbivorous diet of rabbits. instead, electrolyte problems are more likely to be associated with abnormal losses. although rabbits do not vomit, water and electrolyte absorption and secretion are affected by gastrointestinal disease. if facilities are available, electrolyte assays, especially potassium, can be a valuable part of the diagnostic workup for critically ill rabbits. in general, changes in sodium concentrations reflect the osmolality of extracellular fluid rather than the total body sodium content. increased blood sodium concentrations (hypernatraemia) can be the result of water deprivation or the loss of low sodium fluids. decreased sodium concentrations (hyponatraemia) may occur as a result of chronic renal failure when the kidney cannot concentrate urine and fast urine flow through the renal tubules prevents effective sodium/ potassium exchange. lipaemia or hyperproteinaemia can artefactually reduce affect sodium concentrations if certain laboratory methods are used. at the present time, there are few data available on clinical conditions that affect sodium concentrations in rabbits. about 95% of the total body potassium is intracellular, so measurement of extracellular potassium in blood samples does not give a true reflection of the potassium status of the patient. the balance between intracellular and extracellular potassium is regulated by aldosterone, insulin and catecholamines and is affected by blood ph. aldosterone stimulates renal excretion of potassium. insulin promotes the movement of potassium into cells. the effects of these hormones prevent large diet-induced changes in plasma potassium concentrations. potassium is an important ion in the maintenance of membrane potential. abnormally high or low potassium concentrations can have life-threatening consequences due to impaired electrical activity of cells. high blood potassium concentrations can result in cardiac arrest. alterations in blood potassium levels can be due to alterations in dietary intake and 154 textbook of rabbit medicine key points 6.3 • stress of handling can cause a marked elevation of blood glucose levels in rabbits. levels as high as 15 mmol/l can occur in association with handling. higher levels (> 20 mmol/l) may be seen in association with stressful or painful diseases such as intestinal obstruction • there is debate about the occurrence of diabetes mellitus in domestic rabbits. it has been induced in laboratory rabbits and a genetically susceptible laboratory strain has been bred • large individual variations in blood cholesterol and triglyceride values can occur in rabbits • jaundice is unusual in rabbits but may be seen in association with cholestasis in diseases such as hepatic coccidiosis or neoplasia • there is a wide variation between laboratory reference ranges for alkaline phosphatase values • blood urea and creatinine values can be high in cases of prerenal azotaemia in rabbits and do not always signify renal failure. causes include dehydration or water deprivation. excretion, or redistribution across cell membranes. to maintain electroneutrality, potassium ions shift from intracellular to extracellular fluid in exchange for hydrogen ions. in other species, hypoadrenocorticism (addison's disease) reduces the exchange of sodium and potassium ions across the cell membrane and results in increased serum potassium and decreased serum sodium concentrations. hyperkalaemia can be the result of impaired renal excretion of potassium due to kidney disease or from tissue trauma such as crushing injuries that release large amounts of potassium into the circulation. acidosis causes a redistribution of potassium across the cell membrane. artefactually high levels of potassium can result from leakage from red cells in haemolysed samples or those that have not been separated until several hours after the blood was taken. low blood potassium can cause muscular weakness and depression. hypokalaemia can be the result of dietary potassium deficiency or as a result of potassium loss from the gastrointestinal tract. diuresis or the use of potassium-free intravenous fluids also cause hypokalaemia. alkalosis can cause redistribution of potassium and sodium across the cell membrane and result in hypokalaemia. artefactually low potassium concentrations are uncommon although they can occur secondarily to hyperlipidaemia or hyperproteinaemia (willard et al., 1999) . blood collection through a catheter that contains residual potassium-free fluids can lead to erroneously low results. in rabbits, the effect of blood collection methods on plasma potassium levels has been investigated. discrepancies in results were found between blood collected from the ear and from the carotid artery when the blood was collected with a plastic catheter but not with a 21 g needle . general anaesthesia with pentobarbitone depressed plasma potassium values but sedation with chlorpromazine did not affect results. serum potassium concentrations were found to be higher than plasma and in venous rather than arterial blood . low serum potassium values have been found in unanaesthetized rabbits in conjunction with signs of muscular weakness (harcourt-brown, unpublished data). affected animals can still eat and drink but are unable to move. it is not known whether hypokalaemia is the cause of the muscular weakness. possible causes of hypokalaemia are discussed in section 12.6.1.1. further investigations of serum potassium concentrations of rabbits are required to know the clinical significance of measured values and the influence of various physiological states. in horses, blood potassium concentrations can fall to 2.0 mmol/l after prolonged exercise due to potassium loss in sweat and to 2.5 mmol/l while eating hay due to potassium loss in saliva. during moderate exercise concentrations can rise to 4.0 mmol/l due to potassium release from muscle cells (kerr, 1989) . similar physiological variations could occur in the rabbit. calcium is an essential element that is involved in many body systems. most of the body's calcium is stored in bone in conjunction with phosphate. calcium is an essential part of the structure of bones and teeth. it is an important cation in intracellular and extracellular fluid where it is required for muscle metabolism, enzyme activation, blood coagulation and osmoregulation. calcium is found in the blood in three forms: ionized, bound to other anions (especially phosphate) and bound to protein (especially albumin). because of the protein binding capacity of calcium, total serum calcium concentrations are proportional to albumin concentrations. ionized calcium is the physiologically active component of blood and is involved in the permeability of cell membranes. hypocalcaemia is a life-threatening condition. in many species, a high demand for calcium during late pregnancy and lactation can result in hypocalcaemic tetany. there are also some metabolic disorders that can result in alterations in serum calcium concentrations in other species. examples include renal, pancreatic and neoplastic diseases. the rabbit has a different calcium metabolism from other domestic species (see section 1.6.7). dietary calcium is readily absorbed from the intestine and total plasma values reflect dietary intake. total blood calcium levels are higher and can vary over a wider range than other species. an erroneous diagnosis of hypercalcaemia is often made because of the rabbit's high total serum calcium levels in comparison with other animals. parathyroid hormone (pth) regulates calcium metabolism in a similar manner to other animals, but a reduction in plasma pth level occurs at a higher plasma calcium concentration than in other species . the kidney plays an important role in calcium regulation and has a high fractional excretion for calcium when blood levels are high. calcium is excreted in the urine in which it forms calcium carbonate precipitate. some authors have suggested monitoring blood calcium concentrations as part of the protocol for treating 'sludgy urine'. however, high blood calcium levels are not the sole cause of urinary tract disease in rabbits (see section 14.4.1). there are differences between published reference ranges for total serum calcium values in rabbits. variations in dietary calcium intake could account for some of the discrepancies. the peak blood level that was obtained by increasing dietary calcium intake in laboratory rabbits was 5.42 mmol/l (21.7 mg/dl) in a study by chapin and smith (1967a) . experimental calcium restriction resulted in minimum serum concentrations of 3.22-3.5 mmol/l (13-15 mg/dl) before a rapid decline just before death in a separate study by chapin and smith (1967b) . a reference range of 3.2-3.7 mmol/l taken from eight laboratory reference sources has been made by goad et al. (1989) . values outside this range are encountered in otherwise healthy individuals and a range of 3.0-4.2 mmol/l is acceptable for pet rabbits on a varied diet. total blood calcium levels in rabbits are also affected by age and reproductive status. kamphues et al. (1986) found that increased calcium intake only resulted in higher total plasma calcium concentrations in adult rabbits and not young ones of 5-19 weeks. serum calcium concentrations in growing rabbits are fixed at a value of approximately 3.5 mmol/l (14 mg/dl) (kamphues et al., 1986 , gilsanz et al., 1991 . blood calcium levels decrease during pregnancy (assane et al., 1993) . due to the protein binding properties of calcium, albumin levels can also affect total calcium concentrations. albumin concentra-tions in pet rabbits are variable and appear to be affected by the manner in which they are kept (harcourt-brown and baker, 2001) . in dogs and man, total serum calcium values can be adjusted by using a mathematical formula that takes albumin concentration into account. (adjusted calcium concentration = measured serum total calcium concentration -serum albumin (g/dl) + 3.5). this formula is unreliable in cats (flanders et al., 1989) and has not been investigated in rabbits. at present, most published reference ranges for pet rabbits refer to total serum calcium concentrations. ideally, ionized calcium should be measured for an accurate assessment of calcium status but special sample handling and equipment is required that precludes its measurement in most practice situations. however, affordable equipment is now becoming available that can be used in the practice laboratory. measurements of ionized calcium have been made during experimental investigations using rabbits. warren et al. (1989) found a linear relationship between total serum calcium and ionized calcium values. a group of 29 non-pregnant female and male rabbits were found to have ionized serum calcium levels of 1.71 + 0.11 mmol/l. kamphues et al. (1986) reported ionized calcium values of 6.94 + 0.21 mg/dl (1.73 + 0.05 mmol/l) in adult rabbits on an average dietary calcium intake (0.85%). hypocalcaemic tetany has been reported in lactating does (barlet, 1980) . hypercalcaemia is seen in rabbits with chronic renal failure and impaired calcium excretion (see section 14.5.3.1). in a study by tvedegaard (1987) , experimentally induced chronic renal failure resulted in total serum calcium concentrations of 4.82 + 0.77 mmol/l. levels in excess of 4.25 mmol/l have been recorded in association with neoplasia (voelkel et al., 1978) . inorganic phosphate is involved in many enzyme systems and is important in carbohydrate and muscle metabolism as well as forming a major component of bone. phosphate is obtained from the diet. vitamin d and pth influence intestinal absorption of phosphorus in a similar manner to calcium. pth stimulates renal excretion of phosphate and renal conservation of calcium. the ph of intestinal contents and the presence of cations such as calcium and magnesium can affect the availability of dietary phosphate. abnormalities of phosphate metabolism are complex and interdependent with many other factors. blood phosphate values can be difficult to interpret and need to be examined alongside other parameters. in other species, physiological activities such as feeding and exercise reduce serum phosphorus concentrations (aitken and allen, 1994) . there are drug interactions that alter serum phosphorus values. examples include phosphate binders, anaesthetic agents, bicarbonate, parenteral glucose, anabolic steroids, diuretics and tetracyclines (willard et al., 1999) . phosphorus can shift between the intracellular and extracellular space in response to alterations in acid-base metabolism. in addition to these problems of interpretation, phosphate values are subject to artefactual error caused by poor sample handling. haemolysis releases phosphate from erythrocytes, which results in elevated values. in rabbits, there is little information about the clinical relevance of serum phosphate concentrations. rabbit blood clots easily and good quality non-haemolysed samples can be difficult to obtain. hyperphosphataemia can be due to impaired renal phosphorus excretion due to kidney disease. hypophosphataemia may result from dietary deficiency, impaired intestinal absorption or metabolic disorders. blood lead concentrations are given in different units depending on the source. the conversion factor for µg/dl to µmol/l is 0.0483. a study by roscoe et al. (1975) evaluated three diagnostic tests for lead toxicity in rabbits. whole blood lead concentrations of greater than 0.03 mg/dl (1.45 µmol/l) were considered a reliable indicator of lead ingestion. measurement of urinary delta-aminolevulinic acid (␦ala) was considered unreliable. erythrocytes from rabbits that were given lead fluoresced red when exposed to light rays of 320-400 nm. the fluorescent erythrocyte test (fet) was considered a convenient and reliable test for lead ingestion. swartout and gerken (1987) described two clinical cases of lead poisoning that had blood levels of 70 µg/dl (0.07 mg/dl) and 40 µg/dl (0.04 mg/dl). they gave a range of 2-27 µg/dl (0.002-0.027 mg/dl) as a normal range for laboratory rabbits. pth is released by the parathyroid gland in response to both a fall in blood calcium and low serum 1,25-(oh) 2 d 3 levels. it is responsible for the minute to minute regulation of calcium, due to its quick, short duration response. pth stimulates conversion of (25-oh-d) to the active from of vitamin d (1,25-(oh) 2 d 3 ) that, in turn, stimulates intestinal absorption of calcium. pth also stimulates osteoclastic resorption of bone to release calcium, phosphorous and magnesium into the circulation. pth stimulates renal conservation of calcium but not phosphorous, which results in an increase in blood calcium without an increase in phosphorous concentrations. in animals with disturbances in calcium metabolism that result in bone demineralization, pth levels are high, and pth can be used to investigate metabolic bone disease that is often nutritional in origin. dietary calcium deficiency, or a failure to absorb calcium are reasons for metabolic bone disease. failure to absorb calcium can be due to vitamin d deficiency or unavailability of calcium due to binding with substances such as oxalates, fats or phosphates in the gut. pth assays are available in commercial laboratories that specialize in endocrinological investigations. pth assays have been performed on pet rabbits as part of an investigation of the possibility of metabolic bone disease as a cause of poor tooth and bone quality and the development of dental disease in rabbits (harcourt-brown and baker, 2001) . sample handling is of paramount importance as the hormone is labile and haemolysis interferes with the assay. samples require separating and freezing immediately after collection and must be shipped in the frozen state to a laboratory. sufficient, non-haemolysed blood to harvest 1-2 ml of serum or plasma needs to be collected. as pth is responsible for the minute to minute regulation of blood calcium, values can vary over a wide range making interpretation of a single result difficult. diet, age, pregnancy, lactation, diurnal rhythms cause physiological variations in results. warren et al. (1989) reported pth values of 59.6 + 41.2 pg/ml in a group of 29 nonpregnant farm and laboratory rabbits and harcourt-brown and baker (2001) reported values of 40.3 + 10.7 pg/ml in a group of 12 pet rabbits kept outside under free-range conditions all year round (see figure 6 .1). values as high as 100-200 pg/ml have been recorded in baseline samples from laboratory rabbits . values in excess of 230 pg/ml have been found in pet rabbits, one of which was found to have a liver tumour on subsequent post-mortem examination (harcourt-brown, unpublished data). in the uk, serological tests are available for encephalitozoon cuniculi, toxoplasma gondii, myxomatosis, viral haemorrhagic disease and treponema cuniculi as part of the commercial health screening of laboratory rabbits. commercial laboratories may accept individual samples from pet rabbits for serological screening. it is advisable to consult a veterinary laboratory in the first instance. in the usa, serology and a pcr test are also available to detect pasteurella multocida infection (sanchez et al., 2000) but these tests are not available in the uk at the present time (september, 2000) . serological testing for encephalitozoon cuniculi antibodies can be useful in the differential diagnosis of neurological diseases such as vestibular syndrome or paraplegia (section 12.4) or uveitis (section 11.7.3.1). it is also indicated in animals with mild renal insufficiency (section 14.5.1). serological and histological tests from naturally infected rabbits have demonstrated the presence of antibodies before the organism can be seen in the kidney. lesions were not seen in the brain until at least 8 weeks after the first detectable antibodies, suggesting that serology is a sensitive procedure for early diagnosis (cox and gallichio, 1978) . therefore animals with clinical signs that are seronegative are unlikely to be suffering from encephalitozoonosis, although experimental infections with encephalitozoon cuniculi have shown the presence of granulomas in the brain of animals that had become seronegative (kunstyr et al., 1986) . conversely, asymptomatic rabbits can be seropositive. therefore serology can only be used as a guide in the diagnosis of encephalitozoon cuniculi infection. antibody titres can be helpful in distinguishing between recent and chronic infection. simultaneous detection of igm and igg suggest recent infection. urinanalysis is summarized in table 6 .4. urine collection is described in section 3.12.3. in common with other herbivorous species, rabbits excrete alkaline urine. urinary ph is approximately 8-8.2. rabbit urine is normally turbid due to the presence of calcium carbonate that can be seen as sediment in collected samples. the urine from anorexic, pregnant, lactating or young rabbits is often clear. the colour of normal urine can vary from pale yellow, to orange, brown or red, mimicking haematuria. plant porphyrin pigments are the cause of red coloured urine and can be distinguished from haematuria with urinanalysis dipstick tests. alternatively, a wood's lamp can be used, as urinary pigments fluoresce when exposed to ultraviolet light (benson and paul-murphy, 1999) . in addition to the presence of calcium carbonate crystals, oxalate or ammonium magnesium phosphate crystals can also be found in normal rabbit urine. the specific gravity of rabbit urine is difficult to evaluate accurately due to the presence of mineral deposits but is approximately 1.003-1.036. traces of glucose and protein can be present in normal rabbit urine. as in other species, rabbit urine can be spun and the sediment examined microscopically for the presence of crystals, red cells, inflammatory cells and bacteria. cultures can be taken to confirm bacterial infection and aid antibiotic selection. examination of urine sediment stained with gram stain can reveal encephalitozoon cuniculi spores that are oval, strongly gram-positive with a coiled filament inside (pye and cox, 1977; patton, 2000) . ketones may be detected in the urine of anorexic rabbits and is a poor prognostic sign as it is associated with the development of hepatic lipidosis. rabbits produce two types of faeces: hard dry pellets that are composed of compressed indigestible fibre and soft caecotrophs that are composed of a smooth paste rich in bacteria and other microorganisms. the first step in faeces examination is to determine which type of faeces has been collected. it is often samples of soft faeces or caecotrophs that are collected for examination because their consistency is abnormally loose or the owner has mistaken uningested caecotrophs for diarrhoea. microscopically the two types of faeces are completely different. hard faeces contain indigestible fragments of plant debris and little else. caecotrophic material contains a wide range of microorganisms including large gram-negative bacilli, bacteroides, large metachromic staining bacilli and many other bacteria including oval and fusiform rods. in some types of diarrhoea, a mixture of indigestible fragments of plant material can be seen alongside a range of typical caecal microorganisms. this signifies a failure of the proximal colon to separate the indigestible and digestible fractions of the diet. coccidial oocysts or eggs of the non-pathogenic oxyurid passalurus ambiguus can be found in both hard and soft faeces of infected rabbits. coccidial oocysts can be confused with a nonpathogenic saccharomyces, a budding sporogenous yeast that can be present in large numbers in rabbit faeces (see figure 6 .2). clostridium spiroforme is a large grampositive, semicircular or spiral-shaped bacterium that may be seen in faecal smears from diarrhoeic rabbits or those that have died from enterotoxaemia. centrifuging faecal material at 20 000 rpm for 15 minutes and gram staining the residue after the supernatant has been removed improves the chance of diagnosis (langan and o'rourke schaeffer, 2000) . although the presence of semicircular bacteria in the faeces or caecal material is suggestive of clostridial enterotoxaemia, it is not a reliable diagnostic criterion. clostridia species can be present in the normal caecal flora and proliferate after death. the demonstration of the toxin and anaerobic culture of the organism is required for positive diagnosis (carman and borriello, 1983) . clostridium piliforme, the causative organism of tyzzer's disease, is not detected in faeces. a pcr test is available in the usa for detection of this organism. escherichia coli is not a normal inhabitant of the rabbit gut flora 159 clinical pathology (a) coccidial oocysts and saccharomyces (ϫ 100). shows large numbers of coccidial oocysts interspersed with saccharomyces guttulatus. the faecal sample was from a 14-week-old, thin rabbit that was suffering from diarrhoea. faecal material was emulsified with water before being passed through a fine sieve to remove the coarse debris. the homogenate was then centrifuged and the supernatent discarded. the residue was mixed with saturated salt solution and centrifuged again. after the sample had been spun, more saturated salt solution was added to the test tube until a meniscus formed. a cover slip was placed over the meniscus and the sample left for approximately 30 minutes for the oocysts or worm eggs to float to the top of the tube. at the end of this period, the cover slip and surface film was removed from the test tube, placed on a microscope slide and examined under low power. (b) eimeria steidae (high power) (image kindly supplied by dr sheelagh lloyd, division of animal pathology, university of cambridge). several eimeria spp. affect rabbits and mixed infections occur. the species can be differentiated by the morphological characteristics of the oocysts. the most pathogenic species is eimeria steidae, which causes hepatic coccidiosis (see section 16.4.1). e. steidae invades the epithelial cells of the bile ducts and can cause severe liver damage. oocysts may be seen in faeces from infected rabbits or in smears of bile collected from rabbits during post mortem examination. the relative sizes of coccidial oocysts and saccharomyces guttulatus can be seen in (a,b). worm eggs are larger than coccidial oocysts. the most common helminth infestation of pet rabbits is passalurus ambiguus. adult worms or ova from p. ambiguus may be seen in rabbit faeces. the ova are ovoid, slightly flattened and asymmetrical with a cap at one end. (c) saccharomyces guttulatus (high power) (image kindly supplied by idexx laboratories, wetherby). saccharomyces guttulatus is a budding yeast that is commonly found in the faeces of rabbits, chinchillas and guinea pigs. it is not believed to be pathogenic. some texts call the yeast cyniclomyces guttulatus. although small numbers may be present in some animals. enteropathogenic strains can be found in association with diarrhoea in weanling rabbits. pathogenic salmonella spp. may be isolated although post-mortem material is usually available in these cases. infectious enteritis is rare in the individual pet rabbit. plucked hair samples may be examined visually for the presence of mites that are just visible with the naked eye. under good illumination, cheyletiella mites can be seen in scale and skin debris that has been brushed out of the coat. after a few minutes, individual mites can be seen moving into the warmth of the illuminating light. egg cases and cuticles from developmental stages of the fur mite leporacus gibbus (formerly known as listrophorus gibbus) may also be seen on visual examination of hair brushings. they remain attached to hair shafts after hatching or moulting and give the fur a characteristic 'salt and pepper appearance' in heavy infestations. visual evidence of l. gibbus is seen more easily on white or light coloured areas of fur, especially if it is wet. occasionally lice may be found. microscopic examination of acetate strips applied to the skin of alopecic areas can be used to detect cheyletiella parasitovorax. all stages of the life cycle of c. parasitovorax can be seen by this method. skin brushings can also be examined microscopically. fleas, flea dirt, cheyletiella parasitovorax and leporacus gibbus can be seen in skin brushings examined under low magnification. a trichogram is useful in differentiating between conditions that cause alopecia. for this technique a sample of hair is plucked from as close to skin as possible using forceps. the hair is placed on a microscope slide, taking care to ensure the hairs remain orientated in the same direction. a drop of mineral oil and a cover slip are applied before examining the shafts of hair under the microscope. abrupt, fragmented, distal ends of the hair shaft suggest barbering by a companion. egg cases, cuticles or adult mites, usually leporacus gibbus, may be seen attached to hair shafts. the presence of fungal spores in broken hair shafts plucked from a lesion is diagnostic of dermatophytosis. dermatophyte infection can be demonstrated by the presence of mycelia or ectothrix arthospores in potassium hydroxide preparations of macerated scale. asymptomatic infections can be detected by brushing the entire body with a sterile toothbrush and incubating the brushings at 25°c on dermasel agar (oxoid). plates that do not show fungal growth within 3 weeks can be considered negative (vangeel et al., 2000) . dermatophyte infections are usually due to trichophyton mentagrophytes that does not fluoresce under ultraviolet light. microsporum canis infections can also occur that are evident from the characteristic apple green spores that fluoresce under a wood's lamp. clinical pathology key points 6.5 • rabbit urine may be coloured red due to the presence of plant pigments. dipsticks can be used to differentiate haematuria from red urine • normal rabbit urine contains calcium carbonate sediment. small quantities of triple phosphate and oxalate crystals may be present • traces of glucose and protein can be present in normal rabbit urine • rabbits produce two types of faeces that differ in composition. the first step in faeces' examination is to determine which type of faeces has been collected • hard faeces are composed of compressed strands of indigestible fibre • soft faeces or caecotrophs are composed of a strong smelling paste rich in bacteria • some rabbits with diarrhoea excrete a mixture of hard and soft faeces signifying a failure of the proximal colon to separate the indigestible and digestible fractions of the diet • coccidial oocysts and ova from passalurus ambiguus may be seen in both types of faeces • a non-pathogenic yeast saccharomyces guttulatus may be seen in large numbers and can be mistaken for coccidial oocysts (see figure 6 .2) • clostridium spiroforme can sometimes be seen in large numbers on a gramstained smear of faeces collected from a rabbit suffering from enterotoxaemia. exudate from crusty lesions may be examined for the presence of mites. although psoroptes cuniculi normally inhabits the ear canal, the mite can be found in other areas of the body such as the perineal skin folds and can be seen on microscopic examination of the exudate from affected areas. skin scrapings may be required to demonstrate sarcoptic mange mites in scabies cases that are characterized by intense pruritus and crusty lesions. dark field microscopy can be used to look for treponema cuniculi organisms in crusty lesions suggestive of rabbit syphilis (section 16.5.9). the organism is a motile corkscrew-shaped spirochaete. lesions are found on the mucocutaneous junctions of the anus and genitalia or on the nose, lips and eyelids (see plate 10). the lesion is abraded with a sterile saline-soaked swab. serum from the lesion is then expressed on to a slide and covered with a cover slip before being examined immediately (digiacomo et al., 1984) . the slide can be placed in a moisturized chamber. the differential diagnosis of exudative skin lesions in rabbits can be difficult and histopathological examination of biopsy specimens may be required. cerebrospinal fluid can be collected from the cisterna magna of rabbits in a similar manner to other animals. some normal parameters are summarized in table 6 .5. depressed glucose concentrations (< 56 mg/dl) may be indicative of purulent inflammation (kusumi and plouffe, 1980) . key points 6.6 • cheyletiella parasitovorax, fur mites (leporacus gibbus), lice (haemodipsus ventricosus), fleas or flea dirt can be seen in skin brushings from affected animals • acetate tape strips can be used to detect all stages of the life cycle of cheyletiella parasitovorax • a trichogram can differentiate between dermatophytosis and barbering. mites, cuticles and egg cases may be seen attached to the hair shafts • psoroptes cuniculi, the rabbit ear mite, can sometimes be seen on microscopic examination of smears from skin lesions in other parts of the body such as the perineum • dark field microscopy may be used to detect treponema pallidum organisms. minerals and electrolytes part 1 influence of dietary calcium/phosphorous ratio on blood calcium, phosphate and magnesium during gestation in the rabbit. (article in french, english abstract) plasma calcium, inorganic phosphorus and magnesium levels in pregnant and lactating rabbits clinical pathology of the domestic rabbit comparative vertebrate endocrinology physiology interpretation of laboratory results for small animal clinicians laboratory diagnosis of clostridium spiroforme-mediated diarrhoea (iota enterotoxaemia) of rabbits glycosylated haemoglobin levels in a colony of spontaneously diabetic rabbits (abstract) the calcium tolerance of growing rabbits calcium requirement of growing rabbits common diseases and medical management of rodents and lagomorphs clinical chemistry serological and histological studies on adult rabbits with recent naturally acquired encephalitozoonosis leucocyte subpopulations in cerebrospinal fluid of normal rabbits (abstract) clinical course and treatment of venereal spirochaetosis in new zealand white rabbits viral diseases untersuchungen zur klinik und therapie der encephalitozoonose beim heimtierkaninchen (article in german recent findings and future perspectives of digestive physiology in rabbits: a review effects of t-2 toxin on ovarian activity and some metabolic variables of rabbits adjustment of total serum calcium concentration for binding to albumin and protein in cats: 291 cases (1986-1987) biochemical parameters of clinical significance in rabbits. ii. diurnal variations rabbit hematology selected drug dosages and clinical reference data effect of dietary calcium on bone density in growing rabbits total serum calcium concentrations in rabbits effect of ascaris suum infection on blood picture of rabbits in relation to the production of immunity (abstract) textbook of medical physiology parathyroid hormone, haematological and biochemical parameters in relation to dental disease and husbandry in pet rabbits clinical biochemistry and haematology clinical pathology values in pregnant and non-pregnant rabbits rabbit and ferret parasite testing vascular access ports for chronic serial infusion and blood sampling in new zealand white rabbits isolation of encephalitozoon cuniculi from urine samples evaluation of the effect of pentobarbitone anaesthesia on the plasma potassium concentration of the rabbit and the dog effect of intraosseous saline infusion on hematological parameters chronic plumbism in rabbits: a comparison of three diagnostic tests (abstract) interpretation of selected clinical pathology values in ferrets and rabbits ferret and rabbit endocrine disease spontaneous diabetes mellitus in the new zealand white rabbit rabbits. in an atlas of laboratory animal haematology pasteurellosis in rabbits. compendium on continuing education lead induced toxicosis in two domestic rabbits redistribution of lymphocytes after cortisol administration (abstract) the redistribution of lymphocytes during adrenaline infusion. an in vivo study with radiolabelled cells (abstract) alteration of sleep in rabbits by staphylococcus aureus infection haematological effects of exposure to three infective agents in rabbits physiological stabilization of rabbits after shipping arterial disease in chronic renal failure. an experimental study in the rabbit prevalence of dermatophytes in asymptomatic guinea pigs and rabbits changes in plasma parameters in rabbit does as a function of their physiological state and feed rationing acute phase reactants ceruloplasmin and haptaglobin and their relationship to plasma prostaglandins in rabbits bearing the vx2 carcinoma (abstract) regulation of calciotropic hormones in vivo in the new zealand white rabbit neoplastic diseases small animal clinical diagnosis by laboratory methods biochemical values of normal rabbit serum medicine of rabbits and rodents, 4th edn. williams and wilkins. hinton, m., jones d.r.e., festing m.f.w. (1982). haematological findings in healthy and diseased rabbits, a multivariate analysis. lab anim., 16, 123-129. hoefer, h.l. (2000). rabbit weisbroth s.h., flatt r.e., brewer n. (1984).biology and diseases of rabbits. in laboratory animal medicine. pp 207-237. academic press. krishna l., dawra r.k., vaid j., gupta v.k. (1991). an outbreak of aflatoxicosis in angora rabbits (abstract). key: cord-023168-cd7adns8 authors: thachil, jecko; owusu-ofori, shirley; bates, imelda title: haematological diseases in the tropics date: 2013-10-21 journal: manson's tropical infectious diseases doi: 10.1016/b978-0-7020-5101-2.00066-2 sha: doc_id: 23168 cord_uid: cd7adns8 nan haematological disorders are common in low-income countries. they make a substantial contribution to morbidity and mortality of individuals in these regions and have a negative impact on the growth and development of under-resourced nations. genetic red cells abnormalities are common in lowincome countries because they provide protection against malaria and they often co-exist with other causes of anaemia such as malnutrition and chronic illnesses. there is a close association between haematological abnormalities and infections which are a major cause of illness and death in these populations. morphological abnormalities of blood can often provide clues about the underlying diagnosis and blood film examination is particularly important where diagnostic facilities are limited. abnormal blood counts can manifest as various combinations of alterations of numbers of red cells, white cells or platelets. this section will outline some of the most common causes of abnormal blood counts likely to be encountered in clinical practice in low-income countries. anaemia is one of the most common causes of morbidity in the world and its impact is reflected in several of the health-related millennium development goals. although anaemia by itself is not a diagnosis, it suggests that there is an underlying disease state which needs to be recognized and treated. it is also a useful indicator of the general health of the population. the causes of anaemia may be identified systematically by considering the life cycle of the red cells (figure 65 .1). nutrients necessary for red cell production are absorbed from the gastrointestinal tract and carried through the portal vein to the liver and ultimately reach the bone marrow where erythropoiesis occurs. this process is regulated by erythropoietin, a hormone released from the kidneys mainly in response to hypoxia. mature • africa and asia have more than 85% of the world's anaemic populations and anaemia burden is highest among children and women of reproductive age. • the accurate diagnosis of anaemia has been neglected; clinical assessment of anaemia is unreliable unless the anaemia is severe. • in low-income countries, anaemia in an individual is often due to multiple interdependent factors. removing or treating a single factor may not resolve the anaemia. • early diagnosis of sickle cell disease and rapid access to a specialist centre for emergencies such as severe pain crises, strokes and acute chest syndrome, can help to prevent permanent long-term complications. • beta-thalassaemia major is fatal in the first few years of life unless regular blood transfusions are given; unless they are accompanied by iron chelation, these transfusions will eventually cause death due to irreversible organ damage from iron overload. • malarial anaemia is a particular problem for children and pregnant women and severe anaemia can be caused by p. falciparum and p. vivax. malarial anaemia can be reduced with chemoprophylaxis and intermittent treatment, and by anti-mosquito measures such as insecticidetreated bed nets and vector control. • anaemia occurs in 70% of hiv-infected patients and is an independent risk factor for death. prompt treatment of factors associated with anaemia, such as infections and poor nutrition, and commencement of antiretroviral treatment will reduce deaths. • blood shortages are common in tropical countries. to increase the availability of blood, transfusions should be prescribed in accordance with guidelines and efforts made to encourage blood donors to donate regularly as repeat donors are the safest type of donor. reaction' , characterized by circulating myelocytes and metamyelocytes, can be mistaken for leukaemia but, unlike leukaemia, there is an orderly maturation and proliferation of neutrophils. leukaemoid reactions have also been described in patients with tuberculosis, juvenile rheumatoid arthritis and dermatitis herpetiformis. 2, 3 decreased margination of neutrophils with egress of cells into the circulation can occur with exercise, adrenaline (epinephrine) injection, emotional stress and postoperatively or in response to drugs (e.g. steroids, β-agonists). other drugs, such as lithium and tetracycline, produce neutrophilia through increased production. neutrophilia is also a feature of bone marrow proliferation which occurs in myeloproliferative neoplasms, particularly chronic myeloid leukemia and myelofibrosis. teardrop cells and nucleated red blood cells are features of myelofibrosis on the blood film; basophilia and eosinophilia are common with chronic myeloid leukaemia. molecular testing for the jak-2 mutation or bcr-abl fusion gene can also help to differentiate between myeloproliferative neoplasms. rebound neutrophilia can occur following treatment of megaloblastic anaemia or after recovery from neutropenia induced by drugs. acute haemorrhage can cause neutrophilia, especially if bleeding occurs into the peritoneal cavity, pleural space, joints or adjacent to the dura. this is possibly due to the release of adrenaline and chemokines in response to local inflammation. the presence of neutrophilia can be useful in raising suspicions about the onset of complications in infections that are not primarily associated with neutrophilia. examples include meningitis in tuberculosis, orchitis in mumps, bowel perforation in typhoid fever and superadded bacterial infection in measles. the absence of neutrophilia can be helpful in differentiating typhoid and paratyphoid fever from pyogenic infections. neutropenia is defined as an absolute neutrophil count <1.5 × 10 9 /l. it is usually classified into severe (<0.5 × 10 9 /l), moderate red cells are released into the circulation from the bone marrow and percolate through the tissues and organs. anaemia can result from defects in any of these stages. inadequate production of red cells in the bone marrow can be due to lack of nutrients (e.g. iron, b 12 , folate, vitamin a, copper or zinc), abnormal haemoglobin synthesis (i.e. haemoglobinopathies) or ineffective erythropoeisis from myelodysplasia or infections. red cells can be lost from the body (e.g. gastrointestinal bleeding) or removed prematurely if they are abnormal or the spleen is enlarged (i.e. haemolysis). kidney disease can result in decreased erythropoietin. anaemia of chronic disease (or 'anaemia of inflammation') is due to an inadequate response to erythropoieitin or to increased cytokine-induced hepcidin release in inflammatory states which interferes with iron absorption or iron utilization. diagnostic algorithms to determine the cause of anaemia are usually based on a combination of the mean cell volume of the red cells, the reticulocyte count and blood film appearance (figures 65.2, 65.3 ). this approach is based on the availability of a haematology analyser and an experienced microscopist. several conditions which cause anaemia may co-exist in the same individual (e.g. intestinal parasites, malaria and sickle cell disease) and hence a thorough investigation is crucial to identify all potential causes of anaemia. neutrophils released from the marrow after maturation can either enter the 'circulating pool' or they can remain in the 'marginal pool' where they are loosely attached to the blood vessel wall. cells in the marginal pool are not sampled when blood is taken for a full blood count. 1 neutrophilia can therefore result from increased bone marrow synthesis and also from decreased margination which increases the circulating pool. there are many causes of neutrophilia (box 65.1) but the commonest is bacterial infection in which there is increased bone marrow production of neutrophils and release of neutrophil precursors into the peripheral blood. this 'leukaemoid (0.5-1.0 × 10 9 /l) or mild (1.0-1.5 × 10 9 /l). the propensity to develop infections is related to the degree and duration of neutropenia, with higher risk associated with counts below 0.5 × 10 9 /l. africans, african americans, yemenite jews, palestinians and saudi arabians generally have slightly lower neutrophil counts compared with other races. this is thought to be due to an increase in the bone marrow storage pool as ethnic neutropenia is associated with good neutrophil responses to infections. neutropenia can be due to impaired or ineffective (intramedullary death of neutrophil precursors despite normal bone marrow production) synthesis by the bone marrow (e.g. myelodysplasia, megaloblastic anaemia, treatment with phenytoin or methotrexate); a shift from the circulating pool to marginated pool (pseudoneutropenia) and increased peripheral destruction (e.g. secondary to antibodies against the neutrophils or increased reticulo-endothelial activity in sepsis or haemophagocytic syndrome) (box 65.2). increased consumption of neutrophils can result from increased attachment of cells to endothelium or other leukocytes in inflammatory states. neutropenia is often the result of a combination of several of these mechanisms. infants of hypertensive mothers may have moderate to severe neutropenia, which can last for several days. this is probably related to bone marrow suppression. moderate to severe neutropenia can also occur in newborn infants as a result of the transfer of maternal igg anti-neutrophil antibodies in a manner similar to rhesus haemolytic disease of the newborn. 4 although neutropenia has been described with typhoid fever, minimum neutrophil count seldom falls below 0.6 × 10 9 /l and the box neutropenia may not develop until after the first week of illness. infectious hepatitis and yellow fever can both cause neutropenia. overwhelming infections can lead to a failure of bone marrow production of neutrophils, especially in undernourished individuals and alcoholics. individuals with severe neutropenia can develop lifethreatening septicaemia, often from endogenous flora (e.g. oral cavity), and stringent measures should be taken to avoid situations which may predispose these individuals to infections. they may need prophylactic antimicrobials and should have rapid access to medical care. fungal infections are less common than bacterial infections in neutropenic individuals, and viral or parasitic infections rarely occur with isolated neutropenia. granulocyte colony stimulating factor (gcsf) injections can be helpful in raising the neutrophil count in patients with complicating infections since it stimulates the release of neutrophils from the marrow, but gcsf is only useful if there is some bone marrow reserve. patients with some congenital or immune forms of neutropenia can tolerate persistently low counts without any increase in the incidence of infections. monocytosis occurs in chronic infections and inflammatory conditions. protozoan infections such as typhus, trypanosomiasis and kala-azar may be associated with monocytosis. chronic and juvenile myelomonocytic leukaemias are malignant disorders in which monocytosis may be severe; acute monocytic leukaemias may present with mild to moderate monocytosis. monocytosis, and particularly a monocyte : lymphocyte ratio greater than 0.8-1.0, may indicate active progression of tuberculosis and an unfavourable prognosis. the normal ratio of 0.3 or less is restored when the healing process is complete. a decreased absolute monocyte count occurs in bone marrow failure states such as aplastic anaemia or after chemotherapy. low monocyte counts can occur with overwhelming sepsis and with splenomegaly. monocytopenia is a characteristic feature of hairy cell leukaemia and is considered to be a diagnostic hallmark of this disease. peripheral blood contains only around 2% of the total body lymphocyte population since these represent the cells present in the blood during their transit into secondary lymphoid organs. wide variations exist in lymphocyte counts between individuals especially in childhood. lymphocyte counts exhibit a diurnal pattern; peaking at night with a nadir in the morning. lymphocytosis is characteristic of infectious mononucleosis and many atypical and large lymphocytes can be seen in the peripheral blood film. these atypical cells can also occur in cytomegalovirus infection and infectious hepatitis. absolute lymphocytosis can occur with chronic infections such as brucellosis and in the recovery stages of tuberculosis. lymphocytosis is unusual in bacterial infections except in the case of pertussis. heavy smoking is also an often overlooked cause of lymphocytosis and is probably one of the commonest reasons for a mild to moderate increase in the lymphocyte count. malignant bone marrow disorders, predominantly acute lymphoblastic and chronic lymphocytic leukaemia and non-hodgkin's lymphomas, can cause lymphocytosis. these lymphocytes may have characteristic morphological changes identifiable in the blood film (e.g. smear cells with chronic lymphocytic leukaemia) and the correct diagnosis can be confirmed by immunophenotyping for specific combinations of cell markers. lymphopenia is due to decreased production, redistribution or increased rate of death of lymphocytes. decreased production usually results from cytotoxic drugs and radiotherapy, while increased lymphocyte death can occur in infections such as influenza and hiv. occasionally, an isolated low lymphocyte count in the context of an otherwise normal full blood count can be a clue to the diagnosis of hiv. this reflects the destruction of cd4+ t cells by the virus although an expansion of cd8+ t cells may raise the total lymphocyte count to normal levels. redistribution rather than depletion of total body lymphocyte numbers occurs with steroid treatment or with endogenous secretion of corticosteroids during acute illnesses due to the retention of lymphocytes in secondary lymphoid organs. eosinophilia eosinophils are involved in innate immunity and hypersensitivity. their number in the circulation is relatively small compared to other leukocytes because they predominantly reside in tissues such as the gut, skin and lungs which are entry points for allergens and infections. the commonest causes of eosinophilia are helminthic infections, atopy and allergic diseases, and adverse drug reactions. less common causes are classified under the umbrella term of hypereosinophilic syndromes (table 65 .1). since parasitic infections are likely to be the commonest cause of eosinophilia in the tropics and in returning travellers, an extensive search for such infections should be undertaken in patients with persistent eosinophilia; initial investigations should be determined by the patient's history of geographical exposure (figure 65.4) . [5] [6] [7] the absolute number of eosinophils in the peripheral blood may not correlate with their tissue distribution or with their potential to cause tissue damage from their granule release. this is because the degree of eosinophilia depends on the extent of tissue invasion and is therefore modest with tapeworms and roundworms resident in the bowel but much higher where invasion occurs, for example with, toxocara canis or filaria. schistosomiasis almost always causes eosinophilia. strongyloides stercoralis has the capacity to remain in the host for decades after initial infection and causes varying degrees of eosinophilia, with or without other symptoms. steroid treatment, which may be necessary in cases of eosinophilic tissue damage, can exacerbate clinical problems in patients with strongyloides infection so this parasitic infestation should be excluded before starting steroids for hypereosinophilia. mild to moderate eosinophilia is common in asthma although a very high count should prompt a search for churg-strauss syndrome or allergic bronchopulmonary aspergillosis. most drugs including penicillins can cause eosinophilia but the diagnosis can only be made by noting recovery when the drug is discontinued. eosinophilia can be a feature of hodgkin's lymphoma. it signifies a more favourable prognosis and may precede the original diagnosis of lymphoma or relapses. in immunocompromised patients, such as those with hiv infection, the finding of eosinophilia may be crucial since the success of antiretroviral treatment may depend on concomitant eradication of parasites. thrombocytopenia is often discovered incidentally in patients during full blood count estimation. a platelet count above 20-30 × 10 9 /l is usually not associated with any symptoms such as bleeding. if clinically evident haemorrhage does occur at counts above this level, other conditions such as coagulation defects, vascular problems or rarely platelet dysfunction should be suspected. although the prime role of platelets is in haemostasis, several other important roles have been recognized in recent years including wound repair, tissue healing, antimicrobicidal properties, lymphangiogenesis, tumour metastasization and maintenance of blood vessel integrity. congenital platelet disorders are often part of a syndrome. patients with wiskott-aldrich syndrome have small platelets in association with eczema and recurrent infections. other congenital platelet disorders, such as myh9-related disorders, can present with deafness or cataracts while skeletal deformities and oculocutaneous albinism are common in other syndromic presentations. blood film morphology can provide important clues about the causes of thrombocytopenia (figure 65 .5). fragmented red cells (schistocytes) increase the possibility of microangiopathic haemolytic anaemia, where an altered vessel wall and fibrin formation in the blood vessels shred the erythrocytes and consume platelets. thrombotic thrombocytopenia purpura, haemolytic uremic syndrome and disseminated intravascular coagulation can all present with thrombocytopenia. dysplastic red or white cells should raise the suspicion of myelodysplasia which can be confirmed by bone marrow examination and cytogenetic analysis. it is important to exclude in vitro platelet agglutination as a cause for apparent thrombocytopenia. this can be an anticoagulant (edta)-dependent phenomenon so a repeat sample should be examined using citrate anticoagulant. rarely, platelet satellitism where the platelets clump round the neutrophils, can cause artefactual thrombocytopenia. anaemia affects nearly two billion people globally with a much higher prevalence in developing countries compared with more wealthy nations (43% vs 9%). 8 the continents of africa (highest prevalence) and asia (greatest absolute burden) account for more than 85% of the anaemic population. anaemia burden is highest among children and women of reproductive age. anaemia contributes to more than 115 000 maternal deaths and 591 000 perinatal deaths globally per year. 9 who have defined anaemia according to various haemoglobin concentrations (table 65. 2) 10 but the appropriateness of these thresholds has been questioned because there are wide variations in haemoglobin concentration among people of different races. 11 the prevalence of anaemia can be a useful indicator of public health status of a nation because: • the prevalence of anaemia is objective and quantifiable • anaemia is a major complication of several infections, including malaria, hiv, tuberculosis, and the neglected tropical diseases, which are among the commonest problems in most tropical countries • the incidence of anaemia changes in a predictable fashion with alterations in disease burden • the prevalence of anaemia can be used to assess whether an intervention has reached the poorest communities. haemoglobin concentration of <90 g/l has been recommended for disease surveillance in high-prevalence countries where changes in haemoglobin are used for monitoring the impact of interventions. 11 anaemia in tropical countries (box 65.3) is often due to infections but chronic health problems, such as diabetes and chronic respiratory disease, and cancer and related complications are increasing as causes partly due to lifestyle changes. the body to compensate for the drop in haemoglobin content. for this reason the haemoglobin level can drop to extremely low levels before symptoms develop. anaemia presents with symptoms such as exertional breathlessness, palpitations and in some cases, syncopal attacks. patients with chronic anaemia may also have a multitude of nonspecific symptoms including poor concentration, decreased work performance and easy exhaustion (table 65. 3). a thorough history and clinical examination may provide clues about the cause of anaemia but further investigations are often necessary to confirm the diagnosis and guide treatment. however, in many resource-poor settings, access to routine biochemical and haematological testing is scarce, so much reliance is placed on clinical examination. the international guidelines for the integrated management of childhood illness recommend that a diagnosis of anaemia in sick children is based on the assessment of palmar pallor. for pregnant women, symptoms of fatigue and dyspnoea, combined with signs of conjunctival and palmar pallor, and increased respiratory rate suggest anaemia. however, making a diagnosis of anaemia based on clinical assessment alone is unreliable unless the anaemia is severe. 12 no specific anatomical site is particularly accurate for the prediction of anaemia 11 though sensitivity may be increased by using multiple sites. 12 most central laboratories in low-income countries have automated haematology analysers and several manual methods exist for assessment of haemoglobin concentration, which are suitable for rural areas where there is no mains electricity (e.g. haemoglobin colour scale; hemocue technique). [13] [14] [15] haemoglobin colour scale 16 principle. the colour of a finger-prick blood sample, soaked into special chromatography paper, is compared with the clinical symptoms and signs of anaemia vary and depend on the cause and the speed of onset. a rapid drop in haemoglobin is much more likely to cause symptoms of anaemia than chronic anaemia. slowly developing anaemia allows time for in many cases, there will be more than one of these conditions coexisting in the same individual. an adequate response to the treatment of anaemia requires management of all the contributory factors. intrauterine growth. thus, low birth weight and prematurity are both associated with iron depletion in the postnatal period. several interventions have been suggested to improve infantile iron deficiency, including: [17] [18] [19] • delayed cord clamping at delivery; the short delay of 2-3 minutes allows a small but important amount of blood to continue to flow to the foetus from the placenta • improvement of infant feeding practices • prevention and treatment of infectious diseases • interventions to prevent low birth weight, such as maternal nutritional supplementation, the control of infections and chronic health problems in pregnancy. anaemia in young children can be due to increased nutrition requirements during periods of rapid growth; these requirements may be up to 10 times higher per kilogram of body weight than that of an adult male. in addition, infant and toddler diets often lack bio-available iron. a case-control study of preschool children in malawi with severe anaemia (haemoglobin concentration, <50 g/l) identified bacteraemia, malaria, hookworm, hiv infections and deficiencies of vitamins a and b 12 as the commonest causes of anaemia. lack of folate and iron were uncommon. in low-income countries multiple interdependent causes of anaemia often operate in one individual so rectifying a single factor is unlikely to make a big impact on resolving anaemia. interventions which are useful in preventing anaemia in younger children include micronutrient supplementation (food fortification), de-worming, prevention and treatment of infectious diseases, school nutrition programmes and community-based nutrition promotion. who defines anaemia of pregnancy as a haemoglobin level less than 110 g/l, or haematocrit less than 33%, at any time during pregnancy. about one-fifth of maternal mortality is attributable to anaemia in pregnancy 20 and anaemia affects nearly half of all pregnant women worldwide. maternal anaemia is associated with many factors that might also be causally associated with mortality including poverty, infections and inadequate health-seeking behaviour. globally, the most important cause of anaemia in pregnancy is iron deficiency although hookworm, malaria, hiv infection, and deficiencies in folate and other micronutrients may contribute. pregnancy-associated complications, including septicaemia, pre-eclampsia and other obstetric problems can precipitate anaemia. 21 it is important to note that a diagnosis of iron deficiency in pregnancy which relies on ferritin measurements may be misleading because of high-quality digital examples of known haemoglobin concentration. the colours are represented in 20 g/l increments from 40 g/l to 140 g/l. this method is inexpensive, does not depend on skilled scientists, is durable in dusty, hot, dry and humid conditions and is probably better than clinical diagnosis for detecting mild and moderate degrees of anaemia. the disadvantages are that it requires specific chromatography paper and good natural light and it cannot detect changes in haemoglobin less than 10 g/l. this is a small battery-or mains-operated machine, which uses a drop of blood in a plastic cuvette to produce a direct read-out of haemoglobin in a few seconds. it is simple to use, produces accurate and consistent results to one decimal place and it has an in-built quality-checking mechanism. the hemocue hb-301 has been specifically designed for tropical conditions and operates in temperatures up to 50°c, in dusty and humid conditions. however the recurrent costs associated with disposable plastic cuvettes mean there is little opportunity for cost-saving with high-volume workloads. the iron status of an infant is directly proportional to its body mass and blood volume, both of which are reflections of the major cause of anaemia in most of these cases is iron deficiency. some of the effects have been described in individuals with iron deficiency without obvious features of anaemia. there are three intervention strategies recommended by who to prevent anaemia in pregnancy: 1. weekly iron and folic acid supplementation in women of reproductive age 22 2. daily iron and folic acid supplementation during pregnancy 3. presumptive treatment of hookworm infection during pregnancy in areas where hookworm infection is known to be endemic. 23 several factors may interfere with the efficacy of these interventions. under-participation in antenatal care may be common due to factors such as geographic distance, low motivation and poor interpersonal skills of health staff, poor quality of supplies and facilities, insufficient supply of iron and folic acid pills and womens' poor understanding about the daily use of supplements, especially in the face of common side effects. 23 in sub-saharan africa, the acute shortage and high turnover of health workers, and lack of time have also been shown to contribute to ineffective antenatal measures for reducing anaemia. 24 interestingly, a study from bangladesh showed that the first 20 pills (whether taken on a daily basis or less frequently) yielded most of the benefit for raising haemoglobin levels, which suggests that currently recommended doses may be higher than necessary to achieve optimal outcomes, except when anaemia is very severe. 25 the global burden of iron deficiency has been estimated from anaemia prevalence surveys, which include many different causes of anaemia so data may be unreliable as they are often not based on proven cases of iron deficiency. who estimates that globally 41% of women and 27% of pre-school children are affected by iron-deficiency anaemia, making it number 15 of selected risk factors for preventable death and disability worldwide. 20 iron deficiency begins in childhood, worsens during adolescence in girls and is aggravated during pregnancy. poor iron stores at birth, low iron content of breast milk and low dietary iron intake throughout infancy and childhood result in high prevalence of anaemia in childhood. anaemia is exacerbated by increased requirements during adolescence and iron loss from menstruation and is often compounded by the lack of adequate nutrition. the situation is worsened by pregnancy when iron requirement is approximately two times higher than in a nonpregnant state. iron deficiency should not be considered a diagnosis but a secondary outcome due to an underlying medical condition. although it may be a physiological response to rapid growth or increased requirements during childhood and pregnancy, it still requires treatment due to potential deleterious consequences. many of the chronic effects of iron deficiency may develop before the clinical and laboratory evidence of anaemia becomes apparent. the biochemical evidence for iron deficiency occurs in several steps. 26 initially, iron stores in the bone marrow are depleted as reflected by a decreased serum ferritin. the total iron-binding capacity then starts to rise, while the serum iron saturation begins to fall before microcytosis and a drop in haemoglobin ensue. there have been attempts to identify this early iron deficiency before anaemia develops in order to improve neurological and psychomotor functions in children and work performance in adults through widespread iron supplementation. however, there are concerns that iron excess may promote infections, especially in malarious areas. a range of laboratory investigations are usually necessary if iron deficiency is suspected (table 65 .4) 27-30 because once the diagnosis is confirmed, a search for the precise cause is necessary. a systematic approach to the investigation of iron deficiency (see below) is required based on an understanding of alterations in the iron absorption and transport cycle. • deficient intake (cow's milk has poor iron content and can cause gut blood loss in some infants) • rare defects of haem biosynthesis and iron transport. iron-deficient individuals may have no symptoms. excessive fatigue and other nonspecific signs of anaemia become more pronounced as anaemia develops. consumption of unusual 'foods' such as ice and paint or 'pica' only occurs in a minority of individuals. physical examination may reveal stomatitis, glossitis, koilonychia (spoon-shaped nails) and hair loss. oesophageal webs have been described in the plummer-vinson syndrome but are rare and may respond to iron replacement. since iron is important in neuromuscular development, several features of anaemia described in table 65 .3 may be related to iron deficiency. treatment of iron deficiency is with dietary modifications and oral or parenteral iron. 31 blood transfusions should be reserved for those with severe symptoms especially if the anaemia developed rapidly. haemoglobin levels alone should not be considered as a criterion for transfusion since very low levels (e.g. 10-30 g/l) may be appropriately treated with oral iron if anaemia has developed slowly. intravenous iron should only be considered in cases of poor response or intolerance to oral iron. cereals, poultry and green leafy vegetables, contain non-haem iron, which is often poorly absorbed. if dietary history suggests a deficiency, diet with foods rich in haem iron, such as red meat or liver should be recommended if social and religious customs and financial status allow, ideally with a drink containing vitamin c to facilitate iron absorption. absorption is also facilitated by taking supplements on an empty stomach although side effects of dyspepsia may not always allow this strategy. heavy tea intake can interfere with iron absorption and should be avoided. multivitamin or dietary supplements containing calcium, zinc or copper can also interfere with iron absorption. absorption may be delayed by tetracyclines, milk and soft drinks. since acid is necessary for iron absorption, antacids may account for a poor response to oral iron. iron is usually prescribed as a daily dose of 150-200 mg of elemental iron, commonly ferrous sulphate, 1 tablet three times daily. the dose in children is 3-6 mg/kg per day split into divided doses. assuming good compliance and absorption, this should result in an increase in haemoglobin within 4 weeks. once the haemoglobin is normalized, iron should be continued for 3 months to replenish the iron stores. the major problem with oral iron is upper gastrointestinal side-effects, which can be dose-dependent. a reduction in the dose or change in the formulation to gluconate or fumarate or even liquid forms, may be successful. liquid iron preparations may stain the teeth and should therefore be taken through a straw. oral iron can also cause constipation or diarrhoea which is not dose-dependent. parenteral iron is best given intravenously because intramuscular iron is painful and has been associated with development of soft tissue sarcomas. 32 high-molecular-weight iron dextran carries a low but significant risk of anaphylaxis, but the newer formulations including low-molecular-weight iron dextran, iron sucrose, ferumoxytol and iron gluconate have minimal risks. vitamin b 12 or cobalamin deficiency is a well-recognized cause of macrocytic anaemia (box 65.5). although some microorganisms can synthesize cobalamin, humans need to obtain this essential vitamin from foods, mainly meat, poultry and dairy products. vitamin b 12 is an essential co-factor in dna synthesis, serving as a co-factor in two key biochemical processes involving methylmalonic acid and homocysteine as precursors. consequently vitamin b 12 deficiency can interfere with dna synthesis. clinical manifestations include haematological (megaloblastic anaemia and pancytopenia), and neuropsychiatric disorders (paraesthesia, peripheral neuropathy, psychosis and dementia) and an increased risk of cardiovascular disease because of hyperhomocystinaemia. [32] [33] [34] a systematic approach to the investigation of vitamin b 12 deficiency requires an understanding of the absorption cycle. 35 ingested vitamin b 12 is broken down in the acidic environment of the stomach. it binds to r-binders in gastric secretions and saliva which stabilize the vitamin b 12 . in the alkaline environment of the small intestine, vitamin b 12 is released from r-binders to bind to intrinsic factor, synthesized in the gastric parietal cells. this vitamin b 12 -intrinsic factor complex is absorbed from the terminal ileum. recently, an alternative absorption system independent of intrinsic factor and the terminal ileum has been postulated which provides a rationale for mean cell volume useful as a diagnostic clue but not confirmatory can also be low in thalassaemia, sideroblastic anaemia and rarely lead poisoning can be falsely normal in the presence of iron deficiency in older people or with coexistent megaloblastic anaemia anaemia of chronic disease can occasionally cause microcytosis serum ferritin the most useful laboratory measure of iron status low value is diagnostic in the presence of anaemia very high values (>100 µg/l) usually exclude iron deficiency' being an acute-phase protein, it increases in inflammatory conditions, and certain malignancies, making it unreliable also increased in tissue damage especially of the liver levels are falsely decreased in vitamin c deficiency and hypothyroidism erythrocyte zinc protoporphyrin an intermediate in haem biosynthesis and elevated concentrations indicate interrupted haem synthesis due to iron deficiency when zinc is incorporated in place of iron can be measured on a drop of blood with a portable haematofluorometer small sample size makes it very useful as a screening test in field surveys, particularly in children, and pregnant women where inflammatory states may not co-exist red cells should be washed before measurement (serum bilirubin and fluorescent compounds like some drugs can give falsely high values) although not often done lead poisoning can give falsely high values rarely acute myeloid leukaemia and sideroblastic anaemia give slightly high values useful in that it is not increased in thalassaemias who recommends normal level >70 µmol/mol haem iron studies serum iron concentration represents the iron entering and leaving the circulation. its range varies widely with age, circadian rhythm, infections and iron ingestion total iron binding capacity measures iron bound to transferrin. raised levels are suggestive of iron deficiency transferrin saturation is the ratio of serum iron and the tibc expressed as a percentage -it is probably more useful in detecting iron overload rather than low levels. sensitive indicator that falls within days of onset of iron-deficiency reduced levels shown to be predictor of iron deficiency especially in the setting of renal insufficiency false normal values can occur when mcv is increased or in thalassaemia serum transferrin receptor it is not increased in inflammatory conditions may be upregulated by increased erythropoiesis (haemolytic diseases) giving falsely high values -serum transferrin receptor to ferritin ratio has been suggested in these cases bone marrow examination with special iron staining (perl's) absence of stainable iron in a sample that contains particles can establish the diagnosis without other laboratory tests a simultaneous control specimen containing stainable iron should also be assessed useful in differentiating from anaemia of chronic disorders or α-thalassaemia or milder forms of thalassaemia can help in identifying the sideroblastic anaemias (ring sideroblasts with perls stain), and some forms of congenital dyserythropoietic anaemia which can also cause microcytosis. an improvement in haemoglobin and clinical symptoms with iron replacement is probably the simplest way to diagnose iron deficiency. peripheral smear may help by demonstrating pencil cells, anisopoikilocytosis and high platelet number in cases of blood loss. the treatment of vitamin b 12 deficiency can be by the oral or parenteral route. 37 increasing evidence suggests that oral supplementation may be adequate even in the presence of malabsorption or pernicious anaemia. 38, 39 the recommended initial oral replacement dosage is 1-2 mg but higher doses may be needed for malabsorption or pernicious anaemia. 40 for patients with severe anaemia and/or neurological disease, daily or alternate day intramuscular injections should be initiated for the first 2-4 weeks before reverting to the maintenance threemonthly dose. reticulocytosis is an early marker of response to treatment and is noticeable within 1-2 weeks. folic acid deficiency causes similar haematological manifestations to vitamin b 12 deficiency though neuropsychiatric manifestations are less common. the ability of nerve tissue to concentrate folate to levels five times greater than those in the plasma has been suggested as a reason for the absence of neuropathy in folate deficiency. folic acid deficiency is associated with fetal neural tube defects, and possibly with an increase in atherosclerosis and arteriovenous thrombosis, dementia and colonic cancer. dietary folic acid is present in the form of polyglutamates, which are converted to folate monoglutamates by the enzyme folate conjugase in the intestinal brush border, prior to absorption. the monoglutamates function as a carbon transporter and are essential for dna biosynthesis. folate is found in green vegetables and fruits and deficiency can result from decreased intake, impaired absorption and increased utilization, although the commonest cause is dietary insufficiency. 41 in some wealthy countries, cereals have been fortified with folic acid to successfully prevent vitamin deficiency. however folate deficiency continues to be a problem in less wealthy countries and particularly among children and pregnant women. 42, 43 exclusive feeding of goat's milk to infants can lead to folate deficiency. other causes include alcoholism, excessive cooking of vegetables, and malabsorption (e.g. abnormalities of the small bowel). increased demand for folic acid occurs in pregnancy because the growing foetus has a high avidity for folate. for this reason, folate supplementation has been widely recognized as an essential part of routine antenatal care to reduce the risks of neural tube defects. high folate utilization also occurs in haemolytic anaemias such as sickle cell disease due to high red cell turnover and exfoliative dermatitis. several drugs, including sulfasalazine, trimethoprim, methotrexate, pyrimethamine and phenytoin, can also interfere with folate metabolism. folate-deficient individuals develop a macrocytic anaemia with peripheral blood and bone marrow findings similar to that found in vitamin b 12 deficiency. 32 diagnosis of folate deficiency is confirmed by the presence of low serum folate. red cell folate levels decrease more slowly than serum levels during the 120-day turnover of the red cells. red cell folate levels may be a better indicator of tissue folate levels than serum folate, although red cell folate can be more expensive and falsely low in vitamin b 12 deficiency. 45, 44 treatment of folate deficiency is with oral folate (5 mg daily) which is sufficient even in malabsorptive states. it is crucial that any co-existing vitamin b 12 deficiency is ruled out before initiating folic acid therapy, otherwise the neurological manifestations of b 12 deficiency may deteriorate rapidly. it is also important increasingly popular oral replacement therapies. once absorbed, vitamin b 12 binds to transcobalamin ii to be transported around the body. the diagnosis of vitamin b 12 deficiency is based on the measurement of serum vitamin levels in a patient with clinical evidence of deficiency. a note of caution is that folic acid deficiency can cause falsely low serum vitamin b 12 levels. diagnostic clues for vitamin b 12 deficiency include marked macrocytosis (often >130 fl), neutrophil nuclear hypersegmentation and oval macrocytes in the peripheral blood film. blood tests may demonstrate increased lactate dehydrogenase and low haptoglobin levels due to haemolysis within the bone marrow. the cause of the macrocytosis can be confirmed by bone marrow examination which reveals a megaloblastic picture. although macrocytic anaemia is a typical feature of vitamin b 12 deficiency, it can be absent in older individuals who may only have neuropsychiatric features. measurements of methylmalonic acid and homocysteine levels, two markers which are very sensitive for detecting b 12 deficiency, have shown that vitamin b 12 deficiency can occur with normal haemoglobin levels and without macrocytosis. 36 pernicious anaemia is probably the commonest cause of vitamin b 12 deficiency. the presence of parietal cell or intrinsic factor antibodies supports a diagnosis of pernicious anaemia. [33] [34] [35] [36] schilling tests are rarely performed because of the unavailability of the radio-labelled vitamin b 12 and the difficulty in interpreting the results in the presence of renal insufficiency. • pernicious anaemia (begins after 40), increased risk of gastric carcinoma and carcinoid tumours • rare congenital disorders, e.g. imerslund-grasbeck syndrome. the neglected tropical diseases are a group of infections which are endemic in developing countries. several of these neglected tropical diseases cause anaemia and many can be managed using inexpensive interventions to treat the underlying parasitic infections. 14 the mechanisms of anaemia in these conditions are predominantly blood loss from the gastrointestinal or genitourinary tracts but also poor nutrition, bone marrow suppression, inflammation, hypersplenism and haemolysis. 58 anaemia is a common consequence of infections with soiltransmitted helminths or schistosoma with a strong correlation between haemoglobin level and worm load or faecal egg count. even mild infections can lead to anaemia. 59 polyparasitism (i.e. infection with several parasites simultaneously) can be responsible for unresponsiveness of the anaemia to eradication of one organism. 60 treatment of communities at high risk of soiltransmitted helminths improves growth and iron stores in children and reduces anaemia in pregnant women. 61 the treatment of anaemia due to neglected tropical diseases depends on eradication of the parasite with drugs such as albendazole and praziquantel though anaemia resolution may be less successful if it is due to trichuriasis. 62-64 the addition of iron to anthelmintic treatment has met with variable success rates probably because there is associated anaemia related to inflammation. however it is still generally recommended that iron supplementation should be included with anthelmintic therapy in treatment programmes for neglected tropical diseases. [65] [66] [67] introduction haemoglobin s (hbs) has a prevalence of 25-30% in many parts of africa and also some areas in the middle east ( figure 65 .6). hbs tends to be common among ethnic groups that have traditionally had high exposure to plasmodium falciparum malaria. in sub-saharan africa approximately 230 000 infants are born with sickle cell disease each year, mostly with hbss. sickle cell disease (scd) is an autosomal recessive disorder characterized by production of an abnormal haemoglobin, sickle haemoglobin. sickle haemoglobin (hbs) arises from a mutation in codon 6 of the β-globin gene resulting in replacement of the normal glutamic acid residue by a valine. 68 scd is most commonly caused by the co-inheritance of two sickle cell genes (homozygous hb ss disease) but patients who are heterozygous for hbs and for another haemoglobin mutation such as hbc (haemoglobin sc disease) or β-thalassaemia (sβ 0 and sβ + ) can also present with features of scd. 69 ss disease and sβ 0 disease are more severe than sc disease and sβ + disease (box 65.6). 70 scd can affect multiple organs and its clinical course is punctuated by episodes of acute illness on a background of progressive organ damage, especially of the central nervous system and the lungs. 70 the first description of scd was in 1910 in an anaemic grenadian dental student 71 and over the next 30 years it was that the underlying cause of folate deficiency is identified and treated. vitamin a is important in erythropoiesis, iron metabolism (enhances iron absorption and its release from stores to the bone marrow) and for decreasing the risk of infections. 45 vitamin a deficiency is a major public health problem in lowincome countries, with an estimated 200 million preschool children affected. 47 pregnant women and women of childbearing age also constitute high-risk groups for vitamin a deficiency. 46 vitamin a given to thai school children with conjunctival xerosis led to a significant increase in haemoglobin level 47 and in anaemic school children in tanzania, vitamin a supplementation produced a marked increase in haemoglobin which was enhanced by co-administration of iron. 48 vitamin a can also improve anaemia in pregnant women, depending on the local prevalence of deficiency [49] [50] [51] [52] though the response may be suboptimal in pregnant women infected with hiv. 53 copper is a trace element necessary for normal haematopoiesis and myelopoiesis. anaemia in copper deficiency is due to decreased activity of the copper-dependent enzymes, hephaestin, ceruloplasmin and cytochrome c oxidase. these are important in ferrous-ferric iron conversions and their decrease leads to abnormalities in iron absorption and its incorporation into the haemoglobin molecule. acquired copper deficiency occurs with malnutrition and gastrointestinal malabsorption syndromes. coeliac disease, cystic fibrosis and individuals who have had gastrectomy or surgery resulting in 'short bowel' are also at risk. copper deficiency has also been described in persons ingesting excessive amounts of zinc-containing supplements and those who have swallowed zinc-containing coins. 54, 55 anaemia related to copper deficiency is normocytic or macrocytic and can be associated with neutropenia; thrombocytopenia is rare. bone marrow findings are characteristic with cytoplasmic vacuolization of both erythroid and myeloid precursor cells with ringed sideroblasts and an unusual finding of iron granules in plasma cells. these findings may be misdiagnosed as myelodysplastic neoplasm. measurement of serum copper levels is helpful in confirming the diagnosis although the test is fairly insensitive. since almost complete haematological recovery can occur with copper replacement, this may be a useful diagnostic test. oral copper supplements can be started with 8 mg of elemental copper a day slowly decreasing over the next few weeks to 2 mg until a good response is noted. although low zinc levels do not cause anaemia they have been linked to growth retardation, heightened susceptibility to infection and male hypogonadism in relation to sickle cell disease. 56 zinc deficiency has been described in nearly half of children and 70% of adults with sickle cell disease possibly due to increased loss of zinc in the urine and high cell turnover with decreased dietary intake. 57 in contrast zinc excess can cause anaemia through interference with copper absorption by sequestering it in the gut lumen. for this reason, zinc compounds have been used to treat wilson's disease which is characterized by copper excess. however repeated sickling and unsickling eventually causes irreversible changes, 77 so early management to avoid repeated crises is important to prevent disease progression. polymerization, and therefore the clinical features of scd, are influenced by three main factors 78 ; hypoxia, the intracellular hbs concentration and the co-existence of other genetic haemoglobin abnormalities (e.g. α-thalassaemia or hereditary persistence of fetal haemoglobin-haemoglobin f). 79 sickled red cells lead to vaso-occlusion and haemolysis due to the entrapment of sickled erythrocytes in the microvasculature and upregulation of adhesion receptors. 76, 80, 81 white blood cells contribute to this process by providing an inflammatory discovered hypoxia led to red cell sickling 72 scd arises from the tendency of hbs to polymerize in hypoxic states. this phenomenon occurs where there is deoxygenation and is due to the binding between β1 and β2 chains of two haemoglobin molecules, a property unique to haemoglobin variants that have the glu-6-val substitution. 74 the polymerized haemoglobin fills the erythrocyte and deforms its architecture and flexibility to form a sickle shape. this alteration in the structure promotes cellular dehydration, 70, 75, 76 upon reoxygenation, the polymers dissolve thus reversing the sickling process. 90 exposure to cold, fever, menstruation, alcohol intake and dehydration can precipitate pain crises. unlike acute pain crises, chronic pain in scd usually has an identifiable basis such as femoral head necrosis, osteoarthritis or chronic skin ulcers. sickle erythrocytes have an average life span of 17 days and anaemia can be due to several causes (box 65.8). red cell haemolysis causes anaemia and gall stones and can cause fatigue out of proportion to the anaemia. 91, 92 there are suggestions that patients with low haemoglobin concentrations and high haemolytic rates are more likely to develop vascular problems compared with those with higher haemoglobin concentrations. splenic sequestration with a sudden rapid drop in haemoglobin occurs in those who have not yet developed autosplenectomy so it can occur in young children with hbss and adults with hbsc disease or sickle cell-β + -thalassaemia. treatment may require blood transfusion and in rare cases, sequestration can be fatal. splenectomy may be needed for recurrent severe sequestration. parents can be taught to feel their infant's abdomen for an enlarging spleen and report to hospital if there is a sudden increase in spleen size. red cell aplasia can develop due to secondary parvovirus infection which has a predilection for erythroid progenitors. alloimmunization is common in scd patients who have had frequent transfusions so, if possible, extended red cell phenotyping should be undertaken. hyperhaemolytic crisis is suspected when there is sudden exacerbation of anaemia with increased reticulocytosis and bilirubin level. infectious complications of scd are a major cause of morbidity and mortality, even with adequate vaccination and prophylactic antibiotic regimens. this propensity to infection is related to impaired splenic function although tissue ischaemia, especially in the lungs and renal system, can contribute. hyposplenism is demonstrable in the peripheral blood film by the presence of howell-jolly bodies. most children with scd have undergone autosplenectomy by the age of 5 years and therefore have increased risk of infection from encapsulated microorganisms. 93 typical infectious complications include pneumococcal sepsis, neisseria meningitis, osteomyelitis caused by salmonella species, urinary tract infections and pyelonephritis due to escherichia coli. anatomical abnormalities such as renal papillary necrosis can predispose to urinary complications which may require long-term antibiotics. acute chest syndrome (acs) is defined as a new pulmonary infiltrate on the chest radiograph combined with one or more environment. activation of platelets and the coagulation system also contribute to the vaso-occlusion in scd. [82] [83] [84] [85] [86] infants with scd are protected during the first few months of life by the high levels of haemoglobin f in the red cells. anaemia usually develops by 3 months. at all ages, chronic haemolysis of abnormal red cells means that scd is associated with steady state haemoglobin levels of 60-80 g/l. although any organ can be affected by scd and complications can occur at any age, certain features tend to predominate in different age groups (box 65.7). pain is the hallmark of scd 87 and four different patterns of pain have been described with scd each with different underlying mechanisms: 88 • vaso-occlusive (acute and intermittent) • pain from bone and tissue necrosis (chronic) • neuroplasticity (chronic, neuropathic) -functional brain changes • opioid-induced hyperalgesia (acute or chronic). painful crises often start in young children as dactylitis or handfoot syndrome, in which painful swelling of the hands and feet results from the inflammation of metacarpal and metatarsal periosteum. these crises are the result of vaso-occlusion of the bone marrow causing bone infarction and release of mediators that activate pain receptors. 82 the number, severity and frequency of painful episodes vary widely in individuals. half may never have any episodes whereas about 3% may need hospital admission up to 6 times a year. 89 more than three pain episodes requiring hospitalization per year is associated with increased mortality among patients over 20 years old. in under-resourced settings, hospital visits underestimate the frequency of pain box manifestations such as fever, cough, sputum production, tachypnoea, dyspnoea or new-onset hypoxia. 94 acs is the most common cause of death in scd patients and a frequent cause of hospitalization, second only to painful crisis. mortality in patients with acs in a wealthy country setting is 1% in children and 4.3% in adults. 95 the peak incidence for acs is 2-4 years of age and gradually declines to 8.8 per 100 patient-years in subjects older than 20 years. 96, 97 fever and cough are more common in children with acs and chest pain and dyspnoea are more common in adults. 96 acs is often preceded by febrile pulmonary infection in children and by vaso-occlusive pain crisis and lung infarction in adults. it is important to note that although tachypnoea, wheezing and features of chest infection may be identified, a third of the patients may have a normal physical examination. more than one-third of patients with acs are hypoxaemic (oxygen saturation <90%). 98 chest radiography is essential although infiltrates may lag behind clinical symptoms by up to 3 days. repeat chest x-rays are recommended if there is a strong clinical suspicion of acs. bilateral infiltrates or involvement of multiple lobes may predict a poorer prognosis. risk factors for acs (box 65.9) include fat embolus which can be confirmed by finding stainable fat in pulmonary macrophages. 99 chronic complications such as pulmonary hypertension occur in as many as 60% of patients and do not appear to be associated with prior episodes of acs. high serum phospholipase a2, and the surrogate marker c-reactive protein, have been noted in patients admitted with vaso-occlusive crisis 24-48 hours before the development of acs. 100, 101 stroke neurological complications occur in at least 25% of patients with scd and scd is one of the most common causes of stroke in children. 102, 103 in scd, the risk of having a first stroke is 11% by the age of 20, 15% by age 30 years and 24% by age 45 years. both thrombotic and haemorrhagic strokes occur, although the former is more common in children and those over 30 years of age, whereas the latter is more common between the ages of 20 and 30 years. 108 this age-specific pattern may be related to the higher cerebral flow rates in early childhood. although the prevalence of clinically overt stroke is of the order of 11%, clinically silent infarction, detectable by magnetic resonance scans, affect nearly double this number by the age of 20. silent infarcts are associated with cognitive impairment and the majority of these children require lifelong specialist care. 104 cerebral thrombosis, which accounts for 70-80% of all strokes in scd, results from large-vessel occlusion whereas silent infarcts are the result of microvascular occlusion or thrombosis or hypoxia secondary to large-vessel disease. in a third of scd patients, major-vessel stenosis is accompanied by collateral vessels that appear as 'puffs of smoke' (moyamoya) on angiography. risk factors for ischaemic strokes in scd include increased cerebral blood flow velocity, previous silent infarcts, nocturnal hypoxaemia, severe anaemia, acute chest syndrome and elevated systolic blood pressure. an elevated leukocyte count is a risk factor for haemorrhagic stroke. [105] [106] [107] [108] diagnosis often the family history and clinical findings clearly point towards a diagnosis of scd and during an acute crisis, abundant sickled red cells can be seen on a blood film. white cell counts are higher than normal in scd disease, particularly in patients under age 10 years. the presence of sickle haemoglobin in different sickle syndromes (e.g. hbas, hbss, hbsc) ( table 65 .5) can be confirmed by a simple sickle slide or solubility test. haemoglobin electrophoresis will distinguish between many of these variants but high-performance liquid chromatography and iso-electric focusing are preferred for a definitive diagnosis. haemoglobin mass spectrometry and dna analysis are being increasingly used. antenatal screening is available to women in some countries to help to identify couples who are at risk of having a baby with scd. community acceptance of reproductive genetic services however depends on the effectiveness of education and counselling. the use of prophylactic penicillin and the provision of comprehensive medical care during the first 5 years of life have reduced mortality related to scd from 25% to less than 3%. 109 management (box 65.10) individuals with scd are best managed by a multidisciplinary team as they may require a variety of specialist inputs including haematology, ophthalmology, nephrology, obstetrics, orthopaedics and physiotherapy. the cornerstones of scd therapy are disease modification and prompt and effective management of crises. severe pain crises generally require intravenous fluids and adequate, often opiate, analgesia (box 65.11), while disease modification is based on interventions to increase hbf levels. in steady state it is usual practice to give sickle cell patients folate supplements (1-5 mg/day) because their high rates of haemopoiesis put them at risk of deficiency. scd is associated with functional asplenia so patients should also receive prophylactic oral penicillin (250 mg twice a day) and vaccinations against encapsulated organisms. hydroxycarbamide is the main agent used to increase hbf (box 65.12) and is associated with significant reductions in acute pain crises, hospitalization rate, time to first and second pain crises, episodes of acute chest syndrome, and the need for transfusions and the number of units transfused. 110 other beneficial effects of hydroxycarbamide, which are independent of the increase in hbf, include reduced neutrophil count, increased cellular water content, decreased hbs concentration, changing expression of adhesion molecules and nitric oxide generation. 111 hydroxycarbamide may also be an alternative to frequent blood transfusions for the prevention of recurrent stroke in children as it can lower transcranial doppler velocities. 112, 113 under-use of this cheap, effective drug is related to concerns about leukaemogenicity but this has not been shown to be a problem when used for a non-malignant condition like scd. the two main approaches to transfusion 74 in scd are simple top-up transfusion and exchange transfusion. target haemoglobin level in scd therapy is 100 g/l or a haematocrit of 30%; higher target levels are associated with hyperviscosity and box 65.10 management of complications of sickle cell disease • inability to maximally concentrate urine (hyposthenuria) in response to water deprivation is an early finding • renal tubular acidosis • increased urinary tract infections • glomerular hyperfiltration, increased creatinine secretion, and a very low serum creatinine are characteristic of young patients with sickle cell anaemia, so renal dysfunction can be present even with normal serum creatinine values • microalbuminuria is common in childhood and up to 20% of adults develop nephrotic-range protein loss • gross haematuria can develop due to microthrombin in renal vessels, renal medullary carcinoma, and nocturnal enuresis • treatment is based on the early use of hydroxycarbamide and angiotensin-converting enzyme inhibitors in children with clinically significant albuminuria. • noted in up to 60% of scd cases • no relationship to acute chest syndrome (different pathophysiology) • mortality risk with even mild pulmonary hypertension is high • regular blood transfusions and long-term anticoagulation have been tried • hydroxycarbamide may decrease the risk • prostacycline analogues (epoprostenol, and iloprost), endothelin-1 receptor antagonists (bosentan), phosphodiesterase inhibitors (including sildenafil), and calcium channel blockers are being evaluated. • brief but recurrent (stuttering); may occasionally last for many hours and can lead to impotence • usually ischaemic, or low-flow, priapism • patients should be educated to seek medical attention if more than 2 hours duration • detumescence within 12 hours is necessary to retain potency • intravenous hydration and analgesia initially with consideration for α-adrenergic agonists (etilefrine or phenylephrine) • penile aspiration and irrigation with saline and α-adrenergic agents or shunting may be required in severe cases in combination with an exchange transfusion. • assess pain intensity • choose the analgesic, dosage, and route of administration • paracetamol and hydration should be considered in all patients • oral, sustained-release morphine is as good as intravenous morphine infusion in children and young adults • manage mild pain with rest, hydration, and weak opioids (such as codeine). admit patients in whom pain that does not subside promptly or require opioid treatment; fever, pallor, or signs of respiratory compromise; a low likelihood of receiving appropriate care at home • pain management should be individualized and dosing should take into account prior pain management and use of opioids • the pain pathway should be targeted at different points with different agents, avoiding toxicity with any one class • always look for a cause, e.g. infection, dehydration, etc. • education about avoiding exposure to precipitants • be empathetic, reassuring, and supportive • benzodiazepines may be helpful to reduce anxiety • re-examine the patient often to ensure adequate pain relief, to assess sedation and respiratory rate (to avoid opioid overdose). in assessing patient responses to conventional doses of analgesia, it must be remembered that those with sickle cell disease metabolize narcotics rapidly • re-search for evidence of any complications such as acute chest syndrome or anaemia • always look for a cause, e.g. infection. of multi-organ failure. 116 both simple transfusion and exchange transfusions have been used and neither appears to be superior. a short course of steroids may attenuate acs but it may also increase the risk of re-hospitalization. 117 bronchodilators may help patients with wheezing 118 but inhaled nitric oxide has not shown any clear benefits. 119 since coagulation activation is important in the pathophysiology of acute chest syndrome, treatment with low-molecular-weight heparin may reduce clinical complications. 120 transcranial doppler measurement of cerebral blood flow has been a major step forwards in identifying individuals with an increased risk of ischaemic stroke. a value more than 200 cm/ second imparts a 40% risk of stroke within the next 3 years. 121 regular blood transfusions can reduce the incidence of stroke in children. 105 due to a high recurrence of stroke (60%) on stopping transfusions, continuation of transfusions should be guided by transcranial doppler measurements. 122, 123 once a stroke has developed, the best therapeutic strategy is exchange transfusion which probably needs to be done monthly. 70, 73 neurosurgical re-vascularization should be considered for moyamoya-like syndromes when new strokes occur despite transfusion. haemoglobin sc results from the co-inheritance of hbs and hbc and has its highest prevalence in west africa. clinical features and disease management are similar to those of hbss disease but splenomegaly, splenic infarcts and splenic sequestration may occur into adulthood. proliferative retinopathy necessitates regular ophthalmic review in those aged over 10 years. compared with hbss, anaemia is less marked in hb sc (8-140 g/l) and there are fewer sickle cells and more target cells on the blood film. the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focussing. worsening of complications. in exchange transfusion, the aim is to achieve an hbs% of <30%. complications of transfusion in scd include alloimmunization, 114 delayed haemolytic transfusion reactions and iron overload. the high rates of red cell antibody formation (30%) noted in wealthy countries are due to minor blood group incompatibilities between the recipient and the blood donor who is often of a different ethnicity. leukocyte reduction of transfused blood, routine abo, rh and kell matching for all patients and extended phenotype matching for those with alloantibodies may be useful for reducing transfusion reactions. treatment for acs is predominantly supportive and includes adequate pain relief, antibiotics (e.g. a macrolide with a cephalosporin), continuous pulse oximetry and delivery of supplemental oxygen to patients with hypoxaemia. incentive spirometry can prevent atelectasis and infiltrates 115 and blood transfusion is indicated when a patient develops respiratory distress, a clinically significant fall in the haematocrit or signs the following predict a more severe clinical course and are additional reasons to consider offering hydroxyurea: hb <70 g/l, wbc >15 × 10 9 /l, hbf <6% and renal insufficiency due to scd. • start at 10-15 mg/kg per day (to the nearest 500 mg/day) • if no or poor response, increase dose by increments of 5 mg/ kg per day every 4 weeks (max: 30 mg/kg per day). most good responses require about 1-2 g/day in adults • monitor fbc, hbf%, and reticulocytes every 1 or 2 weeks initially, then every 4 weeks when on a stable dose • monitor biochemistry profile (hydroxyurea has renal excretion and hepatic toxicity). • less pain • persistent increase in hbf (usually measured every 6-8 weeks) or mean cell volume • persistent increase in haematocrit if severely anaemic • decrease in ldh • acceptable toxicity. improvement in symptoms and blood parameters may take 3-4 months of therapy, but can be seen after approximately 6 weeks. if the reticulocyte count is less than expected for the degree of anaemia, erythropoietin deficiency should be considered. • aim in all cases to reduce hbs level to <30% • exchange transfusions may be considered in cases of stroke, acute chest syndrome not responding to top-up transfusion and major surgeries • target haemoglobin concentration of 100 g/l may be considered in cases of organ failure and surgery. individuals with sickle cell trait (hb as) have 10-fold protection against severe malaria compared to individuals with normal haemoglobin (hbaa) probably due to both innate and immune-mediated mechanisms. individuals with sickle cell trait (hbas) are generally asymptomatic and they have a normal haemoglobin and normal life expectancy. uncommonly, complications such as poor perfusion of the renal papillae and increased bacteruria may occur. 124 the blood film is generally normal and the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focusing. the original descriptions of thalassaemia originated from areas round the mediterranean and the term derives from the greek thalassos (sea) and haima (blood). [125] [126] [127] epidemiology thalassaemia is one of the most common single gene disorders and approximately 5-7% of the global population are carriers. α + -thalassaemia occurs throughout the tropics, whereas α 0thalassaemia, which is responsible for haemoglobin bart's hydrops fetalis, is concentrated predominantly in south-east asia and to a lesser extent around the mediterranean. 128, 129 β-thalassaemia is common in the mediterranean countries, parts of africa, throughout the middle east, the indian subcontinent and south-east asia. haemoglobin e prevalence is highest in cambodia, laos and thailand and can reach 50-60% with lower prevalence rates in indonesia, malaysia, singapore and vietnam. β-thalassaemia β-thalassaemia is an inherited quantitative deficiency of β-globin chains which are required to make normal adult haemoglobin. 130 more than 200 mutations have been associated with the development of β-thalassaemia (a complete list is available at the globin gene server website, at: http://globin.cse. psu.edu) and they affect protein synthesis 130, 131 leading to reduced (designated β + ) or absent (designated β 0 ) production of the β-globin chains. the clinical severity of thalassaemia can be lessened by co-existing haemoglobin abnormalities such as the co-inheritance of α-thalassaemia and increased production of haemoglobin f. 132, 133 α-thalassaemia normal α-globin synthesis is regulated by duplicate α-globin genes on chromosome 16. the genotype is usually represented as αα/αα and α-thalassaemia usually results from deletion of one or both α-genes. occasionally point mutations in critical regions of the α-genes may cause non-deletional α-thalassaemia (α t ). 130 mutations can completely abolish expression of the αgenes (i.e. α 0 -thalassaemia) or partially down-regulate expression (α + -thalassaemia). 130 both α 0 and α + thalassaemias can occur in the heterozygous or homozygous state or as a compound α 0 /α + heterozygote form (table 65 .6). underproduction of α-globin chains due to three or four gene deletions gives rise to excess γ (fetal) or β (adult) globin chains which form tetramers, called hb bart's (fetal) or hbh (adult). 134 rare forms of α-thalassaemia occur in association with other conditions such as mental retardation and myelodysplastic/ leukaemia syndrome. 135, 136 pathophysiology β-thalassaemia (figure 65 .8) thalassaemias 131, 137, 138 cause an imbalance of αand β-globin chain synthesis. in homozygous β-thalassaemia, excess α-chains precipitate in the red cell precursors and up to 75% of cells are destroyed in the bone marrow resulting in ineffective erythropoiesis and a shortened red cell survival. the red cells released from the bone marrow contain abnormal α-chains and these inclusions promote destruction of the cells by the spleen leading to clinical symptoms and signs of haemolysis. in heterozygotes, the α-chain excess and the degree of inadequate erythropoiesis is much less than in homozygous β-thalassaemia. hbf production normally tails off within a few months of birth but in β-thalassaemia hbf production can continue into adulthood. the effect of increased hbf production is to prevent precipitation of the excess globin chains and consequent ineffective erythropoiesis. however hbf has a high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion. the pathophysiology of α-thalassaemia, and hence the clinical manifestations, is quite different from β-thalassaemia. the excess non-α-globin chains form soluble tetramers rather than precipitates so there is only minimal ineffective erythropoiesis. the only clinical abnormality in those with hbh may be splenomegaly secondary to increased work load from destruction of red cells containing inclusions. rarely anaemia may be severe enough to require blood transfusions. classification of α-thalassaemia divide β-thalassaemia into thalassaemia major (transfusiondependent), thalassaemia intermedia (able to maintain adequate haemoglobin without transfusions or requiring less than 8 units/year) and thalassaemia minor (asymptomatic). 139 infants with β-thalassaemia are protected from severe anaemia by the presence of haemoglobin f and are usually asymptomatic. clinical manifestations of thalassaemia major depend on whether adequate blood transfusions are available and the stringency with which iron chelation is undertaken. untreated patients with thalassaemia major will die in late infancy or early childhood from the effects of severe anaemia. those who receive sporadic transfusions may survive longer but suffer from the secondary effects of anaemia, bony deformities and growth retardation. the clinical features of β-thalassaemia major are divided into those resulting from anaemia, bony changes and iron overload. anaemia from defective erythropoeisis, decreased red cell survival and increased haemolysis in thalassaemia major leads to cardiac decompensation, failure to thrive and growth retardation in children. splenomegaly, from the increased work load of culling red cells with inclusion bodies, can cause dilutional anaemia and a further drop in haemoglobin. compensatory extra-medullary haematopoiesis can lead to hepatomegaly and occasionally vertebral compression and neurological defects. haemolysis from increased red cell destruction is associated with gall stones in up to 20% of individuals with β-thalassaemia. 140 another consequence of accelerated haemolysis is the increased incidence of thromboembolism (4% in thalassaemia major and 10% with intermedia) from the exposure of negatively charged phospholipids on the red cell membrane and the generation of red cell and platelet microparticles. 141 splenectomy with postoperative thrombocytosis is a risk factor for thrombosis especially if combined with endothelial oxidative stress from iron overload, or procoagulant co-morbid conditions such as diabetes mellitus, hormone therapy, thrombophilic mutations and atrial fibrillation. 142 folate deficiency, hyperuricaemia and occasionally gout have been observed in thalassaemia major due to the high turnover of red cells. the enhanced erythropoietic drive from anaemia in thalassaemia can lead to increased marrow expansion with in homozygous β-thalassemia, β-globin synthesis is markedly reduced or absent. the excess α-chains cannot form a tetramer but form a precipitate in the red cell precursors leading to intra-medullary destruction of these cells. this destructive process of the red cell membrane occurs from the formation of α-chain hemichromes (shown as red cell inclusions) and degradation products of the excess α-chains. the red cells which may be released from the bone marrow are destroyed by the spleen leading to clinical symptoms and signs of haemolysis. since only the β-chain is affected in these individuals, the synthesis of hbf and hba 2 continues unabated. these haemoglobins have very high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion splenomegaly, which may be massive, and growth retardation in children. bony changes are unusual. other complications include infections, leg ulcers, gall stones and acute haemolysis in response to drugs and infections. the severity of the clinical features is related to the molecular basis with non-deletional types of hbh disease more severely affected. haemoglobin bart's (−/−) occurs almost exclusively in asians, especially chinese, cambodian and thai populations. an infant with hb bart's hydrops fetalis syndrome has pallor and gross oedema with signs of cardiac failure, marked hepatosplenomegaly and skeletal and cardiovascular deformities. there is often gross hypertrophy of the placenta. many of the clinical manifestations of this condition can be explained by the characteristic bossing of the skull and overgrowth of maxillary region, radiologically noted as 'hair on end' or 'sun-ray' appearance. metatarsal and metacarpal bones are the first to expand so measurement of the metacarpal bones has been considered a good indicator for initiation of transfusion therapy. 143 other skeletal deformities include shortening of long bones due to early epiphyseal fusion and overgrowth of the maxilla causing dental malocclusion. the marrow expansion can also lead to pathological fractures, early bone thinning and osteoporosis 144, 145 while ineffective drainage of the sinuses and middle ear from skull bone overgrowth can cause chronic sinus and ear infections. growth retardation is primarily the result of anaemia with contributions from iron overload, hypersplenism, deficiencies of thyroid and growth hormone, hypogonadism, zinc deficiency, chronic liver disease, malnutrition and psychosocial stress. 146 patients with β-thalassaemia have increased iron absorption mediated by reduced hepcidin and those who receive regular transfusions may also develop transfusion siderosis if they are inadequately chelated. the iron is deposited in the parenchymal tissues with a variety of clinical consequences (box 65.14), [147] [148] [149] [150] [151] [152] [153] [154] a process which may be modulated by variants in the haemochromatosis (hfe) gene. 155 thalassaemia intermedia is characterized by haemoglobin concentrations of 70-100 g/l and children usually present at around 2-4 years of age with symptoms of anaemia, jaundice and hepatosplenomegaly. 156 there may also be skeletal changes such as expansion of the facial bones and obliteration of the maxillary sinuses. 157 several molecular factors including: (a) coinheritance of α-thalassaemia; (b) hereditary persistence of haemoglobin f; (c) δβ-thalassaemia and (d) the specific gγxmn1 polymorphism contribute to the 'conversion' of thalassaemia from major to intermedia type. 158 in contrast to patients with thalassaemia major, iron loading in thalassaemia intermedia occurs mainly as a result of increased intestinal iron absorption rather than transfusion therapy. ineffective erythropoiesis with resultant chronic anaemia and hypoxia can suppress hepcidin, the regulator of iron metabolism, leading to increased iron absorption. 158 the excess iron tends to accumulate in the liver rather than the heart. 159 other clinical complications in thalassaemia intermedia include gallstones, extramedullary haemopoiesis leg ulcers, thromboembolic events and pulmonary hypertension, which is the major cause of heart failure in these individuals. 160 although individuals with thalassaemia intermedia do not usually need regular blood transfusions, there is some evidence that complications, particularly later in life, may be less common in regularly transfused patients. 159 α-thalassaemias 130, 134 carriers of α-thalassaemia (traits, with loss of 1 or 2 α genes) are usually asymptomatic and may only be detected through a routine blood count which shows mild to moderate microcytic, hypochromic anaemia. antenatal counselling may be indicated if the mother has αα/− as there is a possibility that the fetus may be at risk of having haemoglobin bart's. haemoglobin h disease occurs mainly in asians and occasionally in the mediterranean population. it is the result of deletion of three α genes (α−/−) and can produce anaemia varying from 30-130 g/l. there is usually associated • hypogonadism is the most frequent complication in patients with prevalence over 50% in both males and females. it is usually hypogonadotrophic suggesting iron damage to the anterior pituitary or hypothalamus. the features range from total absence of sexual development to delayed puberty. in females with normal menstrual function, fertility is normal with the ovarian function preserved in most although secondary amenorrhoea can develop. damage of the ovaries is rare and is more likely to appear in older women (around 30) because of high vascular activity on the ovaries at this age. secondary hypogonadism is common (50%) in older men. serum ferritin >2000 ng/ml is a risk factor. • hypothyroidism is the second most common endocrine disorder (about 20%) although many of them may have the subclinical variety. most commonly hypothyroidism is of the primary type with secondary, central hypothyroidism increasingly being diagnosed in recent years. • the prevalence of diabetes mellitus is around 5% with the mean age of diagnosis being 18 years. impaired glucose tolerance occurs first with microvascular damage like retinal changes being less common than the conventional form. erythroid precursor destruction. osmotic fragility is reduced, sometimes strikingly so since in some cases the red blood cells do not haemolyse even in distilled water. 161 for this reason, if sophisticated tests are not available, osmotic fragility can be used as a screening test for thalassaemia trait. serum zinc levels may be low and this may be related to abnormal growth. 162 vitamin c levels may also be low due to its increased conversion to oxalic acid in the presence of iron overload. 163 care may be needed if folic acid is commenced on a background of bone marrow failure due to folate deficiency as it may precipitate painful erythropoietic crises. 164 management a comprehensive management plan for patients with thalassaemia may involve transfusion therapy, iron chelation, splenectomy, prevention or early treatment of complications and stem cell transplant. the mainstay of treatment for the severe forms of thalassaemia is blood transfusion with the aim of reducing anaemia and erythropoietic drive. however, in many low-income settings blood supplies are inadequate and many thalassaemic patients are chronically under-transfused (table 65 .7). 165 transfusion frequency should be guided by clinical symptoms and signs such as poor growth and facial or other bone abnormalities, and should take into account any potential disease-modifying comorbidities. 156 although the decision to transfuse should not be based purely on haemoglobin levels, a value of <70 g/l is often used as a trigger for regular transfusions. to prevent alloimmunization, extended red cell antigen typing for c, e and kell in addition to abo and rh(d) typing should be carried out prior to the first transfusion, and before each transfusion, full cross-match and screening for new antibodies should be undertaken. 166 the risk of alloimmunization appears to be greater in patients who begin transfusion therapy after the first few years of life. 128 development of alloantibodies and autoantibodies may result in increased transfusion requirements or haemolysis. use of leukodepletion techniques can result in less alloimmunization and fewer febrile transfusion reactions. since storage of red cells in anticoagulant solutions may decrease their efficacy, the use of blood that has been stored for less than 7-10 days may be beneficial for patients who require frequent transfusions. the use of 1st-degree relatives as blood donors should be discouraged, especially if the patient is a candidate for stem cell transplant. patients with thalassaemia major need lifelong regular blood transfusions, 15 ml/kg per month or 1-2 units of blood every 2-5 weeks, to maintain the pre-transfusion haemoglobin level above 90-105 g/l. the clinical benefits of this regular transfusion programme include normal growth, suppression of erythropoiesis and bone marrow expansion, reduced hepatosplenomegaly and an overall sense of wellbeing, which allows normal age-appropriate activities. a higher target pretransfusion haemoglobin level of 110-120 g/l may be necessary for patients with heart disease or other medical conditions and for those patients who do not achieve adequate suppression of bone marrow activity at the lower haemoglobin level. shorter intervals between transfusions may reduce overall blood requirements but need to be balanced against the patient's work or school schedule and other lifestyle issues. iron chelation therapy 167, 168 has improved survival rates for thalassaemic patients, and prevented hepatic fibrosis and ironinduced cardiac disease; most patients who are compliant with chelation therapy have normal growth and sexual development. iron chelators (box 65.16) are usually initiated in children over 2 years who have received 10 units of blood and/or have a steady-state serum ferritin level above 1000 ng/ml on at least two occasions. this level of iron overload typically occurs after 1-2 years of transfusions. desferrioxamine is started at 25-30 mg/kg per day in these children initially, to avoid toxicity due to over chelation. marked splenomegaly, often treated with splenectomy, was common in thalassaemia patients before the advent of regular transfusion programmes. severe haemolysis in thalassaemia is related to a hyperactive spleen, which aggravates anaemia and can increase transfusion requirements. although early the initiation of regular transfusion therapy for severe thalassaemia usually occurs in the first 2 years of life. some patients with thalassaemia intermedia who only need sporadic transfusions in the first two decades of life may later need regular transfusions because of a falling haemoglobin level or the development of serious complications. haemoglobin should be monitored to assess the rate of fall in the haemoglobin level between transfusions and this can be used to indicate the frequency of transfusions. exchange transfusions have been tried as a way of reducing iron loading and are associated with a reduction in blood requirements by about one-third. (table 65. since each unit of red cells can contain up to 200 mg of iron, cumulative iron burden is an inevitable consequence of a longterm transfusion programme. in addition there is increased iron absorption from the gut (0.3-0.6 mg/kg per day) as a response to severe anaemia and down-regulation of hepcidin. transfusion therapy can avert splenomegaly, hypersplenism still can develop, usually in children between 5 and 10 years of age. in these individuals, splenectomy can limit the complications from extramedullary hematopoiesis. splenectomy should be considered when the annual transfusion requirement reaches 200-250 ml red blood cells/kg per year and usually results in a halving of the transfusion requirements. splenectomy complications include opportunistic infections with encapsulated organisms. patients should therefore receive appropriate vaccinations preoperatively and should be advised to seek medical advice at the first sign of infection. it is advisable to delay splenectomy until patients are at least 5 years old because of the increased risk of overwhelming sepsis below this age. thalassaemia patients can develop thromboembolic complications and pulmonary hypertension after splenectomy so partial splenectomy and splenic embolization have been attempted to minimize these complications but have not been studied in large trials. iron overload can occur in any organ in thalassaemia patients but particularly affects the heart, liver, the endocrine system, the bone and occasionally the pancreas and lungs. iron overload needs to be detected early and treated to prevent long-term damage. annual assessment of the iron loading of the liver and heart can be achieved using non-invasive methods such as magnetic resonance scanning to detect early changes. children should have regular growth and endocrine assessments and appropriate investigations should be carried out if there are any signs of developmental delay or hormonal deficiencies. osteoporosis is increasingly being recognized and should be prevented by ensuring adequate dietary calcium intake and sun exposure. vitamin supplementation with folic acid, zinc, vitamin e and vitamin c may be useful although the combination of vitamin c and desferrioxamine carries a risk of cardiac toxicity. allogeneic stem cell transplant 169, 170 is currently the only means of curing thalassaemia. the outcome in carefully selected patients, measured by overall event-free survival, is around 90% with a transplant-related mortality of 3%. hepatomegaly, liver fibrosis, and inadequate iron chelation therapy predict a poor outcome. the best results from transplant have been obtained with hla-matched siblings. umbilical cord blood is a useful source of stem cells for young children. other potential treatment options for thalassaemia are outlined in box 65.16. [171] [172] [173] [174] prevention of severe thalassaemia births by prenatal diagnosis and termination of pregnancies has been successful in countries with a high prevalence of thalassaemia. 175 early identification of couples at risk and culturally sensitive genetic counselling facilitate decision-making for termination or continuation of pregnancy. the mean corpuscular haemoglobin (mch) is used to screen for the presence of thalassaemia using a cut-off of less than 27 pg. rarely, silent β-thalassaemia mutation may present with an mcv over 27 pg and should be considered in those with a positive family history. at-risk couples should be referred for detailed counselling on the options for prenatal diagnosis. these include chorionic villous sampling or amniocentesis, which are used to obtain fetal dna samples for genetic analysis. polymerase chain reactions and precise hybridization assays to detect single point mutations using very small dna samples have also been developed. a less invasive and less risky option is to isolate fetal dna circulating in the maternal blood for genetic analysis. pre-implantation genetic diagnosis is a newer technique where dna from the blastomere is used for genetic diagnosis. ultrasound can be used from the 2nd trimester for fetuses suspected of having α-thalassaemia to detect signs of hydrops fetalis and enlarged placenta (figure 65.9) . 175 • hydroxyurea -helpful in some patients with β-thalassaemia intermedia, but not as effective in thalassaemia major • histone deacetylase inhibitors -derivatives of butyric acid; intermittent pulses with hydroxycarbamide has been tried • kit ligand • decitabine • knockdown of bcl11a (regulator of γ-globin expression) • erythropoietin. • vitamins c and e • fermented papaya preparations. • successful in β-thalassaemia animal models using a retroviral vector transferring the human β-globin gene sequence and its promoter region into mice stem cells • β-globin gene transfer into progenitor hematopoietic cells of humans is also being studied • other molecular approaches being tried include using different mutations of stop codons and aberrant splicing. partner testing in all cases β-thalassaemia trait protecting red cells. red cells lack any other source of nadph and are solely dependent on the pentose phosphate pathway so g6pd deficiency leaves these cells with no defence against oxidative damage. oxidative damage results in denatured haemoglobin aggregates which form heinz bodies (denatured haemoglobin precipitates). these damaged cells bind to the membrane cytoskeleton resulting in decreased cellular deformability, and are also destroyed in the spleen, resulting in haemolysis. the level of enzyme activity is higher in young erythrocytes than in more mature cells so older cells are more susceptible to haemolysis. the global distribution of g6pd deficiency mirrors that of malaria, and where malaria has historically been prevalent, and it provides a degree of protection against malaria. 181 • class iv -normal (60-150% enzyme activity) • class v -increased activity (>150% enzyme activity). g6pd enzyme variants can be distinguished by their electrophoretic mobility. 184 g6pd b, the wild-type enzyme, and g6pd a + , a common variant in populations of african descent, demonstrate normal enzyme activity and are not associated with haemolysis. g6pd a − is the most common variant associated with mild to moderate haemolysis with approximately 10-25% hb e is caused by a substitution of glutamic acid by lysine at codon 26 of the β-globin gene. 177 this causes reduced synthesis of the β-e chain and leads to a thalassaemia phenotype. hb e β-thalassaemia affects at least a million people worldwide and is an important health problem particularly in the indian subcontinent and south-east asia. in some areas, it has replaced β-thalassaemia as the most common thalassaemia disorder. the frequency of hbe reaches 60% in many regions of thailand, laos and cambodia with estimates of at least 100 000 new cases of hbe β-thalassaemia expected in the next few decades in thailand alone. the natural history of hbe thalassaemia is highly variable; some patients are asymptomatic (e.g. heterozygotes, hbe 20-30% or homozygotes hbe, 80-90%) while others (e.g. hbe with β-thalassaemia) may be transfusiondependent. pathophysiology glucose-6-phosphate dehydrogenase (g6pd) deficiency was originally recognized through its association with haemolysis related to eating fava beans ('favism') and primaquine ingestion. 178 g6pd deficiency is the most common enzyme defect in humans and is present in about 400 million people worldwide (figure 65 .10). 179, 180 it is an x-linked, hereditary defect caused by mutations in the g6pd gene. g6pd is an enzyme that catalyses the first reaction in the pentose phosphate pathway, to produce nadph, which is an important antioxidant used to preserve the reduced form of glutathione. 178, 181 reduced glutathione acts as a scavenger for oxidative metabolites thereby <0.5% 0.5-2.9% 3-6.9% 7-9.9% 10-11.9% 15-25% of africans carrying this variant. g6pd mediterranean, present in all countries surrounding the mediterranean sea, middle east, india and indonesia, has the same electrophoretic mobility as g6pd b but the enzyme synthesis and catalytic activity are reduced. in several populations, g6pd a − and g6pd mediterranean co-exist. the clinical manifestations of g6pd deficiency can be classified into: (i) asymptomatic; (ii) acute haemolytic anaemia; (iii) favism; (iv) neonatal jaundice; and (v) chronic non-spherocytic haemolytic anaemia. acute haemolytic anaemia in g6pd deficiency can be secondary to infection (e.g. pneumonia, hepatitis a and b, and typhoid fever) or oxidant drugs, or may be precipitated by diabetic ketoacidosis, myocardial infarction and strenuous physical exercise. 185, 186 a list of the drugs which may cause haemolysis in g6pd-deficient individuals (table 65 .9) 187 can be obtained from: http://www.g6pd.org/favism/english/index.mv. a drug which is deemed to be safe for some g6pd-deficient individuals may cause haemolysis in others due to the heterogeneity of the underlying genetic variants. haemolysis typically occurs within 1-3 days after commencing the drug and can produce intense haemoglobinuria. fortunately, the disorder is self-limiting and most patients do not develop renal impairment or anaemia requiring transfusion. the spontaneous recovery reflects replacement of the older, enzyme-deficient red cells by younger reticulocytes which can withstand oxidative injury. 186 if the precipitating cause has been removed the haemoglobin begins to recover after 8-10 days. acute renal failure due to acute tubular necrosis and tubular obstruction by haemoglobin casts can develop as a complication of haemolysis in g6pd deficiency. this occurs more often in adults than children and may require haemodialysis. this occurs predominantly in boys aged 1-5 years in mediterranean countries, but it has also been observed in the middle east, asia and north africa. both intravascular and extravascular haemolysis, occasionally severe enough to cause renal impairment, can occur after eating fresh or cooked fava beans, and favism has been reported in breastfed babies of mothers who have eaten fava beans. 179 divicine and isouramil have been implicated as the toxic components of fava beans. 188 neonatal jaundice this occurs in one-third of male babies in areas where g6pd deficiency is common and is likely due to g6pd deficiency. 179 it presents 1-4 days after birth and can lead to kernicterus. 189, 190 maternal exposure to oxidant drugs, and even naphthalenecamphor mothballs, can precipitate haemolysis in affected babies. breast-feeding mothers should therefore be warned to avoid offending drugs, umbilical potions containing fava, triple dye or menthol, and should not apply henna to the skin or use clothes that have been stored in naphthalene. 190 premature infants and babies who have co-inherited the mutation for gilbert's syndrome are at particular risk. phototherapy and exchange transfusion therapy may be required to reduce the level of unconjugated bilirubin. the diagnosis may be easily missed so assessment of g6pd status should be undertaken for any jaundiced infant whose family history or ethnic or geographic origin suggest the likelihood of g6pd deficiency, and in infants who respond poorly to phototherapy. 191 this is an unusual manifestation of g6pd deficiency and usually presents in childhood. 179, 184 there may be a history of severe neonatal jaundice, episodic or worsening anaemia which requires blood transfusions, and complications from gallstones. although these individuals usually have a well-compensated anaemia, and require transfusions only for exacerbations, rarely some may become transfusion-dependent. antioxidants such as vitamin e and selenium may be of benefit in some cases. the haemolysis does not resolve following splenectomy. folic acid supplementation is necessary to support the increased compensatory erythropoiesis. the diagnosis of g6pd deficiency is usually suspected when neonatal jaundice occurs in an area where g6pd deficiency is drugs which may cause haemolysis in g6pd-deficient individuals common or when an episode of non-immune haemolytic anaemia occurs in association with an infection or drug. the appearance of the red cells on the blood film is characteristic because denatured haemoglobin concentrates in one area within the cell creating 'helmet' or 'bite' cells. 192 denatured haemoglobin precipitates in peripheral red blood cells as heinz bodies which can be detected by staining with methyl violet. definitive diagnosis of g6pd deficiency is by quantitative spectrophotometric analysis of the rate of nadph production. point of care tests for g6pd deficiency are being developed but have not yet been validated for routine use. measuring enzyme activity during an episode of acute haemolysis is not helpful since reticulocytosis, which is a feature of acute haemolysis, produces a false-negative result because of the high enzyme levels in younger erythrocytes. 179, 186 management the most effective management strategy for g6pd deficiency is to prevent haemolysis by avoiding triggering agents like infections, drugs and fava beans. for the milder variants (e.g. class iii and iv), drugs known to trigger haemolysis may be given to individuals with g6pd deficiency if the benefits outweigh the risks and the blood count is closely monitored (e.g. use of low-dose primaquine for individuals with g6pd avariant). screening programmes have been established in some mediterranean and other populations where g6pd deficiency is prevalent. 193 haematological complications of malaria (see chapter 43) the pathophysiology of anaemia in malaria is multi-factorial and influenced by the age of the individual and their antimalarial immune status. anaemia mechanisms in malaria involve: • haemolysis with increased red cell destruction of both infected and bystander erythrocytes • dyserythropoiesis • hypersplenism • haemolysis • co-existent conditions which can cause anaemia. haemolysis is more common in non-immune individuals with acute malaria, whereas dyserythropoiesis is the predominant mechanism for anaemia in recurrent falciparum malaria. 207, 194 haemolysis is the result of red cell phagocytosis by the reticuloendothelial system and is triggered by damage to the red cell membranes and exposure of abnormal surface antigens on their surface. [195] [196] [197] [198] ten uninfected red cells are removed from the circulation for each infected red cell destroyed, 199 possibly related to loss of red cell complement regulatory proteins and increased levels of circulating immune complexes. 200 this may partly explain the persistent or worsening anaemia following parasite clearance and the poor correlation between parasitaemia and the severity of anaemia noted in some studies. 207 an increased incidence of anaemia has been noted in malaria vaccine trials possibly due to enhanced clearance of uninfected red blood cells. 201 decreased erythropoeisis with abnormalities in red cell precursors and reticulocytopenia is found consistently on examination of bone marrow from malaria-infected patients. 202 the decreased erythropoiesis is due to many factors including low levels of tnf-α, high levels of interleukin-10, abnormalities of erythropoietin, a decrease in burst colony forming units, cytokine-induced suppression of red cell production and the inhibitory effect of the malarial pigment haemozoin. [203] [204] [205] [206] epidemiology malaria-related anaemia is most commonly seen in children and pregnant women. the prevalence of malarial anaemia in sub-saharan africa in children is 30-90% and in pregnant women it is 60-80%. 207 the highest prevalence is in infants and children less than 3 years of age. infants may acquire malaria through the placenta. 208, 209 individuals living in malarious areas may have multiple reasons for anaemia such as bacteraemia, hookworm infections and vitamin a deficiency 208 making it difficult to assign anaemia solely to malaria. however, animal studies and the fact that anaemia improves with anti-malarial treatment suggest a direct relationship between malaria infection and anaemia. 210, 211 for example, in tanzanian children about 60% of anaemic episodes were thought to be caused by malaria. 212 who defines severe anaemia attributable to malaria as: (i) haemoglobin concentration <50 g/l or haematocrit <15%; (ii) parasitaemia with >10 000 parasites/µl of blood and (iii) normocytic blood film (to exclude other common causes of anaemia). 213 however, aspects of this definition have been criticized because blood films are not examined routinely and parasite density varies with endemicity and age. 207 although traditionally it is p. falciparum that has been associated with the most severe malaria-related anaemia, p. vivax is also a major risk factor for severe anaemia especially in young children or those with chronic and recurrent infections. p. vivax anaemia is associated with recurrent bouts of haemolysis of predominantly uninfected erythrocytes with increased fragility. 214 symptoms of malarial anaemia can vary from negligible to profound depending on the degree of anaemia and the rapidity of onset. splenomegaly is a common feature of malarial anaemia because of the role of the spleen in the removal of both infected and uninfected red cells. 215 blackwater fever, characterized by intense intravascular haemolysis with haemoglobinuria and occasionally renal failure in a patient with malaria, may be related to underlying glucose-6-phosphate deficiency. 216, 217 factors such as poor nutrition, deficiencies of vitamins and micronutrients, bacteraemia, and hookworm or hiv infection may co-exist with malaria and contribute to anaemia 209 so nonmalarial causes of anaemia should be considered in patients whose anaemia does not respond to malaria treatment. the management of severe malarial anaemia involves supportive care and treatment of the malaria and any other underlying conditions. recovery from malaria-associated anaemia can be slow, taking 6 weeks or even longer if there are episodes of re-infection. 212 in children, blood transfusion is usually reserved for those with haemoglobin levels of less than 40 g/l (<50 g/l if there are complications such as respiratory distress 207 ). there of parasitized red cells 227 and the release of von willebrand factor multimers which cause widespread platelet aggregation leading to thrombocytopenia 228, 84, 231 platelet synthesis by the bone marrow is relatively well maintained during infection 231, 229 but antiplatelet antibodies, immune complexes and splenomegaly all contribute to thrombocytopenia. 230 thrombocytopenia occurs in 60-90% of individuals infected with malaria irrespective of the species of plasmodium. 214, 231 thrombocytopenia in febrile patients in an endemic area increases the probability of malaria by a factor of 5 232 and in individuals returning from tropical countries with a fever, thrombocytopenia is highly specific for malaria infection. 233 profound thrombocytopenia is unusual and malaria-associated thrombocytopenia is rarely associated with haemorrhagic manifestations. 234 the clinical consequences of platelet aggregation and endothelial binding are primarily microvascular ischaemia. this may manifest as renal impairment, cerebral ischaemia, and occlusion of retinal vasculature or even in some cases, skin necrosis. bleeding is unlikely, although in severe thrombocytopenia, petechiae or purpura may develop which denotes extravasation of red cells into the subcutaneous tissue. 235 continued platelet activation and consumption can exacerbate bleeding and decreased circulating platelets are associated with increased vascular leakage and the development of oedema. 236 platelet transfusions are rarely required because the platelet count generally rises rapidly on treating the underlying malaria. coagulopathy is a disturbance of the whole coagulation system involving not just coagulation factors but platelets, anticoagulant factors, fibrinolytic system and, in the case of malaria, the parasitized red cells and the vascular endothelium. parasitized red cells induce expression of tissue factor on endothelial cells and monocytes, release of microparticles, cytokine release and platelet clumping, all of which initiate blood coagulation and tilt the balance towards the pro-coagulant state ( figure 65.12) . 234, [237] [238] [239] [240] [241] [242] anticoagulant factors are severely depleted in malaria. protein c and antithrombin levels are inversely correlated with severity of falciparum malaria and return to normal with treatment of the malaria. 245 have been some concerns about a possible increased risk of infection associated with iron supplementation for children in malarious areas 218, 219 but current recommendations advocate that where iron deficiency and malaria are common, iron supplements should not be withheld and appropriate anti-malarial treatment or prevention should also be offered. 220 the best way to prevent malarial anaemia is to prevent malaria infection by avoiding mosquito bites (e.g. through the use of bed nets) or through chemoprophylaxis. malaria chemoprophylaxis during infancy can reduce both malaria and anaemia. 221 children who have been hospitalized with severe malarial anaemia may benefit from intermittent preventive malarial therapy after discharge to prevent recurrence of anaemia. 222 daily co-trimoxazole prophylaxis which is used for hiv-infected individuals has been shown to reduce malaria parasitaemia and anaemia. 223 the normal platelet life span of 7-10 days is reduced to less than 4 days in malaria infection. 224 several factors are responsible for thrombocytopenia in malaria infection, the most common being increased platelet activation and aggregation ( figure 65 .11). 225 platelet activation is by parasitized red cells which express surface tissue factor and initiate coagulation and platelet aggregation. the resultant activated endothelium binds platelets and sequesters them in vascular beds including in the cerebral vasculature. 226, 234 these platelets facilitate the adhesion and to 46% after a year. 253, 247 although transfusions may be required in severe life-threatening cases of anaemia, aggressive transfusion therapy has been associated with fatal pulmonary emboli due to accelerated haemolysis and disseminated intravascular coagulation. 254 in those who do not respond to art, erythropoietin may be considered since reduced responsiveness to this hormone and antierythropoietin antibodies have been noted in hiv patients. erythropoietin is particularly useful in individuals whose erythropoietin levels are less than 500 iu/l 255 because in addition to increasing the haemoglobin it can also improve the quality of life. 256 erythropoietin may take several weeks to achieve full effect and patients should be replete in haematinics. erythropoietin can very rarely be associated with thrombosis or pure red cell aplasia. thrombocytopenia is a common finding in hiv-infected patients and it may be the initial manifestation of hiv infection in as many as 10% of patients. data from wealthy countries demonstrate platelet counts less than 150 × 10 9 /l in 11% of patients, and less than 50 × 10 9 /l in 1.5%. overall the 1-year coagulopathy in malaria infection is unusual, occurring in less than 5% of cases. it appears to be most common in adults with cerebral malaria who may present with gastrointestinal bleeding 243 or with microvascular ischaemia in the brain, kidneys, retina and occasionally, the dermal vasculature. 244 prolongation of prothrombin time and activated partial thromboplastin time only occur in 4-8% of patients with p. falciparum infection and coagulopathy does not appear to be a feature of p. vivax infection. 245 since coagulation factors need to be depleted to less than 20% of normal to prolong the clotting times, these tests can be normal despite active coagulopathy. management of coagulopathy aims to restore the balance between pro-and anticoagulant processes. this is complex and requires input from a coagulation specialist and ideally, access to plasma, heparin and factor concentrates and a well-equipped coagulation laboratory. anaemia anaemia is very common in hiv-infected individuals occurring in up to 20% at initial presentation and about 70% at some stage during their disease. 246 thirty-seven percent of patients with clinical aids have a 1-year incidence of anaemia (haemoglobin <100 g/l) 246 and high rates of anaemia persist despite combination anti retroviral treatment (art). 247 anaemia is directly related to mortality in hiv infection and is independent of other risk factors including cd4 count. 248 there are multiple reasons for anaemia in hiv-infected patients (box 65.17), which often co-exist in individual patients. bone marrow infection by mycobacteria species, histoplasma, cryptococcus and penicillium marneffei can all decrease red cell production 249 and can be detected by bone marrow examination and cultures. parvovirus has a predilection for the erythroid progenitor cells and can cause severe anaemia in hiv-infected patients. serological tests for parvovirus are unhelpful in hivinfected patients and viral polymerase chain reaction is needed to confirm the diagnosis. 250 the likelihood of parvovirusinduced anaemia increases with the severity of anaemia and has been found in 31% of individuals with hiv and haemoglobin less than 70 g/l. 251 haemophagocytosis occurs in hiv infections and may be secondary to co-infection with mycobacteria, cytomegalovirus, epstein-barr or other herpesviruses. poor nutrition due to socioeconomic reasons, hiv-related anorexia, malabsorption from conditions affecting the gastrointestinal tract, and achlorhydria may contribute to anaemia. haemolytic anaemia occurs secondary to drugs or concomitant glucose-6 phosphate dehydrogenase deficiency and because reticulocytopenia is common in those with hiv infection, reticulocyte counts cannot be used to exclude haemolysis. although the direct coombs test may be positive in patients with hiv infection, autoimmune haemolysis is not a common cause of anaemia. a reduction in red cell precursors has also been noted in children in africa with severe anaemia. 252 treatment of hiv-related anaemia should focus on starting art and eliminating any other factors, such as infections or vitamin deficiencies, which may contribute to the anaemia. in wealthy countries art has been shown to reduce anaemia prevalence from 65% to 53% within 6 months of starting treatment, non-hodgkin's lymphoma (nhl) was noted to be associated with hiv infection early in the epidemic and is an aidsdefining illness. 270 the incidence of nhl is up to 200 times greater in hiv-infected adults than in those who are not infected, and it is responsible for nearly one-sixth of the deaths attributable to aids. since the introduction of haart, the incidence of all types of nhl has decreased by approximately 30-50% 271, 272 and the outcome of hiv-infected patients with lymphoma has improved. in the setting of clinical trials, the 60% 1-year survival rate is comparable to those without hiv infection. 273 the incidence of hodgkin's lymphoma has increased in the post-haart era, possibly due to immune reconstitution and increased cd4 cells. 274, 275 evidence of epstein-barr virus (ebv) infection can be found in virtually all cases of hodgkin's disease. 276 hiv-related lymphomas (box 65.18) 277 (see also lymphomas, below), are broadly divided into systemic lymphomas (80%) and primary central nervous system lymphomas. 278 the incidence of highly aggressive lymphomas, either burkitt's lymphoma (approx. 25%) or diffuse large b-cell lymphoma (approx. 75%), is much higher in hiv-infected patients than in those without infection. 279 although t-cell lymphomas are uncommon in hiv disease (1%), there has been an increase in recent years. the incidence of primary central nervous system lymphoma in hiv-affected individuals is 2-6% and it is 1000 times more common than in the general population. 280 the pathogenesis of nhl in hiv infection is related to the inadequate host immune responses to viruses with oncogenic potential, predominantly ebv and human herpesvirus 8 (hhv8)/kaposi's sarcoma-associated herpesvirus. this allows unregulated lymphoid growth and an accumulation of genetic abnormalities in b cells. 272 markers of b-cell activation such as serum immunoglobulins and free light chains, and cd4 cell count have been suggested as predictive markers for the development of nhl in hiv infection. 281, 282 extranodal and leptomeningeal involvement, and b-symptoms occur in the majority of hiv-infected patients with nhl and the bone marrow is commonly involved. the most common extranodal site to be involved is the incidence of moderate thrombocytopenia (<50 × 10 9 /l) is 3.7%, though this is higher in those with clinical aids (8.7%). 257, 258 thrombocytopenia is more common in those who abuse drugs, have opportunistic infections and malignant disorders of the bone marrow (e.g. lymphoma), and it may also be a side-effect of therapeutic drugs. 259 the most common cause of thrombocytopenia in hiv infection is immune thrombocytopenia which may be associated with hepatitis c co-infection, and produces decreased platelet survival, particularly at cd4 counts below 200/µl. the anti-platelet antibodies, immune complexes and cross-reacting antibodies to hiv envelope proteins and platelets, which occur in hiv-associated thrombocytopenia 259, 260 may also contribute to generation of reactive oxygen species. 261 platelet production can also be affected in hiv infection and may explain the high levels of thrombopoietin that have been documented in hivrelated thrombocytopenia. 262 some cases of hiv-related thrombocytopenia may undergo spontaneous remission so treatment of thrombocytopenia is usually only initiated if it is associated with bleeding, which is unusual. 259 the first line of treatment involves antiretroviral therapy with the aim of achieving undetectable plasma hiv viraemia. 263, 264 any drugs that may be associated with causing thrombocytopenia should be withdrawn and opportunistic infections or secondary malignancies treated. the treatment of immune thrombocytopenia is the same as in non-hiv cases and options include a short course of steroids, intravenous immunoglobulin (short-lived response), anti-d, interferon-α or splenectomy. although there are multiple causes of thrombocytopenia in hiv-positive individuals, one of the most devastating is the thrombotic microangiopathy of thrombotic thrombocytopenic purpura (ttp). this is because the combination of haemolytic anaemia and microthrombi has a very poor prognosis. symptoms are nonspecific and may include fever, headache, bleeding and changes in consciousness. if ttp is suspected, an urgent blood film should be requested and the combination of thrombocytopenia with red cell fragmentation is highly suggestive of ttp. ttp associated with hiv infection was more frequent before the introduction of art and is more common if adherence to treatment is poor or resistance to therapy has developed. 265 ttp is thought to be due to endothelial damage, but unlike the situation in non-hiv-infected individuals, low levels of adamts-13 are not a useful predictor of outcome. 266 treatment of ttp involves plasma exchange, and although refractoriness may occur, this can be corrected by art in some cases. 266 if art is administered in these cases it is important to maintain adherence throughout the period of plasma exchange. if apheresis facilities are limited, plasma infusions alone (30 ml/ kg per day) may also produce a response. 267 art should also be administered immediately after plasma exchange to minimize drug removal. patients with a viral load of less than 500 000 copies/ml generally require fewer plasma exchanges for remission than those with a higher load. 265 survival of patients with hiv-associated ttp in the pre-art era was rarely longer than 2 years, even with plasma exchange and steroid treatment, but for patients who are compliant with art the mortality is around 4%. 268 which immediately limits the amount of blood loss. exposure of the subendothelial space releases factors such as von willebrand factor multimers which bind to platelets and initiate platelet adhesion to the endothelium. the adherent platelets release their granules and attract more platelets, which in combination with fibrinogen, form an aggregate. the activated platelets also attract coagulation factors thereby promoting the clotting process. the critical parts of clot formation are the conversion of prothrombin to thrombin and the thrombinfacilitated conversion of fibrinogen to fibrin (figure 65.13) . haemostatic control mechanisms operate throughout the clotting process to prevent excessive clot formation and involve proteins c and s, and anti-thrombin and antifibrinolytic systems. any alteration in these regulatory pathways can lead to either bleeding or thrombotic complications. bleeding can result from: • inadequate vasoconstriction, due to vascular problems which can be acquired (e.g. viral haemorrhagic fevers or immune vasculitis) or congenital (e.g. collagen vascular disorders) • qualitative or quantitative abnormality of von willebrand factor causing von willebrand's disease • decreased number or function of platelets which can be either acquired (e.g. aspirin, nsaids) or congenital (e.g. platelet function defects) • qualitative (e.g. caused by inhibitors to coagulation factors, commonly factor viii) or quantitative (e.g. haemophilia) abnormality of coagulation factors • increased fibrinolysis (e.g. viral haemorrhagic fevers, snake bites). acquired bleeding disorders are commonly caused by vitamin k deficiency, disseminated intravascular coagulation (dic) or platelet disorders (box 65.19) but may sometimes be due to acquired inhibitors of coagulation factors. the initial laboratory tests in a patient with excessive bleeding should therefore include a platelet count, clotting screen (prothrombin time (pt) and activated partial thromboplastin time (aptt)), and gastrointestinal tract, often the stomach or the perianal region. 283 hepatic involvement, seen in a quarter of cases, is associated with a particularly poor prognosis. cns disease may be asymptomatic so diagnostic lumbar puncture may be required. 284 hiv-related lymphomas frequently present with poor prognostic features such as elevated serum lactate dehydrogenase levels. 285, 286 older age, lowest nadir cd4 cell counts prior to nhl diagnosis, developing nhl while on art, and cumulative hiv viraemia are also poor prognostic features. 282 a formal prognostic scoring system has been developed which takes into account the cd4 count (<100 cells/µl). 287 some types of hiv-related lymphoma are associated with characteristic clinical and laboratory features. primary effusion lymphoma is an aggressive lymphoma characterized by effusions in serosal cavities in the absence of any other tumour masses. 288, 289 it is strongly associated with hhv8 infection and the virus can be identified in the nuclei of the malignant cells. plasmablastic lymphoma mainly affects the oral cavity and the mucosa of the jaw and is typically associated with epstein-barr virus. 290 histological examination of biopsied tissue is necessary to confirm the diagnosis and type of lymphoma. diagnostic difficulties may arise because hiv-related hyperplasia in lymph node biopsies may be confused with lymphoma, the histological appearance of hiv-related lymphomas may be different from those of non-infected individuals 291 and many opportunistic pathogens may mimic the appearances of nhl, or co-exist with it, and will need to be identified or excluded before making a diagnosis of lymphoma. prior to the widespread use of art, conventional lymphoma chemotherapy resulted in considerable toxicity, increased opportunistic infections and high mortality. art has facilitated the use of conventional doses of chemotherapy in conjunction with haematopoietic growth factor support. this has markedly improved the outcome of patients with hiv-related lymphomas who now have overall response rates of 60%. 292 the concomitant use of art and chemotherapy is therefore recommended, especially in those with cd4 counts of less than 100/µl. anti-cd20 antibody is now included in treatment regimens for nhl, and studies that include patients with hivrelated lymphomas all report favourable outcomes. 293, 294 some antiretroviral agents such as zidovudine are best avoided in combination with chemotherapy, because it adds to the myelosuppression of chemotherapy. didanosine may worsen the peripheral neuropathy caused by taxanes and vinca alkaloids. hiv-infected patients undergoing chemotherapy should receive adequate anti-infective prophylaxis due to the high risk of opportunistic infections such as pneumocystis, herpes simplex and zoster and candida. consolidation chemotherapy and stem cell transplant have been used successfully in relapsed hivrelated lymphomas. haemostasis is maintained by interactions between vessel walls, platelets and a balance between pro-and anticoagulant factors. although the process of haemostasis is usually considered to occur in a stepwise fashion, in vivo the steps happen virtually simultaneously. activation of the lining of the endothelium by trauma, cancer cells or cytokines triggers vasoconstriction, the tests for each pathway is given with arrows corresponding to each box complications, malignancies and infections and is a serious condition with a high mortality. patients present with spontaneous bruising or excessive bleeding from minor wounds such as venepuncture sites, and they may also have signs of complications such as renal failure, acute respiratory distress syndrome and microangiopathic haemolytic anaemia. dic is associated with a combination of depleted clotting factors (i.e. prolonged pt and aptt), a falling platelet count, red cell fragments on the blood film, raised d-dimers or fibrin degradation products, and reduced fibrinogen levels. management of disseminated intravascular coagulation includes treating or removing the underlying cause, and correcting the haemostatic abnormalities with combinations of platelets, cryoprecipitate and fresh frozen plasma. although bleeding due to thrombocytopenia is unusual unless the platelet counts falls below 10-20 × 10 9 /l, bleeding may occur with a normal platelet count and normal clotting screening tests (i.e. pt and aptt) if platelet functions are impaired (e.g. myelodysplastic syndromes). platelet transfusions are generally not required unless there is active bleeding or prior to surgery. idiopathic thrombocytopenic purpura. idiopathic thrombocytopenic purpura is due to immune destruction of platelets. it is usually primary but can be associated with conditions such as lymphomas and infections including hiv. it may present incidentally or with petechiae, bruising or bleeding from the nose or gums, especially if the platelet count is less than 20 × fibrinogen levels, which may be helpful in cases of excessive fibrinolysis (table 65. 10). a difficult venepuncture can cause in vitro activation of the clotting system resulting in a shortened pt or aptt. similar findings may occur in chronic dic due to in vivo activation. the pt and aptt are not necessarily good predictors of the bleeding risk because some clotting disorders associated with thrombosis (e.g. anti-phospholipid antibodies) can prolong the aptt. a shortened aptt can be associated with marked elevation of factor viii levels (e.g. pregnancy) and may be a predictor of deep vein thrombosis. a prolonged thrombin time is caused by quantitative or qualitative fibrinogen deficiency, heparin and fibrin degradation products. reptilase time is helpful to distinguish between fibrinogen abnormalities (prolonged reptilase time) and heparin therapy (normal reptilase time). deficiency of vitamin k can be due to poor diet, small bowel disease or bile flow obstruction. clotting factors (ii, vii, ix and x) are dependent on vitamin k which is a fat-soluble vitamin. vitamin k deficiency therefore causes prolongation of the pt and aptt. in newborn infants, vitamin-k-dependent clotting factors can drop precipitously within a couple of days of birth. this causes haemorrhagic disease of the newborn which particularly affects infants that are premature, exclusively breast fed or have been exposed to drugs for tuberculosis, convulsions or anticoagulation in utero. these babies develop bleeding into the skin and gut, or bleeding from the umbilical stump or circumcision. vitamin k deficiency will respond to intravenous vitamin k (10 mg/day for 3 days orally or by intravenous injection) and in severe bleeding the clotting abnormality can be treated with fresh frozen plasma. haemorrhagic disease of the newborn can be prevented with 1 mg of intramuscular vitamin k given at delivery. disseminated intravascular coagulation (dic) is characterized by activation of haemostasis with widespread fibrin formation, activation of fibrinolysis and consumption of platelets and clotting factors. it may be precipitated by tissue injury, obstetric desmopressin (ddavp) is a relatively inexpensive drug that increases fviii levels and vwf activity within 30 minutes of administration. it is useful in mild haemophilia and mild von willebrand's disease. 299 the major side effects are headaches and hyponatraemia so fluid intake should be restricted to 1.5 l/ day. tranexamic acid mouthwashes may be helpful for oral mucosal bleeding. danazol can increase both factor viii and ix levels within 5-7 days 300 and has therefore been recommended for patients with recurrent haemarthrosis or with central nervous system bleeding which both carry a high risk of recurrence. most thromboembolic episodes are single events and may be associated with precipitating events or underlying risk factors. thrombophilia is the clinical state of hypercoagulability and should be suspected in patients who have a strong family history of thrombosis, or who have recurrent or unusual thromboses. increasing affluence and consequent lifestyle changes mean that the prevalence of thromboembolism is rising in some low-and middle-income countries. risk factors such as sedentary work, obesity, excessive alcohol intake, smoking and additional cardiovascular risk factors are compounded by other 10 9 /l. spontaneous recovery occurs less commonly in adults than in children. it is important to exclude other causes of thrombocytopenia such as drugs, dic or sepsis. the diagnosis can be suspected from a bone marrow examination which shows increased numbers of platelet precursors. treatment with prednisolone (0.25-0.5 mg/kg) is usually only necessary if there is bleeding or excessive bruising and the dose should be reduced slowly once the platelet count improves. second-line treatments include immunosuppressive agents and danazol. splenectomy may also be beneficial but carries an increased risk of infection. platelet transfusions or intravenous gammaglobulin can temporarily increase the platelet count in an emergency or prior to surgical procedures. inherited bleeding disorders can be classified broadly into coagulation factor deficiencies (e.g. factor viii and factor ix deficiencies), von willebrand's disease and platelet disorders. the frequency of genes for inherited bleeding disorders is the same throughout the world. haemophilia a has a prevalence of about 10/10 000, von willebrand's disease of >10/10 000 and haemophilia b of <0.1/10 000. these conditions occur more frequently among populations where consanguineous marriage is common and where prenatal diagnostic facilities are unavailable. in general, individuals with inherited coagulation factor deficiencies present with soft tissue bleeds such as haemarthroses or intramuscular bleeds. those with platelet disorders or von willebrand's disease tend to present with mucosal bleeds, however severe (type iii) von willebrand's disease can present with severe soft tissue bleeds. many of these conditions are diagnosed following excessive and uncontrolled bleeding after trauma or surgical procedures. menorrhagia and delayed severe postpartum haemorrhage may be presenting features of bleeding disorders, particularly von willebrand's disease or hypothyroidism, which can cause decreased synthesis of von willebrand factor. some inherited platelet function disorders are associated with characteristic syndromes (e.g. oculocutaneous albinism or skeletal defects) which may provide a clue to the diagnosis. early recognition of symptoms by clinicians, teachers and the public is important so that early treatment can be established. patients with inherited bleeding disorders are usually managed with blood products (box 65.20) or chemotherapy designed to reduce bleeding and associated complications. 295, 296, 298, 299 clotting factor concentrates may be imported or produced locally by fractionation of plasma and are included in the who list of essential medicines. 297, 298 one international unit (iu) of fviii clotting factor concentrate per capita is recommended as the minimum requirement for countries wishing to achieve optimal survival for their haemophilia population but only about 25% of the estimated 400 000 people in the world with haemophilia receive adequate treatment. management of patients with bleeding disorders relies on a wellequipped and quality assessed laboratory for accurate diagnosis and monitoring of treatment and access to plasma and components for replacement therapy. appropriate support services such as physiotherapy, orthopaedics and counselling should also be available. in many countries inherited bleeding disorders are associated with stigma, which is particularly directed against the mothers of affected children, 297 acute and chronic leukaemias are usually associated with a high white cell count but acute leukaemias can present with normal or even sub-normal white cell counts. morphology of peripheral blood and bone marrow specimens is crucial to confirm the diagnosis. this is particularly important in the case of acute leukaemia in children which may be mistaken for an acute viral infection. staining methods including sudan black b, myeloperoxidase and nonspecific esterase are important to distinguish between the different subtypes of acute myeloid and lymphoid leukaemias and therefore to guide treatment. acute myeloid leukaemia (aml). prevalence of this increases with age and the success rate with chemotherapy protocols is not high even in the most sophisticated centres. neutropenia and myelosuppression requiring intensive blood component support occur during chemotherapy 314 and bone marrow transplantation offers the best option for cure for patients who relapse. management of aml is therefore complex and expensive. hydroxycarbamide or subcutaneous cytarabine may be used as a palliative treatment. acute promyelocytic leukaemia (aml subtype m3). this must be distinguished from other types of acute myeloid leukaemia because it has a high cure rate with early treatment. it predominantly affects young adults and it has a high incidence in certain ethnic groups especially those of latin american descent. 315 a treatment protocol which includes all-transretinoic acid with combination chemotherapy has been developed which is feasible in low-income countries. 315, 315 another regimen based on intravenous arsenic trioxide has been developed in india, 316, 317 which has an 86% response rate with good diseasefree and overall survival. conditions that are associated with thrombosis such as hiv infection, and chronic infections including tuberculosis 301, 302 and helminth-induced eosinophilic myocarditis. 303 african americans are more likely to be diagnosed with pulmonary embolism rather than deep-vein thrombosis compared to other racial groups 304 and african patients with thrombosis tend to be younger than those reported in literature 305 with higher mortality rates (around 28%) possibly due to late presentation and poor access to health facilities. asian populations 306-308 seem to have a lower prevalence of symptomatic venous thrombosis compared to african americans. 304 very little is known about the prevalence of prothrombotic factors such as mutations of the prothrombin gene or deficiencies of antithrombin, protein c and protein s in tropical countries, although high rates of factor v leiden, a risk factor for venous thrombosis, have been described in tunisia. 309, 310 lupus anticoagulant and anti-phospholipid syndrome, which are associated with increased thrombosis risk, are increased in afro-caribbean populations, especially in the presence of hiv, and have also been described in nigerian women with pre-eclampsia. 311, 312 the management of venous thrombosis is initially with heparin and then with warfarin for 3-6 months. compliance may be difficult in low-resource settings because of the requirement for regular monitoring of warfarin. it is therefore important to try to prevent thromboses by removing any underlying risk factors and by treating individuals at risk of thrombosis with a short course of prophylactic heparin to cover procedures known to be associated with thrombosis risk. this can present as venous or arterial thromboembolism and it may be inherited (e.g. deficiencies of thrombin, protein s or protein c) or acquired (e.g. antiphospholipids). the patient's personal and family history, and the results of clinical and imaging examinations to confirm thrombosis, may suggest the diagnosis. the laboratory tests needed to determine the cause and classify the type of thrombophilia, and their interpretation, are complex, so patients with recurrent or unusual thromboses should be referred to a specialist centre. haematological malignancies are predominantly leukaemias, lymphomas and myelomas. some of the general approaches for managing these conditions in low-income countries are outlined in box 65.21 313 but definitive treatment should be undertaken by a specialist haematology unit. leukaemias can be broadly classified as acute or chronic, and lymphoid or myeloid. the presenting symptoms and signs are related to the disturbed blood cell production from the bone marrow due to the effects of the malignant cell clone (box 65.22) . acute leukaemias are characterized by rapid progression and poor prognosis if left untreated whereas chronic leukaemias generally follow a much slower course. • mobilization of the community (especially parents and families) to raise awareness among local councils and government bodies about the treatability of the cancers and benefits from curing them • find an external partner unit locally, nationally or internationally which is already well-established and willing to help but will not dictate terms • improvement of supportive care facilities, especially protection from those with infectious diseases • development of a safe and reliable blood transfusion service • provision of subsidized travel, and satellite clinics to lessen the burden • development of appropriate protocols for each disease entity which is locally practicable with minimum cost and maximum efficacy • development of medical, nursing and paramedical expertise in the diseases to be treated -initially by offering visiting fellowships and in the long term for the trained individuals to arrange regional and local teaching programmes • formation of a cooperative group bringing together all the professionals involved in the speciality within a country or region to share expertise and develop training programmes. acute lymphoblastic leukaemia (all). this is the most common type of leukaemia in children. it has a good prognosis when treated with modern chemotherapy protocols with cure rates in the best centres exceeding 80%. 318 in low-income countries, cure rates are much lower at around 35% 319 primarily because of failure to complete therapy and deaths caused by treatment. considerable improvements in all outcomes have been achieved by twinning institutions in developing countries with specialist centres elsewhere in the country or internationally. 313 measures that may improve outcomes focus on preventing abandonment of therapy (e.g. providing funding for transport, satellite clinics and support groups) and prompt treatment of infection. 320 treatment in a dedicated paediatric oncology unit using a comprehensive multidisciplinary team approach and protocol-based therapy, is also associated with improved outcomes in resource-poor settings. 321 chronic myeloid leukaemia (cml). management has been revolutionized by tyrosine kinase inhibitors (e.g. imatinib) which can produce complete remission in over 80% of cases. once the diagnosis of cml is established, hydroxycarbamide can be used to reduce the white cell count, followed by treatment with a tyrosine kinase inhibitor. manufacturers will provide the drug free of charge to patients in low-income countries with confirmed cml and generic forms of tyrosine kinase inhibitors are now becoming available. chronic lymphocytic leukaemia (cll). this occurs predominantly in older people and usually presents with lymphadenopathy and recurrent infections. treatment is with chlorambucil and prednisolone although aggressive forms require combination therapy with rituximab, fludarabine and cyclophosphamide. treatment is generally not curative but the disease may be indolent and drugs may only be required if the patient has symptoms or if there is a risk of hyperviscosity from a very high lymphocyte count. approximately 30 000 cases of non-hodgkin lymphoma (nhl) occur in the equatorial belt of africa each year (table 65 .11). 322 there are marked geographical variations in prevalence but up to 50% are thought to be related to hiv infection. 322 burkitt's lymphoma, a b-cell nhl, was originally described in children from africa and has an estimated incidence of 30-70 per million. lymphomas are broadly classified into hodgkin's lymphoma and nhl; nhl are divided into b-cell, t-cell and nk-cell, and immunodeficiency-associated types. the clinical presentation of lymphomas is characterized by enlargement of the lymphoid organs and subsequent compression of the adjacent structures, infiltration of organs by the malignant lymphoid cells and a dysfunctional immunological system which can manifest as immunosuppression or excessive but dysregulated immune activation associated with, for example, autoimmune conditions. the diagnosis and management of the various types of lymphomas are complicated and should be undertaken in a specialist box 65. 22 clinical features of leukaemias • fatigue and cardiac symptoms from anaemia • bleeding from thrombocytopenia • increased risk of infections despite a higher number but dysfunctional white cells • lymphadenopathy and hepatosplenomegaly occur with all although lymphadenopathy may be observed in the monocytic variety of aml • blindness due to hyperviscosity from hyperleukocytosis • tumour lysis syndrome due to spontaneous cell lysis presents as renal failure • pustules or pyogenic infections of the skin from minor wounds • bleeding gums are a characteristic feature of acute monocytic leukaemia • disseminated intravascular coagulation can occur with acute promyelocytic leukaemia • gout can arise from breakdown of the excess white cells and release of uric acid • oral aphthous ulceration is seen with severe neutropenia in both aml and all • granulocytic sarcoma or chloroma represent extramedullary deposits of leukaemic cells in any organ but mainly the skin. this may occur in the absence of peripheral blood involvement and is more common with chromosomal translocation (8; 21) of aml • central nervous system manifestations due to sludging of the cerebral circulation by the malignant cells or increased intracranial pressure due to ventricular blockade can occur. monocytic myeloid leukaemia can also involve the meninges • intracranial haemorrhage can occur in all with very high white cell counts (>400 × 10 9 /l) • bone pain and arthralgia can be a presenting feature of all in children in more than a quarter. these children may present with a limp or unwillingness to walk due to marrow infiltration by leukaemic cells. rarely, they may have normal blood counts delaying the diagnosis of all • anterior mediastinal mass (thymus enlargement) can also occur in children and young adults with all which may present as superior venocaval obstruction • painless enlargement of scrotum is a sign of testicular leukaemia or hydrocele from lymphatic obstruction. priapism can result from hyperleukocytosis rarely. • most often asymptomatic and usually suspected on blood counts • the chronicity of cml or cll tends to cause gradual-onset symptoms since the patients get adjusted to the slowly developing anaemia • abdominal discomfort and early satiety are a feature of cml due to excessive splenomegaly compressing the stomach and reducing the luminal volume • sternal tenderness may be noted in cml • hyperleukocytosis in cml can occur more often than with aml or all due to the gradual increase in white cells. this can cause symptoms like hyperuricaemia and gout, tinnitus, priapism or central nervous system disturbances • left shoulder tip pain can arise from splenic infarction from the massive splenomegaly in cml • cml can rarely present with features of thyrotoxicosis (heat intolerance, weight loss and excessive sweating) due to hyper-metabolism • cll is often associated with lymphadenopathy and rarely with mild to moderate splenomegaly. all, acute lymphoid leukaemia; aml, acute myeloid leukaemia; cll, chronic lymphocytic leukaemia; cml, chronic myeloid leukaemia. centre. diagnosis depends on clinical history and examination, radiological investigations to document the extent of disease, and morphology, immunohistochemistry and molecular studies on tissue samples to confirm the lymphoma subtype. guidance on the diagnosis and treatment of lymphoma in settings where resources are limited includes recommendations about panels of immunostains and chemotherapy regimens that minimize the need for supportive care. 322 tele-pathology, which involves transmitting histological images via the internet to experts overseas, may be helpful in certain circumstances though it is dependent on the quality of the histology preparations and the images of appropriate diagnostic regions in the sample. treatment regimens for lymphomas differ according to the subtype but may involve chemotherapy and radiotherapy. high remission rates can be achieved in burkitt's lymphoma with a combination of cyclophosphamide, vincristine and methotrexate and progressive disease can be managed with ifosfamide, mesna and cytosine arabinoside. 322, 323 adult t-cell leukaemia-lymphoma (atll) adult t-cell leukaemia-lymphoma (atll) is an uncommon lymphoid malignancy which occurs in patients infected with human t-lymphotropic virus type i (htlv-i). 324 htlv-1 is endemic in the caribbean, western africa, peru and southern japan. less than 5% of those infected with htlv-i develop atll and up to 30 years can elapse between the primary infection and the development of atll suggesting additional factors are needed for malignant transformation. 325 atll presents acutely in approximately 60% of cases, although chronic forms have also been described. 326 the clinical presentation is with generalized lymphadenopathy in most cases and hepatosplenomegaly in over half. 324 atll is associated with a high risk of hypercalcaemia which occurs in more than two-thirds of patients during the course of their disease and may be associated with central nervous system disturbances and renal impairment. lytic bone lesions occur as a para-neoplastic types of lymphomas identified from selected countries in sub-saharan africa phenomenon due to production of parathormone-like peptides. as with other t-cell disorders, atll can involve the skin, producing, e.g. erythrodermic plaques. the diagnosis of atll can be suspected from a high peripheral blood white blood cell count in combination with hypercalcaemia and characteristic lymphocytes with convoluted and hyperlobulated nuclei. 324 the diagnosis is confirmed by histological examination of a tissue (lymph node or bone marrow), immunophenotyping for specific cell markers and proof of htlv infection, usually by serological methods. management of atll is primarily with combination chemotherapy with intrathecal prophylaxis. 327,328 a combination of zidovudine and interferon, as agents against htlv, has also been tried with some success. 329 hypercalcaemia and opportunistic infections should be sought and treated early in these patients. the high white cell count is associated with a significant risk of tumour lysis syndrome and should be prevented by adequate hydration and the judicious use of allopurinol and other urate-reducing agents. myeloma is a monoclonal proliferation of plasma cells and it particularly affects older people. myeloma appears to be less common in asian countries than elsewhere, although during the last 25 years, an almost four-fold increase in incidence of myeloma has occurred in taiwan. 330 in the united states, the incidence of multiple myeloma in the black population is twice that of the white population. the abundant plasma cells infiltrate the bone marrow and interfere with normal haematopoiesis. this leads to anaemia, which is a presenting feature in 70% of individuals. bony infiltration by the malignant plasma cells can produce osteoporosis, lytic lesions and pathological fractures in 60% of patients with myeloma. involvement of the bones can lead to hypercalcaemia, which may be a presenting feature, and vertebral fracture leading to spinal cord compression. the malignant plasma cells produce a paraprotein which can cause renal impairment in 20-50% and hyperviscosity may ensue in 10% of patients if the paraprotein production is not controlled. patients with myeloma may need a variety of supportive interventions including management of anaemia, renal failure, hypercalcaemia, hyperviscosity, infections and bone pains. specific anti-myeloma treatment should be managed within a specialist unit and has undergone a radical change in the last decade with the use of thalidomide and its newer formulations, and the more expensive, proteasome inhibitors (e.g. bortzomib). thalidomide is relatively safe and effective although somnolence and constipation can sometimes be troublesome. there is a risk of thrombosis with thalidomide especially at the initiation of therapy, and prophylaxis with heparin, warfarin or antiplatelet agents, depending on an assessment of the risk, may be warranted. melphalan may also be useful, particularly if resources are limited and there is no specialist centre. however it is myelosuppressive, so regular monitoring of the blood count is essential. maintaining an adequate blood supply is a major challenge for low-income countries. only 39% of the global blood supply is donated in the poorest countries where 82% of the world's population lives. 331 blood transfusion is a vital component of every country's health service. it can be a life-saving intervention for illnesses such as severe acute anaemia, but mistakes in the transfusion process can be life-threatening, either immediately or years later through transmission of infectious agents. clinicians need to understand how blood is acquired and its risks and benefits, and to use it appropriately. governments and transfusion services need to put measures in place to ensure that blood is safe for transfusion and that it reaches those who need it in a timely manner. only 16% of member states meet all the world health organization's (who) recommendations for a national quality blood transfusion system. 331 at the national level the transfusion service should have a director, an advisory committee and clear transfusion policies and strategies (table 65 .12). 331 who recommend standardization of blood collection, testing and distribution. although centralization of these services may offer the best guarantee of quality, it is often not practical in countries with poorly developed communications and transport infrastructure. two systems, centralized and hospital-based, exist in lowincome countries for managing blood supply. in the centralized system, voluntary blood donors are recruited, screened and bled by regional centres and the blood collected is distributed to peripheral hospitals. hospital-based systems are the predominant source of blood across sub-saharan africa. hospital-based systems obtain blood predominantly from relatives of patients, and blood is screened and used within the local vicinity. 332 blood from the centralized system costs at least three times as much per unit as that from a hospital-based system. 333 although centralized systems can save costs through batching and bulk purchasing, the quality assurance processes and donor recruitment components are expensive and difficult to maintain without dependence on external funds. in hospital-based transfusion services, testing quality is variable and the families of patients bear the cost of finding blood donors. the vast majority of blood in low-income countries is transfused as whole blood. in high-income countries it is standard practice to optimize the use of each donation of blood by separating it into individual components but whether this approach is cost-effective in low-income countries, where indications for transfusion are different, is not known. these components, which may include plasma, platelets and cryoprecipitate, are prepared by centrifugation using a closed, sterile system and each component has different storage requirements. plasma and cryoprecipitate are kept frozen, red cells are stored at 1-5°c, and platelets at 18-22°c with constant agitation. recent evidence suggests that warm, fresh, whole blood may be better than component therapy for resuscitation of acidotic, hypothermic and coagulopathic trauma patients 334 and for patients needing massive transfusions. 335 many infections can be transmitted through blood transfusions and transfusion of infected blood causes morbidity and mortality in the recipients, and has an economic and emotional impact on their families and communities. those who become infected through blood transfusion are infectious to others and contribute to the spread of disease thereby increasing the burden on health services and reducing productive labour. strategies for recruiting blood donors have to provide blood for all who need it in a timely manner while ensuring that the blood is as safe as possible. the safest type of blood donor is one who donates regularly (i.e. repeat donors). who states that the safest source of blood is altruistic, voluntary, unpaid donors. only 32% of who member states report having at least 90% of their blood supply from voluntary donors, and low-income countries have not been able to increase the recruitment of voluntary donors for several years. 331 recent evidence from sub-saharan africa indicates that the focus on voluntary donors may be misplaced since first-time voluntary donors have a similar prevalence of transfusion-transmitted infections as family replacement donors. 336 in order to limit blood shortage and maintain constant blood supply in poorer countries, both voluntary and replacement donors should be accepted and encouraged to donate regularly. mechanisms to convert family replacement donors into repeating voluntary donors have the potential to significantly increase blood donations in africa. political will and open-mindedness about ways to improve the supply and safety of blood are essential to promote more evidence-based approaches to blood transfusion practice in poorer countries. 337 supporting strategy in wealthy countries, the majority of transfusions are carried out electively. by contrast, in poorer countries, and particularly those where the malaria transmission rate is high, most transfusions are given for life-threatening emergencies. in low-income countries, 50-80% of transfusions are administered to children, predominantly for malaria-related anaemia, and pregnant women. transfusion can significantly reduce the mortality of children with severe anaemia within the first 2 days of hospital admission 347 and successful malaria control can reduce paediatric transfusion requirements. 348 in sub-saharan africa, 26% of in-hospital maternal deaths from severe bleeding were due to lack of blood for transfusion. 349 other specialities which are significant users of blood are surgery, trauma, emergency medicine and general medicine. in low-income countries the most effective way to avoid transfusions is to reduce the prevalence of anaemia. more studies on the efficacy and cost of combinations of interventions including insecticide-treated bed nets, nutritional supplements and anthelmintic drugs to prevent anaemia are needed. when resources are very limited, governments may need to make some difficult decisions in order to achieve an equitable balance between investing in a transfusion service and public health measures to reduce anaemia. whether a patient needs a blood transfusion or not is ultimately a clinical decision. emergency transfusions can be lifesaving for patients in whom anaemia has developed too quickly to allow physiological compensation, as in severe malariarelated anaemia in children, and sudden, severe obstetric bleeding. in contrast, if the anaemia has developed slowly, for example due to hookworm infestation or nutritional deficiency, patients can generally be managed conservatively by treating the cause of the anaemia and prescribing haematinic replacements. iron supplements should be continued for at least 3 months after the haemoglobin has returned to normal, so that body stores can be replenished. clinical guidelines. it is possible to avoid unnecessary transfusions by adhering to clinical transfusion guidelines. most institutions have developed guidelines to help clinicians make rational decisions about the use of blood transfusions (box 65.23) 344, 350 and strict enforcement of transfusion protocols can significantly reduce avoidable transfusions. 351 the principles underlying most transfusion guidelines are similar and combine a clinical assessment of oxygenation, with haemoglobin measurement being used as a surrogate measure for intracellular oxygen concentration. increasingly, transfusion guidelines are making use of evidence which shows that adequate oxygen delivery to the tissues can be achieved at haemoglobin levels that are significantly lower than the normal range. 352 implementation of transfusion guidelines is particularly difficult if clinicians do not have access to reliable haemoglobin high-risk donors, such as commercial sex workers and their contacts, intravenous drug abusers, or those with an itinerant lifestyle such as traders, drivers and military personnel, should be deterred from donating. 338 even in areas where hiv infection rates in the general population are high, donor deferral can be effective in excluding hiv-infected donors. 339 the whole donation process, including tests for hiv and other infections, should be explained to the donor before blood is collected and donors should have the option of knowing the results and receiving counselling. it is imperative that complete confidentiality is maintained throughout all procedures. infections with organisms such as hiv, hepatitis viruses, cytomegalovirus, syphilis, lyme borreliosis, malaria, babesiosis, american trypanosomiasis (chagas disease) and toxoplasmosis can all be acquired through blood transfusions. some 5-10% of hiv infections worldwide are thought to have been transmitted through the transfusion of infected blood and blood products. there have also been reports of transmission of variant creutzfeldt-jakob disease through blood transfusion and there is a theoretical risk of transmission of severe acute respiratory syndrome (sars). 340, 341 who recommends that all donated blood should be screened for hiv, hepatitis b and syphilis and, where feasible and appropriate, for hepatitis c, malaria and chagas disease. malaria can be transmitted by blood transfusion and, depending on the local infection prevalence, 2-55% of blood donors in africa screen positive for malaria. 342 however, there is very little evidence to suggest that these donors transmit malaria to transfusion recipients. although who recommends screening donors in endemic areas for malaria, none of the screening methods that would be practical for transfusion services are sufficiently sensitive. furthermore, in some countries with high malaria transmission, exclusion of parasitaemic donors could result in deferral rates exceeding 50% which would have a major impact on blood supply. 343 there is no evidence to support the widespread practice of routine treatment of transfusion recipients for malaria. fresh blood is potentially infectious for syphilis, but storage at 4°c for more than 5 days can inactivate treponema pallidum. the high demand for blood in low-income countries means that blood is generally not stored for long enough to inactivate t. pallidum and syphilis seroconversion associated with transfusion has been reported from africa. 344 globally, the prevalence of hepatitis c, htlv-1 and -2 and chagas disease is variable and the decision to introduce donor screening for these infections should be based on local assessments of the risks, benefits, feasibility and costs. blood should not be separated into components if the residual risk of infection is high, as this will increase the number of potentially infected recipients. a unit of blood is usually stored until screening tests for infections have been completed. this means that potentially infected blood may be mixed up with units that have already been screened, and costly blood collection bags are wasted. screening potential donors before venesecting a unit of blood may therefore be a more cost-effective way of ensuring safe blood. 345 tests for screening blood donors need to be highly sensitive, and infected blood should be rejected. before informing the donor of the outcome, all positive results should be confirmed using a test with a high degree of specificity. where blood or that the blood may become infected with bacteria during the process. intraoperative blood salvage. this involves collecting blood lost during the operation and reinfusing it into the patient either during or after surgery. although this technique is practical and safe, and reduces the need for donor blood by 27-53%, 355 it requires specialized equipment and training, and may be more expensive than routinely donated blood. 356 other measures. normal saline or intravenous replacement fluids can be used judiciously in acute blood loss, and in certain circumstances may be as effective as whole blood, red cells or plasma. erythropoietin, which stimulates endogenous red cell production is well-established for use in chronic anaemias such as those due to renal failure, cancer and hiv infection but its delayed action makes it unsuitable for use in acute anaemias. synthetic oxygen carriers, such as perfluorocarbons, are not yet routinely available. 357 in low-income countries, the recommended haemoglobin threshold for transfusions is often well below that which would be accepted in more wealthy countries. randomized controlled studies in wealthy countries indicate that for most adults and children undergoing critical care, a haemoglobin threshold of 70 g/l for transfusion is safe 358 whereas paediatric blood transfusion protocols in sub-saharan africa often recommend transfusions for stable children only when the haemoglobin level is less than 40 g/l. 351 complications such as cardiac failure or infection may necessitate transfusion at a higher haemoglobin level. transfusion should be combined with adequate haematinic replacements and underlying conditions should be treated. 359 early evidence suggests that intermittent preventive treatment with anti-malarials may reduce the high hospital readmission rates experienced by children post-transfusion. 360 complications can occur immediately during transfusion, within a few hours of its completion, or be delayed for many years, as in the case of viral infections (box 65.24). measurements. when they doubt the haemoglobin result, clinicians rely entirely on clinical judgement to guide transfusion practice which can lead to significant numbers of inappropriate transfusions. 353 a lack of investment in the quality of a critical test, such as haemoglobin measurement, can waste significant resources downstream in the transfusion process, and unnecessarily expose recipients to the risk of transfusion-related infections. minimizing surgical blood loss. where blood is in short supply, it is particularly important to ensure that the best anaesthetic and surgical techniques are used, to minimize blood loss during surgery. drugs which improve haemostasis or reduce fibrinolysis, such as aprotinin and cyklokapron, and fibrin sealants, can be effective in reducing perioperative blood loss. these drugs can therefore reduce the need for blood transfusion but they may be too expensive for use in low-income countries. a cost-effectiveness study of surgical bleeding in four sub-saharan countries indicates that the antifibrinolytic, tranexamic acid, could save lives in countries with blood shortages, reduce healthcare costs and prevent transmission of infections. 354 preoperative autologous blood deposit. patients undergoing planned surgery who are likely to require a blood transfusion can have units of their own blood removed and stored in case they have significant intraoperative blood loss and need a transfusion. this process, known as preoperative autologous donation, can reduce the need for allogeneic transfusions by 46-74% 355 but it requires careful organization: the surgeon needs to predict how much blood will be required, the patient has to be fit enough to withstand removal of one or more units of blood over the weeks preceding the surgery and the surgery must take place within the shelf-life of the blood. as the blood has to be stored in the blood bank there is still a risk that the patient may receive blood which is not their own box 65.23 prescribing blood: a checklist for clinicians always ask yourself the following questions before prescribing blood or blood products for a patient: 1. what improvement in the patient's clinical condition am i aiming to achieve? 2. can i minimize blood loss to reduce this patient's need for transfusion? 3. are there any other treatments i should give before making the decision to transfuse, such as intravenous replacement fluids or oxygen? 4. what are the specific clinical or laboratory indications for transfusion in this patient? 5. what are the risks of transmitting hiv, hepatitis, syphilis or other infectious agents through the blood products that are available for this patient? bacterial contamination and should be investigated and managed accordingly. allergic reactions are due to infusion of plasma proteins and manifestations include erythema, rash, pruritus, bronchospasm and anaphylaxis. the transfusion should be stopped and the patient treated with antihistamines. if the reaction is mild and the symptoms and signs completely disappear, the transfusion can be restarted. if this type of mild reaction occurs repeatedly with more than one unit of blood, the red cells can be washed before transfusion. this should only be done if absolutely necessary, as it carries the risk of introducing potentially fatal bacterial infection. severe allergic reactions with evidence of systemic toxicity should be managed as acute anaphylaxis. blood should always be transfused slowly to avoid overloading the circulation, unless the patient has active and severe bleeding. fluid overload may be a particular problem when paediatric blood bags are not available, as children may be over-transfused due to miscalculation of the required volume, lack of accurate infusion devices or inadvertent administration of an adult-sized unit of blood. four units of blood contain the equivalent amount of iron stored in bone marrow (approx. 1 g). repeated transfusions for chronic haemolytic anaemia, as in thalassaemia major and sickle cell disease, lead to iron deposition in parenchymal cells. eventually failure of the heart, liver and other organs supersedes. adequate doses of iron chelators, such as injectable desferrioxamine or oral deferiprone, are able to maintain acceptable iron balance in patients with chronic anaemia who need regular transfusions. it is not usually necessary to warm blood unless large quantities are transfused rapidly. this may lower the temperature of the sino-atrial node to below 30°c at which point ventricular fibrillation can occur. if blood needs to be warmed, an electric blood warmer specifically designed for the purpose should be used. this keeps the temperature below 38°c and avoids the haemolysis associated with overheating blood. graft-versus-host disease occurs when donor lymphocytes engraft in an immune-suppressed recipient. the lymphocytes recognize the recipient's bone marrow as foreign and induce aplasia. graft-versus-host disease is almost universally fatal and can be prevented by irradiating the donor blood, which inactivates the donor lymphocytes. transfusion of blood into a recipient who possesses antibodies to the donor's red cells can cause an acute, and occasionally fatal, intravascular haemolysis. this could occur, e.g. if group a cells are transfused into a group o recipient who has naturally occurring antibodies to group a cells. the profound haemolysis induces renal vasoconstriction and acute tubular necrosis. treatment involves stopping the transfusion, cardiorespiratory support and inducing a brisk diuresis. in addition to abnormalities indicating renal failure, laboratory findings include haemoglobinuria and haemoglobinaemia. proof of the diagnosis involves rechecking the whole transfusion process including all documentation stages, regrouping the donor and the recipient, and screening for antibodies on red cells with a direct antiglobulin test. these tests are usually available in any hospital laboratory capable of providing a transfusion service. delayed haemolysis has a similar physiological basis to acute intravascular haemolysis but it tends to be less severe, it occurs 7-10 days after the transfusion and it is less likely to present as a clinical emergency. limited data from sub-saharan africa show rates of bacterial contamination in donated blood of around 9% 361,362 but the clinical consequences for transfusion recipients are unknown. bacteria can enter the blood bag during venesection or if the bag is breached, e.g. when reducing the volume for a paediatric recipient or during component preparation. gram-negative bacteria, including pseudomonas and yersinia, grow optimally at 4°c and infected blood may not necessarily appear abnormal to the naked eye. reactions following infusion of infected blood are often due to endotoxins and may occur several hours after the transfusion has finished. although these reactions are rare, they can be severe and fatal. if bacterial contamination is suspected, the transfusion should be stopped and samples from the patient and the blood bag sent to the laboratory for culture. cardiorespiratory support may be needed and broad-spectrum antibiotics should be started immediately and continued until culture results are available. non-haemolytic febrile reactions are episodes of fever and chills associated with transfusion and for which no other cause can be found. they are due to the recipient's antibodies reacting against antigens present on the donor's white cells or platelets. these reactions are most common in patients who have had transfusions in the past and have therefore 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transfusion on survival among children in a kenyan hospital changing trends in blood transfusion in children and neonates admitted in kilifi district hospital maternal mortality in sub-saharan africa: the contribution of ineffective blood transfusion services press release who/25. 2000. whd/3 information sheet for clinicians development and evaluation of a new paediatric blood transfusion protocol for africa electrocardiographic st-segment changes during acute, severe isovolemic hemodilution in humans use of clinical judgement to guide administration of blood transfusions in malawi giving tranexamic acid to reduce surgical bleeding in sub-saharan africa: an economic evaluation autologous transfusion techniques: a systematic review of their efficacy intraoperative autologous blood management artificial o2 carriers: status in 2005 red blood cell transfusions in acute paediatrics survival and haematological recovery of children with severe malaria transfused in accordance to who guidelines in kilifi intermittent preventive therapy for malaria with monthly artemether-lumefantrine for the post-discharge management of severe anaemia in children aged 4-59 months in southern malawi: a multicentre, randomised, placebocontrolled trial bacterial contamination of pediatric whole blood transfusions in a kenyan hospital bacterial contamination of blood and blood components in three major blood transfusion centres in accra, ghana access the complete references online at www.expertconsult.com key: cord-339705-x8l3zgfd authors: patil, vijaykumar; ingle, d. r. title: an association between fingerprint patterns with blood group and lifestyle based diseases: a review date: 2020-08-18 journal: artif intell rev doi: 10.1007/s10462-020-09891-w sha: doc_id: 339705 cord_uid: x8l3zgfd in the current era of the digital world, the hash of any digital means considered as a footprint or fingerprint of any digital term but from the ancient era, human fingerprint considered as the most trustworthy criteria for identification and it also cannot be changed with time even up to the death of an individual. in the court of law, fingerprint-proof is undeniably the most dependable and acceptable evidence to date. fingerprint designs are exclusive in each human and the chance of two individuals having identical fingerprints is an exceptional case about one in sixty-four thousand million also the fingerprint minutiae patterns of the undistinguishable twins are different, and the ridge pattern of each fingertip remain unchanged from birth to till death. fingerprints can be divided into basic four categories i.e. loop, whorl, arch, and composites, nevertheless, there are more than 100 interleaved ridge and valleys physiognomies, called galton’s details, in a single rolled fingerprint. due to the immense potential of fingerprints as an effective method of identification, the present research paper tries to investigate the problem of blood group identification and analysis of diseases those arises with aging like hypertension, type 2-diabetes and arthritis from a fingerprint by analyzing their patterns correlation with blood group and age of an individual. the work has been driven by studies of anthropometry, biometric trademark, and pattern recognition proposing that it is possible to predict blood group using fingerprint map reading. dermatoglyphics as a diagnostic aid used from ancient eras and now it is well established in number of diseases which have strong hereditary basis and is employed as a method for screening for abnormal anomalies. apart from its use in predicting the diagnosis of disease; dermatoglyphics is also used in forensic medicine in individual identification, physical anthropology, human genetics and medicine. however, the machine and deep learning techniques, if used for fingerprint minutiae patterns to be trained by neural network for blood group prediction and classification of common clinical diseases arises with aging based on lifestyle would be an unusual research work. the study of fingerprint patterns was introduced by dr. harold cummins in 1926 but it is already in use before several hundred years ago. fingerprint patterns have been normally used for identification of an individual. now a days every organization or even may government institutes in india, use fingerprint verification to identify everyone uniquely and it also have been used as a biometric modality for gender and age identification. an individual is their own key; behind this catchy principle biometrics have become an attractive alternative to traditional identification methods such as tokens or passwords (fernandes et al. 2013) . current fingerprint matching methods were started in the sixteenth century. it was henry fauld in 1880 who first experimentally proposed the singularity and uniqueness of fingerprint. herschel (ravindran et al. 2017) added to the establishment of current fingerprinting identification. in the nineteenth century sir francis galton (mcbean et al. 2014 ) directed broad investigations and ordered the sorts of fingerprints relying on essential example as loops, whorls and arches. it was cummins (ferraz et al. 2010 ) who authored the expression "dermatoglyphics (derma ¼ skin, glyphic ¼ bends), to dermal edge arrangements on the digits, of palms and sole and furthermore demonstrated that edge design are resolved incompletely by heredity or natural impact which produce pressure and strain in their development during fetal life. the fingerprint design whorl might be winding, oval, roundabout or any assortment of a loop and record for around 30%. arches are the basic type up till now uncommon (about 5%). the fingerprint design has edges running from one side to the opposite side of the print without having any re-bend. the term composite is utilized for mix of type example that doesn't fit into any of the above characterization (azhagiri et al. 2018) . till date, analysts or researches have generally used fingerprint details as perspectives to build up any individuals uniqueness. the ridge patterns have been comprehensively classified into five different kinds called as arch, tented arch, whorl, ulnar and radial loop. an individual can have any of the above type in any of the its fingers. all things considered, dominant part of fingerprints found in populace review shows that 70 percent of the prints are loops, 20-25 percent being whorls though just 5 to 10 percent consider arch or tented arch patterns or designs (singh and majumdar 2015) . some examinations done on twins have presumed that monozygotic or indistinguishable twins have comparable however not indistinguishable examples found. dermatoglyphics as a diagnostic aid used from ancient eras and now it is well established in number of diseases which have strong hereditary basis and is employed as a method for screening for abnormal anomalies. fingerprint minutiae patterns of ridges are determined as unique through the combination of genetic and environment factors. person identification using fingerprint algorithms are well sophisticated and are being established all over the world for security and authentication. the fingerprint also used to classify gender and age group but very few manual attempts have been made to explore relationship between fingerprint patterns with blood group and common clinical diseases like hypertension, type-2 diabetes and arthritis. it will be helpful for anthropologists to predict blood group and classify common clinical diseases than conventional pathological techniques from the fingerprints those are obtained from mined articles using deep learning techniques for early reminder to prevent such common clinical diseases those arises with aging and also crime investigators to minimize the range of the suspects it would be predicted using deep neural network. the dermatoglyphics and its important role in the diagnosis of different diseases like hypertension, type-2 diabetes and arthritis with genetic bases. apart from its use in predicting the diagnosis of disease; dermatoglyphics is also used in forensic medicine in individual identification, physical anthropology, human genetics and medicine. the research work is aimed in developing the deep neural network algorithms for accurate classification of the fingerprints obtained which include: • to enhancement fingerprint image during sampling or in data set preparation step fingers of an individual recorded using fingerprint scanner. to enhance the fingerprint images precisely, the research focuses to develop various pre-processing algorithms like-segmentation, normalization, orientation estimation, ridge frequency estimation, gabor filter and binarisation and thinning etc. • to extraction of features from fingerprints and finding similarity vector to build similarity vector using features of captured sample images of fingerprint required a feature extraction algorithm. the implementation of the biometric features extraction algorithms needs to extract features like-the ridge count, ridge thickness to valley thickness ratio (rtvtr), white lines count, ridge count asymmetry, minutiae map(mm) orientation collinearity maps(ocm), gabor feature maps(gfm), orientation map (om) for pattern type, 2d wavelet transform (dwt) • to predict blood group by using extracted feature of fingerprints the unsupervised machine learning technique will apply for classification of blood group which helps to identify relationship patterns of different features of fingerprints with abo blood type and then prediction will perform with the application of machine learning and convolutional neural network (cnn) technology with the help of rigid frequency count and distance formula to conclude blood group from feature vector. • to classify and analyse lifestyle diseases like hypertension, arthritis and diabetes normally common clinical diseases arise with the age but, now in current era these are no more only relevant to the age; due to busy schedule or lifestyle of an individual they arise at any stage of life. with the fingerprint images and blood group of an individual, the dataset include the external attributes like age, weight, height, skin color, eyes color, work nature, eating habits (vegetarian or non-vegetarian), region (rural or urban), addiction (if any like drink, smoke), etc. the rest of the paper is organized as follows. the conceptual background discussed in sect. 2. the literature review specificity discusses all the methods used in sect. 3 and the evaluation and discussion included in sect. 4 which illustrates the summary of different dataset/samples and methodologies used. finally, in sect. 5, we conclude the paper. the common types of fingerprint are as arch, tented arch, whorl, ulnar and radial loop, the fig. 2 shows the different types of whorl patterns from fingerprint design. a whorl is portrayed by two deltas and one focal roundabout center. the center may have various examples. it might be winding, concentric circles, vertically compacted circles or even of the state of eye of a peacock quill. the edges start from one end, rise and hover towards the middle and go down towards the opposite end. recent advanced studies in genetics and developmental biology have guaranteed that the various projections of the human mind are physiologically associated with various fingers of both the hands. the practical coordination represented by the left half of the cerebral side of the equator is identified with the fingers of the right hand and the other way around. consequently, the focalized left half of mind is associated with the fingers of right hand, the thumb is facilitated by the unrivaled frontal projection, index is associated with the mediocre frontal flap, middle finger with parietal projection, ring finger with the fleeting projection and little finger with the rear piece of cerebrum, which is the occipital flap. correspondingly, the left half of the mind is associated with similar flaps of the cerebrum. each projection zone is liable for a portion of the other impression of the encompassing (singh and majumdar 2015) . tented arch is a pattern that is portrayed by a straight upstanding edge at the center of a straightforward arch pattern. loops are the most ordinarily up-to-the-minute highlights on a person's fingerprints just as in a subjective example space of a few fingerprints shown in figs. 1 and 2. they are described by edges that start trickling out of a crosswise of the fingertip, circle from place to place the focal point of finger cushion, and back to a similar course where they began from. these loops can either flee from the thumb. because of an individual area of arm bones-radia and ulna, any loop opening endlessly from thumb is an ulnar loop, and the one which opening near the thumb is a radial loop. a spiral whorl is described round patterns that are fit as a fiddle at the core or center. this pattern has two deltas at the two corners. the concentric whorl pattern is showed by having concentric rings of edge patterns. lengthened whorl pattern is described by a long oval whorl flanked by two triradial on either side. every single other component of a whorl is likewise present right now a pattern. it is one of the uncommon fingerprint patterns. it appears to be a tai-chi pattern at the inside or the center, encompassed by different roundabout layers of edges. since the image has two symmetric yet oppositely situated arrangements, the subjects having imploding whorls grandstand doublemindedness. composite whorl or twofold circle is one of the uncommon fingerprint patterns. it is either present on thumb or at most, the index finger. it is once more, one of the uncommon whorl patterns that contain a peacock's eye-molded circle contained inside a whorl. the center comprises of more than one spiral which are lined by a straight line at one of the corners. it to some degree seems as though the pattern on a peacock's tail quills. this pattern ought to have one triradius on either the left or right side. at the point when a pattern can't unmistakably have the option to sort into any of the above pattern types, comprises a blend of at least two above talked about patterns like a combination of concentric whorl and transformed loop and so on., it is professed to be a variation pattern. they don't contain any plain arch or loop, be outspread or ulnar. they do exclude any regular pattern. although people have been utilizing fingerprints as a methods for recognizable proof for quite a while however right now, have put forth an attempt to make stride further to "study a connection between pattern of fingerprint and abo rh blood group", with the goal that one can get a thought regarding the normal blood group from the investigation of finger impression pattern and the other way around. natural attributes like fingerprints and blood groups can't be overlooked and imitated like keys, passwords, and so on subsequently are viewed as increasingly dependable, true and solid in scientific sciences. other than an investigation of "blood group" commonness in itself isn't just significant for transfusion medication yet additionally for organ transplantation and hereditary research, forecast of specific malignancies/infections for certain blood groups just as in advancement contemplates that help researchers to comprehend the spot person involve in development's stretching tree (fayrouz et al. 2011) . different types of fingerprint patterns are as follows: there are four unlike whorl patterns as: the plain whorl, the central pocket loop, the double loop, and the accidental whorl also it has different kinds of form shown in figs. 3, 4 and 5. their normal highlights are that they have at any rate two deltas and at least one of the ridgelines bends around the center to shape a circle or winding or other adjusted, continually bending structure. the accidental whorl can be any pattern or blend of patterns that don't fit into any of the above characterizations. the expression "composite" is utilized to portray such patterns. positive distinguishing proof utilizing fingerprints can be set up just if 16 to 20 purposes of closeness exist in the minutiae (kanchan and chattopadhyay 2006; vij 2005; subrahmanyam 1999 ). arches are the most straightforward patterns and furthermore the rarest. there are two sorts: plain arches and tented arches. in these two types, the ridgelines stream into the print from one side, an ascent in the pattern, and stream out to the opposite side of the print. loops are shaped by ridgelines that stream in from one side of the print, clear up in the middle like a tented arch, and afterward bend back around and stream out or will in general stream out as an afterthought from where they entered. loops are assigned as being either spiral or ulnar, contingent upon which side of the finger the lines enter. the loop is the most well-known of the considerable number of patterns. target/concentric whorl spiral whorl concentric whorl elongated whorl imploding whorl imploding whorl accidental whorl or variant accidental whorl or variant most programmed frameworks for unique fingerprint examination depend on minutiae coordinating minutiae are neighborhood discontinuities in the finger impression pattern. an aggregate of 150 distinctive minutiae types have been recognized some of the minutiae forms shown in fig. 6 . the ridge closure and ridge bifurcation minutiae types are employed as unique mark of acknowledgment. now a day's biometric applications are designed for • authentication or identification of an individual applications like e-records security, cellular phones access, medical records management, library access and virtual learning etc. • e-governance: like, digital signature, aadhar cards, driver's licenses, border travel control, passport control, and welfare-disbursement etc. • digital forensic: such as, corpse identification, criminal investigation, terrorist identification, parenthood determination, and missing children (ramasubramanian and alexander 2009 ). blood group structures were discovered way back in 1900 by karl landsteiner. total 19 foremost groups have been identified which vary in their occurrence of spreading various races of mankind. clinically, only 'abo' and 'rhesus' groups are of major importance. 'abo' system is additional discriminated as a, b, ab, o blood group types according to presence of corresponding antigen in plasma ). yet another biological record that remains unchanged throughout the lifetime of an individual is the blood group. determining the blood group of a person from the samples obtained at the site of crime, helps identify a person. landsteiner classified blood groups under the abo blood group system (imaq 2004) . dermatoglyphics as a diagnostic aid used from ancient eras and now it is well established in number of diseases which have strong hereditary basis and is employed as a method for screening for abnormal anomalies. the study of possible predilection of certain disease and malignancies from blood groups are some of the factors which encourages one to carry the study further. fingerprint minutiae patterns of ridges are determined as unique through the combination of genetic and environment factors. the identification of minutiae shown in fig. 6 , it shows minutiae like ridge ending & bifurcation. dermatoglyphics and its important role in the diagnosis of different diseases like hypertension, type-2 diabetes and arthritis with genetic bases. machine learning is getting popular in all industries with the main purpose of improving revenue and decreasing costs; by using machine learning technique they automate and optimize their process to solve challenging tasks very efficiently. the proposed research work aims in creating a system that finds the relationship between blood group and minutes patterns of fingerprints which will be helpful to predict blood group and common clinical diseases of an individual by analyzing its fingerprints. the fingerprint having basic four categories which are loop, whorl, arch and composites but also there are more than 100 interleaved ridge and valleys which explore unique characteristics of an individual which will help to design deep neural network or convolutional neural network (cnn) which predict blood group and common clinical diseases like hypertension, type 2-diabetes and arthritis. all ten fingerprints will be acquired in real time of both male and female from different age group and from various locations of country by using optical fingerprint scanner with external characteristics to from the large dataset as process. the fingerprint data will be acquiring using fingerprint scanner. all fingers of an individual are scanned to build dataset for training model with other necessary data collected by simple registration form where external attributes like age, weight, height, skin color, eyes color, work nature, eating habits (vegetarian or non-vegetarian), region (rural or urban), addiction (if any like drink, smoke), etc. will be recorded. the obtained fingerprint from database goes through various preprocessing stages for enhancement and removing the noise before feature extraction process which include segmentation, normalization, orientation estimation, ridge frequency estimation, gabor filter, binarisation and thinning from which the orientation estimation and ridge frequency estimation. after the preprocessing of fingerprints, it goes through four steps of feature extraction, one is frequency domain feature vector obtaining by undergoing image through different levels of processing to build feature vector from acquired finger dataset. the all combined vectors within the dataset will then be allowed to pass for unsupervised training model. it uses clustering technique which helps to find similarity measure or relationship between features of fingerprint and external attributes. the clusters formed by unsupervised learning attempts to build neural network model which is deep neural network technique including features extracted from fingers and external attributes of an individual, it will be used to generate predictive model to know the blood group of an individual as well as it used to analyses disease arises with aging. the deep neural network model uses similarity measures or minimum distance for the entire combined feature vectors database. the proposed study divided into four different cases as prediction of blood group, analysis and classification of hypertension disease, analysis of arthritis disease and analysis of diabetics disease. to work out the blood group of an individual, red cells of that individual are blended in with various neutralizer arrangements. if, for instance, the arrangement contains hostile to b antibodies and the individual has b antigens on cells, it will cluster together. on the off chance that the blood doesn't respond to any of the counter an or hostile to b antibodies, it is blood group o. a progression of tests with various kinds of antibodies can be utilized to distinguish blood group. on the off chance that the individual has a blood transfusion, the blood of the individual will be tried against an example of contributor cells that contains abo and rhd antigens. if there is no response, contributor blood with a similar abo and rhd type can be utilized. it shows that the blood has responded with certain antibody and is hence not perfect with blood containing that sort of counteracting agent if the blood doesn't agglutinate, it demonstrates that blood doesn't have antigens restricting the extraordinary immune response in the reagent. in the current framework, the blood group is resolved physically. right now, arrangements, for solutions such as anti-a, anti-b, anti-d to the three samples of blood occurred. after some time, agglutination might happen. contingent on the agglutination, the blood group can be controlled by the individual physically. the weaknesses of this framework are more odds of human blunders are conceivable. only specialists can tell the blood type by observing at the agglutination procedure. the traditional method of distinguishing the blood group is usually the plate test and the tube test . both of which are performed by under comprehensive analog procedures with human observation. in the current era of digitization, it is not an efficient way to handle such a basic yet indispensable medical technique in a full analog atmosphere. there are also a few techniques such as micro plate testing and gel centrifugation ramasubramanian and alexander 2009) . fernandes et al. (2015) presented result in his research paper allow concluding that abo, rh phenotype, reverse, and crossmatching individuals blood group is possible with the developed device and procedure. they proposed device that allows blood type identification near the patient, outdoor a conventional laboratory, without the need of to be a specialized assistant to interpret the test result of blood, and in a very short time (5 min). the fast response time by device enables us it will be used in emergency situations, which is an advantage compared with the automatic commercial systems used in clinical laboratories (in average, response time of 30 min). in addition, the methodology and test protocol applied to the sample's preparation is simple, without the need of sample dilutions or incubations. the prototype was implemented with noncomplex electronic components for a low-cost device. the implemented device distinguishes agglutinated from non-agglutinated samples using a classification algorithm (developed by the authors), based on the variation of od discrete values of samples, for each blood test. the device operation was validated for abo, rh phenotype, reverse, and crossmatching human blood typing based on donor's blood samples provided by the ipst and test results agreed with their typing using their gold standard commercial and automatic systems. the examination group is working in the advancement of programmed and scaled down gadgets for clinical applications. a case of this work is the advancement of a scaled down, minimal effort, versatile and programmed framework to blood typing in crisis circumstances, considering a spectrophotometric approach and within the sight of agglutination (cooperation between red blood cells' surface and explicit reagents). the use of a basic and quick exploratory convention permits deciding blood typing and empowers the structure of an electronic programmed framework. this framework will be helpful to decrease a few impediments of the current frameworks and techniques to blood typing. the outcomes can be influenced by a few variations that makes more enthusiastically the structure of a programmed framework, for example, the trial framework utilized for spectrophotometric estimations; the agglutination quality, which influence the contrasts among control and test samples; the time spent in test readiness since it is important blood and reagents weakening; and perform spectra estimation as quickly as time permits in light of the fact that the agglutinated cells continuously will in general settle in the base of the cuvette. proposed method by fernandes et al. (2013) depends on the examination of the rh phenotypes in human blood type dependent on the plate test and utilizing a spectrophotometric approach. this examination will be remembered for the versatile gadget recently created by the exploration group for deciding abo human blood type. in this way, this paper presents the rh phenotype assurance, including the d, c, c, e and e antigens, utilizing optical retention estimations, in the obvious range, to recognize an agglutinated test (cooperation among antigens and antibodies) from a non-agglutinated test (no association). to decide the nearness or nonappearance of every antigen five samples were set up by setting 50 f. ll of the particular reagent and 12.5 f.ll of entire blood in the plate, as depicted in the reagents manual. every arrangement was blended for around one moment in a region of 2.5 cm2. at that point, the plate was situated in the estimating set-up of the spectrophotometer. an o.d. range estimation of the 50 f.ll reagents was likewise important to set the pattern and to additionally adjust the necessary gadgets. the authors presents the standards for the improvement of a scaled down, ease, compact and programmed framework, in view of a spectrophotometric approach, are introduced. the framework will have the option to decide abo and rh blood types in a brief timeframe and in situ, which is reasonable to crisis circumstances and permit the blood typing outside an ordinary clinical research facility. for that, the essential components of the framework ought to be: a light source, a light receptor and a microcontroller. 1. approval of the general test convention the convention applied in the framework use blood samples (from the portuguese blood institute) and business antibodies as reagents (monoclonal anti-an, anti-b, anti-ab and polyclonal anti-d from hos lab diagnostic). four test samples should be set up for each blood test. each test is acquired by blending blood in with a immune response. blending blood in with the reagents it tends to be acquired two kinds of samples: agglutinated, if there is antigen-counter acting agent connection; or non-agglutinated if there is no association. for instance, blending a positive blood type with anti-a, anti-ab or anti-d it is gotten an agglutinated test since this blood type has the a and d antigens. with the anti-b, it is gotten from a non-agglutinated test. 2. scaling down of the test framework after the approval of the spectrophotometry estimations to human blood typing, with the use of a quick and straightforward convention, the following stage was the execution of a particular light source framework by utilizing light emitting diodes (leds) and a photodiode estimating gadget (s1336-5bq photodiode from hamamatsu), e.g., keeping away from the massive and costly framework dependent on a light source and monochromator. ramasubramanian and alexander (2009) a coordinated fiberoptic a microfluidic gadget for the location of agglutination for blood type crossmatching has been portrayed. the gadget comprises of a straight microfluidic channel through with a responded rbc suspension is siphoned with the assistance of a syringe siphon. the stream meets an optical way made by a producer got fiber optic pair incorporated into the microfluidic gadget. a 650 nm laser diode is utilized as the light source and a silicon photodiode is utilized to recognize the light power. the separating between the tips of the two optic filaments can be balanced. at the point when fiber separating is enormous and the centralization of the suspension is high, the dispersing wonder turns into the prevailing system for agglutination identification while at low focuses and little dividing, opto-interruption turns into the predominant component. an agglutination quality factor (asf) is determined from the information. studies with an assortment of blood types demonstrate that the detecting technique effectively distinguishes the agglutination response in all cases. a dispensable coordinated gadget can be intended for future usage of the strategy for the close bedside pre-transfusion check. stomach muscle positive blood type will respond with against a, hostile to b, and against d antibodies and cause agglutination. henceforth, we just present outcomes for ab positive sort in detail right now. the information is illustrative of the outcomes acquired mouad. ali et al. (2016) proposed fingerprint recognition framework is separated into four phases. first is the acquisition stage to catch the fingerprint picture, the second is the preprocessing stage to enhancement, binarization, thinning fingerprint picture. the third stage is the feature extraction stage to remove the element from the thinning picture by use minutiae extractor strategies to separate ridge ending and ridge bifurcation from thinning. the fourth stage is coordinating (identification, verification) to coordinate two minutiae focuses by utilizing the minutiae matcher technique in which closeness and distance measurements is utilized. the calculation is tried precisely and dependably by utilizing fingerprint pictures from various databases. the fingerprint acknowledgment framework is separated into three phases that are fingerprint picture pre-processing, include extraction and coordinating. the coordinating stage is partitioning into two procedure id and confirmation. at the hour of catch the fingerprint picture, the pre-processing stage is applied to it. the yield of this stage will be passed to the component extraction organize which separates the minutiae point (ridge ending, bifurcation) from thinning fingerprint picture, at that point the bogus minutiae evacuation is applied to remove genuine minutiae. at long last, the genuine minutiae are put away in tangle lab record. at that point if the fingerprint is as of now selecting? at that point send it to the coordinating stage in any case do the enrolment stage and store it in the database as a format. in id case (one-to-many coordinating), the info include set, which is coordinating with n format from the database, n coordinating will be finished. the outcome will be considered as a coordinating score. if coordinating score more like 1, at that point the two fingers from a similar client. if coordinating score close to zero, at that point the two fingers from deferent clients. in confirmation case (coordinated coordinating), the info includes set, which is coordinating with one layout from the database, one coordinating will be done and chosen either the information fingerprint checked or unsubstantiated. siva sundhara raja and abinaya (2019) proposed work comprises of the accompanying stages as pre-processing, feature extraction and classification, as portrayed in fig. 7 . the input pre-processing is the method to play out certain tasks for improving pictures preceding computational processing. it is a system that is utilized to conceal the data that isn't appropriate to the picture for additional processing. the pre-processing steps incorporate the accompanying: picture enhancement, picture resizing procedure, and thinning process. the picture enhancement process amends the lucidity of ridges and valley structure in the fingerprint picture. right now, the histogram evening out technique is utilized. they have taken two types of blood gatherings. mage resizing is utilized to extend or compress the all outnumber of pixels. with the goal that it has the predetermined number of lines and sections. the focal point bending is done when we zoom the focal point, it will transform into a bent shape rather than the keener shape. thinning is a morphological activity that is utilized to dispose of picking front picture components from double pictures. the feature extraction is a technique for catching the visual substance of pictures for ordering and recuperating. the methodologies depend on glcm, wavelet features, laws of surface features, minutiae extraction. the major features of the fingerprints like ridge endings, ridge bifurcations are called minutiae shown in fig. 8 . minutiae states the distinction between one fingerprint from another fingerprint. a ridge ending is the place the ridge suddenly ends while ridge bifurcation is the place the ridge isolates into at least two branches. the extraction of minutiae turns out to be even more testing on account of the commotion present and lack of difference in the picture. ravindran et al. (2017) proposed work taken blood sample images and pre-processed it by using various techniques such as color plane extraction, color to gray image conversion. these blood image pre-processing terms can dramatically increase the uniformity of a visual investigation of collected samples. also, they are applied several filter processes which strengthen or reduce certain image details enable an easier or faster assessment. operators can augment a camera image with just a few clicks. filtering encompasses several image filters for image optimization mixed filter for edge detection improvement, noise suppression, character alteration, etc. image processing includes it includes several functions for image processing. contrast increase by static or dynamic binarization, lookup tables or image plane separation. resolution reduction via binning. the methodology proposed by rhiannon s. mcbean et al. (2014) having two different genetic technologies: single nucleotide variant (snv) mapping by dna microarray and second method was massively parallel sequencing (mps), concerning blood bunch genotyping. the steadiest transmissible change related with blood group bunch antigens are snvs. to perform prediction of the blood type antigen phenotypes, snv mapping which includes profoundly multiplexed genotyping can be performed on business microarray stages. microarrays recognize just known snvs, along these lines, to type uncommon or novel alleles not represented in the cluster, further sanger sequencing of the district is frequently required to determine genotype. a model talked about right now the recognizable proof of uncommon and novel rhd alleles in the australian populace. enormously equal sequencing, otherwise called cutting edge sequencing, has a high throughput limit and maps all purposes of variety from a reference grouping, taking into consideration recognizable proof of novel snvs. instances of the use of this innovation to determine the hereditary premise of vagrant blood bunch antigens are presented here. in general, the assurance of a full profile of blood bunch snvs, notwithstanding serological phenotyping, gives a premise to the arrangement of perfect blood in this manner offering improved transfusion security. ferraz et al. (2010) has developed technique that allows to analyses an image captured by a ccd camera detecting the occurrence of agglutination, through image processing techniques developed for determine the occurrence of agglutination. secondly allows determine the blood type of the patient through the classification algorithm developed. finally, allows store the information in a database built. the built database can store images captured and used in image processing techniques (each image contain four samples of blood and reagent), the standard deviation calculated in each four samples of the image, the result based by the value of standard deviation obtained for each of the samples (if agglutinated or not agglutinated in the sample of blood and reagent) and the result obtained by the classification algorithm (corresponding of blood type).the image will be processed by image processing techniques developed with the imaq vision software from national instruments (imaq 2004) . the descriptions of all the functions presented are presented in the references mentioned (imaq 2004; relf 2003) . the strategy proposed in tejaswini and mallikarjuna swamy (2014) caught pictures of slide tests were a camera comprises of a shading picture made from three examples of blood and reagent. the picture preparing technique is probed the few pictures gained. these pictures are prepared utilizing matlab programming. the picture preparing strategies, for example, shading plane extraction, thresholding, and morphological activities were performed on the pictures. the picture got in the wake of applying auto thresholding grouping capacity it very well may be seen that the item and foundation are isolated. in the following stage, neighborhood limit activity utilizing niblack work is applied it ascertains a pixel-wise edge and it very well may be seen just the outskirt portioned picture. the narkis banu and kalpana (2018) and relf (2003) present the blood group identification using blood cell images which takes from slide tests. picture acquired by the utilization of cutting-edge morphology; it very well may be seen that the portioned picture is filled utilizing shutting activity. progressed morphological activity opening is performed it tends to be seen that it smoothens the shapes of cells by evacuating little articles. at that point the pictures acquired by applying the shading plane extraction hsl luminance plane and measure work. at long last, the blood gathering can be resolved. the utilization of picture preparing procedures empowers programmed discovery of agglutination and decides the blood sort of the patient in a short interim of time. the strategy is appropriate and accommodating in crisis circumstances. keerthana and ranganathan (2017) build up an inserted framework that utilizes an image preparing calculation to perform blood tests dependent on blood composing frameworks. along these lines, the framework permits us to decide the blood sort of an individual killing customary transfusions dependent on the rule of the all-inclusive contributor, decreasing transfusion response dangers and capacity of result without human mistakes. this paper helps in lessening human intercession and perform total test independently from adding antigens to definite age of the outcome and gives the outcomes in most limited conceivable term with exactness and precision alongside capacity of result for additional references. actualizing a quality framework in the lab limits mistakes and guarantees that the correct test is performed on the correct example, the correct outcomes got, and the correct blood item gave to the correct patient at the ideal time. the proposed framework presents the plan and usage of a smart compact gadget that gives the best possible data that we require for the investigation with the decreased expense and the profoundly prepared administrators are not required. this framework utilizes an ai calculation like a neural system that underpins matlab programming for blood bunch recognizable proof and identification of blood check examination. this framework additionally discovers an answer utilizing various calculations and strategies which gives us the most extreme precision in blood bunch distinguishing proof and tallying. the work proposed by berlitz et al. (2012) utilized protein a covering of the gold surface of qcm biosensors for the immobilization of antibodies against blood bunch antigens an and b, which allows the recognizable proof of the four principle blood bunches a, b, ab and 0 with two estimations. the rhesus framework was inspected with various examinations on and the fruitful recognition of rh-d, rh-c, rh-c, rh-e and rh-e antigens on human erythrocytes. the brisk, simple and dependable identification of blood bunch antigens an and b offers the chance of deciding the patient's association to the ab0 blood bunch framework by two estimations on hostile to an and against b sharpened quartz sensors. in satoh and itoh (2004) author also proposed a method which uses genetic analyzer for blood group prediction is recognized by the presence of three common representative alleles such as a, b, and o, in satoh et al. (2001) the author proposed the analysis of four snps at nucleotide positions 261, 796, 802 and 803 to reflect serologic specificity. sensor cells organized in equal and working on weakened entire blood without earlier planning of the blood tests will lessen the necessary time to approx. 3 min. swapping the quartz sensors, encouraged by the card mounted sensors and the licensed module holder, broadens the blood bunch investigation into the rhesus, kell and further blood bunch frameworks. in any event, for blood bunch antigens with a low antigen number for every cell, techniques are accessible to get ready biosensor coatings with satisfactorily upgraded affectability. the proposed method utilized by dalvi and kumar pulipaka (2018) three samples of blood are blended in with three distinct reagents namely anti-an, anti-b and anti-d is taken on a slide. after some time, agglutination may or may not occur. after the occurrence of agglutination, the slide containing three samples of blood blended in with three distinct reagents is captured as an image and allowed to process in matlab image processing toolbox. this framework lessens the chances of false detection of a blood group. image processing techniques utilized for blood group detection are 1. pre-processing techniques, 2. thresholding, 3. morphological operations, 4. hsl plane 5. quantification. the color plane contains color information in image s. 'comparing' sections in an image is the concept utilized in image processing. comparison in grayscale involves straightforward scalar algebraic operators. in color plane extraction, they first convert the rgb image into a gray image and then channel the obtained outcome utilizing median separating. thresholding operation in image processing is utilized to create binary images. the grayscale samples are grouped into two parts as background and object. right now, thresholding is performed utilizing otsu's method. more than one threshold is resolved for a given image and segmentation is done creating certain regions. one background with many objects is the consequence of this staggered thresholding. it is a bunching based image thresholding. morphological is a tool for extraction image components that are valuable in the representation. in morphological operation, there are two fundamental operations, for example, dilation and erosion as far as the union of an image with a translated shape called an organizing component. here, closing operation is performed where dilation is followed by erosion. also, edge detection utilizing the canny edge detection strategy is performed. morphological operations are utilized to eliminate noise spikes and ragged edges. hsl plane stands for hue, saturation, and luminance. it is the representation of the rgb color model. shade is expressed in a degree around a color wheel, while saturation and brightness are set as a percentage. quantification is expressed as a number or measure of quantity. it measures power only in the region of the intrigued area. area (percentage of surface examined for full image), mean (average value of the pixel), standard deviation, least and maximum values of pixel power are resolved. also, region properties are extracted. utilizing the value of standard deviation, the occurrence of agglutination is recognized and accordingly the blood group is resolved. fayrouz et al. (2011) study reveals an association between the pattern of the unique mark and abo blood group. with ongoing advances in unique mark detecting technology and improvement in the accuracy and matching velocity of the finger impression matching algorithms, automatic personal identification is becoming an attractive/complement to the traditional methods of identification. as biometric technology matures, there will be an increased interaction among the biometric market and its identification application, since fingerprints will remain an integral part of the preferred biometric-based identification solutions in the years to come, a relationship of unique finger impression pattern to blood group presents scope for additional identification data which can be utilized for personal identification purpose, also investigation of possible predilection of certain disease and malignancies from blood groups are some of the factors which encourages one to carry the examination further. chosen randomly having distinctive abo blood groups, with the objective to a) study the distribution of unique finger impression pattern among the subjects having diverse abo and rh blood group b) correlate any relation between their characters and blood group. the data from the investigation showed that the male: female ratio was 1.2:1. most subjects (48.9%) right now of blood group o followed by blood group a (33.1%), b (12.8%) and ab (5.2%). rh-positive cases constitute about 87.2% of all considered cases. the general distribution of the pattern of finger showed a high recurrence of loops enlisting 50.5%; followed by whorls (35.1%) and arches (14.4%). in rhþve cases of blood group an and o loops occurrences were the most elevated (52% and 4.3% individually) at that point whorls (33.4% and 30.6% separately), while in blood group b whorls were predominance in both rhþve and rh_ve cases. in all blood groups, there was the high recurrence of loops in thumb, record and little fingers. utilizes the slide test and image processing techniques utilizing the imaq vision from national instruments. the image captured after the slide test is processed and recognizes the occurrence of agglutination. next, the classification algorithm decides the blood type in the analysis. finally, all the information is stored in a database. in this way, the framework allows deciding the blood type in a crisis, eliminating transfusions based on the rule of universal donor and diminishing transfusion reaction dangers. this framework is based on a slide test for deciding blood types and the software developed utilizing image processing techniques. the slide test consists of the blend of one drop of blood and one drop of each reagent, anti-an, anti-b, anti-ab, and anti-d, being the outcome interpreted according to the occurrence or not of agglutination. the agglutination reaction means that occurred reaction between the antibody and the antigen, indicating the presence of the antigen appropriate. the combination of the occurrence of agglutination, or nonoccurrence, decides the blood kind of the patient (datasheet of diameddiaclon anti-a 2008; fingerprint identification -project 2). accordingly, the software developed based in image processing techniques allows, through an image captured after the procedure of the slide test distinguish the occurrence of agglutination and consequently the blood sort of the patient. thakar and sharma (2016) process remarkable highlights which they found inside the fingerprint designs which make us equipped for offering input. in any case, different examinations have demonstrated that even the prepared and experienced specialists submit different sorts the fingerprint, these might be a result of the utilization of discretionary/nonstandard phrasing like clockwise/anticlockwise or bearings and so forth recorded as a hard copy a report. the traditional technique for fingerprint correlation with the focal point to find details in bearings, which is a tedious system should be changed. right now, a framework, optical sensor-based per users are utilized to peruse and gained fingerprint pictures in the accompanying three phases: firstly, picture handling calculations are utilized to get dark tone impressions of the fingerprint picture. also, the prepared picture is accordingly used to extricate the details (just bifurcations and ridge finishing). the third step is examining the position of various details (bifurcations and ridge finishing) on fingerprints, with the assistance of situation coordinated calculations. the separated pictures are contrasted, and the databases present in the framework and results are gotten. in any case, as opposed to the abovementioned, in the present examination, a semi-self-enough method has been utilized. in this way, in the present examination, a far-reaching endeavor has been made to extricate physically all the particulars present in the unique mark with the assistance of adobe photoshop (cs5) software and to build up an adjusted lattice which can be utilized to methodically discover the position of the details alongside estimating certain extra element like angle. azhagiri et al. (2018) study uncovers a huge relationship of blood groups o, a, b, ab to hypertension, peptic ulcer, anemia, rheumatoid arthritis, gastritis, diabetes, and bronchial asthma. the transcendence of the circle was most noteworthy among all blood groups. as indicated by this investigation, following outcomes were watched, loops were the most well-known unique finger impression example and arches were the least normal, whorls and blended were moderate, highest quantities of loops were found in blood groups o, b contrasted with an and ab, blood bunch o positive is the most well-known, o negative and ab negative is the rarest, loops, whorls, blended and arches were most elevated in females, group a was the most widely recognized blood bunch among guys, blood bunch o, b, were the most regularly observed blood groups in females, some basic clinical grumblings were found in all the blood groups. kanchan and chattopadhyay (2006) the result shows that each fingerprint is exceptional; loops are the most normally happening fingerprint design while arches are the least normal. guys have a higher occurrence of whorls and females have a higher frequency of loops. loops are transcendent in blood groups a, b, ab and o in both rh-positive and rhnegative people except for in o pessimistic where whorls are progressively normal. they can reason that there is a relationship between the conveyance of fingerprint designs, blood gathering, and sexual orientation and along these lines expectation of sex and blood gathering of an individual is conceivable dependent on his fingerprint design. rastogi and pillai (2010) present investigation shows that there is an association between the appropriation of fingerprint designs, blood gathering and sexual orientation. the discoveries of the examination can be finished up as follows: each fingerprint is one of a kind consequently it tends to be successfully utilized as a proof for distinguishing proof in the courtroom. loops are the most generally happening unique mark example and arches are the least normal. blood bunch o positive is the most widely recognized and a negative is the rarest. loops are overwhelming in blood bunch a, b, ab and o in both rh positive and rh-negative people except for in o adverse where whorls are progressively normal. whorls are progressively normal in blood bunch o negative. loops and arches are most extreme found in blood bunch some time whorls are increasingly regular in blood bunch o. blood groups an and b were the most widely recognized (similarly dominating) among guys, blood bunch o was the most generally observed blood bunch in females. guys have a higher frequency of whorls and females have a higher rate of loops. hence prediction of sexual orientation and blood gathering of a for every child is conceivable dependent on his fingerprint design. comparative examinations ought to be led to a bigger example in order to build the precision of prediction. joshi et al. (2016) present examination uncovers that there was an association between dissemination of fingerprint (dermatoglyphic) design, gender and blood groups. the general circulation example of the essential fingerprint was of a similar request in people with abo; rh blood groups for example high recurrence of loops, moderate of whorls and low of arches. the discoveries of the examination can be finished up as the loops are the most normally happening unique mark example and arches are the least normal, blood bunch o positive is the most well-known and a negative is the rares, loops are prevalent in blood bunch a, b, ab and o in both rh positive and rh negative people aside from in o antagonistic where whorls are increasingly normal and males have a higher occurrence of whorls and females have a higher frequency of loops. jha et al. (2015) study uncovered that blood bunch ab and o had a most noteworthy rate of loops (76.92% and 71.05% separately) trailed by whorls (23.08% and 28.95% individually), comparatively in blood bunch an and b the design of the loop was normal with 57.14% in the two cases. the study inferred that there is a solid connection between blood groups and fingerprint design. from the examination, it was presumed that the recurrence appropriation loops design were most noteworthy in blood bunch ab (76.92), o (71.05%) and b (57.14%) individually. so also, the examination likewise inferred that the dispersion of whorls was most elevated in blood bunch a with 42.86% circulation, then in blood bunch b with 39.28% dissemination and for blood bunch ab it was discovered 23.08% and arches were least in blood bunch b with 3.70% appropriation. further investigation ought to be completed by expanding the example size to get the progressively exact portrayal of the populace and need increasingly comparative examinations in different districts as well with the goal that near examination should be possible. the study proposed by sudikshya et al. (2018) corresponds to the connection between different patterns of fingerprints and "abo" blood groups and "rh" blood types in nepalese guys and females. in spite of the fact that they realize that fingerprints are rarely indistinguishable and they never show signs of change from birth till death, this examination is an endeavor made to connect fingerprints with sex, diverse blood groups, and rh blood types which may thus improve the legitimacy of fingerprints in distinguishing proof and legal medication and can be utilized for conceivable prediction of specific illnesses. from the present investigation, the accompanying ends are drawn: (1) loops are the most regularly discovered fingerprint pattern and arches are minimal normal in the two guys and females and furthermore in "abo" blood groups. (2) the recurrence of loops is most noteworthy followed by whorls and arches in rh +ve blood types, while the frequency of whorls is most elevated followed by loops and arches in rh -ve blood types. (3) our outcomes uncover the most elevated rate of loops in the center and little finger in all blood groups, while the whorls are usually found in ring fingers in all blood groups. the frequencies of whorls are likewise most elevated in forefinger and thumb in all blood groups aside from in blood bunch "o" where loops are as often as possible present. (4) from this investigation, they can infer that circulation of essential pattern of the fingerprint isn't identified with sexual orientation and abo and rh blood gathering, yet its conveyance is identified with singular digits of two hands. narayana et al. (2016) they present the examination is an endeavor to relate fingerprint patterns with sexual orientation and blood gathering of a person. fingerprint patterns can be of help in anticipating the sexual orientation and blood gathering of a person. it might help in expanding the legitimacy of fingerprints in recognizable proof of people and tackling of wrongdoings. they got results areas: loops were the most generally discovered pattern and composite the least. in the loop pattern, the commonest pattern was an ulnar loop, which was measurably huge right now. blood bunch o positive was the most wellknown and ab negative was the rarest. rh-positive blood groups were more contrasted with rh-negative blood groups, which is demonstrated right now and critically dependent on the information too. blood bunch b was the most widely recognized among rh-positive blood groups followed by o, an and ab blood groups. among rh-negative b and a blood groups were similarly predominant followed by o and ab. loops were most elevated in guys, whorls and arches were most noteworthy in females. loops were prevalent in all the blood groups with the exception of a positive where whorls were prevailing. the most elevated number of the considerable number of patterns was found in blood bunch o and least in ab among rh-positive blood groups and factually demonstrated critical right now. composites were least generally found in all the blood groups. the study by sangam et al. (2011) they found the loops were connected more with o gathering, whorls with ab gathering and arches with b gathering. thumbs introduced a high recurrence of whorls in a + ves. record and ring fingers were related to the high recurrence of whorls in a-ves and ab + ves. so the prediction of blood gathering somewhat might be conceivable with the investigation of unique finger impression patterns which might be of extraordinary incentive in criminological medication, however, impact provincial varieties, sex and hereditary variables ought not to be disregarded. deopa et al. (2014) has an endeavor has been made in the present work to examine their connection with sexual orientation and blood gathering of a person. loops were the most widely recognized (58.29%) fingerprint pattern while whorls were moderate (37.00%) and arches were the least normal (4.71%). guys had a higher rate of whorls and females had a higher frequency of loops. loops are overwhelming in blood bunch a, b, ab and o in both rh-positive and rh-negative people aside from in 'a' constructive blood bunch where whorls prevail marginally. whorls were most noteworthy in an and ab positive blood gathering, and loops were most elevated in o and b blood gathering. arches were least in all blood groups. there is an association between conveyance of fingerprint patterns, blood gathering, and sexual orientation and in this way prediction of sex and blood gathering of an individual is conceivable dependent on his fingerprint pattern. shivhare et al. (2017) does the investigation which uncovered the association between dermatoglyphic, blood gathering and sex: most subjects have a place with rh-positive and o blood gathering. loops are the regular and arches are extraordinary fingerprints. loops were most noteworthy in b blood gathering and least in ab blood gathering. whorls most elevated in an and least in b blood gathering. arches were most elevated in ab and least in b. loops higher in female and most reduced in male, whorls most noteworthy in male and least in female and arches most elevated in male and most reduced in the female. loops were most noteworthy in rh-positive and least in rh-negative. whorls most elevated in rh-negative and least in rh-positive. arches were most noteworthy in rh-positive and least in rh-negative. the investigation does by radhika (2016) uncovered that there is an association between the dissemination of fingerprint patterns, blood gathering, and sexual orientation. loop was most as often as possible seen fingerprint followed by whorl curve and composite. o positive is the most regular blood gathering and ab negative is missing. loops are dominating in blood bunch o followed by b and an in rh-positive subjects, trailed by whorls. curve and composite were basic among o and a positive subjects. morris et al. (2016) proposed method having outcomes that fingerprint asymmetry could be formed into a significant instrument for anticipating the danger of type 2 diabetes mellitus and type 1 diabetes mellitus and that wavelet examination is a technique that can be utilized to evaluate asymmetry in fingerprints. the benefit of fingerprints scored utilizing wavelet-based strategies over hereditary testing, is that it can demonstrate gestational condition and would be considerably less costly. the expense is significant, given late reports that both hazard mindful and chance unconscious people were keen on hereditary testing, however distinguished the requirement for minimal effort tests. they propose an increasingly far-reaching examination of fingerprint asymmetry as a predictor of both t2dm and t1dm chance, scoring asymmetry with wavelet investigation and contrasting with the prescient capacity of hereditary qualities alone, is justified. the propose work by used information digging veena vijayan and anjali (2015) which can be used for evaluating distinctive disease patterns, remedial data extraction, quiet support and organization and finding of clinical parameters. here a choice emotionally supportive network is recommended that predicts diabetes which utilizes choice stump as a base classifier in adaboost calculation. the framework utilizes a worldwide dataset taken from uci vault of ai for preparing which contains 768 cases and 9 characteristics and utilizations nearby dataset for approval which had been gathered from better places in kerala. the pc data framework with the adaboo-choice stump classifier gives a precision of 80.729% for anticipating diabetes with an extremely low estimation of blunder rate. tafa and pervetica (2015) study the dataset which comprises of 402 examples taken from three unique areas in kosovo. the characteristics of the database are bmi (weight file), glucose level before dinner and after the feast, the systolic and diastolic blood pressure, the genetic factor, the customary eating regimen, and day by day physical exercises. the last two qualities are assessed as follows. with respect to the issue of customary eating routine, while depending on contributions from the clinical clinicians, patients were inquired as to whether they took their dinners in roughly the same equidistant everyday interims, in any event, three times each day and furthermore if their suppers were not voluminous. the introduced approach depends on the joint usage of two calculations in matlab that have been executed on the recently gained dataset with the various credits when contrasted with the past work right now. the calculations are executed and assessed freely however the dynamic depends on the joint results from the two calculations. the point of this methodology is to settle on the choice progressively dependable. mehta and mehta (2015) study the one hundred type ii diabetes mellitus patients (50male and 50 female) were chosen for study and contrasted and equivalent number of controls. fingerprints were acquired by a printing technique. parameters contemplated were arches, whorls, loops. circulation of fingertip patterns indicated a noteworthy contrast between diabetics and controls. the appropriation of fingertip patterns on both ways submit male diabetics and controls. the whorls were altogether expanded though loops and arches were essentially diminished in male diabetics when contrasted with controls. the whorls were fundamentally expanded while loops were essentially diminished in female diabetics when contrasted with female controls. nonetheless, arches were fundamentally diminished in the left hand of female diabetics. ameer et al. (2014) investigation right now spellbinding. an aggregate of one hundred patients partook right now was a completely known instance of diabetes mellitus. out of these one hundred patients, most of the patients were having a place with a whorl pattern of fingerprints i-e. fifty half while the number of patients having a place with the loop pattern was forty-five 45%, the composite was just 2,2%, and no specific patient having a place with arch pattern. there is having to build up a definite and tremendous examination to investigate the association of fingerprint pattern with diabetic disease. this investigation offered reasonable weighting on the circulation of fingerprint pattern among diabetic disease patients. confinements of study where it was just restricted to clinical opd patients and limited uniquely to diabetes mellitus patients. the investigation by roshani et al. (2016) concluded whorls are the most regular pattern in both right and left hands of both male and female diabetic subjects and loops are the most normal pattern in both right and left hands, in the two guys and females in non-diabetic subjects. the arches were fundamentally diminished in both the right and left hand of males and females in diabetics and non-diabetic subjects. this examination shows a huge association between fingerprint patterns and diabetes in both genders. study might be helpful to distinguish the high-chance people in the populace, for type-2 diabetes mellitus; the most punctual prediction and finding of patients with type-2 diabetes mellitus will improve the aftereffect of treatment and further entanglements. notwithstanding; there are a few investigations that appeared inverse outcomes to our examination; subsequently, there is having to do additionally contemplates and bigger examples ought to be analyzed in detail to additionally approve the discoveries of this examination and arrive at a complete resolution. smail et al. (2019) examination demonstrated that the type of loop in typical guys is 37.50%, even with the diabetes female gathering the type of loop is 18.75%. this investigation demonstrated that the control male arches bunch is no worth which is zero, while in the patient female the arches bunch esteem is high to 25%. confinements of the examination gave us that the control male whorls run is 6.25%, however for the female patients is 12%. 50 which expanded worth. their investigation demonstrated that the type of loop in typical females is 31.25%, yet in the diabetes male gathering, the type of loop is 0%. this investigation indicated that the control female arches bunch is 6.25%, yet in the patient's male, the arches bunch esteem is high to 25%. another impediment of the examination gave us that the control female whorls run and the diabetes male or equivalent worth which is about 18.75%. for the all-out typical and diabetes guys, the loop bunch in the ordinary male is higher than diabetes (68.75% > 18.75%). for the all-out ordinary and diabetes male the arches bunch in the typical male is lower than diabetes (6.25% < 50%). for the allout typical and diabetes male, the entire gathering in the ordinary male is underestimating than diabetes (25% < 31.25%). arches are found in five percent of fingerprint patterns. the ridges run starting with one side then onto the next of patterns, making no retrogressive turns. usually, there is no information in a curve pattern. igbigbi et al. (2001) have inspected the plantar and advanced prints of the sole of 99 indigenous malawian patients matured 25-66 years going to the clinical outpatient center for diabetes mellitus, fundamental hypertension and a mix of the two conditions at lilongwe and queen elizabeth focal emergency clinics. the gathering comprised of 27 diabetics (15 guys, 12 females), 21 hypertensives (9 guys, 12 females) and 51 diabetics with hypertension (21 guys, 30 females). all patients were analyzed as type 2 diabetics after the age of 20 years. their outcomes indicated that soles of all patients had a greater number of loops than arches and a greater number of arches than whorls, which were limited to the distal zones. in hypertension, whorls were found in zones i, ii and iii though, in patients with diabetes and hypertension, the whorls were found in zones i, iii and iv. in digits, the most prevalent ridge pattern was arches in all patients, trailed by loops and whorls were missing. in the principal digit, diabetic patients had no arches, yet ladies' hypertensives demonstrated arches. in patients with diabetes and hypertension, arches were available in both genders yet in men it was limited to the correct foot. loops were discovered distinctly in the first digit in quite a while. the recurrence of loops was most noteworthy in diabetic patients, high in diabetics with hypertension and least in patients with hypertension alone. tarca and tuluc (2005) have considered a complete number of 133 patients with type 1diabetes mellitus, out of which 58(33 guys and 25 females) were youngsters and adolescents of ages somewhere in the range of 4 and 18 years. the disease showed in these cases between the age of 2 and 17 years. among the female patients, the loop was found in around equivalent extents in both the hands (58.95% on the left hand and 60.60% on the correct hand) while if there should arise an occurrence of typical subject the loops were found on the left side as it were. the loop dissemination on the five fingers indicated an expanded recurrence on the fingers from v and iii. whorls were increasingly visited in the male arrangement and on the correct hands. arches were increasingly visiting in the female arrangement. the presence of these markers, before the clinical sign of the disease, makes workable for their utilization in anticipation of insulin subordinate diabetes mellitus. udoaka and lawyer-egbe (2009) have considered an absolute number of 90(50 guys and 40 females) grown-up diabetic patients and the contrasted and the same number of ordinary subjects as controls. there was no huge contrast in the computerized patterns in the two groups. the atd point, dat edge, the absolute ridge include were fundamentally more noteworthy in the diabetic patients contrasted with the typical subjects. the pattern force record was higher in the diabetic guys however it was lower in the female diabetics. their perceptions can be utilized for distinguishing proof of diabetics. nezhad and shah (2010) have considered 30 patients of diabetes type 1 and 30 typical subjects as control gathering. the mean period of patients and control bunch was 22 ± 11 and 38 ± 8 individually. among these 42% were guys and 58% were females. they have discovered that the state of loop and whorl are heterogeneous, and their number varies altogether contrast with a control gathering (p = 0.001, p = 0.004.). the a-b 32 ridge includes demonstrated an expansion in the ridge considers as a real part of the diabetic men than control gathering. the atd point size in both the experimental group and control gathering of females was more than guys. these creators are of the sentiment that dermatoglyphics can be an appropriate strategy for hereditary examinations and diabetes type 1. sumathi and desai (2007) have considered a sum of 100 patients of diabetes mellitus type 2 and hypertension of either sex or age gathering of 35-55 years. they were coordinated with hundred controls. they discovered diminished a-b ridge include in female diabetics. the accompanying huge parameters have been found in their investigation in the palmar dermatoglyphics in type 2 diabetes with hypertension. in both male and female patients, there is the nearness of diminished i1 pattern and nearness of expanded i3 pattern in the left hand. the nearness of diminished whorls saw in two hands of male patients. the nearness of expanded ulnar loops and whorls in two hands of female patients. padmini et al. (2011) their exploration concentrate on dermatoglyphics in diabetes mellitus underlined that however dermatoglyphics by and large don't assume any significant job in clinical analysis yet, it can fill in as a pointer to pick out subjects from a huge gathering of individuals for additional examinations to affirm or preclude diabetes mellitus have studied fingerprints and palmar prints from 200 subjects, 100 guys and 100 females in the age gathering of 25 years to 80 years, of which 95% of cases were non-insulin subordinate diabetics and 5% of cases were insulin subordinate diabetics contrasted and 200 controls. higher occurrence of variety in methods for ulnar loops (83.2), composite whorl (1.8), all-out finger ridge tally 33 (108.6), total finger ridge check (138.55), dat points of the right hand (59.77) and left hand (62.3) in diabetics than in controls was seen by them. the rest of the parameters were low in diabetics than in controls. in male diabetics increment in methods for ulnar loops (41.6), outspread loops (1.7), complete finger ridge check (106.25), supreme finger ridge tally (137.58),atd edge of right hand (41.68) and left hand (41.67), dat edges of left hand (62.42) and adt of right hand (80.45) was seen than in controls. in female diabetics, huge increment in basic arches (5.7), all-out finger ridge tally (110.94), total finger ridge tally (139.52), dat points of two hands right (61.6) and left hand (62.17) was seen than in controls. sharma and sharma (2012) their investigation of 50 diabetic cases and 50 controls chose from the sms hospital, jaipur, india, found that the complete finger ridge check, total finger ridge tally, and the a-b ridge include were higher in all the patients. the atd points in the hands of the two sides in the patients were expanded in all the groups aside from left side in guys. in any case, they varied fundamentally on the correct side and on the left side in females, p < .001. in the general groups right tda edge was huge. the smidgen and the tda edges on the two sides of the hands in all the groups were lower in the patients aside from left tda edge in 34 guys. be that as it may, they varied just all together in the left bit, right tda in females. the aftereffects of their examination work demonstrated that dermatoglyphic variations from the norm might be utilized as an indicative instrument for anticipating the chance of the advancement of diabetes sometime in the future. taiwo and adebanjo (2012) have completed an examination to explain whether fingerprint pattern of dermatoglyphics is related to withtype2 diabetes or not. dermatoglyphic information was acquired from controls and type 2 diabetic subjects going to the diabetic clinic of lagos university teaching hospital. they saw all-out finger ridge tally was fundamentally higher (p < 0.05) in diabetic subjects than in nondiabetics. considering the association between fingerprint pattern and type2 diabetes, dermatoglyphics might be utilized for the early id of hazard bunch people for reconnaissance purposes so as to forestall disease beginning. rakate and zambare (2014) have looked at the distinctions in the complete finger ridge tally, a-b ridge includes and atd point in patients with type 2 diabetes mellitus with a control gathering. their examination was completed on 75 type 2 diabetic patients (51 male and 24 female) of 30 to 60 years and 75 non-diabetic persons (47 guys and 28 females) of the equivalent 35 age bunch as a benchmark group. in their examination, they found an expansion in the number of whorls, absolute finger ridge tally, a-b ridge tally alongside more extensive atd point in type 2 diabetes mellitus patients. desai and hadimani (2013) have opined that dermatoglyphics is a developing order and its simple and prepared pertinence renders it as a valuable device to the clinician. the dermatoglyphics isn't to analyze and not for characterizing a current disease yet to forestall by anticipating a disease and to distinguish individuals with hereditary inclination to build up specific diseases. they have attempted to decide noteworthy palmar dermatoglyphic parameters if there should be an occurrence of sputum positive tuberculosis, diabetes mellitus type 2 with basic hypertension, dermatitis, innate coronary illness, and down disorder and contrasted and the benchmark group. their investigation demonstrated that there were some hereditary components that were engaged with the causation of different diseases referenced previously. itis conceivable to anticipate from dermatoglyphics people's possibility of gaining disease. noteworthy discoveries they watched were:1. the nearness of diminished whorls, 2. the nearness of expanded ulnar loops,3. the nearness of expanded simian line in the left hand of considered patients. shivaleela et al. (2013) have done an investigation to discover the recurrence of different fingerprint patterns in type 2 diabetes mellitus with and without ischemic coronary illness. their examination likewise planned to discover the recurrence of fingerprint patterns in type 2diabetes mellitus patients having the family ancestry of cardiovascular disease. twenty-five type 2diabetes mellitus male patients in the age gathering of 38-65 years were chosen, of which 18 had an ischemic coronary illness and 16 patients had the family ancestry of cardiovascular occasions. there was a higher recurrence of whorls in type 2diabetes mellitus patients than different patterns. less recurrence of arches, high recurrence of whorls and ulnar loops were seen in type 2diabetes mellitus patients contrasted and type 2diabetes mellitus patients without ischemic coronary illness. the thing that matters was not factually huge. dermatoglyphics in type 2diabetes mellitus and in patients with family ancestry of cardiovascular disease didn't show dominance of any of the fingerprints in ischemic coronary illness. therefore, they opined dermatoglyphics might be symptomatic apparatus in type 2diabetes mellitus however not in recognizing the hazard class for ischemic coronary illness. umana et al. (2013) completed an examination to decide the association between fingerprints patterns and diabetes mellitus utilizing subjects in zaria, nigeria. their consequences of 101 type 2 diabetic patients were contrasted and 126 typical subjects. from their outcomes, there was an association between fingerprint patterns of guys with diabetes mellitus. from the above investigation, they reasoned that the male with a arch pattern of fingerprint in their correct hand is inclined to create diabetes mellitus at a later stage. mittal and lala (2013) have endeavored to discover an association of the dermatoglyphics patterns of the sound people and diabetes mellitus patients. an aggregate of 200 subjects took part in their investigation of which 100 were diabetic patients (50 guys and 50 females) and 100 were solid people utilized as controls (50 guys and 50 females). they signify 'atd' point in both the hands of both the genders of diabetic patients was fundamentally more extensive when contrasted with that of the controls. contrasted with that of control, the mean tda edges in both the hands of both the genders of diabetics were restricted. they signify 'dat' edges were essentially thin just in the left hand of diabetic females when contrasted with left hands of female controls. burute et al. (2013) have meant to contemplate the different dermatoglyphic patterns in the patients of the development beginning diabetes mellitus (type 2 diabetes mellitus) and contrasted and the dermatoglyphic patterns of controls. they did their investigation on 101 (51 male and 50 female) clinically analyzed patients of development beginning diabetes mellitus. for correlation, solid controls (total = 100, 50 guys and 50 females) were considered. in female diabetics, essentially higher recurrence of arches and lower recurrence of whorls were seen than in controls. in diabetic female's outright finger ridge tally and complete finger ridge include were essentially lower than in controls. discoveries of their examination feature on the potential markers to foresee type 2 diabetes mellitus on a bigger example size after a fastidious investigation of various fingertip dermatoglyphic factors. rakate and zambare (2014) have looked at the distinctions on the fingertip patterns to be specific, curve, loop, and whorl in patients with type 2 diabetes mellitus with a control gathering. test for their investigation included palmar prints of 350 type 2diabetic patients old enough gathering between 30 and 60 years out of them 240 were guys and 110 were female contrasted and same age gathering of 350 non-diabetic people as control the gathering, out of the 240 were guys and 110 were females. in the example of 350 type 2 diabetes mellitus patients, they watched an expansion in the number of whorls in two hands of guys and females. the p-esteem was 0.001. the ulnar loop and curve patterns were available in less incentive in diabetic patients which were measurably inconsequential. the plain whorl was available essentially higher in esteem in diabetic patients of guys and females. in diabetic guys in right hand 482 whorls where present. yet, in charge just 326 whorls were available; this distinction was critical at 0.000 levels. in the left hand of diabetic, whorl was altogether more 418 than control 267, p-esteem was 0.000. diabetic females additionally indicated higher recurrence of whorl pattern in two hands; on right hand 158 contrasted and control 121 and 184 on left hand contrasted and 130. the p values were 0.010 on the right hand and 0.000 on the left hand. the central pocket loop whorl pattern watched more in diabetic patients. in the diabetic male they 40 discovered 96 on the right hand and on the left hand 99 central pocket loop whorl pattern which was more contrasted and control right hand 82 and left hand 82. the p-values were 0.138 and 0.094 separately. in diabetic females additionally central pocket loop whorl was watched all the righter hand 36, left hand 40 than control bunch right hand 29 and left hand 36; p-values were 0.237 and 0.320. the twofold loop whorl was watched more in diabetic patients. in diabetic guys on the right hand, 54 twofold loop whorls were available which were more than control 38. likewise, on left hand 69 in diabetic though 67 in charge. the p-values were 0.044 and 0.430 separately. in diabetic females, likewise, more recurrence of twofold loop whorl was seen on right hand 28 contrasted and control 15 and left hand 25 contrasted and 20 in charge. the p-values were 0.021 and 0.223. at the point when they thought about a wide range of whorls together among diabetic and control gathering, noteworthy contrasts were seen in the two guys and females. in diabetic guys on the right hand, 632 whorls were available which were more than 446 in ordinary. comparative discoveries were seen on left hand 586 in diabetic while 416 in charge. the p-values were 0.000 and 0.000 separately. in diabetic females, likewise, more recurrence of whorl was seen on right hand 222 contrasted and control 165. the p values were 0.000. karim et al. (2014) have looked at the distinctions in the fingerprint patterns and finger ridge include in patients with type 2 diabetic mellitus with a control bunch in erbil city, kurdistan area, iraq. in their examination, 50 non-insulin subordinate diabetes mellitus patients, 25 guys, and 25 females were contrasted and 50 (25 guys and 25 females) sound controls. the appropriation of fingertip patterns of male patients indicated no noteworthy contrast in ulnar loops, spiral loops and rose arches while plane arches expanded altogether (p < 0.05) in diabetic type 2 patients contrasted and controls, whorls diminished fundamentally (p < 0.05). higher frequency of ulnar loops, spiral loops and plane arches in female diabetics contrasted and control females. they saw that essentially expanded (p < 0.05) center finger ridge include in the left hand of male diabetic patients. fundamentally expanded (p < 0.05) file and little finger ridge check of the right hand was seen in female diabetic patients contrasted and control female groups. bala et al. (2015) have considered an all-out 210 subjects out of which 70 subjects having diabetes (32 guys and 38 females), 70 subjects having diabetes with hypertension (32 guys and 38 females) and 70 typical sound people (32 guys and 38 females) as control having a place with gangtok area of sikkim. all were clinically analyzed and affirmed by examinations as diabetic and diabetic with hypertensive patients. in their investigation, an examination of diabetic with control bunch indicated the mean estimations of atd and dat edges in two hands of diabetic patients lower than control, though mean estimations of adt edges were higher than control bunch on both right and left sides. the huge distinction was found in the correct hands of diabetes mellitus gathering. in both right and left hands of males and females, the mean estimations of atd point and dat edge of the diabetic gathering were lower than in charge. the mean estimations of adt edge were higher than control. no noteworthy contrast was found. the mean estimations of a-b ridge include in two hands were higher in diabetic male and female aside from in the left hands of male and profoundly noteworthy distinction was found in both hands of the female. mehta and mehta (2015) have analyzed fingertip patterns of type 2 diabetic patients with controls. one hundred type 2 diabetes mellitus patients (50 male and 50 female) were chosen for study and contrasted and equivalent number of controls. in two hands of guys and females' diabetic patients' frequency of whorls was essentially expanded. frequency of loops was fundamentally diminished in two hands of male and female diabetics contrasted with controls. arches were essentially diminished in both ways' hands of male diabetes mellitus patients. arches were fundamentally decreased in left hand of female diabetics. in their investigation, they expressed that dermatoglyphics can be utilized as a screening device for the conclusion of people who are progressively inclined to create diabetes mellitus and, in this way, forestalling the future diabetic intricacies. bala et al. (2016) have examined a sum of 100 type 2 diabetic patients (50 guys and 50 females) were contrasted and 100 diabetics with hypertension patients of hilly district. the mean estimations of all-out finger ridge check and total finger ridge include were higher in male and lower in female diabetic gathering than diabetic with hypertension gathering. the mean estimations of a-b ridge include were lower in males and higher in females in diabetic gathering and a critical contrast was found. the mean estimations of atd edge were higher in diabetic gathering than diabetic with hypertension gathering. the mean estimations of dat edge were lower in the right hands and higher in the left hands of diabetic gathering. the mean estimations of adt point were higher in guys and lower in female diabetic gathering than diabetic with hypertension gathering. in the right hands, the mean estimations of fingertip ridge include were lower in all digits aside from in 2nd, fourth, and fifth digits in the male diabetic gathering. in left hands, the mean estimations of fingertip ridge include were lower in all digits of diabetic gathering apart from in 2nd, fourth and fifth digits and no critical contrast were found. in their examination, they watched an expansion in ulnar loops in the correct hand of male diabetic and diminished frequency in the left hand of male and in two hands of female diabetics. tafazoli et al. (2013) collected samples for this exploration was gotten from 40 patients with acquired essential hypertension disease and 20 ordinary subjects with no indications of essential hypertension disease until 2 ages prior. print of their palm and fingers was acquired in two groups by printing ink. patterns of fingertips, atd edges, a-b ridge and the various types of details were found, recognized and measured outwardly within four divisions of fingerprints. the consequence of this investigation indicated two patterns of fingertips; whorl and curve in diseases are more than ordinary individuals. the most widely recognized tips in ordinary individuals is loop. in normal the quantity of the a-b ridge in female diseases and male-typical are individually more on normal than female ordinary and male sicknesses. the atd point is lower in patients than in typical. consequences of the overview on details demonstrated that frequency of the ridge ending (e) is between with the most elevated frequency. the frequency of bifurcation (b) is lower than the ridge finishing which it is between % in the left hands. additionally, in some part of the fingers noteworthy distinction was found. 32-52.52 17.39-35 but in the right hand the second most patterns intermingling (c) which is between 14.13 and 38.66%. the third most patterns in the left hand was seen in pattern unions (c) with a rate between 8.62 and 32.6, however, in the right hand, the third most patterns are bifurcations (b), which is between 3.57 and 24.81%. the method proposed by chakravathy et al. (2018) does observational examination included a correlation of palmar dermatoglyphic parameters among cases and controls. parameters were dissected quantitatively-atd edge; subjectively outspread loop, ulnar loop, curve, whorl and composite. as appeared in (graph i) out of all-out of 250 cases, 120 (48%) were guys and 130 (52%) were females. of the absolute 250 controls, 137 (54.8%) were guys and 113 (45.2) were females. as indicated by (graph ii), they found that signify "atd" edge was higher in cases than controls and there was the factually critical association of signifying "atd" point in cases contrasted and controls. they likewise discovered both ways signify "atd" point was higher in cases than controls with solid factually huge association of right signify "atd" edge in cases than controls. by dissecting subjective parameters in study gathering, conveyance of dermatoglyphic patterns was measurably noteworthy in situations when contrasted and controls. the outspread loop was increasingly visiting in cases while ulnar loop was more ordinarily found in normotensive controls. the examination of subjective parameters in each hand of cases and controls demonstrated an appropriation of dermatoglyphic parameters that were factually huge. as appeared, looking at subjective parameters in each finger of the two cases and controls for the conveyance of dermatoglyphic patterns indicated factually noteworthy association. lahiri et al. (2013) used digital and palmar dermatoglyphic investigation of 145 normotensive subjects and 131hypertensive subjects was performed. the parameters utilized were advanced ridge pattern, complete ridge tally, and atd edge. the outcome demonstrated that the twofold loop whorl patterns are available with higher frequency in hypertensives. if there should be an occurrence of the hypertensive people, frequency of twofold loop whorl pattern and curve are 4.57% and 5.79% individually yet those are simply 0.44% and 1.33% in normotensives. in spite of the fact that the distinctions of occurrences of whorl just as ulnar loop between two groups are not all that obvious, yet factually huge (p < 0.05). the occurrences of explicit patterns in the hypertensive and the normotensive populace have appeared. the all-out ridge tally unmistakably expressed that it is horribly raised if there should be an occurrence of the hypertensive populace contrasted with the normotensive populace. the various estimations of average total ridge count of hypertensive and normotensive people have appeared with their comparing frequency. subsequent to processing the remedied atd edge, a normal of the atd points of the two hands is made. the mean worth, most extreme and least qualities and model estimation of rectified atd edge (normal of both hands considered) in hypertensive gathering and normotensive gathering. the general pattern of this parameter is best comprehended by measurable examination (t trial) of all out arrangement of information which shows contrasts in estimations of atd edges of the hypertensive and normotensive gathering are of factual hugeness (p < 0.05). tafazoli et al. (2013) does the examination and conclude an observational, systematic and handy examination utilizing a case-control observational methodology with straightforward irregular testing and without substitution did on two groups of solid subjects and patients experiencing hypertension; individuals with no other explicit hereditary diseases which thus influence dermatoglyphic readings. that loops were the most widely recognized patterns on left digits in all patients (guys and females). from a factual perspective, there is an important distinction in frequency of various patterns on the left digit 4 (p = 0.05) between genders yet other left digits indicated no measurably noteworthy contrast. on the correct digits, loops were higher in rate in the two sexual orientations however no huge factual distinction was seen between sexes for frequency of various patterns on right digits (p < 0.05). the higher occurrence of whorls on digit ii and of loops on different digits. their is no huge distinction in the frequency of the patterns between two hands (p < 0.05). as illustrated, frequency in the dissemination of loops were 70 and 58.8% for the left hand and 63.3 and 66.7%, for the correct submit guys and females, separately. the distinction between frequencies of patterns was not measurably huge on any of the two h ands in both genders neither the right nor the left nor both frequency of loops on the left and the correct digits were 70-63.3% in guys and 57.8-66.7% in females, separately. the contrast between frequencies of the patterns was not factually critical on two hands of both genders. the information shows that the frequency appropriations of whorls in the control and test (hypertensive) bunch were 75 and 63% on the correct hand and 79 and 70% on the left hand of guys, individually. no factually critical contrast was seen between the frequency appropriation of patterns on two hands of guys in the two groups (p = 0.04, p = 0.02) level of whorl and arch is more in charge bunch than intolerant gathering while the level of loop is more in-understanding gathering than solid gathering in the two sexual orientations. the percent dispersion of whorls on digits of guys was as high as 77 and 66.5% in the control and the hypertensive gathering, individually. there has been no factually noteworthy contrast between two groups for frequency of patterns on digits in guys. level of whorl digit patterns in females was 53.7 and 62.5% in the benchmark group and in the hypertensive gathering, individually. there has been no factually huge distinction between the two female groups in the frequency of patterns on digits. the dataset utilized and examination by wanga et al. (2015) was gathered from the behavior risk factor surveillance system (brfss) of centers for disease control and prevention (cdc) and is openly accessible and downloadable from the brfss site. brfss is the world's biggest and persistently led phone-based wellbeing study in regard to social hazard factors, incessant wellbeing conditions and utilization of preventive administrations. built-up in 1984 with 15 states taking an interest in the study, it has a long history in conduct and incessant disease observation. the essential point of brfss is to track and measure singular wellbeing conditions and hazard practices that add to the main source of high grimness and death rates in the grown-up populace who are matured 18 years and the older in united states. the review covers a wide scope of wellbeing hazard factors, preventive wellbeing practices and wellbeing conditions, including hypertension, diabetes and carcinoma related things. by gathering an assortment of data and sharing them with general society, brfss empowers scientists to examine the connections between interminable diseases and their hazard factors. we propose to anticipate hypertension just utilizing the surveys other than clinical test information, anthropometric information or hereditary information. its adequacy exhibits the practicability of building up a hypertension observation framework for an enormous size of the populace in a non-obtrusive and prudent way. what's more, the outcomes from this examination might be utilized to manage the advancement of projects outfitted towards forestalling and relieving explicit hypertension chance elements. (2) they propose to coordinate calculated relapse examination and fake neural systems for synchronous hazard factor determination and hypertension prediction. in spite of the fact that theyt now, the proposed approach is basically a general system that can encourage specialists to break down other ceaseless diseases and different types of information. (3) they detail the choice of fake neural system engineering and the setting of applicable parameters, which is a troublesome and testing task in model learning. this can conceivably assuage analysts of the mind-boggling model determination issue and empower them to concentrate on the issues under scrutiny. (4) to manage the class irregularity issues, they propose a viable under-examining method. based on a bunch calculation and choosing the delegate tests from each group in the extent of the group size, the proposed strategy can choose the most discriminative examples from the greater part class while making us lose the minimal measure of data. ravindranath et al. (2003) proposed qualitative dermatoglyphics involving identical fingerprint pattern, interdigital pattern, hypothenar pattern and palmar wrinkle was concentrated on 26 female and 11 male rheumatoid joint pain patients. examination between understanding male and control male; and patient female and control female has been finished. 'chi' square test was performed. in male patients, with hands together, arches were expanded, loops/whorls were diminished. incomplete simian wrinkle was fundamentally expanded. in the correct hand, patterns were expanded in the third interdigital zone. then again, in female patients there was a noteworthy increment in whorls and diminishing in loops on the principal finger on both the hands, increment in arches on the third finger; the two arches and whorls on the fourth finger of left hand. the present examination has accentuated that dermatoglyphics could be applied as a symptomatic device to patients with rheumatoid joint pain. mazumdar et al. found there is an association between rheumatoid joint pain and dermatoglyphics. the conceivable relationship amongst ra and dermatoglyphics may empower dermatoglyphics as a marker device in the analysis of rheumatoid joint pain. the present examination has been attempted to discover the likelihood that the fingerprints and palmprints assume a significant job in the analysis of rheumatoid joint pain. the frequency of twofold loop whorl in the rheumatoid joint pain bunch was seen in pointing finger and center finger of right hand and left hand separately (mazumdar 2015) . the ulnar loops were altogether present in right and little finger of the left hand of rheumatoid joint pain patients contrasted and control. the complete fingerprint ridges were progressively various in right and left hand of rheumatoid joint inflammation gathering. narayanan et al. (2017) investigation was a case-control concentrate with 60 cases with rheumatoid joint pain and 60 controls. the result appears out of the all-out 60 cases, 12(20%) were male and 48(80%) were females. of the complete 60 controls, 12(20%) were guys and 48(80%) were females. the subjective parameters in female was a measurably critical increment in the number of whorls morally justified and left hands of female patients contrasted with the controls. there was a factually noteworthy reduction in the outspread loops in both the hands of female patients contrasted with the controls and the abatement is increasingly huge in the correct hand. there was no measurably huge distinction in the ulnar loop pattern circulation in either hand of female there was a factually critical increment in the finger ridge includes of right to deliver male patients contrasted with controls. there was a critical increment in the ridge check of patients. there was a factually noteworthy reduction in arches in the left hand of females with rheumatoid arthritis contrasted with the benchmark group. the left hand in male patients contrasted with the controls. absolute finger ridge check (included ridge tally of both ways hand) was fundamentally expanded in male rheumatoid joint pain patients contrasted with the controls. the ridge includes of the correct submit female patients was altogether higher than that of controls. the ridge includes left-hand fingers in female patients was fundamentally higher than that of controls. the complete finger ridge tally (right-hand ridge tally + left-hand ridge tally) was essentially expanded in female patients contrasted with controls. the expansion in all-out finger ridge include was increasingly huge in female patients contrasted with the male patients. there was no critical contrast in the pattern force in male patients contrasted with the controls. there was a measurably critical increment in the pattern power in the correct hand of females in examination with controls. rajangam et al. (2008) perform examination of male patients indicated a pattern towards criticalness for 'all-out finger ridge tally', centrality in left hand for 'total finger ridge check', and morally justified for 'a-b ridge tally'. then again, in the female patients, 'supreme finger ridge check' was seen as critical for the right hand and 'a-b ridge means' left hand. the watched contrasts between the male and female patients just as with that of the control might be a direct result of the expanded whorl pattern adding to two tallies and the width of the palm and fingers, along these lines a more noteworthy number of ridges might be available. obviously, the detail of spreading the fingers and the palm, likewise should be remembered. hwang et al. (2004) concluded that the outspread loop and whorl were progressively visit while the curve and ulnar loop were less continuous; these attributes of the spiral loop and whorl were unmistakable in the correct hand and fifth finger. the frequency of the spiral loop was turned around in their left hands and the third fingers. the complete fingerprint ridges were increasingly various in the ra gathering. contrasts of both palmprint ridges and palmprint point atd between the ra and the benchmark groups were not conspicuous, then again, palmprint ridges c-d was progressively various in the ra gathering. the shut wrinkle was progressively visit though open and meeting wrinkles were less incessant in the ra gathering. the typical wrinkle was less continuous though simian and sydney wrinkles were progressively visit in the ra gathering; the general attributes were unmistakable in their correct hands. the general attributes of sydney's wrinkles were turned around in the left hands of female ra gatherings. the all-out level of palm wrinkle transversely was lower in the ra gathering; the attributes of the sydney wrinkle were progressively conspicuous. swati and sujata (2016) used conventional radiograph which has been a standard path for distinguishing the jsw in ra since bones are obviously noticeable in x-beam. toward the beginning of the disease, 90% of the side effects of arthritis are found in hands. manual reviewing strategies are not ready to separate a little contrast in jsw figuring. electronic appraisal of joint space has an effect in dynamic and observing the treatment of joint pain patients. the info hand x-beam picture of the typical hand is of 1000 × 2000 pixels, this picture is resized in the preprocessing step, resized picture goals is 500 × 500 pixels. the division utilizes asm strategy, the quantity of emphases required is 1000. the state of hand i followed flawlessly after the 1000 emphasis. the consequence of the asm division after 900 emphasizes. to separate bone and non-bone districts binarization activity is performed. figure 6 shows the aftereffect of binarization. binarization is finished by otsu's calculation. at that point, the skeletonization of the paired picture is completed. skeletonization brings about midline discovery it utilizes a diminishing procedure. at that point, the pinnacle and valley focuses are recognized along with the skeleton. the key focuses that is the specific joint area is followed by llm. ra tolerant joint area estimation process is appeared. at that point the different factual highlights are determined like mean, middle, change. joint area precision is determined by considering the number of exact joints recognized partitioned by the absolute number of joints that are 14. gobikrishnan et al. (2016) collected patient's data with rheumatoid joint pain in knee locale with disease span short of what one year and ordinary people with no knee disease utilized for this investigation. what's more, this examination was endorsed by the institutional moral council. absolutely 5 patients and 5 control subjects warm picture information was gathered. the mean period of patients utilized for the investigation was 40 ± 10. what's more, the disease length was 5 ± 2. the benchmark group mean age was 40 ± 10. the patients with no clinical proof of knee inclusion was dismissed for this examination. the information was gathered by directing a camp at srm organization of clinical science. before the picture obtaining method composed endorsement from the patient to take part in the investigation was taken. the patients who had knee torment other than rheumatoid joint inflammation were dismissed for this investigation. the highlights like standard deviation, mean, skewness and kurtosis were extricated for patients and control subjects fragmented picture. the acquired outcome indicated essentialness. the standard deviation was beneath 10 for control bunch above 10 for patients experiencing rheumatoid joint pain. mean worth was underneath 5 for control gathering or more 5 for patients experiencing rheumatoid joint inflammation. kurtosis was beneath 10 for control gathering or more 10 for patients experiencing rheumatoid joint inflammation and skewness was underneath 10 for patients experiencing rheumatoid joint pain or more 10 for control gathering. the worth discovered higher for patients because of higher temperature variety. the table 1 shows the different methods of blood group identification, which includes some of the traditional as well as unusual those are build using electrical or electronics components such as diode, sensors. the few researchers tried a software-based approach by processing image of blood sample, but only few used method called fingerprint pattern analysis to predict blood group with limited accuracy because thy apply this method with traditional paper and ink as sample collection mechanism, so it not provide high accuracy. in current era of digitization there are several image (fingerprint) computation techniques which explore a greater number of features from fingerprint image which extends the accuracy of prediction process. as per literature, there are many methods are available for determination of blood group from those some having pros and cons, but the popular and most traditional one is to take blood sample of an individual and test it against various antibodies to determine blood type 3 to 5 min but it not convenient to the small children's and individual having blood phobia. the fingerprint having lots of potential which explore different unique patterns those may leads to identify blood group very quickly and accurately. the table 2 show various methods and dataset or samples which are used for analysis and prediction of lifestyle-based diseases such as diabetes, blood pressure/hypertension, rheumatoid arthritis. the diseases arise with age but not all humankind suffers from such agebased disease. the methods and dataset used to study age-based disease are limited only daily activity, x-ray samples and some are the techniques used after arrival of such diseases. the age, blood group, daily activity, lifestyle of individual and fingerprint patterns analysis helps researchers to generate indication or risk prediction in early age of an individual. as per literature, all authors attempt traditional method for sample collection as ink and paper, so they were only analyses the fingerprint patterns visible to human eyes those are like loops, arches and whorls. above literature shows relation between blood group and finger-print pattern summaries as follows: • loops were the determined common finger-print design and arches were the least common. • whorls and mixed were moderate. • more no of loops was originating in blood groups o, b related to a and ab. • blood group o +ve is the maximum found in samples, o −ve and ab −ve is the fewest. • loops, whorls, mixed and arches were uppermost in females. • group a was the utmost common group among sampled males. • blood group o, b, were the record usually seen in females. in type 1 dm there is increased frequency in whorls, and decreased ulnar loop, increased frequency of sydney line, and increased incidences of arches in females (ravendranath and thomas 1995) . in maturity onset diabetes mellitus, there is decrease in mean value of tfrc, afrc, increase in arches and decrease in whorls (ravindranath et al. 2003) . the fingerprint of an individuals with t2dm would be more irregular than an individual without t2dm, regulatory for gender and age. diabetes regulate if wavelet analysis, a technique already used in forensics for fingerprint archival and matching, but not in previous studies of fingerprints as disease markers, would give results like the traditional ridge count or pattern analysis. the fingertips with whorls or double loops, applied a which rc formula that comprised half-unit values for those ridges situated between the core and delta point or between multiple cores. all ridge counting was as blinded to the diabetic and anthropometric status of the participants. the type 1 diabetes, there is increased frequency in whorls, and decreased ulnar loop, increased frequency of sydney line, and increased incidences of arches in females (roshani et al. 2016 ). • surge in arches in diabetes in both sexes • growth in rate of recurrence of loops and arches and a lessened frequency of whorls especially in mid finger • reduced number of arches in the right hand of male and left hand of female having diabetics, it was more in diabetic males and females than in the controls • growth in radial loop, ulnar loop in both male and female diabetics. • increase in frequency of whorls in both types of gender in diabetics there is increase in tfrc, decreased frequency of axial triradius 't' in right palm of females and 't and t' in right palm of male, decreased atd angle and absence of axial of triradial in 10% cases (mandasescu et al. 1999) . above literature shows relation between patients having hypertension and not a hypertension finger-print pattern summary as follows: • higher prevalence of whorls and loops are associated with higher level of blood pressure • whorls and loops are prime ridge patterns in hypertensive patients • atd angle showed the mean of angle in patient surge rather than in control group • larger frequency of ridge endings in the thumbs and index fingers • amplified frequency in bifurcations and convergences in the middle, ring and little fingers there is increase in arches and decrease in loops and whorls in males, whereas in females there is increase in whorls and decrease in loops on the 1st finger of both hands (sengupta and boruah 1996) , with increase in arches on 3rd digit and whorls on 4th digit of left hand (bala et al. 2015) . above literature study shows change in fingerprint patterns of patients having summarized as follows: • ulnar loop was the most prominent digital pattern in both genders, • decrease in the radial loop in both male and female patients • loops were significantly decreased in the third finger of males and a first and fourth finger of females • decrease in the ulnar loops in both the hands of male and female patients. • increase in the whorl pattern in the right hand of male patients and in both the hands of female patients • decrease in the arches of the left hand of female patients. the strength of the present research work is that fingerprint itself having lots of unique and hidden patents and it also currently used as a traditional, effective, and unique identification method of an individual. the dermatoglyphics as a diagnostic aid used from ancient eras and now it is well established in number of diseases which have strong hereditary basis and is employed as a method for screening for abnormal anomalies. there are more than 100 fingerprint minutiae patterns of ridges are determined as unique through the combination of genetic and environment factors. the weaknesses are in acquisition of fingerprint and finding different unique patterns from people of different age group due to the human digital fingerprint varies in texture as person ages, so it is very difficult to classify fingerprints because there are the fingerprints having the characteristics of two or more patterns changes with age of an individual. normally common clinical diseases like hypertension, arthritis and diabetes arises with aging, but due to busy schedule or lifestyle of an individual, it arises at any stage of life. so, it may lead to increase sample size and distribution of features required. the large datasets of fingerprint images acquired in real operational conditions are, rightly so, secured under data protection regulations that severely restrict the access to these data, even for research purposes. the fingerprints are having immense potential to have an effective method of identification. in this research, it investigates the problem of blood group identification and analysis of disease those arises with aging or disease called as lifestyle-based like hypertension, type 2-diabetes and arthritis from fingerprint by analyzing their patterns correlation with blood group and age of an individual. with the literature review study, it is observed that fingers of an individual are having multiple unique patterns those are need to be extracted with computerized method with fingerprints image captured using digital device which allow to find known association of fingerprints patterns which may enhance the authenticity of the fingerprints in blood group identification and early indication of lifestyle-based diseases of an individual. the fingerprint used as a traditional, effective, and unique identification method of an individual, in future it allows researchers to investigate with various diseases other than those are arised with age but also helps to explore different antibodies or reactive process of human body in several diseases. also, similar study helps to predict the risk of any kind of diseases in early age of an individual. the analysis and classification of community based on age, blood group, fingerprint patterns and lifestyle diseases help to tackle any pandemic in future like covid-19 in which mankind may suffer a lot having lifestylebased diseases like hypertension, type 2-diabetes. fingerprint recognition for person identification and verification based on minutiae matching finger prints pattern variation in diabetic patients analysis of left thumb print pattern among different human blood groups palmar dermatoglyphics patterns in diabetes mellitus and diabetic with hypertension patients in gangtok region comparative study of dermatoglyphic patterns of diabetes mellitus and diabetic with hypertension patients of hilly region rapid automated blood group analysis with qcm biosensors a role of dermatoglyphic fingertip patterns in the prediction of maturity onset diabetes mellitus (type ii) a handy tool for hypertension prediction: dermatoglyphics determination of blood group using image processing a study of fingerprint in relation to gender and blood group among medical students in uttarakhand region dermatoglyphics and health relation between fingerprints and different blood groups rh phenotypes analysis by spectrophotometry in human blood typing a complete blood typing device for automatic agglutination detection based on absorption spectrophotometry automatic system for determining of blood type using image processing technique automatic system for determination of blood types using image processing techniques development of a human blood type detection automatic system diagnosis of rheumatoid arthritis in knee using fuzzy c means segmentation technique. international conference on communication and signal processing ieee dermatoglyphic characteristics of patients with rheumatoid arthritis plantar and digital dermatoglyphic patterns in malawian patients with diabetes, hypertension and diabetes with hypertension an approach for minutia extraction in latent fingerprint matching fingerprint pattern examination of right hand thumb in relation to blood group efficacy of fingerprint to determine gender and blood group distribution of fingerprint patterns among medical students dermatoglyphics study of fingerprints pattern's variations of a group of type ii diabetic mellitus patients in erbil city design and development of blood sample analyzer using intelligent machine vision techniques a study on relationship between dermatoglyphics and hypertension detection of pre-diabetics by palmar prints: a computer study leading to a low-cost tool a case study on dermatoglyphics in rheumatoid arthritis approaches to determination of a full profile of blood group genotypes: single nucleotide variant mapping and massively parallel sequencing study of fingerprint patterns in type ii diabetes mellitus study of fingerprint patterns in type ii diabetes mellitus dermatoglyphics: an economical tool for prediction of diabetes mellitus a new method to assess asymmetry in fingerprints could be used as an early indicator of type 2 diabetes mellitus study of fingerprint patterns in relation to gender and blood group use of palmar dermatoglyphics in rheumatoid arthritis: a case-control study an automatic system to detect human blood group of many individuals in a parellel manner using image processing application of dermatoglyphic traits for diagnosis of diabetic type 1 patients spectrophotometric approach for automatic human blood typing development of an automatic electronic system to human blood typing the study of dermatoglyphics in diabetics of north coastal andhra pradesh population pattern of fingerprints and their relation with blood groups dermatoglyphics-quantitative analysis in rheumatoid arthritis fingertip patterns: a diagnostic tool to predict diabetes mellitus an integrated fiberoptic-microfluidic device for agglutination detection and blood typing a study of fingerprints in relation to gender and blood group finger ridge count and fingerprint pattern in maturity onset diabetes mellitus determination and classification of blood types using image processing techniques dermatoglyphics in rheumatoid arthritis dermatoglyphics in rheumatoid arthritis comparative study on the dermatoglyphic pattern among diabetic (type-2) and non-diabetic adults in north indian population dermatoglyphic patterns among type 2 diabetic adults in north indian population finger print pattern in different blood groups forensic abo blood grouping by 4 snps analyses using an abi prismr 3100 genetic analyser evaluation of abo subtyping by dna sequencing finger dermatoglyphic patterns in diabetes mellitus dermatoglyphics: a diagnostic tool to predict diabetes utility of dermatoglyphics in type ii diabetes mellitus (t2dm) to assess the risk for ihd: apilot study a study of dermatoglyphic pattern in relation to abo, rh blood group and gender among the population of chhattisgarh dermatoglyphics: blueprints of human cognition on fingerprints a cost-effective method for blood group detection using fingerprints comparative study of the fingerprint pattern among diabetic (type 1) & non-diabetic children in koya city modi"s medical jurisprudence and toxicology qualitative analysis of primary fingerprint pattern indifferent blood group and gender in nepalese study of dermatoglyphics in patients with type ii diabetes mellitus essential hypertension in the age group between 35-55 years international conference on automatic control and dynamic optimization techniques (icac-dot). international institute of information technology (i 2 it), pune, 2016 ieee tafa z, pervetica n (2015) an intelligent system for diabetes prediction the study of dermatoglyphic patterns and distribution of the minutiae in inherited essential hypertension disease comparison of dermatoglyphic patterns between healthy and hypertensive people evaluation of association between digital dermatoglyphic traits and type-2 diabetes in lagos dermatoglyphics in insulin: dependent diabetes or diabetes mellitus type 1 (t1 dm) determination and classification of blood types using image processing techniques digital grid method for fingerprint identification and objective report writing dermatoglyphic patterns of diabetic mellitus patients of ijaw origin in port harcourt dermatoglyphic and cheiloscopic patterns among diabetic patients: a study in ahmadu bello university teaching hospital zaria prediction and diagnosis of diabetes mellitus -a machine learning approach textbook of forensic medicine and toxicology predicting hypertension without measurement: a noninvasive, questionnaire-based approach key: cord-306798-f28264k3 authors: walsh, geraldine m.; shih, andrew w.; solh, ziad; golder, mia; schubert, peter; fearon, margaret; sheffield, william p. title: blood-borne pathogens: a canadian blood services centre for innovation symposium date: 2016-02-23 journal: transfus med rev doi: 10.1016/j.tmrv.2016.02.003 sha: doc_id: 306798 cord_uid: f28264k3 testing donations for pathogens and deferring selected blood donors have reduced the risk of transmission of known pathogens by transfusion to extremely low levels in most developed countries. protecting the blood supply from emerging infectious threats remains a serious concern in the transfusion medicine community. transfusion services can employ indirect measures such as surveillance, hemovigilance, and donor questioning (defense), protein-, or nucleic acid based direct testing (detection), or pathogen inactivation of blood products (destruction) as strategies to mitigate the risk of transmission-transmitted infection. in the north american context, emerging threats currently include dengue, chikungunya, and hepatitis e viruses, and babesia protozoan parasites. the 2003 sars and 2014 ebola outbreaks illustrate the potential of epidemics unlikely to be transmitted by blood transfusion but disruptive to blood systems. donor-free blood products such as ex vivo generated red blood cells offer a theoretical way to avoid transmission-transmitted infection risk, although biological, engineering, and manufacturing challenges must be overcome before this approach becomes practical. similarly, next generation sequencing of all nucleic acid in a blood sample is currently possible but impractical for generalized screening. pathogen inactivation systems are in use in different jurisdictions around the world, and are starting to gain regulatory approval in north america. cost concerns make it likely that pathogen inactivation will be contemplated by blood operators through the lens of health economics and risk-based decision making, rather than in zero-risk paradigms previously embraced for transfusable products. defense of the blood supply from infectious disease risk will continue to require innovative combinations of surveillance, detection, and pathogen avoidance or inactivation. • through improvements in screening, testing, and real time surveillance, the residual risk of transmissible diseases in the canadian blood supply remains very low. • we continue to deal with infectious diseases which emerge or reemerge, such as chikungunya, babesiosis, and hepatitis e, as well as infectious diseases such as influenza, that threaten the security of the blood supply despite not being transfusion-transmissible. • new paradigms for transmissible disease prevention must become more cost effective in their scope, using targeted surveillance, donor screening, and risk-based decision making. dr margaret fearon, cbs medical director, medical microbiology, and assistant professor, university of toronto, discussed the current prevalence of classical transfusion-transmissible infections (ttis) in cbs blood donors, new and emerging infectious diseases, how cbs prepares for and manages new risks, and also addressed new paradigms for risk management. dr fearon began by emphasizing that several layers of protection in the canadian blood supply have likely reduced the risk of the classical ttis, (hepatitis b virus [hbv] , hepatitis c virus [hcv] , human immunodeficiency viruses [hiv] 1 and 2, human t-lymphotropic viruses [htlv] i and ii, and syphilis). the success can be largely attributed to intensive donor testing for ttis, supplemented by donor education and deferral of donors with risk factors. the two latter approaches have reduced the number of donors with window-period infections and contributed to a decrease in confirmed transmissible disease-positive allogeneic donors over the last decade in canada, most notably for hbv and hcv [9] . thus, the residual risk of ttis is low by any standard. the estimated residual risk in canada calculated in 2012, using incidence rates from observed donor seroconversions 2006 to 2009, is 1 per 8 million donations for hiv, 1 per 6.7 million donations for hcv, and is 1 per 1.7 million donations for hbv [9] . dr fearon noted that updated residual risks are currently being calculated, but that compared to the 2012 report, the risks are not expected to change dramatically. dr fearon next turned her attention to newer and emerging infectious diseases that threaten the blood supply. some of these diseases have led to the introduction of new tti testing paradigms at cbs (ie, seasonal and selective testing for west nile virus [wnv] and chagas disease). other emerging infections are being monitored (eg, babesiosis, hepatitis e, chikv) while others such as influenza feature in contingency planning, in spite of not being transfusion-transmitted, due to their potential to disrupt blood donation and the health care system. she emphasized her opinion that transmissible disease testing must be context-specific, and account for local disease prevalence, environmental factors, and resource allocation. wnv is a mosquito-borne zoonotic arbovirus that emerged in north america in 1999 and was found to be transfusion-transmissible in 2002 [10] . in humans, febrile illness occurs in 20% to 30% of wnv cases and 1% of patients have serious neurologic symptoms. since most cases are asymptomatic, tti testing is the primary means of preventing transmission [11] . universal donor testing was adopted in 2003 using nucleic acid testing (nat). however, given the seasonal nature of wnv outbreaks, a more nuanced testing methodology was introduced by cbs in june 2015 that recognizes the lack of local transmission during the winter months. now, all donors are tested from june 1 to november 30 and only donors who travel outside of canada are tested during the rest of the calendar year. selective testing is also conducted for chagas disease. chagas is caused by the protozoan parasite trypanosoma cruzi and is endemic to central and south america and mexico, where it is estimated that 6 -7 million people have been infected [12] . with increasing northward immigration of people from these regions, it is estimated that n 300,000 people are infected in the united states, and most american blood services have implemented universal donor testing [13, 14] . the rates of immigration from endemic countries are lower in canada and thus cbs tests donors who are identified to be at risk based on the donor questionnaire. those considered at high risk include those who were born or lived in an endemic country, or had a mother or maternal grandmother that was born or lived in an endemic country. the safety of this approach was demonstrated in a recent study that identified no 60 61 61 63 63 65 66 66 66 66 evidence of infection amongst donors without risk factors identified on the questionnaire (with the exception of one very unusual transfusion transmissionvertical transmission case) [15] . interestingly, the selective testing approaches used for wnv and chagas disease at cbs represent a change from the universal testing approach of the last three decades, which can be summed up as "test everyone for everything". the newer approach to certain ttis takes into account geographic location, seasonal effects, and other risk factors in setting the optimal testing strategy. dr fearon called attention to other infectious outbreaks that can impact the security of the blood supply despite not being transfusiontransmissible, and cited severe acute respiratory syndrome (sars) and pandemic influenza as examples. outbreaks of these diseases can lead to shortages of staff and donors due to illness and shortages of critical supplies. contingency planning is necessary to guard against these risks. staff and donor education, infection control procedures in the clinic, and reassessment of donor deferral criteria are key steps that must be taken to protect the blood supply in this context. other transfusion-transmissible diseases are currently being monitored as potential emerging threats to the safety of the blood supply, including babesiosis, hepatitis e, chikv, and dengue virus. babesiosis is caused by the protozoan parasites b microti, b duncani, and others in this genus, and spread by infected ticks. most infections are asymptomatic or unrecognized, but the spectrum of clinical severity also includes flu-like symptoms, ranging to more severe illness and death in the immunocompromised [16] . babesia microti is the most frequently transfusion-transmitted microbial pathogen in the united states, especially in the northeast and upper midwest states [17] . there were 160 transfusion-transmitted cases reported from 1979 to 2009 in the united states with one case reported in canada [18] . hepatitis e is clinically similar to hepatitis a and it causes water-borne outbreaks in developing countries. in canada, where it was previously thought to be primarily a disease of travelers, the actual prevalence of endemic hepatitis e is unknown. no cases of transmission by transfusion have been reported in north america, but transfusion transmission has been reported in endemic countries and recently in the united kingdom [19] . chikv and dengue are two viruses common in the tropics that are spread by mosquitos. both lead to similar acute illnesses with fever, rash, and muscle/joint pain. transfusion-transmitted cases of dengue have been reported. chikv arrived in the caribbean in 2013 and was thus identified as a threat to north america [20] . however, no transfusion transmitted cases of chikv have been reported to date. current malaria travel deferral provides some protection with respect to many but not all of the affected areas, particularly in the caribbean. how can blood operators best prepare for emerging threats? surveillance is conducted by multiple health agencies, including the world health organization (who), centers for disease control and prevention, and the international society for infectious diseases, which operates the promed (program for monitoring emerging diseases). available to any subscriber, promed is an internet-based reporting system dedicated to rapid global dissemination of information on transmissible diseases. the public health agency of canada is the federal agency responsible for transmissible disease surveillance in canada. public health agency of canada encompasses the national microbiology laboratory and in collaboration with the provincial public health laboratories, provides diagnostic testing and surveillance data that is useful in guiding cbs decision-making. testing data provided by the national microbiology laboratory on travel-acquired chikungunya was used by cbs to calculate an estimated risk of a case of transfusion transmitted chikv in canada of less than 1 in 11 million. collaboration with veterinarians, etymologists, and ornithologists may provide additional information to inform preparative and reactive strategies for emerging agents. for example, active tick surveillance reports provide risk data for lyme disease, but are also relevant to other tick-borne, transfusion-transmissible diseases such as babesiosis [21] . a recent babesia seroprevalence study for b microti in canadian blood donors demonstrated that donor testing is not warranted in canada at this time [22] . dr fearon presented as-yet-unpublished data on a collaborative cbs and héma-quebec (the transfusion service for quebec province) hepatitis e seroprevalence study that indicated that age is the only significant factor for increasing seroprevalence of hepatitis e. the absence of polymerase chain reaction (pcr)-positive results suggests that the risk of transfusion-transmission of hepatitis e in canada is extremely low; however, further prevalence data needs to be collected. a cbs donor travel survey from 2014 also provided data that is used to inform risk assessment. while the united states remains the most popular travel destination for cbs blood donors, nearly 10% of respondents reported travel to the caribbean. such donor travel survey data allows cbs to estimate potential donor loss when assessing the risk/benefit of deferring donors who have travelled to countries with outbreaks. the challenge facing all blood operators is to synthesize all of the available information on existing and emerging threats in order to rapidly make decisions that balance risks, costs, and safety. to meet this challenge, dr fearon suggested utilization of the alliance of blood operators' risk-based decision making framework for blood safety (fig 1: risk-based decision making framework) [23] [24] [25] . this framework has a health sector focus, can aid evidence-based decisions using risk assessment tools, and accounts for multiple sectors included in the decision making process [23, 26] . the use of this approach also represents a paradigm shift for blood operators, away from "zero-risk" to one that uses a decision-making process that integrates evidence, ethics, social values, economics, public expectations, and historical context with broader health care priorities [26] . • the emergence of infectious diseases is unpredictable. • emerging infectious diseases (eids) is a global issue that demands international surveillance efforts. horizon scanning is important. • the eid tool-kit provides a useful framework for managing infectious threats to the blood supply. dr roger dodd, secretary general of the international society of blood transfusion, presented his perspectives on past and current pathogens affecting the safety of the blood supply. the objectives of his presentation were 4-fold: (1) to define what eids are and why they occur; (2) to discuss why some eids impact blood safety; (3) to review how the impact on the blood supply is managed; and 4) to examine some current examples of emerging infections and how they are being managed. dr dodd began with the institute of medicine's definition of an emerging infectious disease as one "whose incidence in humans has increased within the past two decades or threatens to increase in the near future". the institute of medicine further elaborates that "emergence may be due to the spread of a new agent, to the recognition of an infection that has been present in the population but has gone undetected, or to the realization that an established disease has an infectious origin. emergence may also be used to describe the reappearance (or reemergence) of a known infection after a decline in incidence" [27] . eids often originate from animal-human interactions. a prime example is variant creutzfeldt-jakob disease (vcjd) which probably results from human consumption of meat from animals infected with bovine spongiform encephalopathy (also called mad cow disease) [28] . it is estimated that approximately 60% to 70% of current eids are zoonoses, and they can be caused by any class of pathogenic agent (viruses, bacteria, parasites, and prions) and spread through several modes of transmission (fecal-oral, sexual contact, etc). infections emerge for a variety of reasons. pathogens may undergo a "species jump" as was the case in hiv [29] and sars [30] . environmental change, such as global warming, may increase the incidence and range of eids such as dengue, malaria, and babesiosis. drug resistance and mutations may lead to challenges in controlling malaria and hbv and subsequent spread. human migration and travel contributes to the dissemination of t cruzi (the chagas disease pathogen) and chikv. the migration patterns of birds, reservoirs for wnv, are also associated with the spread of this disease. certain parts of the world are considered to be "hot spots" for the emergence of infectious disease for a variety of reasons. for example, china is considered a prime site for the emergence of new strains of viruses (influenza and sars) due to the close proximity of human-human interactions and human-animal interactions, which can lead to the evolution of animal viral strains into novel strains that can infect humans. in a general sense, this evolution can be potentiated by the consumption of wild meat (meat from non-domesticated mammals, reptiles, amphibians, and birds). through careful phylogenetic analysis of simian and human viruses, africa has been recognized as the "hot spot" for hiv emergence, probably due to consumption of primate bush-meat by humans, [29] . urbanization, poor sanitation, and crowding in the developing world have also been linked to hepatitis e emergence [31] . although our understanding of the factors contributing to specific eid transmission has improved, these transmissions are likely multifactorial in nature. dr dodd emphasized that eids are both a local and a global issue. some infections may emerge explosively in new areas if appropriate conditions (eg, the vector or environment) are met, as is the case with wnv, dengue, and chikv. other eids, such as chagas disease, may expand slowly as a result of population movements, but can become constrained in their new environment. tick-borne infections, such as those caused by babesia in the united states, may be constrained regionally. infections that are characterized by direct human to human transmission may spread worldwide but at differing rates, depending on the mode of transmission such as the rapid respiratory spread of influenza and sars and slower sexual transmission of hiv. despite our understanding of infectious disease transmission, the emergence of these diseases remains largely unpredictable. understanding that eids may be spread by human travel and through animal contact allows us to understand the probability of acquiring an infection if specific conditions are met, but it does not allow us to predict which infectious disease will emerge and when. such unpredictability requires regular surveillance and hemovigilance efforts to be in place around the world. disease surveillance has many challenges, but warning signals may help focus efforts to better monitor disease spread. these warning signals may include disease outbreaks in particularly susceptible populations (such as the immunocompromised), and/or the blood-borne nature of a disease. why do some emerging infections impact blood safety? dr dodd noted that when an epidemic occurs, only a minute proportion is attributable to transfusion. for example, only 2% of hiv cases were transfusion-transmitted during the hiv epidemic [32] . similarly, only 23 of 400,000 wnv cases (0.006%) were established as ttis [33] . in other words, outbreaks are not likely to start from a transfusion, and transfused patients are not necessarily more likely to acquire an infection than the general population. for an infectious disease to be considered a transfusion-transmissible disease, certain pathogen-related and recipient-related characteristics have to be present. first, the pathogen must have an asymptomatic blood-borne phase (such as hepatitis b), which may either be acute or chronic. second, the pathogen must be able to survive the donated blood processing and storage procedures, including temperature changes, leukoreduction, and centrifugation. third, the disease must be transmissible by the intravenous route. fourth, the recipient has to be susceptible to infection. fifth, the disease must be a recognizable entity in the recipient once symptoms appear. next, dr dodd turned his attention to how the impact of eids on the blood supply is managed. in 2009, the aabb published a list of 68 eids of interest. each eid was categorized based on the threat it poses to transfusion safety, the level of regulatory concern, the lack of effective intervention, and the amount of public concern. the top priority was assigned to 3 pathogens towards which intellectual and future resources should be focused: dengue viruses; babesia species; and prions causing human vcjd [34] . the list also included chikv, plasmodium species, t cruzi, human parvovirus b19, hiv, and hepatitis e. an eid tool-kit (fig 2) was subsequently developed as a framework to guide health professionals and public health officials in triaging and managing infectious threats to the blood supply [35] . the eid tool-kit presents: a variety of methods for eid surveillance; key questions to be asked when a threat is suspected; and potential courses of action based on the situation. the risk-based decision-making framework. the risk-based decision-making framework was designed by the alliance of blood operator to help blood operators identify, assess, act on, and communicate risk in decisions related to blood safety. it is a flexible tool, and its objectives are to optimize the safety of the blood supply while recognizing that elimination of all risk is not possible; allocate resources in proportion to the magnitude and seriousness of the risk and the effectiveness of the interventions to reduce risk; and assess and incorporate the social, economic, and ethical factors that may affect decisions about risk [23] [24] [25] . included among the 68 pathogens identified in the 2009 aabb report, several agents are being actively monitored both on a regional and global level. due to increasing reports of transfusion-transmitted cases, their poor prognosis in immunocompromised transfusion recipients, and the lack of effective prevention strategies, babesia species have been classified as a top priority for future blood supply safety efforts in the united states. prions such as vcjd are being monitored closely and donor screening has been successful in preventing their spread into the donor pool [28] . prions are currently being investigated for their potential relationship with other protein-folding diseases such as alzheimer's disease [36] . although respiratory infections (eg, middle east respiratory syndrome coronavirus) are not transfusiontransmissible, they do disrupt donor availability and organizational aspects of the blood collection and donation system. dr dodd expanded upon wnv, adding american insights into those earlier provided in the canadian context by dr fearon. west nile fever is caused by a flavivirus transmitted by culicine mosquitoes. the virus spread from southern europe, africa, and the middle east to india, and arrived in the united states in 1999. by 2004 wnv was endemic in most of the continental us and canada [33] . the experience of wnv in the us demonstrated that imported infections can be overwhelming and unpredictable. while wnv was considered a stable disease elsewhere in the world, in north america it was experienced as an explosive outbreak in 2002 and 2003 infecting over 400,000 individuals. public concern was high as wnv, previously unknown in north america, spread rapidly across the continent via infected birds, and then to humans via mosquitoes. although human to human transmission is not possible, there is potential for transfusion-associated transmission if the donation occurs during periods of pre-symptomatic viremia. nat of pooled donor samples offered a rapid route to testing. like other north american blood operators, cbs tests donors in pools. if a pool tests positive, individual donor testing is initiated. after nat was initiated in the us in 2003, cases of transfusion-transmitted wnv have only rarely been encountered [33] . horizon scanning efforts are actively monitoring other potential eids. dr dodd described several emerging agents for which there is some evidence that that have or an assumption that they may be transfusion-transmitted including severe fever with thrombocytopenia syndrome virus (no reported transfusion transmission [tt]), q fever (one report of tt, but no definitive evidence), hepatitis e virus (good evidence of occasional tt), vcjd, and other prions (evidence for tt) [35] . dr dodd focused on two viruses that are current causes for concern. dengue virus is an important arbovirus. like wnv, it is a flavivirus and is spread by mosquitos (aedes genus). humans are the amplifying host, and while a vaccine is under investigation there is currently no vaccine or specific treatment. vector control is the only effective intervention. there are an estimated 390 million infections per annum worldwide [37] . in 50% to 80% of cases infection is asymptomatic. dengue viruses have been found to be transfusion-transmitted in 7 separate geographic clusters in hong kong, singapore, puerto rico, and brazil. currently, there is no fda-licensed test for dengue rna [37, 38] . chikv, which has caused recent massive outbreaks in the caribbean and co-exists with dengue virus, is a potential threat to the north american blood supply due to its geographic proximity and donor travel emerging infectious disease toolkit. the emerging infectious disease toolkit is a framework developed by the aabb transfusion-transmitted diseases, emerging infectious diseases subgroup to guide health professionals and public health officials in triaging and managing infectious threats to the blood supply. the eid tool-kit presents: a variety of methods for eid surveillance; key questions to be asked when a threat is suspected; and potential courses of action based on the situation [35] . patterns [39] . chikv recently appeared in the caribbean. should it arrive in north america, donors could be deferred for exposure or symptoms, nat testing for chikv rna could be initiated, and red cell and plasma collections could even be stopped. dr dodd stated that pathogen inactivation will likely become increasingly important in preventing transfusion-transmission of emerging agents. the american red cross in puerto rico is currently involved in a trial to investigate and monitor the safety of intercept pathogen inactivation technology (cerus corporation). intercept was recently approved by the fda for use with apheresis platelets. use of intercept to make available pathogenreduced apheresis platelets could prevent interruptions in the local platelet supply in areas where viruses like chikv emerge [40] . • the 2014 ebola outbreak in west africa was the largest in history. • the difficulties in identifying the virus in west africa and in containing its spread were related to poverty, growing populations and deforestation, lack of healthcare infrastructure and resources. these fundamental issues must be addressed in order to prevent and/or contain future outbreaks. dr allison mcgeer, director of infection control at toronto's mount sinai hospital, used the recent ebola outbreak in west africa to provide a thought-provoking global perspective regarding pathogens and their spread. as a consultant on a who-initiated mission to liberia, dr mcgeer obtained first-hand information and impressions of the situation on the ground in west africa, where she navigated issues related to policy and healthcare set-up. ebola virus is difficult to study because of its high mortality rates, and because it often occurs in areas that are difficult to access due to poor infrastructure or because of conflict or political turmoil. ebola infections seem to emerge due to the interconnection of enzootic and epizootic cycles [41] . the first sequence involves bats as the most likely reservoir host for the virus, which is spread by enzootic transmission within the bat population. from this pool of virus carriers, transmission to other non-human species is thought to happen in an epizootic cycle. initial transmission to humans involves contact with infected bats or other species through hunting or accidental contact with ill wild animals. humanto-human spread via direct close contact is then very efficient. once humans are infected, the ebola virus first appears to target the immune system and subsequently destroys the vascular system, leading to blood leakage. initial attacks on dendritic cells lead to decreased interferon production and macrophage and endothelial cell degradation [42] . this pattern results in clinical presentations of hemorrhage, hypotension, drop in blood pressure, followed by shock and death [43] . the 2014 ebola outbreak, which primarily affected guinea, sierra leone, and liberia in west africa, is the most recent in the history of ebola epidemics [44] . since 1976, more than 20 outbreaks have been recorded in sub-saharan africa leading to hundreds of cases and deaths [45] . historically, infection has been controlled by local communities with the isolation of any patient showing symptoms. however, in 2014 in west africa, the outbreak was on an unprecedented scale. dr mcgeer provided an eye-opening overview of health infrastructure in the affected west african countries. guinea, liberia, and sierra leone have populations of 11.5, 4.2, and 6 million, respectively (vs 35 million in canada). spending on health (total expenditure per capita) is us$59, us$88, us$228, and us$4759 in guinea, liberia, sierra leone, and canada, respectively [46] . this imbalance is mirrored in the number of doctors per 100,000 population: 10, 1.4, 2.2 for guinea, liberia, sierra leone, respectively, compared to 245.2 in the united states of america [47] . under-resourcing of public health and healthcare delivery was an important contributor to the unprecedented scale of the epidemic. the first step in this epidemic was the spread of the virus from a reservoir in west africa, but the outbreak was aided by many economic, political, and geographic factors. being now relatively stable after periods of civil war, these countries have increasing birth rates and increasing populations. the median age in guinea, liberia, and sierra leone is 18.5, 18.4, and 20, respectively (vs 40 in canada). the ever-growing population and deforestation is believed to have accelerated the frequency of the epizootic cycle. once rich in forests, west africa has been intensively logged over the last decade. guinea's rainforests have been reduced by 80%, while liberia has sold logging rights to over half its forests. some analysts have predicted the complete deforestation of sierra leone within the next few years [48] . the forests are the habitat for fruit bats, ebola's probable reservoir host. with the loss of their habitat, the bats escape to urban environments to hunt for food, coming in contact with humans and triggering more frequent transmissions of the virus. dr mcgeer personally witnessed the huge barriers to effective control of the epidemic in hospitals in liberia. she pointed to the rudimentary nature of facilities and equipment, poor hygiene, and a lack of infrastructure for disposing of hospital waste. dr mcgeer commented that due to the lack not only of resources but also of any form of emergency plan, the base from which to fight the epidemic was completely lacking. the lack of resources for infection control and personal protective equipment are the main reasons for nosocomial transmission [44] , and affected healthcare workers can act as amplifiers spreading the virus into the community. in the 2014 epidemic, 1 in 20 healthcare workers were infected, and of those, 1 in 10 are believed to have died [43] . this devastated the ability of front-line healthcare workers to control the epidemic, and led to hospital closures. poor general infrastructure hindered transportation of medical supplies and expertise, isolating rural areas and limiting access. dr mcgeer highlighted the absence of public health and health care infrastructure as a fundamental issue in being able to fight this disease, not just on the ground in west africa, but globally. this issue is illustrated by a fragmented global health system is which the institutions, laws, and strategies are not interconnected. experience of this outbreak has led to calls to reform the worldwide health systems architecture and the who [49] . some media reports led to widespread misunderstanding of the ebola outbreak as an "african problem," and unhelpfully perpetuated prejudicial colonial-era stereotypes. dr mcgeer took the audience through the evolution of the 2014 epidemic in liberia which peaked in august/september 2014. during july and august 2014 the number of confirmed cases per day increased from about 10 to about 50, and ebola treatment units and burial systems were overwhelmed. in monrovia, the liberian capital city, about 80% of patients with ebola virus disease were being managed at home. despite this, hospitals were overwhelmed with ebola patients, filling to 300% of its capacity. many health care workers were infected, which ultimately led to the decision to close hospitals. at the peak of the outbreak, the president of liberia quarantined west point, a township particularly badly affected. this decision led to riots in that area. similar actions took place in other parts of liberia [50] . as a consequence of the deteriorating situation in west africa, international responses were initiated [49] . by september 2014, more than 100 non-governmental organizations were on the ground in liberia to contribute to the fight against ebola. their work spanned a wide range of activities including: setting up medical care; contact tracing; opening orphanages; providing food for people in quarantine; building roads for improved access to cemeteries; identifying how to de-sludge septic tanks from ebola treatment units; and sourcing and supplying personal protection equipment for ebola treatment units. the crisis response also involved a coordinated international response of unprecedented scale to accelerate vaccine development. no vaccine had ever been tested in humans prior to 2014, but several were fast-tracked through phases i and ii, and in june of 2015, an international group of scientists published the interim results of an open-label, cluster-randomized ring vaccination trial of an engineered vesicular stomatitis virus-based ebola vaccine developed at canada's national microbiology laboratory [51] . ring vaccination seeks to create a buffer of protection around each case, so that the virus cannot continue to spread. this initial trial found that 100% of recipients were protected from the virus; further study of this vaccine and trials of other vaccines are on-going. with improvements in the coordination of the overall emergency response, more than a year later, the outbreak is under control. occasional cases were still appearing in liberia in november 2015, but on november 7, 2015, who declared the end of ebola virus transmission in sierra leone, as 42 days had passed since the second negative test of the last confirmed patient with ebola in the country [52] . control of the outbreak was only achieved through the institution of effective control and quarantine measures and an understanding of local practices and challenges that impacted the spread of the virus. dr mcgeer added her voice to the chorus of experts recommending coordinated national and international efforts to prevent future outbreaks. early warning systems should be developed in connection with local communities in high-risk areas, and provision of clearly defined response recommendations specific to the needs of each community [53] . recent advances in diagnostics, risk mapping, mathematical modeling, and pathogen genome sequencing have the potential to improve substantially the quantity and quality of information available to guide the public health response to outbreaks [54] . however, prevention remains extremely difficult [55] . the world bank estimated that an~$26 m investment in public health in west africa would have prevented more than 90% of cases in west africa. this missed opportunity eventually led to a costly emergency response estimated by the un mission for ebola emergency response at about $1.6 billion. future epidemic control measures in poverty-stricken areas, including worldwide response teams and pre-approved emergency funds may improve outbreak response, but addressing the fundamental issues of poverty, infrastructure, education, healthcare, workforce development, and communications will be needed if outbreaks are to be prevented [56] . • several approaches for the production of red blood cells (rbcs) ex vivo exist and have demonstrated feasibility; however, none can yet be conducted on a scale that would allow replacement of donor-derived rbcs • to improve the chances of success in bringing ex vivo rbcs to the clinic, a multidisciplinary approach and an integrated plan are required to navigate the long path from concept to commercial product. an alternative means to keep the blood system safe from pathogens is to move away from the current paradigm of donor-derived products. this topic was addressed by dr marc turner, medical director at the scottish national blood transfusion service and professor of cellular therapy at the university of edinburgh. dr turner began by mentioning some milestones in the history of blood transfusion, highlighting a number of events that took place in his adopted home city of edinburgh, scotland. for example, in 1816 the first systematic experiments in intra-species transfusion were carried out by john henry leacock, who was studying in edinburgh at the time. james blundell, a university of edinburgh medical school graduate, is credited with conducting the first successful human transfusions several years later. since early in the last century, transfusion has become a mainstay of clinical practice with around 92 million rbc transfusions conducted annually worldwide [57] . for the most part, and particularly in developed countries, blood transfusion is safe. however, limitations remain, including sufficiency of supply, immunological compatibility of the donor and recipient, the risk of ttis, and the risk of other complications such as iron overload. many of these limitations could potentially be overcome by the production of rbcs for transfusion in the laboratory. dr turner's talk focused on human stem cells and their potential use for the ex vivo generation of rbcs for transfusion. dr turner discussed the biological and engineering limitations that must be overcome in order for ex vivo generated cells to become viable alternatives to donor-derived rbcs. the first conclusive observation of stem cells was in 1960 by mcculloch and till in the host city of this symposium -toronto [58] . stem cells have the capability for self-renewal and can differentiate into multiple lineages, ultimately resulting in the generation of differentiated cells or tissues. there are various types of stem cells, including those derived from embryos (eg, human embryonic stem cells [hescs]) and from adults. not all stem cells are the same. for example, hescs are pluripotent and have unlimited ability to replicate, whereas adultderived hematopoietic stem and progenitor cells (hspcs) can differentiate into cells of the hematopoietic and immune systems and have limited ability to replicate. found in the bone marrow, hspcs are morphologically indistinct from other bone marrow cells, and are distinguished as cd34 + cells using flow cytometry. in vivo, hspcs are found in a hypoxic environment at the edges of the bone marrow. as they differentiate, they move more centrally in the marrow into an environment with a higher o 2 tension. it has long been known that when placed into agar/semisolid medium and provided with the right cytokine support, hspcs can differentiate along different hematopoietic lineages, forming granulocyte/monocyte, and erythroid colonies with 7 to 14 days [59] . culturing hspcs in suspension using a two-phase culture system has several advantages over solid-phase culture [60] , the in vitro environment can be more precisely controlled and can be separated more easily [61, 62] . dr turner provided an overview of a two stage culture system which uses combinations of cytokines to control cell differentiation and proliferation and can be used to produce erythroid cells from cd34 + adult peripheral blood cells. during the enucleation process, which moves the cells into the early reticulocyte stage, the nucleus is extruded from the cell, and engulfed by macrophages. by days 10 to 14 of the two-stage culture, the population consists of approximately 50% normoblasts and 50% early (r1) reticulocytes. other 3-stage systems have had demonstrated success expanding cd34 + hspc from peripheral blood, bone marrow, or cord blood into functional rbcs [63] . these proofs-of-principle demonstrated that rbc generation ex vivo is possible, but processes described to date have limited scalability and do not yet constitute a workable approach to generate rbcs for transfusion. dr turner spent the remainder of his talk discussing the two major types of challenges facing the ex vivo generation of rbcs for transfusion: biological challenges and engineering/logistical challenges. from the biological point of view, one major challenge is to determine the best source from which to derive the cells. adult hematopoietic stem cells (hscs), one potential source of ex vivo rbcs, have a limited replication capacity; they could generate a small number of units of rbc but the reliance on donors remains. unlike hscs, hescs have indefinite expansion capabilities and can self-renew, and were a source of much excitement when their derivation was first described [64] . hescs are pluripotentthey can be cultured indefinitely as cell lines and are able to differentiate into all the cells of the body including hematopoietic cells [65] . in 2006, a considerably less ethically-controversial source of pluripotent stem cells was discovered-induced pluripotent stem cells (ipscs) [66, 67] earning the discoverer the nobel prize for medicine in 2012. initially generated by takahashi and yamanaka from human somatic skin fibroblasts by the use of four genes (oct4/sox2/klf4/myc), ipscs are very similar to hescs, can be differentiated into all three germ layers and have huge potential for regenerative medicine applications and disease modeling. like hescs, human ipscs can be differentiated into hematopoietic cells in vitro [68] [69] [70] [71] , and this is an area of intensive research [72] . dr turner provided an overview of a system by which rbcs can be derived from human pluripotent stem cells (hpsc) in vitro using a feeder/serum free approach and cytokine mixes that drive the hpsc to hspcs, then erythroblasts, normoblasts, and eventually reticulocytes over an approximately 30-day time frame. the differentiation process is complex and uses mixes of multiple cytokines and small molecules. several issues remain including the long differentiation time, and the fact that while hpsc-derived rbcs can enucleate, the resulting cells are fragile and difficult to maintain in culture. the hpsc-derived rbcs express α/γ globin chains, the same combination of hemoglobin polypeptides expressed in fetal rbcs (as opposed to α/β globin chains in adult rbcs); however, the oxygen-delivering characteristics of the hpscderived rbcs are acceptable and therefore this would not preclude their use. efforts are underway to optimize erythrocyte differentiation and enucleation and modify the hemoglobin to a more adult form [73, 74] . another approach that holds promise is to immortalize multipotent stem cells using well-understood and established methods. conditional immortalization from hpscs or adult hspc can be achieved using human papilloma virus e6/e7 or combinations of transcription factors. this approach can establish immortalized human erythroid progenitor cell lines [75] . switching off the immortalization allows the production of enucleate rbcs ex vivo. this approach has several benefits over psc-as a starting material: the process is less complex and the differentiation time is shorter; there are reduced costs related to cytokines, growth factors, and media; and the final rbc phenotype is closer to an adult phenotype. the feasibility of this approach to produce enucleated rbcs has been shown [75] . potential drawbacks of this approach include concerns regarding: the introduction of oncogenes into the cells; cell line stability; and the likelihood of being able to meet good manufacturing practice standards and produce clinical grade products. notwithstanding the difficulties involved, the potential now clearly exists to allow culturing of rbc with rare phenotypes or modification of the rbcs [76] to help meet the needs of hard-to-match transfusion recipients. the first proof-of-principle human transfusion with ex vivo generated rbcs cultured from peripheral cd34+ hsc was conducted in 2011, and showed that post-transfusion survival of ex vivo generated rbcs in a single, healthy subject was comparable to that of donorderived rbcs [77] . dr turner then turned his attention to the engineering technology needed to make ex vivo generated rbcs a viable alternative to donorderived products. the key issues here are scale-up, process control, and intensification. dr turner described the use of the ambr bioreactor technology (from tap biosystems, part of the sartorius stedim biotech group) as an approach to optimising the in vitro culture environment. stirred tank bioreactors are a mature technology that is scalable and well-established for production of biotherapeutics. they allow for precise control of several physico-chemical parameters and economic/ rapid development screening at a variety of scales (10 ml, 250 ml, multi-liter). there are two gross limits to system efficiency. the first is the absolute density limit, which is calculated from specific oxygen uptake rate of cells and the mass transfer coefficient of the system. in this regard, the bioreactor system performs well; high density can be achieved, and the potential is certainly there to succeed in scaling it up to the needs of producing erythroid cells. the second limit is media volumetric productivity (liters of media/units of blood). volumetric productivity is precisely determined by cell growth rate and specific support capacity of the media-consumption and supplementation. specific rates are unstable and supplementation with glucose and glutamine does not improve growth, suggesting these are not limiting factors. in order for potential scale-up of this system, the limiting factors need to be identified. currently, dr turner estimates that the costs of ex vivo-derived rbcs stand at many times the cost of donor-derived rbcs. at this stage this technology is therefore only likely to be considered for "boutique" applications in patients for whom a donor-derived matched product is difficult or impossible to source. for more generalized application, many challenges remain, including control over the genetic and epigenetic stability of cell lines; optimization of the differentiation pathway to allow stable enucleation; efficiency of the differentiation pathway; process control over multiple physical and biochemical factors; scale up and intensification to control the cost of goods; detailed characterization of the product; demonstration of preclinical safety and efficacy; quality control and regulatory compliance; and the design and execution of pivotal clinical trials [78] . many of the challenges dr turner mentioned are common to all cellbased therapeutics. regarding quality control and product characterization, one advantage in this field is that there is more than 50 years of rbc product characteristic information, knowledge, and experience on which to draw. dr turner ended by noting that a sea change in the level of process control would be required to make the production of the ex vivo rbcs a reality. dr turner outlined the environment, resources, and approaches he believes are necessary in order for this type of innovation to take place and achieve commercial success [78] . this includes multidisciplinarity, integrated planning, and lengthy time lines and in these regards dr turner acknowledged the commitment of the research teams and funders working in this space. • pathogen testing in canada is centralized, automated, and closely regulated by the federal government. ms. nancy angus, director of testing at cbs, provided an overview of the current state of testing at this organization, with an emphasis on donor testing. focusing on tests in use in canada, ms. angus described laboratory tests for the detection of blood-borne pathogens and summarized the differences in sensitivity among the tests currently in use. cbs testing sites include donor testing, diagnostic services, the national testing laboratory, national reference laboratories, quality control product laboratories, and the human leukocyte antigen laboratory. donor testing, which includes transmissible disease testing, is performed at two sites: calgary, alberta, where all blood collected west of ontario is tested; and toronto, where all blood collected in ontario and east of ontario is tested. each site is a mirror-image of the other in terms of equipment, and can act as a back-up for the other site for business continuity reasons should the need arise. all blood collected by cbs is tested for a number of blood-borne pathogens: syphilis; hiv-1 and − 2; hbv; hcv; and htlv i and ii (table 1 ). since implementation in 2003, wnv testing has been performed on all collected blood; however, as of 2015, that testing is now performed seasonally. in addition to mandatory testing performed on all collected blood, there are tests that are performed based on risk. approximately 40% of collected blood is tested for cytomegalovirus (cmv) in order to supply hospital demand and based on an algorithm identifying which donors are most likely to be cmvfree. testing for chagas is performed selectively, based on risk; donations from donors indicating on their questionnaires that they or their mothers or maternal grandmothers originate in a chagasendemic country or that they have had extended stays in endemic areas for greater than six months. chemiluminescent assays are performed using the abbott prism platform to detect the surface antigen of the hepatitis b virus (hbsag), total antibody to hepatitis b core antigen, antibodies to hepatitis c virus, antibodies to hiv-1 groups m and o and/or antibodies to hiv-2, antibodies to human t-lymphotropic virus type i and/or human tlymphotropic virus type ii, and antibodies to t cruzi. agglutination assays are performed on the beckman coulter pk7300 platform: syphilis infection is identified using a micro-hemagglutination assay to detect treponema pallidum antibodies, and cmv infection is detected using a passive particle agglutination assay to detect total cytomegalovirus antibodies. nat is performed using the roche cobas 201 platform to detect hiv-1 rna (groups m and o), hiv-2 rna, hcv rna, hbv dna, and wnv rna. samples are screened in pools of 6, and single unit testing is performed on selected donations from the same geographic region when a positive donation for wnv is identified. in canada, all testing platforms require approval by health canada. other platforms currently available include the immucor neo platform, which is a solid phase system that allows for the serological detection of syphilis and cmv, and the grifols tigris platform, in use at hema-quebec, which performs nat to detect hiv rna, hcv rna, hbv dna, and wnv rna. if a blood donation is found to be serologically reactive, confirmatory testing is performed to determine if the donor is a "true" positive. confirmatory testing is either performed at cbs or at an external agency. within cbs, confirmatory tests include hbsag neutralization assay, immunoblots for hiv-1, htlv i, htlv ii, and hcv, and enzyme immunoassay to detect hiv-2. syphilis confirmatory testing is performed at either the alberta public health laboratory or the ontario public health laboratory and chagas confirmatory testing is performed at the national reference centre for parasitology in montreal. the medical services and innovation division at cbs performs surveillance to identify blood-borne pathogens that may pose a threat to the blood supply. if it is decided that a new test needs to be implemented to identify a new pathogen or new equipment is required to replace equipment at the end of its life, health canada licensure is required, unless there is an emerging threat. currently on the horizon, cbs is considering the possibility of introducing hepatitis e virus nat, and testing for babesia next generation sequencing: the future of pathogen testing? • next generation sequencing (ngs) is an advanced technology approach that involves sequencing all dna found in a clinical sample. • innovative bioinformatic approaches are required to minimize the computational time required to find pathogen dna in the sample and to maximize accuracy. • ngs is being increasingly used for otherwise indeterminate clinical diagnoses, for tracking of infectious disease outbreaks, and to detect novel pathogens. • ngs is unlikely to play a role in screening the blood supply for infectious disease markers in the near to medium term. dr samia naccache, associate specialist, department of laboratory medicine, university of california san francisco (ucsf) school of medicine, introduced attendees to the use of next generation sequencing, metagenomics, and bioinformatics for the detection and identification of infectious agents. dr naccache first pointed out that she was a member of the laboratory of dr charles chiu, which is home to the ucsf/abbott viral diagnostics and discovery center (ucsf/avddc), and that the center works closely with the ucsf clinical microbiology laboratory to provide advanced technology assistance with the most challenging problems in infectious disease diagnosis and tracking. dr naccache commenced her presentation by contrasting "classical" dna sequencing methods with next generation sequencing (ngs). anti-hiv 1 and 2, antibodies to hiv-1 groups m and o and antibodies to hiv-2; nat, nucleic acid testing; anti-hbc, total antibodies to hepatitis b core antigen; anti-hcv, antibodies to hepatitis c virus; anti-htlv i/ii, antibodies to human t-lymphotropic virus i and human t-lymphotropic virus type ii. ⁎ only selected units are tested. § single unit limit of detection; theoretical sensitivity is calculated by multiplying single unit limit of detection by 6. frederick sanger and colleagues invented a method of dna sequence determination in the 1970s [79] that was intensively used by scientists for the next 25 years [80] . sanger sequencing was based on the selective and partial incorporation of chain-terminating dideoxyribonucleotides into copies of the dna strand being sequenced, and their separation by denaturing electrophoresis into a readable sequence "ladder". dr naccache stressed that sanger sequencing, even in later, high throughput versions, was employed on one limited piece of dna (typically a small dna sector amplified using pcr) at a time to yield a single output sequence. although this method was sufficiently advanced to be used to sequence first the human mitochondrial genome [81] and then the entire human genome [82, 83] , its robustness pales in comparison to ngs approaches. ngs yields huge amounts of dna sequence in parallel; in other words, many sequences in a sample can be analyzed simultaneously [84] . for this reason, ngs is also called deep or massively parallel or high throughput sequencing. its information output is such that thousands or millions of sequences can be provided concurrently, in a matter of hours. ngs has made possible metagenomic approaches, in which all dna sequences from all genomes present in a clinical sample can be identified. this approach can now be used to determine if a blood sample contains only human dna, or human dna plus the dna of an infectious pathogen, as well as to identify that pathogen if its sequence is known. a brief technical overview of ngs methodology was provided by dr naccache. all dna/rna present in a sample is first rapidly extracted (or copied using pcr) and fragmented into pieces of uniform length, providing a library. short artificial dna sequences are then bonded onto either end of all library dna fragments. these adaptors allow hybridization of one strand of the modified dna to complementary, tethered pieces of dna in a flow cell. they are then "sequenced by synthesis" using fluorescent deoxyribonucleotide triphosphate building blocks. as each base is added to the growing chain, its position is noted via imaging and the positional information is captured in parallel. ngs can therefore provide 10 to 100 gigabytes of data-much of it "redundant" in that the same sequence has been detected and read multiple times-to ensure sufficient coverage of all dna sequence present in a sample. this huge amount of data is first processed using algorithms that detect and remove low quality reads, and then the "host" or human genome sequence information is subtracted. the remaining dna is assembled into contiguous arrays by alignment of overlapping sequences, and compared to reference genomes of known pathogens. if an identified pathogen differs slightly from reference genomes, taxonomic classification can then be done to determine how recently the variant has diverged from known sequences. dr naccache stressed that this approach will work on bacterial, viral, fungal, and parasitic pathogens, all encoded by rna-or dna-based genomes, but not on prions, which are protein-based pathogens that infect by causing host proteins to take on pathological conformations. ngs is an advanced technology approach fully dependent on the characteristics of the instrumentation employed. dr naccache surveyed the rapid development of commercial deep sequencing machines [85] . first to market in 2005 was roche, with its 454 sequencer, capable of 500 000 reads of 350 to 550 bp. illumina and ion torent produced instruments that generated millions to billions of reads of slightly shorter sizes of 50 to 300 bp, with similar overall run times, between 2009 and 2014. these products all worked on the paradigm of sequencing by synthesis. the most recent entries into this instrumentation field work on a different principle, called nanopore sequencing: pac bio's rs apparatus (2011); and oxford nanopore technology's minion (2014). both instruments are capable of a smaller number of reads than earlier machines (up to 100,000) but the reads are much longer, up to 40,000 bp. the technology works on the principle of detecting a growing dna chain electrically when the chain is extruded through an engineered protein pore of nanometer diameter. the minion instrument is amazingly small to essentially the size of a large memory stick [86] . effective exploitation of the "mountain" of dna information produced by ngs from a clinical or blood bank sample requires minimizing computation time and maximizing the accuracy of the diagnostic output. dr naccache and co-workers developed a bioinformatics platform for these tasks called surpi, sequence-based ultra-rapid pathogen identification [87] . such platforms are necessary given the size of the ngs output dna sequence, the size of pathogen reference sequence databases, and the fact that pathogen sequences typically constitute no more than 0.001% to 1% of reads in the ngs output. this presents a needle in a haystack-type problem. to achieve rapid and robust detection, surpi employs two analyzer programs that work on both dna and translated protein alignments, using bacterial and viral databases. the platform can be employed in either fast or comprehensive mode; in fast mode pathogenic sequences can be identified in a clinical sample in minutes to hours, while comprehensive mode is more appropriate for detection of a novel pathogen's entire genome or to rule out infection in a clinically complex case. dr naccache noted that 20% to 30% of clinically significant respiratory infections are currently of unknown etiology. dr naccache presented data comparing the time of completion of analysis of ngs data using either surpi mode on a variety of clinical sample types. serum took less time to analyze than biological materials open to the environment (eg, stool samples); hiv spiked into plasma could be detected in minutes in either mode, down to 100 viral copies per milliliter, with successful identification of strain specificity. surpi was extensively tested and optimized using such clinical samples prior to its employment in a prospective clinical case series carried out at the ucsf between april 2013 and december 2014. this was a single-site study with respect to analysis, but included cases referred from across the united states and also from europe. the study included acutely or chronically ill, hospitalized patients with clinical features suggestive of an infectious disease but who tested negative for all candidate agents. dr naccache highlighted two of the diagnoses achieved by ngs in the clinical series. the first involved an adolescent boy whose fever and headaches evolved over the course of four months to hydroencephalopathy that forced his physicians to induce a coma to stabilize him [88] . following over a hundred inconclusive laboratory tests, ngs revealed the presence of leptospira santarosai, a pathogenic bacterium, in cerebrospinal fluid samples. in view of the patient's poor status and the safety of the specific treatment for leptospira had the ngs-based diagnosis been incorrect, intravenous penicillin treatment was commenced before confirmation of leptospira infection was received from the center for disease control. following 2 weeks' treatment followed by rehabilitation, the patient made a full recovery. in the second case highlighted by the speaker, a 42-year-old man underwent a bone marrow transplant, with immunosuppression, for treatment of chronic lymphocytic leukemia [89] . a month later he developed tinnitus and partial deafness, which progressed rapidly and was accompanied by increasing mental deterioration. brain biopsy tissue was assessed by ngs, which detected neuroinvasive astrovirus infection, for which there is no known efficacious therapy; the patient died 4 months post-ngs diagnosis and 7.5 months after the onset of symptoms. ngs sample to answer turnaround times were reported to be 48 hours in the first case [88] and 96 hours in the second [89] . a substantial list of the different pathogens detected by the ucsf/ avddc group using ngs in different biological fluids such as csf, respiratory secretions, and blood, was next presented by the speaker. in the latter category, the agents included pathogens familiar to the transfusion medicine-oriented audience such as the viruses epstein-barr virus, cytomegalovirus, hiv-1 and -2, west nile virus, hepatitis viruses a through e, and chikv virus (chikv), the bacterium pseudomonas aeruginosa (which can be transferred from donor skin into blood products by venipuncture) and the parasite plasmodium falciparum (one of the causative agents of malaria). also included on the ngs detection list were less familiar agents, such as rna viruses enterovirus d68, hantavirus, pegivirus, and rhinovirus c, double-stranded dna viruses such as four variants of human herpesviruses, and the bk and jc viruses, and various single-stranded dna viruses of the cycloviridae and anelloviridae families, the bacterium salmonella typhi, and the parasite leishmania infantum. the sensitivity of ngs is underlined by the detection of viruses that are not usually associated with disease (anelloviridae) or are only associated with disease in immunosuppressed individuals (the bk and jc viruses) that may be part of the "background flora and fauna" in humans that must be discounted in arriving at a bona fide ngs diagnosis. dr naccache continued with a consideration of recent results from the ucsf/avddc group employing nanopore sequencing. the ucsf/ avddc participated in a research program sponsored by oxford nanopore technologies designed to probe and optimize the capabilities of the minion instrument. dr naccache reported that, using minion and a surpi-like bioinformatics platform called metapore, plasma samples from individuals separately infected with chikv, ebola virus, and hepatitis c were rapidly identified in real time [90] . for chikv and ebola virus, samples contained 10 7 to 10 8 copies/ml and were detected within 4 to 10 minutes of data acquisition; lower-titer hepatitis c virus (10 5 copies/ml) was detected within 40 minutes. the analyzer algorithms successfully identified these viruses despite the relatively error-prone nature of the sequence data. the total sample to answer time was less than 6 hours, a feat apparently unprecedented in the ngs area. dr naccache then provided a cautionary tale illustrating the extreme sensitivity of ngs which involved parvovirus-like hybrid virus, a previously undescribed novel virus related to both circoviridae and parvoviridae families, initially detected in chinese patients with chronic seronegative hepatitis of unknown etiology [91] . the ucsf/avddc group also found parvovirus-like hybrid virus by ngs in their hepatitis cohorts, but eventually realized that the virus was present in commercial silica-type spin columns used for dna purification/concentration, but not in any original patient sample [92] . the silicates are typically sourced from cell walls of diatoms and a 100% concordant pvh sequence was found in environmental metagenomics databases of samples taken from north american coastal waters. this instance of laboratory contamination illustrates the extreme sensitivity of ngs and the methodological stringency that must be brought to its application. in wrapping up her presentation, dr naccache discussed the likelihood that ngs would contribute directly to blood donation screening. she assessed this outcome as somewhat improbable over the near term, in part because blood donation screening is currently highly effective. ngs is currently quite expensive in its most accurate form, leading dr naccache to estimate the cost of a well-covered ngs study of around 10 samples using a total of 300 million sequences on the hiseq platform at us$3000 , and a more shallow study, of the kind described above for rapid detection of ebola and chikv, of around 6 samples using a total of 30 million sequences on the miseq platform, at us$1500. however, these estimates do not truly take into account the substantial infrastructure in place at ucsf/avvdc, which comprises specialized equipment for sample extraction, library generation, and validation, ngs sequencing, and extensive computational analysis, not to mention the skills of medical technologists, researchers, and bioinformatics specialists. the relevance of ngs with respect to keeping the blood system safe from emerging pathogens will more likely lie in determining patterns of disease transmission and providing the confidence necessary to re-qualify a previously deferred blood donor. suspected transmission by transfusion can be identified rapidly with great sensitivity and specificity using ngs of all implicated donors and recipients. it can also be used in a general, epidemiological sense, to trace patterns of infection and adaptation and speciation of infectious agents. while the contributions of ngs to clinical diagnosis of intractable cases and to public health, with respect to tracking and understanding disease outbreaks are already substantial, and will likely continue to accumulate exponentially, the prospects of using ngs for routine blood donation screening remain remote due to cost and throughput considerations. the biological impact of pathogen inactivation on blood product quality • pathogen inactivation (pi) of blood products may be advantageous as they overcome some of the limitations of current strategies (eg, assay sensitivity and threats of emerging pathogens). • pi techniques bring two sides of a coin into blood banking: improved safety versus a negative effect on blood component quality (damage to~20%-25% of platelets). • in order to improve the quality of pathogen-inactivated blood products, molecular mechanisms triggered by these technologies need to be identified. • pi-treatment of whole blood might be the emerging method of choice in this field. dr peter schubert, research associate at the canadian blood services' centre for innovation and a clinical associate professor in the department of pathology and laboratory medicine at the university of british columbia, focused his talk on the need for pi to ensure the safety of the blood supply. he compared the currently available pi technologies and their mechanisms of action, and discussed the potential impact of this technology on product quality. dr schubert noted that blood safety has historically been achieved by mitigating known risks with interventions such as donor screening and universal donor testing for specific pathogens. however, risks remain, as all tests have a detection limit and current testing does not account for unknown or unexpected pathogens. thus, pi has the potential to improve the safety of blood products by preventing ttis, especially in platelet products, where bacterial contamination is a particular risk [93] . in the united states, the intercept blood system from cerus corporation is currently licensed for platelet products, and both intercept and the octaplas product from octapharma are licensed for plasma (table 2 , [94] ). in canada, the only currently licensed pi product is octaplas plasma. three different pi systems for platelet concentrates are currently on the market ( table 2) . these exploit the fact that pathogen proliferation occurs by replicating dna or rna, a mandatory step for all pathogens except prions. all use uv light, with or without a photosensitizer, to damage nucleic acids and subsequently prevent proliferation of pathogens. many of these systems are in routine use, mostly in europe and the middle east [94] , and in many other jurisdictions regulatory approval is initiated and under investigation. worldwide, the cerus intercept blood system is the most adopted system, and has been in routine use for over 10 years. hemovigilance data from jurisdictions that use these products are highly favorable and support their safety and efficacy [95] . several clinical trials of pi technologies have taken place or are underway, including eurosprite (looking at intercept-treated platelet concentrates [96] ), sprint (looking at intercept-treated apheresis platelets [97] ), and miracle (looking at mirasol-treated apheresis platelets [98] ). although safety and levels of adverse transfusion reactions were favorable with pi-treated platelets, one observation was that approximately 20% to 25% of the platelets appear to be damaged by the pi treatment. the sprint and miracle trials demonstrated a lower mean 24-hour post-transfusion count increment, increased number of platelet transfusions, and lower 1-hour corrected count increment, respectively, for patients treated with pi platelets [98, 99] . the prepares (pathogen reduction evaluation and predictive analytical rating score) trial is a recently completed prospective, randomized, single-blinded, multicenter non-inferiority trial comparing mirasol-treated and standard of care pooled platelet products in hemato-oncological patients [100] . initiated in the netherlands in november 2010, prepares is sponsored by the sanquin blood supply foundation, the national blood operator in the netherlands, and financially supported by terumobct. the canadian arm of the trial involved cbs producing mirasol-treated pooled platelets at its ottawa manufacturing site and several hospitals in ontario. to account for the observed damage seen in pi techniques in other trials and to be consistent with the preparation of platelet pools in the netherlands, mirasoltreated platelets contain the donation of five donors, rather than four, which is the standard buffy coat platelet product prepared by cbs. although simply increasing the platelet dose in this setting is a straightforward solution to the issue of platelet damage seen with pi, dr schubert noted that the mechanisms of this pi-associated damage are unknown. currently in the literature, there is debate regarding the clinical efficacy of pi platelets. a meta-analysis of bleeding complications in randomized controlled trials using the intercept system suggests an increased risk of clinically significant bleeding [101] while a meta-analysis from the cochrane collaboration suggests no difference in bleeding with pi platelets, although this conclusion is limited by significant heterogeneity between studies [102] . thus, elucidating potential mechanisms is important to further finetune these systems and dr schubert provided an overview of the effect on quality parameters of platelet products by the three different pi systems. the common trend is a negative impact on routine quality measures as well as newer tools introduced to further characterize platelet quality and functionality; however, the magnitude of the effect can be different dependent on the pi technology. amongst other effects, pi treatment increases metabolism, apoptosis development, and platelet activation. these features lead to decreased platelet responsiveness and in vitro clot formation [103] . furthermore, the effect of pi treatment on novel aspects of platelet function has also been extensively investigated. these studies show pi-dependent effects on cytokine [104] , mitochondrial dna and microparticle release [105] [106] [107] , generation of reactive oxygen species and initiation of signaling cascades [108] , and expression profiles of mrna and mirna [109, 110] . these "novel" platelet features might assist with explaining pi-effects associated with increased inflammation, cell damage, and apoptosis, transfusion-related acute lung injury, and modulation of endothelial cell functions. in addition, studies of the effects of pi on the proteome of platelets could provide further insights into mechanisms, although the overall impact on the protein expression profile is relatively small [111] , suggesting pi-triggered modulations of protein activities. finally, some of dr schubert's own work on elucidating signaling cascades in mirasoltreated platelets revealed a central role of p38mapk in regulating platelet activation and apoptosis development [112, 113] . besides platelet concentrates, pi-treatment of plasma is used routinely in some jurisdictions [94] ; however, pi of rbc concentrates has been challenging due to their high optical density, requiring a large amount of uv light, accordingly leading to significant rbc damage. the cerus s-303 system for pi of rbcs demonstrated similar 24-hour recovery to standard rbcs, but these products would not meet canadian standards due to their decreased shelf life [114] . dr schubert then discussed how pi of whole blood may be a more efficient approach compared to pi of individual components. potential benefits of whole blood pi include early removal of pathogens, protection against transfusionassociated graft versus host disease due to wbc inactivation, and increased safety of all blood product components. the application of the mirasol technology on whole blood has demonstrated efficacy against hiv [115] , trypansoma cruzi [116] , and b microti [117] . most recently, a study using mirasol for malaria inactivation in whole blood has been performed in ghana [118] . however, reduction of ttis must be balanced against the effect that whole blood pi has on blood component quality. a study of in vivo viability of stored rbcs derived from mirasol-treated whole blood suggests decreased viability that correlates with quality variables such as hemolysis and atp concentration [119] . this approach was complemented by a study by schubert and colleagues further demonstrating that platelet product quality seems to be less affected when produced from whole blood illumination compared to platelet component treatment [120] . dr schubert concluded by acknowledging that there are two sides to the pi coin, and that a balance must be achieved between safety and quality. further research is needed to understand molecular mechanisms that lead to changes in quality with pi and to balance potential reductions in component effectiveness against reduction in ttis. ultimately, clinical trials are essential when it comes to making decisions regarding the implementation of pathogen inactivation into blood banking. economic and health outcome implications of introducing new pathogen testing and inactivation technologies • in order to be cost-effective, implementation of broad spectrum interventions such as pi are likely to require discontinuation of some current interventions. • the risk-based decision-making framework is a useful set of guiding principles for health economic assessments of blood safety interventions. to end the day, dr brian custer, a senior investigator with blood systems research institute in san francisco and an adjunct professor at the department of laboratory medicine at ucsf, gave an informative lecture on the economic and health outcome implications of introducing new blood safety interventions with a focus on pathogen testing and inactivation technologies. dr custer began his presentation by introducing the audience to the three main concepts that are the basis of economics in general, and of health economics in particular. the first concept, scarcity, stresses the current reality of limited resources and budgets, which may restrict the scope of services health care systems and practitioners are able to provide. the second concept, choice, has to do with choosing how to allocate the limited resources available. the third basic economic concept is opportunity cost, which relates to the next best alternative use of resources. in other words, opportunity cost is the benefit willingly forgone when we do not choose the next best alternative. these three notions summarize the challenge of the decision making process when it comes to economic information. in most jurisdictions, system-level health care decisions are made using a different decision-making framework. district health authorities resolve problems based on the determinants of health priorities such as: national and regional targets; clinical and research data; health experts' opinions and views; and the public's participation. for example, the fda annually tracks data on fatalities related to blood collection, transfusion reactions, and transmissible diseases (http://www.fda.gov). microbial fatalities from transfusion are tracked using variables that experts deem important for focusing future pathogen testing and inactivation efforts such as by the type organism and blood product. these fatality reports help determine the relative burden of fatalities from different causes. as a result, it was shown that in the united states, plateletrelated deaths are mainly caused by bacteria and red cell-related deaths are mainly attributable to babesia. data sources, such as the fda fatality data, help health authorities to understand the most important infection risks blood recipients face. with respect to health economics of the blood supply, dr custer suggested that the blood supply aims to serve the public good. achieving this aim necessitates that scarce resources be allocated in ways that maximize social welfare. the objective enumeration of costs, benefits, and consequences of alternate health programs must be weighed in comparison to society's ethical beliefs and expectations. if resource allocation does not align well with the values that society allocates to certain health outcomes, this may lead to friction between the determinants of health priorities, and ultimately to difficulties in decision making [121] . the risk-based decision-making framework developed by the alliance of blood operators is intended as a guide for the assessment of blood safety interventions. as an integral part of any blood system's risk-management program, the risk-based decision-making framework has two components as shown in figure 1 : policy foundations; and the decision making process. adherence to the framework increases consistency in methods and results, and it integrates contextual issues (social concern, legal considerations) into the decision making process. the concept of quality adjusted life years (qaly) has been developed to facilitate decision making by establishing a balance between public good (subjective qualitative outcome) and resource allocation (objective quantitative outcome). qaly is a tool that measures disease burden, including both the quality and the quantity of years lived. it is a measure of a medical intervention's "value for money". scientists may make various assumptions when measuring health resource outcomes, from choosing the wrong outcome to choosing an inappropriate test to measure it. these limitations, as well as an incomplete knowledge base, lead to uncertainty in data used for health economic analyses. when measuring health costs, one assumes that some expenditures are more important than others for the health care system; when measuring consequences from an intervention, one assumes that there is a sufficient understanding of the risks and benefits associated with that intervention. cost per qaly provides a common denominator for comparing interventions, but cannot reduce uncertainty introduced by unavoidable assumptions. established thresholds for what is considered "cost-effective" in health care interventions are in the range of $50,000 to $100,000/ qaly (all values in us dollars). blood safety interventions, however, consistently fall well above these thresholds. good examples are nat for hiv, hcv, and hbv ($1,500,000-7,500,000/qaly; broad range reflects uncertainty and testing strategy used) serology and pcr for babesia species ($251,000-6,582,000/qaly) and wnv nat testing ($520,000-897,000/qaly), and t cruzi antibody testing for chagas disease ($757,000-1,360,000/qaly). when it comes to considering pathogen testing and pi, some outcomes of interest may not be available to aid in decision making. for example, qaly data on the utility of testing for chikv, dengue viruses, and hepatitis e virus in the blood safety context are not available to inform an economic analysis. hence, understanding the budget impact of such tests becomes difficult. on the other hand, data regarding babesia testing are available. babesia screening in endemic american states was compared to universal screening using qaly and taking into account the number of deaths prevented and the testing methodology used [122] . there are also qaly data available to support the practice of bacterial testing of platelets, and pathogen inactivation of plasma [123] . the data estimate that platelet bacterial testing is more cost-effective ($91,000/qaly) than pathogen inactivation ($497,000/qaly) [123] . when implementation of a cost-effective blood donor test is needed, qaly has been applied to several patient populations and several interventions. bell et al compared platelet pi using intercept in leukemia, lymphoma, orthopedic, and cardiac patients [124] . the authors found that the new method's cost effectiveness ($/qaly) is similar to other accepted blood safety interventions. this means the method can be considered cost-effective in the blood safety context while preserving patient quality of life. dr custer and colleagues have modeled the costeffectiveness of pi as an addition to current pathogen testing based on canadian data. comparing mirasol treatment of whole blood, and of platelet and plasma, costs per qaly of $1,276,000 and $1,423,000, respectively (cdn, in this instance), were shown [125] . a more recent cost-utility analysis of implementing pi in poland suggest high costs, but better cost-effectiveness than found in previous analyses of pi and nucleic acid testing in north america [126] . pi may replace other interventions that currently incur large costs to the health care system such as blood irradiation, bacterial culture, and maintenance of cmv negative inventory [127] . if pi is implemented, removing costs associated with bacterial culture and irradiation could result in a reduction in the cost/qaly of pi by about 14 % [128, 129] . other operational gains may further offset investment costs, including reduction in product wastage [130] . these analyses are based on assumptions, and while there is a high degree of uncertainty in the results, their potential usefulness, as blood operators move into the era of pi, is substantial. health economic analysis allows quantification of alternatives and comparison of interventions in different areas of medicine. broad implementation of pi for blood safety may thus prove unpalatable to funders without compensatory cost reductions in other aspect of component production. either a more focused application of pi or lack of implementation until lowercost alternatives are developed may ensue. decision making based on health economics and on risk assessments is likely to become the new norm in blood transfusion. this would comprise a strikingly different approach to blood safety implementation and decision making than in the past, when novel interventions were added to older ones, sequentially increasing costs and overall blood safety budgets. this shift represents a move towards effectively and transparently managing process change, rather than the traditional approach of reactive change in response to an emerging situation or disaster. defending the blood supply from the threat of transfusiontransmissible infections remains a high priority for transfusion services in canada and around the world. the residual risk from established pathogens is exceptionally small. the emerging agents of greatest concern in the north american setting are arboviruses and babesia. the 2014 west african ebola virus epidemic reinforced concerns about environmental change fueling the rapid emergence of pathogens not previously thought to have potential global significance. pathogen inactivation of blood products may further reduce residual risk from established agents and provide protection from new agents in the medium term; however, the most likely driver for a paradigm shift to the use of pathogen inactivation is to reduce the ever-present risk of bacterial contamination of platelet products. longer term, the possibility of donor-free cellular products and rapid next generation sequencing could drive down transfusion-transmission rates even further than the impressive safety margins now in place in canada and other developed countries. health economic analysis and risk-based decision making will be required to determine if anticipated benefits outweigh the considerable costs of these potential steps. the 13th annual international symposium received funding from the canadian blood services centre for innovation. among the authors of this report, dr geraldine walsh, dr mia golder, dr margaret fearon (author and speaker), and dr william sheffield have no actual or potential conflict of interest in the context of the subject of this program over the past five years. dr peter schubert, author and speaker, discloses grant/research support from terumobct and macopharma. among the speakers, dr allison mcgeer, nancy angus, and dr samia naccache have no actual or potential conflict of interest in the context of the subject of this program over the past five years. the following relationships that could be perceived as a related or apparent conflict of interest in the context of the subject of this program over the past five years are disclosed by dr roger dodd: grant/research support 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decision-making i. health economics and outcomes methods in risk-based decision-making for blood safety costs, consequences, and costeffectiveness of strategies for babesia microti donor screening of the us blood supply costs and benefits of bacterial culturing and pathogen reduction in the netherlands costeffectiveness of transfusion of platelet components prepared with pathogen inactivation treatment in the united states the cost-effectiveness of pathogen reduction technology as assessed using a multiple risk reduction model introducing pathogen reduction technology in poland: a cost-utility analysis pathogen reduction: state of reflection in ireland model calculations to quantify clinical and economic effects of pathogen inactivation in platelet concentrates reducing the financial impact of pathogen inactivation technology for platelet components: our experience variation of platelet production and discard rates in 17 blood centers representing 10 european countries from cerus corporation. the intercept blood system: one system -two components, cerus corporation mirasol pathogen reduction technology system. terumobct main properties of the theraflex mb-plasma system for pathogen reduction macopharma. the theraflex mb-plasma system is able to efficiently remove bacteria and bacterial spores from plasma canadian agency for drugs and technologies in health. cadth optimal use reports. use of solvent/detergent-treated human plasma (octaplas): pilot project. ottawa (on): canadian agency for drugs and technologies in health characteristics of the theraflex uv-platelets pathogen inactivation system -an update we would like to acknowledge the 2015 key: cord-317250-a5ni1s9e authors: jackson, ronald s. title: wine, food, and health date: 2020-04-10 journal: wine science doi: 10.1016/b978-0-12-816118-0.00012-x sha: doc_id: 317250 cord_uid: a5ni1s9e wine has historically been associated with religious rights, used as a salubrious beverage, employed as a medication as well as a medicinal solvent, and consumed as a food accompaniment. it is the last use that is most intimately associated in the minds of most modern consumers. despite this, there is little flavor commonality on which pairing could be based. the first section of the chapter examines this feature and wine's primary role as a palate cleanser and food condiment. the synergistic role of food and wine in suppressing each other's least pleasant attributes is also explained. the final section deals with the latest evidence relating to the many beneficial health effects of moderate wine consumption, shortfalls in the data, headache induction, dental erosion, and conditions under which wine intake is contraindicated. wine and its association with food have generated an incredible cornucopia of literature. amazingly, few of the views and assumptions have been subjected to scientific scrutiny. only a few research papers deal with the topic. this may relate to their subject disciplines (enology and food science) being in separate departments. also, the funding is different. that for enology and viticulture comes largely from government or industry grants, often based on levies on vineyards and wineries. funding in the food sciences comes largely from commercial firms, much of it done internally by food companies, and such research remains proprietary. funding from disparate industries provides little incentive for collaboration on interdisciplinary projects, such as food and wine combination. much of what is written is by those with an arts rather than a science background. sommeliers, for whom the combination of wine and food is their field, are more practitioners than researcher/academicians. thus, the topic has experienced languor, devoid of scientific rigor. only recently have scientists ventured into a subject normally the prerogative of the epicurean. scientists, being inherently doubters, demand experimental verification. the latter is difficult as designing adequate controls is next to impossible. in addition, the laboratory conditions, required for experimentation, make the relevance of finding to "real life" situations dubious. with food and wine combinations, the social context is often more important to perception than actual sensory clues. finally, social pressures often lead to acceptance of supposed norms. what follows is a brief discussion of the issue of food and wine combination and what can be said with a degree of confidence. in addition, it is hoped that it will provide "food for thought" to those seriously interested in wine. the traditional view is that wine is in some fundamental way designed to be consumed with food. however, when one searches for evidence, it seems to be a mythda view so oft repeated as to be taken as gospeld an example of social contagion of collective memory. wine appears to have been the standard food beverage in the greek or roman worlds. in the near east and egypt, wine was almost the exclusive preserve of the secular and religious oligarchy. in contrast, beer was the beverage of the laboring classes. wine is mentioned in the bible but recommended primarily in relation to religious ceremonies and weddings. it is only in regions where wild grapes grew indigenously that vines came to provide the drink for the masses. its acidity, alcohol, and phenolic contents enhanced its antimicrobial property, making it considerably safer to drink than water. wine's mildly antiseptic properties became increasingly important as population numbers grew, hygienic conditions deteriorated, and water supplies became more polluted. although production was seasonal (unlike beer based on dry grains), wine's comparative resistance to spoilage (isolated from air) permitted wine to become the preferred beverage. in addition, wine's higher alcohol content could temporarily numb the afflictions of a peasant life. although safe and nutritious (calorie rich), there is nothing in its attributes that inherently makes wine ideal as a food beverage. it is actually better suited for one of its ancient secondary roles, a solvent in preparing extracts from medical herbs. the sharp acidity of many ancient wines, often extended with aged seawater and vinegar (especially for slaves), can hardly be considered an ideal food accompaniment, at least from a modern perspective. the resinous flavor donated by storage in standard amphoras (made watertight with an inner coating of pitch) would not have facilitated detecting any subtle fragrances they might have had. the quality of the ordinary wine available to the average roman is probably best left to the imagination. finer wines were available for the patrician classes. these were probably stored in amphoras with a vitreous inner surface, without the need for a pitch lining. the most famous seem to have been or became highly concentrated with age, being almost syrup-like, usually diluted before being consumed. several roman poets eulogized the wonders of particular vintages. even modern wines, with their predominant acidic, bitterish, and astringent character have little to suggest food compatibility. these characteristics are simply the natural consequence of grape chemistry, not conscious intent. admittedly, consuming wine with food does mollify its less pleasant aspects, unless one develops an appreciation for sour beverages with a bitter/astringent aspect. some humans seem adept at coming to appreciate, even crave, perceptions initially repulsive to painful. black coffee, capsicum peppers, durian, and limburger cheese are classic examples. in some instances, the iron content of the wine can induce a metallic sensation, by catalyzing lipid oxidation. this may be masked in combination with food. however, this appears to be unrelated to the fishy aftertaste associated with some white wines taken with seafood (tamura et al., 2009) . in most "compatible" combinations, both the cheese and wine appear betterdnot by directly enhancing their respective qualities but by mutually suppressing their less pleasing attributes (nygren et al., 2002 (nygren et al., , 2003a (nygren et al., , 2003b . other studies have extended these findings, lending support to the view that red wines pair better with cheeses, due to wine tannins appearing more silky (bastian et al., 2010) . the fatty acid content of the cheese may also contribute to reduced bitterness (homma et al., 2012) , possibly by coating taste receptors. lipoproteins, typically found in foods, can react with taste receptors, suppressing the perception of bitterness (katsuragi et al., 1995) . however, the bitter-suppressing aspect of cheeses may also relate to its salt content (frijters and schifferstein, 1994; breslin and beauchamp, 1997; keast et al., 2001) or the presence of glutamate and adenosine monophosphate (keast and breslin, 2002) . the fruity aspect of wine may also be enhanced when sampled with cheese (galmarini et al., 2016) . although most cheese/wine associations seem mutually beneficial, some are not, for example, sweet wines and cheese (bastian et al., 2009) . that compatibility originates from the negation of unpleasant sensations may not be what wine pundits and sommeliers profess, but it seems closer to the truth. this phenomenon may also apply to many supposedly food and wine "marriages." salt's suppression of bitterness (nakamura et al., 2002) might explain the seemingly ancient habit of adding seawater to wine, e.g., oinos thalassikos (younger, 1966, p. 130) . pliny (historia naturalis, 14.120) also records that salt enhanced the perceived smoothness of wines. columella (de res rustica 12.41) recommends the addition of salt, apparently to avoid moldy tastes. he also notes salt addition in a recipe for preparing "greek" wine. salt water was even used until recently in preparing new barrels to receive wine (nègre and françot, 1955) . salt is well known as a flavor enhancer. this may involve disrupting weak, nonvolatile complexes between matrix and aromatic compounds, promoting their liberation and retronasal detection (linscott and lim, 2016) . in addition, sodium ion hydration may decrease "free water," changing solution polarity. although salt increases aromatic volatility, saltiness is by itself appreciated (bolhuis et al., 2016) . when one searches for affinities among the attributes of food and wine, one comes up empty-handed. in contrast, there is extensive incongruity. table wines possess gustatory attributes predominantly characterized by sourness, bitterness, astringency, and burning sensations. although pronounced sour tastes are inherently unpleasant, wine's acidity is of value when used as a marinadedpromoting acid-induced hydrolysis of food proteins. wine phenolics can also act as antioxidants, reducing the toxicity of heterocyclic amines (viegas et al., 2012) and acrylamide (qi et al., 2018) generated during frying (viegas et al., 2012) . phenolics are also antimicrobial (nisiotou et al., 2013) . by comparison, sourness is a rare attribute in most world cuisines (see moskowitz et al., 1975 for a marked exception). acids typically are added only as a component in some condiments or flavorants, notably vinegar, lemon juice, or tamarind. they can enhance the flavor of otherwise bland foods. the bitterness and astringency of most red wines also find no equivalent in meat and fish. the protein content of food reacts with both wine acids and phenolics, limiting their activation of taste and touch receptors. in comparison with wines, solid foods are characterized salty, savory (glutamate), sweet, and sebaceous (fatty acids) sensations. sour, bitter, astringent, and hot spicy attributes are (or have been) less common in western cooking and then usually in condiments. the inherent, aversive reactions to such sapid sensations probably arose as a protective response to avoid or limit the consumption of potentially toxic (or rotten) foods. conversely, bitter/astringent/toxic compounds were probably selected during plant evolution to discourage their consumption, with the principal exception of ripe fruit. during domestication, crop variants with reduced aversive and enhanced pleasant-tasting constituents have been propagated. thus, lettuce and other vegetables became less bitter; apples, cherries, and other fruits sweeter and less sour or astringent, citrus fruit less acidic, and legumes less flatulent. food preparation, notably cooking, further facilitated the inactivation or removal of potential food toxins and antimetabolites. examples include fungal toxins, potato alkaloids, and casava cyanogenic glycosides. cooking meat also facilitates digestion (promoting collagen and protein fiber breakdown) and enhances flavor. disappointingly, some cooking processes generate their own toxins, notably roasting and searing. examples are acrylamide (a maillard by-product) and a variety of toxic, pyrolytic, smoke by-products. fermentation is another ancient technique that helped destroy antimetabolites. an example is the action of rhizopus oligosporus degrading soybean flatulence compounds during tempeh production. lactobacillus can also destroy soy saponins. fermentation also has the potential to break down difficult-to-digest oligosaccharides as well as help preserve perishable foods. the aromatic aspects of food and wine equally show little similarity, on which supposed compatibility could be based. wine aromas are most frequently described in terms of fresh fruit, jam, or flowers. none of these is characteristic of the main components of a meal and would be considered odd if present. the hints of "apple" in chardonnay wine may be compatible with chicken, the "pepper" of a shiraz pair with pepper steak, and the "walnut" of some sherries combine with nutcontaining salads (without vinaigrette). however, does the box wood or cat urine of sauvignon blanc, the rose of riesling, and the black currant of cabernet sauvignon really match with any main course? in addition, does the vanilla/coconut of oak or the leather aspect of aged red wines have any inherent compatibility with food? the supposed spiciness of gewü rztraminer wines is given as a reason for its combination with spicy asian foods. however, the wine has more litchi aroma than spiciness, making the stated logic dubious. it is only its intense aroma that may permit its presence to remain noticeable. personally, the best aspect of wine and food association is not their complementary natures but their contrasts. each cleanses the palate between alternate samplings, allowing fresh perceptions of each. thus, wine permits swift shifts in savory sensitivity. without comparable tastes or flavors, where is the supposed inherent rapport between food and wine? is its only justification the reduction of undesirable aspects of the partnership and as a palate cleanser? certainly, wine's ability to partially rinse the palate between food samples is important. it offers both gustatory, olfactory, and trigeminal receptors time to reestablish their native receptive state, countering adaptation and loss of sensory appeal. thus, food appreciation is enhanced by being sampled afresh. wine can, unromantically, be viewed as a savory mouthwash. in addition, volatility of food and wine flavorants is influenced by the dynamically changing concentrations of ethanol, phenols, carbohydrates, etc. supplied with the wine. conversely, food dilutes, masks, and eliminates most wine flavors. the result being that the next sample can be appreciated to its full, adaptation having been avoided. the other aspect of potential compatibility arises from their dissimilar attributes. they act in concert, in a manner similar to condiments, providing flavor accents to enhance and maintain flavor interest throughout the meal. the result can be the creation of a stimulating holistic experience. the typical starchy elements in a meal (rice, potatoes, pasta, bread) supplement the synergy, helping to cleanse the palate and generate a feeling of satiation. however, statements like "wine cuts through fat" are unsupported. if this view has any validity, the partial fat solubility of ethanol may reduce the oily mouth-feel of fats as well as limit the activation of fatty acid taste receptors (running et al., 2015) , but this is no more than speculation. the acids in wine might also have a similar effect. in addition, wine tannins may denature the g protein receptors responsible for detecting fatty acids in the oral epithelium, giving the impression of less fattiness. conversely, fatty acids can reduce the perception of sourness, astringency (peyrot des gachons et al., 2012) , and bitterness (mattes, 2007) , possibly improving mouth-feel. wine phenolics can either reduce (jung et al., 2000) or enhance (mitropoulou et al., 2011; villamor et al., 2013) volatility, depending on the phenolic and aromatic involved. carbohydrates also have the ability to bind aromatics, reducing volatility and significantly modifying wine flavor and its retronasal attributes (voilley and lubbers, 1998; villamor et al., 2013) . thus, both synergies of flavor as well as suppression could be involved in "ideal" pairings. saliva-induced changes in flavor chemistry is another compounding issue little investigated. whether such potential reactions are sufficiently marked and rapid to have significance, within the time frame of food consumption, is currently terra incognita. in all the standard discussions of wine and food combinations, the obvious is rarely if ever mentioned. it limits the rate and maximum amount of alcohol reaching in the blood ( fig. 12.1) . correspondingly, it reduces breath-alcohol content (sadler and fox, 2011) and diminishes alcohol's performance impairment (millar et al., 1992) . in addition, by reducing alcohol uptake, its tendency to promote overeating is represseddby limiting activation of hypothalamic agrp neuron activity (cains et al., 2017) . many aspects of food preference appear to develop in utero, based on what the mother ate during pregnancy (mennella et al., 2004) and infancy. thus, early exposure can play a significant role in developing personal preferences. subsequently, peer pressure and cultural influences may combine to modify these early dispositions. habituation may not be imbued with the effusive and social appeal associated with the image of predestined food and wine marriages but far better fits the facts than any supposedly heaven-inspired pairings. thus, there is little wonder why there has been scant inclination to investigate lovingly held shibboleths about food and wine associations. however, a dose of reality does not have to destroy long-held views. knowing that michelangelo's sistine chapel consists of no more than brush strokes of pigment on plaster, perceived by photoreceptors in the eye, converted to synaptic impulses reconstructed piecemeal into a perception at the back of the brain, need not ruin art appreciation. ideally, it should enhance the wonder it instills. scientific understanding augments adds new layers of appreciation. if supplemental knowledge were not considered to augment appreciation, why would connoisseurs be so concerned with vintage dates, wine geography, cultivars, or details of vineyard sites and wine producers? with knowledge, it really is the more the merrier. admittedly, scientific realism injected at the wrong time may be a despoiler. for special events, psychological appeal can be more significant than sensory reality. as in other aspects of life, science can occasionally be set aside to permit spontaneity and anticipated pleasure presides. the intrigue of magic is being fooled so effectively. under most circumstances, though, some basic rules of pairing should be kept in mind, to avoid glaring mistakes. in most fine cuisine, flavor balance, combined with suitable complexity, is central. nonetheless, the vagueness of this concept is evidenced by the almost infinite variety of combinations seen in cookbooks, food magazines, and culinary shows. accepted norms also vary markedly among cultures (rozin, 1977 (rozin, , 1982 . thus, it should not be surprising that almost any wine can pair with almost any meal. there are limits, however, for example a dry gewü rztraminer with dessert or a riesling auslese with bouillabaisse. to almost everyone, these would not be considered "marriages made in heaven." cabernet sauvignon and dark chocolate is another clash but seeming appreciated by some connoisseurs. except where there are clear flavor or intensity disparities, notably sweet/ acid or sweet/bitter-astringent, almost any combination will be found pleasing to some and acceptable to most. of the generalities oft quoted, the "white with white, red with red" dictum bears logic, within the context of balanced flavor intensity. nonetheless, it is often the food preparation mode (e.g., poached, fried, baked, broiled, barbequed) or the condiments added (e.g., chilies, curry, relish, olive oil, tomato sauce, garlic, herbs) that often have the greatest influence on flavor intensity, the basic character of the meal, and correspondingly a compatible wine. in most instances, premium-quality table wines are best sampled prior to the meal and premium-quality dessert wines after the meal. because of their aromatic complexity, detection of these attributes is compromised by combination with food or dessert. alternatively, the food or dessert should be designed to be a foil for the wine and be mild in character. in contrast, the more markedly acidic or bitter/astringent the wine, the more effectively these features will be mollified by association with food. might not this be the most justifiable reason for the pairing most wines with food (other than reduced alcohol uptake)? in addition, a lack in the wine's aromatic interest can be camouflaged by flavors from the food. where little attention is likely to be paid to the wine, inexpensive, nondescript red or white wines are both financial and logical choices. if some food and wine pairings are ill-conceived, are there seraphic duets? clearly some do pair better than others, but transcendental experiences? published accounts of such paradisaical experiences may be no more than figments of a fertile imagination, created to sell wine, newspapers, or magazines. admittedly there is an incredible range in human sensory sensitivityd those having higher than average acuity tending to prefer milder flavored foods, and those with below average acuity tending to prefer more intensely flavored foods. but these are no more than tendencies, with experience and social pressures capable of inducing significant shifts in preference, if not perception. psychological influences and a desire to be influenced can distort perception, creating impressions that are "real," only because of the conditions under which the experience occurred. the more unexpected and astounding the sensation, the stronger the memory trace created. the more the mind is studied, the more we come to realize how the brain can distort perception, based on past experiences. they generate mental models of reality, against which sensations are judged, interpreted, and potentially modified. thus, it is wise to doubt perceptions and attempt to separate experience-based memory patterns from actuality, unless it is a selective choice to allow the mind to potentially deceive us. in addition to the sensory pleasure wine can supply when taken with a meal, it also has health benefits. among other direct benefits noted below, dining with wine can reduce the absorption of oxidized lipids and thereby limit their cardiovascular damage (natella et al., 2011) . the contrasting social and antisocial effects of alcohol consumption must have become evident shortly after the discovery of winemakingdthe negatives being noted early in the old testament (genesis 9.21) and vividly described by pliny (historia naturalis, 14.28). time has only expanded our understanding of this dr. jekyllemr. hyde relationship. it is clear that excessive alcohol consumption, both acute and chronic, can have devastating effects on physical and mental well-being. abusive ethanol consumption can cause cirrhosis of the liver, increase the likelihood of hypertension and stroke, favor the development of breast and digestive tract cancers, induce fetal alcohol syndrome, among others. many of these effects seem to arise from excessive alcohol intake activating of free-radical release and associated immune perturbations (meagher et al., 1999) . other, more severe, negative consequences may arise indirectly, via the accumulation of acetaldehyde, a major breakdown product of ethanol metabolism (lachenmeier et al., 2009) . because the problems associated with alcoholism (abrams et al., 1987; schmitz and gray, 1998) and its eventual, irreversible, chemical modifications in the brain (nestler and malenka, 2004; heinz, 2006) have been extensively reported, they need not be elaborated here. on the other hand, it is becoming equally clear that moderate wine consumption (approximately 250 ml/day) can potentially have health benefits. this is considerably less than the two bottles per day that brillat-savarin (1848) considered a healthy man could consume and live long; or the amounts considered appropriate in times past (younger, 1966, p. 367) . mark twain, in characteristic style, crystalized moderation in his view of the temperance movement: "temperate temperance is best" (mark twain's notebook, 1896) multiple epidemiological studies suggest that daily, moderate, alcohol consumption (thun et al., 1997; doll et al., 2005) , and notably wine (grønbaek et al., 2000; renaud et al., 2004) , is associated with a reduction in allcause mortality. this is expressed in the now famous jshaped (hormesis) curve ( fig. 12 .2), with earlier mortality being associated with both excess alcohol intake and abstinence. this is particularly evident in the reduced incidence of cardiovascular disease in moderate alcohol consumers. in addition, it reduces the likelihood of type 2 diabetes, combats hypertension, and is correlated with reduced frequency of certain cancers (boffetta and garfinkel, 1990) . although encouraging to those who enjoy wine, the sharp rise in death risk at much above moderate consumption is of concern. presumably, this relationship would apply equally to morbidity figures, were such data available. however, morbidity unlike mortality is qualitative rather than quantitative and thus its measurement fraught with difficulty. additional dangers associated with anything more than moderate consumption, especially alone, comes from alcohol's progressive enhancing of the memory circuitry involving addictive cravings. these epidemiological correlations are supported by in vivo studies that provide potential molecular explanations for these associations. the principal elements missing, in confirming a causal relationship, involves detailed information on the dynamics of absorption, metabolism, and elimination of the proposed active ingredients. faced with a chemical and beverage that can be not only salubrious but also addictive, the fluctuations in society's attitude toward alcohol are not surprising (musto, 1996; pittman, 1996; vallee, 1998) . thankfully for those in the wine industry, wine drinkers appear less likely to become heavy drinkers (jensen et al., 2002) or to illustrate those alcohol-related problems that have given alcohol a bad reputation (smart and walsh, 1999) . in addition, wine has a more positive social image than other alcohol-containing beverages (klein and pittman, 1990; unwin, 1992) . the major caveat is the derogatory epithet, wino, ascribed to some unfortunate members of society. the use of wine as a medicine or carrier for herbal extracts has an extensive history. it goes back at least to pharaonic egypt (lucia, 1963; soleas et al., 1997) . ancient greek and roman society used wine extensively as a solvent for medicinal infusions. this practice continued largely unabated until the beginning of the twentieth century. the excessive abuse of distilled alcoholic beverages, combined with religious and political conservatism, created a backlash against all beverages containing alcohol, notably in north america. alcohol was viewed as an agent of corruption to be annihilated. following the failure of prohibition, humans themselves, not alcohol, came to be viewed as the source of iniquity. alcoholism is now viewed as a developmental, multistage, chronic dependence, possessing a complex etiology (nurnberger and bierut, 2007) , with both genetic and environmental aspects. in this regard, it is similar to other addictions (ersche et al., 2012) . thus, the social climate is changing and the relationship between wine (as opposed to alcohol) and health is again being reassessed and investigated seriously. it is unlikely that doctors will soon be prescribing wine for its health benefits. too often, people have difficulty recognizing the limits of rational use and differ markedly in their metabolism (gross et al., 2010) . in addition, detrimental influences rapidly counter any benefits at more than light to moderate consumption (often viewed as<30 mg ethanol/day) (rehm et al., 2010) . erring on the side of restraint seems judicious, without excessively assuaging pleasure, especially if combined with food. even dietary flavonoid supplements (one of the benefits of wine consumption) can be detrimental, if taken in excess (skibola and smith, 2003) . wine can be wonderful in moderation but is no panacea. alcohol is the primary by-product of fermentation in many organisms. ethanol is also an energy source for an even larger number of species. thus, it is not surprising that enzymes involved in ethanol metabolism are found in most life forms. in humans, ethanol enters the bloodstream either via the consumption of beverages containing alcohol, and/or from ethanol synthesized by members of the intestinal flora. when the concentration of alcohol is low, most of it is metabolized in the liver before it enters the systemic blood supply. most of the blood coming from the digestive tract passes through the liver before being dispersed to the rest of the body. the liver metabolizes about 95% of the blood alcohol content, at about 15 ml/h. the rest tends to be lost via the breath or secreted in the urine and other bodily fluids. the rate of alcohol loss is relatively constant over time, with w50% reduction within 5 h ( fig. 12 .3). the liver possesses two ethanol metabolizing pathways. the primary, constitutive mechanism involves the oxidation of ethanol to acetaldehyde, via cytoplasmic alcohol dehydrogenases (adhs). of the seven known adh genes (crabb et al., 2004) , three function in the liver. the others act in the gastric epithelium and other tissues. subsequent metabolism converts acetaldehyde to acetic acid. this occurs principally under the action of mitochondrial acetaldehyde dehydrogenase (aldh2). the cytoplasmic acetaldehyde dehydrogenase (aldh1) is less active. acetic acid is subsequently secreted into the blood or directly converted to acetyl coa. from this point, metabolism may flow along any standard biochemical pathway (see fig. 7 .20). alcohol metabolizing enzymes frequently occur in allelic forms (isozymes). their relative occurrence also tends to vary among ethnic groups. some isozymes possess distinct physiological attributes. for example, adh1b*1 codes for a subunit that oxidizes ethanol slowly, whereas adh1b*2 encodes a highly active subunit of the dimeric enzyme (about 30 times more efficient) (thomasson et al., 1995) . correspondingly, those individuals who are homo-or heterozygous for the adh1b*2 subunit, eliminate alcohol from the blood more rapidly. rapid alcohol oxidation may donate a degree of protection against alcoholism, by quickly converting ethanol to acetaldehyde. however, if combined with slow-acting alleles for acetaldehyde dehydrogenase (aldh2) (crabb et al., 2004) , the accumulation of toxic acetaldehyde is enhanced, predisposing the bearer to cancers of the oropharynx and esophagus. those also possessing malfunctional glyoxalase and methylglyoxalase repair enzymes are those most susceptible to such damage (see dingler and patel, 2017) . a second, ethanol-degradation hepatic pathway becomes activated when blood-alcohol levels become elevated. it involves a microsomal cytochrome, p4502e1 (cyp2e1). it oxidizes ethanol to acetaldehyde, using molecular oxygen rather than nad þ . regrettably, the microsomal pathway generates free oxygen radicals (meagher et al., 1999) dmolecules (or ions) with one or more unpaired electrons. these highly reactive oxidants, or reactive oxygen species (ros), can be generated long after alcohol intake ceases. another variant of cyp2e1 occurs in the gastric mucosa, seemingly being more active in men. although most ros are inactivated by glutathione, superoxide dismutase, and catalase, long-term exposure to their presence may induce the slow, progressive accumulation of irreparable cellular damage. metabolizing ethanol to acetate (acetic acid) has the advantage that tissue cells can regulate its transport. this is not the situation with ethanol, which can diffuse freely across cell membranes. transport control is a central tenet in proper cellular function. the ability of ethanol to displace water and its unregulated passage across cell membranes explains much of alcohol's toxicity. in addition, its oxidation to acetaldehyde tends to be more rapid than acetaldehyde's oxidation to acetate. thus, acetaldehyde may accumulate in the blood and other bodily fluids. this is often viewed as an important contributor to the toxicity associated with excessive alcohol consumption (lachenmeier et al., 2009) . differentiating between these direct and indirect toxic effects of excessive ethanol intake has proven difficult. one of the first physiologic effects of alcohol consumption is a suppression of cognitive brain function. this is most noticeable in enhanced sociabilitydby blocking social inhibitions regulated by higher brain functions. for others, it quickly induces drowsiness (stone, 1980) . this probably explains why taking a small amount (90e180 ml) of wine before sleeping often helps those suffering from insomnia (kastenbaum, 1982) . this amount often provides the benefits of sleep induction, without causing subsequent agitation and sleep apnead often associated with greater alcohol consumption. the effect on sleep may arise from alcohol's modulating the action of inhibitory g-aminobutyric acid (gaba) receptors, while suppressing the action of excitatory glutamate receptors. gaba and glutamate are estimated to be involved in about 80% of the neurocircuitry of the brain. the level of melatonin in wine is well below those prescribed as an insomnia medication (rodriguez-naranjo et al., 2011) . another effect on brain function results from a reduction in the secretion of vasopressin. as a consequence, urine production increases, producing the frequently reported diuretic effect associated with alcohol consumption. less well known is how alcohol acts as a crucial regulator of the hypothalamicepituitaryeadrenal axis, modulating the release of hormones such as adrenocorticotropic hormone and corticosterone (haddad, 2004) . although alcohol has a general depressive action on brain function, the levels of some brain modulators show transitory increases. examples are serotonin and histamine. the latter may activate a cascade of reactions leading to headache production. another of the multiple influences of alcohol is the conversion of hepatic glycogen to sugar. this results in a short-lived increase in plasma glucose content. this, in turn, can cause glucose loss in the urine as well as an increase in insulin release by the pancreas. both result in a drop in blood sugar content. if sufficiently marked, hypoglycemia results. this apparently causes the temporary weakness occasionally associated with alcohol consumption, especially excess intake. in addition to direct effects, the accumulation of acetaldehyde, as a by-product of ethanol metabolism, may have several undesirable consequences. at low rates of alcohol intake, acetaldehyde metabolism is sufficiently rapid to limit its accumulation and liberation from the liver. at higher concentrations, acetaldehyde production rapidly consumes the liver's glutathione reservesda central cellular antioxidant. this coincides with activation of the microsomal ethanol oxidation pathway that generates toxic free-oxygen radicals. in the absence of sufficient glutathione, free-oxygen radicals can accumulate, disrupting mitochondrial function. elsewhere in the body, acetaldehyde can bind with proteins and cellular constituents, forming stable complexes (niemela and parkkila, 2004) . these can lead to the production of immunogenic determinants, which can stimulate antibody production against acetaldehyde adducts (romanazzi et al., 2013) . this may induce some of the chronic tissue damage associated with alcohol abuse (niemela and israel, 1992) . the binding of acetaldehyde to the plasma membrane of red blood cells is known to increase rigidity. by limiting their ability to pass through the narrowest capillaries, oxygen supply to tissue cells may be restricted. this could participate in suppressed brain function. it is estimated that the brain consumes up to 20% of the blood's oxygen supply but constitutes only about 2.5% of body mass. in addition, acetaldehyde can disrupt dna repair mechanisms. fortified wines (notably sherries) can be a significant source of acetaldehyde (lachenmeier and sohnius, 2008) . although ethanol and acetaldehyde can produce severe, progressive, and long-term damage to various organs, and incite alcohol dependence, these consequences are minimal to undetectable when alcohol consumption is moderate and taken with meals (serianni et al., 1953) . as the sections below demonstrate, moderate, daily, wine consumption can have health benefits for the majority of people. wine's major nutritional value comes from its rapidly metabolized, ethanolic, caloric content. alcohol does not need to be digested, prior to being absorbed through the intestinal wall. in rural viticultural areas, wine historically provided a significant source of metabolic energy for the adult population. the caloric value of ethanol (7.1 kcal/g) is nearly twice that of carbohydrates (4.1 kcal/g). thus, it constituted a valuable caloric source. it is estimated that alcohol may supply about 6% of the energy in the average american diet (halsted, 2003) .wine was also a potable beverage and helped disinfect water to which it was addeddsome bacterial inactivation occurs within seconds (vaz et al., 2012) . wine contains small quantities of several vitamins, notably several b vitamins, such as b 1 (thiamine), b 2 (riboflavin), and b 12 (cobalamin). however, wine is virtually devoid of vitamins a, c, d, and k. in excess, ethanol can impair vitamin uptake. wine contains various minerals in readily available forms, especially potassium and iron (in the ferrous state). nevertheless, excessive alcohol consumption can disturb the uptake of calcium, magnesium, selenium, and zinc and increase the excretion of zinc via the kidneys. the low sodium/high potassium content of wine makes it one of the more effective sources of potassium for individuals on diuretics. although wine contains soluble dietary fiber, especially red wines (díaz-rubio and saura-calixto, 2006) , it is insufficient to contribute significantly to the daily recommended fiber content in the human diet. wine has several direct and indirect effects on food digestion. its phenolic (hyde and pangborn, 1978) and alcohol (martin and pangborn, 1971 ) contents activate the release of saliva. in addition, wine promotes the release of gastrin as well as gastric juices. the principal constituent activating the release of gastric juices in red wines is apparently succinic acid, whereas in white wines it is malic acid (liszt et al., 2012) . they do not activate gastrin release, however. the substance(s) involved in stimulating gastrin secretion are unknown. wine also significantly delays gastric emptying, both on an empty stomach (franke et al., 2004) or when consumed with food (benini et al., 2003) . the latter favors digestion by extending the duration of acid hydrolysis. delayed gastric emptying may be a consequence of wine phenolics activating stanniocalcin-1 cells in the stomach. these possess the same tas2r system as bitter-sensitive receptors in the mouth (see finger and kinnamon, 2011). on stimulus, they release cholecystokinin, a peptide hormone that reduces gut mobility. in addition, wine slows plasma glucose uptake, independent of any insulin response (benini et al., 2003) . furthermore, at the levels found in most table wines, ethanol activates bile release. wine acids and aromatics also have the same effects. in contrast, the high alcohol contents (e.g., distilled spirits) can suppress digestive juice flow, the release of bile, and induce stomach spasms. wine also aids digestion indirectly by inactivating gastrointestinal pathogens. despite the general beneficial effects of moderate amounts of alcohol on digestion, the phenolic content of red wine may counter some of these influences. for example, tannins and phenolic acids can interfere with the action of certain digestive enzymes, notably a-amylase, lipase and trypsin (rohn et al., 2002; gu et al., 2011) . digestion may be further slowed by phenolics polymerizing with food proteins. these effects may be mollified by the presence of ionic carbohydrates found in food (gonçalves et al., 2011) as well as by salivary proteins. both monomers and proanthocyanidins bind with basic, proline-rich, and histatin proteins in the saliva. their bonding is reversible, depending on equilibrium conditions. they can become irreversible, though, in the presence of metal ions, upon oxidation, or with ph changes (luck et al., 1994) . normally, these insoluble saliva/tannin complexes remain stable in the stomach and upper alimentary tract (lu and bennick, 1998) . thus, the inactivation of digestive enzymes or the disruption of mineral uptake by tannins may be limited. degradation of tannin-protein polymers and their moieties subsequently occurs in the colon. in contrast, some pepsin-activated protein breakdown is activated by monomeric phenolics, notably quercetin, resveratrol, catechin, and epigallocatechin gallate (tagliazucchi et al., 2005) . clearly the action of wine phenolics is complex and much more needs to be known. not only are the effects potentially different in the stomach from that in the small and large intestines, but also the chemical composition of wine phenolics changes during passage through the digestive tract. the wine's phenolic content can also decrease iron and copper absorption in the intestinal tract (cook et al., 1995) . although nutritionally undesirable, limiting iron bioavailability may reduce the formation of toxic lipid hydroperoxides during digestion. the antioxidant effect of polyphenolics also applies to peroxide generation in the stomach kanner and lapidot (2001); fig. 12.4 . the activation of gastric juice release not only aids food digestion but also inactivates enzymes involved in ulceration. even more significant may be the antibiotic action of wine constituents against helicobacter pylori (fugelsang and muller, 1996) . h. pylori is often considered the primary causal agent of stomach ulceration. thus, moderate wine consumption may have a prophylactic effect in limiting ulcer initiation (brenner et al., 1997) . the bacterium has also been implicated in gastritis, vitamin b 12 malabsorption, and gastric adenocarcinoma. however, chronic secretion of gastric juice can produce irritation and may provoke ulceration, heartburn, and favor the development of adenocarcinomas in the lower esophagus. wine may further aid human sustenance by increasing nutrient uptake. congeners in wine combine with metallic ions, vitamins, and fatty acids, facilitating their transport across the intestinal wall. consuming wine with food slows the rate of alcohol uptake in the blood ( fig. 12.1) . in the absence of food, w 80% of the alcohol is absorbed through the intestinal wall. although the absolute proportion absorbed via the intestines increases when wine is jointly consumed with food, uptake is dispersed over a much longer period. this results primarily by food retarding gastric emptying. consequently, alcohol transfer into the intestines is delayed. this gives the liver more time to metabolize the alcohol, lowering the maximal blood-alcohol level reached. however, taking sparkling wine on an empty stomach can increase short-term alcohol uptake by about 35% (ridout et al., 2003) . because the same wine, with its carbon dioxide removed, did not have the same influence, it is suspected that carbon dioxide was the active ingredient (ridout et al., 2003) . it has occasionally been proposed that carbon dioxide relaxes the pyloric sphincter, allowing earlier transfer of fluids from the stomach into the duodenum and thereby its absorption into the blood. the rate of alcohol metabolism differs considerably among individuals, with rates commonly varying between 90 and 130 mg/kg/h. a person's hormonal and nutritional state also affects their ethanol metabolic rate. gender is also an influencing factor (kaltenback et al., 2001) . the tendency of women to have a higher body fat content (into which ethanol does not infiltrate), results in their being more rapidly influenced by similar amounts of alcohol (kalant, 2000) . additional benefits that may accrue from wine consumption are derived from metabolic by-products of proanthocyanin degradation in the colon. they assist protecting the colonic mucosa from the toxic effects of p-cresol production (generated from l-tyrosine) (wong et al., 2016) . furthermore, wine phenolics may prevent or delay intestinal diseases associated with inflammation and oxidative stress (e.g., inflammatory bowel disease) (biasi et al., 2014) . finally, wine can have a beneficial cultural/psychologic effect on food intake and digestion. the association of wine with refined eating promotes slower food consumption, potentially permitting biofeedback mechanisms to regulate food intake. in addition, wine consumption can promote a more relaxed lifestyle, something increasingly valuable in our overly compulsive society. whether this explains the reported improved appetite of many elderly and anorectic patients, when wine is taken with the meal, is unknown. wine taken with a meal can enhance the pleasures derived from both but not necessarily those suffering gastroesophageal reflux (acid reflux). this is apparently correlated more with the consumption of white than red wines (pehl et al., 1998) . most investigations on the health benefits of moderate wine consumption have involved population (epidemiologic) and tissue-culture studies. however, to be confident in their interpretation, intermediate stages needs to be known. this involves details on the uptake and degradation in the intestinal tract, metabolism in the liver, transport, binding, and modification in the blood and lymph, elimination by the kidneys, and cellular uptake and metabolism. although absorption via the intestinal tract is a priori requirement for most activity, it alone does not imply bioavailability at the cellular level. however, this does not apply to influences in the oral cavity and digestive tract. in the mouth and upper intestinal tract, a wine's phenolic constituents remain largely unmodified, except for binding with proteins. in contrast, considerable degradation occurs in the colon. here, large phenolics tend to be metabolized, depending on an individual's colonic flora. for example, hydrolyzable tannins are converted to more easily absorbed, antiinflammatory/anticarcinogenic urolithins (tomás-barberán et al., 2014) . if absorbed, most phenolics are quickly metabolized in the liver, conjugated with various moieties (e.g., methyl or sulfur groups) by plasma enzymes, and/or eliminated via the kidneys. amounts found in the plasma are often 1% of that consumed, although this may increase with repeated daily exposure. the proportion of wine-derived flavonoids is estimated at about 4 mg/day/person in the united states (chun et al., 2007) . this compares with about 200 mg/day/person from all sources. this value would increase to about 37 mg flavan-3-ols and 47 mg procyanidin dimers, based on 180 ml of red wine per day (forester and waterhouse, 2009 ). that volume is near the upper limit of what is typically considered moderate wine consumption. in the mouth, mid-sized flavonoid polymers often bind to salivary proteins, forming stable complexes (de freitas and mateus, 2003; pizarro and lissi, 2003) , slightly delaying their transport to the stomach. passage through the stomach does not modify the majority of wine phenolics. among wine flavonoids, anthocyanins appear to be those that most quickly traverse the stomach and pass into the blood (passamonti et al., 2003) . flavonoid glucoside uptake is facilitated by gastric glucose transporters (oliveira et al., 2015) , whereas aglycone uptake occurs by passive diffusion. flavonoids are also effectively translocated across the small intestine lining (talavéra et al., 2005) . phenolic acids, such as caffeic acid (simonetti et al., 2001) and resveratrol (soleas et al., 2001) also readily pass into the plasma via the intestinal tract. in contrast, flavonoid polymers tend to remain in the intestine, until degraded to phenolic acids and aldehydes by the colonic flora (aura, 2008, fig. 12.5) . also metabolized in the colon are any anthocyanins or catechins monomers that have not already been absorbed and/or microbially degraded. these may enhance the growth of beneficial bacteria (e.g., bifidobacterium and lactobacillus spp.) or their attachment to the intestinal wall (bustos et al., 2012) . depending on the compound, variable amounts may be absorbed into the blood (ward et al., 2004) . studies on the bioavailability of phenolics, once in the bloodstream, are still preliminary (williamson and manach, 2005) . although many simple flavonoids are quickly absorbed into the plasma, most appear to be rapidly conjugated (methylated or sulfated), bound to proteins, transformed to glucuronides, or otherwise modified (williams et al., 2004; forester and waterhouse, 2009; xiao and högger, 2015) . hydroxycinnamic acids are also rapidly absorbed and metabolized into glucuronide and sulfate conjugates (nardini et al., 2009 ). this both reduces their toxicity (potential carcinogenicity) as well as facilitating their excretion by the kidneys. however, the latter reduces their potential beneficial effects. in contrast, anthocyanin seems to be efficiently absorbed via the stomach wall, and their metabolites appear to remain in the plasma for several days (kalt et al., 2014) . short-term studies primarily detect the uptake of phenolic metabolites, whereas long-term studies detect more parental constituents (sandoval-ramírez et al., 2018) . small amounts of cinnamic acid-tartrate esters are also found in the plasma. these transformations could significantly affect their antioxidant and other attributes as well as their ability to move into tissue cells and their surrounding fluids. most phenolic metabolites retain one or more hydroxyl groups and thus may still possess antioxidant properties. nevertheless, there is growing evidence that phenolic metabolites act primarily as signaling molecules, notably in oxygen-stress-related pathways (williams et al., 2004) . consequently, smaller amounts are needed than for direct antioxidant reactions. this might explain the discrepancy between the low levels of free phenolics in the plasma and their apparent effects. an example may be the increased activity and the presence of phenolics in the plasma permits their likely uptake into most body tissues. this generality does not necessarily apply to the brain. except where there are specific transport proteins, most compounds above a molecular weight of 500 da are excluded from the brain by the bloodebrain barrier. the barrier consists of tight connections between the endothelial lining of cerebral capillaries. it prevents the diffusion of most molecules from the blood into the cerebrospinal fluid. however, with anthocyanins (passamonti et al., 2005) and simple flavonols (youdim et al., 2004) , access to the brain apparently can occur within minutes of consumption. initial animal studies suggest uptake levels in the brain occur at about 10% that of other tissues (wu et al., 2012) . although fascinating, consumers are more interested (if at all) in what wines provide the maximal health benefits and under what conditions. regrettably, data on these vital concerns are lacking. grape cultivar, maturity, wine production, maturation and aging conditions all influence the amounts and types of phenolics present and thus their activity. the prophylactic action of wine against gastrointestinal diseases has been known for millennia, long before their microbial origins were ever suspected. in spite of this, the mechanism(s) by which this occurs remain poorly understood. the antimicrobial effect of alcohol was discovered in the late 1800s. nevertheless, alcohol is not particularly antimicrobial, certainly at the concentrations found in wine (its sterilant action is optimal at about 70%). the antimicrobial action of wine is closely related to that of crushed grapes (ö ncü l and karabiyikli, 2016). thus, the antibiotic action of wine likely relates more to its phenolic and acidic (vaz et al., 2012) contents, although wine's alcohol content undoubtedly augments their effectiveness. anthocyanins, which are weakly toxic to viruses, protozoans, and bacteria, become more so as a consequence of fermentation. other phenolic compounds in wine are bacteriostatic and fungistatic. for example, p-coumaric acid is particularly active against grampositive bacteria (e.g., staphylococcus and streptococcus), whereas compounds, such as quercetin, inhibit pathogenic gram-negative bacteria (e.g., escherichia, shigella, proteus, and vibrio) (vaquero et al., 2007) . phenolics may also be inhibitory to intestinal pathogens such as clostridium difficile, c. perfringens, and bacteroides (lee et al., 2006) . despite wine being more effective than mildly antimicrobial agents, such as bismuth salicylate (weisse et al., 1995) , full action may take several hours (møretrø and daeschel, 2004; dolara et al., 2005) . although most studies have involved bacteria grown on culture plates, wine has also been shown to be antimicrobial under simulated gastrointestinal conditions (vaz et al., 2012) . an indirect effect, limiting intestinal problems, is illustrated by the action of the colon flora on anthocyanin structure. it favors the growth of bifidobacterium and lactobacillus-enterococcus spp. (hidalgo et al., 2012) . these have been associated with a healthy gut microflora (hord, 2008) . there is also considerable variation in the effects of different flavanols and procyanidins, both promoting and inhibiting the adhesion of probiotic lactobacilli to the intestinal wall, depending on their metabolic modification during passage through the intestinal tract (bustos et al., 2012) . in addition, viniferin (a resveratrol derivative) can inhibit biofilm formation by pathogenic pseudomonas aeruginosa and escherichia coli (cho et al., 2013) . red wine can suppress biofilm formation by oral pathogens (muñ oz. in most instances, the mechanism by which phenolics have their action is unknown. however, in the case of quercetin, the effect may be partially attributed to its inhibition of dna gyrase, whereas with epigallocatechin, disruption of cell membrane function appears central to its antibiotic action. alternative methods of action may involve suppression of cell adherence and colony formation on the gut lining (selma et al., 2012; truchado et al., 2012) . adherence is often a prerequisite for the cascade of events leading to disease development. low ph and the presence of various organic acids appear to accentuate the antimicrobial action of both wine phenolics and ethanol. organic acids may themselves be antimicrobial, as is the case with bacillus cereus (vaz et al., 2012) . wine is also active against several viruses including the herpes simplex virus, poliovirus, hepatitis a virus as well as rhinoviruses and coronaviruses. the effect on the latter two groups appears reflected in the reduced incidence of the common cold in moderate alcohol consumers (cohen et al., 1993) , particularly those drinking red wines (takkouche et al., 2002) . if you have to gargle, port is certainly one of the more pleasant. the antioxidant action of wine phenolics not only appears to play an important role in limiting low-density lipoprotein (ldl) peroxidation (maxwell et al., 1994; rice-evans et al., 1996) but also the action of lipoxygenases and enzymes generating ros. phenolics can also directly scavenge (quench) these radicals (e.g., superoxide and hydroxyl radicals), contributing to the action of cellular antioxidants. the oxidized flavonoid byproducts are much more stable (nonreactive) and tend to be quickly metabolized or eliminated by the kidneys. a flavonoid's quenching ability is largely dependent on the location and number of its oh groups as well as its glycosylation, sulfation, methylation, and acylation status (plaza et al., 2014) . in addition, phenolics can chelate iron and copper, limiting their involvement in radical formation (morel et al., 1994; rice-evans et al., 1996) or access to bacterial pathogens. phenolic can also limit the influx of calcium ions associated with oxidative stress (ishige et al., 2001 ). an antioxidant relatively unique to wine is resveratrol. it is a stilbene phenolic produced in response to plant stresses. it has greater antioxidant activity than common dietary antioxidants, such as vitamin e and ascorbic acid (frankel et al., 1993) . there is also direct evidence that resveratrol can enter the blood system at levels sufficient to suppress cyclooxygenase and 5lipoxygenase pathways. these are involved in the synthesis of proinflammatory mediators (bertelli, 1998) . in addition, resveratrol can activate proteins involved in nerve cell differentiation, synaptic plasticity, and neuronal survival (tredici et al., 1999) . supplemental protection may result from ethanol activating the cellular biosynthesis of hydroxytyrosol (a dopamine metabolite) (de la torre et al., 2006) . hydroxytyrosol is an important antioxidant and antiinflammatory agent. the most clearly established benefit of moderate alcohol consumption, notably wine, relates to a nearly 30%e35% reduction in death rate due to cardiovascular disease (klatsky et al., 1974 (klatsky et al., , 2003 renaud and de lorgeril, 1992, fig. 12.6) . alcohol consumption is also correlated with a decrease in the likelihood of intermittent claudication (pain or cramping in the calf of the leg). claudication is a common indicator of peripheral arterial disease. recent studies have confirmed that incidental factors, such as gender, race, lifestyle, educational level, etc. do not affect these results (see mukamal et al., 2006) . studies have also demonstrated that daily consumption of alcohol significantly reduces the incidence of other cardiovascular diseases, such as hypertension (keil et al., 1998) , heart attack (gaziano et al., 1999) , stroke (truelsen et al., 1998; hillbom, 1999) , and peripheral arterial disease (camargo et al., 1997) . those who consume wine moderately live, on average, 2.5e 3.5 years longer than teetotalers and considerably longer than heavy drinkers. the prime area of contention is the degree to which these benefits accrue from the effects of ethanol vs. phenolic and/or other constituents (rimm et al., 1996) . atherosclerosis is the principal cause of most cardiovascular disease (libby, 2001) . it apparently results from chronic injury to the arteries ( fig. 12.7) . although associated with several independent factors, most damage is correlated with lipid oxidationda subgroup of cholesterol-apoproteins complexes (ldls). because of the hydrophobic nature of cholesterol and triglycerides, their transport in the plasma requires a special structure. as illustrated in fig. 12 .8, lipoprotein complexes consist of an outer membrane of phospholipids, within which apoproteins and free cholesterol occur. they enclose a hydrophobic core possessing numerous triglycerides and cholesteryl esters. metabolism of the enclosed lipids is regulated by the apoproteins in the outer membrane. normally, ldls supply cholesterol for cellular membrane repair and steroid synthesis. however, in high concentrations, they may accumulate in the artery wall. if they remain there for an extended period, their lipid content tends to become oxidized. in an oxidized state, lipids are cytotoxic and indirectly irritate the artery wall. as a consequence, special adhesion proteins attach to the artery wall. monocytes and helper t-cells of the immune system bond to these proteins. in addition, affected endothelial cells may secrete compounds, such as endothelin-1. endothelin-1 activates monocyte and t-cell migration into the artery wall. procyanidins, principally found in red wines, are particularly effective in suppressing the production of endothelin-1 (corder et al., 2001) . anthocyanin metabolites are also effective modulators of endothelial function (edwards et al., 2015) . in the layer just underneath the endothelial lining (intima), accumulated monocytes mature into macrophages. both macrophages and t-cells may release a range of cytokines that further activate the immune system, involving localized inflammation. activated macrophages tend to engulf oxidized ldls. however, as the ldls are not degraded, their progressive accumulation gives the macrophage the appearance of being full of bubbles (termed foam cells). they are the first clear evidence of localized arterial swelling (plaques). occasionally plaques bulge into the vessel. more frequently, they initially enlarge outward into the surrounding tissue. action of immune cells in the plaque also induces migration of smooth muscle cells from the artery wall into the intima. here they proliferate and produce collagen, forming a fibrous cap over the plaque. additional ldls slowly collect, provoking further rounds of inflammation and plaque enlargement. these accretions may develop their own vasculature, becoming fibrous and inelastic. as the plaques enlarge, they may produce irregular protrusions into and block the artery lumen. even without restricting blood flow, plaques set the stage for platelet aggregation, clot formation (thrombus) and the blockage that can precipitate a heart attack or stroke. in the later phases of plaque formation, unknown factors enhance inflammatory changes in the plaque. these disrupt the integrity of the cap. for example, collagenases secreted by macrophages inhibit collagen synthesis by smooth muscle cells. sudden rupture of a plaque permits blood infiltration into the plaque. because plaques contain potent blood clotting factors, thrombus development is almost instantaneous. it is currently thought that plaque rupture is the principal factor inducting thrombus formation and precipitating a heart attack, stroke, or other cardiovascular trauma. atherosclerosis appears to be at least partially reversible, if risk factors such as smoking, high blood pressure, high dietary sources of cholesterol, and possibly infection by pathogens such as chlamydia pneumoniae and cytomegalovirus are eliminated. part of the reversal process involves the action of high-density lipoproteins (hdls). of the two principal forms, ethanol augments the presence of hdl 3 , whereas exercise increases the level of hdl 2 . the effect of ethanol on hdl concentration appears independent of beverage type (van der gaag et al., 2001) . either form of hdl favors the removal of cholesterol from the arteries, transferring it to the liver for metabolism. hdls also appear to interfere with ldl oxidation. because the hdl/ldl ratio affects the degree and rate of cholesterol turnover, the slower the rate, the greater the likelihood of oxidation (walzem et al., 1995) and eventual plaque formation. the beneficial effect of moderate alcohol consumption on the hdl/ldl ratio is now relatively clearly established. less well understood is its effect in lowering the concentration of c-reactive protein (crp) (levitan et al., 2005) . crp is an indicator of inflammation. its level usually rises in correlation with the risk of atherosclerosis. moderate alcohol consumption also reduces the incidence of another risk factor for cardiovascular diseased type 2 diabetes. chronically high values of circulatory glucose, associated with type 2 diabetes, appear to generate high plasma triglyceride and ldl levels. however, the benefits of wine's alcohol content on glucose and insulin metabolism appear not to occur if intake is not coincident with meal consumption (augustin et al., 2004) . phytoestrogens, such as resveratrol, have a similar effect in reducing triglyceride and ldl contents in the circulatory system (see bisson et al., 1995) . another of alcohol's beneficial influences involves disruption of events leading to clot formation. platelets are less "sticky" in the presence of alcohol and thus less likely to aggregate, limiting clot formation. alcohol also increases the level of prostacyclin (interferes with clotting) and raises the level of plasminogen activator (a clot-dissolving enzyme). clots, adhering or becoming stuck to the roughened surfaces of narrowed atherosclerotic vessels, may block blood flow. the oxygen deficiency and cell death that result are central to the damage caused by a heart attack or stroke. thus, it is not surprising that inhibitors of platelet aggregation reduce the frequency of these cardiovascular crises and their sequelae. it is the rationale for recommending the daily consumption of acetylsalicylic acid (an inhibitor of platelet aggregation). ethanol (renaud and ruf, 1996) as well as wine phenolics, such as resveratrol and anthocyanins, have similar effects (fig. 12.9 ). an additional example of the beneficial effects of limited alcohol intake is the relation between alcohol dehydrogenase (adh) genotype and the incidence of myocardial infarction. individuals homozygous for adh1c*2 (slow metabolizers of ethanol) are significantly less likely to have a heart attack than heterozygous individuals and even less likely than homozygous individuals for adh1c*1 (fast metabolizers of ethanol) (hines et al., 2001) . individually, many phenolics, such as resveratrol, catechin, epicatechin, and quercetin appear to have inhibitory effects on platelet aggregation (keli et al., 1994) . in an in vitro study, though, monomeric or low-molecular-weight flavonoids and hydroxycinnamic acids enhanced platelet aggregation and ldl oxidation, with only large polymers being inhibitory (shanmuganayagam et al., 2012) . in another investigation, the combined effect of several phenolics was superior to single compounds (wallerath et al., 2005) . the action partially results from the enhanced synthesis and release of nitric oxide by endothelial cells. this has been found to occur at resveratrol concentrations associated with moderate wine consumption (gresele et al., 2008) . chlorogenic acid also appears to activate nitric oxide production (mubarak et al., 2012) . nitric oxide induces vasodilation (by relaxing vascular smooth muscle), reduces blood pressure, and limits platelet adhesion to blood vessel endothelia. indicative of the complexities of such interactions is the observation that flavonoids may also inactivate nitric oxide (verhagen et al., 1997) . in addition, nitric oxide, notably as peroxynitrite, oxidizes ldls. clearly, much more still needs to be known before a clear picture emerges. in addition to affecting platelet aggregation, wine phenolics can bind directly with ldls (limiting their oxidation), indirectly reduce macrophage-mediated oxidation and preserve the action of paraoxonase (further protecting ldls from oxidation) (aviram and fuhrman, 2002) . furthermore, red wine phenolics directly or indirectly limit the migration of smooth muscle cells into the intima of artery walls. these influences probably explain some of the added benefits of wine vs. other alcoholic beverages in reducing the incidence and severity of cardiovascular disease. although flavonoids tend to suppress inflammation, conflicting observations put the clinical significance of their antiinflammatory action to atherosclerosis in question. whether this might also apply to the antiinflammatory effects of wine phytoprostanes (degradation products of linolenic acid) is unknown. red wines usually have been credited with superior health-related benefits than white wines, especially relative to cardiovascular disease. this presumably results from their higher flavonoid content (tian et al., 2011) . this view is supported by studies where white wine has shown the same effects as red wine, when supplemented with grape polyphenolics (fuhrman et al., 2001) . nevertheless, prolonged skin contact, or choice of particular cultivars, can enhance the presence of phenolic acids in white wine. common phenolics in white wine, such as caffeic and coumaric acids as well as flavonols such as quercetin, are well-known potent antioxidants. the low sodium content of wine is an incidental benefit. it may permit wine consumption by those on a low-sodium diet, for example those with high blood pressure or heart attack victims. the high potassium to sodium ratio of wine (20:1) is also advantageous. as noted, many of the beneficial influences of alcohol and wine consumption show a j-shaped curve ( fig. 12.2 ). this also applies to its effect on age-related macular degeneration (obisesan, 2003; fraser-bell et al., 2006) . the disease expresses itself as a progressive degeneration of the central region of the retina (macula), leading to blurred or distorted vision. it results as a consequence of local atherosclerosis that deprives the retina of oxygen and nutrients. it is the leading cause of blindness in adults over the age of 65. a similar relationship has been found for cataract development. in both conditions wine antioxidants are suspected to be the active protective agent. in this regard, quercetin appears to be more protective (against light-induced lipid peroxidation) than either anthocyanin-or phenolic acid-rich constituents (liu et al., 2016) . however, wines high in ethanol content may undo these benefits, by promoting pro-oxidant action. alzheimer's, a devastating neurodegenerative disease, afflicts more than 15 million people. not surprisingly, researchers have investigated whether wine consumption affects the incidence of this and other neurodegenerative diseases (barnham et al., 2004) . flavonoids not only activate key respiratory enzymes in mitochondria (schmitt-schillig et al., 2005) , but also decrease the production of reactive oxygen species, by stimulating the production of catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase . a pattern appears to apply here, as with most health-related benefits of wine and alcohol consumptiondmoderate intake bing beneficial, whereas high consumption or abstinence is deleterious. alzheimer's disease has been correlated with the accumulation of extracellular amyloid b-peptide (plaque) and the formation of intracellular neurofibrillary tangles containing tau-protein. many in vitro studies have shown that antioxidant compounds, such as vitamin e, protect neurons from b-amyloid accumulation. tannins also inhibit the formation of and destabilize preexisting b-amyloid fibrils (ono et al., 2008; guéroux et al., 2015) , whereas resveratrol promotes the degradation of amyloid b-peptides (marambaud et al., 2005) . wine consumption is also linked to a reduction in the incidence of alzheimer's disease (truelsen et al., 2002; letenneur, 2004; luchsinger et al., 2004) . even mild cognitive impairment and the progression of idiopathic dementia may be reduced with moderate alcohol consumption (solfrizzi et al., 2007) . grape juice has also been found to be effective in this regard (krikorian et al., 2012) . like other health benefits, these finding may not, in and by themselves, justify wine consumption, but they are encouraging to those who choose wine as part of their preferred lifestyle. age-related bone mass loss affects both sexes but is more frequent in postmenopausal women. many risk factors including dietary influences and hormonal supplements, can affect its progress and severity. of these factors, moderate alcohol consumption has been found to favor bone retention (ganry et al., 2000; ilich et al., 2002) . tucker et al. (2009) found data consistent with higher benefits from wine than other alcoholcontaining beverages. the source of these benefits may be a combination of enhanced calcium uptake, associated with alcohol consumption (ilich et al., 2002) , the phytoestrogen effects of phenolics, such as resveratrol and kaempferol, or other unsuspected influences. a number of drugs used in treating arthritis tend to irritate the stomach lining. this side-effect may be counteracted by the mildly acidic, dilute alcohol content of table wines. other beneficial effects connected with moderate wine consumption may accrue from its mildly diuretic and muscle relaxant properties. the diuretic action of wine can help reduce water retention and minimize joint swelling. wine can also directly reduce muscle spasms and the stiffness associated with 12. wine, food, and health arthritis. the antiinflammatory influences of wine phenolics, notably resveratrol (yang et al., 2018) , may also play a role in diminishing the suffering associated with arthritis and other diseases associated with chronic inflammation (schueller et al., 2015) . wine consumption has been shown to attenuate insulin-resistance in type 2 diabetes (dixon et al., 2001; napoli et al., 2005) . this may result from wine phenolics quenching oxygen radicals, thought to be pivotal in the damage associated with the disease. type 2 diabetes appears to result when body cells fail to respond properly to the presence of insulin. the incidence of metabolic syndrome is also lower in wine drinkers (rosell et al., 2003) . these effects may be due to one or more of the following: the influences of alcohol on metabolism; the antidiabetic properties of the element vanadium (for which wine is a significant source) (brichard and henquin, 1995; teissèdre et al., 1996) ; the hypoglycemic and hypolipidemic effects of phenolics such as resveratrol (su et al., 2006) ; and/or through some effect on endothelial nitric oxidase synthase (leighton et al., 2006 ). it appears there may be considerable specificity. for example, in a comparison between malvidin-and delphinidin-3-o-glucosides, only the predominant anthocyanin in grapes (malvidin) seems to have a significant hypoglycemic effect (lida et al., 2012) . relative to diabetes mellitus (type 1 diabetes), moderate consumption of dry wine was found to present no adverse effects on sugar control (gin et al., 1992; bell, 1996) . although wine does contain residual sugars, the most common, fructose, is poorly transported across the gastrointestinal tract. most of what is absorbed is rapidly removed from the blood by the liver, where it is metabolized into glycerol and often stored as fat. it does not stimulate pancreatic insulin release. red wine (and a diet rich in antioxidants) appear to slow the progression of kidney damage (necropathy), occasionally associated with diabetes (zhu et al., 2017) . in an epidemiological study, knudsen et al. (2001) found a strong link between alcohol consumption and a reduced prevalence of goiter and solitary thyroid nodules. the origin of this apparent protective effect is unknown. drinking water has long been associated with reducing the development of kidney stones. increased urine production is thought to limit calcium oxalate crystallization. what is new is the observation that wine consumption further reduces the production of these painful and dangerous inclusions (curhan, 2007) . moderate wine consumption has been correlated with reduced incidence in some cancers (see bianchini and vainio, 2004 ) (e.g., the kidney) but increased risk for others (notably the throat and gastrointestinal tract) (ebeler and weber, 1996; parry et al., 2011) . this potential varies markedly, primarily based on the amounts habitually consumed. for other cancers, moderate consumption appears to be neither protective nor a risk factor (e.g., prostate cancer) (chao et al., 2010) . conversely, excessive consumption of alcoholic beverages increases the risk of a range of cancers, notably those of the oropharynx, larynx, esophagus, liver, colon, rectum, and breast (connor, 2017) . because ethanol is not directly carcinogenic, negative associations with alcohol consumption presumably relate to carcinogens potentially present in wines (e.g., ethyl carbamate). thankfully, its carcinogenicity is reduced at the alcohol concentration typical of table wines. more significant, though, maybe acetaldehyde, a common denominator in gastrointestinal cancers (salaspuro, 2009) . depending on the type of wine consumed, short-term but high concentrations of acetaldehyde may occur in the saliva and gastric juice (lachenmeier and sohnius, 2008) . acetaldehyde may also accumulate due to the action of alcohol dehydrogenase in the digestive tract, microbial alcohol metabolism (notably the colon), and malfunction of cellular acetaldehyde dehydrogenase. certain wine phenolics can be protective, whereas others mutagenic, especially at high concentrations. for example, quercetin can induce mutations in laboratory tissue cultures but is a potent anticarcinogen in whole-animal studies (fazal et al., 1990 ). this apparent anomaly may result from differences in the concentrations of quercetin used, and/or the low levels of metal ions and free oxygen found in the body (vs. tissue culture). in addition, quercetin, along with several other phenolics that are potential carcinogens, lose this attribute when present as a glycoside. most phenolics in the plasma occur in some conjugated state, not as free phenolics. in addition, phenolics may detoxify the small quantities of nitrites commonly found in food. however, in the presence of high nitrite concentrations (a preservative found in smoked and pickled foods), nitrites are converted into diazophenols (weisburger, 1991) . these appear to favor the development of oral and stomach cancers. potential health issues several phenolics can limit or prevent cancer development through a diversity of effects, such as dna repair, carcinogen detoxification, enhanced apoptosis (programmed cell death), disrupted cell division (hou, 2003; aggarwal et al., 2004) , or enhanced immunostimulation (tong et al., 2011) . for example, resveratrol induces the redistribution of the fas receptor. it is a cellular attachment site for tnf (tumor necrosis factor). its action is part of a sequence that can lead to cancer cell apoptosis (delmas et al., 2003) . resveratrol is an inhibitor of angiogenesisdthe production of new vasculature essential for most tumor growth. other effects of resveratrol include inhibition of cyclooxygenase-2 (subbaramaiah et al., 1998) and cytochrome p450 1a1 (chun et al., 1999) . cyclooxygenase-2 is thought to be involved in carcinogenesis, whereas p450 1a1is an important hydroxylase. it can convert several environmental toxicants and procarcinogens into active carcinogens. flavones and flavonols strongly restrict the action of common dietary carcinogens, notably heterocyclic amines (kanazawa et al., 1998) . it is estimated that these amines, produced during cooking, are consumed at a rate of approximately 0.4e16 mg per day (wakabayashi et al., 1992) . antitumor activity is also associated with acutissimin, a flavono-ellagitannin found in oakmatured red wine. the antiallergic and antiinflammatory properties of flavonoid phenolics probably also contribute to the anticancer aspects of these flavonoids (see middleton, 1998) . the major exception to the general benefit of moderate wine consumption may be breast cancer (viel et al., 1997) . the connection is more evident in those with the adh1c*1 (fast metabolizers of ethanol to acetaldehyde) (terry et al., 2006) . however, findings from the long-duration framingham study indicate no relationship between moderate alcohol consumption and the incidence of breast cancer (zhang et al., 1999) . ethanol, although not itself a carcinogen, can enhance the transforming effect of some carcinogens. another example of a negative effect of wine consumption, at least in excess of moderate intake, is to increase the incidence of mouth and throat cancers (barra et al., 1990) . alcoholic beverages may also induce a diversity of allergic and allergy-like reactions. in sensitive individuals, these may express as rhinitis, itching, facial swelling, headache, cough, or asthma. the primary culprit inducing bronchial constriction, at least in some asthmatics, is sulfur dioxide (dahl et al., 1986) . wine containing an abnormally high sulfur dioxide concentration (300 ppm sulfite) induce a rapid drop in forced expiratory volume (vally and thompson, 2001) , recovery taking about 15e60 min. the same individuals did not respond to wine containing 20, 75, or 150 ppm sulfite. fig. 12 .10 illustrates the range of sulfur dioxide contents potentially found in californian wine. thus, the sulfite levels normally found in wine seem not to be a major factor in wine-induced asthmatic responses (vally et al., 2007) . why sensitive asthmatics episodically react to wines with low so 2 content may be related to changes in their asthma control. surprisingly, red wines appear to provoke more asthma problems than white wines, even though red wines typically have lower sulfur dioxide contents than white wines. californian wines (mg/l). reprinted from peterson, g.f., kirrane, m., hill, n., agapito, a., 2000 . a comprehensive survey of the total sulfur dioxide concentrations of american wines. am. j. enol. vitic. 51, 189e191, permission conveyed through copyright clearance center. 12. wine, food, and health the rapidity of the reaction to sulfite suggests some malfunction in the amount of glutathione in lung tissue or the activity of glutathione s-transferase in reducing sulfite to glutathione s-sulfonate. normally, sulfite is rapidly converted to sulfate by sulfite oxidase in the blood. however, low levels of this enzyme could permit sulfite to persist, provoking a heightened response in hypersensitive individuals. at greater risk are individuals afflicted with a rare, autosomal, genetic disease, caused by a deficiency in sulfite oxidase (shih et al., 1977; crawhall, 1985) . affected individuals must live on a very restricted diet, low in sulfur-containing proteins. it is estimated that the synthesis of sulfite, associated with normal food metabolism, generates approximately 2.4 g sulfite/day. the sulfites in wine contribute only marginally to this amount but may be temporally significant. because of the gravity of sulfite oxidase deficiency, most affected individuals do not reach adulthood. another allergy-like reaction provokes rapid facial and neck flushing (cutaneous erythema). it develops shortly after alcohol consumption. other symptoms often include peripheral vasodilation, elevated heart rate, nausea, abdominal discomfort, and bronchoconstriction. the syndrome is associated with a malfunctional form of mitochondrial acetaldehyde dehydrogenase (aldh2*2) (enomoto et al., 1991; eriksson et al., 2001) and is particularly pronounced in the homozygous state. aldh2 is the principal enzyme oxidizing acetaldehyde to acetic acid. it is estimated that up to 50% of eastern asians possess at least one malfunctional aldh2 allele and express some degree of allergy-like reaction to alcohol consumption. it has been suggested that the aldh2 mutant, frequently found in eastern asians, may reflect an evolutionary adaptation to the endemic occurrence of hepatitis b (lin and cheng, 2002) . the mutation could have induced alcohol aversion, thereby avoiding synergism between alcohol and hepatitis b-induced liver damage. elevated levels of acetaldehyde appears to activate the release of histamine from mast cells. it subsequently induces vasodilation and an associated influx of blood (flushing). the connection between acetaldehyde and histamine is supported by the action of antihistamines in reducing the reaction, if taken in advance of an alcohol challenge (miller et al., 1988 ). an alternative proposal is that this flushing reaction results from a direct, cutaneous, alcohol-induced vasodilation. the phenomenon tends to be suppressed by acetylsalicylic acid (aspirin), if taken in advance (truitt et al., 1987) . the unpleasant side-effects of acetaldehyde accumulation is used in treating alcoholism. it involves taking disulfiram (a potent inhibitor of aldh) prior to alcohol consumption. a facial flushing, concomitant with alcohol consumption but devoid of other symptoms, is occasionally experienced by caucasians. whether this is related to an aldh malfunction is unclear. the histamine content of wine has frequently been thought to contribute to several allergy-like reactions. however, wine is typically low in histamine content. fig. 12 .11 illustrates the range found in some wines. thus, it seems unlikely that a wine's histamine content is a major inducer of allergy-like reactions. this is supported by a double-blind study of people, selfreportedly wine intolerant. reactions to two pinot noir wines, differing in histamine content (13.8 vs. 0.4 mg/ l) were not significantly different (kanny et al., 2001) . other common foods are considerably higher in histamine content, for example cheeses. this does not necessarily exonerate biogenic amines from being somehow involved. people vary in diamine oxidase activity (a histamine inactivator) (wantke et al., 1996) and how alcohol (via acetaldehyde) suppresses its action . alcohol also can enhance the permeability of the intestinal lining to histamine. in addition, acetaldehyde accumulation can activate histamine release (harada et al., 1981) . this may explain the benefit antihistamines have in diminishing the rhinitis occasionally associated with wine consumption (andersson et al., 2003) . in addition, antihistamines counteract the broncho constriction in individuals showing histamine intolerance. idiopathic allergic and other immune hypersensitive responses to wine are difficult to predict or diagnose. reactions may include the induction of headaches, nausea, vomiting, general malaise, or a combination of these. in a few instances, ige-related anaphylaxis reactions have been reported to grape pr proteins (endochitinase and thaumatin) (pastorello et al., 2003) . the effects may involve urticaria/angioedema (red patches or wheals on the skin/swelling) and occasionally shock. residual amounts of fining agents, such as egg whites, have also been implicated in some allergic reactions (marinkovich, 1982) . in a double-blind, placebocontrolled trial, wines fined with egg white, isinglass, or nongrape derived tannins presented "an extremely low risk of anaphylaxis" to egg-, fish-, or peanutallergic consumers (rolland et al., 2006) . in an elisa analysis, only egg white and lysozyme could be detected in wine samples (weber et al., 2007) . nevertheless, with more than 1000 compounds potentially occurring in wine, it is not surprising that some individuals may occasionally show some form of adverse reaction to some wines. in addition to physiological reactions to wine constituents, there is a wide range of equally important psychological responses (rozin and tuorila, 1993) , both positive and negative. traumatic memories, associated with the first exposure to, or excessive consumption of, a particular beverage can create an association that lasts a lifetime. other people have come to associate certain products with social groups, lifestyles, or behaviors. such attitudes can make the beverage either unacceptable or desirable as the case may be. in the 1800s, there were many reports linking gout with wine consumption, notably port. gout is caused by the localized accumulation of uric acid crystals in the synovium of joints. their presence stimulates the synthesis and release of humoral and cellular inflammatory mediators (choi et al., 2005) . gout is also associated with reduced excretion of uric acid in the kidneys. mutations in the gene that encodes urease, the enzyme that metabolizes uric acid to allantoin (a soluble by-product), is often implicated in gout. dietary predisposing factors for gout include red meat, seafood, and beer. this is presumably because purines, the principal source of uric acid, are found in higher concentrations in these products than many other foods or beverages. alcohol consumption may occasionally aggravate gout by increasing lactic acid synthesis. it, in turn, favors uric acid reabsorption by the kidneys. despite this, wine consumption appears not be associated with an increased risk for gout. in contrast, it seems to favor reduced serum urate levels (choi and curhan, 2004) . medical historians suspect the nineteenth century gouteport association was connected with leadinduced kidney damage (yu, 1983; emsley, 1986 emsley, /1987 . samples of port from the nineteenth century show high lead contents. lead contamination probably came from the stills used in preparing the wine spirits added in port production. in addition, the former use of pewter and lead-glazed drinking cups and prolonged storage of port in lead crystal decanters or stemware could have further augmented lead content (falcone, 1991; guadagnino et al., 1998) . people occasionally avoid wine because it induces headaches. regrettably, the wine/headache connection is still poorly understood. however, effective differentiation between wine-induced headaches may be pivotal to discovering their causes and possible solutions. one of the most severe headache syndromes, potentially associated with wine consumption, is the migraine. migraines appear to be induced, but inconsistently, by a wide range of environmental stimuli, often in tandem. an association between it and red wine consumption has been noted since roman times. the dilation of cerebral blood vessels, partially associated with histamine release, appears to be a common element in many headache syndromes. migraines may be one of them, although current thought suggests a neurological rather than a vascular origin. in addition, a double-blind study seemingly has exonerated histamine in most redwine-induced migraine headaches (masyczek and ough, 1983) . in addition, migraine attacks are more often associated with consuming spirits and sparkling wines, both lower in histamine content that table wines or beer (nicolodi and sicuteri, 1999) . however, the former are often taken alone, leading to more rapid and higher spikes in blood alcohol content. alcohol may be directly involved in migraine induction through vasodilationdby activating meningeal vessel-associated trigeminal neurons (nicoletti et al., 2007) . alcohol's potential to reduce cerebral glucose metabolism (volkow et al., 2006) could also be a contributing factor. other potential disruptive aspects on brain function may relate to the slow rate of alcohol metabolism by cerebral adh. thus, more alcohol may be metabolized by cytochrome p4502e, a process that generates acetaldehyde as well as ros. the more frequent association of red wines with several headache sequelae may be due to their higher phenolic content. on average, red wines contain about 1200 mg/l phenolics, vs. 200 mg/l for white wines. some phenolics can suppress the action of platelet phenol sulfotransferase (pst) (jones et al., 1995; yeh and yen, 2003) , several isozymic forms (m and p) of which detoxify biogenic amines and phenolics via sulfation. low levels of platelet-bound pst-p have been correlated with migraine susceptibility (alam et al., 1997) . the accumulation of small phenolics (those readily absorbed) in the blood could prolong the action of potent hormones and nerve transmitters (e.g., histamine, serotonin, dopamine, adrenalin, and noradrenaline). small phenolics can also promote platelet aggregation and blood vessel dilation. the associated increase in intercranial pressure may participate in a migraine attack (pattichis et al., 1995) . abnormal and cyclical patterns in platelet sensitivity to 5-ht release in migraine-prone individuals (jones et al., 1982; peatfield et al., 1995) may explain the inconsistent association of wine consumption with migraine induction. in the treatment of cluster-headaches, small doses of lithium have been suggested as preventive (steiner et al., 1997) . because some red wines have a higher than average lithium content, the possibility exists that they might limit the development of, rather than induce, this headache syndrome. another recognized headache syndrome is called the red wine headache (kaufman, 1986) . it may develop within minutes of consuming red wine, often being dose-related. the headache reaches its peak within w2 h, tends to fade but returns roughly 8 h later. the headache seems related to the release of type e prostaglandins, important chemicals involved in dilating blood vessels. their release can be activated by phenolics (padilla et al., 2005) as well as alcohol (parantainen, 1983) . prostaglandins may also activate pain receptors around blood vessels (wienecke et al., 2009 ). this association may explain why prostaglandin synthesis inhibitors (e.g., acetylsalicylic acid, acetaminophen, or ibuprofen) may limit the development of some winerelated headaches (if taken about 1 h before consumption) (kaufman, 1992) . a separate wine-related headache has been dubbed the red head (goldberg, 1981) . it develops within an hour of waking, after drinking no more than two glasses of red wine the previous evening. the headache, associated with nausea, is particularly severe when reclining. although the headache is somewhat relieved by standing, it itself exacerbates the nausea. the headache usually lasts a few hours before dissipating. a similar phenomenon has been reported with some white wines, or mixtures of white wine, taken alone or with coffee or chocolates. its chemical cause is unknown (kaufman, 1986) . because tannins are poorly absorbed in the upper digestive tract, in contrast to monomeric phenolics, the latter are likely the primary phenolic headache activants. this may explain why aged red wines (in which most phenolics occur as large polymers) tend to be less associated with headaches than their younger counterparts. large tannin polymers remain largely unmodified until reaching the colon, where bacteria degrade them (déprez et al., 2000) . because this can take up to 2 days, they presumably are not (or not recognized to be) involved in wine-induced headaches. phenolic absorbed into the blood are primarily detoxified by being methylated or sulfated but may also become more "toxic" (to o-quinones). the latter can retard the breakdown of dopamine and restrict access to m-opioid (painkilling) receptors, exacerbating the pain associated with cerebral blood vessel dilation. nonetheless, some phenolics (e.g., resveratrol) limit, rather than augment, headache development. it inhibits the expression of cyclooxygenases, involved in the synthesis of prostaglandins (jang and pezzuto, 1998) . although red wines are generally associated with headache production, white wines are occasionally associated with their production (relja et al., 1993) . their characteristics and etiology are even less well understood than those evoked by red wines. in some individuals, this situation may be associated with a sensitivity to sulfites but atypically. one of the most recognized alcohol-related headache phenomena is associated with binge drinkingdthe hangover (veisalgia) (wiese et al., 2000) . although not consistently associated with a headache, it is frequently part of the sequelae. hangovers are characterized by tremulousness, palpitations, tachycardia, sweating, loss of appetite, anxiety, nausea, and possibly vomiting and amnesia . when accompanied with a headache, it possesses symptoms resembling a migraine. the headache may be global but frequently concentrated anteriorly, associated with heavy, pulsesynchronous throbbing. it usually starts a few (w3 h) after the cessation of drinking, when blood alcohol level is declining and other hangover symptoms have already developed (sjaastad and bakketeig, 2004; verster et al., 2010) . duration is seldom more than 12 h. despite its all-too-frequent occurrence, the causal mechanism(s) remains unclear. most data suggest that alcohol-induced cerebral inflammation is the primary cause . this may operate directly via tissue dehydration and electrolyte imbalance (due to vasopressin enhanced urination) or indirectly via the toxic effects of acetaldehyde (quertemont et al., 2005) . ethanol can also promote hepatic glycogen breakdown, glucose release, loss via the kidneys, and induction of hypoglycemia. in addition, activation of the liver's microsomal ethanol oxidation pathway releases ros, causing cellular damage and multiple metabolic disruptions. that the severity of a hangover may be reduced by prostaglandin synthesis inhibitors suggests that they may also play a role in hangover sequelae (kaivola et al., 1983) . as the old spanish proverb noted: wine hath drowned more men than the sea because glutathione inactivates free radicals, taking an amino acid supplement (n-acetyl-cysteine) has been suggested as a partial remedy. it is rich in cysteine, an amino acid that forms the core of glutathione. in addition, glutathione facilitates the conversion of acetaldehyde to acetic acid. disruption of membrane function and cerebral neurotransmitter action by acetaldehyde is presumably the rationale for commercial products, such as hangover helper and rebound. they are designed to counter the effects of acetaldehyde. congeners (such as fusel alcohols and methanol) could exacerbate the effects of ethanol and acetaldehyde. however, because their content in wine is low, they are unlikely to be involved in wine-induced hangovers. some purported remedies, such as artichoke extract, have not stood up to rigorous clinical testing (pittler et al., 2003) , but others, such as an opuntia fiscus-indica (prickly pear) extract, apparently reduced the severity of some hangover symptoms (wiese et al., 2004) . that hangovers have been associated with deregulation of cytokine pathways (kim et al., 2003) , may explain the reported value of pyritinol (a vitamin b 6 derivative) as a treatment (khan et al., 1973) . mineral deficiencies have also been correlated with hangovers (min et al., 2010) . combined with restraint, taking wine with a meal is probably the best means by which to avoid a hangover. food delays the movement of alcohol into the intestinal tract, thereby slowing alcohol uptake ( fig. 12.1 ) and correlating uptake closer to the liver's ability to metabolize ethanol. in addition, delayed transfer to the intestinal tract slows phenolic uptake (and other potential provocateurs). wine tasting is not normally considered a hazardous occupation. however, recent studies show that dental erosion is an occupational hazard (mok et al., 2001; mandel, 2005; mulic et al., 2011) . damage results from the frequent and extended exposure to wine acids, correspondingly, white wines are generally more corrosive than reds (willershausen et al., 2009) . saliva is diluted and washed away, resulting in the oral ph falling to that of the wine (obreque-slier et al., 2016) . this causes calcium to dissolve out of tooth enamel, softening and making it susceptible to erosion by masticatory forces and tooth brushing. exposure times as short as 2 min can be harmful (lupi-pegurier et al., 2003) . demineralization commences at about ph 5.7. dental erosion is unlikely to be a significant problem for the typical consumer who takes wine with meals. food and salivary secretion limit, if not prevent, tooth enamel demineralization. after many years, professional wine tasters may experience tooth disfiguration, affecting both tooth shape and size. cupping, a depression in the enamel, exposing dentine at the tip of molar cusps, is a frequent clinical sign. erosion can also contribute to severe root abrasion at the gum line. the good news is that not all tasters are equally at risk (mulic et al., 2011) . protection is partially achieved by rinsing the mouth with an alkaline mouthwash after tasting, application of a fluoride gel (such as apf) and refraining from tooth brushing for at least 1 h after tasting. the delay permits minerals in the saliva to rebind with enamel. for more protective protocols see ranjitkar et al. (2012) . the use of remineralizing agents, such as tooth mousse, also helps prevent dental erosion (piekarz et al., 2008) . in contrast to this risk factor, consuming red wine may have some direct oral benefits. proanthocyanidins can limit the adherence and biofilm-forming activity of caries-inducing streptococcus mutans (daglia et al., 2010) . gibbons (2013) provides a fascinating insight into the association of this bacterium with changes in human diet which resulted from a switch from a hunter-gather to an agriculture lifestyle. mark twain also made pronouncements about dental health, which might be equally applied to wine: "i always take it (scotch whiskey) at night as a preventive of toothache. i have never had the toothache; and what is more, i never intend to have it". from europe and elsewhere. fetal alcohol syndrome refers to a set of phenomena including suppressed growth, mild mental retardation, and subtle facial abnormalities (wattendorf and muenke, 2005) . it was first described in 1973 and appeared most markedly in the children of alcoholic mothers. they tended also to be heavy smokers, users of illicit drugs, consumers of large amounts of coffee, had poor nutrition, or a combination of these (scholten, 1982; whitten, 1996) . although associated with alcohol uptake, the accumulation of acetaldehyde may be the principal toxicant. even more subtle effects have now been associated with alcohol consumption, generating the fetal alcohol spectrum disorders. because the consequences may be lifelong, it is generally recommended that pregnant women, or those desirous of becoming pregnant, refrain from alcohol consumption. although abstinence may be unnecessary (kesmodel et al., 2012) , erring on the side of caution can supply desired peace-of-mind. this also applies to breast feedingd alcohol in breast milk could be detrimental to infant development. the presence of toxins in wine is seldom mentioned, outside academic circles, presumably because of their minimal presence. the only mycotoxin for which there may be regular analysis is ochratoxin a (o'brien and dietrich, 2005; varga and kozakiewicz, 2006) , produced by several black aspergilli (notably aspergillus carbonarius) (somma et al., 2012) . preliminary data suggests that most ochratoxin a is eliminated (destroyed/precipitated) during and after fermentation/maturation (fernandes et al., 2007) . other potential mycotoxins that could occur in wine include isofumigaclavine, festuclavine, and roquefortine, all produced by penicillium spp. (moller et al., 1997) , aflatoxins (el khoury et al., 2008) from aspergillus flavus, fumonisins from aspergillus niger (mogensen et al., 2010) , and trichothecenes by trichothecium roseum (schwenk et al., 1989) . because these fungi are secondary saprophytes, they typically occur only on rotted grapes (thankfully, unlikely on noble-rotted grapes). although the exclusion of all diseased grapes is essentially impossible, their inclusion is limited as much as feasibly possible. pesticide residues are other potential toxins. their levels are usually below those known to be toxic, partially due to regulations limiting their use, precipitation or metabolism during winemaking and degradation during maturation. in addition, most importing countries possess regulations on permissible levels and systems to check for compliance. achieving a zero concentration is probably impossible, if only because of our increasing technical ability to detect their presence at increasingly infinitesimal levels. methanol is present but in amounts insufficient to have any known negative consequences. the same also appears to be true for diacetyl and other potentially toxic compounds. ethyl carbamate, a carcinogen, is no longer likely to occur, since its origin during wine production can be effectively avoided. it may initially be disconcerting to think of trace amounts of toxins in wine, but this situation applies to all food, water, and air. xenobiotics are an inescapable aspect of life, both modern and ancient. their universal presence in the natural environment presumably provided the selective pressures that favored the evolution of organs of detoxification (e.g., the liver and kidneys) and the presence of multiple detoxifying enzyme systems. thankfully, our bodies inactivate most xenobiotics rapidly and effectively, without our conscious knowledge. in addition, governmental agencies set regulations and assess compliance to limit most toxicants to well below known safe limits. as long as exposure to toxicants is kept to a bare minimum, consumers can basically forget they exist. the most important wine contraindication relates to those with a past history of alcohol abuse. for the majority of adults (except pregnant women), moderate wine consumption appears to have significant health benefits. nevertheless, there are several situations in which wine consumption, even in moderate amounts, can complicate or diminish the effectiveness of disease treatment. the acidic nature of wine can aggravate inflammation and slow the healing of ulcers in the mouth, throat, stomach, and intestinal tract. other constituents in wine may also be detrimental in this regard. thus, all beverages containing alcohol are usually contraindicated in cases of gastritis, gastric cancer, and bleeding in the upper digestive tract. nevertheless, the prophylactic action of red wine against helicobacter pylori and the suppression of histamine production by the gastric mucosa (masquelier, 1986 ) may require a reconsideration of the old prohibition in mild cases. in the presence of pancreatitis, alcohol is absolutely contraindicated. wine, along with other alcoholic beverages, may provoke gastroesophageal (acid) reflux in individuals prone to this syndrome. with liver disease, the consumption of wine is normally contraindicated. the presence of alcohol puts additional stress on an already weakened vital organ. chronic alcohol abuse can lead to cirrhosis of the liver. in acute kidney infection, wine should be avoided. the consumption of alcohol increases the burden on an organ essential to eliminating toxic metabolic wastes. with prostatitis or genitourinary infections, the consumption of alcohol can complicate matters. the diuretic action of wine may increase the frequency of urination, or conversely it may induce highly painful urinary retention. in epilepsy, the consumption of even moderate amounts of wine may increase the frequency of seizures. consumption should be strictly limited in most situations of hypertension, hemorrhagic stroke, or atrial fibrillation. patients, about to undergo surgery, are advised to avoid all alcoholic beverages well before surgery. this avoids increasing any tendency to enhance intra-and postoperative bleeding (wolfort et al., 1996) , due to alcohol's reduction of platelet clotting. the consumption of alcohol is also ill advised when eating certain mushrooms. the most well-known example is the antabuse reaction associated if alcohol is consumed with coprinus atramentarius (inky cap). another mushroom generating the same response is boletus luridus (budmiger and kocher, 1982) . the antabuse reaction derives its name from the trade name of disulfiram, a medication used in the treatment of alcoholism. it functions as an inhibitor of acetaldehyde dehydrogenase. even small amounts of alcohol consumed while taking disulfiram can generate very unnerving reactions (e.g., flushing, sweating, weakness, vertigo, blurred vision, difficulty breathing, nausea, chest pain, palpitation, and tachycardia). in severe cases, the reaction can provoke acute congestive heart failure, convulsion, and death. similar symptoms may develop in sensitive individuals when alcohol beverages are consumed while taking certain medications (e.g., cephalosporins, griseofulvin, chloramphenicol, sulfonylurea, metronidazole). in addition to the reactions noted above, consumption of alcohol while taking certain medications can generate dangerous conditions. most of the literature comes from studies on alcoholics or binge drinkers. this limits the potential applicability of the data to conditions of moderate consumption and when taken with food. nevertheless, even small amounts of alcohol may cause loss of muscle control in people taking tricyclic antidepressants. in addition, red wines can reduce the effectiveness of mao (monoamine oxidase) inhibitors, used in controlling hypertension. long-term acetaminophen use can enhance alcohol-induced kidney damage. other contraindications involve the intensification of the effects of barbiturates and narcotics. in combination with certain antidiabetic agents, such as tolbutamide and chlorpropamide, alcohol can cause dizziness, hot flushes, and nausea. mild reactions may occur with a wide range of other medications, such as sulfanilamide, isoniazid, and aminopyrine. additional details may be found in adams (1995) , fraser (1997) , and weathermon and crabb (1999) . in conclusion, mark twain crystalizes what so often seems to be the relationship between food, wine, and health: the only way to keep your health is to eat what you don't want, drink what you don't like, and do what you'd druther not." mark twain in following the equator. interactions between alcohol and other drugs role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies platelet sulphotransferase activity, plasma sulfate levels, and sulphation capacity in patients with migraine and tension headache effects of loratadine in red wineinduced symptoms and signs of rhinitis alcohol consumption and acute myocardial infarction: a benefit of alcohol consumed with meals? epidemiology 15 microbial metabolism of dietary phenolic compounds in the colon wine flavonoids protect against ldl oxidation and atherosclerosis the relative antioxidant potencies of some polyphenols in grapes and wines neurodegenerative disease and oxidative stress type of 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wine consumption: a review of recent results association between consumption of beer, wine, and liquor and plasma concentration of high sensitivity creactive protein in women aged 39 to 89 years complementary approaches to gauge the bioavailability and distribution of ingested berry polyphenolics why can't chinese han drink alcohol? hepatis b virus infection and the evolution of acetaldehyde dehydrogenase deficiency retronasal odor enhancement by salty and umami tastes identification of organic acids in wine that stimulate mechanisms of gastric acid secretion visible light-induced lipid peroxidation of unsaturated fatty acids in the retina and the inhibitory effects of blueberry polyphenols interaction of tannin with human salivary proline-rich proteins alcohol intake and risk of dementia a history of wine as therapy. lippincott, philadelphia williamson, 1994. polyphenols, astringency and proline-rich proteins in vitro action of bordeaux red wine on the microhardness of human dental enamel dental erosion due to wine consumption resveratrol promotes clearance of alzheimer's disease amyloid-beta peptides allergic symptoms from fining agents used in winemaking human parotid secretion in response to ethyl alcohol neuroprotective properties of spanish red wine and its isolated polyphenols on astrocytes azione portettrice del vino sull'ulcera gastrica the red wine reaction syndrome effects of linoleic acid on sweet, sour, salty, and bitter taste thresholds and intensity ratings of adults wine antioxidants and their impact on antioxidant activity in vivo red wine and antioxidant activity in serum alcohol-induced generation of lipid peroxidation products in humans prenatal and postnatal flavor learning by human infants effect of plant flavonoids on immune and inflammatory cell function reduction of alcoholinduced performance impairment by prior ingestion of food antihistamine blockade of alcoholinduced flushing in orientals the application of minerals in managing alcohol hangover: a preliminary review aroma release of a model wine solution as influenced by the presence of non-volatile components. effect of commercial tannin extracts, polysaccharides and artificial saliva widespread occurrence of the mycotoxin fumonisin b2 in wine dental erosion: in vitro model of a wine assessor's erosion toxin-producing species of penicillium and the development of mycotoxins in must and homemade wine role of flavonoids and iron chelation in antioxidant action acute effects of chlorogenic acid on nitric oxide status, endothelial function, and blood pressure in healthy volunteers: a randomized trial alcohol consumption and risk for coronary heart disease in men with healthy lifestyles red wine and oenological extracts display antimicrobial effects in an oral bacteria biofilm model wine is bactericidal to foodborne pathogens the effect of various substances on the suppression of the bitterness of quinine-human gustatory sensation, binding and taste sensor studies red wine prevents the postrandial increase in plasma cholesterol oxidation products: a pilot study manuel practique de vinification et de conservation des vins the addicted brain ethanol causes neurogenic vasodilation by trpv1 activation and cgrp release in the trigeminovascular system of the guinea pig wine and migraine: compatibility or incompatibility? hemoglobinacetaldehyde adducts in human alcohol abusers alcoholic macrocytosis e is there a role for acetaldehyde and adducts? effect of wine-based marinades on the behavior of salmonella typhimurium and background flora in beef fillets seeking the connections: alcoholism and our genes perceived flavour changes in white wine after tasting blue mould cheese effects of tasting technique e sequential tasting vs. mixed tasting e on perception of dry white wine and blue mould cheese perceived flavour changes in blue mould cheese after tasting white wine wine: protective in macular degeneration wine ph prevails over buffering capacity of human saliva experimental and theoretical data on the mechanism by which red wine anthocyanins are transported through a human mkn-28 gastir cell model effects of grape seed-derived polyphenols on amyloid b-protein self-assembly and cytotoxicity ochratoxin a: the continuing enigma relationship between vasodilation capacity and phenolic content of spanish wines alcohol consumption and noncommunicable diseases: epidemiology and policy implications the stomach as a site for anthocyanins absorption from food fast access of some grape pigments to the brain identification of grape and wine allergens as an endochitinase 4, a lipid-transfer protein, and a thaumatin 5-hydroxytryptamine release from platelets by different red wines: implications for migraine prostacyclin, tyramine and red wine different effects of white and red wine on lower esophageal sphincter pressure and gastroesophageal reflux a comprehensive survey of the total sulfur dioxide concentrations of american wines an in vitro assessemt of the ole of tooth mousse in preventing wine erosion effectiveness of artichoke extract in preventing alcohol-induced hangovers: a randomized controlled trial crosscultural aspects of drinking, alcohol abuse and alcoholism red wine antioxidants. evaluation of their hydrophobicity and binding extent to salivary proteins substituent effects on in vitro antioxidizing properties, stability, and solubility of flavonoids epicatechin adducting with 5-hydroxymethylfurfural as an inhibitoury mechanism against acrylamide formation in maillard reactions is ethanol a pro-drug? acetaldehyde contribution to brain ethanol effects the relation between different dimensions of alcohol consumption and burden of disease: an overview wine alcohol, platelets and the french paradox for coronary heart disease alcohol and mortality from all causes effects of alcohol on platelet functions structure-antioxidant activity relationships of flavonoids and phenolic acids. free radic the effects of carbon dioxide in champagne on psychometric performance and blood-alcohol concentration review of moderate alcohol consumption and reduced risk of coronary heart disease: is the effect due to beer, wine, or spirits? melatonin: a new bioactive compound in wine inhibitory effects of plant phenols on the activity of selected enzymes potential food allergens in wine: doubleblind, placebocontrolled trial and basophil activation analysis immune response to acetaldehyde-human serum albumin adduct among healthy subjects related to alcohol intake low prevalence of the metabolic syndrome in wine drinkers e is it the alcohol beverage or the lifestyle? the structure of cuisine the use of characteristic flavourings in human culinary practice simultaneous and temporal contextual influences on food acceptance oleogustus: the unique taste of fat intra-individual and inter-individual variation in breath alcohol pharmacokinetics: the effect of food on absorption acetaldehyde as a common denominator and cumulative carcinogen in digestive tract cancers anthocyanin tissue bioavailability in animals: possible implication for human health. a systematic review flavonoids and the aging brain alcoholism: the health and social consequences of alcohol use moderate drinking in pregnancy proceedings of wine, health and society. a symposium. grt books sulfated and glucuronated trans-resveratrol metabolites regulate chemokines and sirtuin-1 expression in u-937 macrophages untersuchungen zur bedeutung toxischer stoffwechselprodukte des pilzes trichothecium roseum link ex fr. fü r den weinbau resveratrol and some glucosyl, glucosylacyl, and glucuronide derivitives reduce eschericia coli o157:h7, salmonella typhimurium, and listeria monocytogenes scott a adhesion to colonic epithelial cell lines sulfite oxidase deficiency plasma levels of caffeic acid and antioxidant status after red wine intake hangover headache: various manifestations and proposal for criteria. vågå study of headache epidemiology potential health impacts of excessive flavonoid intake. free radic wine as a biological fluid: history, production, and role in disease prevention ultrasensitive assay for three polyphenols (catechin, quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection alcohol consumption, mild cognitive impairment, and progression to dementia diversity of black aspergilli and mycotoxin risks in grape, wine and dried fruits doubleblind placebo-controlled trial of lithium in episodic cluster headache sleep and low doses of alcohol resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin induced diabetic rats effect of some phenolic compounds and beverages on pepsin activity during simulated gastric digestion intake of wine, beer, and spirits and the risk of clinical common cold anthocyanins are efficiently absorbed from the small intestine in rats iron is an essential cause of fishy aftertaste formation in wine and seafood pairing contribution to wine in vanadium dietary intake: geographical origin has a significant impact on wine vanadium levels adh3 genotype, alcohol intake, and breast cancer risk adh2 gene polymorphism are determinants of alcohol pharmacokinetics alcohol consumption and mortality among middleaged and elderly us adults red and white wines inhibit cholesterol oxidation induced by free radicals ellagic acid metabolism by human gut microbiota: consistent observation of three urolithin phenotypes in internention trials, independent of food source, age, and health status immunomodulatory and antitumor activities of grape seed proanthocyanidins is dopamine behind the health benefits of red wine? inhibition of quorum sensing (qs) in yersinia enterocolitica by an orange extract rich in glycosylated flavonones intake of beer, wine, and spirits and risk of stroke: the copenhagen city heart study amount and type of alcohol and risk of dementia: the copenhagen city heart study aspirin attenuation of alcohol-induced flushing and intoxication in oriental and occidental subjects effects of beer, wine, and liquor intakes on bone mineral density in older men and women alcohol consumption stimulates early steps in reverse cholesterol transport alcohol in the western world role of sulfite additives in wine induced asthma: single dose and cumulative dose studies changes in bronchial hyperresponsiveness following high-and low-sulphite wine challenges in wine-sensitive asthmatic patients antibacterial effect of phenolic compounds from different wines ochratoxin a in grapes and grapederived products the antimicrobial effect of wine on bacillus ceresus in simulated gastro-intestinal conditions nitric oxide radical scavenging by wines the alcohol hangover research group consensus statement on best practice in alcohol hangover research inhibitory effect of antioxidant-rich marinades on the formation of heterocyclic aromatic amines in pan-fried beef alcoholic calories, red wine consumption and breast cancer among premenopausal women effect of ethanol, tannin and fructose on the headspace concentration and potential sensory significance of odorants in a model wine flavorematrix interactions in wine low doses of alcohol substantially decrease glucose metabolism in the human brain a blend of polyphenolic compounds explains the stimulatory effect of red wine on human endothelial no synthase atherosclerotic cardiovascular disease and antioxidants older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease histamine in wine e bronchoconstriction after a double-blind placebocontrolled red wine provocation test supplementation with grape seed polyphenols results in increased urinary excretion of 3-hydroxyphenylpropionic acid, an important metabolite of proanthocyanidins in humans fetal alcohol spectrum disorders alcohol and medication interactions investigation of the allergenic potential of wines fined with various proteinogenic fining agents by elisa nutritional approach to cancer prevention with emphasis on vitamins, antioxidants, and carotenoids wine as a digestive aid: comparative antimicrobial effects of bismuth salicylate and red and white wine wine in context: nutrition, physiology, policy prostaglandin e2 (pge2) induces headache in healthy subjects effect of opuntia fiscus indica on symptoms of the alcohol hangover the alcohol hangover prolonged in vitro exposure to white wines enhances the erosive damage on human permanent teeth compared with red wines flavonoids: antioxidants or signaling molecules? free radic bioavailability and bioefficacy of polyphenols in humans. ii. review of 93 intervention studies alcohol and preoperative management deleterious effect of p-cresol on human colonic epithelial cells prevented by proanthocyanincontaining polyphenol extracts from fruits and proanthocyanin bacterial metabolites pharmacokinetics and blood-brain barrier penetration of (þ)-catechin and (e)-epicatechin in rats by microdialysis sampling coupled to high-performance liquid chromatography with chemiluminescence detection stability of dietary polyphenols under the cell culture conditions: avoiding erroneous conclusions resveratrol alleviates rheumatoid arthritis via reducing ros and inflammation, inhibiting mapk signaling pathways, and suppressing angiogenesis effects of phenolic acids on human phenolsulfotransferase in relation to their antioxidant activity flavonoid permeability across an in situ model of the bloodbrain barrier. free radic gods, men, and wine. the wine and food society ltd lead nephropathy and gout alcohol consumption and risk of breast cancer: the framingham study revisited association of alcohol intake with risk of diabetic retinopathy: a meta-analysis of observational studies measuring sensory perception in relation to consumer behavior on food and cooking e the science and lore of the kitchen molecular gastronomy: exploring the science of flavor flavor in food potential mechanisms by which polyphenol-rich grapes prevent obesity-mediated inflammation and metabolic diseases antibacterial, antiviral, and antifungal properties of wines and winery products in relation to their flavonoid content anthocyanins: from sources and bioavailability to cardiovascular-health benefits and molecular mechanisms of action wine and headache the effects of grape and red wine polyphenols on gut microbiotada systematic review wine consumption and renal diseases: new perspectives resveratrol as an anti-cancer agent: a review moderate alcohol consumption and the immune system. a review resveratrol and health e a comprehensive review of human clinical trials bioavailability of wine-derived phenolic compounds in humans: a review allergic and asthmatic reactions to alcoholic drinks effect of flavonoids on learning, memory and neurocognitive performance: relevance and potential implications for alzheimer's disease pathophysiology the alcohol hangover intracellular polyphenols: how little we know key: cord-010088-s9tfvtao authors: nan title: oral abstracts date: 2013-11-01 journal: vox sang doi: 10.1111/vox.12100_1 sha: doc_id: 10088 cord_uid: s9tfvtao nan tadokoro k and satake m japanese red cross society, tokyo, japan it is estimated that there are 2 billion hbv-infected people including 350 million hbv-carriers in the world. it is highly endemic in south africa, amazon, and southeast-central asia. the genotype of hbv is geographically characteristic, e.g. genotype b and c are the major in east asia. hbv transmission remains the most frequent transfusion-transmitted viral infection despite the implementation of various screening tests applied in different settings. the residual risk is mainly related to donations either in the pre-sero (or pre-dna)-conversion window period or occult hbv infection (obi) where blood test is hbv-dna-positive and hbs-ag-negative. infectivity of hbv depends on the transfused blood (viral load, phase of infection, genotype, and anti-hbs in the concurrent blood) and immune status of the recipients (anti-hbs, immunocompetence). it was shown that infectivity is dependent on viral load. allain et al reported that ffps is more infectious than pcs or rbcs. the minimal infectious dose of blood in late acute infection phase in chimpanzee and chimeric mice is approximately 10 times higher than that of pre-acute phase. satake et al reported that transmission rate of obi-derived components with low titer anti-hbc was 1/33(3%), whereas that of anti-hbc-negative components was 11/22(50%), which was verified in the lookback programme conducted in japan. allain et al showed in the study conducted in europe that adjusted transmission rate of obi blood was 28%, and the rate was higher without anti-hbs(63.8%) and lower with anti-hbs(15.4%). discrepancy of transmission rate of obi-derived blood between above two reports might be related to the different cutoff levels of anti-hbc and presence or absence of anti-hbs. dna-positivity rate among obi-derived components is higher in those with the higher levels of anti-hbc and lower in those with the presence of anti-hbs. there has been no report of transmission by obi-derived blood with anti-hbs of 200miu/ml or more. screening test for hbv is different between countries. low endemic countries screen blood for hbs-ag, anti-hbc and mini-pooled nat, while highly endemic countries test for hbs-ag without anti-hbc, because high prevalence of anti-hbc-positive donation hamper securing necessary blood. japan as a moderately endemic country had tested for hbs-ag, mini-pool nat and anti-hbc/anti-hbs where anti-hbs of more than 200 miu/ml irrespective of anti-hbc and low anti-hbc with agglutination-inhibition titer of no more than 2 5 is qualified. transfusion-transmitted hbv cases related to window period donations declined by increasing the sensitivity of mini pool nat, whereas those related to blood with low titer anti-hbc remained stable with around 10 cases annually. in order to decrease such transmission japanese red cross implemented a novel strategy to eliminate all anti-hbc-positive donations with anti-hbs <200 miu/ml. considering the frequency of donations with low titer anti-hbc has decreased to 1.3%, loss of those donations was estimated to be covered by promoting donations, each country should establish its own hbv screening strategy considering the prevalence of hbv, residual risk of transmission, balance between safety and securing blood, and cost-effectiveness. implementation of individual donation nat and universal vaccination could further reduce further the risk of hbv transmission. our blood service is to motivate other population groups to diversify the age and gender composition of our donors. forty-two percent of the whole blood donors participate in blood donation only once a year, so we need develop programs to motivate them to re-join blood donation. also, we have to solve the annually recurring problem of blood shortages for transfusion during the winter and summer season. in the long term, our donor base is going to decrease gradually because of the low birth rate in korea and our rapidly aging population. therefore we should prepare a sustainable solution for a stable blood supply. aims: present methods to recruit blood donors based on age, gender and occupation groups for a stable blood supply. methods: to make blood donation more accessible to individual donors, fixed donation sites are continuously developed. facilities of our fixed and mobile sites are improved to provide safe and comfortable environment to donors. since 1999, we have been operating the 'registered donor system' for registered donors who agreed to donate their blood on a regular basis. to raise awareness of the importance of blood donation among youth, high-school students blood donor groups called 'red campaigners' and groups of university students called 'blood donation supporters' are actively participating in blood donation campaigns. to increase participation of the middle-aged group, agreements were made not only with enterprises and organizations but also with the government and public institutions. we have developed a computerized system for scheduling group donations according to demand and supply and for performance management. to recognize the necessity of blood donation, every 13th was designated as 'blood donation day' in 2012. to deal with donor complaints and requests, a customer relationship management center has been established. results: by increasing the number of fixed donation sites and making donation more accessible, rate of individual donations is getting increased. as of the end of june 2013, 250 agreements for blood donation had been signed with enterprises and organizations. every year more than 100 thousand donors agree to donate on a regular basis, and so far about 600 thousand donors have been registered in the 'registered donor system'. the campaign 'every 13th is blood donation day' has contributed to spread a positive perception about blood donation. conclusions: we have been very successful in engaging youth in blood donation. diversification of the donor group will take time and constant effort. however, with more participation of female donors and retention of our first-time donors, it will be possible to be self-sufficient in blood supply for transfusion and plasma fractionation. the provision of sufficient safe blood products to patients requiring transfusion is the common goal of blood transfusion services and the public expectation of absolute safety continues to be a challenge. although the focus of blood safety falls on laboratory testing, the role of pre-donation donor selection cannot be underestimated. transfusion-transmitted infections (ttis) are blood-borne microbes that can be spread from blood donor to recipient via transfusion. to prevent tti, donated blood must go through validated laboratory testing. in most countries, blood is tested for hiv, hbv, hcv, and syphilis. screening for other ttis may also be implemented in some countries after individual assessment of the prevalence of the infection in their general populations, for example, west nile virus in the united states. with the advance in testing technology, in particular the nucleic acid test, the window periods for the detection of various ttis have been significantly shortened. however, the risk of window donation still exists. furthermore, there are known or emerging ttis such as the variant creutzfeldtjakcob disease (vcjd) where there is still no suitable testing system for routine screening of donated blood. therefore, blood transfusion services have to continue practicing effective pre-donation donor selection to mitigate the tti risks. who recommends selecting voluntary, non-remunerated donors from low-risk populations for blood collection as the first step in reducing the risk of ttis. donor selection is usually conducted by a health history questionnaire to be completed by potential donors and a confidential interview. the questions asked should be effective in assessing whether the respondent's health status is suitable to donate and there is no tti risk factor. people who are deferred should be counseled and given the reason for and duration of the deferral. who has published a document titled 'blood donor selection -guidelines on assessing donor suitability for blood donation'. it recommends the following: national donor selection guidelines and criteria should be based on epidemiological and/or scientific evidence or, where evidence is limited or lacking, on best practice. donor acceptance and deferral policies for the prevention of tti should be based on up-to-date information on the local epidemiology of infections, the markers screened for, the availability of suitable blood screening and confirmatory assays, and the technologies in use. national donor selection criteria should define conditions of acceptance and deferral for each criterion. adequate resources, including a sufficient number of qualified and trained staff, should be made available for the consistent and reliable assessment of donor suitability for blood donation. quality systems should be in place for donor selection, including selection criteria, staff training and documentation. blood transfusion services should establish mechanisms for monitoring and evaluation to assess the implementation and effectiveness of donor selection criteria. in conclusion, pre-donation selection is essential to protect the safety and sufficiency of blood supply, and safeguard the health of recipients and donors. background: blood components may be contaminated by a variety of commensal, pathogenic or environmental bacteria during collection, manufacture or storage. the outcome of transfusion is dependent on the ability of the specific strain to multiply to clinically-relevant titers during storage, the pathogenicity of the strain and the patients' situation. platelets in particular are stored in conditions conducive to bacterial growth and septic reactions to these products are the most frequently documented infectious risk of transfusion. aim: the objective of this update is to review estimates of risk of bacterial sepsis and contamination of platelets, and recent findings describing interventions designed to safeguard patients. methods: this update will use recent reports and unpublished data to describe our current understanding of the role of bacteria in transfusion safety. results: historical data suggests that~1:1-3000 platelet products are contaminated with bacteria, and septic transfusion reactions occurred in 1:15-100,000 transfusions. clinical awareness of the danger and a 2004 aabb standard to 'limit and detect bacteria' in platelet products have driven the development of safety systems. the last decade has seen substantial progress in the implementation of optimal skin disinfection techniques and sample diversion strategies to reduce contamination, and many centers implemented either bacterial culture testing or pathogen inactivation processes to reduce the risk. culture testing can reduce but cannot eliminate the risk of exposure to contaminated components and sepsis. the majority of contaminated products do not cause adverse reactions, however, at the time of collection and manufacture the only means to prevent serious and fatal reactions is to ensure that that the component is functionally sterile. pathogen inactivation technologies show variable efficacy at killing bacteria, suggesting a need for strict adherence to the manufacturers suggested protocols to ensure optimum performance. alternatively, assays performed on the day of transfusion prevent the transfusion of high concentrations of bacteria that are associated with the most severe adverse reactions. conclusions: bacterial contamination and sepsis remain the greatest infectious risks of transfusion. enhanced testing or pathogen inactivation should be implemented to ensure patient safety. the australian red cross blood service, sydney, australia background: pathogen reduction technologies have been developed as a means of reducing the risk of transfusion transmission of blood-borne pathogens. published literature indicates that systems currently in use or under development effectively inactivate a range of pathogens in platelets and plasma, with the exception of some non-lipid enveloped viruses, bacterial spores and prions. development of pathogen reduction technology (prt) is ongoing, and many countries have adopted prt as part of their routine blood component processing. the aim of this update will be to review technologies currently in use or under development for treatment of labile blood components during processing. the impact of prt on blood component quality as well as ongoing challenges will be reviewed. platelets: there are currently three systems for prt treatment of platelets. these are the mirasol tm system (terumobct), the intercept blood system tm (cerus corporation) and the theraflex uvc tm system (macopharma). the intercept and mirasol systems are used widely in blood centres in europe, asia and the middle east for the treatment of both platelets and plasma, whereas clinical trials of platelets treated using the theraflex uvc system are ongoing. in vitro data suggests prt treatment leads to some loss of platelet function. however, haemovigilance reports from sev-eral countries where intercept treated platelets are transfused indicate no change in component usage, and indeed a reduction in transfusion related adverse events. similarly, there have been no reports to date of serious adverse events relating to clinical use of mirasol-treated platelets. plasma: the intercept and mirasol systems can be used to treat both plasma and platelets, and the theraflex system utilises methylene blue (mb) with visible light for prt treatment of plasma. there are some losses of coagulation factors following treatment with each of these systems. theraflex mb plasma has been transfused world-wide since 1999, and haemovigilance data indicates there is not a higher incidence of allergic reactions or other adverse events with mb-treated plasma. red cells/whole blood: commercial prt systems for red cell or whole blood components are under development. cerus corporation has continued development of a second-generation s-303 system for red cells, demonstrating in vivo recovery after 35 days storage. a mirasol prt system for treatment of whole blood is also being developed by terumobct. preliminary in vitro quality and in vivo recovery and survival data indicate that this technology may eventually become available, but further development and clinical trials are still required. challenges: concerns still exist regarding long term effects on patients receiving prt treated blood components, particularly the potential toxic effects of residual products following photochemical treatment. ongoing post-marketing surveillance and clinical trials are required to address these concerns. ideally, prt should provide proactive protection against emerging pathogens and reduce the need to introduce additional pathogen testing, minimise bacterial contamination and potentially replace processing steps such as gamma irradiation. the challenge for blood services is to understand and rationalise the costs, risks and benefits of prt compared to removing any of these tests or processes, whilst aiming to provide the highest level of blood safety. steps in getting a paper published/abstract accepted 1b-h4-01 no abstract available. 1b-h4-02 how to get your abstract accepted and how to present it daniels g many abstracts are submitted to the isbt for presentation at international and regional congresses. all abstracts are refereed by a large panel of international experts, who decide which abstracts should be accepted and which ones should be rejected. they also decide which of the accepted should be presented orally or as a poster. most of the submitted abstracts are accepted, though there is plenty of room for improvement in the quality of many of those abstracts. in this session i will describe why some abstracts are rejected and discuss how you might avoid this pitfall. i will also discuss some methods for improving your abstracts so that they represent the quality of the work you are describing and enhance the reputation of yourself and your institution. once your abstract has been accepted you will have to present it at the congress. if it is accepted for poster presentation, you will have to make a poster. the easiest way to produce a poster is to design it on a single powerpoint slide and then send the file to a company that will turn it into a poster on paper, or even on cloth for easier transportation. if your budget does not run to that, there are cheaper ways, such as printing it on single pages of a4 with the text printed in a very large font. the best posters do not have too much written information (bullet points are often best), contain diagrams and/or pictures, and are colourful and visually attractive. if, however, your abstract is accepted for oral presentation, then you will have to give a talk, usually of 10 min, and then be prepared to answer questions. this may be a daunting prospect, especially if you are not experienced in public speaking, and particularly if english is not your first language. i will discuss techniques for improving your presentation skills, both from the points of your spoken presentation and your visual aids, which will usually be a powerpoint presentation. if you are thoroughly prepared before you stand up in front of an audience, you will feel more confident and, consequently, less nervous. devine d publication of research findings and novel concepts in the biomedical literature is the mainstay of knowledge mobilization. the communication of scientific work as papers follows a well-established framework. a clear understanding of the nature of this framework and how to assess one's own work against it is critical to successful acceptance and subsequent publication of manuscripts. in this session, we will review the standard framework for scientific communication. communicating your findings is a form of scientific story telling. one must clearly explain why it was important to write the paper so that the reader will be interested and want to keep reading it. generally, an author must capture the interest of a potential reader right at the abstract which is sometimes the only way a reader sees the paper if they have discovered the work using a search engine such as medline. in the main body of the paper, the author must clearly explain how the study was designed and carried out with careful attention paid to any important details and to the statistical analysis, if appropriate. results must be presented in a manner that is clear and easily interpreted by the reader. then the work should be discussed in the context of other work in the area, emphasizing the novel findings. in this session, we will also address the following questions: how do i know if i have enough information to write a paper? where should i try to publish the paper? how should my paper be put together to give it the best chance of being accepted? what happens to my paper after i submit it? if it is returned to me with comments from the reviewers, what should i do? what is my next step if my paper is rejected, either without review or with review? we will also consider how to prepare papers in a language other than one's native language. the various sorts of scientific communications (original papers, review articles, reports, letters to the editor) will be discussed. the session is intended to provide general guidance for the publication of papers in the biomedical literature rather than be specifically focused on publication in the society's journal vox sanguinis. the membership survey commissioned by the isbt central office in 2012 gave a good insight into what members and non-members expect from the society. the main message was that members and potential members would like more educational and training resources to be available. many respondents requested that isbt should write international guidelines and develop standards. developing international guidelines when so many are already available and when countries and regions have different requirements would be a titanic task. the isbt board decided that the society should put together a repository (library) of guidelines, standards and regulatory documents that are already currently available. the repository is now ready and launched during the 24th regional congress of the isbt in kuala lumpur. for practical purposes the repository contains recent guidelines in the english language that are freely available, however following consultation with experts some manuals are included that are either relatively old or are not yet freely available on line. approximately 250 documents are in the repository from around 25 countries. the repository is a work in progress and further documents will be added in other languages. the repository is available via a link on the isbt home page www.isbtweb.org to the isbt academy e-learning portal. it can be accessed by country and by subject and a search facility is available. the guidelines are organised by six main subjects; donor, clinical, laboratory, quality/haemovigilance, processing and regulatory. there are sub topics for each main subject. isbt anticipates that you will find the repository a useful resource. background: blood services in africa operate at different levels of development from those comparable to the first world to those operating at a basic level with just hospital based blood banks and no national coordination. in recent years, countries have made efforts to improve their blood services based on voluntary non-remunerated blood donation. major challenges include low knowledge levels, inadequate funding, weak regulatory framework and poor quality systems. many international standards are too stringent for most economies in sub-saharan africa. to address these challenges, the africa society for blood transfusion (afsbt) started a program to develop blood transfusion standards relevant to africa. the afsbt step-wise accreditation standards: these standards were drafted by an afsbt task team for accreditation with guidance from the aabb and input from a team of experts. they were initially based on the who aide memoire for blood safety. an evidence-based decision-making process, where possible, was used to modify existing requirements or create new specific requirements. the goal of the standards is to provide a benchmark for accreditation of facilities and to maintain and enhance the quality and safety of blood transfusion in africa. the development process started in 2009. there is one standard with 3 progressively more rigorous steps of achievement as follows: level 1: minimum quality and operational requirements level 2: intermediate quality and operational requirements level 3: full accreditation at international standard a facility chooses the level to be assessed at. however accreditation at any grade is attained if facilities meet standards of the grade but also comply with specific requirements in section 11 which deals with legal and regulatory requirements, blood supply, equipment and supplies and clinical use of blood and blood products. application of the standards: they apply to facilities that perform any or all of the following functions: mobilization, recruitment and selection of blood donors; blood collection; components preparation; blood group and serological testing; compatibility testing; storage, handling, transportation and distribution of blood products. requirements do not apply in cases where the blood transfusion service is not responsible for an activity e.g compatibility testing. guidance document: there is a guidance document which clarifies and enhances understanding of the requirements of some of the standards. other standards are straight forward and do not need guidance. the guidance document is being updated as queries are received from the field. training: to facilitate meeting the requirements of the standards, there is a training committee, tasked with supporting training for blood services. a full training program is currently being developed. piloting: the standards were piloted in namibia and malawi. the namibia piloting was completed in 2012 while the malawi piloting will be completed in 2013. comments from these pilot sites have provided valuable input into the standards development process. conclusion: stepwise accreditation program is relevant in encouraging improvements for developing blood transfusion services. accreditation standards need to be commensurate with local needs. training is very important in helping developing blood transfusion services attain accreditation status. blood transfusion has become an integral part of modern healthcare. when it is required, it is an essential element of therapy, helps safe lives and improves quality of life. however, it is not without risk. being of human tissue origin, this risk is related to its source as well as the process involved in its provision. to ensure that blood transfusion is safe and does not cause harm to patients, these risks have to be monitored, evaluated and managed appropriately. therefore it is essential to develop system of ensuring safe blood supply and safe transfusion. quality systems should be developed covering the whole transfusion chain. policies, standards and guidelines are important tools. to ensure that the system is effective, there are various mechanisms to monitor, evaluate and analyse in order to bring about improvement. these include developing indicators, quality and clinical or transfusion audits, quality assessment programmes and haemovigilance programme. indicators can be used to monitor transfusion practice such as prevalence of transfusion transmitted disease among blood donors, crossmatch transfusion ratio, expiry fate of blood components and transfusion error. these indicators not only measure safety but also efficiency of the blood service. in countries where the blood supply is not consistent, the rate of blood requirement not met is an important indicator to monitor. performance of laboratories providing blood can be measured using quality assessment programmes. this is an important element that links the source of blood to the patient. audits covering all processes and procedures ensure that the quality and safety of blood is maintained. while these processes and procedures are controlled in ensuring safety and quality of the blood supply, the process involved in the transfusion process is sometimes not controlled by documented procedures and poorly supervised. auditing blood usage which is more complex and laborious provides an important mechanism for ensuring that this precious national resource is utilised efficiently. guidelines whether national or international are required against which these audits are performed. transfusion audits looks at the quality of care of patients besides the use of blood. it analyses the process of diagnosis, the decision making in treatment and the use of available resources. over the last two decades, haemovigilance has evolved and expanded worldwide. it focuses on adverse events that occurred throughout the transfusion chain, analysis of the facts and provide an avenue for corrective actions to be taken to prevent further recurrence. initially, its focus was more on adverse events that occurred to patients, it is now used to monitor adverse events relating to blood donors and blood donating process. in resource limited economies these tools can be used effectively when a stepwise approach is adopted. the aim is to create a culture of professionalism in delivering quality of care to patient with efficient use of available resources. however, its benefit can be extended to influence change and improvement in healthcare in general. only when deficiencies are demonstrated that request for resources can be shown to be justified. serological methods have the advantage of being fast, simple, and determining the expression of antigens directly. however, genotyping and molecular diagnostic methods have their advantages in determining subtypes and variants, as well as in detecting other rare blood groups. commercial blood group genotyping kits have been widely used, not only in clinical blood banks but also in transfusion services. these commercial kits mainly aim to analyze coding genes of abo and rh blood group systems. common alleles found in local populations are included in these kits. at present, many kinds of molecular diagnostic methods are employed in detecting blood group alleles by transfusion laboratories in china. these methods include multiplex pcr, multiplex pcr combined with pool system, sequencing, and gene chip technology. considering the genetic background of several rare phenotypes with clinical importance in chinese persons, one multiplex pcr system was developed to detect fy a , s, and ok a antigens, and the other system was developed to detect di b , k, and js b antigens. using the existing multiplex pcr-ssp assays, only one sample can be detected in a single pcr tube because the primers used in these assays are specifically devised for corresponding high-frequency alleles, and aim to screen for the negative results. therefore, the positive results would mask the negative results by adding more samples in one pcr tube. to improve the efficiency of screening, recently we established a novel method combining the multiplex pcr-ssp assays with the dna pooling strategy. the primers were designed to amplify the corresponding low-frequency snp sites. in each multiplex pcr-ssp assay, every dna pool is tested for the presence of the low-frequency snp sites. a pool is released for further processing when the positive results were found in any site. after then, each individual dna sample of the positive pool was detected respectively to determine the positive sample. finally, pcr-ssp methods were used to determine whether the positive result was caused by homozygous or heterozygous alleles. normally, the homozygous low-frequency alleles would lead to rare phenotypes. at the same time, the control system based on site-directed mutagenesis solves the problem of the lack of experiment controls due to unavailable negative or positive dna control samples. with large scale screening of population samples and diagnosis of various clinical special samples, the relationship between blood group coding genes and the expression of proteins is being clear. the frequency of multiple blood group alleles has also been investigated. the specific molecular events found in asians and chinese populations are revealing the difference among races and regions, as well as the expression diversity of blood group alleles. 1c-h6-02 diagnosis and treatment of autoimmune haemolytic anaemia autoimmune haemolytic anaemia (aiha) occurs as a result of antibodies directed against self-red blood cell (rbc) antigens, leading to the premature destruction of rbc. rbc destruction may occur within the vasculature, often mediated by the membrane-lysing complex, which is generated upon complement activation or occur extravascularly, within the reticulo-endothelial system that expresses fcî¥r and c3 receptors, that bind antibody and complement coated rbc. the laboratory hallmark of aiha is a positive direct antiglobulin test (dat) although it has to be recognized that occasional cases of dat-negative aiha may occur. secondary causes for aiha such as an underlying autoimmune disorder, infections or malignancies should be considered as part of the investigation process for aiha. immunohaematology investigations for aiha should always include a dat using monospecific anti-igg and anti-c3d. on occasions, further testing with anti-iga or -igg subtypes may be necessary. while the dat provides information on bound rbc antibodies, the indirect antiglobulin test (iat) using screening and antibody identification cells will provide information on the specificity of circulating antibodies. often this will give a pan-reactive pattern although it is not unusual to identify coincident autoantibodies with defined red cell specificity. negative iat with screening and identification cells in the presence of a positive dat should raise suspicion of drug-induced aiha. in patients who may have been potentially alloimmunized, further procedures should be performed to exclude the coincident presence of alloantibodies as failure to recognize alloantibodies may result in an immediate or delayed haemolytic transfusion reaction if red cell transfusions are initiated. elution and auto-or alloadsorbtion techniques are useful to separate allo-and autoantibodies. complete red cell phenotyping should be concurrently performed to aid in identifying antibody specificities. heavily antibody-coated red cells may be difficult to phenotype with some anti-sera, in which case, molecular based red cell genotyping should be initiated. molecular based red cell typing is also particularly useful where the patient has recently been transfused and the initial red cell phenotype of the patient is not known. the primary management of the patient with aiha is amelioration of the underlying condition and suppression of immune mediated haemolysis. this is usually achieved with administration of steroids or immunosuppressive agents. splenectomy may be an effective second line treatment. rituximab is increasingly becoming a promising treatment option in patients who are steroid-refractory and not suitable for splenectomy. other treatment modalities for the refractory patient include intravenous immunoglobulin and danazol. red cell transfusions in patients with aiha should be undertaken carefully although they should never be denied blood transfusions because of inability to find compatible units. as far as possible, phenotype matched red cells, cross-match compatible with the patient's autoadsorbed serum should be transfused. if coincident alloantibodies are identified, antigen-negative red cells will need to be selected. clinical immunology and transfusion medicine, university & regional laboratories, lund, sweden what is the value of discussing case studies? they provide us with a forum for sharing our combined experience and permit the development of ideas and techniques as well as the possibility to see a given situation from another perspective. cases that illustrate the following will be discussed: (1) the presence of polyagglutination in a patient with a bacterial infection in europe and the usa, polyagglutination is rarely seen since monoclonal blood grouping reagents are the norm; however across asia, a broad spectrum of blood typing reagents are used and polyagglutination maybe encountered. (2) investigation of an antibody to a low-prevalence antigen in a case of haemolytic disease of the foetus and newborn antibodies to low-prevalence antigens occur not infrequently. how can they be investigated and what should be considered? (3) antibodies to a high-prevalence antigen the discussion will consider how to resolve such a case in laboratories with different levels of resources. what should be considered in different clinical situations? the goal is provide useful tips for investigation of difficult serological cases and information on how to proceed when the investigation is beyond the resources of the laboratory. haemovigilance is an important element of blood safety. it aims to identify, monitor and prevent adverse reactions, incidents and adverse events related to transfusion for both donors and patients (from 'vein to vein'). various local, regional and national haemovigilance models exist, which reflect the range of health systems and blood systems in different countries; for example, some haemovigilance systems are coordinated by professional bodies, some by blood suppliers, and some by health authorities (health departments or regulators). participation may be voluntary or mandatory, and may differ depending on whether all events or only serious ones are reportable. some systems capture events with all levels of imputability, whereas others record only those which are confirmed or highly probable. 'near miss' events are captured by some systems, and many valuable lessons can be learned from these cases. from an early stage of haemovigilance reporting it has been identified that processrelated problems are a major cause of serious transfusion complications. these include 'incorrect blood component transfused' events, where the blood component was intended for another recipient (frequently due to errors in patient identification at the time of collection of the pre-transfusion sample, or at the time of bedside administration), or did not meet the patient's special needs (such as a patient with a red cell antibody who did not receive the required antigen-negative unit). human and system factors, such as lack of awareness or training, working environment, interruptions and inadequate communications between clinical teams, or between the clinical teams and the transfusion laboratory, are very important contributors to these events. investigation of transfusion reactions, incidents and events at the hospital level is essential to identify clinical consequences, contributing factors and to develop and implement plans to prevent recurrence. reportable incidents should be notified to the haemovigilance programme. transfusion safety officers, transfusion nurses and similar roles have been introduced in many countries and they play important roles in haemovigilance, especially at the hospital level. adequate medical and transfusion laboratory support for hospital haemovigilance activities is also essential for success. hospital transfusion committees should oversee haemovigilance activities and reporting, and should ensure that hospital senior management is aware of and responds to serious reactions and events, especially where systems issues are identified to be contributory. at an international level, isbt's working party on haemovigilance brings together isbt members with an interest in haemovigilance. isbt works closely with ihn, an international collaboration of regional or national haemovigilance programmes, and other partners. ihn operates the istare database for international data sharing and benchmarking. haemovigilance reporting can identify priority areas for action (either where the events have serious clinical consequences, and/or occur frequently) and can help identify and monitor the implementation of solutions. an important feature of haemovigilance programs is the sharing of experiences and results. haemovigilance reports can both provide valuable feedback to clinical teams and hospitals locally, as well as share experiences nationally and internationally to improve patient outcomes. wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: haemovigilance systems capture data on adverse reactions and infections in recipients of blood transfusions as well as on errors and incidents in the transfusion chain. the objective is to analyse them and make recommendations for improving transfusion safety. many haemovigilance systems also collect data on complications in blood donors with a view to monitoring and improving blood donor safety. standardised definitions are necessary for classifying and comparing data in all these domains and at all levels. method: since 2004, at international meetings of blood transfusion professionals, members the international haemovigilance network (ihn) and the haemovigilance working party of the international society for blood transfusion (isbt) have collaborated in developing and validating definitions for non-infectious transfusion complications, errors and incidents in the transfusion chain as well as adverse reactions in blood donors. from 2012 contacts with other groups including the who have been put in place to ensure wide consultation as well as awareness and use of the definitions. results: standardised definitions have been published for recipient adverse reactions, for complications of blood donation and for a limited number of types of incident in the transfusion chain. the isbt haemovigilance working party and ihn are committed to ensuring that the definitions remain up-to-date and that revisions and improvements are conducted with wide consultation of professionals in relevant organisations worldwide. the haemovigilance working party and working party on transfusion-transmitted infections are collaborating on the development of definitions and criteria for assessing suspected transfusion-transmitted infections. conclusion: internationally agreed definitions are available for registration and surveillance of complications of blood donation and most types of adverse reaction in patients receiving blood transfusions. for errors and incidents in the transfusion chain, further work is necessary to improve comparability of data between hemovigilance systems. 1d-h7-03 distler pb and ashford p critical to patient safety is the capability of rapidly tracing a medical product of human origin (mpho) from donor to recipient and vice versa. traceability requires that each product be uniquely identified in order to provide a clear, unambiguous path. historically, uniqueness was defined only in the context of a single organization. for example, a blood product identifier was unique only to the blood bank that assigned it. because some medical products of human origin (mpho), especially cells and tissues, are frequently distributed across international borders, it is becoming increasingly important that identifiers of mpho need to be unique not only within an organization, but globally as well. in 2010, the world health assembly urged member states 'to encourage the implementation of globally consistent coding systems for human cells, tissues, and organs as such in order to facilitate national and international traceability of materials of human origin for transplantation.' more recently who has recognized the need for common strategies for global governance of all mhpo, including the global use of isbt 128, to ensure unique identification, optimal traceability, and interoperability between countries and across all mpho for both routine and emergency use. this requires a globally consistent coding system that can provide: a mechanism to allow distinct items to be uniquely identified and consistently characterized to all participants within the system, the means to allocate identifiers in a manner that avoids duplication, and the information infrastructure on which effective traceability can be built. isbt 128 is an international terminology, coding, and labelling system that supports the assignment of unique identifiers to support global traceability of mpho. it is currently in use by many blood banks, tissue banks, and cellular therapy facilities around the world and the who global forum on blood safety has recognized promotion of the use of isbt 128 as a priority for action in improving quality management and haemovigilance. 1d-h8-01 the university of tokyo, tokyo, japan antibodies directed to human platelet antigens (hpa) play important roles in the pathogenesis of neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). presently, six hpa biallelic systems, namely hpa-1 to -5 and -15, which are involved in immune mediated thrombocytopenia, are characterized. there are important ethnic differences in the frequency distribution of these hpa systems, the incompatibility of the hpa-1 system being the mostly involved in thrombocytopenic conditions in caucasian, whereas in japanese, the hpa-4 system is the mostly involved. the frequency distribution of hpa systems reported in other parts of asia seems to be different from caucasian as well as japanese, especially related to hpa-1 and -4, respectively. in addition to hpa, antibodies to human leukocyte antigen (hla), blood group abo, and human neutrophil antigens (hna) have also been shown to be involved in immune mediated thrombocytopenia. in fact, the majority of the cases of ptr are dependent on anti-hla antibodies, and anti-hpa antibodies comprise only a small proportion. the identification of the causative antibody is very important for the implementation of preventive/therapeutic measures for ptr, such as the selection of hla-and/or hpa-compatible platelets. on the other hand, the involvement of anti-hla antibodies in the pathogenesis of nait is questioned, but cases in which the causative antibody cannot be determined still remain relatively high. thus, for the implementation of preventive and therapeutic measures for the immune mediated thrombocytopenia, the detection and identification of the causative antibody is essential. the standard methods applied varies among the regions, the monoclonal antibody-specific immobilization of platelet antigens (maipa) and the platelet immunofluorescence test (pfit) being the preferred methods in the us and europe, whereas in japan, the mixed-passive hemagglutination is the most applied one. neither of the methods alone, however, is able to detect all the clinically significant antibodies, thus, improvement of the available methods as well as the development of new technologies is required. considering the ethnical differences of the hpa frequency distribution, we considered important to develop the research of this field also in asia, and for this purpose, the isbt platelet immunobiology working party, asia regional (isbt piwp-ar) was launched in 2010, during the xxxist international congress of the isbt in berlin, which aims the sharing of knowledge and improvement of technology in asia. a training course on platelet immunology methods and genotyping was provided in tokyo in 2010, and the first workshop of the piwp-ar was organized in taipei in 2011. in may 2013, the second training course on platelet immunology methods and genotyping was organized in guangzhou, china, and the 2nd workshop of the piwp-ar is going to be held in kuala lumpur, malaysia, during the 24th regional congress of the isbt. the presently available methods for the antigen/antibody testing, the problems related with antibody detection, and the activities of the platelet working party will be presented. nanning institute of transfusion medicine, nanning, china background: immunization against human platelet antigens (hpa) is associated with a number of clinical syndromes, including neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr), post-transfusion purpura (ptp), and other platelet immune disorders. the detection and identification of the clinical relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia. aims: the aims of this study is to investigate the characteristic of the detection of clinical relevant platelet antibodies in the asian population, and to evaluate the ability for the detection and identification of platelet antibodies in the platelet immunology laboratories in asian countries. methods: total of 378 cases that were diagnosed and studied as naitp (70), ptr (305) and ptp (3) in asian platelet immunology laboratories were reviewed. of the 378 cases, 167 were japanese in japan, 142 were chinese in china, 30 were in india, 24 were chinese in taiwan, china, 5 were in south korea, 4 were in thailand, 3 were in kuwait, and 1 case each for oman, lebanese and palestinian. the specificities of platelet antibodies in these cases were investigated and compared with the data from western country's laboratories. the methodology of detection and identification of antiplatelet antibodies in asian labs were also reviewed. results: among 378 cases, the immune thrombocytopenia associated antiplatelet antibodies were two cases of anti-hpa-1(in kuwait), 2 of anti-hpa-2 (1 in china and 1 in japan), 5 of anti-hpa-3 (three in japan, one in china and one in taiwan, china), 5 of anti-hpa-4 (all in japan), 4 of anti-hpa-5 (1 in china and 3 in japan), 1 of anti-hpa-7new(antihit a )(in japan), 1 of anti-hpa-15 (in japan), 2 of anti-hpa-21bw (in japan), 309 of anti-hla(101 in china, 147 in japan, 5 in korea,3 in thailand, 23 in taiwan, china and 30 in india), 1 of anti-a (in japan) as well as 15 cases of anti-cd36(nak a ) isoantibody (eight in china, three in japan and one case each in thailand, oman, lebanese and palestinian). thirty one cases of antibodies could not find the specificities (30 in china and 1 in kuwait). the methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (maipa), mixed passive hemagglutination (mpha) assay, platelet immunofluorescence test (pift), modified antigen capture elisa (mace), and solid phase red cell adherence (spaa) have been used in asian laboratories summary/conclusions: platelet alloantibodies are found with variable frequencies in different ethnic groups in asian population. anti-hpa-4 is mainly found in japanese individuals, while anti-cd36 (nak a ) isoantibody that occurred in cd36 type i deficient individuals is most frequent found in asian population especially in chinese population. only two cases of anti-hpa-1 antibodies were found in asian population (all in kuwait), while anti-hpa-1 is the most frequent antibody associated with severe complications in caucasian populations. however, anti-cd36 isoantibody is of a risk factor of immune thrombocytopenia in asian population, as the incidence of cd36 deficiency is 3-11%. the human platelet antigens (hpa) are a group of polymorphic antigens, expressed relatively specific on platelets, capable of eliciting an immunological response with development of alloantibodies. hpa directed alloantibodies have been implicated in neonatal alloimmune thrombocytopenia (nait), post-transfusion purpura and refractoriness to platelet transfusions. twenty-seven hpa are recognized, of which antibodies to both the given alloantigen and the antithetical alloantigen has been reported for hpa-1 to 5 and hpa-15. the remaining hpa are designated with a 'w' as an alloantibody to the antithetical antigen has yet to be reported. most hpa polymorphisms are a result of a mis-sense single nucleotide alteration and are readily detectable using standard molecular typing methods. dna based population wide genotyping have revealed considerable variation in hpa allele frequencies among different ethnic groups. the 'b' forms of hpa-1, -2, -3, and -5 are common among caucasians while hpa-4b is extremely rare. in the chinese however, hpa-1b is extremely rare while uncommon in malays. hpa-4b and hpa-21bw meanwhile are more commonly seen among asians compared to caucasians. consequent to this, alloanti-hpa-4b is the most common cause for nait in the asian population as compared to alloanti-hpa-1a among caucasians. several cases of nait due to anti-hpa-21bw have also been reported in asia. in contrast, nait due to anti-hpa-6b is rare despite the alloantigen being more common among asians. although platelet transfusion refractoriness is more commonly associated with hla antibodies, anti-hpa antibodies have been implicated in some cases. management of nait and platelet transfusion refractoriness include the supply of antigen negative platelet units. a platelet donor registry with a critical mass of hla and hpa typed blood donors is therefore necessary for effective management of these conditions. ready availability of low-cost high-throughput snp genotyping platforms allow for establishment of large hpa genotyped donor pools. knowledge of hpa genotype prevalence in the local population as well as its implications on nait and platelet refractoriness is however crucial before deciding on donor screening strategies, careful considerations would also need to be made with regards to the cost-effectiveness of such ventures in light of alternative management strategies and local incidence rates of nait. clinical -improving patient outcomes 2a-s01-01 setting up a patient blood management programme wood e 1 , engelbrecht s 1,2 and robinson k 2 1 transfusion research unit, monash university, melbourne, australia 2 australian red cross blood service, adelaide, australia patient blood management (pbm) aims to minimise unnecessary transfusions, and also to ensure that if transfusion is required it is managed appropriatelyby individualising care so that patients receive what they need when they need it. pbm is comprehensive and patient-centred, with active participation by patients and a multidisciplinary approach from the hospital team to achieving these aims. 'three pillars' of pbm have been suggested, to optimise a patient's red cell mass, reduce bleeding and improve tolerance of anaemia. in the perioperative setting, important elements of pbm include attention to medical, surgical and anaesthetic interventions and techniques to improve haemostasis and reduce blood loss. where significant intraoperative blood loss is anticipated, use of cell salvage techniques can be very valuable. pbm concepts also apply outside the perioperative setting, and the broad principles can be applied to a wide range of clinical settings, including obstetrics, trauma, critical care and haematology/oncology and other medical settings (e.g. gastroenterology). an effective hospital pbm programme requires planning and communication, with a stepby-step approach to implementation, identifying priority areas for action, engagement, education and training of staff, and on-going monitoring against plans to demonstrate progress and identify areas for improvement. feedback to staff on progress provides a sense of achievement and helps engagement. adequate resources are required, including from medical, nursing and laboratory staff from a range of disciplines who have important contributions to make in clinical practice, education and training, and audit and review. minimisation of unnecessary transfusions saves money for hospitals and the community, and other resources such as staff time, and therefore offsets the costs of establishing and maintaining a pbm programme. effective implementation requires change at individual and organizational levels, and therefore support of hospital executive management, local health authorities and the blood supplier are also very valuable. oversight of the program can be by the hospital transfusion committee or a pbm programme committee, but the particular structure and governance arrangements should be developed to suit local needs. general practitioners can play key roles in preparing patients for surgery by identification and management of anaemia, as well as other pbm activities. ultimately an effective pbm programme can optimise care and outcomes for patients, make better use of limited and precious blood supplies, and reduce risks and costs. trauma is a leading cause of death around the world, with haemorrhage accounting for more than a third of the preventable mortality, with the majority of these deaths occurring within the initial 24 h. massive blood transfusion is generally defined as the replacement bytransfusion of more than 10 units of red cells over 24 h. massive transfusionprotocols (mtps) have evolved over the past decade and are especially importantin facilitating the early delivery of copious amounts of blood products topatients who have major injuries and severe haemorrhage. various studies havealso demonstrated that with mtps, there is a more efficient use and lesswastage of blood products. however, for trauma patients, stopping thehaemorrhage and resuscitating the patient does not only involve the expedientdelivery of red cells to the injured patient. mtps have also emphasized theneed for a more balanced ratio of delivery of blood products, includingplatelets and plasma, to patients who sustain massive blood loss and havedeveloped acute traumatic coagulopathy (atc). often times, mtps also stress theimportance of the consideration of use of haemostatic adjuncts, such astranexamic acid, activated factor vii, level one transfusion units and the useof blood warmers to reverse the potential effects of hypothermia with sustainedtransfusions of large amounts of blood products. ultimately, in tandem withdamage control resuscitation, which allows permissive hypotension whilstsecuring haemostasis, mtps have been shown to also improve survival of theseseverely injured patients. a more recent paper describes the development of amassive haemorrhage protocol to aid in the identification of patients who wouldbenefit from the mtp and this may be the next step in the evolution of a workprocess by which resuscitation for severely injured patients may be optimized. background: rh blood group antigens are highly immunogenic and transfusion of rh d positive blood in rh d negative recipients is avoided. platelets do not express rh antigens, however they may contain significant amount of contaminating red cells to illicit an immune response in the patient. due to limited shelf life of platelets and inventory issues, rh d positive platelets which are not visibly contaminated with red blood cells maybe transfused to rh d negative patients including children and females of child bearing age. there has been increased focus on whether rh immunoglobulin should be routinely administered after such transfusions. in saudi arabia no clear guidelines exist on the administration of rh immunoglobulin after d positive transfusions in d negative patients. aim: the purpose of this study was to determine whether the transfusion of rh d positive platelets to patients who are rh d negative, results in d alloimmunization and whether rh immunoglobulin should be routinely administered in such patients. methods: eligibility criteria for inclusion in the study included the following: transfusion of rh d positive platelets, no anti d detectable before transfusion, no previous exposure to rh d positive blood components, and results of follow-up testing of anti-d in patients serum available. the patients blood group and antibody screening was done using liss-iat gel technology (diamed). results: one hundred rh d negative patients who received rh d positive blood components were identified. out of this 63 (63%) patients received rh d positive platelet transfusions. in 47 (74%) patients out of this, the results for post transfusion antibody screening were available. the mean age of the patients was 46.5 years and included 30 males and 17 females. average number of rh d positive platelets transfused per patient was 11.3 units and total number of platelet units transfused was 523. anti d was detectable in 4 (8.5%) patients post transfusion and included one male and three female patients. of the female patients one was a 3 year old child who received 50 units of random donor platelets and second a 21 year old women who presented in the emergency department as a case of trauma. the third female was 54 year old post-menopausal woman. conclusion: we conclude that the chances of developing d alloimmunization after receiving rh d positive platelets is generally low. however keeping in view the antenatal complications which can arise in the future in females due to this alloimmunization, it is highly recommended that rh immunoglobulin be administered to all rh d negative women in the reproductive age group and female children who receive rh d positive platelets. background: hemovigilance is a quality process which takes into account all the activities of a blood transfusion chain with the aim to improve quality and enhance safety of blood transfusion. we have implemented a transfusion feedback reporting mechanism in our hospital as a part of hemovigilance. the current study aims to collect and analyze this data to improve our transfusion system. aim: to systematically analyze the transfusion process from issue of blood components to completion of the transfusion material and methods: the transfusion feedback forms received back at the transfusion medicine department during a 3 months period were systematically analyzed for documentation related to patient identification, product identification, documentation, completeness, etc. results were analyzed statistically for specific co-relation with patient's location, time of transfusion and type of component transfused. results: of 3474 blood components were issued during the study period. transfusion feedback form were received for 2000 (57.5%) blood components, as follows: prbc-800 (40%), platelets-651 (32.5%), ffp -441 (20.7%). patient identification number /wristband check was not done in 25 (1.25%) cases. pre-transfusion verification of blood group, patients name and patient's identification number was done in 1963 (98.15%) cases. cross checking of component unit with request form was missed in 16 (0.8) cases. pre-transfusion and post-transfusion monitoring of blood pressure was documented in 698 (34.9%) episodes, monitoring of pulse in 696 (34.8%) episodes and patient's temperature was monitored in 681 (34%) episodes. signature of nurse was missing in 86 (4.3%) and that of medical officer in 11 (05%) of the form. adverse transfusion reaction was documented in 1/2000 forms, whereas the transfusion reactions notified at the blood bank during the same period were 2. of 553 (27.65%) transfusions were carried out during non routine work hours. the mean time between the issue of the components and start of transfusion was 28 min for rbc components, 21 min for platelets and 16 min for ffp. patient identification and monitoring and product identification related non-compliances significantly correlated with out of routine transfusions (p = 0.002). conclusion: though overall compliance with established procedure for transfusion was evident, activities related to unit identification, patient monitoring and identification and reporting of adverse reactions were not well documented. this emphasizes the need for ongoing training of nursing staff and medical doctors in safe blood administration practices. introduction and method: matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (maldi-tof ms) is an ideal tool for high-throughput blood group genotyping. using this technique, this swiss (bts zurich) -german (sequenom gmbh, hamburg) cooperation aims to genotype swiss blood donors for blood groups rh, kell, kidd, duffy, mnss (n = 6000), hpa and hna (n = 3000) and high frequency antigens (hfa, n = 36,000) within 3 years. specificities are grouped into single multiplex reactions (mpx, n = 10) with up to 17 snps tested simultaneously in one tube. results: using the kel-jk-fy mpx, more than 4000 donor dnas have been tested and compared to serological pre-values. concordance between geno-and phenotypes reached 100% for k/k, 99.98 for kp a 99.93% for jkand 99.23% for fy a/b/x/0 . only one discrepancy each for kp, jk and fy could be attributed to genetics, the others were erroneous serotypes revealed by genotyping. genetic discrepancies were three new variants: kel*02.03(r700g)nulla kp a relative, jk*b(mutation n.d.) and fy*b (g261r)null. serology for js a/b of some few kel*02.06 positives confirmed validity of genotyping. numbers of detected known kel*mod and null, jk*null and fy*null (gata) alleles were 3, 2 and 51, respectively. call failures (no result) were observed in less than 2% of all mpxs. genotyping for rhd, rhce (5 mpx), gypa and b (mnss, 1 mpx) on more than 4000 samples, delivered results with discrepancy rates for mnss comparable to above, and better rates for rhdce. call failure were at approximately 2% again. reproducibility, robustness and analytical accuracy of the technique allowed measurement of gene copy numbers with relevance for rhd zygosity estimation and detection of the gypb deletion in u negatives. rhd category, partial, weak, del and null alleles and the genetic correspondents of vw, mg, mi(a), he, and uvar were observed. genotyping for hpa and hna (1 mpx) showed expected results among 2300 samples with the exception of hna-1a, b and c, where frequent duplications and deletions of fcgriiib pose difficulties for all genotyping approaches in general. in hfa ('rare') genotyping (2 mpx), currently, more than 13,000 blood donors were analyzed, and delivered: 29 kk, 2 kp(a+bã�), 48 js(a+bã�), 41 kel11+kel17+, 20 lu(a+bã�), 3 lu14+lu08ã�, 52 yt(aã�b+), 17 co(aã�b+), 11 kn (aã�b+), 98 lw(a+b+), and >10 others. specificities for vel negativity and scianna have been included into hfa typing, recently. conclusion: analysis for kell, kidd and duffy showed that genotyping worked qualitatively better and to costs comparable to serology. consequently, genotyping kell, kidd and duffy instead of routinely performing a second round of serotyping as mandatory for donors in switzerland, is recommended. ahead of comparable suggestions with regard to rh and mnss, a more detailed statistical analysis of existing raw data is needed. however, genetically identified donors with rare blood phenotypes, e.g. such as yt(aã�b+), are already selected for respective transfusions and are a strong indicator for the value of the presented project. the serological data suggest that retention of the 208-210 tcc (51s in glycophorin bs) codon from the gypb pseudoexon, prior to the gene conversion insertion of gypa sequence and coding for serine in the hybrid glycophorin, is the basis of 'anek-like' activity for both hybrid glycophorins. it should be considered that these antisera react with a new kipp-related mns system antigen. genetic studies revealed the same crossover for gp.kipp and gp.yak that have been independently reported in conference abstracts, suggesting that these are in fact the same hybrid glycophorin. background: with the increasing knowledge of the genetics of blood group antigens, molecular immunohaematology is gaining popularity. molecular immunohaematology refers to the use of genotyping to encode red blood cell antigens, representing an indirect method used to predict one's phenotype. there are certain advantages of genotyping, namely, typing of red blood cells with autoantibodies, prenatal testing and patients with multiple transfusions. molecular methods are also useful in phenotyping donors as it enables the prediction of numerous phenotypes in one single test. however, there are several misgivings about the cost of molecular methods used to genotype antigens. aim: to evaluate and compare the cost of conventional serological phenotyping and molecular genotyping. methods: a batch of 30 donor samples was typed using both serological and molecular methods. the test kit used for molecular methods, from gen-probe, included the typing for the following antigens -kell (k, k, kpa, kpb, kpc, jsa and jsb), kidd -(jka, jkb, jk), duffy (fya, fyb, fyx, fygatasil) , mns (m, n, s, s, s-s-uvar), rh (c, c, e, e) and dombrock (doa, dob). negatives that are obtained using this method were confirmed using serology. serological typing was performed with available antisera according to the various manufacturers' instructions. this includes kell (k, k), kidd (jka, jkb), duffy (fya, fyb), mns (m, n, s, s) and rh (c, c, e, e). the cost for the different tests were tabulated and compared. the cost includes labour, consumables and equipments. results: the results show that molecular method of typing red blood cells is more expensive than the traditional serological method. the cost of consumables is comparable for both methods. the consumables make up about 80% of the total cost for serological methods, and about 82% for molecular methods. the cost of labour is about 9% for serological methods and <1% for molecular. equipment cost contributes to <1% of the cost using serological methods and about 14% using molecular methods. conclusion: molecular methods may seem more expensive, about two times the cost of serology but results are obtained faster and are less labour intensive which could prove to be an advantage when phenotyping samples in large quantities, especially for donor phenotyping. molecular immunohaematology is a relatively new process, thus kits for these methods are expensive at the moment. as development and adoption of genotyping progresses, consumables and equipment costs are expected to become more affordable. serological methods do have their limitations even for patient testing, especially for patients with autoantibodies and patients who have had multiple transfusions. molecular methods may serve useful in overcoming these limitations. background: transfusion dependant patients often develop multiple antibodies to red cell antigens on exposure to red cells and require antigen-negative red blood cells for further transfusions. finding suitable red cell units for such patients is often difficult and time consuming, requiring phenotyping of large numbers of red cell units in inventory. the increasing cost and scarcity of anti-sera also makes this an expensive exercise. aim: in order to improve provision of phenotype-matched blood for such patients, we studied the feasibility of large-scale snp-based genotyping of common blood groups for establishment of an antigen-negative red blood cell inventory. methods: genomic dna was extracted from donor blood samples using an automated platform. samples were subjected to snp-typing for common local polymorphisms of rhce (ccee), fy (fy a /fy b ), jk (jk a /jk b ), mns (s/s) and co (co a /co b ) using taqman 㢠snp genotyping assays in 384-well plates at 5 ll final reaction volume, on a lightcy-clerii 480 real-time instrument. identification of the cc polymorphism was by probes targeted to the 48c>g and 307t>c of the rhce allele while probes targeted to 676c>g was utilised for ee detection. for the remaining alleles, probes were designed only to detect the most common mutation for the polymorphism within an east-asian population. results were analysed and integrated into the blood donor software system. results: the 48c>g probe designed for identification of c was unsuccessful with poor discrimination between genotype calls. the remaining probes showed satisfactory discrimination between genotypes, with 462 of the 505 (91%) samples analysed, fully genotyped for the five alleles studied. relatively rare genotypes were successfully identified using this strategy: ccee (15/505, 3.0%), fya-/fyb+ (19/505, 3.8%), ss (5/505, 1.0%). the co2 allele responsible for co b was identified in the heterozygous state in four of 494 evaluable samples giving an allele frequency of 0.4% in our population. the estimated reagent and consumables cost for sample dna preparation and snp testing was usd12.00 compared to usd27.15 for phenotyping using commercial anti-sera. we did not factor in the cost of capital acquisition of instruments for genotyping as we used facilities which were common for other molecular tests in the hospital. conclusions: results of this study indicate that snp genotyping would be a costeffective strategy for screening and establishment of an antigen-negative red cell inventory and genotyped whole blood donor pool. the cost of genotype screening was effectively reduced by use of small final reaction volume and extensive use of automation. in addition, genotyping allowed us to identify local prevalence of blood groups which were previously unidentifiable due to lack of commercially available anti-sera. background: over the past 20 years the molecular, biochemical and serological basis of almost all blood groups have been determined, highlighting the different frequencies of antigen, related to different ethnic groups. since october 2012, in our transfusion center (asl caserta) in order to have a blood bank that ensures the different transfusion needs, extended erythrocyte typing is practiced on periodic donors with specific features such as age, group and rh phenotype. aims: the aim of our study was to test the validity of the molecular method of erthrocyte phenotype typing with the serological technique by comparing the results obtained by each procedure. we also compared each method in regards to reliability, ease of use and reproducibility of results. methods: we selected 250 donors aged <45 years old, group o and a, rh homozygous phenotype. samples from each patient were tested by serological typing in solid phase (capture r immucor) and then dna was extracted and each sample was tested by molecular typing in microarray (bioarray solutions immucor) results: see table 1 . table 1 : summary/conclusions: the results show that the frequencies of more immunogenic antigens (k, fya, jka) reflect the specific and ethnic frequencies of the donors. we emphasise the absence of donors having an antigenic structure that is defined as rare (that is present with a frequency of 1:1000 according to american rare donor program (ardp), council of europe, international donor panel (idp), international society of blood transfusion (isbt), council of europe, japanese red cross). we only found one case of a donor expressing weak fyb which is due to mutation in fy 265c> t. the results were confirmed in 99.6% of cases by serological technique. however for the phenotype fyb weak, the result was discordant in serology, where the result was fyb-. in conclusion we can confirm the validity and the need to use both techniques in order to obtain a reliable and reproducible result. the molecular technique is able to identify mutations in particular genes, especially in specificity whilst the serological technique excels in sensitivity and speed but fails to confirm the data obtained. background: pathogen inactivation of blood components promises a new layer of transfusion safety, enhancing existing strategies and providing proactive protection against emerging infectious risks. processes for plasma and platelets are well established in many blood centers and those for red cells and whole blood are in development. clinical acceptance of new technologies depends on the demonstration of product safety, a minimal effect on product efficacy both in vitro and in vivo, and the range and degree of pathogens inactivated. aims: to review the major processes of pathogen inactivation of plasma and cellular blood components with a focus on the range and degree of inactivation of relevant pathogens, especially with regards to platelet pathogen inactivation. methods: this review will use recent reports and unpublished data to describe our current understanding of the potential efficacy of pathogen inactivation in improving transfusion safety. results: the major indication for platelet pathogen inactivation is bacterial contamination, a persistent problem despite multiple innovations to minimize and detect contamination. pathogen inactivation processes need to cover a wide range of possible bacterial concentrations and species. there have been few published reports using the current commercially available systems: optimized in vitro testing documents the ability of the terumo bct mirasol tm , cerus intercept tm and macopharma theraflex tm uv systems to effect a 10 2 ->10 6 log reduction of various bacterial species. most systems are less effective at inactivating bacterial spores, a particular problem as bacillus spp. is common platelet contaminant. testing the level of pathogen inactivation under clinical conditions at very low and high concentrations of bacteria reveals further weaknesses in pathogen inactivation strategies. conclusions: clinical trials of pathogen inactivated platelets have focused on the safety and clinical efficacy of pathogen inactivated treated platelets, but little has been reported on the efficacy of pathogen inactivation to reduce the risk of infection transmission. blood centers should focus on this aspect of efficacy as they decide whether to implement, and to favor those commercially available systems that best meets the clinical need for pathogen protection. for pathogen inactivation (pi) using amotosalen and uva light induces the formation of covalent adducts and interstrand crosslinks between amotosalen and nucleic acids, thus preventing dna replication and rna translation. pi technology has been adopted into routine use in some european countries and is under fda review for licensure in the us. current documentation of pi efficacy relies on illuminator sensors that measure the uva light dose delivered. an indirect methodology is utilized for the validation and qc of the process in some centers that measures the amount of amotosalen consumed during illumination in platelets and before removal with the compound removal device. the% amotosalen remaining is a direct measurement of the uva light delivered and photochemical conversion of amotosalen. however, a functional qc method that measures a direct target within the treated blood product has not been introduced into clinical use. residual leukocytes, platelets and potentially plasma contain mitochondrial dna (mtdna) which is a collateral target of the pi process. aims: the goal of this study was to quantify the impact of intercept treatment on platelet-derived mtdna by real-time pcr. methods: to evaluate the feasibility of detecting pi-induced mtdna modifications by real-time pcr, we spiked purified human leukocyte dna into human plasma, 35% plasma/65% intersol, or pbs. each sample (3.6 ml) was treated with 150 lm amotosalen and 3 j/cm 2 uva (n = 2). control samples were either untreated or treated in the absence of amotosalen or uva. dna was extracted from each sample (0.2 ml) in duplicate and assessed by measuring the inhibition of real-time pcr amplification in duplicate wells using mtdna-specific primers and sybr greenbased detection. amplification of sequences ranging in size from 73 to 1065 bp was evaluated over 45 cycles of amplification. subsequent to the initial feasibility tests, a pilot validation was performed by treatment of platelets in 35% plasma/65% inter-sol (30 ml). six different platelet units were tested as outlined above for inhibition of amplification of endogenous mtdna sequences. results: treatment of purified dna with amotosalen plus uva resulted in 1.3 log to >6.0 log inhibition of pcr amplification (results shown in table) . the extent of pcr reduction roughly correlated with the size of the amplicon, as well as with the type of diluent in the following order: pbs > 35% plasma > 100% plasma. exposure of platelets to amotosalen and uva showed an average of 2.5 log to 3.6 log reduction in pcr signal, with increasing inhibition observed for larger amplicons. in all cases, no pcr inhibition was observed in the absence of amotosalen and/or uva. conclusions: a quantitative real-time pcr assay specific for mtdna is capable of documenting pi-induced collateral nucleic acid modification in platelets. based on this work, this assay can be developed further for use as a quality control method for pi efficacy. background and aims: pathogen reduction technology (prt) provides a proactive approach to improving transfusion safety for platelet concentrates (pcs). however, prt treatment is known to exacerbate the effects of the platelet storage lesion, leading to increased platelet activation and secretion of immunomodulatory factors. little is known regarding how prt-treated platelets may affect the cells of the recipient's immune system once transfused; therefore the aim of this study was to examine the cytokine responses of a recipient's inflammatory cells after exposure to prt-treated platelets using an in vitro whole blood model of transfusion. methods: two abo/rhd matched buffy coat derived pcs were pooled and split to form matched pairs on day-1 post-collection (n = 11). one unit was treated with the mirasol prt tm system (terumo bct), while the other unit remained as an untreated control. all units were stored at 22â°c with agitation and samples were taken on day 2 and 7 post-collection for 'in vitro transfusion' experiments. to represent a transfusion in vitro, 10% v/v platelets were incubated with abo/rhd-matched fresh whole blood, with or without lipopolysaccharide (lps; 1 mg/ml) for 6 h at 37â°c/5% co 2 . protein secretion was inhibited using brefeldin (2 lg/ml) to allow detection of intra-cellular cytokine production in monocytes (cd45 + /cd14 + ) and neutrophils (cd45 + / cd16 + ), using multi-colour flow cytometry. the fold change of cytokine production was calculated by comparison to a 'no-transfusion control' (whole blood without addition of platelets). data was analysed using a one way anova with post-hoc tests for pair-wise comparisons. results: in the absence of lps, both prt-treated and untreated platelets stored for 7 days significantly increased monocyte mip-1b expression (1.5-fold; p = 0.047), whereas exposure to day 2 platelets did not result in any significant differences in mip-1b expression. as expected, lps stimulation significantly increased monocyte production of both il-12 (5.0-to 7.4-fold) and mip-1b (1.8-to 2.4-fold). however, lps-induced monocyte il-12 production was significantly reduced by exposure to prt-treated or untreated platelets stored for 2 days (prt-treated: 3.0-fold, untreated: 2.5-fold; p < 0.0001) and 7 days (prt-treated: 4-fold, untreated: 2.1-fold; p < 0.0001). furthermore, il-12 production was significantly lower following exposure to day 7 prt-treated platelets compared to untreated platelets (p = 0.006); however there was no significant difference following exposure to day 2 platelets. lps-induced mip-1b production was not significantly differentfollowing exposure to either day 2 or day 7prt-treated or untreated platelets. exposure to platelets (untreated or prt-treated) did not significantly modulate monocyte production of ip-10, mcp-1, mip-1a, il-1a, il-1b, il-6, il-8, il-10, or tnf-a. co-culture of platelets and whole blood did not result in any significant changes to cytokine expression in neutrophils. conclusion: using an in vitro whole blood transfusion model, we have demonstrated that exposure to prt-treated platelets stored for 7 days results in significant changes in the il-12 production by monocytes. these changes may reflect the way prt-treated platelets interact with immune cells upon transfusion. therefore, the effect of stored prt-treated platelets, especially in recipients with underlying inflammation, should be further examined. backgrounds: hepatitis c virus (hcv) infection is one of the major causes of chronic hepatitic disease. hcv has six genotypes and more than 80 subtypes. the epidemiology of hcv subtypes vary with different geographic distribution. understand the subtypes prevalent in certain area will help to understand the transmission modes and the spreading trend of hcv and thus help to make effective precautionary measures. hcv subtypes are also closely related with clinical therapeutic effect. it is important for guiding clinical therapy and prognosis and predicting the possible burden of hcv infection and treatment in the future. aims: to investigate and compare the prevalence of hcv subtypes in clinical patients and blood donors in guangdong china. methods: of 191 samples ofclinical patients and 222 samples of blood donors whose hcv rna were positive were collected from guangdong province. hcv ns5b gene was amplified by rt-nested pcr and then sequenced. hcv subtypes were assigned by constructing phylogenetic trees with mega5 software. moreover, spss16.0 software was applied to compare the difference between these two groups. results: of 191 clinical patients, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 2 (1.05%), 127 (66.49%), 17 (8.90%), 5 (2.62%), 5 (2.62%), 34 (17.80%) and 1 (0.52%), respectively. of 222 blood donors, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 1 (0.45%), 92 (41.44%), 1 (6.76%), 19 (8.56%), 11 (4.95%), 83 (37.39%) and 1 (0.45%), respectively. the proportion of hcv 1b was higher in clinical patients than in blood donors(v 2 = 25.866, p = 3.66e-07), while the proportion of 3a and 6a subtypes were higher in blood donors than in clinical patients(v 2 = 6.602, p = 0.010; v 2 = 19.398, p = 1.06e-05). one possible cause was the transmission modes varied with different hcv subtypes. hcv 1b is more related with blood transfusion while 3a and 6a are more relevant to intravenous drug abuse and sexual behavior. with the anti-hcv screen implemented from 1993, the risk of hcv infection by transfusion is diminishing. the other reason was the average time from hcv infection to serious pathological lesions is about 20 years and hcv 6a was transmitted into guangdong later than 1b. conclusions: in guangdong province, hcv 1b and 6a were the predominant subtypes in clinical patients and blood donors. the proportion of hcv 1b, 3a and 6a subtypes were significantly different between clinical patients and blood donors. the reason may relate with the time of hcv transmission into guangdong area and the hcv propagation modes. blood safety and increased public health initiatives to reduce hcv infection from those in these high risk groups to the general population remain a priority. background: in japan, we routinely evaluate the presence of transmission of hbv, hcv and hiv in all transfused patients at three months after the last transfusion. although the sensitivity of detection of hepatitis b virus (hbv) in blood donation improved during recent years, transfusion transmitted hbv infection is left as a serious problem. the japanese society of transfusion medicine and cellular therapy (jstmct) conducts the nationwide questionnaire survey about the clinical blood transfusion activities, supported by ministry of health, labour, and welfare, every year. in this survey, we can collect detailed characteristics of patients, who showed a positive result of post-transfusion hbv-marker-test. aims: the aim of this study is to elucidate the cause of hbv positive in transfused patients. materials and methods: data concerning to transfused patients with positive hbvmarker were collected using results of the nationwide questionnaire survey in 2007-2011. in this questionnaire, if there is a patient showing a positive result of hbsag and/or hbvdna evaluated by post-transfusion-test, it is requested that detailed patient's characteristics, including a total amount of transfusion, disease (hematologic or non-hematologic), therapeutic methods (surgery, use of anticancer drugs, use of immunosuppressive drugs, use of molecular target drugs, blood stem cell transplantation), and results of hbv-marker-test prior to the first transfusion, should report to the jstmct office. a number of hbsag and/or hbvdna positive patients were 234 cases in 2007-2011. among them, 19 patients were not eligible because of the absence of results of hbv-related markers. finally, a total number of 215 patients (36, 37, 41, 43, 45 patients in 2007, 2008, 2009, 2010, 2011, respectively) were enrolled for the present study. results: all the eligible patients showed positive results of hbsag and/or hbvdna in samples obtained from three months after the last transfusion. we classified the cause of these results into five categories according to results of hbv-markers tests prior to the first transfusion. (i) if a result of hbsag and/or hbvdna performed prior to transfusion is positive, a patient is categorized as the hbv carrier group. (ii) a patient showing both a negative result of hbsag and positive results of anti-hbs and/or anti-hbc are categorized as the hbv past-infection group. (iii) if a patient shows no hbv-related marker prior to the first transfusion and patient's hbvdna is identical to donor's hbvdna, we categorized as the transfusion transmitted infections (tti) group. (iv) if a patient does not show any hbv-related marker prior to the first transfusion and hbvdna is not detected in donor's blood by single nat, we categorized as the unknown cause group. results were summarized in table 1 . conclusion: although the positive result of hbsag and/or hbvdna of transfused patients has been considered the sequel of blood transfusion, we showed that hbv reactivation was also an important cause of it. to distinguish hbv reactivation from transfusion transmitted infection, we have to perform hbv-marker-test prior to the first transfusion, or we should freeze pre-transfusion patient's serum. plenary session: it's all about red cells 2b-pl1 the english dictionary provides several definitions for the word 'myth'. one definition is, 'a widely held but false belief'. this seems suitable for the purposes of this presentation. but who decides what is false? as we shall see in the course of this presentation, some 'myths' of blood groups may not be myths at all, and some established facts may indeed be myths. perhaps a more scientific definition would be, 'a widely held but unproven belief'. abo, the original and most important blood group in transfusion and transplantation medicine, has engendered many 'myths'. these have mostly arisen through associations between abo groups and other characteristics such as personality, dis-ease, psychological traits, and ideal diet. although many may indeed be myths, or even fabrications advanced for political reasons or financial gain, some are not. for example, the statistical associations between abo type and thrombosis, where a biochemical basis involving clearance of von willebrand factor from the blood by enzymatic cleavage appears to be affected by abh glycosylation. in rh, the first myth was that the human antibody, now called anti-d, was the same as the antibodies made by immunising rabbits with rhesus monkey red cells. hence the name rhesus, now rh, for the blood group system. in the 1940s, early serological work with rh antibodies led to two genetic theories, involving either one or three rh genes. both theories have now been rejected because molecular genetics revealed two rh genes. but was the three-gene theory really wrong, when the boundaries of genes were not understood at the time? since the 1960s it has been commonly understood that there are two types of variant d antigens: weak d and partial d. policies for transfusing patients with these variants have often been based on this dichotomy. but is this a myth? the difficulty we have in defining the terms weak d and partial d suggests that it might be. it is always tempting to dismiss anything that we don't agree with as a myth. as wiener, one of the discoverers of the 'rhesus' antigen, wrote several papers on 'blood group mythology', doing just that. scientists should beware of falling into this trap. perhaps 'myth' is a term best avoided in science. five new blood groups -what next? the past 2 years has seen the discovery of five new blood groups. at the isbt meeting in 2012, fors, jr and lan were ratified as blood group systems and since then, the molecular basis of the vel blood group antigen has been elucidated, and the complement regulator protein, cd59 has been shown itself to be a blood group antigen. these last two discoveries will no doubt lead to their elevation to blood group systems. how has this happened? it turns out to be a mixture of old and new techniques. rapid advances in molecular biology and in our understanding of the human genome have opened new fields of discovery within human blood groups. the development of comprehensive snp arrays, exome sequencing and rapid sequencing techniques, e.g. next-generation sequencing, has provide us with tools for rapid discovery. combining these with sophisticated algorithms for database mining has resulted in the identification of the molecular bases behind the hitherto unresolved, clinically relevant blood group antigens jr a and vel. however classic biochemistry and subsequent peptide and dnasequencing still play an important role and lie behind the (simultaneous) discoveries of jr a and vel, but also of lan and fors. a rare cd59-deficient patient produced an alloantibody to a high-prevalence antigen that was shown to be targeted at the cd59 protein, which was confirmed by routine dna-sequencing. the jr a and lan antigens were assigned to already well-investigated abc-transporter proteins (abcg2 and abcb6 respectively) whose presence on the red blood cell (rbc) had not been established previously and for which the function on rbcs is still not known. fors antigen was shown to result from the reactivation of the human pseudogene gbgt1. this enzyme builds the carbohydrate forssman antigen on sheep and dog rbcs but is normally inactive in humans. a mutation that reactivated the enzyme explained the unusual a pae phenotype in two families. vel was shown to be carried on a hitherto unknown protein on the rbc, smim1. the function of the protein remains unknown although the protein is highly conserved across all species, which is both intriguing and hints at a fundamental function. absence of cd59 whose function in complement regulation is well-understood, resulted in production of an alloanti-cd59, demonstrating immunogenicity of this protein for the first time. these simultaneous discoveries have shown that regardless of the route of identification, assignation of orphan antigens to their blood group 'home' continues at a rate unparalleled since the 1990s. as the '-omics' fields identify new erythroid genes and proteins, and next generation sequencing permits rapid genome sequencing of individuals, we can anticipate that many more currently unassigned antigens will find their genetic and molecular home. 2c-s04-01 teo d blood services group, health sciences authority, singapore, singapore an emerging infectious disease (eid) is defined as one that has appeared in a population for the first time, or that may have existed previously but is rapidly increasing in incidence or geographic range. in 1992, an institute of medicine report predicted continued emergence and re-emergence of microbial pathogens, facilitated by changes in human populations, environment and infectious agents. east and southeast asia, with 30% of global population, has a reputation as a hot spot for eid. within the region, the forces of rapid social, economic and environmental change have resulted in factors such as urbanization, deforestation, agricultural intensification, rapid population growth and mobility which contribute to the increased exposure and efficient transmission of new pathogens. the emergence of severe acute respiratory syndrome (sars) exactly 10 years ago has dramatically transformed individual and national awareness and capabilities for identifying and responding to regional eid threats. the re-emergence of highly pathogenic avian influenza a(h5n1) virus in 2004, isolation of novel bat-associated reoviruses in 2006, emergence of artemisinin-resistant malaria and discovery of a tick-borne bunyavirus associated with fever and thrombocytopenia in 2009 are some examples of eid emerging within the region since sars. at the time of writing, the situation involving human cases infected with avian influenza a(h7n9) virus is evolving, and there has been a recent report of human infection with avian influenza a(h6n1) virus as well. additionally, there are imported eids such as influenza a(h1n1) virus, west nile virus, and the present cause for concern in middle east respiratory syndrome coronavirus. climate changes in recent years have also accelerated the increase in incidence and range of existing diseases such as dengue and chikungunya. 40% of the world's population is now at risk of dengue, with the majority living in the asia-pacific region. the scourge of dengue is sufficiently high in the region for the association of southeast asian nations (asean) in 2011 to designate 15 june as asean dengue day. hepatitis e is another infection that is widespread in some parts of the region, and reports of silent infection in blood donors are cause for further study. the impact of eids on the blood supply may be direct through the potential for transmission through transfusion, the effect on blood donor attendance and eligibility and the effect on blood demand. blood products such as hyper-immune plasma preparations may be useful treatment options in some eids. there is a need for constant surveillance and the capacity to identify, assess and manage eid risks to the blood supply. in recent years, the strengthening of regional and international partnerships and the availability of new diagnostic tools has improved our ability to respond to infectious threats. nonetheless, the volatile and ever-changing nature of eids will remain a constant challenge to the vigilance and response capabilities of the transfusion medicine community. summary: the residual transmission risk is relatively high in hbv followed by hcv and hiv-1. this finding is not new as malaysia is a country of medium seroprevalence for hbv which ranges from 1.5% to 9.8% in the general population, but is relatively low in blood donor population (0.04%). the obi nat yield was higher than acute phase wp in hbv-nat yield. these obi positivity have shown inconsistent nat results on repeat testing and also low viral loads ranging from <12 to 317 iu/ml. in contrast for hiv and hcv nat yields, their viral loads were consistently high (34,220-424,310 cp/ml and 42,780-5,980 ,350 iu/ml respectively). implementation of id-nat is probably the best option to improve blood safety especially for the detection of low viral load such as in obi and sero-negative wp donation in malaysia scenario. blood centers reported to kcdc. repository samples of these donors were tested for anti-hav igm/igg and hav-rna. if any of these test result was positive, the recipients of the blood components generated from these donations were traced. transfusion records of hospital were reviewed to identify recipients of blood suspected to be contaminated with hav. recipients were contacted by telephone. if the recipients agreed participating in the investigation, laboratory test was performed. results: from may 2007 to december 2011, 15 donors notified the blood center of having been diagnosed with hepatitis a. the median interval from donation to diagnosis in donors was 18.2 days (table) . eleven (73%) of these 15 were male donors, and the median age was 28 years (range 19-42 years). the pcr for hav rna was positive in all of 15 depository samples of these donors. none of the repository samples showed positive result of anti-hav igm. a total of 44 blood components (rbc 15 units, pc 14 units and ffp 15 units) were delivered to hospitals. twenty six products were transfused to 26 patients, and the rest 18 blood components (4 rbcs, 1 pc, and 13 ffps) were recalled immediately and discarded. twelve recipients (46%) were already expired. fourteen recipients agreed to participate in the lookback procedure through testing. twelve recipients did not showed viremic for hav, however two recipients showed positive either on the test for anti-hav igm or hav-rna. these two recipients who were 35 and 37 years old developed symptoms on 46 and 48 days after blood transfusion respectively. they were treated for hepatitis a successfully. summary/conclusions: recipients who had anti-hav igg were not infected with hav, even though they were transfused with blood suspected to be contaminated with hav. however ttha cases were those who did not have anti-hav igg and all of them were 30s. in korea, most people under 40 years old are susceptible to hav because of low immunity. there is a risk of transfusion transmitted infection, if recipients received blood contaminated with hav. 2c-s04-04 cable rt 1 , pistorius c 1 , andersson m 2 , maponga t 2 , lopez t 2 , preiser w 2 and tedder r 3 1 hav seroprevalence was highest in the black donors (86%), lower in coloured donors (62%) and lowest white donors (36%). all were hav igm negative. there was an age-related increase from 44.8% (52/116) in those 16-25 years old to 81% (47/58) in those >46 years. although no active hev infection was identified by pcr on pooled samples, 25% of donors were hev igg positive. rates were highest in whites at 33%, followed by coloured at 23% and lowest in black at 19%. again prevalence increased with age from 12.1% in those 16-25 years to 48.3% in those >46 years. discussion: the results show a lower hav seroprevalence in the white population compared to the black and coloured population groups, likely to be due to socioeconomic living conditions such a contrast is striking given the introduction of democracy in south africa almost 20 years ago. the reduction in anti-hbc (as a marker of past hbv infection) with age is reassuring, suggesting that hbv vaccination is impacting hbv population prevalence. the pattern of hev exposure appears to implicate a zoonotic transmission route rather than being related to socio-economic circumstances. given the subclinical nature of hev infection in healthy donors, larger studies are urgently needed to establish the prevalence of active infection in blood donors. background: hbv demonstrates remarkable genetic variability, with eight genotypes and more subgenotypes. in addition, mutations in the polymerase region may lead to drug resistance and changes in the pres region (including deletions and mutations such as t31c and t53c) and prec/bcp region (mutations including a1762t/g1764a, g1896a, t1896a, c1766t, t1768a) are associated with higher risk of hepatocellular carcinoma (hcc). aims: to study the hbv subgenotype distribution and analyze the changes in pres region, prec/bcp and the polymerase region of hbv in chinese blood donors. methods: of 245 blood samples were selected randomly from hbsag positive blood donors from five blood centers in china. the pres plus s region or the whole genome of hbv was amplified and sequenced and hbv subgenotype was determined. the amino acid sequences of the polymerase region were aligned and the mutations related to drug resistance were determined. the nucleotide sequences of pres region and prec/bcp region were aligned and the mutations related to hcc were determined. distribution of genotype, subgenotype, and mutations by different regions were examined using chi-square statistics. results: of 200 samples (81.6%)were subgenotyped successfully. the predominant subgenotypes were b2, c2, d1 and a1 which accounted for 50.5%, 19.2%, 5.3%, and 3.4% respectively. deletions and mutations (t31c and t53c) in pres region were found in 28 (28/200, 14.0%) samples. five of these 28 samples (2.5% of all samples) have deletions and no deletions specific to genotype d was detected. the prevalence of mutations in pres region was significantly higher in genotype c than in genotype b (p < 0.001). mutations in polymerase region were found in 14 samples (7%, 14/ 200), most of which were related to resistance to adefovir and lamivudine. mutations in prec/bcp region were found in 68 samples (29.8%, 68/228). the prevalence of hbeag was significantly lower in samples with mutations in prec/bcp region than that in the samples with no mutation (p = 0.02). more a1762t/g1764a mutations were found in c than b genotype while the opposite was observed for g1896a mutation (p's < 0.01). conclusions: subgenotype b2 was the most frequent strain circulating in hbv infected chinese blood donors, followed by c2. hcc related mutations were found less in pres region but more in prec/bcp region in blood donors. the prevalence of mutations in pres region and a1762t/g1764a mutations was higher in genotype c than in genotype b while the opposite is the case for g1896a mutations. this is consistent with the distribution of hcc related mutations in general hbv carriers in china. since all donors in this study reported not having received hbv treatment, it is not clear whether drug resistance mutations occurred spontaneously in the hbv-infected blood donors or were acquired by the donors from hbv infected patients who underwent antiviral therapy. 2c-s05-01 a fresh look at measuring quality in blood components devine d confidence in the quality of blood components produced for transfusion, particularly with respect to their safety and efficacy, is a necessity for clinicians and patients alike. the assurance of blood product quality is dependent upon the collection of data that can demonstrate products are within specification. however, the linkage between confidence that an individual blood component unit will perform as expected and the conduct of quality testing is imperfect. this begins with the manner in which we conduct these tests. although we describe our practice as 'quality control', it is, in fact, a type of process control testing in which we test a small proportion of our production inventory to ensure that the process was conducted properly. this testing is often conducted at product outdate, long after problem products have been issued and used in hospitals. in addition, the standards used to assess blood components often have 'wiggle room'. for example, the north american standards for the number of platelets in a whole blood-derived platelet concentrate require that at least 75% of tested products meet or exceed the required platelet count. in practice, this means that up to 25% of individual platelet components issued to hospitals may have fewer platelets than the user specification requires. there are other examples in component quality standards of this same phenomenon. in an utopian blood production laboratory, there would be real time quality control measures that would be made prior to release of a unit to the hospital blood bank or transfusion service, and these measures would be highly predictive of product efficacy. as a community, we have considerable work to do to get to this utopian ideal. first we must identify better product characteristics to use as standards for blood component production. modern science, especially in the field of cell biology, has made huge strides since quality standards were first introduced some 50 years ago, yet we have not applied these advances to quality assessment for blood products. those studying blood component quality have begun to collect data sets that will help to inform this change over to new standards. production methods are only one way to impact component quality; another is the actual features of the donors themselves. this biological variation includes not only well known phenomena such as the range of platelet counts in normal healthy humans or the distribution of plasma factor viii or fibrinogen levels, but also appears to extend to storage characteristics of components made from individual donations. this presentation will review the state of the science of product quality and the regulation of blood products, including new information arising from clinical trials, and the application of modern scientific methods such as proteomics and metabolomics to the broad question of blood component quality. background: blood transfusion has been shown to be associated with poorer surgical outcomes such as higher incidence of infection, higher mortality, and increased number of serious adverse events. microparticles (mp) released in packed red cells (pc) in storage have been suggested to be mediators for transfusion-related complications. however, the underlying mechanism for mp release during storage is mostly unresolved. aims: to examine mp released in pc in storage for procoagulant and proinflammatory activity and define the role of residual platelets in pc in generation of mp during storage. methods: (i) leukoreduced (lr) and non-lr (nlr) packed cells (pc) were stored according to blood bank standards and sampled at day 0, 10, 20, 30, and 40. assay of mp was by flow cytometry using mab to label cd235a, cd45, cd41, and cd62e. thrombin generation (tg) and cd11b expression were used as measures of procoagulance and proinflammation, respectively. (ii) the impact of platelets was further evaluated by reconstituting lr pc with increasing concentrations of platelets at day 0. results: (i) multiple species of mp were released in a time-dependent manner. using nlr pc, we found that, relative to day 0, red cell mp (rmp) were increased by 2.59 at day 20, and by 8.59 by day 40. small amounts of mp from leukocytes (lmp), platelets (pmp), and endothelia (emp) were detected, generally <20% as many as rmp. levels of pmp rose rapidly from day 0 and peaked at day 20. lmp began rising at 20 days, increasing to 1.59 at day 30 and 2.49 at day 40. emp changed little over 40 days. (ii) comparing mp in nlr vs lr pc. as expected, pmp and lmp were higher in nlr pc. unexpectedly, however, the rate of rmp production was <50% as fast in lr vs nlr pc. the levels of rmp were found to be significantly associated with residual platelet counts. therefore, we investigated the possible role of platelets in rmp production. (iii) effects of residual platelets on rmp release. when lr pc were incubated with increasing numbers of platelets (0-200,000 per ll), rmp as well as pmp generation increased. rate of increase of rmp was closely associated with platelet count and storage time. this shows that residual platelets catalyze rmp generation. (iv) procoagulant and proinflammatory activities. mp-mediated thrombin generation and cd11b expression increased from day 0 to day 40 in both lr and nlr pc, but in lr pc, it was only 30-50% magnitude of nlr pc. the time course curves did not match any specific mp species. conclusions: procoagulant and pro-inflammatory mp were generated in a timedependent manner. the new finding is that residual platelets markedly augment release of rmp, which is a known indicator for storage lesion. the benefits of leukoreduction may be due to reduction of platelets and mp production in addition to reduction of wbc. reducing platelet count in pc may be beneficial in reducing storage lesion and transfusion related complications. background: it has been reported the soluble cd40 ligand (scd40l, scd154) that was accumulated during platelet storage induce polymorphonuclear leukocyte (pmn) mediated damage of human pulmonary microvascular endothelial cells(hmvecs), was a potential cofactor in developing the transfusion-related acute lung injury (trali). however the amount of scd40l was only slightly elevated in the recipient by transfused blood components as it was fully diluted in the recipient's blood circulation. the mechanism by which cd40l exert its effect is still needs to be elucidated. aim: to determine the effect of platelet derived microparticles (pmp) on pmn mediated hmvecs damage, and its correlation with pmp bounded cd40l. method: the pmps were isolated by centrifugation of the platelet-free plasma from 10 apheresis platelet concentrates (a-plts) at 20,000 g for 1 h. the pmps were counted by flow cytometric analysis, followed by western blotting that was performed on isolated pmps. the scd40l was assayed with elisa. the priming of the formyl-met-leu-phe (fmlp) activated pmn respiratory burst was measured with the hydrogen-peroxide production. a two-insult in vitro model of pmn-mediated hmvecs damage was used to investigate the effect of pmp. result: the pmp priming activity to pmn are correlative with pmp accumulations and their level of scd40l during 5 days storage (correlation was significant at the 0.05 level); pmn respiratory burst are declined by removing pmp with 0.1 lm pvdf membrane filtration or depletion pmp with cd154 monoclonal antibody combining magnetic dynabeads pan mouse igg; the lipopolysaccharides(lps) activated hmvecs were damaged more significantly by pmp isolated from 5-day stored a-plts compared with 1-day stored a-plts (p < 0.05). conclusion: platelet-derived microparticles carry concentrated cd40l signal, promote pmn mediated hmvecs damage, may be relative to developing of trali. background: continuous efforts has spared on improving the quality of platelets harvested from plateletpheresis. little is known about the contribution of donors on the variation of platelet quality, particular the effect of frequent platelet donation on donor's platelet function. aims: aim of this study is to investigate the effect of frequent platelet donation on the state of in vivo platelet activation in platelet donors. material and methods: of 107 whole blood donors and 335 platelet donors with vary donation history from 1 to 74 times (mean at 11.5 ae 12.7) were recruited. they were stratified into three subgroups according to their previous plateletpheresis history (g1: 0-1 time, g2: 2-10 times and g3: >10 times). blood sample were collected from each participant for the determination of plasma levels of soluble p-selectin (sp-selectin), marker of platelet activation and total platelet p-selectin (tp-selectin), as well as platelet count and platelet indices. results: following the increase of donation times, sp-selectin levels were steady increase (g1: 20.50 ae 5.76, g2: 23.21 ae 6.70 and g3: 25.33ae7.67 ng/ml respectly, p = 0.001) and mean platelet volume (mpv) was decrease (g1: 9.29 ae 0.89, g2: 9.17 ae 0.84 and g3: 9.02 ae 0.91 fl respectly, p = 0.039) as estimated by the analysis of covariance adjusted for sex and gender. no significant changes in tp-selectin, platelet count, platelet distribution width (pdw) were observed. further multivariate regression analysis including variables of abo blood groups as well as donation history, sex, age indicated that increased plateletpheresis donations are positively associated with the elevated sp-selectin levels in blood donors (t = 4.16, p < 0.0001). conclusion: our data suggested that frequent plateletpheresis result in the increased level of sp-selectin in platelet donors, implicating a higher state of platelet activation in vivo in frequent platelet donors. the potential effects of frequent plateletpheresis on the quality of platelet harvested and the donor complication are worthy of attention. background/aims: structural and functional changes in erythrocytes occur during ex vivo storage, including the accumulation of bioactive substances in the supernatant of red cell concentrates (rccs). many of the constituents of the supernatant fraction, which are potential mediators of transfusion-related complications, may be reduced by washing of rccs. with emerging paediatric clinical data supporting a beneficial effect of rcc washing prior to transfusion, the aim of the current study was to characterise the effects of rcc age and post-washing storage on erythrocyte structure, function and the accumulation of bioactive substances in paediatric-sized washed rccs. methods: two units of abo/rhd-and age-matched rccs (either 1-or 4-days old; n = 11 each) were pooled and equally split to obtain matched pairs (day 0). one unit was washed with 0.9% saline by repeated centrifugation then resuspended in 100 ml sag-m, while the other remained unwashed. subsequently, both rcc units were divided equally to produce 4 units of paediatric-sized washed or unwashed rccs. all units were stored at 2-8â°c and samples were taken on days 0, 1, 2, 7, and 14 of storage to measure metabolic activity and quality of rccs, as well as the concentration and activity of bioactive substances in the rcc supernatant. the overall effects of washing and storage were compared using repeated measures anova with posthoc paired t-tests as required, with p < 0.05 considered significant. results: the washing process resulted in reductions in red cell count (9.3%), haemoglobin (9.2%) and haematocrit (5.9%) compared to unwashed rccs. overall, washing and subsequent storage of 1-and 4-day old rccs significantly reduced the ph (p < 0.0001), lactate production rate (p < 0.0001), and 2,3-diphosphoglycerate concentration (p = 0.046). although the atp content of the rcc decreased during storage, it was not changed by washing (p = 0.570). haemolysis in the rccs was increased by the washing process, but remained <0.15% on day 14 for all products. extracellular potassium was significantly reduced by washing (p < 0.0001), but increased during storage in both washed and unwashed red cells (p < 0.0001 for both). washing significantly reduced the number of microparticles in the supernatant of both 1-and 4-day-old rcc compared to unwashed rccs (p = 0.01 and 0.012 respectively). however, the microparticle number in the supernatant of all rccs increased during storage. washing of both 1-and 4-day old rcc also markedly reduced the supernatant concentration of monocyte chemoattractant protein-1, scd62p, rantes, anaphylatoxins (c3a, c4a, and c5a) and iga to levels below or near the limit of detection. incubation of cultured human umbilical vein endothelial cells (huvecs) with supernatant from unwashed rcc led to endothelial cell activation, with increased cell-surface expression of e-selectin and vcam (p < 0.0001 for both). however, little or no activation was observed when huvecs were incubated with supernatant from washed rcc. conclusion: although washing affected some aspects of the in vitro quality of rccs, it effectively reduced the concentration and activity of bioactive substances in the supernatant of rccs, leading to reduced endothelial cell activation. such a reduction may be clinically beneficial in selected patient groups. blood services group, health sciences authority, singapore, singapore many of the critical issues associated withbiobanking have been effectively addressed in blood banking. blood transfusion therapy with its emphasison traceability has developed robust systems for inventory and product release. the ethos of proper quality management hasalso been an integral part of blood banking. the lessons learnt have been applied into the biobanking of cord blood, stem cells, tissue and organs. biobanking includes both banking of tissuefor research only as well as public cord blood banks that play a vital rolesupporting clinical stem cell transplantation. the growth of regenerative medicine will only increase the scope, variety and numbers in biobanking. similarly, the discovery of induced pluripotent stem cells (ips) and itspotential myriad applications has highlighted the central role of biobanking inboth diagnostics, research and therapeutics. principles and key considerations in biobanking including biospecimenprocurement, consent, processing, preservation and traceability will beaddressed. introduction: cell therapy generally includes the extraction, processing, manipulation and implantation of characterized cells effectuating specific functions in a patient. however, adjacent fields like tissue banking or tissue engineering should be incorporated. all together have donor selection and validated core procedures in common. production cycles are carried out in gmp clean rooms. furthermore, quality control includes assays which are common in transfusion medicine. it might be tempting to speculate, that cell therapy is closely related with transfusion medicine and requires minor adaptions. the big moat surrounding cell therapy: but there are huge differences: cell therapy is a domain of specialists rooted in patient care with profound knowledge about specific pathologies and how to target them. cell therapy derives from individual clinical needs and rarely is a prefabricated procedure. this is in stark contrast to transfusion medicine, which focuses on standardized products for any patient in need. today, complex regulations for cell therapy surpass those in transfusion medicine. this distracts clinicians in a cell therapy program. this may aspire transfusion medicine specialists to engage in cell therapies. however, only a small niche is left for blood centers, as two major trends arise: one is the more or less 'academic gmp' setting, utilizing the hospital exemption status. the other is the commercial arena, where companies produce standardized cell therapies to achieve maximized market shares. 'academic gmp' entities promote individualizedmainly autologoustherapies, which require a constant flow of financial assets to keep underused clean rooms running. therefore it is serious to ask why and where blood centers should engage in cell therapies. strategy first: transfusion medicine has been heavily influenced by external factors such as viral safety, blood usage, cost pressures and low resources. the term 'transfusion medicine' misleads outsiders to suppose, that it deals with transfusions, nothing else. a wise strategy must include a change in the mind-set of all. this is the most crucial issue as many clinicians are needed to collaborate and co-develop cell therapies. without deeply rooted partnerships it is impossible to establish sustainable cell therapy programs in a blood center. furthermore, a thorough evaluation includes possible products, quantities, as well reimbursement schemes. cell therapy is one of the most expensive treatments and financial assets must be secured first. second: establishing a cell therapy facility: a mock-up facility, without clean room status is highly recommended. processes will be developed, staff is enabled to learn and define procedures, before a cell therapy unit is planned. planning and establishing a cell therapy unit is very complex and specific expertise is scarce. a basic prerequisite is a project manager carrying out final decisions with a profound knowledge about processes interacting with different technologies (hvac, controls, microbiology). cell therapy units fail if project management has flaws and deep involvement of engineering with medical expertise is ignored. a cell therapy unit is an endless, stressful, path riddled with expensive failures, but rewards in the long run a blood center with exciting future prospects integrating grateful clinicians and patients. any kind of accreditation, either by regulatory authorities or any professional entity, like jacie/fact, is an important milestone in the time line of a new cell therapy and a possible showstopper of a long, expensive and enduring project. honest and thorough preparations before accreditation should start before an application may be sent. three phases are distinguishable: keeping the basics on track: running a cell therapy unit is a high wire task. fundamental knowledge about the medical background, processes, quality control, specifications is interwoven with engineering and controlling tasks. it is inevitable for anyone working in this environment to know about air quality, hygiene, staff education and additional technical features. especially controls and the design of engineering and its qualification should be documented continuously. maintenance, re-qualifications, adjustments in the control-system of a clean room facility offer chances to learn the interplay of systems. build a sufficient knowledge base and freeze the process: protocols for cell therapy are often introduced on primary events and work well in first shot experiments. as further patients are included it is very probable, that the whole process and specifications will be modified and sometimes fundamentals and documentation are out of focus. altering and scaling methods, processes and assays may or may not change the product or its intended use. slippage is often not detected and therefore first steps aim at the build-up of as much information as possible. firstly, current literature has to be collected, reanalyzed and mirrored onto the own processes. then the regulatory framework has to be analyzed. the main question is, how the product fits into the regulatory system. is it an atmp or non-manipulated cell product, is it a blood product or a pharmaceutical? pros and cons about alterations should be meticulously discussed and alternative procedures highlighted in this phaseespecially those which are already accredited. it will be very probable that the same inspectors, who have inspected similar cell therapy units, know about alternative procedures and will raise comparative questions. after building up the knowledge base it is advisable to finish a risk assessment focused on the intended use in patients and to revamp the process. after adjusting all methods, processes and assays the whole production has to be frozen. further changes are prohibited and the documentation has to be refreshed. the last test trial: in the last phase before accreditation all aspects of quality management have to be finished. risk assessments should focus on the safety and effectivity of the cell therapy. donor/patient eligibility criteria, quality control of incoming cells and tissues, production processes and their internal quality control criteria as well storage conditions have to be well documented and validated. under certain circumstances a file of clinical studies has to be prepared. all documents and clinical studies should be reviewed, regulatory aspects should be clear. a preparation project gives better chances to pass the last milestone before patients can be treated on a regular cell therapy. 2d-s07-01 burnouf t plasma fractionation is a complex biotechnological process exhibiting unique features compared to downstream technologies used for recombinant proteins, vaccines, and animal-derived antisera. in human plasma fractionation, by contrast to other biological products, multiple end-products (typically 5-10 or more) are obtained from the same manufacturing pool. some of the targeted proteins are present in plasma in large amounts, as is the case for albumin and immunoglobulin, whereas, by contrast, others, such as coagulation factors and anticoagulant proteins are in trace amounts. with the emergence of selective hemotherapy, plasma fractionation has, over the years, turned into a highly integrated protein separation process carefully designed to isolate various proteins under optimized conditions of yield and purity. current plasma fractionation methodologies combine diverse protein purification tools based on 'crude' precipitation techniques and refined chromatographic procedures. some proteins are stable and not prone to degradation, while others, with specialized functional activity, are fragile and prone to enzymatic degradation, activation, or aggregation. contamination of plasma products with harmful residual plasma protein impurities (such as activated coagulation factors or proteases) can lead to serious adverse events in some patients. in addition, while a few plasma products, like albumin and immunoglobulin, can be formulated as liquid preparations, all others products are freeze-dried to ensure long-term stability, which increases cost and technical difficulties. the diversity of protein products made from plasma explains why plasma fractionation facilities have complex design. the manufacturing lines of the various fractions should be strictly segregated from one another. in addition, the risk of viral contamination of plasma pools requires that each product be subjected to several (typically two or more) dedicated viral reduction treatments, the goal being to gradually increase the degree of viral safety along the downstream process. production zones should therefore be physically segregated to limit risks of cross-or downstream contaminations, adding to the complexity of the plant design, flows of product, personnel and wastes, and working procedures. the plasma fractionation industry has a long history from the years 1940's, when cohn and co-workers developed a sequential cold ethanol precipitation process. this method which evolved over the years, remains the core fractionation process, albeit integrated with cryoprecipitation and multiple chromatographic steps. combined with modern viral removal treatments, the current fractionation process ensures therapeutic protein products of established quality and safety, at more or less acceptable yields. the current safety record of plasma products, which contrasts with that of earlier product generations, somehow represent an impediment to the emergence of new fractionation technologies. novel plasma fractionation processes based on integrated chromatographic steps, membrane electrophoresis, aqueous two-phase system, mini-pool fractionation in disposable equipment, are being developed at pilot-scale and represent interesting alternatives. to reach the market, such technologies should be integrated with robust viral reduction steps and proven to achieve at least equal, if not superior, products yield, quality, safety, and productivity to justify the regulatory load, clinical trials, and licensing of what would be regarded by most regulatory agencies as new plasma products requiring full validation. effective specific antiviral agent is generally not available for emerging infectious disease agents such as sars-coronavirus and middle-east-respiratory-syndromecoronavirus. passive immunotherapy with plasma or plasma derived hyperimmune globulins have been used for treatment or prophylaxis against many exotoxin mediated bacterial or viral diseases such as viral hemorrhagic fever and 1918 influenza despite the lack of data from randomized control trial. antigenically shifted influ-enza a virus causes pandemics and antigenically drifted viruses are associated with seasonal outbreaks. poultry to human transmission of avian influenza a h7n9 and h5n1 virus can cause acute community acquired pneumonia with 25 to 50% mortality. risk factors including extremes of age, pregnancy, underlying medical illness and low serum igg2 are associated with severe pneumonia with delayed clearance of viral load and excessive proinflammatory response. though treatment with neuraminidase inhibitors within 48 hours after onset of symptoms should be effective, those with severe disease and respiratory failure usually present later than 5 days after symptom onset. during the 2009 pandemic of h1n1 influenza, we harvested convalescent plasma from a small percentage of recovered adults sufficient for a case control study for treating severe cases during the pandemic in hong kong. plasma supply is constrained by plasmapheresis capacity during most stages of the epidemic. between august 26 to october 31, 2009, a total of 9101 persons were successfully contacted. a total of 1309 screening and 619 whole blood donation appointments were made. in the former 786 (60.0%) attended screening but only 301 could donate plasma by apheresis because of failure to meet blood donation eligibility criteria, failed laboratory tests, insufficient neutralization antibody titers, and inability to make the apheresis appointment. for those who opted for whole blood donation, 379 (61.2%) had attended and donated. 10.5 l (21 units) and 276 l of convalescent plasma with sufficient neutralization antibodies titers was collected for passive immunotherapy as convalescent plasma or h-ivig production respectively. we recruited 93 patients with severe h1n1 2009 infection already put on neuraminidase inhibitors and requiring intensive care. twenty patients (21.5%) received convalescent plasma. mortality in the treatment group was significantly lower than the demographically matched control nontreatment group (20.0% vs 54.8%; p = 0.01). convalescent plasma treatment was associated with significantly lower day 3, 5, and 7 viral load, compared with the control group (p < 0.05) and corresponding lower serum levels of il6, il10, and tnf (p < 0.05). between 2010 and 2011, 35 patients were randomized to receive h-ivig (17 patients) or ivig (18 patients). no adverse event related to treatment was reported. serial respiratory viral load demonstrated that h-ivig treatment was associated with significantly lower day 5 and 7 posttreatment viral load when compared to the control (p = 0.04 and p = 0.02 respectively). the initial serum cytokine level was significantly higher in the h-ivig group but fell to similar level 3 days after treatment. subgroup multivariate analysis of the 22 patients who received treatment within 5 days of symptom onset demonstrated that h-ivig treatment was the only factor which independently reduced mortality [or:0.14, 95% ci, 0.02-0.92; p = 0.04]. background: in recent years, with the global spread of the west nile virus (wnv), dengue virus (denv) and chikungunya virus (chikv) that are transmissible by mosquitoes, concern has arisen regarding their entry to japan. furthermore, chikv as well as wnv and denv are potentially transfusion-transmissible, posing a serious risk for transfusion medicine. of these, wnv and denv belong to the flavivirus genus, as does the japanese encephalitis virus (jev), and they have similar antigenicity. since most japanese people are periodically vaccinated against jev, there is a possibility that anti-jev antibodies cross-react with wnv and denv and induce a protective immune response. however, because wnv and denv have similar antigenicity to jev, there is concern that the anti-jev antibodies are present in japanese plasma against wnv and denv owing to antibody-dependent enhancement (ade) in fccr-expressing cells. furthermore, if the anti-jev antibodies present in japanese plasma have high ade activity, these antibodies may act in an infection-enhancing manner rather than an infection-neutralizing manner against wnv and denv in vivo. aims: using intravenous immunoglobulin (ivig) prepared from pooled plasma from japanese donors, we evaluated its neutralizing activity and ade activity against these viruses for the purpose of estimating the potency of the japanese individuals to protect themselves against these viruses. methods: neutralizing activity (tcid 50 ) and ade activity were compared among three types of ivig, nisseki polyglobin n (made in japan), gammagard (made in germany) and sanglopor (made in the usa). tcid 50 was calculated from the results of cytopathic effect (cpe) assay using vero cells as target cells. ade activity was measured by plaque assay using bhk cells and fccr-expressing bhk cells as target cells. results: nisseki polyglobin n showed a significantly higher neutralizing activity against jev than gammagard and sanglopor. the neutralizing activity of nisseki polyglobin n against wnv was approximately a log reduction factor of 2.3 higher than that of sanglopor. furthermore, the neutralizing activity of nisseki polyglobin n showed approximately the same neutralizing activity as gammagard against wnv. none of the ivig preparations showed significant neutralizing activity against denv or chikv. nisseki polyglobin n showed only marginal ade activity against any of the viruses. conclusion: although the neutralizing activity of plasma from japanese individuals is not known, it is suggested that the japanese population as a whole has a potency to protect themselves from infection by wnv to some extent, probably due to the cross-reaction of anti-jev antibodies to wnv as a result of jev vaccination and natural infection. therefore, if wnv invades japan, a pandemic may not occur and the risk of wnv infection by blood transfusion may be low. it was suggested that plasma from the japanese individuals has almost no neutralizing activity against denv and chikv. nisseki polyglobin n showed only marginal ade activity against wnv and denv, suggesting the low possibility of the anti-jev antibodies present in japanese plasma acting as infection-enhancing agents against wnv and denv as a function of ade. background: platelet-rich-plasma (prp), platelet gel (pg), and platelet lysate (pl), are used in regenerative medicine to stimulate the healing of soft and hard tissues. in addition to their tissue regenerative properties, platelet materials are claimed, mostly through anecdotal observations, to limit post-surgical inflammation and decrease pain. the anti-inflammatory properties are thought to be due to the release of platelet components, including transforming growth factor-b1 (tgf-b1) and hepatocyte growth factor (hgf), which are known to inhibit some inflammatory pathways in vitro. however, there is a large diversity in the type of platelet fractions used in clinics. they differ significantly in protein composition and content of proinflammatory and anti-inflammatory molecules and may therefore not be equally effective in controlling inflammation. one needs to elucidate the factors responsible for the possible anti-inflammatory properties of platelet materials to standardize the preparation and clinical use of these products when an anti-inflammation effect is clinically beneficial. aims: to investigate the potential anti-inflammatory effect of various plasma/ platelet fractions using an established in vitro model of raw 264.7 mice macrophages stimulated by lipopolysaccharide (lps), and studying the production of nitric oxide (no), inducible nitric oxide synthase (inos), and cyclooxygenase-2 (cox-2). methods: apheresis platelet concentrates (pc) were obtained from three donors and separated within 3 days into pc, platelet poor plasma (ppp), platelet gel releasate (pg), frozen-thawed platelet lysate (pl), and solvent-treated platelet lysate (s/d-pl). proteins were determined, sds-page patterns obtained, and growth factors quantified by elisa. raw 264.7 cells were grown in medium supplemented with 10% of fetal bovine serum (fbs) or plasma/platelet fractions, with or without lps (500 ng/ ml added after 24 h of culture). cell growth and cytotoxicity were checked by cell count determination and mtt assay. no was determined in cell culture supernatants by colorimetric assay and inos and cox-2 in cell extracts by western blot. prp from mice and quercetin, a known anti-inflammatory compound, were used as controls. results: pc and s/d-pl had the highest mean tgf-b1 content ranging from approximately 100-200 ng/ml, and ppp the lowest (5-10 ng/ml). cell count analysis and mtt assays showed consistent cell growth and viability in all conditions evaluated but were slightly lower in the presence of lps and quercetin, as expected. there was no no, inos, cox-2 detected in absence of lps stimulation. the plasma and platelet fractions were all found to reduce no production and inos expression, when compared to fbs, after lps stimulation. interestingly, inhibition of no production and inos was generally more pronounced with s/d-pl. cox-2 synthesis was lower in the presence of s/d-pl compared to other plasma/platelet fractions and higher with pg. the mice prp did not exert any stronger anti-inflammatory action in this mice cellular model suggesting absence of species specificity. conclusions: the plasma and platelet fractions evaluated exert, to various degrees, an anti-inflammatory effect mostly revealed by inhibition of no production and inos. impact on cox-2 synthesis is less obvious. the fact that s/d-pl exhibits stronger global anti-inflammatory activity requires further studies. 2d-s08-01 tani y japanese red cross kinki block blood center, ibaraki, japan rare blood is generally defined as one that occurs at a frequency of 1:100~1000 individuals or less, and it is sometimes difficult to provide such blood types to patients because of their rarities. the japanese red cross (jrc) society lists 46 rare blood phenotypes that are divided in two categories as shown in table 1 . the rare blood types listed in category i occur much less frequently than those listed in category ii. we screen for rare blood cells using monoclonal antibodies (moabs). since 1987, our blood center has established 93 moabs (32 human and 61murine), and has provided them to the other blood centers. many of igg moabs are available on the machine such as beckman coulter pk-7300 blood grouping analyzer by saline or bromelin method by cross-linking with anti-human or anti-mouse igg. in addition, we routinely screen for antigen negative-cells (rhc, c, e, e, jk a , jk b , fy b , di a , m, le a , s to which antibodies are believed to be clinically significant). thus, more than 10,000 donors with rare blood phenotypes (category i, 748; category ii, 9314) are registered in japan, but the number of donors with some category i blood types are insufficient. we freeze rare blood, particularly category i types, and domestically and sometimes internationally supply these units of blood. since 1977, a total of 576 units of rare blood with phenotypes di(b-), d-, jr(a-), ko, jk(a-b-), (para-)bombay, p, en(a-), m k m k , lan-and rhnull have been supplied to 23 international countries. thus, the jrc contributes to the international panel of donors of rare blood type (idp) which is maintained by the international blood group reference laboratory (ibgrl) in bristol, uk. the idp provides information on the location of rare blood donors when they cannot be found in their respective countries. the jrc has encountered difficulties in finding rare donors with rhnull, p k , m k m k , en(a-), u-and ge-phenotypes. we also joined the isbt working party on rare donors which handles all matters related to rare blood. our rare blood donor program is successful because of international cooperation. 2d-s08-02 the china national rare blood group screening program started in 2008. the program has screened more than 1,500,000 donors in thirteen regional blood centers by using large-scale serological tests, gene diagnosis, and other different specific screening technique. now, more than 1300 donors with rare blood phenotypes have been found. including rh null, d --, wrb-, lu(a-b-), jk(a-b-), vel-, lan-, coa-, k 0 , dib-, s-as well as yta-. the primary target for the project is to screen the negative blood antigens whose conjugational antigens have high frequency, and the blood groups which is easy to produce antibodies and the negative antigen in the system is very rare in chinese population (for example, fya-, whose frequency was 1/400 in chinese han population). a professional website for rare blood groups (http://www.chinarareblood.cn)was set up and serviced in jan, 2010. the information of blood donors with rare blood types is registered into the national registering system for blood donors with rare blood types by professional technician from organizations join the program. the information includes the specificity of the rare blood types, other common blood groups, personal data of donors, and the information of contacts. except the network administrator, other visitors could only see the specificity of the rare blood type and the number of the rare blood in rare blood bank storage. application for the rare blood products should communicate with contacts through administrator. according to the standard of the pretransfusion test in china, all the donors and recipients must be identified the abo and rh systems and done the cross-match test, in addition, all the donors must pass the test for contagious marks. nowadays, more and more chinese blood centers use nucleic acid technique to detect hiv and hcv. the results of infection test must be added to the information of the blood products at every time for collecting the rare blood. in recent 2 years, 21 units (1 unit = 200 ml) blood products with different rare blood types have been used in clinical treatment. background: anti-emm is a rare specificity detecting the high-prevalence red blood cell (rbc) antigen emm (isbt 901008). five cases were reported by daniels et al, (transfusion, 1987; 27:319) and two by reid et al, (transfusion 1998; 38 (suppl) :101s). six of these were in untransfused males and anti-emm had not been implicated in a transfusion reaction, most likely because the patients were not transfused. case study: a 58-year-old, untransfused, japanese man with group a, d+, datnegative rbcs, urgently needed transfusion due to an abdominal stab wound. pretransfusion testing using gel column agglutination and peg-iat, demonstrated an antibody reactive with all panel cells, but non-reactive with autologous rbcs; anti-le a was detected by papain methods using gel column. unavoidably, one bag of crossmatch-incompatible le(a-) rbcs had to be transfused. thirty minutes after transfusion, he experienced a drop in blood pressure and hematuria. however, as his hb level was 5.5 g/dl, another two rbc bags were transfused, and his vital signs became stable. on day 3, he was transfused one rbc bag without a hemolytic transfusion reaction. on day 6, after receiving 30ml of rbcs, the patient vomited and had cola-colored urine (t-bil 6.1 mg/dl, ldh 912 u/l) and the transfusion was stopped. following transfusion, his rbcs reacted in the dat: 1+ on day 1, 2+ on day 3 and negative on day 7. thereafter his anemia improved gradually by iron medication and he was discharged on day 24. aims: to identify the antibody in the patient's plasma that caused the transfusion reaction. methods: serological testing was performed by standard methods. result: the patient's plasma reacted in saline at 4c (2+), by albumin iat (2+), peg-iat (2+), and papain-iat (3+) with all panel cells, and by peg-iat with 30 samples lacking high-prevalence antigens; the autologous rbcs were non-reactive. testing his plasma against phenotypically-similar enzyme or chemically treated rbcs showed that the antigen detected was resistant to bromelin, papain, ficin, trypsin, a-chymotrypsin, pronase, dtt, aet, and acid. two examples of emm-rbcs were non-reactive and the antibody was identified as anti-emm. the reactivity of enzyme and chemically treated rbcs is consistent with anti-emm. his rbcs reacted with antibodies to 17 high-prevalence antigens, but could not be confirmed as emmbecause anti-emm was not available. the anti-emm was igg1 and igg3 with a titer of 16 by peg-iat before transfusion rising to 128 on day 10; his serum demonstrated hemolysis after day 6. no other underlying antibodies were detected in the patient's plasma alloadsorbed to remove the anti-emm. conclusions: we report the first japanese case of anti-emm and the first to cause an acute hemolytic transfusion reaction (ahtr). in previous reports, six people with anti-emm were untransfused men and one was in a woman whose transfusion history was unknown, suggesting that the antibodies may be 'naturally-occurring'. the anti-emm reported here, also in an untransfused man, also may be considered a 'naturally-occurring' antibody. similar to the first reported example of anti-emm, the antibody reacted, albeit weakly, at 4c. our case suggests that anti-emm is clinically-significant as the patient experienced an ahtr. 2d-s09-01 background: to recruit blood donation volunteers and provide stable blood supply according to demand, it is more important to change the overall social perception than to carry out one-time event or short-term campaign. the social understanding of blood donation is formed as valuable and honorable service over certain level in korean society. nevertheless, there still are many people who don't even try to participate in blood donation because of fear, health concern, and expectation for reward. to change this culture and social awareness, it is important for the present and future blood donors to have a perception that the blood donation is the sharing one's life and a genuine service which helps other people for nothing. aim: the main purpose of this article is to introduce korean red cross' recent efforts (operation of the red campaigner and blood donation supporters, the construction of virtual blood donation experience center, the blood donation promotion program) to change the blood donation culture and widen the donor base in korea and to present their effect and improvement direction. (1) this article is comprised of the case study and analysis on the operation of red campaigner and blood donation supporters, the construction of virtual blood donation experience center and blood donation promotion program (2) this article outlines the red campaigner and the blood donation supporters and the related programs and addresses their significance in addition, describes the effect and direction for improvement, presenting research related data. (3) this article outlines virtual blood donation experience center construction and presents the exhibition in it and it suggests the effect and possibility on the authority of the research case that the education with fun has more considerable impact than learning by rote. result: to change current culture and increase positive understanding about blood donation, korean red cross is making various efforts. the red campaigner, consisting of high school students and the blood donation supporters, consisting of college students, aim to influence the youth, the potential blood donors, to have a positive understanding of blood donation and to carry out continuous and organized publicity of its importance and safety. in addition, korean red cross is making a progress in the construction of the virtual blood donation experience center which is going to be completed by the end of 2013. we expect that we can achieve the educational purpose that sends a message that the blood donation is a volunteer work to save life and make future donors to recognize the blood donation as an object of not fear, but interest. finally, 'the blood donation promotion program' which began in 2009 is designed to encourage for general groups or organizations to participate in the blood donation campaign and to create the voluntary blood donation culture. conclusion: various projects operated by korean red cross contribute to widen blood donor based by changing blood donation culture in korea and are expected to make continuous contribution. but these projects have a limitation that the partici-pant is restricted and continuous participation isn't progressing in terms of national participation. continuous and positive endeavor to foster these projects as a national campaign should be encouraged. although it is possible to increase the blood donor temporarily through one-time event when blood shortage recurs, widening the donor base by changing blood donation culture should be the fundamental solution. the changing blood donation culture and donor understanding may not be optimistic for a short-term blood shortage problem but will be useful to make conditions that expand the donor base and increase voluntary donors in the medium to longer term. understanding our future donors is of critical importance to blood collection agencies worldwide. not only do we need to know who they are, but also why they do or do not engage in the required behaviour of blood (product) donation. the surge in psychological research into blood donation in the last decade has provided significant insight into understanding the psychological factors underpinning the commencement and continuation of blood donation. this review will summarise the state of our current understanding of knowing how to effectively recruit the non-donor and the complexities that lie ahead for all involved. at the descriptive level, and stemming from the systematic application of various psychological theories and models within blood donation contexts, we now know more than ever about the key factors that non-donors report impact on their blood donation intentions and behaviour. from this, certain psychological elements such as perceived control or self-efficacythe individuals' self perception that they can cope with donationhave emerged as key determinants of donor recruitment. drawing on these results, research psychologists have worked collaboratively with blood collection agencies to develop and evaluate recruitment materials designed to target these central constructs. both laboratory and in field trials have been undertaken which have consistently shown positive effects. for example, participants receiving these materials are more likely to volunteer to donate blood than those receiving standard recruitment materials. the collaboration of researchers and blood collection agencies has furthered understanding and recruitment efficacy generating measurable, operational deliverables. however, this collaborative research has also served to highlight the challenges that lie ahead both in terms of the diversity of our non-donor population as well as the limitations of our current theoretical models. further, there is an increasing need for large scale randomized controlled field trials to systematically evaluate interventions designed on the basis of psychological research. while these needs may provide substantial challenges for researchers and blood collection agencies alike, the promise of psychology in providing the 'who' and 'how' to effectively recruit remains. background: developed countries rely solely on voluntary non-remunerated donors to ensure adequate blood supply but ageing has brought in significant pressure on the health care system including blood supply. in hong kong, blood demand has recorded a 17% increase in blood demand from year 2008 to 2012 with almost 60% blood being transfused to patients aged >60. therefore, sourcing for more blood to meet demand is one of the most urgent issues in blood service. in this report, we report how we successfully modeled donation preference into the development of a new collection site to leverage the blood demand. material and methods: demographic information of all donors with successful donations was retrieved from blood bank computer system for the years 2004-2007. statistical analyses were performed to determine how frequent they came back to donate, whether residential location affected their donation frequencies and lastly identify potential district in hong kong to build a new donor center against the donor and general population distribution. results: of 775,690 donations made by 191,302 male and 201,446 female donors were analyzed. on average 75.8% of donors donated only once during the calendar year but donors were more likely to make repeated donations at donor centers than mobile venues. significantly more donors would come back for second or more donations at donor center (36.1% vs 20.1%, p < 0.05). male were more likely to come back for repeated donations than female (43.3% vs 30.9%, p < 0.05). a total of 466,121 donations made at donor centers were further studied. upon matching with their residential address, distance from donor centers was shown to be a determining factor on their choice of donation through linear regression analysis. reduced donation frequencies were observed with increasing distance from donor centers. regression analysis then identified several districts where many donors had to travel a long distance to the nearest donor center. the district with the highest expected increase in donations, adjusted by the expected growth rate, was then chosen as the site for building the new donor center. a fixed donor center was so selected at yuen long district and open to donors by 29 july 2011. by the end of year 2011, 6160 donations (consisting of 3047 males and 3113 females) were made with more than 88.1% collection given by donors residing at yuen long and the nearby district, tuen mun. when the whole year figure was reviewed in 2012, 16,990 donations were collected which already exceeded the planned collection target of 15,000 by year 3. interestingly, same 88.1% donations were given by donors residing at these two districts. conclusion: despite being a small city, the retrospective analysis of donation behavior has provided valuable information in the service planning in hong kong. the new donor center was able to reach the planned third-year collection target by 15 months earlier. further work is being done by using the more recent data to identify the next optimal collection sites for future expansion under most best cost effective way. singapore red cross, singapore, singapore background: singapore is moving towards providing more fixed blood donation sites with the aim of enhancing donation experience and encouraging repeat donations. it was recognized that the choice of location and an understanding of the human traffic in the vicinity are elements of success. a 2-prong approach was undertaken to: collect information on the footfalls, the social profile and demographics in the designated location. collect information from potential donors on their preferences and sentiments in relation to operating hours and outreach channels for marketing communications. method: a month-long study was conducted in the vicinity by observing and counting the human traffic, the crowd density at various exit/entry points at various timings, the flow and direction of the general foot traffic, overall make up of the surrounding district such as types of corporate, civic & religious organizations operating in the vicinity and number of educational institutions. in addition, an on-site survey to determine potential donor preferences and sentiments in relation to operating hours, tactical outreach and design mechanism of the blood centre was also conducted to help develop a tactical marketing communications strategy. the results indicated that during week days, about 85% of people visiting that vicinity were youths aged 16 to 25 (50.5%) and young adults aged 26 to 39 (33.4%). the main purposes of the visit were for work related activities (22%), attending school (19.1%) and shopping (38.1%). on weekends, 76% of these age groups visited the vicinity for leisure activities or to church. another 24% who visited there were adults age 40-60 years old. most of the respondents had a specific destinations, and most of them indicated lunch time and after office hours as their preferred donation periods. a tactical marketing communications strategy targeting youth and young adults was developed to meet the behavior and preferences of the target group. social media platforms such as online mobile app advertising and locational media buys were employed. in addition, partnerships were developed with nearby educational institutions and churches to host road shows and blood donation awareness activities to engage youths and to foster fun and excitement in the social media atmosphere for the blood collection center. the strategies undertaken proofed favorable as the daily attendance at the new blood collection center has surpassed its baseline target collection of 50 units of blood a day within the 3 months of its operation (jan 2013); and now, has a daily average collection of 60 units of blood. 3a-s10-01 setting up haemovigilance programme from the very first step background: national haemovigilance programmes wherever established have yielded significant data to implement blood safety initiatives. settting up a national haemovigilance programme requires meticulous planning and the following issues need to be addressed; whether reporting will be voluntary or mandatory, what is to be reported and by whom, reporting formats and data submission, resources to sustain the programme, staff training and responsibilities. india is a country of 1.18 billion people, 2700 blood centres, one-third each in public, charitable and private healthcare sectors and annual blood collection of 9 million. given the diversity of blood centre management, setting up such a national programme is a complex task. aim: to set up a national haemovigilance programme in india. method: the ministry of health and family welfare (mohfw), govt. of india had launched the pharmacovigilance programme of india (pvpi) in july 2010, with oversight by the indian pharmacopoeia commission (ipc). adverse drug reaction (adr) monitoring centres were setup in 90 medical institutions in the country and trained staff was recruited, for data collection and submission. after the successful launch of pvpi, the mohfw, initiated the haemovigilance programme, distinct from pvpi with the co-ordinating centre at national institute of biologicals (nib), but in close collaboration with ipc. the broad organizational structure of the programme is as follows; reports will be generated in the medical institution based blood centressubmitted online to the haemovigilance national co-ordinating centre at nibreports will be reviewed by the national advisory committee which will make recommendations to the national co-ordinating centre, ipc for onward transmission to the regulatory authoritythe central drugs standards control organisation to formulate safety related regulatory changes and communicate to blood centres. the programme was formally launched in december 2012. terms of reference of the national advisory committee are: to finalize transfusion reaction reporting form (trrf) for the country. to give expert opinion for collection, collation and analysis of data and development of software for the same. to monitor the quality of data collected. to develop training modules and guidelines for implementation of haemovigilance programme. results: based on recommendations of the committee, the initial focus is on reporting serious adverse reactions as defined by the working party of the international society of blood transfusion, reporting is voluntary and a guidance document and trrf have been made available to the medical institution blood centres, which are the designated reporting centres. the reporting is online through a software -haemo-vigil accessible on the nib website. each centre has been given a unique username and password for login. the security of data submitted through the software has been validated. reporting commenced from february 2013 and till date 434 reports of adverse reactions have been submitted. the data is yet to be analysed. a series of awareness workshops are planned countrywide, five have already been organized. specific funds have been allocated by the mohfw for this programme. conclusion: a well structured programme of haemovigilance has been initiated in india and is now in a phase of development. 3a-s10-02 background: congenital haptoglobin (hp) deficiency being homozygous for a deleted allele of the hp gene, hpdel, was identified in a japanese pregnant woman who had experienced severe anaphylactic transfusion reactions (trs) after infusion of red blood cells (rbcs) and human serum albumin in 1998. in addition, the allelic frequency of hpdel was calculated to be 0.015 by a genetic study of a limited number of the japanese individuals, suggesting that hp deficiency might distribute among the japanese population as a phenotype of serum hp. aims: in this report, we present the results obtained from a hemovigilance survey carried out between 1998 and 2012, in which hp deficiency was identified among japanese patients who had experienced nonhemolytic trs (nhtrs), and those obtained from a screening of hp-deficient japanese healthy blood donors. materials and methods: patients with nhtrs: a total of 19,675 patients who had experienced nhtrs, reported by hospitals to the japanese red cross society between january 1998 and december 2012, were examined. healthy blood donors: a total of 272,068 blood donors who visited the japanese red cross blood centers in the tokyo area between june and august 2010 were examined. testing for identification of hp deficiency: (i) serum hp concentration was determined using peak-rate nephelometry with the detection limit of 6 mg/dl followed by simplified sandwich elisa with the detection limit of 3 lg/dl. individuals who showed a negative result of elisa were assessed as hp-deficient. (ii) the presence of the hpdel allele was analyzed using an allele-specific pcr method. testing for anti-hp antibody: the anti-hp antibody produced in all the hp-deficient individuals was measured by elisa and western blot analysis. it was further analyzed using ouchterlony or surface plasmon resonance technology in some cases. results: thirty-one individuals were identified as hp-deficient among the 19,675 patients who had experienced nhtrs (0.16%). all the patients, except three who could not be tested, were homozygous for the hpdel allele. they were transfused blood products [pc, 17; ffp, 1; rbcs, 9; mixed, 4] . nineteen of them (61%) experienced anaphylactic trs accompanied by severe hypotension and the other patients (39%) experienced milder nhtrs. all the patients except one had a history of transfusion. the anti-hp igg antibody was detected in 28 patients (90%). in addition to the igg antibody, the anti-hp ige antibody was detected in 20 patients (64%). hpdeficient individuals were identified among japanese blood donors with a prevalence of 105/272,068 (0.039%). all the donors were homozygous for hpdel, except one who was heterozygous for hpdel, hpdel/hp2. the anti-hp antibody was not detected in 104/105 (99%) of the hp-deficient donors. conclusions: the higher prevalence of hp deficiency caused by hpdel -homozygosity producing the anti-hp antibody among the patients with nhtrs than in the normal donors (p < 0.001), suggests a higher risk of such trs in hp-deficient patients. hp-deficient individuals are present among normal healthy donors with a prevalence that is expected from its allele frequency. they might be expected as suitable donors for hp-deficient patients to prevent hp-related anaphylaxis. hlaing aa background and objective: collection of the correct blood sample from the correct patient is a vital step in the process of safe blood transfusion. transfusion laboramethods: a study on all reports of mislabeled and miscollected specimens from january 2010 to december 2012 was undertaken and the results were analyzed. mislabeled samples were defined as samples that were incorrectly labeled at the time of collection and miscollected samples were 'wrong blood in tube' samples due to patient misidentification. errors resulting in discrepancies in blood group between the current blood sample and historical records were identified by program flagging during validation of blood group results. these discrepancies were resolved by requesting a second sample from the patient collected by another person. some errors detected at the ward level, were reported by the staff member who had sent the blood sample. workload data for group and screen samples received during 2010-2012 was collected from the annual transfusion laboratory records. using these data, ratio of errors from mislabeled and miscollected samples, to number of group and screen samples received was calculated. results: between january 2010 and december 2012 a total of 162,999 samples were received for abo grouping. of 77 incidents were recorded relating to errors in either sampling or labeling. the overall sample error rate was 0.047% or 1 in 2116 samples. of 52 cases resulted from wrong labeling during collection, 17 cases were due to patient misidentification, five were errors that had occurred during the initial request, in two incidents the cause could not be identified and one labeling error occurred in the laboratory. all errors in labeling resulted from failure to check the pre printed name label with the label on the patient's identity band. in 14 incidents, labeling was performed away from the bedside, in 11 cases name labels of a different patient were found in the correct patient's medical file, in 25 cases labels were taken from wrong patient's file and two errors were due to using prelabeled tubes. of 17 patients had been misidentified and blood taken from the wrong person. root cause for these errors was not following hospital polices in patient identification and sample collection. sixty-four percent of the errors occurred out of normal working hours, mainly during the night while the rest 36% had occurred during normal hours. conclusion: we conclude that mislabeling and miscollected sample errors represent a potential for mistransfusion in our institution. the rates of mislabeled and miscollected samples can be used as key performance indicators in this important step in the clinical transfusion process. this baseline data will be used in formulating standards of performance for sample collection and patient identification and, for implementing risk -reducing strategies. background: haemovigilance is a surveillance programme dedicated for the practice of blood transfusion. it is an important part of the quality system of the blood programme. haemovigilance programme in malaysia was initiated as a national programme in 2003 under the ministry of health (moh). since its inception in 2003 the programme has evolved and has become an integral part of our transfusion service. all adverse events and near misses were reported to the national coordinating haemovigilance unit at the national blood centre (nbc) using a standardised form and predefined criteria. these were compiled and analysed into an annual report for the national background: the process of blood transfusion from blood collection and processing to issue and bedside transfusion of blood components involves several areas of human participation. human error is therefore inevitable in this chain of events. transfusion laboratories have long focused their attention on quality control methods and quality assessment programmes dealing with analytical aspects of blood testing. however, there is enough evidence to suggest that the steps most prone to error are in fact in the pre and post analytical phases. various international accreditation bodies now require clear and effective incident reporting protocols to enhance measures for error trapping and error avoidance. aims: this study aims to quantify and characterize transfusion errors in a joint commission international (jci) accredited tertiary care centre in india. methods: all reports of transfusion related errors, registered in the blood bank or outside, between january 2008 till december 2012 were reviewed and categorised into pre-analytical, analytical and post-analytical events. the process adopted at our centre for assessing transfusion related events at the patient's side uses widely tested criteria of: (1) incident reporting (2) root cause analysis (3) identification of corrective actions results: during the study period 89,156 requests for blood and blood components were received and total of 1,98,505 blood components were issued within the hospital. a total of 16,834 reported errors were analysed. pre-analytical errors comprised a large majority (13,676; 81.24%), most of them being errors of inadequate patient information on request forms (10976; 65.2%) followed by sampling errors (1756; 10.4%). analytical errors comprised (563; 3.3%) and post analytical errors accounted for (2595; 15.4%) of the total errors reported. there was no incidence of acute haemolytic transfusion reaction or direct patient harm during the study period but on several occasions near miss events were reported which, if missed could have background: in australia, we rely on non-remunerated, voluntary donors to provide sufficient blood to meet patients' needs. for fresh components, the australian red cross blood service (blood service) is unable to import components for routine use, so is 100% self-sufficient. hospitals and pathology laboratories are under increasing pressure from government/s to improve value for money for blood and blood products, which is resulting in extra demands being placed on the blood service, especially in relation to lower age at issue and a continuing trend to hold more group o stock and less of the other groups, especially ab. with a typically seasonal inventory pattern for red cells, the blood service has focussed on closer management of blood inventory. aim: the aim of the inventory program was to improve reliability of blood coming into the supply chain and therefore improved reliability in delivery to customers. this is measured by average and variance in the number of whole blood collection packs being receipted at the processing centre. the aim was to reduce the variability in this metric, which would naturally lead to decreased inventory holding requirements, greater control and efficiency, and increased reliability and service to the customers. order fulfilment is another measure used to demonstrate improvement. methods: in order to manage blood inventory effectively, the first step was to introduce a minimum and maximum inventory level, by blood type, by state. this provided a transparent target to aim for. the bands were calculated by firstly setting a minimum inventory level using traditional supply chain safety stock calculations. the next step was to develop a 12 week inventory forecast, using a number of planning assumptions. one of the core assumptions is the number of appointments booked in the lead up to a donation. in order to improve reliability, minimum tar-gets were agreed at 3 months out (re-booking time) through to one week out. a 'traffic light' style appointments portal was developed to provide improved visibility of appointment levels for each collection mode and by state. results: results show that the quarterly standard deviation of blood coming in to inventory has improved from 1711 to 1285 in the last four financial yearsa 25% reduction. in addition, order fulfilment has improved from 82% to 95%, demonstrating that, with improved planning systems and processes, it is possible to manage inventory effectively. the results are demonstrated in the two graphs attached. summary/conclusion: the blood service in australia set a goal to improve reliability of fresh components, in particular, red cells entering finished goods inventory, to improve order fulfilment and provide service excellence to customers. by implementing robust and disciplined planning and reporting systems, reliability has improved which shows that there are methods available to improve the effectiveness of inventory management for blood components. 3a-s11-02 wooi-seong k one of the fundamentals of a blood collection center that procures, processes, stores and supplies blood and blood components to hospitals or other blood banks is an effective and sound management of blood inventory. as blood supply is dynamic, blood supply management requires continuous monitoring and interfacing between blood procurement and inventory management and with hospitals. in an effort to provide adequate, safe and equitable blood supply from voluntary non-remunerated blood donors in the face of increasing demand and decreasing donor population, blood collection centers are also challenged with blood shortages, which unless managed, could impact the healthcare delivery and negatively affect patient care. in order to provide a consistent and reliable blood supply blood centres will have to resort to creative and innovative measure. malaysia, a unique multicultural and multiethnic country celebrates significant religious and historic events as well as commemorations. as such, malaysia observes numerous national and state holidays. in fact, malaysia is ranked as the seventh country in the world in the number of observed holidays. by virtue of its geographical location, malaysia is not exempt from natural and man-made disasters, the most severe being seasonal monsoon floods and flash floods. these and the poor response to blood donation campaigns as a result of 'balik kampung' phenomenon during major holidays due to mass exodus of malaysians to their hometowns, contribute to acute seasonal blood shortages in blood collection centers around the country as well as within the region. adopting a proactive approach to blood shortages includes embracing new measures to recruit and retain blood donors, establishing a blood forecast system, developing a strong network among blood collection centers, being transparent with the blood inventory levels which will lead to greater trust and increased confidence in bts and having a contingency plan. at national blood centre (nbc), the blood action team was formed in 2010. it is a multidisciplinary team comprising of members from the donor education and publicity, donor recruitment, blood procurement, component and processing and inventory divisions as well as medical officers, transfusion medicine specialists and consultant. meetings are held regularly and this has greatly improved the communication interdepartmentally, and has fostered a team whose members are committed to improving blood supply management and preventing blood shortages through discussion and brainstorming sessions. also, blood forecasting is carried out as far ahead as months in advance. the blood stock forecasts are also communicated to blood banks from public and private hospitals which are supplied by nbc, a measure to increase transparency. since the implementation of these measures, nbc has successfully and effectively overcome the annual seasonal blood shortages for the last 3 years. evidently, blood shortages are largely preventable by adopting a proactive approach. 3a-s11-03 . the c/t ratios were calculated and analyzed for each major department. nbc and hkl had continuously introduced several interventions to reduce c/t ratios during the period of this study. results: the overall c/t ratio for hkl had been reduced from 2.2 in year 2005 to 1.9 in year 2012. the four major departments in hkl that showed high reductions in c/t ratio for the same period were obstetrics and gynecology (6.4 reduced to 2.5), surgical (2.6 reduced to 2.1), orthopedic (2.6 reduced to 2.1) and neurology (3.8 reduced to 2.2). in this study, interventions that had contributed to the drastic reduction in c/t ratio were compliance to the maximum surgical blood order schedule (msbos) which was periodically updated within each department, effective communication between clinician and blood bank staff, and continuous medical education (cme) for house officers and clinicians. the active hospital transfusion committee (htc) and hospital transfusion team (htt) also played an important role in reducing the c/t ratio by creating awareness among the paramedics and medical officers regarding the judicious use of blood and blood products, and regular monitoring and audits of the whole transfusion process starting from blood sampling to monitoring of patients during and after transfusion. summary/conclusions: this study showed that several interventions that have been introduced by hkl and nbc such as continuous medical education, compliance with updated msbos, active role of htc and htt, and effective communication between clinician and blood bank staff have successfully reduced c/t ratio in four major departments in hkl. this successful achievement needs continuous monitoring and evaluation to ensure consistency. this can also be a role model that is shared with other hospitals to ensure that the c/t ratio is within the set target. 3a-s11-04 fusion) were collected. during second step, a modeling and simulation were used to define the optimal rcl inventory for the metropolitan area. average rc cell shelflife of regional inventory as well as the number of transfused rcs were calculated. in addition it was hypothesized that an efficient turnover of rc inventory will result in inventory reduction and relatively fresh blood for the transfusion reducing the blood utilization and frequency of transfusion among non-surgical patients especially those with chronic transfusion. results: dynamic inventory management and application of inventory index at regional level (four referral hospitals providing direct health care services to 1.4 and specialized services to 2.0 million population) reduced the regional rc inventory by 32% (1100 rcs to 750 rcs; optimal hospital inventory index of 7.5). this change in inventory was accompanied by a reduction in shelf-life of transfused red cells at 36% (average shelf-life of transfused rcs reduced from 3.45 to 2.21 weeks). the total annual rc utilization and specific categorical data of patients prior to and after the implementation of bump (2010 and 2012) included in table 1 . conclusion: optimization of rc inventory by application of inventory index improved the pattern of regional blood utilization. red cell utilization among chronically transfused patients was decreased by 20% (average). chronically transfused hematology and renal patients showed the highest reduction on rcu (21-26%, p value < 0.0001). that was no change in amount of surgical transfusions. non-surgical (medical) category showed a mild reduction (4%) but statistically was not significant. the results indicate that implementation of bump could significantly improve red cell utilization among chronically transfused patients. this change may also result in reduction of transfusion associated adverse reactions and long term complications like as iron overload. 3a-s12-01 no abstract available. 3a-s12-02 burnouf t 1 , tzeng ys 2 , deng sc 2 , wang ch 2 , tsai jc 3 and chen tm 2 1 taipei medical university, taipei, taiwan 2 tri-service general hospital, taipei, taiwan 3 tatung university, taipei, taiwan background: approximately 15% of diabetic patients develop chronic ulcers, and 25% of those may undergo foot amputation. activated platelet gel, which contains growth factors, has been proposed as an adjunct to promote healing of small diabetic foot ulcers. for large un-healing diabetic ulcers, skin graft is usually needed. we have demonstrated that single-donor allogeneic platelet gel and fibrin glue improve skin grafting to achieve successful persistent healing of large ulcers [1]. however, it is not known whether autologous platelet gel can be beneficial in this application. aims: to evaluate in a prospective study the safety and efficacy of using autologous platelet gel to enhance skin graft take in non-healing diabetic lower extremity ulcers. methods: approval was obtained from the institutional review board of our hospital. eight consecutive diabetic patients aged 25-82 with nine non-healing lower extremity ulcers (median size of 50 cm 2 ; range 15-150 cm 2 ) were enrolled. their median duration of diabetes and ulcer was 10.6 years (range 5-25 years) and 6.5 months (range 3-24 months). none of the patients had received conventional skin grafting in the past. prp was prepared from 100 ml of venous blood using sep-ax-vgr protocol (biosafe sa, switzerland). autologous thrombin was prepared by activating 10 ml of plasma (tgd-001; merries international inc., taiwan). skin ulcer was debrided to remove the infected and necrotic tissues and covered with moist saline dressing. daily dressing change without additional treatment was performed. the wound was sprayed after 7 to 10 days with equal volumes (5 to 7 ml) of autologous prp and autologous thrombin to form the platelet gel within 5-10 s. thin-splitthickness skin graft with multiple slits was then applied on the wound bed and fixed with staples or cat-gut sutures. each patient was placed on antibiotics during the course according to wound cultural results. bolster dressing with sofa-tulle were used to avoid post-graft hematoma formation. negative pressure wound therapy (vac) was not used in this study. results: there was no adverse reaction during the study. eight out of nine skin grafts took well (88% healing rate). the interval between skin graft and complete wound healing ranged from 2 to 3 weeks in the eight successful cases. no ulcer recurrence was noted during the 2-19 months follow-up period. the non-successful case was an attempt to treat an ulcer that was deep to the periosteum of calcaneus bone. free tissue transfer would have been required, but the patient refused the microsurgery, due to age and medical condition, which led to skin graft loss. conclusion: this study shows for the first time, to our knowledge, the possibility to use platelet materials in combination with skin graft procedures to treat large nonhealing diabetic ulcers of lower extremity recently, human platelet lysates (pl) rich in growth factors were shown to replace fbs for ex vivo expansion of various cells, but whether they can be used for cec expansion is unknown. aims: to evaluate the possibility to isolate and propagate cecs ex vivo using a xenogeneic-free, recombinant growth factor-free medium supplemented exclusively with human pl. methods: pl was prepared by cacl 2 activation of apheresis platelet concentrates from three volunteer donors, and centrifuged to obtain a fibrin-free supernatant that was heat-treated (56â°c/30 min; hpl) or not. pl was characterized for proteins, platelet growth factors (pdgf-ab, bdnf, egf and vegf) and chemical composition. cecs were obtained from over 10 bovine corneas (bcecs) using standard procedures and grown in a dmem-f12 medium (containing sodium bicarbonate, selenium, and antibiotics) supplemented either with (i) 5% fbs, 0.5% dmso, 2 ng/ml rhu-egf, 5 lg/ml insulin, 5 lg/ml transferrin, and 1 nm cholera toxin (termed '5% fbs medium'), or with (ii) 2.5%, 5%, 7.5%, or 10% pl or hpl as the only source of protein nutrients and growth factors. cells were grown in duplicates in 6, 24 or 96-well plates at 37â°c in a controlled atmosphere containing 5% co 2 , with medium changes every two days. viable cells were counted for 7 days and cell viability was determined by mtt assay. bcec phenotype was determined by immunostaining using anti-phospho-connexin 43, anti-na/k atpase alpha-1 subunit, anti-zo-1 and purified anti-n-cadherin. anti-mouse and anti-rabbit igg fitc were used as the second fluorescent antibodies. results: pl or hpl contained 55-60 mg/ml total proteins, and a range of approximately 30-40.5, 23.1-32, 0.44-1.82, and 0.24-0.39 ng/ml of pdgf-ab, bdnf, egf, and vegf platelet growth factors, respectively. cecs could be expanded in a med-ium supplemented with 2.5-10% pl or hpl. interestingly, better cell bcec morphology and adherence was found when using hpl compared to pl. cell growth and mtt equivalent to that of the '5% fbs medium' could be achieved only using 10% hpl. in addition, bcecs could be isolated from bovine corneas and subsequently expanded using the dmem/f12 medium supplemented with 10% hpl. bcecs expanded in the hpl-medium maintained their typical morphology, adherence, transparency and phenotype. conclusion: bcecs can be isolated and expanded ex vivo in a growth medium supplemented solely with human platelet lysate material. although further studies using cec from human origin are mandatory to confirm these conclusions, such findings open a possible new paradigm for gmp-compliant, clinical-grade ex vivo propagation of cec and regenerative therapy protocols of human corneal endothelium. platelets are the smallest and second most abundant circulating cells in the blood and their primary role is to maintain the integrity of the vasculature. when blood vessel injury occurs, platelet adhesion and activation receptors recognize subendothelial matrix proteins such as collagen and this can initiate a coordinated series of reactions leading to the formation of a fibrin clot to arrest bleeding. it appears, however, that in addition to hemostasis, platelets also have important inflammatory and immunological functions. as early as the 1960's, reports began to demonstrate that platelets may play an active role in the stimulation and regulation of immune responses. for example, platelets can store and secrete several pro-and anti-inflammatory chemokines (e.g. platelet factor 4 and rantes) and cytokines (e.g. interleukin-1b and transforming growth factor-b) that can affect local immune responses such as chemoattracting neutrophils to sites of tissue damage. on the other hand, platelets may be able to directly regulate adaptive immune responses via their ability to express and secrete cd40/ cd40l co-stimulatory molecules. more recent reports have also suggested that depending on their activation state, platelets may be able to either suppress cd8+ t cell responses or under certain circumstances, present mhc class i associated peptides to activate cd8+ t cells. these studies have suggested that platelets represent a critical link between innate and adaptive immunity. platelet mhc class i expression may also have a detrimental role by conferring tumor cell resistance against immune attack. of perhaps greater interest, platelets have been shown to express the entire family of tolllike receptors (tlr) and this may allow them to act as circulating sentinel cells that first encounter bacterial products for presentation to the innate immune system. in particular, surface expression of platelet tlr4 enables platelets to present lipopolysaccharide to mononuclear cells and neutrophils which modulates their phagocytic capabilities and this has implications for the development of immune platelet disorders. furthermore, tlr9 appears to be contained within a unique platelet granule underneath the cell surface that can be expressed by platelet activation. thus, elucidating the role of platelets in sepsis and a better understanding of the apparent central role that they play as immune cells may be important for the potential development of efficient therapeutic modalities against infections. this lecture will highlight the many characteristics of platelets as immune-like cells will discuss how platelets may be the major controllers of immune responses. macquarie university, sydney, australia malaria remains a major health problem in most of the tropics, and is especially burdensome in economically underprivileged areas. our ability to reduce the high rates of morbidity and mortality due to malaria are hampered by wanning efficacy of current antimalarial drugs and the spread of insecticide-resistant mosquitoes. we desperately need a greater understanding of how the plasmodium parasite succeeds in invading and growing within red cells, how the host responds to an infection, and importantly, the protective mechanisms employed by the host to combat the infection. platelets regulate blood haemostasis, but are now also regarded as an important component of the body's early innate defense against invading microbial pathogens. recently, my laboratory discovered that platelets are able to protect against a malaria infection. in mouse models of malaria, survival to a chronic infection is reduced when platelet levels are artificially depleted. purified human platelets directly bind to p. falciparum-infected red cells in culture and kill parasite within. our current work is exploring how platelets can kill intrerythrocytic malaria parasites. i will present our current understanding of the platelet and red cell molecules involved in the killing mechanism. these include the platelet cytocidal molecule, platelet factor 4 (pf4) and the erythrocyte duffy-antigen molecule, which binds pf4 and mediates the platelet killing effect. the critical requirement of duffy has lead us to propose that platelet-mediated protection against p. falciparum infection is compromised in individuals homozygous for the common duffy-antigen negative allele. 3c-s13-01 graduate school of medicine, the university of tokyo, tokyo, japan although bleeding is a major side effect of heparin, which is used for treatment of thrombosis, heparin also causes a prothrombotic adverse drug reaction called heparininduced thrombocytopenia (hit). hit is caused by the development of platelet-activating antibodies (hit antibody), which is directed against the heparin and platelet factor 4 (pf4) complex. these reactions accelerate platelet activation and coagulation, leading to thrombosis. thus, if hit is strongly suspected clinically in cases of thrombocytopenia and thromboembolism that occur during or after heparin therapy, it is vital to stop all heparins and start administering an alternative antithrombotic drug immediately. in japan, a test to screen for hit antibody (the automated immunoassays based on two types of chemiluminescent immunoassay and a latex-enhanced immunoturbidimetric assay) was approved as a clinical laboratory test in the medical insurance system, in september 2012. only the latex agglutination test is now widely used clinically because of its simplicity, convenience and cost-effectiveness. however, these immunological methods, including enzyme immunoassays (eias), which detect binding of antibodies to immobilized pf4/heparin complexes, may not be employed suitably. the immunological hit tests are useful in diagnosing hit because of their high sensitivity; however, they also often cause overdiagnosis of hit. the value of the selected cut-offs is the key element in ruling out hit. consequently, hit should be confirmed through laboratory detection of platelet-activating antibodies by using functional assays for the hit antibody; it must also be diagnosed based on careful consideration of the clinical picture. in order to diagnose hit properly, our laboratory asks clinicians to assess the pretest probability of hit by using the scoring system (the '4t' scoring: thrombocytopenia, timing, thrombosis, and other explanations). furthermore, our expert staff ensures that the diagnosis is correctly performed, since hit has not been fully recognized in clinical practice in japan as compared to in western countries. the functional assay for hit antibody has been regarded as the gold standard for diagnosing hit in patients in spite of its disadvantages. the platelet activation test procedure is cumbersome, the tests are technically challenging, and limited to specialized laboratories. additionally, the most important requirement for the test is the selection of platelet donors with high reactivity to the platelet activation antibodies. accordingly, in japan, there are very few places where the functional assay is conducted, whereas many institutions still assess patients with only the immunological assay. in our laboratory, the heparin-induced platelet aggregation method is performed, as isotopes such as radiolabeled serotonin release assay should not be commonly used in routine laboratories. in an attempt to improve the sensitivity of the functional assay, we developed two methods for increasing hit antibody reactivity in donor platelets. one is the cooling donor platelet method, used for improving reactivity, and the other a way of donor selection by using monoclonal hit antibody. further studies are necessary to introduce a simple assay method in ordinary laboratory testing to detect platelet-activating antibodies. 3c-s13-02 autoimmune or immune thrombocytopenia (itp) is an acquired bleeding disorder with a low platelet count mediated by immune-mediated mechanisms. this condition is seen in patients with various associated diseases, such as systemic lupus erythematosus, and can also occur without an underlying disease. production of igg autoantibodies to platelet surface glycoproteins, such as gpiib/iiia and gpib, is the hallmark of the disease. it has been thought that anti-platelet autoantibodies promote platelet clearance in the reticuloendothelial system, but recent findings indicate that anti-platelet antibodies also suppress megakaryogenesis, resulting in impaired platelet production. the diagnosis of itp continues to be one of exclusion. several antigen-specific assays for detection of anti-gpiib/iiia and anti-gpib antibodies are reported to be useful in identifying itp patients, but these assays require complicated procedures such as platelet solubilization, and use of commercially unavailable monoclonal antibodies. to solve these problems, we have developed an enzyme-linked immunospot (elispot) assay for detection of circulating b cells secreting igg anti-gpiib/iiia and gpib antibodies, which is a sensitive, specific, and convenient method for evaluating the anti-platelet autoantibody response. in addition, reticulated platelets and circulating thrombopoietin (tpo) are useful in evaluating platelet production status. these findings led us to propose preliminary diagnostic criteria for itp based on a combination of itp-associated laboratory findings, including erythrocyte and leukocyte counts, anti-gpiib/iiia antibody-producing b cells, platelet-associated anti-gpiib/iiia antibodies, percentage of reticulated platelets, and plasma tpo. although the etiology of itp remains unknown, complex dysregulation of the immune system is observed in itp patients. based on a series of experiments using cd4+ t cells reactive with gpiib/iiia derived from itp patients, we have proposed a 'continuous pathogenic loop' model as a mechanism that explains ongoing antiplatelet antibody response in itp patients. this model includes b cells that produce anti-platelet antibodies, reticuloendothelial macrophages that phagocytose opsonized platelets via fcc receptors and present platelet-derived antigenic peptides, and platelet-reactive cd4+ t cells that exert their helper activity upon recognition of the antigenic peptides. once this pathogenic loop is established, anti-platelet antibody production would go on endlessly. recently, regulatory systems that control this pathogenic loop are attracting a great deal of attention. a series of studies in itp patients have found that foxp3+ regulatory t cells are reduced in circulation, bone marrow, and spleen, and are deficient in their suppressive function. in addition, a critical role of regulatory t cells in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in regulatory t cells, which spontaneously develop anti-platelet autoantibody-mediated thrombocytopenia. in addition, our recent analysis indicates that the eradication of helicobacter pylori leads to up-regulated expression of inhibitory fccgriib on macrophages, resulting in the attenuation of the pathogenic loop. therefore, therapeutic strategies aimed at interrupting this pathogenic loop would inhibit anti-platelet autoantibody production and subsequent increase in platelet count. in fact, current treatment regimens for itp, including corticosteroids, splenectomy, and rituximab, are able to suppress the pathogenic loop. interestingly, tpo mimietics have a potential to induce peripherally induced regulatory t cells, resulting in suppression of the pathogenic loop. 3c-s13-03 seguchi s 1 , maeda t 1 , kanaumi y 1 , kawamura s 1 , kodama m 1 , kawai t 1 , okazaki h 2 and miyata s 1 1 national cerebral and cardiovascular center, osaka, japan 2 the university of tokyo hospital, tokyo, japan background: heparin-induced thrombocytopenia (hit) is a devastating immunemediated thromboembolic complication of heparin therapy. heparin administration can cause conformational changes in platelet factor 4 (pf4), resulting in the production of anti-pf4/heparin antibodies. a subset of these antibodies can activate platelets and monocytes (hit antibodies), leading to thrombocytopenia and a thrombininduced hypercoagulable state. up to half of hit patients suffer from arterial or venous thrombosis. if platelet concentrates are transfused into hit patients, it is conceivable that the transfused platelets can be activated by the same mechanisms that affect the patient's own platelets and trigger the onset of new thromboembolism or exacerbate hit-associated thromboembolism. thus, platelet transfusion is thought to be contraindicated in acute hit patients. however, it remains uncertain whether platelet transfusion is a risk factor for thrombosis in hit patients since only a few studies have investigated this issue systematically. aim: the goal is to clarify whether platelet transfusion increases the risk of thrombosis in hit patients. methods: we constructed a nationwide registry for hit with the approval of the ethical review committee. between august 2008 and may 2013, 329 patients from 185 hospitals clinically suspected of having hit were retrospectively included in the registry with clinical information such as changes in platelet count, timing of heparin administration, episodes of transfusion, thromboembolic events, and the results of serological assays for hit antibodies. hit was definitely diagnosed by the detection of anti-pf4/heparin igg with platelet-activating properties at a therapeutic heparin concentration, but not at a high heparin concentration or with anti-fccriia antibodies. the assay was performed using washed platelets prepared from hit antibody-sensitive healthy volunteers at a reference laboratory. we examined patients who received transfusions of platelet concentrates after hit was suspected. results: of the 329 patients, 85 patients were ultimately diagnosed with hit (25.8%). optical density values of anti-pf4/heparin antibodies detected by elisa were significantly higher in hit patients than in non-hit patients (2.3 ae 0.95 vs 0.36 ae 0.53 for igg/a/m, p < 0.001; 1.68 ae 0.80 vs 0.25 ae 0.37 for igg, p < 0.001). the incidence of thromboembolic events was significantly higher in hit patients (51.8%) than that in non-hit patients (28.3%; p < 0.001). among the 85 hit patients, 20 patients received platelet transfusions after the onset of hit. only two of them experienced a thromboembolic event after platelet transfusion, one within 24 h and the other after 9 days. notably, neither patient was being treated with a thrombin inhibitor at the time. the incidence of thromboembolic events in hit patients who received platelet transfusions was not significantly higher compared to hit patients who did not receive platelet transfusion or non-hit patients who received platelet transfusions after the suspicion of hit arose, respectively. conclusions: to our best knowledge, this is the first systematic report that clarifies the clinical impact of platelet transfusion on the occurrence of thromboembolic events in acute hit patients whose diagnosis was confirmed by a washed plateletactivating assay. even in acute hit patients who possess platelet-activating antibodies, the transfusion of platelet concentrates does not appear to increase the risk of thromboembolism, especially while on thrombin inhibitor therapy. 3c-s13-04 lu p, ling b and li rs background: transfusion platelet matches with antigenic similarity would evoke less allorecognition and immune activation. strategies have been based on the theory that selection for hla-a and hla-b cross-reactive groups (cregs) compatible donor as well as abo/hpa-matched donor will predict good increment in platelet corrected count after platelet transfusion. aim: establish large-sized platelet donor registry with hla class i,hpa,abo-typed to meet the needs of immunized patients with platelet transfusion refractoriness. evaluate the effectiveness of platelet transfusion therapy in ptr patients. progressive management to ptr patients maintain a long-term platelet transfusion strategy. methods: to establish platelet aphaeresis donor registry in shanghai, 1931 repeat donors were typed for hla-a, -b and hpa-1,-2, -3, -4, -5, -6 and -15 using standard pcr-ssp method. eighteen patients with hematologic or oncologic diseases which refractoriness to platelet transfusions from random donors who are receiving 36 units of apheresis platelet products transfusion were studied. results: eighteen patients(eight male, 10 female)showing platelet refractoriness to random donor platelets [1 h corrected count increment (cci) <7500 ml/m 2 , percentage of platelet recovery (ppr) <20%] before. patients phenotyped for both hla-a,b and hpa-1,-2, -3, -4, -5, -6 and -15. apheresis platelets from donor registry in shanghai matched to patients abo, hpa and hla-a and hla-b cross-reactive groups (cregs) are transfused. ten patients ( show 24 h ppr >20%. the mean 1, 24 h cci and ppr values from the best donors were significantly higher than those from random donors they transfused before. conclusion: the use of hla-a,-b and hpa,abo-compatible aphaeresis platelet improves posttransfusion 1, 24 h cci values and percentage of platelet recovery in refractory patients. transfusion with hla-a,-b and hpa,abo-matched platelets is mandatory to reduce the risk of bleeding in ptr patients. refractoriness to platelet transfusions developed at least in 50% of the patients we observed and to maintain a long-term platelet transfusion strategy. establish large-sized platelet donor registry with hla class i, hpa, abo-typed may be needed to circumvent platelet-specific antibodies of unknown specificity in all chronically transfused patients. the optimal strategy for platelet substitution in immunized patients remains a challenge. 3c-s13-05 xia w 1 , xu x 1 , ye x 1 , fu y 1 , deng j 1 , liu j 1 , ding h 1 , chen y 1 , shao y 1 , wang j 1 , li h 2 and santoso s 3 1 guangzhou blood center, guangzhou, china 2 department of biotechnology, guangdong food and drug vocational college, guangzhou, china 3 he institute for clinical immunology & transfusion medicine, justus-liebig univ., giessen, germany background: immunization against cd36 leads to the production of anti-nak a antibodies associated with fetal/neonatal alloimmune thrombocytopenia (fnait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). however, no data regarding the clinical relevance of cd36 immunization is currently available for chinese population. study design and methods: platelets and monocytes derived from 998 healthy blood donors were typed for cd36 deficiency using flow cytometry. in addition, four patients with suspected fnait (one case) and ptr (three cases) were investigated. nucleotide sequencing was performed to identify the mutations underlying the cd36 deficiency. transfection in mammalian cells (hek-293t) with cd36 mutated constructs was conducted to confirm these results. anti-nak a antibodies were screened by the use of platelet solid-phase kit (pak-plus, gti diagnostics). results: of 18/998 blood donors failed to express cd36 on their platelets surface. in 5/12 individuals no cd36 expression was detected both on platelets and monocytes, suggesting that the frequencies of type i cd36 deficiency (platelets and monocytes) and type ii cd36 deficiency (platelets only) were approximately 0.5% and 1.3%, respectively. nucleotide sequencing analysis of type i cd36 deficient individuals revealed eight different mutations; four of them were not described so far. however, 1228-1239del attgtgcctatt and 329-330delac appeared to be the most common mutations related to type i cd36 deficiency in south chinese population. further analysis showed that the presence of anti-nak a antibodies in one healthy donor (donor 1) as well as in three cases of ptr (patients 2-4) and one case of fnait (patient 1). these results could be confirmed by immunoprecipitation using biotinylated platelets and by antigen capture assay with stable transfected cd36 cell lines. in all ptr patients, transfusion with platelets derived from cd36 negative donors resulted in good increment (24 h, ppr >20%). table 1 shows the mutations found in these five gpiv defective individuals. conclusions: more than 0.5% of cd36 type i deficient individuals are at risk to be immunized through blood transfusion or pregnancy in china. in this study, we could demonstrate that this immunization is of clinical relevance for the development of ptr and fnait. therefore, testing of anti-nak a antibodies should be considered in suspected immune mediated thrombocytopenia. a national registry of cd36 deficient blood donors should be established to maintain bleeding disorders associated with anti-nak a antibodies. since immunization against cd36 is conceivable for other asian populations an international network within laboratories in south asian region should be established in the future. 3c-s14-01 do we really need ffp? the evolving role of pf24 and pre-thawed plasma devine d fresh frozen plasma (ffp) is defined as plasma frozen within 8 hours of collection. while this product maintains a high functional activity of both coagulation factors and anticoagulant proteins, there has been recent movement in some jurisdictions away from reliance on ffp. in many blood systems, an increasing role for plasma frozen within 24 h of collection (fp24) is seen. such plasma shows little difference in functional protein levels when compared to ffp, with the exception of fviii levels which a show time related decay of activity. even factor viii loss can be consistently minimized if whole blood is held on controlled rate cooling plates prior to preparation of fp24. in addition, the activity profiles of coagulation proteins in fp24 prepared in routine production closely resemble those of commercial pooled plasma products. taken together, these observations have led many blood systems to move from the exclusive use of ffp to a mix of inventory of ffp and fp24, if not to the complete removal of ffp from their menu of offerings. the preparation of cryoprecipitate has also been a driver for the retention of plasma frozen within 8 h of collection. since the most common labeling of cryoprecipitate has focused on the content of both fibrinogen and factor viii, in part owing to original role of the latter in the treatment of hemophilia a, collection of ffp has persisted as the starting material for cryoprecipitate production. in jurisdictions where hemophilia or other factor viii deficiencies are treated with factor concentrates, the labeling of cryoprecipitate to emphasize its antihemophilic factor activity is no longer warranted. as data began to accumulate on fp24, similar studies began to appear that investigated the effect of prolonged cold storage of plasma that had been thawed. this led to the introduction in some jurisdictions of the extension of the allowable period of use for thawed plasma from 24 h to up to 5 days, if stored at 4â°c. such practice is increasingly widespread and there is no evidence that patients receiving such products are compromised. from the perspective of health resources management, the use of both fp24 and pre-thawed plasma reduces discard of products or prevents the use of these products in non-group specific recipients. with the advent of massive transfusion protocols which may require pre-thawed plasma at the ready, it also allows better use of relatively scarce but high demand products such as ab plasma. this presentation will focus on a review of the relevant studies of plasma quality for ffp, fp24 and pre-thawed plasma. we will review the appropriate uses of these different components as well as groups of patients for whom specific products should be restricted or supplemented. 3c-s14-02 background: in 2008, the australian red cross blood service (blood service) began a programme of process improvement aimed at maximising the manufacture of clinical plasma components from male donors, which is a key mitigation to the risk of trali (transfusion related acute lung injury). the challenge of sourcing all clinical plasma from male donors is exacerbated in australia due to its adherence to the council of europe guidelines that stipulate a maximum allowable time between collection and freeze of apheresis-derived clinical plasma of six hours, which is considerably more stringent than for most other blood services despite the greater tyranny of distance that exists in australia. aim: the aim of the programme was to achieve a result of 100% of clinical plasma sourced from male only donors. methods: work began in early 2008 to gather detailed quantitative data that linked information on donor panel to collection centre to production facility, and that could be broken down by blood group and by day of the week. this was then formulated into a suite of reports, highlighting opportunities and variance in performance. based on those reports, a cross-functional team designed a range of improvement initiatives across disciplines such as transport, systems enhancements, donor acquisition and processing, such as: incoming blood donation shippers were marked with colour coded labels that notified the receiving production facility of clinical suitability. this assisted with prioritisation and workflow management. additional deliveries of blood from collection centres to processing centres. a range of targeted campaigns and marketing collateral were produced to attract male donors to apheresis plasma donation donor centre collection staff were trained to convert male ab donors over to plasmapheresis donation activity. changes to progesa to prevent manufacture of female plasma were made (after a time). results: the first report in july 2008 showed that the blood service were issuing 87.71% male clinical plasma; the group ab rate was 57.86%. the results improved dramatically by november 2010 in groups o, a and b (99.64%, 99.72% and 99.62% respectively), allowing for progesa to be configured to prevent the routine manufacture of female plasma from those groups, whilst still allowing supervisor over-ride. by march 2012, the results in group ab had improved to 97.77% (chart below) and the directive was given to manufacture male clinical plasma only as routine. january 2013 was the first month ever where 100% of all clinical plasma was sourced from male donors only. in 2012/13, 99.98% of clinical plasma was male onlythe 0.02% constituted short lead time requests for iga deficient plasma. subsequent to a system change that allowed for a donor identification marker for iga deficient donors, inventory levels of this sub-product have increased by 34%, negating the need to turn female production on to accommodate sporadic demand. summary/conclusion: the multi-disciplinary efforts over an extended period of time have resulted in the practical removal of the risk of trali in australia. this is an achievement that many thought impossible and one that many other blood services have been unable to attain. results: quality control (qc) parameters were measured in the prepared blood components and were listed in table 1 . by standardising bc volume and haematocrit in the primary separation, recovery of red cells and plasma was optimised in both the red cells in sagm and plasma units while wbc and rbc contamination levels in pc and plasma were maintained low. all parameters were well within the blood component specifications set out in the council of europe guide (16th edition), the standard adopted by hkrcbts. qc parameters of the pathogen reduction-treated platelet concentrates in pas so produced were also within the hkrcbts blood component specifications. low contamination with red cells and white cells were demonstrated and the ph range was acceptable after 5 days of storage at 20-24â°c. the new t&b production method for the separation of 450-ml quadruple wb and the preparation of intercept platelet concentrates in ssp+ pas was successfully developed and can be applied to the production of high quality blood components in preparation for clinical evaluation of the pathogen reduction-treated platelet concentrates. 3c-s15-01 blood donors are healthy volunteers who give whole blood or blood components by apheresis for altruistic motives. they should be managed in a way that ensures high standards of care. nevertheless, there are recognized adverse reactions that can occur during blood donation. the overall incidence of complications directly related to blood donation is 1%. they are generally more common in women, in younger and in first-time donors. although the incidence seems to be small, it is of great importance considering the large number of people giving blood each day worldwide. adverse blood donation reactions can generally be minimized or avoided by appropriate donor selection and care, and appropriately trained staff. vasovagal episodes and soft tissue injuries (bruises/haematomas at the venepuncture site) are the most common donor reactions. the majority of these are minor and donors usually recover quickly; however, these reactions can be of concern to donors and reassurance should be provided. other reactions include nerve injury and arterial puncture which, although less frequent, may require medical care outside the blood service and may lead to prolonged symptoms or incapacity. staff should be trained to recognize and manage such adverse reactions, including the provision of first aid. the incidence of bruising should be monitored so that further venepuncture training may be provided to staff as necessary. iron deficiency in regular blood donors has been a top donor health and safety concern in many countries. as each donation causes a loss of 213-236 mg of iron, repeat donation can lead to a continuous depletion of body iron stores. studies have shown that donation frequency had the greatest impact on iron deficiency and further risk factors were lower weight and female gender. to address this issue, many blood services encourage donors to take iron-rich food and/or give them iron supplements. adverse reactions such as delayed faint may occur after the donor leaves the donation venue. donors should be advised to inform the donor centre staff of any ill-effects they suffer after donating. a system for the reporting and investigation of adverse donor events and reactions should be in place as part of the donor haemovigilance system. all adverse events and reactions in donors should be identified, documented and reported. these data should be regularly analysed for possible corrective and preventive actions. the goal of donor haemovigilance is to reduce the occurrence of adverse events and reactions and improve the outcomes both for donors and patients. for various reasons, even today, donors often do not receive detailed information on the blood donation procedure and possible complications. not only do blood services have the ethical duty to inform donors of possible adverse events to enable them to give informed consent and take action for preventing adverse effects, protecting the safety of donors is also important for donor retention because a safe and good donation experience ensures donors will return regularly. 3c-s15-02 wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: without blood donations and the availability of blood transfusion, many important therapeutic advances could not have been achieved. donor hemovigilance is the systematic monitoring of adverse reactions and incidents in the whole chain of blood donor care, with a view to improving quality and safety for blood donors. method: this 'global update' draws on work by the international haemovigilance network and international society for blood transfusion haemovigilance working party, experience in the netherlands, as well as a pubmed search using terms blood donor and adverse reaction. results are discussed for vasovagal reactions, needlerelated complications, long-term morbidity, donor iron status and frequent apheresis. results and discussion: the occurrence of vasovagal reactions is associated with young, female donors, lower body weight and estimated blood volume, first-time donor status. a reduction of vasovagal reactions has been documented with use of a water drink before donation, muscle tensing, social distraction and lower collection volume for donors with small estimated blood volume. needle injury is relatively frequent as a cause in cases of long-term morbidity; needle injury is associated with traumatic phlebotomy and in some cases nerve damage is documented. repeated whole blood donations lead to reduction of body iron stores and in some cases anaemia. some blood services adjust donation intervals to avoid or reduce this, while others have or are considering a policy of iron replacement therapy. fewer studies on acute complications in plasma and other types of apheresis have been published. preliminary studies of bone density and protein levels in non-commercial frequent plasma donors have not substantiated any specific hazard despite theoretical concerns of calcium or protein depletion. international collaboration in strengthening donor vigilance definitions and data analysis may in future increase potential for study of risk factors and measures to improve donor care worldwide. conclusion: donor vigilance is gaining international interest and has increased knowledge of risk factors for vasovagal reactions associated with blood donation. there remains a need of research and of developing preventive measures, including prevention and treatment of needle injury as well as possible long-term effects of frequent donation. assuming that these donors were newly infected, it is crucial for bts to monitor the prevalence of this category of donors in order to strategize specific measures to these targeted groups to improve blood safety. aims: this study aimed to profile blood donors who donated during the hiv serological window period and to identify the risk factors of these blood donors. methods: past donor records of blood donors who had donated blood during the hiv serological window period (nat ultrio and discriminatory detected and negative for both anti-hiv and p24 antigen) at nbc or at blood mobiles organized by nbc from november 2007 until july 2013 were retrieved and analyzed using spss 15.0. results: a total of 18 donors were nat detected and negative for anti-hiv and p24 antigen (none in 2007, 2 in 2008, 2 in 2009, 1 in 2010, 6 in 2011, 4 in 2012 and 3 in 2013 introduction: in pakistan, the predominant reliance for blood supply is on the replacement donors, as sufficient numbers of voluntary blood donors are not available. an increase in the proportion of voluntary donors following the promotion of the concept of voluntary non-remunerated blood donation (vnrbd) will enhance safety and will also help to shift the responsibility for arranging blood availability from the patients to the health care system. objective: the objective of the current study was to promote vnrbd through a public awareness campaign (pac) based on a thorough analysis of the knowledge, attitudes and practices (kap) of a key segment of the society, i.e. 18-25 years old college and university students. material and methods: a cross-sectional, descriptive study was conducted over a period of three months (jan-mar 2012). multi-stage random cluster sampling approach was followed and college and university students were targeted through 20 university based blood donor organisations (bdos) out of a total 56 bdos identified. all the participants voluntarily participated in the study and informed consent was obtained orally. a pre-tested questionnaire comprising of 28 questions related to knowledge, attitudes and practices was applied. the questionnaire was kept anonymous and each question included multiple options or statements. statistical analysis was conducted by the assistance of statistical package for social sciences (spss) software version 17. results: a majority (65%) of the students had heard about blood donation through family/friends and a minority (9%) through the internet, although 45% preferred internet as spare time activity. majority (+85%) of the students had access to the internet and mobile phone. more than 50% of the respondents had donated blood: 22% donated for family, relatives or friends, 4% donated voluntarily as an act of altruism, and 25% donated voluntarily once and then stopped donating, but 55% of these respondents still considered themselves as volunteer blood donors. 35% indicated that important people in their environment had an influence on crucial decisions that they made. motivation for blood donation was a desire to help other people (73%), 28% followed friends invitations, in 21% cases respondents family or friends had received blood transfusions, 16% followed the example of fellow students. restriction for blood donation: 40% generally feared donation, 25% had a specific fear of the needle, 20% had no confidence in the public (health) sector, 25% condemned blood selling practice, 14% had no confidence in the donation procedure (hygiene), 13% experienced parental discouragement. conclusion: to overcome the apprehensions and fears of the donors it is important to provide adequate information about donation to potential donors. this strategy will help convince family replacement donors to become vnrbd and also recruit healthy individuals to become a vnrbd. the approaches and strategies for this transition can be based on the findings of the study. the reported preference for internet as leisure time activity suggests that internet can be utilized as an important tool for information dissemination in a pac, for which detailed study is required. 3d-s16-01 murphy mf platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. there are immune and non-immune causes of platelet refractoriness. the main immune cause is hla alloimmunisation which occurs predominantly in females with a history of pregnancy. other immune causes include hpa alloimmunisation, abo incompatibility, platelet autoantibodies and drug-related platelet antibodies. the incidence of alloimmune platelet refractoriness due to hla antibodies has declined due to leucocyte-reduction of blood components and more aggressive treatment for patients with haematological malignancies and other cancers. in current practice, platelet refractoriness is mainly due to shortened platelet survival associated with non-immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, dic and splenomegaly. if there are poor responses to hla-matched platelet transfusions, the reasons should be sought including hla incompatibility which is most likely to occur in patients with unusual hla types with few well-matched donors, non-immune platelet consumption, and hpa and abo incompatibility. further serological investigations including testing for hpa antibodies may be used to differentiate between these possibilities. depending on the results, the appropriate management could be the use of abo-identical or hpa-matched platelet concentrates if the specificity of the hpa antibodies can be identified. platelet crossmatching may be helpful in some patients with non-specific hpa antibodies. the management of patients with hla and/or hpa alloimmunisation and no compatible donors may be very difficult. there is no evidence that alloimmunised patients benefit from incompatible platelet transfusions which do not produce an increase in the platelet count, and prophylactic platelet support should be discontinued. if bleeding occurs, platelet transfusions from random donors or the bestmatched donors, despite being incompatible, may reduce the severity of haemorrhage although increased doses of platelets may be required. other management approaches such as the use of high-dose intravenous immunoglobulin, splenectomy, and plasma exchange have not been shown to be effective. the management of patients with non-immune platelet consumption is similarly problematic. the usual practice is to continue with daily platelet transfusions as prophylactic platelet support, but it is not known whether this approach is effective, or whether platelet transfusions should be discontinued or the dose of platelets increased. 3d-s16-02 managing bleeding in cardiac surgery: despite major advances in the management of perioperative blood conservation, transfusion rates in cardiac surgery remain very high, with large variations among individual centres. among all major surgical procedures, cardiac surgery with cpb still consumes a large part of the available blood supply. in england indicated that 10-15% of the blood units supplied by the national blood service is used in cardiac surgery units. in the usa, nearly 20% of blood transfusions are associated with cardiac surgery. during the early history of cardiac surgery, patients received large amounts of allogeneic blood. in the 50's, most operations were performed to correct congenital heart disease. during the 60's and 70's, the introduction of satisfactory valve prosthesis and direct grafting for atherosclerotic coronary artery disease led to rapid growth in the scope and number of patients having open heart surgery. in the 80's pharmacological methods to reduce bleeding were introduced and the focus of blood conservation was expanded to include blood components as well as red cells. with increasing application of cardiac surgery in acutely ill older patients with more comorbidities as well as the increasing safety of blood supply have contributed to an increasing incidence of allogeneic transfusions. not surprisingly, physicians, surgeons and anaesthesiologists have shown a great interest in the promotion of safe and effective alternatives to the transfusion of allogeneic blood in cardiac surgery. perioperative risk factors for allogeneic transfusion can be regrouped in three main categories: factors affecting the patient's preoperative rbc mass, factors affecting the perioperative blood losses, and factors affecting the transfusion practice. the ability to predict a patients risk for transfusion allows modification of patient management with the goal of decreasing allogeneic transfusions. using the trac and trust scoring system predicts candidates likely for transfusion. diminished rbc mass appears to be one of the strongest predictors of transfusion. the acceptance of a lower postoperative haematocrit (in ijn the hct on bypass is 20-25% and post bypass is >25%) or haemoglobin concentration represent an important element in current blood conservation practice. the decision to transfuse a patient cannot be based only on haematocrit concentration. optimizing preoperative rbc mass involves the early detection of anaemia and its correction before surgery. preoperative autologous blood donation can be used to conserve allogeneic blood. besides economic concerns, one essential argument against pad is the lack of sufficient time because of the uncertainty of waiting list. erythropoietin has also been used to augment pad in elective cardiac surgery. acute normovolaemic haemodilution (anh) aims at reducing allogeneic blood exposure through a reduction in the net red blood cell mass lost during or just after surgery. perioperative cell salvage (cs) also aims at reducing allogeneic blood exposure through a reduction in perioperative blood loss. antifibrinolytics (ltranexamic acid or epsilon aminoaproic acid) or serine protease inhibitors (aprotininnow unavailable) may reduce excessive fibrinolysis and platelet dysfunction. the use of activated f vii has been reported in intractable bleeding post cardiac surgery. 3d-s17-01 the university of tokyo, tokyo, japan antibodies against human neutrophil antigens (hna) are involved in the pathogenesis of immune neutropenia, such as neonatal alloimmune neutropenia (nan), refractoriness to granulocyte transfusions, and transfusion reactions, such as febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury (trali). the hna systems are assigned to five antigen groups, namely hna-1 to 5. hna-1, -2, and -3a alloantibodies have been implicated in the pathogenesis of trali, and especially hna-3a alloantibody has been found in the severe cases requiring artificial ventilation or with fatal reactions. besides alloantibodies to hna-1, -2 and -3, those against hna-4a and hna-5a have been implicated in nan. the identification of the causative antibodies is essential for the diagnosis as well as for the prevention of these disorders. the detection of hna antibodies has been mainly dependent on cell-based assays so far. among them, the granulocyte agglutination test (gat), the granulocyte immunofluorescence test (gift) and the monoclonal antibody immobilization of granulocyte antigens (maiga) are the most commonly applied. according to the isbt working party on granulocyte immunobiology, the combination of gat and gift is presently the best means of hna antibody detection. gift is usually more sensitive than gat, however, hna-3a antibodies associated with severe cases of trali are better detectable by gat. in gat and gift, the presence of hla antibodies with broad specificities may affect the detection of hna antibodies. on the other hand, the maiga assay allows the differentiation between hna and hla antibodies. these classical methods, however, require fresh neutrophils from hna-typed donors. also, these assays are time-consuming, which makes them not appropriate for the large-scale antibody screening. in our lab, we modified the mixed-passive hemagglutination (mpha) assay, the method largely applied in japan for platelet antigen/antibody detection, for the detection of hna antibodies. recently, alternative assays have been developed, including elisa with recombinant hna (rhna), and immunofluorescence tests with transfectant cells of hna (hna-1, -2, -4, and -5). more recently, the molecular basis of hna-3 antigen has been elucidated, and stable cell lines expressing hna-3 antigens became available. these cell lines seem to have low background, and do not express detectable levels of hla antigens, which make the identification of hna antibodies easier. additionally, kits that use luminex microbeads coated with hna antigen are being developed. these kits, however, do not include hna-3 antigens. these new technologies significantly help improving the detection and identification of hna antibodies, and allow the large scale screening of hna antibodies, contributing for the reduction of the risk of the pathological conditions associated with hna antibodies, especially trali. however, these new technologies significantly increase the cost of the tests. presently, although many assays have been developed, the standard hna antisera are not necessarily available in every lab, which makes their validation difficult to be conducted. thus, the collaborative study among the various labs, by exchanging the available antisera, and comparing the test results, is essential for the improvement of this field. 3d-s17-02 one of the main sites where pmns carry out vital to surveillance functions is in the lungs. the large surface area of the lung is needed for gaseous exchange but lungs also present a vital direct mechanical barrier to the external environment. to patrol and protect this interface, about 28% of the body's total pmns are located in the pulmonary microvasculature. illness may increase the number of lung pmns as well as change their phenotype from quiescent to primed. in trali, the transfusion of blood products with either pmn reactive antibodies or biological response modifiers can activate this concentration of primed pmns to produce an augmented respiratory burst. this causes injury to the pulmonary microvasculature and consequently the symptoms of trali. circulating antibodies to pmns also can compromise their numbers and function. pmns carry human neutrophil antigens (hna) as well as class i hla, which can become targets for pmn reactive antibodies. the granulocyte immunofluorescence test (gift) and granulocyte agglutination test (gat) are primary tools for investigating these pmn reactions, as they are able to detect reaction of hna as well as some hla class i antibodies. immune neutropenias: alloimmune neonatal neutropenia (ann) occurs when a neonate's pmns are destroyed by transferred maternal antibodies developed against an inherited paternal neutrophil antigen. this is similar to haemolytic disease of the newborn, but importantly can occur with the first pregnancy. in early childhood, some children develop severe neutropenia as a result of pmn auto-antibodies. although the pathogenesis of such chronic benign autoimmune neutropenia is still not understood most of these autoantibodies demonstrate specificity for the hna system. passively acquired autoimmune neutropenia, wherein pmns are destroyed by maternal pmn auto-antibodies crossing the placenta are a rare finding. hna specificity is unlikely. autoimmune neutropenia in adults is either primary, secondary to another autoimmune disease or drug related. it can present a clinical and diagnostic challenge as many adults invariably have alloantibodies to neutrophils and the patient's neutrophil count is too low to make a definitive identification of a self reactive autoantibody. hna specificity is extremely rare. the severity of trali and immune neutropenias demand rapid and precise diagnosis with reliable neutrophil serology. the isbt granulocyte immunobiology working party maintains a list of granulocyte immunobiology reference laboratories around the world. 3d-s17-03 when seven sera from 778 donors were screened for neutrophil specific antibodies, 9% samples showed positive reaction. these results, however, could not be confirmed by gift and gat. conclusions: in this study, we found alloimmunization against hla class i and ii in 4.6% male,~4.3% nulliparous and~13.9% parous females. in contrast, alloimmunization against hna was not detectable in this cohort. these results indicate that the use of plasma containing blood products from parous females without hla antibodies pre-testing may increase the risk of trali reaction. although alloimmunization against hna seems to be a rare event in china, further observation is necessary to exclude the necessity of hna antibodies screening in our blood products. it is becoming clear that the ccn family of extracellular proteins play an important role in the health and function of several cells of the hematopoietic lineage. ccn2, also known as connective tissue growth factor, ctgf, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting a role in blood clotting and/or wound healing. on the other hand, ccn3, also known as nephroblastoma-overexpressed, nov, has been found to play an important role in hematopoietic stem cell health and function. in fact, treatment with ccn3 has recently been shown to promote hematopoietic potential, a discovery with dramatic clinical potential. initially using bioinformatics, our laboratory has discovered a signaling pathway that connects these two discoveries and appears to be a key functional node within the development of blood cells. specifically, we have found that the myeloid zinc finger protein 1, mzf1, is a transcription factor that trans-activates both ccn2 and ccn3, in distinct cell types. for example, we have shown that mzf1 can stimulate ccn2 production and secretion in stromal fibroblasts, which is then taken up by megakaryocytes and loaded into platelets. this is the first time that ccn2 loading into developing platelets has been directly achieved and observed in vitro. secondly, we have discovered that mzf1 also regulates the synthesis of ccn3 in several hematopoietic cell types. putting these results together with previous data suggests a new and immediately testable clinical treatment. it is known that both vitamin d (calcitriol) and vitamin a (all-trans retinoic acid) stimulate transcription of the mzf1 gene. (we also have new data exploring the mechanism and suggesting other pharmacological ligands). we have confirmed that treatments with either vitamin a or d activate this pathway and results in increased production of ccn2 in stromal fibroblasts, which in turn results in enhanced loading of ccn2 into developing platelets in vitro. similarly, we have observed that both vitamin a and vitamin d induce ccn3 expression, through mzf-1, and we are currently testing if this will lead to enhanced hematopoietic potential. this work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias. 3d-s18-05 background: foxp3 + t regulatory cells (tregs) consisting of natural and induced treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. while recent studies demonstrated that natural tregs are instable and dysfunctional in the inflammatory condition, induced tregs (itregs) may have a different feature. furthermore, it was reported that tolerogenic dendritic cells (tdcs) could expand itregs in vitro and this action designed to correct defects in numbers or functions of itregs may be therapeutic in the treatment of autoimmune diseases. in this study, the suppression efficacy of tgf-beta-induced tregs expanded by tdcs in vitro and in mouse model of autoimmune arthritis was determined. method: in vitro, first, cd4+ cd25ã� t cells were purified from splenocytes of d1 mice and stimulated by anti-cd3/cd28 in the presence of tgf-b1 for 5 days, which were termed 'itreg'. and tdcs derived from bone marrow of d1 mice were induced by gm-csf, il-10 and tgf-b1 and harvested after 10-day cultivation. then, itregs were expanded by tdcs at the ratio 5:1 and collected after 4 days (termed 'itreg tdc '). the phenotype, proliferation, suppression of cd4t proliferation, induction of foxp3 + tregs from foxp3ã� t cells and suppression of th17 cell differentiation were assessed. for in vivo experiments, the animal models of ra were established. in this model, arthritis was induced in d1 mice after immunized with bovine type ii collagen (cii) on day 0 and day 21, termed collagen-induced arthritis (cia). and 1 9 10 6 itregs or itreg tdc cells were transferred <iv. into cia recipient mice when cia onset. control mice were treated with pbs. clinical and histopathologic scores, cytokine and anti-cii antibody secretion in serum and cd4 + th subsets were analyzed. results: after 5-day induction in vitro, the percent of foxp3 + itregs increased from 9.8 ae 1.5% to 54.9 ae 2.5%. and these itregs could be expanded effectively by tdc in additional 5-day co-incubation, which continued to express higher levers of foxp3 (82 ae 3.6%) and increased 4.17 ae 0.17 fold. compared with itregs, the itreg tdc cells showed stronger inhibitory effect, and could prompt cd4t cells converting to foxp3 + itreg and resistant to th17 cell differentiation more effectively. during the 4-weekobservation-period, a more remarkable anti-arthritic activity, improved clinical scores and histological end-points were found in the itreg tdc -treated group than those in itreg-treated group. serological levels of tnf-a, il-6, il-17 and anti-cii antibodies were also significantly lower and tgf-b production were higher in cia mice following adoptive transfer of itreg tdc cells as compared with itregs. moreover, the itreg tdc treatment resulted in an increase in the number of tregs (cd4+ foxp3+) and a decrease in the percentage of th17 cells (cd4+ il-17+) more obviously. conclusion: these results indicated that itregs expanded by tdcs showed more effective suppression in vitro, and reduced more markedly the severity and progression of cia than itregs. this reduction was associated with modulating inflammatory cytokine and anti-cii igg secretions to lower levels and polarizing the treg/ th17 balance to treg more obviously. this study highlights the potential therapeutic utility of itregs expanded by tdcs in autoimmune arthritis and should facilitate the future design of itreg tdc immunotherapeutic strategies against ra. in may 2010 the world health assembly passed a resolution, wha 63.12, on the availability, safety and quality of blood products. this urges member states to 'take all necessary steps to establish, implement and support nationally co-ordinated bts with the aim of achieving self-sufficiency'. the resolution applies to both fresh blood components and plasma derived medicinal products (pdmps). who defines self-sufficiency as meeting national needs for six products from blood and plasma collected locally. these comprise red cells, platelet concentrates, fresh frozen plasma, and factor viii, immunoglobulin and albumin solution. the definition and overall goals were established by a who expert consensus statement on 'achieving self-sufficiency based on vnrbd' developed at a meeting of who experts in geneva switzerland in 2011. blood services develop in line with the health care system that they support. typically this involves a transition from a supply for whole blood mainly in the context of emergency management of trauma and child birth to the implementation of blood component therapy. blood component production will likely lead to an excess of recovered plasma. effective utilisation of this requires access to plasma fractionation facilities. if this can be achieved then this will reduce reliance on commercial pdmps and ensure maximal utilisation of the donated whole blood. increasing demand for pdmps will potentially lead to a requirement for introduction of plasmapheresis programmes if the goal of self-sufficiency is to be maintained. there are a number of significant barriers to both utilisation of recovered plasma and the implementation of effective plasmapheresis programmes. individual countries will need to consider these as part of their long term strategic planning process. the main barriers for low and medium hdi index countries are access to suitable plasma fractionation facilities. few countries will be able to justify creation of a national fractionation plant. regional co-operation and development of facilities will be needed. secondly plasma for fractionation is seen as a critical material in the manufacture of a medicine. quality system requirements, including full compliance with a code of good manufacturing practice will be a pre-requisite for acceptance of the plasma as suitable for fractionation. there is an urgent need to develop support programmes to raise standards to achieve this. in contrast the main barrier for most high hdi countries is cost and the easy availability of pdmps produced by commercial countries from paid plasma. the safety profile of pdmps is currently excellent since the integration of a number of specific viral inactivation steps in the manufacturing process overcomes any infectious burden associated with payment for plasma. the critical question is then how a country can assure ongoing supply at a reasonable cost. ethical principles will play an important role in defining the strategy and individual countries should be able to define their own approach without unnecessary intervention and competition from the commercial plasma industry. 4a-s19-02 background: blood transfusion service of nepal red cross society was established in the year 1966. since the beginning nrcs has been running the service exclusively with assurance of government of nepal. in 1991 the government of nepal elaborated the mandate with clear policies and declared nepal red cross society as the sole authority to conduct the national blood program through out the country. at present it consists of 86 centers in 62 districts. national leadership and oversight of management and quality standards is the responsibility of the central blood transfusion service, who in partnership with regional and district centers and aims to provide a safe and secure supply of blood/blood products to all needy in the country. in the initial years the service was made possible through collection of blood from professional donors but since 1982 collection of blood was emphasized from voluntary non-remunerated donors only. aims: blood service is provided according to the need base and with the expansion of health services, establishments of medical colleges, government hospitals, private hospitals and nursing homes the demand of blood is rapidly going up through out the country. nationwide more than 250,000 units of blood were served to the needy and around 60% of it is served through the central blood transfusion service. results: with the view of providing safe blood and bloodproducts to the needy patients all collected blood is routinely tested for hiv, hbsag, hcv, syphilis besides grouping, cross-matching and anti-d antibody identification. blood components service is limited in few centers. conclusion: blood program is run through cost recovery basis with material and nominal service charge nationwide. no defined budget is allocated for blood program. there is on and off support provided from nepal government, un agencies, national societies and international non government organizations. currently, the total revenue raised for the blood program is insufficient to meet the total cost of production of blood and blood products. 4a-s19-03 background: blood transfusion services in cambodia face a number of significant challenges in providing a supply of safe blood and blood products sufficient to meet the clinical needs of patients. these challenges include access to adequate and sustainable financing, trained staff, sufficient voluntary non-remunerated blood donors (vnrbd) and appropriate equipment and facilities. aims: a 2011 programme funded by the us presidents emergency plan for aids relief (pepfar) and managed by the us centres for disease control (cdc) was initiated by the cambodian based us cdc office, in a collaborative partnership with the nbts and ministry of health (moh). specific blood program needs were identified and presented in a logical task order framework. the task order required support at both the national and provincial level across the whole blood sector; and priorities three key deliverables: comprehensive assessment of the country's current practices and infrastructure including national policy, legislation, collection, testing and the clinical use of blood. vnrbd recruitment strategies/protocols. a new 5 year national strategic blood plan. methods: the selected technical assistance partner (ta) developed a proposal that addressed the areas of the task order, and utilised a number of strategies to deliver the technical program; including: a critical initial design phase with an introductory visit to map prior local and development partner work and draw on in-country. seeking ongoing engagement and advocacy from local thought leaders. tailoring assessment and strategy planning tools to local requirements. use of inclusive communications with all stakeholders. strengthening of local governance infrastructure (blood safety working group) . the 1 year program resulted in development and delivery of an assessment report on cambodia's blood transfusion services, a knowledge, attitudes and practices survey and a five year national strategic blood plan (nsp background: indonesia has recognized that blood transfusion plays a key role in their modern health care; yet ensuring the availability of safe blood is a challenge for a population of over 250 million living on 6000 islands with limited health care resources with a high background prevalence of hepatitis b virus (hbv) of 9.4%, human immunodeficiency virus (hiv) of 0.2%, and hepatitis c virus (hcv) 0.4%. aim: a health economic study was performed by the irc to evaluate the economic impact of individual donor (id) nat on the burden of disease (hbv, hcv, hiv) associated with transfusion transmitted infections (ttis) and understand the comprehensive costs to the ministry of health (moh) of providing id-nat-tested blood. method: a health economic analysis was performed using a markov method, which predicts the probability of an outcome based on a chain of outcomes and defined transition probabilities. the two arms of the analysis compared the testing costs and treatment costs of: (i) serology alone, and (ii) serology and nat. testing costs were defined as the total costs to the indonesian moh of purchasing the reagents used in conducting either serological testing alone or serological and id-nat testing on donor blood units. the treatment costs were defined by the outcomes associated with each of the three viruses (hbv, hiv, hcv) and the likelihood of various clinical outcomes for patients infected by transfusion. indonesia-specific data drawn from peerreviewed published sources was used to populate the model. in cases where published local data was not available, proxy published data from other geographies and data collected from interviews with local transfusion experts were used. cost information for id-nat and serology testing was sourced from test manufacturers. results: previous effectiveness of nat testingin annual collection of 2.1 million donations determined that id-nat could prevent 3187 infections missed by serology screening. if id-nat assays were not available ttis represents a calculated usd $73 million in treatment costs for the indonesian healthcare system. the introduction of id-nat could saveusd $27 million per year (or~20% savings) due to reduced treatment costs by preventing ttis. the estimated savings exclude costs associated with lost productivity and litigation. conclusion: sincethe initial analysis, additional testing has been performed in variousdemographic locations and it is now estimated that 8500 whole blood units andup to 15,000 blood components could be interdicted with id-nat. these new datarepresent even more favorable economics to the moh. while id-nat represents an additional cost tokeep blood as safe as possible, the incremental cost comparison of serology andid-nat compared to serology testing alone shows a minimum estimated savings ofusd $27 million. based on these results, id-nat blood screening appears to be aclinically and cost-effective way to prevent transmission of infection to patients. 4a-s20-01 yee-sin l tan tock seng hospital, singapore, singapore arbovirus is an acronym for a group of viruses that are transmitted by arthropod vectors, commonly mosquitoes and ticks. person-to-person transmission of arboviruses is not common. however, transmission can occur through transfusion of blood and blood products, and organ transplantation, if the virus is present in the donor's blood or organ. the majority of arboviral infections in humans are asymptomatic. if symptomatic, the symptoms generally occur 3-15 days after exposure. clinical features of arboviral infections can be classified into four groups: acute central nervous system illness, acute benign fever with or without exanthem, hemorrhagic fever, and polyarthritis. there have been published reports of transfusion-transmitted infections of a number of arboviruses, including west nile and dengue viruses, as well as colorado tick fever and tick-borne encephalitis viruses. the risk of transmission depends largely on the burden of the disease in a particular geographical location, the proportion of asymptomatic infections in which clinical diagnosis is not possible, and the availability of diagnostics to detect low levels of viruses in the blood. west nile virus (wnv) entered the united states (us) in 1999 and has spread across the entire north american continent. wnv is now the most significant arboviral infection in the us. one out of every five exposed individuals will develop illness and 1 out of 150 may suffer from severe infection with significant neurological sequelae. in 2002, 23 transfusion-transmitted wnv infections were described. since 2003, all blood donations in the us have been routinely screened for wnv rna using wnv nucleic acid test (nat). since the implementation of screening, 13 transfusion-associated transmissions of wnv have been documented (mmwr 2012) . dengue is the most common arboviral infection in the world. the world health organisation (who) estimates that 50 million dengue infections occur annually and approximately 2.5 billion people live in dengue endemic countries (who 2009 ). the ratio of asymptomatic to symptomatic infections vary widely. singapore reported three cases of transfusion-associated dengue infections involving one blood donation episode. an asymptomatic repeat blood donor informed the national blood bank that he had fever the day after blood donation. by then, packed red cells and other blood products from his donation had already been transfused to three recipients. all three recipients were infected; two of the recipients' blood and the donor's stored serum sample were tested positive for dengue serotype 2 virus by pcr. wilder-smith et al. estimated the risk for transfusion-associated dengue transmission in singapore to be 1.6-6/10,000 blood transfusions in the year 2005, assuming a ration of asymptomatic-to-symptomatic infections of 2:1 to 10:1. subsequently, teo et al. reported the practice of deferring blood donation from recently returned travellers from dengue endemic areas. the massive outbreaks of chikungunya fever in the indian ocean and southeast asia again raise concerns about blood safety. although there has yet to be a reported case of transfusion-associated chikungunya virus infection, such a transmission route remains a distinct possibility. liumbruno gm et al. reported an economic loss of approximately 2 million euros, resulting from the suspension of blood donations at the peak of the chikungunya outbreak in italy in 2008. 4a-s20-02 australian red cross blood service, perth, australia background: arthropod-borne viruses (arboviruses) are an expanding global threat to human health. among them dengue (denv) and west nile virus (wnv) have both been documented to be transfusion-transmissible and transfusion is a plausible route of infection for several others including chikungunya virus (chikv) and ross river virus (rrv). addressing the transfusion risk associated with arboviral outbreaks poses new challenges. unlike established viral threats to the blood supply (e.g. hiv, hcv and hbv) in which the risk is generalized to the population-at-large and chronic infection is common, arboviruses are characterised by focal and often seasonal outbreaks of acute, predominantly self-limiting infection. thus, unlike traditional viral threats the risk is not 'continuous' in nature complicating risk mitigation strategies. aims: to describe possible risk management strategies for arboviral transmission including; geographical donor deferral/restrictions on labile component manufacture, donation testing and pathogen inactivation (pi) which can be applied individually or in combination. methods: how strategies are applied depends on the nature of the risk i.e. whether internal (i.e. local outbreak) or an external (i.e. potentially viraemic donors returning from an outbreak affected country). geographical donor deferral can take several forms ranging from deferral of donors returning from precisely defined 'outbreak risk' areas (e.g. wnv deferral for travel to n. america) to a 'blanket' deferral of all donors returning for a defined period after return from overseas (e.g. the netherlands). while this can be an effective risk mitigation strategy it can lead to the deferral of significant numbers of donors and the impact on sufficiency needs careful consideration. in some cases (e.g. for local dengue outbreaks in australia) this can be partially addressed by restricting manufacture to 'plasma for fractionation' since pi within the fractionation process satisfactorily addresses the transmission risk. triggering implementation of deferrals for specific outbreaks is problematic because a high proportion of arboviral infections are asymptomatic so asymptomatic donor viraemia poses a risk before the first symptomatic case is apparent. instituting a deferral only during the 'transmission season' is complicated because of the difficulty in precisely defining the beginning and end donors for major transfusion-transmitted infectious diseases (ttids; hiv, hbv and hcv) has evolved from performance of serologic assays only to include nucleic acid testing (nat) for detection of window phase and occult infections in most developed and some developing countries. nat screening has also been implemented for other viruses known to be transmitted by blood components and/or plasma derivatives (parvovirus b19, hav), and for several emerging/expanding infectious disease (eid) agents that threaten blood safety in some regions (e.g., wnv in the us, hev in japan, and dengue virus in puerto rico). despite this dramatic progress, eid threats continue to be identified and cause pressure to further enhance the safety of transfusions. the concept of a 'global village' has emerged, which reflects the fact that a potential blood-borne infectious agent present in any region of the world could 'traffic' to other regions overnight. there are increasing examples of zoonotic transmissions of infectious agents, with the potential for rapid adaptation to humans and subsequent spread to blood donors and recipients. there is also intense research to identify new blood born infectious agents using novel molecular discovery strategies. although recent examples of putative new agents have proven to be non-pathogenic (hgv/gbvc, ttv and sen-v), not transmitted at significant rates by transfusions (hhv-8), or contaminants (xmrv), every newly discovered agent requires serious investigation to assess its relevance to transfusion safety. this presentation will highlight recent progress in developing approaches for systematic assessment of eid risks and for applying rational approaches to respond to new pathogens. in an effort to proactively assess transfusion safety threats in the united states and canada, the transfusion-transmitted diseases committee of the aabb has systematically reviewed relevant data and prioritized the risks of individual established and emerging disease agents; this program includes aabb website accessible fact sheets for~100 agents which are updated as new information becomes available or new agents of concern are identified. recently a research program has been started using data of id-nat users for analyses of the efficacy of various testing strategies this allows comparison of the yield and risk reduction obtained by nat and serology assays in countries with differing incidence and prevalence rates of the three major viral infections. furthermore the aabb eid sub-group and the european cdc have developed 'toolkits' for assessing the risk of eids with respect to potential impact on blood safety and availability, and for evaluating and validating the effectiveness of proposed interventions. the toolkit process, including methods to monitor eid agent emergence, identify and recognize a transfusion-transmission threat, processes to quantify risk, and the appropriate management of such threats using formal decision analysis methodologies, should be considered for implementation by all blood systems. the main challenges for hospital transfusion are patient safety, the effective use of blood, robust methods for documentation and monitoring practice, good blood stock management and low wastage, good staff training, and the rapid availability of blood for those patients who need it urgently. patient safety is paramount, but haemovigilance schemes worldwide continue to demonstrate that errors in the transfusion process result in patient morbidity and mortality. this talk will indicate where progress has been made in improving transfusion safety in hospitals in the uk, and use the implementation of an electronic transfusion management system in oxford as an example of improving patient safety. the project involved the development of an end-to-end electronic transfusion process involving barcode patient identification, handheld computers at the bedside and electronically controlled blood fridges linked to the blood transfusion laboratory information system. it was taken through pilot stages between 2001 and 2006 through to its full implementation across the acute hospitals in oxfordshire in 2006/07, and its maintenance and further development since then. there was an improvement from 11.8% to 100% of staff following the process for correct bedside patient identification; no abo incompatible red cell transfusions or serious wrong blood events in over 10years; a reduction in wrong blood in tube events; reduced nursing (one nurse rather than 2 and half the time to administer blood) and laboratory workload; and more rapid delivery of urgently required red cell units (from a median of 18 min to 45 s). the electronic system provided a simple mechanism for compliance with uk/eu regulatory requirements for the traceability of blood, and the documentation of transfusion and training. feedback from both staff and patients was positive. our group wrote a national specification and provided the material for a national recommendation for hospitals to adopt the electronic transfusion process, but wider implementation by other hospitals has been slow. an additional module into the electronic transfusion process is being implemented to provide decision support for doctors ordering blood to minimise inappropriate use of blood as part of a patient blood management programme. there are considerable barriers to improving the safety of transfusion practice in hospitals, but they can be overcome by 're-engineering' the process with the support of a multidisciplinary team with shared, achievable and measurable objectives, engaging with senior hospital management, and having a determination to see the development of a new process through to completion and its implementation as routine practice. whats new with trali? in the last 20 years there have been substantial efforts to minimise or eliminate trali. despite these activities, in 2012 the fda still reported trali as the top cause of transfusion-associated mortality. the pathophysiology of trali follows a two event mechanism. the patient's illness leads to activation of the pulmonary endothelium with the sequestration of primed neutrophilsthe first event. the transfusion is the second event, and leucocyte antibodies or biological responsive modifiers (brms), present in the blood product, activate the microbicidal arsenal of the primed neutrophils to cause injury to the pulmonary microvasculature and consequently acute lung injury (ali). hence, trali may be antibody-mediated or brm-mediated. antibodies to human neutrophil antigens (hna), hla class i and class ii have been associated and implicated with trali. interestingly, a large prospective case-controlled study of over 400,000 transfusions reported that recipients with cognate antigens to hna and hla class ii antibodies had increased risk of trali but found no statistical significance with hla class i (toy et al. 2012 blood) . that hna-3a antibodies are associated with more severe trali indicates that antibody specificity is another determinant of trali injury (reil et al. 2008 vox sang) . additionally, murine models demonstrated that antibody titre also determined the severity of injury (fung et al. 2010 blood) . while the use of male predominant plasma has significantly reduced the incidence of antibody-mediated cases, trali still occurs likely due to brms. brms are generated during the routine storage of blood products. a meta-analysis of cardiac and trauma patients concluded that older blood is associated with a significant risk of death (wang et al. et al. 2012 blood) . given the success of male predominant plasma in managing the risk of antibody-mediated trali, ongoing efforts to reduce the risk of trali are likely to focus on brm-mediated trali. developing a better understanding of the how specific brms may contribute, either individually or in combination, to the development of trali, and how brm concentrations vary between donors, will be crucial to the future development of strategies to manage the risk of brm-mediated trali. they were all thais and their forebears have lived in northeast thailand for at least two generations. hna-1,-3, and -4 were typed by the pcr-sequence specific primer (pcr-ssp). results: the gene frequencies of hna-1a, -1b, -1c were 0.697, 0.302 and 0.001, respectively. hna-1 null allele was found in one sample. the gene frequencies of hna-3a and -3b were 0.780 and 0.220, respectively. hna-4a and -4b gene frequenfrom asian populations aims: to assess whether symptoms consistent with dengue are more common in transfused patients who test positive for denv rna compared to patients who do not. methods: in 2012 and rio de janeiro, patients who were transfused were enrolled and interviews inquiring about symptoms (fever, headache, bone or joint pain, body or muscle pain, vomiting, rash, painful eyes, and bruising) were conducted between 3 to 7 days, and about 30 days following transfusion. inpatients and outpatients were included. blood samples were collected at the time of first interview. subsequently, coded samples were tested for denv rna using the hologic/novartis tma assay. we used two definitions for denv disease: the who clinical definition consisting of fever with at least two contemporaneous symptoms, and a more general definition of having â�¥3 symptoms of any kind. we calculated odds ratios (or) and 95% confidence intervals (ci) for symptomatic infection comparing transfused denv rna+ to rnaã� patients. results: of 595 patients with samples and interviews 3-7 days following transfusion 54% were inpatients while it is possible that viremia was missed in some recipients who had symptoms consistent with dengue, we did not find evidence of significantly increased rates of symptomatic infection in denv rna+ compared to rnaã� recipients. investigation into whether any of these patients acquired denv through transfusion and mie prefectures) where there are many immigrants from latin america including japanese-latin americans. methods: we conducted a questionnaire survey on chagas disease for the subjects who were borne or grew up in latin america. since jan. 8th 2013 we have performed testing for anti-t. cruzi for donors from whom informed consent for epidemiological study was obtained. tests used were ortho t. cruzi elisa assay and an immunochromatographic assay donors who were aware of chagas disease and kissing bug were 67% and 56%, respectively. nobody has ever been bitten by kissing bug. 14% of the donors had been tested for anti-t. cruzi in their country. four donors had a family member with chagas disease and they were all brazilian. four donors had been diagnosed as having a cardiac or digestive system disease before. all of the 287 blood samples obtained from the subjects were anti-t. cruzi-negative except for one false positive result. conclusions: none of the subjects surveyed were anti-t. cruzi-positive. most of latin american blood donors in tokai region of japan are brazilian and about twothirds of them were younger than 40 years. we consider that the risk of transfusion background: leukocyte antibodies presence in the blood product(s) was responsible for the development of immune mediated transfusion related acute lung injury (trali) in recipients. several studies had demonstrated that hla antibodies (class i and class ii) as well as hna antibodies (hna-1, -2, and -3) were capable to induce trali reaction. among them, hla class ii and hna-3a antibodies seemed to be the most frequent antibodies responsible for severe trali reaction. therefore, screening of hla and hna antibodies in our female blood donors may reduce the risk of trali. however, the prevalence of leukocyte antibodies among parous female blood donors in china is currently not available. study design and methods: blood samples were collected from 142 nulliparous females, 343 parous females and 453 male donors. pregnancy history was obtained from consenting donors. hla class i and ii antibodies were screened by the use of lifecodes lifescreen deluxe (lmx), and antibody specificities were determined with lifecodes lsatmclass i & ii (single antigen). antibodies against neutrophil antigens hna-1 and -2 were screened by the labscreen assay (one lambda, inc) and rapid elisa detection (bayat et al, 2009; werth et al, 2013). hna-3 antibodies were analyzed by antigen capture assay (bayat et al, 2012). the specificity of positive samples was reanalyzed by the granulocyte immunofluorescence test (gift), the granulocyte agglutination test (gat). results: screening of the total cohort of 938 donors (453 males and 485 females) showed higher prevalence of hla antibodies production in female compared with male donors (18.9% vs 4.63%). we found in 61 out of 485 females donors alloantibodies against hla class i (12.58%) and hla class ii (10.10%). analysis among female additional immunosuppression may be used in patients with low adamts13 due to autoantibodies. aims: to investigate differences between idiopathic and secondary ttp with regard to clinical presentation, management and outcomes. methods: the australian ttp registry was established in 2009, and expanded in 2012 to include all tma. of 34 hospitals currently participate in the registry. of 103 cases in 94 patients were registered from 18 sites; eight were excluded for incomplete data, leaving 95 episodes in 86 patients. results: median age was 46 years (15-83 years), 53% were female, 16% were relapses. of 51% were idiopathic and 49% had â�¥1 identified precipitant, including infection 23%, autoimmune 18%, malignancy 12%, medication 14%, pregnant/postpartum 4%, solid organ transplant 3%, and allograft 3%. adamts13 levels were available in 66 cases (37 idiopathic and 29 secondary). adamts13 was <10% in 22/37 (59%) idiopathic and 9/29 (31%) secondary cases. precipitants in cases with adamts13 <10% included infection (n = 5), autoimmune (n = 5), medication (n = 1), and pregnancy (n = 1). neurological, gastro-renal, bleeding and thrombotic presentations were similar in idiopathic and secondary cases. data on plasma therapy were available in 88 cases (table 1) . of 84/88 (95%) received pex, by centrifugation in 28/84, membrane filtration in 25/84 (30%), and unknown method in 29/84 (35%). plasma infusions were used in 11/88 cases (without pex in 4). data on additional therapy was available for 89 cases (table 2) . rituximab was given in 12/45 cases with â�¥1 precipitant, including infection (n = 5), non-haematological malignancy (n = 1), haematological malignancy (n = 3), autoimmune (n = 5), medication (n = 2), and pregnancy (n = 1). death rates were lower in idiopathic than in secondary cases (8% vs 25%, p = 0.05). conclusion: our data confirm that very low adamts13 does occur in cases with secondary causes identified. clinical presentation did not differ between idiopathic and secondary cases. plasma therapy was similar in both groups and additional immunosuppression was not only used in idiopathic cases, but also in secondary cases. mortality remains high, and more data to support improved diagnostic and therapeutic approaches are needed. background: living donor liver transplantation (ldlt) is a curative treatment for patients with end-stage liver disease. ldlt is associated with major blood loss and allogenic blood transfusions. although transfusion practices in adult liver transplant (lt) has been widely studied, the same in pediatric lt has not been well documented in india. aims: the purpose of the study was to evaluate the intraoperative transfusion requirements in pediatric ldlt recipients (<192 months) against the diagnosis, preoperative clinical and laboratory variables. methods: the data was collected retrospectively from blood bank and patient records. demographic and important preoperative variables namely clinical diagnosis, peld score, hemoglobin (hb), platelet count, inr, albumin, and fibrinogen levels were reviewed. data on intraoperative transfusion of packed red cells (prc), fresh frozen plasma (ffp), platelet concentrates (plt), cryoprecipitate was computed as ml/kg bw. massive transfusion (mt) was defined as >80 ml/kg of blood components during the surgery. analysis was done using spss software version 16. median levels were compared between mt and non-mt group using mann whitney test. results: between august 2010 to july 2013, 60 pediatric ldlt were performed in a single center in south india. thirty five (58.3%) of them were females and 25 (41.7%) males; 32 (54%) of the recipients blood group were o positive, 14 (23%) b, 9 (15%) a and 5 (8%) ab. recipient characteristics are given in table. transplant indications were biliary atresia and cirrhosis in 28, metabolic /hereditary liver disease in 24, hepatic tumors in 5 and acute liver failure in 3 recipients. of 56 (93.3%) patients received prc, 40 (70%) ffp, 17 (28.3%) plt and 34 (56.4%) cryoprecipitate. sixteen (26.7%) patients received massive transfusion (mt) with a median peld score of 22 (ã�7 to 43) compared to 9 (ã�9 to 46) recipients without mt (p < 0.005). also, mt group had significantly lowered median levels (preoperative) compared to non-mt group, viz. hb (8.6 vs 10.3 mg/dl), platelet count (96 vs 186 9 10 3 per mm 3 ), fibrinogen (137 vs 248 mg/dl), and a higher bilirubin (16.5 vs 3.7 g/dl). transfusion requirements of ffp was higher in acute liver failure (53 ae 17.3 ml/kg) compared to metabolic liver disease (11.1 ae 2.2 ml/kg) and biliary atresia (19.5 ae 4.2 ml/kg); (p = 0.001). conclusion: to conclude, massive blood transfusion requirement in pediatric recipients during ldlt was associated with higher peld score, and more deranged preoperative hematological and coagulation status. in depth analysis of recipient disease status, controlling for the effect of surgical interventional variables on larger samples are recommended to develop predictive models of transfusion therapy. conclusions: this study is the first report of hna gene frequencies in ethnic northeast thais. it could be used for the risk prediction of alloimmunization to hna and estimation of alloimmune neutropenia and trali in the ethnic northeast thai population. 3d-s18-01 distler pb 1 , slaper-cortenbach i 2 and ashford p 1 1 iccbba, san bernardino, united states of america 2 university medical center utrecht, utrecht, the netherlandsbackground: standardized isbt 128 terminology is used by cellular therapy organizations in many countries. as products evolve and new products are created, terminology is required to support the new products. changes to the terminology are managed by the cellular therapy coding and labeling advisory group (ctclag), a committee of experts representing international professional cell therapy societies, technical experts, and regulatory liaisons. since the early nomenclature was devel-oped, the ctclag has approved classes and terminology for very innovative products, including some for which therapeutic benefit has yet to be clearly demonstrated. because the term 'therapeutic cell' was used in the terminology, this became a great concern to the us fda, even to such an extent that the use of isbt 128 for cellular therapy products in the usa could be problematic. aims: the ctclag recognized the concerns raised by regulators and determined it needed to revise nomenclature to address these concerns and to be consistent with isbt 128 nomenclature used in related fields. methods: the ctclag held a face-to-face meeting and reconsidered the use of tc (therapeutic cells) terminology for products and proposed new nomenclature for the problematic terms. a draft of new nomenclature was developed and made available for public comment as well as review by the boards of ctclag sponsoring organizations (aabb, apbmt, asbmt, asfa, ebmt, fact, isbt, isct, jacie, nmdp and wmda). following this review, terminology was updated. results: major changes are:(1) the class name will comprise the type of cells and, where appropriate, the source (eg. 't cells, cord blood').(2) hyphenated class names will be replaced (3) tc and therapeutic terminology will be replaced (4) modifiers will be replaced with attributes providing the same information (5) new attributes will be added the terminology remains compatible with the single european coding system. summary/conclusions: changing terminology will create rework for facilities that have implemented isbt 128 and delay for those in the process of implementation. however, it was felt the revised terminology will provide a strong foundation for consistent nomenclature as new products are developed and address regulatory concerns. an appropriate timescale for implementation of the revised terminology in facilities already using isbt 128 will be developed. this presentation will describe the revised terminology and explain the reasoning supporting the changes.3d-s18-02 kordelas l 1 , rebmann v 2 , ludwig a-k 2 , radtke s 2 , beelen dw 1 , giebel b 2 and horn p 3 1 department of bone marrow transplantation, university hospital essen, essen, germany 2 institute for transfusion medicine, university hospital essen, essen, germany 3 university hospital essen, essen, germanybackground: graft-versus-host disease (gvhd) is a major cause of morbidity and mortality after allogeneic stem cell transplantation. a number of studies reported positive impacts of systemically applied mesenchymal stem cells (mscs) for preventing or treating acute gvhd. in contrast to the initial paradigm that mscs intercalate into injured tissues and thus reduce tissue damage, it is now widely assumed that mscs secrete a number of immune-modulatory factors, which impair inflammation and thus help to suppress gvhd. exosomes are secreted cell organelles, which exert immune-modulatory properties. these small membrane vesicles are released by a huge variety of different cell species, including mscs. methods: here, we enriched exosomes from bone-marrow derived mscs of four different unrelated stem cell donors and compared their immune modulatory properties in vitro. next, we administered immunosuppressive msc-derived exosomes in escalating doses into a 22-years female gvhd patient. this patient suffered a severe and therapy-refractory cutaneous and intestinal gvhd grade iv. we monitored the clinical effects on an in-hospital basis and correlated this with the levels of inflammatory cytokines measured in the patient's plasma. results: we show that even though all propagated msc lines released exosomes, exosome-enriched fractions differed in their potential to modulate immune responses in vitro. administration of the exosome-enriched fraction with the strongest immune suppressive in vitro effect into the gvhd patient was well tolerated and appeared to be safe. during the course of the exosome therapy a clear reduction of the proinflammatory cytokines il-6, il-8 and il-17 was observed in the patient's plasma. in line with that, the clinical cutaneous and intestinal gvhd symptoms improved significantly and the dosage of the immunosuppressive agentsparticularly of steroidscould be reduced. in total the patient was stable for 5 months. interpretation: msc exosome-enriched fractions exert immune suppressive functions in vitro and in vivo. since the in vivo administration seems to be safe, msc exosome administration appears as a promising new treatment option for steroid refractory gvhd patients. key: cord-006391-esnsa4u5 authors: nan title: abstracts 5(th) tripartite meeting salzburg/austria, september 9–11,1982 date: 1982 journal: langenbecks arch chir doi: 10.1007/bf01279099 sha: doc_id: 6391 cord_uid: esnsa4u5 nan the gastric secretion capacity increases during the first year after all types of vagotomy which is assumed to be important for the development of recurrent ulceration. however, the cause of this reestablishment of the preoperative gastric function is not adequately explained. the purpose of this study was to investigate the vagal innervation of the parietal cell mass in dogs 11/2 year after truncal vagotomy (tv), selective gastric vagotomy (sgv) and parietal cell vagotomy (pcv). material and methods: mongrel dogs supplied with a gastric fistula were used. the groups comprised 5 dogs with tv, 6 dogs with sgv and 6 dogs with pcv. the acid secretion was measured before and one month and one year after vagotomy following insulin and pentagastrin stimulations. operative technique: a gastroduodenostomy was performed in addition to the gastric fistula operation in the dogs randomised for either tv or sgv. the vagotomy operations were performed after the prevagotomy secretion experiments. tv by a 4 cm resection of the main truncs through a left side thoracotomy and sgv as described by amdrup [1] . the pcv dogs had no gastroduodenostomy added but the technique at the lower esophagus was identical with the sgv and the dissection of the lesser curvature was extended 1 cm distal of the sharp physiological and histologically determined borderline between the fundus and the antrum. about 11/2 year after the vagotomy the dogs were sacrificed by an acute experiment. the persistent or the regenerated vagal innervation of the parietal cell area was mapped out by neutral red excretion elicited by electrical stimulation of either the thoracic or the cervical vagal nerves [2] . results: one year after vagotomy no increase in insulin response could be demonstrated in the tv and sgv groups, but a gradual increase to about 50 % of prevagotomy output occured in all pcv dogs. reliable neutral red experiments were performed in 2 tv, 6 sgv and 4 pcv dogs. the stimmulated neutral red excretion was scanty to the same extent at the cardia region in all three groups, but a distinct antralfundic border was outlined in all 4 pcv dogs. the border was, however, only suggested in one of the sgv dogs. conclusion: pcv including denervation of 1 cm distal of the antral-fundic border do not prevent a functional important reinnervation of the distal part of the parietal cell area. no important reinnervation seems to occur at the cardia region after any of the three vagotomy procedures. 1 and insulin (0.2 ugkg -1) i.v. bolus; after uni-and then bilateral truncal vagotomy. each study was 120 minutes and blood was taken at 1, 3, 5, 7 and then 10 min intervals. gastric acid was measured by autobiuret titration. plasma was stored at -20°c until assayed for pp by ria using an antibody sensitive to 3 fmol ml-1. results are expressed as mean total increment _+ se. significance: p < 0.01". bombesin-stimulated gastric acid secretion was not significantly altered by vagotomy (p < 0.05), whereas that stimulated by insulin was significantly inhibited by bilateral truncal vagotomy (p < 0.01). bilateral and right hemi-vagotomy significantly inhibited pp release by bombesin (17 < 0.01), however; only bilateral truncal vagotomy significantly inhibited pp release by insulin (p < 0.01). these results suggest that the measurement of pp release by insulin or bombesin is a sensitive index of vagal integrity and that bombesin-released pp may specifically delineate the integrity of the right vagus. since the measurement of gastric acid secretion after operation is both uncomfortable and often difficult to interpret, the value of a simple blood test to determine vagal integrity may be of considerable clinical relevance. glucose (g) or oleate (o) empty more slowly from the stomach than 0.15m nac1 (s). this chemoregulation may be achieved by resistance beyond the proximal stomach [1] . we sougth to define the site of this resistance. expt. 1: gastric emptying of s and 600 mm g was measured in 5 dogs with intestinal cannulas 45 cm distal to the pylorus while gastric pressure was maintained at 10, 18 or 26 cm h20 with a barostat. the dogs were studied either with an uninterrupted intestinal stream or with duodenal effluent diverted through a second barostat prior to its return downstream. since the latter ensured a constant pressure at the ligament of treitz, any observed effects on emptying could not be due to resistance further distally. expt. 2: emptying of s, g, and 40 mm oleate emulsion was measurd in 5 dogs without cannulas after control antroduodenal transection and after antrectomy while gastric pressure was controlled at 11, 17 and 23 cm h20. emptying rates expressed as the average of the volumes emptied at the 3 pressures. under both conditions in expt. 1 emptying rose with pressure yet s emptied faster than g. thus a major site of chemoselective resistance to emptying of liquids lies proximal to the ligament of treitz. after antrectomy s and o emptied faster than before, however, the differences between saline and nutrients were maintained. resistance, therefore, does not reside in the antrum or pylorus and clearly the duodenum is implicated. gastric emptying of liquids may be abnormally rapid in du. this abnormality my be due to a selective loss of inhibition in response to acid since previous studies [1] have demonstrated rapid emptying of acid solutions but normal emptying of glucose and fat. previous investigations, however, used meals of only one concentration, so differences between dus and normals (n) may have been minimized by selection of a supra-maximal inhibitory dose. furthermore intragastric ph was not maintained constant so emptying could have been slowed by higher rates of acidification in du. we therefore studied emptying of graded concentrations of citric acid, glucose and oleate in 500 ml meals. gastric volumes were measured by the george technique in dus and n at 5 rain intervals for 30 min after ingestion. gastric ph was maintained constant by intragastric titration (3.0 for acid; 7.0 for nutrients). subjects received one type of meal on separate days and the doses were randomly administered. dose dependent inhibition of emptying in response to acid and nutrients occured in n and du, but emptying was faster in du (p<0.05). with glucose and acid the difference occured within 0-5 min whereas with fat the difference occurred between 5-30 min. thus (1) dus do not have a selective loss of inhibition of emptying in response to acid, and (2) the mechanisms which control emptying of fat and which are disturbed in du differ from those which control emptying of glucose and acid. 1 gross ra, isenberg ji, hogan d, samloff im (1978) the effect of fat on meal-stimulated duodenal acid load, duodenal pepsin load and serum gastrin in duodenal ulcer and normal subjects. a means of reducing the requirement for postoperative nasogastric intubation would be advantageous. we report the effect of cimetidine on postoperative nasogastric aspirates. thirty patients undergoing elective abdominal aortic surgery or colonic resection were entered into a double blind randomised study, receiving cimetidine 200 mg 4 hourly or placebo (saline) by continuous intravenous infusion from the morning of the first postoperative day. volumes of 4 hourly aspirates were recorded and ph and (h ÷) of samples measured. nasogastric tube removal accorded to standard clinical practice. it has been shown, that the duodenum has a better ability to resist acid than jejunum or ileum. this resistance was attributed to alkaline secretion (a.s.) of the mucosa, because pancreatic and bile secretion were excluded. recent in vitro studies demonstrated an energy dependent hco3-transport (flemstroem 1980 am j physiol g 239: 198). we were interested in studying the role of blood flow (bf) in the production of alkali in an in vivo preparation. min. three groups of animals were studied: i) controis with normovolemia for 120 min. ii) normovolemia for 60 min, thereafter 60 min of shock induced by bleeding to 40 mmhg mean arterial blood pressure. iii) 60 min normovolemia and 60 min vasopressin (0.2 i. u./kg/min) under normovolemia. in controls a small decrease in bfand a.s. as well was observed. both shock and vasopressin induced a significant reduction in a.s. and bf. the relation ofbf to a.s. was found to be exponential (y = 2.71 + 0.21x-0.003x 2 r= 0.71; p<0.05). the reduction in a.s. during shock was much less, when shock induced acidosis was compensated by i.v.-infusion of hco 3 (0.3 ,uequ/kg/min). conclusion: the alkaline secretion of the proximal duodenum is dependent on blood flow and the arterial [hco~. there has been considerable controversy as to the nature of the offending agent in the etiology in reflux oesophagitis in man. in rat studies, surgically induced reflux oesophagitis was shown to be correlated with the presence of active trypsin in the reflucting juice. reflux of bile and gastric juice with a ph of 4 did not induce oesophagitis. even short periods of oesophagitis (1-6 weeks) showed an increasing amount ofpanmural fibrosis which even further increased after a reflux abolishing roux-y operation. the pattern of the collagen content of the full thikkness oesophageal wall was similar to that found in human reflux oesophagitis. the hypothesis that active trypsin is also in man responsible for reflux oesophagitis prompted to the following investigation. 24 patients with typical gastrooesophageal reflux symptoms and 17 control patients were endoscopically examined. on entering the stomach with the endoscope samples of gastric juice were taken for ph and active trypsin determinations. trypsin activity was determined with a kinetic method using s-2160 (kabi vitrum b.v. amsterdam) as substrate. results: in all patients some trypsin could be detected in the gastric juice. in those patients with endoscopically erosive and/or ulcerative changes (n = 14) the trypsin content was significantly elevated compared to the controls (/7<0.05 wilcoxon). conclusions: the composition of gastric juice is determined by gastric secretion and duodenogastric reflux. duodenogastric reflux is increased in patients with symptoms of reflux oesophagitis [1] . trypsin in gastric juice (ph 3.5-7) will stable and partly active over prolonged periods [2] . so from our study in man one may conclude that the high concentration of trypsin in the gastric juice in patients with reflux oesophagitis, may well be the etiological factor of the oesophagitis. disinfection, using artificial contamination of hands with escherichia coil and 'surgical' disinfection directed against the resident microflora of the hands. the authors who developed the procedure have reported unexpectedly high potency for n-propanol compared to iso-propanol, and, in turn, for iso-propanol compared to povidone-iodine and chlorhexidine preparations [1] . however, using the same procedure we have been unable to find any significant differences in activity between these agents. we have identified several potential sources of error including the method of applying e. coliand its spontaneous loss of viability, the use of neutralizers before disinfection, the differing surfactant effects of the agents, and the absence both of a control untreated area and of a cross-over of disinfectants studied sequentially. in our parallel tests using an excision-sample technique [2] which is considerably more sensitive than the dghm procedure, we have observed the following mean reductions in the counts of accessible bacteria: iodine in ethanol, 96%; povidone-iodine, 89%; chlorhexidine in ethanol, 88%; iso-propanol, the purpose of this study was to compare radiation injury in guinea pig small bowel (1) devoid of contents (2) containing bile (3) containing pancreatic juice. one group was intact control animals not radiated. another was intact animals radiated. in group 3 a roux y cholecystojejunostomy was constructed and the bile duct ligated. thus one limb contained bile, the other pancreatic juice. in group 4 a blind roux y was constructed such that one limb was empty of contents and the other contained both bile and pancreatic juice. each animal was subjected to a single dose of 1600 rads via an abdominal port and sacrificed four days later. the severity of injury was judged by counting the number of surviving crypts per circumference. damage was further evaluated by histologic criteria -mucosal loss, edema, inflammation, hypervascularity and loss of mucus. the maximum histologlc grading score on this scale was 20. the microscopist was ,,blinded" as to the origin of each tissue section. for histologic grading all radiated groups were significantly different from control (p<0.02) and from one another (/r<0.05) except pancreatic juice vs. empty. intestine devoid of contents sustains a mild, but significant radiation injury. the presence of pancreatic juice enhances the damage. pure bile makes for yet more severe injury but still significantly less than normal whole intestinal contents which contain both bile and pancreatic juice. the main problem facing patients with ulcerative colitis after mucosal proctectomy and ileo-anal anastomosis is severe frequency of bowel action. our hypothesis was that either an artificial valve [1] or reversed ileal loop might improve intestinal absorption and slow transit. we tested it in dogs after colectomy and low ileo-rectal anastomosis (ira). body weight, xylose absorption, serum albumin, folate, vitamin b12, calcium, urea and electrolytes, full blood count, faecal weight and mouth to anus transit time [2] were measured before operation. the dogs then randomly underwent either ira alone (control, c, n = 7), ira with a 10 cm reversed loop (ira + rl, n = 6) or ira with an artifical valve (ira + v, n = 6). body weight, haematological estimations and faecal chemistry were measured weekly for 3 months post operatively. xylose absorption and transit time were measured a minimum of 2 months after operation. body weight decreased significantly after each type of operation. there was no difference however, between (ira + rl) and c or (ira + v) and c. likewise, haematological and faecal measurements after both test operations did not differ significantly from c or from pre-op, measurements. the grosfeld valve prevented the reduction in transit time that occurred after the control operation, whereas the reversed loop did not. use of such a valve in combination with mucosal proctectomy might slow transit, but is unlikely to improve absorption. it seems worthy of further study. histologic grading___ sem crypt count-+-sem we have examined the effects of changes in intestinal blood flow induced by hypovolaemia, the mesenteric vasoconstrictors somatostatin and vasopressin and the mesenteric vasodilator prostaglandin e1 (pge1), on intestinal absorption, electrical activity and volume. in each of 7 dogs a 75 cm jejunal segment was isolated and serosal electrodes attached. in absorption experiments the segments were perfused at 2.9 ml/ rain with a solution of 90 retool/1 sodium and 100 retool/1 glucose. to measure segment volume, a solution of 50.7 g/1 mannitol was perfused. this solution shows zero net absorption but does not alter electrical activity. intestinal motility and absolute volume were monitored by gamma camera. experiments were performed after a steady state was reached. absorption and transit time were measured using non-absorbable markers. electrical fast wave activity was reduced by (a) intravenous somatostatin 2.5 mcg/kg/h (28+1.4 to 11_+3.4; mean_+sem/5 rain) and abolished by (b) intravenous vasopressin 1.2 u/kg/h (p<0.001). motility was inhibited and mean transit time was prolonged (a) 7.4 to 15.3 rain and (b) 4.4 to 19.9 rain (/r<0.001). intestinal volume rose by (a) 12.3___ 3.7 ml and (b) 15.4-+4.7 ml (p<0,001). absorption was unchanged by somatostatin and fell with vasopressin (5.58---0.7 to 4.35-+0.68 ml/5 min). acute blood loss sufficient to lower the central venous pressure by 5 cm of water produced identical changes to somatostatin. pge1 produced no alteration in electrical activity or absorption. intestinal absorption is resistant to changes in intestinal blood flow whilst electrical activity is depressed and intestinal volume increased by mesentric vasoconstriction. the accepted views on the pathogenesis of amoebic lesions in complicated amoebic colitis are that amoebae produce a toxin resulting in cytolysis and necrosis. this concept may adversely affect the management of patients with complicated amoebic colitis by implying that once the amoebae are killed, the disease process is arrested and colonic perforation is unlikely. we present an alternative hypothesis that 'complicated amoebic colitis is the result of vascular compromise'. transmural disease is caused by amoebic invasion of vessels supplying a segment of the colon with subsequent thrombosis and ischaemic necrosis of the affected area. the ischaemic nature of the necrosis is suggested by its shape, and the demonstration of vascular thrombosis on dissection ofresected colons which have perforated. amoebic invasion of blood vessels can be demonstrated histologically. the ischaemic nature of the lesions can be confirmed by angiographic examination of the resected colon. vascular occlusion can be demonstrated pre-operatively with the use of selective mesenteric angiography, which clearly delineates the ischaemic segment of the colon. selective rnesenteric angiography in 27 patients with fulminating amoebic colitis aided the preoperative diagnosis of colonic infarction before signs of visceral perforation had developed and permitted life saving surgical intervention. there is accurate correlation between angiographic and operative findings. in postdysenteric colitits associated with amoebic strictures of the colon, mesenteric angiography will demonstrate the ischaemic nature of the stricture and accurately define the extent of resection. after ca. the mean age at operation was 33 and 61 years respectively. anal canal length was 3.5___0.1 cm (mean + s.e.m.) after ia and 3.7___0.1 cm after ca. a resting tone of 66___ 3 cm water after ia and 53 -----10 cm water after cawas recorded. squeeze pressure was 123___9 cm water after ia and 79-----3 cm water after ca. the recto-anal reflex was present in 54% of the ia patients and in 75 % of the ca patients. a mean daily bowel frequency of 3.5___0.2 followed ia and 4+_1 after ca. these findings show that after ia anal pressures are within the normal reference range for our laboratory [2] . lower pressures were found after ca, probably due to the older age of the patients in this group [3] . normal anal canal length, the integritiy of the striated sphincter and in most cases preservation of the recto-anal reflex contribute to satisfactory continence following peranal anastomoses. perineal descent is found in patients with idiopathic faecal incontinence (ifi) and patients presenting with symptoms of the descending perineum syndrome (dps), who have no incontinence. manometric, radiological and neurophysiological studies were performed in 17 patients with ifi, all of whom leaked during rectal infusion of 1500 ml saline, 18 patients with dps, who were continent of rectally infused saline, and 14 matched controls. both patient groups exhibited similar degrees of perineal descent below the pubococcygeal line of straining (ifi, -5.1___2.1 cm; dps, -5.9 + 1.7 cm) compared with controis (-2.4__+ 1.5 cm;p<0.005 in both cases), and similar prolongation of both the latency of the cutaneoanal reflex (ifi, 13___5 ms; dps, 16+6 ms; controls, 9+4 ms;/,<0.005 in both cases), and motor unit potential duration (ifi, 9.1___2.6 ms; dps, 8.4___2.0 ms, control, 6.7+1.5 ms; p<0.01 in both cases). moreover, both groups had an abnormal ano-rectal angle (/,<0.005), though this was more obtuse in ifi than dps (120___21 ° vs 107___ 15°;p<0.05). however, while patients with ifi had lower sphincter pressures than normal (basal; 39___17 vs 81___29 cm water; /7<0.005; squeeze, 96___18 vs 208___84 cm water; /7<0.001), and required a lower rectal volume to inhibit sphincter tone for more than one minute (32 + 18 vs 76___29 ml; p<0.00l), these values were normal in patients with dps (basal; 87___ 30 cm water; squeeze, 198_+90 cm water; rectal volume, 52___46 ml). these findings suggest that perineal descent and neuropathy are not necessarily associated with incontinence if sphincter pressures remain normal. aminoacids administered either enterally or intravenously stimulate gastric secretion in both dog and man. it has been suggested that absorption of amino acids from the gut into the circulation might contribute to the intestinal phase of gastric secretion. the mechanism of this stimulation by amino acids remains uncertain but in an earlier communication we reported the direct action on the parietal cell of phenylalanine, glutamine and alanine [1] . in the present study using (14c) aminopyrine uptake as an index of dispersed parietal cell secretory function, the interaction between histamine and individual amino acids and the effect of the histamine h2-receptor blocker ranitidine (10-4 molar) on these interactions have been examined. results (percentage of maximal stimulation produced by 10-4 molar histamine) results (percentage of maximal stimulation produced by 10-4molar histamine) methionine glutamine alanine amino acids only (10-2molar) 26 32 29 amino acids and histamine (10-4molar) 175 -137 amino acids and ranitidine (10-4molar) 10 17 7 histamine and ranitidine -complete inhibition -25 125 1. the response to the combination of amino acids and histamine was greater than the sum of the maximal responses to each of these agents alone. 2. the histamine h2-receptor antagonist ranitidine completely inhibited the histamine stimulated response but only partially inhibited secretion stimulated by amino acids. we conclude that amino acids act directly on the parietal cell by a mechanism which is not solely dependent on histamine. 1 sex related differences in gastric acid secretion have been documented (lilja et al, 1967) . it has been also reported that male rats have a significantly higher serum gastrin concentration than females and that oophorectomy increases serum gastrin to male levels (lichtenberger et al., 1976) . estimation of serum gastrin levels after administration of androgens has not been reported. aim. the aim of this study was to investigate the serum gastrin levels before and after oophorectomy and administration of estrogens and testosterone in female guinea pigs. method. 4 groups of guinea pigs were used for this study, 8 animals as controls, 6 were given estrogens for 2 weeks, 8 were given testosterone for 2 weeks and 5 underwent oophorectomy. results. the mean serum (-+sem) gastrin value in controls was found 35+ 1.63 pg/ml, after estrogens 13.33-+1.22 pg/ml, after testosterone 55_+1.89 pg/ ml, after oophorectomy 46-+2.45 pg/ml. conclusion. it is concluded that estrogens decrease the serum gastrin, and that the increase of serum gastrin after administration of androgens and oophorectomy is statistically significant (/r<0.01). the mechanism by which the ovarian hormones influence gastrin levels may be a sex-dependent change in the synthesis or secretion of gastrin. the reported inhibitory influence of estrogen on food consumption may be also the predominant factor in serum gastrin alterations. mean caloric intake was 2336--+427 kcal/day. moderate to severe dumping occurred in one patient. faecal fat was normal in 11 while 7 patients had greater than 10 grams fat loss per day. weight loss was 14.6_+8.6% of recall weight. muscle mass measured by arm muscle circumference (25.7_+3.9 cm) and creatinine heigth index (99___26%) was normal for our patients but triceps skin fold was 30% less than normal (8.6_+2.2 mm) indicating that in these patients fat stores were reduced. in six patients haemoglobin was less than 13 grams per cent but serum iron, iron binding capacity and serum folate were normal. red cell folate was depressed in six patients whose haemoglobin values were normal. it is concluded that with this reconstruction total gastrectomy produces satisfactory digestive and "nutritional results. examination of factors affecting faecal fat loss and folate metabolism may help improve the nutritional status of these patients. in order to assess the optimal conditions for segmental pancreatic graft viability the following experiment was conducted. the duct obliterated splenic lobe of the canine pancreas was autotransplanted by end to side fashion to the femoral vessels (n = 10). the graft was lodged in a subcutaneous pocket. a month later total pancreatectomy was completed. the dogs were supplemented with pancreatic enzymes (viokase). mean survival was 42___ 12 days. deaths were due to hyperglycemia when grafts lysed from local infection and thrombosis. in the second experimental group (n= 10) the autografts were anastomosed to the iliac vessels and placed intraperitoneally followed by total pancreatectomy one month later. construction of a distal splenic arterio-venous fistula increased blood flow from 31.4 cc/min to 108 cc/min. the pancreatic duct was obliterated with neoprene (n=4) silastic (n=3) or left open (n=3). three dogs died one month to four months due to graft fibrosis and failure. seven dogs had been followed from four months up to seven months when sacrificed. all were normoglycemic. mean k values for ivgtt were 1.5. the normoglycemic dogs had mean plasma concentrations of amino acids similar to healthy controls. a two to threefold elevation was observed in pancreatectomized diabetic dogs for plasma leucine, isoleucine and valine. immunoreactivity of pancreatic polypeptide was measured by radioimmunoassay with an antibody raised against the human hormone by re. chance, lilly research laboratories. mean plasma levels rose form 256--+28 s.e. pg/ml to over 1000 pg/ml following protein meal (20g ground lean beef/kg) in all normal control dogs (n=4), and to levels in the range of 390 to over 1000 pg/ml in the same dogs following infusion ofbombesin (1 pg/kg/ h) for 15 min. mean basal levels were lower (42___ 2.2 s.e. pg/ml) in dogs with autotransplants and did not increase significantly during any stimulatory test. the levels of pancreatic polypeptide did not distinguish between viable and failing grafts. in successful grafts histology showed fibrosis of the exocrine component. transmission electron microscopy revealed the presence of clusters of functional islet cells in the six-month implants. a and b cells were common. d cells were much less frequent. release of secretion granules into the perivascular connective tissue space was observed. the results indicate that vascularized segmental pancreatic graft comprising 25 % to 30 % of the pancreatic mass maintains normal carbohydrate and amino acid metabolism. graft survival was extended by intraperitoneal location and creation od distal splenic a-v fistula, but chronic graft fibrosis occurred regardless of the method of pancreatic duct treatment. the results further suggest the necessity of an intact vagal innervation for the physiological or pharmacological stimulation of pancreatic polypeptide release from thendocrine pancreas which is otherwise apparently functional. besides, these considerations rule out pancreatic polypeptide levels as a useful marker for evaluation of pancreas graft. polyisoprene ductal occlusion allows segmental pancreatic transplantation in man with satisfactory early islet cell function. however acute rejection remains a problem as the diagnosis, based on hyperglycaemia and glycosuria, represents a late manifestation of rejection. although llqndiumlabelled platelets have been used in the diagnosis of renal rejection [1] , their use in pancreas transplants has not been studied. ~ in 6 patients, autologous llqn-labelled platelets were injected on the second, sixth and tenth days following combined renal and pancreatic transplantation. graft radioactivity was expressed as the ratio of counts from the pancreas over a reference area on daily gamma images for 14 days. one patient developed hyperglycaemia coinciding with renal rejection on day 5. over the previous 24 h pancreas radioactivity had increased by 94 %. in a further patient whose pancreas continued to function, a perigraft haematoma was recognised on the gamma image. the remaining 4 patients had good pancreatic function and no 11xin-platelet accumulation: mean (_s.c. mean) pancreatic radioactivity was 1.76+0.190 on the third postoperative day and 1.75_+0.135 at the end of study. these results demonstrate that ductal occlusion with polyisoprene does not cause significant platelet accumulation. hence min-platelets may potentially be used for the earlier diagnosis of pancreatic rejection. isograft models of pancreatic transplantation, methods involving closure of the duct system result in severe inflammatory changes and eventual fibrosis [1] . these changes can be avoided by formal drainage of the duct into the bowel of urinary tract. inflammatory changes initiated by duct closure may contribute to and enhance allograft rejection. pancreas transplants were performed in rats using lewis (rt11) donors and streptozotocin-induced diabetic da(rt1 a) recipients, using duct-ligation, open duct and ureteric duct drainage. cyclophosphamide produced significant prolongation ofnormoglycaemia in all groups and although there was a tendency towards longer function in the unligated groups there was not statistical difference (wilcoxon's unpaired test) between the methods of duct management. management of the pancreatic duct did not seem to have any immunological consequences for graft survival but septic complications, associated with the normoglycaemic deaths, were more common in immunosuppressed animals with draining ducts. between july 1, 1980 and march 30, 1982 combined pancreatic and kidney transplantation was performed in 9 patients with type i diabetes who had all been on insulin therapy for at least 16 years. age at the time of transplantation was 28 to 45 years. the pancreatic segment used for transplantation consisted of body and tail of the organ based on a vascular pedicle of the celiac axis and the splenic vein. imediately prior to intraperitoneal transplantation to the iliac vessels the ductal system was filled with 4 to 6 ml of prolamine, a rapidly solidifying alcoholic protein solution. simultaneous kidney transplantation was performed through a separate incision. five pancreas transplants were lost for non-immunological reasons. four of them never had any useful endocrine function, one graft was lost of venous thrombosis after 48 hours of excellent function. in none of these patients did failure of the graft lead to any substantial complication. in the 4 remaining patients initial graft function was excellent with plasma glucose normalizing within 12 hours and subsequent normoglycemia on a regular diet without exogenous insulin administration. plasma glucose did not rise spontaneously during rejection episodes of the kidney and eleva-tions due antirejection treatment were promptly; reversed as high dose prednisolone was discontinued. in oral glucose tolerance test (ogtt) performed at 4 to 6 weeks in 3 patients median glucose rose from a basal 5.1 mmol/l to 8.6 mmol/1 and was back in normal range (4.7 mmol/1) at 3 h. basal insulin was 43-237 pmol/1 and also returned to the normal range after 3 h after a peak of 201-531 pmol/1 at 50-210 rain. c-peptide showed a significant 2 peaked rise over basal values (230, 233 pmol/1) in 2 patients while all values were above 4500 pmol/l in the third. two of these patients have since rejected both organs. two tranplants still function very well after 12 and 21 months, respectively. in one of these patients the ogtt after 13 months is even slightly better than the above -mentioned first test, and he shows a norreal insulin and c-peptide response. in the fourth patient an ivgtt performed at 7 months and an ogtt at sixteen months were normal with insulin peaking at 251 pmol/1 and a c-peptide peak of 1267 pmol/1 at 60 min in the latter. -results at 26 and 17 months, respectively, will be presented. three problems persist in clinical organ preservation. these are failure of current systems to replenish the ischemically injured organ, to reliably extend the period of organ preservation and to definitely determine organ viability. previous studies have documented the value of electrochemical redox control in rejuvenation of the ischemically injured kidney during perfusion preservation. this study was undertaken to develop the cost effective technique applicable to current organ preservation systems, to test the reliability of redox measurement in prediction of organ viability and to determine the ability ofredox maintenance to safely extend the period of organ preservation. a disposable cell has been developed using reticulated vitreous carbon as an electrode which is driven by a potentiostat powered by a nine volt transistor battery. short term preservation studies using ischemically injured dog kidneys (60 rain in-situ warm ischemia) were auto transplanted after 24 h of pulsatile preservation to determine the optimum redox level of the hypothermic kidney. this was determined to be a -20 mv vs the standard calomel electrode. twenty-one adult mongrel dogs were equally divided into three groups. pulsatile perfusion preservation was extended to four days in group a with redox level monitored only. group b was treated with electrochemical reduction for four days and group c for six days. three of seven dogs survived in group a following auto transplantation and immediate contralateral nephrectomy. the kidneys of all survivors were able to bring perfusate redox potential under control and maintain this level throughout the preservation period. six of seven dogs in group b survived with a mean posttransplant peak serum creatinine of 4.5 rag/100 ml. in group c four of seven kidneys supported life immediately. all redox controlled kidneys made copious amounts of urine. our data indicate that perfusate potential may be either monitored as a reliable index of organ viability or controlled to allow extended safe preservation. antithymocyte globulin (atg) has been shown to be an effective agent in combination with increased doses of steroids in reversing renal allograft rejection. since it is frequently undesirable to employ raised doses of steroids, the following study evaluated the effect of 15-21 i.v. daily doses of horse atg alone without additional steroids, on 2nd-5th rejection episodes in 10 recipients of cadaveric renal allografts (28 rejections: 25 biopsy-proven) detected within 3-18 mos. of transplantation. of 28 rejection episodes, 25 were successfully reversed, with return of serum creatinine to pre-rejection levels in 18 episodes (7 patients). three patients had primarily humoral rejections and returned to dialysis 2-4 mos. after treatment of the last rejection. levels of circulating horse immunoglobulins were obtained in all 10 patients during and follwing administration of atg. recurrent rejections (3rd-5th) following last treatment with atg (3-11 mos.) were seen in 5/10 patients. all 5 patients had rapid immune eliminiation of atg (1/2 life 2-4 days) as compared to the 5 patients who had no more than 2 rejection episodes (1/2 life 6-14 days). atg without additional steroids is an effective agent for reversal of multiple renal allograft rejections which by biopsy are primarily cell-mediated. to be effective, heterologous atg must be given in adequate total doses and/or from appropriate heterologous source, to prevent rapid immune elimination by the recipient. the use of atg alone for treatment of recurrent allograft rejections is particularly recommended for its steroidsparing effect in treatment of multiple rejections and for those patients at high risk from steroid side effects. the significance of the monocyte crossmatch in lrd recipients of hla identical kidney grafts j. cerilli, l. brasile and s. rogers ohio state university hospitals, columbus, ohio, usa in a preliminary study from our transplant center, the presence of pre-formed antibody in recipient sera directed against monocytes from their respective living-related donors correlated with a poor clinical course. a poor prognosis for graft survival was found regardless of the hla match grade. to minimize the role of the hla system, only those living-related recipient/donor pairs who were hla identical at the a, b, c, d and dr antigen loci, and who exhibited severe immunological types of rejection were evaluated. due to the small numbers found in this category at any one center, this abstract represents an international study from 12 different transplant centers. 26 patients who met the criteria were studied for the presence of antibody directed against their respective donor's monocytes both pre-and posttransplant. in eighteen of these patients, cytotoxic antibody against their donor's monocytes was found in their pre-transplant sera. there was no detectable cytotoxic activity against their donor's t or b lymphocytes. two additional transplant recipients exhibited this antibody in post-transplant sera. again, no t or b lymphocyte cytotoxicity was detected. a control group of hla identically matched siblings who incurred no or minimal rejection demonstrated no anti-donor monocyte antibody. the results of this international study points towards a correlation between a high incidence of graft rejection and the presence of antibody directed against their respective donor's monocytes. therefore, in our view, the presence of anti-monocyte antibody to the prospective donor pre-transplant is a contraindication to transplantation. in patients with different liver diseases the reduced concentration of the peripheral blood t-cells and altered immune response were observed. the purpose of our studies was to investigate the mechanism of the immunological modification of the lymphoid system in the hepatic injury. studies were carried out in 3 groups: 1) lew rates were treated with dimethylnitrosamine (dmna), 35 mg/kg b.w., i.v. for acute liver necrosis induction, 2) cc14 (0.1 ml/100g b.w., i.v.) twice weekly, over 4 weeks for chronic liver damage, 3) ccl 4 over 10 weeks for liver cirrhosis (histologically examined). serum and thymus, spleen, mesenteric lymph nodes (ln) were removed 2 and 32 days after dmna treatment and 5 and 35 days after last cc14 injection. the total number of thymocytes and spleen cells was counted and the reactivity to pha and cona was measured. normal liver perfusate (lp) was prepared 6 h after removing (20°c) by 5 times perfusion in 30 rain intervals (flow rate 1 ml/g/min with 2 ml/g of ringer). the effect oflp, sera, dmna and cc14 on the viability and pha response of normal thymocytes was tested in vitro. liver enzymes were evaluated, we found the decreased total number of thymocytes immediately after the stopping of medication (group 1: 9,2___ 1,8 %, group 2: 4,6___ 1%, group 3: less than 1% of control). proliferative response of the remaining cells in thymus to pha was much higher than normal thymocytes (group 1:325 ± 30 % group 2: 608___164%, group 3: nd). the total number of spleen cells was not changed but their response to cona was altered in the all groups and to pha only in group 3. pha response of ln-cells was decreased in the all groups. liver perfusate was cytotoxic (53___3 % of viable cells) and suppressive for pha response of thymocytes (6_+5% of control). the sera of rats with the hepatic damage showed an enhanced suppressive activity. cc14 and dmna did not have any effect on thymocyte in vitro. one month after last medication we observed the recovery of thymus involution (total i in the acute and partial in the chronic hepatic damage and cirrhosis). our results suggest that the liver origin cytotoxic and immunosuppressiv e factor(s) can be released from the damaged liver into the circulation (like to lp) and can destroy the thymus leading to the secondary changes in the other lymphoid organs. the grade of thymus alteration is dependent on the degree and the duration of hepatic damage and is reversable. the hepatic factor (s) showed the similar effect on the cortical population of thymocytes like the steroid immunosuppressants. liver grafts in the rat are in certain strain combinations not rejected and in this situation there is evidence for spontaneous donor specific tolerance [1] . we have developed a model of auxiliary liver transplantation which would allow us to study the immunosuppressive properties apparently produced by a liver allograft. the portal vein is anastomosed to the left renal artery, the i.v.c. to the renal vein and the bile duct to the ureter. simultaneous kidney or heart allografts were performed. conclusions:auxiliary liver grafts are rejected. survival of heart or kidney grafts is not influenced by a simultaneous kidney or heart allograft. heart and kidney grafts are prolonged by simultaneous auxiliary liver grafts. [1] . the role of suppressor cells in the initiation and propagation of malignant tumours in man, is less clearly defined. the present study, using in vitro mitogen assays (pha, cona, pwm) and various rosetting assays [2] with specific monoclonal antibodies to lymphocytic helper (okt4) and suppressor (okt8) cells, has revealed the presence of such suppressor lymphocytes in women with clinically localised (breast and axilla) mammary carcinoma. lymplaocyte hyporeactivity to mitogens was found in 30% of lymphocyte preparations from blood and axillary lymph nodes of patients with breast cancer. in 10% of patients nodal lymphocytes were totally anergic. the most profound hyporeactivity, however, was detected in the lymphocyte subsets (<75 %) of specimens isolated from the breast carcinomas by collagenase digestion and sephadex g-10 column passage [3] . the lymphocyte preparative techniques were not responsible for the low levels of responses detected. also, in situ prostaglandin synthesis and release did not appear to be involved in depressing lymphocyte reactivity [4] . comparable percentages of suppressor cells (okt8+) were detected within these different lymphocyte preparations. suppressor cells were not found in the lymphocyte preparations from the blood and lymph nodes of appropriate controls. from clinical and experimental studies it is known that blood transfusions may have immunosupressive as well as immunostimulating consequences. the effect of transfusions on graft survival has been extensively studied by our group in the bn to wag rat model. in this donor-host combination it was found that a donor specific pre-transplant blood transfusion could lead to a marked prolongation of heart and kidney graft survival, whereas the similar pretreatment resulted in accelerated rejection of bn skin allografts. this specific model was used to investigate the influence of a single bn transfusion on the growth of 2 different syngeneic transplantable tumors in wag rats. the first tumor was a radiation induced basal cell carcinoma of the skin (t 1), the second tumor was a chemically induced adenocarcinoma of the duodenum (t 2). the antigenicity of both tumors was assessed in vivo, using classical challenge-protection experiments. it was observed that t i exhibited strong immunogenetic properties, whereas t 2 was only weakly immunogenic. the doubling time oft i was 2.5 days, the doubling-time of the adenocarcinoma was 14 days. intravenous inoculation of isolated t 1 cells led to development of lung nodules which could be counted after 14 days. wag rats were injected i.e. with 1 ml of bn blood or syngeneic blood (controls) at 7-14 days before tumor challenge. t l was given in two different ways: a) sc. implantation of+2x2 mm pieces, b) i.e. injection of 106 isolated tumor cells. t 2 was implanted sc. only. each experimental group consisted of 8 animals. for t 1 it was found that allogeneic blood transfusions caused a slight (but not significant) inhibition of subcutaneous tumor growth. however, in the t 1 lung-metastasis model it was observed that a single bn blood transfusion led to a 50 % reduction of nodules, counted at 3 weeks after inoculation. this reduction in number and size of nodules was highly significant (p<0.01). for tumor t 2, the bn blood transfusions evoked a strong inhibitory effect on the growth of the sc. implanted tumor. at 8 weeks after implantation all tumors in the control group had grown to a diameter of 10-16 rnm (average diameter 12.2 ram). in the group pretreated with bn blood, only 3 of 8 tumors were palpable at that time (average diamter 4 mm). for t 1 it was further investigated whether a single bn transfusion, given one week after i.e. tumor cell inoculation, would have any influence on tumor growth. no significant effect on number or size of the lung nodules could be noticed, if anything, the transfusion appeared to have a stimulatory effect. the results indicate that allogeneic transfusions can lead to a substantial modification of tumor growth, depending on tumor type and site of implantation. this observation may have important clinical implications. we report here the serial study of circulating immune complexes (cic) in two human tumor systems, colorectal cancer and gestational trophoblastic neoplasia (gtn). cic were assayed by antigen nonspecific insolubilization induced by 3.75 % polyethylene glycol (peg) and monitored as a od45o changes by spectrophotometry. all 11 of the serially studied colorectal cancer patients presented with elevated cic levels (mean = 610 + 396 zt od450) as compared to our standard cic level for pooled normal human sera (202--+_4 aod450, p<0.05). initial values in these patients range from 304 to 1407 a od450 with no correlation to tumor load, site of presentation, or subsequent clinical course. in 6/7 patients who underwent ,,curative" resection of primary or metastatic colorectal cancers, serial cic elevations occured only when antigen excess (measured by simultaneous carci-noembryonic antigen [cea] assay) decreased. immunoglobulin components of fractionated cic showed predominantly iga subclass. in 30 gtn patients followed with serial cic and simultaneous human chorionic gonadotropin (hcg) assay, only those patients documented to enter hcg remission after molar evacuation showed significant elevation of cic. chromatographic fractionation of peak cic in one such patient defined three irnmunoglobulin containing fractions showing immunoreactivity to one of four paternal hla haplotypes (aw32). one ,,antigen" fraction (<25.000 mw) from this complex completely inhibited reference anti-aw32 binding. as in the colorectal cancer patients, these data show that cic rise only when antigen excess decreases (reflected in the gtn patients by hcg normalization). in addition, some gtn patients may react to immunogenic paternal hla haplotypes as part of their response to molar pregnancy. dfmo, an enzyme-activated irreversible inhibitor of ornithine decarboxylase (odc), reduces tumor polyamine levels, inhibits growth of emt6 sarcomas and hepatomas in experimental animals, and induces remission in human leukemia. a renal adenocarcinoma (ra) cell suspension, or a 1 mm segment ofwilms' (wm) tumor was transplanted intrarenally into balb/c mice (n--105) or subcutaneously into wistar-furth rats (n=80) respectively. dfmo (2 %) in drinking water was administered to half the animals in each group throughout the experiment. at 28 days ra tumors in dfmo-fed mice weighed 72 % less than tumors in control animals (p<0.001); wm tumor weight at 28 days was not affected by dfmo feeding. the mean number of lung metastases in dfmo-fed r.a-bearing mice was 0.1 and in ra-bearing control mice was 1.4 (p<0.001). dfmo caused 72-75 % inactivation of tumor odc, reduced ra putrescine levels by 710/o (p<0.001), reduced wm putrescine levels by 65% (d7<0.01), reduced wm spermidine levels by 58% (p<0.001) and increased wm spermine levels by 37% (p<0.01). dfmo feeding did not alter dna content of ra or wm tumors. although final carcass weight was similar in all animals, dfmo feeding progressively reduced total body weight (tbw) of mice, but not rats, until at day 20 the tbw of dfmo-fed mice was 10.5% less than tumor-bearing control mice (p<0.01). dfmo-fed mice bearing ra tumors survived 3.0-t-0.8 days longer than control mice (p<0.05). reduction of polyamine levels in wilms' tumors does not affect tumor growth. lowering of renal ade-nocarcinoma putrescine levels by continuous feeding of dfmo to tumor-bearing animals decreases tumor growth, reduces lung metastases, and increases host survival. we have previously demonstrated that vagal nerve stimulation releases 5-ht into the lumen of the feline gut. this study was initiated to: 1. determine if substance p (sp) and motilin (mt), other enterochromaffin cell products, are released simultaneously, 2. to evaluate if this release is under cholinergic or adrenergic control. in 6 cats, 15cm isolated in situ segments of proximal jejunum were perfused with saline at 1 ml/min (37 ~c). perfusate samples were evaluated at 5 rain intervals and concentrations of 5-ht, sp, motilin were measured by ria's developed in our laboratory. after two 5-min basal periods, the supradiaphragmatic sectioned vagus nerves were stimulated electrically (10v, 5m s, 10hz). the output from the loop in ng/5 min (5-ht) and pg/5 rain (sp and mt) was: introduced into the jejunum of conscious dogs through an external small bowel fistula. the gut was perfused at 5 ml min-1 with a physiological electrolyte solution containing the non-absorbable marker polyethylene glycol (mol. wt. 4000; 5 g 1-1); water and electrolyte absorption and transit time (tt) were measured during intravenous (i.v.) administration of each peptide, and during preceding and succeeding i.v. control infusions of 0.15m naci. separate studies showed jejunal absorption and tt to be constant over prolonged periods during i.v. nac1 administration. bombesin (10 pmol kg-lmin-1) and neurotensin (1 pmol kg-~min 1) significantly reduced jejunal water absorption; bombesin and enkephalin (0.5 nmol kag-1rain-1) significantly prolonged tt; and enkephalin encreased water absorption (/7<0.05 in all cases). measurement of plasma neurotensin during i.v. infusion indicated that physiological blood levels were not exceeded during these studies. conclusion: a number of peptides may be involved in the regulation of small bowel function. the effect of neurotensin on jejunal water transport provides a possible mechanism linking raised blood neurotensin levels with intestinal intraluminal fluid accumulation in the dumping syndrome. in order to establish whether this release was under adrenergic control, 4 cats had cervical ganglionectomies. using the same electrical paramenters, stimulation of the cut cervical vagus nerves resulted in identical 5-ht responses as above. in 4 additional cats atropine administration (lmg/kg iv) totally abolished the 5-ht responses to vagal nerve stimulation. the paralled release of 5-ht, sp, and mt following vagal nerve stimulation, strongly suggest that the ec cell is the source of these luminal hormones. this release appears to be under cholinergic control. since diabetes mellitus is markedly improved immediately after jejunoileal bypass before significant weight loss, but only gradually and often incompletely changed after gastric bypass, it seemed appropriate to investigate the effects of intestinal exclusion on experimental diabetes. studies were performed on alloxan diabetic sprague-dawley rats. two days after jejunal exclusion (je) in 8 rats (resection of proximal 1/3 of small intestine), fasting blood sugars (fbs) decreased 580, from 344 to 146mg/dl, and averaged 164 mg/dl at 3 weeks. after ileal exclusion (ie) in 7 rats (resection of distal 1/3 of small intestine), fbs fell 39% in 2 days, from 250 to 153 mg/dl, but increased 40% above preoperative levels to 350 mg/ dl at 3 weeks. sham operated rats responded similarly to ie rats. after alloxan and operations, all groups lost weight, but only je rats began to increase weight at a normal rate. increased water intake, polyuria (140 ml/24 h), and glycosuria (2756 rag/all), were present in ie rats; je rats were normal (urinary output <20 ml/24 h, and urinary glucose 36 mg/dl). oral glucose tolerance tests (gtt) were extremely abnormal in ie rats, similar to those in non-operated alloxan rats, while gtt curves in je rats were similar to normal animals, with some elevation at 30, 60 and 120 min. serum insulin levels remained low in all alloxan treated rats after jejunal exclusion. possible mechanisms, currently under study, relate to carbohydrate malabsorption and changes in enteric chemical mediators. the purpose of the present study was to investigate the changes in somatostatin release and somatostatin-containing cells of the pancreas and stomach of the streptozotocin (stz) -induced diabetic rat after the amelioration of diabetes by whole pancreatic transplantation. highly inbred lewis rats were divided into three groups: (1) normal rats, (2) stzinduced diabetic rats and (3) transplanted rats. diabetes was induced by the administration of stz (60 mg/kg). on the seventh day after stz treatment, pancreatic transplantation was performed. four weeks after the transplantation, in vivo and in vitro, studies were performed. pancreatic d cells and gastric somatostatin-containing cells were stained with antibody enzyme method. studies in vivo showed marked improvement of the impaired arginine-induced insulin release by the transplantation. studies in vitro employing isolated perfused rat pancreas and stomach revealed following results: mean basal pancreatic somatostatin release in normal, diabetic and transplanted rats were 12___3, 24-t-7, and 17__+4 pg/ml, respectively. total amount of pancreatic somatostatin release in each group during arginine stimulation (19.2. mm) were 946--+200, 2321___266, and 1013-+126 pg/15 min, respectively. significantly higher somatostatin release was obtained from the diabetic pancreas, which was, however, reduced to normal after the whole pancreatic transplantation. on the other hand, insulin release from the diabetic pancreas was severly impaired and pancreatic transplantation had not effect on insulin release from the host pancreas in the transplanted rats. as to the glucagon release, there was not significant difference among them. mean basal gastric somatostatin release in normal, diabetic and transplanted rats were 179-t-5, 173___5, and 135 + 5 pg/ml, respectively. there was no significant difference between normal and diabetic rats, though the significant decreased value was obtained in the transplanted rats. (vs. normal;/7<0.01, and diabetes; /7<0.01). on the other hand, glucagon-stimulated peak values in these groups were 498_ 36, 662___47, and 412+25pg/ml, respectively. glucagon-stimulated gastric somatostatin release in diabetic rats was significantly increased, but reduced to normal value by pancreatic transplantation. also, a number of pancreatic d cells and gastric somatostatin-containing cells were markedly increased on the diabetic rats. on the other hand, a number of these cells in the transplanted rats were descreased to normal levels. in summary, enhanced pancreatic and gastric somatostatin release and cells in the diabetic rats were both normalized after the amelioration of diabetes by the whole pancreatic transplantation. from these results, it is suggested that pancreatic and gastric somatostation are regulated by circulation and/or metablic of nutrients. doppler velocity recordings are widely used for the non-invasive diagnosis of carotid arterial disease. although detailed analysis of carotid doppler spectral information has been suggested as a method for improving diagnostic sensitivity, the accuracy of the relationship between the doppler recording and the true instantaneous velocity profile has not been established. the purpose of this study is to determine if a cw doppler velocitymeter can accurately transduce the true instantaneous blood flow velocity information. methods: a pulsatile flow model has been constructed in which it is possible to record the instantaneous doppler spectrum and simultaneously photograph and measure the true velocity profile. a computer controlled pump generates a carotid waveform in tubes without stenoses and with, symmetrical stenoses. flow is visualized using the photochromic dye tracer technique. a short burst of uv light from a laser is passed across the tube. a narrow band of the fluid turns blue, its movement is photographed, and the instantaneous velocity profile determined every 10 msec throughout the pulse cycle. at the same time, the instantaneous doppler spectral information is recorded by a frequency analyzer. the results follow. for pulsatile laminar flow, the doppler spectrum correctly recorded the true velocity spectrum, including the instant/~neous maximum velocity and mean velocity. for disturbed flow, it was not possible to show the same direct relationship between the doppler spectral recordings and the blood flow velocity. in conclusion doppler velocitymeters accurately transduce velocity information when flow is laminar but when flow is disturbed there is not a direct relationship between doppler recordings and the true velocity profile. consequently, one should be cautious in attempting to relate doppler measurements of disturbed flow directly to the true changes in the velocity pattern. early failure of arterial reconstruction may originate in poor patient selection. in aorto-iliac stenosis (ais) selection for operation relies upon clinical examination of the femoral pulse and radiology. since single plane arteriography is inadequate for accurate definition ofiliac stenosis [1, 2] , this paper compares clinical examination, doppler ankle systolic pressure (aspi) and femoral signal analysis (laplace transform damping, ltd, and pulsatility index, pi) [3] with biplanar contrast studies. i at biplanar angiography 66 of 102 ischaemic lower limbs had ais with diameter reduction from 25-84 %, of the remainder, 25 were normal (<25 % stenosis) and 11 were ,,occluded" ('_--850/0). nearly two thirds (61%) of limbs with a clinically normal femoral pulse had identifiable arteriographic stenosis (~-__.250/0), upstream abnormality was predicted incorrectly in 15 % and the overall accuracy of clinical examination was 67 %, both for detecting stenosis and predicting its severtiy. aspi (median 0.61; 95% confidence ,limits 0.3-1.0) and pi (5.4; 2.3-0.05) although correlated with stenosis (aspir = 0.65; p = 0.05, pir = 0.62; p=0.05 variance analysis for linear regression), did not aid the clinician further (accuracy 62 %). however ltd (0.72; 0.37-1.0) was well correlated (r=0.76, p=0.001) and did improve assessment ofiliac stenosis (accuracy 84 %). the need for biplanar arteriography is reiterated and its use with doppler signal analysis should improve the evaluation of aorto-iliac disease. in the absence ofa non-invasive method for estimating volume flow in an individual artery, local blood pressure measurement has proved, with certain limitations, useful in assessing the cardiovascular system. now ultrasound technology has progressed to enable blood flow in an artery to be measured noninvasively. we report the results o four evaluation ofa 5 mhz, computerized, 30 channel, pulsed doppler vessel imaging and flow measuring instrument in in-vivo experiments. computed blood flow was compared to actual blood flow (calculated by timed collection) in 17 anaesthetised dogs. correlation between computed and actual blood flow was stronger in the larger abdominal aorta than in the smaller common carotid arteries. from the regression plot, the coefficients of determination, p, were: 0.927 (exposed aorta scans); 0.857 (transcutaneous carotid scans); and 0.784 (exposed carotid scans). stepwise regression analysis showed the computed flow values to be independent of probevessel angle, depth and lumen diameter for vessels greater than 2.5 mm in diameter. these results suggest that this pulsed doppler instrument has the versatility and accuracy essential for diagnostic flow measurements in the main conducting arteries of the neck and limbs and in vascular bypass grafts. in the assessment of patients undergoing carotid artery surgery, many laboratory methods are available in addition to angiography. in a series of 210 patients experience has been gained with the use of eeg, tc 99m isotope scanning, opg, ct scanning and doppler. in a 10 year follow up over 85% of patients had a satisfactory outcome. an early mortality of 2% in the beginning of the series has been eliminated due to improved selection. in this report the application of multi-gated pulsed doppler techniques is reported. this allows a non invasive measurement of mean volume flow in the common carotid artery with a method reproducibility of + 5 %. mean volume flow in 50 undiseased arteries (25 subjects mean age 46 years) was found to be 536± 104 (s.d.) ml/min. from this a lower range for normal flow of 300 ml per minute (2 x s.d.) was selected. 30 patients were investigated before surgery and a follow up examination was performed at a mean interval of 4v2 months post operatively. 3 groups were defined. group a >300 ml/min; group b 200-300 ml/min; group c <200 ml/min, of 14 arteries in group a before surgery, 11 remained in the group and 3 dropped to group b. in group b, of 8 arteries, 5 go to group a, 2 remain and 1 dropped to group c. in group c, 7 out of 8 arteries moved to group a and 1 to group b. thus of 16 arteries with below normal volume flow before surgery, 12 were returned to normal range and further 1 improved. 2 remain unchanged and 1 disimproved. of the 30 arteries examined 4;/2 months after surgery, 23 are in the normal range and a further 1 improved. 2 remain unchanged and 1 disimproved. of the 30 arteries examined 41/2 months after surgery, 23 are in the normal range in flow values and a further 2 remain unchanged. the non-invasive and isotope techniques have a valuable and practical application in assessment of carotid artery surgery. timing is influenced by the finding fo infarction on ct or isotope scanning. doppler techniques are useful not only in defining severity of diseas and sub-radiological plaques, but valuable flow information can be obtained by pulsed doppler pre and post operatively. this may help in identifying patients who need further medical or surgical treatment. stepwise logistic regression-amodel for predicing success of femorai-popliteal bypass grafts the objectives of this study were to identify the preoperative factors that influenced postoperative patency of femoral-popliteal grafts and to develop a model that could be used prospectively to determine the probability of successful outcome. data base material consisting of history, physical examination, laboratory data, angiography, and operative findings in 199 patients undergoing femoral-popliteal bypass grafting was entered into a computer programmed for stepwise logistic regression analysis. the computer identified and ranked 24 factors that influenced outcome. the top five factors (other than technical problems) included quantity of runoff, previous ipsilateral femoral-popliteal bypass, preoperative prediction of potential amputation level, concurrent proximal vascular reconstruction, and the location for distal graft anastomosis. having established the computer data base, it is now possible to enter information from new patients into the computer which will weigh all factors and indicate the likelihood of surgical success. in addition, tables can be generated which will look at simple combinations of variables to predict patency. for example, in a patient about to undergo a primary femoral-popliteal bypass with no anticipated technical problems, the likelihood of success as a function of runoff and preoperative amputation level is as follows: irreversibility of shock and ischemic injury is generally considered a consequence of extensive cellular injury. to study the role of intravascular coagulation in shock, 56 rats were bled to a mean arterial pressure of 50 mm hg for 3 hrs or 25% uptake of shed blood, whichever occured first. return of shed blood with these data provide the patient and the surgeon with a quantitative prediction for success and permit an informed decision when considering therapeutic alternatives. potential cytoprotection by heparin was studied by similarly bleeding 52 additional rats; controls were only cannulated. twenty pairs were heparinized (h); 32 were not (nh). paired-bled and control rats were sacrificed following hemorrhage, liver (l) and kidney (k) mitochondria were isolated, and the isolates were studied by the polarographic technic with glutamate and succinate to determine the respiratory control index (rci) as a measure of cellular injury. results were: an equal volume of isotonic saline resulted in a 43 % survival; % uptake of blood during shock allowed prediction of survival. an additional 55 rats were then randomized (coin toss) to heparinization versus no heparin prior to shock, and were similarly bled and resuscitated. significantly improved survival (p<0.025) was seen in heparinized (17/23; 740/0) versus nonheparinized rats (13/32; 41%). uncoupling and inhibition of mitochondrail function were noted in both h and nh rats with rci being significantly reduced from control. however, there was no difference between h and nh compared to each other. heparin does not provide cytoprotection during shock; improved survival with heparin may rather be a consequence of improved reperfusion of tissues following the shock episode. fl-endorphin (b-end) has been postulated to play a role in the pathogenesis of shock because the opiate "antagonist naloxone improves the macrohemodynamics in various shock models [1] . however, plasma levels ofopioid peptides have not been determined as yet. the aime of our study was to measure the plasma concentrations of various peptides and to evaluate the influence of naloxone particularly on the plasma concentration of r-end. in 16 anesthetized foxhounds, the adrenolumbar vein was cannulated and hemorrhagic shock (map = 4 mm hg for 3 h) was induced according to wiggerstechnique. the plasma levels offl-end, methioninenkephalin (m-enk), and leucine-enkephaline (l-enk) were simultaneously determined in c.v. and/ or adrenal venous blood by a specific ria. crossreactivity of r-end withfl-lipotropin was about 4 %. the enk-antibodies crossreacted with less than 5 %. five dogs received an i.v. bolus of naloxone (2 mg/ kg) and a subsequent naloxone infusion of 2 mg/kg/ h after 2 h of hypovolemia. eleven dogs served as control and received equivalent volumes (1 mg/kg per h) of ringer solution. hemorrhage resulted in a sharp rise of central venous plasma levels particularly of m-enk and l-enic this effect was even more pronounced in the adrenal's effluent system.fl-endorphin levels remain elevated whereas the enk secretion began to decrease 1 h after hemorrhage. naloxone treatment inhibited any spontaneous fall of adrenal enkephalin release during the shock phase and the values remained elevated 10-15 fold. volume substitution with autologous blood resulted in a normalization of all peptide levels. these data demonstrate that hemorrhagic shock will cause stimulation of endogenous opioid peptides. the high levels of enkephalins in the adrenolumbar vein indicate that the adrenal gland is the main source of these peptides in the circulation. in addition toil-end, the enk seem to play a role in the pathogenesis of shock as well. at our present state of knowledge, however, it is difficult to design a coherent concept of mechanisms involved. this shows that cp treated cells bound nearly as much [125t]-acth analog as control cells but there was very little specific binding to sp treated cells. low concentrations of acth effectively displaced the acth analog whereas exposure of adrenocortical cells to sp resulted in a significant decrease in acth receptors. this suggests that sp has a factor(s) that binds to acth receptors of adrenocortical ceils which may adversely affect the stress response of shock. f-43 has been proposed as superior to other asanguinous fluids due to increased oxygen carrying capacity. evaluation to date has been largely uncontrolled and at extremes of hemodilution (hct. <2 %) rarely seen clinically. near infrared spectrophotometric monitoring of brain cytochrome a, a3 redox state, a sensitive indicator ofintramitochondrial oxygen availability, offers a unique opportunity to contrast f-43 with balanced salt-albumin (bsa) and whole blood saline (wbs) as a resuscitative regimen in a clinically relevant model. fifteen rats were subjected to 30 minutes of hypoxia (fio2 = 7,5%) and hemorrhagic hypotension (map = 30 mmhc), then randomly allocated to one of three groups and resuscitated by fio2 = 100% and infusion of either f-43, bsa, or wbs. cytochrome a, a3 redox state was monitored continuously at 813 run. thirty additional rats were sacrificed at baseline, end shock and 20 and 120 minutes post resuscitation for cerebral cortical atp and lactate assay. despite hematocrits as low as 15 % in the bsa and f-43 groups, there were no significant differences /7<0.05 between groups in the parameters of oxygen sufficiency; atp, lactate, and cytochrome a, a3 redox state. we assume differences in cardiac output compensated for differences in arterial oxygen content. on this basis we suggest perfluorochemical utilization should be limited to situations in which hematocrits are <15 % and when cardiac reserve is limited. metabolites of the prostaglandin endoperoxide h2 (pgh2) affect both vascular tone and platelet aggregation and thereby may influence blood flow. we, therefore, determined the metabolites formed from pgh2 by microsomes isolated from human saphenous vein used for aortocoronary bypass surgery. in the absence of reduced glutathione (gsh), the enzymatic metabolism of 14c-pgh2 produced only prostacyclin (pgi2) as measured by the formation of its stable breakdown product 6-keto-pgfla. the amount of pgi2 formed varied from 10-50% of the substrate depending upon the microsomal protein and pgh2 concentrations. in addition, the nonenzymatic breakdown of pgh2 resulted in the formation of pgf2a, pge2, pgd2 and heptadecatrienoic acid (hht). there was no formation of thromboxane a2 (txa2) as measured by the absence of its stable breakdown product txb2. in the presence ofgsh, a required cofactor for microsomal pge2 isomerase activity, the formation of pge2 was augmented 2 fold (up to 35 % of the substrate) indicating enzymatic for-mation of pge2. the gsh either did not alter or augmented (less than 1 fold) the formation of pgi2. the increased formation of pge2 in the presence of gsh was at the expense of decreased nonenzymatic breakdown of pgh2 to pgf2a, pgd2 and hht. these data suggest that prostacyclin synthetase activity may serve to protect the vessel graft from platelet aggregation and/or vessel spasms and may possibly serve as an indicator of graft viability. thrombosis is a frequent cause of early arterial bypass graft failure and platelets are known to be major determinants ofthrombus formation on arterial surfaces. pgi2 and flbriolytic activators from the vascular wall counteract intravascular thrombosis. the aim of this work was to study the effect of arterial grafting on the aforementioned mechanisms. 6 cm lengths of tanned human umbilical vein grafts (huvg) with an internal diameter of 5 mm were interposed end-to-end in the carotid arteries (c.a.) and jugular veins (j.v.) of sheep. placed in the c.a.'s, 12 grafts with restricted flow (50 cc/min) were removed on the 10th postoperative day (group i); 18 grafts with unrestricted flow (140___20 cc/min after placement) were taken out 90 days later (group ii) and 4 grafts placed in the j.v.'s were removed 10 days after surgery (group iii). upon removal, the grafts were checked for patency and sections from the proximal and distal anastomoses and midgraft were obtained for determination of pgi2 production (ria) fibrinolysis activators activity (histochemical method) and for light and scanning electron microscopy. sections from the femoral arteries were also obtained. the results of pgi2 generation are expressed in ng/ml/cm% all grafts showed fibrinolytic activity in the adventitia but 4 grafts in group ii also showed fibrinolytic activity in the intima. early neointimal fibrous hyperplasia (nfh) characterized by proliferation of smooth muscle cells was present in group ii. the, occluded grafts showed organizing thrombus material and inflammatory cells and the patent ones showed fibrin and scattered inflammatory cells. in groups i and iii, sem revealed numerous platelets and rbc's incorporated into a proteinaceous material overlying the anastomoses and in some specimens obvious thrombus material was present. in group ii, the anastomotic areas were covered with large endothelial cells, nonetheless, some areas were denuded and small thrombi were occasionally noticed. in conclusion: 1. anastomotic sites create a strong stimulus for thrombus formation despite a high production of pgi2. this suggests that antithrombotic therapy may be necessary to prevent early failures. 2. huvg develop the capacity to produce pgi2 and fibrinolytic activators and 3. although the etiology of nfh remains obscure, the decreased levels of pgi2 in group ii suggest that exhaustion of pgi2 generation from the endothelium might occur leading to proliferation of smooth muscle cells (nfh). these cells will in turn supply pgi2 if a persistent stimulus exists. permeability of intestinal capillaries to fibrinolytic products d. manwaring and p. william curreri department of surgery, university of south alabama college of medicine, usa fibrin/fibrinogen degradation product d (fdp-d) is significantly elevated in the serum of patients after trauma or sepsis. purified fdp-d infused into nontraumatized rabbits precipitates thrombocytopenia, complement depletion, pulmonary dysfunction and increased permeability of lung capillaries to i12s-albu -composite fibrin plate assay size oflytic zones (mmx) after 17 h incubation at 37 °c rain. in order to determin of products of fibrinolysis alter fluid filtration or permeability, either purified fdp-d or fdp-e were tested in an isolated, autoperfused cat ileum preparation. steady-state lymphatic: plasma protein concentration ratio (cl/cp) and lymph flows (ql) were measured at a venous outflow pressure of 5 mmhg. data was analyzed for each animal group by the paired student t test for ql, cl/cp and protein clearance (ql x cl/cp). in cats which received fdp-d (n=7), ql and clearance increased five-fold (p<0.002), but cl/cpwas not altered, which suggests a permeability change. ileal mucosal biopsies prepared for histology had villi that were de-epithelialized and platelet clots in blood vessels. fdp-e (n=7) provoked a slight increase in ql (p<0.01), but not in cl/cp or clearance. histology was normal. (fdp-e causes no pathological lung change in awake rabbits). fdp-d may contribute to various organ pathologies after trauma. the effect of aspirin on the fibrinolytic activity of viable granulocytes r.c. franz, w.j.c. goetzee, b. rotunno and r. anderson department of surgery, university of pretoria, rsa although several influential authors have suggested that low dose (150 rag) acetyl salicylic acid (asa) represents a balanced daily antithrombotic regimen probably by both inactivating thromboxane a2 production and enhancing prostacyclin synthesis little is known about the effect of aspirin on the fibrinolytic activity of live granulocytes [1] . the present study was designed to evaluate this effect in vivo. methods: granulocytes from fasting samples of heparinized venous blood taken from male volunteers were separated from monomuclear cells and platelets by density gradient centrifugation (ficoll: sodium metrizoate). viable granulocyte suspensions and plasma samples were placed as drops on a coinbefore aspirin after aspirin p = [2] . the experiment was repeated 3 h after each subject had ingested 1.8g of aspirin. the results are summarized in table 1 . 1. there appears to be a significant increase in granulocyte fibrinolytic activity 3 h after the ingestion of 1.sg of aspirin. 2. this increment is insufficient to overcome the resting inhibitor potential of plasma on granulocyte fibrinolysis. 3. aspirin does not evoke a significant increase in plasminogen activator-(urokinase) induced flbrinolysis in platelet free plasma or in the combined system of granulocyte-plasma mictures. 4. the optimal dosage of aspirin as a fibrinolytic agent requires further study. the terminal vascular bed of malignant tumors is characterized by a lack of organization, differentiation and sufficient developement of nutritional capillaries. as a result, malignant tumors reveal consistently small regions of low or even no perfusion. pre-vious data in a melanoma indicate that due to the rarefication of capillaries, the full impact of tumor treatment ±st diminished by an elevated microvascu lar resistance, which could significantly affect the impact of tumor therapy. since the improvement of the blood's fluidity has been shown as one therapeutic modality to increase significantly the capillary blood flow, it was assumed that this measure might enhance the accessibility of tumor tissue for bloodborne drugs. this study was aimed to investigate the effects of the improvement,of microcirculatory flow on tumor growth and tissue oxygenation. moreover, the response of the melanoma to chemotherapy was evaluated when isovolemic hemodilution was employed in conjunction with chemotherapy. a transparent chamber technique, intravital microscopy, a platinum multiwire electrode (local po2 measurement) and quantitative television image analysis (capillary blood cell velocity and diameter) were employed to study the microvasculature in the amelanotic melanoma a-mel-3 of 18 hamsters in the event of hemodilution without and in conjunction with chemotherapy (200 mg/m 2 dtic, dimethyl-triazeno-imidazol-carboxamid). permanent indwelling catheters in carotid artery and jugular vein served for measuring systemicpressures, heart rate, for withdrawing blood and the infusion of dtic and/or dextran 60. after inoculation of 4 x 104 cells of the amelanotic hamster melanoma a-mel-3 into s.c. tissue in the preparation, this tumor re~ched a diameter of approx 3 mm within five days. the reduction of systemic hematocrit from 0.45 to 0.31 (1.3 ±0.2 ml blood vs dextran 60, 7 animals) at a tumor diameter of 3 mm increased the growth rate of the melanoma by about 30 % while enhancing significantly the volume flow through capillaries and the mean local po2. table 1 control hemodilution capillary velocity (ram/s) 0.39 _ 0.12 0.50 ± 0.12 capillary blood flow (ml/min x 10 -5) 5.4 ± 1.7 8.9 ± 1.2 mean local po2 (mmhg) 9.4 (0 -28) 15.7 (0 -60) the frequency distribution of local po2 on the tumor's surface showed a distinct shift toward higher po2 values with still some hypoxic regions remaining. intravital microscopy, however, revealed petechial bleeding and localized, interstitial edema which compressed a small number of capillaries. by contrast, the tumor's diameter remained at app. 3 mm for a period of ten days with chemotherapy alone (3 animals). in one of the animals, a complete stop in the melanoma microcirculation was seen within four hours after infusion of dtic followed by a significant decrease of tumor diameter. when chemotherapy was initiated in hemodiluted animals, neither retardation of tumor developement nor vascular obstruction was observed (5 animals). conclusion: capillary blood flow of the melanoma can be enhanced by hemodilution thus diminishing tissue hypoxia. this measure, however, was associat-ed with an increase in melanoma diameter of 30%. at the present, we investigate whether, in hemodiluted animals, a reduction of tumor size can be obtained with a higher dose of dtic. ). however, the etiology of stress hyperglucagonemia in the immobilized rat is only poorly defined. since during restraint stress, catecholamines (ca) are elevated and stimulation ofglucagon by ca is accepted (woods sc d jr [1974] physiol rev. 54: 596), we decided to study by surgical means the the relative contribution to glucagonemia of different sources of ca, i.e. peripheral sympathetic nervous system and adrenal medulla. methods: male sprague-dawley gastric fistula rats (n=74), weight approx. 250 g, were subjected to either sham-op or various sympathectomies [microsurgical splanchnicotomy = s-sx; chemical sympathectomy = c-sx (150 mg/kg 6-oh-dopamine ip two days prior to the experiment); adrenal demedullation = amx; combinations: s-sx + amx; c-sx + amx]. gastric acid secretory trials (duration 8 h), preceded by a 24 h fasting period were carried out 6-7 days following the operation. at the start of the experiment an intraperitoneal polyethylene tube, was inserted into the abdominal cavity of the rats, allowing a constant infusion of physiological saline (4 ml/ kg/h). in addition, stress was performed by pairwise restraint of the extremities and small electric shock waves applied by a tail electrode. at the end of the experiment, blood was drawn from the vena portae and the abdominal aorta for plasma and serum. hormones (glucagon, insulin) were measured by radioimmunoassay, glucose enzymatically, volume was read to the next 0.1 ml, acidity by microtitration. results (see table) : volume and acidity are not changed by the various sympathectomies, when 179 compared to the sham group. the same is true for acid secretion, except in s-sx + amx, where it is elevated. glucagon in peripheral plasma is elevated in c-sx, amx and c-sx + amx. in the portal vein, glucagon is dccreased in s-sx + amx (408___99 pg/ ml) and elevated in c-sx + amx (2625___405 pg/ml) when compared with sham rats (930--.195 pg/ml). the portal/aortal glucagon ratio is significantly decreased only in c-sx and amx (1.7--+0.9, 1.7--.1.0, resp.) when compared with sham (3.8--_ 2.1). insulin is increased only in amx, glucose decreased in amx, s-sx + amx and c-sx + amx, insulin and glucose are unchanged in the other groups. conclusion: 1. stress hyperglucagonemia in the rat is confirmed (levels during zero stress 30-180 pg/ml) and also the rise in insulin following removal ofadrenomedullary ca (but not other sympathectomy); 2. the blood glucose fall (amx; s-sx + amx; c-sx + amx) is not uniformly paralleled by hyperglucagonemia, but in the case of amx it may be secondary to relative stress hypoglycemia owing to removal of adrenal medullary ca or reactive insulin release; 3. mechanism underlying the increased systemic glucagon despite partial (c-sx; amx) or total (c-sx + amx) sympathectomy are yet unknown. 4. during stress the enterogastrone component of hyperglucagonemia may be of minor importance. evlw showed good agreement with gravimetric lung water determinations. significant lung water accumulation was produced by pressure elevations over 20 mmhg. reductions in plasma oncotic pressure significantly increased transvascular fluxes at each level of pressure elevation. however, fluid accumulation was not significantly greater during hypoproteinemia. we conclude that a 40-50% reduction in plasma oncotic pressure does not contribute to increased high pressure edema because the lymphatic safety factor is augmented. this phenomenon may explain the well tolerated state of hypoproteinemia in patients after hemorrhagic shock. computerized gamma scintigraphy is a new technique for the analysis of albumin flux in the acute respiratory distress syndrome (ards). the objectives of this study were to obtain normal control values and to determine the method's validity in patients with cardiogenic vs. permeability pulmonary edema. methods: following 10 mci99mtechnetium-human serum albumin, lung :heart radioactivity ratios were determined. this ratio remains constant unless there is a leak o falbumin, when a rising ratio is seen, called the ,,slope index" (si). si's were determined in 5 control individuals who had :> 50 % left ventricular ej ection fraction and < 7s pulmonary circulation. thirtythree studies were obtained in 27 patients using a portable gamma camera. fourteen patients had clinical evidence of ards. results: studies were considered positive if the si was 2 s.d. > control mean ( -0.4___0.5 x 10-3 units/ min). among 15 positive studies, all had diffuse air space disease on chest radiographs. their average pulmonary capillary wedge pressure (pcwp) was 14.6_+4.6 mmhg. the average artertial: alveolar oxygen tension ratio (a/a)o2 was 0.31+--0.15 on 10.8 cm h20 peep, which were both significantly (p<0.01) different from patients with normal si's. positive si's were present from 24 hours to 6 days following the apparent onset of ards in 6 patients. recovery of gas exchange was associated with normal si's on repeat studies in 4 patients. of 6 patients with cardiogenic pulmonary edema, 5 had negative studies (18-29 mmhg pcwp) and i a positive study (40 mmhg pcwp). conclusion: gamma scintigraphy was a sensitive, non-invasive tool for the detection of a pathological increase in pulmonary protein flux, which was usually normal in cardiogenic pulmonary edema. positive scintigraphy was associated with significantly impaired gas exchange. the method documented that the leak of albumin in ards may last for days but resolves with recovery. cancellous bone thermocoagulation ph. dumontier, r. benichoux and a. vidrequin institut de recheres chirurgicales -c.h.u. de brabois 54511 vandoeuvre les nancy cedex, france electrocoagulation can not stop bleeding from the cancellous part of a sectioned bone. therefore we tested the efficiency ofthermocoagulation by hot air. the hot air generator delivers a flow, of non illtercd air, at 25-30 1/min at a fixed temperature of 300°c measured at the exit of a 3 mm diameter pipe and 60 °c at the site of bleeding. the generator sustains usual steam sterilization. 14 dogs were operated on both patellae, femoral short segments and iliac crests giving 53 different site of cancellous bone. in vitro sterility studies: the conduit of hot air was applied at various distances, from 10 cm to 2 meters, above a petri plate containing culture material. thus the turbulence in the atmosphere around the zone of thermocoagulation has been bacteriologically controlled and a particle counter used. in vivo thermocoagulation: three sites of cancellous bone were used in 14 anesthetized dogs, using a sterile procedure: the iliac crest, a small segment of the femoral bone and the divided patella. 1. five iliac crests were divided and bleeding measured after thermocoagulation. 2. the 2 segmental femoral resections were thermocoagulated. 3. the patellae were vertically divided and each section submitted separately either to thermo or electrocoagulation. the pipe of thermo-181 coagulation was directed to the bleeding surface at a 2 cm distane, sweeping it during 5 to 6 seconds. the bleeding was compared by photography and the two fragments were approximated by a wire synthesis to provide a bone fusion. in few cases both sides were thermocoagulated. results: in vitro: no contamination was found in the atmosphere, up to a distance of 2 meters. there was a significant decrease of particles around the operating site (60 % less, at 80 cm and 30 % less, at 2 meters). in vivo: the bleeding was weighted around 50% less than with conventional coagulation. thermocoagulation did not delay or disturb the healing of the patella after wire synthesis. the in vitro nucleation time of cholesterol crystals from gallbladder bile of patients with gallstones is more rapid than that from normal persons, (holan rt [1979] gastroenterology 77: 611). this study determined whether this was due to a gallbladder or liver defect and wether the defect was the addition of a nucleating factor or the deletion of an antinucleating factor. hepatic and gallbladder bile were gathered at surgery in stone patients and gallbladder bile in patients with a normal biliary tract. after ultracentrifugation, the isotropic phase was observed daily by polarizing microscopy until cholesterol crystals appeared. in gallstone patients, the nucleation time of gallbladder bile was significantly more rapid, 2.5 days+0.9 sem, than that of hepatic bile 7.9+2.1 days, although hepatic bile was significantly more saturated with cholesterol [cholesterol saturation index (csi), 2.12_+0.36], than gallbladder bile, (csi, 1.31_+ 0.12). thus the characteristic short nucleation time of stone formers is due to an alteration in bile after it enters the gallbladder. to determine whether the gallbladder defect was due to addition of a nucleating factor or the deletion of an antinucleating factor, isotropic phases of normal gallbladder bile and that from stone formers were mixed and nucleation time determined. mixtures of up to 95 % normal bile had pathological nucleation times demonstrating that the defect is the addition of a nucleating factor by the gallbladder, and that this factor is potent. the rate of formation ofgaustone precursor crystals in bile, although faster in gallstone patients than in controls, is unrelated to the degree of cholesterol supersaturation [1] , implying that other factors are involved. two competing factors seem likely; (a) secondary seed crystals in bile may trigger and accelerate gallstone crystal formation from supersaturated solution; (b) "poisons" in bile may retard or inhibit crystal growth. because of the complexity of bile itself, experiments were performed in highly purified mixtures of bile salt, lecithin and cholesterol, in concentrations closely resembling those of gallbladder bile. (a) lipid solutions were seeded with calcium carbonate, hydroxy-apatite, calcium bilirubinate and biliary mucus, all of which are found in gallstones [2] . cholesterol crystal formation was significantly faster in_ the presence of all of the seed compounds tested (x= 221.7/~g ml-lh-1) than in unseeded controls (128.0/ag ml-~h-1) (/7<0.05). (b) substances with "crystal-poisoning" properties included heparin, chondroitin sulphate and bile salt. these and changes in ph altered the quantitiy (20-80 % decrease) and rate of calcium carbonate and calcium phosphate crystal formation. we suggest that gallstone precursor crystal formation may be affected by a subtle balance between crystal seeding and crystal growth-inhibition, both due to the presence of other compounds in bile. at the last tripartite meeting we reported experimental data on a new method to destroy concrements of the kidney in situ by shockwaves allowing for spontaneous excretion via the urinary tract [1] . the shockwaves are generated externally by underwater discharge of a condensor with sparking electrodes which are localized in a focus o fan elliptic cavity. for treatment the renal concrement must be positioned exactly in the second, virtual focus opposite to the elliptic cavity. since then, altogether 126 patients were subjected to this form of treatment in our institute by the colleagues of the department of urology at the university of munich. 90% of the patients got rid of their concrements within a few days, in 8.5 % small remnants remained in the renal pelvis, and in 1.5% (2 cases) additional surgery became necessary. meanwhile this technique is employed on a routine basis in the department of urology of the university of munich [2] . the experimental as well as clinical results were considered encouraging enough to extend the technique for the treatment of biliary concrements. for this purpose, human gallbladder concrements of different composition (bilirubin, cholesterol) were implanted into the gallbladder of dogs for exposure to shockwave treatment after wound healing. under in vitro-conditions the biliary concrements could be crushed into any size desired, irrespective of their composition, while only in 8 out of 10 experiments this was accomplished under in vivo-conditions. blockage of the biliaiy duct after shockwave exposure was never observed. concrements which were experimentally implanted into the bile duct could be visualized without difficulties by contrast medium. here, destruction by shockwaves was accomplished as well. currently experiments are conducted to dissolve remnants of biliary concrements after treatment by administration of desoxycholic acid. precise positioning of the gallbladder concrements in the second virtual focus is a problem which has not been satisfactorily solved so far, because the concrements cannot be visualized by conventional x-ray techniques. alternatively it is attempted to employ ultrasound, or visualization by retrograde injection of xray contrast medium through a catheter. in experimental animals, we leave a t-formed drain in the gallbladder for injection of contrast medium. in animal experiments conducted so far, histological or clinical evidence for tissue damage has not been obtained, as is the case with shockwave treatment of kidney stones. we are convinced that treatment of biliary concrements by shockwave exposure can be employed under clinical conditions in the near future. exploration of the common bile duct (cbd) for calculi, particularly in the presence of obstructive jaundice, is a procedure with considerable mortality and 183 morbidity. to avoid the problem of retained stones, choledochoduodenostomy and transduodenal sphincteroplasty have been recommended, but have their own complications. this morbidity might be reduced by removal of cbd calculi prior to surgery. endoscopic sphincterotomy (es) allows this. a review of 123 cases of es performed for calculi indicated that this was a safe (complications 8% no deaths) and reliable procedure (85 % success rate). a study was conducted of patients with known cbd stones who had either preliminary es followed by operation at a later date (group i) or operation alone (group ii). this study showed a lower morbidity in group i. a prospective randomised controlled study has begun on the basis of these findings and the data from both studies are shown in the table. these results suggest that pre-operative endoscopic sphincterotomy my reduce the morbidity of cbd stones. group ii n = 28 two controversies regarding the physiology of the biliary sphincter (bs) concern its functional independence from the duodenum [1] and those aspects of its acitivity which control bile flow [2] . the rabbit was chosen as the experimental animal as it has an easily identifiable sphincter. during anaesthesia induced by intravenous pentobarbital sodium, recordings of the electrical and mechanical activity of the bs and duodenum were made from (a) starved, (b) fed and (c) starved animals during administration of cholecystokinin, pentagastrin, secretin and glucagon. spike complexes (sc) were ordinarily associated with mechanical acitivity of the sphincter and duode-num. of 2.172 sphincter sc, recorded in 18 animals, 775 (36%) were not associated with duodenal acitivity, whereas 1.397 of 1400 (99.8 %) duodenal sc were accompanied by synchronous bs activity. this supports the hypothesis that the rabbit's bs can contract independently of the duodenum but that duodenal contraction is usually accompanied by simultaneous contraction of the bs. sphincter scs correspond to its phasic acitivity. food and cholecystokinin increased the number of sc without altering the baseline pressure of the perfused common bile duct. pentagastrin produced a transitory increase in sphincter activity whereas :secretin and glucagon were without effect. phasic activity of the spincter may influence bile flow through the choledochoduodenal junction. natural blood coagulation finally results in the formation of fibrin, which is one of the most important components of hemostasis in the human organism and thus provides the basis of all reparative procedures that are part of wound healing. it stands to reason to utilize the properties of fibrin for hemostasis during surgery and for joining severed tissue. first attempts of this kind were made at the beginning of this century. but only after greater insight had been gained into the coagulation proc-185 ess and the manufacturing techniques of blood derivatives had become more sophisticated, the essential breakthrough was made. a biological adhesive system has been developed, which consists of highly concentrated fibrinogen, thrombin and clotting factor xiii. this tissue sealant is completely resorbable and of high adhesive property. further advantages are elasticity of consistence and excellent tissue compatibility. after extensive animal experimentation, first clinical experience was made in 1973. in the meantime the fibrin-adhesive-system (fas) has been introduced into numerous surgical disciplines with excellent results. the outstanding properties are: atraumatic tissue synthesis; enhancement of fibroblast proliferation and promotion of rapid wound healing; obtaining of local hemostasis by sealing bleeding surfaces, which is of special importance in the treatment of patients suffering from hemophilia or during operations under heparinization. the authors experience in using the fas within the last 10 years is reported and a review over indications, techniques and advantages of this method is given. bile salts have been shown to enhance the stability and prolong the activity of intraluminal pancreatic enzymes and may therefore influence the effects of impaired exocrine secretion in patients with pancreatitis [1] . individual bile salts in the peak 15 min collection of duodenal fluid following cck/secretin administration have been quantitated by high performance liquid chromatography in 7 patients without pancreatic or hepatic impairment (group c), 6 patients with acute pancreatitis (group ap) and 8 patients with chronic pancreatitis (group cp) all with functioning gallbladders, and 4 patients with gallstone related acute pancreatitis (group gs). the peak total bile salt output in moles and the trihydroxy: dihydroxy (tri:di), primary:secondary (p:s) and glycine:taurine (g:t) bile salt ratios are shown below (mean___ sem). the duodenal aspirates contained detectable amounts of taurine and glycine conjugates of cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate but not lithocholate or free bile salts. the low total bile salt output in groups cp and gs were due to decreased levels of all the individual bile salts. although the bile salt pattern in groups c and ap were similar, the relative proportions of trihydfoxy and secondary bile salts were higher in groups cp and gs respectively. these results indicate that patients with chronic pancreatitis without obvious large bile duct obstruction have an impaired bile salt output into the duodenum and this may exacerbate the effects of pancreatic exocrine insufficiency. an elevated amylase creatinine clearance ratio (accr) was considered a specific test for the diagnosis of acute pancreatitis (ap). however, it has been found elevated in other diseases as well as after surgery. the aim of this study was to evaluate prospectively the accr levels in patients with ap and in several groups of surgical patients. we studied 197 subjects divided into 6 groups: group a: acute pancreatitis (n=67). group b: patients undergoing biliary tract surgery (n=38). group c: peptid ulcer patients undergoing gastric surgery (n=25). group e: patients undergoing cardiac surgery under extracorporeal circulation (n = 16). group f: control group of healthy subjects (n = 40). the accr was determinated using the levitt method. in the surgical groups the accr was measured before and after the operation. amylase was determinated by the phadebas amylase test. ap was diagnosed on the basis of both clinical and radiological findings and the presence of high serum amylase levels. this diagnosis was confirmed through laparotomy in 18 cases (26%). the accr was 1.96 + 0.8 % (mean___ sd) in group f, control group, and 7.5+5.7% in group a, acute pancreatitits p<0.01. accr values below 4 % were found in 21 cases of acute pancreatitis (31%). among those patients whose ap diagnosis was confirmed through surgery the accr was 10.6+7.9% (mean_+sd), higher than in the rest of the ap patients, 6.4±4.2% (p<0,05). in groups b,c,d and e (surgical groups) the accr before the operation was 1.83±0.8%, 2.34±0.9%, 2.28+ 1.6% and 1.54+ 1.06% (mean___ sd), respectively. after the operation it was: 3.85+2.2%, 3.18+2%, 2.83± 1.5% and 2.05+ 1.6%, respectively. on the average, we found an increase in the accr levels after the operation in the biliary tract group (p<0.01), but not in the other surgical groups. in 22 patients (24 %), the accr after operation was above the upper limit of normal. none of these patients had symptoms compatible with clinical pancreatitis. in conclusion: 1. the accr increase in acute pancreatitis (sensitivity: 69o/0) 2. the average accr increase after biliary tract surgery, but not after either gastric or thyroid surgery or after cardiac surgery under extracorporeal circulation. however, it is possible to find isolated cases with high accr after any type of surgery without any symptoms of pancreatitis, suggesting that an increase of accr may be an unspecific finding in postoperative patients which require further investigations. out of 217 patients with acute pancreatitis 25 developed a fulminant type. 18 were males and 7 females, mean age 45 years (range 23-83 years). all 25 patients were primarily treated by peritoneal lavage applied at laparotomy. indication for laparotomy was sudden deterioration with (20) or without (5) organ failure. a necrotic or hemorrhagic pancreas was found in every patient. the pancreas was exposed and 2 soft and large catheters were placed close to the pancreas. mean duration of lavage (ll/h) was 9 days. due to secondary deterioration a pancreas resection was performed 2-10 days later in nine patients. 7 patients with acute fulminant pancreatitis died, a mortality of 27.7%. all 192 patients with the mild type of acute pancreatitis survived, thus the overall mortality was 3.2%. none treated by peritoneal lavage only developed diabetes mellitus, whereas 4 out of 5 surviving patients with an additional pancreas resection had this complication. 19 patients with acute fulminant pancreatitis displayed 3 or more ranson criteria [1] and six died. however, no less than 32 of those with a mild type of acute pancreatitis fulfilled 3 or more of these criteria and they all survived. a laparotomy -not a laboratory test -is necessary to confirm the diagnosis of acute fulminant pancreatitis. the indication for laparotomy is mainly clinical and therefore such a patient should preferably be handled by surgeons or physicians experienced in this disease. after confirmation of a correct diagnosis peritoneal lavage is one of the methods by which the mortality of acute fulminant pancreatits -which by conservative means is 80-100% -can be reduced. coincidences ofhyperparathyroidism and pancreatitis have been given up by different author varying between 1% and 19%. frequently a causal relationship has been defended. recently, however, any causality has been queried [1] . we analysed our own series of patients with surgically and histopathologically confirmed primary hyperparathyroidism (phpt) (n=686) and found a coincidence with a coexisting or prior pancreatitis of 1.5 % (n = 10). from this a causal relationship cannot be concluded. however, 3 out of these 10 patients (i.e. 0.4%) had an acute onset or exacerbation of a pancreatitis immediately following the parathyroidectomy, which is strikingly more than one would expect after an operation without any anatomical relation to the pancreas (<0.01%) [2] . none of these 3 patients had another cause of the pancreatitis such as cholelithiasis or alcohol abuse. hence a causal relationship cannot be excluded. it is imaginable that excessive amounts of parathyroid hormone are released during the surgical manipulation of the pathologically altered parathyroids. the postoperative pancreatitis may be caused by the acute elevation of parathyroid hormone levels in the presence of hypercalcemia. in our series the parathyroidectomy was combined with a partial or total thyroidectomy in 27 patients. in another 7 patients a thyroid operation was performed prior to the parathyroidectomy. although calcitonin has been employed as a possible therapy in patients with acute pancreatitis (ap), the normal levels of this substance in ap are not well documented and have not been correlated with pth. furthermore no definitive role in human calcium homeostasis is accepted for calcitonin, whereas high pth levels have previously been correlated with hypocalcaemia [1] . in 21 patients with ap the mean serum calcitonin on admission was 121 ng/1, and the peak level mean 173 lag/1 (upper limit of normal 75 ng/1). calcitonin levels were higher in severe than mild ap, and 5 of 6 patients with levels >200 ng/1, were objectively graded as severe ap [1] . in the 3 hypocalcaemic patients, with corrected serum calcium <2.0 mmol/1 all recorded calcitonin levels >100 ng/1 and had elevated pth. nine of the 21 patients had significantly elevated pth ('>700 rig/l) and of these only 1 did not have an associated elevation in calcitonin. the high calcitonin levels in patients with ap suggest that supplementary calcitonin intended to inhibit pancreatic secretion is unnecessary. at present it is merely speculative to suggest a role for calcitonin in ap but intriguing to report a tendency to parallel the elevations of pth. nuclide labelled microspheres were used to measure pancreatic and other visceral blood flow in two groups of conscious dogs before and after intravenous alcohol infusion. blood alcohol concentrations at the time of blood flow measurements were similar to those encountered in intoxicated humans. thus 8 dogs (groups a) were given a somewhat low dose of alcohol to produce a mean blood alcohol level of 0.16 gm d1-1, while 6 dogs (group b), received substantially greater doses of alcohol, and reached a mean blood alcohol of 0.32 gm dl-1. wilcoxon matched pairs signed rank test confirmed a biphasic, concentration-related, response of pancreatic blood flow after alcohol infusion; no such response was found in blood flow to other viscera. moderate alcohol levels (group a) were associated with a decrease in pancreatic blood flow (p<0.03), while high blood alcohol concentrations resulted in increased pancreatic blood flow (p'<0.02). colonic blood flow increased in both groups a and b, but blood flow to the gallbladder, small intestine and the parotid gland increased in group b only. gastric, duodenal, renal, hepatic (arterial) and cerebral perfusion did not change. in addition, direct observations of the surface of the pancreas showed occasional haemorrhagic areas and mottling. these findlings however could not be confirmed by objective attempts to measure blood flow in such discrete areas in conclusion pancreatic blood flow shows a biphasic, concentration-related, response shortly after intoxication. this response appears to be peculiar to the pancreas and does not occur in other viscera. we recently showed that acute ethanol (e) and/or aspirin (a) ingestion increased the permeability of the pancreatic duct to large molecules, this suggested that pancreatic enzymes might leak from the duct into the parenchyma, causing pancreatic disease. this is a new concept in the study ot he pathophysiology of this organ (to be presented at aga, may, 1982, plenary session). in the present experiments wie studied the effects of chronic e/a ingestion on pancreatic function in dogs. methods: 6 dogs were fitted with duodenal and gastric cannulas. after recovery, baseline secretory sutdies were performed by cannulating the pancreatic duct and collecting pancreatic juice during secretin infusion (0.1 (submax) or 2.0 u/kg-hr. (maximal) iv). at least 2 studies were performed in each dog at each dose. after baseline studies, 3 dogs (group e) were given daily intragastric ethanol (2 gm/kg-day). 3 dogs (group a/e) were given e and a (100 mg/kgday). after 36-48 weeks, secretory studies were repeated. results: all dogs gained weight (x = 1.28 kg). the pancreases appeared normal by light microscopy. drug treatment increased volume and hco3 output by 47 and 67%, respectively, in group e, submax secretin, but decreased them by 29 and 34 % in group a/e animals. (p=0.0001 vs. predrug values and group e vs. a/e values). drug treatment decreased volume and hco3 output in both groups by 5-10°/c (p=0.0001) after maximal secretin stimulus. (manova test for all statistical analyses). conclusions: consumption of e alone increased volume and hco 3 output after submaximal and decreased them after a maximal secretin stimulus. this confirms the work of sarles. consumption of e and a reduced volume an i-ico3 output after secretin at both doses. thus chronic a ingestion further impaired pancreatic function in these animals. since only a small proportion of chronic alcoholics develop clinically significant pancreatic disease, an aggravating "cofactor" may be operating in this group. chronic asa ingestion, not uncommon in alcoholics, may represent such a cofactor. the development of diabetes mellitus in pancreatic cancer is well known but the standard oral glucose tolerance test is not recognised as a useful diagnostic indicator. glucose homeostasis, insulin and c-peptide secretion in response to intravenous glucagon were studied prospectively in patients with suspected pancreatic cancer and assessed as to their diagnostic value. 34 fasting patients were given glucagon, 1 m.g., i.v., and serial measurements of blood glucose, plasma insulin and c-peptide concentrations made for 60 min. subsequently it was shown that 12 patients had pancreatic cancer and the remainder constituted a control group. there was not significant difference in the rise in blood glucose between the groups after glucagon. the mean plasma insulin concentrations rose rapidly in both groups peaking between 2 and 5 rain but the values were sginificantly lower in the pancreatic cancer group (p<0.01 at 5 min: p<0.001 at 15 min: p<0.01 at 60 min). a similar pattern was observed with c-peptide. in patients with obstructive jaundice the plasma insulin response was a better discriminator of pancreatic cancer. we conclude that abnormal pancreatic beta cell funktion exists in patients with pancreatic carcinoma, detectable before any change in glucose homeostasis, particularly in patients with obstructive jaundice. the glucagon stimulation test may have a useful role in the diagnosis of pancreatic cancer. t-suppressor cells (t~) have previously been implicated as mediators of graft surival in baboons tolerant to their renal grafts [1] . in addition, it seems that t-helper cells (th) are also affected by totallymphoid irradiation in that they are unable to provide help in mitogen-induced t-cell responses or pokeweed mitogen induced immunoglobulin synthesis in bcells. in this study the evolution of t~ and th is followed in baboons undergoing graft rejection at different rates. peripheral blood was collected from the animals at weekly intervals after renal transplantation, defibrinated and the lymphoid cells isolated by flotation on ficol hypaque. the t-lymphocyte fraction was purified by filtration through a column packed with nylon wool. th, ts and total t-cells were enumerated using monoclonal antibodies okt4, 8 and 11 respectively (ortho). the th/ts rations reported were taken immediately before a rapid rise in serum creatinine occurred. in longlived baboons who maintained normal creatinine values, a mean of the ratios over the last month was used. b rats are produced by sublethal (750 rad) x-irradiation of thymectomized animals, reconstituted with bone marrow from syngeneic, thymectomized, thoracic duct drained donors. in these lew rats, (lew x bn)f1 cardiac allografts survice indefinitely (>100 days); unmodified lew rats acutely reject such allografts (8-+ 1 days). in this study, we have tried to restore the processes of acute rejection in b recipients. graft survival appeared independent of blocking factors or suppressor cells, as transfer of serum or lymphocytes from b recipients into syngeneic normal animals failed to increase survival of test allografts, placed subsequently. similarly, immunogenicity of long surviving grafts was unchanged; such grafts functioning >100 days and retransplanted into normal animals were rejected acutely. adoptive transfer of 108 unseparated spleen cells (sl) from nonimmune syngeneic animals produced slow rejection (28--+5 days) in b rats; 108 sensitized slwas somewhat more effective (22_+ 1 day). transfer of 2 x 107 syngeneic peritoneal exudate (pe) cells plus 108 sensitized sl caused acute rejection in 50 % orb recipients (11 _ 1 days), the remainder experiencing rejection at c 3 weeks. pe cells harvested from rats injected ip with thioglycollate 3 days previously, were primarily macrophages/adherent cells, as the cells used were that fraction sticking to plastic dishes and removed with lidocaine (purity >90 %). in vitro, b rat macrophages were abnormal, having only 50 % of capacity of normal macrophages to promote production of interleukin 2 (il2) when co-cultured with purified t lymphocytes. however, b recipients experienced acute graft rejection (10-+ 1 days) after transfer of 108 sensitized sl plus semipurified il2, thus bypassing the above defect. addition ofll2 to the t cell (purity >95°/0) equivalent of 108 sl (purified over degalan bead columns coated with rabbit antirat lgg, nonadherent fraction) failed to reestablish acute rejection (17-+7 days), while further addition of b lymphocytes (degalan bead adherent fraction, purity >90%) or macrophages was uninfluential (16___2 days and 16___3 days, respectively). transfer of il-2 alone never produced rejection. acute rejection can be re-established, however, by increasing the number oft lymphocytes (108, transferred concomitantly with il2). thus, the state of unresponsiveness in b rats can be reversed in vivo by adoptive transfer of particular cellular elements in the presence of growth factors; increased graft survival seems dependent ultimately upon il-2 production by sensitized t cells, presumably t helper cells. the relative inability of b rat macrophages to promote production of il-2 by t cells may be primarily responsible for the immunological deficit of the b rat. one of the most intriguing findings ofcyclosporin a (cya) immunosuppression is that in some species a short course of treatment will produce very prolonged allograft survival. we have tested the ability of cya to prolong the survival ofvascularized heart, kidney and pancreas allografts by direct comparison in a da (rt1 a) to lew (rt1 ~) rat allograft model. accessory abdominal heart and orthotopic left kidney transplantation were performed using standard microsurgical techniques. in renal transplantation the left kidney was removed at the time of transplantation, the remaining right kidney 5 days thereafter. streptozotocin-diabetic animals received ductligated pancreas whole organ grafts isolated on the portal vein and a segment of the aorta giving offthe coeliac axis and the superior mesenteric artery. rejection was taken as complete stop of palpable pulsations in heart transplantation, the day of death in renal transplantation and recurrance of hyperglycemia above 14 mmol/1 in pancreas transplantation, respectively. cyclosporin a 15 mg/kg body weight, dissolved in olive oil, was administered intramuscularly for 14 days starting with the day of transplantation. in all instances functional demonstration of rejection was confirmed by histological examination. cyclosporin a is effective to prolong the survival of vascularized heart, kidney and pancreas allografts. while cya is administered none of the grafts has been rejected. however, following withdrawal of the drug pancreas grafts are rejected within 14 days and heart grafts within 33 days. none of the kidney grafts has been rejected so far. the differential susceptibility of vascularized heart, kidney and pancreas allografts to cya immunosuppression may be caused by differences in immunogenicity due to organ specific alloantigens or a differential representation of spezialized antigen presenting cells. it may also reflect different patterns of rejection of the various organs. during cya administration all rejection processes are effectively suppressed. in the maintaince phase after withdrawal of cya such immune responses may prevail and ultimately lead to rejection of pancreas and to a lesser degree of heart allografts. the venous allograft still remains an attractive alternative for the reconstruction of small caliber vessels. however, when the venous graft is introduced to a non-histocompatible host, rejection and early occlusion is the rule. this study evaluates the use of cyclosporin a (cya) as a graft pretreatment, or systemic immunosuppressant for venous allografts. in addi-tion, cryopreservation techniques for pretreated venous allografts was investigated. 45 adult mongrel dogs, weighing between 12 and 24 kg, were used as recipients for donor jugular vein segments (5-6 cm) which had been excised and flushed with 100 cc of plasma protein fraction (ppf) at 4°c. these venous allografts were anastamosed end-to-end into a carotid artery of the recipients. the animals were divided into five groups as follows: group i (n=6) received untreated venous allografts without subsequent immunosuppression, group ii (n = 5) was the same as group i with minimal immunosuppression (azathioprine 2.5 mg/kg/day). in group iii (n= 10) the animals were transplanted with venous grafts stored in 25 cc of plasma protein fraction (ppf) containing cy a (50 mg/1) at 4 ° c for 24 hours, immunosuppression was as in group ii. in group 1v (n=9) the animals received allografts that had been cryopreserved in a 15% dmso solution at -196 ° c for 25-35 days and then the animals had azathioprine as in group ii. in group v (n= 15) venous allografts recipients were treated with systemic cya (20 mg/kg/day x 2 weeks, followed by 15 mg/kg/day x 2 weeks) as the only immunosuppression. the patency of the allografts was evaluated at 1, 2, 3, 4 and 8 weeks post transplantation. patency results at one month showed that azathioprine alone failed to improve the patency rate (gr. i and ii = 0% patency). cya graft pretreatment, however, significantly improved the one month patency (gr. iii = 57%). in addition, cryopreservation appeared to enhance the graft pretreatment effect of cya (gr. iv; one month patency = 85.7%) of the allografts. finally, systemic cya proved very effective in preventing rejection and occlusion (gr. v; one month patency = 100 %). grafts that remained patent for a initial critical period of 4-6 weeks, all showed long term patency. the effect of cya in preventing graft rejection was further documented by histiological studies of the allografts which showed a marked cellular infiltration and degenerative changes in all the grafts of the control group as compared to minimal or no cell infiltration in the patent grafts of the treatment groups. in summary, it appears that cya used as a graft pretreatment with minimal immunosuppression of the recipient, in conjunction with cryopreservation or given systemically as the sole immunosuppressant can significantly improve the survival of venous allografts. in our previous reports, it was shown that isolated hepatocytes transplanted into the splenic parenchyma of syngeneic rats, proliferated markedly and recomposed the hepatic tissue. this experimental system provided a new model to elucidate the mechanism of hepatic regeneration which could not be obtained in in vitro cell culture experiments. in the present paper, fetal hepatic tissue instead of isolated adult rat hepatocytes were transplanted into the rat spleen. we document briefly long-term morphological observations on the transplanted fetal hepatic tissue with special reference to proliferation of the hepatocytes and bile ducts. materials andmethods:wistar rats were mated in our laboratory for a fetal liver source. gestation day 0 was when a plug or sperm were observed in the vaginal smear. fetuses used were of 18 to 20 days gestation. about ten fetal livers which were obtained from one maternal rat, were minced with scissors. the liver fragments were washed three times with saline solution. transplantation was carried out by direct injection into the spleens of syngeneic adult rats using a 15 gauge needle. half of the liver fragments obtained from one maternal rat were innoculated into the spleen of one animal. a total of approximately 30 rats with transplanted liver fragments were killed 1, 3, 7, 14 and 30 days and then every two to three months until one year after transplantation. the spleens removed were stained by h.e., pas and silver nitrate for histological examination. results: fetal livers exhibited no lobular architecture or hepatic cord structure. the very sparse cytoplasm of the hepatocytes and many hemopoetic cells among the hepatocytes were characteristically found only in the fetuses. one week after transplantation, the survived hepatocytes revealed almost the same morphological features as in fetal liver except for the presence of several proliferated bile ducts around the hepatocytes. two weeks later, the hepatocytes formed apparent hepatic cord structures and the extoplasm of each hepatocyte increased abunduntly and became acidophilic as seen in normal neonatal hepatocytes. hemopoetic cells disappeared. four weeks later, hepatocytes began to proliferate sporadically among the markedly proliferated bile ducts, groups of survived hepatocytes with cord structure were very similar to a neonatal liver except for the lack of the glisson's area. two or three months later, proliferation of the hepatocytes became prominent. there seemed to be no interrelationship between proliferated hepatocytes and bile ducts. one year after transplantation, a white nodule was observed on the spleen macroscopically and it consisted of numerous bile ducts and hepatocytes with or without cord structure on histology. summary: 1. fetal hepatocytes transplanted into the spleen, differentiated to almost normal neonatal hepatocytes two weeks after transplantation. 2. hepatocytes began to proliferate about 4 weeks after transplantation. 3. three days after transplantation, proliferation of bile ducts was already observed independent of the transplanted hepatic tissue. 4. when comparing the difference in proliferation between fetal hepatic tissue and isolated hepatocyte transplantation, marked proliferation of the bile ducts in fetal hepatic tissue was observed and fetal hepatocytes proliferated more rapidly, while there were no proliferated bile ducts in isolated hepatocyte transplantation. pretransplant splenectomy (sx) has been of disputed benefit since its introduction two decades ago. 220 of 315 patients with first cadaver transplants treated at our institution between dec. 1968 and dec. 1981 have had pretransplant sx. at six monts, sx patients had 10% better kidney survival, but this benefit was lost shortly after 1 year and by 5 and 10 years was 10% and 15% worse in sx patients. patient survival for sx and no sx was identical for the first year but was 10% and 20% worse by 5 and 10 years respectively in sx patients. thus, the early improvement in kidney survival was more than offset by a late high mortality. a rational basis for selecting patients who might benefit most from pretansplant splenectomy is urgently needed. since july, 1978, 34 patients ages 16-45 have received first cadaver transplants after having been tested for reactivity to dncb. nine of 20 dncb negative patients had splenectomy as did 6 of 14 dncb positive patients. kidney survival at 1 year for dncb negative patients without sx was 79%; for dncb negative with sx, 29%; for dncb positive without sx, 21%; for dncb positive with sx, 62%. rejection was the sole cause for kidneyloss in dncb positive patients without sx. however, 5 of 9 dncb negative patients with splenectomy died, primarily of septic complications. since 1978 survival of sx patients has been 60% compared to 95% in non sx patients (p<0.04). sx appears to be beneficial in dncb positive patients but has an adverse effect in dncb negative patients because of an increased susceptibility to fatal infections. prior blood transfusion improves renal graft survival [1] . plasma from uraemic patients suppresses the in vitro responses of normal lymphocytes to antigen (plasma suppressive activity, psa) and this effect is mainly attributable to the plasma protein macroglobulin (a2m) [2] . the aims of the present study were: a) to identify changes in psa and a2m concentration in uraemic subjects following primary blood transfusion. b) to correlate the psa of transfused renal transplant recipients with subsequent graft survival. a) ten potential transplant recipients were studied before and after their first blood transfusion. following blood transfusions the psa increased significantly (p<0.01) reaching a maximum at two months. there was no significant change in the plasma a2m concentration over the same period. b) the plasma of 137 consecutive chronic renal failure patients was tested for psa prior to renal transplantation and before institution of immunosuppressive therapy. all but two patients had received previous blood transfusions. after transplantation patients were followed for a minimum of 3 months and a maximum of 42 months. 16 grafts failed for non-immunological reasons and were excluded from the study group. patients were divided into two groups according to the degree of suppressive activity of their plasma. a volume of5/t 1, producing a 50% inhibition of normal lymphocytes was used as a treshold to differentiate those with a high or low suppressive activity. graft survival in the first three months was significantly better, 92% (/7<0.05) for those recipients with a high psa as compared to 74% for those with a low psa. we conclude that blood transfusion causes a significant increase in psa although not a2m concentration and that patients with high psa have a better graft survival. the effect of in vitro steroid on antibody dependent cellular cytotoxicity (adcc) was studied in patients awaiting renal allotransplantation and the results were correlated with transplant outcome. 85 recipients of primary cadaveric allografts were classified as steroidsensitive or steroid-resistant from the degree of adcc suppression induced in vitro by methylprednisolone, 36 patients being steroid-sensitive and 49 steroid-resistant. following transplantation patients received azathioprine and prednisone, and rejection crises were treated with bolus doses of methyl-prednisolone. graft failure occured in 5 of the 36 steroid-sensitive patients, and in 34 of the 49 steroid-resistant patients. the observed one year graft survival rate was 57.6% for the whole group, 83.1% for the patients with steroid-sensitive adcc and 36.9 % for those with steroid-resistant adcc, the difference between the two groups being highly significant (xz=23.6). a high incidence of early graft failure was seen in steroid-resistant adcc patients, 49.7% of grafts being lost in the three months after transplantation, as compared with only 2 of 36 graft failures in the steroid-sensitive adcc group in the same period. analysis of hla-a, hla-b and hla-dr incompatibilities showed no significant difference between the groups, and since all patients had received deliberate pregraft blood transfusion, the difference in survival rates between the two groups appears to be independent of these two variables. these findings confirm our preliminary observation that pregraft assay of adcc response to in vitro steroids identifies those patients who are unlikely to respond to steroid therapy in the treatment of rejection, and in whom alternative forms of therapy may be appropriate. post-operative dxt, whilst not influencing survival, protected patients from loco-regional recurrence 07<0.001, hazard ratio (hr) = 3.1). interestingly it was found to be most effective against axiallary node recurrence (p<0.001, hr = 4.0), reasonably effective against chest wall recurrence (/7<0.001, hr=2.1) but conferred no protection against supraclavicular node recurrence (hr = 1.2) in spite of a supraclavicular field being routinely employed in the radiotherapy technique. with such large numbers involved, this trial has facilitated the study of the prognostic significance of sub-groups of patients with different patterns oflocoregional recurrence as first evidence of treatment failure (see table) . of those patients developing loco-regional recurrence who have since died (222 out of 356 in wp group; 73 out of 128 in dxt group) 63% in the wp group and 69% in the dxt group did so with evidence of persistent loco-reglonal disease. however, the incidence of uncontrolled local disease at death was higher in the wp group overall. stress as well as dietary fatty acids have been shown to prolong allograft survival in rats [1] . poly unsaturated fatty acids (linoleic acid, arachnoidic acid) have been reported to depress immune response [2] . depressed immune response was suggested to correlate with a higher incidence of spontaneous tumor [3] as well as with an increased growth rate of inoculated tumors [4] . the objective of this study was to elucidate the effect of two environmental factors i.e. chronic stress (change in light/dark pattern) and diets low and high in linoleic acid on immune response and growth of transplantable tumors in bn rats. immune response: four experimental groups (n >10) were used in immune response studies. group i: high linoleic acid dietl; group i1: low linoleic diet 2, group iii: l/d shift weekly, normal diet and group iv: controls on normal diet, normal lighting. seven weeks after the start of the experiment the immune response was measured. the results showed that corticosterone levels were slightly increased in all experimental groups, although only the high linoleic group showed statistic significant difference with the control group. cellular immune response (con a stimulation and popliteal node assay) was decreased in all experimental groups compaired to controls. transplantable tumors: 1 x 106 leukemia cells were injected i.v. and pieces of 1 mm 3 of an spontaneous adrenal cortical carcinoma, a urethral squamous cell carcinoma and a round cell cervix sarcoma were implanted subcutaneously. all tumors were inoculated in groups of 10 animals each. spleen weight as a measure of leukemia growth was high in the control group and low in the experimental group. the same pattern was seen in the growth of the subcutaneously implanted adrenal cortical carcinomas. both the urethral squamous cell carcinoma and the round cell cervix sarcoma, being non-immunogenic, did not show any difference in growth. so far, it can be concluded, that the immunosuppression as induced by mild chronic stress or dietary fatty acids does not lead to enhanced tumor growth. in contrary, the results of both leukemia and adrenal cortical carcinoma show a possible reserve effect. little is known of the derivation or content of human breast cysts. recent reports have shown wide variations in the content of steroid hormones, particularly dehydroepiandrosterone sulphate (dhas) [1, 2] . no explanation for this is apparent. to confirm the large variation in dhas concentrations and to further define the contents of cyst fluids, 100 cysts from 85 patients have been analysed for dhas, sodium and potassium. dhas concentrations ranged from 1.5-1155 pmol/1. both sodium and potassium content also varied widely (sodium 30-200 pmol/1 and potassium 3-158 ~umol/1). there was a significant direct correlation between the content of potassium and dhas in cyst fluid (p<0.001) and a significant negative correlation with sodium content (/7<0.001). three separate subpopulations of cysts could be identified according to the sodium and potassium content and these were, predominantly potassium cysts (47), predominantly sodium cysts (44) and mixed cationic cysts (9). the median dhas concentration of the potassium cysts was 215 pmol/1 similar to the levels found in human breast secretions [3]. in contrast the median concentration of dhas in the predominantly sodium cysts was 14 pmol/1 and significantly different (p<0.0005), with many of these cysts having dhas concentrations in the same range as those found in plasma. the remaining mixed cysts had a median dhas concentration intermediate between the two main groups. it may be that the variation in cationic content and dhas concentration in these two major subpopula-tions of human breast cysts represents either, derivation from two different sources, namely breast secretions and plasma or marked differences in the secretory activity of the epithelium lining these two groups of cysts. there is no uniform agreement on the correct management of patients with invasive lobular carcinoma (ilc). it is widely considered that in ilc there is an increased risk of developing a contra-lateral carcinoma and the major controversy surrounds the management of the second breast. the survival of patients with ilc was significantly better than that of idc fp<:0.025). six patients had bilateral carcinomata at diagnosis and a further 14 developed a contra-lateral carcinoma during the period of follow-up (10 to 21 years). survival data showed poor survival for patients with simultaneous bilateral disease, but no difference in survival for patients with metachronous bilateral or unilateral disease. this suggests that the later development of a second carcinoma does not necessarily reduce the probability of survival for patients with ilc. the major factor predicting patients at risk of developing a contralateral carcinoma was histologi-195 cal type. of 22 patients with a particular histological pattern of ilc [4] with a classical pattern of spread but showing nuclear pleomorphism and cellular cohesion, 10 developed a contralateral carcinome, compared with a further 10 in the remaining 81 patients (p<0.001). if bilateral mastectomy is justified it ought to be restricted to patients with this histological type of ilc. both the anti-oestrogen tamoxifen and cyclical combined chemotherapy will provide significant palliation in advanced breast cancer. the optimal use of these agents requires further evaluation and thus this trial was designed to compare a combination ofcytotoxic therapy and tamoxifen, against cytotoxics alone in patients with advanced breast cancer. post-menopausal patients presenting with metastatic breast cancer, locally advanced cancer extending beyond the breast and regional nodes, or with tumor recurrence following primary local treatment were allocated to the 3 treatment arms via sequential manner. doxorubicin, cyclophosphamide, 5-fluouracil, and vincristine were given intravenously once every 3 weeks. tamoxifen was prescribed in a dose of 20 mg. b.d. on failure or relapse from one of the single modality arms, a crossover of those arms occurred. the combination consisted of both the above therapies. assessment of therapies was made in terms of objective response (uicc criteria), duration of response, and survival. we have previously reported that the combination results in a significantly greater response rate [1] . as a result of stenosis reducing flow or by platelet embolisation [1] . as neither aniography nor ultrasound can identify thrombotic activity we have evaluated gamma camera neck imaging using n 1indium platelets. labelled platelets on endarterectomy specimens were also measured and the activities found were then examined in a theoretical model. twentyfive patients with tia received rain platelets and sequential gamma images were interpreted by two observers, carotid endarterectomy in 11 patients allowed measurement of specimen radioactivities. angiography and doppler spectral analysis [2] were also performed. all endarterectomy specimens contained labelled platelet deposits with the most active equivalent to platelets from 1.8 ml of blood. this activity level was at the threshold of resolution in the theoretical model. both observers agreed that 22 of the 50 carotid bifurcations showed platelet accumulation on imaging. of the 12 atheromatous ulcers demonstrated by angiography 11 were visualised, but only 5 of 10 stenoses greater than 80 per cent were detectable~ since ultrasound identified all stenoses only one angiographically diseased carotid was not detected by combining doppler and platelet imaging. diseased carotids accumulate rain platelets with the more thrombogenic ulcerated plaques identified more frequently than stenoses. long term follow-up is required to establish the clinical relevance of platelet deposition. major problem in vascular endoscopy is the existence of blood which prevents clear visualization. we devised a new technique using a combination of balloon catheter and slender fiberoptic endoscope, by which clear visualization was obtained experimentally and clinically. three to four pairs of orifices of intercostal arteries were also visualized in one visual field. in some dogs, acute aortic dissection was experimentally created by means of blanton's method. the entry, which was located at the descending aorta just distal to the left subclavian artery, was clearly identified. complete occlusion of blood flow and clear visualization could be obtained when balloon pressure exceeded systemic blood pressure. clinical study: in six patients requiring major vascular reconstruction of the aorta (abdominal aneurysm 4, leriche's syndrome 1, dissecting aneurysm 1), vascular endoscopy was performed intraoperatively. in five patients, balloon catheter was introduced through the one of the limbs of y graft after proximal anastomosis. in each case, orifices of the major abdominal aortic branches were clearly observed. irregular orifices and atheromatous plaque of the aortic intima which were not expected from aortogram, were also identified in all patients. intimal tears by vascular claps were more extensive than expected and anastomotic suture lines were able to be checked from inside. in a case of dissecting aneurysm, balloon catheter was advanced through the 11 mm graft which was sutured to the common femoral artery with finding the entry just above the left renal artery. using fiberoptic endoscope and balloon catheter was useful to observe orifices of the major aortic branches, unexpected intimal tears by vascular clamps and atheromatus plaques. it was particularly usbful to check the anastomotic suture line from inside of the aorta and to identify the exact location of the entry in dissecting aneurysm. vascular endoscopy could be one of the invaluable methods to examine, diagnose and treat the patients requiring aortic, caval and other major vascular surgery. (3) produced endothelial injury and a local increase in shear stress in 14 cynomolgus monkeys by suture plicating and constricting the aorta and then feeding an atherogenic diet for 6 months. our findings reveal that carotid plaques localize on the outer wall of the internal carotid (plaque thickness 0.7--+0.1 mm) which is an area of low flow velocity ( -4___ 1 cm/s at re 800) and shear stress (0-+2 dynes/cm 2) and not at the flow divider (thickness 0.1___0.1 mm, p<0.01) which is an area of high flow velocity (81---3 cm/s) and shear (161-+48 dynes/cm2). distal to the carotid bulb, velocity and shear increased on the outer wall and little or no plaque was observed. in experimental coarctations, no endothelial damage was observed (sem and tem) within the high-shear coarct channel and the channel was noted to be free ofatherosclerotic plaque despite the development of extensive diet-induced lesions proximally and distally. thus, high flow velocity and shear stress do not appear to produce endothelial damage in vivo. in addition, plaques were minimal in high shear areas in the human carotid bifurcation and high shear appears to have an inhibitory effect on experimental plaque formation. these data contradict previous investigations implicating high shear stress in plaque pathogenesis. in contrast, host aortic endothelium (ae) fails to cover large vp by pannus ingrowth even over much longer times. to see if iaes succeeds because of inherent differences in growth potential between ae and ve, we used ae to seed 8 cm x 6 mm diameter dacron velour infrarenal vp in dogs. an average of 4 x 105 cells obtained by trypsin/collagenase digestion of the bypassed aortic segment was used to seed each vp by a 4 step preclotting method. the identity of ae was confirmed by stains for factor viii antigen. viability of seeded ae was verified by growth of subaliquots in tissue culture. six weeks after surgery central segments of aeseeded (n = 9) and control unseeded (n = 9) vp were compared by light and scanning electron microscopy using an endothelial coverage score range of -1 (for fibrin/platelet thrombi) to +1 (for confluent endothelial coverage). ae-seeded vp had a score of+0.89 ___ 0.33 (mean___ sd) versus. -0.67___ 0.70 for controls (p<0.001). in addition to endothelial coverage, the subluminal smooth muscle and intramural vasa vasorum previously reported in ve-seeded vp were also seen in ae-seeded vp. since ae and ve seeding give identical results, the success of iaes with ve cannot be due to inherent biological differences in mitotic potential between ae and ve. iaes must instead achieve additional endothelial growth either through a) the action of the proteolytic enzymes used for cell harvest or b) mitogenic stimuli to nonconfluent cells at the edges of seeded cell clusters on the vp. further improvement of the efficiency of iaes to allow use of less harvested vein per cm 2 of vp should come from enhancing one or both of these effects. pyrolytic carbon is a crystalline form of carbon that has been extensively used in the construction of cardiac and bone prostheses. since it has also been suggested that pyrolytic carbon will prevent thrombosis from occuring in vascular prostheses, the aim of the present study performed in dogs was to test the immediate blood compatibility of this material and to evaluate its biocompatibility when inserted as vascular substitute. after pryolysis of a gazeous hydrocarbon, the carbone crystalite was deposited on a knitted textile surface or tube. its surface examined by scanning electron microscopy (sem) was rough and porous to a depth of 40p. this material was tested 1 °) for immediate hemocompatibility as inserts within the vascular lumen (aorta and inferior vena cava). the specimens were examined sequentially by sem and histology at 10, 20, 30, 180 s and 10 min after reestablishment of the blood flow, 2 ° ) for long term biocompatibility as vascular cylinders (7 mm id) inserted either in the aorta or inferior vena cava or as intraatrial (left or right) implants. patency of vascular cylinders was tested during 2 postoperative month by doppler ultrasound investigations, specimens were examined by histology, electron microscopy (scanning transmission) at 15, 30 and 60 days following implantation. satellite lymph nodes were examined by histology. already 10 s after establishement of the blood flow, platelet adhesion and limited fibrin mesh with few erythrocytes developed on the material. platelet aggregates of limited extent were only observed on intravenous implants. plasmatic protein deposition, an early event on polymeric vascular material was not observed. after 30 s a fibrino-erythrocytic membrane recovers completely the material. except in the case of intravenous insert, no thrombosis developed at the contact of intraarterial or intracardiac implant. after 15 days it was completely recovered by a 5-7 fibrocellular layer consisting of large myofibroblasts with microfilaments, newly synthetized collagen and elastin. the blood interface was of fibrous nature. at one month by sem, endotheliallike cells developed in a mosaic-like pattern, characterized by transmission e.m., by microvillous projections, numerous pinocytic vesicles and intercellular tight junctions. this endothelial-like cell lining was complete 2 months after implantation. their immunocytochemical properties are now under investigation using specific anti-dog factor viii-rag sera. although preliminary, the present results suggest that among the numerous vascular biomaterials tested, pyrolytic carbon may represent a unique feature of rapid cell development and differentiation of endothelial lining at the blood material interface. department of connective tissue biology, institute of anatomy, university of aarhus, aarhus, denmark in the surgical clinic a significant number of patients report that their incision wound has burst, even though the scar appears to be intact. by mechanical testing of strips from skin wounds we have noticed a breaking pattern, in which the deepest layer of the wound ruptures earlier than the superficial part. therefore, we have investigated the strength and extensibility of rat skin wounds at different levels (superficial-deep) of the epidermis (0.03 mm), dermis (1.8-2.2 mm) and m. panniculus carneous (0.6-1.5 mm), average thickness is indicated. 10, 20 and 60 day old standardized skin wounds from the dorsal region of rats have been used. strips were punched out at right angle to the wound line and mounted in a materials testing machine. the strips were stretched until rupture and load-strain curves registered continuously. simultaneously, the strips were transluminated and the breaking pattern was studied by taking photographs of the wound specimens during the mechanical testing (30-35 photographs of each specimen). the photographs were marked on the load-strain curve by means of a connection between camera and x-y-recorder. from the load-strain curves the maximum load and the failure energy were calculated. the breaking patterns of 10, 20 and 60 day old wounds were found to be similar. the deep part of the wounds ruptured first. the force required to break the deep part was less than that required to break the superficial part of the wounds. the musculus panniculus carneous was very extensible and did not break. however, it possessed only minimal strength. quantitative measurements of the strength of the combined superficial-deep layers were performed on 2 mm wound strips. specimens contained the superficial 0.5, 1.0 and 1.5 mm of the wound area and were produced by cutting off the deep layer parallel to the skin surface. specimens containing the total wound area down to the musculus panniculus carneous were produced by cutting off the muscular tissue. these specimens were mechanically tested as described above. the present studies demonstrate the mechanical inhomogeneity of incision wounds. a new method for testing the mechanical properties of the tissue of incision wounds at various levels (superficial-deep) is presented. the superficial layer of an incision wound contributes a major part of the strength of the wound and is more extensible than the deep layers. these results may explain the clinical observations. the effect on wound healing of different kinds of vitamins is worth investigating, since the efficiency of vitamin c has been dearly demonstrated. the possible action of vitamin bs-whose trophic effect on skin is well known -has been experimentally studied on skin and aponeurosis healing after a standard laparotomy. materials and methods: experiments were carried out on 42 five months old rabbits which were randomly divided into three groups: in group i, 18 animals served as controls (3 animals in sequence of 5 days from the 5th to the 30th post-operative day), group ii, 12 animals injected with vit b5 (25 mg/kg of body weight/24 h) and group iii, 12 animals injected with a placebo (2 animals in sequence of 5 days for each group). in each case four samples were tested of skin and aponeurosis for determinating tensile strength, directly recorded with an original technic [1] : this new apparatus allowed us to obtain simultaneously two dynamic parameters, the healing tensile strength and stretching of the scar. results: 1. no significant difference was found between controls (group i) and the placebo group (group iii) both for resistance of skin and the aponeurosis. 2. as far as vitamin b5 treated animals were concerned (group ii) there was no significant difference regarding skin resistance when compared with the other two groups. 3. inversely aponeurosis resistance become significantly greater when measured on the 20th (p<0.050), 25th (/r<0.025) and 30th (p<0.050) post-operative day. in 17 mongrel dogs (7x 15 cm cranial based rectus abdominis) mc and corresponding rp flaps were raised. in group i (9 dogs) skin bf was determined from the clearance curve for ~33xenon injected intradermally and measured with a computer-linked gamma camera. in group ii (8 dogs) subcutaneous pro2 was determined by a recently developed method using a silastic tonometer, subcutaneously implanted. the pro 2 inside the tonometer was measured in infused saline, by a platinum oxygen needle electrode and a silver/silver chloride reference electrode. b f and pto2 were measured before and after the flaps were raised and on postoperative days (pod) 1, 3, 6 and 15. pto2 were taken at 6 various inspiratoric oxygen levels (f~o2) ranging from 21% (air) to 100 % oxygen. intact areas lateral to the flaps and in flap regions prior to surgery served as controls. immediately after surgery bf in the mc increased while in the rp flaps was 24 %, 23 % and 31% of the flow in the mc flaps, in lateral intact area and in the preoperative areas (p<0.001). during pod 1-2 bf in the rp flaps increased to the preoperative level, but not to the increased levels found in the mc flaps and the lateral intact areas. by pod 6 there were no differences in bf between the two types of flaps and the lateral areas, but all were higher than corresponding preoperative values (/'<0.005). tissue oxygen tension showed a dramatic fall pod 1, 3 and 6 in the rp flaps for all fio2, and for all days the values were lower than the preoperative level (p<0.001). one rp developed pod 3 distal necrosis and the pro 2 was then 0 even with a fio2 of 100%. the mc flap showed an increased pro2 on the operative day but at pod 3 the values were slightly lower than the preoperative level, but pod 1, 3 and 6 the values for all fio2 were higher than for rp flaps (p<0,005). at pod 15 the pro2 reached preoperative level for rp as well as mc flaps. lateral intact areas showed comparable changes to that observed in the mc flaps. it is concluded that the mc flap demonstrates superior bf as well as pro2 when compared to the rp flap. early postoperative pro 2 in the distal part of the rp flaps is critically low despite of increasing f~o2 to 100 % and increasing bf. differences in bf and pto2 may be the biologic factors responsible for the superior healing characteristics of the mc flap. (1) atp~adp + pi (inorganic phosphate) (2) pcr + adp~atp the net result ofreaotjoxas 1 and 2 is a fall in pcr and a rise in pi while atp ~'emains relatively constant. all of the phosphorus metabolites are easily measured in gastrocnemiaas muscle using 31pnmr spectroscopy. normal volunteers and patients with angiographically documented arterial occlusions were studied in a 101/4" bore oxford research systems tmr-32 spectrometer at rest and after exercising each limb separately. normal resting values ofpcr/p iwere >10 and the nmr index = p/(p~+pcr) was 0.07_+0.03 (s.d.). limbs with femoral arterial occlusions whose ankle systolic pressure index was <0.5 had nmr index which was significantly elevated above norreals (0.18 + 0.09p<0.01) indicating a failure of metabolic compensation for reduced bloodflow and oxygen delivery, although atp concentration was norreal. exercise produced a five-fold rise in nmr index in both normal and diseased legs. spectra were taken over one minute intervals during the recovery period and in normal limbs returned to resting values within 2 rain. the recovery period was considerably slower in the diseased limbs indicating abnormal mitochondrial oxygen delivery and impaired mitochondrial formation of atp. these data demonstrate the feasibility of using 31pnmr to non-invasively probe the biochemical abnormalities of energy metabolism in patients with peripheral vascular disease. the incidence of urinary calculous disease (ucd) in the south african black population is very low in comparison with the white population group. no biochemical differences in serum nor urine account for this discrepancy and no other measurable parameters have demonstrated any difference between the two groups. urinary particulate activity measurements have demonstrated differences between normal persons and those with ucd who are otherwise biochemically similar, and it would therefore seem rational to expect such measurements to demonstrate differences between the two population groups. urinary particulate activity was measured in the urin of 100 normal whites and 100 normal blacks, the two groups being matched for age, height and weight, and monitored under normal dietary hydrational and environmental conditions. the three parameters of particulate nucleation, growth and aggregation were measured and the two groups compared. particulate nucleation demonstrated the most significant contrast between the two groups with the production of new particles through nucleation being far greater in the white group than that which occurred in the black group (p<0.001). particulate growth occurred at similar rates in the two groups although at slightly higher rates in the white group. particulate aggregation occurred at a greater rate in the white group but the difference between the two groups was not statistically significant. the differences between the two groups are shown to occur as a consequence of differing rates of particulate nucleation although the rates of particulate growth and aggregation are parallel. whilst the factors responsible for the low nucleation rate in black person remain unknown their effect can now be measured quantitatively through the parameters of urinary particulate activity. blood levels of ketone bodies appear to determine skeletal muscle amino acid release; high levels conserve protein and attenuate gluconeogenesis. starvation indt/ced ketosis is suppressed by infection [1] . to determine if the relative hypoketonaemia following sepsi s in turn contributes to increased glucogenesis, arterial substrates and glucose production (constant infusion 6-3h(n)-glucose) were measured before and after infusion of na-dl-./3-hydroxybutyrate (/3oh) to raise levels three-to fourfold in fed (n= 11), in fasted (n = 8) and in fasted-infected (n= 9) animals. in fasted-infected animals before infusion ketosis did not occur (/3oh 0.81±0.1 mm/1 fasted; 0.45 ±0.55 fasted-infected) and basal glucose turnover was increased (5.05±0.18 /tm/kg/min fasted; 9.5±0.83 fastedinfected). with infusion of glucose and alanine concentrations decreased as expected in fed and fasted animals but not in fasted-infected (glucose 2.33±0.17 ram/1 befor; 2.44+0.11 mm/1 after). glucose production also fell significantly in the fed (10.11±1.33 /~m/kg/min before; 8.44±1.05 after) and fasted (5.05±0.28 v. 4.11±0.33) groups but was unaffected by infusion in the fasted-infected group (9.5+-1.83 v. 9.11+1.44). the accelerated rate of gluconeogenesis in infection is thus not a consequence of hypoketonaemia. the usual reciprocal relationship between glucose and ketone utilisation during feeding and fasting has not been demonstrated~in sepsis. preliminary experiments in a hindlimb model support the hypothesis 201 that during infection amino acid release from muscle is not affected by ketone levels. we have developed a technique for measuring the total body carbon of the living subject which is suitable for measuring the critically-ill as well as the ambulatory patient. by combining this measurement with that of total body nitrogen and calcium [1] an estimate of total body fat is derived. measurement at the beginning and end of a given period enables the changes in total body protein and fat to be obtained, as well as the patient's energy expenditure if energy intake is also known. the method is a radiation technique. the supine patient is irradiated laterally with a horizontal beam of fast neutrons and the resulting gamma rays from the body are detected by a radiation detector placed unterneath the subject. the nuclear reaction employed is the inelastic scattering of fast neutrons by the carbon nuclei of the body with the emitted gamma rays having an energy of 4.43mevi in the initial application, measures of total body fat obtained using the technique were compared with those derived from skinfold thicknesses in six volunteers: there was no significant differences between the two measurements, (see table) . the method is being employed in studying the changes in total body protein and fat, and the energy requirements of surgical patients receiving nutrition. in order to investigate the mechanism of this effect, normal monocytes were incubated at 37°c for 30 min (with intralipid 40/a/ml) and their function assessed by three different techniques (chemotaxis, phagocytosis and chemiluminescence). all three methods showed impairment of function following exposure to intralipid. in order to try and prevent this potentially damaging effect, heparin was added to the various in vitro tests and found to cause marked impairment of phagocytosis. (p<0.01) to assess its effect in vivo, 11 volunteers were given 5,000 units of subcutaneous heparin 2 h prior to intravenous intralipid (as above). although the use of heparin did not affect either immunological function, it completely prevented the fall of monocyte chemotaxis following intralipid alone. these findings suggest that monocyte function may be impaired by the presence ofintracellular lipid particles. the use of s.c. heparin may help to alleviate this problem and could, therefore, be beneficial to ill and often septic patients requiring intravenous nutrition. to investigate the effect of elevated glucocorticoids of stress and trauma on peripheral glutamine metabolism, 0.44 mg/kg bw dexamethasone was injected daily intramuscularely in adult mongroel dogs over a period of 2 weeks. at least 3 weeks prior to the experiments catheters were placed into the animal's abdominal aorta (2) and caval vein (1) in order to measure a-v differences and hindquarter blood flow. during dexamethasone treatment nitrogen balances were negative, -7.1-4-1 g n per day, whereas slightly positive n-balances were observed during the control period (3.1 +__ 1.7 g n/day). muscle glutarnine concentrations declined constantly from 22.1 +2.3 mmol/1 intracellular water to 10.0-4-1.2 by 56% within two weeks. whole blood arterial and venous plasma concentrations remained constant. to test the hypothesis of increased peripheral glutamine utilisation or decreased glutamine formation, the activities of glutaminase and glutamine synthetase were measured in a muscle homogenate obtained before and 8 days after dexamethasone treatment. both enzyme activities were found to be unchanged. hindquarter glutamine efflux increased from 20.3+22.6 pmol/min in the control state to 80.0+24.1 during dexamethasone treatment indicating a 4 fold muscle glutamine output. this increased glutamine output was enirely due to increased a-v differences and despite decreased hindquaarter blood flow during dexamethasone. it is concluded that dexamethasone reproduces the metabolic response of trauma and sepsis in terms of negative nitrogen balance and muscle glutamine depletion. muscle glutamine is shifted from peripheral tissues to visceral organs with muscle compensating for visceral demands rather than skeletal muscle being the primary target of corticoid action. it has been suggested that there is abnormal glucose utilisation in malnourished patients and that this may explain the adverse clinical sequelae of high rates of glucose infusion during intravenous feeding. we have investigated the hypothesis that there is a depression of the key enzymes of glucose oxidation in the muscle of malnourished patients which is due to an alteration of muscle fibre type proportions. 14 malnourished patients (p) (9m,5f 56+12 yrs) our results demonstrate that there is a positive correlation between preoperative cp and stage of cancer 0' =34.4+-3.88x; r=0.62;/9<0.001). nevertheless before surgery there is no difference between cp values in the two groups considered (g.c.= 46.2___9.7 mg %;p.u. =44.4----9.0 mg%), but after surgical trauma cp presents a positive response in patients with p.u. (mean increase +14.9 %), whereas it acts as a negative ap protein in g.c. patients (mean decrease 5-60/0) (p<0.02). moreover malnourished g.c. patients present a reduction of cp values ( -10 %) which is greater than g.c. patients with albumin >3.5 g% ( -1.5%); this difference is not statistically significant. cancer patients undergoing palliative (n=10) or radical surgical procedure (n=31) show parallel decrease of preoperative cp ( -6%), the first group presenting higher preoperative values in relation to the tumor diffusion. in conclusion our results demonstrate that cp is not only a positive ap protein, but in some circumstances it may act as a negative pa protein depending on the underlying disease and the preoperative nutritional status. in this study the free aa concentration in liver tissue of 4 non septic patients (cholecystectomy) were compared with those of 7 septic patients (abdominal sepsis). the liver specimens were taken intraoperatively..the nature and possible risk involved in this study were explained to the patients and their consent obtained. the data presented in this abstract are part of a metabolic screening program of septic patients including the determination of aa (plasma, muscle), hormones (insulin, glucagon, cortisol), nutritional parameters (prealbumin, retinol-binding protein, transferrin), and of energy metabolism (atp, adp, glucose, free fatty acids). for the determination of the free aa the intra-and extracellular water content (chlorid method) and of fat content of the liver specimen were analysed. a membrane potential o f -45mv was assumed. the aa analysis were performed with an automatic aa analyser (kontron, svitzerland) by means of an ionexchange resin (durrum dc-4) and a lithium buffer system (durrum-pico buffers). conclusions: 1. this study reveals decreased concentrations of nearly all aa in liver tissue of septic patients (exception: phenylalanine, tyrosine, cystathionine). 2. the significantly decreased concentrations of the gluconeogenetic aa (thr, ser, ala) indicate that the gluconeogenetic capacity of the liver is not exhausted through an increased uptake of those aa as shown earlier by wilmore et al. [4] 3. an increased administration of gluconeogenetic and basic aa (lys, his) may normalise the aa pattern in the liver of septic patients. the liver is being increasingly recognized as a critical organ in postoperative multiple organ failure. the principle factors precipitating postoperative multiple organ failure were sepsis, hypotension and injury to the liver. previous studies from our laboratory have shown that hepatic failure, which has a high mortality rate, is linked to the marked decrease in energy charge. in order to evaluate the possible presence of metabolic blocks, the changes in the ratio of acetoacetate to fl-hydroxybutyrate (ketone body ratio), which reflects the hepatic mitochondrial redox potential, were analyzed in relation to energy charge in hepatectomized, jaundiced, hemorrhagic-shokked and septic animals, as well as patients with postoperative multiple organ failure. experimental: 1. in hepatectomized rabbits, mitochondrial phosphorylative activity increased to 170 % of the control and the energy charge level decreased from the normal level of 0.87 to 0.76 at 24 h after hepatectomy (/7<0.001). afterward, these values returned to preoperative levels within a week. the ketone body ratio in arterial blood was positively correlated with hepatic energy charge (r=0.696, p~0.01). 2. in jaundiced rabbits, the hepatic energy charge decreased rapidly after the bile duct ligation along with the decrease of mitochondrial pbospborylative activity. the hepatic energy charge fell from 0.87 to 0.73 at 48 h postoperatively with a maximum incidence of mortality. moreover, changes in the blood ketone body ratio were positively correlated with the hepatic energy charge (r= 0.844,/~0.01). the decrease in the blood ketone body ratio was attributed to the restricted mitochondrial reoxidation of nadh due to an inhibition of oxidative phosphorylation. 3. in hemorrhagic-shocked rabbit with a mean arterial blood pressure of 40mmhg, the changes in the blood ketone body ratio were correlated with hepatic energy charge (r=0.772, p<0.01). 4. in septic pigs subjected to the ligation and perforation of the cecum, the hepatic energy charge level decreased gradually from 0.86 to 0.80 and the mitochondrial phosphorylative activity was enhanced to 150% of controls in the hyperdynamic state. in the hypodynamic state, the hepatic energy charge level fell drastically 0.69 concomitant with the decrease in mitochondrial phosphorylative activity and blood ketone body ratio. from these results, the blood ketone body ratio may be regarded as a reliable indicator for assessing the degree of decreased energy charge. clinical: changes in the blood ketone body ratio were measured in 55 patients who underwent major surgery such as hepatectomy. these patients were classified into 4 groups according to the postoperative changes in blood ketone body ratio: group a without decrease to below 0.7, group b with transient decrease to 0.4, group c with progressive decrease to below 0.4 and group d with terminal decrease to below 0.25. all 35 group a and b patients tolerated the operation well. by contrast, the 20 group c patients showed multiple organ failure with 85 % mortality rate, which involved pulmonary failure (70%), hepatic failure (70%), gastrointestinal bleeding (30%), renal failure (600/0), cerebral failure (70%) and coagulopathy (65%). all patients who transitioned to the terminal stage of group d died of cardiogenic decompensation. in 5 patients of group c, the decreased blood ketone body ratio was restored with the amelioration of clinical symptoms after ex vivo pig or baboon liver crosshemodialysis and 3 patients of them were later discharged. evidence presented indicates that the decreased blood ketone body ratio has a direct bearing on multiple organ failure. conclusion: sepsis, hypotension or injury to the liver are a metabolic burden to the liver mitochondria which can result in mitochondrial impairment leading to a marked decrease in hepatic energy charge. such impairment ultimately leads to multiple organ failure as a result of the critically decreased energy and substrate store and the reduced protein synthesis relative to demand in the various organs. 110 in interferon-treated cells, the 2'-5'a synthetase, activated by double stranded rna, polymerizes atp into a series of oligonucleotides characterized by 2'-5' phosphodiester bonds and collectively designated 2'-5'a. these activate an endoribonuclease which cleaves rna. other regulatory functions of this enzyme may be expected because of its wide occurence in mammalian cells (untreated with interferon), where its activity appears, in vitro, to be dependent on the growth conditions, hormone responses regenerating liver after partial hepatectomy is often used as a model for the study of control growth and cell proliferation in vivo. in order to evaluate the role of the 2'-5'a synthetase in the processes leading to initiation of cell division, we measured this enzymatic activity in the rat liver during the first 40 h after partial hepatectomy. partial hepatectomy (50 rats) was performed under neuroleptanalgesia by removing the median and the left lateral lobes of the liver according to the method of higgins and anderson. control animals (3 rats) were subjected to a sham operation. after selected time intervals, the animals were sacrificed and the enzymatic activity in the regenerated liver was measured. the 2'-5'a synthetase activity present in the two first removed lobes was defined as 100%. we observe a very rapid decrease of enzymatic activity which reaches 50 % already 10 h after partial hepatectomy. the lower level of enzymatic activity (25 %) is measured between 20 and 24 h after partial hepatectomy. this minimum is followed by a slow restoration of the activity (at 40 h: 60 %). during this early phase of liver regeneration, a maximal incorporation of tritiated thymidine in dna takes place 24 h after surgery. so well differentiated liver cells have elevated levels of2'-5'a synthetase. but, after partial hepatec-tomy, the 2'-5'a synthetase activity decreases dramatically before the first wave of cell mitosis. these observations clearly illustrate the relationship between 2'-5'a synthetase activity and the growth status. moreover, this drop of enzymatic activity may be a trigger for the initiation of cell division. the primary event or events setting in motion the process of liver regeneration after partial hepatectomy (ph) remain unsettled. regarding the so called hepatotrophic factors, i.e. insulin, glucagon and recently egf, present evidence suggests that they would play mainly a promoting rather than a initiating role. early changes such as glycogen breakdown, fat infiltration and changes in adenine nucleotides and mitochondrial phosphorylative activity are usually, at least the first two, ascribed to metabolic overload of the remaining liver (bucher et al (1969) johns hopkins med, j, 125: 250). so far, however, little attention has been paid to a possible involvement of this phenomenon in the initiation of liver regeneration. attempts were therefore made to modify metabolic overload through early changes in energy metabolism in order to study their influence on the pattern of dna synthesis. fed or 16 h fffsting male wistar rats weighing 200+20 g were examined after ph at 4, 12, 18, 24, 30 and 36 h for adenine nucleotides, oxidative phosphorylation and dna synthesis, based on the rate of~h thymidine incorporation. fasting animals received continuous infusion of 20 % dextrose for 24 h at the rate of 0.45 ml/100 g/h. in addition to the above mentionned parameters hepatic glycogen and fatty acids were measured. within 12 h partial hepatectomy caused a decrease in hepatic atp which was maximal at 3 h (from 2.99___0.13 to 2.36+0.07 /~ moles/g p<0.001); in energy charge (atp + 0.5 adp/atp + adp + amp) from 0.851-+0.01 to 0.816___0.03 (p<0.01) and increase in phosphorylation potential which was maximal at 3 h (from 76+3 to 106_2p<0.001). dna synthesis began at 18 h reaching a peak by 24 h. glucose infusion to ph rats suppressed the decrease of hepatic atp, 2.97___ 0.07/1 mole/g at 3 h vs 2.99_ 0.13 in the control group, prevented glycogen depletion (histochemical estimation) and the increase in fatty acids (two folds increase in ffa and triglycerides (tg) at 24 h vs 10 folds in ffa and 8 folds in tg in the control group),with little effect on mitochondrial activity. the initiation of dna synthesis was delayed and the whole pattern was considerably modified. cessation of glucose infusion restored the usual rate of 3h thymidine incorporation after a late fall of hepatic atp. in conclusion, glucose infusion was shown by one of us to modify the hormonal response to ph, but insulin and glucagon administration to glucose treated animals failed to normalize the pattern of dna synthesis. it is suggested that metabolic overload as estimated on the basis of early changes in energy matabolism may account for one of the events involved in the initiation of dna synthesis after ph. infusion on liver cell regeneration after partial hepatectomy in the rat b. de hemptinne, j.f. ngala and l. lambotte university of louvain, laboratory of experimental surgery ucl 5570, 1200 brussels, belgium after partial hepatectomy (ph) the portal and the peripheral blood serum concentration of immunoreactive insulin suffers a drastic fall and levels ofglucagon show a rapid increase which is maximal 4 h after the liver resection. as these changes appear closely correlated to the blood glucose levels which show a 30 % decrease at 4 h and progressive restoration towards normal values up to 24 h, attempts have been made to alter the insulin/glucagon ratio by glucose infusion after ph and study its relation to liver regeneration. the purpose of this work is thus to determine after ph and hypertonic (20%) glucose infusion: 1. the effect of glucose on insulin and glucagon blood levels over 24 h; 2. the repercussion of the insulin/glucagon modified ratio on dna synthesis; 3. the possible improvement of dna synthesis by extensive glucagon infusion. male fisher rats underwent a standard 70 % ph. a 20 % glucose solution or isotonic salin was infused at a constant rate of 0.9 ml/h through a cannula placed in the iliac vein. the rats were sacrified at 0, 4, 8, 12, 22 and 24 h. (3h) thymidyne (27..3/~ci/mmol) was injected 2 h prior to sacrifice and the liver caudate lobes removed for analysis of (3h) thymidine uptake into nuclear dna. blood samples were withdrawn from the portal vein, the inferior vena cava and the aorta for glucose, insulin and glucagon assays. compared to the salin treated group, the infusion of glucose while keeping a normal steady blood glycemia was responsible of a marked increase of insulin (0.38--+0.01 ng vs 2.24_+0.4 ng,/7<0.01) and decrease of glucagon (0.615-+0.085 ng vs 0.335+0.037 ng, p<0.01), with a major switch of the insulin/glucagon ratio at 4 h after ph (from 6.68 to 0.61). dna synthesis started at 22 h in both series, but was very significantly impaired at 24 h in the glucose infused group (12800-+2280 cpm/mg dna vs 27450+2050 cpm/mg dna, p<0.01). infusion of increasing doses of glucagon (from 0.01 to 2 mg/kg/day could not restore the impaired dna synthesis. only a slight improvement was recorded at 0.5 mg/kg/day as the insulin/glucagon ratio tended to approach that of the control group. fractionation of various doses of glucagon over the 24 h perfusion time, in such a way that the changes in concentration of glucagon after partial hepatectomy was imitated, remained unsuccessful to improve thymidine incorporation. in conclusion: 1. the infusion of hypertonic glucose which impairs dna synthesis after ph, modifies markedly the insulin and glucagon secretion. 2. if a specific insulin/glucagon ratio after ph is important to sustain normal regeneration, its modification does not seem to be the major factor contributing to the blunted dna synthesis response in the hypertonic glucose infusion model. operative mortality of emergency shunt operations or esophageal transection during acute hemorrhage from ruptured esophageal varices in cirrhotic patients (child's category c) has been intolerably high. hence, the emergency operations in these patients should be avoided when the bleeding could be stopped by non-operative measures. when the emergency operation eventually becomes inevitable, the operative procedures should be simple and of short duration. in 1976, we have introduced the endoscopic balloon tamponade method for the management of esophageal variceal bleeding. the new balloon tube used in this method has essentially the similar structure as the sengstaken-blakemore tube, but is made of translucent plastic materials, and has a larger internal diameter so that a small caliber optic fiberscope (ex. bronchofiberscope) can be passed through the tube. by this method, the endoscopic observation of the esophagus is possible through the translucent balloon tube during tamponade of the ruptured varices. it is possible to know directly whether the bleeding from varices has been successfully stopped or not, which seems to be an advantage over the blind tamponade method using the sengstaken-blakemore tube. this endoscopic tamponade method has been used for emergency hemostasis of 166 acute bleeding from ruptured esophageal varices in 66 patients. the mean initial tamponade pressure by the esophageal balloon necessary to stop bleeding was 30_+12 mrnhg and the average duration of tamponade was 50 h. the location of the ruptured v~ices observed by this method was in the lower esophagus within 10 cm of the esophagocardiac junction in 86% of the cases. the bleeding has been stopped in 156 occasions (93.9 %) by this method. no significant complications other than atelectasis in 2 patients have been observed. in 4 patients, the bleeding was initially stopped by the new translucent balloon tube, but recurred within 24 h after decompression of the esophageal balloon. tamponade was repeated for several times since these patients were in the category c of the child's classification, however, the emergency operation eventually became necessary. transthoracic esophageal transection was performed using autosu-ture apparatus, eea, in these patients. one patient died of liver failure in the 6th postoperative day, but others survived the operation and recovered. the use of eea in transthoracic esophageal transection simplifies the esophageal anastomosis, and shortens the duration of operation by 40 rain. we have so far used the autosuture apparatus, eea (28 mm cartridge) in elective esophageal transection of 33 patients. neither anastomotic bleeding or insufficiency has been encountered. acute variceal bleeding in category c patients should be treated initially by endoscopic balloon tamponade, when emergency operation is inevitable, transthoracic esophageal transection using eea is the operation of choice. arterialisation of the portal vein in conjunction with an end-to-side portacaval shunt has been proposed as a method of improving survival following shunting [1] . however, there is little experimental evidence to support this suggestion and so we have examined the effects of arterialisation of the portal stump in cirrhotic rats. 24 rats with dimenthylnitrosamine-induced cirrhosis were used in this study. 8 of the rats received an end-to-side portacaval shunt, 8 had a portacaval shunt and the portal stump arterialised with the left gastric artery, eight were sham-operated. liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before and after shunting. three weeks after surgery lbf and whvp measurements were repeated, the animals bled and the liver removed and weighed. an end-to-side portacaval shunt led to an immediate fall in lbf (31.3_+5.6 to 15.5_+4.3 mls/min/ 100g-~) and whvp (7.6_+0.8 to 3.1-+0.5 mmhg). however, if the portal stump was arterialised lbf (34.6-+2.9 to 45.3+5.7) and whvp (7.9_+1.0 to 7.3_+1.3) did not change significantly. no further changes in lbf and whvp were observed three weeks after operation in any group of animals. arterialisation of the portal stump prevented the loss in body weight, loss in liver weight deterioration of liver function tests and hyperammonemia observed in animals with a portacaval shunt alone. these findings suggest that arterialisation of the portal stump may prevent some of the deleterious effects after shunting. departments of surgery and pathology, university of virginia hospital, charlottesville, virginia 22908, usa we have hadexperience with operative restoration ofhepatopedal portal blood flow in five patients intolerant of total splanchnic shunting. hepatopedal flow was reestablished by takedown of the total shunt and construction of a selective, distal splenorenal shunt, or by isolation and arterialization of the hepatic limb of the shunted portal vein. in two patients, shunt revision was undertaken electively for chronic encephalopathy, unresponsive to low protein diet, intestinal antibiosis and oral lactulose. each individual had been hospitalized more than eight times for encephalopathy or coma. nine and 34 months postoperatively, both patients have had no encephalopathy on unrestricted protein intake, and work. actively as homemakers. serial liver needle biopsies have shown bilobed nuclei and enhanced mitotic activity suggesting hepatocyte regeneration. in three patients, shunt conversion or arterialization was undertaken in desperate circumstances, characterized by liver failure (bilirubin >10 mg/dl, albumin <2.5 girl, prothrombin time >1"6 s)~ coma and respirator dependency. although two patients showed immediate, marked improvement in mentation, all three died of intraabdominal hemorrhage in the first few postoperative days in spite of prolonged attempts to achieve hemostasis and maximum blood product support. three conclusions can be drawn from this limited experience: 1. total shunt procedures which are anatomically suitable for subsequent conversion to a selective configuration or for hepatic limb arterialization should be favored over those not offering such potential; 2. at a time of election, restoration of hepatopedal portal flow can be accomplished in patients with side-to-side portacaval or hemodynamically equivalent shunts with considerable benefit; and 3. similar procedures in patients with fulminant liver failure are unlikely to succeed. peritoneal-venous (leveen) shunts are associated with a significant incidence of disseminated intravascular coagulation (d.i.c.). this study identifies a site of origin, a pathogenic mechanism, and the hemostatic pathway which accounts for the thrombogenicity of human ascites. 500 ml of peritoneal fluid were removed percutaneously from individuals with malignant and cirrhotic ascites. ficoll-hypaque column chromatography and ultracentrifugation were utilized to prepare four fractions: cellular; a low speed cell-free fluid; a high speed supernatant; and the precipitate from the high speed centrifugation. the cellular fraction from both types demonstrated an ability to shorten a one stage clotting time by 60 % relative to saline and endotoxin controls. similarly, low speed cell-free fluid shortened the clotting time of pooled normal plasma by 61%; was also effective in factor viii (required for intrinsic pathway of coagulation) deficient plasma; but had no effect on factor vii (required for extrinsic pathway of coagulation) deficient plasma or platelet aggregation and release. the high speed supernatant was demonstrably less thrombogenic. the resuspended precipitate shortened the clotting time of pooled normal plasman by 67 % and of factor viii deficient plasma from infinity to 99 s. in contrast, this material was ineffective on factor vii deficient plasma. we conclude that the thrombotic potential of human ascites derives from peritoneal cells, either leucocytes or malignant cells. consistently, the thrombotic factor exists in suspension and is thromboplastin-like in its behavior, operating through the extrinsic pathway of coagulation. thus, a site of origin and a pathway of activity for the thrombotic agent in human ascites is identified. the effect of somatostatin in hepatic haemodynamics in the cirrhotic rat s. jenkins, p. devitt and r. shields department of surgery, university of liverpool, liverpool, u.k. although somatostatin has been suggested as an alternative treatment to vasopressin in the emergency control ofbleeding oesophageal varices [1] , recent studies on its effect on wedged hepatic venous pressure in cirrhotic patients have provided conflicting results [2, 3] . therefore we have examined the effect of somatostatin on hepatic heamodynamics in cirrhotic rats. rats with dimethylnitrosamine-induced cirrhosis received a bolus injection of 2, 4 or 8 pg/kg body weight of somatostatin followed by a 30 min infusion of either 2, 4 or 8 pg/h/kg body weight. control rats received saline only. portal venous flow (pvp) portal pressure (pp), liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before, during and after somatostatin infusion. at the lowest rate of somatostatin administration (bolus injection of 2 pg/kg body weight followed by an infusion of 2 pg/h/kg body weight)there was a rapid decrease in pp (9.8+0.5 to 8.0--+0.4 mmhg), whvp (7.5___0.4 to 5.8---0.3 mmhg) and pvf (31.2 ___ 4.9 to 21.0___ 3.8 ml/min). 30 rain after the start of the infusion pp, whvp and pvf were still signaflcantly lower than preinfusion levels. at higher rates of somatostatin infusion there was no furhter decrease in pp, whvp and pvf. lbf was significantly reduced at all the doses of somatostatin. the highest rate of infusion of somatostatin (8 pg) produced a significantly greater reduction in lbf than either 2 or 4 pg. these data suggest that somatostatin may have a role in the management of portal hypertension, but that higher doses may have a detrimental effect on lbf. er positive breast cancers preferentially metastasize to bone (1) prostaglandin e2 (pgez) is synthesized by breast cancers and potentiates bone resorption in vitro (2) . this study investigates the relationship between er status and pge2 synthesis in breast cancer cells. pge2 was measured by radioimmunoassay in 75 primary breast cancers: a) after ethanol inhibition of further prostaglandin (pg) production -,,basal pg" b) after stimulating pg production with excess arachidonic acid -,,total pg". ,,total" minus ,,basal" = ,,synthesized pg". in order to relate pg production to breast cancer cells, measured pge2 values have been corrected for the epithelial cellularity of each tumour. pge2 x 100 corrected pge2 = actual (%) cellularity cellularity was evaluated by a proportional count of cancer cells expressed as a percentage against non cellular material in all fields of 3-5 histological sections at 63x magnification. we conclude that er positive tumour cells synthesize greater amonts pge2 than er negative cells. this may account for the greater tendency of er positive cancers to recur in bone. oestrogen receptor activity (er) is of prognostic value in breast cancer [1] . however, the chosen cutoff between er-negative and -positive is arbitrary and likely to affect its prognostic value. in 201 patients with invasive breast cancer treated by mastectomy, tumour er was determined [2] and the patients were followed up until first recurrence. using a computer program to perform repeated logrank analysis, the effect of varying the cut-off on prognostic value of erwas studied between 1 and 25 fmol/mg protein. er levels were higher in postmenopausal patients (0-1301, median 72, n=121) than in pre-menopausal patients (0-201, median 25, n=80). for the whole group, optimal prognostic discrimination was achieved with a cut-off 6 fmol/mg protein. in premenopausal patients, the effect of varying cut-off was complex, leading to an optimum of 17 fmol/mg protein, whilst in post-menopausal patients the optimum was 3 fmol/mg protein. these findings were unexpected and may relate to the relationship of er to tumour cellularity [3] . it is concluded that: 1. the failure of some centres to relate er to prognosis may be due to the inappropriate cut-off point chosen; 2. in our patients, the optimal cut-off was 6 fmol, which agrees with the level suggested by the british breast group [4], though it differed between pre-and post-menopausal patients; 3. optimal cut-off for prognosis may differ from that for predicting response to endocrine therapy. previous studies have reported that the use of adjuvant chemotherapy improves relapse free survival following mastectomy. the effect on overall survival is less certain. 290 patients with histologically confirmed axillary node metastases were randomised to receive postoperative radiotherapy (rt), chemoterapy (cmf) or radiotherapy plus chemotherapy (rt + cmf). 244 patients have now been followed for between 6 months and 5 years. patients initially treated with rt received chemotherapy on failure where possible. of 79 patients receiving rt alone, 28 have developed distant recurrence and 20 have died. of 78 receiving cmf alone 22 have developed distant recurrence and 15 have died (see table) . although the use of adjuvant chemotherapy significantly prolongs the relaps free interval there is no corresponding improvement in overall survival. a sample of 89 patients with histologically proven prostatic carcinoma underwent transrectal find needle aspiration at six month intervals, for a mean follow-up of 18 months, as an out-patient procedure, without significant side effects. 49 patients were followed from time of initial diagnosis. the others had been on treatment for a mean period of 36 months before the study commenced. in 25 of the new patients, cytology was positive at the time of histological diagnosis, and correlated with the histological grade. there were no false positives. repeat cytology on 15 patients after 12 months showed 6 positive and in 5 of these the disease was progressing, and in one it was stable. of the 9 who were negative, 8 were stable and one progressing. 16 of the 48 patients already on therapy had positive cytology at their initial aspiration -9 showing progressive disease and 7 being stable. 5 patients whose cytology was initially negative became positive 12 months later and all had disease progression at this time_ 19 patients had negative cytology on 3 or more occasions and in only 2 cases was there evidence of disease progression. cytology showed evidence of squamous metaplasia on follow-up in 13 of the patients whose condition was stable. the prognosis for all patients was assessed on the basis of gleeson's score and aspiration biopsy has been shown to be a safe and useful procedure for monitoring disease activity and response to treatment. the optimum method of restoring the ability to swallow in patients with unresectable carcinoma of the oesophagus remains controversial. this study evaluates the palliative potential of pulsion intubation versus retrostemal gastric bypass of the excluded oesophagus in unresectable carcinoma of the upper thoracic segment (20-32 cm from the incisor teeth). patients and methods: 71 patients were prospectively randomised for treatment by pulsion intubation (37 patients) or gastric bypass (34 patients). non-resectability was indicated by (1) tumour lengths greater than 6 cm, (2) tracheal or bronchial invasion, (3) disrant dissemination, (4) mediastinal invasion. the operative mortality, morbidity, palliation of dysphagia and postoperative nutritional status was compared in the two groups. results: mortality: intubation resulted in 2 deaths, a mortality rate of 5,4%. gastric bypass resulted in 3 deaths, a mortality rate of 8,8 %. complications: intubation was complicated by respiratory infection (8), tube migration (1), respiratory obstruction (1), oesophageal perforation (1), bleeding (1). gastric bypass was complicated by chest infection (9), pneumothorax (3), wound infection (5), subphrenic abscess (1), anastomotic leak (4), pulmonary embolism (1), purulent neck discharge (3). ability to swallow: palliation of dysphagia was achieved in 92 % of patients following intubation and 91% of patients following bypass. postoperative nutritional status: improvement in nutritional status was more rapid following intubation. conclusion: pulsion intubation is the preferred palliative procedure because of fewer complications and lesser degree of postoperative catabolism. the current technique for investigating the response of vascular prosthetic materials to infection is by challenge with a sub-lethal dose of bacteria, usually an intravenous infusion of 108 organisms in an animal model. this large bacterial innoculum, however, obscures any difference in the infectibility of prostheses that may be inherent in the material, its incorporation into host tissues, or its resistance to infection. we have developed a sensitive method for determining the susceptibility to infection of vascular prostheses based on calculation of the number of bacteria required to infect a specific prosthesis in 50% of trials (ids0). following implantation of the prosthesis to be tested in the canine infra-renal aorta, a dose-response curve was generated by the intravenous injection of known innocula of s. aureus (at log intervals from 102 to 108 bacteria). at six weeks the prosthesis was harvested and cultured to document infection with s. aureus. a characteristic sigmoid curve resulted from which the ids0 was determined. to test this method, a comparison was made between commercial human umbilical vein grafts (huvg) and huvg impregnated with silver sulfadiazine in 35 dogs. although both prostheses were infected by the standard 108 innoculum, a greater than tenfold increase in the resistance to infection by the treated huvg (<102 to 101) was demonstrated in the dose-response curves. since the number of bacteria in a postimplant bacteremia rarely exceeds 102 organisms/ml, such differences in infectibility are clinically significant. ids0 determination provides a sensitive, reproducible method for quantitating resistance to infection in vascular protheses. prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. it is accepted that infection arises from operative contamination, bacteraemic seeding and abscesses or viscus eroding into the graft. this study investigates the role played by distal sepsis on groin grafts. ten dogs had a specific strain of staphyloccocus aureus inoculated onto a surgical wound in the right foot pad. five days later interposition 6 mm dacron grafts were implanted into the ipsilateral and contralateral groin in continuity with the superficial femoral artery. ten days following this the grafts were removed for bacteriological and histological examination. blood cultures and lymphnode cultures were also taken at this time. in seven dogs the specific staph, aureus, was grown from both grafts. two dogs failed to grow the specific staph, aureus from either graft. these results are significant at the 1% level using fischer's exact test. blood cultures grew staph, aureus from only one dog. ipsilateral lymphnode cultures yielded the specific staph, aureus in seven dogs. we believe that distal sepsis plays an important role in the estabishment of graft sepsis. the mechanism of spread would appear to be via the lymphatics. the influence of tumor growth on wound healing p.w. de graaf and a. zwaveling laboratory experimental surgery, state university, leyden, the netherlands leakage of a colonic anastomosis is a complication each surgeon fears, because it usually leads to a prolonged hospital stay and is accompanied by a high mortality. carcinoma as an indication for operation and preoperative malnutrition are considered to be high risk factors. the subject of this study was to measure the influence of malignant tumor growth at a distant site on woundhealing in colon and skin, and to find a correlation between the influence of the nutritional state of the experimental animal, and the influence of malignant tumorgrowth on woundhealing. we also tried to find ways to compensate the possible unfavourable influence a malignant tumor might have on woundhealing. the study was performed in rats, the tumor used was a rhabdomyosarcoma, transplanted subcutaneously in the rats right flank. parameters for woundhealing were tensile strength of the skin-incision, and bursting pressure of a colonic anastomosis. six groups of rats were studied, each rat undergoing a standardized colonic operation after two or four weeks. the groups consisted of: control animals (20); tumorbearing animals (20), without tumor removal; malnutrltioned animals (20); tumorbearing animals receiving intravenous hyperalimentation (20), without tumor removal; animals undergoing tumor removal and colonic operation in one session (10); animals undergoing the colonic operation two weeks after tumor removal (10). woundhealing was negatively influenced in an early stage of tumorgrowth (f<0.05). four weeks of tumor growth did not impair woundhealing to a greater degree than two weeks. laboratory determinations showed no deviations. malnutrition, leading to less weight gain than in control and tumor bearing animals took much longer to influence woundhealing than tumor pearing. this is in accordance with publications by irvin and hunt (1974), devereux et al (1979) and garattini and guaitani (1981) , which led us to the conclusion that the negative effect of tumor bearing on woundhealing was not solely the result of anorexia. hyperalimentation in tumorbearing rats resulted in undisturbed woundhealing, despite the fact that hyperalimentation as practised by us (with amino-acids and carbohydrates) stimulated tumorgrowth. we concluded from these experiments that tumorgrowth caused a metabolic chaos in the animal, which in turn led to a disturbance in woundhealing. intravenous hyperalimentation could apparently compensate this. woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in two sessions (p<0.05). irvin an hunt (1974) have demonstrated however that extraabdominal trauma had no influence on colonic healing. this led to the conclusion that the diminished woundhealing found in rats operated in one session was an effect of the tumor. obviously this influence is not instantly removed with tumor excision. this study offers a theoretical foundation for the clinical observation that in operations for colonic carcinoma with a substantial operative trauma, the anastomosis needs extra protection and/or needs to be performed in a second session. the aorta of the blotchy mouse undergoes dilatation during adult life, resulting in the formation of fusiform aortic aneurysms. the mutation is x-linked, so only the males are affected. we have found a shift in the fluorescent spectrum of insoluble, hydrolyzed, dermal collagen in these mice; suggesting the presence of an abnormal crosslinkage compound. the purpose of this report is to describe additional studies carried out on the soluble collagen fraction in these animals, which lend support to the hypothesis of a crosslinkage abnormality. dermal collagen was prepared from one-monthold mutant mice and their normal male littermates by sequential extractions in salt solution, acetic acid, and urea. amino acid analyis and sds gel electrophoreses of the salt-soluble fractions revealed similar profiles of amino acids and collagen-chain types, suggesting that there is no abnormality of the basic building blocks of collagen in the mutants. fluorescent spectroscopy of the acid hydrolysates of these fractions also demonstrated similar absorption and emission peaks. however, reduction with sodium porohydride abolished fluorescence in the collagen fraction from the mutants. results (2emission m a x ± sem): normal blotchy before reduction 424.3 ± 3.0 420.7 -----2.9 after reduction 420.0 + 2.1 none sodium borohydride is used experimentally to stabilize labile collagen cross-linkages. the present studies indicate that the salt-soluble collagen from the blotchy mice is borohydride-reactive and is thus chemically ,,unstable". these studies suggest a rational for developing probes based on these fluorescent croperties of investigate collagen from human subjects affected with aneurysms. one third of testes are removed after torsion (1, 2) because of delay in presentation and diagnosis. in a retrospective study of 89 patients with torsion our salvage rate was 64%. nineteen percent were misdiagnosed as epididymitis by a junior doctor and 7 % by a surgical registrar. doctors could not distinguish between torsion of the testis and its appendages. in the literature, eight factors have been reported to be of discriminant value (table 1) . a computer program, using these parameters and bayes' theorem, was written to calculate the most likely diagnosis. data from the 89 patients with torsion and 96 patients with acute epididymitis were analysed by this program ( table 2) . the program correctly diagnosed all 96 cases of epididymitis. of 75 patients with torsion of the testis . in this study we determined the critical ischemic time for the development of an arrhythmogenic potential. egg and bipolar electrograms were recorded from the his bundle, ventricular endocardium and epicardium in twelve anesthetized dogs. short bursts ofventricular pacing (3 beats; 240-420 beats/min) were used to induce ventricular arrhythmias before and after lidocaine administration (2, 4, 6 mg/kg). previously, lidocaine has been shown to exacerbate conduction delay in ischemic myocardium leading to reentrant ventricular arrhythmias. in the control state, ventricular pacing with or without lidocaine induced either no response or single or repetitive ventricular responses. sustained ventricular tachycardia was never evoked in the control studies. in six dogs, after 20 min of left anterior descending coronary artery ligation and reperfusion, ventricular pacing with and without lidocaine produced sustained ventricular tachycardia in one animal. in another six dogs, after 30 rain ofligation and reperfusion, one dog showed sustained vertricular tachycardia in response to ventricular pacing alone. the other 5 showed sustaine~l vertricular tachycardia after ventricular pacing with lidocaine. sustained ventricular tachycardia averaged 400/min and degenerated into ventricular fibrillation within 1-2 rain. in conclusion 20-30 min of ischemia appears to be the critical period for development of malignant arrhythmogenic potential in the canine heart. administration of lidocaine during this critical period enhances the inducibility of cardiac arrhythmias, which may have clinical relevance in the management of acute myocardial infarction. untreated coronary artery air embolism in the experiments without ecc (group i) resulted in a transmural myocardial ischemia with a mortality rate of 28 % in contrast there were no death in the experiment when extracorporeal circulation was used (group ii to v). the best therapeutic effect with regard to the reversibility of temporary myocard ischemia following coronary artery air embolism was achieved by increasc of the postembolic perfusion pressure (group v), a finding being significantly different (p<0.05) to groups i through iv. also volume depletion of the left ventricle on ecc (group ii) resulted in a faster regression of the left ventricular hypothermic area compared to group i (p<:0.05). the application of dipyridamole (group iii) and st.thomas cardioplegic solution (group iv) was not able to produce a significant therapeutic effect. the increase of perfusion pressure following coronary artery air embolism has demonstrated to be the most effective procedure to achieve reversibility of myocardial ischemia. to transfer these experimental findings into clinical application is suggested because of its practicability and convenience. transmural biopsies were obtained before and 5, 15, 30, 60, 90, 120 min after acc for biochemical and structural analysis. myocardial temperature (t) ph (mph), and pco2) were measured continously with a thermistor, a new ph electrode, and a mass spectrometer respectively. in group i (n=8) acc was under normothermia. in group ii mean t was reduced to 19°c by the administration of cold potassium cardioplegia immediately after acc and consecutively every 30 min. mph at the end of acc reached 5.39-t-0.08 group i) and 6.49___0.13 (group ii) (/7<0.001); pmco2 rose from 41___4 to 234_+13 mmhg in group i and did not change in group ii. tissue content of atp decreases by 51% group i) and by 14°/0 (group ii) (f<0.001); tissue creatine phosphate fell by 98 % (group i) and by 48 % (group ii) (p<0.001). changes in atp and creatine phosphate as well as in tissue glucose and glycogen related ton concomitant changes in ph. the degree of ischemic damage assessed histologically by a mean ischemic score was 2.5+--0.06 in group i and 0.75_+ 0.19 in grup ii (p<0.01). irreversible structural demage assessed by electron microscopy occurred in group i 60-90 min after acc and was associated with a mph below 6.2. no such damage was observed in group ii. we conclude: 1. irreversible damage during normothermic arrest occurs considerably later than has been reported for regional ischemia in the beating heart; 2. during 2 h of acc cold potassium cardioplegia does not completely protect against id when mean t is 19°c; and 3. on-line oaeasurement of mph reflects the inadequacy of' mp more accurately than do either pmco2 or t and thus, provides a useful potential method for intraoperative monitoring of mp. in rabbits, infusion cardioplegia was installed at 37, 27, and 17°c respectively. the infusate contained na 27, k 5, 30, 120, 160 or 240, ca 0, 1 or 5 mmol/1, and varying amounts of glucose. measured osmolality varied from 305 to 500 mosmol/kg. the hearts were excised at the end of a 5 rain infusion period; one half was immediately frozen, the other half was incubated for varying periods of time. specimens of left ventricular myocardium were analyzed for metabolite and electrolyte contents. at termination of the cardiplegic perfusion, total adenine nucleotides (tan) and total creatine (tcr) were within control ranges under all conditions. however, atp and ecp = (atp + 0.5 adp)/tan as well as creatine phosphate (crp) and the ratio crp/tcr decreased significantly with increasing dosages of k and ca and higher temperatures. there were corresponding increases in adp, amp, and free creatine. at 17°c, there were no such changes except in hearts perfused with 240 retool/1 k and 5 mmol/1 ca. on the contrary, crp and the ratio crp/ tcr were elevated above the control values in hearts perfused with ca-free solutions containing between 30 and 120 mmol/1 k. the decrease in atp served as the parameter of the ischemia-induced metabolic alterations and the energy deficit developing during cardiac arrest. at 37°c, it averaged 1.67 and 2.55/zmol/g (p<0.02) after 10 min of arrest induced by 30 and 240 mmol/l k respectively. ca, 1 or 5 retool/1 respectively, significantly increased the atp-decay to 2.41 and more than 3.63 pmol/g/lo min respectively (/7<0.02 and p<0.05). hypothermia of 27°c reduced the rate of metabolic deterioration and suspended the influence of the k-dosage. however, the ca-effect was still visible: induction of cardiac arrest by 30 and 240 mmol/l k without/with ca decreased atp by 2.07/ 2.58 pmol/g (p<0.025) and 2.07/2.41 pmol/g (n.s.) respectively within 20 rain. at 17°c, the rate of metabolic alterations was further reduced. the effects of k-dosage and of ca were completely abolished. the atp-decreased 2.2 pmol/g during 40 min of arrest. although higher concentrated k-solutions for infusion cardioplegia do not enhance the ischemiainduced myocardial metabolic alterations in deep hypothermia, they are not recommended for practi-despite the advantages of cbk carrdioplegia, the severely impaired myocardium and/or long ischemia time continue to be a challenge. because of the association of ca ~ with cell injury and death the use of ca ~ channel blockers is logical. investigation of cbd revealied no advantages over cbk. the combination of k and d is appropriate because of their different mechanisms of action. ten dogs had one hour of myocardial ischemia (mi) with topical ice (temp 7___2°c) after coronary perfusion with 200 ml cb (5___ l___c) containing k, 30 meq/l and d, 400 pg/kg. eight dogs had two hours of mi after perfusion with 200 ml cb containing k, 30 meq/l and d, 200/zg/kg. six dogs received the same treatment as the previous group except that d was increased to 1600/zg/kg for all four perfusions. baseline studies were repeated after 60 min ofreperfusion without the use of ca ~ or inotropic agents. heart rate, peak systolic pressure, vc¢, v~,~x, peak + dp/dt, peak -dp/dt, dp/dt over common peak isovolumic pressure, left ventricular compliance, stiffness and elasticity and great water were unchanged from control. coronary vascular resistance was unchanged in groups one and two but declined in group 3. creatine phosphate returned to control but atp, adp and the adenosine pool were depressed. ultrastructure was well preserved. in 16/24 dogs defibrillation was not required whereas 48/48 dogs with cbk and 13/13 with cbd required defibrillation. these data suggest that d is a worthwhile addition to cbk. early one-stage repair of some congenital vascular anomalies might be accompleshed readily if the material used for grafts grew along with the reconstructed tissues. we have evaluated the capacity of tubular grafts constructed from anterior rectus sheath to serve as growing segmental replacements of the descending thoracic aorta of puppies (age 9 weeks). grafts measuring 3 cm in length were placed in 17 animals; 10 were implanted with attention to tissue preservation (live grafts) while 7 received grafts subjected to prior freezing and thawing in order to kill the donor tissue cells (devitalized grafts). grafts were measured initially and at sacrifice 1, 2 or 3 months later. each months 3 animals with live grafts and 2 with devitalized grafts were sacrificed. no aneurysmal dilatations or grafts ruptures were observed in any of the specimens. by 3 months, the live grafts had increased 26.9-1-1% in length and 27.3___ 3 % in diameter, while length and diameter of the devitalized grafts showed minimal growth (2.1___20/o and 5.3___5o/o respectively). during the same period the length of the thoracic aorta increased 4.5___5% in the live graft group and 41.1___ 8 % in the devitalized graft group. grafts were lined by endothelium and organized into 3 transmural zones. the thickness of each zone in the live grafts remained static from 1 to 3 months, whereas thickness of the middle and outer zones in the devitalized grafts increased 4 to 6 fold. in living grafts the layers consisted principally of newly formed fibrocellular tissue; the rectus sheath was reduced to a atrophic fibrous band. by contrast, the rectus sheath remained prominent in the devitalized graft specimens. cellularity (cells/field) of the live grafts was much greater than that of the devitalized grafts (ratio 15.9___3 : 6.0-----2). a newly formed, fibrocellular layered structure replaces the rectus sheath in the live grafts. these findings indicate that rectus sheath grafts are capable of growth in the young animal, providing that at implantation the tissue is well preserved. it is well known that durability of valvular bioprostheses (vbp) depends mainly on the structure of constituent biomaterial and long-term preservation of the collagen network. to our knowledge, no studies on ultrastructure of tissues currently used for vbp has been reported so far after a long time of chemical preservation. the presentation of a new anysotropic tissue, xenogenic cervical duramater (xcd), with a collagen tridimensional network, and a comparative study of scanning (sem) and transmission (tem) electron microscopy of bovine pericardium (bp), human duramater (hd) and procine aortic-cusp (pao) represent the purpose of the present communication. samples of the tissues were obtained in semisterile fashion, rinsed and fixed in karnovsky medium at 4°c for 24 h. materials were then minced on paraphine plates and washed with 0.2m cachodylate buffer for 1 h and dehydrated in increasing concentrations of ethanol (30 %-100 %) and intermediate exposure to 1% uranyl acetate for the in-bloc contrasts. were conducted, and results are exposed in table 1 . as a general rule, histologic evaluation was by far, more satisfactory for tissues preserved with 0.5 % glutaraldehyde (xcd, pap) than those with 98 % glycerol (hd) and 4 % formaldehyde (bp) in combination. it is concluded that the new anysotropic tissue herein presented, xcd, can be appropriately preserved with 0.5 % glutaraldehyde as collagen preservation is concerned, and it's anysotropic and ultrastructural features, maintained for as long as 12 months as assessed by sem and tem. albeit, clinical use of this new biomaterial is subjected to further experimental and animal trials. tetralogy of fallot and absent pulmonary valve (tapv) is associated with massively dilated pulmonary arteries which cause tracheobronchial compression in the newborn and heart failure and cyanosis in older patients. corrective operations have been attended by mortality rates exceeding 40% due to pulmonary insufficiency causing right heart failure (rhf) and pulmonary complications. pulmonic valve insertion (pvi) with complete repair has resulted in improved survival. the purpose of this paper is to assess the results of total correction and pvi in ten patients. during the last five years, 152 patients with tetralogy were corrected. of these, ten patients (ages 51 days to 34 years) had absent pulmonary valve. one patient (51 days) present with severe rhf and pul-monary insufficiency and nine patients presented with mild rhf and cyanosis. chest roentgenographs showed increased cardiothoracic ratio and pulmonary prominence in all. arteriography revealed massively enlarged pulmonary arteries with a mean right pulmonary artery to aorta size ratio of 2.7 to 1. associated pulmonic stenosis and insufficiency was present in all. seven patients underwent closure of ventricular septal defect (vsd) and pvi. of these, three had pvi (2 tissue and 1 prosthetic) with outflow patch and four had right ventricle to pulmonary artery (rv-pa) tissue valved conduits. two patients had repair without pvi, and one had repair with a monocusp pericardial valve patch. nine patients have done well with no episodes of thromboembolism or infection. death occurred in a 51 day old infant who had vsd closure and relief of pulmonic stenosis. pulmonary valve insertion seems indicated in these patients at it lowers peak pulmonary artery pressure and thus reduces compression effects on the trachea and bronchi. as well, when pvi was used right heart failure was not noted postoperatively. although in experimental acute myocardial ischemia intraaortic balloon pumping (iabp) appears to increase regional contractility of ischemic segments of the left ventricle by increasing the collateral flow, evidence for this effect of iabp in patients with refreactory myocardial ischemia is not available. this study was done to analyze the effect of labp on global and segmental left ventricular performance in patients with refractory, acute myocardial ischemia using gated blood pool scanning with tc-99 albium tracer. eight patients were studied on-and-off iabp prior to and following coronary bypass surgery. left ventricular (lv) wall motion analysis was undertaken and both cardiac output (co) and left ventricular filling pressure (lvfp) were determined from thermistor-tipped pulmonary artery catheters. when placed on iabp, mean preoperative global ejection fraction (gef) increased (0.264-0.05 (off) to 0.354-0.05 (on); p<0.01) abut no changes in co or lvfp were observed. when comparisons were made praend postoperatively, co increased after operation (p<0.05), due chiefly to an increase in heart rate (824-6 to 1074-8 beats/rain; p<0.01), but gef was unchanged whether or not iabp was on. only segmental wall motion analysis pre-and postoperatively revealed that iabp increased regional ejection fraction (ref) and contraction velocity by 50% (mean) in the angiographically determined regions of myocardial ischemia and these regional changes were maximal during the last one-third of the lv contraction cycle. it appears that ref is a more sensitive indicator of changes in lv performance than co and gef in these patients. further, this study indicates that iabp does increase the regional wall contracility in the ischemic areas of the myocardium in man. the use of aspirin (asa) to inhibit platelet thromboxane synthesis (tbx2) in patients undergoing coronary artery bypass grafting (cabg) has been advocated to reduce the potential for graft occlusion. however, asa in conventional doses also inhibits the venous synthesis of prostacyclin (pgi2) a potent anti-thrombotic factor, and may contribute to excessive surgical bleeding. to investigate the relationship between asa dose, blood loss and inhibition of venous synthesis of pgi2, 27 patients undergoing cabg were studied. control patients (no asa) were compared to those receiving 80 or 325 mg. asa as a single dose 8-16 hours prior to surgery. production of pgi2 by saphenous vein specimens removed at the time of surgery was measured by radioimmunoassay (ria) of 6-keto pgf~a following incubation with 25 um na arachidonate. blood loss was assessed by chest tube drainage and transfusion requirement in the perioperative period. serum tbx2 was measured by ria following asa ingestion. transfusion (units), drainage (cc), vein pgi2 (pg/mg tissue) and serum tbx2 (ng/ml) were (mean 4-sem): therefore, asa 325 mg or 80 mg did not increase blood loss during cabg. however, asa 325 mg, significantly reduced saphenous vein pgi2 synthesis (/7<0.01) while the lower dose asa 80 mg spared the production of pgi2. asa is both doses inhibited tbx2 (p<0.001) and blocked platelet aggregation. low dose asa deserves further investigation as an anti-thrombotic agent since it does not appear to increase operative bleeding or significantly inhibit venous pgi2 production. transfusion postoperatively, igg increased progressively in patients in group a, reaching preoperative levels on the 6th or 7th day, whereas in patients in group b, igg remained at lower than preoperative levels during the first week. the number of patients with abnormally low lavels of igg during the whole po period was significantly higher in group b (p"~.01). no significant differences were found in the others studied variables. one patient in group a had infection. three patients in group b had infections. conclusion: high doses of fab2igg administered during ohs and the first po days are effective in lessening the po decrease oflgg and thus may be beneficial in preventing po infection. a water insoluble but very hygroscopic polyvinyl alcohol powder, zy-15044", is capable of significantly stimulating the cicatrization of open, contaminated skin wounds in rats. when it is compared to other substances in clinical use such as powders containing antibiotics, antiseptics, amino-acids, enzymes or a dextranomer, no other agent tested was capable of producing a similar beneficial effect. these excellent experimental result justified pilot clinical trials on 30 patients: 18 varicose vein ulcers, 4 atherosclerotic leg ulcers, 4 infected traumatic wounds and 4 infected postoperative wounds. whether in rats or in patients, zy-15044 powder removed secretion, bacteria and tissue debris; it produced an early and increased proliferation of fibroblasts with the appearance of dense granulation tissue; it reduced wound size after less than three applications permitting very early grafting of the wound of eventually, cicatrization advanced to complete epithellzation. regardless of whether the treatments were applied daily in the hospital bed or every other day in outpatients, there were not complaints of unpleasant sensations such as itching, burning or pain. few studies have dealt with long term healing in stomach and none is available for duodenum. this study evaluates morphology and development of mechanical strength and collagen concentration in and adjacent to wounds after 20 to ]g0 days of healing. incisions were made in the non-glandular (rumen) and in the glandular oxyntic (corpus) part of the stomach and in duodenum of 64 male wistar rats, 23 intact rat served as controls. the wounds were dosed with 6-0 polypropylene sutures using a single sutur technique. after 20, 40 and 80 days of healing complete load deformation curves were determined on strips perpendicular to the incision line after the sutures were cut. collagen distribution was measured as hydroxyproline in strips parallel to the incision line. wound tissue continued to gain strength up to 80 days of healing (breaking energy significantly increased in all tissues from 20 to 80 days of healing). no such increase was found for wounds tested togetherwith adjacent tissue, because the "point of maximum weakness" had moved laterally from the incision line with healing time. wound collagen concentration increased in corpus and duodenum between 20 and 40 days of healing, but was unchanged between 40 and 80 days. the biochemical active zone around the incision line was unchanged 4-5 mm on each side from day 20 to 80. conclusion: while the wound tissue itself continues to gain strength during the 80 days ofheaiing studied, the increase seen after 20 days does hot,enhance the functional properties of the organ as a'whole. the "point of maximum weakness" has at that time moved to the borderline of the biochemically active zone, which lies outside the tissue area enclosed by sutures. myocutaneous flaps based on either the inferior or superior epigastric artery may be used to cover extensive soft tissue defects from the mid thigh to the clavicle. we report our experience with 33 rectus abdominis flaps, 29 based on the superior epigastric artery and 4 based on the ingerior epigastric artery. the flap is easily elevated, no skin loss has occurred and primary closure of the donor defect has been achieved in every case. in a follow-up period of between 1 month and 15 months no patient has developed an incisional hernia. the flaps have been used to cover extensive chest wall resection for recurrent carcinoma of the breast (6 patients), extensive radionecrosis of the chest wall (1 case) as part of a primary breast reconstructive procedure (22 patients) and to replace soft tissue overlying the femoral vessels after radical dissection of the groin for metastatic tumour (4 patients). of the 22 patients undergoing primary reconstruction 20 required additional subpectoral prosthesis but in 2 adequate bulk was provided by the flap itself. primary healing occurred in all cases. this easily raised and reliable flap will shorten hospital stay after major ablative surgery for recurrent disease and provides a useful addition to methods of breast reconstruction. a. watson department of surgery, royal lancester in girmary, lancaster, u.k. occult gastro-intenstinal bleeding which defies readiological and endoscopic diagnosis provides one of the greatest diagnostic challenges in surgery. lesions producing this clinical situation are often very small by definition and not infrequently mucosal angiodysplastic lesions, localisation of which may be extremely difficult pre-operatively and indeed at laparotomy. various methods have been described in an attempt to improve pre-operative localisation of such lesions. string tests an dthe detection of sicr labelled red cells in the faeces are notoriously inaccurate. arterioghraphy is invasive and usually necessitates a bleeding rate in excess of 500 ml per day. scintiscanning after red cell tagging with 99mtc gastro-intestinal blood loss, but localisation is difficult. a method oflocalisation of such lesions using slcr labelled red cells and intestinal intubation is described. after labelling of the red cells, a miller-abbot intestinal tube with the balloon inflated and rendered radio-opaque is passed and samples of gastrointestinal secretions aspirated at each 5 cm during passage down the gastro-intestinal tract. samples are counted on a well scintillator counter and when a sample of high radioactivity is obtained, localisation is assessed both by the length of tube passed and by instilling dilute barium down the miller-abbot tube underfluoroscopic control. this method has been used successfully in five patients which were subsequently treated surgically with localised resection resulting in cessation of bleeding. case histories together with photographs of the method and respected specimens will be presented in poster form. postoperative sepsis is tlae most frequent complication of surgery and is the commponest cause ofprolungation of hospital stay. purpose of the study is to prospectively evaluate incidence predisposing factors, bacteriology and costs of postoperative infections. 758 consecutive surgical patients admitted to our institute from may 79 to july 81 were studied. patients undergoing minor surgical procedures (wound less than 2 cm) were excluded from the study. patients were evaluated daily during hospital stay for onset of infection and results recorded in a data sheet. hemocultures in septic patients and free plasma, activated partial thromboplastin time, prothrombin time, and thrombin time, ristocetin test for soluble monomer complexes and fibrin degradation products (fdp, welco test) euglobulin lysis time (elt) and platelet counts. conclusion: 1. thrombocytopenia is not a typical feature of boomslang coagulopathy. 2. the demonstration of soluble monomer complexes is blood samples (positive ristocetin test) appears to be an early and consistent indication of intravascular coagulation. 3. despite unequivocal evidence of advanced consumption, platelet function seems to be maintained, thus preventing complete heamostatic failure. 4. support for this view is found in the clinical observation that bleeding is essentially mild and late in onset. 5. thrombelastographic hyperretractility the "spinning top" appearance is a constant feature and is probably mediated via the platelets. 6. although d. (vpus venom is extremely potent and often fatal, the antivenom is rapidly effective despite advanced consumption. this mechanism which may involve the platelet release reaction has not been fully elucidated and deserves detailed study. in most cases artificial ventricles are driven pneumatically with the advantage of easy handling and the disadvantages of regulation problems, high weight and volume of the driving units and the danger of air embolism in the case of membrane rupture. the driving unit developed at innsbruck universiy consists of a microprocessor-controlled electromagnet driving a pump that functions as a safety chamber (sk). force transmission to the artificial ventricle (ellipsoid heart-eh) is effected hydraulically. thus there is a fixed connection between armature stroke and membrane movement in the eh. the armature position is measured by the microprocessor by means of a position sensor with programmable switch-over points determining the change from systole to diastole so that idling and beat volumes, respectively, of the eh can be programmed. the sk is a newly designed double rolling membrane pump for pressure and suction with very low compliance. pressure and suction are determined by the armature force which is proportional to the microprocessor-controlled current in the solenoids. thus a very positive control of the pumping action is possible. in mock circulation experiments with the hydraulic safety driving unit the cardiac output (co) was between 21/min and 121/min at beat frequencies of 40 to 130 bpm. with constant piston-stroke a direct relation between frequency and co was observed. the influence of preload (starling's law) and afterload decreases with increasing armature force (decreasing periods of systole and diastole, respectively) which is due to friction in the hydraulic transmission system. improvements in the geometry of the next system should reduce these losses. in vivo experiments confirmed the hemodynamic efficiency of the system. applied as lvad the system proved to relieve the beating heart considerably. in the case of heart fibrilation the circulation could be maintained by the safety driving unit. the variable height differences between driving unit and eh as they occur in long-term experiments (animal lying or standing) affect only the ratio systole/diastole periods with the co remaining constant. mechanically assisted circulation is indicated in patients with cardiac failure after open heart operations. the clinical experiments show that left ventricular assist devices are capable only in a few cases to maintain full circulation. the limiting factor is the additional right heart failure syndrome. in patients with postoperative cardiac failure, a distinction must be made between isolated left heart failure and total heart failure. in patients with total heart failurebased on diffuse coronary sclerosis or on an increased pulmonary resistance -left ventricular assistance alone is not effective and right ventricular support is desired. therefore, a simple, quick and safe biventricular assist device is necessary. with the biventricular bypass it is possible to maintain complete circulation in cases of cardiac insufficiency. for a successful outcome in patients with low cardiac output after cardiopulmonary bypass, the e-bvad was evaluated in animal experiments (with 9 calves in 4 acute and 5 survival experiments) in cardiac failure situations. the cannulas for the inflow are put into the left and right ventricle, the outflow tracts into the descending aorta and the pulmonary artery. both parts are connected with the ellipsoid hearts. with the e-bvad it is possible to have a replacement of the heart -a total heart assistance similar to the total artificial heart. in cases of clinical emergency this device can be recommended because of the satisfactory hemodynamic effects achieved and the small degree of traumatic hemolysis (1,8 mg% free hemoglobin). it represents an easy and quick implantable system for total functional heart replacement. the realtionship between acute pancreatitis (ap) and the enzyme and hormone level in blood and gastric and duodenal juices is the factor that had driven us to do this experiment that would complete the study of the physiopathology of this illness. material and methods. we used eight 5-year-old dogs. a) production of two antiperistaltic fistula in the mann and bullman way; one gastric and the other duodenal (first part). 1. blockage of the gastric and duodenal compartments: by the gastric fstula we introduced one foley's bucket with closed point and inflatable globe to block the pylorus. by means of the duodenal fistula we introduced another foley's bucket into the duodenum. once inflated, the ball blocks the first duodenal portion. 2. juice extraction: by adjacent openings in the gastric bucket blocking the pylorum, we extracted the gastric juice separately. with another thin foley's bucket, that was introduced by the duodenal fistula near the already blocked one, to block the third part of duodenum when the globes inflates. b) production of ap: 15 days after the first surgical operation, by the morandeira method with intraparenchymal injection of a mixture of autologous bile and olive oil. results. one of the animals died on the fifth day after the first operation. + no animal died spontaneously after the second operation. they were killed on the 4th day. in each one acute necrotizing pancreatitis could be verified. + the radiograph showed that the gastric and duodenal compartments were sealed. -it was easy to separately extract blood and gastric and duodenal juices. conclusion. we believe that this method is complete, simple, with good results and very useful for the study of the physiopathology of ap. liver samples were taken form 18 patients operated for cholelithiasis and common bile duct obstruction by gallstones or pancreatic tumour. early evident histoenzymatic deficiency was found even without jaundice or liver structural lesions. in the bilio-obstructive jaundice, the histochemical methods revealed more marked liver impairment, as compared to the histological picture. the degree of the enzymatic depression could explain the occurence of the postoperative liver failure in some patients. experimentally, the effect of common bile duct ligation was studied in 80 rats. group i = 10 healthy controls. group ii = aspartate (1 ml 5% sol.) was injected i.p. in 10 rats daily, for a week. group iii = the common bile duct was ligated under other anes-thesia, in 20 rats 48 h before killing, and group iv=in another 20 rats 7 days before killing. group iv =in 20 rats aspartate was administered 3 h before an twice after choledocus ligation, and group vi = in another 20 rats aspartate was injected daily for a week after surgery. the liver slices frozen in liquid nitrogen were cut in a slee-type cryostat and the histochemical reactions were performed according to arnold, chayen-bitensky and lojda-gossrau-schiebler's methods. the biochemical reactions were performed using merckotests and merz-dade test-kits. after 48 h, and still more after 7 days, very severe structural, histochemical and enzymatic lesions developed. the liver cell glycogen and rna, as well as the "marker" enzymes of infrastructures markedly diminished: nach2-tetrazolium reductase -21.33%, glucose-6-phosphatase -48.33%, alphanaphthylacetat esterase -31.33%, atp-ase -71.0%, 5'-nucleotidase -38.67%, pas-reaction -56.0%, mgp-staining -30.67%. aspartate treatment seems to exert a protecting effect against the noxious action of retained bilirubin and conjugated bile salts upon the liver cells: nadh2-tetrazolium reductase +17.37%, gsp-ase +40.64%, esterase + 24.27%, atp-ase +67.81%, 5'n-ase +41.85%, pas + 25.05%, mgp +29.32% and lipids -33.12%. but it does not influence the biochemical signs ofcholestasis: bilirubinemia, alkaline phosphatase, leucinearylamidase, gamma-glutamyltransferase, lactate dehydrogenase. aspartate prevented partially but significantly only the increase of cytolysis enzymes: asat -40.48% and alat -52.97%. the histochemical and enzymatic results are in agreement with the severity ofmorphologic changes. therefore, aspartate treatment might be adequate for the preoperative improvement of the liver function in cases of obstructive-jaundice, in order to reduce the incidence of liver failure, without influencing cholestasis. oral glucose tolerance tests (ogtt) y. yamoaka, a. sugitani, ic kimura, ic ozawa and y. tobe department of surgery, kyoto university medical school, sakyo-ku, kyoto, 606 japan two patients with cholecystographic evidence of septate gallbladder underwent dynamic hepatobiliary radionuclide scanning with 99mtc-ehida. when a steady state of emission had been reached from both gallbladder lobes, cholecystokinin was infused incrementally in four doses (0.005, 0.01, 0.03 and 0.06 ivy-dog-units kg-lmin -1) and counts from the gallbladder analysed by computer. in each an obvious functional difference was revealed between the lobes: both distal lobes ceased emptying abruptly during the third hormone infusion whereas the proximal lobes continued to empty predictably [2] . in one case, histology revealed the septum to be a well-developed smooth muscle ring with cholecystitis glandularis proliferans confined to the distal lobe; the other case awaits surgery. the cause of the difference in response was probably contraction of the muscle in the septum acting as a sphincter. this study provides indirect confirmation that pain in septate gallbladder is due to septum contraction and raised distal intralobe pressure. it is known that the gallbladder stone passes into the common bile duct. furthermore, common bile duct stones pass to the duodenum. these gallstone movements were analysed in 334 cases during the past six years. the phenomenon in which gallbladder stones pass through the cystic duct down to the common bile duct are seen in cases where stones are small and numerous, the cystic duct is wide and connected with the common bile duct angularly or in parallel. in 13.75% of our cases common bile duct stones passed into the duodenum. there were neither inflammatory stenosis of the terminal choledouchous, high grade pappilitis nor severe obstruction of the common bile duct in these cases. it appears that the movement of gall stone is related to size and number of stone and morphological variations in the biliary system. the study was conducted in order to investigate whether intraoperative mano~etry of the bile ducts could be of additional valtie in diagnosis of afflictions of biliary tract or of the papilla. in contrast to current methods of water manometry it seemed important to the authors to use a simple and safe method which could deliver precise results. method: electromanometrics studies were carried out with a cannula placed in the common duct. three manometric parameters were obtained for evaluation. 1. resting pressure (rp); 2. pressure increase after injection of 10 ml saline (lml/s) (pi); 3. time in seconds needed for return of initial pressure (bile flow) (tr). for pressure measurements a pressure transducer with a recorder was used. results: (table) manometry results in patients with calculi in the common duct or with organic stenosis of the papillawere significantly changed as compared with normal findings in the bile ducts. false positive results (proven by cholangiography and biopsies of the papilla) were found in 3.5% of cases, false negatives in 6 % 97 % of all patients with patho-histological changes in the papilla had pathological bile pres-. sures. although false positive and negative results were mainly caused by methodical errors, the described method delivers a high percentage of correct results in agreement with the final diagnosis. it is avery sensitive method in indicating impaired and reduced bile flow and discovers early pathological changes in the papilla as proven by histological examinations. a functional anhepatic state in experimental animals for biochemical studies and as a model for treatment of acute liver failure can be achieved by different surgical procedures like portocaval shunt and complete arterial devascularization or by replacement of the liver with vascular prosthesis and protocaval shunt. we developed a simple hepatectomy procedure using t/4 inch silastic tubing, carbon-y-connector and specially designed "vascular spirales" to restore normal portal and caval blood flow. this model was used for auxiliary liver perfusion studies in heparinized animals; the operation takes 15 min, requires no surgical skill, vascular occlusion is less than 1 min, no signs ofsplanchic hypertension are present and blood loss in fully heparinized animals over 24 h is insignificant. one of the main problems in atypical and anatomical liver resections consists of achieving appropriate hemostasis. also because bile capillaries are opened when liver tissue is separated there is the danger of infection and subphrenic or parahepatic abscesses. the authors have used the human fibrinogen clue to seal the surface of the resected liver in 7 cases (3 hemihepatectomies and 4 resections). even in anatomical resections exists, in the majority of cases, a diffuse bleeding or oozing of the resected area. this can be completely controlled by sealing the surface with the fihrinogen adhesive. also one of the main advantages of the fibrinogen consists of having the possibility to avoid the insertion of numerous tubes for drainage. all our cases were drained by a single silicon tube. the postoperative courses were uneventful in all cases no major complications were observed. liver regeneration is thought to be stimulated by changes in portal blood constituents, or flow or by a substance in the hepatocytes. rice et al. have documented increases in total hepatic perfusion in rats during liver regeneration. this study has measured the portal and arterial components sequentially in large animals. young pigs (6-8 weeks) were subjected to shamoperation or 50% partial hepatectomy (ph). blood flow in the portal vein or hepatic artery were measured pre-operatively and at intervals of 24 h postoperatively using cuff probes of an s.e.m. electromagnetic flowmeter; these were positioned daily under light anaesthesia. systemic arterial and portal venous pressures were measured via indwelling catheters. previous studies have shown that the peak of regenerative response occurs in pigs 3 to 4 days after ph. the mean pre-operative total liver blood flow increased from 1.02+0.36 (s.e.m.) ml.g-lmin-1 to a peak of 226+ 33 % on day 2. thereafter flow declined towards normal by day 6. the pre-operative portal component was 71 ___3 %; this increased to 85+2.5 % on day 2 and returned to normal level within 4 to 6 days. it seems that both total hepatic flow and the portal component increase markedly just preceding the peak of regeneration. this response may stimulate regeneration, or merely accompany it, or may be proyoked by similar stimulators. the analysis of individual bile acids is relevant to several clinical investigations although established techniques have a number of disadvantages. to overcome these we have developed a simple hplc method using a waters associates rcm100 radial compression module with a radial-pak c18, 10,u, 8mm id, reverse-phase cartridge (column). bile acids were eluted from the column at a flow of 2.0 ml min-1 with a mobile phase of methanol:water (75:25 v/v) containing 2.5 % (v/v) acetic acid and adjusted to ph 5.25 with 10 m naoh. a mixture of standards of cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids and their glycine and taurine conjugates were resolved, while the 10 conjugates alone were completely separated in under 20 rain. the technique has been applied to the study of human bile from the common duct, gallbladder, tube and duodenal fluid, and serum from patients with hepatobiliary disorders. bile acids in these samples were rapidly extracted using a sep-pak c18 cartridge before being applied (10/a to 200pl) to the hplc system, although bile could be analysed directly without extraction. quantitation (~mol ml-1 sample) of separated bile acids was achieved by comparison with standards using an on-line integrating computer and less than 5 nmol could be detected using a refractive index detector. acute hepatic failure induced by total liver devascularization in pigs -amino acid uptake by combined charcoal -resin hemoperfusion support system where losses averaged 38 %. jib patients had progressively greater losses with increasing preoperative weight (/250 lbs-30 %, 250-299 lbs-35%, 300-349 lbs-38 %, 350-399 lbs-43 %, 1400 lbs-50 %). weight loss in patients dbs was statistically and clinically greater with jib. 2. diabetics, especially insulin-dependent, were rapidly cured after jib. normal plasma glucoses, serum insulins, and oral glucose tolerance curves were usually seen within l postoperative mouth, unrelated to weight loss. all 13 patients requiring insulin or oral medication preoperatively could discontinue mediation usuallywithin a weekpostoperatively. improvement after gb was gradual, appeared related to weight loss, and was often incomplete. 3. hypercholesterolemic patients had 51% decreases, from 275 to 134 mg/dl, after jib, but only 19 % decreases after gb. all serum cholesterols were normal after jib, but 23 % remained elevated after gb. because of complications after jib, some needed reoperation and conversion to gb. fortunately, most benefits were retained after conversion to gb. we suggest considering jib for "superobese", diabetic and hypercholesterolemic patients. in the past 9 years 140jenunoileal bypass procedures were performed. the most common complications and side effects were frequent abdominal pain, or discomfort and flatulence in almost half of the cases. in addition kidney stones, blind loop invagination, electrolyte and liver dysfunction problems could be observed. the over all complication rate was 20%, the postoperative mortality rate 2 %. to eliminate the blind loop of the small intestine, to maintain enterohepatic circulation of bile, and to diminish undesirable side effects in the conventional jejunoileal bypass, biliointestinal bypass was introduced in 1980. twenty patients with a mean weight of 128 kg were subjected to primary biliointestinal bypass within the last two and a half years. three additional patients had secondary biliointestinal bypass due to side effects, especially diarrhea and flatulence, of jejunoileal bypass, performed two to four years previously. the surgical procedure entailed establishment of an end-to-side jejunoileostomy. 25 cm of the jejunum and 25 cm of the ileum were left in the continuity. the blind loop of the jejunum was anastomosed to a functioning gallbladder. so far the above mentioned complications of conventional jejunoileal bypass could be remarkebly diminished. there have been no deaths in this material and no metabolic side effects. frequent diarrhea is avoided by reduced bile spill-over to the colon. the weight reduction has been satisfactory. in summary: due to a lesser complication rate biliointestinal bypass seems to be superior to the simple jejunoileostomy in the treatment of morbid obesity. hyperplasia or neoplasia g.w. geelhoed, c.j. schaeffer and p. daudu department of surgery, george washington university medical center, washington, usa patients with various thyroid disorders who were undergoing thyroid operation were studied for the presence of absence of estradiol and progesteronebinding proteins in thyroid tissue. steroid receptor assays were carried out using similar techniques and standards as those routinely employed for study of breast cancer specimens. quantitative data were collected by coded specimen number by an observer unaware of the patients' clinical diagnoses, and diagnostic correltions were drawn following de-coding. there was no correlation with age or sex and the presence or quantitative value of steroid-binding site. specimens with the histologic diagnosis of follicular adenoma had estradiol binding sites, and had them at high levels (14.19 to 143.83) femtomoles/mg cystosol protein). patients with the histologic diagnosis of thyroid hyperplasia had marginally positive estradiol-binding and lacked progesterone binding. patients with adenocarcinoma of the thyroid exhibited neither estradiol nor progesterone-binding in significant quantity. presence or absence of steroid binding sites in thyroid tissue appears independent of sex or age, but correlates with benign neoplasia of the thyroid, suggesting a possible etiologic association for thyroid adenomas. 77 patients with esophageal cancer underwent resection and primary reconstruction of the esophagus by posterior invagination esophagogastrostomy which we devised. the anastomosis was made in the cervical level in 58 cases and in the left thoracic cavity in 19 cases. functions of stomach placed in the posterior mediastinum were examined in 7 patients surviving more than 5 years and in 13 patients surviving less than 3 years. within one year postoperatively, the absorption ofvitamine b12 decreased markedly. one and a half year after operation, however, the secretion of castle's instrinsic factor recovered and it showed normal values in patients surviving over 5 years. in secretion of gastric acid by tetragastrin, both total acidity and free hydrochloric acid increased in the progress of the postoperative period and showed normal values in all 4 patients surviving over 9 years. this fact was also confirmed by endoscopic observation of color development by congo-red in the gastric mucosa; its coloring area was scattered in 2 patients surviving 5 years, but extended to the whole surface in patients surviving over 9 years. roentgenographycally in head-down position, the surgically created fornix with a sharp angle of his was effective in preventing gastroesophageal reflux completely. the strain combination of donor and recipients. the nature of the mechanisms determing the different fate of allografts remains obscure and all interpretations of the above findings must be speculative. however, the long-term acceptance of hepatic allografts in the rat appears to be similar to that in other species and emphasizes the role of the liver as an immunologically favoured organ. footnote: part of the work (intrahepatic bile duct proliferation!) contained in this abstract will be presented at the xvii essr meeting 1982. peoples' friendship university, tamojenii pr. 4, department of operative surgery, moscow, ussr a transplanted kidney, besides tissue incompatibility reaction, is influenced by non-specific injuring factors, including decentralisation. for normal functioning of a kidney transplant over a long period of time we carried out an experimental study. the purpose of the study was the investigation of operative reinnervation possibilities and its influence upon the functional state of a transplanted kidney. for the kidney reinnervation kirpatovski's method for suturing perivascular fascial tissue flaps of anastomosed vessels with branches of vegetative plexus nervosus on them was used. experiments were carried out on 175 mongrel dogs, both male and female. in the first group of experiments a kidney was transplanted into the pelvis while the opposite kidney was preserved or ablated. in the second group the kidney autotransplanted into pelvis was reirmerved by the described method with preserving the contralateral kidney in part of the animals and ablating the kidney in the rest of them. the third, fourth and fifth were control groups. in the third group the kidney was denerved, in the fourth it was denerved and reinnerved by the mentioned method, in the fifth group unilateral nephrectomy was performed. after the operation the animals, in different periods of time (from 3 days to 2 years), were studied by isotopic renography and by scanning kidney's by jbl-hippuran and neohydrin-hg 2°3 intravenous injections. results of the isotopic renography were estimated qualitatively and quantitatively by universal renographic index (hirakawa-corcoran, 1963). results of the experiments indicate that autotransplantation and denervation of a kidney without its reinnervation were followed by lowering of a kidney vascularisation and its secretory and excretory functions. during the first 2 or 3 months after the operation a tendency to improvement of the kidney function took place; later those functions gradually oppressed and the renographic index lowered down to 13.9+__2.1, 16.6+--.5.5 (p<0.001) respectively according to the groups. after operative reirmervation of a kidney autotransplant and denervation of a kidney without its transplantation gradual improvement of separate functions of the kidney took place: in 1 or 2 months the kidney circulation was restored; in 2 or 4, and in 4 or 6 months respectively, secretory and excretory functions of the kidney restored; renographic index reached its norm after 4 or 6 months and remained stable during 2 years (according to the groups 54.9+___5.5, 52.3-1-4.3, p>0.05 respectively). thus, the operative reirmervation of a kidney prevented depression of its functions and provided its normal functioning during a long period of time. introduction of fine needle aspiration cytology in renal transplantation gives the possibility not only to distinguish acute tabular necrosis, arterial and venous obstruction and viral infection from acute rejection, fnc also seems to allow judgement of the effectiveness of various types ofimmunosuppressive therapy during rejection episodes. method: after sterile fine needle biopsy the cytological evaluation of the aspirate was performed on cytocentrifuged smears after staining the cells by the method of may-grtinwald-giemsa. thus normal and "activated" mononuclear cell populations as well as parenchymal cells such as tabular and endothelial cells and their pathological changes could easily be recognized. the white blood cell types in fnc were compared with those of the peripheral blood. the difference in number due to the graft invading cells was expressed as "increment". the findings of fnc were correlated with clinical signs and serum creatinine values. results: fnc allows to judge the in situ inflammation in the rejecting kidneys within 2 h. non-rejection grafts show cell counts comparable to peripheral blood. with onset of rejection (grade 1) t-and bblasts and monocytes appear in the graft. with sever-ity of the rejection (grade 2) monocytes and lymphocytes as well as blood cells increase in numbers. acute rejection (grade 3) is heralded by high numbers of monocytes and macrophages. acute tubular necrosis (atn) and virus infections can be distinguished from rejection episodes to be treated. in all cortisone and azathioprine resistant cases where alg was used in order to suppress inflammation during rejection episodes it reduced not only the peripheral white blood cells, but also within 12 h after start of alg therapy the number of in situ cells. beside reduction of inflammatory cells typical changes in shape of the nucleus and density of chromatine in up to 10 % of the lymphocytes and granulocytes could be detected. these changes look closest to what has been described as apoptoses. conclusion: cortisone and azathioprine resistant rejection episodes could be monitored within 12-24 h using fine needle aspiration cytology. this method is safe, cheep, and it provides good information about the in situ situation of the graft, it allows to distinguish acute rejection from atn and other situations where higher immuno-suppressive therapy, especially with cortisone, could only be harmful for the patient. criteria of the border of normothermic ischemic tolerance in dog kidneys are first normal pah-and exogenic creatinine-clearances compared with unilateral nephrectomized dogs in neuroleptic analgesia under excessive water and sodium load and second perfectly normal kidneys after two weeks in pathological examination. the protective solution htk ® by bretschneider has a superior buffering capacity compared to euro-collins-solution ® , which, by itself, enhances anaerobic glycolysis by substrate stimulation (glucose) especially between 15 ° and 35°c. without preservation in normothermia oligo-anuric arf is observed at the border of ischemic tolerance (15-20 min; 36°c). the obligo-anuric arf is dependend on ischemia which will result in necrosis of the kidney after 45 rain and 36°c ischemic temperature. the border ofischemic tolerance corresponds to an intrarenal ph of less than 6.2 (outer stripe of medulla), and medullary lactate levels of 60 to 80 pmol/gdw. substrate stimulation of anaerobic glycolysis limits the effectiveness of euro-collins-solution ® above 15°c earlier than anaerobic glycolysis in pure ischemia. after warm ischemia (>--__..27°c; 60 min) all kidneys protected with euro-collins-solution ® show anuric arf, whereas all htk®-protected kidneys (60 to 240 min of warm ischemia; 32°c) are having polyuric arf. under these conditions, renal ph will not increase above 250 nmol/1 h+-concentra -tion. the border of ischemic tolerance for htk ®protection ofkidneys is 120rain-31°c and seems to be limited by a transitory secondary postischemic oliguria (3-48 h). in summary: intrarenal anaerobic glycolysis limits tolerance of pure warm ischemia by inducing anuric arf. in contrast polyuric renal failure is observed at the border ofischemic tolerance by using the protective procedure with the htk®-solution mentioned above. at least 60 % of unrelated pigs respond to liver allografting with a rejection episode which they overcome without immunosuppression; kidney transplants are rejected uniformly within 9 to 14 days. in this study, mlc responses were measured at weekly intervals in such animals. non-litter mate pigs were subjected to autograft, exchange liver allograft, or exchange renal allograft. blood from mlc responses were taken from unaesthetised pigs before and at weekly intervals after operemained the same for each pair of transplants. in 5 of 8 pairs of liver allografts, mlc responses were depressed up to 10 fold for 3 weeks post-operatively. in all these pigs, the mlc responses were initially high. in 2 pairs where the mlc response was initially low, there was no change after the transplant, and in one pair with a low pre-operative response, there was a marked increase post-operatively. in 3 pairs of kidney allografted pigs, there was a similar depression in the single post-operative sample available. in 3 liver autografted pigs, there was a slight post-operative rise in mlc response. it appears that liver and kidney transplantation but not hepatic autografting, markedly depress sequential post-operative mlc responses. the process of ischaemic kidney degeneration was estimated by measuring the adenine nucleotide (atp)levels and the effect of inosine and naftidrofuryl (praxilene) was assessed. 50 min ischaemia was induced on both dissected kidneys of wistar male rats. then, either both clamps were removed and the right kidney was excised after i0 rain reperfusion the role of mononuclear phagocytes in various disease states has been extensively studied by phagocytosis, fc and complement receptor sites, and enzyme content. chemotaxis of peripheral blood mononuclear phagocytes, however, has not been studied to any great extent. we have therefore investigated a method for quantifying chemotaxis by mononuclear phagocytes. mononuclear phagocytes were purified from peripheral blood by centrifugation over ficoll-hypaque and a discontinuous percol gradient. chemotaxis was quantified by the 'under-agarose method'. mononuclear phagocytes were allowed to migrate towards the chemotactic agent zymosan activated serum (zas), and the control non-chemotactic agent eagles medium. the distance of cell migration was measured after 20 hours incubation at 37°c in 5 % co2 with the aid of a microscope eyepiece graftcule. using non-sepcific esterase staining the purity of mononuclear phagocytes obtained was 85%___ 10% and the viability by trypan blue dye exclusion was at least 99 % preliminary results have shown selective migration by purified human peripheral mononuclear phagocytes towards zas. this method may therefore represent a means of investigating an important role of these cells in disease states. the intrathoracic pressure rises when a person exhales into a manometer to such an extent that finally the venous blood-flow to the heart stops. it is supposed that this venous stop flow pressure (vsfp), measured by an ultrasound device, is equal to the central venous pressure. in a prospective clinical trial by two independant examiners in 97 % of the cases (n= 100) the correlation was within 5 cm h20. therefore, the central venous pressure (cvp) can be measured non-invasively with an ultrasound device. we have previously reported that opiate receptor blockade with naloxone (nal) significantly improves cardiovascular function and survival in canine hemorrhagic shock [1, 2] . since species differences do exist, we investigated the effects of nal in cynomulgus monkeys anesthetized with n20/o2. blood was withdrawn to achieve a mean arterial pressure (map) of 45 mmhg (t---0) which was maintained until t= 1 h when the reservoir was clamped and the animals treated with either nal at 2 mg/kg bolus plus 2 mg/kg.h infusion i.v. or 0.9% nac1 in equivalent volumes. there were no significant differences between nal (n=5) and con (n=5) in map, left ventricular contractility (lv dp/dt max, mmhg.10vs), and survival; the nal animals, however, were acidotic (pha 7.24-----0.09) and colder (37.95-0.3°c) than con (7.42+__0.02, 38.5-+-0.1). map responses to nal were proportional to ph a complications of vascular bypass grafts, especiallyin the femoro-popliteal region are common. the most frequent of these by far is occlusion. other late complications iclude leaking and false aneurysm formation at the anastomotic site, dilatation of the graft material and stenosis ofanastomotic sites. the most commonly employed non-invasive studies render little information of value in the diagnosis of these complications. we have recently undertaken a study to evaluate arterial grafts at various intervals after implantation with the use of a duplex ultrasonic scanner. imaging of graft material with this system ist excellent and patency of the lumen can easily be established. graft diameter can be measured and accumulation of material within the lumen can be measured and quantitated. although anastomoses are not always clearly visualised they are often seen satisfactorily for diagnostic purposes. graft dilatation, false aneurysm formation and occlusions can be accurately diagnosed with this method. we have also studied the behaviour of femoro-popliteal grafts across the knee joint using the duplex scanner. as a non-invasive technique scanning with a duplex system has many advantages enabling frequent investigations and therefore early recognition of complications where synthetic material vascular grafts have been used for arterial bypass. an experimental model for dissecting aortic aneurysm has been designed to obtain much better understanding of the disease, leading to more effective methods of surgical treatment. dissection of the descending thoracic aorta was successfully performed by modified blanton's prodedure in 85 % of more than 100 mongrel dogs. fals lumens ruptured distally into true lumens to form a doublebarreled aorta in 70% of the dogs. the method of surgical treatment performed were (1) closure of entry by direct suture, (2) closure of entry by inserting dacron vascular prosthesis with a stainless steel ring, and (3) bypass grafting with vascular prosthesis and ligation of the thoracic aorta. in 10 dogs treated by methods (1) or (2) at the time of performing the dissection, cine angiography revealed that false lumens hat thrombosed within 1 month and dissection was found to have completely healed on autopsy. in 10 chronic cases treated by methods (1) or (3), false lumens were all patent with various degrees of formation of mural thrombi at observation of 3 to 6 months after surgical treatment. these results indicate that closure of entry for acute dissecting aneurysm should be a curative procedure. studies are now continuing on chronic dissecting aneurysm. the lack of controlled trials not rarely resulted in the incorrect acceptance o fan ineffective operation as an effective one 0igation of internal mammary arteries for relief of angina pectoris, for example). this failure also explains the non-stop controversies over the appropriate operation (and whether the question operate at all) for various myocardial revascularization procedures, over the thymectomy for myasthenia gravis, the limited vs radical mastectomy for breast cancer etc. several clinicians and surgeons assert that controlled clinical trials for new operations are unrealistic and naive because of some important differences between operations and drugs. compared with medical trials surgical ones are often really more difficult to be carded out. they are subject to more restrictions, ethical, statistical and practical, and may require longer or even remarkably long times. in any case, patients must nevertheless be sheltered from harmful and ineffective surgical operations since luckily not all the undoubted difficulties are unsurmountable, and prospective controlled trials of surgery, including random allocation of patients, are quite feasible. comparison between surgical and non-surgical treatment is a really particularly suitable field for these studies. comparison of effectiveness of two operations of the same type or of different types can also be carried out, but only on certain conditions and using suitable and appropriate expedients. also the doubleblind approach may be feasible, although remarkable restrictions and adequate cooperation are necessary. acceptance in trials of surgery as placebo is objectively very difficult, and obviously it has to be considered only when an effective operation for the considered disease does not exist. the ethical sound of this delicate aspect of clinical research can be minimized enough only if (1) there are substantial doubts about the effectiveness of the intended operation and (2) requested placebo-surgery is of very slight importance. posters for scientific meetings mary evans and a.v. pollock scarborough hospital, north yorkshire y012 6ql1, u.k. the display of information in posters antecedes even the,art of writing. they have been used to advertise, to educate and to inform and their function is to disseminate information to a wide audience quickly, simply and effectively. in medicine they have been used since the thirteenth century for the promotion of health education. in recent years poster sessions have been introduced into scientific meetings as an alternative to the spoken paper for the presentation of original work. following this experimental procedure hepatic coma supervenes in 6-8 h and animals die after ca. 24 h. five male pigs, aged 3-4 months, weighing 30-35 kg were studied ca. 20 h after operation. the detoxifying system consisted of activated charcoal (norit ¢ (lmm x 2ram) and ionexchange resin (dowex lx2), subsequently inserted in an extracorporeal circulation. plasma amino acids were determined serially (3, 20, 40, 60 rain) in and out the detoxifying system. plasma extraction of individual amino acids (means_ se in/zmol/min) is reported in the table a remarkable extraction was present in the first 40 rain and chiefly involved aromatic amino acids and tryptophan which did not further increase. during hemoperfusion, the molar ratios between neutral amino acids improved. the ratio of branched-chain to aromatic amino acids increased from 1 late hepatic effects of small intestinal bypass (sibp) for obesity plasmafl-endorphin (/3-end, pg/ml, by ria) was related to t:fl-end = 334 (t-37) + 597, r=0.67, p<0.05. when acid-base balance and temperature were maintained, nal (n=6) significantly (*p<0.05) improved map and lv dp pg/ml in nal and 969 + 94 pg/ml in con). r-end is released in and contributes to the cardiovascular depression of primate hemorrhagic shock. nal reverses this depression and improves survival but only when arterial ph and core temperature samples of exudates at site of infection were taken wherever possible for aerobic and anaerobic cultures. in following infections were recorded: wound, respiratory tract, thrombophlebitis, indwelling intravenous catheter, unidentified origin fever (>38 °c lasting more than 3 days) (fuo) and miscellaneous. incidences: 310 patients (41%) had postoperative septic complications. wound infection was recorded in 145 patients (19.1%), respiratory tract infection in 105 (13 %), urinary tract infection in 100 (13 %), fuo in 55 (7.2%) thrombophlebitis in 18 (2.3 %) and miscellaneous infections in 49 (6.4o/0). predisposing factors: wound infections were 53/413 (12.8 %) in clean operations, 41/215 (22.7 %) in potentially contaminated, 19/65 (29%) in contaminated and 24/65 (36.9o/0) in dirty.wound infections were diagnosed later (mean 9th day) in clean than in dirty operations (mean 5th day) (p<0.05). a statistically significant correlation was found between wound infection and leght of preoperative hospital stay: from 5.4% in patients operated on within 0-6 days to 42.8% for patients operated after 28 days or more (p<0.001). no correlation was found between wound infection and age. urinary infections were more frequent when the patients were catheterized at least once in the postoperative period (34 % vs 11%). a statistically significant correlation was found between the incidence of respiratory infections and duration of anaesthesia (3.2 %----<60 min, 120/0>60<120 min, 270/0>120 min;/~0.05). bacterology: 181 out of 295 cultures gave positive results (61%): aerobes were isolated in 129 samples (490/0); mixed aerobes and anaerobe in 29 (9.8o/0); anaerobes in 23 (7.7 %). bacteroides fr. was the commonest isolated anaerobe in all types of sample except hemocultures were propionibacterium aches was the most frequent. a statistically significant correlation was found between the incidence of recvery of anaerobes and intraoperative contamination (7.4 % in clean operations, 13.5 % in potentially contaminated, 12.5% in contaminated and 20.8% in dirty; p<0.05).in wound infections the most frequent aerobes were staphylococcus in clean and e. coli in contaiminated operations. costs: mean postoperative hospital stay of patients with septic complications was 15 days, whereas patients with no postoperative sepsis were discharged after an average of 10 days (p<0.05). mean daily cost in our hospital was extimated $100; accordingly, the mean postoperative hospital stay of patients with sepsis costs $1500 vs $1000 of that o~ the patients without postoperative infections. overwhelming postsplenectomy sepsis/infection has been accepted the highest risk with more than 50 percent mortality due to pneumococcus species. however, e. coli, pseudomonas and staph. aureus have been reported a deadful threat to the splenectomized individual also. pneumococcal challenge after splenectomy in animal experiment and after partial salvage has been well examined. staph. aureus-challenge has not been tried after splenic repair, so far. material and method: a total of 150 nmri-mice (18-20 g) have been subjected to: 1 sham-operation, 2 splenectomy, 3 peritoneal splenosis, 4 controls as non-operated group.the technique was similar to the one performed in 90 rabbits, prior reported. 6 weeks postoperatively, the mice were exposed to intraperitoneal injection of staph.aureus concentrations of 101°c/ml down to 104c/ml. the peritoneal cavity of mice has a high resistance to staph.aureus, as only no. 36835 proved lethal. results: whereas the macroscopic view post mortem seemed promissing, showing almost total salvage of the splenic particles 6 weeks postoperatively bacterial challenge with staph.aureus was contradictory: 3 x 10 s c/ml was survived by all splenectomized mice, by only 25percent of the splenosis micecand by none of the sham and nonoperated mice. due to the small number of only 48 mice within this mice-pathologenic staph. aureus species, we may not draw definite conclusions. as for one, the intraperitoneal application may not be the natural way of septicemia. on the other hand, splenic remnants may not be as effective in the protection of staph.aureus-sepsis as in a pneumococcal challenge. therefore, further studies with staph.aureus species in other animals and by other application route will be needed! studies of the coagulant effects of boomslang (dispholidus typus) venom have indicated that the coagulant effect was mainly due to its ability to activate prothrombin. it also activates prethrombin i, factor x and possible factor ix as well [1] . although boomslang envenomation is said to represent a classic model of disseminated intravascular coagulation, certain features are worthy of critical thought. the coagulation profile of a nine year old lad, who was admitted to the h.f. verwoerd-hospital, 48 h after a reputed boomslang bite, provided the impetus to explore the in vivo effect ofcurde. d. typusvenom on the thrombelastographic and other haemostatic changes in the chacma beboon. methods: 0.0005 mg, 0.0015rag and 0.05mg respectively of crude. d. typus venom were injected subcutaneously in 3 adult baboons. serial determinations of the flow parameters were done over 3-6 days. thrombelastography on whole blood an platelet this report presents the ten year survey of liver mitochondria analized in 385 patients.1. two mechanisms were of major importance in the augmentation of mitochondrial ability to synthesize atp: a) the enhancement of atp-generating capacity per unit of respiratory assemblies and b) the increase in respiratory enzyme contents. those compensatory mechanisms were functional within a certain range of contents in cytochrome a (0.5-1.5 nmol/mg protein, normal; 0.8). hepatic insufficiency was observed in patients with cytochrome a contents less than 0.5 or more than 1.5.2. in all patients with cytochrome a of 0.7-1.0 nmol/mg, the blood glucose level after an oral glucose load (50g) returned toward normal within 2 h (parabolic pattern). in 25 patients with cytochrome a of more than 1.5, the blood glucose level did not return toward normal within 2 h (linear pattern). parabolic and linear patterns were intermingled in the patients with cytochrome a form 1.0 to 1.5.in this report, we will emphasize the importance of preoperative ogtt and measurement of cytochrome a contents during operation for the prediction of operative prognosis, better than any other currently used index of liver function, in the patients receiving major surgery such as hepatectomy or operation for esophageal varices in severe cirrhosis. the reticulo-endothelial system (res) has largely been ignored in studies of liver regeneration. in this study, the res was "blocked" with colloidal carbon, or was stimulated with olive oil or with glucan. indices of liver regeneration were the thymidine kinase acitivtiy (tk) and the number of mitotic figures (mi) in liver biopsies. fibronectin was measured as a putative index of kupffer cell function.four animals in each group were sacrified at 6, 12, 24, 36, 48, 72 and 96 h after sham-operation or 68% partial hepatectomy (ph). group 1: carbon suspension (pelikan ink, wagner) 16 mg/100 g rat single injection pre-operative; group 2: olive oil (10 % in 5 % dextrose water + 0.1% tween 20) 0.25 ml/20 g rat single injection pre-operative; group 3: as group 2 given at intervals of 24 h; group 4: glucan 1.0 rag/ 100 g rat pre-op, and at intervals of 24 h.results: glucan or a single dose of oil increased tk in ph and sham-operated rats but did not influence m.i. carbon inhibited mi but did not influence tk. fibronectin levels were increased in sham-operated and ph rats after daily oil or glucan.in conclusion, no administration enhanced both tk and mi but stimulation of the res with glucan or oil did increase fibronectin levels in both sham and ph rats. it appears that the res does not have a role in liver regeneration as assessed above, but fibronectin appears to have been an indicator of res activity. anatomical abnormalities of the gallbladder include multiseptate and bilobed organs, and the more common transverse septate variant. patients with the latter type often have typical biliary symptoms which are thought to be caused initially by raised intracystic pressure and subsequently by inflammation and calculi formation in the distal lobe [1] .this study evaluate~ the long term effects of sibp on hepatic lipid and fibrous tissue accumulation. liver was obtained at the time of abdominal operation from 6 normal weight patients, 18 morbidly obese patients (mean body weight 163+21 kg) and 27 patients 4 or more years (mean 69-+ 19 months) postoperative from sibp. from wedge liver biopsies hepatic total lipid, triglyceride, cholesterol, phospholipid and protein contents were determined as well as the activities of acetyl coa carboxylase and fatty acid synthetase. histologic evaluation of needle biopsies of the liver was used to quantitatively estimate the amount of hepatic steatosis and fibrosis present.there was also a significant histopathologic increase in hepatic fibrosis present in the liver tissue from sibp patients when compared to hepatic fibrosis in liver from obese patients as well as when compared to liver specimens obtained from the sibp patients at the time of their sibp. the effect of reversal of the sibp on hepatic fat content and fibrosis was determined by transcutaneous liver biopsy 6-22 mos. following take down of sibp. post-reversal histologic quantitation of hepatic fibrosis and inflammation was significantly decreased from pre-reversal values and the improvement was greater in patients who lost weight. patients year after sibp have persistent hepatic steatosis with hepatic fibrosis and inflammation. improvement in these parameters may be anticipated following reconstruction of the intestinal tract particularly if weight loss is maintained. the aim of this paper is to study the metabolic effects of gastric bypass in dogs. we used dogs weighing between 21 and 25 kg. the animals were divided into three groups: a) five dogs without surgical operation (control); b) six dogs with a fundoj ejunal bypass, and c) six dogs with a gastroplasty. in group b and c the exclusion was 9/10 of the stomach. in all of the animals and groups were determined: cholesterol , live r function tests, calcium, magnesium-and electrolyte levels, the body weight evolution and the apparent-digestibility coefficient (adc) of fat, protein, minerals and glucosides for a type of diet. the composition of this diet in dry substance was: fat 35.4%, protein 35.2%, glucosides 26.5% and minerals 2.9%. these analyses were determined five times during the twelve months of control. the animals were kept in individual metabolism cages which allow feces and urine to be gathered separately as well as the food consumed to be controlled. the cages were housed in a room thermoregulated at 18 °c±2. the cholesterol, calcium, magnesium and electrolyte levels were normal during the twelve months of control in the three groups. the liver function tests were normal in all groups with the exception of serum aspartate transaminase initially deteriored in group b. the body weight evolution was significantly diminished in groups b and c compared with the control group (p<0.002 and p<0.002). the results of adc expressed in % ± sd were.by 15roviding a functional gastric capacity of less than 100 ml and consequently a forced reduction in food intake the gastric bypass produces an effective loss of weigth. our results suggest that the gastric bypass can be considered as an effective and safe alternative to intestinal bypass for treatment of morbidly obese subjects who have failed nonsurgical treatment; however, a great and deeper research is necessary to discover the possible side effects of 227 gastric bypass surgery; then its ultimate benefits will be fully understood. there was no operative mortality. the percent of the excess weight lost following the two operations is shown and compared with weight lost following gastric bypass. it is concluded that: 1. complications occured more often following gastroplasty, 25.5 % compared with 10.4 % of the patients treated by gastrogastrostomy. 2. the amount of excess weith lost was greater after gastroplasty, a mean of 61% for 30 patients followed for two years, than after gastrogastrostomy, a mean of 41% for 13 patients followed for two years. 3. because of the incidence of stomal obstruction requiring reoperation following gastroplasty, 14.8 % compared with 1.4% after gastrogastrostomy, is our preferred operation. 4. to improve weight loss after gastrogastrostomy the ta-55 is now used instead of the ta-90 stapling instrument. the proximal gastric pouch is reduced in size to 20-25 ml and the stoma made smaller to 9 ram. an artifical esophagus was made of silicone rubber tube covered with a dacron mesh. a segment of thoracic esophagus of 16 dogs was replaced with this graft using three different types ofamastomosis, i.e., overlayer end-to-end anastomosis, two layer end-toend anastomosis both using a flanged tube, and monolayer end-to-end anastomosis with noflange tube. seven of 16 dogs (44%) survived more than 12 months without complications, and 4 of them more than 6 years. in 6 of 7 of the prolonged survivors, extrusion of the graft was recognized in the 3rd to 6th month after operation. esophageal stenosis increased slightly up to the 6th month after extrusion of the graft, but it did not further advance until sacrifice. in these dogs, mucosal regeneration of the neoesophagus was complete with muscle layers and mucous glands in the submucosa recognized microscopically. proximal esophagus from the replaced portion was apparently dilatated more than that of the distal portion. there was no definite difference between the anastomotic techniques with regard to complication or prognosis. these results suggest a possibility for clinical trials. paper withdrawn 171 attempts to reduce nephrovascular hypertension by surgical techniques deviating renal venous blood and renin directly to the hepatic filter are at present discouraging. experimental data with portocaval transposition are contradictory, due mostly to the use of heterogeneous biological models (mongrel dogs) and collateral circulation developement. renal to portal vein end-to-side (ets) and end-to-end (ete) anastomoses in the rat were therefore tested as possible experimental models. we observed that even after right kidney decapsulation, collateral vein circulation develops through the periureteral plexus, particularly after ets shunts, one month after surgery. nevertheless, collateral cirulation in the male rat can be prevented by ligature and cutting offthis plexus near the kidney. in the female rat instead, collateral circulation is still possible. in fact, newly formed pericapsular veins join the right kidney to the right ovaric vein and therefore preventive ligature and eradication of these vessels is also necessary. in conclusion, both models (ete-ets shunt) appear feasible in the rat, and reliable for studies dealing with nephrovascular hypertension and hepatic metabolism of renin. the handling of the exocrine system is one of the main problems inpancreas transplantation. one trial to overcome this problem consists of the occlusion of the pancreatic duct system. a the theoretical disadvantage is the induction of a secretion oedema which may lead to blood flow disturbance followed by venous thrombosis. the aim of the present experiments was to study the effect of an anti-oedematous drug (cumarin and rutin sulphate (venalot ®) on the blood flow of autologous segmental pancreatic transplantation was tested by chosing the cervical region of the dog as the site of grafting. material and methods: 10 dogs weighting 20-30 kg were used. these dogs were divided into two groups (control group, n=5; treated group, n=5, venalot ® dose: 1 rag, cumarin/kg/b.w.). the body and the left limb of the pancreas was removed, perfusion with eurocollins was started wia the splenic artery and the duct system was injected with prolamine. a distal a. v. fistula of the splenic vessels was performed. this conditioned graft was transplanted at the neck of the same dog, performing the anastomoses between the carotid and the splenic vessels. the blood flow of the pancreatic graft was measured with radioactively labelled microsperes of 15/~m immediately after, as well as 4 days after grafting. the developing oedema of the graft was estimated by weight gain, histological and electron microscopic investigation. residual exocrine function of the graft was measured by determining the serum lipase and amylase levels.results: the autotransplantation of an segmental pancreatic graft to the neck of a dog is a feasable and technically easy surgical procedure, without major venalot -treated dogs local complications. in the ® there was a higher weight increase of the graft in comparison to the controls. as tested so far there was no difference in the behaviour of the i~lood flow of the grafts in both experimental groups. there was a significant increase of serum amylase and lipase values in the venalot®-trea'ted group. conclusion: the technique of cervical segmental pancreatic transplantation in dogs is recommended in cases where immunological monitoring (aspiration cytology, biopsies) is the aim of the study (good access to needles). the technique of microspheres injection is a useful method for the examination of the blood flow in segmental pancreatic grafts in dogs. the prolamine-induced secretion-oedema of ductoccluded pancreatic grafts is resistant to venalot ®treatment. graft pretreatment has been used in various organs to prolong auograft survival. we have recently demonstrated that graft pretreatment of canine renal allografts with cyclosporin a (ca a) led to improved animal and graft survival. our present study assesses the effect ofcy a as a graft pretreatment on pancreatic islet cell allografts. pancreases were removed from unrelated donor mongrel dogs and placed in iced saline (4°c) after the collagenase digestion. the tissue fragments were washed once more and then another 5 mg cy a was added to the preparation prior to intrasplenic injection of the islet cell allografts. three groups were studied: group i (n= 15) served as pancreatectomized non-transplanted controls, group ii (n = 15) received a non-pretreated islet cell allograft after total pancreatectomy and group iii (n= 10) received a cy a graft pretreated islet cell transplant after total pancreatectomy. all animals were given minimal immunosuppression with azathioprine (5 rng/kg/day x 3, followed by 2,5 mg/kg/ day). blood glucose values were monitored to determine engraftment and subsequent rejection. hyperglycemia was considered when plasma glucose values rose above 150 mg/dl. all pancreatectomized controls (group i) became hyperglycemic by the first post-operative day. non-pretreated islet cell allografts in group ii had variable function and became hyperglycemic between one and nine days after transplantation. only five of ten animals in group iii, receiving cy a pretreated islet cells, became hyperglycemic levels greater than 90 days and one died of unknown causes immediately after transplantation. the following table presents the animal survival data for the 90 day follow-up period.although the exact mechanism of action is not known, this study indicates that cy a graft pretreatmerit can be beneficial in prolonging pancreatic islet cell allograft survival, further studies will optimize the use ofcy a in this application and hopefully contribute to the improvement of pancreatic islet cell transplantation. grafting of vascularised organs has become a standard procedure in surgical research. however, only few data of the fate oforthotopicliver transplantation in the rat are available, probably because of the difficult ~iargery involved. this communication gives an account of the outcome of 40 liver allografts and 16 liver isografts in four inbred strains of rat. the following groups of allo and isografts are formed; the survival time is given in days postoperatively.the technical details of the operative procedure of orthotopic liver transplantation in the rat are described elsewhere (eur surg res 13:236 (1981). the distribution of death among allallograft groups show 3 blocks of survivors. the first block includes animals surviving up to 30 days. acute rejection and infection are the diagnosed causes of death in this compartment. the second block includes animals surviving up to 110 days. in the grafts, which belong to this compartment a intrahepatic bile duct proliferation is a frequent and dominant histological feature. because of the development of identical lesions in isograft surviving the same periode and the induction of this lesions by common bile duct ligation experiments, chronic rejection is excludet as the cause of death. the third block includes all so called long-term survivors, in which the structure of the grafts are remarkably well preserved and very few abnormalities are present. the occurence of fatal acute rejection and long-term survival in each allograft group demonstrates, that the fate of the orthotopically transplanted livers in not dependent upon key: cord-023528-z9rc0ubj authors: wilkins, pamela a. title: disorders of foals date: 2009-05-18 journal: equine internal medicine doi: 10.1016/b0-72-169777-1/50021-4 sha: doc_id: 23528 cord_uid: z9rc0ubj nan before the 1980s, intensive management of the compromised neonate was unusual and little was known regarding many of the problems of this special patient population. although some specific conditions had been described by astute clinician-researchers, most notably the "dummy" foal syndrome 1 and respiratory distress syndrome caused by primary surfactant deficiency, 2 little information regarding the diagnosis and management of conditions of the foal during the neonatal period was available, although at least one active group was investigating fetal and neonatal physiology of the horse in great britain. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] when treatment of compromised foals was undertaken, the approach most commonly resembled treating them as small adults with little understanding of the different physiology of the equine neonate. the advent of improved management of reproductive efficiency of mares led naturally to increased interest in preservation of the conceptus to parturition and the foal thereafter. interested clinicians, taking their lessons from the field of human perinatology/neonatology and sometimes working hand-in-hand with their counterparts in the human field, pioneered investigations into these small patients and created the fields of equine perinatology and equine neonatal intensive care. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] because of the foresight and energy of these early investigators, the field of veterinary perinatology/neonatology exploded in the 1980s, leading to the creation of equine neonatal intensive care units throughout the united sates and the world. from these units information about the normal and abnormal physiology of foals, the medical conditions affecting them, and methods for treatment and management of these problems has been developed through observational, retrospective, and prospective studies. this veritable explosion of information over the last 20 years has improved greatly the ability of all practitioners to provide appropriate care for these patients, whether in the field or at an equine neonatal intensive care unit. the ability not only to save the lives of these patients but also to treat them in such a manner as to allow them to fulfill their purposes, whether as pleasure animals or racing athletes, has improved almost exponentially from those early days. [20] [21] [22] [23] this chapter aims to provide the clinician with some of the most current information regarding the management of these patients, recognizing that much still remains unknown and that advances will continue to be made in this dynamic field. the reader is cautioned that much of this chapter is flavored by the experiences of the author and that variation in approach and treatment of specific problems exists between neonatal intensive care units (nicus) and between clinicians in the same nicu and that each year results in change. in some cases, information that is presented has been gleaned from human nicu studies, essentially using the critically ill infant as the experimental model. many of the problems of the newborn foal have their genesis in utero. identification of high-risk pregnancies is an important component of prenatal care of the foal, and some of the most commonly encountered problems of the dam resulting in abnormal foals include previous or concurrent disease, poor reproductive history, poor perineal or pelvic conformation, poor general health, poor nutritional condition, prolonged transport, history of previous abnormal foals, placental abnormalities, and twins. 24 some of the more common causes of abortion can result in the birth of severely compromised foals of variable gestation lengths (box 19-1). these include pa m e l a a . wi l k i n s infectious causes such as equine herpesvirus (ehv) types 1 (most commonly) and 4 (rarely), equine infectious anemia, equine arteritis virus, bacterial and fungal placentitis, leptospirosis, equine ehrlichiosis, and gram-negative septicemia/endotoxemia. [25] [26] [27] noninfectious causes of abortion include twinning and noninfectious placental abnormalities such as extensive endometrial fibrosis, body pregnancy, and abnormal length (long or short) of the umbilical cord. 24, 28 to the equine neonatologist opportunities for intervention may appear limited, and in the case of many of the aforementioned causes of fetal loss, this is true. however, one can do much in an attempt to preserve the pregnancy and in effect treat the fetus. when one is faced with a threatened pregnancy, one has various ways of evaluating the fetus and its environment and may use many potential therapies. once one identifies a pregnancy as high risk, one should evaluate the fetus for viability. evaluation should include as thorough an evaluation as possible of the reproductive tract, placenta, and fetal fluids. prepartum disorders in the mare usually are readily recognizable, but disorders of the fetus and placenta can be more subtle and difficult to determine. the first step is to take a thorough history of the mare. of particular interest is any history of previous abnormal foals, but the history taking should include questions regarding transportation; establishment of an accurate breeding date (sometimes more difficult than one would suspect); any pertinent medical history including any diagnostic testing performed for this pregnancy such as culture, endometrial biopsy, and cytologic results; and any rectal and ultrasound examination results. additionally, one should obtain information regarding possible ingestion of endophyte-infected fescue or exposure to potential infectious causes of abortion. 29, 30 a complete vaccination and deworming history is requisite, as is a complete history of any medications and supplements administered during pregnancy. after obtaining a history, one examines the mare per rectum. this examination should include palpation of the cervix, uterus, fetus, and all palpable abdominal contents. one should note any abnormalities. the cervix should be tight throughout gestation; the late gestation uterus will be large and distended with fluid and usually pulled craniad in the abdomen. palpation of the fetus frequently results in some fetal movement; however, one should interpret lack of movement with caution, for some normal fetuses do not respond. ultrasonographic evaluation of the uterus and conceptus per rectum can provide valuable information, particularly regarding placental thickness if placentitis is a concern. one may evaluate fetal fluids and estimate fetal size from the size of the eye later in gestation. 31 in the author's hospital the practitioners choose not to perform vaginal examinations or speculum examinations because of an association between these examinations and the subsequent development of placentitis. unless placentitis is recognized with ultrasonograhic evaluation per rectum and culture is desirable, these types of examinations are generally not necessary. following examination per rectum, one performs transabdominal ultrasonographic evaluation of the uterus and conceptus. 28 one can generate a biophysical profile of the fetus from this examination in the late-term fetus and readily determine viability. 32, 33 one also readily can determine the presence or absence of twins in the late pregnant mare in this manner. one performs the sonogram through the acoustic window from the udder to the xiphoid ventrally and laterally to the skinfolds of the flank. imaging of the fetus usually requires a lowfrequency (3.5-mhz) probe, whereas examination of the placenta and endometrium requires a higher-frequency (7.5-mhz) probe. a complete description of this examination is beyond the scope of this chapter, but the reader will find several complete descriptions of the technique and normal values for specific gestation lengths within the relevant veterinary literature. 33 the utility of this examination lies in its repeatability and low risk to the dam and fetus. sequential examinations over time allow the clinician to follow the pregnancy and to identify changes as they occur. a companion to transabdominal ultrasongraphy is evaluation of the fetal electrocardiogram (ecg). one can measure fetal ecgs continuously using telemetry or can obtain them using more conventional techniques several times throughout the day. 24, 28, 34 one places electrodes on the skin of the mare in locations aimed at maximizing the magnitude of the fetal ecg. because the fetus frequently changes position, multiple sites may be needed in any 24-hour period. to begin, one places an electrode dorsally in the area of the sacral prominence with two electrodes placed bilaterally in a transverse plane in the region of the flank. the fetal ecg maximal amplitude is low, usually 0.05 to 0.1 mv, and can be lost in artifact or background noise, so one commonly must move electrodes to new positions to maximize the appearance of the fetal ecg. the normal fetal heart rate during the last months of gestation ranges from 65 to 115 beats/min, a fairly wide distribution. the range of heart rate of an individual fetus can be narrow, however. bradycardia in the fetus is an adaptation to in utero stress, most commonly thought to be hypoxia. by slowing the heart rate, the fetus prolongs exposure of fetal blood to maternal blood, increasing the time for equilibration of dissolved gas across the placenta and improving the oxygen content of the fetal blood. the fetus also has altered the distribution of its cardiac output in response to hypoxia, centralizing blood distribution. 35, 36 tachycardia in the fetus can be associated with fetal movement, and brief periods of tachycardia should occur in the fetus in any 24-hour period. persistent tachycardia is a sign of fetal distress and represents more severe fetal compromise than bradycardia. the author has recognized dysrhythmias in the challenged fetus, most commonly as atrial fibrillation but also apparent runs of ventricular tachycardia. the ability to monitor the fetus in a high-risk pregnancy inevitably has led to questions of whether, how, and when to intervene. most equine neonatologists would agree that removal of the fetus from the uterus before its attainment of readiness for birth is not desirable. one of the difficulties in determining fetal preparedness for birth is that prediction of parturition is difficult in these mares. many of the parameters used in normal mares are unreliable in the high-risk pregnant mare. one must have an accurate history of any previous gestation length in terms of days for the specific mare in question to allow a more accurate estimate of her usual gestational length. evaluation of the usual mammary gland parameters, including size, the presence of "wax," and alteration of electrolyte concentrations, is not generally predictive in the high-risk mare, for in the author's experience many of these mares have changes predictive of parturition for weeks before actual parturition. 37, 38 this circumstance may be related to the observation that many high-risk pregnant mares, particularly those with placentitis, are presented for a primary complaint of early onset lactation. although pulmonary system maturity in human beings can be assessed with some degree of accuracy using measurement of lecithin/sphingomyelin ratios, this measurement-along with sphingomyelin, cortisol, and creatinine concentrations in the amnionic fluid-has proved to be of no benefit in the horse. [39] [40] [41] amniocentesis carries a high risk of abortion in the horse, even with ultrasound guidance, and is not a clinically useful technique at this time. 41 currently, no clear-cut guidelines are available as to when to intervene, but the presence of persistent fetal tachycardia or prolonged absence of fetal movements, including breathing movements, as determined by transabdominal ultrasound evaluation, should initiate discussion regarding the appropriateness of induction of parturition or elective cesarean section. the goal of induction or cesarean section is to remove a pregnancy that is threatening the survival of the dam with no thought to fetal survival or to remove the fetus from a threatening environment to improve its likelihood for survival. preterm induction is ill advised if fetal survival is desirable because of the limited ability to treat severely immature neonates. timing of intervention in these circumstances remains an art, not a science. the approach to management of the high-risk pregnancy is dictated to some degree by the exact cause for concern, but for many mares therapy is similar. many high-risk mares have placentitis, primarily caused by ascending bacterial or fungal infections originating in the region of the cervix. these infections can cause in utero sepsis or compromise the fetus by local elucidation of inflammatory mediators or altered placental function. 42, 43 premature udder development and vaginal discharge are common clinical signs. treatment consists of administration of broad-spectrum antimicrobial agents and nonsteroidal antiinflammatory drugs (table 19 -1). in the author's clinic, trimethoprim-sulfonamide drugs have been the antimicrobial of choice based on unpublished studies performed at the facility demonstrating increased concentration of these agents in the fetal fluids compared with penicillin and gentamicin. however, if culture and sensitivity results are available, one should institute directed therapy. nonsteroidal antiinflammatory agents such as flunixin meglumine are useful to combat alterations in prostaglandin balance that may be associated with infection and inflammation. although the efficacy of these agents is best when administered before the development of clinical signs, to date no detrimental effects have been reported in the fetus or dam when chronically used at low doses in well-hydrated patients. tocolytic agents and agents that promote uterine quiescence have been used and include altrenogest, isoxuprine, and clenbuterol. [44] [45] [46] [47] [48] altrenogest usually is administered, although its need in late gestation has been challenged. the efficacy of isoxuprine as a tocolytic in the horse is unproven, and bioavailability of orally administered isoxuprine appears to be highly variable. 48 the long-term use of clenbuterol is inadvisable because of receptor population changes associated with chronic use and its unknown effects on the fetus at this time. clenbuterol may be indicated during management of dystocia in preparation for assisted delivery or cesarean section. 46 the intravenous form of clenbuterol is not currently available in the united states. one can use three additional strategies in managing high-risk pregnancy patients. in mares with evidence of placental dysfunction, with or without signs of fetal distress, the author provides intranasal oxygen supplementation in the hope of improving oxygen delivery to the fetus. intranasal oxygen insufflation of 10 to 15 l/min to the mare significantly increases pao 2 and percent oxygen saturation of hemoglobin. 49 because of the placental vessel arrangement of the horse, improvement of these two arterial blood gas parameters should result in improved oxygen delivery to the fetus. blood gas transport is largely independent of diffusion distance in the equine placenta, particularly in late gestation, and depends more on blood flow. information from other species cannot be extrapolated to the equine placenta because of its diffuse epitheliochorial nature and the arrangement of the maternal and fetal blood vessels within the microcotyledons. 50,51 umbilical venous po 2 is 50 to 54 mm hg in the horse fetus, compared with 30 to 34 mm hg in the sheep, whereas the maternal uterine vein to umbilical vein po 2 difference is near 0. also unlike the sheep, the umbilical venous po 2 values decrease 5 to 10 mm hg in response to maternal hypoxemia and increase in response to maternal hyperoxia. [52] [53] [54] vitamin e (tocopherol) is administered orally to some high-risk mares as an antioxidant. administration of large doses of vitamin e before traumatic brain injury improves neurologic outcome in experimental models and has been examined as possible prophylaxis for human neonatal encephalopathy. [55] [56] [57] extrapolation of that information to the compromised equine fetus suggests that increased antioxidant concentrations in the fetus may mitigate some of the consequences of uterine and birth hypoxia, but no evidence is available to date demonstrating that protection occurs or that vitamin e accumulates in the fetus in response to supplementation of the mare. finally, many high-risk mares are anorectic or held off feed because of their medical condition. these mares are at particularly great risk for fetal loss because of their lack of feed intake, which alters prostaglandin metabolism. 58 therefore one should administer 2.5% to 5% dextrose in 0.45% saline or water (5% dextrose) intravenously at maintenance fluid rates to these patients. perhaps the most important aspect of managing high-risk pregnancy mares is frequent observation and development of a plan. one should observe mares at least hourly for evidence of early-stage labor and should put them under constant video surveillance if possible. depending on the primary problem, the team managing the mare should develop a plan for handling the parturition once labor begins and for fetal resuscitation following delivery. any equipment that might be needed should be readily available stallside, and a call sheet, listing contact numbers for all involved, should be posted on or near the stall. the plan should include a decision as to how to handle a complicated dystocia, should it occur, with permission for general anesthesia and cesarean section obtained before the event so that time is not wasted. an important question to be posed to the owner at the outset is which is most important to the owner, the mare or the foal, for the answer may dictate the direction of the decision tree once labor begins. 59 early recognition of abnormalities is of utmost importance for successful management of critically ill foals. to recognize the abnormal, one must know the normal. immediately following birth, foals effect several important physiologic and behavioral changes. chief among these changes is the adaptation of the cardiovascular and respiratory systems to extrauterine life. the normal transition of the respiratory tract involves opening closed 1384 part ii disorders of specific body systems alveoli and absorption of fluid from the airway, accomplished by a combination of breathing efforts, expiration against a closed glottis (grunting), and a change in sodium flux across the respiratory membrane from net secretion to net absorption. [60] [61] [62] [63] [64] the transition from fetal to neonatal circulatory patterns requires resolution of the pulmonary hypertension present in the fetus, normally shunting blood flow through the lower resistance ductus arteriosus in the fetal state, to direct cardiac output to the pulmonary vasculature for participation in gas exchange. this change is achieved by the opening of alveoli, decreasing airway resistance and providing radial support for pulmonary vessels, functional closure of the ductus arteriosus, and increasing the oxygen tension in the lung, reversing pulmonary vasoconstriction mediated by hypoxia. 65, 66 pulmonary tree vasodilators (prostacyclin, nitric oxide [no] ) and vasoconstrictors (endothelin-1, leukotrienes) play apparently well-coordinated, but as yet not fully elucidated, roles. in the normal newborn this change is smooth and rapid. these critical events are undermined by factors such as inadequate lung development, surfactant deficiency (primary or secondary), viral or bacterial infection, placental abnormalities, in utero hypoxia, and meconium aspiration. spontaneous breathing should begin in the neonate within 1 minute of birth, many foals attempt to breathe as their thorax clears the pelvic canal. during the first hour of life, the respiratory rate of a healthy foal can be as high as 80 breaths per minute but should decrease to 30 to 40 breaths per minute within a few hours. similarly, the heart rate of a healthy newborn foal has a regular rhythm and should be at least 60 beats/min at the first minute. 67, 68 one usually can auscultate a continuous murmur over the left side of the heart, although its loudness may vary with position. this murmur is thought to be associated with some shunting through the ductus arteriosus. one may auscultate variable systolic murmurs, thought to be flow murmurs, during the first week of life. 69 one should investigate more thoroughly murmurs that persist beyond the first week of life in an otherwise healthy foal, along with any murmur associated with persistent hypoxia. auscultation of the thorax shortly after birth reveals a cacophony of sounds as airways open and fluid is cleared. end-expiratory crackles are consistently audible in the dependent lung during and following lateral recumbency. for a normal newborn foal to appear slightly cyanotic during this initial adaptation period is not unusual, but this should resolve within minutes of birth. the equine fetus, as do all fetuses, exists in a moderately hypoxic environment, but the equine fetus has a greater partial pressure of oxygen, around 50 mm hg. 70 because the fetus is well adapted to low oxygen tensions, cyanosis is rarely present in newborn foals once adaption occurs, even those with low oxygen tensions. although in many species the fetal blood oxygen affinity is greater than the maternal blood, in the equine fetus the oxygen affinity of its hemoglobin is only about 2 mm hg greater than the maternal blood because of decreased levels of 2,3-diphosphoglycerate compared with other species. 71 the result is enhanced oxygen unloading in the equine fetus compared with others. 2,3-diphosphoglycerate concentration increases after birth in the foal and reaches mature levels by 3 to 5 days of age. the major blood adaptation of the equine fetus to chronic hypoxia is an increase in packed cell volume of up to 20%, increasing the oxygen content of the blood as compensation for decreased oxygen delivery at the placenta. 72 a larger than expected packed cell volume in any newborn foal should alert the clinician for possible sequelae from chronic hypoxia. the presence of significant cyanosis that persists should prompt the clinician to evaluate the foal thoroughly for cardiac anomalies resulting in significant right-to-left shunting or separated circulations, such as transposition of the great vessels. the chest wall of the foal is compliant, facilitating passage through the pelvic canal during parturition. this compliance requires that the foal actively participate in inspiration and expiration with several potential consequences. first, restriction of the thorax or the abdomen can result in impaired ventilation, which can occur easily when one restrains a foal and may result in spuriously abnormal arterial blood gas values (see the discussion on arterial blood gas evaluation, respiratory diseases associated with hypoxemia in the neonate). second, foals with primary pulmonary parenchymal disease resulting in poorly compliant lungs develop paradoxical chest wall motion, with the thorax moving inward during inspiration. [73] [74] [75] [76] the work of breathing can increase greatly, resulting in respiratory failure because of respiratory muscle fatigue. a foal that appears suddenly to improve a previously abnormal respiratory rate and pattern may in fact be in greater respiratory difficulty because of fatigue. one can observe a reduction in respiratory rate or abnormal breathing pattern in premature/dysmature foals or foals subjected to peripartum hypoxia/asphxia. although the genesis of these patterns is not understood fully, cheyne-stokes (lengthy periods of apnea interrupted by short breaths that wax and wane in depth), cluster (short periods of apnea interspersed with long periods of breathing), and biot's breathing (periods of apnea and breathing with no discernible pattern) may occur in these cases. foals attempting to maintain an adequate lung volume expire against a partially closed glottis, called valsalva's maneuver, producing an audible grunt. foals are normally nonresponsive while in the birth canal but should respond to stimulation immediately after birth. 67 the lack of responsiveness while in the birth canal has lead to presumption of fetal death during dystocia. because of this, one should attempt other tests before determining that a foal is dead intrapartum. one possibly may detect pulses in the tongue, neck, or any presented limbs or palpate the thorax for a heartbeat. in the author's facility, nasotracheal intubation of the foal combined with measurement of co 2 tensions in the exhaled gas aids practitioners in cases where they can reach the nose. nasotracheal intubation of foals under these circumstances actually can be performed readily with minimal practice. having long endotracheal tubes available of several different diameters (7 to 12 mm outer diameter) with an inflatable cuff is important. one can pass the tube blindly using a finger in one nostril for guidance and can check the position frequently by palpation of the throatlatch region. one inflates the cuff and begins manual ventilation with 100% oxygen or room air using an ambu-bag or equivalent. one can obtain continuous measurement of co 2 tension using a capnograph or single-use disposable end-tidal co 2 monitor attached to the ambu-bag or the nasotracheal tube. in a dead foal the end-tidal co 2 measurement will be negligible after the first 10 to 20 breaths. one must ensure tube placement and seal integrity and allow for multiple breaths. some co 2 will "wash out" with the first few breaths and can result in false hope initially. end-tidal co 2 varies in living intrapartum foals, depending on cardiac output and ventilation frequency, but should be consistently greater than 20 mm hg and is usually closer to 30 mm hg. once one establishes manual ventilation of a living foal, one must continue ventilation until the foal is delivered satisfactorily. the author has resuscitated and maintained many foals successfully in this manner throughout induction of general anesthesia in the mare and cesarean section delivery of the foal. the nasotracheal tube also provides a convenient site for administration of intratracheal medications such as epinephrine used for extrauterine intrapartum resuscitation of the foal. the reader is cautioned that intratracheal epinephrine increases endtidal co 2 measurements transiently, even in a dead foal, because of local actions on tissues. one should allow a washout period after intratracheal administration of epinephrine. the righting reflex is present as the foal exits the birth canal, as is the withdrawal reflex. cranial nerve responses are intact at birth, but the menace response may take as long as 2 weeks to develop fully. one should not consider lack of a menace reflex diagnostic of visual deficits in the newborn foal. within an hour of birth the normal foal will demonstrate auditory orientation with unilateral pinna control. the normal pupillary angle is ventromedial in the newborn foal; this angle gradually becomes dorsomedial over the first month of life. foals should begin attempting to stand shortly after birth and should be able to achieve this on their own within 2 hours of birth. 67 the normal newborn foal has a suck reflex shortly after birth and should be searching for an udder even before it stands. the expectation is that a normal foal will be sucking from the dam unaided by 3 hours post partum; many foals are overachievers and will be sucking well before this time. the normal foal may defecate shortly after standing but may not attempt defecation until after it first successfully sucks from the dam. urination varies more, with filly foals usually urinating before colt foals, but both usually do not urinate for several hours following birth, up to 12 hours for some colts. 67 for colt foals to fail to drop their penises when urinating over the first few days of life is not unusual. the gait of the newborn foal is hypermetric and the stance is base wide. extreme hypermetria of the forelimbs, usually bilateral but occasionally unilateral, has been observed in some foals and is associated with perinatal hypoxic/ischemic insults, but this gait abnormality usually resolves without specific therapy within a few days. spinal reflexes tend to be exaggerated, whereas the crossed extensor reflex may not be fully present until 3 weeks of age. 77 foals also exhibit an exaggerated response to external stimuli (noise, sudden visual changes, touch) for the first few weeks of life. foals are not bonded strongly to their mother for the first few weeks of life and will follow any large moving object, including other horses and human beings. orphan foals bond with surrogate mothers until they are several months of age; their primary motivation appears to be appetite. conversely, mares strongly bond with their foals shortly after parturition; the process begins once the chorioallantois ruptures and is driven more by olfaction and taste than by vision or hearing. interference with this process, by medical intervention or excessive owner manipulation of the foal, can disrupt normal bonding and result in foal rejection by the dam. 78 most newborn foals make the transition to extrauterine life easily. however, for those in difficulty, recognition of the condition immediately and institution of appropriate resuscitation is of utmost importance. a modified apgar scoring system has been developed as a guide for initiating resuscitation and assessing probable level of fetal compromise (table 19 -2). 79 one also must at least perform a cursory physical examination before initiating resuscitation, for issues of humaneness are associated with with serious problems such as severe limb contracture, microophthalmia, and hydrocephalus, among others. the initial assessment begins during presentation of the fetus. although the following applies primarily to attending the birth of a foal from a high-risk pregnancy, one can perform quiet and rapid evaluation during any attended birth. the goal in a normal birth with a normal foal is to disturb the bonding process minimally. this goal also applies to high-risk parturitions, but some disruption of normal bonding is inevitable. the lead clinician should control tightly the number of persons attending, and the degree of activity surrounding, the birth. one should evaluate the strength and rate of any palpable peripheral pulse and should evaluate the apical pulse as soon as the chest clears the birth canal. bradycardia (pulse <40 beats/min) is expected during forceful contractions, and the pulse rate should increase rapidly once the chest clears the birth canal. persistent bradycardia is an indication for rapid intervention. the fetus is normally hypoxemic compared with the newborn foal, and this hypoxemia is largely responsible for the maintenance of fetal circulation by generation of pulmonary hypertension. the fetus responds to conditions producing more severe in utero hypoxia by strengthening the fetal circulatory pattern, and the neonate responds to hypoxia by reverting to the fetal circulatory pattern. 80 during a normal parturition, mild asphyxia occurs and results in fetal responses that pave the way for a successful transition to extrauterine life. if more than mild transient asphyxia occurs, the fetus is stimulated to breathe in utero; this is known as primary asphyxia. 81 if the initial breathing effort resulting from the primary asphyxia does not correct the asphyxia, a second gasping period occurs in several minutes, known as the secondary asphyxia response. if no improvement in asphyxia occurs during this period, the foal enters secondary apnea, a state that is irreversible except with resuscitation. therefore the first priority of neonatal resuscitation is establishing an airway and breathing pattern. one should assume that foals not spontaneously breathing are in secondary apnea and should clear the airway of membranes as soon as the nose is presented. if meconium staining is present, one should suction the airway before delivery of the foal is completed and before the foal breathes spontaneously. one should continue to the trachea if aspiration of the nasopharynx is productive. overzealous suctioning worsens bradycardia as it worsens hypoxia. one should stop suctioning once the foal begins breathing spontaneously, as hypoxia will worsen with continued suction. if the foal does not breathe or move spontaneously within seconds of birth, one should begin tactile stimulation. if tactile stimulation fails to result in spontaneous breathing, one immediately should intubate the foal and manually ventilate the foal using an ambu-bag or equivalent. one can use mouth-to-nose ventilation if nasotracheal tubes and an ambu-bag are not available. the goal of this therapy is to reverse fetal circulation, and hyperventilation with 100% oxygen is the best choice for this purpose. however, recent evidence suggests that no clinical disadvantages are apparent in using room air for ventilation of asphyxiated human neonates rather than 100% oxygen. 82, 83 human infants resuscitated with room air recovered more quickly than those resuscitated with 100% oxygen in one study as assessed by apgar scores, time to the first cry, and the sustained pattern of breathing. 84 in addition, neonates resuscitated with 100% oxygen exhibited biochemical findings reflecting prolonged oxidative stress, present even after 4 weeks of postnatal life, which did not appear in the group resuscitated with room air. thus the current accepted recommendations for using 100% oxygen in the resuscitation of asphyxiated neonates needs further discussion and investigation. 85, 86 almost 90% of foals requiring resuscitation respond to hyperventilation alone and require no additional therapy. one can initiate nasotracheal intubation while the foal is in the birth canal if the foal will not be delivered rapidly, such as with a difficult dystocia. this technique is "blind" and requires some practice but may be beneficial and lifesaving. once spontaneous breathing is present, one apgar score in the foal should provide humidified oxygen via nasal insufflation at 8 to 10 l/min. one should initiate cardiovascular support in the form of chest compression if the foal remains bradycardic despite ventilation and a nonperfusing rhythm is present. one should make sure the foal is on a hard surface in right lateral recumbency with the topline against a wall or other support. approximately 5% of foals are born with fractured ribs and an assessment for the presence of rib fractures is in order before initiating chest compressions. 87 palpation of the ribs identifies many of these fractures, which usually are multiple and consecutive on one side of the thorax and located in a relatively straight line along the part of the rib with the greatest curvature dorsal to the costochondral junction. unfortunately, ribs 3 to 5 frequently are involved, and their location over the heart can make chest compression a potentially fatal exercise. auscultation over the ribs during breathing results in a recognizable click, identifying rib fractures that may have escaped detection by palpation. one should initiate drug therapy if a nonperfusing rhythm persists for more than 30 to 60 seconds in the face of chest compression. epinephrine is the first drug of choice (table 19-3) . practitioners pose various arguments regarding the best dose and the best frequency of administration for resuscitation. however, most of the data are acquired from human cardiac arrest studies and are not strictly applicable to the equine neonate because the genesis of the cardiovascular failure is different. 88, 89 vasopressin is gaining attention as a cardiovascular resuscitation drug, and although the author has used this drug in resuscitation and as a pressor, experience is limited at this time. 90 the author does not use atropine in bradycardic newborn foals because the bradycardia usually is caused by hypoxia, and if the hypoxia is not corrected, atropine can increase myocardial oxygen debt. 89 the author also does not use doxapram because it does not reverse secondary apnea, the most common apnea in newborns. because birthing areas are generally cold, one should dry the foal and place it on dry bedding once resuscitation is complete. the fetus has some homeothermic mechanisms, but its size in relation to its mother and its position within her body means that it is in effect a poikilotherm. the body temperature of the foal generally reflects that of its environment, namely its mother, although the human fetal temperature directly measured at cesarean section, induction of labor, or during labor is approximately 0.5â°c higher than the mothers. 91, 92 adaptation from poikilothermy to homeothermy normally takes place rapidly following birth. the fetus is capable of nonshivering thermogenesis, primarily through the oxidation of brown fat reserves, but this type of thermogenesis is inhibited in utero, probably by placental prostaglandin e 2 and adenosine. 93, 94 immediately after birth the foal must adapt to independent thermoregulation. local physical factors, including ambient temperature and humidity, act to induce cold stress, and the newborn must produce heat by metabolic activity. in response to the catecholamine surge associated with birth, uncoupling of oxidative phosphorylation occurs within mitochondria, releasing energy as heat. this nonshivering thermogenesis is impaired in newborns undergoing hypoxia or asphyxiation and in those that are ill at birth. infants born to mothers sedated with benzodiazepines are affected similarly, a consideration in the choice of sedative and preanesthetic medications in mares suffering dystocia or 1388 part ii disorders of specific body systems undergoing cesarean section. [95] [96] [97] heat losses by convection, radiation, and evaporation are high in most areas where foals are delivered, resuscitated ,and managed, and one must take care to minimize cold stress in the newborn and the critically ill foal. supplementary heat, in the form of radiant heat lamps or warm air circulating blankets, may be required. one should use fluid therapy conservatively during postpartum resuscitation, for the neonate is not volume depleted unless excessive bleeding has occurred. some compromised newborn foals are actually hypervolemic. fluid therapy of the neonate is discussed in more detail later in this chapter. because the renal function of the equine neonate is substantially different from the adult, one cannot simply scale down fluid therapy from adult therapy. [98] [99] [100] if intravenous fluids are required for resuscitation and blood loss is identified, administration of 20 ml/kg of a non-glucose-containing polyionic isotonic fluid over 20 minutes (about 1 l for a 50-kg foal) once intravenous access is established can be effective. the author stresses non-glucose-containing polyionic intravenous fluids because hyperglycemia, but not hypoglycemia, immediately after fetal or neonatal asphyxia interfered with the recovery of brain cell membrane function and energy metabolism in neonatal piglets in one recent study. 101 these findings suggest that post-hypoxic-ischemic hyperglycemia is not beneficial and might even be harmful in neonatal hypoxic-ischemic encephalopathy. indications for this shock bolus therapy include poor mentation, poorly palpable peripheral pulses, and the development of cold distal extremities, compatible with hemorrhagic shock. one should reassess the patient after the initial bolus and administer additional boluses as necessary. ideally, one should follow up on blood pressures and ecg readings and initiate appropriate pressor therapy if needed. again, these procedures are discussed in detail later in the chapter. one can administer glucose-containing fluids after resuscitation at a rate of 4 to 8 mg/kg/min (about 250 ml/hr of 5% dextrose or 125 ml/hr of 10% dextrose) to the average 50-kg foal, particularly in the obviously compromised foal. this therapy is indicated to help resolve metabolic acidosis, to support cardiac output because myocardial glycogen stores likely have been depleted, and to prevent postasphyxial hypoglycemia. under normal conditions, the fetal-to-maternal blood glucose concentration gradient is 50% to 60% in the horse, and glucose is the predominant source of energy during fetal development. 102, 103 glucose transport across the placenta is facilitated by carrier receptors (glucose transporter [glut] receptors), and a direct relationship exists between maternal and fetal blood glucose concentration when maternal glucose is in the normal range. 102 the glut receptors in the placenta are stereospecific, saturable, and energy independent. 104 although the enzyme kinetics for glut isoform 1 suggest that they are not saturable under conditions of euglycemia, equine maternal hyperglycemia results in increased fetal glucose concentration to a plateau point, likely caused by glut saturation. at term, the net umbilical uptake of glucose is 4 to 7 mg/kg/min, with most of the glucose being used by the brain and skeletal muscle. [105] [106] [107] the fetus only develops gluconeogenesis under conditions of severe maternal starvation. a certain percentage of the delivered glucose is used to develop large glycogen stores in the fetal liver and cardiac muscle in preparation for birth, and at birth the foal liver produces glucose at a rate of 4 to 8 mg/ kg/min by using these stores. fetal glycogen stores also are built using the substrates lactate, pyruvate, and alanine; fetal uptake of lactate across the placenta is about half that of glucose. 102, 108 the transition to gluconeogenesis, stimulated by increased circulating catecholamine concentration from birth and by stimulation of glucagon release at the time the umbilical cord breaks takes 2 to 4 hours in the normal foal, and glycogenolysis supplies needed glucose until feeding and glucose production are accomplished. 109 in the challenged foal, glycogen stores may have been depleted and gluconeogenesis delayed, so provision of glucose at rates similar to what the liver would normally produce during this period is requisite. persistent pulmonary hypertension (pph) also is known as reversion to fetal circulation or persistent fetal circulation, and its genesis lies in the failure of the fetus to make the respiratory and cardiac transition to extrauterine life successfully or reversion of the newborn to fetal circulatory patterns in response to hypoxia or acidosis. differentiating this problem from other causes of hypoxemia in the newborn requires some investigation, and multiple serial arterial blood gas analyses are necessary to confirm suspicion of this problem (see the section on arterial blood gas analysis, respiratory diseases associated with hypoxemia in the neonate). however, one should suspect the condition in any neonate with hypercapnic hypoxemia that persists and worsens; these foals are in hypoxemic respiratory failure. the fetal circulatory pattern, with pulmonary hypertension and right-to-left shunting of blood through the patent foramen ovale and ductus arteriosus, is maintained in these cases. pulmonary vascular resistance falls at delivery to about 10% of fetal values, while pulmonary blood flow increases accordingly. 110 early in the postnatal period these two changes balance each other, and mean pulmonary and systolic pressures remain increased for several hours. systolic pulmonary pressures can remain equivalent to systemic pressure for up to 6 hours of age in human infants, although diastolic pulmonary pressures are well below systemic diastolic pressures by 1 hour. 111 mean pulmonary artery pressures fall gradually over the first 48 hours. 112 the direct effects of lung expansion and increasing alveolar oxygen tension probably provide the initial stimulus for pulmonary arteriolar dilation and partly result from direct physical effects, but vasoactive substances are released in response to physical forces associated with ventilation, for example prostacyclin. 110 other vasoactive mediators thought to play a role in regulating pulmonary arteriolar tone include no, prostaglandins d 2 and e 2 , bradykinin, histamine, endothelin-1, angiotensin ii, and atrial natriuretic peptide. the increase in alveolar and arterial oxygen tensions at birth is required for completion of resolution of pulmonary hypertension. much of this increase is thought to be mediated by no, evidence for this being the parallel increase during gestation of the pulmonary vasodilation response to hyperoxia and the increase in no synthesis. 113 however, inhibition of no synthesis does not eliminate the initial decrease in pulmonary artery resistance occurring because of opening of the airways. 114 when these mechanisms fail, one can recognize pph. right-to-left shunting within the lungs and through patent fetal conduits occurs and can result from many factors, including asphyxia and meconium aspiration, but in many cases the precipitating trigger is unknown. inappropriately decreased levels of vasodilators (no) and inappropriately increased levels of vasoconstrictors (endothelin-1) currently are being examined as potential mechanisms. chronic in utero hypoxia and acidosis may result in hypertrophy of the pulmonary arteriolar smooth muscle. 115 in these cases, reversal of pph can be difficult and cannot be achieved rapidly. treatment of pph is twofold: abolishment of hypoxia and correction of the acidosis, for both abnormalities only bolster the fetal circulatory pattern. initial therapy is provision of oxygen intranasally at 8 to 10 l/min. some foals respond to this therapy and establish neonatal circulatory patterns within a few hours. failure to improve or worsening of hypoxemic respiratory failure following intranasal oxygen administration should prompt intubation and mechanical ventilation with 100% oxygen. this serves two purposes, one diagnostic and one therapeutic. ventilation with 100% oxygen may resolve pph and, if intrapulmonary shunt and altered ventilation-perfusion relationships are causing the hypoxic respiratory failure, arterial oxygen tension (pao 2 ) should exceed 100 mm hg under these conditions. failure to improve pao 2 suggests pph or large right-to-left extrapulmonary shunt caused by congenital cardiac anomaly. the vasodilators prostacyclin and telazoline (an î±-blocking vasodilator) cause pulmonary vasodilation in human infants with pph, but the effects on oxygenation vary and the sideeffects (tachycardia, severe systemic hypotension) are unacceptable. 116 recognition of no as a potent dilator of pulmonary vessels has created a significant step forward in the treatment of these patients, for inhaled no dilates vessels in ventilated portions of the lung while having minimal effects on the systemic circulation. 117 based on evidence presently available, use of inhaled no in an initial concentration of about 20 ppm in the ventilatory gas seems reasonable for term and near-term foals with hypoxic respiratory failure and pph that fails to respond to mechanical ventilation using 100% oxygen alone. 117, 118 the author has used this approach in the clinic, administering a range of 5 to 40 ppm no with success. hypoxic ischemic encephalopathy (hie), currently referred to as neonatal encephalopathy in the human literature, is one systemic manifestation of a broader syndrome of perinatal asphyxia syndrome (pas), and management of foals with signs consistent with a diagnosis of hie requires the clinician to examine other body systems fully and to provide therapy directed at treating other involved systems. 119 although pas primarily manifests as hie, the gastrointestinal tract and kidneys frequently are affected by peripartum hypoxia/ischemia/ asphyxia, and one should expect complications associated with these systems. hypoxic ischemic encephalopathy also may affect the cardiovascular and respiratory systems, and one also may encounter endocrine disorders in these patients. hypoxic ischemic encephalopathy has been recognized as one of the most common diseases of the equine neonate for generations. 1, 10, 12 in the past hie has been known as dummy foal syndrome and as neonatal maladjustment syndrome. the designation hie, although not perfect, attempts to describe the syndrome in terms of the suspected underlying pathophysiology. a wide spectrum of clinical signs is associated with hie and can range from mild depression with loss of the suck reflex to grand mal seizure activity. typically, affected foals are normal at birth but show signs of central nervous system abnormalities within a few hours after birth. some foals are obviously abnormal at birth, and some do not show signs until 24 hours of age. hypoxic ischemic encephalopathy commonly is associated with adverse peripartum events, including dystocia and premature placental separation, but a fair number of foals have no known peripartum period of hypoxia, suggesting that these foals result from unrecognized in utero hypoxia (box 19-2). severe maternal illness also may result in foals born with pas. in human beings, ascending placental infection now is suspected of being a major contributor to neonatal encephalopathy in infants, and the incidence of neonatal encephalopathy increases with the presence of maternal fever, suggesting a role for maternal inflammatory mediators. 120 the underlying pathophysiologic details of hie in the foal are unknown, and to date accurate experimental models of hie and pas in the foal have not been described. however, a great deal of attention has been paid to peripartum hypoxia/asphyxia by human counterparts because the effects of adverse peripartum events in the human neonate have far ranging implications for the affected human neonate and for society. therefore equine neonatologists have long looked to human studies and models of the human disease for understanding of the syndrome in the equine neonate. perinatal brain damage in the mature fetus usually results from severe uterine asphyxia caused by an acute reduction of uterine or umbilical circulation. the fetus responds to this challenge by activation of the sympathetic adrenergic nervous system, causing a redistribution of cardiac output that favors the central organs: brain, heart, and adrenal glands. 121, 122 if the hypoxic insult continues, the fetus reaches a point beyond which it cannot maintain this centralization of circulation, cardiac output falls, and cerebral circulation diminishes. 122 the loss of oxygen results in a substantial decrease in oxidative phosphorylation in the brain with concomitant decreased energy production. the na + /k + pump at the cell membrane cannot maintain the ionic gradients, and the membrane potential is lost in the brain cells. in the absence of the membrane potential, calcium flows down its large extracellular/intracellular concentration gradient through voltage-dependent ion channels into the cell. this calcium overload of the neuron leads to cell damage by activation of calcium-dependent proteases, lipases, and endonucleases. protein biosynthesis is halted. calcium also enters the cells by glutamate-regulated ion channels as glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles following anoxic cellular depolarization. once the anoxic event is over, protein synthesis remains inhibited in specific areas of the brain and returns to normal in less vulnerable areas of the brain. loss of protein synthesis appears to be an early indicator of cell death caused by the primary hypoxic/anoxic event. 123 a second wave of neuronal cell death occurs during the reperfusion phase and is thought to be similar to classically described postischemic reperfusion injury in that damage is caused by production of and release of oxygen radicals, synthesis of no, and inflammatory reactions. 124 additionally, an imbalance between excitatory and inhibitory neurotransmitters occurs. 123 part of the secondary cell death that occurs is thought to be caused by apoptosis, a type of programmed cell death termed cellular suicide. secondary cell death also is thought be caused by the neurotoxicity of glutamate and aspartate resulting again from increased intracellular calcium levels. 125, 126 in human infants the distribution of lesions with hypoxic-ischemic brain damage following prenatal, perinatal, or postnatal asphyxia falls into distinct patterns depending on the type of hypoxia-ischemia rather than on postconceptual age at which the asphyxial event occurs. 126 periventricular leukomalacia was associated with chronic hypoxia-ischemia, whereas the basal ganglia and thalamus were affected primarily in patients experiencing acute profound asphyxia, providing direct evidence that the nature of the event determines the severity and distribution of neurologic damage in human beings. these remarkably selective patterns of injury in children, with differential variability in the damage caused to regions anatomically located within millimeters of each other, resulted in the hypothesis that location within neurotransmitter-specific circuitry loops is important. this hypothesis has important implications in the design of neuroprotective strategies and therapies for neonates experiencing hypoxic-ischemic-asphyxial events. now the evidence is overwhelming that the excitotoxic cascade that evolves during hie extends over several days from the time of insult and is modifiable. 125, 126 in brain injury, traumatic or hypoxic, the mechanisms underlying delayed tissue injury still are understood poorly. many believe that neurochemical changes, including excessive neurotransmitter release, are pivotal in the pathophysiology of secondary neuronal death. excitatory amino acid neurotransmitters and magnesium are known to play at least a minimal role in secondary cell death following brain injury; a fair body of literature regarding these factors has been generated over the last 10 years. the activation of the n-methyl-d-aspartate (nmda) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury and is suspected to play a role in hie. [125] [126] [127] mechanically injured neurons demonstrate a reduction of voltage-dependent mg 2+ blockade of nmda current that can be restored partially by increasing extracellular mg 2+ concentration or by pretreatment with calphostin c, a protein kinase c inhibitor. 128 this finding suggested that administration of mg 2+ to patients with brain injury could lead to improved outcome. subsequently, magnesium sulfate solution was shown to improve dramatically the immediate recovery of rats from hypoxia. 129 however, although pretreatment with magnesium sulfate protected against hypoxic ischemic brain injury, postasphyxial treatment worsened brain damage in 7-day-old rats, suggesting an age-related response in the rat. 130 delayed magnesium treatment of mature rats following severe traumatic axonal brain injury improved motor outcome when administered up to 24 hours after injury, with early treatments providing the most benefit. 131 maternal seizure in rats is associated with fetal histopathologic changes that are abolished by administration of magnesium sulfate to the mother, and magnesium sulfate has been demonstrated to protect the fetal brain from severe maternal hypoxia. 132 clinical trials investigating the efficacy of magnesium treatment following hypoxia in infants are under way, with few reports currently in the medical literature. magnesium sulfate was used to treat nine infants after perinatal asphyxia in one study (no control group), and all children were neurologically normal at 1 year of age. seizures did not occur in any of these children, nor were any adverse side effects noted. 133 magnesium sulfate administration failed to delay the global impairment in energy metabolism after hypoxia ischemia, characteristic of severe brain damage, in newborn piglets; at 48 hours after hypoxia ischemia, no difference could be found in the severity of injury in piglets treated with magnesium compared with piglets treated with placebo, suggesting magnesium may not be protective with severe acute injury. 134 in developing countries, birth hypoxia frequently is associated with hie, and although this finding is attributed most frequently to inadequate obstetric care, poor nutrition also may play a role. red blood cell magnesium levels were measured in more than 500 women in labor at a teaching hospital in south africa. 135 fifty five of the women delivered infants with hie and had significantly lower levels of magnesium than controls; the infants with hie also had significantly lower magnesium levels than controls. the large majority (54 of 55) of the women giving birth to hie infants were from poor social circumstances, suggesting nutrition might play a role in some cases of hie, with maternal magnesium levels affecting outcome in the infants. the authors suggested an early pregnancy intervention study may help determine the role of magnesium in the pathogenesis of hie in human infants born to at-risk mothers. therapy for the various manifestations of hypoxiaischemia involves control of seizures, general cerebral support, correction of metabolic abnormalities, maintenance of normal arterial blood gas values, maintenance of tissue perfusion, maintenance of renal function, treatment of gastrointestinal dysfunction, prevention and recognition and early treatment of secondary infections, and general supportive care. control of seizures is important because cerebral oxygen consumption increases fivefold during seizures. one can use diazepam for emergency control of seizures (table 19 -4). if diazepam does not stop seizures readily or one recognizes more than two seizures, then one should replace diazepam with phenobarbital given to effect. the half-life of phenobarbital can be long in the foal (100 hours), and one should keep this in mind when monitoring neurologic function in these cases after phenobarbital administration (j.e. palmer, personal communication, 1998). 136 earlystage, preseizure administration of phenobarbital has been advocated by some investigators for prevention of neonatal encephalopathy. however, one recent study in asphyxiated human infants demonstrated that early phenobarbital treatment was associated with a threefold increase in the incidence of subsequent seizures and consequently a trend toward increased mortality. seizures per se were associated with almost a twentyfold increase in mortality. their findings suggest that early phenobarbital administration may produce adverse rather than beneficial effects following asphyxia. because this was an observational study; the results need to be confirmed by appropriate randomized trials in similar clinical settings. 137 if phenobarbital fails to control seizures, one may attempt phenytoin therapy. in cases of hie, one should avoid ketamine and xylazine because of their association with increased intracranial pressure. one must protect the foal from injury during a seizure and also ensure the patency of the airway to prevent the onset of negative pressure pulmonary edema 138 or aspiration pneumonia. probably the most important therapeutic interventions are aimed at maintaining cerebral perfusion, which is achieved by careful titration of intravenous fluid support, neither too much nor too little (see fluid therapy in neonates) and judicious administration of inotropes and pressors to maintain adequate perfusion pressures (see pressor and inotrope therapy in neonates). cerebral interstitial edema is only truly present in the most severe cases 139, 140 ; in most cases the lesion is intracellular edema and most of the classic agents used to treat cerebral interstitial edema (e.g., mannitol) are minimally effective treating cellular edema. occasionally the author uses thiamine supplementation in the intravenous fluids to support metabolic processes, specifically mitochondrial metabolism and membrane na + ,k + -atpases, involved in maintaining cellular fluid balance. 141, 142 this therapy is rational and inexpensive but unproven in efficacy. only if cellular necrosis and vasogenic edema are present are drugs such as mannitol and dimethyl sulfoxide indicated, and again these cases are usually the most severely affected. in the author's clinic, practitioners rarely have used dimethyl sulfoxide in neonates for the last several years and have recognized no change in outcome by discontinuing its use. when the practitioners use intravenously administered dimethyl sulfoxide, they do so within the first hour after an acute asphyxial insult and use it primarily for its hydroxyl radical scavenging effects and its theoretical modulation of postischemic reperfusion injury. 143 naloxone has been advocated for treating hie in human beings and in foals, [144] [145] [146] perhaps based on a study suggesting that postasphyxia blood-brain barrier disruption was related causally to poor neurologic outcome in a lamb model of hie and that naloxone prevented disruption and neurologic dysfunction among those survivors with an intact blood-brain barrier. 145 however, other studies have demonstrated that naloxone exacerbates hypoxic-ischemic brain injury in 7-day-old rats subjected to unilateral common carotid artery ligation and hypoxia. moreover, systemic acidosis and cellular edema were no different in naloxone-treated animals compared with animals treated with saline solution. the authors concluded that high doses of naloxone in fact may reduce the resistance of the fetus to hypoxic stress. 146 the use of naloxone in human neonatal resuscitation remains controversial, for whether the contradictory effects are related to a reduction in acute neuronal swelling by osmotic effects or by a more direct receptor-mediated mechanism is currently unknown. 147 naloxone is most effective in resuscitation of compromised human infants born to mothers addicted to drugs. some practitioners are using î³-aminobutyric acid adrenergic agonists to manage hie in foals, based on evidence showing neuroprotection when used in ischemia alone and combined with nmda antagonists. [148] [149] [150] the author currently has no experience with these compounds and cannot comment regarding their efficacy in foals. regional hypothermia also is being investigated as a potential therapy for global hypoxia/ischemia; published data are consistent with the theory that cooling must be continued throughout the entire secondary phase of injury (about 3 days) to be effective. 151 experimentally, this approach has resulted in dramatic decreases in cellular edema and neuronal loss; its practical application remains to be demonstrated. despite a lack of consensus regarding the use of magnesium to treat infants with hie, the author has used magnesium sulfate infusion as part of the therapy for selected foals with hie for the past several years. the rationale is based primarily on the evidence demonstrating protection in some studies and a failure of any one study to demonstrate significant detrimental effects. the clinical impressions of the author to date suggest that the therapy is safe and may decrease the incidence of seizure in patients. the author administers magnesium sulfate as a constant rate infusion over 1 hour after giving a loading dose. the author has continued the infusion for up to 3 days without demonstrable negative effect beyond some possible trembling. given the current evidence, a 24-hour course of treatment may be effective and all that is necessary. postasphyxial treatment certainly may be beneficial in foals with hie, and maternal magnesium therapy may be beneficial in certain high-risk pregnancy patients. foals with pas often have a variety of metabolic problems including hypo-or hyperglycemia, hypo-or hypercalcemia, hypo-or hyperkalemia, hypo-or hyperchloremia, and varying degrees of metabolic acidosis. although one needs to address these problems, one should not forget the normal period of hypoglycemia that occurs postpartum and should not treat aggressively so as to avoid worsening the neurologic injury. foals suffering from pas also have frequent recurrent bouts of hypoxemia and occasional bouts of hypercapnia. intranasally administered oxygen is generally needed in these cases as a preventative therapy and as direct treatment, for the appearance of the abnormalities can be sporadic and unpredictable. additional respiratory support, particularly in those foals with centrally mediated hypoventilation and periods of apnea or abnormal breathing patterns, include caffeine (per os or per rectum) and positive pressure ventilation. caffeine is a central respiratory stimulant and has minimal side effects at the dosages used (10 mg/kg loading dose; 2.5 mg/kg as needed). 152 the author purchases whatever oral form of caffeine is available at the local convenience store or drug store and administers it dissolved in warm water per rectum. foals treated with caffeine have an increased level of arousal and are more reactive to the environment. adverse effects generally are limited to restlessness, hyperactivity, and mild to moderate tachycardia. mechanical ventilation of these patients can be rewarding and generally is required for less than 48 hours. one must monitor and maintain blood ph within the normal range. metabolic alkalosis can develop in some of these foals and requires clinician tolerance of some degree of hypercapnia. ph is important in evaluation and consideration of alternatives for treatment. if the respiratory acidosis is not so severe as to affect the patient adversely (generally >70 mm hg), and the ph is within normal limits, the foal may tolerate hypercapnia. 153 the goal is to normalize ph. foals with respiratory acidosis as compensation for metabolic alkalosis do not respond to caffeine. metabolic alkalosis in critically ill foals frequently is associated with electrolyte abnormalities, creating differences in strong ion balance. one handles this ph perturbation best by correcting the underlying electrolyte problem. maintaining tissue perfusion and oxygen delivery to tissues is a cornerstone of therapy for pas to avoid additional injury. one should maintain the oxygen-carrying capacity of the blood; some foals require transfusions to maintain a packed cell volume greater than 20%. adequate vascular volume is important, but one should take care to avoid fluid overload in the foal. early evidence of fluid overload is subtle accumulation of ventral edema between the front legs and over the distal limbs. fluid overload can result in cerebral edema, pulmonary edema, and edema of other tissues, including the gastrointestinal tract. this edema interferes with normal organ function and worsens the condition of the patient. one maintains perfusion by supporting cardiac output and blood pressure by judicious use of intravenous fluid support and inotrope/pressor support. the author does not target therapy to a specific systolic, mean, or diastolic pressure but monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for these patients to require pressor therapy is not unusual, but in some cases the hypoxic damage is sufficiently severe to blunt the response of the patient to the drugs. the kidney is a target for injury in patients with pph, and for renal compromise to play a significant role in the demise of these foals is not unusual. clinical signs of renal disease are generally referable to disruption of normal control of renal blood flow and tubular edema leading to tubular necrosis and renal failure. these foals have signs of fluid overload and generalized edema. one must balance urine output and fluid therapy in these cases to prevent additional organ dysfunction associated with edema. although evidence has accumulated that neither dopamine nor furosemide play a role in protecting the kidney or reversing acute renal failure, these agents can be useful in managing volume overload in these cases. [154] [155] [156] the aim is not to drive oliguric renal failure into a highoutput condition but rather to enhance urine output. overzealous use of diuretics and pressors in these cases can result in diuresis requiring increased intravenous fluid support and can be counterproductive. the author's approach is more conservative. low doses of dopamine administered as a constant rate infusion of 2 to 5 âµg/kg/min are usually effective in establishing diuresis by natriuresis. one should avoid large doses of dopamine (>20 âµg/kg/min) because high doses can produce systemic and pulmonary vasoconstriction, potentially exacerbating pph. 157 one can administer a bolus (0.25 to 1.0 mg/kg) or constant rate infusion (0.25 to 2.0 mg/kg/hr) of furosemide, but once furosemide diuresis is established, one must evaluate electrolyte concentrations and blood gas tensions frequently because potassium, chloride, and calcium losses can be considerable and because significant metabolic alkalosis can develop from strong ion imbalances. the author does not aim for urine production rates of 300 ml/hr, as has been presented by other authors as a urine output goal for critically ill equine neonates. 158 rather the author looks for urine output that is appropriate for fluid intake and does not attempt to drive urine output to an arbitrary goal by excessive fluid administration or pressor use. although the average urine output for a normal equine neonate is about 6 ml/kg/hr (~300 ml/hr for a 50-kg foal), these values were obtained from normal foals drinking a milk diet with a large free water component. [98] [99] [100] the urine of normal newborn foals is dilute, reflecting the large free water load they incur by their diet. expecting critically ill foals to produce such large volumes of urine, particularly those on restricted diets or receiving total parenteral nutrition, is an exercise in futility, and manipulating fluid, pressor, or diuretic therapy in attempt to meet an artificial goal is inappropriate. fluid therapy in the critically ill neonate is discussed later in this chapter. one final caveat regarding renal dysfunction in pas is that one should perform therapeutic drug monitoring when it is available. many antimicrobial agents used to manage these cases, most notably the aminoglycosides, depend on renal clearance. aminoglycoside toxicity occurs in the equine neonate and exacerbates or complicates the management of renal failure originally resulting from primary hemodynamic causes. the author monitors aminoglycoside concentrations for 30-minute peak and 23-to 24-hour trough values in these cases and adjusts dosage and frequency of drug administration based on these results. the author considers a trough value of less than 2 âµg/dl as desirable for gentamicin and amikacin. foals with pas suffer from a variety of problems associated with abnormalities within the gastrointestinal tract. 159 commonly they have ileus, recurrent excessive gastric reflux, and gas distention. these problems are exacerbated by constant feeding in the face of continued dysfunction and continued hypoxia. frequently, enteral feeding cannot meet their nutritional requirements, and partial or total parenteral nutrition is required. one must give special attention to passive transfer of immunity (see failure of passive transfer) and glucose homeostasis in these cases. although some practitioners use prokinetic agents as therapy for ileus in these cases, the author's approach is again more conservative. appearance of damage to the gastrointestinal tract can be subtle and lag behind other clinical abnormalities for days to weeks. low-grade colic, decreased gastrointestinal motility, decreased fecal output, and low weight gain are among the most common clinical signs of gastrointestinal dysfunction in these case, but more severe problems, including necrotizing enterocolitis and intussusception, have been associated with these cases. the return to enteral feeding must be slow in many of these cases. a currently debated topic is constant versus pulsed enteral feeding. [160] [161] [162] the author uses pulsed feeding through an indwelling small-gauge feeding tube. in many foals these tubes stay in place for weeks and cause no problems as the foals are returned to their dams for sucking or are trained to drink from a bottle or bucket. foals with pas are also susceptible to secondary infection. treatment of recognized infection is covered under sepsis in this chapter. if infection is recognized in these patients after hospitalization, one should give attention to the likelihood of nosocomial infection and should direct antimicrobial therapy based on known nosocomial pathogens in the nicu and their susceptibility patterns until culture and sensitivity results become available. one should make repeat determinations of immunoglobulin g (igg) concentration; additional intravenous plasma therapy may be required. nosocomial infections are often rapidly overwhelming, and acute deterioration in the condition of a foal with pas should prompt a search for nosocomial infection. the prognosis for foals with pas is good to excellent when the condition is recognized early and aggressively treated in term foals. up to 80% of these neonates survive and go on to lead productive and useful athletic lives. [20] [21] [22] [23] the prognosis decreases with delayed or insufficient treatment and concurrent problems such as prematurity and sepsis. in human nicus the survival rates of low-gestationlength infants has increased dramatically since the 1980s concurrent with improvements in obstetric and neonatal care. the now routine, well-validated use of antenatal steroid and artificial surfactant therapies has contributed greatly to the enhanced survival of this patient population, although the use of these particular therapies is not common or frequently indicated in the equine nicu. 163, 164 however, with improved care, outcomes in the equine nicu population have improved also, with survival of premature patients in many nicus exceeding 80%. 21 in the equine population, gestation length is much more flexible than in the human population; however, the definition of the term prematurity needs reexamination. traditionally, prematurity is defined as a preterm birth of less than 320 days of gestation in the horse. given the variability of gestation length in the horse, ranging from 310 days to more than 370 days in some mares, a mare with a usual gestation length of 315 days possibly could have a term foal at 313 days, whereas a mare with a usual gestation length of 365 days may have a premature foal at 340 days, considered the normal gestation length. foals that are born postterm but are small are termed dysmature; a postmature foal is a postterm foal that has a normal axial skeletal size but is thin to emaciated. dysmature foals may have been classified in the past as small for gestational age and are thought to have suffered placental insufficiency, whereas postmature foals are usually normal foals that have been retained too long in utero, perhaps because of an abnormal signaling of readiness for birth, and have outgrown their somewhat aged placenta. postmature foals become more abnormal the longer they are maintained, also may suffer from placental insufficiency, and are represented best by the classic foal born to a mare ingesting endophyteinfested fescue. 165 box 19-3 compares the characteristics of premature/dysmature foals with those of postmature foals. the causes of prematurity/dysmaturity/postmaturity include the causes of high-risk pregnancy presented in box 19-1. additional causes include iatrogenic causes such as early elective induction of labor based on inaccurate breeding dates or misinterpretation of late-term colic or uterine bleeding as ineffective labor. most causes remain in the category of idiopathic, with no discernible precipitating factor. despite lack of an obvious cause, premature labor and delivery does not just happen, and even if undetermined, the cause may continue to affect the foal in the postparturient period. all body systems may be affected by prematurity, dysmaturity, and postmaturity, and thorough evaluation of all body systems is necessary. respiratory failure is common in these foals, although the cause usually is not surfactant deficiency. immaturity of the respiratory tract, poor control of respiratory vessel tone, and weak respiratory muscles combined with poorly compliant lungs and a greatly compliant chest wall contribute to respiratory failure in these cases. most require oxygen supplementation and positional support for optimal oxygenation and ventilation. one must extend effort to maintain these "floppy foals" in sternal recumbency. some foals may require mechanical ventilation. these foals also require cardiovascular support but are frequently unresponsive to commonly used pressors and inotropes: dopamine, dobutamine, epinephrine, and vasopressin. careful use of these drugs and judicious intravenous fluid therapy are necessary. the goal should not be one of achieving specific pressure values (e.g., mean arterial pressure of 60 mm hg) but of adequate perfusion. renal function, reflected in low urine output, is frequently poor initially in these cases because of delay in making the transition from fetal to neonatal glomerular filtration rates. 166 the delay can result from true failure of transition or from hypoxic/ischemic insult. one should approach fluid therapy cautiously in these cases; initial fluid restriction may be in order to avoid fluid overload. many premature/dysmature/postmature foals have suffered a hypoxic insult and have all of the disorders associated with pas, including hie. treatment is similar to that of term foals with these problems. these foals also are predisposed to secondary bacterial infection and must be examined frequently for signs consistent with early sepsis or nosocomial infection. the gastrointestinal system of these foals is not usually functionally mature, which may result from a primary lack of maturity or from hypoxia. dysmotility and varying degrees of necrotizing enterocolitis are common. one commonly encounters hyperglycemia and hypoglycemia. hyperglycemia generally is related to stress, increased levels of circulating catecholamines, and rapid progression to gluconeogenesis, whereas hypoglycemia is associated with diminished glycogen stores, inability to engage gluconeongenesis, sepsis, and hypoxic damage. 167 immature endocrine function is present in many of these foals, particularly regarding the hypothalamic-pituitary-adrenal axis, and contributes to metabolic derangements. 168, 169 one should delay enteral feeding when possible until the foal is stable regarding metabolic and cardiorespiratory parameters. on intiating enteral feeding, one should provide small volumes initially and slowly increase the volume over several days. one frequently encounters musculoskeletal problems, particularly in premature foals, that include significant flexor laxity and decreased muscle tone. postmature foals frequently are affected by flexure contracture deformities, most likely because of decreased intrauterine movement as they increase in size. premature foals frequently exhibit decreased cuboidal bone ossification that predisposes them to crush injury of the carpal and tarsal bones if weight bearing is not strictly controlled. physical therapy in the form of standing and exercise is indicated in the management of all these problems, but one should take care to ensure that the patient does not fatigue or stand in abnormal positions. bandaging of the limbs is contraindicated because this only increases laxity, although light bandages over the fetlock may be necessary to prevent injury to that area if flexor laxity is severe. the foals are predisposed to angular limb deformity and must be observed closely and frequently for this problem as they mature. 170 the overall prognosis for premature/dysmature/ postmature foals remains good with intensive care and good attention to detail. many of these foals (up to 80%) survive and become productive athletes. 21 complications associated with sepsis and musculoskeletal abnormalities are the most significant indicators of poor athletic outcome. the last 20 years have seen an explosion of new therapeutic agents purportedly useful for treating sepsis. unfortunately, clinical trials investigating these new therapies have failed to demonstrate a positive effect, have shown negative results, or have resulted in diametrically opposed study results, one showing a benefit and another showing no benefit or a detrimental effect. on a positive note, the survival rate of foals being treated for sepsis has improved. work was done regarding foal diseases and their treatment in the 1960s, but the field did not attract much serious attention until the 1980s. since that time almost every major veterinary college and many large private referral practices have constructed nicus or their equivalent. next to hypoxic ischemic asphyxial syndromes, sepsis is the number one reason for presentation and treatment at these facilities. neonatal septicemia of the horse has been the subject of three international workshops, 171-173 and a perinatology lecture covering some aspect of neonatal sepsis has been presented at almost every large continuing education meeting attended by equine veterinarians. concensus criteria conferences 1 in the early 1990s defined sepsis and septic shock for human beings. 174, 175 sepsis was defined as the systemic response to infection manifested by two or more of the following conditions as a result of infection: a) temperature >38â°c or <36â°; b) heart rate >90 beats/min; c) respiratory rate >20 breaths per minute or paco 2 <32 torr; and d) white blood cell count >12,000 cell/âµl, <4,000 cell/âµl, or >10% immature (band) forms. septic shock was defined as sepsis induced hypotension or the requirement for vasopressors/ionotropes to maintain blood pressure despite adequate fluid resuscitation along with the presence of perfusion abnormalities that may include lactic acidosis, oliguria, or acute alteration in mental status. these definitions are broadly acceptable and applicable to neonatal sepsis in foals, and many of the treatment modalities in human medicine have been applied in some manner to the equine neonatal patient. additional definitions that have come into vogue that are actually useful at times, include the following: sirs, the systemic inflammatory response system; mods, multiple organ system dysfuction; and mofs, multiple organ failure syndrome. (sirs is sick, mods is sicker, and mofs is dying.) the compensatory response syndrome (cars) ideally balances sirs and keeps it from becoming detrimental. if balance is achieved, recovery is possible. imbalance progresses to septic shock, mods, and mofs. in horses, mods is manifested most commonly as renal failure, hepatic failure, central nervous system dysfunction, and disseminated intravascular coagulation. managing the septic patient involves early recognition of all the potential alphabet combinations and supporting the patient or intervening in the face of multiple clinical consequences, termed chaos (cardiovacular compromise; homeostasis; apoptosis; organ dysfunction; suppression of the immune system). 176 inflammatory mediators are involved in all these processes and can be beneficial or detrimental, depending on timing and opposing responses. neutrophils, platelets, lymphocytes, macrophages, and endothelial cells are involved, and the implicated inflammatory molecules grow daily in numbers. sepsis in the foal initially can be subtle, and the onset of clinical signs varies depending on the pathogen involved and the immune status of the foal. for the purposes here, the discussion is limited to bacterial sepsis, but the foal also is susceptible to viral and fungal sepsis, which can appear similar to bacterial sepsis. failure of passive transfer (fpt) of immunity can contribute to the development of sepsis in a foal at risk. 177, 178 testing for and treating fpt has received attention in the veterinary literature. it remains true, however, that foals presented to nicus that have an ultimate diagnosis of sepsis have fpt. 16, 19 the current recommendation is that foals have igg levels greater than or equal to 800 mg/dl for passive transfer to be considered adequate. other risk factors for the development of sepsis include any adverse advents at the time of birth, maternal illness, or any abnormalities in the foal. although the umbilicus frequently is implicated as a major portal of entry for infectious organisms in the foal, the gastrointestinal tract may be the primary site of entry. 179 other possible portals of entry include the respiratory tract and wounds. early signs of sepsis include depression, decreased suck reflex, increased recumbency, fever, hypothermia, weakness, dysphagia, failure to gain weight, increased respiratory rate, tachycardia, bradycardia, injected mucous membranes, decreased capillary refill time, shivering, lameness, aural petechia, and coronitis. if sepsis is recognized early, patients with sepsis may have a good outcome, depending on the pathogen involved. gram-negative sepsis remains the most commonly diagnosed, but increasingly gram-positive septicemia is being recognized. 180 foals in intensive care units and at referral hospitals have an additional risk of nosocomial infection. an attempt to isolate the organim involved early in the course of the disease becomes important. if possible, one should obtain blood cultures, and if localizing signs are present, one should obtain samples as deemed appropriate. cultures should be aerobic and anaerobic. recently, work has been done evaluating real-time polymerase chain reaction technology in sepsis in the foal as a means of identifying causative organisms. 181, 182 until one obtains antimicrobial sensitivity patterns for the pathogen involved, one should initiate broad-spectrum antimicrobial therapy (table 19 -5). intravenously administered amikacin and penicillin are good first-line choices, but one should monitor renal function closely. other first-line antimicrobial choices might include high-dose ceftiofur sodium or ticarcillin/clavulanic acid. one should treat failure of passive transfer if present. one should provide intranasal oxygen insufflation at 5 to 10 l/min even if hypoxemia is not present to decrease the work of breathing and provide support for the increased oxygen demands associated with sepsis. 183 should arterial blood gas analysis reveal significant hypoventilation, one may administer caffeine orally or per rectum to increase central respiratory drive. mechanical ventilation may be necessary in cases of severe respiratory involvement such as with acute lung injury or acute respiratory distress syndrome. if the foal is hypotensive, one may administer pressor agents or inotropes by constant rate infusion (table 19-6) . inotrope and pressor therapy generally is restricted to referral centers where these drugs can be given as constant rate infusions and blood pressure can be monitored closely. some practitioners use nonsteroidal antiinflammatory agents and, in specific circumstances, corticosteroids. use of these drugs should be judicious because they may have several negative consequences for the foal including renal failure and gastric/dunodenal ulceration. [184] [185] [186] nursing care is one of the most important aspects of treating septic foals. foals should be kept warm and dry. they should be turned at 2-hour intervals if they are recumbent. feeding septic foals can be a challenge if gastrointestinal function is abnormal, and total parenteral nutrition may be needed. if at all possible, foals should be weighed daily and blood glucose levels monitored frequently. some foals become persistently hyperglycemic on small glucose infusion rates. these foals may benefit from constant rate low-dose insulin infusions (table 19-7) . recumbent foals must be examined frequently for decubital sore development, the appearance of corneal ulcers, and for heat and swelling associated with joints and physis. the prognosis for foals in the early stages of sepsis is fair to good. once the disease has progressed to septic shock the prognosis decreases, although short-term botulism is a neuromuscular disease of foals characterized by flaccid paralysis. 187 although the disease is discussed in detail elsewhere in this text, the form most commonly observed in foals, the toxicoinfectious form, deserves some specific comments. the causative organism is clostridium botulinum, an anaerobic organism. although affected adults usually acquire the disease by ingestion of preformed toxin elucidated from the organism, in the foal less than 8 months of age the organism can survive and multiply in the gastointestinal tract and produce necrotic foci within the liver, giving the foal constant exposure to newly formed toxin. the horse is exquisitely sensitive to the toxin, and only small quantities of toxin are required to produce clinical signs and death in affected animals. the îµ-toxin of c. botulinum binds to the presynaptic membrane of motor neurons and prevents transmission of impulses by blocking the release of acetylcholine from the presynaptic vessicles. this block produces the clinical signs of muscle weakness, manifested in foals as trembling (shaker foals) or acute recumbency. 188 pupillary dilation, dysphagia, tremors, recumbency, and terminal respiratory distress caused by respiratory muscle paralysis occur. foals can be found acutely dead. in endemic areas (the northeast and mid-atlantic regions of united states), for these foals to be evaluated first as having colic is not unusual. treatment aims to neutralize the toxin by administration of botulinum antitoxin and to provide antimicrobial treatment of the infection with penicillin, metronidazole, and/or oxytetracycline. 189, 190 at a minimum, feeding of milk replacer via indwelling nasogastric tube at 20% of the body weight of the foal per day divided into every 2-hour meals is required. many of these foals require respiratory support (in the form of intranasal oxygen insufflation), because of respiratory muscle paralysis. respiratory acidosis is present on arterial blood gas analysis in most of these foals because of hypoventilation and lateral recumbency, but they can tolerate some degree of hypercapnia (paco 2~7 0 mm hg) if the ph is normal and oxygenation (pao 2 >70 mm hg; percent oxygen saturation of hemoglobin, >90%) is adequate. metabolic alkalosis can accompany the respiratory acidosis, but this is a compensatory change and resolves once gas exchange is normalized. some of these patients require mechanical ventilation, which may be lifesaving. one may discontinue mechanical ventilation as clinical signs resolve and the respiratory muscles gain strength. nursing care is important, and these foals should be turned every 2 hours. they should be maintained in sternal recumbency if possible and kept warm and dry. with good nursing care, good nutritional support, and adequate respiratory support, the prognosis for these foals is good. the limiting factor in the prognosis for life is often financial. 190 foals that recover from the acute stage of this disease eventually fully recover. botulism is an expensive disease to treat and is also an entirely preventable disease. 189, 190 all pregnant mares in endemic areas should be vaccinated against c. botulinum. vaccination does not prevent all cases of botulism, particularly if the foal has failure of passive transfer or acquires the disease after maternal immunity wanes and before its own vaccination. nutritional muscular dystrophy or white muscle disease is a vitamin e/selenium-responsive muscle disease of horses of all ages probably caused by a dietary deficiency of selenium and vitamin e. 191 the condition occurs most commonly in geographic areas with low selenium levels in the soil, generally the northeastern, northwestern, great lakes and mid-atlantic regions of the united states. two forms of the disease are described in foals: the fulminant form, in which the foal is found acutely dead, and the subacute form. in the fulminant form, death usually is attributed to myocardial lesions resulting in cardiovascular collapse. the subacute form is characterized by dysphagia and gait abnormalities primarily caused by stiffness of the muscles of locomotion. paralysis, if present, is not flaccid as in botulism. abnormal function of respiratory muscles may complicate the clinical situation. aspiration pneumonia may be present following problems associated with swallowing; the tongue and pharyngeal muscles frequently are affected in the early stages of disease. 191 foals with severe disease may have widespread muscle necrosis leading to hyperkalemia, which can be severe and result in death of the foal. serum activities of the muscle enzymes creatine kinase and aspartate aminotransferase may be greatly increased. diagnosis is confirmed at necropsy or ante mortem by determination of decreased vitamin e, selenium, and glutathione peroxidase concentrations in the blood of the foal before supplementation. myoglobinuria and acute renal failure are not uncommon in these foals. treatment of foals with nutritional muscular dystrophy is primarily supportive. one should address all metabolic abnormalities. some foals require intranasal oxygen insufflation. affected foals are unable to suck effectively, and one should provide enteral (via an indwelling nasogastric tube) or parenteral nutritional support. because of the high likelihood of aspiration pneumonia, one should administer broad-spectrum antimicrobial therapy parenterally. the patient should be kept quiet and should be stimulated minimally. affected foals should receive parenteral (intramuscular) vitamin e and selenium supplementation. selenium is toxic in large doses. the prognosis for severely affected foals is guarded. for less severely affected foals the prognosis is good with appropriate treatment. the disease is preventable by ensuring that mares receive sufficient vitamin e and selenium while pregnant and by supplementing foals with parenteral injections of vitamin e and selenium at birth in endemic areas. a more complete discussion of the pathophysiology of this disease and the nutritional management is presented elsewhere in this text. primary liver disease is uncommon in the foal and occurs primarily as a sequela to sepsis. clinical signs of severe liver disease may include depression, ataxia, and seizures. in affected foals, increases in serum liver enzyme activities and concentrations of ammonia and bile acids frequently can be identified. the mechanism(s) underlying hepatoencephalopathy are not delineated clearly, although increased excitatory neurotransmitters, or compounds that mimic their activity, are implicated. hepatoencephalopathy is discussed in more detail elsewhere in this text. tyzzer's disease (clostridium piliformis infection) rarely causes primary liver disease in foals from 4 to about 40 days of age. this disease is almost uniformly fatal. the incubation period is short, and the mare is thought to be the carrier. [192] [193] [194] [195] [196] clinical signs range from acute death to depression, fever, and pronounced icterus. the feces of affected foals may appear white to grey because of the lack of bile. clinicopathologic abnormalities include leukopenia, hyperfibrinogenemia, metabolic acidosis, and hypoglycemia. 197, 198 liver lesions at postmortem are characterized microscopically by multiple foci of necrosis. one usually can demonstrate variable numbers of elongated, slender intracytoplasmic bacilli within hepatocytes bordering the necrotic foci. infiltration of the portal triads with inflammatory cells and biliary duct hyperplasia and degeneration are observable. the bacillus also occurs in association with myocardial lesions. lesions in the intestine are characterised by mucosal necrosis with inflammatory cell infiltration, increased mucus production, submucosal lymphoid hyperplasia, and submucosal hemorrhage. necrosis of lymphoid follicles, congestion, and hemorrhage can be present in the spleen and mesenteric lymph nodes. 196 affected foals may have a profound metabolic acidosis that is unresponsive to treatment. the clinical course is short, and most affected foals die within a few hours of developing neurologic signs. primary liver disease has been reported in association with ferrous sulfate administration in a probiotic compound. 199 the lesion was massive hepatocellular necrosis and liver failure. the product is no longer commercially available. portosystemic shunt is rare in the foal but has been reported in foals as young as 3 months of age. [200] [201] [202] most infectious causes of neurologic abnormalities in foals are associated with sepsis. although rarely reported, halicephalobus gingivalis (deletrix) infection has been reported in three foals; in one case the foal was 3 weeks of age. 203, 204 possibly transmission in these cases was transmammary; the dam in one case died 1 year later with confirmed h. deletrix infestation of her udder. listeria monocytogenes has been reported as a cause of neurologic disease in foals. 205 recently, sarcocystis neurona was identified as the causative agent of central nervous system disease in a foal, and equine herpes myeloencephalitis has been diagnosed in individual foals and in herd outbreaks involving foals. 206, 207 neospora also was reported in one foal recently. 208 rhodococcus equi abscesses can form in the central nervous system or cause neurologic signs associated with compression, as with vertebral body abscesses. [209] [210] [211] cerebellar hypolasia, occipitoatlantoaxial malformation, and agenesis of the corpus callosum with cerebellar vermian hypoplasia have been reported in foals. [212] [213] [214] [215] [216] [217] ivermectin toxicity and moxidectin toxicity have been reported. 218, 219 electrolyte abnormalities such as extreme hypo-or hypernatremia may result in neurologic manifestations of disease. 220, 221 cervical stenotic myelopathy and degenerative myelopathy also have been reported in foals, although the age at onset is usually more than 4 months. 222 idiopathic epilepsy of arabian foals usually is associated with another infectious disease and is thought to be temporary and self-limiting. causes, diagnosis, and treatment of fpt of immunity are covered in detail elsewhere in this text. failure of passive transfer occurs when a foal fails to ingest a significant quantity of good-quality colostrum. failure of passive transfer may occur by several mechanisms: failure of the foal to suck from the dam for any reason and failure of the dam to produce sufficient quantity of quality colostrum. box 19-4 presents causes of fpt. several methods are available for measuring igg concentration in blood; the most reliable are enzyme-linked immunosorbent assay and single radial immunodiffusion technology-based tests. [223] [224] [225] [226] [227] [228] [229] foals usually are tested at 24 hours of age, but one may test the foal earlier if colostrum ingestion has occurred and a concern exists regarding the passive transfer of immunity status of the foal, recognizing that additional increases in igg concentration may occur with additional time. 230, 231 the concentration of igg in the blood of the foal has been used as an indicator of the adequacy of passive transfer, but the actual blood concentration at which fpt is diagnosed has been challenged in recent years. [232] [233] [234] foals with sepsis commonly have a serum igg concentration of less than 800 mg/dl. 16, 19 foals with fpt are more likely to die from sepsis. 177, 178, [235] [236] [237] one should consider the igg concentration only as a marker for adequacy of colostral absorption. all the measured igg is unlikely to be directed against the specific pathogen affecting any particular neonate, and igg is not the only immune protection afforded the foal by colostrum. many factors that confer local and more general immunity to the newborn are present in colostrum; these include growth factors, cytokines, lactoferrins, cd14, leukocytes, and other yet to be described proteins. [240] [241] [242] [243] [244] by considering igg a marker of adequacy for passive transfer, similar to î³-glutamyltransferase in calves, the clinician can make choices for replacement that are more beneficial to the patient. 245 after one identifies fpt in a foal, treatment depends on the current condition of the foal and its local environment. foals not presently ill and on well-managed farms with low population density and low prevalence of disease may not require treatment if their igg concentration is between 400 and 800 mg/dl. critically ill neonates with fpt in an equine nicu are by definition ill and in an environment with high disease prevalence. these patients require immediate treatment of fpt and frequent reassessment of their passive immunity status. critically ill foals often fail to demonstrate the expected increase in blood igg concentration based on grams of igg administered per kilogram of body mass compared with healthy, colostrum-deprived foals. 235, 246, 247 sick foals also demonstrate a more rapid decline in igg concentration than do healthy foals because they use and catabolize available protein. one may treat foals with fpt by oral or intravenous administration of various products containing igg. one can attempt oral administration of additional colostrum or igg-containing products such as plasma, serum, or lyophilized colostrum in foals less than 12 to 24 hours of age. [248] [249] [250] depending on the age of the foal and the maturity and function of the gastrointestinal tract, this treatment may be effective. many nicus and large breeding farms maintain colostrum banks for this purpose. one should administer plasma intravenously if the foal is not expected to absorb additional colostrum or if the enteral route is unavailable. commercially available hyperimmune plasma products designed for use in foals are available and can be stored frozen. plasma and banked colostrum should be stored in a non-frost-free freezer to minimize protein loss associated with freeze-thaw cycling. 251 one should administer plasma through special tubing with an in-line filter and should monitor patients closely for transfusion reactions. 252 one may use serum and concentrated igg products, but the practitioner should be aware that many of these products focus on igg retention and not on other factors associated with passive transfer of immunity. one should measure igg concentration after transfusion and provide additional plasma as necessary. administration of plasma to critically ill foals without fpt may be beneficial through provision of other factors present in the plasma. in these situations, fresh frozen plasma or fresh plasma may be best, particularly if transfusion of clotting proteins is desired. neonatal isoerythrolysis is a hemolytic syndrome in newborn foals caused by a blood group incompatibility between the foal and dam and is mediated by maternal antibodies against foal erythrocytes (alloantibodies) absorbed from the colostrum. the disease most often affects foals born to multiparous mares and should be suspected in foals less than 7 days of age with clinical signs of icterus, weakness, and tachycardia. a primiparous mare can produce a foal with neonatal isoerythrolysis if she has received a prior sensitizing blood transfusion or has developed placental abnormalities in early gestation that allowed leakage of fetal red blood cells into her circulation. many are the causes of jaundice in newborn foals, including sepsis, meconium impaction, and liver failure, but these usually can be differentiated readily from neonatal isoerythrolysis by measuring the packed cell volume, which is usually less than 20% in foals with neonatal isoerythrolysis. foals with neonatal isoerythrolysis are born clinically normal then become depressed and weak and have a reduced suckle response within 12 to 72 hours of birth. the rapidity of onset and severity of disease are determined by the quantity and activity of absorbed alloantibodies. affected foals have tachycardia, tachypnea, and dyspnea. the oral mucosa is initially pale and then becomes icteric in foals that survive 24 to 48 hours. hemoglobinuria may occur. seizures caused by cerebral hypoxia are a preterminal event. the salient laboratory findings are anemia and hyperbilirubinemia. most of the increased bilirubin is unconjugated, although the absolute concentration of conjugated bilirubin generally is increased well above normal. urine may be red to brown and is positive for occult blood. the natural development of neonatal isoerythrolysis has several prerequisites. first, the foal must inherit from the sire and express an erythrocyte antigen (alloantigen) that is not possessed by the mare. blood group incompatibility between the foal and dam is not particularly uncommon, but most blood group factors are not strongly antigenic under the conditions of exposure through previous parturition or placental leakage. factor aa of the a system and factor qa of the q system are highly immunogenic, however, and nearly all cases of neonatal isoerythrolysis are caused by antibodies to these alloantigens. the exception is in the case of mule foals in which a specific donkey factor has been implicated. [253] [254] [255] mares that are negative for aa or qa or both are considered to be at risk for producing a foal with neonatal isoerythrolysis. the risk involves approximately 19% and 17% of thoroughbred and standardbred mares, respectively. second, and perhaps most important, the mare must become sensitized to the incompatible alloantigen and produce antibodies to it. the mechanism for this is not known in many instances but generally is believed to result from transplacental hemorrhage during a previous pregnancy involving a foal with the same incompatible blood factor. 255 sensitization via transplacental contamination with fetal erythrocytes earlier in the current pregnancy is possible, but an anamnestic response is generally necessary to induce a pathogenic quantity of alloantibodies. 256 ten percent of thoroughbred mares and 20% of standardbred mares have antibodies to the ca blood group antigen without known exposure to erythrocytes. 255 some common environmental antigen is postulated possibly to lead to production of anti-ca antibodies. data suggest that these natural antibodies may suppress an immune response to other blood group antigens because mares negative for aa that have anti-ca antibodies often do not produce antibodies to aa of the erythrocytes in their foals that also contain ca antigen. this antibodymediated immunosuppression is thought to result from the destruction of fetal cells before the dam mounts an immune response to other cell surface antigens. natural alloantibodies have not been associated with neonatal isoerythrolysis in horses. after the mare becomes sensitized to the erythrocytes of her foal, alloantibodies are concentrated in the colostrum during the last month of gestation. unlike the human neonate, which acquires alloantibodies in utero and thus is born with hemolytic disease, the foal is protected from these antibodies before birth by the complex epitheliochorial placentation of the mare. thus the final criterion for foal development of neonatal isoerythrolysis is ingestion in the first 24 hours of life of colostrumcontaining alloantibodies specific for foal alloantigens. immunoglobulin-coated foal erythrocytes are removed prematurely from circulation by the mononuclear phagocyte system or are lysed intravascularly via complement. the rapidity of development and severity of clinical signs are determined by the amount of alloantibodies that was absorbed and their innate activity. alloantibodies against aa are potent hemolysins and generally are associated with a more severe clinical syndrome than antibodies against qa or other alloantigens. the highest alloantibody titers are likely to be produced by mares that were sensitized in a previous pregnancy and then subsequently reexposed to the same erythrocyte antigen during the last trimester of the current pregnancy. prior sensitization of a mare by blood transfusion or other exposure to equine blood products may predispose to neonatal isoerythrolysis. 256 one can make a tentative diagnosis of neonatal isoerythrolysis in any foal that has lethargy, anemia, and icterus during the first 4 days of life. blood loss anemia caused by birth trauma is attended by pallor. icterus caused by sepsis or liver dysfunction would not be associated with anemia. one must base the definitive diagnosis of neonatal isoerythrolysis on demonstration of alloantibodies in the serum or colostrum of the dam that are directed against foal erythrocytes. the most reliable serodiagnostic test for neonatal isoerythrolysis is the hemolytic cross-match using washed foal erythrocytes, mare serum, and an exogenous source of absorbed complement (usually from rabbits). 5 although this test is impractical in a practice setting, a number of qualified laboratories routinely perform this diagnostic service. the direct antiglobulin test (coombs' test) may demonstrate the presence of antibodies on foal erythrocytes; however, false negatives occur frequently. most human or veterinary hematology laboratories can perform routine saline agglutination cross-match between mare serum and foal cells. because some equine alloantibodies act only as hemolysins, agglutination tests may be falsely negative. most field screening tests of colostrum have not proved to be reliable enough for practical use. if one recognizes neonatal isoerythrolysis when the foal is less than 24 hours old, one must withhold the dam's milk and feed the foal an alternative source of milk during the first day of life. one can accomplish this by muzzling the foal and feeding it via nasogastric tube. the minimum necessary amount of milk is 1% of body mass every 2 hours (e.g., a 50-kg foal should receive 500 ml or 1 pint of mare's milk or milk replacer every 2 hours). the udder of the mare should be stripped regularly (at least every 4 hours) and the milk discarded. in most instances, clinical signs are not apparent until after the foal is 24 hours old, when colostral antibodies have been depleted or the absorptive capacity of the foal's intestine for immunoglobulin has diminished. withholding milk at this point is of minimal benefit. supportive care to ensure adequate warmth and hydration is paramount. the foal should not be stressed and exercise must be restricted. confining the mare and foal to a box stall is a best. intravenous fluids are indicated to promote and minimize the nephrotoxic effects of hemoglobin and to correct any fluid deficits and electrolyte and acid-base imbalances. antimicrobials may be necessary to prevent secondary infections. one should monitor foals carefully for the necessity of blood transfusion, although transfusion should be used only as a lifesaving measure. when the packed cell volume drops below 12%, blood transfusion is warranted to prevent life-threatening cerebral hypoxia. erythrocytes from the dam are perfect in terms of nonreactivity with the blood of the foal; however, the fluid portion of the blood of the mare has to be removed completely from the cells to prevent administration of additional harmful alloantibodies to the foal. one can pellet the erythrocytes of the dam from blood collected in acid-citrate-dextrose solution by centrifugation or gravity and then aseptically draw off the plasma by suction apparatus or syringe and replace it with sterile isotonic (0.9%) saline. one thoroughly mixes the cells with the saline and then repeats the centrifugation or sedimentation, followed by aspiration and discarding of the saline. one should perform this washing process at least three times. one then can suspend the packed erythrocytes in an equal volume of isotonic saline for administration. erythrocyte washing by centrifugation is more desirable than gravity sedimentation because antibody removal is more complete and packed cell preparations can be prepared more quickly (each gravity sedimentation requires 1 to 2 hours). packed red blood cells are advantageous in overcoming the problem of volume overload. when equipment or conditions do not allow the safe use of dam erythrocytes, an alternative donor is necessary. because the alloantibodies absorbed by the foal generally are directed against aa or qa and because the latter are highly prevalent among most breeds of horses, a compatible blood donor is difficult to identify. the odds of finding a donor without aa or qa are higher in quarter horses, morgans, and standardbreds than in thoroughbreds and arabians. previously blood-typed individuals negative for aa and qa and free of alloantibodies are optimal. one should give 2 to 4 l of blood or 1 to 2 l of packed erythrocytes over 2 to 4 hours. these allogeneic cells have a short life span and represent a large burden to the neonatal mononuclear phagocyte system, which may cause increased susceptibility to infection. in addition, these cells sensitize the foal to future transfusion reactions. one must measure all potential harm against the benefit in each situation. if a mule foal is the patient, one should not use blood from a female previously bred to a donkey. in cases in which transfusion will be delayed, one cannot identify a compatible donor, or the packed cell volume is so low as to be life-threatening (hemoglobin <5 mg/dl), one may administer polymerized bovine hemoglobin products at a dose of 5 to 15 ml/kg. 257 one may use dexamethasone (0.08 mg/kg) to treat peracute neonatal isoerythrolysis if the packed cell volume is less than 12% and transfusion may be delayed or is not fully compatible, but dexamethasone has detrimental effects on blood glucose regulation in the neonate, and because the antibody in question is of maternal origin, corticosteroid therapy in immunosuppressive doses probably is not indicated. intranasal oxygen insufflation (5 to 10 l/min) may be beneficial. most foals with neonatal isoerythrolysis have adequate passive transfer of immunity, but antimicrobial therapy is indicated to protect against secondary sepsis resulting from the compromised condition of the foal. supportive care and good nursing care, including keeping the foal warm and quiet are essential. one should expect the packed cell volume to decline again 4 to 7 days after transfusion. 258 the prognosis for neonatal isoerythrolysis in foals depends on the quantity and activity of absorbed antibodies and is indirectly proportional to the rate of onset of signs. in peracute cases the foal may die before the problem is recognized, whereas foals with slowly progressive signs often live with appropriate supportive care. like most diseases, neonatal isoerythrolysis is much more effectively prevented than treated. 259 any mare that has produced a foal with neonatal isoerythrolysis should be suspect for the production of another affected foal; thus one should provide all subsequent foals with an alternative colostrum source and discard the colostrum of the dam unless she is bred to a stallion with known blood type compatibility. mares negative for aa and qa alloantigens are most at risk of producing affected foals, thus they should be identified by blood-typing. subsequently, breeding of these mares may be restricted to aa-and qa-negative stallions, thus eliminating the possibility of producing an affected foal. in breeds with a high prevalence of aa or qa alloantigens (e.g., thoroughbreds and arabians), a stallion negative for these and suitable based on other criteria may be difficult to identify. if these "at risk" mares are bred as desired, their serum should be screened in the last month of pregnancy for the presence of erythrocyte alloantibodies. one must test mares with low or equivocal titers closer to the time of parturition. if one detects alloantibodies, the colostrum of the dam should be withheld and the foal then should be provided with an alternative colostrum source. maternal alloantibodies to ca do not appear to mediate neonatal isoerythrolysis in foals and actually may be preventive by removing potentially sensitizing cells from the circulation 56 ; therefore one should not deprive foals of colostrum from mares possessing anti-ca antibodies, even when ca is present on their erythrocytes. rarely, the antigens de, ua, pa, and ab have been associated with neonatal isoerythrolysis in foals; however, to consider mares without these alloantigens to be at risk for neonatal isoerythrolysis is not practical. these syndromes recently have been recognized and described within the veterinary literature, although they have been recognized widely in human neonatology for many years. [260] [261] [262] [263] affected foals demonstrate these hematologic abnormalities within the first week of life, and the mechanism is similar to neonatal isoerythrolysis following ingestion of maternal antibody directed against the platelet or the neutrophil. in general, affected foals are healthy but may demonstrate bleeding tendencies if thrombocytopenia is severe or they may be more susceptible to sepsis. one confirms the diagnosis by appropriate testing for platelet-and neutrophil-associated antibody. 264 one must rule out other causes of neonatal thrombocytopenia and neutropenia, particularly sepsis. foals born to the mare in the future seem likely to be at risk for developing similar problems, and one should treat future foals as one treats neonatal isoerythrolysis foals: prevent sucking from the dam and provide an alternate source of passive immunity in the form of banked colostrum or intravenous plasma. one should provide an alternative nutritional source, such as foal milk replacer, to the foal for the first 48 hours of life and should muzzle the foal while it is in the company of its dam for that period of time. treatment is primarily supportive, but in the case of severe thrombocytopenia, transfusion of platelet-enriched fresh plasma may be indicated. granulocyte colony-stimulating factor has been used in foals with neutropenia, but substantial efficacy has yet to be demonstrated. broad-spectrum antimicrobial therapy may be prudent in cases of alloantibody-associated neutropenia. treatment with immunosuppressive doses of corticosteroids is probably unwarranted, given the increased risk of infection, because the antibody in question is of maternal origin. other specific diseases of the immune system of foals, severe combined immunodeficiency, selective igm deficiency, transient hypogammaglobulinemia, agammaglobulinemia, and other unclassified immunodeficincies are covered in detail elsewhere in this text. the neonate can experience respiratory distress immediately after birth because of several congenital respiratory tract or cardiac anomalies. chief among these causes are bilateral choanal atresia, stenotic nares, dorsal displacement of the soft palate caused by anatomic deformity or neurologic impairment, accessory or ectopic lung lobes, lung lobe hypertrophy, lung lobe dysplasia, cardiac anomalies with right-to-left shunting, and miscellaneous causes such as subepiglotic cysts and severe edema of the larynx. [264] [265] [266] [267] [268] [269] [270] [271] one must evaluate and treat these situations immediately and should consider them true emergencies. one readily can recognize foals with airway occlusion by the lack of airflow through the nostrils despite obvious attempts to breathe and by respiratory stridor. these foals may demonstrate open-mouth breathing and their cheeks may puff outward when they exhale. one foal with congenital bilateral choanal atresia was recognized during extrauterine intrapartum resusucitation because of an inability to pass a nasotrancheal tube. one can establish an effective airway by orotracheal intubation in these cases under most circumstances, but some foals require an emergency tracheostomy. one diagnoses the underlying problem by endoscopy or radiography in most cases. treatment of choanal atresia and cystic structures is surgical, whereas severe laryngeal edema and laryngeal paralysis frequently respond to medical management. until the underlying problem is resolved in these cases, one should administer broad-spectrum antimicrobial therapy and feed the foal by intubation or total parenteral nutrition. one can give colostrum, but these foals frequently develop aspiration pneumonia if allowed to suck from their dams, so intravenously administered plasma also may be necessary to provide sufficient passive immunity. arterial blood gas determinations are the most sensitive indicator of respiratory function readily available to the clinician. the most readily available arteries for sampling are the metatarsal arteries and the brachial arteries. portable arterial/venous blood gas analyzers now are making arterial blood gas analysis more practical in the field, and the technique is no longer reserved for large referral practices. managing a critically ill equine neonate without knowledge of arterial blood gas parameters is veritably impossible. pulse oximetry is useful, but these monitors only measure oxygen saturation of hemoglobin. desaturation can occur rapidly in critically ill neonates. the utility of these monitors in the foal has yet to be demonstrated clearly, particularly in cases of poor peripheral perfusion. 272 the most common abnormalities recognized with arterial blood gas analysis are hypoxemia with normo-or hypocapnia and hypoxemia with hypercapnia. hypoxemia is defined as decreased oxygen tension of the arterial blood (decrease pao 2 ), and hypoxia is defined as decreased oxygen concentration at the level of the tissue, with or without hypoxemia. hypoxia results from hypoxemia, decreased perfusion of the tissue bed in question, or decreased oxygen-carrying capacity of the blood resulting from anemia or hemoglobin alteration. five primary means by which hypoxemia may develop are (1) low concentration of oxygen in the inspired air such as in high altitude or in an error mixing ventilator gas; (2) hypoventilation; (3) ventilation/perfusion mismatch; (4) diffusion limitation; and (5) intrapulmonary or intracardiac right-to-left shunting of blood. hypoxemia is not an uncommon finding in neonates but must be evaluated in terms of the current age of the foal and its position. 15, [273] [274] [275] [276] one also must consider the difficulty encountered in obtaining the sample because severe struggling can affect the arterial blood gas results. table 19 -8 presents normal arterial blood gas parameters for varying ages of foals. the normal foal has a small shunt fraction (~10%) that persists for the first few days of life and contributes slightly to a blunted response to breathing 100% oxygen compared with the adult. hypoxemia frequently occurs in foals with prematurity, pas, and sepsis, although other conditions also result in hypoxemia in the neonate. in the early stage of sepsis associated hypoxemia, paco 2 may be within normal limits or decreased if the foal is hyperventilating for any reason. if the lung is involved significantly in the underlying pathologic condition, such as with severe pneumonia, acute lung injury, or acute respiratory distress syndrome, increased paco 2 may well be present, representing respiratory failure. 277 hypoxemia usually is treated with intranasal humidified oxygen insufflation at 4 to 10 l/min. hypercapnia is not a simple matter to treat. one must try to distinguish between acute and chronic hypercapnia. acute hypercapnia usually is accompanied by a dramatic decrease in blood ph of 0.008 ph units for each 1 mm hg increase in paco 2 . this acidemia can promote circulatory collapse, particularly in the concurrently hypoxemic and/or hypovolemic patient. the effects of more chronic co 2 retention are less obvious because the time course allows for adaptation. the ph change is less, about 0.003 ph units per 1 mm hg increase in paco 2 , because it is balanced by enhanced renal absorption of bicarbonate by the proximal renal tubule. most foals with acute respiratory distress are in the acute stages of respiratory failure, but chronic adaptation begins to occur within 6 to 12 hours and is maximal in 3 to 5 days. one will note an increase in bicarbonate, particularly if the acidemia is primarily respiratory in origin. intravenous administration of sodium bicarbonate to correct respiratory acidosis/ acidemia should be done cautiously in these foals because co 2 retention may only be increased. also, one should remember that 1 meq of sodium is administered with each meq of bicarbonate and hypernatremia has been seen in foals treated exuberantly with sodium bicarbonate. foals with hypercapnia of several days' duration also may develop a blunted respiratory drive to increased co 2 . in these foals, oxygen administration, although essential to treat hypoxemia, may further depress ventilation and further decrease ph. this effect is caused by a loss of hypoxic drive following oxygen therapy. one should consider these foals candidates for mechanical ventilation if the paco 2 is greater than 70 mm hg or is contributing to the poor condition of the foal, such as causing significant ph changes. if hypercapnia is caused by central depression of ventilation, as frequently occurs in foals with pas, one can administer caffeine (10 mg/kg loading dose; then 2.5 mg/kg as needed) per rectum or orally in foals with normal gastrointestinal function. other clinicians may recommend continuous rate infusions of doxapram hydrochoride (dopram; 400 mg/total dose at 0.05 mg/ kg/min) for these foals. if this therapy fails, one should consider mechanical ventilation. mechanical ventilation of foals with central respiratory depression is rewarding and may be necessary only for a few hours to days. a special category is the foal with botulism exhibiting respiratory failure caused by respiratory muscle paralysis. these foals do well with mechanical ventilation, although the duration of mechanical ventilation is more prolonged, frequently more than 1 week. foals with primary metabolic alkalosis usually have compensatory respiratory acidosis. treatment of hypercapnia is not necessary in these cases because it is in response to the metabolic condition. these foals do not respond to caffeine, and they should not be ventilated mechanically if this is the only disorder present. in the neonate, bacterial pneumonia usually results from sepsis or aspiration during sucking. foals with sepsis can develop acute lung injury or acute respiratory distress syndrome as part of the systemic response to sepsis, and this is frequently a contributor to the demise of foals in septic shock. the best way to diagnose bacterial pneumonia is by cytologic examination and culture of a transtracheal aspirate, but blood culture may aid in early identification of the causative organism and allow for early institution of directed antimicrobial therapy. a second frequent cause of bacterial pneumonia in the neonate is aspiration caused by a poor suck reflex or dysphagia associated with pas, sepsis, or weakness. one must take care to ensure that aspiration is not iatrogenic in foals being bottle fed. auscultation over the trachea while the foal is sucking helps identify occult aspiration. one should suspect occult aspiration pneumonia in any critically ill neonate that is being bottle fed or is sucking on its own that has unexplained fever, fails to gain weight, or has a persistently increased fibrinogen level. older foals develop bacterial pneumonia, frequently following an earlier viral infection. 278 bacterial pneumonia is discussed in depth elsewhere in this text, but a few comments specific to the foal are necessary. one should auscultate and percuss the thorax of the foal, but results may not correlate closely with the severity of disease. the most commonly isolated bacterial organism in foal pneumonia is streptococcus zooepidemicus, and one may isolate it alone or as a component of a mixed infection. [278] [279] [280] transtracheal aspirate for culture and cytologic examination is recommended because mixed gram-positive and gram-negative infections are common, and antimicrobial susceptibility patterns can be unpredictable. one should split the obtained aspirate and submit samples for bacterial culture, virus isolation, and cytologic examination. additional diagnostics include radiography, ultrasonography, and serial determination of white blood cell counts (with differential) and blood fibrinogen concentrations. treatment includes administration of appropriate antimicrobial therapy. some foals may benefit from nebulization with saline or other local products. ascarid larval migration through the lung can mimic bacterial pneumonia. 281 in these cases the foal may not respond to antimicrobial therapy and should be dewormed with ivermectin. deworming the mare within 1 month of parturition and frequent deworming of the foal prevent ascarid migration pneumonia in most foals. a special category of bacterial pneumonia in foals is rhodococcus equi bronchopneumonia. this pneumonia of young foals was described first in 1923. 282 the organism originally was known as corynebacterium equi and is a gram-positive pleomorphic coccobacillus usually less than 1 âµm in diameter and 2 âµm in length. the organisms frequently are associated in l-and v-shaped clusters that have been termed chinese character formations. r. equi has an acid-fast staining characteristic under some growing circumstances because of the presence of mycolic acid in its cell wall, similar to mycobacterium and nocardia species. mycolic acid promotes granuloma formation. the organism is able to multiply in and destroy macrophages as it prevents phagosome lysosome fusion. 283, 284 much attention has been paid to this organism in recent years, given its propensity to produce enzootic and epizootic outbreaks of disease. the organism is thought to be primarily an opportunistic pathogen, and it lives in the soil of most geographic areas. foals are affected most frequently between the ages of 1 and 6 months, when maternally derived immunity has begun to wane. the disease is insidious, and foals may have significant pulmonary involvement before developing noticeable clinical signs. phagocytosis of r. equi by equine macrophages is not associated with a functional respiratory burst and, at least in human beings, the l-arginine-no pathway is not required for intracellular killing of this organism. 285, 286 optimal binding of r. equi to mouse macrophages in vitro requires complement and is mediated by mac-1, a leukocyte complement receptor type 3 (cr3, cd11b/ cd18). 287 opsonisation of r. equi with specific antibody is associated with increased phagosome-lysosome fusion and enhanced killing of r. equi, suggesting that the mechanism of cellular entry is important. 283 neutrophils from foals and adult horses are fully bactericidal, and killing of r. equi is enhanced considerably by specific opsonizing antibody. 288 the ability of r. equi to induce disease in foals likely depends on host and microbial factors. knowledge of the virulence mechanisms of r. equi was speculative until the discovery of the virulence plasmid. 289 as opposed to most environmental r. equi organisms, isolates from clinically affected foals typically contain 85-to 90-kb plasmids encoding an immunogenic virulence-associated protein (vapa) that is expressed on the bacterial surface in a temperature-regulated manner. 290 plasmid-cured bacteria lose their ability to replicate and survive in macrophages and are cleared from the lungs within 2 weeks of intrabronchial challenge without producing pneumonia. 291 however, expression of vapa alone is not sufficient to restore the virulence phenotype. six other genes have approximately 40% overall amino acid identity with vapa, and the identification of multiple genes with considerable homology suggests these genes constitute a virulence-associated gene family in r. equi. 292 other candidates for virulence factors include capsular polysaccharides and cholesterol oxidase, choline phosphohydrolase, and phospholipase c exoenzymes ("equi factors"), but their roles have not been defined clearly. the primary manifestation of disease caused by r. equi infection is severe bronchopneumonia with granuloma, abscess formation, or both. up to 50% of foals diagnosed with bronchopneumonia also have extrapulmonary sites of infection. 293 as the pneumonia progresses, clinical signs may include decreased appetite, lethargy, fever, tachypnea, and increased effort of breathing characterized by nostril flaring and increased abdominal effort. cough and bilateral nasal discharge are inconsistent findings. a smaller percentage of affected foals may have a more devastating, subacute form. these foals may be found dead or have acute respiratory distress with a high fever and no previous history of clinical respiratory disease. hyperfibrinogenemia is the most consistent laboratory abnormality in foals with r. equi pneumonia. neutrophilic leukocytosis (>12,000 cells/âµl), with or without monocytosis, is common. 294 thoracic radiography is a useful diagnostic aid, frequently revealing a prominent alveolar pattern with poorly defined regional consolidation and/or abscessation. ultrasonography is a helpful diagnostic tool when the disease involves peripheral lung tissue. although a number of serologic tests have been described, serologic diagnosis of r. equi infections is controversial and difficult because exposure of foals to this organism at a young age leads to production of antibody without necessarily producing clinical disease. 295, 296 serologic tests may be more useful at the farm level to detect overall exposure than at the individual level. bacteriologic culture combined with cytologic examination of a tracheobronchial aspirate remains the most definitive method for accurate diagnosis of r. equi pneumonia. however, foals without clinical disease exposed to contaminated environments may have r. equi in their tracheae from inhalation of contaminated dust; therefore one should interpret culture results in the context of the overall case presentation. 297 culture results in one study were as sensitive as polymerase chain reaction-based assays and offered the advantage of allowing in vitro antimicrobial susceptibility testing. 298 however, polymerase chain reaction is likely to be a useful tool, and results from a second trial suggest the assay is more sensitive and specific than culture of tracheobronchial aspirates for diagnosis. 299 the combination of erythromycin and rifampin has become the treatment of choice for r. equi infections in foals, and the combination reduces the likelihood of resistance to either drug. the recommended dosage regimen for rifampin is 5 mg/kg every 12 hours or 10 mg/kg every 24 hours orally. the recommended dose of estolate or ethylsuccinate esters of erythromycin is 25 mg/kg every 8 or 12 hours orally. 300 recently, azithromycin has been recommended for treatment of r. equi infection at a dosage of 10 mg/kg orally every 24 hours for 5 to 7 days and then every other day. 301 alternatively, clarithromycin at 7.5 mg/kg every 12 hours orally, in combination with rifampin, may be therapeutically effective. severely affected foals may require intranasal oxygen insufflation, intravenous fluid support, and nutritional support. treatment generally continues for 4 to 10 weeks until all clinical and laboratory evidence of infection is resolved. although well tolerated by most foals, erythromycin can result in soft feces. this diarrhea is generally self-limiting and does not require cessation of therapy, but one should monitor affected foals carefully. an idiosyncratic reaction characterized by severe hyperthermia and tachypnea has been described in foals treated with erythromycin during periods of hot weather. 302 affected foals should be moved to a colder environment and treated with antipyretic drugs and alcohol baths if necessary. clostridium difficile enterocolitis has been reported in the dams of nursing foals treated with erythromycin given orally. 303 the dam is exposed to active erythromycin by coprophagy or by drinking from a communal water source where the foal has "rinsed" its mouth. prevention of r. equi pneumonia on farms with recurrent problems is problematic. the most clearly demonstrated prophylactic measure to date has been the administration of plasma that is hyperimmune to r. equi to foals within the first week of life and then again when maternal immunity begins to wane at around 30 days of age. [304] [305] [306] [307] [308] [309] [310] [311] no effective vaccination protocols for the dam or foal have been described to date. farm management is important in preventing disease, and control measures include frequent manure removal, avoidance of overcrowded conditions, and planting of dusty or sandy soils. 304 the prognosis for r. equi bronchopneumonia is fair to good in foals with the more chronic form of the disease. foals with acute respiratory distress have a more guarded prognosis, as do foals with sites of significant extrapulmonary infection. the long-term prognosis for survival for foals with r. equi bronchopneumonia is good, and many foals perform as expected as athletes. 312 the most commonly identified causes of viral pneumonia in foals are equine herpesviruses 1 and 4 (ehv-1 and ehv-4), equine influenza, and equine arteritis virus (eva). equine herpesvirus 1 is probably the most clinically important, but outbreaks of eva in neonates have occurred and are devastating. 27, [313] [314] [315] [316] [317] [318] adenovirus is reported sporadically and as a problem in arabian foals with severe combined immunodeficiency. [319] [320] [321] in the neonate, infection with ehv-1 or eva is almost uniformly fatal and antemortem diagnosis is difficult, even once an outbreak on a particular farm is identified. several factors appear common to foals with ehv-1, including icterus, leukopenia, neutropenia, and petechial hemorrhage, but these problems also are identified in foals with severe sepsis. 315, 322, 323 the antiviral drug acyclovir (10 to 16 mg/kg orally or per rectum 4 to 5 times per day) has been used in cases of ehv-1 in neonates, with some evidence of efficacy in mildly affected foals or foals affected after birth. 323 if viral pneumonia is a possibility, one should collect blood and tracheal aspirates at presentation for bacterial and virus isolation. the lungs of foals with ehv-1 or eva are noncompliant, and pulmonary edema may be present. mechanical ventilation of these cases may prolong life, but death is generally inevitable because of the magnitude of damage to the lungs. foals suspected of having ehv-1 or eva should be isolated because they may be shedding large quantities of virus and pose a threat to other neonates and pregnant mares. foals with eva generally are born to seronegative mares, and intravenous treatment with plasma with a high titer against eva may prove beneficial because passive immunity appears to have a large role in protection against this disease in neonates. 318, 324 older foals and weanlings may be affected by herpesviruses. disease is usually mild, although a fatal pulmonary vasculotropic form of the disease has been described recently in young horses. 325, 326 the clinical signs of disease are indistinguishable from influenza and include a dry cough, fever, and serous to mucopurulent nasal discharge, particularly if secondary bacterial infection occurs. rhinitis, pharyngitis, and tracheitis may be present. treatment of affected foals is primarily supportive. foals also may become infected with ehv-2. the predominant clinical signs are fever and lymphoid hyperplasia with pharyngitis. 327, 328 diagnosis is by virus isolation. rib fractures have been recognized in 3% to 5% of all neonatal foals and can be associated with respiratory distress. 87 potential complications of rib fractures include fatal myocardial puncture, hemothorax, and pneumothorax. rib fractures frequently are found during physical examination by palpation of the ribs or by auscultation over the fracture sites. one can confirm the diagnosis by radiographic and ultrasonographic evaluation. often multiple ribs are affected on one side of the chest. specific treatment is generally unnecessary, but direct pressure on the thorax should be avoided in all cases. some specific patients may benefit from surgical stabilization of some fractures, particularly those fractures overlying the heart. pneumothorax can occur spontaneously or following excessive positive pressure ventilation 329 or following tracheostomy surgery or trauma. any foal being ventilated mechanically that suddenly has respiratory distress and hypoxemia should be evaluated for pneumothorax. diagnosis is by auscultation and percussion of the thorax, but one can confirm the diagnosis with radiographic and ultrasonographic evaluation of the thorax. needle aspiration of air from the pleural space also confirms the diagnosis. treatment is required in cases in which clinical signs are moderate to severe or progressive and involves closed suction of the pleural space. subcutaneous emphysema can complicate treatment of this problem. idiopathic or transient tachypnea has been observed in clydesdale, thoroughbred, and arabian breed foals. in human infants, transient tachypnea can be related to delayed absorption of fluid from the lung, perhaps because of immature sodium channels. 330 in foals, tachypnea generally occurs when conditions are warm and humid and is thought to result from immature or dysfunctional thermoregulatory mechanisms. clinical signs of increased respiratory rate and rectal temperature develop within a few days of birth and may persist for several weeks. treatment involves moving the foal to a cooler environment, body clipping, and provision of cool water or alcohol baths. these foals frequently are treated with broad-spectrum antimicrobial drugs until infectious pneumonia can be ruled out. a syndrome of bronchointerstitial pneumonia and acute respiratory distress has been described in older foals and appears to be a distinct entity from acute respiratory distress syndrome in neonatal foals in association with sepsis. 331 the underlying cause has not been identified, but the genesis is probably multifactorial with several potential pathogens being implicated. affected foals have acute respiratory distress with significant tachypnea, dyspnea, nostril flare, and increased inspiratory and expiratory effort. auscultation reveals a cacophony of abnormal sounds including crackles and polyphonic wheezes in all lung fields. loud bronchial sounds are audible over central airways, and bronchovesicular sounds are lost peripherally. affected foals are cyanotic, febrile, and unwilling to move or eat. foals may be found acutely dead. laboratory abnormalities include leukocytosis, hyperfibrinogenemia, and hypoxemia with hypercapneic acidosis. foals can be dehydrated severely and have coagulation changes consistent with disseminated intravascular coagulation. hypoxic injury to other organs, primarily the kidneys and liver, can occur. chest radiographs reveal a prominent interstitial pattern overlying a bronchoalveolar pattern that is distributed diffusely throughout the lung. this syndrome is a respiratory emergency. treatment is broad-based and includes administration of oxygen, nonsteroidal antiinflammatory agents, broad-spectrum antimicrobial therapy, nebulization, judicious intravenous fluid therapy, nutritional support, and corticosteroid therapy. one must manage hyperthermia in the foal. corticosteroid therapy appears to have been lifesaving in most of the reported surviving foals. because this syndrome is associated with high environmental temperatures in some areas, prevention involves control of ambient temperatures, not transporting foals during hot weather, and keeping foals out of direct sun on hot days, particularly foals being treated with erythromycin for suspected or confirmed r. equi infection. 332 uroperitoneum has been recognized as a syndrome in foals for more than 50 years. 333, 334 classically, affected foals are 24 to 36 hours old at the time clinical signs first are recognized. [334] [335] [336] previous reports had a proportionately larger affected male than female population. 334, 335, 337 the hypothesis was that colts were more at risk because their long, narrow, high-resistance urethra was less likely to allow bladder emptying, resulting in rupture of a full bladder during parturition when high pressures were applied focally or circumferentially around the bladder. 333 more recent reports suggest that such extreme sex bias may have been an artifact of small case numbers in the early reports. rupture or disruption of any structure of the urinary tract can occur. the dorsal wall of the bladder has been reported to be a frequent disruption site, with the ventral wall less likely to be involved. 336 the urachus appears to be the next most commonly affected structure. a few cases of ureteral and urethral defects have been reported. 336, 337 sepsis does not appear to favor one site over the others. 338 the pathophysiology of uroperitoneum is not yet understood fully. the high pressure exerted on a full bladder during parturition once was thought to be the main cause. full bladder and obstruction caused by a partial umbilical cord at parturition, strenuous exercise, and external trauma have been reported as causes. 339 a few reports describe smooth and noninflamed edges of torn tissue, suggesting the possibility of congenital bladder wall defects. 338, 340, 341 sepsis leading to urinary tract rupture and uroperitoneum may occur in foals hospitalized for a variety of unrelated problems. the onset of clinical signs of uroperitoneum may be insidious in these foals, and diagnosis may be less obvious. 338 clinical signs associated with uroperitoneum in the neonatal foal typically include straining to urinate, dribbling urine, and a stretched-out stance. weakness, tachycardia, tachypnea, and not sucking well are also common. a distended abdomen may be evident, and one may feel a fluid wave on ballottement of the abdomen. occasionally, urine accumulates in the scrotum and should not be confused with hernia. foals also may show signs of sepsis, including fever, injected mucous membranes, diarrhea, and disease of other body systems. laboratory findings vary depending on the duration of the uroperitoneum and on the presence and severity of sepsis. classic findings include hyperkalemia, hyponatremia, and hypochloremia arising from equilibration of urine electrolytes and water with blood across the peritoneal membrane. [335] [336] [337] the usual foal diet of milk, which is high in potassium and low in sodium, promotes the electrolyte abnormalities. foals that develop uroperitoneum while receiving intravenous fluids may not have classic electrolyte imbalances at the time clinical signs are recognized. 338 increased serum creatinine concentration is often present, whereas blood urea nitrogen concentrations occasionally, but not consistently, are increased. [335] [336] [337] metabolic acidosis and hypoxemia may be present. some patients also have serum hypoosmolality. 335 one should test foals for failure of passive transfer. one of the most sensitive laboratory tests for uroperitoneum is the ratio of peritoneal to serum creatinine. a ratio greater than or equal to 2:1 is considered diagnostic of uroperitoneum. one should collect peritoneal fluid and test it for creatinine concentration, as well as for cytologic findings, culture, and sensitivity. cytologic evaluation of peritoneal fluid is necessary to identify concurrent peritonitis or other gastrointestinal compromise. one should perform an electrocardiogram on initial evaluation of a foal with suspected uroperitoneum because hyperkalemia may result in bradycardia, increased duration of the qrs complex, a shortened q-t interval, increased p-wave duration, prolonged p-r interval, or atrioventricular conduction disturbances. other possible cardiac sequelae to hyperkalemia include cardiac arrest, third-degree atrioventricular block, ventricular premature contractions, and ventricular fibrillation. 337, 340 for any foal exhibiting signs of dypsnea, tachypnea, or hypoxemia, one should have thoracic radiographs taken before induction of anesthesia to rule out pleural effusion, pneumonia, or acute respiratory distress syndrome, which could complicate ventilation and oxygenation during anesthesia and the postoperative period. ultrasonography has become the tool of choice in the diagnosis of uroperitoneum and is a useful tool available to the practitioner. 342 one can image free peritoneal fluid readily, and tears within the bladder are readily visible. the empty bladder with a significant defect, in a fluid-filled abdomen, will collapse on itself and often have a u shape. one also can visualize urachal and urethral lesions. six of eight foals in one study had urinary tract lesions identified sonographically, and all 31 foals of another study underwent sonographic evaluation, and a significant correlation between ultrasonographic findings and location of the lesion at surgery existed. 336, 338 initial treatment aims to stabilize the patient and correct any electrolyte and acid-base abnormalities and provide fluid volume replacement. one should use 0.9% or 0.45% saline with 5% dextrose until laboratory data are available. a potassium concentration of greater than 5.5 meq/l can be life threatening. one can manage hyperkalemia by peritoneal drainage to decrease whole-body potassium stores using teat cannulae, foley catheters, large-gauge (16 or 14) intravenous catheters, or human peritoneal dialysis catheters. fluid replacement at least should equal the amount of fluid removed from the abdomen to prevent acute hypotension caused by expansion of previously collapsed capillary beds. abdominal drainage also helps ventilation and decreases the work of breathing by decreasing pressure on the diaphragm. one may administer calcium gluconate, glucose, sodium bicarbonate, or insulin intravenously to decrease serum potassium concentrations. these maneuvers do not correct the whole-body potassium overload, however, and once therapy is discontinued, hyperkalemia can reappear until the urine is removed from the abdomen. one should correct hyponatremia slowly. because of the real possibility of concurrent sepsis, one should obtain blood cultures before preoperative administration of antimicrobials. broad-spectrum coverage (penicillin and amikacin or ceftiofur sodium) is recommended until culture results become available. one should perform therapeutic drug monitoring when using aminoglycoside therapy. however, the peak value may be depressed because of the increased volume of distribution represented by the volume of urine in the abdomen, so one should not make dose adjustment based on a low peak until obtaining a new peak after surgical correction of the uroperitoneum. one should treat foals with failure of passive transfer with adequate volumes of intravenously administered plasma. after one has addressed the metabolic abnormalities, one may consider surgical management. medical management using an indwelling foley catheter has been described. 343 preoperative medical stabilization reduces anesthetic risk. safer inhalant agents such as isoflurane also have decreased risk. removal of the internal umbilical remnant at the time of surgery is usual. one should consider culturing any removed umbilical remnant and submitting the remnant for histopathologic evaluation. recurrence of urinary tract rupture can occur. sepsis, hypoxemia, pneumonia, peritonitis, and acute respiratory distress syndrome complicate the management of uroperitoneum. many affected foals are persistently oxygen dependent for several days following surgical correction, and one should perform serial arterial blood gas analyses before discontinuing intranasal oxygen supplementation. prognosis is associated closely with concurrent illness, especially septicemia. uncomplicated uroperitoneum from a defect in the bladder has a good prognosis. if the location of the lesion is other than the bladder, the prognosis is not as favorable. 337 foals with septicemia have a much poorer prognosis. 338, 339 acute renal failure most often occurs as a complication of prenatal asphyxial syndrome, sepsis, or aminoglycoside therapy. acute renal failure also has been reported following oxytetracycline administration in foals. 344 the dose of oxytetracycline commonly used to treat flexural deformities in foals is approximately 10 times the antimicrobial dose. many foals treated in this manner also have suffered some degree of perinatal asphxia, which also damages the kidney, because of prolonged parturition precipitated in part by the flexural deformity. evaluation of renal function in these foals before the administration of the first dose of oxytetracycline and continued monitoring of serum creatinine concentrations before administering subsequent doses of this nephrotoxic compound would seem reasonable. hemodialysis has been used as therapy in one of these cases, but prevention is important because these foals may fail to respond to usual therapy for oliguric renal failure and are euthanized. 344 the most commonly reported congenital deformity of the kidney of the foal is renal hypoplasia and dysplasia, which may have a heritable component. 345, 346 renal arteriovenous malformations have been reported also. 347 ectopic ureters and fenestrated ureters have been described in the foal. [348] [349] [350] congenital renal defects, among others, were reported in three weak, recumbent neonatal foals born to mares being treated for equine protozoal myeloencephalitis. 351 mares received sulfadiazine or sulfamethoxazoletrimethoprim, pyrimethamine, folic acid, and vitamin e orally. the foals were anemic, leukopenic, azotemic, hyponatremic, and hyperkalemic. serum folate concentrations were lower than those reported in the literature for clinically normal brood mares. treatment was unsuccessful. necropsy revealed lobulated kidneys with thin cortices and a pale medulla. the authors postulated that oral administration of sulfonamides, 2,4-diaminopyrimidines (pyrimethamine with or without trimethoprim), and folic acid to mares during pregnancy is related to congenital defects in newborn foals. the umbilicus serves as the conduit for nutrition and gas exchange between the dam and the fetal foal. the urine from the foal is expelled via this structure into the allantoic cavity. the author has recognized cases of in utero bladder distention in the fetus that were associated with multiple twists decreasing urine flow or focal stenosis creating the same effect. foals born with this condition did not have bladder rupture associated with parturition but did have other severe abnormalities that eventually resulted in their demise, primarily premature delivery with failure to adapt to extrauterine life (p.a. wilkins, j.e. palmer, and f.t. bain, unpublished data). at birth the umbilicus breaks, leaving a small external remnant and a large internal remnant. the umbilicus long has been regarded as the primary site of entry of pathogens into the neonate, although this has been challenged recently. treatment of the umbilicus after birth involves dipping it (preferably just the most distal component) with various caustic compounds. the most current recommendation is to treat the umbilicus with dilute chlorhexidine, povidone-iodine, or dilute iodine solutions for just a few times following birth. exhuberant treatment of the umbilical stump with caustic solutions can lead to scalding of the ventral abdomen and may promote patency of the urachus. the ultrasonographic appearance and measurements of the umbilical arteries, urachus, and umbilical vein of foals from 6 hours to 4 weeks of age have been described in detail. 342 a 7.5-mhz sector scanner transducer placed across the midline of the ventral portion of the abdominal wall of the foal works best because of the superficial location of these structures. the mean (â± sd) diameter of the umbilical vein was 0.61 â± 0.20 cm immediately cranial to the umbilical stalk, 0.52 â± 0.19 cm midway between the umbilicus and liver, and 0.6 â± 0.19 cm at the liver. the urachus and umbilical arteries of normal foals have a mean total diameter of 1.75 â± 0.37 cm at the bladder apex. the umbilical arteries scanned along either side of the bladder have a mean diameter of 0.85 â± 0.21 cm. one can use these measurements and the ultrasonographic appearance of the internal umbilical structures from clinically normal foals as references to diagnose abnormalities of the umbilical structures in neonatal foals. 352, 353 the most common abnormalities of these structures are focal abscess formation, hematoma, and urachal tear. herniae traditionally have been thought to develop from failure of closure at the umbilical stump after birth. however, the closure of the body wall defect at the umbilicus was studied in relation to the development of umbilical herniae in a large group of normal foals followed from birth until 5 months of age or from birth until 11 months of age. 354 at birth, approximately half of these foals had a defect in the body wall at the umbilicus that was termed a palpable umbilical ring. in 18 foals this defect disappeared within 4 days, but in one foal the ring did not close and a hernial sac with abdominal contents was palpable. this foal was considered to be the only foal to have a truly congenital umbilical hernia. twelve foals developed an umbilical hernia between 5 and 8 weeks of age. the prevalence of umbilical herniae was much higher than in other studies, possibly because of the prospective nature of the study. based on this study, the large majority of umbilical herniae would appear not to result from failure of closure but rather to be acquired after birth. one should consider the palpable ring structure within the body wall at the umbilicus a variant of normal in the foal and should not call it a hernia until the foal is at least 1 month of age. in one study of 147 horses treated for umbilical herniae over a 13 1 / 2 -year period, only 8.8% developed complications in association with umbilical defects. 355 six horses had intestinal incarceration; the incarceration was reduced manually in 3 horses before admission and resolved without treatment in 2 others. the hernia was surgically reduced in 1 horse. herniorrhaphy was performed on 4 of the 5 horses in which the incarceration did not require surgical reduction, and the fifth was managed conservatively. the study confirmed that complications of umbilical herniae are rare in horses; however, when they do develop, they may be one of various forms, some of which are insidious in onset. the primary differential diagnosis for an external swelling in the umbilical stump region is an external abdominal abscess, which will be firm, variably painful, warm, and nonreducible. ultrasonographic evaluation readily can confirm either possibility. one report describes a 3-day-old foal that died from intestinal strangulation caused by a remnant of vitelline vein that extended between the umbilicus and the portal vein. 356 patent urachus frequently is recognized in the abnormal neonate, probably because of the increased recumbency and decreased movement of these patients. cauterization of a patent urachus is no longer recommended except in cases that persist for long periods of time (>1 month) after the foal becomes more active. surgical resection may provide relief in some foals, but most cases resolve without treatment if given enough time. foals with a patent urachus may posture and strain frequently to urinate, some of this may be associated with irritation or local infection of the urachus. one can alleviate this by administration of broad-spectrum antimicrobial therapy such that the drug has a high concentration in the urine (e.g., trimethoprim-sulfa drug combinations) and by oral administration of phenazopyridine hydrochloride (pyridium), a dye that anesthetizes the urinary tract epithelial surfaces (see table 19 -7). this dye turns the urine orange and stains everything yellow-orange that it or the urine touches but can provide a great deal of relief to foals with this problem. the umbilicus has been considered the traditional point of entry of bacteria into the septic neonate, and septic foals have been referred to as having "navel ill" and "joint ill" in the past. although current thought suggests that the gastrointestinal tract may be the route of entry in most septic neonates, infection of the umbilicus-termed omphalitis, or omphalophlebitis if the vessels are involved-still occurs as a single focus of infection or along with more generalized infection. external signs, such as swelling, heat, pain, ventral edema, or purulent discharge may be present in some foals, but more usually external signs are minimal and one suspects infection because of infection in another site (e.g., an infected joint), fever, or otherwise unexplained increased blood fibrinogen concentration. one confirms the diagnosis by ultrasonographic evaluation of the internal umbilical remnant. any of the umbilical structures may be involved. a complete description of the evaluation is available within the relevant veterinary literature, but the examination is performed best with the foal standing using a 7.5-mhz probe with a standoff. 353 the usual finding is that the affected structure is larger than expected. a fluid-filled core and echogeneic shadows consistent with gas may be apparent in some cases. interpretation requires some experience, and the examiner should be familiar with variants of normal, such as gas shadows associated with a patent urachus and enlarged vessels caused by hematoma formation, so that treatment is not initiated inappropriately. two options for treatment are surgical and medical. medical treatment is preferable in cases in which the lesion is well localized and small and in foals with a medical condition that is not amenable to anesthesia and surgical intervention. one should institute broad-spectrum antimicrobial therapy, and one may need to continue therapy for 2 to 3 weeks. most affected foals respond to medical therapy. frequent reevaluation of the abnormality is necessary, every 5 to 7 days initially, and one should measure blood fibrinogen concentrations at reevaluation because they should stabilize and decrease with effective treatment. failure to respond to therapy within 10 days to 2 weeks suggests that an empiric change in the antimicrobial used may be necessary. in foals that are refractory to medical management or where the lesion is large, surgical excision of the entire umbilical remnant may be desirable. colic in the foal can be difficult to diagnose accurately because one cannot perform an examination per rectum. however, many diagnostic aids, most importantly ultrasonography, are available to help differentiate medical from surgical causes of abdominal discomfort in the foal. intestinal accidents of all types described in adult horses, with the possible exception of enteroliths, occur in foals. intussusception, volvulus, displacement, diaphragmatic hernia, and intra-and extraluminal obstruction have been reported in foals. abdominal ultrasonographic and radiographic evaluation greatly aids diagnosis. treatment is primarily surgical. foals with pas and intestinal dysmotility are at increased risk of intussusception and displacement, and miniature breed foals appear to be at increased risk for fecolith and enterolith formation. meconium retention or impaction is a common cause of abdominal discomfort in newborn foals. most foals defecate shortly after their first meal. the usual practice for most owners or veterinarians attending the birth of a foal is to administer an enema to aid this process. in the past, phosphate-based commercially available enemata (fleet) were used frequently, but if used excessively these types of enemata can create problems of their own, including rectal irritation and hyperphosphatemia. the best enema is warm soapy water made with a mild soap such as liquid ivory soap that can be administered through soft rubber tubing using gravity flow. foals with significant meconium retention become colicky within the first few hours of life as gas accumulates within their bowel. frequently, one can palpate the meconium through the abdominal wall. additional diagnostics can include abdominal ultrasonography and radiography, particularly if one must rule out other, more serious types of colic. these foals assume a classic stance with an arched back. one must differentiate this stance from the stance assumed by foals with uroperitoneum, which is more extended. foals with meconium retention have had simultaneous ruptured bladder, however, so the clinician must be sure to evaluate the foal fully for both problems. foals that do not respond rapidly to enema administration need additional treatment, which can include giving mineral oil (2 to 4 ounces) by nasogastric tube. one can treat persistent meconium retention resulting in significant abdominal distention by muzzling the foal to prevent further milk intake and administering intravenous fluids at an appropriate maintenance rate. if continuous rate infusion is possible, 5% to 10% dextrose is the preferred fluid to use to provide calories to the foal. one should not use dextrose as a bolus fluid. more aggressive treatment would include administration of retention enemata made using acetylcysteine, which serves as an irritant and increases secretion. extreme cases of meconium retention may require surgical intervention, but this is usually not necessary and most cases resolve with medical management alone within 12 to 24 hours. some foals require pain managment. one should avoid nonsteroidal antiinflammatory drugs in the neonate because of their effects on renal function and gastric mucosal blood flow (see gastric ulcers). many foals respond well to butorphanol administered intramuscularly at a dose of 3 to 5 mg to an average 50-kg foal. intranasal oxygen insufflation is beneficial in foals with significant abdominal distention. one should evaluate foals with meconium impaction/ retention for evidence of pas because intestinal dysmotility is common in pas. colostrum is a laxative, and these foals also may suffer from failure of passive transfer, with meconium retention resulting from the lack of adequate colostrum. these foals are also at risk of sepsis because the mucosal intestinal barrier probably has been disrupted and translocation of bacteria can occur. one should obtain blood cultures on these foals and should monitor them closely for signs of sepsis. atresia within the gastrointestinal system of the foal occurs infrequently, but clinical signs are characteristic. 357 acute colic occurs within the first few hours and is accompanied by abdominal distention similar to meconium retention. three primary types of atresia are described in the foal: membrane atresia, cord atresia, and blind-end atresia. antemortem diagnosis of atresia, short of abdominal exploratory surgery, is aided by the lack of meconium staining of the rectum or any administered enema fluids. additional diagnostic tests may include administration of a barium enema for a radiographic study, colonoscopy, and abdominal ultrasonography. abdominocentesis is usually normal until bowel rupture is imminent or has occurred. one can make affected foals more comfortable by muzzling them to prevent further milk intake and by supplying them with fluids and nutrition intravenously. if one attempts surgical correction, one first should initiate broad-spectrum antimicrobial therapy and determine passive transfer status. frequently, these foals are hypoxemic because of the abdominal distention, and oxygen supplementation is desirable. solid white foals born to overo-overo matings of american paint horses may suffer from congential aganglionosis of the ileum, cecum, and colon. these foals present similarly to foals with meconium impaction or atresia in that colic develops shortly after birth and involves progressive abdominal distention with feeding. the inherited defect is in the endothelin receptor gene. [358] [359] [360] [361] no effective treatment exists, but the clinician should be aware that not all white foals of this mating are affected, and some simply may have meconium retention, so a short period of treatment may be warranted. necrotizing enterocolitis is considered the most common acquired gastrointestinal emergency of human infants. 362, 363 the 1500 to 2000 infants that die every year from this disease in the united states and the large number of infants who develop short gut syndrome from this disease only represent the tip of the iceberg of the problems necrotizing enterocolitis causes. the widespread fear of necrotizing enterocolitis among neonatologists and pediatric surgeons has contributed in large part to the use of the intravenous route rather than the gastrointestinal tract for nourishing these infants for long periods. the pathogenesis of necrotizing enterocolitis is unknown but may result from a disturbance of the delicate balance among gastrointestinal perfusion, enteric organisms, and enteral feeding. risk factors for necrotizing enterocolitis in human infants include prematurity, hypoxic-ischemic insult, and formula or breast milk feedings. the clinical spectrum of necrotizing enterocolitis is multifactoral and ranges from temperature instability, apnea, lethargy, abdominal distention, bilious residuals, septic shock, disseminated intravascular coagulation, and death. medical management is usually adequate treatment for necrotizing enterocolitis. in the neonatal foal, necrotizing enterocolitis is probably one of the most underrecognized causes of gastrointestinal dysfunction and in the past has been attributed only to infection with anaerobic organisms including clostridium perfringens type c and c. difficile. 364 although a specific form of enteritis is associated with intestinal infection by these organisms, most necrotizing enterocolitis is associated with prematurity or pas in the infant and the foal. one should suspect necrotizing enterocolitis in any foal that is having difficulty tolerating oral feeding, demonstrating signs of ileus, or having episodes of colic and in any foal with occult blood or frank blood in the stool. foals exhibiting any of these clinical signs should not be fed orally if possible and should receive parenteral nutrition until gastrointestinal function returns to near normal. the mucosal barrier of the intestine is unlikely to be fully intact, and these foals are at risk for sepsis from bacterial translocation. one should institute broadspectrum antimicrobial therapy in these foals and, if any evidence of coordinated gastrointestinal motility is apparent, should administer sucralfate orally as a protectant. gastric ulcer disease has been recognized in foals, and lesions vary in anatomic distribution, severity, and cause. in clinically normal neonatal foals (<30 days of age), gastric ulcers and mucosal desquamation have been documented. [365] [366] [367] [368] because of these reports and other early reports of death following ruptured clinically silent ulcers in neonatal foals, for years many clinicians felt it necessary to treat critically ill neonates with antiulcer medication prophylactically. [369] [370] [371] recently, this paradigm has been challenged. the pathophysiology of gastric ulcer disease is described most reasonably as an imbalance in protective and aggressive factors. [372] [373] [374] these protective factors are responsible for maintaining a healthy gastrointestinal tract by promoting adequate mucosal blood flow, adequate mucus and bicarbonate production, prostaglandin e 2 production, epithelial growth factor production, gastric afferent innervation, epithelial cell restitution, and gastroduodenal motility. probably the most important factor is maintenance of mucosal blood flow. hypoxia, no, prostaglandins, and gastric afferent innervation influence mucosal blood flow. the aggressive factors include gastric acid, bile salts, pepsin, and enzymes. few specific causes have been found for gastric ulcer disease in foals. excessive administration of nonsteroidal antiinflammatory drugs can result in ulceration of the glandular and squamous epithelium because of an inhibition of prostaglandin production, which leads to a decrease in mucosal blood flow and an increase in acid production. nonsteroidal antiinflammatory drugs also can impair the healing of lesions and rarely are indicated in neonatal equine medicine. 372, 373 in the critically ill neonate the suspected cause of gastric ulcers has shifted away from an excessive amount of intraluminal gastric acid toward gastric mucosal ischemia caused by hypoxia, low blood flow conditions, or both. 375 perforating gastric ulcers are more likely a manifestation of necrotizing enterocolitis than of excessive gastric acid. shock, sepsis, or trauma can result in gastric mucosal ischemia, allowing for the disruption of epithelial cell integrity and permitting damage by aggressive factors or providing an environment suitable for the establishment of bacteria colonization. 375,376 impairment of mucosal blood flow also may result in reperfusion injury, allowing the formation of gastric ulcers. in the sick neonatal foal (<7 days of age) a wide variability in the intragastric ph has been documented depending on the type of disease, severity, and milk intake frequency and volume, suggesting that in the critically ill equine neonate, ulcer prophylaxis using histamine antagonists or proton pump inhibitors is not only unnecessary but unlikely to work. 377 clinically significant gastric ulcers can occur in the squamous, glandular, or both portions of the stomach as a primary problem or resulting from another problem. clinical signs include diarrhea, abdominal pain, restlessness, rolling, lying in dorsal recumbency, excessive salivation, and bruxism. in the neonatal foal the only clinical signs present may be depression or partial anorexia until a more catastrophic event, such as perforation, occurs. some lesions in the gastric mucosa extend from the pylorus into the proximal duodenum and can result in stricture of the pylorus and proximal duodenum. these foals are usually older (>1 month of age) and have a greater volume of reflux. bruxism and ptyalism are also more prominent in these older foals. the most sensitive and specific method for diagnosing gastric ulcers is visualization by endoscopic examination. 365 unfortunately, the use of gastric endoscopy has led to recognition of relative nonlesions and ulcers resulting from other problems and of clinically significant disease states. the clinician should not stop simply when ulceration of the stomach is recognized with endoscopy but should examine that patient fully for other potential sources of the clinical signs. other diagnostic tests may help in determining the severity of the ulcers, including fecal occult blood or gastric blood assessments, contrast radiography, abdominal ultrasound, and abdominocentesis. endoscopy of the foal stomach carries an additional risk of exacerbating colic in the short term, unless the examiner ensures that as much introduced air as possible is evacuated from the stomach at the end of the procedure. the presence of a brown gastric reflux fluid may indicate the presence of bleeding ulcers. blood in the feces of the neonate is more consistent with a diagnosis of necrotizing enterocolitis, which can be associated with gastric ulcers. contrast radiography is useful if one suspsects delayed gastric emptying or pyloric or duodenal stricture in older foals. if a stricture has occurred, one will note a delay in complete emptying of barium from the stomach (>2 hours). 367 abdominal ultrasound may be useful to visualize free abdominal fluid and gastric or small intestinal distention if one suspects a perforation. one can visualize portions of the descending duodenum, and a thickened duodenum should increase the index of suspicion for duondenal stricture. abdominocentesis also may confirm perforation. traditional therapy for gastric ulceration includes mucosal adherents, histamine type 2 receptor antagonists, proton pump inhibitors, and antacids. 378 the most widely used mucosal adherent is sucralfate, which is a hydroxy aluminum salt of sucrose. the main therapeutic action of sucralfate is to bind to the negatively charged particles in the ulcer crater. 378, 379 at a ph less than 2, sucralfate is converted to a sticky viscous gel, which adheres to the ulcer crater and remains adhered for 6 hours, but at a higher ph, sucralfate remains in a suspension. sucralfate is still effective because it inhibits pepsin and buffers hydrogen ions. other important actions of sucralfate include stimulating production of prostaglandin e, which maintains mucosal blood flow; increasing bicarbonate secretion; stimulating mucous secretion; decreasing peptic activity; and binding epidermal growth factor. the histamine type 2 receptor antagonists include cimetidine, ranitidine, and famotidine. these compounds block the interaction of histamine with the histamine type 2 receptor on the parietal cell, resulting in inhibition of gastric acid secretion. clinically normal neonatal foals have a highly acidic gastric fluid that is influenced by sucking. intravenous and oral administration of ranitidine increases intragastric ph in normal foals but critically ill neonatal foals have a blunted response to ranitidine administration. 377, 380 one possible conclusion reached from these studies is that in critically ill neonatal foals, gastric ulcers may not be caused by an increased intraluminal gastric acidity. the most commonly used proton pump inhibitor is omeprazole. this drug has not as yet been approved for use in foals under 30 days of age. omeprazole inhibits the secretion of hydrogen ions at the parietal cell by irreversibly binding to the h + ,k + -atpase proton pump of the cell. most of the lesions in older foals were healed after daily administration of omeprazole for 28 days according to one report. 381 table 19 -9 summarizes the therapeutic agents for treating gastric ulcers in foals. prophylactic treatment of critically ill neonates for gastric ulcers has been standard therapy for years because of the evidence of clinically silent ulcers. this approach may not be appropriate for several reasons. an increased incidence of nosocomial pneumonia and systemic sepsis is associated with high gastric ph in human patients in intensive care. [382] [383] [384] patients in intensive care units treated prophylactically with histamine type 2 receptor antagonists are more likely to develop pneumonia during ventilation therapy and gastric colonization with potentially pathogenic bacteria or yeast. 382, 385 an acidic environment appears to protect against airway colonization by bacteria of intestinal origin and bacteria translocated across the gastrointestinal tract. pathogenesis of ulcers in the neonatal foal most likely does not involve increased intraluminal gastric acid but instead may be caused by decreased mucosal perfusion associated with shock, hypoxia, and hypoxic/ischemic insult to the gastric mucosa. a recent report revealed that gastric ulcer disease in equine nicu patients is independent of pharmacologic prophylaxis. 386 in this study, despite decreased treatment, the incidence of gastric ulcers found in these foals at necropsy had decreased significantly. the decrease was attributed to overall improvement in management of these cases. similarly, in a human intensive care unit, the incidence of stress ulcers decreased independent of the use of prophylaxis. 375, 387 early treatment of sepsis, sufficient oxygenation, improved monitoring, institution of enteral feedings, and improved nursing care may contribute to the reduction in gastric ulcers in the neonatal patient. use of histamine type 2 receptor antagonist and proton pump inhibitors apparently may not be necessary; however, in some instances sucralfate may be useful. sucralfate reduced the rate of bacterial translocation in a rat model during hemorrhagic shock and also may prohibit the generation of acute gastric mucosal injury and progression to ulcer formation induced by ischemia-reperfusion. 388, 389 in a human medical intensive care unit, airway colonization by new pathogens occurred more frequently in patients receiving agents that increased gastric ph than in those receiving sucralfate. 382, 390 in the critically ill neonatal foal, risk factors for gastric ulceration have not been identified clearly, although foals treated routinely with nonsteroidal antiinflammatory drugs may be at increased risk for gastric lesions. prophylactic treatment for gastric ulcers in critically ill neonates may not be necessary, and one should consider carefully the pros and cons of their use before their administration. foal heat diarrhea is a mild, self-limiting form of diarrhea that occurs in foals between 5 and 14 days of age, about the time of the "foal heat" in the dam. the definitive cause of foal heat diarrhea has yet to be described, but the condition may be associated with dietary changes or changes in gastrointestinal function that occur around that time. this form of diarrhea is not caused by stongyloides westeri infestation as previously thought. 391 foals with foal heat diarrhea are not systemically ill and should not require therapy. one should evaluate fully any foals with diarrhea at this time for other possible causes of diarrhea, particularly if they are unwell or exhibit anorexia or dehydration. viral diarrhea occurs most commonly in large groups of mares and foals that are housed together. rotavirus is an isolate from the feces of up to 40% of foals with diarrhea worldwide, alone or with another pathogen. 392, 393 the virus infects and denudes the microvilli, resulting in increased secretion combined with decreased absorption. the virus interferes with disaccharidase function and alters the function of the intestinal sodium-glucose cotransport proteins. the initial clinical signs are anorexia and depression, with profuse watery diarrhea occurring shortly thereafter. severely affected foals may become significantly dehydrated and have electrolyte abnormalities, primarily hyponatremia and hypochloremia with metabolic acidosis. these foals generally require intravenous fluid support, whereas less severely affected foals may require only symptomatic therapy. definitive diagnosis is by detection of the virus in the feces of foals with diarrhea. however, none of the available tests are particularly sensitive, and the virus also may be found with other intestinal pathogens. recently, vaccination of pregnant mares has been suggested as a means of prevention, with preliminary results suggesting efficacy. 394,395 although a definitive role for adenovirus has not been established in the foal, adenovirus is a common co-isolate from foals with rotaviral diarrhea. 396 a specific equine coronavirus recently has been identified from an immunocompetent foal with diarrhea, and a second report of cornavirus diarrhea was published recently. 397,398 one case report suggests a parvovirus caused diarrhea in the foal. 399 treatment of viral diarrhea in foals is primarily supportive. intravenous fluid and parenteral nutritional support may be necessary in severe cases. very young foals may benefit from intravenous plasma administration and broad-spectrum antimicrobial coverage to limit bacterial translocation. one can administer sucralfate orally in these cases as a gastrointestinal protectant and to discourage bacterial translocation. foals with moderate to severe metabolic acidosis may benefit from sodium bicarbonate administration if their ventilatory function is normal. one administers sodium bicarbonate at half the calculated deficit (0.5 ã� standard base excess ã� body mass in kilograms) as an isotonic solution at the maintenance fluid rate. one should reevaluate sodium and bicarbonate (or standard base excess) concentrations regularly. nonspecific therapy of diarrhea is discussed elsewhere in this text. diarrhea is frequently the primary presenting complaint in foals with sepsis, so one should rule out this differential diagnosis in foals less than 1 week of age. one should evaluate all neonatal foals with diarrhea for possible sepsis and should include a blood culture whenever possible. clostridium perfringens and c. difficile are recognized increasingly as serious pathogens of the foal. 400-403 foals with either pathogen generally have abdominal pain, dehydration, and profuse watery diarrhea. some foals may have red-tinged or frankly bloody feces, which carries a poorer prognosis. most foals with this type of diarrhea require intensive care or, at the minimum, intravenous fluid administration. outbreaks of this type of diarrhea on farms occasionally occur, and the suggestion is that the dam has a role in transmission of the bacteria. diagnosis is by recognition of the offending organism by gram stain of the feces, by bacterial isolation from the feces, and by detecting the presence of toxins associated with the organisms. specific treatment includes oral administration of metronidazole and broad-spectrum antimicrobial coverage as prophylaxis for bacterial translocation associated sepsis in younger foals. foals with severe blood loss in their feces may require transfusion of whole blood. salmonella spp., escherichia coli, bacteroides fragilis, and aeromonas hydrophila have been implicated in diarrhea in foals. salmonella generally is associated with septicemia in foals, and although some convincing evidence exists for a role for e. coli in foal diarrheal disease, the extent of e. coli as a pathogen of the gastrointestinal tract in foals has yet to be described fully. 371, [404] [405] [406] [407] proliferative enteropathy is a transmissible enteric disease caused by lawsonia intracellulare. 408,409 most foals have been weaned before the appearance of clinical signs of depression, rapid and significant weight loss, subcutaneous edema, diarrhea, and colic. poor body condition with a rough hair coat and a pot-bellied appearance are common in affected foals. clinicopathologic abnormalities included hypoproteinemia, leukocytosis, anemia, and increased serum creatine kinase concentration. postmortem reveals characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells of the intestinal mucosa. antemortem diagnosis of equine proliferative enteropathy is based on clinical signs, hypoproteinemia, and the exclusion of other common enteric pathogens. fecal polymerase chain reaction analysis may be positive for the presence of l. intracellulare, and affected foals develop antibodies against l. intracellulare. 410 treatment with erythromycin estolate alone or combined with rifampin for a minimum of 21 days is recommended with additional symptomatic treatment when indicated. cryptosporidium spp. cause gastroenteritis and diarrhea in many animal species and are not host-specific. cryptosporidium has been implicated as the casual agent of diarrhea in foals, but the organism is isolated from the feces of diarrheic foals and normal foals with the same frequency and concentration, making a clear role for the organism difficult to elucidate. 411-413 diarrhea caused by cryptosporidium in other species and that described for foals is generally self-limiting, with a clinical course of between 5 to 14 days. immunosuppressed patients, including foals compromised by concurrent disease, are thought to be at increased risk for complications resulting from infection with this organism. 411,412 treatment is symptomatic. cryptosporidiosis is a disease with zoonotic potential, and one should take appropriate precautions, including use of gloves and frequent hand washing, if organisms are identified in the feces of any patients so as to prevent spread to other patients and personnel. eimeria leukarti, trichomonas equi, and giardia equi have been identified in the feces of normal horses and horses with diarrhea. transmission studies have failed to produce reliable clinical signs, and the prevalence and significance of these organisms in the genesis of foal diarrhea remain unknown. strongyloides westeri is a common parasitic infection of foals. 392, 414 transmission is transmammary, and patent infection is recognizable in the foal by 8 to 12 days of age. this nematode previously was associated anecdotally with foal heat diarrhea, but the association has not been demonstrated clearly. the diarrhea is generally mild and is treated effectively by deworming with benzimidazole or ivermectin anthelmintics. 391 strongylus vulgaris fourth-stage larvae cause diarrhea in young foals during migration through the arterioles of the cecum and descending colon. clinical signs may resemble thromboembolic colic. 414 the prepatent period is about 6 months, and diagnosis is based on clinical examination, clinicopathologic changes, and farm deworming history. patients with diarrhea associated with this parasite may have peripheral leukocytosis, neutrophilia, eosinophilia, and hypoproteinemia. appropriate deworming with ivermectin (label dose), fenbendazole (10 mg/kg/day orally for 5 days), or thiabendazole (440 mg/kg/day orally for 2 days) is recommended, with the last two drug dosages being larger than the label dose. cyathostomiasis, or diarrhea resulting from the sudden emergence of encysted cyathostome larvae, is an unusual cause of diarrhea in the foal. the clinician managing critically ill neonates must recognize that intravenous fluid therapy simply cannot be scaled down from adult management approaches. fluid management of the ill neonate, particularly over the first few days of life, must take into consideration that the neonate is undergoing a large transition from the fetal to the neonatal state and that important physiologic changes are taking place. 166 these transitions include shifts in renal handling of free water and sodium and increased insensible losses because of evaporation from the body surface area and the respiratory tract. the newborn kidney has a limited ability to excrete excess free water and sodium, and the barrier between the vascular and interstitial space is more porous than that of adults. water and sodium overload, particularly in the first few days of life, can have disastrous long-term consequences for the neonate. 416, 417 in the equine neonate, excess fluid administration frequently manifests as generalized edema formation and excessive weight gain, frequently equivalent to the volume of excess fluid administered intravenously. in cases in which antidiuretic hormone secretion is inappropriate, as in some foals with pas, generalized edema may not form, but the excess free water is maintained in the vascular space. this syndrome of inappropriate antidiuretic hormone secretion is recognized in the foal that gains excessive weight not manifested as edema generally, with decreased urine output and electrolyte abnormalities such as hyponatremia and hypochloremia. 418 the foal manifests neurologic abnormalities associated with hyponatremia. the serum creatinine concentration varies in these cases, but urine always is concentrated compared with the normally dilute, copious amounts of urine produced by foals more than 24 hours of age on a milk diet. if measured, serum osmolarity is less than urine osmolarity. the treatment for this disorder is fluid restriction until weight loss occurs, electrolyte abnormalities normalize, and urine concentration decreases. if the clinician is unaware of this differential diagnosis, the neonate can be assumed mistakenly to be in renal failure, and the condition can be exacerbated by excessive intravenous fluid administration in an attempt to produce diuresis. the problem of appropriate fluid management in critically ill neonates has been recognized by medical physicians for years and has resulted in changes in fluid management of these patients. the approach taken has been one of fluid restriction, in particular sodium restriction but also free water restriction, and has resulted in improved outcome and fewer complications, such as patent ductus arteriosus and necrotizing enterocolitis. 416, 417 the calculations used for maintenance intravenous fluid support in these patients takes into consideration the ratio of surface area to volume and partially compensates for insensible water losses. maintenance fluids are provided as 5% dextrose to limit sodium overload and provide sufficient free water to restore intracellular and interstitial requirements. the calculation for maintenance fluid administration is as follows: first 10 kg body mass 100 ml/kg/day second 10 kg body mass 50 ml/kg/day all additional kilograms of body mass 25 ml/kg/day as an example, the average 50-kg foal would receive 1000 ml/day for the first 10 kg of body mass, 500 ml/day for the next 10 kg of body mass, and 750 ml/day for the remaining 30 kg of body mass for a total of 2250 ml/day. this translates to an hourly fluid rate of about 94 ml/hr. one should adjust the fluid and sodium requirements for ongoing losses exceeding the maintenance requirements. these losses can take the form of diarrheal losses and excessive urine output, such as those with glucose diuresis and renal damage resulting in an increased fractional excretion of sodium. the normal fractional excretion of sodium in neonatal foals is less than that of adult horses, usually less than 1% (j.e. palmer, unpublished data). in the critically ill foal the sodium requirement can be met with as little as 140 meq of sodium per day, about that administered in a single liter of normal equine plasma. one can address sodium deficits by separate infusion of sodium-containing fluids, although this may not be necessary if one considers the sodium being administered in other forms, including drugs administered as sodium salts and any constant rate infusions (pressors, inotropes, etc.) that are being provided as solutions made with 0.9% sodium chloride. the author has used this approach to fluid therapy in her nicu for the last few years and believes that the percentage of foals suffering from generalized edema and related problems has decreased. if one takes this approach to fluid therapy, one should take the weight of the patient once daily, or even twice daily, and monitor the fluid intake and output as closely as practical. one should evaluate any larger than anticipated weight gains or losses. one should not expect urine output to approach the reported normal of 300 ml/hr for a 50-kg foal because the free water administered is limited, unless the patient is experiencing diuresis (glucosuria, resolution of the syndrome of inappropriate antidiuretic hormone secretion, resolution of previous edematous state, renal disease). one should obtain the urine specific gravity several times daily and should determine fractional excretion of sodium at regular intervals. if the volume of urine produced by the patient is measured accurately, one can determine sodium losses accurately and can obtain creatinine clearance values. one should obtain blood pressure measurements at regular intervals throughout the day because hypotension can be a problem in these patients, particularly in septic foals and foals suffering from pas, and one may need to increase fluid therapy to maintain adequate vascular volume. patients with hypotension may need inotrope and pressor support. inotrope and pressor therapy generally is restricted to referral centers where these drugs can be administered as constant rate infusions and blood pressure can be monitored closely. blood pressure can be monitored directly or indirectly by the use of cuffs placed on the base of the tail. both techniques have advantages and disadvantages. although direct blood pressure measurements are considered the gold standard and are generally more accurate, the difficulty in placing and maintaining arterial catheters and lines in these patients severely restricts the utility of this method. indirect techniques can be inaccurate and are affected by cuff size and placement. however, indirect techniques are easier to use in the nicu and can be useful if trained staff are using the equipment. in the author's nicu, once practitioners identify the appropriate cuff size, they dedicate that cuff to that patient for the duration of the hospitalization to decrease variability caused by using different cuffs. one should monitor the blood pressure of all recumbent patients at regular intervals, and trends upward or downward should prompt the clinician to make necessary adjustments. foals suffering from pas and sepsis are the patients most at risk for significant hypotension and perfusion abnormalities. perfusion is maintained by supporting cardiac output and blood pressure with judicious use of intravenous fluid support and inotrope/pressor support. the author does not aim for any specific target systolic, mean, or diastolic pressure. instead the author monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for nicu patients to require inotrope and pressor therapy is not unusual, but in some cases hypoxic and septic damage is sufficiently severe to blunt the response of the patient to the drugs. one must approach each patient as an individual, and no single inotrope/pressor protocol will suffice for all patients. dobutamine is a î²-adrenergic inotrope that is frequently used as first choice therapy in nicu patients. its effects are î² 1 at the lower dose range. neonates have a limited ability to increase stroke volume in an effort to maintain cardiac output, and one may observe tachycardia in these patients as heart rate increases to maintain cardiac output and vascular pressure. dobutamine is useful after patients are volume replete for support of cardiac output. the dose range is between 2 to 20 âµg/kg/min provided as a constant rate infusion. dopamine has dopaminergic activity at low doses, î² 1 and î² 2 activity at moderate doses, and î± 1 activity at high doses. dopamine causes norepinephrine release, which has lead to the suggestion that this is its major mode of action at higher doses. at doses greater than 20 âµg/kg/min, intrapulmonary shunting, pulmonary venous vasoconstriction, and reduced splanchic perfusion may occur. dopamine also produces natriuresis at lower doses through a direct effect on renal tubules. for these reasons, dopamine has fallen out of favor at some referral institutions. norepinephrine has î± 1 and î² 1 activity but variable î² 2 activity, resulting in potent vasopressor effects; it has inotropic and chronotropic effects, but its chronotropic effect usually is blunted by vagal reflexes slowing the heart rate induced by the increase in blood pressure. in many critical care units, norepinephrine has become a pressor of choice and frequently is used along with dobutamine. evidence suggests that splanchic perfusion is maintained better with norepinephrine than with some other pressors. 419 the dose range is 0.2 to 2.0 âµg/kg/min, although larger doses have been used when necessary in certain patients. epinephrine has î± 1 , î± 2 , î² 1 , and î² 2 activity; î² activity predominates and results in increased cardiac output and decreased peripheral resistance at low doses. epinephrine has been associated with hyperglycemia, hypokalemia, lipolysis, increased lactate concentration, and increased platelet aggregation. the effect on renal function is controversial. use of epinephrine usually is limited to those patients not responding to other pressors. vasopressin (antidiuretic hormone) is a pressor gaining a great deal of attention in the critical care literature. vasopressin appears to be depleted from the neurohypophysis in septic shock, 420 and short-term administration of vasopressin spares conventional vasopressor use, in addition to improving some measures of renal function. 421 low-dose vasopressin infusion increases mean arterial pressure, systemic vascular resistance, and urine output in patients with vasodilatory septic shock that are hyporesponsive to catecholamines. these data indicate that low-dose vasopressin infusions may be useful in treating hypotension in patients with septic shock. 422 the author has been using low-dose vasopressin in patients in her nicu for the past few years and has the clinical impression that blood pressure is defended more readily using this agent in concert with other management strategies. the author commonly uses low-dose vasopressin constant rate infusion with dobutamine constant rate infusion as the initial inotrope/pressor therapy in cases requiring pressure defense, although no prospective studies are yet available regarding this drug in veterinary medicine. 20 for quality health care for animals, advances in medical science, and in some breeds the increasing value of the juvenile equine athlete. equine veterinarians that encounter pediatric orthopedic problems are only beginning to get the information needed to make appropriate treatment decisions. the equine neonate has specific differences in structure and physiology from adults that one must consider when designing an optimal therapeutic or management strategy. few investigations have focused on the equine neonatal musculoskeletal system, 1-6 but a large body of clinical information exists, and one can make cautious extrapolations from work in other species. 7 neonatal equine bones have accelerated modeling and remodeling processes 5 that result in accelerated fracture healing and an increased susceptibility to deformation caused by excessive loading. contralateral limb varus deformities of the growth centers (most commonly distal radius and metacarpus/ metatarsus) are common in overloaded limbs. the increased plasticity of the skeletal structure also is mirrored in the soft tissue support system, for these units become flaccid within 2 weeks of immobilization. 4 this laxity is important, because it further compromises the use of the fractured limb and can last as long as the coaptation was in place. additional divergences from adult physiology include musculoskeletal immaturity (generalized or focal) and immune system differences. finally, foals are lighter and can tolerate and will assume recumbency more readily than adults. the net results of these differences are that one must consider the use of external coaptation carefully, fractures heal quickly, one must consider damage to the contralateral limb from overstress, reducing weight bearing is possible, and infection is always lurking. stresses can affect the musculoskeletal system of the foal at any time, including in utero. although rare, reports describe in utero fractures (k. sprayberry, personal communication, 2003) that result in foal locomotor problems and even maternal uterine damage from sharp bone ends. the cause is presumably from vigorous muscular activity of the foal, but one cannot rule out direct trauma. the fractures result in foal lameness and can increase the likelihood of dystocia and caused colic in one mare when the broken bones damaged the uterus. treatment depends on how long the fracture has been present and on the fracture location and configuration, but if the fracture is repairable, internal fixation probably is necessary. fractures occurring during foaling result from aggressive obstetric manipulation (mandibles) or the advances in medical care of equine neonates in the last 20 years have resulted in the survival of many foals that previously would have died from sepsis, asphyxia, and prematurity; and the successful management of their musculoskeletal system can be a major challenge. major factors adding to the challenge are the immaturity of components of the musculoskeletal system and the demands placed on them by a growing and active foal. additional pressures to treat orthopedic conditions in foals have come from an overall increase in the demand chest compression. one should stabilize unstable mandibular fractures. appendicular fractures usually do not occur during parturition because of the robust character of the bones of the foal. after birth, foals are susceptible to external trauma from many sources. the dilemma is that younger foals with fractures are more likely to heal but also are more likely to develop contralateral limb problems because of excessive weight bearing and affected limb flexor tendon laxity if the limb is immobilized fully. as a result, internal fixation is often the best choice for neonatal fractures to keep the fractured limb in use. proximal sesamoid bone fractures result from hyperextension of the fetlock joint. foals are lame after the fracture, but the lameness can be mild and often diminishes quickly. soft tissue swelling occurs over the sesamoids. fractures are usually simple, can occur uniaxially or biaxially, and can be apical, midbody, or basilar. fractures can occur in any joint and can affect multiple sesamoids in one foal. however, they most commonly are single forelimb fractures 8 and in thoroughbreds are most frequent in the left front medial proximal sesamoid (j.p. morehead, personal communication, 2003). of particular interest to neonatologists is that proximal sesamoids fractures often occur in recovered neonatal patients that are allowed too much exercise too soon. foals from the nicu need a gradual introduction to pasture turnout to allow their musculoskeletal system to adjust. mares are often in need of turnout, but in the interest of their foals, they must wait. treatment of proximal sesamoid fractures in foals is stall confinement with support bandaging. healing occurs, albeit with some distortion of the shape of the sesamoid. severely displaced fragments result in large and misshapen sesamoids, and surgery may be considered for these foals, because restriction of fetlock flexion can occur after conservative therapy. third phalangeal fractures are also common in foals. these foals have a lameness that worsens with hoof compression. hoof abscesses are uncommon in young foals but should be considered. most commonly, radiographs reveal nonarticular small fractures on the wings on the third phalanx. the fractures are associated with hard ground and exercise. the fractures heal with stall confinement, and unlike adults, leave no discernable radiographic fibrous union. avulsion fractures of the proximal insertion of the peroneus tertius and the origin of the long digital extensor tendon have been reported. 9,10 both soft tissue structures attach to the extensor fossa of the distal femur. the two affected foals had lameness of a hindlimb associated with swelling, pain, and crepitation. radiographs revealed multiple avulsion fractures of the extensor fossa. because of the intraarticular fragments in the femoropatellar joint, and the fear of later degenerative joint disease, fragments were removed arthroscopically. both foals were juveniles at last follow-up; one foal was considered normal, and one had a mild residual lameness. tendon and ligament damage is uncommon in neonates probably because of their low body weight. extensor tendon damage following flexural deformities is the most common tendon problem and is discussed in congential flexural deformities of foals. gastrocnemius ruptures are one of the most devastating problems and have occurred after forced extraction because of a breech presentation, severe flexor tendon laxity, and tarsal contracture. loss of gastrocnemius function usually results in a non-weight-bearing limb, although an intact superficial digital flexor tendon may make some weight bearing possible. complete loss of support is difficult to treat successfully. coaptation of the limb is logical but difficult to obtain. schroeder-thomas splints have been used but are difficult to manage. tube casts also are used but must be changed frequently, and cast sores are inevitable (l.r. bramlage, personal communication, 2003) . the prognosis for athletic function is guarded. treatment for ligamentous injuries is usually some form of coaptation, although surgical repairs have been performed when coaptation was unworkable. 11 coaptation in proper limb alignment allows the ligaments to heal and should be used if the injury will destabilize a joint and cause damage to growing epiphyses or cuboidal bones. one can achieve coaptation with casts or splints under a bandage. casts are initially a greater expense, and cast sores and their resulting white hairs are a risk, but the rigid immobilization and the lack of the requirement for daily adjustment makes them preferable. important to musculotendinous health is some measure of weight bearing to avoid laxity after coaptation removal, which one can achieve by using tube casts and splints that allow weight bearing. following coaptation, bandaging and a gradual return to exercise are recommended for ligamentous injuries. patellar luxation can affect foals in one or both hindlimbs, and the luxation can vary from a laxity in the medial attachments to complete luxations that cannot be replaced in the patellar groove of the distal femur. 12, 13 medial luxations have not been reported. clinical signs vary from a slight discontinuous motion during stifle flexion to an inability to stand. many foals have a crouching stance on the affected limb because of an inability to extend the stifle. the pathophysiology of patellar luxations is unknown. congenital bilateral luxations are common in miniature horse foals and are believed to be genetic. luxations are rarer in other breeds and are occasionally traumatic. the affected limbs are usually not grossly abnormal except for effusion of the femoropatellar joint and the luxation. a shallow trochlear groove has been reported to be a cause of patellar luxation, but objective evidence is lacking. one should evaluate foals for the ability to stand. once the appropriate supportive care is provided, if a foal cannot stand, euthanasia is recommended. most bilateral luxations in horses fit in this category. however, miniature horse foals often can stand sufficiently to nurse despite bilateral luxations, and one may consider treatment. treatment consists of replacing and stabilizing the patella and sometimes surgically deepening the patellar groove. delaying surgical repair until the foal is approximately 30 days old is recommended to avoid neonatal problems, allow the musculoskeletal system to mature, and provide good anchors for suture. some surgeons worry that delay may cause further femoropatellar developmental abnormalities, but in a small number of cases, this has not been an issue. the prognosis for miniature horse foals appears to be good because of their low body weights and modest performance expectations. too few reports about the correction of unilateral luxations in light horses exist to make a definitive statement about prognosis except that success and failure have been experienced. 12, 13 congenital flexural deformities in foals can be classified as severe (rarely correctable), moderate (correctable with therapy), or mild (self-correctable). examples of severe flexural deformities include arthrogryposis (deformities of multiple limbs and often the head and neck), severe carpal deformities (flexor angle of the carpus less than 90 degrees), and tarsal contractures (rare). extraordinary methods have been used to correct severe deformities 14 but are often unsuccessful. mild flexural deformities are those that result in an upright conformation to the limb, but the foal can bear weight on the limb and load the flexor structures. these foals require no specific treatment and will self-correct with controlled exercise. moderate flexural deformities are those that make bearing weight on the limb and loading the flexor structures and ligaments difficult for the foal. when these deformities occur bilaterally (most common), the foals cannot rise to suckle or does so with great difficulty, and the lack of weight bearing worsens the flexural deformity. examples of moderate flexural deformities include carpal and forelimb fetlock flexural deformities that usually occur together, hindlimb fetlock flexural deformities with coronopedal flexion or hyperextension, and the uncommon coronopedal flexural deformity alone. treatment of moderate flexural deformities aims to place the solar surface of the foot on the ground so that the weight of the foal can stretch the flexor structures. splints are useful for restoring the limb to normal orientation but require attention to detail because the splints often exert an extreme amount of tension on the soft tissues, and the skin of the foal is thin. pressure sores are easy to create and at a minimum result in an extended convalescence. the first step in splint application is to apply a separate heavy bandage to the limb, which should be reapplied as necessary because the bandage can slip and cause focal constriction. commercial gauze over cotton bandage material works better than sheet cotton as a bandage. the splint is made of polyvinyl chloride pipe cut in half or thirds. using 50% of the diameter of the pipe results in less splint rotation but is bulkier and leaves more splint exposed to cause trauma. one cuts off the corners of the splint and pads the ends with gauze or roll cotton covered with tape. palmar or plantar placement of the splint is preferable, but severe deformities may require initial dorsal placement. as the limb straightens, one can bend the splint to tape the fetlock into the bend to extend it. one can tape the splint tightly to the limb over the bandage with nonelastic (white or duct) tape. this procedure requires at least two persons, one to extend the limb firmly and hold the limb and one to tape. one should leave the splint on for 8 to 12 hours and then remove it for 8 to 12 hours. one can reapply splints as necessary. in addition to splints, some medications are of value for treating flexural deformities. oxytetracycline (40 to 50 mg/kg) given intravenously appears to relax the soft tissues. 15 the mechanism of action is unknown, and the drug is most efficacious when given in the first 3 days of life. this dose is high but appears to be safe for healthy foals and can be repeated at 24-hour intervals. foals should be normovolemic during tetracycline administration. one should use the drug with caution in foals with renal impairment. foals should be urinating and have reasonable urinary parameters (serum urea nitrogen, creatinine, and urinalysis) before tetracycline use. diarrhea is an uncommon sequela to tetracycline use. one should monitor the unaffected limbs closely because all limbs experience a relaxation of the palmar/plantar support. 15, 16 discontinuation of tetracycline therapy before affected limbs are normal but after they can bear weight is common because of worsening laxity in the "normal" limbs. one also can use phenylbutazone (4 mg/kg) for a short time when the splints are used. some analgesia appears to help the foals use the limbs and stretch the soft tissues. one should not use phenylbutazone for long periods of time because of the potential of inducing gastric ulcers. surgical treatment of congenital flexural deformities rarely is indicated. severely affected foals rarely respond favorably to surgery, and mildly affected foals do not need it. surgery is most appropriate for foals with moderate flexural deformities that are neglected or have not responded to splinting and tetracycline. the most common surgical therapy performed for congenital flexural deformities is the inferior check ligament desmotomy for fetlock or coronopedal flexural deformities. ruptures of the extensor tendons commonly occur with congenital flexural deformities and result from the foal overloading the extensor tendons. no specific therapy for the ruptures is necessary. if the rupture is extensive, it can interfere with the ability to extend the fetlock and to place the foot flat. these foals then tend to knuckle over, even after correction of the flexural deformity. a firm fetlock bandage extends the digit and assists in foot placement until the extensor tendons heal. foals commonly are born with hyperextension deformities of the fetlock of varying degrees of severity. all but the worst deformities self-correct as muscle tone improves. a deeply bedded stall is all that is usually necessary to protect the soft tissues, but one can apply a light bandage to the coronary band and pastern if trauma is a problem. severe deformities are more problematic but rare, so therapeutic recommendations are not available. hyperextension of the carpus occasionally occurs and usually is treated conservatively. however, a tube cast to align the limb may be necessary to protect the dorsal surface of developing carpal bones. neonatal foals exhibit three categories of forelimb conformational deviations: angulation, rotation, and carpal offset. angular deviations most commonly are centered in the metaphysis and epiphysis, but their location is described by the closest joint, usually the carpus and fetlock. when the deviation of the distal limb is lateral to the long axis, the deviation is valgus, and when the deviation is medial, the deviation is varus. more than one joint can be affected, and although rare in neonates, valgus and varus can occur in different joints in one limb. rotational deformities appear to originate most commonly in the diaphysis or metaphysis of the radius or the metacarpus. in neonates the direction of rotation of the distal limb at both sites is almost exclusively outward. associated angular and rotational deviations occur. 17 in neonates, limb deviations occur in foals with narrower chests and less developed pectoral muscles than in straight foals, and they appear to have an initial greater overall weakness in the musculoskeletal system because it first interacts with gravity, body mass, and ground reaction forces. however, after the first few days of life, the asymmetric loading of the growth centers does affect limb deviations. angulation results from a compressive load that is asymmetric in a frontal plane but is uniform in the sagittal plane, and rotation occurs when the compressive load is asymmetric in both planes and the limb develops around an overloaded axis point. considered this way, valgus and outward rotation deviations in young foals are coupled, as are varus and inward rotation in older foals. the loading asymmetry for valgus/outward rotation foals is accentuated as foals assume a base-wide posture that is more stable side-to-side but promotes a lateralization of the limb load. the specific effects of intermittent versus static loads, strain magnitude versus strain rate, and shear and hydrostatic stress on growing bones is only beginning to be understood. however, clinical experience supports the general observation that excessive cartilage compression is deleterious to bone growth. offset carpal conformation describes a joint that appears to deviate outwardly and then inwardly, all within the carpus. the deformity is thought to be centered at the radiocarpal joint, but the specific structural cause of offset has not been determined. this conformation is more common in older foals but occasionally occurs in neonates. the deviation is particularly common when incomplete ossification of the carpal bones is present. the causes of conformational deviations are a matter of some debate. as always, the major factors are genetics or environment. genetic influences include the assortment of alleles that controls bone form and growth and the assortment that modulates bone remodeling. many in the horse industry believe that genetics is a strong determiner of limb conformation. environmental influences are many and include the intrauterine environment, the postnatal limb load, nutrition, and bad luck. suffice to say, the situation is complex, but one must consider biologic and mechanicobiologic influences when evaluating the growth of long bones. 18 several factors may contribute to the common occurrence of deviations in the carpus. first, the carpus is in the middle of the limb and is subject to the greatest bending forces. second, the carpal anatomy is complex and perhaps is not understood completely. the carpus has seven cuboidal bones, two long bones, and two epiphyses (distal radial and lateral styloid); and cartilage surrounds all. the ligamentous support includes collateral ligaments, innumerable intracarpal ligaments, and a palmar carpal soft tissue ligament. the distal radial physis is not flat transversely, but undulates in the frontal and sagittal planes. 3 a separate center of ossification for the lateral styloid process is found at its palmar-lateral aspect. because of this separate center of ossification, more cartilage and less bone are in the lateral aspect of the distal radial growth center, suggesting it may be more susceptible to growth alterations from load. less common conformation deformities in young foals include hindlimb deformities, windswept conformation, diaphyseal deviations (usually of the metacarpus/ metatarsus), gross congenital malformations such as agenesis and polydactyly, and acquired varus deformities of the carpus and fetlock. hindlimb conformational deviations can manifest as tarsal and fetlock angular deformities and external limb rotation, usually centered above the tarsus. windswept foals have limbs (usually both forelimb or both hindlimbs) that are curved in the same direction in the frontal plane. diaphyseal deviations, agenesis, and polydactyly are rare and have various presentations. acquired varus deformities are caused by excessive loading, which appears to be focused medially on the growth plates. one should evaluate the limbs to determine the location, extent, and potential cause of the deviation. evaluation consists of observation and then palpation for heat, swelling, or ligament laxity. ligamentous laxity of the medial carpal ligaments is an important cause of carpal valgus and should be evaluated carefully. lameness is not a characteristic of uncomplicated angular limb deformities and suggests further evaluations are necessary. radiography is indicated for foals with severe deviations (all tarsal valgus), ligamentous laxity, lameness, or joint effusions. ultrasonography may be valuable for selected soft tissue evaluations. conservative therapy is by far the most commonly used therapy in foals less than 30 days of age. 19 mild to moderate carpal valgus and external rotation of the carpus and fetlock are common and normal in neonates, particularly light breed horses. most congenital limb deviations improve with age, if the developing musculoskeletal system is protected from overuse and abnormal loads. approximately 90% of thoroughbred foals with congenital carpal valgus self-correct. those foals that do not most often have abnormal bone (incomplete ossification) with normal stress or normal bone with abnormal stress (ligamentous laxity or contralateral limb lameness). correction continues for several months, and on average, foals reach their straightest conformation (regarding angulation) at approximately 10 months of age (e.m. santschi, unpublished data). determination of the appropriate treatment for foals with angular limb deformities is based on the age of the foal, the severity and location of the deviation, and its causes. one must evaluate the entire foal and the affected limb. if the carpal collateral ligaments have no laxity and carpal incomplete ossification is not suspected, one may use an exercise program such as in table 19 -10, assuming that the foal has no contradicting additional problems. exercise is essential for the robust development of almost every body system for neonates, and fresh air and good ventilation reduce the occurrence of respiratory disease. appropriate limb loading along with growth and maturity is what straightens limbs, but excessive amounts of loading can be deleterious. for example, one should use exercise cautiously in foals with very asymmetric deviations. when one limb is much more deviated than the other, it appears to be loaded excessively and compromised more than if both limbs were affected similarly. and finally, limb deviations are additive. foals with external rotation and carpal valgus improve more slowly than those with one type of deviation. incomplete ossification of cuboidal bones and focal ligamentous laxity are complicating matters of great potential impact on adult conformation. they generally manifest as a moderate to severe limb deviation. physical examination indicates laxity because angular limb deviations are reducible. radiographs are the best way to evaluate the extent of carpal bone ossification. incomplete ossification of the cuboidal bones can be focal or widespread. focal immaturity is not common but can result in severe angulation. generalized immaturity is more frequent and initially often manifests as an offset conformation with valgus angulation. when the foal becomes heavier, assumes a base-wide stance, and is allowed exercise, crushing of the bones of the lateral carpus (usually the lateral styloid process of the radius, the ulnar, the fourth and the intermediate facet of the third carpal bone) results in a permanent intracarpal valgus deviation. the same result occurs when significant medial carpal ligament laxity goes untreated. in the forelimb, foals with collateral ligamentous laxity and moderate to severely immature cuboidal bones should have external coaptation placed on the affected limb to maintain axial orientation. tube casts that allow weight bearing on the digit are preferred to splints. ligamentous laxity in the carpus usually responds to tube casting for 7 to 10 days followed by bandaging and cautious exercise. the duration of similar coaptation necessary for immature carpal bones depends on the degree of immaturity and the speed with which the bones mature. because casts cannot be left on neonatal limbs for more than 7 to 10 days because of their fast growth, more than one cast may be necessary. treatment of tarsal valgus and rotational deformities is much less common than in the forelimbs because deviations are less common than in the forelimb, because some breeds prefer an outward position to the hindlimb, and perhaps because owners recognize it less frequently. 20 hindlimbs generally are unaffected by ligamentous laxity, but tarsal incomplete ossification is common and often is associated with tarsal valgus. treatment of tarsal incomplete ossification is important because tarsal crushing results in an unfavorable prognosis for athletic performance. 20, 21 hindlimbs require a slightly different approach to coaptation than forelimbs because of their anatomy. foals can rise to stand if their forelimbs are fixed in extension but cannot do so if their hindlimbs are extended. the multiple bony protuberances of the hock make cast sores more likely than in the forelimb, so casts are problematic. gutter splints are not useful because of the angle of the hock. severely limiting exercise is part of allowing the tarsus to mature without cartilage crushing, but foals cannot always be recumbent. extra small articulated anterior cruciate ligament splints for human beings (playmaker wraparound, dj orthopedics, vista, california) have given the best results. for small foals, a padded bandage is necessary under the splint, which is reversed to conform to the angle of the hock. the splints allow enough flexion in the hock for the foal to rise but appear sufficient when combined with stall rest to protect the cartilage from crushing. splints are left on the hocks until the cuboidal bones have ossified as shown by radiography. fetlock conformational deviations in neonates that are treated best conservatively are rare. outward rotation is the most common deviation but is thought to have minimal effect on the performance and improves with maturity. the only therapy used is to rasp the toe square to promote central breakover. severe outward rotation can promote a fetlock valgus conformation, so one can use a medial hoof wall extension of epoxy to bring the limb load medially. the most commonly treated fetlock deviations are inward but usually occur in foals older than 30 days. however, if the deviation is noticed in neonates, one can use small lateral hoof wall extensions that generally are made of epoxy with fiberglass cloth embedded to prevent chipping. windswept foals are born with multiple deviations. evaluating the foal as a whole is best rather than focusing on individual joints. most of these foals become straight over time with conservative therapy. no surgical procedures are commonly accepted for direct treatment of rotational or carpal offset deviations, so angular deviations are described. surgical procedures to correct carpal and fetlock valgus include periosteal transection and elevation and transphyseal bridging. periosteal elevation is thought to accelerate growth on the concave side of the metaphysis, and transphyseal bridging is used to restrict the growth on the convex side of the physis. studies indicate an approximately 80% improvement of carpal valgus foals after periosteal transection and elevation, but unfortunately they do not compare foals that had surgery with controls that did not. 22, 23 recently, some have suggested that most of the correction was unrelated to the surgery, 24 and one experimental study supports that conclusion. 25 as a result, at this time making firm recommendations about the indications for periosteal transection and elevation is difficult. however, periosteal transection and elevation has a low likelihood of complications and may be effective. the procedure is inexpensive and can be done in the field and therefore may be an option for clients with foals with carpal valgus in which a transphyseal bridging is undesirable or unnecessary. one indication is the very young foal born with a notably asymmetric epiphysis that results in a severe carpal valgus. this distal radial appearance is not particularly common, but the lack of ossification in the epiphysis can make a firm hold with a transphyseal bridging difficult to achieve. however, one can use distolateral radial periosteal elevation at an early age in an attempt to accelerate correction of the valgus and protect developing carpal bones. often a degree of anxiety exists about correction of fetlock angulations because of the much shorter time period for physeal growth. most fetlocks are in their final conformation by 60 days of age, so correction is best accomplished with earlier treatment, usually by 4 weeks of age. one can perform periosteal elevation on the medial (for varus deviations) or lateral (for valgus deviations) aspect of the distal metacarpus/metatarsus. the definitive treatment of limb angulation at a growth plate is transphyseal bridging. one should consider using the procedure at about 3 weeks of age for all moderate to severe fetlock deviations, at about 4 weeks for severe carpal deviations, and 6 to 8 weeks for mild fetlock deviations, moderate carpal deviations, and any worsening angular deformities. one must perform bridge removal when the limb straightens to prevent overcorrection. diaphyseal deviations are rare but can occur in varying degrees of severity. if the foal can bear weight on the limb, a conservative approach is indicated. one can consider periosteal elevation of the length of the concave surface of the long bone. if the foal cannot bear weight on the limb because of the severity of deviation, euthanasia is probably the best option. however, a revision osteotomy and internal fixation may be appropriate for selected foals. 26 polydactyly is also rare and sometimes can be corrected surgically. the outcome is based on the degree of articular involvement. bacteria may invade the foal musculoskeletal system and cause orthopedic infection after delivery by the circulation, by direct extension from another system, or by direct inoculation. hematogenous delivery is by far the most common and results in infection of synovial structures (joints, tendon sheaths, bursae) and bone. extension from another site without hematogenous delivery is rare. direct inoculation almost exclusively results from traumatic rather than surgical wounds. much is still to be learned about the pathophysiology of orthopedic infection, including the source of the infecting bacteria. the umbilicus commonly is accepted as a possible source of bacteria, 27 but many believe that the gastrointestinal and respiratory tracts are at least equally responsible. associated conditions in foals with septic arthritis include failure of passive transfer, pneumonia, and enteritis. 28 the classification of orthopedic sepsis in foals into infection of bones and joints is probably irrelevant because most foals with septic arthritis also have infectious osteitis or osteomyelitis. 27, 29 septic arthritis is more readily recognizable because the reactivity of the synovium to the bacteria causes joint effusion and lameness and because early radiographic signs of bone infection in foals are equivocal. also unclear are the reasons for the apparent site predilection for orthopedic infection in foals. the femoropatellar joint and the tarsocrural joint are affected most frequently, followed by the carpal and fetlock joints, and finally an assortment of miscellaneous joints such as the elbow, shoulder, and hip. 28 the common association of osteomyelitis of the distal femoral, tibial, and metacarpal/metatarsal physes with a newly recognized septic arthritis suggests that the infection in that area started at the growth center (epiphysis, physis, or metaphysis). the localization of the apparent initial site of infection to the growth center has been suggested to result from "looping" metaphyseal vessels with sluggish blood flow that allow pathogens more time to escape the circulation. 29, 30 however, transmission electron microscopy indicates that osteogenic cells and the vascular endothelium are a continuous network in developing embryos, 31 indicating that the relationship between circulation and bone is more intimate than previously suspected. a possible association between osteomyelitis and thickened or traumatized cartilage exists. focal osteomyelitis lesions occur commonly at the bone cartilage junction 27, 29 and particularly in areas where cartilage is attached at an angle to the long axis or where thickened. 29 an association also exists between incomplete ossification of the central and third tarsal bones and osteomyelitis. 32 trauma to the metaphysis is a known predisposing cause of osteomyelitis in young bacteremic rabbits. 33 a trend exists for foals with more than one joint affected to be affected bilaterally in the same joint, rather than in random joints. this trend suggests that a "window" exists when a joint may be more susceptible to infection and that trauma to the developing cartilage may be a contributing factor. in neonates, cartilage is vascular, 34 and possibly small traumatic cartilage lesions with associated hemorrhage and exposure of bacterial binding sites might be the inciting cause for the location of infection. the pathogens most commonly associated with septic arthritis in young foals are also those that frequently are implicated in neonatal sepsis. the most commonly isolated gram-negative organisms are escherichia coli and other enterobacteriaceae, actinobacillus equuli, and salmonella spp. frequently isolated gram-positive organisms include streptococcus spp., staphylococcus spp., and rhodococcus equi. 28 anaerobic bacteria and fungi are rare but should be considered in refractory cases. the diagnosis of orthopedic sepsis can be challenging. the most common clinical sign is lameness, followed by swelling around a joint or metaphysis. joint effusion alone may cause the swelling, but edema is also common, especially if metaphyseal osteomyelitis is present. but effusion and edema can be difficult to detect because of the tissue surrounding the focus of infection in the shoulder, elbow, hip, and coffin joints. one should evaluate lame foals carefully by palpation to localize pain and swelling. if one can find no pain or swelling, one should obtain a complete blood count and fibrinogen level. although a complete blood count is not always abnormal in foals with septic arthritis, abnormalities should raise the index of suspicion of infection. elevations in fibrinogen are fairly common in septic arthritis, 28 and fibrinogen almost always is elevated if the infection involves bone. if hematologic values are normal, the lameness could be caused by trauma, but the foal should be monitored closely for improvement, and closer evaluation is indicated if improvement is not rapid. an arthrocentesis is the diagnostic test of choice for confirmation of septic arthritis. one should perform joint puncture in a sterile fashion, and sedation is indicated to get an atraumatic tap. short-term anesthesia is preferable when joints have effusion because one may perform joint lavage at the same time. normal joint fluid should be clear to slightly yellow, should be viscous, and should contain less than 2500 nucleated cells per deciliter. the cell ratio should be roughly 50:50 polymorphonuclear and mononuclear. the total protein content should be less than 2.5 mg/dl. one should consider joints to be infected if the nucleated cell count is greater than 10,000 cells/dl. for joints falling between 2500 and 10,000 cells/dl, if the polymorphonuclear cell count is >90%, one should consider the joints infected. cytologists are often reluctant to diagnose infection when nuclear degeneration or bacteria are not visible. this is overly conservative and results in delay in treating infections because bacteria and nuclear degeneration are rare in early cases of joint infection. out of an abundance of caution, one should treat lame foals with suspicious joints as infected unless they are clearly normal. one should always culture joint fluid in an attempt to identify the offending organisms, but because of difficulties in culturing pathogens from joint fluid samples, absence of growth does not mean absence of infection. one obtains the best culture results if the foal has not been treated with antimicrobial agents beforehand. one should obtain as much joint fluid as possible for culture and should incubate it overnight in blood culture media before plate inoculation. as always in potentially septic foals, blood culture may assist in the isolation of the organism. other orthopedic infections that do not involve the joint may be more difficult to detect. often these are not apparent until infection breeches the joint and causes lameness. however, astute caretakers may notice early clinical signs such as mild lameness, fever, or edema centered at a growth center. radiography and advanced imaging modalities such as magnetic resonance imaging are the best diagnostic tools for the localization of areas of osteitis and osteomyelitis. one should examine the area of concern carefully, giving particular attention to the growth centers and subchondral bone. interpretation of radiographs may be difficult because these areas are complex and normally have irregular bone margins in the growing foal. if a normal contralateral joint is available, comparison radiographs may be useful. because of the high metabolic turnover in growing foal bone, changes occur faster than with adults, so radiographs at the earliest sign of potential infection of bone and joint are recommended. if evidence of osteolysis is clear, aspiration of the area may yield material for culture. the goals of treatment are to eliminate infection immediately and then resolve inflammation. bacteria and products of inflammation elicited by infection are responsible for destruction of bone and cartilage. the ultimate aim of treatment is to protect the structures critical to athletic performance such as subchondral bone and cartilage in weight-bearing areas. advances in the treatment of sepsis have resulted in hospital discharge rates of 78% for foals with septic arthritis, but their rate of high performers is 30%, 28 indicating a need for improvement. equine veterinarians cannot replace what has been destroyed, so early identification and aggressive therapies are presently the best methods to improve performance rates. one achieves the goals of treatment by physical removal of bacteria, products of inflammation, and debris and by medications to kill the bacteria and reduce inflammation. one should optimize the physiology and general health of the foal to assist this process; one should include other treatments and supportive therapies for septic foals, especially treatment of failure of passive transfer, in the therapeutic plan. intravenous administration of antimicrobials (see chapter 4) is the cornerstone of treatment of orthopedic infection, and if the drug is administered early in the course of infection and bacteria are susceptible, intravenous administration may be sufficient to eliminate the organisms. however, treatment of many foals does not begin until disease is advanced. if treatment begins after bacteria have had a chance to establish themselves, one should bring all appropriate methods to bear to end the infection. additional therapies for septic arthritis include joint lavage, arthrotomy (for drainage), 35,36 debridement (arthroscopically or arthrotomy), 37 intraarticular administration of antimicrobials, intravenous regional perfusion, 38 and antimicrobial beads. 39, 40 one can use any sterile isotonic solution to flush a joint, and additives do not appear to give significant additional benefit. if radiographs do not indicate osteomyelitis, lavage, intraarticular antibiotics, and if possible, regional perfusion are recommended. if osteitis or osteomyelitis is present, debridement is indicated arthroscopically or via arthrotomy (one should culture the debris if the pathogen is unknown). if the joint is closed, one may use antibiotics intraarticularly. if the joint is left open to drain, regional perfusion is useful. antimicrobial beads theoretically are best to use if the wound is closed, but they appear to give benefit even if the wound is open under a bandage. because of concerns about the use of beads in a joint, 41 beads often are used in tissue defects and the surrounding tissues. the major goal is to remove material that is compromising healthy tissues and to obtain high concentrations of antimicrobials in infected tissues. high antimicrobial concentrations are necessary because adhered bacteria are difficult to kill and may require many times the in vitro bacterial minimum inhibitory concentration. intraarticular administration of antimicrobials has been used for many years and has great value. 35 regional perfusion of diluted antimicrobials recently has come into use and may be administered intraosseously 42 or intravenously. intravenous perfusion is preferable because no special equipment is needed, but intraosseous perfusion may be valuable where intravenous access is impossible. the concept behind both procedures is to fill the venous vasculature in the area of the infection with antimicrobials diluted by a sterile balanced electrolyte solution. one isolates the anatomic area of interest using one or two tourniquets. the perfusate diffuses into all tissues and achieves much higher concentrations than are possible using intravenous therapy. this technique has shown excellent results as an adjunct therapy for orthopedic infection. 43 for foals, 12 to 20 ml total of perfusate containing 250 mg amikacin is useful for most single joint sites. amikacin has given consistently good results without complication and is a good choice based on its concentration-dependent activity. one may use a higher volume for the stifle, but the thigh musculature makes an effective tourniquet difficult to achieve. because of concerns that perfusion might dislodge bacteria and renew systemic sepsis, high concentrations of systemic antimicrobials are recommended at the time of the perfusion. if joint lavage and intraarticular administration of antimicrobials are not sufficient to resolve infection, one may perform arthrotomy to assist the joint to drain. passive and active drains add foreign material and so are not useful. maintaining the joint under a sterile bandage is critical and can be difficult to do in proximal joints such as the stifle and elbow. tie-over bandages can be useful in this application. the best measure of success is the resolution of lameness and local inflammation. radiographs may be helpful, but the most common sign of success is a failure of the infection to progress, rather than radiographic healing. one should continue intravenously administered antimicrobials for at least 1 week after the resolution of lameness. if an appropriate drug is available, one should give foals antimicrobials orally for at least 2 weeks more. a total of at least 4 weeks of antimicrobials is recommended for most foals with orthopedic infection. treatment failures usually result from an inability to kill bacteria adhered to isolated tissue (usually dead bone). sometimes this failure is caused by incomplete debridement or an inability to access a known site of infection, but more frequently it is because infection has flourished in an unknown site. for this reason, multiple imaging modalities (radiographs, ultrasound, computed tomography, and magnetic resonance imaging) used multiple times are recommended for all refractory cases of septic arthritis. osteomyelitis not associated with a joint still involves a growth center. the ideal treatment for these infections is surgical debridement, systemic antimicrobial therapy, and some form of local antibiotic delivery. 44, 45 even in the face of large initial osseous defects, infection may resolve, the defect may heal, and the foal may regain normal limb anatomy and 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perinatal asphyxia current options in the management of apnea of prematurity hypocapnia and hypercapnia in respiratory management of newborn infants acute renal failure in children: aetiology and management low-dose dopamine in the intensive care unit: dnr or dnrx? use of dopamine in acute renal failure: a meta-analysis the effects of dopamine and epinephrine on hemodynamics and oxygen metabolism in hypoxic anesthetized piglets initial experience with norepinephrine infusion in hypotensive critically ill foal multiple organ involvement in perinatal asphyxia continuous enteral feeding impairs gallbladder emptying in infants trophic feeding of the preterm infant continuous nasogastric milk feeding versus intermittent bolus milk feeding for premature infants less than 1500 grams functional residual capacity and passive compliance measurements after antenatal steroid therapy in preterm infants current surfactant use in premature infants effects of the fungal endophyte acremonium coenophialum in fescue on pregnant mares and foal viability postnatal renal adaptation in preterm and term lambs risk factors of hypoglycemia in premature infants studies on equine prematurity. 4. effect of salt and water loss on the reninangiotensin-aldosterone system in the newborn foal studies on equine prematurity. 5. histology of the adrenal cortex of the premature newborn foal angular limb deformities in premature/ dysmature foals neonatal septicemia workshop 1, dorothy havemeyer foundation dorothy havemeyer foundation american college of chest physicians/society of critical care medicine consensus conference definitions of the systemic inflammatory response syndrome and allied disorders in relation to critically injured patient definition of sepsis to sirs with love failure of passive transfer in foals: incidence and outcome on four studs in new south wales a prospective study of septicaemia in colostrum-deprived foals care of the gut in the surgical intensive care unit: fact or fashion? bacterial isolates from blood and their susceptibility patterns in critically ill foals: 543 cases (1991-1998) development of real-time taqman pcr systems to facilitate the diagnosis and research of septicemia in foals expressed sequence tags (ests) isolated from blood of a septic thoroughbred foal early goal-directed therapy in the treatment of severe sepsis and septic shock chronic flunixin meglumine therapy in foals clinical and pathological effects of flunixin meglumine administration to neonatal foals gastric ulcers in foals experimentally induced toxicoinfectious botulism in horses and foals toxicoinfectious botulism in foals and adult horses botulism in the horse white muscle disease of foals naturally-occurring tyzzer's disease (bacillus piliformis infection) in horse foals suspected tyzzer's disease in two foals four cases of tyzzer's disease in foals in england bacillus piliformis infection (tyzzer's disease) in foals in northwestern united states: a retrospective study of 21 cases tyzzer's disease in a foal: light-and electron-microscopic observations clinical and clinicopathologic findings in two foals infected with bacillus piliformis serum biochemical and haematological findings in two foals with focal bacterial hepatitis (tyzzer's disease) toxic hepatopathy in neonatal foals the diagnosis and surgical correction of congenital portosystemic vascular anomalies in two calves and two foals clinical signs and radiographic diagnosis of a portosystemic shunt in a foal clinical and diagnostic features of portosystemic shunt in a foal evidence for transmission of halicephalobus deletrix (h gingivalis) from dam to foal halicephalobus (micronema) deletrix infection in two half-sibling foals listeriosis in an arabian foal with combined immunodeficiency suspected protozoal myeloencephalitis in a two-month-old colt pathological findings in horses dying during an outbreak of the paralytic form of equid herpesvirus type 1 (ehv-1) infection central nervous system neosporosis in a foal cauda equina syndrome, diskospondylitis, and a paravertebral abscess caused by rhodococcus equi in a foal vertebral body osteomyelitis due to rhodococcus equi in two arabian foals rhodococcus equi vertebral osteomyelitis in 3 quarter horse colts agenesis of the corpus callosum with cerebellar vermian hypoplasia in a foal resembling the dandy-walker syndrome: pre-mortem diagnosis by clinical evaluation and ct scanning cerebellar hypoplasia and degeneration in a foal cerebellar hypoplasia and degeneration in the young arab horse: clinical and neuropathological features imaging diagnosis: occipitoatlantoaxial malformation in a miniature horse foal occipitoatlantoaxial malformation with duplication of the atlas and axis in a half arabian foal occipitoatlantoaxial malformation in two non-arabian horses ivermectin toxicosis in a neonatal foal presumed moxidectin toxicosis in three foals hypovolemic hyponatremia and signs of neurologic disease associated with diarrhea in a foal extrapontine myelinolysis with involvement of the hippocampus in three children with severe hypernatremia relationships between radiography of cervical vertebrae and histopathology of the cervical cord in wobbling 19 foals assessment of colostral transfer and systemic availability of immunoglobulin g in new-born foals using a newly developed enzyme-linked immunosorbent assay (elisa) system evaluation of a test kit for determination of serum immunoglobulin g concentration in foals relationships among serum immunoglobulin concentration in foals, colostral specific gravity, and colostral immunoglobulin concentration measurement of igg in equine blood by immunoturbidimetry and latex agglutination a rapid, specific test for detecting absorption of colostral igg by the neonatal foal practical methods of determining serum immunoglobulin m and immunoglobulin g concentrations in foals passive immunity in the foal: measurement of immunoglobulin classes and specific antibody gammaglobulin and antibody variations associated with the maternal transfer of immunity and the onset of active immunity immunoglobulin metabolism in the neonatal foal prevalence (treatment days) and severity of illness in hypogammaglobulinemic and normogammaglobulinemic foals factors associated with failure of passive transfer of colostral antibodies in standardbred foals failure of passive transfer in foals failure of passive transfer of colostral immunity in the foal: incidence, and the effect of stud management and plasma transfusions the incidence and consequences of failure of passive transfer of immunity on a thoroughbred breeding farm failure of colostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals secretion and composition of colostrum and milk immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses interleukin-18 in human milk improved recovery of insulin-like growth factors (igfs) from bovine colostrum using alkaline diafiltration cytokine-inducing activity of a proline-rich polypeptide complex (prp) from ovine colostrum and its active nonapeptide fragment analogs measurement of betacellulin levels in bovine serum, colostrum and milk in vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specific defence activities of gammaglutamyltransferase, alkaline phosphatase and aspartateaminotransferase in colostrum, milk and blood plasma of calves fed first colostrum at 0-2, 6-7, 12-13 and 24-25 h after birth efficacy of intravenous plasma to transfer passive immunity in clinically healthy and clinically ill equine neonates with failure of passive transfer evaluation of intravenous administration of concentrated immunoglobulin g to colostrumdeprived foals lyophilized hyperimmune equine serum as a source of antibodies for neonatal foals absorption of bovine colostral immunoglobulins g and m in newborn foals comparison of freezing and lyophilizing for preservation of colostrum as a source of immunoglobulins for calves a comparison of the reduction in immunoglobulin (igg) concentration of frozen equine plasma treated by three thawing techniques use of blood and blood products neonatal isoerythrolysis in mule foals characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis prevalence of anti-red blood cell antibodies in the serum and colostrum of mares and its relationship to neonatal isoerythrolysis polymerized hemoglobin therapy in a foal with neonatal isoerythrolysis post-transfusion survival of 50cr-labeled erythrocytes in neonatal foals strategies for prevention of neonatal isoerythrolysis in horses and mules detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia neonatal alloimmune thrombocytopenia in a quarter horse foal neonatal thrombocytopenia: new insights into pathogenesis and implications for clinical management thrombocytopenia in horses detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia reef vb: cardiovascular disease in the equine neonate congenital polyalveolar lobe in three foals critical pulmonary stenosis in a newborn foal bilateral hypoplasia of the soft palate in a foal pulmonary lobar hypertrophy in a foal equine congenital defects congenital bilateral choanal atresia in a standardbred foal evaluation of pulse oximetry in anaesthetised foals using multiple combinations of transducer type and transducer attachment site electrocardiographic findings during parturition and blood gas tensions immediately after birth in thoroughbred foals blood gas and acid-base changes in the neonatal foal blood gas tensions and ph values in the normal thoroughbred foal at birth and in the following 42h mixed venous blood gases in recumbent and upright positions in foals from birth to 14 days of age ventilatory support of the neonatal foal report of foal pneumonia panel association of microbiologic flora with clinical, endoscopic, and pulmonary cytologic findings in foals with distal respiratory tract infection microbiologic changes during antimicrobial treatment and rate of relapse of distal respiratory tract infections in foals pathologic changes and pathogenesis of parascaris equorum infection in parasite-free pony foals spezifische infektiose pneumonie beim fohlen. ein neuer eiterreger beim pferd interaction of rhodococcus equi with phagocytic cells from rhodococcus equi-exposed and non-exposed foals electron microscopic investigation of intracellular events after ingestion of rhodococcus equi by foal alveolar macrophages influence of rhodococcus equi on the respiratory burst of resident alveolar macrophages from adult horses rhodococcus equi infection of monocytes/macrophages from human immunodeficiency (hiv)-infected patients and healthy individuals: evaluation of intracellular killing and nitric oxide production the intracellular bacterium rhodococcus equi requires mac-1 to bind to mammalian cells rhodococcus (corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses identification of 15-to 17-kilodalton antigens associated with virulent rhodococcus equi virulence-associated 15-to 17 kilodalton antigens in rhodococcus equi: temperature-dependent expression and location of the antigens role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein a in intracellular survival and virulence of rhodococcus equi characterization of avirulence-associated gene family in rhodococcus equi corynebacterium equi infections in horses, 1958-1984: a review of 131 cases clinical manifestations, diagnosis, treatment, and prevention of rhodococcus equi infections in foals detection of corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay rhodococcus equi pneumonia in foals: comparison of elisa and agid serology on a commercial thoroughbred breeding farm studies of naturally occuring and experimental rhodococcus equi (corynebacterium equi) pneumonia in foals detection of virulent rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of r. equi pneumonia in foals comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of rhodococcus equi pneumonia in foals use of erythromycin-rifampin combination in treatment of rhodococcus equi pneumonia pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals hyperthermia in foals treated with erythromycin alone or in combination with rifampin for respiratory disease during hot environmental conditions clostridium difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for rhodococcus equi pneumonia strategies for the control of rhodococcus equi infections on enzootic farms immunoprophylaxis of rhodococcus equi pneumonia in foals failure of hyperimmune plasma to prevent pneumonia caused by rhodococcus equi in foals rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals protection against naturally acquired rhodococcus equi pneumonia in foals by administration of hyperimmune plasma methods of implementation of an immunoprophylaxis program for the prevention of rhodococcus equi pneumonia: results of a 5-year field study effect of prophylactic administration of hyperimmune plasma to prevent rhodococcus equi infection on foals from endemically affected farms valuation of equine immunoglobulin specific for rhodococcus equi virulenceassociated proteins a and c for use in protecting foals against rhodococcus equi-induced pneumonia associations between physical examination, laboratory, and radiographic findings and outcome and subsequent racing performance of foals with rhodococcus equi infection: 115 cases (1984-1992) endoscopic and virological observations on respiratory disease in a group of young thoroughbred horses in training epidemiology of ehv-1 and ehv-4 in the mare and foal populations on a hunter valley stud farm: are mares the source of ehv-1 for unweaned foals? clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus-1 on a stud farm foetal and neonatal foal losses on equine herpesvirus type 1(ehv-1) infected farms before and after ehv-1 vaccination was introduced an outbreak of foal perinatal mortality due to equid herpesvirus type 1: pathological observations equine viral arteritis in newborn foals: clinical, pathological, serological, microbiological and immunohistochemical observations involvement of adenovirus in pneumonia in a thoroughbred foal adenoviral infection of arab foals with respiratory tract disease isolation of an adenovirus from an arab foal with a combined immunodeficiency disease clinical, haematological and biochemical findings in foals with neonatal equine herpesvirus-1 infection compared with septic and premature foals neonatal equine herpesvirus type 1 infection on a thoroughbred breeding farm passive transfer, rate of decay, and protein specificity of antibodies against equine arteritis virus in horses from a standardbred herd with high seroprevalence fatal nonneurological ehv-1 infection in a yearling filly pulmonary vasculotropic ehv-1 infection in equids virological and molecular biological investigations into equine herpes virus type 2 (ehv-2) experimental infections equine herpesvirus type 2: prevalence and seroepidemiology in foals subcutaneous emphysema in a neonatal foal immature epithelial na+ channel expression is one of the pathogenetic mechanisms leading to human neonatal respiratory distress syndrome bronchointerstitial pneumonia and respiratory distress in young horses: clinical, clinicopathologic, radiographic, and pathological findings in 23 cases (1984-1989) risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: 143 cases (1986-1996) disease of foals du plessis jl: rupture of the bladder in the newborn foal and its surgical correction metabolic abnormalities associated with rupture of the urinary bladder in neonatal foals exploratory celiotomy for suspected urinary tract disruption in neonatal foals: a review of 18 cases uroperitoneum in the foal uroperitoneum in the hospitalized equine neonate: retrospective study of 31 cases repair of a defect in the bladder of a foal rupture of the urinary bladder in neonatal foals bladder defects in newborn foals ultrasound of the urinary tract nonsurgical management of ruptured urinary bladder in a critically ill foal hemodialysis for treatment of oxytetracycline-induced acute renal failure in a neonatal foal bilateral renal hypoplasia in four young horses bilateral renal dysplasia and hypoplasia in a foal with an imperforate anus clinical vignette: renal arteriovenous malformation in a quarter horse foal percutaneous ultrasound-guided pyelography aided diagnosis of ectopic ureter and hydronephrosis in a 3-week-old filly bilateral ureteral tears in a foal multiple ureteral defects in a belgian foal congenital defects in newborn foals of mares treated for equine protozoal myeloencephalitis during pregnancy ultrasonography of umbilical structures in clinically normal foals clinical, ultrasonographic, and surgical findings in foals with umbilical remnant infections closure of the abdominal wall at the umbilicus and the development of umbilical hernias in a group of foals from birth to 11 months of age complications of umbilical hernias in horses: 13 cases persistent vitelline vein in a foal atresia coli in the foal: a review of six cases endothelin receptor b polymorphism associated with lethal white foal syndrome in horses a dinucleotide mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome (lwfs): a horse variant of hirschsprung disease a missense mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome: an equine version of hirschsprung disease incidence of the endothelin receptor b mutation that causes lethal white foal syndrome in white-patterned horses necrotizing enterocolitis presenting in the emergency department: case report and review of differential considerations for vomiting in the neonate new concepts in necrotizing enterocolitis hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals endoscopic appearance of gastric lesions in foals: 94 cases endoscopic evaluation of changes in gastric lesions of thoroughbred foals gastroduodenal ulceration in foals prevalence of gastric lesions in foals without signs of gastric disease: an endoscopic survey gastric ulcers in foals exsanguination due to gastric ulceration in a foal gastrointestinal diseases of foals pathophysiology of peptic disorders in foals and horses: a review clinical syndromes of gastric ulceration in foals and mature horses gastroduodenal ulceration in foals stress ulcer: is routine prophylaxis necessary? peptic ulcer pathophysiology intragastric ph in critically ill neonatal foals and the effect of ranitidine a review of medical treatment for peptic ulcer disease effect of sucralfate on healing of subclinical gastric ulcers in foals effect of ranitidine on intragastric ph in clinically normal neonatal foals effects of omeprazole paste on healing of spontaneous gastric ulcers in horses and foals: a field trial incidence of pneumonia in mechanically ventilated patients treated with sucralfate or cimetidine as prophylaxis for stress bleeding: bacterial colonization of the stomach the protective role of gastric acidity in neonatal bacterial translocation upper gastrointestinal tract bleeding in critically ill pediatric patients gastric colonization as a consequence of stress ulcer prophylaxis: a prospective, randomized trial is prophylaxis for gastric ulcers necessary in critically ill equine neonates? a retrospective study of necropsy cases 1995-1999 stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration influence of stress ulcer prophylaxis on translocation of bacteria from the intestinal tract in rats effects of sucralfate on acute gastric mucosal injury and gastric ulcer induced by ischemiareperfusion in rats stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration efficacy of ivermectin in controlling strongyloides westeri infections in foals foal diarrhoea between 1991 and 1994 in the united kingdom associated with prevalence of and risk factors for fecal shedding of cryptosporidium parvum oocysts in horses overwhelming strongyloidosis in a foal verminous arteritis in a 3-month-old thoroughbred foal randomised trial of fluid restriction in ventilated very low birthweight infants restricted versus liberal water intake for preventing morbidity and mortality in preterm infants hyponatraemia in children with acute cns disease: siadh or cerebral salt wasting? comparison of norepinephrinedobutamine to dopamine alone for splanchnic perfusion in sheep with septic shock depletion of neurohypophyseal content of vasopressin in septic shock hemodynamic and metabolic effects of low-dose vasopressin infusions in vasodilatory septic shock references 1. firth ec, poulos pw: blood vessels in the developing growth plate of the equine distal radius and metacarpus microangiographic studies of metaphyseal vessels in young foals physeal form of the longbones of the foal comparison of splinting and casting on the degree of laxity induced in thoracic limbs in young horses histological features of the dorsal cortex of the third metacarpal bone mid-diaphysis during postnatal growth in thoroughbred horses effect of oxytetracycline on metacarpophalangeal and distal interphalageal joint angles in newborn foals the blood supply of the growth plate and the epiphysis: a comparative scanning electron microscopy and histological experimental study in growing sheep fractures of the proximal sesamoid bones in thoroughbred foals avulsion of the origin of the peroneus tertius tendon in a foal avulsion fracture of the origin of the extensor digitorum longus muscle in a foal surgical reconstruction of a ruptured medial collateral ligament in a foal correction of patellar luxation by recession sulcoplasty in three foals lateral patellar luxation in miniature horse foals anatomy and therapeutic resection of the peroneus tertius in a foal preliminary observations on oxytetracycline treatment of congenital flexural deformities in foals effect of oxytetracycline on metacarpophalangeal and distal interphalangal join angles in newborn foals torsion in quadrapeds and its impact on mammalian joints computer model of endochondral growth and ossification in long bones: biological and mechanobiological influences observations on the evaluation and selection of foal limb deformities for surgical treatment treatment response and athletic outcome of foals with tarsal valgus deformities: 39 cases incomplete ossification of the tarsal bones in foals: 22 cases periosteal transection and periosteal stripping for correction of angular limb deformities in foals periosteal transection and stripping for treatment of angular limb deformities in foals: clinical observations restricted exercise and transphyseal bridging for correction of angular limb deformities effect of hemicircumferential periosteal transection and elevation in foals with experimentally induced angular limb deformities diaphyseal angular limb deformities in three foals current concepts of infectious polyarthritis in foals factors associated with prognosis for survival and athletic use in foals with septic arthritis: 93 cases (1987-1994) the site of focal osteomyelitis lesions in foals pathological features of multiple bone infection in the foal stromal cell culture and relationships in perimedullary spaces of chick embryo shaft bones tarsal osteomyelitis in foals cartilage canals in equine articular/epiphyseal growth cartilage and a possible association with dyschondroplasia a histological study of acute hematogenous osteomyelitis following physeal injuries in rabbits open drainage, intra-articular and systemic antibiotics in the treatment of septic arthritis/tenosynovitis in horses comparison of various treatments for experimentally induced equine infectious arthritis a retrospective study of 192 horses affected with septic arthritis/tenosynovitis regional intravenous perfusion of the distal limbs of horses with amikacin sulfate treatment of sepsis in the small tarsal joints of 11 horses with gentamicin-impregnated polymethylmethacrylate beads use of antibioticimpregnated polymethyl-methacrylate in horses with open or infected fractures or joints: 19 cases (1987-1995) the effect of implanting gentamicin-impregnated polymethylmethacrylate beads in the tarsocrural joint of the horse regional perfusion of the equine carpus for antibiotic delivery how to perform equine digital intravascular perfusion surgical management of rhodococcus equi metaphysitis in a foal intraosseous regional perfusion for treatment of septic physitis in a 2-week-old foal key: cord-022561-rv5j1201 authors: boes, katie m.; durham, amy c. title: bone marrow, blood cells, and the lymphoid/lymphatic system date: 2017-02-17 journal: pathologic basis of veterinary disease doi: 10.1016/b978-0-323-35775-3.00013-8 sha: doc_id: 22561 cord_uid: rv5j1201 nan within the marrow spaces, a network of stromal cells and extracellular matrix provides metabolic and structural support to hematopoietic cells. these stromal cells consist of adipocytes and specialized fibroblasts, called reticular cells. the latter provides structural support by producing a fine network of a type of collagen, called reticulin, and by extending long cytoplasmic processes around other cells and structures. both reticulin and cytoplasmic processes are not normally visible with light microscopy but are visible with silver reticulin stains (e.g., gordon and sweet's and sometimes with periodic acid-schiff). bone marrow is highly vascularized but does not have lymphatic drainage. marrow of long bones receives part of its blood supply from the nutrient artery, which enters the bone via the nutrient canal at midshaft. the remaining arterial supply enters the marrow through an anastomosing array of vessels that arise from the periosteal arteries and penetrate the cortical bone. vessels from the nutrient and periosteal arteries converge and form an interweaving network of venous sinusoids that permeates the marrow. these sinusoids not only deliver nutrients and remove cellular waste but also act as the entry point for hematopoietic cells into blood circulation. sinusoidal endothelial cells function as a barrier and regulate traffic of chemicals and particles between the intravascular and extravascular spaces. venous drainage parallels that of the nutrient artery and its extensions. • bleeding time (template bleeding time, buccal mucosal bleeding time). this assay assesses primary hemostasis (platelet plug formation) by measuring the time interval between inflicting of standardized wound and cessation of bleeding. sedation may be required. in small animals the test is usually performed on the buccal mucosa; in large animals it may be performed on the distal limb. prolonged bleeding time may be because of a platelet function defect, von willebrand disease, or a vascular defect. the sensitivity of this test is low; reference intervals are species and site dependent (can perform test on a normal animal as a control). this test is contraindicated in cases of thrombocytopenia because significant thrombocytopenia can cause a prolonged bleeding time (invalidates interpretation of test results). • clot retraction test. this assay assesses retraction of a clot, in which platelets play an essential role. this is a crude test that is rarely performed. different protocols are described. significant thrombocytopenia invalidates interpretation of test results. • tests to characterize platelet function abnormalities more specifically are available through specialized laboratories. • aggregometry-to assess platelet aggregation in response to different physiologic agonists. • adhesion assays-to assess the ability of platelets to adhere to a substrate (e.g., collagen). • flow cytometry-to assay for expression of surface molecules. • pfa-100-an instrument that simulates a damaged blood vessel, by measuring time for a platelet plug to occlude an aperture; to date, this instrument has mainly been used in research applications. • thromboelastography (teg)-global assessment of hemostasis (platelets, coagulation, and fibrinolysis) based on viscoelastic analysis of whole blood. • tests for immune-mediated thrombocytopenia (imt). • flow cytometry-to detect immunoglobulin bound to the platelet surface, using a fluorescent-labeled antibody. • bone marrow immunofluorescent antibody (ifa) test-to detect bound immunoglobulin. sometimes referred to as the "antimegakaryocyte antibody test," this assay actually detects the presence of immunoglobulin nonspecifically: a smear of a bone marrow aspirate is incubated with a fluorescent-labeled antibody to species-specific immunoglobulin. other components of the marrow include myelinated and nonmyelinated nerves, as well as low numbers of resident macrophages, lymphocytes, and plasma cells. of note, the macrophages play an important role in iron storage and erythrocyte maturation. the following basic concepts provide a framework for understanding the mechanisms of injury and diseases presented later in the chapter. • hematopoietic tissue is highly proliferative. billions of cells per kilogram of body weight are produced each day. • pluripotent hematopoietic stem cells are a self-renewing population, giving rise to cells with committed differentiation programs, and are common ancestors of all blood cells. the process of hematopoietic differentiation is shown in fig. 13 -2. • hematopoietic cells undergo sequential divisions as they develop, so there are progressively higher numbers of cells as they mature. cells also continue to mature after they have stopped dividing. conceptually, it is helpful to consider cells in the bone marrow as belonging to mitotic and postmitotic compartments. examples of developing hematopoietic cells are shown in fig. 13 -3. • mature cells released into the blood circulation have different normal life spans, varying from hours (neutrophils), to days (platelets), to months (erythrocytes), and to years (some lymphocytes). • the hematopoietic system is under exquisite local and systemic control and responds rapidly and predictably to various stimuli. • production and turnover of blood cells are balanced so that numbers are maintained within normal ranges (steady-state kinetics) in healthy individuals. • normally the bone marrow releases mostly mature cell types (and very low numbers of cells that are almost fully mature) into the circulation. in response to certain physiologic or pathologic stimuli, however, the bone marrow releases immature cells that are further back in the supply "pipeline." the composition of the marrow changes with age. the general pattern is that hematopoietic tissue (red marrow) regresses and is replaced with nonhematopoietic tissue, mainly fat (yellow marrow). thus in newborns and very young animals the bone marrow consists largely of hematopoietically active tissue, with relatively little fat, whereas in geriatric individuals the marrow consists largely of fat. in adults, hematopoiesis occurs primarily in the pelvis, sternum, ribs, vertebrae, and the proximal ends of humeri and femora. even within these areas of active hematopoiesis, fat may constitute a significant proportion of the marrow volume. immature hematopoietic cells can be divided into three stages: stem cells, progenitor cells, and precursor cells. hematopoietic stem cells (hscs) have the capacity to self-renew, differentiate into mature cells, and repopulate the bone marrow after it is obliterated. progenitor cells and precursor cells cannot self-renew; with each cell division, they evolve into more differentiated cells. later-stage precursors cannot divide. stem cells and progenitor cells require immunochemical stains for identification, but precursor cells can be identified by their characteristic morphologic features (see fig. 13 -3). control of hematopoiesis is complex, with many redundancies, feedback mechanisms, and pathways that overlap with other physiologic and pathologic processes. many cytokines influence cells of different lineages and stages of differentiation. primary growth factors for primitive cells are interleukin (il) 3, produced by t lymphocytes, and stem cell factor, produced by monocytes, macrophages, fibroblasts, endothelial cells, and lymphocytes. interleukin 7 is an early lymphoid growth factor. lineage-specific growth factors are discussed in their corresponding sections. hematopoiesis occurs in the interstitium between the venous sinusoids in the so-called hematopoietic spaces. there is a complex functional interplay among hematopoietic cells with the supporting connective tissue cells, extracellular matrix, and soluble factors, which form the hematopoietic microenvironment. behavior of hematopoietic cells is influenced by direct cell-to-cell and cellmatrix interactions and by soluble mediators, such as cytokines and hormones that interact with cells and with matrix proteins. cells localize to specific niches within the hematopoietic microenvironment via adhesion molecules, such as integrins, immunoglobulins, lectins, and other receptors, which recognize ligands on other cells or matrix components. cells also express receptors for soluble molecules such as chemokines (chemoattractant cytokines) and hormones that influence cell trafficking and metabolism. iron is essential to hemoglobin synthesis and function. it is acquired through the diet and is transported to the bone marrow via the iron transport protein, transferrin. central macrophages either store iron as ferritin or hemosiderin, or transfer the iron to erythroid precursors for hemoglobin synthesis. hemosiderin is identifiable in routinely stained marrow preparations as an intracellular brown pigment. however, perls's prussian blue stain is more sensitive and specific for iron detection. the earliest erythroid precursor identifiable by routine light microscopy is the rubriblast, which undergoes maturational division to produce 8 to 32 progeny cells. late-stage erythroid precursors, known as metarubricytes, extrude their nuclei and become inhibiting apoptosis of developing erythroid cells. the stimulus for increased epo production is hypoxia. within the bone marrow, erythroid precursors surround a central macrophage in specialized niches, termed erythroblastic islands . the central macrophage, also known as a nurse cell, anchors the precursors within the island niche, regulates erythroid proliferation and differentiation, transfers iron to the erythroid progenitors for hemoglobin synthesis, and phagocytizes extruded metarubricyte nuclei. although erythroblastic islands occur throughout the marrow, those with more differentiated erythroid cells neighbor sinusoids, whereas nonadjacent islands contain mostly undifferentiated precursors. as erythroid cells mature from a rubriblast to a mature erythrocyte, their nuclei become smaller and more condensed. the nucleus is eventually extruded to form a polychromatophil. erythroid cells also become less basophilic and more eosinophilic as more hemoglobin is produced and as rna-rich organelles are lost during maturation. (hemoglobin stains eosinophilic, and rna stains basophilic with routine romanowsky's stains.) as granulocytes (e.g., neutrophils, eosinophils, and basophils) mature from a myeloblast to their mature forms, their nuclei become dense and segmented. granulocytes acquire their secondary or specific granules during the myelocyte stage and can be morphologically differentiated starting at this stage. neutrophils have neutral-staining secondary granules, eosinophil secondary granules have an affinity for acidic or eosin dyes, and basophil secondary granules have an affinity for basic dyes. monoblasts differentiate into promonocytes with ruffled nuclear boarders and then into monocytes. in most mammals, mature erythrocytes have a biconcave disk shape, called a discocyte. microscopically, these cells are round and eosinophilic with a central area of pallor. however, the central concavity may not be microscopically apparent in species other than the dog. camelids normally have oval erythrocytes, termed ovalocytes or elliptocytes, which facilitate better gas exchange at high altitudes. the erythrocytes of some animals are prone to in vitro shape change, including those of cervids, pigs, and some goat breeds (e.g., angora). erythrocyte size during health depends on the species, breed, and age of the animal. in dogs, some breeds have relatively smaller (e.g., akitas and shibas) or larger (e.g., some poodles) erythrocytes. akitas and shibas also have a high concentration of potassium, unlike erythrocytes in other dogs. juvenile animals may have larger erythrocytes because of the persistence of fetal erythrocytes, which is followed by a period of relatively smaller cells before reaching adult reference intervals. mature mammalian erythrocytes lack nuclei and organelles and are thus incapable of transcription, translation, and oxidative metabolism. however, they do require energy for various functions, including maintenance of shape and deformability, active transport, and prevention of oxidative damage. red blood cells generate this energy entirely through glycolysis (also known as the embden-meyerhof pathway). except in pigs, glucose enters erythrocytes from the plasma through an insulin-independent, integral membrane glucose transporter. within circulation the erythrocyte mean life span varies between species and is related to body weight and metabolic rate: approximately 150 days in horses and cattle, 100 days in dogs, and 70 days in cats. when erythrocytes reach the end of their life span, they are destroyed in a process termed hemolysis. hemolysis may occur within blood vessels (intravascular hemolysis) or by sinusoidal macrophages (extravascular hemolysis). during intravascular hemolysis, erythrocytes release their contents, mostly hemoglobin, directly into blood. however, during extravascular hemolysis, macrophages phagocytize entire erythrocytes, leaving little or no hemoglobin in the blood. normal turnover of erythrocytes occurs mainly by extravascular hemolysis within the spleen, and to a lesser extent in other organs such as the liver and bone marrow. the exact controls are not clear, but factors that likely play a role in physiologic hemolysis include the following: • exposure of membrane components normally sequestered on the inner leaflet of the erythrocyte membrane, particularly phosphatidylserine. reticulocytes, and subsequently mature erythrocytes. the normal transit time from rubriblast to mature erythrocyte is approximately 1 week. reticulocytes start maturing in the bone marrow but finish their maturation in the blood circulation and spleen. horses are an exception in that they do not release reticulocytes into circulation, even in situations of increased demand. unlike mature erythrocytes, which lack organelles, reticulocytes still contain ribosomes and mitochondria, mainly to support completion of hemoglobin synthesis. these remaining organelles impart a bluish-purple cast (polychromasia) to reticulocytes on routine blood smear examination. the resultant cells are termed polychromatophils. because older reticulocytes do not exhibit polychromasia, more sensitive laboratory techniques must be used for accurate reticulocyte quantification. when a blood sample is incubated with new methylene blue stain, the reticulocytes' ribosomal rna precipitates to form irregular, dark aggregates . cats also have a more mature form of reticulocyte, termed punctate reticulocyte, which is stippled when stained with new methylene blue. punctate reticulocytes indicate prior, not active, regeneration and do not appear polychromatophilic on routine blood smear evaluation. storage pool, which consists of a reserve of fully mature neutrophils. the size of the storage pool varies by species; it is large in the dog, but small in ruminants. in homeostasis mostly mature segmented granulocytes are released from the marrow into the blood. the first monocytic precursor identifiable by morphologic features is the monoblast, which develops into promonocytes and subsequently monocytes (see fig. 13 -3). unlike granulocytes, monocytes do not have a marrow storage pool; they immediately enter venous sinusoids upon maturation. after migrating into the tissues, monocytes undergo morphologic and immunophenotypic maturation into macrophages. within blood vessels there are two pools of leukocytes: the circulating pool and the marginating pool. circulating cells are free flowing in blood, whereas marginating cells are temporarily adhered to endothelial cells by selectins. in most healthy mammals there are typically equal numbers of neutrophils in the circulating and marginal pools. however, there are threefold more marginal neutrophils relative to circulating neutrophils in cats. only the circulating leukocyte pool is sampled during phlebotomy. the concentration of myeloid cells in blood depends on the rate of production and release from the bone marrow, the proportions of cells in the circulating and marginating pools, and the rate of migration from the vasculature into tissues. the fate of neutrophils after they leave the bloodstream in normal conditions (i.e., not in the context of inflammation) is poorly understood. they migrate into the gastrointestinal and respiratory tracts, liver, and spleen and may be lost through mucosal surfaces or undergo apoptosis and be phagocytized by macrophages. lymphopoiesis. lymphopoiesis-from lympha (latin, water)refers to the production of new lymphocytes, including b lymphocytes, t lymphocytes, and natural killer (nk) cells. b lymphocytes primarily produce immunoglobulins, also known as antibodies, and are key effectors of humoral immunity. they are distinguished by the presence of an immunoglobulin receptor complex, termed the b lymphocyte receptor. plasma cells are terminally differentiated b lymphocytes that produce abundant immunoglobulin. t lymphocytes, effectors of cell-mediated immunity, possess t lymphocyte receptors that bind antigens prepared by antigen-presenting cells. a component of innate immunity, nk cells kill a variety of infected and tumor cells in the absence of prior exposure or priming. main growth factors for b lymphocytes, t lymphocytes, and nk cells are il-4, il-2, and il-15, respectively. lymphocytes are derived from hscs within the bone marrow. b lymphocyte development occurs in two phases, first in an antigenindependent phase in the bone marrow and ileal peyer's patches (the site of b lymphocyte development in ruminants), then in an antigendependent phase in peripheral lymphoid tissues (such as spleen, lymph nodes, and mucosa-associated lymphoid tissue [malt] ). t lymphocyte progenitors migrate from the bone marrow to the thymus, where they undergo differentiation, selection, and maturation processes before migrating to the peripheral lymphoid tissue as effector cells. unlike granulocytes, which circulate only in blood vessels and migrate unidirectionally into target tissues, lymphocytes travel in both blood and lymphatic vessels and continually circulate between blood, tissues, and lymphatic vessels. also in contrast to nonlymphoid hematopoietic cells, blood lymphocyte concentrations in adult animals are primarily dependent upon extramedullary lymphocyte production and kinetics, and not lymphopoiesis by the marrow. in healthy nonruminant mammals, lymphocytes are the second most numerous blood leukocyte. according to conventional wisdom, • decreased erythrocyte deformability. • binding of immunoglobulin g (igg) and/or complement to erythrocyte membranes. complement binding may be secondary clustering of the membrane anion exchange protein, band 3. • oxidative damage to erythrocytes. macrophages degrade erythrocytes into reusable components, such as iron and amino acids, and the waste product bilirubin. bilirubin is then exported into circulation, where it is transported to the liver by albumin. the liver conjugates and subsequently excretes bilirubin into bile for elimination from the body. intravascular hemolysis normally occurs at only extremely low levels. hemoglobin is a tetramer that, when released from the erythrocyte into the blood, splits into dimers that bind to a plasma protein called haptoglobin. the hemoglobin-haptoglobin complex is taken up by hepatocytes and macrophages. this is the major pathway for handling free hemoglobin. however, free hemoglobin may also oxidize to form methemoglobin, which dissociates to form metheme and globin. metheme binds to a plasma protein called hemopexin, which is taken up by hepatocytes and macrophages in a similar manner to hemoglobin-haptoglobin complexes. free heme in the reduced form binds to albumin, from which it is taken up in the liver and converted into bilirubin. the concentration of circulating erythrocytes typically decreases postnatally and remains below normal adult levels during the period of rapid body growth. the age at which erythrocyte numbers begin to increase and the age at which adult levels are reached vary among species. in dogs, adult values are usually reached between 4 and 6 months of age; in horses, this occurs at approximately 1 year of age. granulopoiesis is the production of neutrophils, eosinophils, and basophils, whereas monocyte production is termed monocytopoiesis. granulocytic and monocytic cells are sometimes collectively referenced as myeloid cells. however, the term myeloid and the prefix myelo-can be confusing because they have other meanings; they may reference the bone marrow, all nonlymphoid hemic cells (erythrocytes, leukocytes, and megakaryocytes), only granulocytes, or the spinal cord. the main purpose of granulocytes and monocytes is to migrate to sites of tissue inflammation and function in host defense (see chapters 3 and 5 ). briefly, these cells have key immunologic functions, including phagocytosis and microbicidal activity (neutrophils and monocyte-derived macrophages), parasiticidal activity and participation in allergic reactions (eosinophils and basophils), antigen processing and presentation, and cytokine production (macrophages). neutrophils are the predominant leukocyte type in blood of most domestic species. primary stimulators of granulopoiesis and monocytopoiesis are granulocyte-macrophage colony-stimulating factor and il-1, il-3, and il-6 (granulocytes and monocytes), granulocyte colonystimulating factor (granulocytes), and macrophage colonystimulating factor (monocytes). in general, these cytokines are produced by various inflammatory cells, with or without contribution from stromal cells. the earliest granulocytic precursor identifiable by routine light microscopy is the myeloblast, which undergoes maturational division over 5 days to produce 16 to 32 progeny cells (see fig. 13 -3). these granulocytic precursors are conceptually divided into those stages that can divide, including myeloblasts, promyelocytes, and myelocytes (proliferation pool), and those that cannot, including metamyelocytes, and band and segmented forms (maturation pool). within the neutrophil maturation pool is a subpool, termed the platelet aggregation and adherence to subendothelial collagen. expansion of surface area and release of granule contents is aided by a network of membrane invaginations known as the open canalicular system. this system is not present in horses, cattle, and camelids. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. mechanisms of bone marrow disease are summarized in box 13-1. hematopoietic cells' response to injury is dependent upon whether the insult is on the marrow or within extramarrow tissues. in general, marrow-directed injury or disturbances result in production of abnormal hematopoietic cells (dysplasia), fewer hematopoietic cells (hypoplasia), or a failure of hematopoietic cell development (aplasia). dysplasia, hypoplasia, and aplasia may be specific for one cell line, such as pure red cell aplasia, or affect multiple lineages, as seen with aplastic anemia. accordingly, decreased blood concentrations of the involved cell types are expected with hypoplasia or aplasia. erythroid, myeloid, and megakaryocytic hypoplasia or aplasia causes nonregenerative anemia, neutropenia, and thrombocytopenia, respectively. bicytopenia is used to describe decreased blood concentrations of two cell lines, whereas pancytopenia indicates decreased blood concentrations of all three cell types. bicytopenia or pancytopenia may indicate generalized marrow disease, such as occurs with aplastic anemia or marrow malignancies (leukemia), necrosis, fibrosis (myelofibrosis), or inflammation (myelitis). replacement of hematopoietic tissue within the bone marrow by abnormal tissue, including neoplastic cells, fibrosis, or inflammatory cells, is termed myelophthisis. cattle normally have higher numbers of lymphocytes than neutrophils in circulation. however, recent studies suggest that is no longer the case, most likely due to changes in genetics and husbandry. in most species the majority of lymphocytes in blood circulation are t lymphocytes. the concentration of blood lymphocytes decreases with age. thrombopoiesis. thrombopoiesis-from thrombos (gr., clot)refers to the production of platelets, which are small (2 to 4 µm), round to ovoid, anucleate cells within blood vessels. platelets have a central role in primary hemostasis but also participate in secondary hemostasis (coagulation) and inflammatory pathways (see chapters 2 and 3). thrombopoietin (tpo) is the primary regulator of thrombopoiesis. the liver and renal tubular epithelial cells constantly produce tpo, which is then cleared and destroyed by platelets and their precursors. therefore plasma tpo concentration is inversely proportional to platelet and platelet precursor mass. if the platelet mass is decreased, less tpo is cleared, and there is subsequently more free plasma tpo to stimulate thrombopoiesis. the earliest morphologically identifiable platelet precursor is the megakaryoblast, which undergoes nuclear reduplications without cell division, termed endomitosis, to form a megakaryocyte with 8 to 64 nuclei. as the name suggests, megakaryocytes are very large cells, much larger than any other hematopoietic cell ; also see fig. 13 -1). megakaryocytes neighbor venous sinusoids, extend their cytoplasmic processes into vascular lumens, and shed membranebound cytoplasmic fragments (platelets) into blood circulation. orderly platelet shedding is partially facilitated by β 1 -tubulin microtubules within megakaryocytes. platelets circulate in a quiescent form and become activated by binding platelet agonists, including thrombin, adenosine diphosphate (adp), and thromboxane. platelet activation causes shape change, granule release, and relocation of procoagulant phospholipids and glycoproteins (gps) to the outer cell membrane. specific procoagulant actions include release of calcium, von willebrand factor (vwf), factor v, and fibrinogen, as well as providing phosphatidylserine-rich binding sites for the extrinsic tenase (factors iii, vii, and x), intrinsic tenase (factors ix, viii, and x), and prothrombinase (factors x, v, and ii) coagulation complexes. platelet gp surface receptors include those for binding vwf (gpib-ix-v), collagen (gpvi), and fibrinogen (gpiib-iiia), which facilitate increased destruction hemorrhage (especially erythrocytes) consumption (platelets) neoplasia altered distribution abnormal function bone marrow is not routinely sampled during postmortem examinations. however, indications for bone marrow evaluation include suspected leukemia, metastatic neoplasia within bone marrow, or infectious myelitis, as well as cytopenia(s) or hematopoietic dysplasia of unknown cause. multimodal evaluation is ideal, including a recent (<24 hours) complete blood count with bone marrow cytologic and histopathologic examination. however, antemortem blood analyses are not always available, and interpretation of hematopoietic cytomorphologic examination results becomes difficult to impossible shortly after death. postmortem bone marrow should be collected as soon as possible after death or euthanasia, preferably within 30 minutes. samples may be collected from the proximal femur, rib, sternum or vertebrae. when collecting from the femur, the femoral neck is removed with a bone saw, or a fragment of the shaft is removed with bone-cutting shears. cytologic samples are first collected using the paintbrush technique: gently sample the red marrow with a clean, dry, naturalbristle brush, and then carefully brush the material onto a clean glass microscope slide in two to four parallel wavy lines. the brush should be cleaned and dried before its use on a different animal. the slide is then air dried, stored away from formalin fumes, and then stained with a routine (romanowsky) stain. for histologic evaluation the entire femoral head or femoral shaft or rib fragment with exposed red marrow is submersed in 10% neutral buffered formalin. for cosmetic necropsies, samples may be obtained by antemortem techniques, such as needle biopsies for cytologic examination and core biopsies for histopathologic examination. the complete blood count (cbc) is the cornerstone for diagnosis of hematologic disturbances and is often part of a minimum database in sick patients. the cbc includes numeric data indicating the concentration of different cell types, as well as other estimations of red blood cell mass (hemoglobin concentration, packed-cell volume, and hematocrit), red blood cell volume (mean cell volume), and red blood cell hemoglobin content (mean cell hemoglobin and mean cell hemoglobin concentration). cell morphologic features and the presence or absence of hemic parasites are assessed upon microscopic review of a blood smear and are also included in a cbc report. (note: some parasites may infect blood cells, such as hepatozoon organisms within circulating neutrophils or monocytes or bartonella organisms within erythrocytes, but mainly cause disease in other body systems and are therefore not discussed in this chapter.) learning to evaluate blood smears is a valuable skill for any practicing veterinarian. the cbc also may include the plasma protein concentration, as measured with a refractometer. it is important to remember that changes in hydration status and in the distribution of body fluids between the vascular and extravascular compartments affect the concentration of both cells and proteins in the blood. other tests that may help with evaluation of the hematopoietic system include cell or tissue biopsies, the direct antiglobulin test, flow cytometry, immunophenotyping, and polymerase chain reaction (pcr). aspiration cytology and/or histopathology of organs other than the bone marrow can be pursued to assess for the presence of emh, increased destruction of erythrocytes, neoplasia, or infection. the coombs test, or direct antiglobulin test, detects excessive antibody or complement bound to red blood cells' surfaces and is the standard assay for immune-mediated hemolytic anemia. flow cytometry and immunofluorescent antibody tests may also be used to detect autoantibody bound to erythrocytes or other hematopoietic cells. immunophenotyping and pcr are further discussed in the section on hematopoietic neoplasia. structural or functional abnormalities of blood vessels, platelets, or coagulation factors may result in a tendency toward hypocoagulability (bleeding), hypercoagulability (inappropriate thrombosis), or both. in veterinary medicine there has been a great deal of work on specific mechanisms of hypocoagulability, whereas mechanisms of hypercoagulability are less fully characterized. disorders of primary hemostasis typically result in "small bleeds" (e.g., petechiation, mild ecchymosis, bleeding from mucous membranes, bleeding immediately after venipuncture), whereas disorders of secondary hemostasis typically result in "big bleeds" (e.g., hemorrhage into body cavities/ joints, marked ecchymosis, large hematomas, delayed bleeding after venipuncture). this chapter concentrates on primary disorders of hemostasis and also covers disseminated intravascular coagulation, which is the secondary condition. however, it is important to note that coagulation disorders can also result from other underlying disease processes. for example, advanced liver disease can lead to abnormal hemostasis through decreased or defective synthesis of coagulation factors or impaired clearance of fibrinolytic products that inhibit coagulation reactions and platelet function. vascular disorders may also result in a bleeding tendency because of abnormalities of endothelial function or collagen-platelet interactions. specific diseases involving abnormal structure or function of hematopoietic or hemostatic elements are discussed later in this chapter. the cbc provides basic information about platelets, including numeric values for platelet concentration and mean platelet volume (mpv), subjective assessment of platelet morphologic features (size, shape, and granularity), and a rough estimation of platelet numbers based on examination of a blood smear. some laboratories measure reticulated platelets (platelets recently released from the bone marrow), although this test is mostly used in the research setting at present. increased mpv and increased numbers of reticulated platelets tend to indicate increased thrombopoiesis. bone marrow examination is indicated with any unexplained cytopenia, including thrombocytopenia, to evaluate production. tests to evaluate the components of the hemostatic process are described and listed in e-appendix 13-1. secondary myelofibrosis is the enhanced deposition of collagen within the marrow by nonneoplastic fibroblasts and reticular cells. disease pathogenesis is unclear, but there are two leading theories. first, it may represent scar formation after marrow necrosis, as previously presented. and second, high concentrations of growth factors present during times of marrow injury or activation may stimulate fibroblast proliferation. in particular, stimulated megakaryocytes and macrophages produce fibrogenic cytokines, including plateletderived growth factor, transforming growth factor-β, and epidermal growth factor. early in disease there is reticulin deposition without reduction of hematopoietic elements. however, fibrous collagen replaces hematopoietic cells with disease progression. histologic identification of reticulin and collagen fibers can be aided with reticulin silver and masson's trichrome stains, respectively. in animals, secondary myelofibrosis occurs most commonly with leukemias, extramarrow malignancies, and chronic hemolytic anemias, but many cases are idiopathic. experimental whole-body gamma irradiation, dietary strontium-90 exposure, and certain drugs and toxins can also induce myelofibrosis. the responses of marrow adipocytes to systemic and localized disease are under current investigation, especially in relation to energy metabolism, inflammation, and bone trauma. during times of severe energy imbalance, such as cachexia, the marrow may undergo serous atrophy of fat, also known as gelatinous marrow transformation (efig. 13 -1). the pathogenesis of this phenomenon is unknown, but it is characterized by adipocyte atrophy, hematopoietic cell hypoplasia with subsequent cytopenias, and replacement of the marrow with extracellular hyaluronic acid-rich mucopolysaccharides. positive alcian blue staining identifies the extracellular material as mucin. marrow adipocytes secrete adipose-derived hormones, termed adipokines, including leptin and adiponectin. in general, leptin is proinflammatory, prothrombotic, and mitogenic for various cell types, including lymphocytes, hematopoietic progenitors, and leukemic cells. conversely, adiponectin has antiinflammatory and growth inhibitory properties. during times of inflammation and infection, leptin production is increased. in response to marrow trauma, such as orthopedic surgery, fat may enter the vasculature, embolize to various tissues, and cause tissue ischemia. the severity of tissue injury caused by fat embolism is dependent upon the quantity of fat entering circulation and the tissue's susceptibility to ischemia (see chapter 2). responses of circulating blood cells to injury include decreased survival (destruction, consumption, or loss), altered distribution, and altered structure or function (see box 13-1). these responses are not mutually exclusive-for example, altered erythrocyte structure may lead to decreased survival. often, but not always, these responses result in decreased concentrations of blood cells in circulation. abnormal concentrations of blood cells. the concentration of blood cells may be decreased, termed cytopenia (from kytos [gr., hollow vessel] and penia [gr., poverty]) or increased, designated cytosis (from osis [gr., condition]). a specific blood cell type is denoted as being decreased by using the suffix -penia (table 13-1) . a decreased concentration of erythrocytes is the exception and is termed anemia (from a [gr., without] and haima [gr., blood]). decreased concentrations of blood basophils are not recognized in domestic animals because the lower reference interval is typically zero. an increased blood cell type is denoted with the suffix -osis or -philia (see table 13 -1). postmortem quantification of blood cell insults to extramarrow tissues and cells tend to cause increased production of the involved cell types (hyperplasia) with or without dysplasia. loss of erythrocytes from blood vessels (hemorrhage), or premature destruction of erythrocytes (hemolysis) causes erythroid hyperplasia. tissue inflammation may cause neutrophilic, eosinophilic, basophilic, and/or monocytic hyperplasia, depending on the type of inflammation. megakaryocytic hyperplasia may occur with increased platelet use during hemorrhage or disseminated intravascular coagulation (dic) or with immune-mediated platelet destruction. exceptions to these generalizations, such as anemia of chronic disease, iron deficiency anemia, and anemia of renal failure, are discussed in more detail later. endothelial cell response to injury specifically within the marrow is poorly characterized, but it is likely similar to that of endothelial cells elsewhere, playing active roles in coagulation and inflammation (see chapters 2 and 3). however, one potential sign of marrow sinusoidal injury is the presence of circulating nucleated erythrocytes in the absence of erythrocyte regeneration, termed inappropriate metarubricytosis. it is proposed that injured marrow endothelial cells allow premature passage of metarubricytes into blood circulation during times of stress. however, a conflicting theory proposes that marrow stress causes decreased metarubricyte attachment to central macrophages, and subsequent release into circulation. specific causes of marrow injury-induced metarubricytosis include sepsis, hyperthermia, malignancies, hypoxia, and certain drugs and toxins. inappropriate metarubricytosis may also occur with erythroid dysplasia and splenic disorders. in addition to a suspected role in inappropriate metarubricytosis, marrow macrophages are integral to altered iron metabolism, including anemia of chronic disease and hemosiderosis. anemia of chronic disease is a mild to moderate nonregenerative anemia observed in animals with a variety of inflammatory and metabolic disorders. this anemia is discussed in more detail later, but briefly, it is primarily a result of iron sequestration within macrophages. hemosiderosis is the excessive accumulation of iron in tissues, typically macrophages. accumulation of iron in parenchymal organs, leading to organ toxicity, is termed hemochromatosis. in animals, iron overload due to blood transfusions or chronic hemolytic anemias may cause marrow hemosiderosis and hemochromatosis. myelitis can take different forms. granulomatous myelitis occurs with systemic fungal infections (e.g., histoplasmosis) or mycobacteriosis. acute or neutrophilic myelitis may occur with lower-order bacterial infections or those with an immune-mediated component. dogs and cats with nonregenerative immune-mediated hemolytic anemia (imha) often have myelitis, in addition to myelofibrosis and necrosis. the inflammation is evident as fibrin deposition, edema, and multifocal neutrophilic infiltrates; immune-mediated cytopenias may also concurrently occur with bone marrow lymphocytic and/or plasma cell hyperplasia. bone marrow necrosis is the necrosis of medullary hematopoietic cells, stromal cells, and stroma in large areas of bone marrow. potential causes include leukemias, extramarrow malignancies, infection (bovine viral diarrhea virus [bvdv] , ehrlichia canis, and feline leukemia virus [felv]), sepsis, drugs or toxins (carprofen, chemotherapeutic agents, estrogen, metronidazole, mitotane, and phenobarbital), and irradiation. direct hematopoietic or stromal cytotoxicity and altered marrow microvasculature (disseminated intravascular coagulation) are proposed pathogeneses. extensive marrow necrosis results in decreased hematopoiesis and subsequent blood cytopenias, including anemia, neutropenia, and thrombocytopenia. if the animal survives the initial insult, the marrow may recover and resume normal hematopoiesis, or it may undergo scar formation, termed myelofibrosis. concentrations is not possible due to perimortem coagulation. however, a complete blood count (cbc) with microscopic blood smear evaluation is the foundation for antemortem assessment of blood cells. anemia. anemia causes clinical signs referable to decreased red hemoglobin pigment (e.g., pale mucous membranes), decreased oxygen-carrying capacity (e.g., depression, lethargy, weakness, and exercise tolerance), and decreased blood viscosity (e.g., heart murmur). recumbency, seizures, syncope, or coma may occur with severe anemia. anemia is confirmed by identifying a decreased hemoglobin concentration or reduced erythrocyte mass, as measured by the packed-cell volume, hematocrit, or red blood cell concentration. the three general causes of anemia are blood loss (hemorrhage), red blood cell destruction or lysis (hemolysis), and decreased red blood cell production (erythroid hypoplasia). classifying anemia as regenerative or nonregenerative is clinically useful because it provides information about the mechanism of disease; regenerative anemia indicates hemorrhage or hemolysis, whereas erythroid hypoplasia or aplasia causes nonregenerative anemia (table 13 -2). the hallmark of regenerative anemias, except in horses, is reticulocytosis (i.e., increased numbers of circulating reticulocytes [immature erythrocytes]), which is evident as polychromasia on a routinely stained blood smear (see fig. 13 -5). reticulocytosis indicates increased bone marrow erythropoiesis ( fig. 13-7) and release of erythrocytes before they are fully mature. reticulocytosis is an appropriate marrow response to anemia and is often seen with hemorrhage or hemolysis. on a cbc a strong regenerative response may produce an increased mean cell volume (mcv) and decreased mean cell hemoglobin concentration (mchc) because reticulocytes are larger and have a lower hemoglobin concentration than mature erythrocytes. horses are an exception to this classification scheme because they do not release reticulocytes into circulation, even with erythroid hyperplasia. horses with a regenerative response may have an increased mcv and red cell distribution width (an index of variation in cell size). but definitive determination of regeneration in a horse requires demonstration of erythroid hyperplasia via bone marrow examination or an increasing red cell mass over sequential cbcs. in addition to reticulocytosis there may be increased numbers of nucleated red blood cells (nrbcs) in circulation with erythrocyte regeneration, termed appropriate metarubricytosis. when nrbcs are present as part of a regenerative response, they should be in low numbers relative to the numbers of reticulocytes. however, the presence of circulating nrbcs is not in itself definitive evidence of regeneration and may signify dyserythropoiesis (e.g., lead poisoning or bone marrow disease) or splenic dysfunction. these processes should be suspected when nrbcs are increased without reticulocysuch as into the peritoneal cavity, because iron is not lost from the body and can be reused for erythropoiesis. in hemolytic anemia, erythrocytes are destroyed at an increased rate. whether the mechanism is intravascular or extravascular, or a combination, depends on the specific disease process (specific diseases are discussed later in this chapter). some clinical indicators of hemolytic anemia and their pathogeneses are summarized in fig. 13 -9 and are further described in the following discussion. a classic sequela of hemolytic anemias in general is hyperbilirubinemia, which is an increase in the plasma bilirubin concentration. bilirubin is a yellow pigment, which explains why hyperbilirubinemia, if severe enough, causes icterus-the grossly visible yellowing of fluid or tissues ( fig. 13-10) . icterus, also known as jaundice, is usually detectable when the plasma bilirubin concentration exceeds 2 mg/dl. however, it is important to note that hyperbilirubinemia and icterus are not pathognomonic for hemolysis and may also occur with conditions of impaired bile flow (cholestasis), such as hepatopathy or cholangiopathy. in addition to icterus, hemolytic anemia often results in splenomegaly , which is secondary to extravascular hemolysis and macrophagic hyperplasia within the spleen, as well as splenic emh. splenomegaly may also occur in other conditions, as discussed elsewhere in this chapter. intravascular hemolysis is grossly evident as pink-tinged plasma or serum, termed hemolysis or hemoglobinemia. hemolysis is not apparent until the concentration of extracellular hemoglobin is greater than approximately 50 mg/dl. cell-free hemoglobin is scavenged by haptoglobin until haptoglobin becomes saturated with hemoglobin at a concentration of approximately 150 mg/dl. when haptoglobin is saturated, any remaining free hemoglobin has a low enough molecular weight to pass through the renal glomerular filter into the urine. this imparts a pink or red discoloration to the urine, called hemoglobinuria. thus extracellular hemoglobin can cause gross discoloration of the plasma, where it is bound to haptoglobin, before becoming grossly visible in urine. the half-life of haptoglobin is markedly decreased when bound to hemoglobin, so when large amounts of haptoglobin-hemoglobin complex are formed, the concentration of haptoglobin in the blood decreases and hemoglobin can pass through the glomerulus at even lower concentrations. hemoglobinuria is a contributing factor in the renal tubular necrosis (hemoglobinuric nephrosis) that often occurs in cases of acute intravascular hemolysis (see chapter 11). a similar lesion occurs in the kidneys of individuals with marked muscle damage and resulting myoglobinuria (see chapters 11 and 15). hemoglobinuria cannot be distinguished grossly from hematuria (erythrocytes in the urine) or myoglobinuria (myoglobin in the urine), and all three processes cause a positive reaction for "blood protein" on urine test strips. comparing the colors of the plasma and the urine may be informative. in contrast to hemoglobin, myoglobin causes gross discoloration of the urine before the plasma is discolored. this is because myoglobin is a low-molecular-weight monomer, freely filtered by the glomerulus, and does not bind plasma proteins to a significant degree. hematuria can be distinguished from hemoglobinuria on the basis of microscopic examination of urine sediment (i.e., erythrocytes are present in cases of hematuria). in addition to red plasma and urine, hemoglobinemia may also be identified by increased mch or mchc values on a cbc. this is because the hemoglobin concentration is measured by lysing all erythrocytes in the sample and then measuring the total hemoglobin via spectrophotometry. by this method, hemoglobin that originated within or outside of erythrocytes is measured together. however, calculations for mch and mchc, which include results for the hemoglobin and red blood cell concentrations, assume that all of tosis, or their numbers are high relative to the degree of reticulocytosis, termed inappropriate metarubricytosis. in ruminants, reticulocytosis is often accompanied by basophilic stippling (fig. 13-8) . however, like metarubricytosis, basophilic stippling without reticulocytosis is concerning for lead poisoning or other causes of dyserythropoiesis. recall that the stimulus for increased erythropoiesis is increased secretion of epo in response to tissue hypoxia. although the action of epo on erythropoiesis is rapid, evidence of a regenerative response is not immediately apparent in a blood sample. one of the main effects of epo is to expand the pool of early-stage erythroid precursors, and it takes time for these cells to differentiate to the point where they are released into circulation. in a case of acute hemorrhage or hemolysis, for example, it typically takes 3 to 4 days until reticulocytosis is evident on the cbc and several more days until the regenerative response peaks. the term preregenerative anemia is sometimes used to describe anemia with a regenerative response that is impending but not yet apparent on the cbc. confirming a regenerative response in such cases requires either evidence of erythroid hyperplasia in the bone marrow or emergence of a reticulocytosis on subsequent days. hemorrhage results in escape of erythrocytes and other blood components, such as protein, from the vasculature. as a result, a decreased plasma or serum protein concentration, termed hypoproteinemia, may be evident on a cbc or chemistry panel. if the hemorrhage is into the gastrointestinal lumen, some of the protein may be resorbed and converted to urea, resulting in an increased urea nitrogen concentration relative to creatinine in plasma. hemorrhage within the urinary tract may cause red urine with erythrocytes observed in the urine sediment. causes of hemorrhage include trauma, abnormal hemostasis, certain parasitisms, ulceration, and neoplasia. hemorrhage may be acute or chronic, or internal or external. during acute hemorrhage, there are ample iron stores within the body for hemoglobin synthesis and erythrocyte regeneration. however, with chronic external hemorrhage, continued loss of iron may deplete the body's iron stores. as iron stores diminish, so does erythrocyte regeneration, eventually leading to iron deficiency anemia. iron deficiency anemia is either poorly regenerative or nonregenerative and is discussed in more detail later in the chapter. iron deficiency anemia does not occur with chronic internal hemorrhage, spherocytes form when macrophages (mainly in the spleen) phagocytize part of an erythrocyte plasma membrane bound with autoantibody ( fig. 13-12) . the remaining portion of the erythrocyte assumes a spherical shape, thus preserving maximal volume. this change in shape results in decreased deformability of the cells. erythrocytes must be extremely pliable to traverse the splenic red pulp and sinusoidal walls; spherocytes therefore tend to be retained in the spleen in close association with macrophages with risk for further injury and eventual destruction. in the dog, spherocytes appear smaller than normal and have uniform staining ( fig. 13-13 , a), in contrast to normal erythrocytes, which have a region of central pallor imparted by their biconcave shape. this difference in staining between spherocytes and normal erythrocytes is not consistently discernible in many other domestic animals (including horses, the hemoglobin originated within erythrocytes. in the case of hemoglobinemia, the excess extracellular hemoglobin may cause an artifactual increase in the calculated mch and mchc. it is important to remember that similar artifactual increases may also occur with lipemia. once hemolytic anemia has been identified, the specific cause for hemolysis should be investigated based on signalment, clinical history, and microscopic blood smear evaluation. the most common causes of hemolytic anemia in domestic animals are immunemediated, infectious, oxidative, and mechanical fragmentation (i.e., microangiopathic) disorders (table 13-3) . spherocytosis and autoagglutination are hallmarks of immunemediated hemolytic anemia, either primary (also known as idiopathic) or secondary to infectious disease, drugs/toxins, or neoplasms. several initiating processes can cause intravascular hemolysis; formation of the complement membrane attack complex is pictured. with intravascular hemolysis, free hemoglobin is release directly into the plasma, where it is scavenged by haptoglobin and hemopexin. when haptoglobin and hemopexin are saturated, the cell-free hemoglobin causes red discoloration of the plasma (hemolysis) and is excreted in the urine (hemoglobinuria; dark red urine). the liver clears haptoglobinhemoglobin and hemopexin-methemoglobin complexes from plasma and converts hemoglobin to unconjugated bilirubin and then conjugated bilirubin. conjugated bilirubin is normally excreted in the bile and then converted to urobilinogen (yellow) and subsequently stercobilinogen (dark brown). however, excessive bilirubin will spill over into the plasma, resulting in hyperbilirubinemia, icteric plasma (if severe enough), and urinary excretion of bilirubin (bilirubinuria; icteric urine). extravascular hemolysis: during extravascular hemolysis, erythrocytes are phagocytized by macrophages, which digest erythrocytes, and convert hemoglobin to unconjugated bilirubin. excessive bilirubin in plasma causes hyperbilirubinemia with or without icteric plasma. unconjugated bilirubin is processed and excreted by the liver (as previously described) and in dogs, the kidney. kidney u-bilirubin cattle, and cats), whose erythrocytes differ from those of the dog in that they are smaller and have less pronounced biconcavity and therefore less pronounced central pallor. autoagglutination occurs because of cross-linking of antibodies bound to erythrocytes (see . autoagglutination is evident macroscopically as blood with a grainy consistency (see fig. 13-13, b) , and microscopically as clusters of erythrocytes (see fig. 13 -13, c). autoagglutination may also result in a falsely increased mcv and decreased red blood cell concentration when clustered cells are mistakenly counted as single cells by automated hematology analyzers. when autoagglutination is present, the packed-cell volume is the most reliable measurement of red blood cell mass. ghost cells are ruptured red blood cell membranes devoid of cytoplasmic contents (see a) . they indicate intravascular hemolysis and may be seen with a variety of hemolytic disorders, including those with immune-mediated, infectious, oxidative, or fragmentation causes. in the case of immune-mediated hemolytic anemia, antibody or complement binds to red blood cell membranes and activates the complement membrane attack complex (see fig. 13 -12). this causes pore formation in the red blood cell membrane and release of cytoplasmic contents into the plasma. ghost cells are eventually cleared from circulation by phagocytic macrophages, mainly within the spleen. oxidative damage to erythrocytes occurs when normal antioxidative pathways that generate reducing agents (such as reduced nicotinamide adenine dinucleotide [nadh] , reduced nicotinamide adenine dinucleotide phosphate [nadph] , and reduced glutathione [gsh]) are compromised or overwhelmed, resulting in hemolytic anemia, abnormal hemoglobin function, or both. hemolysis caused by oxidative damage may be extravascular or intravascular, or a combination. evidence of oxidative damage to erythrocytes may be apparent on blood smear examination as heinz bodies or eccentrocytes or on gross examination as methemoglobinemia. heinz bodies are foci of denatured globin that interact with the erythrocyte membrane. they are usually subtly evident on routine wright-stained blood smears as pale circular inclusions or blunt, rounded protrusions of the cell margin but are readily discernible on smears stained with new methylene blue . cats are particularly susceptible to heinz body formation and may have low numbers of heinz bodies normally. there is no unanimity of opinion, but some clinical pathologists believe that the presence of heinz bodies in up to 10% of all erythrocytes in cats is within normal limits. this predisposition is believed to reflect unique features of the feline erythrocyte, whose hemoglobin has more sulfhydryl groups (preferential sites for oxidative damage) than do erythrocytes of other species and may also have lower intrinsic reducing capacity. it is also possible that the feline spleen does not have as efficient a "pitting" function (splenic structure and function are discussed in more detail later in this chapter). eccentrocytes, evident as erythrocytes in which one side of the cell has increased pallor ( fig. 13-15, a) , are another manifestation of oxidative damage. they form because of cross-linking of total hemoglobin), methemoglobin imparts a grossly discernible chocolate color to the blood. by itself, mechanical fragmentation hemolysis tends to cause mild or no anemia. mechanical fragmentation results from trauma or shearing of erythrocytes within blood vessels. normal erythrocytes may be flowing through abnormal vasculature, such as with heart valve defects, intravascular fibrin deposition (e.g., disseminated intravascular coagulation), vasculitis, or hemangiosarcoma. alternatively, the red blood cells may be particularly fragile within normal blood vasculature, as occurs with iron deficiency. in either instance, microscopic evidence of mechanical fragmentation includes the presence of erythrocyte fragments (schistocytes [see fig. 13 -15, c]), erythrocytes with irregular cytoplasmic projections (acanthocytes), erythrocytes with blister-like projections (keratocytes), or ghost cells (see figs. 13-13, a, 13-15, d, and 13-15, e). membrane proteins, with adhesion of opposing areas of the cell's inner membrane leaflet, and displacement of most of the hemoglobin toward the other side. the fused membranes may fragment off of the eccentrocyte, leaving a slightly ruffled border; this cellular morphologic abnormality is called a pyknocyte (see fig. 13-15, b) . oxidative insult may also result in conversion of hemoglobin (iron in the fe 2+ state) to methemoglobin (iron in the fe 3+ state), which is incapable of binding oxygen. methemoglobin is produced normally in small amounts but reduced back to oxyhemoglobin by the enzyme cytochrome-b 5 reductase (also known as methemoglobin reductase). methemoglobinemia results when methemoglobin is produced in excessive amounts (because of oxidative insult) or when the normal pathways for maintaining hemoglobin in the fe 2+ state are impaired (as in cytochrome-b 5 reductase deficiency). when present in sufficiently high concentration (approximately 10% of degradation. antierythrocyte antibodies bind rbc surface antigens, resulting in rbc opsonization by immunoglobulins (mainly immunoglobulin g [igg] ) and complement (primarily c3b). immunoglobulin-or c3b-bound rbcs are phagocytized and digested by sinusoidal macrophages. 2, spherocytes. spherocytes form when the membrane of immunoglobulin-or c3b-bound rbcs are phagocytized by macrophages, without removing the entire rbc from circulation. compared to normal erythrocytes, spherocytes appear smaller, more eosinophilic, and lack central pallor. 3, rbc aggregation (agglutination). rbc aggregation occurs when antierythrocyte immunoglobulins (immunoglobulin m [igm] or high concentrations of igg) bind multiple erythrocytes simultaneously. 4, ghost cells. antierythrocyte antibodies bind rbc surface antigens, resulting in complement activation and formation of the membrane attack complex (mac). macs form membrane pores, resulting in rupture of rbcs, and the release of hemoglobin into the circulation. ghost cells are rbc membrane remnants that lack cytoplasm (hemoglobin normocytic, normochromic anemia). it has long been known that patients with inflammatory or other chronic disease often become anemic, and that this condition results in increased iron stores in the bone marrow. sequestration of iron may be a bacteriostatic evolutionary adaptation because many bacteria require iron as a cofactor for growth. in recent years, investigators have begun to elucidate the molecular mechanisms underlying anemia of inflammation. hepcidin, an acute phase protein and antimicrobial peptide synthesized in the liver, is a key mediator that limits iron availability. hepcidin expression increases with inflammation, infection, or iron overload and decreases with anemia or hypoxia. hepcidin exerts its effects by causing functional iron deficiency. it binds to and causes the degradation of the cell surface iron efflux molecule, ferroportin, thus inhibiting both absorption of dietary iron from the intestinal epithelium and export of iron from macrophages and hepatocytes into the plasma ( fig. 13-16 ). anemia of inflammation involves factors besides decreased iron availability. inflammatory cytokines are likely to inhibit erythropoiesis by oxidative damage to and triggering apoptosis of developing erythroid cells, by decreasing expression of epo and stem cell factor, and by decreasing expression of epo receptors. in addition, experimentally induced sterile inflammation in cats resulted in shortened erythrocyte survival, indicating that anemia of inflammation is likely also a function of increased erythrocyte destruction. other causes of decreased erythropoiesis are listed in table 13 -2. specific examples of diseases causing nonregenerative anemia by these mechanisms are discussed later in this chapter. neutropenia. neutropenia refers to a decrease in the concentration of neutrophils in circulating blood. neutropenia may be caused by decreased production, increased destruction, altered distribution, or a demand for neutrophils in tissues that exceeds the rate of granulopoiesis. decreased production is evident on bone marrow examination as granulocytic hypoplasia. this usually results from an insult that affects multiple hematopoietic lineages, such as chemical insult, radiation, neoplasia, infection, or fibrosis, but may also be caused by a process that preferentially targets granulopoiesis. in marked contrast to erythrocytes, neutrophils have a very short life span in circulation. once released from the bone marrow, a neutrophil is in the bloodstream only for hours before migrating into the tissues. when neutrophil production ceases, a reserve of mature neutrophils in the bone marrow storage pool may be adequate to maintain normal numbers of circulating neutrophils for a few days; however, after the bone marrow storage pool is depleted, neutropenia rapidly ensues. immune-mediated neutropenia is a rare but recognized condition in domestic animals. bone marrow findings range from granulocytic hypoplasia to hyperplasia, depending on where the cells under immune attack are in their differentiation programs. neutropenia with no evidence of decreased production and in which other causes of neutropenia have been excluded may be a result of destruction of neutrophils before they leave the bone marrow, a condition known as ineffective granulopoiesis. like other forms of ineffective hematopoiesis, this condition is often presumed to be immune mediated; in cats this condition may occur as a result of infection of hematopoietic cells with felv. as presented in the earlier section on granulopoiesis and monocytopoiesis (myelopoiesis), neutrophils within the blood vasculature are in two compartments: a circulating pool, consisting of those cells flowing freely in the blood, and a marginating pool, consisting of those cells transiently interacting with the endothelial surface. (in reality, neutrophils are constantly shifting between these two pools, but the proportion of cells in either pool normally remains fairly schistocytes are the only red blood cell morphologic abnormality specific for mechanical fragmentation because all other morphologic abnormalities can be seen with other disease processes. for example, ghost cells may be observed with other types of hemolysis. nonregenerative anemia is characterized by a lack of reticulocytosis on the cbc; however, reticulocytosis does not occur in horses even in the context of regeneration. most often this is a result of decreased production in the marrow (i.e., erythroid hypoplasia). erythrocytes circulate for a long time, so anemias caused by decreased production tend to develop slowly. the most common form of nonregenerative anemia is known as anemia of inflammation or anemia of chronic disease. in this form of anemia, erythrocytes are decreased in number but are typically normal in size and hemoglobin concentration (so-called c or because of one specifically depressing thrombopoiesis. in either case, decreased thrombopoiesis is evident as megakaryocytic hypoplasia upon bone marrow examination. general causes of decreased hematopoiesis outlined earlier in the sections on anemia and neutropenia also apply to thrombocytopenia. increased platelet destruction due to immune-mediated thrombocytopenia (imtp) is a fairly common disease in dogs and may also occur in other species. thrombocytopenia with immune-mediated thrombocytopenia is often severe (e.g., <10,000 platelets/µl), resulting in spontaneous multisystemic hemorrhage. increased use of platelets occurs with hemorrhage and disseminated intravascular coagulation. thrombocytopenia secondary to hemorrhage is often mild to moderate, whereas disseminated intravascular coagulation may cause mild to severe thrombocytopenia, often with evidence of mechanical fragmentation hemolysis (e.g., schistocytes). disseminated intravascular coagulation is a syndrome in which hypercoagulability leads to increased consumption of both platelets and coagulation factors in the plasma, with subsequent hypocoagulability and susceptibility to bleeding. risk factors for developing disseminated intravascular coagulation include severe inflammation, such as sepsis or pancreatitis, neoplasia, and organ failure. the spleen normally contains a significant proportion of total platelet mass (up to one-third in some species), and abnormalities involving the spleen may result in changes in the number of circulating platelets. for example, splenic congestion may result in platelet sequestration and thrombocytopenia, and splenic contraction may cause thrombocytosis. lymphopenia. lymphopenia refers to a decreased concentration of lymphocytes in blood. it is a common hematologic finding in sick animals. usually the precise mechanism of lymphopenia is not clear but is often presumed secondary to endogenous glucocorticoid excess that occurs with stress. excess glucocorticoids, either endogenous or exogenous, cause an altered distribution of lymphocytes; there is increased trafficking of lymphocytes from blood to lymphoid tissue, and decreased egress of lymphocytes from lymphoid tissue to blood. at higher concentrations of glucocorticoids, lymphocytes are destroyed. other causes of lymphotoxicity include chemotherapeutic agents, radiation therapy, and some infectious agents. lymphopenia may occur with various mechanisms, including loss of lymphocyte-rich lymphatic fluid (e.g., gastrointestinal disease, repeated drainage of chylous effusions), and disruption of the normal lymphoid tissue architecture because constant in any given species.) circulating neutrophils are part of the blood sample collected during routine venipuncture and are thus counted in the cbc, whereas marginating neutrophils are not. pseudoneutropenia refers to the situation in which there is an increased proportion of neutrophils in the marginating pool. this may occur because of decreased blood flow or in response to stimuli, such as endotoxemia, that increase expression of molecules promoting interaction between neutrophils and endothelial cells. this mechanism of neutropenia is rarely observed in clinical practice. neutropenia may also result from increased demand for neutrophils in the tissue. how rapidly such a situation develops depends not only on the magnitude of the inflammatory stimulus but also on the reserve of postmitotic neutrophils in the bone marrow. the size of this reserve, or storage pool, is species dependent. in dogs this pool contains the equivalent of 5 days' normal production of neutrophils. cattle represent the other extreme in that they have a small storage pool and thus are predisposed to becoming neutropenic during times of acute inflammation. horses and cats are somewhere between the two extremes, closer to cattle and dogs, respectively. it stands to reason that the clinical significance of neutropenia because of a supply and demand imbalance is also species dependent. in dogs, neutropenia as a result of inflammation is an alarming finding because it is evidence of a massive tissue demand for neutrophils that has exhausted the patient's storage pool and is exceeding the rate of granulopoiesis in the bone marrow. however in cows, neutropenia is commonly noted in a wide range of conditions involving acute inflammation and does not necessarily indicate an overwhelming demand. eosinopenia/basopenia. eosinopenia and basopenia are decreased concentrations of blood eosinophils and basophils, respectively. in many laboratories, cbc reference values for eosinophils and basophils are as low as zero cells per microliter, precluding detection of eosinopenia or basopenia. when detectable, eosinopenia is often a result of stress (i.e., glucocorticoid mediated). monocytopenia. monocytopenia denotes a decreased concentration of monocytes in blood; it is of little to no pathologic significance by itself. thrombocytopenia. thrombocytopenia refers to a decrease in the concentration of circulating platelets. mechanisms of thrombocytopenia include decreased production, increased destruction, increased consumption, and altered distribution. decreased production may occur because of a condition affecting cells of multiple hematopoietic lineages, including megakaryocytes, is often used interchangeably with erythrocytosis, but technically and for the purposes of this chapter, polycythemia refers to a specific type of leukemia called primary erythrocytosis or polycythemia vera. causes of erythrocytosis are either relative or absolute. relative erythrocytosis results from a fluid deficit or an altered distribution of erythrocytes within the body (i.e., the body's total erythrocyte mass of generalized lymphadenopathy (e.g., lymphoma, blastomycosis). some hereditary immunodeficiencies, such as severe combined immunodeficiency or thymic aplasia, can cause lymphopenia due to lymphoid aplasia. erythrocytosis. an increase in the measured red cell mass above the normal range is known as erythrocytosis. the term polycythemia rarely, an epo-secreting tumor may cause inappropriately elevated levels of epo in the absence of hypoxia. absolute erythrocytosis, whether primary or secondary, causes increased viscosity of the blood, resulting in impaired blood flow and microvasculature distention. affected individuals are at increased risk for tissue hypoxia, thrombosis, and hemorrhage. clinical signs of hyperviscosity syndrome may include erythematous mucous membranes ( fig. 13-17) , prolonged capillary refill time, prominent scleral vessels, evidence of thrombosis or hemorrhage, and secondary signs related to specific organ systems affected (e.g., neurologic and cardiovascular signs). neutrophilia. neutrophilia, an increased blood concentration of neutrophils, occurs in response to a number of different stimuli, which are not mutually exclusive. major mechanisms of neutrophilia are shown in fig. 13-18 . understanding the cbc findings characteristic of these responses is an important part of clinical veterinary medicine. inflammation can result in neutropenia, as discussed earlier, or neutrophilia, as discussed next. however, before moving on to a discussion of inflammatory neutrophilia and the so-called left shift, it is important to mention two other common is not increased). it occurs most frequently with dehydration, when the decreased proportion of water in the blood results in hemoconcentration. it is observed less frequently with epinephrine-mediated splenic contraction, wherein erythrocytes move from the spleen into peripheral circulation. erythrocytosis from splenic contraction occurs to the most pronounced degree in horses and cats, especially in young, healthy animals. absolute erythrocytosis is a true increase in red blood cell mass due to erythroid neoplasia or hyperplasia and includes causes of primary and secondary erythrocytosis. primary erythrocytosis, or polycythemia vera, is a neoplastic proliferation of erythroid cells with a predominance of mature erythrocytes. diagnosis is based on a marked increase in red cell mass (hematocrit in normally hydrated dogs ranges from 65% to >80%), an absence of hypoxemia, an absence of other tumors, and a normal or decreased plasma epo concentration. secondary erythrocytosis refers to epo-mediated erythroid hyperplasia causing an increased red blood cell mass. the erythroid hyperplasia may be an appropriate response to chronic hypoxia, such as occurs with right-to-left cardiac shunts or chronic pulmonary inflammatory mediators, including interleukin-1 (il-1), interleukin-6 (il-6), interferon (inf), and tumor necrosis factor (tnf), cause anemia of inflammatory disease due to oxidative hemolysis, iron sequestration within enterocytes and macrophages, and impaired erythroid responsiveness to erythropoietin (epo). during homeostasis the membrane transport molecule, ferroportin, transports iron from the cytosol to the extracellular space. the iron is then used for various physiologic processes, including hemoglobin production within bone marrow erythroid precursors. during times of inflammation the liver increases production of hepcidin, which binds ferroportin and causes its internalization and lysosomal degradation. with fewer membrane ferroportin molecules, less iron is absorbed from the diet and mobilized from macrophages. rbc, red blood cell. ( of course, neutrophilia may also indicate inflammation, and inflammatory stimuli of varying magnitude and duration produce different patterns of neutrophilia. a classic hematologic finding in patients with increased demand for neutrophils is the presence of immature forms in the blood, known as a left shift. not all inflammatory responses have a left shift, but the presence of a left shift almost always signifies active demand for neutrophils in the tissue. the magnitude of a left shift is assessed by the number of immature cells and their degree of immaturity. the mildest form is characterized by increased numbers of band neutrophils, the immediate predecessor to the segmented neutrophil normally found in circulation. progressively immature predecessors are seen with increasingly severe inflammation. a left shift is considered orderly if the number of immature neutrophils in circulation decreases as they become progressively immature. the term degenerative left shift is sometimes used to describe cases in which the number of immature forms exceeds the number of segmented neutrophils. as with glucocorticoidmediated neutrophilia, the typical magnitude of neutrophilia caused by inflammation varies by species, with dogs having the most pronounced response. it might be useful to think of neutrophil kinetics in terms of a producer-consumer model in which the bone marrow is the factory, and the tissues (where the neutrophils eventually go) are the customers. the bone marrow storage pool is the factory inventory, and the neutrophils in the bloodstream are in delivery to the customer. within the blood vessels, circulating neutrophils are on the highway, and marginating neutrophils are temporarily pulled off to the side of the road. during health, there is an even flow of neutrophils from the factory to the customer. thus the system is in steady state, and neutrophil numbers remain relatively constant and within the normal range. however, disease states may perturb this system at multiple levels. decreased granulopoiesis is analogous to a factory working below normal production level. ineffective granulopoiesis is analogous to goods that are produced at a normal to increased rate but are damaged during manufacturing and never leave the factory. a left shift is analogous to the factory meeting increased customer demand by shipping out unfinished goods. cases of persistent, established inflammation are characterized by bone marrow granulocytic hyperplasia and mature neutrophilia, analogous to a factory that has had time to adjust to increased demand and is meeting it more efficiently by increasing its output. eosinophilia/basophilia. eosinophilia and basophilia are increased concentrations of blood eosinophils and basophils, respectively. they may occur with parasitism, hypersensitivity reactions, paraneoplastic responses (e.g., lymphoma, mast cell neoplasia, or leukemia), and nonparasitic infectious disease. eosinophilia has also been documented with hypoadrenocorticism and rare idiopathic conditions (e.g., hypereosinophilic syndrome). most cases of eosinophilia and basophilia are due to eosinophilic and basophilic hyperplasia within the bone marrow in response to inflammatory growth factors. however, cortisol deficiency is thought to cause eosinophilia in dogs with hypoadrenocorticism. monocytosis. monocytosis is an increased concentration of monocytes in blood. it most commonly occurs with excessive glucocorticoids or inflammation and uncommonly to rarely with monocytic leukemia, immune-mediated neutropenia, and cyclic hematopoiesis. with excessive endogenous or exogenous glucocorticoids, monocytes shift from the marginating pool to the circulating pool. this stress monocytosis is most common in dogs, less frequent in cats, and rare in horses and cattle. inflammatory diseases cause monocytosis by cytokine-mediated monocytic hyperplasia in the bone marrow. causes of neutrophilia: glucocorticoid excess and epinephrine excess. less common causes of neutrophilia, such as leukocyte adhesion deficiency and neoplasia, are discussed later in the chapter. glucocorticoid excess, either because of endogenous production or exogenous administration, results in a cbc pattern known as the stress leukogram, characterized by mature neutrophilia (i.e., increased concentration of segmented neutrophils without immature neutrophils) and lymphopenia, with or without monocytosis and eosinopenia. mechanisms contributing to glucocorticoid-mediated neutrophilia include the following: • increased release of mature neutrophils from the bone marrow storage pool • decreased margination of neutrophils within the vasculature, with a resulting increase in the circulating pool • decreased migration of neutrophils from the bloodstream into tissues the magnitude of neutrophilia tends to be species dependent, with dogs having the most pronounced response (up to 35,000 cells/µl) and in decreasing order of responsiveness, cats (30,000 cells/µl), horses (20,000 cells/µl), and cattle (15,000 cells/µl) having less marked responses. with long-term glucocorticoid excess, neutrophil numbers tend to normalize, whereas lymphopenia persists. epinephrine release results in a different pattern, known as physiologic leukocytosis or excitement leukocytosis, characterized by mature neutrophilia (like the glucocorticoid response) and lymphocytosis (unlike the glucocorticoid response). this phenomenon is short lived (i.e., <1 hour). neutrophilia occurs primarily because of a shift of cells from the marginating to the circulating pool. physiologic leukocytosis is common in cats (especially when they are highly stressed during blood collection) and horses, less common in cattle, and uncommon in dogs. of a regenerative response in patients recovering from thrombocytopenia, as a result of redistribution after splenic contraction, or within the several weeks after splenectomy. in these cases, thrombocytosis is transient. in the case of splenectomy, thrombocytosis may be marked but normalizes after several weeks. because the body's total platelet mass regulates thrombopoiesis, and a significant portion of the platelet mass is normally in the spleen, it makes sense that splenectomized animals develop thrombocytosis. however, the reason that the number of circulating platelets normalizes in these individuals in the weeks after splenectomy is not thrombocytosis. thrombocytosis, or an increased concentration of platelets in the blood, is a relatively common, nonspecific finding in veterinary patients. in the vast majority of cases, thrombocytosis is reactive-a response to another, often apparently unrelated, disease process. examples of conditions having reactive thrombocytosis include inflammatory and infectious diseases, iron deficiency, hemorrhage, endocrinopathies, and neoplasia. factors that may contribute to reactive thrombocytosis include increased plasma concentration of thrombopoietin, inflammatory cytokines (e.g., il-6), or catecholamines. thrombocytosis may also occur as part 1, neutrophils and their precursors are distributed in five pools: a bone marrow precursor pool, which includes mitotically active and inactive immature cells; a bone marrow storage pool, consisting of mitotically inactive mature neutrophils; a peripheral blood marginating pool; a peripheral blood circulating pool; and a tissue pool. the relative size of each pool is represented by the size of its corresponding wedge. the peripheral blood neutrophil count measures only neutrophils within the circulating peripheral blood pool, which can be enlarged by (2) increased demargination, (3) diminished extravasation into tissue, (4) increased release of cells from the marrow storage pool, and (5) cells. the preceding section focused on abnormalities in the number of blood cells. there are also various acquired and congenital conditions involving abnormal structure or function of blood cells. this section briefly discusses abnormal blood cell structure or function occurring secondary to other underlying disease. primary disorders of blood cells are discussed later in the chapter in the section on specific diseases. morphologic abnormalities detected on routine microscopic examination of blood smears may provide important clues about underlying disease processes. poikilocytosis is a broad term referring to the presence of abnormally shaped erythrocytes in circulation. etable 13 -1 lists conditions with and mechanisms involved in the formation of a number of specific types of erythrocyte morphologic abnormalities, and fig. 13-15 shows some examples. the acquired neutrophil morphologic abnormality known as toxic change (fig. 13 -20) reflects accelerated production of neutrophils as part of the inflammatory response. features of toxic change include increased cytoplasmic basophilia, the presence of small bluegray cytoplasmic inclusions known as döhle bodies (often noted incidentally in cats), and in more severe cases, cytoplasmic vacuolation. although not causing impaired neutrophil function, toxic change occurs during granulopoiesis and thus is technically a form of dysplasia (e.g., döhle bodies are foci of aggregated endoplasmic reticulum). toxic change may accompany any inflammatory response, but in general the more marked the toxic change, the higher the index of suspicion for infection or endotoxemia. other secondary changes to neutrophils may not be evident morphologically. for example, studies in human beings and dogs have shown that individuals with cancer have abnormal neutrophil function (including phagocytic activity, killing capacity, and oxidative burst activity) before initiation of therapy. the clinical significance of this finding is not clear. platelet function disorders, also known as thrombopathies or thrombopathias, may be primary or secondary. many conditions are known or suspected to cause secondary platelet dysfunction (hypofunction or hyperfunction) by altering platelet adhesion or aggregation or by mechanisms that are not fully understood. box 13-2 shows underlying conditions having secondary platelet dysfunction. clear. there is also a rare form of megakaryocytic leukemia known as essential thrombocythemia, which is characterized by marked thrombocytosis. lymphocytosis. lymphocytosis refers to an increase in the concentration of lymphocytes in blood circulation. there are several causes of lymphocytosis, including age, excessive epinephrine, chronic inflammation, hypoadrenocorticism, and lymphoid neoplasia; lymphoid neoplasms are presented later in the chapter. young animals normally have higher concentrations of lymphocytes than older animals, and normal healthy young animals may have counts that exceed adult reference values. because this is not pathologic lymphocytosis, but normal physiologic variation, it is often termed pseudolymphocytosis of young animals. as discussed earlier in the section on neutrophilia, lymphocytosis is also a feature of epinephrine-mediated physiologic leukocytosis, resulting from redistribution of lymphocytes from the blood marginating pool into the blood circulating pool. epinephrine-mediated lymphocytosis may be more marked than neutrophilia, particularly in cats (lymphocyte counts of > 20,000/µl are not uncommon). antigenic stimulation may result in lymphocytosis, which may be marked in rare cases (up to approximately 30,000/µl in dogs and 40,000/µl in cats); however, this is not usually the case, even when there is clear evidence of increased immunologic activity in lymphoid tissues. in cases of antigenic stimulation, it is common for a minority of lymphocytes to have "reactive" morphologic features-larger lymphocytes with more abundant, deeply basophilic cytoplasm and more open chromatin ( fig. 13-19 ). just as glucocorticoid excess can cause lymphopenia, glucocorticoid deficiency (hypoadrenocorticism) can cause lymphocytosis, or lack of lymphopenia during conditions of stress that typically result in glucocorticoid-mediated lymphopenia. a condition known as persistent lymphocytosis (pl) occurs in approximately 30% of cattle infected with the bovine leukemia virus (blv). the condition is defined as an increase in the blood concentration of lymphocytes above the reference interval for at least 3 months. this form of lymphocytosis is a nonneoplastic proliferation (i.e., hyperplasia) of b lymphocytes. in the absence of other disease, cattle with persistent lymphocytosis are asymptomatic. however, cattle infected with blv, especially those animals with persistent lymphocytosis, are at increased risk for developing b lymphocyte lymphoma. aplastic anemia (aplastic pancytopenia) aplastic anemia, or more accurately aplastic pancytopenia, is a rare condition characterized by aplasia or severe hypoplasia of all hematopoietic lineages in the bone marrow with resulting cytopenias. the term aplastic anemia is a misnomer because affected cells are not limited to the erythroid lineage. many of the conditions reported to cause aplastic anemia do so only rarely or idiosyncratically; more frequently, they cause other hematologic or nonhematologic abnormalities. a partial list of reported causes of aplastic anemia in domestic animals includes the following: most of these causes, especially the chemical agents, are directly cytotoxic to hscs or progenitor cells, resulting in their destruction. however, another proposed mechanism is disruption of normal stem cell function because of mutation or perturbation of hematopoietic cells and/or their microenvironment. this pathogenesis is mostly recognized in retroviral infections. aplastic anemia occurs in both acute and chronic forms. most of the chemical causes result in acute disease. grossly, affected animals may show signs of multisystemic infection and hemorrhage due to severe neutropenia and thrombocytopenia, respectively. severe neutropenia typically develops within 1 week of an acute insult to the bone marrow, and severe thrombocytopenia occurs in the second week. this sequence is a result of the circulating life spans of each cell type; in health, neutrophils have a blood half-life of 5 to 10 hours, whereas platelets circulate for 5 to 10 days. the development of signs of anemia, such as pale mucous membranes, is more variable. the presence and severity of anemia depends on how rapidly the marrow recovers from the insult and the erythrocyte life span of the particular species. microscopically, bone marrow is hypocellular with markedly reduced hematopoietic cells. hematopoietic cells are replaced with adipose tissue, and there is a variable inflammatory infiltrate of lymphocytes, plasma cells, and macrophages. in addition, there may be necrosis, hematopoietic cell apoptosis, and an increase in phagocytic macrophages. fig. 13 -21 shows bone marrow aspirates from a dog with pancytopenia from acute 5-fluorouracil toxicosis, before and during recovery. many inherited or presumably inherited disorders of blood cells have been recognized in domestic animals, including rare or sporadic cases and conditions that are of questionable clinical relevance. this section and the later sections covering species-specific disorders are invading cells or microorganism gain access to the bone marrow or blood circulation either hematogenously or by trauma. trauma may be as obvious as a gaping wound or as subtle as the bite of an insect. portals of entry for the bone marrow are summarized in box 13-3. diseases that arise from the bone marrow, such as leukemia, typically spread to other tissues hematogenously. the bone marrow is encased by a protective shell of cortical bone, and blood supply to the marrow provides access to systemic humoral and cellular defenses. of course, leukocytes themselves function as an essential part of inflammation and immune function, as discussed briefly in the section on granulopoiesis and monocytopoiesis (myelopoiesis) and in greater detail in chapters 3 and 5. biochemical steps in the glycolytic pathway or linked to it generate antioxidant molecules that enable erythrocytes to withstand oxidative insults throughout their many days in circulation. in addition to producing energy in the form of adenosine triphosphate (atp), glycolysis generates nadh, which helps convert the oxidized, nonfunctional form of hemoglobin, known as methemoglobin, back to its active, reduced state. another antioxidant erythrocyte metabolic pathway, the pentose shunt or hexose uremia antiplatelet antibodies (also cause immune-mediated thrombocytopenia) infection (bvdv, felv) hyperglobulinemia increased fibrinolytic products hypoammonemia snake envenomation platelet inhibitors nsaids-irreversible (aspirin) or reversible inhibition of cyclooxygenase colloidal plasma expanders (e.g., hydroxyethyl starch) other drugs and exogenous agents (many) hematogenously direct penetration (trauma) including hypercellular glomeruli, thickened glomerular and tubular basement membranes, and tubular epithelial lipidosis, degeneration, and necrosis. other porphyrias have been diagnosed in cattle, pigs, and cats but are not known to cause hemolytic anemia. pyruvate kinase deficiency. pyruvate kinase (pk) deficiency is an inherited autosomal recessive condition due to a defective r-type pk isoenzyme that is normally present in high concentrations in mature erythrocytes. to compensate for this deficiency, there is persistence of the m2-type pk isoenzyme, which is less stable than the r-type isoenzyme. the disease is reported in many dog breeds and fewer cat breeds (e.g., abyssinian, somali, and domestic shorthair). erythrocyte pk deficiency results in decreased atp production and shortened erythrocyte life spans. in dogs the hemolytic anemia is typically chronic, moderate to severe, extravascular, and strongly regenerative. with chronicity, hemolytic anemia causes enhanced intestinal absorption of iron and subsequent hemosiderosis, especially of the liver and bone marrow. dogs typically die at 1 to 5 years of age of hemochromatosis-induced liver and bone marrow failure. however, cats with pk deficiency typically show no clinical signs, have milder anemia, and do not develop organ failure. grossly, affected animals have lesions attributed to hemolytic anemia, including splenomegaly, pale mucous membranes, and rarely icterus. dogs with end-stage disease have cirrhosis, myelofibrosis, and osteosclerosis. dogs with pk deficiency do not necessarily have the same genetic defect, so mutation-specific dna-based assays are required. in contrast, a single dna-based test is available to detect the common mutation affecting abyssinian, somali, and domestic shorthair cats. cytochrome-b 5 reductase deficiency. deficiency of cytochrome-b 5 reductase (cb5r, also known as methemoglobin reductase), the enzyme that catalyzes the reduction of methemoglobin (fe 3+ ) to hemoglobin (fe 2+ ), has been recognized in many dog breeds and in domestic shorthair cats. it is probably an autosomal recessive trait. affected animals may have cyanotic mucous membranes or exercise intolerance but usually lack anemia and clinical signs of disease. life expectancies are normal. glucose-6-phosphate dehydrogenase deficiency. deficiency of glucose-6-phosphate dehydrogenase (g6pd), the ratecontrolling enzyme of the pentose phosphate pathway (ppp), has been reported in an american saddlebred colt, its dam, and one male dog. the ppp is an antioxidative pathway that generates nadph, which maintains glutathione in its reduced form (gsh). therefore in animals with g6pd deficiency, oxidants are not scavenged, and erythrocyte oxidative injury occurs. the colt with g6pd deficiency had severe oxidative hemolytic anemia with eccentrocytes on blood smear evaluation. however, the colt's dam only had eccentrocytes, and showed no hematologic signs of disease. leukocyte adhesion deficiency. leukocyte adhesion deficiency (lad) is a fatal autosomal recessive defect of leukocyte integrins, in particular the β 2 chain (also known as cluster of differentiation [cd] 18 [cd18]). disease has been recognized in holstein cattle (known as bovine leukocyte adhesion deficiency [blad] ) and irish setter dogs (known as canine leukocyte adhesion deficiency [clad]) (see chapter 3). without normal expression of this adhesion molecule, leukocytes have severely impaired abilities to migrate from the blood into tissues. as a result, animals with leukocyte adhesion deficiency have marked neutrophilia with nonsuppurative multisystemic infections. blood neutrophils often have nuclei with greater than five nuclear segments, termed not comprehensive but instead focus on the more common, wellcharacterized, or recently reported conditions. erythropoietic porphyrias. porphyrias are a group of hereditary disorders in which porphyrins accumulate in the body because of defective heme synthesis. inherited enzyme defects in hemoglobin synthesis have been identified in holstein cattle, siamese cats, and other cattle and cat breeds, resulting in bovine congenital erythropoietic porphyria and feline erythropoietic porphyria, respectively. accumulation of toxic porphyrins in erythrocytes causes hemolytic anemia, whereas accumulation of porphyrins in tissues and fluids produces discoloration, including red-brown teeth, bones, and urine (see fig. 1 -59). because of the circulation of the photodynamic porphyrins in blood, these animals have lesions of photosensitization of the nonpigmented skin. all affected tissues, including erythrocytes, exhibit fluorescence with ultraviolet light. histologically, animals may exhibit perivascular dermatitis, as well as multisystemic porphyrin deposition, hemosiderosis, emh, and marrow erythroid hyperplasia. cats may show evidence of renal disease, a b syndrome exhibit oculocutaneous albinism (due to altered distribution of melanin granules) and are prone to infection and bleeding. blood smear evaluation reveals granulocytes with large cytoplasmic granules. glanzmann thrombasthenia. glanzmann thrombasthenia (gt) is an inherited platelet function defect caused by a mutated α iib subunit of the integrin α iib β 3 (also known as glycoprotein iib-iiia [gpiib-iiia]). the disorder has been recognized in great pyrenees and otterhound dogs and several horse breeds, including a quarter horse, a standardbred, a thoroughbred-cross, a peruvian paso mare, and an oldenburg filly. the α iib β 3 molecule has multiple functions but is best known as a fibrinogen receptor that is essential for normal platelet aggregation. bleeding tendencies vary widely between affected individuals but mainly occur on mucosal surfaces. the condition is characterized by an in vitro lack of response to all platelet agonists and severely impaired clot retraction (i.e., whole blood samples without anticoagulant often fail to clot). molecular testing is available to detect diseased or carrier states in dogs and horses. thrombopathia. calcium diacylglycerol guanine nucleotide exchange factor i (caldag-gefi) is a molecule within the signaling pathway that results in platelet activation in response to platelet agonists. mutated caldag-gefi has been documented in basset hound, eskimo spitz, and landseer dogs, and simmental cattle. all reported mutations have a bleeding tendency. in vitro platelet aggregation responses to platelet agonists, such as adp, collagen, and thrombin, are absent or impaired. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. oxidative agents. a variety of oxidative toxins cause hemolytic anemia and/or methemoglobinemia in domestic species. more common or well-characterized oxidants are listed here: • horses-acer rubrum (red maple) • ruminants-brassica spp. (cabbage, kale, and rape), copper hypersegmented neutrophils, due to neutrophil aging within blood vessels ( fig. 13-22 ). these animals are highly susceptible to infections and usually die at a young age. pelger-huët anomaly. pelger-huët anomaly (pha) is a condition of hyposegmented granulocytes due to a lamin b receptor mutation. it has been described in dogs, cats, horses, and rabbits, especially in certain breeds. in australian shepherd dogs the mode of inheritance is autosomal dominant with incomplete penetrance. most cases of pelger-huët anomaly are the heterozygous form and of no clinical significance. however, skeletal abnormalities, stillbirths, and/or early mortality may accompany pelger-huët anomaly in rabbits and cats, especially homozygotes. in pelger-huët anomaly the nuclei of neutrophils, eosinophils, and basophils fail to segment, resulting in band-shaped, bean-shaped, or round nuclei. although the nuclear shape is similar to that of an inflammatory left shift, healthy animals with pelger-huët anomaly do not have clinical signs or other laboratory findings indicating inflammation. for example, neutrophils in healthy animals with pelger-huët anomaly have mature (clumped) chromatin and do not show signs of toxicity ( fig. 13-23 ). an acquired, reversible condition mimicking pelger-huët anomaly, known as pseudo-pelger-huët anomaly, is occasionally noted in animals with infectious disease, neoplasia, or drug administration. chédiak-higashi syndrome. chédiak-higashi syndrome (chs) is a rare autosomal recessive defect in the lysosomal trafficking regulator (lyst) protein. the syndrome has been identified in hereford, brangus, and japanese black cattle, persian cats, and several nondomestic species. the defective lyst protein results in granule fusion in multiple cell types, including granulocytes, platelets, and melanocytes, as well as abnormal cell function. individuals with chédiak-higashi syndrome have severely impaired cellular innate immunity because of neutropenia, impaired leukocyte chemotaxis, and impaired killing by granulocytes and cytotoxic lymphocytes. platelets lack the dense granules that normally contain key bioactive molecules involved in hemostasis, including platelet agonists, such as adp and serotonin. in vitro platelet aggregation is severely impaired. as a result, animals with chédiak-higashi 746.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system von willebrand disease (vwd) is the most common canine hereditary bleeding disorder and has also been described in many other domestic species. the disease actually refers to a group of inherited conditions characterized by a quantitative or qualitative deficiency of vwf. this factor is a multimeric glycoprotein that is stored in platelet α-granules and endothelial cells and circulates as a complex with coagulation factor viii. its primary functions are to stabilize factor viii and mediate platelet binding to other platelets and subendothelial collagen. although not technically a platelet disorder, von willebrand disease is often classified as such because it results in a loss of normal platelet function. different types of von willebrand disease vary in terms of mode of inheritance and severity of clinical disease. type i von willebrand disease is characterized by low plasma vwf concentration but normal multimeric proportions and a mild to moderate clinical bleeding tendency; it has been reported in many dog breeds. type ii von willebrand disease is characterized by low vwf concentration, absence of large multimers, and a moderate to severe bleeding tendency; it has been reported in german short-haired pointer and german wirehaired pointer dogs. type iii von willebrand disease is characterized by absence of vwf and a severe bleeding tendency; familial and sporadic cases have been reported in numerous dog breeds. the buccal mucosal bleeding time is prolonged with von willebrand disease, often with adequate concentrations of platelets and normal prothrombin time and partial thromboplastin time (ptt). however, ptt may be mildly prolonged because vwf stabilizes factor viii, and deficiency of vwf results in enhanced factor viii degradation. grossly, affected animals exhibit bleeding tendencies, especially in the form of inherited coagulation factor deficiencies have been documented in most domestic species, including deficiencies of prekallikrein and factors i, ii, vii, viii, ix, x, xi, and xii. of these disorders, hereditary coagulation factor viii (hemophilia a) and factor ix (hemophilia b) deficiencies are most common. hemophilia a has been recognized in horses, cattle, dogs, and cats, and hemophilia b occurs in dogs and cats. both disorders have an x-linked recessive mode of inheritance, meaning that clinical disease is more common in males. affected males have variable tendencies to bleed, depending on the severity of the deficiency, exposure to trauma, and size and activity level of the affected individual. carrier females are usually asymptomatic. laboratory tests often reveal adequate platelets, normal prothrombin times, and prolonged partial thromboplastin times. hereditary defects in γ-glutamyl carboxylase, the enzyme required for normal carboxylation of vitamin k-dependent coagulation factors, have been recognized in a flock of rambouillet sheep and two devon rex cats from the same litter. the genetic defect is not known in cats, but in sheep it is an autosomal recessive trait that results in a premature stop codon and truncated γ-glutamyl carboxylase. in sheep there is increased lamb mortality with excessive bleeding during parturition, especially through the umbilicus or into subcutaneous tissues. gingival bleeding, epistaxis, and hematuria or at sites of injections, venipuncture, or surgery. of loss is through the gastrointestinal tract (e.g., neoplasia in older animals or hookworm infection in puppies). chronic blood loss may also be caused by marked ectoparasitism (e.g., pediculosis in cattle or massive flea burden in kittens and puppies), neoplasia in locations other than the gastrointestinal tract (e.g., cutaneous hemangiosarcoma), coagulation disorders, and repeated phlebotomy of blood donor animals. rapidly growing nursing animals may be iron deficient when compared with adults because milk is an iron-poor diet. in most cases this has little clinical significance (and in fact is normal). an important exception is piglets with no access to iron, which may cause anemia, failure to thrive, and increased mortality. neonatal piglets are routinely given parenteral iron for this reason. copper deficiency can cause iron deficiency in ruminants and may occur because of copper-deficient forage or impaired usage of copper by high dietary molybdenum or sulfate. it is believed that copper deficiency impairs production of ceruloplasmin, a copper-containing enzyme involved in gastrointestinal iron absorption. iron deficiency causes anemia by impaired hemoglobin synthesis. iron is an essential component of hemoglobin, and when it is absent, hemoglobin synthesis is depressed. because erythrocyte maturation is dependent upon obtaining a critical hemoglobin concentration, maturing erythroid precursors undergo additional cell divisions during iron-deficient states. these additional cell divisions result in small erythrocytes, termed microcytes (see fig. 13 -15, g). however, erythrocytes with low hemoglobin concentrations are produced when microcyte formation can no longer compensate for iron deficiency. the classic hematologic picture with iron deficiency anemia is microcytic (i.e., decreased mcv), hypochromic (i.e., decreased mchc) anemia. microcytes and hypochromasia (see fig. 13 -15, g) may also be discernible on blood smear examination as erythrocytes that are abnormally small and paler-staining, respectively. early iron deficiency anemia is poorly regenerative, whereas continued hemorrhage and iron loss cause nonregenerative anemia. additional hematologic changes may include evidence of erythrocyte mechanical fragmentation (e.g., schistocytes) and reactive thrombocytosis. hypophosphatemic hemolytic anemia. marked hypophosphatemia is recognized as a cause of intravascular hemolytic anemia in postparturient dairy cows and diabetic animals receiving insulin therapy. in postparturient cows, hypophosphatemia results from increased loss of phosphorus in their milk. insulin therapy may cause hypophosphatemia by shifting phosphorus from the extracellular space to the intracellular space. in either case, marked hypophosphatemia (e.g., 1 mg/dl in cows, or ≤ 1.5 mg/dl in cats) is thought to decrease erythrocyte production of atp, leading to inadequate energy required for maintenance of membrane and cytoskeletal integrity. an accompanying decrease in reducing capacity and increase in methemoglobin concentration have also been noted in experimental studies of hypophosphatemic hemolytic anemia in dairy cattle, suggesting that oxidative mechanisms may also contribute to anemia. affected animals are anemic and hemoglobinuric. gross postmortem findings include pallor, decreased viscosity of the blood, and lesions arising from the underlying metabolic derangement (e.g., discolored pale yellow and swollen liver due to hepatic lipidosis). renal tubular necrosis and hemoglobin pigment within the tubules is evident microscopically. this section covers infectious agents within the same genus that are recognized to cause disease in multiple species. other infectious agents with more limited host specificity (e.g., cytauxzoonosis in cats, feline and equine retroviruses) are covered in later sections on species-specific diseases. throughout both sections, diseases are • dogs-acetaminophen, propofol, zinc • cats-acetaminophen, propofol, propylene glycol • all species-allium spp. (chives, garlic, and onions) in horses, red maple leaves and bark are toxic, especially wilted or dried leaves. the toxic principle is believed to be gallic acid. plants that contain high concentrations of nitrates, such as cabbage, kale, and rape, may cause oxidative injury to erythrocytes; cattle are more susceptible than sheep and goats. however, sheep are more prone to copper toxicosis relative to other ruminants. the condition occurs in animals that have chronically accumulated large amounts of copper in the liver through the diet. the copper is then acutely released during conditions of stress, such as shipping or starvation. continuous rate infusions of the anesthetic propofol may cause oxidative hemolytic anemia in dogs and cats, but single or multiple single doses are not expected to cause clinical hemolysis. zinc toxicosis has been identified in a wide range of animals; however, it is most common in dogs due to their indiscriminate eating habits. common sources include pennies, batteries, paints, creams, automotive parts, screws, nuts, and coating on galvanized metals. propylene glycol is an odorless, slightly sweet solvent and moistening agent in many foods, drugs, and tobacco products. although it is "generally recognized as safe" for animal foods other than for cats by the food and drug administration, it has been banned from cat food since 1996. grossly and microscopically, animals show varying signs of oxidative hemolysis and/or methemoglobinemia, as previously presented in the section discussing anemias (see bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in sheep with copper toxicosis, hemoglobinuric nephrosis, frequently described as gunmetal-colored kidneys with port wine-colored urine, is a classic postmortem lesion. snake envenomation. hemolytic anemia from snake envenomation has been reported in horses, dogs, and cats. it is most commonly reported with viper and pit viper envenomations, including those from rattlesnakes. hemolysins within viper venom directly injure erythrocytes, causing intravascular hemolysis. other mechanisms of hemolysis include the action of phospholipase a 2 on erythrocyte membranes and erythrocyte mechanical fragmentation due to intravascular coagulation and vasculitis. nonhemolytic lesions depend on the venom's additional components and may include hemorrhage, paralysis, and/or tissue edema, inflammation, and necrosis. on blood smear evaluation, animals with snake envenomation may have ghost cells, spherocytes, and/or echinocytes (see figs. 13-13 and 13-15). information on this topic is available at www.expertconsult.com. severe malnutrition is probably a cause of nonregenerative anemia in all species attributable to combined deficiencies of molecular building blocks, energy, and essential cofactors. by far the most commonly recognized specific deficiency that results in anemia is iron deficiency. other specific nutritional deficiencies causing anemia in animals are uncommon or rare. acquired cobalamin (vitamin b 12 ) and folate deficiencies are recognized as causes of anemia in human beings but are rare in animals. iron deficiency anemia. iron deficiency is usually not a primary nutritional deficiency but rather occurs secondary to depletion of iron stores via chronic blood loss. the most common route 747.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system antagonism of vitamin k leads to production of a nonfunctional form of some coagulation factors and resulting coagulopathy; a similar condition results from vitamin k deficiency. conditions with avitaminosis k include poisoning with coumarin-related molecules, fat malabsorption (vitamin k is a fat-soluble vitamin) caused by primary intestinal disease or impaired biliary outflow (uncommon), dietary deficiency (rare), and antibiotics that interfere with vitamin k absorption or usage. a number of coagulation factors-factors ii, vii, ix, and x (collectively known as the vitamin k-dependent factors), as well as the regulatory molecules protein c and protein s-must undergo carboxylation to be functional. this posttranslational modification is catalyzed by the enzyme γ-glutamyl carboxylase, and requires vitamin k as a cofactor. vitamin k is oxidized during the carboxylation reaction and is converted back into its active reduced form by the enzyme vitamin k epoxide reductase. coumarin-related rodenticides, such as warfarin, act by inhibiting vitamin k epoxide reductase, resulting in an absence of vitamin k in its active reduced form (efig. 13 -2). this inhibition lasts until the rodenticide is metabolized and cleared. how long this takes depends on the half-life of the rodenticide and dose, but it may take many weeks. secondgeneration rodenticides, such as bromadoline and brodifacoum, are more potent than warfarin, with longer half-lives. spoiled sweet clover contains dicumarol, which causes coagulopathy by the same mechanism. efigure 13 -2 mechanism of anticoagulant rodenticide toxicity. anticoagulant rodenticides inhibit the enzyme that converts vitamin k back to its active reduced form. active factors (carboxylated) inactive factors (ii, vii, ix, x) = inactivation of vitamin k epoxide reductase, the enzyme that maintains vitamin k in the active form vitamin k epoxide (inactive) x x laboratory findings include prolonged coagulation times (prothrombin time [pt] , ptt, and activated clotting time [act] ). early in the course of rodenticide and related toxicoses, pt may be the only one of these tests that is prolonged because factor vii has the shortest half-life of the vitamin k-dependent factors. however, the other tests become prolonged as nonfunctional forms of the other factors accumulate. in uncomplicated cases, patients are not thrombocytopenic. a wide range of hemorrhagic lesions may occur in affected individuals, including ecchymoses, epistaxis, gingival bleeding, hematomas, hemoptysis, melena or hematochezia, hematuria, and other forms of hemorrhage. there are also lesions with regenerative anemia, such as pale mucous membranes and splenomegaly. histologically, there is hemorrhage, emh, and marrow erythroid hyperplasia. the treatment of cases of rodenticide and related toxicoses is regular administration of exogenous vitamin k 1 until the toxin is cleared (determined by repeat coagulation testing after withholding treatment). babesiosis may cause intravascular and extravascular hemolytic anemia via direct red blood cell injury, the innocent bystander effect, and secondary immune-mediated hemolytic anemia. infection with highly virulent strains may cause severe multisystemic disease. in these cases, massive immunostimulation and cytokine release cause circulatory disturbances, which may result in shock, induction of the systemic inflammatory response, and multiple organ dysfunction syndromes. babesia organisms can usually be detected on a routine blood smear in animals with acute disease. infected erythrocytes may be more prevalent in capillary blood, so blood smears made from samples taken from the pinna of the ear or the nail bed may increase the likelihood of detecting organisms microscopically. buffy coat smears also have an enriched population of infected erythrocytes. pcr-based tests are the most sensitive assay for detecting infection in animals with very low levels of parasitemia. at necropsy, gross lesions are mainly related to hemolysis and include pale mucous membranes, icterus, splenomegaly, dark red or black kidneys, and reddish-brown urine. the cut surface of the congested spleen oozes blood. the gallbladder is usually distended with thick bile. less common lesions include pulmonary edema, ascites, and congestion, petechiae, and ecchymoses of organs, including the heart and brain. parasitized erythrocytes are best visualized on impression smears of the kidney, brain, and skeletal muscle. microscopic findings in the liver and kidney are typical of a hemolytic crisis and include anemia-induced degeneration, necrosis of periacinar hepatocytes and cholestasis, and hemoglobinuric nephrosis with degeneration of tubular epithelium. erythroid hyperplasia is present in the bone marrow. in animals that survive the acute disease, there is hemosiderin accumulation in the liver, kidney, spleen, and bone marrow. in chronic cases there is hyperplasia of macrophages in the red pulp of the spleen. theileriosis (piroplasmosis). theileria spp. are tick-borne protozoal organisms that infect many domestic and wild animals worldwide. numerous theileria spp. have been documented, but only the more economically or regionally important species are mentioned here. diseases with the greatest economic impact in ruminants are east coast fever (theileria parva infection) and tropical theileriosis (theileria annulata infection). • horses-theileria equi (formerly babesia equi) • cattle-theileria annulata, theileria buffeli, t. parva • sheep and goats-theileria lestoquardi (formerly theileria hirci) like babesiosis, theileriosis is generally restricted to tropical and subtropical regions, including parts of africa, asia, the middle east, and europe. except for t. buffeli, all previously listed species are exotic to the united states. infection is characterized by schizonts within lymphocytes or monocytes, and pleomorphic intraerythrocytic piroplasms (merozoites and trophozoites). within host leukocytes the parasite induces leukocyte cellular division, which expands the parasitized cell population. infected cells disseminate throughout the lymphoid system via the lymphatic and blood vessels. the infected leukocyte may block capillaries, causing tissue ischemia. later in infection some schizonts cause leukocyte lysis and release of merozoites. merozoites then invade and parasitize erythrocytes, causing hemolytic anemia. possible mechanisms of anemia in theileriosis include invasion of erythroid precursors by merozoite stages and associated erythroid hypoplasia (as occurs with t. parva infection), immune-mediated hemolysis, mechanical fragmentation because of vasculitis or microthrombi, enzymatic destruction by proteases, and oxidative damage. gross and microscopic lesions are similar to those of babesiosis, except that cattle with east coast fever tend not to develop organized by taxonomy (protozoal, bacterial and rickettsial, and viral). babesiosis (piroplasmosis). babesia spp. and theileria spp., presented in the next section, are members of the order piroplasmida, and are generally referenced as piroplasms. these organisms are morphologically similar but have different life cycles; babesia spp. are primarily erythrocytic parasites, whereas theileria spp. sequentially parasitize leukocytes and then erythrocytes. both are protozoan parasites spread by ticks, but other modes of transmission are possible (e.g., biting flies, transplacental, and blood transfusions). evidence is accumulating that dog fighting also transmits babesia gibsoni infection. babesia organisms are typically classified as large (2 to 4 µm) or small (<2 µm) with routine light microscopy ( fig. 13-24 ). over 100 babesia species have been identified, some of which are listed here, along with their relative microscopic size in parentheses: geographic distributions vary with the species, but most have higher prevalences in tropical and subtropical regions. for example, equine and bovine babesiosis are endemic in parts of africa, the middle east, asia, central and south america, the caribbean, and europe. both were eradicated from the united states and are now considered exotic diseases in that country. of the previously mentioned species, only agents of canine babesiosis are thought to be endemic in the united states. fever. anaplasmosis, ehrlichiosis, heartwater, and tick-borne fever are tick-borne diseases caused by small, pleomorphic, gramnegative, obligate intracellular bacteria within the order rickettsiaceae, also colloquially known as rickettsias. as a group, rickettsias primarily infect hematopoietic cells and endothelial cells. rickettsias that predominantly infect endothelial cells (e.g., rickettsia rickettsii [rocky mountain spotted fever]), or cause gastrointestinal disease (e.g., neorickettsia helminthoeca [salmon poisoning disease] and neorickettsia risticii [potomac horse fever]) are discussed elsewhere (see chapters 4 and 7). less commonly, transmission may occur via blood transfusions or blood-contaminated medical supplies. rickettsias that infect erythrocytes include the following species (the disease name follows in parentheses): • cattle-anaplasma marginale, anaplasma centrale (bovine anaplasmosis) • sheep and goats-anaplasma ovis (ovine and caprine anaplasmosis, respectively) anaplasma marginale and a. ovis have worldwide distributions, but a. centrale is mostly restricted to south america, africa, and the middle east. hemolytic anemia. in acute east coast fever, lymph nodes are enlarged, edematous, and hemorrhagic. but with chronic cases they may be shrunken. there is often splenomegaly, hepatomegaly, and hemorrhagic enteritis with white foci of lymphoid infiltrates (pseudoinfarcts) in the liver and kidney. microscopically, infected leukocytes may block capillaries. african trypanosomiasis. trypanosomes are flagellated protozoa that can infect all domesticated animals. the most important species that cause disease are trypanosoma congolense, trypanosoma vivax, and trypanosoma brucei ssp. brucei. disease is most common in parts of africa where the biologic vector, the tsetse fly, exists. however, t. vivax has spread to central and south america and the caribbean, where other biting flies transmit the parasite mechanically. in africa, cattle are mainly affected due to the feeding preferences of the tsetse fly. african trypanosomiasis must be distinguished from nonpathogenic trypanosomiasis, such as trypanosoma theileri infection in cattle. animals become infected when feeding tsetse flies inoculate metacyclic trypanosomes into the skin of animals. the trypanosomes grow for a few days, causing a localized chancre sore, and then sequentially enter the lymph nodes and bloodstream. trypanosomal organisms do not infect erythrocytes but rather exist as free trypomastigotes (i.e., flagellated protozoa with a characteristic undulating membrane) in the blood ( fig. 13 -25, a) or as amastigotes in tissue. the mechanism of anemia is believed to be immune mediated. cattle with acute trypanosomiasis have significant anemia, which initially is regenerative, but less so with time. the extent of parasitemia is readily apparent with t. vivax and t. theileri infections because the organisms are present in large numbers in the blood. this is in contrast to t. congolense, which localizes within the vasculature of the brain and skeletal muscle. chronically infected animals often die secondary to poor body condition, immunosuppression, and concurrent infections. gross examination of animals with acute disease often reveals generalized lymphadenomegaly, splenomegaly, and petechiae on serosal membranes. an acute hemorrhagic syndrome may occur in cattle, resulting in lesions of severe anemia (e.g., pale mucous membranes) and widespread mucosal and visceral hemorrhages. main lesions of chronic infections include signs of anemia, lymphadenopathy (e.g., enlarged or atrophied lymph nodes), emaciation, subcutaneous edema, pulmonary edema, increased fluid in body cavities, and serous atrophy of fat. trypanosoma cruzi is the flagellated protozoal agent of american trypanosomiasis. infections have been reported in more than 100 mammal species in south america, central america, and the southern united states, but dogs and cats are among the more common domestic hosts. infected triatomine insects, or "kissing bugs," defecate as they feed on their mammalian host, releasing infective t. cruzi organisms. the parasite then enters the body through mucous membranes or breaks in the skin. like the other trypanosomes described previously, t. cruzi lives in the blood as extracellular trypomastigotes (see fig. 13 -25, b) and in the tissues as intracellular amastigotes. trypanosoma cruzi primarily causes heart disease. lesions of acute disease include a pale myocardium, subendocardial and subepicardial hemorrhages, and yellowish-white spots and streaks. there may also be secondary lesions, such as pulmonary edema, ascites, and congestion of the liver, spleen, and kidneys. in chronic disease the heart may be enlarged and flaccid with thin walls. microscopically, there is often myocarditis and amastigotes within cardiomyocytes. most of these rickettsias have worldwide distributions. however, e. ewingii has been reported only in the united states, and e. ruminantium is endemic only in parts of africa and the caribbean. although a. phagocytophilum has a wide geographic distribution, strain variants are regionally restricted. for example, a. phagocytophilum causes disease in ruminants in europe, but it has not been documented in ruminants in the united states. reservoirs of disease vary, depending upon the rickettsial species. cattle are the reservoir host for e. ruminantium, canids are the reservoir host for a. platys and e. canis, and the other rickettsias have wildlife reservoirs. pathogenesis of disease involves endothelial cell, platelet, and leukocyte dysfunction. those agents that infect endothelial cells cause vasculitis and increased vascular permeability of small blood vessels. if only plasma is lost, then there is hypotension and tissue edema. however, more severe vasculitis causes microvascular hemorrhage with the potential for platelet consumption thrombocytopenia, disseminated intravascular coagulation, and hypotension. infection of platelets may cause thrombocytopenia by direct platelet lysis, immune-mediated mechanisms, or platelet sequestration within the spleen. pathogenesis of leukocyte dysfunction is unclear, but may involve sepsis, inhibited leukocyte function, endothelial cell activation, and platelet consumption. chronic e. canis infection may cause aplastic anemia with pancytopenia by an unknown mechanism. some studies indicate that german shepherd dogs with ehrlichiosis are predisposed to have particularly severe clinical disease. some breeds of cattle (bos taurus), sheep (merino), and goats (angora and saanen) are more susceptible to heartwater. upon blood smear evaluation, thrombocytopenia is the most common hematologic abnormality; anemia and neutropenia occur less frequently. in early stages of infection, blood cells may contain morulae, which are clusters of rickettsial organisms within cytoplasmic, membrane-bound vacuoles ( fig. 13-27 ). examination of buffy coat smears increases the probability of detecting the organism. chronic infection may cause lymphocytosis, particularly of granular lymphocytes. anaplasma platys causes recurrent marked thrombocytopenia. in general, more common gross lesions are splenomegaly, lymphadenomegaly, and pulmonary edema and hemorrhage. more severe cases may also exhibit multisystemic petechiae, ecchymoses, and bovine anaplasmosis causes anemia mainly by immune-mediated extravascular hemolysis. the severity of disease in infected animals varies with age. infected calves under 1 year of age rarely develop clinical disease, whereas cattle 3 years of age or older are more likely to develop severe, potentially fatal, illness. the reason for this discrepancy is not clear. indian cattle (bos indicus) are more resistant to disease than european cattle (bos taurus). surviving cattle become chronic carriers (and thus reservoirs for infection of other animals) and develop cyclic bacteremia, which is typically not detectable on blood smears. splenectomy of carrier animals results in marked bacteremia and acute hemolysis. pcr testing is the most sensitive means of identifying animals with low levels of bacteremia. grossly, acute disease causes lesions of acute hemolytic anemia, including pale mucous membranes, low blood viscosity, icterus, splenomegaly, hepatomegaly, and a distended gallbladder. in animals with acute disease it is usually easy to detect a. marginale organisms on routine blood smear evaluation ( fig. 13-26 ) or impression smears from cut sections of the spleen. however, in recovering animals, the organisms may be difficult to find. rickettsias that infect leukocytes are broadly divided into those that preferentially infect granulocytes ( in addition to anemia, common findings in animals with leptospirosis-induced hemolysis include hemoglobinuria and icterus. on necropsy, renal tubular necrosis, which occurs in part because of hemoglobinuria (hemoglobinuric nephrosis), may also be present. hemotropic mycoplasmosis (hemoplasmosis). the term hemotropic mycoplasmas, or hemoplasmas, encompasses a group of bacteria, formerly known as haemobartonella or eperythrozoon spp., that infect erythrocytes of many domestic, laboratory, and wild animals. hemotropic mycoplasmas affecting common domestic species are as follows: • cattle-mycoplasma wenyonii • camelids-"candidatus mycoplasma haemolamae" • sheep and goats-mycoplasma ovis • pigs-mycoplasma suis (efig. 13-4) • dogs-mycoplasma haemocanis, "candidatus mycoplasma haematoparvum" • cats-mycoplasma haemofelis, "candidatus mycoplasma haemominutum," "candidatus mycoplasma turicensus" like other mycoplasmas, hemoplasmas are small (0.3 to 3 µm in diameter) and lack a cell wall. they are epicellular parasites, residing in indentations and invaginations of red blood cell surfaces. the mode of transmission is poorly understood, but blood-sucking arthropods are believed to play a role; transmission in utero, through biting or fighting, and transfusion of infected blood products are also suspected. effects of infection vary from subclinical to fatal anemia, depending on the specific organism, dose, and host susceptibility. most hemoplasmas are more likely to cause acute illness in individuals that are immunocompromised or have concurrent disease. however, m. haemofelis is an exception and tends to cause acute hemolytic anemia in immunocompetent cats. anemia occurs mainly because of extravascular hemolysis, but intravascular hemolysis also occurs. although the pathogenic mechanisms are not completely understood, an immune-mediated component is highly probable, as well as direct red blood cell injury by the bacteria and the innocent bystander effect. hemotropic mycoplasmas induce cold agglutinins in infected individuals, although it is not clear whether these particular antibodies are important in the development of hemolytic anemia. when detected on routine blood smear evaluation, the organisms are variably shaped (cocci, small rods, or ring forms) and sometimes arranged in short, branching chains ( fig. 13-28) . the organisms may also be noted extracellularly, in the background of the blood smear, especially if the smear is made after prolonged storage of the blood in an anticoagulant tube. in animals dying of acute hemoplasma infection, the gross findings are typical of extravascular hemolysis, with pallor, icterus, splenomegaly, and distended gallbladder ( fig. 13-29 ). additional lesions documented in cattle include scrotal and hind limb edema and swelling of the teats. microscopic lesions in the red pulp of the spleen include congestion, erythrophagocytosis, macrophage hyperplasia, emh, and increased numbers of plasma cells. bone marrow has varying degrees of erythroid hyperplasia, depending on the duration of hemolysis. immune-mediated hemolytic anemia. immune-mediated hemolytic anemia is a condition characterized by increased destruction of erythrocytes because of binding of immunoglobulin to red blood cell surface antigens. it is a common, life-threatening condition in dogs but also has been described in horses, cattle, and cats. immune-mediated hemolytic anemia may be idiopathic (also called edema, cavitary effusions, and effusive polyarthropathy. hydropericardium gives heartwater its name but is more consistently found in small ruminants than in cattle. chronically infected dogs are emaciated. the bone marrow is hyperplastic and red in the acute disease but becomes hypoplastic and pale in dogs with chronic e. canis infection. equine anaplasmosis is often mild but may cause edema and hemorrhages. disease in cats is rare and poorly documented. histologic findings include generalized perivascular plasma cell infiltration, which is most pronounced in animals with chronic disease. multifocal, nonsuppurative meningoencephalitis, interstitial pneumonia, and glomerulonephritis are present in most dogs with the disease. rickettsial organisms are difficult to detect histologically; examination of wright-giemsa-stained impression smears of lung, liver, lymph nodes, and spleen is a more effective method for detecting the morulae within leukocytes. heartwater is often diagnosed by observing morulae in endothelial cells of giemsastained squash preparations of brain. rickettsial diseases are often diagnosed on the basis of serologic testing, but pcr testing is more sensitive. clostridial diseases. certain clostridium spp. may cause potentially fatal hemolytic anemias in animals; nonhemolytic lesions are presented elsewhere (see chapters 4, 7, 8, and 19) . clostridium haemolyticum and clostridium novyi type d cause the disease in cattle known as bacillary hemoglobinuria. (the phrase "red water" has also been used for this disease and for hemolytic anemias in cattle caused by babesia spp.) similar naturally occurring disease has been reported in sheep. in cattle the disease is caused by liver fluke (fasciola hepatica) migration in susceptible animals. ingested clostridial spores may live in kupffer cells for a long time without causing disease. however, when migrating flukes cause hepatic necrosis, the resulting anaerobic environment stimulates the clostridial organisms to proliferate and elaborate their hemolytic toxins, causing additional hepatic necrosis. the mechanism of hemolysis involves a bacterial β-toxin (phospholipase c or lecithinase), which enzymatically degrades cell membranes, causing acute intravascular hemolysis. bacillary hemoglobinuria also occurs with liver biopsies in calves. clostridium perfringens type a causes intravascular hemolytic anemia in lambs and calves-a condition known as yellow lamb disease, yellows, or enterotoxemic jaundice because of the characteristic icterus. the organism is a normal inhabitant of the gastrointestinal tract in these animals but may proliferate abnormally in response to some diets. c. perfringens causes intravascular hemolytic anemia in horses with clostridial abscesses, and clostridial mastitis in ewes. c. perfringens type a produces hemolytic α-toxin, which also has phospholipase c activity. leptospirosis. leptospirosis is recognized as a cause of hemolytic anemia in calves, lambs, and pigs. specific leptospiral organisms that cause hemolytic disease include leptospira interrogans serovars pomona and ictohaemorrhagiae. leptospira organisms are ubiquitous in the environment. infection occurs percutaneously and via mucosal surfaces and is followed by leptospiremia; organisms then localize preferentially in certain tissues (e.g., kidney, liver, and pregnant uterus). proposed mechanisms of hemolytic disease include immune-mediated (immunoglobulin m [igm] cold agglutinin) extravascular hemolysis and enzymatic (phospholipase produced by the organism) intravascular hemolysis. leptospirosis can also cause many disease manifestations besides hemolysis (e.g., renal failure, liver failure, abortion, and other conditions) that are not discussed here. primary immune-mediated hemolytic anemia or autoimmune hemolytic anemia) or secondary to a known initiator, termed secondary immune-mediated hemolytic anemia. although the cause of idiopathic immune-mediated hemolytic anemia is unknown, certain dog breeds (e.g., cocker spaniels) are predisposed to developing disease, suggesting the possibility of a genetic component. causes of secondary immune-mediated hemolytic anemia include certain infections (e.g., hemoplasmosis, babesiosis, and theileriosis), drugs (e.g., cephalosporins, penicillin, and sulfonamides), vaccines, and envenomations (e.g., bee stings). immune-mediated hemolysis directed at nonself antigens, such as in neonatal isoerythrolysis, is presented later. in most cases of idiopathic immune-mediated hemolytic anemia, the reactive antibody is igg, and the hemolysis is extravascular (i.e., erythrocytes with surface-bound antibody are phagocytized by macrophages, mainly in the spleen). igm and/or complement proteins may also contribute to idiopathic immune-mediated hemolytic anemia. complement factor c3b usually acts as an opsonin that promotes phagocytosis and extravascular hemolysis. however, formation of the complement membrane attack complex on red blood cell surfaces causes intravascular hemolysis; this mechanism more commonly occurs with igm autoantibodies. most immunoglobulins implicated in immune-mediated hemolytic anemia are reactive at body temperature (warm hemagglutinins). a smaller portion, usually igm, are more reactive at lower temperatures, young (hours to days old) with typical gross and microscopic changes of immune-mediated hemolytic anemia. pure red cell aplasia. pure red cell aplasia (prca) is a rare bone marrow disorder characterized by absence of erythropoiesis and severe nonregenerative anemia. primary and secondary forms of pure red cell aplasia have been described in dogs and cats. primary pure red cell aplasia is apparently caused by immune-mediated destruction of early erythroid progenitor cells, a presumption supported by the response of some patients to immunosuppressive therapy and by the detection of antibodies inhibiting erythroid colony formation in vitro in some dogs. administration of recombinant human erythropoietin (rhepo) has been identified as a cause of secondary pure red cell aplasia in dogs, cats, and horses, presumably caused by induction of antibodies against rhepo that cross-react with endogenous epo. experimentation with the use of speciesspecific recombinant epo has produced mixed results. dogs treated with recombinant canine epo have not developed pure red cell aplasia. however, in experiments reported thus far involving cats treated with recombinant feline epo, at least some animals have developed pure red cell aplasia. parvoviral infection has been suggested as a possible cause of secondary pure red cell aplasia in dogs. infection with felv subgroup c causes secondary erythroid aplasia in cats, probably because of infection of early-stage erythroid precursors. grossly, animals with pure red cell aplasia have pale mucous membranes without indicators of hemolysis (e.g., icterus). microscopic examination of the bone marrow shows an absence or near absence of erythroid precursors with or without lymphocytosis, plasmacytosis, and myelofibrosis; production of other cell lines (e.g., neutrophils and platelets) is normal or hyperplastic. immune-mediated neutropenia. immune-mediated neutropenia is a rare condition that has been reported in horses, dogs, and cats. this disease is characterized by severe neutropenia from immune-mediated destruction of neutrophils or their precursors. the range of causes is presumably similar to that of other immunemediated cytopenias (e.g., immune-mediated hemolytic anemia, pure red cell aplasia, and immune-mediated thrombocytopenia). affected animals may have infections, such as dermatitis, conjunctivitis, or vaginitis, which are secondary to marked neutropenia and a compromised innate immune system. microscopically, there may be neutrophil hyperplasia, maturation arrest, or aplasia in the bone marrow, depending on which neutrophil maturation stage is targeted causing a condition known as cold hemagglutinin disease. this results in ischemic necrosis at anatomic extremities (e.g., tips of the ears), where cooling of the circulation causes autoagglutination of erythrocytes and occlusion of the microvasculature. typically immunemediated hemolytic anemia targets mature erythrocytes, causing a marked regenerative response. however, as discussed earlier in the chapter, immune-mediated destruction of immature erythroid cells in the bone marrow may also occur, resulting in nonregenerative anemia. pathogenesis of secondary immune-mediated hemolytic anemia is dependent upon the cause. erythrocytic parasites may cause immune-mediated hemolysis by altering the red blood cell surface and exposing "hidden antigens" that are not recognized as selfantigens by the host's immune system. alternatively, the immune attack may be directed at the infectious agent, but erythrocytes are nonspecifically destroyed because of their close proximity-this is called the innocent bystander mechanism. certain drugs, such as penicillin, may cause immune-mediated hemolytic anemia by binding to erythrocyte membranes and forming drug-autoantigen complexes that induce antibody formation, termed hapten-dependent antibodies. other proposed mechanisms include binding of drugantibody immune complexes to the erythrocyte membrane, or induction of a true autoantibody directed against an erythrocyte antigen. hematologic, gross, and histopathologic abnormalities are typical of those of hemolytic anemia, as presented in the earlier section on bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in brief, there may be spherocytes and autoagglutination on blood smear evaluation, icterus and splenomegaly on gross examination, and emh, erythrophagocytic macrophages, and hypoxia-induced or thromboemboli-induced tissue necrosis on histopathologic examination. dogs with immune-mediated hemolytic anemia also frequently develop an inflammatory leukocytosis and coagulation abnormalities (prolonged coagulation times, decreased plasma antithrombin concentration, increased plasma concentration of fibrin degradation products, thrombocytopenia, and disseminated intravascular coagulation). intravascular hemolysis plays a relatively insignificant role in most cases of immune-mediated hemolytic anemia, but evidence of intravascular hemolysis (e.g., ghost cells, red plasma and urine, dark red kidneys) is noted occasionally, presumably in those cases in which igm and complement are major mediators of hemolysis. neonatal isoerythrolysis. neonatal isoerythrolysis (ni) is a form of immune-mediated hemolytic anemia in which colostrumderived maternal antibodies react against the newborn's erythrocytes. it is common in horses ( fig. 13-30 ) and has been reported in cattle, cats, and some other domestic and wildlife species. in horses, neonatal isoerythrolysis occurs as a result of immunosensitization of the dam from exposure to an incompatible blood type inherited from the stallion (e.g., transplacental exposure to fetal blood during pregnancy or mixing of maternal and fetal blood during parturition). a previously mismatched blood transfusion produces the same results. some equine blood groups are more antigenic than others; in particular, types aa and qa are very immunogenic in mares. in cattle, neonatal isoerythrolysis has been caused by vaccination with whole blood products or products containing erythrocyte membrane fragments. neonatal isoerythrolysis has been produced experimentally in dogs, but there are no reports of naturally occurring disease. in cats the recognized form of neonatal isoerythrolysis does not depend on prior maternal immunosensitization but on naturally occurring anti-a antibodies in queens with type b blood. affected animals are s l rather a secondary complication of many types of underlying disease, including severe inflammation, organ failure, and neoplasia. it is included in the section on inflammatory disorders because the coagulation cascade is closely linked to inflammatory pathways. information on this topic, including efig. 13 -5, is available at www.expertconsult.com. as well as in chapter 2. the term hematopoietic neoplasia encompasses a large and diverse group of clonal proliferative disorders of hematopoietic cells. historically, numerous systems have been used to classify hematopoietic neoplasms in human medicine, some of which have been applied inconsistently to veterinary species (examples include the kiel classification and national cancer institute working formulation). the world health organization (who) classification of hematopoietic neoplasia was first published in 2001 (updated in 2008) and is based on the principles defined in the revised european-american classification of lymphoid neoplasms (real) from the international lymphoma study group. the who classification system is considered the first true worldwide consensus on the classification of hematopoietic malignancies and integrates information on tumor topography, cell morphology, immunophenotype, genetic features, and clinical presentation and course. a veterinary reference of the who classification system, published in 2002, was later validated in 2011 using the canine model of lymphoma. this project, modeled after the study to validate the system in human beings, yielded an overall accuracy (i.e., agreement on a diagnosis) among pathologists of 83%. currently this classification system is accepted as the method of choice in both human and veterinary medicine. the who classification broadly categorizes neoplasms primarily according to cell lineage: myeloid, lymphoid, and histiocytic. this distinction is based on the fact that the earliest commitment of a pluripotent hsc is to either a lymphoid or nonlymphoid lineage. many pathologists and clinicians distinguish leukemias from other hematopoietic neoplasms. leukemia refers to a group of hematopoietic neoplasms that arise from the bone marrow and are present within the blood. leukemia may be difficult to differentiate from other forms of hematopoietic neoplasms that originate outside of the bone marrow but infiltrate the bone marrow and blood. for simplicity, cases of secondary bone marrow or blood involvement may not be considered leukemia but rather the "leukemic phase" of another primary neoplasm. it is now recognized that certain lymphomas and leukemias are different manifestations of the same disease (e.g., chronic lymphocytic leukemia and small lymphocytic lymphoma), and the designation of lymphoma or leukemia is placed on the tissue with the largest tumor burden. based on their degree of differentiation, leukemias are classified as acute or chronic. acute leukemias are poorly differentiated or undifferentiated, meaning that there are high percentages of early progenitor and precursor cells, including lymphoblasts, myeloblasts, monoblasts, erythroblasts, and/or megakaryoblasts. in contrast, well-differentiated cells predominate in chronic leukemias. because well-differentiated cells also predominate with nonneoplastic proliferations, chronic leukemias must be differentiated from reactive processes, such as those cells that occur in chronic and/or granulomatous inflammation. diagnosis of chronic leukemia is often made by excluding all other causes for the proliferating cell type. for example, causes of relative and secondary erythrocytosis are excluded to be able to diagnose polycythemia vera. furthermore, the designation of acute or chronic also refers to the disease's clinical course. acute leukemias tend to have an acute onset of severe and rapidly progressive clinical signs, whereas animals with chronic leukemia for destruction. marrow lymphocytosis and plasmacytosis may be marked (e.g., >60% of nucleated cells). the diagnosis may be supported by flow cytometric detection of immunoglobulin bound to neutrophils but is most often made on the basis of exclusion of other causes of neutropenia and response to immunosuppressive therapy. immune-mediated thrombocytopenia. immune-mediated thrombocytopenia (imtp) is a condition characterized by immunemediated destruction of platelets. it is a fairly common condition in dogs and is less frequent in horses and cats. the disease is usually idiopathic but may be secondary to infection (e.g., equine infectious anemia and ehrlichiosis), drug administration (e.g., cephalosporins and sulfonamides), neoplasia, and other immunemediated diseases. when immune-mediated thrombocytopenia occurs together with immune-mediated hemolytic anemia, the condition is called evans's syndrome. the thrombocytopenia is often severe (e.g., < 20,000 platelets/µl), resulting in varying degrees of bleeding tendencies, mainly in skin and mucous membranes. microscopically, there are multifocal perivascular hemorrhages in multiple tissues, and the bone marrow exhibits megakaryocytic and erythroid hyperplasia. rarely, immune-mediated destruction of megakaryocytes may cause megakaryocytic hypoplasia, termed amegakaryocytic thrombocytopenia. neonatal alloimmune thrombocytopenia. a form of immune-mediated thrombocytopenia, known as neonatal alloimmune thrombocytopenia, is recognized in neonatal pigs and foals. the pathogenesis of this disease is virtually identical to that of neonatal isoerythrolysis as a cause of anemia: a neonate inheriting paternal platelet antigens absorbs maternal antibodies against these antigens through the colostrum. in principle, a similar situation may occur after platelet-incompatible transfusion of blood or blood products containing platelets. gross and microscopic changes are similar to those of immune-mediated thrombocytopenia except that the animal is young (e.g., 1 to 3 days). hemophagocytic syndrome. hemophagocytic syndrome is a term used to describe the proliferation of nonneoplastic (i.e., polyclonal), well-differentiated but highly erythrophagic macrophages. the condition is rare but has been recognized in dogs and cats. unlike hemophagocytic histiocytic sarcoma, which is a neoplastic proliferation of phagocytic macrophages, hemophagocytic syndrome is secondary to an underlying disease, such as neoplasia, infection, or an immune-mediated disorder. the primary disease process causes increased production of stimulatory cytokines, which results in macrophage proliferation and hyperactivation. these activated macrophages phagocytize mature hematopoietic cells and hematopoietic precursors at an enhanced rate, resulting in one or more cytopenias. affected animals usually have lesions of the primary disease, as well as signs of the anemia (e.g., pale mucous membranes), neutropenia (e.g., bacterial infections), and thrombocytopenia (e.g., petechiae and ecchymoses). microscopically, phagocytic macrophages are found in high numbers in the bone marrow and commonly in other tissues, including lymph nodes, spleen, and liver. additional bone marrow findings reported in animals with hemophagocytic syndrome vary widely, ranging from hypoplasia to hyperplasia of cell lines with peripheral cytopenias. disseminated intravascular coagulation. disseminated intravascular coagulation is a syndrome characterized by continuous activation of both coagulation and fibrinolytic pathways and is also known as consumptive coagulopathy. it is not a primary disease, but 754.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system disseminated intravascular coagulation is a consumptive coagulopathy resulting from activation of both coagulation and fibrinolytic pathways. it is a secondary complication of many types of underlying disease, including many infectious diseases, trauma, burns, heat stroke, immune-mediated disease, hemolysis, shock, neoplasia, organ failure, obstetric complications, and noninfectious inflammatory disease, such as pancreatitis. it is common in critically ill domestic animals. disseminated intravascular coagulation involves an initial hypercoagulable phase, resulting in thrombosis and ischemic tissue damage, and a subsequent hypocoagulable phase as a result of consumption of coagulation factors and platelets, resulting in hemorrhage (efig. 13-5) . the pathogenesis of disseminated intravascular coagulation typically involves the release of tissue factor (thromboplastin) and subsequent activation of coagulation pathways and platelets but may also involve defective normal inhibition of coagulation or defective fibrinolysis. classically diagnosis of disseminated intravascular coagulation is based on clinical evidence of hemorrhage and/or thromboembolic disease and a triad of laboratory findings: thrombocytopenia, usually moderate (below the lower reference value but above 50,000/µl); prolonged coagulation times (prothrombin time and/or partial thromboplastin time); and decreased fibrinogen or increased concentration of plasma fibrin degradation products or d-dimer. milder forms of disseminated intravascular coagulation that do not meet all of the diagnostic criteria also occur. decreased plasma antithrombin (antithrombin iii) concentration and schistocytosis are other laboratory abnormalities often found in patients with disseminated intravascular coagulation. dysplasia of myeloid cells, and fewer than 20% myeloblasts and "blast equivalents." 3 acute myeloid leukemia. acute myeloid leukemia (aml) is uncommon in domestic animals but most frequently occurs in dogs and cats. in veterinary species, acute myeloid leukemia is most commonly of neutrophil, monocyte, and/or erythroid origin, with rare reports of eosinophil, basophil, or megakaryocytic lineages. it is caused by felv infection in cats. evaluations of blood smears show many early myeloid precursors, including myeloblasts and blast equivalents ( fig. 13-31, a) . in dogs the total leukocyte concentration averages approximately 70,000/µl; anemia, neutropenia, and thrombocytopenia commonly occur. grossly, animals show lesions attributed to anemia, neutropenia, and thrombocytopenia, such as pale mucous membranes, secondary infections, and multisystemic bleeding, respectively. neoplastic cells often infiltrate tissues, resulting in splenomegaly, hepatomegaly, and lymphadenomegaly. microscopically, myeloid cells efface (replace) the bone marrow and infiltrate extramedullary tissues, especially lymphoid tissue. chronic myeloid leukemia. chronic myeloid leukemia (cml), also called chronic myelogenous leukemia or myeloproliferative neoplasia, is rare in animals. most reported cases occur in dogs and cats. there are various subclassifications of chronic myeloid leukemia, including excessive production of erythrocytes (polycythemia vera), platelets (essential thrombocythemia), neutrophils (chronic neutrophilic leukemia), monocytes (chronic monocytic leukemia), neutrophils and monocytes (chronic myelomonocytic leukemia), eosinophils (chronic eosinophilic leukemia), or basophils (chronic basophilic leukemia). complete peripheral blood count analysis often reveals very high concentrations of the neoplastic cells, such as greater than 50,000 to 100,000 leukocytes/µl (see fig. 13-31, b) or 2,000,000 platelets/µl. cellular morphologic features are often normal, but slight dysplasia may be observed. later in the disease there may be cytopenias of nonneoplastic cell types. animals with polycythemia vera often have red mucous membranes and lesions of hyperviscosity syndrome, such as bleeding and dilated, tortuous retinal vessels. essential thrombocythemia results in multisystemic bleeding due to dysfunctional platelets, or multisystemic infarcts from hyperaggregability and excessive platelets. chronic myeloid leukemias of leukocytes often result in splenomegaly, hepatomegaly, and lymphadenomegaly because of infiltration by the neoplastic cells. histologically, the bone marrow shows proliferation of the neoplastic cell type characterized by dysplasia and low numbers (e.g., <20%) of myeloblasts and blast equivalents. mast cell neoplasia. mast cell tumors (mcts) of the skin and other sites are common in animals (see chapters 6, 7, and 17), but mast cell leukemia is rare. in cats, mcts are the most common neoplasm in the spleen (efig. 13-6) . mast cells normally are not present in the blood vascular system, but the finding of mast cells in the blood (mastocytemia) is highly suggestive of disseminated mast cell neoplasia (systemic mastocytosis) in cats. however, mastocytemia does not necessarily indicate myeloid neoplasia in dogs. in fact, one study found that the severity of mastocytemia in dogs was frequently higher in animals without mcts than those with mcts and that random detection of mast cells in blood smears usually is not the result of underlying mct. granulocytic sarcoma. granulocytic sarcoma is a poorly characterized extramedullary proliferation of myeloid precursors, most typically have indolent, slowly progressive disease. this classification scheme is summarized in table 13 -4. subcategories exist within each of these groups, as discussed further later. information on this topic is available at www.expertconsult.com. this section discusses examples of myeloid neoplasms, including myelodysplastic syndrome, myeloid leukemias, and mast cell neoplasms (technically a form of myeloid neoplasia), and lymphoid neoplasms, including lymphoid leukemias and multiple myeloma. other lymphoid neoplasms, such as the numerous subtypes of lymphoma and extramedullary plasmacytomas (emps), as well as histiocytic disorders are described in the section on lymphoid/lymphatic system, disorders of domestic animals, neoplasia. additional discussion of hematopoietic neoplasia occurs in the species-specific sections at the end of this chapter. myelodysplastic syndrome. myelodysplastic syndrome (mds) most commonly occurs in dogs and cats and may be caused by felv infection in cats. the disease refers to a group of clonal myeloid proliferative disorders with ineffective hematopoiesis in the bone marrow, resulting in cytopenias of more than one cell line. hematopoietic proliferation in bone marrow with concurrent peripheral blood cytopenias is likely a result of increased apoptosis of neoplastic cells within the bone marrow, before their release into circulation. clinical illness and death often result from secondary manifestations, such as secondary infections or cachexia, attributable to the effects of cytopenias and/or transformation of the neoplasm into acute myeloid leukemia. gross lesions are dependent upon the type and severity of the cytopenias. however, essential microscopic findings within the bone marrow are normal or increased cellularity, 3 "blast equivalents" include other stages of immature myeloid cells, such as abnormal promyelocytes, monoblasts, promonocytes, erythroblasts, and megakaryoblasts. before the discussion of specific diseases, it is worthwhile to describe the diagnostic techniques required to classify hematopoietic neoplasms that are becoming increasingly available for routine use in veterinary medicine. immunophenotyping refers to the use of antibodies recognizing specific molecules expressed on different cell types to determine the identity of a cell population of interest. immunophenotyping on the basis of these lineage-specific or lineage-associated markers can be performed on histologic sections (immunohistochemistry [see fig. 13 -86]), air-dried cytologic examination smears (immunocytochemistry), or by laser analysis of cells in suspension in blood or buffer solutions (flow cytometry). in cases of lymphoid neoplasia, immunophenotyping most routinely refers to determination of b or t lymphocyte origin. clonality assays, pcr for antigen receptor rearrangement (parr), can help identify neoplastic lymphoid proliferations on the basis of clonal rearrangements of genes encoding lymphocyte antigen receptors. in terms of practical application the parr assay is most useful in helping to distinguish lymphoid neoplasms from those nonneoplastic lymphoid proliferations mimicking neoplasia. cytogenetic testing has not been routinely used in veterinary medicine, though several genetic mutations have been identified in dogs. for example, breakpoint cluster region-abelson (bcr-abl) translocations have been identified in some canine leukemias, including acute myeloblastic leukemia and chronic monocytic leukemia. dogs with burkitt-like lymphoma have a translocation leading to constitutive c-myc expression. a b thrombocytopenia commonly occur. gross and microscopic lesions also are similar to those that occur in cases of acute myeloid leukemia, except that neoplastic cells may differentiate into morphologically identifiable lymphoid cells. chronic lymphocytic leukemia. chronic lymphocytic leukemia (cll) is uncommon in veterinary medicine. it is predominantly a disease of middle-aged to older dogs but is also documented in horses, cattle, and cats. most canine chronic lymphocytic leukemia cases are of t lymphocyte origin, typically cytotoxic t lymphocytes expressing cd8. in cats the majority of chronic lymphocytic leukemia cases have a t helper lymphocyte immunophenotype. a cbc often shows very high numbers of small lymphocytes with clumped chromatin and scant cytoplasm. proliferating cytotoxic t lymphocytes frequently contain a few pink cytoplasmic granules when stained with most methanol-based romanowsky stains (e.g., wright-giemsa). however, these granules may not be appreciated with some aqueous-based romanowsky stains (e.g., diff-quik). although the number of total blood lymphocytes is often greater than 100,000/µl, relatively mild lymphocytosis (e.g., 15,000/µl) has been reported. seventy-five percent of affected dogs also have often of eosinophilic or neutrophilic cell lines. although rare, there are reports of granulocytic sarcoma in dogs, cats, cattle, and pigs, and it may arise in a number of sites, such as lung, intestine, lymph nodes, liver, kidney, skin, and muscle. acute lymphoblastic leukemia. acute lymphoblastic leukemia (all) is uncommon in dogs and cats, and rare in horses and cattle. in a recent immunophenotype study of 51 cases of acute lymphoblastic leukemia in dogs, 47 arose from b lymphocytes and 4 arose from double-negative t lymphocytes that were immunonegative for cd4 and cd8 markers. in the blood of animals with acute lymphoblastic leukemia, there are typically many medium to large lymphoid cells with deeply basophilic cytoplasm, reticular to coarse chromatin, and prominent, multiple nucleoli (see fig. 13-31, c) . in affected dogs the mean blood lymphoid concentration is approximately 70,000/µl, but cats with acute lymphoblastic leukemia often have low numbers of neoplastic cells in the circulation. as with animals with acute myeloid leukemia, anemia, neutropenia, and c d m the light chains deposit as nonamyloid granules, it is termed light chain deposition disease. light chains are low-molecular-weight proteins that pass through the glomerular filter into the urine, wherein they are also known as bence jones proteins. they tend to not react with urine dipstick protein indicators and are most specifically detected by electrophoresis and immunoprecipitation. in addition to aiding in the diagnosis of multiple myeloma, paraproteins have an important role in pathogenesis of disease. these proteins may inhibit platelet function, increase blood viscosity, deposit in glomerular basement membranes (see chapter 11; see figs. 11-27 and 11-28, or precipitate at cool temperatures, which results in bleeding tendencies, hyperviscosity syndrome, glomerulopathies, and cryoglobulinemia, respectively. hyperviscosity syndrome refers to the clinical sequelae of pathologically increased blood viscosity, which are slowed blood flow and loss of laminar flow. clinical signs include mucosal hemorrhages, visual impairment due to retinopathy, and neurologic signs, such as tremors and abnormal aggressive behavior. cryoglobulinemia is the condition in which proteins, typically igm, precipitate at temperatures below normal body temperature (cold agglutinins). precipitation often occurs in blood vessels of the skin and extremities, such as the ears and digits, and results in ischemic necrosis. in multiple myeloma the neoplastic proliferation of plasma cells results in osteolysis. work with human cell cultures has shown that anemia, and 15% have thrombocytopenia. autopsy findings depend on the stage of disease. in advanced cases with marked infiltration of organs with neoplastic cells, there is often uniform splenomegaly, hepatomegaly, and lymphadenomegaly, and the bone marrow is highly cellular (efig. 13-7 ; see fig. 13-31, d) . other lesions depend on whether there are concurrent cytopenias, such as anemia, neutropenia, and thrombocytopenia, and if the neoplastic cells produce excessive immunoglobulin. lesions caused by excessive immunoglobulin are further discussed in the section on multiple myeloma. histologically, the bone marrow is densely cellular with welldifferentiated lymphocytes. small lymphocytes infiltrate and often efface in the architecture of the lymph nodes and spleen. the liver may have dense accumulations of neoplastic cells in the connective tissue around the portal triad. plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone; the latter is discussed in the section on lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia. multiple myeloma. multiple myeloma (mm) is a rare, malignant tumor of plasma cells that arises in the bone marrow and usually secretes large amounts of immunoglobulin. the finding of neoplastic plasma cells in blood samples or smears is rare. dogs are affected more frequently than other species, but multiple myeloma has also been reported in horses, cattle, cats, and pigs. diagnosis of multiple myeloma is based on finding a minimum of two or three (opinions vary) of the following abnormalities: • markedly increased numbers of plasma cells in the bone marrow ( fig. 13-32 , a) • monoclonal gammopathy • radiographic evidence of osteolysis • light chain proteinuria the classic laboratory finding in patients with multiple myeloma is hyperglobulinemia, which results from the excessive production of immunoglobulin or an immunoglobulin subunit by the neoplastic cells. this homogeneous protein fraction is often called paraprotein or m protein. paraproteins produced from the same clone of plasma cells have the same molecular weight and electric charge. therefore they have the same migration pattern using serum protein electrophoresis, which results in a tall, narrow spike in the globulins region, termed monoclonal gammopathy (see fig. 13-32, b) . the term gammopathy is used because most immunoglobulins migrate in the γ-region of an electrophoresis gel. however, some immunoglobulins, especially immunoglobulin a (iga) and igm, migrate to the β-region. occasionally, biclonal or other atypical electrophoretic patterns may be seen with multiple myeloma as a result of protein degradation, protein complex formation, binding to other proteins, or when the tumor includes more than one clonal population. it is important to note that monoclonal gammopathy is not specific to multiple myeloma but has also been reported with lymphoma, chronic lymphocytic leukemia, canine ehrlichiosis, and canine leishmaniasis. definitively distinguishing monoclonal from polyclonal gammopathy requires immunoelectrophoresis or immunofixation using species-specific antibodies recognizing different immunoglobulin subclasses and subunits. occasionally, multiple myeloma cells produce only the immunoglobulin light chain. an immunoglobulin monomer consists of two heavy chains and two light chains connected by disulfide bonds. these light chains may deposit in tissues and cause organ dysfunction, especially renal failure. when the light chains form amyloid deposits, the disease is called amyloid light chain amyloidosis. but if marrow is dark red as a result of replacement of fat by hematopoietic tissue; the extent of replacement is an indication of the duration of the anemia. the severity of microscopic lesions is dependent on the chronicity of the disease, and they are most significant in the spleen, liver, and bone marrow. as would be anticipated, microscopic findings of the spleen are predominantly influenced by the number and activity of macrophages, which is a reflection of the duration of the disease and the frequency of hemolytic episodes. hemosiderin-laden macrophages persist for months to years; therefore large numbers are consistent with chronicity. kupffer cell hyperplasia with hemosiderin stores and periportal infiltrates of lymphocytes are the most significant changes in the liver. bone marrow histologic findings vary depending on the duration of the disease. in most animals the marrow is cellular because of the replacement of fat by intense, orderly erythropoiesis. granulocytes are relatively less numerous, and plasma cells are increased. as in the spleen, hemosiderin-laden macrophages are present in large numbers in chronic cases. emaciated animals with chronic disease have serous atrophy of fat (see efig. 13-1) . clinical findings with viremic episodes include fever, depression, icterus, petechial hemorrhages, lymph node enlargement, and dependent edema. equine infectious anemia infection is diagnosed on the basis of the coggins test, an agarose gel immunodiffusion test for the presence of the antibody against the virus. congenital dyserythropoiesis in polled herefords. a syndrome of congenital dyserythropoiesis and alopecia occurs in polled hereford calves. the cause and pathogenesis of this often fatal disease are unknown. early in disease there is hyperkeratosis and alopecia of the muzzle and ears, which progresses to generalized alopecia and hyperkeratotic dermatitis. histologically, there is orthokeratotic hyperkeratosis with dyskeratosis, as well as erythroid hyperplasia, dysplasia, and maturation arrest in the bone marrow. ineffective erythropoiesis results in nonregenerative to poorly regenerative anemia. erythrocyte band 3 is integral membrane protein that connects to the cytoskeleton and aids in erythrocyte stability. a hereditary deficiency of this protein has been identified in japanese black cattle, resulting in increased erythrocyte fragility, spherocytosis, intravascular hemolytic anemia, and retarded growth. affected calves show lesions consistent with hemolytic anemia, including pale mucous membranes, icterus, and splenomegaly. histologically, there are bilirubin accumulations in the liver, and hemosiderin in renal tubules. bovine leukemia virus. bovine leukemia virus is discussed in the later section on lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). bovine viral diarrhea virus. bvdv infection may cause thrombocytopenia in cattle, and a thrombocytopenic hemorrhagic syndrome has been specifically caused by type ii bvdv infection. investigations of the mechanism of bvdv-induced thrombocytopenia have resulted in varying, sometimes conflicting, conclusions. more than one study has shown viral antigen within bone marrow megakaryocytes and circulating platelets. evidence of impaired thrombopoiesis (megakaryocyte necrosis, megakaryocyte pyknosis, osteoclasts support the growth of myeloma cells, and that direct contact between the two cell types increases the myeloma cell proliferation and promotes osteoclast survival. increased osteoclast activity causes osteolysis, but the exact mechanism is not known. osteolysis often results in bone pain, lytic bone lesions on radiographs, hypercalcemia, and increased serum alkaline phosphatase activity. later in disease, osteolysis may cause pathologic fractures. morphologically, myeloma cells tend to grow in sheets that displace normal hematopoietic cells in the bone marrow. a proposed diagnostic criterion of multiple myeloma is that plasma cells constitute 30% or more of the nucleated cells in the marrow. welldifferentiated plasma cells are round with abundant basophilic cytoplasm (due to increased rough endoplasmic reticulum) and a perinuclear pale zone (enlarged golgi apparatus for the production of immunoglobulin); anisocytosis and anisokaryosis are often mild but may be marked. some plasma cell neoplasms have a bright eosinophilic fringe due to accumulated iga (see fig. 13-32, a) . nuclei are round with clumped chromatin and often peripherally placed with the cytoplasm; binucleation and multinucleation are common. poorly differentiated myeloma cells may lack and/or display less characteristic features. osteolysis of bone may be present microscopically. common sites of metastasis include the spleen, liver, lymph nodes, and kidneys. flavin adenine dinucleotide deficiency. flavin adenine dinucleotide (fad) is a cofactor for cytochrome-b 5 reductase, the enzyme that maintains hemoglobin in its functional reduced state, and for glutathione reductase, an enzyme that also protects erythrocytes from oxidative damage. reported in a spanish mustang mare and a kentucky mountain saddle horse gelding, erythrocyte fad deficiency is a result of an abnormal riboflavin kinase reaction, which is the first reaction in converting riboflavin to fad. clinicopathologic changes include persistent methemoglobinemia of 26% to 46%, eccentrocytosis, a slightly decreased or normal hematocrit, and erythroid hyperplasia in the bone marrow. equine infectious anemia virus. equine infectious anemia virus (eiav), the agent of equine infectious anemia, is a lentivirus that infects cells of the monocyte-macrophage system in horses (also ponies, donkeys, and mules). the virus is mechanically transmitted by biting flies, such as horseflies and deer flies. less common routes of transmission include blood transfusions, contaminated medical equipment, and transplacentally. disease may present in acute, subacute, and chronic forms and is potentially fatal. after an acute period of fever, depression, and thrombocytopenia that lasts 1 to 3 days, there is a prolonged period of recurrent fever, thrombocytopenia, and anemia. in most cases, clinical disease subsides within a year, and horses become lifelong carriers and reservoirs of eiav. eiav causes anemia by both immune-mediated hemolysis and decreased erythropoiesis. hemolysis is typically extravascular but may have an intravascular component during the acute phase. decreased erythropoiesis may result from direct suppression of earlystage erythroid cells by the virus, as well as anemia of inflammation. thrombocytopenia likely results from immune-mediated platelet destruction and suppressed platelet production. animals dying during hemolytic crises are pale with mucosal hemorrhages and dependent edema. the spleen and liver are enlarged, dark, and turgid, and they and other organs have superficial subcapsular hemorrhages. petechiae are evident beneath the renal capsule and throughout the cortex and medulla. the bone affected animals are not necessarily anemic. however, acute intravascular hemolytic episodes may occur with hyperventilationinduced alkalemia. lesions are typical of hemolytic anemia and include pale mucous membranes, icterus, hepatosplenomegaly, and dark red urine with microscopic emh and marrow erythroid hyperplasia. a single dna-based test is available to detect the common mutation. erythrocyte structural abnormalities. congenital erythrocyte structural abnormalities may occur with abnormal membrane composition or defective proteins within the membrane or cytoskeleton. some of these morphologic changes occur concurrently with clinical disease, but others do not. hereditary stomatocytosis is recognized in alaskan malamutes, drentse patrijshonds, and schnauzers. the specific defects are not known, but they are likely different in the various dog breeds. however, all affected dogs have stomatocytes on blood smear evaluation, as identified by their slit-shaped area of central pallor. erythrocytes also have increased osmotic fragility and decreased survival. schnauzers are clinically healthy and not anemic but do have reticulocytosis, suggesting that the hemolytic anemia is compensated by erythroid hyperplasia. mild to marked hemolytic anemia is documented in alaskan malamutes and drentse patrijshonds. alaskan malamutes have concurrent short-limb dwarfism, and drentse patrijshonds have hypertrophic gastritis and polycystic kidney disease. other (presumably heritable) erythrocyte abnormalities in dogs that do not have clinical signs include elliptocytosis caused by band 4.1 deficiency or β-spectrin mutation, and familial macrocytosis and dyshematopoiesis in poodles. scott's syndrome. an inherited thrombopathy resembling scott's syndrome in human beings, in which platelets lack normal procoagulant activity, has been recognized in a family of german shepherd dogs. the specific defect in these dogs has not been identified on the molecular level but involves impaired expression of phosphatidylserine on the platelet surface. affected dogs have a mild to moderate clinical bleeding tendency characterized by epistaxis, hyphema, intramuscular hematoma formation, and increased hemorrhage with surgery. macrothrombocytopenia. macrothrombocytopenia is an inherited condition in cavalier king charles spaniels in which there are lower than normal concentrations of platelets with enlarged and giant platelets. the condition is caused by defective β 1 -tubulin, which results in impaired microtubule assembly. affected dogs are asymptomatic but may have abnormal platelet aggregation in vitro. canine distemper. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells and is presented in greater detail in the lymphoid section. canine distemper virus may also infect other hematopoietic cells, including erythrocytes, nonlymphoid leukocytes, and platelets ( fig. 13-33) , and can cause decreased peripheral blood concentrations of neutrophils, lymphocytes, monocytes, and platelets during viremia. the thrombocytopenia is a result of virus-antibody immune complexes on platelet membranes and direct viral infection of megakaryocytes. increased erythrocyte osmotic fragility. a condition characterized by increased erythrocyte osmotic fragility has been described in abyssinian and somali cats. the specific defect has and degeneration) and increased thrombopoiesis (megakaryocytic hyperplasia, increased numbers of immature megakaryocytes) in the bone marrow has been reported in type ii bvdv-infected animals, including concurrent megakaryocyte necrosis and hyperplasia in some experimental subjects. calves infected with type ii bvdv also have impaired platelet function. cattle with the hemorrhagic syndrome are severely thrombocytopenic and neutropenic with multisystemic hemorrhages, particularly of the digestive tract, spleen, gallbladder, urinary bladder, and lymph nodes. histologic lesions include hemorrhage, epithelial necrosis of enterocytes, intestinal erosions, crypt proliferation with microabscesses, and lymphoid depletion of the gut-associated lymphoid tissue, peyer's patches, and spleen. lesions of the bone marrow are variable, as previously described. bovine neonatal pancytopenia. bovine neonatal pancytopenia (bnp) is caused by alloantibodies absorbed from colostrum, resulting in a hemorrhagic syndrome in calves. the syndrome was first recognized in europe in the early 2000s and has since been experimentally correlated with prior vaccination of affected calves' dams with a commercial bvdv vaccine (pregsure bvd; pfizer animal health). the vaccine has since been voluntarily recalled from the market. it is thought that vaccination induces alloantibody formation by the dam. the alloantibodies are ingested by the calf and bind to the calf's hematopoietic progenitor cells, resulting in functional compromise of those cells. acutely affected calves are less than a year of age and have peripheral thrombocytopenia and neutropenia. death results from thrombocytopenia-induced hemorrhages or neutropenia-induced secondary infections, including pneumonia, enteritis, and septicemia. within the bone marrow there is erythroid, myeloid, and megakaryocytic hypoplasia. cyclic hematopoiesis. cyclic hematopoiesis (also known as lethal gray collie disease) is an autosomal recessive disorder of pluripotent hscs in gray collie dogs. a defect in the adaptor protein complex (ap3) results in defective intracellular signaling and predictable fluctuations in concentrations of blood cells that occur in 14-day cycles. the pattern is cyclic marked neutropenia, and in a different phase, cyclic reticulocytosis, monocytosis, and thrombocytosis. production of key cytokines involved in regulation of hematopoiesis is also cyclic. neutropenia predisposes affected animals to infection, and many die of infectious causes. affected animals have dilute hair coats and lesions with acute or chronic infectious disease, especially of the lungs, gastrointestinal tract, and kidneys. dogs older than 30 weeks of age have systemic amyloidosis, which occurs because of cyclic increases in concentration of acute phase proteins during phases of monocytosis. phosphofructokinase deficiency. inherited autosomal recessive deficiency of the erythrocyte glycolytic enzyme, phosphofructokinase (pfk), is described in english springer spaniel, american cocker spaniel, and mixed-breed dogs. there are three genes encoding pfk enzymes, designated m-pfk in muscle and erythrocytes, l-pfk in liver, and p-pfk in platelets. a point mutation in the gene coding for m-pfk results in an unstable, truncated molecule. erythrocytes in pfk-deficient dogs have decreased atp and 2,3-diphosphoglycerate (2,3-dpg) production and increased fragility under alkaline conditions. the disease is characterized by chronic hemolysis with marked reticulocytosis. the marked regenerative response may compensate for the ongoing hemolysis; therefore erythrophagocytosis, thrombosis, and histologic changes of ischemia are common, especially within the spleen, liver, and lungs. affected cats typically become acutely ill with fever, pallor, and icterus and usually die within 2 to 3 days. for many years, cytauxzoonosis was considered to be almost always fatal. however, a recent not been identified, but pk deficiency (which has been reported in these breeds) was excluded as the cause. affected cats have chronic intermittent severe hemolytic anemia and often have other lesions secondary to hemolytic anemia (e.g., splenomegaly and hyperbilirubinemia). cytauxzoonosis. cytauxzoonosis is a severe, often fatal disease of domestic cats caused by the protozoal organism, cytauxzoon felis. disease is relatively common in the south central united states, particularly during summer months. bobcats (lynx rufus) and other wild felids are thought to be wildlife reservoirs of disease. c. felis is transmitted by a tick vector, dermacentor variabilis, which is probably essential for infectivity of the organism. cytauxzoonosis has a schizogenous phase within macrophages throughout the body (especially liver, spleen, lung, lymph nodes, and bone marrow) that causes systemic illness. these schizontcontaining macrophages enlarge and accumulate within the walls of veins, eventually causing vessel occlusion, circulatory impairment, and tissue hypoxia. later in disease, merozoites released from schizonts enter erythrocytes, resulting in an erythrocytic phase of infection. infected domestic cats often have nonregenerative anemia, but the pathogenesis for the anemia is unclear. however, it likely represents preregenerative hemolytic anemia because erythrocyte phagocytosis is a prominent finding in many organs. infected cats often also develop neutropenia and thrombocytopenia, which likely result from inflammation and disseminated intravascular coagulation, respectively. on blood smear evaluation, signet ring-shaped erythrocytic inclusions (piroplasms) may be observed during the erythrocytic phase of disease ( fig. 13-34) . these inclusions closely resemble small-form babesia (see fig. 13-24, a) and some theileria organisms. postmortem examination typically shows pallor, icterus, splenomegaly, enlarged and red lymph nodes, diffuse pulmonary congestion and edema, and multisystemic petechiae and ecchymoses. vascular obstruction may cause marked distention of abdominal veins. cavitary effusions are present in some cats. microscopically, large, schizont-laden macrophages accumulate within venous and sinusoidal lumens and often completely occlude the lumens (fig. 13-35) . which the b and t lymphocytes proliferate, differentiate, and mature. in mammals, lymphocytes arise from hscs in the bone marrow, and b lymphocytes continue to develop at this site. ruminants also have b lymphocyte proliferation and maturation within their peyer's patches. progenitor t lymphocytes migrate from bone marrow to mature and undergo selection in the thymus. the spleen, lymph nodes, and lymph nodules are secondary lymphoid organs and are responsible for the immune responses to antigens, such as the production of antibody and cell-mediated immune reactions. at these sites, lymphocytes are activated by antigens and undergo clonal selection, proliferation, and differentiation (see also chapter 5). in addition, the spleen and lymph nodes contain cells of the monocyte-macrophage system and thus also participate in the phagocytosis of cells and materials. the bone marrow is described in the first section of this chapter. the remaining primary lymphoid organ, the thymus, is described first in this section, followed by the secondary lymphoid organs: spleen, lymph nodes, and diffuse and nodular lymphatic tissues. errors from selection of inappropriate sampling sites and artifacts from compression and incorrect fixation for histopathologic and immunohistochemical examinations are common in routine veterinary pathologic analysis. the identification and remedies for these problems are discussed in e-appendix 13-2. the thymus is essential for the development and function of the immune system, specifically for the differentiation, selection, and maturation of t lymphocytes generated in the bone marrow (see also chapter 5). the basic arrangement of the thymus in domestic animals consists of paired cervical lobes (left and right), an intermediate lobe at the thoracic inlet, and a thoracic lobe, which may be bilobed. the cervical lobes are positioned ventrolateral to the trachea, adjacent to the carotid arteries, and extend from the intermediate lobe at the thoracic inlet as far cranially as the larynx. the intermediate lobe bridges between the cervical and the thoracic lobe. the right thoracic lobe is usually small or completely absent. the left lobe lies in the ventral aspect of the cranial mediastinum (except in the ruminant, where it is dorsal) and extends caudally as far as the pericardium. horse-the cervical lobes in foals are small, and the thoracic lobe constitutes the bulk of the thymus. ruminant-the cervical lobes are large. the left and right thoracic lobes are fused and unlike other domestic animals, lie in the dorsal aspect of the cranial mediastinum. pig-the cervical lobes are large. dog-the cervical lobes regress very early and thus appear absent. the thoracic lobe extends caudally to the pericardium. cat-the cervical lobes are small, and the thoracic lobe, which forms the majority of the thymus, extends caudally to the pericardium and molds to its surface. the thymus is referred to as a lymphoepithelial organ and hence is composed of epithelial and lymphoid tissue. formed from the endoderm of the third pharyngeal pouch in the fetus, the thymic epithelium is infiltrated by blood vessels from the surrounding mesoderm, resulting in the development of the thymic epithelial reticulum. the lymphocyte population consists of bone marrow-derived progenitor cells, which fill spaces within the epithelial network. a connective tissue capsule surrounds the thymus, and attached thin septa subdivide the tissue into partially separated lobules. each report, in which numerous cats from a subregion of the endemic area in the united states survived infection with an organism with greater than 99% homology to cytauxzoon felis, suggests the emergence of a less virulent strain. felv is an oncogenic, immunosuppressive lentivirus that causes hematologic abnormalities of widely varying types and severity. manifestations of disease caused by felv infection vary depending on dose, viral genetics, and host factors, but normal hematopoiesis is probably suppressed to some degree in all cases. felv infects hematopoietic precursor cells soon after the animal is exposed and continues to replicate in hematopoietic and lymphatic tissue of animals that remain persistently viremic. the virus disrupts normal hematopoiesis by inducing genetic mutations, by other direct effects of the virus on infected hematopoietic cells, or by an altered host immune system. hematologic changes include dysmyelopoiesis with resultant cytopenias or abnormal cell morphologic features, and neoplastic transformation of hematopoietic cells (leukemia). a notable form of dysplasia is the presence of macrocytic erythrocytes (macrocytes) and metarubricytosis in the absence of erythrocyte regeneration (inappropriate metarubricytosis). the relatively uncommon subgroup c viruses cause erythroid hypoplasia, probably because of infection of early-stage erythroid precursors. felv may be detected in megakaryocytes and platelets in infected cats and may result in platelet abnormalities, including thrombocytopenia, thrombocytosis, increased platelet size, and decreased function. proposed mechanisms of felv-induced thrombocytopenia include direct cytopathic effects, myelophthisis, and immunemediated destruction. platelet life span and function have been shown to be decreased in felv-positive cats. persistently viremic cats are immunosuppressed and are prone to developing other diseases, including infectious diseases, bone marrow disorders, and lymphoma. cbc abnormalities attributed to felv infection include various cytopenias, especially nonregenerative anemia, which may be persistent or cyclical. regenerative anemia may also occur with felv infection, often because of coinfection with m. haemofelis. hematopoietic cell dysplasia or neoplasia may also be evident. grossly, infected cats are often pale, but other lesions are dependent upon the presence of other cytopenias or concurrent disease. microscopically, the bone marrow is hypocellular, normocellular, or hypercellular. there may be erythroid hypoplasia, erythroid hyperplasia with maturation arrest, or acute leukemia. feline immunodeficiency virus. feline immunodeficiency virus (fiv), another feline lentivirus, causes anemia in a minority of infected cats. immunosuppressive effects of fiv from thymic depletion are discussed elsewhere. it is generally accepted that anemia does not result directly from fiv infection but instead develops because of concurrent disease such as coinfection with felv or hemotropic mycoplasma, other infection, or malignancy. the severity and type of anemia in fiv-infected cats depends on the other specific disease processes involved. the thymus, spleen, lymph nodes, and lymph nodules, including malt, are classified as part of both the lymphoid and immune systems. the lymphoid system (also known as lymphatic system in some texts) is broadly categorized into primary and secondary lymphoid organs. the main primary lymphoid organs include thymus, bone marrow, and bursa of fabricius in birds and are the sites at 761.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system because the thymus involutes after sexual maturity, evaluation of whether it is smaller than normal is difficult to discern unless the change is extreme or age-matched control animals are available. before sexual maturity the thymus is easily identified as a lobular white to gray organ with a thin capsule. after sexual maturity the gland is often grossly indistinguishable from adipose connective tissue within the cranial mediastinum, although microscopic remnants may remain. an extremely small thymus in a neonatal animal should be considered abnormal and may indicate a primary immunodeficiency or secondary lymphoid depletion caused by extreme stress, often due to infectious diseases. enlargement of the thymus is most often due to neoplasia. serial sectioning of the thymus allows for gross identification of neoplasms, cysts, or hematomas. spleens vary in size within the same species and among the different species of domestic animals. the spleen can be enlarged (splenomegaly), normal in size, or small (atrophy), and the surface can be smooth, wrinkled, or nodular. the appearance of the cut surface of the spleen in normal animals depends on the amount of stroma (e.g., trabeculae are prominent in ruminants); the size and visibility of the white pulp, which reflects the amount of lymphoid tissue; and whether the red pulp is congested with blood. during an autopsy (syn: necropsy), the spleen is dissected free and checked for torsion of the gastrosplenic ligament (in nonruminants). the spleen is then sliced transversely at approximately 5-mm intervals (serial sectioning), and the cut surfaces are checked for lesions. specimens are taken for tests that require fresh tissue (e.g., bacteriologic and virologic examinations), and the remaining cross-sections are placed in fixative (10% buffered neutral formalin). a diffusely enlarged spleen should be serially sectioned to determine if the splenomegaly is due to congestion. the cut surface of severely congested spleens is red to bluish-black and exudes blood (bloody spleens), whereas cut surfaces of noncongested enlarged spleens ooze little blood (meaty spleens) and the color depends on how much of the normal parenchyma is replaced by inflammatory cells, stored materials, or neoplastic cells (see splenomegaly and table 13 -5). the spleen may be measured and weighed, but because of the wide variation in the dimensions and weight of normal spleens and the amount of blood stored, this information is difficult to interpret. it is essential that spleens with one or more nodules also be serially sectioned and the nodules evaluated for size, shape, and consistency. nodules may be dark red and ooze blood on cut surface, white-tan with a more firm texture, or a mixture of both. multiple wedge sections that include the interface between a nodule and the adjacent nonmass spleen should be collected, because the center of the nodules often consists only of hemorrhage and necrosis, and neoplasms may be missed. the color of the capsular surface of the spleen also varies among species of domestic animals and depends on the opacity or translucence of the splenic capsule. the degree of opacity of the capsule is a function of its thickness and the amount of collagen. the splenic capsules of horses and ruminants are thick and usually appear gray because the color of the red pulp is not visible through the capsule. in the pig, dog, and cat, the splenic capsule is thin, and thus the surface of the spleen is red. the tenseness of the capsule depends on how much the splenic parenchyma is distended; storage spleens devoid of blood usually have a wrinkled surface. irregular contraction of storage spleens is common, especially in dogs, and consists of nonuniform areas of congestion with intermingled contracted and wrinkled regions. lymph nodes should be dissected free of fat and connective tissue, and any firm attachment to adjacent tissues should be noted because these attachments may indicate neoplastic infiltration through the capsule. gross examination includes evaluating size (measurement or weight), shape, and whether the capsule is intact. the cut surface is examined for the presence of bulging tissue, edema, congestion, exudate, discoloration (see pigmentation), obscuration of the normal architecture, and masses such as abscesses, granulomas, and discrete neoplasms. cytologic evaluation of superficial lymph nodes through fine-needle aspirates provides excellent cellular detail and often yields a diagnosis. however, diagnosis of certain diseases (including lymphomas for complete world health organization [who] classification) requires architectural assessments, and therefore cytologic or small histologic samples are not sufficient. tru-cut biopsies are not ideal, but a 2-mm tru-cut needle may provide adequate tissue. the surgeon or pathologist must handle lymph nodes carefully to minimize artifacts. compression (e.g., squeezing with forceps) may cause crush artifacts, usually resulting in nuclear "streaming." immediately after removal, imprints/impression smears should be prepared and then kept away from formalin fumes. formalin fixation (in this case by formaldehyde fumes) destroys the differential staining seen with romanowsky stains such as wright's and giemsa and results in diffuse blue staining. prompt transfer of biopsy or postmortem specimens into fixative is crucial because delayed fixation can lead to numerous artifacts, including an artificial decrease in mitotic index (up to 40% reduction with a more than 12-hour delay in fixation); this reduction can alter tumor classification and grade. the current recommendation for the duration of formalin fixation is 16 to 32 hours; complete fixation of 2-to 4-mm thick tissues is likely to be achieved after 24 to 48 hours. both underfixation and overfixation may lead to difficulties with antigen retrieval for immunohistochemistry, though underfixation is considered the more common and serious problem. thinly slicing some nodes may be difficult, and allowing the node to fix for 1 hour before slicing may help. some pathologists prefer not to incise very small lymph nodes to avoid compression artifacts, but instead nick the capsule to allow formalin penetration. however, fixation of unincised lymph nodes can also cause compression artifacts because the fibrous capsule contracts in the fixative. once fixed, nodes should be cut in uniformly thick cross section to include both the cortex and medulla. transverse sections are usually sufficiently small to allow the entire cross section of most lymph nodes to fit on one microscopic slide, which facilitates histologic interpretation. the longitudinal plane is preferred for porcine lymph nodes because the location and amount of cortex and medulla vary at different sites in transverse sections. molecules) but not self-antigens are permitted to mature by a process called positive selection. cells that do not recognize mhc molecules are removed by apoptosis. those t lymphocytes that recognize both mhc molecules and self-antigens are removed by macrophages at the corticomedullary junction, a process called negative selection. because of the rigid differentiation requirements attributable to mhc restriction and tolerance (positive and negative selection, respectively), only a small fraction (<5%) of the developing t lymphocytes that arrive at the thymus from the bone marrow survive. mature naïve t lymphocytes exit the thymus through postcapillary venules in the corticomedullary region, enter the circulation, and recirculate through secondary lymphoid tissues, primarily located in the paracortex of lymph nodes and the periarteriolar sheaths of the spleen. in these specialized sites, the mature naïve t lymphocytes are activated upon exposure to their specific antigens and undergo additional phases of development to differentiate into effector and memory cells. the thymus attains its maximal mass relative to body weight at birth and involutes after sexual maturity; the rate of involution may vary among domestic species. the lymphoid and epithelial components are gradually replaced by loose connective tissue and fat, although remnants remain histologically, even in aged animals. lobule is composed of a central medulla and surrounding cortex (fig. 13-36) . the thymic cortex consists mainly of an epithelial reticulum and lymphocytes ( fig. 13-37) . the stellate cells of the epithelial reticulum have elongate branching cytoplasmic processes that connect to adjacent epithelial cells through desmosomes, thus forming a supportive network (cytoreticulum). the lymphoid component is composed of differentiating lymphocytes derived from progenitor (also known as precursor) t lymphocytes in the bone marrow. the medulla is composed of similar epithelial reticular cells, many of which are much larger than those in the cortex and have a more obvious epithelial structure. some of the epithelial reticular cells form thymic corpuscles, also called hassall's corpuscles, which are distinctive keratinized epithelial structures (see fig. 13-37 ). interdigitating dendritic cells (dcs) are also present within the medulla, but there are far fewer lymphocytes than in the cortex. the progenitor t lymphocytes released from the bone marrow into the blood enter the thymus in the subcapsular zone of the cortex and begin the differentiation and selection processes, developing into mature naïve t lymphocytes as they traverse the thymic cortex to the medulla. in the cortex, t lymphocytes that recognize self-molecules (major histocompatibility complex [mhc] the responses of the thymus to injury and causes are listed in boxes 13-4 and 13-5. the most common change is lymphoid atrophy caused by physical and physiologic stresses, toxins, drugs, and viral infections. atrophy. because the thymus does not contain any lymphopoietic tissue, it depends on the bone marrow for the supply of progenitor t lymphocytes. thus thymic lymphoid atrophy can be the result of either an inadequate supply of lymphocytes from the bone marrow or lysis of lymphocytes (lymphocytolysis) in the thymus. thymic aims to define the terms used in this chapter. the term splenic sinusoid is used to describe a vascular structure present in the sinusal spleen (also known as sinusoidal spleen); dogs are the only domestic animal with true splenic sinusoids. the term red pulp vascular spaces is used (as opposed to "sinus") to describe the vascular spaces in the red pulp of both the nonsinusal and nonsinusoidal spleens of all domestic animals. 4 the other terms used here include marginal sinus, marginal zone, periarteriolar lymphoid sheath (pals), periarteriolar macrophage sheath (pams), and splenic lymphoid follicles. the spleen is located in the left cranial hypogastric region of the abdomen, where it is typically suspended in the gastrosplenic ligament between the diaphragm, stomach, and the body wall. the exception is in domestic ruminants, where it is closely adhered to the left dorsolateral aspect of the rumen. the gross shape and size of the spleen vary markedly among domestic animals, but generally it is a flattened, elongated organ. some species, notably birds, demonstrate seasonal variation in splenic shape and size. the spleen is covered by a thick capsule composed of smooth muscle and elastic fibers, from which numerous intertwining fibromuscular trabeculae extend into the parenchyma. these trabeculae and reticular cells form a spongelike supportive matrix for the parenchyma of the mammalian spleen in all domestic species. in cattle and horses the three muscular layers of the capsule lie perpendicular to each other, forming a capsule thicker than that of carnivores. carnivores, small ruminants, and pigs have interwoven smooth muscle within the splenic capsule, and pigs also have abundant elastic fibers within the capsule. the spleen differs from many other organs in the organization of its parenchyma. instead of a cortex and medulla, the spleen is divided into two distinct structural and functional components: the red pulp and white pulp (fig. 13-38) . with hematoxylin and eosin (h&e) staining, red pulp appears red-pink because of the abundance of red blood cells, whereas white pulp appears blue-purple because of the heavy concentration of lymphocytes. the white pulp consists of splenic follicles, populated by b lymphocytes; the pals, inhabited by t lymphocytes; and the marginal zone at the periphery of follicles. macrophages, antigen-presenting cells, and trafficking b and t lymphocytes populate the marginal zone. the radial arteries, branches of the central artery (also known as central arteriole), and capillaries from both red and white pulp drain into the marginal sinus of the marginal zone, although the latter has not been shown to be the case in all species to the same degree (e.g., the cat has a small marginal sinus but a well-developed pams) (figs. 13-39 and 13-40). the red pulp consists of cells of the monocyte-macrophage system, pams, sinusoids (dogs, rats, and human beings only), red pulp vascular spaces, and associated stromal elements such as reticular cells, fibroblasts, and trabecular myocytes. the labyrinth of the splenic red pulp vascular spaces serves as both a functional and physical filter for circulating blood cells. the blood circulation of the spleen is particularly suited to enable its functions, namely, (1) filtering and clearing the blood of atrophy must be differentiated from involution, which normally begins at sexual maturity. this distinction is difficult to make, unless the change is extreme or age-matched control animals are available for comparison. inflammation. inflammation of the thymus is rare. neutrophils and macrophages are often present within keratinized hassall's corpuscles during involution and should not be mistaken for a true thymitis. thymitis has been reported in salmon poisoning disease of dogs (see chapter 7), epizootic bovine abortion (see chapter 18), and in pigs infected with porcine circovirus type 2 (pcv2). necrosis and secondary infiltrates of neutrophils and macrophages may be seen in other infectious diseases (e.g., equine herpesvirus 1 [ehv-1]). the main portal of entry to the thymus is hematogenous. portals of entry used by microorganisms and other agents and substances to access the lymphatic system are summarized in box 13-6. these portals include the blood vessels (hematogenous spread by microorganisms free in the plasma or within circulating leukocytes or erythrocytes), afferent lymphatic vessels (lymphatic spread), direct penetration, or through m (for "microfold") cells and dcs in malt. defense mechanisms used by the thymus to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. viruses, bacteria, and particles arriving in the lymph and blood interact with cells of the monocyte-macrophage system through phagocytosis and antigen processing and presentation. hyperplasia of the macrophages often occurs concurrently. antigen processing and presentation are followed by an immune response resulting in proliferation of b lymphocytes, plasma cells, and the subsequent production of antibody; proliferation of t lymphocytes may also occur. the relationships between anatomic structures and the different functions of the spleen are complicated. there are also anatomic differences among domestic animal species and confusion about the correct and up-to-date terminology. the following brief discussion malt, mucosa-associated lymphoid tissue. 4 there are numerous synonyms and misuse of terms within the literature, which have contributed to the confusion over terminology for red pulp vascular spaces. these terms include reticular space, red pulp, splenic cords, sinuses, red pulp sinuses, sinus spaces, pulp spaces, mesh space of the spleen, reticular cell-lined meshwork, interstices of the reticulum network, bloodfilled reticular meshwork of the red pulp, chordal spaces, splenic cords, and cords of billroth. the latter two terms are defined as the red pulp between the sinusoids, which most domestic animals do not have (except the dog). therefore the term red pulp vascular spaces is more appropriate. as a result of this pattern of blood flow, macrophages in the marginal sinus have the first opportunity to phagocytize antigens, bacteria, particles, and other material before macrophages in the sinusoids (in the dog) or in the pams and red pulp vascular spaces (all other domestic animals). in the dog the marginal sinus drains into the sinusoids, but in other domestic animals it drains into the red pulp vascular spaces. the central arteries leave the white pulp, enter the red pulp, and branch into smaller penicillar arterioles. each arteriole is surrounded by a sheath of macrophages known as periarteriolar macrophage sheaths (pams, previously known as ellipsoids), which are notably prominent in pigs, dogs, and cats. in horses, cattle, pigs, and cats the terminal branches of the penicillar arterioles empty into the red pulp vascular spaces lined by reticular cells. because the red pulp vascular spaces are not lined by endothelium, this type of circulation is known as an open system. this system is in contrast to the sinusoidal spleen of the dog (also of the rat and human beings), where the branches of the central artery of the white pulp and vessels from the marginal sinus enter into the sinusoids, which are lined by a discontinuous endothelium, and these empty into splenic venules. this type of circulation is known as a closed system because the blood flow is through blood vessels (arterioles, capillaries, sinusoids, and venules), all of which are lined by endothelium. although circulation in the red pulp is anatomically open in nonsinusoidal spleens, under certain conditions (e.g., during splenic contraction) the circulation is functionally closed, and the blood in the red pulp is particulate matter and senescent cells; (2) transporting recirculating lymphocytes and naïve b and t lymphocytes to the follicle and pals, respectively, to fulfill their specific immune functions; and (3) storage of blood in some domestic animal species (dog, cat, and horse) (fig. 13-41) . phagocytosis is particularly effective in the spleen because blood flows through areas within the red pulp that are populated with increased concentrations of macrophages, namely, within the marginal sinuses, in cuffs around the penicillar arteries (pams), diffusely on the reticular walls of the red pulp vascular spaces, and along the sinusoids in dogs. trafficking of naïve and recirculating lymphocytes is facilitated by the proximity of the marginal sinus to the follicular germinal centers and pals. maps of the vascular blood flow in sinusoidal and nonsinusoidal spleens are illustrated in figures 13-41 to 13-43. the celiac artery is the major branch of the abdominal aorta from which the splenic artery arises. the splenic artery enters the splenic capsule at the hilus, where it branches and enters the fibromuscular trabeculae as trabecular arteries to supply the splenic parenchyma. trabecular arteries become the central arteries of the white pulp and are surrounded by cuffs of t lymphocytes forming the pals. the splenic follicles, populated by b lymphocytes, are eccentrically embedded within or just adjacent to the pals. the central arteries send branches-the radial arteries-to supply the marginal sinus surrounding the splenic follicles. thus the cells at the circumferences of the follicles are brought into intimate contact with blood-borne antigens and trafficking b and t lymphocytes in the marginal sinus. diverted into "channels" lined by reticular cells. because the dog has both sinusoids and red pulp vascular spaces, it has both open and closed splenic circulations, which may allow for both fast and slow flows of blood depending on the physiologic need of the animal. blood flowing through the sinusoids or red pulp vascular spaces is under the surveillance of macrophages. in dogs the pseudopodia of these perisinusoidal macrophages project into the sinusoidal lumen through the spaces in the discontinuous endothelium. in all domestic animals, blood in the red pulp vascular spaces is under surveillance of macrophages attached to the reticular walls. blood from the red pulp vascular spaces and sinusoids then drains into the splenic venules, splenic veins, and ultimately into the portal vein, which empties into the liver. the spleen filters blood and removes foreign particles, bacteria, and erythrocytes that are senescent, have structural membrane abnormalities, or are infected with hemotropic parasites. as a secondary lymphoid organ, its immunologic functions include the activation of macrophages to process and present antigen, the proliferation of b lymphocytes and production of antibody and biologic molecules, and the interaction of t lymphocytes and antigens. in some species the spleen stores significant quantities of blood (box 13-7). the functions of the spleen are best considered on the basis of the two main components of the spleen: the red and white pulp and the anatomic systems contained within them (monocyte-macrophage system, red pulp vascular spaces, and hematopoiesis in the red pulp, and the b and t lymphocyte systems within the white pulp). monocyte-macrophage system. within the red pulp, macrophages are located in the marginal sinus, pams, and attached to the reticular walls of the red pulp vascular spaces. in the dog, macrophages are also located perisinusoidally. the supportive reticular network of the red pulp vascular spaces is composed of a fine meshwork of reticular fibers made of type iii collagen, on which macrophages are dispersed. exactly in which of these concentrations of macrophages phagocytosis of blood-borne particles takes place depends upon (1) the sequence in which they are exposed to the incoming blood, (2) the concentration of macrophages in these areas (e.g., the cat marginal sinus is small and thus not a major site of clearance; there is a compensatory increase in pams for phagocytosis), and (3) the functions of the macrophages. some of the macrophages in the marginal sinus and marginal zone are responsible for phagocytosis of particulate matter and others for the trapping and ingestion of antigens and antigen-antibody complexes. macrophages responsible for phagocytosis of blood-borne foreign material (fig. 13-44) , bacteria, and senescent and/or damaged erythrocytes (e.g., as seen in immune-mediated anemias and infections with hemotropic parasites) are also found in the red pulp. in the dog, sinusoidal macrophages remove entire erythrocytes (erythrophagocytosis), as well as portions of an erythrocyte's membrane and cytoplasmic inclusions, such as nuclear remnants like heinz bodies, by a process called pitting. as such, the presence of large numbers of nuclear remnants in erythrocytes in canine blood smears may indicate malfunction of the sinusoidal system. the normal rate of removal of senescent erythrocytes from the circulating blood does not cause an increase in size of the spleen; however, splenomegaly can be observed when large numbers of defective erythrocytes must be removed, as in cases of severe acute hemolytic anemia. nonsinusoidal spleens lack the fenestrated endothelium and perisinusoidal macrophages of canine sinusoids that allow for slow processing of red blood cells to determine which are to be returned to the equine, canine, and feline spleens all have considerable storage and contractile capacity because of their muscular capsule, increased numbers of trabeculae, and the relatively small amount of splenic parenchyma devoted to white pulp. the storage capacity in dogs and horses is remarkable: it has been claimed that the canine spleen can store one-third of the dog's erythrocytes while the animal sleeps and the equine spleen holds one-half of the animal's circulating red cell mass (which is considered advantageous because it reduces the viscosity of the circulating blood). storage spleens expand and contract quickly under the influence of the autonomic nervous system, via sympathetic and vagal fibers in the trabeculae and reticular walls of the red pulp vascular spaces and other circulatory disruptions, such as hypovolemic and/or cardiogenic shock. thus storage spleens may be either grossly enlarged and congested or small with a wrinkled surface and a dry parenchyma depending on whether the spleen is congested from stored blood or shrunken from contraction (see uniform splenomegaly and small spleens). hematopoietic tissue. in the developing fetus the liver is the primary site of hematopoiesis, with the spleen making a minor contribution. shortly before or after birth, hematopoiesis ceases in the liver and spleen, and the bone marrow becomes the primary hematopoietic organ. under certain conditions, such as severe demand due to prolonged anemia, splenic hematopoiesis can be reactivated; this outcome is called extramedullary hematopoiesis (emh). studies have indicated that splenic emh in dogs and cats most commonly occurs with degenerative or inflammatory circulation, pitted, or phagocytized. instead, the macrophages of the red pulp perform these functions, and phagocytized cells remain in the red pulp vascular spaces. the location of the primary sites of pitting in nonsinusoidal spleens is unclear, but it is likely that most erythrophagocytosis takes place in the red pulp vascular spaces. the cat's spleen is deficient in pitting, and removal of heinz bodies is slow; however, some erythrophagocytosis does occur in the marginal sinus. the macrophages of the sinusoids, marginal sinus, and red pulp vascular spaces are of bone marrow origin. from the bone marrow these cells circulate in the blood as monocytes and migrate into the spleen. some macrophages are replenished by local proliferation. for example, after phagocytizing large amounts of material from the blood, the macrophages of the pams migrate through the wall of the cuff into the adjacent red pulp, denuding the pams of macrophages. after 24 hours, local residual macrophages have proliferated to repopulate the pams. the fixed macrophages elsewhere in the body, namely, those in connective tissue, lymph nodes (sinus histiocytes), liver (kupffer cells), lung (pulmonary intravascular macrophages and pulmonary alveolar macrophages), and brain (resident and perivascular microglial cells), are also derived from bone marrow (see chapters 5, 8, 9, and 14) . storage or defense spleens. spleens are also classified as either storage or defense spleens, based on whether or not they can store significant volumes of blood. the ability to store blood in the spleen depends on the fibromuscular composition of the splenic capsule and trabeculae. splenic capsules and trabeculae with a low percentage of smooth muscle and elastic fibers cannot expand and contract and are designated as defense spleens. these are found in rabbits and human beings. the spleens of other domestic animal species have distention of the red pulp from stored blood separates the foci of white pulp (pals and lymphoid follicles), making white pulp appear sparser. splenic white pulp is organized around central arteries in the form of pals, which are populated primarily by t lymphocytes (see . primary splenic follicles are located eccentrically in pals and are primarily composed of b lymphocytes. when exposed to antigen, the splenic lymphoid follicles develop germinal centers (see lymphoid/ lymphatic system, lymph nodes, function) . macrophages in the white pulp follicles remove apoptotic b lymphocytes not selected for expansion because of low binding affinity for antigen. failure of these macrophages to phagocytize has been experimentally correlated with decreased production of growth factors like tgf-β and increased production of inflammatory cytokines that predispose the animal to autoimmune conditions. the marginal zone surrounds the marginal sinus at the interface of the white and red pulp and consists of macrophages, dcs, and t and b lymphocytes. the blood supply of the marginal sinus is from conditions (e.g., hematomas, thrombosis) and may occur without concomitant hematologic disease (see uniform splenomegaly with a firm consistency). it is also found in splenic nodular hyperplasia (see splenic nodules with a firm consistency). in some species, such as the mouse, emh is a normal function of the adult spleen and not necessarily a response to disease or hypoxic challenge. the splenic red pulp also contains large numbers of monocytes, which function as a reserve for generating tissue macrophages in response to ongoing tissue inflammation in the body. white pulp. white pulp consists of pals, each with a splenic lymphoid follicle surrounded by a marginal zone. normally these foci of white pulp are so small that they may not be visible on gross examination of a cross section of the spleen. however, if nodules are enlarged either by lymphoid hyperplasia, amyloid deposits, or a neoplastic process (e.g., lymphoma), they can become grossly visible on the cut surface, initially as 0.5-to 1.0-mm white circular foci scattered through the red pulp. in animals with storage spleens, the the splenic artery enters at the hilus and divides into arteries, which enter the trabeculae. when a trabecular artery emerges from a trabecula it becomes the central artery and is encased in a periarteriolar lymphoid sheath (pals), which is composed of t lymphocytes. it then enters the splenic follicle and gives off branches-the radial arteries, which supply the marginal sinus and marginal zone. the central artery emerges from the splenic follicle to enter the red pulp and branches into the penicillary arterioles, which are enclosed in a cuff of macrophages-the periarteriolar macrophage sheath (pams). the emerging penicillar arteries branch into arterioles and capillaries that supply the red pulp vascular spaces (see fig. 13-43) . the red pulp vascular spaces also receive blood from capillaries draining from the marginal sinus and drain into the splenic venules and then into the trabecular veins and splenic vein. b, sinusoidal spleen, dog. the blood flow is essentially the same but with the additional feature that arterioles from the marginal sinus drain into the sinusoids and some blood from the red pulp vascular space passes through slits in the sinusoidal wall to enter the sinusoid (see fig. 13-42) . this is the site of pitting and erythrophagocytosis. note that the major flow in a is sequentially past concentrations of macrophages in the marginal sinus, pams, and red pulp vascular spaces. in b there is the additional route from the marginal zone into the sinusoids. dog. red pulp vascular spaces macrophages in the marginal zone are phenotypically distinct from those in the red pulp. the red pulp macrophages function primarily to filter the blood by phagocytizing particles and by removing senescent or infected erythrocytes and pathogenic bacteria and fungi. marginal zone macrophages are divided into two types based on their location and the type of cell surface receptors they possess. the first group is positioned toward the periphery of the marginal zone, whereas the second group, the marginal metallophilic macrophages (so called for their silver staining positivity), is at the inner margin of the marginal zone closer to the splenic follicle and pals. it has been difficult to generate mammalian models that eliminate one of the two classes of marginal zone macrophages, so the degree to which one group specializes in a particular function is not clear. some marginal zone macrophages actively phagocytize particulate matter or bacteria (e.g., septicemias caused by streptococcus pneumoniae, listeria monocytogenes, campylobacter jejuni, or bacillus anthracis) in the blood (see fig. 13-40) . they also play a similar role in limiting the spread of viral infections. other marginal zone macrophages phagocytize and process antigens. thus macrophages of the marginal zone serve to bridge the innate and adaptive immune responses by secreting inflammatory cytokines to activate other immune cells and providing receptor-based activation of marginal zone lymphocytes. studies have shown that a loss of marginal zone macrophages coincides with decreased antigen trapping by resident b lymphocytes of the marginal zone and consequently a decrease in the early igm response to antigens. the responses of the spleen to injury (box 13-8) include acute inflammation, hyperplasia of the monocyte-macrophage system, hyperplasia of lymphoid tissues, atrophy of lymphoid tissues, storage of blood or contraction to expel reserve blood, and neoplasia. these responses are also best considered on the basis of the two main components of the spleen, the red and white pulp, and the anatomic systems associated with each. monocyte-macrophage system. the distribution and function of macrophages in the spleen is described earlier in the section on structure and function. these interactions are complex, and their relationships to both innate and adaptive immunity are areas of intense study (see also chapter 5). to facilitate filtering, all of the blood in the body passes through the spleen at least once a day, and 5% of the cardiac output goes to the spleen. in dogs, blood flow and transit time depend on whether the spleen is contracted or distended; blood flow is slower in the distended spleen. the extent to which macrophages of the monocyte-macrophage system phagocytize particles depends to a large degree on the sequence in which they receive blood. in most species, macrophages of the marginal sinus are the first to receive blood, and consequently phagocytized particles and bacteria tend to be more concentrated here initially. however, there are differences among domestic animal species; the cat, for instance, has a comparatively small marginal sinus, and thus the pams play a larger role in phagocytosis. the spleen is able to mount a strong response to blood-borne pathogens, which has been demonstrated in several studies. the blood of immunized rabbits injected intravenously with pneumococci cleared 98% of those bacteria within 15 minutes and 100% within an hour. the blood of dogs injected with 1 billion pneumococci per pound of body weight into the splenic artery was cleared of all bacteria in 65 minutes. after splenectomy, blood-borne the radial branches of the central artery, and it serves as the portal of entry into the spleen for recirculating b and t lymphocytes. from here, t lymphocytes migrate to the pals and b lymphocytes to the germinal centers. macrophages in the marginal zone capture bloodborne antigens, process them, and present them to the lymphocytes. senescent erythrocytes damaged erythrocytes (e.g., immune-mediated anemias) parasitized erythrocytes (e.g., hemotropic parasites) storage of blood (in storage spleens) extramedullary hematopoiesis severe demand (e.g., anemias) degenerative/inflammatory conditions without concomitant hematologic disease incidental (e.g., within nodules of hyperplasia) monocytes within splenic cords reserve for generating tissue macrophages in response to inflammation homing of circulating lymphocytes in the blood phagocytosis and processing of antigen macrophage activation inflammation, which may be diffuse or multifocal/focal (e.g., blastomycosis and tuberculosis, respectively). red pulp vascular spaces. the main response to injury of the red pulp vascular spaces is congestion (see uniform splenomegaly with a bloody consistency), as well as the storage of blood or contraction to expel reserve blood. white pulp. the responses to injury within the white pulp are most pronounced in the splenic lymphoid follicles. lymphoid follicular hyperplasia is a response to antigenic stimuli and results in the formation of secondary follicles; marked hyperplasia may be grossly evident. hyperplasia of splenic lymphoid follicles follows a similar sequence of events and morphologic changes as seen in other secondary lymphoid organs and is discussed in more detail in lymphoid/lymphatic system, lymph nodes, dysfunction/responses to injury. similarly, atrophy of splenic lymphoid follicles has similar causes as lymphoid atrophy in other lymphoid organs (see box 13-5). briefly, atrophy occurs in response to lack of antigenic stimulation (e.g., from regression after antigenic stimulation has ceased), from the effects of toxins, antineoplastic chemotherapeutic agents, microorganisms, radiation, malnutrition, wasting/cachectic diseases, or aging, or when the bone marrow and thymus fail to supply adequate numbers of b and t lymphocytes, respectively. the follicles are depleted of lymphocytes, and with time, germinal centers and follicles disappear. the amount of the total lymphoid tissue is reduced, and the spleen may be smaller. the response to injury of the monocyte-macrophage system in the marginal sinus and marginal zone is also phagocytosis and proliferation. capsule and trabeculae. lesions in the capsule and trabeculae are uncommon and include splenic capsulitis secondary to peritonitis, and complete or partial rupture of the splenic capsule, usually due to trauma. the two main portals of entry to the spleen for infectious agents are hematogenous spread and direct penetration. the splenic capsule is thick, and thus direct penetration is less common. inflammation from an adjacent peritonitis is unlikely to penetrate the capsule into the splenic parenchyma. cattle with traumatic reticulitis may have foreign objects migrate into the ventral extremity of the spleen, causing a splenic abscess. splenic abscesses also develop secondary to perforation of the gastric wall in horses, due to foreign body penetration, gastric ulcers, or gastric inflammation. portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box 13-6. defense mechanisms used by the spleen to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsule of the spleen. lymph nodes are soft, pale tan, round, oval or reniform organs with a complex three-dimensional structure. on gross examination of a cross section of lymph nodes, two main areas are visible: an outer rim of cortex and an inner medulla (fig. 13-45) . to understand the pathologic response of the lymph node, it is important to consider its anatomic components and their relationship with antigen processing (fig. 13-46) : organisms multiply rapidly and may disseminate widely in the body to cause an overwhelming postsplenectomy infection. studies have also shown that the phagocytic function of the spleen is critical in the control of plasmodium (causative agent of malaria) in human beings and babesiosis in cattle. if the number of pathogenic bacteria in the circulation exceeds the capacity of the splenic macrophages, as in cases of severe septicemia, it may result in acute splenic congestion (see uniform splenomegaly with a bloody consistency). this may be followed by inflammation with areas of necrosis, fibrin deposition, and infiltration by neutrophils in bacteremias of pyogenic bacteria. the marginal zone can be the initial site of response to blood-borne antigens and bacteria delivered by the radial branches of the central arteries to the marginal sinus. similar to the response of the red pulp vascular spaces, the marginal zone can become congested and with time (only hours with highly pathogenic organisms) may contain aggregates of neutrophils and macrophages. histologically, the congestion and inflammation form a complete or partial concentric ring around the circumference of the splenic nodule (see anthrax). hyperplasia of the red pulp macrophages is also seen in chronic hemolytic diseases, because there is a prolonged need for phagocytosis of erythrocytes. similarly, chronic splenic congestion, usually the result of portal or splenic vein hypertension, can lead to proliferation of the macrophages present on the walls of the red pulp vascular spaces and results in thickening of the reticular walls between the red pulp vascular spaces. macrophages in the red pulp also proliferate in response to fungi and facultative intracellular pathogens (e.g., mycobacterium bovis) arriving hematogenously to the spleen. the number of red pulp macrophages may be augmented by monocytes recruited from the blood to form granulomatous acute inflammation with fibrin and necrosis abscesses, microabscesses granulomatous inflammation (diffuse, multifocal, focal) fibroblastic reticular cells and fibers. besides providing structural support, this reticulum helps form a substratum for the migration of lymphocytes and antigen-presenting cells to the follicles and facilitates the interaction with b and t lymphocytes. cortex. the outer/superficial cortex contains the lymphoid follicles (also referred to as lymphoid nodules) (see . the follicles are designated as primary if they consist mainly of small lymphocytes: mature naïve b lymphocytes expressing receptors for specific antigens exit the bone marrow and circulate through the bloodstream, lymphatic vessels, and secondary lymphoid tissues. on their arrival at lymph nodes, b lymphocytes exit through hevs in the paracortex and home to a primary follicle (which also contains follicular dcs in addition to the resting b lymphocytes). lymphoid follicles with germinal centers are designated as secondary follicles: b lymphocytes that recognize the antigen for which they are expressing receptors are activated and proliferate to form the secondary lymphoid follicles characterized by prominent germinal centers. germinal centers are areas with a specialized • stroma-capsule, trabeculae, and reticulum • cortex-"superficial" or "outer" cortex (lymphoid follicles, b lymphocytes) • paracortex-"deep" or "inner" cortex (t lymphocytes) • medulla-medullary sinuses and medullary cords • blood vessels-arteries, arterioles, high endothelial venules (hevs), efferent veins • lymphatic vessels-lymphatic afferent and efferent vessels; lymphatic sinuses (subcapsular, trabecular, and medullary) • monocyte-macrophage system-sinus histiocytes stroma. the lymph node is enclosed by a fibrous capsule penetrated by multiple afferent lymphatic vessels, which empty into the subcapsular sinus (see also . at the hilus, efferent lymphatic vessels and veins exit, and arteries enter the node. fibrous trabeculae extend from the capsule into the parenchyma to provide support to the node and to house vessels and nerves. the lymph node is also supported by a meshwork of medulla. the medulla is composed of medullary cords and medullary sinuses (see . the medullary cords contain macrophages, lymphocytes, and plasma cells. in a stimulated node the cords become filled with antibody-secreting plasma cells. the medullary sinuses are lined by fibroblastic reticular cells and contain macrophages ("sinus histiocytes"), which cling to reticular fibers crossing the lumen of the sinus. these macrophages phagocytize foreign material, cellular debris, and bacteria from the incoming lymph. vasculature: blood vessels, lymphatic vessels, and lymphatic sinuses. the blood vessels of the lymph node include arteries, arterioles, veins, and postcapillary venules (hevs) lined by specialized cuboidal endothelium (see figs. 13-45 and 13-46) . microenvironment that support the proliferation and further development of b lymphocytes to increase their antigen and functional capacity (see lymphoid/lymphatic system, lymph nodes, function). the mantle cell zone surrounds the germinal center and consists of small inactive mature naïve b lymphocytes and a smaller population of t lymphocytes (approximately 10%). paracortex. the diffuse lymphoid tissue of the paracortex (also referred to as the deep or inner cortex) consists mainly of t lymphocytes, as well as macrophages and dcs (see . this region contains the hevs through which b and t lymphocytes migrate from the blood into the lymphoid follicles and paracortex, respectively. t and b lymphocytes may also enter the lymph node via the lymphatic vessels. and larger molecules, small molecules and free antigens, and antigen within dcs. it is helpful to consider the paths taken by particles, molecules, antigens, and cells arriving at a lymph node. the following account describes the journey of an antigen as it enters a lymph node to trigger an immune response. antigen in the lymph arriving in the afferent lymphatic vessels empties into the subcapsular sinus. hydrostatic pressure here is low, and reticular fibers crossing the sinus impede flow, and thus particles tend to settle, which facilitates phagocytosis by the sinus macrophages. lymph then flows down the trabecular sinuses that line the outer surface of fibrous trabeculae, to the medullary sinus, and eventually exits via efferent vessels. as antigens within the lymph travel through the sinuses, they are captured and processed by macrophages and dcs. alternatively, dcs charged with antigen can migrate within blood vessels to the node and enter the paracortex via the hevs. circulating b lymphocytes also enter across the hevs, and if they encounter antigen-bearing dcs, there is a local reaction involving the appropriate t helper lymphocytes, b lymphocytes, and dcs. this results in the migration of the activated b lymphocytes to a primary follicle, where they initiate formation of a germinal center. germinal centers, upon migration of antigen-activated b lymphocytes, develop a characteristic architecture. distinct polarity composed of a superficial or light zone and a deep dark zone is present in cases of antigenic stimulation. the light zone, orientated at the source of antigen, consists mainly of small lymphocytes, called centrocytes, which have moderate amounts of pale eosinophilic cytoplasm. the cells of the dark zone, called centroblasts, are large, densely packed lymphocytes with scant cytoplasm, giving this area a darker appearance on h&e staining. the centroblasts undergo somatic mutations of the variable regions of the immunoglobulin gene, followed by isotype class switching (from igm to igg or iga). during this process most centroblasts undergo apoptosis, and cell fragments are phagocytized by macrophages, which are then termed tingible (stainable) body macrophages. the cells that have survived the affinity maturation process are now called centrocytes and along with t lymphocytes and follicular dcs, populate the germinal center light zone. these post-germinal center b lymphocytes leave the follicle as plasma cell precursors (immunoblasts or plasmablasts) and migrate from the cortex to the medullary cords, where they mature and excrete antibody into the efferent lymph. some of these cells may colonize the region surrounding the mantle cell zone to form a marginal zone. marginal zones are apparent only in situations of prolonged and intense immune stimulation and serve as a reservoir of memory cells. the elliptical mantle cell cuff is wider over the light pole of the follicle, though in instances of strong antigenic stimulation, the cuffs can completely encircle the germinal center. responses to injury are listed in box 13-9, and the responses are discussed on the basis of the following systems: sinus histiocytes of the monocyte-macrophage system, cortex, paracortex, and medulla (medullary sinuses and medullary cords). generally, enlarged lymph nodes can be distributed in several different patterns in the body. first, all lymph nodes throughout the body (systemic or generalized) may be enlarged (lymphadenopathy or lymphadenomegaly). this pattern is usually attributed to systemic infectious, inflammatory, or neoplastic processes. if a single lymph node or regional chain of nodes is enlarged, then the area drained by that node should be checked for lesions (e.g., evaluate the oral cavity if the mandibular lymph nodes are enlarged). thus it is important to know the area drained by specific lymph nodes. mesenteric lymph nodes are normally larger because of follicular approximately 90% to 95% of lymphocytes enter lymph nodes through the hevs, which also play an important role in lymph fluid balance. the lymphatic vasculature consists of afferent lymphatic vessels, which pierce the capsule and drain into the subcapsular sinus. lymph continues to drain through the trabecular sinuses to the medullary sinuses and finally exits at the hilus via efferent lymphatic vessels. all lymph nodes receive afferent lymphatic vessels from specific areas of the body. the term lymphocenter is often used in veterinary anatomy to describe a lymph node or a group of lymph nodes that is consistently present at the same location and drains from the same region in all species. for example, the popliteal lymph node, caudal to the stifle, drains the distal hind limb. the tracheobronchial nodes (bronchial lymphocenter), located at the tracheal bifurcation, collect lymph from the lungs and send it to the mediastinal nodes or directly to the thoracic duct. because lymph from a single afferent lymphatic vessel drains into a discrete region of a lymph node, only these regions of the node may be affected by the contents of a single draining lymph vessel (e.g., antigen, infectious organisms, or metastatic neoplasms [ fig. 13-47] ). the lymph node of the pig has a different structure. the afferent lymphatic vessels enter at the hilus instead of around the periphery of the node and empty lymph into the center of the node. the lymph drains to the "subcapsular" sinus (the equivalent of the medullary sinuses of other domestic animals) and then into several efferent lymphatic vessels, which pierce the outer capsule. this reversal of flow is the result of an inverted nodal architecture, with the cortex in the middle of the node surrounded by the medulla at the periphery. thus a pig lymph node that is draining an area of hemorrhage will have blood accumulate in the periphery (subcapsular) instead of in the center of the node (which may be grossly visible). the functions of the lymph node are (1) to filter lymph of particulate matter and microorganisms, (2) to facilitate the surveillance and processing of incoming antigens via interactions with b and t lymphocytes, and (3) to produce b lymphocytes and plasma cells. material arriving in the lymph can be subdivided into free particles that is undergoing follicular hyperplasia is enlarged and has a taut capsule, and the cut surface may bulge. histologically, the follicles contain active germinal centers with antigenic polarity (light and dark zones) (figs. 13-49 and 13-50; also see fig. 13-46) . depending on the duration and continued exposure to the antigen, there may also be concomitant paracortical hyperplasia and medullary cord hyperplasia and sinus histiocytosis, because these nodes continuously receive and respond to barrages of antigens and bacteria from the intestinal tract. sinus histiocytes (macrophages) are part of the monocyte-macrophage system and the first line of defense against infectious and noninfectious agents in the incoming lymph. in response to these draining agents, there is hyperplasia of the macrophages ("sinus histiocytosis"), most notable in the medullary sinuses ( fig. 13-48) . leukocytes, often monocytes, may harbor intracellular pathogens (e.g., mycobacterium spp., cell-associated viruses such as parvovirus), arrive in the blood or lymph, infect the lymph node, and then are disseminated throughout the lymphoid tissues of the body via the efferent lymph and circulating blood. cortex (lymphoid follicles). follicular hyperplasia of the cortex is discussed in the section lymphoid/lymphatic system, lymph node, function. an antigenically stimulated lymph node the two main portals of entry to the lymph node for infectious agents and antigens are afferent lymphatic vessels (lymphatic spread) and blood vessels (hematogenous spread). portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box 13-6. infectious microorganisms, either free within the lymph or within lymphocytes or monocytes, are transported to regional lymph nodes through lymphatic vessels. agents may escape removal by phagocytosis in one lymph node and be transported via efferent lymphatic vessels to the next lymph node in the chain and cause an inflammatory or immunologic response there. this process can continue serially down a lymph node chain, and if the agent is not removed, it may eventually be transported via the lymphatic vessels to either the cervical or thoracic ducts and then disseminated throughout the body. although most pathogens are transported to lymph nodes via afferent lymphatic vessels, bacteria can be transported to lymph nodes hematogenously (free or within leukocytes such as monocytes) in septicemias and bacteremias. direct penetration of a lymph node is uncommon, because it is protected by a thick fibrous capsule. occasionally, inflammatory cells or neoplasms can extend directly into nodal parenchyma from adjacent tissues. defense mechanisms used by the lymphatic system to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsules of lymph nodes. hemal nodes are small, dark red to brown nodules found most commonly in ruminants, mainly sheep, and have also been reported in horses, primates, and some canids. their architecture resembles that of a lymph node with lymph follicles and sinuses, except that in the hemal node, sinuses are filled with blood (efig. 13-8) . because erythrophagocytosis can be present, it is presumed that hemal nodes can filter blood and remove senescent erythrocytes, but as their blood supply is small, their functional importance is not clear. malt is the initial site for mucosal immunity and is crucial in the protection of mucosal barriers. malt is composed of both diffuse lymphoid tissues and aggregated lymphoid (also known as lymphatic) nodules, which can be subcategorized based on their anatomic location: (1) bronchus-associated lymphoid tissue (balt), which is often at the bifurcation of the bronchi and bronchioles; (2) tonsils (pharyngeal and palatine) form a ring of lymphoid tissue at the oropharynx; (3) nasal-, larynx-, and auditory tube-associated lymphoid tissues (nalt, lalt, and atalt, respectively) within the nasopharyngeal area; (4) gut-associated lymphoid tissue (galt), which includes peyer's patches and diffuse lymphoid tissue in the gut wall; (5) conjunctiva-associated lymphoid tissue (calt); (6) other lymphoid nodules (e.g., genitourinary tract) (fig. 13-51) . diffuse lymphoid tissue consists of lymphocytes and dcs within the lamina propria of the mucosa of the alimentary, respiratory, and genitourinary tracts. these cells intercept and process antigens, which then travel to regional lymph nodes to initiate the immune response, leading ultimately to the secretion of iga, igg, and igm. plasmacytosis. less florid follicular reactions will have smaller separated germinal centers, whereas nodes receiving persistent high levels of antigen stimulation may have coalescing germinal centers (termed "atypical benign follicular hyperplasia"). in such cases of chronic strong antigenemia, the highly reactive nodes may also exhibit colonization of lymphocytes into perinodal fat, and germinal centers may contain irregular lakes of eosinophilic material, known as follicular hyalinosis. as the immune response declines, there is follicular lymphoid depletion and the concentration of lymphocytes in the germinal centers is reduced, allowing the underlying follicular stroma (including dcs and macrophages) to become visible. with ongoing lymphocyte depletion, the mantle cell zones are thinned, less populated, and discontinuous. eventually, residual mantle cells collapse into the follicular stroma, forming clusters of small dark cells within the bed of dcs and macrophages, referred to as fading follicles. paracortex. paracortical atrophy may result from a variety of causes, including deficiency in lymphocyte production in the bone marrow, reduced differential selection of lymphocytes in the thymus, or destruction of lymphocytes in the lymph node by viruses, radiation, and toxins directly on the lymphocytes in the lymph node (see box 13-5). examination of h&e-stained sections allows evaluation of follicular activity in the cortex and the concentration of plasma cells in the medullary cords, which serve as a reasonable estimate of b lymphocyte activity for comparison. paracortical hyperplasia may have a nodular or diffuse appearance depending on which and how many afferent lymphatic vessels are draining antigen. this reaction may precede or be concurrent with the germinal center reaction of follicular hyperplasia. proliferation of t lymphocytes has been reported in the paracortex (and pals of the spleen) in malignant catarrhal fever (mcf) in cattle and in pigs with porcine reproductive and respiratory syndrome. pcv2 can cause a diffuse proliferation of macrophages within the paracortex. responses to injury by the medullary sinuses are dilation of the sinuses and proliferation of histiocytes ("sinus histiocytosis"). sinus macrophages proliferate in response to a wide variety of particulate matter in the lymph, including bacteria and erythrocytes (erythrophagocytosis) draining from a hemorrhagic area (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, pigmentation of lymph nodes). dilation of the sinuses due to edema occurs with many underlying conditions, including chronic cardiac failure or drainage from an acutely inflamed area. as the inflammation progresses, the sinuses become filled with neutrophils, macrophages, and occasionally fibrin, in addition to the hyperplastic resident sinus histiocytes (see fig. 13-48) . depending on the intensity of the inflammation, the adjacent parenchyma may become affected (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly], acute lymphadenitis). as pointed out in the section on lymph nodes, function, after activation and proliferation of b lymphocytes in the follicle, the immunoblasts formed there move to and mature in the medullary cords, which as a result are distended with plasma cells that secrete antibody into the efferent lymphatic vessels ("medullary plasmacytosis"). the concentration of medullary plasma cells correlates with the activity of the germinal centers. as the immune response subsides, the number of plasma cells decreases and the medullary cords return to their resting state populated by few lymphocytes and scattered plasma cells. chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system composition. for instance, m cells increase in animals transferred from pathogen-free housing to the normal environment. m cells may also be exploited as a portal for entry by some microbes (see lymphoid/lymphatic system, portals of entry/pathways of spread). table 13 -5 lists the interactions of the malt with different microorganisms. the responses of malt to injury are similar to those of other lymphoid tissues: hyperplasia, atrophy, and inflammation (box 13-10). hyperplasia. hyperplasia of lymphoid nodules is a response to antigenic stimulation and consists of activation of germinal centers with subsequent production of plasma cells (see fig. 13-51, b) . lymphoid nodule hyperplasia is often present in chronic disease conditions, such as balt hyperplasia in chronic dictyocaulus spp. (horses, cattle, sheep, and goats) or metastrongylus spp. (pigs) associated bronchitis or bronchiolitis. mycoplasma spp. pneumonias of sheep and pigs display marked balt hyperplasia that can encircle bronchioles and bronchi ("cuffing pneumonia"). hyperplastic lymphoid nodules can be so enlarged that they become grossly visible as discrete white plaques or nodules (see fig. 13 -51, a). they can be seen in the conjunctiva of the eyelids and the third eyelid in chronic conjunctivitis, the pharyngeal mucosa in chronic pharyngitis, the gastric mucosa in chronic gastritis, and the urinary bladder in chronic cystitis (follicular cystitis). the normal fetus has no detectable balt, though it may be present in fetuses aborted due to infectious disease. atrophy. atrophy of the diffuse lymphoid tissue and lymphoid nodules has the same causes as atrophy affecting other lymphoid tissues (see box 13-5) and includes lack of antigenic stimulation, cachexia, malnutrition, aging, viral infections, or failure to be repopulated by b lymphocytes from the bone marrow or t lymphocytes from the thymus. lymphocytolysis of germinal center lymphocytes of peyer's patches is a characteristic lesion in bvdv infection in ruminants and canine and feline parvovirus infections ("punchedout peyer's patches) (see chapters 4 and 7). the main portals of entry to malt for infectious agents are hematogenous spread and through migrating macrophages, dcs , and m cells. pathogenic bacteria such as escherichia coli, yersinia pestis, mycobacterium avium ssp. paratuberculosis (map), l. monocytogenes, salmonella spp., and shigella flexneri can invade the host from the lumen of the intestine through dendritic or m cells. some viruses (e.g., reovirus) may be transported by m cells. the scrapie prion protein (prp sc ) may also accumulate in peyer's patches. many viruses, such as bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus, cause lymphocyte depletion within the malt. portals of entry used by microorganisms and other agents and substances to access the lymphoid system are summarized in box 13-6. defense mechanisms used by malt to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters 3, 4, and 5. congenital disorders of the thymus are discussed in detail in chapter 5. summaries of the gross and microscopic morphologic changes are solitary lymphoid nodules are localized concentrations of lymphocytes (mainly b lymphocytes) in the mucosa and consist of defined but unencapsulated clusters of small lymphocytes (primary lymphoid nodule). they are usually not grossly visible in the resting or antigenically unstimulated state, but upon antigenic stimulation, they proliferate and form germinal centers and surrounding mantle cell zones (secondary lymphoid nodules). aggregated lymphoid nodules consist of groups of lymph nodules, the most notable of which are the tonsils and peyer's patches. the aggregated lymphoid follicles of the peyer's patches are most obvious in the ileum. the latter are covered by a specialized epithelium, the follicle-associated epithelium (fae). the fae is the interface between the peyer's patches and the luminal microenvironment and consists of enterocytes and interdigitated m cells. m cells transport (via endocytosis, phagocytosis, pinocytosis, and micropinocytosis) antigens, particles, bacteria, and viruses from the intestinal lumen to the underlying area rich in dcs , which deliver the material to the lymphoid tissue of the peyer's patches. m cells also express iga receptors, which allows for the capture and transport of bacteria entrapped by iga. the proportion of enterocytes and m cells within the fae is modulated by the luminal bacterial a b mercury have a suppressive effect on the immune system. halogenated aromatic hydrocarbons cause dysfunction of dcs through several mechanisms that lead to atrophy of the primary and secondary lymphoid organs. heavy metals, such as lead, mercury and nickel, are immunosuppressive and generally affect the levels of b and t lymphocytes, nk cells, and inflammatory cytokines. other metals, such as selenium, zinc, and vanadium, may be immunostimulatory at low doses. the immunotoxic mechanisms may differ and include chelation of molecules and effects on protein synthesis, cell membrane integrity, and nucleic acid replication. the toxic effects of mycotoxins such as fumonisins b 1 and b 2 (secondary fungal metabolites produced by members of the genus fusarium) and aflatoxin (produced by aspergillus flavus) include lymphocytolysis in the thymic cortex. box 13-10 responses of mucosa-associated lymphoid tissue to injury described in the sections on disorders of horses and disorders of dogs. thymic cysts can be found within the developing and mature thymus and in thymic remnants in the cranial mediastinum. thymic cysts are often lined by ciliated epithelium and represent developmental remnants of branchial arch epithelium and are usually of no significance. thymitis is an uncommon lesion and may be seen in pcv2 infection (see disorders of pigs and also chapter 4), enzootic bovine abortion (see chapter 18), and salmon poisoning disease of dogs (see chapter 7). infectious agents more commonly cause thymic atrophy. variable degrees of acquired immunodeficiency can be also be caused by toxins, chemotherapeutic agents and radiation, malnutrition, aging, and neoplasia. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv-1 in aborted foals (fig. 13-52) , classic swine fever virus, bvdv, canine distemper virus, canine and feline parvovirus, and fiv; severe thymic lymphoid depletion is an early lesion in fiv-infected kittens. environmental toxins, such as halogenated aromatic hydrocarbons (e.g., polychlorinated biphenyls and dibenzodioxins), lead, and thymic hyperplasia. asymptomatic hyperplasia may occur in juvenile animals in association with immunizations and results in symmetrical increase in the size of the thymus. autoimmune lymphoid hyperplasia of the thymus has germinal center formation and occurs with myasthenia gravis. asplenia or the failure of a spleen to develop in utero occurs rarely in animals, and the effect on the animal's immune status is uncertain. (splenic aplasia is present in certain strains of mice, but because these are usually maintained under either germ-free or specific pathogen-free [spf] conditions, the effect of asplenia cannot be evaluated.) congenital immunodeficiency diseases are described in detail in chapter 5, and in the sections on disorders of horses and disorders of dogs. gross examination of the spleen involves deciding whether the spleen is enlarged (splenomegaly), normal, or small (see e-appendix 13-2). diffuse enlargement of the spleen may be due to congestion (termed bloody spleen) or other infiltrative disease (termed meaty spleen). the cut surface of congested spleens will exude blood, whereas meaty spleens are more firm and do not readily ooze blood. the diseases and disorders having splenomegaly are discussed using the following categories, which list the common causes of uniform splenomegaly (table 13-6): • uniform splenomegaly with a bloody consistency (bloody spleen) ( fig. 13-54 , a) • uniform splenomegaly with a firm consistency (meaty spleen) (see fig. 13-54 , b) • splenic nodules with a bloody consistency • splenic nodules with a firm consistency uniform splenomegaly with a bloody consistency-bloody spleen. the common causes of a bloody spleen are (1) congestion (due to gastric volvulus with splenic entrapment, splenic volvulus chemotherapeutic drugs inhibit the cell cycle through various mechanisms, and thus all dividing cells, including lymphocytes, bone marrow cells, and enterocytes, are sensitive to their effects. as such, bone marrow suppression, immunosuppression, and gastrointestinal disturbances are common side effects of anticancer drugs. purine analogues (e.g., azathioprine) compete with purines in the synthesis of nucleic acids, whereas alkylating agents like cyclophosphamide cross-link dna and inhibit the replication and activation of lymphocytes. cyclosporin a specifically inhibits the t lymphocyte signaling pathway by interfering with the transcription of the il-2 gene. methotrexate, a folic acid antagonist, blocks the synthesis of thymidine and purine nucleotides. the immunosuppressive effects of some of these agents is desirable for the treatment of immune-mediated disease (e.g., immune-mediated hemolytic anemia) or to prevent allograft rejection after transplantation. corticosteroids may be given at an immunosuppressive dose, though the degree of suppression is highly variable among species. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissue is within the therapeutically irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. malnutrition and cachexia, which may occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. thymic function may be impaired in young malnourished animals, resulting in a decrease in circulating t lymphocytes and subsequent depletion of t lymphocyte regions of secondary lymphoid organs. lymphoid atrophy may result from physiologic and emotional stress, which can cause the release of catecholamines and glucocorticoids. as part of the general effects of aging in cells (see chapter 1), all lymphoid organs decrease in size (atrophy) with advancing age. in the case of the thymus this reduction in size occurs normally after sexual maturity and is more appropriately termed thymic involution. the term involution should be reserved for normal physiologic processes in which an organ either returns to normal size after a period of enlargement (e.g., postpartum uterus) or regresses to a more primitive state (e.g., thymic involution). because the thymus has both lymphoid and epithelial components, neoplasms may arise from either component. thymic lymphoma arises from the t lymphocytes in the thymus (and very rarely b lymphocytes). it is most often seen in young cats and cattle and less frequently in dogs (fig. 13-53 ) (see hematopoietic neoplasia). thymomas arise from the epithelial component and are usually benign neoplasms that occupy the cranial mediastinum of older animals. histologically, these neoplasms consist of clustered or individualized neoplastic epithelial cells, often outnumbered by nonneoplastic small lymphocytes ("lymphocyte-rich thymoma"). thymomas are common in goats and often contain large cystic structures. immunemediated diseases, including myasthenia gravis and immunemediated polymyositis, occur with thymomas in dogs, and also rarely in cats. myasthenia gravis is caused by autoantibodies directed toward the acetylcholine receptors, which lead to destruction of postsynaptic membranes and reduction of acetylcholine receptors at neuromuscular junction. megaesophagus and aspiration pneumonia are common sequelae to this condition. (syn: necropsy) in horses and dogs that have been euthanized or anesthetized with barbiturates. grossly, the spleen is extremely enlarged (fig. 13-55) , and the cut surface bulges and oozes copious blood. because of the splenic distention, the splenic capsule can be fragile and easily ruptured. histologically, the red pulp is distended by erythrocytes, and the lymphoid tissues of the white pulp are small and widely separated (fig. 13-56 ). electric stunning of pigs at slaughter may result in a large congested spleen; the mechanism is unknown, but it should not be confused with a pathologically congested spleen. splenic congestion in acute cardiac failure is rarely seen in animals. acute congestion/hyperemia. acute septicemias may cause acute hyperemia and concurrent acute congestion of marginal zones and splenic red pulp. microbes are transported hematogenously to these sites, where they are rapidly phagocytized by macrophages. enormous numbers of intravenous bacteria can be cleared by the spleen from the blood in 20 to 30 minutes, but when this defensive mechanism is overwhelmed, the outcome is usually fatal. the response of the spleen depends on the duration of the disease. in acutely fatal cases, such as anthrax and fulminating salmonellosis, distention by blood may be the only gross finding. if the animal survives longer, as in swine erysipelas and the less virulent forms of [all of which compress the splenic vein], and barbiturate euthanasia, anesthesia, or sedation), (2) acute hyperemia (due to septicemia), and (3) acute hemolytic anemia (due to an autoimmune disorder or an infection with a hemotropic parasite). splenic torsion. torsion of the spleen occurs most commonly in pigs and dogs; in dogs this usually involves both spleen and stomach and is seen more often in deep-chested breeds (see chapter 7). in contrast to ruminants, in which the spleen is firmly attached to the rumen, the spleens of dogs and pigs are attached loosely to the stomach by the gastrosplenic ligament. it is the twisting of the spleen around this ligament that results initially in occlusion of the veins, causing splenic congestion, and later in occlusion of the artery, causing splenic infarction. in dogs the spleen is uniformly and markedly enlarged and may be blue-black from cyanosis. it is often folded back on itself (visceral surface to visceral surface) in the shape of the letter "c." treatment for this condition is most often splenectomy. barbiturate euthanasia, anesthesia, or sedation. intravenous injection of barbiturates induces acute passive congestion in the spleen due to relaxation of smooth muscle in the capsule and trabeculae. this phenomenon is seen most dramatically at autopsy only histologic lesion may be marked congestion of the marginal sinuses and the splenic red pulp vascular spaces. at low magnification, congestion of the marginal sinus may appear as a circumferential red ring around the splenic follicle, and there is marked lymphocytolysis of follicles and pals. intravascular free bacilli are noted and may be seen in impression smears of peripheral blood, presumably because death is so rapid from the anthrax toxin that there is insufficient time for phagocytosis to take place. if the animal lives longer, scattered neutrophils are present in the marginal sinuses and red pulp vascular spaces (fig. 13-57 ). anthrax cases are not normally autopsied because exposure to air causes the bacteria to sporulate-anthrax spores are extremely resistant and readily contaminate the environment. acute hemolytic anemias. hemolytic diseases, including acute babesiosis, hemolytic crises in equine infectious anemia, and immune-mediated hemolytic anemia, can cause marked splenic congestion. the splenic congestion is due to the process of removal (phagocytosis) and storage of large numbers of sequestered parasitized and/or altered erythrocytes from the circulation. histologically, there is dilation of the red pulp vascular spaces with erythrocytes and erythrophagocytes. with chronicity there is hyperplasia of the red pulp macrophages, hemosiderosis, and reduced congestion because the number of sequestered diseased erythrocytes is diminished. spleen. the three general categories of conditions leading to uniform splenomegaly with a firm meaty consistency are (1) marked phagocytosis of cells, debris, or foreign agents/material; (2) proliferation or infiltration of cells as occurs in diffuse lymphoid and histiocytic hyperplasia, diffuse granulomatous disease (e -table 13 -2), emh, and neoplasia; (3) storage of materials in storage diseases or amyloidosis. it is important to recognize that more than one of these processes can occur in the same patient (e.g., dogs with immunemediated hemolytic anemia may have both marked erythrophagocytosis and emh). the appearance of the cut surface of a meaty spleen depends on the underlying cause. in diffuse marked lymphoid hyperplasia, large, disseminated, discrete, white, bulging nodules are visible. spleens with diffuse infiltrative neoplasms, such as lymphoma, are pink-light purple on cut surface. diffuse lymphoid hyperplasia. lymphoid hyperplasia has been described in detail in the section on dysfunction/responses to injury. in cases of prolonged antigenic stimulation the lymphoid salmonellosis, there may be sufficient time for neutrophils and macrophages to accumulate in the marginal sinuses, marginal zones, and splenic red pulp vascular spaces. anthrax. b. anthracis, the causative agent of anthrax, is a grampositive, large, endospore-forming bacillus, which grows in aerobic to facultative anaerobic environments. anthrax is primarily a disease of ruminants, especially cattle and sheep (see chapters 4, 7, 9, and 10) . once the spores are ingested, they replicate locally in the intestinal tract, spread to regional lymph nodes, and then disseminate systemically through the bloodstream, resulting in septicemia. b. anthracis produces exotoxins, which degrade endothelial cell membranes and enzyme systems. grossly, the spleen is uniformly enlarged and dark red to bluishblack and contains abundant unclotted blood. in peracute cases the 782.e1 chapter 13 bone marrow, blood cells, and the lymphoid/lymphatic system diffuse granulomatous disease. chronic infectious diseases may cause a uniformly firm and enlarged spleen, mostly due to macrophage hyperplasia and phagocytosis, diffuse lymphoid hyperplasia, or diffuse granulomatous disease. diffuse granulomatous diseases (see etable 13 -2) occur in (1) intracellular facultative bacteria that infect macrophages (e.g., mycobacterium spp., brucella spp., and francisella tularensis); (2) systemic mycoses (e.g., blastomyces dermatitidis, histoplasma capsulatum) (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly]) ( fig. 13-59, a and b) , and (3) protozoal infections that infect macrophages (e.g., leishmania spp.). some of these organisms may also produce nodular spleens with the formation of discrete to coalescing granulomas (e.g., m. bovis) (see splenic nodules with a firm consistency). follicles throughout the splenic parenchyma can become enlarged and visible on gross examination (fig. 13-58 ), leading to diffuse splenomegaly. in contrast to b lymphocyte hyperplasia of the lymphoid follicles, certain diseases (e.g., malignant catarrhal fever in cattle) may lead to t lymphocyte hyperplasia of the pals. diffuse histiocytic hyperplasia and phagocytosis. splenomegaly from hyperplasia and increased phagocytosis of splenic macrophages is a response to the need to engulf organisms in prolonged bacteremia or parasitemia from hemotropic organisms. whereas acute hemolytic anemias cause splenomegaly with congestion (bloody spleen), with chronicity there is decreased sequestration of diseased erythrocytes and hence less congestion. therefore in cases of chronic hemolytic disease, splenomegaly is attributed to diffuse proliferation of macrophages, phagocytosis, and concurrent hyperplasia of the white pulp due to ongoing antigenic stimulation. for example, equine infectious anemia has cyclical periods of viremia, with immune-mediated damage to erythrocytes and platelets, and phagocytosis to remove altered erythrocytes and platelets. these cycles result in proliferation of red pulp macrophages, hyperplasia of hematopoietic cells (emh) to replace those lost, and hyperplasia of lymphocytes in the white pulp. in animals less than 1 year of age. in general, these substrates are lipids and/or carbohydrates that accumulate in the cells, the result of the lack of normal processing within lysosomes. major categories of stored materials include mucopolysaccharides, sphingolipids, glycolipids, glycoproteins, glycogen, and oligosaccharides. macrophages are commonly affected by storage diseases, and thus extramedullary hematopoiesis. emh is the development of blood cells in tissues outside the medullary cavity of the bone (efig. 13-9 ). the formation of single or multiple lineages of hematopoietic cells is often observed in many tissues and commonly in the spleen. the ability of blood cell precursors to home, proliferate, and mature in extramedullary sites relies on the presence of hscs and pathophysiologic changes in the microenvironment (i.e., extracellular matrix, stroma, and chemokines). in the spleen, hscs have been found within vessels and adjacent to endothelial cells to form a vascular niche; thus splenic emh occurs in the red pulp, both within the red pulp vascular spaces and sinusoids (of the dog). the predilection for emh to occur varies among species (for instance, splenic emh persists throughout adulthood in mice), and the underlying mechanisms are not completely understood, but four major theories to explain the causes of emh are (1) severe bone marrow failure; (2) myelostimulation; (3) tissue inflammation, injury, and repair; and (4) abnormal chemokine production. because splenic emh is often observed in animals without obvious hematologic abnormalities, tissue inflammation, injury, and repair is the most likely mechanism of emh in this organ. in dogs and cats emh occurs most frequently with degenerative and inflammatory disorders, such as lymphoid nodular hyperplasia, hematomas, thrombi, histiocytic hyperplasia, inflammation (e.g., fungal splenitis), and neoplasia. emh in multiple tissues may be observed in chronic cardiovascular or respiratory conditions, chronic anemia, or chronic suppurative diseases in which there is an excessive tissue demand for neutrophils that exceeds the supply available from the marrow (e.g., canine pyometra). primary neoplasms. primary neoplastic diseases of the spleen arise from cell populations that normally exist in the spleen and include hematopoietic components, such as lymphocytes, mast cells, and macrophages, and stromal cells, such as fibroblasts, smooth muscle, and endothelium. the primary neoplasms that result in diffuse splenomegaly are the round cell tumors, including lymphoma ( fig. 13-60) , leukemia, visceral mast cell tumor, and histiocytic sarcoma. it is important to note that all of these types of neoplasms can produce nodular lesions instead of-or along with-a diffusely enlarged spleen. the different types of lymphoma in domestic animals are discussed in the section on hematopoietic neoplasia. secondary neoplasms of the spleen are due to metastatic spread and most often form nodules in the spleen, not a uniform splenomegaly. amyloid. the accumulation of amyloid in the spleen may occur with primary (al) or secondary (aa) amyloidosis (see chapters 1 and 5). rarely, severe amyloid accumulation may cause uniform splenomegaly ( fig. 13-61) , in which the spleen is firm, rubbery to waxy, and light brown to orange. microscopically, amyloid is usually in the splenic follicles, which if large enough, are grossly visible as approximately 2-mm-diameter gray nodules. amyloid deposition can also be seen within the walls of splenic veins and arterioles. plasma cells tumors within the spleen may also be associated with amyloid (al) deposits. lysosomal storage diseases. storage diseases are a heterogeneous group of inherited defects in metabolism characterized by accumulation of storage material within the cell (lysosomes). genetic defects, which result in the absence of an enzyme, the synthesis of a catalytically inactive enzyme, the lack of activator proteins, or a defect in posttranslational processing, can lead to a storage disease. acquired storage diseases are caused by exogenous toxins, most often plants that inhibit a particular lysosomal enzyme (e.g., swainsonine toxicity due to indolizidine alkaloid found in astragalus and oxytropis plant spp.). storage diseases typically occur diagnosis, it may be difficult (and futile) to determine the primary site. histologically, hemangiosarcomas are composed of plump neoplastic endothelial cells, which wrap around stroma to form haphazardly arranged and poorly defined blood-filled vascular spaces ( fig. 13-66 ). splenic nodules with a firm consistency. the most common disorders of the spleen with firm nodules are (1) lymphoid nodular hyperplasia, (2) complex nodular hyperplasia, (3) primary neoplasms, (4) secondary metastatic neoplasms, (5) granulomas, and (6) abscesses. lymphoid and complex nodular hyperplasia. see lymphoid/ lymphatic system, disorders of dogs. primary neoplasms. the primary neoplastic diseases of the spleen that result in firm nodules include lymphoma (multiple subtypes), histiocytic sarcoma, leiomyoma, leiomyosarcoma, fibrosarcoma, myelolipomas, liposarcomas, myxosarcomas, undifferentiated pleomorphic sarcomas, solid hemangiosarcomas, and rare reports of primary chondrosarcomas. these locally extensive neoplasms may be solitary or multiple, raised above the capsular surface, but usually confined by the capsular surface. the consistency and cut surface appearance varies depending on the type of neoplasm; spindle cell tumors like leiomyosarcomas and fibrosarcomas will be white and firm, liposarcomas and myelolipomas are soft and bulging, and myxomatous neoplasms are gelatinous. it is important to remember that many round cell neoplasms, such as lymphoma, mast cell tumors, plasma cell tumors, myeloid neoplasms, and histiocytic sarcomas, can form nodules or diffuse splenic enlargement (or both). metastatic neoplasms. neoplasms that metastasize to the spleen usually result in enlarged nodular spleens (fig. 13-67 ) and include any number of sarcomas, carcinomas, or malignant round cell tumors. metastatic sarcomas can include fibrosarcomas, leiomyosarcomas, chondrosarcomas, and osteosarcomas. mammary, prostatic, pulmonary, anal sac gland and neuroendocrine carcinomas may metastasize widely to abdominal viscera, including the spleen. granulomas and abscesses. microorganisms that cause diffuse granulomatous splenitis and uniform splenomegaly may also cause focal to multifocal nodular lesions (e.g., mycobacterium spp., fungal organisms) (see diffuse granulomatous diseases of the spleen and also enlarged lymph nodes). although there are a large number of abscesses bulge from the capsule and cut surfaces, and the exudate can vary in amount, texture, and color depending on the inciting organism and the age of the lesion. the most common diseases or conditions that have small spleens are (1) developmental anomalies, (2) aging changes, (3) wasting and/or cachectic diseases, and (4) splenic contraction. splenic hypoplasia. primary immunodeficiency diseases can result in splenic hypoplasia, as well as small thymuses and lymph nodes (which may be so small as to be grossly undetectable in some diseases). these diseases affect young animals and involve defects in t and/or b lymphocytes (fig. 13-70) . spleens are exceptionally small, firm, and pale red and lack lymphoid follicles and pals. these diseases and their pathologic findings are discussed in chapter 5 and in the sections on disorders of horses and disorders of dogs. congenital accessory spleens. accessory spleens can be either congenital or acquired (see splenic rupture). congenital accessory spleens are termed splenic choristomas, which are nodules of normal splenic parenchyma in abnormal locations. these are usually small diseases and conditions commonly caused by bacteremia (e.g., navel ill, joint ill, chronic respiratory infections, bacterial endocarditis, chronic skin diseases, castration, tail docking, and ear trimming and/ or notching), these rarely result in visible splenic abscesses. pyogranulomas and abscesses in the spleen (multifocal chronic suppurative splenitis) that do develop after septicemia and/or bacteremia are usually caused by pyogenic bacteria such as streptococcus spp., rhodococcus equi (fig. 13-68) , trueperella pyogenes ( fig. 13-69) , and corynebacterium pseudotuberculosis. cats with the wet or dry form of feline infectious peritonitis virus may have nodular pyogranulomatous and lymphoplasmacytic inflammatory foci throughout the spleen. splenic abscesses due to direct penetration by a migrating foreign body are reported in cattle (from the reticulum) and less commonly in the horse (from the stomach). perforating gastric ulcers in horses due to gasterophilus and habronema spp. have also reportedly led to adjacent splenic abscesses. granulomas and a b and may be located in the gastrosplenic ligament, liver, or pancreas (see fig. 13-74, b) . splenic fissures. fissures in the splenic capsule are elongated grooves whose axes run parallel to the borders of the spleen. this developmental defect is seen most commonly in horses but also occurs in other domestic animals and has no pathologic significance. the surface of the fissure is smooth and covered by the normal splenic capsule. aging changes. as part of the general aging change of cells as the body ages, there is reduction in the number of b lymphocytes produced by the bone marrow and decline of naïve t lymphocytes due to age-related thymic involution. consequently, there is lymphoid atrophy in secondary lymphoid organs. the spleen is small, and its capsule may be wrinkled. microscopically, the white pulp is atrophied, and splenic follicles, if present, lack germinal centers. sinuses may also collapse from a reduced amount of blood, possibly because of anemia, which makes the red pulp appear fibrous. wasting/cachectic diseases. any chronic disease, such as starvation, systemic neoplasia, and malabsorption syndrome, may produce cachexia. starvation has a marked effect on the thymus, which results in atrophy of the t lymphocyte areas in the spleen and lymph nodes, which is in part mediated by leptin. b lymphocyte development is also diminished, because b lymphocytes require accessory signals from helper t lymphocytes to undergo somatic hypermutation and immunoglobulin isotype switching. splenic contraction. contraction of the spleen is a result of contraction of the smooth muscle in the capsule and trabeculae of storage spleens. it can be induced by the activation of the sympathetic "fight-or-flight" response and is seen in patients with heart failure or shock (cardiogenic, hypovolemic, and septic shock) and also occurs in acute splenic rupture that has resulted in massive hemorrhage (hemoabdomen/hemoperitoneum). the contracted spleen is small, its surface is wrinkled, and the cut surface is dry. hemosiderosis. hemosiderin is a form of storage iron derived chiefly from the breakdown of erythrocytes, which normally takes place in the splenic red pulp. thus some splenic hemosiderosis is to be expected, and the amount varies with the species (it is most extensive in the horse). excessive amounts of splenic hemosiderin are seen when erythropoiesis is reduced (less demand for iron) or from the rapid destruction of erythrocytes in hemolytic anemias (increased stores of iron), such as those caused by immune-mediated hemolytic anemias or hemotropic parasites. excess splenic hemosiderin may also occur in conditions such as chronic heart failure or injections of iron dextran or as focal accumulations at the sites of old hematomas, infarcts, or trauma-induced hemorrhages. hemosiderin is also present in siderofibrotic plaques. siderofibrotic plaques. siderofibrotic plaques are also known as siderocalcific plaques and gamna-gandy bodies. grossly, they are gray-white to yellowish, firm, dry encrustations on the splenic capsule. usually they are most extensive along the margins of the spleen but can be elsewhere on the capsule (fig. 13-71 ) and sometimes in the parenchyma. with h&e staining these plaques are a multicolored mixture of yellow (hematoidin), golden brown (hemosiderin), purple-blue (hematoxylinophilic calcium mineral), and pink (eosinophilic fibrous tissue) (fig. 13-72 the diseases or conditions with small lymph nodes are (1) congenital disorders, (2) lack of antigenic stimulation, (3) viral infections, (4) cachexia and malnutrition, (5) aging, and (6) radiation. congenital disorders. primary immunodeficiency diseases are described in detail in chapter 5 and in the sections on disorders of horses and disorders of dogs. neonatal animals with primary immunodeficiency diseases often have extremely small to undetectable lymph nodes. in dogs and horses with severe combined immunodeficiency disease (scid), lymphoid tissues, including lymph nodes from affected animals, are often grossly difficult to identify and characterized by an absence of lymphoid follicles. congenital may represent sequelae to previous hemorrhages from trauma to the spleen. splenic rupture. splenic rupture is most commonly caused by trauma, such as from an automobile accident or being kicked by other animals. thinning of the capsule from splenomegaly can render the spleen more susceptible to rupture, and this may occur at sites of infarcts, hematomas, hemangiosarcomas, and lymphoma. in acute cases of splenic capsular rupture, the spleen is contracted and dry and the surface wrinkled from the marked blood loss (fig. 13-73) . in more severe cases the spleen may be broken into two or more pieces, and small pieces of splenic parenchyma may be scattered throughout the omentum and peritoneum (sometimes called splenosis) (fig. 13-74, a) . clotted blood, fibrin, and omentum may adhere to the surface at the rupture site. if the rupture is not fatal, the spleen heals by fibrosis, and there may be a capsular scar. occasionally there are two or more separate pieces of spleen adjacent to each other and sometimes joined by scar tissue in the gastrosplenic ligament. the functional capabilities of the small accessory spleens are questionable, although erythrophagocytosis, hemosiderosis, hyperplastic nodules, emh, and neoplasia can be present in these nodules. accessory spleens due to traumatic rupture should be distinguished from peritoneal seeding of hemangiosarcoma and the developmental anomaly splenic choristomas (see fig. 13-74, b) , which are nodules of normal splenic parenchyma in abnormal locations (such as liver and pancreas). chronic splenic infarcts. in the early stage, splenic infarcts are hemorrhagic and may elevate the capsule (see splenic nodules with a bloody consistency). however, as the lesions age and fibrous connective tissue is laid down, they shrink and become contracted and often depressed below the surface of the adjacent capsule. conditions causing lymphadenomegaly include (1) lymphoid hyperplasia (follicular or paracortical), (2) hyperplasia of the sinus histiocytes (monocyte-macrophage system), (3) acute or chronic lymphadenitis, (4) lymphoma, and (5) metastatic neoplasia. macrophage system. detailed descriptions of lymphoid follicular hyperplasia, paracortical hyperplasia, and hyperplasia of sinus histiocytes are in the sections on lymph nodes, function, and lymph node, dysfunction/responses to injury. follicular lymphoid hyperplasia can involve large numbers of lymph nodes, as in a systemic disease, or can be localized to a regional lymph node draining an inflamed or antigenically stimulated (e.g., vaccine injection) area. acute lymphadenitis. lymph nodes draining sites of infection and inflammation may develop acute lymphadenitis (e.g., retropharyngeal lymph nodes draining the nasal cavity with acute rhinitis, tracheobronchial lymph nodes in animals with pneumonia ( fig. 13-75 ), and mammary [supramammary] lymph nodes in animals with mastitis). grossly, affected lymph nodes in acute lymphadenitis are red and edematous, have taut capsules, and may have necrotic areas ( fig. 13-76 ). in some instances the afferent lymphatic vessels may also be inflamed (lymphangitis). the material draining to the regional lymph node may be microorganisms (bacteria, parasites, protozoa, and fungi), inflammatory mediators, or a sterile irritant. in septicemic diseases, such as bovine anthrax, the lymph nodes are markedly congested and the sinuses filled with blood. examination of these lymph nodes should include culturing for bacteria and the examination of smears and histologic sections for bacteria and fungi. pyogenic bacteria, such as streptococcus equi ssp. equi in horses , streptococcus porcinus in pig, and trueperella pyogenes in cattle and sheep, cause acute suppurative lymphadenitis (see disorders of horses and disorders of pigs). hereditary lymphedema has been reported in certain breeds of cattle and dogs. grossly, the most severely affected animals have generalized subcutaneous edema (see fig. 2 -10) and effusions. in severe cases the peripheral and mesenteric lymph nodes are hypoplastic and characterized by an absence of follicles. nodes draining an edematous area may be grossly enlarged from marked sinus edema. lack of antigenic stimulation. the size of the lymph node depends on the level of phagocytosis and antigenic stimulation; lymph nodes that are not receiving antigenic stimuli (e.g., spf animals) will be small with low numbers of primary lymphoid follicles and few, if any, secondary follicles or plasma cells in the medullary cords. conversely, nodes receiving constant antigenic material (such as those draining the oral cavity or intestines) are large with active secondary lymphoid follicles. the number of follicles increases or decreases with changes in the intensity of the antigenic stimuli, and the germinal centers go through a cycle of activation, depletion, and rest, as described previously (see lymph nodes, function). as the antigenic response wanes, germinal centers become depleted of lymphocytes, and lymphoid follicles become smaller. viral infections. many viral infections of animals target lymphocytes and cause the destruction of lymphoid tissue. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv-1 in aborted foals, classic swine fever virus, bvdv, canine distemper virus, and canine and feline parvovirus. although some viruses destroy lymphoid tissue, others can lead to lymph node hyperplasia (e.g., follicular b lymphocyte hyperplasia in fiv and paracortical t lymphocyte hyperplasia in malignant catarrhal fever virus) or cause neoplasia (e.g., felv, blv, and marek's disease). cachexia and malnutrition. malnutrition and cachexia, which occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. starvation has marked effect on the thymus with resultant atrophy of the t lymphocyte areas in the spleen and lymph nodes and may also affect b lymphocyte development. lymphoid atrophy may result from physiologic and emotional stress and the concurrent release of catecholamines and glucocorticoids. glucocorticoids reduce b and t lymphocytes via redistribution of these cells and glucocorticoidinduced apoptosis. t lymphocytes are more sensitive to glucocorticoid-induced apoptosis than are b lymphocytes. aging. as part of the general aging change of cells as the body ages, there is reduction in the number of lymphocytes produced by the bone marrow and regressed thymus, and consequently a reduction in the b and t lymphocytes in secondary lymphoid organs, resulting in lymphoid atrophy. consequently, lymph nodes are small, with loss of b and t lymphocytes and plasma cells in the cortical follicles, paracortex, and medullary cords, respectively. radiation. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissues are within the irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. fibrosis of tissues within the irradiated field also occurs as mainly a late effect of chronic radiation. bouts of chronic lymphadenitis (e.g., regional lymph node draining chronic mastitis in cows) lead to fibrosis and lymphoid hyperplasia, in addition to chronic abscesses. the classic example of chronic suppurative lymphadenitis with encapsulated abscesses is caseous lymphadenitis, a disease of sheep and goats caused by c. (also see disorders of ruminants). it is also the cause of ulcerative lymphangitis in cattle and horses and pectoral abscesses in horses. classic examples of focal to multifocal granulomatous lymphadenitis are mycobacterium tuberculosis complex, which includes m. bovis among others. members of m. avium complex cause similar lesions and have been described in a number of species, including dogs, cats, primates, pigs, cattle, sheep, horses, and human beings. infection may begin by inhalation of aerosol droplets containing the bacilli, which may spread via the lymphatic vessels to regional lymph nodes, resulting in granulomatous lymphangitis and lymphadenitis (fig. 13-81) . initially lesions in the lymphatic system are confined to the lymphatic vessels (granulomatous lymphangitis) and regional lymph nodes (e.g., the tracheobronchial lymph nodes in the case of pulmonary histologically, the subcapsular, trabecular, and medullary sinuses and the parenchyma of the cortex and medulla have focal to coalescing foci of neutrophilic inflammation, necrosis, and fibrin deposition ( fig. 13-78 ). if inflammation in the lymph node continues for several days or longer, the lymph node is further enlarged by follicular hyperplasia and plasmacytosis of the medullary cords from the expected immune response. chronic lymphadenitis. the types of chronic lymphadenitis include chronic suppurative lymphadenitis, diffuse granulomatous inflammation, and discrete granulomas. in chronic suppurative inflammation, abscesses range in size from small microabscesses to large abscesses that occupy and obliterate the whole node. recurrent figure 13 -76 acute lymphadenitis, lymph node, dog. acute lymphadenitis usually occurs when a regional lymph node drains a site of inflammation caused by microorganisms and subsequently becomes infected. the lymph node is firm and enlarged with a tense capsule. the cut surface bulges and is wet with blood, edema, and an inflammatory cell infiltrate. histoplasmosis (see disorders in dogs). in feline cryptococcosis (most often cryptococcus neoformans), the inflammatory response may be mild due to the thick polysaccharide capsule, which has strong immunomodulatory properties and promotes immune evasion and survival within the host. therefore the nodal enlargement is due mainly to a large mass of organisms (see efig. 13-12) . pigs with pcv2 infection may have a multifocal to diffuse infiltrate of macrophages and multinucleated giant cells of varying severity (see disorders of pigs). secondary (metastatic) neoplasms. carcinomas typically metastasize via lymphatic vessels to the regional lymph node. other common metastatic neoplasms include mast cell tumor and malignant melanoma. although sarcomas most often metastasize hematogenously, some more aggressive sarcomas (e.g., osteosarcoma) may spread to regional lymph nodes. histologically, single cells or clusters of neoplastic cells travel via the afferent lymphatic vessels and are deposited in a sinus, usually the subcapsular sinus (see fig. 13 -47). here the cells proliferate and can ultimately occupy the whole lymph node, as well as drain to the next lymph node in the chain. tuberculosis), but once disseminated in the lymph or blood, lymph nodes throughout the body will have lesions. well-organized granulomas consist of a central mass of macrophages with phagocytized mycobacteria, surrounded by epithelioid and foamy macrophages and occasional multinucleated giant cells (langhans type). these inflammatory nodules are surrounded by a layer of lymphocytes enclosed in a fibrous capsule. over time the center of the granuloma may undergo caseous necrosis due to the high lipid and protein content of the dead macrophages (see chapter 3). in bovine johne's disease the mesenteric lymph nodes draining the infected intestine can have noncaseous granulomas (fig. 13-82 ). diffuse granulomatous lymphadenitis. coalescing to diffuse granulomatous lymphadenitis is seen in disseminated fungal infections such as blastomycosis, cryptococcosis (efig. 13-12) , and extent of the emphysema. in severe cases the lymph node is light, puffy, and filled with discrete gas bubbles, and the cut surface may be spongy. histologically, the sinuses are distended with gas and lined by macrophages and giant cells. this change has been considered a foreign body reaction to the gas bubbles. macrophages and giant cells are also seen in afferent lymphatic vessels (granulomatous lymphangitis). vascular transformation of lymph node sinus (nodal angiomatosis). vascular transformation of the sinuses is a nonneoplastic reaction to blocked efferent lymphatic vessels or veins. this pressure-induced lesion results in the formation of anastomosing vascular channels and may be confused with a nodal vascular neoplasm. these proliferative but noninvasive masses usually begin in the subcapsular sinuses and may be followed by lymphoid atrophy, erythrophagocytosis/hemosiderosis, and fibrosis. the blockage may be caused by malignant neoplasms of the tissues that the lymph node drains (e.g., thyroid carcinoma with nodal angiomatosis of the mandibular lymph node). see section on bone marrow, disorders of domestic animals, hematopoietic neoplasia for a discussion of the who classification of hematopoietic neoplasia that predominantly arise and proliferate within bone marrow. this section will cover neoplasms of lymphoid tissue(s) arising outside of bone marrow. lymphoma. the term lymphoma (also known as lymphosarcoma) encompasses a diverse group of malignancies arising in lymphoid tissue(s) outside of bone marrow. grossly, there may be diffuse to nodular enlargement of one or more lymph nodes (fig. 13-83) , and the cut surface is soft, white, and bulging with loss of normal corticomedullary architecture. there is great variation in the clinical manifestations and cytopathologic features of lymphoma, which underlie the importance of classification to better predict the clinical behavior and outcome. an understanding of lymphocyte maturation is crucial, because the who classification of lymphoma postulates a normal cell counterpart for each type of lymphoma (when possible). in other words, lymphoma can arise at any stage in the development/maturation of a lymphocyte-from precursor lymphocytes (b or t lymphoblasts) to mature lymphoid b and t, lymphocytes and nk cells (table 13 -7 and box 13-11). pathologists use gross features, histomorphologic features, immunophenotype (b or t lymphocyte), and clinical characteristics to red discoloration is caused by (1) draining erythrocytes from hemorrhagic or acutely inflamed areas, (2) acute lymphadenitis with hyperemia and/or hemorrhage, (3) acute septicemias with endotoxininduced vasculitis or disseminated intravascular coagulation, and (4) dependent areas in postmortem hypostatic congestion. blood in pig lymph nodes is especially obvious due to the inverse anatomy (the equivalent of the medullary sinuses are subcapsular and thus readily visible in the unsectioned node). initially erythrocytes fill trabecular and medullary sinuses and then rapidly undergo erythrophagocytosis by proliferating sinus macrophages. hemosiderin deposition occurs within 7 to 10 days in these macrophages, imparting a brown discoloration of the node. black discoloration is often present in the tracheobronchial lymph nodes due to draining of carbon pigment (pulmonary anthracosis, see chapter 9). black ink from skin tattoos will drain to the regional lymph node. these pigments are usually noted within the medullary sinus macrophages. brown discoloration may be due to melanin, parasitic hematin, or hemosiderin. melanin pigment is seen in animals with chronic dermatitis when melanocytes are damaged and their pigment is released into the dermis and phagocytized by melanomacrophages (pigmentary incontinence) and drained to the regional lymph node. the mandibular lymph nodes often contain numerous melanomacrophages in animals with heavily pigmented oral mucosa, presumably due to chronic low levels of inflammation. this must be distinguished from metastatic malignant melanomas. lymph nodes draining areas of congenital melanosis may have melanin deposits. parasitic hematin pigment is produced by fascioloides magna (cattle) and fasciola hepatica (sheep) in the liver and then transported via the lymphatic vessels to the hepatic lymph nodes. hemosiderin, an erythrocyte breakdown product, may form in a hemorrhagic node or arrive in hemosiderophages draining from congested, hemorrhagic, or inflamed areas. drainage of iron dextran from an intramuscular injection may also cause hemosiderin pigment accumulation within the draining lymph node. green discoloration is rare and may be caused by green tattoo ink (often used in black animals); ingestion of blue-green algae, which drain to mesenteric lymph nodes; massive eosinophilic inflammation; and in mutant corriedale sheep, which have a genetic defect that results in a deficiency in the excretion of bilirubin and phylloerythrin by the liver. the phylloerythrin or a metabolite stains all the tissues of the body a dark green, except for the brain and spinal cord, which are protected by the blood-brain barrier. miscellaneous discolorations of lymph nodes may be seen with intravenously injected dyes (e.g., methylene blue or trypan blue) or subcutaneous drug injections. lymph nodes may be yellow in severely icteric patients. the pigmented strain of map (johne's disease) may impart an orange discoloration in the mesenteric lymph nodes of sheep. inclusion bodies. many viruses produce inclusion bodies, and some of these occur in lymph nodes. these viruses include ehv-1 in horses, bovine adenovirus, cytomegalic virus in inclusion body rhinitis and pcv2, herpesvirus of pseudorabies in pigs, and rarely parvovirus in dogs and cats. emphysema. emphysema in lymph nodes is a consequence of emphysema in their drainage fields and is seen most frequently in tracheobronchial lymph nodes in bovine interstitial emphysema and in porcine mesenteric lymph nodes in intestinal emphysema (see chapter 7). the appearance of the lymph node varies with the immunohistochemistry (mum1/irf4 is particularly sensitive and specific for plasma cell neoplasms). histiocytic disorders. histiocytic disorders are frequently diagnosed in dogs and occur less often in cats. briefly, histiocytes are categorized as macrophages and dcs, the latter of which are subdivided into langerhans cells (lcs), found in skin, gastrointestinal, respiratory, and reproductive epithelia (mucosae), and interstitial dcs (idc), located in perivascular spaces of most organs. the term interdigitating dcs describes dcs (either resident or migrating) found in t lymphocyte regions of lymph nodes (paracortex) and spleen (pals); interdigitating dcs consist of both lcs and idcs. these lineages can be differentiated using immunohistochemical stains. histiocytic disorders that are diagnosed in veterinary medicine at this time include the following: canine cutaneous histiocytoma, canine lc histiocytosis, canine cutaneous and systemic histiocytosis, feline pulmonary lc histiocytosis, feline progressive histiocytosis, dendritic cell leukemia in the dog, and histiocytic sarcoma and hemophagocytic histiocytic sarcoma in both dogs and cats. lymph node involvement is seen in many of these conditions. rare reports of regional lymph node metastasis in cases of solitary canine cutaneous histiocytoma have been published. lymphatic invasion with subsequent regional nodal involvement may be seen in dogs with lc histiocytosis, which is a poor prognostic indicator and likely reflects systemic infiltration. the normal architecture of tracheobronchial lymph nodes is often effaced in cats with pulmonary lc histiocytosis. canine reactive histiocytoses are not clonal neoplastic proliferations but likely reflect an immune dysregulation consisting of activated dermal idcs (and t lymphocytes). they are categorized as cutaneous histiocytosis (ch), involving skin and draining lymph nodes, and a more generalized systemic histiocytosis (sh), affecting skin and other sites (e.g., lung, liver, bone marrow, spleen, lymph nodes, kidneys, and orbital and nasal tissues). histiocytic sarcoma complex. histiocytic sarcomas (hss) are neoplasms of idcs and therefore can arise in almost any tissue, frequently the spleen, lung, skin, meninges, lymph nodes, bone marrow, and synovium. secondary involvement of the liver is common as the disease progresses. this neoplasm is most commonly diagnosed in dogs, and a lower incidence is seen in cats. localized histiocytic sarcoma may be a focal solitary lesion or multiple nodules within a single organ. disseminated histiocytic sarcoma describes classify lymphomas. the morphologic features used in histopathologic classification are the following: • histologic pattern-nodular or diffuse. • cell size-the nuclei of the neoplastic lymphocytes are compared to the diameter of a red blood cell (rbc ≅ 5 µm). small is less than 1.5 times the diameter of an rbc; intermediate is 1.5 to 2.0 times the diameter of an rbc; large is more than 2.0 times the diameter of an rbc. • grade-mitotic figures are counted in a single high-power (400×) field. indolent is 0 to 1; low is 2 to 5; mid is 6 to 10; high is more than 10. although there are numerous subtypes of lymphoma recognized under the who system, a detailed discussion of each subtype is outside the scope of this textbook. however, a select number of subtypes are more commonly seen in domestic animals (see table 13 -7) and currently best described in the dog (see disorders of dogs, neoplasms, lymphomas). the most common types in dogs are large cell lymphomas and include diffuse large b cell lymphoma and peripheral t cell lymphoma. t cell-rich large b cell lymphoma is thought to be a variant of diffuse large b cell lymphoma with a distinctive reactive t lymphocyte infiltrate. intermediate cell lymphomas include b or t lymphocyte lymphoblastic lymphomas and burkitt-like lymphoma (both high grade), marginal zone lymphoma, and the intermediate cell variant of t zone lymphomas (both indolent and nodular). small cell lymphomas most commonly diagnosed in domestic animal species include enteropathy-associated t cell lymphoma, commonly seen in the cat, t zone lymphoma (small cell variant), and small cell lymphoma. cutaneous lymphomas are most often of t lymphocyte origin and may be epitheliotropic or nonepitheliotropic, and a distinct entity of inflamed t cell lymphoma has been recently described in dogs (see chapter 17). plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone. multiple myeloma. see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, plasma cell neoplasia. extramedullary plasmacytomas. extramedullary plasmacytomas are most commonly diagnosed in the skin of dogs (also cats and horses), where they constitute 1.5% of all canine cutaneous tumors (see chapter 17). the pinnae, lips, digits, and chin are the most commonly affected locations, and most lesions are solitary, though multiple plasmacytomas are infrequently diagnosed. other tissues affected include the oral cavity, intestine (colorectal in particular), liver, spleen, kidney, lung, and brain; of these, the oral cavity and intestine (colorectal) are involved most often. in one study, extramedullary plasmacytomas represented 5% of all canine oral tumors and 28% of all extramedullary plasmacytomas diagnosed. most cutaneous extramedullary plasmacytomas are benign, and complete excision is usually curative; oral cavity and colorectal extramedullary plasmacytomas are likely to behave in a similar manner. more aggressive forms may occur at any site. as with multiple myeloma, the neoplastic cells composing the tumor may vary from well differentiated to pleomorphic, often within the same tumor. the cells often have a characteristic perinuclear golgi clearing or "halo," and the more pleomorphic cells exhibit karyomegaly and binucleation (fig. 13-84) . extramedullary plasmacytomas may produce monoclonal immunoglobulins with resulting monoclonal gammopathy. amyloid deposition (which may mineralize) is also observed in a proportion of cases. differentiation from other round cell tumors may be aided by severe combined immunodeficiency disease of arabian foals is an autosomal recessive primary immunodeficiency disorder characterized by the lack of functional t and b lymphocytes caused by a genetic mutation in the gene encoding for dna-dependent protein kinase catalytic subunit (dna-pkcs). this enzyme is required for receptor gene rearrangements involved in the maturation of lymphocytes, and the resulting loss of functional t and b lymphocytes leads to a profound susceptibility to infectious diseases. though normal at birth, these foals develop diarrhea and pneumonia by approximately 10 days of age, often due to adenovirus, cryptosporidium parvum, and pneumocystis carinii infections. affected foals often die before 5 months of age. lymph nodes and thymus are small and often grossly undetectable, and the spleen is small and firm due to the absence of white pulp (see fig. 13-70) . the development of genetic tests to identify carriers of the disorder has led to a decrease in the prevalence of severe combined immunodeficiency disease. recently, severe combined immunodeficiency disease was diagnosed in a single caspian filly, though the exact genetic defect was not determined. congenital immunodeficiency diseases are also discussed in detail in chapter 5. streptococcus equi ssp. equi, the etiologic agent of equine strangles, is inhaled or ingested after direct contact with the discharge from infected horses or from a contaminated environment. the bacteria attach to the tonsils, penetrate into deeper tissues, enter the lymphatic vessels, drain to regional lymph nodes (mandibular, retropharyngeal, and occasionally parotid and cervical lymph nodes), and cause large abscesses (see fig. 13 -77). retropharyngeal enlargement from abscesses may lead to compression of the pharynx and subsequent respiratory stridor and dysphagia. abscesses may rupture and discharge pus through a sinus to the skin surface or spread medially into guttural pouches, where residual pus dries and hardens to form chondroids (which serve as a nidus for live bacteria to persist in carrier animals). in up to 20% of these cases, ruptured abscess material may spread via blood or lymph to other organs (metastatic abscess formation, bastard strangles), including lung, liver, kidney, synovia, mesenteric and mediastinal lymph nodes, spleen, and occasionally brain. purpura hemorrhagica, a type iii hypersensitivity reaction, may result in necrotizing vasculitis in some horses with repeated natural exposure to s. equi ssp. equi or after vaccination in horses that have had strangles. the typical manifestation of r. equi infection is chronic suppurative bronchopneumonia with abscesses (see chapter 9). approximately 50% of foals also develop intestinal lesions characterized by pyogranulomatous ulcerative enterotyphlocolitis, often over peyer's patches, and pyogranulomatous lymphadenitis of mesenteric and colonic lymph nodes (see chapter 7; see fig. 13-68) . large abdominal abscesses may be the only lesion in the abdomen and presumably originate from an infected mesenteric lymph node. the diffuse lymphatic tissue in the lamina propria may contain granulomatous inflammation with the phagocytized bacteria. mediastinal pyogranulomatous lymphadenitis may compress the trachea, causing respiratory distress. r. equi lesions also can develop in the liver, kidney, spleen, or nervous tissue. lymphoma is the most common malignant neoplasm in horses and mostly affects adult animals (mean age 10 to 11 years) with no lesions that involve distant sites and has replaced the term malignant histiocytosis. breed predispositions to histiocytic sarcoma complex are seen in bernese mountain dogs, rottweilers, golden retrievers, and flat-coated retrievers, though the disease can occur in any breed. histiocytic sarcoma complex is considered to have a rapid and highly aggressive course, and the clinical signs depend on the particular organ(s) involved. grossly, affected organs may be uniformly enlarged and/or contain multiple coalescing white-tan nodules. tissue architecture is effaced by sheets of pleomorphic round to spindle-shaped cells. there is marked cellular atypia with numerous karyomegalic and multinucleated neoplastic cells (fig. 13-85) . hemophagocytic histiocytic sarcoma. hemophagocytic histiocytic sarcoma is seen in dogs and cats and is a neoplasm of macrophages of the spleen and bone marrow. clinically, dogs present with hemolytic regenerative anemia and thrombocytopenia, thus mimicking evans's syndrome, though they are coombs negative. this form of histiocytic sarcoma carries the worst prognosis of the histiocytic sarcomas, which is likely in part related to the severe anemia and coagulopathy. it is characterized by a non-mass forming infiltrate of histiocytes within the bone marrow and splenic red pulp, causing diffuse splenomegaly. the neoplastic cells exhibit marked erythrophagocytosis, but the severe cellular pleomorphism seen in the histiocytic sarcoma complex may be lacking. the neoplastic cells are often intermixed with emh and plasma cells. metastasis is frequently to the liver, where the cells concentrate within the sinuses. tumor emboli within the lung are often present. malt is involved in a variety of ways with bacteria and viruses, and these are summarized for large animals in table 13 -5. these interactions include being a portal of entry for pathogens (e.g., salmonella spp., yersinia pestis, map, and l. monocytogenes); a site of replication for viruses (e.g., bvdv); a site for hematogenous infection (e.g., panleukopenia virus and parvovirus); and a site of gross or microscopic lesions in some viral diseases. bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus cause lymphocyte depletion within the malt. anthrax is caused by b. anthracis, a gram-positive bacillus found in spore form in soil. cattle, sheep, and goats become infected when grazing on infected soil, and infection causes fulminant septicemia. the spleen in infected animals is markedly enlarged and congested (see uniform splenomegaly with a bloody consistency; see also chapter 4). bovine viral diarrhea is caused by bvdv, a pestivirus. cattle are the natural host, but other animals such as alpacas, deer, sheep, and goats are also affected. bvdv preferentially infects cells of the immune system, including macrophages, dcs, and lymphocytes. the associated lesions in lymphoid tissues are severe lymphoid depletion in mesenteric lymph nodes and peyer's patches, whose intestinal surface may be covered by a fibrinonecrotic membrane. histologically, there is marked lymphocytolysis and necrosis of germinal centers in peyer's patches and cortices of lymph nodes. there is thymic atrophy because the thymus is markedly depleted of lymphocytes and may consist of only collapsed stroma and few scattered lymphocytes. bvd is discussed in detail in chapters 4 and 7. apparent breed or sex predisposition. the most frequent anatomic locations of equine lymphoma are multicentric, cutaneous, and gastrointestinal tract. multicentric lymphoma, defined as involving at least two organs (excluding the regional lymph nodes), is the most common manifestation, followed by skin and gastrointestinal tract types. solitary locations have been reported in the mediastinum, lymph nodes, ocular/orbital region, brain, spinal cord, oral cavity, and spleen. of the multicentric lymphomas, the most frequently observed type is t cell-rich large b cell lymphoma (tcrlbcl), reportedly in one study affecting 34% of the cases. peripheral t cell lymphoma (ptcl) was the second most common, followed by diffuse large b cell lymphoma (dlbcl). the most common lymphoma type in the gastrointestinal tract is also t cell-rich large b cell lymphoma, followed by enteropathyassociated t cell lymphoma. cutaneous lymphomas in horses account for up to 3% of all equine skin tumors. t cell-rich large b cell lymphoma is again the most common lymphoma subtype in the skin, representing up to 84% of all cutaneous lymphomas, and most frequently presents clinically as multiple skin masses. cutaneous t cell lymphoma (ctcl) is the second most common form and arises as smaller solitary nodules. thoroughbreds may have a higher incidence of cutaneous t cell lymphoma compared to other breeds. overall, horses with cutaneous t cell-rich large b cell lymphoma appear to have a longer survival time than horses with other types of lymphoma of the skin. progesterone receptor-positive lymphomas have also been identified in horses, and there is one report of subcutaneous tumor regression following removal of an ovarian granulosa-theca cell tumor. there may be an increased frequency of lymphoma in horses diagnosed with equine herpesvirus 5 (ehv-5, gammaherpesvirus), when compared to healthy horses, although the exact cause-effect role of this observation in lymphomagenesis 5 is not yet known. histologically, the hallmark features of t cell-rich large b cell lymphoma include a majority of small (nuclei approximately the size of an rbc), reactive, mature t lymphocytes admixed with a neoplastic population of large b lymphocytes whose nuclei are two to three times the diameter of an equine rbc. these large atypical cells are often binucleated and have prominent eosinophilic nucleoli ( fig. 13-86 ). the large cells may be observed in mitosis or in necrosis as single cells with retracted cytoplasm and pyknotic nuclei. t cell-rich large b cell lymphoma is often accompanied by the presence of a dense fibrovascular network. johne's disease primarily affects domestic and wild ruminants (and rarely pigs and horses) and is due to infection by map. the characteristic lesions include granulomatous enteritis usually confined to the ileum, cecum, and proximal colon; lymphangitis; and lymphadenitis of regional lymph nodes (see fig. 13-82) . the bacteria are ingested, engulfed by the m cells overlying peyer's patches, and then transported to macrophages in the lamina propria and submucosa. among cattle, sheep, goats, and wild ruminants, there is wide variation in the severity, distribution of lesions, primary inflammatory cell type (lymphocytes, epithelioid macrophages, multinucleated giant cells), and numbers of bacteria within lesions (multibacillary or paucibacillary). histologically, the architecture of the ileocecal lymph nodes may be partially replaced by aggregates of epithelioid postweaning multisystem wasting syndrome pcv2, a small single-stranded dna virus, is highly prevalent in the domestic pig population. several clinical syndromes are attributed to pcv2 infection and collectively termed pcv-associated diseases (pcvads). these include postweaning multisystemic wasting syndrome (pmws), porcine respiratory disease complex (prdc), porcine dermatitis and nephropathy syndrome, and enteric disease (see chapters 4 and 9). the major postmortem findings of postweaning multisystemic wasting syndrome are poor body condition, enlarged lymph nodes, and interstitial pneumonia. the lesions of the lymphoid system are commonly observed in the tonsil, spleen, peyer's patches, and lymph nodes. some pigs have all lymphoid tissues affected, whereas others may have only one or two affected lymph nodes. the characteristic microscopic lesions are lymphoid depletion of both follicles and paracortex with replacement by histiocytes, mild to severe granulomatous inflammation with multinucleated giant cells, and intrahistiocytic sharply demarcated, spherical, basophilic cytoplasmic inclusion bodies. necrosis of prominent lymphoid follicles (necrotizing lymphadenitis) is occasionally observed, and pcv2 can be detected within the necrotic regions. the loss of lymphocytes may be due to reduced production in the bone marrow, decreased proliferation in the secondary lymphoid organs, or necrosis of lymphocytes. splenic abscesses can be the result of bacteremia (see fig. 13 -69) or direct penetration by a foreign body from the reticulum (see spleen and also portals of entry/pathways of spread). c. pseudotuberculosis is a gram-positive intracellular bacterium that causes caseous lymphadenitis, a chronic suppurative disease of sheep and goats. the bacterium may enter through skin wounds (e.g., shearing cuts in sheep, tagging, tail docking, or castration), drain to the regional lymph node, and then be disseminated in lymph and circulating blood to external and internal lymph nodes, as well as other internal organs, including lung. external abscesses are most often detected in the "jaw and neck" region, specifically in the mandibular and parotid lymph nodes. on gross examination the abscesses are encapsulated and filled with greenish semifluid pus due to an infiltrate of eosinophils (see fig. 13-79) . over time the abscesses lose the greenish hue, and contents become inspissated to form the characteristic concentric laminations (see fig. 13 -80); old abscesses may reach a diameter of 4 to 5 cm. bovine lymphoma is broadly classified into enzootic and sporadic forms. the enzootic form, called enzootic bovine leukosis (ebl), is caused by blv, a retrovirus common in cattle. there is a higher prevalence in dairy cattle compared to beef breeds. blv is transmitted horizontally (e.g., blood, milk/colostrum, saliva) or iatrogenically (e.g., rectal sleeves, instruments/equipment). following infection, blv invades and integrates into the genome of infected b lymphocytes, resulting in a polyclonal b lymphocyte lymphocytosis in approximately 30% of cattle. in approximately 1% to 5% of blv-infected cattle, a single clone will emerge, leading to the development of b lymphocyte leukemia/lymphoma. the average incubation period between infection and development of lymphoma is 7 to 8 years, and this low conversion rate suggests that the latency period may be longer than the life span of most animals (dairy cattle seldom live to the 7-to 8-year peak incidence of lymphoma occurrence). other contributing variables, such as genetic background, coinfections, and environmental factors, may also play a role in lymphomagenesis. the exact mechanism of blv-induced tumorigenesis is poorly understood. recently blv micrornas (mirnas) were identified in preleukemic and malignant b lymphocytes, which showed repression of structural and regulatory gene expression. these findings suggested that mirnas may play a key role in tumor onset and progression. grossly, multiple tissues may be affected in cattle that develop lymphoma, including peripheral lymph nodes (cephalic, cervical, sublumbar) ( fig. 13-87) , abdominal lymph nodes, retrobulbar region, abomasum, liver, spleen, heart, urogenital tract, bone marrow, vertebral canal ( fig. 13-88) , and spinal cord. one study indicates most of these high-grade lymphomas are diffuse large cell lymphomas (66%), and approximately 20% are intermediate cell lymphomas (burkitt-like and lymphoblastic lymphomas). the sporadic form of bovine lymphoma is most often of t lymphocyte immunophenotype and has three subcategories: cutaneous, calf, and thymic. there is no known viral cause for the sporadic form, and each subcategory has a much smaller prevalence compared to the enzootic form. of the three sporadic forms, the cutaneous form seems to be the most common and manifests itself as multiple skin nodules in 1-to 3-year-old cattle. the calf form presents as generalized lymphadenopathy with weight loss, lethargy, and weakness in calves less than 6 months old. the thymic form is reportedly more common in beef cattle, 6 to 24 months of age. lymphoma is the most frequently reported cancer of pigs based on abattoir surveys. affected pigs are typically less than 1 year of age, and there is no reported breed predisposition, although a hereditary basis is suspected in cases arising in inbred herds. the two main forms of porcine lymphoma are thymic/mediastinal and multicentric; the latter is more common. spleen, liver, kidney, bone marrow, and lymph nodes are affected in the multicentric form, with visceral lymph nodes reportedly more commonly involved than peripheral nodes. a recent study of lymphoma in 17 pigs found the majority to be multicentric, and subtypes included the following: b lymphoblastic leukemia/lymphoma, follicular lymphoma, diffuse and intestinal large b cell lymphoma, and peripheral t cell lymphoma. one case each of thymic b cell and t cell lymphomas were also described. several types of severe combined immunodeficiency diseases have been described in dogs. a mutation in dna-pkcs (similar to arabian horses) with an autosomal recessive mode of inheritance is seen in jack russell terriers. an x-linked form of severe combined immunodeficiency disease is well described in basset hounds and is caused by mutations in the common γ-chain (γc) subunit of the receptors for il-2, il-4, il-7, il-9, il-15, and il-21. a similar disease is seen in cardigan welsh corgi puppies, though it is an autosomal mode of inheritance in this breed. the mutation inhibits the signal transduction pathways initiated by any of these cytokines, which are critical for the proliferation, differentiation, survival, and function of b and t lymphocytes. affected dogs have normal numbers of circulating b lymphocytes that are unable to class switch to igg or iga and reduced numbers of t lymphocytes, which are nonfunctional due to the inability to express il receptors. affected puppies are remarkably susceptible to bacterial and viral infections and rarely survive past 3 to 4 months of age. the thymus of these dogs is small and consists of only small dysplastic lobules with a few of hassall's corpuscles. tonsils, lymph nodes, and peyer's patches are often grossly unidentifiable due to the severe lymphocyte hypoplasia. congenital immunodeficiency diseases are also discussed in detail in chapter 5. thymic hemorrhage and hematomas have been reported in dogs and are most often seen in young animals. a variety of causes are described, including ingestion of anticoagulant rodenticides (warfarin, dicumarol, diphacinone, and brodifacoum), dissecting aortic aneurysms, trauma (e.g., automobile accident), and idiopathic/ spontaneous. histologically, hemorrhage variably expands the thymic lobules and septa, and in severe cases the lobular architecture is obscured by hemorrhage. in cases of anticoagulant rodenticide toxicosis, the medulla appears to be the main site of hemorrhage. see uniform splenomegaly with a bloody consistency (also see figs. 7-72 and 7-73). see the section on splenic nodules with a bloody consistency for discussion on splenic hematomas (including those induced by nodular hyperplasia or occurring with hemangiosarcoma), incomplete splenic contraction, acute splenic infarcts, and hemangiosarcomas. porcine reproductive and respiratory syndrome (prrs) is caused by an arterivirus and causes two overlapping clinical syndromes: reproductive failure and respiratory disease. the virus is transmitted by contact with body fluids (saliva, mucus, serum, urine, and mammary secretions and from contact with semen during coitus), but often it first colonizes tonsils or upper respiratory tract. the virus has a predilection for lymphoid tissues (spleen, thymus, tonsils, lymph nodes, peyer's patches). viral replication takes place in macrophages of the lymphoid tissues and lungs, though porcine reproductive and respiratory syndrome virus antigen is found in resident macrophages in many tissues and may persist in tonsil and lung macrophages. the result of this infection is a reduction in the phagocytic and functional capacity of macrophages of the monocytemacrophage system. as a consequence, there is reduction in resistance to common bacterial and viral pathogens. most porcine reproductive and respiratory syndrome-infected pigs are coinfected with one or more pathogens, including streptococcus suis and salmonella choleraesuis. infection with bordetella bronchiseptica and mycoplasma hyopneumoniae appear to increase the duration and severity of the interstitial pneumonia. the major lesions are interstitial pneumonia and generalized lymphadenopathy, and tracheobronchial and mediastinal lymph nodes are most commonly affected. coinfections often complicate the gross and histopathologic changes. lymph nodes are enlarged, pale tan, occasionally cystic, and firm; some strains of virus also cause nodal hemorrhage. microscopically, the lesions in the lymph nodes, tonsils, and spleens consist of varying degrees of follicular and paracortical hyperplasia and lymphocyte depletion in follicular germinal centers. streptococcus porcinus causes jowl abscesses in pigs. the bacteria colonize the oral cavity and spread to infect tonsils and regional lymph nodes. the mandibular lymph nodes are the most often affected and have multiple, 1-to 10-cm abscesses; the retropharyngeal and parotid lymph nodes may also be involved (fig. 13-89 ). this once-prevalent disease is now rare, presumably due to improvements in husbandry, feeder design, and hygiene. it is occasionally isolated in pigs with bacteremia. disseminated histoplasmosis include wasting, emaciation, fever, respiratory distress, diarrhea with hematochezia or melena, and lameness. the clinicopathologic changes of disseminated histoplasmosis may include neutrophilia, monocytosis, nonregenerative anemia in chronic infections, changes in total serum protein level, and liver enzyme level elevations with hepatic involvement. the anemia is likely a result of chronic inflammation, histoplasma infection of the bone marrow, and/or intestinal blood loss in dogs with gi disease. cytologic examination is useful for the diagnosis of histoplasmosis (tracheal wash preparations, aspirates of bone marrow and lymph nodes), where organisms are often visible in macrophages (efig. 13-15) . grossly, there is hepatosplenomegaly, the intestines are thickened and corrugated, and the lymph nodes are uniformly enlarged (fig. 13-91) with loss of normal architecture (somewhat similar to siderofibrotic plaques, splenic rupture, and accessory spleens see the section on miscellaneous disorders of the spleen for discussions on siderofibrotic plaques, splenic rupture, and accessory spleens. splenic nodular hyperplasia is common in dogs and categorized based on their cellular components as lymphoid nodular hyperplasia or complex nodular hyperplasia. hematomas may arise within nodules of hyperplasia (see splenic nodules with a bloody consistency). lymphoid (or simple) nodular hyperplasia consists of a focal well-demarcated mass composed of discrete to coalescing aggregates of lymphocytes. the lymphocytes may form follicular structures with germinal centers and/or consist of a mixture of lymphocytes with mantle and marginal zone cell morphologic features. the intervening tissue is often congested and may contain plasma cells, but stroma is not observed (fig. 13-90; e-fig. 13-13) . complex nodular hyperplasia is a focal mass that contains two proliferative components: lymphoid and stroma (efig. 13-14) . the lymphoid component resembles lymphoid nodular hyperplasia described above. there is proliferation of the intervening stromal tissues with fibroplasia, smooth muscle hyperplasia, and histiocytic hyperplasia; emh and plasma cells may also be present. it has recently come to light that the entity splenic fibrohistiocytic nodule (sfhn), first described in 1998, is not a single condition, but in fact a complex group of diseases. our better understanding of the spectrum of diseases once described under the term splenic fibrohistiocytic nodule is due to increasing knowledge of histiocytic disorders and immunochemistry. the original definition of splenic fibrohistiocytic nodule is a nodule characterized by a stromal population of histiocytoid and spindle cells intermixed with lymphocytes. grading was based on the lymphocyte percentage of the population (e.g., > 70% lymphocytes = grade 1; < 40% lymphocytes = grade 3); dogs with grade 1 splenic fibrohistiocytic nodule had a much better 1-year survival rate, and dogs with grade 3 nodules may develop sarcomas (often malignant fibrous histiocytoma, a now outdated term). with our increasing knowledge of histiocytic disorders and additional immunohistochemical stains, diseases that likely were encompassed by the term splenic fibrohistiocytic nodule include the following: complex and lymphoid nodular hyperplasia (see earlier), stromal sarcoma, histiocytic sarcoma, marginal zone hyperplasia, marginal zone lymphoma, and diffuse large b cell lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). histoplasma capsulatum can cause a disseminated fungal disease that is widely endemic, particularly in areas with major river valleys and temperate or tropical climates (e.g., midwestern and southern united states). free-living organisms in the mycelial phase produce macroconidia and microconidia that are inhaled and converted to the yeast phase in the lung. yeasts are phagocytized and harbored by macrophages of the monocyte-macrophage system. in some dogs the disease is limited to the respiratory tract and causes dyspnea and coughing. however, in most dogs, the disease is disseminated throughout the body, predominantly affecting the liver, spleen, gastrointestinal tract, bone marrow, skin, and eyes; primary gastrointestinal disease is also reported. the clinical signs in cases of and hepatosplenomegaly (efig. 13-16) . histologically, the lymph node sinuses and splenic red pulp are filled with macrophages that contain intracytoplasmic, round, 2-µm-diameter organisms with a small kinetoplast. though there is an initial stage of lymphoid hyperplasia in the spleen and lymph node, subsequent lymphoid atrophy occurs with chronicity. the atrophy is due to impairment of follicular dcs, b lymphocyte migration, and germinal center formation. there may be lymphoid atrophy of the spleen and lymph nodes in severe chronic infections. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells (see chapter 14). dogs are exposed through contact with oronasal secretions, and the virus infects macrophages within the lymphoid tissue of the tonsil and respiratory tract (including tracheobronchial lymph nodes) and later disseminates to the spleen, lymph nodes, bone marrow, malt, and hepatic kupffer cells. the virus causes necrosis of lymphocytes (especially cd4 t lymphocytes) and depression of lymphopoiesis in the bone marrow, leading to severe immunosuppression. dogs are therefore susceptible to secondary infections, including bordetella bronchiseptica, toxoplasma gondii, nocardia, salmonella spp., and generalized demodicosis. canine parvovirus type 2 (cpv-2) is a highly contagious disease of dogs spread through the fecal-oral route or oronasal exposure to contaminated fomites. the virus has tropism for rapidly dividing cells, and replication begins in the lymphoid tissues of the oropharynx, thymus, and mesenteric lymph nodes and then is disseminated to the small intestinal crypt epithelium. by infecting lymphoid tissues, canine parvovirus type 2 causes immunosuppression directly through lymphocytolysis and indirectly though bone marrow depletion of lymphocyte precursors. there is marked lymphoid atrophy of thymus and follicles of the spleen, lymph nodes, and maltparticularly of peyer's patches to produce the classic gross lesion of depressed oval regions of the mucosa (so-called punched-out peyer's patches). thymomas. see lymphoid/lymphatic system, disorders of domestic animals: thymus, neoplasia. lymphomas. lymphoma is the most common hematologic malignancy in the dog. using the who classification scheme, several lymphoma subtypes are identified in dogs and clinically range from slow-growing indolent tumors to highly aggressive tumors. of all domestic animal species, lymphoma is the most extensively studied in dogs. the most common clinical presentation in dogs is generalized lymphadenopathy, with or without clinical signs such as lethargy and inappetence. the majority of lymphomas in dogs are large cell mid-to highgrade lymphomas, and up to half of all lymphoma cases are subtyped as diffuse large b cell lymphoma. diffuse large b cell lymphomas are further subdivided into centroblastic or immunoblastic based on nucleolar morphologic features (see table 13 -7 and box 13-11), although it is unclear if this difference has any prognostic significance. histologically, lymph node architecture is most often completely effaced by sheets of large neoplastic cells, which may invade through the capsule and colonize the perinodal tissue. these dogs are often treated with chemotherapy and achieve remission. the overall median survival time for dogs with diffuse large b lymphocyte lymphoma is approximately 7 months, although this number lymphoma, though the nodes tend to be more firm in histoplasmosis). histologically within the node, there is a multifocal to coalescing infiltrate of epithelioid macrophages with intracytoplasmic, small (2 to 4 µm in diameter) yeast organisms with spherical basophilic central bodies surrounded by a clear halo (fig. 13-92) . leishmaniasis is a disease of the monocyte-macrophage system caused by protozoa of the genus leishmania. it occurs in dogs and other animals and is endemic in parts of the united states, europe, mediterranean, middle east, africa, and central and south america. the protozoa proliferate by binary fission in the gut of the sand fly and become flagellated organisms, which are introduced into mammals by insect bites, where they are phagocytized by macrophages and assume a nonflagellated form. cutaneous and/or visceral forms of the disease are observed. in the visceral form, dogs are emaciated and have general enlargement of abdominal lymph nodes numerous other subtypes of lymphoma have been reported in dogs, including several forms of cutaneous lymphomas, most often of t lymphocyte origin and epitheliotropic (see chapter 17). hepatosplenic t cell lymphoma, thought to be of γ/δ t lymphocyte origin, affects the liver and spleen without significant nodal involvement. hepatocytotropic t cell lymphoma is a distinct form of lymphoma with tropism for the hepatic cords; clusters or individual neoplastic lymphocytes invade the hepatic cords, without hepatocyte degeneration. intravascular lymphoma is a proliferation of large neoplastic lymphocytes within blood vessels of many tissues, leading to progressive occlusion and subsequent thromboses and infarcts. this neoplasm does not form an extravascular mass, and neoplastic cells are not found in peripheral blood smears or bone marrow. indolent lymphomas constitute up to 29% of all canine lymphomas. indolent lymphomas in dogs, in descending order of frequency, include t zone lymphoma (tzl), marginal zone lymphoma (mzl), mantle cell lymphoma (mcl), and follicular lymphoma (fl). mantle cell lymphoma and follicular lymphoma are less commonly diagnosed than t zone lymphoma and marginal zone lymphoma; therefore the reader is referred to the suggested readings to learn more on mantle cell lymphoma and follicular lymphoma. t zone lymphoma. t zone lymphoma is the most common indolent lymphoma in dogs ( fig. 13-93) . it presents as a solitary or varies based on the study and the grade of the tumor (as determined by mitotic figures). peripheral t cell lymphomas-not otherwise specified are the second most common subtype in dogs. this category includes all t cell lymphomas that do not fit into the other categories (e.g., t zone lymphoma, enteropathy-associated t cell lymphoma, and hepatosplenic t cell lymphoma). peripheral t cell lymphoma also effaces nodal architecture, and when compared to diffuse large b cell lymphoma, there is more variation in nuclear size and morphologic features. dogs with this subtype tend to have shorter survival times. intermediate cell size, high-grade lymphomas are less common in dogs, and the two most frequently encountered subtypes are lymphoblastic lymphoma (lbl) and burkitt-like lymphoma (bll). lymphoblastic lymphoma may be of b or t lymphocyte origin, though t cell lymphoblastic lymphoma is more common of the two. it is important to recognize a common misuse of the term "lymphoblast" in lymphoblastic lymphoma-by definition in lymphoblastic lymphoma, lymphoblasts are intermediate-sized cells with a distinct dispersed chromatin pattern, and not the large lymphocytes seen in cases of diffuse large b cell lymphoma or peripheral t cell lymphoma. t lymphocyte lymphoblastic lymphoma is an aggressive disease that is often resistant to treatment. burkitt-like lymphoma is a high-grade lymphoma of b lymphocytes. consequently there is marked lymphoid atrophy of thymus, spleen, lymph node, and malt (particularly peyer's patches). see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, myeloid neoplasia, mast cell neoplasia and see efig. 13-6 . lymphoma is the most commonly diagnosed neoplasm in cats, and the incidence is reportedly the highest for any species. mediastinal or multicentric lymphomas are seen in young, felv-infected cats (see fig. 13 -53). with the advent of felv vaccine and routine testing, the prevalence of felv-associated lymphoma is decreased. currently the alimentary tract is the most commonly affected site, and typically occurs in cats greater than 10 years of age . other miscellaneous sites commonly affected are brain, spinal cord, eye, kidney, and nasopharynx. the retrovirus felv has long been recognized as a cause of lymphoma in cats-the risk for lymphoma is increased sixtyfold in infected cats. before the advent of a vaccine in 1985, approximately 70% of cats (mainly young animals) with lymphoma were felv positive. felv infects t lymphocytes and can cause myelodysplastic syndrome, acute myeloid leukemias (see myeloid neoplasia), and t lymphocyte leukemia/lymphoma. in the latter the mediastinum (thymus, mediastinal, and sternal lymph nodes) is the site most commonly involved, although a multicentric distribution also occurs. routine felv vaccination has led to a significant decrease in the prevalence of felv infection, which has resulted in a decrease in the proportion of mediastinal lymphomas. the risk for developing lymphoma in fiv-infected cats is fivefold to sixfold higher than in uninfected cats. cats that underwent kidney transplantation and thus received immunosuppressive drug therapy had a similar risk for developing lymphoma. both fivinfected and posttransplantation cats predominantly developed extranodal, high-grade, diffuse large b lymphocyte lymphomas. this form is also the most common subtype in human immunodeficiency virus and posttransplantation patients caused by the epstein-barr virus (ebv). therefore it is reasonable to question whether these two groups of immunosuppressed cats may be more prone to infection by a gammaherpesvirus similar to ebv, leading to lymphoma. recently a novel feline gammaherpesvirus (fcaghv1) was multiple peripheral lymphadenomegaly (often mandibular lymph nodes) in otherwise healthy-appearing dogs. the characteristic histopathologic architecture is a nodular expansion of the paracortex by neoplastic cells, which push atrophied "fading" cortical follicles against the thinned capsule and trabeculae. this unique architectural feature is best highlighted with immunohistochemical stains (often cd3 for t lymphocytes and cd79a, pax5, or cd20 for b lymphocytes). the neoplastic cells are small to intermediate in size with pale eosinophilic cytoplasm and oval nuclei with sharp shallow indentations. mitotic figures are rare. dogs with this lymphoma subtype tend to be diagnosed with an advanced stage of the disease, likely because they present clinically healthy, without loss of appetite or activity level. even so, dogs with t zone lymphoma have a relatively long survival time compared to other lymphomas: reports on median survival time range from 13 to 33 months, and data suggest that dogs who do not receive chemotherapy actually have longer median survival times. marginal zone lymphoma. marginal zone lymphoma is an indolent b lymphocyte neoplasm derived from the cells of the marginal zone of lymphoid follicles. most marginal zone lymphomas (and mantle cell lymphomas) are assumed to originate in the spleen with slow spread to lymph nodes and often present as a mottled white-red smooth spherical splenic mass. histopathologic assessment of tissue architecture is needed for a diagnosis of marginal zone lymphoma and is characterized by a distinct nodular pattern in which the lighter-staining neoplastic marginal zone cells form a dense cuff around small foci of darkly stained mantle cells (fading follicles). the neoplastic marginal zone lymphocytes are intermediate in size and have a single prominent central nucleolus. mitotic figures are often rare or absent early on and increase with disease progression. differentiating between marginal zone lymphoma and marginal zone hyperplasia (which refers to a proliferation of marginal zone cells and contains a mixture of small and intermediate lymphocytes) is challenging because marginal zone lymphoma arises on the background of marginal zone hyperplasia. additionally, lymphoid and complex nodular hyperplasia are common in the dog spleen (see disorders of dogs), and it is possible that many cases of nodular hyperplasia contain areas of marginal zone lymphoma. therefore immunophenotyping and molecular clonality are ultimately required for a definitive diagnosis of marginal zone lymphoma. the overall median survival time in dogs with splenic marginal zone lymphoma after splenectomy is approximately 13 months (even longer if it is diagnosed as an incidental finding). plasmacytomas. see disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia, extramedullary plasmacytomas (see fig. 13 -84). feline panleukopenia, caused by the single-stranded dna virus feline parvovirus (fpv), is a highly contagious and often lethal disease of cats and other felidae, as well as other species (including raccoons, ring-tailed cats, foxes, and minks). fpv is transmitted by the fecal-oral route through contact with infected body fluids, feces, or fomites. following intranasal or oral infection, the virus initially replicates in the macrophages in the lamina propria of the oropharynx and regional lymph nodes, followed by viremia, which distributes the virus throughout the body. because fpv requires rapidly multiplying cells in the s phase of division for its replication, replication occurs in mitotically active tissues (lymphoid tissue, bone marrow, and intestinal mucosa). by infecting lymphoid tissues, fpv causes immunosuppression directly through lymphocytolysis and of the small intestine. the neoplastic cells are small (nuclei are equal to the diameter of a feline rbc), mitotic figures are infrequent (low grade), and mucosal and crypt epitheliotropism is common (see fig. 13-95) . a diagnosis of this subtype of lymphoma may be difficult (particularly in endoscopic biopsy samples), because this disease often is multifocal and concurrent with or arises within lymphoplasmacytic inflammatory bowel disease (ibd). the neoplastic lymphocytes are morphologically similar to the inflammatory lymphocytes. early small cell mucosal t cell lymphomas often require additional diagnostic testing, namely, immunohistochemistry and molecular clonality testing (pcr for antigen receptor rearrangement [parr]) to confirm a clonal neoplasm. transmural t cell lymphomas also occur focally or multifocally in the small intestine of cats (best classified as enteropathy-associated t cell lymphoma type i) and by definition must extend into the submucosa and muscularis. some tumors invade the serosa and adjacent mesentery. t cell large granular lymphocyte (lgl) lymphoma is often diagnosed, and the intestinal segments orad and aborad to the transmural mass may also have mucosal lymphoma. gastrointestinal b cell lymphomas are less prevalent in cats but occur in the stomach, jejunum, and ileocecocolic region as transmural lesions. most are diagnosed as diffuse large b cell lymphomas. lymphomas in other sites also occur less frequently in cats. the upper respiratory tract (nasal and/or nasopharyngeal region) is a relatively rare site for lymphoma. however, lymphoma is the most common primary nasal tumor, and diffuse large b cell lymphomas (of immunoblastic type) are the predominant subtype. both cutaneous (cutaneous t cell lymphoma) and subcutaneous lymphomas (usually large cell lymphomas) are rare. presumed solitary ocular lymphomas have also been reported. t cell-rich large b cell lymphoma, also referred to as feline hodgkin-like lymphoma in some studies, is composed of a mixture of reactive small lymphocytes and large neoplastic b lymphocytes, many of which may be binucleated and/or have prominent nucleoli (thus resembling the reed-sternberg cells of human hodgkin's lymphoma). this disease is typically characterized by a distinctive clinical presentation of an indolent unilateral neoplasm of the cervical lymph nodes, which spreads slowly to adjacent nodes within the chain. however, a proportion of cases may go on to develop into a more aggressive multicentric large to anaplastic b lymphocyte lymphoma that can affect peripheral and central nodes and multiple organs. suggested readings are available at www.expertconsult.com. a b discovered in domestic cats with a 16% prevalence in north america, and further studies to investigate its role in lymphomagenesis are needed. the overall incidence of feline lymphomas has increased, mainly due to an increase in gastrointestinal lymphomas. mucosal t cell lymphoma, also known as enteropathy-associated t cell lymphoma (eatcl type ii), is the most common and arises from diffuse malt partial thromboplastin time (aptt or ptt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of a contact activator, calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in intrinsic and/or common coagulation pathway (all factors except for vii and xiii) causes prolongation of ptt • insensitive test-prolongation requires 70% deficiency. • other causes of prolongation include polycythemia (less plasma per unit volume, so excess amount of citrate is available to chelate calcium) and heparin therapy activated clotting time (act) • required sample: nonanticoagulated whole blood in special act tube (diatomaceous earth as contact activator) used in practice setting-performed by warming sample to body temperature, monitoring for clot formation; normal clotting times are within 60 to 90 seconds in dogs, 165 seconds in cats • less sensitive version of ptt-prolongation requires 95% deficiency severe thrombocytopenia may cause prolongation. • one-stage prothrombin time (ospt or pt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of tissue factor (tf; thromboplastin), calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in extrinsic (factor vii) and/or common coagulation pathway cause prolongation of pt • insensitive test-prolongation requires 70% deficiency proteins induced by vitamin k antagonism or absence (pivka) test • required sample: citrated plasma • essentially a version of the pt using an especially sensitive thromboplastin reagent • pivka are inactive (uncarboxylated) vitamin k-dependent factors; an increase in pivka is not specific for vitamin-k antagonism but may be an earlier and more sensitive detector than pt or ptt • thrombin time (tt) • required sample: citrated plasma • measures time for fibrin clot formation after thrombin (factor iia) is added • defects directly involving formation and/or polymerization of fibrin prolong this test (i.e., if the lesion is upstream of the conversion of fibrinogen to fibrin, the tt will be normal). hypofibrinogenemia or dysfibrinogenemia causes prolongation of the tt required sample: citrated plasma. • fibrinogen concentration measured based on time to clot formation after addition of thrombin; this is essentially the same as the tt mentioned earlier and is a more accurate method than the heat precipitation method • decreased fibrinogen may be because of increased consumption (disseminated intravascular coagulation) or decreased production (liver disease) increased fibrinogen is associated with inflammation, renal disease, and dehydration required sample: special fdp tube. • used in the practice setting. • performed by adding blood to a special tube containing thrombin and a trypsin inhibitor (sample clots almost instantly in normal dogs and cats) and incubating two dilutions of serum (1 : 5 and 1 : 20) with polystyrene latex particles coated with sheep anti-fdp antibodies (should be negative in normal dogs and cats required sample: citrated plasma. • latex agglutination test. • to date, only validated in dogs and horses assay detects a specific type of fdp resulting from breakdown of cross-linked fibrin; concentration of plasma d-dimer indicates the degree of fibrinolysis; often used as part of a disseminated intravascular coagulation panel • required sample: citrated plasma. • decreased because of decreased production (liver disease), loss (protein-losing nephropathy or enteropathy) • specific factor assays • required sample: citrated plasma. • performed at specialized laboratories intermediary spleen microvasculature in canis familiaris-morphological evidence of a closed and open type molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states characteristics, diagnosis, and treatment of inherited platelet disorders in mammals making sense of lymphoma diagnostics in small animal patients canine granulocytic anaplasmosis: a review lymphoid tissues and organs lymph nodes and thymus diagnostic cytology and hematology of the dog and cat two hundred three cases of equine lymphoma classified according to the world health organization (who) classification criteria hepcidin: a key regulator of iron metabolism and mediator of anemia of inflammation changes to bovine hematology reference intervals from 1957 to atlas of veterinary hematology: blood and bone marrow of domestic animals pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses feline leukemia virus infection and diseases tumors of the hemolymphatic system proposed criteria for classification of acute myeloid leukemia in dogs and cats immunobiology: the immune system in health and disease extramedullary hematopoiesis: a new look at the underlying stem cell niche, theories of development, and occurrence in animals lineage-specific hematopoietic growth factors hepatosplenic and hepatocytotropic t-cell lymphoma: two distinct types of t-cell lymphoma in dogs histology and cell biology: an introduction to pathology lymphoid neoplasms in swine jc: robbins & cotran pathologic basis of disease the spleen ehrlichiosis and related infections hemotropic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential clinical, laboratory, and histopathologic features of equine lymphoma classification and clinical features in 88 cases of equine cutaneous lymphoma histologic and immunohistochemical review of splenic fibrohistiocytic nodules in dogs feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality current state of knowledge on porcine circovirus type 2-associated lesions molecular pathology of severe combined immunodeficiency in mice, horses, and dogs hematologic abnormalities associated with retroviral infections in the cat pathology of the pig: a diagnostic guide pathologic and prognostic characteristics of splenomegaly in dogs due to fibrohistiocytic nodules: 98 cases splenic myeloid metaplasia, histiocytosis, and hypersplenism in the dog (65 cases) fundamentals of veterinary clinical pathology feline parvovirus infection and associated diseases novel gammaherpesviruses in north american domestic cats, bobcats, and pumas: identification, prevalence, and risk factors palmer's pathology of domestic animals veterinary comparative hematopathology histologic classification of hematopoietic tumors of domestic animals. in world health organization international histological classification of tumors in domestic animals, second series canine lymphomas: association of classification type, disease stage, tumor subtype, mitotic rate, and treatment with survival classification of canine malignant lymphomas according to the world health organization criteria canine indolent nodular lymphoma the 2008 revision of the world health organization (who) classification of myeloid neoplasms and acute leukemia: rationale and important changes the coombs' test in veterinary medicine: past, present, future a retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital (1996-2004) schalm's veterinary hematology chronic lymphocytic leukemia in dogs and cats: the veterinary perspective grossly, incompletely contracted areas are characterized by multiple, variably sized and irregularly shaped, dark red to black, raised, soft, blood-filled "nodules." these areas are usually at the margins of the spleen, and the intervening tissues are depressed and pink-red, corresponding to the contracted portions of red pulp devoid of blood. incompletely contracted areas may be confused with acute splenic infarcts or hematomas on gross examination.acute splenic infarcts. splenic infarcts are wedge-shaped or triangular hemorrhagic lesions that occur primarily at the margins of the spleen. in dogs, splenic infarcts most often occur with hypercoagulable states (e.g., liver disease, renal disease, cushing's disease), neoplasia, and cardiovascular disease. splenic vein thrombi may occur in association with traumatic reticulitis, splenic abscesses, portal vein thrombosis, and arterial thrombosis in bovine theileriosis in cattle. valvular endocarditis may also lead to multiorgan infarcts, including the spleen. splenic infarcts are common in pigs with classical swine fever.acute splenic infarcts may not always be grossly visible in the early stages but develop into discrete, dark red and blood-filled, bulging, wedge-shaped foci with the base toward the splenic capsule ( fig. 13-64, a) . with chronicity the lesion becomes gray-white and contracted due to fibrosis (see fig. 13 -64, b).hemangiosarcoma. hemangiosarcoma is a malignant neoplasm of endothelial cells and is a common primary tumor of the spleen, especially in dogs. benign splenic hemangiomas are extraordinarily rare. grossly, hemangiosarcomas may appear as single, multifocal, or coalescing dark red-purple masses and cannot be easily differentiated from a hematoma ( fig. 13-65 ). on cut surface they are bloody with varying amounts of soft red neoplastic tissue; in more solid areas the neoplasm can be slightly more firm and whitetan. metastatic spread occurs early in the disease process. seeding of the peritoneum results in numerous discrete red-black masses throughout the omentum and serosa of abdominal organs, and hematogenous spread to liver and lung are common. hemangiosarcomas in dogs also occur in the right atrium of the heart, retroperitoneal fat, and skin (dermal and/or subcutaneous) and multiorgan hemangiosarcomas are described in horses, cats, and cattle. because hemangiosarcomas have often metastasized at the time of initial accumulations of macrophages within several organs, including splenic macrophages, kupffer cells of the liver, and macrophages in the brain are often observed.splenic nodules with a bloody consistency. the most common disorders of the spleen with bloody nodules are (1) hematomas, including those induced by nodular hyperplasia or occurring with hemangiosarcoma, (2) incompletely contracted areas of the spleen, (3) acute splenic infarcts, and (4) hemangiosarcomas. the term nodule has been applied rather loosely here. in some of these conditions, such as incompletely or irregularly contracted areas of the spleen, the elevated area of the spleen is not as well defined as the term nodule would imply.hematomas. bleeding into the red pulp to form a hematoma is confined by the splenic capsule, and produces a red to dark red, soft, bulging, usually solitary mass of varying size (2 to 15 cm in diameter) (fig. 13-62 ). resolution of a splenic hematoma progresses over days to weeks, through the stages of coagulation and breakdown of the blood into a dark red-brown soft mass (fig. 13-63, a) , infiltration by macrophages that phagocytize erythrocytes and break down hemoglobin to form hematoidin and hemosiderin (see fig. 13-63 , b), and repair leading to fibrosis. on occasion the capsule (splenic capsule and visceral peritoneum) over the hematoma can rupture, resulting in hemoperitoneum, hypovolemic shock, and death.the origin or cause of many hematomas is unknown. some are due to trauma, and others may also be induced by splenic nodular hyperplasia. it is postulated that as the splenic follicles become hyperplastic they distort the adjacent marginal zone and marginal sinus, which compromises their drainage into sinusoids and red pulp vascular spaces. the result is an accumulation of pooled blood surrounding the hyperplastic nodule, which leads to hematoma formation. splenic hematomas can also occur secondary to the rupture of hemangiosarcomas within the spleen.incompletely contracted areas of the spleen. incompletely or irregularly contracted areas of the spleen are caused by failure of the smooth muscle to contract in response to circulatory shock (hypovolemic, cardiogenic, or septic) or sympathetic "fight-or-flight" response, resulting in a lack of splenic evacuation of stored blood. key: cord-344131-e7phs0jd authors: ford, richard b.; mazzaferro, elisa m. title: section 4 diagnostic and therapeutic procedures date: 2012-12-31 journal: kirk & bistner's handbook of veterinary procedures and emergency treatment doi: 10.1016/b978-1-4377-0798-4.00004-9 sha: doc_id: 344131 cord_uid: e7phs0jd nan for anorexic dogs or when pills must be given without food, give medications quickly and decisively so that the process of administering the medication is accomplished before the dog realizes what has happened. with cooperative dogs, insert the thumb of one hand through the interdental space, and gently touch the hard palate. this will induce the cooperative dog to open its mouth (figure 4-1) . using the opposite hand (the one holding the medication), gently press down on the mandible to open the mouth further (figure 4 -2). note: oral medication frequently is dispensed to owners without regard for the client's knowledge of how to administer a pill or tablet or without asking whether the client is even physically able to administer medications. clear instructions that include having the client perform the technique in the hospital significantly improve compliance. quickly place the tablet or capsule onto the caudal aspect of the tongue. quickly withdraw the hand and close the dog's mouth. when the dog licks its nose, the medication likely has been swallowed. dogs that offer more resistance can be induced to open their mouths by compressing their upper lips against their teeth. as they open the mouth, roll their lips medially so that if they attempt to close the mouth, they will pinch their own lips. alternatively, dripping water onto the nostrils or blowing into the patient's nose sometimes encourages the patient to accept and swallow oral medications (tablets or capsules). pilling syringes are also available and in some dogs seem to work well. critical to the oral administration of medication is the ability of the owner to effectively administer the medication at home. animals that aggressively resist oral medication should be treated by alternative methods-for example, parenteral administration of medication. it is inappropriate, and unsafe, to delegate treatment responsibilities to the owner of a dog (or cat) that might injure the individual who is attempting to treat the patient. none required. caution: only experienced individuals should attempt this technique of administering tablets or capsules to cats. even cooperative cats that become intolerant will bite. therefore, this is not a technique recommended for most owners to try at home, even if specific instructions have been given. two methods of pill administration are used in cats. in both methods the cat's head is elevated slightly with the nose pointed upward. success in administering pills and tablets to a cat entails a delicate balance between what works well and what works safely. in cooperative cats, it may be possible to use one hand to hold and position the head (figure 4 -3) while using the opposite hand (the one holding the medication) to open the mouth gently by depressing the proximal aspect of the mandible (figure 4-4) . press the skin adjacent to the maxillary teeth gently between the teeth as the mouth opens, thereby discouraging the alternatively, some cats will tolerate a specially designed "pilling syringe" in an attempt to administer a tablet or capsule. the pilling syringe works well as long as it is inserted cautiously and atraumatically into the cat's mouth. however, if resistance ensues, the rigid pilling syringe may injure the hard palate during the ensuing struggle. subsequent attempts to use the syringe may be met with increasing resistance and increasing risk of injury. success with a pilling syringe depends largely on the cat. pill pockets treats are also available for use in cats and are manufactured in chicken and fish flavors. in addition, as is the case in dogs, some cats will respond to the application of water drops on the nostrils or blowing into the nostrils to encourage swallowing. when dispensing oral medications for home administration to cats, do not expect clients to force a tablet or capsule into a cat's mouth. although some clients are remarkably capable and confident with their ability to administer oral medications to cats, the risk of injury to the client can be significant. whenever feasible, liquid medications or pulverized tablets should be mixed with the diet or an oral treat readily accepted and consumed (see the following discussion). none required. technique is appropriate for owners to perform at home. small amounts of liquid medicine can be given successfully to dogs and cats by pulling the commissure of the lip out to form a pocket . deposit the liquid medication into the "cheek pouch, " where it subsequently flows between the teeth as the head is held slightly upward. patience and gentleness, along with a reasonably flavored medication, contribute to the success. spoons are ineffective, as fluids are easily spilled. a disposable syringe can be used to measure and administer liquids orally. depending on the liquid administered, disposable syringes can be reused several times, assuming they are rinsed after each administration. in addition, disposable syringes can be dispensed legally to clients for home administration of liquid medication. mixing of medications in the same syringe is not recommended. however, dispensing of a separate, clearly marked syringe for each type of liquid medication prescribed for home administration is recommended. compounding pharmacies are also available and can mix many medications into palatable flavors to help facilitate the oral administration of medications. dogs with swallowing disorders should not be treated at home with liquid medications because this could cause complications associated with aspiration. none required. administration of medications, contrast material, and rehydrating fluids can be accomplished with the use of a well lubricated feeding tube passed through the nostrils into the stomach or distal esophagus. when a feeding tube is placed for long-term use (multiple days) and repeated use (described under gastrointestinal procedures later), it is generally recommended to avoid passing the tip of the tube beyond the distal esophagus. the reason for recommending nasoesophageal intubation over nasogastric intubation is based on the fact that reflex peristalsis of the esophagus against a tube passing through the cardia can result in significant mucosal ulceration within 72 hours. this is not a factor in patients receiving a single dose of medication or contrast material. note: this procedure is reserved for in-hospital use only. the technique should be performed only by individuals trained to perform this procedure. the narrow lumen of tubes passed through the nostril of small dogs and cats limits the viscosity of solutions that can be administered through a tube directly into the gastrointestinal tract. nasoesophageal intubation can be done with a variety of tube types and sizes (table 4 -1). newer polyurethane tubes, when coated with a lidocaine lubricating jelly, are nonirritating and may be left in place with the tip at the level of the distal esophagus. when placing the nasogastric tube, instill 4 to 5 drops of 0.5% proparacaine in the nostril of the cat or small dog; 0.5 to 1.0 ml of 2% lidocaine instilled into the nostril of a larger-breed dog may be required to achieve the level of topical anesthesia needed to pass a tube through the nostril. with the head elevated, direct the tube dorsomedially toward the alar fold (figure 4-6) . pushing dorsally on the nasal philtrum and pushing the nostril from lateral to medially will help facilitate passage of the tube into the ventromedial nasal meatus. caution: the tip of the feeding tube can be inadvertently introduced through the glottis and into the trachea. topical anesthetic instilled into the nose can anesthetize the arytenoid cartilages, thereby blocking a cough or gag reflex. after inserting the tip 1 to 2 cm into the nostril, continue to advance the tube until it reaches the desired length. if the turbinates obstruct the passage of the tube, withdraw the tube by a few centimeters. then readvance the tube, taking care to direct the tube ventrally through the nasal cavity. occasionally it will be necessary to withdraw the tube completely t a b l e 4 -1 the french catheter scale equivalents * millimeters inches from the nostril and repeat the procedure. in particularly small patients or patients with obstructive lesions (e.g., tumor) in the nasal cavity, it may not be possible to pass a tube. do not force the tube against significant resistance through the nostril. gavage, or gastric lavage and feeding, in puppies and kittens can be accomplished by passing a soft rubber catheter or feeding tube into the mouth, tilting the puppy's or kitten's head, and watching it swallow the tube. most puppies or kittens will struggle and vocalize. they usually will not vocalize if the tube has been placed into the trachea. a 12f catheter is of an adequate diameter to pass freely, but it is too large for dogs and cats less than 2 to 3 weeks of age. mark the tube with tape or a pen at a point equal to the distance from the mouth to the last rib. merely push the tube into the pharynx and down the esophagus to the caudal thoracic level (into the stomach). verify the placement of the tube using the same dry syringe aspiration technique to ensure that the tube is positioned in the esophagus or stomach rather than the trachea. attach a syringe to the flared end, and slowly inject medication or food. depending on the feeding tube type, the end of the tube may or may not accommodate a syringe. for example, soft, rubber urinary catheters are excellent tubes for single administration use. however, the flared end may not accommodate a syringe. to affix a syringe to the outside end of a tapered feeding tube or catheter, insert a plastic adapter (figure 4-7) into the open end of the tube. none required. intranasal administration of liquids in dogs and cats is usually limited to a single dose of a vaccine specifically labeled for intranasal administration. there is little indication for routine instillation of liquids into the nostrils of dogs and cats. rarely, administration of isotonic solutions directly into the nostrils is indicated. in contrast to singledose vaccines, lavage solutions applied intranasally are usually multiple-dose containers. therefore the tip of the administration device should not be allowed to directly contact the patient's skin or nose. doing so may result in contamination of the entire bottle. oily drops are not advised because they may damage the nasal mucosa or may be inhaled. the technique for intranasal administration of vaccine is straightforward and usually works quite well…the first time. some animals, dogs more than cats, will aggressively resist intranasal administration of vaccine. attempts to overcome this resistance include covering the eyes with a towel or otherwise distracting the patient with noise or other visual cues. concerns expressed over the loss of vaccine immediately after intranasal administration are generally unfounded. manufacturers of intranasal vaccines typically include a greater antigen (virus or bacteria) titer per dose than is necessary to induce a protective immune response. if the patient resists aggressively and the vaccine is indicated, parenteral preparations are available for all intranasal vaccines and should be considered. several objectives should be considered when treating dermatologic disorders with topical medication: (1) eradication of causative agents; (2) alleviation of symptoms, such as reduction of inflammation; (3) cleansing and debridement; (4) protection; (5) restoration of hydration; and (6) reduction of scaling and callus. many different forms of skin medications are available, but the vehicle in which they are applied is a critical factor (box 4-1). technique in all cases, apply topical medications to a clean skin surface in a very thin film, because only the medication in contact with the skin is effective. in most cases, clipping hair from an affected area enhances the effect of medication. when dispensing medications to owners for home administration, the owner should be instructed to wear disposable examination gloves if using fingers and hands to apply the medication. with the widespread availability of compounding pharmacies, prescribing compounded medications for topical and oral administration recently has become a popular dispensing technique for dogs and cats requiring long-term, daily medication. caution is warranted. some compounding pharmacies that serve the veterinary profession are using inappropriate or ineffective vehicles in which the drug has been compounded, or the drug itself, purchased in bulk, is of a lower grade and possibly an ineffective product once compounded. studies on the quality and efficacy of compounded drugs for use in veterinary patients are limited. however, of those studies that have been performed, serious questions are being raised over the bioavailability of the drug administered. it would be admirable to prepare the skin surgically before making needle punctures to administer medications. because such preparation is not practical, carefully part the hair and apply a high-quality skin antiseptic such as isopropyl alcohol. place the needle directly on the prepared area, and thrust the needle through the skin. although the use of antiseptics on the vial and skin is not highly effective, the procedure removes gross contamination and projects an image of professionalism. before aspirating medications from multiple-dose vials, carefully wipe the rubber diaphragm stopper with the same antiseptic used on the skin. observe this basic rule with all medication vials, even with modified live virus vaccines. technique dogs and cats have abundant loose alveolar tissue and easily can accommodate large volumes of material in this subcutaneous space. the dorsal neck is seldom used for subcutaneous injections because the skin is somewhat more sensitive, causing some patients to move abruptly during administration. a wide surface area of skin and subcutaneous tissue over the dorsum from the shoulders to the lumbar region makes an ideal site for subcutaneous injections. administration of drugs, vaccines, and fluids by the subcutaneous route represents the most commonly used route of parenteral administration in dogs and cats. for small volumes (<2 ml total), such as vaccines, a 22-to 25-gauge needle generally is used. the site most often used is the wide area of skin over the shoulders. the large subcutaneous space and the relative lack of sensitivity of skin at this location make it an ideal injection site. cleaning of the skin with alcohol or other disinfectant generally is performed before injection. several injection techniques are used. a common technique entails grasping a fold of skin with two fingers and the thumb of one hand. gently lift the skin upward. using the opposite hand, place the needle, with syringe attached, through the skin at a point below the opposite thumb. aspiration before injection is not typically necessary when using this route of administration. after administration and on removal of the needle from the skin, gently pinch the injection site and hold it for a few seconds to prevent backflow of medication or vaccine onto the skin. when larger volumes are to be administered-fluids in dehydrated dogs and cats-the skin directly over the shoulders is the injection site most commonly selected. generally, only isotonic fluids are administered by the subcutaneous route. depending on the patient's 4 size, needles ranging from 16 to 22 gauge can be used. because of the larger volumes of fluid involved, warming of the fluids before administration is recommended. doing so can enhance significantly the patient's tolerance for the displacement of skin during the period of administration and, in small patients, prevent hypothermia. depending on the rate of administration and breed of dog, relatively large volumes of fluid generally can be given in one location. cats typically tolerate 10 to 20 ml/kg body mass in a single location. large dogs can tolerate volumes greater than 200 ml of fluid in a single location. when administering large volumes, it is usually not necessary to use multiple injection sites for purposes of distributing the total fluid volume. doing so actually may increase the risk of introducing cutaneous bacteria under the skin. because the administration time required to deliver larger volumes is longer, and the injection needle will be placed in the skin for extended periods, it is appropriate to cleanse and rinse the skin carefully before actually inserting the needle. isotonic, warmed fluids may be administered by large syringe or through an administration tube attached to a bag. monitor skin tension and the patient's comfort tolerance throughout the procedure. although fluid absorption begins almost immediately on subcutaneous administration of fluids, significant pressure caused by the bolus of fluid delivered can develop within the fluid pocket. on removal of the needle, firmly grasp the injection site with the thumb and forefinger for several seconds. the procedure is not complete until one has verified that back-leakage of fluid from the subcutaneous space onto the skin is not occurring. depending on the patient's hydration status and physical condition, fluid absorption may take from 6 to 8 hours. the rate of absorption of fluid administered by the subcutaneous route largely depends on the patient's hydration state and vascular and cardiac integrity. for that reason, the subcutaneous route is not recommended to manage patients in hypovolemic shock. exceptions to this do exist-for example, when in a life-or-death situation access to a vein is simply not possible. subcutaneous or intraosseous (see the following discussion) fluid administration may be the only option available. recently, implantable subcutaneous ports* have been introduced for use in patients requiring regular administration of subcutaneous fluids at home. a 9-inch silicon tube is preplaced under the skin and is sutured in place by a veterinarian. objectively, this offers easy access to the subcutaneous space without the need for needle penetration. owners simply attach a syringe or extension tube tip to the port and administer the appropriate volume of fluids at an appropriate rate and frequency. because of the usual requirement for long-term placement of an implantable fluid administration tube, there is some risk of infection under the skin and around the incision site. some cats do not tolerate the device. *gif-tube single implant kit for subcutaneous fluid administration, various models available. phoenix, arizona, www.practivet.com (owner instruction guide is also available). note: not all parenteral medications can be administered safely by the subcutaneous route. when administering any compound by the subcutaneous route, verify that the product to be administered is approved for subcutaneous administration. serious reactions, including abscess formation and tissue necrosis, can occur. none required. because the tightly packed muscular tissue cannot expand and accommodate large volumes of injectables without trauma, medications given by the intramuscular route should be small in volume. these medications are often depot materials that are poorly soluble, and some may be mildly irritating. unless the animal is extremely thin, give injections into the lumbodorsal muscles on either side of the dorsal processes of the vertebral column. after proper preparation of the skin, insert the needle through the skin at a slight angle (if the animal is thin) or perpendicularly (if the animal is obese). when injecting any medication by a route other than the intravenous one, it is imperative to retract the plunger of the syringe before injecting to be certain that a vein was not entered by mistake. this is especially crucial with oil suspension, microcrystalline suspension, or potent-dose medications. never give intramuscular injections in the neck because of the fibrous sheaths there and the complications that may occur. also, intramuscular injections into muscles of the rear legs can cause severe pain, lameness, and occasionally peroneal nerve paralysis because of local nerve involvement. intracutaneous (or intradermal) injections are used for diagnostic testing purposes. prepare the skin by carefully clipping the hair with a no. 40 clipper blade. if the skin surface is dirty, gently clean it with a moist towel. scrubbing and disinfection are contraindicated because they may produce iatrogenic trauma and inflammation, which interfere with the test. stretch the skin by lifting a fold, and use a 25-to 27-gauge intradermal needle attached to a 1-ml tuberculin syringe. insert the point of the needle, bevel up, in a forward lifting motion as if to pick up the skin with the needle tip. advance the needle while pushing the syringe (levered) downward until the bevel is completely within the skin. inject a bleb of 0.05 to 0.10 ml of fluid. if the procedure is done correctly, the small bleb will appear translucent. intradermal injections generally are used in patients subjected to intradermal skin testing for allergenic antigens. administration of compounds by the intradermal technique is not necessarily simple. inadvertent administration of medications into the subcutaneous tissues is easy when attempting intradermal injection. for that reason, specific training and experience are recommended before attempting intradermal skin testing of allergic patients. none required. intradermal administration of vaccine and drugs in veterinary and human medicine largely has been limited to the complexities of accurately delivering the desired dose into, and not under, the skin. in 2004 a transdermal administration system* was introduced for cats (recombinant feline leukemia virus [felv] vaccine) that was designed after a similar device *vet-jet transdermal administration system, merial, duluth, georgia. used in human (pediatric) medicine. recently the transdermal administration system used for administration of the recombinant felv vaccine has been re-designed. this same administration system is now used for the transdermal administration of the oral melanoma vaccine. the transdermal administration system consistently delivers a precise volume of vaccine into the skin, subcutaneous tissues, and muscle. use of the transdermal administration system should only be used to administer those vaccines approved for this method of delivery. administration of vaccine using the transdermal administration system requires training to understand proper procedure for loading and administering vaccine. at this writing, sale of the transdermal administration system for delivery of the canine oral melanoma vaccine is limited to select specialists in veterinary medicine. see section 1. none required. generally, two techniques are used. oscillometric blood pressure (bp) measurement entails use of an automated recording system. a cuff is applied to the base of the tail or a distal limb for access to an artery. this technique generally is regarded as being most accurate in dogs. when oscillometric bp measurements are performed in dogs, the patient should be in lateral recumbency. this places the cuff at approximately the same level as the heart. in cats the patient generally remains in sternal recumbency (and minimally restrained). most patients experience a brief acclimation period to the cuff placement. for this reason, at least three to five separate readings are obtained at 1-to 2-minute intervals. this technique can be used on awake or anesthetized patients (figure 4-8) . the doppler-ultrasonic flow detection system is most accurate in cats for measuring systolic bp. again, the ventral tail base or a dorsal pedal artery (hindlimb) or the superficial palmar arterial arch (forelimb) can be used. apply and inflate an occluding cuff. the readings are obtained by a transducer as the pressure on the cuff is reduced. caution is recommended in interpreting results from dogs that are reported as hypertensive but have no overt clinical disease. the higher reported occurrence of falsely elevated bp in normotensive dogs measured by this method justifies additional scrutiny when interpreting doppler bp results in dogs. clinically, the most common use of indirect bp measurement is in assessing cats for the presence (or absence) of systemic hypertension caused by renal insufficiency or hyperthyroidism (thyrotoxicosis). a common finding among untreated hypertensive cats is retinal detachment and blindness. early detection and therapeutic intervention (e.g., enalapril and or amlodipine) is critical. in dogs, bp measurement is indicated in patients with chronic renal insufficiency and/or protein-losing nephropathy, hyperadrenocorticism, and diabetes mellitus. in veterinary medicine, interpretation of bp centers on the systolic bp reading, not the diastolic reading (table 4-2) . stepien rl: blood pressure assessment. results dictate that even routine bacterial cultures and identification are best reserved for the commercial laboratory equipped to carry out these increasingly complex procedures and experienced in doing so. what follows are fundamental methods and techniques used to properly collect diagnostic specimens and the appropriate methods for transporting samples to a laboratory in order for the best possible diagnostic result to be obtained. before actually collecting and submitting a sample to a laboratory for bacterial culture, it is appropriate (whenever feasible to do so) to prepare, stain, and examine, under direct microscopy, exudates or fluid from the suspect material or tissue. staining the air-dried sample with a rapid romanowsky-type stain (e.g., diff-quik stain) or a gram stain may reveal evidence of neutrophilic inflammation (neutrophilia, especially with a left shift) and occasionally degenerative neutrophils with intracellular bacteria visible. these findings greatly facilitate patient management by documenting the immediate need for interventive empiric antimicrobial therapy until definitive culture and antimicrobial susceptibility results are obtained. the absence of cytologic evidence of bacterial infection does not rule out the possibility that the patient is infected or bacteremic (table 4-3) . collecting diagnostic samples for bacterial culture should be attempted as early in the disease process as possible. it is also critical to accomplish the sample collection under aseptic conditions. it is appropriate, therefore, to perform a surgical scrub of the skin or tissue from which the sample is to be collected in advance. this is especially true for tissue biopsies and fluid samples collected by needle aspiration through intact skin. failing to adequately prepare the collection site can result in significant contamination and complicate diagnostic interpretation of results. in addition, it is recommended to collect the diagnostic sample before the administration of antibiotics in order to minimize the risk of false-negative culture results. in the event antimicrobials have been administered to a patient with a suspected infection, and that is not responding to treatment, discontinuing treatment for 48 hours before attempting sample collection is generally recommended. collection of an adequate amount, or volume (fluid), is equally important in obtaining meaningful result. for example, a single sterile cotton-tipped swab of contaminated tissue should be considered inadequate sampling and inappropriate for any patient. multiple specimens are always recommended when feasible. also, biopsy material, surgically removed tissue, and several milliliters of fluid (e.g., urine) should be collected and placed in a sterile container that can be appropriately sealed (leak-proof container) before transport. a "clean catch" of urine in a "clean cup" is not appropriate. inexpensive commercial containers specifically designed for the transport of infectious material are readily available today and should be used. many containers designed to hold bacterial samples contain buffered, nonnutritive transport media to sustain the growth of pathogenic bacteria yet minimize overgrowth of bacterial contaminants during the time required to transport the sample. most commercial laboratories provide appropriate containment devices for the transport of bacterial samples. because most diagnostic specimens collected for bacterial culture are submitted to commercial laboratories for bacterial isolation, identification, and antimicrobial susceptibility testing, it is important to prepare the sample properly for shipping. special transport media are generally not required for routine aerobic culture specimens as long as the sample can remain moist and relatively cool and the sample can t a b l e 4 -3 common bacterial culture results-cont'd be inoculated onto culture medium within 3 to 4 hours only. for samples that must be shipped overnight to a laboratory, it is imperative that the specimen be kept cool (not frozen) and moist. elevated temperatures during shipping contribute to bacterial overgrowth of nonpathogenic bacteria, making isolation and identification of diseaseproducing organisms difficult. special transport media may be required. contact the individual laboratory regarding information pertaining to shipping of specimens for bacterial culture. specimens submitted for anaerobic culture need to be inoculated onto culture media within minutes after collection. although special anaerobic transport media are available, they may not be well suited for extended shipping times (>4 hours). among the most frequently tested fluids for bacteria, urine supports the growth of several types of bacteria. therefore it is necessary that the genitalia be cleaned before collection of urine (free-catch specimen) or cystocentesis (preferred). use of a urinary catheter to collect urine is likely to introduce urethral bacteria and may result in false-positive culture results. bacteria will survive for only a limited time in urine. samples collected must be sealed, and unless processed within 2 hours the sample must be refrigerated. samples held for longer than 8 hours may not contain viable bacteria. if extended transportation times are required to reach a laboratory, a urine reservation tube (vacutainer brand urine transportation system, bd, franklin lakes, new jersey) will allow storage for up to 48 hours at room temperature (table 4-4) . collection from fluid filled compartments (e.g., abscesses, seromas) requires collection with a needle and syringe. the maximum quantity possible should be collected and submitted. the skin or tissue overlying the area from which the sample is to be collected should be surgically prepared. if it becomes necessary to flush an open lesion (or perform tracheatranstracheal aspiration or bronchoalveolar lavage [bal]), it is recommended that a buffered solution of sterile ringer's lactate be used. use of fluids that contain preservative may actually inhibit the growth of bacteria. if it is necessary to submit fecal material for specific bacterial isolation, at least 2 to 3 g of feces should be submitted. a single cotton-tipped swab inserted rectally is unlikely to yield meaningful results. multiple (up to three) samples are recommended when attempting to isolate specific pathogens (e.g., salmonella). samples should be submitted in a sealed, leakproof container (always appreciated by the lab). the containerized sample should refrigerated if there is a significant delay (several hours) involved in submission to the laboratory. confirmation of the presence of bacteria in the blood (bacteremia) can be difficult and requires some patient preparation before collection of a series of samples. in addition, samples should be collected only in vials clearly marked for the collection of blood. furthermore, there are several reasons for an infected patient to have a negative blood culture result-for example, prior or concurrent antimicrobial therapy, chronic (low-grade) infection, and intermittent shedding of bacteria into blood. sample volume, numbers of samples submitted, skin preparation, and timing of collections are variables that can directly affect results. clip and surgically prepare the skin over the cephalic, recurrent tarsal, and/or jugular veins. do not draw blood for culture through an indwelling intravenous or intraarterial catheter. collection vials are available for aerobic and anaerobic bacterial culture. it is generally recommended that three blood samples be collected from separate veins over a 24-hour period. there is no advantage to collecting arterial blood. it has been suggested that samples collected during times when the patient is febrile may improve the likelihood of isolating bacteria. the volume of blood collected is determined by the size of the patient, the collection vials (adult, pediatric, infant) used, as well as the laboratory equipment used to propagate the culture. in addition to adult human blood culture collection vials (10 ml), pediatric blood collection vials (5 to 10 ml) and infant collection vials (0.5 to 1.0 ml) are available. it is appropriate that sterile technique be adhered to during collection of all samples. this includes the use of sterile gloves by the individual collecting the sample. once blood has been collected, air should not be allowed to enter the collection vial. the vial should be gently inverted (never shaken) two to four times. vials may be maintained at room temperature (the laboratory maintains samples at 37° c). the opportunity to submit complementary cultures (e.g., from urine) from patients in which blood cultures are being collected can help to confirm the infecting bacteria and may lead to identification of a likely source (boxes 4-2 and 4-3). samples collected from patients suspected of having fungal infections of the nasal cavity (e.g., aspergillosis) or systemic (also called "deep") mycotic infections (e.g., histoplasmosis, cryptococcosis) are usually assessed by cytopathology or serology (see section 5) or with tissue biopsy and histopathology involving special stains. direct cytologic assessment of samples from patients suspected of having fungal infections is always indicated. you certainly get credit for trying! however, experience in recognizing diagnostic elements of individual fungi and spores is essential, as is the availability of special stains for wet mount (10% potassium hydroxide) cytopathology. scrapings of skin and plucked hair shafts are commonly selected for fungal culture. the area of skin and hair to be sampled should be cleaned with 70% alcohol. iodine-based soaps and solutions should not be used. hair shafts, particularly those immediately adjacent to the lesion, are removed from skin with a sterile hemostat. skin scrapings can be collected with a sterile surgical blade or the edge of a clean (unused) microscope slide. scrapings from healthy, normal-appearing skin as well as abnormal skin should be collected. skin biopsy may be required if results of attempts to culture hair and skin scrapings are negative. sterile cotton-tipped swabs should not be used to collect samples for fungal culture. hair and skin scrapings can be placed directly into a sterile, dry container without need for any type of media as long as the sample can be processed within hours. refrigeration is generally not required. if transport times are extended, it is reasonable to place samples in a vial containing bacterial transport medium and refrigerate for up to 15 hours. samples should never be frozen. skin and hair samples from patients suspected of having superficial fungal infections can be inoculated directly on a commercially available substrate called dermatophyte test medium (dtm). because samples can remain at room temperature and do not require special handling, the use of dtm is ideal for in-hospital use. the medium contains phenol red as a ph indicator. if a dermatophyte is present, characteristic colony morphology will be observed and the medium underlying the colonies will turn red. vials are unreliable after 2 weeks; color change noted 2 weeks or more after inoculation of the dtm should be disregarded. ultraviolet light filtered through nickel oxide produces a beam called wood light. if an animal is taken into a dark room and its hair and skin are exposed to a wood lamp, fluorescence may occur for several reasons. hair shafts affected by some species of microsporum fluoresce a bright yellow-green (like the color of a fluorescing watch face). however, iodide medications, petroleum, soap, dyes, bacteria, and even keratin may produce purple-, blue-, or yellow-colored fluorescence. the positive fungal fluorescence is a valuable aid in selecting affected hairs for culture inoculation. however, a negative fluorescence does not preclude a possible diagnosis of fungal infection. false-negative and false-positive interpretations are common. various laboratory techniques are currently available for the identification of viral infections in dogs and cats. excellent qualitative testing platforms are commercially available for in-practice use. molecular diagnostic tests, viral culture, histopathology, and serology, all of which are routinely available to veterinarians, require that samples be submitted to commercial laboratories for assessment. among the in-hospital test systems used to identify virus, the enzyme-linked immunosorbent assay (elisa) is the most common testing platform used. elisa testing can be performed quickly (minutes) with little or no patient preparation and with relatively high sensitivity and specificity. virus (antigen) detection tests are available as point-of-care tests for felv antigen in blood or serum and canine parvovirus (cpv) antigen in feces. in addition, these point-of-care tests for viral infections are capable of identifying patients that have not been exposed, enabling the clinician to rule out infection and viral shedding. test sensitivity refers to the likelihood that a patient with known infection will have a positive test result (a test with high sensitivity is expected to have few false-positive results). test specificity refers to the likelihood that a patient that is free of the infection will have a negative test result (a test with high specificity is expected to have few false-negative results). in addition, many commercial and point-of-care nonquantitative serologic assays are available that detect antibody to many of the viruses that affect dogs and cats. however, the positive predictive value of antibody tests is typically lower than that of antigen tests. for example, a positive antibody test result to a particular viral pathogen typically does not constitute a diagnosis of infection, especially in the absence of clinical signs. it may merely reflect recent vaccination (e.g., feline immunodeficiency virus). on the other hand, a negative antibody test result generally does indicate that the patient has had no prior exposure to the virus (or vaccine). serology refers to the use of serum to detect the concentration of antibody and is widely used in veterinary medicine. the value of antibody titers in diagnosing a viral infection is dependent on a number of factors, including the infecting virus, vaccination history, and time since exposure. use of acute and convalescent antibody titers in a patient suspected of having an acute viral infection can be a reliable diagnostic tool if a fourfold or greater increase in titer can be demonstrated over 2 to 4 weeks. acute and convalescent viral titers in individual patients are rarely performed in veterinary medicine. virus isolation, however, is a valuable diagnostic tool that is underused in veterinary medicine, perhaps because of the limited number of commercial and university laboratories that provide viral isolation services and the increased availability of molecular diagnostic testing services. diagnosis of viral upper respiratory infection in cats (herpesvirus 1 and/or calicivirus) is perhaps among the situations for which virus isolation can be most useful, especially in cluster households where many shedding carrier cats exist and kittens may be at risk. to obtain a sample for viral isolation from the oral cavity of a cat, quickly insert a sterile cotton swab into the oral cavity to the level of the tonsil or oropharynx. by rolling the swab across the epithelium, it is possible to harvest cells and virus from infected cats. immediately place the swab into a virus transport medium (usually provided by the laboratory). antibiotics added to the solution prevent bacterial overgrowth of the sample. for short-term transit (5 days or less), hold specimens for viral isolation at 4° c rather than frozen. on reaching the laboratory, the specimen will be inoculated into a suitable tissue culture. within a few days it is usually possible to establish, based on the cytopathic effect on the tissue culture, whether a virus infection is present. fluorescent antibody testing can be done subsequently to confirm the isolate. although availability is limited, direct assessment of specimens (e.g., feces for cpv or canine or feline coronavirus) can be accomplished by electron microscopy. these methods can be useful for infections in which the virus concentration in the specimen reaches 10 6 to 10 7 organisms per milliliter. specimens such as feces, vesicle fluid, brain tissue, urine, or serum can be submitted for electron microscopy. tissue specimens and exfoliative cytologic preparations can be submitted for viral identification by histopathology, immunohistochemistry, and direct fluorescent antibody testing. such testing has limited application in patients with active disease because of the limited availability of these types of services and the time required for samples to be processed and reviewed by a pathologist. these tests can be particularly useful in postmortem diagnostics when multiple animals are potentially at risk. molecular diagnostics refers to the use of nucleic acid-based tests for the detection of viral dna or rna. polymerase chain reaction (pcr) is a laboratory technology that offers exceptional test sensitivity. through its ability to amplify trace amounts of dna or rna from pathogenic organisms millions of times, pcr facilitates identification of the "target" sequence of nucleic acid and therefore the infecting organism. this technology is also available commercially for the detection of dna from selected bacteria and rickettsiae. pcr technology is particularly useful in the very early stages of a viral infection, when the level of antibody has not yet reached levels that are detectable with conventional antibody tests. in addition, pcr testing may detect healthy virus carrier animals that pose a risk to a larger population of susceptible animals yet cannot be identified by conventional virus isolation or identification technologies. it should be noted, however, that pcr technology is still subject to false-positive and false-negative test results. therefore such testing is not necessarily indicated as a primary or exclusive test method for an individual patient. serology serum, plasma, or other fluids (e.g., cerebrospinal fluid [csf]) can be tested for the presence of antibodies to selected pathogenic viruses. whole blood samples should be allowed to thoroughly clot and retract (or the sample should be centrifuged) before serum is collected. samples are submitted in a leak-proof vial. refrigeration is appropriate for samples that must be held for several hours before testing. samples are limited to tissue obtained during surgical biopsy. as with conventional histopathology, samples (no more than 5.0 mm thick) should be placed in 10% buffered formalin and submitted in a leak-proof vial. it is recommended that the volume of formalin used be at least 10 times greater than the tissue sample submitted. testing can be performed on tissues collected during surgical biopsy or from tissue impressions (exfoliative) made from tissue imprints on a clean microscope slide. it is recommended that tissue impressions on slides be fixed in alcohol or acetone before submission. fresh tissue is submitted on wet (not dry) ice and is not subjected to formalin fixation. small amounts of tissue suitable for electron microscopy should be no larger than 1 × 2 mm thick. fixation in 2% to 4% glutaraldehyde for 24 hours at 20° c is required. feces and body fluids collected for electron microscopy should be submitted fresh, not frozen or fixed in preservative. if shipping is required, feces and body fluids may be refrigerated or shipped on wet ice. samples should be viable for 48 to 72 hours. sterile swabs may be used to collect samples for viral culture and isolation. samples should be inoculated into a sealed vial containing viral transport medium (usually provided by the laboratory). samples should not be frozen or fixed in preservative. laboratories offering pcr testing typically accept serum or anticoagulated (ethylenediaminetetraacetic acid [edta]) whole blood in leak-proof vials. samples should be refrigerated and shipped on wet ice. samples should not be frozen. in most instances, a 3-to 5-ml sample of anticoagulated whole blood is adequate for routine hematology; some laboratories will accept as little as 1 ml. for routine biochemical analyses, the volume of serum requested can vary from 1 to 2 ml, depending on the number and type of tests requested. plan ahead which samples are required to prevent the need for further venipuncture at a later time. in small dogs and cats, using the jugular veins facilitates collection of an adequate volume of blood. if smaller samples are required, the cephalic, lateral saphenous, or medial saphenous vein can be used for sample collection. do not use the jugular vein if a coagulopathy is suspected, as hemorrhage may be difficult to control after venipuncture. for successful venipuncture, proper restraint of the animal is important. details for the proper restraint for various venipuncture locations are discussed with each specific topic throughout this text. the patient must remain comfortable yet relatively motionless to avoid iatrogenic vessel laceration. stretch the skin tightly over the selected vessel without causing vascular occlusion to help anchor the vessel in place during penetration by the needle. the specific venipuncture will vary somewhat depending on the specific vein selected. the following sections describe venipuncture technique for each of four commonly accessed veins: the cephalic vein, jugular vein, lateral saphenous vein, and medial saphenous vein. to restrain a dog or cat for venipuncture of the cephalic vein, place the dog or cat on the table, sitting or in sternal recumbency. if the right vein is to be tapped or catheterized, the assistant should stand on the left side of the animal and place the left arm or hand under the animal's chin to immobilize the head and neck. the assistant should reach across the animal and grasp the leg just behind and distal to the right elbow joint. the assistant should use the thumb to occlude and rotate the cephalic vein laterally while the palm of the hand holds the elbow in an immobilized and extended position. make sure that the animal stays on the table if struggling occurs. the person performing the venipuncture then grasps the leg at the metacarpal region and begins the venipuncture on the medial aspect of the leg, just adjacent to the cephalic vein proximal to the carpus. for a jugular venipuncture in the dog, place the patient in sternal recumbency, with the hands of the assistant placed around the patient's muzzle to extend the neck and nose dorsally toward the ceiling. in short-coated dogs, the jugular vein usually can be seen coursing 4 from the ramus of the mandible to the thoracic inlet in the jugular furrow. the vessel may be more difficult to visualize in dogs with long hair coats or if excessive subcutaneous fat or skin is present. the person performing the venipuncture should place the thumb of the nondominant hand across the jugular vein in the thoracic inlet or proximal to the thoracic inlet to occlude venous drainage from the vessel and allow it to fill. with the dominant hand, the person performing the venipuncture should insert the needle and syringe or vacutainer (bd, franklin lakes, new jersey) into the vessel at a 15-to 30-degree angle to perform the venipuncture. for smaller and very large animals, the jugular vein also can be tapped by placing the patient in lateral recumbency. the assistant should pull the animal's front legs caudally and extend the head and neck so that the jugular vein can be visualized. the venipuncture then can be performed as previously described. a jugular venipuncture is contraindicated in patients with thrombocytopenia or vitamin k-antagonist rodenticide intoxication. place cats in sternal recumbency. the assistant should stand behind the patient so that the patient cannot back away from the needle during the venipuncture. the assistant should extend the cat's head and neck dorsally while restraining the cat's front legs with the other hand. the cat's fur can be clipped or moistened with isopropyl alcohol to aid in visualization of the jugular vein as it stands up in the jugular furrow. the person performing the venipuncture should occlude the vessel at the thoracic inlet and insert the needle or vacutainer apparatus into the vessel as previously described to withdraw the blood sample. alternately, place the cat in lateral recumbency as described in the previous paragraph. to perform a lateral saphenous venipuncture, place the patient in lateral recumbency. the lateral saphenous vein can be visualized on the lateral portion of the stifle, just proximal to the tarsus. the assistant should extend the hindlimb and occlude the lateral saphenous vein just proximal and caudal to the tarsus. the person performing the venipuncture should grasp the distal portion of the patient's limb with the nondominant hand and insert the needle or vacutainer apparatus with the dominant hand to withdraw the blood sample. to perform a medial saphenous venipuncture, place the patient in lateral recumbency. move the top hindlimb cranially or caudally to allow visualization of the medial saphenous vein on the medial aspect of the tibia and fibula. the assistant should scruff the patient, if the patient is small, or should place the forearm over the patient's neck to prevent the patient from getting up during the procedure. with the other hand, the assistant should occlude the medial saphenous vein in the inguinal region. the person performing the medial saphenous venipuncture should grasp the paw or hock of the limb and pull the skin taut to prevent the vessel from rolling away from the needle. the fur may be clipped or moistened with isopropyl alcohol to aid in visualization of the vessel. the needle or vacutainer apparatus can be inserted into the vessel at a 15-to 30-degree angle to withdraw the blood sample. incorrect proportions of blood to anticoagulant may result in water shifts between plasma and red blood cells (rbcs). such shifts may alter the packed cell volume (pcv), especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. evaporation of plasma water and adherence of the cells to the surface of the container can produce artifactual changes in hematologic results. refrigerate liquid blood mixed with anticoagulant after collection if the sample is to be held before being transported to a laboratory. white blood cell (wbc) and rbc counts, pcv, and hemoglobin level can be measured within 24 hours of sample 4 collection. platelet counts, however, should be done within 1 hour of collection. dried, unfixed blood smears can be stained with most conventional stains 24 to 48 hours after being made. if a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least 5 minutes. blood smears fixed by this method are stable indefinitely. never place unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. take care to leave unfixed blood smears face down on a countertop or in a closed box. special stains, such as peroxidase, may require fresh blood films. the anticoagulant of choice for hematologic testing is edta. heparin is especially to be avoided if blood films are to be made from blood mixed with anticoagulant because contact with whole blood will distort the morphology of cells significantly. heparin is acceptable for most procedures requiring blood plasma. the anticoagulant effect of heparin is transitory. specimens still may clot after 2 to 3 days. make blood films immediately after collection because cell morphology rapidly deteriorates after sample collection. although blood films can be made after introducing blood to edta, a better practice is to make blood smears (films) immediately from the collection needle before the blood comes in contact with any anticoagulant. never use blood exposed to heparin to make blood smears. incorrect proportions of blood to anticoagulant may result in water shifts between plasma and rbcs. such shifts may alter the pcv, especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. evaporation of plasma water and adherence of the cells to the surface of the container can produce artifactual changes in hematologic results. refrigerate liquid blood mixed with anticoagulant after collection if there is a delay in making the laboratory determinations. wbc and rbc counts, pcv, and hemoglobin level can be measured within 24 hours of sample collection. platelet counts, however, should be done within 1 hour of collection. dried, unfixed blood smears can be stained with most conventional stains 24 to 48 hours after being made. if a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least 5 minutes. blood smears fixed by this method are stable indefinitely. never place unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. take care to leave unfixed blood smears face down on a countertop or in a closed box. special stains, such as peroxidase, may require fresh blood films. prepare the selected vein as described earlier. most clinical chemistry procedures are performed on serum. the serum is obtained by collecting blood without any anticoagulant and allowing the blood to clot in a clean, dry tube. separate serum from cells within 45 minutes of sample collection (venipuncture). special vacuum vials are available that produce a gel barrier between the clot and the serum (serum separator tubes) which avoid the need to draw off the serum into a separate vial. clotting of the blood and retraction of the clot occur best and maximum yields of serum are obtained at room or body temperature. refrigeration of the sample delays clot retraction. some samples clot and retract faster than others. if a serum separator tube is not used, it is recommended to free the clot from the walls of the container by rimming with an applicator stick. after the clot is freed, allow clot retraction to occur, and then centrifuge and draw off the clear supernatant serum using a pipette or suction bulb. allow whole blood samples to completely clot before attempting to remove serum. failing to do so may result in a mixture of plasma and serum in the submitted sample. serum yield is usually one third of the whole blood volume. patients that are hypovolemic or dehydrated can have a significantly lower serum yield. many clinical chemistry procedures can be performed on plasma and on serum. the advantage of using plasma is that separation of cells can be accomplished immediately after centrifugation or sedimentation, without the need to wait for clot formation and retraction. the disadvantage of plasma is that the presence of the anticoagulant interferes with many of the chemistry assay procedures. plasma is less clear than serum, which may be an additional disadvantage for colorimetric assays. plasma and serum are virtually identical in chemical composition except that plasma has fibrinogen and the anticoagulant. for many procedures in which plasma or whole blood is to be used, heparin is the anticoagulant of choice. heparinized blood is the only acceptable specimen for blood ph and blood gas analyses. although blood containing edta is acceptable for certain chemical procedures, it cannot be used for determination of plasma electrolytes because it contributes to and sequesters them from the specimen. in addition, edta can interfere with alkaline phosphatase levels, decrease total carbon dioxide, and elevate blood nonprotein nitrogen. refer to the tube selection guide in section 5 to assure use the proper collection tube is used for the appropriate test requested. separate serum or plasma and remove it from the cells as soon as possible after blood is collected, because many of the constituents of plasma exist in higher concentrations in rbcs. with time, these substances leak into the plasma and cause falsely elevated values (positive interference) and falsely lower values (negative interference) ( table 4 -5) . under no circumstances should whole blood be sent via the mail; serum derived from such specimens usually is hemolyzed, and results are often inaccurate. separate serum and transfer it to a clean, dry tube for shipment. bone marrow aspiration collection of bone marrow may prove valuable in diseases of the blood in which examination of the peripheral blood reveals abnormal cells or cell counts. conditions such as leukopenia, thrombocytopenia, nonregenerative anemia, agranulocytosis, pancytopenia, leukemias, other bone marrow cancers, and infectious diseases (e.g., histoplasmosis, ehrlichiosis) may be confirmed only by assessment of bone marrow cytology. bone marrow in the young animal is cellular and exists in the flat bones (sternum, ribs, pelvic bones, and vertebrae) and in the long bones (humerus and femur). as the animal ages, the cellular content of the marrow decreases, especially in the long bones. in older animals, bone marrow cells still exist in the flat bones; however, in conditions of stress in which new blood cells must be produced in large numbers, primitive cells in the bone marrow of the long bones again become active. interpretation of the bone marrow smear may be limited by (1) technique used to obtain a bone marrow specimen or (2) the specialized knowledge necessary to interpret bone marrow cells. bone marrow aspiration is much underused in clinical practice. the procedure does require some degree of skill if high-quality samples are to be obtained, but the procedure is of low risk to the patient and can be highly valuable in establishing a diagnosis or prognosis. a short-acting anesthetic occasionally may be needed, but tranquilization together with infusion of local anesthetic is usually sufficient. the site selected for aspiration or biopsy must be shaved and surgically prepared. bone marrow aspiration or biopsy is a percutaneous procedure conducted using sterile technique. the techniques involved include marrow aspiration and bone marrow core biopsy alone or in combination. when aspiration biopsy fails to produce adequate cytology (as in advanced myelofibrosis, neoplasia, or marrow aplasia), a core biopsy of bone marrow is indicated. the bone marrow aspiration needle may be a 16-gauge rosenthal needle or illinois needle for a medium-sized dog; an 18-gauge rosenthal needle for a small dog or a cat; or a jamshidi (pronounced yam-she-dee) bone marrow biopsy needle, 12 gauge for most adult dogs and 14 gauge for small dogs and cats. the selection of needles for aspiration biopsy of bone marrow is based on the biopsy site, the depth of the biopsy site, and the density of cortical bone. for bone marrow aspiration, the modified disposable illinois sternal-iliac bone marrow aspiration needle works well (figure 4-9) . for a core biopsy of bone marrow, the jamshidi bone marrow biopsyaspiration needle (pediatric, 3.5 inches, 13 gauge) can be used (figure 4 -10). the iliac crest is a commonly used site for marrow aspiration in dogs. place the animal in lateral recumbency, and prepare the aspiration site. to aspirate marrow, have the needle enter the widest part of the iliac crest and stop the needle just after penetration of the bone. remove the stylet, place a 12-ml syringe on the needle, and aspirate 0.2 ml of marrow. alternatively, the head of the humerus offers easy access to abundant bone marrow. sedation may be required. with the patient in lateral recumbency and the humerus flexed (the humerus is positioned parallel to the patient's thorax), instill local anesthetic into the skin and subcutaneous tissues to the level of the head of the humerus. the site of needle insertion is on the most proximal facet of the humoral head ( figure 4 -11). direct the needle into the bone toward the elbow and parallel to the humeral shaft. if the needle is positioned too far medially over the humeral head, it is easy to penetrate the joint capsule. although this is a common occurrence, it does not pose a risk of injury to the patient (assuming the skin was surgically prepared). if joint fluid contaminates the bone marrow aspirate, the sample will be rendered useless. contamination of the bone marrow with peripheral blood results if (1) the marrow is not aspirated immediately after the needle enters the marrow cavity or (2) if aspiration time is sustained and a large volume of blood enters the syringe subsequent to the rupture of small blood vessels in the bone marrow. perhaps the least desired technique is to obtain marrow from the proximal end of the femur by insertion of the bone marrow needle into the trochanteric fossa. make a small skin incision over the trochanteric fossa just medial to the summit of the trochanter major. insert the bone marrow aspiration needle medial to the trochanter major, and place the long axis of the needle parallel to the long axis of the femur. once the site has been selected, grasp the needle firmly. apply steady, slight pressure while alternately rotating the needle tip against the bone (fast, 180-degree clockwise and then counterclockwise movements). begin with gentle pressure until the needle begins to seat into the bone. gradually increase the pressure as the needle penetrates into the bone. insert the bone marrow needle 1 ⁄2 inch into the femoral canal. remove the stylet from the needle, and aspirate using a 12-or 20-ml syringe that contains a small volume (approximately 0.1 ml) of 4% edta. use significant negative pressure, for example, by withdrawing the plunger of a 12-ml syringe to the 8-or 9-ml mark. collection of more than 1 ml of bone marrow is unnecessary. collection of larger volumes may cause greater amounts of peripheral blood to enter the syringe, leading to hemodilution of the sample. once collection is complete, immediately transfer the aspirate to a watch glass containing approximately 0.25 ml of 4% edta. immediately mix the sample well using the end of the syringe. this is also a good time to remove the bone marrow needle from the patient. prepare slides in a manner similar to that used for peripheral blood smears. preparation of five to eight high-quality slides for submission is customary. smears are air-dried. slides may be stained using the same stains used for peripheral blood smears. bone marrow biopsy samples, usually obtained as a core, should be placed directly into 10% buffered formalin. it is generally recommended not to roll the core across a microscope slide (exfoliative cytology), as this may significantly disrupt the architecture of the sample and influence histopathologic interpretation. when submitting a bone marrow aspirate or bone biopsy, a complete blood count (cbc) should also be collected from that patient on the same day. the bone marrow sample and the cbc should be submitted together in order to obtain maximum diagnostic information. a thorough patient history should accompany the submitted samples. depending on the volume of bone marrow aspirate obtained, any additional aspirated bone marrow remaining after slides have been made can be mixed with edta in the same type of tube used to collect whole blood for a cbc. tubes may be refrigerated for short periods but never frozen. prompt shipping and processing of liquid samples of bone marrow is encouraged, as these cells tend to rapidly undergo degeneration. bone marrow biopsy core samples, after fixation in 10% buffered formalin, require decalcification before processing and interpretation. when feasible, a short-acting anesthetic administered to a cat before bone marrow aspiration or biopsy is recommended owing to the difficulty of adequately restraining a cat, even if sedated. the site selected for aspiration or biopsy must be shaved and surgically prepared. infusion of local anesthetic at the aspiration or biopsy site is appropriate. supplemental oxygen may be indicated. accessible sites for bone marrow sampling and biopsy in the cat are the iliac crest, the head of the humerus, and the proximal end of the femur via the trochanteric fossa. the techniques described for the dog can be used. smears of bone marrow are made immediately after aspiration. extrinsic thromboplastin present in bone marrow tissue will cause the marrow to clot within 30 seconds. unstained slides should be submitted. a core of bone marrow can be fixed in 10% buffered formalin before submission for decalcification and histologic preparation. another method is to aspirate the sample of bone marrow into a syringe containing 0.25 ml of 4% edta solution. expel the aspirate, up to 0.5 ml, into a sterile petri dish, from which the marrow particles can be isolated easily by aspirating an aliquot with a glass pipette, placing an appropriate volume onto several glass slides, making the appropriate number of smears. slides prepared from bone marrow aspirates should be allowed to air-dry and then labeled appropriately. slides should never be refrigerated, as moisture from condensation can alter or destroy the appearance of individual cells. cytology collection techniques (see also section 5 for additional information on slide preparation of samples to be submitted for cytopathologic examination.) cytopathology involves a simple, direct, and inexpensive technique that can yield significant diagnostic information within a short time at minimal direct cost. cytologic examination can be made of material obtained from pustules, vesicles, or the raw, ulcerated, or cut surfaces of a lesion. to make the smear, press a clean microscope slide firmly against a raw or ulcerated lesion to transfer cellular material to the slide. exudates may be collected by sterile swab or may be aspirated into a sterile syringe. roll the swab gently across the slide, or place a drop of fluid from the syringe onto the slide and carefully spread the fluid in a uniform film. transfer material from a block of tissue to the slide by gently pressing the tissue onto the slide in several locations. use various stains for different conditions. rapid stains such as new methylene blue or a quick romanowsky-type stain (e.g., diff-quik) are useful and convenient for office procedures. even wright and gram stains for evaluation of bacteria in tissues and fluids are easy to use. the presence of many bacteria, especially mixed types, may mean only surface contamination, whereas single types of bacteria, abundant polymorphonuclear wbcs, and especially phagocytosis support the diagnosis of infection and the host response to it. a few acantholytic cells (loose epidermal cells) in the smear may be compatible with infectious processes, but large numbers, or "rafts, " of acantholytic cells are highly suggestive of pemphigus and imply the need for more complex tests for positive diagnosis. large numbers of eosinophils sometimes are found in stained smears. contrary to popular opinion, they usually do not mean allergy. these cells are seen most commonly with furunculosis and may be associated with the eosinophilic granulomas, eosinophilic plaques, sterile eosinophilic pustulosis, pemphigus complex, and ectoparasites. yeasts (usually malassezia, rarely candida) commonly are found as budding cells in masses of wax and debris from ear smears. tumor cells may be recognized in some impression or aspiration samples where giemsa is a preferred stain. although special expertise is needed, cases of mastocytoma, histiocytoma, and lymphoma are recognized most easily. always prepare formalin-fixed tissues for histologic diagnosis in tumor evaluations (box 4-4). fine-needle aspiration, the use of needle and syringe to remove cells from normal and abnormal tissue, apply them to a glass slide, stain the smear, and review the results immediately is among the most useful, cost-effective procedures available in clinical practice. in most cases there will be no specific requirements for patient preparation. shaving hair over the aspiration site is generally not required. surgical preparation of the site is optional. lymph node aspiration is a procedure that can, and should, be performed routinely in clinical practice. follow proper technique to maximize the diagnostic utility of this procedure. lymph node aspiration typically is indicated (1) in patients with generalized lymphadenomegaly, (2) to evaluate abnormally enlarged solitary lymph nodes, and (3) in suspected instances of tumor metastases to lymph nodes. surgically prepare the skin over the node from which a biopsy specimen is to be taken. with one hand, localize and immobilize the lymph node; with the other hand, guide the aspiration biopsy needle into the affected node. affix a 6-ml syringe onto a 22-to 20-gauge needle (a 25-gauge needle can be used when the site to be aspirated is particularly small), and advance the needle into the lymph node. withdrawal of the syringe to approximately 0.5 ml before inserting it into the tissue is recommended. doing so helps to prevent expelling material when removing the sample from the tissue. when the needle is in position in the approximate center of the node, gradually draw negative pressure on the syringe to a level of 4 to 5 ml. hold the negative pressure in place for a few seconds. release, and then repeat two or three times. before removing the needle from the tissue, release the negative pressure in the syringe (this is why it is recommended to have 0.25 ml of air prepositioned inside). do not remove the syringe from the tissue while maintaining negative pressure, because this can enlargement of nucleus or nuclei larger than 10 nm decreased nuclear/cytoplasmic ratio multinucleation because of abnormal mitosis abnormal or frequent mitosis variations in size and shape of nuclei increase in size and number of nucleoli increased basophilia of cellular cytoplasm; increased rna content anisokaryosis or pleomorphism multinucleated giant cells box 4-4 cytologic features of malignancy 4 result in the aspiration of significant amounts of blood from the skin, thereby significantly diluting the sample with peripheral blood. eject cellular material within the needle onto clean glass slides. handle all aspirates gently. to make slides, place two slides together and pull the slides apart to avoid shearing the cells. do not compress or force slides together. in addition, a biopsy of the lymph node can (and usually should) be performed as a means of confirming or supporting diagnostic decisions made on aspirates. lymph node biopsy samples can be obtained easily and safely by punch (core) techniques (e.g., 4-mm skin biopsy punch) or tru-cut biopsy needle. the most significant limiting factors are (1) the technical ability to prepare high-quality slides and (2) the ability to interpret the cytologic findings. some experience is needed to obtain the skills needed to aspirate cells and make diagnostic preparations. significant training is required to interpret the slides adequately. however, access to cytopathologists affiliated with diagnostic laboratories today makes fine-needle aspiration a highly useful diagnostic tool. the lymph node aspiration technique, described next, illustrates the finer points of the fine-needle aspiration technique. also called "touch impression cytology, " exfoliative cytology entails preparing cytologic slides directly from the cut surface of biopsy samples. requirements for patient preparation depend on the location of the tissue from which the sample is taken. the number and quality of cells collected are best when the procedure involves the freshly cut surface of tissue. attempting to collect samples directly from skin lesions on the patient is much less likely to yield diagnostic cytology. preparation, therefore, depends on the target tissue from which slides are needed. preparation may entail local anesthesia and collection of a tissue biopsy specimen from a lesion or suspect tissue (e.g., lymph node or cutaneous tumor) or general anesthesia and an exploratory abdominal procedure (e.g., liver biopsy). once the tissue has been collected, a scalpel blade is used to make a full-thickness linear cut through the biopsy specimen. a fresh surface of the tissue of interest is exposed. using forceps or a sterile needle, gently lay the tissue on a clean glass slide. do not force the tissue onto the slide, because this can significantly damage cells. several imprints can be made from the same surface. as needed, make new cuts to obtain a fresh surface from which to exfoliate cells. allow the slide to air-dry completely. apply conventional staining, and examine the specimen when it is dry. the remaining tissue, if not significantly damaged, can be submitted for histopathologic examination (recommended). once slides have air-dried and are labeled, they may be stained and reviewed immediately or submitted for review and interpretation by a pathologist. unstained slides should not be refrigerated, as moisture from condensation can alter the cytology of the preparation. several slides should be submitted. any remaining tissue may be placed in 10% buffered formalin and submitted for histopathology. the number of diagnostic cells obtained when making slides by way of exfoliative cytology depends on the tissue. epithelial cells (e.g., carcinoma), mast cells (cutaneous mast cell tumor), and lymph nodes readily exfoliate abundant numbers of cells. excessively thick slides can make interpretation difficult. on the other hand, biopsy specimens of tissue composed predominantly of mesenchymal cells (e.g., granuloma, fibrosarcoma) do not readily exfoliate cells, and slides made from these types of tissues are typically hypocellular. contamination of the cytology specimen with peripheral blood is a common mistake that can make interpretation of the sample difficult. it may be appropriate to gently blot the cut surface of the tissue sample, thereby removing excessive blood, before making slides. depending on the tissue type and lesion, it may be possible to obtain diagnostic cytologic samples from scrapings (e.g., conjunctival epithelium for virus inclusions), brushes (e.g., material obtained during endoscopy), and swabs (e.g., ear and vaginal swabs). the cells, once harvested, can be applied delicately directly to a clean glass slide by carefully rolling or even by just touching the material to the slide to create a thin layer. allow the sample to airdry thoroughly before staining. cytologic examination of fluids obtained with needle and syringe from body cavities, cysts, and urine typically requires additional preparation to obtain adequate cell concentration to make diagnostic decisions. analyze fluid specimens with respect to protein and nucleated cell count and a morphologic description of the cells. if overall cell counts are low, centrifugation will be required to concentrate cellular material for analysis. after centrifugation, remove the supernatant (and save it). resuspend the cells in two or three drops of the supernatant. apply a single drop of the mixture to a glass slide and allow it to air-dry. i prefer not to smear the liquid onto the slide; instead, i allow the liquid to run, by gravity, from one end of the slide to the other. after the liquid is thoroughly air-dried, it can be stained and reviewed. none required. skin scrapings frequently are obtained to find and identify microscopic parasites or fungal elements in the skin. material required includes mineral oil in a small dropper bottle, a dull scalpel blade, glass slides, coverslips, and a microscope. select undisturbed, untreated skin for a scraping site. the best method is to scrape the periphery of skin lesions and avoid the excoriated or traumatized center areas. in scraping for demodectic mange, pinch a small fold of affected skin firmly and collect the surface material for examination. this procedure forces the mites out of the hair follicles and onto or near the skin surface. for sarcoptic mange, scrape large areas. select sites on the elbows, hocks, and ear margins when searching for sarcoptic mange. many or frequent scrapings may be necessary to demonstrate sarcoptic mange mites or their fecal pellets or eggs. place the accumulated material on a microscope slide and mix it with mineral oil. examine the entire area with a ×10 objective thoroughly and carefully. none required. acetate tape preparation is one of the simplest diagnostic procedures to perform when looking for the presence of ectoparasites, especially the nits of cheyletiella. use clear (not frosted) acetate tape. bend the tape into a loop around the fingers with the sticky side facing out. part the animal's hair coat, and press the tape firmly onto the skin and hair around suspect lesions. the sticky tape picks up loose particles with which it makes contact. cut the loop of tape and place the strip of tape sticky side down on a clean microscope slide. use a low-power microscope to look through the tape at the collected particles. this technique is excellent for trapping and identifying biting and sucking lice, otodectes and cheyletiella mites, flea dirt and larvae, fly larvae, and dandruff scales. acetate tape also is useful for studying hair abnormalities. use a strong hemostat to securely clamp and quickly avulse a group of 10 to 20 hair shafts. press the pointed distal ends onto sticky acetate tape (lined up like pickets in a fence), and cut the hair shafts off in the middle with scissors. likewise, press the butt ends with the hair roots onto another piece of tape. then press the tape holding the hair onto a microscope slide to allow low-power examination of the hairs through the clear tape. the tips of the hairs will be well oriented and controlled; thus, it is easy to evaluate whether the hairs are split, broken, or bitten off and whether the hair roots are in the anagen or telogen growth stage. urine can be removed from the bladder by one of four methods: (1) voided (the "free catch"), (2) manual compression of the urinary bladder (expressing the bladder), (3) catheterization, or (4) cystocentesis. for routine urinalysis, collection of urine by voiding (micturition) is satisfactory. the major disadvantage is risk of contamination of the sample with cells, bacteria, and other debris located in the genital tract and the perineal hair coat. the first portion of the stream is discarded, as it is most likely to contain debris. voided urine samples are not recommended when bacterial cystitis is suspected. compressing the urinary bladder is occasionally used to collect urine samples from dogs and cats. critical: do not use excessive pressure; if moderate digital pressure does not induce micturition, discontinue the technique. excessive pressure can culminate in forcing contaminated urine (bladder) into the kidneys, or, worse, in patients with a urethral obstruction the urinary bladder can rupture. the technique is most difficult to accomplish in male dogs and male cats. several types of urinary catheters are currently available for use in dogs and cats. the catheter types most often used today are made of rubber, polypropylene, and latex-free silicone. stainless steel catheters are occasionally used but unless placed with care these can cause damage to the urethra and/or urinary bladder. generally, urinary catheters serve one of four purposes: 1. to relieve urinary retention 2. to test for residual urine 3. to obtain urine directly from the bladder for diagnostic purposes 4. to perform bladder lavage and instillation of medication or contrast material the size of catheters (diameter) usually is calibrated in the french scale; each french unit is equivalent to roughly 0.33 mm. the openings adjacent to the catheter tips are called "eyes. " human urethral catheters are used routinely in male and female dogs; 4f to 10f catheters are satisfactory for most dogs (table 4-6) . polypropylene catheters should be individually packaged and sterilized by ethylene oxide gas. equipment needed to catheterize a male dog includes a sterile catheter (4f to 10f, 18 inches long, with one end adapted to fit a syringe), sterile lubricating jelly, povidone-iodine soap or chlorhexidine, sterile rubber gloves or a sterile hemostat, a 20-ml sterile syringe, and an appropriate receptacle for the collection of urine. proper catheterization of the male dog requires two persons. place the dog in lateral recumbency on either side. pull the rear leg that is on top forward, and then flex it ( figure 4-12) . alternatively, long-legged dogs can be catheterized easily in a standing position. before catheter placement, retract the sheath of the penis and cleanse the glans penis with a solution of povidone-iodine 1% or chlorhexidine. lubricate the distal 2 to 3 cm of the appropriate-size catheter with sterile lubricating jelly. never entirely remove the catheter from its container while it is being passed because the container enables one to hold the catheter without contaminating it. the catheter may be passed with sterile gloved hands or by using a sterile hemostat to grasp the catheter and pass it into the urethra. alternatively, cut a 2-inch "butterfly" section from the end of the thin plastic catheter container. this section can be used as a cover for the sterile catheter, and the clinician can use the cover to grasp and advance the catheter without using gloves. if the catheter cannot be passed into the bladder, the tip of the catheter may be caught in a mucosal fold of the urethra or there may be a stricture or block in the urethra. in smallbreed dogs, the size of the groove in the os penis may limit the size of the catheter that can be passed. one also may experience difficulty in passing the catheter through the urethra where the urethra curves around the ischial arch. occasionally a catheter of small diameter may kink and bend on being passed into the urethra. when the catheter cannot be passed on the first try, reevaluate the size of the catheter and gently rotate the catheter while passing it a second time. never force the catheter through the urethral orifice. effective catheterization is indicated by the flow of urine at the end of the catheter, and a sterile 20-ml syringe is used to aspirate the urine from the bladder. walk the dog immediately after catheterization to encourage urination. equipment needed to catheterize a female dog includes flexible urethral catheters identical to those used in the male dog. the following materials also should be on hand: a small nasal speculum, a 20-ml sterile syringe, lidocaine 0.5%, sterile lubricating jelly, a focal source of light, appropriate receptacles for urine collection, and 5 ml of povidone-iodine or a dilute chlorhexidine solution. use strict asepsis. cleanse the vulva with a solution of povidone-iodine or dilute chlorhexidine. instillation of lidocaine 0.5% into the vaginal vault helps to relieve the discomfort of catheterization. the external urethral orifice is 3 to 5 cm cranial to the ventral commissure of the vulva. in many instances the female dog may be catheterized in the standing position by passing the female catheter into the vaginal vault, despite the fact that the urethral papilla is not visualized directly. in the spayed female dog, in which blind catheterization may be difficult, the use of a sterilized otoscope speculum and light source (figure 4 -13), vaginal speculum, or anal speculum with a light source will help to visualize the urethral tubercle on the floor of the vagina. in difficult catheterizations it may be helpful to place the animal in dorsal recumbency (figure 4-14 and 4-15 ). insertion of a speculum into the vagina almost always permits visualization of the urethral papilla and facilitates passage of the catheter. take care to avoid attempts to pass the catheter into the fossa of the clitoris because this is a blind, possibly contaminated cul-de-sac. before attempting urinary bladder catheterization of the male cat, administer a short-term anesthetic (e.g., ketamine, 25 mg/kg im), but only after a careful assessment of the cat's physical, acid-base, and electrolyte status (see treatment of hyperkalemia in section 1). in some cases, drugs to treat hyperkalemia may be required before anesthetic induction. once the patient's electrolyte status has been evaluated and hyperkalemia, if present, addressed appropriately, anesthesia can be induced with a combination of propofol (4 to 7 mg/kg intravenously [iv]) and diazepam (0.1 mg/kg iv); then the patient is intubated and maintained on gas anesthesia. place the anesthetized patient in dorsal recumbency. gently grasp the ventral aspect of the prepuce and move it caudally in such a manner that the penis is extruded. withdraw the penis from the sheath and gently pull the penis backward. keeping sterile catheters in a freezer will help them become more rigid to facilitate passage into the urethra. pass a sterile, flexible plastic or polyethylene (pe 60 to 90) catheter or 3-to 5-inch, 3.5f urethral catheter into the urethral orifice and gently into the bladder, keeping the catheter parallel to the vertebral column of the cat. caution: never force the catheter through the urethra. the presence of debris within the urethral lumen may require the injection of 3 to 5 ml of sterile saline to back-flush urinary "sand" or concretions so that the catheter can be passed. in some instances the presence of cystic and urethral calculi will prevent the passage of a catheter into the urethra. for this reason a lateral radiograph of the penis, with the patient's hindlimbs pulled caudally, may help document the presence of a urethral stone. urinary bladder catheterization of the female cat is not a simple procedure. when indicated, and after a preanesthetic examination has been performed, attempt the technique only in the anesthetized cat. urinary bladder catheterization can be accomplished with the use of a rubber or plastic, side-hole (blunt-ended) urinary catheter. the same catheter type used in male cats is effective in female cats. instilling lidocaine 0.5% has been recommended as a means of decreasing sensitivity to catheter insertion in sedated (not recommended) cats. cleanse the vulva with an appropriate antiseptic. catheterization can be accomplished with the cat in dorsal or ventral recumbency. experience and size of the cat dictate which technique works best. after cleansing of the perineum and vaginal vault, place the patient in sternal recumbency, and gently pass the catheter along the ventral floor of the vaginal vault. conversely, if the patient is placed in dorsal recumbency, direct the catheter dorsally along the ventral vaginal floor. if a catheter cannot be placed blindly, a small otoscopic speculum can be placed into the vagina, and the catheter pushed into the urethral papilla once it is visualized directly. for continuous urine drainage in the awake, ambulatory patient, use a closed collection system to help prevent urinary tract infection. a soft urethral or foley catheter can be used, and polyvinyl chloride tubing should be connected to the catheter and to the collection bag outside the cage. the collection bag should be below the level of the animal's urinary bladder. place an elizabethan collar on the animal to discourage chewing on the catheter and associated tubing. the urinary bladder is catheterized as described previously. despite the quality of care of the catheter, urinary tract infection still may develop in any patient fitted with an indwelling urinary catheter. ideally, remove the catheter as soon as it is no longer necessary, or if there are clinical signs of a urinary tract infection or previously undiagnosed fever. a urinary catheter is generally changed after it has been in place for more than 48 hours. observe the patient for development of fever, discomfort, pyuria, or other evidence of urinary tract infection. if infection is suspected, remove the catheter and submit urine for culture and sensitivity or determination of minimum inhibitory concentration (mic). previously, culture of the catheter tip was recommended to diagnose a catheterinduced infection. however, culture of the catheter tip is no longer recommended, as it may not accurately reflect the type of microorganisms in a urinary tract infection. the empiric use of antibiotics to help prevent catheter-induced infection is not recommended, as their use can allow colonization of resistant nosocomial bacteria in the patient's urinary tract. cystocentesis is a common clinical technique used to obtain a sample of urine directly from the urinary bladder of dogs and cats when collecting a voided, or free-catch, aliquot is not preferred. the procedure is indicated when necessary to obtain bladder urine for culture purposes. urine that is collected by free catch has passed through the urethra and may be contaminated with bacteria, thereby making interpretation of the culture results difficult. cystocentesis also is performed as a convenience when it is desirable to obtain a small sample of urine but the patient is not ready or cooperative. cystocentesis involves insertion of a needle, with a 6-or 12-ml syringe attached, through the abdominal wall and bladder wall to obtain urine samples for urinalysis or bacterial culture. the technique prevents contamination of urine by urethra, genital tract, or skin and reduces the risk of obtaining a contaminated sample. cystocentesis also may be needed to decompress a severely overdistended bladder temporarily in an animal with urethral obstruction. in these cases, cystocentesis should be performed only if urethral catheterization is impossible. warning: penetration of a distended (obstructed) urinary bladder with a needle could result in rupture of the bladder. to perform cystocentesis, palpate the ventral abdomen just cranial to the junction of the bladder with the urethra, and trap the urinary bladder between the fingers and the palm of the hand. use one hand to hold the bladder steady within the peritoneal cavity while the other guides the needle. next, insert the needle through the ventral abdominal wall into the bladder at a 45-degree angle (figure 4-16) . although this procedure is relatively safe, the bladder must have a reasonable volume of urine, and the procedure should not be performed without first identifying and immobilizing the bladder. for the procedure to be performed safely and quickly, the patient must be cooperative. if collection of a urine sample by cystocentesis is absolutely necessary, sedation may be indicated to restrain the patient adequately for the procedure. generally, cystocentesis is a safe procedure, assuming the patient is cooperative and the bladder can be identified and stabilized throughout the procedure. however, injury and adverse reactions can occur. in addition to laceration of the bladder with the inserted needle (patient moves abruptly), the needle can be passed completely through the bladder and into the colon, causing bacterial contamination of the bladder or peritoneal cavity. there is also risk of penetrating a major abdominal blood vessel, resulting in significant hemorrhage. obtaining a skin biopsy from abnormal skin only to receive a nondiagnostic result as reported from a pathologist suggests that improved biopsy technique may result in collecting a specimen with higher diagnostic value. the following guidelines apply when skin biopsies are performed: ❏ consider obtaining multiple samples from multiple sites, which is especially useful when different stages of similar lesions are identifiable. ❏ do not perform a surgical scrub before collecting the sample; shaving the hair away is fine, but surgically prepared skin removes superficial lesions that, had they been left in place, might have been diagnostic. ❏ biopsy of lesions that are depigmenting should be done before they have turned white; the absence of color usually denotes absence of active skin lesions. biopsies from completely depigmented skin are less likely to demonstrate active lesions. ❏ biopsy of lesions associated with alopecia should be done in the center of the most alopecic area. ❏ also, biopsy of lesions associated with alopecia should be done at junctional (between normal-and abnormal-appearing) skin. ❏ consider submitting biopsy samples from completely unaffected, normal-appearing skin. ❏ avoid biopsies of ulcerated skin areas. biopsy samples may be obtained with a scalpel blade (incisional or excisional) or via a dermatologic punch biopsy. punch biopsy instruments are circular blades available in 4-mm, 6-mm, and 8-mm diameter sizes (figure 4-17) . hold the punch perpendicular to the skin site of interest. a back-and-forth motion that rotates the circular blade cuts through the skin. when the skin no longer moves as the punch is rotated, the biopsy is complete and the skin sample may be removed (from the skin or from the biopsy instrument). avoid grasping the dermis or epidermis of the sample with any instrument to prevent crushing of the sample and causing artifact. if the sample must be lifted, use the attached subcutaneous fat only. if the lesion of interest is deep, the punch biopsy technique may not be effective. in this situation, an incisional or excisional biopsy using a sterile no. 10 or no. 15 surgical blade is indicated. biopsies of ulcerated skin and solitary nodules are best done by removing a wedge of skin (incisional biopsy). in some cases it is possible surgically to remove all visible, palpable parts of the lesion (excisional biopsy). place each sample of skin in buffered formalin, using a volume that is at least 10 times that of the sample size. if particularly large areas of skin are harvested during biopsy, cut these into 1-cm thick pieces before placing into formalin. (note: placing larger tissue samples into formalin may result in inadequate or incomplete fixation of the tissue and the inability to properly prepare the tissue for examination). alternatively, it is possible, and in many cases important, to evaluate a biopsy specimen of skin or subcutaneous tissue at the time of collection. when the lesion of interest is suspected to be neoplastic, quickly differentiating between inflammatory cells and neoplasia may be possible by simply performing an exfoliative cytologic examination (described in this section) on one of the biopsy samples in addition to fixing a separate sample in formalin and submitting that for histopathologic examination. small biopsy samples that have been subjected to the additional handling required to make impressions on a glass slide are not good candidates for subsequent fixation and histopathologic examination. it is generally recommended to perform exfoliative cytologic and histopathologic examinations on separate samples. among the most common diagnostic procedures carried out on the skin of dogs and cats is a routine skin scraping. yet despite the frequency with which this test is used, doing a skin scraping in such a manner that the sample recovered maximizes the opportunity to establish a diagnosis can be anything but routine. a skin scraping, properly done, does require using consistent techniques appropriate to the suspected diagnoses, and as such, superficial or deep scrapings, or both, may be indicated. skin scraping is indicated whenever ectoparasite infestation is suspected. superficial scrapings are appropriate for detecting mites that live on the skin surface, such as cheyletiella species and otodectes cynotis, as well as mites that burrow within the outermost layers of skin (stratum corneum), such as sarcoptes species and notoedres cati. because the area to be scraped is relatively large (≥2 cm 2 ), shave dogs and cats with long hair coats before attempting the procedure, unless cheyletiella infestation is suspected. make the scraping over healthy-appearing skin. do not cleanse the skin of superficial scale or crusts. the technique for superficial skin scraping entails the use of mineral oil or pyrethrin ear drops applied to a clean scalpel blade and directly onto the area of skin to be scraped. scraping begins as a gentle motion made in the direction of the hair coat. gradually increase the pressure of the blade against the skin with repetitive scrapings over the same area. take care not to lacerate the skin, although minor capillary bleeding at the site is common. transpose material collected on the edge of the blade to a clean glass slide, cover it with a coverslip, and thoroughly examine the material under low magnification for evidence of ectoparasites. note that for mites such as cheyletiella or scabies, finding just one mite or one egg is diagnostic and justifies implementing treatment. same as described for superficial skin scraping (earlier). a slightly different technique is indicated in dogs and cats suspected of having an infestation that includes demodex canis mites. the mites are known to live predominantly in sebaceous glands and hair follicles. they can survive in the skin of animals without manifesting lesions. hair loss and skin lesions develop where overgrowth of the mite population occurs. demodex infestations can be localized or generalized; infestations can occur in either dogs or cats but the most severe, generalized infestations are much more likely to occur in young dogs. although both superficial and deep skin scrapings may reveal the presence of mites on the skin, deep scrapings may reveal demodex mites in some patients when superficial scrapings are negative. the technique for deep skin scraping targets a small area of skin (<2 cm 2 ). it may be helpful to apply gentle pressure to the skin or actually to squeeze the area of interest between the thumb and a finger in an attempt to force mites from the deeper to the more superficial skin. in some breeds (e.g., old english sheepdogs and shar-peis) recovering mites on a skin scraping can be particularly difficult. in such cases, when demodex infestation is highly suspected but the results of repeated skin scrapings are negative, a skin biopsy is appropriate. alternatively, a procedure called a trichogram that involves pulling (plucking) a few hairs from the hair follicles using a hemostat may be diagnostic. once the hairs have been plucked from the skin, place them on a glass slide that has been preprepared with a drop of mineral oil, add a coverslip, and examine the hair shaft under low magnification. half of all dogs with demodex infestation will have a positive trichogram. not all dogs and cats with otitis externa require comprehensive ear flushing and debridement before or as part of otic therapy. in many cases, home treatment is sufficient to resolve the problems effectively, assuming the underlying diagnosis has been established. however, in patients with chronic or particularly severe infections, topical treatment administered by the owners at home may not be sufficient. in such cases, the external ear canal requires a careful and comprehensive cleaning before administration of topical medications. properly performed, flushing and cleaning of the external ear canals is not a quick procedure. anesthetize the patient. attempting to perform a thorough ear cleansing under sedation usually will not be successful. once the animal is anesthetized, perform a careful otoscopic (or video otoscopic) examination to establish the integrity of the ear canal, evaluating, for example, for the presence or absence of tumors or parasites. in severe cases the tympanic membrane (ear drum) may not even be visible. with the patient in lateral recumbency, flush the ear canal (figure 4 -18) or lavage it with warm saline initially, and then aspirate the material from the canal. if this procedure is not successful in removing the debris attached to the epithelium of the ear canal, use 4 ceruminolytic ear solutions to facilitate breakdown and removal of this material. a 5-minute instillation and soak is recommended, followed by thorough flushing to remove debris and the ceruminolytic material. remove hair growing inside the ear canal with forceps. a suction apparatus is recommended for removal of debris and liquid remaining. reintroduce an otoscope to examine the integrity of the skin in the ear canal and to look for any evidence of stenosis, foreign body, or tumor. the flushing process is not complete until it is possible to visualize the tympanic membrane. carefully remove any remaining debris with an otologic loop (figure 4-19) , not a cotton-tipped swab. repeat the procedure on the opposite ear as indicated. at the conclusion of the examination, apply appropriate topical medication into the ear canal before allowing the patient to recover from anesthesia. systemic therapy or surgical intervention may be required in some patients for complete resolution of the problem. however, a thorough examination and cleaning is critical before actually making decisions regarding medical versus surgical intervention. use of a cotton-tipped swab to remove debris from the external ear canal, although commonly done, is not generally recommended. repeated attempts may ultimately force debris deeper into the external ear canal. general anesthesia and otic lavage or flushing may be required to effectively correct the problem created by the use of cotton-tipped swabs. in selecting an appropriate endotracheal tube, consider the size of the animal and select a tube that has the largest diameter that can be inserted without force (table 4 -7). the length of tube selected must not extend beyond the bifurcation of the trachea (carina). always check the cuff of the endotracheal tube to ensure there are no leaks and that the cuff is working properly before intubation. lubricate the selected endotracheal tube with sterile lubricating jelly before inducing anesthesia. after induction, place the patient in sternal recumbency and elevate the head. the individual inserting the tube should grasp and extend the tongue with a piece of gauze. the tongue is extended to facilitate visualization of the larynx. avoid excessive downward pressure on the tongue in order to prevent inadvertent laceration or injury from the lower incisors. if a laryngoscope is used, place the tip of scope at the base of the tongue. gently press the tip of the laryngoscope ventrally to move the epiglottis and expose the glottis. contact during an attempt to intubate, one or two drops of 2% lidocaine can be applied to the arytenoid cartilages. once the tube has been inserted into the trachea, never advance it farther than the carina. doing so may result in intubation of either the right or the left main bronchus (endobronchial intubation). once the tube is in place, secure it in place with a loop of 1 ⁄2-inch white tape or muzzle gauze. overinflation of the endotracheal tube cuff can cause tracheal ulceration, tracheitis, hemorrhage, tracheomalacia, fibrosis, stenosis, and subcutaneous emphysema. abdominal paracentesis refers to the aspiration of fluid from the abdominal cavity for both diagnostic and therapeutic purposes. always weigh the animal before and after removing abdominal fluid. any subsequent gain in weight indicates a reaccumulation of abdominal fluid. place the animal in left lateral recumbency and restrain it in this position. clip and surgically prepare a 1-to 3-inch square between the bladder and the umbilicus just lateral to the midline. if the bladder is distended, empty it before performing paracentesis. infiltrate the paracentesis site with lidocaine 0.5% using a 22-to 25-gauge needle. in most cases, local anesthesia is not necessary. abdominal puncture can be made with an 18-to 20-gauge needle (figure 4-20) . gently insert the needle through the skin and external abdominal oblique muscles while simultaneously pushing and twisting, to push viscera away from the tip of the needle. blind abdominocentesis without the use of ultrasound to guide needle placement into a fluid pocket can have negative results if there is less than 5 ml of fluid per kilogram within the peritoneal cavity. when the abdominal puncture has been made, allow the animal to rest quietly to 4 facilitate drainage of the fluid. some clinicians recommend tapping while the patient is in a standing position in the hope of obtaining more complete drainage. changing the patient's position after the tapping may result in needle-tip laceration of intraabdominal organs. aspiration may be easier if a specially adapted needle with multiple holes drilled in the shaft is used because it is less likely to become plugged with omentum. ideally, tap four quadrants of the abdomen. if four-quadrant abdominocentesis has been performed and no fluid has been obtained, and if there is suspicion for peritonitis, diagnostic peritoneal lavage can be performed. the abdomen is clipped and aseptically scrubbed as previously described. next, insert an over-the-needle catheter or hypodermic needle just caudal to the umbilicus, and instill 10 ml of warmed 0.9% sterile saline or lactated ringer's per kilogram. after instillation of the fluid, walk the patient around or gently roll the patient from side to side to distribute the fluid throughout the abdominal cavity. next, perform four-quadrant abdominocentesis as previously described. only a small amount of the fluid infused will be obtained. the fluid will be diluted by the saline or lactated ringer's infused, so biochemical analysis will not be accurate. however, the fluid can be examined microscopically for the presence of plant material, bacteria, wbcs, or bile pigment to help diagnose various forms of peritonitis. hackett numerous biopsy techniques are available, and the selection of the appropriate technique is based on the tissue to be examined, the condition of the patient, and the skill of the examiner. inserted cranial and to the right/left of the umbilicus, and caudal and to the right/left of the umbilicus. when the needle is inserted, it should be twisted while penetrating into the abdominal cavity, to prevent iatrogenic puncture of hollow organs. in some cases it may be necessary to place more than one needle before fluid will flow from the needle hub. appropriate locations are marked on the skin with an "x." excisional biopsy refers to the surgical removal of the entire lesion or organ with subsequent histologic examination. excisional biopsy is used most frequently for skin lesions and cases in which an entire organ may have to be removed (such as an eye or an internal organ that has developed a tumor). incisional biopsy refers to the surgical removal of a portion of a lesion with subsequent histologic examination. choose a representative area of the lesion for biopsy. include lesion margins, if possible. needle aspiration refers to the use of needle and syringe to remove representative cells from the tissue or organ of interest. specialized needles are available that allow removal of very small biopsy specimens that can be submitted for histopathologic examination. (see also fine needle aspiration.) needle biopsy techniques: general considerations needle biopsy or aspiration techniques refer to a variety of techniques used to obtain diagnostic tissue or cells from internal organs, including the lung, liver, spleen, pancreas, abdominal lymph nodes, and mass lesions within the abdomen and thorax. in contrast, fine-needle aspiration is a technique generally used to recover cytopathologic samples (cells only) from skin or subcutaneous tissues (e.g., superficial lymph nodes). the advantage of needle biopsy is related directly to how well the abnormal tissue has been characterized and how easily it can be identified during the procedure. in addition, depending on patient cooperation, most procedures can be performed safely with the patient sedated only. shortterm intravenous anesthesia and general anesthesia eliminate undesired patient movement during the biopsy procedure. potential lesions or abnormal tissues from which aspirate or biopsy samples are to be taken are located using palpation, radiographs, or ultrasound-guided imaging techniques. shave the skin over the site of needle penetration and surgically prepare it. the type of sedation or anesthesia depends on the temperament of the animal and the site on which the biopsy will be performed. attach a 22-gauge needle without stylet to a 12-ml syringe prefilled with 0.5 to 1.0 ml of air. optionally, affix a flexible extension set to the needle and connect it proximally to the syringe. needle length may vary from 1 to 3 1 ⁄2 inches depending on the required depth of penetration and size of the patient. guide the needle into the tissue or organ of interest. stabilize the tip of the needle to avoid random movements through organs, especially highly vascular tissue such as liver and spleen. once the needle has been inserted, the aspiration technique entails withdrawing the plunger of the syringe to the 7-or 8-ml level. hold that position for 1 to 2 seconds, and then release. repeat the procedure. depending on the nature of the lesion, it may not be indicated to thrust the needle into the tissue at multiple and different angles. neutralize the pressure in the syringe, and withdraw the needle rapidly. expel any material within the needle onto glass slides using the air in the syringe. this same procedure can be repeated with a new needle to obtain an additional three to five samples from alternative sites. this technique allows samples to be obtained without applying negative pressure to the syringe, which may damage cells. ultrasound guidance for needle aspirations from abdominal tissues greatly enhance the safety of this technique, especially when obtaining samples from smaller animals. automatictrigger needles such as cook or temno biopsy needles (14 to 18 gauge) are available for use in human beings but are seldom used in veterinary medicine. the risks associated with fine-needle aspiration include rupture of an encapsulated inflammatory process, dissemination of an infectious agent, seeding of neoplastic cells in the needle tract, and hemorrhage. larger volumes of fluid and cells can be placed directly into a vial containing edta to prevent clot formation. prepare and examine direct and sedimentation specimens. needle biopsy of internal organs using the tru-cut needle is particularly useful in patients with subcutaneous ( figure 4 -21) or cutaneous masses and for localized abdominal and thoracic mass lesions and diffuse liver, kidney, and splenic disease. serious complications, usually hemorrhage or laceration of the gallbladder (during liver biopsy), can occur when the procedure is performed blindly. therefore ultrasound-guided needle biopsy is strongly recommended whenever a percutaneous biopsy of internal organs is performed. additional safety factors provided by ultrasound guidance include the ability to image, and avoid, large aberrant blood vessels. risk of complications associated with needle aspiration of the lung is considerably higher than for most abdominal procedures. pneumothorax can occur after a single, "clean" aspiration attempt. see respiratory tract procedures for a detailed description of performing fine-needle aspiration of the lung. histologic examination of diseased skin can serve as a means for diagnosis of cutaneous lesions. the causative agent often is found in acute and chronic skin infections. punch biopsy of the skin is a quick and accurate means of removing a small sample of diseased skin for histopathologic examination. select a site that is well developed but not traumatized or excoriated. the sample should include little or no normal tissue. if the lesion (pustule, vesicle) can be identified early in its development and if the biopsy sample is taken only from the lesion, one may obtain a superior specimen. it is best not to take too large a sample that contains much normal skin; by mistake, the technician might take a section that misses the lesion. proper selection of the biopsy site is crucial to accurate diagnosis. carefully clip the hair from the lesion. lightly blot the skin with 70% alcohol. avoid superficial trauma while cleaning the skin. inject a small subcutaneous bleb of 2.0% lidocaine to deaden the area. special equipment needed for the biopsy includes a 4-mm, 6-mm, or 8-mm biopsy punch and 10% buffered formalin solution. after the area has been anesthetized with lidocaine, press and rotate the biopsy punch through the skin until the subcutaneous tissue is penetrated. remove the biopsy specimen by "spearing" the subcutaneous fat with a fine needle. do not grasp the specimen with a forceps. blot the specimen gently between two paper towels. spread the tissue out gently (like a pancake), place the specimen epidermal side up on a piece of cardboard or tongue depressor, press the specimen gently to cause adhesion, and drop the specimen into the formalin fixative. the skin defect may be closed with one or two simple interrupted sutures. if deep subcutaneous tissue or large biopsy samples are needed, a punch biopsy is inadequate. use a small (no. 15) scalpel blade to obtain an appropriate sample. in all cases in which skin biopsies are made, take multiple samples to increase the odds that at least one will have diagnostic lesions. specimens submitted to laboratories should be accompanied by extensive, detailed clinical information, including a differential diagnosis. skin biopsies routinely are stained with hematoxylin-eosin; however, periodic acid-schiff, gomori methenamine silver, and verhoeff stains are used for special problems. the diagnosis of liver disease is generally confirmed on the basis of the patient's clinical signs coupled with laboratory findings, radiography, and abdominal ultrasound. the development of a more specific diagnosis and prognosis in liver disease may be aided greatly by information obtained in a liver biopsy. percutaneous liver biopsies are of much greater value in generalized liver disease such as cirrhosis, generalized acute hepatic necrosis, or amyloidosis than in focal hepatic disease. the major indications for performing a liver biopsy are (l) to explain an abnormal liver profile, (2) to define reasons for abnormal liver size, (3) to identify a possible liver tumor, (4) to arrive at a prognosis and rational approach to management, and (5) to identify the cause of ascites. the procedures for obtaining liver tissue are numerous; however, needle biopsy of the liver, when performed properly, can be helpful. careful physical and clinicopathologic examination should precede a liver biopsy. a normal coagulation profile should be documented on every patient undergoing liver biopsy. detect and correct abnormalities in normal hemostatic mechanisms, if feasible, before needle biopsy of the liver. liver biopsy should be performed only in the fasted patient and only after removal of ascitic fluid. percutaneous needle biopsies and fine-needle aspirations of the liver can be performed with local anesthesia in the sedated, and cooperative, patient. general anesthesia is a reasonable alternative whenever feasible. biopsy sites in the liver can be selected best when needle biopsy techniques are used along with laparoscopy or ultrasound techniques. blind percutaneous needle biopsies of the liver can be performed with relative safety if the liver is significantly enlarged and easily palpated. however, blind biopsies do carry the risk that the operator will be unable to determine the impact of penetrating the liver if only an abdominal radiograph and impression of abdominal palpation are available. in cases in which the liver is not palpable, blind biopsy carries significantly higher risk and should be performed only when no alternative exits. a modified percutaneous liver biopsy can be performed by the following method. place the animal in dorsal recumbency, and place a local block in the midline of the skin and abdomen at the caudoventral aspect of the left hepatic lobe. the incision into the peritoneal cavity should be large enough to accommodate the gloved index finger. make a separate skin puncture site in the abdominal wall to accommodate the biopsy needle. use the index finger manually to fix the left hepatic lobe (or other desired hepatic lobe) against the diaphragm or other adjacent structures, and insert the outer cannula and stylet through the abdominal wall in the isolated hepatic lobe. remove the stylet, and rapidly insert the cutting prongs. if properly placed, the cutting prongs should not go through the entire hepatic lobe. advance the outer cannula over the blades of the cutting prongs, thus entrapping the hepatic tissue material within the cutting prongs. remove the biopsy needle. using a wooden applicator stick, carefully place the biopsy specimen into fixative. biopsy samples can be used to prepare slides for cytologic examination, and the biopsy needle may be cultured. close the abdominal incision in the routine manner. another liver biopsy technique entails use of a tru-cut biopsy needle. place the dog in dorsal recumbency. clip a 5-cm 2 area over the triangle formed by the xiphoid cartilage and left costal arch, and prepare the area as for aseptic surgery. make a small paramedian incision large enough to accommodate a sterile otoscope head 7 mm in diameter. use a sterilized halogen-illuminated otoscope speculum to visualize the liver. pass a tru-cut biopsy needle through the otoscope cone to directly obtain a biopsy specimen of the liver. the technique for performing diagnostic nasal biopsies is sufficiently complex (and bloody) that it is generally recommended to refer patients in need of this procedure to a specialty or referral hospital that has rhinoscopic and/or computed tomography capabilities. blind biopsies of dogs and cats with chronic nasal disease, especially if associated with bleeding, can be associated with significant risk, including penetration of the cranium. no one likes nasal biopsy results that indicate "normal brain. " renal biopsies can be valuable in confirming or eliminating a diagnosis of renal disease that is based on history, physical examination, and radiographic and laboratory data (box 4-5). in addition, biopsy may be a way of arriving at a prognosis in generalized renal disease and a better means of evaluating the type of treatment to be instituted. ultrasonographic guidance can prove valuable during renal biopsy for placing the needle into the tissue desired and avoiding complications. the liver biopsy, although a critical diagnostic tool in patients with laboratory evidence of liver disease, can be a fatal event, even in the hands of the experienced clinician. abdominal ultrasound imaging of the liver before and during biopsy is strongly recommended. before renal biopsy, the animal should have a baseline coagulation profile that includes, at the very least, an activated coagulation time and platelet count. a buccal mucosal bleeding time may be indicated if there is any history of spontaneous bleeding in a patient with a normal platelet count. obtain biopsies from the renal cortex. administer fluids to patients before and after biopsy. many patients with generalized renal disease are critically ill and debilitated, and general anesthesia is contraindicated. in these cases, a neuroleptanalgesic agent may be used for sedation. if the animal is a good anesthetic risk and renal function will permit it, use inhalation anesthesia. when bilateral renal disease is documented, select the left kidney for biopsy because it is more accessible than the right kidney. with the anesthetized patient in right lateral recumbency, surgically prepare the skin behind and below the junction of the costal arch at the level of the second and third lumbar vertebrae. make a 2-inch paralumbar incision parallel to, but just behind, the costal arch. dissect muscle and fascia until the peritoneum is visible. carefully open the peritoneal cavity. digitally feel for and examine the caudal pole of the left kidney. guide the needle toward the posterior pole of the kidney with the index finger. immobilize the kidney against the body wall and insert the tru-cut biopsy needle, with the biopsy notch exposed into the parenchyma of the kidney. capture the biopsy specimen by sliding the outer sleeve of the needle over the (now embedded in the kidney) biopsy notch. remove the needle and gently lift the biopsy sample from the needle and place it into formalin. evaluate the site for hemorrhage. once bleeding is controlled, a second biopsy specimen may be collected. once bleeding from the biopsy site has stopped, the incision can be closed. in dogs, renal biopsy can be performed under ultrasound guidance using probes with channels for biopsy needle insertion. evaluation of bone marrow is indicated in patients with evidence of persistently diminished cell counts of any or all cell lines (wbcs, rbcs, platelets) or evidence of morphologically abnormal cells in peripheral blood. bone marrow aspiration and bone biopsy are extremely helpful but underused diagnostic procedures. the availability of inexpensive, high-quality biopsy needles makes these procedures safe and easy to perform (once experience is gained). conventional practice today is to obtain a bone marrow aspirate (cytopathologic examination) and a bone biopsy specimen from the same patient during the same procedure when changes in the peripheral blood justify this level of diagnostic testing. bone marrow aspiration technique is described earlier in this section. two types of bone biopsy needles are available. the most commonly described procedure involves use of the jamshidi biopsy needle, an 11-to 13-gauge needle that ranges in length from 5 to 10 cm (see figure 4 -11). the needle contains a stylet that extends beyond the needle tip by 3 to 4 mm. because of the size of the jamshidi needle, its use is limited to medium and large dogs. for bone biopsies in cats and small dogs, the illinois bone marrow aspiration needle is preferred (see figure 4 -10), which is a 15-to 18-gauge needle available in lengths ranging from 2.5 to 5.0 cm. the patient usually is sedated or anesthetized for the procedure. although some patients will tolerate this procedure when performed under local anesthesia only, the additional manipulation required to obtain a high-quality sample justifies sedation. in some cases the patient is sufficiently obtunded that sedation is neither indicated nor required. the technique for bone biopsy is the same regardless of the needle used. once the site has been selected (usually the same sites selected for bone marrow aspiration: head of the humerus, wing of the ileum, ischial tuberosity, proximal femur), clip the hair and surgically prepare the skin. make a small stab incision in the skin over the site selected. pass the needle, with stylet in place, through the incision and subcutaneous tissues until the needle tip makes firm contact with bone. advance the needle using steady, increasing pressure and stable rotation. rotation, in this case, means rotating the needle back and forth to the left 180 degrees and then to the right 180 degrees. once the needle is situated in the bone (about 0.5 cm penetration only), stop. carefully remove the stylet. continue the penetration by gradually applying additional pressure and simultaneously rotating the needle. the usual depth of penetration varies from 1 inch to as much as 3 inches. on reaching the desired depth, remove the needle by continuing to rotate as described but gradually withdrawing the needle from the bone. an obturator is provided to push the sample out of the bone. place the core of bone directly into buffered formalin and submit it for histopathologic examination (decalcification will be required, which takes a little longer). some authors recommend carefully rolling the bone core across a glass slide (for cytopathologic examination) before placing the bone in formalin. most pathologists do not recommend this because additional handling of the biopsy sample can sufficiently disrupt the architecture of the tissue and compromise the quality of the biopsy (besides upsetting the pathologist). note also that the needle can, with a little gentle manipulation, be reinserted into the hole from which the biopsy sample was obtained. because the illinois needle and the jamshidi needle accommodate a syringe, it is possible to obtain (if done quickly, to prevent clotting) a bone marrow aspirate from the same site. place that sample directly onto glass slides or (recommended) into 4% edta and mix it before making slides. there are no specific requirements for postbiopsy care of the patient. clean the blood from the skin using hydrogen peroxide; sutures generally are not required. the femoral and dorsal pedal arteries can be punctured to obtain an arterial blood sample for blood gas and electrolyte analyses (see section 1) for information on indications and interpretation of results). to obtain a sample of arterial blood gas, place the patient in lateral recumbency and restrain the patient in a manner similar to that for a medial saphenous venipuncture. a 25-gauge needle affixed to a tuberculin syringe is preferred for arterial puncture. prepare the tuberculin syringe by coating it with heparin and forcing all the heparin out except for that left in the hub of the needle. pull back on the plunger of the syringe slightly to facilitate visualizing the point at which the artery is entered. arterial blood initially will enter the syringe without the plunger being drawn back. for collection of blood from the femoral artery, and once the patient is sufficiently restrained, the individual collecting the arterial blood sample should palpate the medial aspect of the limb over the proximal medial femur until the femoral pulse is palpated. direct the needle at a 30-to 45-degree angle, inserting the needle slowly, watching for a flash of blood in the hub of the needle (figure 4-22) . gradually withdraw the plunger to facilitate blood entering the syringe. collect 0.4 to 0.5 ml and immediately submit the blood for analysis, or place it on ice until the analysis can be performed. to obtain blood from a dorsal pedal artery, place the patient in lateral recumbency and extend the rear limb as for a medial saphenous blood sample collection. the person obtaining the blood sample should pull the paw of the down leg in the nondominant hand toward his or her body, rotating the limb slightly in a medial direction to palpate the arterial pulse. palpate the pulse in the dorsal pedal artery on the dorsomedial aspect of the tarsus. gently insert the needle at a 30-degree angle into the artery, watching carefully for a flash of blood into the syringe. when the necessary amount of blood has filled the syringe, remove the needle and place pressure over the site of arterial puncture for a minimum of 2 minutes. evacuate excess air from the syringe and needle, and cap the needle with a red rubber stopper to prevent air from entering the needle and syringe. place the sample on ice until analysis, if arterial blood gas analyses cannot be performed immediately. in the event that percutaneous access to a peripheral artery is not possible, the femoral artery can be isolated and prepared for surgical cutdown. after appropriate aseptic skin preparation, make a 4-to 5-cm incision in the skin over the femoral artery. find the caudal edge of the sartorius muscle by blunt dissection and then reflect it anteriorly to expose the underlying femoral artery, vein, and nerve. taking care to avoid tearing any vessel branches, gently isolate up to 2 cm of the femoral artery from the surrounding fascia. visually direct the needle into the artery at this point. alternatively, catheterize the artery in the event repeated arterial samples are required. elevate the femoral artery by preplacing two stay sutures beneath the artery and then elevating the vessel to the level of the skin. insert a long catheter-overthe-needle system into the lumen of the artery without penetrating the deep wall. gently insert the catheter into the vessel, remove the needle, and cap and flush the catheter. close the incision and affix the catheter to the skin via a tape tag sutured to the skin. the collection of csf is an important diagnostic procedure indicated for patients suspected of having significant intracranial or certain spinal diseases. however, it is our opinion that the technique to safely perform this procedure requires hands-on training and, preferably, prior experience before attempting the procedure in a clinical patient. attempting to perform csf collection from a written description in a text is not recommended. although this procedure is generally safe when performed correctly, significant injury and even death are possible, despite the experience of the individual performing the procedure. the electrocardiogram provides a fast, efficient way to obtain considerable data about a patient's cardiovascular status. electrocardiography is a clinical test and must be correlated with clinical findings (box 4-6). keep in mind that an electrocardiogram measures only electrical activity of the heart as seen on the body surface at any one instant. electrical disorders of the myocardium can be transient or intermittent and, as such, can be missed on a single electrocardiogram. if the answer to any of these questions is no, proceed to identify the abnormality. next, determine the rate, rhythm, and wave character-that is, evaluate measurements of the p wave, pr interval, and qrs complex. evaluate the st segment, t wave, and qt interval. use all leads to determine the axis and any miscellaneous criteria. depending on the type of electrocardiographic equipment used, there are several methods for determining heart rate from the electrocardiographic tracing. many electrocardiographs compute the heart rate and print that on the tracing. however, in patients with a significant dysrhythmia, these calculations can be flawed and should be verified manually when a question exists. small linear lines or demarcations at the top of the electrocardiogram paper can be used to determine the heart rate. at a paper speed of 50 mm/second, the time between adjacent marks is 1.5 seconds. counting the number of qrs complexes (or r waves) between just two of these divisions and multiplying by 20 equals the heart rate in beats per minute (figure 4-23) . for those inclined to higher mathematics, the heart rate also may be determined by counting the number of small squares between r waves (at a paper speed of 50 mm/sec) and then dividing into 3000 (box 4-7) . the normal heart rhythm is sinus in origin. for every qrs complex there is a p wave (figure 4 -24). the p waves are related to qrs complexes (p-p interval is constant). sinus arrhythmia, sinus arrest, and wandering pacemaker are normal rhythm variations. in sinus arrhythmia, the p-p interval is irregular. the pauses are never longer than twice the usual p-p interval (figure 4-25) . a wandering pacemaker means that the p waves vary in height and may even be negative temporarily (figure 4-26) . sinus arrest is defined as a prolongation of the p-r interval longer than twice the usual p-p interval. the normal p wave is 0.04 second × 0.4 mv (two boxes wide × four boxes tall) for the dog and 0.04 second × 0.2 mv for the cat. in p mitrale (left atrial enlargement), the p wave is wider than 0.04 second. in p pulmonale (right atrial enlargement), the p wave is taller than 0.4 mv for the dog and 0.2 mv for the cat. the pr interval is measured from the beginning of the p wave to the beginning of the qrs complex. the normal interval is 0.06 to 0.13 second (3 to 6.5 boxes wide) for the dog and 0.06 to 0.08 second for the cat. in first-degree atrioventricular heart block, the pr interval is prolonged. the pr interval is sometimes useful in monitoring the effects of digitalis therapy. the qrs complex duration is measured from the beginning of the q wave to the end of the s wave. normal duration is up to 0.04 second in cats, 0.05 second in small dogs, and 0.06 second in large dogs. a qrs complex that is too wide indicates left ventricular enlargement (figure 4-27 ). an r wave that is too tall indicates left ventricular enlargement. the amplitude is measured from the baseline to the top of the r wave (figure 4-28) . the normal r wave can be up to 0.8 mv tall in cats, 2.5 mv in small dogs, and 3.0 mv in large dogs. figure 4 -26: a, the wandering pacemaker in this recording is suggested by the slightly negative p waves in some of the complexes. negative p waves of this nature result from vagal depression of the sinoatrial node and the development of a junctional atrioventricular nodal rhythm. b, marked sinus arrhythmia and a wandering pacemaker result in a decreased heart rate (increased r-r interval) and negative p waves in the fifth complex. as the pacemaker returns to the sinoatrial node, the rate increases, and positive p waves of varying amplitude result in the sixth and seventh complexes. the st segment is between the end of the s wave and the beginning of the t wave. the qt interval is measured from the beginning of the q wave to the end of the t wave. the normal interval is 0.14 to 0.22 second (7 to 11 boxes wide) in dogs and up to 0.16 second in cats. a lengthened qt interval may be seen with hypokalemia or hypocalcemia. the qt interval varies with heart rate and tends to be prolonged when bradycardia occurs. a decreased qt interval may be seen with hypercalcemia. the mean electrical cardiac axis measures the direction (vector) of the cardiac ventricular impulse during depolarization. therefore the qrs complex is examined in leads i, ii, iii, av r , av l , and av f . these six leads determine the axis. they are arranged in a manner known as bailey's hexaxial lead system (figure 4-29) . the procedure is as follows: 1. find an isoelectric lead-that is, a lead for which the total number of positive (upward) and negative (downward) deflections of the qrs complex is equal to zero (figure 4-30) . when there is no perfectly isoelectric lead, use the one that comes closest. 2. find the lead that is perpendicular to the isoelectric lead: lead i is perpendicular to av f ; lead ii is perpendicular to av l ; and lead iii is perpendicular to av r . 3. determine whether the perpendicular lead is positive or negative on the patient's electrocardiogram. if the perpendicular lead is negative, the axis is at the negative end of that lead (each lead has a plus and a minus pole marked). if the perpendicular lead is positive, the mean electrical axis is at the positive end of the perpendicular lead. for example, if avl is isoelectric (normally it is), lead ii is its perpendicular. if lead ii is positive on the electrocardiogram, the axis is +60 degrees. if lead ii is negative on the electrocardiogram, the axis is −120 degrees. the mean electrical axis in the normal dog is +40 to +100 degrees; for the cat it is more variable, at 0 to ±180 degrees. right axis deviation (axis more than +100) indicates right ventricular enlargement in the dog (figure 4-31) . left axis deviation (axis 0 to +40 degrees) indicates left ventricular enlargement in the dog. when there is biventricular enlargement, the axis usually remains normal. axis determinations are of less value in the cat because the normal range is so wide (boxes 4-8 to 4-10). inappropriate use of endoscopic equipment not only can damage expensive equipment but also can cause serious injury to the patient. endoscopy of the upper respiratory tract is among the most important advanced diagnostic and therapeutic tools used in the evaluation of patients that have stertor (snorting), reverse sneeze, stridor (wheezing), and chronic cough. laryngoscopy is of value in the diagnosis of upper airway obstructions such as eversion of the lateral ventricles, collapsed arytenoid cartilages, hyperplasia of the vocal cords, nodules on the vocal cords, elongated soft palate, collapsed proximal trachea, and traumatic injuries to the neck. note also, however, that a careful visual examination of the larynx in the anesthetized patient (only) can be highly valuable even without the use of endoscopic equipment-for example, for assessment of laryngeal movement in patients with laryngeal paralysis. suspected lesions inside the larynx may be difficult to visualize with or without endoscopic equipment. examination of the trachea and main stem bronchi requires endoscopic evaluation to assess the integrity of the airway for conditions such as collapsed trachea, mediastinal tumors, hilar lymph node enlargement, parasitic nodules (filaroides osleri), and foreign body aspiration. in addition, tracheobronchoscopy is a valuable technique that permits culturing and cytologic examination of material from bronchi involved in chronic respiratory disease. upper airway obstruction that is not responsive to conservative therapy is an indication for more extensive diagnostic procedures, such as bronchoscopy. note: the discussion that follows centers around indications and capabilities of endoscopy in clinical practice. the discussion is not intended to be used as a "how-to" instruction guide on performing endoscopic procedures in dogs and cats. today, numerous types of endoscopes and accessory materials are available for use in clinical practice. specific hands-on training and complete familiarity with the equipment package available is essential before attempting to perform any of the procedures outlined. endoscopes of varying sizes are appropriate for use in examining the larynx and trachea. however, in cats and small dogs, examination of the trachea using equipment as small as a (human) bronchoscope may limit the examination because the endoscope nearly occludes the tracheal diameter. additional training and/or experience is recommended for performing tracheoscopy in small patients. one of the most important endoscopic techniques performed in dogs and cats involves examination of the nasopharynx, the upper respiratory compartment above the soft palate. sometimes called pharyngoscopy, examination entails retroflexion of a small-diameter endoscope (e.g., bronchoscope) 170 to 180 degrees to allow visualization of the space between the posterior nares (choanae) and the larynx (figure 4-32) . this is a common location for foreign body entrapment and occasional tumor development in cats and dogs (figure 4-33) . pharyngoscopy is the only effective means of examining this portion of the upper respiratory tract in patients that have a history of stertor (snorting) and so-called "reverse sneeze." lower respiratory tract: bronchoscopy endoscopic examination of the bronchi and lower airways is a highly diagnostic, occasionally therapeutic procedure indicated in patients presented with persistent cough. as in all endoscopic procedures, the patient is anesthetized for the examination. however, examination of the lower respiratory tract requires considerable attention to patient oxygenation and respiratory status during the examination. the requirement for oxygen to be administered throughout the procedure may be a significant limiting factor unless special accessories are used. in the ideal situation, the patient is a medium-to large-sized dog and the endoscope can be passed through the endoscope using a t adaptor while oxygen and anesthetic are administered simultaneously. however, in cats and small dogs it is usually not possible to pass an endoscope through the endotracheal tube. the procedure must be done by passing the endoscope directly into the trachea to the level of the right and left main bronchi and probably not much farther. supplemental intravenous anesthetic is likely to be required because of the time required to complete the examination. training and/or experience is essential before performing bronchoscopy, particularly in cats and small dogs. the greatest advantage in performing bronchoscopy is to visualize the integrity of the trachea and, to a limited extent, the lower airways. airway collapse, not visible on conventional radiography, can be strikingly apparent. foreign body entrapment, tumors, respiratory parasites, and airway trauma also can be identified with bronchoscopy. in addition, the bronchoscopic examination allows for collection of cytologic samples from discrete areas (airways) within the lower respiratory tract. the ability to perform bal in patients with reactive airway disease, subclinical or clinical infections, and certain types of tumors can be highly diagnostic. flexible fiberoptic endoscopy is a noninvasive, atraumatic means of visualizing the mucosal surfaces of the esophagus, stomach, and colon. flexible endoscopes are available from several companies at a wide range of prices. to minimize the risk of injury to the animal and to reduce the possibility of damage to the endoscope, place animals undergoing endoscopic examination under general anesthesia after routine preanesthetic preparation. a fast of 12 to 24 hours is recommended for most patients undergoing upper gastrointestinal endoscopy. however, for patients with indications of delayed gastric emptying, a longer fast (24 to 48 hours) may be needed to empty the stomach completely. in preparation for colonoscopy, a 24-to 48-hour fast is recommended. give a high warm-water enema the evening before and again 2 to 4 hours before the procedure. give such enemas until the return is clear. the clinical signs indicating esophageal disease and a potential benefit of esophagoscopy include repeated regurgitation, excessive drooling, ballooning of the esophagus, anorexia or dysphagia, and recurrent pneumonia. esophagoscopy allows visualization of the mucosal lining of the esophagus and makes it possible to detect inflammation, ulcerations, dilatations, diverticula, strictures, foreign bodies, tumors, and parasite infestations. endoscopic examination of the mucosal aspect of the stomach is indicated when the clinical signs or physical findings suggest the presence of gastric disease or when there is a need for confirmation or clarification of radiographic findings. in most cases, persistent vomiting is the chief complaint. other clinical signs suggestive of serious gastric disease include hematemesis, melena, weight loss, anemia, and abdominal pain. gastroscopy allows visualization of the mucosal lining of the stomach and enables detection of inflammation, ulceration, foreign bodies, and tumors. in most dogs and cats the endoscope can be passed into the proximal duodenum. depending on the patient size and length of the scope, it may be possible to evaluate as much as 12 inches or more of the proximal duodenum. colonoscopy is endoscopic examination of colon, rectum, and anus. the technique is helpful in the definitive diagnosis of lower bowel lesions, such as granulomatous colitis, foreign bodies, tumors, lacerations, and other mucosal abnormalities. the primary indication for colonoscopy is the presence of signs of large bowel disease, which typically include tenesmus and the passage of small, frequent stools containing fresh blood or excess mucus. endoscopic examination of the colon allows direct visualization of the effects of mucosal inflammation, ulceration, mucosal polyps, malignant neoplasia, and strictures. histologic examination of mucosal biopsy material will confirm the diagnosis of colonic disease. the large bowel must be empty for the colonic mucosa to be visualized. the bowel can be emptied by withholding food for 24 hours and performing a colonic irrigation the evening before and again 2 hours before the examination. the material used for the enema must be nonirritating and nonoily. mildly hypertonic saline solutions such as fleet enemas work well if given 2 hours before examination so that gas and fluid can be passed completely. however, do not use fleet enemas in cats or small dogs. if the general physical condition of the animal is poor and withholding food is not possible, feeding a low-residue diet for 12 to 18 hours before colonoscopy can be helpful. this diet could consist of cooked eggs, small amounts of cooked beef or chicken, and small amounts of carbohydrates, such as a slice of toast or 1 ⁄4 to 1 ⁄2 cup of moist kibble. maintain good hydration. if all food is contraindicated, oral electrolyte solutions such as gatorade (pepsico, purchase, new york) can be used to maintain hydration without moving solids through the intestinal tract. give the animal a short-acting anesthetic and place the animal on a tilted table in lateral recumbency with the hindquarters elevated. perform a digital examination of the rectum and pelvic cavity to ensure that there are no strictures, polyps, or other obstructions. lubricate the proctoscope thoroughly with water-soluble jelly and pass it gently through the anal sphincter. press the proctoscope forward slowly and carefully with a spiral motion. if any resistance is encountered, stop the motion, remove the obturator, and inspect the 4 bowel to determine the cause of the resistance. if possible, replace the obturator and continue forward motion until the instrument is passed its full length. withdraw the obturator, and observe the mucosa. the major portion of the examination is conducted as the instrument is withdrawn. to view the colonic and rectal walls completely, one must move the anterior end of the proctoscope around the circumference of a small circle while withdrawing the proctoscope. occasional insufflation with the inflating bulb is helpful in smoothing out folds of tissue. repeated instrumentation may produce petechiae and minor hemorrhages that are not pathologic. for examination of the terminal rectum and anus, the hirschman anoscope provides adequate, convenient visualization. newer techniques for visualizing the upper and lower gastrointestinal tract are being used in dogs. the flexible fiberoptic endoscope enables one to visualize and photograph the esophagus, colon, and stomach. one is able not only to visualize lesions of the gastrointestinal tract directly but also to assess motility, take biopsies of lesions, and remove foreign bodies. the ability to visualize directly the vestibule, the vagina to the level of the cervix, and the urethral orifice in female dogs is of particular value in evaluating patients with known or suspected congenital urinary tract disorders, such as incontinence or ectopic ureters and vaginal strictures (congenital or traumatic). numerous vaginal malformations and chronic infections cause visual changes that are identified easily during endoscopic examination. frequently the procedure can be conducted in the standing awake patient. sedation or general anesthesia is indicated when extensive manipulation, catheterization of the bladder, or a vaginal biopsy are indicated. position the sedated or anesthetized patient in dorsal or ventral recumbency to facilitate orientation during the procedure. if catheterization of the urinary bladder is required during the procedure, dorsal recumbency seems to facilitate visualization of the urethral papilla and insertion of the catheter. vaginoscopy entails use of a relatively small, flexible endoscope 4 to 6 mm in diameter or a 2-to 3-mm rigid scope. the flexible scope offers the advantage of a larger biopsy channel and the ability to view the lateral vaginal wall easily. vaginoscopy is considered an invasive procedure and should be conducted under sterile conditions. before insertion of the sterilized endoscope, the vulva should be free of obvious debris, should be clipped if necessary, and should be cleaned gently with a surgical soap and rinsed. insert the scope such that initial position of the tip of the scope is directed toward the anus. as insertion proceeds, the tip of the endoscope reaches the horizontal portion of the vestibule and vagina. when feasible, pass the scope to the level of the cervix. slight insufflation of the vagina may be useful in dilating the vagina, greatly facilitating the examination. conducting the examination from the level of the cervix caudally is recommended. this maximizes the ability to visualize critical anatomic features. the relatively recent introduction of very small (2-mm diameter) flexible and rigid endoscopes into veterinary medicine allows visual examination of the urethra, trigone, urinary bladder, and right and left ureterovesicular junctions of female dogs and even cats. such examinations are most useful when obstructive lesions (tumor or calculi) of the urethra or trigone are suspected. visual examination of the interior surface of the bladder and the capability of collecting biopsy samples make this a particularly useful diagnostic tool in the hands of the experienced clinician. numerous techniques are described for administering calories and nutrients to patients that are unable or unwilling to take in, chew, or swallow food. one method, intravenous hyperalimentation, is reserved for patients that are not able to tolerate any food being introduced via the gastrointestinal tract and represents a radical, and ideally transient, departure from normal. however, enteral feeding, which is always preferable to intravenous hyperalimentation, allows the clinician several options for administering food directly into the gastrointestinal tract. consideration of several variables is critical when one is initiating enteral feeding programs, such as the patient's diagnosis and attitude, the status of the gastrointestinal tract, and the ability of the patient to digest and absorb food once introduced. in addition, consideration of the type and constituency of the diet provided is important. although the options available for enteral nutrition are much greater that those for intravenous hyperalimentation, the clinician must consider dietary requirements carefully when planning enteral nutritional support. when evaluating enteral feeding for the individual patient, the clinician has four basic options: nasoesophageal tube, pharyngostomy tube (least recommended), esophagostomy tube, and percutaneous gastroscopy tube (which can be introduced using an endoscope or with the so-called "blind" technique). all techniques involve use of a polyurethane or silicone feeding tube. the nasoesophageal tube placement technique does not require general anesthesia, and the tube may be inserted using a topical anesthetic only. each of the other techniques described requires that the patient be anesthetized to ensure proper and safe placement. for temporary, short-term feeding, nasoesophageal intubation is a simple technique that works well in cats, puppies, and adult dogs. patients that are comatose; have severe, persistent vomiting; have esophageal disease or dysfunction; or are unable to swallow are not candidates for this procedure. the objective of the procedure is to place a small-diameter tube (8f to 10f for dogs weighing more than 15 kg and 5f to 8f for small dogs and cats) through the nasal cavity into the distal esophagus. the tube does not have to enter the stomach. when measuring the tube length, measure from the tip of the nose to the eighth or ninth rib (figure 4-34) . administer 3 to 5 drops of a topical ophthalmic solution (0.5% proparacaine) directly into one nostril. hold the head gently upward for a few seconds to allow the solution to reach the back of the nasal cavity. in most patients, it is desirable to wait 1 to 2 minutes and then to repeat the instillation in the same nostril. for larger dogs, 2% lidocaine solution (0.5 to 2.0 ml) gradually instilled into the nostril is an alternative technique to achieve topical anesthesia. lubricate the tube with a thin coat of a water-soluble lubricant, such a 2% lidocaine lubricating gel. pass the tube into the nasal cavity while directing the tube tip medially and ventrally into the ventral meatus. the anatomic shape of a dog's nostril usually requires directing the tip medially but almost perpendicular to the plane of the nasal cavity to facilitate insertion. initial resistance (pressure, not pain) usually is perceived, and the patient's head as expected quickly retracts, leaving the operator holding the tube tip some inches away from the patient's nose. be persistent. repeat the procedure, as necessary, by quickly inserting the first inch or more of the tube into the nostril. with the other hand, push the nasal philtrum up, and with a finger, push the lateral portion of the nostril medially. this will help facilitate movement of the tube into the ventromedial nasal meatus. once started, the remainder of the technique is relatively straightforward. as the tube reaches the caudal aspect of the nasopharynx, it should pass directly into the esophagus with little or no resistance. affix the tube remaining outside the patient to the head or face using a "butterfly" tape, gauze, suture (figure 4-35) , or skin glue (skin glue [superglue] generally is not recommended because this can result in loss of hair and skin pigment when the glue becomes dislodged). caution: the tip of the tube can be introduced inadvertently through the glottis and into the trachea. topical anesthetic instilled into the nose can anesthetize the arytenoid cartilages, thereby blocking a cough or gag reflex. i prefer to check the tube placement with a dry, empty syringe. attach the test syringe to the end of the feeding tube. rather than inject air or water in an attempt to auscultate borborygmus over the abdomen, simply attempt to aspirate air from the feeding tube (figure 4-36) . if there is no resistance during aspiration and air fills the syringe, it is likely that the tube has been placed in the trachea. completely remove the tube and repeat the procedure. however, if repeated attempts to aspirate are met with immediate resistance and no air enters the syringe, the tube tip is positioned properly within the esophagus. if there is any question regarding placement, a lateral survey radiograph is indicated. less invasive and not requiring endoscopy equipment, esophagostomy tube placement in dogs and cats is an alternative technique to use in patients that have long-term feeding needs. use a 14f to 20f rubber, polyurethane, or silicone feeding tube placed at the level of the middle of the cervical esophagus to the level of the eighth rib. the technique does require general anesthesia or, in the hands of an experienced individual, shortterm intravenous anesthesia. the technique has been described in detail in textbooks (see marks sl; additional reading). to place an esophagostomy tube, first assemble the necessary supplies: large curved rochester-carmalt forceps, clipper and clean blades, antimicrobial scrub, gauze squares, red rubber tube, permanent marker, scalpel blade and handle, needle holder, suture scissors, and nonabsorbable suture (0 nonabsorbable, cutting needle. after placing the patient under general anesthesia and intubating the patient, place the patient in right lateral recumbency and clip the lateral left side of the neck from the ramus of the mandible caudally to the thoracic inlet, and dorsally and ventrally to midline. note that the left side of the neck is preferred because of the normal anatomic location of the esophagus. however, if there is injury, infection, or mass that prevents placement of the esophagostomy tube in the left lateral cervical region, the right lateral side of the neck can alternately be used. next, aseptically scrub the clipped area, and push the rochester-carmalt forceps through the mouth into the esophagus. direct the curved tips of the instrument laterally, so the tips can be visualized under the skin. use care to note where the external jugular vein lies, to avoid laceration of the jugular vein. measure the tube from the proposed site of tube entrance to the mid thorax, then label the tube with a permanent marker. next, open the curved tips of the instrument, and make a stab incision through the skin, through the open tips of the instrument, into the esophagus. push the tips of the instrument through the skin incision. grasp the distal end of the tube with the instrument, and clamp the instrument. pull the tube through the skin incision and rostrally out of the front of the mouth. if the tube does not come easily, usually the hinges of the forceps are caught on tissue within the oropharynx or pieces of the endotracheal tube. once the distal end of the tube is through the front of the mouth, push the distal end of the tube caudally into the esophagus with a finger or the instrument. as the distal end of the tube is pushed into the esophagus, pull the proximal end of the tube, to add tension to the tube. the proximal end of the tube will flip toward the patient's nose when the tube is situated in the esophagus. the tube can be taped in place while radiographs are taken to confirm placement. after radiographs confirm placement in the esophagus, suture the tube in place with two sutures, one purse-string and finger-trap around the tube entrance site, and another deep suture near the atlas, with a second finger-trap. finally, place antimicrobial ointment over the tube entrance site, and a loose bandage around the neck. unlike gastrostomy tubes, esophagostomy tubes can be used immediately, and removed immediately, if the patient chooses to start eating voluntarily after tube placement. it is important that one first observe the technique being performed by someone with experience before attempting to place an esophagostomy tube for the first time. although postplacement complications generally are limited to local irritation or minor infection at the site of the stoma in the midcervical region, tube placement into the mediastinum or subcutaneously can occur. percutaneous gastrostomy tube placement percutaneous gastrostomy tubes are used routinely to administer nutrients and medications orally over days or weeks to cats and dogs that cannot have nutrients administered by mouth or that will not eat (e.g., because of feline hepatic lipidosis, oropharyngeal neoplasms, maxillary or mandibular fractures, oral reconstructive surgery, esophageal masses or foreign bodies, or severe pharyngitis). the percutaneous gastrostomy tube is placed so that it extends through the skin and left cranial abdominal wall of the abdomen into the body of the stomach. catheter preparation for percutaneous gastrostomy tube is as follows: 1. use the french-pezzar mushroom-tipped catheter. stomach tube preparation for percutaneous gastrostomy tube is as follows: 1. use a smooth-ended vinyl stomach tube. 2. measure the length of the tube needed to reach the stomach by laying the tube along the animal's side with the rounded end 1 to 2 cm caudal to the last rib. 3. mark the tube with an indelible marker or adhesive tape at the tip of the muzzle and cut off the excess tube. 4. put the tube in the freezer for 30 minutes to stiffen the tube before beginning the procedure. placement of a percutaneous gastrostomy tube is as follows: 1. clip and surgically prepare the skin over the left abdominal wall. 2. place the mouth speculum between the right canine teeth. 3. place the stomach tube in the esophagus to the level of the cardia. 4. rotate the tube counterclockwise while carefully advancing it through the cardia. 5. turn the tube back clockwise and advance the tube until it can be visualized through the abdominal wall 1 to 2 cm caudal to the last rib (figure 4-37 ). 6. rotate the tube so that the tip lies against the stomach and abdominal wall one third of the distance between the epaxial muscles and the ventral midline. 7. make a 2-to 3-mm skin incision directly over the lumen of the stomach tube. 8. use a sovereign catheter (over the needle) and puncture the abdominal and stomach walls, placing the catheter inside the lumen of the stomach tube. remove the needle (figure 4-38) . 9. thread a long, rigid suture through the catheter and advance it through the stomach tube until the end is observed at the mouth end of the tube (figure 4-39) . 10. carefully remove the plastic catheter from the stomach tube opening and place a hemostat clamp at the end of the suture material. 11. remove the stomach tube over the oral end of the stiff introduction suture line. 12. attach the open, beveled end of the french-pezzar catheter stomach tube to a plastic sovereign catheter using a mattress suture (figure 4-40) . 13. force the tip of the rubber stomach tube into the large end of the sovereign catheter. 14. advance the catheter tube through the mouth and esophagus into the stomach by placing traction on the abdominal end of the introduction line. 15. the catheter will emerge through the skin incision, followed by the rubber tube. grasp the tube with forceps and pull it through the incision opening (figure 4-41, a) . 16. remove the catheter by cutting it off 2 cm below the beveled tip. pull the rubber tube through the abdominal wall until slight resistance is felt (figure 4-41, b) . 17. slide the outer flange over the end of the tube down to the skin level (figure 4-42) . 18. apply antimicrobial ointment and a sterile gauze sponge over the skin incision. 19. bandage the gastrostomy tube in place (figure 4-43) . rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. tear production comes predominantly from the tarsal and conjunctival glands and from the accessory tarsal glands. the reflex tear secretors are the main lacrimal gland and the accessory lacrimal glands. the production of normal lacrimal secretions can be tested by using the schirmer tear test, a standardized filter paper (figure 4 -44) that effectively measures the rate of tear production in millimeters per minute. schirmer tear strips now are impregnated with a blue dye to facilitate visualization of the distance (in millimeters) that the tear migrates during the 1-minute test. none required. technique each eye can be tested independently, or both eyes can be tested simultaneously in the cooperative patient. carefully fold the notched end of the test strip before removing it from the plastic package. insert the folded end into the lower conjunctival cul-de-sac (figure 4 -45) and begin the timing. maintain the schirmer test strip in position by gently holding the eyelids closed but not touching the paper. at the end of 1 minute, note the degree (distance) of wetting that occurred and record it in the medical record. the normal dog and cat should produce wetting over 10 to 25 mm in 1 minute for each eye. amounts less than that are consistent with keratoconjunctivitis sicca. amounts greater than 25 mm may be normal or may be consistent with excessive tear production, or epiphora. the cornea is composed of various layers of specialized avascular epithelium and stroma. the outer layer, the corneal epithelium, is a highly sensitive, thin layer overlying the corneal stroma, the thickest layer. descemet's membrane is a distinct, thin layer of tissue beneath the stroma. the innermost layer of the cornea is the endothelium. damage to the corneal epithelium occurs frequently in dogs and cats. clinical presentation typically is characterized by blepharospasm of the affected eye with or without a visible ocular discharge or conjunctivitis. whenever superficial corneal injury is suspected, assessment of the integrity of the corneal epithelium is indicated. fluorescein dye-impregnated test strips can be used to determine whether the epithelial barrier overlying the corneal stroma has been disrupted and thus can establish the presence or absence of a corneal ulcer (figure 4-46) . none required. the test is simple to accomplish. moisten the dye-impregnated tip of the test strip with a drop of balanced saline solution (or commercial ocular irrigation solution). gently allow the tip of the test paper to touch the cornea, or sclera, of the affected eye. (in patients with particularly painful, sensitive eyes, use a topical anesthetic to moisten the test strip or apply the anesthetic directly to the cornea before testing.) immediately rinse the eye with a sterile irrigation solution to remove the excess dye (the test strip has a lot of dye; be prepared to catch the excess fluid with 2-× 2-inch gauze). promptly examine the eye with a direct, focal light source. evidence of green dye uptake in the stroma indicates that an ulcer is present. the absence of staining generally indicates that the corneal integrity is intact. one exception exists. the descemet membrane will not take up fluorescein dye. a patient with a deep corneal ulcer that penetrates through the corneal stroma and allows herniation of the descemet membrane (descemetocele) will not demonstrate a positive stain. careful visualization of the cornea, however, is likely to reveal the presence of a such a serious, deep ulcer. none required. fluorescein dye can also be used to assess patency of the nasolacrimal duct. to perform this examination, place a drop of fluorescein dye from a sterile fluorescein strip into the eye and add 1 or 2 drops of a sterile eye wash. after 2 to 5 minutes, examine the external nares with the aid of a cobalt blue filter or wood light for the presence or absence of fluorescence. a clean, 2-× 2-inch white gauze square touched against the nasal planum also will pick up the greencolored dye if the duct is patent. if dye is present, the lacrimal excretory system is patent and functioning. if epiphora exists but the primary dye test indicates that the lacrimal excretory system is patent, hypersecretion of tear fluid may be implicated as the cause of the epiphora. irrigation of the nasolacrimal system is indicated if the primary dye test result is negative. in the dog the nasolacrimal puncta are located 1 to 3 mm from the medial canthus on the mucocutaneous border of the upper and lower lids. in the dog, use a 20-to 22-gauge (in the cat, a 23-gauge) nasolacrimal cannula (figure 4-47) . topical anesthesia often is required. fill a 2-ml syringe with saline, and attach the lacrimal cannula and pass it into the lacrimal puncta of the upper lid. several points should be made about evaluating the nasolacrimal system. brachycephalicbreed dogs and cats occasionally may have a negative primary dye test result, although no blockage in the nasolacrimal system exists. in flushing the nasolacrimal system of some animals, fluid may not appear at the nose; however, the animal may gag and exhibit swallowing movements, indicating that the fluid has entered the mouth and the system is patent. none required. this procedure is preferably performed without administration of topical anesthesia. topical anesthetics not only are bacteriostatic but also may distort the cells and compromise the cytologic examination. in performing conjunctival scrapings, use a platinum spatula (kimura spatula), the tip of which has been sterilized. gently scrape the inferior conjunctival cul-de-sac (figure 4-48) . place the material on two glass slides. fix one slide in acetone-free 95% methanol for 5 to 10 minutes, then stain the slide with giemsa stain. heat-fix the other slide, and apply gram stain. to culture the conjunctiva, use sterile cotton-tipped applicators, fluid thioglycolate medium, and blood agar medium. evert the palpebral conjunctiva of the lower lid, and pass one side of a sterile cotton applicator, previously moistened with sterile broth or thioglycolate medium, over the palpebral conjunctival surface. streak the swab onto a sterile blood agar plate, then place the plate in a tube of thioglycolate broth. no topical anesthesia is used before culturing because preservatives present in anesthetics can inhibit the growth of bacteria. glaucoma is an increase in intraocular pressure incompatible with normal ocular and visual functions. one method used to measure intraocular pressure is tonometry, in which the tension of the outer coat of the eye is assessed by measuring the impressibility, or applanability, of the cornea. because the measurements based on tonometry involve calculations that have a wide base of variations, tonometry readings are always approximations. the schiøtz tonometer consists of a corneal footplate, plunger, holding bracket, recording scale, and 5.5-, 7.5-, 10.0-, and 15.0-g weights. the principle of the schiøtz tonometer is that the amount that the plunger protrudes from the footplate is related to the indentability of the cornea, which in turn is related to the intraocular pressure. however, use of applanation tonometry today has virtually replaced use of the schiøtz tonometer. in applanation tonometry, a very small area of the cornea is flattened by a known force, usually a calibrated burst of air. the advantage of this technique over the indentation (schiøtz) method is that the errors resulting from ocular rigidity and corneal curvature are greatly reduced. special equipment is required to perform applanation tonometry (figure 4-49) . the presence or absence of glaucoma, an increase in intraocular pressure, can be determined using applanation tonometry. on the other hand, gonioscopy permits one to visualize and examine the iridocorneal angle and potentially establish the cause of glaucoma. however, specific training and equipment are required not only to perform gonioscopy but also to interpret the result. this procedure is most appropriately performed by an ophthalmologist. barnett kc, crispin sm: feline ophthalmology, philadelphia, 1998, wb saunders. barnett kc, sansom j, heinrich c: canine ophthalmology, philadelphia, 2002, wb saunders. gastrointestinal studies when considering a contrast study of the gastrointestinal tract, it is not unreasonable to question the value of doing the procedure. at issue is the fact that abdominal ultrasound and/or gastrointestinal endoscopy has largely replaced contrast radiography of the gastrointestinal tract and for good reason. diagnostic modalities such as ultrasound (in the hands of an experienced individual) and endoscopy have a much greater diagnostic yield than the less sensitive contrast study. so why even try? endoscopes are not available in every practice, and limited access to ultrasound equipment, much less someone who is qualified to use it, puts routine use of advanced diagnostic modalities out of reach for many practices. however, it must be appreciated that with regard to diagnostic value, a radiographic contrast study of the gastrointestinal tract is a far less sensitive diagnostic modality than abdominal ultrasound or endoscopy. the procedure for the gastrointestinal radiographic contrast study is outlined next. contrast agents available for gastrointestinal studies include barium suspension preparations or micropaque (guerbet, villepinte, france), and water-soluble agents (gastrografin [bracco diagnostics, princeton, new jersey], which is 60% meglumine and 10% sodium diatrizoate). water-soluble agents are used if bowel perforation is suspected. undiluted water-soluble agents are hypertonic and should be diluted at a ratio of one part gastrografin to two parts water. no single procedure is appropriate for all gastrointestinal cases. the clinician must select procedures based on the clinical history and physical findings, apparent location of the lesion within the gastrointestinal tract, endoscopic findings, and results from other imaging studies, such as abdominal ultrasound. the contrast esophagram also is called barium swallow. the decision to perform a contrast esophagram is based on physical evidence of dysphagia (difficulty or pain while attempting to swallow) and/or persistent regurgitation (reflux of swallowed food without effort). the procedure necessitates that the animal fast for 12 hours before radiography. remove all leashes from around the animal's neck, and obtain survey radiographs of the thorax. in esophageal contrast studies, administer barium suspension contrast medium, 2 to 5 ml/kg body mass. administration of barium as a contrast material is contraindicated if a perforation of the esophagus is suspected. when the esophagus has been coated with radiopaque material, take lateral, ventrodorsal, and right ventrodorsal oblique thoracic radiographs to visualize the esophagus. properly prepared, the barium should be relatively thick and of a pastelike consistency. position the patient and cassette, and have the radiographic technique set. give a tablespoonful of barium orally. make the exposure when the animal takes its second swallow after the barium has been given. for esophageal studies and barium swallows, sedation with acepromazine and buprenorphine (iv, im, sq) will produce no adverse alteration in gastrointestinal motility. for cats, ketamine 10 mg iv and midazolam 0.2 mg/kg (combined) can be administered intramuscularly (im) with no significant effect in esophageal motility. caution: patients with significant swallowing disorders have a risk of aspiration if contrast material is regurgitated. sedation can increase that risk. in some cases of incomplete esophageal stricture, barium liquid will pass through the esophagus unobstructed, whereas food will not. veterinarians should mix kibbled food with the barium in this case and allow the patient to eat the mixture just before the radiograph is taken. ideally, contrast esophagrams are performed using fluoroscopy rather than conventional radiographs. in this manner it is possible not only to identify strictures and dilatations, if present, but also to obtain a dynamic study of the esophagus that provides valuable information pertaining to swallowing and esophageal motility and function and an opportunity to evaluate sphincter activity at the level of the cardia. contrast studies of the upper gastrointestinal tract are used to facilitate diagnosis of persistent vomiting, hematemesis, unexplained and chronic diarrhea, suspected enteric foreign bodies, and suspected neoplasms and obstructions and for confirmation of displaced intestinal organs, as may be seen in diaphragmatic hernias. that said, abdominal ultrasound has become sufficiently available to largely replaced the upper gastrointestinal series. with an experienced ultrasonographer, the diagnostic value of abdominal ultrasound far exceeds that derived from evaluating sequential radiographs of a patient after oral administration of a contrast medium such as barium. in the event that ultrasound capability is not available, a contrast study of the upper gastrointestinal tract still can be performed. however, the clinician must appreciate that a barium contrast study of the stomach, duodenum, jejunum, and ileum has a low sensitivity as a diagnostic test. that is, negative findings are not expected to correlate well with the absence of clinical disease. a negative study does not rule out disease. likewise, a contrast study of the upper gastrointestinal tract is not recognized for its ability to confirm a diagnosis of gastrointestinal tract disease, even when disease is present. perhaps the greatest value in performing the upper gastrointestinal series in a dog or cat today centers on the need to identify a displacement of the stomach and/or small intestine because of an extraluminal mass lesion or congenital defect in the patient. in addition, the use of a microfine barium suspension may facilitate identification of intestinal ulcers, irregularities (e.g., intraluminal neoplasia), and radiolucent foreign bodies. however, variable-diameter, solid-phase radiopaque markers called barium-impregnated polyethylene spheres (bips) can be used to assess gastric emptying time, gastrointestinal transit times, and, to some extent, obstructive disorders. if an upper gastrointestinal study is indicated, follow the technique described: 1. ensure that the hair of the animal is free from dirt, paint, and foreign material. bathe the animal if necessary. 2. withhold food for 18 to 24 hours. 3. if the colon is filled with feces, administer a cleansing enema the evening before performing the procedure. in dogs, give a second enema 3 to 5 hours before the start of the gastrointestinal series. 4. at the start of an upper gastrointestinal series, obtain survey radiographs of the abdomen. administer a barium sulfate (micropulverized) preparation by stomach tube, or induce the animal to swallow the fluids. flavored, prepared barium suspensions are available, but they taste bad (personal experience). dosage levels vary, but for barium suspensions, give approximately 10 ml/kg. as an alternative to barium, use an organic iodide liquid preparation. administer 0.5 ml/kg by stomach tube. obtain lateral and dorsoventral radiographs of the abdomen immediately after administration of the contrast material and at 30-minute, 1-hour, and 2-hour intervals. watersoluble contrast material passes through the gastrointestinal tract in 30 to 90 minutes. barium suspensions take 60 to 180 minutes to traverse the intestine. the colon usually is filled with barium 6 hours after oral administration and may contain barium for 2 to 3 days after administration. barium contrast radiography is contraindicated if perforation of the stomach or upper gastrointestinal tract is suspected. in these cases, use water-soluble contrast media such as the oral diatrizoates because leakage into the abdomen will produce no foreign body granuloma. in addition, do not administer barium sulfate when an obstruction of the lower bowel may be present. in these cases, barium may only contribute to the obstipation. the following radiographic views are recommended after administration of radiographic contrast material: 1. immediately after administration of contrast material, obtain ventrodorsal, right lateral, and left lateral views. the right lateral view shows the pylorus of the stomach filled with barium, and the left lateral view shows the cardia and fundic portion filled with barium. the objective is to evaluate the distended stomach and initial gastric emptying. 2. twenty to 30 minutes after administration of contrast material, obtain ventrodorsal and right lateral views to assess the stomach, pyloric emptying, and the proximal duodenum. 3. sixty minutes after administration of contrast material, repeat the ventrodorsal and right lateral recumbency views to assess the small intestine. 4. two hours after administration of contrast material, repeat the ventrodorsal and right lateral views to evaluate passage of contrast material into the colon and complete emptying of the stomach; contrast material should be in the terminal portion of the small intestine. the passage of contrast material through the normal gastrointestinal tract is variable; however, the following guidelines have been suggested: 1. contrast material is in the duodenum within 15 minutes in most patients. excitement can delay gastric emptying time to 20 to 25 minutes. 2. contrast material reaches the jejunum within 30 minutes and is within the jejunum and ileum at 60 minutes. 3. contrast material reaches the ileocecal junction in 90 to 120 minutes. 4. at 3 to 5 hours after administration, contrast material has cleared the upper gastrointestinal tract and is within the ileum and the large intestine. in evaluation of gastrointestinal contrast studies, consider the following criteria: (1) the size of the intestinal mass, (2) the contour of the mucosal surface, (3) the thickness of the bowel wall, (4) the flexibility and motility of the bowel wall, (5) the position of the small intestine, (6) the continuity of the opaque column, and (7) the transit time. clinical disorders for which the barium enema is indicated in dogs include ileocolic intussusception and cecal inversion (intussusception), mechanical and functional large bowel obstruction, invasive lesions of the large bowel, a mass outside the large bowel compressing the bowel, and inflammation of the lower intestinal tract. barium sulfate enemas are contraindicated in suspected obstruction of the colon and rupture or perforation of the colon. however, these same disorders also can be identified by ultrasonic examination or colonoscopy, either of which is the preferred diagnostic modality over a barium enema. twenty-four hours before radiographs, administer a liquid diet only, preferably water. during the 18 to 24 hours before the radiographs, administer a mild high colonic enema or give a saline laxative orally. do not give any irritating enemas within 12 hours of the scheduled radiographic examination; however, administer isotonic saline solution or plain water enemas before the examination to ensure that the bowel is clear. obtain survey radiographs of the abdomen, and examine the colon to ensure that this portion of the bowel is clear. sedation or anesthesia may be indicated. barium may be infused through a catheter into the colon or allowed to flow in by gravity through an enema bag. do not force barium into the colon under pressure. do not elevate the enema bag more than 18 inches above the animal. cuffed rectal catheters (bardex cuffed rectal catheters, 24f to 38f, and the bardex cuffed pediatric rectal catheter, 18f [c.r. bard, murray hill, new jersey]) can be used in dogs (figure 4-50) . for very small dogs and cats, use smaller catheters. a plastic catheter adapter and a three-way stopcock are needed. various barium sulfate preparations can be used; however, the final concentration should be 15% to 20% w/v. a commercially available barium enema kit is helpful. place the cuffed rectal catheter so that the inflated bulb is cranial to the anal sphincter. place the animal in right lateral recumbency and fill the colon with contrast material at a dose of 20 to 30 ml/kg. take the radiographs after infusion of a two-thirds dose of barium. if the colon is not filled, infuse more contrast agent. obtain radiographs in the ventrodorsal and lateral positions, and determine whether the colon is distended adequately. remove as much of the contrast material as possible from the colon, and repeat the radiographs. insertion of room air at 2 ml/kg into the colon facilitates the evaluation of the colonic surface. deflate the cuff on the catheter, and remove the catheter from the rectum. throughout the procedure of filling the colon with contrast material or air, take care not to overdistend the colon, which may lead to rupture. when reviewing individual radiographs, look for the following radiographic lesions: (1) irregularity of the barium-mucosal interface; (2) spasm, stricture, or occlusion of the bowel . the arterial phase demonstrates renal blood flow; the nephrogram demonstrates the accumulation of contrast agent in the renal tubules and is used to evaluate renal parenchyma; the pyelogram phase evaluates the urinary collecting system, including the ureters; and the cystogram reveals the collection of contrast agent in the urinary bladder. excretory urography does not result in any quantitative information about renal function and is not a substitute for renal function tests. the degree of visualization of contrast material within the renal excretory system depends on the concentration of iodine in the contrast medium, the technique of excretory urography performed, the state of hydration of the patient, renal blood flow, and the functional capacity of the kidneys. an intravenous catheter is prepositioned. the contrast medium most commonly used is a diatrizoate or iothalamate compound. administer 850 mg/kg of an iodine compound iv by syringe. continuous iv "push" is indicated. obtain a ventrodorsal radiograph at 10 seconds after injection, and repeat ventrodorsal and lateral radiographs 1, 3, 5, 15, 20, and 40 minutes after injection. this method is the current standard technique. if the patient's blood urea nitrogen level is greater than or equal to 50 mg/dl or the creatinine level is greater than 4 mg/dl, double the dose of contrast material. lesions that can be detected by using intravenous urography are renal mass lesions; neoplasia; renal cysts; renal and ureteral traumatic lesions; pyelonephritis; hydroureter; hydronephrosis; renal agenesis; hypoplasia; pelvic and ureteral obstructions (calculi, blood clots); renal parasites; ectopic ureter; and duplication of the collecting system. retrograde urethrography is a diagnostic tool used to localize diseases of the lower urinary tract of dogs and cats. this method can reveal conditions such as urethral neoplasms, strictures, trauma, calculi, or other anomalies. the procedure involves the injection of an aqueous iodine contrast medium into the urethra through a ureteral or balloon-tipped catheter. the radiopaque contrast material is mixed to a threefold to fivefold dilution with sterile lubricating jelly to increase the viscosity. a dilution of 1:3 contrast medium with sterile distilled water or saline also can be used. before retrograde contrast urethrography is performed, give the animal a cleansing enema. sedation or anesthesia may be necessary. inject 5 to 10 ml of contrast medium. near the end of the injection, while the urethra is still under pressure, obtain a lateral radiograph. if the urinary bladder is to be distended with contrast material or air, remove urine from the bladder. in the male dog, position the catheter so that the tip of the catheter is distal to the os penis. inject lidocaine 1 to 2 ml into the urethral lumen to anesthetize the urethra adjacent to the balloon-tipped catheter. in male cats, retrograde contrast urethrography can aid in defining the extent of urethral damage (stricture) or the presence of urethral calculi. in male cats, use a 4f balloon catheter or a 3.5f tomcat open-ended urethral catheter. insert the catheter 1.5 cm into the penile urethra. if the urethra is patent, 2 to 3 ml of contrast material will enable visualization of the urethra, but increased amounts of contrast material (2 to 3 ml/lb) injected into the bladder are needed for maximum distension of the preprostatic urethra. a voiding positive contrast urethrogram is necessary to visualize the distal (penile) urethra. apply external pressure to the bladder (using a wooden spoon or other external compression device), and radiograph the distal urethra. take extreme care with the amount of fluid placed in the bladder if the urethra is occluded by a balloon catheter. overdistension of the bladder results in hematuria, pyuria, urinary bladder rupture, and mild to severe bladder inflammation. palpate the bladder carefully during distension, and note the backpressure on the syringe used in filling the bladder. cystography refers to contrast radiographic procedures that facilitate visualization of the lumen and/or contents of the urinary bladder and trigone (box 4-12). three procedures can be used to image the urinary bladder: positive contrast cystography, negative contrast cystography (also called pneumocystography), and double-contrast cystography (combination of positive and negative cystography performed in the same patient). note: many of the indications for performing contrast cystography are also indications for ultrasound examination. contrast cystography is most useful for characterizing congenital and acquired alterations in the normal anatomy and function of the ureters and lower urinary tract, such as ectopic ureter. abdominal ultrasound, when available, remains the preferred method for imaging abnormalities within the bladder lumen (e.g., calculi and tumors) and changes within the bladder wall. pneumocystography, also called negative-contrast cystography, involves the insufflation of a soluble gas into the lumen of the urinary bladder to facilitate imaging of any material or tissue within the bladder lumen that otherwise would be obscured by the presence of urine or positive contrast material. prepare the patient as described previously. once a urinary catheter has been placed and the urethra is occluded, use a syringe and a three-way stopcock to inject 4 to 10 ml of carbon dioxide or nitrous oxide per kilogram. palpate the bladder while filling it with gas to avoid overdistension or rupture. inject air until there is pressure on the syringe barrel or leakage of air around the catheter. replace any air that escapes during the procedure. take lateral and ventrodorsal views of the abdomen. caution: room air is the most accessible contrast material for pneumocystography and generally can be found in most practices. however, an increased risk of air emboli is associated with the placement of room air into the bladder under positive pressure, particularly in patients with hematuria. pneumocystography is not an innocuous procedure; fatal venous air emboli have occurred in dogs and cats. this complication is seen most commonly in cases of severe hematuria. ultrasound or positive contrast cystography is preferred over pneumocystography in such cases if a soluble gas is not available. if possible, use a gas that is readily soluble in blood (such as carbon dioxide or nitrous oxide) for bladder insufflation. the injection of radiographic contrast material into the urinary bladder is referred to as contrast cystography or positive contrast cystography. when ultrasound examination is not available or not feasible, the clinical and radiographic findings noted in box 4-13 justify the use of contrast radiography to image the bladder. the same principles of preparation apply as for obtaining a pneumocystogram. use a urethral catheter with a three-way valve or a small foley catheter with an inflatable cuff. organic iodides are the contrast material of choice and should be used in 5% to 10% concentrations. double-contrast cystography also can be performed in patients for which a positive contrast study is not diagnostic, yet there is reasonable indication for an intraluminal lesion. in this case, the same urinary catheter as used for the contrast study, remove all remaining urine and contrast material. if necessary, inject 2 to 5 ml of an aqueous organic iodine contrast material into the bladder. gently roll the patient over in an attempt to coat the bladder with contrast material. then distend the bladder with air in the same manner as described for pneumocystography. some of the lesions routinely diagnosed with the aid of cystography are calculi ( vaginal examination is indicated for collection of material from the mucosal wall for culture and exfoliative cytologic examination and for vaginoscopic examination of vaginal and cervical mucosa (box 4-14). examination of the vagina for culture and cytologic or vaginoscopic examination occasionally can be performed in the cooperative patient without the use of sedation or anesthesia. an assistant is used to restrain the patient on an examination table. bitches that can be restrained for other minor examinations (ears, teeth, toenails, anal sacs, and blood samples) often will tolerate vaginal examinations. those that need further restraint may require sedation or administration of a short-acting barbiturate anesthetic. trim long perivulvar hair and cleanse the perineum with a germicidal or surgical scrub such as povidone-iodine. water and germicidal soap usually will not control surface contamination by pseudomonas and proteus species, which frequently contaminate culture swabs. in dogs with long tail hair, it is appropriate to wrap the tail with gauze before the procedure to prevent bacterial contamination. if vaginal culture is indicated, this procedure should be conducted first to avoid contamination induced by the general examination. pass a sterile, warm vaginal speculum with only a thin coating of lubricating gel into the posterior vagina while an assistant spreads the vulva. guide the speculum into the vagina by placing the speculum into the vulva just at the dorsal commissure of the vulva and applying pressure up and out against the commissure. direct the speculum dorsally toward the rectum until meeting resistance, and then direct it horizontally into the cranial vagina. this procedure bypasses the clitoral fossa and enables visualization of the urethral opening and pelvic arch. take a guarded culture swab (swab covered by a protective plastic pipette) from its individual sterile bag and pass it inside the vaginal speculum to the anterior vagina or cervical area. then expose the swab from the protective plastic tubing and rotate it against the mucosa. retract the swab into the protective plastic tubing and carefully remove it from the vagina. the protected swab then may be placed back in its original sterile bag until it is processed for culture (30 minutes) or placed in amies transport medium with charcoal. amies transport medium with refrigerator packs and a styrofoam-insulated mailing box will retain fastidious organisms for 72 to 96 hours. process bacterial, mycoplasma, and ureaplasma cultures for potential infectious agents. viral transport medium can be used for a separate sterile swab if viral agents such as the genital form of canine herpesvirus are suspected. immediately after the swabbing for culture, while the vaginal speculum is still in place, advance a clean or sterile swab moistened with sterile physiologic saline solution carefully into the anterior vagina to make a smear for cytologic examination. gently scrape vaginal epithelial cells from the ceiling of the vagina at or cranial to the region of the external urethral orifice. collect samples from the region of the clitoral fossa, which is lined by stratified squamous epithelium at all stages of the estrous cycle. gently rub the swab on the vaginal mucosa. remove the swab and roll it smoothly onto two or three clean glass slides. sterile vaginal speculum (e.g., adjustable spreading, stainless steel, or disposable plastic; cylindric; glass, plastic, stainless steel, or nylon) sterile otoscope heads of variable size for small dogs sterile protected culture swabs (teigland type or other) sterile culture swabs (culturettes) amies transport medium with charcoal viral transport media glass slides and coverslips sterile proctoscope (welch allyn, human pediatric type) or other endoscope, flexible or rigid sterile offset biopsy punch 4 the smears may be fixed immediately in 95% alcohol, sprayed with a commercial fixative or hair spray, or left to dry in air. a drop of new methylene blue stain placed on a coverslip and inverted on the smear can be used to examine a wet mount preparation immediately. this stain is not permanent and precipitates when it dries, and new methylene blue-stained smears cannot be used for comparison with other smears made later in the cycle. the diff-quik or leukostat stain is a permanent stain that can be submitted for review by a pathologist. examine the smear for stage of estrous cycle and evidence of active inflammation. compare these findings with culture results and vaginoscopic findings to interpret evidence for an active genital tract infection, a carrier state of a potential infectious agent, or a possible contaminant at culture. a diagnostic laboratory with the ability to isolate specific infectious agents should indicate the number of organisms (few, moderate, many, or heavy) and report whether the isolates are pure or mixed and their significance. the vagina of the bitch is long in comparison with that of other domestic animals, hence digital examination of the cervix, and in many cases the urethral orifice, is not feasible. the mucosa forms longitudinal folds. the clitoris is in a well-developed fossa in the floor of the vestibule. the vagina can be visualized completely with a small, sterile proctoscope or flexible endoscope. lubricate the warmed, sterile instrument, and pass it to the region of the cervix. examine first without insufflation for true color and vaginal fluids or discharge. when insufflation is performed while the vulva is compressed around the sterile proctoscope, the vagina expands and its entire wall can be viewed completely as the instrument is withdrawn. the normal canine vagina has a uniform light pink color and longitudinal folds. during proestrus and estrus, the folds become more prominent and cross-striations give the surface a cobblestone appearance. this cobblestone appearance remains smooth when estrogen levels are high but quickly becomes angular (cobblestone appearance) when estrogen levels drop during the luteinizing hormone peak (ovulation), and progesterone levels increase. this change can be used to indicate ovulation and the ideal time for breeding. the hyperemia causes the vagina to appear reddish and congested. the pressure of air insufflation balances the mucosa. the canine vulva has a large cranial dorsal median fold that may obscure the cervix. in fact, ridges near the dorsal fold may give a false impression that this fold is the cervix. during estrogen stimulation, the cervix may be open and uterine blood may be escaping. in the management of dystocia, the vaginoscope can be used to detect puppies in the birth canal and to diagnose malpositions and aid in the correction of these conditions. during the endoscopic examination, small tumors or polyps can be removed or large masses can be sampled with the biopsy punch. ulcers or erosions can be cauterized, and foreign bodies can be removed. a complete vaginal examination must include careful palpation of the vaginal wall and pelvic canal. this palpation is accomplished by digital examination through the vulva (using a sterile glove) and is assisted by palpation through the posterior abdominal wall. incomplete hymen rings, vaginal fibrous stenotic rings, or pelvic malformation can be diagnosed. a digital rectal examination may be needed for vaginal masses or pelvic deformities. the canine reproductive cycle begins at the age of 6 to 12 months and repeats at intervals of 4 to 12 months. in the average bitch, ovulation occurs spontaneously 1 to 3 days after the onset of estrus; in normal bitches ovulation may occur 3 days before to 11 days after the onset of estrus. sperm live in the uterus of the estrous bitch up to 11 days, and the ovum lives up to 5 days after ovulation. the fertilized ovum takes 4 to 10 days to reach the uterus, and implantation takes place 18 to 20 days after ovulation. the gestation period from the first breeding is 57 to 72 days and from the luteinizing hormone peak is 64 to 66 days. anestrus is characterized by dryness of the mucosa and a thin vaginal wall with stratified squamous epithelial cells a few cells to several layers thick but without cornification. noncornified epithelial cells and wbcs are present in a ratio of 1:5 in the vaginal smear. the wbcs are polymorphonuclear. the noncornified epithelial cells are 15 to 51 nm in diameter and have round free edges, granular cytoplasm, and large nuclei with distinct chromatin granules. the period of anestrus is 2 to 3 months or longer in some breeds. in proestrus the vaginal wall is thicker than in anestrus, and the mucosa shows prominent cornified squamous epithelium (20 to 30 cells thick) with rete pegs. the longitudinal and transverse vaginal folds are thick, smooth, and round. the vaginal wall becomes impervious to wbcs, but there is extravasation of rbcs to the surface epithelium. the rbcs are discharged. vaginal smears show predominantly rbcs and noncornified epithelial cells, which become cornified as proestrus progresses. wbcs are present, but their numbers decrease as estrus approaches. debris and bacteria are abundant for 7 to 10 days. the vagina is thick with longitudinal and transverse folds that become angular as estrogen levels decrease and progesterone levels increase. fluid is abundant, often tinged with blood. noncornified epithelial cells and wbcs are absent. cornified epithelial cells, which are polyhedral and contain pyknotic nuclei or no nuclei, are predominant; their presence seems to be related to the appearance of flirting by the bitch and acceptance of the stud. wbcs reappear about 36 to 96 hours after ovulation. bacteria and debris are absent during estrus, but they are seen again in the smears after ovulation when wbcs reappear 7 to 10 days later. the number of wbcs increases rapidly, the number of cornified epithelial cells decreases, and the number of noncornified epithelial cells increases. after 5 to 7 days, the number of wbcs may decrease to 10 to 30 per field. after parturition, much cellular debris, wbcs, rbcs, and a few epithelial cells are present for several days, until placental sloughing is complete. the presence of masses of degenerate wbcs (and bacteria) indicates metritis or endometritis. the continued presence of blood-tinged fluids containing abundant rbcs, a few noncornified epithelial cells, and occasional wbcs (nontoxic) plus necrotic cells for months postpartum is evidence of subinvolution of placental sites. most of the characteristics just discussed that apply to bitches also pertain to queens. however, the small size of the feline vagina precludes palpation and early vaginoscopy. a sterile, warm, small-animal otoscope speculum enables fairly good visualization of the vaginal mucosa and can be used with a small, 4-mm-diameter sterile swab to obtain smears for culture procedures. use of the speculum is easiest after parturition or during estrus. vaginal cells for cytologic examination can be obtained with a moistened 3-mm cotton swab (calgiswab) inserted 2 cm into the vagina. in some cases, flushing the vagina with sterile saline injected and aspirated with a clean glass eyedropper is more successful. use of an eyedropper may trigger ovulation, as it simulates coitus. unlike the bitch, the queen shows no diapedesis of rbcs during proestrus or throughout the estrous cycle. cytologic examination of feline vaginal smears reveals the following by stage of the estrous cycle. cytologic examination reveals scarce debris and numerous small, round epithelial cells with a high nuclear/cytoplasmic ratio, frequently in groups (seasonal: from september to january in the northern hemisphere). cytologic examination reveals increased debris and fewer but larger nucleated epithelial cells with a low nuclear/cytoplasmic ratio (0 to 2 days). cytologic examination reveals markedly less debris and numerous large polyhedral cornified cells with curled edges and small dark pyknotic nuclei or loss of nuclei (6 to 8 days) after coitus or induced ovulation. cytologic examination reveals hazy, ragged-edged cornified cells and zero to numerous wbcs with numerous bacteria and increased debris. cytologic examination reveals increasing numbers of small basophilic cells with wbcs still present (total period of metestrus, 7 to 21 days). if ovulation does not occur, the smear will return to an anestrous stage with few to no wbcs. the feline estrous cycle is continuous every 14 to 36 days if 12 to 14 hours of light are present daily. ovulation is induced 24 to 30 hours after coitus. sperm require 2 to 24 hours for capacitation in the uterus. implantation is expected 13 to 14 days after coitus. the procedure for artificial insemination in dogs includes the following steps: 1. determine the correct time to inseminate by test-teasing with a stud, by cytologic examination of vaginal smears, or by vaginoscopic examination to determine the day when vaginal folds change from round to angular. breed the day after the bitch first stands staunchly to accept service and "flags" her tail or during cytologic indications of estrus (complete cornification of vaginal epithelial cells) but before wbcs reappear in the smears. breed at 48-hour intervals until the female dog goes out of heat or for three or four inseminations. 2. if the vulva is soiled, clean it thoroughly with alcohol swabs (box 4-15). 3. gently aspirate semen through the inseminating pipette into the warm syringe. applying the artificial vagina to the shaft of the penis, apply pressure with the thumb and forefinger proximal to the exposed glandis. this usually can be done with one motion as the stud is thrusting. if the stud is shy and not interested, massage the penis slightly in the prepuce or in the artificial vagina to cause erection. when erection of the bulbus is felt, reflect the prepuce posteriorly to free the bulbus. apply pressure with the thumb and forefinger behind the bulbus, circling the shaft of the penis. after completion of the most rapid pelvic thrusting and ejaculation of the sperm-rich fractions of semen (1 to 3 ml), twist the penis 180 degrees backward in a horizontal plane, between the hind legs, so that the penis remains in the same plane as in the forward position, with the thumb and forefinger still applying pressure around the circumference of the penis proximal to the bulbus. the penis cannot be twisted unless the prepuce is reflected posterior or proximal to the bulbus glandis. twisting the penis in this position simulates a natural "tie" and allows the person collecting the semen to better visualize the collection (artificial vaginas are widely available now and are much preferred because they simulate the natural pressure of the vagina). the first drops of ejaculate may be discarded, especially if any urine is present. collect the sperm-rich fraction separately. a clear ejaculate is prostatic fluid, which may be collected separately for examination. 6. after semen collection, place the penis in the forward position, straighten out the prepuce to avoid paraphimosis, and remove the bitch from the room. allow the stud to lick the erect penis and lose the erection. check the stud for evidence of paraphimosis before it is released or caged. the ejaculate consists of three fractions: first fraction: urethral secretion (usually clear fluid)-0.1 to 2 ml within 50 seconds, ph 6.3. if evidence of urine is present, discard this fraction and do not add it to the sperm-rich fraction. in most ejaculates collected from dogs, the first and second semen fractions are collected together. second fraction: sperm-containing secretion (milky opaque fluid)-0.5 to 3 ml within 1 to 2 minutes, ph 6.1. third fraction: prostatic secretion (usually clear fluid)-2 to 20 ml within 30 minutes, ph 6.5. the total specimen is 0.3 to 20 ml, ph 6.4. because the first and third fractions are clear, waterlike material and the second fraction is milky-opaque, the clinician can separate them by changing collecting tubes as each fraction is ejaculated. collection of only enough prostatic fluid to rinse the sperm fraction into the test tube is best. too much prostatic fluid may be detrimental to the longevity of sperm in storage. collecting individual fractions may be important in determining the site of an inflammatory reaction, but for artificial insemination only the sperm-rich, low-volume ejaculate is needed for insemination, dilution, or freezing. 7. return the stud to his cage. retain the bitch until the semen is examined, if insemination is to be performed. immediately after semen collection, slowly invert the tube several times to mix the semen gently. determine the motility of sperm by placing one drop of semen on a warmed microscope slide. cover the slide with a coverslip, and observe the specimen under low power for progressive motility. there will be no "waves, " but general vigorous forward motion should be evident. if the sample is too concentrated for individual sperm to be found, mix one drop of semen with one drop of saline at body temperature on a warmed microscope slide. using high power, count 10 different groups of 10 sperm, observing the numbers of motile and nonmotile sperm. total motility for a suitable sample should be 80% or greater. motility less than 60% is not satisfactory. determine the number of sperm in the total ejaculate. sperm concentration may be determined in a hemocytometer with a 1:100 blood cell dilutor kit (unopette), and concentration then is multiplied by volume to determine sperm numbers per ejaculate. remember that more dilute samples will be obtained when prostatic fluid is collected, but total sperm numbers in the ejaculate will be only marginally influenced by dilution with prostatic fluid. total sperm per ejaculate should exceed 300 million in a normal male dog and may approach 2 billion in large dogs. a minimum number of 200 million sperm per insemination is needed on average for conception. determine morphology. make a smear of a drop of semen like a blood smear and allow it to air-dry. then stain the smear with diff-quik stain; dip the slide into the fixative and solutions 1 and 2 for 2 to 3 minutes each. then count 100 sperm at ×1000 magnification, noting normal and abnormal sperm. if there is any question about abnormality, examine 500 sperm cells. normal canine sperm are 63 nm long; the heads are 7 nm long. the percentage of abnormal sperm should be less than 20%. differential abnormality is important, and the following abnormalities should not be exceeded in any sperm count: abnormality of the head, 10% to 12%; midpiece abnormalities, 3% to 4%; tail abnormalities, 3% to 4%; and retained protoplasmic droplets, 3% to 4%. figure 4 -51 shows abnormalities that should be counted and recorded. the presence and location of distal or proximal protoplasmic droplets, which may indicate cell immaturity, are important to note. defects of the cells within the testes are generally more serious than defects that occur in the sperm during epididymal transport or after ejaculation (such as fractured heads, retained protoplasmic droplets, or bent tails). usually a biopsy should not be done on material from testes unless the testes are azoospermic. damage produced after the sperm have left the testes may indicate epididymal disease or may be the result of cold, trauma, or osmotic or urinary contamination. when abnormalities are found, it is wise to obtain two or three semen samples within a few days for baseline evaluation and then repeat the studies in 4 to 6 weeks to determine whether there is a healing or regressing trend. there are usually 64 days from the date of sperm formation to the date of ejaculation: 54 days in the testes and 10 days in transport and maturation in the epididymis. normal male dogs can be used at stud once every other day indefinitely or once every day for 7 to 9 days, after which sperm numbers in the ejaculate will decline but not to less than the numbers needed to achieve conception. although castration is a common first recommendation for any male dog with known or suspected prostatic disease, a number of prostatic disorders are recognized for which cytopathologic and histopathologic examination, rather than castration, is indicated. benign prostatic hyperplasia is recognized as the most common prostatic disorder of male dogs. in half of the dog population, changes consistent with benign prostatic hyperplasia are present by 4 to 5 years of age, especially in older intact dogs. because benign prostatic hyperplasia is androgen dependent, routine castration is the recommended treatment. however, at least three differential diagnoses justify additional diagnostic tests: prostatic neoplasia (usually adenocarcinoma), acute and chronic bacterial prostatitis, and prostatic cysts (septic and nonseptic). in male dogs with prostatomegaly and associated signs (dysuria and/or dyschezia), further evaluation of the prostate is indicated. several techniques have been described. abdominal ultrasonography is the preferred technique for evaluating prostate size, shape, and consistency. distension retrograde contrast urethrocystogram has been described as a means for evaluating the internal integrity of the prostate and is moderately effective in distinguishing normal from abnormal. however, this technique is not known to distinguish among various types of prostate disease. cytologic examination and quantitative bacterial culture of the ejaculate (especially the third fraction) of a male dog is recommended in any patient with prostatomegaly. however, sample collection can be difficult and is frequently not successful. in addition to lumbar radiographs and abdominal ultrasonography, performing a prostatic wash is a simple, noninvasive technique that may yield diagnostic information. using aseptic technique, place a conventional urinary catheter into the bladder and remove all urine. lavage of the urinary bladder with up to 5 ml of sterile saline is recommended. recover the saline and save it (sample no. 1). subsequently, retract the catheter tip, but only to the level of the prostate gland (immediately caudal to the trigone). position of the tip usually can be verified by tactile placement and the detection of increased resistance to catheter movement during retraction. position can be confirmed with a lateral radiograph of the pelvis. with the catheter in place, identify the prostate on a digital rectal examination and gently massage for approximately 1 minute to force prostatic fluids into the urethra. infuse 5 ml of sterile saline through the catheter. the objective is to wash prostatic fluids and cells into the urinary bladder and recover the saline from the bladder (sample no. 2). examine fluid from both samples cytologically by distributing a drop of fluid across a glass slide, air-drying, and staining; submit a small aliquot (0.5 ml) for bacterial culture. cytologic examination is used to detect the presence of inflammatory cells versus neoplastic cells. low numbers of neutrophils (<5 cells per high-power field) are present in ejaculates and prostatic washes from normal dogs. quantitative bacterial culture, with a yield of greater than 2 log 10 , of one or more bacterial species in sample no. 2 confirms bacterial prostatitis. complications from this procedure are unlikely, but conceivably a patient with septic prostatitis and prostatic abscesses could become bacteremic after this procedure, which in some patients could lead to sepsis. ultrasound examination is an important first step, when available, in assessing the size, shape, and internal integrity of the canine prostate gland and for detecting any changes in structures adjacent to the prostate. however, ultrasonography generally will not distinguish among different types of prostatic disease. further diagnostic tests are especially indicated in castrated, middle-aged to older male dogs with evidence of prostatomegaly. percutaneous fine-needle aspiration and/or prostatic biopsy are indicated. fine-needle aspiration of the prostate is performed through a ventral abdominal approach. use aseptic technique, and surgically prepare the skin at the level of needle insertion. because needle movement, once the needle has been inserted, could damage the urethra or 4 adjacent structures, perform the procedure in the sedated or anesthetized patient. use an approach similar to that used for cystocentesis in a male dog with the exception that needle entry is at a point caudal to that used to enter the urinary bladder but is cranial to the pubis. the procedure can be performed with or without ultrasound guidance. in the absence of ultrasound guidance, determine needle position by tactile placement and detection of resistance as the needle enters the prostate. multiple needle penetrations and aspirations are attempted without withdrawing the needle from the skin. relieve negative pressure in the syringe before removing the needle. apply any material collected to a glass slide and allow it to air-dry before staining. any conventional stain used for peripheral blood is appropriate. a transrectal approach to fine-needle aspiration of the prostate has been used in dogs and is performed routinely in men. however, the distance from the anus to the prostate, visualization, and the risk of infection generally are cited as reasons for not performing this technique in dogs. fine-needle aspiration may not be diagnostic, particularly in patients with isolated, discrete lesions (cysts or neoplastic nodules) within the prostatic parenchyma. in such cases, ultrasound-guided needle (tru-cut) biopsy of the prostate is indicated. specific training and experience are indicated for performing this procedure because significant complications can result. complications associated with prostate biopsy and fine-needle aspiration are not insignificant. hematuria and periprostatic hemorrhage are described. postaspiration and postbiopsy abscess also have been described. consider the risk of urethral penetration and subsequent stricture at the site of penetration. kutzler ma, yeager a: prostatic diseases. in ettinger sj, feldman ec, editors: textbook of veterinary internal medicine, ed 6, st louis, 2005, elsevier. upper respiratory tract for purposes of this discussion, the anatomic limits of the upper respiratory tract of the dog and cat extend caudally from the nasal planum to the first tracheal ring. key anatomic structures that principally can cause clinical signs include the anterior (external) nares, nasal cavity, nasal turbinates, frontal sinuses, maxillary recesses, upper dental arcade (especially the roots of the maxillary canine teeth), choanae (posterior nares), nasopharynx, soft palate, arytenoid cartilages, glottis, larynx, and vocal folds (see table 4 -9). clinical signs related to the upper respiratory tract in dogs and cats are among the most common presenting complaints encountered in small animal practice and, interestingly, are frequent reasons for referral to specialty practices and veterinary teaching hospitals. the oral and nasal cavities are important portals of entry for foreign body entrapment and infectious agents. in addition to the occurrence of nasal neoplasia and trauma, it is not surprising that upper respiratory tract diseases in dogs and cats are common presentations. however, upper respiratory signs can be associated with significantly different underlying causes. localizing the problem amid a variety of clinical signs in an anatomically complex area presents significant diagnostic and therapeutic challenges to even the most astute clinician. the presentation addresses upper respiratory disease in the dog, with specific emphasis on clearly defining the presenting clinical signs, localizing the problem, and establishing the diagnosis. the first and most important step in establishing a diagnosis of canine upper respiratory disease is to define the presenting sign. experience has shown that an owner's ability to describe the patient's clinical signs accurately, particularly when signs are not present at the time of examination, is usually inconsistent and inaccurate, although it can be most entertaining. the four localizing clinical signs characteristically associated with upper respiratory diseases are sneezing and/or nasal discharge, stertor, stridor, and cough. each sign, considered independently, will focus the examination to the appropriate anatomic region of the upper respiratory tract. definition of the clinical signs sneezing and nasal discharge may seem intuitive. this is the most common presenting sign in dogs with upper respiratory disease. owners that present a dog with sneezing are likely to be accurate in their description of the problem. however, the presence or absence of a nasal discharge may be more difficult to establish. volume, character, and frequency of the discharge ultimately determine whether the owner will have even observed this sign. the astute owner will report whether the discharge is unilateral or bilateral. in the patient that has a history of sneezing and nasal discharge, instillation of a topical nasal decongestant into each nostril occasionally will provoke sneezing and elicit the nature of any discharge that is present. sneezing and/or nasal discharge localizes the problem to the nose, nasal cavity, and paranasal sinuses. however, thorough examination of the nose and nasal cavity can be difficult, even with the availability of appropriate endoscopy equipment. in addition to careful examination of facial symmetry, the first part of the examination begins in the oral cavity, with emphasis on the maxilla, the hard palate, and the canine teeth. examine the hard palate for evidence of trauma (penetrating or nonpenetrating) and congenital cleft palate (puppies). carefully probe the medial aspect of the maxillary canine teeth for evidence of oronasal fistulas. despite normal-appearing teeth and gingiva, severe, occult periodontal disease with resulting necrosis of bone does result in a septic communication between the oral and nasal cavities. the owner characteristically describes paroxysms of sneezing associated with a sanguineous nasal discharge or spray. if these findings are negative, radiographs of the skull are indicated. three views, obtained in the anesthetized patient, are indicated: lateral, ventrodorsal, and occlusal (open mouth) view. radiographic interpretation of the nasal cavity and sinuses dictates that the clinician have a thorough understanding of the anatomy of the upper respiratory tract. subsequently, with the patient still anesthetized, attempt a visual examination of the nasal cavity. radiographs are always performed before visual examination of the nasal cavity. manipulation of the tissue may result in intranasal bleeding, which will significantly complicate radiographic interpretation. a simple otoscope speculum placed into each nostril allows an adequate examination of the proximal 20% to 25% of the nasal cavity in most dogs. visual examination of the caudal 75% of the nasal cavity can be attempted only with a small-diameter endoscope. flexible and rigid scopes are available; each has advantages and disadvantages that will be discussed. computed tomography and magnetic resonance the second most common clinical sign associated with upper respiratory disease in dogs, stertor is intermittent, yet persistent or continuous snorting, also called stertorous breathing. paroxysms of stertor, typically called reverse sneezing, are characterized by rapid, consecutive inspiratory bursts through the nose. seldom actually seen during examination, reverse sneezing is likely to result from the patient's attempt to displace matter trapped in the nasopharynx and move it into the oropharynx, where it can be swallowed. visualization of the nasopharynx and choanae is essential in the patient that has chronic or persistent stertor. the examination can be accomplished only in the anesthetized patient. sedation is not sufficient to conduct the examination. a flexible endoscope with the ability to flex approximately 170 to 180 degrees is recommended. examination allows visualization of the nasopharynx and associated mucosa, the choanae (posterior nares), and the top of the soft palate (see figure 4 -34). nasopharyngeal foreign bodies are by far the most common finding. sticks, plant material (grass and juniper twigs), peas, cotton balls, and thread are just a few examples. neoplasia is the second most common finding. in cats, lymphoma (felv related) obstructing the choana most commonly is observed (see figure 4 -35). in dogs, neoplasia is uncommon, but (in my experience) sarcomas in young dogs have been seen most frequently. the least commonly encountered of the upper respiratory signs is stridor, or stridulous breathing. stridor is audible wheezing and is associated with restriction to airflow, usually at the level of the larynx. therefore stridor is the most critical and potentially life-threatening upper respiratory sign. this is especially true when stridor is continuous. the patient that has continuous stridor deserves immediate attention. make every effort to discern the cause once the clinical sign is characterized. in obtaining the history, owners generally describe wheezing accurately; however, some patients actually may have severe dyspnea or orthopnea. careful questioning of the client is indicated to determine whether wheezing is associated with the additional effort to breath. the clinician also should make an effort to discern whether the owner has observed any change in the ability of the dog to vocalize or bark. simply listening to the patient breath in a quiet room is the first step in assessing stridor. a stethoscope is not required to hear wheezing but should always be used to examine the cervical trachea, the larynx, and the lungs. any restriction to airflow in the larynx or cervical trachea can cause stridor. however, in the majority of cases the stridor will be significantly louder at the level of the larynx, indicating a restrictive lesion at that level. if any indication of respiratory distress is reported or manifests during the examination, subject the patient to a visual examination under general anesthesia. sedation is not sufficient to conduct the examination. be prepared. these patients are not routine. emergency resuscitation may be required on induction of anesthesia, including the need to perform a tracheostomy. on induction, carefully place an endotracheal tube. if there are no complications associated with inserting the tube, once anesthesia has been effectively induced and the patient's condition is stable, lateral and dorsoventral radiographs of the larynx and cervical trachea are indicated. metallic objects (e.g., fish hooks) can become buried in the mucosa and may not be observed during a visual examination. remove the endotracheal tube in order to conduct a visual examination. a focal, handsfree light source directed into the oropharynx is strongly recommended. carefully examine the epiglottis, arytenoid cartilages, glottis, and vocal folds using a cotton-tipped applicator. careful observation of the symmetry and function of the arytenoid cartilages is essential. the left and right cartilages normally respond to tactile stimuli when the patient is in a light plane of anesthesia; both sides should move to the medial plane rapidly and at the same time. they may not close, depending on the depth of anesthesia. it should be possible to visualize the cartilage on the inside of the tracheal rings while looking through the glottis. in large breed, middle-aged and older dogs, laryngeal paralysis is the most common cause of stridor. associated signs may include exercise intolerance and collapse during exertion. laryngeal paralysis and stridor also may be observed in young breeds as a congenital disorder (dalmatian, rottweiler, bouvier des flandres, siberian husky, and bull terrier). foreign body penetration of the laryngeal tissues can cause serious and lifethreatening obstruction because of infection and swelling. neoplasia may cause obstructive mass lesions involving the larynx, especially squamous cell carcinoma and lymphoma. granulomatous laryngeal disease and fungal mycetoma have been reported. the presence of a mass lesion, assuming there is no foreign body detected, warrants biopsy of the lesion. additional effort to control postbiopsy bleeding is important. i use a cotton-tipped applicator saturated with a 1:10,000 dilution of epinephrine held against the biopsy site for 30 to 60 seconds. this is time well spent. postbiopsy administration of systemically effective dexamethasone has been suggested to control laryngeal swelling, but i have not found this to be effective or important. the following diagnostic procedures are elective and are indicated in patients with chronic disorders of the lower respiratory tract that are not considered life-threatening. transtracheal aspiration is a safe and clinically useful method for obtaining material for cytologic and bacteriologic examination from the lower respiratory tract of medium-sized to large dogs without invading the oval cavity. this procedure is not indicated in cats. the technique can be performed on the unanesthetized animal, although some sedation may be indicated. the hair overlying the larynx is clipped and prepared surgically. in small dogs and cats, tracheal aspirates are collected by passing the catheter through sterile tracheal tubes. light levels of anesthesia are used to accommodate coughing and tracheal intubation. place the animal in sternal recumbency or in the sitting position. elevate and extend the head. locate the cricothyroid membrane by moving the finger along the proximal trachea until the large ventral ridge of the cricoid cartilage is felt. use a 16-gauge, 1 ⁄2-inch intravenous catheter to collect material through the trachea (figure 4-52) . puncture the cricothyroid membrane with the 16-gauge needle, and pass the catheter into the trachea until it reaches the distal trachea or main stem bronchus. (alternatively, in large dogs, insert the catheter between the tracheal rings at the junction of the middle third and distal third of the cervical trachea.) withdraw the needle, and leave the catheter in place. attach a 12-ml syringe containing sterile saline solution to the catheter. expel 1 to 2 ml of saline from the syringe. when the animal coughs, aspirate with the syringe to collect cells and mucus for bacteriologic and cytologic examination. when material has been collected, remove the catheter and bandage the animal's neck. culture material present in the syringe in blood agar and in thioglycolate medium. prepare material from aspiration for cytologic examination. press large plugs of mucus between two clean glass slides, and stain thin smears with wright or giemsa stain. complications of transtracheal aspiration biopsy include catheter trauma to the lower airway or needle trauma to the larynx, resulting in bleeding, subcutaneous emphysema, pneumomediastinum, pneumothorax, or airway obstruction. in cats and small dogs and in dogs for which general anesthesia is not contraindicated, tracheal aspiration (or tracheal wash) is a relatively safe, easy-to-perform procedure that can yield excellent diagnostic cytologic and culture specimens. the procedure has some advantages over transtracheal aspiration in that it allows sample collection from airways beyond note: cats and dogs with acute, severe dyspnea must be regarded as having a life-threatening condition until proved otherwise. immediate therapeutic and diagnostic intervention is indicated. section 1 describes appropriate interventive procedures for the management of these patients. the bifurcation of the trachea (carina) and avoids complications associated with patient discomfort and movement during the procedure. however, cough reflexes are eliminated completely, thereby decreasing potential sample yields from deep in the airway structure. in either case, transtracheal and tracheal aspirations provide the best diagnostic material from large airways, not small airways and alveoli. the anesthetized dog or cat usually is placed in sternal recumbency. lateral recumbency (affected side down) may facilitate recovery of specimens from patients with focal or regional lung disease. use a sterile endotracheal tube to administer the anesthetic and oxygen. introduce a sterile red rubber catheter (long enough to extend beyond the carina) through the endotracheal tube (figure 4 -53). (note: disposable adapters for use with endotracheal tubes are available that allow continuous administration of anesthetic gases while passing the rubber catheter through the tube [ figure 4 -54].) introduce the catheter blindly until resistance is met as the tube attempts to enter smaller airways. use aliquots of warmed, sterile saline in prepared syringes to wash and retrieve samples. aliquots of 3 to 5 ml can be used per collection attempt in small dogs and cats, whereas volumes up to 10 and 20 ml are appropriate for larger dogs. with the catheter positioned as deep as practical in the airway, infuse the entire volume of saline. gentle agitation (intermittent aspiration and injection) may facilitate sample collection. if a 10-ml quantity is infused, retrieval of only 1 to 2 ml as a final volume per collection attempt is not unusual. the remaining fluid is rapidly (seconds) absorbed into the pulmonary vasculature. important: when performing this procedure, do not withdraw the rubber catheter while maintaining a high negative pressure on the syringe. doing so actually may tear mucosa away from the airway and could lead to pneumothorax or pneumomediastinum. the procedure can be repeated safely in the same patient several times. collection of three to five samples is routine. more samples may be indicated depending on the patient's condition and response to the procedure. monitoring of patients undergoing a tracheal wash procedure for oxygen saturation (pulse oximetry) throughout the procedure is recommended. in some patients with reactive airways, infusion of saline may cause significant bronchoconstriction, detected by a rapid decline in oxygen saturation. process collected samples immediately. submit at least one sample of liquid (not a swab of the liquid) for bacterial culture and sensitivity or mic. quantitative cultures are impractical because specimens will be diluted. if the sample appears to be highly cellular (characterized by turbidity), place aliquots into tubes containing edta. syring rs: tracheal washes. in king lg, editor: textbook of respiratory disease in dogs and cats, st louis, 2004, elsevier. bronchoalveolar lavage bal is an alternative diagnostic procedure to transtracheal aspiration and endotracheal wash. bal has the advantage of retrieving fluid samples from distal airways and alveoli. this is a highly diagnostic procedure indicated in patients with generalized lung and regional (interstitial and/or airway) disease that are not in respiratory distress. patients suspected of having allergic or infectious respiratory disease or neoplasia are candidates for bal. although bal is used as a therapeutic procedure in human beings with chronic lung disease associated with accumulations of surfactant in the alveoli, there is no therapeutic indication for bal in dogs or cats. bal must be performed in the anesthetized patient and consequently may be contraindicated in some patients with severe respiratory disease. technique bal entails instilling sufficiently large volumes of fluid into the distal airways to reach, and recover, reasonable cytologic samples representative of small airways and alveoli. several variations on the technique have been described, but all recommend blind or visual placement of a catheter or bronchoscope into an airway of a lung lobe such that the airway is occluded. sterile, nonbacteriostatic 0.9% saline, warmed to approximately body temperature and drawn into prepared syringes, is the fluid of choice. the volume of fluid varies with the size of the patient. defined doses of saline per kilogram of body mass have not been described. in large dogs, two 25-ml aliquots (50 ml total) can be infused into each lobe sampled. in small dogs and cats, total volumes per lobe generally are restricted to 10-ml aliquots. recovery may be as low as 2 to 5 ml with each attempt. ensure that the animal's hair is free of dirt and debris try to limit the animal's fluid intake in the 12 hours preceding radiography empty the animal's bladder immediately before taking radiographs understanding the most commonly diagnosed causes of sneezing and nasal discharge is especially helpful in patient management. in no particular order, the most common differential diagnoses for sneezing and/or nasal discharge include the following: 1. oronasal fistulas: especially common in middle-aged to older dogs, despite a history of recent dental prophylaxis. empiric treatment with an orally administered antibiotic typically results in rapid and complete resolution of clinical signs no breed is predisposed, but the condition is uncommon in brachycephalic breeds. persistent nasal discharge, sneezing, and intermittent epistaxis are common presenting signs. nasal radiographs may demonstrate lytic bone lesions. lysis of the vomer strongly supports neoplasia versus mycotic rhinitis. exposure to tobacco smoke has been associated with 2.5 times greater risk in long-nosed dogs. no or minimal response of the discharge to antibiotics occurs. eighty percent of nasal tumors are malignant. adenocarcinoma is most common, followed by squamous cell carcinoma persistent and voluminous mucoid nasal discharge, with or without sneezing, and nasal pain are reported. erosion of the external nares is an important physical finding. discharge is not responsive to antimicrobial treatment. occlusal view radiographs of the nasal cavity may demonstrate evidence of turbinate destruction and/or increased fluid density on the affected side. forty percent of patients are 3 years of age or younger; 80% are 7 years of age or younger. the diagnosis is uncommon in brachycephalic breeds lymphoplasmacytic rhinitis: poorly described clinical syndrome associated with chronic sneezing and nasal discharge (bilateral or unilateral) nebulization is used (l) in combination with oxygen therapy upper respiratory disease of cats, pneumonia, and postoperative atelectasis and pneumonia; and (4) in chronic respiratory diseases such as chronic bronchitis, bronchopneumonia, collapsed trachea with secondary tracheobronchitis, emphysema, and bronchiectasis. aerosol therapy, however, is a limited-use therapeutic technique used in dogs and cats to administer antimicrobials, bronchodilators (aminophylline, 100 mg), or corticosteroids. the advantage of doing so is to achieve relatively high levels of drug in the respiratory tract in patients with defined lower respiratory tract disease. in addition, administration of potentially toxic antimicrobials (aminoglycosides) by this route has been shown to be associated with minimal or insignificant uptake into the general circulation drugs that can be applied by jet nebulizer (figures 4-56 and 4-57) include the following: 1. bronchodilators: always use bronchodilators when administering drugs that may be irritating and constricting systemic administration of most antibiotics produces adequate pulmonary concentration for antibacterial effect. for bordetella species that are located at the tips of bronchial cilia, topical contact via nebulization may be useful. antibiotics that have been used successfully and safely include kanamycin (250 mg in 5 ml saline twice daily); gentamicin (50 mg in 5 ml saline twice daily) bland solutions: use these in large volume for prolonged mist effect: 0.9% sterile saline (5 to 200 ml as needed); glycerin (5% in saline) detergents and mucolytics: these compounds are irritating and currently are not recommended by most authors antifoaming agents: administer ethyl alcohol (70% solution, 5 to 10 ml twice daily) figure 4-55: disposable jet nebulizer used to administer humidified air and/or medication drugs affecting the respiratory system advanced and expensive techniques recently have been described: laparoscopic-assisted cystotomy, use of the ellik evacuator, use of stone "baskets" (through a cystoscope), and lithotripsy are examples. however, for removal of uroliths from dogs and cats with partial or complete urinary obstruction, urohydropulsion is among the more effective yet inexpensive techniques available. urohydropulsion is a therapeutic procedure for removal of foreign material, namely, uroliths, from the bladder and/or urethra of dogs canine lower urinary tract diseases canine and feline nephrology and urology 4. using a gloved left index finger (not lubricated) as a guide, insert the pipette through the vulva and dorsally into the vagina and forward to the cervix. elevate the bitch's rear quarters to a 45-degree angle by having an assistant pick up the bitch by holding the hock region so that no pressure is applied to the ventral abdomen and uterus. eject the semen gently and slowly. eject a bubble of air to push all the semen through the pipette. deposit the semen in the anterior vagina. 5. remove the pipette, and hold the bitch in an elevated position for 5 minutes. during this time, use the finger encased in a sterile glove to "feather" the ceiling of the vagina to stimulate constrictor activity. this may be important to simulate a "tie" and transport semen into the uterus. 6. lower the bitch to the normal position, and immediately walk her for 5 minutes so that she does not sit down or jump up on a person and allow semen to run back out of the vagina. 7. for best conception, inseminate undiluted fresh semen immediately. 8. refrigerated extended semen is best used within 24 to 48 hours if possible. however, refrigerated semen has been kept viable for up to 9 days with proper care. skim milk has been used as an economical and adequate extender. heat milk to 92° to 94° c for 10 minutes, cool it, and skim it at room temperature. to each milliliter, add 1000 units of crystalline penicillin. if pseudomonas species affect the semen, polymyxin b may be added at 200 units/ml of extender. dilute semen with extender at a semen/extender ratio of 1:1 to 1:4. extend canine semen for freezing with a diluent containing 11% lactose, 4% glycerin, and 20% egg yolk. refrigerate the 1:4 diluted semen; then pipette 0.05-ml portions into depressions in a block of dry ice and hold them for 8 minutes to freeze. store the frozen pellets in liquid nitrogen. frozen semen can be thawed in buffered saline at 30° to 37° c. good semen may be stored in liquid nitrogen for many years without significant loss of motility. conception is best when large numbers of thawed motile sperm are deposited in the cervix or uterine cavity. conception is poor when thawed semen is placed in the anterior vagina, as done in artificial breeding with raw semen. memon ma, sirinarumitr k: semen evaluation, canine male infertility, and common disorders of the male. in ettinger sj, feldman ec, editors: textbook of veterinary internal medicine, ed 6, st louis, 2005, elsevier. semen collection: canine semen is collected for examination for breeding soundness, for investigation of infertility or prostatic disease, and for artificial insemination (box 4-16).the following steps outline the procedure for collecting semen from a male dog: 1. take the stud and an estrous teaser bitch (if available) to a quiet room where there will be no distractions and where there is good traction (rubber mats or rug) for mounting by the stud. 2. hold the bitch, and allow the stud to "flirt" (become aroused) for several minutes. if the bitch is in heat, a brief period of foreplay ("foreplay" may not be the appropriate word to describe the mating behavior of dogs, but it illustrates the point) with both dogs unrestricted will help the process. 3. if necessary, have assistants restrain the muzzled bitch and control the stud by a collar and leash. bring the stud up to the rear end of the bitch, and allow him to mount her or keep his nose in the region of her perineal area. 4. attach the artificial vagina to the semen collection tube, and apply a scant amount of lubricant to the opening of the artificial vagina. 5. if mounting occurs, allow the stud to grasp the bitch and start to thrust his pelvis in an attempt to copulate. gently, from the side of the sheath, grasp the penis by the prepuce and move the prepuce back over the engorged bulbus glandis; while for dogs undergoing bal, particularly when reactive (allergic) airway disease is suspected, pretreatment with a bronchodilator is appropriate and is recommended. aminophylline can be administered at 5 mg/kg (cats) or 11 mg/kg (dogs) orally 1 to 2 hours before the procedure. alternatively, terbutaline, 0.01 mg/kg, can be administered subcutaneously to cats 30 minutes before the procedure.bronchoscopic bal allows direct visualization of the airway or lobe of interest. in medium to large dogs, place the bronchoscope directly through a sterile endotracheal tube. use of an inexpensive, disposable endotracheal tube adaptor permits simultaneous administration of oxygen and anesthetic throughout the procedure. saline can be infused from a syringe directly through the biopsy channel of the endoscope. the bronchoscope serves as the infusion catheter. using this technique, samples can be collected effectively from multiple lobes. blind placement (nonbronchoscopic) bal using a rubber end-hole catheter is required in cats and small dogs. blind placement is also appropriately used in patients with generalized lung or airway disease when discrete placement of the bronchoscope cannot be accomplished reliably. as with the endotracheal wash procedure described before, gentle agitation with the syringe (intermittent aspiration and injection) may facilitate sample collection. do not withdraw the bronchoscope or catheter while maintaining significant negative pressure because this may lacerate the airway, leading to pneumothorax or pneumomediastinum.bal is an invasive diagnostic procedure that is not without risk of injury or death. after completion of bal, administration of 100% oxygen for 5 to 10 minutes via endotracheal tube is recommended for all patients. evaluate the patient carefully for breathing effort and oxygen saturation (pulse oximetry) during recovery. although significant quantities of fluid remain in the airways after bal, most of the volume is absorbed rapidly. residual amounts of fluid, however, can be retained for 24 to 48 hours after the procedure. during this time, some patients will manifest cough. crackles may be auscultated. percutaneous aspiration needle biopsy can be helpful in establishing a diagnosis in conditions such as (l) chronic inflammatory disease of the lung-for example, granulomatous lung disease caused by mycotic organisms; (2) chronic inflammatory disease; (3) metastases to the lung; and (4) primary lung tumors. the biopsy may provide enough diagnostic information to preclude performing an exploratory thoracotomy. lung biopsy is contraindicated in animals with hemorrhagic disease or thoracic disease that produces forceful breathing and coughing. clip and surgically prepare the biopsy site. infiltrate the skin, subcutaneous tissue, muscle, and parietal pleura with 1% to 2% lidocaine. in patients with diffuse parenchymal lung disease, taking biopsy material from the diaphragmatic lobes is recommended. the dorsal portions of the seventh to ninth intercostal spaces are preferred for percutaneous biopsies. in diffuse lesions, take biopsy material from the right or left thorax. caution: understanding of the risks associated with performing fine-needle aspiration of the lung is important, and these risks must be clearly communicated to owners whose pets undergo this procedure. lung aspirates will yield only cells, fluid, and trace amounts of tissue, yet there is a significant risk of inducing pneumothorax with the procedure, even when performed without difficulty or complications. for the procedure, a 22-to 25-gauge disposable needle (such as a 1-inch spinal needle) with stylet is preferred. leave the stylet within the needle until the lung has been penetrated. then quickly remove the stylet and immediately attach a sterile 6-to 12-ml syringe. the amount of air that might enter the lung between the time the stylet is removed and the syringe is attached is negligible. holding the syringe carefully and steadily against the patient's thorax, establish negative pressure in the same manner as when obtaining an aspirate from a lymph node. as much as the patient will permit, attempt three to four aspirations without withdrawing the needle.alternatively, insert a conventional 25-gauge needle attached to a 6-ml syringe subcutaneously over the area of interest. then establish significant negative pressure while the tip of the needle is still positioned in the subcutaneous tissues outside the parietal pleura. while maintaining the same amount of negative pressure in the syringe, direct the needle into the lung, leave it in place for 1 to 2 seconds, and withdraw it completely. apply any material collected directly to glass slide. this procedure is best conducted in patients that are awake. attempting the procedure in anesthetized dogs or cats could result in an unsuccessful aspiration, or, if the lungs were under positive pressure (ventilation or bagging), the risk of causing pneumothorax could be increased. other reported complications include hemothorax (always exciting), lung laceration caused by patient movement during the procedure, pulmonary hemorrhage, and hemoptysis. contraindications to fine-needle aspiration include patients with a known bleeding diathesis and coagulopathy, thrombocytopenia, uncontrolled coughing, pulmonary hypertension, pulmonary cysts, and bullous emphysema.ultrasound-guided techniques for fine-needle aspiration or biopsy of the lung recently have been described and generally are associated with fewer procedural complications. however, additional training and experience, in addition to having access to the proper size and type of ultrasound probe, are critical. cole sg: fine needle aspirates. in king lg, editor: textbook of respiratory disease in dogs and cats, st louis, 2004, elsevier. inhalation therapy can be defined as nebulization (humidification of the inspired air) and aerosol therapy (the process whereby drugs are vaporized in a solution and delivered directly into the respiratory tract). in companion animals, inhalation therapy is most useful for humidifying air in the respiratory tract and moistening the mucous membranes (nebulization). sustained inspiration of dry air or gases causes irritation to the respiratory epithelium, which in turn results in swelling, bronchial gland hypertrophy, goblet cell proliferation, and loss of ciliary epithelium over time. respiratory secretions become thick and tenacious, and efficient bronchial drainage is impaired. the objectives of inhalation therapy include the following: 1. humidification of bronchial mucous membranes 2. deposition of miniscule amounts of potent drugs in smaller airways to achieve optimal topical therapeutic effects with minimal systemic side effects (e.g., bronchodilators) 3. deposition of moderate amounts of potent agents or agents that are effective only topically (e.g., antibiotics and mucolytics) 4. deposition of relatively large quantities of bland substances that promote bronchial drainage with minimal irritation (e.g., saline, propylene glycol, glycerin, and detergents) the objective of voiding urohydropulsion is to induce forceful voiding of urine by manually compressing the bladder to facilitate removal of cystic uroliths in female dogs. caution: do not perform this procedure until it can be confirmed by catheterization or cystoscopy that the urethra is patent.with the bladder filled with urine or saline (via catheterization), lift the patient (preferably sedated or anesthetized, although this procedure can be done in the awake patient) into a position such that the tail and perineum are ventral and the head is upright. the spine should be approximately perpendicular to the working surface. using one or both hands, gradually increase pressure on the bladder to induce and maintain a forceful stream of urine. objectively, small uroliths will be extruded. if the procedure is only partially successful, it can be repeated as necessary. obviously, voiding urohydropulsion has limitations and cannot be used in male dogs or in dogs with urethral obstructions or strictures. this procedure is indicated for male dogs and cats with partial or complete urethral obstruction caused by uroliths or accumulations of "sand. " this procedure should be performed in the anesthetized patient.note: there are discrepancies in the literature regarding whether to empty the urinary bladder of urine before performing this procedure. because patients with urethral obstructions may have a significant volume of urine in the bladder at the time of presentation, some authors recommend performing cystocentesis to relieve the internal pressure before attempting urohydropulsion. however, in patients that have had a profoundly distended bladder for several hours (even days), penetrating the urinary bladder with a needle presents significant risk of rupturing a fragile bladder. the next step, of course, is abdominal surgery. i recommend avoiding cystocentesis whenever possible. the volume of saline required to flush uroliths into the bladder is inconsequential considering the total volume already present.with the patient positioned in lateral recumbency, retract the prepuce and expose the penis as for conventional bladder catheterization technique. use sterile technique to pass an appropriately sized flexible catheter, which is advanced to the point of obstruction. attach a catheter-tipped 60-ml syringe filled with warmed (my preference) sterile saline and a water-soluble lubricant mixture (approximately two parts saline to one part lubricant) to the urinary catheter. an assistant places a gloved (always preferred) finger into the rectum to identify and occlude the lumen of the pelvic urethra at the level of the pubis. subsequently, infuse saline forcefully into the catheter to dilate the urethra proximal to the obstructing urolith. at that point, release the digital pressure on the proximal urethra while the solution continues to be infused through the catheter. objectively, the pressure within the urethra forces small stones retrograde into the urinary bladder, thereby relieving the obstruction. the objective of this procedure is not to push the calculi into the bladder with the catheter, because this can substantially injure the urethral mucosa, nor is it to force the calculi around the catheter and move it antegrade. key: cord-017248-a37t31u1 authors: nan title: alphabetic listing of diseases and conditions date: 2010-05-17 journal: handbook of autopsy practice doi: 10.1007/978-1-59745-127-7_17 sha: doc_id: 17248 cord_uid: a37t31u1 part ii begins with a list of special histologic stains, their for use and their corresponding references. at the end of this list is a procedure for removal of formalin precipitate from tissue sections. diseases. there may also be a list of possible associated conditions. these entities are generally linked pathogenetically to the main disease entry. any asterisk after a related disease indicates that that disorder is also listed as a disease entry. many disease entries will be followed by a three-column table that provides the reader with a listing of the pathologic findings to be expected with the disease as well as the prosection and dissection procedures necessary to demonstrate those findings. it is expected that routine hematoxylin-eosin stains will be done on all sections submitted for histologic examination. special stains will be recommended in the procedures column of the tables, when indicated. any table immediately following the two columns of disease entries always refers to the disease in the right column. prepare smears of undiluted blood. obtain blood for molecular studies for preservation of small intestinal mucosa and for preparation for study under dissecting microscope, see part i, chapter 2. submit sample for histologic study. submit stool for chemical analysis. record weight and submit sample for histologic study. freeze liver for molecular studies record appearance of spine (see also chest roentgenogram). for removal and specimen preparation, see chapter 4. request luxol fast blue stain. for removal and specimen preparation, see chapter 5. below-normal weight in infants. kyphoscoliosis. very low concentrations of cholesterol and decreased triglycerides; serum~-lipoprotein or absent; a.-lipoproteins present. acanthocytosis (spiny red cells). gene mutations (4) . abnormal shape of villi; vacuolation of epithelial cells. fatty stools fatty changes. gene mutations (4) systemic manifestations of malabsorption syndrome* and of vitamin a deficiency. * kyphoscoliosis. axonal degeneration of the spinocerebellar tracts; demyelination of the fasciculus cuneatus and gracilis (2) . possible involvement of posterior columns, pyramidal tracts, and peripheral nerves. atypical retinitis pigmentosa (2) with involvement of macula. angioid streaks (3) . synonym: cerebral abscess. note: for microbiologic study of tissues and abscesses, see part i, chapter 7. include samples for anaerobic culture. it is best to study the brain after fixation but if specimen is examined fresh, aspirate and prepare smears of abscess content. photograph surface and coronal slices of brain. request giemsa stain, gram stain, pas stain, and grocott's methenamine silver stain for fungi. external examination if there is evidence of trauma, see also under "injury, head." prepare roentgenograms of chest and skull. submit for microbiologic study. for removal and specimen preparation, see chapter 4. for microbiologic study, photography, and special stains, see under "note." for exposure of venous sinuses, see chapter 4. sample walls of sinuses for histologic study. for exposure of paranasal sinuses, mastoid cells, and middle ears, see chapter 4. for removal and specimen preparation, see chapter 5. procedures depend on suspected lesions as listed in right-hand column. skin infections in upper half of face. edema of forehead, eyelids, and base of nose, proptosis, and chemosis indicate cerebral venous sinus thrombosis. * trauma; craniotomy wounds. skull fracture and other traumatic lesions. for possible intrathoracic lesions, see below under "other organs." the national transportation safety board (ntsb)* has authority over aircraft wreckage and the legal authority to investigate and to determine the cause of air crashes. (1) the dead are the responsibility of the medical examiner or coroner. local police will seal off the area of the crash. other than for the purpose of determining that death has occurred, no one should be allowed to approach the bodies or any objects until the identification teams and the medical examiner or coroner have taken charge. the sudden influx of bodies after a commercial air carrier accident and the request for speedy identification of the victims would overburden almost any institution. managing such a disaster is eased by writing a contingency plan beforehand. temporary morgue facilities may have to be established near the scene of the crash. refrigerated trucks may serve as storage space. a practical approach is to deal first with those bodies that seem to be the easiest to identify, in order to narrow the field for the more difficult cases. if bodies are scattered, their locations can be referenced to stakes in the ground or spray paint on pavement; only then should these bodies (or parts) and personal effects be collected. for large-scale crashes a locations can be referenced to a string-line grid benchmarked to gps coordinates. records and diagrams of the relative positions of victims are prepared during this phase. if bodies are still within the airplane, their positions are recorded, and photographed. the personnel of the medical examiner or coroner can augmented by d-mort team staffed by forensic pathologists, anthropologists, dentists, morgue technicians, and investigators supplied by the national disaster medical system. ** the airline will provide a list of the passengers and the federal bureau of investigation (fbi) disaster team will make itself available to take and identify fingerprints and aid in the acquisition of other identifying data such as age, race, weight, height, and hair color and style. if dental records can be obtained, this provides one of the most certain methods of identification. a medical history indicating amputations, internal prostheses, or other characteristic surgical interventions or the presence of nephrolithiasis, gallstones, and the like will be helpful. fingerprints (and footprints of babies) should be taken in all instances. wallets with identification cards,jewelry, name tags in clothing, or other personal belongings may provide the fastest tentative identification. the medical examiner may elect to autopsy only the flight crew but not the passengers of an aircraft crash. however, the grossly identifiable fatal injuries should be described, photographed, and x-rayed. this may reveal identifying body changes. if comparison of somatic radiographs, dental records, fingerprints, or photographs do not identify the victim, dna comparison must be considered. burned or fragmented bodies of passengers and the bodies ofcrew members, and particularly the pilots, must have a complete autopsy, including roentgenographic and toxicologic examinations, which must always include alcohol and carbon monoxide determinations. internal examination might reveal a coronary occlusion, or roentgenograms may disclose a bullet as evidence that violence preceded the crash. in some airplane crashes, particularly in light airplane accidents, suicide must be considered. in such cases police investigation is required to determine if the pilot exhibited suicidal ideation in the recent past.. when resources permit, autopsies should be performed on all deceased occupants of aircraft crashes, including passengers, in order to distinguish among blunt impact trauma, smoke inhalation, and flash fires as causes ofdeath, and to answer future questions concerning pain and suffering, intoxication, and sequence of survivorship. after a crash victim has been identified, the coroner or medical examiner will issue a death certificate. if remains of a decedent cannot be found, a judge can, upon petition, declare a passenger dead and sign a death certificate prepared by a medical examiner. *phone # ofntsb command center: 202-314-6000 **phone # of dmort: 800-872-6367. entry should be followed. usually, the circumstances that led to drowning are not apparent from the autopsy findings but can be reconstructed from reports of witnesses and the police. because the reflex drive to seek air is triggered by hypercarbia, not hypoxia, loss of consciousness and drowning can ensue after hyperventilation and breath-holding by experienced swimmers who then drown without a struggle. there are no specific autopsy findings. a search for trauma, including a posterior neck dissection, should be made in all instances. head and cervical injuries may be responsible for loss of consciousness and drowning, usually in individuals diving into shallow water. toxicologic examination as described below for scuba diving accidents is always indicated. with scuba diving fatalities, investigation of the equipment and circumstances is usually more important than the autopsy. scuba fatalities should be studied by or with the aid of diving experts-for instance, members of a diving club or shop (not the one providing the gear used by the decedent) or the u.s. navy. (1) careful investigation of the scene and study of reports of witnesses and the police are essential. the investigation should ascertain the site of diving (currents and other underwater hazards), the estimated depth, the water temperature (exposure to cold), and a description of water clarity. electrocution should be considered if the site has electric underwater cables (see "injury, electric"). cerebral concussion should be considered if explosives were used in the vicinity. knowledge of the method of recovery of the body and the type of resuscitation efforts can aid in the interpretation of apparent wounds. the medical history of the diving victim should be sought, as it may lead to a diagnosis for which the autopsy is typically silent, such as seizure disorder, or may reveal asthma, emphysema, or chronic bronchitis, all of which increase the risk of air trapping and arterial air embolism. although drowning may be the terminal event in some scuba deaths, the investigation should be focused on the adverse environmental and equipment factors that place a capable swimmer at risk of drowning (see "embolism, air" and "sickness, decompression"). because scuba divers risk arterial air embolism if they ascend with a closed glottis, on can attempt to document gas bubbles at autopsy, but their interpretation is problematic: bodies recovered immediately are subjected to resuscitation efforts, which can by themselves produce extra-alveolar air artifacts, and bodies not recovered immediately tend to be found in a putrefied condition, full of postmortem gas. in the remaining cases, the pathologist must consider the potential of introducing artifactual gas bubbles by the forcible retraction of the chest plate and by sawing the calvarium. the following procedures apply primarily to scuba diving accidents. interrogation of witnesses is important; the behavior and complaints of the decedent, if any, might help distinguish between a natural death by heart disease and an unnatural death by air embolism. external examination eyes and ears head (skull and brain) chest blood (from heart and peripheral vessels) heart tracheobronchial tree and lungs a procedures photograph victim as recovered and after removal of wet suit and other diving gear. record condition of clothing and gear. impound all diving equipment for study by experts, particularly scuba tank, breathing hoses, and regulators. residual air in tank should be analyzed. record color of skin (including face, back, soles, palms, and scalp). palpate skin and record presence or absence of crepitation. record extent and character of wounds. prepare histologic specimens. record appearance of face (including oral and nasal cavities) and of ears. prepare roentgenograms. if air embolism must be expected, as in the presence of pneumomediastinum, follow procedures described under "embolism, air." for evaluation of findings, see also above under "note." if decompression sickness (caisson disease) is suspected, also prepare roentgenograms of the elbows, hips, and knees. otoscopic examination. funduscopic examination. save vitreous for possible toxicologic and other studies. for removal of brain, see chapter 4. record contents of arteries of the circle of willis and its major branches and basilar artery. strip dura from base of skull and from calvarium. for removal and specimen preparation, see chapter 4. for demonstration of pneumothorax, see under "pneumothorax". if gas is visible in coronary arteries, photograph. photograph and aspirate gas in heart chambers. submit samples of heart blood and peripheral blood for toxicologic study and drug screen. examine lungs in situ. save bronchial washings for analysis of debris. fresh dissection is recommended. if decompression sickness is suspected, prepare sudan stains from fresh-frozen lung sections. complete toxicologic sampling should be carried out (see chapter 13). record nature of gastric contents. remove neck organs toward end of autopsy. for posterior neck dissection, see chapter 4. incise tongue. for removal, see chapter 4. for removal, see chapter 4. for removal, prosthetic repair, and specimen preparation, see chapter 2. consult roentgenograms. in decompression sickness, fatty change of liver, and ischemic infarctions of many organs. interstitial emphysema. aspiration (see above). trauma to cervical spine. mottled pallor of tongue after air embolism. contusion of tongue after convulsive chewing. nitrogen bubbles in spinal cord arteries may occur after rapid ascent. air embolism;' cerebral edema in decompression sickness. aseptic necroses (infarcts, "dysbaric osteonecrosis"), most often in head of femur, distal femur, and proximal tibia. infarcts indicate repeated hyperbaric exposures. nitrogen bubbles in and about joints and in periosteal vessels ("bends") occur during rapid ascent. related terms: automobile accident; motorcycle accident. note: a visit to the scene can make the interpretation of the autopsy findings easier. the vehicle can also be inspected in a more leisurely fashion at the impound lot. this is particularly useful for correlating patterned injuries with objects in the vehicle. most vehicular crashes occur as intersection crashes or because a vehicle with excessive speed left a curved road. the medical examiner or coroner should gain a basic understanding of the crash mechanism so that informed descriptions can be rendered, e.g., "impact to the b pillar of the decedent's automobile by the front of a pickup truck which failed to stop for a stop sign at an intersection, resulting in a 2-feet intrusion into the cabin; restraint belts not employed; air bag deployed; extrication required which took 15 minutes." police are responsible for determining mechanical and environmental risk factors for the crash and for determining some human risk factors such as suicidal or homicidal intent. the pathologist determines other risk factors for crashes such as heart disease, a history of epilepsy, and intoxication by carbon monoxide, drugs, and alcohol. suicide as a manner of death should be considered when a single-occupant vehicle strikes a bridge abutment or a large tree head-on, with no evidence of evasive action or braking. in such a situation, the standard police traffic investigation should be supplemented of interviews of the victim's family and friends. the ambulance run sheet is an invaluable source of observations that often are not available from the police. this document should be acquired in all instances, even if the paramedics determined that death occurred and did not transport. the basic autopsy procedures are listed below. most traffic victims who die at the scene or who are dead on arrival at the hospital died from neurogenic shock caused by wounds of the head or vertebral column, or from exsanguination from a tom vessel or heart. as such, they have little lividity, and little blood is found in the vehicles. presence ofintense lividity may indicate suffocation or heart disease as a cause of death. if postural asphyxia is suspected, the first responders to the scene should be interviewed to determine the position of the decedent in the vehicle, and the vital signs, ifany, ofthe decedent from the time of the crash to the time of extrication. posterior neck dissection is indicated in these instances. if manifestations of heart disease, intense lividity, and absence oflethal wounds suggest that a crash occurred because the driver was dead, other drivers on the road may have observed that the victim was slumped at the wheel before the crash. the determination of heart attack at the wheel is usually simple, because most such victims realize that something is wrong, and bring the vehicle to a stop at the side of the road, or coast gently into a fixed object. in such instances, damage to the vehicle is minor, and wounds to the decedent are usually trivial. while pattemed wounds can often be matched to objects (see below), patternless wounds usually cannot be visually matched to specific objects, although an opinion can sometimes be given as to what object was struck, based on the direction of motion and position ofthe body with respect to the vehicle. impacts with the a-pillar produce narrow vertical zones of facial laceration and fractures extending from forehead to jaw. tempered glass shatters into small cubes on impact, and leaves so-called "dicing" wounds, which are abraded cuts arranged in a somewhat rectilinear pattern. windshield glass leaves shallow, abraded, vertically oriented cuts on the face or scalp. with pedestrians, the lower extremities are of particular forensic interest, to determine the height and direction of impact from vehicles that left the scene. scalp hair and blood should be collected from such "hit and run" victims and from occupants of a suspect car if police have a question as to which occupant was the driver; these exemplars can be compared to fibers and tissue recovered from the vehicle in question. likewise, foreign material in wounds can sometimes be matched to suspect vehicles, and should be sought and retained as evidence. for pedestrians, the distance between the impact point on the lower extremities and the soles of the feet should be recorded. the legs should be opened to inspect tibial fractures; cortical fractures initiate propagation opposite to the side of impact, where they usually have a pulled-apart appearance, and then splinter the cortex at the side of impact. abrasions are better impact markers than contusions, because subcutaneous blood extravasation can be caused not only by impact to the skin, but also from blood extravasating from underlying fractures. if no cutaneous abrasions or fractures of the leg bones are found, the skin of the legs should be incised to expose contusions. fracture descriptions should include location in the bone (e.g., proximal metaphysis or shaft), whether the fracture is complete or incomplete, and whether the fracture is displaced or distracted. lacerations of intervertebral disks, facet joint capsules, and ligamenta flava should not be loosely termed "fractures." the presence or absence of blood extravasation in soft tissue adjacent to the fractures should be recorded, and its volume estimated if it appears severe enough. venous air embolism from tom dural sinuses cannot be diagnosed without a pre-autopsy chest radiograph or an in situ bubble test. if an x-ray machine is readily available, an anterior-posterior chest radiograph should be obtained in every traffic victim who dies at the scene or after a failed resuscitation attempt. if a hemothorax is suspected, the rib cuts should be placed further lateral and the chest plate reflected so that the internal mammary vessels can be inspected before the chest plate is removed. after measuring and removing the bloody effusion, the underlying serosal surfaces should be inspected for defects. lacerations of the heart and aorta will be obvious. tamponaded lacerations of the aorta, around which the adventitia still holds, must be noted as such. if no lacerations are found at the usual sites, lacerations of the azygous veins must be considered, especially in association with fracture dislocations of the thoracic vertebral column; other sites are the internal mammary arteries, especially with fractures of ribs i and 2 or of the sternum, and intercostal arteries with displaced rib fractures. only after the serosal defect is identified should the organs be removed, because that procedure creates many more holes in the serosa. for that reason, as much information as possible should be gained by in situ observation. the only evidence of concussion of the heart may be a cardiac contusion or a sternal fracture. the usual clinical history suggests cardiovascular instability that is not associated with craniocerebral trauma and which does not respond to the infusion of intravenous volume agents. the autopsy assistant may saw but should not retract the skull cap and remove the brain. the pathologist should observe in situ whether shallow lacerations of the pontomedullary junction with stretching of the midbrain are present. these lesions cannot be distinguished from artifact by examining the brain later. thus, only after appropriate in situ inspection should the pathologist remove the brain. a posterior neck dissection is required if no lethal craniocerebral or cardiovascular trauma is found, or if suffocation is suspected; neck trauma must be ruled out to diagnose suffocation in a traffic fatality. sudden death in a patient with seemingly trivial wounds may be caused by undiagnosed trauma of the craniocervical articulation. a posterior neck dissection is required in these instances. the diagnosis of diffuse axonal injury of the brain in victims with no appreciable survival interval requires that suffocation be ruled out and that no resuscitation from a cardiac arrest has been attempted. clinicians are quick to apply the label "closed head injury" when a victim of a traffic crash has cerebral edema on a computerized axial tomogram of the head, even if no cerebral contusions, scalp contusions, or skull fractures are evident. this may be a misinterpretation, because cerebral edema can be caused by hypoxic encephalopathy made evident after resuscitation from a cardiac arrest, or from hypoxia caused by suffocation. procedures possible or expected findings record presence of lividity. photograph all external wounds; measure all lacerations and any abrasions or contusions with a pattern. collect scalp hair and blood (see below) from victims of hit and run accidents. collect foreign material in wounds. intense lividity and absence of lethal wounds may indicate that the crash occurred because the driver was dead from heart disease or suffocation. wound documentation. patterned injuries often sometimes be matched to objects in or about the vehicle (the most common patterned wound is that from tempered glass; see above under "note"). impact patterns in pedestrians may help to reconstruct the accident. hair and blood of the victim may be matched to transfer evidence on a vehicle suspected of having left the scene. part ii / diseases and conditions internal examination of body cavities heart and great vessels abdomen skull and brain; neck soft tissue compartments at any location prepare roentgenograms of chest is cases with head impact and skull fractures. collect samples for toxicologic study from all victims, including passengers. create pleural window to detect pneumothorax. if blood is seen, examine internal mammary vessels (see under "note"). measure volume of blood in cavity bleeds, and note whether chambers of heart and great vessels are collapsed or filled. record evidence of cardiac contusion, sprain of intracardiac inferior vena cava, laceration of pericardial sac, and fracture of sternum. laceration of heart or great vessels (measure volume of blood). follow routine procedures for dissection of heart and great vessels (see chapter 3) . in situ bubble test to confirm venous air embolism. record evidence of trauma and volume of blood in peritoneal cavity; estimated volume of blood in retroperitoneal soft tissues. autopsy assistant may saw the skull but pathologist should inspect brain in situ and remove it personally. for removal and specimen preparation of brain, see chapter 4. record brain weight. posterior neck dissection is indicated if there is no craniocerebral or cardio-vascular trauma, or if suffocation is suspected. record evidence of trauma and estimate volume of blood. venous air embolism.' evidence of alcohol or drug intoxication. pneumothorax, hemothorax, e.g., after laceration of internal mammary vessels. evidence of significant hemorrhage. indirect evidence of cardiac concussion. evidence of exsanguinating wounds. evidence of cardiovascular disease that may have felled the driver before the crash. in european countries, the concentration is expressed in promille (grams per liter). in the united states, it has become customary to refer to concentration by percentage (grams per deciliter), and values in these units have been written into legislation and included in the uniform vehicle codes. unless qualified, the use of promille or percentage does not indicate whether the result of the analysis is weight/weight, weight/ volume, orvolume/volume. another common way ofexpressing concentration, milligrams per deciliter, has also been used to indicate alcohol concentrations. the method ofexpressing concentration must be clearly specified whenever the alcohol level is mentioned. the desired expression canbe derived from the toxicologic report by using the following equation: i,000~g/ml =100mg/dl =0.10g/dl =21.74 mmolll =1.0 promille =0.10% what is the legal interpretation of alcohol (ethanol) intoxication? objective impairment of driving ability is observed at threshold blood alcohol concentrations of .035-.040 g/dl. as of august 2005 all states and the district of columbia have adopted laws that make it criminal offense for a driver to operate a motor vehicle with a blood alcohol concentration of 0.08 g/ dl or greater. many states have an enhanced penalty for high concentrations such as 0.15 g/dl or above. several states have zero tolerance laws, under which drivers who are minors are legally operating only if their blood alcohol concentration is 0.02 g/dl or less, and in some states, not detectable at all. blood alcohol concentrations obtained at autopsy are valid until putrefaction begins. specimen tubes with sodium fluoride should be used, and the specimen should be stored in the refrigerator. if the air space above the blood samples in the container is large, alcohol can evaporate and a falsely low blood alcohol level can result. putrefactive changes before autopsy or during storage may cause a falsely high blood alcohol concentration. ethanol can be produced in the specimen container; this is more likely in the absence of a preservative. because fluoride inhibits bacteria far more than fungi, higher fluoride concentrations are required for the inhibition of fungal growth (4) . although there is no major difference in the alcohol concentrations ofblood samples from the intact heart chambers and the femoral vessels (5), autopsy samples from pooled blood in the pericardial sac or pleural cavity are unsatisfactory. we therefore recommend that blood be withdrawn from peripheral vessels. is there normal "endogenous" blood alcohol (ethanol) in a living person? blood alcohol concentrations are generally believed to be negligible in the absence of ingested alcohol. "endogenous" ethanol in human blood exists at a concentration of about 0.0002 g/dl, which is below the limit of detection for most methods (6) . first in such a list would be postural asphyxia, for example, in drunks who fall asleep face down. also, depressant drugs in the tricyclic, analgesic, barbiturate, and benzodiazepine classes all potentiate the effect of alcohol (7) . also included in such a list would be infancy and childhood; ischemic heart disease;' chronic bronchitis and emphysema;' other chronic debilitating diseases; poisoning with carbon tetrachloride' or carbon monoxide;' and other causes of hypoxia.' how can one estimate blood alcohol (ethanol) concentrations from vitreous, urine, or tissue alcohol levels and from alcohol in stomach contents? the ratio of serum, plasma, urine, vitreous, and various tissues has been compiled by garriot (8) . the values may vary considerably. for vitreous, the ratios varied from 0.46-1.40. these variations may depend on whether blood alcohol concentrations were increasing or decreasing at the time of death. most other body fluids and tissues showed ranges closer to 1. most urine values were above the blood alcohol concentrations. in another study (9) , the blood/vitreous (bn) ratio in the early absorption phase was 1.29 (range, 0.71-3.71; sd 0.57) and in the late absorption and elimination phase, the bn ratio was 0.89 (range, 0.32-1.28; sd 0.19). blood ethanol concentrations probably can be estimated using b =1.29v for early absorption and b = 0.89v for later phases. a urinelblood ethanol ratio of 1.20 or less indicates that the deceased was in the early absorption phase. how can one use alcohol (ethanol) concentrations in postmortem specimens to estimate the blood alcohol concentration at various times before death? with certain limitations, one can base calculations of this kind on the assumption that the blood alcohol level decreases from its peak at a fairly constant rate of 0.015-q.018g/dl/h until death (10) . if blood is not available, conversion factors (see above) must be used. alcoholics have been reported to metabolize at a rate of up to 0.043 g/dl/h (6) . example: the driver of an automobile drinks at a party until midnight. he leaves his host at about 1:30 a.m. and is involved in a head-on collision at 2:15 a.m. he dies in the emergency room at 6:35 a.m. there are multiple injuries and the patient exsanguinates. the autopsy is done at 1:30 p.m. although this appears quite unlikely, let us assume that no satisfactory blood sample was obtained before death and that no blood or plasma expanders were given. if under such circumstances the alcohol concentration in the vitreous was found to be 0.157 g/dl, what was the alcohol concentration in the blood at the time of the accident? vitreous and blood alcohol concentrations may be assumed to have remained unchanged after death. therefore, the blood alcohol level at the time of death must have been approx 0.157 (vitreous humor alcohol) x 0.89 (conversion factor, see above) = 0.14g/dl. the time interval between the accident (2:15 a.m.) and death (6:35 a.m.) is 4 hand 20 min or 4 1/3 h. if we assume that the decedent was not an alcoholic and that the blood alcohol concentration was decreasing from its peak at a constant rate of 0.015 g/dl/h, then the concentration at the time ofthe accident is estimated to have been 0.14 (concentration at time of death) + (4 1/3 x 0.015) = 0.140 + 0.065 = 0.205 g/dl or 0.2%. the blood alcohol concentration at the time of the accident could have been lower if the victim stopped drinking later than 1h or 1 1/2 h before the accident. in the latter case, the peak alcohol level would have occurred after the accident, reflecting the time to absorb the latest drink. the blood alcohol concentration at the time of the accident could have been lower or higher if the time when the patient stopped drinking, the time of the accident, or the time of the death is uncertain. the blood alcohol concentration at the time of the accident could have been higher if the victim was a chronic alcoholic. the elimination rate in such persons may be as high as 0.040 mg/dl, which would change the figures in our example above to 0.140 + (4 1/3 x .040) =0.140 + 0.173 = 0.313 g/d1 or 0.3%. only rough estimates are possible. first, the peak blood alcohol level must be determined or calculated, as described in the previous paragraphs. tables (see below) are available that relate blood alcohol level to the minimal amounts of whiskey, wine, or beer that must have been consumed (10) . however, tables of this type are often based on the minimum amount of alcohol circulating in the body after specific numbers of drinks; such tables do not yield reliable results if used conversely. furthermore, inasmuch as drinking and elimination of alcohol may take place concomitantly, over a longer period the total amount of alcohol consumed may have been much greater than the tables would indicate. it cannot be lower. according to these tables, 6 pints of ordinary beer or 8 fl oz of whiskey would be the minimal amounts needed to produce a blood alcohol level of about 200 mg/dl in a person weighing 140-180 pounds. the total body alcohol can be calculated from the blood alcohol level by using widmark's formula: average concentration of alcohol in entire body = .68 concentration of alcohol in the blood in a person weighing 70 kg, the blood alcohol concentration would be increased 50 mg/dl (0.05%) by the absorption of 1oz of ethanol (20z of 100-proof whiskey). strength of alcohol is measured in "proof'; absolute alcohol is 200 proof. therefore, in the united states, alcohol content as volume percent is half the proof (for example, 100-proof whiskey contains 50% alcohol by volume). the alcohol content of various beverages is shown in the following table. approximate alcohol content in various beverages t toata from glaister, rentoul e. medical jurisprudence and toxicology, 12th ed. e & s livingstone, edinburgh, 1966 with permission. twithin 1 h after consumption of diluted alcohol (approx 15%) on an empty stomach, assuming body weight of 140-180 pounds (63.6-81.7 kg) reproduced from (11) with permission. *one ounce (about 30ml) of whiskey or 120z (about 355ml) of beer. what is the toxicity of alcohol other than ethanol? in general, the toxicity increases as the number of carbon atoms in the alcohol increases. thus, butyl alcohol is two times as toxic as ethyl alcohol: but isopropyl alcohol is only twothirds as toxic as isobutyl alcohol and one-half as toxic as amyl alcohol. primary alcohols are more toxic than the corresponding secondary isomers (10) . anemia, hemolytic synonyms and related terms: acquired hemolytic anemia; extracorpuscular hemolytic anemia; hereditary hemolytic anemia (hereditary elliptocytosis, pyropoikilocytosis, stomatocytosis. spherocytosis); immunohemolytic anemia; intracor-puscular hemolytic anemia; microangiopathic hemolytic anemia; spur cell anemia. possible associated conditions: disseminated intravascular coagulation;* eclampsia;* glucose-6-phosphatase deficiency (g6pd); hemolytic uremic syndrome;* malignant hypertension; lymphoma* and other malignancies; paroxysmal nocturnal hemo-globinuria; sickle cell disease;*thalassemia;* thrombotic thrombocytopenic purpura.* (see also below under "note.") note: hemolysis also may be caused by conditions such as poisoning with chemicals or drugs, heat injury, snake bite,* or infections or may develop as a transfusion reaction* or be secondary to adenocarcinoma, heart valve prostheses (see below), liver disease (see below), renal disease, or congenital erythropoietic porphyria. * procedures prepare skeletal roentgenograms. jaundice; skin ulcers over malleoli. in young patients: thickening of frontal and parietal bones with loss of outer table ("hairon-end" appearance); paravertebral masses caused by extramedullary hematopoiesis; deformities of metacarpals, metatarsals, and phalanges. osteonecrosis* of femoral heads. remove and place in fixative as early as possible in order to minimize autolysis (alternatively, formalin can be injected in situ; see below). samples should include oxyntic corpus and fundus mucosa. record weights. submit tissue samples for histologic study. record weight of thyroid gland. for removal and specimen preparation, see chapter 4. request luxol fast blue stain. for removal and specimen preparation, see chapter 5. if there is a clinical diagnosis of anemia-related amblyopia, follow procedures described under "amblyopia, nutritional." jaundice. manifestations of malnutrition. * stomatitis with cheilosis and perianal ulcerations due to folic acid deficiency. chronic exfoliative skin disorders. vitiligo. macrocytosis; poikilocytosis; macroovalocytes; hypersegmentation of leukocytes; abnormal platelets. atrophic glossitis with ulcers. pharyngoesophagitis (folic acid deficiency). previous total or subtotal gastrectomy. carcinoma of stomach. autoimmune gastritis (diffuse corporal atrophic gastritis) with intestinal metaplasia. crohn's disease;* sprue;* other chronic inflammatory disorders; jejunal diverticula; intestinal malignancies; fish tapeworm infestation; previous intestinal resection or blind intestinal loop; enteric fistulas. hepatosplenomegaly. alcoholic liver disease. * giant epithelial cells. hyperthyroid goiter; thyroiditis. demyelination of cerebral white matter (in advanced cases). demyelination in posterior and lateral columns of spinal cord, most frequently in thoracic and cervical segments. demyelination of peripheral nerves. retinal hemorrhages; demyelination of optic nerves. hypercellular; megaloblastic. myeloproliferative disorder. brain other organs if mycotic aneurysms are expected and microbiologic studies are intended, follow procedures described below under "aneurysm, mycotic aortic." request verhoeff-van gieson, gram, and grocott's methenamine silver stains. for cerebral arteriography, see chapter 4. if arteriography cannot be carried out, rinse fresh blood gently from base of brain until aneurysm can be identified. record site of rupture and estimated amount of extravascular blood. for paraffin embedding of aneurysms, careful positioning is required. expected findings depend on type of aneurysm. mycotic aneurysms are often multiple and deep in brain substance. berry aneurysms are the most frequent types and often are multiple. most frequent sites are the bifurcations and trifurcations of the circle of willis. saccular atherosclerotic aneurysms are more common than dissecting aneurysms, which are very rare. with congenital cerebral artery aneurysm: coarctation of aorta;* manifestations of hypertension;* and polycystic renal disease. with mycotic aneurysm: infective endocarditis;* pulmonary suppurative processes; and pyemia. aneurysm, dissecting aortic (see "dissection, aortic.") aneurysm, membranous septum of heart note: for general dissection techniques, see chapter 3. most aneurysms ofthe membranous septum probably repre-sent spontaneous closure of a membranous ventricular septal defect by the septalleafiet of the tricuspid valve. aneurysm, mycotic aortic note: (i) collect all tissues that appear to be infected. (2) request aerobic, anaerobic, and fungal cultures. (3) request gram and grocott methenamine silver stains. (4) no special precautions are indicated. (5) no serologic studies are available. (6) this is not a reportable disease. chest and abdominal organs aorta other organs submit blood samples for bacterial culture. en masse removal of adjacent organs is recommended. photograph all grossly identifiable lesions. aspirate material from aneurysm or para-aortic abscess and submit for culture. prepare sections and smears of wall of aneurysm and of aorta distant from aneurysm. request verhoeffvan gieson and gram stains. septicemia and infective endocarditis. * streptococcus, staphylococcus, spirochetes, and salmonella can be found in mycotic aneurysm. para-aortic abscess. septic emboli with infarction or abscess formation. aneurysm, syphilitic aortic part ii / diseases and conditions heart and aorta other organs en masse removal of organs is recommended. for coronary arteriography, see chapter 10. request verhoeff-van gieson stain from sections at different levels of aorta, adjacent great vessels, and coronary arteries. see also under "syphilis." aneurysm usually in ascending aorta. may erode adjacent bone (sternum). syphilitic aortitis may cause intimal wrinkling, narrowing of coronary ostia, and shortening of aortic cusps. disruption of medial elastic fibrils. aortic valvulitis and insufficiency;* syphilitic coronary arteritis; syphilitic myocarditis. external examination aorta prepare chest and abdominal roentgenograms. open aorta along line of blood flow, or bisect into anterior and posterior halves. photograph tear(s). measure bloody effusions in body cavities. measure or estimate amount of blood in mediastinum. request verhoeff-van gieson stain. cutaneous impact trauma. mediastinum widened by hemorrhage in case of tarnponaded dissection. a bleed into a body cavity of less-thanexsanguinating volume should point to an alternate mechanism of death such as neurogenic shock or lethal concussion; a posterior neck dissection may be required in such instances. microscopy may show transmural rupture, false aneurysm, or localized dissection. angiitis (see "arteritis, all types or type unspecified.") angina pectoris note: see under "disease, ischemic heart" and chapter 3. angiokeratoma corporis dittusum (see "disease, fabry's.") angiomatosis, encephalotrigeminal (see "disease, sturge-weber-dimitri.") angiopathy, congophilic cerebral synonyms and related terms: beta amyloid angiopathy due to~-amyloid peptide deposition (~a4) (associated with alzheimer's disease; hereditary cerebral hemorrhage with amyloid angiopathy of dutch type; or sporadic beta amyloid angiopathy); hereditary cerebral amyloid angiopathy, due to deposition of other amyloidogenic proteins such as cystatin c (icelandic type) and others (e.g., transthyretin, gelsolin) (1). procedures possible or expected findings request stains for amyloid, particularly congo red, and thioflavine s (examine with polarized and ultraviolet light, respectively). request immunostain for~a4. some tissue should be kept frozen for biochemical studies. multiple recent cerebral cortical infarctions or small cortical hemorrhages, or both, or massive hemispheric hemorrhages, both recent and old. amyloid deposition in leptomeninges and cortical blood vessels. senile plaques are usually present. in some cases, angiopathy is part of alzheimer's disease. * other organs a prepare material for electron microscopy. electron microscopic study permits definite confirmation of diagnosis. organs and tissues may be minimally affected by amyloidosis. anomaly, coronary artery possible associated conditions: with double outlet right ventricle; persistent truncal artery; tetralogy of fallot;* and transposition of the great arteries.* note: coronary artery between aorta and pulmonary artery, often with flap-valve angulated coronary ostium. coronary artery may communicate with cardiac chamber, coronary sinus, or other cardiac veins, or with mediastinal vessel through pericardial vessel. saccular aneurysm of coronary artery with abnor-mal flow, infective endarteritis of arteriovenous fistula, and myocardial infarction may be present. ifone or both coronary arteries originate from pulmonary trunk, myocardial infarction may be present. heart perform coronary angiography. if infective endarteritis is suspected, submit blood sample for microbiologic study. ectopic origin of coronary arteries or single coronary artery. sudden death. for a detailed description of possible additional findings, see above under "note." anomaly, ebstein's (see "malformation, ebstein's") anorexia nervosa note: sudden death from tachyarrhythmias may occur in advanced cases and thus, autopsy findings may not reveal the immediate cause of death. external examination all organs record height and weight, and prepare photographs to show cachectic features. record abnormalities as listed in righthand column. follow procedures described under "starvation." record weight of endocrine organs and submit samples for histologic study. cachexia, often with preserved breast tissue; hirsutism; dry, scaly, and yellow skin (carotenemia). mild edema may be present. parotid glands may be enlarged. manifestations of starvation.* ovaries tend to be atrophic; other endocrine organs should not show abnormalities. synonyms: cutaneous anthrax; gastrointestinal anthrax; pulmonary (inhalational) anthrax. note: (1) collect all tissues that appear to be infected. this is a reportable disease. bioterrorism must be considered in current cases. external examination and skin blood photograph cutaneous papules, vesicles, and pustules. prepare smears and histologic sections. submit samples for bacteriologic study. submit sample for serologic study. disseminated anthrax infection may occur without skin lesions. edema of neck and anterior chest in nasopharyngeal anthrax. anthrax septicemia. see above under "note." part ii i diseases and conditions lungs gastrointestinal tracts and mesentery neck organs record character and volume of effusions. after sampling for bacteriologic study (see above under "note") perfuse one or both lungs with formalin. extensive sampling for histologic study is indicated. extensive sampling for histologic study is indicated. photograph meningeal hemorrhage in situ. pleural effusions;* hemorrhagic mediastinitis; anthrax pneumonia (inhalational anthrax; woolsorter's disease). histologic sections reveal hemorrhagic necrosis, often with minimal inflammation and gram-positive, spore-forming, encapsulated bacilli. gastrointestinal anthrax with mucosal edema and ulcerations. hemorrhagic mesenteric lymphadenitis. tongue, nasopharynx, and tonsils may be involved. hemorrhagic meningitis (hemorrhage tends to predominate). external examination distal colon and rectum photograph perineum. measure depth of anal pit, if any. dissect distal colon, rectum, and perirectal pelvic organs in situ (as much as possible). search for opening of fistulous tracts from lumen. use roentgenologic study or dissection, or both, to determine course of tract. absence of normally located anus; anal dimple. abnormal termination of the bowel into the trigone of the urinary bladder, the urethra distal to the verumontanum, the posterior wall of the vagina, the vulva, or the perineum. aortitis note: see also under "arteritis" and "aneurysm, ascending aortic." heart and aorta other organs and tissues remove heart with whole length of aorta and adjacent major arteries. record width and circumference of aorta at different levels. describe and photograph appearance of intima and of orifices of coronary arteries and other aortic branches. submit multiple samples for histologic study and request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. secondary aortic atherosclerosis or intimal fibroplasia. widening of aorta; syphilitic aneurysm. * giant cell aortitis; rheumatoid aortitis; syphilitic aortitis; takayasu's arteritis.* manifestations of rheumatoid arthritis, * syphilis,* systemic sclerosis,* hodgkin's lymphoma, and many other diseases associated with vasculitis. external examination brain spine and spinal cord other organs prepare roentgenogram of spine. for removal and specimen preparation, see chapter 4. for removal of spinal cord and specimen preparation, see chapter 4. expose nerve roots. record appearance and photograph spinal cord in situ. submit samples of spinal cord and inflamed tissue for histologic study. request gram, gomori's iron, and grocott's methenamine silver stains. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. signs of previous spinal surgery or lumbar puncture (myelography). evidence of previous trauma or previous myelography. cerebral arachnoiditis. fibrous arachnoidal adhesions and loculated cysts. tuberculosis;* syphilis;* fungal or parasitic infection. systemic infection (see above). ascending urinary infection or other manifestations of paraplegia. arch, aortic, interrupted synonym: severe coarctation. note: the basic anomaly is a discrete imperforate region in the aortic arch, with a patent ductal artery joining the descending thoracic aorta. type a interruption is between the left subclavian and ductal arteries; type b between the left subclavian and left common carotid arteries; and type c (rare) between the left common carotid and brachiocephalic (innominate) arteries. for general dissection techniques, see part i, chapter 3. possible associated conditions: bicuspid aortic valve (with type a); di george syndrome* with thymic and parathyroid aplasia (with type b); hypoplasia of ascending aorta (with all types); persistent truncal artery (truncus arteriosus); ventricular septal defect. arrhythmia, cardiac note: see also under "death, sudden cardiac." toxicologic studies may be indicated, for instance, if digitalis toxicity (see "poisoning, digitalis") is suspected. if a cardiac pacemaker had been implanted, the instrument should be tested for malfunction. arteriosclerosis (see "atherosclerosis.") arteritis, all types or type unspecified synonyms and related terms: allergic angiitis and granulomatosis (churg-strauss);* allergic vasculitis; anaphylactoid purpura* and its synonyms; angiitis; buerger's disease;* cranial arteritis; giant cell arteritis;* granulomatous arteritis (angiitis); hypersensitivity angiitis; infectious angiitis; necrotizing arteritis; polyarteritis nodosa;* rheumatic arteritis; rheumatoid arteritis, syphilitic arteritis; takayasu's arteritis;* temporal arteritis; thromboangiitis obliterans; and others (see also below under "note"). note: autopsy procedures depend on (1) the expected type of arteritis, such as giant cell arteritis,* polyarteritis nodosa,* or thromboangiitis obliterans (buerger's disease*); and (2) the nature of suspected associated or underlying disease, such as aortic arch syndrome,* beh~et's syndrome,* cogan's syndrome, degos' disease,* dermatomyositis,* erythema nodosum and multiforme,* goodpasture's syndrome,* polymyositis, rheumatic fever, * rheumatoid arthritis,* syphilis,* and other nonspecific infectious diseases, systemic lupus erythematosus,* systemic sclerosis (scleroderma),* or takayasu's disease. for histologic study of blood vessels, verhoeff-van gieson stain or a similar stain is recommended. temporal and ophthalmic arteritis. arteritis of ciliary and retinal vessels. clinically, polymyalgia. anemia. arteritis, takayasu's synonyms: aortic arch syndrome; pulseless disease. external examination heart, aorta, and adjacent great vessels kidney eyes and optic nerve brain for in situ aortography, clamp distal descending thoracic aorta and neck vessels as distal as possible from takeoff at aortic arch. remove heart together with aorta and long sleeves of neck vessels. for coronary arteriography, see chapter 10 (method designed to show coronary ostia). test competence of aortic valve. open aortic arch anteriorly and measure (with calipers) lumen at origin of great neck vessels. photograph aorta and neck vessels and submit samples for histologic study. request verhoeffvan gieson stain. submit tissue for histologic examination. for removal and specimen preparation, see chapter 5. for removal and specimen preparation, see chapter 4. facial muscular atrophy and pigmentation. narrowing at origin of brachiocephalic arteries. dilated ascending aorta. narrowing of coronary arteries at origins. myocardial infarction. aortic insufficiency. * aortic atherosclerosis. thromboses of brachiocephalic arteries. giant cell arteritis. * diffuse mesangial proliferative glomeulonephritis (1) . atrophy of optic nerve, retina, and iris; cataracts; retinal pigmentation. ischemic lesions. artery, patent ductal synonym: patent ductus arteriosus. note: the basic anomaly is persistent postnatal patency of the ductal artery, usually as an isolated finding (in 75% of cases in infants, and in 95% in adults). it is more common in premature than full-term infants and at high altitudes than at sea level. possible complications in unoperated cases include congestive heart failure, * plexogenic pulmonary hypertension,* ductal artery aneurysm or rupture, fatal pulmonary embolism,* or sudden death. in some conditions, such as aortic atresia* or transposition with an intact ventricular septum,* ductal patency may be necessary for survival. possible associated conditions: atrial or ventricular septal defect;* coarctation ofthe aorta;* conotruncal anomalies; necrotizing enterocolitis in premature infants; postrubella syndrome; and valvular or vascular obstructions. artery, persistent truncal synonym and related terms: type i, pulmonary arteries arise from single pulmonary trunk (in 55%); type 2, pulmonary arteries arise separately but close-by (in 35%); type 3, pulmonary arteries arise separately but distal from one another (in 10%). note: the basic anomaly is a common truncal artery, with truncal valve, giving rise to aorta, pulmonary arteries, and coronary arteries, usually with a ventricular septal defect. interventions include complete rastelli-type repair, with closure of ventricular septal defect, and insertion of valved extracardiac conduit between right ventricle and detached pulmonary arteries. possible associated conditions: absent pulmonary artery (in 15%); atrial septal defect (in 15%); absent ductal artery (in 50%); coronary ostial anomalies (in 40%); di george syndrome;* double aortic arch; extracardiac anomalies (in 25%); interrupted aortic arch* (in 15%); right aortic arch (in 30%); truncal valve insufficiency (uncommon) or stenosis (rare); trun-cal valve with three (in 70%), four (in 20%), or two (in 10%) cusps. heart and great vessels if infective endocarditis is suspected, follow culture procedures for endocardial vegetation described in chapter 10. request verhoeff-van gieson stain. infective endocarditis,* usually of truncal valve. late postoperative conduit obstruction. postoperative late progressive truncal artery dilation with truncal valve insufficiency. hypertensive pulmonary vascular disease. cerebral abscess,* if right-to-ieft-shunt was present. arthritis, all types or type unspecified note: for extra-articular changes, see under the name of the suspected underlying conditions. infectious diseases that may be associated with arthritis include bacillary dysentery, * brucellosis, * gonorrhea, rubella,* syphilis, * tuberculosis, * typhoid fever, * and varicella. * noninfectious diseases in this category include acromegaly,* beh<;et's syndrome,* felty's syndrome,* gout,* rheumatoid arthritis,* and many others, too numerous to mention. remove synovial fluid and prepare smears. submit synovial fluid for microbiologic and chemical study. for removal of joints, prosthetic repair, and specimen preparation, see chapter 2. for removal and specimen preparation, see chapter 5. in the polyarticular variant, facial asymmetry may be noted. rheumatoid factor positive in some cases. pericarditis.* interstitial pneumonitis; pleuritis. (see also under "arthritis, rheumatoid.") lymphadenopathy. splenomegaly. monarthritis or severe, erosive polyarthritis; see also under "arthritis, rheumatoid" and above under "externalexamination and skin." ankylosing spondylitis* may be present. chronic iridocyclitis. see "arthritis, rheumatoid." arthritis, rheumatoid synonyms and related terms: ankylosing spondylitis;* felty's syndrome;* juvenile rheumatoid arthritis* (still's disease); rheumatoid disease; and others. possible associated conditions: amyloidosis;* polymyositis (dermatomyositis*); psoriasis;* sjogren's syndrome;* systemic lupus erythematosus;* systemic vasculitis, and others. subcutaneous rheumatoid nodules on elbows, back, areas overlying ischial and femoral tuberosities, heads of phalangeal and metacarpal bones, and occiput. deformities and subluxation of peripheral joints (see also below under "joints"). subaxial dislocation of cervical spine may be cause of sudden death. pneumothorax;* pleural empyema.* t-cell abnormalities (1) . bacteremia. positive rheumatoid factor. rheumatoid granulomas in myocardium (septum), pericardium, and at base of aortic and mitral valves; constrictive pericarditis;* aortic stenosis;* coronary arteritis. systemic vasculitis (arteritis*). rheumatoid granulomas in pleura and lung (with pneumoconiosis*); bronchopleural fistula; rheumatoid pneumonia with interstitial pulmonary fibrosis and honeycombing; bronchiectasis;* bronchiolitis with cystic changes; pulmonary arteritis. pneumoconiosis* in caplan arthrogryposis (2) may be a primary muscle disease, or it may involve abnormalities of the brain, spinal cord, and/or peripheral nerves. etiologies are numerous, as are the modes of inheritance. critical to making the appropriate diagnosis is the collection of muscles from various sites for routine histology, muscle histochemistry, and electron microscopy. portions of peripheral motor nerves must also be prepared for histology and electron microscopy. abdominal cavity intra-abdominal lymphatic system puncture abdominal cavity and submit fluid for microbiologic study. record volume of exudate or transudate and submit sample for determination of fat and cholesterol content. prior to routine dissection, lymphangiography (see below) may be indicated. possible associated conditions: with pulmonary aspergillosis-bronchiectasis; * bronchocentric granulomatosis;* sarcoidosis;* tuberculosis. * with systemic aspergillosisleukemia;* lymphoma;* and other conditions complicated by immunosuppression (l, 2) . other organs a carefully make multiple parasagittal sections through the unperfused lungs. culture areas of consolidation. if diagnosis was confirmed, perfuse lungs with formalin. prepare histologic sections from walls of cavities, cavity contents, and pneumonic infiltrates. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. assault note: all procedures described under "homicide" must be followed. asthma note: spray death* may occur in asthma sufferers from pressurized aerosol bronchodilators. record thickness and position. perfuse one lung with formalin. because mucous plugs may block bronchial tree, attach perfusion apparatus to pulmonary artery or to bronchus and pulmonary artery. monitor perfusion to ensure proper inflation. prepare photograph of fixed cut section. submit samples of pulmonary parenchyma and bronchi for histologic study. request azure-eosin and verhoeff-van gieson stains. record weight and thickness of walls. leave attached to stomach. photograph and submit samples for histologic study. eczema. conjunctival hemorrhages and subcutaneous emphysema may be present after fatal attack. pneumothorax;* mediastinal emphysema. low diaphragm (see below). increased igeconcentrations in fatal asthma; postmortem tryptase determination is of doubtful value in this regard (1) . hypertrophy. low position of diaphragm. hyperinflated lungs. thick-walled bronchi with prominent viscid mucous plugs. typical microscopic inflammatory changes (2) . asthmatic bronchitis with eosinophilic infiltrates. bronchocentric granulomatosis.* pulmonary atherosclerosis with breakup of elastic fibers. paucity of ecosinophils in mucous (6) . cor pulmonale. refl ux esophagitis (3) . peptic ulcer. * pneumatosis of small intestine; emphysema of colon. centrilobular congestion and necrosis. petechial hemorrhages in hypothalamus; necrosis of cerebellar folia; anoxic changes in cortex, globus pallidus, thalamus, sommer's sector of hippocampus, and purkinje cells of cerebellum. suspected changes in anterior hom cells of spinal cord in patients with asthma-associated poliomyelitis-like illness (hopkins syndrome) (4). allergic polyps and other allergic inflammatory changes (5) . increased erythropoiesis. atresia, aortic valvular synonym: aortic atresia; aortic atresia with intact ventricular septum; hypoplastic left heart syndrome. note: the basic anomaly is an imperforate aortic valve, with secondary hypoplasia ofleft-sided chambers and ascending aorta. for possible surgical interventions, see two-stage norwood and modified fontan procedures in chapter 3. possible associated conditions: atrial septal defect* (or patent foramen ovale, usually restrictive); dilatation of myocardial sinusoids thatcommunicate with coronary vessels; dilatation of right atrium, right ventricle, and pulmonary trunk; fibroelastosis ofleft atrial and left ventricular endocardium; hypertrophy of ventricular and atrial walls; hypoplastic left atrium, mitral valve, left ventricle, and ascending aorta; mitral atresia* with minute left ventricle; patent ductal artery (ductus arteriosus); small left ventricle with hypertrophic wall; tubular hypoplasia of aortic arch, with or without discrete coarctation. synonyms and related terms: congenital biliary atresia; extrahepatic biliary atresia; infantile obstructive cholangio-pathy; syndromic (alagille's syndrome) or nonsyndromic paucity of intrahepatic bile ducts ("intrahepatic" biliary atresia). possible associated conditions: alpha]-antitrypsin deficiency;* choledochal cyst;* congenital rubella syndrome;* polysplenia syndrome* (1); small bowel atresia; trisomy 17-18; trisomy 21; turner's syndrome;* viral infections (cytomegalovirus infection;* rubella*). dissect extrahepatic bile ducts in situ or leave hepatoduodenalligament intact for later fixation and sectioning (see below). record appearance and contents of gallbladder and course of cystic duct. in postoperative cases, submit sample of anastomosed hepatic hilar tissue for demonstration of microscopic bile ducts. remove liver with hepatoduodenalligament. prepare horizontal sections through ligament and submit for histologic identification of ducts or duct remnants. prepare frontal slices of liver and sample for histologic study. request pas stain with diastase digestion. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. jaundice. congenital rubella and other viral infections. alpha]-antitrypsin deficiency;* defects in bile acid synthesis. chromosomal abnormalities. in atresia of the hepatic duct, the gallbladder will be empty. in isolated atresia of the common bile duct, the gallbladder contains bile but it cannot be squeezed into the duodenum. atresia or hypoplasia of bile duct(s); choledochal cyst(s). biliary drainage created by kasai operation. obliterative cholangiopathy (2) . intrahepatic cholelithiasis; postoperative ascending cholangitis; secondary biliary cirrhosis; giant cell transformation; paucity of intrahepatic bile ducts. pas-positive inclusions in alphal-antitrypsin deficiency.* polysplenia syndrome* (1) with malrotation, situs inversus, preduodenal portal vein, absent inferior vena cava, anomalous hepatic artery supply, and cardiac defects. for other abnormalities outside the biliary tree, see under "possible associated conditions"). nephromegaly (3) . atresia, cardiac valves (see "atresia, aortic valvular," "atresia, mitral valvular," "atresia pulmonary valvular, with intact ventricular septum," "atresia, pulmonary valvular, with ventricular septal defect," and "atresia, tricuspid valvular.") atresia, duodenal possible associated conditions: with membranous obstruction of the duodenum-annular pancreas; atresia of esophagus* with tracheoesophageal fistula; congenital heart disease; cystic fibrosis;* down's syndrome;* hirschsprung's disease; imperforate anus* or other congenital obstructions of the intestinal tract (1); intestinal malrotation; lumbosacral, rib-, and digitllimb anomalies; single umbilical artery; spinal defects; undescended testis (1). see also under "atresia, small intestinal." the basic anomaly is an imperforate pulmonary valve, with a hypoplastic right ventricle. in unoperated cases, ductal closure is the most common cause of death. for possible surgical interventions, see modified blalock-taussig shunt, mod-ified fontan procedure, and pulmonary valvulotomy in chapter 3. for general dissection techniques, see chapter 3. possible associated conditions: dilated myocardial sinusoids that may communicate with epicardial coronary arteries or veins; patent ductal artery (ductus arteriosus); patent oval foramen (foramen orale); tricuspid atresia with minute right ven-tricle; tricuspid stenosis with hypoplastic right ventricle (in 95%); tricuspid insufficiency with dilated right ventricle (in 5%). synonym: tetralogy of fallot with pulmonary atresia. note: the basic anomaly is atresia of the pulmonary valve and ofvariable length ofpulmonary artery, and ventricular septal defect (membranous or outlet type), with overriding aorta, and with pulmonary blood supply from ductal or systemic collateral arteries. for possible surgical interventions, see rastelli-type repair and unifocalization of multiple collateral arteries in chapter 3. possible associated conditions: right ventricular outflow tract a short blind-ended pouch (70%) or absent (30%); atresia of pulmonary artery bifurcation, with nonconfluent pulmonary arteries; right aortic arch (40%); atrial septal defect (50%); persistent left superior vena cava; anomalous pulmonary venous connection; tricuspid stenosis or atresia; complete atrioventricular septal defect; transposed great arteries; double inlet left ventricle; asplenia, polysplenia, or velocardiofacial syndromes; dilated ascending aorta, with aortic insufficiency. related term: jejuno-ileal atresia. possible associated findings: esophageal atresia* with tracheoesophageal fistula; lumbosacral, rib-, or digit/limb anom -alies; undescended testes (l) . note: see also under "atresia, duodena1." fascia lata, blood, or liver these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter 9) . intestinal tract for mesenteric angiography, see chapter 2. leave mesentery attached to small bowel, particularly to the atretic portion. trisomy 21. multiple atresias; proximal dilatation; volvulus; malrotation; meconium impaction; other evidence of cystic fibrosis. anorectal malformation (l) . annular pancreas (1). atresia, tricuspid valvular note: the basic anomaly is an absent right atrioventricular connection (85%) or imperforate tricuspid valve (15%), with a hypoplastic right ventricle (100%), muscular ventricular septal defect (90%) that is restrictive (85%), and a patent oval atresia, urethral foramen (80%) or secundum atrial septal defect (20%). for possible surgical interventions, see modified fontan or glenn procedures in chapter 3. for general dissection techniques, see chapter 3. possible associated conditions: juxtaposed atrial appendages; large left ventricular valvular orifice; large left ventricular chamber; persistent left superior vena cava; pulmonary atresia; transposition of the great arteries (25%), with aortic co-arctation (35% of those); anomalies of musculoskeletal or digestive systems (20%); down's,* asplenia, or other syndromes. heart aorta and cervical arteries brain if infective endocarditis* is suspected, culture using the method described in chapter 7. for dissection of carotid and vertebral arteries, see chapter 4. for removal and specimen preparation, and cerebral anteriography, see chapter 4. if a foreign body is discovered during a medicolegal autopsy or if the discovery of a foreign body may have medicolegal impli-cations (e.g., presence of a surgical instrument in the abdominal cavity), the rules of the chain of custody apply. for the handling of bullets or bullet fragments, see "injury, firearm." if analysis offoreign material is required, commercial laboratories may be helpful. bolus (see "obstruction, acute airway!') burns note: fatal bums should be reported to the medical examiner's or coroner's office. the questions to be answered by the pathologist depend on whether the incident was accidental, sui-cidal, or homicidal, and whether the victim survivied to be treated in the hospital. a pending death certificate should be issued if the fire and police investigators are not sure of the circumstances at the time of the autopsy. for electrical bums, see under "injury, electrical." for victims who were treated at the hospital, autopsy procedures should be directed toward the discovery or confirmation of the mechanism of death, such as sepsis or pulmonary embolism.* death can be caused primarily by heart disease, with other-wise minor bums and smoke inhalation serving as the trigger that leads to lethal ventricular arrhythmia. because carbon monoxide concentrations are halved approx every 30 min with 100% oxygen therapy, the pathologist must obtain the first clinical laboratory test results for co-hemoglobin. soot can be detected with the naked eye 2 or 3 d after inhalation of smoke. ambulance records should be examined to determine whether a persistent coma might have been caused by hypoxic encephalopathy following resuscitation from cardiac arrest at the scene. admission blood samples should be acquired to test for cohemoglobin and alcohol. this may not have been done in the emergency room. persons suffering from chronic alcoholism succumb to fire deaths more often than persons who do not drink. a very high initial serum alcohol concentration suggests a risk factor for the fire and presence of chronic alcoholism. patients with chronic alcoholism typically are deprived of alcohol when they are in the bum unit and this can cause sudden, presumably cardiac, death,just as it occurs under similarcircum-stances, not complicated by bums. under these circumstances, the heart fails to show major abnormalities. this mode of dying seems to have no relationship to the presence or absence of liver disease. if the body is found dead and charred at the scene, prepare whole body roentgenograms, before and after removal of remanants of clothing. see also under "identification of the body" and "external examination" in chapter 13). one or two fingerpads may yield sufficient ridge detail for identification. if this is not possible, ante-and postmortem somatic and dental radiographs must be compared for identification, or dna comparison must be used. external examination, heart and lungs abdominal cavity and liver see below under "cardiomyopathy, dilated." record volume of ascites. record actual and expected weight of liver. request iron stain. see below under "cardiomyopathy, dilated." alcoholic cirrhosis and alcoholic cardiomyopathy rarely coexist. however, in genetic hemochromatosis,* cirrhosis and heart failure are common findings. cardiomyopathy, dilated (idiopathic, familial, and secondary types) note: for general dissection techniques, see chapter 3. external examination heart other organs and tissues record actual and expected weights. record ventricular thicknesses and valvular circumferences. evaluate relative atrial and ventricular chamber sizes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. note: huntington's disease maps to the short arm of chromosome 4. the gene is widely expressed but of unknown function; it contains a cag repeat sequence, which is expanded (range, 37 to 86) in patients with huntington's disease. a sensitive diag-nostic test is based on the determination of this cag sequence, which can be done on fresh-frozen tissue or blood (1) . in the absence of genetic confirmation, sampling of organs and tissues cannot be excessive because a complex differential diagnosis must be resolved. note: disseminated intravascular coagulation (dic) often is a complication of obstetrical mishaps such as abruptio placentae or amniotic fluid embolism,* or it complicates malignancies (such as adenocarcinomas or leukemia*) or bacterial, viral, and other infections. other conditions such as aortic aneurysm* or hemolytic uremic syndrome* are known causes also. ifthe nature of the underlying disease is known, follow the procedures under the appropriate heading also. note: this is a cause of diarrhea. microscopic colitis is associated with older age; collagenous colitis is associated with female sex (1). the colon is grossly normal but microscopically, increased lymphocytes in the lamina propria and a subepithelial band of collagen is found. if only the lymphocytic infiltrate is found, the term "lymphocytic colitis" or "microscopic colitis" should be applied. a trichrome stain should be ordered in all instances, because the collagen band may be difficult to see without the special stain. i death, anaphylactic synonym: generalized anaphylaxis. note: autopsy should be done as soon as possible after death. neck organs should be removed before embalming. if death is believed to be caused by drug anaphylaxis, inquire about type of drug(s), drug dose, and route of administration (intravenous, intramuscular, and oral or other). this will determine proper sampling procedures-for instance, after penicillin anaphylaxis. allergy to bee stings, wasp stings, fire ants, and certain plants may also be responsible for anaphylaxis. however, envenomation also can be fatal in the absence of anaphylaxis. external examination search for injection sites or sting marks. if such lesions are present, photograph and excise with 5-cm margin. freeze excised tissue at -70â°c for possible analysis. prepare chest roentgenogram. foam in front of mouth and nostrils. swelling of involved tissue. antigen-antibody reaction in involved tissues. antibodies against suspected antigen. laryngeal edema may recede soon after death. foamy edema in trachea and bronchi; diffuse or focal pulmonary distention ("acute emphysema") alternating with collapse; pulmonary edema and congestion; accumulation of eosinophilic leukocytes. eosinophilic leukocytes in red pulp. death, anesthesia-associated. note: there are many possible causes of anesthesiaassociated death that are not drug-related, such as acute airway obstruction* by external compression, aspiration, arrhythmia of a heart not previously known to be diseased, tumor, or an inflammatory process. some ofthe complications are characteristically linked to a specific phase of the anesthesia, and many are not revealed by customary morphologic techniques. the task for the pathologist charged with investigating an anesthesia-associated death is to reconstruct the chain of physiologic events culminating in cessation of vital signs. autopsy morphology plays a supporting role; the main investigations center around the record left by the anesthesiologist, testing of anesthesia equipment, and toxicological testing. a consulting anesthesiologist can divine much more information from the anesthesia and recovery room records than can the pathologist, and can suggest avenues of further investigation. therefore, the most important step in these autopsies is to obtain the anesthesiaassociated records and to secure the consulting services of an independent anesthesiologist. the changes in the vital signs during and after anesthesia will help to focus the investigation toward a cardiac mechanism ofdeath or depression ofbrainstem function as a terminal mechanism. when information is gathered about drugs and chemical agents that have been administered or to which the victim may have had access, the pathologist must keep in mind that some non-medical chemicals and many drugs are known to affect anesthesia. drugs and their metabolic products, additives, stabilizers, impurities, and deterioration products (one of which can be carbon monoxide) may be present and can be identified in postmortem tissues. therefore, all appropriate body fluids and solid tissue should be submitted for toxicological examination. if the anesthetic agent was injected into or near the spinal canal, spinal fluid should be withdrawn from above the injected site into a standard toxicologist's collection tube with fluoride preservative. if the anesthetic agent was injected locally, tissue should be excised around the needle puncture marks at a radius of 2-4 em. serial postmortem analysis of specimens may permit extrapolation to tissue concentration at the time of death. the time interval between drug administration and death sometimes can be calculated from the distribution and ratio ofadministered drugs and their metabolic products. for a review of anesthetic death investigation, see ref. (1) . halothane anesthesia and some other anesthetic agents may cause fulminant hepatitis and hepatic failure. the autopsy procedures suggested under "hepatitis, viral" should be followed. note: for special autopsy procedures in postoperative deaths, see chapter 1. in some instances, procedures described under "death, anesthesia-associated" may be indicated. for a review of investigational procedures and autopsy techniques in operating-room-associated deaths, see ref. (1) . if the autopsy will involve anatomy or dissection techniques that are unfamiliar, the pathologist should not hesitate to invite the surgeon to the autopsy. in patients who develop a cerebral infarction after open heart surgery, arterial air embolism should be considered as a possible cause. the diagnosis often must be based on excluding other causes because the air has been absorbed prior to death. if a patient dies rapidly, the hospital records may be incomplete or scanty. for example, if a patient bleeds to death despite attempted repair of hepatic lacerations, hospital records may not suffice to reach the correct cause-of-death opinion; personal accounts from the surgeon and anesthesiologist may be needed. autopsy data on patients dying following thoracic surgery may be found in ref (2) . d death, restaurant (see "obstruction, acute airway.") death, sniffing and spray related terms: glue sniffing; sudden sniffing death syndrome. note: no anatomic abnormalities will be noted at autopsy. sudden death may occur after cardiac dysrhythmia or respiratory arrest. procedures possible or expected findings lungs brain if poison had been inhaled at the time when death occurred, tie main bronchi. submit lungs in glass container for gas analysis. submit samples of small bronchi for histologic study. for removal and specimen preparation, see chapter 4. submit samples of fresh or frozen brain for toxicologic study. submit samples in glass containers (not plastic) for toxicologic study. trichloroethane, fluorinated refrigerants, and other volatile hydrocarbons are most often involved in the "sudden sniffing death syndrome." spray death may occur in asthma sufferers using pressurized aerosol bronchodilators. freons and related propellants may also be responsible for sudden death. toxic components of glue-such as toluene-accumulate in the brain of glue sniffers. also present in various glues are acetone, aliphatic acetates, cyclohexane, hexane, isopropanol, methylethyl ketone, and methylisobutyl ketone. aerosols may occlude the airway by freezing the larynx. carbon tetrachloride sniffing may cause hepatorenal syndrome (see also under "poisoning, carbon tetrachloride"). death, sudden unexpected, of adult note: medicolegal autopsies are usually indicated, and appropriate procedures should be followed. ifanaphylactic death is suspected, see also under that heading. for all unexpected deaths, the pathologist should learn the circumstances of the death, in order to determine whether the mechanism of death was rapid or slow, and to guide the selection of ancillary tests. whenever paramedics attended a person, the run sheet should be obtained to look for a history of recent drinking or ofchronic alcoholism may be an important clue. the combination of a history ofalcoholism, a negative test for ethanol, and absence ofcardiovascular disease, should suggest alcohol withdrawal as the cause ofa sudden death. the list of"possible or expected findings" below is not complete. for general toxicologic sampling, see chapter 13. possible associated conditions: atrial septal defect;*bicuspid aortic valve;* coarctation,* hypoplasia, or interruption (type a) of aortic arch; coronary artery from main pulmonary artery; right atrial arch; patent ductal artery;* right pulmonary artery from ascending aorta; subaortic stenosis;* tetralogy of fallot;* ventricular septal defect. * (in approx 50% of the cases, one or more of these associated conditions are found.) defect, atrial septal note: the basic anomaly is a defect of the atrial septum, usually at the oval fossa (in 85%). possible complications in unoperated cases include atrial arrhythmias, congestive heart failure; paradoxic embolism; plexogenic pulmonary hypertension â«10%), and pulmonary artery aneurysm. possible surgical interventions include surgical and transcatheter closure of defect. for deficiency, vitamin c synonyms: hypovitaminosis c; scurvy. external examination and skin other organs bones, joints, and soft tissues record extent and character of skin lesions; prepare sections of skin. describe appearance of gums, and prepare sections. record evidence of bleeding. for removal, prosthetic repair, and specimen preparation of bones and joints, see chapter 2. hyperkeratotic hair follicles with perifollicular hemorrhages (posterior thighs, anterior forearms, abdomen); petechiae and ecchymoses (inner and posterior thighs); subcutaneous hemorrhages. gingivitis. in rare instances, gastrointestinal or genitourinary hemorrhages. hemorrhages into muscles and joints. subperiosteal hemorrhages occur primarily in distal femora, proximal humeri, tibiae, and costochondral junctions (scorbutic rosary). deficiency, vitamin d synonyms: hypovitaminosis d; rickets. note: features or rickets may be found in familial hypophosphatemia (vitamin d-resistent rickets; fanconi syndrome). vitreous or blood (serum) other organs prepare skeletal roentgenograms. in infants with suspected rickets, record size of anterior fontanelle and shape of head; state of dentition; and shape of costochondral junctions, wrists, long bones, and spine. submit samples for calcium, magnesium, and phosphate determination. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. weigh parathyroid glands and submit samples for histologic study. submit samples of intestine for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter 2. in infantile rickets, diagnostic sites for histologic sampling are costochondral junctions, distal ends of radius and ulna, and proximal ends of tibia and humerus. for adults, see under "osteomalacia." in infants, rachitic changes at costochondral junctions; in adults, osteoporosis* and osteomalacia*-with or without pseudofractures (milkman's syndrome (1) . note: the term spinocerebellar degeneration encompasses a variety of lesions whose classification is controversial. a new approach has come from linkage analysis and molecular biology. for instance, friedreich's ataxia, the classic form of hereditary ataxia, is due to an intronic expansion of a gaa tri-nucleotide repeat. other forms are also identified by their specific gene loci. neuropathologic examination still is important and ample sampling is suggested, which should include cerebral cortex, basal ganglia (caudate nucleus, putamen, and globus pallidus), thalamus, subthalamic nucleus, midbrain (red nucleus and substantia nigra), pons (pontine nuclei), spinal cord (at cer-vical, thoracic, and lumbar levels), optic tract, optic nerves with lateral geniculate nucleus, and sensory and motor peripheral nerves. for removal and specimen preparation, see chapter 5. enlargement of head. poor demarcation between cortex and gelatinous white matter. extensive demyelination and vacuolation of white matter, particularly subcortically. optic atrophy. degeneration, striatonigral (see "atrophy, multiple system.") related term: thirst. note: possible underlying conditions not related to inaccessibility of water include bums, exposure to heat, gastrointestinal diseases, recent paracentesis, renal diseases, and use of diuretic drugs. see also under "disorder, electrolyte(s)." external examination vitreous urine prepare histologic sections of blisters, ulcers, or skin abrasions. submit sample for sodium, chloride, and urea nitrogen determination. skin turgor may be decreased and eyes may be sunken. microscopic changes help to decide whether skin lesions are antemortem or postmortem. sodium concentrations more than 155 meqll, chloride concentrations more than 130 meq/ and urea nitrogen concentrations between 40 and 100 meq/dl indicate dehydration. absence or minimal amount of urine. dementia (see "disease, alzheimer's.") drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." drug abuse, cocaine note: cocaine is spontaneously hydrolyzed by blood esterases, even after death. however, one of its major metabolite, benzoylecgonine, is routinely identifiable by immunoassay screening tests. when cocaine is abused concurrently with heroin or other depressant drugs, it may be difficult to ascribe deth to a single agent, unless circumstances clearly point to a rapid cardiac mechanism or a slow brainstem depression mechanism. note: if narcotic paraphernalia and samples of the drug itself are found at the scene of the death, they should be submitted for analysis. helpful information about the nature of a drug may be obtained from witnesses. state crime laboratories may provide much assistance. if name of drug is known, see also under "poisoning,..." the slang name of a drug may be insufficient for identification because these names often are used for different compounds at different times of places. opoid narcotics can be injected intravenously, or subcutaneously, or snorted. death may occur with such speed that the bodies may be found with needles and syringes in the veins or clenched in the hands. drug abuse may be associated with a multitude of local (see below) or systemic complications, including malaria* and tetanus. * as stated in chapter 13, for a growing number of analytes, most notably tricyclic antidepressants, peripheral blood is preferred over central blood. peripheral blood is aspirated by percutaneous puncture before autopsy, from the femoral vein or the subclavian vein. the authors prefer the femoral approach in order to avoid any question of artifact in the diagnosis of venous air embolism. it may be pru-dent to add naf to some of the samples. related term: childhood dermatomyositis (or polymyositis) associated with vasculitis; dermatomyositis (or polymyositis) associated with neoplasia or collagen vascular disease; primary idiopathic dermatomyositis; primary idiopathic polymyositis. possible associated conditions: carcinoma (lung, stomach, intestine, and prostate in males; breast, ovary, and uterus in females; miscellaneous sites in both sexes); lymphoma* (rare) and other malignancies (1); lupus erythematosus;* mixed connective tissue disease; progressive systemic sclerosis;* rheumatoid arthritis;* sjogren's syndrome;* and others. vasculitis of childhood polymyositis (dermatomyositis). external examination and skin heart lungs esophagus and gastrointestinal tract photograph grossly involved skin. prepare sections of involved (anterior chest, knuckles, knees) and grossly uninvolved skin and subcutaneous tissue. prepare roentgenograms. submit samples from myocardium for histologic study. perfuse one lung with formalin. submit samples from all segments for histologic study. arteritis* and phlebitis* with thrombosis, fibrosis, and infarctions. steatohepatitis and manifestations of diabetes mellitus* may be found (2) . myositis with muscular atrophy and fibrosis; vasculitis in childhood cases. polyneuropathy (rare) (5). arthritis. diabetes mellitus synonyms: type i (insulin-dependent or juvenile-onset) diabetes mellitus; type ii (insulin-independent or adult onset) diabetes mellitus; secondary diabetes mellitus (e.g., due to drugs or pancreatic disease). note: in infants of diabetic mothers, macrosomia and congenital malformations must be expected. record size and weight of placenta and total weight and length, crown to rump length, and crown to heel length of infant. compare with expected measurements (see part iii). expected histologic finding in-clude hyperpla-sia with relative increase ofb cells of the islands of langerhans with interstitial and peri-insular eosinophilic infiltrates, decid-ual changes of the endometrium, enhanced follicle growth in the ovaries, and leydig cell hyperplasia. possible associated conditions: acanthosis nigricans; acro-megaly;* amyotrophic lateral sclerosis; * ataxia telangiectasia;* fanconi's anemia;* friedreich's ataxia;* gout;* hemochro-matosis; *hyperlipoproteinemia; * hyperthroidism;* obesity;* turner's syndrome;* and many others, too numerous to mention. note: the term "caroli's syndrome" often is used for cases that also show histologic features of congenital he-patic fibrosis or other manifestations of fibropolycystic liver disease,* whereas the name "caroli's disease" refers to idiopathic dilatation of intrahepatic bile ducts, without associated abnormalities. possible associated conditions: choledochal cyst* and related extrahepatic biliary abnormalities (1); congenital hepatic fibrosis; * cysts of kidneys (renal tubular ectasia or medullary sponge kidney; autosomal-recessive polycystic kidney disease, and rarely, autosomal-dominant polycystic kidney disease [2] )* and of pancreas. record volume of effusions. prepare smears of fresh blood or of buffy coat, or make thick-drop preparation. submit sample for xenodiagnosis or animal inoculation and for serologic study. record weight. in chronic chagas' disease, perfuse intact heart with formalin (chapter 3) and slice fixed heart in a frontal plane so as to create anterior and posterior halves. prepare photographs. histologic samples should include conduction system. include several sections of atrial (auricular) walls for histologic study of autonomous ganglia. perfuse at least one lung with formalin. leave affected hollow viscera intact and fill with formalin. cut fixed organs in half, photograph, and cut histologic sections on edge. record liver weight and submit samples for histologic study. record weight. prepare photographs of abnormalities. weigh and examine. prepare histologic sections. for removal and specimen preparation, see chapter 4. autopsy is desirable in suspected cases because the diagnosis can only be firmly established after neuropathologic examination. serologic studies are not available. unfortunately, all tissues (not just the brain and spinal cord) may remain infectious even after prolonged fixation and histologic processing. thus, the autopsy recommendations for most other infectious diseases do not apply here. this is a reportable disease in some states. special precautions are indicated and therefore, the procedures described here should be followed strictly (1) (2) (3) (4) : all persons in the autopsy room must wear disposable long-sleeved gowns, gloves, and masks. contamination of the autopsy table should be prevented by covering it with a disposable, non-permeable plastic sheet. autopsy generally should be restricted to the brain. if organs in the chest or abdomen need to be examined, this is best done in situ. to prevent aerosolization of potentially infectious bone dust, a hood or other protective device should be used while opening the skull with a stryker saw. after completing the autopsy, instruments and other potentially contaminated objects should be autoclaved in a steam autoclave (1 h at 134â°c). porous load is considered more effective than gravity displacement autoclaves. immerse autopsy instruments in distilled water before and during autoclaving, in order to protect them from corrosion. ifno autoclave is available, chemical disinfection (see below) is a satisfactory alternative. disposable items should be put in a container for infectious hospital waste and ultimately incinerated. contaminated objects not suitable for autoclaving (such as the stryker saw) should be soaked with a2nnaoh solution for 1 h (alternatively, 1 nnaoh may be used for 2 h). contaminated surfaces should be thoroughly washed with the same solution. aluminum should be treated for 2 h with a fresh 5% naoci (sodium hypochlorite) solution with at least 20,000 ppm free chloride. wash waters should be collected; if no autoclave is available, 2 n naoh or >4 volumes of 5% sodium hypochlorite bleach should be added to the water and left for a minimum of 2 h before being discarded. before removing the body from the autopsy room, it should be sponged with 5% sodium hypochlorite. to deactivate cjd infectivity, tissue blocks, 5 mm or less in thickness, should be fixed in formalin in a formalin-totissue ratio of at least 20: 1 for at least 48 h and then soaked in concentrated formic acid (95-100%) for i h, followed by another 48 h of formalin fixation. the fixation fluid should be collected and decontaminated, as described earlier for wash water. glassware and tissue carriers should also be decontaminated as previously described. after this deactivation, the tissue blocks can be processed in a routine fashion. at any stage of these procedures, special care must be taken to avoid cuts with potentially contaminated glassware, blades, or other objects. parenteral exposure to potentially contaminated material also should be avoided. remains of patients who have died of the disease should not be accepted for anatomy teaching for students. if specimens are prepared for pathology collections, they should be handled with great caution. morticians and mortuary workers should be warned of possible hazards posed by tissues of patients with transmissible spongiforme encephalopathies; they should be advised about proper use of disinfectants. clinical laboratories that receive autopsy tissues or fluids must be warned about the infectious nature of the material. if possible, decontamination should be done at the site where the autopsy was done. for the shipping of potentially infected material, see chapter 15. increased concentrations of nse (5). spongiforme changes, astrocytosis, neuronal loss, amyloid plaque formation, prp deposition, and proliferation of activated microglia (6). cerebrospinal fluid brain submit sample for neuron-specific enolase (nse). for removal and specimen preparation, see chapter 4 and above under "note." submit fresh-frozen material for confirmation of diagnosis by histoblot technique on protease k-digested frozen tissue or western blot preparations on brain homogenates. immunohistochemical localization ofprp and hla-dr protein on paraffin-embedded tissue is possible. disease, demyelinating (see "degeneration, spongy, of white matter," "encephalomyelitis, all types or type unspecified," "leukodystrophy, globoid cell," "leukodystrophy, sudanophilic," "sclerosis, multiple;' and "sclerosis, schilder's cerebral.") disease, diffuse alveolar synonym: diffuse pulmonary disease. note: autopsy procedures are listed under the more specific diagnoses, such as "hemosiderosis, idiopathic pulmonary," "lipoproteinosis, pulmonary alveolar," "microlithiasis, pulmonary alveolar," "pneumonia, lipoid," and "syndrome, goodpasture's." glycosphingolipid storage in cornea; lens opacities; dilated vessels in conjunctiva and lens; thrombi in blood vessels (5). disease, fibropolycystic, of the liver and biliary tract note: "fibropolycystic disease of the liver and biliary tract" comprises a group of well defined conditions, which may occur together and hence need a collective designation. the conditions include autosomal-recessive (infantile) and auto-somal dominant (adult) polycystic disease of the liver; caroli's disease or syndrome;* choledochal cyst,* congenital hepatic fibro-sis,* multiple biliary microhamartomas, and related disorders. for autopsy procedures, see also under more specific designations. disease, glycogen storage synonyms: andersen's disease or brancher deficiency (glycogenosis, type iv); cori's or forbes' disease (glycogenosis, type ill); cyclic amp dependent kinase (type x); glycogen synthetase deficiency (type 0); hers' disease (glycogenosis, type vi); mcardle's disease (glycogenosis type v); phosphorylase b kinase deficiency (types ixa, b, and c); pompe's disease (glycogenosis, type it); tarui disease (glycogenosis type vii); von gierke's disease (glycogenosis, type ia); x-linked glycogenosis (type vill). note: if the diagnosis had not been confirmed prior to death, samples of liver, skeletal muscle, blood, and fascia (for fibroblast culture, see below) should be snap-frozen for enzyme assay, which will determine the specific deficiency. types ia and b, iii, vi, and hepatic phosphorylase b kinase deficiency (types ixa, b and c) are hepatic-hypoglycemic disorders, whereas types v and vii affect muscle energy processes. type ii also affects the musculature, whereas type iv may cause cirrhosis and death in infancy from extreme hypotonia. determination of type of glycogenosis usually can be based on (i) pattern of glycogen storage in liver, (2) presence or absence of nuclear hyperglycogenation in liver, (3) cytoplasmic lipid in liver, (4) presence or absence of liver cirrhosis, and (5) presence or absence of glycogen and basophilic deposits in skeletal muscles. possible associated conditions: fanconi syndrome* or gout* with type ia glycogenosis; neutropenia, recurrent infections, and crohn's disease with types ib or ie. glycogen primarily in retinal ganglion cells and ciliary muscle. glycogen in sympathetic nerve ganglia and neurons of cranial nerves in type vii. gouty arthritis. disease, graft-versus-host note: this disease occurs most commonly after bone marrow transplantation. the disease has also occurred after transfusion of viable lymphocytes, for example, to patients with cancer or leukemia. * in patients with graft-versus-host disease (gvhd), autopsy also may reveal recurrence of the underlying disease such as leukemia. possible associated conditions: alphal-antitrypsin deficiency;* amyloidosis;* ankylosing spondylitis;* primary sclerosing cholangitis;* sjogren's syndrome. * see also below under "possible or expected findings." note: in many instances, either chronic ulcerative colitis or crohn's disease* had been diagnosed clinically, but sometimes, the distinction is difficult to make, even at autopsy. many features described below occur in chronic ulcerative colitis but some manifestations of crohn's disease or conditions that may occur in all types of inflammatory bowel disease also are listed so that both positive and negative findings can be recorded properly. osteoporosis;* ankylosing spondylitis;* arthritis of peripheral joints; periarthritis; hypertrophic osteoarthropathy;* tendinitis (particularly of ankle and achilles tendons). disease, iron storage (see "hemochromatosis.") related terms: atherosclerotic heart disease. note: the most common anatomic finding at autopsy in subjects older than 30 yr is coronary atherosclerosis. unusual under-lying or associated conditions include chronic aortic stenosis or regurgitation; coronary artery anomalies; coronary artery dissection; coronary embolism; coronary ostial stenosis (due to calcification of aortic sinotubular junction or, rarely, to syphilitic aortitis); coronary vasculitis (for instance, in polyarteritis nodosa* or acute hypersensitivity arteritis); hyperthyroidism,* gastrointestinal hemorrhage; * hypothyroidism, * idiopathic arterial calcification of infancy; intramural coronary amyloidosis; pheochromocytoma, polycythemia vera; * pseudoxanthoma elasticum,* radiationinduced coronary stenosis; severe pulmonary hypertension (with right ventricular ischemia); sickle cell disease;* and others. if bypass surgery had been performed, see "surgery, coronary bypass." macular rash (4). multifocal fibrinopurulent pneumonia with sparing of the bronchi and bronchioles. exudate is rich in phagocytes, fibrin, and karyorrhectic debris. synonym: lyme arthritis note: this infection is caused by the spirochete, borrelia burgdoiferi, which is transmitted from rodents to human by the hard deer ticks, ixodes dammini, 1. ricinus, and others. brain and spinal cord for removal and specimen preparation, see chapter 4. request luxol fast blue stain for myelin. symmetric and zonal demyelination in corpus callosum, anterior commissure, optic chiasm, optic tracts, and white matter of frontal lobes. external examination and skin; oral cavity lungs aorta record distribution of skin lesions and submit tissue samples for histologic study. for preparation of angiograms of the pulmonary arterial and venous vasculature, see chapter 2. if aneurysm or dissection is present, follow procedures described under those headings. telangiectatic (often papular) lesions most commonly found in cheeks, scalp, nasal orifices, oral cavity, ears, neck, shoulders, fingers, toes, and nail beds. cyanosis and clubbing may be prominent. arteriovenous malformations/fistulas. aneurysm; * aortic dissection. * if cirrhosis is present, prepare angiograms of hepatic arteries and veins (chapter 2). photograph and prepare sections of angiomatous lesions. note: parkinson's syndrome is caused by conditions that may simulate parkinson's disease; these include carbon monoxide* and manganese poisoning, corticobasal degeneration, druginduced parkinsonism, huntington's disease, multiple system atrophy,* progressive supranuclear palsy* (steele-richardson-olszewski syndrome), space-occupying lesions (rare), trauma (dementia pugilistica), and causes related to tumors and vascular diseases. brain for removal and specimen preparation, see chapter 4. histologic sections should include midbrain (substantia nigra), upper pons (locus ceruleus), medulla, nucleus basalis (substantia innominata), and basal ganglia. if parkinsonian syndrome was diagnosed, follow procedures described under the name of the suspected underlying condition (see above under "note"). depigmentation of substantia nigra and locus coeruleus; neuronal loss and reactive gliosis; eosinophilic intracytoplasmic inclusion bodies (lewy bodies) in some of the surviving neurons; no significant changes in basal ganglia. disease, pelizaeus-merzbacher synonyms: sudanophilic (orthochromatic) leukodystrophy. brain and spinal cord for removal and specimen preparation, see chapter 4. request luxol fast blueipas stain for myelin and bielschowsky's stain for axons. prepare frozen sections for sudan stain. brain generally atrophic. myelin loss in centrum ovale, cerebellum, and part of brain stem, with a tigroid pattern of residual myelin near vessels. axons are preserved. diffuse gliosis with relatively few lipoid-containing macrophages, compared to the myelin loss. lipoid material stains with sudan. brain and spinal cord for removal and specimen preparation, see chapter 4. request silver stains (bielchowsky or bodian stain). histochemical stains in pick's cells and bodies reveal phosphorylated neurofilaments, ubiquitin, and tubulin. some tissue should be kept frozen for biochemical studies. severe cerebral atrophy, involving primarily frontal and anterior temporal lobes (knifeblade atrophy; walnut brain). microscopically, severe neuronal loss accompanied by astrocytosis. characteristic argyrophilic, intracytoplasmic inclusions (pick's bodies), particularly in hippocampus and swollen, distended "ballooned" neurons (pick's cells). these changes are not always present. external examination, skin, and adipose tissue blood cerebrospinal fluid heart liver and kidneys brain, spinal cord, and peripheral nerves eyes submit sample for determinaion of phytanic acid concentration and for molecular studies. for obtaining a sample, see chapter 7. sample for histologic study. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 5. ichthyosis. phytanic acid accumulation in adipose tissues. phytanic acidemia, mutation of phyh or pex 7 (2). increased protein concentrations. cardiomyopathy.* phytanic acid accumulation. axonal neuropathy. retinitis pigmentosa. hypoalphalipoproteinemia. lymphadenopathy with diffuse deposition of cholesterol esters. premature atherosclerotic cardiovascular disease (1). hepatosplenomegaly with foam cells. enlarged tonsils with characteristic orange discoloration. polyneuropathy (2) . in adults, corneal infiltrates. foam cells. request pas stain. in granulomas, bacilli are not always pas positive (2) . section all grossly involved tissues for histologic examination. submit section for electron microscopy. emaciation. hyperpigmentation, particularly of exposed skin and in scars. hyperkeratosis. arthritis involving ankles, knees, shoulders, and wrists. ascites; fibrinous peritonitis. * nodules in peritoneum containing sickle-form particlecontaining cells (spc cells submit sample for determination of sodium, potassium, chloride, glucose, urea nitrogen, and creatinine concentrations. calcium and phosphate concentrations can also be tested. if sample is small, indicate priority for testing. if indicated, submit sample for chemical study. submit tissue samples for histologic study. considerably increased or decreased values for sodium (more than 155 meqll or less than 130 meqll) and chloride (more than 135 meqll or less than 105 meqll) indicate that changes were present before death. for further interpretation, see chapter 8. postmortem electrolyte concentrations are quite unreliable. may be useful for calcium determination. vacuolar nephropathy (vacuolar changes in proximal convoluted tubules) in potassium deficiency (may also occur after infusion of hypertonic solutions). disorder, hemorrhagic (see "coagulation, disseminated intravascular," ''disease, christmas:' ''disease, von willebrand's," "hemophilia," and "purpura,.â�¢â�¢") disorder, inherited, of phagocyte function note: several conditions represent phagocyte function disorders. autopsy procedures for one of these disorders can be found under "disease, chronic granulomatous." consult this entry for other phagocyte function disorders. synonyms and related terms: fabry's disease* (angiokeratoma corporis diffusum); gangliosidosis;* gaucher's disease;* glycogenosis,* type ii; leukodystrophies (krabbe's or globoidcell,* metachromatic leukoencephalopathy*); mucopolysaccharidoses* (hunter, hurler, morquio, and sanfilippo disease); mucolipidosis; niemann pick disease* (type a, b, c, or sphingomyelinase deficiency); neuraminidase deficiency; neuronal ceroid lipofuscinosis (batten's disease or kufs' disease). hypopharyngeal pulsion diverticulum (zenker's diverticulum) at lower margin of inferior constrictor muscle of pharynx. traction diverticulum at midesophagus after an inflammatory process-for instance, tuberculous lymphadenitis. epiphrenic diverticulum may also occur. luxtacardiac or juxtapyloric diverticulum. heterotopic tissue in meckel's diverticulum, with or without peptic ulceration. colonic muscular hypertrophy and stenosis, usually in sigmoid colon. diverticulitis with perforation, fistulas, or peritonitis. * diving (see "accident, diving (skin or scuba).") related terms: dry drowning; fresh-water drowning; near-drowning; salt (sea)-water drowning (see the following table). primary drowning ("immediate drowning") deaths occurring within minutes after immersion, before or without resuscitative measures deaths from hypoxia and acidosis caused by glottal spasm on breath holding. there may be no evidence of water entering stomach or lungs and no appreciable morphologic changes at autopsy. note: the diagnosis is one of exclusion. the pathologist should help the police to determine: i) how did the person (or dead body) get in the water, and 2) why could that person not get out of the water? it is not enough to ask if a person could swim but investigators should find out how well (what strokes did the victim know?) and how far he or she could swim. the inquiry must include the depth of the water and must address hazards such as undertow or underwater debris, and the behavior deaths occurring from within 30 min to several weeks after resuscitation, because of metabolic acidosis, pulmonary edema, or infective or chemical pneumonitis deaths from hypoxia and acidosis caused by obstruction of airway by water related to: hypervolemia hemolysis hyponatremia hypochloremia hyperkalemia of the victim immediately before submerging. deaths of adults in bathtubs and swimming pools are usually from natural, cardiac causes, or they are suicides, unless the victim was drunk. diatom tests (1) have not proven useful in the united states but there is enthusiasm for such tests among european pathologists. the distinction between hyponatremic deaths in fresh water and hypernatremic deaths in salt water derives from experimental studies; in practice, one cannot reliably predict the salinity of the immersion medium from autopsy studies. because many bodies of drowning victims are recovered only after the body floats to the surface, decomposition will often obscure even the nondiagnostic findings such as pleural effusions, which are often associated with drowning. external examination and skin (wounds) organ samples for diatom search serosal surfaces and cavities if identity of drowning victim is not known, record identifying features as described in chapter 13. prepare dental and whole-body roentgenograms. submit tissue samples for histologic study of wounds. inspect inside of hands. collect fingernail scrapings. record appearance and contents of body orifices. record features indicative of drowning. photograph face from front and in profile. take pictures of all injuries, with and without scale and autopsy number. remove vitreous for analysis. if diatom search is intended, clean body thoroughly before dissection to avoid contamination of organs and body fluids with algae and diatoms (see below). submit sample for toxicologic study. sample early during autopsy, before carrying out other dissections. use fresh instruments for removal of specimens to avoid contamination. submit subpleural portion of lung: subcapsular portions of liver, spleen, and kidneys; bone marrow; and brain. store samples in clean glass jars. for technique of diatom detection, see below. record volume of fluid in pleural spaces. photograph petechial hemorrhages. photograph layerwise neck dissection if strangulation* is suspected. open airways posteriorly, and photograph, remove and save mud, algae, and any other material in tracheobronchial tree. record size and weight of lungs. there may be wounds that were inflicted before drowning occurred-for instance, in shipwrecks or vehicular and diving accidents. other wounds may be inflicted after deathfor instance, from ship propellers or marine animals. sometimes, premortem and postmortem wounds can be distinguished histologically. object (hair?) held by hands in cadaveric spasm. cutis anserina and "washerwoman" changes of hands and feet are of no diagnostic help. foreign bodies; semen (see also under "rape"). foam cap over mouth and nose. in the autopsy room, water running from nose and mouth is usually pulmonary edema or water from the stomach. high concentrations of alcohol indicate intoxication (see under "alcoholism and alcohol intoxication"). evidence of alcohol intoxication may be found. diatoms may occur in the liver and in other organs of persons who have died from causes other than drowning. comparison with diatoms in water sample from area of drowning may be helpful. penny-sized or smaller hemorrhages may indicate violent respiratory efforts or merely intense lividity. presence of pleural fluid suggests drowning. for diatom detection (l) , boil 2-5 g oftissue for 10--15 min in 10 rnl of concentrated nitric acid and 0.5 rnl of concentrated sulfuric acid. then, add sodium nitrate in small quantities until the black color of the charred organic matter has been dispelled. it may be necessary to warm the acid-digested material with weak sodium hydroxide, but the material must soon be washed free from alkali to avoid dissolving the diatoms. the diatoms should be washed, concentrated, and stored in distilled water. for examination, allow a drop of the concentrate to evaporate on a slide, and then mount it in a resin of high refractive index. all equipment must be well-cleaned, and distilled water must be used for all solutions. there are several variations and adaptations of this method. drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." ductus arteriosus, patent (see "artery, patent ductal.") synonyms and related terms. achondroplastic dwarf; asexual dwarf; ateliotic dwarf; micromelic dwarf; normal dwarf; pituitary dwarf; true dwarf; and many other terms, too numerous to mention. external examination bones and joints record height and weight. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter 2. growth retardation. abnormal growth of epiphyseal cartilage with enlargement of metaphysis. long bones and pelvis most commonly affected. cavernous hemangiomas (maffucci's syndrome). see above under "external examination." chondrosarcoma. dyscrasia, plasma cell note: these conditions are characterized by abnormally proliferated b-immunocytes that produce a monoclonal immunoglobulin. multiple myeloma, * plasma cell leukemia, plasma-cytoma, and waldenstrom's macroglobulinemia* as well as heavy-chain diseases and monoclonal gammopathies of unknown type belong to this disease family. amyloidosis* is closely related to these conditions. for autopsy procedures, see under "amyloidosis," "macroglobulinemia," or "multiple myeloma" and under name of condition that may have caused the plasma cell dyscrasia. such conditions include carcinoma (colon, breast, or biliary tract), gaucher's disease,* hyperlipoproteinemia, * infectious or noninfectious chronic inflammatory diseases, and previous cardiac surgery. synonym: shigella dysentery. note: (i) collect all tissues that appear to be infected. blood bowel eyes joints submit sample for culture and for serologic study. submit sample of feces or preferably bloodtinged mucus for culture. if bacteriologic diagnosis has already been confirmed, pin colon on corkboard, photograph, and fix in formalin for histologic study. submit sample of vitreous for study of sodium, potassium, chloride, and urea nitrogen concentrations. for removal and specimen preparation of eyes, see chapter 5. for removal, prosthetic repair, and specimen preparation, see chapter 2. escherichia coli septicemia. colitis with microabscesses; transverse shallow ulcers and hemorrhages, most often in terminal ileum and colon. dehydration* pattern of electrolytes and urea nitrogen. serous arthritis* of knee joints is a late complication. external examination record extent of pigmentation, facial features, and primary and secondary sex characteristics. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter 2. record size of apertures of cranial nerves in base of skull. unilateral skin pigmentation and precocious puberty in females (albright's syndrome), less commonly in males. synonyms and related terms: becker's muscular dystrophy; congenital muscular dystrophy; duchenne's progressive muscular dystrophy; dystrophinopathy; em-ery-dreifuss mucular dystrophy; facioscapulohumeral dystrophy; limb girdle dystrophy; myotonic muscular dystrophy. external examination record pattern of scalp hair. record status of skeletal musculature. obtain sections for histologic examination. dystrophin staining of the sarcolemma is absent in duchenne's muscular dystrophy and patchy in becker's dystrophy. frontal baldness (in myotonic muscular dystrophy). atrophy and wasting of muscles (generalized or local: predominantly distal in myotonic muscular dystrophy). pseudohypertrophy of calf muscles in duchenne's muscular dystrophy. dystrophic changes include variations in fiber size, fiber degeneration and regeneration, peri-and endomysial fibrosis, and fatty replacement of muscle. the liver, especially the right lobe, is the most common site of involvement. secondary infection or calcification may be present. the lung is the second most common site of involvement. fluid and air may be visible on the roentgenogram. cysts may be present in the abdominal cavity, muscles, kidneys, spleen, bones, heart, and brain. eosinophilia. edema, angioneurotic synonym: angioedema. note: possible causes and suggested autopsy procedures are described under "death, anaphylactic." related term: silo-filler's disease. n<yfe: this condition is causedby inhalationoftoxic gases, such as oxides of nitrogen (silo-filler's disease) and phosgene (coci 2 ). see also "bronchitis, acute chemical" and "poisoning, gas." (1) gunshot and shotgun wounds of the head involving dural sinuses; (2) injury to large veins, particularly cranial sinuses (during neurosurgical procedures) and veins ofthe neck (knife wounds, surgery) or uterus (criminal abortion); (3) insufflation offallopian tubes (particularly in pregnancy or during menstrual period); (4) infusion of blood components or crystalloid; (5) malfunctioning of dialysis machines; (6) subclavian vein catheterization in the semi-fowler position; and (7) fracture in the hub of a central venous catheter used for parenteral nutrition. possible causes of arterial air embolism include: (1) open heart surgery involving the aorta, left atrium, or left ventricle; (2) positive-pressure ventilation in newborn infants; and (3) underwater ascent with closed glottis (see accident, diving) ifair embolism is suspected, the autopsy should be performed as soon after death as possible. decomposition gases may be produced within a few hours. roentgenography of the whole body may detect large quantities of air, and the roentgenograms may serve as a guide to the most advantageous way of dissection. the postmortem diagnosis of arterial air embolism is made largely on the basis of medical history and circumstances. the autopsy serves to rule out competing conditions. the diagnosis of venous air embolism is made by chest roentgenography before the autopsy (1). the air in the right chambers of the heart can be confirmed at autopsy by aspiration into a syringe. the formerly recommended procedure of clamping the internal mammary vessels below the sternoclavicular joints, and then cutting across the sternum distal to these clamped vessels so that the sternoclavicular joint area remains intact, is designed to prevent the production of a vacuum that pulls air into the veins. the procedure is not necessary if the air is welldocumented by roentgenography before autopsy. at autopsy, a large fatal pulmonary air embolism is readily apparent. the right atrium and ventricle are distended with fine, frothy, brightred blood, which also may distend the pulmonary arteries and superior vena cava. microbiologic examination ofblood and pericardial sac contents may help to rule out the presence of gas-forming bacteria that may simulate air embolism (fig. ii-i). however, the presence of putrefactive emphysema in any part of the body points toward a bacterial origin of the gas. differentiation of air and decomposition gases at the autopsy table with the pyrogallol test is described below. a 2% pyrogallol solution is prepared (it should be waterclear). two lo-ml syringes (syringe a and syringe b) are loaded with 4 ml of the pyrogallol solution in each, without permitting any air to enter the system. immediately before the solution is used, 4 drops of 0.5 n naoh is aspirated through the needle of syringe a to adjust the ph to about 8 (1 drop per 1ml of solution); the mixture will turn faint yellow. six ml of gas is then aspirated from the heart or blood vessels. the needle is immediately sealed with a cork or replaced by a cap, and the syringe is vigorously shaken for about 1 min. in the presence of air, the pyrogallol solution will turn brown. if the solution remains clear, decomposition gases were present. in the latter instance, 4 drops of 0.5 n naoh and 6 ml of room air should be aspirated into syringe b, which is then also sealed and shaken for 1 min. the mixture should turn brown, thus serving as a fig. 11â·1 . gas-forming bacteria simulating air embolism. the pericardial sac is opened and filled with water. the heart is kept submerged with a pair of scissors. the coronary arteries have been incised with a scalpel. note gas bubbles and foam on the water surface. no discoloration of 2% pyrogallol was noted. blood cultures were positive for enterococcus organisms. microscopically, gram-negative rods were found in most tissues. control that the pyrogallol solution had been properly prepared. syringe b may also serve as a reserve. if only one syringe is used, the decomposition gas can be expel1ed and room air can be aspirated for the control test. if only small amounts of gas can be aspirated, the volume of the pyrogallol solution should be decreased so that the gas-fluid volume ratio is at least 3:2. if no bacterial gas formation is present, the edges of the pericardial incision are elevated and the pericardial sac is fil1ed with water. clamping of the ascending aorta and venae cavae prevents the escape of gas into these vessels. the heart is held under water while the coronary arteries are incised, and the escape of bubbles is recorded. when the right coronary artery is opened, care must be taken that the right atrium is not incised. air in the coronary arteries indicates systemic embolism. the heart chambers are then incised. when there is gas in any of the arteries or heart chambers, gas bubbles rise to the surface of the water in the pericardial sac ("bubble test"). sometimes the vessels have to be somewhat compressed in order to cause the gas to escape. because large amounts of air or other gases cause the heart to float, it must be kept submerged before the vessels and chambers are incised. basically the same procedure is used for demonstrating the presence of gas in the superior or inferior vena cava and the pelvic veins (for example, in cases of criminal abortion). in this situation, the abdominal cavity is filled with water and the inferior vena cava and its tributaries are incised. in support of the diagnosis of systemic arterial air embolism, the skull vault may be removed without puncturing the meninges, so that the cerebral arteries can be inspected for gas bubbles. the demonstration of gas bubbles in the meningeal vessels and in the circle of willis is meaningful only when the neck vessels are still intact and the internal carotid artery and basilar artery have been clamped before the brain is removed. in acute cases, gas bubbles will be visible within the cerebral vessels. they are released under water when the clamps are removed and the vessels are slightly compressed. for the collection of gas from blood vessels or cavities, a system oflittle quantitative reliability is an air-tight, water-filled glass syringe with a needle. the needle is inserted into the vessel or cavity in question and gas is carefully aspirated. a combined qualitative and quantitative method has been described by kulka (2,3) (see fig. 11 -2). he devised an apparatus for gas collection and described it as shown in fig. 11 -2 and caption. the entire system is filled with mineral oil so that, when the funnel is level with the upright bottle, the oil fills only about half of the funnel. in operation, the funnel is first raised to a position 30-40cm above the level of the upright bottle. all the cocks are opened and the position is held until every trace of gas has been driven from the system through the needle, which is thereby coated on the inside by a film of oil. after all air has been expelled, the cocks are closed and the funnel is lowered to its original position. as a precautionary measure and control, the air-tightness of the whole system should be tested before operation. this is done by inserting the needle into musculature or skin and attempting aspiration in the manner described in the next paragraph. to make the test, the bottle is inverted and the needle is inserted into the cavity in question. when the needle is in position, all cocks are opened. the funnel is lowered about 70-90 cm, or until adequate suction is created. this aspirates the contents of the cavity, which may consist of air or other gases, either pure or mixed with blood or other liquid. any gas or liquid entering this system may be observed through the wall of the short bent glass tubing. in a positive test, gas bubbles will collect in the bottle above the level of the oil. if desired, this gas can be saved for further examination by closing all the cocks and returning the bottle to its upright position (4). the volume ofintravenous air needed to cause death in adults depends of the cross sectional area of the cardiac cavity or great vessel that is the site of the gas lock, which in tum depends on the position of the decedent at the time of the embolism (5). 100 ml of gas can pass through an intravenous hub in just a few seconds. small amounts of air entering the systemic circulation may cause death within minutes. delayed air embolism with fatal outcome may also occur. other organs if purpura is present, prepare photographs and record extent. submit sample (from right atrium) for microbiologic study. collect blood from right atrium and right ventricle. after the heart has been removed, allow blood in pulmonary vessels to pool in the pericardial sac. centrifuge this blood and submit sample of flocculent layer above buffy coat for microscopic study (1) . submit a section of lung for bacteriologic study. dissect pulmonary arteries; prepare histologic sections of all lobes; request mucicarmine stain and the aldan blue and phloxinetartrazine stain of lendrum. also request sudan stain on frozen sections. skin purpura. vernix, lanugo hairs, and meconium can be found in pericardial blood pool. meconium-type material in blood vessels. in histologic sections, squamous epithelium, meconium, and fat from vernix caseosa. complete or incomplete lower uterine tear; chorioamnionitis. manifestations ofdisseminated intravascular coagulation* and fibrinolysis. intrauterine pneumonia. large amounts of debris in the blood vessels of all sections of the lungs may be considered to be lethal if there is no other cause of death. small amounts in one or more blocks of pulmonary tissue are more likely incidental (1). a small uterine tear is more likely followed by a fatal amniotic fluid embolization than is a large tear, which may result in fatal hemorrhage or fibrinogen depletion. chorioamnionitis, intrauterine pneumonia, and positive lung cultures of the mother indicate infection of the amniotic fluid. record weight and sample for histologic study. for removal and specimen preparation, see chapter 4. photograph horizontal sections through brain, brain stem, and spinal cord. prepare frozen sections and request sudan stain. prepare frozen sections of one-half of gland and paraffin sections of the other half. petechial hemorrhages of skin (chest, neck, and face). wounds; other traumatic lesions. bone fractures. petechiae of conjunctivas and retinas. fat may accumulate on surface ofblood pool. no useful technique is available for estimating the amount of fat globules in the blood. fat emboli in lumen of small vessels and in pulmonary air spaces. severe fatty changes may be the cause offat embolism. fractures are the most common cause of fat embolism. petechial hemorrhages, fat emboli (2). abdominal cavity submit sample of subphrenic exudate for gram stain and cultures. record location and volume of subphrenic exudate. possible causes of subphrenic empyema include appendicitis, cholecystitis, * diverticulitis, intrahepatic abscess, pancreatitis,* ruptured viscus; penetrating abdominal wound(s), perforated ulcer of stomach or duodenum,* and other conditions. pleural cavities and lungs e procedures record volume of effusion or exudate in pleural space. basal pleuritis and pneumonia, adjacent to empyema. encephalitis, au types or type unspecified synonyms and related terms: acute disseminated encephalomyelitis;* acute hemorrhagic encephalitis; acute infective encephalitis or encephalomyelitis; acute poliovirus encephalitis or encephalomyelitis; amoebic encephalitis; arbovirus encephalitis (japanese encephalitis; eastern encephalitis, western encephalitis, venezuelan equine encephalitis, st. louis encep-halitis); bulbar encephalitis;* brain stem encephalitis;* herpes encephalitis (cytomegalovirus encephalits, epstein-barr virus encephalitis, varicella-zoster encephalitis); herpes simplex ence-phalitis; hiv encephalitis; measles encephalitis; measles inclusionbody encephalitis; postinfectiousencephalitis; postvac-cinal encephalitis; progressive multifocal leukoencephalitis or leukoencephalopathy; rabies* encephalitis; subacute encephalitis; subacute sclerosing panencephalitis; viral encephalitis, west nile encephalitis and other terms (1), too numerous to mention. see also under "note" and under "possible or expected findings." note: if the condition that caused the encephalitis is known, see also under that heading. if the cause of the encephalitis is unknown, submit samples of tissue for microbiologic and toxicologic study, particularly if there is a suspicion of lead poisoning.* see also under "encephalitis, brain stem," "encephalomyelitis,...," "encephalopathy" and "myelopathy, myelitis." encephalitis, brain stem synonyms and related terms: brain stem abscess; infectious brain stem encephalitis; listeria monocytogenes brain stem encephalitis; viral brain stem encephalitis. note: see also under "encephalitis, limbic." brain for removal and specimen preparation, see chapter 4. necrotizing encephalitis, with or without abscess formation (1 enterocolitis, other types or type undetermined note: a multitude of infectious and noninfectious agents may cause inflammation of the small bowel, large bowel, or both. if the condition is not listed under "colitis," "enteritis," or "enterocolitis" or under another specific heading such as "dysentery, bacillary," obtain sufficient material for microbio-logic and histologic study to identify organisms such as clos-tridium, chlamydia, shigella, salmonella, yersinia, helicobacter, verotoxic e. coli, and others. if lymphogranuloma venereum,* or tuberculosis* are suspected, see also under these headings. see also under "disease, inflammatory bowel" and "disease, crohn's." synonyms and related terms: c. difficile colitis; darmbrand; hemorrhagic necrosis (gangrene; infarction) of intestine; ischemic enteritis or enterocolitis;* neutropenic enterocolitis;* pseudomembranous colitis. note: the name "pseudomembranous enterocolitis" is descriptive; the condition may be infectious, ischemic, or both. if the intestinal changes are clearly ischemic, see above under "enterocolitis, ischemic." if the cause is in doubt and if pseudomembranes can be identified, follow the procedures described here. (l) collect all tissues that appear to be infected. for other infectious intestinal diseases, see under specific names, such as "enterocolitis, neutropenic" or "enterocolitis, staphylococcal." intestinal tract and mesentery other organs collect material from pseudomembranes for culture and for c. difficile toxin assay. sample intestinal wall with pseudomembranes for histologic study. if the condition is suspected to be caused by ischemia, follow procedures described above under "enterocolitis, ischemic." note: myoclonic seizures also have been described in a number of progressive encephalopathies with complex neurological symptoms, such as gmi and gm2 gangliosidosis,* and niemann-pick* and krabbe's disease but also acquired disorders, including alzheimer's disease,* creutzfeldt-jakob epilepsy, myoclonus (continued) disease,* posthypoxic encephalopathy, and subacute sclerosing panencephalitis. mitochondrial encephalomyopathy also can present with myoclonus epilepsy and a mitochondrial myopathy with ragged red fibers (merrf syndrome) in skeletal muscles. lafora-body-type material in the heart, liver, retinas, peripheral nerves, skeletal muscles, and sweat gland ducts (especially axillary). ragged red fibers in mitochondrial myopathies. epilepsy, symptomatic note: possible causes or underlying conditions include cerebrovascular diseases, congenital malformations ofthe brain, degenerative and demyelinating diseases of the brain, head injury,* intracranial and cerebral infections, toxic or metabolic disorders (alcoholism,* barbiturate,* carbon monoxide,* and lead poisoning,* hemodilution, hypocalcemia, or hypoglycemia),* and tumors of the brain. * f failure, congestive heart note: coronary atherosclerosis and manifestations of ischemic heart disease, * valvular heart disease, congenital cardio-vascular diseases, and manifestations of systemic or pulmonary hypertension are the most frequent findings in patients dying of or with congestive cardiac failure. other causes include cardiomyopathies* and secondary myocardial disease (such as amyloid or pericardial constriction). if the cause of the congestive cardiac failure is unknown or not immediately evident after dissection of the heart and of the great vessels, myocardium and other appropriate tissues may be submitted for microbiologic study-including viral cultures-and for electron microscopy. specimens can also be snap-frozen for possible immunofluorescent, biochemical, or histochemical studies, particularly of the myocardium. possible causes of congestive cardiac failure are too numerous to mention. dilatation of heart, with or without mural thrombosis. myocardium may be soft, normal, or firm. pulmonary congestion, with or without hemosiderosis; congestion of viscera with organomegaly. other organ manifestations include bowel edema or hemorrhagic enteropathy (without mechanical vascular occlusion) and zonal hepatic steatosis, fibrosis, or necrosis, with or withoutevidence of liver failure. acute renal tubular necrosis may be present also. synonyms and related terms: acute kidney failure; chronic kidney failure; renal failure; uremia. note: if acute kidney failure had been diagnosed, the autopsy procedures will depend on the expected causes, such as poisoning with ethylene glycol,* lead,* mercury,* or methyl alcohol;* disseminated intravascular coagulation* and its various underlying conditions; glomerulonephritis* and its various underlying conditions; diabetes mellitus;* or multiple myeloma. * the procedures described below deal primarily with chronic renal failure. if the patient had had dialysis, see also under "dialysis (for chronic renal failure )." if transplantation had been carried out, see also under "transplantation, kidney." lassa fever is a highly communicable disease and autopsy studies are not recommended in the usually available surround-ings. if lassa fever is suspected, contact the state health department and the centers for disease control and prevention, atlanta, ga, for disposition and further studies (1).ifan autopsy is done, disinfection can be accomplished by washing instruments with 0.5% phenol in detergent (i.e., lysol), 0.5% hypochlorite solution, formalin, or paracetic acid. for shipping procedures, see chapter 15. this is not a reportable disease. synonyms: borreliosis; louseborne (epidemic) relapsing fever; tickborne (endemic) relapsing fever. note: (i) collect all tissues that appear to be infected. (2) rat/mouse inoculation with infected blood is the most sensitive method for the detection of the organism. consult the state health department. (3) before the autopsy, consultation with the microbiology laboratory is advised. (4) request direct dark-field examination and giemsa or wright stain. (4) no special precautions are indicated. (5) serologic studies are of questionable value due to the antigenic variability of the organism. (6) fever, scarlet note: usually, this disease is a nonfatal group a streptococcal tonsillitis and pharyngitis with skin rash. potentially fatal complications include suppurative otitis media,* mastoiditis, and pharyngeal abscess (1), with or without septicemia. (1) collect all tissues that appear to be infected. (2) atresia, but may also be seen with viral myocarditis, type ii glycogen storage disease (pompe disease) and carnitine deficiency. thus, a metabolic disorder must be considered. it is ideal to perform the autopsy as soon after death as possible (1) . urine plasma for removal, prosthetic repair, and specimen preparation, see chapter 2. malnutrition (1) muscle necrosis (clostridial myonecrosis) and accumulation of gas; little leukocytic infiltration. other organs g procedures depend on expected findings or grossly identified abnonnalities as listed in right-hand column. see also above under "note" and under "skeletal muscles." synonyms and related terms: acute postinfectious glomerulonephritis (nonstreptococcal postinfectious glomerulonephritis;*minimalchangedisease;mesangialproliferativeglomerulonephritis;* focal and segmental glomerulosclerosis with hyalinosis (focal sclerosis); poststreptococcal glomerulonephritis; idiopathic nephrotic syndrome; iga nephropathy (berger's disease); membranous glomerulonephritis; membranoproliferative glomerulonephritis; mesangial proliferative glomerulonephritis; rapidly progressive glomerulonephritis (associated with systemic infectious or immunologic multisystem diseases; drug idiosyncrasy; or as primary crescentic glomerulonephritis or superimposed on another primary glomerular disease). possible associated conditions: acquired immunodeficiency syndrome (aids);* alport's syndrome;* amyloidosis; anaphylactoid purpura; bee stings; chronic allograft rejection; dennatomyositis;* dennatitis herpetiformis; diabetes mellitus;* drug dependence;*fabry's disease;* goodpasture's syndrome;* guillain-barre syndrome;* henoch-schonlein purpura;* hemolytic uremic syndrome;* infective endocarditis;* leprosy;* malig-nancies;mixedconnectivetissuedisease;myxedema;polyarteritis nodosa;* rheumatoid arthritis;* preeclamptic toxemia; renovascular hypertension; sarcoidosis;* serum sickness;* sjogren's syndrome;*syphilis;*systemiclupuserythematosus;*systemic sclerosis;* thrombotic thrombocytopenic purpura;* thyroiditis;* vasculitis; viral hepatitis;* wegener's granulomatosis;* and many other conditions. urate deposits in scleras and corneas. granulocytopenia note: midline granulomas may belong to the angiocentric immunoproliferative lesions, which are related to lymphomatoid granulomatosis* and malignant lymphoma. the name "idiopathic midline granuloma" should be reserved for the few cases without evidence of malignant lymphoma or wegener's granulomatosis* (1). the diagnosis of idiopathic midline granuloma also can be ruled out if studies reveal fungal organisms or features of leishmaniasis;* leprosy,* rhinoscleroma, pseudotumor of the orbit or tuberculosis. * complications of nasal cocaine abuse also may mimic midline granuloma (2). eosinophilic pneumonitis (degenerating eosinophils with charcot-leyden crystals) and granulomas. angiitis (mostly arteritis), typically with giant cells in tunica media (1) . findings resemble those in polyarteritis nodosa. * heart (2), grastrointestinal tract (3), skin, muscles, and joints are commonly involved. however, renal disease is often (but not always) mild or absent. necrotizing vasculitis of small arteries and veins is present, with extravascular granulomas and eosinophilic infiltration of vessels and perivascular tissues. optic neuritis may be found (4). may be affected by the vasculitis (4). pcr studies on paraffin sections via rna in situ hybridization may confirm presence of epstein-barr virus-positive b-cell proliferations combined with dense t-cell accumulations (3) (4) (5) . the condition closely resembles angiocentric t-/nk cell lymphoma (3). infiltration of lymphocytoid cells, plasma cells, and macrophages with necroses and granulomatous features, which are found primarily in the vicinity of blood vessels. special stains may reveal evidence of infection. lymphoreticular and granulomatous infiltrates (see "lung"). lymphoreticular and granulomatous infiltrates (see "lung"). characteristic infiltrates may be present in all organs and tissues. involvement of spleen, lymph nodes, and bone marrow is uncommon. in rare instances, the disease is confined to the abdomen. in most instances, characteristic infiltrates are present (6). 4 septicemia; circulating immunoglobulin complexes, and antiendothelial antibodies (7). angiocentric granulomatosis (4) hemorrhage, intracranial (see under "hematoma, subdural" and under name of suspected underlying condition, such as "aneurysm, cerebral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemorrhage, subarachnoid (see under name of suspected underlying condition, such as "aneurysm, cere-bral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemosiderosis, idiopathic pulmonary note: this diagnosis is made by exclusion; involvement of organs other than the lungs (see below under "kidneys") suggests another disease. hepatitis, chronic synonyms and related terms: autoimmune hepatitis; chronic viral hepatitis b (with or without d) and c. note: the term "chronic hepatitis" is not a complete etiologic diagnosis and related names such as chronic active (aggressive) hepatitis, chronic active liver disease, and chronic per-sistent hepatitis are obsolete. most cases in these categories represent autoimmune hepatitis or chronic viral hepatitis b or c. many other liver diseases, including drug-induced hepatitis, inborn errors ofmetabolism (e.g., alphal-antitrypsin deficiency* or wilson's disease*), developmental disorders, and chronic biliary diseases such as primary biliary cirrhosis or primary sclerosing cholangitis also may present as chronic hepatitis. ifchronic hepatitis was the cause ofdeath, submassive hepatic necrosis or cirrhosis* is usually present, often with manifestations ofportal hypertension,*hepatic encephalopathy, hepatorenal syndrome,* and with ascites or spontaneous bacterial peritonitis. possible associated conditions: see also below under "possible or expected findings." conditions that may be associated with hepatitis c include (1): autoimmune hepatitis; behcet's disease; diabetes mellitus (type 2);* glomerulonephritis;* guillain-barre syndrome;* idiopathic pulmonary fibrosis; idiopathic thrombocytopenic purpura; iga deficiency; lichen planus; mixed essential cryoglobulinemia; mooren's corneal ulcers; polyarthritis; porphyria cutanea tarda;* thyroiditis. * note: coinfection with other hepatitis viruses (e.g., c and g) and/or systemic viruses such as the immunodeficiency virus are common, particularly in drug addicts (1, 2) . in many cases of fulminant hepatitis, tests for known hepatitis viruses are negative (3, 4) . homicide note: not all of the following procedures can be carried out or will be required in all cases, nor will this checklist be sufficient in all instances. consult also the entries indicating the cause of death, such as "injury, firearm" or "injury, stabbing." if the victim is an infant, see all under "infanticide." before the body arrives or before autopsy is begun: 1. investigate the scene where the body was found and where the crime may have been committed (these may be two separate locations). if this is not possible, study the report and photographs submitted by the investigator or the police. study all available information. request previous medical records and roentgenograms of the victim. 2. emphasize to first responders and autopsy personnel that the body of the victim should not be undressed, washed, or otherwise disturbed until it has been inspected by the pathologist. embalming is not permitted before completion of the autopsy. advise first responders or transporters to put paper bags over the hands of the victim. this will protect possible evidence, such as hair from the assailant. 3. prepare record sheets to document the time of arrival and release of the body; chain of custody for the body and specimens; the name, age, sex, and other information about the victim; the names ofthe technician, pathologist and guests; and the specimens taken. 4. prepare roentgenographic and photographic equipment. after body arrives but before autopsy is begun: 1. if the body is left unguarded-for instance, overnight-a lock should be placed on the refrigerator or cool room where the deceased is kept, and the key should be retained by the technician. 2. if the pathologist is not accustomed to a busy autopsy room, he or she should feel free to ask all persons other than the pathologist(s), technicians, and law enforcement personnel who are not immediately involved in the actual performance of the autopsy to leave the morgue. 3. the body temperature of the victim can be recorded at the autopsy facility, but this is rarely useful, particularly if the victim seems to have been deadfor longer than a day ortwo or ifthe body had previously been stored in arefrigeratoror cool room. insert thermometer deep into the anus (about 7-8cm). record temperature and manner and time of procedure. 4. aspirate vitreous from one or both eyes and store the specimen in a refrigerator. 5. prepare roentgenograms. in each case, a decision must be made whether roentgenograms should be made of the entire body or only of parts of it-or not at all-and whether they should be made before the victim is undressed, after the victim is undressed, or at both times. 1. take overall survey photographs that depict the anterior surfaces of the body as it is on arrival to the facility, before any undressing, manipulation for roentgenographs, or cleaning. 2. take close-up photographs of any trace evidence such as fibers or flakes of gunpowder before undressing or cleaning. 3. photograph the face of the victim for identification purposes after it has been cleaned of any blood and foreign matter. to avoid perspective distortion from wide angle lenses, the lens should be about 4 ft from the face. 4. photograph all injuries and other forensically significant findings after blood and foreign matter have been cleaned off the body. a moist towel is useful; brushes are too abrasive. two photographs should be taken of each finding. one should include the autopsy number and a scale. a second photograph should be taken without any extraneous objects. collection and documentation of evidence: 1. fingerprinting can be done before or after the autopsy but should be done in all homicide cases. in cases involving close contact between assailant and victim, fingernail scrapings or clippings (use a new clipper) and hair with roots should be collected, and its source should be identified (hair pulled from scalp, axillae, and pubis is identified as that of the victim; hair in hands or under fingernails or on clothing of victim may be from the assailant). hair exemplars can be collected in cases of homicide by firearm but are rarely of use. if the body is decomposed or mutilated or for any other reason has not been identified, prepare roentgenograms ofthe head, neck, and torso before the autopsy (the radiographic appearance is altered by the autopsy). these can be used for comparison purposes if antemortem films are located. if films of the extremities are needed they can be prepared after the autopsy. dental roentgeno-.grams are taken, usually after the autopsy, when investigators have located antemortem dental records. detailed descriptions and sizes of clothing, with photographs of clothing, can be useful then there is no putative identity. 3. in cases in which sexual assault may have been involved, collect pieces of clothing that may contain seminal fluid stains. follow procedures described under "rape." 4. preserve clothing or part of clothing as evidence. wet clothing should be dried before it is stored in labeled and sealed bags. the autopsy: 1. the measured length and weight, extent of rigor, and color and distribution of livor should be recorded, along with the state of preservation, nutrition, and hydration. do not omit examination of the hands, particularly of the volar surfaces. 2. if air embolism is suspected, see procedure described under part ii "embolism, air". if pneumothorax is suspected, see procedure described under part ii "pneumothorax". if there is evidence of strangulation or other neck injury, perform a layerwise dissection of the neck that includes the hyoid bone and tongue. 3. prepare diagrams of wounds and identify their location by anatomic region. for wounds of the torso, state the distance from the soles of a foot and the distance laterally from to the right or left of the midline. 4. submit samples of wounds for histologic study when there is any question of the age of the wounds. 5. the records should show when body fluids and tissues were collected for laboratory analysis, the volume and appearance of such specimens, and what was done with them. if most of the toxicology specimens are collected immediately after the internal examination has commenced, the time of the internal examination serves as the collection time. in all instances, the following items should be collected: blood ofvictim for dna comparison and toxicologic study (determination of alcohol, carbon monoxide, and drug concentrations will be requested most frequently); vitreous; urine; gastric contents; at least one solid organ specimen for toxicologic study (liver and brain have the most comparison data if an extracerebral congenital malformation is suspected, follow procedures described under "malformation, arnold-chiari." if cerebrospinal fluid is aspirated with a syringe, record volume. record weight of brain; record size of brain in relation to inner dimensions of skull. describe size of ventricles. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. if a surgical shunt is present, its location should be recorded and both ends of the implanted specimen that was used for shunting may be submitted for microbiologic study. if the shunt is not patent, record site and nature of obstruction. related terms: antibody-mediated hydrops fetalis; erythroblastosis fetalis;* nonimmune hydrops fetalis. note: the classic example of antibody mediated (rh incompatibility) hydrops fetalis is hemolytic disease of the newborn (erythroblastosis fetalis*). however, nonimmune hydrops fetalis also may be caused by hematologic disorders, e.g., alpha-thalassemia* (1) or it may have no known cause. infec-tions, e.g., with human parvovirus bl9 (2), cytomegalovirus, or syphilis;* heart and vascular diseases (3) (cardiac tumors, cardi-omyopathy,* myocarditis,* arterial calcification, and others); storage disease (4); tumors (including neonatal leukemia*); and many other fetal (e.g., congenital chylothorax* or lymphatic dysplasia; pulmonary sequestration and cystic adenomatoid malformation) or maternal conditions, e.g., maternal thyrotoxicosis, also may cause nonimmune hydrops fetalis. if coronary insufficiency or myocardial infarction is suspected, follow procedures described under "disease, ischemic heart." for coronary arteriography, see chapter to. record distribution of atherosclerotic lesions in aorta. samples for histologic study should include aorta, coronary arteries, and peripheral arteries. see also above under "heart." submit samples of head, corpus, and tail for histologic study. if applicable, see also under "diabetes mellitus." for special procedures and stains, see above under "heart." other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter 4. obesity* is a common finding. eruptive xanthomas (palms, elbows, knees) in some but not all types of hyperlipoproteinemia. most prominent in apoprotein cii deficiency. xanthomas of tendons (knees, elbows, dorsum of hands), xanthelasmas, and arcus comeae in familial hypercholesterolemia. gangrene of lower extremities (see below under "arteries"). severe coronary atherosclerosis and myocardial infarcts in type 3 hyperlipoproteinemia and multiple lipoprotein-type hyperlipidemia. atherosclerosis of abdominal aorta and its branches and of carotid arteries. coronary atherosclerosis (see above). pancreatitis* in familial apoprotein cii deficiency. foam cells with triglycerides in liver, spleen, and bone marrow in familial lipoprotein lipase deficiency. manifestations of diabetes mellitus* and hypothyroidism* in some cases of type 3 hyperlipoproteinemia. cerebral infarct (stroke*) in type 3 hyperlipoproteinemia. hypernatremia (see "disorder, electrolyte(s).") hyperoxaluria synonyms and related terms: oxalosis; primary hyperoxaluria type i (a1anineglyoxylate aminotransferase deficiency); primary hyperoxaluria type ii (d-glyceric acid dehydrogenase deficiency); secondary hyperoxaluria (see under "note"). note: in ethylene glycol poisoning,*oxalatecrystals in media ofsmall arteries, with associated ischemic lesions. similardeposits may occur after long-term hemodialysis (1). these conditions must be distinguished from the genetic disease. also, oxa-iate nephropathy may be a complication of short bowel syndrome. if patient with congenital hyperoxaluria underwent liver or combined liverlkidney transplantation (2) , see also under these headings. remove vitreous for biochemical evalvation. submit tissue (i.e., fascia lata) for karyotype analysis. polycystic ovaries, testicular adrenal rests (4 note: there are no diagnostic autopsy findings for hypoxia. possible causes of acute hypoxia include anesthesia-associated death,' compression of the torso by a vehicle, diving accident,' exposure to toxic gases-forinstance, carbon monoxide poisoning;' mechanical failure ofan oxygen supply system, as in an airplane; and sudden mechanical airway obstruction,' as in aspiration ofa foreign body orstrangulation. causes ofchronic asphyxia include prolonged exposure to high altitude and chronic pulmonary disease. profound medial hypertrophy of small pulmonary arteries and of pulmonary veins from chronic exposure to hypoxia. acute pulmonary edema may also occur in chronic hypoxia. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. malnutrition. empty and collapsed colon; small amounts of gray and dry meconium in terminal ileum; meconium masses in dilated and hypertrophied mid-ileum; liquid contents in proximal intestine (1); appendicitis; bowel perforation. glandular inspissation and atrophy of intestinal mucosa. volvulus, infarction, necrosis, perforation, peritonitis, and acquired intestinal atresia may be present. absence of neural plexuses in congential megacolon* (hirschsprung's disease). manifestations of cystic fibrosis, with or without cirrhosis;* biliary atresia* (3). immunodeficiency (see "syndrome, acquired immunodeficiency" and "syndrome, primary immunodeficiency.") impression, basilar note: basilar impression constitutes an upward bulging of the margins of the foramen magnum. when occipital condyles are displaced above the plane of the foramen magnum, basilar invagination is present. possible associated conditions: klippel-feil syndrome;* osteogenesis imperfecta;* osteomalacia;* paget's disease of bone;* rheumatoid arthritis; rickets; syringobulbia; syringomylia. * compression and secondary ischemic injury to lower brain stem and spinal cord. see also above under "skull and spine" and under "possible associated conditions." incompetence,â�¢â�¢â�¢ (see "insufficiency,..â�¢") related terms: battered-child syndrome; child-abuse or child-neglect death. note: vitreous should not be aspirated until the skull has been opened and trauma ruled out, because there is a risk of artifactual damage to the retina. in the presence of craniocerebral trauma the pathologist can opt to examine the eyes by removal, fixation, and histologic study; or retinal photography. follow general procedures described under "homicide" and, if necessary,. the procedures described under "rape." external examination see above under "note." prepare roentgenograms of entire body. see above under "note." in other situations, or after study of the fundus, submit sample of vitreous for chemical and possible toxicologic study. umbilical cord attachment prepare histologic sections of umbilicus. and end of umbilical cord blood submit sample for toxicologic study. consider retaining dried blood on filter paper for possible dna testing for paternity investigation. if there is a possibility that the cadaver represents a stillbirth, see part ii, "stillbirth". if infant is thought to have died shortly after birth, determine the location of air in the intestinal tract; roentgenograms may be helpful. save stomach contents and record amount. other organs see also under "homicide." in the abused child, bruises, hematomas, burn marks, or other patterned injuries. in any child, diaper rash, mongolian spots, self-inflicted fingernail scratches, skin infections, and emaciation. in the previously battered child, fractures in various states of healing. in hypertonic dehydration,' sodium concentrations more than 150meq/l. this may be caused by organic disease, improper medical treatment, or physical neglect. if infant was born alive, inflammatory changes may be found, depending on survival interval. for the hydrostatic lung test, see "stillbirth." the test is unreliable. extraneous material in air passages indicates that child was alive. air reaches the stomach after 15 min, the small intestine after 1-2 h, the colon after 5-6h, and the rectum after l2h. there is no difference in the speed of gas propulsion between full-term and premature infants. bacterial gas production and previous resuscitation attempts are potential sources of errors. in questionable stillbirth, presence of milk proves that infant was alive and had been nursed. traumatic lesions and signs of neglect may be present. unilateral ulcers in visceral zoster. in rare instances, diffuse meningoencephalitis may occur. limited necrotic and inflammatory lesions in the cord or brain stem at the level of the affected ganglion are common. posterior hom myelitis (3). ganglion cell necrosis; lymphocytic infiltration, hemorrhage, and, later, fibrosis. as a rule, only one ganglion is severely involved, but less severe lesions may occur in the ganglia that are immediately adjacent. diffuse lymphocytic infiltration; demyelination; axonal destruction; fibrosis. conjunctivitis; keratitis; iridocyclitis; retrobulbar neuritis; neuroretinitis; occlusion of retinal vessels. evidence of hematologic malignancies (2) or other conditions, as listed above under "note." infection, middle ear (see "otitis media.") infection, pneumocystis carinii synonym: pneumocystosis. note: (1) collect all tissues that appear to be infected. (2) this organism cannot be cultured at present but is frequently associated with viral, bacterial, or fungal infections that can be diagnosed by culture. (3) for rapid staining of aspirates, use gram-weigert stain, which will stain cysts of pneumocystis carinii and also fungi and bacteria. pneumocystis carinii is best demonstrated with grocott's methenamine silver stain. (4) no special precautions are indicated. (5) usually, no serologic studies are available. (6) this is not a reportable disease. injury, fire (see "burns.") injury, firearm note: protect all drains of autopsy table, which will prevent accidental loss of bullets or bullet fragments. recovery of bullet fragments and pellets can also be improved by passing tissue fragments, blood, and other appropriate materials through a fine nonmetallic sieve. caution must be exercised because sharp edges andjagged projections ofbullets and bullet fragments may cause injury (1). this applies particularly to the black talon bullet. bullets and bullet fragments should not be touched with forceps or other metal instruments that may produce artifactual markings; they must be placed in properly labeled evidence containers, and the chain of custody must be preserved. from a birdshot wound, at least 10 pellets should be recovered. the firearms examiner will divide the total pellet weight by 10 and derive median pellet weight. from a buckshot wound, all pellets should be recovered. in many of these cases, procedures described under "homicide" must also be followed. do not excise wounds before completion of autopsy (see below). as an academic exercise, excised firearm wounds may be used later for analysis of metal traces by neutron activation or for tests for carboxyhemoglobin. in practice, however, a high quality photograph is adequate to establish the presence or absence of gunpowder stippling or soot deposition toward the end of establishing the range of fire. the dimensions of the stippled areas and soot deposits should be recorded. after completion of external examination, dry the wet clothing and keep as evidence. clothing may also be used for possible test firing or for stain, powder, or particle analysis. external examination if identity of victim is unknown, follow procedures described in chapter 13. prepare initial roentgenograms of the body regions that have been shot, before disrobing of body, and then lateral films of body regions with bullets. follow up with films of other body regions as needed in the course of autopsy. if the body is decomposed to the extent that the external examination cannot be relied upon to determine cutaneous gunshot perforations, prepare roentgenograms of the entire body before autopsy. record location and number of bullet holes in all layers of garments, indicating whether the involved area is bloodstained or shows gunpowder or soot. if there are several cutaneous perforations, number or letter each consecutively and refer to these numbers in all records. location of bullet holes should be described by recording distance from soles of feet or top of head, and from midline of body. prepare diagrams and photograph holes, with and without labels. photographs should show surrounding garments and extent of blood stains. in hairy areas, shave around bullet wounds for better photographic documentation. for evaluation of distance from where a shot had been fired, see fig. 11 additional roentgenograms during the autopsy may be needed to find bullets embolized to the femoral veins or down the spinal canal. systemic roentgenograms may reveal bullets from obscure entrance wounds, particularly in decomposed bodies, old bullets, bullets in the skin flaps reflected at autopsy, or bullets external to the body in clothing. bullet(s) may have been arrested in hollow viscus or blood vessel and may have been transported to distant sites by peristalsis or bloodstream ("bullet embolus"). bullets may be deflected in unexpected directions from bony surfaces. soot ("smudging") and gunpowder stippling may occur around entrance wound, depending on the muzzle-to-skin distance. there may also be an impression from a recoiling handgun or from a power piston. wounds may be stellate, round, jagged, or slit-like, with or without wide margins of abrasion. primer residues on hand of suicide victim after use of a handgun. fatal secondary injuries, particularly hemorrhages. alcohol intoxication is a frequent finding. fig. 11â·3 . bullet wounds. differences in the appearance of cutaneous wounds of entrance and of exit and differences in wounds of entrance according to the distance between muzzle and skin at the moment of fire. entrance wounds often differ from exit wounds in that the former are usually surrounded by a narrow zone of abrasion. if the muzzle of a gun is in close contact with the skin at the moment of fire, the combustion products will be blown into the wound and will not be visible on the surface. for close-range wounds, the stippling of the skin around the wound, produced by particles of burned and unburned powder, becomes progressively more dispersed as the range of fire increases and is ordinarily not perceptible if the range has been greater than 24 inches ( synonyms and related terms: acute radiation syndrome; chronic (delayed) radiation injury; radiation enterocolitis; radiation nephritis; radiation pneumonitis. note: procedures and expected findings depend on type of radiation damage, whether acute or chronic, localized or whole-body irradiation. if suspected radiation injury was associated with the administration of radionuclides such as 32p, 131 1, or 198au, follow procedures suggested in chapter 11. in fatal 355 whole-body irradiation, findings are most likely related to bone marrow injury, and the suggested procedures and expected findings are those described under "pancytopenia." record extent of oropharyngeal and intestinal ulcerations and hemorrhages. late complications include malignant tumors (carcinoma, leukemia,* lymphoma*), manifestations of hypothyroidism,* and cataracts. organ changes in localized radiation injury depend on site of irradiation (lung, brain, kidney, intestine). the skin is involved in both acute (erythema) and chronic (atrophy, epilation) radiation injury. related terms: cutting injury; knife injury. note: in many of these cases, procedures described under "homicide" must also be followed. insuffiency, adrenal synonyms and related terms: adrenocortical insufficiency; polyglandular autoimmune syndrome (type i, usually in childhood, with parathyroid insufficiency and chronic mucocutaneous moniliasis; type ii, usually in adults, with two or more autoimmune endocrine disorders such as thyroiditis and diabetes mellitus*); primary adrenal insufficiency (addison's disease); secondary adrenal insufficiency (in hypothalamic-pituitary disease or steroid induced); x-linked adrenal hypoplasia. is caused by anatomic changes (see below under "adrenal glands"), drugs (enzyme inhibitors or cytotoxic drugs), or acth-blocking antibodies. possible associated conditions: aids with systemic infections (1); antiphospholipidantibody syndrome (2); autoimmune hepatitis; chronic lymphocytic thyroiditis; type i diabetes mellitus;* hypoparathyroidism;* hypothyroidism;* megaloblastic anemia;* surgical procedures, such as heart surgery or orthopedic procedures. large cell lymphoma (5). should be removed as soon as possible (before embalming), either from cervical midline incision or from chest (neck of cadaver must be well-extended). photograph obstruction before larynx is opened, and inside of larynx and epiglottis after larynx is is opened in posterior midline. make gram-stained touch preparations. make histologic sections of larynx and epiglottis. hemophilus injluenzae (cannot always be isolated from larynx synonyms and related terms: acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, and hairy cell leukemia. multiple subtypes have been identified; they are characterized by cell surface markers, chromosomal abnormalities, staining reactions, and morphologic features. note: at the time of autopsy, most leukemias have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent of the disease and the presence of complications. if the leukemia had not been classified or if the features might have changed from the time of the last work-up (e.g., in suspected cases of superimposed diffuse large cell lymphoma [richter's syndrome]), material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation,* see also under that heading. possible associated conditions: agnogenic myeloid metaplasia with myelofibrosis; bloom's syndrome;* cutaneous mastocytosis; down's syndrome;* immunodeficiency syndromes* (bruton's disease; louis-bar syndrome; wiskott-aldrich syndrome); infantile genetic agranulocytosis; klinefelter's syndrome;* lymphoma;* multiple myeloma;* polycythemia;* and many others. for sampling and specimen preparation, see chapter 4. increased protein concentration. material in sediment stains red with toluidine blue. metachromatic material. arylsulfatase-a deficiency. excessive loss of myelin with large amounts of metachromatic material (see above under "urine") in white matter and also in some neurons. demyelination with metachromatic material (see above under "urine"). leukoencephalopathy, progressive multifocal possible associated conditions: aids* and other immunosuppressed states; carcinoma; malignant myeloproliferative or lymphoproliferative disorder; sarcoidosis;* tuberculosis. * procedures submit portion of fresh brain for viral culture. fix remainder of brain and spinal cord in formalin and submit samples for histologic study. in situ hybridization for ic virus is available on paraffin-embedded tissue. small patches of demyelination with tendency to form confluent areas in the cerebral white matter. white matter necrosis may be present. eosinophilic intranuclear inclusions occur in affected oligodendroglia cells. subsequently, large, bizarre astrocytes develop. no inflammatory (lymphocyte) cell reaction is present. lightning (see "injury, lightning.") lipoproteinemia (see "hyperlipoproteinemia.") lipoproteinosis, pulmonary alveolar synonym: idiopathic alveolar lipoproteinosis. note: the condition has been described in allografts (1); in such cases, see also under "transplantation, lung." elephantiasis of penis, scrotum, or vulva (in chronic cases); skin rash (l) and conjunctivitis (in acute cases); genital ulcers. suppurative or fibrosing inguinal, iliac, or pelvic lymphadenitis, with or without sinus tracts. rectal strictures and fistulas in chronic cases (2) . systemic involvement in the acute stage with pericarditis,* and arthritis,* and meningitis,* proctitis (3) lymphoma synonyms and related terms: aids-related lymphoma; adult t-cellieukemiallymphoma; angioimmunoblastic lymphadenopathy; b-celllymphoma; burkitt's lymphoma; cutaneous t-cell lymphoma (includes mycosis fungoides and sezary syndrome); hodgkin's disease; non-hodgkin's lymphoma (low grade, intermediate grade, high grade, all with multiple subtypes too numerous to list, which vary in natural history); natural killer cell neoplasms; post-transplant lymphoproliferative disorders (ptld); t-cell lymphoma. note: at the time of autopsy, most lymphomas have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent ofthe disease and the presence of complications. if the lymphoma had not been classified or if the features might have changed from the time of the last work-up, material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation, * see also under that heading. for current terminologies of hodgkin's disease and non-hodgkin lymphomas as well as for staging criteria, appropriate hematologic textbooks should be consulted. possible associated conditions: acquired immunodeficiency diseases (e.g., after organ transplantation; human immunodeficiency virus-l infection); autoimmune disease (e.g., celiac sprue,*rheumatoid arthritis,* systemic lupus erythematosus,* or sjogren's syndrome*); chemotherapy with or without radiation treatment; epstein-barr virus infection; human t-cell leukemia virus infection; inherited diseases with immunodeficiency (e.g., klinefelter syndrome; * common variable immunodeficiency disease, wiskott-aldrich syndrome); radiation. the lesions are found most commonly in the urinary bladder and other parts of the urinary tract, but may also occur at many other sites, including skin (1) and upper respiratory tract (2). note: (i) collect all tissues that appear to be infected. (2) usually, cultures are not indicated. (3) request giernsa or wright stain. tissue blocks should be rinsed of blood and be as thin as possible before fixation in refrigerated buffered and neutral 10% formalin solution. this is to avoid precipitationofformalin pigment that may be confused with malaria pigment. the formalin solution should be used in a ratio of 100 parts formalin to one part tissue and should be agitated every few hours. if one is dealing with tissues that contain formalin pigment, this pigment may be removed with a bleaching solution. (4) usually, no special precautions are indicated. (5) possible associated conditions: histoplasmosis* and other chronic fungal infections; immune connective tissue diseases such as rheumatoid arthritis* (l) ; malignancies (l); sarcoidosis;* silicosis;* tuberculosis* (l) . note record location and extent of skin changes or skin defects on back. prepare skeletal roentgenograms. if meningitis is suspected, submit sample of cerebrospinal fluid for culture. dse the posterior approach to remove the spinal cord. atrophic skin over meningocele, lacking rete pegs and skin appendages; skin defect in complete rachischisis. bony defects of spine. in meningocele and spina bifida occulta, arachnoid and dura herniate through the vertebral defect. spinal cord and roots are generally not involved. lumbosacral mass in meningomyelocele, with a highly vascular mass (area medullovasculosa) in spina bifida aperta. neural defects in complete rachischisis. diastematomyelia, hydrocephalus (1) . pyelonephritis;* enlarged urinary bladder ("neurogenic bladder"). external examination heart lungs procedures prepare chest roentgenogram. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. record weights. prepare photographs and roentgenograms of fresh and fixed lung specimens. perfuse one lung with formalin. for preparation of paper-mounted sections, see chapter 2. submit one lobe for microbiologic and chemical study. decalcify tissue for histologic study. request van gieson's, hale's colloidal iron, pas, and sudan stains. miliary mottling in lower lung fields. mitral stenosis* may be a cause of microlithiasis (mostly intra-alveolar ossification). * cor pulmonale and heart failure (1) (l) , see also under that heading. (1) collect all tissues that appear to be infected. (2) viral isolation, especially with ebv, is not diagnostically useful, due to long incubation periods. (3) note: these diseases are characterized by a deficiency of a variety of hydrolases, resulting in accumulation of gly-cosaminoglycans (mucopolysaccharides) and glycolipids within lysosomes of fibroblasts, macrophages, white cells, and parenchymal cells of many organs (1, 2) . mucopolysaccharides are also excreted in the urine. the general approach is similar in all types. formalin may dissolve all of the stored material and leave empty vacuoles in the involved cells. therefore, frozen sections should be utilized or tissues should be fixed in absolute alcohol. the accumulated material will show intense metachromasia if stained with toluidine blue. it will also stain with pas, alcian blue, and colloidal iron. oil red a will also stain the material from frozen sections. for specimen preparation for elec-tron microscopy, see chapter is. characteristic external features include dwarfism; thickened long bones; coarse facial features; coarse hair; macrocephaly; prognathism; hypertelorism; malformed teeth, and scaphocephalic skull with hyperostosis of sagittal suture; short neck; chest deformity; umbilical and inguinal hernias; lower thoracic and lumbar gibbus; genu valgum and coxa valga; pes planus and other joint deformities; wide hands (clawhands) and feet. coarse thickened skin, covered with lanugo-like hair. hyperlordosis; ovoid deformities of vertebrae; odontoid hypoplasia; large, shoe-shaped sella turcica; kyphosis; hypoplasia of femoral heads; osteoporosis.* if patient underwent bone marrow transplantation (3), see also under that heading. chronic upper respiratory infections. large cytoplasmic granules in neutrophils. storage of mucopolysaccharides in osteocytes, chondrocytes, and fibroblasts of periosteum, tendons, fasciae, and other connective tissues; dysostosis multiplex. other tissues histologic sampling cannot be excessive. (1) collect all tissues that appear to be infected. (1), see also under that heading. blood; other organs and tissues record extent of skin lesions and prepare photographs; prepare sections of affected and unaffected skin. request gram stain of sections and smears. submit samples of blood and grossly affected organs or tissues for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. extensive epidermal necrosis. shedding of granular and horny layers of epidermis. septicemia; staphylococcal infection of nose, throat, ears, eyes, heart valves, urogenital tract, and other sites. bronchial epithelial detachment and bacterial pneumonia (2). synonyms and related terms: acute kidney failure; *acute tubular necrosis; lower nephron nephrosis. note: the morphologic diagnosis of this condition may be difficult to discern from autolysis. the features that suggest tubular necrosis are: tubular dilation with epithelial flattening, intertubular edema and necrotic epithelial cells in the collecting ducts. the autopsy should be performed as soon as possible. needle specimens of the kidneys obtained in the immediate postmortem period may yield acceptable material. ifnephrotoxic drugs or chemicals are thought to be responsible for tubular necrosis, submit samples for toxicologic study (see also under synonyms and related terms: multiple endocrine neoplasia (men), type 1 (parathyroid hyperplasia or adenoma; pancreatic islet cell hyperplasia, adenoma, or carcinoma; pituitary hyperplasia or adenoma); or type 2a (medullary thyroid carcinoma, parathyroid hyperplasia or adenoma; and pheochromocytoma); or type 2b (medullary thyroid carcinoma, pheochromocytoma, mucosal and gastrointestinal neuromas, and marfanoid features). note: in men, type 1, foregut carcinoids and subcutaneous and visceral lipomas also may be found. in type 2a, cutaneous lichen amyloidosis may be observed. mixed syndromes include (i) familial pheochromocytoma and islet cell tumor, (2) von hippel-lindau syndrome, pheochromocytoma and islet cell tumor, (3) neurofibromatosis* with features of men1 or 2, and myxomas, spotty skin pigmentation, and generalized endocrine overactivity (carney complex). in all instances, the autopsy should be done as soon as possible so that tissues for biochemical study can be frozen without delay. nephritis note: see under specific designation, such as "glomerulonephritis" and "pyelonephritis," or under name of suspected underlying condition, such as "gout" or "lupus erythematosus, systemic." nephroblastoma (see "tumor of the kidney(s).") synonyms and related terms: renal stones; urolithiasis. possible associated conditions: carcinomatosis; cushing's syndrome;* cystinuria;* fanconi syndrome;* hyperoxaluria;* hypervitaminosis d;* gout;* multiple myeloma;* osteoporosis;* polycystic renal disease;* primary hyperparathyroidism;* rheumatoid arthritis* (1); sarcoidosis* (2). kidneys ureters, urinary bladder, and urethra other organs remove kidneys, ureters, and urinary bladder en bloc. excise and bivalve kidneys to reveal renal pelves. open ureters. record size, number, and appearance of stones, and save for chemical analysis (4). record heart weight. dissect and record weights of all parathyroid glands. other procedures depend on suspected underlying conditions, as listed above under "possible associated conditions." nephropathy note: see under name of suspected underlying condition, such as "diabetes mellitus," "disorder, electrolyte(s)" (hypercalcemia, potassium depletion), "gout," "hypertension (arterial), all types or type unspecified," or "poisoning,.. ." (heavy metal). if kidney failure was present, procedures under that heading should also be followed. renal tissue may need decalcification or fixation in water-free solution. (2) note: in this condition, lesions in the brain and cranial nerves, spinal cord, and spinal roots may be schwannomas (including bilateral vestibular schwannomas); multiple meningiomas; gliomas (generally of spinal cord), mostly ependymomas (75%) and pilocytic astrocytomas; or schwannosis of spinal dorsal root entry zones. intracortical meningioangiomatosis; glial hamartia (intracortical, basal ganglia, thalamus, cerebellum, and dorsal horns of spinal cord) and cerebral calcifications also may be found. cutaneous abscess (j). acute necrotizing nocardial pneumonia with abscesses or sinus formation into surrounding tissues; empyema. pulmonary and mediastinallymph nodes other organs submit samples for culture and histologic study. submit samples from all organs and tissues with suspicious gross lesions for culture and histologic study. poorly encapsulated abscesses of any organ; endocarditis (2) . related term: total parenteral nutrition. note: air embolism* or blood loss may have occurred if line became detached from the catheter hub. metabolic complications such as fluid overload or disturbances of acid-base and electrolyte balance often cannot be diagnosed reliably at autopsy. the disease(s) that may have necessitated parenteral nutrition therapy are not considered here; they include the acquired immunodeficiency syndrome (aids);* cancer cachexia; inflammatory bowel disease; liver or kidney failure;* severe pancreatitis;* short bowel syndrome, and others. obstruction, acute airway synonyms and related terms: aspiration; bolus death; "cafe coronary"; croup; restaurant death. note: ifpermission has been obtained, remove neck organs through straight incision from chest to chin to avoid dislodging a foreign body. ifdissection has to be accomplished from chest, neck of unembalmed cadaver should remain well extended during procedure. inspect larynx and trachea from above and below, respectively, before opening them carefully along the posterior midline. for removal, prosthetic repair, and specimen preparation, see chapter 2. submit samples of osteocartilaginous and synovial tissues for histologic study; sagittal or frontal saw section through spine provides for best routine evaluation. heberden's nodes at interphalangeal joints of fingers. degenerative changes, primarily of spine, hip joints, and knee joints. histologic and macroscopic degeneration of cartilage; exposure of subchrondral bone; formation of marginal osteophytes; bone cysts; synovial fibrosis; hypertrophic synovitis. o procedures if artificial joints (e.g., hip or knee) had been implanted, record state of implant site. loose joint prostheses. synonyms: hypertrophic pulmonary osteoarthropathy; pac-hydermoperiostosis (idiopathic hypertrophic osteoarthropathy [1j). note: in all instances, an underlying disease must be identified; they include cardiac disease (cyanotic congenital heart dis-ease with right-to-left shunt; infective endocarditis*); gastroeso-phageal reflux (3); neoplasms of lungs, esophagus, intestine, and liver; chronic liver disease; inflammatory bowel disease;* pulmonary disease (e.g., abscess;* bronchiectasis;* cystic fibrosis;* emp-yema;* emphysema;* lipoid pneumonia* (4); pneumocystis pneumonia; sarcoidosis;* tuberculosis*); hyperthyroidism. * external examination elastic arteries other organs prepare roentgenograms of extremities. for removal, prosthetic repair, and specimen preparation, see chapter 2. record presence of aneurysms (thoracic aorta, subclavian) or arteriovenous fistula of brachial vessels. if abdominal aortic aneurysm had been surgically repaired, search for evidence of graft infection (2) . procedures depend on expected findings or grossly identified abnormalities as listed above under "note." clubbing of fingers or toes (see below under "elastic arteries"); swelling of extremities. periosteal new bone formation in distal shafts of bones of forearms and legs. all bones of extremities may be involved. see above under "external examination." unilateral clubbing. clubbing of toes but not of fingers. swelling; fistulas. bone defect(s) and focal osteosclerosis. bacterial or fungal infections, most common in metaphyseal region of bones; paravertebral or psoas abscess in osteomyelitis of spine. bacterial or fungal osteomyelitis, with or without cavitation and fistulas. trauma, surgery, insertion of prosthesis, generalized (1) or contiguous infection, inflammatory bowel disease (2), diabetes mellitus,* and peripheral vascular diseases, deep vein thrombosis (3). synonyms and related terms: aseptic necrosis of bone; avascular necrosis of bone; idiopathic osteonecrosis; perthes' dis-ease; postfracture osteonecrosis; renal transplant associated osteonecrosis. note: possible underlying conditions include chronic alcoholism,* decompression sickness,* diseases that have been treated with high doses of corticosteroids (1), gaucher's disease,* human immunodeficiency virus infection* (2), tuberculosis* (1), sickle cell disease, * systemic lupus erythematosus,* tuberculosis* (2), and bisphosphonate therapy (3). other organs submit sample for biochemical study. for removal, prosthetic repair, and specimen preparation, see chapter 2. if osteonecrosis is suspected because one of the possible underlying conditions (see above) is present, prepare saw sections through femoral heads, medial femoral condyles, and heads of humeri. procedures depend on suspected underlying conditions, as listed above under "note." osteopetrosis synonymsand related terms: albers-schonberg disease; autosomal-dominantosteopetrosis; carbonic anhydrase-it deficiency; malignant infantile osteopetrosis (1); marble bone disease. note: manifestations of renal tubular acidosis may have been a clinical complication. possible associated conditions: bone marrow transplantation* (for infantile osteopetrosis). anemia, recurrent infections, bleeding, and bruises may have resulted from myelophthisis associated with osteopetrosis. upper airway obstruction in malignant infantile osteopetrosis may have necessitated tracheostomy (1). in heritable osteoporotic disorders of connective tissue (osteogenesis imperfecta,* marfan' s syndrome,* and others) are pre-sented separately. for"osteomalacia," see above. for "osteodystrophy," see under "failure, kidney." possible associated conditions: acromegaly;* chronic alco-holism;* chronic obstructive pulmonary disease; chronic kidney failure* (1); cushing's syndrome;* debilitating disease (various kinds, often with immobilization); diabetes mellitus* (2); epilepsy;* hyperthyroidism (2);* hypogonadism; malabsorption syndrome;* malnutrition;* primary biliary cirrhosis;*rheu-matoid arthritis;* scurvy;* steroid therapy or anticonvulsant medication. normal parathyroid glands in uncomplicated cases. hyperparathyroidism* (1) (without osteitis fibrosa) may be present, however. features of osteitis fibrosa (osteoclastic osteoporosis; see "hyperparathyroidism") or osteomalacia* exclude the diagnosis of uncomplicated osteoporosis. osteoporosis, which may be localized, occurs in various neoplastic (e.g., mastocytosis) and inflammatory diseases. external examination brain and base of skull with middle ears examine external ear canal. for removal and specimen preparation, see chapter 4. culture any lesion appearing to be infectious. draining, foul-smelling greasy material associated with acquired cholesteatoma. bacterial or viral infection, with or without mastoid osteitis. neck abscess, sinus thrombosis (1) and brain abscess* and meningitis* may complicate otitis media. otosclerosis (1,3) note: otosclerosis may be a measles-virus-associated disease (1) and therefore, studies to rule out a past infection may be indicated. for removal and specimen preparation, see chapter 4. for current methods of temporal bone studies, see ref. (2) . spongy bone in the capsule of the labyrinth. trabeculae of woven bone show pagetoid changes. if stapedectomy had been done, a prosthesis may be in place. brain is externally normal or mildly atrophic. characteristic is midbrain atrophy with aqueduct dilatation and depigmentation of the substantia nigra. neuronal loss with reactive gliosis, associated with neurofibrillary tangles (globose tangles). primarily affected are globus pallidus, subthalamic nucleus, red nucleus, substantia nigra, tectum, periaqueductal gray matter, and dentate nucleus (grumose degeneration). palsy, pseudobulbar (see "disease, motor neuron.") related terms: acute pancreatitis; alcoholic pancreatitis; chronic (or chronic fibrosing) pancreatitis; interstitial pancreatitis. note: some causes of pancreatitis such as adverse drug reactions (e.g., due to azathioprine, furosemide, or estrogens) cannot be diagnosed at autopsy. possible associated conditions: acute fatty liver of pregnancy; apolipoprotein cli deficiency; cystic fibrosis; *hyperparathyroidism;* kidney transplantation. * status post endoscopic retrograde cholangiopancreatography. synonyms and related terms: calcifying panniculitis; histiocytic cytophagic panniculitis; mesenteric panniculitis (mes-enteric lipodystrophy); "sclerema neonatorum." note: the term weber-christian disease described systemic panniculitis with fever, bleeding, pulmonary and pancreatic lesions, and other abnormalities also involving lungs and pancreas, among others. however, this condition is not a specific entity and thus, the name has become obsolete. the term histiocytic cytophagic panniculitis is more descriptive and preferred. leukemia* and subcutaneous t-celllymphoma* may closely simulate panniculitis. possible associated conditions: alphaj-antitrypsin deficiency;* dermatomyositis;* rheumatoid arthritis;* scleroderma and morphea; sjogren's syndrome;* systemic lupus erythematosus.* external examination, skin, subcutaneous tissue, and breasts chest heart lungs liver and spleen record extent and character of skin lesions. record size, location, and gross appearance of subcutaneous nodules. submit tissue samples for histologic study of grossly unaffected skin, skin lesions, and subcutaneous nodules. request verhoeff-van gieson, gram, and grocott's methenamine silver stains. request s-l 00 protein stain. ascertain that cell infiltrates are not leukemic or lymphomatous (1) . dimpling of skin; necroses; fistulas. panniculitis with subcutaneous nodules in trunk, breasts, and thighs (5) . calcification may occur in breast tissue but also at other sites (e.g., in kidney failure*). pancreatic enzymeinduced fat necroses associated with severe pancreatitis. widespread hardening of fat tissue with rupture of fat cells and crystal formation in "sclerema neonatorum." focal traumatic panniculitis also may occur in newborns. s-ioo stain negative in panniculitis but positive in rosai-dorfman disease (sinus histiocytosis with massive lymphadenopathy). mediastinal panniculitis. pericarditis;* interstitial myocarditis. * pneumonia;* pleuritis. fat embolism. * features of alphal-antitrypsin deficiency* (2) . fatty changes of liver; splenitis. intestinal mucosal erosions and ulcers, with or without perforation. massive gastrointestinal hemorrhage in histiocytic cytophagic panniculitis. blind intestinal loop (3). for sampling and specimen preparation, see chapter 4. prepare samples for electron microscopy. vacuolar myopathy. (1) . external examination, skin, and mouth record extent of skin lesions; photograph; submit multiple samples for histologic study, preferably of areas that are free of secondary infection. prepare sections for immunofluorescent staining. bullous and other lesions of scalp, eyelids, nose, axillae, umbilicus, inframammary areas, back, hands, groins, genitalia, anus, knees, and feet. similar lesions may occur in the mouth. acantholysis is suprabasal or near granular layer. igg deposits on the surfaces of keratinocytes. note: (i) collect all tissues that appear to be infected. collect inguinal, popliteal, axillary, and supraclavicular lymph nodes for aerobic culture. photograph enlarged lymph nodes, and prepare smears of fresh cut surfaces. submit consolidated areas for microbiologic study (see above under "note"). perfuse both lungs with formalin. submit samples of pulmonary tissue and bronchial lymph nodes for histologic study. submit samples of grossly abnormal organs, exudates, or drainage fluids for culture and gram stain. hemorrhagic necrosis; variable suppuration. severe hemorrhagic edema; pneumonia with lobular to lobar features. large areas of necrosis teeming with organisms and minimal to suppurative inflammation. plasmacytoma (see "myeloma, multiple.") platybasia note: possible causes or associated conditions include arnold-chiari malformation;* basilar impression* orinvagination; fusion ofatlas to the foramen magnum; klippel-feil syndrome;* malpositioning of odontoid process; osteitis deformans (paget's disease of bone*); osteogenesis imperfecta;* osteomalacia;* rickets; syringobulbia, syringomyelia. * skull and spine prepare roentgenograms of skull and cervical spine. flattening of the base of the skull (angle formed bythe planeoftheclivus andtheplane of the anterior fossa exceeds 135â°). pleuritis (see "effusion(s) and exudate(s), pleural.") pleurodynia, epidemic synonyms: bornholm disease; devil's grip; epidemic myalgia; epidemic myositis. blood heart and pericardium submit samples for viral culture. sample for histologic study and for viral cultures (coxsackievirus a and b; echoviruses). if pericardia! fluid can be obtained, submit for viral culture also. cultures may be negative and search for viral rna by in situ hybridization may be indicated. chest organs other organs dissect thoracic duct and its tributaries (see chapter 3). perfuse one or both lungs with formalin. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit samples of cystic lesions for histologic study. submit samples of cystic lesions for histologic study. gas in thoracic duct. emphysema* of lungs. necrotizing enterocolitis in premature infants. pyloric stenosis, colitis (5), redundant sigmoid colon, ischemic bowel disease. mucosal pseudolipomatosis (6) . usually, cysts are subserosal in adults and located between muscularis mucosae and muscularis propria in infants and children. mucosa may be inflamed. extraintestinal cysts, as listed above under "abdomen." cysts may also occur in vaginal mucosa. include bright-field and polarized light microscopy, transmission electron microscopy, scanning or transmission electron microscopy with energy dispersive x-ray analysis (2), ion beam instrumentation, and secondary ion mass spectrometry. the last three methods are time-consuming, require much skill and expensive equipment, and do not lend themselves well to quantitation. ideally, bulk analysis and in situ microanalysis should be used in combination. analyses can be conducted in specialized laboratories at the following centers (for charges and other information, consult the appropriate laboratories): prepare chest roentgenogram. submit sample for microbiologic study. refrigerate sample for possible immunologic study. submit one lobe or samples of all lobes for bulk analysis and for in situ microanalysis (see above under "note"). microincineration may permit preliminary dust analysis. for demonstration of asbestos fibers, see chapter 2 and ref. (4) . submit one lobe or segment for microbiologic study. for pulmonary arteriography and bronchography, see chapter 2. prepare roentgenograms of entire lungs and slices. perfuse one lung (or both, if only routine studies are intended) with formalin. submit samples for histologic study of nodular dust lesions, diffuse fibrotic lesions, grossly uninvolved areas, bronchi, and bronchopulmonary lymph nodes. for preparation of paper-mounted sections, see chapter 2. prepare samples for transmission and scanning electron microscopic study. for removal, prosthetic repair, and specimen preparation, see chapter 2. alveolar rupture with dissection of air into the mediastinum in neonates with respiratory distress (see "syndrome, respiratory distress, of infant") and in adult patients ventilated on volume respirators. external examination prepare chest roentgenogram. photograph mediastinum. explore major veins and record compression in cases of tension pneumomediatinum. roentgenograms provide the best permanent record of a pneumomediastinum. air bubbles in mediastinal soft tissues. part ii / diseases and conditions abdomen and neck organs perfuse one lung with formalin. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. search for evidence of trauma of other lesions that may have allowed air to enter soft tissues. intubation injury (j); perforation or rupture of major bronchus, trachea, or esophagus. alveolar rupture, e.g., in asthma* (2), may not be discernible. bronchiolitis obliterans (3), interstitial pneumonia* (4) and other lung conditions also may lead to pneumomediastinum. dissection of air from lesions in abdomen (e.g., retroperitoneal colonic perforation [5] ) or in neck area. pneumonia, all types or type unspecified note: if the type of underlying infection is known, follow procedures suggested under the name of the infectious disease. if the etiologic agent of the pneumonia is unknown, proceed as follows: (l) collect all tissues that appear to be infected. and grocott's methenamine silver stains. (4) special precautions may be required (see chapter 6). (5) serologic studies may be helpful once a specific etiologic agent is suspected. thus, collect serum at the time of autopsy or procure serum that was collected prior to death. (6) this may be a reportable disease. related terms: acute interstitial pneumonia (hamman-rich syndrome); desquamative interstitial pneumonia (dip); idiopathic organizing pneumonia (or "bronchiolitis obliterans with patchy organizing pneumonia [boop]" or "cryptogenic organizing pneumonitis"); idiopathic pulmonary fibrosis; lymphoid interstitial pneumonitis (lip); nonspecific interstitial pneumonia (nsip) (or "cellular interstitial pneumonia" [eip]); usual interstitial pneumonia (uip); fibrosing alveolitis; pulmonary alveolitis; and many others. note: the conditions listed under "related terms" are histologic variants of idiopathic interstitial pneumonia. vip is synonymous with idiopathic pulmonary fibrosis (ipf) (1). diffuse alveolar damage is the histologic finding in patients with the adult respiratory distress syndrome* (ards) causing interstitial and intra-alveolar fibrosis. this is an acute condition, referred to as acute interstitial pneumonia when it occurs as an idiopathic form of rapidly progressive interstitial pneumonia. possible associated conditions: acquired immunodeficiency syndrome* (aids) in patients with with lip or nonspecific interstitial pneumonia (2,3); trauma and shock in ards. secondary pulmonary fibrosis from inhalants (extrinsic allergic alveolitis or pneumonia; pneumoconiosis*), in drug-induced pneumonia, hypersensitivity pneumonitis (microgranulomatous hypersensitivity reaction of lung), rheumatoid arthritis,* sjogren's syndrome,* and other collagen-vascular diseases; primary biliary cirrhosis in patients with nonspecific interstitial pneumonia (2). external examination postmortem chest roentgenograms provide the only reliable and permanent record of a pneumothorax and its main complication, mediastinal shift due to a tension pneumothorax (fig. 11-4) . if roentgenograms cannot be prepared, a reasonably reliable diagnosis still can be made if the prosector inserts a needle through the lateral chest wall. the needle should be connected to a water-filled flask. ifa pneumothorax is present, gas bubbles appear in the flask as shown in fig. 11 -5. one can also expose the intact parietal pleura and observe if the lung tissue is separated from the parietal pleura by gas. incising the thorax at the base of a water-filled skin pocket is the least reliable method. infants can be totally submerged under water before the chest cavity is incised. however, care must be taken not to make an accidental incision into the underlying lung tissue. poisoning, all types or type unspecified note: if a specific substance is suspected-for instance, arsenic or ethylene glycol-follow procedures described under the appropriate entry. similar entries can be found for poisons whose general character or source is known-for instance, gas or mushroom poisoning. for some substances, the appropriate entry can be found under "abuse,...," "death,...," or "dependence, ..." in all instances, routine sampling of toxicologic material should be done as described in chapter 13. if no specific substances can be incriminated, the toxicologist must be provided with all available clinical information, as shown in chapter 13. in most cases of fatal poisoning, the coroner or medical examiner must be notified. poisoning, alkaloid note: see under specific name of alkaloid-for instance, "dependence, cocaine," "poisoning, atropine," "poisoning, digitalis," or "poisoning, strychnine." only a fraction of this large group of plant poisons has been listed. whether the specific name of the alkaloid is known or unknown, complete toxicologic sampling is recommended. poisoning, antimony ote: toxicologic material should be submitted for anal ysis, as suggested under "poisoning, arsenic." iatrogenic antimony toxicity may occur after treatment with antimony compounds for conditions such as filariasis, fungal infections, and schistosomiasis.* external examination and eyes pharynx and gastrointestinal tract heart, liver, and kidneys record skin changes and eye abnormalities. for toxicologic sampling of contents, see chapter 13. submit tissue samples for histologic study. request frozen sections for sudan stain. if exposure was from du t in smelting work, dermatitis and conjunctivitis may be present. severe gastroenteritis in acute poisoning. if victim drank antimony trichloride, ulcerative pharyngitis and gastritis may be present. fatty changes of myocardium and hepatic and renal parenchyma. poisoning, arsenic note: toxicologic material will be contaminated by bringing it in contact with fluids. keratinized tissues take up arsenic from solutions. put plastic bags over hands of victim. if exhumed bodies are investigated for arsenic poisoning, include material from surrounding soil and coffin along with tissues submitted for chemical analysis. interpretation of toxicologic findings: after fatal poisoning, arsenic concentrations in the liver tend to exceed 0.5-1.0 mg/ioo g wet tissue. in acute poisoning, arsenic concentrations in hair may reach 3 !1g/g (1) and in nails 8 !1g/g. organs collect all contents, particularly in accidental poisoning in children. body dry and warm after death. mydriasis. iatrogenic atrioventricular block (1) . fruits of atropa belladonna or seeds of datura stramonium may be found. no characteristic findings. poisoning, barbiturate(s) note: this type of poisoning has become uncommon. barbiturates may cause sudden death. in all instances, concomitant alcohol intoxication* must be ruled out. standard toxicologic sampling is sufficient. blood bile urine esophagus and stomach liver and brain procedures submit samples of blood from portal vein and peripheral veins or heart for toxicologic study. refrigerate for possible toxicologic study. record total volume and ph value. request tests for protein, glucose, and ketones; request drug screen. submit all contents and record their character. analyze for barbiturates and alcohol. submit samples for toxicologic study. evidence of alcohol intoxication.* poisoning by other addictive drugs. gritty residues of unabsorbed tablets, powder, or capsules. mucosal corrosion, ulceration, and discoloration from capsules may occur. concentration of barbiturate in parenchyma important for interpretation. poisoning, bismuth note: accidental poisoning is common (industrial exposure or drugs with soluble bismuth compounds). search also for other heavy metals. acute kidney failure* may be the cause of death. external examination and oral cavity stomatitis with bluish black discoloration of gums; loose teeth; sticky white membranous patches in mouth and throat. jaundice. protein casts and tubular epithelial cells in sediment. gray or black mucosal membranes; swelling of mucosa; intestinal ulcers that may be perforated. hemosiderosis. fatty changes; hemosiderosis. fatty changes; renal tubular degeneration with amorphic basophilic deposits in epithelium of convoluted tubules. hemosiderosis. see above under "external examination." for removal and specimen preparation, see chapter 4. peripheral neuritis. synonyms: bromine poisoning; bromism. note: the lethal dose is about 0.2 g in children and i g in adults (ingested). after fatal methyl bromide poisoning, headspace gas chromatography revealed a subclavian blood concentration of 3.0 microgram/ml whereas inorganic bromide concentrations were 530 micrograms/ml in the blood (1). tissue concentrations were lower than those in the blood. toxicologic samples should include liver and kidneys. chemical bums on face and conjunctivitis indicate direct exposure; skin pustules over body and nodose bromoderma of the legs indicate bromism. blood is best suited for bromide determination. after ingestion of bromide, necrosis with brown discoloration of mucosa of upper gastrointestinal tract may be present. after inhalation of bromide, swelling and inflammation ofmucous membranes in upper and lower respiratory tracts may be present. there may be pulmonary edema. pneumonia occurs in bromism. organs to be analyzed for cd should have no contact with water or be contaminated with blood; they should be sealed in polyethylene bags. cd leaks into fixation fluid. postmortem blood concentrations are very high and no indicator of the antemortem values (1). external examination and oral cavity blood urine gastrointestinal tract other organs procedures submit sample for toxicologic study. submit sample for determination of cadmium concentration. submit sample for toxicologic study and a portion of one lobe for microbiologic study. perfuse one lung with formalin. fix bowel as soon as possible. collect renal tissue for light microscopic and electron microscopic study. sample for toxicologic study. submit samples for histologic study also. poisoning, carbon monoxide note: if the victim had been in a fire, see also under "bums." carbon monoxide poisoning may rarely be responsible for automobile accidents. relatively low carboxyhemoglobin concentrations may contribute to death if there is concomitant poisoning-forinstance, with alcohol or drugs, particularly sedatives. anemia, atherosclerotic heart disease, and chronic pulmonary disease also increase sensitivity to carbon monoxide. ifblood had been withdrawn at time of hospital admissionfor instance, for crossmatching-submit this for carboxyhemoglobin determination. if no blood can be obtained, see under "heart, kidneys, and other organs." for quick-orienting qualitative tests, for quantitative methods of carbon monoxide determi-nation, and for interpretation of toxicologic findings, see below. request also determination of hemoglobin concentrations and of blood alcohol. request drug screen. for shipping of blood and tissues for carbon monoxide determination, see chapter 15. it should be noted that losses of up to 60% of the original saturation occurred when blood was kept in uncapped container at room temperature for 2 yz wk or at 4â°c for 3 wk. external examination blood heart, kidneys, and other organs brain record color of fingernails, particularly in heavily pigmented persons in whom lividity is difficult to discern. record appearance of blood and submit sample of postmortem blood for toxicologic study. see also above under "note." if no blood can be obtained, prepare water extract of spleen, kidneys, or other organs. request determination of carbon monoxide content and of carbon monoxide-binding capacity of this mixture. submit tissue samples for histologic study. for removal and specimen preparation, see chapter 4. pink skin and fingernails; bullous edema of skin; decubital ulcers. blood tends to be cherry red. for interpretation of toxicologic findings, see below. necrosis of papillary muscles in the heart or myocardial infarction may occur. renal tubular degeneration may also be found. acute kidney failure* has been observed after rhabdomyolysis complicated by compartment syndrome (2) . hemorrhagic necrosis of basal ganglia (lenticular nucleus in globus pallidus); diffuse petechial hemorrhages in white matter; cerebral edema. acute hydrocephalus in infants (3) . many methods of carbon monoxide determination have been described. currently, carboxyhemoglobin is detected in most medical examiner toxicology laboratories by visible spectrophotometry or gas chromatography. in hospitals, carboxyhemoglobin is frequently detected and reported in the course of routine arterial blood gas analysis. pink discoloration of skin and organs usually indicates the presence of more than 30% carboxyhemoglobin (but rule out cyanide poisoning* and exposure to cold*). in a healthy, middle-aged person, a carboxyhemoglobin concentration greater than 50-60% is usually fatal. if the victim was anemic or suffered from chronic lung disease, particularly emphysema* or atherosclerotic heart disease, the concentration may be lower. in association with alcohol, sedatives, and other drugs, carboxyhemoglobin levels may also be much lower and yet fatal. a heavy cigarette smoker may have a carboxyhemoglobin concentration of 8-10%, and higher levels may occur in police officers and other persons exposed to automobile exhaust in dense traffic. if the victim survived the carbon monoxide poisoning for several hours, postmortem blood samples usually will fail to show the presence of carboxyhemoglobin. in these instances, blood taken at the time of admission to the hospital may still be available and of particular value. if the victim had spent 1 h in fresh air before death, 40-50% of the carbon monoxide will have been removed, and 8-10% will have been removed during each subsequent hour. even though clearance may be complete, death may still occur-primarily from brain damage and infectious complications in prolonged coma. or delayed by only a few hours, particularly after inhalation of carbon tetrachloride. sudden death probably is caused by cardiac dysrrhythmia. alcohol concentrations should be determined in all cases or, if death was delayed, evidence of drinking at the time of exposure should be sought. alcohol considerably increases the hazards of carbon tetrachloride. note: see also under "bronchitis, acute chemical" and under "poisoning, gas." hydrochloric acid is sold by plumbing supply houses and pool supply companies as muriatic acid. it is a liquid. chlorine is a water-soluble gas. as supplied for pool sanitation, liquid chlorine is usually acidic. for convenience, chlorine and hydrochloric acid are discussed here together. prepare photographs of face. conjunctivitis and cyanosis in chlorine gas poisoning; burns of lips from hydrochloric acid. see also above under "larynx and trachea." photograph opened esophagus and stomach. sample for histologic study, particularly if there is doubt whether a perforation was antemortem or postmortem. for removal and specimen preparation, see chapter 4. severe pulmonary edema, bronchopneumonia, and swelling of mucous membranes in chlorine poisoning. arterial thrombosis may occur. pulmonary fibrosis may develop after prolonged survival. swelling and ulceration of mucous membranes in chlorine poisoning; acute laryngotracheitis. tracheoesophageal perforation. corrosion of mucosa with thickening, hemorrhage, and blackish discoloration after ingestion of hydrochloric acid. antemortem and postmortem perforation may occur. glomerular capillary thromboses. hemorrhages in white matter (1). poisoning, cyanide synonym: hydrocyanic acid (hydrogen cyanide) poisoning. note: hydrocyanic acid (hydrogen cyanide, hcn) is a water-soluble gas. its salts, sodium cyanide and potassium cyanide are sold as "eggs" to the jewelry industry. hydrocyanic acid is formed when cyanide salts are dissolved in acidic solutions. containers from which the poison might have been ingested or inhaled should also be submitted for toxicologic examination. for cyanide screening tests in the autopsy room and for interpretation offindings, see below. caution: stomach may still contain cyanide gas, formed by acidic reaction of cyanide salt. it may be best to open the stomach under a hood (1). the odor is quite characteristic for cyanide poisoning but most persons are unable to smell this odor. it is helpful to know in advance if any person in an office or laboratory can smell cyanide. forensic pathologists who can smell the compound state that it has its own specific odor, which differs from the often quoted smell of bitter almonds. autopsies also can be done in a negatively pressured isolation room (1). for odor, see above under "note." bright red skin color is not always present. corrosion around mouth and in oral cavity may be found after ingestion of potassium or sodium cyanide. blood is fluid and sometimes bright red. if potassium or sodium cyanide was ingested, brown-red mucosal corrosion may be present in stomach or in upper digestive tract. pulmonary edema. for odor, see above under "note." if death was not instantaneous, there may be hyaline thrombi in small blood vessels, minute hemorrhages, and necroses of lenticular nuclei. this test can be used for blood and gastric contents (2) . dip squares of filter paper in a small amount ofsaturated picric acid. let these squares dry until barely moist. place a drop ofthe material to be tested-e.g., blood or gastric contents--on a piece of paper. let material dry for a moment, and then place one drop of 10% sodium carbonate in the center of the material to be tested. ifcyanide is present, a reddish purple color will chromatograph out from the material. the higher the concentration of cyanide the more blue the color will be. it is possible to recognize whole blood because the blood turns a rather dark brown and the reddish to purple color is clearly visible. high concentrations of sulfide interfere by giving a false-positive test. another screening test is done as follows (3) . dip filter paper into normal blood. then treat the paper with potassium chlorate, whereupon brown methemoglobin forms. place this preparation into the fluid suspected of containing cyanide (e.g., blood, gastric contents, pulmonary edema fluid). if bright red cyanmethemoglobin forms, the reaction is positive. if the concentration of cyanide in the stomach is high and the concentration in the lungs is low, cyanide was ingested. alternatively, if the pulmonary cyanide concentration is high and the concentrtaion in the gastric contents is low, hydrogen cyanide most likely was inhaled. occasionally, minimal cyanide levels will be present in decomposed bodies. related term: digoxin toxicity. note: certain drugs may interfere with correct digitalis determination. blood heart vitreous procedures submit sample of peripheral blood for digoxin radioimmunoassay. freeze fresh myocardium for digitalis extraction. submit sample for digoxin radioimmunoassay. poisoning, drug(s) (see "dependence, drug(s), all types or type unspecified" or under "poisoning,â�¢â�¢â�¢" followed by specific name of drug.) poisoning, ethanol (ethyl alcohol) (see "alcoholism and alcohol intoxication," "cardiomyopathy, alcoholic," "disease, alcoholic liver," "syndrome, fetal alcoholic," and syndrome, wernicke-korsakorr.") poisoning, ethylene glycol related term: antifreeze poisoning. note: pulmonary and cerebral manifestations are the main findings in acute poisoning, and renal tubular necrosis is the primary finding in chronic poisoning. for general toxicologic sampling, see chapter 13. calcium oxalate crystals can be demonstrated in routine histologic sections but also in scanning electron micrographs of thick deparaffinized sections. eyes blood urine for removal and specimen preparation, see chapter 5. in acute cases, submit sample for ethylene glycol determination; in chronic cases, request determination of calcium concentrations. prepare sediment. papilledema; optic nerve atrophy. ethylene glycol in serum (i) . protein casts; calcium oxalate crystals (2) . crystals are light yellow and birefringent, arranged as sheaves, rhomboids, or prisms. poisoning, food related terms: bacillary dysentery* (shigella food poisoning); botulism;* clostridium perfringens food poisoning; favism; mushroom poisoning;* salmonella food poisoning; staphylococcal food poisoning. note: if cause of food poisoning is unknown, submit suspected food for aerobic and anaerobic cultures, gram stain of smears, and routine toxicologic study. this should include tests for heavy metals (antimony, cadmium, and lead) that may have leaked from old cooking utensils. test for the presence of staphylococcal enterotoxin are done only in specialized laboratories. if botulism is suspected, follow procedures described under that heading. mushroom poisoning also is listed as a separate entity. if salmonella food poisoning is suspected, see under "fever, typhoid." see also under "enteritis" or "enterocolitis" or under another specific heading such as "dysentery, bacillary." obtain sufficient material for microbiologic and histologic study to identify organisms such as chlamydia, clostridium (type f strains), salmonella, shigella, verotoxic e. coli, yersinia, and others. external examination gastrointestinal tract submit contents for aerobic and anaerobic cultures (see above under "note"); prepare smears of contents for gram stain. submit samples for histologic study. debilitated states; patients in extremes of life. enteritis or enterocolitis. poisoning, gas note: anesthesia-associated death, * carbon monoxide poisoning,* and sniffing and spray death* are presented under the appropriate headings. procedures discussed here deal with other volatile substances, including chemical irritants such as ammonia (nh 3 ), chlorine or hydrochloric acid poisoning (see also under that heading); methylene chloride, phosgene (coci 2 ), sulfurous acid (h 2 s0 3 ), or sulfur dioxide (s02) ' gases from body cavities, heart chambers, or blood vessels can be removed as described under "embolism, air." gases can also be trapped with a rubber dam after cutting organs under water. samples from various organs should be shipped in hermetically sealed nonplastic containers or in analyzing solutions. external examination, and oral cavity blood larynx and trachea record extent of chemical bums. submit sample for gas analysis. in many instances, inhaled gases can be demonstrated chromatographically in gas from head space above sealed blood specimen. chemical bums in and around mouth or conjunctivas. chemical bums. other organs if gas was inhaled and is to be analyzed, submit intact lungs with bronchi ligated in airtight, nonplastic container to laboratory that can conduct gas analysis. if survival was short, formalin perfusion of lungs is not recommended; it may cause artifactual ballooning and internal ruptures of organ. submit samples of tissue for routine histologic study. see above under "note." chemical pneumonia; pulmonary edema. after longer survival, obliterating fibrous bronchiolitis, chronic bronchitis, and saccular bronchiectasis* may occur. poisoning, glycol (see "poisoning, ethylene glycol.") poisoning, halogen (see "fluorosis," "poisoning, bromide," "poisoning, chlorine or hydrochloric acid," "poisoning, gas?, and "poisoning, iodine!') poisoning, heavy metal (see "poisoning, antimony," "poisoning, arsenic," "poisoning, cadmium," "poisoning, lead," poisoning, mercury," "poisoning, thallium!') poisoning, insecticide (see "poisoning, organophosphate(s)*) poisoning, iodine related terms: lugol's solution; tincture of iodine. for toxicologic sampling, see chapter 13. external examination and eyes urine, blood, and parenchymal organs lungs and upper respiratory tract stomach record color of skin and extent of corrosive lesions. submit samples for toxicologic study. submit samples for histologic study. submit contents for toxicologic study; photograph mucosa; prepare histologic sections. see above under "stomach." perioral corrosive lesions; yellow discoloration of skin; conjunctivitis after exposure to vapors. acute inflammation of respiratory tract after inhalation of vapors. corrosive gastritis; if starch was used as antidote, gastric lining will be bluish. histologically, well-preserved mucosa is present because of in vivo fixation. mucosa may show same changes as stomach. swelling of tubular epithelium. synonyms: propanol; rubbing alcohol. procedures see under "alcoholism and alcohol intoxication." nonspecific autopsy findings: visceral congestion; pulmonary and cerebral edema. poisoning, lead note: lead-free syringes and lead-free polyethylene containers should be used. blood lead concentrations can be determined by inductively coupled plasma mass spectrometry (1). for screening methods, see ref. (2) . this is a reportable disease in some states. external examination, oral cavity, and hair bluish lead line at gingival margin in victims with poor oral hygiene. external examination, oral cavity, and hair for diagnosis of chronic plumbism, analysis of scalp hair may be useful. analysis is done by neutron activation (see also under "poisoning, arsenic"). remove samples with lead-free syringe (see above under "note") or 20-ml vacutainer tubes (becton, dickinson and company). do not add anti-coagulant or preservative. for coliection procedures, see above under "blood" and under "note." request also determination of coproporphyrin concentrations. submit sample for histologic study; submit remaining tissue for toxicologic analysis. submit with contents for toxicologic study, particularly in acute poisoning. submit sample from each kidney for histologic study; submit remaining tissue of both kidneys separately for toxicologic study. submit at least 10 g of fresh bone for toxicologic study. for removal and specimen preparation, see chapter 4. poisoning, lsd (d-lysergic acid diethylamide) (see "abuse, hallucinogen(s).") poisoning, lye related terms: ammonium hydroxide poisoning; calcium oxide or quicklime poisoning; poisoning by alkaline corrosives; potassium hydroxide poisoning; sodium hydroxide poisoning. external examination and oral cavity blood neck organs, esophagus, trachea, and lungs record extent of oral, perioral, and other facial corrosive injuries. photograph lesions. prepare histologic sections of tissue from inside of lips or mouth. submit sample for toxicologic study. after removal of heart, remove neck organs with hypopharynx, esophagus, larynx, and trachea. leave stomach attached to esophagus. open pharynx and esophagus along posterior midline. in acute cases, formalin perfusion of lungs is not recommended; it may cause artifactual baliooning and internal ruptures of organ. lye bums on face and chest in acute cases; scars and manifestations of malnutrition* in chronic cases. sweliing, edema, and necrosis of mucous membranes in acute poisoning. fibrosis and strictures in chronic cases. bronchitis* and bronchopneumonia. submit specimen from pharynx for histologic study. for removal and specimen preparation, see chapter 4. submit sample of brain for toxicologic study. exfoliative dermatitis. blue line at gingival margin; hypertrophy of gum; acute and chronic gingivitis; exfoliation and loss of teeth (2) . degeneration of myocardium. increased concentrations of mercury (1) . congestion. erosive gastritis and colitis. increased concentrations of mercury (1) . degeneration of proximal tubules; calcifications. chronic kidney failure* may be the cause of death. induration of mucosa. increased concentrations of mercury (1) . cortical hemorrhages. cholinesterase activity will be low. pulmonary edema if poison was inhaled. airways may contain aspirated material. cholinesterase activity can be determined reliably even after decomposition and embalming. clinically, guillain-barre syndrome has been observed after poisoning with organophosphate. in acute poisoning, the cholinesterase levels may be 25% of the normal values (see below). the cholinesterase levels in the blood are not affected by the duration of the postmortem interval; measurement may be attempted even on decomposed or exhumed bodies. severe fatty changes; periportal necroses. fatty changes in myocardium, skeletal muscles, and other organs. for toxicologic sampling (gastric contents, urine, blood, brain, and other organs), see chapter 13. rigor mortis after fatal strychnine poisoning may occur very soon after death and may be very severe (opisthotonos); it may persist until decomposition sets in. congestion of viscera; no characteristic morphologic autopsy findings. acute pancreatitis has been observed (1). high strychnine concentrations (also demonstrable in exhumed bodies [2] ). poisoning, thallium note: for toxicologic sampling, see below and chapter 13. thallium is used as a rodenticide and pesticide, and has some industrial applications. retrobulbar neuritis. chapter 4 and 5. submit brain for toxicologic study. submit sections of brain and optic nerves for histologic study. submit specimens for toxicologic study (take from lower extremity). for removal, prosthetic repair, and specimen osteomalacia;* osteomyelofibrosis. preparation of bones, see chapter 2. for preparation of sections and smears of bone marrow, see chapter 2. submit samples for toxicologic study. synonym: acute anterior poliomyelitis. note: the disease has been nearly eliminated in the usa but not in many other countries. (1) collect all tissues that appear to be infected. ( for removal and specimen preparation, see chapter 4. in acute cases, submit portions of brain and spinal cord for viral culture. neurogenic atrophy of skeletal muscles in areas of paralysis. electrolyte disorder. * hypertensive heart disease; myocarditis.* aspiration or bronchopneumonia (or both); edema; atelectasis; embolism;* alveolar wall necrosis (acute or organizing diffuse alveolar damage) after oxygen toxicity. acute ulcers. acute gastric dilatation; acute gastroduodenal ulcers; gastrointestinal erosions and hemorrhages. dilatation of colon; perforation of cecum. urolithiasis and nephrolithiasis,* pyonephrosis and pyelonephritis. * phlebothrombosis of legs, most commonly on left side. necrosis of anterior hom cells of spinal cord, with neuronophagia and perivascular inflammatory reaction. old lesions show neuronal loss and gliosis. medulla ("bulbar polio") and other areas of brain stem, cerebellum, and cerebrum, particularly the motor cortex, may be affected in various degrees. part ii / diseases and conditions pregnancy note: in some instances, procedures described under "death, abortion-associated," "embolism, amniotic fluid," or "toxemia of pregnancy" may be indicated. synonym: werner syndrome. external examination and skin abdomen procedures record volume of intraperitoneal fluid; prepare smears; submit samples of peritoneum for histologic study. colloid carcinoma of stomach or colon. well-differentated adenocarcinoma of ovary or, rarely, of appendix; ruptured appendiceal mucinous adenoma is the most common cause of peritoneal adenomucinosis. pseudotumor cerebri synonym: benign intracranial hypertension; meningeal hydrops. note: this condition, which generally affects young, obese females, is characterized by symptoms and signs of increased intracranial pressure without a demonstrable cause. hence, intra-cranial mass lesions, infections, and related conditions should be excluded. such conditions include adrenal insufficiency;* guillain-barre syndrome* (increased colloid-osmotic pressure); hyperadrenalism; hypervitaminosis a* (e.g., after treatment of acne); hypoparathyroidism;* hypothroidism;* infectious mono-nucleosis;* lyme disease; pregnancy; sydenham's chorea; throm-bus ofthe lateral or superior sagittal sinus (otitic hydrocephalus); and wiskott-aldrich syndrome. submit samples of all accessible organs and tissues, with or without gross lesions. submit material for electron microscopy. for removal and specimen preparation, see chapter 5. for removal, prosthetic repair, and specimen preparation, see chapter 2. heart small bowel liver procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. fix the bowel as soon as possible. record weight. submit samples for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter 2. aortitis involving ascending aorta and aortic valve with regurgitation; mitral valve and adjacent myocardium and conduction system also may be involved. sprue-like changes with loss of villi. fibrosis or cirrhosis and other abnormalities, particularly in patients who had been taking methotrexate. psoriatic arthritis and spondylitis. synonyms and related terms: allergic purpura; anaphylactoid purpura; hypersensitivity vasculitis. external examination and skin rabies note: (i) in most instances, an autopsy limited to the brain is sufficient to confirm the diagnosis. (2) rabies virus is especially infectious and thus, universal precautions should be strictly followed (see chapter 6) . avoid the use of scalpels whenever possible. the generation ofaerosols should be assidusee above under "note." if a complete autopsy is done, sample heart, lungs, kidneys, pancreas, submaxillary salivary glands, and adrenal glands; freeze or refrigerate sampled tissues and submit for viral study (see below under "brain and spinal cord"). obtain serum for serologic studies. freeze or refrigerate cerebral tissue immediately after removal and submit frozen to special laboratory for fluorescent antibody staining. take these sections from hippocampus or brain stem. place the refrigerated tissues in 50% neutral glycerol saline solution for preservation. fix remaining brain and spinal cord tissue in 15% formalin and submit for histologic study. as an alternative to freezing of tissue, immunoperoxidase methods for detection of rabies viral antigens can now be applied to formalin-fixed tissue (1 in almost all instances, the procedures described under "assault" and under "homicide" must also be followed. see also refs. (1) and (2) . for the evaluation of bite marks, photographs with and without scales and black and white film should be used. a forensic dentist is required to compare the victim's bite marks to the teeth of suspects. swabs of possible sperm or other fluids must be sent to the crime lab for proper evaluation. external examination other organs and tissues genital organs comb pubic hair over a towel and then pull sample; also, pull samples of hair from head. collect fingernail clippings and place in containers marked "right" and "left." collect blood, hair, fibers, and residues of urine or saliva and/or semen that may have stained the victim's clothing or that may be found on the skin of the victim. for study of stains and scrapings, see below. introduce sterile dry cotton swab into the posterior vault of the vagina or-preferablyinto the cervical canal. prepare smear on glass slide and send to crime laboratory for identification of sperm (see also above under "note" using a sterile cotton swab moistened with sterile saline, lift suspected dried spermatic fluid from hair or skin of external genitalia, perineum, buttocks, or other sites. stains on clothing will be extracted by forensic laboratory personnel. let specimens air dry in absence of sunlight and then place in paper bags or envelopes and seal. smears are made for the demonstration of spermatozoa, cytologic changes, bacteria, and other possible findings (some crime labs prefer to make their own smears from the swabs). other samples are used for the determination of acid phosphatase, as described below. usually, the survival time of morphologically recognizable spermatozoa in the vagina is less than 24 h, but on occasion this may be much longer. thus, within the first 24 h after coitus, 64% of cervical smears have been found to be positive for spermatozoa. at day 10, spermatozoa have been found in 13% of the smears. obviously, if the assailant had had a vasectomy, spermatozoa will not be found at any time. spermatozoa have been found in the vagina of some women several days or weeks after death. dried stains (see above) may give positive results after longer intervals. acid phosphatase activities greater than 5 bodansky units (for method, see above) indicate that probably less than 12 h have elapsed since the time of intercourse (i) . in another study, vaginal swab specimens showed acid phosphatase activities of more than 2,000 king if there is evidence of parotid involvement, other salivary glands should also be studied. parotid gland can be biopsied from scalp incision. submaxillary gland can be removed with floor of mouth. for sampling and specimen preparation, see chapter 4. for removal, prosthetic repair, and specimen preparation, see chapter 2. chronic meningitis; space-occupying lesions (submeningeal nodular granulomas). granulomatosis. iridocyclitis; chorioretinitis; papilledema; posterior uveitis; keratoconjunctivitis sicca; conjunctival follicles; cataracts. involvement of lacrimal glands and other orbital tissues (2) . sarcoidosis of parotid gland is common. sarcoid neuropathy and sarcoid myopathy. cystic changes, primarily of small bones of hands and feet. sarcoidosis of joints (3). synonyms and related terms: bilharziasis; schistosoma haematobium infection; schistosoma japonicum infection; schis-tosoma mansoni infection. note: unless specifically stated, the changes listed below refer to chronic schistosomiasis mansoni and japonica. (1) collect all tissues that appear to be infected. (2) request direct examination for schistosoma. the following procedures have been described and recommended (1). for demonstration of schistosoma eggs, compress 4-mm tissue fragments-for instance, mucosa of the urinary bladder-between glass slides. if this gives negative results, digest a 5-g portion of tissue in potassium hydroxide. for the recovery of adult worms of schistosoma mansoni and schistosoma haematobium, remove the viscera en bloc and rinse with water. separate the intestines from the mesentery. subsequently, perfuse the portal vein system, the liver, and one lung with saline (2) . then pass the perfusion fluid through a monofilament nylon cloth with an aperture size of 180 11m. submerge the cloth in water and examine with a dissecting microscope. fix worms in formalin solution. examine the intestinal mucosa directly. from the urinary bladder, ureters, and surrounding connective tissue, worms can be recovered as follows. inject water into the tissue until the tissue increases 2 or 3 times in thickness. then compress the tissue gently with a glass plate. cut slices 0.1-0.2 cm in thickness. compress these slices between the glass plate and the stage of a dissecting microscope and examine for the presence ofadult worms. many worms also will be present in the fluid expressed from the tissue as it is cut. for the counting of eggs in tissues, urine, and feces, see ref. (1) . immunodiagnostic methods (elisa and imrnunoblot) also have been developed prepare skeletal roentgenograms. there are no diagnostic laboratory tests but it still is advisable to save a blood sample. record heart weight. test competence of valves (chapter 3). open heart in line of blood flow. photograph and measure valvular lesions. prepare sections of valves, myocardium, conduction system (if there was a history of heart block), and ascending aorta. perfuse at least one lung with formalin. submit any consolidated area for microbiologic study. fix intestines as soon as possible. abnormalities as listed in right-hand column. sample for histologic study and request amyloid stains or renal parenchyma. prepare roentgenograms of spine, sacroiliac joints, symphysis ossium pubis, and manubriosternal, sternoclavicular, and humeroscapular joints. if spine cannot be removed in its entirety, it can be split in midline and one half can be removed, with costovertebral and costotransversal joints. hip joints should be exposed. histologic sections should include synovia and periarticular tissue. secondary and peripheral osteoarthritis* may be present. leukemia* developed in some patients who had had radiation treatment (1950 or earlier). acute anterior uveitis. (2) request fungal culture. (3) request grocott's methenamine silver stain. (4) record weight of all organs (for expected weights, see part iii). submit samples of all major organs, including endocrine glands, lymphatic and fat tissue, bone, and bone marrow for histologic study. hunger edema; skin changes secondary to vitamin deficiencies. hypoproteinemia. atrophy; hemosiderosis of spleen. degree of atrophy varies from organ to organ; atrophy of fat tissue, lymphoid tissue, and gonads usually is most pronounced. severe infections, such as tuberculosis,* may be present without having been apparent clinically. steatohepatitis, nonalcoholic (nash) note: the morphologic findings in the liver are indistinguishable from those in alcoholic liver disease* (1). follow autopsy procedures described under "disease, alcoholic liver." if the patient had received a liver transplant, procedures described under "transplantation, liver" should be followed also. possible associated conditions: celiac sprue (rare); diabetes mellitus* (type 2); hyperlipidemia; malnutrition* from bulemia (rare); morbid obesity with liver failure particularly after episodes of rapid weight loss (e.g., after recent gastroplasty); lipodystrophy; mild obesity;* short bowel syndrome (rare); total parenteral nutrition. * stenosis, congenital valvular pulmonary related terms: isolated (pure, simple, or dome-shaped) pulmonary stenosis. note: the pulmonary valve is usually acommissural in isolated congenital pulmonary stenosis. with other coexistant congenital heart disease (such as tetralogy), the pulmonary valve is usually bicuspid and hypoplastic, but may be unicommissural or dys-plastic and tricuspid. heart and great vessels; peripheral pulmonary arteries brain procedures culture any grossly infected valve. for general dissection techniques, see chapter 3. record weight of heart, thickness of ventricles, and annular circumferences. for removal and specimen preparation, see chapter 4. infective endocarditis* of pulmonary valve and tricuspid valve; infective endarteritis at bifurcation of pulmonary trunk. it is preferable to have autopsies of fetuses and stillborns performed by pathologists experienced in perinatal pathology. if such personnel are not immediately available, the attending pathologist may nevertheless collect important information. if attempts to induce abortion appear to have caused the death of the mother, see "death, abortion-associated." the placenta should be immediately procured from the delivery room should it not arrive in the laboratory with the fetus. this will avoid the possibility of the placenta being discarded by the delivery room staff. no fetal autopsy is complete without a careful examination of the placenta (see part i, chapter 2). pathologic changes of placenta that are causative of stillbirth are summarized in ref. 1 . fascia lata or other aseptically obtained tissue should be collected for tissue culture for karyotype analysis. if the fetus is at all autolyzed, then the fascia lata cells may not grow in tissue culture. therefore, if the fetus shows any evidence of autolysis, tissue should be taken aseptically, from placenta immediately beneath the chorionic plate. a portion ofplacenta and liver should also be snap-frozen for possible molecular analysis. the initial stage of the autopsy should include photography and radiography, taking anterior-posterior and lateral views. the photographs will record the degree of maceration, which can be roughly correlated with the duration of fetal demise before delivery [1] external measurements should include body weight, circumference ofhead, chest and abdomen, crown-rump length, crown-heel length, and foot length. these measurements are compared with tables of standards for normal fetuses (see part iii of this book). assessment of growth retardation may be based on these data. a careful external examination should search for abnormalities such as jaundice, bulging fontanel, cranial bone softening, hyper-or hypotelorism, choanal atresia, external ear anomalies, cleft lip, cleft palate, macroglossia, micrognathia, colobomata, cystic hygroma, shortened neck, contractures, omphalocele, gastroschisis, abnormal external genitalia, anal atresia, absent vagina, sacral pits, open neural tube defects, hemihypertrophy, syndactyly, clinodactyly, transverse palmar creases, or incomplete descent of testes. the organ bloc may be removed in the manner similar to the adult, using the technique of letulle (see chapter 2) . if the thyroid gland is noted to be in its usual location and if it appears normal, then the tongue may be left in the body. in macerated fetuses, it is suggested that the organ bloc be fixed overnight in formalin solution prior to dissection. to aid adequate fixation, the following simple steps may be done: i) wash the organ bloc thoroughly prior to fixation; 2) place multiple transverse cuts through the liver and lungs; 3) dissect the posterior leaves of the diaphragm away from the adrenal glands and kidneys; 4) bivalve the adrenal glands and kidneys in the coronal plane; 5) instill formalin in the lumen of the intestine, using a syringe; and 6) gently instill formalin into both ventricles of the heart, being careful to avoid the ventricular septum. the procedure for dissection of the organs is similar to that of the adult except: 1) the venous and arterial connections of the heart, including the patency of the ductus arteriosus must be determined before the heart is removed; 2) the esophagus must be opened posteriorly prior to its complete removal so that esophageal atresia or tracheo-esophageal fistula may be recognized and photographed prior to further dissection; 3) the location of the appendix and of the testes should be recorded. until the intestine has been examined for stenosis or atresia, the mesentery should be left attached. the degree of autolysis as seen grossly and with histologic examination of both the fetus and the placenta can be used to estimate the duration of time between fetal death and delivery (2, 3, 4) . trichrome stain is useful for better visualizing histologic features in severely autolyzed tissue. to remove the brain, benecke's technique of one of its modifications may be used. stillbirth vs livebirth. a decision has to be made whether the infant was born alive or was stillborn. the hydrostatic lung test, described in the previous edition, appears unreliable. the presence of gas in the lungs does not rule out stillbirth. after death, air can be introduced into the lungs, or putrefaction gases might be present. however, air artificially introduced after death will not distend the alveoli and can be squeezed out, whereas this does not seem to be the case after active ventilation. the distribution of fat in the fetal zone of the adrenal cortex may indicate whether intrauterine death was acute, more prolonged, or chronic (3). this is particularly helpful if the stillborn baby is macerated. if the mother died also, see under "death, abortion-associated" strangulation note: in many instances, procedures described under "hom-icide" must also be followed. if a rope or some other material had been used (see also under "hanging"), leave ligature in place until autopsy can be done. see also under "hypoxia." toxicologic sampling, particularly for alcohol, should be done in all instances (chapter 13). results than body fluids. (2) universal precautions should be strictly followed. the generation of aerosols should be minimized. to sterilize tissue surfaces in preparation for culture, swab the surface with povidone iodine. avoid searing the tissue surface since this will produce an aerosol. keep no more than one scalpel in the dissecting area at anyone time. have an assistant available with clean, gloved hands to receive specimens in containers, so as to minimize the degree of contamination on the outside of the containers. for cleaning procedures and related information, see chapter 6. (3) hiv-l in tissue may be demonstrated using polymerase chain reaction (pcr), immunohistochemistry, in situ hybridization, or immunofluorescence. (4) for immunocytochemical and molecular studies, fix tissue in ethanol or carnoy's solution. the virus can be identified using pcr in most tissues, whether fresh, frozen, or fixed in ethanol. (5) serologic tests as well as direct fluorescent antibody tests are avail-able for many of the expected infections. (6) this is not a reportable disease. external examination and skin record body weight and evidence of lipodystrophy following aids medications. record and photograph any skin lesions. examine oral cavity. if the diagnosis is in doubt, samples can be submitted for enzyme immunoassay. cachexia. severe loss of subcutaneous fat in face and extremities, associated with large fat deposits on upper back and upper abdomen ("protease paunch"). cutaneous kaposi's sarcoma (l) . test may be positive for many weeks after death (2) . syndrome, myelodysplastic synonyms and related terms: chronic myelomonocytic leukemia;* refractory anemia; refractory anemia with excess of blasts; refractory anemia with excess of blasts in transformation; refractory anemia with ringed sideroblasts; refractory dysmyelopoietic anemias. note: the myelodysplastic syndromes are represented by a heterogeneous group of normocytic anemias, often with neutropenia, thrombocytopenia, and monocytosis. for expected bone marrow changes, see above under "synonyms and related terms." autopsy procedures are similar to those recommended for most cases of leukemia, with particular attention paid to intercurrent infections and thrombocytopenic hemorrhages. in all instances, material should be collected using aseptic technique for tissue culture for chromosome analysis. common findings in these conditions are deletion of the long arm ofchromosome 5, deletion of chromosome 5 or 7, or trisomy 8. syndrome, nephrotic note: see under name of suspected underlying condition, such as amyloidosis,* anaphylactoid purpura,*diabetes mellitus,* glomerulonephritis,* goodpasture's syndrome,* heavy metal poisoning, hemolytic uremic syndrome,* infective endo-carditis,* polyarteritis nodosa,* syphilis, * or systemic lupus erythematosus. * if accelerated hypertension or constrictive pericarditis is the suspected underlying condition, see under "hypertension (arterial), all types or type unspecified" or "pericarditis," respectively. in all instances, the renal veins and the inferior vena cava should be opened in situ. "en masse" removal of organs is recommended for this purpose. if thrombosis is found, record exact location and size of clot and submit sample of clot with wall of veins for histologic study. see also under "thrombosis, venous." coronary atherosclerosis and its complications seem to be increased in patients with the nephrotic syndrome. submit sample for bacteriologic, viral, and serologic study. record weight and submit samples for histologic study. if heart block was present, submit samples of conduction system for histologic study (chapter 4). submit consolidated areas for microbiologic study. perfuse at least one lung with formalin. for removal of urethra, see chapter 2. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter 5. consult roentgenograms (see above under "external examination, skin, and oral cavity"). keratoderma (keratosis blennorrhagica) with vasculitis (2) of sole of feet, of palms, and of circumcised glans penis. arthritis* of knees, ankles, and metatarsal and midtarsal joints, with or without ankylosis. osteoporosis.* usually, sterile culture. pericarditis; * myocarditis.* aortic valve lesions. pleuritis and pneumonia.* pulmonary fibrosis involving upper lobes. erosions, papules, and plaques in urethral or bladder mucosa. enteritis. thrombophlebitis. conjunctivitis; multifocal choroiditis; acute anterior uveitis; iritis; keratitis. arthritis* (see above under "external examination, skin, and oral cavity") resembling rheumatoid arthritis. * nonspecific synovitis. for removal and specimen preparation, see chapter 5. syndrome, reye's note: hypoglycemia* may have been present, but that condition is difficult or impossible to confirm after death. an immediate autopsy is indicated (see below under "liver" and "pancreas"). vitreous blood urine heart lungs submit sample for sodium, chloride, and urea nitrogen determination. submit sample for microbiologic (viral) and serologic study, for ammonia and bilirubin determination, and for determination of salicylate levels if there is a history of treatment with this drug. obtain sample for biochemical and toxicologic analysis. record weight. request frozen sections for sudan stain. submit fresh tissue for bacterial and viral culture. request paraffin sections and frozen sections for fat stain (see above under "heart"). influenza;* varicella.* manifestations of liver failure. evidence of salicylate administration. assay for organic acids should rule out acylcoenzyme a dehydrogenase deficiency, and toxicity, e.g., of acetaminophen and valproic acid (l) . cardiomegaly; fatty changes of myocardium and patchy myocytolysis. viral pneumonia rarely present. acute interstitial pneumonia;* bronchitis;* hemorrhages. lipid-laden histiocytes in alveoli. record body weight and length, stature, and distribution of head, axillary, and pubic hair. record and prepare photographs of features of face and neck. prepare skeletal roentgenograms. submit samples of breast tissues for histologic study. these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter 9) . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit for histologic study. submit samples of all portions for histologic study. if biliary atresia is suspected, follow procedures described under that heading. dissect kidneys and ureters in situ and record findings. submit samples of gonads or-if gonads cannot be identified-equivalent ridges on mesosalpinx for histologic study. also submit samples of endometrium, cervix, and vagina. record weight and submit sample for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter 5. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for sampling and specimen preparation, see chapter 4. peroxidase-labeled gastrin antibodies seem to react with cells in all gastrinomas. aberrant gastrinoma may occur at hilus of spleen. metastases are found in regional lymph nodes and liver. there may be manifestations of multiple endocrine neoplasia. * secondary syphilis. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody techniques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. (4) in adult autopsies, no special precautions are indicated (see below under "liver"). (5) serologic studies are available from local and state health department laboratories. (6) this is a reportable disease. external examination record and prepare photographs of all abnormalities listed in right-hand column. prepare smears and sections of acute lesions; prepare sections of older skin lesions or anogenital mucosal lesions. hunterian chancre in primary syphilis; condylomata lata. noduloulcerative gummas and scarring in later stages. cerebrospinal fluid lymph nodes heart and aorta other organs and tissues prior to 20 wk gestation, the destructive effects of syphilis may not be seen. gummata are rare in neonates. tabes dorsalis is also uncommon. serologic diagnosis is difficult in the neonate because of transplacental transfer of maternal igg antibodies. acquired syphilis (syphilis in adulthood) is presented above under a separate heading. (1) collect all tissues that appear to be infected. (2) culture methods are not available, but animal inoculation can be performed. consultation with a microbiology laboratory is recommended. (3) special stains for treponema pallidum rarely are positive except with material from fresh lesions of primary or secondary syphilis and of syphilitic hepatitis of the newborn. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody tech-niques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. (4) in neonates, special precautions are indicated (see below under "liver") (5) serologic studies are available from local and state health department laboratories. (6) this is a reportâ· able disease. (i) collect all tissues that appear infected. (2) request aerobic and anaerobic cultures. however, the presence of tetanus bacilli established in culture is not diagnostic, since spores of c. tetani frequently contaminate wounds. record body weight and length and appearance of wound(s); photograph and excise wound(s) for histologic study. record evidence of parenteral drug abuse, especially subcutaneous injection (i.e., "skin popping"). prepare chest roentgenogram. evidence of weight loss; subcutaneous abscesses. tetanus may occur in drug addicts. tension pneumothorax* after mechanical ventilation. submit sample for microbiologic study. submit any consolidated area for microbiologic study. bacterial meningitis must be ruled out. aspiration and bronchopneumonia; embolism;* atelectasis. tetany note: see under name ofsuspected underlying conditions, such as hyperparathyroidism,* malabsorption syndrome,* or vitamin 0 deficiency. * respiratory or metabolic alkalosis* cannot be confirmed after death; determination of blood ph is not helpful because acidity increases rapidly after death. the concentration of serum phosphates also increases after death. synonym: large ventricular septal defect with pulmonary stenosis* or atresia.* note: the basic anomaly consists of subpulmonary stenosis, ventricular septal defect, overriding aorta, and secondary right ventricular hypertrophy. for general dissection techniques, see chapter 3. surgical interventions include modified blalock-taus-sig subclavian-to-pulmonary arterial shunt; complete repair with patch closure of ventricular septal defect, and reconstruction of right ventricular outflow tract (with a patch or with an extracardial conduit). possible associated conditions: origin of left pulmonary artery from aorta; minor abnormalities of the tricuspid valve; absent ductal artery (25%); atrial septal defect* (in 20%; pentalogy of fallot); bicuspid pulmonary valve;* dextroposition of aorta; double aortic arch; hypoplastic pulmonary arteries; patent ductal artery;* patent oval foramen; complete atrioven-tricular septal defect* (usually with down's syndrome*); persis-tent left superior vena cava; pulmonary valve atresia (see "atresia, pulmonary valve, with ventricular septal defect"); right aortic arch (25%); second ventricular septal defect; origin ofleft anterior descending (lad) or right coronary artery (rca) from contralateral aortic sinus or coronary artery (5%); syndrome with absent pulmonary valve and massively dilated pulmonary arteries (rare). chest cavity heart lungs other organs record course of superior vena cava and of its tributaries. record course of thoracic aorta and of its main branches. record origin of pulmonary arteries. if infective endocarditis is suspected, follow procedures described under that heading (chapter 7). for coronary arteriography, see chapter 10. perfuse both lungs with formalin. request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. persistent left superior vena cava. right aortic arch with or without right-sided ductal artery; double aortic arch; absent ductal artery. origin of the left pulmonary artery from descending aorta. for possible additional findings, see above under "note" and under "possible associated conditions." infective endocarditis* (usually of pulmonary or aortic valve). see also above under "possible associated conditions." marked right ventricular hypertrophy. right ventricular dilatation and fibrosis with late postoperative right-sided heart failure or sudden death due to arrhythmia. old in situ thrombosis of small pulmonary artery branches. paradoxic embolism; cerebral abscess. * thalassemia synonyms and related terms: congenital hemolytic anemia; alpha-thalassemia; beta-thalassemia major (cooley's anemia); beta thalassemia minor (beta-thalassemia trait). note: the changes described below are observed primarily in beta-thalassemia major. unless the cause of the renal vein thrombosis and the nature of the complicating or underlying renal disease are known, follow procedures described under "glomerulonephritis." procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. in infants, manifestations of dehydration. * pulmonary embolism. * tumor of the kidney* (renal cell carcinoma); aortic aneurysm; lymphadenopathy; other mass lesions. inferior vena cava thrombosis involving orifice or whole length of renal veins. tumor thrombus (e.g., from renal cell carcinoma). femoral vein thrombosis. rare venous malformations also may cause renal vein thrombosis (l) . hemorrhagic infartion. parenchymal renal disease with nephrotic syndrome,* including amyloidosis* and vasculitis* (these last conditions may be associated with renal vein thrombosis). acute gastroenteritis in infancy. hyperparathyroidism,* pregnancy,* trauma, and other conditions. thrombosis, venous note: for peripheral venous thrombosis, the autopsy procedures are essentially similar to those described under "phlebitis." see also under "hypertension, portal," "syndrome, budd-chiari," "thrombosis, cerebral venous sinus," "thrombosis, lateral sinus," and "thrombosis, renal vein." thymoma (see "'fumor of the thymus.") thyroiditis synonymsand related terms: chronic fibrosing thyroiditis (riedel's struma); chronic thyroiditis with transient thyrotoxicosis; hashimoto's thyroiditis; pyogenic thyroiditis; subacute (granulomatous, giant cell, de quervain's) thyroiditis. possibleassociatedconditions: withhashimoto's thyroiditisautoimmune (chronic) hepatitis,*megaloblastic anemia,*rheumatoid arthritis,*sjogren's syndrome,*and systemic lupus erythematosus.* with riedel's struma-retroperitoneal fibrosis,* sclerosing cholangitis,* sclerosing mediastinitis,*and other conditions with idiopathic fibrosis. with subacute thyroiditis-viral infection. submit sample for determination of protein concentration; record appearance of sediment. for removal and specimen preparation, see chapter 4. thromboses of small vessels. glomerulonephritis.* hemorrhagic necroses. proteinuria; abnormal sediment. abruptio placentae, small placenta with accelerated maturation of villi; infarcts; fibrinoid necrosis of decidual arterioles. thrombotic occlusion of small vessels with hemorrhagic necroses; anterior lobe of pituitary gland may contain necroses (see also "syndrome, sheehan's"). submit samples for histologic study. if infection is expected, submit material for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. transposition, complete, of the great arteries note: the basic anomaly is the origin of the aorta from the right ventricle, and of the pulmonary artery from the left ventricle, with a shunt, and usually with a right anterior aorta. there are four major types: (1) with an intact ventricular septum (65%), (2) with a ventricular septal defect* (20%), (3) with a ventricular septal defect and subvalvular pulmonary stenosis* (10%), and (4) with an intact ventricular septum and subvalvular pulmonary stenosis* (5%). for general dissection techniques, see chapter 3. interventions include atrial septostomy or septectomy; mustard or senning atrial switch procedure; rastelli-type repair with a valved extracardiac conduit; and jatene arterial switch procedure with lecompte maneuver. possible associated conditions: abnormal origin ofcoronary arteries (10%); atrial septal defect* (5%); coarctation of the aorta;* interrution of the aortic arch;* juxtaposition of atrial appendages (4%); overriding aorta (5%); overriding pulmonary artery (10%); patent ductal artery;* patent oval foramen; subvalvular pulmonary stenosis* (15%); tubular hypoplasia ofthe aortic arch;* ventricular septal defect* (30%), often mal-alignment type (50%). synonyms: atrioventricular and ventriculoarterial discordance; l-transposition. note: the basic anomaly is a mirror-image ventricular inversion, with a left anterior aorta, and with blood flow from right atrium to left ventricle to pulmonary artery, and from left atrium to right ventricle to aorta. for general dissection techniques, see chapter 3. interventions include patch closure of the ventricular septal defect; relief of pulmonary stenosis; relief of tricuspid insufficiency; and insertion of pacemakers. possible associated conditions: anterior and posterior av nodes (100%), prone to develop complete heart block; dysplasia or epstein's malformation* ofleft-sided tricuspid valve (40%); mirror-image epicardial coronary artery distribution (100%); right-sided mitral valve anomalies; subvalvular pulmonary stenosis* (40%); ventricular septal defect* (65%). kinyoun's, or other acid-fast stains. polymerase chain reaction for mycobacterial dna may be helpful in the differential diagnosis ofgranulomas (1). (4) universal precautions should be strictly followed and aerolization should be avoided. (5) reliable serologic studies are not available. a definite diagnosis requires isolation of the organism. (6) this is a reportable disease. cavitary, fibrocalcific, miliary, bronchial, and other types of pulmonary tuberculosis; granulomas in hilar lymph nodes. most organs and tissues may be involved, including liver, pancreas, spleen, kidneys, adrenal glands and other endocrine glands, gonads, and lymph nodes. tuberculous meningitis; tuberculoma. iridocyclitis or panophthalmitis. tuberculous arthritis and synovitis (hips, spine, knee); tuberculous osteomyelitis (anterior aspect of vertebrae; metaphysis of long bones). part ii / diseases and conditions procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. chronic ulcerative colitis may be associated with carcinoma of bile ducts, typically arising in psc.* metastases common in regional lymph nodes and lungs. i for removal, prosthetic repair, and specimen preparation and decalcification procedures, see chapter 2. if osteoblastic metastases appear to be present, search for primary tumor in prostate and breast, carcinoid tumors and hodgkin's lymphoma. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. cachexia. evidence of hypercalcemia (particularly in presence of osteolytic metastases). metastases to bone (e.g., from carcinoma of prostate, breast, bronchi, thyroid gland, kidney, or urinary bladder) usually involve red bone marrow. therefore, distal extremities are rarely involved by metastatic tumors in adults. metastases, commonly in lungs. metastatic calcification in patients with hypercalcemia. eosinophilia. myopathy.* metastases in liver and regional lymph nodes. see also above under "possible associated conditions." malakoplakia (rare) (2) . part ii / diseases and conditions other organs and peripheral arteries tumor(s). if tumor is within a heart chamber (usually a myxoma), record extent of mobility and capability of tumor to obstruct a valvular orifice. submit samples of tumor(s) for histologic study and, particularly if the tumor is of unknown type, for immunohistochemical study and electron microscopy (chapter 15). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. metastatic carcinoma or melanoma. secondary cardiac effects by tumors include carcinoid heart disease, amyloid heart disease in multiple myeloma, and lymphocytic myocarditis in pheochromocytoma. treatment effects such as adriamycin cardiotoxicity may be noted also. tumor (myxoma) emboli in pulmonary or peripheral vessels (1) . multiple aneurysms* of cerebral and other arteries may rarely be associated with atrial myxomas. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. part ii / diseases and conditions if there was evidence of endocrine activity (see above), snap-freeze portion of fresh tumor for hormone assay. perfuse lungs with formalin. sample neoplastic and non-neoplastic tissue for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 4. for removal and specimen preparation, see chapter 2. see above under "possible associated conditions." note that hypoglycemia* generally cannot be confirmed at autopsy. nonbacterial thrombotic endocarditis.* carcinoma may be associated with asbestosis or other types of pneumoconiosis,* chronic bronchitis,* emphysema,* interstitial pneumonia,* and many other bronchopulmonary diseases. see above under "possible associated conditions." metastases (regional lymph nodes, liver, bones, brain, and many other sites) and metastatic calcification. migratory thrombophlebitis.* encephalomyelitis;* cerebellar corticoid degeneration;* subacute spinocerebellar degeneration. * crooke cell hyperplasia. myasthenic syndrome (eaton-lambert syndrome); myopathy;* dermatomyositis.* peripheral neuropathy. see above under "external examination and skin." bones (with red marrow) are common sites of metastases. record presence or absence of testes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. thmor of the soft tissues the possible sites and characteristics of soft tissue tumors vary so much that no universally applicable autopsy techniques can be presented. in all instances, the size, weight, and location of the tumor(s) must be recorded and tissue must be sampled for histologic study. if the tumor had not been classified prior to death, samples should be snap-frozen for immunohistochemical study. other samples should be prepared for electron microscopic study. evidence of paraneoplastic syndromes (see below) may require additional procedures. possible associated conditions: only a few paraneoplastic syndromes or systemic complications can be presented here. for the type of soft tissue tumor that was associated with each condition, see title of reference. kasabach-merritt syndrome (1) (thrombocytopenia, microangiopathic hemolytic anemia, and acute or chronic coagulopathy associated with a rapidly enlarging hemangioma); liver function abnormalities (2); neurofibromatosis (3); osteomalacia (4). heart esophagus, stomach, and duodenum other organs and tissues t in most instances, detennination of vitreous sugar concentration and of blood group is not indicated. inspect valves and prepare photographs and sections of vegetations. leave esophagus and part of duodenum attached to stomach. submit samples of tumor and of grossly uninvolved stomach for histologic study. request pas stain and warthin-starry stain for identification of h. pylori. portions of endocrine gastric tumor should be snap-frozen for immunohistochemical study and possible hormone assay. record location of tumor metastases. other organs procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for dissection of the thoracic duct (for search of tumor cell clusters), see chapter 3. tumor of the uterus (with cervix) possible associated conditions: acquired immunodeficiency syndrome* (aids) (1) if peritonitis is present, submit exudate for bacteriologic study. record volume of exudate and location of perforation. see "disease, ischemic heart." other procedures depend on grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. perfuse at least one lung with formalin. for celiac arteriography, see chapter 2. open stomach and duodenum in situ and record site of perforation or penetration. record measured or estimated volume of blood in gastrointestinal tract. rinse ulcer with saline to locate eroded vessel(s). pin stomach and duodenum on corckboard (serosa toward board) and fix specimen in formalin before sectioning. prepare histologic sections of ulcer(s) and of remainder of stomach. request warthin-starry stain for h. pylori. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. free air in abdomen suggests perforation of ulcer. peritonitis. * perforation of ulcer. coronary atherosclerosis and manifestations of coronary insufficiency are commonly associated with peptic ulcer disease. in rare instances, pericardial fistula may be present from ulcer in hiatus hernia. peptic ulcers may be associated with emphysema,* tuberculosis,* and other chronic pulmonary diseases. vasculitis (see "aortitis," "arteritis, .â�¢.," "phlebitis," and "purpura,â�¢â�¢â�¢") ventricle, double inlet left (1) synonyms: single functional ventricle; univentricular heart; univentricular atrioventricular connection; holmes heart. note: the basic anomaly is the connection of both atrioventricular valves to the left ventricle, often with transposed great arteries and a restrictive ventricular septal defect. there are four major types, based on the ventriculoarterial connection: (1) with congenitally corrected transposition (60%); (2) with complete transposition (30%); (3) with normally related great arteries (holmes heart; 5%); and (4) double outlet, persistent truncal artery, or pulmonary atresia (5%). for general dissection techniques, see chapter 3. possible associated conditions: bicuspid pulmonary valve; bilateral mirror-image mitral valves (without tricuspid morphology); subvalvular aortic stenosis,* often with hypoplasia, coarctation, or interruption of the aortic arch; subvalvular pulmonary stenosis;* dual av nodes and progressive heart block in patients with congenitally corrected transposition. ventricle, double outlet right synonym: origin of both great arteries from right ventricle; taussig-bing anomaly. note: the basic anomaly is the origin of the aorta and pulmonary artery primarily from the right ventricle, usually with a ventricular septal defect, and often with subpulmonary stenosis. for general dissection techniques, see chapter 3. possible associated conditions: complete atrioventricular septal defect (often with asplenia syndrome); muscular discontinuity between aortic and mitral valves; right ventricular infundibular stenosis; ventricular septal defect* that may be subaortic, subpulmonary, doubly committed, or remote. examine histologically with h&e and oil red a stains. hepatomegaly and splenomegaly, jaundice. deficiency of acid lipase (1). foam cells in the lamina propria. hepatomegaly, severe steatosis with cholesterol clefts in hepatocytes and kupffer cells. fibrosis. splenomegaly with lipid-laden foam cells. symmetric enlargement with dystrophic calcifications, giving the adrenals a gritty texture. vacuolated cells of the inner zona fasciculata and entire zona reticularis with cholesterol clefts. foam cells may be found in the lungs, bone marrow, lymph nodes, vessel walls, as well as schwann and ganglion cells. new insights into lipoprotein assembly and vitamin e metabolism from a rare genetic disease angioid streaks associated with abetalipoproteinemia familial hypobetalipoproteinemia: genetics and metabolism aviation pathology scuba diving accidents: decompression sickness, air embolism aetiology and occurrence of diving injuries. a review of diving safety scuba decompression illness and diving fatalities in an overseas military community diving-related emergencies bodies found in water achalasia and squamous cell carcinoma of the esophagus: analysis of 241 patients esophageal achalasia and adenocarcinoma in barrett's esophagus: a report of two cases and a review of the literature a technique for necropsy evaluation of stenosis of the foramen magnum and rostral spinal canal in osteochondrodysplasia mutation analysis of the men i gene in multiple endocrine neoplasia type i, familial acromegaly and familial isolated hyperparathyroidism. i increased incidence of neoplasia in females with acromegaly benign and malignant tumors in patients with acromegaly pathology of acromegaly ethanol in sequestered hematomas stages of acute alcoholic influence/intoxication electrolyte imbalance in alcoholic liver disease collection and storage of specimens for alcohol analysis analysis for alcohol in postmortem specimens disposition of alcohol in man medicolegal aspects of alcohol. garriott jc blood, urine and other tissue specimens for alcohol analysis relationship between postmortem blood and vitreous humor ethanol levels larson cpo alcohol: fact and fallacy gradwohl's legal medicine left ventricular hypertrophy is more prominent in patients with primary aldosteronism than in patients with other types of secondary hypertension intracranial aneurysm and hemorrhagic stroke in glucocorticoid-remediable aldosteronism association of adrenal medullae and cortical nodular hyperplasia: a report of two cases with clinical and morpho-functional consideration amyloidosis: recognition, confirmation, prognosis, and therapy electron microscopy of primary and secondary cutaneous amyloidosis and systemic amyloidosis ocular amyloidosis current status review: cerebral amyloid spinal cord compression by amyloid deposits the burden of "sticky" amyloid: typing challenges quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with congo red histochemical stain kidney involvement in takayasu arteritis pathogenesis of rheumatoid arthritis splenic involvement in rheumatic diseases aspergillus sinusitis in patients with aids: report of three cases and review invasive aspergillosis in systemic lupus erythematosus aspiration (see "obstruction, acute airway aspergillus endocarditis, myocarditis and pericarditis complicating necrotizing fasciitis. case report and subject review aspergillus-related lung disease biliary atresia and the polysplenia syndrome: its impact on final outcome biliary atresia nephromegaly and elevated hepatocyte growth factor in children with biliary atresia coccidiomycosis pneumonia in a nonendemic area associated with inftiximab codeine (see "dependence, drug[s], all types or type unspecified all types or type unspecified (see "enterocolitis, other types or type undetermined chronic ulcerative (see "disease, inflammatory bowei cytologic diagnosis of cryp -tococcus neofonnans in hiv-positive patients cryptococcosis as an opportunistic infection in immunodeficiency secondary to paracoccidioidomycosis hypereosinophilia in disseminated cryptococcal disease cryptococcosis-a review of 13 autopsy cases from a 54-year period in a large hospital cryptosporidiosis in persons with hiv infection broncho-pulmonary cryptosporidiosis in four hiv-infected patients cryptosporidiosis and inflammatory bowel disease sclerosing cholangitis associated with chronic cryptosporidiosis in a child with a congenital immunodeficiency disorder cyanide (see "poisoning, cyanide cyst(s), choledochal choledochal cyst-dinical features and classification cyst(s), liver (see "disease, fibropolycystic, of the liver and biliary tract pulmonary related terms: congenital cystic adenomatoid malformation; congenital pulmonary lymphangiectasis; intralobular bronchopulmonary sequestration. references infection or calcification of cysts; pyelonephritis;* perinephric abscess. obstructive uropathy in recessive polycycstic renal disease, congenital hepatic fibrosis is present and cystic bile ducts may be present in dominant cases (5). see above under "possible associated conditions the pathology ofhuman renal cystic disease cystic kidney: genetics, pathologic anatomy, clinical picture, and prenatal diagnosis hepatic fibrosis associated with hereditary cystinosis: a novel form of noncirrhotic portal hypertension ophthalmic manifestations and histopathology of infantile nephropathic cyctinosis: report of a case and review of the literature pathological investigations of deaths following surgery, anaesthesia and medical procedures clinical outcome versus post-mortem finding in thoracic surgery: a lo-year experience order from american registry of pathology sales office intra-alveolar pulmonary siderophages in sudden infant death: a marker for previous imposed suffocation a contribution to a possible differentiation between sids and asphyxiation sudden infantdeath syndrome (sids)-standardized investigations and classification: recommendations investigation of the sudden deth of infants: a multicenter analysis alpha l-antitrypsin deficiency-associated panniculitis: resolution with intravenous alpha i-antitrypsin administration and liver transplantation clinical manifestations of alpha l-antitrypsin deficiency risk of hepatobiliary disease in adults with severe alpha i-antitrypsin deficiency (pizz): is chronic viral hepatitis b or c an additional risk factor for cirrhosis and hepatocellular carcinoma? alpha i-antitrypsin deficiency and inflammatory bowel disease severe alphal-antitrypsin deficiency (piz homozygosity) with membranoproliferative glomerulonephritis and nephrotic syndrome, reversi-bleafterorthotopic livertransplantation klippel-feil syndrome associated with malfonned larynx. case report klippel-feil syndrome in association with a craniocervical dennoid cyst presenting as aseptic meningitis in an adult: case report klippel-feil syndrome accompanied by an aneurysm of the non-coronary sinus of valsalva the hereditary ataxias clinical and genetic characterizations of 16q-linked autosomal dominant spinocerebellar ataxia (ad-sca) and frequency analysis of ad-sca in the japanese population cocain-related medical problems. consecutive series of 233 cases direct cocaine cardiotoxicity demonstrated by endomyocardial biopsy the pathology and etiology of cocaineinduced heart disease ischemic colitis related to cocaine abuse hepatic dysfunction accompanying acute cocaine intoxication high incidence of malignancies in patients with dennatomyositis and polymyositis: an li-year analysis partial lipodystrophy associated with juvenile dennatomyositis: report of two cases chronic pneumomediastinum and subcutaneous emphysema: association with dennatomyositis spontaneous esophageal rupture in adult dennatomyositis polyneuropathy in juvenile dermatomyositis combined glucose and lactate values in vitreous humor for postmortem diagnosis of diabetes mellitus diabetes is related to cerebral infarction but not to ad pathology in older persons hepatic lesions resembling alcoholic liver disease relationship between cardiomyopathy and liver disease in chronic alcoholism cyanamide-associated alcoholic liver disease: a sequential histologic evaluation caroli's disease: 1977-1995 experiences caroli 's disease and autosomal dominant polycystic kidney disease: a rare asso-ciation? spontaneous rupture of a bile duct and its endoscopic management in a patient with caroli's syndrome bartonella henselae endocarditis in an immunocompetent adult cat-scratch disease and bacillary angiomatosis attack, transient cerebral ischemic" and "infarction, cerebral!') cholesteryl ester storage disease: hepatopathology and effects of therapy with lovestatin clinical, biochemical and histological analysis of seven patients with cholesterol ester storage disease cutaneous manifestations of chronic granulomatous disease. a report of four cases and review of the literature aspergillus endocarditis in chronic granulomatous disease colitis complicating chronic granulomatous disease. a clinicopathological case report hypertrophic cranial pachymeningitis with spinal epidural granulomatous lesion ocular pathologic findings of chronic granulomatous disease of childhood review: creutzfeldt-jakob disease survival of creutzfeldt-jakob disease virus in formol-fixed brain tissue guidelines for high risk autopsy cases: special precautions for creutzfeldt-jakob disease tissue handling in suspected creutzfeldt-jakob disease and other human spongiforme encephalopathies (prion diseases) cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of creutzfeldt-jakob disease autopsy case of sporadic creutzfeldt-jakob disease presenting with signs suggestive of brainstem and spinal cord involvement metastatic crohn's disease: a rare cutaneous manifestation crohn's disease with pulmonary involvement in a 3 year old boy mesenteric fibromatosis asso-ciated with crohn's disease cholangiocarcinoma and crohn's disease the pancreas as a site of granulomatous inflammation in crohn's disease dermatomyositis associated with crohn's disease 1994 cardiocyte storage and hypertrophy as a sole manifestation of fabry's disease. report on a case simulating hypertrophic non-obstructive cardiomyopathy an atypical variant of fabry's disease with manifestations confined to the myocardium pulmonary involvement in fabry disease cerebrovascular complications offabry's disease high incidence of thrombosis in fabry's disease oral involvement in chronic graft versus host disease after allogenic bone marrow transplantation massive pericardial effusion complicating the course of chronic graft-versus-host disease (cgvhd) in a child with acute lymphoblastic leukemia following allogeneic bone marrow transplantation the histological spectrum of pulmonary graft-versushost disease in bone marrow transplant recipients bronchiectasis in bone marow transplantation oncocytic metaplasia of bile duct epithelium in hepatic gvhd immune-mediated myelopathy after allogeneic marrow transplantation gunther's (see ''porphyria, congenital erythropoietic heavy-chain synonyms and related terms: gamma heavy-chain inflammatory bowel disease (first of two parts) takayasu's arteritis associated with idiopathic ulcerative colitis cerebrovascular complications ofinflammatory bowel disease pathology of inflammatory bowel disease legionnaires' disease source of infection for sporadic legionnaires' disease: a review legionnaire's disease associated with macular rash; two cases severe skin loss after meningococcal septicemia: complications in treatment osteonecrosis following meningococcemia and disseminated intravascular coagulation in an adult: case report and review rhabdomyolysis during the subacute stage of meningococcal sepsis primary meningococcal arthritis in a child: case report and literature review chondrosarcoma as a complicating factor in paget's disease of bone lymphoma arising in paget's disease osteoarthritis and paget's disease sickle cell disease sickle cell disease and sudden death: a retrospective/prospective study of 21 autopsy cases and literature review vascular lesions of the liver in sickle cell disease. a clinicopathologic study in 26 living patients whipple's disease and "tropheryma whippelii periodic acid-schiff-negative granulomatous lymphadenopathy in patient with whipple's disease. localization of the whipple bacillus to noncaseating granulomas by electron microscopy serial imaging studies of cerebral whipple's disease: from onset to end the wilson's disease gene is a putative copper transporting p-type atpase similar to menke's gene the liver biopsy diagnosis of wilson's disease. methods in pathology hepatolenticular degeneration (wilson's disease) venous air embolism in homicidal blunt impact head trauma: case reports a practical device for demonstrating air embolism amniotic fluid 3. kulka w. laboratory methods and technical notes: a practical device for demonstrating air embolism proof of fatal air embolism venous air embolism from head and neck wounds hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease liver involvement in hemorrhagic fever with renal syndrome hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hanta virus pulmonary syndrome lassa fever in the united states. investigation ofa case and new guidelines for management lassa fever: review of virology, immunopathogenesis, and algorithms for control and therapy early diagnosis of lassa fever by reverse transcription-pcr rheumatic pneumonia: reappearance of a previously recognized complication of rheumatic fever acute rheumatic fever and poststreptococcal acute glomerulonephritis caused by t serotype 12 streptococcus scleritis, uveitis, and glaucoma in a patient with rheumatic fever comparison of clinical features and pathologic findings in fatal cases of typhoid fever during the initial and later stages of the disease typhoid fever complicated by acute renal failure and hepatitis: case reports and review hemophagocytosis and granulomas in the bone marrow of a child with down syndrome diseases involving intrahepatic bile ducts idiopathic eosinophilic esophagitis with stricture formation in a patient with longstanding eosinophilic gastroenteritis eosinophilic gastroenteritis presenting with colitis and cholangitis eosinophilic gastroenteritis presenting with acute pancreatitis idiopathic midline destructive disease: does it 3. mendenhallwmetai.lethalmidlinegranoloma-nasalnaturalkillerff-celi exist? nasal cocain abuse mimicking midline granuloma pulmonary lymphomatoid granulomatosis in acquired immunodeficiency syndrome: lesions with epstein-barr virus infection recurrent bilateral spontaneous pneumothoraces associated with pulmonary angiocentric immunoproliferative lesion lymphomatoid granulomatosis: pathogenesis, pathology and clinical implications wegener's granulomatosis: histological documentation of common and uncommon manifestations in 216 patients necrotizing granulomatosis of the breast wegener's granulomatosis with gangrene of toes pathology of pulmonary granulomatous vasculitis. sarcoidosis gunshot (see "injury, firearm splenic involvement in rheumatic diseases wegener's granulomatosis, acute laryngotracheal airway obstruction and death in a 17-year-old female: case report and review of the literature antiendothelial antibodies in patients with wegener's granulomatosis: prevalence and correlation with disease activity and manifestations. i rheumatoi2007 transfusion-transmitted disease references flattening, broadening, and possible fusion of villi. phlegmonous inflammation and edema, mainly in ileocecal region encephalitis.* leukopenia; thrombocytopenia; aplastic anemia (4) (pancytopenia*) synovitis (arthritis*) hepatitis g virus infection in hepatitis c virus-positive patients co-infected or not with hepatitis b virus and/or human immunodeficiency virus hepatitis g and c coinfection in children tumor of the liver diaphragmatic related terms: congenital diaphragmatic hernia fulminant hepatitis in patients undergoing liver transplantation: evidence for anon-a, non-b, non-c marrow transplantation for hepatitis-associated aplastic anemia: a follow up oflong-term survivors langerhans cell histiocytosis research. past, present, and future an update on c1onality, cytokines, and viral etiology in langerhans cell histiocytosis langerhans cell histiocytosis in adults pulmonary langerhans cell granulomatosis central nervous system disease in langerhans cell histiocytosis immunohistochemical analysis oflangerin in langerhans cell histocytosis and pulmonary inflammatory and infectious diseases pathological approach to the diagnosis of hydrocephalus thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report infectious causes ofhydrops fetalis human parvovirus b19 infection associated with hydrops fetalis cardiac abnormalities associated with hydrops fetal is gmi-gangliosidosis presenting as nonimmune hydrops fetalis: a case report a case of recurrent infantile polycystic kidney associated with hydrops fetalis the pathologist and the hydropic placenta, fetus, or infant chronic diarrhea associated with thymoma and hypogammaglobulinemia (good's syndrome) systemic amyloidosis in a patient with hypogammaglobulinemia. i hcv infection in patients with primary defects of immunoglobulin production encepahomyelitis in primary hypogammaglobulinemia retinitis pigmentosaand hypogammaglobulinemia intestinal obstruction in the newborn with congenital syphilis meconium ileus and its equivalent as a risk factor for the development of cirrhosis: an autopsy study in cystic fibrosis biliary atresia and meconium ileus associated with nieman-pick disease postmortem cranial mri and autopsy correlation in suspected child abuse cytomegalovirus-associated pseudotumor simulating gastric malignancy in acquired immuno-deficiency syndrome: a case report with review of literature bone marrow fibrin ring granulomas and cytomegalovirus infection cutaneous reactions following herpes zoster infections: report of three cases and a review of the literature herpes zoster and internal malignancy posteriorhorn varicella-zoster virus myelitis death or injury caused by electrocution myocardial hemorrhagic necrosis in delayed death from electrocution safety in bullet recovery procedures: a study of the black talon bullet lightning injuries: prognostic signs of death lightning injuries adrenal insufficiency, recurrent bacteremia, and disseminated abscesses caused by nocardia asteroides in a patient with acquired immunodficiency syndrome ards and adrenal insufficiencyassociated with the antiphospholipid antibody syndrome adrenal insufficiency from bilateral adrenal hemorrhage after total knee replacement surgery non-hodgkin's lymphoma presenting with adrenal insufficiency and hypothyroidism: an autopsy case report a case ofprimary unilateral adrenal burkitt-like large cell lymphoma presenting as adrenal insufficiency effect of growth hormone on fatty liver in panhypopituitarism increased fracture frequency in adult patients with hypopituitarism and gh deficiency pituitary abscess the liver: an atlas and text of ultrastructural pathology intubation (see "injury, intubation iodine (see "poisoning, iodine attack, transient cerebral ischemic" and "infarction, cerebral heart (see "disease, ischemic heart isomorism (see "syndrome, polysplenia and asplenia a study on performance of two serological assays for diagnosis of leprosy detection of mycobacterium leprae infection by pcr pathology of eye in leprosy references intervillous abscesses containing grampositive, non-acid-fast bacilli bacteria are predominantly intracellular. enterocolitis. listeria monocytogenes can be cultured from meconium. generalized lymphadenitis. disseminated abscesses and/or granulomas, particularly after transplacental infection listeria monocytogenes empyema in an hiv infected patient fatal listeria meningitis, endocarditis and pericarditis in a patient with haemochromatosis liver abscesses due to listeria monocytogenes yust 1. brainstem abscess and meningitis due to listeria monocytogenes in an adult with juvenile chronic arthritis pulmonary hypertension associated with systemic lupus erythematosus: predominantly thrombotic arteriopathy accompanied by plexiform lesions the liver in systemic lupus erythematosus: pathologic analysis of 52 cases and review of japanese autopsy registry data chronic pancreatitis: a complication of systemic lupus erythematosus disseminated perivenous necrotizing encephalomyelitis in systemic lupus erythematosus: report of an autopsy case skeletal muscle lymphocytic vasculitis in systemic lupus erythematosus: relation to disease activity clinically occult avascular necrosis of the hip in systemic lupus erythematosus storage histiocytes and hemophagocytosis: a common finding in the bone marrow of patients with active systemic lupus erythematosus extensive medium vessel vasculitis with sle: an unsual association cutaneous manifestations of sexually transmitted ing cause of proctitis in men who have sex with men lymphogranuloma venereum as a cause cutaneous malakoplakia in a patient with acquired immunodeficiency syndrome (aids) extensive malakoplakia ofthe nasopharynx: management of a rare disease malakoplakia and colorectal adenocarcinoma coinfection is common in measles associated pneumonia giant cell pneumonia: light microscopy, immunohistochemical, and ultrastructural study of an autopsy case subacute sclerosing panencephalitis manifesting as viral retinitis: clinical and histopathologic findings intestinal neuronal dysplasia thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report disease, meningococcal encephalitis, all types or type unspecified" and "meningitis mercury (see "poisoning, mercury with myelofibrosis synonym: idiopathic myelofibrosis acute cardiac tamponade associated with pericardial extramedullary hematopoiesis in agnogenic myeloid metaplasia methyl alcohol) (see "poisoning, methanol (methyl alcohol) thrombotic thrombocytopenic (see "purpura, thrombotic thrombocytopenic epstein-barr virus in inflammatory diseases of the liver and liver allografts: an in situ hybridization study fulminant hepatitis due to epstein-barr virus infection the mucopolysaccharidoses: a clinical review and guide to management biochemical diagnosis of mucopolysaccharidoses: experience of 297 diagnoses in a 15-year period (1997-1991) mucormycosis synonym: phycomycosis; zygomycosis. note: diseases that may be complicated by mucormycosis include bums,* diabetes mellitus,* leukemia,* lymphoma,* and tuberculosis cardiac involvement in mucopolysaccharidosis: effects of allogeneic bone marrow transplantation mitral and aortic regurgitation in 84 patients with mucopolysaccharidosis toxic epidermal necrolysis possibly linked to hyperacute graft-versus-host disease after allogeneic bone marrow transplantation pulmonary complications in toxic epidermal necro-iysis: a prospective clinical study references possible or expected findings pyelonephritis;* nephrocalcinosis; granulomas; tumor infiltrates; manifestations of the conditions listed below under "other organs renal stones in patients with rheumatoid arthritis nephrolithiasis as a presenting feature ofchronic sarcoidosis: a prospective study nephrolithiasis and risk of hypertension posterior lens opacities; retinal hamartomas. peripheral neuropathy with focal schwannomatous changes or onion-bulb-like schwann cell or perineural cell proliferation neurofibromatosis type i. in: pathology and genetics of tumours of the nervous system neurofibromatosis type 2. in: pathology and genetics oftumours ofthe nervous system neurological complications involving the central nervous system in neurofibromatosis type i primary cutaneous nocardia asteroides infection after heart transplantation native valve endocarditis due to nocardia-like organisms hepatobiliary complications of total parenteral nutrition total parenteral nutrition: a histopathologic analysis of the liver changes in 20 children obesity and breast cancer risk nonalcoholic steatohepatitis obesity cardiomyopathy: pathogenesis and pathophysiology bennett cpo pachydennoperiostitis in childhood unilateral hypertrophic osteoarthropathy associated with aortofemoral graft infection gastroesophageal reflux and esophagitis-associated hypertrophic osteoarthropathy hypertrophicosteoarthropathy caused by lipoid pneumonia achondroplasia" and dyschondroplasia, oilier's osteogenesis imperfecta synonyms and related terms: lobstein's syndrome osteogenesis imperfecta congenita; 01 type i, ii, and iii; osteogenesis imperfecta cystica hypercalcemia in osteogenesis imperfecta treated with pamidronate gastrointestinal problems in patients who have type-iii osteogenesis imperfecta osteomalacia related terms: osteoporosis;* renal osteodystrophy; rickets. note: many possible causes of osteomalacia may not be apparent at autopsy, e.g., sodium fluoride or diphosphonate toxicity or use of anticonvulsant drugs osteomalacia associated with adult fanconi syndrome: clinical and diagnostic features epstein-barr virus in b-celllymphomas associated with chronic suppurative inflammation osteomyelitis and osteonecrosis in inflammatory bowel disease references hyperlipidemia; hyperuricemia. fracture or traumatic dislocation of hip see above under "note osteomyelitis and osteonecrosis in inflammatory bowel disease osteonecrosis and human immunodeficiency virus infection report of fifteen cases. therapeutic recommendations malignant infantile osteopetrosis: otolaryngological complications and management renal tubular acidosis and osteopetrosis with carbonic anhydrase ii deficiency: pathogenesis of impaired acidification spondylolysis in children who have osteopetrosis osteopetrosis with arnold chiari malformation type 1 and brain stem compression osteoporosis and atherosclerosis in chronic renal failure endocrine disorders and osteoporosis otosclerosis: a measles virus associated inflammatory disease correlations between pathologic changes in the stapes and conductive hearing loss in otosclerosis the aetiology of otosclerosis: a review of the literature etiologic links between chronic pancreatitis and pancreatic cancer hyperparathyroidism and pancreatitis during pregnancy pancytopenia related terms: agranulocytosis;* aplastic anemia; fanconi's anemia.* note: some causes of pancytopenia such as adverse drug reactions (e.g., due to antimetabolites, sulfa drugs thrombotic thrombocytopenic purpura/hemolytic uremic syndrome secondary to pancreatitis retinopathy as asys-temic complication of acute pancreatitis references mesenteric panniculitis necrotizing pancreatitis. * vasculitis with thrombi; adrenocortical infarctions. vasculitis with thrombi and infarctions. relapsing polychondritis (4). see also above under "possible associated conditions subcutaneous panniculitic t-cell lymphoma is a tumor of cytotoxic t lymphocytes abecassism. alpha i-antitrypsindefi-ciencyassociated panniculitis: resolution with intravenous alpha l-anti-trypsin administration and liver transplantation blind loop syndrome: an unusual cause of panniculitis. j am acad paracoccidioidomycosis synonyms: paracoccidioides brasiliensis infection cutaneous panniculitis and relapsing polychondritis: two cases normal subcutaneous fat, necrosis of adipocytes and classification of the panniculitides paraneoplastic pemphigus a case of pemphigus vulgaris with esophageal involvement rheumatoid constrictive pericarditis (clinical conference) benign paroxysmal (see "fever, familial mediterranean pneumatosis intestinalis in pediatric acquired immunodeficiency syndrome pneumatosis cystoides intestinalis: an unusual complication of systemic amyloidosis pneumatosis intestinalis in children with primary combined immunodeficiency pneumatosis cystoides intestinalis with abdominal free air in a 2-year-old girl after allogeneic bone marrow transplantation pneumatosis intestinalis: a review pneumatosis coli: a proposed pathogenesis based on study of 25 cases and review of the literature collagenous inorganic dust pneumoconiosis, diffuse type: aluminosis; asbestosis; chronic pulmonary berylliosis; talcosis; collagenous inorganic dust pneumoconiosis, nodular type: silicosis; noncollagenous inorganic dust pneumoconiosis (includes mixed-dust fibrosis): baritosis; china clay pneumoconiosis; chromite pneumoconiosis; coal worker's pneumoconiosis; fuller's earth pneumoconiosis; hematite or magnetite miner's lung; siderosis or welder's lung; stannosis; organic dust pneumoconiosis: byssinosis. note: in addition to the aforementioned classic forms of pneumoconiosis, many other airborne substances have been impli-cated in recent years, for example, cerium uncommon causes ofoccupational interstitial lung diseases rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review diagnostic criteria for chronic beryl1ium disease (cbd) based on the uk registry 1945-1991 pneumocystis carinii (see "infection, pneumocystis carinii this condition is diagnosed by inspection, palpation, and roentgenography. tension pneumomediastinum may autopsy evaluation of asbestos exposure: retrospective study of 135 cases with quantitation of ferruginous bodies in digested lung tissue atypical mycobacteriosis as a complication of talc pneumoconiosis silicosis, mixed dust pneumoconiosis, and lung cancer complications associated with the use ofthe esophageal-tracheal combitube pneumomediastinum: an unusual complication of asthma in a young man spontaneous pneumomediastinum in a patient with bronchiolitis obliterans after bone marrow references i. katzenstein a-l, myers j. idiopathic pulmonary fibrosis: clinical relevance of pathologic classification update on lymphoid interstitial pneumonitis lymphocytic interstitial pneumonitis and nonspecific interstitial pneumonitis histologic diagnosis of extrinsic allergic alveolitis role of electron microscopy in interstitial lung disease detennination of arsenic in hair using neutron activation arsenic intoxication associated with tubulointerstitial nephritis atrioventricular block after administration of atropine in patients follow-ing cardiac transplantation death following intentional methyl bromide poisoning: toxicological data and literature review pulmonary edema, alveolar wall damage, and interstitial pneumonia after acute inhalation. severe pulmonary fibrosis may develop in chronic cases. gastroenteritis after nonlethal food poisoning. degeneration of proximal tubules and proteinuria in acute poisoning degenerative changes of liver and myocardium problems in the analysis of cadmium in autopsied tissues elevated urinary cadmium concentrations in a patient with acute cadmium pneumonitis cadmium-associated renal disease the effects of storage conditions on the stability of carbon monoxide in postmortem blood acute renal failure, poisoning, carbon tetrachloride synonym: tetrachloromethane poisoning. note: toxicologic sampling of body fluids and organs should be done routinely in all cases. in many instances, however, death occurs i wk to 10 d after exposure, and by this time no carbon tetrachloride is demonstrable. death may be sudden compartment syndrome, and systemic capillary leak syndrome complicating carbon monoxide poisoning southern je acute hydrocephalus following carbon monoxide poisoning prevention of occupational cyanide exposure in autopsy prosectors gradwohl's legal medicine references possible or expected findings digoxin values in digitalis toxicity are greater than 2 ng/ml (1) digoxin concentration may be higher or lower than concentration in serum, depending on how long before death drug was taken (i) digoxin concentrations in postmortem specimens after overdose and therapeutic use a fluorimetric determination ofmyocardial digoxin at autopsy, with identification of digitalis leaf, digitoxin and gitonin ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis ethylene glycol poisoning: case report of a record-high level and a review lead concentrations in human plasma, urine and whole blood trace metals (lead and cadmium screening) an emg case report oflead neuropathy 19 years after a gunshot injury aminoaciduria and glycosuria following severe childhood lead poisoning risk of nervous system cancer among workers exposed to lead demonstration of mercury in the human brain and other organs 17 years after metallic mercury exposure spontaneous exfoliation of teeth following severe elemental mercury poisoning: case report and histological investigation for mechanism. oral surg oral med oral pathoi1997 acute chemical pancreatitis associated with nonfatal strychnine poisoning homicide by strychnine poisoning coronary arteritis, with or without aneurysms and infarctions, primarily in childhood. myocardial hypertrophy secondary to hypertension. * minimal or no involvement by polyarteritis nodosa; considerable involvement in other types ofnecrotizing vasculitis (see "arteritis, all types or type unspecified") with or without formation of aneurysms and infarctions, may occur in all organs. the liver may show bile duct injury and rarely, nodular regenerative hyperplasia (8) more frequently involved in giant cell elastic arteritis.* polyarteritis of small muscular arteries, including vasa nervorum. arthritis* may rarely be present with swollen joints. infarctions;* subarachnoid hemorrhage; arteritis of cerebral arteries. papillitis; retinal hemorrhages polyarteritis nodosa-like vasculitis in human immunodeficiency virus infection the coexistence of familial mediterranian fever and polyarteritis nodosa: report of a case microscopic polyarteritis during polymyalgia rheumatica remission development of polyarteritis nodosa in the course of inactive systemic lupus erythematosus bone marrow pathology in relapsing polychonditis: high frequency of myelodysplastic syndrome relapsing polychondritis associated with subclinical sjo-gren's syndrome and phlegmon of the neck giant cell arteritis and polymyalgia rheumatica posterior scleritis and polymyalgia rheumatica polymyalgia rheumatica in patients with ankylosing spondylitis: a report of 5 cases inflammatory changes of hip synovial structures in polymyalgia rheumatica polymyositis (see "dermatomyositis polyneuritis (see "syndrome, guillain-barre familial, and related syndromes related terms: cowden's disease (multiple hamartoma syndrome); familial colonic polyposis gardner's syndrome; non-polyposis syndrome (hereditary nonpolyposis colorectal cancer syndrome) sampling for histologic study depends on expected findings or grossly identified abnormalities as listed in right-hand column. (see also below under "soft tissues syndrome; mucocutaneous pigmentations (buccal, perioral, priorbital, distal extremities) in peutz-jeghers syndrome;* papules in face and oral mucosa in cowden's disease; tumors of skin and subcutis (see below under "soft tissues gardner's syndrome. osteomas or exostoses (mandible, calvara desmoid in familial adenomatous polyposis malignant potential in intestinal juvenile polyposis syndromes ampullary carcinoma in familial adenomatous polyposis adenoma of the common bile duct in gardner's syndrome may cause relapsing acute pancreatitis orbital osteoma in gardner's syndrome myelopathylmyelitis," and "syndrome, guillain-barre.") references possible or expected findings hypertrichosis; erythrodontia; scarring and mutilation of hands and face. hemolytic anemia. erythrocytes contain large amounts of uroporphyrin i; nonnoblasts and reticulocytes exhibit intense red fluorescence uroporphyrin i and coproporphyrin i in high concentrations. splenomegaly (may have been treated by splenectomy) correction of congenital erythropoietic porphyria by bone marrow transplantation successful cord blood stem cell transplantation for congenital erythropoietic porphyria (gunther's disease) congenital erythropoietic porphyria associated with nephrotic syndrome atypical red cell inclusions in congenital erythropoietic porphyria possible associated conditions: adverse drug reaction; chronic alcoholism;* chronic hepatitis c (1); human immunodeficiency virus infection porphyria cutanea tarda and human immunodeficiency virus: two cases associated with hepatitis c porphyria cutanea tarda and antibodies to hepatitis c virus an unhappy triad: hemochromatosis, porphyria cutanea tarda and hepatocel1ularcarcinoma-a case report variegate porphyria possible associated conditions: ebstein's malformation of tricuspid valve. references possible or expected findings chronic eczematous skin lesions or superficial scarring; nail changes. perivascular pas-positive hyaline (2) brown protoporphyrin deposits with red to yellow birefringence with a maltese cross configuration in hepatocytes, kupffer cells, and bile canaliculi erythropoietic protoporphyria cytofluorometry as a diagnosis of protoporphyria recessive inheritance of erythropoietic protoporphyria with liver failure pseudogout synonym: calcium pyrophosphate dihydrate (cppd) deposition disease puncture grossly affected joints and submit sample of synovial fluid for crystal analysis under compensated polarized light (2) references possible or expected findings punctate calcium deposits in kneejoints and, less commonly, in joints of hips, ankles, shoulders, or wrists or in symphysis ossium pubis and intervertebral disks. arthritis* with synovitis. crystals of calcium pyrophosphate dihydrate in periarticular tissue (1) and synovial fluid. cartilage contains calcium salts of pyrophosphate, hydroxyapatite, and orthophosphate. alkaptonuria;* gout calcium pyrophosphatedihydratedeposition disease presenting as tumoral calcinosis (periarticularpseudogout) calcifications in dermis; calcifications of blood vessels. diaphragmatic hernia.* hypertensive cardiomegaly. characteristic plaques in pericardium and endocardium, with or without mitral valve involvement. coronary atherosclerosis may have been a causeofangina(2).coronarythrombosisand myocardial infarction may be present also. accelerated atherosclerosis; calcification of peripheral vessels. gastrointestinal hemorrhage;* peptic ulcer degeneration of bruch's membrane with retinal hemorrhages; sclerosis of choroid vessels calcification of elastic fibers in pseudoxanthoma elasticum new report of severe coronary artery disease in an eighteen-year-old girl with pseudoxanthoma e1asticum. case report and review of the literature hivrelated thrombotic thrombocytopenic purpura: report of 2 cases and a review of the literature beham-pyelonephritis synonyms and related terms: acute ascending pyelone angiotropic large cell lymphoma presenting as thrombotic micro-angiopathy (thrombotic thrombocytopenic purpura) splenic pathology in thrombotic thrombocytopenic purpura emphysematous pyelonephritis in diabetic patients nephrobronchialfistulaandlungabscessresulting from nephrolithiasis and pyelonephritis a case ofan enterorenal pyothorax (see "empyema, pleurai.") fistula and pyelonephritis with airin renal pelvis systemic amyloidosis secondary to pyonephrosis. resolution after nephrectomy medicolegal investigation of death the laboratory's role in detecting sexual assault toluidine blue in the detection at autopsy of perineal and anal lacerations in victimes of sexual abuse sarcoidosis presenting as heart disease sarcoidosis and its ocular manifestations rheumatic features of sarcoidosis cutaneous sarcoidosis: differential diagnosis schistosoma mansoni and s. haematobium infections in egypt. i. evaluation of techniques for recovery of worms and eggs at necropsy. am i a quantitative post-mortem study of schistosomiasis mansoni in man immunodiagnosis of schistosomiasis. screen with fast-elisa and confinn with imrnunoblot hepatic connective tissue changes in hepatosplenic schistosomiasis schistosomiasis in women: manifestations in the upper reproductive tract sclerosis, systemic amyotrophic lateral (see "disease, motor neuron diffuse (see "sclerosis, schilder's cerebrai multiple synonyms and related terms: acute and subacute variants: acute multiple sclerosis (marburg type), acute necrotizing myelopathy; concentric sclerosis (ba16 type); concentric lacunar leukoencephalopathy; encephalitis periaxialis diffusa (schilder's type; see also next entry silicone ganuloma in acral skin in a patient with silicone-gel implants and systemic sclerosis organizing diffuse alveolar damage associated with progressive systemic sclerosis bayle 0, fiessinger in. brain involvement in scleroderma: two autopsy cases skeletal muscle pathology in systemic sclerosis mechanisms of scleroderma-induced lung disease thberous sclerosis complex and subependymal giant cell astrocytoma bilateral temporal arachnoid cysts associated with tuberous sclerosis poisoning, alkaloid accident, diving [skin or scuba scurvy (see "deficiency, vitamin c shigellosis (see "dysentery, bacillary shock related terms: anaphylactic shock; bacteremic shock; many others. note: see also under name of suspected underlying condition, such as "bums intestinal histological and ultrastructura1 inflammatorychanges in spondyloarthropathy and rheumatoid arthritis the sacroiliac joint in the spondyloarthropathies granulomatous ileitis in a patient with ankylosing spondylitis sporotrichosis synonym: sporothrix (sporotrichum) schenckii infection. note: (1) mckiernan 1. partial lipodystrophy in coeliac disease coeliac disease and lymphoma: current status review: nonalcoholic steatohepatitis alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease non-alcoholic fatty liver disease in the metabolic syndrome the placenta in stillbirth estimating the time of death in stillborn fetuses: i. histologic examination of fetal organs: an autopsy study of 150 stillborns estimating the time of death in stillborn fetuses: ii. histologic evaluation ofthe placenta; a study of71 stillborns estimating the time ofdeath in stillborn fetuses: iii. external fetal examination; a study of86 stillborns fat distribution in the adrenal cortex as an indication of the mode of intrauterine death stimulant(s) (see "dependence, drug(s), all types or type undetermined cutaneous manifestations of the acquired immunodeficiency syndrome aids: cardiac findings from 115 autopsies the liver in hiv infection neuropathology of the spinal cord in the acquired immunodeficiency syndrome postmortem enzyme immunoassay for human immunodeficiency virus bk virus-associated renal failure among hiv patients -2100 or write to american registry ofpathology sales office pathology of acquired immunodeficiency syndrome (aids) in children thymus involution in the acquired immunodeficiency syndrome micrencephaly in children congenitally infected with human immunodeficiency virus-a gross-anatomical morphometric study a rapid method for detecting the predominant ashkenazi jewish mutation in the bloom's syndrome gene bloom syndrome: multiple retinopathies in a chromosome breakage disorder severe eczema in a patient with di-george's syndrome digeorge's syndrome orthe iii -iv pharyngeal pouch syndrome: pathology and a theory of pathogenesis basal ganglia calcification and psychosis in 22q 11.2 deletion syndrome down's synonyms and related terms: mongolism possible associated conditions: acute lymphocytic, my references epicanthal folds; cleft lip/palate; high arched palate; furrowed tongue; rhagades. depressed nasal bridge small, broad or fiat nose; slanted palpebral fissures; fiat occiput; brachycephaly; palmar "simian crease"; short fifth middle finger ventricular septal defect;* complete atrioventricular septal defect* (1). duodenal stenosis and atresia; imperforate anus. microcephaly; poorly developed secondary gyri hypoplastic brain stem, medulla and cerebellum; polymicrogyria; neurofibrillary tangles; meningomyelocoele. acute lymphocytic leukemia; acute myelocytic leukemia; acute megakaryocytic leukemia cardiovascular disease in down syndrome possible or expected findings hyperelasticity of skin, bruises or scars, hemorrhages, and hyperpigmentation. lipomatous pseudotumors that may be calcified or ossified. normal or abnormal amounts and fragmentation of elastic tissue. abnormalities of collagen. dislocation of hip, shoulder, patella, radius, or clavicle. loose-end clavicles; spondylolisthesis mitral valve prolapse; aortic insufficiency.* aortic dissection, aneurysm, ectasia, occlusion (1). various congenital anomalies. congenital anomalies of gastrointestinal and genitourinary tracts. bleeding from various organ sites vascular ehlers-danlos syndrome: imaging findings note: this syndrome belongs to a large family (with more photograph abnormal features as listed in right-hand column. record appearance of external genitalia. prepare skeletal roentgenograms. procure long bones and vertebral bodies short-limb dwarfism* with narrowing of the rib cage; polydactyly; dysplasia of fingernails; thin and sparse hair; premature eruption of teeth; defective dentition; eye abnormalities; upper lip bound down by multiple frenula. cryptorchidism; epispadias; hypospadias. chondrodysplasia with acromelic micromelia (shortening of the distal segment of the limb); fusion of capitate and hamate bones of wrist; defects of lateral aspect of proximal tibia see lesions listed above under "external examination empty sella synonyms and related terms: primary empty sella syndrome; secondary empty sella syndrome (e.g., after surgical removal or spontaneous infarction of pituitary adenoma) chorea, philic pneumonia: histopathologic features in nine cases. am rev resp hereditary ascariasis and hookworm chronic eosinophilic pneupophosphatemic vitamin d-resistant rickets; galactosemia;transport defect; tyrosinemia. * excludes fan-1988 weight and external measurements; record and photograph abnormalities as listed in right-hand column. prepare skeletal roentgenograms. radiographs of long bones. submit for tissue culture for possible enzyme analysis. possible or expected findings redlblond hair, fair skin (diminished pigmentation); dehydration;* stigmata of hypothyroidism;* delay of sexual maturation. rickets; osteomalacia.* positive rheumatoid factor. various types of pneumonia after splenectomy, presence of accessory spleen may account for treatment failure. manifestations of portal hypertension.* lymphadenopathy of mediastinal and paraaortic lymph nodes. anemia;* neutropenia sinusoidal lymphocytosis of the liver in felty's syndrome with a review of the liver involvement in felty's syndrome splenic involvement in rheumatic diseases felty's and pseudo-felty's syndrome nodular regenerative hyperplasia of the liver in rheu-matic diseases: report of seven cases and review of the literature fetal alcohol syndrome: craniofacial and central nervous system manifestations goodpasture's syndrome anti-glomerular basement membrane glomerulonephritis: a morphologic study of 80 cases gronblad-strandberg (see "pseudoxanthoma elasticum acute inflammatory polyradiculoneuropathy; guillain-barre-strohl syndrome hemolytic uremic syndrome/thrombotic thrombocytopenic purpura: pathophysiology and treatment cyclosporin-induced hemolytic uremic syndrome: factors that obscure its diagnosis enterohemorrhagic escherichia coli 0157:h7: an emerging pathogen hemolytic uremic syndrome as a presenting form of hiv infection hemolytic uremic syndrome in prostatic carcinoma diabetes secondary to genetic disorders. baillieres suprasellar tumors of maldevelopmental origin in klinefelter's syndrome. a report of two cases klippel-feu synonym: congenital fusion of cervical vertebrae primary yolk sac tumor of the prostate in a patient with klinefelter's syndrome extragonodal germ cell tumors are often associated with klinefelter syndrome organs and tissues procedures possible or expected findings external examination prepare roentgenograms of chest, neck, and head skull, spine, brain, and spinal cord myasthenia gravis disorders with dysraphia (see below), fusion of cervical vertebrae. congenital elevation of the scapula (sprengel's deformity) chiari malformation;* basilar impression;* meningomyelocele; platybasia;* spinal cord compression; weight and length; record and photograph abnormalities as listed in right-hand column. record weight and sample for histologic study. if renal transplantation (3) had been carried out, see also under that heading. follow procedures described under "glomerulonephritis retinal dystrophy (1) and other retinal changes the cardinal manifestations of bardet-biedl syndrome, a form of laurence-moon-biedl syndrome laurence-moon-biedl syndrome accompanied by congenital hepatic fibrosis pediatric renal transplantation in laurence-moon-biedl syn-drome possible or expected findings typical skeletal proportions. arachnodactyly; pectus excavatum genu recurvatum; dislocation of joints diaphragmatic hernia. * infective endocarditis. * atrial septal defect. * myxomatous transformation of mitral ring. mitral valve prolapse. aortic and pulmonary dilatation and valvular insufficiency.* aortic dissection* with dissection of adjacent vessels. ascending aortic aneurysm multiple cysts (see "cyst(s), pulmonary") aortopulmonary fistula: an uncommon complication in dystrophic aortic aneurysm peripartum acute myocardial infarction in marfan's syndrome aneurysm of the cervical internal carotid artery associated with marfan's syndrome--ease report noonan's syndrome in association with acute leukemia lymphatic dysplasia in a neonate with noonan's syndrome noonan syndrome: a clinical description emphasizing the cardiac findings cerebral arteriovenous malformation in noonan's syndrome clinical variability in a noonan syndrome family with a new ptpnii gene mutation peutz-jeghers syndrome bilateral large-cell calcifying sertoli cell tumor of the testes in peutz-jeghers syndrome: a case report ovarian tumors associated with the peutz-jeghers syndrome robin sequence and a deficiency of the left forearm in a girl with a deletion of chromosome 4g33-gter congenital heart disease in the pierre robin syndrome glossoptosis-apnea syndrome chiari i malformation, caudal regression syndrome, and pierre robin syndrome: previously unreported combination risk factors for squamous cell carcinoma of the esophagus heterotaxy syndrome; ivemark's syndrome possible associated conditions: with asplenia: right isomerism (bilateral mirror-image right-sided symmetry) of heart, lungs, and abdominal viscera; common atrium; total anomalous pulmonary venous connection; absent coronary sinus; complete atrioventricular septal defect;* subpulmonary stenosis;* pulmonary valve atresia;* midline symmetric liver; malrotation of bowel; absent spleen. with polysplenia: left isomerism (bilateral mirror-image left-sided symmetry) ofheart and lungs, with variable sidedness ofabdominal viscera primary immunodeficiencies reiter's syndrome-like patternin aids-associated psoriasiformdermatitis reference pneumothorax;* pneumomediastinum;* pneumoperitoneum. septicemia. right ventricular hypertrophy and/or dilatation. intubation trauma and mural edema and hemorrhage in trachea; hyaline membrane disease intubation trauma. hemorrhages or other evidence of birth trauma; germinal matrix hemorrhages in premature infants; infarctions of subcortical white matter in term infants coalson 11. pathology of bronchopulmonary dysplasia pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia inborn errors of metabolism and reye's syndrome: differential diagnosis prevalence and non-specificity of microvesicular fatty changes liver histopathology in clinical reye syndrome ultrastructural study of intranuclear inclusions in the exo-crine pancreas in reye's syndrome neuropathologic findings in reye syndrome sanfilippo's (see "mucopolysaccharidosis scheie's (see "mucopolysaccharidosis segawa's (see "syndrome, dystonia severe acute respiratory (sars) note: this highly contagious illness, caused by a coronavirus (sars-cov)details, see biosafety level 3 laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects pathology and pathogenesis of severe acute respiratory syndrome pulmonary involvement in primary sjogren's syndrome polymyositis and sjogren's syndrome associated with bronchiolitis obliterans organizing pneumonia the gastrointestinal manifestations of sjogren's syndrome clinicopathological factors relating malignant lymphoma with sjogren's syndrome acute transverse myelopathy and cutaneous vasculopathy in primary sjogren's syndrome primary localized cutaneous amyloidosis with unusual clinical features in a patient with sjogren's syndrome disease, parkinson's erythema multiforme man related terms: armadillo disease; continuous muscle fiber activity; neuromyotonia autoimmune central nervous system paraneoplastic disorders: mechanisms, diagnosis, and therapeutic options sudden infant death (sids) (see "death, sudden unexpected, of infant.") syndrome, superior vena cava related term: superior vena cava obstruction continue dissection of veins into neck and axillas. record and photograph site of thrombosis or of compression by surrounding pathologic conditions. submit samples for histologic study. benign or malignant tumors; fibrosing mediastinitis;* postradiation fibrosis; infectious disease (tuberculosis,* histoplasmosis*); thoracic aortic aneurysm;* chronic constrictive pericarditis;* chest trauma; arteriovenous fistula between ascending aorta and superior vena cava toxic shock synonyms and related terms: staphylococcal scarlet fever. note: (1) collect all tissues this is a reportable disease. premature aging; increased number of pigmented nevi; infantile sex organs; webbed neck; broad chest with wide spacing of nipples. short fourth metacarpal; abnormal epiphyseal fusions; osteochondrosis-like changes of spine bicuspid aortic valve;* coarctation of aorta. * rarely other anomalies (1) decreased/absent follicles. intestinal telangiectases. biliary atresia. * horseshoe kidney; double ureters. streak gonads without germ cells or follicles hashimoto's thyroiditis. * manifestations of diabetes mellitus,* hypertension,* or thyrotoxicosis. neuroblastoma and related tumors (2). keratoconus acute aortic dissection, aortic insufficiency, and a single coronary artery in a patient with turner's syndrome neuroblastoma and related tumors in turner's syndrome turner's syndrome associated with bilateral retinal detachments weil's (see "leptospirosis related terms: alcoholic wernicke's encephalopathy; korsakoff's psychosis; nonalcoholic wernicke's encephalopathy (1,2); and specimen preparation, see chapter 4. for selection of histologic samples, see right-hand column melissas 1. wernicke's encephalopathy after vertical banded gastroplasty for morbid obesity syndrome, respiratory distress, of infant.") syndrome, wiskott-aldrich (see "syndrome, primary immunodeficiency malformation, ebstein's" and "preexcitation, ventricular alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease cerebro-hepato-renal syndrome; infantile refsum's disease. * note: this congenital familial cholestatic syndrome results from impaired assembly of peroxisomes and has its main manifestations in the brain, liver, and kidneys postmortem findings and prenatal diagnosis ofzellwegersyndrome. case report alcoholism and alcohol intoxication" and "disease, alcoholic liver possible associated condition: multiple endocrine neoplasia primary cardiac gastrinoma causing zollinger-ellison syndrome localization, syphilis, acquired synonym: treponema pallidum infection. note: congenital syphilis is presented below under a separate heading and surgical management of gastrinoma fundic argyrophil carcinoid tumor in a patient with sporadic-type zollinger-ellison syndrome nonimmune hydrops fetalis due to congenital syphilis associated with negative intrapartum mater-nal serology screening syringomyelia synonyms and related term: hydromyelia; idiopathic syringomyelia; secondary syringomyelia; syringobulbia. possible associated conditions: with idiopathic syringomyelia-arnold chiari malformation, type i;* basilar impression;* 2. oppenheimer eh, dahms bb. congenital syphilis in the fetus and neonate congenital syphilis: detection of treponema pallidum in stillborns with secondary syringomyelia-intramedullary gliomas (ependymoma, pilocytic astrocytoma) and vascular tumors; spinal arachnoiditis and pachymeningitislisted in right-hand column. prepare roentgenograms of spine and joints. for removal and specimen preparation, see chapter 4. for histologic sampling, see right-hand column. hypertrophy of body parts. muscle atrophy of upper extremities and hands cervical spinal cord is swollen and tense, with cavitation (syrinx) containing clear fluid. wall of cavity consists of degenerated glial and neural elements, with marked gliosis. spinal cord parenchyma is markedly compressed. see also above under hydrops fetalis caused by alpha-thalassemia: an emerging health care problem limb defects in homozygous alpha-thalassemia: report of three cases poisoning, thallium thirst (see "dehydration thromboangiitis obliterans (see "disease, buerger's purpura, thrombotic thrombocytopenic" and "syndrome, hemolytic uremic the pathological manifestations of pulmonary toxoplasmosis in the acquired immunodeficiency syndrome a spectrum in the pathology of toxoplasmosis in patients with acquired immu transfusion (see "reaction to transfusion bone marrow note: if the patient had symptoms of acute or chronic graft-versus-host disease (gvhd), see "disease, graftversus-host morphology and diagnostics of human toxoplasmosis fatal myocardial coinfection by toxoplasma gondii and parvovirus b 19 in an hiv patient if there is evidence of infection, submit material for microbiologic studies. if there is evidence of recurrent leukemia or lymphoma, sample as described under those headings. if the patient had symptoms of thrombotic thrombocytopenic purpura (tip) or hemolytic uremic syndrome (hus), see these headings. record average size. fix specimens in b-plusâ®. make touch preparations. request giemsa or wright stain. for preparation of sections and smears (imprints), see chapter 2. manifestations of graft-versus-host disease (e.g., scleroderma-like changes).* septicemia. interstitial pneumonia* and cytomegalovirus infection* or human herpesvirus 6 infection (1). bacterial, fungal bacterial or fungal infections in gvhd.* other manifestations of gvhd. * recurrent leukemia,* lymphoma* (including posttransplant, epstein-barr virus associated lymphoproliferative disorder). recurrent solid tumor, e.g., carcinoma of breast, lung (small cell carcinoma), ovary lymphomatous or leukemic infiltrates myeloid cells may be absent in marrow graft rejection or nonimmunologic marrow graft failure. references hematomas; hemorrhagic necroses; infarcts; bacterial or fungal infections leukoencephal-opathy; vascular siderocalcinosis; neuro-axonal spheroids (6) human herpesvirus 6 infection and associated pathogenesis following bone marrow transplantation pulmonary veno-occlusive disease in an adult following bone marrow transplantation. case report and review of the literature acute renal failure following bone mar-row transplantation transplantation-associated thrombotic thrombocytopenic purpura and hemolytic uremic syndrome thrombotic microangiopathies associated with drugs and bone marrow transplantation record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record weight, ventricular thickness, and valve circumferences. take multiple sections from all areas of myocardium. cut coronary arteries in cross sections; request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. left ventricular hypertrophy, from cyclosporine-related hypertension. acute transplant rejection (for grading, see ref. 1). chronic transplant vasculopathy with coronary artery stenosis (2) opportunistic infection (1). pneumonia a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group autopsy findings in cardiac transplant patients: a lo-year experience fulminant toxoplasmosis following heart transplantation banff schema for grading liver allograft rejection: an international consensus document record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record lung weights separately. if lung infection is suspected, submit material for microbiologic possible or expected findings cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. opportunistic infection (in patients with a single lung transplant, infections may involve references the nontransplant lung, as well). chronic bronchitis* and bronchiectasis.* acute rejection (rare); chronic airway rejection (obliterative bronchiolitis); chronic vascular rejection. (for grading of rejection, see ref 1.) post-transplant lymphoprolijerative disorder a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group recurrent salmonella enteritidis and hepatic tuberculosis tularemia synonyms and related terms: francisella tularensis infection; pasteurella tularensis infection cerebral abscesses complicating tularemia meningitis endocrine (see "neoplasia, multiple endocrine any type note: ifthe tumor had been treated by surgery, irradiation, chemotherapy, or other means (the most common situation possible associated conditions: with adrenocortical adenoma-conn's syndrome with adrenocortical carci-noma-cushing's syndrome;* hypoglycemia;* virilization. with pheochromocytoma-cerebellarhemangioblastoma ery-throcytosis; hypercalcemia; hypertension,* multiple enarteries record body weight and abnormal features. photograph skin changes. record heart weight and thickness of ventricles. procure multiple sections of myocardium possible associated conditions.") focal myocarditis. * hypertensive cardiovascular disease (see "hypertension myocardial infarction without severe coronary artery disease (with pheochromocytoma) asystematicreviewoftheassociationbetween barrett's esophagus and colon neoplasm malakoplakia and colorectal adenocarcinoma an unusual case of colonic mixed adenoendocrine carcinoma: collision vs composite tumor. a case report and review of the literature submit lymph nodes for histologic study. remove neck organs with tongue together with esophagus. open pharynx and esophagus in posterior midline. if fistulas and abscesses are suspected, dissect esophagus but leave attached to mediastinum, stomach and diaphragm. record width of lumen of esophagus at various levels. photograph and record size and location of tumor; sample tumor and uninvolved esophagus for histologic study. request pas stain of uninvolved esophagus (sample from all levels). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. eczematoid dermatitis with adult renal cell carcinoma. cushing's syndrome,* galactorrhea or feminization or masculinization in some cases of renal cell carcinoma. evidence of hyperalcemia. pulmonary tumor embolism after invasion of renal vein and inferior vena cava. acquired renal cystic disease prolactin, renin, and prostaglandins in some renal cell carcinomas. see above under "possible associated conditions wilms tumor associated with polycythemia: case report and review of the literature renal medullary carcinoma: a potential sickle cell nephropathy of children and adolescents acquired cystic kidney disease byler's disease, carcinoid syndrome,*cirrhosis* of any type (mostly nonbiliary), congenital hepatic fibrosis, * cushing's syndrome,* erythrocytosis, genetic hemochromatosis, * glycogen storage disease (type 1),* hepatitis b or c virus infection, hereditary tyrosinemia (type i),* hypercalcemia, hypercholesterolemia, hypoglycemia,* neurofibromatosis,* osler-rendu-weber disease* (hereditary hemorrhagic telangiectasia), polycythemia,* porphyria (acute intermittent and porphyria cutanea tarda), * pseudohyperparathyroidism,* and thorium (thorotrast) deposition. with bile duct carcinoma (cholangiocarcinoma}-clonorchis sinensis or opisthorchis viverrini infection, fibropolycystic liver and biliary tract disease,* hepatolithiasis, hypercalcemia, pri-mary sclerosing cholangitis,* and thorium (thorotrast) deposition. with hepatoblastoma-alpha-fetoproteinemia, cardiac and renal malformations, cleft palate, diaphragmatic hernia,* down's syndrome,* familial colonic polyposis,* hemihypertrophy, nephroblastoma. with angiosarcoma-thorium (thorotrast) deposition. see also below under "possible or expected findings describe and photograph cut surfaces. sample tumor and nonneoplastic liver for histologic study. if thorium deposition is suspected, prepare roentgenograms of liver slices and submit samples for energy-dispersive x-ray microanalysis of paraffin sections. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. cachexia. precocious puberty. feminization and gynecomastia in rare cases of hcc. spider angiomas; clubbing of fingers. see above under "possible associated conditions thorotrast storage may cause cirrhosis, hcc, bile duct carcinoma, or angiosarcoma. see above under "possible associated conditions tumor of the lung or bronchus possible associated conditions: abnormal concentrations of hormons or other metabolites in blood and tumor tissue (adrenocorticotropic, antidiuretic, growth hormone, parathyroid-like substances, or 5-hydroxyindolacetic acid); acanthosis nigricans s syndrome;*dermal hyperpigmentation; feminization; hyperglycemia; hypercalcemia; hypoglycemia;* hypokalemia; hyponatremia; precocious puberty. for syndromes affecting the brain, peripheral nerves or muscles, see below under "possible or expected findings organs and tissues procedures possible or expected findings external examination and skin record body weight. record abnormal features as listed in right-hand column; prepare photographs. prepare histologic sections of normal and grossly abnormal skin clubbing of fingers; spider angiomas. for other rare tumor-related changes, see above under "possible associated conditions thmor of the pancreas possible associated conditions: with carcinoma of the exocrine pancreas--cushing's syndrome;* diabetes mellitus* (rare); hypercalcemia; hyperglycemia with islet cell tumor-abnormal concentrations of hormons in blood and tumor tissue (see below under "pancreas"); features. dermal hyperpigmentation. for other rare tumor-related changes, see above under "possible associated conditions biliary obstruction caused by tumor in head of pancreas. islet cell tumor with adrenocorticotropic hormone, gastrin, glucagon, insulin, or other peptide hormones. see above under "possible associated conditions paraneoplastic pemphigus associated with pancreatic carcinoma pancreatic cystadenocarcinoma in peutz-jeghers syndrome testes may have been surgically removed to achieve androgen deprivation. urinary obstruction and hydronephrosis. * osteoblastic metastases hemolytic uremic syndrome in prostatic carcinoma thmor of the small intestine possible associated conditions: acquired immunode oncogenic osteomalacia associated with prostatic cancer gardner's syndrome) submit sample of non-neoplastic small bowel for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above. cachexia. mucocutaneous pigmentations associated with peutz-jeghers syndrome. * carcinoid tumor. lymphoma (see also above under "possible associated conditions"). familial polyposis or related syndrome. * celiac sprue. * crohn's disease. * see above under "possible associated conditions kasabach merritt syndrome in infants paraneoplastic hepatopathy associated with soft tissue sarcoma neurofibro-thmor of the spinal cord possible associated conditions: with angioma-cerebellar hemangioblastoma; segmental cutaneous vascular nevi; with matosis in children with soft tissue sarcoma tissues record abnormal features; photograph and submit nevi for histologic study. for removal and specimen preparation, see chapter 4. see also below under "vertebral column bone erosion and calcification of spinal canal. vertebral hemangiomas may be associated with arteriovenous malformation of spinal cord. manifestations of von hippel-lindau disease. * thmor of the stomach possible associated conditions: hypoglycemia;* megaloblastic anemia;* skin changes (as listed under "possible or expected findings)weight. record abnormal features; photograph and submit samples of normal and abnormal skin for histologic study. possible or expected findings cachexia; lower leg lymphoedema (3) metastases common in liver, retroperitoneal and supraclavicular lymph nodes (virchow's node), ovaries (krukenberg tumor), and peritoneal cui de sac (blumer's shelf') carcinoma of the stomach with hyperkeratosis palmaris and plantaris and acanthosis of the esophagus gastric lymphoma ofmucosa-associated lymphoid tissue and helicobacter pylori gastric signet-ring cell carcinoma: unilateral lower extremity lymphoedema as the presenting feature thmor of the testis possible associated conditions: demyelinating neuropathy record location, size, and weight of both testes and of testicular tumor. submit samples of tumor and of ininvolved testis and epididymis for histologic study. snap-freeze tumor tissue for hormone assay. possible or expected findings cachexia. cryptorchism; hypospadia. gynecomastia. increased concentrations of alphafetoprotein and human chorionic gonadotropin testicular microlithiasis. secondary testicular tumors (metastases to the testes) are rare, except in association with leukemia* in children. references pulmonari embolism.* paraneoplastic diseases as listed above under "possible associated conditions chronic inflammatory demyelinating polyneuropathy (clop) associated with seminoma dermatomyositis associated with intratubular germ cell tumor and metastatic germ cell cancer testicular cancer in association with developmental renal anomalies and hypospadias submit samples of lymph nodes, spleen, peyer's plaques, and bone marrow for histologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. dermal hyperpigmentation. pemphigus foliaceus (rare). hypogammaglobulinemia and other immunoglobulin abnormalities. usually, thymoma presents as an infiltrating, anterior mediastinal mass that rarely metastasizes. carcinoid tumor, malignant lymphoma* (hodgkin's disease), and metastases from carcinomaofthe breast* and other tumors also may occur in this location herpes simplex*) and fungal infections (e.g., candidiasis*) due to thymoma-related immunodeficiency (3). manifestations of paraneoplastic diseases and conditions as listed above under "possible associated conditions thrombotic thrombocytopenic purpura accompanied by transient pure red cell aplasia and thymoma glomerulonephritis associated with myasthenia gravis thymoma and cellular immune deficiency in an adolescent possible associated conditions: with papillary carcinoma-familial adenomatous polyposis thyroid carcinoma associated with familial adenomatous polyposis an association between acromegaly and thyroid carcinoma thyroid carcinoma causing fatal laryngeal obstruction prostate cancer metastasis to thyroid gland schistosomiasis and the risk of bladder cancer in human papilloma virus infection (2) or herpesvirus infection (3) with invasive cervical carcinoma. erythropoietin may be found in some tumors. thrombosis. * myopathy* may be associated with carcinoma of the cervix. cerebellar cortical degeneration* may be associated with carcinoma of the uterus. manifestations of uremia (see "failure, kidney") invasive cervical cancer in human immunodeficiency virus-infected and uninfected hospital patients association of risk factors for cervical cancer and human papilloma viruses in invasive cervical cancer association of herpesvirus infection with the development of genital cancer tyrosinemia type ii: a challenge for ophthalmologists and dermatologists hereditary tyrosinemia type i (chronic form): pathologic findings in the liver richner-hanhart syndrome (tyrosinemia ii): early diagnosis of an incomplete presentation with unusual findings acute pancreatitis-associated acute gastrointestinal mucosal lesions: incidence, characteristics, and clinical significance uncinariasis (see "ancylostomiasis uremia (see "failure, kidney from: handbook of autopsy practice urticaria pigmentosa of childhood (see ''mastocytosis, systemic acute aortic dissection;* aneurysma(s) of cerebral arteries; aortic insufficiency;* calcific aortic stenosis,* coarctation of the aorta;* infective endocarditis;* shone's syndrome; turner's syndrome. * organs and tissues procedures possible or expected findings heart if infective endocarditis is suspected, see chapter 7. open heart in cross-sections (see chapter 3). photograph aortic valve and test valvular competence note: reye's syndrome* is a possible postviral complication of varicella that must be distinguished from varicella encephalitis. varicella may also cause exacerbation of tuberculosis.* (i) collect all tissues that appear infected bicuspid aortic valves are associated with aortic dilatation out of proportion to coexistent valvular lesions varicella-zoster virus infection in children with underlying immunodeficiency virus infection group a beta-hemolytic streptococcal epiglottitis as a complication of varicella infection optic neuritis: a rare complication of primary varicella infection hsv-l-induced acute retinal necrosis syndrome presenting with severe inflammatory orbitopathy, proptosis, and optic nerve involvement varicella with acute motor axonal neuropathy descending necrotizing mediastinitis in a child with chicken pox double outlet right ventricle with intact ventricular septum respiratory syncytial (see "pneumonia, all types or type unspecified vitamin a (see ''deficiency, vitamin a" and "hypervitaminosis a vitamin bt (thiamine) (see "syndrome, wernicke-korsakott vitamin bt2 (see "anemia, megaloblastic deficiency deficiency, vitamin d" and "hypervitaminosis d waldenstrom's macroglobulinemia (see ''macroglobulinemia, waldenstrom's.) waterhouse-friderichsen syndrome (see "disease, meningococcal wegener's granulomatosis (see "granulomatosis, wegener's werdnig-hottman disease (see "disease, motor neuron acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using 4-methylumbelliferyl palmitate which may be secondarily infected by bacteria. dark field microscopy will identify the organism expected findings mucosal ulcers, resembling those of typhoid fever, primarily of distal ileum and colon. villous shortening, crypt hyperplasia with mucosal microabscesses. microabscesses in regional mesenteric lymph nodes zellweger's syndrome (see "syndrome mucormycosis" and procedures under "diabetes mellitus abscesses and granulomas may occur in all organs and tissues. granulomatous lesions in central nervous system (4) and eyes (5). fungal osteomyelitis* that may be multifocal, including sites such as metacarpals and metatarsals. external examination other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. osteosclerosis of vertebrae, ribs, clavicles, pelvic bones, scapulae, skull, and metaphyseal ends of femur and humerus. osteomyelofibrosis or osteoreticulosis; rarely, panhyperplasia of bone marrow; increase of megakaryocytes.changes associated with chronic granulocytic leukemia* or with polycythemia vera* may imitate idiopathic myelofibrosis. widespread extramedullary hematopoiesis (1) with splenomegaly. infectious diseases, including tuberculosis;* gouty arthritis; ascites and other manifestations of portal hypertension;* or hepatic vein thrombosis (budd-chiari syndrome*). cardiac tamponade (1) . synonyms and related terms: hepatic porphyria; protoporphyrinogen oxidase deficiency (1) .note: the manifestations of the disease closely resemble those in porphyria cutanea tarda and hereditary coproporphyria. measurements of porphyrins and porphyrin precursors are the only clearly distinguishing features. for autopsy procedures, see under "porphyria cutanea tarda." fibrin and platelet thrombi, with or without microaneurysms and purpura in kidneys, adrenal glands, pancreas, heart, and brain; lesions may also be present in liver; spleen (3), lymph nodes, muscle, bone marrow, and synovium. fibrin thrombi can be demonstrated with labeled antihuman fibrin antibodies. lesions in precapillary arterioles. hypertrophic osteoarthropathy* (with pleural mesothelioma).pleural effusions. * asbestosis may be complicated by pleural mesothelioma. other organs and tissue sample bladder tumor and uninvolved urinary bladder for histologic study.cachexia. hydronephrosis* (obstructive uropathy) and hydroureter, usually caused by distal ureteral obstruction. recurrent nephrolithiasis* or pyelitis may have been present and is a risk factor for urinary bladder carcinoma.chronic urocystitis; infestation with schistosoma haematobium (1) (uncommon in north america). manifestations of uremia (see "failure, kidney"). key: cord-032181-gmcugd8h authors: song, jian-xin; zhu, lin; zhu, chuan-long; hu, jin-hua; sun, zi-jian; xu, xiang; xin, min-you; zhang, qiong-fang; zhang, da-zhi; shang, jia; huang, jia-quan; xu, dong title: main complications of aechb and severe hepatitis b (liver failure) date: 2019-05-21 journal: acute exacerbation of chronic hepatitis b doi: 10.1007/978-94-024-1603-9_2 sha: doc_id: 32181 cord_uid: gmcugd8h this chapter describes the clinical features, and diagnosis of complications in aechb including secondary bacterial infections, coagulation disorder, water electrolyte disorder, hepatorenal syndrome, hepatic encephalopathy, hepatopulmonary syndrome and endotoxemia: 1. patients with severe hepatitis have impaired immunity and are therefore vulnerable to all kinds of infections. after infection, these patients may experience shock, dic and multiple organ failure, all of which seriously affect their prognosis and are major causes of death. concurrent infections consist primarily of infections of the lungs, intestines, biliary tract, and urinary tract, as well as spontaneous bacterial peritonitis and sepsis. 2. severe hepatitis may reduce the synthesis of coagulation factors and enhance their dysfunction and increase anticoagulants and platelet abnormalities, leading to coagulopathy. infection, hepatorenal syndrome and complications can further aggravate coagulopathy, resulting in dic and seriously affecting patient prognosis. 3. hepatorenal syndrome, which is characterized by renal failure, hemodynamic changes in arterial circulation and abnormalities in the endogenous vascular system, is a common clinical complication of end-stage liver disease, and one of the important indicators for the prognosis of patients with severe hepatitis. 4. water electrolyte disorder (water retention, hyponatremia, hypokalemia, hyperkalaemia) and acid-base imbalance are common in patients with severe hepatitis. these internal environment disorders can lead to exacerbation and complication of the illness. 5. hepatic encephalopathy is a neurological and psychiatric anomaly syndrome based on metabolic disorder, and an important prognostic indicator for patients with severe hepatitis. 6. the hepatopulmonary syndrome is an important vascular complication in lungs due to systemic hypoxemia in patients with cirrhosis and portal hypertension. the majority of patients with hps are asymptomatic. long-term oxygen therapy remains the most frequently recommended therapy for symptoms in patients with severe hypoxemia. 7. endotoxemia, an important complication of severe hepatitis, is not only a second hit to the liver, but also leads to other complications including sirs and mods. (nacseld) for survival analysis of prospectively enrolled cirrhosis patients hospitalized with infections, bajaj found that there is 15.8% of nosocomial infection in all 507 patients [1] . domestic data showed significant difference of infection incidence in different times. it is related to raising diagnosis and treatment level. according to the data from shanghai public health clinical center, in 372 patients who were treated as severe hepatitis from january 2007 to december 2009, there were 68 patients obtained nosocomial infection with a rate of 18.3% [2] . the incidences of infection complicated with acute liver failure, acute on chronic liver failure or chronic liver failure are similar. patients with old age or long hospitalization are more easily to get infection. there was rare report about infection in children patients with severe hepatitis. in 2011, godbole et al. from uk reported that 25% in 145 children patients with severe hepatitis were complicated with infection, and mostly are sepsis, respiratory tract infection and urinary infection. most of the infections occurred within 2 weeks of admission, while patients with infection had longer hospitalization [3] . reduced innate immunity the innate immune system is the first line of defense against premier environmental challenges and injury. in liver, it is a complex system that includes nk cells, nkt cells, kcs, neutrophils, eosinophils and complement components. the innate immune response acts much more rapidly compared with adaptive immunity. monocytes and macrophages: the liver plays an important role in defense and immune function. the main cellular components of the innate immune system within the liver are the kupffer cells. kupffer cells represent 80-90% of the tissue macrophages in the human body [4] . in normal condition, kupffer cells in liver help to clear the macromolecular substance such as pathogen, endotoxin, heteroantigen and immune complex to defense infection. in severe hepatitis, due to massive hepatocytes necrosis, the number and function of monocytes/macrophages are impaired, thus the activity of fibronectin, which is opsonin of macrophages, is decreased. therefore, bacterium, endotoxin and other poison from gut directly access into circulation. subrat kumar acharya from india reported that the plasma fibronectin (fn) level in severe hepatitis patients was significantly lower than that in healthy controls (85.6 μg/ml ± 75.8 μg/ml, vs. 295.5 μg/ ml ± 88.5 μg/ml). the fn level was remarkably correlated with incidence of infection and mortality [5] . complement: liver is the organ where complements are mainly produced, such as c2, c3, c4, c5. the complements help to expand phagocytosis of phagocytes by chemotaxis, opsonization or adhesion, as well as help antibody to kill or solute some gram negative bacilli [6] . report from wyke showed that the defect of complement closely correlated with impaired opsonization [7] . defect of c3 or c5 can result to weakened movement of polymorphonuclear leucocyte. in severe hepatitis, the ability of liver to produce complement has been weakened due to massive injury of parenchymal hepatic cells, which leads to decreased activity of complement to 40% of normal condition. meanwhile, serum opsonization to e. coli or saccharomycetes are also diminished. besides, high ammonia level in severe hepatitis also restrains complement activity to impact germicidal effect. the most direct and also the most important result for decreased complement production and reduced opsonization is the susceptibility to infection. it was also reported that complement and the alternative pathway play an crucial role in lps/d-galn-induced fulminant hepatic failure [8] . neutrophils: a majority of patients with severe liver disease have altered function of neutrophil granulocytes [9] . the most common manifestation include abnormal ultrastructure or function of neutrophil granulocytes, as cytoplasm degeneration, organelle reduction, mitochondrial swelling, pyknotic nuclei, etc. decrease filtration and phagocytosis of reticuloendothelial system, as well as impaired chemotaxis of blood cells, making immunity weakened, lead to invasion of bacterium. therefore, patients with severe hepatitis are vulnerable to be infected with bacterium or fungi due to decreased phagocytosis and germicidal effect of neutrophil granulocytes, and impaired adhesion of macrophages and white cells [10] . data from liu h demonstrated pretreatment neutrophil-lymphocyte ratio was associated with the prognosis of patients with hbv-aclf, and elevated nlr predicted poor outcome within 8 weeks [11] . natural killer and natural killer t cells: natural killer (nk) and natural killer t cells (nkt) are important components of the innate immune response. natural killer cells have potent cytolytic activities that are exerted through the death receptor and perforin/granzyme pathways. activated nkt cells have both perforindependent and fas-ligand dependent cytotoxic function that are triggered upon tcr recognition of an antigen [12] . nk cells and nkt cells play an important role in many experimental models of liver injury, such as viral hepatitis, alcoholic liver disease, and autoimmune liver disease [13] . however, their role in aclf has not yet been clearly elucidated, it was reported the median percentage of nk cells in the lymphocytes of patients with acute and fulminant liver failure were significantly lower compared to healthy controls. meanwhile, patients with acute and fulminant liver failure had significantly high and comparable nkt cells compared to control group [14] . the important pathophysiological role of innate immune dysfunction in patients with acute-on-chronic liver failure (aclf) has been investigated in recent years. however, dysregulation of adaptive immunity remains poorly elucidated [15] . patients with severe hepatitis has varying degrees of impaired cellular immunity, manifested as decreased cd4+ cell number and declined cd4+/cd8+ ratio, which is pathogenesis of opportunistic infection. it was reported that there exists a reduction in cd4(+) t lymphocytes in hbv-aclf patients. these cd4(+) t cells predominantly are cd4(+) tconv, and the development of suppressive cd4(+) tregs greatly surpass tconv, which constitutes important characteristics of adaptive immune dysfunction of hbv-aclf [15] . a report from china showed total amount of lymphocytes, cd4(+) t cells, cd8(+) t cells and nk cells in circulation were lower in the hbv-aclf patients compared to the chb patients [16] . hbv-specific cd8(+)t-cell responses are considered to be of great importance in viral control and immune-mediated liver damage [17] . however, cd8(+) t cell has seldom been studied in aclf. ye [18] reported decreased activated cd8(+) t cells may be related to poor outcomes in patients with sh. the frequency of circulating th17 cells increased with disease progression from chb to aclf patients compared to healthy control. th17 cells were also found largely accumulated in the livers of chb patients. the increases in circulating and intrahepatic th17 cells were positively correlated with plasma viral load, serum alanine aminotransferase levels and histological activity index. in addition, the serum concentration of th17-associated cytokines was also augmented in both chb and aclf patients [19] . in process of diagnosis and treatment for severe hepatitis, repeatedly abdominal paracentesis, retention catheterization, venous cannula, hemofiltration, or trachea cannula are usually necessary. unthoroughly sterilization or nonstandard sterile operation will lead to pathogen invading to develop an infection. in addition, artificial liver treatment is also an important cause of infection. it was reported that the incidence of fungal infection in severe hepatitis correlated with the number of artificial liver treatment. application of broad spectrum antibiotics is also a major cause of infection in severe hepatitis. antibiotics inhibit or kill sensitive normal bacterium as well as pathogens, especially normal bacterium colonized in natural orifice, leading to flora disproportionality. this time, nonpathogen could cause infection, or mass produced pathogen become dominant colony to develop infection. it was proved that dosage, exposure time, or varieties of antibiotics used in patients was closely correlated with severity of dysbacteriosis and incidence of sbp [20] . nosocomial origin of infection, longterm of norfloxacin prophylaxis, history of recent infection by multiresistant bacteria and recent use of β-lactams were independent inducements associated with the development of multiresistant infections. bacterial translocation is defined as the migration of viable microorganisms or bacterial products (i.e., bacterial lps, peptidoglycan, and lipopeptides) from theintestinal lumen to the mesenteric lymph nodes and other extraintestinal sites [21] . there are multiple mechanisms which are involved in defective gut functions and altered microbiota in patients with cirrhosis or liver failure. these include small intestine bacterial overgrowth (sibo), increased intestinal permeability, and impaired antimicrobial defense. additionally, decreased bile acids, due to decreased synthesis and defective enterohepatic circulation, contribute to altered gut microbiota [22] . small intestinal bacterial overgrowth (sibo) was defined as ≥10 5 total colonyforming units per milliliter of proximal jejunal aspirations, which presents in 59% of cirrhotic patients and is associated with systemic endotoxemia [23] . in the condition of hepatic failure, due to impaired immunity, bacterium overgrowth and translocation happened, which increased incidence of infection [24] . in vivo study from wang showed phagocytosis of monocytes were prominently and immediately diminished after resection of liver tissue up to 90%, accompanied by decreased oxygen ingestion [24] . mass propagation of e. coli in the lower part of the small intestine and bacterium translocation were all happened 2 h after resection. in rats with ascites in severe liver disease induced by carbon tetrachloride, the bacteria easily transferred from the intestine to extra-intestine, including local lymph nodes and blood circulation. these evidences proved that liver failure prompted the proliferation of intestinal bacterial overgrowth and translocation, which may be the main reason for the endogenous infections in patients with liver failure. another reason for bacterial translocation is that patients with cirrhosis or liver failure display increased intestinal permeability. in patients with severe hepatitis, gastrointestinal mucosa membrane has impaired ability for regeneration and repair, leading to dysfunction of intestinal barrier and declined anti-infection ability. patients with severe hepatitis have weakened regeneration and repair capacity of gastrointestinal mucosa, as well as decreased gastrointestinal barrier function and resistance to infection. the intestinal mucosa often manifested as congestion, edema or erosion. especially when intestine get infection, bacteria can invade to abdominal cavity directly or through slight mucosal defect [25] . besides, ascites often present in severe hepatitis patients, which provide a good medium for bacteria to multiply. patients with severe hepatitis often have ascites combined with portal hypertension. due to lobular structural damage, abnormal construct of liver sinusoids or blood vessels, bacteria directly access into the systemic circulation without filtration and phagocytosis by liver, leading to formation of bacteremia. meanwhile, bacteria in blood circulation may access into the abdominal cavity and cause abdominal infections. hence, patients with severe hepatitis are not only prone to get endogenous infection but also prone to have intestinal endotoxemia. patients with severe hepatitis have decreased bile secretion, changes of bile composition, so that infection are prone to occur in epithelium of bile duct and gallbladder. in patients with severe hepatitis, intestinal edema and cellulitis are obvious, some may develop acute serositis. for patients with portal hypertensive gastrointestinal disease, regeneration and repair capacity of gastrointestinal mucosa are decreased, accordingly, the natural immune barrier function is reduced. once esophageal and gastric venous bleeding occur, gastrointestinal ischemia aggravate, which result in decreased resistance to infection. invasive procedures, complications, prophylactic use of broad-spectrum antibiotics, underlying diseases, long hospital stay, and old age are risk factors for infection in patients with severe hepatitis. elderly patients with other underlying diseases, decreased immune function, more severe primary disease, have a high risk of infection and may predispose to severe infections. in addition, albumin level is closely related to ascites production and sbp occurrence. irrational uses of immunosuppressive agents such as corticosteroids suppress immune function, lead to flora, and promote the formation of drug-resistant strains and pathogenic bacteria. mortality of severe hepatitis is closely related to infection, which directly affects the prognosis of severe hepatitis. to reduce the incidence of infection in patients with severe hepatitis, we should aim at a variety of risk factors, improve patients' immunity, rectify hypoproteinemia, treat the primary disease, prevent complications, strengthen disinfection and isolation, operate with strict aseptic technique and strict indications for invasive procedures, and use antibiotics rationally. once patients with severe hepatitis are infected, the consequences are often serious, as infection easily diffuse, which is difficult to control. after infection, the bacteria produce more toxins, which aggravate liver disease and cause a series of adverse effects, finally lead to severe complications (hepatic encephalopathy, hepatorenal syndrome, and etc.), which is the major cause for death in patients with severe hepatitis. bacterial endotoxin plays a major role in development and prognosis of severe hepatitis when infection happened [26] . endotoxemia may directly or indirectly worsen liver injury. intrahepatic cholestasis positively correlated with endotoxin levels. endotoxin can trigger hepatorenal syndrome. for kidney, endotoxin is a potent renal vasoconstrictor substance, which can make a strong contraction of the renal artery and renal blood flow reduction. in addition, endotoxin can cause kidney capillary thrombosis and acute renal tubular necrosis, or even renal cortex necrosis. endotoxin can activate coagulation factor vii and factor vi, making the intrinsic coagulation system startup, as well as directly damage hepatocytes which release tissue thromboplastin to start the extrinsic coagulation system. meanwhile, endotoxin can also damage the vascular endothelial cells to cause bleeding, especially upper gastrointestinal bleeding. in the event of gastrointestinal bleeding, infections are more prone to happen or primary infection aggravates. in recent years, due to extensive use of broad-spectrum antibiotics, the mortality of severe hepatitis has been significantly reduced. however, infection is still an important cause of death. nosocomial infection control directly affects the prognosis of patients with severe hepatitis, which is an important part for the treatment of severe hepatitis. once the etiology and other evidence of infection appear, patients should be treated with antibiotics. effective prevention and treatment for nosocomial infections are positive to promote recovery and reduce mortality. bacterial translocation (bt) is the key mechanism in the pathogenesis of sbp, which is because of the concurrent failure of defensive mechanisms in liver failure [30] . investigators have demonstrated pronounced impairment of gastrointestinal tract motility in cirrhosis [31] . the disturbance of gut flora microecology was associated with changes in the (ultra)structure of the gastrointestinal tract [32] . meanwhile, reduced cellular and humoral immunity make it easier for the free flow of microorganisms and endotoxins to the mesenteric lymph nodes [33] . besides, it was reported that hypoalbuminemia was related to sbp. a prospective study from runyon reported that the incidence of sbp in patients with ascites protein less than 1.0 g/l was 15%, while the number in patients with ascites protein more than 1.0 g/l was only 2%. after 3 years of follow-up, sbp incidence in patients with ascites protein higher than 1.0 g/l were negligible [34] . the level of ascites protein correlates with opsonin activity, which means ascites protein <1.0 g/l was an independent risk factor for sbp. the clinical manifestations of sbp are diverse. most patients have subtle and insidious onset, which usually manifested as abdominal pain and fever. acute onset may burst chills, fever and abdominal pain. generally the body temperature of patient is around 38 °c, sometimes as high as 40 °c. fever type usually presents as remittent fever. abdominal pain is always around the navel or lower abdomen, paroxysmal or persistent. nausea and vomiting are obvious, sometimes with diarrhea. ascites can occur in patients without ascites, or increased significantly. patients may also have significant bloating, abdominal muscle tension, tenderness, rebound tenderness, diminished or disappeared bowel sounds, and so on, or even shock in severe cases. according to clinical manifestations, sbp can be divided into five types: 1. common type: acute onset, protruding abdominal pain, followed by fever. or irregular fever take place followed by abdominal pain, abdominal tenderness and rebound tenderness, mild to moderate abdominal tension and growing ascites. increased wbc count and nuclear left shift. routine examination of ascites showed acute inflammatory changes. 2. shock: septic shock break out in a few hours to one day after abdominal pain or fever. the clinical manifestations include low temperature, lip cyanosis, abdominal tenderness, and hardly relieved shock. wbc count increases, blood culture is positive. 3. encephalopathy: fever and abdominal pain are often obscure, the early emergence of trance and other neuropsychiatric symptoms rapidly develop into a coma. careful examination of the abdomen in patients with light coma may still find in patients with pain expression. jaundice is deep, liver damage is serious. 4. refractory ascites: ascites progressively increase, which is difficult to subside with invalid diuretic therapy. abdominal distension is obvious, often without pain. carefully check the abdomen may still find slight peritoneal irritation. 5. asymptomatic: symptoms are mild, exhibited as slight bloating, occasional fever. mild tenderness can be found by deep palpation. in addition, a considerable part of the patients showed non-specific symptoms and signs, such as deepened coma, deepening jaundice, oliguria, azotemia or dramatically increased ascites. diagnosis of sbp should be considered if following conditions exist: (1) fever, which can't be explained by other reasons or other parts of infection; (2) abdominal pain, abdominal tenderness or rebound tenderness, but not serious; (3) sudden increased ascites or poor diuretic effect manifested as refractory ascites; (4) sudden onset of septic shock; (5) rapid deterioration of general condition, or deteriorated liver and kidney function in a short term, deepening jaundice, or hepatic encephalopathy. the diagnoses of sbp mainly rely on ascites puncture. easl clinical practice guidelines recommend that a diagnostic paracentesis should be performed in all patients with new onset grade 2 or 3 ascites, and in all patients hospitalized for worsening of ascites or any complication of cirrhosis (level a1) [34] . for patients with severe hepatitis, diagnostic paracentesis indications are: 1. in liver cirrhosis patients with ascites that paracentesis should be performed after admission to determine whether sbp exist. 2. if the following conditions happened during hospitalization, diagnostic paracentesis should be performed: (1) abdominal sighs suggest abdominal infections, such as abdominal pain, rebound tenderness and gastrointestinal symptoms (such as vomiting, diarrhea, intestinal paralysis); (2) systemic infection signs such as fever, leukocytosis, or septic shock; (3) no clear incentive for hepatic encephalopathy, or rapidly emerged renal dysfunction. 3. in patients with ascites and gastrointestinal bleeding, paracentesis should be performed before prophylactic antibiotics. once ascites was acquired, polymorphonuclear cells (polymorphonuclears, pmn) count and ascites culture should be performed. diagnosis of sbp is made according to international guidelines [34, 35] in patients with liver cirrhosis if the ascites polymorphonuclear (pmn) cell count exceeds 250 cells/μl and other forms of peritonitis have been excluded. an ascites fluid polymorphonuclear (pmn) leukocyte count ≥250/mm 3 should be considered as sbp, if pmn >500/mm 3 can be confirmed as sbp. if there is bloody ascites (erythrocytes more than 10,000/mm 3 ), pmn count are as 1/250 of red blood cells. the leukocyte esterase reagent strips (lers) test is based on the esterase activity of the leucocytes. since 2000, 26 studies have examined the validity of using leukocyte esterase reagent strips (lers) in sbp diagnosis. lers appeared to have low sensitivity for sbp. on the other hand, a high negative predictive value (>95% in the majority of the studies)supported the use of lers as a preliminary screening tool for sbp diagnosis [31] . there is bacteria colonization in ascites but no inflammation. the diagnosis is based on positive ascites culture, ascites pmn countless than 250/mm 3 , without evidence of systemic or local infection. bacterascites have two outcomes: a shortterm or transient bacterial ascites (mostly asymptomatic), or the development of sbp (mostly with symptoms). once the diagnosis was established, paracentesis examination should be conducted again after 2-3 days. take appropriate action under the circumstances. if the second sample has a pmnl count >250/mm 3 , treat as for sbp. if the pmnl count is<250/mm 3 and a second set of cultures is positive, treat as for sbp. if the pmnl count is <250/mm 3 and the second set of cultures is negative, no further action is recommended [36] . if ascites culture is positive but ascites pmn <250 /mm 3 and there were signs of abdominal infection, it usually progress to sbp within a few days. these patients should be given appropriate antibiotic therapy. brolin first proposed the concept in 1982. the mechanism: in early stage of severe hepatitis, ascites has not been exist yet, however because of necrosis of hepatocytes, dysfunction of kupffer cell, impairment of liver immune barrier, intestinal bacteria easily invasive into the systemic circulation through liver, causing spontaneous bacteremia, and then sbp. diagnostic criteria: (1) primary disease was severe hepatitis, no ascites was detected by strict examination and ultrasound. (2) clinical manifestation include fever, varying degrees of abdominal pain, diarrhea, diffuse abdominal tenderness and rebound tenderness, increased peripheral blood leukocyte, positive rivalta's test, ascites fluid leukocyte count>500/mm 3 , or pmn > 250/mm 3 . (3) exclude abdominal organ perforation or primary foci. it was used to describe the clinical situation when the ascitic pmnl count is >250/mm 3 but cultures fail to grow any bacteria. however, the term is now considered obsolete. in severe hepatitis with secondary infection, pneumonia is most common. patients with hepatic encephalopathy are susceptible to pulmonary infections as pneumonia since bed-ridden, impaired cough reflex and inadequate ventilation, especially comatose patients with intubation and tracheotomy. inpatients underwent thoracentesis, paracentesis and other invasive procedures, non-specific damage to immune barrier provide conditions for the bacterial invasion. reported in patients with invasive procedures, lung infection risk increased significantly, which demonstrated blood infection is an important way for pulmonary infection. furthermore, there was a significant increase of pulmonary infection in patients with intestinal infections or abdominal infection. pathogen: in recent years, pneumonia was classified as "community acquired pneumonia" (community acquired pneumonia, cap) "and" nosocomial pneumonia (hospital acquired pneumonia, hap). cap is the pneumonia acquired outside hospital [37] . although cap can be caused by a wide variety of micro-organisms, the pneumococcus, mycoplasma pneumoniae and chlamydophila pneumoniae, staphylococcus aureus and certain gram-negative rods are more usual pathogens encountered [38] . hap is the pneumonia that develops 48 h or more after hospital admission and that was not incubating at hospital admission. hap infected with gram-negative bacilli accounts for more than 60%, of which pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, acinetobacter baumannii, other aeruginosa are common bacteria. the primacy is pseudomonas aeruginosa, while gram-positive cocci accounted for about 20%, mainly are staphylococcus aureus, coagulase-negative staphylococci, viridans and streptococcus pneumoniae. anaerobic is rare. clinical manifestations and diagnosis: clinical manifestations are characterized as fever, cough, expectoration, dyspnea, and cyanosis. diagnostic criteria: 1. chest percussion of dullness, auscultation of rales, along with one of the following conditions: (1) purulent sputum or change of phlegm; (2) positive pathogens in sputum culture. 2. the chest x-ray and/or ct examination revealed new or progressive exudative lesions, and the emergence of one conditions above. urinary tract infection is divided into upper urinary tract infection and lower urinary tract infection. upper urinary tract infections are mainly pyelonephritis, which can be manifested as acute pyelonephritis and chronic pyelonephritis. lower urinary tract infections include urethritis and cystitis. the most common pathogen of urinary tract infection is escherichia coli, followed by enterococcus faecalis, klebsiella pneumoniae and streptococcus agalactiae. urinary tract infections often occur in patients with indwelling catheter, therefore, aseptic urethral catheterization and management and timely replacement of catheterization are effective to prevent such infections [39] . 1. acute pyelonephritis: (1) acute onset; (2) chills, chills; (3) fever; (4) malaise, headache, fatigue; (5) loss of appetite, nausea, vomiting; (6) urinary frequency, urgency, dysuria; (7) low back pain, kidney area discomfort; the (8) tenderness of upper ureter point; (9) tenderness of rib waist point; (10) percussion pain in kidney area or bladder. 2. chronic pyelonephritis: (1) acute onset of acute pyelonephritis with the same, but usually much lighter, even without fever, malaise, headache and other systemic manifestations, urinary frequency, urgency, dysuria and other symptoms are not obvious; (2) edema; (3) hypertension. 3. urocystitis and urethritis: frequent urination, urgency, dysuria, urinary bladder pain. urethral secretions. laboratory tests and diagnosis: diagnosis elements include: (1) tenderness in rib waist point, percussion pain in kidney area. (2) urine leukocytosis, pyuria. (3) urinary sediment smear find bacteria. (4) positive in urine culture. (5) urinary colony counts>10 5 /ml; in patients with urinary frequency and other symptoms, colony counts>10 2 /ml are meaningful; counts 10 3 -10 4 /ml also helpful in diagnosis; (6) one hour urine wbc count>200,000. (7) blood test showed leukocytosis, neutrophil nucleus left. (8) increased esr. biliary tract infection is a common complication in severe hepatitis, which is often in company with cholelithiasis to reinforce each other. hepatitis b virus can directly violate bile duct cells and cause cholecystitis. on this basis, cholelithiasis and secondary bacterial infections are easy to happen and become a major focus, which can cause other parts of infection in severe hepatitis patients. furthermore, severe hepatitis patients often have reduced gastric acid secretion, so that e. coli in duodenum easily reproduce and cause ascending infection. biliary tract infection is usually a mixed infection of aerobic and anaerobic infections. enteric gram-negative bacteria include escherichia coli, klebsiella, enterobacter, proteus and enterococcus. anaerobic bacteroides include clostridium and fusobacterium, in which bacteroides is common, about 80-90%, particularly bacteroides fragilis. common symptoms of biliary tract infection include chills, fever, nausea, vomiting, right hypochondrium pain and tenderness in gallbladder area. clinical performance of concurrent biliary tract infection in severe hepatitis is not always very clear, often unconfirmed by pathogen test. symptoms were epigastric or right upper quadrant pain, radiating to the right shoulder, hours after a heavy meal or a high fat meal. the patient may have severe colic, often accompanied by nausea and vomiting. patients with chronic biliary tract infection have indigestion symptoms as heartburn, belching, acid reflux and bloating, or sometimes fever and right upper quadrant pain. severe hepatitis patients have weakened intestinal resistance, especially for reduced immunoglobulin a secretions, which creates good invasion opportunities for bacterium. in addition, as mentioned above, intestinal flora are prone to happen in severe hepatitis patients, which makes intestinal infection very common [24] . few patients exhibit cellulitis like colitis, which can further result in peritonitis and septicemia, and finally lead to death. pathogens in intestinal infections could be shigella spp., salmonella, campylobacter jejuni, clostridium difficile, and salmonella typhi etc. clinical manifestations are nausea, vomiting, abdominal pain, diarrhea, watery stool or bloody mucopurulent stool. some patients may have fever and tenesmus. depending on the pathogenesis and clinical manifestations, intestinal infections are classified as enterotoxigenic bacterial enteritis and invasive bacterial enteritis. pathogenesis of enterotoxigenic bacterial enteritis is that bacteria adhere but not invade intestinal mucosa. intestinal toxins are secreted in the process of bacteria growth and reproduction, which stimulate small intestinal epithelial cells secreting large amount of water and electrolytes. overuptake of water and electrolyte and retention in intestine makes watery stool, which is called "secretory diarrhea." secretory diarrhea is exhibited as frequent, large amount stool with no pus, usually without pain or tenesmus. it is often accompanied by vomiting, which is prone to bring out dehydration, electrolyte imbalance and acidosis, but less severe systemic toxic symptoms. stool examinations show less erythrocytes or leukocytes. invasive bacterial enteritis refers to pathogenic bacteria adhere and invade the intestinal mucosa and submucosa, causing significant inflammation. different pathogens violate different parts of intestine, small intestine, or colon, and sometimes cause inflammation both of small intestine and colon. the basic clinical manifestations include obvious systemic sepsis, high fever, and even septic shock in severe patients. stool can be mucus bloody or purulent, less amount and more frequency. abdominal pain are often severe, paroxysmal colic. if the lesion invades the rectum and distal colon in particular, there may be tenesmus. sigmoidoscopy examination showed diffuse inflammation and ulceration. if only the small intestine or upper part of colon are invaded, the stool is more moisture, and without tenesmus. stool examination show large amount of leukocytes. although diagnosis of intestinal infection is not difficult, it should be careful to distinguish the site of infection, make sure of pathogens, and pay particular attention to water, electrolyte imbalance and acid-base imbalance. therefore, in addition to routine examinations and stool culture, keeping abreast of the general condition is also important to avoid disturbance of the internal environment which aggravate liver damages. in severe hepatitis, the more severe hepatic damage and immune dysfunction, the higher of incidence of sepsis. bacterium most commonly enters through intestine into the portal vein and then into the systemic circulation, followed by skin, respiratory tract, urinary tract or other intrusion. pathogens are often opportunistic bacteria, in which gram-negative bacteria are more than gram-positive bacteria, especially escherichia coli. clinical manifestations of nosocomial infection concurrent with severe sepsis are not specific, easily masked by the primary disease and complications. in some cases primary focus is not obvious, therefore the diagnosis mainly rely on blood culture. clinical manifestations include: (1) unexplained sudden chills, fever, shock, increased peripheral blood leukocytes or neutrophils; (2) deepening jaundice, an increase in ascites, or appearance of hepatic coma, hepatorenal syndrome in a short term. when complications appear in severe hepatitis patients, sepsis should be alert. the mortality of sepsis in severe hepatitis is high. nosocomial infection not only aggravates liver damages, but also induces hepatic coma, hepatorenal syndrome, upper gastrointestinal bleeding and other serious complications, leading to multiple organ failure. nancy rolando reported mortality of septicemia in patients with liver failure was as high as 59%, in which 98% with septic shock [40] . fungal infections can be classified into endogenous and exogenous infection. according to the source of the fungus, the former belongs to opportunistic pathogens, while the latter is in the environment, being infected through various routes of exposure. fungal infections in severe hepatitis are invasive fungal infection in most occasions, and the majority are nosocomial infection and endogenous pathogenic fungi infection [27] . candida infection is most common, followed by aspergillus, again neoformans and histoplasma monocytogenes. candida albicans is widely present in normal human digestive tract. other fungi such as cryptococcus neoformans, aspergillus are widely found in nature, which can colonize in body surface, or non-enclosed cavity. they also exist in hospital work environments, increasing the chance of nosocomial infection in hospitalized patients. clinically, severe hepatitis with fungal infections are mostly superinfection. the rate of fungal infection in severe hepatitis is increasing in recent years. nancy rolando reported fungal infection was present in 16 of 50 acute severe hepatitis patients (15 candida, 1 aspergillus) and in seven cases was considered the major cause of death [41] . domestic data reported that in a group of patients with liver failure, fungous infection was found in 143 cases in which the rate of nosocomial infections was 86.71%. in 155 separated fungous strains, 90 strains (58.06%) were candida albicans, 17 strains (10.97%) were aspergillus fumigatus and 25 (16.13%) strains were non-candida albicans. the main sites of fungus infection were lungs (94 cases) and oral cavity (53 cases) [29] . for severe hepatitis complicated by fungal infection, the mechanism is complex. there are many influencing factors such as immune dysfunction and reduced defensing ability, besides, application and abuse of broad-spectrum antimicrobial drugs are also related [42] . because of broad-spectrum antibiotics destroy the imbalance between bacteria and fungi in digestive system, which inhibit the growth some gram-negative bacteria that had an anti-fungal effect and some bacteria that are able to synthesize vitamin b family. lack of vitamin b can lead to inhibition of oxidation coenzyme, enabling weakened immunity, which is conducive to fungal growth. research and experience have demonstrated that repeatedly use of glucocorticoid is also an important factor to induce fungal infection in patients with severe hepatitis [28] . and therefore, the use of corticosteroids also need special care of. currently the use of glucocorticoids in patients with severe hepatitis is still controversial. the majority do not advocate glucocorticoids, or consider a short-term use in the early stages of the disease and be removed as soon as possible. otherwise it will cause a large increase of possibility of fungal infection. fungal infections are among the leading causes of death in patients with severe hepatitis. according to an analysis from rolando, among 11 cases of severe hepatitis, seven cases of deaths directly related to fungal infections [41] . a recent domestic research analyzed outcomes of 115 patients with severe hepatitis, it was found that the mortality of patients with fungal infection was significantly higher than those without fungal infections (59.1% compared to 34.8%) [28] . according to statistics, fungal infections often occur eight days after admission (0-24 days) or 5.5 days after broad-spectrum antibiotics usage (1-14 days). symptoms of fungal infection are often covered by severe liver injury related symptoms, while clinical systemic manifestation such as fever is also difficult to identify with the bacterial infection. the site of infection often occurs in mouth, respiratory, digestive or urinary tract. severe immunocompromised persons may appear disseminated infection. oropharyngeal is the site that fungal infections mostly occur, and candida albicans is the most common pathogen, followed by non-candida albicans and aspergillus infection. bedridden patients with severe hepatitis can not properly maintain oral hygiene, which results in change of oral local environmental ph and blood flow. candida albicans easily retains in the mouth, and multiplies, causing oral flora and opportunistic infections. usually general symptoms are mild. patients often have abnormal taste or loss of taste, followed by xerostomia, mucosal burning and other symptoms. candida stomatitis may have pseudomembrane formation which is not easy to peel, accompanied by angular cheilitis, or sometimes manifests as mucosal congestion, erosion or clumps shrink of tongue papillae, thickening of coating on the tongue. there are clear lines between oral pseudomembranous damages. if remove the pseudomembrane it will leave a bright red base, sometimes thick film is like a layer of cheese. take the film directly under microscopic examination shows hyphae and spores. oral fungal infection is often a prelude of deep fungal infection, which should be on the alert. simple oral candida albicans infection is not always accompanied with fever. oral fungal infection is easy to be found, therefore, if oral fungal infection exists with fever and increased leukocytes, it should be paid attention to merger of deep fungal infections such as the lungs or other organs [43] . aspergillus is ubiquitous in nature which can be spread through air flow. the most common pathogen that causes invasive aspergillosis is aspergillus fumigatus, while aspergillus flavus, aspergillus niger and aspergillus soil are less common. inhalated aspergillus spores can reproduce in healthy or immunity weakened people. respiratory tract is an infective route of invasive aspergillosis, accounted for 95%. once tissue infection exists, blood vessels violation and bloodstream invasion are common. invasive aspergillosis infection has three characteristics: tissue necrosis, hemorrhage, dissemination [44] . the mortality of invasive pulmonary aspergillosis accompanied by severe hepatitis is up to 90% [44] . it is lack of specific clinical manifestations. in most patients fever is the first arisen symptom, mostly with middle or low heat, sometimes with sudden high fever. hemoptysis occurs with fever simultaneously or 1-8 days later, often accompanied with purulent tan sputum or bloody sputum, sometimes with a pinhead size of gray-green particles in it. shortness of breath and chest tightness often exist in late stage of infection, as well as dyspnea, cyanosis, hypoxemia, and expectoration of a lot of bright red bloody sputum or blood clots. pulmonary signs appear later, manifests as pulmonary wet or dry rales, occasional pleural friction sound. in some cases there are no pulmonary signs. x-ray examination of invasive pulmonary aspergillosis showed patchy infiltrates and (or) nodular lesions. typical nodules were like cotton, which can occur in unilateral or bilateral lobes. the lesion progresses rapidly to cause expanded infiltrates, and segmental or lobar consolidation. ct examination showed mass shadow, nodules, or exudative lesions. there are two typical imaging performance: (1) a characteristic finding on chest ct is the halo sign: a solid nodule surrounded by a halo of ground-glass attenuation [45] . (2) the formation of voids, which appear in late infection. signs are hollow cavity lesions, the balloon "crescent" sign was seen around necrosis tissue [46, 47] . in order to prevent hepatic encephalopathy, patients can be given high doses of laxatives such as lactulose, which can reduce the intestinal ph value, creating an environment for growth of fungi, thereby increasing the chances of fungal infection of the intestine. in particular, some antibiotics combination increases the incidence of intestinal candidiasis. patients usually have watery or jerry-like diarrhea, with foam or blood. diarrhea is accompanied by bloating, sometimes with vomiting and fever, however, abdominal pain is not obvious. in patients with severe hepatitis, due to decreased intestinal mucosal defense capability, candida may invade the muscle and cause intestinal bleeding, or even perforation. in part of patients, it may even progress to fungal peritonitis, which is similar to clinical manifestations of bacterial peritonitis. in patients with oral candidiasis, if there is dysphagia or pain, especially retrosternal burning, it should be considered that esophagus is invaded. incoordination motor of the upper and lower esophageal can be found by esophageal barium enema. gastroscopy helps to confirm the diagnosis. urinary fungal infection usually involves bladder and kidney, among which candida infection is the most common. however, all pathogenic fungi (such as cryptococcus neoformans, aspergillus species, mucor species, histoplasma, blastomycete, coccidioides) can spread to the urinary system as a systemic or part of disseminated fungal infection, which is more related to usage of broad-spectrum antibiotics and indwelling catheters. clinical manifestations include fever and urinary tract irritation, while some patients were asymptomatic with candidiasis urine. candida and bacterial infections often occur simultaneously. candida infections of the kidney, mostly secondary, are caused by the spread of blood candida. renal cortex and medulla abscesses can occur, which affect renal function in severe cases. patients may have low back pain, abdominal pain, fever and chills, accompanied by urgency, urinary frequency, proteinuria and hematuria. urine tests may find hyphae and fungal spores, culture for candida is positive. candida albicans is common, but now there is a growing trend of candida glabrata [27] . the main pathogen of fungal sepsis is saccharomyces. most patients have high fever, often above 39 °c. the thermal type varies, of which intermittent fever or remittent fever is more common. wbc count and neutrophil are usually increased. disease in patients with fungal sepsis followed by severe hepatitis is deteriorating rapidly, even progressing to shock. fungal septicemia may invade all the tissues and organs. involved organs have corresponding performance, such as fungal pneumonia, oral fungal infections, intestinal and urinary tract infections. disseminated fungal infection often occurs in severe immunocompromised patients who have long-term use of antimicrobial agents. candida, cryptococcus, aspergillus can disseminate along with blood to all of the organs, such as kidneys, lungs, heart, and liver. the condition is dangerous, which often lead to death in a short term. in addition to common bacterial and fungal infections, severe hepatitis can also be complicated by other pathogens, such as viruses, tuberculosis, protozoa and others. cmv (cytomegalovirus, cmv), herpes simplex virus (herpes simplex virus, hsv), or varicella -zoster virus (varicella-herpes and zoster virus, vzv) infections are three of common herpes viruses infections in severe hepatitis. their common characteristics rely on that once the host is infected, the virus can persist for long periods in the host. when the host immunity is weakened, the virus can re-proliferative, which leads to disease resurgence [48] . hsv infections manifest as perioral or external genital herpes, oral and esophageal mucosa inflammation and ulcers, also viremia which leads to pneumonia and encephalitis. common clinical symptoms of herpes simplex are mild, only a few people show fatigue, fever and other symptoms. local manifestations are single or multiple blisters on skin or mucous membrane, with tingling. due to reduced immunity, skin rashes in patients with severe hepatitis perform as varicella-like rash, vaccination herpes, herpes keratoconjunctivitis and disseminated herpes simplex. severe herpes usually manifests as herpes simplex virus encephalitis with high mortality. there are fever, headache, mental disorders, coma and other clinical symptoms, often without skin herpes lesions. the sites of infection are commonly in the frontal and temporal lobes. elevated serum antibodies help confirm the diagnosis. cytomegalovirus infections are common in cirrhosis of the liver [49] . interstitial pneumonia is the most common clinical manifestation in severe hepatitis concurrent with cmv infection. chest ct examinations mainly perform as diffuse interstitial or alveolar infiltrations, very few case show as nodular shadows, occasionally as pleural effusion [50] . pulmonary consolidation reminds complicated bacterial or fungal infections. pathology manifestations show alveolar interstitial edema, with varying degrees of fibrosis, lymphocyte infiltration and epithelial cell proliferation. blood examination shows leukopenia. because viral pneumonia shares a certain similarity in clinical manifestations, diagnosis mainly depends on pathologic examination. pathogenic examinations for cmv often use methods below: (1) detect of cmv inclusion body cells and viral particles: eosinophilic nuclear inclusions giant cells are found in respiratory secretions and bronchoscopy lung biopsy specimens. respiratory secretions, saliva, urine, cervical secretions, liver or lung biopsy specimens were inoculated to human embryonic fibroblast cell culture medium, where cytomegalovirus was separated. (2) immunological methods: cmv antigen from secretions was detected by fluorescent antibody assay, or elisa, which is conducive to the early diagnosis. serum antibodies can also be detected by complement combined experiment, in which acute and convalescent serum antibody titer more than 4 times are positive. (3) molecular biology methods: pcr technology and nucleic acid hybridization, which helps to make distinction between a variety of different subtypes of the virus. most of vzv infections in patients with severe hepatitis are due to latent virus reactivated. in patients who have had chickenpox, there is a small amount of virus lurking in the spinal cord dorsal root ganglia or cranial nerve sensory ganglia. when severe hepatitis happened, latent virus is reactivated in ganglia due to decreased immunity. the activated viruses spread along with sensory nerve axons to downstream disposal areas, proliferate and cause shingles. in early time, there is paresthesia, itching, and pain in local skin. and then rashes and herpes break out, chaining into a strip, which distribute in denomination or trunk, unilateral, with duration of about three weeks or several months. part of patients with severe hepatitis show severe disseminated herpes zoster. varicella-like rashes appear in a few days, often accompanied by fever, which may be complicated by lung, brain damage with a high mortality rate. latent tuberculosis can burst to tuberculosis and extrapulmonary tuberculosis when cellular immune function gets weakened. under normal host immune function, lymphocytes, macrophages and common langerhans cell may promote granuloma formation and make infection localized. when host immunity is dysfunctional, tissue reaction is very small or even disappeared, leading to mycobacterium growing rather than formation of granulomas, nor any effective defense against infection. severe hepatitis patients with m. tuberculosis infection may develop acute miliary tuberculosis that manifest as fever, cough, expectoration, bloody sputum, chest pain, and shortness of breath in addition to deteriorated liver function. moreover, tuberculosis easily spread in patients with severe hepatitis, with poor anti-tuberculosis efficacy. common extrapulmonary tuberculosis can be lymphatic tuberculosis, intestinal tuberculosis, bone tuberculosis, renal tuberculosis, epididymal tuberculosis, genitourinary tuberculosis, nervous system tuberculosis, tuberculous meningitis [51] . in patients with severe hepatitis concurrent with tuberculous mycobacterial infections, tuberculin reaction is negative in about half of patients, especially in those applied with glucocorticoid. diagnosis relies on sputum acid-fast staining or pcr, but the diagnostic yield is not high. interferon gamma release assays (igras), including quantiferon ® -tb gold in-tube, and the t-spot tb, have been extensively used for the auxiliary diagnosis of tuberculosis infection in adults. igras detect circulating t-cells responsive to specific mycobacterium tuberculosis antigens, which are absent in bcg and other non-tuberculosis mycobacteria, and exhibited similar sensitivity and higher specificity than tst in adults [52, 53] . however, these igra tests are also affected by host immune status [54] . in addition, the decision regarding whether to treat ltbi should be dependent not only on igras results but also on clinical histories. ntm generally causes local wound infections. however, in severe hepatitis patients with impaired immune function, non-tuberculous mycobacteria can invade the lungs, causing tuberculosis-like diseases, but rarely causes hematogenous dissemination. histological examination of the lesion is mainly characterized by epithelioid cell granulomas and foam cell-like balls of tissue proliferation, detection of non-tuberculous mycobacteria [55] . protozoa and worms, such as toxoplasma gondii, giardia lamblia, cryptosporidium and stercoralis, can also infect patients with severe hepatitis, especially those with immunosuppression drugs or combined with cancer. main lesions of toxoplasmosis manifest as lymphadenopathy, hepatosplenomegaly, encephalitis and pneumonia [56] . clinical manifestations of giardia lamblia infection are chronic diarrhea and malabsorption, also fever and cholecystitis [57] . pathological changes are deformation of small intestine villi and lymphoid hyperplasia. parasites present in small intestinal surface and gallbladder, and the detection of parasites from the stool and duodenal drainage fluid that is eligible confirmed. strongyloides stercoralis is a weak virulent worm, there is few clinical stercoralis infection [58] . but this worm infection in patients with severe hepatitis is a serious threat, even causing death. clinical manifestations include long-term nausea, vomiting, diarrhea, bloating, intestinal paralysis, dehydration, electrolyte imbalance, edema, weight loss and difficulty breathing in cases with extensive lung lesions. all patients have hypoproteinemia and anemia. patients with increased eosinophils have good prognosis, whereas eosinophils reduction often is a dangerous signal. varieties of complications, such as system or local infections, coagulopathy, hepatic encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, acid-base imbalance and water-electrolyte imbalance, are main causes of hbv-associated mortality during deterioration of liver function of aechb. reducing and effective treatments of these complications are still nodules in therapy of severe phase of aechb. infection is one of usual complications of aechb, which shows 18-52% morbidity in civil china. the most common localities are respiratory system, especially lungs, and abdomen, which shows the incidence of spontaneous bacterial peritonitis (sbp) as much as 49%. others include urinary tract and bloodstream. gram-negative bacteria are the predominant causes including aerobic gram-negative bacilli, escherichia coli, klebsiella, enterococcus and anaerobic bacteroides fragilis, etc. however in recent years, gram-positive bacterial infections are found increasing in patients with aechb, including pneumococcus and other streptococcus, but staphylococcus aureus infection is relatively infrequent. fungal infections are usually secondary to bacterial infection. candida, aspergillus, sporotrichosis, histoplasmosis and coccidioidomycosis infection, especially candida albicans, are most frequent in occurrence. but, infections of cryptococcus and mucormycosis are relatively rare. beyond of bacterial and fungal infection, other infectious pathogens in aechb are virus, mycobacterium tuberculosis, mycoplasma, chlamydia, parasites and protozoa. bacterial infection is the most frequent complication in aechb. infections are more likely occurred in abdomen, including abdominal cavity, biliary tract, gastrointestinal tract, or other parts like respiratory tract, and urinary tract. keeping oral and perineal asepsis, unobstructed respiratory tract and urine tract, anti-bedsore care for patients with limited mobility, rational use of antibiotics, avoiding long-term use of broad-spectrum antibiotics, strict controlling usage of glucocorticoid, aseptic practices in invasive operation, parenteral nutrition and protecting the intestinal mucosa are all necessities to prevention of bacterial infection in aechb. empirical use of antimicrobial agents could be determined by the localities of infection without antibiotic susceptibility testing results. gram-negative infections are more frequent in peritoneal or biliary tract, in which cephalosporin or quinolone could be the first choice. penicillin and vancomycin could be considered in pulmonary infection. azithromycin and quinolone could be adopted in urinary tract infection. metronidazole or tinidazole could be used in anaerobic infection. broad-spectrum antibiotics can be used in serious infection, such as ceftriaxone, cefoperazone, cefepime, and carbapenems, but the secondary infections need to be highly concerned. initial antibiotics should be adjusted according to antibiotic susceptibility testing results as soon as possible. non-specific immune enhancer agents, such as thymosin, are well used in treatment of infection during aechb, but it currently still lack of evidence-based support. the incidence of fungal infection ranks secondly in aechb associated infection. respiratory tract, gastrointestinal tracts and genitourinary system are the major sites. keeping wards dry and ventilated can help to reduce environmental fungal growth. oral and perineal cleaning and regularly dental check are necessary. if oral fungal spots are found, alkaline mouthwash and nystatin with glycerol can be used in treatment. for patients with limited mobility, anti-bedsore care is terribly necessary. rational use of antibiotics, especially avoiding long-term usage of broadspectrum antibiotics, can prevent secondary fungal infection. glucocorticoid should be used strictly according to indication. during invasive procedures, aseptic operations are obligated. regular check of sputum, lungs, urine and feces will facilitate early diagnosis. empiric antifungal treatment is generally not recommended, but to patients with aechb with aids, especially with count of peripheral blood cd4 + t cell-less than 200/μl or oropharyngeal candidiasis found, the sulfamethoxazole (smz-tmp) should be chosen to prevent pneumocystis pneumonia. fluconazole should be considered when moderate amount of candida albicans has been indicated by sputum culture. empirical treatment. if candida albicans infection is highly susceptible, the initial treatment choice is fluconazole (200-400 mg/days). but if aspergillus infection is preferred, amphotericin b liposome, itraconazole, caspofungin or voriconazole could be considered as the initial treatment, in which process liver and kidney function should be intensively monitored. to patients with cryptococcal encephalopathy combining severe liver damage, fluconazole or flucytosine combined with intrathecal injection of amphotericin b treatment could be implemented. evidence based treatment. initial antifungal strategy should be adjusted according to antibiotic susceptibility testing results. for certain invasive pulmonary aspergillosis, combination treatment of several different types of antifungal agents should be considered under monitoring of liver and kidney function. however, for pulmonary mucormycosis infection, the combination of amphotericin b and flucytosine is the only effective strategy. amphotericin b is a type of polyene antifungals, mainly for aspergillus, candida, cryptococcus, histoplasma infection, but is invalid in aspergillus terreus or ringworm fungus infection. intravenous or intrathecal injection of amphotericin b should be administrated because of non-digestive absorbance. the recommended initial dose of intravenous administration is 1-5 mg/days, and gradually increases to 0.5-1 mg/kg.days. the infusion track needs to be dark and process needs not less than 6 h. the initial dose of intrathecal injection is 0.1 mg/days and gradually increases to 0.5-1 mg/days. amphotericin b has an extra toxicity to liver and kidney, and also causing hypokalemia. intensive monitoring of liver and kidney function and serum potassium levels is necessary during treatment, and be sure largely avoiding combination with other agents with liver and kidney toxicity. itraconazole is one of triazole antifungal agents, mainly for aspergillus, candida, cryptococcus and histoplasma infection, but it is invalid in fungi fusarium or zygomycetes infection. the intravenous dose for the initial 2 days is 400 mg/days, administrated by twice, and then followed by 200 mg/days for 2 weeks. the oral doses of 200 mg bid could be subsequent. the total curative course could be determined by the improvement of symptoms and largely absorption of radiographic lesions of infection. the monitoring of liver function is necessary and recommended, especially in long duration treatment. furthermore, combination treatment with other hepatotoxic drugs should be avoided. fluorocytosine is one of bacteriostatics, mainly for cryptococcosis and candida infection, frequently based on the combination with amphotericin b. the daily dose for adults is 2.5 g with intravenous dripping speed of 4-10 ml/min. a half dose should be conducted in renal insufficiency. the inhibited administration includes severe liver or kidney dysfunction and allergy to fluorocytosine, and also cautious treatment for pregnant or breastfeeding women. no combination treatment of fluorocytosine with cytarabine or bone marrow suppression agents is recommended. fluconazole is a triazole antifungal, mainly for candida albicans or cryptococcus infection, but not as good in candida glabrata infection, totally invalid in aspergillus or candida krusei infection. the recommended daily dose for adult is 200-400 mg, and the initial dose should be doubled. voriconazole belongs to triazole antifungals, mainly for candida, cryptococcus, aspergillus, fusarium and histoplasma capsulatum infection, especially used for invasive aspergillosis and invasive fluconazole-resistant candida infection, but is invalid for mucor or rhizopus infection. the recommended intravenous initial dose in adult is 6 mg/kg, q12h, with infusion rate of 3 mg/kg within 1-2 h. the maintenance dose from the second day is 4 mg/kg, q12h. to patients without tolerance to normal dose, it could be reduced to 3 mg/kg, q12h. the reduction of daily dose could not be necessary in mild to moderate liver dysfunction. but intravenous administration should be avoided in patients with severe renal dysfunction. the side effects of voriconazole include transient visual disturbances, mental disorders, thrombocytopenia and so on. caspofungin belongs to echinocandin antifungals with antibiotic spectrum of pathogenic aspergillus, candida and pneumocystis, without in cryptococcus neoformans, fusarium and mucor. it is mainly used for invasive aspergillosis. the initial dose for adults is 70 mgqd, and with subsequent 50 mg qd. the infusion time is no less than 1 h. no fixation curative course is suggested. caspofungin should be avoided for patients with severe liver function, because of highly hepatic distribution and metabolic pathways during treatment. liver is the largest solid organ in the body, and it plays a central role in the clotting process, except for general function such as metabolism, detoxification and choleresis [59] . a majority of the coagulation factors are synthesized almost exclusively in the liver. in the pathogenesis of severe hepatitis, massive hepatic necrosis leading to reduced production and dysfunction of coagulation factor. in addition, the increased levels of anticoagulation and platelet abnormalities also contribute to occurrence of coagulopathy, which were further exacerbated by severe complications such as spontaneous bacterial peritonitis (sbp) and hepatorenal syndrome (hrs). coagulation abnormalities, even disseminated intravascular coagulation (dic) often occur in those patients with severe hepatitis. therefore, the changes in coagulation factors were usually used to evaluate prognosis of severe hepatitis [60] . the normal procedure of coagulation includes two independent coagulation process, namely intrinsic and extrinsic pathway, which initiated by coagulation factor xii, and factor viia/tissue factor, respectively. the two pathways reach a confluence at the points of factor xa and va. wherein factor xa activates prothrombin to thrombin in the presence of ca 2+ and factor va bound to membrane surfaces, and then thrombin converts fibrinogen to fibrin. thus, blood becomes clotted and the "y" shaped pathway was established [61] . simultaneously, there are some anticoagulation systems existing in our body, which prevents the formation of thrombus, then keeps normal circulating of blood flow. it also participates in maintaining of normal permeability of the blood vessel [62] . among anticoagulation systems, the most important one is the fibrinolytic system, whose basic process can divided into two stages, i.e. plasminogen activation and fibrin degradation [63] . plasminogen activators include tissue-type plasminogen activator (tpa) and urokinase-type plasminogen activator (upa), and the principal function of these two plasminogen activators is to convert plasminogen to plasmin, then plasmin cleaves fibrin into soluble small peptides, namely fibrin degradation products [64] . moreover, the process of fibrinolysis was affected by fibrinolysis inhibitors, such as plasminogen activator inhibitor (pai) and alpha2antiplasmin (α2-ap). those fibrinolysis inhibitors can either inhibit plasminogen activation, or reduce the function of plasmin [65] . thus, there is a dynamic balance between coagulation and anti-coagulation systems under physiological conditions ( fig. 2 .1) [66] . many factors involved in coagulation process, i.e. clotting factors, thrombin inhibitor, fibrinolytic system and so on, are synthesized almost exclusively in the liver. thus, liver plays a pivotal role in remaining the balance between hemorrhage and coagulation under physiological conditions (table. 2.1) [67] . it is the important basis for pathogenesis of coagulopathy that massive hepatocyte necrosis occurs in severe hepatitis patients, resulting in reduced production and dysfunction of those blood clotting factors [68] . coagulation abnormalities, such as decreased production and dysfunction of coagulation factors, are commonly found in severe hepatitis, there are at least 14 types of factors participated in the coagulation process, including 12 classical coagulation factors, namely factor i/fibrinogen, factor ii/prothrombin, factor iii/tissue factor, factor iv/calcium ions, as well as factor v, vii, viii, ix, x, xi, xii, and factor xiii. some bradykinin factors, such as rk (prekallikrein) and hmwk (high molecular weight kininogen) are also associated with coagulation. among above factors, the others belong to proteins excepting factor iv. plasma coagulation factors are synthesized exclusively in the liver, excepting factor iii/tissue factor, factor iv, factor vi/activated factor v, factor viii and factor viiia [69] . massive hepatic necrosis leads to the decreased production of clotting factor in patients with severe chronic hepatitis. moreover, the coagulation factors are sensitive indicators for clinical evaluation of severe hepatitis. it is common that the serum levels of factor v, vii, ix, x, xi and prothrombin decreased in the patients with severe hepatitis. on the contrary, clotting factor viii is synthesized, and then excreted increasingly by the mononuclear phagocytic cells with stimulation of various inflammatory cytokines in the patients with severe hepatitis [70] . anorexia and antibiotics overuse lead to obstacles in assimilation, absorption, and application of vitamin k in these patients. vitamin k dependent coagulation factors includes factor ii, vii, ix, and x. the function of γ-hydroxylation was weaken by the deficiency of vitamin k and damage of hydroxylase, then the abnormal clotting factors without γ-hydroxy glutamate were synthesized. finally, these abnormal clotting factors lead to dysfunction of the vitamin k dependent coagulation factors [71] . coagulation factor vii eliminated significantly in the initial stage of liver injury was usually used as an early diagnosis index [72] . in addition, coagulation factors ii and x, then factor ix decreased markedly, along with exacerbation of liver injury. the deficiency of vitamin k induced by obstructive jaundice, malabsorption syndrome, can be rescued after intravenous injection of vitamin k, which is different from coagulopathy caused by liver injury. the plasma level of fibrinogen is within normal range in patients with compensatory cirrhosis. however, patients with severe hepatitis or liver failure have a significant reduction in the level of fibrinogen, and then develop dysfibrinogenemia [73] . fibrinogen, as the main hydrolysis substrate of thrombin, is the crucial factor in the coagulation process. thus, decreased levels or abnormal structure of fibrinogen leads to coagulation abnormalities in patients with severe hepatitis [74] . there are two clotting factors associated with thrombin except for fibrinogen, i.e. factor v and factor xiii. factor xiii induce a crosslink of sfm (soluble fibrin monomer), resulting in the formation of insoluble fibrin [75] . it reports that the levels of circulating coagulation factor xiii were decreased in 30% of patients with liver disease, resulting in fibrin decline or abnormality. these patients with liver disease will receive a bad prognosis if the plasma level of fibrin was at level below 35% of normal concentration [76] . it is uncommon that the plasma level of plasma factor v decreased markedly in patients with severe hepatitis, except for those complicated with dic or hyperfibrinolysis. the low level of factor v cannot induce the formation of the enzyme-substrate complex, which delay the activation of prothrombin [77] . contact factors include classical coagulation factor xii and xi, as well as some bradykinin factors, i.e. pk and hmwk. these factors contact with vascular intima damage or abnormal surface in blood vessel walls, then active the intrinsic coagulation pathway [78] . moreover, the contact factors can connect with bradykinin, fibrinolysis and complement system. in addition, coagulation factor xiii also activates fibrinolysis system as the initiating factor. factor xii deficiency does not cause excess bleeding, but induce thrombus due to its decreasing activation of fibrinolytic system [79] . there is a dynamic balance between pro-and anti-coagulation systems in physiological conditions, which keep normal blood circulation in the body. the anticoagulation system includes physiological anti-coagulation factors (e.g. antiprothrombin-iii, protein c, and so on) and fibrinolysis factors (e.g. plasminogen, α-2 plasmin inhibitor, and so on) [80] . at-iii, with a half-life period of 2.8 days, synthesized mainly in hepatocytes and partly in endotheliocyte, is responsible for about 70% of anticoagulation in plasma. it is the main reason for low plasma level of at-iii that decreased production and increased consumption of at-iii caused by hepatocytes necrosis in patients with severe hepatitis. the activity of at-iii obviously decreased in severe hepatitis [81] . therefore, heparin, thrombin, activated coagulation factors (e.g. factor x, ix, xi, xii) cannot be inhibited, due to rare at-iii combining with them [82] . there is a negative correlation between at-iii: a (the activity of at-iii) and pt (prothrombin time), which indicate that the level of at-iii: a decreased obviously along with exacerbation of liver injury [83] . the plasma levels of pro-and anti-coagulant factors are low in patients with liver injury. when the necrotic tissue and cytolysis are released in the blood, the balance of pro-and anti-coagulant is destroyed. finally the depletion of at-iii by massive activated coagulation factors will lead to dic [84] . massive hepatocytes necrosis, vitamin k deficiency, and protein c without γ-hydroxyglutamic acid result in blocking activation of protein c. thus, va, viiia and pai cannot be degraded, and the plasma level of them will rise up, due to reduction of activated protein c (apc) in the patients with severe hepatitis [85] . plasminogen synthesis will decreased by about 70% in the liver when sever hepatitis occurs. as the activators of plasminogen, tpa and upa are synthesized by vascular endothelial cells, and their production will increase after vascular endothelial cells initiated with virus, immunocomplex or endotoxin in the patients with severe hepatitis b [86] . with exacerbation of liver injury, pai synthesis decreased significantly in the liver. the main physiological activity of pai is to inhibit tpa induced plasminogen activation. therefore, the activity of tpa will increase with reduction of pai synthesis, resulting in promoting the conversion of plasminogen into plasmin [87] . plasmin, as a kind of powerful proteolytic enzyme, can hydrolyse fibrinogen into fdps, degrade coagulation factors, and inhibit platelet aggregation, resulting in the aggravation of bleeding [88] . hyperfibrinolysis can be caused by congenital or acquired reason, and it commonly leads to a rapid depletion of coagulation and anticoagulation factors, especially in those patients with dic [89] . synthesis and secretion of tpa and upa markedly increase, while pai, namely tpa or upa inhibitor, has been a decrease in the plasma of severe hepatitis patients, resulting in hyperfibrinolysis [90] . at the same time, mononuclear phagocyte system cannot degrade plasminogen activator, also leading to hyperfibrinolysis. it is not necessarily accompanied by hemorrhagic tendency, although there are many risk factors that can cause hyperfibrinolysis, even hyperfibrinolysis occurred in severe hepatitis [91] . in addition, fdps inhibit fibrin monomer polymerize function, and also block platelet aggregation, then further worse the deficiency of hemostasis and coagulation, finally leading to the aggravation of bleeding tendency [92] . studies have shown that low-level endogenous small molecule heparin in patients with chronic hepatitis may be associated with many factors, such as increased mastocyte, decreased production of heparinase in the liver and reduced activity of ph4 (platelet factor4) [93] . when the disease progress to cirrhosis or chronic severe hepatitis, pt was significantly prolonged, while endogenous heparin wasn't increased markedly, indicating that low-level endogenous heparin has little effect on pt elongation and hemorrhagic tendency [94] . however, if the patient is undergoing the following condition together, the endogenous small molecule heparin will increase significantly. (1) esophageal variceal bleeding with serious infections (such as abdominal infection, biliary tract infection and pulmonary infection) or portal hypertension. (2) combining with the significant increased white blood cell count. (3) percentage of neutrophils >75%. the level of endogenous small molecule heparin in plasmin will decrease after those infections were cured. taken together, these data indicate that increased level of endogenous small molecule heparin in blood circulation was closely related to severe hemorrhagic tendency when combining with infection [95, 96] . platelet significantly promotes blood coagulation through connecting with many coagulation factors (such as fibrinogen, factor v, factor xi, factor xiii and so on). α-granule includes fibrinogen, factor xiii, and some platelet factor such as platelet factor2 (pf2) and platelet factor3 (pf3), which promote coagulation activation process of factor xii and factor xi can be accelerated by activated platelets [97] . it is estimated that pf3 provided by platelets could accelerate activating of thrombin by twenty thousand times. xa and factor v could not be inhibited by at-iii and heparin if they were linked with pf3. when platelets aggregated and formed platelet plugs, the process of coagulation was initiated at this site, and then platelets reveal a large number of phospholipid surfaces, which were helpful for activating of factor x and fibrinogen [98] . various platelet factors were released from α-granule after platelet aggregating, and then hemostyptic fibre was produced increasingly. those hemostyptic fibre finally produced blood clot formation after capturing the other hemocytes. thus, platelet plugs would progress independently of platelet disintegrating gradually [99] . two aspects of platelet abnormalities consist of depletion and dysfunction. in patients with chronic hepatitis and cirrhosis, the occurrence of decreased platelet level is usual through hypersplenism accompanied with other hemocyte decreasing [100] . the ratio of which reaches 37-77%. its incidence rate in severe hepatitis and explosive hepatic failure also reaches approximately by 50%. when severe hepatitis occur, myelosuppression, decreased megacaryocyte replication leading short-life platelet, lacking of hemopoietic material such as vitamin b, folate and so on, all of these can initiate thrombocytopenia [101] . other reasons leading to thrombocytopenia contain low expression and metabolic disturbance of thrombopoietin (tpo), as well as platelet antibody production. researches show that the serum level of tpo related positively to platelet expression [102] . we next studied platelet associated immunoglobulin (paig) and its sorts such as paigg, paiga, paigm etc., with chronic hepatitis. in line with previous reports, we found that serum levels of paig and paigg negatively correlated with blood platelet count, corroborating the crucial role of the paig-mediated autoimmunization in controlling thrombocytopenia in viral hepatitis [103] . various factors impact platelet function forwardly or passively. increased expression of oxide and prostacyclin, two kinds of platelet inhibitor derived by endothelium, may inhibit platelet activation in vivo [104] . on the other hand, increased serum level of von willebrand factor (vwf) can promote platelet adhering and aggregation in patients with cirrhosis. when severe hepatitis occur, 66.7% and 77.8% of patients have decreased levels of platelet adhesion rate and platelet aggregation rate (par), respectively. in addition, reduced effectiveness of pf3 and abnormal clot contraction occur in patients with severe hepatitis by an incidence of 65.6% and 77.8%, severally [105] . patients with terminal liver disease significantly exert hemorrhagic tendency, especially in the digestive tract [106] . the latest report indicated that basic laboratory examinations for coagulation function testing in common use at present, such as pt, aptt, international normalized ratio (inr) etc., have little correlation with occurrence of gastrointestinal bleeding in these patients, thereby revealing the importance to search and pay close attention to those complicating disease upregulating bleeding risk, such as bacterial infection, renal failure, hemodynamic change after portal hypertension, dysfunction of endotheliocyte as well as macrophagocyte and so on [107] . it is common to see renal failure occurrence in advanced hepatopathy. when it happened, acquired platelet dysfunction, abnormal activation of platelet and vascular wall, anemia and so on, all of them significantly promote hemorrhage [108] . as another severe complication, bacterial infection is also very important and common [109] . when tumor necrosis factor (tnf) were injected into healthy individuals, we found that endotoxin have an important role to exert its function directly in clotting cascade reaction. early researches indicated that the body can express endogenous heparin-like substance through stimulation by endotoxin in patients with cirrhosis [110] . furthermore, some studies revealed the relevance of endotoxin with prothrombin fragment, indicating that infection promoted occurrence of dic-like status in laboratory examination in cirrhosis [111] . coagulation system became weaker in patients with chronic hepatitis. it can hardly mediate factors due to the relative deficiency of pro-and anticoagulation factors. when the balance was destroyed, it may tend to hemorrhage or thrombosis depending on related risk factors which were in the ascendant. to assess abnormality of blood coagulation in patients with liver disease, we should be in consideration of hypercoagulability, one state may easily be overlooked. otherwise, it will be unfair. current literatures indicated that, unlike previous concept that the body of patients initiated anticoagulation state automatically when infected by the hepatitis virus. various clinical evidences revealed that hepatitis virus infecting cannot inhibit thrombus forming. furthermore, it may increase dangerous to thrombus forming especially in the portal system, for existence of individuals having hereditary mutation to promote thrombus forming [112] . slower bloodstream, abnormal fibrinolysis initiated by blood stasis, and decreased activity of anticoagulant accelerate formation of venous thrombosis. moreover, the changes of platelet phospholipids membrane activity were also helpful to the formation of thrombosis in patients with chronic liver disease [113] . it is reported that the incidence of periphery deep venous thrombosis and pulmonary embolism was 0.5 and 1.0% in patients with cirrhosis, respectively [114] . the rate of portal vein thrombosis is about 1% in patients with compensated cirrhosis, but it reaches 8-25% in the candidates waiting for liver transplantation [115] . for mutations (such as leiden mutation of factor v, g20210a mutation of prothrombin, c677 mutation of methylenetetrahydrofolate reductase) or the existence of antiphospholipid antibody syndrome, the hypercoagulable state will be represented in patients with liver disease [116] . the hypercoagulable state represents diverse modes in the body of patients with chronic hepatitis. among them, thrombosis is more traditional and common. the mechanism of dic complicated with severe hepatitis may contain several aspects as follows. 1. with attacks from endotoxin, virus and immune complex etc., endotheliocyte was injured, and then it activated plasmakinin system and complement system, leading to the aggravation and participation of dic progress. damaged endotheliocyte can simultaneously activate factor vii, intrinsic coagulation pathway and platelets, and participate in micro-thrombosis with platelets adhesion and aggregation beneath endothelium [117, 118] . 2. in patients with severe hepatitis, massive necrotic hepatocytes activated extrinsic coagulation system with the releasing of various tissue thromboplastin-like substances [119] . 3. hepatocellular necrosis or dysfunction decreased expression of anti-coagulation factors such as at-iii, pc, protein s and so on, and then it enhanced activation of thrombin and plasmin [120, 121] . 4. impaired function of the mononuclear phagocyte system. mononuclear phagocytes can express activated tissue factor with the releasing of tnf, il-1 and platelet activating factor (paf) on its surface after stimulation by endotoxin, inflammatory cytokines and complement activation. tnf and il-1 can decrease the activation of protein c through its function to increase the expression of plasminogen activator and plasminogen activator inhibitor-1 and to inhibit the production of thrombomodulin (tm) [122] . activated clotting factor and other factors with promoting coagulation can lead to the occurrence and aggravation of dic, since it cannot be promptly removed [123] . 5. the onset and enhancement of fibrinolysis. massive clotting factors and platelets were depleted in the extensive formation in vivo microthrombus. thrombin promoted the conversion of fibrinogen into fibrous protein [124] . simultaneously, it activated fragments which dropped in the formation of activated factor xiii, factor xa and factor xiia. all of them can activate plasmin, and it enhanced fibrinolysis with tpa, one factor released by damaged blood vessel endothelium [125] . fibrinogen and fibrous protein were degraded after plasminogen activation to generate the corresponding fdps, one factor inhibit blood coagulation and also block platelet aggregation [126] . taken together, the above process exacerbates bleeding initiated by coagulation factors depletion and platelet deficiency. coagulopathy, characterized by prolongation of blood clotting time, tendency of hemorrhage, or even dic, were commonly found in severe hepatitis patients. it may cause uncontrolled external or internal hemorrhage. as common clinical symptoms, the external hemorrhages include gingivale or nasal mucosal bleeding, skin petechiae, the punctures or injection-site ecchymosis, and so on. the internal hemorrhages include esophagogastric varices, intracranial, subcutaneous, and muscle interval bleeding. except for esophagogastric varices hemorrhage, the other internal hemorrhages rarely occur in those patients. in addition, massive hemorrhage from esophagogastric varices is a serious medical emergency, can potentially cause death and cardiac arrest if proper medical treatment is not received quickly [127] . when dic occur, a wide range of thrombogenesis in microvasculature can cause circulatory collapse, characterized by low blood pressure and shock. microcirculatory dysfunction occur after microthrombosis producing, resulting in hypofunction of multiple organs (e.g. kidney, liver, lung and pancreas), which perform from dysfunction to failure, with illness progressing [88] . crushed by the fibrous protein in the vessels, red blood cell destruction can lead to intravascular hemolysis. at the early stage of disease, it shows hypercoagulability. the blood in the needle is easy to coagulate, when getting blood sampling from the vein. after that, it comes with the stage of consumed hypocoagulation. the consuming of a great number of blood coagulation factors leads to significant tendency of hemorrhage [107] . it is difficult to stop bleeding in many parts of the body, including visceral organs, operative sites, injection sites, puncture sites, and mini-invasiva sites [128] . when the third stage, namely secondary fibrinolytic stage comes, a great number of the blood coagulation factors have already been used up, resulting in severe bleeding under the condition of low coagulation. shock, acidosis and mods make the patient's condition continue deteriorating, and are also the main reasons for death [129] . the latest study indicates that thrombosis is also a noticeable state in cirrhosis and end-stage liver disease. portal thrombosis and peripheral vein thrombosis (e.g. deep vein thrombosis and lung embolism) are commonly seen. the deep vein thrombosis is more danger than lung embolism. anyway, the incidence rate of thrombosis in cirrhosis and end-stage liver disease is still very low. the clinical symptoms depend on the embolizing position after the thrombosis occurs [130] . presently blood clotting factors test is the most maturely and frequently used test in the term of blood coagulation function in the world. the indicators including prothrombin time (pt), international normalized ratio (inr) and prothrombin time activity (pta) have always been chosen in the patients with severe hepatitis. prothrombin time (pt) reflects whether there are anticoagulant substances in the extrinsic blood coagulation system and blood circulation or not. the elongation of prothrombin time (pt) presents the declined activity of several blood clotting factors including factorii (fii), factorv (fv), factor vii (fvii), factorx (fx) or the existence of anticoagulant substances. in severe hepatitis patients, the incidence rate of prothrombin time (pt) elongated can reaches to 90%, thus it is regarded as a sensitive and frequently used indicator in the term of liver function [131] . another index international normalized ratio (inr), a reckoned ratio calculated from prothrombin time (pt) and international sensitivity index (isi), making prothrombin time (pt) between different laboratories and different reagents comparable, is an international general indicator. the guides about acute-on-chronic liver failure and acute liver failure take the index, inr ≥ 1.5, as one of the most significant diagnostic criteria in the american association for the study of liver diseases (aasld) and the asian pacific association for the study of liver (apasl). international normalized ratio (inr) can also be used as a monitor on blood coagulation function [132] . hepaplastin test (hpt), reflecting not only blood coagulation mechanism in hepatitis patients but also the function of hepatocytes to synthetize vitamin k dependent clotting factors including factorii (fii), factor vii (fvii), factorix (fix), factorx (fx) synthetically, is a test about liver reserve function; but this indicator can't reflect the change of factorv (fv). when severe hepatitis occur, the function of liver to synthetize above-mentioned clotting factors declines and the time of hepaplastin test (hpt) elongates. with illness progressing, hpt continues elongating. survivors undergoing effective therapies can have gradual recovery in the time of hepaplastin test (hpt). so this test is regarded as the specific test of liver diseases or the optimal indicator reflecting liver reserve function [133] . the severity of liver damage is positively correlated with the decline degree of prothrombin time activity (pta): the more severe damage occurs in hepatocytes, the more significantly prothrombin time activity (pta) will decline. therefore prothrombin time activity (pta) <40% and total bilirubin (tbil)>171 μmol/l have always been used as the main laboratory indicators to diagnose severe hepatitis domestically. pta <40% is also regarded as diagnostic criterion of blood coagulation dysfunction in the guideline of acute-on-chronic liver failure in the asian pacific association for the study of liver (apasl) [134] . in severe hepatitis, total bilirubin (tbil), total cholesterol, prothrombin time activity (pta) and complications (e.g. rectory hyponatremia, hepatic encephalopathy, hepatorenal syndromes and so on) are all independent risk factors to evaluate prognosis. the lack of any factors including factori (fi), factorii (fii), factorv (fv), factorvii (fvii), factor x (fx), can lead to the decline of prothrombin time activity (pta). moreover the half-life time of those factors are extremely short, factorii (fii) 50 -80 h, factorv (fv) 12 -24 h, factorv (fv) 2 -6 h, factorv (fv) 48-60 h, respectively. it means that when hepatocytes suffer from severe serious damage and necrosis in severe hepatitis, prothrombin time activity (pta) will have dramatic decline just in a few days. as a result, prothrombin time activity (pta), characterized by significant advantages in evaluating patients condition and judging prognosis over other laboratory indicators, has been widely used. elongation of aptt prompts the lack of any clotting factor belonging to intrinsic coagulation system or the existence of anticoagulant substances. the incidence of the elongation of aptt reaches to 80-100% in severe hepatitis patients [135] . fxii:c reflects liver synthetic function. fvii, characterized by the shortest halftime 6-8 h, is the first one to be affected when facing liver synthetic dysfunction. on the contrary, fv:c, characterized by a relatively long half-time, is one of the latest factors to be affected and is correlated with the degree of liver damage. it prompts severe liver failure, bad prognosis and even easy death when plasma levels of fv:c under 20%. some literatures report that fv:c and pta can be used as significant prognostic factors in liver failure and significant screening indicators in liver transplantation. however, the indicators-fxii:c and fv:c-are not listed as routine examination items. they are just selected on the condition of illness demand [136, 137] . the main test about anti-clotting factors is the determination of antithrombin iii activity (at-iii:a). in the state of pathosis, the decline of antithrombin iii activity (at-iii:a) is not parallel with the decline of antithrombin iii (at-iii) content, namely the depletion of antithrombin iii activity (at-iii:a) more apparent. owe to this, it has more clinical value to determine antithrombin iii activity (at-iii:a) rather than antithrombin iii (at-iii) content. moreover, anti-clotting factors tests include the determination of protein c activity (pc:a) as well [138] . serum levels of fdps are very low in normal people. significantly elevating of fdps indicates the existence of hyperfibrinolysis and reflects the occurring of dic indirectly. there are many assay methods including immunization fi test (namely latex particle agglutination test, normal titer<1:8), fdps flocculation test, radial immunodiffusion staphylococcal clumping test, indirect hemagglutination inhibition test, enzyme-linked immunosorbent assay (elisa) and so on. if the serum levels of fdps elevate, it indicates acute dic may occur [92] . plasma protamine paracoagulation test (3p test) and ethanol gel test (egt) reflect the soluble fibrin complexes in plasma. soluble fibrin complexes, combination of fdps and fibrin monomer, can't be solidified by thrombase. but protamine is able to make the complexes isolate and then fibrin monomers separate out again. the paracoagulation test means self-polymerization between fibrin monomers and fdps, then forming macroscopic flocks. ethanol gel test (egt) has the same principle with plasma protamine paracoagulation test (3p test), whereas the former has a lower positive rate. the two methods may have false negative results and false positive results. in contrast, egt has a relatively lower sensitivity, while 3p test has a relatively lower specificity. for example, relative small molecular mass of the shreds of fdps may lead to negative result using 3p test. so it is more valuable to compare the two indicators simultaneously [139, 140] . euglobulin, a protein (including fibrinogen, plasminogen and other activins except for fibrinolysis inhibitor) separating out from plasma in acid circumstances, can be used to determine whether levels of plasminogen activators increase or not [141] . when hyperfibrinolysis occurs, plasma levels of plasminogen decline, plasma levels of plasmin increase, and euglobulin suffer from accelerated dissolution by a great number of plasmin. the normal value of euglobulin lysis time (elt) is above 2 h. that is to say dissolution within 2 h means the occurrence of hyperfibrinolysis. domestic population data report the positive rate of elt test reaches 25-42.9%, when dic occur [142] . furthermore there are other tests about fibrinolysis including tissue-type plasminogen activator test (t-pa), plasminogen activator inhibitor test (pai), plasminogen antigen test (plg:ag), plasmin activity test (pl:a), α2-plasmin inhibitor test (α2-pi) and so on [143, 144] . blood platelet count reflects the absolute number of platelet in peripheral blood circulation. according to the reports at home and abroad, platelet count have a significant decline in patients with chronic severe hepatitis. moreover, studies on domestic population find that platelet count ranges from 68 × 10 9 /l to 130 × 10 9 /l in peripheral blood of severe hepatitis patients. some studies have already compared the platelet count among early stage, typical stage and late stage in severe hepatitis, and they have turned out to be 130 × 10 9 /l, 109 × 10 9 /l and 87 × 10 9 /l respectively. as a result, it indicates that platelet count is positively correlated with the severity of hepatitis [145] . except for platelet count, other routine indicators including mean platelet volume (mpv), plateletcrit (pct) and platelet distribution width (pdw) also have significant reference value. when severe hepatitis occurs, the above-mentioned three indicators will be dramatically lower, and they have the tendency to continue declining with illness progressing. what's more, platelet quality tests include platelet aggregation rate, platelet factor 3 validity tests and blood clot retraction test (table 2. 2) [146] . diagnosis of blood hypercoagulability in the early stage of dic relies on several molecular marks including plasma thrombinogen segment 1 + 2 (f1 + 2), thrombinantithrombin complex (tat) and d-dimer, due to no significant changes in general laboratory tests. this stage is characterized by the elevated levels of those three molecular marks, and levels will increase more significantly with the occurrence of typical dic symptoms. dynamic monitoring of above-mentioned indicators is helpful for early diagnosis of dic [147] . the stages are mainly characterized by the decline of blood clotting factors (including factorv (fv), factorvii (fvii), factorxii (fxii), factorix (fix), fac-torx (fx)), platelet count and plasminogen, and increasing of fibrinolytic the elevating levels of fibrin(−ogen) degradation products (fdps) and d-dimer; the shortening of euglobulin lysis time (elt) and the positive reaction of 3p test [148, 149] . with the occurrence of dic, there will be a wide range of blood coagulation and highly-activated fibrinolysis in the patient's body [150] . what's more, abnormal increased soluble fibrin monomer and fdp fragments will exist in plasma [151] . the level of soluble fibrin monomer complex (sfmc), a complex combined fibrin monomer with fdp, is determined by 3p test. the 3p test shows positive with the occurrence of secondary fibrinolysis, whereas it shows negative with the occurrence of primary fibrinolysis. this means 3p test is negative when there is no blood coagulation. domestic population data report that the positive rate of 3p tests reach 50-60%. however, the test can't be used as the ideal diagnostic indicator for dic, owing to many affected factors. false positive reactions are mainly found in the following conditions: gastrointestinal bleeding, massive hemoptysis, malignant carcinoma or blood sampling reserved improperly, whereas false negative reactions are usually found at the late period of the stage of secondary fibrinolysis [152, 153] . as mentioned above, plasma thrombinogen segment 1 + 2 test (f1 + 2), thrombinantithrombin complex test (tat) directly reflects production of intracorporeal thrombase, which increased in the early stage of dic. plasmin degrades the crosslinked fibrin to release fibrin degradation products and expose the d-dimer antigen, which reflects production of intracorporeal plasmin. d-dimer will have an apparent increase with the occurrence of secondary fibrinolysis, but d-dimer test shows negative when primary hyperfibrinolysis occurs. monitoring the above molecular markers dynamically is helpful to estimate therapeutic efficacy and guide treatment [154] . as mentioned above, clinical manifestations of blood coagulopathy in patients with severe hepatitis are lack of specificity. the most common manifestation is bleeding, not only little hemorrhage from superficial sites, but also massive hemorrhage from internal sites, such as esophagogastric varices [155] . the diagnosis of severe hepatitis complicated with blood coagulopathy is mainly based on the results of laboratory tests and clinical manifestations [156] . according to current guidelines, basic diagnose conditions of dic contain the following points [157] . 1. severe or multiple bleeding tendency. 2. microcirculation collapse or shock which is difficult to explain using protopathy. 3. a wide range of embolism in skin and mucosa, focal ischemic necrosis and ulcer, or unexplained dysfunction of kidney, lung, brain. 4. anticoagulant therapy is effective. if severe hepatitis b patients have one of the above-mentioned points except for (1) and exhibit blood coagulating easily or prothrombin time (pt) shortening over 3 s simultaneously, it can be considered as the early stage of dic in which the tendency of bleeding is not obvious. in addition, if severe hepatitis b patients have two of the above-mentioned four points, dic can be considered as the preliminary clinical diagnosis. furthermore, it can be definitely diagnosed when combined with the aforementioned items of laboratory tests ( table 2. 3). firstly, we should use anti-viral therapy as a mainly method to treat primary disease of severe hepatitis b [158] . then, we must eliminate the incentive, maintain the balance of water and electrolyte, and correct hypoxia and acidosis. it is very important to focus on massive upper gastrointestinal hemorrhage and disseminated intravascular coagulation when treating coagulation disorders [159] . gastrointestinal bleeding, including esophageal varices bleeding and non-bleeding esophageal varices, were correlated with coagulation dysfunction and portal hypertension. prevention is still focused on improving the coagulation function, including adequate vitamin k1 supplements, coagulation factors, fibrinogen, fresh plasma or platelets supplements. it is particularly critical to control diet in patients with severe hepatitis. in which, light and easily digestible diet was recommended, but rough, hard and too greasy food was prohibited. for these patients, appropriate antacids can be used to protect the gastric mucosa [160] . 1. general treatment: bed rest is necessary, meanwhile vital signs were closely monitored. the patients can eat liquid diet when bleeding mildly or having no active bleeding. however, abrosia is required when the patients have a heavy bleeding. 2. fluid resuscitation: firstly, intravenous access should be established rapidly in patients with gastrointestinal bleeding, then, the patients were given intravenous infusion of normal saline, lactated ringer's solution, plasma, whole blood or plasma substitute [161] . 3. vaso-active agents: vaso-active agents such as dopamine and alamin may be given to maintain normal blood pressure, if blood circulation is still not stable after fluid resuscitation [162] . 4. hemostatic: if mucosal bleeding was caused by portal hypertension, oral administration of norepinephrine and ice normal saline can promote mucosal vascular contraction and hemostasis. in addition, oral administration of yunnan baiyao may be effective [163] . 5. acid suppression therapy: the proton pump inhibitors, such as omeprazole, pantoprazole and esomeprazole, are commonly used to inhibit gastric acid secretion [164] . 6. reduction of portal pressure: patients with severe hepatitis complicated by gastrointestinal bleeding often accompanied with portal hypertension, so it may be considered to give them drugs to decrease portal pressure. especially in patients with portal hypertension gastropathy, reduction of portal pressure is more important than acid suppression therapy [165] . 7. compression hemostasis via using three-chamber double-balloon catheter: after the above treatment, if there is still active bleeding in patients with bleeding esophageal varices, it can be considered to use three-chamber double-balloon catheter [166] . 8. endoscopic hemostasis: if the above treatments have no effect in upper gastrointestinal bleeding, endoscopic hemostasis, including endoscopic hemostatic agents spray, endoscopic ligation or endoscopic injection sclerotherapy may be used [167] . 9. others: surgery or interventional therapy may be considered, if internal medicine therapy is ineffective [168] . firstly, we should treat original disease, then improve coagulative function in those patients. in addition, prevention and control of infection, correction of electrolyte disturbance, avoiding the hemorrhage and reduction of allergies and transfusion reactions should be considered [169] . the key is early diagnosis and early treatment. 1. anticoagulant drugs: low molecular weight heparin is the most commonly used drug in earlier stage of dic. it is recommended to periodic test blood routine and coagulation, and dynamically observe coagulation status during medicine therapy. others such as dextran, anti-platelet aggregation ticlopidine, salvia injection, urokinase may be effective, but it should be support by more evidence-based medicine [156] . 2. plasma and blood products: during the process of dic formation, the patients should be transfused with fresh plasma, each 10-20 ml/kg. when they developed the stage of secondary fibrinolysis, prothrombin complex containing coagulation factors, cryoprecipitate and platelets can be supplemented due to a large consumption of coagulation factors [170] . 3. others: such as hemodialysis, anti-fibrinolytic 6-aminocaproic acid and tranexamic acid should be supported by more evidence-based medicine. hepatorenal syndrome (hrs) is a common and serious complication occurring in patients with decompensated cirrhosis and liver failure, who have overt circulatory dysfunction. the 1-year incidence of hrs in patients with ascites is about 20% [171] . hrs may predict a poor prognosis in spite of it being functional reversible [172, 173] . the clinical characteristics of hrs were first described in 1963 [174] . in 1979, hrs was defined by a group of international investigators as a progressive renal dysfunction that occurred in severe liver diseases, with features of prerenal failure (low urine sodium concentration and hyperosmolar urine) but without any improvement following volume expansion [175] . the international ascites club (iac) developed hrs definition in 1996, as a syndrome that occurs in patients with cirrhosis, portal hypertension and advanced liver failure, characterized by impaired renal function with marked abnormalities in the arterial circulation and activity of endogenous vasoactive systems [176] . hrs is a potentially reversible syndrome that occurs mainly in patients with liver cirrhosis, ascites and all kinds of liver failure [172] . it's clinical features include impaired renal function, marked changes in cardiovascular function and over activity of the renin-angiotensin systems and the sympathetic nervous. progressive hrs with severe renal vasoconstriction is able to cause a decrease of gfr [177] . clinically, hrs can be divided into two types (1 and 2). type-1, so-called acute hrs is a rapid progressive form of renal failure defined by doubling of the initial serum creatinine concentrations to a level higher than 220 mmol/l (2.5 mg/dl) or a 50% reduction of the first 24 h creatinine clearance to <20 ml/min within 2 weeks [178] . it appears mainly in patients with acute liver failure, but often develops after a precipitating event, such as bacterial infections (especially spontaneous bacterial peritonitis, sbp). the prognosis of type 1 is poor with the median survival about 1 month [177] [178] [179] . type-2, so-called chronic hrs is a steady or slowly progressive form of renal failure defined by serum creatinine from 133 to 226 mmol/l or from 1.5 to 2.5 mg/ dl [175] . type-2 hrs is mostly related to refractory ascites. survival of patients with type-2 hrs is generally around 6-7 months, which is better than that of patients with type-1 hrs but shorter than that of non-azotaemic cirrhotic patients with ascites. patients with type-2 hrs tend to develop type-1 hrs while infections or other trigger events occurred [171, [177] [178] [179] ]. portal hypertension is the essential factor of haemodynamic changes, which resulted from the development of cirrhosis associated with distortion, compression and even obliteration of the hepatic sinus and vessels [175] . in patients with portal hypertension, bacterial translocation is increased and intrahepatic hypercontractile stellate cells activated [180, 181] . this overall increased resistance to portal hypertensional causes increased local production of various vasodilators such as nitric oxide, leading to splanchnic vasodilation [182, 183] . there are several other factors contributing to the splanchnic vasodilation, including hyporesponsiveness of the splanchnic vessels and mesenteric vascular hyperplasia [181] . in addition, portal hypertension per se can cause renal vasoconstriction by activating sympathetic nervous. for example, when tips is used to reduce portal hypertension and improve renal blood flow, a corresponding reduction in sympathetic nervous activity has been observed [184, 185] . as a result of splanchnic vasodilation, blood is accumulated in the splanchnic vascular bed just like a splanchnic steal syndrome [186] . the combined effect leads to reduction in the effective arterial blood volume (relative hypovolemia) causing a relative inadequacy in the systemic circulation, which triggers a hyperdynamic circulation in these patients [187, 188] . vasodilatation induces activation of neurohumoral systems including the reninangiotensin-aldosterone system (rass); sympathetic nervous system (sns); and non-osmotic release of antidiuretic hormone (adh) [189] . relative hypovolemia initially causes sodium and water retention, increases intravascular volume, and simultaneously increases cardiac output. as cirrhosis progresses, vasodilatation aggravates, which activated vasoconstrictive systems, causing renal vasoconstriction and decreased renal blood flow [181, 190] . local release of potent vasodilators such as nitric oxide (no) leads to splanchnic visceral vasodilation, as well as enables the splanchnic circulation against a variety of vasopressor agents, including norepinephrine, vasopressin, angiotensin ii and endothelin [191] . the resistance of the splanchnic circulation to these vasopressor agents makes the control of arterial pressure during cirrhosis dependent on the extra-splanchnic effects produced by the endogenous vasoconstrictor systems. the role of vasoconstrictors in maintaining haemodynamic stability becomes pivotal as arterial vasodilatation increases during cirrhosis, which makes clear why cirrhotic patients with hrs are prone to develop renal, hepatic and cerebral vasoconstriction [189] . the reduction of effective arterial blood volume leads to the compensatory activation of various vasoconstrictor systems. normally, the kidneys increase the production of renal vasodilators including prostaglandins and kallikrein to maintain blood flow. however, renal vasodilator production is generally reduced in patients with cirrhosis, thus contributing to renal vasoconstriction. this type of renal hypoperfusion further increases the production of various intrarenal vasoconstrictors such as angiotensin ii and endothelin, causing further decline of renal haemodynamics and renal function, occasionally accompanied by glomerular ischaemia and mesangial constriction [178] . when blood pressure fluctuates, renal auto-regulation regulatory mechanisms initiate to make sure that the kidneys receive a relatively constant blood supply. when the critical threshold is below 65 mmhg, renal blood flow is proportional to renal perfusion pressure which, in turn, is dependent on mean arterial pressure. in cirrhosis, with the development of liver disease in patients of cirrhosis, the renal auto-regulation curve gradually shifts to the right -which means as liver disease advances, the renal blood flow gradually decreases for each given renal perfusion pressure [178] . furthermore, lumbar sympathetic blockade increases renal blood flow in patients with hrs, suggesting that the renal sympathetic activity is involved in this outgoing hepatorenal arm. insufficient cardiac output is considered one of the leading causes for renal hypoperfusion in patients with hrs in recent years. despite control of infection, the cardiac output of cirrhotic patients with sbp who developed progressive renal failure was lower than that in similar sbp patients without renal failure. similarly, when patients with non-azotaemic cirrhotic patients who developed hrs are compared with similar patients who did not, it is observed that low cardiac output and high plasma renin activity (pra) were independent predictors of hrs. in addition, in patients developing hrs, the evolvement of circulatory dysfunction leading to arterial hypotension and renal failure occurs in the setting of a continued decline in cardiac output and increase in pra. therefore, effective hypovolaemia occurs when cardiac output decreases, resulting in renal hypoperfusion and hrs [192] . to summarize, the principal mechanisms leading to renal vasoconstriction include systemic circulation changes, accompanying portal hypertension which are characterized by decreased peripheral vascular resistance with subsequent vasodilatation, hyperkinetic circulation and the activation of compensatory mechanisms, i.e., the sns, raas, and adh. with the progression of cirrhosis, the combined effective of all the above factors will result in the gradual deterioration in renal function. any event that leads to a sudden deterioration in hemodynamics can cause a rapid renal dysfunction, precipitating type 1 hrs [193] . the diagnostic criteria for hrs have been first defined by iac in 1996 [176] . the main findings include reduced glomerular filtration (creatinine clearance) less than 40 ml/min or serum creatinine increased more than 135 μmol/l after excluding the other causes of renal dysfunction. however, estimation of renal function by using creatinine clearance is not reliable, because these patients have lower levels of serum creatinine and higher renal tubular creatinine secretion compared with filtered creatinine. furthermore, it is often incomplete for the collection of a 24 h urine. iac developed the new definition and diagnostic criteria for hrs in 2005, which (1) excludes creatinine clearance because of its inaccuracy of renal function estimation and the complicity to perform; (2) includes renal failure at the time of combined bacterial infection (but absence of septic shock), indicating that hrs can be diagnosed before antibiotic treatment; (3) determines by using albumin for plasma volume expansion better than saline. (4) excludes minor diagnostic criteria (urinary indices) because of its poor sensitivity and specificity for the diagnosis [178, 189, 194] . the diagnostic criteria of hrs for patients with liver cirrhosis are as follows: 1. cirrhosis with ascites; 2. serum creatinine >133 mmol/l (1.5 mg/dl); 3. no improvement in serum creatinine (decrease to a level of ≤133 mmol/l or 1.5 mg/dl) after at least 2 days of diuretic withdrawal and volume expansion with albumin. the recommended dose of albumin is 1 g/kg body weight/day up to a maximum of 100 g/day; 4. absence of shock; 5. no current or recent treatment with nephrotoxic drugs; 6. absence of parenchymal kidney disease as indicated by proteinuria >500 mg/ day, microhematuria (>50 red blood cells/high power field) and/or abnormal renal ultrasonography [177, 194] . there are some other causes of renal failure in patients with cirrhosis that were not included, such as membranoproliferative glomerulonephritis and/or iga nephropathy in patients with chronic liver diseases. these chronic forms of kidney disease can cause acute rises in serum creatinine. determining whether it is a potential kidney disease or hrs that causes a sudden increase in serum creatinine in patients with cirrhosis and chronic kidney disease could be difficult [195] . naturally, liver transplantation is the only rational solution in cases of advanced liver disease while it is also the treatment of choice for both type-1 and type-2 hrs. as calcineurin inhibitors (ciclosporin and tacrolimus) may induced gfr impairment, it is recommended to delay their administration until a partial recovery of renal function is recorded, usually 48-72 h after transplantation [177] . clinically, the haemodynamic associated with hrs as well as neurohormonal abnormalities fade away within one month of transplantation, and the patients recover their ability to excrete sodium and free water. compared with patients without hrs, patients with hrs tend to have more complications, take more days in intensive care units and have higher in-hospital mortality rates after liver transplantation. nevertheless, their 3-year survival rate is acceptable (60% vs. 70-80% in liver transplant patients without hrs [196] . the primary limitation of liver transplantation is that most patients with type-1 hrs die before transplantation due to the shortage of donor liver and their extremely short survival time. reference to the model of end-stage liver disease (meld, including scr, tbil, inr) for organ prioritisation has partially addressed this problem, since patients with hrs have a high priority on the waiting list. in addition, the use of vasoconstrictors and albumin in the treatment of type-1 hrs can improve survival rate of these patients, and increase the likelihood of their transplantation [177] . in patients with advanced liver disease and the renal insufficiency, simultaneous liver and kidney transplant (slkt) is taking into consideration. however the reversibility of renal function in some patients when they receive slkt should be taken into account. therefore, to ensure allocation of transplants only to those truly in need, the transplant community proposed an evaluation algorithm in 2006, whose purpose is to determine the presence of kidney disease with structural damage (preferably on biopsy) before giving slkt. in the case of chronic kidney disease, a decreased creatinine clearance at 30 ml/minute or less is considered an indication of slkt. slkt should not be performed for the patients with simple hrs, but for the patients with hrs who become dialysis dependent and without any recovery after 6 to 8 weeks of dialysis [179, 189] . vasoconstrictors combined with albumin are the first line of therapy for type-1 hrs patients. it was recognized long time ago that the effective plasma volume was reduced when patients of advanced liver diseases complicated with hrs, and this led to many attempts to improve the patients' renal function by expanding their plasma volume, including a large dose of albumin or saline perfusion. with the advent of safer compounds including terlipressin, a vasopressin analogue with longer activity, and the α2-agonist midodrine combined with octreotide the analogue, vasoconstrictors is widely used in the patients with hrs. these vasoconstrictors are able to ameliorate vasodilatation while increasing effective arterial blood volume, improving renal vasoconstriction and improving renal flow. in order to further increase effective blood volume, vasoconstrictors have been used in conjunction with intravenous albumin. the clinical results from 12 uncontrolled studies including 258 patients with hrs (type-1, 240) showed that a total of 60% were observed complete response (mostly defined as a decrease in scr to 1.5 mg/dl). interestingly, once the treatment is stopped, hrs relapses only in a few "responders" [177, 189] . there were several randomized controlled trials (rcts) published, suggesting that terlipressin was associated with an increase in gfr compared with albumin alone or with a placebo. the rate of hrs reversal in the terlipressin group was higher than that in the control group (46% vs. 11%). as survival rate was not improved in the two largest rcts, liver transplantation is still the preferred treatment for hrs, but terlipressin seems to serve as a "bridging" treatment. two recent small, open-label rcts suggested that the incidence of hrs reversal and the rate of side effects showed no significant difference between the two groups of norepinephrine and terlipressin [189, 197, 198] . the initial dose of terlipressin recommended in many studies ranged from 0.5 to 1 mg per 4-6 h [199, 200] . if the creatinine level did not decrease by 25% on the third day, the dose could be increased to 2 mg every 4 h or 12 mg/days by continuous intravenous infusion, respectively. in some studies, the daily dose of albumin was generally 20-40 g by a load of 1 g/kg body weight. some mentioned central venous pressure to establish albumin doses and to prevent body fluid from overloading. this treatment was maintained until hrs is reversed, but did not exceed 2 weeks [177] . about 20% of patients relapsed after the treatment withdrawal. however, these patients should be given repeated treatment with terlipressin, which is often effective [177] . several studies have evaluated the role of transjugular intrahepatic portosystemic stent-shunt (tips) in hrs. these studies show that tips help decreasing in scr in most patients, even in a minority of organic renal failure, but it is slower compared to those obtained using terlipressin combined with albumin [201] . recrudescence of hrs is rare provided the shunt remains patent, while hepatic encephalopathy often comes. it is worth noting that the vast majority of patients included in these studies suffered from alcoholic cirrhosis, many of whom have active alcoholism, and therefore the improvement observed may be caused by the improvement in an acute-on-chronic process. in addition, since all these studies excluded patients with a child-pugh score ≥ 12, resulting in a lack of data, the efficacy of tips should be further explored in rcts [189] . extracorporeal albumin dialysis molecular adsorbent recirculation system (mars) is designed for making clearance of water-soluble cytokines (i.e., il-6) and albumin-bound toxins (i.e., bile acids) which is implicated in the pathogenesis of hrs. two studies showed that mars was ineffective in improving survival rate and emic haemodynamics in type-1 hrs [193, 202] . another clinical observation including 32 patients with type-1 hrs reported a rate of complete renal response of 28% [201] . extracorporeal albumin dialysis (ecad) reduces serum creatinine levels, but it is not clear whether this effect is due to a real improvement of renal function or merely a filtration process. several studies demonstrated that patients' systemic haemodynamics improved after ecad, manifested as an increase in systemic vascular resistances and arterial pressure, as well as a decrease in cardiac output, pra and levels of norepinephrine. however, there were too few studies on the effect of ecad on survival in type-1 hrs patients to draw any definitive conclusions [203, 204] . in addition, ecad is very expensive and therefore not suitable for wide and rapid clinical application [177] . the treatment of type-2 hrs should take into account the survival rate as well as controlling the ascites. both hrs-1 and hrs-2 are indications for the tips treatment. the therapeutic effect of tips is excellent for its better controlled of complications of portal hypertension compared with other treatments. tips have been reported not only to improve renal function in patients with type-1 hrs but also to treat refractory ascites in patients with type-2 hrs [205] [206] [207] . the contraindications to the creation of tips are shown in the followings [195] . • contraindications to placement of a tips: there were only a few studies evaluated the role of tips in type-2 hrs and the number of cases was quite low. in most patients, tips could decrease scr, even in some with organic renal failure [208] . hrs recurrence is rare as long as the shunt remains patent, but hepatic encephalopathy often occurs [189] . nine patients were followed-up for 1 month after the treatment of tips in a study, eight cases were found with decreased scr decreased and notably controlled ascites. four patients died, two of them died within 1 month, the other two died at 12 months and 14 months respectively. the remaining five patients survived for a long time. although tips can be used in improving refractory ascites which often contributes to type-2 hrs, data on the effect of tips on survival are still insufficient. therefore, the efficacy of tips should be further explored in randomized controlled trials (rcts) [189] . the information about combining albumin and vasoconstrictive agents treated in type-2 hrs is limited. only a few patients with type-2 hrs have been specifically treated with terlipressin and albumin. in one clinical study, 39 patients with hrs-2 were assigned to receive this treatment and 21 of them achieved improvement of renal function. however after the treatment withdrawal, 11 hrs patients showed relapsed during the follow-up. the most common side effects during terlipressin therapy are cardiovascular and ischemic and reported as an incidence of nearly 12%. the high recurrence rate of hrs after terlipressin and albumin treatment discontinuation suggests that they are less effective in treating type-2 hrs compared to type-1hrs [209] . prevention of hrs is important because it develops at a constant frequency in cases of spontaneous peritonitis (sbp) and advanced liver disease [210, 211] . it becomes possible to prevent hrs if sbp is diagnosed and treated promptly [212] . according to current data, using albumin in combination with antibiotics for the treatment of patients with sbp seems to be warranted but only for those with jaundice or renal dysfunction. the prophylactic use of antibiotics in cirrhosis with gastrointestinal bleeding also seems to be necessary, because the use of antibiotics contributes to reducing incidence of infection and rebleeding whereas improving survival rate. furthermore, the incidence of hrs in sbp patients decreases by albumin administration, and prevention of hrs can also be related to increased survival. the recommended dose of albumin is 1.5 g/kg body weight on the first day then 1 g/kg body weight on the third day, a maximum of 150 g and 100 g, respectively. albumin administration is strongly recommended in sbp patients with serum bilirubin levels higher than 68.4 mmol/l (4 mg/dl) or serum creatinine more than 88.4 mmol/l (1 mg/dl). a placebo-controlled rct that enrolled the patients with low (<1.5 g/l) ascites protein who also had advanced liver diseases or "renal dysfunction"(defined as scr ≥ 1.2 mg/dl or blood urea nitrogen≥25 mg/dl, or serum sodium level ≤ 130 meq/l) suggested that oral norfloxacin contributed to a reduced hrs incidence within 1 year (28% vs. 41%) and an improvement in survival at the end of 3 months [213] . norfloxacin may ameliorate or prevent vasodilatation by reducing bacterial translocation and overt infections, as well as suppressing plasma renin activity, thereby prevent these patients from developing hrs. the concept that the severity of the clinical course of patients with cirrhosis complicated with serious bacterial infection is related to the degree of an impairment of circulatory function, which has led to new and effective approaches in the prevention and treatment of these complications. in patients with severe hepatitis, multiple causes may lead to disorders of internal environment, mostly manifesting fluid and electrolyte imbalance as well as acidbase imbalance, usually resulting in deterioration, greater complexity and even death. accurately recognizing the occurrence of severe hepatitis with complications such as fluid and electrolyte imbalance and/or acid-base imbalance, and therefore giving appropriate treatment to maintain balance of internal environment, is of great importance for improving prognosis of the patients [214] . water is the major component of human body. electrolytes are substances that dissociate in solution to form charged particles, orions. body fluid comprise mainly of water and electrolytes and electrolytes comprise mainly of na + , k + , ca 2+ , mg 2+ , cl − , hco 3 − , hpo 4 2− and so 4 2− . the primary function of electrolyte include: (1) to maintain osmotic pressure and acid-base balance of body fluids; (2) maintain nerve, muscle, cardiac cells resting potential, involved in the formation of action potentials; (3) involved in metabolism and physiological activities [215] . body fluid include intracellular fluid and extracellular fluid, the latter can be divided into plasma and interstitial fluid. intracellular and extracellular fluid differ in ion components. na + is major cation in extracellular fluid and its main anions are cl − and hco 3 − . k + is major cation in intracellular fluid and its major anion is hpo 4 2− . the total number of ions in body fluids is called osmolality, its unit is mosm/l. if the osmolality on both sides of a semipermeable membrane is not equal, water moves toward the side with the higher osmolality. this phenomenon is called osmosis [216] . osmosis of water can be opposed by applying a pressure across the semipermeable membrane in the direction opposite to that of the osmosis. the amount of pressure required to oppose the osmosis is defined to be osmotic pressure. in spite of various solute concentrations are different in extracellular and intracellular fluid, the osmotic pressure remain equal. normal plasma osmotic pressure is 280-310 mosm/l. steady osmotic pressure is the basic guarantee to maintain the fluid balance across the cell membrane. multiple mechanisms in nervous and hormonal system are involved in the regulation of body fluid and electrolyte balance [217] . (1) there exists sensation of thirst in central nervous system, which plays an important role in regulating body water. (2) there is a powerful feedback system for regulating plasma osmolarity and sodium concentration that operates by altering renal excretion of water independently of the rate of solute excretion. a primary effector of this feedback is called antidiuretic hormone (adh) which plays an important role in regulation of renal concentration and dilution to maintain the body fluid homeostasis. (3) reninangiotensin-aldosterone system (raas) is an important regulator of sodium reabsorption and potassium secretion by the renal tubules. human body fluid environment must be suitable ph value for maintaining normal metabolism and physiological function, under normal conditions, human body take in acidic and basic food and drinking water, produce acids and bases during metabolism and eliminate acidic or basic substances by the kidneys and lungs. in the plasma, the normal ph value ranges from 7.35 to 7.45 with an average value at 7.40. the regulation of the acid-base balance is accomplished by the buffer system of the body fluid, the respiration of the lungs and the excretion of the kidney [218] . (1) blood buffer system is composed by a weak acid and its corresponding buffer base, includes bicarbonate buffer system, phosphate buffer system, plasma protein buffer system, hemoglobin and oxygen synthetic hemoglobin buffer system. (2) the role of the lung in acid-base balance is to adjust the concentration of plasma carbonic acid by changing the amount of co 2 , so that the ratio of hco 3 − and h 2 co 3 in the plasma is close to normal, so that the ph is relatively constant. (3) the major role of the kidneys in maintaining acid-base balance is to conserve circulating stores of bicarbonate and to excrete h + . the kidneys maintain ph by increasing urinary excretion of h′ and conserving plasma hco 3 − when the blood is too acidic, or increasing urinary excretion of hco 3 − , and decreasing urinary excretion of h + when the blood is too alkaline. patients with severe hepatitis are prone to develop water retention, with the main manifestation of seroperitoneum (ascites) as well as body weight gain. with the acatharsia of water becoming more serious, oliguria and edema of lower extremities occur. sbp (spontaneous bacterial peritonitis) can also occur, which is manifested with symptoms such as fever and abdominalgia. several factors contribute to ascites include an increase in capillary pressure due to portal hypertension, obstruction of venous and lymph flow through the liver, decrease in colloidal osmotic pressure due to impaired synthesis of albumin by the liver, salt and water retention by the kidney [219] . some theories have been used to explain the increased salt and water retention by the kidney. because of vasodilation or an actual loss of fluid into the peritoneal cavity, the effective blood volume maybe reduced, which may in turn decrease the renal blood flow leading to a lower glomerular filtration rate (gfr) and an activated rennin-angiotensin-aldosterone system (raas). the diagnosis of water retention depends on typical clinical symptoms such as ascites, pleural effusion, and edema of lower extremities, when the body begins to excess water, blood pressure is increased, which leads to many complications such as congestive heart failure and pulmonary edema. hyponatremia is a common complication in severe hepatitis patients, and always incorporate with the retention of water and sodium, but the total sodium can be decreased, normal or even increased, that is dilutional hyponatremia. in laboratory test, serum sodium is below 135.0 mmol/l. the mechanism of hyponatremia probably depends on the following factors: (1) the decreased function of adh inactivation in liver brings adh increase, enhancing the reabsorption of water in renal tubule, which causes the formation of water retention. this is the main cause of dilutional hyponatremia. (2) water retention brings about volume extension, causing the aldosterone secretion decrease, which leads to the sodium egestion increase in urine. (3) some severe hepatitis patients frequently vomit, and can not eat, bringing about a major loss of body fluid and electrolyte. (4) severe hepatitis patients, serum albumin reduces, combining with the factors such as poor appetite, anorexia, fasting or limit sodium, bring about a state of low permeability in the cell, which causes the extracellular na + moving into the cell. (5) iatrogenic factors, exhaust potassium diuretic such as hydrochlorothiazide and furosemide and spironolactone have a strong role in the excretion of sodium, so a large number diuretic is liable to hyponatremia in ascites patients, and in the treatment of cerebral edema, a large infusion of mannitol may cause hyponatremia either [220] . hyponatremia due to the osmotic pressure of extracellular fluid decreased, water moves to the cells, causing cell edema, especially brain edema. so the symptoms of nerve system are the main manifestation in hyponatremia patients [221] . generally dilutional hyponatremia in severe hepatitis patients develops slowly and progressively, and the symptoms are often covered by primary disease symptoms, like weak, feeble, nausea, vomiting, lethargy, significant body weight increase, pale and moist skin and sometimes saliva, tears increase. improper treatment will bring about a sharp serum sodium decrease in the short term, such as serum sodium rapidly decreasing to below 125 mmol/l, acute hyponatremia syndrome comes, the manifestation include convulsions, coma, hypotension, pulse narrowing, tachycardia, oliguria even respiratory arrest and death. if cerebral hernia happens, corresponding nerve location signs will follow. hypokalemia refers to the condition in which the concentration of potassium (k + ) in serum is less than 3.5 mmol/l. it could occur during the whole period in severe hepatitis patients and is more common in early metaphase of disease. hypokalemia can be the result from one or more following medical conditions: (1) insufficient intake of potassium due to the poor appetite or anorexia in the patients with severe hepatitis. (2) frequent vomiting leads to excessive loss of stomach acid, which causes alkalosis and extracellular potassium is transferred into cells. (3) the reduce of effective circulating blood volume can cause to high aldosterone levels and excessive urinary losses of potassium. (4) the decreased function of aldosterone inactivation in liver brings aldosterone increase, enhancing urinary losses of potassium. (5) some medications such as diuretics can also cause urinary losses of potassium. the clinical syndromes of hypokalemia are related to the degree of the shortage of intra/extracellular potassium and disorders of other electrolytes and acid-base, but more depends on how soon it occurs. in the early time it shows muscle weakness, first in limbs, then develops to the torso and respiratory muscle. deficiency of potassium also can lead to weak peristalsis, poor appetite, sick and constipation in mild hypokalemia but abdominal distention and paralytic ileus in severe situation. in cardiac syndromes, it mainly presents atrioventricular block and arrhythmia which including premature ventricular contraction or atrial premature beats, sinus bradycardia, paroxysmal auricular tachycardia or junctional tachycardia, even ventricular fibrillation. in hypokalemia state an increasing shift of potassium from extracellular fluids into cells and an obligate loss of potassium from kidney can cause metabolic alkalosis and abnormal acidic urine. long-term hypokalemia also can lead to hypokalemic nephropathy with proteinuria and cylindruria syndrome. the changes of ecg [222] : in the early stage, flattened t wave and an obvious u wave, st-segment depression can be found, qu interval is widen. in severe situation, a wide pr interval, low voltage, wide qrs interval and ventricular arrhythmia can occur. hyperkalemia refers to the condition in which the concentration of potassium (k + ) in serum is higher than 5.5 mmol/l. it is more common in the middle and late period of severe hepatitis. the mechanism of hyperkalemia: (1) the most usual way lead to hyperkalemia is oliguria or uroschesis which are caused by renal dysfunction among the patients have severe hepatitis with hepatorenal syndrome. (2) metabolic acidosis and na + -k + -atp enzyme inactive lead to a shift of potassium out of cells also contribute to develop hyperkalemia. (3) long-term and high dose potassium-sparing diuretics applied during the treatment lead to hyperkalemia is not rare, and easy to get sudden death in patients. hyperkalemia mainly influences myocardium and skeleton muscle. the most dangerous situation is fatal arrhythmia. when the concentration of k + is higher than 5.5-6.5 mmol/l, there are peaked t waves. when it is over 7-8 mmol/l, pr interval is widen and p wave is flattened even vanish. when it is up to 9-10 mmol/l, t waves and qrs complex can evolve to sinusoidal shape and cardiac arrest. hyperkalemia is a common critical and severe symptom in clinic. when it happens, all of the potassium-sparing diuretics and potassium uptake should be stopped. at meantime, the treatment against the toxicities to myocardium and skeleton muscle should be taken to accelerate a shift into cells and potassium excreting. there also can happen hypocalcemia, which shows neuromuscular excitability, cardiac electrical instability and instable emotion. the concentration of calcium in serum under 2 mmol/l is significant in diagnosis. hypomagnesemia can be found too. it mainly presents similar symptoms as hypocalcemia such as weakness, muscle cramps, increased irritability, tetany and chvostek positive. hypomagnesemia can cause cardiac arrhythmia, when it occurs, the concentration of magnesium is less than 0.75 mmol/l, and it should be urgently treated. severe hepatitis patients prone to acid-base imbalance [223] , mainly to alkalemia. the main types of acid-base imbalance include respiratory alkalosis, metabolic alkalosis, respiratory alkalosis plus metabolic alkalosis, secondly include respiratory alkalosis plus metabolic acidosis, triple acid-base disorders (tabd), metabolic alkalosis plus metabolic acidosis [224] . history and clinical manifestations provides important clues for the judgment of acid-base imbalance. the result of blood gas monitoring is the decisive basis for judging the type of acid-base imbalance. serum electrolyte examination is an important reference. anion gap (ag) has important diagnostic value in determining the type of acidbase imbalance [225] . ag = na + -(cl − + hco 3 − ) is a simple formula for the value between the number of cations and anions in serum. its normal value was 12 ± 4 mmol/l. ag can not only help diagnose "potential" metabolic acidosis and to distinguish different types of metabolic acidosis, can also help determine special types of mixed acidosis, and has its unique role in the judgment of tabd. sometimes the indicators of blood gas analysis are normal, the calculation of ag value become the only evidence of diagnosis of metabolic acidosis. in addition, ag value can be used as reference for correction of acid-base imbalance. in severe hepatitis, the change of ag values can be used as an indicator to estimate the complications and prognosis. clinical observations indicate: ag value significantly increased often suggestive of severe infection, kidney dysfunction or severe bleeding, and the prognosis is poor. respiratory alkalosis refers to arterial paco 2 decrease and ph > 7.45 as well as compensatory decrease of blood hco 3 − . respiratory alkalosis occurred in early stage of severe hepatitis. in severe hepatitis, respiratory alkalosis related to hyperventilation: accumulated ammonia and other vasoactive peptides excited respiratory center, ascites and pleural fluid increase respiratory rate, hypoxemia excited respiratory center. compensatory mechanisms: co 2 reduction, breathing shallow and slow, so that co 2 retention, h 2 co 3 compensatory rise; when last longer, reduce renal row h + , hco 3 − excretion increased, hco 3 − /h 2 co 3 equilibrium at a low level. most patients have the performance of shortness of breath and heart rate increase. can have vertigo, hand, foot and mouth numbness, muscle tremor, hand and foot convulsions. convulsions associated with low calcium. dysfunction of nervous system is related to the damage of brain function and cerebral blood flow decrease. respiratory alkalosis diagnosis relies on the following: (1) ph is normal when fully compensated, increased underdecompensation. (2) paco 2 lower (typically <35 mmhg or 4.67kpa). (3) hco 3 − compensatory decline. (4) ag value may have a slight increase. (5) blood cl − may increase. metabolic alkalosis refers to the type of acid-base imbalance characterized by an increase hco 3 − in extracellular liquid. the inappropriate application of basic drugs, potassium-sparing diuretics, dehydrating agents, hormones can often induce or aggravate metabolic alkalosis. severe gastrointestinal symptoms, anorexia, vomiting or diarrhea are also the reasons for the occurrence of metabolic alkalosis. compensatory mechanisms: when alkaline substances increased in body, buffer system instantly transfer strong base into weak base, increase hco 3 − consumption, h 2 co 3 increased. inhibit respiratory center, decrease pulmonary ventilation, co 2 retention, hco 3 − compensatory increase. renal carbonic anhydrase activity decreased and h + formation and excretion decreased, nahco 3 reabsorption is also reduced, so hco 3 − /h 2 co 3 compensatory restore to 20:1, ph value is normal. patients with mild metabolic alkalosis usually have no obvious symptoms. many disorders can occur in severe metabolic alkalosis. (1) functional changes in the central nervous system, the patient may have irritability, confusion, delirium, consciousness disorders. (2) slow and shallow breathing, hypoxemia. brain tissue is particularly sensitive to hypoxia, thus neurological symptoms first appeared. (3) hypocalcemia and neuromuscular stress increased, the performance of tendon hyperreflexia, face and muscle twitching, and limbs twitching. (4) hypokalemia can cause neuromuscular symptoms and arrhythmias. according to ph value, paco 2, hco 3 − , level of k + and cl − , effective circulating blood volume and performance of primary disease, diagnosis of metabolic alkalosis is no difficult to make. metabolic alkalosis should be divided into two categories based on the urinary level of cl − . (1) chloride positive metabolic alkalosis: supplement sodium chloride can correct the alkalosis. it indicates that the body has cl − deficiency, urinary cl − < 10 mmol/l. (2) chlorine negative metabolic alkalosis: alkalosis can not be corrected by supplement sodium chloride, urinary cl − >20 mmol/l. respiratory alkalosis plus metabolic alkalosis tend to occur on the early stage of severe hepatitis. in most cases, there is no obvious complication, more often metabolic alkalosis happens on the basis of respiratory alkalosis, or the other way around. due to respiratory and metabolic factors are inclined to alkaline change, name as reduce paco 2 and elevated plasma hco 3 − , there is no mutual compensation between them, so it is easy to present as severe decompensation and poor prognosis. main point of diagnosis: (1) ph value of blood increase significantly. (2) paco 2 decrease. (3) hco 3 − increases, the value should be greater than 0.5×(40-paco 2 ) + 2.5. (4) hypokalemia and hypochloremia are common phenomenon. respiratory alkalosis plus metabolic acidosis is relatively rare. metabolic acidosis can be divided into 3 types: (1) value of ag is normal (high chlorine acidosis), commonly seen at long-term diarrhea, combined with renal tubular acidosis and a large amount of physiological saline input in patients or water intoxication. (2) high ag value type (normal blood chlorine acidosis), regularly present in combination of hepatorenal syndrome, lactic acidosis and patients with ketoacidosis. (3) a hybrid type (high ag merged high blood chlorine), mainly in patients with severe diarrhea following lactic acid or ketoacidosis. when respiratory alkalosis plus metabolic acidosis happens, paco 2 and plasma concentration of hco 3 − are higher than scope of compensation to each other. its characteristics as follows: (1) the range of blood ph change is not large, normal, slightly higher or slightly lower. (2) paco 2 reduce to less than 1.5 × hco 3 − + 6 or hco 3 − < 24-(40-paco 2 ) × 0.5-2.5. (3) ag values can be normal or elevated, the latter is more common. if the elevated blood cl − value is equal to the hco 3 − decrease, ag value normal metabolic acidosis type can be diagnosed; the cases that the rising value of ag is equal to the decline of hco 3 − values can be diagnosis as ag increased metabolic acidosis type. on the third occasion, the increase value of ag is equal to the sum of hco 3 − and cl − drop, the diagnosis is mixed metabolic acidosis. for this type of offset mixed acid-base balance disorders, treatment should be moderate, the measure of the metabolic factors correcting should be precede to respiratory factors, avoid paco 2 quickly returning to normal in the process of treatment, which would lead to blood ph drop rapidly and acidosis more worse. to patients with severe hepatitis, the harm of alkalosis is greater than acidosis, thus the blood ph should be kept slight acidic in a normal state. generally, the target of alkali supplement can be arterial blood ph value recovered to 7.20. respiratory alkalosis tabd refers to respiratory alkalosis, metabolic acidosis and metabolic alkalosis three primary imbalances coexist in the same patient, which is one sort of serious acid-base imbalance, mostly develops in the late stages of severe hepatitis, fatality rate is high. respiratory alkalosis tabd characteristics as follows: (1) blood ph value depends on the relative severity of these three primary imbalances, which can be normal, or slightly high generally. (2) reduce paco 2 , its value is less than 1.5xhco 3 − + 6. (3) hco 3 − can raise, normal or lower. (4) value of ag rise significant, and extent of ag raise is greater than the hco 3 − lower. (5) cl − often lower than normal. the occurrence of metabolic alkalosis plus metabolic acidosis in patients with severe hepatitis is not uncommon. usually it is accompanied with existing lactic acidosis or ketoacidosis, and the patient may manifest frequent vomiting. since the causes for raising and lowering hco 3 − coexist, they tend to cancel one another. the ph and hco 3 − concentration can be normal, increased or decreased, depending on the relative severity of the two kinds of imbalances. severe hepatitis can also be accompanied by metabolic acidosis, metabolic acidosis plus respiratory acidosis, tabd of respiratory acidosis, and etc. pure metabolic acidosis refers to arterial blood ph < 7.35 and compensatory decline of paco 2 due to primary decrease of hco 3 − . typical manifestation is known as kussmaul breathing, characterized by deeper and faster breathing, as well as obvious contraction of respiratory muscle, and also ketone-smelled exhaled breath. the patients often flush, companied by increased heart rate and decreased blood pressure. there may be reduced or disappeared tendon reflexes, confusion or stupor. due to respiratory and metabolic factors both towards to acidic changes, there is no respiratory compensation for decrease in hco 3 − , nor renal compensation for increase in paco 2 , hence presenting severe decompensated status. the resulting distinct decrease in ph and vicious circle are the characteristics for metabolic acidosis plus respiratory acidosis. the characteristics for tabd of respiratory acidosis include significantly increased paco 2 , elevated hco 3 − , ag > 16 mmol/l, and significantly decreased cl − . the incidence of the above types of acid-base imbalance is very low in patients with severe hepatitis. once it happens, it should be actively treated with corresponding methods, so that the blood ph quickly restores to the safety range. during therapeutic process against various pure acid-base imbalance, interactions among various treatments need to be taken into consideration, in order to avoiding the possibility that the treatment for one type of acid-base imbalance causes or aggravates another type. in summary, water-electrolyte imbalance and acid-base imbalance have a relatively high morbidity in patients at various stages of severe hepatitis. this often results in deterioration and complication of the disease, evermore, the death of the patient. therefore, the functions of heart, lung, kidney, blood circulation as well as changes of body weight in the patient need to be intently monitored. regular detection of k + , na + , cl − , carbon dioxide combining power (co 2 cp), blood urea nitrogen, creatinine, ph, data about arterial blood gas analysis, and also detailed records of patient's input and output are demanded. during the process of diagnosis and treatment, careful analysis of the history, clinical manifestations and laboratory examination are necessary to achieve correct diagnosis, early prevention and prompt treatment. in normal condition, proper amount fluids can make a lubrication action on organs in peritoneal cavity. but in those patients who have severe hepatitis, especially with cirrhotic portal hypertension, too many fluids over 200 ml can lead to ascites. the ascites can be categorized into uncomplicated ascites and refractory ascites. there is no infection in uncomplicated ascites and won't lead to hepatorenal syndrome, but refractory ascites is in the contrast. the refractory ascites includes diureticresistant and diuretic-intractable ascites. the diuretic-resistant ascites shows no response to diuretic and diuretic-intractable ascites limits the application of diuretic due to the complications induced by diuretic. 100 mg antisterone per day as an initial dose can be given to those patients with moderate ascites, if it goes to no satisfied effect, 100 mg can be added after every 7 days till the maximum dose to 400 mg/d. if the patients show a hyperkalemia or aldosterone antagonist-resistant, nicorol can be combined and with an increasing dose from 40 to 160 mg/d gradually. the patients without edema losing weight should be less than 0.5 kg and those with edema should be less than 1 kg per day to avoid electrolyte disturbance or hepatorenal syndrome during the whole treatment period. diuretics should be withdrawn on the patients with severe hepatic encephalopathy, severe hyponatremia, progressive renal failure or severe muscle spasm. the patients should only take the minimum dose of diuretics to maintain the state after the syndrome controlled, or withdraw when it is necessary. due to the poor outcome and living quality, the median survival time of refractory patient is half year. so liver transplantation can be considered in the patients with refractory ascites induced by cirrhosis, which required more cautious before make a diagnosis. generally, if the patients meet the following conditions when they are receiving 400 mg/days antisterone and 160 mg/days nicorol treatment over 1 week and sodium uptake limited in 90 mmol/days, refractory ascites can be diagnosed. (1) losing weight less than 0.8 kg/days over 4 days (2) sodium uptake is more than elimination (3) grade 2-3 ascites is arisen again after 4-week long treatment (4) hepatic encephalopathy, hepatorenal syndrome and severe electrolyte disorder induced by diuretics are shown up. refractory ascites patients without complications can be treated with abdominocentesis, but it will possibly induce circulation failure, and increases the risk of hepatic coma and hemorrhage. so low rate infusion of albumin with abdominocentesis is combined to avoid circulation failure, meantime diuretics also need to give after abdominocentesis. aldosterone antagonist combined with nicorol is a suitable strategy: the dosage of antisterone is increased from 100 to 400 mg/days and nicorol from 40 to 160 mg/days gradually. the goal of this strategy is to maintain the situation without ascites under the minimum dosage. but when severe hepatic encephalopathy and electrolyte disorder show up, which means serum sodium concentration is less than 120 mmol/l, serum potassium concentration is less than 3.0 mmol/l, nicorol should be withdrawn, if serum potassium concentration is more than 6.0 mmol/l, antisterone should be withdrawn. to those patients who need abdominocentesis repeatedly, transjugular intrahepatic portosystemic shunt (tips) can be considered, but the risk of hepatic encephalopathy will be higher and the outcome is poor. so the patients with severe liver and renal failure, cardiorespiratory function failure or active infection should be in cautious. the mean survival time of refractory ascites patients complicated with hepatorenal syndrome is 3 months, preventive antibiotics combined with albumin is an option for these patients. to those patients who have already showed hepatorenal syndrome, terlipressin combined with albumin could be useful. meantime, abdominocentesis, tips or artificial liver supporting treatment can improve patients' living quality in short term, but long-term outcome won't be good, so liver transplantation should be execute as soon as possible. in general, medicine can improve the symptoms in short term but with poor outcome, liver transplantation is more meaningful. patients with hypovolemic hyponatremia can have a supplement with sodium and decrease the dosage of diuretics, patients with hypervolemic hyponatremia can restrict fluids uptake (less than 1000 ml/d) and combined with vasopressin v2 receptor blocker or antidiuretic hormone receptor blocker. currently, vaptans, tolvaptans, conivaptan and satavaptan have already applied in clinical practice. there was research showed that vaptans could improve 45-82% patients' symptoms significantly after patients took it for 1 week to 1 month, and main side reaction was thirsty. the patients complicated with hepatic encephalopathy should be used vaptans with cautious due to its high risk in dehydration and hypernatremia. meantime, vaptans is metabolized by cyp4a, so rifampin, barbital and phenytoin can decrease its effect, and ketoconazole, clarithromycin can increase its plasma concentration. tolvaptans can give some relief but increase the risk of hemorrhage. satavaptan will decrease patients' survival rate. so the proper treat period and side reactions of these drugs in long-term using need to make clear. potassium uptake should be withdrawn immediately after hyperkalemia occurs, emergency treatment for detoxicating potassium should be taken to protect cardiac. the treatment depends on the plasma concentration of potassium. the treatment for patients with fulminant hepatitis b with cirrhosis complicated with hyperkalemia is to restrict the uptake of potassium, improve the microcirculation, correct renal filtration decrease induced by hepatorenal syndrome and increase the elimination of potassium. there are also some patients have abnormal distribution of potassium due to hypoxia, acidosis, catabolism, and deficiency of energy supplies, which leads to intracellular potassium is transferred into extracellular. the treatment for these patients is to correct hypoxia and acidosis, high glucose, insulin and atp are administered to boost glycogen synthesis to transfer potassium from extracellular to intracellular. peritoneal dialysis and plasmapheresis can be given to the patients with intractable hyperkalemia. hypokalemia can present during the whole period of fulminant hepatitis b, it will occur more often on the early and middle stage. long-term inappetency and abdominal distension lead to insufficient potassium uptake. nausea, vomiting and diarrhea lead to increase of potassium losing. renal filtration rate decreasing and aldosterone increasing lead to potassium elimination increase. complicated with alkalosis and anabolism increase also can cause hypokalemia. the treatment for hypokalemia should focus on comprehensive therapy, correcting alkalosis, increasing potassium supply, improving microcirculation. disturbance of acid-base balance occurs quit often in hepatitis b patients, especially with alkalosis. during the early stage, it can present in pure respiratory alkalosis, also can complicated with metabolic alkalosis. during the middle and late stage, both of above symptoms and metabolic acidosis occurs concurrently. treating idiopathy and correcting hyperventilation is a treatment for respiratory alkalosis. arginine hydrochloride injection is used to treat metabolic alkalosis to avoid secondary metabolic alkalosis. the general regulation is prefer acid to base, till ph value of arterial blood back to 7.2. in prevention of disturbance of acid-base balance, the effective strategy is to correct hypokalemia and hypochloremia, control vomiting. meantime, controlling infection, endotoxemia and upper gastrointestinal hemorrhage are necessary. hepatic encephalopathy (he) due to metabolic disturbance is a complex neuropsychiatric syndrome caused by severe liver dysfunction or disorder and is one of the common complications and causes of death in severe liver diseases. patients with he mainly present with neuronal or mental abnormalities and disturbance of consciousness, even coma and death. the clinical manifestations and the severity of the disease vary because of its complex pathogenesis. hepatic encephalopathy is the result of acute and chronic hepatic failure caused by cirrhosis or various kinds of portosystemic shunt (pss) created. a diagnosis of he can be made after excluding encephalon diseases. the syndrome is caused by metabolic disorders and is potentially reversible. he clinical features differ due to the wide degree and range of neuropsychiatric symptoms that vary from subtle abnormalities detected only by intelligence tests or electrophysiological methods geared for detecting personality changes to abnormal behavior, intellectual impairment, and even different degrees of consciousness disorders. he was previously known as hepatic coma, but that is only one of the worst severe signs of he and does not represent all types of he. in 2003, the world congress of gastroenterology (wcog) suggested that based on the cause he can be divided into three types (a, b, and c) [226, 227] . type a: type a is acute liver failure-related he and the symptoms occur within 2 weeks. in subacute liver failure-related he, the symptoms of he occur within 2-12 weeks with or without predisposing factors. type b: patients with type b he have obvious pss and normal liver histology without associated intrinsic liver disease. these clinical manifestations are similar to those in patients with he and cirrhosis. the pss may be spontaneous or caused by surgical or interventional procedures [228] . common causes of pss include congenital vascular malformation, intrahepatic or extrahepatic portal vein obstruction (including trauma, carcinoid, and bone marrow hyperplastic disease caused by a high coagulation state due to portal vein branch embolization and thrombosis) and generation of portal hypertension by oppression of lymphoma, metastatic tumors, and bile duct carcinoma. type c: type c he is related to chronic liver diseases, with cirrhosis being the most common type, and is generally accompanied by portal hypertension and pss. type c he is mainly caused by liver function failure, rather than by pss. according to the clinical manifestations, duration and characteristics, type c can be divided into three types: episodic he, persistent he, and minimal he [226] . episodic he, related to chronic hepatic disease, is defined as a disturbance of consciousness and cognitive change in a short time and can be alleviated by spontaneous remission or drug treatment in the short term, which cannot be explained by a relevant preexisting mental disorder. episodic he can be divided into three types according to the presence of known risk factors: (1) incentive type: there is a clear history of predisposing factors; (2) spontaneous type: there is no history of predisposing factors. (3) recurrent type: he attacks more than two times within a year. persistent he related to chronic hepatic disease is defined as an occurrence of continuous neural mental abnormality, including cognitive decline, disturbance of consciousness, coma and even death. persistent he can be further divided into three types according to the severity of the disturbance in the patient's self-control and self-discipline: 1, mildest type, namely west haven level 1; 2, severe type: namely west haven level 2-4; and 3, therapeutic resistance type: medication can alleviate he quickly, but withdrawal can aggravate he rapidly. patients with minimal he, with normal clinical manifestations and routine biochemical tests, have mild cognitive and psychomotor deficits detected by neuropsychology and neural physiology tests, and these patients usually have a history of chronic hepatic disease [229] . the prevalence of minimal he in patients with cirrhosis is 30-80%. patients with minimal he with reduced physical and mental ability have gained more and more attention recently because they have a high risk of accidents when engaged in occupations involving mechanical, or driving work. the pathogenesis of he has not been fully elucidated so far, and many theories have been put forward. it is generally believed that he is caused by acute and chronic liver failure and/or pss. when toxic substances absorbed by the intestines cannot be detoxified and cleared by (or through) the liver, they directly enter into the systemic circulation and pass through the blood-brain barrier to reach the brain tissue and cause central nervous system dysfunction. a variety of the risk factors mentioned above can result in he. hyperammonemia is still recognized as one of the most important factors, especially in he related to chronic liver disease, liver cirrhosis and/or pss. according to the ammonia intoxication theory several factors including false neurotransmitters, such as γ-aminobutyric acid/benzodiazepine (gaba/bz) receptor complex, an imbalance in the ratio of branched chain amino acids to aromatic amino acids, brain cell edema, astrocyte dysfunction, mercaptan, short chain fatty acid toxicity and manganese deposition are all involved in the occurrence of he [230] . ammonia intoxication caused by an ammonia metabolism disorder is the most important factor in the pathogenesis of he [231] . ammonia comes mainly from the gut and the generation and absorption of ammonia increase in a serious liver disease when excess ammonia cannot be cleared sufficiently by ornithine cycle due to serious damage to liver parenchyma. when pss occurs, intestinal ammonia directly enters the systemic circulation without liver detoxification, resulting in increased blood ammonia. high levels of blood ammonia can enter the brain through the blood-brain barrier and generate central nervous system toxicity by interfering with cerebral energy metabolism, neurotransmitter and nerve cell membrane ion transport; increasing cerebral edema; and changing gene expression (such as stellate cell glutamate carrier, stellate cell structural protein, glial fibrillary acidic protein, peripheral benzodiazepine receptor and aquaporin-4) and inducing the mitochondrial permeability transition (mpt). the main way of removing ammonia from the brain is through urea cycle. during glutamine synthesis, glutamic acid is formed from ammonia and α-ketoglutaric acid and the glutamic acid combines with ammonia to generate glutamine. this process requires atp and consumes a large amount of α-ketoglutaric acid, which interferes with the brain energy metabolism and causes an energy supply shortage in brain cells. glutamate is an important excitatory neurotransmitter in the brain, and lack of glutamate increases inhibition in the brain. glutamine synthetase is present in astrocytes, where glutamic acid is detoxified to glutamine. glutamine is a strong intracellular osmotic agent, and increases in glutamine can lead to brain cell swelling. reports have identified a strong correlation between the content of glutamine in cerebrospinal fluid (csf) and the degree of he [232] . during he, excess ammonia under the effect of glutamine synthetase, not only reduces the formation of active glutamate but also consumes a lot of energy, leading to the accumulation of glutamine, which increases intracellular osmotic pressure and causes brain cell swelling. swollen astrocytes with impaired function further affect ammonia metabolism, reduce the ability of neurons to efficiently uptake or release extracellular ions and neurotransmitters, and stimulate glial cell synthesis of neurosteroids by upregulating their expression of the peripheral-type bz receptor (translocator protein, 18 kda). neurosteroid is an endogenous bz that can enhance gaba nerve tension and cause symptoms in patients with he [233] (fig. 2.2 shown that the metabolic rate of cerebral ammonia in he patients is increased. increased levels of blood ammonia enter the brain through the blood-brain barrier. brain dysfunction also occurs even if blood ammonia levels appear normal; this partially explains the occurrence of he in the case of normal blood ammonia and invalidates he treatment by simply reducing blood ammonia. in addition, increasing evidence suggests a synergistic effect between blood ammonia and its metabolic disorders with systemic inflammation, nerve steroids, oxidative stress, nitrification stress, manganese poisoning, and gaba/bz [233] . the main inhibitory neurotransmitter in the mammalian brain is gaba. plasma gaba is derived from the conversion of glutamic acid by glutamate decarboxylase in intestinal bacterial. notably, gaba has dual role. on one hand, during liver function failure and pss, the removal of gaba in liver is significantly decreased; on the other hand, gaba can directly enter the systemic circulation bypassing the liver, resulting in increased concentration of gaba in blood. the concentration of gaba in csf and brain tissue increases as more gaba crosses the abnormal blood-brain barrier. in addition, endogenous bz was found in the blood and csf, and the gaba receptor on the membrane surface of the brain's postsynaptic neurons increased significantly in some patients with he and in animal models. this receptor not only combines with gaba but also binds to barbiturates (barb) and bz on different parts of the receptor surface; thus, it has been named the gaba/bz complex receptor or the super receptor complex. when liver function is severely impaired, the binding affinity of this complex receptor to its three ligands is also increased. binding of gaba, barb, or bz with the complex receptor can promote entry of chloride ions from neuronal membrane ion channels into the cytoplasm of postsynaptic neurons, causing membrane hyperpolarization and nerve conduction inhibition. he symptoms were relieved in about 30% of patients treated with a gaba receptor antagonist or bz receptor antagonist, and gaba/bz and ammonia were reported to act synergistically in he. recently, some studies focused on peripheral type bz receptors, which are different from central gaba [234, 235] . some questions, including the source of endogenous bz, and the correlation between the increased degree of gaba or bz and the disease, remain to be answered. therefore, therapy targeted at reducing the blood ammonia concentration in patients with he and significantly reducing the increased gaba nerve tension seems reasonable [236] , but may not be completely effective. treatment effects of reducing ammonia vary, because of the different levels of ammonia in he patients that can be produced by the interaction between various known or unknown factors and the different effects of bz receptor antagonists. this theory is related to the metabolism of aromatic amino acids (aaa), the precursors of true neurotransmitters, including norepinephrine and dopamine. due to the reduction in the liver's detoxification function or formation of pss, the amines (phenylethylamine and tyramine) produced in the intestine cannot be cleared completely, resulting in elevated concentrations of these amines in the systemic circulation and increased levels in the brain through the blood-brain barrier. under the effect of β-hydroxylase, phenethanolamine and β-hydroxytyramine (β-dopamine) are generated from phenylethylamine and tyramine, respectively and are similar to norepinephrine and dopamine in chemical structure. these amines can be taken up, stored and released by adrenergic neurons in the brainstem reticular structure. phenethanolamine and β-hydroxytyramine are called false neurotransmitters because of their low physiological effects on the postsynaptic membrane, which is about 1/10 of norepinephrine. when these false neurotransmitters accumulate in the nerve synapse, they can outcompete or replace normal neurotransmitters, resulting in a disorder of nerve conduction. it was reported that plasma aaa (such as phenylalanine, tyrosine, and tryptophan) increased and branched-chain amino acids (bcaa, such as valine, leucine, isoleucine) decreased in patients with decompensated liver cirrhosis, leading to an imbalance of amino acid metabolism. aaa are decomposed and metabolized in the liver, and liver failure decreases aaa decomposition resulting in an elevated concentration of aaa in the plasma. insulin can promote bcaas entering muscle, which is then broken down and metabolized in the skeletal muscle instead of the liver. insulin inactivation is decreased in patients with liver failure, promoting a large number of bcaas entering the muscle tissue and decreasing the concentration of bcaas in plasma. finally, the bcaa/aaa ratio is reduced from a normal 3-3.5:1 to 1:1 or lower. the above process reduces the bcaa concentration, but increases the aaa concentration, leading to an increase in synthesis of false neurotransmitters and reduction of the normal neurotransmitter [237] [238] [239] . the epidemiological data suggests that manganese poisoning and he extrapyramidal have common clinical symptoms. the liver is an important organ for manganese excretion. the concentration of blood manganese can be increased when liver function is affected, during pss, or when excretion of bile is reduced. manganese content in plasma was sharply increased in more than 80% of patients with acute hepatitis and liver cirrhosis and the density of globus pallidus increased in the brain basal ganglia of he patients (partially 2-7 times higher by mri). based on histological results, the above changes were caused by manganese deposition, which disappears after liver transplantation. it has been suggested that manganese deposition may cause dopamine dysfunction. deposition of manganese not only cause direct brain injury, it can influence the function of 5-hydroxytryptamine (5-ht), norepinephrine and gaba neurotransmitters; impair astrocyte function; and have a synergistic effect with ammonia. however, there is no reliable correlation between the concentration of serum manganese and he severity, which may be due to the chronic deposition of manganese [240] . the characteristic change in mri imaging as the deposition of manganese remains to be verified. the effectiveness of manganese removal to improve the symptoms and neurological signs of patients with he needs further validation. the synergistic toxic effects between toxins (ammonia and mercaptan) and short chain fatty acids [241] , the 5-ht hypothesis, the effect of helicobacter pylori urease, opioids, endotoxin, tumor necrosis factor, melatonin, and hepatitis b virus termed additional theories of he syndrome. this theory also suggests the same hypothesis mentioned in the above theories. due to the extensive amount of liver cell damage caused by acute liver failure in type a he, the residual liver cells cannot effectively remove toxins leading to central nervous system dysfunction. type a he, known as non-ammonia encephalopathy, is endogenous he without clear causative agents. simple type b he is rare in mainland china; the liver can clear limited metabolic toxins in patients with chronic liver failure or pss, but once these toxins exceed the compensatory capacity of the liver, type c he occurs. the occurrence of type c he is largely related to the following risk factors, which are the most important factors in the prevention and treatment of he. patients with chronic liver failure or pss are less tolerant to the protein found in food, especially animal protein. a large amount of ammonia and aaa are produced by the decomposition of intestinal bacteria, which can induce he. oral ammonium salts, urea, and methionine can induce he by increasing the absorption of nitrogenous substances and elevating blood ammonia. intestinal production of ammonia can be increased by hemorrhage in the intestine (100 ml of blood contains 15-20 g protein). at the same time, because of the lack of isoleucine in the blood, after digestion and absorption of a hemorrhage, extra blood leucine and valine increase bcaa decomposition by enhancing the activity of bcaa dehydrogenase, thereby exacerbating the imbalance in the bcaa/aaa ratio. loss of blood volume, cerebral ischemia and hypoxia also increase the sensitivity of the central nervous system to ammonia and other toxic substances [230] . infections such as spontaneous peritonitis, pneumonia, and urinary tract infection can increase tissue decomposition and production of ammonia. secondary sepsis or sirs induce he through tnf-α, il-1, il-6 and other inflammatory factors, exacerbates oxidative stress, and increases the blood-brain barrier permeability of ammonia and other toxic molecules to liver and brain [242] . studies have shown that sirs is directly related to the deterioration of he in patients with liver cirrhosis, and its extent and mortality increase with the deterioration of sirs [243] . similarly, sirs is a common factor in triggering chronic liver failure characterized by he and renal failure. in a study of patients with liver cirrhosis, artificially-induced hyperammonemia by oral administration of glutamine may have worsened the results of psycho-mental testing in 10 cases of sepsis patients; while brain toxicity was not obvious after the inflammation was relieved, the observation of decreased cytokine levels indicated that infection and induced inflammatory mediators enhanced brain toxicity of hyperammonemia. accordingly, some researchers suggested that sirs could be an independent pathogenesis of he rather than a risk factor [243] . hyponatremia can affect the intracellular osmotic pressure and lead to brain edema, which induces he. hypokalemia is often associated with metabolic alkalosis [244] . mass use of diuretics or extraction of ascites can also cause alkalosis. ammonia is easily absorbed by the intestinal tract or through the blood-brain barrier inducing he [245] . a variety of reasons can cause pre-renal azotemia such as hypovolemia, anorexia, diarrhea, limiting the amount of liquid, mass use of diuretics, or extraction of ascites. hepatorenal syndrome or other causes can result in renal azotemia. pre-renal azotemia and renal azotemia caused by hepatorenal syndrome or other causes can increase the concentration of ammonia in the blood. several other predisposing factors can contribute to he such as constipation, hypoglycemia, the use of sedatives and proton pump inhibitors, and epilepsy. after the occurrence of constipation and intestinal obstruction, the patient's intestinal mucosa is exposed to ammonia longer thus increasing the absorption of ammonia. hypoglycemia can reduce brain deamination. the binding of sedatives, hypnotics and the brain gaba/bz receptor produce an inhibitory effect on the brain. it was reported that proton pump inhibitors increase the risk of he in patients with cirrhosis in a population study [246] . another study also suggested that epilepsy was associated with an increased risk of he in patients with cirrhosis [247] . patients with type a he often have no obvious anatomical abnormalities in their brains, but 38-50% of patients have brain edema, which may be a secondary change of the disease. hypertrophy and hyperplasia of the original plasma astrocytes in gray matter and subcortical tissue can be found in patients with type c he. patients with longer course of the disease will exhibit brain atrophy (especially in patients with alcoholic cirrhosis) of different degrees, thinning of the cerebral cortex, loss of neurons and nerve fibers, and deep cortical sheet necrosis, even the cerebellum and the base may also be involved. the majority of patients with cirrhosis may have different degrees of he at some stage in the course of the disease. the incidence of he in patients with liver cirrhosis is at least 10-50% in mainland china while the incidence of post-tips (transjugular intrahepatic portosystemic shunt) he is 25-45%. if patients with chronic liver disease have he, the outcome is poor; the one year survival rate is lower than 50% and the 3 year survival rate is less than 25% [248, 249] . the incidence of mild he is 39.9% in mainland china in patients with liver cirrhosis, 24.8% in patients with child-pugh a, 39.4% in patients with child-pugh b, and 56.1% in patients with child-pugh c. the incidence of mild he is not significantly associated with cirrhosis; however, with the increased degree of decompensated liver cirrhosis, the incidence of mild he increase. several studies have found that the incidence of depression and anxiety in patients also increased, with the increase of liver function damage, the incidence also increased, and the outcome is poor [250, 251] . the clinical manifestations of he vary, because of the difference in the nature of underlying disease, the degree of liver cell damage, the speed of injury and incentives. they are not specific to he compared with other metabolic encephalopathies. early pathological changes of he are mild he. the neuropsychological and intelligence tests detect mild form of he, which exhibit no clear clinical symptoms and often develop symptomatic he. the main clinical manifestations seen in acute liver failure induced by type a he are rapid-onset jaundice, bleeding, decrease in prothrombin, and eventually, change in mental status that can start as mild confusion but progress to coma and even death. type c he is characterized by chronic recurrent episodes of changes in personality and behavior [252] , stupor and coma, which is often accompanied by increased muscle tone, hyperreflexia, hepatic flap, ankle clonus or positive babinski sign and nervous system abnormalities. most patients in the early stages relapse, but then their symptoms become persistent. he often has a variety of risk factors such as consuming a high-protein diet or discontinuing treatment of he. patients with type c he not only have the clinical manifestations of encephalopathy, they also have chronic liver injury, cirrhosis and other clinical manifestations [226] . observation of encephalopathy dynamic changes is beneficial for early diagnosis, treatment and analysis of treatment efficacy. he can be graded and quantified according to the degree of disturbance of consciousness, nervous system performance and eeg changes. according to the 2009 edition of the "consensus on the diagnosis and treatment of hepatic encephalopathy" in china, he is divided into 0-4 periods, but each period can be overlapping or distinct but each period can be overlapping (table 2 .4). at present, scholars have stressed that the occurrence of he is a continuous progression of the disease and should be viewed as a continuum of a wide range of neuropsychiatric abnormalities, rather than isolated clinical stages. according to the traditional west haven criteria diagnosing grade 1 he is based on clinical signs and physician assessments, resulting in diagnostic criteria confusion [253] . (fig. 2.3) . covert he is diagnosed by a variety of neuropsychological and intelligence tests; the evaluation of overt he widely uses the modified west haven semi-quantitative grading table for the analysis of patients with neuropsychiatric state (table 2 .5), the glasgow coma scale for the analysis of the degree of consciousness of patients, and the simple he severity rating scale for the disease in addition to abnormal liver function (such as increased bilirubin, enzyme bile separation, and decreased prothrombin activity) commonly used auxiliary examinations for he diagnosis include: determination of ammonia, amino acid analysis of plasma and csf, psychological intelligence test, neurophysiological test, electroencephalogram and neuroimaging. the normal level of fasting venous ammonia is 6-35 μg/l (serum) or 47-65 μg/l (whole blood) and arterial ammonia concentration may be 0.5-2 times that of venous ammonia. generally, the determination of arterial ammonia is common in clinical practice than intravenous determination; however, if venous blood has been transported on ice box and detected in a timely manner after proper collection, the result is expected to be as effective as arterial detection. ammonia levels are increased in type b and c he, but are normal in type a he. thus, he cannot be ruled out based on having a normal ammonia level. the increased level of ammonia was reported to be associated with the degree of type a he, but significant overlaps in different clinical stages of patients were also found [255, 256] . therefore, ammonia detection is not routinely recommended in the diagnosis of he. notably, we need to rule out falsely elevated levels of ammonia caused by lab error, renal failure, complete parenteral nutrition, gastrointestinal bleeding, the use of steroid hormones and other extrahepatic factors. the fischer ratio (bcaa/aaa) is used as a marker of he, the plasma bcaa levels decrease while aaa levels increase; resulting bcaa/aaa: < 1 (normal >3). it was reported that the concentration of glutamate in csf in he patients is increased compared to healthy controls. the concentrations of phenylalanine and tyramine in csf were also significantly increased, and the level of phenylalanine was closely related to the degree of he [257] . recently, it was reported that h-nuclear magnetic resonance spectroscopy could select biomarkers for these diseases, such as in patients with he [258] , but this is not commonly used clinically because the the characteristic manifestations of cognitive dysfunction in patients with covert he are lack of attention, working memory problems, and deficits in executive function. therefore, various intelligence tests are used to assess the subtle changes in a patient's cognitive or precise movement, which is important for the diagnosis of covert he, but not for overt he. [259] . at present, computer-aided psychological tests such as information and communication technology (ict), cognitive drug research test (cdr), and critical flicker fusion test (cff) are not influenced by the factors mentioned above and easily operated, which can be used as an alternative choice for pen and paper tests. ict with sensitivity 87% and specificity 77% was one of the most commonly used tests to diagnose minimal he. cff was originally used to detect the critical flicker frequency of alert patients, reflecting brain conduction dysfunction. based on a spanish study of 217 cases, including patients with cirrhosis and healthy controls, cff was a sensitive method to diagnose covert he with sensitive, simple and reliable advantages [260, 261] . because the diagnosis of minimal he has just started, the related diagnostic value still needs to be further evaluated. an abnormal eeg is often observed before biochemical abnormalities or mental abnormalities [262] . the main abnormalities by eeg are slowed rhythm, sporadic or universal θ wave (4-7 times/s) and the occasional α wave (1-3 times/s). with the deepening of consciousness, symmetrical δ-wave and three-phase waves appear on both sides simultaneously. this change usually occurs on both sides of the forehead and the top, gradually moving backwards. although these eeg changes are not specific to he and can appear in uremic encephalopathy and other metabolic encephalopathy, the severity of changes have a good correlation with clinical stages of he. computer analysis of eeg frequency distribution, such as artificial neural network-expert system (aness) and short epoch dominant activity cluster analysis (sedaca), is more objective and valuable in diagnosing minimal he than conventional eeg [263] . there are many kinds of evoked potential tests, including visual evoked potential (vep), brainstem auditory evoked potential (baep), somatosensory evoked potential (ssep) and endogenous event evoked potential (event-related potentials, erps) p300, of which the p300 is the most sensitive test for the diagnosis of he. compared with intelligence tests, neurophysiologic tests, independent of age and education background, are more objective. however, they are only used in clinical studies and are limited by equipment, and professional operation. based on cerebral ct and mri, brain edema can be found in patients with type a he while brain atrophy in the frontal cortex, and the t1-weighted signal enhancement in the globus pallidus can also be found, which may be associated with manganese deposition. detected by h-magnetic resonance spectroscopy (h-mrs), the metabolic changes of he patients in the brain include increased levels of glutamate and glutamine, and decreased levels of inositol, taurine and choline [263] . using fluid attenuation inversion recovery (flair) and diffusion weighted imaging (dwi) techniques, diffuse t2-weighted signal enhancement is found in the hemisphere white matter and corticospinal tracts, which may be associated with cerebral ischemia. however, the sensitivity and specificity of the above-mentioned imaging abnormalities remain unknown, and the correlation with he staging is not clear. therefore, the main significance of cranial nerve imaging is to exclude cerebrovascular accident, intracranial tumors and other diseases, rather than diagnose he. there is no gold standard diagnostic criteria of he, and diagnosis is mainly based on the exclusion of other diseases. but one should consider the following five factors [232]: several forms of hepatic diseases may lead to different kinds of he. type a he is caused by acute hepatic failure, but without chronic hepatic disease. type b is caused by pss, but without any history of hepatic diseases. type c is caused by serious hepatic diseases and/or widespread pss, such as cirrhosis, liver cancer, post-tips and so on. psychiatric symptoms can be found such as change of mood and personality, dementia, behavior disorder and disorientation. drowsiness alternating with excitability, hypermyotonia, asterixis, ankle clonus, insanity and coma are physical signs could be present in progressed patients. some patients may lack related physical signs and psychiatric symptoms, but have deficits in ability of learning, understanding, concentration, and quick verbal response. upper gastrointestinal hemorrhage, ascites tapping, excessive diuresis, high protein diet, medicine (such as sedatives) and infection could lead to he. previous he symptoms could be helpful for the diagnosis. type a usually does not have any risk factors. metabolic encephalopathy includes ketoacidosis, hypoglycemia, uremia, pulmonary encephalopathy, serious electrolyte disturbances and toxic encephalopathy. nervous system diseases include intracranial hemorrhage, infection or tumors, mental diseases and excessive use of sedatives [264] . but one should also watch out for the coexistence of he in these situations. an overt he should be considered if (1), (3), (4), and (5) coexist; and covert he is based on (2), (3), (4), and (5) [228] . then, based on the degree of neuropsychiatric symptoms, determine the stage of he, or for he classification refer to the west have semi-quantitative classification table or ishen scores. the flow chart of diagnosis is shown in fig. 2.4. he is a complex metabolic disorder caused by many factors and comprehensive measures should be taken to cure it from different aspects. according to the clinical type, inducements and the severity of the disease, different plans of treatment should be designed for he. at present, the treatment of overt he generally includes the following aspects: (1) supportive treatment; (2) identification of possible concurrent encephalopathy and removal of other precipitants; (3) cause of treatment; and (4) empirical treatment (fig. 2.5 ). the point of nutritional therapy is to promote anabolism, inhibit catabolism, and maintain a positive nitrogen balance, rather than simply limiting protein intake. to reduce the source of ammonia, it has been suggested that patients with he should limit protein intake. in critically ill patients, it has been suggested that they should stop all protein intake and, after the disease improves, gradually increase protein intake to the maximum clinical tolerance. these recommendations are now being questioned because most cirrhotic patients are malnourished and all long-term protein-restricted diets increase the severity of malnutrition. in addition, a negative nitrogen balance increases mobilization of skeletal muscle, resulting in a reduction in ammonia metabolism that may increase blood ammonia levels. recent studies have shown that normal ingestion of protein 1.2 g/(kg • d) can also improve the health-related quality of life (hrqol), especially in mhe [265] ; and have no adverse effects on the recovery of blood ammonia and he fig. 2.4 the flow chart of diagnosis of he compared with the restricted protein intake. according to the guidelines of the european society of enteral nutrition in 2009, the intake of protein should be guided by the following principles: patients with acute phase he on the first day should be put on a prohibited protein diet and given glucose to ensure energy supply and those who cannot eat food may be fed through a nasogastric tube without short-term fasting; patients with chronic he do not need to fast and their intake of protein should be 1-1.5 g/(kg • d); oral or intravenous use of bcaa and essential amino acid preparations can be administered to adjust the balance of aaa/bcaa, promote the balance of nitrogen, and also reduce the risk of he recurrence [266] ; probiotics and prebiotics can enhance the body's tolerance to protein; plant protein is superior to animal protein because it contains methionine, has less aaa, and more bcaa, but it also contains cellulose, which is conducive to maintain the normal flora in the colon and acidize the intestinal tract, shortening the transit time of the colon and reducing absorption of ammonia. the above points need further verification. additional supportive treatments include: maintaining adequate hydration, electrolytes and acid-base balance; ensuring an energy supply of 30-35 kal/(kg • d), which should be composed of 50-60% sugar, 20-30% protein, and 10-20% fat; administering appropriate vitamins and trace elements; treating for hypokalemia, hyperkalemia, hyponatremia, hypocalcemia, hypomagnesemia and metabolic alkalosis as needed; strengthening the basis of treatment with the appropriate infusion of fresh plasma or albumin, increased plasma colloid osmotic pressure; treating for hypoxemia and cerebral edema; and preventing and treating any bleeding and bacterial infection. type c he has a variety of precipitents. actively finding and eliminating triggers can effectively prevent the development of he, such as esophageal variceal bleeding that can develop into he. active hemostasis, anemia correction, and removal of intestinal blood are also conducive to controlling he. in addition, active control of infection, correction of water and electrolyte imbalance, elimination of constipation and improving renal function are essential to control he. anesthetics, painkillers, sedatives, sleeping pills, and other drugs should be strictly controlled. patients with mania or convulsions can reduce the use of diazepam and scopolamine, and the frequency of administration can also be reduced for promethazine, chlorpheniramine, and other antihistamines. toxic substances causing he mainly come from the intestine. thus, in order to prevent and control he, it is very important to clean the intestinal tract to reduce the generation and absorption of ammonia and other toxic substances. saline or weak acidic solution enemas (such as a dilute acetic acid solution), or oral or nasal feeding of 25% magnesium sulfate (30-60 ml) can be used to clear intestinal hemorrhage, intestinal impaction, and other toxic substances. an enema composed of non-absorbable lactulose (300-500 ml) plus water (500 ml) is also useful, especially when applied for type b he. a recent clinical trial suggested that polyethylene glycol was more effective than the current standard first-line therapy in treating these patients [267] . another two studies showed that polyethylene glycol was more effective than the standard lactulose therapy in treating patients with acute he by cirrhosis [268, 269] . other available drugs include pear liquors, mannitol, rhubarb, and so on, but excessive use of these substances may lead to dehydration and aggravate he. non-absorbable disaccharides include lactulose and lactitol. lactulose, a kind of synthetic ketone disaccharide, cannot be broken down in the stomach and small intestine due to a lack of enzymes that can break down galactose in the digestive tract. after entering the colon, lactulose can be broken down into acetic acid and lactic acid with the help of gut bacteria, leading to a reduction in the colonic ph and inhibition of the absorption of ammonia in the intestine. these non-absorbent disaccharides are decomposed into organic particles in the intestinal tract, which can increase the osmotic pressure of the intestine, and their acidic products stimulate the intestinal wall and can slightly promote intestinal excretion. these non-absorbent disaccharides, acting as prebiotics in the intestine, can inhibit the growth of bacteria, which can produce ammonia and urea, finally reducing the production of ammonia and reversing low-grade cerebral edema when combined with rifaximin [270] . however, probiotics can benefit patients in the long-term [271] . oral or nasal feeding (15-30 ml, 2 or 3 times daily) was recommended to adjust the daily defecation appropriately, about 2-3 times daily. main adverse reactions include abdominal discomfort, abdominal distension, abdominal pain, loss of appetite, nausea, vomiting, and diarrhea. lactulose can even be used in patients with diabetes or lactose intolerance when the purity of non-absorbable disaccharide was high (≥ 98%), but is not used in patients with intestinal obstruction. numerous randomized controlled studies showed that lactulose or lactitol can significantly alleviate overt he and improve the patient's cognitive function and quality of life [272, 273] . lactulose is still the first-line therapy of anti-he, although its effect on improving the survival rate of patients is uncertain. antimicrobial agents can be used as a substitute for non-absorbable disaccharides in treating acute and chronic he. in the past, oral aminoglycoside antibiotics, such as neomycin, which are rarely orally ingested, were used to inhibit the overgrowth of bacteria in the intestine. however, recent randomized placebo-controlled studies have shown that neomycin may not benefit patients with he compared with placebo-treated patients and that long-term use of neomycin may lead to increased ear and renal toxicity risk and impair the function of small intestinal mucosa [274] . metronidazole can inhibit anaerobic bacteria in the intestine and alleviate he, but long-term use may lead to disruption in the intestinal flora, gastrointestinal discomfort, or neurotoxicity. rifaximin, a derivative of rifamycin with a broad-spectrum, has a potent inhibitory effect on intestinal bacterial growth, is a minimally-absorbed oral antibiotic and only plays a role in the gastrointestinal part. administration of rifaximin (550 mg, twice a day) can significantly prevent the occurrence of he compared with placebo-treated patients [275] [276] [277] ; rifaximin was equivalent to or better than lactulose and neomycin in treating patients with chronic he [278] . a study indicated that rifaximin-α in combination with lactulose was a cost-effective therapy for patients who had experienced at least two prior overt he episodes [270] , and this therapy could also improve the driving ability of patients with covert he without toxicity to the auditory nerve and renal function [274, 277] . thus, rifaximin has been recommended by the us food and drug administration (fda) for the prevention of recurrent he. the efficacy of rifaximin and relation between longterm use of rifaximin and intestinal flora in the treatment of he needs to be further investigated. however, a recent study in mice showed that rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression in mhe [277] . the study may provide a new opportunity to study intestinal flora in the treatment of he. microecologics with bifidobacterium and lactobacillus can regulate intestinal flora structure to inhibit the growth of bacteria that produce ammonia and urease. in combination with prebiotics, microecologics can reduce the production and absorption of intestinal ammonia and other toxic substances [279, 280] . in a recent openlabel study, 190 patients with cirrhosis were randomized to three groups and treated with lactulose (30-60 ml daily), probiotic capsules, or with both drugs. after a month of treatment, patients with he showed better results in the neuropsychological test, p300 auditory evoked potentials, and blood ammonia. however, there was no difference in the therapeutic effect among the three groups [281, 282] . clinicians commonly use sodium glutamate, potassium glutamate, arginine hydrochloride and potassium magnesium aspartate, but the exact efficacy is highly controversial at present and effective drug reduced ammonia is described below. (a) l-ornithine-l-aspartate lola, a dipeptide, can lower blood ammonia by promoting ammonia consumption and the synthesis of urea, glutamic acid and glutamine in brain, liver and kidney [283, 284] . ornithine, a substrate of the ornithine urea cycle, can increase activity of carbamyl phosphate synthetase and ornithine carbamyl transferase, and promote urea synthesis. n-methyl-d-aspartate (nmda) is a substrate of glutamine synthesis, and the conversion of glutamic acid to glutamine in the body can remove blood ammonia [285] . nmda is also involved in nucleic acid synthesis in liver cells and indirectly improves the metabolism of the krebs cycle process in liver cells to facilitate the repair of liver cells. clinical studies show that, compared with a placebo group, 20 g/days lola intravenously could noticeably reduce fasting blood ammonia (fnh3), postprandial blood ammonia, and improve the mental status of patients with he [286] . patients with oral lola also had improved he examination results for the digital connection test, the flapping tremor, and eeg results [287] . in addition, glycerol phenylbutyrate (gpb) can safely reduce the incidence of he as well as ammonia in patients with cirrhosis and he. the results showed that gpb had therapeutic potential in this population [288] . zinc is an important cofactor in urea cycle enzyme catalysis. a study in he patients showed that serum zinc concentration is reduced, and showed a negative correlation with the blood ammonia concentration; the serum ammonia level is much lower after zinc supplementation in patients, and he can be improved in some patients. a new study suggests that antioxidant and zinc supplementation can improve mhe in patients with liver cirrhosis [289] . oral zinc preparation can also reduce absorption of divalent cations such as manganese in the intestine; however, it has not been determined if zinc has a positive therapeutic effect on he. (c) sodium benzoate sodium benzoate can lower the blood ammonia concentration by activating the urea cycle for ammonia detoxification and promoting urinary ammonia. randomized controlled studies showed that sodium benzoate had the same efficacy as lactulose in treating patients with he. the recommended sodium benzoate dose is 5 g twice a day; nevertheless, few patients can tolerate this dose because of its high gastrointestinal side effects [232] . a recent study showed that tranilast could protect patients from thioacetamide-induced acute liver injury and alleviate he [290] . endogenous bz analogues combine with the inhibitory neurotransmitter gaba receptor to depress the action on the cns, and is one of the occurring hallmarks of he pathogenesis. a large-scale clinical study on 560 he cases showed that the improvement rate in brain function in treatment and control groups were 15% and 3%, respectively [291] . the study showed that treatment of he with receptor antagonists such as fluorine marcie is feasible. a meta-analysis which included 13 casecontrol studies of 805 patients show that fluorine marcie can noticeably improve he, but didn't show any long-term benefits or improve patient survival rate. so fluorine marcie should only be considered for he patients who had used bz. although the reduction of dopamine neurotransmitter activity is also one of the pathogenesis, the application of bromocriptine, levodopa, has been unable to bring more benefits besides partly improving symptoms of patients. oral or intravenous infusion with a bcaa-based amino acid mixture can theoretically correct an imbalance in amino acid metabolism and control false neurotransmitter formation in the brain [292] . a meta-analysis which included five studies showed that intravenous bcaa did not reduce the mortality rate of he. three studies with bcaa did not reduce the mortality rate of he; however, two larger studies (randomized controlled study about patients with liver cirrhosis in 174 cases and 622 cases, respectively) show that the application of bcaa not only reduced the occurrence of he and liver failure, but also improved the nutritional status, liver function and survival rate in patients. another study showed that bcaa could stimulate liver cell regeneration thus reducing the occurrence of liver failure. supplementation with a bcaa-rich amino acid mixture showed improved restoration of the patients' positive nitrogen balance, and increased the patient's susceptibility to protein food, improving cerebral perfusion [293] . considerable progress has been made to understand treatment of he, studies of basic and clinical research are underway using newly discovered treatment strategies, such as toll-like receptor 4 antagonists (with the ability to reduce systemic inflammation and oxidative stress) as well as non-steroidal anti-inflammatory drugs (ibuprofen) [286, 294] , nmda antagonists, anticholinesterase. however, research using gene therapy should not be ignored [295] . after tips, lola can significantly reduce the increase of venous ammonia concentration in patients with he [296] . and the positive dietary intervention can significantly reduce the incidence of he [297] . for patients with refractory he, embolization of pss is a safe and effective treatment strategy [298] . improving liver function antiviral treatment with nucleos(t)ide analogues can reduce or eliminate liver inflammation and necrosis, promote the regeneration of liver cells, and help restore the functions of hepatic metabolism and detoxification in chronic liver failure caused by hepatitis b virus. artificial liver support systems can be divided into three types including nonbiological type, biological type and mixed type. the non-biological liver support system is the most widely used type, and consists of hemodialysis, hemofiltration, plasma exchange, blood perfusion, plasma adsorption, and the molecular adsorption recirculation system (mars) [299] . the artificial liver support system can replace the partial function of the liver, remove the poison accumulated in the body, create conditions that allow for the regeneration of liver cells and provide enough time to wait for liver transplantation for patients with he. an artificial liver support system can be used to treat acute and chronic he, but patients with overt stage 2 he should be especially careful with plasma exchange. liver transplantation remains the only promising therapy for patients with an acute liver failure or endstage liver disease. liver transplantation is an effective means for all kinds of persistent and severe he; however, in patients with he there is a significant increase in mortality among patients awaiting liver transplantation [300] . recently, it has been reported that cognitive function was not fully recovered after liver transplantation in some patients with severe he [301] . the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. theoretically, for patients with serious pss, interventional therapy, surgery or permanently/temporarily and partially/totally blocking the pss can improve the patient's symptoms. the use of this therapy should be carefully weighed because it can increase the risk of gastrointestinal bleeding in case of portal hypertension. covert he has gained an increasing amount of attention in recent years. patients with covert he do not exhibit obvious signs and symptoms; however, their quality of life is reduced because of reduced operational ability or sleep disorders. without treatment, covert he will progress to overt he over time. the population with high risk should be examined and treated early, especially those engaged in potentially dangerous occupations. the following solutions can be referred to: (a) adjusting dietary structure (vegetable protein is the main intake); (b) oral administration of lactulose (15-30 ml, 2-3 times daily); (c) oral administration of rifaximin (550 mg, twice a day); (d) oral administration of lola (6 g, 3 times a day); (e) oral administration of baaa; and (f) oral administration of probiotic preparations [271, 275, 289 ]. although medical technology has made great progress and the research into he is also increasing in recent years, the pathogenesis of he is still unclear. due to a lack of specific methods, combination treatment is still the main therapy for he. it is generally believed that the onset of he may be a result of the synergistic effects of many factors. therefore, it is difficult to implement and draw convincing conclusions from randomized controlled trials with a single intervention targeting a specific pathogenesis and risk factor. ongoing issues remain, such as standardizing the research design of he treatment and evaluating the efficacy of he treatment more scientifically and objectively. some clinical studies may bring new hope for he treatment by new ongoing strategies of targeted systemic inflammation, oxidative stress, and neurosteroids. in addition, the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. it is difficult to popularize the cognitive dysfunction detection methods of latent he. so the key and difficult point is to develop new method of assessments for clinicians in the future. jia shang hepatopulmonary syndrome (hps) is a syndrome of shortness of breath and hypoxemia induced by vasodilation in the lungs of patients with a variety of acute and chronic liver disease. essentially primary liver disease, pulmonary vasodilation and arterial oxygen lack of co-triad constituted. due to abnormal increase of vasodilators、ventilation/ blood flow disproportion and pulmonary hypertension caused by liver disease, the hypoxemia (pao 2 < 9.33 kpa) (70 mmhg) is included in hps. when fluckiger reported a 37-year-old syphilis female patient as early as in 1884, he described cirrhosis, cyanosis and clubbing at the same time, while he was not aware of the intrinsic relationship between these clinical manifestations [302] . in 1935, snell reported decreased arterial oxygen saturation (sao 2 , less than 94%) with abnormal hemoglobin in 38 patients with liver parenchymal lesions and biliary obstruction, and 3 years later, he proposed that such a phenomenon was associated with decreased affinity of o 2 with hemoglobin. in 1956, rydell and hoffbauer reported the detailed clinical diagnostic and treatment process of a 17-year-old male with "juvenile cirrhosis", and found multiple arterial-venous anastomoses in the lungs during autopsy, which he thought contributed to clinical cyanosis mainly. this provided a histological basis for the patient, and people conducted a large amount of studies thereafter. in 1966, berthelot et al. injected opaque glue into the pulmonary vascular beds at the time of biopsy after the patient's death for the first time, and he found abnormal small arterial dilation in the lungs of the patient with cirrhosis, which he termed lung spider nevus. the term hepatopulmonary syndrome (hps) was first proposed by kennedy and knudson in 1977 [303] . after nearly 20 years of studies in a large number, people gradually developed a clear understanding of the mechanisms underlying its pathogenesis. in 1988, eriksson used the term functional hepatopulmonary syndrome for the first time. in 1989, the famous liver disease expert sherlock formally used this diagnostic term in his monograph hepatobiliary diseases, which has been recognized by many scholars [304] . hps can occur in patients of any age groups, and various literature reports show conflicting incidences of hps in patients with cirrhotic portal hypertension, with the average incidence of various chronic liver diseases being about 5-29%. the incidence of cirrhosis in patients is high, and 30-70% of patients can additionally develop mild arterial hypoxia and 12-28% develop arterial hypoxia. in the study by binay on indian cirrhotic populations arising from hepatitis b mainly, the incidence of this disease is relatively low (6.7%) [305] . the differences in incidence were mainly attributable to the different diagnostic criteria adopted. schenk et al. studied the incidence of hps by performing transthoracic contrast echocardiography (ttce), pulmonary function tests and blood gas analysis on 98 patients with cirrhosis patients. the results showed that the incidence of hps patients in whom alveolar-arterial partial pressure of oxygen (aapo 2 ) was used as an indicator of hypoxemia was significantly higher than those in whom arterial partial pressure of oxygen (pao 2 ) was used [306] . when arterial partial pressure of oxygen (pao 2 ) was reduced to reflect hypoxemia, hps incidence was 19% when <80 mmhg and 15% when <70 mmhg, respectively. while when increase in alveolar-arterial partial pressure of oxygen (aapo 2 ) was used to reflect hypoxemia, the incidence of hps was high, with 32% when >15 mmhg and 31% when >20 mmhg, respectively. hps is most common in cirrhosis due to various causes. pulmonary vascular abnormalities and arterial hypoxemia can occur in a variety of acute and chronic liver diseases, and this is true mainly when it comes to cirrhotic patients due to chronic liver diseases, especially cryptogenic liver cirrhosis, alcoholic cirrhosis, hepatitisinduced cirrhosis and primary biliary cirrhosis. besides, hps can also occur in chronic hepatitis, acute severe hepatitis, cholestasis, ɑ-anti-trypsin deficiency [307] , tyrosinemia, wilson disease, and non-cirrhotic portal hypertension (such as idiopathic portal hypertension and schistosomiasis cirrhosis, etc.). arterial hypoxemia can also occur in extrahepatic portal vein occlusion. the observation of these patients suggests that portal hypertension may be the main factor for the pathogenesis of hps. hps can also occur in non-cirrhotic portal hypertension, and even cirrhosis-and portal hypertension-free chronic viral hepatitis. in 2000, binay et al. found that patients with progressive liver failure with hyperdynamic circulation are most likely to suffer from hps, while they did not find the correlation with the severity of liver cirrhosis. hps is, in essence, hypoxemia due to anomaly in pulmonary vascular dilatation and arterial oxygenation when liver disease occurs. arterial hypoxemia occurs as the result of insufficient oxygenation by blood cells in the blood when blood flows through the lungs, or a proportion of blood fail to flow through the alveoli [308] . since primary heart and lung diseases have been excluded when hps occurs, the abnormal pathways that red cells may pass through include: (1) passing through the pleural and hilar bronchial vessels while not reaching the alveoli; (2) blood flows directly into the pulmonary veins due to the high pressure portal system in the mediastinum, thereby bypassing the pulmonary circulation; (3) flowing directly into the pulmonary veins through the expanded alveolar capillaries or the pulmonary-venous fistula. alveolar telangiectasia may be more important to the formation of hypoxemia, and existing study data show that the development of hps is at least associated with the systemic hyperdynamic state, portal hypertension, hepatic encephalopathy, hepatorenal syndrome and pulmonary hypertension [308] . therefore, it is believed that the main causes of hps are systemic metabolism and hemodynamic disorders, and that it is involved in the formation of systemic metabolism and hemodynamic disorders, which is of important pathophysiological significance. 1. the basic pathological change of hps is pulmonary vascular dilatation, which is manifested as: (a) dilation of anterior capillaries in a large number. (b) formation and opening of the pulmonary basilar arterial -venous communicating branches. (c) formation of pleural "spider mole", which is mainly manifested as dilation of anterior capillaries. in autopsies, it was found that the basic pathological changes in patients with liver cirrhosis and other chronic liver diseases were extensive pulmonary vascular dilatation and arteriovenous communicating branches. some people found the pathological changes through vascular shaping, with pleural vasodilation at the basal aspect of the lungs or the formation of subpleural spider nevus. domestic professor gu changhai summarized these pathologic changes in 1997 as arterial dilation within the pulmonary acinus in a pattern of inhomogeneous distribution, thin-walled blood vessels, 60-80 μm in diameter, in the lower lobes of the whole lungs, extensive dilation of pulmonary vascular beds adjacent to the alveolar gas at the anterior capillary level, and significantly expanded pulmonary artery branches and pulmonary capillaries up to 160 μm in diameter. electron microscopy showed thickened pulmonary capillaries, pulmonary arterial walls and the basal layers of small veins. 2. factors that affect the dilation of blood vessels: the mechanisms underlying pulmonary vascular dilatation have not yet fully elucidated, and the possible influencing factors include: (a) increased activity of vascular dilators various acute and chronic liver diseases, liver cell failure and metabolic disorders, particularly reduced inactivation of vasoactive substances which can enter directly into the systemic circulation through abnormal anastomotic collateral vessels, result in disorder of the systemic hemodynamics and increased contents of vasodilators in the blood circulation. just as visceral congestion in patients with portal hypertension, they can act on the intrapulmonary vessels, causing pulmonary vascular dilatation and pulmonary congestion. substances that cause vasodilation include glucagon, prostaglandin, vasoactive intestinal peptide, nitric oxide, angiotensin, bradykinin and endotoxin, etc. (b) reduced vasoconstrictors or decreased sensitivity of intrapulmonary vascular beds to the endogenous vasoconstrictors, such as norepinephrine, endothelin, atrial natriuretic peptide, vasopressin, serotonin and tyrosine, etc. the contents of the substances are not absolutely reduced because maybe their sensitivity is reduced. when chronic liver disease occurs, the anterior communicating branches of the originally closed non-functional capillaries may be opened, and a disorder occurs in the hypoxic pulmonary vascular systolic dysfunction which should have been normal, and it is only 75% of the normal state [309] . (c) neurological factors cirrhotic patients show sympathetic nerve hyperactivity, but after the formation of portal hypertension, their sympathetic nerve function may be damaged, which play an important role. animals with portal hypertension often show abnormal pressure responses and reduced sensitivity of blood vessels to norepinephrine, resulting in increased cardiac output, and dilated pulmonary vascular volumes. besides, hemodynamics within the lungs is also a manifestation of the body's hyperdynamics. (d) decreased reactivity of intrapulmonary blood vessels to hypoxia recent inert gas dispersion tests show that cirrhotic patients with over two spider nevus are manifested as not only liver damage, but also decreased systemic intrapulmonary vascular resistance, decreased reactivity of blood vessels to hypoxia and dilated pulmonary vessels. however, it was also found using pulmonary angiography that in spite of the dilated vessels at the ending of arteries, the responses of vessels to oxygen were almost normal, which did not support this view. (e) intrahepatic angiogenesis or dysplasia may also be one of the factors for the formation of hps. to date, the mechanisms underlying pulmonary vascular dilatation caused by hps is still unclear. however, long-term administration of intrapulmonary vasoactive substances can cause significantly increased intracellular cyclic adenosine monophosphate (camp) and/or cyclic guanosine monophosphate (cgmp), resulting in hypoxic pulmonary vasomotor dysfunction and pulmonary artery dilatation, which may be an important cause of this disease and also pulmonary manifestations of systemic hyperdynamic circulation. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, some of the blood around the capillaries in contact with the alveoli can still undergo exchanges with gases, while the central blood, due to the increased diffusion distance from the alveoli, leads to insufficient gas exchange, resulting in insufficient arterial oxygenation and thus a series of hypoxemic manifestations. to date, the pathogenesis underlying the pathogenesis of hps has not yet been elucidated. in view of the above pathophysiological changes and current studies, it is believed that the disease may be caused by insufficient ventilation, diffusion disorder, ventilation/blood flow imbalance and decreased oxygenated hemoglobin affinity, or the above factors in combination. under normal circumstances, insufficient ventilation due to a variety of reasons causes insufficient oxygen inhaled into the alveoli and reduced blood oxygen exchanges, which can result in hypoxemia [310] , such as chronic bronchitis, foreign bodies in trachea, atelectasis and respiratory muscular paralysis, etc. and the presence of insufficient ventilation in patients with chronic liver disease and cirrhosis or not is still controversial. in 1982, fujiwara studied the lung function in 22 patients with decompensated liver cirrhosis and reported that vital capacity (vc), functional residual capacity (frc) and respiratory reserve volume (evr) in the patients were significantly reduced, that r/t was mildly increased, and that there was no changes in 1 s forced expiratory volume (fev1). therefore, it was believed that mechanical compression and insufficient ventilation due to pulmonary interstitial edema in patients with liver cirrhosis was the main reason for impaired lung function. subsequently, edison et al. studied the pulmonary function of 21 patients with decompensated liver cirrhosis, and found that their vc, maximum ventilation volume (mvv), frc, total lung volume, and r/t were significantly reduced, and they believed that patients with cirrhosis had obvious obstructive and limited insufficient ventilation, which were mainly caused by compression of lung tissue due to increased abdominal pressure, elevated diaphragm and increased chest volume and pressure when patients had additional ascites, and atelectasis [306] . however, decreased fev1 resulted from compression of small trachea due to pulmonary interstitial edema and vasodilation, and early closure of expiration. theoretically, all of the above factors can lead to insufficient ventilation, one of the factors resulting in this disease. this was also substantiated by significantly increased arterial partial pressure of oxygen and decreased co 2 partial pressure in cirrhotic patients with pleural effusion after pleural effusion extraction and recovery from atelectasis. however, there are also some people who do not think that hypoxemia results from insufficient ventilation, but because cirrhotic patients are not complicated by high concentrations of co 2 when their arterial partial pressure of oxygen is decreased [311] . this is likely because when patients have hypoxemia, compensation of hyperventilation causes arterial blood co 2 partial pressure not to increase, and results in decreased paco 2 or even respiratory alkalosis. besides, in some patients without decompensated liver cirrhosis, arterial hypoxemia can also occur. even it has been found that the lung function tests in patients with decompensated liver cirrhosis are normal. therefore, the majority of scholars currently believe that insufficient ventilation is not the main cause of hypoxemia in cirrhotic patients. for patients with hps, the inert gas exclusion technique should be performed to prove that there is a disorder in the diffusion of oxygen, which is determined by the basic pathological changes of hps -pulmonary vasodilatation. pulmonary angiography can show small spider-like to obviously cavernous diffuse vasodilation within the lungs. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, the diffusion distance of the blood flow in central blood vessels and the alveoli is increased, preventing the gases in the alveoli from entering the pulmonary capillaries, thereby affecting the gas exchanges. studies have shown that hypoxemia often occurs in patients with cirrhosis or aggravates during exercises, and it is believed that diffusion disorder or limitation of oxygen occurs in patients. in fact, factors that affect o 2 diffusion do occur in patients with cirrhosis, but they are still not sufficient to explain the apparent hypoxemia. although vascular dilatation occurs at arterial endings in patients with hps, their arterial partial pressure of oxygen can be reduced while inhaling air and increased when they are given oxygen inhalation, which further proves that although diffusion disorder does exist and it plays a role in the formation of this disease, the role is not important. to engage in gas exchanges is the most important biological function of lung tissues, and this gas exchange must be completed when there is an appropriate ventilation/blood flow ratio. under the normal circumstance (normal adult resting state), the most appropriate ventilation/blood flow ratio physiologically is 0.8. changes in the ratio due to any cause can affect the gas exchange, and the imbalanced ventilation/blood flow ratio in hps patients with hypoxemia is mainly because of pulmonary vascular dilatation and arteriovenous shunt [312] . 1. intravascular vascular dilatation: pulmonary vascular dilatation has been confirmed pathologically and by angiography. dilated blood vessels in the lungs leads to gas diffusion disorder. besides, since the oxygen molecules in the air can not be diffused to the central dilated blood for the gas exchange, causing decreased ventilation/blood flow ratio and pulmonary arterial partial pressure of oxygen. this decreased ventilation/blood flow, together with increased amount of reactive cardiac output, shortens the duration of blood that flows through the capillary network and insufficient oxygenation [312] . excessive ventilation can in part enhance patients' pao 2 . if the alveolar oxygen partial pressure is increased at this time, some oxygen molecules can reach the central areas of dilated blood vessels, increasing the arterial partial pressure of oxygen. therefore, it is called the diffusion -perfusion disorder or pulmonary arteriovenous functional shunt rather than the true lung shunt. 2. arterial -venous shunt: intrapulmonary vascular fistula and pleural spider nevus can occur in cirrhosis of the liver and allow the pulmonary arterial blood to circumvent gas exchange and directly flow into the pulmonary vein so that patients may develop hypoxemia. this hypoxemia can not be corrected by oxygen inhalation and represents the true pulmonary shunt, which has been confirmed by pulmonary histopathology, angiography, transthoracic echocardiography and other examinations. it is now believed that pulmonary vascular casting is still the most direct evidence for determining the arterialvenous shunt. this intrapulmonary arterial -venous shunt is the main cause of abnormal ventilation/blood flow ratio and insufficient gas exchange. although pleural spider nevus can also cause arterial -venous shunt, it generally does not suffice to cause significant hypoxemia due to the small amount of shunt. in addition, studies in recent years also show that portal-pulmonary vein shunt in a small amount occurs in some patients with cirrhosis, in whom blood flow circumvents the alveolar gas exchange and enters directly the systemic circulation. this can also cause ventilation/blood flow abnormalities, causing insufficient gas exchange 3. airway closure: in 1971, ruff et al. proved that cirrhotic patients had significantly increased closed volume (cv) and total amount of closed gas (cc) and increased gases trapped in the lower fields of the lungs, resulting in an extremely low ratio of ventilation/blood flow, and they believed these were due to reduced airway ventilation [302, 313] . in 1984, furukawa et al. measured the lung function of 105 patients with liver cirrhosis and did not find abnormalities; however, most patients had flow -volume abnormalities and significantly increased cv, suggesting the closed the airway in advance and decreased ratio of ventilation/blood flow, which might important causes of hypoxemia. 4. decreased affinity of oxygen with hemoglobin: some reports showed that 15 patients with cirrhosis (mostly alcoholic cirrhosis) patients had mild systemic vascular or pulmonary vascular dilatation, normal pao 2 , mild hypocapnia, mild right shift of the oxygenated hemoglobin dissociation curve, normal amount of carbon monoxide diffusion [313] , and mild imbalance of the ventilation/pao 2 blood flow, indicating that the right shift of the oxygen dissociation curve due to decreased affinity of oxygen with hemoglobin in the patients. this was possibly caused by the increased concentration of 2,3-diphosphate glyceride in red blood cells, which, however, is not an important factor in the occurrence of hypoxemia [302] . in summary, hypoxemia can result from many factors, while none of the factors can completely explain the pathogenesis underlying the disease. since the basic pathological changes in patients with hps are intrapulmonary vascular dilatation and opening of arterial -venous communicating branches, together with recent findings, it is suggested that the diffusion disorder of the alveoli and pulmonary capillaries and ventilation/blood flow imbalance may coexist, and are the main cause of hypoxemia in this disease. other factors may aggravate hypoxia and are secondary factors. therefore, it is believed that the disease occurs as result of the above factors. the pathological features of hps are dilation of capillaries in the anterior aspect of the lungs and telangiectasia. autopsies show arterial-venous short circuit within the lungs, vasodilation and thickened pulmonary muscles [314] . at the same time, arterial hypoxemia is common in liver diseases, often attributable to a variety of factors (such as ascites, hepatic pleural effusion, and copd in patients with alcoholism); it shows unique pathophysiological characteristics under specific circumstances of hps. its prominent features are dilation of micro-arteries in the anterior aspect of pulmonary capillaries and true capillaries (the normal diameter of these vessels is 8 to 15 μm, which can reach 15-100 μm when patients rest), with the number of dilated vessels increased macroscopically. some patients show arterial-venous communicating between the pleura and lungs, vascular anastomosis in the liver and the lungs, and thickened walls of small veins and capillaries. pulmonary vascular dilatation is promoted, and mixed venous blood quickly or directly enter the pulmonary veins through the anastomosis in the lungs, leading to oxygenation defects. increased nitric oxide (no) is a key cause for pulmonary vasodilation, and whether other mediators, such as heme oxygenase-derived carbon monoxide, are causes of pulmonary vasodilatation are not yet confirmed. abnormal arterial oxygenation seriously affects the survival of patients, and is an important indicator that determines the timing and risk of liver transplantation as well as an important basis for the grading of severity of hps. causes of deaths associated with hps are often multifactorial and are associated with basic liver diseases, and there are few cases of respiratory failure due to severe hypoxemia. hps is a triad composed by intrapulmonary vascular dilatation and insufficient arterial oxygenation due to primary liver disease, and it is mainly clinically manifested as primary liver disease and pulmonary lesions. hps can occur in various liver diseases, mostly in chronic liver disease, especially cirrhosis caused by various causes, such as cryptogenic cirrhosis, alcoholic cirrhosis, liver cirrhosis, viral cirrhosis, postnecrotic cirrhosis and biliary liver cirrhosis, etc. the most common clinical manifestations include liver palms [314] , spider nevus, jaundice, ascites, hepatosplenomegaly, gastrointestinal bleeding and abnormal liver function, etc., while they are not significantly correlated with hps. some patients with clinically stable liver disease may also develop the clinical manifestation of progressive pulmonary insufficiency. since hps patients have no primary cardiopulmonary diseases, most (80-90%) patients gradually develop respiratory manifestations on the basis of various liver diseases, such as cyanosis, dyspnea, clubbing, orthodeoxidation and platypnea, etc. among them, progressive dyspnea is the most common lung symptoms of hps. binay et al. believed that cyanosis was the only reliable clinical sign, and that platypnea and orthostatic hypoxia are the most characteristic manifestations. pulmonary examinations generally show no obvious positive signs [315] . a small number of patients (about 16-20%) can present complaining dyspnea on exertion in the absence of clinical manifestations of a variety of liver diseases, to which attention should be paid clinically so as to prevent misdiagnosis. the domestic researchers gao zhi et al. reported that 2 patients presented to hospitals with cyanosis, palpitation after exercises and shortness of breath; meanwhile, they found that the patients had clinical manifestations of liver cirrhosis [308] (such as liver palms, spider nevus, hepatosplenomegaly and ascites), which were conducive to the diagnosis of this disease. if liver disease patients have other lung diseases (such as chronic bronchitis, emphysema and pneumonia, and pleural effusion, etc.), then significant respiratory symptoms may occur. therefore, differential diagnosis should be made. data show that the duration of initial dyspnea to the conformed diagnosis in patients with hps average 2-7 years; that is to say, about 18% of patients already have dyspnea at the time confirmed diagnosis. 1. orthodeoxidation: pao 2 is decreased by >10% when patients switch from the supine position to the standing position. 2. platypnea: when patients switch from the supine position to the standing position, they have palpation, chest tightness and shortness of breath, and when patients resume the supine position, the above symptoms are improved [313] . krowka reported that about 80-90% of patients with hps had the above two manifestations because vascular dilatation in the patients was mainly distributed in the middle and low lung fields. when patients switch from the supine position to the standing position, the blood flow in the middle and lower lobes of the lungs is increased under the action of gravity, aggravating hypoxemia [315] . although the two manifestations are not unique to hps, they suggest the significant abnormality in the patients' pulmonary vascular system. if patients with a variety of liver diseases present with the above two manifestations, further examinations are needed for confirmation. patients may present with liver palms, hepatosplenomegaly, spider nevus and ascites; patients show palpitation, chest tightness, shortness of breath when switching from the supine position to the standing position due to hypoxemia. schenk et al. defined the values of pao 2 for the diagnosis of hps, thinking that pao 2 < 70 mmhg suggested a high possibility of hps, and that for pao 2 < 65 mmhg, the diagnosis of hps could be made [306] . pulmonary function tests mainly showed significantly decreased vc, frc, mvv and fev1, but sometimes the total lung volume and fev1 were normal. chest x-ray, ct scan and transthoracic contrast echocardiography (ttce) hps patients are mostly normal on chest radiography or show diffuse small millet shadows predominantly in both lower lobes of the lungs, nodular shadows in both lower lung interstitium, dilated pulmonary arterial trunks, and thickened pulmonary markings, whereas these manifestations have no specific values to the diagnosis of this disease. ct scan shows certain diagnostic values in that it demonstrates distal vasodilation and even pleural blood vessels, and can suggest the presence of hps. arteriovenous communicating occurs in hps patients due to pulmonary vascular dilation, indicating that subclinical pulmonary vascular dilatation and abnormal gas exchanges occur in cirrhotic patients with normal pao 2 [306] . contrast echocardiography: when hps is suspected, transthoracic echocardiography can be used as a preliminary screening to determine whether the intrapulmonary vascular dilation occurs or not. the microbubble contrast material in the right atrium, after intravenous injection of dioxane isotonic saline, will develop images in the left atrium through the dilated vascular beds after 3-6 cardiac cycles, while the microbubbles cannot pass through the normal capillaries (normal capillaries are <8-15 μm in diameter). approximately 40% of patients with cirrhosis had positive changes on contrast echocardiography, while only a small proportion of patients are in line with the diagnosis of hps due to the influence of dilated blood vessels. if contrast echocardiography is positive for liver cirrhosis or portal hypertension patients with hypoxemia and cardiopulmonary diseases in them can be ruled out, then the diagnosis of hps is established. this means is used to confirm the diagnosis of intrapulmonary vasodilatation. the pulmonary vascular abnormalities in hps patients are as follows: (1) diffuse spider nevus images. patients of this type have severe hypoxemia and erectile hypoxia and respond well to inhalation of 100% oxygen; (2) cavernous or spotted arterial dilatation mainly seen in the basal aspect of the lungs. patients during this period respond poorly to 100% oxygen; and (3) intermittent local arterial malformation or communicating branches, isolated earthworm-like or bulk images. in addition to severe hypoxemia and erect hypoxia, patients of this this type respond extremely poorly to the inhalation of 100% oxygen. when hypoxemia is caused by hps and cardiopulmonary diseases concurrently, 99cm tc-maa scan can determine hypoxemia resulting from hps more likely. radiolabeled albumin 99cm tc, which is administered through intravenous injection, is about 20 pm in diameter. when pulmonary vascular shunt occurs, a proportion of the polymerized albumin passes through the lungs and enters the systemic circulation, the intake of albumin by other organs can be simultaneously determined by scintigraphy [308] . therefore, the amount of shunt can be calculated. a study showed that 99 cm tc-maa scan was positive for hps patients with pa02 < 60 mm hg, while the scan was negative for chronic obstructive pulmonary disease (copd) patients with the same degree of hypoxemia, a result indicative of the good specificity of this means. compared with contrast echocardiography, 99cm tc-maa scan, in spite of its low sensitivity, can be used for the diagnosis of hps in patients with copd. pathological examinations are the most reliable means for the diagnosis of hps, whose basic pathological change is pulmonary vasodilation manifested as diffuse anterior capillary dilation or discontinuous formation of arteriovenous branches [312] . in addition, pulmonary perfusion scan and right cardiac catheterization are also valuable for the diagnosis of hps to a certain extent. there is no unified standard for hps diagnosis to date. diagnosis should be based on clinical manifestations plus imaging evidence of pulmonary angiography. (c) the domestic scholars gao zhi et al. thought in 1998 that the diagnosis of this disease should be based on the following manifestations in patients, hepatosplenomegaly, ascites, liver palms, spider nevus, dyspnea on exertion, hypoxia while breathing in the supine and orthostatic positions, increased mesenchyma in the basal aspects of the lungs and vascular markings on chest radiography, patchy or nodular shadows, dilated basilar pulmonary vessels and increased pulmonary vascular branches on ct, severe hypoxemia or not on blood gas analysis, increase in alveolar -arterial oxygen gradient by ≥20 kpa (150 mmhg), and 82% diffusion disorder on pulmonary function tests [316] . in addition, shunt-related examinations should be performed, such as 99cm tc-maa scanning, contrast-enhanced two-dimensional echocardiography and pulmonary angiography, etc., while the last one does not show the same sensitivity as that of the former two because the small blood vessels in the lungs may not develop on angiography. most hps patients have a slow onset of the disease, are difficult to treat, and have a poor long-term prognosis, with a mortality of more than 40% after 3 years. therefore, early diagnosis of the disease and its differential diagnosis are vital to improving the prognosis of patients. first of all, the previous liver and lung diseases in the patients should be ruled out, such as chronic obstructive pulmonary emphysema, pulmonary infection, interstitial pneumonia and silicosis, etc. at the same time, cirrhosis with pulmonary hypertension, infections secondary to pleural effusion, interstitial pulmonary edema, atelectasis and hyperventilation syndrome, etc. need to be ruled out. hps should be differentiated mainly from the following diseases: 1. liver cirrhosis following pulmonary heart disease: this is mainly because pulmonary diseases result in cardiac insufficiency and thus increased pulmonary venous pressure. repeated or long-term existence of liver congestion can lead to central venous hypertrophy and lobular central connective tissue hyperplasia, and further progression of the lesion will lead to the formation of liver cirrhosis following pulmonary heart disease. patients with pulmonary cirrhosis often have a long history of chronic lung disease and signs of cardiac insufficiency, such as edema of lower extremities, palpitation, shortness of breath and other symptoms. this patient has no history of chronic lung disease or edema of lower extremities, making him inconsistent with liver cirrhosis following pulmonary heart disease. 2. left heart insufficiency: both hps and left heart insufficiency can cause severe dyspnea and hypoxemia. a history of liver disease or evidence of chronic liver damage and decreased po 2 can be found in patients with hps, especially such characteristics as orthostatic hypoxemia and intrapulmonary vascular dilatation. patients with left heart insufficiency have a history of heart disease, orthopnoea, pink frothy sputum and moist rales in the lungs, etc. this patient is inconsistent with such manifestations. 3. primary pulmonary hypertension: after inhalation of pure oxygen, hypoxemia in most of patients with hps will be significantly alleviated. the effects of oxygen are poor in patients with hypoxemia [317] , and hps is manifested as orthostatic hypoxemia. the characteristics of hemodynamics of patients with hps are hyperdynamics and normal or decreased pulmonary artery pressure and pulmonary vascular resistance, while those in hypoxemic patients are increased. 4. others: ductus arteriosus, eisenmenger syndrome and pulmonary embolism, etc., need to be differentiated from this disease, and comprehensive judgments should be provided based on other clinical data of the medical history. hps patients can also have the above-mentioned diseases, and careful and meticulous examinations are needed for differentiation. because hps is developed on the basis of original liver disease, the frequency of its occurrence and its severity are mostly associated with the liver cell function of patients, while there are also hps patients in whom chronic liver disease is relatively stable and liver functions are normal. besides, pleural effusion, ascites and infections secondary to pulmonary edema after liver function decompensation can aggravate patients' respiratory function injury. therefore, under the current circumstances in which there are no effective measures for hps, active and effective treatment of primary liver diseases is the basis for the treatment of hps. therapy of primary diseases, including correction of hypoproteinemia, elimination of pleural effusion, improvement of liver function and treatment of complications, etc., can improve tissue oxygenation and improve arterial oxygen saturation. on this basis, the following treatment can be given. oxygen therapy also helps the differential diagnosis of pulmonary shunt: if pao 2 is resumed after oxygen inhalation, then the diagnosis of intrapulmonary vascular dilatation (ipvd) can be made; for patients with partial improvement, pulmonary anatomical shunt and functional shunt may coexist; for patients in whom the oxygen therapy proves inefficacious, pulmonary arteriovenous fistula is a possible diagnosis. it is now believed that once the diagnosis is established, treatment should be given as soon as possible. in the early stage of correcting hypoxemia in patients with mild conditions, even in patients in whom the critical value of hypoxemia (pao 2 , 8-9 kpa (60-67.5 mmhg) is reached and who have ascites, the hemoglobin saturation may still be less than 85% when patients are in activities or even sleep. that is to say, nasal catheter oxygen inhalation at 2-3 l/min is needed so as to improve hypoxemia [318] . with the development of the disease, oxygen flow needs to be gradually increased, and intratrachea oxygen supply can be offered when necessary. during the late stage, patients can receive pressurized oxygen through a ventilator or a hyperbaric oxygen chamber. for patients whose conditions are severe, the efficacy of oxygen therapy alone is not obvious. 2. vasoactive drugs vasoactive drugs for the treatment of patients with hps are most studied; however, since its pathogenesis has not been clarified to date and primary liver disease is difficult to reverse, it is hard to define the clinical efficacy of these drugs. the commonly used drugs include: used aerosolized ephedrine hydrochloride for the treatment of 12 patients with hps, and the preliminary efficacy was significant. the mechanisms were that ephedrine could excite the pulmonary vascular α receptor, resulting in contracted bronchial mucosa and pulmonary capillaries and alleviated bronchial edema, so that the dilated blood vessels within the lungs were contracted and intrapulmonary shunt was reduced. meanwhile, the bronchial β2 receptors were excited and the bronchi were dilated so as to improve the ventilation/blood flow ratio and relieve hypoxia. further studies are merited. (f) others: there have been reports on sympathomimetic drugs (isoproterenol) and β-blockers (propranolol), etc. that improve the symptoms of hps. theoretically, vascular endothelin, estrogen suppressor (tamoxifen) and so on can relieve the spider nevus and pulmonary vascular dilatation in patients with liver cirrhosis and improve their respiratory symptoms, while further studies are needed. no is most studied currently, and there are reports indicating that no synthesis inhibitors can increase pulmonary vascular resistance. alexander et al. used no for the treatment of severe hypoxemia in patients after liver transplantation, and obtained good results. durand et al. also reported that an hps patient was cured by inhaling no, while its mechanisms and clinical efficacy needed to be further confirmed. 3. pulmonary embolism it is generally considered that pulmonary vascular dilatation can vanish after liver transplantation in hps patients who are normal on pulmonary angiography or who have cavernous vessels on imaging [310] ; for patients who show diffuse pulmonary vascular dilation features on pulmonary angiography, embolization is usually not adopted since patients' lesions are extensive and the efficacy is poor; for patients with isolated and severe pulmonary vascular dilation or arterial-venous communicating branches, local pulmonary embolism therapy can yield a satisfactory effect. 4. liver transplantation it is currently considered that liver transplantation is still a possible fundamental measure for the treatment for hps. in the past, it was believed that serious hypoxemia was an absolute contraindication against liver transplantation, while recent studies show that liver transplantation is preferred for patients who have good alveolar gas diffusion function, who can respond well to pure oxygen inhalation and who can undergone oxygenation safely during anesthesia. recent reports further prove that hypoxemia can be cured after liver transplantation [308] . through literature review and case reports, krowka et al. believed that progressive hypoxemia in hps could be used as an indication of liver transplantation. temporary hypoxemia following liver transplantation can be adjusted by using no and taking the head-down supine position and the alternate lateral decubitus position. and for hps patients who fail to respond to the inhalation of pure oxygen, who have direct pulmonary arterial communicating branches on pulmonary angiography and who have severe clinical hypoxia, liver transplantation cannot improve their hypoxic status, has limited efficacy, or even increases intraoperative and postoperative risks. therefore, liver transplantation should not be performed on them. tips was an effective method for the treatment of hps, and its effects of improving symptoms, enhancing oxygenation and reducing intrapulmonary shunt could last up to 4 months. riegler et al. performed tips on an hps patient with diffuse intravascular dilatation who was not suitable for vascular embolization, and the results showed significantly increased pao 2 and significantly improved hypoxemia. however, coley et al. also reported that a patient failed to respond to tips, and therefore, its exact effects remain to be studied. 6. other treatment options one patient with hps was once treated with garlic, and 18 months later, his oxygenation was significantly improved and his symptoms were relieved. there are also patients who receive plasma replacement therapy, which has limited effects on the oxygenation of patients with hps [308] . to sum up, no effective treatment options are currently available for hps. since the basic cause of hps is liver cell failure, the usual cause of patients' deaths is not lung failure, mostly complications such as gastrointestinal bleeding, renal failure, hepatic encephalopathy and sepsis. therefore, we consider that the therapy of primary liver disease is particularly important. oxygen inhalation alone can be given in the early stage of hypoxemia, or conservative treatment can be provided if additional drugs are effective. liver transplantation is the best solution whenever possible. it is generally accepted that liver transplantation is the most promising regimen with confirmed efficacy. if oxygen inhalation is less satisfactory and patients are diagnosed with local intrapulmonary vascular dilatation or arterial -venous fistula by such means as pulmonary angiography, pulmonary embolism should be carried out as soon as possible. for patients with additional obvious portal hypertension, tips treatment can also be given. the interval from chronic liver disease and cirrhosis in patients to the confirmed hps due to such respiratory symptoms as anoxic dyspnea is usually several years or even more than 10 years [average interval, (4.8 ± 2.5) years], and a small number of patients can develop such a disease acutely in the short term. besides, signs of chronic liver disease can be traced in patients complaining breathing difficulties. once hps is established, obvious hypoxemia has occurred already. it confers a poor prognosis, and most patients die within 2-3 years often due to other complications of liver disease [312, 315] . if patients' oxygenation is satisfactory and they have undergone liver transplantation, or with the improvement in liver function, their hypoxemia can be resolved or improved of its own volition with good prognosis. if patients' oxygenation deteriorates severely and they have a very poor prognosis, most of them will die in the short term. hps often progresses slowly. although it is not a direct cause of death in patients with cirrhosis, it can significantly aggravate the disease. therefore, cirrhotic patients, especially those with positive liver palms and spider nevus as well as patients with portal hypertension, should be careful of the possibility of hps. timely detection and symptomatic treatment (such as low flow oxygen inhalation) can improve the prognosis of patients. active and effective treatment of primary liver disease forms the basis for the prevention of this disease. education of common sense should be given to patients with liver diseases so as to avoid factors inducing hps in their life. for patients with liver disease, mild hps should be found as soon as possible and appropriate treatment should be given. jia-quan huang and dong xu lipopolysaccharide (lps) is a constituent of bacteria cell wall which plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as nitrous oxide, cytokines, superoxide anions, and lipid mediators. despite the recent advances in antibiotic treatment and hemodynamic monitoring, septic shock still remains a serious disorder that is associated with a high mortality rate, to more comprehensive definition of the mechanisms that underlie innate immunity against bacterial pathogens, lps has been extensively studied [319] . the pathophysiological consequences of bacterial sepsis are contributed by the dysregulation of these same mechanisms. before we can hope to design effective anti-sepsis therapies, greater insight into the nature of host interactions with lps is extremely essential. the gram-negative bacterial envelope is composed of two bacterial membranes, outer and inner membrane. the outer membrane consists of the following substances, like lipopolysaccharide (lps), several kinds of outer membrane proteins, lipid a and metal ions. for most gram-negative bacteria, lps is a major component in the outer monolayer of the outer membrane which works like a tight shield. the shield is composed by unique molecules, such as polysaccharide, or long chain of sugar, and lipid a. during the process to evoke the signaling events of lps, lipid a plays a pivotal role. the entire lipid component of lps molecule, however, is required for optimal activity [320] . the basic principles of lps bioactivity are nowadays well understood. endotoxins do not elicit their toxic effects -as we might suspect and as it is known for many proteinous exotoxins which can kill host cells or inhibit cellular functions. rather, lps requires the active response of host cells. according to present knowledge we get, lps interacts with various host cell types through lipid a, those cells include mononuclear cells, thrombocytes, endothelial and smooth muscle cells, and polymorphonuclear granulocytes, among which macrophages/monocytes are of particular importance. through the lps-induced activation, macrophages produce many substances, like bioactive lipids, reactive oxygen species, and in particular, cytokines such as tumor necrosis factor a (tnf), interleukin-1, il-6, il-8, and il-10. it appears that when low levels of mediators are produced, beneficial effects (e.g., induction of resistance to infection, adjuvant activity) are elicited and when high levels of mediators reach the circulation that detrimental effects (e.g., high fever, hypotension, irreversible shock) are induced. however, when the host organism is in a hyperreactive state lps, low mediator concentrations may also become harmful. the hyperreactivity to endotoxin may be caused by exo-toxins, chronic infection, and by growing tumors, and interferon-γ. to function properly, organism requires an immune system that must detect pathogens, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. the immune system can be classified into humoral immunity versus cell-mediated immunity or the innate immune system versus the adaptive immune system. when microbes invade organism, the innate response is usually triggered by pattern recognition receptors, which recognize components that are conserved among broad groups of microorganisms, or when injured, damaged, or stressed cells send out alarm signals, many of which (but not all) are recognized by the same receptors as those that recognize pathogens. the innate immune system defenses are nonspecific, meaning that the system responds to pathogens in a generic way. this system does not confer long-lasting immunity against a pathogen. in most organisms, the innate immune system is the dominant system of host defense [321] . they activate innate immune responses by identifying some conserved non-self molecules, so as to protect the host from infection,. bacterial lipopolysaccharide (lps), an endotoxin which is found on the bacterial cell membrane, is considered to be the prototypical pamp. lps is specifically recognized by toll-like receptor (tlr) 4, a recognition receptor of the innate immune system. the interaction of the lipid a moiety of lps with macrophages appears to be especially important because subsequent cellular activation results in the release of systemically active pro-inflammatory molecules, which in turn mediate systemic toxicity. lps has extreme potential in macrophages activated at concentrations of lps as low as 1 pg/ml [322] . host-defense peptides (hdps) could be a possible alternative solution since they possess the antimicrobial, antiseptic, and immunomodulatory properties [323] . endotoxins lipopolysaccharide is released not only from dead gram-negative bacterial, but also from the growing ones. endotoxins are very stable molecules, which are resisted to extreme temperatures and ph values in comparison to proteins. endotoxins are shed largely during cell death as well as growth and division. they are highly heat-stable and are not destroyed under regular sterilizing conditions. endotoxin can be inactivated through exposed at temperature of 250 ° c for more than 30 min or 180 ° c for more than 3 h. acids or alkalis of at least 0.1 m strength can also be used to destroy endotoxin in laboratory scale [324] . gut microbiota is composed of strict anaerobes, facultative anaerobes and aerobes. recent reports suggest the existence of over 35,000 bacterial species in the human gut microbiota. an important characteristic of gut microbes is their heterogeneity [325] . the composition and the frequency of the microbiome changes with the different segments of the elementary tract. the composition is influenced by the environment, consumed diet and host factors. endotoxin to surrounding tissues and organs or blood shift that shift pathway include: (1) via the portal vein, liver into the systemic circulation; (2) through the intestinal tract into the lymphatic system lymphatic; (3) through the intestinal wall into the peritoneal cavity and then absorbed into the bloodstream. under physiological conditions, although a small amount of toxins continued to parenteral shifted via the portal vein into the liver, but it does not cause endotoxemia; mild in gram-negative bacilli infections, although bacteria continue to release to the tissue or blood endotoxin, but it does not give rise to a strong inflammatory response, the above are dependent on the presence of an effective mechanism within the body to remove toxins and detoxification. the liver is the main organ of clearance of endotoxin, and the spleen, also removes toxins. molecules in lps removed include cationic antimicrobial peptides (cationic antimicrobial peptides, cap), acyloxy acyl hydrolase (acyloxyacyl hydrolase, aoah) lipoprotein binding protein and anti-lps antibodies are important endotoxin clearance methods [326, 327] . liver blood endotoxin clearance, primarily through kupffer cells, hepatocytes internal toxin endocytosis achieved, but the specific metabolic process is not entirely clear. mediated by kupffer cells engulf toxins may be scavenger receptor, it may be a molecular weight of 119,000 and 83,000 protein; mediate phagocytosis liver toxin structure may be the lectin-like receptor (lectin-like receptor). endotoxin receptor hepatocyte sinusoidal plasma membrane on the surface: after one to one binding, is taken up within the liver cells endocytosis way to microtubule-dependent vesicular transport through the liver cells, transported to the liver cells bile duct surface to exocytosis into the bile duct, and then discharged into the biliary system through the intestine [328] . splenic macrophages containing approximately 15% of the body to settle within the organization, and macrophages are important endotoxin removal cells. when endotoxin intravenously into the body, except gathering in the liver, a lot of endotoxin can be quickly gathered and taken up into macrophages in the spleen, the spleen and therefore equally important endotoxin removal organs. in addition to its clear role in the performance of its direct effect, but more importantly, spleen macrophages is the precursor cells of kupffer cells in the liver, having a very big impact on removing toxins within the liver [329] . mechanisms for removing toxins in the intestine are related to the intestinal villus tip epithelial cells. under normal circumstances, injected into the intestinal, endotoxin in intestine does not enter intestinal epithelial cells, but after intravenous injection of endotoxin, endotoxin may enter intestinal epithelial cells inside. therefore, endotoxin receptor may identify ways by endotoxin and (or) simple diffusion way into the intestinal epithelial cells. endotoxin way into the intestinal epithelial cells intravenously may have two: (1) displaced from the lamina propria macrophages to intestinal epithelial cells basolateral; and enter intestinal epithelial cells from the side; (2) including lower toxin, intestinal lamina propria macrophages, intestinal epithelial cells release large amounts of no and oxygen free, resulting in intestinal lamina propria microvascular injury, increased permeability, extravasation of endotoxin and ultimately displaced into the outer intestinal epithelial cells. villus tip epithelial cells within a stronger uptake of toxins, thus endotoxin can be started from the crypt, moving along the intestinal villi, and finally to the top of the inner hair cells of the intestinal epithelium. uptake of endotoxin villus tip epithelial cell loss, while the endotoxin into the intestine, which constitutes one of the effective clearance mechanisms of endotoxin [327, 330] . plasma lipoproteins in endotoxin detoxification mechanisms play an important role, in which the lipopolysaccharide binding protein (lipopolysaccharide binding protein, lbp) and high-density lipoprotein (high density lipoprotein, hdl) play a major role. endotoxin into the bloodstream within minutes there were half white blood circulation due to binding to the edge of the pool or the pool is cleared, the remaining residual endotoxin and rapidly bound to plasma lipoprotein is inactivated. in plasma, lipoproteins and endotoxin when hdl plays a major role in its binding of endotoxin to reach more than 50% of the total, hdl, and thus research endotoxin important [331] . cationic antimicrobial peptides are an ancient ingredients in the natural evolution of the immune system, including bactericidal/permeability-increasing protein (bactericidal/permeability-increasing protein, bpi), cathelicidin, lactoferrin, defensins and other substances, with not only the activity against gram-negative bacteria, but also the ability to combine internal toxins. cationic antimicrobial peptides are mainly in regular contact with the pathogen site mammalian skin, digestive tract, respiratory tract, and inherently express or express induced by pathogens and their products in the blood, secretions and neutrophil granules. cationic antimicrobial peptides have two types of three-dimensional structure; one is a α-helix, having such a molecular structure include cathelicidin and lactoferrin; the other is β-fold, including mammals α and β-defensins, etc. [332] aoah aoah is a glycoprotein produced by white blood cells with weight of 5.2-60,000, the large subunit of 50,000 and small subunits of 1.4 million to 20,000, the large and small subunits connected by covalent disulfide bond. aoah as a lipase with hydrolysis for toxin, can selectively hydrolyze the secondary acyl chain on lipid a acyl groups acyloxy. when hydrolyzing endotoxins, both the large and small subunits of aoah play an important role, and both are indispensable. in addition to directly destroying toxin, the deacylated lps after the hydrolysis of endotoxin by aoah is also involved in aoah's detoxification mechanism on endotoxin; the material can accumulate and inhibit endotoxin-induced inflammatory response in the cell. however, due to a limited number of secretion by local infiltration of leukocytes, the internal detoxification of toxins is also limited [333] . when the body respond with endotoxin, one trigger inflammation, on the other hand can be cleared to produce or activate, specific polysaccharide inactivate toxins, including antimicrobial-specific polysaccharide antibody and anti-core polysaccharide antibody. after two antibodies binding with endotoxin, and then with the fc receptors on the cell membrane, inner source of the toxin-mediated, so that the endotoxin inactivated intracellularly. anti-endotoxin antibodies can interfere with toxin within lbp binding, thus preventing lbp endotoxin transport [334] . in the body's defense system, the shift from the inhibition of intestinal endotoxin ingredients include intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, the normal intestinal flora and liver hepatocytes and kupffer cells, in which intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, hepatocytes and kupffer cells play a direct inhibitory effect, while the normal flora plays an indirect inhibition. the intestine is huge "endotoxin library", a special anatomical location determines the intestinal mucosa must be an effective defense barrier. immunological barrier intestinal barrier formed by the epithelial cells of mechanical barrier and secretory iga (siga) and the like components [335] . intestinal epithelial cells and tight junction formation mucosal mechanical barrier, is a significant barrier in the intestine endotoxin translocation defense to maintain its integrity is it to play an important role in defense guarantee. in severe trauma, burns, infection, considerable loss of body fluids, hypovolemia, cause the body ischemia and hypoxia. in order to maintain blood pressure, to ensure that the blood supply to the heart brain and other vital organs, compensatory splanchnic vascular contraction, including gastrointestinal ischemia and hypoxia longer time than other organs, even after shock patients after resuscitation to restore normal hemodynamics, stomach intestinal still in a state of shock occult. therefore, when the intestinal microvascular perfusion recovery, intestinal ischemic/reperfusion injury, epithelial cells produce large amounts of reactive oxygen species and other media, resulting in intestinal epithelial cell apoptosis, destruction of tight junctions between cells, thus rapidly increasing intestinal permeability mechanical barrier function weakens, thus contributing to the intestine of the endotoxin absorbed through the intestinal wall to parenteral tissue displacement [336] . intestinal mucosal intestinal immunology barrier is a defense of the invasion of pathogens and endotoxin important line of defense, siga plays an important role in intestinal mucosal immunity. siga is an important component of the protection of the intestinal mucosa, both to prevent bacteria in the intestine mucosal surface colonization, but also in endotoxin. studies have found that e. coli o157 infection suffered intestinal mucosa, the anti-endotoxin core polysaccharide-specific siga secretion, in convalescent patients has been particularly evident, suggesting siga endotoxin to prevent the transfer of the body has a protective effect. in addition, studies suggest that nitric oxide (nitrogen monoxide, no) preventing endotoxin translocation in intestinal mucosal barrier oxide formed locally. under physiological conditions, nitric oxide synthase (inducible nitric synthase, inos) expression only in the respiratory epithelium, the pregnant uterus and ileal mucosa and a few other parts. induced by endotoxin including but under normal colonic epithelial cells also express inos and catalytic synthesis of no, are formed in the local oxidation barrier to prevent bacterial translocation colon, thus effectively preventing bacterial translocation, also indirectly prevents endotoxin shift [337] . under physiological conditions, intestinal flora forms a relatively balanced microecosystem. flora distribution in the intestine has certain rules: deep close to the intestinal mucosal surface, parasitic anaerobic bifidobacteria and lactobacilli, these anaerobic bacteria are sugar coated, relatively stable, known as membrane flora; middle class bacteria, streptococcus digest, veillonella and excellent bacilli; the surface of e. coli and enterococci, can swim in the intestine, known as cavity flora. the antagonism between the layers flora, mutual cooperation, to maintain a dynamic equilibrium, in which the film anaerobic flora is a very important body's natural defense barrier that can prevent opportunistic pathogens such as e. coli colonization in the mucosa, but also can inhibit the overgrowth of opportunistic pathogens. intestinal flora micro-ecosystem is a very sensitive system, in severe stress or long-term systemic administration of large doses of broad-spectrum antibiotics, etc., the film significantly reduced the number of anaerobic bacteria, e. coli and other bacteria thrive conditions and continuous release of endotoxin to the intestine, since the film flora defense decreased, these opportunistic pathogens to colonize the intestinal mucosa, resulting in intestinal mucosal barrier damage, followed by the occurrence of intestinal bacteria, endotoxin translocation [338] . under normal circumstances, the liver is one of the major barriers preventing endotoxin translocation, via the portal vein into the liver hepatocytes and a small amount of the toxin can be kupffer cell depletion. in conditions such as stress, not only liver cell dysfunction, so the ability to reduce endotoxin detoxification and collaterals between the portal vein and the vena cava, causing an overflow of endotoxin from the liver into the systemic circulation. endotoxin absorbed into the bloodstream, which in turn may increase the intestinal epithelial cells of the intestinal microvascular endothelial cell damage and, in a vicious cycle [339] . when the body's defense system to produce responses in endotoxin, the innate immune system plays a major role. pathogens can be identified conserved receptors called pattern recognition molecules (pathogen-associated molecular patterns, pamps), including endotoxins of gram-negative bacilli, peptidoglycan grampositive cocci, lta and other cell wall composition and gram-negative bacteria, gram-positive bacteria such as dna. although a variety of pattern recognition molecules of different chemical structures, but they have similar characteristics: (1) characteristic structure in which different types of pathogens in a relatively constant conserved; produced by a pathogen, the host body without these molecules; survival or disease-causing pathogen is generally the essential, such as mutations, death or loss of a pathogen will pathogenicity. natural immune system to recognize the receptor molecule called pattern recognition receptors (pattern-recognition receptors, prrs), including cd14, toll-like receptor family (toll-like receptor, tlrs) and scavenger receptors. but in recognition of toxins, some differences exist between the different kinds of cells [340] . macrophages in addition to expressing cd14, tlrs and scavenger receptors and other associated endotoxin receptors on the cell surface, but in the cytoplasm also express the protein molecules nod1 recognizing toxins. cd14, tlrs are key receptors that mediate endotoxin within macrophage activation; and scavenger receptor has relationship with macrophage clearing and inactivating toxins [341] . kupffer cell is the main cell that clears the endotoxin in the liver. under physiological conditions, although there is still a small amount of bacteria and endotoxin via the portal vein into the liver, but kupffer cells will clear. kupffer cells are the most resident macrophages in the liver and are the largest number of resident cells in tissues. there is a theoretical speculation that if kupffer cells and on is very sensitive to endotoxin as other macrophages, the cell will be in constant activation, but in fact when kupffer cell engulfs, removes endotoxin, its itself is not activated by endotoxin, which suggests that in the treatment of endotoxin, kupffer cells have different mechanisms with other macrophages: kupffer cells treats endotoxin mainly depending on its phagocytosis. in the absence of serum, the phagocytic effects of kupffer cell on endotoxin can play a normal; and with the appropriate increase in endotoxin concentration, the phagocytic activity of kupffer cell was enhanced. the effect has something to do with phosphorylation events of two protein tyrosine residue individually weighting 11.8 million and 8.3 million. cd14 is the main receptor that mediates endotoxin activating kupffer cell, scavenger receptor is kupffer cells' important defense of receptor mediated kupffer cell to remove and inactivate endotoxin. there are four stages of the activation of kupffer cells, of which cd14 is the characteristic marker of cellular activation and function change. (1) the stationary phase; the performance of less number of kupffer cells, small shape, a number in the hepatic sinusoids, cd14 staining negative; (2) reaction period: for the local kupffer cells stimulate hyperplasia and systemic mononuclear macrophage intrahepatic accumulation; (3) pre excitation period: kupffer cell phenotype occurred transformation period, expressed cd14 cell membrane receptor, kupffer cell functional changes; (4) the activation period: the performance of nuclear transcription factor nf kb activation, cellular secretion cytokines [342] . the cd14 (cd14 membrane-bound, mcd14) and tlr4 endotoxin were activated by the neutrophil surface to activate the neutrophils by binding with the receptor. in addition to the expression of the high affinity endotoxin receptor cd14, the surface of the neutrophils also expressed the low affinity endotoxin receptor l-and the activated cells were activated by the receptor. in addition, the integrin is considered to be a low affinity endotoxin receptor for the surface of the neutrophils [343] . it is generally believed that the expression of mcd14 was not on the surface of endothelial cells and serum soluble cd14 (soluble cd14 (scd14) is mediated endothelial cell recognition of lps molecules, and tlr4 is involved in lps induced endothelial cell activation. lbp was transported to scd14 by endotoxin, and tlr4 was activated by lps/scd14 and activated endothelial cells in the endothelial cell membrane. scd14 in addition to mediated endothelial cell activation and also mediated by endothelial removal of endotoxin and lps/scd14/lbp form trimers and binding to endothelial cells, following the lps/scd14 endogenise, thus the removal of endotoxin [344] . the epithelial cells of the intestinal mucosa were consistently associated with the bacterial and its products, and the bacteria and its products could stimulate other types of cells and induce inflammatory response, but did not induce intestinal epithelial cell defense, this feature for colonic epithelial cells is particularly important, because if can react to the normal intestinal flora in intestinal epithelial cells, it will cause adverse effects on the body. but this does not mean that intestinal epithelial cells are immune cells, when suffered pathogens and their products invasion, intestinal epithelial cells produce normal response. description: intestinal epithelial cells with normal differentiation of natural bacteria and pathogenic ability, and the recognition system of subcellular localization. there are different endotoxin recognition mechanisms in the intestinal epithelium and the myeloid cells [327] . uncontrolled inflammatory responses (uncontrolled inflammatory response) has a relationship with infection, bacteremia, septicemia, sepsis, systemic inflammatory response comprehensive sign (systemic inflammatory response syndrome (sirs), compensatory anti-inflammatory response syndrome (compensatory antiinflammatory response syndrome, cars) and other related terms used for a long time, but also has an essential difference. out of control including inflammatory reaction syndrome (msas anti-inflammatory response syndrome, mars), a dynamic process of sirs and cars and the mixed antagonistic response syndrome, at present clinical many diseases occurrence and development are closely related. uncontrolled inflammation is a common pathological phenomenon in clinic, which is the important mechanism of the development of the complication after trauma. lps is one of the main factors that induce the uncontrolled inflammatory reaction in the most common. lps receptor on the monocyte/macrophage surface is the he initial factor for the body to recognize and start inflammatory reaction, also is one of the key links for the induction of uncontrolled inflammatory response. the concept of uncontrolled inflammatory response refers to inflammatory disorders then resulting in multiple organ dysfunction syndrome (multiple organ dysfunction syndrome. mods), emphasizes the importance of the of balance inflammatory/anti-inflammatory mechanism in the body, changes the limitations that in the past we only attached importance of the pathogenic effects of inflammatory factors. it is believed that the response of the body to the inflammatory factor is the dominant factor in the development of the whole body inflammatory reaction and mods. this concept is more focused than the previous focus on the dynamic changes of the whole process of inflammation. this can be divided into two types of mods: one is the early stage after the injury, that is, the speed hair style. the main blame is a strong inflammatory reaction induced by proinflammatory factors, and the other is a late phase of the disease, which is "late style", mainly due to the immune paralysis or worse immune disorders caused by cars or mars. the inflammatory is actually a kind of medium disease mainly caused by the chain reaction of cytokine. endotoxin is thought to be one of the most important predisposing factors in the chain reaction and can be referred for chain reaction "trigger" (trigger). endotoxin induced inflammation mechanism is mainly mediated by pamps that can induce cytokines such as il-1, tnf alpha and other active molecules synthesis, the formation of the cytokine network, has a very important role in the occurrence and development of infection. the excessive activation of cytokines can cause septic shock, and is the leading cause of death in patients with bacterial infections. accordingly, prrs plays an important role in innate immunity and inflammation, and it can distinguish the pathogens from self organization through prrs organism, which is characteristic of immune response [345] . inflammatory response syndrome systemic (sirs) is a systemic inflammatory response caused by any pathogenic factor to the body. the concept is first proposed by coris in 1985. 1991 august american college of chest physicians and critical care medicine to present the diagnostic standard of sirs, think to have the following each of the two or more than two, sirs can be established: (1) temperature > 38 deg c or < 36 deg c; ii heart rate 90 beats/min; (3), the breathing frequency > 20 times per minute or arterial blood carbon dioxide into pressure (paco 2 ) < 4.27kpa 32 mmhg; (4) peripheral white blood cell count >12 × 109/l or 4 × 109/l < or immature myeloid cells >10%. what should be paid attention to is that sirs is a common athophysiological state of body with severe inflammatory reactions, and should be differentiated from some abnormal factors such as leukemia or cause increase or reduction of white cells after chemotherapy. although the naming of sirs has been generally concerned, but some scholars have raised objection to the concept, for example sirs has following problems: the sensitivity and specificity of the diagnostic criteria is poor, has the same meaning with the "critical"; can not understand the pathophysiology of the original disease; is difficult to guide clinical trials and practice. the production of sirs can be divided into two cases, the sirs caused by the infection and the non infectious sirs. from the point of view of the clinical development process, sirs can be followed by injury immediately aroused, then known as the "single phase velocity hairstyle; also to start local, and later developed into a systemic sirs, namely after the initial shock is brief period of stability, later gradually intensified when sirs is called" dual phase delayed onset. either of the factors or the clinical development process, the systemic inflammation of the control of the uncontrolled, and ultimately can lead to mods [346] . the intestine is the biggest bacterial and endotoxin warehouse in the body. in severe trauma, systemic infection, intestinal ischemia and liver disease, there may be the occurrence of endotoxin. the main source is due to intestinal gram-negative bacteria in the excessive growth and reproduction, or due to increased intestinal permeability lps entry into the portal vein increased. if hepatic kupffer cell phagocytic function is low, the amount of endotoxin over the liver ability to remove endotoxin can "flood" (spill over) into the body of the loop and the endotoxemia formated. because of the endotoxin from the gut, so it is called intestinal endotoxemia (intestinal endotoxemia, ietm) [347] . hepatitis b patients are often accompanied with ietm. its formation mechanism is: the production and absorption of intestinal endotoxin increased. there is a large number of gram negative bacteria in the body's normal intestinal, so endotoxin in intestinal contents is very high, but the intestinal mucosal epithelial cells have stronger resistance to toxins so that endotoxin is not easy to run through the intestinal mucosa into the blood, even a small amount of endotoxin breaking through the intestinal mucosal barrier into the portal vein, will be swallowed up by the kupffer cells in the liver. severe hepatitis b when the intestinal flora disturbance, endotoxin increased, increased intestinal hyperemia, edema and the permeability of the intestinal mucosa; endotoxin itself can damage the mitochondria and lysosome of intestinal epithelial cells, leading to epithelial cell autolysis; endotoxin can cause intestinal microvascular contraction of the intestinal mucosa, reduce blood, intestinal ischemia, hypoxia, cause the intestinal mucosal barrier function decreased, increased the absorption of endotoxin; severe hepatitis, due to intrahepatic bile acid and bilirubin deposition in kupffer cell phagocytosis was inhibited, resulting in the removal of endotoxin in the endotoxin decreased; through the door body circulation circuit into the systemic circulation, resulting in blood within liver cells to escape kupffer toxin the phagocytosis and clearance, which aggravate endotoxemia; the endotoxin can also pass through the celiac lymphatic system into systemic circulation by thoracic duct. in addition, severe hepatitis patients with sepsis, spontaneous bacterial peritonitis, etc., in the release of endotoxin, so the formation of the endotoxin is exogenous [348] . in viral hepatitis and other basic diseases complicated with ietm and liver function failure are closely related and endotoxin can directly cause arterial vasoconstriction, the organ ischemia; endotoxin can activating endogenous clotting system, coupled with kupffer cell dysfunction, decrease delimination of blood coagulation or fiber soluble substances, easily lead to dic, so as to damage to multiple organs. endotoxin activated phospholipase a2 mediated membrane phospholipid degradation and lipid peroxidation, which is an important part of liver cell damage. nolan has pointed out that the effect of kupffer cell dysfunction induced by intestinal endotoxemia on liver and body, far more than the direct action the endotoxin, and production and release of inflammatory mediators and factors from kupffer cells activated by endotoxin are closely related. the occurrence of ietm affect hepatic energy metabolism, resulting in liver cell damage and necrosis, also caused hepatic microcirculatory disturbance, performing liver hemorrhagic necrosis. on the basis of severe hepatitis, it can accelerate liver function failure [349] . hepatic cellular jaundice the acute and chronic liver function is often accompanied by intrahepatic cholestasis jaundice, and ietm plays an important role in the occurrence of intrahepatic cholestasis.. endotoxin involves in liver cell damage mainly through activation of phospholipase a2, and inhibits the activity of na + − k + − atpase on liver canalicular membrane to make the bile excretion disorder, and then to cause intrahepatic cholestasis in the liver cells. endotoxin can start the peroxidation of liver parenchymal cells mitochondrial membrane lipid so that an increase in the content of oxygen free radical in blood, resulting in the disorder of energy generation, atp was reduced, so that the active uptake, metabolism and secretion of bile acid by liver parenchymal are short of energy, resulting in cholestasis [350] . the liver disease with ietm often causes the coagulation dysfunction, the serious person appears the different degree bleeding, in particular the severe liver disease patient may concurrent dic, endangers the life. violi found that, when liver dysfunction in patients with ietm, the expression of tissue factors on the surface of macrophages and endothelial cell factor induced by endotoxin increased, promoting the synthesis of tumor necrosis factor (tumor necrosis, factor, tnf) to increase, and thrombin generation increasing, activation of coagulation system in about 70% of patients, followed by hyperfibrinolysis, suggesting that ietm in liver dysfunction can be used as the warning signal as the activation of coagulation and fibrinolysis system and activate, and with the increased hepatic lesions, plasminogen activation decreases with endotoxin levels increased, thus plasminogen may decline with endotoxin induced by chronic consumption of dic on the microstructure of ii related factors, new blood can not correct. in fact, before variceal rupture bleeding, patients with liver function severely damaged already have the gastrointestinal mucosa extensive ischemia and erosion, which is the potential causes of bleeding, ietm in the process. and gastric h + at this time can occur abnormal reverse diffusion and stimulate mast cells to release histamine, may lead to mucosal blood vessel dilation and permeability enhanced. as a result, hemorrhage, edema; histamine and directly stimulate the secretion of gastric acid, so that an increase in the number of h + and reverse diffusion, lesions persisted, form a vicious circle [351] . it is important for the formation of ascites in ascites due to the obstruction of the hepatic vascular outflow tract obstruction. the initial vascular response to endotoxin was the rapid obstruction of the hepatic venous outflow tract and increased the portal pressure which may be related to endotoxin induced swelling of kupffer cells, liver cells with microvilli swelling, platelet aggregation and fibrin deposition effect, while others think that is endotoxin of blood vessels of the liver has a direct effect. ietm continuous damage to the liver cells, resulting in albumin synthesis and of hormones such as aldosterone de live function obstacles, thus affecting the renal function, and led to the emergence of the refractory ascites plays an important role in the process [352] . patients with severe liver disease always are complicated with the functional renal failure, hepatorenal syndrome (hrs). the patients with severe liver disease can be associated with the pre-renal azotemia and acute renal tubular necrosis, and there is a certain relationship with ietm. the clinical studies showed that the levels of no 3 --no 2 and endotoxin in serum of patients with liver cirrhosis were significantly higher. at the same time, plasma renin activity, aldosterone and vasopressin levels increased and urinary sodium excretion decreased. the mechanism about ietm induced by hrs is not clear, may be related to the following factors: leukotrienes (leukotrienes, lts) lts can lead to renal vasoconstriction, increased renal vascular resistance, reduced renal blood flow and renal blood redistribution, decreased glomerular filtration rate induced by hrs, in ietm lts generation and release increased obviously. in addition, liver dysfunction, liver's uptake, inactivation and excretory function of lts decline, causing blood concentration increased; the thromboxane a2 (thromboxane a2 txa2)/i2 (prostaglandin i2, prostaglandin pgi2) can contract renal arterioles, decrease glomerular filtration rate, while pgi2 and pge2 (prostaglandin e2, pg e2) is caused by on the role of txa2. in patients with severe hepatitis with ietm, elevated systemic levels of pgi2, reducing the renal vascular resistance, leading to renal vascular resistance txa2, reduce the renal blood flow and glomerular filtration rate, promote the formation of hrs; third, nitric oxide, nitrogen monoxide) nono can through vasodilatation of the systemic, resulting in effective circulating blood volume reduction and evoked hrs; endothelial endothelin (et) et caused renal cortical blood priming hrs; and platelet activating factor (growth factor (paf) endotoxin and platelet activating factor (paf) can lead to a decrease in cirrhotic rats cardiac ejection fraction, reducing blood flow to the kidney, and paf antagonist can improve hemodynamics changes [353] . the mechanism of endotoxin induced hepatic encephalopathy is not clear. we have known that lps can increase the permeability of blood brain barrier, promote intestinal toxic substances through the blood brain barrier (bbb), damage mitochondrial oxidative metabolism in brain cells, reduce oxygen utilization in patients with liver cirrhosis and disrupt energy metabolism of brain cells, induce brain edema. the clinical symptoms of chronic severe hepatitis with endotoxemia included in addition to fatigue, anorexia, tiresome of the oil, nausea, vomiting, yellow skin and sclera and, performance of endotoxemia. endotoxin can cause the release of histamine, 5-hydroxytryptamine (5-ht), prostaglandin, bradykinin, resulting in micro circulation expansion, venous blood volume reduction, decreased blood pressure, inadequate tissue perfusion, hypoxia and acidosis, and main symptoms and signs are: fever, elevated white blood cell count, bleeding tendency, heart failure, renal dysfunction, hepatic injury, nervous system symptoms and shock [354] . improve liver function this is the basic treatment of ietm. liver function improved can strengthen mononuclear phagocyte system function to help the endotoxin removal. it can also decrease portal vein pressure to relieve intestinal congestion, edema, hypoxia, improve the intestinal microenvironment, reduce production and lymph reflux, and lower door shunt. these all contribute to the prevention and treatment of ietm. clean the gut saline is available as enema if severe liver disease, which helps reduce intestinal endotoxin generation and absorption. decompensated cirrhosis is often accompanied by small intestinal bacterial overgrowth and intestinal flora disturbance. thus, the promotion of intestinal flora back to normal state help prevent and treat intestinal endotoxemia. a variety of bifidobacterium, lactobacillus can be selected. a synthetic disaccharide, it is not digested and absorbed in the small intestine, but can be broken down into lactic acid, acetic acid and other small molecules by the bacteria into the colon. such acidification of the intestine reduces the generation and absorption of endotoxin, and promotes the growth of intestinal bacteria, stimulates bowel movements so as to increases tool frequency and so on. in addition, the lactulose may have internal direct inactivation of toxins, prevents activation of macrophages to release cytokines. oral absorption of antibiotics can effectively suppress the generation of intestinal endotoxemia. patients with liver cirrhosis taking oral polymyxin e or neomycin, the level of plasma lps and no 2 -/no 3 -horizontal declines in synchronization. polymyxin b has an internal direct antitoxin effect [355] . it play a role by reducing intestinal absorption of toxins, inactivating toxins and inhibiting those lps-induced media by monocyte macrophages. the new anti-endotoxin therapy including interrupt endotoxin synthesis, binding or neutralizing its activity, preventing its interaction with the host effector cells, or interfering with toxin-mediated signal transduction pathways. therapeutic formulations include endotoxin analogs, antibodies, subunit vaccines, polymyxin combination column, recombinant human protein, small molecule inhibitors of endotoxin synthesis and intracellular signal transduction. bacteriophage producing a piece of short nucleotide sequence which plays the role of an antisense rna, blocking the synthesis of bacterial lps synthase. current clinical studies carried out an experiments in cloning of human anti-endotoxin lipid a light and heavy chain variable region. it laid the foundation for the next screening and expression that recombination between dna of antibodies and phage's succeeded. the clone45 is one kind of anti-polymyxin b (polymyxin b) monoclonal antibody of the igm class. it can play the role of anti-endotoxin shock by imitating the surface antigen structure of lipid a so as to substitute receptor antagonist of lipid a and lps blocking the causative link that the endotoxin induces inflammatory mediators [356] . isolating antibodies having a high affinity of various g-bacteria to prepare a chimeric monoclonal igg1 antibody sdz219-800 which have a therapeutic effect on the human endotoxemia. anti-endotoxin core glycolipid monoclonal antibody (antimonoclonal antibody r595) can prevent and treat the metabolic disorders in peritoneal infection with mods; it plays a significant role in conditioning in high catabolism, and can significantly improve metabolic disorders under the condition of abdominal infection associated with mods. bactericidal/permeability-increasing protein (bpi) bpi is a human endogenous protein, found primarily in neutrophils primary particles. its molecular amino-terminal and carboxy-terminal appear v-shaped structure planar symmetry. many amino acids to form a hydrophobic capsule hold lps's lipid a. it was reported that bpi has an obvious protective effect on intra-abdominal infection induced sepsis, which might be related to its antagonism against endotoxin [357] . reconstructing hdl (high density lipoprotein, hdl) hdl can be used as an endogenous lps scavenging system, binding of bacterial endotoxin with high affinity to form a stable hdl-lps. lps-hdl complexation may contribute to a reduction in endotoxic activities in vivo by preventing lps (lipid a) from generating important transmembrane signals after binding to cells [358] . e5531 is the first generation lipid a analogue, which is derived from the lipid a structure from the endotoxin of rhodobacter capsulatus. it can block lps in cell culture without any endotoxin-like activity. e5531 can protect mice from lethal doses of lps, and viable e. coli infections in combination with antibiotics. in human healthy volunteers who are exposed to intravenous lps. e5531 also blocks the endotoxin response [359] . an anti-cd14 antibody cd14 has a very important role in monocyte-macrophage cell signaling. since epitopes of lps on the cell membrane at the binding site is the same material with soluble cd14, so we can develop a monoclonal antibody interfering with lps binding to cd14 and blocking to pass activation signals from immune effector cell [360] . tyrosine kinase and mitogen-activated protein kinase are involved in lps cellular signal transduction. build anti-endotoxin (lps) single-chain antibody gene and attempt to make it express in e, coli. the scfv gene was successfully constructed and gst scfv fusion protein highly expressed in e. coli was obtained. glucocorticoids, including the synthetic glucocorticoid dexamethasone, are recognized for their anti-inflammatory properties and have the ability to inhibit the production of proinflammatory cytokines such as tnf-α. in intestinal ischemia-reperfusion methylprednisolone pretreatment can prevent endotoxemia. combined lps and dexamethasone treatment at 120 h significantly changed tnf-α [361] . this points that glucocorticosteroids added before or during stimulation of macrophages can prevent tnf release, after which the administration would be invalid. in fact, it is very difficult to use corticosteroids before tnf release. current clinical anti -tnf antibodies and tnf antagonist use exists, and before many scholars have obtained a more satisfactory results of blocking or neutralizing excessive tnf with anti-tnf. although the clinical symptoms improved, yet the survival rate is not higher than expected. the effect of anti--tnf clinical application needs further evaluation. the first proposed concept is sequential organ failure, based on which multiple organ failure (multiple system organ failure, msof or mof) is put forward, and in 1980 diagnostic criteria is developed, but it reflected the end-stage, denied reversibility, ignore the dynamic development from organ dysfunction to failure. therefore, the us accp/sccm proposed to replace the concept with mods. mods emphasized an early phase of organ dysfunction before overt failure occurred. it is defined as "the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention." [362] organ dysfunction may be relative, or can be absolute; with extension of time, mods can increase or reverse. thus, the term. mods was coined to indicate the wide range of severity and the dynamic nature of this disorder, which contributes to early diagnosis and treatment of patients and is more in line with clinical practice. there are two relatively distinct (although not mutually exclusive) pathways by which mods can develop: in primary mods, there is a direct insult to the organ that becomes dysfunctional. examples of such direct insults include gastric aspiration in the lungs or rhabdomyolysis in the kidney. this direct insult causes: an inflammatory response that is localized, at least in the beginning, to the affected organ. secondary mods is a consequence of trauma or infection in one part of the system that results in the systemic inflammatory response and dysfunction of organs elsewhere [363] . secondary mods means not directly caused by damage, but to experience the "second strike", the first blow can make the immune system in a preactivated state under which inflammation lost control and significant sirs appears, then the following second strike can quickly cause multiple distant organ dysfunction and easily form sepsis with the basis of sirs/cars. this type of mods often develops as the following model: the original cause → stress → immune wpw → sirs/cars → infection → sepsis → mods → mof. the process of severe hepatitis, can cause intestinal damage and massive release of endotoxins into the blood, leading to intestinal endotoxemia (ietm). endotoxin is a powerful trigger for complement activation, and then these complement can activate "cascade effect" (cascade) to release oxygen free radicals, prostaglandins, endorphins, paf, cytokines and other inflammatory mediators to cause cytotoxicity, the error of microcirculation and tissue metabolism, eventually leading to the occurrence of mods. in this case, we emphasize mods originated in continuous, uncontrolled inflammation and factors causing systemic inflammatory response can be induced mods, including bacteria, fungi, parasites, viruses, toxins and other infectious agents. it is a debate of long-century problems that whether endotoxin has effects on hearts. the late 1970s a large number of experiments prove endotoxins has a role of heart. it enables reduction of coronary blood flow, decrease of total coronary vascular resistance, and have meanings in heart failure. studies have shown that endotoxin can damage myocardial mitochondria, muscle paddle net, muscle membrane, contractile proteins etc., leading to cell membrane system damage, energy metabolism. past research in cardiac dysfunction under endotoxin shock did not attract enough attention, for they think that the heart is the final failure among all organs so that clinical treatment value is little. now people's awareness has completely changed that heart dysfunction can occur early in sepsis or septic shock. therefore, early identification and prevention of the occurrence and development of cardiac dysfunction may have some clinical value for treatment of septic shock and other serious complications [364] . clinical gram-negative bacteria sepsis often complicated by adult respiratory distress syndrome, or server development of mods. in this pathological process, the high biological activity of endotoxin plays an important role. endotoxins often involving the lungs firstly, and the pathogenesis of non-injury may be associated with the direct damage on endothelial cell by endotoxin through complement pathway and induction of cytokines [365] . the liver is the major site of endotoxin on clearing and detoxifying and the place where clinical gram-negative bacteria sepsis often can be complicated, and it is also the primary organ suffering attacks by toxins. it is generally believed that toxins in the liver circulating mainly from the gut and liver dysfunction is closely related to the formation of endotoxemia. histological examination revealed the inner toxins damage the liver cells, showing sinusoidal congestion, dieldrin expansion chamber, kupffer cell swelling, endoplasmic reticulum、mitochondria swelling, crest destroy and lysosomal activation etc. [366] . the liver is the body's largest metabolic organ, and many cases of acute liver failure often involve other organ complications, which has become an important factor in determining the prognosis. endotoxins may cause the reduction of liver nutritional blood flow, mitochondrial oxygen metabolism, interfering with sugar metabolic pathways in liver leading to metabolic disorders. various damaging factors (such as gastrointestinal disorders, ischemia, immunocompromised, dysbiosis) promote absorb of intestinal bacteria and toxins and displacement via the portal vein, the lymphatic system into the systemic circulation. on the one hand these infectious agents can directly damage liver cells, or mediate hepatic injury whether by kupffer cell; on the other hand it can induce systemic inflammatory response by the monocyte-macrophage cells to release the media, both leading to organ perfusion disorder, affecting protein synthesis and energy metabolism, eventually resulting in severe sepsis, mods and even death. the effect of endotoxin on kidneys is not clear yet. in the early stage, it can affect the kidneys, decreased its blood flow renal via vasoconstriction. when endotoxemia is complicated by renal failure, the mechanism of glomerular filtration rate decreasing is unclear. the pathophysiology of aki in sepsis is complex and multi-factorial and includes intrarenal hemodynamic changes, endothelial dysfunction, infiltration of inflammatory cells in the renal parenchyma, intraglomerular thrombosis, and obstruction of tubules with necrotic cells and debris [367] . the relationship between endotoxin and dic is quite complicated. dic is considered to be an important incentive of mods, especially patients with severe sepsis with dic having a highly possibility of developing mods, what's more, the prognosis is very poor, and the mechanism is multifaceted. endotoxin can start the endogenous coagulation system directly or via activating factor xii (hageman factor) by damaged endothelial cells, also can act on the monocyte-macrophage cells, stimulate the release of tissue factor to trigger the extrinsic coagulation pathway [368] . in clinical practice it has been noted that serious infections is very possible to be complicated by gastrointestinal failure. the intestine is the biggest reservoir of bacteria in the body and leakage of bacteria or microbial products, notably lps, from the lumen of the gut into the systemic compartment, leads to initiation or amplification of a deleterious inflammatory response and mods [369] . after endotoxins challenging the gastrointestinal mucosa, it initially shows mucosal telangiectasia, interstitial edema and hemorrhage. microcirculation leading to damage of lysosomal and release of proteases in the cell, cell degeneration and necrosis. in addition, mucosal cellular energy metabolism decrease, h + reverse diffusion, prostaglandins and bradykinin further aggravate mucosal damage. at the same time destroy of the gastric mucosal barrier also make a lot of bacteria and endotoxins pass through the gastrointestinal mucosa, migrate to the blood circulation, the lymphatic system and the abdominal cavity etc., leading to systemic multi-system organ damage. clinical characteristics of mods 1. organ failure usually do not result directly from the primary injury. there is a certain time interval from the primary injury to organ failure. 2. not all of infection have bacteriological evidence, and more than 30% of patients and autopsy found no infected lesions. thus, to identify and treat the infection may not be able to improve the patient's survival. 3. mods may have perfectly healthy organ involved, and it is ferocious and rapidly progresses. once happened, it is difficult to depress in the event almost, so often with a high mortality rate. 4. in pathology, mods lacks features, the affected organ only showing acute inflammation, such as inflammatory cell infiltration and so on, and these changes are very inconsistent with severe clinical manifestations, and once restored, patients do not have any clinical sequelae. 5. mods is closely with shock and infection. shock, infection, injury (including trauma and surgery, etc.) are the three main causes of mods. 6. generally the later period of shock will typesetting idc and mods, and the order of occurrence of mods usually is the lungs, liver, kidney, gastrointestinal tract, finally the heart. the characteristic clinical manifestations 1. instability of circulation due to a variety of inflammatory mediators have effects on the cardiovascular system, the circulation is most likely involved. almost all cases, at least in the course of the early and middle will be in highpower type of cycle of "high ranked low resistance". cardiac output up to 10 l/ min or more and low peripheral resistance cause shock and need vasopressors to maintain blood pressure. 2. high metabolic systemic infection and mods are usually accompanied by severe malnutrition. its metabolic mode has three salient features: (1) persistent high metabolism, metabolic rate up to 1.5 times more than normal; (2) abnormalities of energy pathway. in starvation, the body obtain energy mainly through the decomposition of. however, with systemic infection, the body will get energy by breaking down proteins while the use of sugar is limited and fat utilization may increase early, fall later; (3) poor response to exogenous nutrient, supplement of exogenous nutrition can not effectively prevent itself consumption, which suggests that a high metabolism itself has a "mandatory" also known as "autophagy metabolism." high metabolic may have serious consequences. first, protein malnutrition result from it will cause serious damage to the structure and function of the enzyme system of organs; secondly, imbalance of branched-chain amino acids and aromatic amino acid which makes the latter formate into a pseudo-neurotransmitter, then further lead to dysfunction of nerve. 3. hypoxia in tissue cells at present many scholars believe that the high metabolic and circulatory disorders often cause oxygen supply and oxygen demand does not match, so that the tissues of bodies are in a hypoxic state, mainly clinically manifesting "oxygen supply dependency" and "lactic acidosis. ". currently mods still lacks an unified diagnostic criteria, and any one of the diagnostic criteria of mods is difficult to reflect the entire contents of organ dysfunction, so in clinical practice we can select one according to our own specific situation. 1. the main contents from national critical care medicine conference standard in 1995 are: (1) respiratory failure: r > 28/min; pao 2 < 6.7 kpa; pco 2 > 5.89 kpa; pao 2 /fio 2 ≤ 26.7 (200 mmhg); p (aa) do 2 (fio 2 1.0) > 26.7 kpa (200 mmhg); x-ray of chest shows alveolar consolidation ≥1/2 lung (which have more than three or three); (2) renal failure: except prerenal factors, little or no urine, serum creatinine, increased blood urea and nitrogen levels, exceeding more than twice the normal value; (3) heart failure: systolic blood pressure < 80 mmhg (10.7 kpa), sustained more than 1 h; ci <2.6 l/(min · m 2 ); ventricular tachycardia; ventricular fibrillation; degree atrioventricular block; resuscitation after cardiac arrest (with which three or more); (4) liver failure: total bilirubin>34 μmol/l; liver enzymes increased more than 2 times compared with the normal; prothrombin time > 20s; with or without hepatic encephalopathy; (5) dic: platelets 100 × 10 9 /l; prothrombin time and partial thromboplastin time prolong 1.5 times, and fibrin degradation products increase; systemic hemorrhage; (6) brain failure: glasgow score below 8 means coma, and less than 3 points means brain death. 2. the sooner the primary diseases or the primary risk factors are eliminated or controlled, the greater the possibility of organ recovery is. 1. to effectively rescue and debride as soon as possible, prevent infection, prevent ischemia-reperfusion injury, use a variety of supportive care; 2. to reduce stress response, mitigate and shorten high metabolism and the magnitude and duration of glucocorticoid receptor; 3. to pay attention to the patient's breathing and circulation, as soon as possible to correct hypovolemia and hypoxia; 4. 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microcirculation in experimental endotoxemia in rats characterization of the effect of lps on dendritic cell subset discrimination in spleen protective effects of transforming growth factor β2 in intestinal epithelial cells by regulation of proteins associated with stress and endotoxin responses native high-density lipoprotein augments monocyte responses to lipopolysaccharide (lps) by suppressing the inhibitory activity of lps-binding protein an altered immune response, but not individual cationic antimicrobial peptides, is associated with the oral attenuation of ara4n-deficient salmonella enterica serovar typhimurium in mice bovine blood neutrophil acyloxyacyl hydrolase (aoah) activity during endotoxin and coliform mastitis importance of antibodies to lipopolysaccharide in natural and vaccine-induced serum bactericidal activity against neisseria meningitidis group b. infect immun effect of tridax procumbens on liver antioxidant defense system during lipopolysaccharide-induced 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evolving history pathophysiology of septic acute kidney injury: what do we really know? sepsis, thrombosis and organ dysfunction bile high-mobility group box 1 contributes to gut barrier dysfunction in experimental endotoxemia key: cord-015126-cyhcbk1j authors: nan title: ps 0036-0344 date: 2007-08-25 journal: intensive care med doi: 10.1007/s00134-007-0820-y sha: doc_id: 15126 cord_uid: cyhcbk1j nan in our 21-bed icu-cum-hdu of a 550-bed tertiary referral cancer centre, medical oncology admissions increased from <5% of total admissions to over 35% in the last 3 years. we audited outcomes in these patients to determine prognostic factors that may aid patient selection and management. methods. 263 consecutive admissions (170 males, 93 females, age > 16years) from february 1, 2006 to february 28, 2007 were prospectively studied. the total sofa score on day1 (sofa1), the highest sofa score of the first three days (max3) and the change in sofa score between day2 and day1 (delta1) and between day3 and day1 (delta2) were calculated. predictors of outcome were identified using univariate and multivariate binary logistic regression. results. 58 patients had solid tumours , 89 had leukemia, 84 lymphoma, 17 myeloma, and 15 had other diagnoses. mean age was 44.8±16 years and apache ii score was 21.2±7.9. icu mortality was 71% and hospital mortality 78.7%. 67/74 patients (90.5%) with icu stay < 1 day died. overall length of icu stay was 3.7±4.3days. in survivors vs. nonsurvivors, sofa1, delta1 and delta2 (median, interquartile range) were 3.0(1.0 to 6.0) vs. 8.0(5.0 to 12.0; p<0.000), -1.0(-2.0 to 0) vs. 1.0(-1.0 to 3.0; p<0.000) and 0(-2.75 to 1.0) vs. 1.0(-1.0 to 5.0; p<0.02), respectively. several factors were associated with mortality on univariate analysis (table 1) . on multivariate analysis, only need for vasopressors (or 9.14, p=0 .002) and max3 (or 1.52, ci 1.19-1.93, p=0.001) were independently associated with hospital mortality, while type of cancer and leucopenia were not. for 141 patients staying >2 days, no factor predicted hospital mortality, but sofa1 (or 1.64, ci 1.01-2.68, p=0.047), delta1 (or 1.57, ci 1.15-2.14, p=0.004) and delta2 (or 1.25, ci 1.01-1.60, p=0.04) predicted icu mortality. usually, the cgr transfused in our icu are old (about 80 % of rbc are stocked more than 14 days). icu outcome is independently associated with the number of rbc transfused, but not with their age. this result in contradiction with previous report could possibly be explained by the systematic leucodepletion performed before storage in france, contrary to precedent studies where rbc were not leukodepleted. we compared them with ≥70 years old and an icu stay < 30 days patients, the differences in icu mortality, apache ii, age, gender and the necessity for renal replacement therapy (rrt) were not significant (see table) . the survivors patients (≥70 years old and an icu stay ≥ 30 days) were more older and 21(65'62%) were still alive one year later. when we analyzed the overall patients, according their stay < or ≥ 30 days, did not find statistically significant differences between both groups in the mortality (p=0'610 conclusion. icu mortality rates in elderly patients with a stay < or ≥ 30 days at icu were comparable. the 1 year-survival of elderly patients with a long-term intensive care unit stay was high. results. seventy patients were admitted to our icu with the diagnosis of acute pancreatitis during study period and 35 of them were later confirmed as having sap. the average icu length of stay in patients with sap was 17 days compared to 5 days in patients with mild form of the disease. pancreatic infection was present in 8 patients. the mortality rate in the group with sap was 31% compared to 2,8% in the group with mild acute pancreatitis, p< 0,05. the most common etiology of patients with sap was biliary and this was similar both in survivors and non-survivors. the most common cause of death in the group with sap was multiple organ dysfunction/failure syndrom(mods/mof) in 80% followed by bleeding complications in 20%. twelve patients with sap (34%) underwent the surgical intervention. mortality in the group of patients who underwent a surgical intervention was 25% (3 patients). 55,0+/-17,5 56,3+-14,08 apache ii score( mean+/-sd) 20,2+/-11,3 10,0+-6,9* necrotising form (%) 90 88 infected necrosis (%) 20 24 ct guided fnab (%) 30 32 * p > 0.05 conclusion. the patients with mild form of acute pancreatitis had low mortality rate (similar to general ward population) despite positive icu admission criteria in our case series with fifty per cent development of severe form with organ dysfunction/failure later on. apache ii score was better predictor of mortality in patients with sap than presence, extent or infection of pancreatic necrosis. patients with higher risk for development of severe form of acute pancreatitis should be admitted to multidisciplinary icu prior to definitive diagnostic evaluation of pancreas. further studies are warranted. conclusion. absi is an aprropiate score for estimating the probability of death in critical brun injury patients. preexisting cardiac and liver diseases have a little influence on mortality and its addition to the absi variables don't predict mortality more accurately. poisoned patients constituted up to 10,5 % of all icu admissions in our hospital. demographic data and specific poisons have been presented at the table. the total poisoned mortality rate was 10,2 %. methyl alcohol poisoning has a higher mortality than others poisoning. conclusion. childhood poisoning is usually accidental and is usually associated with a low morbidity and mortality. in adults, self-poisoning is usually deliberate suicide or parasuicide) and has a higher morbidity and mortality rate. (1) the most important part of the poisoned patient's care are the general supportive management and specific antidotes therapy. it has abundantly been demonstrated that duration of mechanical ventilation can be reduced by the use of protocols for weaning and sedation [1, 2] . utilization of the required sedation scales and adherence to protocols, however, is poor in daily practice, as has been shown in recent studies [3, 4] . it has been proposed to use daily checklists to improve the quality of care [5] . to improve adherence to the established guidelines for weaning and sedation in our icu, we included two questions in a checklist printed on patients' charts which had to be answered daily by the physician on duty: conclusion. the checklist as a daily reminder to observe established weaning and sedation protocols may have significantly accelerated weaning from mechanical ventilation. we carried out a prospective and descriptive study in patients admitted to our icu from 1993 to 2004. we defined tolerance as the need to use more than 300 mg/h, at least for four hours, or the need either to use or to change to other sedatives to obtain a 3 to 5 level on the ramsay scale (1). the appearance of tolerance in the first 48 hours was considered as tachyphylaxis or early therapeutic failure to this sedative. in our sedation protocol we use propofol preferably in patients who need frequent neurological consciousness evaluations, or in patients whose sedation is expected to have short to medium duration, and who have haemodynamic stability. also, we use propofol as a sequential strategy when early weaning from ventilation is expected. all patients received analgesic drugs. during this time, we admitted 5277 patients, 2519 of them needed mechanical ventilation and in 2005 patients we administered continuous analgesic and sedative infusions. continuous propofol infusions were administered in 1651 patients at some point of their sedative strategy, and 683 (34% of the sedated patients) received propofol for more than 48 hours. tolerance development was observed in 48 patients, 3% of the patients sedated with propofol. in thirty-seven of them, this situation was present in the first 48 hours (early therapeutic failure). conclusion. in our sedative protocol for propofol use, the incidence of tolerance in patients sedated with this drug was 3%, which is substantially less than the usual described midazolam tolerance. most of these cases (77%) happened in the first 48 hours. diabetes mellitus (dm) with its chronic and acute complications puts patients suffering from the disease at increased risk. none of the scoring systems used for risk prediction in intensive care units accounts for diabetes as a risk factor, although, in everyday practice, patients with dm admitted to icus may be recognized as those with higher risk. not much data is available on how much risk can be attributed to diabetes. we have compared course and outcome of patients with dm with non-diabetics to try to answer this question. we have analyzed data from the "croicu.net", national pilot-project which collects data on patients from icus in croatia. data collected during the first 26 months (nov 2004 -dec 2006 have been analyzed. adult patients from 14 icus in university hospitals were included; three most frequent admission diagnoses were selected for comparison of diabetic and non-diabetic patients. the diagnosis of dm had to be established prior to admission according to the usual criteria. icu mortality and icu length of stay (los) were primary outcome measures. incidence of organ failure was a measure of disease course. in the analysed period there were 6456 admissions to the analysed icus, 1341 (20.8%) with documented dm prior to admission. patients with md did not differ significantly from non-diabetics in age or sex distribution. overall mortality was higher for dm patients (15.1% vs. 12.7%), as was los (5.8 vs. 4.1 days). three most frequent diagnoses were: sepsis (n=723; 11.2%), pulmonary oedema (659; 10.2%) and myocardial infarction (n=555; 8.6%). patients with diabetes had significantly higher mortality and higher los in all three subgroups. in the sepsis subgroup, patients with diabetes had higher incidence of organ failure and higher number of failing organs. in the other two subgroups, the differences were not significant. in multivariate analyses which was performed separately for all three diagnoses and included dm, age, apache ii score and sofa score, diabetes mellitus was shown to be an independent predictor of mortality and los in all three cases. although some chronic effects of diabetes mellitus can be included in multiparameter scoring systems such as apache ii score, the disease itself is not scored. we have shown on three most common diagnoses in icus of university hospitals that diabetes mellitus is an independent predictor of mortality and los and that it has significantly higher incidence of organ failure in sepsis. patients with dm should be given appropriate attention as high risk patients in the icu. introduction. neuromuscular abnormalities are common in critically ill patients with systemic inflammation and organ failures. we assessed the incidence of a clinically diagnosed critical illness polyneuro-myopathy (cipm), and its potential impact on mortality and long-term neurological outcome. methods. 39 consecutive critically ill patients on mechanical ventilation for 48 hours and with the presence of 2 or more sirs criteria were prospectively studied. based on daily clinical neurological examinations, cipm was defined as symmetric limb muscle weakness [2 or more muscle groups, m3 or less (mrc)] without other explanation than cipm in patients with normal neurology at icu admission. a barthel index (score for activities in daily living) was performed at day 28 and 6 months after icu discharge. after 6 months a neurological examination was also performed. . cipm was diagnosed in 13 patients (33%). patients with suspected cipm had a prolonged icu stay and a high mortality. the barthel index was significantly lower in this group at day 28 but improved over the next six months. 20 of 23 patient who survived could be reached 6 months after discharge and 12 of them were clinically examined. at this time the most compromised activity in daily living is climbing stairs. patients with a clinical diagnosis of cipm have a high mortality. if they survive, they are severely limited in simple daily activities one month after icu discharge, but improve later. host infection by pathogens triggers innate immune response leading to a systemic inflammatory response, often followed by a paradoxical compensatory antiinflammatory response. this immune dysfunction can impair the eradication of primary infections and favor the emergence of nosocomial sepsis. dendritic cells (dcs) have a central role in initiation and control of innate and adaptative immune responses to infectious challenges. dcs might contribute to sepsis-induced immunodepression. indeed, depletion of dcs has been reported in secondary lymphoid organs of patients who died from sepsis and in animal models of lethal sepsis. in order to investigate the mechanisms of sepsis-induced immunodepression, we studied quantitative and functional features of dcs in a murine model of sublethal sepsis. we developed a sublethal murine model of polymicrobial sepsis through cecal ligature and puncture followed by short course of antibiotics and volume resuscitation. we isolated splenic dcs by immunomagnetic procedure and generated bone marrow-derived dcs (bmdcs) by 6-day culture of medullar progenitors in the presence of gm-csf before stimulation with lps to induce maturation. we counted spleen dcs and studied the following functional features of spleen dcs and bmdcs in the early (day 1) and late (day 8) phases of sepsis : maturation (expression of mhcii, cd40 and cd86 through facs analysis), production of cytokines (tnf-alpha, il-12, il-10) and priming of cd3-positive t-cell lymphocytes (3h-thymidine proliferation assay in allogeneic mixed lymphocyte reaction). upon anesthesia induction with isoflurane sepsis was initiated by cecal ligation and double puncture in 3 groups of 4 c57bl/6j-mice per group [18g, 22g, 26g] (clp). control mice underwent laparatomy and manipulation of the cecum only (sham). 24, 48 and 96 hrs post-surgery in 26 and 22g mice and 36 hrs post surgery in 18g mice single cell suspensions of thymus and spleen were analyzed by means of cell surface staining and flow cytometry. fluorescence-labeled antibodies included cd3, cd4, cd8, b220, igm, igd, cd25, cd69. data are presented as mean+sem. results. similar to previous results, thymi primarily demonstrated a time-dependent reduction of cd4+cd8+ double-positive cells which was more pronounced during severe sepsis (18+22g). at 96hrs post-clp cd4+ cells and cd8+ t-cells recovered to values of sham mice in 26g animals, which previously recovered fastest with highest survival rates of about 90%. in contrast cd4+ cells and cd8+ cells, respectively, raised to maximum levels at 96 hrs in 22g animals. concerning spleocytes cd4+ and cd8+ cells were similarly reduced to about 80% and 70% after 48hrs and to 60% and 50% in 22g and 26g mice compared to sham mice. splenocytes of 18g-treated mice, which could only be investigated at 36 hrs postclp showed no difference to sham mice. as far as b cells are concerned no significant differences between the groups or different time points could be detected. relative numbers of peripheral t cells expressing the early activation marker cd69 or cd25 were clearly more pronounced at 24hrs compared to 96hrs in 26 and 22g mice. in 18g treated mice cd69 and cd25 positive t cells were significantly higher at 36hrs compared to sham mice. a mild clp model is more appropriate to study during murine sepsis. the rapid occurrence of peripheral activated t cells suggest a very early function of the adaptive immune system during sepsis. considering a milder disease course of 26g mice they seem to more efficiently use their t cells to fight the infection. thymocyte data suggest a block in lymphopoiesis from cd4-cd8-to cd4+cd8+. b cells are not likely to play a major role in polymicrobial murine sepsis. further studies have to be performed to elucidate the turnover and the homing of lymphocytes during sepsis. endotoxaemia is associated with intestinal perfusion deficits and gut barrier failure. regional sympathetic blockade by means of thoracic epidural anaesthesia (tea) has been shown to positively affect intestinal microcirculation during endotoxaemia. this study tests the hypothesis that the microvascular changes observed with tea go along with an increase in overall gastrointestinal blood flow. in addition we investigated whether the use of tea influences gut barrier function. after approval of the animal care committee rats were anaesthetised (urethane/ketamine), hemodynamically monitored and mechanically ventilated with room air. lidocaine 2% or normal saline were administered as a bolus (30 µl) and subsequent continuous infusion (30 µl x h −1 ) via an epidural catheter (tip at t7/8, spread t4-t10). organ blood flow (n = 30 rats) was measured by the fluorescent microspheres technique at baseline, 30 min after epidural infusion, and 60 min and 120 min after the infusion of endotoxin (e. coli lipopolysaccharide, 1.5 mg x kg-1 x h −1 ) or normal saline. for assessment of gut barrier failure rats (n = 27) received a bolus infusion of endotoxin (50 mg x kg-1) or normal saline and epithelial permeability to low molecular fluorescein isothiocyanate-dextran (4kd) was quantified using a ligated loop of terminal ileum after 5 hours of normotensive endotoxaemia. in hypodynamic shock models pure o2 breathing was shown to redistribute blood flow in favour of hepato-splanchnic organs and to improve survival. in contrast, this therapeutic approach has not yet been evaluated in hyperdynamic septic shock, since an increased production of o2 radicals, which is directly related to the increased o2 partial pressure, is considered as harmful. therefore, we investigated the effects of pure o2 breathing on hepato-splanchnic macro-and microcirculation, energy balance and tissue cell death during porcine fecal peritonitis. after induction of fecal peritonitis, pigs were randomly ventilated for 24h with 100% o2 (n=10) or an fio2 adjusted to yield a sao2>92% (n=10). before as well as at 12 and 24 h of peritonitis we measured cardiac output as well as hepatic artery and portal vein (pv) flows (ultrasound flow probes), microcirculation in the intestinal wall (laser doppler flow), intestinal wall oxygenation, portal and hepatic-venous acid-base status, and lactate/pyruvate (l/p) ratios. apoptosis was analysed post-mortem in liver biopsies with the tunel assay. within group effects were analyzed using a friedman anova on ranks, intergroup differences with an unpaired rank sum test. at the end of experiment the contribution of both pv and total liver blood flow to cardiac output was significantly higher in the hyperoxic animals than in the control group (qliver/co 20 (6;25)% vs. 25 (13;34)%, p=0.022; qpv/co 18 (6;23)% vs. 23 (13;32)%, p=0.043, respectively), which was concomitant with attenuated regional venous metabolic acidosis and lower hepatic-venous l/p-ratios. intestinal wall microcirculation and oxygenation did not significantly differ between the two groups. the hyperoxic animals presented with a markedly reduced number of apoptotic cells in the liver. our results show that early 100% o2 ventilation redistribute blood flow in favour of the hepato-splanchnic system even in peritonitis-induced hyperdynamic septic shock. furthermore, the hepatic energy balance is improved and the morphologic integrity of the liver better maintained under these conditions. grant acknowledgement. supported by the eli lilly-esicm sepsis elite award, the alexander-von-humboldt-stiftung, and the deutscher akademischer austauschdienst glucocorticoids are known as strong modulators of immune response that play an important role in patophysiology of sepsis and inflammation. they have strong influence on the development of immune system, its effector functions, and trafficking of immune cells.the biological activity of glucocorticoids depends not only on their plasma concentration, the number of receptors and the responsiveness of the target cells but also on the local metabolism of glucocorticoids that is predominated by 11b-hydroxysteroid dehydrogenase (11hsd). two isoforms of 11hsd are known. the isoform 11hsd1 operates in vivo predominantly as a nadph-dependent reductase that locally increases glucocorticoid concentration (cortisol, corticosterone) by reduction their 11-oxo derivatives (cortisone, 11dehydrocorticosterone) . the isoform 11hsd2 is a sole nad+-dependent dehydrogenase that inactivates biologically active glucocorticoids to their inactive 11-oxo derivatives. the aim of this study was to investigate peripheral metabolism of glucocorticoids in immune cells and tissues in experimental model of sepsis and inflammation. sepsis was induced in balb/c mice and wistar rats by intraperitoneal administration of lipopolysaccharide or pooled fecal inoculum. in these animals and in healthy controls we measured expression and activity of 11hsd1 in lymphatic nodes, peripheral blood leukocytes and alveolar macrophages. activity was measured by incubation with corticosterone and 11-dehydrocorticosterone, following hplc determination. the abundance of 11hsd1 mrna was measured by semi-quantitative real-time rt-pcr. for years etomidate has been known to cause adrenal insufficiency in the critically ill and is a confounder when studying corticosteroids in septic shock. subgroup analysis of a prospective, randomized, placebo-controlled study of corticosteroids in septic shock. patients underwent a short high dose acth test before study drug administration. patients received 11d treatment with hydrocortisone (hc) or placebo (p). the affects of etomidate administration on acth responsiveness and 28d mortality were studied. results. 499 patients were enrolled. overall 33.5% patients died in the hc group and 31% in the p group (p=0.57). in total 27% of patients received etomidate. 101 received etomidate before baseline [21% hc group + 20% p group] and in 36 after baseline [8% hc group + 6% p group]. overall, more of the patients receiving etomidate were acth nonresponders [58% vs 42%] . no mortality differences was seen between patients receiving etomidate at any time during study and those who did not receive etomidate [34.3% vs. 31 .5%](p=0.61). there was a possible trend towards a difference in mortality between patients who received etomidate in the 72 hrs before randomisation [45% hc vs. 38% p] or not receiving etomidate during this time period [31% hc vs. 30% p](p=0.052). etomidate was commonly used in patients in the corticus study. etomidate was associated with an increased likelihood of adrenal hyporesponsiveness in all patients. there was no increase in mortality associated with etomidate administration at any time, there was a trend towards increased mortality in those who received it in the 72 hours before trial baseline. this result comes from an underpowered subgroup and should be considered exploratory. d. pestaña* 1 , e. martinez-casanova 1 , a. buño 2 , r. madero 3 , a. criado 1 1 anestesia-reanimación, 2 análisis clínicos, 3 bioestadística, hospital universitario la paz, madrid, spain introduction. steroids are indicated in septic shock patients when relative adrenal insufficiency is suspected. our aim was to study if the measurement of total proteins (1) and eosinophil count (2) improves the accuracy of cortisolemia to predict the hemodynamic response to steroid treatment in this setting (3). we analysed data from 66 consecutive surgical patients with criteria of septic shock receiving steroid treatment. four criteria were chosen to define hemodynamic improvement based on the combination of noradrenaline (na) withdrawal (at 24 and 48 h) and an increase of the hemodynamic index (hi = mean arterial pressure/na dose) of 150% at 24 h and of 350% at 48 h. the accuracy of the baseline cortisolemia to predict the hemodynamic response to steroid treatment following the four criteria was determined by roc curve analysis. the largest area under curve (auc) was found for the noradrenaline withdrawal or an increase of the hi > 350% at 48 h after starting the steroid treatment (table 1) . this criteria was met by 35 patients (53%) and was associated with a lower mortality (25.7% vs 67.7%, p=0.001, 70% sensibility and 72.2% specificity). however, no clear cortisolemia cut-off value for the diagnosis of adrenal insufficiency based on the hemodynamic response could be found. neither the basal proteins nor the eosinophils improved the accuracy of cortisolemia to predict a hemodynamic improvement. mortality was also related to age (p=0.017), apache ii (p=0.004) and sofa score (p=0.005). neither basal cortisolemia nor lactate were related with icu mortality. twelve septic shock patients admitted to the icu < 96 hours after family consent were enrolled. we excluded all patients in use of steroids in the preceeding 6 months, etomidate, espironolactone, oestrogens, oral contraceptives, ketoconazole or any other drug known to suppress adrenal function; aids, pregnancy, history of disease of the hypothalamic-pituitaryadrenal axis, shock of other etiologies. after a baseline serum cortisol was obtained, a ld (1 ug) corticotropin stimulation testing was performed. subsequently, serum cortisol at 30 and 60 min was measured. four hours later, another bc was obtained. then, a hd (249 ug) corticotropin stimulation testing test was performed and serum cortisol was again measured after 30 and 60 min. results. both baseline serum cortisols were similar. delta hd cortisol was higher than delta ld cortisol (19.1±15.3 vs. 8.8±6.6ug/dl, p=0.009). five patiens had a bc < 25 ug/dl, but only one showed rai in both tests. concordance between ld and hd tests was 66% (8/12). it was strong for responders (86%,6/7) but weak for non-responders to ld test (40%, 5/2). the preliminary results of our study suggest that a ld test is a more sensitive test than a hd test. a further study comparing treatment of rai defined by a ld or a hd test is still needed. the potassium channels (kc), atp-sensitive k+ (katp) channels and calcium-activated potassium (bk) channels, may be implicated in shock induced vasoplegia. the aim of our study was to demonstrate that the potassium channels are overexpressed in experimental shock independently of the etiology. three rats models of shock were used : peritonitis by caecal ligation and perforation (clp, n=14) observed at 18h, ischemia-reperfusion model (hemorrhagic shock + resuscitation + laparotomy, n=9) observed at 18h, and pressure fixed hemorrhagic shock (n=6) observed at 4h. these three models were compared to a control group. we performed quantitative real-time pcr (lightcycler technology -roche -and sybr green -sigma) and western blot on aorta and mesenteric arteries. we studied the expression of the vascular smooth muscle katp channels -kir 6.1 and sur 2b subunits -and bk channels -bk alpha subunit. we assessed the inflammatory syndrome in studying inos expression. we were able to detect kir 6.1, sur 2b, bk alpha and inos arnm in both vessels. quantitative real-time pcr results (reference gene : beta-actine) clp clp ir ir hs hs aorta mesenteric aorta mesenteric aorta mesenteric inos 3.7 ± 0.7* 12.1 ± 3.5* 4.2 ± 1.2* 21.8 ± 11.6* 14.2 ± 3.1* 22.4 ± 7.5* expression kir 6.1 1.7 ± 0.2 4.8 ± 1.2* 20.5 ± 5.2* 7.1 ± 1.6* 19.5 ± 2.0* 11.5 ± 4.8* expression sur 2b 1.4 ± 0.2 3.2 ± 0.6* 6.8 ± 1.5* 2.3 ± 0.6* 5.3 ± 1.3* 1.6 ± 0.4 expression bk alpha 1.9 ± 0.2* 1.9 ± 0.4* 2.8 ± 0.4* 2.7 ± 0.6* 2.1 ± 0.5 1.1 ± 0.5 expression * : p< 0.05 vs control group conclusion. various potassium channels are activated and up-regulated during shock independently of the etiology. thus, potassium channels likely play a major role in sepsis but also in prolonged and severe hemorrhagic shock and in ischemia reperfusion. (cars) . a predominantly anti-inflammatory reaction induces immunosuppression with impaired host defense. application of gm-csf to patients with major surgery or sepsis has been proposed to improve host-defense. in this study we investigated the differential effects of gm-csf production in an ex-vivo model. and lps on the tnf-a. whole blood of 40 healthy donors (age 16-72 years, mean 54 years) was used to determine optimal concentrations and incubation time for lps. the immunomodulating properties of gm-csf (leukine ® (sargramostim), berlex)) were investigated in whole blood of 28 healthy donors (36-65 years, mean 51 years) and 12 icu patients suffering from sepsis. six of the patients had immunoparalysis as defined according to local standards by a monocytic hla-dr expression of < 150 mfi and an ex-vivo stimulation test of < 175 pg/ml after lps incubation (dpc biermann, bad nauheim , germany), whereas the other 6 displayed a hla-dr expression of > 150 mfi and a ex-vivo stimulation test of > 175 pg/ml. samples were primed either with gm-csf, gm-csf simultaneously or lps prior to incubation. tnf-a and il-8 concentrations were determined with the immulite chemoluminescence immunoassay system (dpc-biermann, bad nauheim, germany). leukocyte phenotyping was performed by dual-colour flow cytometry using whole blood lysis technique and monoclonal antibodies. in healthy donors, ex-vivo stimulation with lps leads to a massive increase of tnf-a production. however, if whole blood is incubated with gm-csf 3 hours prior to the lps challenge, the tnf-a production is significantly increased. the simultaneous incubation with lps and gm-csf leads to a significant decrease in tnf-a levels in the same patient population. gm-csf stimulation of whole blood 3 hours after the production. in patients lps challenge causes no significant change in tnf-a levels of with sepsis and endogenous tnf-a < 30pg/ml, gm-csf pre-incubation production, whereas patients leads to a significant increase in ex-vivo tnf-a had a blunted ex-vivo reaction to lps with higher endogenous levels of tnf-a stimulation. both the sequence of stimulation with either gm-csf or lps and the presence or absence of systemic tnf-a determine the ex-vivo cytokine response of whole blood. hence, it may be speculated that 1. the administration of gm-csf prior to the inflammatory stimulus would be most efficient, and that 2. the lack of stimulation effect in patients with high endogenous tnf-a may mirror endotoxin tolerance. the most common acquired causes of weakness and muscle wasting in the critically ill patient in the intensive care units (icu) are critical illness polyneuropathy and critical illness myopathy. there is significant clinical and neurophysiologic overlap between the two conditions, such that the term critical illness polyneuropathy and myopathy (cipnm) is often used. over a 12-mo period, 22 critically ill patients who needed prolonged intensive care were studied. clinical manifestations include delayed weaning from the respirator not explained by pulmonary complications, muscle weakness and prolonging of the mobilization phase. included patients were classified as having mof, sirs and sepsis according to established consensus definitions. the occurrence of a positive emg for cipnm, as defined by an electrophysiologist who was blinded for treatment allocation, was analyzed during icu stay. variables recorded at baseline and during follow-up included patient demographics, principal diagnosis, routine blood tests and microbiological culture results. levels of tnf-alpha, il-6, il-10, il-4, procalcitonin (pct) and c-reactive protein concentrations were repeatedly measured by elisa. all patients were divided in: patients without cipnm at any time (group a, n=7), with a positive emg during icu stay (group b, n=8), and with a diagnosis of cipnm since the admission (group c, n=7). emg testing demonstrated severe acute denervation with striking involvement of proximal muscles in 15 patients. 6 patients died of complications of sepsis. critically ill patients without cipnm showed serum il-6 levels lower (p < 0,05) than those with a diagnosis of cipnm while no differences were found as concerned serum il-10 levels. il-4 and tnf-alpha did not show any difference between the two groups. il-6 levels resulted higher in groups a and b (p < 0,05) while il-10 levels were higher in group a (p < 0,01). in the group b, we observed a characteristic pattern of il-6 and il-10 serum concentrations that may be important for clinical outcome. il-6 levels were higher than il-l0 in patients with worse clinical outcome. the opposite pattern was observed in those with a good prognosis. no differences in clinical and laboratory variables were observed between patients with and without cipnm. pct appeared to be most helpful in differentiating patients with sepsis from those with sirs (p < 0,001), exhibiting a greatest sensitivity (83%) and specificity (96%). conclusion. the analysis of the serum cytokines il-6, il-10, tnf-alpha and il-4 to standard indicator did not improved the predictive power of detecting cipnm but may contribuite to explain its pathogenesis. high dose glucocorticoids are known to induce muscle weakness. we investigated in a pilot study the occurrence of cip/cim in septic shock patients treated with low dose hydrocortisone (hc). patients were enrolled in the randomized controlled study of hc in septic shock (corticus) and received hc (50 mg q 6h for 5 days, tapered until day 11) or placebo (pl). electrophysiological testing (ep) consisted of the assessment of compound muscle (cmap) and sensory nerve action potentials (snap), spontaneous activity (spa), and muscle membrane excitability investigated by direct muscle stimulation (dms). clinical muscle weakness was defined by a medical research council scale (mrc) below 4. cmap and snap were categorized based upon normal age related values. ep results were categorized as unspecific (cim or cip or both) when cmaps and spas were pathological in >/= 2 muscles. presence of cip was defined by pathological snaps in >/= 1 nerve, and cim by dms values < 3 mv. data are shown as mean and 95%ci, chi square test and mann-whitney-u-test were performed for statistical analysis. from jun 03 -feb 05, 20 patients were enrolled in 9 sites: 9 hc and 11 pl. median time for ep assessment was 12 days (4 -38) after study enrolment. 10 pl and 7 hc patients had unspecific electrophysiological signs; 6 pl patients, but only 1 hc patient had reduced snaps indicating cip. in 16 patients dms could be performed, 7/11 pl and 3/5 hc patients showed reduced muscle membrane excitability indicating cim. in 13 patients (pl 7, hc 6) evaluation of mrc score was possible. muscle strength did not differ between placebo [3.8 (3/4.5)] and hc group [4 (3.2/4.7)]. none of the parameters reached statistical significance. conclusion. the frequency of cip/cim diagnosed by electrophysiological examination was higher in patients who received placebo. the clinical diagnosis of muscle weakness assessed by mrc scale was not different in both groups. with limitations of the small sample size, this first prospective evaluation showed no impact of hc on the development of cip/cim in this cohort of patients with septic shock. surviving sepsis campaign guidelines recommend treatment with hydrocortisone in septic shock patients requiring vasopressor support. however, the association of fludrocortisone remains controversial. the objective of the study was to determine if the association of fludrocortisone in patients with septic shock and adrenal insufficiency treated with hydrocortisone is related to an improved outcome. from a database including 106 patients with septic shock requiring vasopressor support, we retrospectively studied 87 patients who fulfilled criteria for adrenal insufficiency (baseline cortisol less than 20 µg/dl and/or an increase after injecting 250 µg synacthen less than 9 µg/dl). all patients included received treatment with hydrocortisone (h) or hydrocortisone plus fludrocortisone (h+f) for at least 24h. data are presented as mean ± standard deviation. groups were compared by using student's t test for continuous variables and chi-square test for categorical variables. long rank test and kaplan-meier curves were used to analyze time to shock reversal and mortality. forty-eight patients received hydrocortisone (h group) and 39 hydrocortisone plus fludrocortisone (h+f group). overall mortality was 63% (55 patients). both groups were comparable in baseline clinical and demographic characteristics. no differences were found in age (mean age 61±14), gender, weight (75±15 vs 73±13, p 0,48) (kg), infection site and severity scores: saps ii (50±14 vs 47±14, p 0,36), apache ii (22±6 vs 21±6, p 0,57) and sofa max (14±3 vs 13±2, p 0,06). both groups presented no differences regarding baseline (24±6 vs 25±13,p 0,88), stimulated (31±16 vs 31±15, p 0,99) and delta cortisol values (6,8±5,4 vs 6,6±5,2, p 0,76)(µg/dl). we did not find differences between both groups in norepinephrine(ne)maximal dose received(µg/kg/min), time to shock reversal (days of ne use), time of mechanical ventilation, icu and in-hospital length of stay (days) and mortality ( prospective, randomized, double-blind, placebo-controlled study of 28-day mortality in patients with septic shock for less than 72 hr who underwent a short high dose acth test in 52 centres in 9 european countries. patients received 11-day treatment with hc (50 mg q 6h for 5 days, q 12h for 3 days, q 24hr for 3 days) or placebo (p). serum electrolytes levels were obtained at baseline, day1 (d1), day2 (d2), day3 (d3) and day 7 (d7) from randomisation. from mar 02 -nov 05, 499 patients were enrolled. baseline serum sodium were 139 (6) mmol/l and 140 (6) mmol/l in the hc and p group respectively. serum sodium peaked at d3 (143 mmol/l) and remained elevated up to d 7 (143 mmol/l) in the hc group. in the placebo group, serum sodiumpeaked at d2 (142 mmol/l). the mean change in serum sodium were, in hc treated and p treated patients respectively, at d 1: 1.3 (3.9 sd) vs 1.3 (3.7) mmol/l; d 2: 2.4 (5.4) vs 1.7 (5.5) mmol/l; d3: 3.1 (6.7) vs 1.6 (6.5) mmol/l; and at d 7:3.0 (8.0) vs 0.5 (7.5) mmol/l. the difference between groups reached statistical significance at day 7 (p=0.003). there were no significant changes in mean potassium levels over time between the two treatment arms. according to the guidelines for the management of severe sepsis and septic shock, low doses of steroids are recommended in septic shock patients requiring vasopressors, despite adequate fluid replacement. the aim of this retrospective case control study was to assess the effectiveness of low doses of hydrocortisone in patients with late septic shock and mods. the study was held in a 19 bed multidisciplinary icu of a tertiary hospital. twenty four norepinephrine dependent (> 0.5γ /kg/min) patients, fulfilling the criteria of septic shock, were enrolled in the study. patients were divided in 2 groups according to the continuous administration of 300 mg hydrocortisone for 7days (group a:12 pts) or conventional treatment (group b:12 pts). end points of the study were, the within 7 days vasopressors weaning, evolution of mods and 7-day as well as 28-day survival. mods was described by sofa score. statistics : statistical analysis was computed by using paired t-test and linear regression analysis. groups were similar regarding demographics (57±17 vs 64±15 y), initial sofa score (10±3 vs 9,5±2), initial norepinephrine dose (1.9 ± 0.7 vs 1.13 ± 0.6 γ /kg/min) and mean elapsed time from the onset of shock (3.7± 3.1 vs 3.5±2.5 days). an early and significant decrease in norepinephrine dose (p<0.005), was observed in all group a pts, while no difference was detected in group b pts. this decrease was associated with hemodynamic stability. on days 3 and 4 mean abp was significantly higher in group a pts (p<0.001, p<0.005). weaning from vasopressors within 7 days was achieved in 5 pts in group a (41.6%) and 1 pts in group b (0.8%). seven day mortality was 16.6% in group a vs 50% in group b while 28-day mortality was 50% and 91% respectively. in the treatment group a positive correlation between the within 7 days shock reversal and survival (cor coeff = 0.657, r 2 = 0.432, p=0.02) was found. there was no relation between the time elapsed from the onset of shock to the steroid administration and survival (p=0.66). oxygenation parameters (fio2/po2), sofa score and creatinine did not differ between groups. wbc in group a pts were significantly higher (p<0.005) only on day 3. no significant adverse effects were detected. in late septic shock patients with mods the administration of low doses of hydrocortisone is associated with decreased vasopressors requirements, hemodynamic improvement and beneficial effect on survival. the within 7 days shock reversal was a good predictor of survival. introduction. early microcirculatory impairment followed by mitochondrial dysfunction may combine to produce multi-organ failure in sepsis. we recently reported that tissue oxygen tension (tpo2), the balance of local o2 supply/demand, is variably affected in four different organs (kidney cortex, liver, muscle, bladder) at 3h' post-endotoxin challenge (1). we seek to measure temporal changes in tpo2 in these organs in a resuscitated rat model for up to 72h following the onset of faecal peritonitis. here we present our 6-hr timepoint results with assessment of the impact of fluid loading. methods. male wistar rats (approx 300g weight) with tunnelled right jugular venous cannulae in situ received i.p. injection of faecal slurry. fluid (1:1 mixture of 5% glucose/6% hetastarch; 10 ml/kg/h) was started 2h later. at 6h, rats were anaesthetised with isoflurane, and then instrumented with a left common carotid arterial line and tissue po2 probes (oxford optronix, uk) sited in thigh muscle, between right and left liver lobes, in the left renal cortex and within the bladder lumen. after 30-min stabilisation, recordings were made of bp, tpo2, and end-diastolic volume (edv) and cardiac output (co) by echocardiography (vivid 7, ge healthcare, bedford, uk). this was performed before (bi, baseline instrumented) and after fluid challenge (f) of 25 ml/kg bolus of 6% hetastarch given to optimise lv filling. comparisons were made against sham-operated animals that underwent instrumentation but received no i.p. injection. notwithstanding considerable volume resuscitation beforehand, left ventricular filling and output were significantly reduced at 6h in this faecal peritonitis model. despite the 30% reduction in output, baseline tpo2 values were similar in bladder and renal cortex compared to sham animals but showed a decreased trend in muscle and a significant reduction in liver. fluid loading restored cardiac output to control values, however only muscle and liver tpo2 increased, albeit not significantly. these data suggest a combination of microcirculatory and mitochondrial dysfunction with each predominating in different organ beds at this timepoint. confirmation is required using complementary techniques. microcirculatory dysfunction leads to inadequate tissue oxygenation and multi organ failure during sepsis or septic shock. aim of this study was to compare non-invasive assessment of tissue oxygen saturation (sto2) with systemic oxygenation using mixed venous oxygen saturation (svo2) as an indicator in an established model of porcine septic shock. in a prospective animal study 20 anaesthetised, ventilated pigs (28.2 ± 2.1 kg) were investigated. animals received 1g/kg/body weight faeces into abdominal cavity to induce sepsis and were observed over 8 hours. volume therapy was administered to maintain a central venous pressure of 12 mmhg. svo2 measured by co-oxymetry (radiometer, copenhagen) was obtained hourly after induction of sepsis. at the same time quadriceps muscle sto2 was measured by near-infrared spectroscopy (nirs) (inspectra tm , hutchinson, usa). correlation was analyzed by linear regression analysis. a total of 136 measurements were performed in 20 animals. sto2 was significantly correlated with the svo2. r = 0.52 (r 2 = 0.27) (p<0.01) and y = 0,32x + 43,6. comparing the change in sto2 and svo2 of two successive measurements reveals a correlation of r = 0.19 (r 2 = 0.035) (p<0.05). changes in sto2 and svo2 were parallel in 47% of two successive measurements (both measurements changed at the same time in the same direction). although there is a significant correlation between sto2 and svo2 in our experimental septic shock model, paired sto2 and svo2 changed in the same direction only in 47%. thus, svo2 may not be estimated on the basis of sto2 in treatment of experimental septic shock and tissue oxygenation may not be estimated on the basis of svo2 either. whether a combination of sto2 and systemic oxygenation measurements is a useful monitoring approach in sepsis needs to be revealed. grant acknowledgement. inspectra device was provided by hutchinson. systemic immune response syndrome (sirs) frequently develops in critically ill patients and may lead to multiple organ dysfunction or failure even in the presence of normal or normalized global hemodynamic parameters, mainly due to tissue dysoxia and microvascular dysfunction. near infrared spectroscopy (nirs) is a validated method for the assessment of tissue oxygenation but its accordance with routine parameters has not yet been sufficiently studied. aim: to compare nirs parameters to routine monitoring parameters of the critically ill. thirty two consecutive critically ill patients (age=57±17 years, male/female=18/14, length of icu stay=17±12 days) were enrolled. all patients were evaluated with nirs and the occlusion technique within 24 hours of icu admission. all patients were mechanically ventilated and 26 were sedated. routine hemodynamic parameters (mean arterial pressure=83±15 mmhg, central venous pressure=8±3 mmhg, heart rate=85±16), full blood analysis (hemoglobin=11.3±4.4 g/dl, white blood cells=11,345±4,602 /dl) and arterial blood gases analysis were recorded. sofa, apache ii and saps iii (55±13) scores were assigned on icu entry day. tissue oxygen saturation (sto2%) was continuously monitored before, during and after 3-min occlusion of the brachial artery via pneumatic cuff inflated up to 50 mmhg above measured systolic arterial blood pressure. (elwi) has been demonstrated to predict mortality and to correlate to pao2/fio2-ratio and to the compliance of the lungs in patients with sepsis and ards. however, with an increasing number of obese patients, there is the question which body weight should be used for indexation of elwi. therefore it was the aim of our study, to investigate the correlation of elwi to pao2/fio2-ratio and oxygenation index (mean airway pressure* 100 / pao2) using different weight parameters for indexation. in 25 patients of a medical icu with a body mass index >25kg/m2, 260 measurements of extravascular lung water were performed using the picco system (pulsion, munich; 7.0. software). extravascular lung water was indexed using the actual body weight (abw), predicted (pbw), ideal (ibw) and adjusted body weight(adbw) , respectively. these data were correlated to pao2/fio2-ratio and oxygenation index. spearman correlation, spss-software. the highest correlation to pao2/fio2-ratio was found using adbw, the highest correlation to oxygenation index for elwi adjusted to pbw. 3.) although the extent of correlation varied within smaller limits (-0,438 to -0.510 and 0.446 to 0.578, respectively), the distribution of the patients within "normal", "modestly elevated" and "significantly elevated" elwi would have changed markedly using different indices. 4.) with regard to impaired respiratory function in the patients of our study, pbw, ibw and adbw seem to more accurately reflect "functional" extravascular lung water than abw with 71% of the patients in the normal range. our objective is to analyse the hemodynamic profile and the extravascular lung water in the first stages of severe acute pancreatitis (sap) that are admitted at the intensive care unit (icu), through the collected data by transpulmonary thermodilution. observational and prospective study, in which 13-sap-diagnosed patients consecutively admitted at the icu were analyzed. all of them were monitorised at their admission with continuous cardiac output system picco ® (pulsion medical systems). demographic variables, general (apache ii and sofa) and specific (balthazar) severity scores as well as the development or not of respiratory failure, were collected. the ordinary hemodynamic parameters [heart rate (hr), mean arterial pressure (map), cardiac index (ci), vascular resistances (svri)] were determined on days 0, 1, 4 and 7 as well as preload parameters [intrathoracic blood volume index (itbi), global end-diastolic volume index (gedi)], extravascular lung water index (elwi) and pulmonary vascular permeability index (pvpi) according to picco ® methodology. the results are expressed as means±sd and percentages. the non-parametric mann-whitney test for quantitative variables was performed and statistical significant level was established at p<0.05. age was 54±21 years with a majority of males (54%). the biliar was the most frequent cause (46%). apache ii=13±5 and sofa=6±3. all patients showed an alteration determined by ct scan (balthazar grading system) degree c or higher. seven patients (54%) needed mechanical ventilation in the first 48 hours. hospitalary mortality was of 38%. on day 0, the ci (3.5±0.8 l/min/m 2 ) and the rvsi (1885±717 din.seg.cm -5 .m 2 ) were at normal parameters and only 3 patients needed vasopressor support. however, on days 0 and 1, the preload parameters were low (itbi= 765±162 ml/m 2 and gedi =612±130 ml/m 2 ) and improved on the 4th day (itbi= 870±195 ml/m 2 and gedi =706±172 ml/m 2 ). patients with respiratory failure and mechanical ventilation showed neither higher elwi nor higher pvpi than the rest (day 1, elwi: 7.1±1.8 vs 5.8±0.7 ml/kg; pvpi: 1.7±0.5 vs 1.6±0.3; p=ns). in our population, certain hypovolemia degree in the first stages of the disease was found, corresponding to the development of the third space. the respiratory failure associated is not mainly due to an extravascular lung water increase or to a permeability increase. 0.6 (0.5 -1.5) 0.85 (0.8 -1.0) 0.025 cpo after dobutamine (w) 1. 2 (0.8 -1-2) 0.6 (0.5 -0.9) 0.008 poap: pulmonary occlusion arterial pressure, swi: stroke work index. conclusion. cpodelta after dobutamine challenge is a good predictor for mortality in ss. septic shock is a common disorder with a high mortality. recent guidelines for the haemodynamic management of severe sepsis have emphasized the importance of aggressive volume resuscitation in the initial phase. central venous pressure (cvp) and pulmonary capillary pressure (pcp) are common end-points for volume resuscitation, however these cardiac filling pressures are poor predictors of fluid responsiveness in septic patients. 1 right ventricular end diastolic volume index (rvedvi) is a better predictor of preload, and it allows the identification of patients with right ventricular (rv) dysfunction and dilation (> 100-130 ml/m 2 ), as well as predicting mortality. we correlated rvedvi with pcp, cvp and hypoperfusion variables during septic shock initial management. longitudinal, prospective and observational study. demographic, haemodynamic (rvedvi, pcp, cvp) and hypoperfusion (lactate, base deficit) variables were obtained. descriptive statistics with mean ± sd (numerical variables) and frequencies and percentages (categorical ones). comparisons between groups with u mann-whitney test and x 2 and fisher exact test as needed (statistically significant value if p<0.05). results. 30 patients (mean age 64±17)were divided in: survivors n=21 (rvedvi 147±16 ml/mt 2 ) and non-survivors n=9 (rvedvi 111±15 ml/mt 2 ). early dilation of rv predicts survival with a sensibility of 100% sensibility and specificity of 67%. methods. ten patients with severe sepsis 60±6 yr, 25 patients with septic shock 63±8 yr and 10 polytrauma patients with hemorrhagic shock 40±7 yr, who remained in icu more than 24 hours were included in the study. serial bnp measurements were performed for at least 5 days. consecutive hemodynamic measurements were done using a right ventricular ejection fraction (rvef) thermodilution catheter (edwards). transthoracic echocardiography was performed in the first two days. . bnp values (1st day) was dramatically elevated in septic shock (1110±356 pgml-1), significantly elevated in severe sepsis (312±115 pgml-1), but within normal limits in hemorrhagic shock (52±23 pgml-1) (p<0.001). inotropes (noradrenaline) were similar in patients with septic or hemorrhagic shock on day 1. bnp levels did not correlate with pulmonary arterial wedge pressure, right atrial pressure, rvef or left ventricular ef (lvef) measured by echocardiography. eleven patients with septic shock, 2 with sepsis and 1 with hemorrhagic shock died during 28 days. bnp decreased gradually in survivors from septic shock after day 2. septic shock survivors had lower apache ii, and increased rvef and lvef compared to non-survivors (21±4, 39±4 and 73±9 vs 27±5, 32±7 and 65±7 respectively, all p<0.05), but not bnp (960±297 vs 1322±422 pgml-1). in conclusion, bnp is significantly elevated in sepsis, mainly in patients with septic schock, probably indicating the level of inflammation severity. inotropes, shock and myocardial stretch, as it is expressed from hemodynamic parameters, do not seem to be implicated to bnp release. sepsis and septic shock are major causes of mortality and morbidity in the icu. if inflammatory mediators responsible of sepsis remain elevated or if there is a poor cardiac function, septic myocardial dysfunction may occur, increasing morbidity and mortality. brain natriuretic peptide (bnp) is an adequate biomarker for cardiac failure 1 so our objective was to determine its utility in predicting myocardial dysfunction in septic patients. the role of hemofiltration, its dose and biological effects in sepsis remain a contentious issue. although some beneficial effects on systemic hemodynamics and reduced vasopressor requirement were reported, the potential of hemofiltration to prevent sepsis-related disturbances of microcirculation and energy balance has not been evaluated. therefore, we investigated the effects of standard hemofiltration (hf, ultrafiltration rate 35 ml/kg/h) and high volume hemofiltration (hvhf, 100 ml/kg/h) during 22h hyperdynamic porcine septic shock. in 21 mechanically ventilated and instrumented pigs fecal peritonitis was induced by inoculating autologue feces. 12h after induction of sepsis pigs were randomly assigned to three groups: 1) controls (n=7), 2) hf (n=7), 3) hvhf (n=7). before, 12,18 and 22h after the induction of peritonitis we measured, in addition to systemic and regional hemodynamics, ileal mucosal and renal cortex microvascular perfusion (ops and laser doppler flowmetry). energy balance was determined by measuring arterial lactate pyruvate (l/p) and hepatic venous ketone body (kbr) ratios. in the control group hyperdynamic septic shock resulted in a progressive deterioration of intestinal mucosal and renal cortex microvascular perfusion despite well-maintained regional blood flows. altered microcirculation was paralleled by gradually increased l/p and kbr indicating disturbed energy balance. compared to six animals in the control group, only three and two pigs required noradenaline support in hf and hvhf group, respectively. however, neither hf nor hvhf blunted the sepsis-induced alterations in microvascular perfusion and cellular energetics. in this clinically relevant model of septic shock, the protective systemic hemodynamic effects of early hemofiltration did not translate into the improved microvascular perfusion and energy metabolism. hvhf did not confer any additional benefit. the value of hemodynamic improvement as a surrogate marker for efficacy of hf is therefore ambiguous. patients in prolonged septic shock show enhanced pressor sensitivity to vasopressin(vp) yet decreased response to norepinephrine(ne). as both act via g protein-coupled receptors and activate the inositol phosphate cascade to increase vascular smooth muscle(vsm) ca 2+ levels, the reason for this disparity is uncertain. we postulate that these drugs may have diverse effects on different ca 2+ mobilisation pathways during sepsis. we investigated this using specific modulators of ca 2+ release and influx on contractile responses to vp and ne in mesenteric arteries from septic and sham-operated rats. sepsis was induced in 6 awake, fluid-resuscitated wistar rats by ip injection of fecal slurry. paired sham controls received no injection. rats were sacrificed after 24h, and mesenteric arteries mounted on a wire myograph to measure isometric tension responses to vp and ne. the contributions of sarcoplasmic reticulum(sr) ca 2+ release and ca 2+ entry through the store-operated channel(socc) were assessed by removing and returning extracellular ca 2+ respectively. the contribution of the voltage-gated ca 2+ channel(vgcc) was assessed by applying vp/ne in the presence of nifedipine. contractions were significantly enhanced to vp but depressed to ne in septic vessels . in all arteries, constriction to both agonists relied predominantly on extracellular ca 2+ influx rather than sr ca 2+ release. ne responses were more sensitive to extracellular ca 2+ removal in septic vessels. the ca 2+ influx in response to ne was almost entirely vgcc-mediated, with a negligible contribution from soccs in both sham and septic arteries. soccs contributed significantly to vp contraction however, and socc-rather than vgcc-mediated influx of ca 2+ predominated in septic arteries. patients in prolonged septic shock show enhanced pressor sensitivity to vasopressin (vp) yet decreased responsiveness to norepinephrine (ne). we have reproduced this pattern in ex-vivo contractile responses of resistance arteries taken from rats subjected to a clinically realistic septic insult (1). we hypothesise that an underlying mechanism is vp-mediated sensitisation of the vascular smooth muscle contractile apparatus to calcium. to investigate this, we performed simultaneous wire myography and fluorescence microscopy to examine the relationship between contractile response and intracellular calcium concentration ([ca 2+ ]i). sepsis was induced in conscious, tethered, male wistar rats by intra-peritoneal injection of faecal slurry. paired sham controls received no such injection. both groups received 10ml/kg/hr of intravenous fluid. after 24 hours, animals were sacrificed, and 3rd order mesenteric arteries dissected and mounted on a wire myograph (danish myo technology). arteries were loaded with a fluorescent calcium indicator (fura-2, 10mum) for 1 hour and imaged by fluorescence microscopy. [ca 2+ ]i and isometric tension kinetics were measured simultaneously in response to vp (3nm) and ne (10mum). ]i was higher in arteries taken from septic rats. tension responses to vp were significantly enhanced in septic arteries, however the associated increases in [ca 2+ ]i were comparable in septic and sham groups. tension responses to ne were significantly decreased in septic arteries, with a similar degree of depression in delta [ca 2+ ]i. data were analysed for statistical significance using un-paired t tests. conclusion. the higher baseline [ca 2+ ]i in the vascular smooth muscle of septic arteries suggests an abnormality of intracellular calcium storage. the ability of vp to produce a greater contractile response in septic compared to sham arteries, despite an equivalent degree of [ca 2+ ]i elevation, implies sensitisation of the contractile apparatus to the effect of vp. there was contractile hyporesponsiveness to ne in the septic vessels and no evidence of calcium sensitisation to this agonist. these findings provide one potential explanation for the hypersensitivity to vp observed in patients with septic shock. mitochondrial dysfunction and compromised cellular energetic status are associated with poor outcome in septic patients [1] . maintenance of mitochondrial function is mediated in part by activity of transcription factors nrf-1 and nrf-2, the transcriptional co-activator pgc1-alpha and mitochondrial transcription factor alpha (tfam). these markers of mitochondrial biogenesis were elevated in a rodent model of endotoxaemia [2] . in an ongoing study in critically ill patients, we have investigated the relationship between cellular energetics and mitochondrial biogenesis. with ethics approval and appropriate consents, critically ill patients were recruited within 24h of icu admission. age-matched control patients were undergoing elective hip surgery. muscle biopsies were taken from vastus lateralis. atp and creatine compounds were determined by hplc of perchloric acid extracts and standardised to total creatine [total cr = phosphocreatine (pcr) + creatine (cr)]. mrna levels for pgc1-alpha, nrf-1 and tfam were determined by rt-pcr and standardised to 18s mrna. data were analysed for significance using one-way anova. the ratio of pcr/cr was significantly decreased in both survivors and non-survivors. mrna levels of the mitochondrial biogenesis markers pgc-1alpha and nrf1 increased in survivors but not in non-survivors. a similar pattern was observed with the mitochondrial transcription factor tfam, although statistical significance was not reached. (1) the decreased pcr in both survivors and non-survivors indicates increased demand for atp in the acute phase of critical illness. (2) increased levels of markers of mitochondrial biogenesis in survivors indicate that maintenance of mitochondrial function, specifically atp synthesis, may be crucial to recovery. failure to maintain adequate mitochondrial function through biogenesis may contribute to atp depletion and mortality. local metabolic changes are not well investigated in sepsis and sirs. our aim was to describe subcutaneous metabolic changes using microdialysis (md) concurrently with systemic hemodynamics over 7 days in patients with sepsis/sirs and circulatory failure. methods. 25 patients with severe sepsis/sirs were recruited. at inclusion, all patients had circulatory failure despite resuscitation according to the rivers concept. cardiac index (ci), intrathoracic blood volume index (itbvi), extravascular lung water index (evlwi), blood lactate (p-lac), md lactate (md-lac) and md lactate-pyruvate ratios (md-lac/pyr) were analysed 4-6 hourly. data were tested for differences over time using anova. patients were subdivided into sepsis and sirs groups, and intergroup differences were tested using the rank sum test. mean apache scores were 26&24 for sepsis & sirs respectively. sofa decreased from 11.5 to 4.5 with no difference between sepsis & sirs. ci increased over time and itbvi, evlwi, p-lac & md-lac decreased. md-lac & p-lac were maximal at day1. lactate concentrations were generally higher in md than in blood, and in the sepsis group. severe sepsis and septic shock have been recognized as a serious clinical problem that shows an increasing incidence and that is responsible for substantial morbidity and mortality in intensive care units. sepsis has been defined as the systemic host response to infection with an overwhelming systemic production of both pro-and anti-inflammatory mediators. continuous hemofiltration has been suggested as possible therapeutic option that may remove the inflammatory mediators. on the other hand, hemodialysis and hemofiltration were reported to influence cardiac electrophysiological parameters and to increase the arrhythmogenic risk. therefore, in this study we have investigated the effects of hemofiltration on electrophysiological properties of the septic pig heart. methods. 40 pigs of both sexes were divided into 5 groups: 1) control group without hemofiltration; 2) control group with conventional hemofiltration (35 ml/kg/hour); 3) septic group without hemofiltration; 4) septic group with conventional hemofiltration (35 ml/kg/hour); 5) septic group with high-volume hemofiltration (100 ml/kg/hour). in septic groups, the sepsis was induced by fecal peritonitis and maintained for 24 hours. hemofiltration was applied for the second 12 hours of this period. ecg was measured just before and after 24-hours period of sepsis in septic groups and at the same time points in non-septic groups. action potentials were recorded in isolated ventricular preparations obtained from the hearts at the end of experiments. . rr and qt intervals were significantly shortened by sepsis in all 3 septic groups, in non-septic groups they were not influenced by the experiment. action potential duration (apd) was also significantly shortened by sepsis (septic group without hemofiltration vs. control group without hemofiltration) at all cycle lengths tested (500, 1000, 2000 ms). both conventional and high-volume hemofiltration in septic groups shortened apd further at slow pacing rates. hemofiltrate obtained in septic groups by both conventional and high-volume hemofiltration prolonged significantly and reversibly apd at all pacing rates. substitution solution alone had no effect on apd. neither hemofiltration nor hemofiltrate in control, non-septic groups influenced apd. we conclude that the hemofiltration in septic groups and the septic hemofiltrate influence significantly the electrophysiological properties of the heart, probably due to removal/content of various inflammatory mediators in the septic hemofiltrate. introduction. the precise mechanism by which multiorgan failure develops in severe sepsis and septic shock remains unclear. potential mechanisms include alterations of microvascular flow distribution, mitochondrial dysfunction and treatment effects. we investigated the effects of lps and different catecholamines on oxidative respiration of rat skeletal muscle fibers and hepatocytes. muscle fibers (m. gastrocnemius) were isolated from anesthetized male wistar rats (200-300 g). human hepatocytes (hepg2 cells) and human monocytes (monomac 6 -mm6) were also used. to avoid systemic effects of endotoxin and catecholamines, experiments were performed in vitro using the skinned-fiber technique. the mechanically dissected muscle fibers were incubated with lps (10 µg/ml) for 2 h. after 1 h of lps incubation, norepinephrine, dopamine, and dobutamine (100 µm each) were added. monocytes and hepatocytes were treated with different concentrations of lps only. mitochondrial respiration was determined after permeabilization with saponin, using a clark type electrode (oxygraph 2k, orobros instruments, innsbruck, austria). septic shock is associated with severe cardiac dysfunction, whose mechanisms remain only partly defined. recent data suggested that it might be triggered by the direct action of microorganisms and their products on the heart itself. we previously shown that flagellin (flag), the protein monomer from bacterial flagella, is a potent activator of nf-κb-dependent pro-inflammatory signaling in cultured cardiomyocytes. therefore, the aim of the present study was to evaluate whether flag might induce such an inflammation in the heart in vivo and contribute to cardiac dysfunction. h9c2 cardiomyocytes were stimulated with recombinant salmonella muenchen flag (1-100 ng/ml, 10 min to 24 h). in vivo, balb/c mice were injected (tail vein) with 1-5 µg flag (30 min to 6 h). the effects of flag were evaluated by its ability to activate nf-κb, and to induce transcription of tnfα and mip-2 cytokines. in vivo, cardiac neutrophils recruitment was evaluated by myeloperoxidase (mpo) activity. the expression of the flag receptor tlr5 was also determined. in vivo physiological measurements: left ventricular pression-volume curves. a microtip pressure-volume (pv) catheter (spr-839; millar instruments) was inserted into the left ventricle (lv) via the right carotid artery. the pressure and volume signals were continuously recorded and heart rate, cardiac output, end-systolic and end-diastolic volumes, stroke volume, ejection fraction and end-systolic and end-diastolic pressures were measured. load-independent indices of lv systolic and diastolic functions were determined by the slope of the end-systolic, respectively end-diastolic pv relationships in conditions of rapidly reduced preload (transient compression of the vena cava). . flag activated nf-κb in cardiomyocytes in vitro and in vivo, and also upregulated the transcription of tnfα and mip-2. flag also increased cardiac neutrophils recruitment. flag induced significant increases in end-systolic and end-diastolic lv volumes, indicating cardiac dilation, and a significant reduction of the load-independent indices of lv systolic function (end-systolic pv relationship, espvr, and maximal elastance, emax), indicating significant lv systolic dysfunction. in contrast, no change in the slope of the end-diastolic pv relationship (edpvr) was noted. bacterial flagellin induces a prototypical inflammatory response in cardiomyocytes in vitro and in the myocardium in vivo. these effects are associated with a profound alteration of the lv systolic function in vivo, suggesting that flagellin may represent a critical mediator of cardiac dysfunction in septic shock. current guidelines recommend either dopamine (da) or norepinephrine (ne) as the initial vasopressor in septic shock (ss), but the management of moderate to severe ss is still controversial. to explore this issue is important, because pharmacodynamic differences between vasopressors might be irrelevant in mild cases, but could potentially affect outcome in more severe patients. beside clinical implications, there are also economical considerations since these drugs are not cost-equivalent. this subject may be specially important for developing countries. the aim of our study was to compare ne vs da as the exclusive vasopressor for established moderate to severe septic shock (requirements of > 0.1 mcg/k/min of ne or > 10 mcg /k/min of da to maintain map 70 to 80 mmhg) multicentric rct involving nine polivalent icus from argentina, brazil and chile, randomizing moderate to severe ss patients to ne or da titrated to target map or maximal dose of 2 mcg/k/min ne or 100 mcg/k/min da. after inclusion patients were switched blindly to the assigned drug. the study could be stopped if severe hypotension or arrhythmias developed. epinephrine was used as a rescue drug. main outcome criteria were 28 day mortality, organ dysfunctions and adverse effects (ae). the study was stopped early after randomizing 50 patients because of low enrollment rate. only 45 patients were evaluable. main results are shown on the table. adverse effects with da were 4 cases of atrial fibrillation (af) and 6 supraventricular paroxysmal tachycardia (spt), which were considered serious in 3 cases. aes with ne were two af and one spt, which resolved with no drug suspension. aes occurred more frequently with higher doses of da. conclusion. the use of dopamine as exclusive vasopressor for established moderate to severe septic shock appears to be associated with a worst outcome and more adverse effects. this should be explored in a future better powered rct. although arterial blood pressure (abp) is a widely used guide for hemodynamic therapy in sepsis, few data exist on its association with mortality and on critical abp limits that should be maintained. in this retrospective cohort study, clinical, hemodynamic, and laboratory parameters were extracted from a prospectively collected database in 274 sepsis patients. the severity and duration of hypotension was calculated by the area under the curve (auc) of systolic arterial blood pressure (sap), mean arterial blood pressure (map), and mean perfusion pressure (mpp = map -central venous pressure). laboratory parameters included the most aberrant variables during the icu stay. urine output per hour during the first 24 hours and need for renal replacement therapy were recorded. the sepsis-related organ failure assessment (sofa) score was calculated from given clinical and laboratory parameters. binary and linear regression models were corrected for the severity of disease by inclusion of the saps ii (excluding sap count) as a covariate and were used to examine the association between abp and 28 day-mortality or organ function. similarly, a binary logistic regression model including saps ii as a covariate was used to determine the best discriminating cut-off limit of abp in regards of 28 day-mortality. the goodness of fit of each limit was assessed by the r2-value according to the nagelkerke method. . sap and map were recorded for 21.6±3.6 hours, mpp for 18.9±5.2 hours. there was a significant association between 28 day-mortality and the auc of sap (p<0.001, r2=0.262), map (p<0.001, r2=0.269), mpp (p<0.001, r2=0.295). the area under map 60 mmhg and mpp 45 mmhg was associated best with 28 day-mortality. one or more episodes of map <60 or mpp <45 mmhg increased 28 day-mortality by 2.96 (ci 95% 1.06-10.38, p=0.041) and 2.97 (ci 95% 1.19-7.76, p=0.01), respectively. there was a linear association between time under the critical map and mpp limit and 28 day-mortality. while abp was significantly associated with the sofa score, arterial lactate levels, and renal function, no association with liver function or troponin i was observed. the critical map and mpp limits for the need for renal replacement therapy were 75 mmhg (r2=0.127, p<0.001) and 60 mmhg (r2=0.124, p<0.001), respectively. during early sepsis, abp is associated with 28 day-mortality and organ function. mpp shows the best association with mortality and may be a new resuscitation target. animal models of traumatic brain injury (tbi) are used to elucidate sequelae underlying human head injury in an effort to identify potential neuroprotective therapies. although human tbi is a highly complex multifactorial disorder, animal trauma models tend to replicate only single factors involved in the pathobiology of clinical head injury and may thus partly underlie the discrepancy between preclinical and clinical trials of neuroprotective therapeutics. we here present our experience with a large animal model of tbi which was designed to closely resemble the forces impacting the brain in e.g. traffic accidents. anesthetized, mechanically ventilated instrumented sheep (n=26) were placed in prone position with the head resting on a support to allow free lateral movements of the head. a left-temporal head impact was then delivered by mechanical stunning device (mk 1200, schermer, germany), which is approved for euthanasia of domestic lifestock. a captive bolt with a mushroom-shaped head is propelled from the muzzle of the stunner against the skull by the discharge of blank cartridge inserted in a chamber behind the proximal end of the bolt. depending on the charge and the positioning of the stunner, this device delivers an intracranial atmospheric pressure of approximately 11 bar in sheep at a bolt velocity of approximately 49 ms-1. to prevent skull fractures, a steel plate was attached to the left temporal fossa. a fiberoptic intracranial pressure (icp) catheter and a brain tissue oxygen (pbro2) probe were introduced in the parietal white matter. unilateral ultrasound flowprobes were attached to the internal carotid artery to measure cerebral blood flow. after measurements, sheep were killed and the brains removed for neuropathological examination. brain injury was characterized by a marked increase in icp from 10 ± 3 to 23 ± 13 mmhg (mean values ± standard deviations) 2 hours after head impact. intracranial hypertension was accompanied by a significant decrease of cerebral blood flow. pbro2 significantly decreased from 19 ± 10 to 13 ± 12 mmhg. the decrease in sinus venous oxygen saturation did not reach statistical significance. in instrumented control animals (n=2), parameters remained unchanged. neuropathological examinations revealed the presence of multifocal traumatic subarachnoid hemorrhage in 18, and diffuse axonal injury in 23 out of 24 animals. while interstitial brain edema was found in all sheep brains, contusion zones were present only in a minority of the animals. the pathobiological characteristics of the head impact model presented here closely resemble the alterations frequently found in human tbi. the relatively high variability of neuropathological changes after head impact may be seen as a disadvantage of this model. non-neurologic organ dysfunction triggered by infection represents a frequent and independent predictor of poor outcome in traumatic brain injury (tbi) patients admitted to intensive care units (1). because tbi itself significantly increases susceptibility to infection (2) and infection is a potentially modifiable risk factor, we developed a combined experimental model of tbi and sepsis in the rat. controlled cortical impact (cci) was produced in left parietal cortex by using a 3 mm diameter tip (velocity 5 m/sec; depth 2 mm). sepsis was induced contemporarily by cecal ligation and puncture (clp). the outcome was evaluated in terms of mortality, neurological function (via the morris water maze (mwm) and beam balance (bb) tests)and histologically. rats were subdivided into 4 groups: sham, cci, clp, and cci + clp. 15-day mortality was 0% in sham, 4% in cci and 16% in clp group respectively. adding clp to cci increased mortality up to 40% (p<0.01 vs cci and p<0.05 clp alone). at 48h and 1 week post-injury mwm and bb test performance was significantly worse in cci and cci + clp than in sham and clp groups (p<0.05). lesion volume was similar in injured groups. ca3 cell loss in left hippocampus was unaffected in the sham and clp groups, while it was 25% in cci and 34% in cci + clp groups (p<0.05 cci vs cci + clp). our results show that the occurrence of systemic sepsis exacerbates mortality and cerebral damage in rats subjected to traumatic brain injury. t. j. p. lieutaud* 1 , j. rhodes 2 , p. j. d. andrews 2 1 anesthesiology and intensive care medicine, hospices civils de lyon, lyon, france, 2 anesthesiology and intensive care medicine, university of edinburgh, edinburgh, united kingdom introduction. human recombinant erythropoietin (epo) appears promising in different brain injury models but its cellular mechanisms remain poorly understood. following brain trauma injury (tbi), inflammation (il-1b) and chemokine expression (mip-2, neuropath appl neurobiol 1998) are important. the aim of this study was to measure the effects of acutely administered rhepo on il-1b and mip-2 after tbi. methods. with home office approval, under isoflurane anesthesia 9 rats sd were subject to lateral fluid percussion tbi (1.6-1.8 atm) (dixon j neurosurg 1987) of the left parietal cortex. epo (1000, 3000 or 5000 iu/kg) or placebo were injected in a random and double blinded manner by the intra-peritoneal (ip)route. the ipsi-and contra-lateral cerebral cortices were removed 4h later and homogenized. il-1b and mip-2 were measured in the surnageant using elisa kits. results are expressed as pg/mg of protein (mean ± sem). there was a significant increase in il-1b and mip-2 in the ipsilateral cortex in comparison with the contralateral side for both proteins analyzed. neither 1000 nor 3000 and 5000 iu/kg rhepo did not exhibited any significant effect (figure 1). conclusion. this study confirms that inflammation is important and occurs early after lfp-tbi. epo did not display significant effects on two of the main inflammation mediators. the purpose of this study was to evaluate the effects of agmatine on histopathological damage following traumatic injury using a clinically relevant model of diffuse axonal injury (dai) on the rat. a total of 24 male sprague-dawley rats weighing 175-200 g were anaesthetized and subjected to head trauma using marmarou's impact-acceleration model. the rats were then separated into two groups; one group was treated with agmatine and the other group was treated with saline for up to four days immediately after the head trauma. rats from both groups were killed one, three or eight days post-injury. the brains were examined histopathologically and scored according to the neuronal, vascular and axonal damage. there were no significant histopathological differences between the control and agmatine-treated group after one or three days (p>0.05), but evaluation after eight days revealed a significant improvement in the group treated with agmatine (p<0.04). our data indicate that agmatine has a beneficial effect in diffuse axonal injury and should be tried for therapeutic use in the management of this condition. d. morii*, y. miyagatani critical care department, national hospital organization kure medical center, kure, japan the disadvantageous effect of haemorrhagic shock on head trauma related mortality are well known. thus, efficacious shock treatment is a surely significant measure against the development of secondary brain damage. the small volume resuscitation by hypertonic saline has been shown to promote systemic and cerebral haemodynamic benefits. similarly, many clinical studies have demonstrated the effects of long-term mild hypothermia on outcome of traumatic brain injury. in this study, we evaluated the new strategy consisting of therapeutic mild hypothermia and hypertonic saline therapy to the multiple trauma patient with severe traumatic brain injury. severe multiple trauma patients (iss>=33, head ais>= 3) were studied to evaluated the efficacy of therapeutic hypothermia (35.5˚c for 48 h) and hypertonic saline therapy (na+: 200 meq/l for the first 24 h , 180 meq/l for 24 to 48 h, 160 meq/l for 48 to 72 h) which were applied to them in parallel with massive blood transfusion . we evaluated glasgow coma scale (gcs), injury severity score (iss), the probability of survival (ps), the volume of blood transfusion, infusion and urine volume during the first 3days, and glasgow outcome scale (gos). we monitored the extent of brain swelling by head ct. four male patients (age: 39±16 y.o., mean±sd) were examined. the characteristics of injury mechanism were explosion 1, mva 2, fall 1. on admission, gcs, head ais, iss and ps were 8.75±4.5, 4.5±1, 46±11 and 0.67±0.29, respectively. the sum of blood transfusion, infusion, and urine volume during the first 72 h were 2200±1773 ml, 16313±5503 ml, 6985±4109 ml. no patient was died and their gos on posttrauma day 90 was 3.8±0.5. the combined therapy of therapeutic hypothermia and hypertonic saline to multiple trauma patients with brain injury may lead to good outcome in spite of the necessity of a large quantity of blood transfusion and infusion. recent data suggest that commonly used anaesthetic agents, e.g. propofol, cause neurodegeneration in the developing brain. the intention of our study was to investigate the effects of propofol on primary neuronal cultures referring to the cell survival rate. primary cortical neuronal cultures were prepared from wistar rat embryos at 18 days gestation. to test the effect of propofol on neuronal survival, cultures were exposed to 100 µl gibco neurobasal-a medium per well with propofol at a concentration of 1 mg/ml for 3, 6, 9, 12, 24, and 48 hrs. cell viability was assessed using the methyltetrazolium method (mtt) and was related to untreated cells as controls. all cells were kept in normoxia. after three and six hours of exposition to propofol cell viability values of the propofol treated cells were significantly higher (150.1±12.1%, p=0.0033 and 122.6±8.5%, p=0.0297, respectively) compared to untreated control cells (100%). after 9 hours, values were decreasing to levels of the control cells (106.6±9.8%). after 12, 24 and 48 hours of exposition to propofol, in contrast, cell viability was significantly reduced (82.1±6.9%, p=0.0285, 42.8±5.9%, p<0.0001 and 22.4±4.0%, p<0.0001) compared to controls. at high concentrations, propofol has a time-dependent effect on the viability of primary cortical neurons. during the first 6 hrs propofol has a potential neuroprotective effect, whereas it seems to cause neurodegeneration in the period of 12 to 48 hrs of exposition. e. paramythiotou* 1 , j. papanikolaou 2 , p. ntagiopoulos 2 , a. armaganidis 1 , a. karabinis 2 1 icu, attikon university hospital, 2 icu, george gennimatas hospital, athens, greece multiple trauma patients constitute a significant majority of admissions in a general icu. brain injury is often present in those patients. the aim of our study was to investigate demographic, clinical and management characteristics in trauma patients suffering a brain injury in a five year period. in a retrospective study all trauma patients hospitalized in the 10 -bed multivalent icu of a 700 bed -tertiary hospital between 1st jan 2002 and 31th dec 2006 suffering a traumatic brain injury were enrolled. recorded data included age, gender, cause of the injury, icu length of stay, initial glasgow coma score (cgs), submission or not to an emergent neurosurgical intervention, all cause mortality and neurological outcome. a total of 260 trauma patients were hospitalized during the study period. tbi was present in 176 patients (67.7 %). among them, 35 were women (20%) and 141 (80%) were men. their mean age was 37.6 years (range 16 -83). icu length of stay (los) ranged between two and 300 days (mean 35.4days). traffic road accidents were the cause in 156 cases (88.6 %) while 14 tbis (8 %) were due to fall from a height on the ground which happened either accidentally or as a result of a suicide attempt. the rest 6 cases (3.4%) were due to accidents during work. mean glasgow coma score was seven (range 3-13). an extradural hematoma was present in 9p and a subdural one in 30p. intracerebral hemorrhage was noticed in 5p, hemorrhagic contusions in 21p (with or without diffuse axonal injury) and a traumatic subarachnoid hemorrhage in 32 p. twenty nine patients were submitted to craniotomy and 37 p were submitted to unilateral or bilateral decompressive craniectomy. mean los was 37.8 d for p submitted to a surgical intervention versus 39 d for the other group. barbiturates were used in 16 p (9%). a total of 138 patients survived (78.4%). death was due to neurological cause (herniation of brain stem and subsequent cerebral death) in 25 p. other causes of death included sepsis, multi organ failure, severe injury in other organs, and hemorrhage from upper gastrointestinal tract. a poor neurologic outcome (mean glasgow outcome score < 7) was noticed in 10% of patients. almost two thirds of trauma victims suffer from a cerebral injury. most of them are young males, victims of traffic road accidents. the injury is often severe and one third of patients are submitted to a neurosurgical operation. though overall mortality is rather low, long duration of treatment is often required and severe disability is present in a not negligible number of patients. in the majority of the intensive care units (icu), several of the admissions involves patients with primary nervous system illnesses. a great progress of the technologies used in the icu in the last few decades had reduced neurological illnesses mortality and morbidity. since september of 2006 we had beginning an longitudinal e prospective coort study verifying the characteristics of the patients 18 years older that had been admitted in the icu for primary neurological cause (clinical or surgical). the study occurred in a private hospital icu with 46 beds. we recorded 148 patients until the moment. the number of neurological patients corresponds 10% of the admissions in the unit. the average age of this group of patients is significantly lesser of the remain icu patients (65 vs. 72 years), however does not have difference estatistically significant between apache ii (12 vs. 11) and the mortality (11 vs. 12%) of the neurological patients and others. the stay of length in the unit is bigger (8,6 vs. 4,8) . we also recorded mechanical ventilation time length (26% ventilated patients with for average time 14 days). in ventilated patients, 41% was tracheostomyzed (on average in 9 days). 19% developed sepsis (11% with septic shock). the patients were divided and analised in several goups (for example: trauma, surgery, central nervous sistem infection, vascular disease,...). neurology was one of the most benefited specialties with the intensive care units progress and evolution. however, high mortality and morbidity caused by the neurological illness, and the social and economic impact that its sequels cause, still deserve the attention of the involved professionals cares of these patients in the acute illness. n. baffoun* 1 , w. gdoura 1 , h. ouragini 1 , k. baccar 1 , m. lamourou 1 , t. chaoua 1 , r. souissi 1 , c. kaddour 1 , n. ben romdhane 2 , s. mahjoub 2 1 anesthesia and intensive care, national institute of neurology, 2 departement of haematology, chu la rabta, tunis, tunisia trauma victims develop frequently various degrees of haemostatic disorders. the severity of such post traumatic coagulopathie is considered to be major detrimental factor of outcome. the aims of our study were: to identify the origin of such disorders, time course and their correlation with mortality. our aim was identification of coagulopathy disorders and relation to outcome in severely head injured. prospective study,june 2003-march 2004. included:critically ill isolated closed severe head trauma. collected data:demographics,management prior and during icu hospitalization (sedation, catecolamin drug use, blood product transfusion, intra-cranial pressure monitoring, neurosurgical emergency surgery etc.),ct-scan results, daily worst glasgow coma scale, admission simplified acute physiology score ii. we inserted an arterial catheter for invasive pressure monitoring, a central venous catheter and a unilateral jugular bulb in front of the most damaged brain hemisphere(cf. ct-scan). jugular bulb thrombosis was prevented by continuous infusion of 2ml per hour isotonic serum without heparin. blood samples were obtained simultaneously from the central venous line(k) and jugular bulb(b) at admission, 6th, 12th hour, and then in case of neurological aggravationt or daily till 3th day. we measured platelet count,prothrombin time (pt),activated partial thromboplastin time (act),fibrinogen concentration (fib), prothrombin fraction1+2 (f) and thrombin anti-thrombin complex (tat). during the study only central venous blood samples (pt, act, fib and platelet count) could be available if necessary. otherwise blood samples were centrifuged and preserved refrigerated for post hoc analysis. statistical analysis by student's t test, paired t test for paired results and analysis of variance. significance set as p<0,05. results. n=19; 9 survivors(s) and 10 deaths (ns). no differences between s and ns in demographics,management modalities, admission gcs(7±3), ct-scan,saps ii (27±10 vs 30±17, p=0,69). b vs simultaneous k platelet count was significantly lower in all drawn blood samples,with a trend to decrease overtime. s vs ns at day1 and day2: 191±60 vs 125±35 (p=0,017). admission b thrombin fractions was higher in ns(1000±209 vs 460±294, p=0,014). b day1 tat was higher in ns:45±20 vs 9,6±12 p=0,02. no difference for other tests between b vs k and s vs ns for different paired tests. pro-coagulant factors (f and tat) are valuable prognostic factors at day1 in closed isolated severe head trauma. severe traumatic injury is a multisystemic disease where normal homeostatic mechanisms are lost. this situation involves an increase in physiological needs. usually these patients present anormalities in the hypothalamic-hypophyseal axis, which become neuroendocrine dysfunctions with deteriorated physical or neuropsychological secuelae. the aim of this study is to improve our knowledge about this part of the axis in acute phase of politraumatism. methods. an observational prospective study was carried out, with 27 patients who were admitted to our icu with a critical traumatic injury, for six months. demographic and epidemiological data were registered. apache-ii (acute physiology and chronic health evaluation system) and apache-iii scores during the first three days were measured. tiss (therapeutic intervention scoring system) score during the hospital stay was recorded. also gh (grown hormone), igf-1 (insulinlike-grown-factor-1) levels and nitrogen urinary losses in the first three days after traumatic event were measured. statistical data were analysed with the spss 13.0 program. in our study 85,2% (23 cases) were men and 14,8% (4 cases) were women. the average age was 48,15 years old. the hypothalamic-hypophyseal-somatotrophic axis role in the first three days was characterized by a progressive increase in gh levels and a progressive decrease in igf-1 levels. connections between average hormonal levels in the first three days and apache-ii, apache-iii and tiss scores during this time were studied. a good inverse connection between igf-1 and prognosis was shown si (spearman index) -0,524, -0,631 p value 0,006 and 0,001 respectively with apache-ii and apache-iii. this appropriate connection could not be shown with tiss score sp -0,039 p value 0,848; but the connection between gh and tiss was better, sp 0,37 p value 0,063. conclusion. gh levels increase and igf-1 levels decrease in the first three days after acute trauma. lower igf-1 levels can mean a worse prognosis. there are no connections between igf-1 and sanitary resources used (tiss score) but these connections seem to get better when gh levels are higher. trimodal distribution of deaths and the golden hour concepts are in part responsible for the genesis of all modern trauma systems but these concepts have been challenged recently. our aim was to describe distribution of death in trauma using data from a trauma system and discuss what can be done from the organizational point of view to improve outcome. all traumatic deaths occurring between 2001 and 2005 in a trauma system were. data on age, gender, time and place of injury, time of first and second hospital arrival, cause of trauma and type of accident, hospital characteristics, dominant injury and time of death were collected for this study. for mortality distribution the variable time was transformed applying a natural logarithm. results. 1436 deaths occurred over a period of 53 months. 52% at the scene, 18% in the level i trauma centre, 21 % in level iii trauma centre and the remaining in level iv/v trauma centre. death distribution using a logarithmic scale in minutes showed four peaks: deaths at the scene, deaths in the first hours, deaths in the first two days and finally deaths in the second week that we referred as 2 minutes, 2 hours, 2 days and 2 weeks peak (image1). we found statistically significant differences in age and dominant injury concerning timing of death. a tetramodal pattern of death distribution could be described. our data support the need to focus on the treatment of severe head injuries namely in the intensive care environment. anaemia is usually detected in critically ill patients. red bloos cell transfusion is not free of risk. we want to start an alternatives to transfusion protocol but fist we tryed to dercrive our critically ill patients anaemia. our objectives were to: study the red blood cell and iron metabolism in the icu patients at admission. observe changes in these parameters across the first seven days after admission. observe rbc transfusion and his relation whit morbidity and mortality. find transfusion predictors at the admission moment. during tree mounths of 2006, we include all the admissions in a trauma and neurocritical icu of our hospital that stay in unit more than 24 hours. at the moment of admission we determinated haematocrit, (hto), haemoglobin (hb), and reticulocytes (%retic) levels, iron metabolism, folic acid, b12, epo and creatinin (kr) we repeated determinations seven days after admission if patient was still in icu. adverse events occurred during icu stay were also registered (mainly infections) together the number of rbc transfusions (with hb levels before and after administration). we included in the study 73 patients. severe traumas (41%), neurocritical patients (38%), tumoral neurosurgery (6,8%) and other patients (13%) . average age was 54.25 years and apacheii 14.6 ± 6.9 points. 76% were males . results of admission blood determinations and seven days after are exposed in table i . there is a tendency to decrease in hto and hb parameters, but not significant. the only parameter we observe difference statistically significative was the reticulocites rate (%retic), significative lower 7 days after admission. (p<0.05) in graphic 1 we describe anaemia groups in admission and the evolution of anaemia groups seven days after admission. we appreciated that no anaemia group suffers a severe decrease. 30% of patients were transfused during their fist week stay. average levels of pre-transfusional hb were 8.01 g/dl . we analysed transfusion predictors. hto and hb levels at admission predict transfusion. there is no other analytical parameter at admission that predicts transfusion. we also detected tracheal intubated patients at admission and patients with inotropic drugs perfusions at admission were significative more transfused (p<0.02 and p<0.05). conclusion. the high mortality rate in our patients is related to the initial gcs and cranial cat at the moment of admission. it is necessary to continue the study to determine the influence of the rest of the variables in the mortality rate of these patients. introduction. traumatic brain injury, subarachnoid hemorrhage (sah) and spontaneous intracerebral hemorrhage (ich) are associated with systemic inflammatory response syndrome (sirs). early diagnosis of sepsis versus sirs is frequently difficult in neurointensive critical care units. procalcitonin (pct) has been used as a predictor marker of bacterial infection in different groups of patients. there is variable and scarce information about pct in neurocritical patients. the aim of this study was to evaluate the utility of serum pct in the early diagnosis of fever from bacterial infectious origin in patients with acute brain hemorrhage. we made a prospective diagnostic study between july 2004 and january 2005. we analyzed serum level of pct and c-reactive protein (crp) on consecutive patients with diagnosis of sah, ich or tbi who have fever during the intensive care unit admission. we excluded patients with antibiotic therapy previous to admission. pct and crp were blindely measured from samples of serum extracted within 48 hs of fever onset and within 24 hs of antibiotic administration. blinded to pct and crp results and according to previously defined criteria patients were classified in two groups: proved bacterial infection (pbi) and non proved bacterial infection (npbi). serum pct was measured by immunochromatographic semiquantitative method brahams pct-q (brahams diagnostica, berlin, germany). its sensitivity is 0.5 ng/ml. we analyzed sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of serum pct and crp for diagnosis pbi. we defined negative serum pct as <0.5 ng/ml and negative crp as <100 mg/l. we studied 17 patient, 6 with sah (35%) and 5 with ich (29%). ten patients had pbi (59%, 95%ci 33-82%). pbi were pneumonia (6), urinary tract infection (3), meningitis (2) and central line associated blood infection (2). two patients had simultaneous infection sources. there were 2 bacteremic infections. pct was positive in 4 patients in pbi group (3 pneumonia and 1 bacteremic central line associated blood infection) and in 0 of npbi. sensitivity was 40% (95%ci 12-74%), specificity 100% (95%ci 59-100%), ppv 100% (95%ci 40-100%) and npv 54% (95%ci 25-81%). crp was positive in 10 pts, 5 pbi and 1 in npbi. sensitivity was 50% (95%ci 19-81%), specificity 85% (95%ci 42-99%), ppv 83% (95%ci 33-99%) and npv 55 (95%ci 23-83%). in this study serum pct had an adequate ppv to diagnose pbi, without false positives results. however, it has a low negative predictive value to diagnose pbi. due to the results obtained, we consider that the quantitative pct assay with a sensitivity limit of 0.1 ng/ml should be used for the future study to evaluate the role of pct as a predictor marker of acute bacterial infection in patients with acute brain hemorrhage. in different published series cerebral infarction occurs in 30-60% of patients with symptomatic vasospasm after subarachnoid hemorrhage (sah) despite maximal therapy. standard triple-h treatment is associated with life-threatening side-effects (such as myocardial ischemia and pulmonary edema) and has not been properly validated. milrinone, a phosphodiesterase iv inhibitor, has few side effects and exhibits inotropic, vasodilatory and immunomdulatory properties besides inhibiting platelet aggregation and thromboxane a2 synthesis. we present our experience using our m&h protocol (milrinone and homeostasis) in patients with vasospasm. it consists of cvp-guided normovolemia (maintain cvp=or>6), aggressive temperature control, maintenance of normal serum sodium and step-wise interventions based on symptoms (milrinone 0.05-0.1 mg/kg bolus plus infusion, levophed and angiogram plus intra-arterial milrinone). we retrospectively reviewed the charts and imaging studies of 60 patients diagnosed with symptomatic vasospasm based on the development of focal symptoms and the results of angiographic and doppler studies. cerebral infarction was defined as a new hypodensity on ct scan appearing at least 2 days after aneurysm clipping or coiling. conclusion. among the different physiological scores, the sah-pds was most strongly associated with the major outcomes and the h&h score was better than the other aneurysmal bleed scores. the strong association of physiological scores with outcomes suggest that interventions targeting physiological derangements may improve outcomes in sah patients. contrast induced nephropathy (cin) is the acute deterioration of renal function due to parenteral administration of radio-contrast media. cin is defined as an increase in serum creatinine concentration of >44 µmol/l (0.5 mg/dl) or 25% above baseline within 48 hours after contrast administration. [1]epidemiologic data in neurosurgical patients undergoing endovascular coiling are sparse and only one study in stroke patients reported figures of 5% prevalence. [2] cin is associated with increased morbidity, length of hospital stay and costs. pre-existing renal failure and the dose of contrast media are known risk factors for the development of cin in cardiac patients where the condition is well-described. [3]although the pathogenesis of cin is not entirely clear, several mechanisms for contrast-induced renal injury have been proposed, including alterations in renal medullary perfusion, direct cytotoxicity and oxygen-free radical generation. [4] we conducted a twelve month retrospective electronic patient record based review of data from patients presenting to the hospital for endovascular coiling. renal dysfunction was based on increase in serum creatinine of 44 µmol/l (0.5 mg/dl) or 25% above baseline within 48 hours after contrast administration; the incidence of contrast induced nephropathy was investigated. peri-operative care and post-operative management were analysed. a multi-variate analysis of risk factors was conducted and statistical tests done using microsoft excel. . 149 patients visited our hospital neurosciences unit and underwent endovascular coiling over a one year period (sept 2005-sept 2006). the incidence of contrast induced nephropathy was 9%. 1.3% had pre-existing renal disease and 0.6% needed haemofiltration on intensive care for renal failure post-operatively. the odds ratio for developing cin in patients with diabetes mellitus was 3.4 (0.8-14.2) p=0.2. the odds ratio for developing cin with pre-existing kidney disease was 1.89 (0.37-9.4) p=0.54. the development of cin did not show any correlation with patient age, emergency or electively performed procedure or the number of coils used. no anti-oxidants were given for prophylaxis and no protocol for peri-operative hydration was used though fluids were administered intra-operatively. conclusion. cin is a common cause of acute renal functional impairment and accounts for significant morbidity in patients undergoing endovascular coiling. patients with pre-existing renal failure are at high risk; other predisposing factors should be identified. there is some evidence regarding use of peri-procedural hydration and anti-oxidants and, therefore, management protocols should be developed. open prospective observational study. were studied patients treated by embolization after spontaneous intracranial aneurysm rupture. included: embolization complicated by rupture of aneurysm during the obliteration procedure. rupture was ascertained by extravasation of contrast. current results of period ranging from july 2006 till october 2006. thirty two patients embolized for 32 aneurysms. one patient pesented a rupture during the embolization: she was a 41 y.o female; she came to our institution's emergency suffering from acute headache, nausea vomiting and a mild meningism. she got no neurological defect (wfns grade i). ctscan showed a mild sah (fisher class 1). an angiography followed, confirming presence of a 3 mm ruptured pericallosum aneurysm. during embolization procedure, a sudden hemodynamic instability (bradycardia, unstable blood pressure) was noticed and rerupture of aneurysm confirmed by extravasation of contrast medium. this complication occurred during placement of the first coil. the procedure continued successfully and aneurysm was completely obliterated by three coils. ctscan performed immediately after end of the procedure showed no massive cerebral haemorrhage (class 2 fisher). the patient was thereafter transferred to our icu where she was extubated. she developed a transient neurological defect (right hemiparesis). she was discharged alive without any disability. aneurysmal perforation during embolization seems to be a rare event. in our case it doesn't cause much damage, but clinical severity is variable and far from being predictable. re-bleeding can result in severe intracranial hypertension and ultimately brain death. aneurysm thrombosis complicated 2 procedures, and was fatal for both (respectively 3 and 5 days after embolization) due to massive ischemia (aneurysm of the internal carotid artery) and refractory intracranial hypertension (aneurysm of anterior communicating artery). those two patients got respectively wfns grade / fisher classification: iii/3 and ii/2. the patient with wfns grade iv got a successful uncomplicated procedure 14 days after the initial insult and partial clinical recovery. he continued to improve and was discharged alive from hospital without major neurological disability (gos: good, modified rankin scale = 2). conclusion. endovascular coiling could be an efficient therapeutic tool. incidence and outcome of procedures complications is still to be determined. strategy in patients with high wfns grade is certainly try embolization because of too risky surgery. right management timing is still to be determined. the quantitative estimation of blood loss helps in the choice of the best treatment tactics. the purpose of the study is to evaluate the ability of central blood volume index (cbvi, volume in heart and lungs and large vessels divided on body weight) and total end diastolic volume index (tedvi, sum of the end-diastolic volumes of the atria and ventricles divided on body weight) to reflect the magnitude of a hemorrhage. normo-volumic values of cbvi and tedvi were measured in 14 cardiac icu cardiac patients, 2 pigs and 8 rats with weight range of 0.3 kg to 95 kg. blood loss in the order of 25-35 ml/kg (3-4 steps) was applied in 8 rats and 2 pigs. ultrasound dilution technology utilizes the decrease in blood ultrasound velocity caused by injecting isotonic saline, and can be used in species of any size. cardiac index (ci), cbvi and tedvi were measured by hcp101 (transonic systems inc., usa) before and after blood loss. a disposable extracorporeal av loop filled with heparinized saline was connected between an existing artery catheter and central venous catheter. reusable ultrasound sensors were clamped on to the arterial and venous limbs of the loop. a peristaltic pump (nipro, japan) was used to circulate the blood from the artery to the vein at 8-12 ml/min for 5-7 min. measurements were obtained by injecting 0.5-1 ml/kg (max 25 ml) of isotonic saline. at the conclusion, the av loop was flushed with heparinized saline. in normo-volemic situations indexes are in the range of cbvi = 9-15 ml/kg and tedvi = 5-9 ml/kg, despite 30 times differences in weight. a dramatic blood loss of 25-35 ml/kg in experimental animals produces the same magnitude 48-50% decrease in cbvi and tedvi. severe dysphagia associated with silent aspiration and the danger of asphyxia requires translaryngeal intubation or tracheostomy. the aim of the study was to apply the clinical screening test (cst) and fibrooptic evaluation of swallowing test (fest) to determine the best method of upper airway protection. it was a prospective cohort study during the period of 2003-06. it included 1643 patients operated for fpt. all patients were delivered to icu intubated and mechanically ventilated after operation. after full recovery from anesthesia, returning to consciousness and passing spontaneous breathing test (sbt) (if not -mechanical ventilation continued) they underwent cst of 6 points. the patients who passed cst without deficit were considered to have none or low level of dysphagia. the patients who passed cst with some deficit were considered to have dysphagia. all the patients were extubated and underwent fest. in patients with poor cst, icu crew was ready to perform translaringeal intubation immediately if necessary. patients with severe cases of dysphagia underwent tracheostomy and received cuffed tracheostomy tubes to prevent aspiration and ensure free air passage. on the next day after performing tracheostomy, swallowing rehabilitation therapy began. tracheostomiesd patients underwent fest every week. after passing fest with blue dye, decanulation was possible. results. 345 patients of total group who did not recover consciousness or did not pass sbt in 48 hours after operation were determined for prolonged artificial ventilation and were excluded from further study. the 1155 patients who passed cst without any deficit were successfully extubated and showed absent or mild dysphagia in fest. 143 patients passed cst with deficit and after fest were divided into three groups by the level of dysphagia 52-mild, 39-intermediate and 52-severe. the regress of swallowing disorders was evaluated by fest every week. in the first group the earliest recovery was in three days, in the two other groups none recovered earlier than after three weeks. the latest recovery was determined after a year of swallowing rehabilitation therapy. two patients were not decanulated at all. postoperative recovery made possible to reduce rs. but insufficient rs exhaust the patient and may result in secondary impairment of the brain. the aim of the study was the analysis of different respiratory strategies in these patients to choose the best. it was a prospective cohort study of 101 patients after removal of pft with complicated postoperative period during 8 and no significant difference in±2005-06. the age of the patients was 34 severity of complications and neurological status. all patients included into study demanded rs after operation because of low rd. all patients had bulbar palsy syndrome (bps). 64 patients with bps were tracheostomiesed. after full recovery from anesthesia and returning to consciousness ventilation modes were simv+ps or cpap+ps (ventilator pb 7200). rr (respirator and patient), tv, ps, fio2, peep, pao2 and paco2 and neurological status were evaluated and registered daily. the criteria of readiness to wean were determined as: pao2/fio2>200, peep<5, ps<8-10, spo2>90%, rr<30, fio2<30%, gcs>10. weaning was successful if patient could breathe spontaneously for more than 48 hours without neurological deficit arise. patients were divided into 3 groups: 1. simv+ps ventilation (respirator rr 50-80% of total rr) -41 cases; 2. cpap+ps ventilation -33 cases; 3. failed extubation in first 72 hours -27 cases. all patients of the 3 group were ventilated in simv+ps after reintubation. the patients of the 2 group were extremely unstable and the modes of ventilation were corrected 4-10 times per day. duration of ventilation was minimal in the 1 group with maximum replacement of spontaneous breathing with artificial ventilation -simv+ps (table 1 ). in this group was minimal number of breathing disorders (minimal number of ventilator mode corrections) and patients were most stable. in first group was tendency to regress of bps (73 %) and there wasn't cases of arising neurologic deficit. but in the 2 group there was increase of bulbar palsy syndrome in 46% cases and no regression. c. a. eynon* 1 , p. collins 2 1 neurosciences icu, wessex neurological centre, southampton, 2 wessex regional transplant, queen alexandra hospital, portsmouth, united kingdom the management of severe brain injury in the uk is undergoing significant change. national recommendations are that all severely brain-injured patients are referred to specialist centres. protocolised guidelines for the management of brain injury have resulted in improvements in mortality and morbidity. with this has come a reduction in the numbers of brainstem dead patients suitable for solid organ donation. however, there still exists a group of patients for which continued treatment is felt to be futile and who may be suitable as solid organ donors following death by cardiorespiratory criteria. all deaths during a 24-month period were audited prospectively. when patients did not fulfill the requirements for brainstem testing, futility in continuing medical treatment was determined by the supervising consultant neurosurgeon, neurointensivist and senior nurse. in such patients, treatment other than comfort care was withdrawn. patients (<75 yrs) where medical treatment was to be withdrawn were considered for nhb organ donation. . 75 patients died during a 2-year period. 13 patients had death confirmed by brainstem tests of which 5 became solid organ donors. 28 patients were potential nhb donors. nhb donation was considered in 26 cases and offered to the family in 23. in one case the next of kin were untraceable, in one case the coroner refused permission. consent for donation was obtained from the family in 15/23 cases. nhb organ donation occurred in 9 cases. in the remaining 6 cases, 4 patients died outside the time window for organ retrieval, in one the next of kin withdrew permission and in one the coroner did not grant permission. 3 of the patients who died outside the time window for nhb organ donation, subsequently donated tissue. a total of 18 kidney transplants, 5 liver transplants and one double lung transplant were performed from nhb donors. conclusion. the number of brainstem dead patients is declining in the uk. patients in whom continuation of medical care is felt to be futile can provide a source of solid organs suitable for tranplantation. successful transplantation of solid organs from potential nhb donors occurs in a significant proportion of cases. feedback from family members has been supportive regarding the decision to donate. the studies on treatment of patients with head injury and brain damage, with sudden cardiac arrest due to various reasons revealed, that it is very useful to introduce neuroprotective therapy in those patients. it allows to decrease the consequences of local and global brain ischemia. the aim of the study was to present the efficacy and tolerance of treatment with amantadine sulphate (amantix, merz, germany), as a neuroprotective therapy. in the intensive care unit, between 2004 and 2007 we monitored a group of 67 patients with consciousness disorders, in the age of 65.85 +/-12.85, with average bmi of 26.48 +/-3.49. the level of coma's deepness and its reasons were different. the examination plan, methods used, choice and classification of patients were carried out based on previously prepared protocols. the minimal period of treatment with intravenous infusion of amantadine sulphate was 7 days, however, if possible, the therapy was continued for 14 days. after this period the patients received amantix in tablets. many additional therapeutic measures from different groups were used in those patients. an endotracheal intubation and ventilation were necessary in all of the patients. amantix was used as treatment's supplementation in the dose of 2x200mg/day. at the admission the patients were classified with the use of gcs (glasgow coma scale). in order to evaluate the effects of use of the preparation, some specific function of the patients were examined before the use of amantix and after finishing of the therapy. the examination was carried out by the intensive care unit doctors, neurologist and nurses taking direct care of the patients. the results were compared with the control group of 55 patients, age 48,35 +/-25,52. those patients were treated with the use of standard methods. all of the collected data were worked up statistically. the authors revealed statistically important difference in gcs grading between the groups. the average gcs score in amantix group at the admission was: 5.55 +/-1.25, and at the discharge: 12.46 +/-1.21. analogically, in the control group the admission score was: 4.82 +/-2.11, and at the discharge: 8.76 +/-4.21. in 5 patients using amantix we have noted the presence of side effects, usually it was hiperactivity. 55 patients were transferred to different wards. 12 patients died. the average hospitalization period in the amantix group was: 20.08 +/-8.05, and in the control group: 25.58 +/-7.25 days. . this has been fuelled by increasing evidence demonstrating either sub-optimal care or poor end of life decision making as antecedants to cardiac arrest calls on acute wards. outreach and medical emergency teams have developed as a result, but their effectiveness remains unproven [2] . at southend, development of a critical care outreach service began in 2002. the aim of this study was to establish the trends in cardiac arrest call rates from the acute wards in the years prior to, during and after the introduction of the outreach team, to assess any potential impact this may have had. hospital switchboard records were analysed retrospectively to provide data relating to the date, time and location of ward cardiac arrest calls occurring between january 1 2001 and december 31 2006. arrest calls to all acute wards except the critical care unit were included. the data collected was then related to hospital inpatient activity (in terms of completed in-patient consultant episodes, supplied by the hospital's information department) to enable meaningful interpretation of the observed trends. table 1 summarises the results from the medical and surgical wards separately and then together to present data for the hospital's acute wards as a whole. the data shows an upwards trend for the years prior to and during the establishment of the outreach service, and a falling trend subsequently. conclusion. the establishment of a comprehensive outreach service that promotes all aspects of outreach critical care (expediting appropriate and preventing inappropriate critical care admissions, following up patients post critical care discharge and promoting critical care skills throughout the hospital) is likely to lead to a reduced frequency of cardiac arrest calls. however, this effect may take years and not months following introduction to be manifest. we suggest all outreach services should collect and present this simple data locally to demonstrate the potential impact of their activities. intracerebral haemorrhage (ich) represents 10-30% of all strokes. the acute and subsequent blood pressure management presents a therapeutic dilemma. it is necessary decrease high systolic blood pressure, but there is the risk of decrease cereb. objective: can the regional cerebral oximetry helps us to determine individual adequate blood pressure? ral perfusion pressure and risk of ischemia developing. methods. regional oxymetry is the method of measurement the cerebral oxygen content based on near-infrared spectroscopy, which is carried out by means of the invos device (in vivo optical spectroscopy). this method is non-invasive, delivers continuous information and it allows the possibility of emergency therapeutic response. rso2 is transcutaneous monitoring of regional cerebral saturation with hemoglobin oxygen (rso2) in mixed blood in the frontoparietal regions, which represents interface beetween the basin of the anterior and middle cerebral arteries. the normal value of rso2 is beetwen 55-75% in a majority of the population, and every change from the baseline in both directions by more than 20-25% signifies the risk of ischemia for the observed tissue. during a twelve-month period all pacients admitting with ich in our neurointensive care unit (nicu) were managed by regional cerebral oximetry (n = 20). arterial blood pressure was monitored and was corrected farmacologically. the functional outcome of patients when discharged from the nicu and after six month were evaluated by the glasgow outcome scale, barthel index and modified rankin scale. data was collected retrospectively for comparison with pacient which didn't monitor by rso2. we found correlation between discovery of patological rso2 values and age, initial gsc and volume of ich. there are less septic and hemodynamic complications in the group with monitoring rso2. using this method, the probability of successful improving outcome all patients with intracerebral haemorrhage will be estimated. there is the need for guidelines regarding the blood pressure managemet of these patients. elaborated data are available on iccollege.be. 39 of 134 (28,3%) icu directors, representing 637 icu beds completed the extended query. main findings were: visits limited < 2h/day (1h36 + 1h56) ; hcp dedicated to family (68%) children admitted from 10 y of age (89%) ; family accompanied by hcp during resuscitation (74%) ; no witnessed resuscitation procedures (5%) ; scare possibilities for family to stay during night (42%) ; insufficient bad news delivery (65,79%) ; poor team psychological support (5-11%). 147 icu physicians completed the follow-up simplified query. main findings were: psychological support for family (59,86%) and team (27,89%) ; post-resuscitation debriefing (31,97%) ; identification of dedicated hcp (59,86%) ; use of (90,48%) and written (96,60%) dnr-orders ; comprehension of (98,64%) and family witness (97,28%) of patients' will ; structured bad news delivery (97,96%) ; witnessed resuscitation (4,76%) and invasive procedures (2,04%) ; children accepted < 10 y (4,08%). in belgium, although there's obvious concern from the majority of icu's to communicate with relatives, recommendations for psychological team support, teaching bad news delivery, schedule of visits and witnessed procedures are made. sudden death constitutes an important sanitary problem. early diagnosis and advanced cardiorrespiratory live support are considered the most important factors related with short term prognosis. the objective of this study was to analyze the prognosis, clinical characteristics and evolution of patients who initially recovered after an episode of out-of/hospital or in-hospital cardiac arrest and who were admitted to a medical-surgical intensive care unit (icu). sixty three consecutive patients were included and retrostectively studied when they were admitted to a medical-surgical icu. for two years, from april of 2004 until april of 2006, sixty three consecutive patients were included. eighteen of the patients were women (28.6%) and 45 were men (71%). cpr was given out of hospital to 17 patients, and 28 patients suffered sudden death on a conventional hospital ward and 18 patients in special units (surgery, coronary, emergency room, etc.). the etiology of the arrest was considered to be of probable primary cardiac origin in 60% of the episodes and the rest of the origin of arrest was considered secondary to other pathologies (respiratory, sepsis. . . ). mortality in icu was 52.4% and 48,6 % were discharge alive but of that percentage of patients only 20% were released without important neurological damage. patients recovering following cardio-pulmonary arrest out of hospital and hospital ward had greater mortality than those who suffered an event in a monitored area (25%).(p<0,01) the lengthy resuscitation times (greater than 15 minutes), elevated apache ii scores and advanced age is associated with greater mortality. recovered cardiac arrest is a pathology with high mortality and morbidity in intensive care. in our series only 20% were released alive without severe neurological damage. the existing condition of the patient and the excessively long resuscitation times were decisive factors in these results. we conducted a retrospective case-note study in a six-month period at an innercity district hospital (distant from any international airport), and report three patients who deteriorated about the time of overseas travel by air. results. case 1. a retired gentleman of 74-years with progressive idiopathic pulmonary fibrosis requiring home oxygen therapy travelled by air without a medical escort. he deteriorated shortly after his arrival at the family home in the uk. he presented to the emergency department in respiratory failure requiring non-invasive ventilatory support. he died during prolonged hospitalization. case 2. a 51-year old woman with obstructive sleep apnoea reduced her diuretic prescription without her physician's knowledge prior to a long-haul flight. she deteriorated with acute shortness of breath shortly after her arrival at the family home in the uk. she was brought by her family to the emergency department where she was found to be in cardiogenic pulmonary oedema, requiring non-invasive ventilation. she survived hospitalization and was discharged with home oxygen therapy. case 3. a 39-year old man collapsed in the street explaining to passers-by that he had swallowed some packages. he had a travel ticket from the airport in his possession but was able to give no other history. he was taken to the emergency department and required intubation due to extreme agitation. he was found radiographically to have ingested multiple wrapped packets. he required laparotomy to remove 36 differently coloured packs some of which had ruptured releasing their contents. urinalysis revealed cocaine metabolites. he subsequently made an uneventful recovery after extubation and transfer to a surgical ward. patients may present to hospitals distant from international airports with clinical deterioration consequent upon risks associated with long-distance air travel. (2000) prospective observational study of a cohort including every septic patient admitted in a medical icu of an university hospital from may 2004 to december 2005. demographic, clinical, laboratory and therapeutic variables were registered. a clinical examination assessing motor deficit and tendon reflexes was daily performed in order to check cipnm criteria. univariate and multivariate logistic regression tests were used. . 378 septic patients were included with age 60±17, apache ii score 22±7, maximum sofa score 9.7±4, icu mortality 37%, in-hospital mortality 44%. 290 patients survived at least 12 days. 93 patients did not require mv and none of them developed cipnm. finally the analysis was performed with the 197 patients who survived at least 12 days and required mv, with a cipnm incidence of 39%. 73 variables were included in the univariate analysis. after multivariate analysis, it was found that several variables were significantly related with risk for the development of cipnm (odds ratio, or; 95% confidence interval, ic; signification level of change in 2 log likelihood, p): 1. mv length (days): or 1. patients in the icu often develop an acute neuromuscular disorder characterised by difficulty of weaning from mechanical ventilation and associated with variable degrees of muscular weakness including quadriplegia [1] . often associated with steroid treatment, neuromuscular blocking agents (nmba) and septic patients, the pathogenesis of cim is poorly understood [1] . originally thought to be neuropathic in nature, however, today myopathy is more often diagnosed [2] . to further clarify this point we present a series of 12 patients. between 1994 and 2002 a retrospective study was carried out on 12 patients diagnosed with cim and whose muscle samples were analysed in the dept. of neuropathology of chuvi, spain. in the clinical studies special attention was paid to the neuromuscular status apache ii, and treatments with steroids, nmba,total parenteral nutrition (tpn)and insulin. all patients underwent electromyographic studies and biopsy and in those with sensitive neurography an abnormal nerve biopsy. of the 12 patients, 4 were women and 8 were men, all aged between 46 and 86, (mean 66±11). in three of the patients admission to the icu was not necessary. all save two received prolonged high doses of steroids and two were on chronic treatment of steroids. only one was treated with nmba for more than 15 days. two patients were diabetic with no electromyographic signs of neuropathy. seven needed insulin to control glucemia during the critical period. 5 received tpn, and 9 had sings of sepsis. muscle biopsy showed signs suggestive of cim (atrophy of both types, alteration of the intermiofibrilar pattern) and in some cases miofagia and thick filament loss. in two cases there was discrepancy between neurophysiologic and biopsy findings (muscle and nerve). the seven patients that survived the acute illness showed neuromuscular symptoms on release from hospital. follow up was possible on three patients for 5, 8 and 9 years respectively. all recovered muscle strength, the electromyography normalized and currently have normal independent daily life activities. the aim of this clinical trial is to study cip in icu patients (pts) after surgical procedures. we enrolled retrospectively 64 icu pts (49 men (76.6%), 15 women (23.4%) who underwent at least one surgical procedure under general anaesthesia and developed cip. all of them were mechanically ventilated and stayed > 12 days. underlying diseases: multiple trauma 43, complicated surgery 19, pancreatitis 2. mean age: 42.8±18.1 years. operation sites: abdomen 28, cns 26, orthopaedics 12, thorax 4, other 3. mean anaesthesia time: 129±24 min. in all pts an electromyogram was performed twice, as well as daily neurological examination. we analyzed several parameters predisposing to cip. conclusion. 1) sepsis predisposes to cip, but cip can be appeared without sepsis (32.8%). 2) age and serum albumin values do not predispose to cip (p<0.1); however the early implementation of a nutritional protocol is useful. 3) although not well correlated, we try, if possible, to avoid neuromuscular agents. 4) high pgl predispose to cip (p<0.01); it is important to maintain pgl < 110 mg%. 5) cip prolongs lmv (p<0.01), los in icu (p<0.01) and los in hospital (p<0.001), but does not increase mr significantly (p<0.1). s. kjaergaard* 1 , s. e. rees 2 1 intensive care, anaesthesia and intensive care, region north jutland, aalborg, 2 center for model-based medical decision support, aalborg university, denmark (1) is accepted as the gold standard method of describing pulmonary gas exchange. in the clinical setting, if any, only very simple one-parameter models are used. the parameters of these varying upon changing the fio2. in a previous paper we have compared the miget with a simpler model, and shown that this simpler model is a good fit to the inert gas data obtained from the miget experiment (2) . this study explores whether the simpler model can reproduce oxygenation data in an oleic acid lung damage model upon changing the fio2 and compared these results with those obtained using the miget. seven pigs were used for the study. lung damage was induced by an intravenous infusion of oleic acid. six inert gases were infused to estimate the distribution of v/q-ratios of the miget model and dead space, shunt and a parameter describing v/q mismatch, i.e. fa2, of the simpler model (1,2). measurements were taken at five different ventilator settings. the two models were then used to simulate arterial oxygenation data when the model-parameters along with measurements of mixed venous blood gases at different values of fio2 were given as input to the models. both models can be used to simulate sao2 at varying fio2. this is shown in the figure where the models have been used to simulate sao2 at varying values of fio2 (miget "+", simple "squares") ranging from 0.21-1.0. it shows that the models simulate identical values of sao2 with a mean difference = -0.2 +/-1.7. since the miget and the simpler models provide both equally good fit to the inert gas data (2) and precise predictions of arterial oxygenation, they might be interchangeable in a clinical setting where only a limited amount of data are accessible. in addition, the parameters of the simpler model can be obtained quickly and non-invasively (3). the model could therefore have applications a clinical situation. ethanol may be used in the management of toxic alcohol poisonings 1 , or as sedation in alcohol withdrawal. ethanol may be a component within drug formulations, for example nimodipine infusion or chemotherapeutic agents 2 . ethanol flush has also been used to restore the patency of occluded catheter lumens 3 . in clinical practice, ethanol should only be infused via a pcvc and not a peripheral venous cannula, as the high osmolality of ethanol can cause thrombophlebitis. given anecdotal reports of pcvc deterioration during ethanol infusion 2,4 , this study applied a bench testing method and statistical modelling to develop clinical practice guidelines at our institution. the test solutions used were: dextrose (d) 5%; ethanol (e) 5%, 10%, 25%, 50%, 70% and 100%. each test solution was perfused through 3 pcvcs. a total of 21 pcvcs were perfused. (b) 24 hour perfusion. the test solutions used were: d5%, e5%, e10% or e25%. each test solution was perfused through 3 pcvcs. after perfusion, the strength of all pcvcs was assessed. the pcvc was attached to a force gauge. a known force was applied to the pcvc and the pcvc length was measured. this was repeated for increasing forces until the pcvc broke. length-force relationships were plotted and were described statistically using linear mixed effects models. . this bench test model produced reproducible data. the pcvcs were not directly traumatised by the testing apparatus. (a) 30 minute perfusion. pcvcs perfused with e50% , e70% or e100% perished with obvious structural deterioration. two distinct length-force relationships were described on linear mixed effects models: e50%, e70% or e100% weakened the pcvcs , whilst d5%, e5%, e10% and e25% had no effect upon pcvc structure (p<0.05) (b) 24 hour perfusion. the pcvcs did not perish. on linear mixed effects models, e10% and e25% weakened the pcvcs, whilst d5% and e5% had no effect (p<0.0001). conclusion. this model quantifies the effect of ethanol infusion upon pcvcs. this has not been demonstrated previously. the infusion of e50% e70% or e100% via pcvcs should be avoided. infusion of e10% and e25% for 24 hours weakens pcvcs. nimodipine and other drugs using ethanol as a carrier vehicle should be infused via pcvcs with caution. these potential hazards should be outlined in individual pcvc package inserts and drug product information leaflets. (2005) in septic shock patients tissue microcirculation is altered despite an increased tissue oxygen tension (1). microcirculatory distress could be one of the earliest stages in the progress of sepsis to multiple organ failure, and microcirculatory shunting could be an important contributing factor to this development (2) . sofa score has been suggested to clinically assess the level of organ dysfunction(3). we've done a prospective observational study to determine if changes in the rate of thenar muscles tissue deoxygenation during stagnant ischemia in patients with severe sepsis and septic shock are related to changes in organ dysfunction using the sofa score. fourteen septic shock patients were included in a preliminary study during the first days of sepsis evolution. , hutchinson?thenar muscle sto2 was measured noninvasively by nirs (inspectra technology, usa) before and during upper limb ischemia. sto2 decrease (downslope) after limb ischemia were analyzed during first and fifth day after icu admission. changes in sto2downslope, sofa score, cardiac output, lactate and the use of vasoactive drugs between first and fifth days were recorded. we found good correlation between ∆sto2downslope and ∆sofa between the first and the fifth day. (spearman's rho = -0,693; p<0,01). our results are in accordance with those reported by pareznik(4) wich correlated isolated values of sto2 with sofa in septic shock patients but moreover we show that changes in both variables during evolution are also correlated. in septic shock patients, thenar muscle ∆sto2downslope is well correlated with changes in ∆sofa, a clinically accepted tool to measure organ dysfunction evolution during sepsis. ∆sto2downslope monitoring could be not only a good marker of microcirculatory state but also a good indicator of organ dysfunction evolution during sepsis and consequently a potentially therapeutic objective. one of the important tasks that the anesthesiologist should perform is to monitor the functions of body organs; lung airway pressure is among the most important ones. a real-time continuous monitoring device which would be designed in a small volume and is portable could be used by anesthesiologists for this purpose. so, this device could improve the quality of anesthesia care while being efficient and cost containing. the device consists of four consisting parts as follows: sensors (pressure transmitter and gas velocity transmitter), processors (two avr microprocessors), monitor and software. software simulation: the performance of the monitor was controlled through a simulation process with matlab-simulink software (the mathworks inc. ma, usa),(1). the monitoring device demonstrated acceptable results, both clinically and at the lab assessments. the study demonstrated this device as an effective, reliable and cost containing device. a. rodríguez salgado* 1 , a. socias 1 , b. comas 2 , a. llompart 2 , i. losada 3 , p. ibáñez 1 , m. borges 1 1 intensive care unit, 2 emergency department, 3 internal medicine, h. son llàtzer, palma de mallorca, spain since we have a global computerized system on our hospital we used it to develope an integral and multidisciplinary working protocol for the early recognition of sepsis and its appropiatte therapy. prospective study conducted in a four-hundred bed teaching hospital with medical and surgical areas and the support of a global computerized system and on line internet conexion among areas. a computerized protocol to improve management of sepsis was developed. it automatically produces an annotation on the medical chart and a serie of analytics forms when activated. additionally clinical guidelines on sepsis management can be consulted. it was started on january 2006, and here we present all patients included until january 2007. during the study period 313 patient were included in the protocol, with a mean age of 63,92 (16,10) y, 65,4% were male. we have observed an ascending tendence in the number of patients included in the protocol, having arised from 8 patients on january 2006 to 50 on january 2007. the protocol was activated at the icu in 129 (51,8%) cases, at the emergency department in 99 (39,8%) and at hospitalization units in 21 (8,4%). two-hundred and two (65,4%) patients were admited at the icu. though initially the protocol was exclusivelly directed to patient with severe sepsis or septic shock, lately some patients with sepsis have been included. so, 25 (8%) had sepsis, 155 (49,8%) severe sepsis and 131 (42,1%) septic shock. only 158 (50.5%) had fever and 179 (57.2%) had arterial hypotension at the protocol entry. sepsis was community-adquired in 223 (72,4%) cases, nosocomial-non icu adquired 61 (19,5%) cases and icu adquired in 24 (7,8%). the the most frequent site of infection was the lung in 134 (43,1%) patients, followed by the abdomen in 80 (25,7%) patients. isolation of the causal microorganism was achieved in 219 (70%) patients. blood cultures were positive in 192(61.9%) cases. forty seven (15%) had 1 organ disfuntion (od), 81 (25.9%) 2 od, 68 (21.7%) 3 od and 63 (20.13%) 4 or more od. mean lactate levels were 2,66 (2,21) mmol/l, 2,30 (2,11) mmol/l and 2,01 (1,99) mmol/l at the activation moment, at 6 and a 12 hour respectively. mean c-reactive protein levels were 78,8 (22,89) mg/l. eighty-five (27,2%) patients deceased, of whom 2 (8%) had sepsis, 30 (19,4%) severe sepsis and 53 (40,5%) septic shock at the moment of activation. conclusion. it is possible to implement a global multidisciplinary computerized protocol for identification and management of the sepsis, although this is a laborious and continual process. t. kyprianou* 1 , g. panayi 2 , d. zeinalipur-yazti 3 , m. dikaiakos 3 1 intensive care unit, nicosia general hospital, 2 ngo, intensive care forum, 3 dept of computer science, universiy of cyprus, nicosia, cyprus introduction. the physiological condition of icu patients is marked by rapidly evolving and frequently life-threatening derangements as well as 'silent' yet important alterations in homeostasis. reliable monitoring i.e. the capability to collect, store, process, and share inpatient monitoring data along with physicians' remarks can bring tremendous benefits to all aspects of intensive care medicine (practice, research, education). currently, grid infrastructures assemble an extensive collection of resources and expertise (egee grid: 200+ sites around the world with more than 30,000 cpu's -5pb of storage, adequate for storing and managing icu-related data. we present the design and implementation of the intensive care window (ic-window), a software tool that enables the retrieval and integration of data from patient-attached medical sensors. ic-window follows a modular design to retrieve data from different patient monitoring devices. the tool includes a full-edged interaction protocol and graphical user-interface to interact with the phillips intellivue mp70 monitor. ic-window is implemented in the context of icgrid (intensive care grid), a novel data-grid framework that utilizes the egee infrastructure to enable the seamless integration, correlation and retrieval of 'clinically interesting episodes' across intensive care units clusters. we present preliminary data from software's use in 10 icu patients. conclusion. ic window belongs to a new generation of tools that could improve dramatically intensivist's capabilities as offers virtually unlimited storage capacity for every possible type of patient's data. in the future we plan to extend the ic-window application to communicate with other medical devices found within the icu. this will provide an open platform for the aforementioned applications. introduction. strict glycemic control by lowering blood glucose levels to 80 -110 mg/dl reduces the intensive care unit (icu) mortality, morbidity, duration of the hospital stay, and overall medical care costs. to provide an intelligent system for tight glycemic control, the eu-project "closed loop insulin infusion for critically ill patients (clinicip)" was started in january 2004. three different sensor technologies -two based on an enzymatic reaction with immobilised glucose oxidase using either amperometry or fluorimetry as transducer and another based on reagent-free infrared spectroscopy -have been developed to continuously monitor the glucose levels in the subcutaneous interstitial body fluid. monitoring of the subcutaneous interstitial fluid is realized using a microdialysis catheter cma60 from cma microdialysis ab as a body interface to all glucose sensors. experiments were carried out at the center for medical research (graz, austria), lasting up to 28 h with the probands starting under fasting condition, but receiving later their normal diet. after microdialysis probe implantation, the perfusate (either 5 % mannitol solution or elo-mel) flow rates were around 1 µl/min. for reference measurements, dialysate samples were collected. in parallel, blood glucose concentrations in venous blood samples, collected under arterialised conditions with the arm resting in a hot box, were determined using a glucose analyzer from beckman instruments. a clarke error grid analysis of the results from all three sensors has shown all values in clinically acceptable zones. the blood reference and sensor measurements were further compared using bland-altman plots. owing to the tubing connecting the catheter outflow and sensor, the lag times for the sensor readouts were between 10 and 30 min. for the electrochemical and infrared sensors a simultaneous micro-dialysis recovery rate determination has already been implemented for improving the correlation of the sensor readout to the whole blood levels. some observational studies suggest that the use of pulmonary-artery catheters to guide therapy is associated with increased mortality. we performed a randomized trial to study outcome benefit of using pulmonary artery catheter (pac) in ards patients when compared to standard care using central venous catheter (cvc). the subjects were 26 ards patients on mechanical ventilator who were assigned either to pac (pac group), or cvc (cvc group). the base-line characteristics of the two treatment groups were similar. the primary outcome was icu and in-hospital mortality from any cause. the pac group had a significantly lower icu mortality than the cvc group (2 vs 3, p value= 0.004) but there was no difference between the 2 groups in in-hospital mortality (one case mortality in cvc group). there were no significant differences between pac and cvc groups in urine output (1.7± 0.89 vs. 1.7± 0.56), use of vasopressors (0.46 ± 0.51 vs. 0.61± 0.51), and length of hospital stay (4.1 ± 2.1 vs. 4.46 ± 2.7) respectively. our findings suggest that pac can be used in ards patients for better hemodynamic assessment that may result in reduced icu stay and mortality rate. ethanol may be used in the management of toxic alcohol poisonings1, or as sedation in alcohol withdrawal. ethanol may be a component within drug formulations, for example nimodipine infusion or chemotherapeutic agents2. ethanol flush has also been used to restore the patency of occluded catheter lumens3. in clinical practice, ethanol must be infused via a pcvc, as its high osmolality can cause peripheral thrombophlebitis. given anecdotal reports of pcvc deterioration during ethanol infusion2,4 , this study applied a bench test and a statistical model to develop clinical practice guidelines at our institution. each 20cm triple lumen pcvc was perfused with a single test solution only. (a) 30 minute perfusion. the test solutions used were: dextrose (d) 5%; ethanol (e) 5%, 10%, 25%, 50%, 70% and 100%. each test solution was perfused through 3 pcvcs. a total of 21 pcvcs were perfused. (b) 24 hour perfusion. 12 additional pcvcs were perfused with d5%, e5%, e10% or e25%. after perfusion, the strength of all pcvcs was assessed. the pcvc was attached to a force gauge. a known force was applied to the pcvc and the pcvc length was measured. this was repeated for increasing forces until the pcvc broke. length-force relationships were plotted and were described statistically using linear mixed effects models. . this bench test model produced reproducible data. the pcvcs were not directly traumatised by the testing apparatus. (a) 30 minute perfusion. pcvcs perfused with e50% , e70% or e100% perished with obvious structural deterioration. two distinct length-force relationships were described on linear mixed effects models: e50%, e70% or e100% weakened the pcvcs , whilst d5%, e5%, e10% and e25% had no effect upon pcvc structure (p< 0.05). (b) 24 hour perfusion. the pcvcs did not perish. e10% and e25% weakened the pcvcs (p< 0.0001). not been demonstrated previously. the infusion of e50% e70% or e100% via pcvcs should be avoided. infusion of e10% and e25% for 24 hours weakens pcvcs. nimodipine and other drugs using ethanol as a carrier vehicle should be infused via pcvcs with caution. these potential hazards should be outlined in individual pcvc package inserts and drug product information leaflets. (2005) introduction. inadvertent esophageal intubation may lead to serious complications such as hypoxia, cardiac arrythmias and death. auscultation of breath sounds may be an inaccurate method to determine correct endotracheal tube placement of endotracheal tube placement.1 vibration response imaging (vri) is a novel non-invasive technology that measures vibration energy of lung sounds during respiration. as air moves in and out of the lungs, vibrations propagate through lung tissues and are recorded by 36 sensors spacially distributed on the patient's back over the lungs and a dynamic image is created. a 68 year old female patient presented with lung cancer. plain chest radiograph and ct scan revealed a large left lung mass comparable for a neoplasm. she was admitted for left lung lobectomy. after informed consent was obtained, she underwent vri before and after intubation. the esophagus was inadventently intubated and recognized immediately after the vri recording was obtained. the patient went on to have a successful operation. analysis of the vri data obtained during esophageal and tracheal ventilation are compared along with a normal vri image. during esophageal ventilation most of the vibrations (60%) were detected by the upper sensors and the least by the lower sensors (8%) (fig. 1) . following the endotracheal intubation as well as in a normal image, the vibrations were more evenly distributed with the sensors from the middle region receiving more vibrations. quick detection of inadvertent esophageal intubation is crucial to prevent serious complications but commonly used methods of confirmation such as auscultation and plain chest radiograph are inaccurate or do not provide timely results. vri is a novel technology that offers the potential to quickly identify inadvertent esophageal intubation in the or and perhaps other settings. the acapella ® is a small hand-held vibratory device that combines the resistive features of the positive expiratory pressure (pep) and the vibratory features of a flutter valve to mobilize secretions in the airway. vri is a novel dynamic imaging technique that measures vibration energy of lung sounds generated during respiration. in this study, our aim is to determine, using the vri, what regions of the lungs receive the most vibrations when the acapella is being used. a 20 second vri recording was performed on a healthy volunteer during normal breathing (first three breaths) and while using the acapella device (last four breaths). the vri recordings were obtained in 20 second periods of respiration. dynamic digital images and numerical raw values for vibration energy are analyzed and compared any regions of interest. . vri images at maximal expiration while using acapella show increased total vibration intensity. when the distribution of expiratory vibration is examined, it appears that vibration from the acapella goes more to the lower lung regions (figure 1 and 2) . asymptomatic catheter-related central vein thrombosis (cvt) which is diagnosed by venographic studies is mentioned to be as high as 66 %. moreover, when thrombosis occurred, the risk of catheter related sepsis was declared to be 2.6 % higher. in this prospective study we aimed to diagnose cvt early as possible, its incidence and risk factors. icu patients (pts) that needed a central venous access for at least 48 hours without chemotherapeutic agents administration were included in this prospective study. the catheters were inserted via internal jugular or subclavian vein at bedside under aseptic conditions using the seldinger technique. diagnosis of vein thrombosis was detected by color doppler ultrasound examination performed in less than 24 h after catheter removal (picture). the protocol was approved by the ethic committee. three hundred and thirty eight pts (154 f, 184 m), mean 58.8 years old (17-91 years), were included in the study. catheters mean duration time was 11.62 days and duration of insertion mean time was 7.16 min (4-40 min). in 289 pts catheter insertion was performed with a single puncture, in 31 pts with double and in 15 pts with three and more punctures. catheter localization was : in 256 pts right subclavian vein, in 61 pts left subclavian vein, in 15 pts right internal jugular vein and in 6 pts left internal jugular vein. catheter related thrombosis was diagnosed in 23 pts (6.8%) while catheter infection was seen in 13 patient (4.1%) (table). generally the chemotherapeutic agents administered via the central vein catheter have thrombogenic effect. when we study our cvt diagnosed 23 pts we found out that all of them were over 60 years old, the mean catheter duration time was 11.28 days (table) . but these results were not statistically significant when compared with the other pts under 60 years old and more than 11.62 days of mean catheter duration time. out of 275 pts who were not under anticoagulant therapy 14 had cvt while out of 62 pts under anticoagulant therapy 9 had cvt diagnose which was found statistically insignificant (p>0.05). our results show that patients under anticoagulant therapy have a three fold more cvt risk ratio than the others who are not using this anticoagulant therapy. patients under anticoagulant therapy have to be followed more closely regarding to cvt. the provision of good glycaemic control is thought to have some beneficial aspects in critical care patients. we have previously described the introduction of a web-based insulin dose calculator program to support the control of blood glucose in critical care. the aim of this study is to describe a modified version of a calculator program based on van de berghe's studies. this allows nursing staff to enter blood glucose values together with the insulin infusion rate into a calculator. the calculator then provides a recommended insulin infusion rate to control blood glucose with the added ability to recommend small bolus doses of insulin when appropriate, store blood glucose concentrations, insulin rates, bed number and the date and time of calculation. we also modified our feeding protocol to restrict the target enteral feed from 1800 kcal to 1500 kcal per day and removed the night time rest period. we studied the data stored by the program which was used for all patients admitted to a 16-bedded intensive care unit (approximately 40% of whom have neurological injuries) between june 2005 and may 2006. overall there were 661 patients admitted (mean apache ii score 16 [sd +/-7], with a mean age of 51 years [sd +/-17]. 146 patients died prior to icu discharge. there was a total of 5573 patient days with 13029 recorded calculation data points. the mean blood glucose concentration was 6.7 mmol (95ci 3.7 -12.1). there were 34 episodes of treated hypoglycaemia of which 11 were on an insulin infusion. there were two troughs in the time of data entry that corresponded with staff handover. there was no diurnal variation in blood glucose concentration or in insulin infusion rates, although this did peak slightly in the early morning. the mean value of the insulin infusion rate was 4.3 units / hr (sd +/-3.7). in normal subjects there is a decreased level of endogenous insulin in the early morning, that is only partly lost with constant nutrition. from this study we concluded that the web based insulin calculator facilitates the dosing of insulin in critical care in an economic manner. the lack of diurnal blood glucose concentration variation, suggests that once daily estimation of blood glucose may be an acceptable method of monitoring blood glucose concentrations in critical care. systemic inflammatory response syndrome (sirs) is a common entity in the intensive care units. early institution of an appropriate antimicrobial regimen in infected patients is associated with a better outcome. both c-reactive protein (crp) and procalcitonin (pct) are accepted sepsis markers. however, there is still controversy concerning the correlation between serum concentrations, infection and sepsis severity. objective:to determine the clinical aplication of procalcitonin (pct) and c-reactive protein (crp) plasma concentrations in the detection of sirs related to infection and sepsis and the assesment of severity of sepsis. desing: prospective observational study. setting: medicosurgical intensive care unit. patients: over a period of 2 months (january-february 2007), forty seven consecutive adult patients admitted in a intensive care unit for an expected stay >24 hrs.and sris symtoms and signs. informed consent was obtained from all patients. measurements: pct and crp plasma concentrations and white blood cell counts , apache ii y sofa within the first 24 h . each patient was examined at the time of enrollment and was classified in one of the following four categories according to the accp criteria: siris and sepsis group (sepsis, severe sepsis and septic shock). statistical analysis: were performed with spss 12.0. differences in continuous variables between infected and non infected patients were compared with the nonparametric mann-whitney test. and lineal.regressión. pct levels were significantly higher in the severe sepsis(p=0,01) and shock septic group (p<0,01). pct and cpr levels no weren found differences between sepsis of less gravity group and noninfectious sirs. pct and crp levels are significantly correlated to the severity of organ dysfunction (sofa y apache ii). pct and crp levels were significantly higher withing short space of time in patient with infection than in patients with non-infectious sirs, but for sepsis of less gravity, pct and crp plasma values not differentiate between sepsis and non-infectious sirs. investigators have reported microcirculatory alterations in critically ill patients using various techniques. persistent microvascular alterations might be associated with the development of organ failure and death. in this study, microcirculatory blood transit time was measured in intensive care patients using micro-channel flow analyzers and related to the severity score and mortality. thirty-one patients were included in this study. mean apache-ii score was 16.8. patients were divided into two groups, group l (apache-ii<17, n=18) and group h (apache-ii>=17, n=13). in both groups, blood transit time was measured using microchannel flow analyzers (mc fans). the micro-machined silicon chip is utilized in these instruments to simulate human capillary blood flow. microcirculatory alteration was presented as a blood transit time (second) of heparinized blood through micro-channel array under the pressure difference of 20 cmh2o. hematocrit, white blood cell (wbc) count, platelet count, and labolatory data were obtained at the same time. blood transit time was significantly longer in group h comparing that in group l (105.2+/-20.3 sec, 57.6+/-9.5 sec, p<0.05). wbc count was larger in group h comparing that in group l (15800+/-2300 /ul, 10400+/-1300 /ul, p<0.05). triglyceride (tg) and immunogloblin (igg/m/a) levels were significantly higher in group h comparing these in group l. none of the group l patients died, however, hospital mortality rate was 53.8? in group h. blood transit time through micro-channel array was prolonged in patients with high apache score 2)wbc, tg, and immunogloblin levels might be associated with patients blood fluidity. 3) micro-channel flow analysis may become a valuable tool to monitor microcirculation in critically ill patients. a. roman* 1 , t. el mahi 2 , c. hanicq 1 , d. gnat 2 , f. vertongen 2 , e. stevens 1 1 intensive care, 2 clinical chemistry, chu saint-pierre, brussels, belgium bedside glucose monitoring is mandatory for icu patients under tight glycemic control. point-of-care (poc) glucometers are based on glucose-dehydrogenase coupled with pyrroloquinoline-quinone/ferricyanide (gd/pqq)or phenanthroline-quinone/nad (gd/pqnad), or glucose-oxydase/ferricyanide (go) enzymatic methods for whole blood measurements. the laboratory reference method is hexokinase for measuring the plasma glucose levels. some drugs and metabolites can interfere with poc methods. the aim of this study was to evaluate the effect of the uric acid levels on the accuracy of these bedside methods. in this prospective observational study, arterial blood glucose was measured simultaneously on the accu-chek inform roche (gd/pqq), on the precision pcx abbott (gd/pqnad), on the rapidlab1265 bayer (go) and each value was compared with the reference laboratory result.363 measures were done in 75 adult icu patients. uric acid was obtained only once a day. a bland-altman analysis was done. biases were expressed as the poc minus the laboratory result. data were also analysed using linear regression. spearmann's rho squares were calculated to evaluate the uric acid level effect on the difference between poc and laboratory methods. the uric acid level range was 1.2 to 15.8 mg/dl. the biases, the 95% limits of agreement between each poc method and the reference method, the r 2 of spearmann for the correlation between uric acid level and the difference of result glucose level for each poc method are shown in table 1. the accu-chek inform overestimates moderately the glucose level while the precision pcx and the rapidlab underestimate it slightly. the wilcoxon ranked test with bonferroni correction gave a p < 0.001 for comparing the bias from the accu-chek to the bias from the precision pcx, p < 0.001 when compared to the bias obtained for the rapidlab. no statistical difference between the precision pcx bias and rapilab was found. the r 2 of spearmann correlating the effect of the uric acid level and the difference between the accu-chek and the reference method was 0.247. the weak effect of the uric acid level of the patient on the overestimation of the glucose measured by the accu-chek can be summarized as : glucose difference(accu-chek-laboratory) = 2.53 x uric acid (mg/dl) -2.3 . for the other poc glucometers, such correlations were absent. a patient presented with severe acidosis, point-of-care (poc) lactate of 42 mmol/l, suspicion of mesenteric ischemia and potential need for laparotomy. however, plasmalactates was <4mmol/l, and ethylene glycol (eg) ingestion was subsequently diagnosed. we, therefore, wished to determine why discrepant lactates occur and if this "lactate-gap" could be clinically useful. we phlebotomized blood, added various concentrations of eg metabolites, and tested with the five most common lactate analyzers. the pressure-volume(p-v)curve of the respiratory system defines the mechanical properties of the lung and the chest wall by relating airway pressure(paw)in no-flow conditions with lung volume at the same pressure level. objective:to evaluate a new technique for p-v curve tracing. two p-v curves were obtained in 10 ali/ards patients using the continuous positive airway pressure (cpap) method and an automated system built into a commercial ventilator (p-v tool 2, galileo, hamilton). for the cpap method, ventilators were switched to cpap and pressure was raised from 0 to 35 cmh2o in 5 cmh2o steps and then decreased while respiratory inductive plethysmography measured lung volume. for the automated method, we selected the automatic pv mode(galileo, hamilton)with flow 3l/m and maximum pressure of 35 cmh2o. lung-volume and airway-pressure data were recorded. p-v pairs were fitted to a mathematical model. lower (lip) and upper (uip) inflection points on the inspiratory limb and maximum curvature point on the deflation limb were obtained. correlation between methods was calculated using bias and 95% agreement limits for lips and uips and the intraclass correlation coefficient (icc) for absolute agreement for each pressure level. no adverse events were observed. p-v curves were equivalent for each method, with icc >0.75 for each pressure level. bias and precision for lip and uip were:lip 0.51±1.90cmh2o and uip 0.53±3.04cmh2o. the automated method for tracing p-v curves is equivalent to the cpap method. easily applicable at the bedside, it avoids ventilator disconnection and can obtain both inspiratory and deflation limbs of p-v curves. introduction. hypoxic hepatitis (hh) is a common cause of acute hepatic impairment. however, few is known about the degree and duration of the reversal of the liver impairment. therefore we assessed the liver function by indocyanine green (icg) clearance via limon (pulsion medical systems, munich, germany) in patients with hh. icg clearance was assessed in 17 critically ill patients fulfilling the criteria of hypoxic hepatitis. mean apache iii score was 81 ± 31. nine patients were male. icu survival was 47%. icg -plasma disappearance rate (pdr) (normal range: 18-25 %/min) and the retention rate of icg extrapolated to 15 minutes (r15) were obtained on the day of development of hh and till day five. nine patients with decompensated liver cirrhosis child c requiring intensive care therapy served as control group. results. icg-pdr and r15 expressed as mean ± standard deviation were 5.79 ± 1.89 %/min and 42.12 ± 10.13 %, respectively (17 patients), on the day of development of hh. icg-pdr and r15 were 6.07 ± 2.67 %/min and 42.81± 14.50 %, respectively, in the control group and was comparable to the hh group (p=ns). icg-pdr and r 15 improved continuously from time of development of hh to day five (7 patients alive and at icu) and were comparable to the course of laboratory data during observation period (table 1) . exhaled breath condensate (ebc) is a non-invasive means of collecting samples of airway lining fluid from the lower respiratory tract and monitoring respiratory diseases. we have used ebc acidification to study the effects of mechanical ventilation. ebc was collected (30-40 minutes at -20 o c: ecoscreen, jaeger). immediately after collection and as soon as the sample returned to room temperature, we measured conductivity and ph before and after deareation with helium (10 minutes). results are expressed as median (interquartil range). we have applied spsswin with spearman correlation and mann-whitney test. our earlier evaluations of a decision support system for tight glucose control (tgc) in the critically ill utilising model predictive control (mpc) documented clinically acceptable performance with hourly bg sampling. the mpc advises on insulin infusion based on blood glucose (bg) measurements and carbohydrate content of parenteral and enteral nutrition. in the present study, we evaluated an improved version of the mpc (v 1.04.01 to 1.04.03), which extends the advice by suggesting the time of the next bg measurement in the range from half-to four-hourly to reduce nurse workload. patients were admitted at one medical (mug; n=21) and two surgical (kul: n=13; cup: n=19) icus. patients were followed for a minimum of 48 hours and up to 72 hours. we evaluated safety of tgc (hypoglycaemia frequency), efficacy (mean bg; hyperglycaemic index, hgi; and time spent in the target range 4.4-6.1mm), and efficiency (time between bg measurements). nonparametric statistical tests evaluated differences among icus. one hypoglycaemia (bg < 2.2 mm) occurred in one subject at mug and in another at cup. there was no hypoglycaemia at kul. bg was within the target range but differed among icus with values of 5.8 (5.5-6.2), 5.9 (5.6-6.2), and 6.4 (5.9-7.1) mm [median ( strict glycemic control of plasma glucose has become general practice in most icus. frequent glucose control is required to titrate the amount of insulin infused and detect episodes of hypoglycemia. for practical reasons bedside glucometry is often used. aim of our study was to determine the accuracy of several glucose point-of-care (poct) devices in critically ill icu patients. arterial blood samples from unselected icu patients were collected and glucose measurements were performed on a bloodgas analyzer (glucose-oxidase; rapidlab bloodgas analyzer, bayer diagnostics) and three different poct devices (gdh-pqq, accu-chek sensor, roche diagnostics), gdh-nad+ (precision, abbott diagnostics) and modified gdh (hemocue). results of paired measurements were compared in three ways. paired values were plotted on a bland-altman plot. the pearson correlation coefficient (r) between the different methods was determined by linear regression. each pair was also analysed using the international organization for standardization (iso) criteria: -glucose > 4,1 mmol/l value within 20% of reference -glucose ≤ 4,1 mmol/l value within 0.8 mmol/l of reference. comparison between accu-chek and rapidlab 860 of samples from unselected icu patients (n=212) showed a good correlation (r2=0.9431). bland-altman analysis and analysis by iso criteria revealed clinical significant differences in 13.2% of pairs. in all cases the poct values were higher than the values from the bloodgas analyzer. comparable results were found using the precision and hemocue: although correlation was high, analysis by iso criteria showed differences in 9/81 (11.1%) and 3/81 (3.7%) of pairs. a clinically important inaccuracy was found between poct devices and bloodgas glucose measurements in critically ill icu patients. in the most cases values from poct devices were false high, increasing the risk of hypoglycemia. in the context of an insulin infusion protocol for aggressive glucose control in sedated icu patients poct devices are potentially dangerous and should be avoided. acute hyperglycaemia associated with insulin resistance is common in critically ill patients. acute tight control of blood glucose is considered important, although difficult to perform in routine care. we developed a software to implement tight glycaemic control (cgao): after each glucose level measure, the cgao advises a new insulin pump rate and the schedule for the next glucose control, gives indication for correcting any hypoglycaemia episode, and presents numerous parameters describing the quality of glycaemic control. in a retrospective case control study, we compared the software cgao (lk2, igny, france) used routinely in our unit since may 2006 with our previous method for glycaemic control based on daily medical prescriptions. patients without cgao (group pres) were randomly selected from our prospective intensive care database (admission after january 1, 2004) and matched 2:1 for sex, age, simplified acute physiologic score (saps ii), medical or surgical category, history of type 2 diabetes, and length of stay (los) with patients for whom we used cgao. type 1 diabetic patients or patients with los < 3 days were excluded. endpoints were average glucose level, hyperglycaemic index calculated above 6.1 mmoles/l, fractions of time (ft) resp. with normoglycaemia [4.2 -6.1 mmoles/l] and hyperglycaemia [> 6.1 mmoles/l], cumulative duration of hypoglycaemia [< 4,2 mmoles/l], average insuline requirements per day, and mean sampling interval for glucose control. we included 150 patients (mean age: 64 ± 17 years, saps ii: 42 ± 18, surgical: 26 %, type 2 diabetic: 12%), permitting to compare 50 cgao patients with 100 pres patients. a. sigalas*, d. w. patch, a. k. burroughs, j. p. o'beirne liver transplantation and hepatobiliary medicine, royal free hospital, london, united kingdom recently a number of studies have reported that relative adrenal insufficiency (rai) is common in critically ill cirrhotics. depending on the definition used the prevalence of rai in critically ill cirrhotics has been reported to be 50-60%, whilst in patients immediately post liver transplantation the incidence of rai has been reported to be 93%. given the high prevalence of rai in critically ill cirrhotics and patients undergoing liver transplantation, we hypothesised that adrenal function impairment may be a feature of chronic liver disease per se. the aim of this study was to define the prevalence of impaired adrenal function in patients with stable cirrhosis. we also examined whether the use of the 1µg or 250 µg acth tests was associated with different responses. methods. 34 patients with biopsy proven cirrhosis (or compatible imaging and biochemistry) underwent adrenal function testing with the 1 µg (n=20) or 250 µg(n=14) short synacthen tests (sst). patients were those with stable cirrhosis undergoing evaluation for transplantation or assessment for tips insertion for refractory ascites. patients with a recent history of infection or bleeding were excluded. . 34 patients underwent adrenal function testing. the median age of the group was 54 (iqr 48-60). the commonest cause of cirrhosis was alcohol in 40%. disease severity was measured by meld and childs-pugh scores. the median meld was 16 (iqr 13.5-20.5) and the median childs-pugh score was 11 (iqr 8-13). 11 patients (32%) showed a baseline cortisol < 250 nmol/l and an increment < 250 nmol/l following sst. 19 patients (56%) had an increment in cortisol < 250 nmol/l following sst. 23 patients (67%) had a baseline cortisol < 250 nmol/l. overall abnormalities in the sst (low baseline, peak or increment) were seen in 31 patients (90%). there were no significant differences in the frequency of abnormalities in the sst between the 1 µg or 250 µg sst groups. in multivariate analysis only meld score significantly predicted abnormalities in the sst. the above data suggest that adrenal dysfunction is a frequent finding in patients with stable cirrhosis and is correlated with liver disease severity. the underlying mechanism of this finding is unknown but may account for the very high frequency of rai in critically ill cirrhotics. the direct relation between glucose and lactate levels in critically ill patients has hardly been studied. we studied the relation between glucose and lactate in general and during hypoglycemia. intensive insulin therapy was performed with the nurse-centered grip computer system that aimed at a glucose level of 6.5 mmol/l or less. glucose and lactate were routinely measured together. all hypoglycemias detected over a 20-month period at the surgical icu were analyzed. hypoglycemia was divided in mild (3.4 thru 3.9), moderate (2.3 thru 3.3) and severe (<=2.2 mmol/l) hypoglycemia. . 30,000 glucose/lactate measurements were analyzed in 727 patients. glucose and lactate both were not normally distributed. after taking these distributions into account no evident relationship between simultaneous measurements of glucose and lactate was seen. 230 hypoglycemias were identified (141 mild;79 moderate; 8 severe). lactate showed a with a nadir value two hours after the hypoglycemia. the magnitude of hypoglycemia was not related with lactate response. evidence accumulates that improved glucose control in intensive care patients results in better outcome. improved glucose control requires rapid point of care glucose measurement. however, the reliability of point of care glucose measurements has been questioned. this study was done to evaluate the accuracy of accucheck point of care glucose measurement in intensive patients as compared to glucose measurement by the central hospital laboratory. the unit is a 10 bed mixed closed format icu. glucose regulation is performed by nurses for all patients using a computerised protocol(1). for this study, paired glucose measurements were randomly done in patients in the icu, only when glucose measurement was clinically indicated and only if workload permitted the extra task. the accucheck inform device (roche diagnostics) measures whole blood glucose in a single drop of blood. the central laboratory uses glycoseoxidase vitros 950 to measure glucose in serum. from 40 patients 246 paired measurements were obtained (table 1) . central laboratory glucose measurement was generally higher than accucheck glucose measurement. the mean difference was 0,6 mmol/l. correlation coefficient r was 0,935. the difference was more than 1,0 mmol in 24% of cases. blood samples were mostly (98%) derived from arterial lines. the correlation and bland altman plots are presented in figure 1 . related literature was examined for benchmarking purposes. data collection was carried out over a one month period, two days a week, in the icu. each blood sugar level (bsl) was recorded and ensuing action chosen on adjusting the insulin infusion rate, and resultant information analysed. a survey was carried out on nursing staff regarding their views on the protocol. statistical analysis was carried out using microsoft excel ® . the bsls were in the target range of 5.5-6.9mmol/l 39.4% of the time (n=863). the proportion of bsls that complied with the surviving sepsis guidelines target of less than 8.3mmol/l was good at 79.7%. the incidence of severe hypoglycaemia, defined as less than 2.2mmol/l, was low at 0.2%. compliance with the action chosen on adjusting the insulin infusion rate was high at 76.3%. total compliance (action and timing) with the protocol was 22%, and a relationship between compliance and achieving target bsls was shown. in general, a positive view of the protocol was obtained from the nursing staff regarding the protocol. the amnch icu insulin infusion protocol is effective at achieving tight glycaemic control in a safe manner. the low incidence of severe hypoglycaemia and high proportion of bsls complying with the surviving sepsis guidelines illustrates this. compliance with the protocol is achievable, demonstrated by the high level of compliance on action taken on the insulin infusion rate and the survey responses. however the timing of bsl checking needs to be addressed in future drafts of the protocol, as this is an area that needs improvement in terms of feasibility and compliance. further changes and auditing of the protocol are necessary to ensure consistency and improvement of the tight glycaemic control. introduction. intensive insulin therapy might be able to reduce mortality and/or morbidity in critical patients. besides adherence to strict protocols this strategy implies multiple, accurate measurements of glycemia. gold-standard laboratory assessment isn't able to provide immediate readings and capillary or arterial blood samples may differ too much when bedside reflectance meters are used, particularly in shock patients. our aim was to assess the accuracy of two methods of blood glucose analysis (bedside "glucometer" using capillary and arterial blood) in two groups of critical ill patients (shock and non-shock). prospective non-randomized, cohort study, in a university hospital general icu. a group of 18 consecutive icu patients with shock syndrome and vasoactive amines and another 18 contemporary patients without shock, were included (shock-sg and non-shock-nsg groups). for each patient 1 to 4 "triplets" of blood samples were collected in a 48h period, and included concomitant samples of blood drawn from fingerstick (cap) and non-heparinized arterial line(art). drops of capillary and arterial blood were analyzed with a bedside glucometer (glucotouch ® , lifescan), and a sample of arterial plasma was sent to laboratory for glycemia determination (lab). . total group had a median age of 57 years, mean saps ii of 53,4. sg was older (median age -64 vs 48 ys) and more ill (mean saps ii 63,2 vs 43,7) than the nsg. total mortality was 33,3% (sg-55,6%; nsg-11,1%). in the sg 61,1% had septic and 27,8% cardiogenic shock. in the nsg 44,4% had politrauma and 16,7% pneumonia. a total of 102 "triplets" were studied. non parametric wilcoxon test was applied to test agreement between cap-lab and art-lab paired samples. although we've found a highly significant correlation (spearman r>0,8) between cap-lab and art-lab values, agreement were rejected by 2-tailed wilcoxon signed ranks test, both in total, sg and nsg (p=0.000). an error grid-analysis using iso 2003 for blood glucose determination showed that 19,1% of cap and 23,4% of art determinations had a deviation more than 20% the reference lab value in the sg. in the nsg 31% of cap and 38% of art samples had more than 20% deviation. this study show that the glucometer we used had an unacceptable accuracy, both in shock and non-shock patients, far from the iso criteria that imposes only 5% of values can be more than 20% apart the reference value. glucose control is a major issue in the icu and standard procedures for its determination are still lacking. introduction. arginine (arg) is a precursor of the vasodilator nitric oxide (no), while asymmetric dimethylarginine (adma), derived from proteolysis of methylated arg residues, is a no synthase inhibitor. accumulation of adma is related to oxidative stress, impairing its degradation, and to renal-and liver failure. accumulation is associated with increased mortality (1). aim of this study was to evaluate the relation between plasma arg, adma, arg/adma ratio, organ failure and survival in patients with shock. we measured plasma concentrations of arg, adma and lactate, sofa scores and hospital mortality in septic (ss) or cardiogenic shock (cs) patients on d1, d2 and d5 of icu admission. patients were enterally fed with impact (arg-enriched). values are presented in mean ± sd or median (iqr). for regression analysis, arg, adma and arg/adma were log transformed. of the 44 patients, 27 had ss, 17 cs. mean age was 66 ± 14 yrs, sofa 9 ± 3, apache ii 26 ± 7.4. hospital mortality was 36%, predicted mortality was 56 ± 25%. at d1, median (iqr) of arg was 30 (22-48) mumol/l (normal range 30-145 mumol/l), adma 0.42 (0.33-0.55) mumol/l, arg/adma 80 (49-116) and lactate 4.6 ± 3.2 mmol/l. arg and arg/adma at d1 were inversely related to lactate (r = 0.59, p < 0.001, for arg; r = 0.65, p < 0.001 for arg /adma), and to sofa scores. the table presents the relation between arg and arg/adma to sofa score during sampling, and of arg and arg/adma on day 1 to maximum sofa score. apneic oxygenation (ao) is apllied during several operations in thoracic surgery and some procedures in th icu. retention of co2 often leads to hypoxemia, limiting the tolerable time in ao. this experimental study was designed to evaluate the effects of recruitment maneuver on oxygenation, co2 retention and survival times ao. following the ethic committee approval, 15 male sprague-dawley rats were anesthetized, tracheostomized, cannulated via the a. carotis and ventilated with pressure controlled ventilation (peak pressure: 15 cmh2o, frequency: 25/min, 0 cm h2o peep) for 15 minutes. following the basal (t0) arterial blood gas sample, they were randomized into 3 groups and disconnected from the ventilator: in group 1 (n=6), rats underwent ao with a cannula inserted to carina (o2-flow:0.2 l/min), in group 2 (n=6), recruitment maneuver (40 cm h2o (peep) ventilation pressure during 20 seconds) was performed before ao. in control group (group 3, n=3), data were recorded after apnea (this group was stopped after the first 3 subjects have died during the study period). further arterial blood gas samples were drawn in 1st, 3rd and 6th minutes, and ph, po2, pco2, hco3 and be values were recorded. survival times after the initiation of ao were also investigated. kruskal-wallis test was used to compare the values in different times, and mann-whitney-u the values in different groups. there were no significant difference in t0 values. compared to t0 values, there was a significant decrease in po2 and a significant increase in pco2 during 3rd and 6th minutes in all subjects, with a less change in g2. there was a significant difference between g1 and g2 in po2 after 3 and 6 minutes p<0.05; table 1 ), the difference in pco2 was not significant. survival time in g2 was significantly longer (g1:10,3±2,3 min; g2:14,3±3,6 min; p<0.05). to investigate potential prognostic factors and to predict extent of risks for postoperative pulmonary complications by logistic regressive analysis, and to evaluate the role of non-invasive ventilation in reducing the incidence of complications in elderly patients. stair-climbing test was carried out with asa score, fev1, changes of spo2 and hr et al were noted at the same time. logistical regressive analysis based on the parameters above were used to assess the relation between potential prognostic factors and postoperative complications. patients with limited pulmonary reserves were selected using the equation, and protective effect of non-invasive ventilation on these patients was assessed. incidence of postoperative pulmonary complications for high-risk patients with non-invasive ventilation was 33.2%, and incidence of pulmonary complications for high-risk patients without non-invasive ventilation was 67.7%. there was not a significant difference between these two groups with low-risk (p>0.05). conclusion. the mathematical model of logistic regressive analysis using stair-climbing testing combined with other parameters is a simple, reliable method to predict the cardiopulmonary reserved function in elderly patients. non-invasive ventilation can effectively reduce the incidence of postoperative pulmonary complications for high-risk patients, but it has no effect on patients with low-risk. continuous epidural analgesia (ea) and intravenous analgesia (ia) are widely used for postoperative thoracic pain control. the aim of this study is to compare the advantages and the disadvantages of both analgesic techniques. ropivacaine 0.2% to 20 mg/h using thoracic epidural catheters (ea) vs intravenous analgesia with remifentanyl 0.055 µgr/kg/min (ia). one hundred patients, undergoing pulmonary surgery, were recruited and divided, after randomization into 2 groups. patients included in ea group had an epidural thoracic catheter placed at th4 -th6 space, received ropivacaine 0.2% by continuous infusion (rate 10 ml/h). patients included in ia group received an ev continuous infusion of remifentanyl (rate 0.055 µgr/kg/min for 24 hours). rescue medication consisted of morphine 2 mg ev at patients demand. analgesia at rest and while coughing as evaluated by visual analogue scale (vas). haemodynamics, motor blockade (bromage scale) and side effects such as nausea, vomiting and pruritus were observed. the follow-up took place after weaning and every hour to 24 hours at rest and coughing. data are reported to media ± standard deviation (sd). analgesic effects were compared by using chi square statistics (p<0.05). both groups showed good analgesic effects. remifentanyl seems to decrease the incidence of side effects and the need of rescue analgesia. conclusion. 1)our data show that both analgesic techniques are able to guarantee a good pain relief after thoracotomy. 2)epidural analgesia was more difficult to perform and it showed less acceptance by patients. non-invasive ventilation (niv) has become an effective treatment to reduce morbidity and mortality in patients with acute respiratory failure. its application has been restricted to critical care o intermediate care areas, and little data is available on its usefulness in the post-anaesthesia care units (pacu). the aim of this study is to document our experience after eight patients treated in the pacu. we undertook a retrospective audit of patients treated with niv between 1 october and 31 december 2006. data of past medical history, age, asa physical status, surgical procedure, anaesthesia modality, type of respiratory failure, ventilatory mode, and time of niv were recorded. we also recorded side effects related to niv application. descriptive statistical analysis was used. eight patients were included. the mean age was 59.75±19.44 (sd) years. five patients were classified as asa 3 (62.5%), two as asa 4 (12.5%), and one as asa 1 (12.5%). three patients had morbid obesity, two chronic heart failure, and two chronic obstructive pulmonary disease. general and regional anaesthesia were employed in 6 and 2 cases respectively. type of surgery was thoracic (25%), urologic (25%), and plastic (25%). there was one case of abdominal surgery and another one of oral surgery. hypoxemic failure was detected in three patients (37.5%), and cpap was applied in these cases. bipap was applied in cases of hypercapnic (12.5%) or global (50%) respiratory failure. the mean time of niv was 89.37±26.78 (sd) minutes. no complications related to niv occurred. no patient required either intubation or transfer to the icu. all of them were transferred to the surgical wards the same day. conclusion. niv can be safely applied to selected patients in the pacu, to treat respiratory failure after either general or regional anaesthesia. it is an effective method to avoid intubation and icu stays, with minimal side effects. further studies should be conducted to analyze the clinical and economic impact of niv in the pacu. the routine use of volatile anesthetics in intensive care medicine has been limited so far due to technical difficulties and the need for an anaesthetic machine. the new anesthetic conserving device (anaconda)can provide a safe application of isoflurane or sevoflurane under intensive care conditions. this system is a modified heat and moisture exchanger which includes activated carbon fibres and works as a miniaturized vapor with recirculation. we studied the effectiveness of sevoflurane sedation in operative intensive care patients undergoing mechanical ventilation. we included 40 ventilated patients (neurosurgery, septic patients) in our retrospective analysis. the anaesthetic conserving device (anaconda-system) replaces the common heat and moisture exchanger in the ventilator circuit. the volatile anaesthetic is continuously applied in liquid status via a syringe pump to the minivapor where the anesthetic is vaporized. the expired anaesthetic gas is stored in the carbon filter and about 90% are resupplied into the breathing circle. first experiences with sevoflurane at our institution with a mean application time of 90.1 ± 56.3 hours per patient, showed a mean dose of 5.5 ± 1.3 ml sevofluran to achieve the individually targeted sedation level. 13.6 ± 7.1 minutes after the end of sevoflurane application, the patients could be neurologically evaluated or transferred to spontaneous breathing or extubated. no relevant side effects like nausea, vomiting or elevated enzymes were observed. we could demonstrate a safe application route, no development of tolerance as well as short wake-up times after long-term sedation with sevoflurane. the current literature suggest that volatile anaesthetics present an alternative for long-term sedation on intensive care units, providing optimized pathways from a medical as well as from an economical viewpoint. safety and effectiveness of sedation and analgesia in permanent pacemaker implant (ppm) is of special concern, due to age and comorbidity of the implanted patients. remifentanil pharmacological properties appear to be of interest in this setting. to date, there are no reports describing the use of remifentanil in this procedure, without the use of mechanical ventilation. consecutive patients in whom a ppm or other procedures, such as pacemaker battery change, was scheduled were included. a sedation and analgesia protocol for ppm implantation was performed: metoclopramide premedication, remifentanil infusion (20 mg/ml), local anaesthesia with mepivacain 2%, magnesic metimazol administration at the end of procedure, and remifentanil infusion withdrawal 20 minutes later. remifentanil infusion was initiated at a rate of 2 mcg/min, increasing the rate to attain a sedation ramsay scale grade 2 or 3, to a maximum of 6 mcg/min. remifentanil failure was defined as the need to administer a different sedation after the maximum dosage was attained. adverse effects, lenght of infusion and dosage were recorded. .two hundred and thirty-six consecutive patients were included. the men age was 77,5 ± 13,1. procedures: bicameral pacemaker 23,7%, unicameral 64,3%, battery change 5,6%, other 7,3%. infusion description and adverse effects are showed in tables 1 and 2. serious adverse effects were resolved with remifentanil infusion withdrawal. all the procedures were completed. remifentanil is safe and effective as sedation and analgesia for ppm implantation, even for old patients, with the dosages used in our protocol. nausea is the most frequent adverse effect. serious adverse effects are uncommon and can be resolved with infusion withdrawal. glass psa, gan tj, howell s. a review of the pharmacokinetics and pharmacodynamics of remifentanilo. anesth analg 1999; 89: s7-s14. peripheral arterial occlusive disease (paod) can cause intense neuropathic/ischemic limb pain in patients (pts) with end stage renal disease (esrd). although fentanyl may be an excellent choice in esrd due to the absence of active metabolites, the use of fentanyl as pca in esrd has never been reported. we used iv fentanyl pca for ischemic lower extremity pain in 11 esrd patients (9m, 2f), 10 of whom were scheduled for amputation. pts received iv fentanyl pca via a gemstar (abbott) pump. initial settings were 25mcg bolus, 20 min lockout, no basal, and dose was adjusted as needed to achieve visual analogue scale (vas) score < 4. pca started 48 hours preamputation and continued postoperatively for 48 h in 6 pts (4 pts had epidural postoperative analgesia and one terminal cancer pt did not have surgery). pain was assessed twice daily with vas. the mcgill pain questionnaire (mpq) -total ranked rating index (pri(r)), was administered immediately before and 24 h after pca started. sedation was assessed twice daily on a four-point scale: 1) agitated, 2) awake, 3) roused by voice and 4) unarousable. pain scores were compared with paired t-test. group data are presented as mean ± sd. mean sedation score was 2 in men and 3 in women. we did not observe respiratory depression in any patient. the aim of this study was to determine risk factors for relapse, and for icu-mortality in patients with ventilator-associated pneumonia (vap) related to nonfermenting gram negative bacilli (nf-gnb). retrospective case-control study based on prospectively collected data. vap diagnosis was based on clinical, radiographic and microbiologic (endotracheal aspirate ≥ 10 6 cfu/ml) criteria. patients with monobacterial vap related to nf-gnb were eligible. patients with subsequent superinfection or persistent pulmonary infection were excluded. patients with relapse of nf-gnb vap were matched (1:2) with patients without relapse according to duration of mechanical ventilation before vap occurrence. univariate and multivariate analyses were used to determine risk factors for relapse, and for icu-mortality in cases and controls. . 276 patients were eligible. 26 patients were excluded for superinfection. no persistant infection was diagnosed. 30 (11%) patients developed a relapse of nf-gnb vap, and were all successfully matched with 60 controls. pseudomonas aeruginosa was the most frequently isolated bacteria (55%), followed by acinteobacter baumannii (34%) and stenotrophomonas maltophilia (10%). no significant difference was found between cases and controls with regard to age (63±17 vs 64±14), male gender (90% vs 72%, p = 0.061), and surgery (13% vs 27%). however, saps ii at icu admission (40±10 vs 48±14, p = 0.015) was significantly lower in cases than in controls. duration of adequate antibiotic treatment for first vap episode was significantly shorter in cases than in controls (12 ± 3 vs 14 ± 4d, p = 0.031). inadequate initial antibiotic treatment was the only variable independently associated with relapse of vap related to nf-gnb (or [95% ci] = 8.1 inadequate initial antibiotic treatment is independently associated with relapse of vap related to nf-gnb and with icu-mortality. ∆ radiologic score and saps ii at day 7 after vap diagnosis are independent risk factors for icu-mortality in these patients. s. blot* 1 , j. solé-violán 2 , j. blanquer 3 , j. almirall 4 , a. rodriguez 5 , j. rello 5 1 icu, ghent univ hosp, ghent, belgium, 2 icu, dr negrin hosp, gran canaria, 3 respiratory care, clinic hosp, valencia, 4 icu, mataró hosp, barcelona, 5 icu, joan xxiii univ hosp, tarragona, spain practice guidelines suggest processes of care such as timely pulse oximetry monitoring and antibiotic therapy, as quality indicators for the management of communityacquired pneumonia (cap). the objective of this study was to determine whether postponed initial processes of care such as pulse oximetry monitoring delays initiation of antibiotic therapy and adversely affects intensive care unit (icu) survival in patients with severe cap. a prospective observational multicenter study was conducted including 529 patients with cap admitted to the icu in 33 hospitals. a secondary analysis was conducted to evaluate processes of care and icu survival. postponed blood culture sampling, arterial blood gas sampling and pulse oximetry monitoring was predictive for delayed antibiotic administration (p<0.001). linear regression analysis demonstrated that a delay of >1h in blood culture sampling was associated with a delay of 4.7h (95% confidence interval [ci], 2.5-7.0) in antibiotic therapy, a delay of >1h in blood gas sampling with a delay of 5.2h (95% ci, 2.9-7.6), and a delay in pulse oximetry monitoring of >1h with a delay of 4.2h (95% ci, 1.7-6.7). a delay in antibiotic administration of >6h was associated with increased mortality in univariate analysis (relative risk [rr], 1.85; 95% ci, 1.15-2.97), but not after adjustment for disease severity. a delay in pulse oximetry monitoring of >4h was associated with increased mortality in univariate analysis (rr, 1.97; 95% ci, 1.11-3.51) and after adjustment for disease severity (hazard ratio, 1.95; 95% ci, 1.22-3.11). in patients with severe cap timely executed processes of care are associated with a short time to antibiotic administration and reduced risk of death. appropriateness of antibiotic therapy is associated with reduction of bacterial load. c-reactive protein (crp) is a valid biochemical surrogate. our objective was to determine the correlation of bacterial load, measured by quantitative tracheal aspirate (qta), with crp as an indicator of inflammatory response in episodes of lower respiratory tract infection. to evaluate whether appropriateness of antibiotic treatment influences microbiologic (qta), biochemical (crp) and clinical resolution criteria (temperature, wbc, sofa and po2/fio2 fraction). prospective cohort study. sixty-five intubated patients with monomicrobial lower respiratory tract infection were included. crp and bacterial load variation were evaluated through the ratio between d4 and d0 measures. a qta was performed on lower respiratory tract onset (d0) and 96h afterwards (d4). its logarithm value (logqta) was recorded. logqta correlated positively with crp, temperature and wbc. logqta has decreased significantly more from d0 to d4 in patients receiving appropriate empirical antibiotic therapy compared to those with inappropriate treatment (logqta ratio 0.77 vs 1.04, p<0.05). mean crp levels showed a similar pattern, decreasing from d0 to d4 in patients receiving appropriate empirical antibiotic treatment, but not in episodes with inappropriate treatment (crp ratio d4/d0 0.54 vs 1.36, p<0.05). ancova showed that crp level on d4 was significant lower in patients with appropriate antibiotic treatment compared to inappropriate empiric treatment (103±10 mg/l vs 192±14 mg/l, p<0.05). the best cut-off to predict appropriateness of antibiotic therapy is a crp levels reduction of 80% on d4(auc=0.87). conclusion. c-reactive protein correlates with bacterial load and is a valid biochemical surrogate of bacterial burden in lower respiratory tract infection. follow-up measurements of crp anticipate the appropriateness of antibiotic therapy. a. günther* 1 , p. schenk 2 , m. maggiorini 3 , a. betbesé 4 , p. f. laterre 5 , n. fedorovskiy 6 , f. j. h. taut 7 , r. g. spragg 8 1 university of giessen, lung center, giessen, germany, 2 , medical university vienna, vienna, austria, 3 , universitätsspital zürich, zurich, switzerland, 4 , hospital sta cruz y san pablo, barcelona, spain, 5 , hôpital saint luc, brussels, belgium, 6 , city clinical hospital n67, moscow, russian federation, 7 altana pharma ag, a member of the nycomed group, konstanz, germany, 8 , uc san diego, san diego, united states the formal diagnosis of ards requires the acute onset of a severe impairment in oxygenatio(pao2/fio2 <= 200 mm hg), exclusion of a hydrostatic cause, and the presence of diffuse bilateral opacities. pneumonia is one of the most common underlying reasons for development of ards, but when only unilateral opacities are present, these patients fail to fulfil ards criteria. it is currently not known whether fulfilment of the formal ards criteria has any impact on 28-day mortality in patients with pneumonia suffering from severe gas exchange abnormalities. the valid study, a randomised, double-blind study in intubated and mechanically ventilated patients with severe respiratory failure (pao2/fio2 <= 170 mm hg) due to pneumonia or aspiration of gastric contents investigates the effect of rsp-c surfactant (venticute ® ) on mortality. the study does not require a formal diagnosis of ards for patient enrolment. however, the presence or absence of ards is documented. we conducted univariate and multivariate logistic regression analyses using preliminary blinded data from the first 443 patients randomised with a diagnosis of pneumonia. the prognostic value of the formal diagnosis of ards was determined. univariate logistic regression analysis failed to identify a significant correlation (p=0.13) between the formal diagnosis of ards and mortality at day 28. pao2/fio2 was more likely to be associated with mortality (p=0.01) as was the number of quadrants on chest radiograph that showed opacities (p=0.02). age and apache ii score were highly associated with mortality (p<0.0001). multivariate logistic regression identified age (p<0.0001), the number of involved quadrants (p=0.002), and apache ii (p=0.06) as independent factors affecting 28-day mortality. conclusion. the prognosis of ventilated patients with pneumonia is not dependent on the formal diagnosis of ards. instead, age, apache ii score, and the number of lung quadrants with radiographic opacities are more predictive of outcome. bernard gr et al. intensive care med. 1994; 20:225-232 . to determinate the clinical-epidemiological characteristics and risk factors for postsurgical pneumonia (psp) after lung cancer resection in a university hospital. a retrospective case-control paired study (1:2) was performed in 604 cases of lung cancer collected from 1999 to 2004. definition of psp case was a new or changing radiographic infiltrates with two or more of the following criteria: fever > 38 o c, wbc>10000 mm3 or/and purulent secretions. control group was formed by patients matched by age and lung cancer stage. . 81 patients were evaluated (22 psp and 62 controls). overall, data of both groups were: age 64 ± 10 yr, males 79 (94%), smoking habit (active or past smokers) 81 patients (96%), copd 64 patients (76%) and weight loss over 10 kg in 5 patients (6%). incidence of psp was 4%, crude mortality rate and attributable mortality estimated for psp was 32% and 24%, respectively. in the psp group, we found the following isolates (64%): p. aeruginosa (18%), s. viridans (14%), h. influenzae (14%) s. pneumoniae (9%) and undeterminated (36%). psp was associated with low bmi (p=0.007), low fev1 (p=0.012), stage iiia (p=0.004), anaesthetic time (p=0.047), pneumonectomy (p=0.017), thoracic pain (p=0.046), reintubation (p=0.001) and haemorrhage (p=0.018). conclusion. the incidence of psp in our series is low but with a high mortality. identification of risk factors (some of them suitable for medical intervention) may improve the management of lung cancer patients treated with surgery. j. karhu* 1 , h. syrjälä 2 , p. ylipalosaari 2 , j. laurila 1 , p. ohtonen 3 , t. i. ala-kokko 1 1 anesthesiology, division of intensive care, 2 infection control, 3 surgery, oulu university hospital, oulu, finland introduction. scap (severe community acquired pneumonia) and hap (hospital acquired pneumonia) requiring icu treatment have been shown to be associated with significantly higher mortality compared to those not requiring icu treatment (1, 2). we compared pneumonias acquired outside the icu to that acquired in the icu, during mechanical ventilation (ventilator-associated pneumonia, vap). patients admitted into a mixed university level icu during a 14 month period whose icu stay was longer than 48 hours were included. the occurrence of scap, hap and vap were prospectively assessed. the following information was collected: age, severity of underlying disease on admission, underlying malignancy and recent use of immunosuppressive therapy. the length of icu and hospital stay as well icu, hospital and 28 day mortalities were recorded. a total of 335 patients fulfilled the inclusion criteria during the study period. there were a total of 156 pneumonias. majority of the pneumonias were scap (86/156), while there were 44 hap and 26 vap cases. patients with hap tended to be older (61.7, p=0.07) and a larger proportion of them had malignancy (34%, p< 0.001), compared to vap (56 years, 27 %) or scap (24 years, 7 %). there were no significant differences between the mean admission apache ii scores (scap 23.6 vs. hap 22.6 vs. vap 22.2) . the icu length of stay was longest in vap; while the hospital stay was longest in patients with hap (table 1 ). the survival rates were highest in hap, although this did not reach statistical significance. in apache ii and age adjusted multivariate logistic regression analysis vap (or 3.8, 95% ci 1.39-10.47, p=0.009) and scap (or 3.3, 95% ci 1.71-6.41, p<0.001) remained significant risk factors for hospital mortality together with immunosuppression (or 2.3, 95% ci 1.25-4.42, p=0.008). heart surgery in infants is often associated with pulmonary inflammatory process. at the same time, the blood level of pro-inflammatory factors: interleukin-6 (il-6) and interleukin-8 (il-8) is increased. the number of polymorphonuclear leukocytes (pmn-elastase) and neutrophils is raised as well. a qualitative evaluation of the factors, cellular composition analysis of nonbronchoscopic trachebronchial lavage (ntl) combined with clinical findings can help early diagnose pneumonia. the objective of the study was to reveal the peripheral blood level of pro-inflammatory cytokines (il-6, il-8), the activity of pmn-elastase and α1antiprotease inhibitor (α1-pi), as well as examine the ntl cellular composition and cytokine level in infants before and after heart surgery. we studied 24 infants aged from 4 days to 11 months, weighting between 2.3 and 11 kg. 15 patients underwent cardiopulmonary bypass surgery, 9 patients were operated on without cardiopulmonary bypass. in 7 cases a clinical diagnosis of pneumonia was made between 2 and 5 days postoperatively. early postoperative survival was 100%. the peripheral blood cytokine concentration in operated infants pre-and postoperatively is presented in the study (table 1) . a significant increase in pro-inflammatory factors after surgery can be observed. we examined the ntl of 9 infants who underwent heart surgery and who did not develop pneumonia. we noticed that the number of neutrophils increased significantly in all patients after cardiopulmonary bypass surgery, sometimes reaching 80%. we consider it as a sign of pulmonary inflammatory process. the number of nonviable alveolar macrophages before and after surgery exceeded 50%. it indicates a decrease in cellular pulmonary protection. the pmn-elastase peripheral blood activity was 273.2±75.00 iu/ml preoperatively and 339± 55.00 iu/ml postoperatively; the α1-pi level was 32.1±7.0 iu/ml and 32.5±15.00 iu/ml, respectively. conclusion. thus, an increase in the peripheral blood level of pro-inflammatory cytokines was observed in infants who underwent heart surgery. at the same time, the ntl relative number of neutrophils was increased. an early detection of the mentioned factors appears to be a diagnostic marker of the pulmonary inflammation reaction onset. all colistin resistant gram-negative isolates from patients hospitalized in a 7-bed icu during one-year period were retrospectively recorded. demographic data, the underlying disease, prior antimicrobial therapy, microbiological data and the clinical and bacteriological response to treatment were recorded. the antimicrobial susceptibility of the isolates was determined using the disk-diffusion (kirby-bauer) method, the vitek ii system and the etest method (ab biodisk, solna-sweden). interpretation of the susceptibility results was in accordance to the clinical and laboratory standards institute (clsi). nine patients with infections caused by colistin resistant gram-negative isolates were recorded. all patients had prolonged icu stay, were under mechanical ventilation and had a significant exposure to antibiotics including colistin for mdr gram-negative bacteria. three k.pneumonia isolates producing metallo-beta-lactamases (mbl), two k. pneumonia isolates producing extended spectrum b-lactamases (esbl) and mbl, two acinetobacter baumannii isolates susceptible to tetracyclines, one pandrug resistant (pdr) acinetobacter baumannii and one pdr pseudomonas aeruginosa were recorded. the bacteria were isolated from bronchial secretions in four cases and from the blood stream in five patients. in five patients antibiotic treatment was based on susceptibility tests, with clinical and bacteriological success. antibiotic combinations including colistin plus meropenem or colistin plus cefepime were provided in patients harbouring pdr isolates. these patients failed to respond to treatment and had a fatal outcome. the overall clinical success and survival rate was 55.5% at 14 days. conclusion. the development of colistin resistant strains with increasing mortality rates urges for the continuous surveillance on these highly resistant organisms and the strict implementation of infection control practices. ventilator-associated pneumonia (vap) is one of the most severe infections in the icu, continuing to complicate a high percentage of the patients receiving mechanical ventilation and leading to increased morbidity and mortality, especially when it is due to highrisk pathogens. our aim was to study the incidence and outcome of vap due to mdr bacteria in our icu. prospective, epidemiological study, in a mixed icu of a tertiary care hospital. all patients admitted from august 2004 to march 2007 were included. lower respiratory tract samples of all patients with suspicion of vap were cultured. standard diagnostic criteria were followed. statistical analysis was performed with spss v.12. during the 32 months period of the study 330 patients were admitted. their mean age was 66 years and 61% of them were male. their mean apache score was 18 and the average duration of stay in the icu was 18 days. forty-two episodes of vap due to mdr bacteria were recorded in 39 patients. the bacteria isolated from lower respiratory tract samples were 21 acinetobacter baumanii, 16 pseudomonas aeruginosa, 4 klebsiella pneumoniae and 1 enterobacter cloacae, while in 13 cases concomitant bacteremia was recorded. the mean time from admission to the icu to diagnosis of vap was 30 days. positive outcome was noted in 62% of cases and was found to be reversely related to the apache ii score (p=0.02), to days of stay in the icu (p=0.011) and to multi-organ failure (p=0.002). of the 39 patients with vap, 25 had normal renal function before the lung infection. of these, 15 developed renal failure due to the lung infection and had to be started on renal replacement therapy. the mortality of these patients was significantly higher than for the patients who did not develop renal failure (p=0.02). regarding the crude mortality of patients with and without vap, this was found to be 53.8% and 32.5% respectively (p=0.012). (pa) is not a frequent pathogen in this setting but could be associated with poor prognosis. in our population of patients undergoing cs, we compared risk factors and prognosis of pa-eop with eop due to others micro-organisms. this retrospective study performed on 2 years (2005-6) involved 1504 patients (pts) who underwent cs with cardiopulmonary by-pass. diagnostic of pneumonia was based on clinical and laboratory criteria: t˚>38.4, purulent tracheal secretions, wbc>11,000/mm3, chest x-ray changes and microbiological criteria (broncho-alveolar lavage>104 cfu/ml). pre, per and postoperative risk factors, empiric antibiotic, and prognosis of pa-eop were compared with those obtained for eop due to others germs. the 2 groups were compared using chi-square. p<0.05 was considered significant. over the studied period, eop occurred in 62 pts (incidence 4 %), including 14 pts ( conclusion. in our experience, pa-eop following cs seems to be more frequent than what was previously reported. criteria for prediction of pa-eop remain to be assessed. in case of pa eop, empiric antibiotic is often inappropriate with a possible increased risk of mortality. these results lead us to modify our empiric broad-spectrum antibiotic treatment and to take into account pa, especially in severe forms of eop and in copd pts. antibiotic exposure and timing of pneumonia onset influence ventilatorassociated pneumonia (vap) isolates. the first goal of this investigation was to evaluate whether trauma also influences prevalence of microorganisms. a retrospective, single-center, observational cohort study. . vap isolates in a multidisciplinary icu documented by quantitative respiratory cultures and recorded in a 42-month database were compared, based on the presence (t) or absence of trauma (at). causative microorganisms were classified in four groups, based on mechanical ventilation duration (>5 days), and previous antibiotic exposure. one hundred eighty-three patients developed 196 episodes of vap (98 trauma). methicillin-sensitive staphylococcus aureus (mssa) was more frequent (34.5% vs 11.5%, p<0.01) in trauma, whereas mrsa was more frequent (2% vs 11.5%, p<0.01) in nontrauma. no significant differences were found between trauma and nontrauma patients regarding prevalence of other microorganisms. in trauma patients, mssa episodes were equally distributed between early and late-onset vap(51% vs 49%) but no mrsa episode ocurred in the early-onset group. conclusion. trauma influences the microbiology of pneumonia and it should be considered in the initial antibiotic regimen choice. our data demonstrate that patients with trauma had a higher prevalence of mssa, but the overall prevalence was sufficiently high to warrant an s. aureus coverage for both groups. on the other hand, since no mrsa was isolated during the first 10 days of mechanical ventilation on trauma patients, mrsa coverage in these patients is only necessary after ten days of admission. a retrospective study of a hiv patient's cohort that stays in icu with acquired community pneumonia in the period between january 2002 and december 2006. data analyzed included age, clinic stage, years of disease evolution, antiretroviral therapy, cd4 levels and viral charge at the hospitalization, positive hcv and/or hbv, severity scores and microorganism isolated. chi-square analysis was used to compare categorical data. continuous data was compared using student's t-test. prognostic factors of mortality were studied by multivariate logistic regression analysis. . fifty-three patients were studied. 66% were males. the average age was 40±9 years. the most frequently risk practice was intravenous drug addiction (68% we prospectively collected data regarding demographics and microbiology of bacteremias. blood cultures were obtained on clinical suspicion of bacteremia and followed up on days 3, 7, and 14th. severity of illness scores, apache and sofa were recorded at baseline and days 3, 7, and 14th. improving hand hygiene is a cost-effective way of decreasing hospital-acquired infection rates. in this study we recorded opportunities for and compliance to hand hygiene in our icu. four trained nurses and a doctor monitored opportunities for hand hygiene performance (hand antisepsis and glove use) as well as compliance to the cdc guidelines in our icu for 10 days. the procedure was anonymous, involved all icu personnel and was performed in 15-min sessions, throughout all shifts. we collected 829 opportunities for hand hygiene, mostly related to nurses (62%). compliance to hand antisepsis was 45%, higher in nursing and assistant staff (48% and 53%, respectively) compared to doctors (34%). compliance was lowest before contact of healthcare staff with a patient or his inanimate environment (23% and 16%, respectively). the activity index (=the need for hand antisepsis performance) for the nursing staff was high (12 opportunities per hour per nurse in the morning shift, ie 96 opportunities per shift). however, no significant correlation was found between compliance rate and activity index of the staff (r=-0.17, p=0.21). alcohol-based hand-rub was used in 62% of the cases. technique of antisepsis performance was uniformly poor and mean duration of the procedure was low (6.3 seconds). compliance with glove use guidelines was 91% and was high in all staff categories and all types of opportunities. is an aerobic non-fermenting gram negative bacillus. it is generally considered an opportunistic pathogen. s. maltophilia is increasingly recognised as a cause of nosocomial infection among ventilated and immunocompromised patients, and in those receiving broad spectrum antibiotics. s. maltophilia infections are commonly resistant to multiple antibiotics including beta lactams, quinolones, aminoglycosides and carbapenems. reported mortality rates for patients with bacteraemia due to s. maltophilia vary from 10-60%. the mid western regional hospital, limerick, ireland, is a 500 bed hospital located on three sites. the intensive care unit(icu) is a seven bed medical and surgical unit with approximately 450 admissions per year. the s. maltophilia clusters prompted epidemiological investigation, restriction fragment-length polymorphism typing (rflp) of genomic dna of outbreak strains, and finally, instituting revised infection control measures to limit spread. we conducted a retrospective chart review of affected patients noting admission apache ii scores, medical co-morbidity, immunocompetence, antibiotic history, and patient outcome. we collected cultures of icu cubicle/ room surfaces, sinks, ventilatory equipment, and water sources. patients and environmental isolates were examined by rflp typing. this preliminary analysis suggests that pct can be use to accurately early identify sepsis only at levels above 2 ng/ml and then use them to decide to rapidly beginning the use of antibiotic. in patients with pct below 2 ng/ml we cannot use them to exclude the diagnosis of sepsis. with the cutoff 0,5 ng/ml we found the same analysis. other studies with more samples are necessary to confirm this conclusion. during these three years 402 patients were hospitalized in total. one hundred and thirty one (29.8 %) were hospitalized less than 72 h and were excluded. a total of 99 bacteremias were observed. forty -four bacteremias were catheter related bloodstream infections. fifty five were due to gram negative microorganisms (pseudomonas aeruginosa 23%, acinetobacter baumanni16%, klebsiella pneumonia16%). in the following table, resistance to broad spectrum antimicrobials is presented during these three years. infection in patients with severe stroke is an important problem and the sensitivity and specificity of its diagnosis with clinical criteria are deficient. fever is a common event and, as leucocytes or c-reactive protein, its specificity is very low in this kind of patient. our objective was to evaluate the utility of a biological marker such as procalcitonin (pct) in the diagnosis of infection in patients with severe stroke. we followed 27 patients with severe stroke receiving mechanical ventilation because of coma. during the first 4 days of evolution nih and apache ii scales were registered, we measured pct and c-reactive protein on days 1 and 3 and if infection was suspected microbiological samples were collected. infection was diagnosed if the patient fulfilled the cdc criteria. mann-whithney u and x-square tests were used. twenty-six cases corresponded to haemorrhagic stroke. baseline characteristics were: mean age 57 years, 55% males, glasgow scale 6 (3-12), nih scale 30 (13-37), apache ii 20 (9-32), temperature 36.9 o c (33-39.5), leucocytes 10341/mm3 (2400-24800), pct 0.404 ng/ml (0.022-1.311) and c-reactive protein 37.3 mg/dl (3.7-121). on the third day of evolution 3 cases of ventilator-associated pneumonia were diagnosed. when compared with the noninfection group there were no differences in baseline characteristics and on the infection day we only found differences in pct, 3.505 ng/ml in front of 0.552 ng/ml; p < 0.001. seventeen (70%) of the patients without infection presented a temperature 3 38 o c sometime during the follow-up and in all cases pct did not show any change. these results indicate that pct is a useful tool in the diagnosis of infection in patients with severe stroke. the ongoing challenge of accurately diagnosing infection in the icu motivates a search for novel molecular diagnostics. we reported recently that microarray analysis of circulating leukocytes can be used to derive a "riboleukogram", which captures the dynamics of the host response to and recovery from ventilator-associated pneumonia (vap). in the current study, we tested the hypothesis that the informational content of circulating leukocytes differs, thereby allowing one to rank leukocyte populations on their potential to contribute to rna diagnostics for pneumonia. sixteen patients (10 male, 6 female) at risk for vap were entered into our irbapproved study that collects blood and clinical data every 48 hours for up to 21 days. four of the sixteen patients developed vap as diagnosed and treated by the attending icu physician. previously reported blood protocols were used to isolate buffy coat, enriched neutrophil, and enriched monocyte populations by using negative selection. cellular purity was assessed by facs for one of the 4 vap patients. genome-wide expression analysis was performed on rmanormalized signal from affymetrix u133 2.0 plus genechips. edge software (fdr=0.10) was used to determine changes in mrna abundance over time for each cell population. during the 5-day window in which each of the four patients (all males) developed vap, significant changes in gene expression were observed (table) , but the information content (number of genes altered) varied across leukocyte populations. these differences were not due to signal variance (coefficient of variation, cv) or differences in the number of samples available for analysis. moreover, only 0.6% of the monocyte gene list overlaps with the neutrophil list, arguing that neutrophil contamination of monocyte populations is insufficient to explain the 40-fold difference in gene number. the aim of the present study was to evaluate the relationship between the cytokine expression in bronchoalveolar lavage fluid and bacterial burden in mechanically ventilated patients with suspected pneumonia. mechanically ventilated patients with suspected pneumonia admitted in icu from november 2004 to january 2006 were prospectively enrolled. fiberoptic bronchoalveolar lavage (bal) was performed with 150 ml of sterile isotonic saline in 3 aliquots of 50 ml, local anesthetic were not used. bal samples for microbiologic quantitative cultures and bal cytokines: interleukin (il) 6, il 8, tumor necrosis factor-alpha (tnf-alpha), granulocyte colony stimulating factor (g-csf) and granulocyte-monocyte colony stimulating factor (gm-csf) were measured. . 59 patients were included, most of the patients (79.7%) were with prior antibiotic therapy. 22 patients (37.2%) had a positive bacterial culture defined than a diagnostic threshold of > 104 colony-forming unit/ ml. the concentration of tnf-alpha was significantly higher in the group of patients with positive bal (table 1) . it has been demonstrated in a swine model that therapeutic hypothermia (30˚c) facilitated transthoracic defibrillation. however, the mechanisms leading to reduced defibrillation threshold (dft) remain unclear. we hypothesized that therapeutic hypothermia promotes the wavefront organization of ventricular fibrillation (vf), therefore facilitating defibrillation. methods. by using a two-camera optical mapping system, epicardial activation patterns of vf were studied in 7 isolated rabbit hearts at baseline (37˚c), 10-min therapeutic hypothermia (30˚c), and 10-min rewarming (37˚c). in 12 additional hearts, dft50 (voltage required to achieve 50% probability of successful defibrillation, n=6 hearts) and apd (action potential duration)/conduction velocity (cv) restitutions (n=6 hearts) were determined at these 3 stages. results. comparing with at baseline (35±5%) and rewarming (34±7%), there was a higher percentage of vf duration containing organized repetitive activities during hypothermia (73±10%, p<0.001). however, there was no significant difference of dft50 among these 3 stages (151±34, 141±38, and 146±46 v, p=0.556). the electrophysiologic characteristics of ventricles at these 3 stages were summarized in table 1. in brief, hypothermia prolonged apd, decreased cv, and subsequently shortened wavelength. hypothermia also failed to flatten the slope of apd restitution. furthermore, apd dispersion at the epicardial surfaces of both ventricles and cv heterogeneity among 4 epicardial lines were all enhanced by hypothermia. (pt) with acute coroanry syndrome (acs) at admission is a associated with a high mortality. the mechansims are poorly understood. we sought to determine an interrelation between no coronary reflow after percutaneous coronary intervention (pci), the likelihood of developing cardiogenic shock, death in hospital and plasma glucose level at admission. we performed a prospective analysis of 161 consecutive pt presenting with an acs in our emergency room. we recorded basis data (gender, age, bmi), cardiovascular risk factors, burden of coronary artery disease (cad), coronary blood flow after pci, killip-classification, left vetricular ejection fraction, probabilty of developing cardiogenic shock and the likelihood of dying in-hospital. our findings suggest that elevated bs at admission is a useful risk marker to identify pt with a high risk to develop coronary no reflow-phenomenon after pci. this may be due to increased inflammatory activity and hypercoagulability. if one dies in cardiogenic shock, these pt present always with elevated bs at admission. prull mw, trappe hj. activation of blood coagulation in nstemi: does diabetes mellitus matter? intensivmed 2007. we measured serum cortisol levels before and 60 minutes after a 0,25mg corticotropin stimulation test in 15 pts with cs following acute myocardial infarction (mi) and in a control group of 8 pts with uncomplicated mi at day 0, 1, 2, 3, 5, and 7 after onset of shock/mi. rai was defined by an increase in serum cortisol levels in response to corticotropin of less than 9µg/dl. data were correlated to vasopressor-need and interleukin (il) levels (il1,il6,il8,il10). baseline cortisol levels in pts with cs were significantly higher than in control pts especially on day 0 (35±22 vs 15±9, p=0.006). in 5 cs-pts the test-series were stopped at day 1 to 3 because the physician in charge started a therapy-trial with hydrocortisone due to increasing vasopressor need. three other pts died within the seven day period. rai was observed only at day 0 in 5 of the 15 cs-pts but in none of the control pts (p=0.06). these cs pts with rai had higher il-6 and il-10 levels at baseline ( during tidal mechanical ventilation, an end-expiratory pause abolishes the cyclic increase in intra-thoracic pressure. this may produce a transient increase in cardiac preload and then in cardiac output in volume responsive patients. our objective was to test whether the effects of an end-expiratory pause on cardiac index and pulse pressure may help in detecting fluid responsiveness in patients with acute circulatory failure. in 30 mechanically ventilated patients with an acute circulatory failure and no spontaneous ventilator triggering who were deemed at volume expansion, we performed a 15-sec end-expiratory pause. we continuously measured the systemic arterial pressure and the pulse contour-derived cardiac index (picco device) at baseline, during the 10 last seconds of the end-expiratory pause and after a 500ml saline administration. volume expansion induced an increase in cardiac index ≥15% in 20 patients (classified as responders). in these patients, volume expansion increased the cardiac index by 41±35% from 2.1±1.1 l/min/m2. before volume expansion, the end-expiratory pause had induced an increase in cardiac index by 11±11% and in pulse pressure by 14±16% as compared to the baseline values. by contrast in the 10 non-responders, before volume expansion the cardiac index and the pulse pressure did not change during the pause as compared to baseline (2±2 % and 1±3% increases, respectively). importantly, an increase in cardiac index ≥5% during the end-expiratory pause predicted fluid responsiveness with a sensitivity of 84% and a specificity of 90%. a pause-induced increase in pulse pressure ≥4% detected fluid responsiveness with similar sensitivity and specificity (90% and 90%). in responders, a second end-expiratory pause was performed again immediately after volume expansion. in 16 patients, the increases in cardiac index induced by this second pause induced had dropped below 5%. in the 4 remaining responders, the second pause induced an increase in cardiac index still higher than 5% (12±2%). in these patients, the pause-induced increase in cardiac index was abolished by a second 500ml saline administration. conclusion. an increase in cardiac index and in pulse pressure during an end-expiratory pause enables to detect fluid responsiveness in critically ill patients with mechanical ventilation and acute circulatory failure. , and tissue doppler imaging measurements of the mitral annulus velocities like early (ea) peak diastolic velocity. the aim of the study was to examine which echocardiographic index is the best marker of preload by making the hypothesis that a good measure of preload should increase with fluid-induced increase in stroke volume (sv) but not with dobutamine-induced increase in sv. comparison of the capacity of the intra thoracic blood volume index (itbvi) and the central venous pressure (cvp) to predict fluid responsiveness in critically ill patients with acute circulatory failure (systolic blood pressure < 90 mmhg or vasopressor requirement). methods. this prospective interventional study performed in a surgical intensive care unit of a tertiary university hospital included 35 (21 males) mechanically ventilated and sedated patients with acute cardiovascular failure requiring cardiac output measurement (transpulmonary thermodilution technique)and a fluid challenge. intervention: fluid responsiveness was defined as an increase in stroke index (si = cardiac output/heart rate/body surface area) ≥ 15%. receiver operating characteristic (roc) curves were generated for itbvi and cvp. in 48 eligible patients, 10 could not be included because of cardiac arrhythmia (n = 8) or moribund status (n = 2) or protocol violation (n = 3). the cause of acute circulatory failure was septic shock in 24 (69%) patients, haemorrhagic shock in 2 (6%) patients, and systemic inflammatory response syndrome in 9 (26%) patients. fluid challenge induced an si increase ≥ 15% in 18 (51%) patients (responders(r). no statistical difference was shown between responders and non responders for cvp and itbvi. the areas under the roc curves of itbvi and cvp were 0.64 [95% ci: 0.46-0.80], and 0.68 [95% ci: 0.50-0.83], respectively, without any statistical difference (p = 0.73). the best cut of value for cvp and itbvi were 9 mmhg (sensitivity = 61 %; specificity = 82%) and 928 ml.m-2 (sensitivity = 78 %; specificity = 53 %), respectively. the relative changes in si and ci were correlated with relative changes in itbvi (r = 0.59, p = 0.001; r = 0.66, p = 0.0001 respectively) but no correlation was found between relative changes in si and ci and relative changes in cvp (r = -0.07, p = 0.70; r = 0.10; p = 0.54). conclusion. itbvi is similar to cvp to predict fluid responsiveness in critically ill patients with acute circulatory failure. the pulse pressure variation (ppv) is used to predict fluid responsiveness in mechanically ventilated patients. nevertheless false positive of this parameter have been reported especially in patient with right ventricular dysfunction. the peak systolic velocity of tricuspid annular motion (sta) assessed by doppler echocardiography (dec) is a parameter of right ventricular systolic function. the aim of the study was to find out whether sta can discriminate between false and true positive of vpp. methods. 35 mechanically ventilated patients were prospectively included. all patients had a measurement of ppv>12%. a dec was realised before and after infusion of 500 ml of colloid solution. patients were separated into 2 groups as they were responders (r) (at least 15% increase in stroke volume (sv)) or non-responders (nr) to fluid infusion. all data are expressed as mean [standard deviation]. the comparison of demographic, hemodynamic and echocardiographic parameters in r and nr patients was performed using a t-test. a p value < 0.05 was considered statistically significant. roc curves were plotted. a threshold value of sta was calculated with roc curve. in the resting patient, pulse pressure (pp = systolic -diastolic pressure) is mainly related to arterial stiffness and stroke volume index (svi). the dynamic effects of fluid loading on pp are poorly documented and were studied in the critically ill using arterial tonometry. we tested the hypotheses that i) arterial stiffness was unchanged after fluid loading, ii) pp changes paralleled svi changes such that pp increased in fluid-responders only, and iii) aortic pp was more indicative of svi changes than radial pp. twenty-two critically ill patients (9f), mean age(sd), 53(15) years, were prospectively included. radial pressures were calibrated from brachial cuff pressures. radial applanation tonometry (sphygmocor ® ) allowed us to estimate aortic pp, left ventricular ejection time, and the augmentation index which quantifies wave reflection. the svi was calculated by transpulmonary thermodilution. the arterial stiffness was estimated from the aortic pressure curve using standard formula. fluid challenge (500 ml saline 0.9%) was required by the patient's hemodynamic status. data were obtained before and immediately after fluid loading. responders had increases in svi > 15 %. baseline mean values were as follows: svi = 38(13) ml.m-2, heart rate= 89(15) bpm, mean arterial pressure (map) = 75(15) mmhg, radial pp = 51(18) mmhg, aortic pp = 32(11) mmhg. after fluid loading, svi increased from 38(13) to 42(15) ml.m-2 and map increased from to 80(17) mmhg (each p < 0.01). arterial stiffness was unchanged (1.28(0.71) vs 1.33(0.89) mmhg.ml-1. m2 ) as well as heart rate, left ventricular ejection time, radial and aortic pps and augmentation index. there was a positive linear relationship between the svi changes and the changes in radial pp (r2 = 0.50) and aortic pp (r2=0.61) (each p < 0.01), not map (r2 = 0.006). when responders (n=8) and non responders (n=14) were compared, the increases in map were similar while the changes in pp were higher in responders (radial: 13 mmhg, 24%; aortic: 10 mmhg; 35% ) than in non responders. (radial: -1mmhg, -0.5%, aortic: -1mmhg; -0.6%) (each p< 0.01). given the unchanged arterial stiffness throughout the fluid infusion, the changes in aortic pp (and slightly to a lesser extent radial pp) paralleled the changes in svi. both radial and aortic pps increased in responders but not in non responders, while map similarly increased in the two groups. the capability of arterial pp changes to track svi changes during fluid loading appears promising but deserves a further large scale study. new device may be used in intensive care unit to measure cardiac output (co) by arterial pulse pressure waveform analysis , but comparative studies with co thermodilution in cardiac surgery have shown large bias between the methods 1. aim of this study is to evaluate in critical ill patients not submitted to cardiac operation 1-cardiac output (co wave) obtained using flo track tm vigileo .2 -the correlation with co obtained by thermodilution (co therm). methods. 35 critical care patients admitted to a general intensive care were enrolled in the study . all patients were mechanically ventilated ( tv 6-8 ml /kg pl press < 30 cmh20) and connected to an integrated monitoring system ( flow trac tm / vigileo tm , ewdards lifescience ,irvine ,ca, usa ) that attaches to an arterial cannula . a central venous catheter and a pac ( thermodilution catheter ; arrow international , inc ., reading ,pa,usa ) was inserted via the jugular internal vein . after haemodynamic stabilization co wave was calculated from an arterial pressure based algorithm that utilises the relationship between pulse pressure and stroke volume , primarily based on the standard deviation of the pulse pressure waveform. at the same time a co therm. determination was performed by triple injection of 10 ml of iced isotone na cl into the central line of the pac. every patients had two co determination at two time point. for each measurement of co therm corresponding simulataneous co wave was documenteted . a regression analysis and bland altman analysis was used to compare the two methods of co determination. a total of 70 co determination was performed in 35 patients . co vigileo correlated co thermodilution with r = 0.68 , p< 0,001. at table 1 are reported the bland altman's results. the left ventricular ejection fraction (lvef) as measured by echocardiography is considered as the reference estimate of the lv global contractility at the icu bedside. the transpulmonary thermodilution technique (picco system) continuously provides a measure of the cardiac function index (cfi), which is the ratio of cardiac output over global end-diastolic volume. thus it could be considered as a marker of cardiac global contractility and could enable a continuous monitoring of this key parameter. we tested whether cfi could actually behave as an indicator of lv systolic function by testing if it fulfilled the following criteria: (i) increase with inotropic stimulation, (ii) no alteration by fluid loading, (iii) correlation with the echographic lvef and (iv) ability to track the changes in lvef during inotropic stimulation. in 33 patients (40 cases) with an acute circulatory failure, we simultaneously measured the echographic lvef (transthoracic 4-chambers apical view) and the cfi at baseline, after a 500ml saline administration in a group of 22 cases and after 15-min of dobutamine administration in a group of 18 cases. volume expansion did not alter lvef significantly (47±11% vs. 46±10% at baseline) nor cfi (4.4±2.2 vs. 4.5±2.2 min-1 at baseline). by contrast, dobutamine infusion induced a significant increase in lvef from 30±10% at baseline to 39±10%(+33±29%) and in cfi from 3.2±1.7 at baseline to 4.0±1.9 min -1 (+27±22%). considering the whole set of cfi:lvef pairs of measurements (n=80), a significant correlation was observed between cfi and lvef (r=0.65, p<0.0001). importantly, a cfi value <3.2min -1 predicted a lvef value higher than35% with a sensitivity of 76% and a specificity of 87%. in patients receiving dobutamine, there was a significant correlation between the changes in cfi and the changes in lvef induced by dobutamine infusion (r=0.69, p<0.0001). our study demonstrates that cfi fulfilled the criteria that are required from a bedside indicator of lv contractile function: it was increased by inotropic stimulation while it was not altered by volume expansion, it was fairly correlated with the echographic lvef and it was able to track the changes in echographic lvef with reliability. this suggests that the continuous monitoring of cfi provided by transpulmonary thermodilution could help in assessing the effects of inotropic therapy and could alert the physician in case of abrupt lv contractile deterioration. passive leg raising (plr) is a predictive test of preload responsiveness in patients with acute circulatory failure. it could predict fluid response to fluid loading in mechanically ventilated patients. critically ill patients have an increased risk of lower extremity deep venous thrombosis. elastic compression stocking (ecs) is frequently used in association with unfraction or low molecular weight heparin. the aim of this study was to evaluate the effect of the elastic compression stocking on the plr test variations. methods. 20 patients undergoing cardiac surgery were included. all of them were anaesthetised and mechanically ventilated (tidal volume ≥ 8ml/kg). pre-operative left ventricular ejection fraction was > 50% for all patients. they were monitored with central venous pressure (cvp), invasive blood pressure and esophageal doppler. hemodynamics parameters were obtained before and after plr, without and with elastic compression stocking respectively (ssv = systolic stroke volume, co = cardiac output, ppv = pulse pressure variation and sbp = systolic blood pressure). results are presented as median [inter quartile range](iqr) and compared with mann whitney test. . table represents hemodynamics variations after plr without and then with elastic compression stocking. second table represents hemodynamics effects of the elastic compression stocking in supine position (sp). conclusion. this study shows a clear improvement in gut permeability after surgery. the effects of early feeding shall be assessed in a future study. methods. descriptive-prospective study. pre and post-class 12 question -survey (administered one week before and after). the transplant co-ordination team gave informative classes in secondary schools, 2005-06 / 2006-07. . surveys collected; 713 pre/ 581 post-class: 58% of 4eso (16 years old), 38% 2bachiller (18 years old) and 4% ciclo formativo (18 years old) / 581 post-class: 36% 4eso, 59% 2bachiller and cf 4% . 98% had some prior awareness and 67% broad knowledge. massmedia is usually sole information channel (53%), ticked in all cases. other sources were: family, school and peers. regarding attitude to donation: we found no differences in refusals between own donation or relatives'(25%); or in doubts 46% -42%. related to transparency and parity of the health system: 26% believed equality did not exist and 60% had doubts. 55% felt this inequality was worse abroad. 64% are convinced that organ trafficking exists and 26% assume it is possible. pre-course standpoint by course is showed in figure 1 . 9% had prior knowledge about spanish transplant law. following classes the students claim higher awareness (87%). in general they maintain their standpoint on donation,45% have reconsidered their previous attitude. regarding transparency and equality, 26% maintain doubts and 15 % are convinced of its absence. on trafficking: 72% assume it is possible, 13% occurs exclusively abroad, uniform group distribution. post-course attitudes by course are in figure 1 . despite an in-depth discussion about the law and its consequences (presumed consent), they generally disagree and some consider this too extreme , refusing to accept that donation is an obligation (only 8% agree) and believing that it should be an optional act of solidarity (81%). conclusion. knowledge about donation and transplant in urban areas is slanted, due to information sources ( usually mass media ) and a warped (tv-dominated) perception of the health system's transparency and equality. a considerable number of students still refuse donation or maintain their scepticism, despite a decrease following classes. however, our desire is not to convince them to become donors, we simply wish to provide decision-making tools. generally college students ,without gender differences, are the most resistant to the process, having the greatest incidence of refusals and doubts about transparency, equality and organ trafficking. 140 (115-186) 0.18c (pao2/fio2) / peep day 1 12.5 (7.9-25.19) 10.7 (6.2-20.7) 0.33c (pao2/fio2) / peep day 3 14.8 (9.6-20.3) 11.2 (8.4-14.7) 0.01c (pao2/fio2) / peep day 7 13.3 (10.8-27.5) 10.6 (7.8-17.9) 0.02c conclusion. the pao2/fio2 ratio on day one is useful to predict mortality, but not in the subsequent days. the (pao2/fio2)/peep index is a better predictor in later days, specially on the third and seventh day of mv. a. roch* 1 , l. fouché 1 , j. forel 1 , d. blayac 2 , c. aglioni 3 , d. lambert 2 , j. carpentier 3 , l. papazian 1 1 réanimation médicale, 2 dar, hôpitaux sud, 3 réanimation, hôpital laveran, marseille, france introduction. general anesthesia promotes atelectasis of the dependent parts of the lung. we evaluated the differential effects of neuromuscular blocking agents (nmba) on consolidation formation in healthy or injured lungs. methods. 30 pigs (37±2 kg) were anaesthetized with pentobarbital, fentanyl and ketamine in order to prevent spontaneous ventilation and ventilated using volume controlled ventilation (vt 10 ml/kg, fio2 0.4) for 6 hours after randomization into 5 groups: healthy lungs ventilated without (hzeepno) or with nmba (cisatracurium, hzeepnmba), healthy lungs ventilated with nmba and peep 5 (hpeepnmba) and injured lungs ventilated without (tweenpeepno) or with nmba (tweenpeepnmba). lung injury was induced using instillation of 1.5 ml/kg of 7.5% tween 20. injured lungs were ventilated with peep 8 , fio2 0.8 and vt 10 ml/kg. after lung removal, six sections of equal thickness were obtained from the right lower lobe and 4 from the upper. sections were photographed and analyzed using a software (sigmascan pro 5, spss inc). the areas of consolidated, edematous and normal parenchyma were measured on each section and then added to obtain the percentage of consolidated lung. . nmba use induced a two-fold increase of the consolidation (from 20±6 to 39±8 %)that was totally prevented by peep 5. the deleterious effect of nmba on derecruitment did not occur in injured lungs. consolidation was located to the dependent parts in healthy lungs and nmba extended consolidation towards more cephalad parts. in injured lungs, consolidated parenchyma was diffuse and its cephalo-caudal distribution was not affected by nmba. pao2 to fio2 ratio was affected neither by nmba nor by peep. * p<0.001 vs hzeepno and hpeepnmba; **p<0.001 vs hzeepno and hzeepnmba. conclusion. nmba increase dependent lung consolidation during volume-controlled ventilation of healthy lungs. this effect is prevented by a moderate peep level. in contrast, nmba do not increase the extent of pathologic lung areas in injured lungs ventilated during a 6-h period. 20th esicm annual congress -berlin, germany -7-10 october 2007 s81 0306 m. amigoni* 1 , m. scanziani 1 , g. bellani 1 , g. balconi 2 , e. zanotto 1 , s. masson 2 , n. patroniti 1 , r. latini 2 , a. pesenti 1 1 dept of experimental medicine, milano-bicocca university, monza, 2 cardiovascular research, istituto di ricerche farmacologiche mario negri, milano, italy introduction. surfactant dysfunction seems to play a pivotal role in the deterioration of gas exchange and lung mechanics that occurs in ali/ards following aspiration pneumonitis. we investigated the effects of exogenous surfactant administration in a murine model of unilateral acid-induced lung injury. we instilled 1.5 ml/kg bw of 0.1m hydrochloric acid in the right bronchus of anesthetized and mechanically ventilated mice (vt 8-10 ml kg-1 bw, rr 130 min-1, fio2 1 and peep of 2.5 cmh2o). mechanical ventilation was stopped 10 minutes after injury; animals were then placed in an oxygenated chamber (fio2 0.5). after 10', 1hr or 6hrs from acid instillation, the mice were reintubated and received a single bolus of surfactant in the injured lung at a low or high dose. each animal was again mechanically ventilated for 10 minutes, placed in oxygenated chamber until full awakening. acid-injured mice instilled at the same time and with the same volume (1ml/kg bw) of sterile saline (0.9% nacl) were used as controls. lung mechanics, blood gas analysis, and lung myeloperoxidase activity (mpo) were assessed 24hrs after acid aspiration. no effect of surfactant administration was present upon oxygenation 24hrs after the injury. at the opposite the high dose group showed a significantly better compliance at 24hrs, when compared to both the low dose and control groups. this effect was present only in the late (6hrs) administration group. mpo activity did not change after surfactant treatment in the right (injured) lung while in the controlateral, it tended to be lower in both low and high dose when treatment administration occurred at 6hrs (n=4/group: n right lung 38±15 left lung 40.2±19.7; s(low dose) right lung 39.2±17.8 left lung 28.7±15.8; s(high dose) right lung 40.1±16.5 left lung 30.3±13.6). pulmonary aspiration is associated with significant morbidity and mortality 1 . several risk factors for aspiration have been highlighted in the literature 2 . the aims of this study were to: (i) identify specifically which patient factors predispose to aspiration and (ii) determine the outcome of patients admitted to our inner city hospital intensive care unit (icu) with a diagnosis of aspiration. we identified 27 patients with a diagnosis of pulmonary aspiration on our icu over a 2 year period (august 2004-06), by using our institution's icnarc (intensive care national audit and research centre) database. 19 of these patients' case notes were able to be retrieved and reviewed in detail. patient demographics, risk factors for aspiration, number of ventilated days, icu & hospital length of stay and mortality were analysed. we also looked at any documented signs that supported the diagnosis of aspiration. median age of the patients was 58 years (range 30-83). 13/19 patients (68%) were male. the main risk factor was a reduced glasgow coma score (19/19 patients, 100%): the median score was 5 (range 3-13). the following risk factors were also identified: obesity (5/19 patients, 26%), excessive alcohol intake (5/19, 26%), acute cerebrovascular event (4/19, 21%) and cardiorespiratory arrest (4/19, 21%). the following signs were most frequently observed: perioral vomitus (12/19 patients, 63%), acute hypoxaemia (16/19, 84%) and a new radiographic infiltrate (10/19, 53%). one patient exhibited all three markers. all 19 patients required mechanical ventilation. the median duration of ventilation was 11 days (range 1-33). the median length of icu stay was 12 days (1-41) and the median length of hospital stay was 31 days (1-256). icu mortality was 27% (5/19 patients) while hospital mortality was 37% (7/19). patients who presented to our inner city icu with aspiration had risk factors that included impaired conscious level, obesity, a recent cerebrovascular event or cardiorespiratory arrest. signs that supported the diagnosis of aspiration were the presence of perioral vomitus, acute hypoxaemia and a new radiographic infiltrate. icu and hospital length of stay were both prolonged, but icu and hospital mortality were no higher than our institution's overall rate. a high index of suspicion should be applied to these patients at risk of aspiration, to facilitate the early initiation of appropriate care. reference(s). 1. hickling k. a retrospective survey of treatment and mortality in aspiration pneumonia. int care med 1998; 14: 617-22. 2. kozlow j. epidemiology and impact of aspiration pneumonia in patients undergoing surgery in maryland, 1999 -2000 . crit care med 2003 31: 1930-7. t. tagami* 1 , s. kushimoto 2 , t. atsumi 2 , r. oyama 1 , k. matsuda 3 , m. kawai 2 , h. yokota 2 , y. yamamoto 2 1 surgery, tokyo metropolitan saiseikai central hospital, 2 critical care medicine, nippon medical school, tokyo, 3 critical care medicine, yamanashi prefectural central hospital, yamanashi, japan introduction. restoration of intravascular volume by massive fluid administration without pulmonary edema formation is one of the biggest challenges in the early treatment of burn shock. although it is not easy to predict the development of the respiratory failure before the treatment, the hallmark of the edema is increased capillary permeability which may be possible to measure by the pulmonary vascular permeability index (pvpi). the aim of the present study was to clarify whether the pvpi is predictable indicator of pulmonary edema formation in patients with burn. we studied 11 mechanically ventilated patients with burn involving more than 25% of the body surface area that were treated at intensive care burn unit between july 2004 and january 2007. all patients had a central venous catheter and a thermistor-tipped arterial thermodilution catheter (picco system) for hemodynamic management. we measured the extravascular lung water index (evlwi) and the pulmonary vascular permeability index(pvpi) as soon as the picco catheter was inserted. infusion volume was calculated according to the parkland formula. only crystalloid fluid (lactated ringer's) was infused during the first 12 hours after the thermal injury. we investigated the medical records and defined the respiratory failure during the period of burn shock as a clinical syndrome of acute respiratory distress associate with pulmonary rales and radiographic evidence. inclusion criteria were: 1)acute onset and rapid progress, 2)oxygenation index (pao2/fio2 ratio<200 and 3) bilateral infiltrates on chest x-ray. those are the part of the standard criteria of acute respiratory distressed syndrome. the pvpi was significantly higher in the patient with respiratory failure (n=4 pvpi: 2.65±0.98) than in patient without respiratory failure(n=7 pvpi:1.43±0.26) before the fluid treatment. there was no significant difference between the groups in terms of evlwi at the beginning (7ml/kg vs 6.1ml/kg). although the evlwi increased after 48 hours in the patient with respiratory failure, it did not change in patient without respiratory failure(18.2ml/kg vs 6.0ml/kg). the pvpi increased before the evlwi increased in patient with respiratory failure. the pvpi is considered to be the predictable value to identify the risk of respiratory failure during the period of burn shock. ultrasonography allows observation of diaphragm. in healthy subjects, a correlation was found between its excursion and the tidal volume. in addition, diaphragm thickness variation measured in the zone of apposition has been used to evaluate paralyzed diaphragm. we assessed the accuracy of these indexes to assess diaphragmatic function and respiratory workload. five patients were studied in spontaneous ventilation (sv) and during noninvasive ventilation at different levels of pressure support (ps). diaphragmatic excursion (e) was carried out subcostally. diaphragm thickness was measured in the zone of apposition and the thickening fraction (tf) was calculated as tf = (thickness at inspiration -thickness at expiration)/thickness at expiration. diaphragmatic pressure time product per breath (ptpdi) was measured by assessment of esophageal and gastric pressure. ptpdi and tf both decreased as the level of pressure support increased (fig 1 and 2) . a positive correlation was found between ptpdi and tf(r=0.68; p=0.01; fig 3) . in addition, there was also a significant correlation between tidal volume and e (r=0.91; p<0.01; fig 4) . ultrasonography of the diaphragm could be applied in intensive care to assess diaphragmatic function. tf and ptpdi decrease as the level of pressure support increases. these results suggest that tf could help to assess diaphragmatic contribution to respiratory workload. reference(s). (1) fantus g. metformin's contraindications: needed for now frequency of inappropriate metformin prescriptions systematic review and meta-analysis of studies of the timing of tracheostomy in adult patients undergoing arteficial ventilation catheter infection is a common concern in the intensive care unit (icu). recent works have pointed that the site of catheters is related to this problem. we analysed data obtained from our data base to confirm the results of previous works. methods. catheters were inserted in a surgical-medical icu, along five years. semiquantitative cultures were obtained if the catheter was kept in place more than 48 hours and it was no longer necessary, the catheter was withdrawn because of fever of unknown origin or an infection was suspected at the point of insertion. every catheter site, culture and germ was registered in our patient data base. we studied the following variables: type of catheter, site and results of cultures. statistical analysis: variables were compared by chi-square. a p< 0,05 was considered statiscally significant. results. a total of 2.407 catheters were registered (venous catheters 1307, arterial 1100). rate of germs was as follow: gram-positive 67,4%, gram-negative 28,1%, fungi 3%, contaminated flora 1,5%. site and germs were not statistically associated. table 1 shows type, site and rate of infection of cultured catheters. femoral arteries were more frequently cultured than radial arteries (p< 0,001); no differences were found for cultured venous catheters. femoral arteries were infected more frequently than radial (p< o,001); yugular and femoral venous catheters were more frequently associated to infection. (sc) in non neutropenic patients is increasing with a high cost and mortality. we define the clinical and epidemiological profile of patients admitted to our icu and the microbiological aspects of the pathogen. mortality analysis was done, including sevilla score system (sss). we include 36 patients admitted in icu from 2002 to 2007 with candidas ssp (cd) positive blood cultures (bacter system). we analysed demographic factors, reason for admission to the unit, associated risk factors, need of multi-instrumentation or parenteral nutrition, value of apache ii, and length of stay in the icu. the kind of cd diagnosed, its sensitivity profile, and the existence or not of previous wide spectrum antibiotic or antifungic therapy were determined. the sevilla score system was applied and correlated with mortality. chi square, t-test and multivariant analysis were made. there were 69.4% male patients, with 58 years old median age and with a length of stay longer than 14 days. the reason for admission was sepsis (27%), surgery (22.2%), acute respiratory failure (16.7%) and trauma patiens (11%). apache ii median was 22.8 points.risk factors related with fungal infections were diabetes (19.4%), neoplasia (11%), steroid therapy (7,7%), a length of stay longer than 7 days (44%) and antibioticoterapy. none had neutropenia. 94% of patiens received antibioticoterapy previous to diagnosis, 69.4% parenteral nutrition and 88% of them underwent multi-instrumentation. patient isolation was achieved in 36% of them (90% in period 2005-7). candida albicans was isolated in 44.4% of cases against 55.6% of candida nonalbicans, specially c. parapsilosis 30,6%. first antifungal therapy was fluconazole (61%), caspofungin (16.7%) and lipid amphotericins (5.6%). we found a significant increase of sc cases along the years, (36% in 2002-4 vs 63.9% in 2005-7, p<0 .049), being unresponsive to azoles 3.8%. mortality was specially high (41.7%), unrelated with cd type; those with high/moderate sss risk had a significative higher mortality (p<0.035). candida albicans was more frequently found in septic patients while candida nonalbicans was gaining place in patients under parenteral nutrition (c.parapsilosis).conclusion. 1) systemic candidiasis affects men admitted with sepsis or surgery, with a high apache ii index, multiple organ failure, multi-instrumentation and more than two weeks intensive care unit stay. 2) we observe a progressive incidence of non albicans candidiasis (c. parapsilosis). 3) type of candida ssp did not affect mortality. 4) c. albicans was more frequently isolated in septic patients, while candida nonalbicans was predominant in cases with parenteral nutrition. 5) mortality was greater in moderate/higher sss risk group. f. alvarez-lerma* 1 , m. palomar 2 , p. objetive: to present changes of multiresistance markers in icu-acquired infections. a prospective, cohort, multicenter study. all patients admittted to the participating spanish icus between the years 2002 and 2006 were included. patients were followed until discharge from the icu or up to a maximum of 30 days. the following infections were studied: mechanical ventilation-related pneumonia (mv-p), catheter-related urinary tract infection (cr-uti), and primary bacteremia (pb). markers of multiresistance were those defined by the cdc (1) of a total of 39,937 pacientes included in the study,4,050 (10.1%) developed 5,546 infections (13.9%) during their stay inthe icu, in which a total of 6,034 pathogens were identified.multiresistance markers are shown in table 1 . pulse pressure variation greater than 12% predicts fluid responsiveness in patients ventilated with large tidal volumes. the aim of this study is to evaluate the influence of a low tidal volume on the capacity of pulse pressure variation (deltapp) to predict fluid responsiveness.methods. this is a prospective interventional study that took place in a 33-bed university hospital medico-surgical icu. the study included eighteen mechanically ventilated critically ill patients with a low tidal volume (6-7 ml/kg) requiring fluid challenge. fluid challenge was performed with 1,000 ml crystalloids or 500 ml colloids. complete hemodynamic measurements including deltapp were obtained before and after fluid challenge. overall, the cardiac index increased from 3.04±1.54 to 3.80±2.32 l/min/m2 (p <0.05). it increased by more than 10% in 11 patients (responders). pulmonary artery occluded pressure was similar (14.9±5.5 vs. 14.5±6.0 mmhg, p=0.90) but deltapp higher in responders than in non-responders (8±7% vs. 5±7%, p=0.46). fluid responsiveness was equally predicted by deltapp (roc curve area 0.61±0.14), pulmonary artery occluded pressure (0.58±0.14) and right atrial pressure (0.66±0.13) (p=ns). the best cutoff value for deltapp was 4% with a sensitivity of 46% and a specificity of 86%. the preliminary results suggest that deltapp is not a better predictor of fluid responsiveness then paop or rap in mechanically ventilated patients when tidal volume is 6-7 ml/kg. if used, a lower critical value may help to predict fluid responsiveness. svv and ppv are proven influenced by the different airway pressures due to depth of tidal volume and peep. the effect of respiratory rate or respiration frequency on svv and ppv is however unclear. aim of this study was to evaluate the effect of respiration frequency on svv and ppv in mechanically ventilated patients. after obtaining informed consent, 6 (coronary bypass grafting) patients were studied immediately after surgery. cardiac output (co), svv and ppv were assessed by arterial pulse contour analysis (lidco, lidco ltd). all patients were ventilated in pressure controlled mode (settings: fio2 0.40, tidal volume 8 ml/kg, peep 5 cmh2o, frequency 12min-1) and sedated with propofol. in this study svv and ppv were evaluated with fixed ventilator frequencies of 8, 12 and 16min-1. this protocol was repeated 4 to 5 times (before and after volume loading of 500 ml) in each patient. during the study the mean airway pressure was maintained constant by adjusting inspiration time. 135 collected data points are described in means (sd) and evaluated using anova. in six patients (female/male ratio 1/5) after coronary bypass grafting, mean age 62(±11.7) years [range 46-74 years], 135 data points by fixed respiratory frequencies could be analysed (29/8, 30/16 and 76/12). all measurements were performed in hemodynamically stable conditions, hr mean 84(±11.4) min-1, map 84.7(±9.5)mmhg, cvp 10.2(± 2.5)mmhg and co 4.7(±0.84) l/min (p for all ns). mean airway pressure 9.7(±0.81)mbar (levene statistics, p = 0.605), for resp-f8 9.3(±0,71)mbar, resp-f12 9,8(±0,79)mbar and resp-f16 10.1(±0.79)mbar. on fixed respiratory rates svv and ppv were unchanged: for svv (resp-f8) 8.0(±3.3)%, (resp-f12) 7.3(±3.0)%, (resp-f16) 8.2(±4.8)%, p = 0.349, for ppv (resp-f8) 9.4(4.6)%, (resp-f12) 8.6(±3.8)%, (resp-f16) 9.6(±4.8)%, p = 0.499. in ventilated cardiothoracic surgical patients, svv and ppv were not influenced by forced changes in respiratory frequencies between 8 and 16min-1. (svv) has been studied as a dynamic preload marker to predict fluid responsiveness in critically ill patients. patients undergoing major abdominal surgical procedures with the aid of pneumoperitoneum may have a difficult preload management, due to either a preoperative hypovolemic status or an excessive intraoperative fluid loading to maintain an adequate volume and tissue perfusion. the aim of this study was to use the svv to optimize the fluid management in patients undergoing major abdominal robot-assisted laparoscopic surgery. methods. 20 patients (asa score 3-4; mean age 69.3 +/-9.9) were prospectively enrolled. cardiac index (ci), stroke volume variation (svv), and central venous saturation (scvo2) were calculated with the vigileo system. gastric carbon dioxide pressure (pgco2) was measured with a gastric tonometer. before the induction of anesthesia, 6 ml/kg normal saline solution was administered. later, colloids were infused whenever a svv >15% resulted. hemodynamic variables and pgco2 were measured before, during, and after the end of surgery. the total amount of intraoperatively administered fluids (iaf) was calculated. subsequently, the iaf was compared with theoretical iaf using the formula proposed by miller. analysis of variance and student's t-test were applied. mean surgery time was 3.6 +/-0.6 hours. ci ranged from 1.8 to 5.5 liters/min/m2. scvo2 ranged from 62% to 88%. the pgco2 ranged from 4.1 to 15.2 mmhg. anova did not show significant variations of ci, scvo2 and pgco2. mean baseline and postoperative svv% were 17 +/-4.1 and 9.1 +/-4.5, respectively. with respect to preoperative values, anova showed a significant reduction for svv%. moreover, at the end of surgery the svv% resulted less than 15% for each patient. the total amount of fluid was 14.9 +/-2 vs 18.7 +/-2.4 ml/kg per hour (calculated vs theoretical, respectively. p<0.05). no patient showed signs of hypoperfusion. no complication or death occurred.onclusion. the vigileo system seems to be a reliable tool to provide indications for fluid administration and volume responsiveness. it could be useful especially in major surgical procedures at risk of fluid overfilling. svv continuously monitored may help physicians to avoid fluid overloading in patients undergoing major abdominal robot-assisted laparoscopic surgery. recently, the preload parameters global enddiastolic volume gedv and intrathoracic blood volume itbv measured with transpulmonary thermodilution were convincingly shown to be superior to the historically used central venous pressure 1 . the extravascular lung water evlw was shown to be a prognostic marker in critically ill patients 2 . however, in our clinical experience, we failed to achieve the proposed normal ranges for gedv/itbv indexed to body surface area in a substantial number of patients. as hypothesis, we investigated the dependence of transpulmonary thermodilution parameters on the patient's age. we retrospectively analyzed the transpulmonary thermodilution data in a series of 128 patients treated on our neurosurgical intensive care unit. diagnosis was predominantly severe subarachnoid hemorrhage, but included traumatic brain injury and polytrauma, too. itbvi and gedvi were measured with the picco ® system (pulsion medical systems ag, munich, germany). measurements were performed with 20cc iced saline injected repeatedly in a central venous line. all data was stored online and pooled for analysis. mean patient age was 54.2 (sd 14.4) years. 2587 pooled thermodilution measurement sequences consisting of 9405 single injections were analyzed. mean gedvi was 721 (sd 173) ml/m 2 , mean itbvi was 898 (sd 213) ml/m 2 and mean evlwi was 8.0 (sd 3.7) ml/kg. younger patients had lower mean values calculated by linear regression, with an increase of 4.6 ml/m 2 for gedvi and 5.8 ml/m 2 for itbvi per patient year. evlwi was independent of age.conclusion. the thermodilution data from our patient collective contrasts the use of fixed age-independent normal values for gedvi and itbvi but not for evlwi. this data set, however, comprises a neurosurgical patient collective and may not be validly extrapolated to other clinical surroundings. 1. michard f., et al.: chest 2003; 124: 1900 -1908 2. sakka, s., et al.: chest 2002 122: 2080 -2086 thirty mechanically ventilated patients with severe sepsis or septic shock (age 60±15; apache-ii score 31±8; 18 male) requiring invasive hemodynamic monitoring due to cardiovascular instability were included in a prospective observational trial. the study was performed in a university hospital setting with a 24-bed medical intensive care unit (icu) and a 14-bed anaesthesiological icu. volume-based hemodynamic parameters were assessed using the single-pass thermal-dye transpulmonary dilution technique. simultaneously, ivc diameter was measured throughout the respiratory cycle by transabdominal ultrasonography. we found a statistically significant correlation of both inspiratory and expiratory ivc diameter with central venous pressure (p=0.004 and p=0.001), extravascular lung water index (p=0.001, p<0.001), intrathoracic blood volume index (p=0.026, p=0.05), the intrathoracic thermal volume (both p<0.001), and the pao2/fio2 oxygenation index (p=0.007 and p=0.008, respectively).conclusion. sonographic determination of ivc diameter is useful in the assessment of volume status in mechanically ventilated septic patients. this approach is rapidly available, non-invasive, inexpensive, easy to learn and applicable in almost any clinical situation without doing harm. ivc sonography may contribute to a faster, more goal directed optimisation of fluid status and may help to identify patients in whom deleterious volume expansion should be avoided. it remains to be elucidated whether this approach influences the outcome of septic patients. a severe burn injury is associated with hypermetabolism and catabolism that has been shown to persist for over 12 months post injury. propranolol has been shown to reduce hypermetabolism during the acute hospital course. the effect of propranolol, a nonselective beta blocker, on respiratory variables in children with severe burns has not been established. beta-blockade is associated with a known risk of bronchoconstriction in children with hyper-reactive airway disease, but it is not known whether the effects are also seen in severely burned children. the purpose of this study was to determine the effect of propranolol, given during acute hospitalization, on respiratory variables. forty-six patients with burns >40% total body surface area (tbsa) were enrolled into the study and randomized to receive propranolol at 1.0 mg/kg/day (n=23) or placebo (n=23). administration of propranolol was started the day following the first operation and continued for three weeks. respiratory variables were measured by a flow transducer attached to a bicore cp respiratory monitor. all patients were breathing spontaneously and non-intubated. study variables included respiratory rate (rr), minute ventilation (mv), tidal volumes (vt), and peak inspiratory/expiratory flow rates (pifr/pefr). baseline measurements were taken at rest before the drug or placebo was initiated. follow-up measurements were performed at the end of the study period. data were analyzed using paired t-test within groups and un-paired t-test between groups. data are reported as mean ± sd. significance was accepted at p<0.05. the mean age in both groups was 10 ± 4 years. as expected, heart rate was reduced by approximately 20% in the propranolol group compared to placebo (p<0.05). there was a significant increase in pefr from 0.57 ± 0.26 to 0.74 ± 0.36 l/s in the propranolol treated group (p=0.03). in contrast, neither placebo nor propranolol significantly affected rr, vt, ve or pifr. results indicate that short term administration of propranolol showed significant effects on pefr suggesting increased pulmonary conductance. further studies on the effects of propranolol on gas exchange and lung compliance are needed. grant acknowledgement. funded by nih grants p50-gm06338 and ko1-hl70451 a. storesund* 1 , e. wallestad 1 , l. rygh 2 1 postoperative section, surgical department, 2 surgical department, haukeland university hospital, bergen, norway international studies point out that to work with agitated children, described as restless and disorientated are particularly stressful for the child, parents and caregiver. this project is based on the assumption by nurses in the post anaesthetic unit (pau) that there was a noticeable post anaesthetic agitation difference between the children who received long-term opioids initially and in the end of the operation (refill, a) compared to those who only got long-term opioids in the beginning of the operation (no refill, b). the main purpose of this project was to examine whether there were any difference in postsurgical agitation between the refill and no refill group. further, this project seeks to uncover if there are any factors that can be improved per-and postoperative for these patients. we observed 35 post anaesthetic children, lip-(n=8), cleft-(n=0), and palateclosure (n=12), adeno-(n=5), & adeno-tonsillectomy (n=10). these children were recruited using a convenience sampling strategy at the pau at haukeland university hospital, norway, over a 19 week period in 2006-07. a pilot-tested fixed cross sectional designed questionnaire was utilised by the nurse responsible for each patient. several statistical tests by the use of spss made it possible to analyse and answer the research question: are children who only get long-term opioids in the initial anaesthetic phase (b) of the operation more agitated than those who where also given a refill of long-term opioids (a)? we found that 20/28 got refills of long-term opioids (a), 8/28 did not get refills (b), 20% were recorded as missing values. t-test result = 0,735 is greater than 0,05, hence there is no statistically significant difference between the two groups. levene's test tells us that the two variances are not significantly different (levene's test sig=0,167). there were no significant relationships between the parameters recorded. however, there was a tendency that more preoperative anxious children got refills (5/5) compared to non-anxious children (6/10) (fisher's exact test p=0,15). the latter results may conceal the agitation-scores in the two groups; refill and non-refill-group. this possible bias may have been eliminated if the patients had been randomized to either refill or non-refill. the present study confirms previous observations by others indicating no singular factors can explain why some children experience agitations and others do not. analysis of the parameters studied did not discover any statistical significant relationships. thus, how to minimise the cohort of children who experience post anaesthetic agitation still remains a recurrent challenge. pulmonary hypoplasia with severe cardiorespiratory dysfunction is often the leading cause of death in neonates with congenital renal disease and oligo-anhydramnios. aim of the study was to determine whether ino is effective to improve respiratory function in these critically ill neonates. we retrospectively reviewed the charts of all newborns who were admitted between february 1996 and september 2002 with the diagnosis of oligo-anhydramnios of renal origin. during this period all patients were treated according to a standardised algorithm. they were intubated either if post cpr or if fio2 had to be increased above 0.4. 100mg/kg of bovine surfactant were applied for improvement of ventilation. pre-and postductal oxygen saturation were measured simultaneously with target values of 85-93%. if fio2 remained above 0.5 a transthoracic echocardiography was performed. the presence of a ductal or atrial right to left shunt or a difference in oxygen saturation between the pre-and postductal measurements of >15% led to the diagnosis of pulmonary hypertension and to the initiation of ino therapy. further, ino was applied as a rescue therapy if oxygen saturation remained below 80% despite a fio2 of 1.0 and optimization of ventilator settings and therapy with catecholamines. all patients had informed parental consent. the patient population (n=20) included 7 children receiving ino of whom 5 suffered from obstructive uropathy and two had polycystic kidneys, whereas 13 patients did not receive ino treatment. in this group there were 11 children with obstructive uropathy and 2 born with polycystic kidneys. all data are presented as median (range). we concentrated on the group receiving ino. in this group mortality was 57.1%. therapy was started at an age of 10.9 (1-28) hrs. initial dose of ino was 14.9 (4-40) ppm with peak dose of 20.3 (5-57) ppm. ino led to a decrease of oxygenation index (oi) from 28.4 (3.4-44.4) to16.6 (3.2-70.7). five children suffered from obstructive uropathy. three of them had a favourable long-term outcome, one child died immediately, whereas one child was initially stabilized but finally succumbed to its underlying disease. two children demonstrated genetically determined pulmonary hypoplasia due to the presence of polycystic kidneys. both children died within the first three days despite ino treatment. children with obstructive uropathy and severely impaired oxygenation seem to benefit from ino therapy. patients suffering from a hereditary renal and pulmonary hypoplasia did not respond favourably to ino therapy and had a fatal outcome. a. khaldi*, k. menif, a. bouziri, a. hamdi, s. belhadj, n. ben jaballah pediatric intensive crae unit, children's hospital, tunis, tunisia the use of high-frequency oscillatory ventilation (hfov) and ino resulted in a decline in the need for extracorporeal membrane oxygenation (ecmo) in near-term and term neonates with persistent pulmonary hypertension (pphn). association of hfov and ino is actually an accepted treatment modality even in non-ecmo centers. however, because not all neonates respond to hfov + ino, identification of factors related to a poor response is very important for prognosis and for early transfer to ecmo canters if possible. the objective of this study was to identify the risk factors predicting poor shortly outcome in near-term and term neonates with pphn treated with hfov and ino in a tertiary care pediatric intensive care unit in a university hospital. we conducted a prospective clinical study including all neonates with gestational age ≥ 34 weeks with echocardiographic signs of pphn. patients with pulmonary hypoplasia or congenital diaphragmatic hernia were excluded . patients were ventilated with conventional mechanical ventilation (cmv) with ino (10-20 ppm). hfov were instituted if patient required, on conventional ventilation (cmv)+ino, a fraction of inspired oxygen (fio2) 0.5, and a mean airway pressure > 10 cm h2o to maintain adequate oxygenation or a peak inspiratory pressure > 24 cm h2o to maintain tidal volume between 5 and 7 ml/kg of body weight. hfov were used in association with ino in seventy infants (gestational age, 37 ± 1,9 weeks), after a mean duration of cmv of 4 ± 7 hours. arterial blood gases, oxygenation index (oi), and alveolararterial difference in partial pressure of oxygen (p[a -a]o2) were recorded prospectively before and during hfov. there were a rapid and sustained decreases in mean airway pressure (map), oi, and p[a -a]o2 during hfov (p ≤ 0.01). this improvement, along with decreased need for oxygen, was sustained through the subsequent course of hfov. sixty-six infants (93%) were weaned successfully from hfov. five infants (7%) were classified as meeting treatment failure and died from their underlying disease. treatment failure was associated with lack of improvement in p[a -a]o2 and oi at 4 hour of hfov (p < 0.01) and the presence of intractable shock requiring epinephrine or norepinephrine (p=0,01). in near-term and term neonates with pphn, the association of hfov and ino lead to a rapid and sustained improvement in gas exchange in the most cases. the magnitude of improvement of oi and p[a -a]o2 at 4 hrs can predict outcome early. early burn sepsis is notable for the complexity of diagnostics, malignant course and high lethality. the problem remains actual for the children who got a severe burn trauma (more than 20% body surface area). purpose to define procalcitonin test (pct) effectiveness for early sepsis diagnostics for children with thermal trauma. during the period of time from january 2004 up to april 2007 there were 190 children in our clinic with extensive burns from 20%up to 70% body surface area (bsa) at the age from 6 months to 14 years old. 39 patients at the age from 8 months to 13 years old with the burns from 20 % to 70% bsa were included in our research. all the children got surgery in shortest time after trauma (tangential excision with authodermoplastics), antibacterial, and infusion therapy. from the moment of registration in icu all the patients, who were suspected to have sepsis, simultaneously with traditional examinations (blood analysis, bacteriological investigation) were taken pct analysis with the help of "pct-express test" (brahms, germany). . 17 patients (44,7%) were diagnosed sepsis, 3 children died. these patients pct level was from 2 to 10 ng/ml; together with this all the patients had increasing quantity of leucocytes, acceleration the level of c-reactive protein, fever. 22(56%) patients had no sepsis, so pct figures fluctuated in the bounds of 0,5 ng/ml. among these patients traditional markers of inflammation were increased. no trustworthy difference is found as for the level of leucocytes and c-reactive protein figures between the patients without infectious complications and with sepsis. only with the help of pct the beginning of sepsis and sirs manifestation can be differentiated.conclusion. 1. burn trauma itself is not the reason for pct increase. pct level increases in cases of burn injuries as the sign of infectious complications joining. 2. with the help of traditional sepsis markers it is difficult to differentiate sirs manifestation and first stages of infectional complications in case of thermal trauma. 3. in cases of severe burns pct test is a highly sensitive method of sepsis early stages diagnostics. 4. surgery treatment at early stages after trauma allows to avoid development of severe sepsis. h. knoester* 1 , m. b. bronner 2 , a. p. bos 1 , m. a. grootenhuis 2 1 pediatric intensive care unit, 2 psychosocial department, emma children's hospital, amc, amsterdam, netherlands introduction. improved survival in children with critical illnesses has led to new disease patterns due to long-term complications and effects of the original illness and its treatment. as a consequence, health related quality of life (hrqol) has become an important outcome measure in pediatric intensive care unit (picu) survivors. little is known about hrqol in picu survivors,. hrqol evaluation could contribute to improvement of support after discharge. the purpose of this study was to assess hrqol in picu survivors. october 2005 all parents of children, acutely admitted to our picu were invited to complete hrqol questionnaires, 3 and 9 months after discharge. hrqol in children from 1-6 years of age was evaluated with a dutch validated questionnaire, the tno-azl preschool children quality of life questionnaire (tapqol). the tapqol covers 12 domains of hrqol; norm data from the general dutch population are available. data analyses was done by non-parametric testing (patients versus norm group) and by calculating effect sizes (difference in mean scores between the patients and the norm group divided by the standard deviation of the scores in the norm group). effect sizes give an indication of changes in hrqol in comparison with the norm group. . 34 of 75 (45.3%) eligible patients were evaluated. statistically significant differences with the norm group were found on 2 domains, 3 and 9 months after discharge (more lung problems and worse liveliness) and on 1 domain 3 months after discharge (better appetite). moderate (0.5) and large (0.8) effect sizes were found on five respectively four domains 3 and 9 months after discharge: indicating worse hrqol on lung problems, sleeping problems, motor functioning, anxiety, positve mood and liveliness; and indicating better hrqol on problem behaviour. no statistically significant changes over time were found for all domains 3 and 9 months after discharge. our results indicate that hrqol in young picu survivors is decreased in some domains of physical and emotional functioning. these problems do not diminish over time. positive evaluation by parents regarding appetite and problem behavior could be influenced by response shift (changing of internal standards and values due to confrontation with a life-threatening disease). more research is necessary because of the small study group and to determine the influence of risk factors such as length of stay, age of the child at admission, severity of illness and physical sequelae of the disease and its treatment on hrqol. hrqol evaluation can be a useful tool as part of screening after picu survival to determine the necessity for follow-up care. coarctation of the aorta is not an uncommon congenital heart defect. one of the possible postoperative complications is the so-called postcoarctectomy syndrome (mesenteric arteritis). the purpose of the present study is to assess the changes in gut flow through the dual sugar permeability test. five patients have been included in the study until now. median age 1 month (0.3 -24) and median weight 4.1 kg (3 -15). premedication and anaesthesia was the same for all the patients. the test solution contains 3-o-methyl-d-glucose, d-xylose, l-rhamnose and lactulose. patients received 2 ml/kg of the test solution after induction of anaesthesia, at 8 and 24 hours after the initial dose. urine production is measured during a three-hour period after each instillation. the sugar content is analysed by capillary gas chromatography (normal values l/r = 0.05, 3omdg and xylose 10-30%). a. monsel 1 , p. durand 2 , v. haas 2 , c. beaujard 3 , p. rouleau 3 , s. el aouadi 3 , d. benhamou 3 , k. asehnoune* 4 1 anesthesie reanimation, 2 reanimation pediatrique, 3 anesthesie réanimation, hopital de bicetre, bicetre, 4 anesthesie réanimation, chu hotel-dieu, nantes, france pediatric epidural anesthesia (ea) is considered to be without hemodynamic impairment in children. however, when compared with information relating to adults, little is known about the hemodynamic effects of epidural anesthesia on the cardiac output (co) in infants. using transesophageal doppler (ted) monitoring of co, we prospectively studied 14 infants < 10 kg who were scheduled for abdominal surgery. during sevoflurane general anesthesia, ted monitoring of co was performed before and after lumbar ea with 0.75 ml/kg of 0.25 % bupivacaine and 1:200,000 adrenaline. co, arterial blood pressure, and heart rate were measured before and 5, 15, and 20 minutes after performance of ea. in patients anesthetized with sevoflurane and sufentanil, ea resulted in an increase in stroke volume by 29% (p<0.0001) and a decrease in heart rate by 13% (p<0.0001). ea also induced a significant decrease in systolic, diastolic, mean arterial blood pressure and systemic vascular resistances by 11%, 18%, 15%, and 25% respectively. conversely, co remained unchanged. the increase in sv observed is probably explained by optimization of afterload due to the sympathetic blockade induced by ea. these results confirm that ea provides hemodynamic stability in infants weighing < 10 kg and support the use of ea in this pediatric population. bleeding is the most frequent complication during extracorporeal life support (ecls) after pediatric cardiac surgery. we would like to present our experience with ecls and recirculation blood saving, volume auto-regulation system using the law of connected vessels based on converted cpb set in infants after cardiac surgery with significant bleeding. since 2003 to 2005 26 ecls in the postoperative period was performed (1,9 % of all cardiac operations in this period). the significant bleeding (>5ml/kg/h) was noted in 20 pts. in most recent 11 pts the volume recirculation system was implemented, whereas in 9 previous patients blood was sucked out the circuit. the retrospective analysis of data was carried out. there were 16 infants with single ventricle anatomy and 10 with two-ventricle anatomy. there were no significant differences with respect to age, weight and prevalence of single ventricle anatomy between groups. the indication for ecls was cardiac arrest in 14, low cardiac output in 4, hypoxemia in 6 and sepsis in 2 patients. the overall mortality rate was 46%. the mortality did not differ significantly between groups (36,3% versus 55% in non-recirculation group; p=0,35). there was significantly lower number of blood products transfusions(p<0,05), lower number of surgical explorations(p<0,05) lower mean lactate level 8 hours after ecls institution p(<0,05) and shorter ecls duration (p<0,05) in the recirculation group. the system of blood recirculation in children with bleeding on ecls is simple, highly effective in stabilization of the haemodynamics and no-cost consuming. it can reduce necessity of chest exploration, blood product transfusions and duration of support. t. tunc* 1 , t. topal 2 , m. kul 1 ,ö.öngürü 3 , a. korkmaz 2 , s.öter 2 1 neonataloji bilim dali, 2 fizyoloji anabilim dali, 3 patoloji anabilim dali, gülhane askeri tip akademisi, ankara, turkey necrotizing enterocolitis (nec) is the most common gastrointestinal emergency in the premature infant. the major risk factors in nec include prematurity, hypoxia, enteral feeding, and bacterial colonization. these factors predispose at-risk infants to an exaggerated intestinal inflammatory response leading to ischemic bowel necrosis. experimentally induced ischemia and reperfusion (i/r) of the intestine is a model which can be appropriately used to imitate nec. n-acetylcysteine (nac), erdosteine (erd) and alpha-lipoic acid (ala) are well-known antioxidants with similar structural properties. in the present study, the effectiveness of these three sulfur-based antioxidants against intestinal i/r-injury was evaluated.methods. 40 one month old male spraque-dawley rats were randomly divided into five groups (n = 8 for each): i/r (control), i/r+nac, i/r+erd, i/r+ala and sham-operated group without i/r. animals were operated at a temperature of 24 o c under ketamine anesthesia. ischemia was provided by occluding the superior mesenteric artery via a microvascular clamp. collateral vessels of the small intestine were ligated to prevent collateral circulation. 30 min of ischemia was followed by 30 min of reperfusion. nac (100 mg/kg/day, i.p.) was administered first 30 min before operation and followed once daily for 5 days. erd (10 mg/kg/day, oral gavage) administration was begun 2 days before operation and continued 5 daily doses. ala (35 mg/kg/day, i.p.) was injected only one time 24 h before operation. at day 5 after operation the ileum was resected and the rats were sacrificed. protein oxidation (carbonyl content, pco), lipid peroxidation (malondialdehyde, mda), superoxide dismutase (sod) and glutathione peroxidase (gsh-px) were measured in the ileal tissue. oxidative and antioxidant parameters of resected ileal segment (mean ± sd) groups 0 as a clinically relevant model to nec, our experimental i/r protocol resulted with marked rise in oxidative stress levels and fall of antioxidant enzymes activities. these changes were ameliorated with the antioxidants used. among all, ala presented the strongest and nac the weakest effect. this outcome promises beneficial usage of these sulfurbased antioxidants against oxidative stress which plays an important role in nec pathogenesis. a. khaldi* 1 , k. menif 2 , a. bouziri 2 , a. hamdi 2 , s. belhadj 2 , n. ben jaballah 2 1 pediatric intensive crae unit, children's hospital, 2 pediatric intensive crae unit, children's hospital, tunis, tunisia high-frequency oscillatory ventilation (hfov) may significantly improve oxygenation and outcome in newborns with respiratory dysfunction and beyond the neonatal period in patients with a variety of diffuse alveolar diseases. in small airway disease like respiratory syncytial virus (vrs) bronchiolitis, hfov is considered potentially hazardous because of the risk of air trapping. however, a few studies had reported utility of hfov in children with acute hypoxemic or hypercapnic respiratory failure caused by vrs and failing optimal conventional mechanical ventilation (cmv). the objective of the study is to evaluate the effectiveness and safety of hfov in pediatric patients with acute respiratory failure due to rsv and failing cmv. we conducted, over 6-year period (october 2000 to october 2006), a prospective clinical study in a tertiary care pediatric intensive care unit. fourteen (14) patients (ages 12 to 73 days) with acute respiratory failure due to rsv bronchiolitis and failing optimal cmv were included. passage to hfov was indicated for severe hypoxemia in 11 patients (median alveolar-arterial oxygen difference [p(a-a)o2]: 564 [465-624] mmhg, median oxygenation index [io]: 28 [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] ) and for severe hypercarbia in 3 patients (median ph: 7,20 [7,11-7,24] , median paco2: 98 [90-120] mmhg). hfov was instituted after a median length of cmv of 32 (4-48) hours. ventilator settings, arterial blood gases, oi and p(a-a)o2 was recorded before hfov (h0) and at a predetermined intervals during hfov and compared using the one-friedman rank-sum procedure and a two-tailed wilcoxon matched-pairs test. after starting hfov, a distinct decrease in fio2 at 4 hrs that continued to 24 hrs (p<0,05). in all patients, there were significant decreases in oi and p(a-a)o2 at 4 hrs, that were sustained up to 20 hrs (p<0,04). target ventilation was achieved in all cases and paco2 significantly decreases after 1 hr of hfov (p=0,01) and remained within the target range thereafter (40-70 mmhg). the median maximum pressure amplitude used on hfov was 45 (35-60) cm h2o and the median maximal paw was 23 (18-26) cm h2o. no significant complications associated with hfov were observed. twelve patients (86%) survived to hospital discharge without supplementary oxygen. tow patients (14%) died from septic shock. in pediatric patients with either hypoxemic or hypercapnic acute respiratory failure due to rsv bronchiolitis, hfov can be used successfully and safely if conventional ventilation fails to improve gas exchange. however, randomized controlled trials are needed to identify its benefits over conventional modes of mechanical ventilation. and are influenced by numerous factors like patient's disease severity, policies of the treating unit, religious and cultural traits, education and awareness of the patient's family, financial status of the family and legal provisions. majority of published studies on eol reflect either european or american ethos; that is either physician's paternalistic approach about the patient or patient's autonomy and self determination,(1,2) about this sensitive process. studies on eol which reflect the influence and pivotal role of closely knit indian family on eol decision making are scant. we retrospectively analysed the eol decisions taken by the family in our icu as majority of the patients which merit eol care were not in a condition of decision making. setting-50 bedded multidisciplinary icu of a 400 bedded tertiary care teaching hospital in pune in india. case papers of all icu admissions during one year i.e. 1st january to 31st december 2006 where eol decision was documented, were reviewed. data collected included demographics, underlying disease process, duration of aggressive treatment till eol consent, duration between eol decision and death, consenting person's relation with the patient, organ failure & level of life sustaining supports at decision and mode of payment of the treatment. during the study period 524 patients died in our icu of which eol decision and consent was explicitly documented in 95 cases which constitute study population. average age of the patient was 63 years (range 17 to 91), average duration of active treatment till eol consent was 83.35 hours(range1 to 960),average duration between consent and death was 29.03 hours(range 1to 168). 92.7% consents were signed by close relatives( son/daughter, brother/sister, spouse, father/mother) and 7.3% were by other relatives( cousins, son in law/daughter in law). at the time of eol decision 90.7% patients were having glassgow coma scale 8 and below, 69.4 % patients were on mechanical ventilation, 56.84 % were on vasopressors and 6.3% were needing renal replacement. metastatic disease (31.5%) and traumatic or vascular brain injury(22.1%) were the commonest causes of death. only 24.05% patients had medical insurance or employer assistance as a mode of payment for the treatment and in 75.9% cases family members were the payers. withholding of non beneficial life sustaining therapies as eol process was practised in 18.12% of the total icu deaths. all 95 (100%) eol decisions as well as directive requests and consents were signed by patients' relatives, reflecting the importance of close family ties in indian eol practices. our objective was to study frequencies of withholding and withdrawing treatment and time until death in a dutch university hospital icu. between october 2006 and february 2007 we collected data of all patients that died. data were collected from patient files and during interviews with the doctors and nurses who were responsible for the patient at the time of death. we analyzed which treatments were withheld or withdrawn and calculated the time until death following withholding or withdrawal. preliminary results show that of 471 admissions, 74 patients died (16%). nonsurvivor's (median age 64 years [range 15-84]) median length of stay was 3 days (range 30 minutes -5 months). in 57 patients (77%) treatment was withdrawn and in 4 patients (5%) treatment was withheld but not withdrawn. of all patients 71 (96%) were mechanically ventilated of which 6 (8%) were weaned and extubated before death. in 4 of these patients it was decided not to intubate again and 2 other patients not to intubate at all (median time until death: 24 hours). in 45 (61%) ventilator-dependent patients mechanical ventilation was withdrawn; 35 (47%) were extubated. the median time until death after ventilator withdrawal was 30 minutes. when patients were also extubated, it was 22 minutes (p=0,16 [mann-whitney test]). in 6 patients mechanical ventilation was not withdrawn, but fio2 was decreased to 0.21 (median time until death 35 minutes). in 25 patients (34%) inotropic medication was withdrawn (median time until death 30 minutes). in 20 cases, the withdrawal of inotropic medication was combined with the withdrawal of mechanical ventilation. in 8 patients (11%) it was decided not to increase inotropic support (median time until death 6:45 hours). in 45 patients (61%) the decision was made not to resuscitate in case of cardiac arrest. median time of this decision before death was 26 hours. in the patients that died treatment was withdrawn in the vast majority of patients. withdrawal of mechanical ventilation and/or withdrawal of inotropic support were most often used. a considerable number of patients died within 30 minutes following withdrawal of therapy. r. veiga* 1 , g. silva 1 , g. campello 1 , c. dias 2 , c. granja 1 1 intensive care department, hospital pedro hispano, matosinhos, 2 biostatistics and medical informatics, faculty of medicine, porto, portugal the high mortality of critically ill patients underscores the need for icu teams to recognize end-of-life care as an integral component of critical care. besides survival, the success of intensive care should also include the quality of lives preserved and the quality of dying. the aim of this study was to evaluate the incidence and type of end-of-life decisions in critical patients that died in an icu. retrospective analysis of all patients that died in the icu in the period of 1 january to 31 december 2006 and evaluated the following variables: demographic characteristics (age, gender); co-morbidities: (heart failure, chronic obstructive pulmonary disease (copd), diabetes mellitus, neoplasia, chronic renal disease, hiv/aids, alcoholism); reason for admission; saps ii; length of icu stay (icu los) and type of end-of-life decisions. three concepts were defined in order to classify the end-of-life decisions: comfort care: a change from curative therapy to comfort care therapy; limited therapy: maintenance of curative therapy but without escalating it (e.g. not raising rate of vasopressor agents, no renal substitution); without previous end-of-life decisions: when no attitudes toward end-of-life care were considered. given the diminished number of patients in the without previous end-of-life decisions group we decided to evaluate them apart from the other two groups.results. two-hundred and twenty seven patients were admitted in the icu and 62 of them died (27%). reason for admission in those who died was septic shock/ severe sepsis (43%), post-cardiac arrest (22%); cardiogenic shock (9%); acute respiratory distress syndrome (7%). the most common co-morbidity was alcoholism (19%), followed by diabetes mellitus (14%), neoplasia (13%), heart failure (11%) and copd (11%). forty seven patients (76%) died after comfort care decision, eleven patients (18%) after limited therapy decision and four (6%) patients died without previous end-of-life decisions. comparing the groups comfort care and limited therapy we found significant differences in the following variables: hemorrhagic shock at admission (2% vs. 27%) (p=0.04); saps ii (55 vs. 85) (p= 0.008); icu los (4.9 days vs. 0.27 days) (p<0.001). patients in the limited therapy group had more admissions with hemorrhagic shock, a higher severity score and stayed less time in the icu. this analysis suggests that end-of-life decisions in this group express their higher severity. patients of the comfort care group presented less severity and stayed longer in the icu. their shift of curative therapy to one designated to provide comfort care reflects an absence of a clinically favorable response. the low percentage of patients without previous end-of-life decisions is consistent with previous reports and should be seen as a positive issue. non invasive positive pressure ventilation (nippv) is widely accepted as an initial approach to providing ventilatory support to many patients with acute respiratory failure (arf). palliative approaches focused on the quality of life and comfort; represent a challenge for family's physicians and the patients. nippv is an attractive option to treat acute respiratory failure in end stage patients when the failure is irreversible and it is a final outcome of the primary disease. the approach to providing ventilatory support to patients with arf, to relieve them from the sensation of dying suffocate without intubating them because they don't wish it either, is very challenging. after institutional approval and patients consent, we conducted a prospective observational study of patients that fulfilled the criteria.12 cases received nippv (10 with end stage cancer and 2 with pulmonary fibrosis). when nippv was ordered we recorded: respiratory rate, heart rate, arterial blood pressure, neurological status and arterial blood gases, before nippv initiation (baseline data) and then 1st, 4th and 8th hour. at the time of initiation of nippv, all patients were alert and cooperative with nippv. analgesia and/or sedation were used when it was necessary. pao2, pco2 and ph measures were analyzed using statistical methods. percentage changes from baseline (pre-nippv) of these measures were used as dependent variables. (mean value of 2 measurements at different time points was used). dependent variables (percentage of pao2, pco2 and ph) were regressed on time, for each patient. in all cases the results were statistically significant, with p-values ranging from a low of 0.0002 to a high of 0.0185. for all patients, the regression coefficient for the percentage change was positive; indicating that the percentage change was increasing with time. we can remark that pao2 increases over time, pco2 and ph p values > 0.05. we believe that nippv via helmet cpap is a means of potentially ensuring the highest quality of end-of-life care. nippv can be applied for palliative care, and it might be used to keep patients whom developed acute respiratory failure comfortable before the inevitable. decisions regarding the resuscitation status of patients are among the most difficult facing healthcare professionals, patients and families. these groups often need to discuss decisions regarding resuscitation yet their understanding and expectations can differ greatly. this study sought to determine the knowledge and beliefs of doctors, nurses and the general public regarding resuscitation decisions.methods. an observational study was designed. three study groups (doctors, nurses and general public) were interviewed using a face-to-face interview by a single interviewer and questionnaires completed. questions examined opinion, factual knowledge and knowledge of the ethics surrounding hospital resuscitation attempts. . 30 doctors, 25 nurses and 30 general public were randomly selected. 70% doctors, 24% nurses and 0% of public correctly estimated survival to discharge following in-hospital resuscitation attempt. the remainder overestimated survival. 37.9% of doctors and 76% of nurses consider resuscitation decisions to be made too infrequently. deficiencies were identified in doctor and nurse knowledge of the ethics governing resuscitation decisions and public opinion was found to conflict with ethical guidelines. public understanding of the nature of cardiopulmonary arrests and resuscitation attempts, and of the implications of a dnar order is poor. 58.3% of public report television medical dramas as their primary source of information on such matters. knowledge regarding resuscitation principles, outcomes and ethics is poor among both healthcare staff and the general public. these knowledge differences may not be appreciated or addressed in discussions regarding resuscitation and this reduces the likelihood of meaningful discussion and acceptable decisions. there is a need for educational initiatives to address these deficiencies. public apprehension surrounding this subject needs to be identified and corrected during discussions and this could be facilitated with a patient information leaflet. [1] poor communication during this process may lead to unnecessary anger and a delay in the grieving process that could linger for many years to come. giving the family the option to be present during resuscitation offers a more compassionate and family-centred approach to this crisis. this option of family presence however is frequently met with resistance and uncertainty by health care workers who may view the family's presence as increasing their risk of making a mistake or worse, being sued. a study in the uk estimated that out of one-hundred-and-sixtytwo uk emergency departments family witnessed resuscitation was allowed by 79% for an adult patient and 93% for a child. [2] another us study also found that amongst patients in emergency departments, 72% preferred to have their family present during resuscitation. [3] a survey was conducted amongst the doctors, nurses and paramedics who work in two uk eds to assess their attitudes and beliefs. experience, life support training, years in practice, consent issues, ethical factors and concerns regarding medico legal implications were sought for. a 5-point likert scale was used and mean scores analysed using microsoft excel. . 129 staff were surveyed.34% of doctors, 29% of nurses and 35% of paramedics believed in the concept in trauma fwr. in cardiac arrest patients, 55% were in favour of it, 28% opposed to it and 17% undecided. 62% of staff believed that litigation was possible with family witnessed resuscitation. 83% of respondents thought that critical incident de-briefing would be of benefit to assist staff dealing with stress. fewer doctors believed in cardiac fwr compared to nurses (p=0.004) and paramedics (p=0.006). in trauma, difference was non-significant. as health care professionals caring for families in the emergency departments, we need to recognize the need for compassionate family-centered care. with a well trained and motivated team equipped with effective, well thought out guidelines, there is considerable benefit for family members and staff in this difficult situation. thorough information about the events that are going to take place in the icu after an elective procedure might facilitate the awakening process and weaning from the ventilator, mitigating patient's anxiety and increasing their comfort. the aim of this study was to analyze the impact of preoperative information on the patient's perceptions and reactions to the usual inconveniences, such as orotracheal tube (ott), associated with the first postoperative hours in the icu. prospective, cohort study with a group of cases (a) and a control group (b). duration: two months. inclusion criteria: all patients undergoing elective cardiac surgery. there were no exclusion criteria. setting: cardiac surgical icu of a tertiary hospital. the survey was made in the first 24 hours. the study was blinded for the doctors in charge of the patients. the characteristics of both groups are presented as a/b with the p value into brackets. the quantitative variables are shown with the mean value and the qualitative variables as a percentage. the number of patients included was 86: 47 cases (a) and 39 controls (b). age: 62,9/61,3 years (0,4); men: 64/74%(0,3); time receiving sedative drugs: 4,12/4,05 hours (0,7); total hours with ott: 6,53/8,35 (0,01); hours with ott after stopping sedation: 2,5/4,29 (0,004). the first patient's perceptions were: discomfort related to ott in 10,6/38,5% (0,002); surgical pain in 29,8/25,6% (0,6); thirst in 19,15/12,8% (0,4); welfare or calm in 19,15/2,56% (0,01), and nothing in 12,7/17,9% (0,5). additional sedatives were required in 6,38/20,51% (0,05). information was considered very useful in 97,9%. patients valued very positively the provided information. in addition, this information had a significant impact on the tolerance to the ott, requirement of additional sedatives, and in the sense of welfare. there were not differences in the time under sedative drugs or in the perception of thirst or pain. a multiparameter questionnaire was sent to 21 icu. each questionnaire comprised 24 informational topics groupe into 6 categories (table). one relative per patient was asked to quote (yes/no) within 4 days after admission, each item, i.e. if he would like to find information on that item in an ib. if "no" was quoted, he was asked to say why (closed answers). demographic data on patient and relatives were correlated to the scores (nbre of "yes"), in each item category (factor analysis with varimax rotation followed by stepwise multiple linear regression). . 246 questionnaires were analyzed (patients: age 60 ± 18 year, saps2: 48 ± 19, sofa: 7 ± 7). table: % of positive response for each item ("would you like information on this topic in an icu booklet?") grouped into categories. "no" answers were mostly explained by "i trust the team to manage information about this" (median: 61%, range: 16-81). mulitvariate analysis showed that demographics data describing patient condition (age, saps2, chronic disease) correlated (p<0.05) with "yes" score of the items comprized in "icu rules" (table) but not with other items grouped in other information categories. conclusion. interestingly, as a whole, most items were highly wished in a booklet, suggesting that 50-70% of relatives express a plea for transparency in face of "difficult icu issues", without taboo. only the "yes score" to "icu rules" items correlated with patient status whereas items from other topics did not. this sounds, as relatives visiting the most severe patients may consider visiting rules as crucial. other items did not correlate to profiles, and may thereby be considered as societal standard requirements in terms of information. in 10/2003, our 15-bed medical icu signed a convention with the asp iroise association defining hv's role and presence. the association, a member of a national network of hv associations, works with our university hospital. four hvs took alternate turns in the icu one afternoon per week. hv were free to meet any conscious patient or any family member who wished so; icu staff also asked them to meet patients or families who seemed particularly distressed. hv wrote a brief commentary in a special transmission logbook which could be consulted by the staff and gave feedback about their visits whenever needed. patients (pts)and families (fam)who met an hv were sent a questionnaire either in 01/2005 or in 08/2006. 1258 pts were admitted during the period of study: the hv met 128 pts (10,2%) and 209 families (16,6%). 56 people answered the questionnaire (23,6%): 12 pts and 44 fam:27 spouse,7 parents,1 sister,3 children(6 no answer). ethics consultation has been introduced into the practice of medicine during the last decades as a way to help physicians and nurses come to a decision about a medical treatment where value-laden conflicts are involved. the primary goal is helping to identify, analyze, and resolve ethical problems. the aim of this study was to evaluate ethics consultation in a dutch university hospital intensive care. intensivists, residents, fellows and nurses can consult a clinical ethicist specialized in intensive care for advice in value-laden situations. we evaluate ethics consultation on our icu between 1 january 2006 and 1 april 2007. the clinical ethicist was consulted 61 times. in 29/61 cases (48%) advice was asked before withdrawal of life-sustaining therapy. in this category 15/29 (52%) cases concerned palliative care. in 25/29 cases (86%) the independent advice was in confirmation with the physician's view. in 11/61 cases (18%) advice was sought in cases were there was doubts to proceed with intensive care therapy. in four cases relatives wanted to withdraw therapy, where the intensivist did not consider this as futile. in 9/11 cases (81%) the advice was in accordance with the treatment plan. in 6 cases (10%) questions about information asked by non-relatives. all advises were followed. 3 cases concerned triage, 2 cases withholding therapy, 4 brain death declaration, 2 a deadly iatrogenic complication and in 2 patients a question concerning emergency research. in 7 (11%) cases a lawyer specialized in health care was consulted. in the 29 cases about 'withdrawal of therapy', the advise could be given within 30 minutes in 72% of the cases. ethical advise by a clinical ethicist specialized in intensive care can be additional, affirmative and reassuring, and improves quality of care. in most cases advice could be given immediately. . deferred consent has been proposed as a surrogate for a priori subject or proxy consent. the aim of this report is to evaluate the practicality and efficacy of a deferred consent procedure in an ongoing dutch multi-centre clinical trial. screening logs were collected from two participating centres of a clinical trial that is currently conducted to evaluate the efficacy of early lactate-directed therapy and that uses deferred consent. screened patients were analyzed for eligibility and reasons for exclusion. (12%) were not reported to the study investigators, 41 patients (16%) were not included for medical-ethical reasons (e.g. treating clinician deemed risk/benefit ratio of the study intervention unacceptable), in 7 patients (3%) study participation was practically impossible (e.g. unavailable study materials) and the reason was unknown in 2 patients (1%). only 9 patients (or their relatives)(4%) refused informed consent. in an ongoing dutch multi-centre emergency clinical trial using deferred consent, only 4% of patients or their relatives refused informed consent. deferred consent in emergency research is practical and facilitates a high inclusion rate. adult respiratory distress syndrome (ards) and peep have been linked to right ventricular dysfunction (rvd). this has been attributed to elevated pulmonary artery pressure (pap) and pulmonary vascular resistance (pvr) due to ards as well as increased intrathoracic pressure due to peep therapy. we wondered if rvd was a late phenomenon in ards or could also be detected during early peep treatment of hypoxia in patients with multiple ards risk factors. pulmonary embolism is a highly prevalent disease associated with severe morbidity and mortality. although the hemodynamic changes induced by pulmonary embolism are known, the alterations in respiratory mechanics after an embolic event are not completely understood. the aim of this study was to evaluate acute changes in hemodynamics, static and dynamic respiratory mechanics and lung histology induced by an experimental model of pulmonary microembolism. ten large white pigs (weight 35-42 kg) were instrumented with arterial and pulmonary catheters and pulmonary embolism was induced in 5 pigs by injection of polystyrene microspheres (diameter ∼300 µm), in order to obtain a pulmonary mean arterial pressure (pmap) of twice the baseline value. five other animals were injected with saline and served as controls. hemodynamic and respiratory data were collected and pressure x volume (pxv) loops of the respiratory system were performed by a quasi-static low flow method. animals were followed for 12 hours and after death lung fragments were dissected and sent to pathology. the average amount of microspheres necessary to generate microembolism was 6.7 ± 2.2 mg/kg. pulmonary embolism induced a significant reduction in stroke volume (71±18 ml/min/bpm pre vs 36±9 post, p<0.05), an increase in pmap (27±4 mmhg pre vs 39±6 post, p<0.05) and pulmonary vascular resistance (193±122 mmhg/l/min pre vs 451±149 post, p<0.05). respiratory dysfunction was evidenced by significant reductions in pao2/fio2 ratio (480±50 pre vs 159±55 post, p<0.05), dynamic lung compliance (27±6 ml/cmh2o pre vs 19±5 post, p<0.05) and increase in dead space ventilation (20±4 pre vs 47±20 post, p<0.05). pxv curves of the respiratory system were affected by embolism, with shift of the loops to the right and consequent reduction in static compliance and pulmonary hysteresis. pathology depicted inflammatory neutrophil infiltrates, alveolar edema, collapse and hemorrhagic infarctions. pulmonary microembolism induced by polystyrene microspheres is associated with cardiovascular dysfunction, as well as respiratory injury characterized by decrease in oxygenation, dynamic and static lung compliances and pulmonary hysteresis. pathology findings were similar to those verified in inflammatory-induced acute lung injury. the similarities between respiratory and histologic features of this model and those from conditions associated with lung inflammation suggest that pharmacologic and ventilatory interventions already used to treat acute lung injury may also be tested in pulmonary embolism. the presence of patent foramen ovale (pfo) is frequently underdiagnosed in icu patients suffering from refractory hypoxemia. however, it is relatively common in the general population. we examined the prevalence of pfo in mechanically ventilated icu patients with refractory hypoxemia and abnormal chest x-ray findings. over a period of five years, 24 mechanically ventilated patients with refractory hypoxemia and abnormal chest x-ray findings were examined with transesophageal echocardiography (tee) for the presence of pfo as a contributing factor to their hypoxemia (right to left intracardiac shunt). all patients were ventilated with tidal volume 6-8ml.kg -1 and peep between 5-10cmh2o. their mean pao2/fio2 ratio was 102±24 mmhg. the coexisting pathology consisted of: ards (12 cases), massive pulmonary embolism (3 cases), copd (3 cases), cabg surgery with rv infarction (4 cases), cerebrovascular accident (1 case) and pulmonary oedema due to fluid overload (1 case during a two-month period we investigated the possibility of opening of the foramen ovale during a recruitment maneuver in either patients with ards or in patients with atelectasis and a pao2/fio2 ratio<200. we enrolled 10 consecutive patients (ards: 6 cases, patients with atelectasis and hypoxemia: 4 cases), likely to benefit from a recruitment maneuver. mean pao2/fio2 ratio was 112 and mean compliance was 40 ml.cmh2o -1 prior to the maneuver. all data regarding the mechanical properties of the lung were recorded from the ventilators monitor screen. after deficits of intravascular volume had been addressed and hemodynamics had been optimized, a baseline transesophageal echocardiographic study using contrast material was performed to rule out the possibility of a foramen ovale already patent prior to the maneuver. the recruitment inflation pressure was chosen as the lesser of 45 cm h2o or the peak pressure at 12 ml.kg -1 tidal volume. the ventilator was then adjusted to deliver this high inflation pressure for 20 secs. five seconds after the onset of inflation, 20 ml of a contrast material were injected through a central venous line with the transesophageal probe already in place to detect the passage of the material to the left atrium. passage of the contrast material to the left side of the circulation was detected using two dimensional echocardiography. we found that the sustained high inflation pressure resulted in foramen ovale opening in 2 patients, whereas it did not produce such a result in 5 patients. in 3 of the 10 studied patients, the baseline transesophageal study revealed a patent foramen ovale before recruitment was attempted. no adverse effects following the recruitment maneuver were noted. mean pao2/fio2 ratio was 138 and mean compliance was 40 ml.cmh2o -1 twenty minutes after the recruitment maneuver, with only one of the recruited patients showing a significant improvement in oxygenation.conclusion. patent foramen ovale may be a contributing factor of refractory hypoxemia in icu patients. opening of the foramen ovale is not an unlikely event during a recruitment maneuver. acute respiratory distress syndrome (ards) remains a major problem in critically ill patients, with mortality rates of 40-50%. to date, no specific treatment has been shown to decrease mortality, but this may largely be due to the heterogeneity of the populations meeting the ards criteria.objectives: to evaluate patients who died with a clinical diagnosis of ards and who had a postmortem examination in order to: 1-define the pathological alterations associated with the syndrome, with particular reference to the typical pattern of diffuse alveolar damage (dad); 2-evaluate whether etiologies or precipitating factors were missed; and 3-speculate whether a lung biopsy could have guided the clinical management. three year (2002) (2003) (2004) review of all patients with ards (using the aecc criteria) who had a postmortem examination. comparisons between ante-and post-mortem diagnoses were classified as major and minor discrepancies using the goldman classification. results: of a total of 9184 admissions, 376 patients had a clinical diagnosis of ards. of these, 169 died; 69 had a postmortem examination and 64 of these had complete data for analysis. the main causes of death were multiple organ failure in 27 (42%) and refractory hypoxemia in 9 (14%). postmortem lung examination revealed dad in 32 (50%) patients (4 associated with a lung infection), (broncho)pneumonia without dad in 16 (25%), invasive pulmonary aspergillosis without dad in 5 (8%), and other diagnoses in 11 (17%). major unexpected findings were found in 15 (23%) patients, classified as 6 goldman class i errors and 9 class ii errors. the class i errors included 4 cases of invasive pulmonary aspergillosis.conclusion. ards as a syndrome, can be due to various pathological patterns; at autopsy, only half of patients with ards have typical dad. special attention should be paid to the possibility of aspergillosis; in this setting, lung biopsy may have a role. g. s. georgieva*, s. kurata, c. zhu, a. bilali, t. imai critical care medicine, tokyo medical and dental university, tokyo, japan development of efficient lung preservation method has been anticipated and we elucidated that positive pulmonary venous pressure (pvp) (5mmhg) prevented ischemia-reperfusion (i/r) injury in isolated mechanically ventilated rat lungs. the aim of this study is to determine whether cpap accompanied with 5 mmhg of pvp would be effective for prevention of i/r injury. after tracheostomy rats were ventilated at 70 strokes /min with air (5% c02) and with peep of 2.5 cmh20, cannulated to the left atrium and pulmonary arteries (pas), and perfused with krebs -henseleit solution supplemented with albumin (4%) (0.04 ml/g/min). the lungs and heart "en block" were isolated and placed in a chamber; right and left bronchus as well as pas were dissected which permit each lung to be ventilated and/or perfused selectively by selective occlusion of each bronchus and/or pa. after 30 min control condition, the left lung (ll) was maintained under cpap (selective occlusion of left broncus); the control right lung (rl) was ventilated with peak airway pressure of 5 cmh20 above peep;perfusion to the both lungs was stopped (ischemia). pulmonary venous outflow was elevated so as to be applied 5 mmhg to the left atrium during ischemia. after 60-min ischemia, reperfusion with 0 mmhg pvp and both lung normal ventilation were resumed for 30 min. perfusion pressures of rl and ll was measured at the beginning and at the end of the experiment by occlusion either the left or right pulmonary artery, as appropriate. albumin content in bronchoalveolar lavage fluid (balf) separately for each ll and rl, and lung weight were measured. protein content in balf was calculated as (mg of protein)/(ml of balf)/(g of lung dry weight). all the data were compared by wilcoxon's rank-sum or mann-whitney u-test and expressed as mean +/-sd. in i/r lung maintained at cpap, wet/dry and balf as well as perfusion pressure increased compared to the control rl. conclusion. cpap(2.5 cmh20) and 5 mmhg pvp cannot prevent ischemic lung injury despite constant distention of pulmonary vasculature and alveolar space. this suggests that gas exchange during ischemia would be necessary for escaping from i/r injury. potential peripheral airway obstruction is of importance for the choice of ventilatory strategy in acute lung injury (ali). use of a limited expiratory time counteracts early regional expiratory collapse but might cause hyperinflation in case of significant peripheral obstruction. the aim of this study was to assess regional expiratory time constants and gas trapping in early ali. ten anesthetized pigs were ventilated in volume-controlled mode with i:e ratios of either 1:3 or 3:1 at a rate of 15 breaths per minute. starting from the end-inspiratory level, sequential computed tomography (ct) exposures were performed during passive, uninterrupted expiration to the atmosphere. the procedure was performed before and after oleic acid-induced lung injury (oai) had been induced in the lower lobe on one side. the gas volume of bilateral dependent and non-dependent regions of interest (rois) was calculated from radiographical attenuation values. the expiratory time constant was calculated from a mono-exponential decay of roi gas volumes during expiration. gas trapping in injured and non-injured regions were compared. during ventilation with i:e ratio 1:3, oai caused overall compliance to decrease from 31 +/-5.5 to 27 +/-4.4 ml/cmh2o (p<0.05). dependent, injured regions showed a shorter time constant and a lower volume of gas than dependent non-injured regions regardless of whether the preceding end-inspiratory volume had been increased or not by application of a limited expiratory time. in non-dependent, non-injured regions, the gas volume was similar on both sides after both patterns of ventilation. one of the additional approaches in the therapy of the acute respiratory distress syndrome (ards) is the use of a pumpless arteriovenous extracorporeal membrane oxygenator (interventional lung assist (ila)). the aim of our study was to test the effects of an ila system on hemodynamics and gas exchange during resuscitation and to establish whether ila should be kept open or clamped under these circumstances. the study was designed as a prospective experimental study. the experiments were performed on 12 pigs (41 to 58 kg body weight). the pigs were anesthetized and mechanically ventilated. one femoral artery and one femoral vein were cannulated and connected with ila. acute lung injury was induced by repeated bronchoalveolar lavage until arterial partial pressure of oxygen (pao2) was lower than 100 torr for at least 30 min during ventilation with 100% o2. ventricular fibrillation was then induced by an indwelling pacemaker. manual compressions of the thorax were started at once and continued for 30 minutes. in 6 animals, ila was kept open, in the other 6 it was clamped immediately. statistical analysis was performed using graphpad prism. two-way analysis of variance was applied and significance was accepted at p values < 0.05. the data is given as mean ± sd. with a mean systolic arterial pressure in the group with ila open of 76 ± 28 mm hg and 78 ± 23 mm hg with ila clamped and mean blood pressures of 23 ± 5 mm hg with ila open and 25 ± 5 mm hg with ila clamped the blood pressure did not differ between the two groups. endtidal carbon dioxide decreased from 46 ± 23 torr with ila open and 37 ± 9 torr before intervention to 8 ± 5 torr and 8 ± 10 torr, respectively. the arterial partial pressure of carbon dioxide (paco2) was significantly lower in the group with the ila system open (31 ± 25 mm hg versus 80 ± 25 mm hg at 20 minutes) and the pao2 was higher (although significant only at 20 minutes, 191 mm hg ± 140 mm hg versus 57 mm hg ± 14 mm hg). the blood pressure generated with thorax compressions did not differ significantly between the two groups and endtidal co2 was also in the same range. therefore we assume that circulation was not significantly affected by ila and that the shunt caused by the ila system did not deteriorate circulation. paco2 was significantly lower in the group with the ila system open and pao2 was higher. our results indicate that the ila system was not harmful during resuscitation, it even might have a beneficial effect.grant acknowledgement. the study was partially supported by novalung, hechingen, germany. respiratory failure -miscellaneous 0317-0330 increased thorax rigidity and high intraabdominal pressure reduce the stretch ability of the thoracic cage and modify the regional lung function. this phenomenon is often seen in intensive care patients, e.g. with abdominal compartment syndrome. objective of this study was to determine the effect of decreased thoracic cage compliance on regional distribution of spontaneous ventilation in different postures by the non-invasive method of electrical impedance tomography (eit). for this survey we examined ten healthy male spontaneously breathing volunteers (mean age ± sd: 28 ± 3 years; body weight: 81 ± 10 kg, height: 183 ± 9 cm). the compliance of the thoracic cage was restricted by external abdominal and thoracic corsets respectively. the eit examinations were performed with the goe-mf ii eit device (viasys healthcare, höchberg, germany). sixteen self-adhesive electrodes (3m red dot 2239, 3m health care, borken, germany) were applied on the chest circumference in one transverse plane and used for rotating electrical current injection and voltage measurement. the eit data were acquired at a rate of 25 scans/s. impedance data and spirometry were obtained during spontaneous ventilation in three body positions (sitting, left and right side). statistical analysis was performed using repeated anova with bonferroni's multiple comparison test and student's t test. p values <0.05 were considered significant.results. the regional distribution of ventilation in subjects without restrictions revealed a close match with physiologically expected values. thoracic and abdominal restrictions led to reduction of ventilation in the dependent lung areas. the non-dependent lung areas were not affected. the fractional ventilation in the dependent lung areas was reduced in the right side position from 69.3 ± 15.4% to 57.2 ± 9.8% (thoracic restrictions) and 55.7 ± 9.9% (abdominal restrictions), in the left side position from 55.3 ± 11.4% to 36.4 ± 8.4%, and 36.9 ± 8.7%. thoracic and abdominal restrictions of the thoracic cage reduce ventilation only in the dependent lung regions in spontaneously breathing healthy volunteers. eit is a suitable method for non-invasive determination of regional lung ventilation. k. raymondos* 1 , k. vieweger 1 , j. ahrens 1 , m. przemeck 2 , m. homann 3 , s. piepenbrock 1 1 anaesthesiology, medical school hannover, 2 anaesthesiology, annastift, 3 johanniter-unfall-hilfe e.v., ortsverband wasserturm, hannover, germany germany are still performed with ambulances in that only limited monitoring and usually only volume-cycled emergency ventilators can be used. we established an intensive care ambulance system and evaluated the transfers of critically ill patients performed with this system. we prospectively recorded 1847 interhospital-transfers. the ventilatory modes before and during the patients' transfer and further characteristics of the interhospital-transfers were evaluated. transport ventilation was performed with the raphael ® silver ventilator (hamilton medical ag, rhäzüns, switzerland) with that also pressure-support ventilation (psv), airway pressure release ventilation (duopap ® /aprv) and the combination of both could be used. indications for the interhospital-transfers included ischemic (28.1%) and other (6.7%) cardiac diseases, cerebral diseases (24.7%) of which 73% required neurosurgy, pulmonary disease (8.4%) and others (32.1%). 605 (32.8%)% of the transferred patients received ventilatory support, 712 patients (38.6%) breathed spontaneously with and 529 patients (28.7%) without oxygen insufflation. the majority of the mechanically ventilated patients received ventilatory modes supporting spontaneous breathing before (74.4%) and during the transfer (80.6%). the patients were transferred in 55 minutes (5 minutes -11 hours) over a distance of 60 km (2-970 km) (median (range)). at least 2 motor syringe pumps were needed during the transfer of 642 patients (34.8%). monitoring during the transfer was similar or more extended compared to the monitoring in the hospital prior to transfer (ecg 93% vs. 85%, pulse oximetry 89% vs. 70%, non-invasive blood pressure 73% vs 65%, intraarterial pressure 24% vs 22% and capnography 21% vs. 9%). most ventilated patients received weaning techniques and most of these ventilatory modes were continued during the transfer. these ventilatory modes and a more extended monitoring including intraarterial pressure monitoring and capnography cannot be applied in emergency ambulances. the less invasive ventilatory modes and the extended monitoring enable a less invasive and safer interhospital-transfer as the intensive care treatment and monitoring prior to transfer is maintained or even extended during the transport. a. sánchez*, m. palomar, r. alcaraz, a. socias, d. moreira intensive care unit, hg vall d'hebron, barcelona, spain introduction. some series have shown the bad prognosis of patients with pulmonary fibrosis (pf) who require admittance at icu for respiratory failure. there are doubts of the benefit of the ventilatory support if the precipitating cause is not well defined. lung transplant (lt) could be a therapeutical option. the aim of this study was to analyze the prognosis of the patients with pf who are admitted to an icu of a hospital with lt program.methods. case-series, observational study of patients with pf and acute respiratory failure admitted to the icu of a third level hospital with lt programm between january 1998 until june 2006. information about the cause of pf, clinical course, current status, ventilatory support, length of stay, pulmonary functional tests, possibility of trasplantation, complications and mortality was collected. . 22 patients (15 men, 7 women) with pf (14 idiopathic pf, 6 connectivopaty and 2 due to radiotherapy) were admitted for acute respiratory failure (arf) to our icu. mean age was 54,5 (21-65) years. the median duration of illness from diagnosis until admittance was 1,5 (0-17) years. apache-ii score was 15 (6-27). the precipitating cause of arf was identified in 18 patients: bacterial pneumonia was documented in 5 patients; 1 had a pulmonary embolism; 1 fungic infection and 11 cases were due to the progression of the disease. in 4 cases the precipitating cause could not be identified. mechanical ventilation (mv) was required by 18 patients (81,8%) during an average of 11,5 (1-51) days with a mortality rate of 77,7%. pa o2 /fi o2 at admittance 77 (40-260) mm hg; and paco2 at admittance 54 (31-122) mm hg. respiratory functional studies were available in eleven patients with a fev1 of 1.34 (0.52) l and fvc of 1.65 (0.77) l. 16 patients (72%) died during their stay at icu. the cause of death was multi-organic failure in 7 (31.8%); refractary hypoxemia in 6 (27.3%) patients and 3 of them died while the transplantation was being performed. mean length of stay was 14 (1-56) days. 12 patients were included in the urgent lt list and 10 were transplanted. no donor was found in 2 cases and died on the waiting list. there were performed 2 single-lung and 8 double-lt. mean age was 48 (21-65) years. the time from the admittance until transplantation was 6 (3-19) days. 9 of them (75%) required mv with a mortality rate of 66,7%. from this group 8 (66,7%) patients died during their stay at the icu. 3 of the patients died while the transplantation was being performed.conclusion. literature shows a bad prognosis of patients with pf who need admittance to an icu for arf. in our experience the survival was 33% so the existance of a lt programm could offer a chance to these patients. m. e. lugarinho*, p. p. souza intensive care unit, hospital de clinicas mario lioni, rio de janeiro, brazil introduction. acute kidney insufficiency (aki) worsens the outcome in critical ill patients. we investigate whether the presence of aki had any effect on lenght of mechanical ventilation and mortality rate. observational, prospective study in a 12-bed general intensive care unit (icu) from january to december 2006. the inclusion criterion was invasive mechanical ventilation for more than 48 hours. aki was defined as the presence of dialysis during the icu stay. patients were then separated into aki and non-aki patients (control group). the primary end point was duration of total length of mechanical ventilation and the secondary end point was the icu mortality. a total of 114 patients were studied: 28 with aki and 86 non-aki. the groups were similar in regard to age, sex, and apache ii score. the median (interquartile range) duration of mechanical ventilation 15 [6-22] versus 5 [2] [3] [4] [5] [6] [7] [8] [9] [10] days, (p<0,005). the icu mortality rate were significantly greater in the aki patients: 75% versus 56,4% (p<0,005).conclusion. this study shows that renal insufficiency has serious impact on the duration of mechanical ventilation and morbi-mortality in critically ill patient. these data elicits the poor outcomes of mechanical ventilated patients who demands for dialytic methods. it will be useful in end of life discussions and decisions in our icu. introduction. 5-ht1a-r-agonist 8-oh-dpat has been shown to counteract morphine induced ventilatory depression, while opiate antinociception remained unaffected. repinotan-hcl, another 5-ht1a-r-agonist, is unlike 8-oh-dpat suitable for the use in humans. it was hypothesized that repinotan-hcl is capable to antagonize ventilatory depression without impairing anti-nociception in rat. with approval from local animal care committee, 12 rats were anesthetized with sevoflurane and tracheotomized to record respiratory rate (rr), tidal volume (vt) minute ventilation (mv). inguinal vessels were catheterized to monitor arterial blood pressure and apply drugs iv. nociception was assessed by tail-flick reflex. morphine was administered at increments of 5mg/kg until a target 70% reduction of rr was achieved. subsequently, repinotan-hcl was added cumulatively at increasing doses (0.02, 0.2, 2, 20, 200 µg/kg, n=6). another group received nacl 0.9% to serve as control (n=6). morphine (12.5±1.8mg/kg) depressed rr to -81±4%, and tfr was abolished with first dose of morphine in any experiment. repinotan-hcl antagonized ventilatory depression dose-dependently, 20mcg/kg repinotan-hcl re-established ventilation almost at pretreatment level (rr +1.9±14%, p<0.001, 2-anova, compared to control). tfr remained absent throughout repinotan administration. repinotan functionally antagonized morphine-induced ventilatory depression, while suppression of nociceptive reflex sustained. 5-ht1a-r-agonists such as repinotan-hcl appear to be promising candidates to stabilize spontaneous breathing. a. makowski* 1 , b. misztal 1 , c. plowright 1 , k. safranow 2 1 anaesthetics, medway maritime hospital, gillingham, united kingdom, 2 biochemistry, pomeranian medical university, szczecin, poland vapotherm's (vap) patent pending membrane technology makes higher flows from 1 to 40 lpm possible by saturating breathing gases with water vapor at body temperature. fio2 is ranging from 0.21-1.0. heat and humidity allow nasal flow to be well tolerated by the patients. high flow in animal study caused small amount of peep. can we achieve desired therapeutic goal in treatment of respiratory failure (rf) with this very simple, non-invasive method? we investigated effectiveness and hospital outcome of patients with rf treated on vap at surgical hdu between december 2005 and march 2007. data were taken during retrospective investigations. we analysed type and reason of rf as well as respiratory rate (rr), fio2, flow, arterial blood gases (abg). data were collected before (bef) vap was commenced, 1 hour after, and every day of treatment. we also recorded length and outcome of vap therapy and patient satisfaction. data were analysed with wilcoxone and also spearman's rank correlation tests. the 55 patients (49% female, 51% male) at age 33-93 (69.81±14.55) were treated 1-16 (4.5±3.13) days. we applied vap therapy for 75.92% patients with type i rf and 24.07% with type ii rf. the reasons of rf were pneumonia in 45.45%, sepsis in 21.81% pulmonary oedema in 14.5%, copd in 9.08%, others in 9.16%. for 81.82% patients there was a sufficient and definite treatment whereas 18.18% required mechanical ventilation and icu admission. the 87.27% of patients were satisfied with therapy. the 81.82% survived and were discharged from the hospital. high flow and small amount of peep reduce work of breathing and significantly decrease rr. after effective vapotherm therapy we observed in abg significant increase of oxygen saturation and pao2. vast majority of patients were satisfied during the treatment. in critically ill patients who need long-term mechanical ventilation, early tracheostomy may facilitate weaning and shorten the length of stay in intensive care (1). however, there are no clinical tests that identify patients as being at an increased risk for prolonged ventilatory support; clinicians must predict the duration of arteficial ventilation by their clinical experience. in our surgical intensive care unit we conducted a prospective clinical study to determine if there was an association between different clinical parameters (age, body mass index, gcs, saps2 score, vasopressor use, pao2/fio2 ratio) and long-term mechanical ventilation. furthermore, we examined the positive predictive value of clinicians' prediction; to do that, clinicians had to indicate whether they considered prolonged mechanical ventilation as the most likely (but not always certain) outcome or not. we enrolled 50 patients and collected date on days 0-4th and 7th of treatment. prolonged meshanical ventilation was defined as at least 7 more days on respirator. none of the examined parameters could be used alone to predict long-term mechanical ventilation. overall sensitivity of clinicians' prediction was 72.7 %, and positive predictive value was 46.1 %. 27.3 % of patients died, 26.6 % was weaned from respirator (6.2 % extubated) within 7 days despite predicted by clinicians as having prolonged ventilatory need. suprisingly, the best positive predictive value (64.0 %) was found on the day of admission, the worst (25.0 %) on day 7; the difference was not significant (p=0.17 with chi-square test). this result could be explained by the fact that most patients in the study group were ventilated on day 7, but only a few on day 14.conclusion. prediction of prolonged mechanical ventilation was found to be very inaccurate, and did not improve in the course of first week of treatment. however, in our department where many neurosurgical patients are treated, only a minority could be extubated within 7 days when long-term ventilatory support was predicted. as selection of patients who need tracheostomy seems not to be better after one week of treatment than at an early stage, there can be a reason for early tracheostomy if we anticipate prolonged arteficial ventilation. n. abidi 1 , h. thabet* 2 , o. béji 1 , h. elghord 1 , n. brahmi 1 , m. ben othmen 2 , n. kouraichi 1 , m. amamou 1 1 intensive care medicine, 2 emergency medicine, centre d'assistance médicale urgente, tunis, tunisia introduction. acute exacerbation of copd is a frequent cause of admission in icu and usually have a poor outcome. such a patient consume a large amount of resources particulary if they need endotracheal intubation. the aim of this study is to report epidemiological, clinical features,treatment and outcome of patients admitted in icu for acute exacerbation of copd. a retrospective study was carried out of consecutive admisions in icu over a 7 years (from january 1999 to december 2005). american thoracic society criteria are usued to define copd. exacerbation is defined as a worsening of copd symptoms. a total of 144 patients were included in this study with 164 episodes of acute exacerbation. mean age was 66±10,5 years. the sex ratio was 2,64 (m/f: 119/45). eighty percent were current tobacco users. seventy two percent had one or more associated comorbities mainly cardiovascular disease. according to copd severity 53,7% of patients were in stage iii. 25,6% were receiving home oxygen and 53 (32%) were previously mechanical ventilated. on icu admission severity score are apache ii 21±8; igsii 44±19. 51 patients (31%) have a shock and 39 (23,8%) have a coma (gcs<9). treatment consist of starting non invasive ventilation (niv) for 49 patients (30%); 82 patients (50%) need immediate intubation and mechanical ventilation. failure of niv was noted for 27 patients. in the course of hospitalisation in icu main complications were: nosocomial infection for 57 patients (34,8%), barotrauma 10 patients (6,1%) and thromboembolic complications for 3 patients (1,8%). the median icu stay was 17,6 ± 23,5 days and mortality was 36,6% (60 patients). the main cause of mortality were septic shock (37 cases, 61,7%) and ards (9 cases, 15%). in this retrospective study patients admitted for exacerbation of copd need a mechanical ventilation in 66,5%. failure of niv were 55%. main complications were nosocomial infection (34,8% of cases). mortality is high 36,6% but not different for patients admitted in icu for other disease. it is described, that gelatin leads to red blood cell (rbc)-coating, which is protective against shear stress in extracorporeal circuits. (1) an increase of mean corpuscular volume (mcv) without an increase in mean corpuscular hemoglobin content as well as a reduction of red blood cell (rbc) counts can be assumed to reduce pulmonary oxygen transfer. increased rbc aggregability (accelerated blood sedimentation rate, bsr), as could occur due to coating, impairs microcirculation. since adequate oxygen delivery is important in ventilated patients to counteract metabolic acidosis, we compared rbc features in acidotic pigs undergoing hemofiltration. healthy pigs (male, dlxde, 37-46kg) were anesthetized, received acid infusion (0.4 m) and low tidal ventilation with fio2 > 0.9 resulting in normoxic acidosis (ph 7.19-2.4; paco2 80-85 mmhg). tris-hydroxymethylaminomethane (tham) was infused to titrate a ph of 7.19-7.24. either hes 130 or gel (n=6-7/colloid-group) was infused additionally to crystalloids (colloid to crystalloid ratio was 1:4). samples were collected before acid and colloid infusion (bs), after induction of acidosis (baseline acidosis, bsa), and after 3h of continuous acidosis (3ha). thereafter, acid infusion was stopped and tham was infused with 2.1 mol/kg/h for 2h in order to normalize ph-values. final values (fv) were taken. parameters investigated were: paco2, rbc counts, mcv, and bsr. the fio2/pao2 ratio was also determined. compared to hes 130 application, gel infusion was associated with a reduction in rbc count, an increase in mcv and an accelerated bsr from bsa until fv. values did not recover from initial deterioration (bsa) even not after normalization of ph (fv). based on the healthy lungs in this porcine model, these changes did not impair pao2/fio2 ratio. whether increases in mcv were due to gel coating or due to unhampered swelling of rbcs during acidosis could not determined. however, in acidotic pigs gel induced unfavorable effects concerning rbc features with respect to rheology while hes 130 did not. in individuals with impaired pulmonary function and hypodynamic state the described difference between the two types of colloids could become crucial with respect to total oxygen delivery. perctaneous dilational tracheostomy (pdt) has become more common procedure used in intensive care. however, several complications, such as hemorrhage, posterior tracheal wall injury, tracheal stenosis have been recently reported. the aim of this study was to confirm whether the ultrasound can easily and clearly delineate the pretracheal anatomy and identify the potential problems for pdt. we also examined the accuracy in identifying the correct puncture level between 2 and 3 tracheal cartilages blindly (by hand). we studied 10 patients and 40 volunteers. before ultrasound scanning, the circumference of the neck was measured and the puncture level between 2 and 3 tracheal cartilages was marked blindly in each subject. in ultrasound scanning, we examined the relationship of the thyroid to the trachea, aberrant vascular anatomy in the pretracheal region, counted the number of extrathoracic tracheal rings. the distances from the skin to cricothyroid ligament and anterior tracheal wall at the level between 2 and 3 tracheal cartilages were estimated and the relationship between depth of trachea and circumference of the neck was analyzed by simple regression. we also checked the level of trachea pointed by operator blindly was correct or not by comparing the level identified by ultrasound images. the mean age and circumference of the neck were 39±17 years (range: 20-79) and 36±4 cm. ultrasound examination of the trachea and thyroid was easily carried out in each subject except 2 subjects. approximately 6 extrathoracic tracheal rings could be imaged with ultrasound. anterior jugular veins were seen in 19 subjects (38%) and six were near the midline. the depth of trachea between 2 and 3 tracheal cartilages were varied in each subject (0.44-3.58cm) and there were stastistically relatioship between circumference of the neck and depth of trachea (r2=0.33, p=0.0047). the accurate decision of trachea level was made in 88% of the subjects.conclusion. this study showed that: 1) ultrasound can delineate the neck structure and detect variations related to the complication of pdt; 2) blind identification of the puncture level for tracheostomy without ultrasound was not necessarily correct. our results demonstrated that the routine use of ultrasound could be recommended before pdt. introduction. fluid therapy system of critically ill patients is very variable, and it is based in the interpreting of differents physiologic parameters with a double aim, by one hand keep an adequate perfusion of vital organs, and the other hand avoid overload volumen. our objective was analyze changes in critically ill patients fluid therapy when we including evlw in treatment protocol and evaluate response in short time. observational and prospective study in a neurotraumatological icu. we included consecutives patients that were admited with acute lung injury/adult respiratory distress syndrome and/or septic patients who needed monitoring with central venous and arterial catheterization with picco system. we made a therapeutic reassessment of the fluid therapy and/or vasoactives after we knew evlw when one of the following events in the patient evolution hapenned: hypoxemia, hypotension, olyguria/anuria, or its addition. response in short time was also evaluated. our sample included 40 patients and 52 determinations( 7 patients with 2 determinations, 1 patient with 4 determination and 1 patient with 3 determination). after we knew evlw we changed initial therapeutic plan in 55.8 %; this change affected fluids in 96.6 % and vasoactives in 30.4 %. evlw in patients who therapeutic plan was modified was 13.76 ± 4.98 and if therapeutic plan was not modified, evlw was 9.87 ± 4.13 (p< 0.05). association is observed between evlw value and decision about fluids, so when we decided increase fluids was 7.17 ± 1.47; if the decision was decrease fluid, evlw was 12.20 ± 2.77 and in the cases that diuretics were added 16.36 ± 3.99, in all cases statistics significant was found. no differences was observed in evlw values about vasoactives decision. we found improvement of initial event in short time after intervention in 67.3 %.conclusion. evlw determination affects in important way to fluids therapy plan in critically ill patients. we think that inclusion of evlw contributes to a more racional management of these patients. patients who had received ino were identified from icnarc records. hospital notes and icu charts were reviewed. data collected included diagnosis, apache ii and unit and hospital outcome. the pao2/fio2 ratio (in mmhg) was recorded prior to starting ino (day 0) and subsequently on days 1-4 using the data from the time at which oxygenation was best in each 24 hour period. . 29 patients received ino. 28 patients received it for treatment of hypoxaemic respiratory failure, and 1 for treatment of pulmonary hypertension. mean apache score was 19.2 on admission (survivors 14.4; non-survivors 22) . the mean pao2/fio2 ratio was 67.6 on day 0 and improved to 135.5 on day 1. in unit survivors, the mean pao2/fio2 increased from 62.9 to 168.3 on day 1, compared with unit non-survivors in whom it increased from 70.2 to 116.6. 19 (66%) of patients were responders to ino (defined as a >20% increase in pao2/fio2 ratio). unit and hospital survival figures for responders and non-responders are presented below. hospital surviviors (n=9) hospital non-survivors (n=20) responder (n=19) 8 (42%) 11 (58%) non-responder (n=10) 1 (10%) 9 (90%) fisher's exact test (2 tailed) p=0.1 conclusion. ino was used in patients with more severe hypoxia than those included in randomised trials. (2) in this review, responders were found to have a significantly reduced unit mortality and a reduced hospital mortality compared with non-responders. we believe ino may be a valuable therapy in ards patients with severe refractory hypoxaemia, and that studies in this subgroup of patients are warranted. outcome predictors of hfov in severe ards are not well studied. we prospectively evaluated the outcome predictors of hfov in adult ards. methods. ards patients receiving mechanical ventilation as per the ardsnet protocol with po2/fio2≤150 inspite of peep≥12cm and fio2≥0.7,were considered for hfov. continuous distending pressure(cdp),frequency ,amplitude, inspiratory time and bias flow of hfov were optimised with the help of frequent blood gas analysis. weaning from hfov to pressure support ventilation was attempted once po2/fio2 ratio remained ≥200 with cdp≤18 cm &fio2 ≤0.5. responders(r) were defined as patients who were successfully weaned to a state which required no ventilatory support for > 12 hrs. non responders(nr)were defined as patients who could not be weaned off ventilatory assistance. results. 17 out of total 31 patients were r & 14 were nr. both the groups were similar prior to hfov as shown in table. improvement in po2/fio2 ratio and oxygenation index (oi) at 6hrs &24 hrs in r group was statistically significant as compared to that in nr group. we could show that chaotic variation of pressure support improves pressure support ventilation (psv), and named this new mode noisy psv. in this work, we compared noisy psv to conventional biphasic positive airway pressure ventilation (bipap), which has been claimed to be a "gold standard", in experimental acute lung injury. after approval by the local animal care committee, 18 juvenile pigs (22.5-30.7 kg) were anesthetized and mechanically ventilated (dräger evita xl 4lab; volume controlled ventilation, vt = 10 ml/kg; fio2 = 1.0; peep = 5 cmh2o). after induction of acute lung injury by saline lung lavage (30 ml/kg), lungs were recruited and a decremental peep trial was performed to determine the optimal peep according to the elastance of the respiratory system (ers). thereafter, spontaneous breathing was resumed and animals were randomly assigned to noisy psv or bipap groups (n=9 each group). the ventilator settings were as follows -bipap: fio2 = 1.0; plow = according to peep of minimal ers; phigh = titrated to generate vt of 6 ml/kg; thigh = 2s; tlow = 2s -noisy psv: fio2 = 1.0; peep = according to peep of minimal ers; mean pasb = titrated to generate vt of 6 ml/kg. noisy psv was accomplished by means of remote control of the evita xl 4lab by a laptop, which generated a sequence of 600 respiratory cycles with different pressure support levels (mean = pasb; sd = 30 % of mean). gas exchange, respiratory parameters and hemodynamics were measured at baseline, injury, after resuming of spontaneous breathing (baseline 2) and during an observational period of 4h. statistical analysis was performed with general linear model statistics adjusted for repeated measures using baseline 2 as covariate. significance was accepted at p<0.05. bodyweight, peep and number of lavages as well as hemodynamics did not differ significantly between groups. oxygenation and co2 elimination were significantly improved with noisy psv (p<0.05 both). analysis of respiratory parameters revealed significant lower mean airway pressures with noisy psv as compared to bipap (p<0.05), as well as increased mean peak airway pressure, spontaneous respiratory rate, and mean tidal volume (p<0.05 all).conclusion. this study represents the first evaluation of the recently developed noisy psv combined with peep levels titrated according to lowest ers. noisy psv was found superior to conventional bipap with regard to gas exchange and respiratory parameters. further experimental studies are necessary to determine the potential role of noisy psv in intensive care therapy. we investigated if chaotic variation of pressure support (noise) can improve the performance of pressure support ventilation (psv) in experimental acute lung injury (ali). with approval of the local animal care committee, 12 pigs weighing 25 to 30 kg were anesthetized, intubated and mechanically ventilated (volume-controlled mode, fio2=0.5, peep=5 cmh2o, tidal volume=12 ml/kg). following that, ali was induced by surfactant depletion, and biphasic intermittent positive airway pressure (bipap) was initiated with: lower cpap (cpaplow) = 5 cmh2o, higher cpap (cpaphigh) titrated to obtain tidal volumes of 6-8 ml/kg, respiratory rate set to obtain paco2 between 50-60 mmhg. then, depth of anesthesia was decreased to allow spontaneous breathing, and animals were ventilated with two different modes (1 hour each, random sequence): 1) traditional psv, with pressure support level set at cpaphigh -cpaplow; 2) noisy psv, with random variation of pressure support and mean value set at cpaphigh -cpaplow, and standard deviation set at 30 % of the mean value (normal distribution). gas exchange, inspiratory drive (p0.1) and inspiratory pressure time product of esophageal pressure (ptp) were assessed. helical computed tomography (ct) of chest was performed at end-expiration and the hyperaerated, normally aerated, hypoaerated and non-aerated lung compartments were calculated in 8 animals. patients with respiratory failure treated with vm with fio2 0.5 were included. after 30 minutes of oxygen therapy, arterial blood gases were collected and patients were asked to quantify (from 0 to 10) three items: dyspnea, dry mouth and general confort. then, vm was changed for hfnc (optiflowtm, fisher & paykel, new zeland) . the same variables were collected after 30 minutes using hfnc. results are expressed as median (interquartil range). we have applied spsswin v13.0 with wilcoxon test. patients n=10 (4 m), age 54 (33-64). in the moment of inclusion, one patient (10%) presented mods and sofa score was 4 (2.5-5.5). during their evolution, five patients (50%) finally need endotracheal intubation. main results are presented in the following tables: a computer-driven system (cds) has been recently used to optimise psv to patient's needs during weaning. in some pts, the cds fail to find a "comfort window" despite stepwise increase in pressure support (ps) levels. for these pts, cds could further increase respiratory muscle workload. we speculate that failure to adapt respiratory rates (rr) and vt following changes in ps levels might identify a subset of pts unlikely to benefit from the cds.to test this hypothesis, we used a bedside test before switching ventilated pts to a closed-loop algorithm of psv. we studied 23 pts at initiation of weaning with psv using the smallest ps level resulting in rr≤30, vt>5 ml/kg. we collected baseline values and assessed changes in vt (dvt), rr (drr) during 5 min after 5 cmh2o-increase and decrease in ps levels. then, a cds session was started at the baseline ps level. we searched for correlations between dvt, drr, and outcome (failure/success) of the cds sessions. a cds session was deemed successful when the system detected criteria for separation of the ventilator or when psv was efficiently adjusted by the cds within 48 h after starting the session. in pressure support ventilation auto-peep is considered a major contributor to the inspiratory work of breathing. measurement of auto-peep requires esophageal pressure tracings, which are not routinely available. the presence of auto-peep is likely, when flow is interrupted at end-expiration, a pattern well-established in controlled ventilation. we studied expiratory flow-volume relationships as substitute for detection of auto-peep in patients on pressure support ventilation. in 22 patients successively admitted to our icu respiratory mechanics were obtained from 5 consecutive breaths on pressure support ventilation. auto-peep was considered present when in flow-versus-time recordings flow was interrupted at end-expiration. from flow-volume relationships expiratory time-constants were calculated and related to actual expiration times. all measurements were obtained with a nico-computer; for analysis a computer program analysis plus was used (both respironics/novametrix, inc.). in 7 of the 22 patients flow at end-expiration was interrupted suggesting the presence of auto-peep (interrupted flow group). in the remaining patients flow was zero at end-expiration (zero flow group). in the flow-volume curves of patients in the interrupted flow group versus the zero flow group end-expiratory flows varied between 0.14 -0.33 l/s and 0.04 -0.10 l/s respectively. the expiratory time-constants ranged from 1.1 -1.6 s in the interrupted flow group and 0.5 -1.0 s in the zero flow group. the ratios between expiration times and expiratory time-constants varied between 0.8 -1.5 and 1.9 -4.1 for the interrupted and zero flow groups respectively . the means and standard deviations for both groups were:means +/-sd in patients on pressure support ventilation with interrupted flows at endexpiration higher expiratory time-constants and lower ratios between expiration times and time-constants were found, suggesting the presence of auto-peep. these variables can be used as substitute for detection of auto-peep. non invasive ventilation (niv) is the delivery of assisted mechanical ventilation to the lungs, without the use of an invasive endotracheal airway. niv has decreased the need for invasive mechanical ventilation and its attendant complications. acute cardiogenic pulmonary edema (acpe) is defined as an episode of acute heart failure accompanied by severe respiratory distress and oxygen saturation <90% on room air before all treatment. our study aimed to asses the respiratory effects of a device that delivers a continous positive airway pressure via face mask in patients with severe acpe, the feasibility of using this technique in an emergency department (ed) and estimed the need of endotracheal intubation (ei). we evaluated a series of 20 patients consecutively treated in our ed for acpe, from june 2006 to december 2006. a peep level of 10 cm h2o delivered by cpap-boussignac device (vygon, ecouen, france) was used in all patients. fio2 was estimed to range from 60 to 90 %. clinical and blood gas parameters were recorded at entry and also after 30 minute and 1 hour of treatment. all patients were treated with standard medical therapy. the average of age was 67 years (52-82), 13 were male and 7 were female. the inclusion criteria for niv were: ph <7,35 but >7,10, paco2 >45mmhg or an acute augment of 15-20 mmhg, respiratory rate >25/min, pao2/fio2 <250mmhg on room air and score kelly max 3. resolution of respiratory distress occurred from 30 to 50 minute ( media 40 minute). all patients showed an improve of clinical and emogasanalytic impairment. only 5 patients needed ei and were transferred in icu. 15 patients were treated in ed and after normalization and stabilization of their vital signs they were discharged in other medical departments (10 cardiology department and 5 pneumology department). the rate of ei was 25%.conclusion. cpap delivered using boussignac device is feasible in an emergency care setting. it can quickly improve respiratory distress in acpe patients and reduce the need of ei. in clinical practice niv is being used as a sole respiratory support modality or in the weaning period in at least 50% of arf patients admitted to emergency department. the remaining patiens need imv as primary and secondary forms of respiratory support. failure of niv seems to predict higher mortality rates. as a conclusion we need both support modalities and the physician has to use them carefully according to patients condition and their expertise. methods. medline, pubmed, cochrane, & cinahl databases (1982 to 2006 were searched using the terms: aprv, bipap, bilevel & lung protective strategy, individually and in combination. reference lists of identified papers were also examined. two independent reviewers determined eligibility of papers based on predefined criteria. database searching yielded 501 citations, of which 81 were selected on review of title and abstract. data were abstracted onto pre-designed forms from 51 experimental studies and 19 discussion articles on further review. of the 51 experimental studies, 31 used a randomised design, 9 were cohort studies and 11 case series. aprv was the named mode in 39 (76%) studies, bipap in 11 (22%), and inverse mandatory pressure release ventilation in one study. extreme inverse inspiratory:expiratory (i:e) ratio was used in 18 (46%) aprv compared to 0 bipap studies (p =0.001); 8 (20%) aprv and 1(9%) bipap studies used mild inverse ratio (up to 2:1). a 1:1 ratio was used more often with bipap (7, 64% vs 12, 31%, p =0.06) as was a normal i:e ratio (3, 27% vs 2, 5%, p =0.04). in adult studies, mean inspiratory pressure was 24cmh2o (aprv) and 18cmh2o (bipap) (p=0.3). mean expiratory pressure was 5.5cmh2o for both modes (p=0.9). seven aprv studies described synchronisation, 3 (43%) stated the mode did not synchronise to patient effort. all 4 bipap studies that described synchronisation stated it was available.conclusion. aprv assumes inverse ratio ventilation (irv). some studies advocate extreme irv with short release times to improve gas exchange, haemodynamic stability, renal and splanchnic blood flow(1). extreme irv was used in only 46% of aprv studies, 31% described an i:e ratio of 1:1. further, ventilator settings used for studies of aprv may be indistinguishable from bipap studies (2, 3) . given the variation in ventilatory settings described, uncertainty of optimal settings may exist. commercial ventilator branding may further add to confusion. generic naming of ventilatory modes, as with drug prescribing, combined with consistent definitions of the parameters that define the modes, may avoid confusion, improve consistency of patient response and assist the implementation of these modes into clinical practice. pav is intended to normalize neuro-ventilatry coupling by assisting each breath in proportion to patient effort, but requires reliable measurements of elastance (e) and resistance (r). pav+ allows to (a) automatically and non invasively measure e and r, and (b) continuously adjust ventilatory support accordingly. aim of our study was to test the physiological effects of pav+ versus cmv (ardsnet lung protective strategy) in a model of ards. in 12 pigs ards was induced through chloridric acid inhalation (4 ml/kg). at t0 (after damage) each pig was randomly assigned to pav+ or cmv. gas exchange and lung ct scan at 6 (t6) hours were compared with those obtained at t0 (delta = t6-t0). data are mean +/-standard deviation; *) p < 0.05 pav+ versus cmv cmv pav+ ∆ hyperinflated areas (cm2) 1 +/-1 3 +/-8 ∆ normally aerated areas (cm2) -28 +/-46 468 +/-186 * ∆ poorly aerated areas (cm2) 1 +/-4 48 +/-195 * ∆ nonaerated areas (cm2) 10 +/-14 -83 +/-115 * ∆ pao2/fio2-37 +/-11 141 +/-66 * ∆ paco2 (mmhg) 8 +/-6 -15 +/-8 * our data suggest the ability of pav+ to improve gas exchange, principally through an increase in normally aerated areas. the impact of pav+ on ventilator induced lung injury deserves further investigation.grant acknowledgement. university of bari. introduction. the major advantage of high-frequency oscillatory ventilation (hfov) to conventional mechanical ventilation (cmv) is delivery of smaller tidal volumes to an optimally recruited lung. assuming there is a save window in the pressure volume curve of the lung between a lower zone with atelectasis and a upper zone with overdistension, surpassing this zone would result in either cyclic recruitment and decrecruitment, overdistension, or both. in diseased lungs this safe window may be too small to harbor the relatively large tidal volumes of cmv. co2 removal (v'co2) and therefore paco2 is a function of frequency (f) and alveolar delivered tidal volume (vt): v'co2 = f x vt 2 . it is an inherent technical feature of all oscillators that vt at maximal power decreases as frequency increases. in addition, pressure swings fall down the endotracheal tube and the airways. this fall in pressure swings is a function of frequency and mechanical properties of the respiratory system. as a result of both phenomena vt delivered to the alveoli decreases substantially at higher frequencies. up till now oscillation is set at a fixed frequency, in adults at 5 hz, in children and neonates at 10 hz. paco2 is regulated by adjusting the power, and thus the pressure swings (delta p) and the delivered volume. if the maximum power has been reached, decreasing the frequency can lower the paco2 further. we calculated vt required to keep v'co2 constant at different oscillation frequencies and measured the delivered vt at maximal power as function of frequency with the sensormedics 3100a. . vt needed to keep v'co2 constant and maximal delivered vt can be plotted against oscillatory frequency. by increasing frequency, vt needed to keep v'co2 constant and maximal delivered vt both decrease. however, a point is reached at which the required vt to maintain v'co2 equals the maximal delivered vt. at this point vt has its lowest possible value to maintain paco2. at higher frequencies the delivered volume of the oscillator is lower than required and paco2 would rise above the pre-arranged level. we advocate a ventilatory strategy with the oscillator set at its maximal power and the frequency to be adjusted according to the paco2. with this strategy the lowest vt is delivered to the alveoli with the largest safety margins between atelectasis and overdistension. automatic tube compensation (atc) compensates the resistance caused by the endotracheal tube. tube resistance is defined by the equation hagen-poiseuille: r = (128 x x l) / π x r4. (r= resistance, = viscosity, l= length of the tube, r= radius of the tube). atc is designed to lower the work of breathing in intubated spontaneous breathing patients by creating a higher initial flow and therefore a higher peak pressure. the aim of this study was evaluate the consequences of atc during controlled mechanical ventilation without spontaneous breathing activity on peak pressure distal of the tracheal tube, in comparison to the set pressure. moreover, the time needed to reach the set inspiratory pressure distal of the tube with and without atc was assessed. in an experimental laboratory setting using an artificial lung the maximum pressure in the ventilator (draeger evita 4), proximal and distal of the tube with and without 100% inspiratory atc in a tube id 7,0 and a tube id 9,0 were measured. the time needed to reach the set inspiratory pressure distal of the tube with and without 100% inspiratory atc were compared. baseline ventilator settings were bipap, asb 0, peep 8 mbar, i:e-ratio 1:1, fio2 21%, rise time 0 seconds. a set of 20 measurements where performed for each of the following settings: pressure constant group (pcg): frequency of respectively: 10, 30 and 50 a minute at a fixed pinsp of 25 mbar. frequency constant group (fcg): pinsp of respectively: 15, 30 and 45 mbar at a fixed frequency of 15 a minute. no peak pressure were measured at any time distal of the tube regardless of frequency or set pressure. the pressure distal of the tube never exceeded the set pressure level in the ventilator. the time needed to reach the set inspiratory pressure distal of the tube was significant shorter during atc. (see table) conclusion. there is no danger of creating a higher pressure distal of the tube than the set inspiratory pressure at any time during the use of atc 100% with the draeger evita 4. with the use of atc the set inspiratory pressure at the distal end of the tube is reached more quickly. atc creates a faster rise time on the tracheal level, resulting in a higher mean airway pressure. key: cord-005646-xhx9pzhj authors: nan title: 2nd world congress on pediatric intensive care 1996 rotterdam, the netherlands, 23–26 june 1996 abstracts of oral presentations, posters and nursing programme date: 1996 journal: intensive care med doi: 10.1007/bf02316512 sha: doc_id: 5646 cord_uid: xhx9pzhj nan we present the results of a prospective population-based audit of paediatric intensive care activity in two comparable communities with markedly different delivery systems. in the trent region of the uk (4.2 million people), children receive intensive care largely without the supervision of a paediatric intensivist in a variety of hospitals, few of which have designated paediatric intensive care units (picus). critically ill children otherwise receive intensive care in children's wards, special care baby units (scbus) or adult intensive care units. in the australian state of victoria (4.5 million people), children receive intensive care almost exclusively in one centre -a picu staffed by full time paediatric intensivists. the two regions are otherwise demographically comparable. in both groups, data were collected on all children admitted to an intensive care unit between 1/4/94 and 31/3/95 and children who received intensive care (defined by levels of intervention and nurse dependency) in other sites during the same period. values of each variable at first contact with the icu, and the highest and lowest values over the first 24 hours were recorded. the principal outcome was survival to discharge from the intensive care unit. severity of illness was assessed using pim (paediatric index of mortality) and prism. risk-adjusted mortality was compared using flora's z test and logistic regression. the rate of utilisation of intensive care (>1000 admissions in each region) were similar. there was some variation in case mix between the two groups, but crude mortality rates were similar (7.4% in trent and 6.6% in victoria). however severity corrected data and other measures of picu performance were dramatically better in' the centralised delivery system. the substantial excess mortality in the trent region provides strong evidence for the benefits of centralisation of paediatric intensive care services. there are considerable difficulties in evaluating the efficiency and effectiveness oflcare in children presenting with respiratory failure during acute medical illness. optimal outcomes for such episodes include survival and the shortest length of stay (los) in intensive care with negligible risk of readmission. we have tried to determine whether or not the time course of acute severe medical illness with respiratory failure is predictable. study i (n=1000): a retrospective study of intubated and mechanically ventilated children (>28 days, <17 years) with acute severe medical illness. measures: diagnosis, intensive care los in calender days, and survival. results: the underlying diagnosis fell within one of three broad categories: respiratory disease (n=521, mortality 19.2%), central nervous system (cns) disease (n=342, mortality 38.7%), and systemic inflammation or multisystem (sims) disease (n=137, mortality 47.5%. the los in survivors was: respiratory -median (interquartile range) 8(4-16) days, cns 4(3-8) days, £p,4£ 5(7-g) days. 5:i'~'-+cen diag~,~is-rc!ated-grnnp~ (drgs) were identified (8 respiratory, 5 cns, 3 sims disease) and each have been characterised by mortality and los. study ii (n=300): a prospective study of patients supported by the hypothesis that los for the above drgs was predictable (compared with study i data). in certain instances attributable causes for variances in los were identified: e.g. disease severity, timing ofdrug therapy, and associated disease. with daily paediatric risk of morality scoring within each drg, four profiles of instability were identified. discussion: the time course of acute severe medical illness with respiratory failure is predictable and variance may be attributable to specific care or diagnostic factors. we are now developing a means of linking drg-specific clinical care pathways with an integrated computerised decision support and education facility at the bedside. the objective of this open, prospective study was to assess the relation between basic patient characteristics as well as effectiveness of treatment on the one hand and resource utilization in pediatric intensive care on the other. as universal, non-monetary indicators of resource utilization we used the therapeutic intervention score system (tiss) and length-ofstay (los), from which indicators for total resource utilization per admission (tisstot) and average daily resource utilization (tiss-mean = tisstot/los) were obtained. overall 593 admissions, totalling 3130 days, were included. mortality was 8.4%; non-survivors accounted for 14.1% of overall resource utilization. in non-survivors, both total resource utilization per admission and average daily resource utilization were higher, whereas los was not different from survivors'. severity of illness, surgical status, the presence of substantial chronic comorbidity, emergency admission and transfer from another hospital constituted the major predictive determinants of tisstot (r:=0.19) and tissmean (ra=0.45) in multiple regression analysis (p<0.0001). hence these indicators are appropriate non-monetary measures of resource utilization, a considerable proportion of which are determined by a concise set of basic clinical characteristics. subsequently we analysed the relation between effectiveness of care and resource utilization by assessing severity of illness corrected mortality in low, medium and high resource users, respectively. these 3 categories were delineated by percer/tiles of resource utilization (< p20, p20-ps0, > ps0). despite on average long los and high resource utilization in the high risk group, a relatively low standardized mortality was found, probably warranting prolonged intensive treatment in this patient category. summary: objective:the primary purposes of intensive care are to provide treatments to patients with life-threatening physiological dysfunction or to monitor and observe patients perceived to be at significant risk of dying. this collaborative study was performed to describe our patients and their outcome. in order to improve our results we tried to identit~ high risk groups, patients and methods: 13 picus entered the study, the data included all the admissions with >12 hs. during a 60 days period between the l°june and the 30th september 1993. the records included: age, sex, weight, mechanical ventilation (mv), post-operative condition (p.op), malnutrition, diagnosis, length of stay, prism score and outcome. student test, mann-whitney or wileoxon were performed for univariate analysis. fisher exact test or chi square for dicotomic variables. risk group analysis was performed by logistic regression, odds ratio and 95% confidence interval. results: 650 patients entered the study. mean age was 47.6 months (ds hh¢# 60) and median 18 months. we found significant statistical differences in calculated ,is observed mortality rate comparing malnourished with euthrofic patients; mechanical ventilated (mv) with non mv patients. no differences in ter ~,h of stay or di~ noses were found. effect of the un sanctions on the morbidity rate araong the iraqi small children ( below 3 years old of age ) in bagdad. abdulsamad a.abood / institute of medical technology, bagdad. meningitis is essentially a childhood disease (i). the risk of infection are increased by powerty and overcrowding (7). the impah'ed immunity may be an important pathogenic factor underlying the susceptibility to infections in undernourished subjects (5). in general, malnutrition is a man made disease and it begins quite in the womb and ends in the grave (i). 1918 small children, below 3 years of age were admitted to the pediatric hospital in washash with meningitis over 4 cold months in i994, in contrast to only 176 child admitted with meningitis over the same period in1989. all of the children who admitted in 1994 were frankly undernourished, 45% of them were infected with enterobacteriae, because they were exposed to faulty hygiene and lack of asepsis. these facts showed precisely that our small children had suffered at most from the un_ sanctions against iraq, because of food, milk and drug shortage, since 4 years which had resulted a severe undernutrition among them, which impaired their immune status. m wells, of riera-fanego, j lipman. baragwanath intensive care unit, university of the witwatersrand, south africa. background the use of prism or other scoring systems in the icu is of great importance for evaluating the efficacy and efficiency of a particular icu, the prism score was developed and validated in the usa and europe but has recently been shown to be inaccurate in a south american population, a south african population as well as several european studies. part of the poor performance of the prism score is as a result of differences in the case mix between the reference population and other paediatric icus. since scoring systems should generally be used only in populations similar to the reference population from which the prediction model was developed, a modification of the prism score is necessary to improve its discriminatory ability in a wide range of patient groups, aim to improve the predictive power of the prism score in a south african paediatdc icu population. patients & methods we analysed prism, demographic and clinical data collected prospectively from 1528 consecutive paediatrie icu admissions. the prediction of actual mortality by prism was evaluated by standard statistical methodology (goodness-of-fit test and receiver operating characteristic (roc) analysis), the components of the prism logistic regression equation (prism score, operative status and age) and the 14 physiological variables making up the prism score in addition 10 new variables analysed (nutritional index, the need for inotropes and institution of mechanical ventilation) were subjected to discriminant analysis to determine their association with outcome. results the goodness-of-fit test showed a significant failure of prism to accurately predict mortality over a wide range of expected mortality (chi2[8] = 195, p = 0). prism underpredicted mortality at lower prism scores, but overpredicted mortality in patients with high prisms. similarly roc annysis indicated apoor predic~jve power (az = 0.73 ± 0.01), with an area under the curve significantly less than that for the prism reference population (p = 0), prism showed equally poor discriminatory function at all age groups and diagnosfic categories. '~mth the addition of an index of nutrifional status (proportional weight-far-age), and indicators of early respiratory and cardiovascular failure to the logistic regression formula, and a recalibration of the acute physiological score component, the roc can be improved to 0.83 ± 0.02, with a good fit described by the goodness-of-fit test (cn218] = 3, p = 0.89). discussion the prism score is not accurate in our patient population has been recalibrated in view of the poor discriminatory function that we have shown. part of the inaccuracy derives from the different demographic characteristics of our icu population and a different pattern of diseases. in addition to assessments of acute physiological aberrations, an assessment of nutritional status and early respiratory and cardiovascular failure significantly improve the discriminatory ability of the prism score, these parameters have been devised with a view to improving the accuracy of prism in our population, while not decreasing its accuracy in icus similar to the reference population. in interviewing parents regarding how physicians have communicated bad news, the response i have received is that it has not infrequently been done without appropriate care, understanding and compassion. personal experience and the lessons learned from parents, chaplains and others who deal extensively with these situations have provided me with an approach that has been supportive, compassionate, and caring. an especially difficult communication situation for the intensivist occurs when the parents have to be informed of the death of their child. for the parent, death is the hardest loss of all -the ultimate unalterable loss. circumstances surrounding the death are an important consideration (e.g., a fatal crash caused by a drunken driver, a prolonged illness, a suicide, aids). each produces a different grief reaction. the physician needs to inform parents of their child's death sympathetically coming right out with the news and leaving details until later. allow pauses and time for the paren~ to express sorrow and grief, the best communication may be thoughtful silence and a tender touch. there is disbelief that this happened. it is necessary to repeat oneself. acknowledgment of the parent's "feeling terrible" and the physician's acknowledgment of how terrible he/she feels that the life of the child could not be saved is an important first step in the parent's dealing with this tragic loss. with prolonged resuscitation, it is helpful to have a member of the icu team talk to the parents while the resuscitative efforts are ongoing so that the parents are not left unsupported at this time. a progress report should be delivered in a caring, lucid, and sensitive.manner, indicating that every effort is being made to save the life of their desperately injured child. after a child has died, it is helpful to the family if the physician maintains some contact with them. this should take the form of follow-up telephone calls at approximately 6, 12, and 24 months. this can help to screen for depression in the parents. in giving bad news to the family and making every effort to support them through this tragic time, it is necessary to remind oneself that the intensivist has personal needs for dealing with grief and will also require support to pass through this stage. direct evidence that child mortality is lower in specialist pediatric icus comes from 3 studies. a study in oregon (ccm 1981; 19:150-9) found that mortality adjusted for severity of illness was 102% of expected in 3 pediatric units and 139% of expected in 71 general units (p<0.05). a study in holland (ccm 1995; 23:238-45) found that mortality in high risk patients was 85% of expected in 6 tertiary pediatric units, and 143% of expected in 4 nontertiary units (p<0.05). a third unpublished study, has found that children in victoria (who almost all receive intensive care in a pediatric icu) have a much lower standardised mortality rate than children in the trent region of the uk (where many children receive intensive care in adult icus). there is indirect evidence that icus looking after many children are likely, on average, to perform better than icus looking after few children: numerous studies in many specialities have found that units looking after many cases of a particular disease have better results than units with few cases. see luft hs, "hospital volume, physician volume, and patient outcomes", happ, 1990; and farley d, medical care 1992; 30:77-94. compared to general icus, medical and nursing staff in pediatric icus are likely to be better at looking ~fter children, and plcu rmos have greater skills in pediatric intubation, ventilation, iv drip insertion and drug doses. picus are more likely to have appropriate equipment to manage children -especially for uncommon but life-threatening situations. icus in pediatric hospitals are more likely to have physicians and surgeons with pediatric expertise available for consultation at all times. the american academy of pediatrics, the society of critical care medicine, the british paediatric association and the australian nh&mrc have all said that children should receive intensive care in'specialist pediatric units. the weight of authoritative opinion, and direct and indirect evidence is strongly in favour of looking after children in dedicated pediatric icus. neurological deficit showed higher cbf values (125.7/115.2 ml/100g/ rain.) than the 11 patients with good outcome (mean cbf 1 17.5 sd +8.1; cbf 2 19.9 sd _+9.1 ml/100g/rain}. discussion: in asphyxia decrease of ph is due to reduced tissue oxygenation and indicates the severity of metabolic derangements. co2reactivity in newborns with perinatal asphyxia correlates with the lowest ph and therefore may reflect severity of asphyxia. continuous monitoring of cerebral activity is carried out in our unit on all admissions at risk of cerebral dysfunction, a number of monitors are commercially available and we report our experience with the cfam2 which provides in addition to amplitude integrated eeg analysis, continuous raw eeg display and frequency distribution. bilateral recordings are commenced as soon as possible and continued while clinically indicated. forty one children ranging in ages from 4 weeks to 16 years were monitored for periods from 3 hours to i0 days, diagnoses included traumatic brain injury (11), sepsis/meningitis/encephalitis (1 t), status epilepticus (8) and miscellanous others (11). results are tabulated below. patients 13 12 16 status epilepticus 10 4 1 * beta activity 1 8 15 * background voltage 10 3 1 * < i o/zv 2 or more of above 12 2 1 * (*z2 p < 0,001) asymmetry developed in 4 children, all of whom died. positive predictors of good outcome included a mean background activity of >10zzv, the presence of faster frequencies (usually 13) in response to sedative drugs and the absence of seizures. all monitoring is performed by the picu staff and increasing expertise in interpretation has resulted in earlier therapeutic and diagnostic interventions. regional it was previously found that histamine, a vasoactive mediator, accumulated in brain compartments (kov~ics et al 1995 neurosci lett 195:25) , and antihistamines prevented brain edema formation (dux et al. 1987 neuroscience 22:317) in asphyxiated newborn pigs. in the present study we investigated the effect of intracarotid histamine injection on the blood-brain barrier (bbb) permeability, left internal carotid artery of 30 newborn pigs (4-8 h; 1,180-1,530g; ketamine anesthesia, 10 mg x kg 4) was catheterized through the external branch and different doses of histamine (0, 10 -6, 5xi0 -6, 10 -5, 5x104, 104 m, respectively, in 6 groups of animals; n=5 in each) diluted in 1.0 ml isotonic saline was injected into the vessel through 1 rain. bbb permeability was determined for a small (sodium fluorescein, sf, 376 da) and a large (evans blue/albumin, eba, 67 kda) tracer (2%, 5 mlxkg 4, 30 rain circulation time for both dyes) concomitantly in frontal, parietal and occipital cortex, hippocampus, and periventrieular white matter both on left and right sides 1 h after the challenge. then, intravascular dyes were removed by perfusion and bbb permeability for both tracers was quantified by fluorescence spectrophotometry (wavelengths for excitation and emission were 440 nm and 525 nm for sf; and 620 nm and 680 nm for eba, respectively). histamine injection, in doses higher than 10 .6 m, significantly (p<0.05; kruskal-wallis one way anova on ranks followed by dunn's test) increased bbb permeability for both tracers in each brain region. changes in left hemisphere were more intense (p<0.05) than those in right one after the doses of 5xi0 -6 and 10 -5 m in each region, i0 4 m histamine administration induced similar edema in both sides. increased intracarotid histamine levels resulted in a dose-dependent vasogenic brain edema formation. histamine might have a pathogenetic role in neonatal hypoxicischemic cerebral injuries. supported by otka f-12722 and h-u.s,-jfno.392, $162 in coma caused by traumatic brain jnjury, an indication of the likely outcome is provided by the best motor response to pain in the first .$ hours after the insult. in a study in our picu, the proportion of children who died or had a severe disability was 100% in 35 who had no response to pain, 40% in 47 with an extensor response, 14% in 64 with a flexor response, and 1% in 61 who localized in response to pain. the long term outcome of traumatic brain injury appears to be worse in children <4 years old. other risk factors in traumatic brain injury are absent basal cisterns, midline shift or subdural haemorrhage on ct scan (or loss of grey-white differentiation in nontraumatic injury); or an intracranial pressure >30 mmhg despite hyperventilation, mannitol and barbiturate infusion. apart from brain death, there are two findings implying such a poor prognosis that consideration should be given to stopping treatment: first, after traumatic injury, the absence of any motor response to painful stimulus in the cranial nerve distribution (providing drug effects and a post-ictal state have been excluded); and second, in acute brain injury from trauma, infection, hypoxia, or ischaemia, the b{lateral absence of short-latency somatosensory evoked potentials (providing brain stem haemorrhage, subdural and extradural effusions, and decompressive craniectomy have been excluded). in children over 2 months of age, recovery from prolonged coma or a vegetative state is exceedingly rare when more than 12 months have elapsed after traumatic brain injury, and when more than 3 months have elapsed after nontraumatic injury. overproduction of nitric oxide (no) via an inducible isoform of" no synthasc (inos) produces profound vasodilatation in adult septic shock. high nitrate levels have been reported in hypotensive children with sepsis syndrome ]. cardiovascular collapse is a prominent feature of severe meningocoecai disease (mcd). however, systemic vascular resistance (svr) was slightly higher in a group of non-survivors ~ and the rote of no in ivicd remains unclear. children with a presumptive diagnosis of mcd were enrolled. parental consent was obtained. blood was drawn on admission and 12hrly thereafter. plasma was separated immediately and stored at -80°c. the final concentrations reported represent the product of nitrite and nitrate (nox). nox was measured spectrophotometrically using the greiss reaction. 21 children were studied (median age (range); 27m (5-203)). the diagnosis of mcd was confirmed in 18 children, 12 of whom had a glasgow meningococcal score (gms) of" ~8. in this group with severe mcd there were 3 deaths. peak nox was significantly higher (,.54(27-78) vs 96(50-363)nmol/ml, median) and systolic btood pressure was significantly lower in children with severe mcd than mild mcd (p<0.05. wilcoxon rank test). there was a significant correlation between peak nox and gms (spearman's rank correlation r=0.6 (p=0.01)) and prism (r=0.6 (p:0.01)). nox production from adm.ission onwards was also higher in the severe mcd group (p:0.002, kmskal ~wallis). we have demonstrated that plasma nox levels are elevated in children with mcd, correlate directly with the severit 3' of disease and are inversly related to systolic blood presssure. similar to hypotensive septic syndrome, mcd appears to be associated with an up-regulation of the l-arginine-no pathway.. non-survivors with mcd have higher svrs and may be relatively hypovolaemic. in our group of severe mcd there was a significantly lower systolic pressure and increased no formation. excess inos expression at different stages in mcd may contribute to the pathology of the disease. the identification of agents which can boost and/or inhibit no reiease may therefore represent different treatment strategies for mcd. u. merz, th. peschgens, g. kusenbach, m. b6hle, h. h6rnchen in this controlled, prospective study 30 ventilated premature infants with a birth weight < 1250 g were randomized to receive treatment with dexamethasone (dex) either on day 7 of life or on day 14 of life. dex was given over 16 days tapering from 0.5 mg/kg/day to 0.1 mg/kg/day. the infants treated with dex on day 7 of life could be weaned earlier from the ventilator -in median after 14 days (range 10 -34) versus 24 days (range 8 -44) in the [ate treatment group (p = 0.01). the need for supplemental oxygen was shorter in the early treatment group -in median 24 days (range 10 -50) versus 40 days (range 10 -70) (p = 0.2, ns). the incidence of chronic lung disease was lower in the early treatment group -6 of 14 infants (42.9%) versus 10 of 16 patients (62.5%) (ns). to evaluate the long-term efficacy of early dex treatment we performed a respiratory function test in the age of 3 -6 months using an infant whole body-plethysmograph. the intrathoracic gas volume (itgv), the airway resistance (r.w) and the airway conductance (gaw) were measured and no significant differences could be detected between the groups. the frequency of adverse effects due to dex therapy was found to be without significant differences between the early and the late treatment group. we conclude that early dex treatment had short-term improvements in pulmonary outcome in our study population, long-term efficacy however, remained unproven. several factors contribute to the development of chronic lung disease (cld) in premature infants including structural immaturity of the lung, mechanical ventilation, and oxidative stress. reactive oxygen species are formed during normal cellular metabolism but they are generated in higher concentrations during inflammation or inhalation of high oxygen concentrations. to study the relationship between increased oxidative stress, antioxidants and the development of cld we examined 102 ventilated premature infants with birth weights below t500g. 32 infants developed severe chronic lung disease of prematurity (cld), defined by radiological signs of cld and an increased oxygen requirement at a postconceptional age of 36 weeks, and 29 infants had moderate cld with an increased oxygen requirement on day 28 but not at an age of 36 weeks. ventilator settings (fio2, peak inspiratory and mean airway pressure) and the incidence of early-onset-sepsis were significantly higher in the severe cld group than in infants with moderate cld or without cld (n=41) during the first week of life. plasma concentrations of the two antioxidative substances bilirubin and uric acid (ua) were comparable in all groups during the first days of life. however, on day seven bilirubin and ua were significantly decreased in the plasma of infants with severe and moderate cld compared to the non cld group (p<o.001). there was no difference between both cld groups. in serially obtained tracheal aspirate fluids (taf) malondialdehyde (mda) was measured by hplc as an indicator of lipid peroxidation reflecting oxygen injury of the lung. ua was found in higher concentrations in taf than bilirubin. ua has been proven to be a potent antioxidant, capable to react especially with hydroxyl radicals and hydrochlerous acid, which play an important role in the induction of lipid peroxidation. infants with severe cld had significantly higher mdnua ratios in taf from day 5 to 14 compared to the non-cld group (p<o.01). mdnua ratios in taf of infants with moderate cld were significantly higher from day 7 on but still lower than in the severe cld group. in addition, the ratio oxidized / reduced glutathione was significantly increased in taf of infants with cld compared to infants without cld between day 5 and 14. the data support the hypothesis that an imbalance of oxidative stress and antioxidant defense contributs to the onset of cld at the end of the first week of life. introduction. rocuronium bromide (roe), a neuromuscular blocking agent with an onset time dose to that of succinyleholine l'z, has been studied in american society of anesthesiologists' physical status (asa) i or ii pedlatrie patients under non-critical elective conditions. there is a paucity of studies looking at the pharmacodynamics of roc under emergent conditions, involving pediatric patients who are asa iii or iv, without the use of adjuvant inhaled anesthetics ~. methods. with approval of our committee on clinical investigations (irb), roe 1.2 mg/kg was administered to 15 asa iii/iv pediatric intensive care patients (age 40 days to 23 months; m:f=8:7) on intravenous sedation. the time to reach 25% a~d 10% of b~elinc tetanie stimulus response was recorded using a r.elaxogra'ph/qmt-100 nerve stimulator/recorder (datex, helsiaki). depth of clinical relaxation at the 25% level was recorded. results. the time from rapid bolus to 25% was 25.4+12.6 see with a range of 10-50 see; and to 10% was 30.1+17.6 see, with a range of 10-69 see. at the 25% level, excellent clinical relaxation was seen. conduslon. in critically ill children, use of roe at 1.2 mg/kg produces rapid onset of neuromuscular blockade and excellent clinical relaxation, such that roe should be considered for rapid sequence intubation. int anesth clin. 1995;33(1):39-60. anesthesiology. 1994;81 (5) computed tomography of the chest (ct) in mechanically ventilated adults can detect intrathoracic pathology not appreciated on chest radiographs (cxr) and influence intensive care management. no such data exists for ventilated children. aims: 1) to assess if ct provides additional information over cxr regarding extent and nature of intrathoracic disease in ventilated children. 2) to determine whether such information alters clinical management, inclusion criteria :10 ventilated children with cxr changes inconsistent with their respiratory status underwent ct if either a) pao2:fio2 ratio <30 and/or mean paw>15 cm h20 or b) there was an unexplained increase in ventilatory requirement. methods : high resolution ct was performed in 3 patients and spiral ct in 7 patierits, to ensure minimal transport related morbidity, patients were transferred to the ct scanner by a specialised mobile intensive care team. results: in 2/10 patients ct demonstrated greater extent of disease than appreciated on cxr but did not significantly alter clinical management. in 7/10 patients ct provided additional information regarding the nature of disease present, in 2/7 children this involved a further diagnosis and in 5/7 children the exclusion of a suspected pathology. new information led to a positive therapeutic intervention in 2 children, prevented inappropriate manoeuvres in 3, and had no significant effect on acute management in 2 children. conclusions: initial data suggests that in a selected group of mechanically ventilated children chest ct can add to the sensitivity and specificity of intrathoracic diagnosis provided by the chest radiograph and directly influence acute management. case selection criteria and choice of the most appropriate protocol requires further study. pressure control ventilation (pcv) utilizes a decelerating flow pattern which may improve gas distribution and lead to alveolar recruitment. in contrast, volume control ventilation (vcv) employs a constant flow. in children, the effects of pcv as compared to vcv are unclear. the purpose of this study was to determine how these two modes compare in terms of dynamic compliance (cdyn). peak iaspiratory pressure (pip), and mean airway pressure (paw) at equivalent minute ventilation. methods: sixteen infants and pediatric patients ranging in age from 1 day to 13 years were studied. diagnoses included ards (6), postoperative cardiac surgery (7), head trauma (1), and resfrictive lung disease (2). patients were randomized to pcv (9) or vcv (7). initial measurements of gas exchange (abg's) and respiratory mechanics (ventrak, novametrix medical systems) were obtained after a 20 minute stabilizadon period. respiratory mechanics included pip, peep, paw, delivered tidal volume, and cdyn (avolume/apressure). the patients were then crossed over to the alternate mode of ventilation holding delivered tidal volume, peep, inspiratory time, minute ventilation, and fio2 constant. data were collected after 20 minutes, in each mode the absence of intrinsic peep was confirmed. to assure that the measurements were not affected by changes in clinical status, the patients were returned to the initial mode of ventilation and measurements repeated (final) . patients were ventilated with a siemens 900c or sv300. reselts: data were analyzed using 2-way analysis of variance with repeated measures. ~ <0.05 vs. vcv) vcv pcv ~ initial ] final ! cdljn 3.5_+0.7 4.3_+0.8 * 3.7_+0.6 3.9_+0.7 i , pip 32+1.0 30l-_t.0 * 31_+1,0 31+-1,0 paw 9.2_+0.6 10.9i-_0.7 * 9.7+0.7 10.0-!-_0.8 pao2 97_+14 92+-10 87_+9 97_+14 discussion: at the same minute ventilation, the decelerating flow pattern of pcv resulted in a 23% increase in cdyn and an 18% increase in paw while decreasing pip by 6%. the lack of a significant change in oxygenation may be a result of the limited time in each ventilator mode as well as the inclusion of patients with both normal and abnormal lungs. there was no significant difference in initial and final measurements indicating patient stability. the beneficial effects of iecre~l~iug cdyn and paw while decreasing pip indicate that pcv may be a preferable mode of ventilation in patients with lung injury. further randomized studies examining the effect of pcv on respiratory outcome measures in pediatrics are indicated. prolonged positive pressure ventilation following repair of cdh is associated with a high prevalence of iatrogenic lung injury, in our unit dudng 1981-1990 314 late deaths after repair of cdh were due to chronic lung disease. since 1990 babies requiring assisted ventilation for more than 7days following surgery were transferred to a cnep chamber to limit lung injury. cnep of -6cm of h20 was combined with positive pressure ventilation via an endotracheal tube dudng the transition phase. immediate reduction of peak inspiratory and positive end pressures were possible and following extubation respiratory support was maintained by cnep v~th appropriate inspired oxygen. overall outcome: [1981] [1982] [1983] [1984] [1985] [1986] [1987] [1988] [1989] [1990] n=68 deaths before surgery (%) 11 ( ecmo during 1990 -1995 /16 who were ventilated for more than 7 days received cnep and there were no deaths and no chronic lung disease in that group. cnep assisted ventilation may be an important management option for babies who require prolonged respiratory support to avoid the adverse effects of chronic positive pressure ventilation, introduction so far 2 modes of liquid ventilation (lv) have been used in experimental animals and, exceptionally, in humans: 1. total liquid ventilation (tlv)-functional residual capacity (frc) is filled by perfluorocarbons (pfc), and slow tidal volume (tv) breathing is performed by pfc. 2. partial liquid ve,0ti,la~ion (page) -only frc is filled by pfc. gas tv is delivered by conventional mechanical ventilation (cmv), high frequency jet ventilation (hfjv) or high frequency oscillation (hfo). the aim of our study is to present our limited experience with page in newborns and infants. page was used in two groups of infants: 1, in 2 infants with brain death before disconnection from cmv, because recipients for organ transplantation were not available. these infants have relatively normal lungs (fio~ less than 0.4). infants stayed on page for 1 hour, during that period no ventdator manipulations were made. after page, infant were switched to cmv for next 6 hours. 2. very critically diseased infants with ards (rds) -2 on ecmo more than 5 days, 1 before cannulation for ecmo, 4 on hfo because of intractable respiratory failure, preoxygenated rm 101 (miteni, italy) was used in the doses up to 40 ml/kg intratrachealy. blood gases and parameters of pulmonary mechanics were followed (dynamic compliance -c dyn, airway resistance -raw, bicore monitor). page was combined with no inhalation (5-80 p.p.m, in 2 infants). in both groups ad hoc an approvement from e local ethical commission and informed parental consent were obtained. in the first qroud with relatively normal lung parameters of oxygenation drops after pfc instilation intratracheally and stayed depressed for 4-6 hours. slight pco2 retention occured in both cases during page. c dyn increased almost double during page period, raw drops transitorily after pfc instilation but in 10 minutes they were identical like in prepage period, parameters of oxygenation (peo2/fio2) after 4-6 hours after page improved and were better than in prepage period. after that time infants were disconnected and died. in the second group no improvement of oxygenation was seen in one ecmo baby, in spite ()f transient improvement of c dyn. in the second ecmo baby, oxygenation improved and flow of pump could be decreased by more than 20%. none of these babies, however, survived, improvement was only transient in spite of repeated dosis of pfc. in these babies serious problems were to maintain the adequate frc by liquid, because of severe air leak, in 5 babies on hfo/hfjv with severe ards/rds the improvement of oxygenation were seen in all the cases immediately after pfc instiletion for the period of 4-5 hours. after that period, pfc dose had to be repeated. two babies of this group survived. conclusion. page is going steadily from tabs to clinical practice. it is simple, could be performed anywhere, cheaper than tlv. however, because liquivent -perflubren (aliance pharmaceutical) is not available in europe, rm 101 of 82 (mitenti, italy) is the only solution, which could be currently used here. before the widespread use of page in clinics, liquid network among most nicus and picus must be built up, the criteria for page must be defined and ethinal-legal problems resolved as well. after resolution of these particular problems page can be life saving procedure for very special part of critically ill newborns end infants. catherine caronia, peter silver, laura nimkoff, cad quinn, jack gorvoy, and mayer san. division of pediartic critical care, medici,, schneider children's hospital, new hyde park, ny 11040, imroduetiun: cystic fibrosis (cf) patients awaiting lung transplantation present a therapeutic dilenuna when severe respir, aory decompemalion occurs, endotracheal intubation and mechanical ventilation is known to have no long term benefits and is associated with high morbidity and mortality. noninvasive respiratory support appears to be a beneficial alternative. methods: we instituted bipap (respironics, inc,, murrayville, pa) in 9 end-stage cf patients who were admitted to the pediatric icu with severe respiratory decompeusation. all patients were awaiting tung transplantation. after a control period, bipap was applied via a tight fitting nasal or facial mask, using the spo~aneous breathing mode, expiratory pressures were set at 4-8 cm hhzo. inspiratory pressures were started at 8 cm ~i o and increased in 2 cm i-i20 increments until the patient's respiratory comfort was achieved and substantiated by non-invasive monitoring. patients were instructed to use bipap during night sleep and whenever subjectively required, data are reported as mean _+ s.d. results: all 9 patiems utilized nocturnal bipap for 6-10 hours/day during a follow-up period of 2-19 months. compared to their pre-bipap status, the patiems' oxygen requirement and respiratory rate both oz~ cundusion: bipap tl~rapy improves the respiratory status of decompeusatir!g end-stage cf paacnts. it is well tolerated for long term use at home, and provides an extended period of respiratory comfort and stability for cf patients awaiting lung transplantation. l. bindl*, g. kiihl**, p. lasch***, appel**, j.m611er**** and the "arbeitsgemeinschaft ards im kindesalter" background acute respiratory distress syndrome (ards) is a therapeutic challenge in pediatric intensive care in view of the high mortality, in 1992 about 50 german paediatlic hospitals founded a working group aiming on collaborative clinical research in this field. aims and methods the aim of both a prospective and retrospective survey conducted in german pediatric intensive care units in 1993 was to accumulate data on the epidemiology, risk factors, natural history and treatment strategies in a large group of pediatric ards patients who were treated in the tt~ee year period from 1991 to 1993.all patients had acute bilateral alveolar infiltration of noncardiogenic origin and a po2~io2 ratio < 150mmhg. the influence of sex, underlying disease and single organ failure was analyzed using the fischer's exact test, the influence of additional organ failure on mortality was tested with the cochran-mantel-haenszet statistics. results 112 patients were reported giving an incidence of 7 cases per 1000 admissions to pediatric icus. median age was 24 month. in 43% of the cases, ards was associated with a pulmonary, in 39% with a systemic underlying disease. in 20% immunocompetence was impaired. mortality was 46% and not dependent on age, sex and triggering event. the number of associated organ failures, however, strongly influenced mortalib,. mortafity in immuno-compromised patients was 8 t %. the analysis of treatment modalifies employed in the patients revealed a lack of uniform therapeutic strategies. on the other hand, the patients were exposed to interventions not yet supported by controlled trials. conclusions the observation of the lack of uniform treatment strategies led to the elaboration of recommendations on ventilator therapy and patient monitoring within the working group. the data gathered in this survey provide the basis for the design of prospective multicenter studies urgently needed to evaluate innovative treatment modafities in pediatric ards. recurrent apnea and respiratory failnre due to severe lower respiratory tract disorders such as bronchiolitis or pneumonia are the most common reasons for mechanical ventilation during respiratory syncytial virus (rsv) infection. acute respiratory distress syndrome (ards) has been described as a complication of severe rsv infectionj in contrast to the low mortality rates associated with rsv infection (< 5 %), mortality rates in the range of 40-70 % have been reported in pediatric patients with ards. however, studies on ards are usually lumped in respect to causation and the disease course of rsv induced ards has not been previously studied. we examined the lung function abnormalities of 37 infants with rsv induced respiratory failure requiring assisted ventilation, measurements included respiratory mechanics, maximal expiratory flow-volume curves and lung volumes, ards was defined clinically using the criteria which were recently proposed by the american-european consensus conference on ards~: acute disease onset, pao2/fio~ ratio _< 200 mrn hg, bilateral infiltrates on chest radiograph and absence of clinical evidence of left atrial hypertension. we calculated the murray lung injury scores modified for use in pediatric patients 3 from total respiratory system compliance, radiographic findings, ventilator settings and blood gas results. we identified 10 infants with severe restrictive lung disease that fialfilled the clinical criteria fbr classification as ards. all had lung injury scores above 2.5 which is the recommended cut-off for a diagnosis of ards, twenty-seven infants had obstructive disease consistent with a clinical diagnosis of bronchiolitis. the ards patients were significantly younger, had a longer time of assisted ventilation (p <0.05) and a greater proportion of infants with preexisting illnesses (p=0.023, odds ratio =6.67) when compared to the patients with obstructive disease. with the exception of one immunodeficient patient, none of these infants died. given the low mortality despite a clinical picture of severe lung injury, there is evidence that rsv induced respiratory failure may represent a relatively benign cause of ards in pediatric patients, bachmann an audit of patients with severe acute bypoxic respiratory failure (ahrf) receiving highfrequency oscillatory ventilation (hfov) in our unit ( n=32, mortality 75%) revealed that sub-groups with severe underlying disease (n=14, mortality 100%)and those with mu~pie organ failure ( > 2 systems failing, n=7 mortality 100%) accounted for all the deaths beyond the neonatal period. v~ therefore hypothesized that in a modem paedistric intensive care unit (picu): a) children greater than one month of age with ahrf do not die in the absence of severe, pre-existing disease or multi-organ dysfunction syndrome, b) respiratory parameters alone will predict outcome poorly in ahrf. method prospect~/e sty/of all adm~ns to our tertiary picu. data it, citing the respiratory parameters (oxygena~n index [ol] , aiveolar-artedal oxygen tension gradient , pao2/fio2 ratio) were collected hourly from the bedside charts throughout admission. patients were included in the study if ahrf was present at admission either none or in combination with other organ dysfun~on. ahrf was defined as the acute (<48hour) onset of respiratory dysfunctk:~l with a pao2/fio2 ratio.< 200 for six consecutive hours dunng the first 24 hours of admission (with no evidence of left anal hypertension), x-ray review defined a sub-group of patients with acute respiratory distress syndrome (ards) by the presence of bilateral interstitial infiltrates. results to date 59 children (ages 1-168 months, weight 1.2-70 kg) have been admitted in ahrf. 18 of these also had ards. the overall mortality was 23.7% (14/59), and greater in the ards group than the non-ards group (10t18, 55.5% vs, 4141, 9.7%, p< o.01) . it was not possible to predict survivors from non-survivors on the basis of the seventy of the respiratory failure alone, the a-ado2 on the day of admission (best in 24 hours) was not significantly different between survivors and non-survivors: (mean, + sd)(174 mmhg +_108, vs 304 mmhg _+_156). kdl non-survivors were immunodeficient (n=8), previously extmrnsly premature infants (<28140),(n=3) or suffedng fcom chronic metabolic or gastrointestinal disease (n=3). no previously normal child died. conclusion the severity of respiratory failure does not allow predioljon of outcome in our patients. we believe that this reflects that modem picu is so effective at providing respiratory support that pre-existing pathology alone de~ prognosis. this suggests that an abnormally regulated host response or abnormal persistence of a pathogen may be required to induce lung injury of sufficient severity that the resulting respiratory failure cannot be supported in a modem picu. introduction: postural changes (supine to prone) is a therapeutic intervention that could be useful in children with adult respiratory distress syndrome. objective: to determine the effects of postural changes in the oxygenation of young children with ards. method,s: a prospective stud3," was performed in eleven subjects aged 6 to 120 months (mean=33) with the diagnosis of ardsreceiving vendlatory support. (mean peep and fio2 of 9 and 0.75 respectively). postural changes was performed every 8-12 hours, during a period of time ranging from 5 to 16 days. arterial blood gases were determined before and 30-60 n~n after the postural change, no modification in the mechattical ventilation other that changes in the fio2 were performed. the oxygenation was determined by the index pao2/fi02 (p/f). to study the differences between the oxygenation mean, before and after the postural changes the wilcoxon test for paired samples was used, results: 184 changes were performed (104 from supine to prone and 80 from prone to supine). a9% increased p/f ratio was obtained after the change from supine to prune. although, not all the patients receiving postural changes improved their p/f. six of them (group i) showed an improve in the p/f when changed from supine to prone, returning to their base line when positioned from prone to supine. no improvement on the p/f was observed in the remaining 5 subjects (group ii)after postural changes (table 1) . during the maneuver no complications were observed. two patients had a pneumothorax, not related with the postural change. conclusions: postural changes (supine to prone) is an easy way to improve oxygenation in some children with ards. change to prone change to supine introduction: the common noninvasive diagnostic efforts to identify possible obstruction of the intrathorucic airway, are of limited value. invasive procedures such as bronchoscopy and bronchography may also be noncontributory and entail risks. we evaluated the usefulness of 3d-ct in the diagnosis and management of pediatric patients with suspected intrathoracic airway obstruction (itao). methods: we used a diagnostic algorithm (see diagram) in patients with suspected itao resulting in respiratory distress. three-dimensioual imaging of the tracheobronchial tree was reconstructed, following high speed spiral ct scan, by specific computer software (advantage window computer work station, general electric, milwaukee, wisconsin). non-ionic contrast medium was injected, in some patients, to delineate the intrathoracie large vessels.. results: eight patients were studied. in 5 patients the 3d-ct revealed intrathoracic airway abnormalities. these patients underwent further invesive studies which confirmed the following diagnoses: 2 patients had bronchomalacia, 1 had bronchial stennsis due to a dilated pulmonary artery mad 2 patients had subglottie stenosis extending to the thoracic cavity. three patients had no significant disruption in the configuration of the tracheobronchial tree and thus did not require invasive diagnostic procedures. conclusion: computer reconstruction of three dimensional images of the tracheobronehial tree is a safe and reliable diagnostic tool for itao. ards and ecmo; preliminary data from a randomized clinical trial. j fackler, c steinhart, d nichols, d bohn, m heulitt, t green, l martin, k newth, m klein, j ware. many suggest ecmo be considered experimental for ards and undertaken only with careful data collection and reporting. a mtflticenter pediatric rct is in progress to determine whether 1) ecmo and/or 2) permissive hypercapnia, offer significant advantage for the treatment of ards. methods: all patients aged 2 wk to 18 yr (without congenital heart disease) are eligible for study. data collection begins when a patient receives at least 50% oxygen and a peep of 6 cm h20 for 12 hours (stage t). if the predicted mortality reaches 60% within 7 days (stage 2), eligible patients are asked for written consent for randomization. patients are excluded from randomization with significant chronic lung disease, immune compromise, cardiac disease; or profound acute central nervous system damage. the prime outcome variable is survival. at the studies onset, 400 pts were estimated to be required so that 65 pts were randomized per arm. results: 131 patients are enrolled from 9 centers. data are complete on 85. 66 patients never reached stage 2 (i.e. 60% mortality). 47 patients improved and 19 died. of the latter, 13 had randomization exclusion criteria even if stage 2 was reached. 19 patients reached stage 2. 11 had exclusions from randomization and all died. eight patients (4 survivors were eligible for randomization; consent was obtained in no case. two patients received ecmo. overall survival is 60% (51/85). in patients without randomization exclusions, survival is 77% (34/44). morbidity m survivors (discharge -admission popc or pcpc score >_2) was seen in none of the 4 stage 2 surviviors and 15% (7/41) of those who reached only stage !. conclusion: the rct requires completion. the records of hospital in-patients at king faisal specialist hospital and research center who received external cardiac massage as part of their cardiopulmonary resuscitation were reviewed. success of resuscitation was analyzed as (1) short term (restoration of spontaneous circulation), and (2) long term (discharge from hospital). of 234 such patients, 171 (73.1%) survived the initial resuscitation, and 66 (28.2%) were discharged. success of outcome was not related to age, location of patient, time of day, or rhythm at arrest, including asystole. longer resuscitation time was associated with less chance of restoration of spontaneous circulation (p<0.001), but not associated with hospital discharge rate. results for patients with congenital heart disease were similar to those with other medical or surgical conditions. in this series, 36.7% of ward in-patients survived to discharge, compared to two 5"*;'~r ~r;~'9 ,.,.'her,, the r-e~ult~ were 0c/ "'~d ~, ~,°(. overall, 39 7% of patients who survived the initial resuscitation were discharged from hospital. where resuscitation continued for more than 30 minutes, 18.9% of patients had tong term survival. outcome from asystole was no worse than for other cardiac rhythms, we believe that previous reports of poor outcome from asystole in pediatric cardiac arrest should noi influence decisions to stop resuscitation for pediatric in-patients prematurely. successful restoration of spontaneous circulation with long term survival can be achieved after prolonged resuscitation. abdelmoniem~ lindsey jahusou~,mariano fiallos, university of florida, 820 prudential drive, suite 203 jacksonville, florida 32207 usa central acidosis is well recognized as a marker of inadequate tissue perfusiou, and ventilation. however, obtaining central venous blcod is difficult and fraught with complications in the child undergoing cardiopuimonary resuscitation. intraosseous blood may be used instead of central venous blood to judge ph and pcoz during short durations of cardiopulmonary resuscitation and during hemorrhagic shock. the purpose of this study is to compare the ph and pcoz status of intraosseous and central venous during prolonged cardiopulmonary resuscitation after fluid and drug infusion. we hypotbesized that there would be no difference in ph and pco2 values of simultanecusly obtained intraosseous and central venous blood samples. eighteen (18) introduction: cardiopulmonary arrest (cpa) in children is usually preceded by a deterioration of cardiac or respiratory function due to sepsis, dehydration and hypovolemia. early recognition of clinical and laboratory signs followed by immediate intervention are essential for prevention of cpa. the purpose of the present study was to identify factors which contributed to high rates of mortality from cpa in patients admitted to a paediatric intensive care unit (p1cu). methods: a prospective study was done of all non-surgical patients with cpa who were admitted to the picu, hospital baca ortiz, quito ecuador from january to october 1995. clinical and laboratory variables before and after admission to the picu, time from hospital admission to picu admission and the pediatric risk of mortality score (prism) were recorded on a questionnaire designed specifically for this study. results: of the 70 non-surgical patients admitted to the picu, 14 (20%) were admitted after developing cpa on the general pediatric wards. mean age was 16 + 19.1 months, with 13 of 14 patients under 20 months of age. initial diagnoses upon picu admission included meningitis (n=3), respiratory failure (n=2), congenital heart disease (n=2), severe neurological impairment (n=2), end stage neoplastic disease (n=2), hypovolaemic shock (n=l), peritonitis (n=l) and sepsis (n=l). mean time from hospital admission to p1cu admission was 16 _+ 19.2 hours. the mean prism score upon hospital admission was 30+ 13.7 (score > 20 = > 50% mortality). 79% (11/14) of the patients died. one of the three survivors had severe neurologie injury. prior to picu admission, patients experienced tac~,cardia (n=9), hypotension (n=8), neurological deterioration (n=8), respiratory, distress (n=7), oliguria (n=5), bradycardia (n=3), metabolic acidosis (n=7), hyponatremia (n=4), hypokalemia (n=2), hypocalcemia (n=2) and severe hypoglycemia (n=2). there were serious delays from the time of development of clinical and laboratory abnormalities to the time of admission to picu. conclusion: in the critically ill pediatric patient, rapid recognition of clinical and laboratory signs of deterioration, followed by immediate intervention, are required to prevent end stage shock and cpa. we found serious delays in intervention following development of important premonitory clinical and laboratory abnormalities in patients less than 20 months of age on the general pediatric wards, which iikely contributed to the dismal 79% mortality rate. hospitals throughout ecuador should institute immediate improvements in ctinical supervision, and provide training in paediatric advanced life support (pals) to decrease excessively high rates of and mortality from cpa. intraosscous access is recommended by the american heart association and american academy of pediatries as a means of rapid access to the vascular system for childhood emergencies. bone marrow and fat embolism is a concern and has been reported post intraosseous infusion in stable animals but has never been studied in animals subjected to cardiopuimonary resuscitation. we undertook this study to investigate the incidence and magnitude of lat and bone marrow embolism with the use of intraosseous infusion during prolonged cardiopuhaonary resuscitation and after fluid and drug infusion. we hypothesized that there will be no difference in the magnitude of fat embolism between cardiopulmonary resuscitation only and other cxperirnental conditions. thirty-one (31) piglets were anesthetized, mechanically ventilated, and instrumented (carotid artery, pulmonary artery and intraosseous earmulas ). the animals then underwent bypoxic cardiac arrest followed by chest compressions with the mechanical thumper (michigan insmunents) and mechanical ventilation for a minimum of 45 minutes. the animals were divided in groups: a (n=5) which had no intraosseous, ~'oup b (n=6) had intraosscous with no infi~ion, and groups c (n=6), d (n=6), e (n=8) had intraosseous with infusion of adrenaline, normal saline and sodium bicarbonate, at cessation ofcardiopulmonary resuscitation, representative lung samples were collected fi'om upper and lower lobes of each lung, embedded in ocp and firozen immediately. ltmg specimens were stained using oil red-o dye and observed for fat globules and bone marrow elements. the amount of emboli present was rated as a percentage in relationship to iung tissue, by a pathologist blinded to the experimental groups. buffy coat specimens were collected before and at cessation of cardiopuimonary resuscitation, stained with oil red-o dye and observed for fat globules. percentage of fat present were compared using analysis of variance. fat globules were seen in the prebronchial blood vessels and in intravascular areas throughout all lung fields. there was no difference in appearance or distribution of fat globules between groups. quantity varied in the different groups[(a) 45%, (b) 44%, (c) 30% (d) 23%, (e) 25%], but were not statistically significant (p = .097). fat globules in the buffy coat were few and inconsistent with lung findings. fat and bone marrow emboli were present in all experimental conditions, the use of the intraosseous cannula does not increase the magnitude of embolization during cardiopuimonary resuscitation. the decision to use the intraosscous route should not be influenced by the risk of embolization. tzareva iv/,, md*, nedialkova r, md**, *dept. of pathophysiol, *~dept. of child surg. and icu, emergency medical institute pirogov, sofia, among 566 children with blunt abdominal trauma, treated in emi pirogov during the last five years, 79 children had serious disturbances of the basic vital functions, connected with the trauma, and most often with massive haemorrhage, for this reason being an object of reanimation and intensive care. in the group of children who survived -37, predominated the trauma of only one abdominal organ (mainly the spleen, rarely the kidneys, the intestine) and only 15 children had injuries of more than one abdominal organ. in the same group, in 15 children the abdominal trauma was combined with chest or head trauma or bone fractures. in the group of children who died -12, a profound combined trauma was present. the haemodynamic parameters in all children showed a characteristically significant tachycardia along with normal or even high blood pressure, while hypotonia was present in only 64% of the children on the first trauma day. despite the fact that only 13.4% of the children had direct chest injury as well, the gas exchange was considerably disturbed -899'0 of the children were hypoxemic during the first, and 100% during the third trauma day -in 25% significant -below 8.0 kpa (60 mmhg). together with the markable decrease in haemoglobin levels, this determines the pronounced disturbance in oxygen transport. during the first trauma day all the children were acldo~c, and a metabolic alkalosis was present during the following days. twelve of the children with severe combined trauma died within several hours, with the symptoms of irreversible haemorrhagic shock, or in the next 2-3 days, developing multiple organ failure. in conclusion, the intensive therapy of children with severe abdominal and combined trauma, should take in consideration the special haemodynamical trauma answer in children, and requires dynamic monitoring of the most influenced homeostatic parameters -blood gases, acid-base metabolism, haemostasis. introduction: endocrine emergencies, other than diabetic ketoacidosis, are uncommon causes of pediatric intensive care unit (picu) admissions. we report our experience of children diagnosed of adrenal insuficiency (ai) admitted in the picu, during the last four years. subjects: five eases of ai requiring 7 intensive care unit admissions are presented. four females anna 1 male, with ages ranging from 11 days to 7 years, none of them had a previous systemic or endocrine diseases that could suggest al the initial clinical manifestations were: dehydration (5), vomits (3), abdominal pain (2), seizures (2), lethargy (2) and hyperpigmentation in the muco-genitat area in a newborn male and ambigna genitalia in a newborn female. the reason for their admission in the p1cu were: shock in two subjects; three because of hyperkalemia and hyponatremia (k/na: 5.6/123; 9/126; 7,1/134 meq/l); and two with severe hyponatremia (na: 117; 113 meq/l). laboratory findings: severe hyponatremia (5), increased concentration of urinary sodium and chloride (4); metabolic acidosis (4); hyperkalemia (3); increased levels of urea (3) and hypoglycemia (2). in all of them, the electrolytes abnormalities did not normalize with replacement and only normalized after the administration of hydrocortisone. tile ai was due to: autoimmtme disease in two subjects, congenital adrenal hypoplasia, congenital adrenal hyperplasia secondary to 21 alia hydroxylase deficiency and in one no etiology was found, at the present time, comments: aiis an uncommon disease in the pediatric age. anearly diagnosis is crucial, as if the treatment is delayed could lead to patients death. in subjects with arterial hypotension and electrolytes abnormalities refractory to the usual treatment, they should be treated with corticosteroids, if no etiology is found. although, previously samples must be obtained to make the diagnosis, 0: denotes the number of cases. gerbaka b; hakme c; akatcherian c. toxics are frequently involved in domestic accidents during childhood; among non medical products ingestion, carbohydrate poisoning is a serious injury often made possible by inadequate stocking. over 10 years, 43 children aged 10 years and less were examined in the emergency department of hotel-dieu de france hospital for carbohydrate ingestion. 62,8% are boys; age goes from 13 months to 6 years (moan = 2,5years). kerosene is found in 35,8% of cases; all were admitted (mean = 2,8 days). 79,1% were symptomatic on first examination but 93% of all children presented signs of gastric (58%) or respiratory (69,8%) irritation sometime during their history; 37,2% had neurological signs and 41,9% presented some fever. leucocytosis is found in 65% of cases; 25,6% of the children received antibiotics. chest x ray was abnormal in 48,8% of cases: mainly parahilar infiltrates were found, all children survived; 76,7% with a normal course (1,9 days of hospital stay) whereas those who presented complications (severe pneumonia, coma) stayed in the hospital for 6 days (mean) with short course of assisted ventilation for two of them; long term follow up was not possible. we fonnd nick's criteria for hospital admission to be of value: -symptomatic children with normal x ray } 6 to 8 hours monitoring -asymptomatie children with x ray abnormality } -symptomatic children with x ray abnormality: hospital admission -asymptomatic children with normal x ray : no admission. these criteria would have helped to avoid admission in 8 children and would have allowed a short t2 hours stay for 6 more. we found chest x ray to be mandatory in carbohydrate ingestion; other tests were not helpful, aside arterial blood gases measurement in case of respiratory involvement; we now also advocate more restriction in antibiotic use. prevention remains efficient and should be stressed on. severe liver failure [slf] is a rare but severe condition in infants. we report our experience. patients: slf was defined as liver insufficiency with hepatic encephalopathy and a decrease in the level of factor v to below 25 %. between 1984 and 1996, 29 infants (mean : 4 mo) were admitted for slf (neonates excluded). main causes were metabolic disorders (41.3%) (tyrosinemian=5, hemochromatosis n=2, reye's syndrome n=2, other n=3), virus-induced flf (20.6%) and hematologic diseases (13.7%). in 4 cases, the causes remained undetermined. results: olt was contraindicated in 12 cases because of multiple organ failure (n=10), or underlying disease. all of them died within 6 days after admission. 7 patients had no indications for olt, all but one are alive. (1 of them was transplanted later for tyrosinemia and 1 died lately (virus induced-slf). among the t0 infants who underwent emergency olt, 6 are alive and 4 died because of primary non function of the graft. conclusion: slf in infants admitted before their first birthday is a severe condition with an overall mortality rate reaching 60%. inherited metabolic disorders are the first cause of slf at this age. contraindications for olt are frequent because of underlying disease or multiple organ failure. a number of children undergo primary graft failure after liver transplantation. it is unknown if there is any increased morbidity or mortality following retransplantation. this study seeks to explore these issues. methods: a pediatric intensive care/iiver transplant database is in formation. records of all liver transplant patients are reviewed and abstracted. this data is then computerized to allow analysis. this data provides the source for this study. statistical analysis was performed via student's t-test where appropriate. results: of the 350 patients who have thus far received at our center orthotopic liver ransplants, the records of 112 who underwent 140 transplants form the basis for this review. twenty-three patients underwent multiple transplants, 19 required one additional, three required 3 organs, and one patient survived after a fourth organ transplant, there was no significant difference in age at first transplant between those who received multiple organs and those who did not (40 vs, 44 months, p=ns). the anesthesia time for the procedure did not significantly increase tbr subsequent transplants (8.3 vs, 7,3 hours), nor did time in the intensive care unit (t6.6 vs. 22.2 days), nor did time on the ventilator (8.4 vs. 15.3 days) subsequent transplants did not predispose to having more bleeding in the intensive care unit for usage of packed red blood cells or platalets was not significantly altered (299 vs 306 ml and 127 vs 207 ml respectively). patients who required retransplantatior~ did receive mere fresh frozen plasma (ffp)daring their first transplant than in the subsequent ones (275 vs 81 ec, p < 0.05). however ffp use was not significantly different than patients who did not require retransplant. patients who underwent retransplant had a markedly increased mortality (47%) than the overall mortality for liver transplants at our center (20%), conclusion: children who require another liver transplant have a markedly increased mortality. bleeding and prolonged icu stay is not significantly different between the first and subsequent transplants, fulminant hepatic failure and ortothopic liver transplantation.dr.sasb6n,j;centeno,m;entin,e;acarenza,m;ciocca, m:gofii,j;bianco,g;weller, g;imventarza,o. unidad de cuidados intensivos.hospital de pediatria "dr.j.p. garrahan"1245.buenos aires.argentina. introduction:fulminant hepatic failure (fhf) is a clinical syndrome, defined by the development of hepatic encefalopathy within 8 weeks from onset of illness in a previously healthy person.by far,the most comun cause of pediatric fhf in all series, is acute viral hepatitis.we report our experiences with the pediatric fhf and ortothopic liver transplantation (olt) as attemative of treatment. patients:30 childrens with fhf diagnosis were admitted at the picu from 1/1/1993 to 1/12/1995.symptomatic treatment was given to all children and all were put on list for olt,) following the king's college criterion (protrombina time,age,atiologies,bilirrubin,and encefalopathy state). results:etiologic causes corresponded to the 30 childrens were:23, hav (76%); 6, noa nob (20%);1 ,autoinmune (4%).the age was mean:4 years (range:16 month-10 years).seventeen patients were transplanted,13 chidmn were discarded because:no donors:5;withdrow of the list:3,because sepsis in 2 and bleeding of cns 1;and no admission at list:5 because genetic syndrome 1 ,massive intestinal necrosis, 1 ,mitral valvulopathy 1 and sepsis,2. 25 patients (86%) had at least one complication dudng the post operative period.the most frequent was the acute renal insufficiency(ari) and 4 patients requiered continuos hemofiltration.the gtobal mortality rate was 75%.the mortality of patients without olt was 100% and the mortality of patients with olt was 41%,4 patients dayed because sepsis, (2 candidiasis) and the others 3 because mof.the actuarial survival at 1 year is 54% and the follow up of 8 months. conclusions:the fhf is a very severe and frequent disease at picu. supportive treatment only is associated with a very poor prognosis and high mortality rate.the most frequent etiology in our country is the hav. the olt is applicable in this cases and is a valid alternative of treatment (mortality in our series 41%).the ari is the most frequent complication during the post opeative period.in argentina,due the high prevalence of hav,prevention must be considered the main and only way to avoid this catastrophic illness.to assess the efficacy of gastric intramucosal ph (phi) for evaluation of tissular perfusion and prediction of hemodynamic complications m critically ill children. patients and methods: thirty critically ill children (16 boys and 14 girls) whose age ranged from 3 month and 12 years old were studied. a tonometry catheter was placed in the stomach of all patients at their °admission in pediatric icu. intramucosal ph measures were made at the admission and each 6-12 hours during the study: a total of 202 determinations were made. the catheter was removed after extubation and/or checking of hemodyrmmic stability of the patient. the intramucosal ph was derived from application of the henderson-hasselbaeh formula using the pco2 value from the tonometer and the arterial bicarbonate. values of phi between 7.30 and 7.45 were considered normal. the relationship between phi and severity of patient measured through prism, presence of major (cardiorespiratory arrest, shock) and minor (hypotension, hypovolemia or arrhytlmtias) hemodynamic complications, mortality and stay in the picu, was analysed. results: the admission value of phi was 7.48 -t-0.15 (range 7.04-7.68). five patients (16%) had an admission phi < 7.30. no relationship was found between an admission phi < 7.30 and a higher incidence of hemodynamic complications. sixteen patients (53%) showed some values of phi < 730 during their evolution. patients with phi < 7.30 had a higher number of hemodynanuc complications than the rest (p< 0.0001). every cardiorespiratory arrest (cra) and shock cases were related to a phi < 7.30. patients with major complications (cra and shock) had a phi lower (p= 0.03), as well as a higher number of measurements of low phi (p= 0.003) than patients with minor hemodynamie complications. the value of phi lower than 730 presented a 90% of sensibility and 98% of specificity with regard to hemodymanic complications. there was no relationship between phi < 7.30 and prims score and stay in picu. patients with phi < 7.20 presented a prims higher than the rest of patients (p< 0.05). conclusions: the phi value may be an early sign of presence of hem0dyaaimc complications in the critically ill child. we tested the hypothesis that gastric intramural ph (phi) can be used as an early sign of failure m weaning pediatric patients because the blood flow from nonvital areas is diverted to meet the increased demands of respiratory muscles. methods: 24 children (mean age (4.2_+0.3) years + sd) who were thought by their physicians to be weanable from mechanical ventilation (mv.). these patients were ventilated on serve 900c ventilators, receiving ranitidine, and had intestinal tonometer (tonometrics, inc.) 60 minutes before obtaining a sample.. all children were placed on pressure support (ps) at levels judged to overcome the resistance of the endotracheal tube and ventilatory circuit (2 em h.,o). a sample of arterial blood and a sample oftonometer were obtained during vm and weaning (ps). phi, hemodynamic and respiratory data were recorded during vm and weaning we did not interfere with the primary caretaker's decisions regarding extubation. patients were considered to be successfully weaned if they were able to sustain spontaneous ventilation for more than 24 hours after extubation. paired t-test were used to compare the values obtained during mechanical ventilation with those obtained during weaning trials. unpaired ttest were used to compare values from the group that was successfully weaned (a=i5) with those from the group that were not (b=9). results: we did not find statistical differences in any of those variables mesured during mv for patients who were successfully weaned(group a) and those who were not (group b). gastric phi was in group a: 7.35 + 0.03 (vm) and 739 + 0.02 (weaning); in group b: 7.40 _+ 0.04 (vm) and 7.4t _+ 0.02 (weaning). discussion: although we did not find differences in gastric phi during vm, the group a had a lower value than group b because of the number of cardiac patients (70%) and transfusion therapy, in fins group. in group b 75% of patients showed a problem in upper airway (subglottic edema, and enlarged tonsils). we found it after extubation. conclusion: 1) gastric phi is a good predictor of risk in critically ill patients but maybe because of the small size of the sample, in our study is not of practical value as a predictor of failure in weaning pediatric patients from vm. 2) this test is not a predictor of problems in upper airway~ important etiology of failure weaning in children. objectives: i-to determine the prognostic value of the gastric intramueesal phi in mortality and multiple organ dysfunction (sdmo) in critically ill children. 2-to compare this value, with the pediatrics risk index mortality score (prims). methods: aprospective study was performed with 51 critically illcbildren, aged from 1 mouth to 16 years. the athnittiug diagnosis was: 26 post-surgery (13 neurosurgery, 9 spinal fusion and 4 thoracic or abdominal surgery), 7 sepsis, 6 polytraumatism, 5 adult respiratory distress syndrome and 8 with miscellaneous. all the subjects were monitorized on picu admission and treated for their underlying condition. gastric intramucnsal pt{ was measured following the tonometric method, ou admission and every 4-8 hours depending on the patients state. the severity of the clinical condition was evaluated using the the prims, on admission (prims-i) and during the first 24 hours, when the clinical condition deteriorate, the worse score was utilized for the statistical analysis (prims-2). to perform the statistical analysis the subjects were divided in two groups, one with the phi<7.30and the other with phi>7.30.aunivariate analysis (student's tand wilcoxon two tailed test, chi-square) and multivariate analysis were used. results: 12 out of the 51 subjects dyed. of 14 children developing multiorgan failure (mof) 9 expired. 50% of the patients admitted to the picu with sepsis, ards and miscellaneous had a phi < 7.30. in contrast, with 27 % of post-surgical and none of the postqraan~atism. the mortaliry rate, in children with a phi<7.30was 47% (ci 95%:26.16; 69,04) and 11.76% (ci 95%:4,67; 26.62) in children with phi>7.30 (p=0.011). mofwas observed in41,18% of children withphi<7.30v.s, 20.6% with phi >7.30.no relatiouship was observed between the phi and the score of prims-i and 2. perforating an unconditional logistic regression analysis, two independent variables have mortality predictive value: the phi and the prism-2. (table i) following induction of anaesthesia, a laser doppler probe (moorsoft instruments ltd) was inserted 7cm into the patient's rectum, the probe's special design ensuring that the optical prism lay against the mucosa. continuous monitoring of rectal mucosal perfusion ("flux") was continued throughout the operation. after 10 rain cpb at 35°c, "steady state" readings of nasopharyngeal temperature, mean femoral arterial pressure (map) and flux were recorded over a further 5 min before cpbinduced core cooling to 14-24°c. steady state was defined as a 5 rain period with no change in core temperatures or map. other 5 rain steady state recordings were taken immediately prior to low flow, immediately prior to rewarming and after rewarming to 35°c, before initiation of any vasoactive drugs. the cpb flow rate was kept at 100 m l k g -1 min q, the pcv at 25_+3%, the p~co 2 at 5.3+0.5 kpa and the pro2 at 20+5 kpa. results: initial warm and rewarm map (both 46 mmhg) were significantly lower (19=0.008) than during the 2 cold cpb periods (63 & 64 mmhg). the mean cold flux before (152) and after (159) low flow were both significantly lower (p=0.001) than the mean initial warm cpb flux (211). the mean rewarm cpb flux (127) was significantly lower than all other flux values (p=0.001). there were no siglaificant correlations between map and flux except at the first warm cpb period (r=0,33, p=0.04). conclusions: although hypothermia significantly reduces rectal mucosal perfusion, rewarming produces an even greater reduction in gut perfusion which, considering that mucosal oxygen constmaption is highest during this time, may prove crucial in the postoperative development of mof. therapy aimed at improving gut perfusion during cpb should be directed at the rewanning period in particular. abstract this work is aimed at establishing a clinical procedure for the diagnosis of enteritis necroticans (en), even at the communal level, and to define criteria for diagnosis able to distinguish between acute forms. subjects and method : 100 cases admitted at the institute for protection of children's health dpch), having characteristic symptoms, were examined clinically, by roentgenography of the abdominal cavity, with the analysis of the blood (total protein, electrolytes, hematocrite) and cultures of intestinal fluid and faeces. through surgical operations, the pathological lesions were observed and recorded. results: common epidemiological features: the average age is 6-8 years old (3-15) ; male/female : 1.85; in 70% of the cases, the disease occurred after a meal rich in protides. the acute toxic form accounted for 15% : severe shock appearing early, with very severe dehydration associated with profoundly decreased blood protein concentration and lowered natriemia as well. the lesions of the small intestine were expanded, all of them were necrotic. in the surgical form (20%), the predominant feature was an obstruction -peritonitis syndrome, the peritoneal fluid showed a characteristic inflammatory reaction. for the rest of cases 65% were the internal form, the shock syndrome was less severe, the abdominal distention was light and disappears gradually, the inflammatory reaction of the peritoneal fluid was not so characteristic. conclusion (ino) is a selective pulmonary vesodilator that is rapidly inactivated compared to intravenous vasodilators. these qualities make ino an attractive agent for the treatment of pulmonary hypertension (pittn). the efficacy of ino has been studied in persistent fetal circulation, acute respiratory distress syndrome (ards), and congenital heart disease (chd). potential adverse effects oflno include: nitrogen dioxide (no0 toxicity, methemoglobinemia, and platelet dysfimction. our objective was to evaluate the safety of ino in pediatric patients (pts). methods: pediatric pts. with phtn from ards or chd were studied under an established, approved protocol conforming to fda guidelines tbr an investigational new drug. informed consent was obtained for each child prior to treatment. 1no was sequentially titratad from 10 parts per million (ppm) to 20, 40, 60, and 80 ppm at ten minute intervals. parameters monitored before and during therapy included nitric oxide (no) and no~ concentrations (cone.), mean arterial blood pressure (map), and percent methemoglobin (mhg). no and noz levels were continuously monitored using an inline dr~ger electrochemical detection device. ~,litp was continuously measured with an indwelling arterial catheter. mhg was measured by co-oximetry. a mhg level e 5% or no2 cone. ~ 5 ppm were considered adverse effects by study criteria. pretreatment map was compared to map at 40 and 80 ppm ino using paired t-tests. ap value < 0.05 was considered statistically significant. results: thirty-two mechanically ventilated children with phtn (16 with ards, 16 with chd) were studied. five pts. were treated following cardiopulmonary bypass. methemoglobin (met-hb) levels were routinely measured in two prospective clinical studies on no inhalation in 25 pediatric patients with pulmonary hypertension following heart surgery with extracorporeal circulation and in 19 pediatric and neonatal ards patients, the observed differences between the groups prompted in an in vitro study, red blood cells (rbc) of 20 patients sampled before and after surgery with and without extracorporeal circulation (ecc), respectively, were incubated with 32 ppm no for 100 rain, met-hb, atp, and nadht nadph concentrations were compared, during therapeutic exposure no increased met-hb from 0.2 -2-_ 0.1 to 1.2 _+ 0.7 % in cardiac surgery patients and from 0.2 ± 0,1 to 0.5 ± 0.4 % in ards patients (p < 0.01 ). rbc's having undergone ecc were more susceptible to met-hb formation (p< 0,001 ) whereas intracellular coenzymes did not differ neither between the groups (table) nor before and after no exposure. ecc predisposes to increased methemoglobinemia upon exposure to no both in vivo and in vitro. our data suggest a reduced activity of met-hb reducing enzymes rather than diminished availability of energetic substrates, variation of the inhaled nitric oxide concentration with the use of a continuous flow ventilator. anne pmc de jaegere ~, frans im jacobs 2, nico gc laheij 2, john n van den anker t . dept. of paediatrics ~, central instrumentation 2, sophia children's hospital, erasmus university rotterdam, rotterdam, the netherlands. objective: to investigate the homogeneity of nitric oxide (no) concentration in a delivery system with a continuous flow ventilator. design: bench study, setting: biomedical laboratory. interventions: a nitrogen/nitric oxide (njno) gas mixture was injected at three different sites in the patient circuit: just before and just behind the humidifier, and 20 centimetres before the y-connector. ventilator flow (12, 15, 20 l/rain), ventilator rate (40 to 110, increments of 10) and compliance of the testlung (0.36; 0.5; 1.0 ml/cm h20) were changed. carbon dioxide (co2) instead of n2/no was injected at the same points in the circuit. measurements and main results: a) though the flow ratio of the njno and the ventilator gas were kept constant, the no concentration ([no]) raised with increasing ventilator rates. the increase in [no] was up to 40% when the n2/no injection site was close to the y-connector of the ventilator circuit. minimal changes in [no] were noticed when the n~/no was mixed to the ventilator gas before the humidifier. b) analysis of the ventilator flow pattern showed variations at different places in the ventilator circuit. the magnitude cf the p, ow change depended on the meas~:rement site. the closer to the expiratory valve the highest the flow change was. the duration of the flow change was inversely proportional to the adjusted ventilator flow. c) real time measurements of the co 2 concentration ([coz]) showed variations during tile respiratory cycle. these [co2] variations were higher when the co2 gas was blended closer to the yconnector. conclusions: the ventilator flow variations in relation to the fixed side flow of the n2/no gasmixture result in changes of the inhaled [no] during the respiratory cycle. the no concentration during inspiration is always higher then during expiration. this could not be detected with the available monitoring system. to ensure a constant [no] by blending a njno gas balance in a continuous flow ventilator, the site of injection should be as close as possible to the inspiratory outlet. nitric oxide, a potent and selective pulmonary vasodilator, has recently been successfully used to treat pulmonary hypertension of variable etiology in infants and children. side-effects and complications in infants are so far not well known. we describe here two cases in which prolonged (5 and-7 days respectively) high-dose (50 -80 ppm) nitric oxide was used to treat refractor~¢ pulmonary hypertension. one patient was a newborn infant with pulmonary hypertension secondary to a large leftsided diaphragmatic hernia. nitric oxide was begun under conventional ventilation (babylog 8000) at 7 hours of life with a slight initial improvement in oxygenation. he was then placed on oscillation with the same nitric oxide concentration due to worsening respiratory failure. he died on 5th day of life. monitored nitric dioxide concentration never exceeded 4 ppm. the other patient was a 3 months old infant with severe pulmonary hypertension due to a complete atrioventricular septal defect. he required high-dose nitric oxide to come off cardiopulmonary bypass after surgical repair of his heart defect. he slowly improved over the week following surgery but developped suddenly respiratory failure due to massive pulmonary hemorrhage and died. surprisingly, a particular autopsy finding in both infants was a massive acute necrotizing tracheobronchitis. we conclude that nitric oxide is an excellent and sometimes lifesaving treatment of pulmonary hypertension in infants. tracheobronchitis has not yet been reported as a possible complication of nitric oxide administration. we suggest that caution needs to be taken with prolonged high-dose administration and this possible complication to be looked for at autopsy. introduction: permissive hypereapnia (ph) is a beneficial strategy for patients with acute respiratory distress syndrome (ards) to minimize barotrauma by decreasing the peak inspiratory pressure (pip). hypercapnia and hypoxia cause pulmonary vasoconstriction, pulmonary artery (pa) hypertension, and, thus, an increased afterload to the right ventricle. this increased afterload may result in increased right ventricular (rv) work load and subsequent rv dysfunction. one therapeutic approach is the use of inhaled nitric oxide (inn), a selective pa vasodilator. the objectives of this study were to test the hypothesis that in a swine model of ards with ph, inn would improve rv work load and not change intrinsic rv contractility. methods: in 11 swine (25-35 kg), ards was induced by surfactant depletion. hypercapnia was achieved by decreasing the pip while increasing the peep to maintain a constant mean airway pressure, inn was administered in concentrations of 2, 5, and 10 ppm in a random order. pulmonary blood flow (qpa) was determined by an ultrasonic flow probe. rv total power (tp) and stroke work (sw) were calculated by fourier transformation of the pa pressure (ppa) and qpa data. preload recruitable stroke work (prsw), a preload and afterload independent measure of ventriculur contractility, was determined by a shen-subtraction method and vena caval occlusion. respiratory failure with pulmonary hypertension in piglets gerfried zobel*, bernd urlesberger*, drago dacar**, siegfried rtdl*, fritz reiterer* and ingeborg friehs** depamnents of pediatrics* and cardiac surgery**, university of graz,austria objective: to evaluate gas exchange, pulmonary mechanics and bemodynamic data during partial liquid ventilation (plv) combined with inhaled nitric oxide (no) in acute respiratory failure with pulmonary hypertension. design: prospecfive~ randomized, controlled study. setting: university research laboratory. subjects: twelve piglets weighing 9 to 13 kg. interventions: acute respiratory failure with pulmonary hypertension was induced by repented lung lavages and a continuous infusion of the stable endoperoxane analogue of thromboxane. thereafter the animals were randomly assigned either for plv or conventional mechanical ventilation. initially perfhiorocarbon liquid (30ml/kg) was instilled into the endotracheal tube over 5 min followed by 5-10ml/kg~. all animals were treated with different concentrations of no ( 1-10-20 ppm) inhaled in random order. measurements and results: continuous monitoring included ecg, cvp, mpap, map, san2 and svo2 measurements. during plv pao2/fio2 increased significantly from 62_+3.2 mmhg to 193±44 mmhg (p<0.01) within 10 rain, while pao2]fio2 remained constant at 61 -+3.3mmhg. qs/qt decreased significantly from 48-+4% to 25-+5% (p<0.01) during plv and did not change during conventional mechanical ventilation. static pulmonary compliance (cstat) increased significantly ff~m 0.4r±0.07 to 0.75_+0.03 ml/cmh20/kg (p<0.01) during plv and decreased slightly from 0.58_+0.08 to 0.46e0.04 ml/cmh20/kg during conventional mechanical ventilation. the infusion of the endoperoxane analogue resulted in a sudden decrease of pao2/fio2 from 262_+44 to 106_+8.0 mmhg in the plv group and from 71±7 to 52+_2.0 mmhg in the control group. inhaled no significandy improved oxygenation in both groups (pao2/fio2:344_+38 mmhg during plv and 196+_.56 mmhg during conventional mechanical ventilation). during inhalation of no mpap decreased significantly from 57-+2 m 35±2 mmhg (p<0.01) in both groups. there was no significant change in oxygenation and mpap during inhalation of 1 and 20 ppm no. conclusions : plv significantly improves oxygenation and pulmonary compliance in acute respiratory failure. the additional application of inhaled no further improves oxygenation and pulmonary hemodynamics when acute respiratory failure is associated with severe pulmonary hypertension. inhaled no is very effective in improving oxygenation and pulmonary blood flow even at low doses. the work was supported in part by grants of the austrian nationalbank nr 5545. as in neonates, severe respiratory failure in infants and children can be aggravated by pulmonary hypertension, resulting in further deterioration of oxygenation due to increasing intrapulmonary shunting. we analysed the influence of inhalational nitric oxide (ino) in treatment, course and outcome of severe ards in a pediatric population. since 1993 20 infants and children (age: 1-107 months) with ards and oi > 15 (mean value: 32.5± 11) underwent a trial with ino (concentration: 3, 10, 30, 60 and 100 ppm) to prevent further respiratory failure. 11 patients had a significant improvement of their oxygenation (rise of pa09 > 15 mm hg) for at least 24 hours (responders); mean best ~fficient no dose: 24.6 ppm. the non-responders had only a short-term improvement or ino had no effect. in responders and nonresponders there was no significant difference with regard to age, underlying disease, ards severity, time on mechanical ventilation, blood gases and ventilator settings before notrial, nor was there a different grade of pulmonary hypertension (estimated by echocardiography). the only difference was an higher ol in the group of the non-responders: 40.9 ± 9.i vs. 25.6 ~ 6.7, p < 0.002. in the group of the 11 respenders there was a secondary deterioration of lung function after i -6 days on ino in 5 children (transient responders): in these patients, as well as in the group of the non-responders, alternative modalities of treatment (hfov and/or ecmo) became necessary. 6 children (30 %) died: 2 transient respenders and 4 non-responders. in infants and children with ards due to different underlying diseases ino can acutely lead to a significant improvement of oxygenation in about 50 % of the cases. the right selection of patients for no therapy and the influence of ino on the survival rate of ards in childhood has to be evaluated in further studies. and pediatric cardiology, university of graz, a-8036 graz purpose: after fontan procedure cardiac output is critically dependent on the pulmonary vascular resistance. even minor elevations of the pulmonary vascular resistance may significantly decrease cardiac output. inhaled no is an effective, selective pulmonary vasodilator in experimental and clinical situations of pulmonary hypertension. the aim of this study is to evaluate the effects of inhaled no on oxygenation and pulmonm 3, circulation in children after a bidirectional glenn-anastomosis (n-~) or a fontan-like operation (n=9). material and methods: from june t993 to january 1996 13 children with a mean age of 7.1+~2.1 (sem) yrs and a mean body weight of 24.3-+5.8 (sem) kg were treated with inhaled no after glenn-or fontan-like operations. all but one had complex cardiac malformations with single ventricle. all children were mechanically ventilated with an fin2 >0.75. inhaled (no) was applied using a rrdcrdproeessor based system which additionally allowed measurement of no/nox using the chemihimniscence method. methemogtobin concentrations were determined 3 times a day. the major indication for postoperative inhalation of no was a high (>10mmhg) transpulmonary pressure gradient (tpg--cvp-lap). severe myocardial dysfunction of the single ventricle was excluded by echocardiography. results: the mean duration of mechanical ventilation was 8.1_+2.2 (sem) days the. mean dose of inhaled no was 4.4-+0.8 (sem) ppm, the mean duration of no-inhalation was 106_+19 (sem) hours. the mean methemoglobin concentration was 1.2-+0.2 (sem)%. hemodynamic data and arterial oxygen saturation before inhaling no and 15 minutes later are given in table 1 acute hypoxaemic respiratory failure (ahrf) in children occurs in a heterogenous group of diseases with pulmonary pathophysiological processes ranging from reversible physiological intrapulmonary shunting to fixed structural lung damage. we hypothesized that inhaled nitric oxide (ino), a selective pulmonary vasodilator, might identify those patients with potentially reversible disease, i,e, large response may indicate a greater likelihood ef reversibility and thus survival. a retrospective review of the early response to ino in 30 infants and children (aged 1 month to 13 years, median 7 months) with severe ahrf(18 with ards). the mean p(a-a)o2, pao2 / fio2, oxygenation index (oi) and acute lung injury (all) score prior to the commencement of ino were 568 +_9.3, 56 +_2.3, 41 _+3,8 and 2.8 +_0.1 respectively, the magnitude of response to ino was quantified as the % change in oi occurring within 60 minutes of 20 ppm ino therapy. this response was compared to patient outcome data. results. there was a significant correlation between response to ino and patient outcome, kendall tau b r=0,43, p<0.02 (table) conclusion. in ahrf response to ino appears te define a subgroup of patients with improved outcome compared to nonresponders. we speculate that response to ino may be useful in selecting patients with potentially reversible lung disease for special support therapies such as ecmo. randomised controlled trials are needed to define the role of ino in paediatric ahrf. between may 1994 and december 1995, 22 patients (pts) were treated for mas. treatment groups were: group i only 02:6 pts; group i1 conventional mechanioal ventilation (cmv): 11 pts; group ii1 hfo: 1 pt; group iv hfo+no: 4 pts. therapy was stepwise intensified until oxygenation improved ( i -) ii -) iii --) iv). "high volume strategy" was used with hfo (mawp 18-24 cm h20). the initial no-concentration was 20-30 ppm, with rapid reduction down to 5-10 ppm once oxygenation improved. results: one pt (group it) died of hypoxic-ischemic encephaiopathy (termination of therapy); all other newborn babies survived. in group iv pt 1 and 2 showed barotrauma prior to hfo. pt 1,2 and 4 were treated with additional mgci2 (max. mg serum concentration 2.8 -6.5 mmol/i). following the identification of inhaled nitric oxide 0"no) as a selective pulmonary vasodilator (frostell et al 1992) [ 617 .+6,3 626+6.3 data are compared to baseline values within each group. *=p<0.05, **=p<0.03, ***=p<0.0l among 12 patients who fulfilled ecmo criteria, 6 improved with no and did not required extracorporeal life support. tltree out of 6 ecmo patients eventually survived. conclusions: m our study low-dose of irthaled no showed a variable effect on oxygenation in newborns with acute respiratory failure. an acute response to no appeared to be correlated with a better short-term outcome and the avoidance of extracorporeal support in ecmo candidates. differently, lack of acute and/or sustained response was associated with death or need for ecmo. although the nature and severity of the underlying disease or the degree of prematurity may play an important role in these patients, we believe lack of acute response to no may be an early predictor of bad outcome, prompting toward alternative treatments such as ecmo or liquid ventilation. *picea s., °bartuli a.,°dionisi-vici c., *dello strologo l., §villani a., §bianchi r., ^salvatori g.,*rizzoni g, °sabetta g. *div. of nephrology, °div. of metabolism, §intensive care unit, ^div. of neonatology. "bambino gesfl" children research hospital. rome, italy. successful prevention of handicaps or death in newborns with ~ depends on rapidity and efficiency of treatment. poor response to nutritional and/or pharmacological treatment requires extracorporeal removal of nh4. efficiency and cardiovascular tolerance are often difficult to obtain with peritoneal or hemodialysis in neonates. we report the results of cavhd in 3 newborns with hc. methods: vascular access: femoral vessels. blood flow: 10-35 ml/min, dialysate flow: 200-500 ml/h. filter: amicon minifilter plusrm(polysulfone membrane; 0.08 sq.m.). no ultrafiltrate(uf) production, patients: case 1 with carbamoytphosphate synthetase deficiency (body weight -bw-: 3.2 kg) showed hc at day 4, a relapse of hc occurred at day 14 due to an infectious event. case 2 and 3 (bw: 3.0 and 2.8 kg), both affected by propionic aeidemia, showed hc at day 5 and day 7, respectively. plasma nh4 (~tg/dl) decrease is shown in the complications: transitory ischemia of arterial cannulation limb and transitory thrombocytopenia occurred in case 1; surgical repairing of artery after cavt-id was necessary in case 3; no cardiovascular instability was observed during cavhd . outcome,'all patients recovered from hc in less than 1 day: case 1: alive, mild b)iootonia at 34 mos; case 2: dead after 10 days from cavhd withdrawal for pulmonary hemorrhage; case 3: alive, normal development at 7 mos. conclusions: 1) in newborns with hc, ca~q-id provides good cardiovascular tolerance,high efficiency and quick removal of nh4, even without uf production (i.e. only by diffusion). this allows easier management (no need of fluid and electrolyte balance). 2) arterial complications seem frequent in neonates treated by cavhd. venovenous circulation could overcome this problem. vb nguyen, m jokie, c leeaeheux paediatric intensive case service, hospital university centre, avenue c6te de nacre, 14033 caen cedex, france background, the implication of polymorphonuclear neutrophils (pmns) in the physiopathology of children's haemolytic.uraemie syndrome (hus) becomes more and more evident. the purpose of the present study is to role out their impact among other pronostie elements during the course of the disease. patients and methods. diarrheal prodrome and its duration, patient's age, maximal blood nitrogen level, anuria and dialysis time, extra.renal involvements, white enll and pmn counts and thrombopenia duration have been retrospectively analysed in 18 infants with good outcome and in 8 another children with unfavorable outcome. results. neither diarrhoea or its duration, nor children's age, nor blood nitrogen level, nor anuria or dialysis time had any predictive value for the disease evolution in the acute phase of our patients. adversely, extra-nenal involvements was accompanied by severe and complicated courses of the disease (p<0,02). the elevation of white cells and pmns (heyon 20 x 109/i) and pmns (more than 15 x 109/1) as well as its persistence beyon a week were most frequently observed in complicated forms (p<0,001, p<0,001 and p<0,01, respectively). a transient thrombopenia (less than 5 day@ in patients with elevated counts of white cells may be a filrther obvious sign of an unfavorable course of the disease (13<0,02). conclusion. the elevated count of white cells and pmns, either alone or associated to one rapid regeneration of platelets, seems enabled to predict an unfavorable evolution of the hus in children. msud results from an inherited impairement of catabolic pathway of branch chain amino-acids. high leucine blood levels may induce acute brain dysfunction. this dramatic complication led us to propose leucine removal procedures as continuous hemofiltration. patients and methods three newborns in acute msud onset were treated by hf, hdf and hd. extracorporeal circulation was performed through a 6.5 fr catheter, a circuit with a blood pump (priming volume = 40 ml). patients and procedures characteristics are summarized below in the sucralfate (an aluminium salt of sucrose octa sulfate) is used to prevent and treat upper gastrointestinal bleeding in critically ill patients. with minimal absorption, the potential for side effects is thought to be limited, though aluminium toxicity has been reported in patients with chronic renal failure. these patients may already have had high body stores of aluminium. we report 5 critically ill children with high serum concentrations of aluminium following sucralfate therapy. all 5 had renal impairment. the normal aluminium level is < 0.4 gmol/l and in patients with chronic renal failure < 2.2 ].tmol/l. none of these patients had known preexisting chronic renal disease. cpb was conducted under deep hypothermia (t,°16°c) and cardiocirculatory arrest (cca) or under hypothermia (t,°24°c) and low-flow perfusion. continuous holter-electrocardiograms (h-ecg) were recorded from the ilranediate postoperative (po) period on for 72 hours. h-ecg were also recorded prior to the operation and before discharge. following dr were observed: snpraventricutar (sv) and ventricular (v) extrasystoles (es) (>50/24h), sv and v tachycardia (svt and vt), accelerated junctional rhythm (ajr) and junctional ectopic tachycardja (jet), and 2nd and 3rd degree atrioventricular block (avb2 and avb3). the incidence of po dr was 20% in the pre-op h-ecg, 74% on the 1st, 33% on the 2rid, 34% on the 3rd po day and 21% befbre discharge. compared to the pre-op findings, an increased incidence of sves, ves, svt and avb3 on the 1st po day was observed, whereas vt and a jr or jet were exclusively observed po. all types of dr were observed up to the 3rd po day. ty23e of dr before discharge was similar to pre-op findings and there was no definitive avb3. considering patient groups according to the most frequent isolated op-procedure, the incidence of dr on the first po day was 56% after asd ii-closure (n=23), 74% after stthaortal vsd-closure (n=lg), 75% after correction of a complete avsd (n=8), 80% after correction of a tetralogy of fallot (n=20) and 100% after fontan-operation (n=10). incidence and type of dr were not significantly different between groups. longer cpb-dttration and use of cca were risk factors for po ves and vt (p<0,005 and p<0,05, respectively) whereas use of cca and degree of hypothermia were risk factors for the development of a jr and jet (p<0,02 and p<0,0001, respectively). -our results indicate that po dr after cpb in children m'e frequent but mainly transient. in our series, specific cpb-related parameters are of greater influence than surgical procedure itseif for the development of dr and are discriminant risk factors for particular types of dr. the course of anp, cgmp/anp (as indicator for atrial natriurefic peptide biological activity), and no2 and no3 (as indicator for endogenous nitric oxide (no) synthesis) was investigated in i9 infants (median age 4 months) undergoing cardiopulmonary bypass (cpb). patients were divided into 2 groups according to whether they had (group 1, n=13) or not (group 2, n=6) preoperative heart failure (hf) and pulmonary hypertension (pht). group 1 patients had preoperatively significantly higher levels of anp (p<0.005), cgmp (p<0.02) and no2 and no3 (,p<0.02) but had significantly lower cgmp/anp (i0<0.05) than group 2 patients. during cpb, anp was significantly higher in group 1 patients ~<0.02). as compared with prebypass values, cgmp/anp was reduced in both groups during cpb (p<0.0001). cgmp/anp inversely correlated with duration of cpb and aortic clamping time (p<0.001, respectively). no2 and no3 were significantly higher in group 1 than in group 2 patients (p<0.05) without any intraindividual change during cpb. from the early postoperative period on anp, cgmp/anp and no2 and no3 were similar in both groups. after cpb, anp correlated in both groups with blood pressure (p<0,001) and diuresis (p<0.05). no2 and no3 inversely correlated with pulmonary arterial pressure immediately after cpb (19<0.05 patients after a fontan-type of procedure have elevated central venous pressures (cvp) leading to congestion in the gastrointestinal system and often ascites. purpose of this study was to evaluate whether this causes a different postoperative gastric mucosal ph (phi). methods: we evaluated a series of 35 patients, who underwent cardiac surgery with cardiopulmonary bypass (age: 5 days to 16 years (mean 2,2 yrs), weight: 3.2 to 37kg (mean 10.2 kg). a commercially available tonometer (tonometics®) for sigmoidal use in adults was inserted into the stomach after induction of anesthesia. the phi measurements were done according to manufacturer recommendations we compared three groups of patients: 1) aeyanotic (n=20), among them 9 p with vsd and 5 p with avsd; 2) cyanotic (n=10): tof: 6p, tga: 4p; 3) cyanotic after a fontan-type procedure (n=5). phi were measured at picu arrival and after 6h. fudhermore we compared lactat levels at these time points. differences between the groups were evaluated with one way anova on ranks with pairwaise multiple comparisons (dunn's method). the relationship between cvp and phi was investigated by regression analysis. results: the median phi for groups i, 2 and 3 were 7.28, 7.27 and 7.13 at ardval and 7.30, 7.25 and 7.21 after 6h respectively. at picu arrival group 3 was significantly (p<0.05) different from groups 1 and 2. there was no significant difference between the latter two groups, after 6h group 1 was different from group 3, there were no other significant differences. the median lactate levels for groups t, 2 and 3 were 2.2, 3,2 and 4.1 at ardval and 1.6, 3.1 and 3.3 after 6h respectively. at ptcu arrival group 3 was significantly (p<0.05) different from group 1, after 6h there were no significant differences. there was a weak negative correlation between cvp and phi: r= -0.21; p<0.05. conclusion: patients after a fontan-type of procedure have lower phi than patients after other cardiac surgical procedures, however, this is only in part due to the elevated cvp and venous congestion. eleven children were investigated 32 months (median) after postoperative mof. iviof was defined as the failure of at least two vital organ systems (kidney, liver, lung, central nervous system) in addition to cardiac insufficiency and high fever. underlying surgical procedure was repair of tetralogy of fallot (n=3), fontan-(n=7) or seuning procedure (n=l). all patients fulfilled criteria for mof in the 3 first postoperative (po) days. six patients needed peritoneal or hemodialysis for 31 days (median) during the po period. one patient showed cerebral infarction due to thromboembolism in the territory of the right internal carotid artery immediately after the operation. the follow-up protocol consisted of extensive investigations of heart-, renalliver-, and lung functions as well as complete neurological and psychological examinations. all patients had adequate cardiac examination. lung function was normal in all but 2 patients who had an obstructive syndrome. only 1 patient showed an isolated decreased creatinine clearance. abnormalities of the liver ftmction tests were only noticed in patients after fontan procedure. severe neurological sequels such as paraplegia (n = 1) and diplegia (n-i) were observed in 2 of the 11 patients. the remaining 9 children presented with a delayed graphomotorical and speech development associated with normal intelligence. -in our series the most frequent and severe sequels after postoperative mof were neurological. -abnormal liver fimction tests are more likely to be a consequence of the fontan hemodynamics than a sequel of mof. the optimal dosing schedule of surfactant therapy for the treatment of neonatal respiratory distress syndrome (rds) remains unclear. goal: surfaetant function and the concentration of phospholipids (pl) in tracheal aspirates are compared in a prospective randomized trial involving neonates with rds who received either two or more (3 or 4) doses of survanta. methods; ventilated neonates <35w with rds were treated with survanta 1oo mg/kg if fio 2 >_40% or mean airway pressure _>7,5 cm hzo, after 6h a 2nd dose was given (same criteria), if the support still exceeded the criteria 12h after the 2nd dose, the patient was randomized to no extra dose (two}, or to an extra dose of survanta (morel (and a 4th dose 12h later; same criteria), pl was measured in tracheal aspirates and corrected for dilution with the urea method. "active" large aggregates and "non-active" small aggregates of surfactant were separated by centrifugation and quantified. surface tension of the large aggregate fraction was measured by pulsating bubble surfactometer, results: 13 neonates were randomized, 6x two and 7x more (5x3 and 2x4 doses), gestational age was 31,7±2,4w and birth weight 1582±568g. most patients had severe rds with initial ventilation: rate 63.1_+11,1, peak inspiratory pressure (pip) 24,3-+6.4 cm hzo, fio 2 75.3±21.0%. at randomization: rate 63.5±6.9, pip 20.3-+2.5 cm hzo, fio 2 29.5±15.7%, and 24 h after randomization: rate 45.9±17.1, pip 18.7_+2.2 cm hzo, fio 2 26.8±6.6%, without signif, differences between the groups. there was 1 relapse (again fio2_>60% within 72h) in group two and t bpd in group more. in total, 112 tracheal aspirates were analyzed. pl was not signif, different before randomization (two 27.5 ± 15.7 vs more 24.5 ± 11.4 /jmol/ml), but neither after randomization (two 21.2-+ 11.0 vs more 19.3±7,o /~mol/ml). there was no difference in the % small aggregates (two 4.2±1.9 vs more 6.9±5.5%), the surface tensions (ran/m) were not signif, different (each time two vs more): before randomization 10.0±2,3 vs 14.2-+7.2, in the 24h after randomization 12.6±5.0 vs 11.2-+3,8, or 24-48h after randomization 17.0-+5.5 vs 12.8±9.8, or 48-72h after randomization 15.7_+0.4 vs 13.7-+5.6. conclusion: neonates who received more than two doses of survanta did not have higher pl, nor a better surfactant function than neonates who received only two doses of survanta. continuation of the trial is necessary to evaluate clinical outcome. may not indicate need for treatment p.c. clemens s.j. neumann university of hamburg, department of pediatrics, klinikum schwerin, wismarsche str.. 397, d-19049 schwerin. aim of the study: the finding of elevated tsh and decreased t4 in the newborn usually is classified as "transient hypothyroidism", thus the elevation of tsh is classified as consequence of the lowered t4. but on the other hand several data sets show that tsh elevation as well as low t4, one independently of the other one, are associated with different kinds of perinatal stress. each of these laboratory deviations, if not associated with the other value being abnormal too, is generally accepted not to be an indication for treatment. from this we conclude, that more pefinatal stress, as in intensive care neonates, may produce tsh elevation as well as low t4, but only coincidentially, not the tsh elevation being the consequence of low t4, thus not to be classified as "hypothyroidism", thus not indicating treatment. if this hypothesis is right, we should find an association of increasing pefinatal stress with an increasing number of neonates from tsh and t4 normal via tsh or t4 abnormal to high tsh and low t4. method: in the newborn screening program in germa w we determine primarily tsh, and only in the neonates with elevated tsh, in addition we determine t4. thus in our study we asked whether we find an association of increasing perinatal stress with an increasing number of neonates from tsh normal via tsh abnormal while t4 normal to high tsh and low t4. definitions for this study were: tsh elevation = >20 mu/1 (as usual in the german screening programs), t4 lowered = < 6 p_g/dl perinatal stress score was 0 or 1 or 2 or 3 in dependency of the neonate having stress in none to all of the following three categories: (a) forceps or vacuum extraction or sectio co) birth weight below 2500 g (c) at the 5th day existence of a relevant neonatal disorder (rds, ictems gravis, infection/sepsis, vitium cordis with hemodynamic relevance, severe malformation). results: our data of 1131 neonates show a high significant association (chi2 = 84, p <0.001) of, on one hand, perinatal stress score 0 with normal tsh, versus, on the other hand, perinatal stress score 2 or 3 with high tsh and low t4. discussion: facing the background given above, in the intensive care newborn, the constellation of high tsh and low t4 may be only a coincidential addition of two independent abnormalities. in tbese cases -the high tsh not being the consequence of low t4 -the classification as "hypothyroidism" is not justified, thus a therapy not indicated. on the other hand of course there exist rare cases with high tsh as consequence of low t4 thus with hypothyroidism tlms with indication for therapy. unfortunately we have no criteria, that enable a certain discrimination of these two categories thus in respect to the question of therapy or not. conclusion: further research has to be done to learn how to discriminate the coincidential high tsh and low t4 from the causal constellation of high tsh and low t4. until we have certain discrimination criteria we have to treat both groups of neonates. few studies have focused on fa composition of surfactant pc in preterm infants before and after surfactant therapy. methods: tracheal aspirates were collected in 7 venttlated mfants from birth until extubatlon (27/7_1 /twk ga, 859.+ 155g bw). after lipid extraction, t.l.c,, and methylation, fas of pc were quantified by gaschromatography. intralipid a (53.2 % linoleic acid,18:2•6) was started 48h after birth. results: six infants developed respiratory distress syndrome (rds) and received survanta r i00mg/kg (sr), all doses within 18h after birth (ix s r n=l, 2x s r~ n=3, 3x s r n=2). one child did not develop rds. in alt patients, the patmitate % in pc was ~ 65% (before sr<=natural composition), increased to ~ 85% after s r, and remained >80% for i5h after lx s a, 22.3.+i1.8h after 2x, and 38.5.+3.3h after 3 doses. in 4 patients, intubated long enough, the palmitate % decreased with a half-life of 78.7_+42.8h to a new plateau which was still higher than baseline after 1 week. linoleic acid % was 5.85_+2.3 (with rds), decreased after s r~ and returned to baseline due to the decrease in patmitate %. thereafter the linoleic acid % increased linearly with 0.021% per h, in 1 patient even up to 15.1%. other fas did not increase after return to baseline. in neonatal medicine the current parameters, arterial oxygen saturation and arterial oxygen pressure, are poor indicators for oxygen delivery and oxygen demand. the purpose of this study was to obtain venous blood samples from the inferior vena cava in stable neonates with respiratory failure and to determine a parameter that reflects more adequately the balance between oxygen delivery and oxygen demand. "l~e study included 22 neonates requiring mechanical ventilation tbr severe respiratory insufficiency. an umbilical venous and arterial catheter were inserted in the inferior vena cava and in the aorta respectively. paired blood samples were obtained at the time that the patients were hemodynamically stable. fifty paired arterial and mixed venous blood samples were analyzed. 1jnear regression analysis showed the following correlations: in a neonatal intensive care unit adjacent to a delivery room caring for 4000 mothers per year, (with a referral of 400 mostly for preterm delivery), virtually every neonate <ls00g was seen by the same ophtalmologist, j.o. the incidence of retinopathy of prematurity (rop) was in 1976-80 of ii cases (464 preterms <ls00g with 58% surviving to discharge, and 4% rop in survivors; 8% if less than 30 weeks). in 1987-1993, 57 cases of rop were diagnosed by the same ophtalmologist. 679 were born at less than 30 weeks, of which 452 (66%) survived to discharge, and among those survivors, rop was present in 53 (12%), with rates varying from 58% at 24 weeks to 5% at 29. objective: to compare a method of teicoplanine (teico) infusion using an electrical syringe-pump with a manual injection. methods: infusion of teico was simulated through a standart neonatal i.v. system. the infusion system consisted of an life care 4 infusion pump (abbo'it lab.) with its i.v. set for maintenance intravenous fluid (flow < 6 mi/h) connected to a 3-way stopcock. an 1 meter extension tubing (vygon lab.) was placed between the stopcock and a neonatal catheter, an another 1 meter tubing (injection tubing) was connected to the stopcock. the volume of the injection circuit was 2.6 ml. simulations were realised for 2 weights (1 or 3 kg), with a doses of 8 mg/kg and a injected volume of 0,8 ml to 3 ml. our goal was to perform a complete infusion in 10 minutes. we compare one method of infusion with an electrical syringe-pump with 2 manual methods: a: teico was infused with a syringe-pump pilot c (becton & dickinson lab.) according to a protocol using 1) a priming before connecting the syringe to the tubing (for immediate starting of infusion), 2) a programmed volume injected in 5 minutes (the drug volume was greater than the dose prescribed to avoid loss of teico into the syringe), 3) a 5 ml wash out was performed over 5 minutes with the syringe pump. b: teico was manually injected in a few seconds in the tubing followed by a 5 ml wash out over 10 minutes. c: idem as b but a nu site valve (medex) was connected to the proximal end of injection tubing to avoid leakage between removal of the teico syringe and connection of the wash out syringe. during each run, serial samples were collected every ten minutes over a one hour period. the samples were assessed using hplc method. results: the amount of drug delivred at 10 minutes was calculated and expressed as a perventage of the total amount of teico prescribed. results are a mean of 5 to 8 rons. a b c 1kg 94.2+17,9% 86,8+9.1% 94,4-+6.4% 3kg 95-+4,9% 72-+13,4% 90.9-+6% conclusion: with a drug volume injected < circuit volume and a valve, a direct i.v. administration of the drug in the circuit followed by a wash out seems an accurate alternative to drug infusion conbroled by a syringe pump and is easier to perform. doxapram hydrochloride is a central and respiratory stimulant which is used in neonatal intensive care units to treat caffeine-resistant apnoes of the newborn. routinely, doxapram is administered intravenously. oral administration would be much easier but data on bioavailability after oral administration are limited (two studies) and show a large variation. we therefore studied the oral bioavailability of doxapram in 8 preterm infants. doxapram treatment was started with an intravenous loading dose of 2,5 mg/kg during 15 minutes, followed by a continuous infusion of 1 mg/kg/h. after 24 h the intravenous administration was changed to continuous oral administration with the same dosage, blood samples were collected 24 h after both the intravenous and oral administration and analyzed by hplc-assay. apnoea rates per 6 hours were listed before and after starting doxapram treatment. the median apnoea rate per 6 hours declined from 9 to 2 after both the intravenous and oral administration. the oral bioavailability of doxapram is between 43 and 65%, indicating that the oral route can be used effectively in these infants 2. there were no differences in the decline of the apnoea rate after intravenous versus oral administration of doxapram, infasurf r has been comnared with exosurf r in two studies: one as drodhvlaxis and the other a rescue trial. similar. althouah non-sianificant benefits were found for the natural surfactant. in 6 trials there was a sianificant reduction in dulmonarv air leaks for 0.53: 95% ci 00.41-0.641 for babies treated with natural comnared to svnthetic surfactants. for 7 trials (3554 babies~ comdarina natural and synthetic surfactants for rds {6 comdarina survanta r and exosurf r. and one infasurf r and exesurfr% neonatal mortalitv was sianificantlv reduced (or 0.80: 95% ci 0.66-0.971 with natural compared to synthetic surfactant treatment. in two further trials different natural surfactant drenarations were compared. reduced oxvaen needs for 24 hours after treatment were found for r r infasurf and curosurf respectively when each was compared to survanta r. andarent lonaer-term benefits from these surfactants were not statisticallv sianificant. further trials will be needed to ascertain differences between various surfactant preparations. introduction bacterial meningitis is a common cause of admission in the paediatric age group, and one which still carries a high morbidity and mortality. these complications are strongly associated with a delay in antibacterial therapy. a significant proportion of these patients require mechanical ventilation; it is in this particular group of patients that survival is greatly diminished. the identification of risk factors for mechanical ventilation associated with bacterial meninges may help improving survival by shortening the delay to icu referral. the paediatdc risk of mortality score (prism) is the most widely used scoring system in the paediatric icu literature; the accuracy of prism on predicting outcome in meningitis has been shown to be poor. aim we aimed to describe our population of patients with bacterial meningitis requiring ventilation, and then to identify predictors of survival. methods this study was conducted at the baragwanath hospital in south africa which is an universityaffiliated institution with end average annual paediathc admission of 4500 patients. baragwanath icu admits 250 non-neonatal paediatdc patients per year. a retrospective chart review from january 1991 to december 1995 of 42 consecutive paedistdc icu admissions with bacterial meningitis was performed. results approximately 150 cases of bacterial meningitis are admitted to the general paediatric wards every year; this constitutes ± 3% of all admissions. the mortality of these patients is + 14%. during this time 42 (23m, 19f) patients (7%) were accepted for icu admission, with a mortality of 42.2%. the median age was 9 months (range 15 days to 17 years). the median delay to hospital admission was 48 hours, and the median delay to icu admission was a further 4 hours. the median duration of tcu stay was 48 hours (40 for non-survivors, 120 for survivors). the main presenting features were convulsions (34%), altered mental state (40%), fever (50%), respiratory symptoms (24%), headache (18%), and diarrhoea and vomiting (32%). the most common indications for icu admission were seizures (55%), coma (36%), shock (26%), respiratory failure (29%) and acidosis (21%). in 29% of patients there was a cardiorespirstory arrest prior to admission. the most common organisms in the csf was pneumococcus (57%), haemophilus influenza (15%) and e coli (12%). the presence of leucopenia (wcc < 5), a low platelat count (<100), acidaemia (ph<7.2), and the need for inotropic support were strongly associated with nonsurvival. tachycardia and hypotension were not significantly essorjsted with poor outcome, as has been previously reported. discussion early diagnosis of meningitis and prompt institution of antibiotic therapy requires a high level of clinical suspicion. paediatric patients with bacterial meningitis that require mechanical ventilation have a poor prognosis. there is little evidence to suggest that nomsurviva[ can be predicted prior to icu admission, but a rapid deterioration requiring ventilation and inotrepic support with evidence of severe sepsis (low platelet count, leucopenia) is nmost invariably associated with a fatal outcome. the long term functional outcome of these patients make this disease even more devastating; the rote of icu in the management of these patients has yet to be fully estanished. objective and study design: a retrospective study was pertbrmed for all patients diagnosed with hemorrhagic shock and encephalopathy syndrome (hse) during 1984 to 1994. soroka medical center is the only medical facility in the southern negev region of israel serving a population of -400,000 residents, consisting primarily of jews and bedouins. results: 20 patients (17 bedouins and 3 jews) were diagnosed with hse. main features on arrival included profound shock and coma with con~alsions. active bleeding and/or disturbing coagulation tests with falling heuroglobin levels and thrombocytopenia was noted in every case. all infants developed diarrhea shortly after arrival. elevated urea, creatinine and liver enzymes was noted in all cases. annual incidence tbr inlhnts <1 year of age was 5:10,000 for bedouins and 0.6:10,000 for jews. patients ranged in age from 6-32 wks and arrived at the hospital late night/early morning (2:00am-ll:00am), during winter/early spring (november-april). all were healthy prior to admission, with short prodromal symptoms of upper respiratory tract or gastrointestinal infection noted in 10 cases. most infants had markedly elevated body temperature on arrival. a history of overwrapping and/or excessive hearing was obtained in 4 of 20 infants. bacteriological and virological cultures were negative in all infants. one infant died and neurological sequelae were observed in all survivors. the high prevalence of hyperpyrexia during sleep in the presence of negative microbiological results with no evidence of excessive hearing, and the high incidence of rise among a closed, culturally isolated society known to have a high incidence of congenital malformations, may support previous assumptions that hse results from hyqperpyrexia, originating in most cases from a "physiologic" heat induced trigger which starts and peaks during the night in previously healthy infants with susceptible, predisposing genetic underlie. approximately 10% of hospitalised infants with respiratory syaacytial virus (rsv) infection require mechanical ventilation. our general practice is not to use specific antiviral therapy. we have undertaken studies of rsv-infected mechanically ventilated patients without underlying congenital heart disease or immunodeficiency. study i (n=45): a retrospective review of 45 infants (mean post-conceptual age 48 weeks; median duration of intubation was 8 days (interquartile range 5-11 ). blinded review of chest x-rays during the first three days of mechanical ventilation revealed a spectrmn of lower respiratory tract findings from marked diffiase consolidation in all zones without hyper-inflation (n=10) to gross hyperinfiation without consolidation (n=5), with the remaining patients have an intermediate picture (n=30). death occurred only in patients who were at the poles of this continuum (4/10 consolidation and 1/5 hyperinflation). patients at these poles could be differentiated by gender predominance, birth gestation, alveolar-artarial (a-a) oxygen gradient (p<0.0 l), oxygenation index (p<0.0 l), peak inspiratory pressure (p<0.05), and intubation days (p<0.01). study ii (n=28): prospective audit of infants with the aim of verifying the above differentiation. separating patients based on their early x-rays and a-a gradient we confirmed the above predictable difference in duration of supportive therapy (consolidation v intermediate: 6(4-7) v 14( 12-33 ) p<0.01 ). severe lower respiratory tract rsv-infecrion in young infants results in different distinctive partems of disease with characteristic radiological and clinical features, and each has a predictable timecot~se. conflicting reports on special therapy should be interpreted with respect to these observations before making conclusions about the efficacy of any specific treatment. the clinical data of 186 children wich suffered from a severe mycosis between jan.1990-dac.1994 and wich were treated with amphotericin b in 10 spanish hospitals were collected. mean age was 6+3.9 years; 157 were mate (84%) and 29 female (16%). the most common underlying diseases were leukemimymphoma (31%) and congenital heart defects (20%). the major pathogens isolated were candida albieans (61%), other candida (18%) and aspergillus (17%). and liposomal amphotericin (la) in 68 (37%). in the ca group, the starting dose was 0,3~0,1 mg/kg, reaching a maximal dose of 0.9+0,3 mg/kg after 4.7+3 days. maintenance time was 14+10 days and the cumulative dose: 12~17 rag. in the la group, the starting dose was 1.1±1 mg/kg, reaching a maximal dose of 2.9_+2.4 mg/kg (p<0.01) after 5+5 days. maintenance time was 19~16 days (p<0.05) and the cumulative dose 42_+37 mg (p<0.05). the reasons for la use were: elective in 66% of cases, previous renal failure in 29%, and ca inefficacy in 4%. hepatic or renal function impairment was two times more frequent in la group than in ca group, the antifungal efficacy was 62% for ca and 67% for la. therapeutic failure or toxicity induced withdraw of ca in 7% of cases, vs, 1,5% for la (p<0,01). mortality was not different in both groups (21 vs. 22%), adverse effects were related to ca in 28 events vs, only one in la group (p<0,01). the commonest side effects in ca patients were fever (18%), chilis (10%) and nausea (9%). in ca group, 44 cases (37%) developped analitical anormalities related with amphotericin, vs. 10 cases (15%) in la patients (p<0.001). in the first group, the commonest serum alterations were hypokaliemia (25%), raised creatinine (14%) and bilirrubin (6%). in the second one, hypokaliemia presented in 7% of cases (p<0.01), raised bilirrubin in 6% and creatinine in 3% (p<0,05), the antifungal efficacy of la is at least the same of ca. from the clinical and analitical points of view, la is much less toxic than ca. la can be used at a greater dose than ca and is safe in children with renal failure. laurence desplanques -serge gottot -christian dageville d~artement de sant~ publique -facult~ xavier bichat -16 rue henri huchard 75018 paris -france -the french << reaped ~> network was created to implement a nosecomial infections (ni) quality care program in nicu and picu, the first objective was to describe the annual ni incidence rate in each icu population : all patients stayed more than 48 hours in icu. methods : n] criteria were defined by the reaped group according to cdc criteria. all data were collected by a medical and nursing team. all infection data were validated by an external investigator. results : 4525 patients were admitted over a 14 months period. 68% were newborns. 371 ni were identified among 311 patients. the overall ni incidence rate (ir) was 8.2% and 5.9°/00 person day (from 5.0 to 8.2°/00 according to age, lowest rate for newborns). septicemia (50% of ni) and pneumonia (41% of ni) were the two main ni. according to age, the septicemia ir varied from 6.8 to 10.9°/oo catheter day (lowest rate for newborns) and the pneumonia ir from 3.9 to 7.4°/00 ventilator day (lowest rate for newborns). there were very few other infections (uti : 4%, ir : 7.4°/00 catheter day). gram positive cocci were isolated in 73% of septicemia ( 70% of them were coagulase negative staphylococcal). gram negative bacilli were isolated in 53% of pneumonia (40% of them were pseudomonas). 5% of ni were caused by candida, mostly septicemia. the septicemia and pneumonia ir varied according to unit even after adjustment for age. discussion the aminoglycoside antibiotics are frequently used in newborns for the treatment of severe infection and sepsis due to gram-negative microorganisms. the currently recommended dosage schedule for tobra (2.5 mg/kg q18h) does not take into account differences in gestational or postnatal age during the first 4 weeks of life. we questioned the validity of these recommendations and studied the population kinetics of tobramycin to establish predictive equations that enables the clinician to select the appropriate initial dosing schedule. methods tobra trough (t=0) and peak values (t= 1) were taken on day 2-4 after birth in 460 newborns. tobra was administered as a 30-minute intravenous infusion already in an adapted dosage schedule: 3.5 mg/kg q24h in infants with gas < 28 weeks; 2.5 mg/kg q18h in infants with gas between 28-36 weeks and 2.5 mg/kg q12h in infants with gas > 36 wks, tobra concentrations were analyzed by tdx-assay, a one-compartment model was assumed and non-linear mixed effect modelling (using nonmem) was applied to the data, a trough level < 2 mg/l and a peak level between 6 and 10 mg/l was required, with the present dosage scheme 40% of the trough levels were too high and almost 60% of the peak levels too low. calculations showed that the following dosage schedule should result in optimal levels of tobra. preterm infants gas < 28 wks: 6 mg q48h preterm infants gas 28-36 wks: 4.5 mg q36h preterm infants gas > 36 wks: the currently recommended dosage schedules for toeira result in high trough and low peak levels. prolongation of the dosing interval and increasing the amount of drug per dose according to the above scheme will improve tobra level control. since january 1993 british clinicians have been conducting a randomized controlled trial of neonatal ecmo. mature infants (>-35 weeks gestation and birthweight 2 2 kg) with severe cardiopulmonary failure have been randomized to receive continued care in their referring institution or referral to a designated ecmo centre for further management. we now present the preliminary results which have prompted closure of recruitment to this trial. the final outcome will be assessed as intact survival against death or severe disability at one year of age for all the recruited patients. patients were categorised by diagnosis such as isolated persistent fetal circulation, secondary persistent pulmonary hypertension of the newborn or congenital diaphragmatic hernia and by severity of illness at the point of first contact with the clinical coordinators of the trial -judged primarily by the oxygenation index (240 before randomization). 180 patients were randomized (90 in each arm). hospital outcome data are reported for all patients and 1 year outcomes on t18 (65 survivors). at this stage 26 of the babies allocated to ecmo are known to have died compared to 52 of those allocated to conventional management (rr 0.5; 95% ci 0.35-0.72; p=0.0002). fewer deaths have been obsea-ved amongst ecmo allocated babies in all the diagnostic categories used. a 28% incidence of disability and impah~nent has been observed amongst survivors. this rate is similar in both groups and the survival advantage is not offset by an increased rate of disability or impairment following allocation to ecmo. we consider that these data combined with those available from other studies provide conclusive evidence that the survival to discharge from hospital is substantially higher in patients allocated to ecmo than in comparable infants not so allocated. therefore recruitment to this trial has been closed whist awaiting complete one year outcome data. sigston pe, goldman ap. #keating j. crook r. ~e dj~. great ormond street hospital for children nhs trust, and ~biochemistry department, kings college hospital, london, united kingdom. isoflurane is a safe and effective means of long term sedation in both children and adults in the intensive care setting. the use of isoflurane, by adding it to the sweep gas allows the use of this volatile anaesthetic agent in patients on ecmo, enabling rapid control and weaning of sedation. a potential problem with the long term use of isoflurane is fluoride ion accumulation with the possibility of renal toxicity, the purpose of this study was to assess plasma fluoride levels in patients receiving prolonged isoflurane on ecmo. method: fifteen infants and children (aged 1 day -10 years, median 2 weeks) receiving ecmo support for either cardiac or respiratory failure were recruited to this study. the patients were sedated with isoflurane as well as intravenous agents (morphine and midazolam). isoflurane was administered (0% -3%) via a calibrated vaporiser to the sweep gas, adjusting the level to maintain adequate sedation. blood samples were obtained on a daily basis for plasma inorganic fluoride assay. the relationship between plasma fluoride and amount of isoflurane administered, as %-hours (vaporiser setting in % x hours) was calculated by linear regression. results: the duration of ecmo ranged from 42 to 532 (mean 207) hours, during which the amount of isoflurane administered varied from 7 to 418 (mean 168) %-hours. 75 blood samples were anaiysed, demonstrating individual peak plasma fluoride levels of 2.7 to 16.5#mol/1, mean 7,1p.molli (toxic threshold = 50gruel/f). the plasma fluoride positively co;related with the %-hours of isoflurane (r = 0.65, p = < 0.001). conclusion: this study shows that although there is a dose related accumulation of inorganic fluoride ions in patients sedated with isoflurane on ecmq, the peak fluoride levels are well below the suggested toxic threshold. merzel y, lev a, bar yosef g, halbertal m, lorber a ecmo center, picu, emek medical center, israel. the mortality rate of pediatric patients with acute myocarditis is 20-60% according to the severity of myocardial damage. a 15 month old gzrl presented with high fever, respiratory and cardiac failure. diagnosis of acute myocarditis was made and the patient was ventilated with high pressures and fio2 of 1.0. she required high doses of inotropes. echocardiography revealed a dilated la and lv with severe mr. lvedd was 41 mm and lvsf 9%. calculated oxygenation index was 55. she was resuscitated after a cardiac arrest. she was commenced on ecmo (using biomedicus centrifugal pump and avecor 800 oxygenator) at a flow of 100 ml/kg/mm with immediate improvement of hemodynamlcs, oxygenation and pc02. resptratory assistance and vasoactive drugs were reduced. the patient was transported by air, on ecmo, to the ecmo cevter. she developed arf and cvvh-d was performed. cardiac fimction started to improve after 12 days. ecmo was discontinued on day 18. echo revealed lvedd 34 mm and lvsf 24%. ippv was discontinued on day 20. on discharge, a month later, her lvedd was 29 mm and lvsf 28%. she behaves normally for age without neurologic or other medical sequellae. literature search revealed no case of acute myocarditis, as severe, that was treated successfully. survavors of disease this severe usually suffer dilated cardiomyopathy and permanent disability. the use of ecmo allows myocardial rest which prevents long term myocardial damage. introduction ecmo is increasingly used in the care of critically ill newborns. despite the frequent use of betalactam antibiotics in the treatment of these infants there are no data available on the dispbsition of cefotaxime (ctx) and amoxicilfin (am) d0ring ecmo. the purposes of this study were to determine the pharmacokinetics of these two drugs in infants on ecmo and consequently formulate appropriate dosing regimens. we therefore studied the pharmacokinetics of ctx (100 mg/kg ql 2h) and am (50 mg/kg q6h) in 8 term infants on day 3 after birth, blood samples were taken before (t-o) and 0.5,1,2,4,6 (am) and t2 h (ctx) after the intravenous bolus injection and analyzed by hplc-assays. 2. ctx 100 mg/kg q12h results in adequate serum levels of ctx in fullterm infants on ecmo, am 50 mg/kg q6h results in very high serum trough levels. recalculation based on the known volume of distribution and elimination serum half-life of these infants resulted in the following dosage recommendation: 50 mg/kg q12h. persistent pulmonary hypertension of the new-born (pphn) is characterised by rapid fluctuations in pulmonary artery pressure (pap) and a clinical impression of stifflungs. lung mechanics were measured in 35 term infants, mean age 1.5 +_ 0.7 days who were paralysed and ventilated within the first three days of life. fourteen infants had pphn with systemic or suprasystemic pap measured by echocardiography. in these patients, the respiratory system resistance was 29.4% higher (p < 0.001) and compliance 22.4 % lower (p = 0.03) during systemic or suprasystemic pap compared to when the pulmonary hypertension had resolved. in contrast, there were no changes in resistance in the 14 infants with respiratory distress syndrome (rds) and no pulmonary hypertension or in the seven infants with normal lungs, where two readings were taken 24 hours apart. the changes in lung mechanics interfered with mechanical ventilation, resulting in a 12.5 mmhg rise in paco2 (p=0.007) during pulmonary hypertension. inhalation of nitric oxide 10 ppm resulted in a 16% decrease in respiratory system resistance and an improvement in oxygenation. the bronchial and vascular smooth muscle was increased by 120% in postmortem lung samples from eight infants with pphn compared to six age matched post-mortem controls with normal lungs (p<0.001). these findings suggest a co-constriction and co-hypertrophy of bronchial and vascular smooth muscle during pphn. anatomically the pulmonary vasculature and bronchi lie in close proximity to each other. thus mediators such as endothelin-1 released locally may act on both vascular and bronchial smooth muscle to produce the observed vasoconstriction, bronchoconstriction and smooth muscle hypertrophy. prince of wales children's hospital university of new south wales, randwick, n.s.w. australia. introduction an increasing mortality in asthmatic children has been reported. the increased severity of asthmatic illness leads to an increased demand for icu admission, and a corresponding increased need for mechanical ventilation. geographic end environmental factors are thought to be partly responsible for differences in disease sevedty throughout the wodd. for this reason, epidemiological studies from diverse areas are important, risk factors for icu admission, and for the institution of mechanical ventilation should be identified, to optimise icu admission criteria and to avoid unnecessary delays in admitting at-risk patients. aim to document the clinical characteristics of ventilated and non-ventilated asthmatic patients admitted to icu. methods this is a retrospective study of all paediatric asthma icu admissions from january 1990 to december 1995. results there were 65 patients admitted to the icu for acute severe asthma in the study period. the male:female ratio was 33:32, the mean age 76.1 • 57.3 months, the mean prism 8.5 4-11.1%, and the mean duration of admission 135 4. 129 hours. there was no seasonal variation in admissions. only 40% (26/65) patients required mechanical ventilation. in 22% of all patients this was the first presentation with asthma. there were some significant differences between ventilated and non-ventilated patients (see table) . there was a significantly higher incidence of concomitant and nosocomial pneumonias in the ventilated patients (84.0% vs 21.1%) as well as segmental lung collapse (68.0% vs 26.3%). there were no deaths. discussion the need of mechanical ventilation significantly increases the morbidity of and duration of icu stay of asthmatic patients. younger asthmatic paediatdc patients have a significantly higher risk of ventilation. the need for ventilation is predicted principally from a worsening pco2 and respiratory acidaemia, which is often independently interpreted by the clinician as respira4ory exhaustion. this study has shown that icu admission is important in the management of young paediatdc patients with acute severe asthma and respiratgry fa!!ure. intravenous salbutamoi in the emergency, department management of severe asthma in children. g.j.browne,a. perma,x. phung,m.soo westmead hospital, sydney, australia. it is postulate that if an initial intravenous loading dose of salbutamol is given in severe asthma, a more rapid clinical response will occur, reducing requirements for continued high doses of nebulised salbutamoi with fewer side effects. this double blinded study was conducted in the emergency department of westmead hospital a university hospital in sydney, australia. all children with severe asthma had initial nebuliser therapy (5rag of salbutamol with 4ml of saline). if asthma remained severe 20 minutes later, they were given a dose of intravenous hydrocortisone (5mg/kg) and either normal saline or salbutamol 15microgm/kg intravenously. frequent nebulised salbutamoi therapy continued during the initial first hour if clinically indicated. continuous respiratory and haemodynamic monitoring occurred in the first 2 hours. serum potassium and glucose determinations were made at study commencement and 1 hour after intravenous therapy. salbutamol determination was made at study commencement. children remained clinically monitored for the next 22 hours, with their ongoing treatment determined by clinical response. 29 children with severe asthma 12 months to 12 years of age were studied, with 14 given intravenous salbutamol and 15 given intravenous saline. the intravenous satbutamol group (ivsg) showed rapid reduction in asthma severity scale in the first 2 hours, with reduced need for high frequency nebuliser therapy ( _<2 hourly), occurring 8.78 hours.earlier. no clinically significant side-effects were found in either group, although, tremor more frequent in the [vsg. biochemistry and salbutamol concentrations were similar in both groups. the use of intravenous salbutamol (i5 microgm/kg) in the management of severe childhood asthma is a safe and effective therapy with no significant side-effects and the potential to abort severe asthma attacks in the emergency department. intravenous terbutaline in picu piva j., amantra s, rosso a., zambonato s, giugno k, maia t. introduction: the admission to a picu of children with respiratory failure secondary to an acute obstructive lower airway disease is a common event, especially during winter seasons. these diseases have several causes, but most of them (especially asthma and chronic airway disease) have a good response to the administration of b2-adrenergic drugs. objective: to find the dosis of intravenous terbutaline that is safe, efficient and with minimal adverse effects when used in children admitted to a picu with acute obstructive lower airway disease and respiratory failure. material and methods: we study the records of all children that were admitted to our picu during the winter of 1995. only the patients that had respiratory failure and acute lower airway disease and who needed the use of iv terbutaline were selected. the records were divided in two groups: less than 12 months and more than a year old these two groups were compared in the following aspects: the minimal and maximal dosis, and the length of time of use of iv terbutaline, frequency of tachycardia, hypokalemia, and mechanical ventilation. to establish any difference in the two groups we use the t exact test of fisher and x2, with p< 0.05, results: during the period of study were admitted 367 patients to the picu, and 38 (10,3%) of them used of iv terbutaline. the mean age was 14.2 +12.2 month, used iv terbutaline during 7.24 +3.75 days (0.5 to 17 days), the initial rate was 0.55 +0.26p~g/kg/min, and the means of therapeutic dosis was 2.48 +l.181~g/kg/min (ranged from 0.5 to 4.4). twelve (31.5%) patients had tachycardia art obstacle to the increases in the rate of use of iv terbutaline during any time. mechanical ventilation was necessary in 22 patients (57.8%) and 11 (28.9%) patients died. the children under 1 year of age used initial dosis of iv terbutaline lower than the children up of 1 year old (0.45 p.g/ kghnin x 0.57 ~tg &g/rain, p<0.001), but without difference in the length of use, the maximal dosis, the rate of mechanical ventilation and tachycardia. the frequency of hypokalemia was most common in the group of children under year of age. acute respiratory failure during status asthmaticus may require mechanical ventilation. current therapy includes paralysis, pressure control ventilation (pcv) and permissive hypercapnia to limit pulmonary barotranma and its hemodynamic consequences. asthmatic children exert a significant amount of respiratory effort during exhalation. with paralysis, this expiratory effort is lost. unloading the inspiratory work of breathing while maintaining the patient's expiratory eftbrt using pressure support ventilation (psv), may be beneficial. methods: children receiving pcv (peak inspiratory pressure (pip) = 4 kpa. rate 10 breaths/min) and pco2 > 8 kpa were switched to psv. children were initially ventilated with psv 3.7 kpa and peep = 0.3 kpa (servo 900c). all children received beta agonist therapy, ipratropium and anesthesia with ketamine or inhalational anesthesia, and were breathing spontaneously. respiratory parameters and blood gases are shown be~bre psv, within 30 minutes (start) and when the ph had normalized (during). data are presented as median and range, * p < 0.03 compared to before psv. results: children with hypercarbia during pcv responded to psv, normalizing pcos and ph within 6 hours. the mean respiratory rate decreased from a median of 45 (31-46) to 35 (22-35) while the pip was decreased to 3.2 (2.5-4.0) kpa within 6 hours. the i:e ratio also significantly decreased. conclusion: psv permitted patients to active/y exhale while unloading the inspiratory work of breathing. perhaps this strategy shifts the patient's respiratory effort from inspiration to exhalation, thus permitting the child to meet the excess work of breathing caused by bronchoconstriction. maged z. youssef, peter silver, laura nimkoff, and mayer sagv. division of pediatric critical care medicine, schneider children's hospital, new hyde park, ny 11040. introduction: mechanical vemiladon of patients with severe bronchospasm can be difficult, due to poor chest compliance and increased airway resistance. ketarmne is a cormnonly used anesthetic agent that has been shown to have bronchodilator properties. the purpose of this study was to determine ifa continuous infusion of ketamine had an effect on the oxygenation and chest compliance of children with severe lironchospasm who were mechanically ventilated. methods: a retrospective chart review was conducted of pediatric patients in severe bronchospasm who were mechanically ventilated in our picu and treated with a continuous ketamine infusion. all patients were receiving aggressive bronchodilator therapy and adequate sedation prior to keramine. patients were excluded if any new bronchodilator or sedative agents were started within 24 hours of initiation of ketamine treatment. all patients were simultaneously treated with benzodiazepines. for each patient, the pao2/fio ~ ratio and dynamic compliance [tidal volume/(peak imp. pressure -peep)] was determined immediately prior to ketamine, and at 1, 8, and 24 hours post-ketsmine initiation. data are presented as mean ± s.d., and were a~yzed using one way anova and the multiple comparison method of bonferroni. patients (age 6.0 ± 5.7 yrs.) received * p<0.05 ketamine for severe bronchospastu during mechanical ventilation in our picu. both . .xto-* * the pao2/fio2 ratio and dynamic . . -.... . compliance increased significantly following initiation of the ketamine 200infusion (see figure) . the mean ketamine dose was 32 ± 10 mcg/kg/min, and the -, mean infusion duration was 40 ± 31 too-[/ hours. one patient required glycopyrrotate 6 ~' to control excessive airway secretions, and " one patient required an additional dose of o--j i ~-~4 ~/me diazepam to control hallucinations after i 8 cessation of ketamine. all patients were t~n~,mr~ *~am~ successfully weaned off mechanical ~l~s ~,~s~on ventilation and discharged from the picu. conclusion: continuous ketamine infusion to mechanically ventilated pediatric patients with refractory broncliospasm results in a significant improvement in oxygenation and dynamic compliance of the chest. reports of adults with status nsthraaticus document significant morbidity and mortality, whereas studies in children have had more varied results. different centers report mechanical ventilation (mv) in 10 to 33% of admissions, occurrence of pneumothoraces or paeutuomediastinums in 2 to 11%, and mortality in up to 7% of patients ~'t3. we retrospectively reviewed 113 status asthmaticus admissions to the pediatric intensive care unit (picu) between january 1993 and december 1995. seventy-five of these patients were admitted fr~an the emergency department of chla (er admit). the mean length of stay in the picu was 2.1 days and the mean length of stay in the hospital was 4.6 days. based on 95 patients who had arterial blood analyses, 36 patients had hyperoapnia (pco2 > 45). all patients received oxygen, inhaled albuterol (alb), and cortieosteroid therapy. ninety-five percent of patients also received methylxanthine (mx) therapy. of the 113 admissions, 12 patients (11%) required mv. only 4 of these patients were admitted through our emergency department, whereas the remaining 8 patients were intuhated at outside facilities. twenty-three cases required intr:wenous beta-agonist therapy, either isoproterenol osop) or terbutaline (terb). h~ff of the ea.~es re~%wed were complicated with hypokalemia (k+< 3.5). c,', ,~lications ofpoeumothoraces or pneumomediastinums were seen in 10% of ,'r:u~ported patients, but in only 4% of er admit patients. only 2% of these were in mechanic.all, )atients. there were no deaths in the review. respiratory mechanics measurements 'are useful in mechanically ventilated children to optimize ventilator settings. nevertheless, the transducers used to measure flow (f) and pressure (p) remain expensive. objective. to evaluate the performances of piezoelectric p transducers (350 us dollar) in measuring f and p. methods. we used a previously described monitoring system measuring respiratory parameters [ 1] . in this study f was obtained by a differential piezoelectric p transducer (_+ 12.7 cmi-i20, honeywell) whose sensitivity has been reduced to +_ 2 cmh20 by an electronic amplification equipment and p by a piezoelectric p transducer (_+ 7().3 cmhzo, honeywell) connected to a grid pneumotachymeter &nt) ffleisch 0 or 1 ). volume (v) (5 to 400 ml) obtained by numeric integration off (0.125 to 10 l/rnin ) and p (2 to 70 cmh20) were respectively delivered through a calibrated seringe and an electronical manometer (pic 400 premier) and calculated by the computer. bland and altman analysis was used for assessment of results bias. coefficient of repeatability (cr) was estimated by the standard deviation of repeated measurements of the parameters as calculated in a oneway analysis of variance. results. mean difference (mdi 0 between injected v (5 to 50 ml) and measured v using pnt 0 was 0.15 ml, sd = 0.13 ml. difference and mean v were not correlated. sd of repeated v measurements were not correlated to v. cr was 0.4 ml. mdif between injected v (25 to 400 ml) and measured v using pnt 1 was 3 lrd, sd = 6 ml sd of repeated v measurements were not correlated to mean v. cr was 6 ml. mdif between injected p and measured p was 0.3 cmi-i20, sd 0.4 cm h20 sd of repeated p measurements were not correlated to mean p. cr was 0.3 cmh20. conclusion. inexpensive piezoelectrical transducers can be used to measure f and p and evaluate respiratory mechanics in ventilated children. previous studies have already shown the problem of the reproducibility of pft in preterm ventilated babies. were studied 10 preterm ventilated babies {mean weight 1128 gr) in the first week of life in clinically stable condition, measuring flow, airway pressure and esophageal pressure simultaneously. each baby was studied twice with an interval of one hour and each study was done increasing the rate till 60 to inhibit spontaneous breaths. none sedative has been used. only mechanical breaths were analyzed. compliance and resistence were calculated with a computer system using the linear regression method. we expressed quantitatively the intrapatient variability as the percentage of variation of tidal volume, compliance and resistence between the two studies in each baby. then intraclass correlation coefficient test (icc) was applied to confirm qualitatively our results (total agreement =1, good reproducibjtity > 0.75). we h~£ed, an a6eept~ble ~efiabirl¢, ~-~r;= '~ . during mechanical ventilation, an air leak (al) and plateau phase duration (pl) may influence dynamic and static compliance (cdy and cst, respectively). this study evaluated the effect of al and pl on two methods of measuring c.dy and est. methods. 13 intubated, ventilated patients in a pediatric intensive care unit were evaluated after obtaining informed consent. patients were intuhated with a cuffed endotracheal tube and ventilated with a serve 900(2 ventilator. cdy and cst were determined using the serve ands~rmedics 2600. objective: evaluate the repercussion in respiratory mechanics and arterial blood gases and the impact of the ventilator adjustments on the auto-peep magnitude. material and methods: the measurement of the auto-peep was performed using an eletronic-pneumatic controlled device with a oclasion valve installed between endotracheal canutla and the ventilator circuit. the d~'ice was connected to a solenoid to detecte the end of inspiratuo phase and thus, the activation of the oclusion valve. the signs of pressure and flow were monitorized using a diferential transducer and it was processed using a pc computer and tmeumoview® software. the stud3 were divided in 2 phases: phase a. where the ventilator adjustments was performed using the routine of the unit and phase b, where the targets of mechanical ventilation were to minimize the auto-peep. static compliance (crs) was ineasured by the single-breath occlusion technique, using a mean of ten occlusions for analysis. passive respiratory resistance measurements and the tidal breathing flow-volume loops were also obtained., while the ventilatory settings were siguificantly reduced soon atier ecmo was started. before ecmo crs measured in all patienls was 0.23_+t).03 ml/cmh20/kg (mean_+sem). for each patient the ecmo course was divided into four periods, proportional to the duration of the treatment, and the best ~alue of crs in each period was chosen for analysis. as shown on the figure. crs significantly improved (*p<0,05) from the second half of the ecmo course in the group of patient that finally were successfidly weaned from ecmo. no change ill compliance was measured in the group of patients who failed to respond to the extracorporeal hmg support our data suggest that compliance measurements during ecmo can be useful togelher with overall clinical evaluation to predict both outcome and duration of cxtracorporeai support in the neonatal and pediatric population. objectives: brain temperature determines the amount of neuronal damage caused by hypoxic insults. thus measuring brain temperature at standardised conditions is in request. we investigated whether brain temperature of neonates varies with head insulation environmental temperature, body activity and time course. patients and methods: we investigated non-invasive brain temperature analogues in 19 healthy prematures tess than two weeks of age in an incubator (gestational age 31.5 + 2.1 wks; x + sd, weight 1653 + 370 g). we measured nasopharyngeal temperature (tnasoph) by a thermistor placed in the nasopharynx via a feeding tube, zero-heatflux temperature (zht) at the temple by a thermistor and healflux transducer, insulated by two pads, as well as rectal and incubator temperatures. patient activity was documented by video taping. measurements were performed during periods of increased insulation 1) by turning the head with its measuring site on to the mattress ( (5) 3 (5) -2 (6) 4 (5) 0 (6) 4 (3) 2.5 2 4(5) 25(10) 20{12) 8(5) 5(10) -2(7) 5 6 38 (22) 112(34)i70 (48)51(27) 20 (18) 5(15) 7.5 3 38 (19) 125(21) t85(29) 120(30) 70(30) 30(20) 10 4 53 (30) 133(28) 182(33) 157(24) 154(34) 110(45) web 2170 (lmg/kg) 5 at 30 rain 3 3 (5) -4 (6) 5 (6) 4 (3) 5 (4) -3 (6) the vehicle had no effect. paf caused dose dependent rise in ao and pa pressure and reduction in flow to lpa (up to 80% like the vascular endothelium, the endocardial endothelium (ee) has a significant impact on adjacent myocytes, and may critically alter myocardial function.~ we have previously shown that ee cells are capable of sensing and responding to hypoxia by the release of prostacyclin (pgl). 2 potassium channels in other cell types have been reported to be oxygen sensitive. to determine whether potassium channels modulate the ee hypoxic response, we investigated the effects of three potassium channel inhibitors on hypoxia-induced pg] 2 release from ee cells. methods: ovine endothelial cells were harvested and passaged onto 30 ,~ microcarriers. cells were constantly perfused with normoxic and hypoxic kreb's solution, and with three potassium channel blockers: glibenclamide (gb, 3 #g/ml), tetraethyl-antmonium (tea, 10 ram) and 4 aminopyridine (4ap, i0 mm), perfusate was assayed for prostacyclin (ria). data were compared by analysis of variance. * p<.05 compared to 3normoxic control; # p< .05 compared to hypoxic control. adrenaline is extensively used for resuscitation in neonates with rds. however, effects of adrenaline on systemic, pulmonary and cerebral hemodynamics have not been defined in newborns with rds. thirteen anesthetized, and ventilated newborn piglets were subjected to repeated saline lung-lavage series while mean systemic arterial pressure (abp), mean pulmonary arteriat pressure (pap), mean left atrial pressure (lap) and mean central venous pressure (cvp), cardiac output and blood flow in the internal carotid artery (ica) were measured. systemic vascular resistance (s~), pulmonary vascular resistance (pvr) and cardiac index (ci) were calculated. sixty minutes after luug-lavage, the adrenaline group (a) (n=6) received adrenaline as a continuous infusion of 1.2 lag/kg/mi, while the control group (c) (n=7) received saline. none of the varlables were changed by saline. however, significant increases in abp (p<0.0001), pap (p<0.0001), ci (p<0.001) and svr (p<0.01) were observed after administration of adrenaline, whiie pvr and ica were not modified. mean±sd for abp/pap (p/a), fvr/svr (p/s) and ci (ml/mirdkg) were: ratios of pap/abp and pvpjsvr significantly increased following infusion of adrenaline. these data suggest: 1) the cerebral perfusion is preserved during the infusion of adrenaline; 2) effect of the adrenaline infusion on the systemic circulation is more pronounced than its effect on the pulmonary circulation in newborn piglets with surfactant deficiency. s demirak~a, ch knothe, kj hagel, j bauer department of pediatrics, justus-liebig-university giessen, frg inhaled no is a short acting selective pulmonary vasodilator. we studied the effects of 80 ppm no and 100% oxygen during heart catheterization in 16 children (age 1 -6 years, median 9 years) with heart defects and elevated pulmonary vascular resistance index (pvri) in order to asses the value of no as a tool of decision making for corrective cardiac surgery. patients were eligible for testing when they were more than one year old and had a pathologically elevated pvri in a previous heart catheterization. intubation, 'anesthesia and muscle paralysis were performed in all patients during testing of pulmonary reagibility. calculations of pulmonary vascular resistance and flow were based on the fick method. response to no was assumed when pvri declined more than 30%, 9 of the 16 patients were responders to no. effects of no and oxygen on pvri, mean pulmonary arterial pressure (mpap) and pulmonary vascular flow (qp) in all responders are described in the table below. cardiac surgery was offered to all responders, and 5 of them were successfully operated. surgery is planned in another 3 patients and parental consent for surgery was not given in one patient. in ebstein disease, during the first days of life, the ability of right ventricle to propel blood to the pulmonary artery is impaired due to high pulmonary vascular resistances. the flow is mainly directed to left atrium through tricuspid insufficiency, right atrium and foramen ovale. to decrease pulmonary resistances and increase pulmonary blood flow, high frequency oscillations, mechanical ventilation, nitric oxide and prostaglandin are required. after few days, a forward circulation is normally established. we cared two newborns with ebstein disease where this approach was hindered by a large pulmonary valve insufficiency. both of them were diagnosed in utero, showing a large tricuspid insufficiency with a non opened pulmonary valve and a ductal left to right shunt. one fetus was hydropic. at birth, blood stream from the ductus arteriosus was directed to the right ventricle through the pulmonary valve insufficiency then to right atrium, left atrium and ventricle, aorta and ductus arteriosus. a low pulmonary blood flow was demonstrated by low mean velocities (10cm/sec). a high reverse flow was seen in descending aorta with a negative flow in the renal artery. both of these newborns were oliguric because of ductus arteriosus steal. pulmonary blood flow doppler evaluation allowed different strategies of ventilation, switching between hfo and conventional ventilation, modulation of pge1 doses, inhaled pulmonary vasodilators (nitric oxide) and surfactant. the hydropic baby died, the other survived after 3 weeks of intensive care complicated by supraventricular arythmia (wpw). in conclusion, during neonatal period, in ebstein disease, a large pulmonary insufficiency leads to a vicious circle where lungs are excluded, inducing severe asphyxia and high pulmonary resistances. the blood is backward propeled from the aorta through the ductus arteriosus to the right ventricle and atria, then left cavities to aorta. arec must be considered when pulmonary blood flow does not increase despite optimal therapy. guti~rrez-larraya f*, mandoza a*, velasco jm*, zavaneua (3**, gatindo a ~, s&nchez-andrede r, s&nchez jl***, mellon a***, mar f***. pediatric cardiology*, pediatric cardiac surgery**, pediatric intensive care unit***. hospital 12 de octubre. madrid. background: transesophageal pacing (tp) is effective and sate both for diagnosis and treatment of pediatric arrhythmias. material and methods. eleven consecutive patients are included. a tri or quaddpolar 6 or 7f temporal transvenous catheter with an interpolar distance of 13 to 22 mm was advanced through the nares and positioned to the point with the largest amplitude of atrial deflection, surface ecg and a bi or monopolar electregram were recorded simultaneously, selecting filters when needed (5 to 100 mhz). pacing was performed with a programmable stimulator (medtronic 5328) beginning with 2 ms and increasing ma to 10 and then increasing up to 9.9 ms. narula method was selected to diagnose sinusal node disfunction (snd) and overdrive pacing to treat tachyarrhythmias. results. tp was useful in all the 11 patients and no complications were observed: in 3 patients a snd was diagnosed (one needing a definitive pacemaker), in two patients with atrial ratter (ripe 1) sinus rhythm was recovered, in one patient with a postoperative junctional ectopic tachycadia we were able to get atrial synchrony with marked bemodinamic improvement, and 5 patients with paroxysmal supraventricular tachycardia sinus rhythm was easily and quickly restored (2 of them recquirad repited episodes of tp until pharmacelogycal levels of antiarrhythmic drugs were raised). mean age and weight were 31 months and 12.7 kg (one patient had 2.1 kg). there was a close relation between height and depht insertion (r= 0.98). mean stimulation parameters were 9,1 ms and 13.5 ma. discussion. in experiencied hands tp is an effective and safe way to treat and diagnose cardiac arrhythmias even in newborns. it should be tried before endovenous pacing is stablished and it is faster than pharmacologycal treatment. bailing g., eicken a., sebening w., vogt m., schumacher g., bl~hlmeyer k.; kinderkardiologie, deutsches herzzentrum m0nchen, germany to assess the outcome of balloon valvuloplasty in infants with cardiac failure caused by critical aortic stenosis a retrospective study was performed. between 1986 and 1995 33 neonates, aged 1 -28 days (median 9 d), weight 2.t -4,1 kg (median 3,3 kg) with critical valvar aortic stenosis were dilated by balloon (aovp) as the first line treatment. 21 patients received prostaglandin el, 18 needed inotropic drugs and 16 mechanical ventilation. associated cardiac lesions : persistent ductus arteriosus (pda) in 27 patients (restrictive pda in 8 cases), a mitral regurgitation (mivr) in 27 cases (15 severe and 12 moderate or mild mivr), angiographic findings of endocardial fibroelastosis (efe) in 12 patients, mitral stenosis (mivs) in 8, coarctation of the aorta (coa) in 2, and finally a small musculary ventricular septum defect (vsd) in i patient. vascular approach for ballooning : a. axitfaris in 20 cases (61%) a. femoralis in t 0 (30%) and v. femoralis in 3 cases (9%). the median ratio between inflated balloon and aortic valve diameter was 0,99. dilatation was achieved in all 33 cases. the peak systolic gradient across the aortic valve (pre aovp) ranged from 0 to 137 mmhg (median 50 mmhg) and was reduced to 0 to 55 mmhg (median 15; gradient reduction is significant (p < 0,01)). aortic regurgitation (aovr) was absent or mild in 30, moderate in 2 and severe in 1 patient after aovp. 23 children survived (actual suwival rate: 70%; early mortalffy: n = 3; late mortality: n = 7). mid term follow up (0-8,8 years; mean 2,7 years) showed an increase of the systolic peak doppler gradient across the aortic valve (median 41 mmhg) but no increase of aovr. 10 re-interventions (re-aovp: n = 3, commissurotomy: n = 2, mitral valve replacement n = 2, resection of subaortic stenosis: n = 1, resection of coarctation: n = 2,vsd-closura: n = 1 ) were performed in 6 patients. rv contractility and pulmonary vascular mechanics(pvm) in immature animal models are poorly underslood. we developed an acute rv injury model to measure rv contractility and pvm in response to commonly used cateehalamines. ten anesthetized piglets (9-12kg) were instrumented with micromanometers in the lv, rv, pa, and la. a pulmonary artery flow probe was placed to measure cardiac output(qpa). ultrasonic dimension crystals were sutured to the myocardium and dynamic chamber volumes estimated using shell subtraction methodology. rv injury was induced with 3-7 cryoprobe injuries at -50 to -70°c for 3-4 minmes each. da at 10mg/kg/min, db at 10mg/kg/min, and ep at 0.1 mg/kg/min were infused in random order. rv contractility was evaluated by calculating a load independent measure of contractility, the preload recmitable stroke work(prsw), during vena caval occlusions. to describe pvm, input resistances), characteristic impedance(z0), total pewer(tp), and efficieacy 03f=qimo"p) were measured. measurements were made pre-and post-injury, during infusions, and between infusions. clyoablation decreased prsw (22.8_+7.8 to 13.8+4.1, p<0.001). at the end of the experiment, prsw remained depressed to this level indicating stability of the model. one factor contributing to organ dysfunction for infants undergoing repair of congenital heart defects (chd) is their "inflammatory response" to cardiopulmonary bypass (cpb). this response is characterized by an increase in cytokine release, complement activation and endothelial injury. modified ultrafiltration (muf) is a method for removing tissue water and inflammatory mediators by rapid ultrafiltration followin~ cpb, muf may acutely improve post-operative end organ function. in this study, we evaluated the effects of muf on the pulmonary and cerebral function of infants undergoing cpb for repair of chd. we prosnecrivety randomized 30 infants (.~ 5 mos) to either muf (n=16) or no muf (n=14)(control) following correction for chd. the study intervals were 1) before cpb, 2) immediately after cpb, and 3) 20minutes after cpb. pulmonary function was evaluated by measuring dynamic compliance (cdyn) and airway resistance (raw). for 13 pts (mue=6 pts; control=7 pts) exposed to a period of deep hypothermie circulatory arrest (dhca), cerebral metabolism (cmro2) was calculated at each interval using the xe 133 clearance technique for cerebral blood flow measurements and arterial and jugular bulb saturation measurements to calculate cmro2. a reduction in cmro2 has been consistently demonstrated after dhca. the effects of muf on cdyn and on cmro2 are shown below: p<0.05 vs pre-cpb; # p<0.05 vs post-cpb • p--o.06 vs. post-cpb this study demonstrates that immediately following exposure to cpb, muf will improve pulmonary compliance. raw was not different between groups. there was no significant difference in hours of post-op ventilation for either group. in those pts exposed to dhca a trend towards better cerebral metabolic recovery compared to control was demonstrated. this is the first technique applied to infants undergoing dhca where cmro2 after cpb was greater than precpb measm~s. although this may be beneficial to postoperative hemodynamics, ventilatory management and long-term neurologic recovery, more patients and longer follow up will be necessary to verify such an effect. the effects of conventional mechanical ventilation (cmv) on left ventricular (lv). diastolic filling in neonates are not well established. one approach to improve lv filling is the use of cmv to provide a phasic increase in airway pressure {thoracic augmentation). this phasic increase in airway pressure may result in an increase in lv filling similar to that which occurs with cpr. thoracic augmentation has not been evaluated in neonates with ventricular dysfunction who frequently demonstrate increased heart rates. attempts to maintain low peak airway pressures during cmv may result in a prolonged inspiratory time that occurs over multiple cardiac cycles. this may alter lv filling in the later cardiac cycles. to determine the effects of inspiratory time on lv diastolic filling, 10 infants were examined with doppler echocardiography less than 24 hrs after surgery for the arterial switch procedtme. pulsed doppler recordings of the millal valve (mv) were obtained with the inspiratory time adjusted to occur over 3 cardiac cycles (21 sec.). a pressure transducer was placed in line with the ventilator, and the respiratory cycle was recorded superimposed on the doppler tracing to provide accurate determination of inspiration and expiration. doppler recordings were obtained from the apical 4-chamber view and the following measurements were made: peak e and peak a velocities, eia ratio, and deceleration time. compared to the expiratory phase of cmv, the initial beat during the iuspiratory phase of cmv resulted in an increase in mv peak e (.53 +-.06 vs .65 -+ .08 m/s, p<0.05) and peak a (.47 + .07 vs .63 -+ .09 m/s, p<0.05) velocities with no change in mv deceleration times (p<.01). compared to the initial beat during tile inspiratory phase, the third beat during the inspiratory phase resulted in decreased peak e (.65 + .08 vs .40 + .05 m/s, p<0.05) and peak a (.63 + .09 vs .40 + .05 m/s, p<0.05) velocities with no difference in deceleration times. thus, cmv augments lv filling during the initial phase of inspiration. however, as the increase in airway pressure is distributed over multiple cardiac cycles, lv filling falls below baseline levels. these observations indicate that while thoracic augmentation may be beneficial, to optimize lv filling the inspiratory time of cmv must be < 3 cardiac cycles. energy expenditure in pediatric orthotopic liver tranaplantat~on, to determine the actual calorie requirements of critically ill children and evniuate the correlations between measured, stress-p~lictod and repleted energy exponditttm and the severity of illness. des/gn: a prospective, dinlcal study. se~ng: tertiary care pediatric icu in a university hospital. patients: ten patients aged 6 to 210 months with disorders prompting picu admission, including sepsis, respiratory failure, solid organ transplantation, and cardiovascular surgery. inta~entions: all patients were studied within 24 hrs of major surgery or transplantation, or following acute illness. all patienls were severely stressed clinically and all but two were intubated by cuffed tubes, in three of them, still in a stress state, the study repeated on the third day of the disease, energy expenditure mensurements (mee), as well as illness seventy scoring systems, mtfltisystern organ failure scores and various anthropemetric and clinical indices of nutritional status, the stress-predicted energy expenditure (s-pee), the basal metabufie rote (pbmr), the repleted energy (re) and the recommended dietary allowances (rda) were measured or calculated in each patient. multiple regression analysis was used to analyze the data. measurements and main results: although the mean mee was significantly lower than the mean s-pee (37.6+11 kcal/kg/day vs. 50.35:16 kcal/kg/day, p<.002), it did not differ significantly from the pbmr (mean difference -2.62 kcal/kg/day, range -10.07 to +9.06 kcal/kg/day). the s-pee/mee ratio ranged from 1.04 to 2.07, while the re/rda ratio (21.25:4 kcal/kg/day)/(75.85:7 kcal/kg/dny) ranged from only .1 to .5. the prism/tiss ratio was not correlated better with mee than the diagnostic category (r~=.36 vs..38, respectively). the re was positively correlated withthe mee (rz=.65, i)=.07) while negative oarrelatian has been found between mee and age, mid-arm circumference, triceps skinfotd and the use of vaseactive agents (r~.81, -88, -.67, p<.005 and -.71 resp~lively). concl.m~: if s-pee is used for caloric repletion in the stressed oritic~ly fll el~d, these patients will be substantially overfed by as much as 100%. although pbmr appears to approximate the mee by ±10%, other clinical and nutritional indices should also be ennsidered. objective: to deter .mine..t.he metabpli.c and.nutritional state of mechanically ventilated intants and children m relatmn wlm severity or msease. patients and methods: 37 mechanically ventilated infants and children, median age 7 months (range 3 days to 13years), were studied. severity of illness was assessed using prism, prism-ii~ and fiss-scores. oxygen consumption (vo2), energy expenditure (mee) and respiratory quotient (rq) were determmed by mdirect calorimetry. total urinary nitroger(tun) and creatinine excretion, levels of albumin and crp were aetermmed in 16 patients. in these patients daily caloric intake and substrate utilization were assessed. they were categorized in subgroups: a partial feeding (recent admission to p1cu); b complete feeding. results: mee of the total group (n=37) 0a) i=intake g/kg/day (% total intake); u=utilization g/kg/day (% total production). nitrogenba]ance was negative in all patients in group a (mean -227.7 --176:4 mffkg/day) and positive in all but one patient in group b (.mean 84.9±109.d n~g/..kg/day;p=0.001). no significant correlations were round between creatinine height index, crp, albumine, jun vs v u2/kg conclusions: the mean measured energy expenditure does not exceed predicted resting energy expenditure, but ~ere is a wide range. in a majority ot patients with complete feeding h.igh carbohydrate intake resulted, in high kq and lipogenesis. in patients witla partial teeding the highly negatwe nitrogen'balance suggests that in the early phase of diseasean higher protein intake should be provided. severity of illness scores ann oiocnemicm markers of physiologic stress correlatedpoorly with oxygen consumption. leite,hp; iglesias, s; faria, c; ikeda, a; albuquerque, mp; carvalho, wb pediatric icu -s~o paulo federal university -s~o paulo, brazil objectives: 1 ) to evaluate patterns of use and monitoring of nutritional support in critically ill children; 2) to evaluate an education program in nutrition support given throughout the resident physician training in the pediatric icu. patients and methods: records of 37 patients receiving nutritional support during 1993 were reviewed. aider this first phase, knowledge and understanding of the role of nutrition support was conveyed to the residents through didactic lectures. in a second phase thedata were reevaluated in 35 children who were given nutrition support in 1995. results: from a total of 425 days ofthempy, the single parenteral route was utilized in 80,5%, the digestive route (tube feeding or oral route) in 19,5%. of this time. a previous nutr~ional assessment was performed in 3 children; no patient had the nutr~on goals set. the nitrogen to nonprotein calories ratio ranged among 1:80 and 1:250. only 29,7% of the patients had their estimated caloric needs supplied and this goal was achieved only in those patients who were on enteral tube feeding. patients did not achieved their goals for vitamins. the supply ofoligonleme~s was adequate except the zinc. nutritional monitoring parameters including weight, serum albumin and serum triglycerides were performed in almost all the patients but without uniformity. the reevaluation ofthase parameters showed adequacy of protein and micronutrients supply; however deficiency in nutritional monitoring and infrequent enteral feeding were still detected. conclusion: there were lacks in the implementation of nutritional support, which were partially corrected in the 2rid phase of the study, although the training of residents may have contributed to give them cognitive skills, it didn't changed policies and procedures as desired. we recommend reinforcement of the education program concerning basic nutritional aspects, and the organization ofa multidisciplinary team in charge of coordinating the providing of nutritional support. plasme free fatty acids (ffa) are the meier energy source for mast tissues. during fasting ffa are released from the breakdown af triglycefides in edipose lissue (at). lipalysis, le. the rote of release o/ ffa, has been megsured in humans by means of stable isotope techniques using labeled pa or glyeerd as traces. no information is avoilob!e io dale on the ro of la. we infused albumin hound u13c-pa and u13c-la in 7 critically ill infants, receiving 20 kcel/kg/doy of iv glucose end na oral feeding (weight 3.6,i.,3 kg;, range 1.9-5.8; ego 57:64 days, range 1 149) and measured simultaneously the ra of pa and la from (he isotopic enrichment of plasma fea by gas chromatography-mass speclrome|ry ai 1:50, 2:00 and 2:10 hours from tile shod of the infusion. a subcutaneous gluted at biopsy was obtained far fatty acid (fa) composition. we intended to (1) in fie infants sbjdied atipa ~'os hi9her than attla (~p<o.o01)' and ihe ra of pa was higher then thai of la (~p=0.00 §). however the ratio el the re's to the respeclive fa in at was higher for la than far pa (*p=0.02). in conclusion the ra af la was higher then expected from ~he fa composition of at. we speculate [hat la is preferentially released during tipolysis and its contributuion to the energy metnbolism of the sick infonl could io !orger than what: is normai!y assumed from ihe fa composilion of at and of plasma fea. preoperative starvation can be prevented in infants undergoing non gi surgery by continuing feeds till as long as it is safe to do so. some gi disorders require withholding of feeds for a long period. for these and for infants already suffering from pem~ parenteral feeds are given preoperatively. peroperatively nutrition is maintained by ensuring adequate glucose supply in the infusion fluid and its smooth metabolism. proper placement of feeding tubes are also carried out peroperatively. postoperative early resumption of oral feeds is ensured by new techniques of anaesthesia and pain relief and methods to ensure early return of peristalsis. enteral feeds of pre digested substances and feeds through transanastomotic tubes and through gastrostomies and jejunostomies can prevent starvation in many situations. parenteral feeding is carried out only when enteral feeding is not possible, for example in necrotising enterocolitis~ gastroschisis, short gut syndrome, high enteric fistula and acute pancreatltls. the artedal ketone body ratio (akbr) is established as a valuable tool in the management of adult patients undergoing surgery for severe liver disease.the redox theory emphasises the central role of the liver in preserving homeostasis and the importance of the hepatic mitochonddal redox potential (nad+/nadh) in monitoring liver function and integrity.the akbr (plasma acetoacetate/3hydroxybutyrate) is a measure of hepatic mitochonddal redox status, reduced ratios associated with poorer outcome. the venous ketone body ratio (vkbr) is influenced by peripheral ketone utilisation and therefore thought unreliable in assessing hepatic redox its use has been dismissed as no correlation with the ratio in hepatic venous blood was found. inspite of this. vkbr is used in the diagnosis of lactic acidoses and respiratory chain defects. the objective of this study was to determine the relationship between the akbr and vkbr in a paediatnc intensive care population. 31 children admitted to the paediatdc intensive care unit, with indwelling arterial and central venous lines as part of their routine care, were recruited for the study. the median (range) age was 2.0 years (0-16.6 years) with prism score 10 (0-36), simultaneous akbr and vkbr were measured. suppression of ketogenesis was confirmed using a rapid 3-hydroxybutyrate strip test (ketofilm, genzyme diagnostics) pdor to sampling. the median vkbr 0.50 (range 0.13-1.46) was lower than median akbr 0.56 (range 0.11-1,33), there was good agreement between artedal and venous ratios, bland-altman analysis giving a 95% confidence interval for relative bias of -0.13 to -0.03. there was a significant correlation between vkbr and akbr r 2 0,8271, p,0.001 (intercept 0.02, slope 0,85) conclusions; values for akbr tend to be higher than vkbr, however there is a consistent relationship in critically ill children. in paediatdc patients in whom ketogenesis is suppressed, vkbr may be considered equivalent to akbr. this simple test may prove to be a useful adjunct in predicting mortality in cdt{cally ill children. to understand the present status of pediatric intensive care in china, we conducted a survey between october and december, 1993, involving 20 hospitals in 14 provinces and municipalities. the results showed that there were totally 41 icus for pediatric patients, including 19 pediatric icus (picu), 18 neonatal icus (nicu) and 4 pediatric surgical icus (sicu), with total of 403 beds. the physicians to bed and nurses to bed ratios were 1:1.5 and 1:0.91 respectively. the average number of equipment per bed was 0.47 ventilator, 0.34 multi-fnnction monitor and 0.47 infusion pump. very few of the icus had portable x-ray machine, biochemical and blood gas analyzers, and hemodialysis machines. the most frequently treated diseases/conditions were pneumonia, intracraniat infections, post-operation and sepsis in picus, and neonatal pneumonia, hypoxic ischemic encephalopathy and sclerema in nicus. pneumonia and respiratory failure accounted for 33.29% and 26.50% of all the diseases/conditions treated in the icus and the mean case fatality rate of respiratory failure was 24.50%. the results of the survey suggest that there is shortage of tcu beds and modern equipment, and treatment is often delayed due to excessively strict criteria for mechanical ventilation. a set of simple, and nationally acceptable criteria for evaluation of severity and cure of the diseases/conditions is urgently required. the medical reports of 286 children were reviewed and each admission was analysed in terms of age, sex, diagnosis, management within the hosp~al, length of hospital stay and type of poisoning. the youngest age was 23 days and the oldest was 16 years. hundred and twelve (39.5%) of the children were girls and 174 (60.5%) were boys. the most common (26.5%) reason of admission was intoxication among all of the cases and the greatest group (73.7%) of the poisoned children had ingested medication which was followed by another group of patients (9.2%) who had eaten mushrooms. the peak incidence of drug ingestion was salicylate intoxication (20%). forty drug poisoninigs were accidental while 16 was intentional. the majority (82.4%) of the cases that committed suicide was between 12 and t7 years old, and the main cause of suicide was being unsuccessful at school. we conclude that poisoning -especially salicylate intoxication -is still a major problem in turkey and we believe that emphasis on the need to stere all kinds of drugs in a secure place and re-examination of a chin resistant packaging should help to reduce childhood poisoning significantly which is a preventable condition. included were all patients who died in the hospital, except those treated in the neonatal icu (predominantly premature and sga newborns) and those who died in the emergency room. among a total of 7179 hospital admissions (excluding the neonatal icu and emergency room), 99 patients died (1.4%). of these 99 patients 73 (74%) died in the pediatric icu, 18 (18%) in the ward and 8 (8%) in the operating room. a chronic underlying disease was present in 66 (67%). withholding or withdrawal of therapy was implemented in 48 (48%) children, 27 (27%) died due to failed cpr, 20 (20%) were brain dead and 4 (4%) died following a dnr order. justification for therapeutic restrictions in the 52 patients of the dnr and w/w groups was imminent death in 32 (62%), lack of future relational potential in 12 (23%) and excessive health burden in 8 (15%). withdrawal or withholding of therapy was carried out by extubation in 46%, vasoactive drugs were stopped in 25 %, and mechanical ventilation was withdrawn in 21%, analgetics and sedatives were frequently used (in 73% and 79%, respectively). hence decisions concerning restrictions of treatment are common in pediatric practice, mostly due to imminent death. patients in which treatment was restricted were characterised by a longer hospital stay, a worse prognosis, a higher frequency of chronic underlying diseases and a lower acute mortality risk. despite restrictions of intensive therapy, most patients were allowed to die in the pediatric icu. background microalbuminuria (mca), a subclinical increase in urinary albumin, reflects glomerular and overall vascular permeability 1"2. an increase in urinary excretion of albumin occurs after burns and trauma. transcanillary albumin escape rate is also increased in response to elective surgery 3 and in critically ill adult patients 4. we investigated a possible relationship between urinary albumin levels and clinical instability, as measured by pediatric risk of mortality (prism) scores. method we studied 26 consecutive patients (median age of 13 months, range 2-100). patients whith nephropathies or any abnormality of urine analysis were excluded from the analysis. prism scores, mca (mg.1-1) (immunonepbelometric) and urinary creatinine (cu) (mmol.l -!) (jaffb) (48 hour collection sample) were determined within 48 hours from admission to the picu. the mca/cu ratio (mg.mmol.1 "l) was used to correct for urine output variability. diagnoses included respiratory failure (6), postoperative (5), neurologic (5), sepsis (4), trauma (3) and miscellaneous cases (3). pearson's correlation was performed to correlate data. results and conclusions. mean prism score and mca/cu were 16.9 ± 5.9 sd and 1004-39 sd, respectively. a significant correlation was found between mca/cu and prism scores (r=0.80, p<0.001). our observations show that, independently of the initial insult, the paediatric unstable patient may have increased capillary permeability, which is correlated with the degree of physiological derangement, as measured by prism scores. microalbuminuria can be rapidly determined since it is routinely used in the management of diabetic patients, it is inexpensive, simple to measure and blood-spearing. therefore, it might have a role in the clinical assessment of capillary permeability and transcapillary albumin escape worldwide the demand for paediatric intensive care services exceeds the supply. in developing countries sporadic access to such services alters expectation of care and can lead to children being either mechanically or handventilated in general paediatric wards. the outcome of children requiring ventilation in a major teaching hospital in india was reviewed.children were ventilated on an adult intensive care unit (aicu) if a bed was available, otherwise in the general paediatric wards. over a 4 year period 109 children were ventilated on aicu with 54 deaths. yearly mortality rates varied between 43-58%. over a 3 month period 37 patients were ventilated on the paediatric wards. of the 15 p~tients over 4 weeks of age 11 died (chi squared 0.t >p>0.05) reasons for the higher mortality rate on the paediatric ward likely include the higher patient:nurse ratio, and more limited resources. a predictor of mortality based on simple physiological observations without the need for expensive blood tests and including chronic health status would be a useful tool. the establishment of a paediatric intensive care unit is proposed to redress the balance of care. to assess the performance of the pediatric intensive care unit of hospital dona estef~nia by an international standard score, the authors did a prospective study of 1149 consecutive admissions to the unit during a period of 29 months. mean age was 50.63 _+ 54.07 months; mean lengh of stay was 3.16 + 5.59 days. the effectiveness and efficiency were determined by the admission prism. admission efficiency was defined by two criteria: a) mortality risk > 1% or b) the administration of at least one intensive care unit-dependent therapy. the cumulative observed mortality was 5.57% and the expected mortality was 5.97%, with a standardized mortality ratio (smr) = 0.933. the overall performance of the prism score-based predictive model was found to be good (goodness-of-fit test x2 [5] = 6.387;p=0.271). of 1149 patients admitted, combining the two criteria (icudependent therapy and mortality risk) an admission efficiency of 825 (71.8%) was found, equating to 3263 (89.94%) of 3628 1cu days. conclusion: in our study the assessment of the admission efficiency and of the effectiveness of the unit was possible by using the prism score of admission. there was no significant difference between mean values for otiss and ntiss)in level l patients (p=0.12 paired t-test).for level 2 and 3 patients mean value of ntiss was greater than otiss (p<0.0001). there was a significant correlation between levels using either ntiss or otiss (mean difference level 1 and 2, level 2 and 3, ( p < o.oool). conclusions: a new tiss has been developed and used in a picu. nurses were able to accurately score the interventions on their shift. the assignment of patients to intensive care levels correlates with tiss values allowing a quantitative measure of severity. objective : to compare the rate of cerebral palsy (cp) between monochorionic-twins, dichorionic-twins and singletons born at 25 to 32 weeks' gestation. design : two-year prospective cohort study. setting : geographically defined study (region of franche-comt~., france). main outcome measures : type of plasentation was obtained by anatomopathological, or macroscopic examination of placenta and comparison of 6 twins' blood-groups. neurological assessment was performed at two years of age (uncorrected for gestational age) by family doctor (pediatrician or physician), or neonatologist of the icu at tertiary center. sample : 167 of 17i survivors aged of two years (98% follow-up rate), born between 09/30/90 and 10/01192. triplets and chromosomic malformation were non included. results : thirteen (11%) of the 119 singletons had cp.vs 3/29 (10%) of dichorionic twins and 6/19 (32%) of monochorionic twins (p=0.04). four of the 19 monochorionic twins (21%), 2/29 dichorionic twins (10%) and 4/119 (3%) nngletons suffer from quadriplegia (p<0.01).in a multivariate approach, monochorionic twin placentation was the strongest risk-factor of cerebral palsy (or=9.7, ic 95% = 2a-39, p<6.01). others risk-factors of cp were : lack of father's profession (or 11, p<0 .03), maternal antecedent of abortion (or 3.2, 1-10, p<0.04), vaginal delivery (or 3.4, 1-11, p<0.03), hyaline membrane disease (or 3.4, 1.2-t0, ~0.02). discussion : this is the first population-based study to uplight the role of monochorial twin-placentation as a strong risk factor of cp for premature infants. cp is more severe in monochodonic twins than in other infants. mecanism of cerebrat deficiency is not clear since none of our infants with cp was survivor of an in utero cotwin's death, and none of these infants was exposed to twin to twin transfusion syndrome. were these monochorionic-twins affected by an undiagnosed neurological structural defect that could lead both to prematurity and handicap remains an open question, a vital role of the intensivist is to ensure that knowledge and practice are imparted to trainees in the icu so that patients receive optimal care. teaching effectiveness varies widely leaving gaps in knowledge and practice in the trainee. being an effective teacher should not be a "gift" of a privileged few. the icu provides a fertile ground for using a variety of methods for teaching, e.g. didactic, at the bedside, emergencies, and in the performance ofproeeaures. in this environment, much can be learned. we have embarked upon a program to facilitate this learning process. i) teaching needs to be recognized as the foundation of good clinical care, i.e., patient related, and in its ability to generate discussion and research investigation. 2) teaching structurally has many components including the speaker, audience, varying situations, and the message delivered. 3) establishment of a program using these components to enhance teaching abilities at all levels, a) evaluate base-line teaching skills initially, b) individualize interventions to improve teaching skills, e) demonstration of learned skills with re-evaluation. this process is analogous to the analysis of a clinical disorder in a patient which, once recognized, interventions are then instituted and then re-evaluated. 4) instill the desire to use these attained skills to teach and interest others to teach. teaching excellence should be recognized through awards, honors, and academic advancement. a major emphasis of this program is to provide participants with skills necessary to teach thought processes, decision-making skills (what to do, what to avoid) and implementing appropriate management during stressful emergency situations common to the picu. introduction: many" e-mail based discussion groups exist on the internet to provide medical professionals with a rapidly responsive medium for the international exchange of ideas relating to patient care. several such lists each serve more than a thousand professionals in more than 30 countries, each distributing a dozen or more messages each day to every subscriber. there is very little known about the time being spent by professionals interacting with these lists, and very little known about the impact of the discussions on patient care. we wished to test the hypothesis that these discussion groups provide infortuation which is being used to change the care of individual patients and the general approach to patient problems. methods: in early january 1996 a pilot electronic survey was sent to a small fraction (n=63) of the memberships of 2 e-mail discussion groups, picu@its.mew.edu, and nicu-net@u.washington.edu (the full memberships of both. groups (n=t439 for nicu-net, n=1045 for picu) will be surveyed in early february of 1996). participants were asked for demographic information, experience and skill level relating to e-mail, time spent with the discussion groups, perceived usefulness of different types of discussions, and the ways in which the discussions were used clinically. the pilot study was analyzed for construct validity by correlating an overall assessment question with a summary of the specific questions. scale reliability was measured by cronbach's alpha statistic. results: the pilot survey response rate was 30163 (48%). the majority of respondents were male physicians, with an average age of 39+_5 years, who had completed subspecialty training in intensive care, and were working at a university-affiliated hospital. most had been using e-malt for more than 6 months, and considered themselves moderately adept in that use. 63% felt that the list helped weekly to keep them informed about current issues and practices in their field(s), and 57% felt that, at least monthly, they used information from the list(s) that was not readily available in medical journals. overall, 75% agreed that the list improved their professional competency. when asked to compare the value of 6 months of membership on an e-mail discussion group with more traditional educational media, 34% compared it with attending a national conference, and 26% compared it to a journal subscription. cronbach's alpha was .76, construct validity testing yielded coeff=.50, p <.05. conclusior~: internet-based e-mail discussion groups for health care professionals can be an important part of a strategy for maintaining professional competency. despite the very low cost of this medium for most, the value is felt to be comparable to that of t~r more expensive forums for education. further study will include distribution of the full survey in early february of 1996. fronk shann, tony slater, gale pearson and the pim study group we have developed a new score for predicting the risk of mortality in children admitted to intensive care. the score is calculated from only seven variables collected at the time of admission to icu: mechanical ventilation (yes/no), booked admission after elective surgery (yes/no), the presence of any one of 14 specified underlying conditions, both pupils fixed to light (yes/no), the base excess, the pao 2 divided by the fio2, and the systolic blood pressure. most scores used to predict outcome in intensive care require the collection of a large number of variables (so many icus do not calculate them routinely), and they use the worst value of each variable in the first 24 hours in intensive care. this means they appear to be more accurate than they really are (about 40% of child deaths in icu occur in the first 24 hours -so they are diagnosing these deaths rather than predicting them), and they blurr the differences between traits (a child admitted to a good unit who recovers will have a low score; but the same child who is mismanaged in a bad unit will have a high score -the bad unit's high mortality rate will be incorrectly attributed to its having sicker patients). pim was developed in the picu at the royal children's hospital in melbourne, and has been tested in six other picus in australia and one in the uk. objectives: to study the characteristics of the muhiorgan dysfunction syndrome (mds) in children. methods: a retrospective study with all the children with mds diagnosed from january 1990 to june 1995 is presented. 173 children fulfilled the wilkinson criteria (i). in all of them the number of organs affected and the prims score were determined during the first 24 hours. several groups were performed according to the clinical diagnosis, the hospital of origin and the order of organs affected. results: the 173 subjects studied were an 8% of the pediatric intensive care unit admissions. 100 of them expired (58%). no differences in age, sex and weight were observed between the children dying and the survivals. the most common causes of mds were sepsis, both nosocomial (25%) and medingococcal (i4%) and acute respiratory failure. sixty-fivepercent of the patients were from the hospital wards and the remaining were directly admitted to the pigu from the emergency room. the systems affected were: respiratory (93%), cardiovascular (92%), hematologic (61%), central nervous system (52%), renal (43%) and (hepatic) liver (28%). the organs initially failing were: heart (39%), tung (28%) and central nervous system (18%). the children dying had a larger number of organs with failure than the survivors (3.89 v,s. 3.34, p<0.001).the prmis score was higher in the children expiring than in the survivors (22.4 v.s. 17, p <0.001). s.mmary: the mds is a common pathology in picu, with a high mortality, the mortality is higher in children with a larger number of organs affected and a higher prism score. sepsis is the most common etiulogy. methods : from june ist to july 15th 1995, all patients admitted to the pediatric icu were included. the score was measured at day 1 (d1) and day 3 (d3) and we used 10 variables. for each organ system, we defined 2 categories : dysfunction or failure, which we respectively confered 1 or 4 points. results : 56 patients were admitted : 22 newborns, 34 children. 23 were medical and 33 were surgical patients. 36 (64 %) patients had two or more organ failure at the admission, 12 (21,4 %) patients died, which 6 (50 %) in the first 48 hours. the mortality rate was the same for children with two or more organ faiiure at d1 and d3 : 6/36 (16,6 %) at d1, 4/22 (18,2 %) at d3. the mean score is different for children who survived or who died : 8,6 versus 17,9 at d1 ; 10,6 versus 18,2 at 133. when the score is > 15, the mortality rate is significant. conclusion : in this study, there is a good correlation between the score of severity and the mortality rate but we have few included patients. we need a prospective multicentric study to assess these results and we must compare this score to other scores of severity used in picu. back.qround: injury to the central nervous system is the cause of death in the majority of pediatric trauma victims, studies have identified a wide range of factors associated with poor outcome from brain injury. however, when single features are analyzed, they are not sufficiently accurate predictors. few studies have used a multivariate analysis of these factors and pediatric outcome, methods: clinical and radiographic features of 164 comatose children after traumatic brain injury were analyzed, clinical parameters, the initial cranial ct scan, and demographic characteristics were analyzed for an association with death or vegetative survival at 6 months. a tree diagram in which risk factors may differ within the study subpopulations was constructed using recursive partitioning. results: chitdren with a motor score _<2 had an 11-fold increased risk of poor outcome compared to those with motor scores >2. among patients with scores of _<2, those with abnormal pupillary reflexes experienced a 13-fold increased risk of death compared to those with normal pupillary reflexes. among patients with a motor score >2, an intracranial diagnosis code (no pathology, mild shift _<5 mm, swelling, shift >5 mm, surgical mass lesions, or non-operative mass lesions) was highly predicative of poor outcome at 6 months. children with ct findings other than normal or mild swelling had a 4-fold increased risk of poor outcome. of children with swelling, shift or mass lesions, the pupillary light reflex was associated with outcome. children with abnormal pupils had a 6-fold increased risk of poor outcome. discussion: a few clinical and radiographic features stratified comatose children into fairly distinct risk groups. information available early after traumatic brain injury in comatose children provides useful prognostic information on the likelihood of death or devastating injury. a retrospective study of 70 children with the diagnosis of epidural hematoma was made during 1990-1995 period. ages ranged between 7 days and 17 years (18% less than 1 year, 40% between 1 and 10 years, and 42% older than 10 years), 82% of them were admitted at the picu. 51% of the cases were due to falls, 35% to road traffic accident and 14% to other causes. on admission gcs was less than 8 in 19% of the cases and more than 14 in 53%. diagnosis was made during first 4 hours in 63% of patients and delayed more than 12 hours in 28% of them. neurologic impairment was present at admission in 33% of patients, and delayed in 30%. even so,27% remained without impairment. radiological findings at first ct were skull fracture (68%); epidural hematoma localization was: in the right side (63%), frontal area (24%), temporoparietal (66%) and occipital (t0%). associated lesions were: several (13%) or unilateral (51%) cerebral contusions, diffuse brain oedema (10%), unilateral hemispheric oedema (14%) and 38% showed shifted middle line. four patients died, half of them during the first 24 hours. 41 fully recovered (58.6%) and 25 have sequelae of different nature :7 were left with severe motor disability (10%); at the follow-up t3 have some degree of neurodisability. next datas keep correlation with death or neurosurgical impairment: only were significative multiple cerebral contusion (p=0.002) and brain oedema (p=0.05), gcs less than 8 at the admission (p--0.002), shock (p=0.003) and remaining cerebral contusion in control ct correlated with death or diasability at discharge. on the other hand, neither surgical drainage volume nor first or highest levels of icp (12 cases),nor pupillary abnormalities (10 cases) correlated with worse prognosis. conclusion: gcs equal or less than 8 an shock are main factors related to worse prognosis, also multiple cerebral contusions in ct and diffuse brain oedema. the results of a modified gcs were compared to outcome and intensive therapy in 78 children (mean age 8,5t4,7 years) with head and associated injuries (53,6% of all cases) of different causes (traffic accidents, falls). the gcs was regularly used inn the course of intensive therapy. according to our own and other experiences the gcs was divided in 3 stages: stage 1 (4-8 points), stage 2 (9-12 points) und stage 3 (13-19 points) palhuiugy wile sp, tdhlg c~'lcb1al blood ~0w. sabgcqucntl}. rhc slat,: rerltncd to t1011tl,91 iiltlils. the p0st,~pem~v~ b}i~g wij!!,:q1! ,:_a!~p!ica!j0n~:. 4 ri~;¢ ill the level of sensibflizatjou lo tile cerebn~ anhgrns up to 1t.4-o7 was flofcd iu 9 i,alicnts. there wa.~ al~ iuclt~a~e ill cerebral vdociij,. ~m d~;'ati0a il~ p¢fiphc~ai re~ista/isc of the large ce~'bral ve~ds. neur0h;~c ~:yn'.pt,m~at0!a~, (s::mno!en~', _r_uscu!~r l~:pot0ni& !ryper*'flema) was nbserwed tu lt~ese pal~enls o. cbruc~l ~0nnds. rile ple~c.ut abse~vafion~ suggesl ihal die ~tttdy at" ihe stale ~f hematocr~chcplm/itic bm~ic~ in ckil&en with 31110on emergensy is of abviou.~ !?ece~sib; in co~.te ctin g severe pa~0lo2~-i~mnediately f0u0wing ne ,:~per,'~fion. background: reconstruction of the heart by three-dimensional (3d) echocardiography provided new information on anatomy of complex congenital heart defects, we assessed the utility of 3d ultrasound in detecting morphological changes in cerebral anatomy in newborns before and after cardiac surgery. methods: transfontanel cross-sectional ultrasound, scans were obtained in standardized coronal and median sagittal planes. subsequently, rotational scanning was used to acquire the multiple sequential crosssections of the brain. for rotational scanning, a conventional 5 mhz transducer was rotated 180 degrees.scanning took less than one minute and required no sedation, data was stored in the image processing computer which allowed for off-line three dimensional reconstruction of different brain regions.twelve infants aged 3 -21 (median 7) days were assessed before and after cardiac surgery, results: cavity of lateral ventricle, choroid plexus and the periventricular brain parenchyma could be reconstructed in all. accurate estimation of size and volume of lateral ventricle, aqueduct, and other ultrasonographic visible pathological brain lesions could be performed. reconstruction of various brain areas was accomplished in 3-10 minutes. the localisation and extension of severe periventricular hemorrhage which was detected preoperatively in one infants was better visualized than in conventional ultrasonography. epicortical and subarachnoidal space could be reconstructed in all and allowed detection of hemorrhage in one case which was not detected by conventional ultrasound. conclusion: 3d reconstruction of different areas of the brain may provide additional quantitative information on size and volume of the internal ventricle and choroid plexus, and better understanding of the topographical aspects and the extension of intra-and periventricular hemorrhage than conventional cross-sectional ultrasound. introduction: intracranial cerebral blood has been estimated to be 70% venous, the invasive measurment of venous blood saturation in the jugular bulb provides quantitative information on cerebral oxygen supply and consumption. however, routine oxymetric measurement of blood saturation in the jugular bulb by insertion of a catheter line into the internal jugtdar vein is an invasive procedure which has limited use especially in infants and young children. thus the aim of this study was to investigate the correlation between the non-invasive spectroscopic measurement of rso2 and the oxymetric determination of the blood saturation in the jugular bulb in infants and children undergoing routine cardiac catheterization.. methods: during routine cardiac catheterization 30 infants and children (age 5 day-16 year, median 4,5 year) the rso2 was measured continuously using a two chanel cerebral oxymeter (invos 3100a). the sensor was placed in standardized location at the left temporal head side. after the routine oxymetric blood sampling in the superior vena cava the oxymetric catheter was manupilated into the left jugular bulb. after control of the catheter position simultenuous values of the rso2 were documented. results: over a range of (33-87%) sjo2, a significant linear correlation was found between the spectroscopic measurement of rso2 and the oxymetric determination of venous blood saturation in the jugular bulb (r=0,83, p<0,001) and the superior vena cava (r=0,65, p<0,05). no significant correlation was found between rso2 and the arterial blood saturation in the descending aorta and as well as to the standared hemodynamic parameters. conclusion: meusurement of rso2 by mrs may provide continuous non-invasive information on cerebral venous blood saturation and thereby possibly on cerebral oxygen supply and consumption in infants and children. these may be of clinical value particulary during and immediately after heart surgery by means of non-pulsatile cardiopulmonary bypass. information on refractory status epilepticus (rse) from developing countries is scarce. we analysed 43 cases of rse admitted over last 2 yrs. the objective was to study etiology end evaluate efficacy of diezepam infusion. median age of the patients was 1.25 years irange 1.5 months to t 1.5 yrs); 70% were boys. onset of seizures was 1-t44 hours (median 24 hours) prior to hespitalisation. the glasgow coma scale score ranged from 3.11 (mean+sd 5 + 2). the commonest underlying causes were acute cns infections (26/43, 60%; bacterial meningitis, 16, encephalitis, 10) and epilepsy (8/43, 10%). oiazepam infusion in incremental dose (range 0.01-0.025 mg/kg/min) was used in 38 patients over 3.4_+2.1 days. seizures were controlled n 31 (82%), mechanical ventilation was required in 10 (26%)only, while none had hypotension; 84% patients survived. thiopental infusion (holus 5 mg/kg followed by 0.2 mglkg/min, and increments of 0.1 mg/kg/min till seizure control) was used in 8 patients over 1.7_+0.7 days; seizure were controlled in all, but five patients needed mechanical ventilation, six developed hypotension needing infusion of vasopressoi drugs, 3 out of 8 (38%) died, overall mortality was 26%, mainly due to acute cns infections (n-6) and prolonged se. the patient was a 2-year-old gift di~aosed of dov,~'s s~drom¢, tetralogy of fallot. (t.f.) before admission a vasovagal crisis after coughing and vomiting was seen, and she was taken to the emergency room. mother said she had eyanosis in the mucous membranes of the mouth with exercise.on physical examination, she ~as afebrile, normal fundi and neurologic examination was normal. a harsh systolic murmur was hear~ with decrased intensity during bradycardia. chest rx disclosed a decreased pulmonary vascular markings. ecg: synus rhythm, with bradycardia and nodal escape rhyflmas. she was transferred to our picu because of severe h3,pertomc seizure, lost conciousness, and deeembrate poslamng~ ~t cyancx~is. the episode lasted for ~weral seconds, and ceased v~th diazepam. on admission she was lethargy, and neurologlc exammation showed weakness of left leg without babinski, and normal funduscopic. the patient had two episodes of bradycardia and isoproterenol was begun. during those episodes the patient was cyanotic, and the murmur was heard with the same intensity. act scan disclosed a tight parieto-temporai abscess with midline shift, lnmediately after the diagnostic ct, we administered antibiotics, antiedema treatment and it was drained. the abscess culture was negative. a ct control disclosed air and midlme shift. ~ the next two days she had three episodes of h39oxia and c'yauosis ceased with o@gen, morphine and propanolol the patient died during a fourth episode. discussion: arrhytmias are uncommon in patients with tetralogy of fallot before surgery. in our case the first diagnosis was sick sinus syndrome vs bradycardia secondary to cyanotic episodes. the incidence of cerebral abscess in children with congenital heart disease (chd) is approximately 5%. tetralogy of fallot is the most common associated lesion, and is unusual in children under 2 years of age. conclusion: 1) brain abscess is a rare complication of patients with cyanotic chd, but should be suggested in patients with °'apparent" sick sinus syndrome. in patients with down's syndrome, t.f.,with cyanotic episodes, and difficult neurologic exploration, a brain ct scan is recommended. guillain-ba~re syndrome (gbs) is an acute autoimmune reaction, directed primarily toward the myelin encasing the peripheral motor nerves= this reaction causes a delay or block in nerve conduction. the presentation often can be very subtle but is followed by rapid loss of neuromuscular power, leading to acute respiratory distress, resulting from weakness of muscles and aspiration pneumonia. there were 3 boys -4, 8, and i i years old with gbs, treated in our icu. two of them due to the respiratory distress were intubated nasotracheally and ventilated mechanically with servo-9ooc (siemens-elema, sweden) ventilator. duration of ventilation was i i and 34 days, respectively. plasma exchange was performed in all cases. the numbers of plasma exchange sessions were 2-4 in each case. mean amount of plasma exchanged per session was 28,24 ml/kg. plasma was substituted with albumin, plasma or saline. the most important aspect of the management of patients with gbs in the icu involves the airway care, prevention and treatment of aspiration pneumonia and the mechanical ventilation if respiratory distress presents. endotracheal intubation should be performed whenever there is evidence of retention of pulmonary secretions, refractory to chest physical therapy, weakness of protective reflexes of the airway, leading to aspiration pneumonia and (or) atelecr~sis. cardiac arrhithmias too, is a main threat to the circulatory stability in gbs. therapeutic plasmapharesis has been shown to be beneficial, reducing the time for weaning from the ventilator and for achieving independent ambulation. however, plasma exchange is expensive and not without significant risks for the patient. some authors find that plasmapheresis is not effective for patients with fulminant course of gbs and blocking of nerve conduction. recent studies have demonstrated that intravenous high-dose immunoglobulin can be equally effective. there were no significant complications associated with plasma exchange. all presented patients survived without residual disability. tetraparesis associated with long-term paneuronium use in an infant. paneuronium is a muscle relaxant used in ventilatory management of patients with respiratory distress in intensive care unit. after the end of sedation some patients were found to have severe tetraparesis. paresis was accompanied by complete areflexia and diffuse atrophy of alt extremity muscles. this neuromuscular complication is caused by prolonged high-dosage pancuronium treatment. in the last 5 years, numerous reports have linked the use of pancuronium bromide with prolonged paralysis, disuse atrophy and areflexia. this side-effect is well known in adults patients but rare in a pediatric intensive care unit. we describe one pediatric observation of tetraparesis after prolonged pancuronium treatment in a 9-month-old girl, this female infant developed respiratory distress syndrome and was intubated and mechanically ventilated. to decrease chest wall rigidity pancuronium bromide was administered during 11 days. (she received approximately 120 mg of pancuronium bromide). on day 12 the drug was discontinued and the patient had severe tetraplegia and areflexia with normal head movements. electromyograpliy showed absence of any disorder of neuromuscular transmission. this infant showed a recovely of muscles after 3 months. the other causes of peripheral neuropathies were eliminated. electroencephalograms and head scans were normal. the recovery pattern observed in our patient correspond to the process of regeneration after axonal degeneration. it is suggested that these neuromuscular complications were caused by prolonged high-dosage pancuronium treatment (associated with cortieoid and aminoglucosides). polyneuropathy syndrome in adult lc.u. appeared in literature in 1984 and is extremely common in long stay cases. the etiology of these disorders remains elusive. it is tempting to ascribe them to administration of drugs (muscle relaxants, steroids, aminoglycosidea), plolonged immobility, malutrition, sepsis and ischemia associated with reperfusion injury. to our knowledge there is only one case report of similar condition in a children i.c.u. (pascucci 1990) we present a serie of 16 previously healthy children, aged 9 months to 13 years, who admitted in i.c.u with respiratory failure and who following weaning from m.v, remained in profound diffuse hypotonia with proximal and distal muscle weakness for various length of time, recovery of muscle strength occured in a week or months {the longest i0 months), all children, except one, 3-4 days before admission developed symptoms of either respiratory or upper airway infection with fever. on admission viral and bacterial cultures were positive in 2 cases (haemophilus influenze, herpes virus). during treatment 9 patients became septic. muscle histological and neurophusiological investigations have not been done. considering the multifactorial nature of the aquired nmd in adult critically ill pts, is impossible to attribute the muscle weakness of our pts to any specific cause, in conclusion, our findings suggest the need for further investigation of nmd in critically ill children treated in i.c.u. a van esch, ha van steen~l-m011, ir ramtal, g derksen-lubsen, idf habbema. febrile status epilepticus (fse) is a prolonged and serious febrile seizure. little is known about the outcome of fse in neurologically normal children. this survey involved patients between 6 months and 6 years of age who had visited due to their first fse, the sophia children's hospital during the period of january 1981 till december 1991. patients with a history of neurologic disorders were excluded. 57 patients were identified, 65% were male. the cause of the fever remained unknown in 51% of the cases. in all case the fse was generalized and it most frequently occurred at night (47%). the mean age at fse was t.6 years (0.5-4.7), the mean temperature 39.6°c (38.5-40°c). the mean follow up time was 1.7 year. twelve children (21%) had neurologic sequelea. the neurologic sequelae varied from speech deficit (4 case mild, v2 -1 year delayed; 4 case moderate > 1 year delayed) to severe retardation and epilepsy (4 cases). speech deficit was detected after a mean period of 6 months (range 0-18), age, gender, temperature, family history and time of onset were no significant risk factors for neurologic sequelae. duration of seizure [rr 3.0 (0.8-11.3)] and more than two drugs to treat fse (rr 5.2 (t.5-18.1) were related to neurologic sequelae. we recommend that fse children should be followed for at least a year to detect possible speech disorders properly and start early intervention. unusual presentation of myasthenlg gra%qs ibtza e. modesto ,v~ abe~gochea a, sanch]s 1l all, go l varas k folgado s, garcia e. p.1.c.u. la fe, valencia. spain case report: the patient was a 2-year-o!d gift transferred to our pic because of severe respiratory failure. the patient, convaleseem of ehiekenpox, came into contact with horse manure previous afternoon. in the morning, she was lethargy, and irritability, with poor finding, and ~ an episode of coughing, cyanosis and acute respiratory failure after mucous vomiting when she was drinking milk. on admission she had severe respiratory distress, respiratory acidosis, and the sat 02 was 86%. she was mtubated without difficulty, and was transferred to our p.i.c.u. physical examination reveals stable hemodynamies, pupils equal, round, reactive to light, normal fandi, and muscle relaxation. crusted vesicles diseminats~d. rhonehi over both lungs. hepatomegaly (+) and splenomegaly (+). ~lhe urine, hematologic, and c.s.f. laboratory findings were normal. c.t. scan of the brain, e.e.g., and ekg. revealed no'abnormalities. rx chest disclosed a retrocardiac atelectasis. speci~ts of stool and blood were obtained for cultures and study of c. botul#num toxins. pending receipt of these results, a broad-speotmm antibiotic and acyctovir was begun. the initial differennal diagnosis consisted of laryngospasm associated with aspiraqlon, botulism, and postmfecfious varicella encephalitis. after 15 hours, weatm~ was begun. the neurologic examination showed a low modified glasgow coma ~ale (mgcs), generalized hypotouia and muscle weakness. these data suggested three diagnoses, posfnfecfious encephalitis, residual neuroumsoaar blockade, and excessive doses of sedative and analgesic drugs. after 20 hours she regained skeletal muscle poxver and ufltlcient respiratory effort, the mcgs was acceptable, and blood gases were normal. she was given n~-tigmine and atropine, and her tr~ma was extubated. an acute respiratory failure ocurrs 120 ram. after. chest radioga'aph disclosed a left inferior lobe atelectasis. after 20 hours weaning begun~and the same episode w~as seen. at this point her mother stated that the girl showed weakness of the eyelids or extraneular muscles. it suggested myasthenic syndrome vs ~-barr6 syndrome. c. botul#num toxins were negative, chotinesterase level ~as normal. edrofoinum test ~as positive. anti-acetyleholine receptor antibodies were negatives. e.m.g. confirmed myasthenia gravis (congenital vs juvenile serenegative). pyridostigmine was begun and the trachea was extubated without complications. conclusion: din the differential diagnosis of weamng failure we must consider ~c gravis~ 2)myasthenia gravis could resemble encephalitis, because of low ocs, overall if is triggered by viral infection. 3)in some diseases (this case) gcs could not he an aemuate index of mental state. a burguet*, a menget*, e monnet**, a gasca-avanzi*, c fromentin*, h allemand**, jy pauchard*, ml dalphin*. * r4animation infantile potyvaiente chu st jacques 25030 besancon cedex. ** d~padement de sant6 publique 25030 besancon cedex, france, objective : to point out that strabism is) of one-year-old premature is a good predictor of a poor neurological outcome at two years of age. design and setting : two-year prospective cohort study and geographically defined study (region of franche-comte, france). main outcome measures : neurological assessment was performed at one and two years of age (uncorrected for gestationnal age). a mailing questionnaire was sent to the famity and fuu-filled by thefamily doctor (pediatrician or physician), or neonatologist of the icu at tertiary center, s was diagnosed at one year of age by the examinator but s was not used to diagnose cerebral palsy (cp). sample : 161 of 171 survivors (94%) evaluated at one and two years of age. results : correlation of one and two years neurological evaluation is weak (kappa=0.5). correlation of s at one year and cp at two year is fair (kappa=0,72). the goal of this paper is to review evidence related to hypothesis that the "waiting" axons and cells of the transient subplate zone may participate in the structural plasticity of the human cerebral cortex after perinatai brain damage (kostovic et al, metabot brain res4:17, t989) and to correlate this phenomenon with different forms and mechanisms of structural plasticity. it is our basic assumption that all lesions occuring during cortical histogenesis will lead to more or less pronounced structural reorganization. here we show that various components of the subplate zone participate in several forms of the structural "plastic" responses in the human cortex: modification of convolutional pattern, changes in size of cytoarchitecturat areas~ columnar reorganization, dendritic and synaptic plasticity. the etiological factors which induce lesions and subsequent plastic changes act via the following pathogenetic mechanisms: * disturbances of radial unit formation (rakic); * changes in ingrowth of afferent fibres; * changes in the rate of normally occuring reorganisational events, depending on the critical period for a given histogenetic event. in the present study developmental lesions (localized perlventricular leukomalacia and haemorrhages) were demonstrated by ultrasound in live-born infants ranging between 26 to 40 weeks of gestation. in younger infants (24-34 w) who died shortly after birth, examination revealed lesions of the white matter with the preservation of the subplate zone. in infants who died one week of more after the lesion, we have observed localized micropolygyria, cavities, condensed layer vi -subplate zone, and columnations of the cortical plate. these changes are less prominent if the lesion occurs after diminishment of the subplate zone (after 34 w). since in the fetal cortex the subplate zone serves as predominant source of growing fibers, transient neurons, trophic factors and contains cellular substrata for migration, this zone is the most likely candidate for major types of structural plasticity. in conclusion, cerebral cortex of the low -birthweight infants is more susceptible to the various lesions but shows vigorous structural plasticity and conspicuous functional recovery due to the growing, transiently located neuron at elements. the mortality due to meningoccocal sepsis is high in spite of important progress in emergency and intensive care medicine. during the last decade multiple scoring-systems have been developed in order to establish a therapeutic approach and to evaluate the final outcome of a meningococcal infection. different clinical and biological data (shock, ecchymosis, peripheral wbc and platelet count, coagulopathy, acidosis, meningism, etc) are taken into consideration and the importance given to these data depends on the scoring-system used. a review of the different scoring-systems is given and a clinical case is presented. we report the case of a 4 year old male, who was transfered to our icu 12 hours after onset of temperature and skin rash. the parents described a fast deterioration of his condition. the boy presented wide spread ecchymosis, high temperature, no signs of meningism, circulatory insufficiency and shock, coagulopathy and low peripheral wbc and platetet count. disseminated intravascular coagulopathy developed promptly. the glasgow meningococcal septicemia prognostic score (gmss) was used and the obtained score reached the highest level (15/15). this corresponds to a 100% mortality. the patient required mechanical ventilation for 5 days. at admission he received human albumine, fresh frozen plasma, dexamethason, dopamine, dobutamine and a continuous infusion of adrenaline. antibiotical treatment consisted of ceftdaxone. the evolution was favorable and the infant fully recovered. retrospectively the gmss was compared to other meningococcal scoring scales which gave the same mortality (100%). we conclude that the scoring-systems are important to evaluate the seriousness and to assess the therapeutic approach, but they should be used cautiously even when 100% mortality is predicted by several risk evaluations scoring-systems. the aim of this study was to assess the haemodynamic status on admission and the critical care management of children presenting with meningococcat infection. this was a retrospective study of the charts of 46 consecutive admissions. mean age was 3.43 years (+/-3.46). the average duration of symptoms prior to admission was 20.4 hours (+/-14.09). on admission 17.4% were hypotensive, 45.6% had clinical signs of haemodynamic instability and 54.8% of cases that had a blood gas analysis on admission had a metabolic acidosis (bases excess < -5.q): the mortality rate was 10.9%. 80% of patients that died were hypotensive on admission and all had a metabolic acidosis. of the 41 survivors 9.7% were hypotensive on admission, 39% had clinical signs of haemodynamic instability, 25% required invasive pressure monitoring and 7.3% were ventilated and received inotropic support. this study demonstrates that at the time of presentation with meningococcal infection children had a high incidence of established haemodynamic instability. successful management of this infection is dependent on early presentation and initiation of therapy and on aggressive support of the cardiovascular and vital organ systems. dept. of intensive care medicine and dept of infectious diseases, our lady's hospital for sick children, crumlin, dublinl2, ireland. jude. pediatric intensive care unit, ch&u, 59037 lille-france. more than 10% of children surviving sip (defined as purpura with shock) have snli. objective. to search for a specific hemostatic profile in children with snli. patients and methods. between may 1989 and march 1995, 34 children with sip were admitted to our picu : 6 (17.6%) died and 28 (82.4%) ranged in age from 1 to 185 months (mean : 29) survived, 5 of them (17.8%) with snli (defined as the need of a surgical procedure). in survivors, two hemostasis studies (between h0 and h12, and 24 h later) included the determination of coagulation factors (routine tests), protein c (pc : amidolytic activity, biogenic), total protein s (ps : elisa, stago), c4b binding protein (c4bbp : laurell's technique, stago), antithrombin3 (at3 : chomogenic test, stago), and plasminogen activator inhibitorl (pail : chromogenic test, biopool). three severity scores were determined at admission : french group of pediatric intensive care, gedde-dahl, and crp. statistical analysis used the wilcoxon's test. results. at admission (lst sample) severity scores and at3, pc, ps, c4bbp levels were not different between the group with snli and the group without snli ; quick time (22 4-5% vs 35 ± 14% ; p = .025), vti+x (20 4. 3% vs 30 4-10% ; p = .04i) and pall (105 4-157 ui/m! vs 580 4. 570 ui/ml ; p = .028) were lower in the group with snli. on the 2nd sample there was no difference between the two groups. kinetics of hemostatic abnormalities was not different between the two groups. conclusion. in the literature, intravascular coagulation (dic), low fibronectin and at3 were identified as predictors of snli, and a negative correlation was found between the mean size of the skin lesions and pc activity, at3, and total ps. in this series, apart from dic, there were no specific hemostatic abnormalities that support the use of treatments such as pc, at3, and pail antibodies administration to prevent snli. further studies including more children are needed. the aim of study was to investigate the efficacy of intravenous immunglobulin with enriched igm content pentaglob/n /biotest/. in our pediatric intensive care unit ten septic children /group i/-their average age 2,6 years /sd:o,6/, 7 of them with gramm negative and one with gramm positive blood cultures, and two with unindentified bacteria-were treated with basis sepsis therapy and pentaglobin. the application of pentaglobin was as follows: 1,5 ml/kg loading dose for one hour, followed by a continuous intravenous infusion 0,1-0,4 ml/kg/hour depending on body temperatura /lanser scheme/ for 72-96 hours. another ten septic patients /control-group ii/the mean age 2,5 years/sd:o,65/, their blood cultures were gramm negative bacteria 6, positive 2, and the bacteria was not indentified in two cases -were treated with only the basis therapy. results: the duration of intensive treatment decreased from an average 22,7 days /sd:8, min 12-max 38 days/ to 19,5 days /sd:5,2 min 9-max 25 days/ in the group treated wit pentaglobin. the difference was significant /x 2 p<0,01/. in the group i nobody died, but three in the group ii. conclusion: the pentaglobin therapy can improve the efficacy of the basis therapy of sepsis. sinus bradycardia after an episode of sepsis is a rare symptom complex decribed in children with hematologic malignancies. we present a case of postsepsis bradycardia following severe typhlitis and septic shock in a 12 year old boy with relapse common all. blood and ascitic fluid specimen grew clostridium species and pseudomonas aeruginosa. at surgery there was a necrotic gangrenous terminal ileum and cecum, requiring ileocecal bowel resection with ileostoma. while clinically recovering from sepsis he developed bradycardia for 120 hours. extensive diagnositic procedures was given and the heart rate slowly increased to normal range of age. postsepsis bradycardia in children with hematologic malignancies after an episode of sepsis is self-limiting and after careful differential diagnostics warrants an expectative attitude. nitrate level is known to be enhanced during sepsis. serum nitrate is the stable metabolic end-product of endogenous nitric oxide generation. nitric oxide has demonstrated to be a powerful anti microbial final mediator and also a key molecule driving to the lethality of one of the most common complication of sepsis; the endotoxic shock. such facts prompted us to investigate the possible diagnostic and/or prognostic value of monitoring serum level in high risk, presumptive and confirmed sepsis patients. additionally we have explored the usefulness of this mediator as index of therapeutic response. in our study it is demonstrated that there is an important relationship between nitrate level and the occurrence of neonatal sepsis. septic newborn group showed 6 fold higher nitrate level than that of healthy control group. in addition, the group of patients with high risk of sepsis which finally became septics, exhibited 3 fold higher nitrate level at 24-72 hours before the first symptoms appeared, when compare with those who did not develop sepsis. however in the presumptive sepsis group, there was no difference between the patients which finaliy ,&'ere considered septics and those which not. in all septic cases, after 7 days of a successful therapy with antibiotics, the level of nitrate diminish 3 fold. our results suggest the utility of monitoring nitrate as index for the diagnosis of neonatal sepsis. the potential benefits of exchange transfusion, plasma exchange, and haemofiltration have all been described in children with overwhelming sepsis. however, little hard evidence exists to prove the benefits of any of these techniques. i have treated five patients with plasma exchange (pe), having been asked to see all these patients at a point when it was felt death was inevitable. two of the patients had staphylococcal, two meningococcal and one enterococcal septicaemia. all patients showed a dramatic haemodynamic improvement following pe with improvement in blood pressure, reduction in inotrope requirement and improvement in tissue perfusion. three patients survived. one of the patients with staphylococcal sepsis and both of the patients with meningococeal sepsis had developing gangrene of the limbs which showed remarkable reperfusion with pe. in two of the patients measurements of cardiac output (co) and systemic vascular resistance (svr) showed ~a reduction in co and a rise in svr over the course of a pe despite the reduction or cessation of vasoconstricting inotropes. many believe haemofiltration is of value in septic shock. a trial with a no treatment limb is difficult to achieve. i believe we now have enough evidence to justify a controlled trial of haemofiltration versus plasma exchange in patients with septic shock and unstable haemodynamic status whilst on inotropic support. during the next several days, cough and chest pain suggested pulmonary embolism confirmed by radiologic evaluation. echocardiographic examination showed multiple thrombosis of the superior vena cava, right atrium and ventricle and pulmonary artery. estimated protein c level was 50.7 % (normal range 70-140%); identical deficiency was found in patient's mother and elder sister. cvc was removed, and alter 2-month heparin therapy and supstitution of protein c with fresh frozen plasma, there was almost complete thrombolysis of the great vessels and cardiac chambers. we conclude that invasive diagnostic and therapeutic procedures in such patients may result in higher risk for severe thrombosis at unusual sites, and numeuos further complications bronchopulmonary dysptasia (bdp) is a chronic pulmonary disease of preterm and term babies treated with mechanical ventilation for respiratory problems of different origin and requiring oxygen therapy 28 days after birth. bpd is a disease affecting the growth and development of pulmonary tissue. such pulmonary }esions heal by squamous metaplasia leading to scar formation and fibrous tkssue r~growth, the pediatric intensive care unit makes the survival of babies w~h very low birth weight (500 -999 g) possible. with the increase in their aulyival, the number of complications in low birth weight babies increases as well. bdp is a very serious complication. therefore the importance of early diagnosis and treatment of bdp must be stressed in order to reduce the consequences. babies with bdp must be under medical suveillance for at least 3 years as the disease needs at least that long for complete resolution. tn the icu of pediatric department at madbor teaching hospital: during the past two years (1994-95) 154 newborns were treated with mechanical ventilation. the neonatal and postnatal death rate of all newborns admitted to our icu was 7,1%o.ln the two years from 1994 to 1995, 16 newborns were admitted to our icu (2 %~ of all newborn babies at maribor teaching hospital), with birth weight 500-999 g. in the icu, the survival of these babies and parallel to it the number of complications is increasing. during the mentioned 2-year period, 8 babies with very low birth weight (500-999 g) survived: 5 in 1994 and 3 in t995. in 45-50 %, first or second stage bdp was treated,there was no case of third of fourth stage bdp. the treatment consisted of eary removal from mechanical ventilation, oxygen therapy~ intensive treatment of infection, volume and caloric intake contro}, corticosteroid treatment throught 6 weeks with decreasing doses, diuretic end antioxydant therapy. the children are to be reevaluated at the age of 3 and 6 months and again at i and 3 years. oeure j van der, markhorst do, haasnoot k department of pediatrics, pediatric intensive care unit, free university hospital, amsterdam, the netherlands. case summary a 4%-month 6.5 kg girl of african origin was admitted to the pedfatric irtensive care unit with pneumonia and progressive respiratory irlsuffjderey. she was intubated and ventilated by pressure regulated volume controijed ventilation (servo 300c, siemens, soma, sweden). maximum conditions were inspiratory minute volume 3.2 l, peep 10 cm h~o ahd 100% 0~. chest x-ray showed bilateral interstitial consolidation. material obtained by broncho-alveolar lavage showed preumocystis car}nil htv-serology (elisa and westerll blott) and p24-antigerl were positive, confirming the diagnosis of pediatric aids. she was then treated with high dose co-tllmoxazoie, penthamldine, z{(~ovudire and steroids iv. because of thee x-ray features, high need for o 2 (100%, pad 2 56 mm hg), not responding to elevatiofi of peep (max 10 cm h=o) and pao2/fio = <200 (s6). m acute respiratory distress syhdrome (ards) was diagnosed. because conventional ventilation (cv) failure, hfo-v (31ooa, serisor medics,yorba linda, ca) was initiated. starting mean airway pressure (map) of 19 cm h~o was based or map of the cv, oscillatory pressure amplitude (dp) of 47 was, at ii~itial frequency of 7.5 hz, adjusted ur~til chest wall vibrations were visible, it was required to raise map to 26 cm h20 and dp to 66 before optimal lung volume and ventilation were achieved and need for o 2 reduced within hours, this was monitored by frequent blood-gas analysis and chest x-rays. map and dp could slowly be reduced, after a good response the first day, gradually 02demand reduced and the patient could be weaned from the ventilation. map, dp, fi02 and oxygenation index (map x pa0~jfio 2) are shown in table i. chest x-ray follow-up showed gradually improving lung features, with marked improvement of aereation. after 10 days hf0-v she could be succesfully detubated when a map of 10 cm h20 was acmeved. results : sianificant increase in ventilato~ rate and mean airway pressure was noticed after the change to savi. no differences in oxygenation, co 2 partial pressure and systolic, diastolic or mean blood pressure between imv and savi periods were noted. in 6 infants however an improvement in pao2/p43.ol/ and decrease in paco 2 was observed after the switch to savi. these babies had a lower initial a/a oxygen tension ratio and required higher initial ventilator rate /p<o.05/ in comparison to the rest of the studied patients. conclusion:infants with low a/a po2ratio who need high initial ventilator rate are likely to obtain benefit from savi.. fumimare hatori, haruo uchida, masao katayama, and rika muto department of anesthesia and critical care medicine chiba children's hospital, chiba city, japan. purpose: this study was made to find out whether the heat and moisture exchangers (hme) was effective enough to humidify in the respiratory management for children. patients: the study population consisted of 21 patients who were provided with artificial airway. they were divided into the two groups: ii with heated humidifier (a) and i0 with hme (b). methods: hme could be used for those without pulmonary complication and with less leakage around the endotracheal tube. in both a and b groups, (i) we have measured relative humidity, temperature, and absolute humidity at the endotracheal tube connector. for these measurements, humidity sensor system was utilized. (2) any troubles and complications caused by the artificial airway were investigated. results: (i) in group a, we confirmed the mean values as the relative humidity of 96.5%, the temperature of 32.8~c, and the absolute humidity of 34.0mg/l. in group b, they were 92.7%, 29.5°c, and 28.7mg/l respectively. no difference was noted between the two groups as to the relative humidity, but the temperature and absolute humidity were lower in group b with a significant difference (p<0.0001). (2) in neither of the groups, any respiratory complications such as obstruction or stenosis of the artificial airway were noted. in pediatric icu, hme can be used without any problems under given conditions. titei: objective: evaluate the precision, reproductibility and aplicability of a new device to measure auto-peep in pediatric patients during mechanical ventilation. material and methods: it's an electromc-pneumatic eontolled device with an oclusion valve and a pressure monitor installed between endotracheal canulla and the ventilator circuit. the measurements were performed with a sollenoid conected with to the ventilator to detect the end of inspiration phase and the actwation of the oclusion valve. the signs of pressure and flow were moniturized using a difere~rtial transducer and it was processed using a pc computer and a pneumoviaw® software. the auto-peep also displayed in the ventilator pressure monitor. results: we submitted 17 children with neuromuscular disease or respiratory distress in this study. the incidence of auto-peep was 76%, with variability between 1,5 to 13 cmh20. all the measurements were performed 3 times to verify the precision and the reproduetibility and evaluation of the pressure and flow curves. conclusion: the device is low cost, easy to use and can be used without the pneumotacograph. the auto-peep measurement can be done during mechanical ventilation with time cycled, pressure limited and continuous flow, the most commom ventilator used in pediatric patient. resistance was modeled by 3 endotracheal tube (ett) sizes (2.5, 3, 3,5), and compliance by 2 test lungs (1 and 5 ml/crnh20), for each resistance -compliance arrangement, we measured tidal volume (vt) at various frequencies, peak to peak (p-p) and mean airway pressures. results vt increased when ett size increased with all 3 ventilators in accordance with fredberg results (jap, 1987; 62: 2485-90) . the maximum vt delivered was smaller with the hfv-babyiog 8000 than with the ohf 1 or the sm 3100a at a given frequency (figure on the left). increasing p-p resulted in a linear increase in vt delivery in the 0-30% range of maximum p-p (figure on the right). hfv-babylog 8000 didn't provide significant vt increase when p-p was set above 50% and vt deliven/decreased when mean airway pressure decreased. --i 5~ sm 3100a a number of ventilatory parameters had been analyzed with russia-produced monitor humitemp htm 902 (servisinstrument j,-s. co) in 24 newborn infants with rdsn of t-iv stages on cmv, cppv, imv, and cpap steps using respirators of various kinds (stephan-staxel, bear-2001, newport 8rezee, sechrist). parameters investigated were: temperature of inspired mixture (t), relative and absolute humidity (rh and ah, respectively), static compliance (c), expired tidital volume (vte), expired minute volume of ventilation (mve). the monitor read these parameters in following ranges; rh-from 10 to 100%; ah-up to 55mg/i; mve-up to 3.0 i/mini vte-from 3 to 130 ml; t-accurate to 3%; c-from 0.1 to t0 ml/cm h20. continuous monitoring time ranged from 2 to 96 hours, it was noted, that during this time the monitor and transducers had no malfunctions, and it's using didn't require additional calibration, it was found, that humidifiers mr 498 and mr 410 (fisher, paykel) couldn't maintain parameters t and ah continuously (these values varied from 34,3 to 38.2 c and from 37,5 to 47.1 mg/i, respectively, when heating regulator position was unchanged). changes of parameters c depended clearly on cppv conditions and rdsn severity (with rdsn of stages i-ii c value ranged from 2.4 to 1.2 ml/cm h20; and with stages iit-tv-from 0.3 to 1.1). tn 6 newborn suffering from rdsn of stages ii-ltl and ill-lv with pip>25 mbar, fi02>0,7, peep=4-7 mber, c-from 0.3 to 1.2 ml/cm h20, effectivity of exosurf therapy was studied. in 4 newborns in 4-12 hours of therapy pip decreased to 0.3-0.4, and c increased to 1,7-2.4 ml/cm h20. in 2 newborn infants with aad02>500 mmhg and c from 0,3 to 0.8 mltcm h20 positive effects of exosurf on lung compliance were not observed. in 3 newborns the monitor had revealed decreased of c (from 3.4-2.9 to 1,8-1.3 ml/cm h20), manifested clinically by pneumothorax. in general, monitor htm 902 made possible; 1), to estimate the adequacy of cmv-parameters and regimes in newborn infants; 2). to select optimal t and ah values in the respiratory outline in dependence on lung damage severity and infused volume; 3). to reveal rdsn severity; 4), to optimize indications and adequacy of surfactaot therapy; 5). to diagnostieate the air leakage syndrome; 6). to effects to some agents (broncholytics, spasmolytics); 7). to obtain objective indications for imv/simv and cpap regimes. albano communication is an important aspect of human development and existence, and an inability to vocalise can be a problem in ventilatordependent patients. we present our experience with speaking aids as a means of enhancing verbal communication in four ventilatordependent children in our paediatric intensive care unit. the age of the children ranged from 7 months to 5 years, and the period of ventilation ranged from 3 months to 21 months via a tracheostnmy. they require continuous flow generated pressure limited or control ventilation at rates of 13-20 bpm. the reasons for ventilation include tetraptegia following a shrapnel injury; tetraplegia following congenital cervical spine damage; tetraplegia following atlanto-axial subluxation; and critical illness polyneuropathy following adult respiratory distress syndrome from prolonged ventilation for a severe head injury. the first three patients have passy-mnir one-way speaking valves and the final patient has a bivona foam cuffed tmcheostomy tube with a talk attachment in view of recurrent aspiration. an improvement in quaiity of speech has been shown by independent assessment. we will review the present literature on this subject and discuss the advantages and disadvantages of these two types of speaking aids in the light of our experience. the prognosis of antenatally diagnosed cdh is closely related to the degree of ph. there have been attempts to correlate antenatal or postnatal criteria to mortality: none have been demonstrated to be predictive of lethal ph. the aim of this retrospective study was to determine whether antenatal or early postnatal data could correlate with the findings of post-mortem examinations. patients and methods: between july 1990 and july 1994, 32 cdh patients have been antenatally and postnatally managed at our institution. twentythree infants underwent a post-mortem examination. ph was assessed by using the lung weight to body weight ratio (lw/bw) and the radial alveolar count (rac). antenatal results: cdh diagnosis was made at 24 weeks of gestation (wg) (15-37). twenty-eight patients had a left sided cdh, 3 had a right sided cdh, and one had a bilateral cdh. herniated organs were stomach none (n=21), or liver alone (n=4), or both stomach and liver (n=5 the patient was a 3-yenr-old girl with chronic renal insufficiency see~ to renal dysptasm, two months before admission a kidney trar~ptant was performed. one morah later she showed acute graft rejection with serum ereafinine (cr) level of 0.7 mg%. the rejection was unreslxmsive to an increased steroid dosage, and okt3 was begun with resolution of the rejection. one week arer, new rejection episode was seen marestxmsive to an increased steroid dosage, and transp~ ~s performed five days before admission to our ptc. hemedialysis and peritoneal dialysis (p.d.) each other day, was indicated (g.r.f.< 10 ml/rnin). four days before admission t ~ rose to 38°c. "lhe diagnosis of opporttmistic pneumoma was made on the basis of tach3,pr',e~ hypoxi~ and diffuse interstitial infiltrates. senma ~ was positive for cytomegaloviras (cmv), and stool culture for c albicans. pentamidine, ganciclovir (dhpg), arai-cmv gamma globulin, eritromicine and amphotericin b was administered. on admission in our picu, trachea was mmbated, (a-a) o2 gradient was 600, paofffio~: 65, lung injury score > 3 with peep level of 8 cm hzo. she had normal fiver function. during te next days she had fever and developed ards. bal was negative. p.d. was of little efficiency. we adjusted pentanfdine, and dhpg doses for severe renal failure, with supplements after hero, sis, and at~rp.d.. during ~ next days she was afebrile, and the chest became radiologlcally normal. after ten days on menhani~al ventilation (mv.), the patient was extubated. cr. level was 3.2 rag%, (a-a) oz gradient was 20, and paoyfioz was 375, the patiem was discharged with chronic ambulatory p.d. discussion: opportunistic pneumonia is a major complicalaou in imm~romised children, specially after kidney tvansplaraafion. c m.v. infection can result at~r okt3 administration. in the treatment dhik} dose must be adapted to the degree of renal insu~cieney, with supplements after hemedialysis, and after pd. pneu~y~tis cann# tmeumov~ is ehemeterized by ventilafion-perfusion mistmaeh, decreased pulmonary compliance, hypoxia arld elevated (a-a) oz gradient, with diffuse interstitial infiltrates. in our ease bal was negative. although we did not find the etiology the prevoclons eombh~ation of arairmcrobiat therapy, along with m.v., and supportive measures were the most effective trealme~. conclusion: 1) in patients with severe renal failure and life-threatening infections, we must co~ider drug adjuslments. 2) in our patient we gave dhpg supplements at~r pd. with excett~at results, although p.d. was of little effiele~. introduction: endotracheal intubation and mechanical ventilation have become an important treatmem for many diseases accompanied by respiratory failure. with the frequent use of this treatment modality, an increasing number of complications associated with endotracheal intubation have gained clinical significance. material and methods: a transversal study was realized to find the prevalence of pulmonary aspiration with endotracheat tubes in 36 infants and children. aspiration was assessed by applying two dyes (evans blue, er)¢rosine sodic) on the tongue and searching for the dye during suctioning in the endotracheal aspirate. the factors, that potentially have influenced the aspiration, including weight, age, sex, cause of respiratory failure, main pressure airway (map), level of consciousness, presence of swallowing and body position were evaluated. all the variables studied had their association with aspiration tested by chi-square method with relative risk considering a confidence interval of 95%. the results were adjusted by multivariate analysis. results: the overall prevalence of aspiration was 36.1%. among all children who aspirated, compared to those who did not, there was a statistically significant difference in the presence of swallowing (p=0.005). the odds ratio to aspiration in the presence of swallowing was 38.4 (t.75 -100 c.i.95%) and the relative risk 55.5. aspiration was not significantly affected by sex, weight, age, cause of respiratory failure, map, level of consciousness and position of the body during the ventilation. conclusion: the endotracheal intubated children frequently aspirate as intubated adults and that preventive measures are ineffective. the presence of swallowing movements is the main risk factor to aspiration of oropharingeal content in intubated patients. clinical features and shortterm outcome skling, rp gie pneumonia is the second most important cause of death in young south african children. the clinical features, intensive care course and outcome of children being ventilated for pneumonia in the developing world is unreported. aim: to describe the clinical findings, aetiology and shortterm outcome of children younger than 6 months with pneumonia requiring ventilation. the data of all babies under the age of six months with a lower respiratory tract infection admitted to the paediatric icu for ventilation were prospectively collected over a period of 14 months. tracheal aspirates and blood specimens were submitted for viral and bacterial cultures. results: forty-seven babies aged 14 to 174 days were ventilated for pneumonia. twenty-six infants had been born prematurely; t2 had been ventilated during the neonatal period and 4 had bpd. the median duration of symptoms was 1 day, the most common being cough, tachypnoea, apnoea and cyanosis. five babies (10%) died. the mean duration of ventilation was 8 days (range 1-85 days) and of ward stay after icu discharge 19 days (range 1-161 days), blood euttures were positive in 7 children (15%). viruses were cultured in 14 children (30%). conclusion: 1) fifty-five percent of children below 6 months requiring ventilation for pneumonia were premature infants, of whom 46% had been ventilated during the neonatal period. 2) the median duration of symptoms prior to admission was 1 day. 3) ninety percent of the children survived and were discharged from hospital. 4) viral pneumonia was responsible for 30% of the admissions. mechanical ventilation and atrial natriuretic factor release ulloa santamarfa, e, p6rez navero jl, ibarra de la rosa i, espino hernladez m, velasco jabalquinto mj, frfas p6rez m. picu. reina sofia children's llospital. c6rdoba. spain. mechanical ventilation effects on renal function decreased diuresis and natriuresis due several factors including anf. several studies have demostrated anf released due increaasing pressure in right atrium. on the other hand, mechanical ventilation, overall peep modality, inhibits peptide release althougt cvp increased is found. this study was designed to demostrate anf stimulation is due rigth atrium stretch which be higher during mechanical ventilation instead of atrium pressure. we desing a prospective study including 14 patients, age range 16 months-13 years with congenital heart disease. all of them were admitted at pediatric intensive care unit after extracorporeal surgery and were assisted by mechanical ventilation. hemodinamic state was stabilized in all patients and nor renal neither neurological diseases were found. after 24 hours with mechanical ventilation, plasmatic levels of anf were measurement, pvc, pericardical pressure were assessment; all patient were sedated with midazolan and paralized with neuromuscular blocking agent; mechanical ventilation technique was as follow: imv between 20 and 30, tidal volume and fi o2 enough to mantain respiratory parameters in normal range. afterwards, at least twentyfour hours in spontaneous breathing, the study was made again in each patient. atrial stretch was assesssment according to following equation: transmural pressure= cvp -pericardial pressure. cvp were significantly higher with mechanical ventilation than when the patient was breathing by himself. (5.4+__ 2.2 vs 3.8 + 1.8 mm hg; p<0.01). however, transmural pressure during mechanical ventilation were lower than during spontaneous breathing (8.92 +__ 3.86 vs 11.76 +__ 3.32 mm hg; p < 0.01) equal, plasmatic anf levels were lower during mechanical ventilation ( 87.77 + 46.55 vs 108.92 + 49.06 pg/rnl; p<0.01). in conclusion, anf secretion decreases during mechanical ventilation, even with cvp higher. anf release would depend on atrial stretch meassured by transmural pressure, lower in patients with mechanical ventilation and it would not depend on atrial pressure. the paediatric intensive care unit shaikh zayed hospital, lahore is an acute care area devoted to the care of critically sick children upto the age of 13 years. in a 6 bedded unit with limited equipment, constant care is ensured by the presence of at least one nurse aed one doctor round the clock. in this setup we have the facility to ventilate 2-3 children at one time, between sep. 93 and dec. 95, out of 885 patients admitted to icu, 171 (19.32%) were below 1 yr of age, while 48 (28%) were below 1 month of age. life support was discontinued in 17 (9.9%). total mortality was 56 (32.7%), major mortality was in 0-1 month age group 22 (12.8%), and 1 month to 6 month 15 (8.7%). majority of the patients were of sepsis (36.2%), cns disorder (22,2%) followed by respiratory problems (14.6%). it seems therefore that the major indicatiou for ventilation was overwhelming septicemia leading to multiple organ failure, rather than purely respiratory problems. high frequency oscillation (hfo) in the therapy for ards in pediatric patients requiring aggressive conventional mechanical ventilation (cmv) -routine or experimental mode ef pre ecmo therapy. fedora m., nekvasi~ r, vobruba v., srnsky p,, zapadlo m. dpt. critical care medicine, nicu and ecmo center, university children's hospita! brne, nicu of university hospital prague, czech republic. introduction: 9 pediatric patients (8 males, 1 female, average age 4.7 months, average body weight 5,8 kg) with severe ards ventilated with aggressive regimen of pcv or prvc were connected to hfo (sensormedics 3100) as the last "rescue" therapy due to uncontrollable respiratory failure before intended ecmo. in the course of hfo 2 of them were given no in the concentrations of 5-80 p.p.m., 3 were subjected repeatedly to surfactant replacement therapy (alveofact). results: ecmo was needed in no patient, 8 patients survived, 1 patient was disconnected from the ventilator because of brain death in spite of conspicuous improvement of oxygenation and other parameters, some relevant parameters 48 hours before and 48 hours after starting hfo are given in table 1~ in all the cases, the disconnection from hfo was carried out through the simv regimen, never directly to cpap. table 1 : the levels of blood gases, oxygenation index (oi), aado2,map,fio2 and pao2/fio2 ratio 48 hours before and 48 hours after starting hfo. conclusion: although none of the patient had to be subjected to pediatric ecmo, hfo should be carried out only in workplaces having the immediate possibility of using this method in the case of hfo failure. speculation: should not hfo be used ir pediatric patients with ards earlier than aggressive cmv? can hfo ce considered standard, not experimental method of therapy? refractory hypoxemia in premature patients is characterized in a persistent elevation of pulmonary vascular resistance, with right to left shunt through the ductus arteriosus and or foramen oval. we report the case of a vlbw patient (ga 27w, bw 1010g) who present a severe hypoxemia related to hyaline membrane disease and a pulmonary and systemic infection to group b streptococcus, refractory to conventional ventilatory support and surfactant therapy, associated to hemodynamic failure falling in ecmo criteria used for term infants. a rescue therapy with hfov (sensor medics 3100a) is decided at 5 h of live, the table resume the patient's evolution before and after hfov. at 36w of postgestational age the patient present a fio2 of 0.23 with a chest x ray compatible with a cld type l at discharge no oxygen requirements was needed and actually he's doing well. conclusion: hfov, using an adequate alveolar recruitment strategy, was effective in the rescue of a severe hypoxemic respiratory failure with a rapid off of ecmo criteria entry in our vlbw premature patient, during the united nmioffs embargo ~nst yugoslavia the prevalence of the ast}nnafic ~acks in c~dldren aratsed. the mo~t common causes have beem dramm~e worsening of life standard, ecom~c disaster in global community, gr~ number of refugees from the other parts of former yugodavia. it wm obviom that mcio-ecoumnical conditions took a part in the exacerbations of previously known cldldhood asthra~, ~av~ of micro-and m~mclimaflc changes, psychosocis] and emotional cryses, lack of medics-m~nts for p~ve~on and tl~rspy of acute asflanatic attacks. about 10% of d-dldv~ tmslod in our picu for these year~ exp~dvncod ~vcr~ attack for the flint time iu ~jzeir lifts. it has been cu~ 1~%~ children in mspir~ry picu of our hos~mt. the scut~ revere attack (more ~asn ~/o of hight clinical score) was detected in 62% of all children admitted with respirak~ problems. from tl~ mmlysss we exclu&d: bmncldolifis, ~i anomalies, ~eve~ i~ccqions. concerning our drug supplies (which wc~e reduced), we started our therapy by administration of oxygen, ~ta2-ago~dst inhalations (but sometimes we had the solution for jet nebulizcm only for o~e inhalation per p~cnt), mwinophyllin and mefl~ylpr~ini~done in/ravenously. 48% of ih~ asthmatics needed repea~ doses of muinophyl~n pinch.ally, tnch.,ding the fluids. the bronchodilak)r msponm was poor ~r~cl slow, hospital stay in picu was for 4 days and for 14 days in other units sl~rwsvds. tim ~ of their stable condifio~ was hard at borne (or refugees camps), without p~ventkm, so they came bsvk to hospital for morn than 3 times in 27% of cases, dtrdng ~e4je last motlfl~s file dtustion improved, concerning tim drugs supply for prevention, and we hope that these lifc~restening conditions wouldd~ introduction: the incidence of ards is increasing as survival of critically ill patients is higher. the application of new therapeutic modalities have increased the survival rates in (ards) adult patients. objective: to study the therapeutic efficacy of new tleamlents in children with ards material and methods: a retros~ctive study was conducted from 1990 to 1995. 17 children with severe ards, (lung severity score > 2,5) (r), aged 15 days to 16 years, were included. the diagnosis were as follows: 9 interstitial pneumonitis, 5 non interstitial lung infection, 2 with lung aspiration and 1 with clinical sepsis. 5 patients had different tipes of cancer and 4 to suffer inmunodeficiency disease, the first 8 subjects (group t) were treated with conventional measures. from october of 1994new therapeutic modalities were introduced, including: less agressive ventilatory support, postural changes (prone to supine) in 9 subjects, administration of corticosteroids in 8 patients, rfitric oxide in 3, pe~ssive hypercapnia and administration of exogeans sarfactant in one, pao2/fio2, d(a-a)o2, oxigenation index (oi) and the score of respirator), severity disease were similar in both groups. the two groups evolntiou was compared. results: -ten patients died, 6 from group i and 4 from group ii (75% v.s.44:4%,ns). -the evolution time, either to exitus or weaning from ventilatory support was higher in group ii (22.9 v.s. 13.6days in group i, ns), -the incidence of barotrauma was observed in 12 subjects (70.6%), 6 from group i and 6 from ii. of these patients 75% expired. -during the course of the disease, 15 (88%) patients had more than one damaged organ. only in one subjet mof was considered to be the main cause of death. the majority of the patients expired because of their respiratory disease, although, 80% of them met criteria of mof. -fifty percent of the subjects were infected at the time of death. stmmry: a trend toward a higher survival rate is observed in the subjects receiving the new modalifies therapeutic intervention (corticosteroides, postural changes and permissive hypercapnia). our results are not significative,probably because of the small number of subjects studied. a new doubleaurae~t two-stage et-tube (dl-ett) was desig~aed and tested in the rabbits with acute king injury under conventional mechanical ~entilation_ ventilation efficiency of dl-ett was emrrpared with that of canveniionally t~sed single lumen et-tube (sl-ett). meth~s: dl-ett was specially made out of two sl-ett. vertical crosssections at the distal end of two et-tube (td 3_0 rmn portax) were adhered with each other to form a tracheal stage lumen wifu id 3.0mm the two remained uncut parts of the tubes corlntithted the oval s~ge with two separate imnens. dl-ett and sl-ett were randomly applied to five adult paralyzed rabbits with acute lung injury (by 0.1 nffkg oleic acid. iv). a bird inter 3 vetffttator (bird products corporation) was used for time-cycled pressure-limited ventilation at 40/min of respiratory rate, 10 ern h20 of peak i_~piratory pressure, l: 1 of ire ratio, 6 ljmin. of flow rate and 0.21 of fich. peak inspirntory pressure, mean mrway pressure, posi6ve end-expiratory pressure at tip of et-mbe and bemodynamics were measured and recorded continuously. arterial blood and expired gas were measured ~by avl 993 blood gas analyzer) after each stabilization t.~iod of 30 minntes. _analysis w~as by prated t test. result: dl-ett acaltety improve cos removal at all amman. pa(?oz was decreased by t0.6+_t.5 (p<0.0l) and physiologic dead space fraction (v~zvt) reduced by 22% +-1.8% (p<0.0t), compared with dl-ett. there were no significant change in arterial oxygenation. conelus|on: the double-lumen two-stage et-tabe significantly increases ventilation effmiency with simple operation in rabbits v, ith acute hmg injury, lts availability may influence future clinical management of ~ennated patient~. this ~muly was fimded by the science and technology. commiuee of beijing municipality. analis of hemostasis alterations on different coagulation cascades in 46 children with septic shock has shown that coagulation disorder character is dependent on lung affection rate. the initial manifestation of the respiratory distress-syndrome (rds) are characterized by the obvious activation of blood thrombin potential, moderate coagulopathy and not sharply marked endoteliosis, the witlebrand's factor (wf) increase tot 140-220%. progress in the clinical picture of "shock lung" leads to chronometric and structural hypocoagulation with potential hypercoagulation in "mix-test", high level of firbin derivative, thrombocytopenia with thrombocytopaty and the wf increase to 210~315%, terminal stages of the rds, as a rule, are characterized by potential hypercoaguletion absense, depletion of at-lit and plasminogen, prevalence of antithrombin and antiaggregating activity, obvious endoteliosis (the wf to increase250-540%). the arteriowenous difference according to index of the thromboelastography (teg) in the rds ill-iv rates was 69,8% less than in the 1-11 rates, disorder of lung filtering ability in severe rds is confimed also by minimal arterio-venous difference of activated euglobulin lyses (ael) in children with the rds ill-iv rates is only 11,4%, while the patients whit rds i-i1 rates have the ael-activity in arterial blood 2,1 times as much than in venous blood. the use of then allows to determine the potential hypercoagulation rate, the at-ill level and fibrinogen quantity during the anticoagulant therapy and also the character of the x-factor activation and thrombocytic hemostasis. the effective therapy component of septic genesis rds in children is the controled coagulation method with the use of the individual selected heparin doses in according to desagregants, kryoplasma, proteolisis inhibitors and trombolytics. it is necessary to avoid the heparintherapy for children with the rds complicated with producting coagulopaties and termal phases of blood disseminated intravascular coagulation (dic). bronchoseopy has been used for evaluation of the potential problems of the airways and for investigation the bronchial specimens for diagnostic purposes. regent technical advances result in performing this procedure at the bedside manner and in critically ill patients. we have performed 150 hronehoaeopy during last three years on 1362 pediatric patients with respiratory problems, in 90% of cases the opentube hroneh0seopy was performed (for diagnostic as well as for therapeutic reasons) and collected secretions or bioptic material were examined. the indieatiuns were: acute upper respiratory problems, chronic wheezing, inspiratory strider, tracheal or bronchial bleeding, chronic eongh, retractable atelectssis, severe pulmonary infections, lymph node perforation in lung tuberculosis and soquells like bronehiectssis and fibrosis. our results were: anatomical malformations in 10%, mueosal oedema with chronic inflammation and thick secretions in 56%, easuos masses in 11%, granulation tissue and purulent secretions in foreign bodies and bronehieetasis in 16%, and only 7% of eases were normal finding. our exlxdenees pointed that this invasive procedure in carefully selected patients has important role in establishing the diagnosis and in theintroduction: tbg has been a useful investigation in the management of ventilator-dependent infants in our experience. one ml of contrast was hand ventilated into the respiratory tree via their nasotracheal tubes and their anatomy and dynamics demonstrated on radiological screening. case descriptions: three infants who were difficult to ventilate requiring high airway pressures, high peep and a significant oxygen requirement had tbgs. the ages ranged from 3 to 9 months. two cases were complicated by complex cardiac lesions. in all cases there were frequent episodes of desaturation, where hand ventilation proved difficult and various intermittent lobar collapses occurred. microlaryngobronchoscopies (mlb) performed on the infants by experienced paediatric ent surgeons failed to identify the airway problems. more than one mlb was frequently done. concern about introducing contrast into the airways of infants with limited cardiorespiratory reserve combined with an uncertainty about how much extra intbrmafion would be gained often led to a delay in investigation. when performed these fears proved groundless, the anatomy and pathology of the airways were demonstrated in full and the correct therapeutic plan started. in two cases tracheostomy and peep producing patency of bronchomalacic segments allowed weaning to low levels of ventitatory support. in one case tracheal reconstruction was undertaken and in the cardiac cases the respiratory component of the ventilatory dependence was fully assessed. at the age of 4 months, a baby boy with a history of minor respiratory problems, was admitted to hospital with an upper airway infection and severe dyspnoea. shortly after arrival at the icu he had a total airway obstruction. after intubation there were still difficulties to establish a normal gas exchange, and he was tranferred to the regional picu. ct scan and bronchoscopy verified a congenital tracheal stenosis affecting the whole trachea except the upper 15 mm below the vocal cords. the diameter was estimated to less than 2 ram. an unsuccessful attempt was made to dilate the extremely rigid stenosis with a balloon. after the procedure he had a respiratory and circulatory arrest, and he was put on ecmo as a bridge to surgical correction. after 4 stable days on ecmo, surgery was performed during ecmo with a tracheal homograft transplantation. immediately after surgery, ecmo was discontinued. a silastic dumont type stcnt was inserted inside the homogra~, and a nasotracheal tube was placed inside the stent for assisted intermittent mechanical ventilation. repeated bronchoscopies were performed to remove granulation tissue and secretions. at 9 months of age, the stem was removed with an endoscopic procedure. however, the trachea was still soft and collapsable, and another silicon stent was placed inside the trachea for another 4 months period, after removal he had some respiratory problems and he was treated with nebulized salbutamol, mcemic epinephrine and steroids. he was discharged from the hospital at 14 months of age and his condition is now stable. this is the first procedure of its kind in sweden. it was accomplished by international and multidisciplinary collaboration. ecmo may be a bridge to corrective surgery and long time stenting may be necessary in the postoperative period. post mtubation laryngitis ( pil ) is still a frequent complication, occurmg in l -6 % of intubated patients. inhaled racemic epinephrine has for long been used as an accepted therapy, but this drug is not always available. the authors undertook a randomized, double-blind, placebo-controlled trial to determine the efficacy of inhaled l-epinephrine(le) in the treatment of plu in the period between july/93 and may/95, 289 patients were submitted to endotracheal intubation for ventilatory support. atter the extubation procedure patients were considered for enrollement if they met the following criteria: clinical signs of laryngeal estridor and a downes and rafaelly score for upper respiratory obstruction equal to or higher than 4 patients with primary upper respiratory disease were excluded all patients enrolled reeieved either inhaled l-epinephrine 1% or normal saline. dexametasene ( 0,6 mg/kg/day) was given to all patients in both groups. after 2 inhalations, au patients were monitored for a period of 1-20 minutes and monitoring included cardiac and respiratory rate, mean arterial blood pressure, arterial blood gases and the dowries and rafaelly score. statistical analysis included, qui-square with the fisher correction test and the z-test for paired variables. thirty eight patients ( 13,1% ) met the criteria for enrollment, 18 to the le group and 20 to the placebo group.there were no significant differences in both groups in regard to age, sex, initial score ( 5,05 x 5,1 ) and endotracheal tube diameter. the period of ventilatory support and tracheal intubation was significantly higher in the le group (8,06 x 4,54, p = 0,01). the follow-up score showed a significant drop only at 30 minutes after the inhalations (p = 0,03). re-intubation due to laryngitis, occured in 1 patient of the le group and in 4 of the placebo group with no statistical sxgnificance (p = 0,2). no difference was observed on the monitored hemodynamic variables during the 120 minutes, except for the mean arterial pressure at 60 minutes, being heighar on the placebo group (p = 0,05). we concluded that, although the l-epinephrine group showed a trend in better scores post-inhalation and fewer re-intubations due to laryngitis, the results were not statistically significant. we especulate that the period of intubation may have affected our results. similarlly there were no differences in the incidence of adverse effects between both groups. objectives:to evaluate the complications of endotracheal intubation in children with upper airway obstruction due to epiglottitis or croup. methodes: during a 5 year period (1991 -1995) all patients with epiglottifis or croup were reviewed to determine the complications of endotracheal intubation, especially upper airway obstruction due to granulomas. results: 33 patients were reviewed. in 17 children (mean age 2.5 years) with epiglottitis the mean duration of intubation was 4.0 days (3 -5). no complications were seen. in 16 patients (mean age 2.3 years) with croup the mean duration of intubation until the first extubation was 8.1 days (1 -15 days). elective extubation was performed if an airleak was present or after 7 days without airleak but in the absence of fever and obvious secretion. reintubation was not necessary in 10 children (62.5%). in this group the mean duration of intubation was 6.4 days (1 -12). in 6 patients (37.5%) reintubation was necessary because of severe upper airway obstruction due to granulomas. mean duration of intubation until the first extubation was 10.8 days (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . there seems to be a difference in duration of intubation between these two groups with croup, however it is not significant (p > 0.1). all the patients with granulomas could be successfully extubated after microlaryngeal surgery, with a mean intubation period of 35.3 days (21 -47). revealed no complications, where as endotracheal intubation in children suffering from croup showed a high incidence (37.5%) of granulomas. however1 laryngeal steepsis and other serious complications were not sesn~ 3 patients (42 days averagely] was obviously seen in ~he peak =one of fl, f2 resonance and in the zone of high freq,-~ncy :r, ~;~e composition while 12 cases(3 day~ average;y] :~bowed no abnormality both clinically and isryngoscopica!~y. 7/10 patients with catheter placement for more than 6 week~ end 1126 p~tie,~ts for less than 5 weeks had t;~ryngeal abnormal change in their larynges,abnormal changes of sound spectrogram were all seen in 3 patients with placement for mope than 5 weeks. our data suggest= ca] the complication of endotracheal intubation was increases with increasing length of time of catheter placsm. entjbut aeriuoa complication is rare i (b] the time limit of pernasal endotraoheal catheter placement is 5 weeks within which the procedure is • comparatively safe and effective means for maintaining e tong term artificial airway. in a 6-year period (1986) (1987) (1988) (1989) (1990) (1991) (1992) we diagnosed tbm as an apparent dilatation of the trachea and main bronchi ih four premature infants on continued mv for respiratory distress syndrome (rds). the infants were three boys and one girl with gestational age (ga) 26-33 weeks and body weight (bw) 1100-1965 g. mv was provided by bourns 2001 cub time-cycled and pressure-limited ventilator to attain normal gas tensions. no jet ventilation was used. chest radiographs were reviewed for a complete evaluation, and for the evaluation of the airway. after the intial subjective diagnosis of tbm, the width of the tracheal and main bronchial air column was measured at the lower level of the first and the third thoracic vertebal body it1, t3) and near the carina; the width of the main bronchi below the carina was also measured. in all infants, tbm became apparent close to the 20lh day, that is, after 2-3 weeks of mv. therefore, for the time period from birth to the 20th day the following ventilatory parameters were reviewed and analyzed: (1) the percentage of total ventilation time when more than 40% o2 concentration was required, (2) the peak inspiratory pressure, (3) the positive end-expiratory pressure, and (4) the duration of high frequency ventilation (80-160 breaths per minute). also noted were the apgar scores (1 and 5 min after birth), the duration of hypotension (systolic bp below 40 mmhg) and circulatory instability, the presence of systemic or tracheal conatal or later infection, the duration of mv, and the final clinical outcome. the records were also reviewed for other possible pertinent data. rigid respiratory endoscopy in children fraga j, amant6a s, piva j, nogueira a, palombini b. introduction: the respiratory endoscopy is an important procedure to diagnose and treat many airway's diseases in children. although have had advances in radiologic investigation exams and pulmonary function tests, the direct anatomic visualization of airway is important to the management of many respiratory problems. objective: evaluation the respiratory endoscopies performed with a rigid bronchoscope in a pediatric reference hospital. material and methods: we study the records of all children that were submitted to respiratory endoscopy under general anesthesia from march 1989 to march 1992. age, sex, clinical to indicate the procedure, diagnosis and complications of endoscopy were registered. results: three hundred and fifty six respiratory endoscopies were performed. the most common indications for endoscopy were strider (52%), suspected foreign body (16%), atelectasis (16%) and difficult tracheal extubation (8%). the most frequent diagnosis were laryngomalacia (36%) and subglottic stenosis (6%) in the glottic and subglottic areas, and foreign body (9%) and tracheomalacia (7%) in the tracheobronchial area. normal endoscopy was performed in 54 (21%) of the children. only three slight complications of the endoscopy were observed. two patients presented bradycardia during the exam, and the third need tracheal intubation due to post-endoscopic subglottic edema. conclusion: the rigid endoscopy in children is efficient and has no serious complications. near drowning; indicators of acute and long term prognosis bernardien t.mj. thunnissen t, reinoud j.b.j. gemke 1, loes veenhuizer?, krijn haasnoot 3, a.johannes van vugh0 department of pediatrics, ~wilhelmina children's hospital, utrecht, 2sophia hospital, zwolle, and ~free university hospital, amsterdam, the netherlands. in this retrospective study factors that affect short and long term prognosis after submersion were analysed. all patients that were admitted to a tertiary pediatric icu between january i, 1986 and january i, 1992 were included. of 34 patients, aged 0-13 years, 8 died in the icu, one after hospital discharge. survivors and non-survivors showed significant differences with respect to central temperature, pupillary reactions, arterial ph, pediatric risk of mortality (prism) score and therapeutic intervention scoring system (tiss) upon admission (p < 0.05). non-survivors more frequently required mechanical ventilation, bicarbonate administration and active reheating. ards was seen in 22 patients (65 %), invariably within 6 hours after admission. no patients with cardiac arrest on" admission snrvived without sequelae. hypothermia appeared to have no protective effect on hypoxic damage. survivors with persistent sequelae _> 6 months after discharge had significantly higher prism and t1ss scores (mean 27 and 34, respectively) than those with complete recovery (mean 14 and 23, respectively). long term cognitive problems were present in 7/25 survivors (28%) and emotional disturbances in 5/25 (20%). in conclusion, a concise number of clinical and laboratory parameters, representing acute severity of illness, are important prognostic indicators for survival and health status of children after submersion. there were 59 (91%) bronchoscopies, and 6 (9%) were oesophagoscopies.the average age was 2,8 years for bronchoscopies, and 4 years for oesophagoscopies. the outcome of the patients was good. no complications were observed. extraction is recomended in every symptomatic patient. orphenadrine is an anticholinergic drug mainly used to decrease symptoms of parkinson disease. orphenadrine has a peripheral and central effect and overdose can result in athetoid movements, convulsions, cyanosis, coma, arrhythmias, shock and cardiac arrest. physostigmine is a specific antagonist of the peripheral and central effects and can be a useful antidote. we report the case of a two and a half year old female who was transfered to our icu for general convulsions. the little girl had, three hours before admission, accidently ingested 400rag of orphenadrinehydrochlodde (disipal®), which was her grandmothers anti-parkinson medication. three hours after ingestion she presented neurological signs: confusion, unstable walking, and periods of aggression. generalized tonic-clonic seizures appeared who were rebel to administration of multiple anti epileptica but ceased after iv administration of diazepam and endotracheal intubation and ventilation. an episode of ventdcular tachycardia responded well to the iv administration of tidocaine. the levels of orphenaddne in the serum were high at admission (3550pg/l) and were present in the blood up to 96 hours after ingestion. high serum levels are, in the literature, associated to a high mortality rate. physostigmine was administered three times at a 0.02mg/kg dose in the first 24 hours. we decribe the noted effects of physostigmine on the different symptoms. the patient survived and could leave the icu after one week. in conclusion: orphenadrine poisoning is a very complicated medical problem associated with high mortality. in severe intoxication, the benefit of physostigmine more than counterbalances its side effects. objective: to define the optimal volume of dilution for endotracheal (et) administration of epinephrine (epi) design: prospective, randomized, laboratory comparison of four different volumes of dilution of endotracheal epinephrine (1.2, 5, and 10 ml of saline) setting large animal research facility ofa universi~ medical center subjects and interventions: epinephrine (0.02 mg/kg) diluted with four different volumes ( 1, 2.5. and i 0 rot) of normal saline was injected into the et tube of five anesthehzed dogs. each dog served as its own control and received all four volumes in different sequences at ieast one week apart. arterial blood samples for plasma epinephrine concentration and blood gases.were collected before and 0.25, 0.5. 0.75_ 1.2.3, 4. 5. 10, 15.20, 25.30 , and 60 minutes after drug administration. heart rate and arterial blood pressure were continuously monitored. measurements and main results: higher volumes of diluent (5 and i0 ml) caused a significant decrease of pao2, from 147:!:8 tort to 106±i0 torr, compared to the tower volumes of diluent (1 and 2 ml), from 136±10 torr tu135+_7 torr (p<0.05). these effects persisted for over 30 minutes. mean plasma epinephrine concentrations significantly increased within 15 seconds following administration for all the volumes of diluent. mean plasma epinephrine concentrations, maximal epinephrine concentration (cmax), and the coefficient of absorption (ka) were higher in the 5 ml and 10 ml groups. the time interval to reach maximal concentration (tmax) was shorter in the 5 ml and 10 ml groups. yet these results were not significantly different. heart rate. systolic and diastolic blood pressures did not differ significantly between the groups throughout the study. conclusions: dilution of endotracheal epinephrine into a 5 ml volume with saline optimizes drug uptake and delivery, without adversely affecting oxygenation and ventilation. the aetiology and outcome of paediatric out-of-hospital cardiac arrest was studied during a 10-year period in southern finland served by physician staffed emergency care units. the files of 100 prehospital patients less than 16 years old without palpable pulse and spontaneous respiration were analysed retrospectively. fifty patients were declared dead on the scene (dos) and resuscitation (cpr) was initiated in 50 patients. the sudden infant death syndrome was the most common cause of arrest (68%) in the dos patients as well as in patients receiving cpr (36%). asystole was the initial cardiac rhythm in 70% of the patients in whom cpr was attempted. eight of the 13 hospitalised patients were discharged, 6 of them with mild or no disability, 1 with moderate disability and one in vegetative state. in multivariate analysis the short duration of cpr (<16 minutes) was the only factor significantly associated with better survival. due to various aetiologies the survival rate from prehospital paediatric cardiac arrest is quite low. on the other hand, hypothermic near-drowning victims seem to have a relatively good prognosis. duration of cpr less than 16 minutes was the best predictor of intact survival, our study supports the previous findings of the importance of early and effective resuscitation efforts for establishing ventilation and perfusion on the scene. in our system well trained physician staffed emergency care units are able to provide immediate and effective als on the scene. on the other hand, these units also appear to be able to refrain from resuscitation when the prognosis is pessimistic. objective: to assess the normal ,gastric intramucosal ph ~hi) by tonometry in healthy children patients and methods: twelve healthy children (6 males and 6 females) with age rmaged from 6 months to 12 years scheduled for minor plastic or urologic surgery. children were previously medicated with midazolam (0.25 mg/kg) and atropine (0.02 mg~) both i.m.. anaesthetic induction was standardized with 02 -n20 (75%) administered via facial mask and increased halotane concentrations (up to 2%). all patients got an endotraeheal tube after iv. administration of femanile (2 mcg:jkg) and vecuronium (0.1 mg/kg) or suxametonio (1 mg/kg), pmaesthesia was maintained with o 2 -n20 (60-75%) and isofluorane (0.5-1%). during surgery, 8 children needed mechanical ventilation and the others maintained spontaneous breathing. ekg, heart rate, blood pressure, and pulse oximetry were moniterized. after anaesthesia, a sigmoid tenometry catheter (tonometrics, inc.) was inserted in the stomach of the patients by direct visualization with laryngoscope and magyll clamps. children were all maintained normoventilated and with normal cardiorespiratery variables. cadet's balloon was £~led with 2.5 ml of saline. thirty minutes after the insertion 1 rrd was extracted and rejected, just afterwards the remanent 1.5 ml was extracted and immediately analyzed. simultaneously an arterial gasometry by puncture was performed. gastric phi was calculated by the henderson-hasselbalch's equation using the pco 2 obtained from the tenometry catheter and the bicarbonate value obtained from the arterial gasometry. results: average gastric phi was 7.34 -i-0.027, range (7.29-7.46). objective: demons~ating intramucesai ph (phi) alterations during transport of patients from operative room to pediatric intensive care unit (picu), material and methods: phi measurements were performed with gastric tonometer catheter in t4 patients undergoing cardiac surgery with cardiopulmona d" bypass (cpb), there was 9 mate and 5 female, the average age = 3yl0ra, average weight = 12,5 kg, average time of cpb = 70 rain. the measurements were made at the end of the surged' and when the patients had arrived in the picu statistical aualysis: average and ~andart deviation and test "t" student. objetive: to asses the efficacy of gastric iatramucosad ptt (phi) and arterial lactate levels to evaluate splacalc tissular perfusion in an experimental model of intestinal ischemia. suneets ~nd methods: twelve piglets weights t3-20 kgs. undergoing orthot~ie liver trasplantation. the intestinal ischemia was induced by aortic damping. tonometry catheter (tonometrics inc.) w~s placed in the stomach after artaesthesia and ot intubation. phi ~s determined 7 times and lactate levels was determined fi times in 3 stages: i) pre-ae~hepatic stage (twice: before surgery and before aortic clamping ); ii) end anhepatic stage (only phi): iii) reperfusion stage (a 30, 90, 120 and 180 minutes). the phi was derived from application of the henderson-hassdbach formula using the pco2 value from the tonometer and the arterial bic~rbonate. all pipets received raaitidiila before sttrgery. values of phi above 7,35 and lactate levels between 6 and 15 mg/dl were considered nortrm. the results were statistically anaj.izated with anova and bonferroni tests. results: the phi was normal on pre anhepatic stage (> 7,35) and lactate levels were slightly increased (21, 5 +_ 8, 9 and 19, 5 ±5, 9mg/dl ns) . in relalion to we-anhepatics values, phi decreased signncatly at the mid of anhevatic stage (7,39_+0,14 vs 6,94_+0,1 p<0,001), phi remain low in stage iii, at 30 rain (6,86+0,12 p< 0,001) and 90 min(g94-+o, 12 p< 0,001). arterial lactate levels increased significatly in relation to levels in stage i, at 30 rain (63,6_+9,7 p<0,o01) arid 90 rain (65,8±9,9 p<0,001) of reperfusion stage. there is a slight improvement on phi and lactate ievels at 120 and t80 rain althought the differences did not reach significance. cnmments: phi and arterial lactate levels propperly reflect hypoperfusion on the experimental model of acute intestinal isdlemia. b~kground : the paediatrie gallbladder diseases generally described are calculous ¢hol~tstitis, cystic duct obstruction, congenital anomaly of the biliary tract, and inflammation. in the neonatal period, noulithogenie gallbladder disease could be also due to erythroblastosis or hyperalimentation. obieetive : we describe an other type of disease affecting the gallbladder in neonates thought to be related to their vascular vulnerability. methods : four patients with abnormal gallbladder ultrasound not related to classical observations were included. we have studied and reviewed the biological and clinical data, the ultrasound findings and their evolutions. results : four patients, 30 to 32 ~.k-old neonates ~ffth a birthweight be~,een 1,3 and 1,9 kg, were intubated and under total parenteral nutrition for 10 to 35 days. none of them were symptomatic on repeated clinical evaluations. one newborn developped hypotensien on umbilical bleeding at 3 hours of life. in two cases, signs of cholestasis were discovered : the total bilirubin level has risen to 5 mg/dl; the direct bilirubin level was 1,5 mg/dl while the urina were dark and the ~o~,ls :mcolour~. the c~mplct~ ~crology as a!! the culvare~ remained negative. the ultrasound explorations were atypical : in the four eases, an initial increasing broad and thickness of the wall of the gallbladder with an hyperecbogenie inside content, which was not sludge, was discovered. in three eases the images resolved in ten to fifteen days. in one ease, an asymptomatie thrombosis of the vena portu which remained patent was discovered. in this case, at one month, the ultrasound showed images encountered in chronic ebolecystitis and, at one year, the gallbladder appeared atrophic. none of them underwent surgery. conelusiou : the gallbladder diseases are multifactorial. besides the prematurity, the infections, the total parenteral nutrition, the premature neonate is exposed to vascular vulnerability affecting also the gallbladder and this may explain our findings. progress in prognosis of pts with b-nhl had followed the use of multimodality chemotherapy (ct). with the prolonged survival, there are comlications due to myetosupression & desease process. the syndrome of neutropenic enterocolitis (ne) is one of the ominous problems because ofpts increased susceptibility to infection & overwhelming sepsis. this material included 25 neutropenic pts (4-14 years) with the stages iil& iv of b-nhl who were treated with the modifired bfm-90 (mtx 1 g/m 2 in 24-h inf.); 22 males, 3 females. seventeen episodes of ne were observed & only after the first 2 courses of ct (13 of 25 after tst, 53%; 4 of 24 after 2nd, 17%). the symptoms existed 3 to 14 days. wbc ranged from 50 to 600 in l~tl (median, 100). the first signs of ne were directly correlated to the beginning of the neutropenia & the recovery of neutrophils led to the disappearance of abdominal recovery of neutrophils led to the disappearance of abdominal pain. the conservative treatment included gastrointestinal tract decompression, broad spectrum antibiotics initially, volume & electrolyte substitution, nutritional support, correction of acid-base balance, symptomatic treatment. sixteen pts were treated nonoperatively, 1 died. on autopsy the transmural bowel necrosis due to thrombosis of branches of a.mes.sup, was found. the bowel perforation occurred in one patient, he was undergone laparotomy & hemicolonectomy & survived. we conclude that ne is a frequent complication in neutropenic pts with the st. lii& iv of b-nhl. it occurs after the induction courses of ct. close observation by surgeons, oncologists & pediatric intensivists is mandatory. conservative treatment is effective & more preferable until leucopenia resolves. operation is necessary only for those.with perforation. near infrared spectroscopy as a tool for evaluation of intestinal perfusionpresentation of an animal model. c. scheibenpflug, p. buxbaum and a.m. rokitansky the recent development of and investigations in the so called near infrared spectroscopy ( nirs --transcutanous emission and simultaneous registration of intensity of spectralcolours depending upon modulations of tissue perfusion ) enable physicians to measure and qualify organ perfusion and nowadays is mainly used to control cerebral as well as skeleton muscular blood flow in trauma patients at intensive care units ( icu ). today intestinal perfusion, hypoperfusion , cell damage caused by reperfusion injury, bacterial and toxin translocation are serious problems in critically ill patients at an icu. paediatric intensive care physicians put major concern on intestinal perfusion, which for. instance gains more and more importance, especially in the neonatal period for example as an etiologic factor for necrotizing enterocolitis. we established an animal model, in which we measured intestinal perfusion by nirs under various invasive and noninvasive conditions. methods and results will be referred. for preliminary conclusion we propose near infrared spectroscopy ( nirs ) also as a potent diagnostic tool to determine early intestinal malperfusion in order to prevent lethal outcome. fm'ther investigations in animals as well in paediatric iritensive care patients should be done to estimate our efforts. introduction: following the acute phase of necrotising enterocolitis (nec) starvation of the gut for a period up to 3 weeks is a generally accepted treatment modality in many centres. objective criteria to refeed these patients are hardly available. recently the double sugar test has become available as a parameter for (ab)normal gut permeability ~'2. aim of the study: to evaluate the changes in permeability of the small bowel in patients with nec and controls before introduction of enteral feeding. methods: a lactulose! rbarrmose (i/r) test was performed in two groups. group 1 was studied 2-3 times within a 5-week period of starvation (n=5, mean gest. age 35, range 31-40 weeks). in group 2 seven different control patients were studied (mean gest.age 33, range 28-38 weeks). the test was performed by giving a patient after at least a 4 hour fast 1 ml/kg bodyweight l/r solution and determination of the 1/r ratio in a 4-hour urine sample by chromatography. results: objective: to evaluate the prognostic factors in the response to nitric oxide (no) in children with acute respirator/ distress syndrome (ards) and/or pulmonary hypertension (pht). patients and methods: 23 critically ill children received no inhaled for ands and/or pht treatment. 14 patient before and after cardiac surgery (2 cardiac transplants), 5 with bronchopneu~onia, 2 multiple trauma, 1 sepsis and 1 cardiorespiratory arrest. 15 patients showed /j~ds and 8 pht, in 4 with associated ards. we analyzed age, sex, diagnosis, pao2, pa02/fi02, oxygenation index, pht, shock, and sepsis as prognostic factors and response factors to n0. results : after no administration oxygenation did not improve in 2 patients (8.6 %) and pht did not diminishe in one children (12 %). 12 patients survived (52 %), 8/15 (53.3 % with /d%ds) and 4 /8 (50 %) with pht. the four patients with isolated pht survived , and the 4 patients with pht and ards dead. patients after cardiac surgery presented less mortality (35.7 %) than the rest of patients (66.6 %). patients with shock presented higher mortality (64.2 %) than the rest of patients (22.2 %). there are no differences in response to no in respect of sex, age, diagnosis, shock, and sepsis. survivors showed higher increase of pao2/fi02 64.3 ± 58.4 to no than non-survivors 48.4 ± 51.1 (n.s). patients with pht showed higher increase in pa02/fi02 to no administration ( 88 ± 47.1) than patients with ards (43.4 ± 50.8), (n.s), but patients with ards showed a higher increase in 0!, 15 ± 6.7, than patients with pht 4.8 ± 4 (p < 0.05). patients with pa02/fi02 < i00 showed less increase in pa02/fi02, 47.8 ± 46.3, than the rest of patients 82.8 ± 65.5 (n.s) conclusions: i. mortality of isolated pht treated with no is less than patients with ap~s. patients with shock and those with pht and ards showed higher mortality. 2. we have not found any clinical or analytical factor to predict clinical response to no administration. 14 patients showed ards, and 9 severe pht after cardiovascular surgery, in 5 with associated ards. we registered respiratory assistance, blood gases, pao2/fi02, the oxygenation index (oil, and mean pulmonary pressure/ mean systemic pressure (pap/sap) before and after no inhalation. we measured continuous concentration of no and no2 by electrochemical method (noxbox, bedfont, airliquide). results: no administration improved oxygenation mean pao2 from 74 ± 17 tm~g to i19 ± 54 ~g (p < 0.01), mean pa02/fi02 fr<xa 83 ~ 30 to 13£± 72 (p < 0.01)and 0i from 28 ± 14 to 20 ± 12 (p < 0.01). 2 patients did not improved with no administration. the oxygenation improved in the first five minutes and the best oxygenation was achieved with 5 -15 ppm. there is no significant change in pac02. pap/sap diminished from 62 ± 17 % to 42 ± 9 % ( p< 0.05), in one patient there is no response. the no administration was maintained between ~5 minutes to 47 days. the effect of no on pulmonary pressure and oxygenation was maintained without change during all the time it was administrated. n02 concentration were always less than 2 ppm y metahemoglobinaemia less than 3.5 %. there are no side effects secondary to no administration. 12 patients survived (52 %). concluaions:l.administration of no improves oxygenation in children with ~/eds and diminishes pht after cardiac surgery. 2.no effect is fast and maintained during the evolution. high frequency oscillatory ventilation in combination with inhaled nitric oxide g~thberg sylvia, md, edberg k e md, phi), dept of paediatric intensive care, children's hospital, s-416 85 g6teborg, sweden background: for man), years severely sick infants with congenital heart malformations or acute lung disorders have died due to pulmonary hypertension, hypoxia and multiple organ failure. today there are several therapeutic facilities coming up for these infants. ecmo progranunes have been introduced in severul centres as well as improved venlllatory techniques, as high frequency oscillatory ventilatior~ (hfov), which provides adequate ventilation with less risk for hmg injury.. in 1987, the endothelium derived relaxing factor was identified as nitric oxide (no), and extensive studies have sho~-a that inhaled no reduces pnimonm3 o vascular resistance and improves oxygenation in hypoxic infants with pulmona~ hypertension. material.. from july 1994 to january 1996, we have treated 13 severely hypoxic children with combined ttfov-no. seven were newborn, 3 with cdh, one with mas, one with irds, one with paediatric ards due to rsv infection and one with poor oxygenation atler open heart surgery. 6 were between 1 month and 9 years and all had ards of differentorigin but one who was hypoxic after open heart surgery. method: hfov was given by means of sensormedics 3100a oscillatory ventilator to 12 patients, and dr~ger babylog 8000 was used in one case. mean airway pwssures varied from 10-34 cm h20. oscillatory pressures varied from 25-85 cm h20 and ventilatory rates from 6-15 hz. fi o2 varied from 0,21 -1,0. no was administered by nomius classic. no and no2 was measured in the inspiratory limb with an electrcchemieal device. noconcentration varied from 2 to 20 ppm and the no2 -concentration between 0-0,2 ppm. methemoglobin was never more than 3,7 % (average 1,7). duration of treatment varied from 1 to 20 days. combined trealment with hfov and inhaled no improved oxygenation and carbon dioxide elimination in 12 out of 13 treated childretl 5 patients died, 3 due to their underlying congenital heart disease, one of asphyxia due to rsv-infection and one of cdh with progressive hypoxia and multiple organ failure. combined treatment with hfov and inhaled no improves oxygenation in severely hypoxic children. treatment should be slarted early to reduce the risk for chronic lung injury following barotranma and high oxygen concentrations. we speculate that the combined hfov-no may reduce the use of ecmo, and that it may improve outcome in ceotres where ecmo programmes are not introduced. apart from its vasodilative properties nitric oxide appears to act also in physiologic immune defense. intracetlular concentrations of no synthesized by macrophages are in the range of 103 above those produced by vascular endothelium. we investigated the bacteriostatic effect of nitric oxide at concentrations used during inhaled therapy for pulmonary vasodilation in neonates. ten different strains of five species were used (staph. anrens, staph. epidermidis, strop. group b [gbs], e. coil and pseudomenas aeruginosa), which are ~e most often tracheally isolated bacteria in mechanically ventilated premature infants and neonates. we compared bacterial growth of cultures applying three different concentrations of nitric oxide (40 ppm. 80 ppm, 120 ppm) m the growth of the same strains in room air for a duration of 24 hours. no bacteriostatic effect was demonstrable at no concentratioes of 40 ppm. e. coil showed decreased bacterial growth at 80 ppm and 120 ppm, however without reaching statistical significance (p=0.058). at nitric oxide concentrations of i20 ppm staph. epidermidis and gbs grew significantly less as compared to colonies of the same strains in room air. no effects were found regarding the growth of staph. aureus and pseudomonas aneruginosa. we conclude that nitric oxide has a selective bacteriostatic effect on some of the most often trachealb, isolated bacteria in premature infants and neonates. this effect appears to be dose-dependant and occurs in the upper range of dosages used with inhaled no therapy. further research is required in order to examine the mechanisms of action as well as specific interactions between different strains of bacteria and nitric oxide. the no,box monitor (bedfont, kent, uk) is used to monitor nitric oxide (no) and nitdc dioxide (no 2) during no administration in ventilated neonates. according to the manufacturers specifications the monitor can be applied in cases where airway pressures range from 5 to 50 mbar. we investigated in-vitro the accuracy of the no monitor in a range of ventilatory conditions. using a dr~ger babylog 8000 we ventilated an artificial lung. no was administered {800 ppm no in 100% nitrogen) with a mass flow controller to the inspiratory tube, at a distance of 20 cm from the y-piece. the no target value was set to 10 ppm. the ventilator was operated in both cpap and ippv modes at different settings. the no sampling tube was placed on two different locations; in the inspiratory tube dose to the y-piece; in the expiratory tube haft way between the y-piece and the ventilator. the no-monitor was ca)ibrated with 84 ppm no at 30 mbar. fig. 1 and 2 show the sensitivity of the no-monitor for respectively static pressure (cpap) and pulsatile pressure (ippv) with the sampling tube located in the inspiratory tube. fig. 3 shows the resur for pulsatile pressure with sampling in the expiratory tube. we conclude that the accuracy of the bedfont no-monitor is dependent upon airway pressure and sampling site, the latter possibly caused by incomplete mixing, pressure and no are linearly related when sampling occured further away from the administration site. based on this study we suggest to place the sampling tube in the expiratory tube. during neonatal care the clinician should be aware of varying inaccuracies depending on the respiratory pressures. hypothesis: availability of therapy with inhaled nitric oxide (ino) decreases ecmo use in patients referred .to a tertiary care hospital for treatment of respiratory, failure unresponsive to conventional therapies. background: at children's hospital of wisconsin (chw) treatment for patients who have failed conventional treatment for respiratory failure, sometimes called "rescue" therapy, has included ecmo since 1986 and high frequency oscillatory ventilation since 1992. ino has been in experimental use at chw since may of i994 and has resulted in the perception of a decreased need for ecmo therapy in those patients referred for rescue therapy. this study was designed to tbst the validity of that perception. study type: retrospective cohort. methods: the medical records of all 105 patients referred to chw from 1/lj93 to 4/1/95 for rescue therapy for severe respiratory failure were included in the chart review. data were collected regarding diagnoses, illness severity, hospital course including interventions and complications, and outcomes. exclusion criteria w~ere the finding of structural heart disease or congenital diaphragmatic hernia as the basis for hypoxemia. qualification for ino treatment included an a-ado2 -600 or oi ~-40 for two hours or -> 25 for twelve hours and echocardiographic demonstration of persistent pulmonary hypertension of the newborn. patients were classified into two groups based on the availability of ino at the time of their hospitalization. results: in the time period of the study, 105 patients were referred for possible ecmo therapy. twelve patients greater than 4 weeks old, 31 with congenital diaphragmatic hernia and 12 with congenital heart disease were excluded from this analysis, leaving 50 patients for study, ino availability reduced ecmo use from 16 of 34 (47%) patients in the ~ino unavailable" group to 2 out of 16 (12.5%) patients in the "ino available" group, p=&026 by fisher's exact test. the fact that the two groups were composed of patients of similar severity of illness is reflected by comparable rates of ecmo and ino rescue therapy (47% vs. 56%). conclusion: by providing an alternative rescue therapy, ino has reduced the need for ecmo in this group of neonates referred for respiratory failure. introduction: true hepatnrenal syndrome (his) is defined an acute renal failure {arf) in the presence of severe liver disease without other known causes of renal failure. hrs is frequently seen in the course of hepatic cirrhosis• in children, cirrhosis is rare; however, arf can be seen in combination with aseites and liver dysfunction• we describe 3 patients with hepatic dysfunction and aseites in combination with ar~ and abnormal sodium-water handling, leading to the diagnosis of hrs. pathophysiology: three factors are considered in the pathogenesis of hr~: i) hepatic dysfunction, 2) deranged hemodynamics, including abnormal blood pressure, reduced effective arterial blood volume and abnormal blood flew distribution, and 3) neuro-humoral dysrsgulatiom, including elevated levels of aldosteron, renin, angiotensin-ll, ade, vasodilatim 9 nitric oxide and vasoconstrictor peptide endothelin-l. the main pathogenetic feature is decreased cortical renal blood flow, decrease of glomerulur filtration rate (gfr), vastly increased sodium retention, uliguria, and azotemia. treatment: therapy is based on counteracting sodium and fluid retention by highdose aldosteron antagonists and loop diuretics, improving renal perfusion by lowdose dopamin, and strict restriction of fluid and sodium. interventions as paracenteals of aacites or n peritoneo-systemic shunt are associated with high morbidity and poor outcome in children. reversal of hem by conservative measures can only be attained at early stages of hrl liver transplantation is the only definitive treatment that can reverse ere at advanced stages. patients: the described patients developed severe ascites with insidious renal dysfunction and abnormal sodium-water handling during admission at picu and fullfilled clinical criteria fur hrs. treated according to the cited principles, all patients showed improvement of gfr, with increased natriuresis and gradual decrease of ascites. eventually, renal function normalised completly. conclusion: ere deserves greater recogmitimn in the picu population; diagnosis can be suspected on clinical criteria. with this increased awareness, therapy tun be instituted at an early phase, with better prospects for recovery. positive outcome of hem depends on early recognition of the clinical picture, understanding of the pathophysiology, and early institution of consistent treatment. mtx is an antimetatxflite widely used as chemotherapeutic agents. high dose ivitx (i to 30~m2) administered as a prolonged intravenous infusion (over 4-42 hours), is often used to treat malignant paediatric diseases. major complications of this treatment are myelosuppression, orointestinal mucositis, dermatitis and impairment of anal function. we report two cases of mtx overd~age occurred in two children (5-year-old. 14 month-old) t~ted for acute lymphoblastic leukaemia. they were treated by cavh and the mtx bhk~d levels rapidly decreasedavoiding multisystemic involvement. establishment of alkaline diuresis and monitoring of plasma mtx levels during treatment is essential to prevent nephrotoxicity. however. leuco',cnn rescue may not prevent the development of potentially lethal toxicities in patients with mtx concentrations persistantl} exceeding t0mm. in theses cases, em'ly treatment of mtx intoxication may pm~cnt myelosuppression and reducerenal damage. the goal is to lower the concentration to below 10 mmoll, at which time rescue agents aleme would be expected to be cllcctive. respective indications of these remo',at mctny.:is are still discussed : hacmt~ialysis t~ eharc(~l haemoperfusion should be prolx',sed for massive and acute intoxication. however, rebound has been reported after combined hcmodialysis and hemoperfusion. exchange transfusion may be proposed as a treatment for prolonged and moderate intoxication. peritoneal dialysis is an incflbedve method for remo~ al of mtx. cavh was used in our icu. cavh is a simple method for blood purification and n':dy iluid control. use of cavh was never be reported in this indication to our knowledge. simplicity, rap~d application and gco.l clinical tolerance are the main advantages of this technique. the technique presents ~peclal advantages in terms of low priming volume of extracorporeal circuit, low blood flow, low rate heparinisation. our results show a decreaseof plasma mtx concentration and a rapid reduction of halfqite of elimination (t5 hours over the period of cavh). moreover, we didn't delec~d rebound after stopping prc,xedure. small size of the i:ratients may present sometime special problems, but these technical problems can be overcome, no severe complication (needing, inlection) were observed during filtration, in summary, aggressive intravenous fluid hydration and alkaliniaation of the urine coupled with careful monitoring of renal function and plasma mtx concentrations during and al'tcr infusion along with lem~overin rescue has reduced the inndcace of life-threatening toxicity after highdose mtx. however, some mtx inu>xication still occurred, leading to se~em toxicity, particularly nephrotoxicity. in these cases, we think that cavh (or cavhd) is a reliable, rapid method without rcix~und increase in plasma mtx concentration or important adverses effects compared to other procedure removal. gouyon jb, germain jf, semama d, pr6vot a, desgres j preliminary limited data suggested that hemofiltration and hemodiafiltration may be valuable in some neonates with decompensation of maple syrup urine disease (msud). venovenous hemofiltration (vvhf) and hemodiafiltration (vvhdf) were performed with a new neonatal hemo(dia)filter (miniflow 10, hospal) on 8 anesthetized rabbits infused with branched-chain amino acids (leucine, isoleucine and valine) and c~-keto-isocaproate. the bcaa and aketo-isocaproate blood levels were close to those previously observed in neonates with msud when extracorporeal blood purification was required. vvhf and vvhdf performances were assessed with two different blood flows (qb = 8.3 and 16.6 ml/min). vvhdf was performed with 4 dialysate flow rates (qd = 0,5, 1.0, 2.0 and 3.0 l/h). thus, each animal was submitted to 10 successive procedures. within each studied period, clearances of the 3 bcaa were strictly similar. bcaa clearances obtained by vvhf were similar to ultrafiltrate rates (respectively, 0.78 4-0.14 and 1.79 4-0.28 ml/min at high and low qb ; p < 0.05). the ~x-keto-isocaproate clearances obtained by vvhf were 0.39 4-0.17 and 0.92 4-0.43 ml/min at low and high qb (not significantly different). whatever qd value, the vvhdf procedures always allowed higher bcaa and c~-keto-isocaproate clearances as compared with the corresponding v'~hf period with similar qb. bcaa clearances obtained by vvhdf with a 0.5 l/h dialysate flow, were 4.1 4-0.5 mljmin and 5.4 4-0.5 ml/min at iow and high qb, respectively. the concurrent a-keto-isocaproate clearances were 2.5 4-,. 0,8 ml/min and 2.9 _+ 1,0 ml/min. at both qb regimens, bcaa clearances provided by vvhdf were markedly higher than values previously obtained with peritoneal dialysis in human neonates with msud. the management of renal failure in the newborn is difficult. when dialysis is instituted peritoneal dialysis (pd) is usually the technique of choice. this is can be problematic and impossible in some patients with pre-existing intra-abdominal pathology. continuous arterio-venous haemofiltration (cavh) has been described in infants but sick preterm infants are not able to support the circuit. i have devised a means of having pumped haemofiltration in small/preterm infants (phis/pi) and describe its use in nine patients ranging in size from 750 to 3000gms for periods of 1 to 7 days. vascular access was achieved through 24 or 22 guage cannulae in either a peripheral artery and a central vein or through two central veins. blood was pumped out using an ivac 572 infusion pump and through a gambro fh22 haemofilter. a second ivac pump was used to remove haemofiltrate from the filter and a third to infuse replacement solution. removal rate was set to give a clearance of 15mls/min/1.73sq.m and blood flow rate set to between 5 and 10 times the removal rate. heparin was infused into the circuit to prevent clotting of the filter. biochemical and fluid balance control was achieved in all infants. guaranteed fluid removal allowed the administration of full nutritional support. four patients died when treatment was withdrawn because of an untreatable underlying problem. one recovered renal function but died some weeks later from unrelated problems, three survived and recovered renal function and one patient is still on treatment. this system allows a secure means of achieving fluid and electrolyte control in the preterm infant. the use of this technique may allow haemofiltration to become as applicable to preterm infants as it is to older children and adults. unibrtunately, children often receive no treatment, or inadequate treatment for pain and painful procedures. this prospective, multicentric study focuses on the efficacy, safety and side effects of novalgin (metamizol sodium) for this indication. patients and method: novalgin was administered to 56 children, aged between 6-16 years, with acute, postoperative or procedural pain. novalgin (10-15 mg/kg) was given 6-8 hourly iv or im respectively, in some cases (15) in combination with opioids (tramadol 10, piritramid 3, butorphanol 2). the pain relief was assessed by six-step verbal rating scale (vrs) from 0 to 5, vital signs were monitored, the side effects, that occured were recorded. results: pain relief was good (vrs less 2) in 53 children -94.6 % of study patients. novalgin was very well tolerated, only one patient had adverse reaction -hyperpyrexia following intravenous application of the drug. discussion: novalgin (metamizol sodium) is safe and effective drug in the management of acute pain in children with low incidence of side effects. obie~qve: a prostx~tive study comparing simultaneous, indepeadent ratings conducted by intensi~4sts using an american (comfort) and an european chartwig) sedation scale for mechanically ventilated pediatric patients. measurements and results: the study comprised 30 observations in 18 mechanically ventilated pediatric patients (aged 16 days to 5 years) in a pediatric intensive care unit (from march 1995 to january 1996 . each patient was sedated by his/her managing physician with opiates, benzodiazepines, barbiturates, used isolated or in combination. each observation consisted of a 3-mid period of oly~ervatien of the patient in his or her pediatric icu bed, after each observation, the comfort (analyses 8 dimensional physiologic and behavioral subscores -range 8 to 40 paints) and hartwig (analyses 4 dimensional behavioral subsenres -range 8 to 25 points) were performed by the intensivist. we established the comfort scores ~ correspanding to adequate (range 17 to 26), excessive (range 8 to 16), and inadequate (range 27 to 40) sedation; and, hartwlg scores z correslxmding to adequate (range 15 to t8), excessive (range 8 to 141, and inadequate (range 19 to 25). statistical mmlysisj: agreement rate (kappa) and p <.01 was considere d s!l~nificant. comfort 18 (60.9%) 2 (6,6%/ 10 (33.4%) hap, twig , 17 (56.6%) 7 (23.4%) 6 (20.0%) to the comfort score, the average for adequately sedated, inadequately sedated, and too sedated was 20.28+-2.78, 2z5_+0.70, and 15a.+_l10, respectively. and to the ha~twig scorn, the average for adequately sedated, inadequately sedated, and too sedated was 16.35:k-'0.77, 20.85-&l57, and 13.0l-0.89, respectively. conclnsion: in our study there were no significantly statistical difference when you apply a more complex scale (conff'ort) or a less complex scale (hartwig) to assess the sedation of mechanically vemilated pediatric patients. the application of local and intravenous morphine infusion after surgery of urinary tract eva nemeth , m.d. semmelweis medical university , first oepartment of paediatrics , budapest , hungary in±roduction:continuous analgesia with morphine may be ~egaroed as a safe and effective method of pain relief during postoperative period. subjects and methods: 24 children /mean age 2.3 years/ underwent elective ureteroneoimplanta±ion were randomly selected to receive either morphin intravenously of lo ug/kg/h /group one/ or bladder morphineinfusion 50 ug/kg/h /group two/ after surgery. all patients were prospectively evaluated during their s±ay in the postanaesthetic care unit. cardiac and respirafory rates,blood pressure,sa 02 ~,degree of alertness,pain perception and complaints of the paticnto ~cr~ recorded hourly. pruritus,nausea and vomiting,voiding difficul-±ies,sedation,dysphoria were systematically sough and quoted. statistical analysis was performed by chi square test. results:postoperative analgesia was the same in the two groups,but side effects were less in the bladder morphine group,because of the lower se morphine concentration.the differentes weren't significant in two groups. conclusions:the administration of bladder morphine infusion is a safe and effective method in children. objetive: compare the evaluations of sedation level made by physicians and nurses with the visual analog scale (vas) and the comfort scale (cs) in pediatrics patients receiving difforents modes of intravenous sedation. material ~ method." file evaluations were made by an attending physician and nurse with the vas and by another physician (always the same) using the cs. the observations were divided following the sedation mode: one drug (fentanyl or midazolan), two continuous drugs, one continuous and one intermi~ent drug and two intermittent drug (fentanyl and midazolan). the groups were compared using the t-student test. the groups also were compared between the percentual of agreement of the evaluations of sedation level made by physicians and nurses with the cs and vas using the x 2 . results: we didnk find any statistical difference between the observations made by physicians and nurses with the vas in the differmts modes of intravenous sedation, the average of the observations using the cs betwom one drug and two drugs modes didnk exhibit also statistical difference. the observations made by physicians mad nurses using the the vas when compared with the cs didn't show statistical difference between the sedation level. we found statistical difference only in percentual of concordance of sedation level between physicians and nurses when compared the one and two drugs modes of sedation. conclusion: we didn't find differences in the observations made by physicians and nurses in the sedation level, only in concordance pereentua/ of observations when compared two modes of sedation. the observations using the cs (more complex) didnk show differences when compared with the vas. effects of age, concurrent administration of other pharmacologic agents, and disease [cardiac(n=31) & pulmonary(n=22)] on the pk & pd of b were evaluated in volume overloaded infants aged 4 days-6 mo (n=53). single doses of 0.005,0.01,0.015,0.02,0.025, 0,03, 0.035,0.05 & 0.10 mg/kg iv were given over 1-2 min after baseline evaluation. age was used as a continuous vadable to determine its effects on the variability in the pk & pd of b. values for pk parameters were compared between patients in cardiac and pulmonary disease groups. hierarchical multiple regression analyses were used to determine the effects of age, disease and other pharmacologic agents on the variability of bumetanide excretion rate (ber) and pd responses, e.g. urine flow rate (ufr) & electrolyte excretion. cit, cir & cinr increased with age (p<0.05) while t,2decreased markedly in the first monthe of life (p<0.05). ber normalized for dose increased with increasing age. patients with pulmonary disease exhibited significantly greater clearance and shorter t~= (p<0.05) than those with cardiac disease whereas vd~ was similar in both groups. the administered dose of b was the primary determinant of ber but increasing age also contributed. penicillin antibiotics decreased ber. dose response curves for ufr and electrolyte excretion were similar between disease groups. more of the variability in ber and pd responses could be accounted for in the pulmonary group than the cardiac group but this was not statistically significant. conclusion: the pk of bumetanide were influenced significantly by age and disease. differences in pk between patients with pulmonary and cardiac disease were primarily due to differences in total clearance. age and the administered dose of b were positive determinants of ber and pd responses while penicillin antibiotics had a negative impact on both, once b reached its site of action, no differences in pd responses were detected between disease groups. the pharmacodynamic effects of bumetanide were evaluated in volume overloaded infants (n=56) aged 4 days-6 months. single doses of 0.005, 0.01,0.015, 0.02, 0,025, 0.03, 0.035, 0.05 & 0.10 mg/kg iv were given over 1-2 rain. bumetanide concentration in blood (n=l 0) & urine (n=6) samples were quantified by hplc. baseline urine samples were collected over 2-4 hours prior to drug administration. determinations of urine volume, electrolytes (na ", k +, ci, ca ++ and mg++), creatinine and osmolality were performed before and at 0-1, 1-2, 2-3, 3-4, 4-6 and 6-12 hours after bumetanide dosing. changes in urine flow rate and electrolyte excretion were plotted as a function of bumetanide excretion rate which was considered the effective dose of the drug. peak bumetanide excretion rate increased linearly with increasing doses of drug and showed no evidence of approaching a maximum. time course patterns for urine flow rate and electrolyte excretion were similar for all dosage groups. urine flow rate and electrolyte excretion increased lineady up to a bumetanide excretion rate of approximately 7 #g/kg/hr and either plateaued (urine flow rate) or declined at bumetanide excretion rates > 10 #g/kg/hr. bumetanide had no detectable effect on serum electrolyte concentrations, conclusion: maximal diuretic responses occurred at a bumetanide excretion rate of about 7 ;~g/kg/hr. higher bumetanide excretion rates produced no increased diuretic effect. peak bumetanide excretion rate of about 7 #g/kg/hr corresponded to bumetanide doses of 0.035-0.050 mg/kg. neonates using an electrical syringe-pump. authors: tr~luyer j.m., sertin a., bastard v., settegrana, c., bourget p., hubert p. background and objective: many problems can be observed with drug administration by i.v. route, especially in neonates. so we evaluate different protocols of teico delivery using an electrical syringe-pump. methods: we simulate infusion of teico with a syrlnge-pump (pilot c, becton & dickinson lab.) trough d standart neonatal i.v. system. for 2 weights (1 or 3 kg) we used 2 doses of teico (8 mg and 16 mg/kg) and a dose volume _<4.2 ml. our goal was to perform a complete infusion in 10 minutes. the infusion system consisted of an life care 4 infusion pump (abbott lab.) with its lv. set for maintenance intravenous fluid (flow _< 6 ml/h) connected to a 3-way stopcock. an 1 meter extension tubing was placed between the stopcock and a neonatal catheter. an another 1 meter tubing (injection tubing) connected the teicoplanine syringe to the stopc, ock. the volume of the injection circuit (from the syringe to the distal part of the catheter was 2.6 ml 4 methods of injections were assessed: a: injection of the predetermined volume of teico in 10 minutes with no wash out. b: idem as a but the teico was injected in 5 minutes, followed by a wash out (5 ml / 5 minutes). c: twice the required volume was introduced in the syringe and the volume to infuse was programed in 5 minutes, followed by a wash out (5 ml/5 minutes). d: ]dem as c but a priming was performed before connecting theteico syringe to the tubing. during each run, serial samples were collected every ten minutes over a one hour period. the samples were assessed using hplc method. results: the amount of drug delivred at 10 minutes were calculated. the results are a mean of 2 to 6 runs and expressed as the percentage of the total amount of teico prescribed. a 2,8 % 6,4 % b 47 % 62,3 % c 82a % 86,8 % d 94,2% 95 % conclusiom for accurate and reliable intermittent drug infusion with a syringe pump it is mandatory to use a precise protocol of administration and to take in account 1) a priming (for immediate starting of infusion), 2) a drug volume greater than the dose prescribed and a programmed volume injected, 3) a wash out of the tubing (with a volume ~ 1,5 x volume of tubing injection) caz is an antibiotic with activity against the major pathogens responsible for neonatal bacterial infections. we previously reported the pharmacokinetics of caz in 136 preterm infants on day 3 of life which showed that the clearance of caz increased with increasing gestationat age (ga). mean serum half-life of infants with gas < 32 wks was 8.7 h. we wanted to investigate the effect of postnatal age on caz pharmacokinetics, we therefore studied caz pharmacokinetics on day 19-21 of life in 10 preterm infants with gas < 32 wks. caz (25 mg/kg) was administered as an intravenous bolus injection. blood samples were coilected before (t =0), and 0.5,1,2,4,8 and 12 h after the caz dose and analyzed by hplcassay, the pharmacokinetics of caz followed a one-compartment open model. during 1995 11 newborns with complex congenital heart defects requiering either htx or palliative staged single ventricle repair were admitted to our hospital: hlh n=8, unbalanced cavsd, tga with hypopl. rv and hypoplastic aoa. tga with hypopl. rv, sas and dextrocardia. 8/i 1 children had been admitted with cardiogenic shuck and mukiorgan failure due to intermittend closure ofductus arteriosus; in 3/8 stabilization failed. parents were informed about the known and unknown risks of the always palliative surgery; in 2 cases parents denied further therapy. one pafiem with hlh underwent orthotopic htx at the age of 5 month after the ducms art. had been stunted in the newborn period. 9 month later he is still in favourable condition and without any sign of acute organ rejection. 5/11 underwent first stage of palliative single ventricle repair: norwood -op. ( 3 ) ( n=3 ), damus-kaye-stansel -procedure ( 2 ). the clue to adequate postoperative management was to archieve a balanced distribution of flow to systemic and pulm circulation, that is to protect the single ventricle from volume overload and to guarantee sufficient oxygenation and pulmonary development as well. with the centralvenous sato2 at about 50% provided maintaining the arterial sato2 at about 75_+5% is corresponding with a qp/qs of 1:1. using modified bt-shunts of3.5mm resp. a central anrtopulm, shunt of 4mm in one case l severe puim. hypertension, surgery at 6 weeks of age ) there was no excessive pulm. blood flow and no need to increase pvr with inspired co2. one child ( norwood at 5 weeks, preexisting pnim_ edema ) developed severe pulur hypertension and parenchymal pulm. dysfunction after prolonged bypass and multiple transfusions due to intraoperative bleeding: hypoxemia could be managed successfully by implanting a second shunt of4mm 18hh later and temporarily using prostacyclin and no; at sternum closure 6 dd later the second shtmt was banded to 3ram. follow-up ranges 5-5 month: all 5 children are at home being assigned for second stage operation at about 6 month of age. establishing clinical practice guidelines has become increasingly important in the current health care environment. significant effort has been focused upon development of post-operetive critical care pathways. however, benchmark data upon which such pathways should be based has not been well reported. length of mechanical ventilation (lmv) and length of stay (los) for children following cardiac surgery, for example, is poorly described. we prospectively recorded the lmv and los in 168 patients who underwent cardiothoracic surgery between 9/1/93 to 6/30/95. only patients who belonged in any one of five categories of congenital heart disease (ventricular septal defect _+ other septal defects (vsd), atrioventricular (av) canal, tetralogy of fallot (tof), transposition of great arteries (tga), and single ventricle physiology (fontan)) were included. eight non-survivors were excluded from the analysis. all patients were admitted to an intensive care unit 0cu) post-operatively where mechanical ventilation was managed by 4 pediatric intensivists. lmv was defined as the period from post-operative admission to planned extubation. length of stay (los) was defined to be from le from the icu. cytokine patterns during and after cardiac surgery in young children. especially in children, cardiac surgery with cardiopulmonary bypass (cpb) can cause a systemic inflammatory response. this process is thought to be mainly a result of inflammation induced by surgery and exposure of blood to an artificial surface, and of reperfusion injury during weaning of bypass. complement activation, degranulation of granulocytes, induction of free oxygen radicals, endotoxemia and release of cytokines, are important contributing factors. we studied cytokine patterns before, during and after cpb in young children admitted for complex surgery or for septal defect correction. in the first group, significant amounts of il-6 and il-lra could be detected preoperatively. these findings could reflect the already existing hemodynamic dysregutation. in both groups, cpb procedure upregulated the circulating pro-inflammatory cytokines il-6/8, but not il-1b. at the same time, il-lra became detectable. therefore, we suggest that in these patients the production of the anti-inflammatory cytokine il-ira was not induced by the preceding acnvity ot pro-inflammatory cytoidnes. during cpb, we noticed a sharp decline in the capacity of the leucocytes to secrete il-6/8. the ex-vivo production of il-lra however, was only slightly attenuated. we conclude that there is a differential regulatory pathway for the induction of il-6/8 and il-lra. in addition, we studied the influence of dexamethasone administration on the cytokine pattern. administration delayed the appearance of il-6/8 and il-ira in the plasma, interestingly, it did only interfere with the ex-vivo production of pro-inflammatory cytokines. the latter supports our hypothesis that production of il-6/8 and of il-lra is regulated by two independent pathways, (60%) of 43 pts. 82% ofpts < 12 months of age developed metabolic alkalosis as compared with 38% ofpts > 12 months of age.the infants with metabolic alkalosis received more citrated blood products and furosemide. following cardiac pulmonary bypass the highest ph-values and be-values were observed 24-48 hours and 48-72 hours, respectively. ii. prospective study: metabolic alkalosis was registerd in 2t children (70%), 8 of those <12 month (75%) developed metabolic alkalosis and 67% of those elder than 12 monms.durmg the postoperative course patients younger than 12 months developed the highest ph-and base excess values after 102 and t05 hours, in the subset of the older patients maximum ph and base excess was found after 48 and 81 hours, respectively. in one case the top level ofph-value exceeded 7.6, the base excess +20 mvalb. conclusion: children undergoing cardiac surgery with cardiopulmonary bypass often develop metabolic alkalosis.in contrast to previous reports, we did not observe an association between metabolic alkalosis and mortality, nor greater frequency of cardiac arrythmias or prolonged mechanical ventilation. in context with decreasing serum lactate levels, our data show positive correlation of metabolic alkalosis with postoperative improvement of liver function. respirator, mechanics and weaning outcome in children undergoing cardipvascular surgery. vassallo j., cernadas c., saporiti a., landry l., rivello g., buamsha d., rufach d., magliola r. mechanical ventilation (mv) and acute respiratory failure are common events in children unergolg cardiovascular surgery (cvs), the development of new techniques helped to measure some of the main respiratory mechanics (rm) in a non invasive fashion. our goal was to evaluate the predictive value of these measurements in weaning (w) outcome in these patients, patients and methods: we prospectively evaluated children considered clinically to be ready for w with < 20 kg and > 24 hs mv. patients with diaphragm paralysis and those who failed w because of upper airway obstruction were excluded. before patient extubation the following measurements were recorded during spontaneous ventilation (cpap/t piece) using the cp 100 neonatal pulmonary monitor bicore (lrvine, ca): total respiratory system static compliance (cssr) and resistance (rts), rapid shallow breathing index (rsbi). maximal inspiratory negative pressure (pi max) was measured using an unidirectional expiratory valve. threshold values predicting w success (ws) were: cssr > 0.5 ml/cm h20, rts < 75 cm h20 /l/sec, rsbi 160 and pi max > -30 cm t120. w failures (wf) -patient reintubation within the following 48 hs, these values were compared between w success and failures using fisher exact test. an apriori level of statistical significance was chosen at p < 0.05. 4 considered, an increase in tnf-a levels is observed after cardiac surgery (p<0.001) with a return to previous values after 24 hours (p<0.005). 72 hours after cpb, similar values are observed in groups ii and ill, but there is a further increase in serum tnf-a levels in group i when compared with both other groups (p<0.03). we found no statistically differences in any other moment. there was a significant correlation between serum tnf-o levels determined 72 hours after surgery and cpb duration (p<0,003). conclusions: cpb in childhood provokes a significant increase in serum tnfa levels, in newborns the inflammatory response is maintained 72 hours after surgery. this enhancement of serum tnf-e levels indicates the existence of a relevant inflammatory response in these patients. introduction: cardiac surgery appears to induce a systemic inflammatory response. we have investigated the behaviour of il-1 i~ and il-6 before and after cardiac surgery. patients and methods: we studied serum il-1 6 and il-6 levels from 20 children with congenital heart disease (10 boys and 10 girls), aged from 7 days to 14 years, undergoing open heart surgery, before cpb (d we found no statistically differences in the il-i levels in the different groups and moments. there is a significant increase in il-6 immediately after surgery (p<0,01) with similar levels 24 hours after cpb and a significant decrease (p<0.01) 72 hours after cpb. preoperatory il-6 levels were higher in the groups i and tl than in group i11 (p<0.05). 24 hours after cpb serum il-6 levels in group 1 were significantly higher when compared with group 111 (p<0.05). conclusions: cpb in childhood induces a significant transient increase in serum il-6 levels, strongly relevant in newborns. cpb was not associated to a significant modification in serum il-1 6 levels. thus, cpb in childhood induces a dissociated behaviour in the proinflammatory il-6 and il-1 & pathways. obiective, to evaluate the effects of amg receipt on the clinical condition during the first 12 hours after birth (t2), the morbidity and mortality in immature outborn neonates. methods. we studied 44 outborn neonates with ga 26 to 29 wks, admitted during the years 1993 to 1995. eighteen neonates exposed to amg (ga:27,6+lwks, bw: 1066_+195g) and 26 neonates did not (ga: 27,7_+1wks, bw: 1042_+187g). results. amg-exposed neonates compared to those not exposed had lower incidence of apgar score at 5 min _< 3 (6% vs 35%, p<.05), lower incidence of ph t2 <7.20 (11% vs 48%, p<.05), decrease need of bicarbonate 12 (22% vs 54%, p<.05), lower fio212 (fio212min>40: 17% vs 48%, p<.05 and fio212max >80: 17% vs 52%, p<.05), lower incidence of intubation (67% vs 92%, p<.05), lower requirements of surfactant (50% vs 79%, p<.05) and lower mortality (11% vs 50% p<.01). there were no differences between the two groups for the following parameters: type of delivery, hypothermia hypoglycemia and anemia during admission, hypernatremia, hypotension 12 (map<30mmhg), need of dopamine and or plasma 12, incidences of ptx pda sepsis nec severe rop major ivh (plus pvl) and bpd and duration of intubation. conclusions. the main beneficial effects of amg receipt on the immature outborn neonates were the decrease of mortality and the decrease of surfactant need. there was no effect of amg receipt upon other severe morbidity in this high risk group of neonates. premature babies are very sensitive on homeostatic disturbances, and often develope intracranial haemorrhage (ich). ultrasound scan of the bram shows four grades of ich: -grade i -only periventricular hyperechogenic areas -grade ii -haemorrhage ham the lateral ventricles -grade ili-dilated lateral ventricles -gtrade iv -intracerebral haemorrhage. the purposes of this study were: 1 to show the incidence of ich in premature babies and its correlation with the gestational age, 2. to determine the severity of ich 3. to present the outcome &those babies. in the study were included 393 premature babies successively-born at the department of gynecology and obstetrics before 37 gestational week (g.w.) and grouped in three groups: less than 28 g.w., 28-32 g.w., 33-36 g.w. to all of them was performed ultrasound scan of the brain. results : 1. the incidence of ich hi premature babies is 49 % and there is ingh level of correlation with the gestational age: -babies born before 28 t~ g.w. have 100% incidence of ich and graduated : i grade -5%, ii grade -65%, iii grade -25%, iv grade -5% -babies old between 28-32 g.w. have incidence of 61% : i grade -24%, i[ grade -62%, iii grade -14%. -babies older than 32 g.w. have incidence of 33%: i grade -46%, ii grade 48%, iii grade -6% 2. sixty of 393 premature babies have died and it is 15.2% lethality. in all died ilffant was confirmed the grade of ich diagnosed by ultrasotmd scan of the brain. d. maksimo~5c. z.braiko~ic, n.vunjak. p. ivanovski (5~iversi~, children's hospital. belgrade, yugosla~, ia infantile intracranial hemorrhage is the most frequent and serious manifestation of late hemorrhagic disease of the newborn caused by ,,~tamm k deficiency in earl?,, ti~fancy. in the last two years, we recorded five cases of infantile intracranial hemorrhage due to "dtamin k deficiency, despite routine prophylax~s (intramuscular vitamin k, 1 mg) , with bpieal clinical presentation: age was 18 -65 days (average 40 days): vomiting, poor feeding, lethar~'irritabiljty, palor, bulging t0ntanelle and convatsiones were present in most cases.two patients developed signs of hemorrhagic shock, with hemoglobin level less than 70 g.1. in 3~5 f \qi level was less than 30 % of predicted value. there was no evidence of head trauma or liver disease in none of patients. four inlants were breast fed, while one, who had diarrheal disea.se, was on adapted milk formula. routine therapy wa.s given (including vitamin k and fresh frozen plasma). two patients were discharged with no sequellae, one developed posthemon'hagic hydrocephalus as a complication and two patients died. late hemorrhagic diseo.se of the newborn is sill/ a significant cause of morbidib' and mortality in earl3' infancy, despite different approaches to prophylaxis developed in recent years. background: neonatal hearing screening in at risk newborns can detect 50% of the children with a congenital hearing loss. automated abr hearing screening (algo-1) has been introduced for healthy newborns. the aim of this study is to test the validity of this algo-1 screener in at risk newborns in a neonatal intensive care unit. subjects: 250 at risk newborns (median gest.age: 30.0 wks, median birthweight 1350 g) selected according to the criteria of the american joint committee on infant hearing. interventions: algo-i automated abr-hearing screening at a level of 35 db was performed in the neonatal intensive care unit. when bilaterally referred, further audiologic screening and/or therapeutic intervention took place. when passed uni-or bilaterally, children enrolled in a) a nation wide screening programme (ewlng) at the age of 9 months and b) in a half yearly follow-up programme in which hearing and speech-and language development were observed according to egan an illingworth. results: screening without disturbance from ambient noise or from routine technical equipment was possible in the incubator, even during nasal cpap therapy. 245 (98%) newborns passed algo-1 screening. 5 (2%) did not pass bilaterally. 1 of 5 with a congenital rubella died shortly after screening.in 4 of 5 bilateral congenital hearing loss of ->35 db was confirmed. 235 of the newborns passed were still alive at the age of 1 year. ewing screening was performed in 183 of 235 (77,9%). 161/183 passed, 15 of 183 had passagere conductive hearing loss, in 7/183 no further investigation was performed. all 235 children enrolled in the i/2 yearly follow-up programme had normal speech-and language development. in this study all 4 at risk newborns with bilateral congeni "tai hearing loss were detected with algo-1 screening. screening results showed no false negatives at follow-up. the algo-1 infant hearing screener can be used as an valid automated abr-screener to detect hearing loss in at risk newborns in a neonatal intensive care unit. gancia gp, bruschi l pnlito e, ferrari g, rondini g -divisione di patologia nc~matate e turapia intensiva -irccs policlinico s. mattco -pavia, italy latrogenic esophageal perforations (iep) in preterm and term infants are seldom reported in litteraturc, in association with difficult endotracheal (et) intubation (with or without stylets), insertion of gastric tube, and pharyngeal suctioning with stiff catheters. crieopharyngeal muscle spasm caused by instrumentation may also lead m a narrowing of lumen, with increased risk of local injury. we report 4 iep observed in intubatcd, mechanically ventilated newborn infants (2 male, 2 female, all outborn). a common feature of iep was inability to pass a nasogastric (ng) tube into the stomach, mimicking e~)phageal atresia.~se 1: birth weight (bw) 185(i g, gestational age (ga) 37 wk, sepsis. before admission to n1cu, the baby underwent multiple et inmbations, because of inappropriate securing of et robe. bloody secretions in pharynx were observed. the endoscopy showed a large lesion at the end of proximal third of the esophagus, case 2: bw 1080 g, ga 32 wk, rds. chest x-ray (cxr) showed a retrostcrnal air leak: the ng tube was stopped }~etwcen d8 and d9 and soluble contrast was seen in upper mediastinum.case 3: bw 76(/g, ga 26 wk, rds. the endo~opy showed an esophageal lesion. cxr showed a paravertebral route of ng tube and a right pneumothorax.case 4: bw 102(i g, cz 22 ,.v!:. rd c. ~!,'.::;;: ::':'_'rvt!~'2s l" ~k':.rvrx. cwr, d,,,,vs ~,,mr~e, ~n rhe upper mediastinum and abnormal route of ng tube through a false passage. surgical intervention is needed in case of mediastinitis or mediastinal abscess: conservative management included broad spectrum antibiotics, total parenteral nutrition, antireflux therapy and, if necessary, drainage of air leaks. enteral feeding has been stopped lor 15 days and cautiously resumed after radiographic study. [x~cal sequelae and death are uncommon, but iep occur in newborns with high risk of death due to prematurity and other diseases. in our patients, et intubation has been performed by experienced personnel: therefore the lack of skills in resu~itative procedures is not always the main factor of iep. prevention of iep requires appropriate materials (et tubes, laryngoscope blades, suction catheters), and procedures (positioning of the infant with correct neck estension, firm et placement). sedation and pain control may help to prevent the muscle spasm. aggressive treatment has improved the tong-term outcome of extremely low birth weight neonates (elbw) but it has also increased the chances of iatrogenic lesions. reviewing the charts of our neonates we observed a high number of vascular injuries. from 1987 to 1994, 2898 neonates were admitted to the neonatal intensive care unit (nicu); 335 of them were elbw (11.5%). studying the charts of these elbw we observed 9 cases (4 m -5 f) with vascular lesions (2.6%). mean gestational age of these patients was 28.7 weeks (rain 24-max33). mean weight at birth was 880g . mean weight at diagnosis was 1825g (1230-2700). in the same period 10 patients with vascular injuries were reported in the 2563 neonates over 1500g (0.3%). the injuries observed in elbw group were: 6 arteriovenous fistula (2 bilateral) at femoral,level, 1 carotid lesion and 2 limb ischemic lesions. aetiology was in 7 cases by venipuncture, in one case umbilical catheter and in the case of carotid lesion a wrong surgical maneuver. no general simptoms were observed. the vessels were repaired with microsurgical technique in six cases: the carotid lesion and five arteriovenous fistula; one case was solved with thrombolitic drugs; an amputation at knee level was required in one case after a long period of medical treatment. the last neonate with an arteriovenous fistula was only observed for parent's will. at follow-up (clinical and by ecodoppler) 7 out of 9 neonates presented normal vascular function without sequelae. from our experience elbw neonates have more chances than older neonates to develop iatrogenic vascular lesions. we advocate an aggressive microsurgery and/or medical treatment to obtain good results and prevent late sequelae. a retrospective comparison between 2 natural surfactants l.j.i.zimmermang m.c.m,van oosten. dept. pediatrics, div. neonatofogy, sophia children's hospital/erasmus university, rotterdam, the netherlands. aim: retrospective comparison of alvofact (in 1993) versus survanta (in 1994) as rescue treatment for neonatal respiratory distress syndrome (rds). methods: both surfactants were given at an initial dose of 100 mg/kg (except for alvofact 50 mg/kg for mild rds grade mi). repeat doses were attowed (survanta 100 mg/kg, alvofact 50 mg/kg) up to a maximum of 200 mg/kg, all parameters and outcome criteria were strictly defined beforehand. the initial response (good,mild,no response,relapse) to surfactam therapy was defined on the basis of the decrease in fio 2. results: there were no signif. differences in patient population and initial parameters: ga (29.9+_2.2 vs 29a _+2,6 wks), birth weight (t332_+431 vs 1227-+444 g), severity of rds (grade ill-iv: 78.6% vs 80.3%), apgar scores, cord blood gases, initial ventilatory settings. in '93 however, the initial surfactant dose was administered earlier than in '94 (14.4-+ 17.4 vs 6.5_+7.8 hrs postpartum, p= 0.025). although the average total cumulative dose was equal in '93 and '94 (169.3-+65,8 vs 167.4_+69 .4 mg/kg), more doses of alvofact were given compared to survanta {2.3_+1.1 vs 1.7_+0.6, p=o.o01) and more patients in '93 received more than two doses than in '94 (46% vs 18 % of patients). there was no difference in the incidence of non-putmonarycomplications. aivofact ( there was a better initial response to survanta and a better respiratory outcome in 1994: in the group < 1250g the duration of ventilation was half in 1994, and in the group >~125og the duration of extra o 2 need was half in 1994 as compared to 1993. we speculate that the main reason for this difference is the earlier and initially higher dosing used with survanta compared to that used with alvofact which was given in the same total cumulative dose but over a larger time span. background: e×ogerlous sur&ct~t raplacem~t treatmem has become rou~ne k~ the t~eatme~t of respira~"¢ dim'~ syndrome (i~ds) of pr~e~tur~, wh~eas its effica w th odi~ respiratory diseoses is sdi1 being wader mvesugatio~. objective: "eac~ mt ereat isto report ottr results of prospect/re, non-randomized "re~-o.e" study oe suffact~t replacement in outhom premamae infa~t~ with rds reruirmg me~aical ventilatioa (nfv). p~tien~ and metho0.s: from j-aly 1993 to june 1995, 18/58; (31%) out~ ~¢ infaats, at a mesa age of 22 z 2,7 horn's ( 13 boys, 5 ~rls; ~ gestafioan age 32-+2.8 weeks, mera~ birth weight 1846 _+ 544 g, ~ 7.2 i" 17 at 5 minutes) with rds, requiring mv, received bov~e-suff~amt (survanta, ros~/aboti, laboratotie~ columbus, ohio) eadotracheally, as was recomm~aded by maaufacturer. as the c,~:ttrol group 19 o~bom premature infants (ot~ of 49; 39%, admitted with rds from euiy 1991 to eune 1991) were saelected ~d who did not receive surfaaam, compared with ~hctant ~'oup they were admitted for treatmeat e~'li~" aft~" daliv~:y (at the age 6.4::2.2 hours vs. 11.7+-13 hours), but they did not diff~ in othe~ baseline dam'a~eri~cs at ~ti~ion. entry crkeda for ~¢fa~aut ~hcadou were fractional i~firat o~ oxtgem r~emeats -fio2 > 0.50 -0.60, ratio au-lerlal to alveolar oxygea pre~are~ao2~ao2 < 0,20 ~ad oxyge~at,~ i~.dex -ol > 10. primary o~comes were deter~caned by ~hanges m exs'ge~ab, c~ ~r~d vmtilatic~ ~ the following variable~; (1) fi'aaic~ of i~spired oxtge~ (fio2); (2) mesa nnvay presmzre (map) (3) pag2 ~ao2 ratio, (4) oxyge~ion index (oi). commo~ comphcadces of prem,musty ~d con~ol mechamcal v~ati]al~on (pater dumas merios.s, intracr~nlal haemcrn:hage, air leak, br onchop ulmrmm'y dy~pl~a ~d death) were reg~ded as sec~d,~y outcomes. r~suas: in warfactaat group we observed slg~5.c~t improve~aeat (p<0.05) in oxygea~thia md veaatilation at 24 hours all~ e~try k~to the m~dy in compari~ion to nons~fa~m" group. compa~on of secondao' outcomes in ~ts with p,.ds showes table l we did not observe ~y major acute hfe fl:u-eattming complicatlola,s m sxlrlhct~mt grou~ tr/lmediately after stu'~actsmt rcplacemev_t therapy. the duramm of mechmucal ven~ation ~ad oxygen lreau~ent m survivals of both groups did not dafter 51gmficautl y a-ore ead~ other. condusion: l!a premature mthats with rds treated with surfaaaat replacemeaat therapy we observed decrease m mc~de~ce of tme'~m~o~oraces add de~th (p<0.01 and p<0.05), whe~e~s m othe~ observed variables thee was uo ,igmfi~t d~=ecce infectious complications during the therapy of respiratory insufficiency in neonates with birth weight less than 1500 g in the course of 3 yearsretrospective study. zitek infants on cmv, cppv, and imv were administered exosurf in dose of 50-60 mg/kg twice endotracheally (see table) . in 32 newborns (86.4%) 2 hours after surfactant admin fi02 value decreased by 20.8%, and after 6 hours -by 28.1% compared with initial value; pip and peep values decreased by 3-5 cm h20 and 1-2 cm h20 after 6 hours, and by 4-7 cm h20 and 2-3 cm h20 after 1 day, respectively accompanied by mean decrease of aado2 from 486,2 to 240.2 mmhg, qs/qt decrease from 24.9 to 13.2% (see table) . mean time of cmv, cppv was 7.8 days, imv-14-36 hours, cpap -10-24 hours. respiratory therapy in 5 newborns (13.5%) was complicated by pneumothorax (bilateral -in 2 infants chorioangioma is a rela~ively rare placentai malformation associated with considerable mortality and morbidity. a chorioangioma can be regarded as an arterio-venous shunt in the circulatory system of the fetus. this causes volume loading eventually resulting in cardiomegaly and high output cardiac failure. a female neonate (gest age 40 wk, birth weight 2290 g, -2.6 sd) was born with an apgar score of 4 and 7 after 1 and 5 rain respectively. the placenta showed multiple chorioangioma. ultrasound of the heart showed a hypertrophic cardiomyopathy. she developed severe hypertension (100/70 mm hg), treated with nitroglycerine and nitropruside. finally blood pressure decreased when enalaprillic acid was given (0.15 mg.kg4). we measuered the activity of the renin-angiotensinsystem. an elevation in renin-angiotensin system is shown probably to compensate for the low resistance circulation before birth, hypothesis: the instantaneous cut off of a large arteriovenous shunt did not result in a fast downregulation of the renin-angiotensin system resulting in hypertension. hypertension should be added to the list of complications of chorioangioma of the placenta. the authors studied 75 cases of children's septicemia with blood culture yielding staphylocucetts aurens. the age of patients varied from 2 months to 15 years (51,3% from 3 years downward), 74% of the children caught their disease in the hot season (may to october). the deaths also occured in this season: 87,5% (21/24). following were the anatomo-dinical lesions. -skin 42%, muscle 60,0%, bone 21,3%, joint 9.3%. -viscera : lung 50%, heart 33.3%, cerebrum 22.6%, kidney 60.6%, fiver 17,3%. -simple lesion skin-muscle-bone joint: 12%, no death in this group. the concomitant lesions of the soft tissue,bone-joint and viscera : 34% with one viscera, 26% with two viscera, 18% with three viscera and 9% with four viscera. -bone lesion : mainly on the long bones (50% on the tibia, 25% on the femur, the remainder being the mandible (3) and the humerus), inflammation of' the hip joint was the main one. -i,ung lesion had forms pneumatocele (4 cases), bronchopneumonia (6 cases), pleural effusion (7 cases), multimicroabcess bursting into the pleura (8 cases), most multimicroabcesses were lethal : 20/22 (90,9%), -heart: all thethreelay~rs got le@~r~, 20% had 2 or 3 layers alrected and death ensued. -cerebrum : the meninges had three forms of lesions purulent meningitis (13 cases), obturafing embolns of brain vessels (2 cases) and cerebral abcess (one case). the characteristic clinical sign was paralysis and meningismus, phlebothrombosis of eavcrnous finus (13 cases)was mually ther~sultofalxil vdfi:h burst there were 6 cases of death with lesion of the meninges and 2 cases of obturating embolns of brain vessels. -the main sign of lesion of the kidney was a change in the components of urine: 60% got proteinuria, 75% had leucocytes in their urine, 42% had erythrocytes in their urine, the urea in their blood increased (over 60rag%) in 21.4% of cases.the lesion of the kidney seemingly had little relation to death. seven cases of ictertts due to an increase of direct bilirubinemia and a decrease of blood-albumin. -the biological characteristics of the pathogen staphylococci showed that all the 75 isolated specimens had positive coagulaza ; the specimens from the dead patients were less semiti~e to, mad ~t to mali~ overag death rate was 34.7 % (24/75). the fungal infection to fusariun species in immunocompromissed child have been reported in the literature with a rare, severe and high, mortality rate in spite.of the use of antifungal drugs. we report a case of successful treatment of a severe disseminated fusariun infection in a ll-year-old boy with acute lymphocytic leukemia (lla-l3), after use a chemotherapy followed by absolute granuloeytopenia. the patient developed fever, skin lesions, pneumonia and fungaemia. fusariun species was cultured from the blood, necrotic skin lesions and lung secretion. the child developed multiple organ system disfunctiou in spite of use broad spectrum antibiotcs and antimycotic therapy needing. uci during 18 days. the patient receive suport treatment (mechanical ventilation, inotropie d~.ugs, diuretics, imunestimulants, blood components, a broad spectrum antibiotes and antifungal agents). we absorved a gradual recovery in the white blood cell count and regression on the sites of infection. the association of preeoce diagnostic and the terapentic with increase in the white blood cell count was the most important in a successful treatment. a 5 year old african-american child suffered a severe pulmonary injury in a house fire. initial survey revealed 1% total body surface burns, soot on the face, and bloody endotracheal secretions. initial chest radiograph revealed diffuse, bilateral infiltrates. severe respiratory failure with an oxygenation ratio of 73 rapidly developed. he developed a pneumomediastinum and subcutaneous emphysema. although transient improvement occurred with inverse i:e ventilation and surfactant, he became more hypoxic (sac2 as low as 47%) and acidotic. on day 2 post injury, he was placed on venc~venous extracorporeal life support (ecls). on ecls day 30 he was decannulated. chest radiograph on ecls day 15 showed an opacity in the left chest. ultrasound of the left chest was consistent with atelectasis rather than pleural fluid. flexible bronchoseopy failed to reveal any obstruction in the left lung. a computed tomography (ct) seen of the chest, which was performed after decannulation, revealed a large loculated collection of fluid in the left, anterior chest. under ct guidance, a 14 f cope loop catheter was inserted and 40 cc of thick blood was removed, follow-up ct performed immediately after this procedure revealed minimal change in the size of the fluid cavity. over the next 48 hr, we instilled urokinase 20,000 units over 20 minutes every two hours. a 30 minute dwell time was allowed before draining the fluid. repeat ct scan done at the end of the urokinase infusion showed a marked decrease in the size of the fluid cavity. act scan was not performed prior to decarmulation because the ecls circuit tubing was too short to allow appropriate positioning of the child in the ct scanner. after a ct scan revealed loculated pleural fluid, a simple drainage procedure was diagnostic but inadequate treatment. we were able to successfully dissolve the thrombus after 48 hr of urokinase therapy even though the thrombus was > 14 days old. we suggest that large loculated plenral thrombi which develop as a complication of ecls therapy may be successfully managed with urokinase infusion. introduction: haemorrhages, particularly intracranial, are major complications experienced in 10-35% of neonates treated with extracorporeal circulation. an induced thrombocytopenia and impaired platelet function play a key role in the increased bleeding tendency observed in these patients. the aim of the present study was to establish a dose-respons curve for the effect of a synthetic protease inhibiting agent, nafamostat mesilate (fut-175), on platelet membrane glycoprotein density and platelet activation during experimental perfusion. methods: two identical extracorporeal life support (ecls) circuits were primed with fresh, heparinized human blood and circulated for 24 h. four different concentrations of fut-175 (7.12 mg/l blood/h; 14.25 mg/l/h; 14.25 mg/l/h+25% bolus at the start of the perfusion and 2&5mg/l/h+25% bolus) were used in different perfusion experiments. a total of eight paired experiments were performed. platelet count, plasma betathromboglobulin levels and platelet membrane density of glycoprotein ib and lib/ilia were followed as well as plasma concentration of haemoglobin. results: a protective effect of the agent on platelet count, plasma concentration of btg and platelet membrane gpib could be observed during the first 3 hours of the perfusion when a bolus dose was added. no positive effect could be recorded with the two lower doses used. plasma concentration of haemoglobin was higher in all the fut-circuits compared to the control circuits. conclusion: the addition of a bolus dose of fut-175 at the start of the perfusion seem to induce a protective effect on platelets during the first hours of perfusion. extracorporeal membrane oxygenation (emco) is a form of invasive cardiopulmonary support that can provide imporary physiologic stabilisation in reversible circulatory failure and or respiratory failure. we reviewed our expierence with extra corporeal membrane oxygenetion in 4 children aged 1 day to 4 year between 1991 and 1995. two neonates was succesfully decanulated, but died 1-2 well after decanulation due to septic complictions. one child 4 years old, one neonates died on day 5 and day" 7 respectively while still on emco. complication which were and encountered were heavy bleeding in case 1 (child), 4 (neonate) and raceway rupture in case 2 (neonate). problems which are specific developing countries like indonisia are: high cost (20.000 us for 7 days) difficulty in transportation (transporting intubated baby) from the orgin hospital, lack of knowledge and understanding of the primary physician and nm-ses and difficulty organizing in 24 hours emco team. resnratory mon1tor/ng in picu z,zjvkovic, s. mihailovic, o, tosev respiratory monitoring in pediatric intensive care unit 0picu) provide the importartt informations for understanding of the pathophysiology of the clinical signs, aid with the diagnosis, and assist in therapeutic management and predicting prognosis. pien in children's hospital for ~flmonary diseases and tuberentosis remained for the t~s't two end a half years relatively limited for diagnomic tools and therapeutic regimens, mostly because of the poor fmnaeial suptx~rt. the number of children admitted for aurae asthmatic at.lzek~ severe pneumonias, bronehiolitis, complicated pulmonary tuberculosis, foreign bodies and exacerbations of ehronit'. pulatonary diseases was t362. for all patients the respirator' monitoring system means: physie~d examination, ehe~ x rays, capillary bltxxl gas mmlyses (vevv few ehiktren experienced itwasive arterial blt~.~'i gases), noninvasive oxyntctry, measuring of the vital capacity in coopo-able patients, as~d capnography. later on, after the imtial critical illness, a complete hmg fimction tests was performed, as well ,~s bronehoscopy in selected eases, (~lr experience revealed that abotrt 60% of ehil&en heos suecessthl outcome, without s~lllens , instead they had been tremted in limited conditions. ']'he rest of our patients were previously diagnosed ~s ettronie pulmonary patients, with high risk score system ibr having seqnells 'llae mortality rate were 0,5%. the continuous blood gas monitor, pasatrend 7 (biomedical sensors, ltd., high wycombe, bucks, england) has the capability of measuring ph, pco2, and po2 via an indwelling optical absorption optodelclark electrode sensor that is placed through an intra-arterial catheter. we evaluated the accuracy of the sensor in radial and femoral locations in critically ill pediatric patients. methods: the simultaneous values of ph, pcoz, and po2 recorded from the paratrend 7 monitor were compared to values measured by standard arterial blood gas analyzer (coming 278, ciba-corning diagnostics, medfield, ma). criteria for the elimination of data points included a core vs. sensor temp. gradient, and sensor pulled back beyond accepted insertion distance. mean time of monitoring per sensor was 108 hours (range 0.75-403.7 hrs). mean time of radial monitoring was 35 hrs (range 0.75-160.5hrs) and of femoral monitoring was 137.2 hrs (range 12.8-403.7 hrs.). linear regression and bland-altman analysis for bias and precision for each parameter were calculated. results: a total of 49 patients (age range 2 weeks to 18 years) had paired samples of ph, pens, and poz made by the sensor and blood g&s analyzer. the range of measurements were ph 6.99-7.66, pco, 16.0-i14.2 t(n r, and po2 34-480 torr. the paratrend 7 monitor demonstrated accuracy that is comparable to the accepted standard of blood gas analysis in a group of critically ill pediatric patients manifesting wide variation in ph, pen2, and poz..this technique appears m be very useful especially in the extreme values of the parameters measured. funding provided by biomedical sensors. understanding of pulse oximetry d.semple, l.e.wilson. royal hospital for sick children, edinburgh, eh9 1lf, scotland, uk. pulse oximetry is a useful, non-invasive monitor, routinely used on the itu and increasingly often on the general wards. we used a questionnaire incorporating questions on the theory and clinical uses of the pulse oximeter to assess understanding of pulse oximetry in medical and paramedical staff doctors indicated grade, speciality, pulse oximetry tuition and neonatology experience. 45 doctors, 15 itu nurses, t9 medical students and 4 physiotherapists completed the questionnaire. some confusion existed between the principles of pulse oximetry and transcutaneous oxygen measurement. wide variations in the lowest acceptable saturation in fit children were seen (80-95%), with around 20% of respondents in all groups accepting values of 90% or less. some potentially serious mistakes were made in the evaluation of oxygen saturations in the clinical scenarios. there were widespread variations in correct responses at all grades of medical staffing. nurses scored well on more clinically-orientated questions but relatively poorly on theory. only 15% of doctors (mostly senior grades) had received tuition in putse oximetry. neonatology rotations appeared to confer little additional knowledge on pulse oximetry. few doctors and nurses receive tuition in the use of pulse oximetry a significant proportion of nurses and doctors, of all grades, exhibited a lack o{" understanding of the principles of pulse oximetry. this may result in unsafe use of the equipment and put patients at risk. one can see from the table that blood composition in uv and ua differens in some characteristics, and similar in sgp magnitude. venous-asterlal gradients "gas functiomals" between uv and ua represent the measure of difference in this characteristics. the gradient cari be positive, zero -order or negative and change both in value and in sign but not reach apo2 (positive) and apco2 (negative) in absolute significance.minimization of "gas functionals" deviations atom the zero is achieved due to"mutual replacement acts" between po2 and pco2 in uv and ua blood. we suggest that presented tests can be useful in full evaluation of gas exchange in newborns. (pap) in the context of pulmonary hypertension is oft desired but rarely achieved. inhaled nitric oxide (no) has been shown to produce this desirable effect, but is relatively difficult to administer or monitor. we wondered whether np, chemicaily related to no but more stable in solution, would produce similar physiologic effects when administered in the convenient modality of nebulization. methods: 9 piglets were anesthetized, mechanically ventilated, and surgically instrumented. systemic blood pressure (bp), pap, and cardiac output (co) were monitored continuously. after postoperative stabilization, 0.9% nac} nebulization was begun, and pulmonary hypertensiorr was induced by reducing fio2 from 0.30 to 0.07. the piglets were monitored for 15 minutes during this hypoxic phase, next, without altering fio2 or ventilator settings, np (10 mg/ml, dissolved in 0.9% nacl, flow 4 ipm) was substitued for 0.9% nacl in the nebulizer circuit. np was nebulized for 15 mins. results: during hypoxia, pao2 fell from 159 to 29 mm hg. pap rose during hypoxia from 14 to 31 torr (p< 0.01). ,^fhile bp and co did not change significantly. pap fell during nebulized np in each piglet, (mean apap = 31 to 21 torr; p< 0.01; mean reduction of hypoxia-induced rise in pap = 61%; range: 36 to 78%; p < 0.01). pvr/svr fell by 28% during np nebulization (p< 0.01), while bp and co did not fall significantly (90 to 86 tort; 653 to 636 mllkg-min), the reduction in pap began within 2 minutes of the onset of nebulized np, and appeared to reach a plateau by 15 minutes. no tachyphylaxis to nebulized np was noted. nebulized np did not significantly affect pap, bp, or co under normoxic conditions. conclusions: 1) like no, np selectively reduced hypoxia-induced pulmonary hypertension without altering systemic bp, 2 ) unlike no, np can be administered by nebulizer, a technique familiar to virtually all health-care providers, and potentially adaptable to both intubated and non-intubated patients. 3 } nebulized np may be beneficial in clinical contexts where inhaled no is impractical. dang phuong kiet and nguyen xuan thu examining 6 cases of purulent pericarditis with various clinical forms treated by surgery, the authors drew the following experiences for their diagnosis. t. clinical factors. purulent pericarditis appeared like a cardiac tamponade in a septicemia due to staphylococci with dassieal symptoms: severe dyspnea, tachycardia, faint heartsound, big liver, prominent cervical vein ; rentgenography of the chest showing enlargement of the cardiac silhouette, a diminution of ventricular pulsations, ~i clear lung field. by an emergency operation, 500ml of diluted blood were drained. purulent pericarditis and pleural effusion appeared at the same time but at first tile symptoms of purulent pericarditis were masked by the predominant symptoms of plearal efihsion. after the pleura was drained, its pus was no more, the general state was relatively stabilized but there still were big liver, dyspnea, enlargement of the cardiac silhouette while central venous pressure increased. purulent pericarditis appeared late. in the first stage (about 2 weeks) there was no suspected sign. later on gradually appeared such symptoms as dyspnea (during serum transfusion for instance). central veinous pressure also raised. the heart chest diametre increased at first (up to 60-65%) then decreased (down to below 50% ) but the liver kept on swelling together with the particular changes of electroeaediegramme. now the pericardium had no more pus but get fibrous (up to 3ram) thus constricting the heart and its main arteries 0ike pick syndrome). 2. diagnostic values of electrocardiograms : common signs of ecg related of these purulent pericarditis were: a diminution of voltage, a widespread elevation of the st segment, the tf wave flattened and inverted. however, what should be stressed was : the diagnostic values of an electrocardiogram for purulent pericarditis was mainly in the dynamics of their signs: in the first week, the voltage diminished corresponding to a pericardium containing pus, while the st segment went up then seemed parallel to the fibrosis of the epicardium, the liver swelled, the central velnous pressure increased, the heart/chest dimension ratio decreased, the st segment went down, the t wave became more flat and inverted. between 1986 and 1995 23 neonates, aged 2 -23 days (median 5), weight 2,38 -4kg (median 3,28) with critical valvar pulmonary stenosis were scheduled for balloon dilation (psvp), 19 children (83%) were on pge1 and 13 (57%) needed mechanical ventilation. after stepwise dilation a final balloon : pulmonary valve (pav) ratio of 114% (25-150) was achieved, there was a significant correlation (p<0,01) between an adequately sized balloon and freedom of reintervention. two valves could not be passed, four neonates underwent surgical procedures (brock n = 3, commissurotomy n = 1), two children (10%) died of sepsis. 17/23 patients (73%) were successfully palliated by psvp in the first month of life. the rv : systemic pressure value fell from 132% (75-231) to 58% (40-87), complications included 2 transient dysrhythmias, 1 transient hypoxia, 3 vessel occlusions;-1 right ventricular outflow tract perforation. in 16/17 patients follow up data is available. the residual systolic peak doppler gradient over the pav on the last out patient visit (5-103 months after psvp) was 10-41 mmhg (median 20). four children needed repea.ted psvp 26 to 72 months after the initial intervention. conclusion: psvp of critically ill newborns is possible. the risk of mortality is relatively low. psvp in neonates with an adequately sized balloon is a challenging alternative to surgical treatment. post hypoxic-ischemic (hi) reperfusion induces the formation of non protein bound iron (npbi), leading to production of the reactive hydroxyl radical. it was investigated if the ironchelator deferoxamine (dfo) could reduce free radical production and improve neonatal myocardial performance after hi. severe hi was produced in 13 newborn lambs and changes from pre-hl values were measured at 15, 60 and 120 min post-hi for (mean) aortic pressure (mean pao), cardiac output (co) and stroke work (sw). left ventricular (lv) contractility and co were assessed by measuring lv pressure (tip-manometer) and volume (conductance catheter), using inferior caval vein occlusion to obtain slope (ees) and intercept of the end systolic pv relationship (v10). npbi, reduced and oxidized vitamine c ratio (vcred/ox) and lipid peroxidation (mda) were measured from sinus coronarius blood. 7 lambs received dfo (10 mg/kg i.v.) immediately post-hi, control lambs (cont) received a placebo. results: mean pao was stable, co and sw decreased up to 60 and 40% respectively in cont as compared to pre-hi. in both dfo-groups co and sw remained within the normal range. ees and v10 decreased in all groups post hi, but did not differ between groups. npbi and mda were higher at 15 min post hi (p<o.05), vcred/ox was lower at 15 min post-hi (p<o.o5) in cont as compared to dfo-group, indicating more oxidative stress in cont. conclusions: dfo reduced the oxidative stress of the heart and prevented co and sw to drop, suggesting a positive effect of iron chelation on the myocardium after h{. from 1987 we published reports with analysis of performance of the heart as a whole organ.while analysing the heart performance as a whole, we recognize three block in it located intrapericardially: l."atrial"block -left (la) and rigth (ra) atriums; 2."aorta-pulmonary" block-aorta 'bulb (a) and pulmonary artery(pa); 3.ventricular "three-chambered" block (vb) consisting of: -left (lv) and right (rv) myocardial chambers, both frith blood outflow into "aorta-pulmonary" block vessels, and -spongy (venous) myocardial chamber with the blood outflow through coronary sinus (cs) and thebesiau vein (tv) into "atrial" block. the following conceps are introduced for vb functions assessment "common" systole and "common" diastole of "three-chambered" vb. the process of normal "common" vb systole: -begins with blood ejection from vb spongy chamber into the "atrial" block; -continues with blood outflow from rv and lv into "aorta-pulmonary" block wi~ venous minimums -x-coiiapses -~btmation iu "a~riai" block; -completes with "three-chambered" vb general emptying. at this period the following blood volumes are transferring: -two-from rv and lv(their stroke volumes)into"aor~o-pulmonary"block; -two-from "spongy" chamber .into "atrial" block; -two-from systemic and pulmonary veins into "atrial" block during the process of so called "systolic" membrane suction (at pulling atrio-ventricular valves into rv and lv chambers as blood ejects out of them). it appears to be the regulation of blood inflow to "atrial" block by blond outflow from "three-chambered" vb into "aorto-pulmonary" block. objective: to evaluate the efficacy and complications of transpyloric enteral nutrition(ten) in critically ill children patients and methods~ from june 1994 to january 1996 23 children (i0 boys and 13 girls), aged from 5 days to 11 years (mean l.8 ± 3.4years) were treated with ten. 18 patients were included after cardiac surgery, 3 with acute respiratory failure, and 2 after cerebral surgery. the indication of ten was mechanical ventilation in 22 patients and failure of nasogastric nutrition in 1 patient.19 children (82 %) had previously received parenteral nutrition. 8 and i0 fg enteral tubes was inserted into duodenus through nasogastric intubation en 21 patients and by endoscopy in 2 patients. rx and ph determination were used as methods to control tube situation. 18 children received adapted formula, 7 caseine hidrolisate, and 3 enteral formulas. (in 5 patients the nutrition formula was changed during ten). 22 patients were supported with mechanical ventilation during ten, 17 received midazolamperfussion(range 2 to 15 mcg/kg/min), 15 fentanyl (i -12.5 mcg/kg/h), and 6 vecuronium (0.1 -0.3 mg/kg/h). resulte: ten was used during 3 to 73 days (mean 18.3 ± 19.3 days). mean maximmnvolume ad~iniateredwas 132 ± 47 ml/kg/day (range 34 208) .patients with midazolam, fentanyl and vecuronitml tolerated ten similar than children without sedatives. complications were diarrhoea 1 patient, abdominal distension and/or important gastric fed residuals 2 patients. pulmonary aspiration or respiratory complications were no registered. ten was ended in one patient due to diarrhoea, in 18 due to change to oral or nasogastric nutrition, 3 children continue yet with ten and 1 was discharged of picuwith ten. conolusion: ten is an useful method of nutritional support in critically ill children with mechanical ventilation and/or nasogastric intolerance. introduction: the enteral nutritional support of the critically ill child may reduce the morbidity and mortality by attenuating, the hypermetabolic and catabolic response, decreasing the gut translocation and the septic complications, improving the bowel mucosa integrity and the wound healing. the aim of this study was to show the safety and tolerance of en. methods: we enrolled 36 consecutive patients admitted to our unit from may to december 1995, mean age 3.4 years (range imonth to i7 years). the prism, mechanical ventilation, inotropic use, opiates and other drugs were recorded. the en was delivered by continuous infusion across a polyuretane tube.we kept the patients head elevated 30 °, and we used cisapride routinely. the gap betweeen admission and the en beginning, average time to meet the nutritional objective, en duration, selected formula, patients outcome and complications were recorded. results: prism groups were 0-1=0 patients; 1-5=1; 5-10=13; 10-15=7; 15-30=12. two patients with mof (multiorgan failure) died. 72% needed mechanical ventilation.the average time elapsed from picu admission to initiation of en was 2.1 days (range 1 to 7 ) and the average duration of the en was 7.3 days (range 3 to t4). the average time to meet the nutritional objective was 2 days (range 1 to 3). the selected formula were: lactose free infant formula in 10 patients, elemental diet in 8, pediasure in 16 and jevity in 2. we recorded vomiting in 4 patients, diarrhea in 5 and constipation in 4. none of them forced the en suspension. we didn't recorded any case of ~spiration or pneumonia. we found no relation between drugs, selected formula and complications. we think that en may be a safe and well tolerated procedure at the picu setting. generation of free radicals in iipid emulsions used in parenterai nutrition (pn) has been described recently. this seems to be due to high polyunsaturated fatty acid content. we studied lipoperoxidation status and antioxidant parameters presurgically and through the postoperative period (5 days) in a group of twelve children (ranging from 4 months to 12 years of age), who underwent heart surgery and received nutritional support with pn. malondiaidehyde (mda), an end product of lipid peroxidation was used as a marker of oxidative stress. it was determined by the yagi spectrofluorometric method. antioxidant activity was measured with an assay based on the inhibition of spontaneous autooxidation of a brain homogenate, and vitamin e in serum by the technique of hansen and warick. erythrocyte mda release fonowing incubation in hzo 2 in vitro was used as a functional measure of tissue vitamin e levels. data were compared using one-way variance analysis. there was not significant differences in the plasma mda production among the values obtained before surgery (3 :t: 0.5 nmol/ml), postoperative pre-pn (3.5 +_ 0.6 nmol/ml) and through the course of pn (3.5 5:0.6 nmol/ml). levels of vitamin e previous to surgery were 5,1 5:1.1 #g/ml and decreased to 3.2 _+ 1.4 #g/ml at 24 hours post-surgery before starting pn. while receiving pn, concentrations of vitamin e increased progressively up to 7 + 4. l #g/ml (p = 0.08). pre-surgicai plasma antioxidaut activity values (72 :i: 11%) dimiuished in the first postoperative day (56 _+ 9.5%) and then showed a clear increasing tendency, directly correlated with that observed in vitamin e levels. the measurements of the maximal percentage of mda release of erythrocytes in vitro, showed an inverse relationship with plasma vitamin e levels and with the plasma antioxidant capacity: preoperative 5.8 + 2.4 % and postoperative descending from 20.5 + 14% to 12 _+ 9.5%. our results indicate that during surgery and within 24 hours postsurgery, a decline in antioxidant defenses occurs, in the postoperative period, while patients are on pn and taldng into account our obtained data: the unaltered mda values, the rise of plasma vitamin e levels, the recovery of both functional erythrocyte membrane antioxidant protection and the plasma antioxidant activity, they do reflect that during the intravenous administration of lipid infusions, oxidative damage does not occur, probably due to the addition of vitamin e to pn solutions in order to avoid autooxidation of lipid emulsions. garnitine is one of the essential nutrient in the diet of newborn infants. mother's milk represents the natural eamitine source for the newborn infants. we studied enteral earnitine intake in 71 premature infants 35 eutrophic (eu) and 36 hypotrophic (hy), fed with mothers milk trough 5 consecutive days-from fifth to tenth day of life. mean gestational age was 33 (range 32-36) weeks for eu and 34 (range 32-36) weeks for hy. birth weight was ranged 1430-2450 g for eu and 1250-2300 g for hy (p < 0,05). breast milk carnitine concentration was 78,88 i.tmol/i in mothers milk delivered eutrophic babies, and singnificant higher mean coenetration 113,88 l.tmol/i, of hypotrophic premature infants (p<0,05). breast milk earnitine concentration was ranged between 39,15-163,71 ~moi,'l, and daily carnitine excretion was between 9,7-165,48 p.mol/i, mean 60,6 +-38,7i p.molll. the daily mean carnitine intake was from 1-1,93 mgkg/d, in eutrophic and 1,23-1,93 mg/kg/d, in hypotrophic premature infants, in milk volumene intake from 150-200 mllkgld., the results of this study suggest that premature infants should be fed with own mothers mitk in eady neonatal pedod in attempt to prevent camitine deficiency. keto-acids and parenteral lipid admlnistration. castorina m., antuzzi d., ricci r., rendeli c., polidori g. pediatric intensive care unit -catholic university -roma (italy). some metabolic studies have suggested that the administration of mediumchain tltgticerides (mct) might induce a marked increase in ketone body concentrations; the aim of this study is to evaluate the effective ketogenetic risk of infusing mct emulsions in total parenteral nutrition (tpn). two groups of seriously infected children were examined: a-12 septic infants were given to tpn with a 100 % of long-chain tryglicerides (100 lct). b-10 septic infants, in wich the lipid source in tpn consisted in a 50-50 mixture of lct and mct. the children in the different groups showed similar severity and therapeutic scores (prism, tiss, ~, f~2, f~3); the lipid intake was the same, corresponding to 1-1.5 g, ckg bodyweight/day. in all patients a sample of urine was taken six times a day for the whole time of tpn the urine samples were screened by the diphenylhydrazone test, and the ketoacids excretion was confirmed by a chromatography. the urinary excretion of 13-keto-acids equivalent to p-ohphenyl-pymvate (mcg/ml urine) is summarized in the no significant increases in frequence and/or in severity of acid-base disturbances were found in all studied patients in consequence of the lipid infusion. the patients of b-group showed a lower excretion of 13-ketoacids. the c~-keto-acids excretion was ever unsignificant and seemed to be correlated with the illness severity more than with the lenght of the tryglicerides chain. this observations let us suppose that the mct, at employed doses, can be safely administred also in childen with metabolic acidosis and/or serious illness. reye syndrome cayetano heredia hospital, lima, peru five patients with reye syndrome were studied; included were those diagnosed from january 1991 to march 1994 and treated at cayetano heredia hospital. the age of presentation varied from z.5 to 7 months. the syndrome occurred more frequently in males (4/5); the time of illness at the presentation varied from 2 to lo days, and the following features were found: fever (3/5), akeration in mental status (5/5), seizures (4/5), gastrointestinal illnesses (diarrhea) (4/5), and respiratory insufficiency (1/5) --in this case ventilator)-support was needed, in all cases hepatomegaly, intracranial hypertension, hypokalemia hyponatremia, metabolic acidosis, hyperammonemia and elevation of hepatic enzymes were found. coagulation blood tests were abnormal in three patients. cerebrospinal fluid (csf) showed hypocellularity in all cases (less than 8 cells/ram3). the cultives were negative, and the final diagnosis was confirmed by hepatic biopsy. no deaths were due to reye syndrome. a contribuhon to the study of reye's syndrome the aulhors analysed 38 records of patients meeting all the clinical, biochemical and anatomic criteria put forward by hutrealoch~ and samaha (1975) who were ireated at the emergency and resusdlation depammlt tithe ipch in 10 years. the average age of the children was 3 years (the 3~umgest 11 monks and the driest 7 year~ most of lhon (34/38) came from lhe countryside to lhe lmfia~ willfin 24 hours of file oulbreak. a few days previously the patients w¢~ld have fever (some of ahem wilh diarrhea or cough) ,,rod vomit then rapidly fidl into coma and c~nvuksien. they ~me to the imtia~ with the signs of typical increased inlracranial pressure ( stages 2-4 according to lovejiy for most cases) but the liver was unllkely big. the main biocheafical dmnge of the blood was acute hepatic thilure -the bllod glucose decreased : (ha the aver~ 23,46n~% (26) ~ not measured in five cases (glucose a"aces). -the blood ammonia ino~ased riven 158 to 275 microg/dl fin lhe average 220, n = 3) -the percentage of p~in was low, in the average 44,5% (n--9), 11% at the lowest (a case ofmekaa lasling five days was ctwed with vitamin k). -got and gpt enzymes increased 3 -4 times (n=14) besides, tht~re was decompcmated addt~is with lhe average valnes ph = 7.19; pcoz = 2~6mmhg and eli = -1~6 ~, (n~). the charadaislic dumge in the ¢~'ebrospinal fluid was a low glucose tx~acenwalien h the average of 1k65mg/dl (3 cases of glucose it'aces) ; meanwhile protein decreased in the average of 14mg/dl (4 cases below 10mg/dl). besides ~here was no inflammatory lesion ~tion) : very serious brain oedona and fatty degeneration of live~ scatteredly or parllally.microso~ically (biopsy and necropsy) in file brain appeared bright space around blood arteries and glioma extended v'lrdmw -robin space, with no inflammatory reaction. liver.-the sa~dure was intact there was no " "mtlammal~s3' cell, the kupffer cells did not show hyperplasia but show a heterogenous deg~nerafion~eart: also showed a fatty degencralion in some areas of the myocard eellkidney: the ihtty degenerati~l scattetedly in the tubular cell. pano~as:fatty degeneration scattetedly in file pancreatic cell. two cases of liver biopsy for lhe 2rid lime (check before leaving file inslitute) found that fatty degeneration was r~tta~ nearty ff~pletely. the exact muse remained unknown. objectives: to analyse whether there exists serum and urine electrolyte disorder in children with respiratory failure, methods: we have made prospective study of 48 children in our icu during a 12 month period, electrolyte concentrations were measured in serum and urine collected during 24 hours (sodium-na, potassium k, chloride-ci, calcium-ca, magnesium-mg and phosphorus-p). results: average values in serum were: na 139.28 +/-3,20 (rv:133-146) mmol/i, k 4.22 +/-0.55 (3.3-5.2)mmol/i), cl 95.20 +/-3.98 (93-106) mmol/l, ca 2.t2 +/-0.12 (2.20 2.65)mm01/i), mg 0.82 +/-0.22 (0.77-1.12)mmol/i. average electrolyte levels in 24 hours urine were: na 64.23 ~/~ 44.11 (42-202)mmol/day. k 22.14 +/-10.33 (22-112)mmol/day, ci 56.14 +/-27.22 (100-244)mmol/day, ca 1.17 +/-0.13 (1.68-6.8)mmol/day and p 11.89 +/-7.12 (11.7-32)mmol/day. conclusions: average serum ci and ca levels were decreased in children with respiratory failure. in urine, average k, ci, ca and mg levels were decreased and urine p concentration was increased. we concluded that serum and urine electrolite disbalance may be expected in children with respiratory failure. the nurse is going to kill me! (icu syndrome) anita duvndam ~, sonia v.d,sluis 2 ~dpt. of pediatrics, div. pediatric intensive care, sophia children's hospital, rotterdam 2dpt. of pediatrics, div. pediatric tntensive care, juliana children's hospital, the hague. content description: this presentation will provide the critical nurse with background information concerning the identification, the prevention and management of the critical ill child who has developed the icu syndrome. the results of a questionnaire concerning the occurrence of icu syndrome on a picu will be presented. behavioral objectives: at the end of this session the participant will be able to: 1. describe four clinical symptoms of the tcu syndrome, 2. identify major sources of picu environmental stress, 3. discuss nursing's unique role in helping the child to cope with the icu syndrome, 4. discuss nursing's unique role in preventing icu syndrome. content outline: the icu syndrome is a situation in which the individual is not able to deal with changes in observation and interpretation of both quantity and of patterns of sensory perceptions. in other words the borders between the inner and outer world becomes unclear for the person. the exact incidence of the icu syndrome in the critical care setting is unknown, we have limited knowledge of the occurrence of the icu syndrome in critical ill children. yet critical ill children in our hospitals sometimes show the symptoms of icu syndromes. to be able to identify the occurrence of icu syndrome we developed a questionnaire. the thesis of the questionnaire was: does the icu syndrome occur in critical ill children in the age between three and ten, who have been intubated and/or ventilated in a picu? eighteen questionnaires have been send out to parents of critical ill children in two hospitals. the response was 90 percent. conclusions: 1. most of the children were anxious (n=11), showed regression (n=8) or had sleeping problems (n-lo) during the stay on the picu. 2. children showed the same problems after discharge. 3. there is no relationship between environmental factors and symptoms of the tcu syndrome during the stay. 4. there is no relationship between preexistential behavior and symptoms of the icu syndrome during the stay. because the lack of a scientific definition for the icu syndrome in children and good criteria for diagnosis, we were not able to get an answer to our thesis. more research is needed. continuous veno venous haemofiltration (cvvh) was adopted as a first line renal replacement therapy in our paediatric intensive care unit (picu) some three years ago. ~/he process of introduction and development of cvvh proved to be an exciting challenge for nurses as indeed it was for the whole multidisciplinary team involved. we have successfully used cvvh in the treatment of over 20 infants and children, weighing between 3-85 kg. the range of conditions treated is ever increasing. to date we have not only used cvvh for patients in renal failure and fluid overload, but also to gain biochemical control in tumour lysis syndrome-and metabolic disorders. other distinct patient benefits in comparison with more conventional means of renal replacement therapies are that, it is a continuous controlled treatment being extremely effective in creating 'space' for nutrition, which is especially important in the fluid restricted, catabolic patient. of paramount importance in providing this level of service is the education and training necessary to impart the skills and knowledge for nurses to become expert practitioners in this field. we now have a teem of highly advanced practitioners who have developed their nursing skills to provide even more holistic care for their patients. this group are embracing new challenges and responding positively to the developing service whilst expanding their knowledge base. this paper will discuss the advantages and disadvantages of cvvh as we have experienced, relating i t to the wide variety of patients we have treated. we wish to share how cvvh has become an accepted role of the picu nurse and how the service has been successfully implemented. integrated care pathways (icp's) define the optimum course of events in the care of a patient with a specific condition, within a set timescale. aims and methods: icp's and case management (cm) were introduced to evaluate and improve clinical practice. icp's were developed for patients undergoing surgery for atrial septal defect (asd) ventricular septal defect (vsd) and fallots tetralogy. they were based on the median experiences of the last 31 patients. 108 patients have been managed using icp's. results: analysis of variation from the icp's shows a reduction in the following potentially avoidable causes of variation: delay in extubation has been reduced from 29% to 10%, and 31 patients (29%) were extubated earlier than the median. prolonged stay on the intensive care unit (icu) has decreased from 13% to 0%, and 24 patients (22%) were discharged to the ward a day earlier than the median. the number of patients receiving inadequate post operative analgesia has decreased from 35% to 15%. delayed feeding after operation has been reduced from 52% to 28%. unavoidable delays in extubation and discharge from icu occurred in 22 patients (20%) because of haemodynamic instability" or lung problems. cm utilising individualised pathways for these patients has reduced variation in care which can improve patient outcomes. pathways have been developed for other conditions and it is anticipated that approximately, 80% of patients will be treated using an icp revision of icp's results in continuous evaluation and improvement of the care provided, conclusions: the use of icp's and cm has improved patient care and decreased avoidable delays and variations. the future development of other icp's, combined with a case managed model for selected patients, is seen as a viable method of continuously improving patient care. this paper is a retrospective audit of 24 children who received continuous veno venous haemofiltration (cwh) whilst on the paediatrie intensive care unit (picu), data was collected over a 21 month period from may 1994 to january 1996. the 9 girls and 15 boys were aged from 1 day to 15 ½ years (median 4½ years) and weighed between 2,5 and 85 kg (median 17.75 kg). length of admission to picu was 2 to 47 days (median 9 days). sepsis syndrome was diagnosed in 11 cases: 4 of these had bowel necrosis and 3 meningoceccal disease. eight patients had an underlying haematological, oncolngical or immune disorder. the remaining children had inborn errors of metabolism (2), nephrotic syndrome (2), or cardiac failure (1). cvvh was instituted for a variety of reasons. muttiorgan dysfunction was present in 46% of patients, 29% had acute renal failure, 12.5% tumour lysis syndrome, 8.5% uncontrollable metabolic acidosis with hyperammoneemia and 4% required fluid management. treatment was continued for between 4 hours and 26 days (median 4½ days). haemofiltration was successful in achieving its desired effect in all except one patient. 87.5% of children with haematological, ontological or immune disorders died despite successful haemofiltration. survival was higher in cases of septicaemia (45.5%), nephrotic syndrome (50%) and errors of metabolism (50%). overall mortality was 62.5%. this was attributed to the severity of the underlying disease process rather than the effectiveness of cvvh as a renal replacement therapy. asynchronous defibrillation and synchronized cardioversion deliver direct currents of high amplitude through the chest, to stop ventricular fibrillation or to convert life-threatening arrhythmias. since the human body is partially conductant, it should be possible that after a brief delay of time, a derivation of this monophasic defibrillation pulse is measurable in regions of the defibrillated body, other than the traject between anodal ("sternum") and cathodai ("ape~;") paddles. one could also estimate that health care personnel in the immediate environment of defibrillatoty shocks, are always at risk to possible electric injury. accidents might occur when health care personnel do not stand clear from the patient during firing and create an additional electric path from this patient through their own body. most likely, the nonisolated parts creating a transversal eiectric path, will be the hands of the caretaker. since defibrillation generates touch voltages up to 5000 v and 60-70 a in ved, short delay's (5 to 10 millisec), the total body resistance during skin to skin or skin to paddle contact, is calculated to be as [ow as 750 ohm (percentile tank 50 for the entire population). an experimental protocol was developed to evaluate the conducted currents during defibrillation. mature pigs with a weight of 100 up to 150 kg. were used as animal models. the:,, had barbiturate anaesthesia, inotropic support with dobutamine at l 0 micmgrams/kg bodyweight and all had a sinasat rhythm at the begining of the test-rounds. synchronized defibrillation at 100 j (approx. 0.75 j/kg) and asynchronized -at 360 j (whenever ventricular fibrillation or ventricular tachycardia occurred), were attempted, through latero-lateral placed and firmly pressed defibrillation paddles and/or adhesive multi-function electrodes. gel pads were choosen as contactmedium to cross the skinpaddle barrier. subdermal electro-myography needle electrodes were put at different parts and regions of the pig's body, but not between the paddles. these electrodes were coupled to a resistance device of 800 ohm and a measuring computer. we measured monophasic and low current pulses up to 0,10 a during the defibrillation burst. thus conducted currents are high enough to produce accidental electric shocks when additional electric paths are created by the caretaker. "llaese are usually harmless, but in some "worst case" (wet or transpiring hands, rings,...) or in association with defectuous equipment, peak currents might be harmfull, and can produce pain, bums, apnae or cardiac (transient or persistant) arrhythmias. maria skogbv. karin mellgren, lars-g6ran friberg, g6sta mellgren, hans wadenvik. g6teborg, sweden. bleeding complications in extracorporeal circulation is partly due to perfusion-induced platelet dysfunction. the aim of this study was to evaluate an in vitro model for investigation of platelet function parameters in an extracorporeat system. study: two complete extracorporeal life support systems were perfused with fresh heparinized human blood for 24 hours. piatetet membrane glycoprotein changes, platelet granule release and platelet count were followed. eight paired experiments were performed. one of the circuits was perfused by a roller pump (polystan) and the other by a centrifugal pump (biomecicus), other components were identical. results: a continuous increase in glycoprotein (gp) ib-negative platelets was seen in both circuits. a marked increase of plasma beta-thromboglobulin concentration and a decrease of the intracellular pool of serotonin was observed, indicating a marked release of alpha as well as of dense granules. the plasma concentration of glycocalioin increased in parallel with a reduced platelet surface expression of gpib, suggesting that the loss of gpib is caused by proteolysis rather than by a downregulation of this receptor protein. conclusion: 24 hours of ecls perfusion induces pronounced platelet degranutation and causes changes of important membrane receptors. this is in accordance with clinical studies, suggesting that our in vitro model does mirror the platelet exhaustion observed in a clinical reality. no significant difference was observed between the two pumps. the purpose of this study was to examine signs of distress exhibited by several patients following the discontinuation of opioids and benzodiazepines (o & b) in a 7-bed pediatric intensive care unit. the authors compiled a list of all cases of possible withdrawal reactions reported by nurses in the study setting over a one-year period. five cases were selected for study in terms of their wide diversity of relevant circumstances. the 5 cases were examined through a retrospective chart review. data pertaining to analgesic and sedative administration, along with nurses' reports of behavioral distress, were transcribed and coded. a remarkable pattern of behavioral distress was clearly associated with discontinuation of o & b. cessation of benzodiazepines was associated with severe distress. these reactions were characterized by various combinations of crying, irritability, tremors, grimacing, gagging, vomiting, or feeding problems. some of these persisted in spite of large amounts of bolus drug administration. these signs were manifested for 2 to 9 days following cessation of o & b. these findings demonstrate that the rapid weaning of o & b infusions, sometimes following short-term therapy, can cause severe withdrawal reactions. the particular course that a specific patient will follow will likely be modulated by underlying manifestations of "icu psychosis" and unresolved pain and emotional distress. hermana tezano8 mt, pilaf orive j, latorre garcia j, lizarraga azparren ma, lopez bayon j ucip hospital de cruces. bilbao. spain a female child, one year old, was referred to our unit from another hospital because she had ischemic syntomsjn her right forearm wich started three days before. she had ~i downs syndrome and fallot's tetralogy with a systemic-pulmonary by-pass. nine days before the admission in our hospital she was undergone to a cardiac catheterization by arterial and venous femoral punctures with no incidences. on the admission to the picu her forearm was cold and pale with absence of cubital and radial pulses and slow capilary filling. treatment with heparine was started unsucessfully in the firsts 24 hours, so uroquinase was added that later was changed to rtpa plus brachial plexus blockade. by the time me arterial tlux became more and more evident (doppi~) il bccuute aiat) evident a regional aedema witch made necessary a fasciectomy &the wrist. she also developped necrotic delimitted areas that included her first and fifth fingers. she was discharged from the picu after 13 days and a week later she went to the operating room where an amputation of the distal phalange of the fifth finger was made. the etiolgy of this ischemic picture can't be attributed to the catheterization itself, but probably it should be due to some attempts in arterial puncture in its course, attempts that could be guess by the little marks noticed at the elbow flexure when she was admitted. long maintenance of cardiovascular funtion by assisted ventricular device with membrane oxigenator hermana tezanos mt, pilar orive j. latorre garcia j, lizarraga azparren ma lopez bay6n j. ucip. hospital de cruces. bilbao. spain a little girl of three and a half years came to the intensive care unit from the operating room. affected of great vessels transposition with a pulmona~' banding corrected vith rastelli technique, it became impossible to discharge her from the by-pass circulation. it was assumed that this factt was due to a transient myocardial disfunction dfter the surgeon x~ent through the technique and found no wrong step in it. at the picu admission she had an ejection fraction belox~ 20% (echncardiografy), and was manteined on veutricular assistance with a circuit consisting ofa plate's membrane oxigenator, a bio-medicus pump and pvc rubber tubes, with monitoring of pulmonary', right and left atrial pressures. initially she needed a ventncular support of 1.2 l/min with dopamine. dobutamine and adrenaline; gradually the ejection fraction rose to 60% aliowing a decrease in the mechanical and inotropic support. the organic function ,.'.-as preserved but x-ray of the thorax showed images of pulmonary aedema with an increase in the 02 needs until a fi02 of 0.4. even though she was placed in a transplant program, she was remouved when the ejection fraction rose to 50% on the 1 lth da 3 of the evolution. there were no signs of infection (profilactic coverage). on the fifhteen postsurgery day. with a good cardiac sound and an ejection fraction of at least 60%. it was considered the withdrawal of the ventficular support, but unsucessfully despite the increase ofinotropic drugs, so she died. we think that our patient is a good example of the posibiti~ to mantain x~ith ecmo during a prolonged period of time a potential transplant receiver without major complicatios. joan wierema; ma0)an mourik. sophia children's hospital, department of pediatric surger,j, rotterdam, the netherlands. the success of an ecmo program is heavily determined by the organizational structure and the daily availability of both well trained nurses and physicians. until now there is no general guideline to determine the optimal training schedule to set up an ecmo program. objective: to evaluate the set-up of an ecmo program in an area without direct availability of perfusionists of cardiothoracic surgeons. description of the progress: during the preparation of the ecmo program which started november 1991, different phases are identified. first phase, acquisition of experience by training abroad of one physician, staff member of the icu, physician acting as a fellow and one icu nurse with theprimary task to serve as an ecmo coordinator and train the nursing start. in the second phase 12 animal experiments were performed by 2 paediatric surgeons, 2 staff physicians, 2 fellows and 12 nurses. third phase, start of the actual ecmo program, priming by one trained nurse and ecmo fellow, daily care of the patient by 2 nurses, 1 icu nurse taking care of the patient related activities, 1 ecmo trained nurse taking care of the circuit. fourth phase, transition of the tasks for priming the system from the ecmo fellow to the ecmo nurse and changing daily care for the ecmo patient now including the circuit by 1 trained icu nurse with special legislation for ecmo. ongoing training of 6 to 8 nurses working for at least half a year in the icu. during an ecmo run direct contact between the ecmo nurse and one of the staff members; patient data: in a 4 year period 52 neonatal ecmo cases were performed with an overall mortality of 74%, ranging from over 95% in meconium aspiration to 45% in congenital diaphragmatic hernia. in 7 other patients pediatric ecmo (age range 1 month -14 months) mortality was 50%. conclusions: ecmo can be established without perfusionists or cardio surgical back up once a predetermined training schedule is available, resulting in comparable results with the international ecmo registry both for neonatal as well as for pediatric cases. mechanical ventilation at home makes normal development of the child with chronic respiratory failure possible. the parents must be encouraged to accept the treatment at home; therefore, they should be enabled to take care of their children all by themselves and the continuous professional support has to be offered to them. it must be stressed that the nurse is the link between the diseased child and his family introducing them into the process of the medical care at home. all the equipment needed for the mechanical ventilation of the child at home (ventilator, suction device, pulse oxymeter, oxygen tanks) has to be provided before the child is discharged. it is paid by the national insurance company. the maintainance service workers will make the necessary installations available and they will renew oxygen and compressed air weekly. the ventilation assembly must not interfere with the childs activities. the related dispensary and nursing service should be notified of the child's condition and the list of the required material (suction tubes, cannulas, desinfectants etc.) has to be made. our experience is so far -from the nursing and medical point of view -satisfactory, however the social status of the parents needs further regulations, omphalocele. mourik m, sophia children's hospital, department of pediatric surgery, rotterdam, the netherlands. in patients with (giant) omphalocde operative closure is impossible due to the lack of intra-abdominal space and a variable degree of pulmonary hypoplasia. consequently conservative treatment allowing epithelialization is the treatment of choice with a high risk of infection and/or sepsis due to a sometimes very long stay in the icu. this conservative treatment consists of daily application of an antibiotic containing powder on the cele which is covered by sterile gauzes. furthermore the enteral intake is started as soon possible. objective: to evaluate whether this treatment renders a high risk for serious infections. patient characteristics: in a 10 year period 23 patients with a giant omphalocele were admitted; mean birth weight 2800g (1500-3990g) mean gestational age 38.5 (33-42 weeks). overall mortality 6 (19%). 11 patients had to be ventilated for a median duration of t6 days (2,5-164). results: patients were discharged at the mean of 68 days following birth (17-260) following complete epithelialization of the omphalocele. following the initial stabilisation period parents were involved in daily care of all patients even during artificial ventialation. enteral intake was started at a median of 2 days (1-18). colonization of the omphalocele was observed within 2 -4 days in all patients. although infectious periods were observed at regular intervals, sepsis was the primarily cause of death in only 3 of the 6 patients who died, in conclusion: nursing care to prevent sepsis in patients with (giant) omphalocele is feasible for a long period of time and can be performed with simple measures, since june 1994, we have been using nasal cpap in the treatment of respiratory disorders in newborn infants (severe apnea syndrom, tachypnea, hyaline membrane disease). in newborns presenting hyaline membrane disease, we have used cpap and administered surfactant therapy to 25 premature babies out of 70. clinical data were : mean ga : 30.8 + 23 weeks. mean bw : 1686 + 551 g. mean administration time was 11 hours. before administration, mean fio2 was 0.4 + 0.07, mean pao2 6.2 + 1.8 kpa and mean a/ao2 ratio 0.21. all babies were intubated for administration of surfactant (curosurf) and were extubated after half an hour to 3 hours after administration. this treatment failed for seven babies and they were ventilated by oscillation ventilation ; all babies survived. complications : we have observed pneumothorax in 2 cases and 1 cerebral hemorrage. mean duration time of nasal cpap was 71 hours for the 18 babies without assisted ventilation. nurses in charge of babies with nasal cpap should be aware of neonatal care, of the possibility of surfactant administration and of complications during this type of treatment. therefore nurses should know very well the use of nasal cpap, the monitoring during this treatment, the fixation of the system on the baby, nursing of the babies during this treatment and finally should take care of the baby physical and psychological well being. as a conclusion, nurses in intensive care neonatal units should know the possibility of treatment of rds by nasal cpap and should be aware of baby-nursing. emco in the postcardiotomy infan_t : a simplified circuit and reduced management grillet lsabelle, bosson christa, goelz andrea, helmer pascale, icu nurses, tschaut rudy perfusianist aldo castaneda institute, clinique de genolier, suisse to improve postoperative survival of infants with repaired cardiac tessions but severe residual cardiopnlmonary dysfunction, emco was used in 4 infants, ranging in age from l0 days to 9 months and in weight from 2,8 kg to 7,8 kg. diagnosis included tga / intact ventricular septum (2) (both subiected to rapid arterial switch operation because of unsuitable lv function) and tga +vsd (2). "['he emco circuit included a sarns roller 15ump, a medtronic minimax plus hollow fiber oxygenator and tubing (1/4 -1/16 inch) with bioactive surfaces, an arterial canula 10 fr., 2 venous canulas : right atrial 20 fr, left atrial 12 fr., an air oxygen mixer and a sarns heater. the act was kept between 180-200 sec. after the infant was connected to emco by the surgical team and the perfusion technologist, the patients were being cared for and controlled exclusively by the 1cu nurse assigned to the patient and the physician on call for the icu, pump flows of 100-150ml / kg / mln were targeted to insure an adequate urine output, a brisk capillary fill and physiologic left and right atrial pressures. sodium nitroprusside was used to control systemic hypertension while all other inotropic drugs were discontinued. inspired oxygen fraction on the ventilator was reduced to 0,25 at a respiratory rate of 10-15 breaths per minute. body temperature was maintened close to normal. when the heart started to eject again, inspired oxygen was increased to about 0,40. the preparation for weaning from emco was done by the icu nurse: including oxygen fraction and ventilator rate. weaning from emcowas coordinated by the medical / surgical team and the perfusion technologist. the duration of emco in the 4 patients ranged from 2 to 9 days. three patients (75%) were saccessflly weaned from emco. one of the three died within 24 hours from neurologic complications, unrelated to the emco. the other 2 patients ( both with tga + vsd ) are long term survivors and are doing well. neither the survivors nor the patients who died bad hemorrhagic complications, despite the fact that 3 of the 4 were placed on emco because of failure to wean from cardiopulmonary bypass. the simplified emco circuit worked reliably throughout this experience and no complications occurred nor could any shortcoming be ascribed to the use of this reduced, cost effective management team. 10 years of praciical experience wiih exiracorporeal membrane oxygenation (echo) -the viewpoint of the nursing siaef monika schindler in 1985 a training program was started for the introduction of echo in the kinderklinik of mannheim/germsny. 2 years later (1987) the first european echo patient, a 3 days old newborn baby, ~as treated successfully in our institution. with echo, more treatment modalities ~ere available after failure of conventional therapy in eases of severe lung diseases like meeonium aspiration syndrome, congenital diaphragmatic hernia, sepsis/pneumonia, primary persistent pulmonary hypertension of the newborn and -in infants and children -ards (acquired respiratory distress syndrome) due to multiple underlying illnesses. in 1989 the first ards patient, a 5 years old girl, was treated successfully ~ith ecmo in mannheim. until now 137 patients (age: i day -10 years) were treated with echo in our institution; 111 additional patients were transferred to us from other institutions for therapy with echo, but could be treated ~ieh alternative moda]ities of therapy like improved conventional ventilatory support, high frequency oscillatory ventilation (hfov) and inhaled nieric oxide (no), unexpectedly the more ~ide-spread use of hfov and no -also in other ~ntensive care units -did not lead to a reduction of the echo frequency in our hospital. due to this long ecmo practice, which is helpful in many critical situations, a certain routine came up in the nursing staff of our intensive care units; but ecmo continues to be a maximum strain for all co-workers and implies an high personnel and material/financial-expense. but in our opinion the improved survival rates in severe respiratory failure-(70 % in neonates and 50 % in infants and children, estimated survival rates under continued conventional management: < 20 %) by the use of echo justifies this expense, even in times of less financial support for public health. • analyse the differences between each urdt. method : ni criteria were defined by the rt~a ped group according to cdc criteria. all data were collected by a nursing and medical team. data 3vere dealed with epi info software. all u-dections data were validated by an external mvestigatol : 4 525 patients who stay more than 48 hours in an icu were included over a 14 months period. 68 % were newborns, 19 % infants and ,13% children. 371 ni were idenlified among sll patients, the overall ni incidence rates was 8,2 % and 5,9 person day, septicemia was the first cause of ni (50 %). staphylococcus epidermidis were isolated in 60 % of septicemia cases. pneumonia was the other main ni (41%) with gram negatwe bacille isolated m 53 % (40 % of them beei,ng fseudomonas) accordmg to age, the septicemia mcidence rate varied from 6,8 ~., to 10.9 %,, catheter/day and pneumonia incidence rate from 3,9 %, to 7,3 % ventilation/daywith the lowest rate for newborns. : this survey was possible thanks to the collaboration between laboratories, bacteriologist, physicians and nurses, and allowed each concerned unit to work together in~tead of every one in his own field. 'lhe results of this survey bring changes in attlhades and empower the different team work the next step is to analyse the ditferent nurses procedures of indwelling central venous catheter in each unit ar)d implement a ni quatity care. program in all nicu anti picu. v~,~jl~j~'v~y : new born in~ra-v~6nous drugs ~md lheir pr~taration mcounter a lot of risk ~)f utters, due to inadapted conditionn~ng to podia)eric and minimal amount injec~.~d, th~ study t~ed to evaluate degrees of do.~age er~o~ dye tt) dru~ preparaek, n i~ nc,:,~atol~gy and to come out w~th ~ method of dihttion able to reduce risk to minima (errors _< 5 %) at the ~eme t~me, ¢o~,~x-tueaces on cost were studioj methnd~ : amikacine was choosen because this antibiotic is regularly ~,~l in neonatology unit and its dosage is easily used by standardiaud method, 30 amikacine doses (15, 23 mg doses and 15, 12 mg doses) were prepared" by 15 nurse~ form 50 mg for 1 m) vial. each dose w'a~ diluted to obtain 2 mi volume, tallowing usual ward method, for each dilution, amikacine con<entrati('m and it,s volume was measured and used equipment for preparation wa~ regi~tred. 10 "first s01utj(m" were prepared diluting ~t'~ mg amikacine pouder inn 51) ml glucnse solution bag (viaflex baxter bag with transfer cap), from tho~e "first solution" 15 doses at 23 mg and 15 doses of ~2 rng were prepared, amik~cine coc, cenir,)tmn for each preparabon and their volume were measured, ai,~n ntttl~,ber of seringe were tounted. first soiuhon staeiiity was conrroieo aher one week conservation at + 4'>c. amjkacine concentration were measured by fluorescence process (tdx abbott) after sample dilurion. on a 10 mg/l sample, tovhnical reliahility show~ > 9~ % of result mpmductlon and < 5 % of variation due to dilutions. results : when amikacine injection werv pro.pared from araikacme 5/) mg for 1 mt vial > 10 % do~ge, ermr~ were found in 19/40 cases ; ~ 30 % in ,t1,to cases. if preparation is done from amikacine "~it'st soltltion", les.--concenvr~tcd, it i~ more preci,,,e and only one dosage error ~ 5 % (6,3 %} is found in eli 30 studied doses. in add)inn to )hal if 10 doses were wep,m-'d from one "first soiatiol~' bag, the cost economy sl~ouid b~" of 32 fr~, and ii 20 dos~$ were prepared tram the same bag the saving mtmey should be o{ i72 its .cencluslon : .ur survey shows th~t h' ntu)nato|ogy the u~ of a "first sohation which can be kept fi~r one week is enable to reduce dosage erroes and i~ co,~tsavmg, regarding [,v. admimst'rahon method the survey is still on, introduction: so-called vein of galen m~iformations ale rare in~racranial embryologycal anomalies, repl~senti~g tess than 1 of symptomatic intracranied artefiovenoas l~alform~tions. the spontlneous prognosis is ~s~u~lly fatal, because of cardiac frilure due to left-to-right shunt thrq~ugh the fistula. recent developments of new techniques of treatment of the malformation and its cardiac consequence have led to a revolution in the practical approach of children w~th galen malformation. our fukfose is to contribute, with our persoaal series of 7s newborns and infal~ts admitted in our unit after endov~,scular embolization, to a better management of these children. such a management requ!res a rnultidisciplinary approach. intensive care are required prior to embollzation for patients with cardiac failure or cardiogenic shock and after cmbolization in order to insure cardiac and cerebral hemodyna.mic stabilities. this overlooking suppose for the nursing team to understand: prior to embolization : heart failure and cardiogenic shock. after cmbolization : evaluation of neurological and hemodynamic consequences of this proccdure, without forgetting the nursing and psychologic aspects, in concl'iision, this last ten yerrs, these new approaches give to the patients and their famitiy a good reason to hope a total recovew, in our exl)erience, the global mortality is 9 % aad 66 % of children #j-e neurologically normal after embolizafion, ii ii~ i ~ii i ii i i l i iiii~ i ~i iii i background: venous oxygen saturation (svo z) reflects the residuai oxygen after tissue oxygen extraction and represents the relation between tissue oxygen supply and demand. we studied svo 2 and arterial lactate during progressive isovolemic anemia to assess the relation between svo2 and tissue hypoxia. subjects: ten 8-10 day old anesthetized ventilated piglets sao 2 and svq were measured continuously by a fiberoptic catheter (oximetrix, abbott lab.) in the carotid and pulmonary a~epy tissue hypoxia was confirmed by a reduced vo, and an increase in lactate. conclusion: svo 2 reflects better a reduced dp obtained by progressive anemia surfactant replacement improves gas exchange in early-stage adult respiratory syndrome (ards) [1,2], but not in late-stage ards [3] . we report the first case of successfull treatment of ards after repeated instillation of surfactant.a ten year old boy, weighing 32 kg, presented with hemorragic shock. biphasic-positive-airways-pressure ventilation was performed (evita ii, dr~ger, germany). he had recieved nine units of packed red blood cells and underwent surgical exeresis of two bleeding gastric ulcus. post-operatively, a cardiac arrest required cardiopulmonary resuscitation for three minutes. hemodynamic status was subsequently stabilised. the chest-radiograph showed infiltrates of both lungs without signs of cardiac failure. on the third day, the patient became severely hypoxic with a pao2/fio 2 ratio of 30. gas exchange was not improved by high ventilator settings. peak inspiratory pressure (pip) and ventilatory rates were 40 cmh~o and 18 breaths/min respectively. inspiratory:expiratory time was 1:1 and the positive end expiratory pressure (peep) 8 cmh20. after increasing the peep level to 11 cmh20, we instilled over 2 minutes, 80 mg/kg of porcine surfactant (curosurf, serene france), in two equal volumes in both main bronchus,the spo~ rose to 97% within 15 rain, the fie 2 could be reduced to 0.6. twenty four hours later, gas exchange worsened again (pao2/fio2 ratio 90). we increased the peep from 8 to 11 cmh20, and instilled a second dose of surfactant (60 mg/kg). again, fie 2 could be reduced within 15 minutes (spo 2 95; fie 2 0.6.). the patient was weaned from the ventilator and extubated on the tenth day. follow-up at four month showed normal lung function.we demonstrate improvement in oxygenation after repeated exogenous surfactant administrations. we assume that in early-stage ards, surfactant may potentiate shunt-reducing effect of peep as it has been demonstrated in experimental model of ards [4] , and allow decrease in fie2. in case of secondary deterioration, we think that a second dose of surfactant should be administered. 1. weg jg, balk ra, tharratt rs, et al. ,lama 1994 : 272: 1433 -8. 2. spragg rg, gillard n, pdchman p, et al. chest t994: 105: 195-202. 3. haslam pl, hughes da, mcnaughton pd. et al. lancet 1994 343:1009 -11. 4. huang yc, caimulti sp, fawcett ta, et al. jappl physiol 1994 76:991-1001 43% (ref) . the aim of this study was to verify these data: patients/~lethods: all pts admitted to our multidisciplinary nicu/picu in 1995 were included if they were in respiratory failure recruiting conventional mechanical ventilation (cmv) with peep >_ 6 and 'fig2 -:2 50% or high-frequency oscillation ventilation (hfo) with mean airway pressure _> t8cm h20 for 12 or more houm. diagnosis, maximal ventilatory parameters, barotrauma, organ/ system failures, mechanism of death and glasgow oulcome scale (gos) 1 and 6 months after study entry were prospectively collected. results: 685 patients were admitted to the unit, o1 whom 337 required mechanical ventilation for a mean duration of 4.0 days. overall mortality was 5%, 22 patients fulfilled study criteria. 17 survivors had gos 5, 2 pts with preexisting neurological impairment survived with gos 3. neonatal diseases included hyaline membrane disease (7), meconium aspiration syndrome (4) and cardiovascular surgery (1), pediatric diseases included bacterial (1) and viral (5) pneumonia, aspiration (1) and cardiovascular surgery beyond the neonatal period (3). 1990 -1994) . patients and methods: cefotaxim was used as a prophylactic agent in 43 patients in life threatening situations (e.g. multitrauma, neurosurgery atc.). more than 85 % children required cefotaxim for the treatment of severe infections (epiglotitis, meningitis, sepsis, pneumonia mainly in immunodeficient and neutropenic patients) in monotherapy or in the combination with the other antimicrobial agents. results: cefotaxim as a prophylactic drug was successful in all 43 cases (100 %). the effectivity of treatment of infections was 82.8 % (313 patients). the change of antibiotic therapy required 9 patients (2.4 %). 40 patients (10.6 %) died, but only in 12 of them (3.2 %) the obduction confirmed infection. conclusion: we conclude that cefotaxim is very effective and safe antibiotic and represents "golden standard" in the treatment of severe infections in childhood. in order to improve nursing quality, we recently adapted nursing care to the "five nursing functions" (activities of daily living, accompagnment in crisis, treatment, prevention and research) as described by the swiss red cross in accordance to the new educational guidelines of the european community, the aim of this study was to document complications of "treatment nursing function".methods: all treatment complications were prospectively collected by the nursing and medical staff. the nursing staff included patient (pt) name, time of occurence and exact description of complication, proposal for prevention and information of parents. the medical staff reported type of complication together with pt information, diagnosis, medication, treatment and interventions, outcome and referral, all complications were discussed in monthly meetings including nursing and medical staff.results: from january until december 1995, 685 pts were admitted to the picu/nicu for 3233 nursing days (81% of total bed occupancy). 337 pts needed endotracheal intubation for an average of 4.0 days and 47 pts required nasal cpap. 26 complications in 21 pts were noted (1 per 26 pi): inadequate check-up of equipment 11; accidental extubation 4 (1 in 85 intubated pts); bedsores 3; false drug dosing 2; wrong drug 2; umbilical bleeding 2; wrong transfusion setup 1; nasal septal necrosis 1). there was no mortality due to these complications. exact documention of treatment complications and their meticulous discussion within the medical and nursing staff may improve "treatment nursing function". however, documentation and evaluation of nursing within all "five nursing functions" will be nessecary in order to achieve optimal nursing care. cardiac output determination by thermodilution, using iced injectate has been shown to be valid and reliable in pediatric patients. it has been demonstrated in adult patients that there is no difference in cardiac output values when using room temperature injectate as compared to iced temperature injectate. the purpose of this study is to examine the effect of injectate temperature on cardiac output values in pediatric patients. our study consisted of sixteen pediatric patients who had oximetric thermodilution catheters in place after cardiac surgery and who had cardiac output determined using both iced and room temperature injectate. with each patient, cardiac output was measured once on the day of surgery and again the following day. in each case cardiac output was measured using both iced and room temperature injectate. statistical analysis included a two-way, repeated measures analysis of variance for each individual injectate administered and no significant differences were found in cardiac output. no statistically significant differences were found between groups with regard to the order of injectate administration or volume of injectate used (i,e., 3 or 5 cc's). the correlation coefficients between groups for cardiac output measurements at each injectate administration time, and for the average measurements across times, ranged between 0.81 to 0.94 (p < .0005). preliminary data analysis suggests that cardiac output measurements for children are not effected by the temperature of injectate. a lenghty stay at a paediatric intensive care unit will always have sideeffects on a child's well-being and will put a high strain on the parents. in order to minimize the side-effects longterm intensive care unit opened in 1990 at the childrens' hospital. admitted children are all ~ongterm-ill and technically-dependent and the ventilatory support can alter from a tracheostoma to cpap or portable volume ventilator. nutritional support is applied by gastrostomies. a homelike atmosphere surrounds the children, they share a dormitory, a living-room and a dining-room the main purpose is to send the child home with or without technical equipment. this can only be implemented by giving structured education (theory and practice) to all categories involved. the multi-disciplinary team consists of one anaesthesiologist, head nurse, clinical specialist, rn nurses, nurses, one habilitation doctor, one social worker and therapists. twenty-four patients have been admitted to licu during these six years. length of stay was from one day to four years. four are presently staying at the trait. the assessment of pain in children (0-3 yrs) is still difficult, because children of this age have limited language and cognitive skills. to standardize the assessment of postoperative pain and distress in the intensive care unit an observational mstrument was needed that met several criteria. it should be easy to use in daily routine care. be suitable for the i.c. situation, and in children of 0-3 hrs of age. the comfort scale, an observational instrument designed to assess distress in infants in i.c. units, met these criteria. to accommodate the use of the comfort scale in the i.c. units and in research, nurses should be trained to use the scale. an additional requirement was that the inter-rater reliability should be sufficiently high, (cohen's kappa > .60). objectives: 1) to introduce the comfort scale in the i.c.u.; 2) to examine whether this instrument can easily, be incorporated into routine care; 3) to investigate the inter-rater retiabtlity. methods: the comfort scale is an 8-item instrument specifically designed for use in pediatric i,c, units and contains both physiological items (heart rate, blood pressure) and behavioral items (e.g., alertness behavior, calmness/agitation, body movement, facial expression respiratory response, muscle tension). the observation period is 2 minutes. the scale is supplemented with an item on crying tbr children who are not mechanically ventilated. groups of 8 t.c. nurses were trained by means of video's and observations at the wards. after the training, each nurse completed 10 scores with other nurses, after which the cohen's kappa was computed. when the kappa's for the items met or exceeded our .60 criterium, a new group of nurses was trained. results: to date, 30 nurses have been trained. nurses find the comfort scale easy" to administer and a valuable addition to routine care in the i.c. unit. the cohen's kappa's were higher than .60 for all items that the inter-rater reliability was high. the comfort scale is feasible in postoperative care in the i.v. and is considered a valuable instrument to improve and maintain high postoperative quality of care in the i.c. unit. introduction:children with neuro-muscular disease are believed to have a higher resting energy expenditure (ree), because of their increasedwork of breathing.the influence of nocturnal nasal mask ventilation on energy metabolism and nutritional state of these children has not been studied so far.objective:l,ls the ree inereased?2.1s there an influence of nasal mask ventilation on the ree?3.what is the nutritional state?4.what is the estimated total energy expenditure(ete) in relation to the caloric intake? methods:a pilot study of 4 patients(12-16 years) .the following measurements were performed:l.anthropometry.2.bioelectric impedance-3.ree was measured by indirect calorimetry during the day (in bed) with and without nasal mask ventuation,ree was compared with predicted ree according to schofield(pee),4.caloric-intake and activities were recorded during 48 hour before measurement.5.total energy expenditure was calculated as follows:measured ree x estimated activity factor. results:tin all children weight for height was too low, <p3.2.aii children had a low % of fat.3.according to the healthy group, ree is not increased.4,nasal mask ventilation lowered ree in 2 patients, in 1 patient we measured a clear decrease.conclueion:l.anthropometry and indirect calorimetry were helpful in evaluating the nutritional state.2.there is no increased ree,in t patient there is clearly effect of nasal mask ventilation.3.when caloric intake was related to ree and level of activity 2 patients had too low caloric intake.4.the influence of nocturnal nasal mask ventilation and nutritional assessment should be evaluated longitudinally. increasing awareness that the neonate can percept pain and suffer following surgical procedures, has major impact on pain management. in our unit continuous morphine infusion (t0/~g/kg/hr) was introduced from january 1990 as a standard medicatton following surgical procedures. before 1990 morphine was given as a bolus (i.m., i.v., rectal) of 100/~g/kg up till four times a day. repeated gifts of morphine were only gwen following observation by the nurse that the child experienced pain. aim of the study: to evaluate pain management before and tbllowing introduction of a continuous morphine infusion for postoperative pain in our icu. patients and methods: term born babies with isolated oesophageal atresia and tracheo-oesophageal fistula were included in the study. the total amount of morphine (/~£/kg/24 hours); number of days morphine was given; duration of artificial ventilation was evaluated in a case control study. res ults: 1985 -1990 1991 -1994 central venous catheters (cvc) have been increasingly used also at our picu, university medical centre ljubljana. besides advantages, the cvc has brought numerous risks of complications; the catheter sepsis occurs most often and can be fatal for the patient. for example, in 1995 we dealt with 400 children aged 0 -12 years or in other words 1.14 cvp per a child treated at our picu. a specialized unit for preparing parenteral natrition started to function in the university medical centre pharmacy in 1977. after that we equipped a special room for preparing other necessary infusion mixtures and organized a team of nurses -"catheter nurses".their tasks comprise: managing cvcs, replacing and installing infusion systems and mixtures made at pharmacy, preparing drugs and some necessary infusion mixtures in special room, permanent training and co-operation with related disciplines, pharmacy, epidemiology, microbiology, etc. all registered nurses at picu have been trained for this work. the routine is as follows: in the morningafter the doctor's visit, we first check the cvc and change the dressings; according to the nurse's observations, the doctor decides whether there is a need to remove or replace the cvc. if the catheter site is inflamed, swollen, or purulent, a swab is taken for microbiological culture and the area cleaned. in case of systemic infection signs, cvc has to be replaced, blood culture taken and the infection treated with antibiotics. the dressings are always changed by two tmcatheter nurses together. afterwards the infusion mixtures for the next 24 hours are prepared for each child. the infusion mixtures which will not be used immediately are kept on +4°c; before application they are, of course, warmed to at least room temperature. finally the "catheter nurse" supplies the cvc with new infusion mixtures made at our pharmacy. our observations show that such work requires responsibility and is demanding; but our pemanent care lessens the number of complications. thus the described scheme fulfilled our expectations. perl-0perative management of neonates with hypoplastic left heart syndrome muir warren, blondel maryse, foltz rhonda, farine martine, icu nurses, the aldo castaneda institute, clinique de genolier, switzerland both the pre-operative and post-operative physiology of patient with hypoplastic left heart syndrome is critically determined by the puhnonary-tosystemic resistance ratio. a high ratio results in minimum volume work for the right ventricle but is at the expense of important hypoxemia. a low ratio is reflected in minimum hypoxemia but an unfavorable increase in excess ventricular volume work. a resistance ratio of approximately 1 results in an arterial oxygen saturation of 75% -80%, a qp/qs of 1, and ventricular volume work only twice normal. this latter physiology appears best for oxygen delivery and systemic perfusion with tolerable volume work. carbon dioxide (co2) in the inspired gas is known to increase pulmonary vascular resistance without significantly influencing systemic resistance in the range of 1 to 20 tort partial pressure. hyperventilation with no co2 in inspired gas decreases pulmonary vascular resistance. thus, managing co2 may be useful to modulate the pulmonary-to-systemic resistance ratio, in the tast 16 months, 20 patients with hypoplastic left heart syndrome were managed by a norwood procedur e. 10 patients with endotracheal entubation pre-operatively had co2 added to inspired gas to modulate the pulmonary-to-systemic flow ratio. 17 patients had co2 added to inspired gas post-operatively. all but 1 patient had characteristically large pulmonaary-to-systemic flow ratio with oxygen saturation mean 90 % preoperatively, even in those patients receiving co2, co2 was accompanied by elimination of metabolic acidosis pre-operatively. the most comlnon partial pressure of co2 added post-operatively was 14 torr. po2 ranged from 25 mmhg to 40 mmhg. all surviving patients developed a metabolic alkalosis (10 mean) when treated with co2. there were 4 deaths, one from drug toxicity, one from excessive post-operative hemorrhage, two from sepsis or mocardial insufficiency. only one patient benefited from catecholamine support which can increase systemic resistance unfaborably. in this experience co2 was felt to be an important adjunct in some patients pre-operatively and most patients postoperatively to faborably modulate the critically important systemic-topulmonary flow ratio, to maximize oxygen delivery and systemic perfusion, while minimizing excessive right vantricular volume work. key: cord-006860-a3b8hyyr authors: nan title: 40th annual meeting of the gth (gesellschaft für thromboseund hämostaseforschung) date: 1996 journal: ann hematol doi: 10.1007/bf00641048 sha: doc_id: 6860 cord_uid: a3b8hyyr nan the variable molecular weight (mw) of vwf is due to differences in the number of subunits comprising the protein. it is assumed that endothelial cells secrete large polymeric forms of vwf and that smaller species arise from proteolytic cleavage. vwf has two main properties: it stabilizes factor viii protecting it from inactivation by activated protein c or factor xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. each vwf subunit contains binding sites for collagen and for platelet giycoproteins gp ib and gp iib/i~a. multiple interactions of the multivalent vwf lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. only the largest multimers are hemostatically active. lack of the largest vwf multimers was observed in patients with yon wiuebrand disease type 2a. unusually large molecular forms of vwf were found in patients with thrombotic thrombocytopenlc purpura. proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vwf and thus play an important role in the pathogenesis of vwf abnormalities in some patients with congenital or acquired disorders of hemostasis. we have purified (-10,000-fold) from human plasma a vwf degrading proteas¢ using affinity chromatography and gel filtration. the proteolytic activity was associated with a high mw protein (mr -300 kd). vwf was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of 1 m urea. proteolytic activity had a ph optimum at g-9 and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. inhibition by chelating agents was best reversed by barium ions, the observed properties of the vwf degrading enzyme differ from those of all hitherto described pretenses. analysis of cleaved vwf showed that the peptide bond 842tyr-g43met had been cleaved -the same bond that has been proposed to be cleaved in rive. the endothelium releases the vasodilator nitric oxide (no) and the vasoconstrictor endothelin (et)-i. no is formed from l-arginine via the activity of constitutive nitric oxide synthase (cnos or enos). an inducible form of nos (inos) is activated by cytokines. no activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cgmp which induces relaxation or platelet inhibition, respectively. in vessels, no is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, atp and adp. infusion of no-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. within the endothelium, no inhibits et gene expression and release of the peptide via cgmp. hence, no-induced hypertension is associated with increased plasma et levels. et, a 21-amino acid peptide, has potent vasoconstrictor properties via eta-and in part etb-raceptors on vascular smooth muscle. in endothelial cells, et activates etb-receptors linked to no and prostacyclin formation. under basal conditions, little et is formed, but is increased by thrombin, angiotensin ii, arginine vasopressin, cytokines and ox-ldl. et antagonists have been developed and allow to study the effects of et in vivo. et and no most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. clinical trials to further define their role in these disease states are now under way. in summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. in disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. pharmacological tools aiming to inhibit those changes are now being developed. j.m. harlan, r.k. w/nn, s. sharar, and n. vedder universit3, of washington, seattle, washington ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion. this reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. we have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. the current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~4ewed. an amidolytic assay for the determination of activated protein c (apc)-resistant factor va (fva) has been developed. this assay measures the cofactor activity of fva in diluted plasma samples via the rate of thrombin formation. the apc response is calculated from two fv determinations: one performed in the presence (apc-fv) and one in the absence of recombinant apc. the apc-fv activity is expressed as percentage of the initial fv activity and indicates the sensitivity of fva to apc. normal ranges were established by analysing plasma samples of 100 healthy individuals and an apc-fv activity above 60% was found to be indicative for apc-resistance (apc-r). in a control group of 34 patients the apc-r assay gave abnormal results in 15 patients. dna analysis confirmed heterozygous fv r506q mutation in all 15 patients and confirmed the non-carrier status in all of the 19 patients yielding normal results. an aptt-based apc-r assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. one of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. comparison of the assay results demonstrate a correlation of 96% indicating that the assay is independent of the activities of vitamin k-dependent clotting factors. the apc-r amidolytic assays allows specific and sensitive detection of fva-resistant to apc. the assay is performable in plasma samples of all persons in whom the diagnosis of apc-r may be indicated. in patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aptt the apc-r amidolytic assay is helpful to establish the diagnosis of apc-r. dept of pediatrics, university hospitals kiel and mtinster, germany resistance to activated protein c (apcr), in the majority of cases associated with the arg 506 gin point mutation in the factor v gene is present in more than 50 % of patients < 60 years of age with unexplained thrombophilia. to determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (cns, n=4), vena portae (n=4), deep vein thrombosis (n=4), vena caval occlusion (n=4), neonatal renal venous thrombosis (rvi'; n--3), neonatal stroke (n=14), stroke (n=3), arteria femoralis ocdusion (n=2). four ont of these 38 patients showed a positive history of unexplained familial thrombophilia. apcr was measured in an activated thromboplastin time (afit) according to dahlbtick. the results were expressed as apc-ratios: clotting time obtained using the apc/caci2 -solution divided by dotting time obtained with cac12. concerning the special properties of the childhood hemostatic system, infants and children with apcr < 2 were considered to be apc-resistent only when the results were confirmed in a 1: l 1 dilution with factor v deficient plasma (instrumentation laboratory munich, germany). plasma of 268 healthy children served as controls. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mul i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. consistent with the ap'it based method 10 out of 19 children with venous (v) thrombosis and eight out of 19 patients with arterial (a) vascular insults showed the common factor v mutation. additional coagulation defects (autithrombin, protein c type i, enhanced antiphospbolipid igg, enhanced lipoprotein (a)) were found in 30% (v) and 28% (a). furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of 10 (v) and three out of 8 (a) children with thrombosis and apcr. all four patients with a positive family history of thrombophilia (mothers only !) showed the common factor v mutation arg 506 gin. in the control group the prevalence of apcr was 5.1%. the high incidence of additional exengenic factors in children with apcr confirm literature data of previously described inherited coagulation disorders during infancy and childhood: an acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. furthermore, the incidence of 42 % apc resistant children with arterial insults in this study challenge the view that apcr is associated with venous but not with arterial thrombo-st8. 12 activated protein c resistance and plasminogen deficiency in a family with thrombophilia m, zttger 1 , f. demarmels biasiutti 1, ch. mannhalter 2, m. furlan 1 , b.~e 1 1httmatologisches zentrallabor der universit~t, inselspital, ch-3010 bern 2klinisches institut fur medizinische und ehemische labordiagnostik, universit~t wien, a-1090 wien several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. since the recent discovery of activated protein c (apc) resistance due to the factor v rf06q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (tel, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. thus, protein c-or protein s-deficient patients having suffered from te seem to be more likely to carry the factor v r506q mutation than expected from its allelic frequency in the population. we report a family (see figure) in which plasminogen deficiency (0.60 u/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (dvt) at ages of 29 and 31 yrs, respectively, as well as in 5 family members (0.53-0.69 u/ml). out of these 5 plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent dvt at age <20yrs. reinvestigation of this family in 1995 showed factor v r506q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (pe). his father had had postoperative pe; he is deceased and could not be examined. ~, [] plasminogen deficiency ~ ,rll factor v rf06q mutation /~ propositus ~¢ bistory of dvt and/or pe e superficial phlebitis j2~,,~ not investigated "1" deceased even though this family is small for establishing unequivocal association of te with known defects, the two most severely affected individuals with recurrent te at ages <30yrs had combined plasminogen deficiency and apc resistance whereas those with isolated plasminogen deficiency were asymptomatic. these data support the concept of multigenie interactions leading to familial thrombophilia. resistance to activated protein c (apc resistance) is the most common dsk factor for venous thrombosis (vt). in most cases apc resistance is caused by a single point mutation at position arg 506 in the factor v gene (factor v leiden). while ample data in hetarozygous patients have been published, reports in homozygous patients are limited. we studied 29 patients (12 males [m] , 17 females it]) in whom a homozygous mutation had been verified by dna analysis. the median age at the time of the study was 38.8 years (y) (range 9-83 y). twenty-five patients had experienced vt (10 m, 15 f). four patients were discovered dunng family studies and were asymptomatic, three were children (between 9 and 13 y) and one patient was a 62 y old man. in males the first thrombosis occurred at a median age of 38 y (range 21-82 y), in females this was at a significantly younger median age of 26 y (range 17-49 y). twelve of the 15 symptomatic females had taken oral contraceptives (oc, estregen content 0.02-0.ling) for 6 to 150 months (median 71 m) pdor to thrombosis. in 2 women vt occurred during pregnancy, in 1 female it was precipitated by hormone replacement therapy. in contrast, in 8,<10 males the thrombosis happened spontaneously, in 2 males it followed surgery. the sites of thrombosis were dvt in 9 males and 14 females, dvt and pulmonary embolism (pe) in 4 females and 1 male, dvt and caval vein thrombosis in 1 female and superficial thrombophlebitis in 4 males and 1 female. eight females had at least one pregnancy, in total 11 children and 5 abortions. two had thrombotic events during pregnancy and 2 after delivery. all homozygous patients showed apc ratios between 1.12 and 1.68 (mean 1.39 + 0.19). conclusion: patients with homozygous fv leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein c or protein s deficiency. however, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, peg-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. while the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (thromb res 74(3): 193-205, 1994) . while these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of apc is available. sinceapc plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors v and viii, end a pro-fibrinelytic enzyme, by stimulating the release of t-pa fi'om endothelial sites, an inhibition of apc may result in both pro-coagulant state and fibrinolytic deficit. representative thrombin inhibitors (dup 714 -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and feg-hirudin) have been compared for their ability to inhibit apc (american red cross). these bionhemically defined studies in which the remaining activity of apc after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (s-2288, pharmacia, franklin, oh) , demonstrated that dup 714 and efegatran inhibit apc in a coneentrafinn dependent manner (ic~0=0.75 and 8,4 gm resp~tively), hirnlog inhibits apc weakly (60 p2¢i produced only 25% inhibition), while argatroban and ~ have no anti-apc activities, while hirulog, hirudin and argatroban produced no direct enti-apc activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein c, resulting in a functional apc deficit and failure to improve clinical outcomes despite higher dosage. while initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of apc, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and dup 714 also produce direct apc inhibition. endogenous inhibition of formed ape by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of apc and may lead to thrombotic amplification in fully enticoagulated patients. these studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on apc. poor anticoagulant response to activated protein c (apc resistance) causes a significant portion of deep vein thrombosis (dvt) whereas its association with coronary artery disease (cad) and myocardial infarction (md is still controversial. therefore, we investigated 346 recently hospitalised patients suffering from cad with or without previous mi. the cad was proven by coronary angiography. apc resistance was analysed by using the ap'l-i'-based assay coatest apc resistance (chromogenix). eleven patients showed an apc sensitivity index below 2.0 viewed as apc resistance. using pcr technology, the factor v mutation causing apc resistance 11691 g "-) a) has been shown in nine of these eleven patients. this represents 2.6 % (9/346), compared to 8,5 % found in healthy blood donors (8/94). one homozygous carrier (male, age 76) was identified (apc sensitivity index 1.4) who suffered from dvt at age 59. recent angiography demonstrated diffuse cad, no thrombotic events were reported in his family. in contrast, multiple thrombotic manifestations (dvt, mi, stroke) occurred in the relatives of four heterozygous patients. we conclude that the prevalence of apc resistance is rather low in patients with cad. nevertheless, the natural history of coronary manifestation of apc resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. resitanee to activated protein c (apc resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor v leiden pcr based methods are used to identify the crucial point mutation in the factor v g(me. we designed primers in order to identify factor v leiden by allele-specific pcr amplification. amplification specificity for factor v was ensured by the 3'primer fv1001, located at the introng/intronl0 border of the g~ae. one sense and two antisense primers were used in ~vo separate primer mixes specific for factor v arts06 (wildtypo) or factor v otn506 (factor v leiden) yielding 235bp products each. in each pcr reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control (429bp pcrfragment). after an initial denaturation step 10/.tl samples (100rig genomic dna) were subjected to 10 two-temperature cycles fouowed by 20 threetemperature cycles. for visualisation 8p3 of the amplification product were run on a 2% agarose gel presmined with ethidittm bromide. the presence or absence of specific pcr amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. the in~ernal positive control primers it~cate a sucessf-u/pcr amplification, allowing the assignment of homozygosity. in a prospective study 126 p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with pcr -rflp according to bertina et al. the concordance between these techniques was 100 %. in 27 patients a heterozygous factor v ohas06 mutation was detected, whereas one pa6ent with recurrent thrombcembolism was homoz-ygous. no false-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. in addition in 15 samples identified to carry the point mutation by al/ele-specifin pcr anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor v leiden. background: an initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. we compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis. methods: english-language reports of randomized trials vtta'e identified th~ a medline search (1984 through 1995) and a complementary extensive manual search. reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. we assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality. results: twelve of the 21 identified trials satisfied the predetermined criteria. the relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom 20.60% aad were all statistically significantly in favor of low-molecular-wedght hqtmrin. coadusions: low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical im)fil~ the benefits of each ~ shodd ~tabllsbcd separately. unfraetionated hcparin (uh) and low molecular weight heparin (lmwh) are widely used for the prevention and treatment of thrombotic disorders. uh and lmwh induce platelet aggregation in vitro. rgd peptides compete with fibrinogm for the binding to the glycoprotein receptor (gp lib-ilia) of platelets and inhibit platelet aggregation. to inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of rgd peptides, we linked ac-rgdv-ssggs-ahx-yk eovalently to lmwh in a ratio 1:1. the peptide is composed of three regions: a. rgd-gives the specificity for the receptor gp lib-ilia; b..ssggs-ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the gp lib-ills receptor; c. -yk arc functional antino acids for iodination (y) and for covalent attachment (k) to the cattier lmwh. the aggregation achieved with different concentrations of lmwh, lmwh-eonjugate and lmwh/rgd-peptide mixture in a ratio 1:1 was mea.~ared after 20 rain.; maximum aggregation after platelet activation with 10 pm adp was set equal to 100%. platelet aggregation in normal human plateletrich eitrated plasma (prp; 220 000/p.l) was induced by lmwh in a dose ~ndent manner. heparin can induce antbodies which interact with platelets and endothelial cells. this causes thrombocytopenia and thromboembolic complications. hitpatients do need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory prooven hit (hipa-test) with recombinant (r-)hirudin. as these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies himdin antibodies were screened by a sandwich elisa using r-hirudin fixed to the solid phase as antigen. all plasma samples were screened for anti-hirudin antibodies of the igg class, but solar only a suset of samples for lge anti-r-himdin antibodies. 38 of 82 patients (46.3%) developed anti-hirudin antibodies of the igg class. anti-hirudin antibodies were not detectable not before 6 days of r-hirudin administration solar no ige anti-hirudin antibodies were found. none of the patients devdoped thromboeytopenia or allergic symptoms. however, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. in 5 patients the hirudin dosage had to be decreased by 2-3 fold to maintain a stable aptt level, in 3 patients, despite stable r-hirudin maintenance dose the aptt increased to values >100 see.. during the study 4 patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion none of these patients developed any allergic reaction. in conclusion we found a high proportion of anti-hirudin antibodies in hatpatients treated with r-hirudin for more than 7 days. these antibodies seem to have minor clinical relevance in regard to allergic reactions. however, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. therefore, aptt must be monitored closely in patients receiying r-hirudin for more than 5 days a major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. however, thueh a hirudin antagonist is unknown in nature. to prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position 419 is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. d419n-prothrombin was converted to the related molecules d419n-meizothrombin and d419n-thrombin by limited proteolysis by e. carinatue venom and o. scvutellatus venom, respectively. both d419n-thrombin and d419nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. however, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that d419n-thrombin and d419nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. animal model studies confirmed that d419nthrombin and d419n-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. while i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of d419n-thrombin and d419n-meizothrombin resulted in a normalization of these coagulation parameters. elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. however, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (vte). consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-hcys) and juvenile vte. patients: 85 patients (29 men, median age 42 ys; 56 women, median age 35 ys) who had at least one verified episode of vte before the age of 45 ys were investigated in regard to their total plasma hcys levels. none of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. methods: plasma total homocysteine levels were determined by hplc with fluorescence detection. hyperhomocysteinemia was defined as hcys levels exceeding the upper limit of the normal range obtained in our laboratory from 80 healthy control subjects (40 males, median age 25 ys, hcys 95% ci: 2.02-5.67 pmol/l; 40 females, median age 27.5 ys, hcys 95% ci: 2.99-5.40 ,gruel/l). resuits: 13 out of 85 patients had hyper-hcys, giving a prevalence of 15.3 %. of these 13 patients, 9 were male and 4 female, indicating that the relation between elevated plasma hcys levels and vte may not be as strong in woman as in men. discussion: according to previous reports, our study shows that there is a high prevalence of hyper-hcys in patients with juvenile vte. however, the mechanisms by which hyper-hcys can provoke vte and whether hcys is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. some authors suggest hcys-iaduced effect on factor v activation or inhibition of thrombomodalin-dependent protein c activation. in addition an influence on thrombocyte aggregation has been postulated. conclusion: measurement of hcys levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-hcys is easily corrected by vitamin supplementation. detailed determination of the pathogenesis of vte in patients with hyper-hcys should be the aim of further investigations. a deficiency of one of the coagulation inhibitors antithrombin (at), protein c (pc) or protein s (ps) and resistance to activated protein c (apc resistance) are established risk factors for venous thromboembolism (vte). in the majority of patients with apc resistance, the .tug 506 gin mutation (factor v leiden) is present. whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor v leiden is 2-7% in western europe. heterozygous individuals have a 3-7fold, homozygous an 80fold increased risk for vte the typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. the thrombotic risk is low during childhood, but increases considerably after the 13th year of age. a retrospective study in adult patients out of families with a symptomatic deficiency of at, pc or ps revealed that around 30% of surgical interventions and traumas of the lower extremities were complicated by vte. therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than 13 years. pregnancy is associated with a very high risk for vte in individuals with at deficiency and prophylaxis should be initiated already in the first trimester. after delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. oc increase the risk, especially in at deficient and in homozygous factor v leiden females and are therefore contraindicated in these individuals. oc do also increase the risk for vte in patients heterozygous for factor v leiden and females known to have this abnormality should be discouraged from taking oc or should at least be informed on their increased risk. university hospital-', jerusalem, israel, hospital bcan.iou ~. paris, france, increased frequency of thrombocmbolie events have been observed iu patients with b-thalassemia. our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc a: metabolitcs (blood 77:1749 (blood 77: , 1991 suggested an increased platclet activation in tbese patients. we also fouud that isolated thalassemie rbc enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (am j. hematol. 44:33, 1993) . we now show that annoxin v, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by factor xa, by binding to thalassemic rbc (ic~, = 0.32 nm). kerckhoff-klinik, bad nauheim ~, medizinische poliklinik bonn 2, institut for immunologie und transfusionsmedizin universit~ll greifswald a antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with hat. while the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of hat-related venous thrombosis. since resistance to activated protein c (apc) is the most common inherited risk factor for venous thrombosis described the frequency of apc resistance among a population of 68 hat patients has been studied. hat was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~4c-serotoninrelease test. the diagnosis of apc resistance was established by two functional assays and genetic analysis. at time of diagnosis of hat, 38 patients showed venous thromboembolic complications. among these, 24 were found positive for apc-resistance. pulmonary embolism was diagnosed in 18 hat patients, 14 of them were apc resistance positive. none of the 18 hat patients who showed exclusively thrombocytopenia were apc resistance positive. early oral anticoagulation (oa) was initiated in 7 patients after the diagnosis of hat has been established. six of these patients developed serious thrombotic complications including skin necrosis. these results demonstrate that apc resistance is an additional and common risk factor for the development of hat-related venous thrombosis. early initiation of oa during an acute episode of hat dramatically increases the risk of thrombosis. therefore, oa in hat patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved. controlled trials for primary and secondary prevention of stroke g. de (3aetano, c. cerletti and v. bertel~ consorzio mario negri sud, santa maria imbaro, italy this presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. in two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. the incidence of vascular death was not modified by aspirin in any of these trials. this is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in 1000 us physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. a metanalysis of about 150 trials and over 100,000 patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed 25% overall reduction of vascular events including stroke. the optimal dose of aspirin for secondary stroke prevention could not be established. in patients with previous minor strokes or tia there was 22% reduction of vascular events and 23% of non fatal strokes. the avoidance of nine strokes of any cause among the 38 expected in 1000 patients at risk would result from the sum of 10 ischemic events avoided and a haemorrhagic one occurred in excess. ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. we compared the effect of the direct specific thrombin inhibitors, napsagatran (na) and rec. hirudin (rh) with unfractionated heparin (uh) on the further growth of preformed thrombi. as a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate (650/s). fibrin and platelet thrombi were allowed to form during a 10min perfusion period after which the test agents were given iv as a bolus and a continuous infusion (3 and 10pg/kg/min, n=6) and the perfusion continued for 20min. the control groups were perfused for 10 or 30 rain (n=6). fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. the % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the 10 and 30 min control groups (39+8% and 335:6%, respectively, mean:l:sem). platelet thrombus area (ta) in the control groups increased from 24+7pm2/pm after 10min to 97+32pm2/lim after 30rain perfusion. na at 1011g/kg/min reduced ta by 94% to values (6+_2ptm2/ ~m) lower than those of the 10min control group whereas rh at this dose reduced ta by 70% (30-j:141.tm2/i.tm). uh at both doses was ineffective. these findings show that in contrast to uh the direct thrombin inhibitors na and rh inhibit the growth of preexisting thrombi. these results could be explained by the higher potency of na and rh as compared to uh for inhibiting clotbound thrombin (gast et al., blood coagul fibrinol 1994, 5.' 879-87) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. we challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the stagnation point flow adhesio-aggregometer (spaa). the spaa provides flow mediated contact of platelets with the superfused surface. heparinized (3.5 -4.0 u/ml) platelet rich plasma (prp) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (adp 2"10 -6 m). platelet deposition was recorded on-line by video as well as by measuring scattered light. fixed samples were examined by phase contrast and electron microscopy, inhibition experiments were performed with either the tetrapeptide rgds, the non-peptide gpiib/llla-inhibitor ro-43-8857 or a monoclonal antibody directed against the gpilb/llla complex. stimulation with adp prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to 100 micrometer. adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. infusion of rgds or ro-43-8857 into the flowing, adp-stimulated prp completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. when the inhibitor inflow was stopped while adp stimulation persisted, adhesion and aggregation occurred immediately. re-establishing the inflow of the inhibitors -with still continued adp stimulation -led to disintegration of the adhering aggregates. when prp preincubated with the monoclonal antibody against gpllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet gpilb/llla receptor complex. prevent thrombus formation affer ptca i.p. 8tepanova t, g.v. bashkov 2, l.p.kapralova, 2 s.p. domogatsky ~ cardiology research center t and national haematology scientific cettter 2 russian academy of medical sciences, moscow, russia percutaneous transluminal coronary angioplasty (ptca) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. subendothelial collagen type i-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. the anlithrombotic action of rabbit polyclonal antibodies to rat collagen type i-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-pa/rseu-pa), cross reacting with rat tcu-pa/scu-pa was studied both an in vifro and in vivo. anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries. the short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56_+4 % (p<0.001) as well as by the bispecific conjugate (44_+4%, p<0.001). the treatment of collagen-adsorbed conjugate by rtcu-pa did not increase the autithrombotic effect of bifunctional antibodies. the present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after ptca. increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. we therefore assessed fibrinogen, prothrombin fragment 1+2 (fi+2) and yon willebrand factor (vwf) antigen in 80 consecutive patients (aged 59+5 years) with known coronary artery disease (cad) who all underwent coronary angiography. the extent of coronary artery disease was quantified according to modified criteria of the american heart association (total, proximal and distal "score"). furthermore the intima-media thickness (imt) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vwf antigen was found to correlate positively with the total and proximal coronary score (r=029, p<0.05 and r=o.36, p< 0.01). while fi+2 showed no correlation with the coronary scores, it was significantly correlated with the imt values in the carotid arteries (r=0.27. p< 0.05). after differentiating tertiles of the parameters patients belonging to the upper tertile of fi+2 concentrations had significantly higher imt values of the carotid and femoral arteries (0.81_+0.11 mm vs. 0.72+_0.13 mm in the lower tertile, p<0.05:1.38_+0.44 mm vs. i. 15-+0.25 ram, p=0.05) whereas in patients belonging to the upper tertile of vwf antigen concentrations the proximal coronary artery score was significantly higher (!.71-+0.59 vs. 1.31+ 0.92 in the lower fertile, p<0.01). fro correlation of fibrinogen concentrations and extent of cad or imt values of the carotid and femoral arteries could be demonstrated. in conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon willehrand factor antigen and fi+2 may be contributing factors in atherogenesis. we have previously shown that pgei is a potent inhibitor of pdgf-ioducod proliferation of vascniar smooth muscle cells (vscm) and inhibits dna replication by a camp-related mechanism (grol~er et al, 1994) . the present study investigates of whether or not this aatimitogeni¢ activity of pget can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to ptca (maresta et al. 1994) , vsmc were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. cells of passage 4-9 wore incubated in serumfree medium for 24h in the presence of indomethacin (3 p.m). addition of pdgf-bb (10 ng/ml) under these conditions stimulated dna-replication as assessed from 'hthymidin lncm'poration, by 3.-4laid above control level. trapidil at 10 idvl caused a minor reduction of pdgf-induced mitogenesis whereas 10t) of the compound resulted in a marked reduction of dna replication by 69% (p < 0.05, n = 4). pgei at 0.3 nm diminished the incorporation rate by t 1% while the simultaneous administration of both pged and trapidil (100 idyll caused a significantly stronger response as seen from n reduction of ~h-thymidine incorporation rate by 82% (p < 0.05, n = 4). as a possible mechanism of action, trapidil might have inhibited phosphodiesterases. to establish this, we measured the camp-depcudont proteinkiaasc (pk) a activity in cell homogenates. trapfdil increased the basal fka-activity from 19% to 31% of the maximum response while the response to pget (10 am) amounted to 55%. coincubation of pgei with trapidil caused a 65% stimulation of pka activity, sugesting a small though detectable inhibition of vscm phosphodiesterases by trapidil at anttmitogenic concentrations. essentially similar results wore obtained when thrombin was used as the mitogenic agent. the data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. at these concentratrations, pget induced inhibition of mitogenesis is markedly enhanced by trapidil. inc. i~enna, and ~cenlral itematnlogy laboroto~. , university hospital of bern pibrinogen (fg), yon willebrand factor antigen (vwf) and tissue-type plasminogeu activator antigen (l-pal have recently been shown in be independent risk factors for subsequent coronary events in patients with angina pectoris (nejm 1995; 332:635) although paul antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. furthermore, we have recently shown in a study investigating 200 survivors of myocardial infarction that not only are fg, t-pa and pai-i significantly increased in these patients when compared to a heahhy conlro[ group, but pci activity is also elevated (7hrornb. tfaemost. 1995;73:1137 abst.) , hi order to obtain cut.off points for the individual parameters, frequency histogram plotl; were transformed into straight line cumulative frequency (probit) plots (thromb i/aemost. 1995;74:718) . the cut-off valu~ for the four parameters were determined as follows: fg at 2.7 g/l, t-pa at 8.7 ng/ml, pal-i at 25 ng/ml and pc[ at 126% of a normal pooled plasma. utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. when fg w;qs employed alone as the discriminatow factor, it was observed that 47% (941200) of the coronary heart dir, ease (chd) group eilher had the cul..off value or were below it aud 29% (29199) of the normal group were above the cut-off value, thus, resulting in 47% false ne$atives and 29% false positives. when a second additional risk factor, t-pa wa_~ introduced, the number of false negatives dropped to 21% [i.e. 79% (158/200) had two, risk factors elevated] and the number of false positives to 13% to investigate whether a third parameter could discriminate further, pai-i antigen was used to analyse the rcnudning false positives and negatives. an additional 4% could be detected, resuhing in 83% of the chd group having three risk factors elevated. similarly, the number of normal aubjecta with three parameters elevated dropped by 4% to 9% furthermore. when a fourth parameter was introduced, namely pci, it was round to discriminate a further 8% in the chd group, thereby increasing tile di~riminalion to 91%. the number of false positives dropped to 4%, additionally, determination of pci increased the discrimination of patienta having had multiple infarctions from 88°/= when thrce parameters were mcasured to 100%. from these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-pa adds significant discrimination. pai-i antigen which correlated strongly with t-pa did not significantly increase the discriminatory potential of both fg and i-pa. however, by employing pci as a fourth paramctcr, virtually complete separation between the chd and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. to test the hypothesis that oral contraceptives (oc) enhance exercise-induced activation of blood coagulation we examined 11 women (27 + 3 (sd) years, bmi 20.4 + 2.0 kg/m 2, vozm.. 49 + 7 ml/kg/min) without oc between day 18 and 22 of the menstrual cycle and 10 women (24 + 2 (sd) years, bmi 20,6 ± 1,6 kg/m', vo2max 47 + 7 ml/kg/min) taking oc (150 mg desogestrel and 30 mg ethinylestradiol) between day 7 and 21 of drug intake. prothrombin fragment 1 +2 (ptf1 + 2) and fibrtnopeptide a (fpa) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. mean heart rate [191 ± 10 vs. 196 ± 12 min 1) and mean plasma lactate (3.3 ± 1.6 vs. 3.1 + 1.2 mmot/i) wera comparable during exercise between control and oc group, respectively. results for markers of thrombin and fibrin formation were: ptf1 +2 (nmol/i) fpa (ng/ml) control before 0,66 ± 0.19 1.0 + 0.2 after 0.73 + 0.23 2.2 + 1.2" oc before 0.82 + 0.31 1.0 + 0.2 after 1.11 + 0.48* + 5.8 -+ 6.0* + * p < 0.05 vs. baseline, + p < 0.05 between groups. we conclude that oral contraception with 150 mg desogestrel and 30 mg ethinylestradiol enhances exercise-induced thrombin and fibrin formation, our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of oc. a. haushofer +, wm. halbmayer +, j. radek +, m. dittel *, r. spiel *, h prachar *, j. mtczoch *, m. fischer + + zentraltaboratorium mit thrombose-und c~rinnungsambulanz -krankenhaus lai~: * 4. medizinische ab[eilung mtt ka~liolo$i¢ -krankenhaus lainz und ludwig bottzmann-lnstitut ftlr herzinfarktforsohung, wien fifty-one patients (age 61.6 ± 9.2a; 34 m / 171) implanted with 74 coronary stems 03 palm~-schatz, 27 gianturco-roubin, 14 micro stcnts) received a now antithrombotic treatment using a combination of ti¢lopidine (tic) 2 ×250 mg/d for 28 days and acetyl salicylic acid (asa) zoo mg/d for long-term treatment. patients (pat) only received 15000 tu standard hepartn as i.v. bolus immediately before stent implantation (day l ). side effects and changes in hematological (day i to 7, 14. 28 and 35 [= without t[cil liver and kidney parameters (day 7, 14, 28, 35) were monitored. thirty-eight pat (75%) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (teg) and bleeding time (bt) (day 2g and 35). the other pat were monitored externally, side effects were reported. thrombin geucration after stenting was monitored from day i to 7 by prothrombin fragment 1+2 (f 1+2) and thrombin-antithrombin-lll-comptex (tat). "k" of the "leg decreased (day 28 vs 35; p< 0.0l ). bt prolongation was negatively correlated with the bodysurf ace area (tic+asa: p< 0.05, asa: p< 0.0 l) and showed a reduction after withdrawal of tic (2 l0 sec, 180/ 300 so: [median, quartiles] vs. 120 sec, 881157 sec; p< 0.ix)01). f 1+2 and tat of day i (blood collection: 0, 2, 4, 6 h after intervention, f 1+2:0.84 nmol/i, 0.68/i.07 nmol/l: tat: 2.6 pg/1, 2.0/4.6 ilg/1) were lower compared to day 2 to 7 (f i +2: 1.31 nmol/l, ].08/i .62 nmol/l; tat: 4.8 pg/i, 3.2/7.2 ijg/1; p< 0.0001). tic scorns not to be a strong thrombin generation inhibitor. during stenting one pat (i.9%) sustained a non penetrating mci and one (1.9%) an ischaemic stroke. tic+asa were very effective, only with one pat (1.9%) stent thrombosis (acute) occurred. side effects: 8/15.7% gastrointestinal (one lead to hospitalization), 5/9.8% hematomas at the needle site in the groin (one surgical intervention), 5/9.8% leucopcnias (one agranulozytosis with hospitalization), 3/5.9% allergic skin reactions and 2/3.9% increased liver enzymes (got, gpt, "pgt, alkaline phosphatase; > 2 × of the j. ). with one pat with gastrointestinal disturbances and skin reactions tic had to be withdrawn and treatment was changed to oral anticoagulatlon + asa. one pat showed a combination of skin reactions, gastrointestinal distufl~aneas and on day 28 a heavy reaction of the liver enzymes ( j. after 5 weeks). a decrease of the white blood count (day 1:7.19 gh, 6.03/8.2 g/l, day 28:5.46 g/l, 4.63/6.47 g/l; p< 0.000 i) could be observed. the safety of the therapy with tic+asa should be elucidated and extensively discussed. the serpins c1 esterase inhibitor (cllnh), antithrombin iii (atiii), alantitrypsin (slat), and a2-antiplasmin (azap) are known inhibitors of coagulation factor xla (fxla). although initial studies suggested al at to be the main inhibitor of fxla, we recently demonstrated cllnh to be a predominant inhibitor of fxla in vitro in human plasma (wuillemin et el., blood 1995; 85:1517) . the present study was performed to investigate the plasma elimination kinetics of human fxla-fxla inhibitor complexes injected in rats. the amounts of complexes remaining in circulation were measured using elisas. the plasma tl/2 of clearance was 98 min for fxla-alat complexes, whereas it was 19, 1 8, and 1 5 min for fxla-cllnh, fxla-a2ap, and fxla-atiii complexes, respectively. thus, due to this different plasma tl/2, preferentially fxla-alat complexes may be detected in clinical samples. this was indeed shown in plasma samples from thirteen children with meningococcal septic shock (mss), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. fxla-fxla inhibitor complexes were assessed upon admittance to the intensive care unit. fxla-a 1at complexes were elevated in all patients, fxla-c11nh complexes in nine, fxla-atiii complexes in one patient, and no elevated fxla-a2ap complexes were found. we conclude from this study that, (1) although c1 inh is the predominant fxla inhibitor, fxla-alat complexes may be the best parameter to assess activation of fxi in clinical samples, (2) measuring fxla-fxla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of fxla in rive, and (3) fxl is activated in patients with meningococcal septic shock. dudng the coagulation of plasma about 20 % of the (x2ap present is covalently crosslinked to fibrin by factor xiila (aoki und sakata 1980, thomb. res. 19: 149-155) . we investigated the binding of azap by factor xiila to soluble fibrin (desaabb-fibdno) whose polymerization was inhibited by an isolated fibrin ddomain named d=,,, (haverkate and tiemann 1977, thromb. res. 10: 803-812) . d==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (williams et el. 1981, biochem. j. 197: 661-668; hantgan et al. 1983, ann. n. y. acad sci. 408: 344-365) . material and method: soluble desaabb-fibrino was prepared by incubation of (lztl)-fibrinogen (0.48 mg/ml), d=1o (1.91 mg/ml; molar ratio d==o to fibrin 14:1) and 0.4 u/ml thmmbin for 45 min. then q2sl)-c~2ap (16 p.g/ml), faktor ×ill (2 ulml) and ca 2) (5 mmol/i) were added. the crosslinking reaction was stopped at different times of factor xiila-incubation by adding of urea/edtasolution. the suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and sos. re~ultl: the elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of factor xiila-action. the crosslinked fibrin polymers contained about 80% of the fibrin initially added. although factor xiila acted well, crosslinking of azap in the fibrin oligomers could not be observed. conclusl0n: as we already demonstrated (kelach et el. 1994, ann, hematol. 70 (suppll) : a 60) the crosslinking of azap to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. in desaabb-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. thus it is consistent with our theory that we did not observe any binding of aiap to the fibrin oligomers of desaabb-fibrino. human pci is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. it is synthesized in many organs including liver, pancreas, and testis. the physiological role of pci has not been defined yet. recently, we have cloned and sequenced the mouse pci gene (zechmeister-machhart etal., manuscript in prep.) . this enabled us to study pci gone expression in murino tissues using mouse pci edna and crna probes. by northern blot analysis, mouse pci tar.ha was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for pci mkna, indicating that in the mouse pci is not a plasma protein. to determine which cells of the reproductive tract synthesize pci, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. in testis, pci mrna was present in the spermstogonia layer and in leydig cells, while sertoli cells and peritubular myoid cells were negative. these results are consistent with the immunohistological localization of human pc1 (laurell et al,, 1992) . in the mouse ovary, stroma cells of the medulla and around the follicles were positive for pci mrna. no pci expression was detected in theca or granulosa cells. we also studied the regulation of mouse pci gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in pci mrna in seminal vesicles, which was reversible upon the administration of testosterone. in tissues of intact adult male and female mice, pci mrna levels decreased after injection of human chorionic gonedotropin (hcg), while in castrated male mice, hco had no effect on seminal vesicle pci mrna. progesterone and 17-b estradiol decreased ovarian pci mrna levels in immature female mice. these data suggest direct down-regulation of mouse pci by sex steroids. the different tissue specific pci-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. ctr. transgene technology, leuven "[' 1-tissue factor ('it) is a 47 kda glycoprotein mainly known a the primary cellular initiator of blood coagulation. whether tf expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the tf gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development. to determine the significance of "if in ontogenesis, the pattern of tf expression in mouse development was examined and compared to the 'if distribution in human postlmplantation embryos and fetuses of corresponding gestational age. at early embryonic period of both murine (6.5 and 7.5 pc) and human (stage 5) development there is a strong tf expression in both ectodermal and entodermal cells. "if decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "if in the adult organism. surprisingly, during renal development and in adult organism tf expression differs between men and mice. in humans maturing stage glomerali were "if positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were tf positive. in ncuroepithelial cells there was a striking 'if expression indicating a possible role of'if in neumlation. moreover, there was a robust tf expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. in contrast to tf, its physiologic ligand factor vii was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, the temporal and spatial pattern of tf expression during murine and human development support the hypothesis, that 'if serves as an important mo~hojzenic factor darinz embrvozenesis. to serve as an anticoagulant, protein c (pc) must be activated by a complex formed between the enzyme thrombin (t) and its cofactor thrombomodulin (tm). therefore, downregulation of endothelial cell surface expressed tm, for example, triggered by an inflammatory stimulus, could become a critical factor in effective pc activation. in order to develop a recombinant (r) pc mutant which can be activated independently of the tkm-complex, a peptide sequence including p1-7 in the activation peptide of pc was modified to be identical to the factor xa (fxa)-cleavage site in prothrombin. the mutant was expressed in hu 293 cells, purified and its anticoagulant properties characterized. using purified fxa the mutant showed activation rates between 0.02 and 0.13 nmlmin at pc concentrations between 97 and 770 nm, while the rpc wild type was insensitive for fxa activation. the activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of 3 mm and was enhanced to 3.8-fold by addition of anionic phospholipids (pl). in contrast to the wild type pc the rpc mutant was insensitive for activation by the t/i-m complex. addition of the mutant to normal human plasma induces a prolongation of tissue-factor and p-it-based clotting assays. using normal human plasma as a source for fxa the the activation rates of the mutant were found 5-fold higher than in the purified system if tissue factor was used to generate fxa. in conclusion, our data demonstrate that the rpc mutant is effectively activated by fxa in a purified as well as in a plasma system. interestingly, the activation rates are enhanced in the presence of pl and normal human plasma. fudher studies should clarify the potential use of this mutant as a novel anticoagulant. thrombln plays a pivotal role in thrombotic events. the time course of thrombln concentration in blood or plasma after activation is of special interest to answer a variety of questions. with a chromogenic assay developed by hemker et el. [thromb. haemostas, 70, 617, 1993] it became possible to measure the generation of thrombin in activated plasma continuously. inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to immediately block generated thrombin. we have used a test based on hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor xa needs to efficiently block thrombin generation in human plasma. thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate h-~ala-gly-arg-pna (pentapharm ltd.). a series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor xa with different potency were investigated. because of the inhibition of activated factor x the generation of thrombin in plasma is delayed and the amount of the generated thrombin is reduced. the concentrations which cause a 50 % inhibition of thrombin generation (icso) correlate with the k~ values of the inhibitors. low molecular weight inhibitors with k~ values of about 20 nmol/i inhibit the generation of thrombin after extrinsic activation with icso in micromolar range. after activation of the intrinsic pathway tenfold lower concentrations are effective. the strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-tap) with ic~o of 0.049 pmol/i (axtdqsic) and 0.037 pmo/i (intdnsic). in the compadson of synthetic low molecular weight inhibitors of thrombin end factor xa which have similar k= values for the inhibition of the respective enzyme (lowest i<1 20 nmol/i), factor xa inhibitors are less effective tn the thrombin generation assay. in contrast, the highly potent xa inhibitor r-tap shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. background: resistance to degradation of coagulation factor v by activated protein c is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor v. to date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. for this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. the conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. these methods have disadvantages for a large scale dna diagnosis, which include the need for electrophoresis or a high cost and time consumption. methods: an alternative strategy of dna diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor v. following pcr amplification of the target dna, tile procedure was performed completely automatically on a robotic workstation with an integrated elisa reader using a 96-well microtiter plate. allelespecific restriction enzyme site analysis was performed to confirm the genotypes. results: in 10 patients with the mutation and in 20 individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in 100% concordance with the elisabased oligonucleotide ligation assay. discussion: the pck-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor v point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. resistance to the asticoa=m~lant activity of activated protein c (apc resistance) has emerged as the most con'anon inherited thrombophilic state. patients lreterozygous for factor v leiden are more likely to suffer from thromboembolie events than controls. this risk is even more pronounced in homozygotes. due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of factor v leiden mutation by pcr-based methods. re e~tly we presented an aptt-based functional test (acceleria inactivation test ait): 1:20 diluted plasma (50bi) is mixed with factor v deficient plasma (50~tl) and aptt reagent (501.d), incubated at 37°c and then coagulation is induced by caci2 and a.pc (50~d). using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%ai). using this test, the widely used apc-ratio as well as pcr-based factor v leiden detection (confirmed by direct sequencing) we prospectively studied 172 consecutive patients with thromboembolic events. patients without the factor v mntation eonsitently showed more flazm 50% al with the exception of one patient with severe factor deficiencies (including factor v) due to hepatic failure and heterozygous for factor v-leiden resulting in 62*/. ai, there was a complete concordance between the pcrbased method and dysaseelerinemia detected by ait. due to these result a specifity and sensitivity of ait above 95 % was calculated. furthermore, a clear discrimination could be obsoved beween 34 heterozygotes (20%<ai<50%) and 8 (incl. 5 samples obtained from jeaa) homozygotes (ai<20%). in contrast a.pc-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor v leiden. in addition we confirmed thc di.~eulties nsing the apc-rauo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein s deficiency. due to the time and cost-saving featttres the/kit should be considered for the rapid detection of apc resistance without the use of pcr-based techniques. in summary we found, well in accordance with previous reports, that apcr due to the fv-leiden mutation is • highly prevalent defect in vte pts. pts with the fv-leiden mutation were eharacterised by a higher incidence of vte's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent vte's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable. acquired a recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein c (apc), it is now welt recoginzed to be due to a genetic defect in the factor v gene, which results in the syn-,thesis of an abnormal factor v molecule and to be a frequent risk factor for thromb.osis, however apc resistenee can also be found in certain acquired conditions. since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to 1% and thromboembolic events, we determined the prevalence of apc resistance during the course of pregnancy at the city hoslfital ruesselsbeim. germany. blood samples from pregnant woman were drawn at mmester 1, 2, 3 and at term. besides the routine coagulatton screening, protein c and viii:c, the apc resistance were evaluated. a modified aptt method utilizing human plasma fracnonated apc (amencan red cross. bethesda, maryland, ~usa) was used to assay apc resistance. this method relies on the determination of two aptt's in the patient plasma, one in the absence of apc and one in the presence of a fixed amount of apc. clotting tune was determined after mcubation using the aptt reagent ( we investigated the sensitivity of various species towards activated, human protein c (apc) and found an impaired response in the order monkey < horse < pig < dog < rabbit. we assume that this effect is mainly caused by differences in the apc cleavage region of factor v, as recently described for a factor v mutation in bumat~s: factor v-leiden. this was corroborated by the finding that addition of human factor v similar to the factor v added with the animal plasma had only minor influence on test outcome. coagulation assays dependent on apc, such as for protein c and protein s determination, in the standard apc assay, the more factor v-leiden specific apc assay and in the pcat, a new screening assay for the whole protein c system, are based on the inactivation of factor va via apc. thus, in all these assays, 5% of rabbit plasma added to human plasma mimicked a apc response as observed for heterozygous carriers of factor v-leiden and 20% as for homozygous carriers. we exploited this factor v-leiden like behaviour to prepare plasma standards for calibration of apc dependent assays by mixing rabbit plasma with human plasma~ as reference for these standards the 0% value was assigned to the clotting time in the absence of apc, while the 100% value was assigned to the clotting time in presence of apc as obtained with a human, normal plasma pool. then these calibrated standards were used for establishing reference curves. on automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• this not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. in order to establish the methods for activated protein c resistance and the faktor v leiden-mutation we investigated 204 healthy individuals (128 women, 76 men, median age 37 ys) using the assay "contest ® apc-resistance" (chromogenix, sweden). the mean apc-ratio was 2.65, the cut-off level for low response to apc was calculated as 2.15, 46/128 women were oral-contraceptive users. they showed a slight decreased apc-ratio (mean 2.46). 24 of the 204 individuals (11,8%) had a poor response to apc (mean ratio 1.94) as a apc-resistance. for detecting the factor v gene mutation we a used non commercial own modified easy to handle pcr-based test. in 14 of the 204 individuals (6.9%) the factor v-leiden mutation could be found as heterozygotes. in this group, the apc-ratio was significant lower (mean 1.87) as in the group with apc-resistanee and without deteetiun of the factor v mutation (mean 2.04). nevertheless in one individual with beterozygote factor v-leiden, the apc-ratio was found in the normal range (2.24) , also in a few individuals with very iow apc-ratio the factor v mutation could not he detected. finally this data suggests a high prevalence of apc-resistance and factor v-leiden mutation in the population of the southwest of germany. resistance to activated protein c (apc) has been reported to interfere in clotting assays for protein c (pc) and protein s (ps) from various manufacturers, resulting in decreased values or even apparent deficiency (type ii') of pc. or is. in our laboratory, when using functional, avit-based assay kits for pc., is, and apc sensitivity from behring (marburg, germany) on an automatic turbidimctric coagulation analyzer (fibrintiraer a, behring), the interference in the is assay has been observed in a very pronounced manner soon after the introduction of apc sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. yet we were not aware of an interferenca caused by apc resistance in the pc test. we therefore evaluated our results for pc eed ps with respnet to apc sensitivity status in a retrospective analysis. exclusion criteria were: oral anticoagulant therapy with vitamin k antagonists, impaired liver function, borderline result in the apc sensitivity test, known analytical intedercnc* other than apc resistance (e.g. elevated factor viii:c). resu/ts: apparant pc activity was slightly (12 %) lower and apparent ps activity was grossly (45 %) lower in ape resistent patients than in patients with normal apc sensitivity: there was no case with a presumed du~l defect of both at~ rnsistaac¢ and pc deficiency. in addition, none of the few patients diagnosed with pc deficiency before the introduction of the apc sensitivity assay turned out to ix: apc resistent. much more of a problem is the known sensitivity of the pc test to an elevated factor viii concentration which is quite a common finding among hospital patients but rarely known in an individual case. by aeccelerating the clotting process, elevated factor viii can cause apparent pc deficiency that can not be confirmed in a chromogunie pc assay. ,clearly, when combined with apc resistance, this problem is exspacted to be more severe. on the other hand, the comhination of both apc resistance and apparent is deficiency was found on a regular basis with many decreased and some bordeflinc values for is but only few in the lower normal range and virtually none in the upper non~al range. also, several #eats previously diagnosed with is deficiency turned out to be pc resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. conclusion: for, the interpretation of pc and/or 1:"3 values from clotting assays the apc sensitivity status must be considered. when apc resistance and an apparent deficiency of pc or is is found, a type i deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type ii deficiency, especially in the case of ps. therefore, there is a clear need for analytical improvements and the development of functional assays that are less sensitive to interference by apc resistance. the most common inherited risk factor for venous thrombosis is the resistance to activated protein c (apc). the main reason for this resistance is a point mutation in the gene of dotting factor v (f v). a nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off v. the imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor 7 to 10 for heterocygotes and about 80 for homocygote individuals. the prevalence in patients with a history of thrombosis is about 20%, among apcresistant patients as high as 80%. prevalence-off v ,leiden" is reported to be 2% of the normal population in leiden/netherlands and as high as 15% in a certain city in the south of sweden. due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in germany. first attempts to assess the prevalence off v mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of wiirzburg 7.7% of blood donors carry the mutation, which is significantly different (fischer t-test, level of significance 0.05%) from published data observed in freiburg (3.4%). homburg/saar and harmover revealed 5.8% and 8.5%, respectively, and were not significantly different from wfwzborg. further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in germany and might finally help to draw a map era regional risk of venous thrombosis. the regular apc resistance test is not applicable to plasma from orally anticoagulated (oac) or heparinized patients due to decreased levels of vitamin k-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. the former limitation is overcome by prediluting samples 1-1-4 with fv-defident plasma. for this purpose a special quality of fv-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. this fv-deficient plasma has now been evaluated with the coatest apc resistance assay. a complete ape ratio discrimination for the fv q506 mutation is obtained for plasmas from oac and heparin patients as well as from non-treated individuals. ratios above 2.2 are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range 1.6-2.0. the basal apt times for 15 oacplasmas with widely spread inr-values were reestablished into the normal range. .the stability of the reconstituted fv-deficient plasma was found to be' at least 3 hours at room tempetature, indicating its suitability for .routin.e u.~. f .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the apc ratios to any significant extent. our results prove the usefulness of the modified coatest apc resistance kit method as a reliable, simple and rapid tool when screening for the presence of the fv q506 mutation. hereditary resistance against the anticoagulatory action of activated protein c (apc-resistance, apcr) which was first described by dahlb~ick et al. (pnas, 90:1004 , 1993 was identified as a possible new thrombophilic factor in a high percentage (17-60%) of young adults with thrombotic events. a single missense mutation (r506q) due to a g/a transition (g1691a) in exon 10 of the factor v gene (bertina et al., nature, 369:64, 1994) is tightly linked with apcr and is regarded as the causative molecular defect. identification of this mutation by pcr-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for apcr. since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. we compared 125 pediatric patients with thrombosis, divided into 3 different age groups (0 to <0,5 years; >0,5 to <10 years; >10 to <18 years) with a normal population of 159 children. the mutation g1691a was found with an unexpected high prevalenee of 12% in our normal controls. however, the prevalence was significantly higher in the age groups: 0 to< 0,5 years (25%) and >10 to <18 years (30%). in patients between >0,5 to <10 years the overall prevalence was similar to the control (13%). however in patients of this age with spontaneous thrombosis apcr was also a significant risk factor (29%). our results emphasize the impact of apcr for thrombogenesis in children. however, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. activated protein c (apc)-resistanec is a newly reeognised risk factor for thrombosis. in at least 90% of the cases it is caused by a single point mutation in the factor v gene (g->a at nucleotide 1691), which predicts replacement of arginin 506 with ghitamin. one of the apc cleavage sites in factor va is located c-terminal of arginin 506, and mutated factor va (factor v leiden) is resistant to apc-mediated inactivation. from epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. apc-resistance is a major basis for venous thromboembolism and is prevalent in about 5.10% of the general caucasian population. recurrent spontaneous abortion (rsa) affects 1-5% of couples and represents a major concern for reproductive medicine. in spite of extensive endocrine, genetic, serologic and anatomic evaluation some 30-40% of rsa women remain unexplained. a frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space. because of these findings we studied the prevalence of apc-resistance in women with rsa (more than 3 miscarriages) of unknown origin. in 2 of 35 cases we found a pathologic apc-resistance, both patients had a history of recurrent thrombosis and were heterozygous for factor v leiden. the prevalance of apc-resistance is 5,7% and thus equals the prevalence in the general population. our data do not support the hypothesis that apc-resistanee is a risk factor for recurrent spontaneous abortion. h~matologisches zentrallabor der universit~t, inselspital, 3010 bern resistance to activated protein c (apc) due to the mutation 506 arg --~ (]in of factor v (factor v leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of 20 -50 % in patients with idiopathic venous thromboembolism and of about 3 -5 % in the general population. with an allele frequency of 2 % the expected number of homozygous individuals is about 4 in 10000. homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis (80 -fold versus 7 -fold) as well as to the age of the first thrombotic event (31 versus 44 years). deficiency of the vitamin k dependent protein s (p$), an important cofactor of apc, is another hereditary thrombophilia which is, however, much rarer than apc resistance with a prevalence of 5 to 8 % in patients with venous thromboembolism. factor v leiden mutation as well as ps deficiency are associated with impaired anticoagulatory activity of apc, which is most pronounced in case of the combination of the two defects. the combination of ps deficiency (with an assumed prevalence similar to that of pc deficiency) with heterozygous or homozygous apc resistance can be expected with a probability of 1: ~ 5000 or 1: ~ 500000, respectively. it is well known that ps levels decrease towards the low normal or even subnormal range during pregnancy. moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of apc decreases during pregnancy resulting in an acquired apc resistance. these pregnancy associated effects art obviously much more relevant in case of preexisting ps deficiency or apc resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. we describe a young woman with a combination of homozygens apc resistance ( apc ratio 1.10, normal range: 2.02 -3.73), pronounced ps deficiency (free ps 0.ll u/i, total ps 0.30 u/i, normal range: 0.55 -1.20 u/1 and 0.65 -lag u/i, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after 10 rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. examination of her familiy showed heterozygous apc resistance in her asymptomatie father (apc -ratio 1.99) , combination of heterozygous apc resistance (apc -ratio 1.60) and ps deficiency (free ps 0.30 u/i, total ps 0.58 u/i) in her nsymptomatic mother and no defect in her sister. considering the fact that the mother was still thrombosis free at the age of 49 one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for apc resistance. s. ehrenforth, m. adam, b. zwinge, i. scharrer university hospital, dept. of angiology, frankfurt a.m., germany introduction: apc resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (vt). however, most of the present epidemiological studies concerning apc-r prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. this may influence the great differences reported on the prevalence of apc-r among these patients. for example, it has been shown that freezing of plasma specimens prior to analysis of apc-r causes a significant decrease in the assay results.the aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic apc-r assay. patients and methods: blood was collected from 31 patients (t9 women, 12 men; fv gent.type: 13 r/r 506, 14 r/q 506, 4 q/q 506) through veinpuncture into trisodmm ciwat (1:9). platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at 4"c for 10, 20, 30, 40, 50, 60 rain at 1000, 2000, 3000, 4000, 5000 and 6000 rpm. additional, pnp obtained from 14 healthy individuals (7 male, 7 female without hormonal trealraent) was prepared equally. apc-response was determined within one hour after centrifugation using the coatest apc resistance kit from chromogenix. results: for both, pnp and sin/gle plasma samples, we observed continuous higher af'c-ratios after increasing cenwifugation intensity. for example, an increase from 1000 to 6000 rpm resulted m an increased apcratio from 2.7 to 3.26 (20 min), from 2.88 to 3.326 (60 rain) respectively. even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from 10 to 60 minutes we observed a continuous increase in apc-ratio, for example from 2.74 to 3.12 when using 2000 rpm and from 3.09 to 3.36 when using 4000 rpm. the decrease of the ratio after low eentrifugation is the eonse-9nence of the shortening of affft in the presence of apc, without a signhcant influence of basal al:rl~ without apc. conclusion: our results demonstrate that centrifugation conditions are important to consider for the interpretation of apc-r results. supporting our observations, recent studies from sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the apc-response. however, so far the mechanism responsible for the significant effect of both on apc-r assay results is unknown. although technically simple, the biochemical cemplexitiy inherent in the chromogenic apc-r assay necessitates a standardized plasma handling procedure to secure a reproducible determination of apc-il compapjson of different assays for determination of apc-resistance with the geno'fyping factor v (arg -> glu) g. siegert*, s. gehrisch*, e. runge**. r. naumann**, r. kn6fler*** *institute of clinical chemistry, **clinic of internal medicine, *** childrens hospital resistance to apc diagnosed on the basis of prolongated clotting time in the aptt assay" is now considered a major cause of thrombophilia. in the majority apc resistance is ~ted with a point mutation in factor v molecule (arg506glu), but both are not synonym. protongated baseline aptt is a limitation of the assay. following the determination is not possible in risk groups of patients (factor)ill deficiency, lupus anticoagulan0 and in patients under anticoagulant therapy. in these causes a dilution of plasma in factor v deficient plasma is recommended. the immunochrom assay is based on the inactivation of factor villa by apc. the aim of the study was to compare different functional apc response assays with the result of the dna analysis. apc response was tested in 100 healthy probands, 81 thro~ patients and 16 family members using the lmmtmochrem assay, the contest (chromogeaix) and the contest with 1+ 4 dilution of the plasma in native factor v deficient plasma (immune). the dna analysis was performed as described by bertina. one patiem was homozygoas for factor v mutstion~ a hetemzygous result was obtained in 4 members of the control group, in 28 patients and in 6 family members. in all cases with factor v mutation the ratio of the immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (dna arg/arg) and in 17 patients under anticoagulant therapy ( two of this patients are one cloned twins). in the contest a ai~ response was diagnosed in all cases with factor v mutation without anticoagulant therapy and in 40 % of heterozygous patients under anticoaglant therapy. results of the test using the dilution in factor v deficient plasma showed a good agreement vath the results of the dna analysis but the method is obviously only sensitive for the factor v mutation. the reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. the majority of this patients is treated with anticoagulants, a comparison with the contest is not possible. interestingly in one patient under heparin and low ratio in the immunochrom assay' after reduction of hepann the ratio of the coatest was also low. it seems necessary to investigate in which distance to the thrombotic events the apc resistance should be tested. following pathological ratios in ftmctional apc assays must be discussed: high levels of factor viii and or v wiuebrand antigen (acute phase reactien), other mutations in factor v and viii. the factor v dilution assay should be replaced by the dna analysis. due to their differing compositions, the "sensitivities" of various aptf reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. for lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. with a "lupus-insensitive" ai~ reagent there is no or only slight prolongation of the aptt in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. for the meaninof~l use of aptr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. the following 14 apti' reagents were tested: • ptt-reageaz, p'rta, ptta liquid, ptt-la, pti'-lt (boehringer/stago) • pathromtin, pathromfin sl, necthroratin (behring) • platelin s, piatehn excel ls (organon tekinka) • actin-fs, actin-fsl (dade) • aptt silica lye, aptt silica liquid (instntmentation laboratory) the material for investigation consisted of 20 plasmas from patients with lupus anticoagulants. a confirmatory test (lupus anticoagulant test, immune) was positive for all of the patients. measurements were made using the sta coagulation analyser (boehringer/stago). it can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing aptt reagents. low susceptibility to lupus anticoagulants was shown by actirt fs (dade), ptt-reagenz (bcehrlnger) and neothromfin (behring). high susceptibility was shown by platetin excel ls (organon teknika), ptt-la and pti'-lt (boehringer/stago). lupus anticoaguhant screening with the aptt reaction is promising when two aptr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. the resistance to the anticoagulant response of activated protein c (apc) is a major cause of venous thrombosis. apcresistance is due to a single mutation in factor v gene, which predicts replacement of arg 560 in the apc-cleavage site with gln (factor v leiden mutation). in contrast to other known genetic risk factors for thrombosis, this factor v 1691 g-a mutation has a high prevalence in the common population of western europe (average 4-5 %). we have determined the prevalence of the factor v 1691 g-a mutation in a population of 700 probands of north-eastern part of germany. the mutation was found in 7 %. (heterozygoty were found in 48 subjects 1 person was homozygous.) the results are compared with our studies of populations from argentine and poland. me analysed the factor v 1691 g-a mutation in 71 patients with thrombosis from germany and hungary. this mutation has been found in about 60 % of these patients. in contrast, the frequency of this mutation was strongly reduced in a group of 47 patients with thrombosis and pulmonary embolism of argentine (3 heterozygotes in 47 patients; 6 %). the results of these different populations will be described and discussed. past medical history: venous thromboembolic events (re) at 18, 19 and 2i years; intermittent oral anticoagulation (oac) without te's. diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. no other relevant illnesses; family history uneventful. two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (dvt) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from 121 to a nadir of 21 g/l; referral to our department. on admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, i/reduced, +/positive, -/negative): pt t, aptt t, tr n, factor ii, v, viii n, factor vii, ix, xi, xii /,, fibrinogan t, atiii n, protein c, s *, activated protein c sensitivity ratio 1.92 ($), fv-leidenmutation pcr -, fibrinolytic system n, tat t, ft÷2 t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. an immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the dvt oeeured and normalisation of the platelet count was observed. during follow-up under oac ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein c resistance, have returned to normal; no further te's have been observed. in summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein c and heparin induced thrombocytopenia (type ii), entering complete remission under combined immunosuppressive and anticoagulant therapy. in the last 30 years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. in these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral). we studied 50 healthy subjects, aged 21 to 65 years and 118 patients, aged 9 to 93 years using a new aptt reagent (pathromtin $l). the results were compared with those obtained with a routinely used reagent (pathromtin). the reference range, factor-, heparin-and lupus anticoagulant sensitivity were determined. analysis was performed using the behring fibrintimer a (bfa) with optomechanical clot detection, the behring coagulation timer (bct) with op-"dcal clot detection by threshold and the dw test and dw confirm for lupus anticoagulant diagnostic. our results showed that the new pathromtin sl reagent met the demands for a higher factor and lupus anticoagulant sensitivity. it is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. restenosis following percutaneous transluminal angioplasty (pta) continous to be a major clinical problem. neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. anticardiolipin antibodies (act_) have been established as dsk factors for venous or arterial thrombosis. methods: in a cohort of 75 patients (53 men and 22 women, age 68±13 years) undergoing pta of a peripheral artery we prospectively evaluated whether acl could influence 6 months restenosis rate. patients were clinically examined before, 3 and 6 months after pta. noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. restenosis was arbitrarily defined as more than 50% occlusion of the lumen at the site of dilatation 6 months after successful intervention. laboratory investigation at the same time included acl and other known atherosklerosis risk markers, such as fibdnogen (fbg), yon willebrand factor (vwf), homocystein (hcy), c-reactive protein (crp). thrombin generation markers, such as thrombin-antithrombin iii complexes and prothrombin fragments 1+2, as well as thrombomodulin (fm) as an endothelial activation marker, were also measured. results: 31/75 (41.3%) patients were considered to have developed restenosis after 6 months. 9/75 (12%) patients were found to have positive igg-(19-35 gpl) and/or igm-acl (14-103 mpl) at all three measurements. 1/75 was negative before but seroconverted (igm) 3 months after pta. 2/10 (20%) acl-positive and 29165 (44.6%) acl-negative developed restenesis at 6 months (chi-square p-value=0.14). all above mentioned coagulation parameters did not differ between acl-positive and -negative patients, measured before or 6 months after pta. some of them are shown below (values before pta): fbg ( basilar artery stenosis is a rare event in young children. risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. elevated lipoprotein (a) (lp(a)) serum levels in adults can mediate atherosclerosis. in addition, lp(a) might interfere with fibrinolysis. here we report on a 3 year old boy , who presented with acute brain stem symptoms. history revealed neither trauma nor infectious disease. conventional and mr angiography showed stenosis of basilar artery without ischemic lesions. laboratory findings were normal in routine blood and csf tests. global coagulation parameters as well as procoagulant and anticoagulant factors were normal. cardiac and autoimmune disease could be ruled out. lp(a) serum levels were significantly elevated to 104 mg/dl (normal range <30 mg/dl). analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and cve in elderly family members. clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. however, radiological signs of basilar artery stenosis were progredient. in a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. in conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of lp (a). it is unclear, whether apa contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. apa, however, might increase the risk of further thrombotic events in this patient. in 110 patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (aca) aund lupus anticoagulant (la) were ~ea~ured. for aca detecting we use the assays from elias for igg-and ig~}-antibodies. we use as sensitive methods for detecting la in our laboratory the testkits from diagnostlca stago (staclot la with hexagonal array of phospholipids, ,ptt-la a very sensitive pttmethod and staclot p~p-a platelet neutralization procedure) and the ptt from organon teknik~ (platelin excel ls with two incubation times, 1 and 10 minutes). i"~e results of this tests were compared with three new or~e on german market: specktin apot (aktlvated plasma clotting time), specktin aptt (aptt wlth purified soy extract) and 8pecktin la (phospholipid preparation in concentrations between 10 and 500 ~g/ml); all wak chemie. traditional aptt reagents were developed for the sensitive detection of factor vib an ix as a cause of hemorhage. high sensitivity against lupus anticoagulants, which also prolong aptt, was not required for this purpose, with increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. we here report an algorhythm, by which we use an inhibitor (lupus anticoagulant) sensitive (sta aptt, boehringer) and an inhibitor insensitive reagent (actin fs, dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aptt. citrate plasma from 33 patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aptt (>40s). 13 plasmas with factor deficiencies remained abnormal with the insensitive aptt reagent. a regular correction of their defect occured on mixing with normal plasma. by measurement of single coagulation factors five patients with contact factor xii deficiency were found. this condition is associated with thrombosis and very rarly with bleeding. three patients with factor xi deficiency and two patient with factor ix deficiency were also identified. antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. there is no general consensus regarding secondary prevention of cerebral ischemic lesions. aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. european stroke prevention study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (persantln), is also very active. to collect more information, esps 2 was organized and 6602 patients receiving either a placebo,either 50 mg aspirin,either 400 mg sustained release form of dipyridamole (persantin (r)), or the combination aspirin/dipyrldamole, were recruited. it ended march 31st 1995 with the following conclusions: i-aspirin, 50 mg a day, brings about a significant secondary reduction of stroke (18.z %), after a two year follow-up. notwithstanding the low dose of aspirin, haemorrhages remain important. 2-dipyridamole, at 400 mg a day, brings about a significant reduction of stroke (i6.3~), similar to that of aspirin. one could thus substitute 50 mg aspirin by 400 mg dipyridamole. 3-the combination of 50 mg aspirin and 400 mg dipyridamole brings about a significantly greater reduction of stroke (37.0 ~). esps 2 revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. the study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. the conclusions drawn from esp£ 2 underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, the state of activation of circulating platelets in acute cerebral ischemia is controversial. activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (cd62). shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. methods: we investigated 20 untreated patients within one day after cerebral ischemia, 20 patients months after stroke treated with warfarin, and 21 age and sex matched control subjects without vascular risk factors. venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. we determined the fraction of resting discoid platelets by phase contrast microscopy. the expression of p-selectin was measured by flowcytometry. results: the fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia (7.8_+4.7%) than in control subjects (1.9_+1.1%; p<0.001, u-test). patients with stroke (n=15, 7.8+4.5%) and patients with transient ischemic attack (tia; n=5, 7.6-+6.1%) had similar values. patients months after stroke still had higher values (4.3+9,3 %, p<0.05) than control subjects. the rate of discoid platelets was not different between patients with acute ischemia (n=17, 85.6-+8.8 %), patients months after stroke (n=19, 85.7-+5.1%) and control subjects (n=21, 86.7_+5.8 %). platelet count was not significantly different between groups. conclusion: the elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or tia than analysing the reversible shape change. further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. vascular smooth muscle cell (smc) proliferation and migration into neointima are the hallmarks of atherogenesis. the complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, one crucial molecule seems to be the urokinase-type plasminogen activator receptor (upar) recently also assigned as cd87 antigen, upar serves a dual function: (1) it directs upa proteolytic activity to a special location on the cell surface and (2) induces cellular signals leading to various phenotypic changes. we have investigated the signal-transducing capacity of upar in human smcs and provide here a molecular explanation for uparrelated cellular events. activation of these cells with upa (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of upar-associated protein tyrosine kinases (ptks) upon ligand binding. we obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ 55-65 and 35-40 kd. using antibodies against different types of ptks as well as immunoprecipitation-and immunoblotting techniques the ptks involved in the upar-signalling complex were identified to be members of the src-ptk family. the cotocalization of upar and ptks at the cell surface of smcs was further confirmed by confocal microscopy studies. we conclude that the upar-ptk complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. this mechanism may be crucial for the progression of atherosclerotic plaques. activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. simultaneous pancreas-and kidney transplantation (pkt) in type i diabetes has been shown to improve diabetic complications and long term survival. we measured haemostatic vascular risk factors and activation markers in plasma of 18 patients after successful pki', 17 patients after pkt and rejection of the pancreas graft and 5 patients after pkt and rejection of the renal graft. blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. despite individually adjusted insulin therapy hba1 plasma levels increased after pancreas rejection (5,37 vs 7,i2, p<0.001). platelet counts and plasma levels of fibrinogen, f1+2 fragment, tat-, app-complex and-fibrin monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful pkt. one major reason of the increased activation state of haemostasis may be cyclosporin treatment given to all patients, t-pa and pal i plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r.0.049; p<0.005). d-dimer plasma levels were significantly lowered after pancreas rejection (772(375) vs 324(232) nglml; mean(sem) p<0.005), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. in conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. according to the investigations of fowler et al. and pepe et al. the probability of an ards occurring with one risk factor is 5-8%, and in the presence of several risk factors, 25%. goris et al. and johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. all these investigations are however not to be interpreted as typical following coronary surgery. these investigations demonstrated that the kallikrein and factor xii systems are of great importance as intraoperative risk factors. here the factor xii system plays a major role with direct or indirect activation of the kauikrein-kinin system with the splitting products alpha-factor xiia and bfactor xha respectively. all ards scores take the pmn-elastase into account. if the pmn-elastase values (1000 pg/l) are constantly high postoperatively then lung complications are to be expected. patients developing an ards displayed significantly lower alpha2-macroglobulin values. patients who developed a highly significantly raised kallikrein-like acdvity (>60 u/i) after the beginning of bypass and showed constantly high values during ecc are difficult to keep under control due to the blood pressure behaviour. the platelet pal also shows a significant rise and intraoperatively runs analogous to platelet factor 4, only antiparailel, since it attacks the endothelium. we were able to show that pai-1 is suitable as an indirect marker for a possibly developing restenosis. 85% of the patients investigated with lowered pai-1 values in the postoperative phase did not develop a restenosis. however, with patients showing significantly rising pa[-1 values from the 1 st. to 3rd. postoperative day 50% of all the cases had a restenosis. a further risk factor in this respect are significantly raised fibrinogen levels which lay over 800% at the end of surgery. if these fibrinogen values do not fall from the 1st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. the following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: 1) regards the blood pressure behaviour, the kallikrein-like activity (>60 u/i); 2) with regards to the lung complications, aipha2-macroglobulin and pmn-elastase (>900 g/i); 3) and final/y as a possible marker for a developing restenosis pai-1 and fibrinogen (>800%). resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of ldl, and enhanced affinity of lp(a) for fibrin. to our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (pmnl) were followed under incubation with 60, 300, and 600 i.tm (final concentration) dl-homocysteine (hc) in isolated fractions or whole blood, respectively: l) spontaneous mobility of pmnl, measured as migration distance into micropore filters in a modified boyden-chamber, is found to be significantly enhanced by the two smaller hc concentrations. 2) chemotaxis induced by 0.1 i.tm formylmethionylleueylphenylaianine (tmlp) shows no significant differences. 3)monitoring of chemiluminescence signals (autolumat lb953, berthold) is complicated as hc influences the luminol-mediated indicator reaction. adjusting appropriate conditions the following results are obtained: spontaneous chemiluminescence and that induced by zymosane, tmlp, and the ca2+-ionophore a23187 are entranced by the two higher hc concentrations. there are, however, differences between the blood donors as a minority does not respond to hc in repeated measurements. with phorbol 12-myristate 13 acetate the signal is diminished by hc in all cases and with all concentrations. 4) phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized e. coil (cytoflowmetric evaluation) is significantly increased by the two higher hc concentrations. conclusion: the activation of human pmnl is enhanced with respect to the majority of investigated stimuli by hc in concentrations reached under pathophysiological condititions. the effect of pysical exercise on hemostatic parameters was studied in 12 patients (male, mean age: 55 [range 36-68] yrs) with angiographically documented coronary artery disease (cad) and in 11 controls (male, 53 [43-62] yrs) both participating in an 1 hour group exercise session for cardiac rehabilitation. in each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by doppler ultrasound examinations. patients were all under 6-blocking agents and aspirin. plasma levels of prothrombin fragment 1+2 (ptfi+2) and fibrinopeptide a (fpa) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after 1 hour of exercise consisting of jogging, light gymnastics and ball games. training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to 80+6% (mean-+sd) and in controls to 79-+5% of the maximal heart rate previously determined on a bicycle ergometer. baseline values for ptf1 +2 were significantly lower in oatients (0.67-+0.15 nmol/i; mean-+sd) than in controls (1.01-+0.21; p<0.001 i. after exercise we found an increase of ptf1 +2 in controls to 1.14-+0.24 nmol/i (p<0.001) while in patients ptfi+2 remained unchanged (0.67-+ 0.14 after). accordingly, exercise induced r se of fpa was more pronounced in controls (from 0.62-+0.24 to 1.60-+0.62ng/mt; p<0.001) than in patients (from 0.63-+0.26 to 1.20+0.46ng/ml; p<0.00t). we conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with cad. lower baseline values and lack of exercise induced increases of ptf1 +2 in patients with cad might be attributed to medication with aspirin and/or b-blocking agents. periodontitis marginalis (pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. clinical signs of pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. recent epidemiologlcal studies have provided some evidence for an association between pm and atherosclerosis. in the present paper we will summarise some of the results that we have obtained in studies on patients with pm as well as on patients with hypercholesterolaemia (hc) and atherosclerosis. pm was frequently found to be associated with hc (90 % in rapidly progressive pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and paf-induced aggregation). patients with hc and atherosclerosis had a higher frequency of severe pm when compared with data on the community periodontal health. the severity of pm was higher in patients with plasma cholesterol levels _> 6.5 mm when compared to those with plasma cholesterol < 6.5 mm. in patients with coronary atherosclerosis the severity of pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. these results provide further evidence for an association between pm, hc and atherosclerosis. it can be speculated that hc is not only a risk factor for atheroscterosis but also a risk factor for pm and acts by increasing the reactivity of neutrophils and platelets. on the other hand, pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. it has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of ldl. so far, there is no evidence that atherosclerosis may contribute to the pathogenesis of pro protein z (pz) is a vitamin k dependant plasma protein synthesized in the liver. it promotes the association of thrombin with phosphorlipid surfaces. recently it has been shown that a deficiency of pz may lead to a bleeding tendency. in patients undergoing chronic hemodialysis, disorders of hemostasis are common. to examine if plasma levels of pz are altered in patients with end stage renal disease we determined pz in plasma of patients at the beginning of hemodialysis treatment. the results were compared with a group of 50 healthy controls. the difference of pz levels in plasma of patients with end stage renal disease with the control group was not significant. control group was 2900 + 487 ng/ml and in patient group was 3133 + -1394 ng/ml. one patient with marked bleeding tendency after hemodialysis pz was 1387 ng/ml. we concluded that patients with bleeding disorders pz determination may be helpful. the normal range of actin fs was reinvestigated in a multicentric approach. a protocol was developed which requests from each center to assess the aptt with one common and one variable lot of actin fs in 50 samples of suspected normals. inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. results: a total of 1056 results was obtained. the majority of centers in this study used the electra 1000 or 1600 c (mla). results for the electra group (n = 6) showed a precision for the common lot of actin fs with a common lot of a three level control from 1.5 % (level 1) to 1.1% (level 3) with an excellent accuracy between the 6 centers. clotting times with the variable lots of actin fs were very similar. the results from normals, however, showed a somewhat higher dispersion using the common lot of actin fs. 4 of 6 centers had almost identical mean values (range 26.6 to 27.2 sue) whereas one reported shorter and one longer clotting times (25.2 and 29.7 sec). results with the variable lots gave almost identical results as the common one. a total of 556 results of all lots gave a normal range of 23.5 to 31.7 (5 -95 % percentiles) on electra. mean values on acl (n = 200) were 26.3, on bct, 27.65 sec, on amga coagulometric, 29.4 sec, on amga turbidimetric, 26.6 sec (n = 100 each). all centers used sarstedt monovettes with 3.2 sodium citrate. discussion: the results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. interestingly in the large group of electra users the normal ranges showed some differences, though the controls in all centers were almost identical. this confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. direct acting anticoagulant agents such as hirudin (r-h), argatroban (arg), efegatran (efe) and peg-hirudin (ph), represent specific and potent inhibitors of thrombin. blood samples collected in r-h (10 ~g/ml), arg (50 ~tg/ml), efe (25 ~tg/ml) and ph (10 ~tg/ml) do not clot for extended periods (>24 hours), thus allowing for the collection of plasma for analytical purposes. unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. in contrast to citrate, oxalate, edta and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. we have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. plasma samples collected with these agents are obviously not suitable for global clotting tests (pt, aptt, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, tfpi, tnf and other protein mediators. electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with edta blood. unlike other anticoagulants, these agents do not interfere in the cell staining process. washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. these agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in edta, citrate or heparin. antithrombin ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lu determination by abilgaard method in blood of 40 patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. it was founded, that antithrombin level is decreased to 50-60% of normal values in case of pregnancy pathology, to <50% -in case of acute leukemia and thrombocytopenia, to s0% -in case of anemia. obtained results show the strong relationships between named disorders and patient antifhrombin iii level. therefore anfifhrombine iii estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. bm coasys 110 is a complete automated analyzer system for coagulation tests. it is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. analytical performance and practicability were tested by a common evaluation program in five hospital laboratories. within run and day to day cv's were below 5 % in different samples (controls, patients) . comparison in different therapeutic ranges confirms the declaration of the isi-value for calculating inr-values. normal values for coagulation tests with results in pdmary units were checked in 390 samples and confirmed. due to the optical measuring principle of the bm coasys 110 there was a little tendency to shorter times with the thrombin reagent. in conclusion the performance of coagulation tests with the bm coasys 110 was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling. flexibility and stability of the system permit optimal integration and innovation into the w0rkflow of the routine laboratory. the purified thrombin and antithrombin iii (at iii) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. this interaction is used for isolation and purification of thrombin and at ul from native materials, blood plasma or its fractional products. we have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica (1), heparin-gammapropyl-silica (2), heparjn-tozylsilica (3), and heparin-cellulose (4). we founded that thrombin binds with all sorbents, while at iii doesn't binds with sorbents 2 and 3. there wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t m naci. at iii binds more stronger with heparin-ceuulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. thrombin specific activity and purity degree were approximately twice higher on sorbents 2 and 3 in comparison with sorbents ! and 4 (2250-3000 nih units/mg versus 1260-t350 nih unlts/mg). therefore, sorbents 2 and 3 can be used for isolation and purification of thrombin and sorbents t and 4 can be used for isolation and puriiication of at ii1. we used these sorbents for large scale purification of named proteins. purified thrombin was used for production of diagnostic kits for anfithrombine iii, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. after an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. young volunteers (n=54; median age 25 years) were investigeted~ there was an aerobic exercise (achieved heart rate 130 --10/min, lactate < 4 mmol; n= 27) and an anaerobic exercise (achieved heart rate 150 ~ lo/min, lactate > 4 mmol/1; n= 27). in both groups there was a significant shortening of the ptt. under anaerobic conditions hematocrit and quick significantly increased. factor viii activity rose significantly in both groups. indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 only under anaerobic conditions (tat from 2,5 to 5,4 pg/1; f1+2 from 0,87 to 0,93 nmol/1). indicating activation of fibrinolysis t-pa activity increased significantly in the anaerobic group (from 0,1 to 0,4 iu/ml) but not in the other group. this findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load. membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. as far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). so far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. in this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. the units have yielded excellent results, regarding performance and speed of separation as well as capacity. the application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors viii and ix from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. in terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. the period of time required for separation was five times shorter than with corresponding column chromatographical methods. our measurements showed an excellent correlation of the two systems (r=0,99). the maximum amplitudes on the roteg were on average 2.2% higher than on the hteg, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the hteg. we report first results out of the evaluation of sta compact (boehringer mannheim/diagnostica stack)). sta compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [405 nm] and turbidimetric [540 run]) tests. in addition, it does measure ,,derived" fihrinogen. tests as follows were evaluated: prothrombin time (pt), partial thromboplastin time (aptt), fibrinogen (clauss method), thrombin time, at iii (chromogen), hepato quick, as well as the factors ii, v, vii, x, and viii. results: within run cvs of the clotting tests were below 2% (calculated on the basis of seconds) in most cases, day to day cvs below 4% (not measured for factors, yet). at iii yielded within run cvs below 3% in the decision range. measuring ranges: at iii: 20-140 %; fibrinogen: 1.3-9.0 g/l (plasma -dilution 1/20), after rerun with other dilutions: from 0.3 g/1 (dilution: 1/5) to 18 g[l (dilution: 1/40). method comparisons, using sta as reference, yielded slopes close to 1.00 and negligible intercepts. throughput: with routine clotting tests about 100 tests/h, in a sample selective access mode. we conclude, that sta compact allows precise measurement of routine and special coagulation tests. it is also a reliable system for photometric tests and well suited for intermediate workloads as well as stat analyses. we did evaluate ptt lt, a new liquid, silica based ptt reagent. special attention was given to reference interval and heparin sensitivity. the new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than sta aptt [boehringer mannheim = bm]). it is stable for 4 days in the cooled compartment of the sta analyzer. methods: all experiments were made on sta. for comparison, we used three other ptt reagents (a lab. routine, silica based aptt, as well as sta aptt and sta ptt kaolin from bm). in addition, thrombin time (3 u/ml thrombin, sta thrombin reagent) and heparin (chromogenic xa test, rotachrom heparin) were measured. results: within mn imprecision (n=21) was below 0.7 % cv in the normal range and in two controls (mean values: 35 s, 76 s), and 1.4 % in a heparin plasma (mean: 81 s). between day imprecision (d=10) was below 3% in two controls ( mean values: 34 s and 58 s). the upper limit of the reference range is 40 s (97.5 th perc., median: 32 s; 90 patients with normal coagulation status [routine aptt, fib., pt], median age: 54 years); almost identical reference ranges were obtained with sta aptt and the routine ptt reagent, while sta ptt kaolin showed significantly lower values (97.5th perc.: 33 s, median 28 s). method comparison study: good agreement using plasmas from patients without heparin: (y= 0a + 0.98 x, n= 198, range of(x) from 25 to 79 s, r = 0.97; x = sta aptt). the median values from 54 patients under high dose heparin were: routine ptt: 81 s, sta aptt: 75 s, ptt -lt: 82 s0 sta ptt kaolin 54 s, thrombin time 37 s and heparin 0.5 iu/ml in conclusion, results of the new reagent compare well to our routine ptt and to sta aptt system reagent. it allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. the interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the teg amplitude (a're~). the aa'ec is a measure for the mechanical stiffness of the clot and depends on: a) fibrin formation and adequate polymerisatiun of a 3-dlmensional network: measurements with nonrecalcified citrated blood activated with adp or epinephrine (both n=10) did not show any clot formation in the teg this relies on the need for a mechanical coupling between the teg pin and cup over a distance of 1 mm, which is accomplished by the fibrin network therefore, teg can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by teg. b) the attachment of the dot on the surface of the teg pin and cup. according to recent literature we suggest that the attachment of the clot in the teg relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. we could show a complete interruption of the clot attachment by dipping the pin for one second in 30% albumine solution (n=10). c) the fibrinogen concentration (fg) and platelet count (pc) of the sample. in 50 volunteers we found only a poor correlation of the maximum amplitude (ma) with fg alone (r=0.40) or pc respectively (r=0.50), while there was a very good nonlinear correlation to the product of fg and pc. we suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. this effect of the ptatelets can be completely reversed by gplibfllla antagonists. d) adequate coagulation activation: in nonactivated teg even small amounts of inhibitors can lead to a significant reduction of the ateg. conclusion: alterations in teg measurements can be judged more properly when the underlying mechanisms are understood. the consideration of the limitations of the method allows a more specific interpretation of the results. as a response on a customer request we did investigate the sample stability of blood samples for the aptt. the study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. blood was drawn from 10 donors into 3.2 % sodium citrate and mixed well before it was divided into several aliquots which were kept at room temperature. the aliquots were centrifuged after ~ 0,5, 11, 23 and 29 h after venopuncture and the plasma was analyzed immediately with 3 different reagents on electra 1000. results: there was a clear difference in the change of the apttover time with these reagents. also f viii (determined with a chromogenic assay with complete and standardized activation) change considerably. 2 reagent a: ellagic acid, plant phospholipid, reagent b: sulfatide/kaolin, phopholipids, reagent c: ¢llagi¢ acid, plant and rabbit brain phospholipids the increase of aptt was apparently not a function of the decrease of fviii because the in vitro f viii sensitivity of reagent b. was inferior to reagent a though reagent b showed more prolongation of the aptt than reagent a. reagent c, however, showed only minor changes in the aptt. discussion: these data show that the sample stabifity of the aptt is reagent dependent and that it is not simply a function off viii sensitivity. other factors such as the buffer system but also the sensitivitiy towards other factors than f viii seem to contribute. a comparison of the technical principle of the roteg coagulation analyser and conventional thrombelastographic systems an. calatzis, p. fritzsche. al calatzis, +m. kling, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology yechnische universit~t m0nchen thrombelastography (teg) was introduced by hartert in 1948 as a method for continous registration of the coagulation process. in 1995 we presented the roteg coagulation analyser, using a newly developed technical method. in teg systems according to i/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by 4,75 °. a cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. when coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. the angle of the pin is electromagnetically detected, transformed to the teg amplitude and continously recorded. in the roteg the pin is attached to a short axis, which is guided by a ball beating. thus all possible movement is limited to rpotation (r_oteg). the cup is stationary, and the pin is rotated alternately by 5 ° to the right and left by a feather system. when a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. here the reduction of the rotation of the pin, which is detected optically, is tranformed to the teg amplitude. as can be shown by theoretical analysis and by control measurements, the roteg provides the same measuring capabilities as conventional teg systems. the main advantage is the solid guiding of the measuring system, which makes the roteg easily transportable and less susceptible to shock or vibration during measurement. yhrombelastography (teg) is a standard monitoring procedure for evaluation of coagulation. usually only nonactivated native blood teg measurements (nateg) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. our aim was to develop a coagulation monitoring system based on teg providing fast and specific information on the different components of coagulation. methods: the following measurements are performed in paralel using disposable pins/cups (haemoscope): a) extrinsic activated teg (exteg): 3551al whole blood (wb) + 5~tl innovin (recombinant thromboplastin reagent, dade). b) intrinsic activated teg (integ): 3551al wb + 5~tl kaolin (suspension 5g/l, behring). c) aprotinin teg (apteg): exteg + 20 kie aprotinin (trasylol, bayer). d) heparinase teg (hepteg) as decribed in (1). results: exteg and integ provide information on the extrinsic/intrinsic system within 5-10 min and information on the platelet/fibrinogen status within 10-20 min. because of the addition of potent activators integ and exteg can be performed when inhibitors like heparin are present in the circulation. fibrinolysis effects can be seen on exteg and integ and by comparison of exteg and apteg (apteg: invitro-fibrinolysis inhibition by aprotinin). if fibrinolysis is detected by exteg or integ and aprotinin-susceptibility is verified by apteg, aprotinin therapy will be initiated. heparin effects are revealed by hepteg. discussion: by the comparison of parallel teg measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. the presented tests can be easily performed bedside and only a small specimen of whole blood is needed (0,4-1,8 ml). introduction: a severely prolonged aptt (333s; normal: 40~os) was observed during preoperative screening for a planned splenectomy in a 71-year-old man with an 8 year history of osteomyelofibrosis. fellewing neer-normal~atien (71s) of the ap'ci" after 10 rain preincubation in a kaolin based aptt assay, pk deficiency was suspected and studies were performed to further investigate the nature of the pk deficiency as well as the mechanism underlying the normalization of the prolonged aptt by increasing the preincubation time. methods: the apl-r assay was peal'armed using kaolin/inesithin. high molecular weight kininogen clotting activitiy (hk:c), fxii:c end pk:c were measured by an aptt based assay using neothromtin ® (behnng) and 1 rain (pk:c) or 4 min (hk:c, fxli:c) preincubation. pk amidolytic activity (pk:am) was assayed using cosset pk ~ (chromogenix) and pk antigen (pk:ag) by quantitative immunoblotting. fxll and hk proteolysis dunng activation of plasma by kaolin (10mg/ml at 37=c) or ds (12.5~tglml at 4=c) was demonstrated by immunotilotting assays of fxii and hk following sds-page. assay pk:c pk:am pk:a~i fxfi: the propositus had pk:c<5%, pk:am=5% and pi~ag<2.5% as compared to normal pooled human p(asma (nhp). his son and two daughters had pk:c-50% and normal aptt values, incubation of the propositus' plasma with ds did not result in fxii or hk cleavage within 180 rain, whereas jn nhp detectable f×ii and hk proteolysis occurred after 5 rain and complete proteolysis was observed after -120 rain. in contrast, kaolin activation of propositus' plasma led to slow activation of fxii after 10 rain, presumably by autoactivation, and to fxlla-induced hk proteolysis. near-normalization of the propositus' aptt by prolongation of the preincubation time paralleled fxii autoactivation as evidenced by immunobletting. we describe a propositus with severely prolonged aptt due to hereditary, crm negative pk deficiency suffering from omf. activation with a particulate suspension of kaolin led to slow fxii autoactivation and hk proteolysis, whereas ds in solution did not induce fxii or hk cleavage. fxii autoactivation seems to be responsible for the normalization of the prolonged aptt in pk deficiency after prolonged preincubation times. in our study we compared a conventional bag with silicone tubing (a) for blood donation with 2 new ones (]3 from biotrans and c from baxter) with a newly developed y-shaped adapter. this adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. the 3 systems were identical in amount and content of anticoagulant, i. e. 63 ml of cpd per bag resulting in approximately 14% of the final whole blood volume. the purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. in plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. intralndividual control eitrated (.135 m) blood samples were initially drawn from the contralateral cubital vein from the same male donor (34 in each group). in all bag samples we found small but significantly higher levels of the global test parameters ap'it and ti" compared to controls, indicating a higher amount of anticoagulant. pt, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to mann-whimey). increase of procoagulatory activity measured as tat complexes showed elevated levels in bags a and c whereas prothrombin fragments fl+2 decreased only in a. conceming the fibrinolytic system, plasminogen a~tivators and pai-1 values were diminished in all three systems 03 < a < c) compared to controls. d-dimers were lowest in a followed by slightly higher values in c, controls and b. fibrin monomers did not reveal any significant differences: a < c < controls < b. in summary, the quality, of the 3 different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. the activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. all values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. in 20 patients with acute myocardial infarction (ami) and thromholytie therapy (13 patients with rt-pa, 6 patients with streptokinase and one with heparln) with ck, myoglobin and ekg criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . blood samples were taken before, 30 rain, i h, 2 h, 4 h, 8 h,12 h after lysis and than every day till day 10. because of the central role of factor xii in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor xila initiated by ami and the relation of factor xiia to the thrombolytie agent and reoeclusion rate. for the investigatlens we take the kits from shield diagnostics (xiia), behring diagnostica (c~-inactivator, pl~nogen, ~-antip]~n~n, pap), chromogefiilx ab (prekallikrein) and di~nostica stage (vile). the results: there is an increase of factor xiia immediately after starting the fihrinolysis (max. 30 rain after starting); the increase 5/i independently of the thrombolytie agent. parallel to factor xiia raises factor viia without significant changes of c1-1naotor and prekalllkrein. that means: activation of xiia and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by vila-tissue factor. in some patients is an additional rise in the system xiia -viia, when the fibrinolytic system is already in the normal range. there will need further investigations to define the risk of reocclusion as a result of activation of faktor viia by faktor >li ia. autoimmune thrombocytopenic purpura (aitp) is a frequent complication of chronic lymphocytic leukemia (cll] which developes on different stages of the disease and needs special treatment measure. mechanism of autoimmune disorders in cll remains uncleared. we investigated immunologic phenotype of blood lymphoid cells in 22 patients suffering from cll with aitp. in these patients we did not observe disorders in expression of b-lineage markers as compared with cll patients without immune complications (13 patients). but in the 1st group of the patients the greater number of b-celts expressed markers of activation. according to ig heavy chain expression, the lymphocytes in most cases of cll complicated by aitp had more mature phenotype. in all patients with k phenofype of cll lymphocytes we found immune disorders. the development of aitp was accompanied by lowered level of t cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of cdz~cells and increased one of cd~'÷lymphocytes. the results of our investigations undirectly proved that malignant b-cells in cll are involved in production of autoantibodies against blood cells. dysbalance in t-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in cll a24 in women with severe fvii deficency (<10%) hypermenorrhagia may cause life threatening blood loss. therefore, hysterectomy at a young age is reported frequently in the literature. a 12 year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. the initial laboratory data revealed an abnormal quick-test of 30% due to fvll of 9,0%, normal platelet count and hemoglobin level of 7,2 g/dl. antifibrinolytic therapy (tranexamic acid 4x15mg/kg bw/d) and lynestrenol substitution were started to reduce the hemorrrhage. despite treatment the daily blood loss increased to a maximum of 290ml. therefore, substitution therapy with recombinant fvila (rfvila) (novonordisk) was started at a dose of 15 ilg/kg bw q 6 h. subsequently blood loss decreased to 30ml/d, but even with an increasing dose of rfvlla up to 35 i~g/kg bwq 4h (fvil activity max. 7400% 10 min after injection) and additional hormonal support with a lh-fsh-anatgonist some hemorrhage remained. a short .course of methergin was stopped due to severe pain. ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. it decreased slowly over several weeks and hemorrhage stopped completely after 40 d. the total rfvlla dose administered was 118 rag. no side effects were observed. no transfusions of blood products were necessary. currently, menstrual cycle is suppressed by estriosuccinate. conclusion: due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe fvll deficiency hysterectomy was avoided. in three male members aged between 27 and 52 years of a family suffering from inherited bleeding disorders the diagnosis of protein z deficiency was established. plasma protein z evaluated by elisa (asserachrom protein z, diagnostica stago, france) ranged between 200 and 300 ng/ml. the patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-willebrand factor. spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. the use of pcc containing a high level of protein z in these patients is restrained to severe bleeding disorders or major surgery. defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (crinos s.p.a., v"flla guardia, italy). while the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. since defibrotide is found to be effective in ischemic disorders such as paod, vod related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. in vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with 10 mg/kg defibrotide iv exhibited a profound desensitization to the agonist induced contractile process. the time course of these effects was found to be much larger than the plasma half-life of defibrotide. this presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide. a. wehmeier, a. popescu, w. schneider klinik for h,~matologie, onkologie und klinische immunologie der heinrich-heine-universit&t d0sseldorf in chronic myeloid leukemia (cml), evolution of blast crisis is the limiting factor of survival. however, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. we retrospectively evaluated 182 patients with cml during chronic phase (170 cases), accelerated disease (58 cases), and blast cdsis (72 cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. in chronic phase, 28 patients had bleeding complications (8.4%/patient year) and 15 patients thrombotic episodes (4%/patient year). the incidence of bleeding increased significantly in accelerated disease (18 patients, 51.2%/patient year) and blast crisis (37 patients, 347%/patient year), and many patients had repeated complications. contrary to our expectations, the incidence of thrombotic complications also increased to 10.2%/patient year in accelerated phase and 39.8% /patient year in blast crisis, tn chronic phase, 3 patients died because of bleeding events. in accelerated phase, 5 patients died due to bleeding and 1 patient due to thrombotic complications. in blast crisis, bleeding was associated with 21 deaths, and pulmonary embolism with 2 deaths. analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. however, there was no obvious explanation for thrombotic complications. we conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in cml, and that the incidence of such complications increase in advanced stages of the disease. klinik for innere medizin °, klinikum schwerin patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor x deficiency. we report on a 60-years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (quick 50 %). amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. in addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type x). detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor x deficiency (residual activity 4 %). inhibitors of coagulation factors could not be demonstrated. the treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. due to the extremely rapid clearance of infused factor x, no increase of its activity was observed. chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). over the following six months the frequency of major bleeding episodes gradually decreased. however, subclinical occult bleeding continued. the factor x activity was repeatedly found between 10 and 12 %. we support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor x deficiency if the residual activity is below 10 %. sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (sirs), coagulation abnormalities (dic), organ failure (mof) and severe hemodynamic alteration frequently leading to death in a shock. the aim of our studies was to investigate the efficacy of antithrombin iii (kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. pigs, in this model respond to lps with elevated tnflevels, decreased leukocytes and platelets counts, increased tat and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (pap), indicating impaired lung function. a total number of 13 male castrated juvenile domestic pigs (25 -30 kg) were anaesthetized, ventilated mechanically and infused with saimonella abortus equi lipopolysaccharide (s. equ-lps) over three hours (0.5 ~g/kg * h). a swan-ganz-catheter was inserted into the pulmonary artery to measure the pap. animals were allocated to two groups,, the treatment group (n = 7) received antithrombin iii (at iii) according to the following regimen: 250 u/kg (t = 60 -30, i. v. infusion), 125 u/kg (i. v. bolus, t = 0) and 250 u/kg (t = 180 -240 rain, i. v. infusion). the placebo group ( n = 6) received the appropriate amount of human serum albumin: 50 -25 -50 mg/kg (same schedule as with at iii). main objective was defined as the mortality rate at six hours a_~er s. equ-lps infusion. whereas in the placebo group 4 out of 6 animal died (mortality rate: 66 %) all at iii-treated pigs survived the observation period of 6 hours (p < 0.05, x2-test). the at iii group was shown to have a lower pap than the control group, especially the second peak of hypertension was abolished by at iii. it is therefore concluded that at iii should be a useful tool for the treatment of severe sepsis and septic shock. in a nationwide monthly survey all childrens hospitals in germany (esped) were asked to clinical and therapeutical informations about children suffering from pmi. during july 94 till june 95 299 children were registered. from these, 87 had either ecchymoses and/or necroses related to an increased mordibity and mortality (20%), whereas 212 showed no bleeding signs except for petechiae. of these children one died. the therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. from the patients with ecchymoses or necroses, 13/87 received combination therapy (compared to 5/212 with petechiae or no bleeding sign) of at iii, heparin and/or plasma. only t child received protein c concentrate. the data show that children with low risk did in part receive higher doses of heparin and/or at iii concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. furthermore, the wide range of given therapeutics allows no information about the different medications. therefore, controlled studies with respect to the different therapeutic interventions in children with high risk pmi is desirable. a fully automated procedure for the reptilase time assay y. schmitt (1) and h.j. kolde (2) (1) institute for laboratory medicine, st~dtisches klinikum, darmstadt, frg, (2) dade diagnostics, unterschlei6heim the reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. the procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from bothrops atrox. this enzyme cleaves fibrinogen by releasing fibdno peptide a only but not fibdno peptide b. in contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin iii and hepadn batroxobin is not inactivated by physiological inhibitors. at present this assay is mainly performed manually or on mechanical instruments. we have adapted this assay to the electra 1000 fully automated coagulation analyzer (medical laboratory automation, pleasantville, n.y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (dade diathe clot formation is registrated turbidimetrically and the dotting time is pdnted. the within run precision (n= 10) of this procedure was tested with two plasmas from the daily routine and was between 2.8 and 3.4 %. in 25 normal samples we found clotting times from 10.5 to 12.8 sec. in 30 samples with liver disease (confirmed by pseudochlinesterase < 2000 u/ml) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the electra was compared to the semiautomatic method using a kc 10 coagulometer (amelung, lemgo, germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. the two assays agreed very well with a correlation coefficient of r = 0,948 and a regression line according to passing and bablok of y = 1.0 x + 1.7. these data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the electra 1000. introduction: disseminated intravasal coagulation (dic), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. laboratory diagnosis of dic is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute dic objective: in four cases from a cohort of 43 patients with severe sepsis and dic we analysed special haemostaseological parameters (tat, f1-t2, d-dimers, human-leucocyte-elastese (file), catepsin g and heparin cofactor ii (hc ii)) and correlated them with a mof-score in order to test their predictability on the prognosis of these patients. results: all patients were substituted with at iii concentrate. l,1 the investigated patients median time of treatment with at iii concentrate was 8 (6-9) days and median time of dic-duration was 6 (4-8) days. none of the presented patients died during observation period. all analysed parameters, except d-dimers, showed a sufficient correlation with the evaluated mof-score (tat: r= 0,78; f1-f2: r= 0,84; hle: r= 0,71; catepsin g: r=-0,75; hc ii: 1"=-0,88). the d-dimers did not correlate with the mof-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of dic. furthermore, the decrease of the tat-complexes, f1-f2, hle and catepsin g levels were followed by an increase of at hi and hc ii activity. conclusion: in general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the dic. the hyperfibrinolytic activity of dic is sufficiently represented by the d-dimer test, but is of defered reactivity in the course of dic. unfortunately these parameters are not established in the routine monitoring of dic on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. we have previously reported that notoginsenoside r1 (ng-r1) has an effect on counteracting lipopolysaccharide (lps) induced upregulation of plasminogen activator inhibitor-1 and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [fibrinolysis 1994;8:(suppl 1)119]. in this study we investigated the effect of ng-r1 on prevention of lps induced lethal toxicity in mice. because mice are relatively resistant to lps when applied as a single agent, we sensitized them by simultaneous treatment with d-galactosamlne. the 80% lethality induced by lps (1.5 mg/mouse) plus d-galactosamine (8 mg/mouse) in c3hs-ie mice was reduced to 16% by simultaneous administration of ng-r1 (1.5 mg/mouse) with lps/galactosamine (p<0.05 by x 2 test). ng-r1 also significantly delayed lps/galactosamine induced lethal toxicity from 12 hours to 30 hours with all animals surviving beyond 30 hours. because lethality induced by lps involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (tnf)-ct, interleukin (il)-i, interferon 3' etc., we also investigated the effect of ng-r1 on lps induced tnf-ct production from leukocytes in cultured human whole blood cells (hwbcs) ex vivo. the production of tnf--ct induced by lps (1 ng/ml for 24 hours) in the supernatant of hwbcs was inhibited by 46% and 22% respectively, when the cells were incubated 1 ng/ml or 10 ng/ml lps together with 100 i~g/ml ng-r1, respectively (tnf-ct concentration, 1 ng/ml lps treated cells: 297+192 pg/ml, i ng/ml lps plus 100 l.tg/ml ng-ri treated cells: 162+137 pg/ml, p<0.01; 10 ng/ml lps treated cells: 3094_+487 pg/ml, 10 ng/ml lps plus 100 pg/ml ng-r1 treated cells 2423+713 pg/ml, /'=-0.02). the present results suggest that ng-r1 can prevent the onset of lps toxicity as well as the lps induction of cytokines. therefor ng-ri may be effective in preventing the effects of septic shock in gram-negative infections. to elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. this group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. 62 patients with febrile infectious events were accrued to the study. of these, 13 patients progressed to severe sepsis and an additional 13 patients to septic shock. at onset of fever, factor (f) vlla activity, f vii antigen and antithrombin iii (at iii) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: 0.3 versus 1.4 ng/ml, 21 versus 86 u/dl and 45 versus 95%; p < 0.001 ). the decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment 1 + 2 (medians: 3.6 versus 1.4 rim; p=o.05). these differences were sustained throughout the septic episode (p < 0.0001 ). f vlla and at ill levels of <0.8 ng/ml and <70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100 and 85%, and a specificity of 75 and 85%, respectively. in contrast, fxila-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (p=o.001). thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. furthermore, in septicemia both f vlla and at iii measurements are sensitive markers of an unfavourable prognosis. hemostatic parameters in sepsis patients treated with anti-tnfct monoclonal antibodies c. salat 1, p. boekstegers 2, e. holler 1,3, b. reinhardt i, r. pihusch 1, k. werdan 2, m. kaul 4, t. beinert 2, e. hiller 1 med. klinik iii 1 und i 2, klinikum grosshadern der ludwig-maximilians-universitat monchen, h~imatologikum der gsf 3, knoll ag ludwigshafen 4 tumor necrosis factor et (tnfc~) is a central mediator in the pathogenesis of sepsis and septic shock. as administration of anti-tnfct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. tnfct exerts a procoagulant effect, e.g. by enhancing pai-i and activating thrombin as indicated by an increase in tat and pf 1/2 levels. therefore it may be involved in disseminated intravascular coagulation in sepsis. we determined tat, pf 1/2, d-dimers, tpa, upa, pai-i and vwf levels in 30 patients with sepsis or septic shock. 14 patients received the anti-tnfa monoclonal antibody mak 195f (knoll ag, ludwigshafen), whereas 16 patients served as controls. we found a significantly lower level ofupa in anti-tnfc~ treated patients. since the difference existed before onset of treatment it can not be attributed to tnfot antagonisation. all other parameters investigated did not differ significantly between the two groups throughout the study period. failure to detect modulation of hemostasis by anti-tnf~ might be explained by delayed initiation of treatment in clinical sepsis. in animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or 30 minutes after endotoxin challenge, but not when it was administered 2.5 hours later. in addition, beneficial clinical and hemostatic effects of tnfet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. larger studies addressing this point are under way. protease receptors for thrombin and trypsin have been described for different cell lines. we investigated the ability of trypsin to activate human umbilical vein endothelial cells (huvec). cell activation was measured by the increase of intracellular free ca 2* (caff) with help of microscope fiuorometry (fura-2) and by the von willebrand factor release measured by a sandwich elisa. incubation of huvec with thrombin (1u/ml) or trypsin (10nm) showed a 2-10 fold increase of c~ff. a subsequent homologous stimulation after 80 s lead to a 2-5 fold lower concentration of ca~ 2÷ compared to the first stimulation. therefore cells have been desensitised by the first stimulation. inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of ca~ 2÷ concentration. in cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. the same effect was measured with first stimulus trypsin and second stimulus thrombin. trypsin and thrombin induced a release of von willebrand factor (2-5 fold in comparison to unstimulated cells). we found a vwf release dependent on the concentration of trypsin similar to thrombin. an electrophoretic analysis of the released von willebrand factor showed a different multimeric composition of vwf between trypsin and thrombin stimulation. these results indicate, that there might be a protease receptor on huvec for trypsin being different from the thrombin receptor. clinical and laboratory findings of coagulopathy were investigated by an 1-year-survey to 320 children's hospitals. 291 meningococcal infections were evaluable. severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in 42 of these children; 29 of those survived and 13 died. clinical signs of severe coagulopathy were seen in 83 children: ecchymoses (n = 73) and skin necrosis (n = 36) were associated with increased mortality (16% and 20%, resp., compared to 4.5% overall mortality). five of 29 surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). petechiae were frequent (n = 156) and as isolated finding not related to severe disease or fatal outcome (6% mortaliy). platelet counts at admission were lower in non-survivors (10th-90th percentile: 30 -450.000/gl, median: 139.000/i.tl) than in survivors (10th-90th percentile: 140 -480.000/i.tl, median: 242.000/gl). at iii values showed no difference between survivors and non-survivors. protein c was available in few patients (n =14): in this subgroup, protein c was lowered in patients with limited disease (10th-90th percentile: 20 -105%, median: 48%) as well as severe disease (10th-90th percentile: 30 -75%, median: 60%). in conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease. the influence of irradiation on pai-i and vwf levels in human umbilical vein endothelial cell cultures k. fragiadaki, c. salat, r. pihusch, b. reinhardt, m penovici, e. hiller med. klinik iii, klinikum grosshadern der ludwig-maximilians-universitat monchen an elevation of pai-1 in bone marrow transplant recipients developing veno-occlusive disease (vod) of the liver has been described earlier. endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. in order to investigate a possible role of irradiation we studied the influence of several doses (0, 5, 15, 30 gy) on pai-1 and vwf levels in the supematant of human umbilical vein endothelial cell cultures (huvec). pai-1 antigen and vwf were determined by enzyme immunoassays. whereas pai-1 and vwf levels remained unchanged alter irradiation with 5 gy and in control cultures, a rise was observed one day after irradiation with 15 gy (mean day 0"-)day +1) in pai-1 (100,0% --)171,2 %) and vwf (100%--)159,7%) levels. the increase was more pronounced and reached levels of statistical significance after a dose of 30 cry (pai-1 100%--) 278,7% and vwf 100%--)168%). both pai-1 and vwf levels decreased on day 2 after irradiation with 15 and 30 gy. our results indicate that irradiation induces an increase of pal-1 and vwf in endothelial cells. nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive 3x4 gy. additional factors seem to be of significance. cytokines like tnfo~ enhance pai-1 and vwf in endothelial cell cultures and are known to be elevated in bmt-associated complications. it can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tpa and concomitant elevation of pai-i secreted from inflamed endothelial cells. in an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and pal-1 antigen was measured 2h, 4h and 24h after stimulation. in contrast to results published from experiments performed with macrovascular human umbilical vein cells (huve), our results obtained with 3 different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced pal-1 antigen levels. in addition to tnf-a (25ng/ml) and lps (10pg/ml), we found that il-10 (100 u/ml) and gm-csf (100 u/rot) also reduced pai-i levels within the first 2h of incubation (from 120ng/ml to 80-110 ng/mll and the effect was even more pronounced after 4h and 24h (from 380 ng/ml to 250 ng/ml). il-1 (10 u/ml) and lps (10 pg/l) also reduced constitutive levels of pal-1 but the effect occured later than 4h after addition of the stimulator. the strongest synergistic effect was demonstrated with gm-csf plus il-1 resulting in pal-1 suppression of 50% after 2h and 30% after 24h. in contrast, g-csf (300 u/ml) induced the immediate (120 to 140 ng/ml after 2h and 380 to 420 ng/ml after 24h) upregulation of pal-1 antigen. stimulation of pat-1 levels was also observed with tgf-i~ (10 pg/ml), however not earlier than 18h of incubation. interestingly, both stimulatory cytokines, ie. g-csf and tgf-13, alone were able to counteract the decrease of pat-1 antigen by tnf-a but only a combination of g-csf plus tgf-g neutralized the effect by il-1. results indicate that inflammatory cytokines regulate pal-1 fibrinolysis in a synergistic and antagonistic fashion. we established the culture of human brain microvascular endothelial cells (hbmec) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. it is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. the human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. ery throcytes containing later stages of p. falciparum specifically bind to capillary ec in vivo (sequestration). tnf-cq il-1 and il-6 are considerably elevated in severe malaria. coagulation factors such as tissue factor and von willebrand factor are affected by malada suggesting the involvement of the hbmec in cerebral malada. so far, research on the involvement of the hbmec has been performed on ec cultured from human umblilical veins (huvec). the relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. for the~ reasons, our laboratory has established the hbmec as a model to study the pathophysiology of human cerebral malaria. to demonstrate the relevance of this model in the context of malaria, hbmec were challenged with sera from different patients with severe p. falciparum malaria and with serum from a healthy donor. we can demonstrate that in cells challenged with malaria patient sera icam-1 and substance p were upregulated. on the other hand cells challenged with serum from a healthy donor expressed neither icam-1 nor substance p. these results strongly suggest the relevance of this model for vessel involvement in malaria. both, histamine and serotonin have been described as potent stimulators of yon willebrand factor (vwf) release from human umbilical vein endothelial cells (huvec). we performed experiments to differentiate the receptors for histamine and serotonin induced vwf release. absolutely unexpected we don't found any significant vwf release after the addition of serotonin to huvec or human artery endothelial cells (huaec) in concentrations from 0.1 ijm to 50 pm. in the case of histamine (0.1 pm -50 pm) we measured a vwf release 2-5 fold compared to unstimulated cells. this release was in the same order of magnitude as the release induced with 11u thrombin. to verify these results we measured the effect of histamine and serotonin on the intracellular ca 2÷ concentration (ca~ 2÷) in huvec and huaec. cells were labelled with fura-2 and the change in fluorescence after agonist addition was measured with a microscope fluorometer. using the same agonist concentrations as above we found an 5-10 fold increase of caj 2. with histamine or thrombin but no effect by addition of serotonin. this results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vwf release or increase of cay. activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin-2, tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. pal-l and tm were assessed as parameters of activated endothelium following bsct in three to four days intervals from start of conditioning therapy through day +35. data were compared to the occurrence of sepsis, veno-occlusive disease (vod), capillary leakage syndrome (cls) and graftversus-host-disease (gvhd). patients with neither complication served as controls. no *days after stem cell tranplantation pai-1 and tm were increased in all patients with sepsis, cls~ vod and/or gvhd. pai-1 peaked at days 14 to 18 and the increase was highest in sepsis and lowest in cls. the increase in tm values was somewhat delayed (day +24) and was highest in vod and cls and lowest in gvhd. pai-1 and tm are sensitive markers of endothelial activation in sepsis, vod, cls, and/or gvhd, but they do not allow a differention between these complications. endothelin (et) is the most potent vasoconstrictor. it is known that et plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (cm). the contribution of the heart to the production of et is still unknown. to investigate the pathogenetic mechanism in patients without coronary artery disease (cad), we examined 13 patients with hypertension ( . pulmonary capillary wedge pressure (pcwp) was measured in all patients. et and its precursor big-endothelin (bet) were determined at rest and after pharmacological stimulation with dipyridamole (0.5 mg/kg body weight), that increases coronary blood flow by factor 2 -4 on a non endothelial pathway. cardiac coronary et and bet concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). blood samples were collected into ice chilled vacutainer tubes and stored after centrifugation at -70 *c. et and bet were analysed after extraction by a sepal< c 18 cartridge by radio immuno assay technique (immundiagnostik). it is concluded that et is increased with elevated filling pressures of the heart in patients with cm. it is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. there4ore the lung has to be considered as the major organ for the production of et and bet in patients without cad. to characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. it seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase-4-studies. we studied the influence of 21 reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.the following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (wu-gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to breddin and bfirck (1963) and an interactive computer-aided system (ibas, kontron gmbh, manchen, frg) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of 512x512 pixels. results: platelet counts showed significant differences pre and post polymer contact, the wu-grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (pre contact (n=1000): 27:~-6 large forms of platelets, 700~-39 small forms and 275:l-41 spider forms; post contact (n=1000): 6-+-5 large forms, 510a:56 small forms and 484±58 platelets with spider threads). in some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. however, the relative increase of these nonspread spider forms could be stated with beth methods (wilcoxon test). we therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase-4-studies in patients. however, the ibas-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. 6%. in cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. in order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: htk (group 1) and st. thomas (group 2). after randomisation each group consisted of 20 patients who had to undergo aortocoronary bypass surgery. aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures. in the context of this study the classical enzyme-diagnostical methods ck,ck-mb and ldh as most useful, however not as convincing. still, we have in the meanwhile been able to show that the cardiac muscle troponin t proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to conflrm in extensive preliminary trials. cardiac troponin t was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac troponin t. simultaneously the corresponding pre-and postoperative ecg was registered. further, within this context we investigated parameters that indicate cellular damage, such as platelet factor 4 (pf4), t-pa, interleukin-6 and pmn-elastase. in the reperfusion phase in group 2 there is a significant rise in tmponin t while in group 1 these values remain practically unchanged up to the 1st. postoperative day. of special importance is interleucin 6 since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. this pathway must, further, be regarded within the context of free radical formation. on the 1st. postoperative day the 11 6 values in group 2 are significantly higher. the effects of membrane damage is also observed via pf4 and the pmn-elastase to be different in both groups. on the basis of this study we arrive at the conclusion that the htkcardioplegia is essentially less damaging than that of the st. thomas solution. (2) r. hetzer (2) (1) department of hematology and oncology, vimhow klinikum, humboldt university, berlin, germany (2) we investigated the influence of two different vad systems on these hemostatic changes. vads were implanted in 18 patients [11 bi-vad (berlin heart), 7 left vad (novacor n 100)] with end-stage heart disease who were awaiting heart transplantation. the following hemostatic parameters were measured during the first 51 days of bddging or until heart transplantation: thrombin-antithrembin iii (tat) complexes, prekallikrein, factor (f) xll, plasminogen, or2 -antiplasmin, and i?,thremboglobulin. results: during the first week of bridging, significantly higher tat levels were observed in novacor patients compared to berlin heart patients. prekallikrein activity levels were significantly lower in the berlin heart patients in the early bridging period. all other parameters were comparable in both groups throughout the entire observation period. differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the novacor group and more prominent contact activation in the berlin heart group. avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. patients and methods: arer randomization apparative intraoperative autotransfusion was performed in 5x7 patients during elective hip surgery using i-iaemonetics cell saver ill, haemonetics cell saver v, electromedics elmd, haemolite 3 and fresenius continuous autotransfnsion system (cats). at defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at 9 determination points in the reservoir, the retransfused blood and in the patient. results: no significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. increased hemolysis (p<0.01) in the hcs iii group vs. the other groups (lo rain. after application of the retransfnsed blood). low heparin concentrations of retransfused blood in the hcs iii group( 0.32+-0.3 u/ml) vs. high concentrations in the cats group (0.47 +-0.3;p--0.01). parameters of thrombin generation were elevated in the hcs iii group vs. the other groups (p=0.02). conclusions: the use of 5 different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. the activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. however intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. little information is available on the management of patients with factor viii deficiency who require cardiac surgery. we report the case of a 54 year old man with factor viii deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. he had a history of several bleeding episodes following minor surgery. previous factor viii levels were between 8 and 26%. using standard cardiopulmonary bypass, a double valve replacement with a 23 and 29 mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. a high dose aprotinin regime was used (5.5 x 10 a iu). three doses of factor viii concentrate were given in the perioperative period, totalung 7000 1u until the 1st postoperative day. repeated measurements of the factor viii level were performed. the postoperative chest tube drainage was 350 rot. until the 4th postoperative day an additional dose of 3000 iu of factor viii was given to maintain a level of at least 30%. the obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. due to a persistent 3rd degree av block a permanent pacemaker was inserted with additional 2000 iu of factor viii. on the 17th postoperative day warfarin was commenced aiming for an inr of 3.0 -3.5. the patient was discharged home therearer. he was trained to monitor his inr with a coagu chek device. no bleeding episode occurred during the first 3 months follow up. open heart surgery can be performed safely in patients with factor viii deficiency with the use of factor viii concentrates and monitoring of factor viii levels. coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. stents were coated with a thin layer consisting of a polylactide polymer containing peg-hirudin and a stable prostacyclin analogue. these materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. this observations were supported by analysis of coagulatiuon activation markers. unlike coated stents, uncoated stents revealed high levels (>detection limit) of tat complexes and prothrombin fragments (f1-2). in a series of experiments stents were tested in sheep. in 16 sheep stents (coated/uncoated patmaz-schatz stents) were ptaced by conventional techniques in the left anterior descending artery. anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. no ant/coagulation was given thereafter. existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in january 1996). this coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. studies concerning catheters, vascular prosthesis and oxygenators are in progress. the mechanical circulatory support (mcs) is a therapy for patients (pts) with endstage cardiac insufficiency. during mcs thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. activated blood platelcts play a major role in this context. therefore, patient's platelet morphology was investigated. during the period of mcs, using the novacor left ventricular assist system n100, blood samples of 8 pts were observed by means of scanning electron microscopy (sem). blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first 3 months. samples were drawn via an 18gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. additionally, the common blood coagulation parameters were evaluated. preoperatively, 15.0 + 4.6 % of activated platelets were found. within the first postoperative week, the mean level of activated platelets raised to 32.8 + 8.0 % (p<0.05). comparing short-(<30 days) vs. long-term (>30 days) mcs, a significant difference of activated ptatelets (overall mean values) could be seen (24.3 +_ 3.3 % vs. 34.8 _+ 3.4 %, p=0.004). during mcs a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. also, specific platelet deformations and damages appeared during mcs, which could not be found preoperatively. all pts with mcs showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. with regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. during extracorporeal circulation (ecc) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. furthermore leukocyte activation occurs and platelet function is impaired. these humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. the impertance and even perhaps the existence of the damaging effects of cpb have been widely debated in the literature over the past 30 years. many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators. recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during cpb, the here presented study was designed to assess changes in coagulation and flbrinolytie activity in 20 patients undergoing cpb. in this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments fl+2, thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after cpb. the activation of the complement cascade was followed by measuring the concentration of c5a, c4 and c3c. the results demonstrate distinct alterations in above mentioned parameters. in spite of a high dose hepariulzation (act>450s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of cpb followed by an activation of the fibrinolytic system. therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for cpb. during cardiopulmonary bypass blood is exposed to nonphysiologic conditions. the contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. this so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy. our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in 20 patients undergoing cpb. at different time points, pre, during and pest cpb, we determined the levels of interleukin (il) 113, il-2, il-4, il-6, il-8, il-10, tumor necrosis factor ¢z (tnf-a) and interferon "1' (ifn'--/) using elisa-techulques. lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against cd3 (pan t-cell marker), cd4 (surface antigen on t-helper cells), cd19 (surface antigen on b-cells), monocytes were determined by cd14 and platelets by cd41 (act. gpilb/llla) and cd42b (gp ib). single cell activation was analyzed using markers against cd25 (il-2 receptor), cd126 (il-6 receptor), hla-dr (mhc class ii), cd71 (transferrin receptor) and cd69 (activation inducer molecule), platelet activation was monitored with an antibody against cd62 (gmp-140). preliminary results revealed distinct increases in r,-6, il-8, and il-io following cpb whereas tnf-a and ifn--/levels were not significantly influenced. fttnhermore, activation of particular cell populations was observed. finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by cpb and thus might help to develop new strategies to circumvent the negative impacts of cpb. optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (ffp) as well as for the safety of the donation. in the present study the suitability of prothrombin fragment ( ft+2 ) in the assessment of anticoagulation during plasmapheresis was investigated. matarlal and methods: 75 plasmapheresis procedures were performed on 25 donors (10 ~, 15o" ) using 3 different plasmapheresis machines (a 200, baxter; mcs 3p, haemonetics; pph 900, electromedics/medtronic). acid citrate dextrose formula a (acd-a) in a ratio to whole blood of 8 : 92 was used for anticoagulation. the concentration of fi+2 in the donor's blood was measured before and after plasmapheresis and in the prepared ffp. the actual acd-a volume used was also registered. results: there was a significant rise of the ft+2 -concentration in the donors blood after plasmapheresis with each of the three automatons: a 200:1.32 vs 1.14, p < 0.05; mcs 3p: 1.26 vs 0.98, p < 0.05; pph 900:1.20 vs 1.05, p < 0.05. the ffp prepared with each machine showed the following f~+2concentrations: 0.91± 0.18, 1.0:2 ± 0.17 and 0.93 ± 0.11 respectively. the difference between the groups was not significant. the elevation of the ft+2 -concentration in the donor's blood showed a negative correlation with the volume of the acd-a used. during 6 of the 75 procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). after these procedures there was a marked elevation of f~+2 in the donors blood (2.74 ± 0.53), accompanied by an elevated f~+2 -concentration in the prepared ffp's. conclusion: these data show that ft+2 is a suitable parameter for the assessment of anticoagulation during plasmapheresis. several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. prothrombin time derived fibrinogen (df) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. several studies showed that the df values correspond well with the clanss method except in cases such as thrombolytic therapy in which the df results are higher. however, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the df values are also comparable to the established clausss method. the aim of our study was to compare df values to clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (cab(]). measurements of df were performed on an acl 3000 (il) using the pt-fibrinogen-hs reagent. fibrinogen clanss method was done on the acl using fibrinogen c reagent (il) and on a kc4 (amelung) with fibrinogen a reagent (boehringer maanheim). for calibration we used the calibration plasma half volume (it.) with the fihrinogen concentration proposed by the manufacturer. plasma samples were obtained from 24 patients at admission before cabg and postoperatively up to 1 week, and from 23 healthy persons (staff). within assay imprecisious using normal and abnormal controls (il) were comparable with both methods showing cvs between 1.99 and 4.22 %. in normal healthy persons the medians of the df and the clanss method run on the acl were very similar (296 vs 302 rag/all), whereas kc4 values were about 10% lower (268 md/dl). in cabg patients at admission we found the same differences as in normals with the clanss method (acl: 363 vs kc4: 337rag/all), however the df values were siginficantly higher (median 418mg/dl). if we took a cutoff value of 320 mg/dl, as suggested by the results from the northwick park heart study, we would categorize into the high risk group 21 out of 24 patients using the df method, 20 with the clanss-acl method and 16 with the clanss-kc4 method, i.e. nearly 30% more patients were classified in the high risk group using the df method. postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. because of its rapidity and reproducibility the df method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. for fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to clauss (cfib) and the sn called "derived" fibrinogen method (dfib) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (pt) assay. it is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (fdp), there is a discrepancy between the two methods with higher values for dfib than for cfib. yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. the patient (male, 26 years) had ingested the esterase inhibitor parathion (e605) in an attempt o f suizide and was treated with high doses of atmpin. he had no clinical signs or history or family history of bleeding or thrombotic disorders. except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding pt, afft, thrombin time, and factor xiii. pt and aptt did nnt differ between an optical coagulometer (electra 1000c, mla) and a mechanical one (kc..4, amelung). there was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. however, in all 7 blood samples received for dotting tests during a period of 7 days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low dfib on the electra (pt reagent: thromboplastin is, dade) and a normal or high cfib (kc4; thrombin reagent, dade). on admission, values were 57 mgml (derived) vs. 275 mgldl (clauss). cfib rose to s42 mg]dl with dfib at 155 mg/dl in the last sample on day 7. ~ al! samples dfib was about 20 % (ls-23) of cf[b. when the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for dfib and values slightly higher than predicted for cfib. in the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: 1) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (cfib correct, dfib falsely tow); 2) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (dfib correct, cfib falsely high). in either case, the molecular basis could be: a) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; b) an interfering substance. direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. we hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. however. a multitude of methods for fibrinogen determination is available. there is a lack of standardisation among fibrinogen assays. in a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from 340 family members using a functional and an immunochemical assay. the fimctional assay according to clauss was performed on the analyser ca 5000 using the test fibrinogen a from boehringer. the immmmephelometric assay was performed on ~e behring nephelometer system using the reagent and standard from behring. a good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased c-reactive protein (crp). values obtained by both assays correlated similar with total cholesterol, ldl--cbelesterol and apolipeprntein b. the ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-pa, v wiuebrand factor and crp. release of two fibrinopeptides a from fibrinogen generates desaa-fibrin monomer, which rapidly aggregates, forming fibrin complexes. fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain neo-n-termini generated by fibrinopeptide release. although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide a and one fibrin alpha-chain neo-n-terminus has so far escaped analytical procedures. we have employed a monoctonal antibody specific for fibrin alpha-chain neo-n-terminus, mab 2b5, attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. the material was desorbed by sds-urea buffer and subjected to sds-page and immunoblotting. immunostaining with panspecific anti-fibrinogen and anti-fdp-e antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin e-domain. lmmunostaining with monoclonal anti-fibrinopeptide a antibody resulted in a doublet band corresponding in size to fibrin monomer. similar results were obtained with polyclonal antisera against fibrinopeptide a. for a more quantitative approach, desa-fibrin monomer was detected by an elisa procedure using mab 2b5 as capture and monoclonal anti-fibrinopeptide a antibody as tag. a sample with extremely high level of desaa-fibrin monomer, determined by elisa (enzymun®-test fm) was used for calibration, since reference material is not available. a correlation of r=o.g4 was found between desaa-fibrin monomer and relative desa-fibrin monomer levels. detection of desa-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. from these preliminary data it appears that desa-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. fibrinogen has been shown to be a major cardiovascular risk factor. especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. fibrinogen levels are generally measured by clotting assay according to clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derfbg). the clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. the pt-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. we have compared the clauss assay and derfbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. d-direct antigen was measured in parallel using tinaqaant d-dimer lpia. results were correlated with total fibrinopeptide a release by thrombin, measured by elisa. a total of 484 samples were included, of which 29 samples (6 %) were recorded as above measurung range by derfbg. these samples encompassed a range of 5.90-10.40 g/l and 5.21-12.37 g/l in clauss, and turbidimetric assay, respectively. the range of values measured by derfbg assay was 0.72-9.14 g/i, corresponding to 0.26-11.00 gll and 0.24-10.48 g/1 in the clauss and turbidimetric assay, respectively. the correlation of derfbg with the clauss assay was re0.91, correlation with turbidimetric assay was r=0.92 for the values actually detected. the correlation between clauss and turbidimetric assay was r=0.93 for all values. there was no dependency of test results or inter-test variation upon d-direct. correlation graphs displayed a decreased test response of clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. the derfbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. correlation with fibrinopeptide a release was r=0.88 for clauss assay, r=0.89 for turbidimetric assay, and r=0.82 for derfbg. for clinical routine, derfbg appears to be applicable for all samples between 1.00 and 5.00 g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement of high range samples. for inhibition of pk is 0.067 pmol/l the antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• the compounds which inhibit plasmin and pk influence remarkably the streptokinase-induced clot lysis but not lysis induced by uk and tpa. surprisingly, inhibitors of uk and tpa do not influence clot lysis induced by uk or tpa. the structure-activity relationships for inhibition of ptasmin, uk, tpa and pk could help in the design of more potent inhibitors of fibrinotytic enzymes. uk inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/pk inhibitors could be prototypes of a "synthetic aprotinin". in the ecat angina pectoris study t-pa antigen was an indepcndem risk factor of subsequent acute coronary syndromes. pat indicates the risk bat depends on other known risk factors. it should be tested in 183 members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active pal antigen or the whole pai antigen showed a stronger relation to t-pa and metabolic variables. the active pall antigen was determined using elisa actibind pat-1 (technoclone / lmmuno) , the whole pai-i antigen was measured using the f_lisa pat-1 (technoclone i immuno). t-pa activity was determined with the coaset t-pa from chromogenix, the tintelize tpa from biopool was the used test for determination of t-pa antigen. the active pat antigen showed a stronger correlation to t-pa activity and t-pa antigen than the whole pal antigen. circulating t-pa activity was influenced predominantly by the active pal antigen. both pat antigens were correlated in similar manner with metabolic variables, lipoproteins and b/vii. table: correlations of active and whole pal antigen (** p < 0,001) active pal antigen whole pal antigen active pat antigen 1,000 0,851 ** whole pal antigen 0,851 ** 1,000 t-pa activity -0,594 ** -0,492 ** bpa antigen 0,604 ** 0,497 ** body mass index 0,502 ** 0,462 ** triglycerides 0,452 ** 0,441 ** total cholesterol 0,252 ** 0,255 ** ldl-cholesterol 0,263 ** 0,264 ** hdl-cholesterol -0,357 ** -0,355 ** apolipoprotein b 0,428 ** 0,402 ** apolipoprotein a i -0,233 ** -0,211 ** the lower relationship of the whole pat antigen to t-pa is obviously caused by patient samples with high levels of whole pat antigen in contrast to normal values of active pat as well'as of t-pa. possibly, a high ratio of whole pai antigen / active pat antigen is caused by a raise of latent pal the main form of pat in the platelets. the clinical importance of an increased ratio whole pal antigen / active pal antigen remains under investigation. the cyclic antibiotics-polypeptides bacifracin a, bacilliquin from boci/lu~, licheniformis and gramicidin s from bocil/us brevis, var. (3. b., were used for investigation. we studied their influence on the fibrinoly~c and coagulation activity in vitro• me~hods. to solution of human plasmin (thrombin). containing 0.2 mg of protein (1 nih unff)/ml, the analyses' solution of antibiotics (0.1-8,0 mg) was added. then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution. results. in following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity): ki, mm ki --the constant of inhibition. n. d. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. i. --the inhibitor% activity was observed but ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). conc/us/on.~ the results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. these results were used for preparation in our laboratory of biospeciflc sorbents containing c-ramicidin a, bacil}iquirt and gramicidin s.as ligands, they can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. the enzymes are selectively eluted without substantial losses of specific activity in e yield of 60-90%. there is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tpa or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon willebrand factor levels. in vitro clo~/-[ lysis tests, induced by tpa were performed in 41 patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method. the tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, huvec}. clot lysis speed proved to be 1.5-2 times slower with huvec milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in 5-fold reduction of lysis speed. our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. deep vein thrombosis (dvt) is a systemic disease with prolonged clinical manifectation. anticoagulation therapy in dvt is not completely effective. thrombolytic therapy may give rise to a systemic lytic state, the fibrinospesific agents (scu-pa and t-pa) have short half-lives in the circulation. we investigated the potency of the acylated plasminogen streptokinase activator complex (gbpg-sk) to deep vein clot dissolution as compared to well known sk and apsac both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. it was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. stability studies carried out by incubating sk and pg-sk activator complexes in plasma with euglobulin precipitation . total fibrinolytic activity was measured by the fibrin plate method. gbpg-sk possessed the greatest stability in human plasma than apsac or sk because of its prolonged inactivation period (the deacylation half-life for gbpg-sk was 230 :e 21 rain in contrast with 73 -~ 6 min for apsac). the stability degree of two acylated thrombolytics (gbpg-sk and apsac) was in order to inverse proportion of their first order rate deacylation constants (2.9 • 10 -4 and 6.0 • 10-s sec-1 respectively). the fibrinolytic potency of sk, apsac and gbpg-sk was measured by 1251-labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed 1251-labeled fibrin clot inserted into the jugular vein. fibrinolytjc activity of acylated plasminogen activators gradually increased in time. under sk administration, the clot lysis came to the end by 2 hours while apsac and gbpg-sk haven't lost their activity for 5 -6 hours. gbpg-sk possessed significantly more prolonged fibrinolytic activity than apsac, the acyl-enzymes did not significantly influence on plasminogen,,.~2-anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. in opposite, sk produced a significant depletion of plasminogen, ~-2antiplasmin and fibdnogen levels in plasma. it seems, on the basis on in vitro and an animal experimentation, than apsac with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but gbpg-sk with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. it's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. at present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. 125i-labelled partially cross-linked fibrin clots of different volume (0.1-0.35 ml) were immersed in tris-hcl buffer (3 ml) containing plasmin (5-100 nm) at 37°c. the lysis rate was detected by counting of soluble fibrin degradation products (fdp). at all the eases lysis slowed down and stopped in 3 hs though clots dissolved up to only 60-85%. no irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. however the increase of fdp concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to 5% of baseline. the sds-page analysis under non-reduced conditions shown the acoumulation of high-molecular weight fdp at the surrounding buffer. the inhibition phenomenon could be connected with the specific binding of plasrnin with soluble fdp having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. to estimate the kinetic parameters the kinetic curves were linear in the coordinates [p1/t (l/t*ln(isl°{(lslo.lpi)). the obtained parameters were following: keat=l.36 min-l,km=l.33 ixm,kp=0.12 ~tm. the clinical trials have shown that fdp concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range 0.05-0.2 ~tm. therefore the described phenomenon of fibrinolysis inhibition by formed fdp may take place during thrombolytic therapy. al. calatzis, an. calatzis, +m. klmg, +l. mielke, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology technische universit~.t monchen thrombelastography (teg) is an established method for the detection of fibrinolysis. fibfinolysis is usually determined when the teg amplitude decreases by more than 15% atter the maximum amplitude is reached. this takes a considerable amount of time (more than 30 minutes). our approach bases on the understanding of fibrinolysis as a process which runs in paraue[ to coagulation and is not exclusively subsidiary to it. the effect of fibrinolysis on the growing clot in the teg is shown by the comparison of two parallely performed teg measurements: exteg: teg measurement with standardised activation of the extrinsic system. apteg: exteg with in-vitro-fibrinolysis inhibition via aprotinin. exteg-reagent (ex): 1:2 dilution of innovin (recombinant thromboplastin reagent, dade) with aqua dest. apteg-reagent (ap): 5 parts innovin, 2 parts trasy[ol (aprotinin, bayer, i0.000 kie/ml), 3 parts aqua dest. test procedure: l0 p1 ex or ap + 300 ~l citrated blood (cb) + 50 lal cacl2-solution 0,15 m. the only difference of the two reagents is the addition of 20 kie aprotinin in the apteg, leading to an in-vitro fibrinolysis inhibition. the usage of disposable pins and cups (haemoscope, illinois, usa/e.m.s., vienna) is recommended for ensuring standardised conditions for both measurements. results and discussion: when there is a better clot formation in the apteg (corresponding to a lower so-cafled k-value) than in the exteg, fibdnolysis can be suspected. this technique requires only commercially available reagents and is easy to perform on conventional teg systems. due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. according to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l0 minutes. many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. the application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. we suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. these methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). method~. 0.2 ml of studied solution was added to 0,8 ml of azofibrin suspension in certain buffer (5-10 mg/ml) and the mixture incubated at 37 oc for 10-60 rain. after the end of incubation the mixture was filtered, the volume of solution brought up to 4 ml by 0.02 m naoh and the optical density was determined at 440 nm. resuffs. azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. for comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented: activity. with an increase of pal and ldl-and a decrease of hdl-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. also disturbances of lipid metabolism an abnormal whr seems to be of an additional atherogenous factor in dm. plasma concentrations of thrombin-anfithrombin-iii (tat), alpha-2antiplasmin-plasmin (app) complexes and ddimer were investigated in 50 patients treated with thrombolytic therapy for acute myocardial infarction (ami) either with streptokinase (n=24), urokinase (n=16) or recombinant t-pa (rt-pa, n=10). all patients received an intravenous heparin bolus of 5,000 iu on admission, which was followed at once by an infusion of 1,000 iu/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. tat, pap and ddimer were measured by enzyme immunoassay on admission, 1, 2, 4, 6, 8, 12, 24 hours and on day 3 and 7 after admission. groups did not differ significantly in regard to age, sex, delay and infarct location. on admission, no marker differed significantly between groups. thereafter, tat levels increased significantly exclusively in rtpa treated group. from 2 to 6 hours after admission, tat were significantly higher in rtpa treated patients than in streptokinase and urokinase treated group (p<0.02). however, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group tat concentrations decreased below admission values. app were significantly higher only 1 hour after admission in the rt-pa group (p=0.03). ddimer did not differ signifieanfly between groups. our results demonstrate, that rtpa induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. the significant tat decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for ami. thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. endothelium derived relaxing factor-no (edrf-no) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ however, due to the chemical nature of edrf-no few is known about its production and activity as a general index or marker of vascular function in human diseases. one way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular edrf-no production. in this report a self-developed elisa method is described, which was used for this perpose. nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well after a comparison of the excretion values to other markers of angiopathy (yon willebrand factod soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. two major concerns still accompany the application of prothrombin complex concentrates (pcc). viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. the inherent risk of thrombo-embolic side effects has to be considered. to minimize these risks and to achieve good clinical efficiency the quality criteria for pcc's are under pending discussion. it is generally accepted that a modem pcc-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein c and protein s. additionally, the concentration of activated coagulation factors should be kept at a minimum. a present pcc-produedon process mainly consists of a qae-sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. further purification is achieved by subsequent chromatography on deae-sephamse. the aim of this study was to improve product quality by avoiding f viiactivation without implementing major changes to the production process. at the same time, a second virus eliminating step was added to the production process. it could be shown that speeding up the chromatographical process by switching the deae-sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of f viia-genemtion. mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. the relation between f vii/f viia was 10 : 1 or more. in order to investigate the feasibility of virus filtration the eluate of the deae-sepharose column was filtered through a virus removing ultipor vffilter. the analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. preliminary studies showed significant virus reduction values. in the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor viii has been discussed in literature. we observed 121 patients with hemophilia. inhibitors to factor viii:c were revealed in 32.7 % of patients with hemophilia a and fo factor ix --in 1.6 % of patients with hemophilia b. the level of an inhibitor was not higher than 87 befhesda u/ml, that is those patients were not regarded as "high responders". a high incidence of inhibifors in young patients [from 7 to 26 years of age, 51.9 %) compared with older patients (from 27 to 40 years of age, 11.2 %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor viii, ix. inhibitor development in patients (40.5 %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. the investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications --53.9 %, aufoimmune processes --44.9 %, secondary tumours --1.2 %. plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < 10 bu/ ml) and in patients with mean response. thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. in a recent multicenter study 73 previously untreated patientens (pups) with severe hemophilia a were treated with a recombinant factor viii concentrate (rfviii, recombinate©). during fviii treatment 21 (29%) developed inhibitors, 6 high titer (>5 bethesda units (bu)/ml), 4 low titer (<5 bu/ml) and 11 transient inhibitors. plasma samples from before treatment and during treatment but before inhibitor occurrence were available in 12 inhibitor patients. these plasma samples were analyzed by a highly sensitive immunoprecipitation (ip) assay for the presence of anti-tviii antibodies. in 9 (66%) a significant increase of anti-fv]]i antibodies was seen indicating the development of a clinical relevant inhibitor titer. this immune response occurred after 2 to 17 (median 5) exposure days (ed). in the same period only 3 out of 15 inhibitor patients showed a decreased in vivo recovery. in 16 pups who developed no inhibitors plasma samples from the entire treatment period were available. an immune response to rfviii treatment was seen in 7 pups after 2 to 43 ed (median 24 ed). the immune response was later and less pronounced in comparison to inhibitor pups before inhibitor occurrence. with the ip method the detection of an early immune response is possible which might be predictive for a later inhibitor development. the inclusion of the lip method should be considered for future multicenter pup studies. in the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia a and b. we report on 3 severe bleeding episodes in 2 patients with hemophilia a and b, respectively. both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor viii-and factor ix-concentrate, respectively. high purity factor concentrates were tolerated well without any allergic side effects. a 67 years old patient with a moderate form of hemophilia a (f viii 4 %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and 3 different plasma derived factor viii-concentrates of intermediate purity. in all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. replacement therapy with two different recombinant factor viii concentrates was tolerated well without any side effects. a 12 years old haemophiita b patient developed hypersensitivity reactions to prophylactic factor ix substitution, which could be overcome by using a factor ix .concentrate with improved purity. a recent recurrence of hypersensitmty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor ix concentrate. we conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble hla-antigens, are advantageous in patients disposed to allergic reactions. introduction: antibody formation against factor (f) viii remains one of the most severe complications of repeatedly transfused patients with haemophilia a. as reported previously in our study about the incidence of fviii inhibitors, we have observed a high incidence of fviii inhibitors among our haemophilia a patients. it is still not clear why certain haemophiliacs develop antibodies and others do not. a number of previous studies suggest that there is a genetic predisposition for the fviii inhibitor development. thus, the purpose of our study was to examine, if there is a correlation between fviii antibody-formation and genetically determined histoeompatibility antigen (hla) patterns in our haemophiliacs. patients and methods: hla-class i (a, b, c) and hla-class ii (dr, dq) typing was carried out for 51 respectively 44 multi-transfused paediatric haemophilia a patients (fviii:c activity < 5%), including 22 who had developed an antibody to fviii: 19 were high responders (> 5 bu), 3 were low responders (< 5 bu). hla-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (drk frankfurt) using antisera with defiend hla-specifity (biotest diagnostica). results: we found an under-representation of hla-a2 in fviii inhibitor patients when compared with the subgroup without inhibitor. in regard to the hla-b and hla-c antigen frequencies there are no apparent differences between the groups. among the class ii antigens there were higher frequencies of dr1, drw52 and dqwl in the non-inhibitor group. however, the reduction in hla-a1, hla-cw5, hla-dqw3 respectively hla-dr4 frequency for inhibitor patients as reported previously could not be confirmed in our study. conclusion: so far it remains unclear if there is a significant association of a certain hla allels with the development of fviii antibodies. recombinant factor sq (r-viii sq, pharmacia) is a b-domain-deleted recombinant factor viii. it is formulated without albumin (hsa). the product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-viii). the international clinical trial programme was initiated in march 1993. pharmacokinetic studies have shown that the b-deleted r-viii sq should be given according to the same dosage principles as a full length p-viii. at present, the product is being tested in previously treated patients (ptps) and untreated patients (pups) with severe haemophilia a (viii:c < 2 %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery. the long-term study in previously treated patients in germany was started in january 1994. thirteen patients have been included in 8 centers. all patients are still on treatment with r-viii sq, most of them receiving prophylactic treatment. global treatment efficacy has in general been considered excellent or good. no serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor viii or antibodies to mouse-lgg or cho-cell components developed in the patients. further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. on examination of 125 patients (pts) (hemophilia a --110 pts, hemophilia b --11 pts, willebrandt's disease u 4 pfs) the development of immune complications was revealed in 34.4 %. chronic persistent hepatitis (3.2 %), chronic active hepatitis (3.2 %), herpes simplex (1.2 %), chlamidiosis (1.2 %), bacterial infection (7.2 %} were regarded as infectious complications. bacterial infections have a routine course due to preserved phagocytic function of neufrophils. and viral infections, whose ability to resistance is connected with t -cell link immunity, take on a chronic persistent course, mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura --2.4 % of pts, immunocomplex disease --4.9 % of pts, the appearance of immune inhibifors --34.4 % of pts} is connected with the impairment of immunological surveillance over b -cells aufoimmune clones as a result of dysbalance in the system of t -lymphocyfes immunoregulatory subpopulations. lymphadenopathy and splenomegaly (4.9%) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of t -lymphocytes system, or they may be an evidence of virus infection. we observed one episode of acute leukemia. immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. in immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment. control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), pettersson score and social integration. oren, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. in order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint. since 1988 we have established a computer database (haemopat) that contains data from all patients treated in our center. tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. the results of 8 years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. parallelly a new program (haemopat win 1.0) will he introduced allowing for easier handling of data and their evaluation. this program will be used as of december 1995. in combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities. the program will be available for all those interested free of charge. 2) kinderklinik der westf. wilhelms univ. mttuster 3-6) biotest pharma gmbh, dreieich haemoctin® sdh; the fviii sdh (sdh = solvent detergent and dry heat = 100 °c, 30 rain) from biotest pharma is a high purity (specific activity ~ 100) fviii concentrate manufactured from large human plasma pools. virus validation studies have shown virus inactivation/reduction (log 10) during the manufacturing process for lipid coated vints~ such as: h]v-1 > 16.2; psr > 16.8; vsv > 14,5; bvdv > 15.7; hcv > 4.5* and non enveloped vimsas such as: parvo** = 2.7; reo > 5.3*** and hav > 13.9. more than 50 hemophilia a patients (ptps = previously treated patients), baseline fviii activity < 1%, were included in an international drug monitoring study to follow their fviii inhibitur status. the hemophilia centers included were three centers from hungaria (helm pal children hospital and the national inst. of haematology, budapest and regional blood transfusion center, debrecen) and four centers from germany (two from berlin, one fraukfurffmain and one monster). patients were enrolled in the drug monitoring beginning aug. 1993. at the entry none of the patients had a detectable inhibitor. at the end of sept. 1995 there were no side effects or adverse events in connection with the use of haemuetin®. before the haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified fviii products. inhibitor testing was done on patients plasma samples using the bethesda method. repeated fviii recovery determination at one time (between 12 to 24 hrs) after haemoctin® application demonstrated the expected recovery and normal half life time. none of the hemophilia a patients, treated with haemuetin® sdh developed a clinical relevant inhibitor. at the beginning of the stud)', the clinical efficacy of haemuetin® was studied in 16 hemophilia a patients and shown to give an in vivo recovery of 71 + 15 % by one stage assay and 77 + 17 % by a chromogenic assay. t ½ values were 13 + 2.8 and 12.7 + 3.2 hrs respectively. the study for the clinical efficacy of haemoctin® sdh was repeated in a group of 6 patients approximately two years later. although cd4 lymphocyte counts are known as reasonable predictors of prognosis in hiv infection, the cd4 count is not in all cases an infallible indicator of prognosis. therefore several serological markers are used to predict disease outcome, including beta-2 microglobulin (132m), immunoglobulin a (iga), lymphocyte counts (lymph) and others. in this study we followed a cohort of 23 haemophiliacs (19 with haemophilia a, 4 with haemophilia b) and 2 patients with severe von willebrands disease over a period of 28 months (mean, range: 22-34). testing for l~2m, igg, iga, igm, cd4 and cd8 cell counts (abs. and relat.), cd4/cd8 ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least 3 times a year. at the same time clinical examinations and review of history were undertaken. mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data. 1-4 5-8 9-12 13-16 17-20 21-24 cd41 440+956 344+924 278+925 302.-1:240 273.+.220 166+125 cd8 ~ 1165+474 1171+523 1236+1187 1017+439 930±412 873+478 1~2m z 2.0+0.6 3.0+1.0 3.0+1.2 3.0+1.1 3.5±1.0 3.5±1.3 lymph ~ 1.98+0.6 1.83+0.6 1.72+0.7 1.67±0.6 1.46±0.6 1.31±0.5 means/pl ± standard deviation means mg/l ± standard deviation during time ef observation we found significant changes of cd4 (abs. and relat.), abs. cd8 counts, cd4/cd8 ratio, f~2m, leucocytes and lymphocytes. the abs. cd4 and cd8 counts correlated clearly with lymphocytes und leucocytes counts but not with 1~2m. the prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. the availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of fviii or fix to prevent or reduce bleeding in patients with haemophilia. in conliast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about 20% and preventing possible side effects of bolus doses. the new application mode, however, requires stable products which tolerate slow passage through an infusion device. our objective was to test in vitro the fviii concentrate immunate (stim plus) and the fix concentrate immi.ynine (stim plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. infusion rates were chosen to mimic clinical situation. the control samples were not infused through the pump but were otherwise treated identically. test samples were drawn before and at 1, 4, 8, 24 and 48 hours after the onset of each infusion run. fviii (one-stage, two-stage and chromogenic assay) and fix (one-stage) activity were measured using immuno reagents. presence of activated factors were measured by napt'i', while flla, fxa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. the data showed equivalent results between test and control samples with no loss of fviii or fix activity. the potencies of both immunate (stim plus) and immunine (stim plus) remained within 100 + 20% of labeued values within 48 hours after onset of infusion. in conclusion, immunate (stim plus) and immunine (st1m plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. in htanans, circulating half-lives of asparaginase enzymes from e. coli and erwinia chrysanthemi vary within a wide range. moreover, half-lives differ not only among different e. coli strains but also among commercial e. coli preparations. to investigate the possible influence of two different sources of e. coil asparagmase (asn) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating asn pharmacokiuetics (asn activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o.2-antiphismin (ct2ap), tissue-type plasminogen activator (t-pa), tissue type plasminogen activator inhibitor 1 (pal 1), d -i)imer (i)-d)). together with prednisono, vincristine and an anthracycline 20 children received i0000 iu-/m 2 asn medae r (originally purchased: kyowa hakko, kyogo japan) and 20 children 10000 iu/m 2 crasintin r (bayer, leverkusen, germany). blood samples for pharmacokinetic and coagulation analysis were drawn before the first asn administration and every third day whilst on medication. the results are shown in the 0.05 asn activity shows a negative correlation (spearman: rho/p) to plas (-,637/0.0003) and ct2ap (-, 751/0.0001). a positive correlation was found between asn activity and d -dimer formation (0.475/0.01). t-pa and pal 1 showed no relationship to asn activity. all children showed complete aspamgiue depletion at a detection limit of 0.1 um during the course of asn admiatstration. two thrombotic events occurred in the kyowa group, one of the distinctions between the two e. coli asn preparations administered ill this stndy is the absence of cystine in the kyowa asn, which also has a lower isoelectric point and a longer half-life than the bayer type a asn. with respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. along with studies on asn pharmacokinoties dose recommendations need to be tailored to the specific asn preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications. different types of coagulopathy in hepatic veno-occlusive disease (vod) and capillary leakage syn-drome (cls) after bone marrow transplantation w. ntimberger, s. eckhof-donovan, st. burdaeh and u. g6bel department for pediatric hematology and oncology, heinrich heine university medical center, diisseldoff, germany it is generally accepted, that cls, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic vod. we assessed patients with either vod or cls or both vod and cls, in order to analyze the influence of either syndrome on different aspects of hemostasis. vod was diagnosed according to jones et al. [transplantation 44 (1987) 778]. diagnosis of cls was >_3% increase of body weight in the past 24 hours and non-responsiveness to furosemide [niirnberger et al., ann hematol 17 (1993) 67] . patients with vod, cls or both were compared to control patients without either diagnosis. eight patients suffered from both vod and cls, 5 patients only from vod, and 8 only from cls. 61 patients had neither syndrome and served as control population. activation of the coagulation system was assessed by increase of tat-complexes and/or increased consumption of at iil the hemostasis patterns were as follows: no. introduction: lung cancer goes along with coagulation activation and increased thromboembolic risk. acute phase reaction in cancer patients leads to elevated levels of c4b-binding protein (c4b-bp) followed by a shift from free to c4b-bp-bound protein s. we tried to find out whether there is a correlation between alterations of c4b-bp, protein c protein s system and interleukin 6 (il-6), which is one of the most potent inducers of hepatic acute phase reaction. patients: i. 25 patients with lung cancer; 2. control group: 11 patients in complete remission after lung cancer. methods: clotting methods: protein c and s activity; elisa tests: protein c antigen, tat-complexes, prothrombin fragments f i+2, il-6. electroimmuno-diffusion (laurell): free and total protein s, c4b-bp. results: tat-complexes and f i+2 were elevated in cancer patients. c4b-bp levels were slighthly increased (129±19 % of n.), protein s activity was 89±33 % of n. (control group: 108±23 % of n.). il-6 in lung cancer patients was 37.2252.9 pg/l (control: 27.2±8.2 pg/l). conclusion: one source of the hypercoagulable state in lung cancer patients is decreased protein s activity due to elevated c4b-bp levels. this is probably caused by hepatic acute phase reaction which is triggered by increased il-6 levels. these plasma levels correlate with levels of the tumor marker ca 125 and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. plasma levels of d -dimer and ca 125 (determined by sandwich elisa assays} were measured prior to treatment in 36 women with figo stage t to iii ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of 28 months (range 16 to 40 months). levels in 71 healthy women and 27 patients with benign ovarian disease served as controls. the occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. preoperative d -dimer and ca i25 levels in ovarian cancer patients were statistically signfficantly higher than in controls. preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. d -dimer levels above a cut off level of 2060 ng/ml were statistically significantly associated with the rate of relapse but ca 125 levels were not. deep venous thrombosis occurred in 33 % of cases but there was no difference between properafive levels of d -dimer in patients who subsequently did versus did not develop deep vein thrombosis. high levels of d -dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. the high levels of d -dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. thus d -dimer levels may serve as a marker for overall tumor burden as well as "disease activity". a high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of d -dimer are not predictive of this occurence. yon tempelhoff georg -friedrich, michael dietrich, dirk schneider, lothar heilmann. dept. obstet. gynecol. city hospital of ruesselsheim. -germany. an increase of plasminogen activator inhibitor activity (pai act.) in the plasma of cancer patients has been recently discribed. we have longitudinally investigated pai act. in 136 patients with primary breast cancer and compared the results with the outcome of malignancy. patients with untreated primary breast cancer and without proof of metastasis (t 1-4 n0-2 m0) were eligible for this study. in all patients coagulation tests including fibrinogen {method according to clauss), d -dimer (elisa} and pal act. (upa dependent inhibition test) were performed prior to primary operation, 6 months thereafter and at the time of cancer relapse. seventy -two healthy women and 43 patients with benign breast disease served as controls. during a mean follow -up of 32 + 16 months 34 patients (25 %) developed cancer recurrence and 13 (9.6 %) patients died. in all cancer patients preoperative levels of fibrinogen and pai act. were significantly higher compared to healthy women and to patients with benign breast disease. preoperatively only pal act. was significantly higher in patients with vs. without cancer recurrence (4.52 _+ 1.67 u/ml vs. 3.4 1 + 1.55 u/ml; p = 0.002). in patients with later recurrence pai a~t. significantly dropped 6 months after operation (p = 0.02) and was again significantly increased at the time of cancer recurrence (4.90 _+ 2.89; p = 0.001). a preoperative cut off value (calculated via cox model) of pai act. above 3.52 u/ml was significantly associated with the rate of relapse (tog rank: p = 0.0005) and in 70 % of patients who died of cancer preoperative pai act. were also above this cut off. impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. a monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. for further analyses tyramine, which was covalently bound to low molecular mass heparin by endp0int attachment (malsch r et al: anal biochem 1994; 217: 255-264) , was labeled with 125-iodine at the aryl residue. the tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline igg. the mab recognized specifically intact heparin and heparin fractions. the lower detection limit of heparin preparations was 100 ng/ml. no cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate a and c. oversulfated heparin showed lower affinity to the antibody hl.18 than 2-0-and 6-0-desulfated beparin. the method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. sds-page and high pressure capillary electrophoresis prooved the high purity of the received antibody. the biological activity of mab was tested by the chromogenic assay $2222 and remained stabile while purified. in conclusion, the present abstract describes an purified igg 1 monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. the concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. for this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. we developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. tyramine was bound by reductive amination to the 2,5 anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by 13c.nm r spectroscopy. (anal biochem 217: 254-264, 1994) this method was also used to produce a low molecular mass dermatan sulfate (lmmd)derivative after partial deacatylation. in order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (cp) samples with different degrees of pol3~merization and sulphation we applied to pharmaeapea article (a~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the 3rd international heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (a2). the model admits the "kinetics of cp elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: ct=co exp(-i~ x t), where ct is cp concentration at the time moment t; co is cp concentration at the moment of injection; i~ is the elimination constant. besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty a2 : t=kt ct + tin, where t is the time of clot formation at different tlme intervals after of cp injection; t~, is the time of clot formation prior to cp injection. t value was assessed in two tests: in blood coagulation time (bct) and in activated partial thromboplastin time (aptt). no correlation was observed between a1 and a2. at the same time the values of ifm and the period of semieliminatinn (tvz) with the use of the original method that were obtained with the help of the quantitative determination of cp in rabbit's blood taken at different time intervals after injection, showed a close correlation (1"=0,94 p<0,05) between the same parameters, obtained with the help of the of the pharmacokinetic model in bct test. thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for a2 calculation. we recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. in this investigation we compared the biological activity of a low-molecular-heparm (lmw-heparin, mono embolcx®) after intravenous, subcutaneous and oral application in rats. sprague-dawly rats were anaesthetized by ketamine/diazepam and the blood samples were taken from the retro orbital sinuus. 150 axa u/kg body weight of the lmw-heparin were injected intravenously and subcutaneously to 10 rats each. between 3 minutes and 10 hours after injection serial blood samples were taken. 200 mg/kg (20.000 axa u/kg) body weight of the lmw-heparin were applicated orally using a stomach tube. blood samples were taken between 1 and 24 hours after oral application. the antifactor xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances s 2222 and s 2238 (kabi vitmm). after i.v. injection the maximum axa and alia activities were 2.8 axa u/ml and 0.8 aiia u/nil respectively. after s.c. application the antifactor xa activity of the lmw-heparin showed a maximum of 0.5 axa u/ml atter 120 minutes. the antithrombin activity exhibited an eatiier maximum activity of 0.2 alia u/nil 60 minutes after injection. after the oral application no increase of the axa or alia activities was measured. the lmw-heparin has a high antifaetor xa and antithrombin activity after i.v. and s.c. injection. after oral application no activity of the lmw-heparin was measurable. these results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. to improve the activity after oral application modified hepatins have to be synthesized. in an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, cy 222, cy 231, astenose, hexasaccharide, ssh 14) on thrombin-and adp-induced platelet aggregation as well as on adpmediated platelet activation in whole blood was investigated. all heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. calciparin and astenose were found to be the most effective compounds with ic5o values of 0.67 and 1.2 p, mol/l, resp.; higher concentrations (5-30 times) were required for the other compounds. furthermore, the heparin derivatives were studied with regard to their potentiating effect on adp-induced platelet aggregation. in a concenwation range from 1 to 10 u/nil calciparin, fraxiparin, cy 222 and astenose led to a potentiation of the adpinduced aggregation whereas cy 231, hexasaccharide and ssh 14 did not show this effect. the increase in aggregation was associated with an increase in thromboxane a2 lbrmation. in addition, the effect of calciparin, fraxiparin, cy 222 and astenose on adp-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors opiiia (cd-61) and p-selectin (cd-62). at concentrations that caused a maximum potentiation of adp-induced platelet aggregation these substances led to a strong increase of adp-mediated activation of platelets in whole blood. the effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. in conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. heparins are negatively charged polysaccharides and bind protamine forming a stable complex. here we report on the properties of microbeads (4.5 pro) coated by protamine. protamine chloride (0.16 ijm) was covalently bound to 0.5 mg paramagnetic tosyl..activated microbeads m-450 (dynal). the covalent binding of protamine was from 1.0 to 13,0 mg/g beads. protamine-dynabeads were produced in a phosphate buffer at different ph (7,0; 7,5; 8,0 and 8,5). the protamine-dynabeads produced ph 7.5 showed the best properties for flow cytometry analysis. in saline solution they bound lmm-heparin-tyramine-fitc (lmmh-tyr-fitc) dose dependently from 0.001 to 2 u/ml, whereas in plasma and blood they bound lmmh-tyr-fitc from 0.05 to 2 u/ml. dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.the adsorbed proteins, however do not bind lmmh-tyr-fitc dose dependently. the saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). in saline solution the saturation was measured at 380 rfi, in human plasma at 325 rfi and in whole blood at 252 rfi. using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to protamine dynabeads. these data demonstrate that protamine-dynabeads can be used to measure the concentration of lmmh-tyr-fitc in saline solution, plasma and blood because they do not bind to human blood cells. the present study was designed to investigate the anticoagulant action of inhaled low molecular weight (lmw)-heparin in healthy volunteers. 3,000 iu (group t), 9,000 iu (group 2), 27,000 iu (group 3) or 54,000 iu (group 4) lmw-heparin were given to 20 healthy volunteers each at 4 weeks intervals. in group 1 tissue iactor pathway inhibitor (tfpi) antigen and activity, 82222 chromogenic factor xa assay, heptest, aptt and thrombin clotting time (tot) remained unchanged during the 10 days observation period. in group 2 tfpi antigen and activity, aptt, tct and the $2222 method remained uneffected. heptest coagulation times were 18.7 + 2.0 before, 26.1 + 5.2 sec. 6 hrs and to 20.5 + 1.9 sec. 24 hrs after inhalation. in group 3 tfpi antigen increased from 74.1 + 13.9 to 80.5 + 14.2 ng/ml 3 hrs after inhalation. tfpi activity remained unchanged. $2222 method increased from 0.01 to 0.08 + 0.06 iu/ml 6 hrs after inhalation. heptest coagulation values were prolonged up to 42 _+ 7.6 s ec after 6 hrs and returned to normal within 72 hrs after inhalation. aptt and tct remained unchanged. after inhalation of 54,000 iu lmw-heparin, the following changes were observed: tfpi antigen increased to 103 +_. 17.9 ng/ml and normalized within 24 hrs. -i'fpi activity increased to 1.14 _+ 0.23 u 3 hrs after inhalation and was normal after 24 hrs. antifactor xa activity, as measured by s2222 method, increased to 0.343 + 0.196 u/ml after 6 hrs and was normal after 72 hrs. heptest coagulation values increased to 77.5 + 11.8 sec 6 hrs after inhalation and normalized after 144 hrs. aptt and tct did not change throughout the observation period. the data demonstrate a resorption of lmw-heparin by intrapulmonary route in man. no side effects were observed. recently we developed a tritium-labelled arachidonic acid ([3h]aa) release test with high sensitivity to membrane-toxic agents. the assay performed in u937 cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [kloeking et at. (1994) , toxicology in vitro 8, 775-777]. local irritation reactions are described in patients receiving therapeurieat dosages of lmw heparin. this fact prompted us to examine the following lmw hepafins and heparinoids for their membrane toxicity in u937 cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (mps), pentosan polysulfate sodium (pps), polysulfated bis-lactobionic acid amide derivatives lw10082 (aprosulate) and lw10086. for this purpose, [31--1]aa labelled u937 ceils were incubated with different concentrations of lmw heparins and heparinoids at 37°c for 1 hour. compared with untreated cells, the [~h]aa release of cells treated with 5 mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide lw10086, five times higher with pentosan polysulfate, 20 times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. the rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. semi-synthetic glyensaminoglycans (gags) with antithrombotic properties can be prepared from the e. coli k5 polysaecharide by coupled chemical and enzymatic methods. the molecular weight of these semi-synthetic gags can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. in order to compare the biochemical and pharmacologic properties of a semi-synthetic gag (sr 80486a, sanofi/choay) with a commereiany available low molecular weight heparin, fraxiparine (sanofi, paris, france), valid biooheanical and pharmacologic methods were used. the molecular profile of this agent as determined by hplc exhibited a comparable distribution profile (mr=5.7 kda) in comparison to fraxiparine (ma=5.1 kda) . the anticoagulant properties of sr 80486a were comparable to fraxiparine in the aptt and heptest. however, in the usp assay, this agent showed slightly weaker activity. sr 80486a also exhibi~d comparable affinity to atffl and hcii. in comparison to fraxiparine, it produced a much weaker response in the hit screening system. in~ viv0 studies, sr 80486a preduecd strong dose-dependent antithrombotic actions in both the iv and sc studies in the rabbit jugular vein stasis thrombosis model (ed50=i 5-60 gg/kg). additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. the hemorrhagic effects of this agent were comparable to those of fraxipafine as measured in a rabbit ear blood loss model. intravenous administration of sr 80486a also revealed a comparable pharmaeokinetie behavior to fraxiparine. no abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted in primates. at a dosage of i and 2.5 mg/kg iv, this agent also caused a release of functional tfpi which was comparable to the observed responses of other low molecular weight heparins. these studies suggest that sr 80486a is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. limited information on the comparative pharmacoldnetics of low molecular weight heparin (lmwh) is available on the data obtained from aptt, heptest, anti-xa and antmia assays. since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. aptt, heptest and antmia test may be valuable in the assessment of their effects. in order to investigate the relative pharmacokinetics of lmwh using apt'i', heptest, anti-xa and anti-iia methods, certoparin (sandoz, basel, switzerland) was administered to individual groups of healthy male volunteers (52-70 kg) via intravenous (30 mg) and subcutaneous (50 nag) routes in a crossover study. blood samples were drawn at 0, 5, 15, 30, 60, 90, 180, 360, 540 and 720 minutes. using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of certoparin. a non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t 1/2, vd, and clsys. in the intravenous studies, the t 1/2 was found to be dosedependent for aptt, heptest, anti-xa and antm]a. the auc, however, was significantly different for each test and was dose-dependent following the order: aptt<heptest<anti-xa<anti-iia. in the subcutaneous studies, 1the t 1/2 was both test and dose-dependent. the auc was also dose-dependent and followed the order: anti-xa>heptest>aptt>antmia. the clsys of the antma was much faster in comparison to the other tests. the clsys of the aptt and heptest was independent of dose. however, anti-xa clsys by this route was lower than other tests. the apparent vd followed the order aptt>antmia>heptest>anti-xa. the bioavailability of the certoparin as measured by various tests ranged from 81-119%. these studies suggest that beside providing pharmacokinetic data, aptt, heptest and anti-iia assays may provide useful data on thier safety and efficacy at high dosages. the immunological type of heparin associated thrombocytopenla (hat ii) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. only patients with absolute thrombocytopenia have prompted suspicion of hat in clinical practice. we report on a 44 year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of a. brachialis. fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed hat type ii in hipa and elisa (stago-boehringer, marmheim). this special patient had platelet counts within a normal range, when developing the thromboembolic episodes. it appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. from a clinical point of view we recommend the use of a lab panel to exclude hat type ii in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. platelet counts within a normal range are no absolute exclusion criterion for hat ii. low molecular weight heparins (lmwhs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. in this indication, lmwhs are administered as a single or twice a day subcutaneous regimen. usually these agents are administered at 30-40 mg total dose which is equal to 3000-4000 anti-xa (axa) iu. newer methods such as ehromogenic substrate based axa methods and the heptest clotting time can be used to determine the effects of lmwhs during the initial phases of prophylactic therapy. this may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. similarly in the overweight patients, a fixed dose may not be efficacious. thus, monitoring of lmwhs in these patients may be useful in the optimization of their therapy. lmwhs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. high dosages of up to 200 mg sc/day and infusions of up to 30 axa iu/kg/hr have been administered. in these conditions, the monitoring of the circulating lmwh levels may be useful in optimizing the dosage. we have modified the aca heparin (do_pont merck, wilmington, de) method to measure the lmwh levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. this presentation provides an overview of the clinical application of various lmwhs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. a double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of 5000 ie anti-xa of low molecular weight heparin (lmwh) sandoz (certoparin) and 5000 ie unfractionated heparin (ufh) tid. in 288 patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of lmwh or ufh resp., two hours after administration on the first and 7th postop, day and on the last day of prophylaxis (day 10-14), anti-xaactivity was measured by chromogenic substrate assay, heptest and aptt by clotting assays and tissue factor pathway inhibitor (tfpi) and heparin-pf4-antibodies by elisa techiques. as expected, the anti-xa-activity and the heptest values were significantly higher in the lmwh-group at all time points after administration of the drugs; the mean values of heptest were 35 sec in the ufh-and 75 sec in the lmwh-group respectively, the aptt was not different in both groups. at the end of prophylaxis positive antibodies to heparin-pf4complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. a detailed correlation between patients with deep vein thrombosis (dvt) and positive antibodies has still to be done (all patients were screened for asymptomatic dvt between day 10-14 by bilateral phlebography. tfpi was markedly increased in the lmwh-and only slightly elevated in the ufh-group; the differences are statistically significant. summarizing it can be concluded that antibodies to heparin-pf4complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type ii and that tfpi may play a sigificant role for the antithrombotic efficacy of ufh and lmwh. unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. heparin-indueed thrombocytopeala (hit) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (hit type i). hit type ii, however, is immune-related an may result in absolute thrombocytopenia (platelet count <i50/~l) or a relative decrease in platelet count (?_50% reduction from baseline values). unlike most other immune thrombecytopenias in which bleeding complications often predominate, hit type ii may results in severe arterial and/or venous thromboses. the management of patients with hit type ii and thrombosis is often difficult. heparin must be stopped immediately. antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirl plasmapheresis, used in other immune-related disorders, has also been occasionally reported. at our institution this intervention appears to provide significant elininal benefit to hit patients. several recent reports have noted that the heparin-dependent antibodies in hit type ii are directed against a complex of heparin bound to platelet factor 4 (pf4) and an elisa-based test system for hepafin-pf4 eomplexinduced antibodies (hpia; stago) has been develuped~ we report our experience in 3 patients with hit type ii in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. the patients developed profound absolute thrombocytopenia (range, 3-21 k/~tl) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. thromh0cytopenia induced by hepariu (hat-syndrome) typically appears several days after initiation of heparin therapy. this phenomenon is caused by antibodies of the lgg type which are induced by a heparin-pf 4-complex and which also activate platelets. in a recent study (n engl j med 1995; 332:1330-5) the hat-syndrom was diagnosed in 2.7 % (9 of 332) patients who had received unfractionated heparin (ufh) and in none of those 333 patients who had been treated with low-molecular-weight heparin (lmwh). 8 out of the 9 patients treated with ufh presented with at least one thrombotic event (7 times a venous, once an arterial event). the incidence of heparin-dependent igg antibodies (7.8 %) was higher in the ufh treated group than in patients treated with lmwh (2.2 %). our patient was a male caucasian white, 62 years of age, otherwise in good clinical condition with no severe diseases in his pmh. both parents, however, had suffered several thrombotic episodes. the patient was admitted to the hospital in september "95 with clinical signs of a deep vein thrombosis (dvt) of the right leg. the diagnosis was phiebographically confirmed. after having given informed consent the patient was treated with 5 cycles of r-tpa (loco-regional application) and 3 cycles of uhsk as part of a clinical trial (uhsk vs. rtpa, boehringer/m). lysis was successful with complete recanalisation of the veins. after completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (ufh). as a complication the patient suffered from a retroperitoneal and an upper gi4ract bleeding from esophagitis grade ii to hi while being on coumarin prophylaxis. therefore the patient was put on lmwh and then discharged with this prophylaxis. after less than two weeks the patient presented again to the hospital with a relapse of dvt in the same leg. phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ 3, swelling of the whole leg. blood count showed a marked decrease of platelets to 27/nl. the platelet aggregation test demonstrated a hat-syndrome which was confirmed by the hipa-assay. the heparinoid orgaran® was not reactive in the assay. having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (hat study, behring) instead of orgaran®, since hirudin allows a much more precise dose adjustment. after 10 days the patient was discharged with no further complications. risk factors for dvt have not been detected until now. to our knowledge this patient is one of the first cases of hat-syndrome induced by lmveh. under routine conditions aptt is the most frequently used test method for the surveillance of patients undergoing hepann therapy. in literature a 1,5 fold to 2,5 fold prolongation of the initial value is recommended for individual therapy a~ixatian. in contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. when choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalxability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage (0.1 -0.3 u/ml). in the therapeutic range (0.4 -0.8 u/ml) very often no linear relationship between prolongation of the aptr and heparin concentration level is found_ therefore very often coagulation times above the routine measuring range (180 s) can be observed in this range. moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. coagulation limes with different apq't reagents in correlation with the heparin concentration level can be found in the adjoined table. significantiy prolonged coagulation ti.~es of aptt lead to a reduction of the hel~ dose in clinical practice. due to the discrepancy between heparin concentmon level and the prolongation of the afrt in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. therefore the producers should be asked to include precise directions on the heparin sensitivity of the aptt reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. a coordination between clinic and laboratory should be compulsory for the choosing of the aptt reagent. introduction: hat is a rare but sometimes fatal complication of therapy with unfractionated (ufh) and low molecular weight beparins (lmwh). we report on our experience with the clinical coume and management in 12 patients (pts). in all cases a severe decline of platelet count and in 11 cases a positive hipa test (= heparin-induced platelet antibodies) were documented. patients, clinical course and outcome: 8 female and 4 male patients (median age 68 years, range 22-78) were analysed in this retrospective study. 9 pts were referred from outside hospitals. all pts had been treated with ufh (5 pts additionally with lmwh) for prophylaxis (n = 9. in 2 pts pedoperatively) or treatment (n = 3) of venous thromboembolism (v'te). in 11 pts at least one vte occured during heparin therapy, in all cases prior to hat-diagnosis (vte: n = 13; putmonary embolism: n = 6; artenal embolism n = 1; thrombosis of sinus transversus n = 1). hat was suspected 16.5 days (median; range 12 -48) after first heparin application. hepadn therapy was discontinued immediately, at that time the median plateiet count was 35 g/i (range 10 -117). 9 pts were treated with orgaran r, followed by recombinant hirudin (r-hirudin, behdngwerke ag: clinical study) in 4 cases; 3 pts were treated exclusively with r-hirudin. plat~let count normalized in 9 pts within 3 days (median; range 1-10). one patient in the orgaran" group died on recurrent pulmonary embolism. 11 pts recovered without any further thromboembolic complication. conclusion: in our small series of 10ts hat was complicated with vte in 11/12 cases. in many pts hat was diagnosed strikingly late. for early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, both orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, p 179 to prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with lmwh was administered a longitudinal study was perfomed. within a 30 months period 48 children and adolescents (6 -19 years) suffering from malignant bone tumors (ewing's sarcoma or osteosarcoma) received enoxaparin (clexane r/rhone-poulenc rorer, france) for primary propylams. the majority of patients received 0.6 mg/kg bw enoxaparin 12 hours before major surgery (limb salavage). in addition, ten neonates and children received enoxaparin in the same dosage for secondary prophylaxis. in both groups therapy was adjusted to 0.2 -0.4 iu/ml anti xa activity. median (range) duration of therapy was performed for l0 (6 -24) weeks. before ('1"1), one (t2) and 3 weeks (t3) within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of 12 patients, who had to undergo a total end-prosthetic hip implantation (teph) for the first time. criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. at eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. the statistic evaluation ensued with the spss software package. for all three patient collectives an activation of both systems was to be observed with the teph-implantation whereby there were no statistically striking differences between the three groups. thus in the case of the kallikrein-like activity an intraoperative rise of over 37% was registered, while at the same point in time there was only a 4% reduction in the prekallikrein activity. postoperatively a 10% rise in the prekallikrein activity was to be observed. as a reserve inhibitor alpha 2-macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha 1-proteinase inhibitor and cl-esterase-inhibitor yielded a postoperative rise of 135% and up to 100%. the beta-factor xllainhibition showed a rise of 121% during the reconvalescent phase. the 65 year old female caucasian patient presented here, was admitted to a nearby district hospital in december '94 with the acute symptoms of pulmonary embolism. the source of the embolus could not be detected. high dose heparin was administered. pmi-i revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. the first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. following this treatment the platelet count decreased significantly to 10/ni. with an acute thrombosis of the lower caval vein and with leriche's syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. on initial examination we saw a patient in no acute distress, no dyspnea, blood pressure 120/80 mmhg, pulse 94/rain, no peripheral edema or disturbances of sensitivity. neither femoral, popliteal, nor pedic pulses were palpable on either side. a heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (hipaa). the autoantibodies targeting the underlying pf4-heparin complex were determined later by an elisa method in serum samples of the patient (asserachrom hia, diagnostica st,ago). a temporary anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. in the following days, 2 cycles of lysis therapy with 9 mil u streptokinase were administered taking six hours for each cycle. heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values 1.5 to 2 times the individual base line level. at the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. the left leg showed better microperfusion than before. computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. for continuous anticoagulation after lysis we used the heparinoid orgaran® to overlap the period until full anticoagulation was achieved by coumarin. the heparinoid was tested before us non-reactlve in the hipaa. with this prophylaxis the platelet count increased to normal values. hat ii related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (dvt, dic). we present to our knowledge the first case of hat ii during thrombolytic therapy (it) with simultaneous hepatin administration. at admission a 16 year old girl showed complete occlusion of the pelvic veins of the right leg (r common and external iliac v., common and proximal superficial femoral v.). as in children no prospective studies on the thrornbolysis of dvt with rt-pa are available, we started tt according to the frankfurt/m. regimen: rt-pa (actilyse) bolus injection 0.4 nag/kg bw followed by continuous infusion of 1 mg/kg bw/d; simultaneous low dose heparin 100 -200 u/kg bw/d. doppler ultrasonographie control on day nine after the beginning of tr revealed a further distal growth of the thrombus into the popliteal vein. at the same time at ir was decreased to 70% and there was a dramatic drop in platelet count to <50% of the baseline value (216 to 86 gpt/1). the diagnosis hat ]i was confirmed by positive hipa-test. the only heparin preparation not cross-reacting with the antibody was the lmw heparinoid orgaran. heparin therefore was replaced by orgaran: bolus injection 1,500 u/h followed by infusion of 200 u/h. rt-pa-therapy was not discontinued. at ri substitution was performed at levels <70%. under orgaran administration platelet count rose from 77 to 239 gpt/1 within 5 days. on day 12 complete reperfusion was achieved and rt-pa was discontinued ,on day 14. the patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition at admission thrombophilia parameters (protein c and s, apc) were within normal range. heparin induced thrombocgopenia (hit) is a serious but rare side effect of treatment with unfractionated heparin. patients with hit present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. in our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. 15 patients were screened on the 3rd and 6th postoperative day for heparin-antiplatelet 4-antibodies, decrease in platelet count and thromboembolic complications. 3 patients developed antibodies against heparin-platelet factor 4. one of the 3 patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. we conclude that heparin-platelet factor 4-antibodies are often produced in patients under i.v. heparin treatment. only few of these patients actually suffer from clinically s3anptomatic hit. medizinische universit~itsklinik, institut far klinische biochemie und pathobiochemie, d-97080 wtirzburg, germany the important physiological and pathophysiological role of platetets in health and disease is well established. platelets are also the primary target of many vasoactive hormones, factors and drugs. in addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events (1,2). platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor gp iib/iiia. activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. in contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the camp and/or cgmp signal transduction cascades. recent data form our laboratory suggest that the focal adhesion protein vasp, a major target of the intracellular no/cgmp and prostacyclin/camp effector systems, is an important link between signal transduction pathways and elements controlling cell motility (3-6). the implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. however, in vivo truncated "i'po is much less potent than full length tpo due to its rapid clearance. this suggest that the c-terminal glycosylated domain acts to stabilize circulating tpo. tpo stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. in vivo tpo induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that tpo regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. this later notion is supported by the phenotype of ti~ and cmpl knockout mice in which platelet levels are only 10-20% of normal while other blood lineages are unaffected. in animal models of myetosuppresion ,and marrow transplant, tpo reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, tpo also has profound effects on red cell recovery in these models. these results suggest that tpo will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. the haparg-study ( .~a.emostatic parameters as risk factors in healthy volunteers) was sponsored by the german ministry of research and started in 1984. a previous study ( pard, intern. angiology 5, 181-195,1986 ) had shown that spontaneously enhanced platelet aggregation as measured with the pat-ill-test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. it was the aim of the haparg study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. employees of hschst ag, frankfurt aged 40-65 years and personnel of the university of mainz, aged 30-60 y. entered this prospective study. besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm doppler -index and an ecg were recorded and serum cholesterol, hdl, ldl, triglycerides, uric acid and glucose were measured. 1885 men and 988 women entered the study and were followed for 4-6 years. during this period 53 vascular occlusions ocoured ( 36 coronary and 9 cerebral events and 8 peripheral vascular occlusions). only three of these events occured in women. besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p=0.001), smoking (p=0.0013), an elevated diastolic blood pressure (p=0,018),followed by spontaneous aggregation (p = 0.017) and higher hdl-levels (p=0.034) as significant risk factors for men. higher fibrinogen levels just missed significance in this analysis (p=0.059). none of the other hemostatic parameters showed any significant correlation with the vascular events.to our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to adp, pal: and thrombin receptor-derived peptide (trap-6). platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. an abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to gp llxx by immunoblotting. the variant gp differed even from the largest normal polymorphic gp iba form a by an increase in molecular mass, platelets of the patient contain the variant and normal c form in equal amounts. the quantity of gp ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal gp indicated that the defect is located in the c-terminal part of the glycocalicin fragment. measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of 470 angstr6m and a fraction with an average size 0f650 angstr6n~ preliminary results of molecular analysis of the gp iba gene in the patient confirm the localization of the genetic defect by protein studies. in summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofgp ~a. barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat gly-pro-hyp structure, are highly platelet reactive in polymeric form. they additionally have shown that the activity does not involve gpla/lla (integdn c~z~l). platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against gpflb/llla (integfin m~d33). however, platelets of a patient with type i glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical gly-pro-hyp peptide. the lack of an essential involvement of cd36 was shown with cd36deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and cd62 and cd63 expression were measured. to study the involvement of the yon willebrand factor (vwf) platetets from a patient with severe type 3 vwd (vwf not detectable in platelets and plasma) were investigated. the vwf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. addition of pudfied vwf to the vwf-deficient platelets before adding the paptide raised the platelet respond slightly we conclude that neither ~3~ integrin, nor m~ integdn, nor cd36, nor vwf are the pdmary platelet receptor for the collagen-like peptide. there is evidence that native low density lipoprotein (ldl) can be oxidatively modified in vivo and the oxidized ldl may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. by means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized ldl, compared to native ldl, affects platelets at least in the following five aspects. 1) oxidized ldl alters platelet functional morphology in a time and dose dependent manner. 2) oxidized ldl induces secretion of serotonin from both morphological resting platelets and shape changed platelets. 3) oxidized ldl advances, accelerates and enlarges platelet adhesion. 4) oxidized ldl causes cytodamage in platelets and cultured endothelial cells. 5) oxidized ldl stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. further experiments show that oxidized ldl inhibits the activity of ca2+-atpase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. these results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized ldl. since oxidized ldl, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. in this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. confluent cultures of human umbilical vein endothelial cells (hlivecs) were exposed to gc ° for 10, 20, 40, 80, or 160 rain, respectively. control cultures were exposed to 37°c for the same time periods. thereafter medium was removed, fresh medium was added and the cells were incubated for 24 hours at 37°c conditioned media (cm) were harvested and plasminogen activator inhibitor-i (pal-i) antigen was determined by a specific elisa. our data showed that huvecs responded to exposure to 8°c with a time-dependent increase of pai-i in cm. maximum induction of pal-! antigen was seen after 20 minutes exposure to 8°c (pal-! antigen: 754_+.83 ng/105 cells for treated cells; 4202_17 ng/105 cells for control cells). the inereese in pal-1 antigen was also reflected on the level of specific mrna synthesis as evidenced by northern blotting which showed a twofold increase in pai-i specific mrna in huvecs exposed to 8°c as compared to control cells. in conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of pai-i. our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. nitric oxide (no) is formed from intracellular l-arginine by two no synthase (nos) isoforms: a ca+ +-dependent constitutive form (cnos) and a ca+÷-independent inducible form (inos) which is expressed after challenge of cells with immunologic or inflammatory stimuli. endothelial cell derived no is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. the present study investigates the pattern of nos isoforms and no production in actively proliferating low density human umbilical vein endothelial cells (ld-huvec) and in high density monolayers of quiescent cells (hd-huvec). ld-huvec but not hd-huvec expressed inos (measured as ca ++independent citrulline formation from l-arginine in cell lysates) with a maximal activity of 30-40 pmoles/min/mg protein, cnos (ca +÷dependent citrulline formation) was demonstrated in hd-huvec (0.02-0.2 pmoles/min/mg) but not measurable in ld-huvec lysates. lmmunoprecipitation of cell lysate with a mab against murine macrophage inos decreased inos activity of supernatant by 70% in ld-huvec. immunoprecipitation with an anti-ecnos mab did not modify inos activity in ld-huvec but abolished cnos activity in hd-huvec. no release (measured as nitrite + nitrate production in an 18 h incubation in the presence of 1 mm l-arginine) was 1.9 nmol/mg cell protein in the supernatant of hd-huvec whereas it was t0fold higher with ld-huvec. measurement of intracellular cgmp after stimulation of cells with i-2 #m ionomycin (a sensitive index of enos activity) showed a 400-800% increase of basal levels in hd-huvec but only 50-150% increase in ld-huvec. on the contrary, sodium nitroprusside-induced cgmp increase was similar in non-confluent and confluent cells. the expression of inos and the high no production in ld-huvec suggest a role of endogenous no in mediating cell proliferation. indeed, the nos inhibitors l-nmma and l-canavanine inhibited [~h]thymidine incorporation in ld-huvec by 60%. on the ++ nt no r uctlon ar x r d m other hand enos and ca -depende p od " e e p esse " differentiated huvec and correspond to the ability of the cells to regulate vascular tone and platelet activation. the processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. while mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. in the present study the expression of mrna of urokinase receptor in megakaryoblastic cell lines chrf-280, dami and meg01 was found at a low basal level and increased 5-10 fold after pma-treatment. urokinase receptor mrna levels remained differentiation dependent elevated for up to 5 days. functional analysis of upar expression on the cell surface was documented immunologically by facs-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokjnase and mirrawed the increase in urakinase recptar mrna level. as expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase c, since the urokinase receptor belongs to the group of gpi-anchored receptors. in cultered megakaryocytes derived from bone marrow urokinase receptor mrna was demonstrated by rt-pcr, as well. these results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. regulation of am gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. the aim of this study was to investigate the influence of selected antioxidants on tnfa (100 u/ml, 6 h)-induced surface expression of elam-i, icam-i and vcam-i in human umbilical vein endothelial cells. am expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). the following antioxidants were tested: pyrrolidine dithiocarbamate (pdtc), n-acetylcysteine (nac), glutathionemonoethyl ester (gshmee), probucol and the estradiol-derived scavestrogens j824, j861, j901. additionally, 17fi-estradiol was examined. the used concentrations were 1-5 mm for nac and gshmee and 1-100 ~m for all other substances. according to their influence on tnf-induced am expression, the tested antioxidants can be divided into three groups: 1) pdtc, nac and gshmee inhibit the expression of all three am, 2) j861 and j824 reduce vcam-i and icam-i and increase elam-i, 3) j901 and probucol show no effect. 17fl-estradiol elicits a potentiation of tnfa-induced expression of all three am. tnfa-induced vcam-i expression was most effectively inhibited by ptdc and j861 (100 em: 68% and 78% inhibition, respectively). the best inhibitors of icam-i induction were pdtc (100 ~m: 51% inhibition) and j824 (100 t~m: 47 % inhibition). j824 and j861 most effectively increased the elam-t induction (100/~m: 85 % and 62 %, respectively). the results confirm that antioxidant-sensitive mechanisms play a role in tnfct-induced am upregulation. it is suggested that icam-1-and vcam-l-expression is regulated differently from elam-i. the diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. heamophiliacs who continue to produce tilers of f.v11i inhibitors no higher than 5 to 10 bethesda units (bu) when given continuous f,viii replacement therapy are classified as low responders. studies carried out on low responders to assess the effect of immune tole~lcc therapy flit) in these patients. it was found that elimination ofiahibitors in low responders could be achieved using f.vlii at 20 to too u/kg given every 2 to 3 days over a period of 6 weeks. we consider that 1tt is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of f.viii inhibitors. it also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. in view of the improved prognosis, we recontmend starting itt in low respor, ders soon after the diagnosis has been made in order to minimize f.vill exposure before isfi.tiating el'. itr in low responders has a better success rate an.,d requires shorter u'eatment than itt in high responders. w.schramm, med.klin~, innenstadt, lmu, mfinchen following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. the incidence of inhibitors varies between 15 and 35 % of persons with hemophilia a. inhibitor develop, mostly in paticm~ with sovere bemoph/lia a, early in life and afmr few exposure days (9 -12 days). using the bethesdamethod the inh~itom can be derided in low (< 5 bu) and high msponder (>5 bu) hemopb~iacs with high fitcrs have ~ually serious ~ problems. they are resistent to mg,,flary replacement therapy, the ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. in the tream'tmt of acute blcedings in patients with hlhibitors factor viii inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (feiba) or prothrombin complex concentrates (pcc) arc mostly used. the meehani~n of aefiou of theses concentrates is net fully investigated. their effect is usually related to the high coment of activated clotting factors ~d phosphoupids. since some years acdwated recombinant factor vii (f vii a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. in addition porcine factor vii1 is widely used in particular in the uk for the treatment of factor v].ii inhibitor patients and could demonstrade good clir3cal results, in case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (protein a adsorption) and immune suppression with cyclophosphamid (~alm6 protocol). follow~g the first description by h. bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia a patients have ~en propet'ed. these schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'jng in the dosage of factor viii infused. successful rates about 70 to go % can be obtained with ~ and high dose regimens. but is has to be co~sidered that the~ expensive trea.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& the different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. most of the patients with low concentrations of inhibitors cm be managed with factor viii in increased dosage. this is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in germany fi'orn 1994. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of eases, vk deficiency oceured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacental transport of vk in the perinatal period,following studies were carried out. t)hepaplastintest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlations were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastin test value of less than 120% of the normal adult value, the value of the hepaplastintest was less than 30 % of normal adult value in the cord venous blood° we also demonstrated that vk passed through the placenta but only in small qualities. 61hiv-negative patients (median age 4]yrs, 13-70}, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including afp screening and sonography. 31.suffer from severe, 20 from moderate or mild haemophilia a or b, 10 from other severe coagulation factor deficiencies. 52 had been treated with products of the swiss red cross (src) only (28 with small pool cryoprecipitate}, 4 with foreign products only, 5 with both src and foreign products. treatment intensity was variable with>20'000 iu/yr in 26,< 20'000 in ]6, < 1 treatment episode/yr in ]0, a total of only 1-3 treatment courses in 6 patients. 3 afibrinogenemic patients had prophylactic replacement therapy. hcv serology was positive in 56/6] patients (92%), in 47 with detectable hcv rna (77%). the 5 persons who escaped hcv infection, with normal alt-levels and without sonographic alterations, had low intensity treatment with small pool src preparations only. alt-levels were elevated in 33/56 anti-hcv positive patients (59%). 26/56 had abnormal sonographic findings (46%). there was a clear correlation between elevated alt-levels and abnormal sonographies: of 33 patients with elevated alt 23 had abnormal sonography, of 23 with normal alt 3 had abnormal sonography. 8 patients had liver cirrhosis (6 with clinically overt hepatopathy), 4 (4/56 = 1%!) with hepatocellu]ar carcinoma (hcc) with elevated afp-leveis. of these 4 patients 2 had intraarterial embolization with ]ipiodol-epirubicin; in 2 patients hcc diagnosis was made in a late stage. i patient with advanced liver cirrhosis underwent successful liver transplantation. 2 of the 6 patients with hepatopathy had severe haemophilia with temporary high alcohol intake, 4 had mild coagulatlon disorder with few treatment episodes. possible precipitating factors were coinfection with hbv, high alcohol consumation and first exposure to hcv contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for f vlll and vwf. since the factor viii level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections. 22 children with severe form of haemophilia a undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant f viii concentrates. before surgery, patients got bolus injections to raise the factor viii levels to more than 80 %. during continuous infusion factor viii levels were measured two to three times a day and the infusion rate of 4 to 5 iu/kg/h could be reduced on the second or third day to 2-3 iu/kg/h. the clinical efficacy was excellent with no bleeding events. in 5 children with vwd also undergoing elective surgery continuous infusions with humate pr were performed in the same way. no bleeding events were observed in these patients. none of the patients developed postoperative wound infections. the overall doses of f vtll concentrate 'were about 20-30 % lower than those required during replacement therapy with bolus doses. lg8 factor x frankfurt i : molecular and functional characterisation of a hereditary factor x defect (gla +25 lys) huhmann i., holler b., krinninger b., turecek p.l, richter g., scharrer i., forberg e., watzke h. univ. klinik f0r inhere med.i, abteilung for h~tmatologie und h~mostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrombin to thrombin. the congenital fx-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his p'fi (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboll site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exert ii. fx was isolated from plasma of the propositus by monoq ion exchange chromatography. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intdnsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account. we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardiopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64__+9 y). they lost 750+__460 ml blood (mean+sd) into the drains within the first 18 hours after end of surgery. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificity of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptidea (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n = 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other parameters were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrin will provide further insights into fibrin(ogen) metabolism. heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. the initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. conventional diagnostic methods include platelet aggregation responses to patient's serum and ~4c serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor 4 (hpf4-ab) ned-antibodies by using elisa methodology. several other individualized methods are also used to demonstrate platelet activation. to test the diagnostic validity of the platelet aggregation (pa) 14c serotonin release (sr) and the relevance of hpf~-ab 340 serum samples collected from patients with clinically eunfwmed eases of lilt syndrome were compared in parallel in various assay systems. the diagnostic efficacy of these tests varied from 60-74% with the pa test providing better results than others. when the pa test was compared with serotonin release, a poor correlation was noted (r=0.47). in contrast, the correlation between the pa and hp4-ab was somewhat better (r=0.66). in another study, blood samples collected from 50 patients treated with ahigh dose low molecular weight beparin for two weeks (80 mg o.d.) were tested. 20 of these patients showed a high titre of hpf4.ab without any decrease of platelet count. none of these patients were found to be positive in the 14c serotonin release assay. a third study included blood samples from dvt patients administered with iv heparin infusion, high dose sc lmw heparin (certoparin) and iv lmw heparin for the management of dvt. none of these patient groups (20-34) exhibited any hit responses, hmvever, the incidence of high hpf4-ab titre was found to be 53% in heparin, 36% in patients with lmw heparin iv and 26% in lmw heparin sc groups. pa and sr studies revealed 8% and 12% false positive ~ respeetively. these studies clearty suggest that the currently available ~ for laboratory diagnosis of hit syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. heparin-induced thrombocytopenia (hit) is one of the major severe side effects during treatment with heparin. in postoperative medicine clinical studies demonstrated the prevalence of hit with unfractionated over fractionated heparins. few data are available from the non-ope1"ative medicine and from patients without thmmboembolism before heparinization. in a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (lmw) heparin over 10 days in bedridden medical inpatients (haemostasis, in press). 1968 patients were randomized and controlled for the development of thrombocytopenia. thrombocytopenia was defined as a platelet count below 40.000lid at day 10. 4 patients developed thrombocytopenia in the heparin group and no patient in the lmwheparin group (p<0.05). none of the patients with thrombocytopenia developed a thromboembolic complication. in a second prospective case control study 90 patients with side effects on anticoagulants were treated with lmw-heparin once daily subcutaneously for a period of 1 month to 9 years. platelet count was performed every 1 to 3 months. none of these patients developed thrombocytopenia during heparinization with lmw-heparin. it is concluded that hit is a very rare complication in nonoperated bedridden medical patients. a decrease of platetet count may occur in about 0.5% of patients receiving low-dose heparin. the incidence of hit with thrombosis during low-dose heparin and of hit during lmw-heparin in non-operated patients is manyfold lower and remains to be determined. terminology: instead of the term "hemorrhagic disease of the newborn (hdn)" the term vkdb should be used, since neonatal bleeding is often not due to vkdeficieacy and vkdb may occur after the neonatal period (i.e. after 4 weeks). definition: vkdb is a bleeding disorder caused by reduced activity of vkdependent coagulation factors which responds to vk. diagnnsis: in a bleeding infant a prolonged pt (inr > 3.5) together with normal fibrinogen and platelet count is almost diagnostic of vkdb. the diagnosis is proven, if vk shortens the pt (after only 30-60 minutes) and/or stops bleeding. classification: classification by age of onset into early (< 24 h~. classic fdav 2-7) and lale form (> i week <6 months), and by etiology into idionathic and ~ec0nd~'y. in secondary vkdb in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of vk and increased consumption of vk. vk-prophylaxis: benefits: oral and intramuscular (i.m) vk (one dose of i nag) prevents equally well the classic form of vkdb. lm. vk appears to be more effective in preventing the late form (times 15-> 50). the protection achieved by single oral prophylaxis (times 3-5) is improved by triple oral vk (times 15-30). risks: because of poten[ial ri~l~ associated with extremely high levels of vk and the possibility of injection injury, i.m. vk has been questioned as the prophylaxis of choice for normal neonates. since vk is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. proposal: repeated (daily or weekly) small oral doses of vk are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high vk levels. the incidence of intracranial vkdb can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). whether or not the more reliable absorption of the new mixed mieellar (mm~ nrenaral~i0n of vk can reduce the protective oral dose of vk-.prophylaxis has to be evaluated. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of cases, vk deficiency occured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacentel transport of vk in the perlnatal period,followlng studies were carried out. 1)hepaplastlntest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlatlons were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastln test value of less than 120% of the normal adult value, the value of the bepaplastlntest was less than 30 % of normal adult value in the cord venous blood. we also demonstrated that vk passed through the placenta but only in small qualities. the point mutation g to a at nt 449 in exon v of the factor x gene (gin 102 to lys) has previously been found in two independent kindreds with fx deficiency. it occured in both families in an heterozygote state and was associated with two other genetic defect in the fx gene. we have identified another familiy in which this mutation occurs in a homozygote state. in this family the mutation is associated with the previously reported mutation gla 14 to lys which also occurs in a homozygote state. the pt and ptt of the proposita and her siter are markedly prolonged. the fx activity is reduced to <1% in the extrinsic system, to 30% in the intrinsic system and to 18 % after activation with rvv. the fx antigen is reduced to 20%. the coagulation profile of this family thus is identical with that of fx vorarlberg despite the fact that the fx vorarlberg kindred is only heterozygous for the mutation glal02 to lys. haplotype analysis could not rule out consanquinity with the fx vorarlberg kindred. these data suggest that the mutation at nt 449 which leads to a fairly dramatic amino acid change from glu to lys would indeed represent a polymorphism. to further address this question we cloned the fx gene in an expression vector (pcep 4) for transient expression in the human embryonic kidney cell line 293 and introduced the mutation at nt 449 by site directed mutagenesis. hereditary deficiency of factor ixa, a key enzyme in blood coagulation, causes hemophilia b, a severe x-chromosomelinked bleeding disorder; clinical studies have identified nearly 500 deleterious variants. the x-ray structure of porcine factor ixa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fx activation by phospholipid-bound flxa and cofactor villa. the 3.0 a resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (egf)-like modules; the n-terminal gla module is partially disordered. the catalytic module, with covalent inhibitor d-phe-pro-arg chloromethyl ketone, most closely resembles fxa but differs significantly at several positions. particularly noteworthy is the strained conformation of glu-388, a residue strictly conserved in known fixa sequences but conserved as gly among other trypsin-like serine proteinase. flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. most hemophilic mutation sites of surface fix residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flxa-fvilla-fx complex structure: the stabilizing fvilla interactions force the catalytic modules together, completing flxa active site formation and catalytic enhancement. factor x frankfurt i molecular and functional characterisation of a hereditary factor x defect (gla +25 ---, lys) huhmann i., holler b., krinninger b., turecek pi., richter g., scharrer i., forberg e., watzke h.. univ. klinik ftlr innere medi, abteilung for h~matoiogie und hamostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrembin to thrombin. the congenital f×-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his ptt (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular characterisauon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboti site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exon i1. fx was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intrinsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account_ we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective ip both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardlopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64+9 y). they lost 750+__460 ml blood (mean_+sd) into the drains within the first 18 hours after end of sur. gory. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificily of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptide a (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n= 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other paramelers were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrln will provide further insighls into fibrin(ogen) metabolism. this study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {lmwh} -monoembolex sandoz and unfractionated standard heparin glfh) for the perioperative prevention of venous thromboembolie disease (dvt) following major surgms' in patients with gynecologic malignancy.. three hundred and twenty 4 women (six drop outsl werr randomized and received either 3 times daily 5000 [l" s.c. ul.'i-i (sandoz nuemberg germany] (n = 164) or once a day t5000 i~'v'. units s.c. monoembolex (n = 160) plus two placebo injections. heparin therapy was started the morning before opcrati(m and continued until the 7th postoperative day. up to the 10th poatop, day the incidence of dvt was 6.25 % (n = 10; incl. 7 pulmona~ embolisms pe) in the lmwh group and 6.10 % (n = 10; incl. 4 pe} in the ufh group. the overall incidence of clinically hemorrhagic wound complications was significantly decreased in the lmwh group 16.3 % (n = 2hi compared to the ufh group 26.8 % {n = 44; p < 0.0051. the incidence of major hemorrhagic episodes was 9.4 % in = 151 in the lmwh group and 14.0 %/n = 23) in the ufh group. this difference was not statisticauy significant. one case of fatal pe was observed in the lmwh -treated group. five women deaths in the lmwh group were observed during the study and 3 in the ufh group. this study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. however, the incidence of thromboembohc complications is simmilar in both treatment regimes. to explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (hir) was covalently linked to the fab' fragment of fibrin-specific monoclonal antibody 59d8 (fab) resulting in a stable conjugate (hir-fab). in vitro, hir-fab was 9times more efficient than hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p<0.01). to validate these results in vivo, hir-fab was compared to hir in a baboon model. the deposition of ill-in-labeled platelets onto a segment of dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. blood flow rate was 40 ml/min. one hour local infusions of 4500 atu of either hir-fab or hir resulted in deposition of 0.16 x 109 and 2.17 x 109 plate!ets, respectively. equieffective dosages were 2000 atu hir-fab and 9000 atu hir resulting in deposition of 1.06 x 109 and 0.93 x 109 platelets, respectively. based on full dose response curves (n = 14), hir-fab was found to be > 4.5-fold more potent (based on activity) than hir. because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. triabin, a 17 kda protein from the saliva of the assassin bug triatoma pallidipennis, is a new specific thrombin inhibitor (1). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. the recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. aggregation of platelets in tyrode's solution was measured turbidimetrically at 37°c. for the studies on blood vessels rings (2-3 mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. the integrity of the endothelium was assessed by the relaxant response to bradykinin. like hirudin, triabin inhibited the thrombin (0.1 u/ml)-induced aggregation of washed human platelets at nanomolar concentrations (ec50 = 2.6 nmol/l); whereas the adp-and collagen-induced aggregation were not suppressed. in pgf2c~-precontracted porcine pulmonary arteries, the thrombin (0.2 u/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin (1 u/ml). in all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. these studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. dept. of medicine, university hospital benjamin franklin, free university of berlin, dept. of medicine and dept. of surgery, heinrich-heine:university dusseldod after standardized training in home prothrombine estimation using the coaguchek system, 150 consecutive patients (p) who had st. jude medical aortic or mitral valve implantation were allocated to two random arms; 75 p were asked to control the inr themselves every third day. in the remaining 75 p anticoagulation was managed by the home physician without recommending an interval for these controls. all 150 p were monitored during the education period to a target therapeutic range of inr 3.5-4.0. p were asked to contact their home physician immediately if the inr was measured 0.5 below or above the target range (inr-corrider 3.0-4.5). all p had out-patient re-examinations every three months. thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. the following findings were documented during the follow-up period: 4.49 0.9 0 the results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. significant (p<0.001) more inr measurements were found inside the target therapeutic range. moreover. bleeding and thromboembolic complications could be reduced (p = 0.038) in the study group with home prothrombine estimation. life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events (2.72 %/year) were documented in group a. local vascular injury following ptca exposes circulating platelets to prodmmbogenic stimuli. by binding to platelet gp iiblliia fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. fradafiban (bibu52zw) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to gp iib/ii/a on human platelets. in the first double-blinded, prospective phase ii study three escalating doses of bibu52zw as a continuous 24 h-i.v, infusion were tested in comparison to placebo in 65 patients with stable angina pectoris undergoing elective ptca. the mean receptor occupancy with 20rag, 40ms and 60ms per hour were 71.974, 84.5 % and 87.9% at 24 hours, respectively. as compared to placebo breeding time was significantly prolonged (7 vs 20rain) during fi-adaiiban infusion with a weak dose-dependency. platelet aggregation in platetet rich plasma ex vivo with collagen (2.0 and 4.0 gg/ml), adp (2.5 and 5.0 gmol/ml) or ca-ionophor a 23187 (2.5 and 5.0gg/ml) was significantly and dose-dependently inhibited as compared to placebo. using the two upper doses of fradafiban, we observed major bleeding complications in 8 patients requiring blood transfusions or vascular surreal repair. in these patients, too, maximal antiplatelet effects could be documented. these data sugest that bibu52zw is an effective fibrinogen receptor antagonist in patients. the requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. in a placebo controlled interaction study 9 healthy volunteers were randomized to receive either a 24 hour infusion of peg-hirudin ( 0.02 mg/kg/h) after an i.v, bolus of 0.2 mg/kg + placebo, or 325 mg/day acetylsalicylic acid (asa) for three days followed by a placebo infusion or the peg-hirudin infusion + asa. each volunteer received all three treaments. there was a washout period of at least 14 days between the infusions. at short intervals aptt, activated clotting time (act), ecadntime (ect), alia-activity using the chromogenic substrate 2238, collagen-induced aggregation, platelet adhesion and platelet induced thrombin gene,ration time (pitt) were measured, bleeding time (simplate) was studied before drug administration, on day three before the infusion and 6 hours after start of the infusion.the infusion of peg-hirudin after 4 and 8 hours led to a mean hirudin plasma level of 1.8 pg/ml. asa markedly inhibited collagen induced aggregation as expected. the mean bleeding time was prolonged under the influence of peg-hirudin from 5.2 to 6.22 min, after asa from 5.8 -18.2 min and after the combination of peg-hirudin + asa from 5.4 -33.7 min. in each volunteer the bleeding time was longer under the combination than after asa alone. in two volunteers receiving peg-hirudin + asa the bleeding time measurement was stopped after 60 rain. none of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. however the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under asa alone.the combination of hirudin at a higher dosage with asa probably is associated with a relative high risk of bleeding. either the hirudin dosage should be reduced if the combination seems feasabie or asa should be given after the end of hirudin treatment. fibrinogen with the sta/stago and the mla/dade systems correlated well, but neither system correlated well with the acl/il system. at iii, protein c, protein s, and anti-xa heparin assays using stago reagents performed as expected for normals and low abnormals on the sta. factor levels on the sta/stago system were less sensitive than factor levels obtained with the dade reagents on the mla or fibrometer. using the sta/stago system, thrombin time results correlated well with the aptt and heparin levels. the thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the sta reagents do not come into contact with tubing. the sta was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the mla for chromogenic assays. the overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the sta hemostasis analyzer. a. wehmeier, d. s6hngen, c. rieth klinik for h#,matologie, onkologie und klinische immunologie der heinrich-heine-universit~it d0sseldorf hirudin selectively inhibits thrombin by direct interaction. because the effect of hirudin is independent of antithrombin iii and other factors, it seems an attractive alternative to current anticoagulants. however, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and lmw heparin. we investigated the effect of 2 recombinant hirudin preparations (rhein biotech, dt3sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. hirudin was used in concentrations of 0.1-100 i.tg/mi, and was compared to trisodium citrate (0.38%), conventional heparin (50 iu/ml) or lmw heparin (fraxiparin, 500 iu/ml). both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aptt. however, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and lmw heparins. platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about 40% but was more effectively reduced by both heparin preparations and citrate. hirudin had an inhibitory effect on p!atelet aggregation in prp induced by thrombin, collagen, and predominantly epinephrine but not adp and ristocetin. in whole blood, a small effect could only be observed with hirudin concentrations of >25 ~g/ml as compared to citrateanticoagulated blood. in summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. the role of endothelin (et), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. in anaesthesized pigs the effects of i.v. infusion of et (3 /~g/kg) alone (group 1, n=6) and after pretreatment with the potent thrombin-inhibitor hirudin (0,5 mg/kg)(group 2, n=6) on haemodynamics, coagulation parameters (factor viii, antithrombin iii, precallicrein, fibrin monomers, aptt) and prostaglandins were investigated. plasma renin activity (pra)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. et-infusion caused an initial bp-reduction and marked hr-reduction followed by a transient bp-elevation and hr-reduction. activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. in an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, dup 714, recombinant hirudin and peghirudin on platelet activation induced by various agonists was studied in whole blood. blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. for anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of 10 ~tg/ml each. blood samples were then incubated at 37°c either with saline, r-tissue factor (rtf), arachidonic acid (aa), adenosine diphosphate (adp) or collagen. at definite times (1, 2.5, 5, 10 rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors gpiiia (cd-61) and p-selectin (cd-62). the agunists used induced a platelet activation of 37.4 + 15.2 % (rtf), 65.1 + 12.1% (aa), 19.3 + 7.4 % (adp) and 27.1 + 12.8 % (collagen). flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. in contrast, after induction of platelet activation with the other agonists an increased percent cd-62 expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood. in conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. the formation of active serine proteases including thrombin may be effectively inldbked by these agents. the observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. this work was supported by the grant bmft 07nbl01. animal experimental studies on the pharmacokinetics of peg-hirudin e. bucha, a. kossmehl, g. nowak max-pianck-gesellschaft e. v., arbeitsgruppe "pharmakologische h~imostaseologie", jena hirudin, when complexed with polyethylene glycol (peg), increases its molecular weight from 7 to 17 kda, thereby preventing extravasation of this drug. peg-hirudin is distributed almost only in the intravascular blood space. in addition, its increased molecular weight retards the renal elimination. the elimination half-life of hirudin in rats (58 + 12 min, as determined) is increased five-fold (244 ± 18 min). with the same hirudin dose applied, the blood level of hirudin is increased 19-fold, measured in the 13-elimination phase. in the urine of rats, 2 -3% of the hirudin activity were recovered following hirudin administration, but 48% could be detected after peg-hirudin had been applied. after subcutaneous administration of peg-hirudin, the trnaxwalue is reached at 380 rain (r-hirudin: 65 min); the cmax-value is increased 3-fold, compared to that of r-hirudin (2.5 pg/ml). 24 hours later, still one fifth of the maximum concentration (cma,) is present in the blood, and the renal elimination is still retarded. in the urine of rats, 12% of the hirudin activity applied were recovered in the 24-h urine sample. with intact renal function, following subcutaneous administration, peghirudin is abte to produce a constant blood level of hirudin over a long pedod. thrombin inkibitors such as r-hirudin (rh), argatroban (a), efegatran (e), and peghiradin (ph) are currently undergoing extensive clinical trials in such cardiovascular indications as ptca, ami, and treatment of unstable angina. a rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. currently, act and aptt are used to measure the anticoagulant effect of these agents. we have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (plop) to measure the antithrombin effects of various thrombin inhibitors (cv diagnostics, raleight, nc). the heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (a)0 (e), (rh), and (ph). blood samples drawn from patients treated with (a) and (rh) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. in the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. the results obtained are comparable to the act which is conventionally used for the monitoring of these agents. both (rh) and ( period. we would like to present a case of heparin-induced-thrombocytopenia (hit) in a 47 years old woman who underwend open heart surgery. she suffered from a combined aortic valve disease and leading stenosis. laboratory analysis showed constant low platelet counts (50/nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. but platelets also decreased after heparin application. heparin-antibodies were found in the heparin induced platelet activation assay (hipaa). treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. therefore, the only suitable anticoagulant for the necessary aortic valve replacement was hirudin: a bolus injection of r-hirudin of 0,75 mg/kg b.w. was administered 10 min. bevore start of the extracorporal circulation (ecc), the heart-lung machine (hlm) was primed with 6 mg r-hirudin and another bolus of 5 mg of r-hirudin was administered. additionally 10 mg of r-hirudin was applicated to the cell-saver-reservoir. during the period of ecc ecarin clotting time and aptt values were taken every ten minutes for monitoring r-hirudin concentration. the postoperative anticoagulation was performed with a constant infusion of r-hirudin starting eight hours after the end of ecc and monitored by aptt. due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting 3 days postop. the therapy with hirudin showed no side-effects. hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. doses fi:om 2-30 mg/kg gave similar post-op blood loss measurements without s dnseresponse (4-15 oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; 10 q-3 ec/kg). doses >30 mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. the bleeding time test was less elevated than for heparin. platelet counts and hematoerit did not vary except for hemodihition on pump. liver enzymes did not vary significantly pre-op to post. act values showed arg was eliminated (dose-dependently) by 1 hour post-op. dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to arg (as opposed to variable responses to heparin). in a substudy it was demonstrated that hypothermia did not affect the activity of arg, nor did varioos formnlations. this dose finding study strongly suggests that arg may be a safe and effective alternative to heparin for patients undergoing cpb. this is particularly important for the growing population of patients with hit who require cardiac surgery, for which no anticoagulant alternative is presently available. three recent clinical tdals with r-hirudin (timi 9, gusto 2 and hit) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aptt-levels. the large intedndividual variability of the aptt and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. we have therefore evaluated the ecadn clotting time (ect) as descdbed by nowak and bucha (thromb. haemost. 1993; 69: 1306) under conditions which allow conclusions on its reliability in the clinical situation.for this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of peghirudin. measurements of aptt and ect were made in duplicate. in contrast to the aptt, the ect showed a close, linear relationship with peg-hirudin plasma concentrations in the range of 100 and 10 000 ng/ml. the lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to 10 pg/ml. the ect was not affected by fibdnogen concentrations 60% below normal. a somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between 20 and 32%. no significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. the most potent thrombin inhibitor containing a benzamidine moiety is napap (k i = 6 nmol/i). unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of napap as an oral anticoagulant. the application of choice of a synthetic thrombin inhibitor would be the oral one, therefore, we looked for other lead structures. with the nc~-arylsulfonylated piperazides of 3-amidinophenylalanine we found a new group of derivatives which inhibit thrombin with ki-values in the nanomolar range. the piperazides exert anticoagulant activities with high selectivity, leaving activated protein c and components of the fibdnolytic system unaffected. in rats, the piperazides are rapidly eliminated from the circulation (tl/2 ~ 10 min) upon i.v. administration, too. after oral administration, the systemic bioavailability is low. upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. to cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the n~-(2naphthylsulfonyl)-3-amidinophenylalanine n'-acetylpiperazide in rats using hplc-analysis. like other benzamidines the piperazide is excreted via the bile to a high extent. enteral absorption rates of about 20 % are found after blocking the hepatic uptake and biliary excretion. hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. at the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. the piperazides of 3-amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. a 69-year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. in addition, growing thrombocytopenia was observed involving a drop in platelets by 50%, compared to the initial values. the haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. at the end of may 1995 heparin antibodies were detected and the hat diagnosis was confirmed. immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. using steam-stedlised haemophan dialyzers and 0.14 mg/kg r-hirudin (iketon, italy), the minimum therapeutic blood level of hirudin (0.4 pg/ml whole blood) was reached. this provided therapeutically relevant blood level conditions during a 4.5h haemodialysis. more than 80 regular haemodialyses were run without problems. in all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. after the 34th haemodialysis, the frequency was reduced from 3(4) to 2 haemodialyses a week. accordingly, the hirudin dose was increased to 0.2 mg/kg. the creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis. platelet count and haemodialytic efficiency normalized. we could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. capillary electrophoresis methods provide a fast measurement of proteins. thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. for the measurement of r-hirudin we used fused silica capillary and a borate buffer. this buffer was used to detect r-hirudin, but could not be used to measure peg-hirudin. for simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. the buffer was a 20 mm tricine buffer (ph = 8.0 field strength 500 v/cm). it resolved r-hirudin from peg-hirudin at 214 nm using reverse polarity. a linear correlation between the peak area and the concentration was found between 80 pgtml and 10 mg/ml for hirudin (r 2 = 0.99) and between 2,5 and 10 mg/ml for peg-hirudin (r2= 0.99) was found by coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. a linear correlation between the peak area and the concentration was found. the method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. triabin is a thrombin inhibitor from the saliva oft. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. we used sf9 insect cells infected with recombinant baculovirus to produce sufficient triabin for a detailed biochemical characterization. the activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. a 4-fold prolongation of thrombin-clotting time and aptt was achieved with 22 nm and 600 nm triabin, respectively. a kinetic analysis of the thrombin-catalyzed fibrinopeptide a release from fibrinogen showed that triabin is a tight-binding inhibitor. using the graphical method of dixon, the ki was determined to be 3 pm. introduction: thrombocytopenia is a common adverse effect of heparin therapy, in type ii hit platelet decrease induces severe complications. we here present two special cases of type ii hit. case report i: a 66 year old male patient with dvt of the left leg was treated with therapeutic doses of heparin. from the first to the 12th day of therapy, platelet count decreased from 122000 to 54000/ui. hit was confirmed by hipa-test, heparin therapy was s~opped and treatment with the heparinoid orgaran n was started. during the following days, arterial thromboses in the right a. femoralis occurred. several thrombe~tomies were not successful and although orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. during the following days under hirudin-treatment platelet count normalized and no further complications occurred. case report 2: a 74 year old female patients suffering from hip fracture was treated by surgery with tep-operation and received prophylactic heparin treatment. after 6 days, platelet count decreased from initially 170000 to 8000/ul and dvt of the right leg was diagnosed. on the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to 7.8 g% (before surgery 16.0 g%). hit was confirmed by hipa te~t, heparin was stopped and treatment with orgaran started. thrombocyte count normalized and no further complications occured. conclusion: hit type ii can cause severe bleeding as well as thromboembolic complications. because of possible cross-reactivity between heparin and orgaran~,, hirudin should be given in hit patients. currently thrombin time (ti), aptt, activated clotting time (act) or anti ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. the "13" responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. aptt appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. act is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. the ecadn clotting time (ect) is a new clotting assay, recently described by nowak and bucha (thromb.haemost 1993, 69,1306) . it measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. ec.t shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. 0.1-5 pg/ml). in a clinical interaction study healthy volunteers were administered hirudin, asa or both. 15 male volunteers received an i.v. infusion of peg-hirudin (0.02 mg/kg/h) for 24 hours after an initial i.v. bolus of 0.2 mg/kg to compare the sensitivity and reliability of ect with aptt, l-r end act. the act was measured on the hemochron 801, usa, ect on a fibrin timer, aptf using the aptt lyophylized silica reagent by il and alia on an acl (il-milan) with the chromogenic substrate 2238. all tests were performed in duplicate. ect was more sensitive to different hirudin concentrations than aptt, or act. the ect results were better correlated with the alia-activity than ap'l"r and act. the lower detection range for ect is 0.05 pg/ml hirudin. ect is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. the therapeutic effects of these agents are usually monitored by aptt whereas in cardiovascular indications, cefite act and hemotech® act are used. the reliability of both aptt and the act tests in predieting the safety of various thrombin inhibitors has been heavily debated. furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained aptt and act results do not lady refleot the effects of these agents. fcafin is a snake venom derived fi'om echis carinatus which converts prothrombin into mesothrombin, targeting the arg~3-ile tm bond between the a and b chains of prothrombm. while thrombin inhibitors are capable of inhibiting mesothrombin, atiii/beparin complex does not have any effect. using purified ecarin, nowak and bucha (1993 thromb haemost 69:1306) proposed to assay hirudin. since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ect) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. lyphilized eoarin was obtained from knoll ag, ludwigshafen, germany). concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of 0-15 p.g/ml. all of the antithrombin agents produced a concentration dependent prolongation of ect and showed va~angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. on a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. utilizing the ect, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ 10 gt g/rnt, have been measured. unlike such global tests as pt and aptt, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ect. plasma samples from heparinized (aptt 45-90 sec) or coumadinized ('pt 15-25 see) patients, supplemented with argatroban or hiredin did not show any differences m the ect. a medified ecarm act comparable to the celite act has also been developed. initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the inr range of 1.5 -3.0. these results indicate that ecarin based clotting times provide slx~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. r-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. after intravenous administration, r-hirudin is eliminated exclusively with the urine. its plasma half-life is very short, 1-2h. peg-hirudin is a derivative produced by coupling polyethylene glycol (peg) to a specially designed recombinant hirudin mutein. peg-coupling results in a considerable prolongation of the plasma half-life of peg-hirudin, compared to r-hirudin. after intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity (2 -4% of applied activity) was recovered in the urine. in contrast, after peg-hirudin had been administered, more than 30% of the applied activity could be recovered in rat urine. these results suggest differences in the renal metabolism of peg-hirudin and r-hirudin. within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of peg-hirudin after kidney passage in urine. affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. but it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. the isolated biologically active metabolites were purified by reversed-phase hplc and were biochemieally characterized. in previously reported studies we found a hlrud n derivative consisting of the amino acids 1-50 as the main metabolite in rat urine following intravenous administration of r-hirudin. this metabolite was not detected in the urine after administration of peg-hirudin, confirming the suggestion of a different renal metabolism. carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. in previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. only a part of its antithrombin activity is indirectly mediated through antithrombin iii. to investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. the molecular weight has been determined with the aid of size exclusion hplc using dextrans as molecular weight standards. the degree of sulfation has been determined by anion-exchange hplc. we have obtained low molecular weight lambdaearrageenans ranging from 10,000 dalton to 100,000 dalton with degrees of sulfation of 13 -17% and 33 -38%. the anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. the activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. we have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. the anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. the natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors ap-l(jun/fos) and nf-~:b in cultured endothelial cells (ec). when ec were cultured in the presence of curcumin, electrophoretic mobility shift assays (emsa) demonstrated, that binding of endogenous ap-1 to its dna recognition motif was suppressed. inhibition was due to direct interactions of curcumin with the dna-binding motif for ap-i. enhanced ap-1 binding, induced after tnfa stimulation of ec, was decreased in cells pretreated with curcumin. this resulted in reduced transcription and expression of tissue factor, known to be controlled by ap-f and nf-~b. nuclear run on assays proofed, that curcumin directly reduced the tnfa mediated transcription of genes, regulated by ap-1, as tf, endothelin-1 and c-jun. thus, curcumin did not only suppress apl(jun/fos)-binding, but also inhibited tnfa induced jun transcription, transient transfections with tissue factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact ap-i sites. beside its effect on ap-l-binding, curcumin reduced the radical dependent activation of nf-kb due to its antioxidant properties, however, this inhibition was indirect and less prominent. the relevance of the in vitro data was confirmed in vivo in mice bearing meth-a-sarcoma. when mice received curcumin before tnfa was injected, tumors showed reduced ap-1 activation. simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced tissue factor expression. thus, curcumin inhibits ap-t activation and expression of endothelial genes controlled by ap-t in vitro and in vivo. (jung, 1991) . additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. in all patients aptt, bleeding time, platelet adhesiveness, von wiuebrand f~ctor and factor viii concentration and activity were determined. the patients with von willebrand disease showed characteristic morphological changes of capillary geometry. tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. plasma viscosity was significantly low. multiple parameter analysis concerning to galen and gambino (1983) and using the parameters ,,plasma viscosity below 1.25 mpas", ,,torquation index higher than 5", ,,erythrocyte column diameter bigger than 16,9 gin" showed a positive predictive value of 100 %. capillary diameter and capillary tortuosity have a positive predictive value of 91,2 %. additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon willebrand patients. it was quite remarkable, that 16 of 40 of the yon willebrand patients showed significant capillary bleedings. these findings confirm some former observations (e.g. o'brian 1950) and preliminary reports of our group (koscielny 1994). polymerase chain reaction (pcr)-based quantitation of mrna transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the bernard-soulier syndrome. however, for the exact quantitation of mrna a number of methological requirements has to be met. first, a standard (s) mrna must be synthesized which is able to undergo the same processing as the target wild type (wt) mrna. secondly, the quantitation step following the pcr must differentially recognize standard and target dna, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. in order to satisfy these requirements we constructed a s-gpib mrna which is identical to the wt-gpib mrna except a 13 bp long primer recognition site at its 5" end allowing differentiation between the pcr amplified wt-or s-gpib cdna through incorporation of a fluorescein or biotin labelled 5" primer. both standard and w[ gpib mrna showed identical amplification kinetics in the pcr reaction. the amplified dna was quantified using an dna binding assay. in this assay binding of amplified dna to gcn4 fusionprotein-coated microtiterplates is measured. since the gcn4 binding motif is incorporated into the wt-and s-gpib cdna through an identical 3" primer, competition between s-and wt-cdna during amplification has been analyzed. at a given concentration of 250 nm of gcn4. primer no competition between the sdna and wt-dna for the primer was observed during 25 pcr cycles. the sensitivity limit of the assay performed in this way was 250 amol wt-gpib~, dna, and intraassay variability reached from 1.66% to 6.72% calculated for 100 fmol and 5 fmol dna, respectively. to sum up, combination of rt-pcr with the amplified dna binding assay and usage of an internal standard mrna allows sensitive and accurate quantitation of gpiba mrna in human platelets. since upa and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (vsmc), which is part of the pathogenesis of atherosclerosis. we are currently assessing the role of spatial expression of upa and thrombin receptor (tr) on cells with human carotid artery plaques (n=10). we have used a double immunolabeling approach, combining anti-upa and anfi-tr antibodies. to identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-sma) for smooth muscle cells, ulex europaeus agglutinin i (uea i) for endothelial cells, inflammation cell cocktail (cd68+cd45) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (pcna) to stain proliferating cells. in the carotid atherosclerotic plaques, upa immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (fcrr). it was present in distinct patterns: cytoplasmic staining. tr staining was distributed similar to upa staining. with double staining combining anti upa antibodies with anti-tr antibodies, cellular co-localisation of both upa and tr was demonstrated. these cells were identified as smooth muscle cells by -sma. inflammatory cells were mainly localized within the fcrr, they only stained for upa. in conclusion: our data demonstrates that upa and tr are coexpressed in vsmcs in human carotid artery atherosclerotic plaque tissue. we therefore conclude, that the mitogenic activity of upa is associated with the thrombin signalling pathway. in the proficiency test of the ,,deutsche gesellschaft flir klinische chemie" (dgkc) 1/95, 5 lyophilised plasma samples (immuno ag) were sent to participants: a normal plasma and 4 plasmas from persons under oral anticoagulation (oac-plasmas. inr 1.9 to 3.7). the participants (n=552) returned the pt times obtained and in most cases (n=355) also the isi value for the thromboplastin used (isi of pack insert). the inr was calculated using the pt of normal plasma and the isi of pack insert (method i). two additional methods for inr calculation were compared with method i. according to the concept of calibrated plasmas (houbouyan et al., t993), a calibration curve was constructed using the normal plasma and the 30ac-plasmas. the inr calculated using the pt •fn•rma¿ plasma and the laboratory-specific isi value given as 1/slope of the calibration curve (method ii) or was read off directly (method hi). for inr values, calculated by the 3 methods from the participants data (n=355), outlier elimination (2sd, iterative) was performed. the inr mean values for all 3 calculation models remain in a narrow range. using calibrated plasmas (method 1i and m), less outlier were eliminated and cv's obtained were smaller than using the conventional procedure ( i ). obviously, the inr inherited problems, such as accurate isi value, pt value of normal plasma and instrument/laboratory influences on isi, can be reduced using calibrated oac-plasmas. practical approach and educational considera-tions of home prothrombin time estimation a. bernardo, a. bernardo, c. halhuber herz-kreislauf-klinik, bad berleburg, germany specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. the training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. the education program is divided into a theoretical and a practical part. the theory part has group sessions of twenty patients of a time. the practical course is reduced to a maximum of five patients. the sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. on average eight hours of theoretical education and two hours of practical training are sufficient. the contents of the theoretical lessons are: • need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. an alternative is a full-day intensive course which can be held during the weekend. our recently reported (1) observation that oral anticoagulant treatment causes an increase of heparin cofactor ii (hc ii) activity in plasma is now confirmed by a more extensive study. in 43 thrombophilic patients who were on vitamin k antagonist therapy (marcumar r) we found a median hc ii level of 142 % as compared to 119 % for 72 thrombophilic patients without any therapy (p < 0.002" ) and 104 % for 59 healthy controls (p < 0.001" ). moreover we observed that the increase of hc ii level was significantly correlated with increasing inr-values (r = 0.63, p < 0.001). follow-up observations on some patients showed, however, clear differences in the levels of hc ii activity after onset of vitamin k antagonist therapy. thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders"). in conclusion, the determination of hc ii activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (inr > 3.5). after intracoronary stent implantation an aggressive oral anticoagulation (oac) therapy is mandatory. to find out whether coagulation activation occurs after coronary stent implantation during high dose oac therapy markers of plasmatic coagulation and d-dimer were measured. patients 5 male patients (average age 57 years) were examined. blood samples were taken before and right after stent implantation and during the following week. patients got 30 mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (asa) were given. methods ptz, aptt, tz, protein c, tat-complexes, fi+2 and d-dimer were measured. results d-dimer levels increased steadily between day 0 and day 7. tatcomplexes showed a slight increase from day 0 (2.4 bg/i) to day 3 (15.3 ~tg/i). on day 7 tat levels were down again (2.0 p,g/l). fl+2 (day 0:1.0 ng/ml) also showed a slight increase on day 3 (1.3 ng/ml). protein c decreased steadily from day 0 (108%) to day 7 (15%). conclusion during the initial phase of oac therapy a coagulation activation is reported but no significant elevation of tat or fl+2 was found. this result shows that additional heparin and asa therapy was sufficient to avoid systemic coagulation activation. the increase of d-direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. three methods for the determination of prothrombin time from capillary blood in patients under oral anticoagulation have been investigated. two methods were run on coaguchek® monitors (boehringer mannheim) from capillary whole blood. after fingerpuncture the first drop of blood was applied to the well of a coaguchek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. the third method was hepato quick (boehringer mannheim) which was determined out of citrated capillary blood from an earlappuncture. 66 specimen of patients under oral anticoagulation were investigated. the method comparisons between each of the coaguchek® methods and the laboratory method show good results and the correlation between the coaguchek® methods is excellent. mean differences to the lab methods are -0.1 inr in both cases. no mean deviation was detectable between the coaguchek® methods. scattering of coaguchek® versus hepato quick was +/-0.6 inr in the range 1 to 4 inr except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. introduction: haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. recently, we described protein z deficiency in patients with bleeding complications of otherwise unknown origin. thus, we were prompted to measure protein z in patients with coumarin skin necrosis. patients: 4 patients (i man, 4 women; age: 35±10 years) suffering from haemorrhagic coumarin skin necrosis were examined. all patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. method: protein z antigen test, diagnostika stago, france. results: 4 out of the 5 patients examined had diminished protein z levels ( 700, 820, 1080, 1700 ug/l) in comparison to normals (2900 ug/l). in one of our patients, protein z was normal (3020 ug/l). conclusion: low protein z levels are additional risk factors for haemorrhagic coumarin skin necrosis. oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. since oral anticoagulants interfere with the function of vitamin k, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin k. we report about a 59-year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding 21 years. atrial fibrillation has been first diagnosed 18 years ago. latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. in 1993 the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. the resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. since vitamin k free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. as a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. therefore, the decission 1:o discontinuate heparin therapy and initiate oral anticoagulation has been made. because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. over a 4 weeks lasting induction phase inr values were controlled daily. a dosage regime starting with '10 mg warfarin at the day of vitamin application (day 1) followed by 3.75 mg on day 2 and 1.25 mg on days 3, 5, and 6, respectively, was found to be optimal to maintain inr values within the target range (inr: 2.0-3.0). in order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. during an observation period of 6 months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. these data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin k plasma levels. portable monitors for home monitoring of inr are well established for adults on oral anticoagulants. patient's compliance is improved as well as long term outcome. experience concerning accuracy of the procedure in children is limited. 32 inr determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of 4 months. the coaguchek® monitor from boehringer mannheim was used. choosing an arbitrary range of agreement of ,qnr 0.5 for both determinations, 81% of the measurements were within the defined range. 5/6 outliers were due to low inr resulting from difficulties in capillary blood sampling. the degree of agreement increased when the procedure was performed at least once a week. in conclusion: inr determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. a dose adjustment should be done only on the base of inr determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. a stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. if prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. for this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. for the first time a clinical trial was started in 1986 and continues until today at the cardiac rehabilitation center bad berleburg, germany with patients mainly after heart valve replacement. the patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. within six years 600 patients were trained: 216 patients could be followed up with regard to their selfdetermined prothrombin times. the results were within the therapeutic range in 83.1% of the measurements (n=14.812) taken by the patients themselves. on average, the patients who determine their prothrombin time themselves did so at a weekly interval. neither major bleeding nor thromboembolic complications could be observed in the 205 patient-years of home prothrombin estimation. it is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. circulating plasma thrombomodulin (tm) is a novel endothelial cell marker, which may reflect endothelial injury. tm acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein c/s pathway. tm therefore belongs to the anticoagulant defence system against thrombosis. increased tm levels have been described in various diseases such as ards, thromboemboembolic diseases, ttp, diabetes, le and cml reflecting alterations of the vascular system at the endothelial level. to find out to what extent cardiac catheterisation imtates vascular endothelium, tm concentrations (stago, asnieres, france: x 10 3 iu/ml) were investigated prospectively in 58 infants and children (three days -16 years). blood samples were drawn before the intervention, immediately at the end and 24 h later, snap frozen (-70 °c) and investigated serially in dublicate six weeks -3 months later. the results (median and range values) are shown in the enhanced tm concentrations immedately after the operative intervention, followed by normalisation within 24 h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. recently in an al=wl" based method dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein c (apc) in thrombophilic adult patients. apcr is in the majority of cases associated with the arg 506 gin point mutation in the factor v gene. concerning the special properties of the neonatal hemostatic system (low vitamin k dependent coagulation factors, physiological prolongation of the pt and aptf) we adjusted this ap'it based method (chromogenix, m~,lndal, sweden) to neonatal requirements: apcr was measured in 120 healthy infants according to dahlb~ck. the results were expressed as apc-ratios: clotting time obtained in a 1:1, 1:5 and 1:11 dilution with factor v deficient plasma (instrumentation laboratory munich. germany) using the apc/caci2solution divided by clotting time obtained with cac12 in the same i:1, 1:5 and 1:11 dilution. in addition, plasma of 24 neonates with septicaemia were investigated and data of 18 infants aged birth -three months with arg 506 gin +/-were shown. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mnl i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. results are shown in the 1.6(1.4-1.95) neonates and infants were considered to be apcr when the aptt ratio was < or = 2. concerning the special properties of the neonatal hemostatic system, our data show concordance with the pcr method in neonates and infants only, when the aptt based method was performed in the i: 11 plasma dilution. case report: we report on an 8-year old boy with severe hemophilia b and frequent screaming at night. eeg showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. complex partial seizures were diagnosed and therapy with carbamazepine was initiated. as no improvement was seen nmr was performed. this revealed lesions within the right frontal cortex. higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. the patient is now treated with carbamazepine and valproate. he still suffers from one short seizure per day. because of his seizures we started prophylactic replacement therapy with 600 i.e. factor ix twice per week. discussion: in 1992 wilson et al. first detected brain abnormalities in 25 of 124 children and adolescents with hemophilia a or b who were negative for immunodeficieney virus (1). the most common findings (14/25 patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on t2weighed images. similar lesions have been reported in children with sickle cell cerebral infarction (2) . only three of these 14 patients had seizures, all of those having a documented history of intracranial hemorrhage. our patient has similar lesions as those described by wilson et al. but no history of intracranial hemorrhage is documented. even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. conclusions: 1. in patients with hemophilia and seizures nmr might be useful as a high sensitive method for the detection of gray and white matter changes. 2. further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. disseminated intravascular coagulation (dic) is a rare, but foudroyant disease occuring in gram-negative sepsis like meningococcal septicemia. despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about 10 % ), rising to 40 % in patients presenting with severe shock and consecutive dic. as the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with dic. the parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. monitoring the course of hemostaseologicai findings in 26 pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: a prolongation of partial thromboplastin time to an average of 69. 1 sec (range 22 -150 sec, norreal 30 -45 sec), a decrease of prothrombin time to 45.7 % (range 13 -71 %, normal 70 -100 %) and of antithrombin iii to an average level of 16. 8 u/ml (normal 20 -29 u/ml ) was found 1 to 4 (-6) hours after admission. the consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( at iii and heparin in stage of pre-dic ; at iii and fresh frozen plasma in case of advanced dic ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). an early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a waterhouse-friderichsen-syndrome. this study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in 60 children (neonate -16 years) during cardiac catheterisation with low dose flush heparin (10 iu/ml saline). aptt (instrumentation laboratory: see), anti xa activity (xa; chromogenix: iu/ml), prothrombin fragment ft.2 (f1.2; behring werkc marburg: nmol/l) and d -dimer formation (d-d; bnhring werke/vhrburg: ug/l) were investigated before (t1), at the end (t2) and 24 h after cardiac catheterisation (t3). in addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein c (apcr), protein c, protein s and antithrombin were investigated. during catheterisation median (range) hepadn was administered in a total dose of 60 (17-206) iu/kg bw. in addition infants < 6 months of age (arterial catheterisatiun only) or patients with known thrombophilia received 300 -400 iu/kg hepafin for fmther 24 hours. the results (median and range) are shown in the ft.2 was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values 24 h later. in contrast no cfinically relevant fibrinolytic activation was seen: d -dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels 24 h later. three children showed resitance to apc. tn one child stroke occurred before. not knowing the result of apcr in the remaining two patients only one neonate received further prophylactic heparin. the third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ in addition, no protein c, protein s or antithrombin deficiencies were found. although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. if fnrther prophylactic hepariuisation in children with a~r, protein c, protein s or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study. a.sandvoss, w.eberl, m.b0rchert introduction: capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below 1.500 g. septicemia, asphyxia and immaturity seem to be most important risk factors. to determine the influence of c 1-esterase inhibitor (cilna) in preventing contact phase and complement activation we investigated c11na concentrations in normal and symptomatic preterm infants. methods: activity of cilna were measured by chromogenic substrate method (behringwerke), cilna concentration with radial immunodiffusion (behringwerke,germany). results: cllna-activity in asymptomatic preterm infants (n= 14) was 65+/-15% of normal at birth. healthy newborns showed activities of 80+/-20%. cilna reached normal adult values 2-4 days after birth. preterm infants with respiratory distress syndrome(n = 14) showed lower activity on day 2-5, patients with additional septicemia (n=15) had decreasing c1 ina-activities in the first three days of life. individual course of cllna-activity and thrombocyte count correlated in the group with irds with and without septicemia. in children with capillary leakage onset of diuresis went parallel with raising cllna-activity. markers of contact phase (f xlla) and complement activation (c 5al were investigated in single cases and evidence for involvement of both systems was found. conclusion: contact activation and complement system play an important role in capillary leakage in preterm infants. cilna regulates both systems. activity of cilna correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. antiphospholipid antibodies (apa) interfere with hemostasis probably by inhibition of protein c or prothrombinase complex. thereby, apa might lead to thrombosis or increased bleeding. however, incidence and clinical importance of apa has not yet been investigated in children. therefore, we assayed plasma samples of 220 children, aged 0,1 to 19 years (mean 7 years) by elisa detecting igg-and lgm-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. in patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. according to their diagnosis children were devided into 5 groups: i. autoimmune diseases, ii. infections, iii. metabolic diseases, iv. other diseases, v. healthy children. results: apa were found in 69/220 patients. in the respective groups we demonstrated apa in the following proportions: 1. lgg-isotype: activitiy of c1 esterase inhibitor (c11na) is reduced in preterm infants especially if birth weigth is below 1.500 g and respiratory distress syndrome and/or septicemia is present. capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial c1 esterase inhibitor preparation (berinertr,behringwerke), case reports are given. all patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jefficiacy of substitution therapy is dose related, supranormal iactivities of cilna are necessary, reflecting raised consumption of inhibitor in ongoing disease. clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated. or arterial thromboembolic event in children e. lenz, c. heller, w. schr6ter*, w. kreuz johann w. goethe-universit~itskinderklinlk, frankfurt a. main, germany * georg-augast-universit/itskinderidinik, g/3ttingen, germany venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these patients. after the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. anticoagulation after venous thrombosis is generauy recommended for 6 months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. when evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. the hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children. only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. we want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (lmwh) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. from the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients. in this context we would like to present our data on 8 patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. because prophylaxis for re-thrombosis was mandatory in these patients, lmwh was given for long-term anticoagulation in a dally subcutaneous dosage of 100-150 anti-xa u/kgbw. monitoring was done by anti-xa-test (0,4-0,8 anti-xa u/ml). under this regimen none of the patients developed re-thrombosis or bleeding complications. alopecia was seen as a side-effect. this study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin (2x17000 u/kg bw) in 42 infants and children aged 0.1 -15 years, and to correlate these findings to the clinical outcome. prothrombin fragment f 1.2 (f1.2; behring werke marburg: nmol/l), antithrombin-serinesterase -complex (atm; stago: ng/ml), d -dimer formation (d-d; behring werke marburg: ug/l), tissue-type-plasminogen activator ag (t-pa; chromogenix: ng/ml), plasminogen activator inhibitor 1 antigen (pai; chromogenix: ng/ml) and cl-inhibitor (c1; behring werke marburg: x 10-3 g/l) were investigated before the operation (t1), at the end of the operation (t2), and on postoperative days 1 (t3), 4-6 (t4) and 7-9 (t5), respectively. the results are shown in the table (median and median absolut deviation): t1 t2 t3 t4 "1"5 nv fi.2 0.9 +/-0.5 1.7 +/-0.9 1.4+/-1 1.8+/-0.8 1.6+/-0. the platelet (pl) function defect induced by thrombolytic agents has been attributed either to the degradation of pl surface receptors or to the anti-aggregatory effect of fgdps. in contrast to other plasminogen activators scu-pa is intimately inked with pl: they can rapidly incorporate exogenous seu-pa, release it upon stimulation and bind the proenzyme. recently we have reported that exposure of prp to recombinant scu-pa (2.5-t00 um) in timed interval 1-30 min resulted in dose-dependent inhibition of pl aggregation. timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first 3-5 rain with the moderate suppression of pl aggregation in the 30 min period. when tcu-pa (25-100 nm) was exposed to prp in the same conditions dose-and time-dependent inhibition of pl aggregation was also observed. since the effect was obtained no earlier than t0 min after exposure of tcu-pa to prp, and the threshold dose was higher. comparable inhibition of pl aggregation was obtained with 25nm of scu-pa versus 100nm of tcu-pa and the llbrinogen depletion by the end of the 30 min period was 2% and 30% respectively. it's likely that tcu-pa and its precursor have different mechanisms of action on the pl aggregatory function. in a recent study we have shown that recombinant rscu-pa inhibits platelet (pl) aggregation in prp. to exclude the possible influence of rscu-pa/plasma interfere on this process the aggregation of washed pls was under the investigation. pls were washed according to modified mustard's method, suspended in buffer and adjusted to 250,109/1. the resuspended pls were exposed to 5-100 nm of rscu-pa for 30 min at 370(;. at time points 3, 5, 15 and 30 min the aggregation with 0.6 iu/ml of thrombin was measured. it was found that the exposure of pls to rscu-pa (20-100 nm) for 3 man resulted in marked inhibition of their aggregation. since after 15-30 man of incubation with 20-50 nm of rscu-pa the inhibitory effect on pl aggregation became less pronounce or even disappeared. when 5 nm of rseu-pa was used the inhibition of pl aggregation became significant only by 15 rain of exposure period and didn't change for 30 man of investigation. the observed results may be cormeeted with uptake of rscu-pa by pls from surrounding buffer as well as with individual variations of pl response to the same concentration of rscu-pa. loss of glycosylation may result in a reduced platelet (p) survival and perhaps altered function. we analyzed the structural and functional effect of specific deglycosylation (combinations of n/o-glycosidase and neuraminidase treatment) of p and isolated p gpib. washed and formaldehyde-fixed p were digested as follows: 1) with neuraminidase (0.125u/ml) + o-glycosidase (3.1mu/ml) + n-glycosidase (1.25u/ml), 2) with neuraminidase alone (0.2u/ml), 3) with n-glycosidase (2u/rnl) and 4) with neuraminldase (0.2u/ml) + o-glycosidase (5mu/ml). all reactions were performed in the presence of protease inhibitors (pmsf, leupeptin, sbti), after washing x2 the p and identically treated controls were analyzed by flowcytometry with the antibodies 6di (mab: a-gpib), 7i-l2 0vlab: a-gpiiia), and the lectins wheat germ agglutinatinln (wga, for neunac) and peanut agglutinin (pna, for [3dgal(1-3)-galnac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with 6di and 7h2). the botrocetin (13) and ristocetin (r)induced agglutinations showed arer treatment 1) (all enzymes) a full inhibition of r-induced agglutination but only a mildly reduced b-induced agglutination (70% of normal). treatment 2 and 3 (neuraminidase alone, and n-glycosidase alone) affected both agglutinations only mildly (70-80% of normal).treatrnent 4) (o-deglycosylation) however showed a major inhibition of r-agglutination down to 30%, while b-agglutination interestingly was almost fully retained. the results of the rotary shadowing electron microscopy of purified gpib suggested a collapse of the normally stretched, glycosylated, gplb, not only after the treatment with all three glycosidases, but also .after o-deglycosylation alone. we conclude that oglycosylation is most important for ristocetin-induced platelet-von willebrand factor-interaction and responsible for the typical stretched shape. the phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (ptcp) in the presence of calciumchelatization by na-edta and sodium-citrate was studied in blood samples of a patient. initial platelet counts electronically measured were 20000/ul blood anticoagulated with na-edta and sodium-citrate. normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. immunoglobulines of the igg and igm subclass were identified in the patients plasma. by incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of na-edta and sodium-citrate but not in the presence of heparin. the platelet membrane glycoproteins (gp) hb/llia, ix and iiia/vnr g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. on platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. calcium depletion induced by calcium chelators as na-edta and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. the decrease of gp llb/illa platelet surface antigen to 10% (normal >75%) indicated the important role of the gp iib/iiia receptor at ptcp. the saliva of tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. to investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type i in different concentrations was measured by flow cytometry. the same concentrations of pallidipin that inhibited the couagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the prp from the same donor measured contemporaryly. collagen type i-induced platelet aggregation of cd36-deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. there was no inhibition of collagen-induced fibdnogen-binding in the cd36-deficient platelets as well. pallidipin did not cause inhibition of collagen-induced membrane expression of cd62 and cd63 of control and cd36-deflcient platetets as measured by flow cytometry. however eadier studies had shown an inhibition of collagen-induced atp and {3tg secretion by pallidipin. therefore we compared the effect of pallidipin in unstirred and stirred prp samples. while pallidipin had no effect in unstirred samples it showed strong inhibition of ptg secretion in stirred samples. we therefore conclude that pallidipin does not act on collagen-induced aggregation through cd36 and that the inhibition is a post fibdnogenbinding event. pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps. 17-hydroxy-wortmannin does not inhibit the transport of 1nm-gold labelled fibrinogen in resting platelets. e. morgenstem, b. kehrel and k.j. clemetson medical biology, saarland univ., homburg, germany, haemostasis research, univ. muenster, germany and theedor-kocher-lnstitut, univ. bern, switzerland. wortmannin, an inhibitor of phosphoinositide 3-kinase and of myosin light chain kinase blocks reactions of the activated platelet. to obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of 17-hydroxy-wodmannin (hw) on the endocytosis of fibrinogen from the surface of resting platelets was studied. gel filtered platelets (gfp) were incubated for 10 min at 37°c with hw (3x10-6m) or with iloprost. controls and gfp preincubated with hw or ilopmst were incubated with 1.4nm-gold labelled fibrinogen molecules (fg-au; final concentration 40p.g/ml) at 37°c. the experiments were stopped after 5 or 30 min by rapid freezing. after freeze substitution in acetone with 4% osmiumtetroxide, sedal sections were prepared. the sections were examined after incubation with ascorbic acid (5% in h20) for 30 rain at 20°c (to reduce metallic osmium) and silver-enhancement using danscher's (1981) method (to visualize the fg-au). examination of adp stimulated platelets in the presence of 40fg/ml fg-au shows that the ligand is able to mediate aggregation. the examination reveals, that fg-au was present in a low density on the platelet surface, in higher density in the surface connected system (scs), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. after 30rain, the number of labeled granules was increasing. labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. hw or iloprost did not alter the resting gfp and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. we conclude from the results with hwthat the regular contractile function of the cytoskeleton is not necessary to transport the fg-au in resting platelets. methods: edta anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). platelets were also incubated with fluorescent antibodies to gpib, gp lib/ilia and gmp-140 (two colour method). results: surface expression of gpib was the same in young and older platelets. results for gp lib/ilia and gmp-140 (in resting and activated platelets) will be presented. conclusion: young platelets can easily be detected using thiazole orange and flow cytometry. there is no differential expression for gpib. further results will be presented. the influence of erythrocyte and thrombocyte content on the release of atp by different agents in whole blood specimens was tested. the measurement had been performed in the lumi-aggregometer using the principle of the luciferin-luciferase reaction. altogether 39 blood samples were diluted gradually before induction of the release reaction by arachidonic acid (1,25 mmol/i final concentration), adp (30 ijmol/i) and collagen (1,0 and 5,0 tjg/ml). the peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into atp concentrations (= absolute peak) after testing the atp standard in parallel for each dilution step separately. the peak in relation increases by increasing dilution with all inducers. it was identic with the atp standard and with collagen, somewhat lower with arachidonic acid and much higher by adp. a luminescence-optical effect may influence all these results. the absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. under induction by adp no decrease of the absolute peaks by increasing dilution of the samples was abserved. this can be explained only by liberation of atp from the erythrocytes. the atp standard is essential for the quantification of the release reaction. adp doesn't suit for it. collagen with a final concentration of 1 pg/ml was proven as the best inducer. platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. in addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. smooth muscle cells showed the same effect but very less marked. in presence of extracellnlar matrix spontaneous aggregation occured. endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. incubation of endothelium with several cytokines increased its anti-thombotic properties. for example, at a platelet count of 3x105/id in the prp, 10 -6 m adp led to maximal aggregation in uncoated cuvettes, in presence of 5,5x106 endothelial cells aggregation was completely abolished, in presence of 2,75x10 "6 cells aggregation was decreased to 40%. smooth muscle cells diminished the aggregation effect of 0,1 nih thrombin to 67% when only one side of the cuvette was coated and to 63% when both sides were coated. endothelium could not inhibit aggregation induced by 2,5 x 10 -6 m adp but endothelium incubated with 500 u/ml tnf-a or 30 u/ml intedeukin-lfl or lmm l-nitro-arginin for 24 h did completely inhibit aggregation. platelets become sticky and adhere to surfaces or to another without contracting and secreting. during maturation of megakaryocytes finally platelets lost their genomic nuclear message. only mitochondrial dna of platelets can be identified. we focused our attention on the impact of mitochondrial dna and the mitochondrial transscriptive mechausisms during platelet activation in normals. materials and methods: leucocyte free (nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by pall 100 leucocyte filters. the influence of different anticoagulants (commercially available sarstedt tubes containing citrate, heparim edta and 500 atu/ml hirudin wacker) was examined. activation was due to a 60 nun. hemapheresis procedure ( 3-5fold increase of cd 62, cd 63) and ex rive stmaulation due to 4 niy u/ml thrombin, 0.025 m cac12 or combmatious. the guanidiurn method for total rna preparation were used according to t. brown: current protocols in molecular biology 4.21-4.9.14,1991. different primers of mitochondrial genome (e.g. cytochrome b and atpase) were prepared using pcr and mitochondrial transscription was examined using northern-blot-technique. results: 1., there is less activation of mitochondrias using hirudin anticoagnlation, but a 2fold increase of mitochoindrial rna content in heparinized samples. 2., stimulation with thrombin leas to an increase to 5.5 e-l0 rna btg/platelet, compared to 4.7 -4.8 e-10 rna ~tg/platelet under unstimulated conditions.. conclusion: there is evidence for the importance of platelets mitochondrial dna and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. thrombospondin-1 (tsp-1) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. the investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. monoclonal antibodies are one of the most powerful of these reagents. therefore, we purified human tsp-1 from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. a subclass igg 1 monoclonal antibody designated 48.42 was purified from ascitic fluid and further characterised. western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the tsp-1 subunit chain was not recognised. no cross-reactivities with human fibrinogen, fibronectin, vitronectin and von willebrand factor were found. preliminary results indicate that the monoclonal antibody 48.42 can be used to investigate tsp-1 function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for hl-60 cells. in addition, a sandwich enzyme immunoassay was developed using goat-antihuman tsp-1 igg and derivatised monoclonal antibody 48.42 (peroxidase, biotin) as a sensitive method for detection of tsp-1 in human body fluids. in the following study the expression of the platelet antigen (cd62p) and the leukocyte antigen (cdllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (cd45, cd14, cd42a). the measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions (37°c, 1000 rpm, different stirring times). the results are presented as the percent positive events in each gate (platelets, leukocytes -pmnl, monocytes, lymphocytes and rosettes -plateletpositive events in the pmnl, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. stirring (max 15 rain) induced an increase of cd62p on the platelet surface of ca. 10%, without any change in the mean fluorescence. under these conditions increased cdllb on pmnl and monoeytes could be detected. an increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas pmnl showed an increased mean fluorescence, but not an increased number, of platelet-positive events. the time-dependent changes in rosette formation on stirring could be further increased by addition of adp. these results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. 17 itp patients undergoing splenectomy were observed after 1-30 years following operation and divided into 2 groups. first group consisted of 8 patients with normal platelets count and absence of haemorrhagic syndrome. second group was formed of 9 itp-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. in the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. the increase of b-cells, expressing cd22, cd37, hla-dr-antigen has been revealed in the 2nd group. quantity of srfc, cd3 +, cd5 + cells in the blood of recovered patients was lower than in patients of the first group. this group was also characterized by statistically significantly increased level of cd4 + cells while the cd4/cd8 ratio was equal to 1.0 :i: 0.3 % (0,5 + 0,1% in patients of the second group, respectively, p>o,05}. also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. we have observed the opsi-syndrome in 2 patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. extracellular matrix (ecm) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. thus it contains collagens, fibroneetin, von witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (gp) iibiiia, gp ib/ix and gpiiib. incubation of platelets with ecm results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. we studied patients whose platelets showed either a receptor defect in gpiibiiia or gpiiib or a storage pool disease. adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. platelet adhesion of patients with thrombasthenia glanzmann (receptor defect of gpiibiiia) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. the adhesion to ecm was diminished and also no aggregates were detected. patients with a receptor defect in gpiiib showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ecm was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ecm. by centrifugation of citrate blood (250 x g, 10 min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. so the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. if the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. the quotient between thrombocyte counts in prp and whole blood is a measure for thrombocyte activation. we chequed the value of this screening in different groups of patients with arterial occlusions disease (aod), chronical venous disease (cvd), diabetes mellitus (dm] and in healthy control persons (control). variation coefficient of the method is 3.7 (prp) and 4.4 (tc) respectively (coulter counter). differences to the control group are significant. changes in the patient groups in dispensaires follow up 5 years are also significant. nicardipin -induced immunthrombocytopenia p. eichler 1, c. hinrichs 2 , g greinacher l i.institut fur immunologic und transfusionsmedizin, ernst-moritz-arndt-universitat greifswald, 2. deister-s0ntel-klinik, bad m0nder drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. patients are at risk to suffer from severe bleeding complications. especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. we report the case of a 71 year old male patient who received allopurinol, captopril, digitoxin, furosemid, and nieardipin. the patient presented with hematomas (pit. count < 10 g/l) and later developed bone marrow dysplasia. in an elisa using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (antagonil, ciba-geigy) could be demonstrated. the reaction pattern is given in the the enzyme-immunological determination of soluble fibrin (sf) proved to be highly sensitive and specific. this sf-elisa detected fibrin hacking fibrinopeptide a (fpa) via the monoclonal antibody 2t35 specific for the neoepitope generated on the aa-chain after the split of fpa. lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with kscn. this strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. it was the aim of this study, therefore, to compare the two sf-elisa modifications (with and without kscn-pretreatment of specimens) . in order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= 160 samples from 10 courses). the former sf-elisa (no kscn) correlated well with prothrombin fragments, thrombin-antithrombin !11 -complexes and with the release of fibrinopeptide a ( r > 0.96, n= 151). results of the new sf-elisa with kscn pretreatment of patients' plasma, however, correlated conspiciously well with d-dimer levels (r > 0.94) but distinctly less with the markers of thrombin generation (-0.12 < r < 0.29). this good correlation with d-dimer levels was unaccountable since the d-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < 0.05). therefore, kscnpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. nevertheless, the results of the former assay without kscn-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. consequently, further examinations are necessary to specify the effect of kscn on soluble fibrin complexes and the resulting assay specificity. a rapid assay for the determination of the primary hemostasis potential (php) of whole blood has been developed (kundu et al, 1995) from the original method of kratzer and born. the new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. the test procedure is very simple. the cartridge is loaded with -500 p.l citrated whole blood and is inserted into the platelet function analyzer (pfa 100aaw). the test is started automatically after a preincubation phase of 2.5 rain. the reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. under constant negative pressure the sample is aspirated and through the contact ofplatelets and vwf with collagen adherence and aggregation begins. the adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. the result of the php is reported as closure time (ct). additional parameters such as bleeding volumes are possible as well. first results show good reproducibility, normal values in the range of up to 150 sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. the system detects aspirin induced thrombocyte function defects and von willebrand disease. in ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdadp coating can be employed. in the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. the proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in 20 young healthy male volunteers aged 20 to 40 ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of 1000 meters. the purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. we were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. venous blood samples were obtained directly before and directly after the jump. flight time from the departure of the airplane to the landing of the parachutists was approximately 20 minutes. the maximum time that elapsed between the two blood withdrawals were 45 minutes. in a preliminary study with different voinnteem, certain fluid imbalances had been observed. absolute numbers of leukoeytes (6.9 vs. 9. l/n0, erythrocytes (4.6 vs. 5.1/pl), and platelets (246 vs.276/nl) significantly increased (p < .001), as well as the hemoglobin concentration from 145 to 156 g/l (p < .018). even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (.41 vs..42), we noticed a marked drop in aptr (27 vs. 23 sec) and a significant increase in factor viii ~tivity. as a direct stress response, we found a rise in fibrinogen concentration (2.4 vs. 2.8 g/l) which is one of the shortest acting acute phase proteins. concerning reactive fibrinolysis, d-dimers showed an increase in concentration from 115 lag/l to still normal values of 192 lag/l, which was not significant due to low numbers of values (p = .086). we observed similar changes in fibrin monomers and prothrombin fragments fl+2. from other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil7 is not reached until 24 hours after initiation of activation.these investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. to better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. triodena (t) 30/40/30 ug ee, 50/70/100 ug gestodene) were tested for their effect on hemostatic parameters. three groups (n=20) of healthy female volunteers were treated for 6 months with one of these oc. blood was taken before treatment (day 24-28 of pretreatment cycle, 0) and on days 18-22 of the 3 ~ (i) and 62 (ii) treatment cycle. indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment f i+2 and of fibrin split product d-dimer and plasmin antiplasmin complexes were found elevated during treatment. the following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor vii antigen fviiag, fvii clotting activity fviie, circulating activated factor vii cfviia and antithrombin 3 at3 activity, total protein s antigen tps-ag, free protein s antigen fps-ag, protein s activity psact, circulating thrombomodulin etm fviiag, fviie and cfviia significantly increased during treatment; cfviia: 0: c 32.4 mu/ml a prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. the present study includes 161 patients with clinical suspicion of myocardial infarction. blood samples were drawn by the primary care physician, upon arrival in the hospital, and after 2, 6, 12, and 24 hours of hospital stay. patients with myocardial infarction were identified by typical course in 12 lead ecg, and upon sequential determination of troponine t, myoglobin, ck, and ck-mb. patients with primary cpr were excluded from evaluation. soluble fibrin was measured by enzymun®-test fm (boehringer mannheim). patients with acute myocardial infarction display soluble fibrin levels within the normal range (< 5 ~tg/ml) during the initial two hours after onset of symptoms. there was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. in patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately 8 hours is observed. most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. four patients with pulmonary embolism showed soluble fibrin levels in the range of 40-300 [.tg/ml, which remained in the same range during the entire observation period. in conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. high levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. the possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. we investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. in all patients factor viia, xii, xiia and also the fibrinolytic parameters t-pa, pai-1, pap, plasminogen and a2-ap were determined. in addition, f1+2 and apc-resistance with heterocygote factor v-leiden-mutation and confirmed thrombosis. we found increased factor viia which showed partly also an increased fl+2. patients with other pathological results such as a reduced t-pa and/or increased pai-1 showed a low incidence of elevations in factor vii or f1+2. the activation of factor xii seems to be of minor importance in patients with thrombosis. a different picture is found in septic and transplanted patients. obviously factor xii-activation is of major importance in this group. a deterioration of the clinical symptoms is correlated with an increased factor xiia which is paralleled by a decrease of factor xiiactivity. the investigation of fibrinolysis parameters such as pai-1 and pap demonstrate a fibrinolytic disturbance of the balance. statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. in patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. the possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. however, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. molecular markers of blood coagulation activation might add valuable information in individual cases. we investigated 48 patients with thromboembolic manifestations (deep vein thrombosis n=22, pulmonary embolism n= 13, myocardial infarction n= 13) for one year beginning with admission to the hospital. tat, prothrombin fragments f 1 +2, d-dirner and fibrin monomer concentrations were analysed. all markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. patients suffering from venous thromboembolism demonstrated higher concentrations of tat and f 1 +2 in comparison to myocardial infarction (34.6 vs 12.3 pg/1, p=o.009; 2.8 vs 1.3 nmol/i, p=0.0025). f 1 +2, tat and d-dimer concentrations decreased gradually over the first 14 days of anticoagulant therapy reaching values within the established normal ranges in all cases. f 1 +2 and tat concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. we conclude that f 1 + 2 and tat appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. the influence of thyroid failure on haemostasis is controversial. mainly hypoceagulable states have been described in clinically overt hypothyroidism. since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (b, n=42, age 59+13) or overt (c, n=8, age 55-zcj) hypothyroidism, as well as in hypothyroid women under 1"4 treatment (d, n=8, age 57+9) and euthyroid controls (a, n=80, age 50+14). simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (fvii, fviii, von willebrand factor), coagulation inhibitors (antithrombin ill, hepadn cofactor ii, protein c, protein s) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. results factor vii activity (vii:c), factor vii antigen (vii:ag) and their ratio were found increased in hypothyroid patients. factor viii activity showed the same tendency, whereas von willebrand factor ramained unchanged, as did all other parameters with exception of free protein s, which declined in overt hypothyroidism and in t4 treated subjects. these differences tended to diminish after exclusion of 26 women with estrogen replacement therapy for menopause, but the ratio vii:cnii:ag, as well as fvii:c still remained significantly higher in hypothyroid patients. conclusions: subclinical and overt hypothyroidism are associated with significantly higher levels of factor vii:c and vii:ag. the disproportionate increase in vii:c compared to vll:ag, as shown by their ratio, might reflect the presence of activated factor vii (vila), which in turn indicates a hypercoagulable state. this pattern becomes more pronounced with the concomitant estrogen replacement after menopause. exocytosis following platelet activation leads to translocation of cd62p (p-selectin), cd63, and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. we here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. two different methods are compared, i. e. using either methanol or the fix&perm kit (an der grub) for cell membrane permeabilization. in addition, interleukin(il)-ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using fix&perm. whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. this technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. the positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. the influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n=33; median ag~ 61 years, achieved heart rate: 68/min). indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 among the coronary patients (tat from 2,1 to 3,4 pg/1; fi+2 from 0,92 to 1,1 nmol/1). the degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. this must be explained by existence of the coronary heart disease or by the higher age in the patient group. indicating an activation of fibrinolysis t-pa activity increased significantly in coronary patients (from 0,14 to 0,5 iu/ml) resulting in an unchanged balance between coagulation and fibrinolysis. from this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. a prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. the principle of the fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. there are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. we investigated the coagulation system in 20 children and young adults aged 4 to 21 years in a general examination 4 to 61 months after fontan procedure. besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-iii-complex (tat) in 12 patients (60%). as a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha2-antiplasmin-(pap-) levels in 14 patients (70%). less frequently, the concentrations for the prothrombin-fragments 1 and 2 (f1 and 2) (7 patients, 35%) or the d-dimer (2 patients, 10%) were increased. we didn't find significant differences in a clot-lysis-assay between fontanoperated patients and an age-matched control group. there was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. whether the present data can help to estimate the risk for a thrombo-embolic complication following fontan procedure, still has to be investigated. the results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-pa should be used as for other patients. a 2nd generation functional protein s assay p. van dreden* and e. adema** * serbio, gennevilliers france, ** boehringer mannheim, tutzing germany a second generation protein s test was developed with improved sensitivity to protein s and better reagent stability. the test result was found to be unaffected by apc-resistence (10 patients, heterozygote for the mutation with a apti' + apc ratio between 1.4 and 1.9), heparin up to 2 iu/ml and f viii activity between 1 and 250%. in the test, diluted sample is mixed with protein s deficient plasma, activated factor v, activated protein c, phospholipids and an intrinsic pathway activator. this mixture is incubated for 3 minutes. during this time, the activated protein c inactivates part of the f va. the extend of f va inactivation depends on the protein s concentration. after 3 minutes caci2 is added and the time untill clot formation is measured. the clotting time is a linear function of the protein s concentration between 10 and 140% protein s. for the three preproduction lots the difference in dotting time between 10 and 100% protein s was 43-54 seconds. this compares to 30-40 seconds typically obtained with the old test. within run precision (n= i0 on sta) is cv= 2 -7% on the basis of protein s. day to day precision (n=10 on sta) was found to be cv= 4 -11%, again calculated on the basis of protein s concentration. the cv of 11% was obtained for an avk plasma with 13% protein s; it corresponds to a standard deviation of only 1.5% in protein s. the insensitivity to interferences, in particular apc-resistence and better precision and stability are expected to improve the quality/reliability of a protein s determination. in this study we evaluated the use of hormonal contraception on the parameters protein c, protein s and pal. samples from 71 women with, without hormonal contraception and in menopause were assayed by coagulometric (protein s clotting test (behdngwerke, marburg, frg) or chromogenic methods (protein c activity test and pal reagent from behringwerke, marburg, frg) in double determination and were compared with the reference ranges. in addition thromboplastin time (thromborel s reagent) and fibrinogen (multifibrin) from behringwerke, marburg, frg, and aptt (actin fs reagent from dade corp., unterschlei6heim, frg) were determined. in women using hormonal contraceptives (p<0,01) and in menopause (p<0,05) protein s activity was significantly reduced compared to other women (<45 years) while protein c acitivity did not change. in menopausal women a higher susceptibility to thrombosis was supported by an increase of aptt (p<0,05) and fibronogen (p<0,01). while there was no change for pal, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p<0,05). we could not observe a higher turnover of coagulation and fibdnolytjc system with hormonal contraception. noteworthy was the occurence of low (<200 mg/dl) and borderline fibrinogen (max. 220 mg/dl) in 40,9% of women res. in 22,8% of women (together with borderline aptt) who had an individuell risk for arterial disease. protein s protein c fibdno~en aptt plasminog~ without hcc 109,1-+13,6 78,3-+14,1 255,0-+14,0 36, [2] [3] [4] [5] 4 24, [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 2 with hcc 85, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 2 78, 5±12, 0 253, 8.+24, 1 35, [2] [3] [4] 8 14, 9 menopause 90, 3+97, 6 87, 4±41, 4 307, 05:57, 6 39, [8] [9] [10] 0 12, 6 hcc= hormonal contraception hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease c. salat 1, , e. holler t,3, hi. kolbl, 3, b. reinhardt l, r. pihusch 1, p. g0hring 2, s. poley 2, e. hiller 1 l=med. klinik iii, 2 = institut flit klin. chemie, klinikum grosshadern der ludwig-maximilians-universit~tt mfinchen, 3=h~tmatologikum der gsf a 40 year old patient suffering from all received allogeneic bone marrow transplantation (bmt). after an uncomplicated early posttransplant period the patient was dismissed after 4 weeks. a bilirubin rise with subsequent liver failure was observed during the following weeks. according to biopsy proven hepatic veno-occlusive disease (vod) liver transplantation was performed on day 79. unfortunately the patient died on day 140 due to aspergillosis. we monitored levels of protein c (pc) and s (ps) as well as pall during the pre-and posttranspiant period. pal1 level was normal (<43 ng/ml) during the first 4 weeks after bmt but increased with the manifestation of vod (317.5 ng/ml on day 47). it reached its peak immediately before liver transplantation (547.6 ng/ml) and returned to normal levels within the next few days. pc levels which were normal before bmt decreased prior to clinical diagnosis of vod and were normal after liver transplantation. ps levels lay within the normal range at all timepoints. vwf was elevated before bmt (240%) and remained relatively stable during the whole investigatonal period ranging from 170 to 260%. it is assumed that vod is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. our results indicate that the "endothelial cell marker" vwf is not helpful in predicting vod. the kinetics of the investigated parameters underline the significance of pc and pai-1 as described by others and our group earlier, whereas ps does not seem to play a role in the pathogenesis of vod. the budd-chiari syndrome (bcs) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. it frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein c and protein s deficiencies e.g.). only recently, the factor v leiden mutation (fvlm) has also been associated with bcs. we hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (tmaap) are a major risk factor for the precipitation of bcs. we screened our cohort of 27 patients (pts) with bcs for the presence of defects in the tmaap and identified 3 pts with protein s deficiency (psd). these pts were screened for the three point mutations in exon 1 (codon-25; ins t), exon 15 (codon 636; a-->t) and in intron 10 (g-->a + 5) of the ps alpha-gene that have been demonstrated by bertina et al to coseggregate with psd. restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded pcr-products were employed. all living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. no single abnormality in these genes despite presence of pbd in those family members was found. in addition, pts and family members were also screened for fvlm. one pt and two of his family members, in addition to psd, were subject to fvlm. the other two lots and their family members were not subject to fvlm. in contrast to the first family, despite psd, those two pts suffered from morbus crohn and acute myeloid leukaemia as risk factors for bcs. we conclude: psd is one major risk factor for the precipitation of bcs. to precipitate this disease, one additional risk factor is required. psd may be caused by genomic defects in the protein s gene other than those described by bertina. only a few publications describe a thromboembolic disease due to dramatically reduced protein s levels being associated with viral or bacterial infections, autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion. we report on a 5 year old boy who developed purpura fulminans of the left leg during varicella infection. on the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. on admission the boy suffered from a purpura fulminans with central necrosis measuring 15x8 era. suspecting a hereditary thrombophilic disease we started therapy with protein c concentrate and recombinant tissue type plasminogen activator. the fellowing coagulation investigation showed a severe deficiency of protein s (total protein s-antigen < 5 u/ml, free antigen not measurable) in combination with factor v leiden mutation. other thrombophilie and coagulation parameters did not show deviation from normal range. after 4 weeks we saw a slight improvement of the total protein s antigen up to 50 u/ml. the free protein s antigen was still undetectable. during the following weeks the patient recovered slowly and the protein s activity and antigen normalized. because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (fragmin®, 100 ie/kgbw/die) and continued for 6 months. under this therapy there were no further thromboembolic events. these results suggested an autoimmune protein s deficiency in a patient suffering from chickenpo×. an analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (igg 16,1 iu/ml, igm 15,1 iu/ml) which normalized during hospitalisation. we suspect an antibody to protein s probably caused by similar presented viral antigens. we suppose that autoimmune mechanism during different infections in combination with a heterzygous apc-resistance may be a potential risk factor for developing thrombotic disease. in the central nervous system mrna encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. we investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (trap-6, sfllrn) on [ca2+]i, phosphoinositide hydrolysis and protein kinase c activation in the rat glioma c 6 cell line which is a widely used in vitro model for studying glial functions. stimulation of c 6 cells with both c{-thrombin and trap-6 resulted in transient [ca2+]i mobilization, [3h]inositol phosphate response and activation of the pkc isoenzymes c{,i],7,5, and s. these results suggest that ~-thrombin and trap-6 activate at least partially the same intraceltular signaling pathways in rat glioma c 6 cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma c 6 cells. further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in c 6 glioma. thrombin is a potent mitogen in vascular smooth muscle cells (smc). the mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (hutrap-14) was investigated in bovine coronary artery smooth muscle cells. confluent cells (passage 4-8) were incubated for24 h in serum-free medium with thrombin (10 nm) or trap (10-100 pm). the mitogenic response was assessed using [sh]thymidine incorporation. results demonstrated that thrombin induced a three-to fourfold increase in [3h]thymidine incorporation compared to the control, thus being as active as the potent mitogen pdgf-bb (20 ng/ml). the mitogenic effect of thrombin was dependent on its proteolytic activity. when the active site of thrombin was irreversibly inhibited by d-phe-pro-arg-chloromethylketone (ppack) no increase in [°h]thymidine incorporation was observed even at the high concentration of 250 nm ppackthrombin. thrombin-induced [3h]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor nc£-(2naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (napap, lpm) . in contrast to thrombin trap-14 at a concentration up to 100 pm did not increase [3h]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin (10 ijm). in order to demonstrate whether hutrap caused a stimulation of the receptor and signal transduction in bovine smc the activation of protein kinase c (pkc) was measured as translocation of the cytosolic to the membrane bound fraction. it was found that both thrombin (10 nm) and trap (10 pm) induced a translocation of pkc after 20 rain. in conclusion, thrembin and trap activated pkc in coronary smooth muscle cells but only thrombin acted as a mitogen. endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. but after a recovery period of about 90 min the cells are sensitised again for activation by thrombin. in our experiments this resensitisation couldn't be inhibited by actinomycin d or cycloheximide nor by the phosphatase inhibitor ocadaic acid. therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with golgi vesicles. brefeldin a (bfa) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the golgi apparatus. because of this we examined the effects of bfa on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. the vwf release from weibei-patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. the addition of bfa (20 pg/ml -20 ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by bfa at the concentrations used. these results give further evidence that the proposed storage pool of thrombin receptors is iocalised in vesicles of the golgi apparatus. randomized comparison of double bolus reteplase (r-pa) and front-loaded alteplase (rt-pa) in patients with acute myocardial infarction (rapid ii) c. bode, rw. smalling, j. kalbfleisch, g. berg, dj. odenheimer, e. bshm, wd weaver and the rapid ii investigators. med. klinik iii, university of heidelberg, germany the study was undertaken to assess the efficacy of a double bolus regimen of 10 + 10 megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (gusto-regimen) alteplase. there was no age limit and patients were recruited up to 12 hrs after onset of symptoms. the primary endpoint "patency at 90 minutes" was centrally assessed in a blinded fashion. infarct related coronary artery patency (timi grade 2 or 3) and complete patency (timi grade 3) at 90 min for reteplase vs. alteplase were 83.4% vs. 73.3% (p=0.03) and 59.9% vs. 45.2% (p=0.01), respectively. at 60 minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: 81.8% vs. 66.1% (p=0.03). and 51.2% vs. 37.4% (p=0.01). reteplase treated patients required fewer additional coronary interventions within the first 6 hrs of treatment (13.3% vs. 26.5%, p=0.01). there were no significant differences between reteplase vs. alteplase regimens in 35 day mortality (4.1% vs. 8.4%), severe bleedings (12.4% vs 9.7%) • or hemorrhagic stroke (1.2% vs. 1.8%). reteplase, when given as a double bolus of 10+10 mu to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. lysis therapy with uhsk of deep vein thrombosis (dvt) of the lower extremity commoniy results in a relatively high patency rate. in our patients, patency was achieved in approx. 70-80%. these results were supported by several studies. in dvt of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. on the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms 0phlegnmsia cerulca dolens etc.). until september "95, 44 consecutive patients (16 males, mean age 41 ranging from 18 to 63 years) with dvt of pelvic veins were included in this study. after clinical and phiebographic/computer tomographic (ct) diagnosis, a temporary filter system was placed intravenously either by a wambmchial (34 pat.) access route, lransfemorally (6 pat_) or wansjngularly (4 pat.) under mdiological control. the open filter was positioned above the thrembus in the lower caval vein: 26 times an antbeor system, 11 times an anglocor and 10 times a cook filter were used. in the following three days up to 3 cycles of lysis therapy with 9 mli u of streptokinase were administered, each cycle lasted six hours. heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrit values 1.5 to 2 times the individual base line level. effective lysis was controlled by a second phlebography/ct, and the filter system removed through the introducer sheath valve. after lysis therapy all patients were put on coumarin with overlapping heparinization. successful lysis (complete recaanlisation without remaining thrombi and lysis of more than 50 per cent of the original thrombus) could be achieved in 62% (29 patients). in two cases a fatal pulmonary embolism occurred. 5 times great parts of a thrombus were captured by the filter and could be removed with the system. due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. this complication could be resolved by additional administration of streptokinase. in nearly all cases we found extensive hematomas on the catheter insertion sites. 4 patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. one filter system was broken after use. lysis of dvt of the pelvic veins with uhsk and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. the filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. heparin should be administered at least in the remainder of the day of lysis. the insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. we report about seven of our patients (pts.) with bcs treated by fibrinolytic therapy (5 female, 2 male, age 27, 6 years + 8). only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein c deficiency and in another one morbus behcet was detected in five pts. chronical and in two acute bcs was diagnosed. as complications were found: in three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. we treated just one patient with urokinase (3 x 800.000 iu/d for 18 days). four pts. were treated with rt-pa in concentrations of 25 to 50 mg/d (for a period of up to 14 days) and continuous heparin infusion. high doses of rt-pa (80 to 120 mg/d for one or four days) were given in one case of acute and one of chronic bcs. partial (after high-dose rt-pa nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of bcs. no significant recanalisation was proven in hepatical veins of chronic bcs. three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. in one patient partial lysis of v. porta and in another one of v. lienalis was found. furthermore complete recanatisation of v. porta followed local rt-pa lysis during surgical intervention. in our two pts. with acute bcs significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• in chronic bcs hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. the benefit of flbrinolytic therapy of bcs seems to be influenced by the dur~ion of the disease• according to our results fibrinolytic therapy should be considered in cases of acute bcs as well as in the above mentioned thrombotic complications• recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. as the mechanisms of this phenomena are rather unclear we studied fibrin (0.4-1.5 ~tm) clot lysis by plasmin (30 nm) presented outside or inside the clot in purified system (tris-hc1 buffer, 37°c). the clots was formed by addition of cac12 (30 mm), thromboplastin (1 u/ml) and human thrombin (6 nih/ml)t the lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. obtained kinetic parameters of this reaction: kest=99.4 rain -1, km=i.9 ~tm (plasmin inside clot); kcat=l.36 rain -1, km=i.3 lzm (plasmin outside clot). this data indicate that eatalytie efficiency of plasmin from inside the clot is 50-fold higher than from outside as measured from kcat/km. furthermore we studied plasma clot (0.25 ml) lysis in original plasma (3 ml) containing sk (5-100 nm) and seu-pa (5-100 rim) in the final concentrations from inside or outside the clot. the results are presented in the the similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. it may be concluded that fibfinolysis is a surface-dependent process. it's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. for the improvement of the thrombolytic therapy in children more clinical data are needed. therefore we report on our experiences with rt-pa presenting the clinical course in 11 patients (age range: 7 days to 16 years) with venous thrombosis (n=9) and purpura fulminans by meningococcosis (n=2). the patients were treated with rt-pa (actilyse r) for 10,5 hours to 13 days. the venous thromboses were localized in lilac/femoral veins (n=4), brachiocephalic/jugular/subclavian veins (n=3) and the cava superior vein (n=l). central venous catheters were causative in 2 cases. the dosage range was between 0.2 to 0.5 mg/kg for the initial dose and 0.8 to 2.0 mg/kg/d for the subsequent continous infusion. ten patients received simultaneously heparin. complete clot dissolution occured in 6 cases, partial clot dissolution in 1 patient. in 4 patients with a history of the first clinical signs of thrombosis of 3 weeks or more the lysis was not successful. concerning the side effects only small bleeding episodes on former venipuncture sites were observed in 5 patients. systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. in conclusion, the thrombelysis with rt-pa presents an effective and safe therapy in children at: the rt-pa dosage used. in case of a history of the thrombotic event of more than 3 weeks a thrombotytic therapy should not be carried out. d.dimer is widely used in clinical situations associated with thrombotic diseases. the most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. d.dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. quantitative, flexible and sensitive assays are required. we have developed a new automatic method which works on the coagulation walk-away instrument, sta. this assay was developed with two monocloual antibodies specific for d.dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. absorbance is measured at 540 nm and is a direct relationship of d.dimer concentrations. the assay works with 50 ~tl undiluted plasma and is performed in less than 10 minutes. the dynamic range is from 0.2 to 16 [~g/ml and no prozone effect is observed up to concentrations higher than 100 ~tg/ml. the detection threshold is of 0.2 [lg/ml. the intra-assay reproducibility is below 5% and the inter-assay reproducibility is below 6%. recovery studies of purified d.dimer added to normal plasma were between 94 and 105%. no interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of 100 u/ml is observed. there is no effect of high fibrinogen concentrations (> 10 g/l). when compared with conventional elisa techniques (asserachrom ddi), the assay demonstrated a correlation coefficient of 0.97 on 131 samples from normal individuals and hospitalised patients with elevated d.dimer concentrations. slope was of 0.97 and intercept was of -0.07. this new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. it is then well adapted for all the applications of d.dimer measurements in coagulation laboratories. 16 children between an age of 3 days and 11 months ( median 6 weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-pa for thrombolysis. the affected vessels were both sided renal veins or one sided renal vein and v. cava inf. in 8 cases, the v. cava superior in 3, the v. cava inf. plus renal veins plus aorta in 1, the left ventdcle in 1, the aorta in 1, the a. femoralis in 1 and the v. portae in 1 case. 10 out of 16 occlusions were associated with an indwelling catheter. underlying dieseases were sepsis (4), prematudty (3), vitiurn (2), asphyxia (1), short bowel syndrome (1), hus (1), diabetes (1), cmv (1), exsiccosis (1) and m. hirschsprung (1). thrombolysis was performed with an bolus of rt-pa (0.1-0.2 mg/kg) followed by continuous infusion (0.8-2.4 (-9) mg/kg/24h, median 1.8 mg/kg/24h). low dose hepadn (100 ie/kg/24h) was given dudng full dose hepadn (aptt 1,5-2 times normal) after the thrombolysis. in 5 pts. rt-pa was administered locally through the catheter and in 11 cases systemically. in 13 patients the vessels could be recanalised completely, in 2 partially, in 1 patient the therapy had to be discontinued. in 2 vessels a reocclusion occurred. bleedings were noted in three patients, all from recent venous puncture sites. the results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. the aim is to compare efficacy and safety of rt-pa with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. the design is a randomised, notblinded trial with a cross-over option after three days in cases without success. study end points are recanalisations, major bleedings and number of cross-overs. inclusion criteria are age under 1 year, lifethreatening vessel obstruction, age of thrombus up to 10 days, no precaeding fibdnolytic therapy. exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last 7 days and cns injuries during the last 2 months. although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as fxii deficiency. fxii is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in fxii deficient pts..up to date only few and controversial data exist about the frequency of fxii deficiency in pts. with thrombophilia. cons~uently the aim of our study was to evaluate the association between fxii deficiency and juvenile venous thrombosis in a great population. patients and methods: 1554 pts. (851 female, 703 male, aged i to 61 ys, median age 38.2 ys) with venous thromboembolism before the age of 45 ys were studied. one-stage clotting activity assay of fxii (fxii:c) was performed on acl using fxii deficient plasma from instrumentation laborato~. fxii antigen concentration (fxii:ag) was measured by electroimmundiffusion using reagents from behfingwerke, enzym research respectively. the normal ranges are tl~. routine reference values obtained m our labratory from 80 healthy subjects (40 males, 40 females, median age 26.2 ys); 95% range: fxii:c 53-135%, fxii:ag 57-132%). results: 122/1554 pts.were classified as fxi1 deficient (f 60, m 62), giving a prevalence of 7.8%. severe fxii deficiencies with fxii:c below 1% were observed in 7 pts..ll5 pts= proved to have moderate fxii deficiency with fxihc ranging lrom 2 to 51% and fxii:ag ranging from 1 to 53%. in none of them inherited deficiencies of other well established thrombophila risk factors could be detected. none of the fxii deficient pts. had positive lupus anticoagulant tests. familial fxii deficiency was found m 9 cases. discussion and conclusion: the precedences of fxii deficiency amongpts, with venous thromboembolism was previously described to be 7.5-10%. supporting these data, we have shown a praevalence of fxii deficiency of 7.8 %. in comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of fxii deficiency m our study group.these data, from the largest such study reported, strongly indicate that fxii deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. we describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor xii (hageman factor). the first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age 35. since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control the patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. her plasma plasminogen chromogenic activity was 51-59 % compared to a normal plasma pool (reference range 70-130 %), plasminogen antigen was diminished to the same extent. the patient's factor xii exhibited only 28-55 % activity in a factor-deficient plasma assay as compared to a normal plasma pool. other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin iii, protein c, protein s, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aptt prolongation after addition of activated protein c. the proposita's mother had died at age 60 from pulmonary embolisnt no coagulation studies are available. the proposita's sister was first diagnosed deep leg vein thrombosis at age 17, since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. this sister's plasminogen activity was 120%, but factor xii activity was reduced to 55 %. three brothers of the proposita were examined, too, all in their 3rd decade of life. none of them recalled symptoms of or treatment for thromboembolic disease. in one brother, factor xii activity was normal (100-105 %), but plasminogen only about 50 %. in the 2nd brother, factor xii was very variable (64, 42 and 94 %), plasminogen was in the lower normal range, in the 3rd brother, factor xii was about 50 % (repeatedly), plasminogen was normal. current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial. msrgit serbsn,maria cucuruz,dan madras,carmen petrescu, natalie rosiu,rodica costa iii rd psediatric clintc,universtt v of medicine, the unsatisfactory efficiency of entihepetitis b vaccination in our haemsphiliscs suggested the control of the immune status in 52 hiv negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (cd3,cd4,cds,cd&/cd8 ratio and cd19) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (hay, hbv,hcv ebd hdv) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector viii/ix concentrate) and the quantity of their consumption (ui/k9 weight/yesr).the interpretation of the results pointed out • significant lower level of cd3,cd& (p<o,o01) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the cd19 counts (p< 0,001) without an immunoglobulin-deficiency. the same behaviour was found at the group of haemophiliacs carriers of hb~ and hcv markers.the superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis b or c infections or the negative impact of the itarative important alloantigenlc load ? illumination with l~tm methylenblue (mb) is used for inactivation of enveloped viruses in human plasma. via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. fibrinogen is an important mediator in the cell-cell interaction of platelets. the four rgd sequences (arg-gly-asp) of fibrinogen can be recognized by the activated fibrinogenreceptor gphb/iiia . after binding to gpiib/iiia one molecule fibdnogen can link two platelets. the aim of this study was to investigate the influence of mb treatment on the fibrinogen activity in human plasma. furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the gpiib/]iia receptor. human plasma was treated with 1-50~m mb. after illumination fibdnogen was isolated by fl-alanin precipitation. purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. possible receptorblockade were examined in an 125j-fibrinogen competition assay. densitometric scanning of reduced sds-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased mb concentrations. this effect was not detectable for fibrinogen isolated from plasma with 1bm mb. this results were confirmed by two-dimensional gelelectrophoresis. ibm mb treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with adp and fibrinogen. proposed validation of a quantitive quality-assured pcr method f. dorner, j. eibl, g. zerlauth immuno ag we have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (pcr) and on the reverse transcriptase polymerase chain reaction (rt-pcr), to screen for the nucleic acids of human immunodeficiency virus type 1 (hiv-i), hepatitis b virus (hbv) and hepatitis c virus (hcv) in plasma and plasma products. the detection of pcr products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence pcr" (lif-pcr). the procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. a high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. in this report we present validation data for the lif-pcr and discuss them in the light of the recently published ich-guidelines on the validation of analytical procedures. taking into account the pecularitles of the replacement therapy of haemcphiliacs in romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; 142 hsemophlllacs,elasslfled in 3 groups: a-ulth no exposure to blood or bloodproducts, b-treated ~ith factor-concentrates and c-ulth plasma,cryoprecipitate + factor-concentrates in their therapy have been investigated (elisa method) for serological markers of hepatitis a,b,c end d, cvtomegalovirus (cmv) and hiv i and hiu 9 infection. the infections profile of our haemophiliacs~hes some peculsrities. the hepatitis infection is the most extended,lnvolving more than 75% of the patients. the frequency is for hepatitis -bz,z~ , u -"/~,~ , g -bb,7% end o -24% : the multiple infection was diagnosed in more than 50% of cases. the prevalence uas significantly hlgher in the group c. we studied blood count and relevant coagulation system parameters of autologous blood donors. indications for autologous blood donation were divided in 4 categories: a group of diverse (a, n=13), reduction mammoplasty (b, n:5), prostatectomy (c, n=io) and total hip replacement (d, n=74). we performed bleod count (bc), thromboplastin time (tt), partial thromboplastin time (aptt), fibrinogen (fib), f vhi act, vwf rco, vwf ag, f xiii, at iii, d-dimer-fragments (dd) and fibrinmonomers (fm). all parameters were analyzed as initial value, bc, tt, aptt, fib, f xiii and at iii were measured additionally at each donation. the percentage of initial pathologic data was (1) were included in the study. the inclusion criterittm was: an endegen(y.~ red cell reserve (ercr) of less than 400 ml in men and less than 540 ml in women. ercr is the volume of blood that can be withdrawn up to tim hematocrit of 0.34. the selected dosages of rhepo (200, 400 and 800 iu/kg body weight biweekly over 4 weeks) were based on the calculated blood volume and the ercr. a unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least 11.5g/100 ml (or hematocrit was at least 0.34) up to an autologons predeposit of 800 ml red cell volume (rcv). additionaly, platelet concentrations and serum ferritin levels were measured~ results: at the initial clinical examination the ercr was 333 ml in women and 307 in men. this resulted in a weekly dosage of 2x32.000 iu rlaepo in women and 2x25.000 iu rhepo in men. 7 patients donated 5 red cell each (858 ml rcv on an average) within 19.1 days. the targeted rcv could not be reached in 3 patients because the operation was reschednled. the collection from one patient had to be aborted because of angina pcetoris complains. tic following side-cffczts occurred: episodic hypcrtejlsion (2 tim~), chest pain (5), fafigne (8), inflnenza-lihe syndrome (3). platelet count increased from initially 203 4-68 x 10'//d (i00 %) during therapy to a maximum level of 260 + 93 10v~ (128 %) without any clinical signs of thrombeembolism. in the control group (autologous blood donation weekly 3 or 4 times without erythrupoiedn stimulation, n=12) platelets increased from initially 230 __+ 73 10'/fi (100%) to 255 + 71 10v#i (110%) i1]aximlzm colldttsions: the adlninist/'ation of lhepo in the above described manner to autologous donors undergoing heart surgery is well tolerated. it allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. it is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. we retrospectively evaluated vascular complications in 128 patients (33 men, 95 women) with a median age of 61 years in relation to therapy. three major treatment groups were analysed: 17 patients treated with acetylsalicylic acid (ass) alone, 29 patients given alkylating agents, and 62 patients treated with interferon alpha (ifn). there was no significant number of patients treated with hydroxyurea. ifn-treated patients were younger (median age 54 years) than patients in the other groups, but platelet counts before therapy were similar (median 0.84-1.13 x 1012/i). before therapy, 47% of patients treated with ass, 45% treated with alkylating drugs, and 32% treated with ifn had bleeding events, microcirculatory or thrombotic complications. dudng therapy, median platelet count was 0.9 x 1012/i in patients on ass, 0.68 x 1012/i in patients treated with alkylants, and 0.48 x 1012/i in ifn-teated patients. the rate of patients with complications during therapy was 18% (9.5%/patient year) for ass, 14% (6.3%/patient year) for patients on alkylating drugs, and 15% (6.2°/dpatient year) for ifn-treated patients. we conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. the results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: p-selectin (cd62) and fibrinogen (fg) bound to the gp iib/iiia complex. interaction of platelets with teukocytes via cd62 is believed to stimulate the production of reactive oxygen species (ros) in leukocyte*. the role of platetet-bound fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ros production has been described. in the present study we measured ros production in undiluted samples of citrated whole blood (wb) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich wb) or platelet-poor plasma (ptatelet-poor wb). ros production was determined by measuring luminol-enhanced chemiluminescence (cl). stirring wb samples at 1,000 rpm for 10 min resulted in an activation of leukocyte* (increase of cl, upregulation of cd1 lb) and platelets (platelet aggregation, upregulation of cd62). stirring-induced cl was significantly higher in platelet-poor wb when compared to platelet-rich wb. higher cl in plateletpoor wb than in platelet-rich wb was also found when adp (moderate stimulation of platelets and leukocyte*) or fmlp (strong stimulation of leukocyte*) but not when pma (strong platelet and leukocyte stimulation) was added. dextran sulphate that is known to block cd62-mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced cl in wb samples. in contrast, when binding of fg to the platelet surface was blocked by rgds or the fg receptor antagonist gr144053, stirring-induced cl, but not cl in unstirred samples, was significantly enhanced. gr144053 also reduced adhesion of platelets to neutrophils, but did not affect cd62 exposure. the results indicate that interaction of platelets with leukocyte* in whole blood via cd62 and fg may have opposite effects on leukocyte ros production: stimulation by cd62 and inhibition by platelet-bound fibrinogen. late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. the pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. to test the effects of low molecular weight heparin (certoparin, sandoz ag, numberg germany) against late occlusive processes, four groups (groups a-d) of rats (7-10) were anesthetized and the external common carotid arteries were exposed. all groups received a 200 u/kg of unfraetionated heparin through the femoral vein. a 2f balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck vascdar injury was induced by repeatedly (x3) inflating the catheter balloon and withdrawing it to the bifurcation. following this procedure, two hours after, equlgravimetric amounts of various agents were administered via sc route to each of the individual groups. group a was administered with certoparin od for 21 days. group b received a lower low molecular weight heprin (mr 3.4 kda). group c received unfraetionated heparin and group d served as a control. on day 21, perfusion fixation was performed on all animals. the injured and contralateral carotid arteries were excised for histelogic examinatior~ in comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups. however, the degree of inhibition followed the order: heparin<certopann<llmwh_ in contrast to heparin treated group (c), the other groups a, b, and d did not exhibit any hemorrhagic lesions. however, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. in comparison to heparin, certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. the observed efficacy of these agents may be due to better bioavailability and sustained duration of action. in the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. a low-molecularmass heparin (lmmh) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein-5-isothiocyanate (fitc) by endpoint attachmenc. binding to leukocytes was quantified using flow cytometry analysis. the leukocyte populations were identified by their light scatter properties in man by cd4, cd13, and cd14 antibodies, in mice by cd 4 and gr 1 antibodies, and in rat by cd 45, ed 9, and rp 3 antibodies. granulocytes, monocytes and lymphocytes of all species bound dose-dependently lmm-heparin. the binding of lmmh-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. binding of lmmh-tyr-fitc was reversible in the presence of unlabeled heparin or lmmh. more than 99% of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with lmmh-tyr-fitc. this separation was not obtained for mouse and pig granulocytes. the data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled lmm-heparin for biological investigations. background and purpose: ptca is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents 0aeparin). in the present study we examined the time course of changes in blood coagulation and fibrinolytic function following ptca in patients with ischaemic heart disease. 20 patients were randomly assigned to receive either standard therapy (250 nag aspirin p.o.; 5.000 iu hepafin i.e. bolus, and 5-10.000 iu heparin intra-proeadurally)(n=10), or adjunctive therapy (aspirin, hepann; 100 mg sodium pmtosan polysulphate, spp intra-cortmarily and subcutaneously) (n=10). the sensitive markers of thrombin activation (thrombin-amithrombin iii complex ('rat), prothrombin fragmem f 1+2 (pl+2), d-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (pap), tissue type plasminogan activator (t-pa) and plasminogen activator inhibitor (pal)-i activities) were measured before, immediately after, and 6 hs after ptca. results: we found elevated mean levels of tat and fl+2 in both groups during the procedure, while plasma coneentrafiuns of d-dimer and pap remained in normal range. activity of pai-i was not elevated compared to normal comrol. the t-pa activity obtained immediately post-ptca showed significant increase in patients given spp. primary angiologic success was achieved in 80% of both groups. one patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. one patient receiving adjunctive therapy died in cardiogenic shock within 24hs arer ptca. major bleeding was observed in one patient given spp. conclusion: no significant intracoronary haemostatic activation occurred in the majority of patients during and up to 6hs after ptca. treatment with spp was associated with enhanced fibrinolytic activity. the benefits and risks of adding spp to heparm and aspirin should be evaluated by further studies, in july 19,¢1 the paul ehdich institute became responsible for quality and safety of bt~d and blood products. since ~his time, batch m~se of ppsb and factor ix prapamtions were carded out regularly, whereas ihe obligatop i testlng of all other products derived frem pooled plasma will be starling in janualy 1996. the european pharmacopoeia does .or contain directions for the quality control of ppsb preparations. therefore, ifua ~ coneenning factor ix clotting concentrates are applied, ircludmg the non activated ptt and the classical tkrombln test these m~ods are not su'rlable for reliable exclusion of ituombogenk~b/ ,of pp,sfl, because vlla actlvry is not d~. a hllhedo unsolved problem is the high sensitivity of file classical thrombin test often not correlating with ~ data of thrombogenlci~. in ordm td es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of ila, vlla. ixa, and xa (chrornogenic, fluorege~c clolfing reactions) wera instaued. in agreement with the [deralure, considerable differences were found among the ppsb pr~ons registered in germany. cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. however, in severely ill patienls, eg. post operatively, tissue factor may be expressed which pofenllatesthe dsk of ~aclor vlta. in the case of a certain ppsb preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor vii, cotmlaled with fatal comlffcal~ns, evaluating ~ expe~ concenlralbn limits for adjvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. the mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. to determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and 24 hours aider aspirin (650 mg po) ingestion (n=5). blood samples were supplemented with saline and aprotinin (40 ug/ml) and tissue factor activation was studied at 200 pg/ral. platelet activation was investigated using gtycoprotein ffla and gmp 140 specific antibodies on a flow cytometer (epic coulter). tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. however, the relative increase in the aspifinized group was much monger (25% of the baseline). these results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. while these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , 10-20 (group 2) or >20 years (group 3) duration. anticoagulated whole blood was incubated with fluorescent antibodies to gpib and gmp-140 (two colour method) and analyzed with a flow cytometer. thrombomodulin, f1+2, protein s, 13-thromboglobulin were measured according to standard procedures. results: surface expression of gmp-140 was not different in groups 1 to 3, however, there was a tendency to higher acitvation in group 1 (<10 years iddm). results for thrombomodulin, f1+2, protein s, 13-thromboglobulin will also be presented. conclusion: though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. this wilt be correlated with endothelial and plasmatic activation markers. in our clinic four patients with hiv-related thrombocytopenia were treated with a lot of gammagard (93f21abllf), which later turned out to be hcv contaminated. before infusion all patients were negative for hcv antibodies and hcv rna. 2 to 8 months after infusion 2/4 patients, who suffered from arc at the time of hcv infection with cd4 counts >100/pl, seroconverted, whereas in the two other patients, who suffered from aids with cd4 counts below 100/pl, there was no seroconversion. in all cases hcv rna was found. genotyping with inno-lipa (innogenetice) showed hcv genotype l(b) in all patients. liver enzymes and hcv rna copies were measured repeatedly over a period of one year after infection. the 2 patients with arc showed a strong increase of hcv rna titre during the first 3 to 4 months after infection, followed by a rapid decrease within the next months. in the patients with aids hcv rna copies increased moderately within the first 4 to 6 months, followed by a slow decrease. elevation of liver enzymes was mild in the aids patients and seems to be independent from the hcv rna titre. in the arc patients liver enzymes changed parallel to hcv rna titers with a delay of 2 to 3 months. the course of hiv infection was only slightly influenced by the acute hepatitis c as measured by cd4 counts, i%2microglobulin and hiv rna copies. introduction:mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. in experimental animals, administration of monocional antibodies (mab) recognizing cd18 can reduce organ injury following ischemia/repedusion. no data, however, have been reported concerning clinical ischemia situations. patients and methods:we investigated expression of cdt8, cd1 la, cdf l b and cd1 lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. the tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. control samples were drawn from the nonischemic contralataral arm with the same timing, duration ot ischemia ranged between sixty and one hundred minutes (80~16). whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (facscan, becton dickinson, san jose, ca). mean fluorescence intensity (mfi), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. data were compared by the paired student's t-test, p<0,05 was evaluated as significant. results:mfi for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. in contrast, expression of cd18 was significantly enhanced in granulocytes (321_+50 vs. 189_+38), monocytes (653-+54 vs. 426+122) and lymphocytes (299_+45 vs. 228-+36) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. at the same time, an increase of cdf lb on granulocytes (500~_342 vs. 213+150) and monocytes (533+359 vs.237-+206) but not on lymphocytes was found, no modifications of cdlta and cdttc expression could be observed. there was no correlation between duration of ischemia and quantitative expression of these markers, conclusions:our data indicate that relatively short ischemia periods induce an increased expression of ~2" integrins adhesion molecules on leucocytes. these results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. in patients suffering from chronic inflammatory bowel diseases, such as morbus crohn and colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. hereby, the deficiency of coagulation factors, especially of factor xiii in plasma is established. ttowever the influence of factor xiii on the pathomechanism of the underlying disease is still under discussion. therefore we studied the f xiii content in the intestinal mucosa. an immunohistochemicat method was developed using commercially available antibodies against f xiii subunit-a, the detection of mucosal factor xiii depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. with this method, it is possible to locate but not to quantify f xili in the intestinal tissue. therefore we developed an elisa-metbod in homogenized intestinal tissue, using commercially available antibodies. its precision was validated using a standard curve with commercially available factor xiii preparations (fibrogemmin®). the detection limit of this method is > 0.05 i.u. f xiii/ml of tissue solution. freezed dried intestinal tissue (lmg) was homogenized in 1 ml buffer using a potter. specimens of the large bowel revealed f xiii values of 0,21 + 0,0038 i.u. (x __+ sd), tissue solution. with this method it is possible to quantify tissue-bound faxtor xiii. studies are in progress to elucidate the content of f xiii in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of f xiii deficiency. in a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. the question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. therefore, 20 patients were treated with or without aprotinin (2 million kiu loading dose over 15 minutes followed by 500,000 kiu per hour), and citrated blood samples were obtained at the following time points: before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and 24 hours postoperatively. the determinations of plasmin/antiplasmin-complexes, d-dimers, thrombin/antithrombin iii-complexes, and prothrombinfragments 1 +2 were performed by means of test kits from behring, germany (enzygnostrpap micro, enzygnost r d-dimer testkit, enzygnost r tat micro and enzygnost r f 1 +2 respectively). -all markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. however, the values of pap and d-directs of the aprotinin group were below the values of the control group until the end of operation. the markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. as expected, the aptt was significantly prolonged in the aprotiningroup. the aprotinin treatment was also associated with a significantly lower blood loss in these patients. -concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. further blood samples should be analysed between the implantation of the femur shaft and the end of operation. in our laboratory large amounts of human prothrombin are required (30-50 mg/week). as we try to produce meizothrombin and meizothrombin-des-fragment-1 from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. commercially available human prothrombin is expensive and of an unacceptable quality for our applications. in most of these batches we found small amounts of factor x and prothrombin activation products. we now developed a procedure to isolate prothrombin from "prothrombin complex concentrates" (ppsb-250-bulk, drk-blutspendedienst nds.). the concentrate also contains fac-tor vii, factor ix, and factor x. the prothrombin had to be separated from these factors. the concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin-1) as well. for the preparation of prothrombin from ppsb we used anion exchangechromatography (resource-q ®) on an fplc ®. we applied dissolved ppsb directly or after buffer exchange on sephadex g-25 onto the column at room temperature. the prothrombin was eluted with an naci-gradient in trisodium citrate buffer, ph 7.0. the buffer conditions are similar to the conditions used in the preparation of ppsb. the quality of the prothrombin so obtained was sufficient for most of our experiments. a second purification step on ion-exchange resulted in a 99% pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained sds-page electrophoresis and assays for factor x. this prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. we are now able to isolate the amounts of pure prothrombin required for preclinical investigations. most of the commercially available lmwhs such as enoxaparin, fraxiparin, and fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. on the other hand, tiactionated lmwhs retain their native characteristics and are structurally similar to heparin. in addition, the oligosaocharide sequence responsible for atiii binding is not modified. physical methods such as gamma irradiation (~co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (deambrosi et at. in : biomedical and biotechnological advances in industrial polysaccharides, pp. 45-53). utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. this communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. fragments exhibiting molecular weights of 5, 7, 8, and 9 kda were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of 2.5 to 25 mrad under controlled temperatures. unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or atiii affinity. in routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. on a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. these studies suggest that gamma irradiation can be used to prepare lmwhs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ futthermore, lmwhs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. university hospital, dept. of angiology, frankfurt a.m., germany introduction: thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. the plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-p'a) or by high ptusma levels of regulatory proteins, such as plasmino-8en. activator inhibilors (pal). the aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pls. with thrombophilia. patients: a great population of 884 pts. (fenmle 478, male 406; age 21-61 ys median 39.8 ys) with venous thromtx~emolism before the age of 45 years were investigated in regard to their plasma fibrinolytie system. in none of them well established thrombophilia risk factors could be identified previously. methods: plasminogen ~behdngwerke), pai-1 activity (ehromogenic assay, biopool), pal-i anugen coneentration (elisa, biopool), t-pa activity (chromogenic assay, biopool) and antigen concentration (elisa, biopool) were measured before and after venous oeclusion.vo was performed z 12 month after the last thromboembolic epi~xle. 24 healthy subjects (median age 24.7 ys) served as controls. results." 24 pts.(2.7%) were classified as plasminogen deficiencies (activity and antigen). 142 pts.(16%) had significantly elevated levels of pal activity (up to 120 u/ml) and pal antigen (up to 90 ng/ml). none of the pts. with high pal levels had laboratory signs of acute phase reaction. low t-pa activity could be demonstrated and confirmed in 121 pts., aecordingto a prevalence of 13.6% (range: 0-2.7 u/ml; reference limils: 2.8 -21.8 u/ml). however, there was a significant negative correlation between t-pa activity and pal values. in 67 pts. (55.4%) the low t-pa activity was associated with increased pal levels whereas the t-pa antigen concentration was normal. a parallel reduction of t-pa activity and t-pa antigen (range: 0.35-3.5 ng/ml; reference limits: 3.6 -21.0 ng/ml) were determined repeatedly in 54 pts. (f 23, m 31, median age 39 ys). thus, the prevalence of a defective t-pa release was 6.1% in our study group. conclusion." in comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-pa activity, elevation of pal respectively in our study group. our data strongly indicate that besides t-pa and pal acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. the antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin ssh 14 was studied after i.v. and s.c. administration in rats. thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for 20 rain and was assessed by a thrombus score ranging from 0 (no thrombus formation) until 3 (complete thrombus formation). ssh t4 injected either 10 min (i.v.) or 30 rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from 0.25 to 2 mg/kg i.v. and 1 to 4 mg/kg s.c. there were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (edh0 after i.v. injection in females 0.35 mg/kg, in males 0.9 mg/kg). the sex differences were confirmed in studies on the time course of the antithrombotic effect. after i.v. injection of fully effective doses (2 mg/kg i.v. and 4 mg/kg s.c., resp.) the antithrombotic effect disappeared after 8 h in female or after 4 h in male rats. for studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. similar to its antithrombotic action ssh 14 also caused doseand sex-dependent anticoagulant effects. the most sensitive assays were the aptt and the heptest; thrombin time and prothrombin time were less or not influenced by ssh 14. in conclusion, ssh 14 was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. at present there is no explanation for the clear sex differences found in this species. venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. patients and methods: after informed consent 3x30 patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis (30 patients received fraxiparin 36 rag once daily, 30 patients clexane 40 once daily and 30 patients 7500 u calciparin twice daily). there were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. pre surgery, day 1, day 5-7 phiebograms were performed and also tat, dimers, fl+2 prothrombin fragments were examined. results: 1., dvt in 26 patients (28.9%). dvt in 5/30 patients under calciparm prophylaxis, 8/30 patients under fraxiparin and 13/30 patients under clexane treatment. 2., low speciflty (3.4%) of dimers and tat (24%) for detecting a dvt in these special patients undergoing knee replacement therapy, elevated fi+2 fragments in the dvt group at ti and t2 vs the patients without dvt (t1 dvt: 3.24+-1.8 vs. 1.6+-0.3 -p= 0.0042). 3, only 8/26 patients (31%) with dvt had clinical signs of thrombosis. conclusions: 1., there is an increase of thrombin gneeration measured by tat and dimers after knee replacement therapy. there are further studies with more patients necessary to confirm that fl+2 prothrombin fragments can discriminate between patients with and without dvt from a clinician's point of view. 2., phlebographicauy confimled dvt in almost 30% of our patients demonstrate the high thromboembolic risk in these patients. von willebrand's disease (vwd) type 2 is characterized by absence of high molecular weight muitimers. qualitative changes in the structure of the molecule might be associated with enhanced binding of von willebrand factor (vwf) to platelet glycoprotein lb. therefore in some patients vwd type 2 is associated with severe thrombocytopenia. here, we report on a 9 year old boy who presented with severe purpura and platelet counts about 20000/gl at the age of 2 years. thrombocytopenia did not respond to corticosteroids. a normalized platelet count of short duration was observed after high-dose immunoglobulins. in addition, increase of platelets was seen after anti-d treatment. thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. despite platelet counts above 50000/gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. therefore, he needed several transfusions. coagulation analysis revealed vwd. application of ddavp lead to a normalization of partial thromboplastin time (ptt) and an increase of factor viii with subsequent cessation of bleeding symptoms. recently, vwd was typed 2 by lack of high molecular weight multimers. in conclusion, we report a case with vwd type 2 responding to ddavp. however it is unclear, whether thrombocytopenia is part of the vwd type 2 or of autoimmune origin. since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of vwf to glycoprotein lb. von willebrad disease (vwd) with a prevalence of 0,8% (ruggeri 1994, rodeignere 1987) seems to be the most frequent inherited hemostatic disorder. • the diagnostic criteria for vwd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (ptt), level of factor viii:e, vwf, vwf:ag, ristocetin induced platelets aggregation (ripa) and multim~-analysis.the diagnosis ofvwd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types the aim of this study was the evaluation of diagnostic approach to vwd in childhood and diagnostic reliability of all available laboratory tests. all previously mentioned laboratory tests have been done on our own material (51 child who satisfied all criteria for vwd, 23 boys and 28 girls, 1-9 years old) except mulfimer analysis which was unavailable in some cases. majority of laboratory tests proved to be highly specific and necessary for diagnosis. however, the diagnostic reliability of fviii:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while ptt is an unvaiied test. the most specific screening test for vwd is vwf which diagnostic reliability is almost 1,00. the optimal strategy to establish general diagnosis of mild forms ofvwd is use of vwf and vwf:ag plus ripa if necessary and multimer analysis to classify variant types. we report on a new multimeric structural defect of vwf detected in a german family (two sisters and their three children): all members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). wound heeling was impaired in two cases. clotting assays showed slightly prolonged apti" and a mild decrease of f viii:c, vwf:ag and vwf:rcof levels. collagen binding activity was within normal ranges. bleeding time (simplate i) was slightly prolonged. the analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: all multimers were detectable; the structure of vwf was reproducably abnormal in all family members so that the defect must be caused genetically. the thmmbocytic vwf showed neither qualitative nor quantitative alterations. minirin@ (ddavp) was administered as a test dose of 0,3 ~tg/kg bw in 100 ml 0,9% nacl-solution i.v. to evaluate efficacy and tolerance: clotting assays showed normalization of a_vrt, f viii:c, vwf:ag, vwf:rcof in plasma and shortening of bleeding time in three cases. an insufficient rise of vwf:ag and vwf:rcof levels could be observed in one case. one patient had no rise of f vm:c but a corrected bleeding time. multimeric analysis showed no structural change. the administration of ddavp was well tolcrated in all cases. the existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vwf in this family does not cause functional defects so that the defect could be classified as a type i vwd. the response to ddavp was only partially effective. mild von willebrand disease (vwd) is far the most frequent congenital bleeding tendency. its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. following post-operative periods or monitoring the management of haemorrhagic episodes in vwd patients is also strongly recommended. current methods involve complex technologies, are time consuming and require large series. these assays lack the expected flexibility for rapid individual testing in patients. a new and flexible assay which works on the fully automatic walk-away coagulation instrument, sta, has been developed for these applications (liatest vwf). the technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vwf. the assay has a dynamic range from 2 to 420% yon willebrand factor (vwf) concentration, it works with a 2 fold dilution of tested plasma (50 td) and it offers a calibration established with the nibsc international standard. the total assay time is of less than 10 minutes and the detection threshold is of 2% there is no prozone effect up to concentrations higher than 1,000% vwf. intra-assay reproducibility is < 4% and inter-assay one < 5%. in dilution studies a mean recovery of 98% was obtained. in a study on 55 plasma samples from norma~ individuals, patients with high vwf concentrations, and vwd, comparison with the elisa technique demonstrated a correlation coefficient of 0.997 with a slope of 0.978 and an intercept of 3.30. in the low assay range too, a good agreement was obtained with the elisa. we conclude that liatest vwf is a reliable, flexible, sensitive, and rapid automated assay which fits well the vw'f assay applications in coagulation laboratories. fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. for this activity, t-pa is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. with this background t-pa is used for thrembolytic therapy with great success. however, data from t-pa knockout mice indicate that t-pa might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. in contrast, u-pa, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. on the other hand, the major plasminogea activator inhibitor pal-i seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a pai-1 knockout model, but increased levels of pai-1 seem to predict reocclusion after angioplasty). in addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. the u-pa/u-pa-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. for such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. there seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (serpins): serpins have homologies to hormone binding proteins and cleavage of serpins by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the serpins. from these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. in addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. 4g vs. 5g in the pai-i promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation. plasmatic for both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor viic and vwf:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. the same holds for tpa:ag. however, a group analysis in quintiles reveals that particularly low tpa:ag values are connected with a particularly low coronary risk. unexpectedly also the acute phase protein crp is positively associated with increased coronary risk. for clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. the assessment of the pathophysiological significance of these observations remains at dispute. 4 pathways are discussed: 1. the assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. 2. both vwf:ag and tpa:ag are produced in endothelial cells. an increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. the risk association of the two acute phase proteins crp and fibrinogen could be interpreted analogously. 3. first prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. it could also be shown that there is a certain dependance of the gene-polymorphism for co-and 13-fibrinogen chains from the coronary risk. 4. even slightly elevated concentrations of fibrinogen and/or vwf:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. this could mean that an early coronary clot under these conditions could more readily develop to a stable, occlusive thrombus. a newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below 2.8% in a clotting assay. both parents had activities of 71% and 69%, respectively. however, the immunological determination ofprth by elisa revealed normal concentrations in all family members (93%-101%). furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. activation of prth by fxa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. although clotting times still were different, finally, normal levels of fl+2 and tat were generated as determined by elisa. western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. tat was only weakly visible in the serum of the mother and nearly absent in the child.the mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. after reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin 2. these observations let suggest that prothrombin marburg is a deletion mutant lacking the cleavage region arg 320-ile321. upon cleavage by factor xa only prethrombin 2 is formed under liberation of fl+2. this prethrombin 2 is able to cleave chromogenic substrates in the ecarin assay. probably, prethrombin 2 forms a complex with atiii which is detected by elisa, but unstable under denaturing conditions as in the western blot. as a major complication of haemophilia a treatment, up to 30% of the severely affected patients develop antibodies to substituted factor viii. investigating 133 patients and considering the data of further 231 patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. patients with more severe gene defects, like intron 22 inversions, stop mutations or large deletions had a risk of about 35% for inhibitor developement, which was about 7 times higher than for missense mutations or small deletions. besides an influence of mutation type, we investigated other parameters e. g. immune response genes (i-ila-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. to exclude any effect of mutation type, we focussed on 72 patients with an intron 22 inversion. hla-typing showed that some t-ila-alleles (dqb0602, bt) occurred more otten and others (dqa103, dqb603, dr13, c2) less frequent in inhibitor patients. treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. the results presented here, prove that inhibitor development is considerably influenced by the mutation type. this supports the hypothesis that patients with severe molecular defects have no endogenous factor viii protein and that substituted factor viii represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. in addition, the immune response seems to be modified by the hla-genotype. however oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. it is still unsolved why substituted factor viii does not lead to a recognizable immune response in 2/3 of the patients with severe molecular factor viii gene defects. consequently other factors, probably concerning the antenatal phase, must be involved. viia in the treatment of patients with inhibitors against factor viii or ix: a german/swiss/austrian multi~center trial d. ellbriiek*, i. scharrer**, j. dethling***, and the rfviia study group *section h~mostaseology, university ulm **dept. of angiology, jwg-university hospital frankfurt a.m. ***novo nordisk, mainz administration of activated recombinant factor vii (rfviia) can by-pass the fvnlwlx pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. from november 1994 to october 1995, a total of 25 bleeding episodes and 10 surgical interventions in 18 patients were treated with rfviia in a phase iiib multicenter trial. diagnosis was hemophilia a (n = 15) or b (n=l) with inhibitor, and acquired inhibitor against factor viii (n=2). various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. dose regimen was 90-120/zg/kg bw every two to three hours until clinical improvement, with subsequent dose reduction. results: for bleeding episodes, response to rfviia after 24 hours was effective in 72%, partially effective in 12 9"0, ineffective in 129"o and not evaluable in 1 (4%) of the patients. two of the three treatment failures were associated with very long dosage intervals of rfviia. the third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. the efficacy of rfviia for surgery was very good. response to treatment was independent of antibody titer. no signs of dic or activation of coagulation were noted. conduslon: in our experience, rfviia is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. it should be investigated, whether rfviia can be an alternative treatment also for the hometreatment situation. successful immunetolerance therapy of f vih-inhibitor in children after changing from high to intermediate purity f vih concentrate w. kreuz, j. joseph-$teiner, d. mentzer, g. auerswald*, t. beeg, s. becker zentrum der kinderheilkunde, j. w. goethe-universit~itj frankfurt am main *professor hess kinderklinik bremen introduction: inhibitor to f viii is the most severe complication in treatment of patients with haemophilia a. the incidence of f viii inhibitors is estimated to range between 15-33%. several authors reported that the immunetolerance therapy (itr) of f viii-inhibitors can be induced with high dose f viii concentrate. objective: this presentation will show data of four children with haemophilia a and f viii inhibitor (high responder), who had an unsuccessful lit with high dose f viii concentrate (high purity) in the first step. f viii concentrate was changed to an intermediate purity product (haemate hs®) in the subsequent course of h't. all patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (feiba®). results: median age was 13 (9-18) months, when the inhibitor was first detected. in all four patients the f viii inhibitor titre increased under immunetolerance treatment with f viii concentrate (high purity) in the first step of therapy. after changing the f viii concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to 0be within months. median duration of f viii inhibitor elimination time (until first testing of 0 be) was 3 (2-5) months. in all patients the f viii inhibitor was successfully eliminated. until now all patients are under prophylactic treatment with f viii concentrate and had no positive inhibitor testing since. median observation time since the first testing of 0 be is 14 (4-60) months. conclusion: different studies concerning immunetolerance treatment have been successful with f viii concentrates of different purity. according to our experience in these four presented patients, we assume that probably not the purity of the f viii concentrate is important for the induction of immunetoleranee, rather than the type of f viii presentation in the used concentrate. the used preparation (haemate hs®) is a f viii concentrate with high concentration of vwf, which is known to be important for the protection of f viii against degradation by proteases. this may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot itr. long scale trials are needed to prove the above assumptions. thrombasthenia glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (gp) ilbllla expression or a modification of this complex. since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury. here we report about a 60 years old women, who suffered since early childhood from a severe bleeding disorder. life threating bleeding complications occured after tooth extraction and after abdominal surgery. analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased (11 fl). clot retraction was diminished to 17%. platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. adp (i#m) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. cross immunelectrophoresis resulted in an atypical peak of gpiibllla with reduced electrophoretic mobility. in the electroimmunoassay according to laurell 14% of gpiibllla was detected. moreover we observed a markedly diminished 125j-fibrinogen binding. sequence analysis of the gpiib and gpiila cdna after pcr amplification unraveled a g2508 --, a transition in gpiib, substituting gly805 --* glu. the structure/function relationship of this mutation has still to be investigated. we report two new abnormal fibrinogen variants, denoted as bem iv and milano xi, both having an exchange of arginine to histidine in position 16 of the ac~-chain. routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. the onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. release of fibrinopeptide b by thrombin, measured by reversed phase hplc, was normal whereas only one half amount of normal fibrinopeptide a was released. in addition to normal fibrinopeptide a, an abnormal fibrinopeptide a* was cleaved from both dysfunctional fibrinogens. the structural defect was determined by asymmetric pcr and direct sequencing of a gene fragment coding for the nh2-terminus of the aachain. both variants were found to be heterozygous for the transition g to a at nucleotide position 1203, leading to the substitution actl6 arg-->his, resulting in a delayed fibrin polymerization. the simple assay permits detection of the most common amino acid substitutions occuring in the nh2-terminus of the ac~-chain of the functionally abnormal fibrinogen variants. protein c inhibitor (pci) a member of the serpin family is also known as plasminogen activator-3 (pal-3). pci was first described as a component of human plasma, regulating the activity of activated protein c and other sedne proteases of the human coagulation and fibdnolysis system. since then pci was found to be present in extra-plasmatic systems also. high concentrations of pci were detected in human seminal plasma suggesting a role for pci in human fertility. significant concentrations of pci mrna and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for pci in this environment. in this study we present evidence that pci is also present in human pancreas. rna from human pancreas was reverse transcribed and pcr amplified. the resulting pci cdna was identical with pci cdna from human liver. ~p labeled antisense rna probes used in in situ hybridization experiments with human pancreas tissue sections showed that pci rna was located in the acinar ceils. pancreatic fluid was analyzed by sds-page and immunoblotting. using monospecific antibodies directed against human plasma pci, a 57 000 mw protein band was observed which comigrated with purified human plasma pci. our results show that pancreas cells contain a significant concentration of pci mrna. this message is localized in the secretory acinar cells. therefore we conclude that pci antigen found in pancreatic fluid is likely to originate in the pancreas. the role of pancreatic pci is unknown at present. however, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that pci can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that pci may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. these inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. institute for clinical chemistry and laboratory diagnostics and *clinic for cardiology, universi w of duesseldorf p-selectin (cd 62p, the former granule membrane protein 140 or gmp 140) is an integrated membrane protein of platelets and endothelial cells. under inactivated conditions it is stored in the alpha granules of platelets and in the weibei-palade bodies of endothelial cells. endothelial cells covering atherosclerotic plaques show an increased expression of p-selectin. 13-thromboglobulin (13-tg), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. we therefore analysed cd 62p and 13-tg in acute coronary syndromes, healthy subjects (hs, n=l i), patients with stable angina pectoris (sap, n=20), unstable angina pectoris (uap, n=l 2) and acute myocardial infarction (ami, n= 12). plasma samples were obtained by using ctad vacutainer tubes (0.109 m na~-citrate, theophylline, adenosine dipyridamole). patients with cad showed significantly increased plasma concentrations of cd 62p (hs: 98+20 versus sap: 133+38ng/ml, p<0.05; versus uap: 128+28 ng/ml, p<0.01; versus ami: 144+72 ng/ml, p<0.05) independent of the severity of clinical symptoms. in comparison only patients with ami showed significant higher 8-tg concentrations compared with hs (hs: 30+20 versus ami: 39+14ng/ml, p<0.05). although the cd 62p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between cd 62p (r=0.47; p< 0.001; n=55) to the severity of cad classified as i, 2, 3 vessel disease. it is concluded that elevated cd 62p concentrations are correlated with the severity of cardiovascular disease. cd 62p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. the involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased 13-tg concentrations. iklinik nr herz-, thorax-und herznahe gef&schirurgie und 2institut x~tr klinische chemie und laberatodumsmedizin der universint regensburg an increased blood loss following surgery with extracorporeal circulation (ecc) contributes to the morbidity and mortality. postoperative haemorrhage following ecc has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. we investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ecc. g0 patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. on the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. the surface expression of glycoprotein (gp) lib-ilia, gp lb, and p-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (adp) using whole blood flow cytomet~y. platelet counts and platelet factor 4 (pf4), as well as, routine clotting tests were performed. activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-iii complex fiat), fibrinogen fig), d-dimers (dd), cc2-antiplasmin (tz2a), prothrombin fragment 1+2 (fl+2),and tissue plasm~ activator (t-pa). blood loss fxom chest tubes was measured hourly until removal of drains. following ecc the levels of pf4, tat, dd, o~2a, fl+2, and dd were sigulticnatly increased (p<0.0001) compared to baseline values. gp iib-iila, gp ib, p-selectin, platelet count, and fg were significantly reduced (p<0.0001). analysis of variance (anova) revealed that postoperative values of gp ib (p<0.0001), dd (p<o.05), fg (p<0.01),and platelet count (10<0.05) had a significant influence on blood loss. none of the preoperative data was significantly associated with increased blood loss. a subgroup of patients who bled more than the mean + 1 sd (14 cases) was formed_ in logistic regression analysis gp ib expression (p=0.01) and dd (p=0.007) were of value in identifying bleeders. the sensitivity of the equation was 56%, the stx~ificity 97% and the positive predictive value 82%. although the reasons for an increased blood loss following ecc are multifactorial, we were able to identify a small number parameters with a significant infl~nco. the combination of the expression of yon willebrand receptor on the platelet su_rfaco and d-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. this may be helpful to define indications for platelet transfusion and flesh frozen plasma. the most widely distributed receptor for the fc-region of igg is the fcreceptor iia (fcyriia). for this receptor a functionally relevant polymorphism of amino acids argmhis is known. we previously showed hit-antibodies to bind to platelets via feyriia and now whether clinical manifestation of hit is associated with the fe'/riia polymorphism. therefore, we developed a rapid two step allelle-specific pcr (asp) method for genotyping the foyriia. with control samples the asp was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of fctriia (using the moabs iv.3 and 41h16) and the monocyte stimulation assay. complete concordance between the assays was found. we used the asp to determine the allelic distribution of the inst. physiol. chem., marburg; *)univ. of virginia, charlottesville, the functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). in order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via g i-and gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. furthermore, we analyzed the adp-ribosylation of rhoa, which is a lowmolecular weight gtp-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. aggregation was enhanced in low-density platelets (sp i) and less inhibited when cgmp was increased by no, compared to high-density platelets (spiii). sp i possessed a lower basal level of cgmp and exhibited a smaller increase in cgmp after stimulation with no. cyclic amp-dependent phosphodiesterase activity was higher in sp i compared to spiii, whereas cgmp-dependent phosphodiesterase activity was similar in sp i and sp iii. sp i, when compared to sp iii platelets, showed a higher degree of prephosphorylation of proteins in the range of 47, 32-34, and 20-22 kda. after thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. pertussis toxin,induced adp-ribosylation of the 40-41 kda gi-protein was highest in sp i, whereas cholera toxin induced adpribosylation of the gs-protein and botulinum c3 exoenzyme-induced adpfibosylation of rhoa were highest in sp iii. the findings suggest that a) the higher aggregability in sp i compared to sp iii may be caused by a poorer ability of guanylate cyclase to form cgmp and a higher activitiy of campdependent phosphodiesterase and that b) the adp-ribosylation of g-protein subunits and rhea may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of sp i for aggregation. 1) opper et al., atherosclerosis, 113, 211-217, 1995. causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (niddm), known to be associated with increased plasma activity of plasminogen activator inhibitor type 1 (pal-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. this study was designed to determine whether proinsulin and insulin increase pal-1 in vivo thereby potentially accelerating atherogenesis. to simulate hyper(pro)insulinemia seen with niddm, we infused equimolar concentrations of insulin (1 iu/kg), proinsulin, c-peptide, or placebo, all endotoxin-free, i.v. over 1 h in 53 conscious rabbits maintained euglycemic. plasma pal-1 did not increase with placebo (n=16) or c-peptide (n=4), but did with proinsulin and insulin peaking in 3 h at 3.5-and 3.7-fold (n=16 and n=17, p<0.o02 for each) in parallel with pal-1 protein quantified by reverse fibrin autography. the increases were specific as reflected by unchanged activities of o~-antiplasmin and t-pa. pat-1 gene expression (pal-1 mrna) increased with proinsulin and insulin by 2.1-and 2.1-fold in 3 h in aorta, 1.9-and 2.4-fold in liver (p<0.05 for each), but not in heart, lung, spleen, or kidney and returned to baseline in 24 h (n=4 each). in situ hybridization localized the increased pal-1 mrna in aorta to endothelium (n=3 each). thus, both proinsulin and insulin directly augment gene expression of pal-1 in arterial endothelium in vivo, thereby increasing plasma pal-1 activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. the sarco-endoplasmic-retikulum ca+÷atpases (serca) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. recent studies in platelets and rat endothelial cells have identified two forms belonging to the serca 2b and serca 3 types. the monoclonal antibody pl/im 430 raised to human platelet intracellular membranes has been shown to recognise the serca 3 ÷+ isoform and to inhibit ca sequestration in human platelets. the aim of this study was to identify ca atpase distribution in human umbilical vein endothelial cells (huvec) and human polymorphonuclear leukocytes (pmnl). polyclonal antibodies with known specificity towards serca 2b (r 2/88) and serca 3 (89/3) in addition to pl/im 430 were used in histochemical studies of permeabilised cells and in western blotting experiments using cell lysates and mixed membranes. using the alkaline phosphatase anti-alkaline phosphatase (apaap) method r 2/88 and 89/3 reacted strongly with proteins in permeabilised huvec and pmnl, however pl/im 430 showed good staining only with pmnl. in western blotting experiments r 2/88 recognised proteins with molecular sizes of 100 kda (known to be the parent ca++atpase) and 70 kda (which may represent antibody recognised degradation product), in both huvec and pmnl. 89/3 ÷+ recognised bands at 100 kda (parent ca atpase), 74 kda and 46 kda in both huvec and pmnl membranes. in agreement with histochemical results pl/im 430 recognised proteins (100, 74 and 46 45 + + kda) only in pmnl membranes. in ca transport studies pl/im 430 ÷+ inhibited ca uptake in membranes prepared from pmnl but not in huvec. the distinct reaction of pl/im 430 with pmnl and huvec suggests the possible presence of distinct sub-isoforms of serca 3 with the pmnl and platelet proteins being similar and different to the serca 3 isoform present in huvec. microvascular endothelial cell (ec) lines with a limited life span of 2-3 months in vitro were established from human bone marrow, skin or solid tumor tissue. homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. the cultured ec constitutively expressed hla-class i antigens, cd44, procollagen type i, cd62e, cd34, and receptors for granulocyte colony stimulating factor (g-csf). endothelial cells constitutively produced il-6 and il-8 as well as tpa and pai-i as determined by elisa. in the presence of 5 iu/ml of exogeneous il-la, g-csf was secreted at high amounts (lng/ml) and gm-csf at low amounts (15pg/ml) in confluent monolayers and during over night incubation. these ec potently bound to isolated neutrophilic granulocytes. when granulocytes were stimulated by either 10~ 4 or 10 .7 m formyl-methionyl-leucyl-phenylanaline (fmlp) to produce oxygen radicals, the preincubation with a single cell suspension of ec caused oxygen radical scavenging. a ratio of 1 : 100 ec to neutrophilic granulocytes resulted in a 50 __+ 10% inhibition of fmlp-induced and luminol-enhanced chemiluminescence in vitro. this effect was probably due to the constitutive nitrogen oxide (no) production by ec. unexpectedly, preincubation of ec with 50 iu/ml of interferon-y (ifn-y) to increase no-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of ec to neutrophilic granulocytes. according to the surface antigen expression pattern, identical ifn-f treatment upregulated adhesion receptors on ec. concomitantly, binding of neutrophils to ec was significantly increased. thus, the net oxygen-scavenging effect by ec appears to be stable under the condition of ifn-y stimulation which indicates maintenance of ec function and effective neutralizition of non-specific oxygen radical damage. moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on ec function in vitro. atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (smcs), by macrophage infiltration and extracellular matrix remodeling. circumstantial evidence suggests that urokinasetype plasminogen activator (upa) may be relevant in these processes. this study examines upa expression and upa antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. representative segments of macroscopically normal areas (mnas), early lesions (els) and fibrous plaques (fps) were processed in parallel for in situ hybddization and immunohistochemical analysis. in mnas, upa was detected both in the luminal endothelium and in celocalization with ~-actin-positive smcs throughout the tunica media. in els, upa mrna and antigen were substantially reduced in the luminal endothelium. however, cellular upa expression was detected in the subendothelial area in colocalization with tissue-infiltrating cd68positive macrophages. in the deeper intimal and in the medial layers strong upa expression was observed in association with ~-actinpositive smcs, with most of the upa-positive cells being located in areas with a high proliferative activity as detected by pcna staining. in fps, upa mrna was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. in addition, upa antigen was identified in acellular areas of the necrotic core regions. compared to the diseased intima of these lesions, the adjacent media stained only weakly for upa. in conclusion, this study demonstrates an enhanced expression and synthesis of upa by intimal smcs and macrophages in advanced atherosclerotic lesions of human coronary artedes these findings suggest a role for upa in the progression of the coronary atherosclerosis. adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. it was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. when confluent human umbilical vein endothelial cells were incubated with adenosine (0.01-10mm) a significant dose dependent decrease in plasminogen activator inhibitor type-1 (pai-i) antigen production by this cells was seen as compared to control. this effect by adenosine was also time dependent and seen after 2 hours of incubation with adenosine. as determined by elisa, pai-i antigen in conditioned media of endothelial cells incubated with 10mm adenosine decreased to 57% as compared to control. pai-i antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. these results were also reflected on the level of specific mrna as determined by northern blotting. pai-i mrna decreased in endothelial cells after incubation for 4 hours with adenosine (10mm) to 77% (2.2kb) and 85% (3.2kb) of control. in conclusion our data demonstrate that adenosine can downregulate the expression of pai-1 in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. this effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. sowohl die effizienz als such die komplikationen einer antikoagulantien-dauerbehandlung sind in erster linie yon der gore und engmaschigkeit der gednnungskontrellen abh~ngig seit 1986 wird die schulung zur gerinnungs-selbstkontrolte in der herz-kreistauf-klinik bad bedebarg in gruppen eingeobt. die theoretische und praktische anleitung erfolgt in 8 stunden durch eine in der patientenschuiung erfahrene arzthelfedn (oder krankenschwested-pfleger) und einen arzt. die praktische schulung wird durch einen patientennahen theoretischen tell erg~nzt. verglichen mit konventionell ~berwachten oral antikoagulierten patienten (ca. 50% der tpz-werte liegen im theapeutischen bereich) erreichten 216 quickwert-selbstbestimmer, daf$ 83,1% yon 14 812 tpz-werten im therapeutischen bereich lagen (tabelle 1 ). schwerwiegende thromboernbolische ereignisse oder blutungskomplikatienen wurden nicht beobachtet. durchschnittlich kontrolliert der quickwert-selbstbestimmer seine gerineungswerte w6chentlich, itmgstens im zweiwochen-abstand. ob sich blutungs-und thremboembolierisiko langfristig senken lassen bei gleichze~iger verbesserung yon prognose und lebensqualit~it, bedarf des nachweises im rahmen einer randomisierten, kontrollierten und prospektiven studie. tabelle i verteilun~l der selbstbestimrnten gednnur ~swerte zeitraum 1986 zeitraum -1988 zeitraum 1986 zeitraum -1990 zeitraum 1986 zeitraum -1992 to evaluate the potential benefit of an optimized oral anticoagulation management with inr measurements performed by the patients themselves, two prospective randomized studies have been completed. the first study (n=60) was performed to evaluate the accuracy of inr self-testing (inr-st) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by inr-st, e.g. the percentage of measurements found inside the target therapeutic inr-range. the 231 parallel measurements revealed a high accuracy of inr-st with a 4.5% median difference between parallel controls of single measurements. in addition, inr-st resulted in a significant increase of measurements inside the target therapeutic range (from 54 to 80%) in the entire group. however, inr-st performed every 4 weeks did not result in a significant improvement, while selfcontrols every week (from 44 to 84%) and every 2 days (from 51 to 84%) did. in a second study (150 patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed inr-st. during a median follow-up of 1.5 years in the group with standard oral anticoagulation management an incidence for bleedings of 10.9% per year and for thromboembolic complications of 3.6% per year was found, while in the group of patients with inr-st bteedings occured with an incidence of 4.5% per year and thromboembolisms with 0.9% per year. it can be concluded that inr-st can be performed with high accuracy by the patients themselves. inr-st results in more frequent controls of the inr and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic inr range. the more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications. the cell biology of tissue factor (thromboplastin) h. prydz, the biotechnology centre of oslo, norway tissue factor (tf,thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.erwin chargaff (of dna fame) first described that tf consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by deutsch and lechner in vienna and by myself in oslo. in 1973 tf was first purified and shown to be an integral membrane protein of mr about 50 kda (this was a slight overestimate,recent studies indicate 46-48 kda). in 1987 four groupes independently cloned tf edna, later also genomic dna. the structure of tf suggested that it was a membrane spanning receptor, with an extracellular part of about 219 amino acids folding into two domains remotely like the domains of the ig-superfamiliy, and more specifically the eytokine receptors. tf is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. however, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize tf. another possible way tu get tf into contact with blood is thruugh trauma, surgical ur otherwise. the fact that tf is inducible has created a lot uf interest in its cell biology. we reported recently that binding of factor viia tu tf un the cell surface elicited a marked increase in intracenular ca spikes, thus rendering the final proof that tf not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: tf has a transmembrane segment and a cytoplasmic tail tf has a high specificity/high affinity ligaud (factor vii and factor x) tf induces an intracellular signal upon ligand binding recent results regarding tf as a receptor and regarding its intracellular traffic will be reported. both f.ix and f.x are activated by the tf/f.viia complex. we have used a tf-initiated model system to examine the roles played by f.ixa and f.xa in initiating coagulation. the in vitro model contained a cellular tf source; plasma concentrations of f.ii, v, viii, ix and x, tissue factor pathway inhibitor and antithrombin iii. we tested the ability of exogenously added f.ixa or f.xa to substitute for tf/viia activity in initiating platelet activation and thrombin generation. f.xa was 10 times more potent than f.ixa in initiating platelet activation, but f.ixa was more potent in promoting iia generation. in the presence of tf/viia, zymogen f.x was required both for platelet activation and iia generation, while f.ix was only required for iia generation. thus, tf/viia-activated f.ixa and f.xa have distinct physiological roles. the main role of f.xa is to activate platelets by producing an initial small amount of iia. the process of platelet activation is much more efficient if this initial iia is produced on the tf-bearing surface. f.ixa, by contrast, enhances iia generation on platelets by providing f.xa for platelet surface prothrombinase assembly. we conclude that activation of both f.ix and f.x by tf/viia is essential for efficient initiation of coagulation. furthermore, f.x activated on the surface of a tf-bearing cell isnot functionally equi.valent to f.x activated on the platelet surface. therefore, f.x activation by tf/viia does not compensate for a lack of f.ix or viii in hemophilia because the f.xa activated by tf/viia is located on the wrong surface for efficient thrombin generation. our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. the x-ray crystallographic structure of the fviia-tf complex located the areas within fviia in contact with tf to the first egflike and the protease domains. high-affinity binding of fviia to tf depends on ca2+ and previous studies suggest that ca2+ binding to a site in the protease domain is required. however, using surface plasrnon resonance we have shown that removal of the n-terminal gla domain and hydrophobic stack (hs) from fviia results in a decreased affinity for tf. in search for the mechanism by which regions in fvua not in direct contact with tf affect the affinity, various techniques have been employed. a plausible model is one in which the gla domain and hs optimize the interaction with tf by affecting regions that mediate the association with tf. using gel filtration we demonstrated that the hydrodynamic radii of des(1-38)-fviia and, especially, fviia decreased upon ca2+ binding, whereas des(1-44)-fviia was virtually unaffected. this suggested that the gla domain and the hs interact in a ca2+-dependent manner with other regions in fviia, presumably the protease domain, making fviia more globular. using cd spectroscopy, we could detect ca2+-induced changes in the secondary structure of fviia, all assignable to an increased helical content of the gla domain. the amidolytic activity of fviia in the absence of tf has a ca2+ dependency that suggests involvement of the gla domain in obtaining full activity. this activity was very sensitive to guanidine hydrochloride (c50<0.5 m) and its quenching corresponded to unfolding of the ca2+-loaded gla domain as measured by changes in the trp fluorescence of fviia. based on these results it is conceivable that the gla domain and perhaps the hs of fviia interacts with the protease domain and that this globular form mediates the initial contact with "it. several studies have indicated a profound role for fvii(a) in arterial thrombogenesis. in the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant fviia (degr-rfviia) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of 650 s -1. native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where degr-rfviia (0.9 -880 nmol/l final plasma concentration) or buffer were mixed homogeneously with the flowing blood. subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized tf and phospholipids was positioned. fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. degr-rfviia inhibited these thrombus parameters in a dose dependent manner with ic50 values between 0.5 and 0.7 nmol/l the plasma levels of thrombin-antithrombin m complexes were inhibited at an ic50 of 0.6 nmot/l since the concentration of fvii and fviia in normal plasma is about 10 and 0.1 nmol/l, respectively, theic50 values indicate that degr-rfviia is a very efficient inhibitor of thrombus formation in this model. thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach. the anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. the design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin inhibitor. extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. brain abnormalities in male children and adoleescents with hemophilia: detection with mr imaging mri of sickle cell cerebral infarction in addition, portions of the preparations were incubated with 1.4nm-gold labelled fibrinegen molecules (fg-au; final concentration 4gp_g/ml) for 10 see at 37°c. the expenments were stopped al~er 3 or 10 rain by glutaraldehyde fixation. serial sections were prepared and examined after silver-enhancement using danscher's (1981) method to visualize the fg-au. hwdid not alter the resting gfp. after .3,rain, activated hw-platelets changed their shape moderately but in contrast to the controls did not aggregate. moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. fg-au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. only a very small number of hwtreated cells had fg-au on their surface and showed internalization. 10 rain after adp most of the platelets in both preparations had retumod to a discospherecytic shape it is confirmed that hw inhibits the platetet aggregation and in addition, 1) the formation of the contractile cytnskeleton as well as 2) the binding and interalization of the used ligand fibrinogen santoso 2 l.institute for immunology and transfusion medicine, emst-moritz-arndt-university greifswald; 2. institute for transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. activation of the coagulation mechanism by adherent monocytes expressing tissue factor (tf) is expected to be involved in the development of transplant atherosclerosis. in this in vitro study the effect of the immunosuppressant cyclosporine a (csa) on monocyte tf expression and the regulation of the tf gene transcription was analysed. csa was shown to inhibit the induction of monocyte procoagulant activity (pca) by decreasing lipopolysaccharide (lps)-induced tissue factor (tf) expression in monocytes. csa exerted a dosedependent inhibitory effect on monocyte tf expression at concentrations (1,5 txmol/l csa: 50 % inhibition) not decreasing the cellular protein synthetic rate. addition of csa during lpsstimulation prevented activation of the nuclear factor (nf) ~b as demonstrated by electrophoretic mobility shift assay. western blot analysis confirmed reduced nuclear translocation of nf-~b in the presence of csa. inhibition of the lps-induced nf-~b activation by csa resulted in a reduced tf gene transcription as demonstrated by rt-pcr-analysis. thus, cyclosporine a prevents tf expression by inhibiting the induction of tf gene transcription in activated monocytes. csa may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. the n-glyean pattern of recombinant human coagulation factors ii (rf-i~ and ix (rf-ix), derived from both transfected chinese hamster ovary (cho) cells and african green monkey (vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. human plasma-derived coagulation factors 1i (hpf-i1) and ix (hpf-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~(2-6)sialic acid. terminal galactose-b(1-4)-n-acetylglucosamine units were detected in hpf-ix. both cho cell-derived rf-]i and rf-ix exhibited complot-type glycosylafion and contained ~2-3)-sialio acid in addition to terminal galactose-b(1-4)-n-acetylglucosamine. vero cell-derived if-ix exhibited a complex-type glycan structure sinailar to that of cho cell derived rf-ix. in contrast, rf-li produced by vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et(2-3)-sialie acid. galaetose-b(1-4)-n-acetylglucosamine structures and a low concentration of ct(2-6)-sialic acid were detected in both micro-heterogeneity fractions of veto cell-derived rf-li. although different in the'tr carbohydrate structures, coagulation factors ii and ix obtained recombinantly from both transformed cho-cells and vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. exposure of tissue factor (tf) to flowing blood, as a consequence of vascular injury, leads to tf/f.viia-initiated coagulation which may play a key role in triggering intravascular thrombosis. in order to evaluate the potential advantages of tf pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit tf antibody (ap-1) and heparin were compared in a rabbit arterial thrombosis model. thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (cfv) of the arterial blood flow. rabbits received intravenously either ap-1, heparin or vehicle, and cfv were monitored on line for 120 min. activated partial thromboplastin time (aptt), and activated clotting time (act) were measured at the end of the experiment control ap-i heparin <lmg/kg* 0.2mg/kg/hr 0.8mg/kg/hr intraabdominallretroperitoneal, cns) on a named-patient basis. later also mild/moderate bleeds, predominantly joint bleeds were included.in 80-90% of the bleeds novoseven was found to be hemostatically efficacious. also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be 94% since rfvlla is proteotytically active only in complex with tf the risk for systemic activation of the coagulation system should be minimal. also very few side-effects have been seen.doses between 75-120 tjg/kg have been used, which initially have to be given at 2 hours intervals. to achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis and an appropriate dose interval ensuring a plasma level of fvli:c well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. acute myocardial ischemia is usually triggered by coronary thrombosis. heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (ua) and myocardial infarction (mi). hirudin (hiq is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment ua and mi. the oasis phase i pilot study conducted in 24 canadian centres, has evaluated the safety, feasibility and efficacy of hir (hbw 023, behringwerke ag, marburg) compared to hep in 601 patients with mi or suspected non q-wave mi. patients (95 % received aspirin) were randomized to a 72 hr infusion of either hep (5000 u bolus followed by 1200 u/hr; n=253), or low dose hir (0.2 mg/kg bolus then 0.10 mg/kg/hr; n=175) or medium dose lair (0.4 ms/ks bolus then 0.15 mg/kg/hr; n=173). at seven days of follow-up the incidence of major bleeds was 0.4 % in the heparin group, 0.6 % low dose hit, 1.2 % medium dose lair (p=ns), minor bleeds were 10.3 %, 17.1% and 19.1% respectively (p---0.008 hep vs med dose); there were no hemorrhagic strokes. the incidence of the primary outcome of cardiovascular death, new mi and severe recurrent ischemia (refractory or severe angina) was 17.0 %, 15.4 % and 8.7 % respectively (p---0.014 hep vs reed dose). there was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. in a 200 patient substudy, levels of thrombin antithrombin complex, fibfinopeptide a and d-dimer were all suppressed to a greater extent by mr compared to hep. these results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. full clinical and coagulation resuks will be presented. hat is caused by an immunologic response to heparin. affected patients need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory confirmed hat (hipa-test) with r-hirudin (behringwcrke ag, marburg). treatment regimens were: ai acute hat: 0.4 mg/kg b.w., continuous infusion 0a5 mg/kg b.w/h (51 patients); a2 acute hat with thrombolysis: bolus 0.2 mg/kg b.w., continuous infusion 0.1 mg/kg b.w./h (5 patients); b: proph, treatment: continuous infusion 0. i mg/kg b.w.pa (18 patients); c: during cardio-pulmunary bypass. primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. secondary objectives were the incidence of new thromboembolic complications (tecs), major hemorrhage, limb amputations and deaths. results were compared with a historical control of 120 ix-at patients, all confirmed by the hipa test, but treated before rhirudin was available (danaparoid, marcumar, no anticoagulation). primary efficacy analysis: respunders ai: 74%o, a2: 60%, b: 44%, c: 50%, total: 65%. all adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. proportion of patients with event in the obse~wation period; death: a1 5.9%, a2 0%, b 16.774, c 0%, total: 7.3%, during rhirudin treatment: 1.2%; new tec: ai 5.9%, a2 0%, b 27.8%, c 0%, total 9.8%, during rhirudin: 2.4%; limb amputation: ai 3.9%, a2 0%, b 5.6°/0, c 0%, total: 3.7%, during rbirudin: 3.7% eaapoiat we conclude, that r-hirndin is an effective anticoagulant in hat typa ii patients allowing rapid re, coved" of platelet counts. compared to a historical control the r-hirudin group had a strongly reduced incidence of combined endpuint of mortality, limb amputation and new tecs. major bleedings occurred slightly more frequent than in the historical control. however, the dosage used in regimen b might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only 43% and concomitantly had the higl)est incidence of new tecs. key: cord-005497-w81ysjf9 authors: nan title: 40th international symposium on intensive care & emergency medicine: brussels, belgium. 24-27 march 2020 date: 2020-03-24 journal: crit care doi: 10.1186/s13054-020-2772-3 sha: doc_id: 5497 cord_uid: w81ysjf9 nan ventriculostomy-related infection (vri) is a serious complication in patients with hemorrhagic stroke. in such patients, diagnosis of vris is complicated by blood contamination of csf following ventricular hemorrhage. we aimed to evaluate the diagnostic potential of white blood cells count (wbc), c-reactive protein (crp), and procalcitonin (pct) to identify vris in patients with hemorrhagic stroke during the time of external ventricular drain (edv) in situ. this retrospective study was conducted at the neurosurgical-icu, university hospital of zurich. a total of 347 patients with hemorrhagic stroke and an external ventricular drain (evd) were admitted over a 6 years period at the icu. of those, 14 patients with vris ("vri"), defined by positive csf bacterial culture and increased wbc in csf (>250/ul), and 115 patients without vris and with serial csf sampling ("no-vri") were analyzed. patients with csfcontamination or suspected vri (negative csf cultures but antibiotic treatments) were excluded. wbc, crp, and pct were measured daily. csf was sampled routinely twice a week or by t>38°c. for the analysis, mean peak values of wbc, crp, pct during the time of evd in situ were compared between groups (t test). data are expressed as mean with ci 95%. results: between groups, wbc and crp were similar (wbc: 15.13 g/l and 14.55 g/ l, p=0.68 and crp: 115.93 mg/l and 129.44 mg/l, p=0.56 in the group vri and no-vri, respectively) ( figure 1, panel a and b ). in the group vri, pct was low and significantly lower than in the group no-vri (0.16 ug/l and 2.61ug/l, p=0.03 in the group vri and no-vri, respectively) (panel c). wbc in csf were similar between groups (710.14/ul and 675.16/ul p=0.93 in the group vri and no-vri, respectively). in this study, serum-inflammatory markers were not able to screen patients with vris. their routine measurement should be carefully evaluated. introduction: central nervous system (cns) infections constitute a potentially lifethreatening neurological emergency. patients admitted to the intensive care unit (icu) usually present with a severe disease and organ failure, leading to high mortality and morbidity. we have performed a retrospective analysis during a 5-year period of patients admitted to a polyvalent icu. clinical, demographic and outcome data were collected to evaluate its clinical impact on the outcome of patients with cns infections. we identified 30 patients with the diagnosis of meningitis, meningoencephalitis and ventriculitis, where the median age was 57,6 years (range 24-80). upon clinical presentation, their most frequent signs were fever (70%), meningeal signs (40%), seizures (30%), and a glasgow coma scale score <8 (66%). all needed ventilation support and 66% needed cardiovascular support. a definitive microbiological diagnosis was achieved on 22 patients and antibiotic therapy was adjusted on 18 of them. most common microorganisms were streptococcus pneumoniae (n=7), listeria (n=5) and pseudomonas aeruginosa (n=4) (figure 1 ). other gram negative microorganisms were detected and lead to more adverse outcomes. meningitis was the cause of admission on 26 patients and on a minority (n=4) meningitis was considered to be a secondary diagnosis on patients admitted for other causes (traumatic brain injury, subarachnoid or intraparenchymal hemorrhage, postoperatively of neurosurgical tumor). patients that eventually died had at least one risk factor (age>65, immunocompromised due to diabetes, corticotherapy, hiv or heart transplantation). patients admitted to the icu were not so aged, but had some comorbidities and risk factors leading to more uncommon microorganisms, increasing the risk of adverse outcomes. this lead to an increase of mortality: 23% in the icu and an overall of 43%. study of selenium levels in unresponsive wakefullness (uws) patients with systemic inflammatory response syndrome (sirs) e kondratyeva 1 , s kondratyev 2 , n dryagina 2 the objective of this study was to evaluate the pharmacokinetics (pk) of levetiracetam (lev) in critically ill patients with normal and augmented renal clearance (arc), and determine if the recommended dosage regimen provides concentrations in the therapeutic range (12-46 mg/l) [1] . a prospective observational study was conducted in a tertiary hospital. six blood samples were taken during a dose interval at steady state and lev was quantified by hplc. a population pk study was carried out. statistical analysis was conducted to evaluate the differences in pk between patients with and without arc. the suitability of drug concentrations was also assessed. results: seventeen patients were included, 13 with normal creatinine clearance (crcl) (80-129 ml/min) and 4 with crcl≥130 ml/min (arc). ten patients received 500mg q12h, one 1000mg q12h and two 1500mg q12h. the data were best fitted to a two-compartment model. figure 1 shows lev concentrations during the dosing interval. mean clearance (cl) was 4 l/h and mean volume of distribution of central compartment (v) was 44 l. interindividual variability was 38 and 61% for cl and v, respectively. no differences were identified between both groups (p>0.05) in pk parameters. no correlation was found between lev cl and crcl. trough levels were below the minimum concentration (c min ) 12 mg/l of the therapeutic range in all patients except 1. furthermore, between 3-5 h 50% of samples were below the c min . conclusions: administered doses were not able to maintain lev concentrations in the recommended therapeutic range. other dosage strategies, such the extension of infusion time with higher doses, could be evaluated in order to obtain a more favourable profile. no correlation between lev cl and crcl was found. the mechanical properties of muscles such as tone, elasticity, and stiffness are often affected in chronic critical ill (cci) patients. a hand-held device known as the myotonpro demonstrated acceptable relative and absolute reliability in a ward setting for patients with acute stroke [1] . the technology works on the principle of applying multiple short impulses over the muscle bulk via the testing probe. the aim of our study is to assess the feasibility of objective measurement of muscle tone in cci patients with neurological dynamics and serum biomarkers. the study included 23 cci patients with neurological disorders (stroke, traumatic brain injury, neurosurgical intervention for brain tumors) with more than a 3-weeks stay in icu. dynamic measurements of the muscle properties were taken on the deltoideus, brachioradialis, quadriceps femoris, gastrocnemius using the myo-tonpro. to identify the leading factor in impaired muscle tone also were measured neurological (s100, nse), inflammatory (il-6), bacterial load (pct) biomarkers using elecsys immunoassay and the serum level of microbial metabolites using gc-ms (thermo scientific). results: all patients were divided into groups depending on positive and negative clinical dynamics. significant differences were obtained in parameters characterizing changes in muscle tone of lower limbs -f gastrocnemius (tone) -15.5 vs 18.5 hz, r quadriceps femoris (the mechanical stress relaxation time) -16.5 vs 13.6 ms (p < 0.01, respectively). some significant correlations between five parameters of muscle tone biomarkers and microbial metabolites were revealed. the results of a quantitative measurement of muscle tone objectively reflect the dynamics of neurological status, which in the future may be promising technique for the personalized approach cci in patients. introduction: changes in hormonal status in patients with unresponsive wakefulness syndrome (uws) remains poorly understood. methods: 275 patients in uws were examined at the period from 2007 to 2017. 152 patients (115 men) with tbi and 123 patients (63 men) after hypoxia. acth, cortisol, tsh, free t3 and t4, sth, prolactin and natriuretic peptide were studied in the period from 2 to 4 months uws. in men, the level of total testosterone, lh and fsh was additionally studied. the obtained data was compared with the uws outcome in 6-12 months (crs-r scale assessment). none of the studied hormones of the hypothalamic-pituitary-adrenal axis were a reliable criterion for predicting the outcome of uws. most often and consistently was revealed a tendency of disrupt the rhythm of cortisol secretion, with higher rates in the evening hours. the average value of sth was higher in men with the consequences of head injury who had recovered consciousness than in those who remained in uws. significant decrease in testosterone levels, regardless of age, was found in patients with a consequence of tbi. mean levels of lh were higher in patients with tbi and hypoxia who remained unconscious than in patients who later restored consciousness. the average level of fsh was higher in patients who had recovered consciousness . the increase of natriuretic peptide level was observed both in patients who remained in chronic uws and in those who restored consciousness. no certain endocrine background, characterising this category of patients was found. violations of some hormones secretion rhythms, in particular, cortisol can be considered usual for uws patients, especially in patients with tbi. therapeutic hypothermia has not been used before our research in chronically critically ill (cci) patients. temperature decrease in neuronal cells is a strong signal that triggers endogenic cytoprotection programs using early response genes expression. our goal is to determine influences of craniocerebral hypothermia (cch) on level of consciousness in cci patients. we examined 98 patients with different types of brain injuries. 54 males and 44 females, mean age 45.56 ±16.03. patients were divided into 2 groups: main group -47 patients (vegetative state (vs) -28, minimally conscious state (mcs) -19), comparison group -51 patient (vs -32, mcs -19), groups were equal on main parameters (severity, functional state, comorbidity). patients from main group received courses of cch, duration -180 minutes, scalp temperature 5-8°с, cerebral cortex cooling up to 32-34 o c, session end was without slow reheating period, and session's amount was set -until signs of consciousness recovery. cortex temperature check done noninvasively by using detection of brain tissue emi in shf-range. consciousness recovery in vs and mcs patients controlled using crs-r scale. results: cch sessions significantly increased level of consciousness in vs and mcs patient groups. in vs patients vegetative state increased until minimally conscious state and mcs +, and in mcs group until lucid consciousness (p <0.05) (figure 1 ). craniocerebral hypothermia is used in chronically critically ill patients for the first time. our research results demonstrated effectiveness of cch as an additive treatment tool in such patients. this let us optimistically determine the perspective of inclusion of cch method in chronically critically ill patient's rehabilitation to increase level of consciousness. despite the clinical benefit of endovascular treatment (evt) for large vessel occlusion (lvo) in ischemic stroke, space-occupying brain edema (be) represents a common complication during the course of disease. routinely, ct imaging is used for monitoring of these patients, notably in the critical care setting, yet novel and easy bed-side techniques with the potential to reliably predict be without repetitive imaging would be valuable for a time and cost effective patient care. we assessed the significance of automated pupillometry for the identification of be patients after lvo-evt. we enrolled 40 patients admitted to our neurocritical-care unit who received evt after anterior circulation large vessel occlusion. we monitored parameters of pupillary reactivity [light-reflex latency (lat; s), constriction and re-dilation velocities (cv, dv; mm/s), and percentage change of apertures (per-change; %)] using a portable pupilometer (neuroptics®) up to every 60 minutes during the first 72 hours of icu stay. be was defined as midline-shift ≥5mm on followup imaging within 3-5 days after evt. we assessed differences in pupillary reactivity between patients with and without be (u-test) and evaluated prognostic performance of pupillometry for development of be (roc analysis). in 32 patients (19 women, 74.3±12.0 years) without be, 1,224 assessments were compared to 207 assessments in 6 patients (3 women, 71.7±11.8 years) with be. on day 1, day 2, and day 3 after evt, patients with be had significantly lower cvs and dvs, and smaller perchanges than patients without be, whereas lat did not differ between both groups. roc-analyses revealed a significant negative association of cv, dv, and per-change with development of be. conclusions: automated pupillometry seems to identify patients at risk for be after evt. a prospective study should validate whether automated pupillometry harbors the potential to reduce unnecessary follow-up ct imaging. the aim of this preliminary analysis is to detect differences between the qualitative and quantitative evaluation of the pupillary function carried out by doctors and nurses of an intensive care unit (icu) of a tertiary level hospital. secondary purpose is to investigate new indications for the use of pupillometry in a population admitted in icu methods: the study has been conducted (currently in progress) at the intensive care unit and ecmo referral center at careggi teaching hospital (florence; italy). the enrolled patients are adult subjects (> 18 years) with alteration of consciousness defined by a glasgow coma scale (gcs) < 9, following a primary brain injury and/or the use of sedative drugs. the studied parameters, obtained with neurolight pupillometer ® (id-med, marseille, france) are analyzed, integrated and visual/qualitative evaluation of the pupil function shows a lower reliability if compared to automated pupillometry. the estimated error in the proper determination of photomotor reflex is 34.9% (p< 0.01). no significant difference is reported between quantitative and qualitative pupillometry in the detection of anisocoria. our preliminary results are compatible with previously reported data [1] [2] [3] , even if there was no difference in anisocoria determination. interestingly, a longer latency period among patients treated with opioids has been observed. other results are still in progress. introduction: due to the dynamic of critical care disease, a rapid bedside, noninvasive and highly sensitive and specific method is required for diagnosis. in this study we set out our experience with trancranial color-coded duplex ultrasound (dxt) [1] . the dxt study identifies cerebral arteries as well as hemorrhagic phenomenon, hydrocephalus, mass-occupying lesions and midline shift. this is the main difference between dxt and conventional transcranial doppler (dtc) which is a blind study and do not provide any image. descriptive, cross-sectional and observational study from december 2018 to june 2019. 21 patients were included. inclusion criteria: neurocritical patients. exclusion criteria: no acoustic window, presence of ultrasound artifacts. data collection was performed. it was used a lowfrequency transducer from 1.5-3.5 mhz with trancranial duplex preset ( figure) . the patterns were defined as normal, vasospasm, high resistance, hypermedia and cerebral circulatory arrest, depending on the cerebral flow velocity, lindegaard ratio (lr) and pulsatility index (ip). results: 12 men (57.1%) and 9 women (42.9%). average age 55.6(20-79). patients diseases: subarachnoid hemorrhage 6, traumatic brain injury 5, av malformation 4, stroke 2, hemorrhagic cerebrovascular accident 2 and mass occupying lesions 2. normal pattern: 10 patients (rel. freq 0.47). vasospasm: 5 patients (rel. freq 0.23). high resistance: 4 patients (rel. freq 0.19). hyperemia: 1 patient (real. freq 0.04). cerebral circulatory arrest: 1 patient (rel. freq 0.04) conclusions: dxt should be part of the routine of neuromonitoring, it allows real time images especially useful in unstable conditions. although it will be needed a large amount of patients to be statistical significant, dxt is useful considering a non invasive study, bedside and it allows early identification of different clinic conditions. introduction: embolization of the draining vein during endovascular treatment of arteriovenous malformation (avm) may result in venous outflow obstruction and hemorrhage. anaesthesiologist can use deliberate hypotension to reduce blood flow through avm which may be somehow helpful to prevent this scenario. adenosine-induced cardiac arrest may facilitate the embolization too. the goal of our study was to improve the results of endovascular treatment of avm using adenosine-induced cardiac arrest. methods: after obtaining informed consent 13 patients (8 male, 5 female) were selected for adenosine-induced cardiac arrest during endovascular avm embolization. main age was 40,8±6 years old. 9 of them were evaluated as iii class asa, 4 as iv. endovascular treatment in all cases was performed under general anaesthesia. propofol, fentanyl, rocuronium were used to induce anaesthesia, then all the patients were intubated and ventilated with parameters to keep etco2 32-35 mm hg. sevoflurane 2,1-2,6 vol% (12 cases) or desflurane 6 vol% (1 case) were used to maintain anaesthesia. hemodynamic monitoring consisted of ecg, pulsoximetry, non-invasive blood pressure measurement. onyx or/and squid were used as embolic agents. ct was performed to every patient just after procedure as well as neurological examination. results: adenosine dosage was 0.875-1.26 mg/kg. time of consequent cardiac arrest was 10-40 sec. there were 10 cases we administered adenosine for 1 time, in one case we had to administer it twice, in one fig. 1 (abstract p014) . circle of willis and pulsed-wave doppler mode of middle cerebral artery -3 times and 4 times in one more case as well. hemodynamic parameters recovered without any particular treatment in all the patients. embolization has been performed in all the cases uneventfully. postoperative ct showed no hemorrhage. nobody from investigated group had neurological deterioration in postoperative period. our study shows that adenosine-indused cardiac arrest is not very difficult to perform method and it can be useful during avm embolization. a major risk factor for stroke is atrial fibrillation (af). to treat af anticoagulation is needed. there are now several anticoagulants available. however, a lack of head to head data as well as the absence of accurate techniques makes it difficult to compare them and measure determine there efficacy. stroke is known to produce an abnormal clot microstructure which is a common factor in many thrombotic diseases. this pilot study aims to use a functional biomarker of clot microstructure (d f ) and clotting time (tgp) to investigate the therapeutic effects of different anticoagulants in stroke and af. we recruited 114 patients (59 af and 55 stroke & af). two samples of blood were taken: before anticoagulation (baseline) and post anticoagulation (6-10weeks) . patients were either given warfarin (31%) or axipaban (69%). d f and tgp were measured and compared before and after anticoagulation. results: warfarin increased t gp (267±56 secs to 332±78 secs (p<0.05)), and decreased d f (1.71±0.05 to 1.65±0.06 (p<0.05)). apixaban increased tgp (235±66 sec to 410±105 sec (p<0.05)) but did not change df (1.72±0.04 & 1.72±0.05). interestingly we found that in the apixaban group tgp significantly correlated (p=0.05) with blood drug concentration levels. in this study we show that d f and tgp can quantify and differentiate between the therapeutic effects of two different oral anticoagulants. showing that warfarin prolongs clotting and weakens the ability of the blood to form stable clots. conversely apixaban prolongs clotting time but does not affect the bloods ability to form stable clots. this shows the utility of the d f and tgp biomarkers in comparing two different treatment options, something no other current marker has proven able to do. where d f and tgp may prove useful tools in a personalized approach to anticoagulation treatment and monitoring in an acute setting. hospital mortality compared to the model with the original hairscore. patients with poor-grade aneurysm subarachnoid hemorrhage (asah) world federation of neurological surgeons (wfns) grades iv and v, have commonly been considered to have a poor prognosis (70-100% mortality). though early intervention and aggressive treatment in neuroicu has improved outcome in the past years, it is controversial because most of the patients left hospital severely disabled. the objective of this study was to investigate the clinical and social outcomes in intracranial aneurysm patients with poor-grade asah underwent different intervention therapies. a single center observational registry of 25 poor-grade asah consecutive patients, defined as wfns grades iv and v, treated at tertiary chilean referral center from december 2013 to march 2019 were enrolled in this study. the clinical data including patient characteristics on admission and during treatment course, treatment modality, aneurysm size and location, radiologic features, signs of cerebral herniation (dilated pupils), and functional neurologic outcome were collected. clinical outcomes were assessed via gose and and sociooccupational outcome, both at discharge and at 6 months. figure 1 ). 20% mortality is less than previously reported, and survivors had a favorable recovery, confirmed with neuro psychological test. poor-grade asah patients in our study shows a more positive outcome than previously considered. prognosis of subarachnoid hemorrhage (sah) is scarce, indeed almost half patients die or become severely disable after sah. outcome is related to the severity of the initial bleeding and delayed cerebral infarction (dci). infection and more precisely pneumonia have been associated with poor outcome in sah. however, the interaction between the two pathologic events remains unclear. therefore, we hypothesized that dci may be associated to pneumonia in sah patients. thus the aim of our study was to analyze the association between delayed cerebral infarction and pneumonia in patients with sah. in this retrospective, observational, monocentric cohort study, patients included in the analysis were admitted in neurosurgical intensive care unit or surgical intensive care unit in the university hospital of brest (france) for non-traumatic sah. primary outcome was diagnosis of dci on ct scan or mri 3 months after sah. multivariate analysis was used to identify factors independently associated with dci. a total of 224 patients were included in the analysis (female male ratio 141/83, median age 57 [47-65] years). multivariate analysis was adjusted on sedation, intracranial surgery, fisher classification of sah severity, pneumonia occurrence and non-pneumonia infectious event occurrence ( figure 1 ). pneumonia occurred in 66 patients (29.5%) and other causes of infections in 45 patients (20.1%). dci was found in 108 patients (48.2%). factors independently associated with dci were pneumonia (or 3.10 [1.41-7.06]; p=0.006) and non-pneumonia infectious events (or 2.50 [1.20-5.39 ]; p=0.016). interestingly severity table 1 (abstract p023). correlation of safety and efficacy markers of thrombolysis and thrombolysis time with distance from stroke centre results expressed as odds ratio with 95% confidence interval of initial bleeding evaluated by fisher scale was not independently associated with dci. dci is independently associated with the occurrence of pneumonia or other cause of sepsis. those results may highlight the need for rigorous approach for prevention protocol, early diagnosis and treatment of hospital acquired infectious diseases in sah patients. introduction: traumatic brain injury (tbi) can have devastating neurological, psychological and social sequelae. increased psychiatric morbidity after tbi has been shown in both adult and the pediatric population. also, critical illness as such is a risk factor for psychiatric problems in youth. our aim was to assess risk factors for later being prescribed psychiatric medication in survivors of intensive care unit (icu)-treated pediatric tbi. we used the finnish intensive care consortium (ficc) database to identify patients 5-17 years of age, treated for tbi in four icu in finland during the years 2003-2013. we examined electronic health records and ct scans and collected data on drug prescription after discharge. we used multivariable logistic regression models to find statistically significant risk factors for psychiatric drug reimbursement. we identified 248 patients of which 46 patients received psychiatric drug prescription (19%) during follow up. the median time to prescription was 14 months after tbi (interquartile range [iqr] 5-31 months). 33 patients received antidepressants, 9 received stimulants and 18 received antipsychotics. increasing age showed a positive association with all drug prescriptions except for stimulants, where an inverse relationship was observed (table 1) . using multivariable analyses, we could not find any admission or treatment related factors that significantly associated with being prescribed psychiatric medications. teenage survivors with moderate disability (glasgow outcome scale [gos] 4) showed high numbers of psychotropic drug utilization (45% received any medication, 36% received antidepressants, 24% received antipsychotics). our data suggests, that the risk of psychotropic drug prescription after tbi depends on factors other than those related to injury severity or treatment measures. the incidence of drug prescription is especially high in patients with moderate disability. the effects of 1-adamantylethyloxy-3-morpholino-2-propanol hydrochloride on the formation of steroid neurotoxicity in rats with brain injury a. semenenko 1 , s. semenenko 2 , a. solomonchuk 3 , n. semenenko 3 depending on the nature of the brain injury and the severity of the victims, mortality in traumatic brain injury (tbi) ranges from 5 to 65% [1] . one of the targets for pathogenetic influence on the course of tbi is the use of pharmacological agents that are able to counteract the negative effects of excess concentrations of glucocorticoids on brain. the therapeutic effect of new pharmacological derivative 1adamantylethyloxy-3-morpholino-2-propanol hydrochloride (ademol) in rats with tbi was evaluated for 8 days. the pseudoperated animals and control group received 0.9% nacl solution and the comparison group received amantadine sulfate. cortisol levels were used to determine the efficacy of the test drugs in tbi. in rats treated with ademol, the level of cortisol in the blood ranged from 179 to 188 ng/ml (p5-p95) and was 2.58-fold lower (p<0.05) compared to control pathology group on the 8 day of therapy. instead, the effect of amantadine sulfate on the level of cortisol in the blood was significantly less than that of ademol. the concentration of cortisol in rats with amantadine sulfate in the blood ranged from 271-280 ng/ml (p5-p95), was 1.73 times lower (p<0.05), compared with the control pathology group, and by 49.2% (p<0.05) exceeded the corresponding value in animals treated with ademol. therapeutic treatment of rats with severe tbi with a solution of ademol, preferably better than rats in the group with 0.9% nacl and amantadine sulfate protect the brain from the formation of steroid neurotoxicity by cortisol (p<0.05). although cerebrovascular pressure reactivity (prx) well correlate to patient's outcome [1] , it requires continuous monitoring and mobile average calculation for its determination. we therefore hypothesized that a simplified model of variation between mean arterial pressure (map) and intracranial pressure icp over the first three days of admission would have been able to predict patient outcome: we call this new parameter cerebrovascular pressure correlation index (cpc). we performed a retrospective observational study of all adult patients with severe tbi admitted to icu from january 2017 to april 2018 inclusive. all consecutive patients with a clinical need for icp monitoring were included for analysis. both for icp and map data were mean value over 2-hours registration, for a total of 12 observations/day, cpc was therefore calculated as the pearson correlation coefficient between icp values (x axis) and map values (y axis), obtaining one single value every 24 hours. variables included in the model (i.e. cpc, cpp, icp, systemic glucose, arterial lactate, paco 2 , icp, and internal body temperature) were collected for the first 3 days since trauma. for the main outcome only the minimum value of cpc fit the regression analysis (p = 0.004). the correspondent roc curve showed an auc of 0.80. the associated youden criterion was ≤0.26 (sensitivity = 0.90; specificity = 0.68). of all the variables considered for the secondary outcome only cpcmin fit the regression model (p =0.03). table 1 reports the median and iqr range for sg and nsg of all the variables considered in the model. this observational study suggests that cpc could be a simplified model of variation between map and intracranial pressure icp over the first three days of admission predicting patient outcome. introduction: impaired cerebrovascular reactivity (car) after traumatic brain injury (tbi) is a marker for disease severity and poor outcome. it is unclear how dynamic changes in body temperature and fever impact car and outcome. we calculated the pressure reactivity index (prx) using the center-tbi high-resolution intensive care unit cohort, as a moving correlation coefficient between intracranial pressure (icp) and mean arterial pressure (map). minute and hourly values of prx and temperature were averaged in patients with simultaneous recording of icp and abp. demographic data was based the core registry (v2.0). linear mixed models were calculated based on minute-by-minute data using r with lme4 v1.1-21 and ggeffects v0.9.0. generalized estimating equation models were used to analyze changes during effervescence (increase of temperature of >1°c within 3 hours). we assessed high frequency physiological data during 567 days of 102 patients admitted to the icu with predominantly a closed injury type (n=94/102). median age was 46 years (iqr 29-62), baseline gcs was 6 (iqr 3-9), and 27% had at least one unreactive pupil. the main measurement site for temperature was the urinary bladder 55/ 102(54%). half of the patients (49/102) developed fever(>1h with mean t ≥ 38.3°c) with a total of 834h fever and a median of 9h fever(iqr 4-24) per patient. of 110 effervescence episodes 30(27%) reached the febrile threshold of 38.3°c which was associated with an increase in prx from 0.09 (±sd 0.25) at baseline (2h before) to 0.26 (±sd 0.3) during the febrile peak (p=0.014) (figure 1-a) . linear mixed models showed a quadratic relationship between prx and temperature (p<0.001) with an increase in predicted prx with febrile and hypothermic temperatures ( figure 1b ). the association of increasing body temperature with worsening of car supports prevention of fever in severe tbi. prospective studies are needed to further differentiate between mechanisms involved (i.e. inflammation) and central autonomic dysregulation. fig. 1 (abstract p030) . the patients with a good 6-month outcome (gose>3) after severe traumatic brain injury showed an increase in root mean square of successive differences between normal heartbeats (rmssd) (compared to baseline 30 -minutes before tracheal succtioning) acute kidney injury (aki) is relatively common in patients with severe traumatic brain injury (stbi) and it can contribute to morbidity and mortality [1] . nephrocheck is a point-of-care urine test that flags two biomarkers that indicate if a critically ill patient is at risk for aki. we investigated the incidence of subclinical aki in patients with stbi. we performed a prospective observational study of all adult patients with severe tbi admitted to icu from january 2017 to april 2018 inclusive. all consecutive patients with a clinical need for icp monitoring were included for analysis. urine samples of severe tbi patients was collected at icu admission from 33 patients to measure nephrocheck (nc) test [igfbp7] x was performed using the nephrocheck® astute1 40 ™ meter. serum creatinine was collected at admission, during the first three days, at icu dismission and 60-days follow up to assess renal recovery. the diagnosis of aki was based on kdigo criteria. hemodynamics, electrolytes, peep, p/f, kind of fluid administered, fluid balance, % fluid overload, length of stay, the sequential organ failure assessment score, injury severity scores and mortality were collected. a total of 15 patients (45%) presented a median nc higher values at icu admission. one patient with positive nc value experienced aki at 24 hrs. the positive nc group had more plasma transfusion (p-value 0.03) and a lower median hematocrit at 24 hrs (p-value 0.013), but similar hospital length of stay (p=0.17) and mortality rate (p=0.80) conclusions: nc at icu admission identifies subclinical aki in tbi patients and it maight be used to predictclinical aki. hemodilution (but not fluid overload) seems to be associated with development of subclinical aki. higher nc at icu admission is not associated with worst longterm outcome in tbi patients. severe traumatic brain injury (tbi) is considered a serious public health problem in europe. partly because of the heterogeneity of tbi, considerable uncertainty may exist in the expected outcome of patients. the international mission for prognosis and analysis of clinical trials in tbi (impact) and the corticosteroid randomization after significant head injury (crash) prediction models are considered the most widely validated prognostic models [1, 2] . however, studies using these prediction models for benchmarking of outcomes have been scarce. we aimed to compare actual outcomes in a tbi cohort of critically ill tbi patients with predicted outcomes in a quality of care initiative in an academic hospital. in this retrospective cohort study, we included consecutively admitted tbi patients to the icu adults of erasmus mc, university medical center, rotterdam, the netherlands between january 2018 and february 2019. we included 87 patients with tbi. 14-day mortality was 25%, sixmonth mortality was 36% and six-month unfavourable outcome was 50%. the impact core+ct+lab model predicted 34% 6-month mortality (vs 35% actual, p=0.89) and 51% unfavourable outcome (vs 50% actual, p=0.9). the 14-day mortality prediction by crash prognosis calculator was 43% versus actual 14-day mortality of only 25% (p=0.01), whereas 6-month unfavourable outcome prediction by crash was 67% (vs. 50% actual, p=0.02) ( figure 1 ). the impact model, although developed more than a decade ago, seemed appropriate for benchmarking purposes in this single center cohort in the netherlands, while crash predictions were less applicable to our setting. introduction: out of hospital cardiac arrest (ohca) continues to be associated with significant mortality and morbidity. centralisation of care has considerably improved patient survival but has resulted in increased morbidity in the form of neurological deficit. accurate neurological prognostication remains challenging incorporating repeated clinical examination and ancillary investigations [1, 2] . data was collected retrospectively and analysed for 96 patients admitted post ohca from october 2018 to october 2019. patient arrest demographics were collected in conjunction with extensive inpatient investigation findings including ct, traditional pupil assessment, pupillometry and eeg. results: 50% of patients survived to hospital discharge. patients presenting in a shockable rhythm continue to have higher survival rates ( table 1) . 53% of patients who received immediate cpr survived to hospital discharge in comparison to 41% of patients who did not receive immediate cpr. 73% of patients underwent non-contrast ct head. 74% of patients had traditional pupillary examination performed on arrival. pupillometry was introduced in december 2018; 31 out of a possible 85 patients had pupillometry during their inpatient stay. eeg was undertaken in 11% of cases. our data shows receiving immediate cpr and presenting with a shockable rhythm remain positive prognostic factors. ct head as a stand-alone prognostic modality is unreliable with 14% of patients who survived to discharge, with intact neurology, had an admission ct head reported as hypoxic brain injury. a new neuroprognostic strategy is required in our unit that adds further certainty to likely clinical outcome. this includes increased use of tests such as eeg and pupillometry and the introduction of biomarkers such as neuron specific enolase, somatosensory evoked potential testing and magnetic resonance imaging. introduction: post-resuscitation care of patients following an out-of-hospital cardiac arrest (oohca) is set out by the uk resuscitation council [1] . this is in line with the european resuscitation council guideline [2] . the aim of this audit was to review compliancy to this guideline at the intensive care unit at the bristol royal infirmary . a retrospective audit was performed over a six-month period in adults who were admitted to the intensive care unit at the bri following an oohca whom later died during that admission (41 patients). the focus was on whether the neuroprognostication and end-of-life (eol) care received was as per the standards set by the uk resuscitation council. the main neuroloical examinations documented were pupillary reflex (100%), corneal reflex (75%) and motor response to pain (100%). 61.5% of patients received an ssep analysis >72 hours post-rosc, 81.5% underwent an eeg and 66.7% had >2 serum neuron-specific enolase measurements recorded. all patients (100%) underwent a ct head during their admission. 5.6% of patients were referred to palliative care during their admission. 22% of patients were prescribed all eol medications. most common prescriptions included alfentanil (90.2%) and midazolam (58.5%). finally, 100% of appropriate patients were referred to be potential organ donors. the audit reflected our local practice and that some parameters were not being maintained as set by uk resuscitation guideline. multiple introduction: the prognostication of neurological outcome in comatose out-ofhospital cardiac arrest (ohca) patients is an integral part of post cardiac arrest care. biochemical biomarkers released from cerebral cells after hypoxic-ischemic injury represent potential tools to increase accuracy in predicting outcome after ohca. currently, only neuronspecific enolase (nse) is recommended in european prognostication guidelines. in this study, we present the release dynamics of gfap and uch-l1 after ohca and evaluate their prognostic performance for long-term neurological outcome in ohca patients. serum gfap and uch-l1 were collected at 24, 48 and 72 h after ohca. the primary outcome was neurological function at 6-month follow-up assessed by cerebral performance category scale (cpc), dichotomized into good (cpc 1-2) and poor (cpc [3] [4] [5] . outcome prognostic performance was investigated with receiver operating characteristics (roc) by calculating the area under the receiver operating curve (auroc) and compared to nse. results: 717 of 819 included patients had at least one serum gfap or uch-l1 value at 24, 48 or 72 h after ohca. gfap and uch-l1 levels were significantly elevated in patients with poor outcome. gfap and uch-l1 discriminated excellently between good and poor neurological outcome at all time-points (auroc gfap 0.88-0.89; uch-l1 0.86-0.87) and overall predictive performance measured by auroc of gfap and uch-l1 was superior to nse (auroc 0.76-0.85) ( figure 1 ). however, the roc at the highest specificities of uch-l1 and gfap overlap those of nse and comparing the sensitivities for uch-l1 and gfap with those of nse for the highest specificities (>95%) revealed higher sensitivities for nse than for uch-l1 and gfap at 48 and 72 h. gfap and uch-l1 predict poor neurological outcome in patients after ohca excellently and with a higher overall accuracy than nse, but both biomarkers perform inferior to nse at specificities over 95% at 48 and 72 h limiting their clinical use to guide decisions on prognosis. blood pressure after cardiac arrest and severity of hypoxicischemic encephalopathy c endisch 1 , s preuß 2 , c storm 3 introduction: blood pressure management in post cardiac arrest (ca) patients ensures sufficient cerebral perfusion to avoid secondary brain injury. in local chain-of-survival improvements affect p-ohca survival [1] [2] [3] [4] [5] . also initial rhythm in p-ohca is an important predictor of survival [1, 4] . little is known about the relationship between initial rhythm in p-ohca and long-term outcome [6] [7] [8] . our aim was to establish the relation between shockable rhythm and favorable long-term outcome in pohca. all children aged 1 day-18 years who experienced non-traumatic ohca between 2002-2017 and were admitted to the sophia children's hospital in rotterdam were included. long-term outcome was determined using a pediatric cerebral performance category score at the longest available follow-up interval. the primary outcome measure was survival with favorable neurologic outcome, defined as pcpc 1-2 or no difference between pre-and postarrest pcpc. the association between shockable rhythm and the primary outcome measure was calculated in a multivariable regression model, adjusted for the pre-defined variables. from the 329 patients included in the 16 year study period 126 (38%) patients survived to hospital discharge of which 99 patients (30%) had favorable neurologic outcome (median follow-up duration of 24 months). the rate of favorable neurologic outcome rose from 17% in 2002 to 52% in 2017 (p < 0.001 for trend) (fig. 1) the odds of favorable neurologic outcome at the longest follow-up duration were significantly higher after a shockable initial and unknown rhythm. secondly, trend analysis showed an increase in aed defibrillation and shorter cpr duration. this was followed, finally, by a rise in rosc, survival to hospital discharge and favorable neurologic outcome rate. low socioeconomic status is associated with worse outcome after cardiac arrest. this study aims to investigate if patients´socioeconomic status impacts the chance to receive early coronary angiography after cardiac arrest. in this nationwide retrospective cohort study, 4011 patients admitted alive after out-of-hospital cardiac arrest (ohca) and registered in the swedish registry for cardiopulmonary resuscitation were included. individual data on income and educational level, prehospital parameters, coronary angiography results and comorbidity were linked from other national registers. in the unadjusted model there was a strong correlation between income level and rate of early coronary angiography where 35% of patients in the highest income quartile received early angiography compared to 15% in the lowest income quartile. when adjusting for confounders (educational level, sex, age, comorbidity and hospital type) there were still higher chance of receiving early coronary angiography with increasing income, or 1.31 (ci 1.01-1.68) and 1.67 (ci 1.29-2.16) for the two highest income quartiles respectively compared to the lowest income quartile. when adding potential mediators to the model (initial rhythm, location, response time, bystander cardiopulmonary resuscitation and if the arrest was witnessed) no difference in early angiography related to income level where found. the main mediator was initial rhythm (figure 1 ). higher income is strongly related to the rate of early coronary angiography after ohca. this finding is consistent when adjusting for known confounders. however, the association between income and early angiography seems to be mediated by initial rhythm. patients with low income more often presents with non-shockable rhythms which lowers the likelihood to undergo early coronary angiography. a. the total amount of mortality as a stacked bar: in light-red the number of patients who deceased at scene, in green the number of patients deceased during admission, in red patients who died after discharge. the grey line is the total number of inclusions. b. the rate of bystander aed use, rate of initial shockable rhythm, rate of less than 15 minutes of cpr and rate of favorable neurologic outcome over time. p for trend significant for bystander aed use, less than 15 minutes of cpr and favorable neurologic outcome. trend analysis performed using binary logistic regression for dichotomous data (and a kruskal-wallis test for non-normally distributed continuous data) effect of simulation teaching of cardiopulmonary resuscitation for nursing v spatenkova 1 introduction: simulation teaching is a modern type of critical care (cc) education. the aim of this study was to assess the effect of simulation teaching of cc on a comparison of final examination in different model levels of cardiopulmonary resuscitation (cpr) after the first (cc1) and third, final cc3. the success rate of cpr was tested in prospective study (2017) (2018) on two groups with a total of 66 students in cc1 and cc3 at the faculty of health studies. three semester of undergraduate nursing simulation education (lectures and training) used the laerdal simman 3g. quality of cpr was evaluated according to 4 parameters: compression depth, compression rate, chest release and time of correct frequency. we tested if cpr quality differed between the two groups. for the compression depth and compression rate parameters, first the conformity of variance was verified and then two-sample t-test. as the chest release and time of correct frequency are recorded as percentages, the wilcoxon rank-sum test was conducted for these parameters. to ensure good resuscitation, all recorded parameters must be properly performed during resuscitation. thus, pivot tables were used to generate statistics and test if the number of correctly performed resuscitation parameters for cc1 and cc3 differ. the compression depth parameter was statistically significantly higher for the cc3 than for the cc1 (p=0.016). there were no differences in compression rate (p=0.210), chest release (p=0.514) and time of correct frequency (p=0.586). it was also tested how many of the parameters were performed correctly by students at cpr. the chi-square test shows the relative frequency of cpr success is higher for the cc3 group than for the cc1 group. at least 3 out of 4 parameters were correctly performed by 13% of cc1 students compared to 28% of cc3 students. the study showed a significant improvement of cpr in the final cc3 and supported the three semester simulation education. changes in blood gases during intraoperative cardiac arrest jj wang, r borgstedt, s rehberg, g jansen protestant hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, transfusion medicine and pain therapy, bielefeld, germany critical care 2020, 24(suppl 1):p049 introduction: blood gas analysis (bga) is a common approach for monitoring the homeostasis during surgery. while it is well known that cardiac arrest (ca) leads to circulatory collapse and disturbances in homeostasis, little is known about changes of blood gas during peri-operative ca. we retrospectively analysed patients ≥18 years who suffered from peri-operative ca during non-cardiac surgery from 01/2014 to 12/ 2018. peri-operative ca was defined as need for cardiac compression during anaesthesia care. collected data included ph, paco2, pao2, return of spontaneous circulation (rosc) and 30-day mortality after ca. within the study period, we observed 56 peri-operative ca (m=35, f= 21; age 69±16) during 62742 anaesthesia procedures (rosc occurred in 38 patients (68%). 30 days after ca, the mortality was 45% (n=17), 23% (n=13) were discharged, and 14% (n=8) still in hospital. 87% (n= 49) of ca patients had an invasive blood pressure monitoring, 52% (n=29) had bga before and 66% (n=37) during peri-operative ca. prior to ca, the average values were: ph 7.3±0.1, paco2 39±8 and pao2 225±107. during ca, the average values were ph 7.2±0.2, paco2 50±17 and pao2 215±138. table 1 shows the distributions of blood gas before and during ca. there were no statistical differences between the groups (ph: p=0.4; paco2: p=0.19; pao2: p=0.21). hypercapnia and respiratory acidosis is common in peri-operative ca. these data suggests inadequate ventilation during peri-operative resuscitation. further studies should focus on its impact on the outcome. ]. comparing cases with and without rosc, there were significant more diagnostics done in the group without rosc but more therapeutic consequences seen in the rosc-group (table 1) . icu-ca is frequent. diagnostics to detect reversible causes of ca were used rarely in icu-ca (44%), even in patients without rosc. notably, diagnostics often had therapeutic consequences particularly in rosc. further studies are required to define standardized diagnostic algorithms during icu-ca. continuous monitoring of cardiac patients on general ward were improved short term survival of in-hospital cardiac arrest uj go 1 introduction: the importance of early detection in the in-hospital cardiac arrest (ihca) is emphasized. previous studies have reported that clinical outcomes are improved if ihca is witnessed, or if a patient admitted to a monitored location [1, 2] . this study aimed to evaluate the association between continuous monitoring and survival of ihca on general ward. a retrospective cohort study of ihca in patients admitted to ward at an academic tertiary care hospital between january 2009 and december 2018 was performed. the primary outcome was return of spontaneous circulation (rosc). the secondary outcomes were 72hour survival and survival to hospital discharge. (table 1) . cardiac patients with continuous monitoring on general ward showed improving rosc and 72-hour survival but not survival to hospital discharge in ihca. in-hospital cardiac arrest is associated with poor outcomes. although steroids are frequently used in patients with septic shock, it is unclear whether they are beneficial during cardiac arrest and after return of spontaneous circulation (rosc). of 369 cardiac arrest patients evaluated, 100 were enrolled. advanced life support was conducted according to the2015 resuscitation guidelines. forty-six patients were randomly assigned to receive methylprednisolone 40 mg during resuscitation, and 54 to receive saline (placebo). after resuscitation, steroid-treated patients received hydrocortisone 240 mg daily for up to 7 days, followed by tapering . there was no significant difference between the two groups in scvo 2 andall the secondary outcomes (p>0.05 for all comparisons). the present study found no significant physiologic benefit of corticosteroid administration during and after resuscitation in hospitalized patients with cardiac arrest. the experiences of ems providers taking part in a large randomized trial of airway management during out of hospital cardiac arrest, and the impact on their views and practice. results of a survey and telephone interviews m thomas 1 introduction: the aim is to explore ems experiences of participating in a large trial of airway management during out-of-hospital cardiac arrest (air-ways-2), specifically to explore: 1. any changes in views and practice as a result of trial participation. 2. experiences of trial training. 3 . experiences of enrolling critically unwell patients without consent. 4. barriers and facilitators for out-of-hospital trial participation. an online questionnaire was distributed to 1523 ems providers who participated in the trial. in-depth telephone interviews explored the responses to the online questionnaire. quantitative data were collated and presented using simple descriptive statistics. qualitative data collected during the online survey were analysed using content analysis. an interpretive phenomenological analysis approach was used for analysis of qualitative interview data results: responses to the online questionnaire were received from 33% of airways-2 study paramedics and 19 study paramedics were interviewed. paramedics described barriers and facilitators to trial participation and changes in their views and practice. the results are presented in five distinct themes: research process; changes in views and practice regardingairway management; engagement with research; professional identity; professional competence. conclusions: participation in the airways-2 trial was enjoyable and ems providers valued the training and study support. there was enhanced confidence in airway management as a result of taking part in the trial. study paramedics expressed preference for the method of airway management to which they had been randomized. there was support for the stepwise approach to airway management, but also concern regarding the potential to lose tracheal intubation from 'standard' paramedic practice. causes of medical care-associated cardiac arrest on the intensive care unit s entz 1 introduction: cardiac arrest on intensive care unit (icuca) following therapeutic interventions is of imminent importance, because the interventions are comparatively predictable and precautions can potentially be taken. this study investigates medical care associated complications that led to icuca. intensive care database was screened for patients ≥18 years who experienced icuca in a tertiary hospital with five icu (two medical, two surgical, one interdisciplinary, with a sum of 71 icu beds) in germany from 2016-2018. icuca was defined as receiving chest compression and/or defibrillation after admission on icu and classified as "medical care associated" if it was preceded by a therapeutic intervention (i.e. induced by medication, bedding procedures, iatrogenic injuries, procedure associated). subgroups included patients with recurrence of spontaneous circulation (rosc) vs. no-rosc and patients with vs. without vasopressor therapy before intervention. there were 125 icuca in 114 patients of totally 14,264 icu patients. medical care associated complications leading to icuca were detected in 28 cases (22%) [incidence 19.6/10,000(ci95 12.3 -26.9)]. icuca following therapeutic interventions occurred because of circulatory insufficiency [n=20(70%)], respiratory failure [n=5(17%)] and airway associated problems [n=3(10%)]. nine of the 28 patients (32%) with care-associated icuca died. table 1 demonstrates therapeutic interventions followed by icuca. care-associated complications were common reasons for icuca. most of events were induced by circulatory insufficiency due to induction of anaesthesia and bedding procedures. further investigations should focus on preventive strategies, such as vasopressor infusion before therapeutic interventions. in-hospital cardiac arrest (ihca) is a lethal event. however, ihca has received less attention than out-of-hospital cardiac arrest (ohca). there have been some studies on ihca; however, there is a lack of information on the evidence and clinical features of ihca compared with information for ohca. we therefore conducted this study to clarify important aspects of the epidemiology and prognosis of ihca in patients with code blue activation. we carried out a retrospective observational study of patients with code blue events in our hospital during the period from january 2010 to october 2019. we obtained information on the characteristics of patients including age and gender, ihca characteristics including the time of cardiac arrest, event being witnessed, presence of bystander cardiopulmonary resuscitation (cpr), initial shockable rhythm, vital signs 1 h or 6 h before cardiac arrest, survival to hospital discharge (shd), and the cardiac arrest survival postresuscitation in-hospital (caspri) score. the primary endpoint was shd. we performed univariate and multivariate logistic regression analyses. a total of 293 code blue events were activated during the study period. finally, 81 patients were included in this study. overall, the shd rate was 28.4%. the median time of cpr was 14 min (interquartile range, 6-28 min). the rate of initial shockable rhythm was 19.8%. there were significant differences in cpr duration, shockable rhythm, and caspri score between the shd group and non-shd group by univariate-logistic regression analysis. caspri score was found to be the most effective predictive factor for shd (or=0.98, p=0.006) by multivariate-logistic regression analysis. our results demonstrated that caspri score is associated with shd in cpa patients with in-hospital code blue events. caspri score in ihca patients would be a simple and useful adjunctive tool for management of post-cardiac arrest syndrome (pcas). peri-operative cardiac arrest in prematurityincidence and causes at a tertiary care hospital between 2008-2018 g jansen, j popp, e lang, r borgstedt, b schmidt, s rehberg protestand hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, bielefeld, germany critical care 2020, 24(suppl 1):p058 the peri-operative care of premature pediatric patients requires special expertise and is therefore reserved for specialized centers. although premature birth is described as a risk factor for peri-operative complications and cardiac arrest (poca) there are no data on its incidence and causality in this particular population [1] . the present study investigates the incidence and causality of pediatric poca at a tertiary care hospital and level i perinatal center in germany. in the anesthesia database of the study center, all anaesthesiological procedures in patients <16 years of age were examined for poca in preterm infants (gestational age <40th week of gestational age) between 2008 and 2018. the peri-operative period was defined between the beginning of anesthesiological care up to 60 minutes after anesthesia and/or sedation. we defined cardiac arrest as the necessity of chest compressions. the perioperative phase and the cause of the poca, gestational age and birth weight were recorded. between 2008 and 2018, 308 (1.3%) of the 22,650 pediatric anesthesiological procedures were performed on 301 premature infants. in total, 10 poca occurred in 9 of these patients (f=6, m=3; average gestional age 208±27 days; average birth weight 1510±747g (incidence 3.2%, ci 96 1.3-5.2%). the time of occurrence and the causes of poca are shown in table 1 . poca in premature babies is rare and has an incidence of 1.3%, which is significantly higher than the non-premature babies. the main causes are problems or complications associated with the respiratory tract and its management, as well as massive hemorrhage. introduction: peri-operative cardiac arrest (poca) in children's anesthesia care is a dreaded event. depending on the country and population, studies describe incidences between 2.9-20.6 per 10,000 children's anesthetics. there are no data on the current incidence of pediatric poca in germany. the present study investigates the incidence of poca at a tertiary hospital and level i perinatal center in germany. in the anesthesia database of the study center, all anaesthesiological procedures in patients <16 years were examined for poca. the peri-operative period was defined between the beginning of anesthesia care up to 60 minutes after anesthesia or sedation. cardiac arrest was defined as the necessity of chest compressions. age, weight, asa status, cause of death and survival after 30 days were recorded. results: 18 poca (median weight was 2525g [q1 7151;q75(14748)]) were observed in 22,650 anaesthesiological procedures (incidence 7.9±4.2 per 10,000 [ci95 4.3-11.6]). table 1 shows the distribution of the individual age groups, incidences and mortalities of poca. peri-operative 30-day mortality was 3 per 10,000 [ci 95 [1] [2] [3] [4] [5] . three children died intraoperatively as a result of hemorrhagic shock, one on the picu as a result of malignant hyperthermia. 30 days after poca, 4 more children had died on the icu due to their underlying disease. poca is a rare event. risk factors are an age <28 days and an asa status ≥ iii. the main cause of peri-operative death in patients <16 years of age is massive hemorrhage, the 30-day mortality is determined by the underlying disease. in-hospital cardiac arrest -predicting adverse outcomes t partington, j borkowski, j gross northwick park hospital, anaesthesia/critical care, london, united kingdom critical care 2020, 24(suppl 1):p060 introduction: cardiac arrest occurs in 1.2 per 1000 hospital admissions in the uk. return of spontaneous circulation (rosc) is achieved in approximately half of resuscitation attempts, but rate of survival to hospital discharge is substantially lower [1] . in our centre, post-arrest care accounts for 6.4% of icu admissions. premorbid social function is purported to affect outcomes, but comorbidity scores are more often used for risk stratification. using a novel social function score alongside an existing comorbidity scale, we aimed to identify trends to inform management of patients at risk of deterioration. a six-month prospective observational study was conducted in a major uk hospital from october 2017 to april 2018. for all adult inpatient cardiac arrests, medical notes were reviewed and data collected on the following domains: patient demographics comorbidities and functional status admission details post-arrest events statistical analysis was performed using student's unpaired t-test. results: 54 cardiac arrests occurred. 85% were in medical patients, with the majority male (63%) and aged over 75 (63%). 89% were emergency admissions, with mean duration of hospital stay pre-arrest 9 days. in 17 cases (31%) sustained rosc was achieved. however, seven of these (41%) were not subsequently admitted to the icu. only six patients (11%) survived to hospital discharge. pre-admission function and comorbidity were worse in patients who did not survive to discharge ( fig. 1 ), but these were not statistically significant in view of small survivor group size. in an increasingly frail inpatient population, a substantial proportion of patients in whom circulation is restored after cardiac arrest are subsequently considered unsuitable for icu admission. given our understanding of inferior outcomes in patients with poor physiological reserve, we encourage early discussion regarding the appropriateness of cpr in selected patients, guided by social function and comorbidity. references: 1. national cardiac arrest audit 2017/18 introduction: there are studies that determine events related to poor outcome in cardiac arrest [1] . in our study, following parametres were determined ohca patients; age median years, asian/europe/syrian, bystander cpr, bystander aed, ems defibrillation, initial cardiac rhythm, prehospital rosc, corneal and pupillary light reflex and day survival. we determineted poor prognostic sign with post-cardiac arrest patients. in this study, we identified the causes of poor outcome in patients with ohca. this was a single-centre, retrospective study. we determined incidence and epidemiological factors including: demographics, initial cardiac rhythm. our study population were non-traumatic ohca. our icu, all ohca patient were evaluated wtih echo, and fluid, inotrope and vazopressor were added according to cardiac performance. results: during our study, 5970 patients who were admitted to intensive care unit between 2012-2019 were screened. 133 of these patients were out-of-hospital arrest and 41 of them were in-hospital arrest. development of cerebral oedema during treatment in hospital remains a poor prognostic sign. the evaluation of initial cardiac ritm is useful to predict neurological outcome in post-cardiac arrest patients. survival after ohca remains low. the evaluation of initial cardiac ritm is useful to predict mortality and neurological outcome in postcardiac arrest patients. basic life support (bls) education and training for school children is active in japan. however, the bls action by schoolchildren may be limited by school rules. this study aimed to analyse the time factors for basic life support performance and outcome in classmatewitnessed out-of-hospital cardiac arrest (ohca) and to investigate how schoolchildren act when they detect ohca. methods: nation-wide database for 1,068 school children cases with ohca and local extended database for 5,478 ems-unwitnessed ohca, both of which were prospectively collected during the period of 2011-2016, were retrospectively analysed. proportion of schoolchildren-detected ohca was low in classmate cases (16.8%, 179/889) in nationwide database and extremely low in all ems-unwitnessed ohcas (1.6%, 88/5,478) in local database. nationwide database analyses revealed that both emergency call and bystander cpr were delayed when a classmate witnessed the ohca case: median, 1 vs. 0 min and 3 vs. 2 min, respectively. classmate-witnessed cases were associated with higher incidences of shockable initial rhythm, aed use and traumatic causes. the rate of neurologically favourable outcome was 19.6% and 12.3%, respectively in classmate-witnessed and other cases: adjusted or; 99% ci, 1.24; 0.63-2.47. of 88 cases detected by schoolchildren in our prefecture, 8 (34%) cases had presumed cardiac aertiology and 12(13.8%) cases were caused by suicide attempts (hanging and fall). school children placed emergency 119 calls as the first action only in 32 (36.4%) cases. emergency calls were largely delayed when school children dialled other numbers or left the scene to seek adult help. school children were rarely involved in bystander cpr (21%) and aed placement (1%). school children are rarely involved in entire bls. emergency calls and bystander cpr are delayed when schoolchildren act to seek help. because schoolchildren detect suicide-related ohcas, psychological care to schoolchildren involved in bls may be necessary. prognostic value of neutrophil/lymphocyte and platelet/ lymphocyte predicting cardiopulmonary resuscitation with spontaneous circulation recovery c li the affiliated suzhou hospital of nanjing medical university, suzhou, china critical care 2020, 24(suppl 1):p063 to investigate the predictive value of peripheral blood neutrophil-tolymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) on inhospital mortality in patients with spontaneous circulation recovery after cardiac arrest. a retrospective analysis was made of 30 patients who recovered from cardiac arrest in our hospital from april 2012 to november 2018 and were admitted to the intensive care unit for more than 24 hours. they were divided into survival group and death group according to the outcome of discharge.the dynamic changes and differences of nlr and plr in 24 hours and 48-72 hours after admission to icu between the two groups were analyzed and compared. multivariate analysis and roc curve were used to explore the predictive value of nlr and plr for in-patient mortality. compared with the survival group, plr in the dead group was significantly lower within 24 hours of admission to the intensive care department (p < 0.05), while nlr in 48-72 hours was significantly higher (p < 0.05). the nlr of surviving group was significantly lower than that of 24 hours (p < 0.05), while the nlr and plr of death group were not significantly different (p < 0.05) from that of 24 hours (p < 0.05). multivariate logistic regression analysis and roc curve showed that nlr of 48-72 h in icu was an independent risk factor for predicting in-patient mortality, and had high sensitivity and specificity in predicting death outcomes. neutrophil to lymphocyte ratio, platelet to lymphocyte ratio can help to judge the outcome of patients with cardiac arrest and recovery of autonomic circulation after cardiopulmonary resuscitation. [2, 3] patients with sofa score > 12 (vs sofa score ≤ 12) had a higher free iron level (35.5 μmol/l vs 16 μmol/l, p = 0.0333) ( figure 1 ). we found a positive correlation between free iron level at h0 and changes of sofa score between h0 and h48 (r= 0.56 ic95[0.08;0.76]). out-of-hospital cardiac arrest is associated with a significant change of plasma free iron level. free iron level at admission is associated with short term outcome. further research is warranted to better determine the significance of such changes. the optimal level of arterial oxygen in the post-resuscitation period is unknown. recent studies show conflicting results in regard to hyperoxia and its association with survival after out-of-hospital cardiac arrest (ohca) [1] . the aim of this trial is to study the association between early hyperoxia after ohca with return of spontaneous circulation (rosc) and 30-day survival. observational study using data from three swedish national registers (i.e. intensive care, cardiac arrest and national patient registries after a successful resuscitation, a systemic inflammatory response occurs, and the c-reactive protein (crp) level represents the degree of inflammation [1] [2] [3] . this study examined the association between increased inflammation and early-onset pneumonia (eop) in patients treated with extracorporeal cardiopulmonary resuscitation (ecpr) after out-of-hospital cardiac arrest (ohca). this retrospective study included data of patients with ohca treated with ecpr admitted to st. luke's international hospital between april 2006 and april 2019. the exclusion criteria were as follows: age < 18 years, therapeutic hypothermia withdrawal due to death or circulatory failure, or sepsis as a suspected cause of cardiac arrest. patients were diagnosed with eop according to clinical signs and symptoms acquired after a hospitalization period of >48 h and within 7 days of admission. the crp levels were measured daily from admission to day 3. we studied 55 patients with a median age of 55 years (interquartile range: 42-65 years). furthermore, 52 (95%) patients were males, and the median time interval from collapse to adequate flow was 51 (42-63) min. all patients received prophylactic antibiotics, and 18 (33%) of them had favorable neurological outcomes (cpc, 1-2). eop occurred in 32 (58%) patients, with a significantly higher crp level on day 3 than that in those without eop (13. 9 categorizing reasons for death after ecpr is important for comparing outcomes to other studies, assessing benefits of interventions, and better define this heterogeneous patient collective. a categorizing for death after cardiac arrest in both in-hospital (ihca) and outof-hospital (ohca) arrests has been proposed in non-ecpr patients by witten et al. here, we adopt this categorization to ecpr patients. single-center, retrospective, cohort study of patients without rosc after ihca or ohca and ecpr between 2010 and 2017. patients with survival below 24 hours were excluded. patients were allocated to one of five predefined reasons for death. results: 231 va-ecmo patients were included (age 58.6±14.3, 29.9% female, 58% ecpr, 30 day survival 42.9%). reasons for death for patients with va-ecmo for shock (survival 53%) and ecpr (36%) were: neurological withdrawal of care (10% vs 25%), comorbid withdrawal of care (18% vs 4%), refractory hemodynamic shock (16% vs 33%), respiratory failure (3% vs 2%), and withdrawal due to presumed patient will (0% vs 1%) ( figure 1 ). the differences in reasons for death among the two groups were significant (p <0.001), driven by withdrawal due to neuroprognostication, comorbidity and hemodynamic instability. categorizing death after va-ecmo into five categories is feasible. there are significant difference between patients with va-ecmo for shock and ecpr. interestingly, only a quarter of patients after ecpr died due to brain damage. introduction: scarcity of potential dead brain donors and the persistent mismatch between supply and demand of organs for transplantation has led the transplant community to reconsider donation after circulatory death (dcd) as a strategy to increase the donor pool. normothermic regional perfusion (nrp) by extracorporeal membrane oxygenation (ecmo) may be the most effective method for preserving abdominal organs in dcd, especially in liver transplantation [1, 2] . a pitfall of this method is its complexity and the unavailability of this resource in some hospitals, especially in regional hospitals, where potential dcd donors may exist. aim of this study is to report the use of mobile ecmo team in controlled dcd. from june 2018 to november 2019 our group has worked as a mobile ecmo team for cdcd outside our center. portable equipment included cannulation material and the ecmo device. the transplant team consisted of 1 transplant coordinator (anesthesiologist-intensivist, ecmo operator and organ extraction supervisor), 1 cardiac surgeon (cannulation), 1 interventional radiologist (cannulation) and one cardiovascular perfusionist (ecmo operator). twenty-five cdcd donations were performed. characteristics of donors and organs retrieved are summarized in figure 1 . from 25 cdcd, 17 livers, 4 lungs, 45 kidneys were obtained. the evolution of grafts and receptors was favorable at day 30 post-transplant. mobile ecmo teams may enable cdcd in hospitals without these resources, thereby increasing the pool of donors and optimizing graft outcomes. what is the useful coagulation and fibrinolysis marker for predicting extracorporeal membrane oxygenation circuit exchange due to intra-circuit thrombus? y izutani, k hoshino, s morimoto, k muranishi, j maruyama, y irie, y kawano, h ishikura fukuoka university hospital, emergency and critical care center, fukuoka-shi, japan critical care 2020, 24(suppl 1):p071 a thrombus formation is one of the most frequent and adverse complications during extracorporeal membrane oxygenation (ecmo) support. previous studies have reported that increased d-dimer is a useful predictor of thrombus formation within the ecmo circuit. the purpose of this study was to identify coagulation/fibrinolysis markers for predicting the replacement of ecmo circuit due to intra-circuit thrombus during ecmo support. fourteen patients who underwent veno-venous ecmo for acute respiratory failure between january 2014 and december 2018 were enrolled. these patients received a total of 125 days of ecmo support. of these, 9 days (times) on which the ecmo circuits were replaced was regarded as the replacement group, while the remaining 116 days were considered as the non-replacement group. the several coagulation/fibrinolysis markers were routinely measured every day during ecmo support. we compared with the levels of these markers between two group to identify the most relevant marker for ecmo circuit replacement due to thrombus. the mean duration of ecmo support was 9±11 days, and the mean number of ecmo circuit replacement was 0.6±1.0 times per patient. ddimer, thrombin-antithrombin complex (tat), plasmin-α2 plasmin inhibitor complex (pic), and soluble fibrin (sf) were significantly higher in the replacement group rather than in the non-replacement group (p < 0.01, respectively). according to a multivariate analysis, sf was the only independent predictor of ecmo circuit replacement due to thrombus. the odds ratio (95% confidence intervals) for sf (10 μg/ml) was 1.2 (1.1-1.3). the area under the curve and optimal cut-off value were 0.94 and 85 ng/ml for sf, respectively (sensitivity, 100%; specificity, 85%). from these results, we concluded that sf may be the useful marker rather than d-dimer for predicting the replacement of ecmo circuit due to intra-circuit thrombosis. inhomogeneity of lung elastance in patients who underwent venovenous extra corporeal membrane oxygenation (v-v ecmo)-a computed tomography scan study rd di mussi 1 , ri iannuzziello 2 , fm murgolo 2 , fd de carlo 2 , e caricola 2 , na barrett 3 , lc camporota 3 , sg grasso 2 1 università degli studi di bari "aldo moro", department of emergencies and organ transplant, bari, italy; 2 università degli studi di bari "aldo moro", bari, italy; 3 department of adult critical care, guy´s and st thomas´nhs foundation trust, king´s health partners, london, uk critical care 2020, 24(suppl 1):p072 in patients with acute respiratory distress syndrome (ards), nonaerated, poorly aerated, and normally aerated regions coexist to variable degrees in lung parenchyma. the recruitment maneuvers aim to reopen collapsed lung tissue. in a theoretical point view, this strategy may also prevent the normal aerated lung tissue hyperinflation [1] . the objective of our study was to evaluate lung characteristics in terms of hounsfield units (hu), volume and elastance before and after a recruitment maneuver. in 37 patients with severe ards who underwent v-v ecmo, computed tomography scans (ct-scans) at 5 cmh 2 o of continuous positive airway pressure (cpap) and 45 cmh 2 o were performed. the same ct image was selected at the two different levels of pressure. the distribution of lung opacities, in terms of hu, was classified using the "ucla" colour coding table (osirix image processing software, geneva, switzerland). correspondent lung regions of about 1020 voxels were selected. the quantitative analysis, in terms of volume air (vair) was performed with maluna software (version 3.17; maluna, goettingen, germany). elastance was calculated as the pressure(cmh 2 o)/ vair (ml) ratio. results: see figure 1 . lung inhomogeneity occurs also after recruiting maneuvers. our data confirm that the elastance of recruited lung regions is higher than the elastance of the normal aerated lung regions at low positive end-expiratory pressure (peep) (baby lung). on the contrary the "baby lung" frequently develops hyperinflation. the unpredictable pattern of distribution of volume after recruitment maneuverers may explain the controversial role of peep during the ards treatment. . formal recommendations on target, timing, and rate of at supplementation are lacking. we conceived this study to evaluate the effect of prolonged at supplementation in adult patients requiring veno-venous ecmo for respiratory failure on heparin dose, adequacy of anticoagulation and safety methods: before ecmo start patients were randomized to either receive at supplementation to maintain a functional at level between 80 and 120% (at supplementation group) or not (control group) for the entire ecmo course. anticoagulation was provided with unfractionated heparin following a standardized protocol [1] . the primary outcome was the dose of heparin required to maintain the ratio of activated partial thromboplastin time between 1.5 and 2. secondary outcomes were the adequacy of anticoagulation measured with anti-factor xa and the incidence of hemorrhagic and thrombotic complications and amount of blood products fig. 1 b) . conclusions: this retrospective analysis was not able to show a survival benefit for additive pp to ecmo support in general. early initiation of pp could be an important factor for improving survival in this setting and should be considered in a randomized controlled trial for further evaluation. cause-specific mortality during extracorporeal membrane oxygenation, a single center review of medical records m panigada, d tubiolo, p properzi, g grasselli, a pesenti fondazione irccs ca´granda ospedale maggiore policlinico, intensive care unit, milano, italy critical care 2020, 24(suppl 1):p075 introduction: mortality during extracorporeal membrane oxygenation (ecmo) settles around 35% and the occurrence of bleeding during ecmo is associated with a high mortality rate. however, cause-specific mortality is rarely reported, probably due to the difficulty of its classification. the purpose of the study was to evaluate the agreement between two expert icu physician in the classification of the cause of death of patients supported with ecmo for either respiratory or cardiac support. methods: two intensive care unit (icu) expert staff physicians independently reviewed the entire medical records of all ecmo patients who died before icu discharge from january 2011 to september 2019 at fondazione irccs ca' granda, milan. they were asked to choose the cause of patient's death among six categories. in case of disagreement, a third expert adjudicated the case. the two reviewers were also asked whether, in their opinion, bleeding during the last 24 hours contributed to death. elso definition of major bleeding [1] during the last 24 hours was also recorded for each patient. results: two-hundred and two patients were supported with ecmo of whom 70 (34.6%) died. most of these patients (n=53, 75.7%) died during ecmo. interrater agreement for cause-specific mortality between the two expert physicians was substantial (k 0.71, se 0.07, p<0.01) of the 14 discordant cases 6 were categorized as refractory respiratory failure and 4 as multiorgan failure and septic shock respectively. the distribution of cause-specific mortality is shown in figure 1 . major bleeding (elso) was present in 27 (38.6%) patients, only in 9 (33.3%) of them bleeding contributed to death according to the reviewers. patients treated with early pp while ecmo showed a superior survival to patients treated with late pp or without pp while ecmo. optimal cut off value for duration of ecmo initiation to first pp was calculated using roc-analysis (auc = 0.789) and the youden-index. highest sensitivity and specificity for beneficial survival were achieved for a beginning of pp in <0.71 days. (log rank=0.018). pp: prone positioning p076 non-invasive mechanical ventilation in veno-venous extracorporeal membrane oxygenation j rilinger, v zotzmann, x bemtgen, pm biever, d duerschmied, c bode, dl staudacher, t wengenmayer heart center freiburg university, department of cardiology and angiology i, freiburg, germany critical care 2020, 24(suppl 1):p076 introduction: veno-venous extracorporeal membrane oxygenation (ecmo) support can be combined with a variety of different non-invasive ways to deliver oxygen to the patient's lung. several positive effects might be linked to this so called "awake ecmo". so far there is little evidence about indications and outcome of this approach. we report retrospective registry data on all ards patients treated with ecmo support at a university hospital between 10/2010 and 04/ 2019. in a systematic review of medical records, we distinguished between patients with invasive mechanical ventilation (imv) from the initiation of ecmo therapy (imv group) and patients that received any kind of non-invasive oxygen supply (non-imv group). a total of 276 patients could be analysed. 16 (5.8%) patients received non-imv ecmo support. patients receiving non-imv ecmo therapy showed severe underlying pulmonary disease and immunosuppression (fig. 1) . these patients had higher rates of lung fibrosis, long-term oxygen therapy, pulmonary hypertension, renal insufficiency and immunosuppression (p<0.05). 12 of 16 patients (75%) required imv during the hospital stay in average 5.3±5.0 [0.8-17.1] days after ecmo initiation. reasons were hypoxia despite of ecmo, insufficient ecmo-flow, insufficient protective reflexes or patient agitation. patients with initially non-imv ecmo support showed a numerical but not significant lower icu and hospital survival (25.0% vs. 45.4%, p= 0.111). non-imv ecmo support was applied in patients with severe underlying pulmonary disease and/or immunosuppression. in a high proportion of patients the ventilation regime had to be switched from non-invasive to invasive. survival in this very selected cohort was low. in this retrospective analysis no evident benefit for a noninvasive ventilation strategy could be found. the high proportion of patients who switched from non-imv to imv therapy underlines the need for rigorous patient selection. intra-hospital transportation on extracorporeal membrane oxygenation (ecmo) -a single centre experience in ireland. z siddique, s o´brien, e carton, i conrick-martin mater misericordiae university hospital, department of critical care medicine, dublin, ireland critical care 2020, 24(suppl 1):p077 the objective of this study is to evaluate intra-hospital transportation of patients on extracorporeal membrane oxygenation (ecmo). it is a retrospective analysis of prospectively collected database, performed as part of ongoing quality improvement initiatives. the setting of this study is an 18-bed, combined surgical and medical adult intensive care unit (icu) located in a 570-bed hospital that serves as the national referral centre for cardiothoracic surgery, heart & lung transplantation and ecmo in ireland. we reviewed 33 months of data (from 2017 to 2019) regarding patients admitted to our critical care unit who required intra-hospital transfer for diagnostic and/or therapeutic interventions. we also compared the data to available local guidelines. results: 23 patients were transported on ecmo on a total of 28 occasions; the most common indication being ct brain (table 1) . ecmo cannulation sites were peripheral in 20 patients, 3 patients were centrally cannulated. median time from start of the transfer until the patient was returned to icu was 50 minutes (range: 35-195). the ecmo console was placed on a dedicated ecmo trolley apart from two occasions where it was placed on the patient's bed. number of staff required for transport was between 4 to 10; with an icu consultant as team leader. ecmo specialist nurses were always present on the transport team. 27 transfers were during normal working hours with 1 happening on a weekend. a total of 12 complications occurred during the transports, of underlying pulmonary disease or status of immunosuppression in ecmo patients without invasive mechanical ventilation which 1 was significant and 11 were not. the significant complication encountered was ventricular tachycardia in a v-a ecmo patient which required electrical defibrillation. no adverse events related to transport were seen following return to icu. in this single-centre study, we have demonstrated safe intra-hospital transport of ecmo patients. the use of local guidelines, appropriate personnel and performance during normal working hours is recommended. a novel approach for flow simulation in ecmo rotary blood pumps a supady 1 , c benk 2 , j cornelis 3 , c bode 1 , d duerschmied 1 1 heart center freiburg university, cardiology and angiogiology i, freiburg, germany; 2 heart center freiburg university, department of cardiovascular surgery, freiburg, germany; 3 fifty2 technology gmbh, 79108 freiburg, germany critical care 2020, 24(suppl 1):p078 introduction: extracorporeal membrane oxygenation (ecmo) is used increasingly in critically ill patients suffering from acute respiratory failure, cardiogenic shock or cardiac arrest. however, this therapy can have deleterious side effects such as bleeding or clotting complications and hemolysis. these complications are particularly caused by physical stress acting upon the blood components while passing through the ecmo system, especially within the rotary pump. we here present a novel approach to simulate blood flows through rotary blood pumps used in current ecmo systems in order to better understand the genesis of these complications. geometries of the xenios dp3 (xenios ag, heilbronn, germany) rotary pump were reconstructed by ct-scans and manual measurements using computer-aided design (cad). the computational fluid dynamics (cfd) simulation was performed using the software preon-lab (fifty2 technology gmbh, freiburg, germany), which implements a mesh-free lagrangian method requiring minimal preprocessing of the cad data. the geometries are introduced to the simulation model as tessellated surfaces. five operating points have been specified by the rotation of the centrifugal fan and the corresponding inflow and outflow of blood. the blood is approximatively modelled as a newtonian fluid with a density of 1040 kg/m 3 . preonlab allows detailed assessment of the blood flow while passing through the rotary pump including analysis of local flow rates, pressure gradients and shear stress acting upon the blood. dead zones in the fluid flow can be detected which gives reference points for optimizations of the pump design. for the first time, we demonstrate a novel approach for flow simulation in an ecmo rotary pump ( figure 1 ). this approach may help better understand hemodynamics within the extracorporeal system to define optimal operating points or re-design components aiming to limit hemolysis, coagulation disorders and bleeding in seriously ill patients. one-year experience of bedside percutaneous va-ecmo decannulation in a territory ecmo center in hong kong km fong, sy au, pw leung, kc shek, hj yuen, sk yung, hl wu, so so, wy ng, kh leung queen elizabeth hospital, intensive care unit, hong kong critical care 2020, 24(suppl 1):p079 when veno-arterial extra-corporeal membrane oxygenation (va-ecmo) support can be terminated, arteriotomy wounds of the patients of are traditionally closed by open repair in the operation theaters. lots of manpower are involved and timeslots in operating theaters are scarce. transport of the critically-ill is risky. successful va-ecmo decannulation using percutaneous device called proglide has been reported and our group had adopted and modified this approach [1] . methods: this is a retrospective study analyzing the one-year experience of bedside va-ecmo decannulation. our institution is a 23-bed tertiary ecmo referral center in hong kong. our first bedside decannulation was performed in november 2018, and since then, this practice had replaced the traditional open repair, unless contraindicated. data from november 2018 to october 2019 were analyzed. in the study period, 39 patients received va-ecmo. 28 survived to decannulation and 25 received bedside percutaneous decannulation. their median age was 59 (52-67). the default arterial catheter size was 17fr, with 15 fr in 3 cases and 19fr in one. five (20%) failed percutaneous closure and they were subsequently surgically repaired without extra corporeal life support (ecls) continues to be associated with high mortality rates. our ability to predict outcome prior to initiation ecls remains limited. here we take a single cell rnaseq approach in an effort to identify novel immune cell types that are associated with-and may contribute to-survival on ecls. whole genome transcriptomic profiles were generated from~40,000 peripheral blood monocytes obtained from 38 patients at the time of cannulation for veno-arterial ecls (va-ecls). within each subpopulation, differential gene expression analysis was performed to identify new markers associated with survival. findings were validated in a additional cohorts by flow cytometry. surviving patients had significantly higher proportions of cd8 + nkt cells (cd3 + /cd8 + /cd19 -/cd56 + ) that were cd52 + (p = 0.001, fdr < 0.05) ( figure 1 ). to validate this observation, we performed fc analysis of a second cohort of 20 patients. for each patient, we quantified the proportion of cd8 + nkt cells that were cd52 + . using the median proportion as the cutoff, we again found that a high proportion of cd52 + cells among cd8 + nkt cells was predictive of 48 hour survival (p=0.024). we noted that while high levels of cd52+ cells among the cd8+ nkt cells was protective in this cohort of va-ecls patients, this relationship did not hold for patients with sepsis. as only a few the va-ecls patients were septic, we analyzed a third cohort of septic ecls patients. we observed that high levels of cd52+ cells among the cd8+ nkt populations was not protective in this population. the proportion of cd8+ nkt cells that are positive for cd52 is predictive of survival among patients undergoing va-ecls for noninfection related indications. introduction: the use of calcium sensitizers has grown enormously in the last decade, probably due to their interesting pharmacodynamic properties. levosimendan (ls) is frequently administered in patients under mechanical circulatory support. we performed a retrospective evaluation of patients treated with ls prior to weaning from mechanical support. this evaluation was combined with a review of the literature. a query of our icu patient data management system revealed 22 patients receiving ls prior to or during vad/ecls support. outcome data were obtained from the patients medical records. of our 22 patients, 78% was successfully weaned off ecls. fourteen patients (63 %) died before being discharged of whom 5 while on ecls support. of the weaned patients, 9 died afterwards. 4 of the converted patients needed subsequent veno-venous ecls support for right ventricular support after the implantation. survival to discharge ratio for the whole group was 31 %. more detailed demographic results can be found in table 1 . a pubmed search using the terms "(ecmo or ecls) and ls and weaning" resulted in 7 publications which dealt specifically with weaning of ecls support. several weaning approaches are available, however poor outcome has remains a problem. some recent studies show a possible beneficial effect of ls infusion prior to weaning from ecls. however most of these studies are retrospective or observational at best. because ls is primarily reserved for the most severe cases, outcome interpretation is difficult. overall weaning success ranges from 82%-92% and variation is very dependant of inclusion criteria. the calcium sensitizer ls can be used when weaning off patients from ecls, certainly given its low incidence of complications. future, large randomized trials are however needed in order to confirm this strategy. cardiogenic shock is well described in newly diagnosed pheochromocytoma, and crisis may be precipitated by hemorrhage into tumour. v-a ecmo represents a rescue therapy in a subset of these patients refractory to medical management, facilitating cardiac recovery and subsequent definitive surgery. consent to publish: written informed consent for publication was obtained from the 2 patients. during a spontaneous breathing trial respiratory mechanics can worsen, and respiratory muscle effort can increase, leading to respiratory muscle fatigue, pump failure, hypercapnia and an unsuccessful weaning from mechanical ventilation. this case report discusses the possibility of applying extracorporeal co 2 removal (ecco 2 r) to reduce respiratory muscle effort in a liver transplant recipient who already failed three weaning attempts from mechanical ventilation. the ecco 2 r membrane lung was integrated into a conventional renal replacement therapy circuit and blood flow was increased from 150 to 300 ml/min. measurements of respiratory mechanics (including esophageal pressure, as shown in fig. 1 ) were used to assess the reduction of respiratory effort before and during the application of ecco 2 r. was delivered through a 13fr-double-lumen-cannula; 350 ml/min blood-flow with 10lt oxygen sweep-gas-flow and aptt 1.5-2 baseline were maintained (iv-heparin). in all cases respiratory and metabolic parameters improved without complications ( figure 1 ). ecco2r-crrt facilitated extubation (4 out 9 imv pts). in 4 out of 5 pts at risk of niv failure, it avoided imv. treatment mean duration was 73±31 hours, mean lenght of icu stay was 6±4 days. all patients survived to the treatment, nevertheless 2 patients died due to irreversible multiple mof. in our aecopd series prismalung®-prismaflex® facilitated weaning from imv and avoided intubation in patients at risk of niv failure without complications. these positive results may be related to minimal invasiveness of the low-flow device used and may constitute the rationale for a larger randomized controlled trial. consent: written informed consent for data publication has been obtained. extracorporeal the primary outcome findings from the supernova trial [1] demonstrated that the use of extracorporeal carbon dioxide reamoval (ecco 2 r) allows a reduction in tidal volume (tv) to ultraprotective levels (≈4 ml/kg predicted body weight or pbw) during mechanical ventilation in ards patients without significant increases in the arterial partial pressure of carbon dioxide (paco 2 ). unfortunately, it was not feasible to directly measure ecco 2 r rates during the trial. we used a mathematical model of whole-body oxygen (o 2 ) and carbon dioxide (co 2 ) transport and biochemistry [2] to calculate ecco 2 r rates that permit a fit to the data reported for hemolung (alung technologies) and ila (novalung)/cardiohelp (getinge) devices in the supernova trial [3] . the mathematical model was calibrated under baseline conditions where patients were mechanically ventilated at a tv of 6 ml/kg pbw in the absence of an ecco 2 r device; the o 2 consumption rate, co 2 production rate and pulmonary shunt fraction were adjusted to match the measured baseline arterial partial pressure of o 2 and paco 2 . assuming all baseline parameters were fixed, tv was then reduced to 4.1 ml/kg pbw and the mathematical model predicted the ecco 2 r rate to the change in the paco 2 level. model predictions for the devices are shown in table 1 . these predictions suggest that ecco 2 r rates for ila/cardiohelp devices were approximately twice those for hemolung devices during the supernova trial. these results may be useful to evaluate the expected performance of novel ecco 2 r devices. efficiency and safety of a system crrt plus ecco2r to allow ultraprotective ventilation protocol in patients with acute renal failure f maldarelli 1 despite renal function replacement techniques (crrt), a patient who develops acute renal failure(aki) in intensive care unit (icu) has a mortality rate of 5-80%. this risk is partly due to the adverse effect of aki on other organs than the kidney. respiratory complications are frequently associated with the development of aki. new machines combining crrt with a carbon dioxide removal membrane (ecco2r) allows the setting up of an ultra-protective ventilation (4 ml/kg of predicted boby weight (pbw)) to reduce any lung damage from mechanical ventilation (mv). the reduction in tidal volume (vt) is associated with a decrease in lung damage partly triggered by aki. we evaluated the efficacy of a combined system crrt+ecco 2 r to reduce the vt to ultraprotective values in patients with acute respiratory failure and aki. ards is a syndrome with high morbidity and mortality. an emerging treatment option is ecco2r, but the benefit its remains unclear. we assess different degrees of ecco2r and varying dead space (ds) on ventilator settings in order to minimize mechanical power. we calculated mechanical power as (1) power=rr*{δ〖vt〗^2*[1/2*el+rr*(1+i:e)/(60*i:e)*r]+ δvt*peep} (el: system elastance, r: airway resistance, peep: positive end expiratory pressure, i:e: inspiratory to expiratory ratio). we calculated the combination of respiratory rate (rr) and tidal volume (vt) ("optimal rr" and *optimal vt*) leading to minimal applied power for a stable carbon dioxide elimination of 300 ml/min (vco2) for two scenarios: 1) variation of physiological ds from 10 to 40 % of vt at a fixed rate of eccor2. 2) variation of ecco2r of either 80, 120, 160 or 200 ml/min at a fixed physiological ds of 20%. the alveolar ventilation (va) necessary to eliminate the vco2 was calculated as (2) va= (-vco2*σ_co2*r*t*(1+k_c ))/(vco2/q-p_vco2*σ_co2*r*t*((1+k_c ))/760) σco2: co2 solubility in blood, r: gas constant, t: temperature. pvco2: venous partial pressure, kc: function of ph (12.5 for a ph of 7.2), q: blood flow [5 l/min]). increasing ds from 10 to 40% increases the minimal mechanical power from 5.9 to 10.8 j/min, primarily caused by an increase of optimal vt (495 -672 ml). optimal rr was only slightly increased (6.4 -7.5 /min, figure 1 panel a). for varying ecco2r removal, necessary ventilation ranges from 1.6 to 3.6 l/min. this predicts a minimal power between 5.6 and 10.4 j/min with an unchanged optimal vt (540 -543 ml) and an increasing optimal rr (5.4 to 12.3 /min ( figure 1 panel b)). in order to minimize mechanical power, increasing shunt or co2 production should be met with increases in rr while increases in ds should be met with increases in vt. our results indicate that during ecco2r, mechanical power and thus risk for lung injury can be minimized with higher vt compared to conservative ventilation strategies. validity of empirical estimates of physiological dead space in acute respiratory distress syndrome jd dianti, eg goligher, as slutsky university of toronto, interdepartmental division of critical care medicine, toronto, canada critical care 2020, 24(suppl 1):p091 increased physiological dead space fraction (v d /v t ) is a hallmark of the acute respiratory distress syndrome (ards) and has been shown to predict ards mortality. v d /v t is also important in estimating the reduction in tidal volume (v t ) and driving pressure (δp) with extracorporeal co 2 removal (ecco 2 r). v d /v t can be measured with volumetric capnography but empirical formulae using the patient's age, weight, height, gender and paco 2 have been proposed to estimate v d /v t based on estimates of co 2 production (v co2 ). the accuracy of this approach in critically ill patients, however, is not clear. secondary analysis of a previously published trial [1] in which v d /v t and v co2 were measured in ards patients. estimated dead space fraction (v d,est /v t ) was calculated using standard formulae. agreement between methods was evaluated by bland-altman analysis. the predicted change in δp with ecco 2 r was evaluated using both measured and estimated alveolar dead space fraction (v dalv /v t ). results: vd,est/vt was higher than measured vd/vt, with a low correlation between the 2 (r2= 0.21). vco2 was underestimated by the predicted approach (table 1) , accounting for 57% of the error in estimating vd/vt. the expected reduction in δp with ecco2r using vdalv/ vt was in reasonable agreement with the expected reduction using introduction: acute respiratory distress syndrome (ards) is a common condition in critically ill patient. however neuromuscular blockers (nmb) result controvertial in early treatment of ards [1] . we ought to search systematically and realize a meta-analysis on the matter. an electronic search of randomized clinical trials in adult patient treated with early neuromuscular blockers compared without neuromuscular blockers in ards. the primary objective of the analysis was the mortality at 21 to 28 days. secondary endpoints included mechanical ventilation free days, icu acquired weakness and barotrauma. the search obtained 6 studies for the analysis [1] [2] [3] [4] [5] [6] (figure 1 ). the early use of neuromuscular blockers in ards showed no increase in mortality, but the results should be taken with caution. there was no differences in mechanical ventilation free days. barotrauma is less with the use of nmb. ultrasound is fairly sensitive in the detection of lung infiltrates in patients with hematologic malignancies. in patients with pneumonia requiring intensive care (icu) admission, we hypothesise that abnormal right ventricular (rv) function is associated with an increased 90-day mortality. rv dysfunction in critically ill patients has a well-known association with adverse outcomes [1] . however, its impact on mortality in patients with pneumonia has not been directly studied. patients admitted to the queen elizabeth hospital birmingham icu between april 2016 and july 2019 with a diagnosis of pneumonia who had a formal cardiologist tte were included. abnormal rv function was defined by either depressed function, dilated size or moderate to severe risk of pulmonary hypertension (phtn). abnormal lv function was defined by an lv ejection fraction £ 45% or grade ii or more diastolic dysfunction. patients with a clinical suspicion of pulmonary embolism were excluded. the primary outcome was 90-day mortality. continuous data is presented as median (iqr). categorical data is presented as % and analysed using a chi-squared test. results: 942 patients were admitted to icu with pneumonia, of which 347 (37%) had a tte. patients were 59% male, had a median age of 67 (46-88) and 90-day mortality of 31%. abnormal rv function was present in 30% (n=103), with 15% depressed, 15% dilated and 14% with moderate to severe risk of phtn. rv dysfunction was associated with an increased 90-day mortality compared to normal rv patients (62% vs. 18%, p<0.0001). lv function was abnormal in 25% (n=88) and was not associated with a higher 90-day mortality compared to normal lv patients (38% vs 29%, p = 0.20). rv dysfunction was associated with a higher 90-day mortality than lv dysfunction (62% vs 38%, p = 0.001). conclusions: this is one of the first studies to demonstrate that abnormal rv function is associated with an increased mortality in icu patients with pneumonia. interestingly, abnormal lv function was not associated with an increased mortality. rakuno gakuen university, anesthesiology, hokkaido, japan critical care 2020, 24(suppl 1):p097 we previously reported a simple correction method of estimating pleural pressure (ppl) by using central venous pressure (cvp) and that it can be used to estimate ppl and transpulmonary pressure in pediatric patients with respiratory failure. however, it remains unknown that this method can be applied to patients with various levels of chest wall elastance and/or intravascular volume. the objective of this study is to investigate whether our method is accurate in various conditions of chest wall elastance and intravascular volume. the study was approved by the animal care and use committee of rakuno gakuen university. ten anesthetized and paralyzed pigs (43.2 ± 1.8kg) were mechanically ventilated and subjected to lung injury by saline lung lavage. each pig was subjected to 3 different intravascular volume and 2 different intraabdominal pressures; in each condition, the accuracy of our method was tested. specifically, airway flow, airway pressure (paw), esophageal pressure (pes), and cvp were recorded in each condition, then changes in pes (δpes) and δppl calculated using a corrected δcvp (cδcvp-derived δppl) were compared. cδcvp-derived δppl was calculated as κ × δcvp, where κ was the ratio of the δpaw to δcvp during the occlusion test. means and standard deviations of the two variables that reflect δppl (δpes and cδcvp-derived δppl) in all pigs with all conditions were 6.1 ± 4.1 and 6.4 ± 5.3 cmh 2 o. the bland-altman analysis for the agreement between δpes and δcvp showed a bias of -0. 3 the activity and functionality of the diaphragm are difficult to measure in patients ventilated in intensive care. ultrasound can be a useful tool for monitoring diaphragm muscle activity during different ventilation modes. few data currently exist on diaphragm muscle activity in critically ventilated patients [1] . our goal is to evaluate the respiratory muscular work of the diaphragm with different settings of the respirator by means of an ultrasound scan. the ultrasound assessments of the diaphragm were performed with a 10mhz linear probe at the apposition zone. we measured the thickening of the diaphragm with the respiratory acts, through the thickening fraction (thickening fraction, tf), defined as:tf = (tdimax -tdimin / tdi min)% tdimax: diaphragm thickness at the end of inspiration (maximum thickness) tdimin: diaphragm thickness at the end of expiration (minimum thickness). ventilatory support was divided into 4 classes: 1 -spontaneous breathing (sb) or continous positive airway pressure (cpap); 2 -pressure support ventilation (psv) with low pressure support (5-12cmh2o); 3-psv with high pressure support (> 12 cmh2o); 4 -controlled mechanical ventilation (cmv). a total of 223 assessments were performed in 70 patients. the evaluations were all possible at the right hemidiaphragm, while on the left they were not possible in 7% of the cases. the median tf (iq range) of the 4 ventilation classes was respectively: 42% (25-62%) in sb / cpap; 26% (17-31%) in low-psv; 17% (9-22%) in high psv; and 5% (2-13%) in cmv. the kruskal-wallis test confirms a significant difference between the groups (p <0.0001). the ultrasound of the diaphragm can be a valid tool for monitoring respiratory muscle activity during mechanical ventilation. introduction: extubation failure is defined as reintubation after 48 hours of extubation in mechanically ventilated critically ill patients. it is associated with morbidity and mortality. the aim of our study was to assess reintubation rates in a busy district general hospital and evaluate the impact of high flow nasal oxygen therapy (hfno) on reintubation rates. we performed a retrospective observational study looking at patients admitted to our 7 bedded level 3 critical care unit (370 patients a year) for a period of 5 years between 1 st november 2014 and 31 st october 2019. we included patients over 16 years of age who were mechanically ventilated and length of stay was greater than 48 hours. exclusions were age < 16 years, tracheostomy and patients requiring ventilation for < 48 hours. data was collected from ward watcher, a sicsag database and electronic patient records. our study failed to show any impact of hfno on reducing extubation failure. further work is needed to develop a standardized approach to weaning and to consider routine application of noninvasive ventilation to reduce reintubation rates [1] . fig. 1 (abstract p097) . the bland-altman analysis for the agreement between δpes and cδcvp-derived δppl in various conditions. low: low intravascular volume, normal: normal intravascular volume, high: high intravascular volume, abd-: without an abdominal compression band, abd+: with an abdominal compression band oral endotracheal intubation is common to critically ill patients in intensive care unit. oral care for an intubated patient is important to maintain the moisture of oral mucosa. also, the securement method of oral endotracheal tube developed from cloth tape to commercial tube holder. training powerpoint and video for microteaching was prepared to train up 30 icu nurses to perform the new practice. demonstration and re-demonstration was arranged to assess skills of every nurse. afterwards, each nurse answered a quiz to evaluate the understanding of oetth and its special techniques in application. questionnaire was designed to collect the feedback from all nurses too. the result showed there was 21 nurses (72%) out of 30 nurses achieved full marks in the post-quiz which demonstrated their full understanding of the use of oral ett holder and its nursing care. about the feedback from nurse, 72% of nurses claimed that they were confident in using the new oetth in clinical setting after training. 96% of nurses agreed in time-saving of nursing care routine with the use of an oetth. however, only 56% of nurses agreed that the oetth is effective in prevention of oral mucosa injuries and another 24% of nursing staff disagreed on its function in improving the patient's oral care. in conclusion, some of the nurses did not agree the prevention of oral mucosa injuries by the new securement method with oetth while some nurses welcomed the new oetth as more easy and effective in oral care to intubated patients. execution of percutaneous dilatational tracheostomy using the standard laryngeal mask airway for ventilation: a prospective survey study g gagliardi 1 , v gagliardi 2 , c chiani 3 , g laccania 4 , f michielan 3 1 aulss 5 -veneto, anesthesia and intensive care, adria, italy; 2 aulss 5 -veneto, university of padua, adria, italy; 3 aulss 5 -veneto, anaesthesia and intensive care, adria, italy; 4 aulss 6 -veneto, anaesthesia and intensive care, padua, italy critical care 2020, 24(suppl 1):p101 we fulfilled a survey study dealing with bronchoscope-guided percutaneous dilatational tracheostomies (pdt), using the classic laryngeal mask airway (lma) for the airway management [1] . the aim was to verify the safety and the effectiveness of the aforementioned procedure methods: we performed an observational prospective survey study enrolling 150 patients hospitalized in the intensive care unit. before performing the tracheostomy, the endotracheal tube has been replaced by the laryngeal mask airway. arterial blood gases, ventilation pressures and tidal volumes have been monitored, registered and compared. the median peak inspiratory pressure has been detected stable in all patients. furthermore, during the ventilation with the laryngeal mask, the tidal inspiratory and expiratory volume difference observed between before and after the bronchoscope positioning, has shown a statistically significant variation. finally, in all cases etco 2 , spo 2. , pao 2, and blood ph values persisted within the normal range. the standard lma provides for a reliable airway management and allows an effective ventilation while performing the pdt. once positioned in the supraglottic zone, the lma does not need to be moved throughout all the pdt performance, avoiding risks of displacement, glottic harm and airway device damage, and permitting an easy handling of the bronchoscope, which gives an appropriated visualization of the trachea and a more efficient aspiration. in consequence to the large internal diameter of the lma tube, ppeak has continued to be stable in all patients, providing for minor resistance and inspiratory work. eventually, no late complications, such as tracheal stenosis and infections, have occurred. tracheostomies are the most common surgical procedure performed on critically ill patients. randomized control trials comparing tracheostomy timing in intensive care patients have been equivocal. in order to perform non-urgent tracheostomy in our icu, consent is required from the patient or a formal guardian appointed ad hoc by the courts. since tracheostomies are practically the only elective surgery performed in the critically ill, icu requested guardianship almost always indicates a clinical decision to perform tracheostomy. as appointing a guardian and arranging a tracheostomy takes about a week, the decision to appoint a guardian offers a unique "intention to treat" opportunity to evaluate outcomes in patients for whom tracheostomy is planned. we performed a retrospective analysis over 3 years on patients for whom guardianship was sought excluding those requiring urgent tracheostomy and those with a do-not-resuscitate order. patients were divided according to outcome (tracheostomy, extubation or death prior to tracheostomy) and compared. guardianship was sought for 233 ventilated patients. a decision to withhold tracheostomy was made for 13 patients, who were excluded, leaving 220 patients for analysis. tracheostomy was performed for 131/220 (60%) patients, 62/220 (28%) were extubated and 27/220 (12%) died while waiting for tracheostomy (from nonairway related reasons). tracheostomy was performed on mean ventilation day 16±1. comparing extubated patients to those who had tracheostomy (table) shows similar demographics, but significantly lower mortality and hospital length of stay. a significant proportion of patients initially planned for tracheostomy were successfully extubated. despite demographic similarities, mortality in this group was significantly lower than for patients undergoing tracheostomy. for a selected subgroup of possibly difficult to characterize patients, delaying tracheostomy may be beneficial. figure 1 ). ptis were analysed by speciality and by outcome. complications occurred in 6 cases (incidence 6.5%). there were 3 cases of subcutaenous emphysema, 1 pneumothorax (occuring d6 post procedure) and 1 case each of stoma and suture site infection. there was 1 unplanned cannula change within 7 days of insertion. 24% of cases had cuff inflated on discharge from icu. handover of care was suboptimal; follow up care plans were documented in 18% of cases. a supervising consultant was present for all ptis. there was a trend of increased insertion by consultant and increased reliance on theatre, with corresponding decrease in the number inserted by trainees. pti in our training icu appears safe with low incidence of complications and good senior support for tracheostomy insertion. emphasis must continue on training junior intensivists in pti. transition of care beyond icu requires further work where currently there is suboptimal handover of care and safety netting for non-icu colleagues. supplemental oxygen administration is ubiquitous in the critical care environment, yet evidence is mounting for the deleterious effects of hyperoxia [1] . concerns over the adverse effects from hypoxaemia often exceed those of hyperoxaemia in developing world settings, and inconsistent availability of blood gas monitoring may limit judicious oxygen titration. the aim of this project was to audit oxygen delivery practice and introduce qi measures to avoid excess oxygen delivery in a tertiary icu in lusaka, zambia. a prospective snapshot of ventilatory parameters were recorded for critically ill patients over a 5-week period, including positive end expiratory pressure (peep), fio 2 , and time-course spo 2 . systematic education was provided through group and one to one tutorials to empower nursing and medical staff to titrate oxygen safely and appropriately. repeat data collection was then performed over 4 weeks. initially 18/30 patients (60%) were over-oxygenated, as defined by fio2 >0.5 and spo 2 consistently >95%. 12/18 patients with an fio 2 of >0.5 had peep ≤ 5cm (67%). no patient had a pao 2 recorded in the past 24 hours. education was provided as well as implementation of unit protocols above all patient beds documenting a stepwise approach to titration peep and fio 2 . post intervention fewer patients were over-oxygenated: 7/21 (33%) had fio2 >0.5 and spo 2 consistently >95%, and 7/18 with an fio 2 >0.5 (39%) had a peep ≤ 5cm. in addition, 7/21 (33.3%) had a pao 2 recorded within 24 hours. this qi project has shown that nurse engagement and systematic education to titrate fio2 and peep can be achieved in a resource poor setting and may decrease the incidence of hyperoxia in critically ill patients. availability of blood gas monitoring and knowledge of interpretation was a major barrier to oxygen titration tracheal intubation (ti) in adult burn patients might be unnecessary in 30 to 40% of cases [1, 2] . in pediatric burn patients, there is little data on both the rate of ti and the rate of early extubation [3] . it has been common practice for a child with a facial burn and/or a suspected airway injury to be intubated early due to the risk of losing airway patency. however this risk should be mitigated against the potential risks of ti and mechanical ventilation in children. therefore the aim of this study was to describe the airway status of child burn victims taken in charge of in our pediatric burn intensive care unit. focused on patients arriving with ti, we investigated the rate of early extubation. in addition we compared non intubated patients with those with prolonged ti. this retrospective study described a cohort of 1520 patients hospitalized between 2010 and 2018. data was retrospectively recorded from the patient's paper clinical chart. the mean age of our patients was 2.8 ±3.1 years [mean±sd] with an average burn area of 14±11%. 86% had scald burns and 45% had facial burns. 4% of the children were admitted in the burn icu with ti. for 36% of them, tracheal tube was removed within the first 48 hours after admission. the probability of prolonged ti increased independently with the burned skin area (bsa) (p <0.0001), the presence of facial burns (p = 0.001), and in case of flame burns (p = 0.007) ( figure 1 ). among patients with more than 70% bsa, 85% were intubated more than 48h. among patients with less than 20% bsa, 0.5% were intubated more than 48h. according to our retrospective data, it seems appropriate to intubate children with 70% and more bsa, while for patient with less than 70% bsa, it might be relevant to seek guidance from physician of the nearest burn center. under 20% bsa, ti seems rarely required. an analysis of the predictive applicability of initial blood gas parameters for the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury c pirrone 1 , m chotalia 2 , t mangham 1 , r mullhi 1 , k england 1 , t introduction: we hypothesise that initial blood gas parameters have a good predictive applicability in detecting the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury. to the best of our knowledge, this has not been directly studied in the literature. patients with suspected inhalation injury admitted to the icu at queen elizabeth hospital, birmingham between april 2016 and may 2019 were included. the initial blood gas parameters analysed were pao 2 (kpa), paco 2 (kpa), ph, carbon monoxide level (cohb; %) and pao 2 /fio 2 (pf) ratio. receiver operator characteristics (roc) for these parameters were plotted against the need for intubation for more than 48 hours and the presence of inhalation injury as detected by bronchoscopy and laryngoscopy. area under the curve (auc) for each parameter was calculated. results: 85 patients were admitted with suspected inhalation injury to the icu. 68% were intubated for more than 48 hours. of patients who were intubated, 69% had inhalation injury as indicated by bronchoscopy or laryngoscopy. table 1 outlines the auc for initial blood gas parameters in detecting the need for intubation for more than 48 hours and the presence of inhalation injury. ph was the parameter with the most prominent auc, with reverse correlation indicating fair accuracy. no clear inflection point was identified, although all patients with ph < 7.25 required intubation and had inhalation injury. paco 2 had a fair predictive applicability in detecting the need for intubation. pf ratio, pao 2 and cohb had poor accuracy. conclusions: initial blood gas parameters had a broadly poor predictive applicability for the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury. severe acidosis (ph < 7.25) was the most useful blood gas parameter. clinicians should be cautious in using blood gas parameters alone to inform intubation decisions. lung cancer surgery is associated with a high rate of pulmonary complications including ards and mandates lung protective ventilation strategies [1, 2] . such strategies include non-intubated video assisted thoracic surgery (nivats) with spontaneous breathing [3] . currently neither data on respirator settings nor on gas exchange have been reported for applying the latter. this data constitutes a prerequisite for meaningful evaluating the respiratory consequences of non-intubated spontaneous breathing during lung cancer surgery. the aim of this case series was for the first time providing such data from lung cancer surgery including pneumonectomy. during a 12 month period 32 patients without contraindications [3] scheduled for video assisted thoracic surgery (vats) for non-anatomical and anatomical lung resection including one pneumonectomy (px) were offered non-intubated spontaneous breathing. all patients gave informed written consent to the procedure as well as for analysis and publication of data. anaesthetic management included target controlled infusion of propofol and remifentanil, laryngeal mask airway, and pressure support ventilation. we present early data that early trials of cuff deflation within 48 hours of tracheostomy insertion can be achieved using a standardized protocol. its impact on length of stay, duration of ventilation and patient-centered outcomes needs to be investigated in larger multi-centre trials. preventing underinflation of the endotracheal tube cuff with a portable elastomeric device. a randomized controlled study je dauvergne 1 , al geffray 2 , k asehnoune 2 , b rozec 1 , k lakhal 1 1 hopital laënnec -chu de nantes, service d´anesthésie-réanimation, nantes, france; 2 hotel-dieu -chu de nantes, service d´anesthésieréanimation, nantes, france critical care 2020, 24(suppl 1):p112 the management of the endotracheal tube cuff pressure (p cuff ) is routine practice for critical care nursing staff. underinflation could lead to ventilator-associated pneumonia [1] whereas overinflation exposes to tracheal damage [2] . multi-daily check and adjustment is recommended to ensure that p cuff lies between 20 and 30 cmh 2 o [3] . to automate this task some devices exist but may be inconvenient, bulky and/or ineffective. their use is not supported by guidelines. a portable elastomeric device could be appealing for p cuff automated regulation. this prospective randomized controlled study tested whether the tracoe smart cuff manager tm reduced the rate of patients undergoing ≥1 episode of underinflation (p cuff <20 cmh 2 o), as compared with routine manual p cuff adjustment. monocentric, randomized controlled study. patients with acute brain injury and receiving mechanical ventilation were prospectively allocated to one of the two arms: manual reading and adjustment of p cuff at least every 8h (routine care) or adjunction of the smart cuff manager tm (intervention). this study was approuved by an institutional review board. among 60 randomized patients (routine care in 32, smart cuff manager tm in 28), 506 measurements were performed in 48h. with routine care, a higher rate of patients experienced at least one episode of underinflation (62.5 vs. 17.8%;p<0.001). episodes of underinflation episodes (15% vs. 2%;p<0.001) and manual adjustments (77% vs. 56%;p<0.001) were more frequent with routine care. for overinflation, there was no between-arms difference (p>0.99). the adjunction of continuous p cuff control with the tracoe smart cuff manager tm reduced the incidence of p cuff underinflation as compared with manual intermittent adjustments. overinflation was not promoted by this device. direct laryngoscopy as a technique for tracheal intubation is a potentially lifesaving procedure that healthcare professionals in a variety of fields are taught. however, this skill is challenging to acquire and difficult to maintain. poorly performed intubation technique can lead to potentially serious complications [1] . the intersurgical iview video laryngoscope is a new intubation tool which may have advantages over direct laryngoscopes, such as the macintosh, in the hands of novice personnel. a prospective randomized counterbalanced trial of 30 medical students, who did not have previous airway management experience, was conducted. each student received brief didactic teaching,following this, participants were directly supervised performing laryngoscopy and intubation using the macintosh and iview devices in an alternating pattern. students were permitted up to three attempts to successfully intubate under four conditions, three laryngoscopy conditions using alaerdal intubation trainer and one using a laerdal simman manikin. there was no significant difference in the success rate of intubation or time to intubation between the two devices. the iview outperformed the macintosh in time to intubation in the normal airway in the final scenario, once students gained experience with both devices. no significant difference was found in the number of optimisation manoeuvres, or intubation attempts between groups. areas where the iview outperformed the macintosh included severity of dental trauma and participants' perception regarding ease of use ofthe device. the iview may prove to be a useful teaching tool for novice personnel who are acquiring the skills of tracheal intubation. patients with a primary pulmonary pathology were more likely to respond to aprv. this association has not been described before and warrants further multi-centre exploration in a larger patient group. introduction: airway suctioning is common during mechanical ventilation, using either an open endotraqueal suctioning or closed endotracheal suctioning (ces). closed circuits were developed to prevent arterial desaturation and atelectasis associated to ventilator disconnection. however, ces may cause substantial loss of lung volume. the purpose of this study was to investigate the effects of a compensation method to prevent the loss in aeration during ces. the suctioning technique was performed for 15 seconds, negative pressures limited at 150 mmhg. closed suction catheters with 14fr (halyard health, georgia, eua) were used. electrical impedance tomography (eit) monitoring and arterial blood gas were collected. a nihonkoden mechanical ventilator (nkv550, california, eua) was applied, having a newly developed algorithm for suctioning which overcomes any pressure loss during suctioning (inlinesuction-app). when activated, the app delivers pcv ventilation, adding 2 cmh 2 o of end-expiratory pressure above peep, and delivering driving pressures of 15 cmh 2 o. results: pigs (30±5.4kg) with injured lungs and mechanically ventilated. we tested the aspiration procedures using low peep=5cmh 2 o, or high peep=±12.3cmh 2 o with v t2 o), whereas maintenance of compliance was observed when the app was on (from12.2±1.4 ml/cmh 2 o to 12.5±4.5 ml/cmh 2 o. blood gas in a representative animal showed a drop in pao 2 when app was off (from 247, to 149 mmhg after 2 min, and to 176 mmhg after 10 min) ( figure 1 ). with app on the pao 2 changed from 259 (pre-suction), to 223 (2 min), to 253 mmhg (10 min). the new nksoftware, delivering pcv ventilation during suctioning, could prevent atelectasis and functional loss associated to the procedure. tyrosine kinase inhibitor: an effective tool against lung cancer involvement responsible for acute respiratory failure in icu y tandjaoui-lambiotte 1 patients with advanced-stage non-small-cell lung cancer have high mortality rates in the intensive care unit (icu). in the last two decades, targeted therapies have changed the prognostic of patients with lung cancer outside the icu. the fast efficacy of targeted therapies led some intensivists to use them as rescue therapy for icu patients. we performed a national multicentric retrospective study with the participation of the grrroh (groupe de recherche en réanimation respiratoire en onco-hématologie). all patients with non-small-cell lung cancer admitted to the icu for acute respiratory failure between 2009 and 2019 were included in the study if a tyrosine kinase inhibitor was initiated during icu stay. cases were identified using hospital-pharmacies records. the primary outcome was overall survival 90 days after icu admission. results: thirty patients (age: 60+/-14 years old) admitted to a total of 14 icus throughout france were included. seventeen patients (59%) were nonsmoker. adenocarcinoma was the most frequent histological type (n=21, 70%). most patients had metastatic cancer (n=21, 70%). epithelial growth factor receptor mutation was the most common oncologic driver identified (n=16, 53%). during the icu stay, 17 (57%) patients required invasive mechanical ventilation, 13 (43%) catecholamine infusion, 3 (10%) renal replacement therapy and one (3%) extracorporeal membrane oxygenation. eighteen patients (60%) were discharged alive from icu and 11 (37%) were still alive after 90 days (see figure) . moreover, 6 patients (20%) were alive one year after icu discharge. despite a small sample size this study showed that, in the context of lung cancer involvement responsible for acute respiratory failure, the use of tyrosine kinase inhibitor should not be refrained in patients with severe condition in icu. the burned patient is one of the most complex patients whith a very high mortality. those patients with inhalation injury have a worst prognosis, typically associated with respiratory complications. the aim of our study is to evaluate the mortality of burn patientes with inalation injury in a critical burn unit. a prospective, observational and descriptive study was conducted over a period of 3 years. inhalation injury was defined with these criteria (≥ 2): history of injury in an enclosed space, facial burns with singed nasal hair, carbonaceus sputum and stridor. if they were intubated it was diagnosed by bronchoscopy. demographic data, tbsa, absi, baux score, apache ii, sofa, mechanical ventilation (mv), complications, length of stay, hospital course and mortality data were collected. results: 362 burns patients were admitted. 24% (84 patients) had inhalation injury. mortality among patients with inhalation injury was 28,6% (24 patients). most patients were men and those who died were older and with higher severity scores (fig. 1) . we found no significant differences between groups in the need for mv (95% vs. 85%) or in the percentage of tracheostomy performed (33.3 vs. 28.3). however, patients who died had more respiratory complications like ards, and also shock, renal failure and need of renal replancement therapies although infectious complications were similar in both groups. there was no statistically significant difference in volume used during initial resuscitation in the different groups. patients with inhalation injury who died had higher severity scores at the begining. although there were no differences in the need for mv patients who died had more respiratory complications as well as shock, renal failure and need of rrt, but no infectious complications.the volume used during inicial resuscitation, that was always related to the prognosis, was similar in both groups. further studies are needed to see if this greater initial severity corresponds to the degree of inhalation. aerogen, medical affairs, galway, ireland; 2 aerogen, science, galway, ireland critical care 2020, 24(suppl 1):p120 patients with acute exacerbations such as asthma are prescribed aerosol therapy from presentation in the emergency department to progression through to the intensive care unit. however, the variability in dose delivery to the lung across the possible patient interventions is not well characterized. here, we assess the predicted lung dose of a bronchodilator in a simulated spontaneously breathing adult patient via both facemask and nasal cannula, and via tracheostomy during mechanical ventilation. a standard dose of 2.5 mg in 2.5 ml salbutamol was aerosolized using the aerogen solo nebulizer (aerogen, ireland). for facemask testing, the nebulizer was used in combination with the aerogen ultra with 2lpm supplemental oxygen flow. for nasal cannula testing, the nebulizer was used in combination with the airvo 2 system (fisher and paykel, nz) system at both 10 and 50lpm gas flow rate. tracheostomy-mediated ventilation was assessed in combination with a hme, with the nebulizer placed between the hme and the tracheostomy tube. international standard iso27427 adult breath settings (vt 500ml, bpm 15, i:e 1:1) were used across all tests, and generated using a breathing simulator (asl5000, ingmar medical, usa) or mechanical ventilator (servo-u, maquet, sweden). the dose delivered to the lung was assessed using a capture filter at the level of the trachea, with drug mass determined using uv spectrophotometry at 276nm and interpolation on a standard curve. the results of testing are illustrated in figure 1 . the bronchodilator dose delivered to the simulated patient was seen to be relatively consistent between progressive interventions, except during high flow therapy, with the more clinically relevant 50lpm gas flow rate having a profound effect on the dose. these results may go some way towards explaining how different patient interventions can affect aerosol dose. the the mechanical ventilation (mv) have been identified as an independent factor indicating a worse prognosis for lung cancer patients [1] . this study was conducted in order to assess the results of noninvasive mechanical ventilation (niv) and/or invasive mechanical ventilation (imv) modalities in lung cancer patients admitted to the icu with acute respiratory failure (arf). in this study, lung cancer patients with respiratory failure who were admitted to the icu between january 2017 and december 2018 were evaluated retrospectively. results: 93 patients were included in the study. the mortality rate was 18.3%. 83 patients had niv. imv was applied to 10 patients. in the first 24 hours, 39 of the 83 patients who were initially treated with niv were administered imv. the duration of hospital stay, diagnosis of pneumonia and mortality rate were found to be significantly lower in patients treated with niv alone (p≤0.001, p=0.004, p=0.025), but glaskow coma score (gcs) was significantly higher in this group (p≤0.001). the mortality rate was similar between the patients who were initially treated with imv and those who were treated with imv in the first 24 hours. charlson comorbidity index (cci) and mv duration were significantly higher in patients who died (p=0.01, p= 0.021), but gcs was significantly lower in this group (p=0.032). in the linear regression model for the likelihood of mortality, ccl≥9 and unsuccessful niv increased the mortality rate by 3.4 (1.1-10.5) and 5.2 times (12-23.6) respectively (p=0.036, p=0.032). niv has been an effective modality for respiratory support in most lung cancer patients presenting with arf. however, failed niv seems to be a factor for increased mortality. therefore, the choice of respiratory support modality to be applied in this patient group should be decided by considering the gcs, cci and etiology of arf. the interaction between ventilator settings and the occurrence of acute kidney injury is not fully elucidated. this study aimed at investigating the effect of stepwise increase in peep level on the risk of acute kidney injury as evaluated with the renal resistivity index (rri).the primary outcome is to investigate whether increased levels of peep could lead to increase rri and whether rri could predict the occurrence of aki. methods: patients mechanically ventilated for at least 48 hours and without aki at admission were included in the study. rri was calculated at icu admission. posterolateral approach was used for kidney ultrasound. the peak systolic velocity (v max ) and the minimal diastolic velocity (v min ) were determined by pulse wave doppler, and the rri was calculated as (v max -v min )/v max . the exam was performed modifying the peep levels: 5, 10 and 15 cm h 2 o in random order for 15 minutes. occurrence of aki was defined within 7 days according to kdigo criteria. sixty-four patients were enrolled in the study and incidence of aki was 14/64 (22%). demographical and clinical characteristics are reported in table 1 . increase in peep showed a significant increase in rri from peep 5 to peep 10 (p<0.001) and from peep 10 to peep 15 (p=0.001) ( figure 1 ). the area under the roc curve of rri to predict aki was 0.845 at peep 5, 0.898 at peep 10 and 0.894 at peep 15 (all p<0.001). the youden index analysis showed an rri>0.70 as the best cut off for aki with a sensibility of 65% and a specificity of 96%. patients with rri>0.70 were 11/64 (17%), 13/64 (20%) and 22/64 (34%) at peep 5,peep 10 and peep 15 respectively. patients ventilated with a peep value associated with rri>0.70 had higher incidence of aki (11/14 vs 6/50, p<0.001). the application of peep can increase intrarenal vascular resistance,which is associated occurrence of aki; peep level should therefore be balanced taking into account the rri. the rri seems able to predict occurrence of aki in mechanically ventilated patients. alveolar and respiratory mechanics modifications produced by different concentrations of oxygen in healthy rats subjected to mechanical ventilation with protective ventilatory strategy d dominguez garcia 1 , r hernandez bisshopp 1 , jl martin barrasa 2 , d viera camacho 1 , a rodriguez gil 1 , j arias marzan 1 , s garcia hernandez 3 high oxygen can damage tissues [1] . in this study, we analyze the histological and pulmonary mechanics modifications that can occur when identifying different inspiratory oxygen fractions (fio 2 ) in lungs of healthy rats during protective mechanical ventilation. we use sprague-dawley rat. 4 groups were designed, each with 6 animals, the tidal volume (6 ml/kg), peep (3 cmh 2 o) and respiratory rate (90 rpm) were kept constant, changing the fio 2 between the groups. four groups were established: fio 2 0.21, 0.4, 0.6 and 1. after 4 hours, the lungs were removed for histological study and obtaining the wet/dry index. the histological modifications studied were: alveolar septa (as), alveolar hemorrhages (ah), intraalvelolar fibrin (if) and inflammatory infiltrates (ii). each parameter was rated from 0 to 3 [2] . peak pressure (pp) and pulmonary compliance were monitored every 60 minutes. different statistical tests will be used to analyze the data. results: references to the damage produced in the as, ah, if, ii and the global histological pattern were identified in the groups with the highest fio 2 and there was more damage (p <0.00001) ( figure 1 ). the wet/dry index rose significantly as the oxygen concentration increased (p = 0.001). in the groups to which a fio 2 of 0.6 and 1 was administered, the pp selected specific values with respect to the baseline intake from the first 60 minutes, an aspect that was not appreciated in the other groups (p <0.0001). regarding pulmonary compliance, it will be seen that, in the fio 2 0.6 and 1 groups, it decreased from the first 60 minutes, finding differences with respect to the other groups (p <0.0001). conclusions: mechanical ventilation applied for 4 hours in healthy animals produces disorders that are more pronounced as oxygen concentration increase. fio 2 greater than or equal to 0.6 should be avoided without clinical justification. introduction: patients requiring prolonged acute mechanical ventilation (pamv, defined as 4+ days on mv) are sicker and incur disproportionate morbidity and costs relative to patients on short-term mv (stmv, <4 days of mv). we quantified specific clinical outcomes among patients requiring pamv vs. stmv in a contemporary database. we conducted a multicenter retrospective cohort study within~700 hospitals in the premier database, 2014-2018. using icd-9-cm and icd-10 codes we identified pamv and stmv patients, and compared their baseline characteristics and hospital events. because of the large sample size, we omitted hypothesis testing. a total of 691,961 patients met the enrollment criteria, of whom 266,374 (38.5%) received pamv. at baseline, patients on pamv were similar to stmv with regard to age (years: 62.0 ± 15.8 pamv vs. 61.7 ± 17.2 stmv), gender (males: 55.6% pamv vs. 53.9% stmv), and race (white: 69.1% pamv vs. 72.4% stmv). pamv group had a higher comorbidity burden than stmv (mean charlson score 3.5 + 2.7 vs. 3.1 + 2.7). the prevalence of each of the indicators of acute illness severityvasopressors (50.3% vs. 36.9%), dialysis (19.4% vs. 10.3%), severe sepsis (20.3% vs. 10.3%), and septic shock (33.5% vs. 15.9%)was higher in pamv than stmv, as were hospital mortality and combined mortality or discharge to hospice (figure 1 ), extubation failure (12.3% vs. 6.1%), tracheostomy (21.6% vs. 4.5%), development of c. difficile (4.5% vs. 1.7%), and incidence density of ventilator-associated pneumonia (2.4/1,000 patient-days vs. 0.6/1,000 patient-days). conclusions: over 1/3 of all hospitalized patients on mv require it for 4 days or longer. pamv patients exhibit a higher burden of both chronic and acute illness than those on stmv. commensurately, all clinical outcomes examined are substantially worse in association with pamv than stmv. identifying the readiness of patients recovering from critical illness for liberation from invasive mechanical ventilation (imv) is not always straightforward [1] . the scottish intensive care society (sics) trainee audit 2018 conducted a scotland-wide study to understand current practices relating to liberation from imv. data were prospectively collected on patient demographics, indication for intubation, spontaneous breathing trial (sbt) practices, physiological markers, icu outcome and icu los. all patients >18 years ventilated with imv for > 24hrs from the 1 st nov. 2018 -30 th nov. 2018 were eligible for inclusion. exclusion criteria included extubation for end-of-life, death whilst intubated and presence of tracheostomy. logistic regression was performed to detect factors associated with extubation failure (ef). results were analysed via excel 2010 and stata v.14.1. patient benefit and privacy panel approval was granted. total population of 172 patients were included: 108 (63%) male and median apache2 score 19 (iqr 13-23). ef at first attempt occurred on 27 occasions (15.7%), median icu los of 10 days (iqr 7-12), mortality rate 22.2%. the cohort successfully extubated first time had a median icu length of stay of 5 days (iqr 3-9) and mortality rate of 1.4%. methods of sbt and extubation outcomes detailed in table 1 . no sbt prior to extubation had higher odds of ef (or 2.52, ci 1.09-5.84, p=0.03); patient ventilation for < 3 days had a three times higher odds of ef (or 3.31, ci 1.09-10.1, p=0.03). these were independently associated with ef on multivariate analysis conclusions: we found a reintubation rate of 15.7% in scottish icus. type of sbt most commonly used is divergent from the methods advocated in the literature. the lack of sbt and early extubation attempt was associated with failure, which in turn was associated with longer icu los and higher mortality. in patients undergoing prolonged invasive ventilation we hypothesise that abnormal right ventricular (rv) and left ventricular (lv) function are associated with increased 90-day mortality. whether changes in lv or rv function could aid in the prognostication of these patients has not been directly studied. patients admitted to the queen elizabeth hospital birmingham icu between april 2016 and july 2019 who were intubated and ventilated for more than 7 days and had a formal transthoracic echocardiogram (tte) whilst in icu were included. abnormal rv function was defined by the presence of depressed function, dilated size or moderate to severe risk of pulmonary hypertension. abnormal lv function was defined by the presence of lv depression (lv ejection fraction £ 45% or grade ii or more diastolic dysfunction) or a hyperdynamic lv (formally mentioned in tte report). patients who had a neurological cause for prolonged ventilation were excluded. the primary outcome was 90-day mortality. categorical data is presented as % and analysed using a chi-squared test. continuous data is presented as median (iqr). results: 871 patients required prolonged ventilation, of which 350 (40%) had a tte. patients were aged 62 (49-75), were 61% male and had a 36% 90-day mortality. the median ventilator days were 13 (6-20) and 77% required a tracheostomy. abnormal rv function was present in 26% (n=90) and was associated with an increased 90-day mortality compared to normal rv function (68% vs. 25%, rr 2.71 [2.10-3.50], p< 0.0001). lv function was abnormal in 27% (n=95) and was associated with an increased 90-day mortality compared to normal lv function (54% vs 28%, rr 1.91 [1.47 -2.49], p < 0.0001). abnormal rv function had a trend towards an increased mortality compared to abnormal lv function (68% vs 54%, rr 1.26 [1.00 -1.60], p = 0.07). in this study, abnormal rv and lv function were present in a quarter of patients undergoing prolonged ventilation and were associated with an increased mortality. introduction: tidal volume delivered by mechanical ventilation (mv) in sedated patients is distributed preferentially to ventral alveoli, causing overdistention and associated collapse in dorsal alveoli, driving volutrauma, atelectrauma and ventilator-induced lung injury [1] . temporary transvenous diaphragm neurostimulation (ttdn) stimulates diaphragm contraction [2] . when used in synchrony with mv, ttdn encourages increased dorsal ventilation due to the change in pressure gradients with diaphragm contraction, mimicking a more normal physiological pattern. this may improve gas exchange and reduce injury. a pilot study was conducted using 50 kg pigs undergoing mv in a mock icu. deeply sedated subjects were provided lung-protective volume-control ventilation at 8 ml/kg. ttdn diaphragm contractions were delivered in synchrony with inspiration on every second breath, reducing the ventilator pressure-time-product by 15-20% during mv+ttdn breaths. tidal volume distribution was recorded in each condition using electrical impedance tomography, and compared to never-ventilated, spontaneously breathing subjects (nv). results: dorsal ventilation changed from 49% during mv breaths to 54% during mv+ttdn breaths, compared to 60% in the nv group (p=0.035). ventral ventilation changed from 51% during mv breaths to 46% during mv+ttdn breaths, compared to 40% in the nv group (p= 0.042, figure 1 ). conclusions: ttdn diaphragm contraction used as an adjunct to mv yields a more physiological pattern of volume distribution. this translates into less overdistension in the ventral areas and less atelectrauma in the dorsal areas and reduces ventilator-induced lung injury. this technology introduction: by measuring the pes and its derivatives, we can measure the relationship that exist between the diaphragmatic excursion and the oscillation of the esophageal pressure curve: pswing (ps) so we infer that, just as with the pes, the variations of it might be related to a weaning failure [1, 2] . however, no nominal value exists in the bibliography to predict the test result. patients who meet with the inclusion criteria start the weaning process through a test of 30 minutes of spontaneous ventilation, t-tube (tt). and also the respiratory rate (rr) and the tidal volume (tv). from this analysis, an average ps (aps) is determined for each moment of the test (aps1, initial and aps2, final.).a quotient was obtained in relation to these variables using the value previously obtained (quotient dtv/dps x100. a total of 13 patients were included (n=13).regarding the evolution during tt, 9 (n=9) (69%) were successful, while 4 (n=4) (30.76%) failed when analyzing a rate that relates the variables tv and ps, a quotient was obtained in relation to these variables using the value previously obtained (quotient dtv/dps) for patients who were successful and who failed, (dtv/dps)/100 successful patients presented a value of 18.75 while those of the failure group presented a value of 45.83, (or 1,2 -3 p=0.082) ( table 1) . when presenting the relationship between tv and ps through the quotient (dvt/dps)/100, it is observed a tendency to have a higher quotient among patients who failed versus those who did not fail. the process of weaning from mechanical ventilation imposes an additional workload on the cardiovascular system, which may result in impaired myocardial function, increase in left ventricular filling pressure and respiratory distress. among surgical patients, those undergoing heart surgery are particularly susceptible to cardiac dysfunction induced by weaning because of inadequate cardiovascular reserve. the aim of our study was to depict the pathophysiological changes assessed by echocardiography during the steps of weaning and to identify possible predictors of weaning failure (wf). we enrolled 34 consecutive patients undergoing isolated coronary artery bypass grafting in our institution. data were obtained by intraoperative transesophageal echocardiography before sternotomy (t0) and by transthoracic echocardiography at the beginning of weaning (t1) and at the time of extubation (t2). wf was defined as deferral of planned extubation or respiratory failure needing reintubation or non-invasive mechanical ventilation within 48 hours. results: wf occurred in 7 patients (20.6%) and involved manifestations of respiratory distress in 5 (14.7%). we found a significant association between left ventricle outflow tract-velocity time integral (lvot-vti) and ventricular-arterial coupling measured at t1 and wf, with lvot-vti emerging as the best predictor of wf with an area under roc curve of 0.8669 ( figure 1 ); an optimal cutoff value of 15 cm provided 100% sensitivity and 71% specificity. significant increase in e/e' measured at t2 (13.44 vs 9.96, p 0.02) suggested a cardiac etiology of respiratory distress in patients who failed the weaning trial. our study showed that serial assessment of hemodynamic parameters by means of echocardiography is feasible in cardiac surgical patients and can provide insight into pathophysiological changes during weaning. although these preliminary data need to be confirmed in a larger population sample, lvot-vti emerged as a promising predictor of subsequent wf. compliance with guidelines for respiratory therapy in preclinical emergency medicine g jansen, n kappelhoff, s rehberg protestand hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, bielefeld, germany critical care 2020, 24(suppl 1):p131 introduction: current guidelines on pre-hospital emergency ventilation are based on the guidelines for lung protective ventilation in the intensive care unit. the present survey was designed to determine the accordance of actual pre-hospital emergency ventilation by german emergency physicians (gep) with these recommendations. recommendations include a respiratory rate (rr) between 10-16/min, a tidal volume (vt) between 6-8 ml/kg, a maximum pressure (pmax) <30 mbar and a positive end-expiratory pressure (peep) of 5 mbar. an anonymous web-based questionnaire encompassing 7 questions was sent to gep from september to december of 2018. gep were asked to specify their level of education, their preferred ventilation settings and the usually chosen parameters employed to guide mechanical ventilation. statistical analysis was performed using the ch²-test with a significance level ≤0.05. 60% of the questionnaires were completed (159/261). 25% of the participants were trainees (tr), 75% consultants (co). as target parameters for guidance of ventilation, 87% of the tr and 91% of the co use capnometry. the vt controlled 62% of the tr and 54% of the co on the basis of body weight. 81% of the tr and 81% of the co reported to control oxygenation using spo2. table 1 shows our analysis of the given answers. there were no statistically significant differences between the groups. deviations from the guidelines of pre-hospital emergency ventilation settings are common and mainly concern the use of a guidelinecompliant peep. in addition, recommended target parameters for guidance of ventilation were not applied in a significant proportion of gep. prospective observational study including ltx recipients admitted to our icu from february2017 to january2019, who underwent a spontaneous breathing trial (sbt) using a t-piece for 30 minutes. clinical variables and arterial blood gas samples were recorded before starting sbt and after 20 minutes on the t-piece. diaphragmatic excursion (de) and thickening fraction (dtf) were also assessed using ultrasound(us) after 20 minutes on the tpiece. us-dd was defined as de<10 mm or dtf<0.3 of at least one hemidiaphragm. patients who successfully completed a sbt, defined according to clinical criteria,were extubated. extubation failure was defined as the need for reintubation within 48h. results are expressed as medians (iqr) or frequencies (%). 193 ltx recipients were admitted to the icu, 79 of whom underwent an sbt. 51 were male, and the median age was 58y. main indications for ltx were interstitial lung disease (43.0%), copd and cystic fibrosis. 59 were bilateral ltx, and 13 and 7 were left and right unilateral ltx respectively. 69 patients were extubated after sbt and 6 required reintubation within 48h. 53 presented us-dd, though there were no differences between patients who succeeded and those needing reintubation. in contrast, patients who succeeded showed higher pao2/fio2 after 20 minutes on the t-piece (table 1) . similarly, higher reductions in deltapao2/fio2 after 20 minutes on the t-piece were observed in patients who failed. oxygenation after sbt performed using a t-piece may predict extubation failure in ltx recipients with successful sbt. us-dd was not associated with the need of reintubation. descriptive study about the relationship between self-extubation episodes and patient-ventilator interaction s nogales 1 , introduction: to evaluate the relationship between self-extubation and patientventilator interaction, among other physiological variables, in order to predict and to prevent these events. self-extubation (se) are quality indicators in patients under invasive mechanical ventilations (imv) and are related with mortality [1] . planned secondary analysis of a prospective data base of clinical and physiologic signals of patients receiving imv. we included se episodes (2012-2018) with continuous record of ventilator and monitor signals (bclink bettercare®). we analysed demographic data, physiological parameters (peripheral oxygen saturation spo2, heart rate hr, respiratory rate rr and media arterial pressure map) and patientventilator interaction (asynchrony index ai, ineffective efforts during expiration iee and double cycling dc). we studied a period of 12 hours prior to the se episode. we used the wilcoxon non-parametric test and for a proper analysis a linear mixed effects model. we included 21 episodes of se, mean age 62±13years, 76%men, apache ii at admission 17±10, 4,6±3,8days under imv until the episode, reintubation rate 47.6%, icu stay 20,9±17,6days, icu mortality 14%. at the time of the se, 65% were under sedation, 65% with physical restraint. the 67% were in weaning. we observed a trend to increase in spo2, rr, hr, map and asynchronies in the 2-hour period prior to se episode. we compared these variables from this period with a 2-hour period before and we observed a statistically the data presented in this study show that our results are in accordance with the literature with favorable mortality and early postoperative complication rates and support that this procedure is an excellent alternative for surgery in the elderly patients. it is reported that patients with pulmonary hypertension (ph; systolic pulmonary arterial pressure (spap)≥35 mmhg)) have frequent cardiac complications after transcatheter aortic valve implantation (tavi). ph often gets worse in some patients despite the normal cardiac function after tavi. no studies have ever examined prognosis after tavi in patients with or without worsening of ph. therefore, we retrospectively examined the frequency of mid-to long-term heart failure and cardiac death in patients with and without deterioration of ph after tavi. among 113 patients who underwent tavi at our hospital between february 2014 and march 2016, we analysed 27 patients with ph (spap≥35 mmhg) before surgery. spap was measured in transthoracic echocardiography before and within 1 week after tavi. patients were divided into two groups according to whether spap worsened/ did not change or improved after tavi. we examined the frequency of admission due to heart failure or cardiac death (death caused by heart failure, angina, or myocardial infarction) during the period of 3 years after tavi. ph worsened or did not change after tavi in 9 patients, while it improved in 18 patients. the left ventricular ejection fraction measured within 1 week after tavi showed no difference between the two groups (56.6±11.9% vs 58.4±10.0%, p=0.71). the worsened/ no change group was higher in frequency of admission due to heart failure (logrank; p<0.05) and cardiac death (logrank; p<0.04). despite successful treatment for as by tavi, the frequency of heart failure and cardiac death was higher in patients who did not show improvement of ph after tavi, even in the absence of cardiac function decrease. vigorous intervention for ph worsening after tavi may be helpful to improve prognosis. the there are several different anti platelet drugs that can be used to treat acute cardiac events. currently there are no effective markers that can assess how these drugs modify coagulation profile and quality. a new functional biomarker that measures fractal dimension (df ) and clot formation time (tgp) has been developed [1] . df quantifies clot microstructure whereas tgp is a real-time measure of clotting time. we aimed to validate df and tgp in st elevation myocardial infarction (stemi) and assess the effect of two p2y12 inhibitors which have different pharmacological mechanisms: clopidogrel and ticagrelor. we prospectively recruited 72 stemi patients in the emergency setting. venous blood samples were collected 12 hours after admission, following treatment with either ticagrelor or clopidogrel, in accordance with the local guidelines at the time. the blood samples were tested using the df and tgp biomarker, platelet aggregometry, clot contraction and standard markers of coagulation. results: 36 patients received clopidogrel and 36 received ticagrelor. the df for clopidogrel was higher than ticagrelor (1.75±0.05 vs 1.73±0.06, p=0.18 which corresponds to a decrease in clot mass of 20% figure 1 ) and the tgp was reduced (205±91sec vs 257±89 sec, p=0.06 a 20% reduction in time). the results of the study suggest that clopidogrel is less powerful in its effects on clotting characteristics compared to ticagrelor. blood from patients receiving clopidogrel formed quicker and denser clots. this would suggest the risk of secondary events or stent occlusion is lower in those patients on ticagrelor, highlighting that df and tgp may be important in identifying patients at risk of future thrombotic events, the study is ongoing and will investigate the long term outcome in these patients. introduction: new onset atrial fibrillation (noaf) during critical illness frequently resolves prior to discharge. however long-term risks of noaf (i.e. heart failure, ischemic stroke and death)remains high [1] . previous studies noted that nearly half of noaf cases did not have diagnosis recorded [2] . addressing this may reduce post critical illness mortality by increasing af surveillance post intensive care (icu) discharge. retrospective data was collected from an electronic health record for icu admissions over a 10 month period from a biomarker is defined as a measurable indicator of some biological state or condition. combined with a good clinical evaluation, they can enable an early and safe diagnostic, thus a faster management for the patient. cardiac biomarker testing is not indicated in routine in the emergency department (ed) because of low utility and high possibility of false-positive results. however, current rates of testing are unknown. the aim of our study was to evaluate the importance of measuring cardiac biomarkers especially troponins, d-dimer, and btype natriuretic peptide in our daily practice, and to identify the latest recommendations for a better use of these biomarkers in the diagnostic and therapeutic approaches. we conducted a prospective observational study, over a 13 months periods performed in the ed of the university hospital center ibn rochd, casablanca, morocco, including all patients admitted during our study period and having a blood test for at least one biological marker. the dataset was analyzed by spss statistics 21.0. a total of 182 patients was enrolled. troponins were tested in 85.3% patients (high sensitive in 49.5% and troponin i tni in 35.8%), ddimer in 30.9%, bnp 19% and nt pro bnp in 9.5% of cases. the diagnostic impact was significant in 94.4% of cases for troponins, 84.6% of cases for d-dimer and 87.5% for bnp. the therapeutic impact was considered important in 80.6% cases for troponins, 69.2% for ddimer and 87.5% for bnp. cardiac biomarkers have an important role in the ed, not only do they confirm the diagnosis (including the role of troponins in acs) but also eliminate others (with a strong negative predictive value of d-dimer for thromboembolic disease) and prove the cardiopulmonary origin of acute dyspnea (the significant place of bnp in confirming the diagnosis of acute heart failure). a multicenter study on the comparison of inter-rater reliability of a new and the original heart score among emergency physicians from three italian emergency departments the heart (based on history,ecg,age,risk factors,troponin) score is a valid tool to stratify the acs in chest pain. but some reports suggest that its reliability could be low for heterogeneity in the assignment due to the subjective interpretation of the history. we used the chest pain score for the "history". in this study we compare the reliability of the new heartcps and original heart. this is a multicenter retrospective study conducted in 3 italian ed between july and october 2019 using clinical scenarios. ten physicians were included after a course on heart and heartcps score. we used 53 scenarios which included clinical and demographic data. each participant independently assigned scores to the scenarios using the heart and heartcps. we tested the interrater agreement using the kappa-statistic (k), the confidence intervals are bias corrected ; we used stata/se 14.2 statistical software . a p-value of < 0.05 defines statistical significance. the overall inter-rater reliability was good for heart and heartcps: kappa =0.63 (ci 95%;0.57-0.72)and 0,65(ci95%;0.63 -0.67); with good agreement among all the class of risk for heartcps but moderate in the medium class for heart . we found significant differences of inter-rater reliability among the senior and junior physicians who used the heartcps:k=0.56(ci95%;0.52-0.57)and 0.75(ci95%;0.70-0.79). heartcps score increased its history inter-rater reliability specially among the junior physicians from k=0.35 (ci 95%; 0.27-0.43) to k=0.69(ci 95%;0.62-0.71).the junior physicians seem to be more reliable than senior with the heartcps:k=0.75 (0.71-0.79) vs k=0.56 (ci95%;0.52-0.57). the heartcps showed inter-rater reliability better than original heart among the medium class of risk and the junior group. it could be proposed to young doctors to stratify the acs risk of chest pain. limit: we used scenarios rather than real patients. a hybrid approach as treatment for coronary artery disease: endo-cabg or pci first, does it matter? introduction: the aim of this study is to discuss the short-term results of a hybrid approach combining minimally invasive endoscopic cabg (endo-cabg) with a percutaneous coronary intervention (pci). to bypass the disadvantages and potential complications of conventional cabg via median sternotomy, we developed the endocabg technique to treat patients with single-and multi-vessel coronary artery disease (cad). this procedure is performed with three 5-mm thoracic ports and a mini-thoracotomy utility port (3 cm) through the intercostal space. this technique can be combined with pci: the hybrid approach. the sequence of the 2 procedures (endocabg followed by pci or vice versa) may result in different outcomes. from 02/2016 to 12/2017 data from 81 consecutive patients scheduled for a hybrid technique at jessa, belgium, were prospectively entered into a customized database. this database was retrospectively reviewed. subgroup analysis was performed to compare outcomes of patients who first received endocabg with patients who first received pci. a p-value < 0.05 is considered significant, a p-value < 0.1 is considered as a trend toward significance. four patients underwent revision surgery and 2 patients died within the first 30 days. in 79 patients the left anterior descendens artery (lad) was grafted with the left internal mammary artery (lima), the right coronary artery (rca) was the most stented vessel using pci. patients first treated with pci received more units of fresh frozen plasma after endocabg compared to those who were first treated with endocabg (p=0.03). there was also a trend toward significant more transfusion of packed cells in this small subgroup (p=0.07). the hybrid approach is a feasible technique as a treatment option for patients with multi-vessel cad. if cabg follows the pci, patients are more likely to receive transfusion. a possible explanation could be the need for dual antiplatelet therapy prior to surgery in this group, but this needs further investigation. prognostic difference between troponin elevation meeting the mi criteria and troponin elevation due to myocardial injury in septic troponin t (ctnt) elevation in critically ill patients is common and is associated with poor outcome. using common assays, 40-50% of patients in the icu will have elevated troponin level. our aim was to determine whether there is any prognostic difference between troponin elevation meeting the mi criteria (rise and fall more than 20% together with echo and ecg new abnormalities) and troponin elevation due to myocardial injury in septic patients. we enrolled 101 patients with sepsis and mean sofa score 5,2 respectively in which ctnt level was measured more than once and analyzed there ecg and echo findings. patients were classified into three groups:definite mi (rise and fall ctnt ≥ 20% and contemporaneous changes on ecg and/or echo),possible mi (rise and fall ctnt ≥ 20% and no other findings),myocardial injury (ctnt rise less than 20%) results: data from 101 patients were analyzed (49% female; mean age 61.9 (sd 16.9)). a total of 101 patients had at least one elevated ctnt more than 0.03 mkg/l. in 71 (70%) of patients ctnt level rised more than 20% from the first elevated measurement.64 (63%) of patients met mi criteria considering new ecg and echo findings. the overall mortality rate in all patients was 53.9%.the mortality rate didn't differ significantly in three groups: in the definite mi group 62.4%, in the suspected mi group 52%, in the non mi ctnt elevation group 56,4%, p=0,6. coronary angiography was performed in 46 (73%) of patients from the definite mi group,pci was performed in 18 (39%) of patients. the mortality rate in the invasive group was not significantly lower comparing to the nonivasive group 29% vs 37,8%, p=0,06. bleeding complications were significantly more frequent in the definite mi group 13% vs 7% and 8% respectively conclusions: ctnt level elevation is associated with poor outcome regardless coronary or non coronary injury. myocardial revascularization may be beneficial in patients with sepsis and definite mi, but it is also associated with increased bleeding risk. diagnostic interest of "marburg heart score" in patient consulting the emergencies department for acute chest pain chest pain is a common reason for emergency department visits, although this primarily refers to acute coronary syndrome (acs), this symptom may be frequently related to other non-ischemic etiologies. the aim was to validate the marburg heart score as a tool to exclude coronary artery disease in emergency department patients with nontraumatic acute chest pain. methods: a prospective, observational, descriptive and analytic cohort study conducted in the emergency department, from february 1st to march 31st, 2019, collecting patients consulting for nontraumatic acute chest pain, the "marburg heart" score was calculated for all these patients. telephone contact was made after 6 weeks to look for an ischemic cardiovascular event. we included 171 patients. the mean age was 57 +/-13 years, the sex ratio was 0.86. the majority of the patients (78.9%) consulted directly to the emergency department, 21.1% were referred by a primary care physician. the median time to consultation after the onset of chest pain was 24 hours. high blood pressure was the most common risk factor (43.9%), followed by smoking (31%), diabetes (24.8%) and dyslipidemia (23.4%). thirty-five patients (20.5%) had already coronary heart disease, ecg was pathological in 19.3% of patients, 8 patients had an acs with st segment elevation. at six weeks, 20.6% of the patients had an acute coronary event. according to the patients' answers on the 5 questions of the marburg heart score. the area under the roc curve of this score was 0.78 with a negative predictive value of 87.2%; the "marburg heart score" is a simple, valid and reproducible clinical score with a discriminatory power to rule out the diagnosis of coronary artery disease from the first contact with the patient presenting for chest pain in emergencies. the abdominal aortic aneurysm (aaa) surgery is a complex procedure in elderly patients with high cardiovascular risk. anesthesiological techniques should play special attention to the volume status during cross-clamping as well as to the blood loss. goal directed fluid therapies (gdt) in aaa surgery in elderly patients decrease the perioperative morbidity and mortality [1] . aim of this study is to investigate administration of fluid-based on either a gdt approach or a control method (fluid administered based on static preload parameters and traditional hemodynamic) in all phases of aaa surgery and especially in the phase of clamping and de-clamping. a total of 30 patients asa iii, randomly scheduled for elective, open aaa surgery were included in this clinical trial. they were randomly assigned to two groups i -gdt with targeting stroke volume variation (svv) and ii -control group where fluids were administered at the discretion of the attending anaesthesiologist. in both these groups hemodynamic parameters, central venous pressure (cvp), temperature, blood loss and diuresis were registered during the operation and 48 hours postoperatively. each group was assessed for postoperative complications. gdt group received less fluids and had a higher cardiac index (ci) (3.9± 0.6 vs. 2.9± 0.8 l/minute per m 2 , p < 0.01) and stroke volume index (55.1 ± 5.4 vs. 35.1 ± 5.8 ml/m 2 , p < 0.01) than the control group. there were significantly fewer complications in the intervention than control group (3 vs. 9, p = 0.02). gdt fluid administration enables less use of fluids, improved hemodynamic and fewer postoperative complications in elderly patients undergoing aaa surgery. ultrasonography is a valid diagnostic tool, used to measure changes of muscle mass. the aim of this study was to investigate the clinical value of ultrasound-assessed muscle mass, in patients undergoing cardiothoracic surgery that present muscle weakness postoperatively. for this study, 221 consecutive patients were enrolled, following their admission in the cardiac surgery intensive care unit (icu) within 24 hours of cardiac surgery. ultrasound scans, for the assessment of quadriceps muscle thickness, were performed every 48 hours for 7 days. muscle strength was also evaluated in parallel, using the medical research council (mrc) scale. of the 221 patients enrolled, ultrasound scans and muscle strength assessment were performed in 165 patients. the muscle thickness of rectus femoris (rf), was slightly decreased by 2.18% ([95%ci: -0.21; 0.15], n=9; p=0.729) and the combined muscle thickness of the vastus intermedius (vi) and rf decreased by 3.5% ([95% ci: -0.4; 0.22], n=9; p=0.530). patients whose combined vi and rf muscle thickness was below the recorded median values (2.5cm) on day 1 (n=78), stayed longer in the icu (47 ± 74 vs 28 ± 45 hours, p = 0.015). patients with mrc score ≤ 48 on day 3 (n=7), required prolonged mechanical ventilation support compared to patients with mrc score ≥ 49 (n=33), (44 ± 14 vs 19 ± 9 hours, p = 0.006). the use of muscle ultrasound seems to be a valuable tool in assessing skeletal muscle mass in critically ill patients after cardiothoracic surgery. moreover, the results of this pilot study showed that muscle wasting of patients after cardiothoracic surgery is of clinical importance, affecting their stay in icu. prediction of cardiac risk after major abdominal surgery s musaeva, i tarovatov, a vorona, i zabolotskikh, n doinov kuban state medical university, anesthesiology and intensive care, krasnodar, russia critical care 2020, 24(suppl 1):p147 the aim is to assess the incidence of cardiovascular incidents in major abdominal surgery [1] using the revised lee index. a study was conducted of 144 elderly patients who underwent major abdominal surgery in the krasnodar regional clinical hospital no. 2 under combined anesthesia. in the preoperative period, the risk of cardiovascular incidents was assessed using the revised lee index and the functional status was assessed by met. depending on the lee index, 3 groups were identified: group 1 (n = 69) -low risk (index value -1), group 2 (n = 52) -intermediate risk (index value -2); group 3 (n = 23) -high risk (index value> 3). we estimated the incidence of critical incidents in groups: hypo-, hypertension, arrhythmias, and bradycardia. in the general population, cardiac risk was 2.2 ± 0.7 points; functional status -7.7 ± 1 met. the greatest number of critical incidents was recorded in patients with high risk (58.4%), the smallest -in patients with low risk (9.1%), in patients with intermediate risk -26.5% (n <0, 05 between groups according to chi-square criterion). in the structure of critical incidents, hypotension was most often encounteredin 62 (43%) patients, while some patients revealed several incidents from the circulatory system (n = 116). overall, the lee scale showed good prognostic ability (auroc = 0.81) in predicting hemodynamic incidents. the revised lee index is a useful tool to help assess the risk of cardiovascular incidents and determine patient management tactics in the perioperative period. postoperative cognitive dysfunction (pocd) remains an unresolved problem due to lack of consensus on its etiology and pathogenesis. some believe that pocd is the result of the direct toxic effect of general anesthetics on the nervous system. others claim that surgical trauma activates proinflammatory factors that induce neuroinflammation. wistar rats were allocated into 2 groups: 1-minor surgery (n=20), 2major surgery group (n=20). after 5 days of handling and habituation rats undergone surgery under isoflurane general anesthesia (2 vol.%). group 1 rats underwent laparotomy with gentle gut massage followed by wound closure. rats in group 2 undergone left side nephrectomy. starting from the 4th postoperative day spatial memory in rats was studied in morris water maze which is a cylinder metal pool with a diameter of 1.5 and a height of 0.5 m filled with water (temp.26±1 o c) up to half. it has a platform with a diameter of 12 cm and a height of 1 cm below the water level. testing was preceded by a training stage, which included 8 sessions daily for 4 days. thus, rats developed spatial memory to the location of the platform. on the 5th day of the study test stage was conducted to assess spatial memory: rats were launched from 3 points into maze without platform and data were recorded for 60 seconds at each session. time spent on the target quadrant (ttq) and the number of target area crossings (tac) were registered. a second test was conducted 14 days after the first test to evaluate long-term spatial memory. the duration of surgery and anesthesia did not differ significantly between groups. there was a significant difference between groups in average ttq and tac in test 1 (table 1 ). in test 2 minor surgery group showed better results but they were less significant. major surgery is associated with a more pronounced deterioration of spatial memory in rats in early postoperative period compared to minor surgery. cardiac inflammatory markers in icu patients with myocardiac ischemia after non cardiac surgery (a pilot study) p manthou 1 , g lioliousis 2 , p vasileiou 3 , g fildissis 1 1 national kapodistrian university of athens, athens, greece; 2 national kapodistrian university of athens, general thoracic hospital´´sotiria´´, athens, greece; 3 national kapodistrian university of athens, university of athens, athens, greece critical care 2020, 24(suppl 1):p149 patients with known coronary artery disease have higher perioperative risk for myocardial ischemia [1, 2] . mortality is frequent following cardiac ischemia in the intensive care unit (icu) after non-cardiac surgery. the first group includes patients admitted to the intensive care unit for post-operative follow-up without myocardiac ischemia in the first 24 hours. the second group includes patients with myocardiac ischemia postoperatively and needs intensive care monitoring. cardiac risk assessment was made with the lee index,hemorrhagic risk assessment with the has-bled bleeding score and thrombotic risk assessment with cha2ds2-vasc score. postoperatively, pathological test values such as bnp, troponin, crp, calcitonin were estimated. the sequential organ failure assessment (sofa) systeme was used to assess sepsis. the nursing activity score (nas) scale was used to measure the workload of various nursing activities in the icu. according to the pilot study, the sample consists of 35 patients. 31.4% had myocardial ischemia. the lee index was significantly higher in patients with myocardial ischemia. the duration of hospitalization, the high dose of vasoconstrictive drugs, the length of stay in the icu, the duration of mechanical stay and the nursing workload were higher in patients with myocardial ischemia. ck-mb and troponin levels differed significantly between the two groups. creatinine, bilirubin and bnp during the 24 hours were significantly higher. patients with myocardial ischemia had significantly higher mortality. cardiac risk assessment, has-bled score and cha2ds2-vasc score in combination with cardiac enzymes such as troponin could predict myocardiac ischemia in severely ill icu patients. introduction: according to the literature an airway complication followed thyroid gland surgery are: difficult trachea intubation, tracheomalacia, postextubation stridor and bleeding [1, 2] . most common cause of death was problem with respiration and airway obstruction [3] . subsequent hypoxia could require emergency airway and even tracheostomy [3] . aim of our study was to determine the most common of airway complications and their association with type of surgery in our region. the retrospective cohort study included 400 pts., (369 women, 31 men) was performed in odessa regional hospital, oncology centre odessa. there were three types of patients: with euthyroid goiter -170 (43%), polynodos goiter -125(31%) and thyroid cancer -105 (26%) ( table 1) . airway complications were diagnosed after trachea extubation based on indirect laryngoscope, presence of stridor, desaturation. the pearson's criteria was calculated. the ratio of airway complications after thyroid surgery was 9.7% (39 pts). the main reasons of airway complications in thyroid surgery included: laryngeal edema -22 pts (5.5%); recurrent laryngeal nerve injury -12 pts (3.0%) and postoperative bleeding 5 pts (1.2%). thyroid gland cancer and polynodosal goiter associated with laryngeal edema and recurrent laryngeal nerve injury (pearsen criteria were 0.271 -moderate and 0.203 consequentially). it's may require more attention from the anesthetists after extubation and readiness for an urgent airway. serum iron level and development of multiple organ dysfunction syndrome in patients in the perioperative period s tachyla mogilev regional hospital, department of anesthesiology and intensive care, mogilev, belarus critical care 2020, 24(suppl 1):p151 recently there has been attention of researchers to the problem of perioperative anemia. it was found that it increases the risk of death and postoperative complications. threatening complication is multiple organ dysfunction syndrome (mods). the objective was to determine the level of serum iron in the perioperative period in patients with endoprosthetics of large joints, and with the presence of mods in abdominal surgery. a prospective cohort study was conducted in 77 patients, including 18 men and 59 women, age 61.9 ± 15.1 years. two groups were identified: 1st (control) -patients after endoprosthetics of large joints (n = 40), 2nd (main) -patients in abdominal surgery with the presence of mods (n = 37). the presence of mods was established based on the criteria for the 2016 sccm / accp conference. serum iron was monitored using an au 680 analyzer (usa). the study identified several stages: 1st -before surgery, 2nd -1st day after surgery, 3rd -3rd day, 4th -7th day, 5th -10th day. when studying the indicators of serum iron, its significant decrease (p <0.05) in the postoperative period was established. in the 1st group: 1st stage -15.2 (10-19.4) mmol / l, 2nd stage -5.2 (3.9-7.6) mmol / l, 3rd stage -6.6 (5-8.7) μmol / l, stage 4 -9.7 (8.6-12.1) μmol / l, stage 5 -9.4 (7.8-11 9) μmol / l. in the 2nd group: 1st stage -11.9 (10-15) mmol / l, 2nd stage -3.7 (1.7-4.1) mmol / l, 3rd stage -3, 6 (2.4-4.5) μmol / l, stage 4 -6.5 (4.4-8.2) μmol / l, stage 5 -7.6 (6.5-9 4) μmol / l. moreover, in both groups, iron increased at the 4th stage against the 2nd stage (p <0.05). when comparing the level of iron between the groups, significant differences were found (p < 0.05) at the 2nd, 3rd and 4th stages. in patients in the postoperative period, a decrease in serum iron is observed, the level of which rises by the 7th day, but does not reach the initial values. this decrease is more pronounced in patients with the presence of mods after abdominal surgery. kidney and pancreatic graft thrombosis happened in 5.6% and 18.9%, respectively, and bleeding in 21.1%. forty-one (45.6%) developed at least one infection during hospital stay. infection during icu was found in 13.3% and main pathogens were gram negative bacilli sensible to beta-lactam. after icu, the incidence of multi-drug resistant pathogen was 13.5%, predominantly gram negative bacilli. fungal infection was lower 4%. all-cause hospital mortality rate was 5.6%. infectious complications are the main cause of morbidity and mortality following spk transplantation. the administration of broadspectrum prophylactic antibiotics are leading to the appearance of multi-drug resistant pathogens. knowing local microbiological flora may be helpful, allowing more adequate antibiotic prophylaxis. introduction: cardiopulmonary bypass (cpb) is associated with thrombotic complications. occurrence of thrombosis after cpb is 12% which takes the third place between cpb-associated complications. our study determined preoperative predictors of thrombosis in children with congenital heart defects. 138 patients with congenital heart diseases in age up to 11 months 29 days (median age -4,7 months, youngest age -2 days after birth, oldest -11 months 29 days), underwent cardiac surgery with cpb, were enrolled in this study. all patients were divided into two groups: 1 st -without thrombosis, 2 nd -with thrombosis. protein c, ddimer, von willebrand factor and plasminogen plasma levels were assessed directly before surgery. thrombotic cases were proven by performing doppler ultrasound or mri. thrombotic complications were diagnosed in 30 children (21%). between all thrombotic complications ischemic strokes were diagnosed in 73% (22 cases), arterial thrombosis in 17 % (5 cases), intracardiac thrombus in 7% (2 cases) and mechanical mitral prosthetic valve thrombosis 3%(1). receiver operating characteristic (roc) curves are created for the listed indicators. area under the curve (auc) for protein c 0,64 (sensitivity(sn)-65%, specificity(sp) -50%), d-dimer is 0,65 (sn -65%, sp 50%), for plasminogen activity -0,62 (sn 60%, sp 40%) and for von willebrand factor level -0,64 (sn 80%, sp 55%). an roc curve was created for all three indicators, the auc was 0.7 (sn -80%, sp -40%). these parameters can be recommended as predictors of thrombosis in children after cardiac surgery. cpb is related with a large number of life-threatening complications. in our work, preoperative predictors of thrombosis were identified. based on this data, it is possible to create thrombosis risk scale change the tactics of the anaesthetic approach, the prevention of thrombosis in the postoperative period. further studies are needed to identify other possible predictors of thrombosis. introduction: abdominal ischemia occurs in 9% of patients submitted to aortic aneurysm repair. its early diagnosis requires an elevated index of suspiction, particularly in more severe patients. we hypothesized that earlier increase and higher levels of c-reactive protein (crp) may help to predict intra-abdominal ischemia. we performed a retrospective study of patients admitted to the intensive care department (icd) after abdominal aorta aneurism surgery. we included all patients admitted during a two-year period, that survived for more than 48 hours. primary outcome was splanchnic ischemia assessed by abdominal ct-scan. we also evaluated the presence of bacteremia, abdominal compartment syndrome and icd mortality. association between inflammatory parameters and ischemia was evaluated by multivariate logistic regression. introduction: crp (c-reactive protein) has been shown to be a useful biomarker in identifying complications after major abdominal surgery. gastrectomy is a high-risk surgical procedure that requires post-operative critical care support to monitor for complications which are predominantly infective in nature. the aims of this study were to determine whether there is a relationship between post-operative crp levels and patients who developed post-operative infective complications. a retrospective analysis was performed on patients undergoing elective gastrectomy for gastric cancer at a single centre between september 2011 and july 2016. post-operative crp levels for each day following resection were analysed for all patients. roc curve analysis was used to determine which post-operative day (pod) gave the optimal cut-off. of 144 patients included, the majority were male (61.8%), mean age was 68.5 years and 53.5% had node-negative disease. a total of 84 patients (58.3%) had an infective complication, which includes those who experienced an anastomotic leak. crp levels on post-operative day 3 gave the greatest auc for the gastrectomy group (0.765). crp cut-off of 220mg/l was significantly associated with infective complications (or7.29, 95% ci 3.42-15.58, p= <0.001) and gave a sensitivity of 70% and specificity 76% (ppv 67%, npv 78%). more patients with a crp >220 on post-operative day 3 experienced an infective complication (67% vs 21%, p = <0.001) or a leak in particular (17% vs 6%, p = 0.059). a crp level of less than 220 mg/l on pod3 may be useful to predict the development or exclude the likelihood of such infective complications in this group of patients prior to clinical signs (ppv 67%, npv 78%). this may prompt and facilitate decision-making regarding early investigation and intervention or prevent inappropriate early discharge from critical care, whilst providing more assurance in identifying those who could be stepped down to ward level care. vasoplegia is commonly observed after cardiopulmonary bypass surgery (cpb) and associated with high mortality. chronic use of reninangiotensin aldosterone system inhibitors (raasi) is associated with its incidence and ensuing need for vasopressor support after cpb. renin serves as marker of tissue perfusion [1] . we examined the role of renin in the setting of raasi exposure and vasopressor needs in the peri-cpb period. prospective observational study of 31 adult patients undergoing cpb, aged 66.0±10.5 years (22 men, 9 women). blood was collected 1) post induction, pre-cpb; 2) 30 min post cardioplegia, and 3) immediately post bypass. vital signs and perioperative medications were recorded. as control, blood was collected from 5 men and 4 women aged 53.5 ±10.7, not diagnosed with lung disease and not prescribed any raasi. baseline plasma renin in cpb patients tended to be higher than in control subjects (mean=38.5pg/ml±9.2 vs. 17.1 pg/ml ± 3.5, respectively, p=0.670). 30 minutes into cpb, mean renin was increased from baseline (77.6 pg/ml±17.5, p=0.211), and remained elevated immediately post cpb (74.6 pg/ml±19.1). patients using raasi prior to cpb tended to have a larger increase in renin post cpb (delta=55.3 pg/ ml±30.9) vs. those not previously on raasi (26.9 pg/ml±9.5, p= 0.092). renin was elevated in patients requiring vasopressor support in the 24 hours post cpb vs. those not requiring pressors (41.4 pg/ ml±15.7 vs. 25.1 pg/ml±16.1 p=0.0246). in those prescribed raasi and requiring pressors post cpb, there was a tendency toward greater renin increase than those not requiring pressors postoperatively (49.9 pg/ml±49.7 vs. 8.5 pg/ml±3.9, p=0.036). this study suggests a trend toward higher renin levels, particularly during cpb, in patients prescribed raasi, and a positive association between renin and postoperative vasopressor needs. we speculate that increased renin levels may predict postoperative vasoplegia. cardiac surgery is associated with perioperative blood loss and a high risk of allogenic blood transfusion. it has been recognized that high blood product transfusion requirement is associated with adverse clinical outcomes. 1 guidelines on patient blood management therefor aim at reducing blood loss and blood transfusion requirements in cardiac surgery. 2 as there remains controversy about the advantage of minimal invasive techniques on blood loss an transfusion requirements, 2 we wanted to investigate if the average blood loss and transfusion requirement in minimal invasive endoscopic coronary artery bypass graft surgery (endo-cabg) differ from conventional technique. we assessed the influence of pre-operative anticoagulant medication for blood loss. estimated average blood loss after conventional cabg 3 is 400ml (+/-200) and transfusion requirement 3,4 units packed red blood cells 4 . we performed a retrospective cohort study of our cardiac surgical database. from 01/01/2016 to 31/12/2017, we collected data from 423 patients undergoing endo-cabg. we analyzed blood loss, transfusion as well as pre-operative use of anti-coagulants as a risk factor for blood loss. we found that mean total blood loss in endo-cabg does not differ from conventional cabg, nonetheless mean transfusion requirement was lower in our cohort. use of direct oral anticoagulant is aossciated with increased blood loss and transfusion requirements (table 1) . total blood loss is not influenced by minimal invasive technique for cabg (endo-cabg). an explanation for the lower transfusion requirements is the use of a minimal extracorporeal circulation, which is known to reduce the risk of transfusion. another important factor is the implementation of a standardized transfusion-protocol based on available evidence. reducing transfusion requirements is an important component in improving patient outcome after cardiac surgery and is related to multiple factors in perioperative care of our patients. retinal microvascular damage associated with mean arterial pressure during cardiopulmonary bypass surgery v shipulin 1 retinal perfusion corresponds to cerebral perfusion and it is very sensitive to hemodynamic disturbances [1, 2] . we investigated the association between retinal microvascular damage and hemodynamic characteristics in patients undergoing coronary artery bypass grafting surgery (cabg) with cardiopulmonary bypass (cpb). methods: 10 patients with coronary artery disease and systemic hypertension were examined. ophthalmoscopy and optical coherence tomography were performed before and 10-14 days after cabg. the hemodynamic parameters during cpb were analyzed. results: 4 (40%) patients had changes in the retinal vessels and in the ganglionic fiber structure on 10-14 day after surgery: in 30% of patients the foci of ischemic retinal oedema appeared, in 10% the decrease of the thickness of ganglionic fiber were observed. these changes may be associated with intraoperative ischemia of the central retinal artery. in 6 (60%) patients the mean arterial pressure (map) during cpb was increased up to 90 mmhg. in 4 (67%) of them the association between map and foci of ischemic retinal oedema were revealed. the ischemic retinal changes were observed significantly more often if the delta of map during cpb was over then 20 mm hg compared with the patients where the delta of map was less than 20 mm hg (p=0.035). this is probably due to an intraoperative disorders of the myogenic mechanism of blood flow autoregulation in the retinal microvasculature in patients with coronary artery disease [3] . the level of map up to 90 mm hg during cpb is associated with retinal blood flow impairment and the foci of ischemic retinal oedema. delta of map more than 20 mmhg was associated with the foci of ischemic retinal oedema and decreased ganglionic fiber thickness in 67% of cases. atrial fibrillation after cardiac surgery: implementation of a prevention care bundle on intensive care unit improves adherence to current perioperative guidelines and reduces incidence introduction: atrial fibrillation after cardiac surgery (afacs) is a very frequent complication affecting 30-50% of all patients. it is associated with an increase in morbidity, mortality and hospital and intensive care unit (icu) length of stay. we aimed to implement an afacs prevention care bundle based on a recently published practice advisory [1] , focusing on early postoperative (re)introduction of β-blockers. baseline afacs incidence and β-blocker administration practices in our centre were audited for all patients undergoing valve surgery or coronary artery bypass graft (cabg) during a 6 weeks period. the afacs prevention care bundlean easy to follow graphical toolwas subsequently introduced to the cardiac icu by a multidisciplinary team and audited following a model of improvement approach. after exclusion of patients with preoperative af, differences between pre-and post-implementation groups were compared with chisquare and fisher's exact tests for categorical, and one-way anova for continuous variables, using spss. a total of 384 patients were analysed. patient and surgery characteristics did not differ between groups. significantly more patients received postoperative β-blockers after bundle implementation (82.7% pre-vs 91.3% post-bundle, p=0.019) with a higher proportion on day 1 (36.7% pre-vs 67% post-bundle, p<0.001, figure 1 ). the incidence of afacs was significantly reduced from 35.4% to 23.3% (p=0.009), with a particularly marked reduction in the age group 65-75 years and for isolated aortic valve and cabg surgery. there was no significant reduction in hospital length of stay for this cohort. introduction of an afacs prevention care bundle using a graphical tool improved adherence to current guidelines with regards to early β-blocker administration and significantly reduced afacs incidence. future care bundles should include preoperative interventions and might reduce hospital length of stay. in neonates with univentricular physiology, there is a delicate balance between pulmonary and systemic circulations, with a tendency towards generous pulmonary blood flow, and a risk of systemic underperfusion. preoperatively, the use of hypoxic gas mixture (hm) has been advocated as a therapy to increase pvr, with the aim of improving systemic oxygen delivery. it is a therapy which has been routinely initiated in our institution in the setting of signs of pulmonary overcirculation. we performed a retrospective analysis of all patients in our institution who underwent a norwood procedure and who received hm preoperatively. we compared peripheral saturations, arterial blood gas analysis, serum lactate, regional cerebral and renal saturations and invasive blood pressure, prior to, and then 4,8 and 24 hours after hm was commenced. between 2014 and 2018 (inclusive), 49 patients underwent the norwood procedure. 18 patients received preoperative hm. average fio2 was 17% during administration of hm. average peripheral saturations were 96.1% prior to hm, and dropped to 87.4% at 4 hours, and 88% at 8 and 24 hours after initiation (p < 0.05). there was no change in any of the measured markers of systemic oxygen delivery, including regional cerebral and renal saturations, lactate, urine output or blood pressure. there was an association between an extended period of hm (> 48 hours) and the need for pulmonary vasodilator therapy post norwood procedure. hypoxic gas mixture in patients with parallel systemic and pulmonary cicrculations causes desaturation and hypoxia. it does not lead to an increase in systemic perfusion and thus an improvement in systemic oxygen delivery. its ongoing use in this fragile population should be considered. introduction: analgesia in the critical patient, and especially in the neurocritical patient, is a basic goal in all therapeutic practices. patients in the icu are frequently administered prolonged and/or high doses of opioids. multiple serious complications due to the use of infusion of opioids at large doses has been described. to reduce high doses of intravenous opioids, multimodal forms of analgesia can be used. prospective observational study of the use of tapentadol enteral and buprenorphine in transdermal patches, at low doses, for the control of pain and its effect on reducing the use of fentanyl infusion in high doses on 84 patients admitted to neuro icu of indisa clinic during 2 consecutive years (2018-19). enteral tapentadol (through ng tube) 50 mg/6 hours, was considered in patients who required intravenous fentanyl in continuous administration. buprenorphine was also added at low doses (5 ug/hr) in a weekly transdermal patch, in cases of neurosurgical spine patients, fractures and long-term neuropathic pain. pain was controlled on behavioral pain scale (bps) and visual analogical scale (vas) scores, according to the conditions of each patient. their hemodynamic, gastrointestinal complications and the appearance of delirium episodes according to cam-icu scale were recorded. results: 84 patients received tapentadol. 32 of them also received transdermal buprenorphine. all managed to maintain adequate level of analgesia, not requiring fentanyl at doses greater than 0.5 ug / kg / hr. distribution by diagnoses: neurotrauma 21 patients, guillain barre 12, spine surgery 15, hsa 18, hice 10, malignant ischemic acv 8. complications: gastric retention 12 patients (7%), hypotension 1 (1%), acute hypoactive delirium 3 (3.5%), acute hyperactive delirium 8 (9%). no drug interactions were found. the introduction of enteral tapentadol and buprenorphine patches in neurocritical patients was safe and resulted in a decrease in the use of endovenous opioids and its adverse effects. we hypothesized that changing the pain management for our post cardiac surgical patients to an assessment-driven, protocol-based approach using fast acting and easily titratable agents will significantly improve patient satisfaction by reducing pain intensity in the first 24h after surgery as suggested by society of critical care [1] guideline. we prospectively assessed 101 and 99 (05.2018 vs 06.2019) consecutive patients before and after introducing our pain management protocol. the nursing and medical team received rigorous training on the guideline as well as the correct assessment using appropriate pain scores measured at least hourly (numeric pain score, ≥ 2 is timing of beta-blocker (re)initiation versus incidence of afacs before and after prevention care bundle implementation, per post-operative day and for postoperative days 1-5 (insets) moderate to severe or critical care observation tool, > 2 is moderate to severe). we introduced a multimodal approach with a combination of fast acting iv, long acting oral opiates, regular paracetamol and rescue iv boluses for difficult to control situations and we created a prescription bundle on our electronic prescribing record. among other variables we assessed hours spent in moderate to severe pain in the first 24h after surgery and compared to the data collected before the guideline was introduced. we analysed 101 patients from 2018 and 99 from 2019. baseline characteristics were similar between the two groups. in 2018 only 41.6% of the patients spent less than 5 hours and 29.4% spend more than 10 hours in moderate to severe pain. the 2019 data showed significant improvement in that 79.5% of patients spent less than 5 hours and only 5% patients who spent more than 10 hours in moderate or severe pain. (p <0.0001, chi square) ( figure 1 ). only 9% of the patient needed rescue medications. 3% of time was the protocol inadequate necessitating other approach. introducing an assessment driven, stepwise, protocolized pain management significantly improved patient satisfaction by reducing pain intensity in the first 24h on our cardiothoracic intensive care unit. introduction: proximal femur fractures are most common fractures in the elderly and associated with significant mortality and morbidity, with high economic and social impact. perioperative pain management influence outcomes and mortality after surgery with early mobilization being possible [1, 2] . the goal of the study was to compare the efficacy and safety of the compartment psoas block for perioperative analgesia in elderly patients with proximal femur fractures. the randomized controlled study was held in medical center "into-sana" (odesa, ukraine) from january 2018 till july 2019. patients with proximal femur fractures and older than 60 years were included in the study. they were randomly allocated to 2 groupscompartment psoas block group (bupivacaine analgesia was started as soon as possible before surgery and prolonged during and after surgery with additional ischiadicus block before surgery) and general (inhalational) anesthesia with systemic analgesia perioperatively. results: 60 patients were included in this study. perioperative compartment psoas block was associated better pain control, decreased opioid consumption, better sleep quality, earlier mobilization after surgery, decreased incidence of opioid-associated vomiting/nausea and myocardial injury. there were no difference in the incidence of hospital acquired pneumonia and delirium. perioperative compartment psoas block is effective and safe for perioperative analgesia in elderly patients with proximal femur fractures, and is associated with better pain control and decreased complications incidence. parenteral olanzapine is frequently used in combination with parenteral benzodiazepines for hospitalized patients with severe agitation. the fda issued a warning for increased risk of excessive sedation and cardiorespiratory depression with this combination based on post-marketing case reports with overall limited quality of evidence [1] . the purpose of this study is to evaluate the safety and efficacy of concomitant parenteral olanzapine and benzodiazepine for agitation. this retrospective chart review evaluated agitated patients who received concomitant parenteral olanzapine and benzodiazepine within 60 minutes from 1/31/2016 to 9/1/2019 . the primary end points were rate of respiratory depression requiring mechanical ventilation and hypotension requiring vasopressors. the secondary end points were percentage of patients requiring additional sedatives for agitation during the same time frame, cumulative dose of olanzapine and benzodiazepine (midazolam equivalent) received, and rate of cardiac arrest and death. a total of 208 patients were included with notable baseline characteristics: median age of 35 years old, 59% with a history of substance abuse, and 40% with a history of psychiatric illness. for the primary outcomes, 2.9% of patients required mechanical ventilation and 0% required vasopressors. additionally, 27.9% patients received additional sedating agents to control agitation. refer to table 1 for more details. no cardiac arrests or deaths were observed. concomitant use of parenteral olanzapine and benzodiazepine within 60 minutes for the treatment of agitation appears to have a small risk of respiratory depression without significant hypotension. hip fracture is very common in the elderly,it causes moderate to severe pain often undertreated. ficb is a simple safe method, easy to learn and use. the aim of our study is to assess the efficacy and safety of preoperative ficb compared with intravenous analgesia for elderly patients with femoral fracture and hip surgery in terms of opioid consumption and perioperative morbidity methods: after informed consent obtained,54 patients 50-98 yo asa i-iii with hip fracture were randomized to receive either an us guided ficb(40 ml of ropivacaine 0,35%) or a sham injection with normal saline 30' before surgery. both groups were operated under general anesthesia. postoperative analgesia was done according to vas: vas 0-30 mm, paracetamol 1g iv at 8 h, vas 30-60 mm, ketoprofen 100 mg iv at 8 h, vas>60, morphine 0,1mg/ kgbw iv. the primary outcome was the comparison of vas score at rest over the first 30'following the procedure, at the end of the surgery and at 6h intervals for 24h. the secondary outcome were the incidence of the cardiovascular events, of the ponv and of the confusion episodes, the amount of morphine consumption for 24h results: at baseline, ficb group (a) had a lower mean pain score than the sham injection group (b). the same difference was observed over 24 h of follow-up (p<0.05). there was a significant difference between the two groups in total cumulative iv morphine consumption at 24 h and in the incidence of ponv and confusion episodes ( figure 1 ). ficb provides effective analgesia for elderly patients suffering from hip fractures, with lower morbidity and lower opioid consumption compared with intravenous analgesia. pain assessment in chronic disorders of consciousness patients with ani monitoring e kondratyeva, m aybazova, n dryagina almazov national medical reseach centre, minimally conscious research group, st petersburg, russia critical care 2020, 24(suppl 1):p166 pain and suffering controversies in doc to be debated by the scientific, legal and medical ethics communities. methods: ani (anti nociception index) monitor was used to assess pain in patients with chronic disordersof consciousness (doc) age range 22 to 56 years -9 in vegetative state/ unresponsive wakefulness syndrome (vs/uws) and 20 minimal consciousness state (mcs). average age: in mcs group 31,8±11,29 and 42,1±8,46 in vs/uws group. neurological status was assessed using crs-r scale. the average score on the crs-r scale was 5±1.4 in vs/uws and 10.45±4.5 in mcs. pressure on the nail phalanx was used as a pain impulse. ani and nociception coma scale was evaluated before the application of pain stimulus, immediately after and past 30 minutes. prolactin level was measured before the pain stimulus application and 10 minutes after. ani less than 50 indicates pain, 70-100 hypoalgesia, 30 severe pain. the mean value of the ani in mcs patients: before the pain stimulus 66.25±14.11, after the pain stimulus application 45±16.12 and 30 minutes later 66.55±18.13. prolactin level in mcs patients before pain 13.01±9.06 ng/ml; after pain 13.75±8.73 ng/ml (p>0.05). prolactin in vs/uws patients before pain 10.79±7.2 ng /ml, after pain 14.5 ±8.88 ng / ml (p> 0.05). conclusions: ani monitor revealed that vs/uws and mcs patients react equally to the pain impulse. prolactin dynamics showed poor statistical mean and can not be consider as a marker of nociception in this group of patients. it is possible that the level of pain impulse was insufficient neuroendocrine response activation or the increase of prolactin level occurs in the long term (more than 10 minutes). in all patients the total hip arthroplasty tha is one of the most common major surgical procedures associated with significant postoperative pain that can adversely affect patient recovery and could increase morbidity. effective perioperative pain management allows an accelerated rehabilitation and improve the functional status of these patients. multimodal analgesia mma combines analgesics with different mechanism of action which by synergistic and additive effects enhance postoperative pain management and reduce complications. the aim of our study is to assess if perioperative association of very low dose of ketamine, a potent nmda antagonist and dexamethasone, by antiemetic and antiinflammatory properties could decrease opioid consumption and postoperative morbidity of patients with tha. after informed consent, 58 patients scheduled for primary hip joint replacement surgery aged 55-91 yo asa i-iii were prospective randomized in two groups. both groups were operated under general anesthesia fentanyl/sevoflurane. supplementary, patients in group a received 12 mg iv dexamethasone and 8mg at 12 h and ketamine 10 mg iv bolus at induction and 10mg/h iv during surgery. postoperative analgesia was done according to vas, 0-30 mm paracetamol 1 g iv at 8 h, 30-60 mm ketoprofen 100 mg iv at 12h, vas>60 mm morhine 0,1 mg/kgbw iv. we recorded perioperative opioid consumption, the number of intraoperative cardiac events, vas score at the end of surgery and at 24 h, the incidence of ponv and persistance of chronic pain at 3 months. we obtain a significant less pain score at the end of surgery p<0.05 in group a, no significant difference at 24 h, a significant less chronic pain at 3 months, a fewer npvo and cardiovascular events in group a, p<0.05 ( figure 1 ). a multimodal approach with very low doses of ketamine and dexamethasone could be efficent in the treatment of pain for elderly patients with hip arthroplasty, decreasing postoperative side-effects and reducing chronic pain persistance. introduction: treatment in an intensive care unit (icu) often necessitates uncomfortable and painful procedures for patients. chronic pain is becoming increasingly recognized as a long term problem for patients following an icu admission [1] . throughout their admission patients are often exposed to high levels of opioids, however there is limited information available regarding analgesic prescribing in the post-icu period. this study sought to examine the analgesic usage of icu survivors pre and post icu admission. methods: 183 patients enrolled in a post-intensive care programme between september 2016 and june 2018. intensive care syndrome: promoting independence and return to employment (ins:pire), is a 5-week multicentre, multidisciplinary rehabilitation programme for icu survivors and their caregivers. patients' level of analgesia was recorded pre-admission and upon attending ins:pire, their level of prescribed analgesia was categorized using the word health organisation (who) analgesic ladder [2] . results: 33.3% of patients (n=61) were prescribed regular analgesia preadmission; this increased to 60.7% (n=111) post-admission, representing a significant absolute increase of 27.4% (95% ci: 20.2% -34.4%, p<0.001) in the proportion of patients who were prescribed regular analgesia pre and post icu. in addition, pre-admission, 22.4% (n=41) of patients were prescribed a regular opioid (step 2 and 3 of the who ladder) compared to 38.7% (n=71) post-admission, representing an absolute increase of 16.3% (95% ci: 9.8% -22.8%, p< 0.001). this study found a significant increase in analgesic usage including opioids in icu survivors. follow-up of this patient group is essential to review analgesic prescribing and to ensure a long term plan for pain management is in place. introduction: pain, agitation, and delirium (pad) are commonly encountered b patients in the intensive care unit (icu). delirium is associated with adverse outcomes, including increased mortality and morbidity. clinical guidelines suggest that routine assessment, treatment and prevention of pad is essential to improving patient outcomes. despite the well-established improvements on patient outcomes, adherence to clinical guidelines is poor in community hospitals. the aim of this quality improvement project is to evaluate the impact of a multifaceted and multidisciplinary intervention on pad management in a canadian community icu. a pad advisory committee was formed and involved in the development and implementation of the intervention. the 4-week intervention targeted nurses (educational modules, visual reminders), family members (interviews, educational pamphlet, educational video), physicians (multidisciplinary round script), and the multidisciplinary team (poster). an uncontrolled, before-and-after study methodology was used. adherence to pad guidelines in the assessment of pad by nurses was measured 6 weeks pre-intervention and 6 weeks post-intervention. data on 430 patient-days (pd) and 406 pd were available for analysis during the pre-and post-intervention, respectively. the intervention significantly improved the proportion of pd with assessment of pain and agitation at least 4 times per 12-hour shift from 68.0% to 87.5% and from 69.7% to 82.2%, respectively ( figure 1 ). proportion of pd with delirium assessment at least once per 12-hour shift did not significantly improve. a multifaceted and multidisciplinary pad intervention is feasible and can improve adherence to pad assessment guidelines in community icus. quality improvement methods that involve front-line staff can be an effective way to engage staff with pad. oversedation introduction: sedation is a significant part of medical treatment in icu patients. a too deep sedation is associated with a longer time of mechanical ventilation, lung injury, infections, neuromuscular disease and delirium, which can lead to a longer duration of icu hospitalization, as well as an increase of morbility and mortality. many patients spend a considerable amount of time in a non-optimal sedation level. a continuous monitoring system of the sedation level is therefore necessary to improve clinical evaluation. our goal was to evaluate the incidence of non-optimal sedation (under and over sedation) comparing the parameters expressed from ngsedline with clinical evaluations and to correlate oversedation and the incidence of delirium. we have studied a cohort of patients admitted to the icu of spedali civili of brescia university hospital requiring continuous sedation for more than 12 hours. in addition to standard monitoring, the patients have been studied using next generation sedline (masimo). sedation depth was evaluated through rass scale and the presence of delirium was evaluated with cam-icu scale. we collected data from 50 adult patients. our data showed high incidence of oversedation. 45 of our 50 patients had a sr>2 and 48 had a psi level<30. a logistic regression analysis was performed and it showed statistically significant association between incidence of delirium and the age of the patients (p 0.009). the association between delirium incidence and suppression rate time was at the limits of statistics significance (p 0.059) and was statistically significant for non neurocritical patients (p 0.049). our study didn't show an association between delirium and the total time of sedation. non-optimal sedation is an unsolved problem in icu, affecting lot of patients, with a major incidence of over-sedation compared to under-sedation. our study shows an association between sr levels and the incidence of delirium. predictors of delirium after myocardial infarction, insights from a retrospective registry m jäckel, v zotzmann, t wengenmayer, d dürschmied, c von zur mühlen, p stachon, c bode, dl staudacher heart center freiburg university, department of cardiology and angiology i, freiburg, germany critical care 2020, 24(suppl 1):p172 delirium is a common complication on intensive care units. data on incidence and especially on predictors of delirium in patients after acute myocardial infarction (mi) are rare. by analyzing all patients after acute mi, we aim to identify incidence and potential risk factors for delirium. in this retrospective study, all patients hospitalized for acute mi treated with coronary angiography in an university hospital in 2018 were included and analyzed. incidence of delirium within the first 5 days of care attributed to the mi and was defined by a nudesc score ≥2, which is taken as part of daily care three times a day by especially trained nurses. this research is authorized by ethics committee file number 387/19. results: 626 patients with acute mi (age 68.5±13.3 years, 260 stemi, mortality 3.4%) were analyzed. delirium occurred in 70 (11.2%) patients and was associated with a longer hospital stay (12±15.9d vs 5.5±4.3d, p< 0.001). patients with delirium were significantly older than patients without (76.3±11.14 vs. 67.5±13.10 years, p<0.001) and had more often preexisting neurological diseases (24.3% vs. 10.8%, p<0.001) and dementia (11.4% vs. 1.4%, p<0,001). multivariate logistic regression analysis suggested that odds ratio for delirium was higher in patients after resuscitation or 7.5 (95% ci 2.1-26.7), preexisting dementia or 28.9 (ci 3.1-268) and in patients with alcohol abuse or 18 (ci 2.7-120). while maximum lactate was also connected to delirium or 1.4 (ci 1.1-1.9), infarct size or type had no effect on the incidence of delirium. in patients with mi, delirium is frequent. incidence is associated with clinical instability and preexisting neurological diseases rather than infarct size. incidence and risk factors of delirium in surgical intensive care unit ma ali, b saleem aga khan university, anaesthesia, karachi, pakistan critical care 2020, 24(suppl 1):p173 introduction: delirium in the critically ill patients is common and distressing. the incidence of delirium in the icu ranges from 45% to 87%. although delirium is highly common among intensive care patients, it is mostly underreported. to date, there have been limited data available related to prevalence of delirium in surgical patients. in a study published in 2008, the risk was observed 73% in surgical and trauma patients [1] . the purpose of this study was to find out the incidence and associated risk factors of delirium in surgical icu (sicu) of a tertiary care hospital. we conducted prospective observational study in patients with age more than 18 years and who were admitted to the surgical icu for more than 24 hours in aga khan university hospital from january 2016 to december 2016. patients who had preexisting cognitive dysfunction or admitted to icu for less than 24 hours were excluded. delirium was assessed by intensive care delirium screening checklist icdsc. incidence of delirium was computed and univariate and multivariable analyses were performed to observe the relationship between outcome and associated factors. delirium was observed in 19 of 87 patients with an incidence rate of 21.8%. multivariable analysis showed that copd, pain >4 and .76] were also the strongest independent predictors of delirium while analgesics exposures was not statistically significant to predict delirium in multivariable analysis. delirium is significant risk factor of poor outcome in surgical intensive care unit. . there was an independent association between pain, sedation, copd, hypernatremia and fever in developing delirium delirium is an acute mental syndrome which may cause negative consequences if it is misdiagnosed [1, 2] . the aim of this study was to determine the incidence of delirium in different intensive care units and reveal the risk factors. the study was performed with 212 patients hospitalized in intensive care units of anesthesia, neurology and general surgery departments. written informed consent was obstained from patients or relatives. delirium screening test was performed twice daily with camicu (confusion assessment method for the icu). patients who met the study criterias, were evaluated for the possible risk factors of delirium and the data was recorded daily. patients were reevaluated after the treatment. the incidence of delirium was 32.5%. delirium was found to increase with the length of stay (p <0.001). the mean age of the patients with delirium was 67.46. this was higher than the patients without delirium (52.48) (p<0.001). visual impairment (p<0.001), hearing impairment (p=0.001), educational status (p=0.024), hypertension (p=0.002), mechanical ventilation (p = 0.001), oxygen demand (p=0.002), midazolam infusion (p=0.025), propofol infusion (p=0.042), infection (p < 0.001), sofa (p = 0.001), apache ii (p <0.001), nasogastric catheter (p=0.012), aspiration (p <0.001), number of aspirations (p<0.001), enteral nutrition (p<0.025), albumin (p=0.025), steroid (p=0.024), hypercarbia (p=0.039) hypoxia (p=0.039), sleep disturbance (p<0.001) were found risk factors for delirium. oral nutrition (p<0.001) and mobilization (p=0.003) were found to prevent delirium development. various factors are important in the development of delirium. these risk factors should be considered in reducing the incidence of delirium in intensive care units. ). an unplanned and brutal stop of alcohol consumption, as it can occur during icu admission, may lead to an alcohol withdrawal syndrome (aws). the most severe clinical manifestation of aws is described as delirium tremens (dt). there are no current guidelines available for aws treatment in icu. the study's aim was to describe the clinician's practices for dt treatment and the outcome of dt in icu patients. observational retrospective cohort study in two icus of a universityaffiliated, community hospital in france. patient diagnosed for dt during their icu stay, as defined by dsm-v classification, were enrolled in the study. results: 61 patients with dt were included between 2014 and 2017. benzodiazepines was administered to 23% of the patients in order to prevent an aws. as associated measures, vitamin therapy was administered to 83% of the patients and 59% had an increased fluid intake (mean 2.5l+/-0.7). concerning the curative approach of aws, the treatment's heterogeneity was notable. there was a high frequency of treatment's association (66% of the patients), every patient had benzodiazepines and the use of second line treatments such as neuroleptic, alpha-2 agonist, propofol was variable ( figure 1 ). complications of dt were the following: need for mechanical ventilation due to unmanageable agitation or acute respiratory distress (33% of the patients) self inflicted injuries such as pulling out of central lines, tubes, surgical drain (46%) falls (7%). seizures (33%). delirium tremens is a severe complication of an untreated aws, which can lead to serious adverse events in icu. the current lack of evidence concerning the management of aws in icu probably explains the heterogeneity of treatments. given the potential severity of aws in icu, further evidences are required to optimize care of aws in icu patients. the incidence and related risk factor of delirium in surgical stepdown unit s yoon 1 , s yang 2 , g cho 2 , h park 2 , k park 2 , j ok 2 , y jung 2 1 asan medical center, nursing department, seoul, south korea; 2 asan medical center, seoul, south korea critical care 2020, 24(suppl 1):p177 step down units (sdus) provide an intermediate level of care between the icu and the general medical-surgical wards. the critically ill patients who are in recovery after long-term intensive care or who require monitoring after acute abdominal surgery are admitted to sdus. delirium in critically ill patient is common and leads to poor clinical outcomes. it is, however, preventable if its risk factors are identified and modified accordingly. to determine risk factors associated with delirium in critically ill patients to admitted surgical sdu at asan medical center. this is retrospective study conducted on critically ill patients who were admitted to the sdu from september 2018 to april 2019 and able to express themselves verbally. delirium status was determined using the short-cam tool. data were analyzed by spss 13.0 software, using t-test, fisher's exact test and logistic regression. the incidence of delirium was 32.1%(25 of 78 patients) and hypoactive delirium(12case, 48.0%) was the most commonly assessed, followed by hyperactive delirium(9case, 36.0%), mixed type(4case, 5.1%). risk factors associated with developing delirium identified from univariate analysis were age(p=0.048), admission via icu (p= 0.041), tracheostomy (p=0.037), chronic heart failure (chf) (p=0.017), invasive hemodynamic monitoring (p=0.041), heart rate (p= 0.037). after adjusted in multivariate analysis; factors those remained statistically significant were old age (rr we identified risk factors consistently associated with incidence of delirium following admitted to surgical sdu. these factors help to focus on patients at risk of developing delirium, and to develop preventive interventions that are suitable for those patients. patients with sepsis frequently develop delirium during their intensive care unit (icu) stay, which is associated with increased morbidity and mortality. the prediction model for delirium in icu patients (pre-deliric model) was developed to facilitate the effective preventive strategy of delirium [1] . however, the pre-deliric model has not yet been validated enough outside europe and australia. the aim of this study is to examine the external validity of the pre-deliric model to predict delirium using japanese cohort. this study is a post hoc subanalysis using the dataset from previous study in nine japanese icus, which have evaluated the sedative strategy with and without dexmedetomidine in adult mechanically ventilated patients with sepsis [2] . these patients were assessed daily throughout icu stay using confusion assessment method-icu. we excluded patients who were delirious at the first day of icu, were under sustained coma throughout icu stay and stayed icu less than 24 h. we evaluated the predictive ability of the pre-deliric model to measure the area under the operating characteristic curve. calibration was assessed graphically. of the 201 patients enrolled in the original study, we analyzed 158 patients in this study. the mean age was 69.4 ± 14.0 years and 99 patients (63%) were male. delirium occurred at least once during their icu stay in 63 patients (40%). to predict delirium, the area under the receiver operating characteristics curve of the pre-deliric model was 0.60 (0.50 to 0.69). graphically, the prediction model was not well-calibrated ( figure 1 ). to predict delirium in japanese icus, we could not show the well discrimination and calibration of the pre-deliric model in mechanically ventilated patients with sepsis. introduction: delirium is a serious and common complication and in some cases it treatment is difficult. aim of the study was an evaluation of the prevalence, structure of delirium and efficacy of dexmedetomidine and haloperidol sedation in geriatric patients after femur fracture. after local ethic committee approval 207 case-records of geriatric patients with femur fracture in the period from 2017 to 2018 in the institute of traumatology and orthopedics in astana were analyzed. patients was divided for 2 groups: in dpatients with delirium treated by i/v dexmedetomidine (0.2-1.4 mkg/kg per hour), in g group patients with delirium treated by i/v galoperidol (0.10-0.15 mkg/kg). delirium was assessed by rass at day of permission and every day at 8 a.m. the prevalence, structure of delirium and efficacy of sedation were analysed. results: by anthropometric and gender characteristics of the group did not differ. the average age in the d-group with delirium was 81.8±0.9 years old, which was comparable to the g-group -79.7±0.7 years old (p = 0.06). all study participants had similar comorbidities. delirium in all patients debuted at 2.0±1.4 days, with an average duration of 2.2±1.2 days. the effect of dexmedetomidine was better and expressed in 52% decrease in the duration of delirium in compare to haloperidol (p <0.05). dexmedetomidine provided a more controlled and safe sedation compared with haloperidol. the average consumption of narcotic analgesics in the subgroup with dexmedetomidine was two times less than in the subgroup with haloperidol. thus, the average consumption of trimeperidine hydrochloride in patients of group d was 6.9 mg versus 14.1 mg in group g (p = 0.004). in gerontological patients with femur fracture treatment delirium by dexmedetomidine was more effective in compare with haloperidol. when using dexmedetomidine, the consumption of narcotic analgesics in postoperative period was 50% less than with haloperidol. live music therapy in intensive care unit mc soccorsi 1 , c tiberi 1 , g melegari 1 , j maccieri 2 , f pellegrini 2 , e guerra 1 intensive care units (icu) are not comfortable for patients, relatives or next of kin. in the last years many news approaches were described to implement the humanization of medical treatments. the positive effect of music therapy in icu is well described, especially reducing delirium risk [1] . the aim of this paper is describing the effect in patients and their family of a music live performance in icu. after ethical committee approval (procedure aou 0018356/18, italy) for three months (november 2018-january 2019) patients in icu were treated twice a week with live music therapy performed by coral vecchi-tonelli of modena, italy (fig. 1 ). data were collected all awake and conscious patients. vitals parameters, gcs, raas and cam icu were collected before, during and after the treatment, at every performance. after the treatment a feedback questionnaire were given to patients and to next of kin. results: 18 subjects were enrolled in the research with mean age of 66.66 years old, delirium rate before the treatment was 16.38% later 15.38%, raas does not show any difference. over 85% of patients were satisfied, and relatives felt less anxiety. we recorded also a satisfaction also in relatives not enrolled. the study does not demonstrate a delirium risk reduction for the small sample and the length treatment, anyway it was recorded a low delirium rate. the safety and the potential effect of music therapy are well known, surely the research underlines the feeling of patients and their next of kin: icu is the most stressful setting for admitted patients and its humanization is a current topic for medical literature. live performances could be an entertainment moment and probably create a moment of an interaction among patients, their family and medical and nurse: icu become more human. the high level of satisfaction push us to continue this experience. introduction: patients undergoing medical procedures benefit from distraction techniques to reduce the need for drugs alleviating pain and anxiety. this study investigates if medical hypnosis or virtual reality glasses (vrglasses) as adjuvant method reduces the need for additional drugs. in a prospective, randomized, interventional trial, patients undergoing procedures were stratified in four age groups, and randomly assigned into three arms by means of a closed envelope system. all patients received standard care for pain before the procedure; the control group received further drugs for pain and stress as indicated by the visual analog scale (vas; threshold 3/10) and comfortscore (threshold 14/30), two index groups received either medical hypnosis or vr glasses as a plus before and during the procedure. vas and comfort were scored continuously and analysed with the kruskal-wallis test. patients, parents and healthcare providers scored their satisfaction at the end. of 104 included patients 6 to 86 years old, 47% were female. regardless of age, pain and comfort scores were similar before and at the start of the procedure (vas 3.7-4.2; comfort 16-16.7), but as of one minute after starting the procedure, both vas and comfort reduced significantly more in both index groups compared to the control (p< 0.001), remaining far below the threshold for both pain and stress ( figure 1 ). there was no advantage of one index group over the other (p=0.43). there were no adverse effects. patients in the vr group were more satisfied than in the standard group (p=0.02) or in the hypnosis group (p=0.04). there was no significant difference in satisfaction of parents or healthcare providers. from the very start of the intervention, the application of either medical hypnosis or vr glasses significantly reduces pain and anxiety in patients undergoing medical procedures. more studies are needed but both are promising safe adjuvant tools to standard pharmacological treatment. music to reduce pain and distress due to emergency care: a randomized clinical trial ne nouira, i boussaid, d chtourou, s sfaxi, w bahria, d hamdi, m boussen, m ben cheikh mongi slim academic hospital, emergency department, tunis, tunisia critical care 2020, 24(suppl 1):p182 recent clinical studies have confirmed the benefits of music therapy in managing pain and improving quality of care in the emergency department. the aim wasto evaluate the impact of receptive music therapy on pain and anxiety induced by emergency care methods: a randomized controlled study in patients consulting the emergency department. two groups: the music therapy group; patients needed venous sampling, peripheral venous catheter or arterial catheter. will bless ten minutes music therapy by headphones and a second control group of patients with the same care without music therapy. consent was requested from all participants. the level of pain caused by the act of care was assessed by visual analogic scale. heart rate, blood pressure and the mood of the patient were assessed before and after emergency care. we assessed patient satisfaction, adverse events. patients admitted to the emergency room, patients with communication difficulties and non-consenting patients were not included results: two hundred and forty patients were included randomized in both groups, 123 with music therapy and 117 without music therapy, the results showed comparable characteristics between the two groups: demographic data, pathological history, and initial clinical presentation. after the session of music therapy a difference was noted in the evaluation of the mean vas who was in the group with music of 2.87 ± 1.82 versus 3.60 ± 2.06 in the control group p< 0.004 ci 95% [-1.23; -0.23], and the mean of diastolic blood pressure which was 71, 07 mmhg in the first group against 74.27 mmhg for the control group p = 0.048 ci 95% [-6.37; -0.25]. as for the mood, the patients were more smiling after the act of care in the group music therapy. all patients were satisfied with their experience and 93% recommend this therapy to their relatives . music therapy may reduce pain and anxiety in patients during emergency care. the music therapy is the intervention of music and/or its elements to achieve individual goals within a therapeutic.the music has proved to have positive physiological and psychological effects on patients [1] . patients admitted to the intensive care unit (icu) experience anxiety and stress even when sedated, negatively influencing recovery [2] . methods: two groups are established, a music therapy group (mg) and a control group (cg). the first one undergoes music therapy interventions, it consists of 10-minutes sessions of live music. patients of the gc will receive the usual treatment established by the service protocol for weaning management and the data are collected during the same time interval. data collection includes mean arterial pressure (map), heart rate (hr), respiratory rate (rr), oxygen saturation (sao2) and temperature (t). a total of 28 patients were recruited, of which 6 patients had to be excluded for meeting any of the exclusion criteria (n=22). of which 13 (n=13) were randomized in the gm and the rest to the gc (n=9) ic95%. regarding delirium in gm 9 (40.9%) presented a positive cam-icu, while in the cg were 13 (59.9%) (p=0.09). when analyzing the variables in the cg and gm, it was observed that there were no differences with respect to hr, rr and map variable ( figure 1 ). according to the results, we can say that music therapy as a nonpharmacological strategy for management of anxiety and delirium in patients of critical care units, might be an useful tool for the management of patients in weaning of mechanical ventilation introduction: coagulopathy and basopenia are common features of anaphylaxis, but the role of coagulopathy in anaphylaxis remains uncertain. the aim of this study is to evaluate the association between coagulopathy and clinical severity or basopenia in patients with anaphylaxis. we conducted a single-center, retrospective study of patients with anaphylaxis about their coagulopathy. levels of fibrin degradation products (fdp) and d-dimer were analyzed with the cause of anaphylaxis, clinical symptoms, medications and outcomes. we also studied the levels of intracellular histamine as a biomarker of basophil degranulation in the peripheral blood in relation to fdp and ddimer. in total, sixty-nine patients were enrolled to the study, and the levels of intracellular histamine were analyzed in 14 patients. the symptoms included respiratory failure (n=47), shock (n=56), abdominal impairment (n=20), and consciousness disturbance (n=37). thirty-two patients needed continuous intravenous vasopressors for refractory shock. the increase of fdp was significantly associated with consciousness disturbance (p=0.029) and refractory shock (p<0.0001). the increase of d-dimer was also significantly associated with refractory shock (p=0.0066). there was no correlation between the levels of intracellular histamine and either of fdp or d-dimer (p=0.13 and p=0.16, respectively). the increase of fdp and d-dimer were associated with severe symptoms of anaphylaxis, while they were not correlated with intracellular histamine. these results suggest that anaphylaxis is closely associated with coagulopathy in a mechanism which is different from basophile degranulation in anaphylaxis. cardiac manifestations of h1n1 infection in a greek icu population e nanou 1 , p vasiliou 1 , e tsigou 2 , v psallida 1 , e boutzouka 2 , v zidianakis 1 , g fildissis 1 1 agioi anargiroi hospital, attiki, greece; 2 agioi anargiroi hospital, icu, attiki, greece critical care 2020, 24(suppl 1):p185 introduction: cardiovascular involvement in influenza infection occurs through direct effects on the myocardium or through exacerbation of pre-existing cardiovascular disease [1] . the aim was to study cardiac manifestations in all pts admitted to the icu with severe influenza's attack. clinical, laboratory, electrocardiographic, echocardiographic and hemodynamic data were retrospectively recorded in all pts admitted to the icu due to influenza infection (winter 2018-spring 2019). diagnosis was established by pcr on bronchial aspirates the next 7 days after admission. myocardial injury was defined by troponin levels >116 pg/ml (10 fold uln). left ventricular systolic dysfunction was defined as ef <50% and was characterized as either global or regional. hemodynamic monitoring by fig. 1 (abstract p183) . comparison between mg and cg transpulmonary thermodilution method (picco) was recorded in pts with shock (norepinephrine >0.1 μg/kg/min). values are expressed as mean±sd or as median (ir). results: nine pts (5 males) with a mean age 49.78±17.01 years, apache ii 19±5.29 and sofa score 10.50±2.93 were assessed. icu admission was due to ards (7) and copd exacerbation (2) . icu los was 24.44± 14.19 days and mortality rate was 18%. no history of vaccination or coronary heart disease was referred. results are shown in table 1. levosimendan was administered in 2 pts with severe cardiogenic shock. in all survivors, shock and indices of myocardial dysfunction subsided till discharge. coronary angiography was performed in 1 pt showing no abnormalities. mortality was attributed to septic shock and multi-organ failure. myocardial involvement, though common in influenza pts admitted to the icu, didn't contribute to a dismal prognosis. the cardioprotective effects of levosimendan could be related to the modulation of oxidative balance. we aimed to examine the effects of levosimendan in patients with cardiogenic shock or with ejection fraction (ef) lower than 30% on cardiac systo-diastolic function and plasma oxidants/antioxidants (glutathione, gsh; thiobarbituric acid reactive substances, tbars). in 4 patients undergone coronary artery bypass grafting or angioplasty, cardiovascular parameters were measured at t0 (before the beginning of levosimendan, 0.1mcg/kg/min), t1(1 h after the achievement of the therapeutic dosage of levosimendan), t2 (at the end of levosimendan infusion), t3 (at 72 h after the end of levosimendan infusion), t4 (at the end of cardiogenic shock). the same time-course was followed for plasma gsh and tbars measurements. we found an improvement in cardiac output, cardiac index and systolic arterial blood pressure. ef increased from mean 25% to 45%. a reduction of central venous pressure and wedge pressure was also observed. moreover, indices of diastolic function were improved by levosimendan administration (e/e' from 14 to 6; e/a from >1 to <1) at early t2. it is to note that an improvement of gsh and tbars was observed early after levosimendan administration (t1), as well ( figure 1 ). the results obtained have shown that levosimendan administration can regulate oxidant/antioxidant balance as an early effect in low cardiac output patients. the modulation of oxidative condition could be speculated to play a role in exerting the cardio-protection exerted by levosimendan in those patients. table 1 . early administration of vasopressors and their use in the emergency department was associated with survival in septic shock. this seemed to be independent of median map recorded in the ed. we excluded all the traumatic or post-myocardial infarction forms. out of 83 patients, the tuberculous etiology was identified in 15 cases (18,1%), mean age was 34 years, 57,8% were men. 9 patients reported a tb contact in their environment, 5 had a medical history of pulmonary tb. after pericardiocentesis, the liquid was citrine yellow in 6 cases and hematic in 5 patients, no patient underwent surgical drainage in our serie. mycobacterium tuberculosis was found in the expectorations in 4 cases and ada was positive in 4 patients. hiv serology was negative in all our patients. a 6 months anti bacillary therapy with isoniazid, rifampin, pyrazinamide, and ethambutol was initiated in all our patients with a good evolution in 7 cases, 2 deaths, 1 chronic constrictive pericarditis, 2 small pericardial effusion and 3 lost to follow-up. althought cardiac tamponade is rarely caused by tuberculosis, this condition remains common in endemic countries such as morocco and affect younger population, hence the importance of a better knowledge of its prevalence and and multidisciplinary management and more importantly the treatment of the underlying cause using combined antibacillary medication that has shown satisfying results. 1. the main perceived limiting factor is the absence of a standardized didactic program, followed by mentor's availability in residents' perception and by mentor's experience in consultants' one. pocus teaching is present although not optimal and not homogenous in italian acc residency schools. standardisation of residents' ultrasound curriculum is suggested to improve ultrasound teaching. the study included a convenience sample of critically ill patients with supradiaphragmatic cvcs and a cxr for confirmation. us is used for direct confirmation of the guidewire in the internal jugular (ijv) or subclavian (scv) vein and visualizing the guidewire in the right atrium. to evaluate for pneumothorax, "sliding sign" of the pleura was noted on us of the anterior chest. results: 34 patients have been included, 35% of the catheters have been placed in the scv and 65% in the ijv. it was possible to confirm the position of the cvc tip for 70.6% (23 correct, 1 incorrect cxr) of 34 (figure 1 ). overall, it was not possible to identify the guide in the right atrium 11 cases (10 false negatives, 2 of them due to the presence of defibrillator leads). regarding the 1 case where an incorrect position was seen on cxr it was also detected on ultrasound: us of the inserted vein and a negative tte confirmation. in all cases it was possible to exclude a pneumothorax by us. these results show that bedside ultrasound might be a feasible technique to confirm the cvc positioning. it is important to note that the level of the operator's expertise is significant when assessing the feasibility of this method. we only had a limited sample size and the occurrence of only one misplaced catheter. these preliminary results need to be confirmed on a larger scale. central venous catheter (cvc) misplacement occurs more frequently after cannulation of the right subclavian vein compared to the other sites for central venous access. misplacement can be avoided with ultrasound guidance by using the right supraclavicular fossa view to confirm correct guidewire j-tip position in the lower part of the superior vena cava. however, retraction of the guidewire prior to the cvc insertion may dislocate the j-tip from its desired position, thereby increasing the risk of cvc misplacement. the aim of this study was to determine the minimal guidewire length needed to maintain correct guidewire j-tip position throughout an us-guided infraclavicular cvc placement in the right subclavian vein. methods: 100 adult intensive care patients with a computed tomography scan of the chest were retrospectively and consecutively included in the study. the distance from the most plausible distal puncture site of the right subclavian/axillary vein to the junction of the right and left brachiocephalic veins (= vessel length) was measured using multiplanar reconstructions. in addition, measurements of the equipment provided in commonly used 15-16 cm cvc kits were performed. the minimal guidewire length was calculated for each cvc kit. the guidewires were up to 90 mm too short to maintain correct j-tip position throughout the cvc insertion procedure in seven of nine commercial cvc kits. four of these are shown in table 1 . when us guidance is used to confirm a correct guidewire j-tip position, retraction of the guidewire prior to the cvc insertion must be avoided to ensure correct cvc-tip positioning. this study shows that most of the commonly used 15-16 cm cvc kits contain guidewires that are too short for cvc placement in the right subclavian vein. the reliability of lung b-lines to assess fluid status in patients with long period of supine introduction: ultrasound-guided cannulation is usually done using either longitudinal or transverse approach. the oblique approach utilizes advantages of both these approaches allowing visualization of the entire course of needle including tip and lateral discrimination of artery from vein [1] . the reported incidence of the complete overlap of femoral vein by the femoral artery is 8-10 percent [2, 3] . we describe the use of the oblique approach for successful cannulation of such a femoral vein which is not possible by usual approaches (figure 1 ). endothelial cells play a pivotal role in the atherogenic process. endothelial cell dysfunction (ed) is the main risk factor for cardiovascular diseases such as hypertension, coronary heart disease (chd) and peripheral occlusive disease (pod). these diseases significantly increase the risk for perioperative complications. therefore, identifying patients with ed is important and should influence our prospective perioperative strategy. however, sensitive tools to diagnose ed are still missing and do not belong to our standard of care. aim of this study was the validation of a new non-invasive method to detect ed and a correlation with a set of established an new endothelial biomarkers. the cohort includes 20 preoperative patients without anamnestic relevant cardiovascular disease and 20 patients with known peripheral occlusive disease (pod). we used non-invasive endopat® technology from itamar-medical to measure ed by changes in vascular tone before and after occlusion of the brachial artery and calculate a reactive hyperemia index (rhi). in addition, we measured established markers and alternative biomarkers potentially indicate vascular diseases such as substrates and products from the no-metabolism l-arginin, asymmetric/symmetric dimethylarginine (adma/sdma), von-willebrand factor (vwf) and sphingosine-1-phosphate (s1p). rhi was able to identify patients with pod. rhi was significant lower in patients with clinical signs and symptoms of pod (p<0.05). among other markers adma was significant higher in pod patients compared to controls and correlates with rhi. the pad technology is a helpful non-invasive functional test to measure ed and seems able in identify patients with vascular disease. in future, a combination of anamnesis, new diagnostic tools and biomarkers may further increase our sensitivity in identifying risk-patients. single-lumen 5fr and triple-lumen 6fr peripherally inserted central catheters (piccs) for cardiac output assessment by transpulmonary thermodilution s d´arrigo 1 achieving effective critical care in low-and middle-income countries is a global health goal [1] , which includes the provision of effective point of care ultrasound [2] . we sought to establish zambia's first focused critical care echocardiography training programme in a 16bedded icu at university teaching hospital, lusaka. the programme was accredited by the uk intensive care society fice programme, with teaching adapted for local disease patterns such as tuberculous pericardial effusions. parasternal, apical and subcostal windows were used to assess ventricular dysfunction, hypovolaemia, pleural effusion, alveolar interstitial syndrome and pneumothorax. zambian doctors working with critically ill patients received an intensive one-day course, followed by mentored scanning at the bedside. teaching was delivered by visiting fellows from the uk who are accredited in echocardiography and experienced ultrasound educators. patients with abnormal mean ci or hr suffer from increased hospital mortality. abnormality of mean svi was not associated with mortality. these data support accurate measurement of ci as a hemodynamic target and the normal range defined for ci. since ci also carries the hr information, ci seems to be the more important target than svi. our data cannot necessarily be interpolated to less invasive and less precise measurements of ci. an evaluative study of the novelty device with the function of auto-aspirating and pressure indicator for safety central venous catheterization ly lin, wf luo, cy tsao national taiwan university hospital, taipei, taiwan critical care 2020, 24(suppl 1):p203 previous studies have shown that 0.8% of cvc attempts resulted in arterial punctures that were not recognized by blood color. to overcome the problem, our team has developed a concept of pressure detecting syringe that can indicate the artery puncture [1] . based on previous research, different springs, the actuator of the design, have been evaluated to optimize the proposed device and reduce the risk of cvc procedure. tested devices -the inner-spring is set between the pressure indicator and plunger (fig. 1a ). three springs are tested. test condition -blood samples were simulated by glucose solution with absolute viscosities of 2 and 6 mpa-s. different blood pressures were applied to simulate the artery and vein (fig. 1b) . the response time (rt) is defined as the time required to show the indicating signal (is) which is the movement of the piston from the position in fig. 1b : a2-1 to a2-2. the rt is strongly influenced by spring (fig. 1b) but every design can show the is when pressure is higher than 50 mmhg, the assumed minimum artery pressure. the rt of s1, the strongest spring design, is about 10s in the 50mmhg-pressure and high viscosity condition. during our tests we found the user can realize the is before the position be fully changed from fig. ib : a2-1 to a2-2. thus, we believe the 10s rt, the worst case, is still acceptable. we also found the weak spring force may lead to difficulty to empty the syringe because the spring must to overcome the blood pressure and the friction between the piston and barrel. as a result, it was difficult for s3 to absolutely empty the syringe even if the blood pressure is only 30 mmhg. the spring will be compressed as fig. 1b : a2-2 and fail to push the piston when pushing the plunger forwardly, which is not acceptable in clinical use. the results indicate the feasibility of using the device to facilitate cvc and we believe the s1 or s2 are more suitable for the future application. introduction: models using standard statistical features of hemodynamic vital sign waveforms (vs) enable rapid detection of covert hemorrhage at a predetermined bleed rate [1] . by featurizing interactions between vs we can train powerful hemorrhage detectors robust to unknown bleed rates. waveforms (arterial, central venous, pulmonary arterial pressures; peripheral and mixed venous oxygen saturation; photoplethysmograph; ecg) of healthy pigs were monitored 20 min prior and during a controlled hemorrhage at 20 ml/min (n=38) and 5 ml/min (n=13). two sets of vs features were extracted: statistical features [1] and maximal pairwise cross correlations between pairs of vs within a 5s lag over various time window sizes (30s, 60s, 180s, 300s); and normalized with pre-bleed data of each given animal. for each feature set, a tree-based (ert) model [2] was trained and tested in a one-animal-out setting to mitigate overfitting on the 20 ml/min cohort, and another trained on the 5 ml/min and tested on the 20 ml/min cohort. we evaluated models with activity monitoring operating characteristics curves [3] that measure false alert rate as a function of time to detect bleeding. models using cross-correlations show no significant deterioration of performance when applied to detect bleeding at different rates than trained for, while standard models require 64s longer on average to detect hemorrhage at 1% false alert rate in the previously unknown setting ( figure 1 ). correlations between vs data encode physiologic responses to hemorrhage in a way independent of the actual bleed rates. this enables training effective hemorrhage detectors using only limited experimental data, and using them in practice to detect bleeding that occurs at rates other than used in training. we validated a dataset of 634 data lines containing hemodynamic variables and treatment options. we selected nine hemodynamic variables as inputs. furthermore, data were collected regarding underlying conditions: heart failure, septic shock, renal failure or respiratory failure or a combination. we applied datastories regression on the dataset (turnhout, belgium, www.datastories.com). six different interventions were analyzed as kpi: administration or removal of fluids, increasing or decreasing inotropes and increasing or decreasing vasopressors. finally, we elaborated and challenged 63537 predictive models to generate a decision algorithm to predict each kpi. we first looked at how each hemodynamic parameter impacts the prediction of each kpi individually and performed a standard correlation analysis as well as a more involved analysis of the mutual information content between each kpi and all other hemodynamic parameters individually. confusion matrix and variable importance was obtained for each kpi. the baseline hemodynamic parameters were: gedvi 738±218 ml/m2, evwli 11.2±5.5 ml/kg pbw, svv 14.8±8 %, mbp 77.8±16.5 mmhg, hr 94.2±23.5 bpm, ci 3.3±1.2 l/min.m2. the results of the regression analysis identified the different variables of importance for each of the different interventions ( fig 1a) . based on these results the hemodynamic variables (hr, mbp, gedvi, elwi, ci, svv) were used to develop the final hemoguide prediction model ( fig 1b) . the hemoguide app can be used to advise physicians with respect to basic therapeutic decisions at the bedside or as an educational tool for students. with the collection of new data, the accuracy of the system may grow over time. the next step of the project is to develop a more-sophisticated suite: the icu cockpit. feedback function contributes to accurate measurement of capillary refill time r kawaguchi 1 , ta nakada 2 , m shinozaki 2 , t nakaguchi 2 , h haneishi 2 , s oda 2 1 chiba university, department of emergency and critical care medicine, chiba, japan; 2 chiba university, chiba, japan critical care 2020, 24(suppl 1):p206 capillary refill time (crt) is well known as an indicator of peripheral perfusion. however, it has been reported to have an intra-observer variance, partly because of manual compression and naked-eye measurement of the nailbed color change. we hypothesized that a we developed a novel portable crt measurement device with an oled display that feedbacks weather the strength of the nailbed compression is enough and counts the time. we settled the target strength and time as 5n and 5seconds according to the study we reported before [1] . 20 examiners measured crt with and without the feedback function. the pressing strength and time during the measurement were evaluated. there was a significant difference among the pressing strength and time between the crt measurement using the device with and without the feedback function (strength: p<0.001; time: p<0.001). furthermore, intra-examiner variance was significantly reduced with the feedback function (strength: p<0.001; time: p<0.001). in all measurements without the feedback function, 41% was outside the optimal strength while the measurements with the feedback function 100% achieved the targeted range. without the feedback function, 12% could not reach the optimal time, while 100% with the feedback function did. in total, 49% of the measurements could not achieve the optimal pressing strength and time. the feedback function for crt measurements, guiding examiners to an optimal pressing strength and time, fulfilled the required measurement conditions and reduced intra-examiner variance. our novel portable device would assist an accurate crt measurement regardless of personal work experience. introduction: the aim of the study was to detect the difference of conjunctival microcirculation between septic patients and healthy subjects and evaluate the course of conjunctival microcirculatory changes in survivors and non-survivors over a 24 hours period of time. this single-centre prospective observational study was performed in mixed icu in a tertiary teaching hospital. we included patients with sepsis or septic shock within the first 24 hours after icu admission. conjunctival imaging using idf videomicroscope as well as systemic hemodynamic measurements were performed at three time points: at baseline, 6 hours and 24 hours later. baseline conjunctival microcirculatory parameters were compared with healthy control. a total of 48 patients were included in the final assessment and analysis. median apache ii and sofa scores were 16 (12-21) and 10 (7-12) respectively. 44 (92%) were in septic shock, 48 (100%) required mechanical ventilation. 19 patients were discharged alive from the intensive care unit. we found significant reductions in all microcirculatory parameters in the conjunctiva when comparing septic and healthy subjects. we found a significant lower proportion of perfused vessels and microvascular flow index (mfi) of small vessels during all three time points in non-survivors compared with survivors. in nonsurvivors we observed no significant changes in conjunctival microcirculatory parameters over time. however, survivors had significantly improved mfi of small vessels at second and third time points compared to first time point. microcirculatory perfusion in conjunctiva was altered in septic patients. over 24 hours evaluation survivors in comparison with nonsurvivors had better microcirculatory flow with incremental improvement of microvascular flow index. 16 healthy pigs were centrally cannulated for veno-arterial ecmo and precision flow probes were placed on the pulmonary artery main trunk for reference. 10ml boluses of iced 0.9% saline chloride solution were injected into the ecmo circuit and right atrium at different ecmo flow settings (4, 3, 2, 1 l/min). rapid response thermistors of standard pa-catheters in the ecmo circuit and pulmonary artery recorded the temperature change. after calibration of the catheter constants for different injection volumes in the ecmo circuit, the distribution of injection volumes passing each circuit was assessed and enabled calculation of pulmonary blood flow. analysis of the exponential decay of the signals allowed assessment of right ventricular function. calculated blood flow correlated well with true blood flow (r 2 = 0.74, p < 0.001, figure 1 panel a, individual measurements organ congestion is susceptible to be a mediator of adverse outcomes in critically ill patients. point-of-care ultrasound (pocus) is widely available and could enable clinicians to detect signs of venous congestion at the bedside. the aim of this study was to develop prototypes of congestion scores and to determine their respective ability to predict acute kidney injury (aki) after cardiac surgery. this is a post-hoc analysis of a prospective study in 145 patients for which repeated daily measurements of hepatic, portal, intra-renal vein doppler and inferior vena cava (ivc) ultrasound were performed before surgery and during the first 72 hours after cardiac surgery [1] . five prototypes of venous excess ultrasound (vexus) scores combining multiple ultrasound markers were developed (figure 1 ). the association between each score and aki was assessed using timedependant cox models as well as conventional performance measures of diagnostic testing. a total of 706 ultrasound assessments were analyzed. we found that defining severe congestion as the presence of severe flow abnormalities in multiple doppler patterns with a dilated ivc (>2cm), corresponding to grade 3 of the vexus c score, showed the strongest association with the development of subsequent aki compared with other combinations of ultrasonographic features (hr: 3. there is an increasing awareness on the consequences of fluid administration in patients leading to the development of methods that evaluate the effects of fluids loading on the cardiocirculatory system. however, most of methods used in the clinical practice investigate the effects of fluids on the cardiac function, instead of investigating those on the determinants of venous return. besides volume of fluids, the determinants of fluid loading are the blood volume distribution and the availability of vascular bed. in this study we aimed to test non-invasively the effects of fluids administration on the venular compartment in the skeletal muscle. in addition to the mean systemic filling pressure (msfp), we calculated changes in the stressed and unstressed volumes (vs, vu) and the venular bed availability. we enrolled 10 critically ill patients in our intensive care unit. we assessed volumes and pressures by the near infra-red spectroscopy on the forearm using graded venous occlusions in steps of 5mmhg from 50 to 0 mmhg. the msfp, vu and vs were measured as previously reported (microcirculation 2014; 21:606-614). the vascular bed availability was measured by changes in the volume recruited from the occlusion maneuvers. all the measures were done at baseline and after a fluid load ranging from 250 to 500 ml. values were expressed as median and interquartile range. wilcoxon test was used to compare data and a p< 0.05 was considered as significant. introduction: hypotension is a common side effect of general anesthesia (ga) and is associated with organ hypoperfusion and poor perioperative outcome [1] . post-induction hypotension (pih) is caused by the depressant cardiovascular effect of anesthetic drugs and could be amplified by hypovolemia. the aim of this study was to assess the ability of two echocardiographic fluid responsiveness markers to predict pih: the inferior vena cava collapsibility index (ivc-ci) and the velocity time integral change (δvti) after passive leg raising. sixty patients > 50 years of age and scheduled for elective surgery were included. ivc-ci and δvti were measured before ga induction. anesthesia protocol, fluid infusion and vasopressor administration were standardized in all patients. pih was defined as a mean arterial pressure (map) <65 mmhg or a relative decline from pre-induction value of at least 30% within 12 minutes of ga induction. receiver operating characteristic (roc) curve analysis was used. the optimal cutoff was selected to maximize the youden index (sensitivity + specificity − 1). the measurement of ivc-ci and/or δvti were unsuccessful in seven patients (11.6%). pih occurred in 32 patients (incidence 53 %). the areas under the roc curves ( figure 1) preload responsiveness might be detected by the changes of cardiac index (δcimini) induced by a "mini-fluid challenge" (mini-fc) of 100 ml or even by the changes (δcimicro) in response to a "micro-fluid challenge" (micro-fc) of 50 ml. however, the smaller the fluid challenge, the larger the "grey zone" of diagnostic uncertainty. we tested whether (1) micro-and mini-fc monitored by calibrated pulse contour analysis detect preload responsiveness and (2) adding 50 ml when the result of a micro-fc is within the grey zone improves diagnostic accuracy. in 30 patients with circulatory failure, we infused 50 ml saline over 30s followed by 50 ml over 60s. we measured δcimicro and δcimini by the pulse contour analysis (picco2). preload responsiveness was defined by an increase in ci (δciplr) during a passive leg raising test ≥10%. diagnostic uncertainty was described by calculating the grey zone after bootstrapping. δcimicro were larger in responders than in non-responders (5. for the micro-fc, the area under the receiver operating characteristic curve was 0.975±0.03 (threshold 1%), while it was 0.955±0.03 for the mini-fc (threshold 4%). for the micro-fc, the grey zone ranged from 0.82% to 3.47% and included 9 (30%) patients. for the mini-fc, it ranged from 2.8% to 6.8% and included 9 (33)% patients, among which 6 were already in the grey zone of the micro-fc. when evaluated by pulse contour analysis, micro-and mini-fc reliably detect preload responsiveness but with a large diagnostic uncertainty. it seems that adding 50ml more fluid to a micro-fc when its result is within the grey zone does not improve the diagnostic accuracy. the study is ongoing. the starling-sv bioreactance device (cheetah medical) reliably detects passive leg raising (plr)-induced changes in cardiac index (δci). we tested whether it can also track the small and short-time δci induced by the end-expiratory occlusion (eexpo) test, and whether shortening the time over which it averages cardiac output (24 s in the commercial version) improves the detection. in 42 mechanically ventilated patients, during a 15-sec eexpo, we measured δci (in absolute value and in percentage) through calibrated pulse contour analysis (ci pulse , picco2 device) and starling-sv. for the latter, we considered both ci starling-24 provided by the commercial version and ci starling-8 obtained by averaging the raw data over 8 s. we calculated the correlation between δci pulse and both δci starling-24 and δci starling-8 , and the area under the receiver operating characteristic curve (auroc) to detect preload responsiveness, defined by a plr test. when considering absolute values, the correlation coefficient r between δci pulse and δci starling-24 was 0.362 (p=0.02), which was lower than the one between δci pulse and δci starling-8 (rr comparison). when considering percentage changes, no correlation was observed between δci pulse and δci starling-24 . conversely, the correlation coefficient between δci pulse and δci starling-8 was 0.402 (p=0.01), but it was lower than the one obtained for absolute values (p=0.04 for r comparison). eexpo-induced δci starling-8 , both in absolute values and in percentage, detected preload responsiveness with aurocs of 0.90 (sensitivity 83%, specificity 87%) and 0.89 (sensitivity 83%, specificity 79%), respectively. shortening the averaging time of the bioreactance signal increases the reliability of the starling-sv device to detect eexpo-induced δci. moreover, the accuracy of the method is increased when absolute rather than percentage changes of ci are considered. fluids are among the most prescribed drug in intensive care, particularly among patient with circulatory failure. yet, very little is known about their pharmacodynamic properties and this topic has been left largely unexplored. there is a lack of strong scientific evidence in current guidelines for fluid administration in shock. several factors may impact the hemodynamic efficacy of fluids among which the infusion rate. the aim of this study was to study the influence of fluids administration rate on their pharmacodynamics in particular by studying mean systemic pressure (p ms ). we conducted a prospective observational study in 17 patients with circulatory failure to compare two volume expansion strategies. when a patient required a fluid bolus, 500 ml of normal saline were administered and several hemodynamic parameters were recorded continuously: cardiac output (co), arterial pressure (ap), mean systemic pressure (p ms ). infusion rate was let to the discretion of the attending physician and a "slow" and a "fast" group were determined based on the median of the infusion time. fluids effect was measured by the area under the curve (auc), maximal effect (e max ) and time to maximal effect (t max ) for each hemodynamic variable. results: p ms auc was higher in the "fast" group compared to the "slow" group (p=0.043). we observed a shorter t max and a higher e max for p ms in the "fast" group compared to the "slow" group (p=0.039 and 0.02 respectively). regarding co, t max was also shorter in the "fast" group (p=0.041). auc and e max were similar between the two groups. fluid effect dissipated within 60 minutes following the end of fluid infusion for every patient in both groups. the decreasing slope from maximal effect was comparable in the groups, for p ms and co alike. the effect of a 500 ml fluid bolus in septic shock patients vanished within one hour. a faster infusion rate increased maximal effect and shortened the delay to reach it. study is ongoing. fluid management in the control arm of sepsis trials aa anparasan, ac gordon, mk komorowski imperial college london, department of surgery and cancer, london, united kingdom critical care 2020, 24(suppl 1):p219 in the past, high-volume intravenous fluid resuscitation in severe sepsis and septic shock was common. more recently, concerns over the harmful effects of this practice have led some clinicians to adopt less liberal fluid strategies. we sought to analyse temporal trends in fluid administration in the control arms of recent adult sepsis trials and assess any correlation with patient severity and mortality. a literature search was conducted to identify relevant randomized controlled trials that reported fluid administration published post 2000. we recorded 4 outcomes: total amount of iv fluid administered in the control arms of these trials between hospital admission and hour 6 and hour 72 following trial enrolment, mortality rates at the latest reported time point and apache-ii score at admission. we computed the pearson correlation coefficient and linear regression between study dates and the 4 outcomes. we identified 9 relevant trials [1] [2] [3] [4] [5] [6] [7] [8] [9] , which recruited a total of 2,444 patients in their control arms, from 1997 to 2018. the temporal analysis revealed no obvious trend in the in the total volume of iv fluid given by hour 6 following trial enrolment (correlation p=0.94) ( figure 1 ). however, the total volume of fluid given by hour 72 decreased significantly over the period of interest (r=-0.78, p=0.02). in parallel, we observed a decrease in mortality (r=-0.6, p=0.08) but there was no evidence of decrease in illness severity over time (p=0.84). we found that in published rcts over the last two decades, the amount of intravenous fluid given to patients with sepsis in the initial 6 hours did not appear to change, however less intravenous fluid was given over the first three days. upcoming large rcts will test the safety and efficacy of restrictive fluid administration approaches in sepsis. clinical practice guidelines recommend prompt intravenous (iv) fluid resuscitation for pediatric sepsis, including an initial fluid bolus of 20 ml/kg [1] . however, recent evidence is conflicting as to the effectiveness, volume, and consequences of aggressive fluid resuscitation in septic children. therefore, we sought to determine the epidemiology of early iv fluid resuscitation in an integrated health system, specifically at community hospital emergency departments (ed). we studied a retrospective cohort of pediatric patients (ages > 1 month to < 18 years) with sepsis identified in electronic health record data at 11 community eds in southwestern pennsylvania from 2010 to 2014. sepsis was defined as 1) suspected infection (combination of fluid culture collection and administration of antibiotics and 2) organ dysfunction (pediatric sofa score ≥ 1) within 24 hours of suspected infection. fluid bolus therapy was defined as electronic documentation of administration of 0.9% normal saline iv bolus within 1 hour of the time of sepsis onset. results: among 1,247 patients with pediatric sepsis, 513 (41%) received iv fluid bolus therapy within 1 hour of time of sepsis onset. the volume of fluid administered ranged from 2 ml/kg to 67 ml/kg (figure 1 , panel a), corresponding to a median volume of 20 ml/kg (iqr 17-22 ml/kg). patients who received ≥ 20 ml/kg of fluids (n = 258, 50%) were younger (mean age 5 years, sd 5 vs. 9 years, sd 6; p<0.001), more often had blood cultures collected during evaluation (86% vs. 76%, p=0.003), and were more often transferred to another facility (48% vs. 33%, p<0.001) when compared to patients who received < 20 ml/kg of fluids (n = 255, 50%). mean fluid bolus volume within 1 hour of time of sepsis onset by hospital ranged from 12 ml/kg to 24 ml/kg (figure 1, panel b) . in a cohort of community emergency departments, 41% of septic children received intravenous fluid boluses within one hour, and of those, only one half received volumes concordant with guidelines. (figure 1 ). a wide range of fluid balance exists in septic shock patients cared for in icu. trends of serum albumin in septic and non-septic critically ill introduction: the link between hypoalbuminaemia and poor outcomes in critical care is well established [1] . limited data are available on serum albumin trends during critical illness [2] . in this study we assessed trends in serum albumin for up to 7 days in both septic and non-septic critically ill patients. we retrospectively examined the records of 1107 adult patients admitted to critical care at the royal liverpool university hospital between 2008 and 2014. we then excluded patients who did not have albumin data available for the first 7 days, leaving us with 758 patients. 506 patients (66.8%) had sepsis, and of these patients 116 had died by day 28. of the 252 non-septic patients (33.2%), 40 patients had died by day 28. albumin levels were collected for 7 days from admission to critical care, in addition to other demographic and biochemical data. statistical analysis was performed using repeated measures analysis. septic patients had lower serum albumin than non-septic patients throughout the 7 day period (p<0.001). we observed a decrease in albumin by day 2 in all groups, with levels increasing over the subsequent days. there was no difference in daily serum albumin between non-septic patients who survived or died. this is the first study, to our knowledge, to compare albumin trends in septic and non-septic critically ill patients over 7 days. further research is needed to elucidate the optimal recipients and timing of albumin therapy. introduction: burn injury is characterized by marked inflammation, capillary leakage, and profound hemodynamic alterations. early albumin resuscitation is avoided fearing a paradoxical fluid escape into the interstitium. on the other hand, administration of crystalloids in massive amounts causes tissue edema and fluid extravasation, which deteriorates tissue perfusion by increasing oxygen diffusion distance. albumin administration could reduce the amount required to maintain hemodynamic stability in this population. we investigated whether albumin improves tissue perfusion and microcirculation by reducing tissue edema. this is an observational study conducted in the burn unit of maasstad hospital, rotterdam. patients with burns higher than 15% of total body surface area (tbsa) were included in the study. sublingual microcirculation was measured at admission (t0), 4(t4), and 12(t12) hours after burn injury. total vessel density (tvd) and functional capillary density (fcd) were analyzed. fluid management was calculated according to the modified parkland formula. albumin (20%) infusion was started 12 hours after the burn insult. a total of nine patients were recruited between january and december 2019. patients were included in the study after 5.7±2.3 hours of the insult with a mean tbsa of 36±22%. the amount of crystalloid infusion was 2718±3348 ml and 8501±5230 ml at t0 and t12,respectively. within the first 12h (t12) 502±386 ml albumin was given. tvd decreased from 23.6±2.2 at t0 to 20±1.3 at t4 (p<0.05) (figure 1) introduction: spontaneous bacterial peritonitis (sbp) accounts for ≥24% of the bacterial infections that occur in patients with cirrhosis, and sbp has a high mortality rate (20% to 50%). albumin infusion has been shown to improve the outcome of sbp. the aim of this study is to examine the impact of albumin infusion on hospital length of stay (los) for cirrhotic patients with sbp. we utilized a nationwide electronic health record data set (cerner health facts®) to extract real-world data on adult patients (≥18 years old) with cirrhosis and sbp who received antibiotics and admitted between january 1, 2009, and april 30, 2018. international classification of diseases (icd-9/10) codes were used to identify cirrhosis and sbp. we used laboratory data for calculation of the model for endstage liver disease sodium (meld-na) score and vital signs data for calculation of the quick sepsis related organ failure assessment (qsofa) score at baseline for each encounter. a generalized linear model was used to assess the relationship between albumin infusion and hospital los. results: there were 2,131 encounters that identified patients with sbp and cirrhosis, of which 1,661 survived hospitalization. albumin was infused within 24 hours of admission ('early albumin') in 43% (n=718), after 24 hours in 31% ('late albumin', n=517), and not administered in 26% ('no albumin', n=426). meld-na was higher at presentation in early albumin cases versus late-or no-albumin cases (mean 24.0 and 19.5). unadjusted los was lower in patients receiving early albumin (8.7 days versus 10.4 days). risk-adjusted analysis demonstrated that early albumin led to a 17.5% reduction in los (95% ci 12.6%-22.2%, p = <0.0001). in these real-world data, albumin infusion within 24 hours of admission in patients with cirrhosis and sbp was associated with a shorter hospital stay despite more severe illness. early albumin may not only improve clinical outcomes but may also reduce the costs of hospitalization in cirrhotic patients with sbp. early albumin use in patients with septic shock is associated with a shorter hospital stay: real-world evidence in the united states introduction: septic shock is among the most common critical care illnesses and incidence is rising, with mortality in excess of 35%. septic shock predisposes patients to multiple organ failure. while albumin is effective in management of circulatory dysfunction in septic shock, its utilization in this population is understudied in the us. we evaluated the impact of albumin utilization on hospital length of stay (los) among septic shock patients. we used a nationwide electronic health record data set (cerner health facts®) to extract real-world data on adult patients (≥18 years old) with severe sepsis or septic shock, admitted between january 1, 2013, and april 30, 2018, identified by international classification of disease (icd-9/10) codes, and receipt of antibiotics and vasopressors. we calculated the charlson comorbidity index (cci) and the acute physiology score (aps) at baseline. a generalized linear model was used to examine the association between albumin and hospital los, especially accounting for the timing of albumin infusion. we identified 3,156 unique visits for septic shock patients that survived to discharge. albumin was infused within 24 hours of admission ('early albumin') in 15%, after 24 hours ('late albumin') in 20%, and not administered in 65%. both cci and aps were higher, at presentation, in early albumin cases than late-or no-albumin cases (mean: 7.49 and 7.17, and 51.50 and 43.23, respectively). unadjusted los was slightly lower in patients receiving early albumin (11.81 days versus 11.84 days). a risk-adjusted analysis demonstrated that early albumin was associated with 4.92% shorter los (95% ci 0.43%-9.22%, p = 0.0322). albumin infusion within 24 hours of admission was associated with a shorter length of hospital stay. early albumin infusion may lead to better outcomes and reduced costs in patients with septic shock. further research is being conducted to assess other potential benefits of early albumin administration in this patient population. every new septic event follows by hemodynamic instability may lead sequentially to decreased organ perfusion, multiple organ failure. acute renal failure is recognized clinical feature during sepsis (up to 40-50% in all cases). furthermore, urine output close monitoring is a cornerstone diagnostic clinical tool in each septic critically ill patient. in present study, we analyzed the dynamic minute-to-minute changes in the urine flow rate (ufr) and also the changes in its minute-to-minute variability (ufrv) during new septic event in critically ill patients. demographic and clinical data were extracted from the of 50 critically ill patients who were admitted to the icu and developed new septic event (followed by fever and leukocytosis) and analyzed. a foley catheter was inserted into the urinary bladder of each study patient. the catheter was then connected to electronic urinometer, a collecting and measurement system which employs an optical drop detector to measure urine flow. the urine flow rate variability (ufrv) is defined and calculated as the change in ufr from minute to minute. results: ufr and ufrv both decreased significantly immediate after new septic episode until beginning fluid resuscitation (ppvalues <0.001) (figure 1) . statistical analysis by the pearson method demonstrated a strong direct correlation between the decrease in ufr, ufrv and the decrease in the map (r=0.03, p=0.003; r=0.03, p=0.004) ( figure 1 ), and heart rate (r=0.12,p=<0.001) since systemic pressure starts to drop. ufrv and ufr demonstrated good clinical response to fluid administration despite the fact that systemic blood pressure did not improve (figure 1) . we consider that dynamic changes in ufrv and ufr could potentially serve as a more sensitive signals ofclinicaldeterioration during the new septic event in critically ill patients.we also suggest that those parameters mightbeable to identify the optimal end-point of fluid resuscitative measures in septic critically ill patients. diminished urinary output (uo) is largely used as marker of acute kidney injury (aki) in critically ill patients. we aimed to explore the role of urinary output on incidence and mortality of aki developed during icu admission. the study population consists of all patients admitted between 2007 and 2018 to one of the dutch icus included in the nice database with an icu length of stay of at least 48 hours, having daily measurement of creatinine and uo. only patients without renal replacement therapy that have a serum creatinine lower than 1.1 mg/dl (97.5 μmol/l) or a uo above 0.5 ml/kg/h on the day of the index icu admission were considered at risk for aki. patients were followed during their icu stay and classified according to the highest kdigo criteria reached based on creatinine alone (model 1) and creatinine plus uo (model 2) using icu admission serum creatinine as baseline. in both models, patients were classified as: no aki, renal impairment at the first day of icu admission, aki stage 1, aki stage 2, and aki stage 3. we identified 52,863 patients (60% male, mean age 63 years, median icu-los 4 days). of those, 51.2% of patients had renal impairment at the first day of icu admission. among the remaining patients, 44.4% in model 1 and 29.9% in model 2 were classified as having no aki, 2.6% and 1.4% as aki stage 1, 0.7% and 9.3% as aki stage 2, and 1.1% and 8.2% as aki stage 3, respectively. survival at 30-day markedly differed according to the aki classification model used (figure) . similarly, adjusted hrs for 30-day mortality differed among patients with and without aki compared to patients with renal impairment at the first day of icu admission ( figure 1) . among patients admitted to the icu 50% had renal impairment at the first day of icu admission. our findings suggested that uo plays an important role both on aki incidence and mortality and should be carefully interpret in the clinical setting especially in aki stage 2 classification. introduction: acute kidney injury (aki) mostly attributed to renal tubular damage, has a high morbidity and mortality outcome [1] , so a sensitive tool to assess the degree of tubular affection is needed for early detection and management of this condition. we investigated the ability of furosemide stress test (fst) (one-time bolus dose of 1mg/kg or 1.5 mg/kg if on prior furosemide-intake) to predict progression to akin stage-iii in critically ill subjects with early aki. we studied 80 subjects; 40 consecutive patients in group i receiving fst and 40 consecutive patients in group ii receiving standard medical management for aki;15 patients (37.5%) and 20 patients (50%) met the primary endpoint of progression to akin-iii in groups i and ii respectively. patients with progressive aki had significantly lower urine output following fst in the first 6 hours (p<0.033). the area under the roc curves for the total urine output over the first 2 hours following fst to predict progression to akin-iii was 0.87 (p = 0.001). the ideal-cutoff for predicting aki progression during the first 2 fig. 1 (abstract p227) . thirty-day survival according to aki classification model 1 and model 2. hazard ratios (hrs) for 30-day mortality adjusted by sex, age, type of admission, apache iv score, sofa score at day of admission (excluded renal sofa score) for patients with aki classified with model 1 and model 2 fig. 1 (abstract p226) . clinical correlation between urine flow rate variability (ufrv) and ufr and mean arterial blood pressure over new septic event (black arrows) and and after initial fluid resuscitation (red arrows). note: the ufrv and ufr decreased progressively in parallel with the falling mean arterial blood pressure and, than, rose again after the administration of fluids hours was a urine volume of less than 325 milliliters with a sensitivity of 87.1% and specificity 84.1% group receiving fst. on the other hand, statistically significant hypotension, hypo-(kalemia, phosphatemia and magnesemia) occurred in group i. the fst in patients with early aki could predict liability for progression of aki, however it should be performed under adequate monitoring. introduction: ischemia-reperfusion (ir) causes renal dysfunction and damage. ir induces renal tubular injury triggered by hypoxia and hyperoxia, mediated by oxidative stress and inflammation. furosemide inhibits na + -k + -2clcotransporter in the thick ascending limb of the renal medulla to decrease na + reabsorption, reducing oxygen consumption. we investigated if furosemide could improve renal oxygenation, function and damage by reducing o 2 consumption and oxidative stress after ir. methods: 24 wistar albino rats were divided into 4 groups, with 6 in each group; sham-operated control (c), control + furosemide (c+f), ir and ir+f. after anaesthesia (bl), 45 min supra-aortic occlusion was applied to ir and ir+f groups followed by 15 min (t1) and 2 hours of reperfusion (t2). furosemide 50μg/kg/h infusion was simultaneously administered to c+f and ir+f after ischemia. systemic hemodynamic, renal blood flow (rbf), renal vascular resistance (rvr), renal oxygen delivery (do 2ren ), renal oxygen consumption (vo 2ren ), creatinine clearance (ccr), sodium handling, urine output (uo), cortical (cμo2) and medullar (mμo2) microvascular oxygenation were measured. results: rbf was reduced in ir (2.1±1) and ir+f (2.3±1) at t1 (p<0.05) but it was further reduced in ir+f (1.9±1) (p<0.05) at t2 compared to c and c+f. rvr was increased in ir (5338±2860) and ir+f (5123±2517) at t1 compared to c. rvr was normalized in ir (2198±879) but not in ir+f (4232±2636) at t2 compared to c (p<0.05). cμo 2 and mμo 2 did not differ between groups after ir insults (figure 1 ). tissue o 2 was reduced at the medulla, but not at the cortex in ir+f group compared to ir. do 2ren and vo 2ren were reduced in ir (56±17 and 26±12 ml/ min) and ir+f (34±20 and 21±14) at t2 (p<0.05). pc was higher in ir+f (37.33±4.27) compared to ir 29.67±3.39 (p<0.05). vo 2 / tna + was increased in ir+f compared to ir. no change in ccr and uo was observed. furosemide after ir causes further impairment of renal perfusion, energy utilization and renal oxygenation resulting in renal damage. acute renal failure induced by hypoxemia: incidence and correlation study a trifi 1 , h fazzeni 2 , a mehdi 2 , c abdennebi 2 , f daly 2 , y touil 2 , s abdellatif 2 , s ben lakhal 2 1 la rabta hopital, medical intensive care unit., tunis, tunisia; 2 la rabta hopital, tunis, tunisia critical care 2020, 24(suppl 1):p230 introduction: acute renal failure (arr) is a common complication in icus and usually caused by hypoperfusion. arf induced by hypoxemia is a concept rarely reported in icu. its incidence and pathogenesis are not well understood. we aimed to study the relationship between hypoxemia and the occurrence of arf. retrospective cohort study including patients with hypoxemia whatever its etiology between january 2016 and august 2019. patients with chronic renal failure were excluded. arf was defined and ranked according to the kdigo criteria 2012. arterial blood gas, urea, creatinine and clearance were reordered on the first, third and seventh days of evolution. results: 50 patients were included and 2 groups were obtained: group of hypoxemic patients with arf (arf+, n=30): versus group of hypoxemic patients without arf (arf-, n= 20). the incidence of hypoxemie-induced arf was therefore 60%. clinical characteristics were comparable in both groups with a mean age of 47 ± 16 and a sex ratio of 1.77. the comparative study showed in arf+ group: a lower ph (7. .25], p = 0.023). the most significant correlation was showed with mdrd clearance at day 3 and p/f ratio at day 1 (rho = 0.338, p = 0.038). multivariate analysis found that septic shock and non invasive ventilation in hypoxemic patients were the factors related to arf with respectively or=11.08, 95% ci=1.56-83.84, p=0.016 and or=6.18, 95% ci=1.16-34.07, p=0.033. overall mortality was 68% (n=34) and arf was an independent factor of mortality: or=6, and 95% ci=1.35-26.64, p = 0.017. hypoxemia-induced arf is a common complication associated with excess mortality. our study suggests that renal function is correlated with the degree of hypoxemia and that this correlation is rather distinct 48 hours from hypoxemia. in preclinical models of sepsis, we have previously demonstrated that activation of amp activated protein kinase (ampk) using metformin, improves survival and organ function. thus, ampk activation is a potential therapeutic target in sepsis, and we hypothesize that exposure to metformin during sepsis is associated with decreased aki and mortality methods: retrospective analysis of a 13-hospital cohort of adult icu patients with type 2 diabetes mellitus (t2dm) who presented sepsis. we investigated if exposure to metformin during the hospitalization was associated with reduced 90-day mortality and aki. we used 1:4 propensity score matching (psm), propensity score stratification (pss) and propensity score weighting (psw) based on the probability to be exposed to metformin using 55 covariates. for psm an exact match for insulin, amputation, cardiovascular diseases, retinopathy, charlson index, egfr, hba1c, and apache iii, were used. sepsis was defined using sepsis 3 criteria, and aki as kdigo stage 2 or 3. from 164,910 patients, we found 673 diabetic adults exposed to metformin during hospitalization and 14,174 who were not. metformin exposure during hospitalization is associated with decreased 90-day mortality and aki in septic adult patients with t2dm. these findings suggest that metformin may constitute a potential therapeutic strategy in sepsis, and the potential role of ampk activation as a protective mechanism. however, studies are needed to confirm this association and the specific mechanisms of action. introduction: acute kidney injury (aki) may occur up to 50% in the intensive care unit (icu). predicting aki recovery may allow for risk stratification of patients, patient and family counseling, and early post-discharge renal care planning. however, predicting aki recovery at an early stage remains a challenge. methods: this is a retrospective study of the epanic multicenter randomized controlled trial database [1] , which was split into development (n=2194) and validation (n=2446) cohorts, and patients experiencing aki stage 3 and/or renal replacement therapy (rrt) in the icu were included [2] . aki recovery was defined as being alive, without any stage of aki, and without need of rrt at hospital discharge. a logistic regression model with backward feature elimination was developed. the model performance was assessed by discrimination, calibration, and net benefit analysis, and internally validated with ten-fold cross validation. only the results in the development cohort are reported. of the 229 patients who developed aki3, 86 patients (37.55%) recovered from aki. the multivariable model selected age, bilirubin, heart rate, mean arterial blood pressure, surgical diagnostic group on icu admission, mechanical hemodynamic support on icu admission, suspected sepsis on icu admission as aki recovery predictors. the model had a mean area under the receiver operating characteristic curve (auroc) of 0.75 (standard deviation (sd) 0.01), mean calibration slope of 1.02 (sd 0.04), and mean calibration-inthe-large of <0.01 (sd 0.01) (figure 1 ). at the classification threshold that maximized sensitivity and specificity, mean net benefit with respect to treat-none was 0.16 (sd 0.01) and mean net benefit with respect to treat-all was 0.11 (sd 0.01). by using the routinely collected clinical data, the developed prediction model can fairly identify patients with a higher chance of aki recovery at hospital discharge. introduction: acute kidney injury (aki) is a frequent complication in critically ill patients and is associated with increased morbidity and mortality. sepsis is one of the most common cause of aki. a prospective study was conducted over 6 months (january 01-june 30, 2018).we included patients with septic shock at admission or at any time during hospitalization.the aki staging was based on kdigo criteria.patients were divided into two groups, a group with aki (aki+) and a group without aki (aki-).then we compared the baseline characteristics, laboratory and physiologic data. patients with aki (aki+) were subdivided according to their prognosis. were enrolled 75 patients. the mean (sd) age was 56.43(±18) years.sex ratio was 1.91. fifty-two (70%) patients developed aki.sapsii and sofa score in admission were higher in patients with kidney injury [59 vs 44 points (p= 0.002), 6.5 vs 4 points ;(p=0.003)] respectively.the serum lactate level was significantly higher in (aki +) group patients during the first day of septic shock [6.12± 1.38 mmol/l (aki+)vs 4.11± 0.79 mmol/l(aki-);(p=0.002) ] and its clearance was lower [(32±10.99% (aki +)vs 61±13%(aki-);(p=0.001)]. a significant difference was observed in c reactive protein level [224±114 mg/l (aki +) vs 124±77 mg/l (aki-) ; (p=0.004)].among (aki+) patients, kadigo iii was observed in 59.6% of cases.nineteen (36.5%) patients received hemodialysis.a normal kidney function was recovered in 40.4% of cases.aki+ patients had a higher occurrence in disseminated intravascular coagulation (32 vs 3 patients, p=0.002),acute respiratory distress syndrome (18 vs 2 patients; p=0.023) and cardiac dysfunction (20 vs 1 patient, p= 0.001).mortality was higher in aki group (67% vs 9%; p=0.001). the development of septic aki was associated with poor outcomes and prognosis.a better understanding of sepsis induced aki pathway will enable us to develop targeted therapeutic protocols.newer tools,permitting aki early detection, may make these therapies more fruitful. this study aims to show that contrast procedures do not significantly increase the risk of renal injury and should not be deferred. traditionally ciaki is the most important cause of in-hospital renal failure after nephrotoxic drugs and shock. problem is also the non-uniform definition of ciaki proposed by three different initiatives (akin, esur and kdigo). akin, being the most rigorous, defines ciaki as an increase in serum creatinine >0.3 mg/dl or >50% of baseline within 48hours. a retrospective observational single-centre cohort study analyzed 82 patients who underwent a contrast procedure with iomeron 350. the first group underwent a ct pulmonary angiography (ctpa), and the fig. 1 (abstract p232 ). internally validated model performance: (top row) roc curve; (middle row) calibration curve; (bottom row) decision curve second a coronary angiography with pci. no patient was previously prepared (raas blockade removal, crystalloid administration etc). we studied demographics, history of ckd and comorbidities and their impact on the ciaki by the akin criteria. a total of 82 patients were divided into two groups (ctpa and pci). ctpa group (20m, 21f) all had acute pe and the pci group (28m, 13f) were treated for acs. the mean age was 69 and 65 years respectively. ckd was more prevalent in the pci group (8pt vs. 3pt) possibly explained by the more advanced atherosclerotic disease. advanced chd (nyha iii/iv) was found in 3pt (pci) vs. 2pt (ctpa) while diabetes and shock were equally distributed (11pt and 5pt) in both groups. the mean amount of contrast was significantly higher in the pci group (242.3ml vs. 60ml). the mean creatinine/egfr measured before and after contrast in the ctpa group was 87. the goal of this study was to determine whether changing the body mass (bm) with fat-free mass (ffm) in cockcroft-gault (cg) formula could provide a more accurate prediction of aki in obese patients undergoing cardiac surgery. in this retrospective study, we reviewed institutional data of patients who underwent elective cardiac surgery in a tertiary referral university hospital. baseline patient creatinine value was collected and gfr was estimated using the mdrd, ckd-epi and cg formulas. cg formula was further modified by replacing the bm with ffm derived from the bioelectrical impedance analysis. postoperative aki was defined by kdigo creatinine change definitions. accuracy of the egfr values to predict the aki was calculated with roc-auc analysis. all the calculations were performed in different categories of bmi. figure 1 ). the egfr is a poor predictor of aki in obese patients undergoing cardiac surgery. the ffm modified cauckraft-gault formula yield more accuracy in this specific group. retroaki: a ten-year retrospective study of acute kidney injury in intensive and progressive care units introduction: acute kidney injury (aki) is a frequent condition in intensive care units (icu) and progressive care units (pcu), affecting 15% to 70% of the patients, depending on the studied population and aki definition. aki has been identified as an independent risk factor of icu mortality and development of chronic kidney desease. the objective of this study was to describe the incidence of each aki stages as defined by kdigo definition (with evaluation of urine output, serum creatinine and initiation of renal replacement therapy (rrt)), in a mixed medical and surgical population of patients hospitalized in icu and pcu over a 10-year period (2008-2018). we included all patients who stayed more than 12 hours in icu or pcu of edouard herriot hospital from may 2008 to january 2019. data used to classify the patients were the urine output over a sixhour period, serum creatinine and the need for rrt, according to kdigo classification results: 18,882 hospital stays were analyzed. median icu/pcu length of stay was 3 days [iqr: 1.5-6.6]. among icu patients, 74% had at least one aki episode graded 1, 2 or 3 and 49% had at least one severe episode (stage 2 or 3). among pcu patients, 44% had at least one episode of aki and 20% a severe episode of aki. patients had an average of 1.9 episodes of aki per stay. table 1 represents the incidence of maximal aki stage during one stay. we found that urine output was the more frequent criteria to make diagnosis of aki stage 1 or 2 whereas rrt was more frequent for aki stage 3. this retrospective study reports a more important aki incidence in our icu/pcu than in previous studies. the difference could be fig. 1 (abstract p236) . when comparing auc in different categories of bmi, the mcg appeared to be the only statistically accurate formula in patients with bmi 30-34.9 explained by the difficulty to collect urine output from conventional database. serum creatinine and the use of rrt are often the only two criteria used to define and classify aki. these results confirm the high incidence of aki in icu and pcu and the importance to make an early aki screening of patients for whom preventive nephroprotective actions are needed. introduction: icu-patients with acute kidney injury (aki) requiring renal replacement therapy (rrt) are at risk for infections [1, 2] . in this study we evaluated the incidence of infection in icu patients with and without less severe aki. finally, impact on outcomes was explored. this is a retrospective study on the pdms (protection data management system) of the 4 adult icus of a university hospital. aki was assessed on kdigo criteria (creatinine (scr) and urine output), during the first 7-d of icu stay. infection was validated in the pdms by a team of icu specialists. results: during a 4-year period, a total of 7485 subjects were enrolled. aki was diagnosed in 64.7% of patients during icu stay. aki patients were older (63 vs. 59 y, p=0.001), had higher saps 2 (57 vs. 41, p< 0.001), and had more urgent icu admission (64% vs. 48%, p<0.001). more aki patients had mechanical ventilation (55% vs. 41%, p<0.001) and vasopressors on d-1 (47% vs. 23%, p<0.001). aki stage 1, 2, and 3 was present in 25.5%, 28.0% and 11.1% of patients. more aki patients had infection (57% vs. 28%, p<0.001) and increasing aki stages were associated with higher infection rates (aki-0: 28%; aki-1: 55%, aki-2: 55%, aki-3: 69%, p<0.001) (figure 1 ). we observed 2-3 times higher mortality in aki patients with infection, and a stepwise increase of mortality with increasing aki stages. after correction for infection and other confounders we found that all aki stages were associated with in-hospital mortality (ors aki-1: 1.7, aki-2: 2.0, aki-3: 3.6, all p< 0.001). over half of aki patients experienced an episode of infection and increasing aki severity was associated with higher infection rate. aki patients with infection had marked higher mortality, suggesting that infection was an important driver of outcome. however, after adjustment, aki stages had strong association with hospital mortality. several new biomarkers have been introduced to improve early diagnosis of acute kidney injury (aki). "nephrocheck" (nc; astute medical, usa) is a bedside test calculating "akirisk" (product of urinary concentration of the cell cycle arrest-markers timp-2 and igfbp7). several studies suggest the usefulness of nc in selected populations. however, the value of early routine measurement of nc is unclear. methods: therefore, we compared the prediction of a combined endpoint (cep: death <60 days and/or requirement of renal replacement therapy rrt) by nc within 12h of icu admission (nc1) and 24h later (nc2) with admission values of serum-creatinine, bun, cystatin c, urinary ngal, apache ii and sofa (roc-analysis). as a secondary endpoint we investigated the additional value of pathological measurements of nc1≥0. 3 critically ill patients showed increased relative uce in the first days of icu admission, which may be attributed to higher protein catabolism. increased relative uce was associated with arc and both had no effect on 90-day mortality. introduction: this study compared epidemiology, short-and long-term outcomes for patients with community-acquired (ca) and hospital-acquired (ha) acute kidney injury (aki). we retrospectively analyzed all episodes of aki over a period of 3.5 years (2014-2017) on the basis of routinely obtained serum creatinine measurements in 103,161 patients whose creatinine had been measured at least twice and who had been in the hospital for at least two days. we used the "kidney disease: improving global outcomes" (kdigo) criteria for aki and analyzed the first hospital admission. a total of 103161 were admitted in hospital and fulfilled the inclusion criteria. average observation period per patient was 248 days. the incidence of ca-aki among included hospital admissions was 9.7% compared with an incidence of 8.6% of ha-aki, giving an overall aki incidence of 18.3%. patients with ca-aki were younger than patients with ha-aki (64 vs 66.2y) and had significantly less comorbidities, including preexisting cardiac failure, ischemic heart disease, hypertension, diabetes. patients with ca-aki were more likely to have stage 1 aki (69,3 vs 58,4%, p<0.001) and had significantly shorter lengths of hospital stay than patients with ha-aki (14 vs 24d, p< 0.001). those with ca-aki had better survival than patients with ha-aki (figure 1; p<0 the evidence base for management of fluid removal during renal replacement therapy (rrt) is limited. a recent international survey revealed the extent of practice variation worldwide [1] . our aim was to summarise the responses from europe-based healthcare professionals who participated in the survey. the international self-administered, cross-sectional, internet-assisted, open survey was disseminated between january 2018 and january 2019 via website links and emails to members of different critical care societies. results: 485 participants from 31 european countries completed the survey of whom 365 (75%) were intensivists and 306 (63%) worked in university-based hospitals. persistent oliguria / anuria was the most common indication for fluid removal (51% responders). the parameters which guided fluid removal included hemodynamic status (47% responders), cumulative fluid balance since admission (23% responders), and 24-hour fluid balance (17% responders). 90% of participants reported using crrt with a median net ultrafiltration rate 98 ml/hr (iqr 51-108ml/hr) for hemodynamically unstable and a rate of 300 ml/hr (iqr, 201-352ml/hr) for hemodynamically stable patients. only 26% of practitioners checked net fluid balance hourly (70% nurses, 16% physicians). new hemodynamic instability, defined as new onset or worsening tachycardia, hypotension, or need to start or increase the dose of vasopressors was reported to occur in 20% fig. 1 (abstract p242 ). long-term survival patients (iqr 10.0-30.0). different strategies to re-gain hemodynamic stability were used. (figure 1 ) main barriers to fluid removal were patient intolerance (72% physicians, 85% nurses) and interruptions in fluid removal (43% physicians, 64% nurses). the majority of participants agreed that guidelines and protocols would be beneficial. the practice of fluid removal during rrt is very variable across european countries. nurses and doctors identified a need for evidencebased protocols and clear guidelines. introduction: kidney disease improving global outcomes (kdigo) guidelines suggest the use of anticoagulation in continuous renal replacement therapy (crrt) [1] . the effectiveness of the anticoagulation is important because replacing the hemofilter and tube interrupts crrt and increases total therapy time. regional citrate anticoagulation (rca) and unfractionated heparin (ufh) are most commonly using methods for crrt anticoagulation [2] . the aim of this study was to investigate the efficacy, safety and metabolic differences of the patients in icu who underwent crrt and anticoagulation method changed from ufh to rca for different reasons. after ethics committee approval (2019-14/9) 100 patients who underwent crrt between 2018-2019 at bursa uludag university hospital icu have been investigated and 11 patients who underwent crrt by both rca and ufh included in the study. we divided patients in two groups (rca, ufh), demographic data (sex, age), sofa score, creatinine, urea, mean filter life time (flt) and ultrafiltration flow (uf), platelets, electrolytes (na, k, ca, mg), lactate, nahco3 and ph of groups at beginning and ending of first rca and ufh hemodialysis collected. we used t-test and 1000 bootstraps statistic tests. in agreement with other studies [3, 4] , flt and uf was statistically significant lower in ufh group (table 1) . there was no statistically significant difference in efficiency (urea and creatinine decrease), ph, lactate, nahco3 level, platelets count and electrolytes between two groups. to our knowledge, there are no studies comparing these two anticoagulation methods in the same patients. small number of patients and retrospective evaluation are limitations of the study. our results suggest that the implementation of rca method is safe and effective as ufh method with longer flt and uf. regional citrate anticoagulation during crrt in liver failure mj jain, pk kumar g, dg govil, jk kn, sp patel, ms shafi, rh harne, dp pal, sm monanga medanta the medicity, critical care, gurugram, india critical care 2020, 24(suppl 1):p245 continuous renal replacement therapy (crrt) with regional citrate anti-coagulation (rca) is increasingly being used as a treatment modality in critically ill patients. there is limited experience of use of citrate anticoagulation patients with acute liver failure and acute on chronic liver failure who pose a tough challenge of being at a higher risk for bleeding. an institutional protocol was formulated for use of commercially available citrate solutions and the same was studied to assess filter life and safety of citrate in liver disease. the primary objective was to assess safety of citrate anticoagulation in liver disease. this study was a single centre, prospective, non-randomized, single arm, observational study. all adult patients, with acute liver failure and acute on chronic liver failure requiring crrt were included. blood ionized calcium levels of 0.9 to 1.1mmol/l was targeted throughout the therapy and total to ionized calcium ratio of less than 2.4 was maintained. rca was stopped if the ratio was more than 2.4 for 2 consecutive assessments. incidence of citrate accumulation and toxicity were assessed. average filter life was also assessed. metabolic parameters, electrolytes and strong ion gap were followed till 24 hours after completion on crrt. a total of 25 patients were included in the study. nineteen patients of acute on chronic liver failure and 6 patients of acute liver failure underwent crrt with rca. baseline average serum bilirubin, lactate and inr were 11.8 mg/dl, 6.4 mmol/l and 2.1 respectively. the average filter life was 50 hours 3 minutes. citrate accumulation took place in (n=13) patients and rca had to be stopped for ( n=6) patients due to the same. none of the patients had evidence of citrate toxicity. citrate anticoagulation was well tolerated in patients with acute liver failure in patients with or without pre-existing chronic liver disease on crrt. introduction: the intention of this study is to highlight the levels of citrate load for the general population that increases the risk of citrate complications (insufficient trisodium citrate delivery; net citrate overload and citrate accumulation) [1] . this was a prospective data collection between february and march 2019 in a fourteen bedded critical care unit. eleven consecutive episodes of crrt were collected (a new episode characterized if crrt was discontinued for 48 hours and above). one episode was excluded due to short duration (less than 4 hours). patients undergoing rca-crrt received either a fixed 25 or 35 ml/kg/h effluent dose protocol. median patient age was 59, male 100%. average time on crrt was 4.1 days (2-9). 70% of the patients had complications, although 60% were minor ( figure 1 ). all of the patients with net citrate overload had citrate loads of 13.8mmol/h or above. the main risk factors were found to be shock and liver impairment which occurred in 60% of cases of which 40% developed complications. a fixed dose effluent protocol to standardise practice can potentially lead to a higher risk of minor complications. in our experience this is likely due to a lack of appropriate monitoring for rca-crrt complications. despite this, our complication rate of citrate accumulation is in line with that reported in literature. citrate loads in our 25 ml/kg/ hr protocol were 22.6% higher than our 35 ml/kg/hr protocol and strongly related to higher complication rate that worsened in patients with risk factors for poor citrate metabolism. introduction: there is no optimal timing of continuous renal replacement therapy (crrt) in acute kidney injury (aki); however, it is based on volume overload, azotemia, hyperkalemia and severe metabolic acidosis [1] . an important reason for metabolic acidosis in aki is increased unmeasured anions (ua) [2] . delta-ph-ua (δph ua ) detects the degree of metabolic acidosis caused by ua and is calculated by using 'the partitioned ph model' [3] . in this study, we investigated whether δph ua was a predictor to start crrt in patients with aki. the study was designed as a multicentric, prospective, observational study in 2019. patients who were ≥18 years old and diagnosed with aki [1] were included. the moment aki was diagnosed, arterial blood gas, albumin, magnesium, inorganic phosphorus, urea, creatinine and δph ua values were recorded. all patients were divided into two groups as crrt(-) and crrt(+) which consists of patients performed crrt due to traditional criteria. fig. 1 (abstract p246) . incidence of complications introduction: continuous renal replacement therapy (crrt) is labor intensive and requires advanced nursing knowledge and skills. however, 40% of registered nurses (rn) are less than 2-year post-registration experiences in our unit. also there is an increasing demand of crrt from 185 crrt days in 2017 to 248 crrt days in 2018. the obstacles for crrt in our department, includes variation of regimen, complicated workflow and insufficient training of nurses. a continuous quality improvement project is carried out to standardize the regimen, enhance workflow and provide structured training to nurses in the intensive care unit, to enhance nursing competence. methods: introduction: sepsis and septic shock is a leading cause of mortality in the intensive care unit. we tried to evaluate a novel hemoperfusion cartridge through a retrospective evaluation of patient's data in our centre. we used it as an adjuvant therapy in our patients with sepsis and septic shock due to varied causes. the aim of this study was to evaluate the efficacy of therapeutic hemoperfusion cartridge (hc-foshan biosun medical ® ) in the management of patients with sepsis. we retrospectively analysed data of group 1 (n=30 sepsis) and group 2 (n=30 sepsis+hemoperfusison; sepsis treated with hemoperfusion cartridge) admitted between 2015 to 2018. group 2 had received hemoperfusion cartridge as adjuvant therapy along with standard of care. demographic data, procalcitonin [1] and leukocyte levels before and after therapeutic cytokine removal and duration of hc were recorded. while the mean duration of cvvhdf was 96.4 hours, the duration of hemoperfusion cartridge (application was 32.1±16.4 hours). among 30 patients who survived 25 patients were administered hemoperfusion cartridge within 12 hours of icu admission. there was a significant reduction in scores like apache and sofa score post hemoperfusion cartridge therapy procalcitonin and leucocyte levels after therapeutic hemoperfusion cartridge were found significantly lower than the pretreatment values (respectively p=0.001, p=0.001). retrospective analysis showed significant reduction of vasopressors, and improvement in map in group2. therapeutic hemoperfusion cartridge with cytokine removal applied with cvvhdf in septic patients have positive contributions to provide survival advantage. removal of activated leukocytes and endotoxin from the blood is a complex therapeutic effect of the device for removing endotoxin. in the main group (16 patients with abdominal septic shock) after surgery, the traditional treatment was supplemented with two sessions of endotoxin removal (2 hours each with an interval of 24 hours) using "alteco lps adsorber" (sweden). the control group consisted of 8 patients with a similar diagnosis and only traditional treatment. results: 28% of white blood cells were adsorbed in lps adsorber. among them, granulocytes (35%) were maximally extracted, then cd14 + monocytes (cd14 + mo) (33%), hla-dr + mononuclear cells (6%), monocytes (2%). il-6, il-10, procalcitonin (pct) were not adsorbed. the 28-day mortality rate in the main group was 50% and was lower compared to the control group -75%. during monitoring, in the main group 24 hours after the first removal of endotoxin, a decrease in the initially increased amount of activated cd14 + mo by 2.2 times, as well as functionally mature defensin + granulocytes (def + gran) by 1.6 times was observed. il-6, il-10, and pct decreased by 1.9; 17.8; and 1.2 times, respectively. during this period, the control group showed an increase in cd14 + mo and def + gran, while il-6, il-10 did not change, and pct increased 1.9 times. a day after the second removal of endotoxin and then 5 days later, the main group of il-6, il-10, and pct continued to decline. in the control group, only il-10 decreased after 3 days, the rest continued to grow. the cellular adsorption of endotoxin-bound cd14 + mo and mature def + gran is an important part of the mechanism of action of the endotoxin removal device. does the endotoxin adsorption of pmx column saturate in 2 hours? preliminary study c yamashita 1 in the euphrates trial, the polymyxin b-immobilized fiber column (pmx) hemoperfusion (hp) had no significant effect on 28-day mortality. endotoxin (lps) burden by endotoxin activity assay >0.90 may exceed 50 μg [1] , so the dose and duration of pmx-hp could be insufficient to lower the lps burden. to confirm this issue, we experimented in a closed-circuit with 24 h continuous lps addition, and pmx can adsorb > 50μg [2] . further, lps concentration became constant within 2 h in the single lps spike test for determining pmx-hp duration [3] . to prove our hypothesis that the single lps spike test reflects the adsorption equilibrium, and not saturation, we added lps intermittently to reaction. methods: lps (10 ng/ml) was mixed with 125 ml deactivated fetal calf serum as a reflux solution, as previously described [2] ; this concentration is much higher than that observed in septic patients. we created a closed circuit that incorporates pmx-01r at 1/14 th the amount of an adult pmx and performed pmx-hp at 10 ml/min for 5 h. lps was added in two shots (post 2 h: 1250 ng, 10 ng/ml; post 4 h: 3750 ng, 30 ng/ml). lps was measured using the limulus amebocyte lysate test at 0, 0.5, 1, 2, 3, 4 and 5 hr. after an initial decrease between 0 and 1 h, lps concentration did not decrease between 1 and 2 h after pmx-hp initiation. post lps pulse addition at 2 h, it increased and then decreased till 3 h. futher, it did not decrease between 3 and 4 h, but it increased and then decreased again after lps pulse addition post 4 h (figure 1 ). lps adsorption rates were 76.2, 43.4, and 40.7% at 2, 4, and 5 h, respectively. conclusions: lps adsorption capacity of pmx-01r was maintained even after two additional shots of lps, suggesting that the constant lps concentration in the previously reported lps spike test might be indicative of adsorption equilibrium rather than saturation. a coohort study included 65 patients admitted to three intensive care with sepsis / septic shock ( sepsis 3 criteria ) and aki ( akin score). all patients were submitted to cvvhdf with the oxiris filter (baxter, usa) . the main clinical data, il 6, procalcitonin, endotoxin ( eaa ) and sofa score were evaluated at basal time ( t0 ) and at the end of the treatment ( t1 ). all data are expressed as mean ± sd or median and iqr . anova test was used to compare the changes in the time. results: 60 patients were submitted to rrt with the oxiris filter for 46 ± 12 hours . 21 patients had aki 3 stage , 13 patients aki 2 stage and 25 patients had aki 1 stage. at t0 all groups had an high vasopressor fig. 1 (abstract 251) . lps concentration in lps pulse addition test support to maintain map ≥ 70 mmhg. il6, procalcitonin eaa and sofa total were also elevated with no difference between the groups. at t1 creatinine improved better in aki 2 ( p< 0.001 vs. t0 ) and in aki 1 ( p< 0.0001 vs t0) then in aki 3 group. map increased in aki 2 ( p< 0.01 vs t0) and aki 1 ( p < 0.01 vs t0) , but not in aki 3 group. il6, procalcitonin decreased more in aki 1 ( p < 0.0001 vs t0) then aki 3 . at t 2 sofa total was higher in aki 3 then aki 1 ( p< 0.001 ) and aki 2 ( p< 0.01 ). conclusions: aki 2 and aki 1 stage patients submitted to bp with the filter oxiris respond better then aki 3 stage patients . 2 -this transalte in a better clinical course. 3-crrt with oxiris filter is useful in septic patients with aki, but aki 3 stage septic patients represent an high risk group. a non-interventional, multicenter, non-randomized patient registry for multiple organ dialysis with the advos system multiple organ failure is a challenging problem in the icu. as an advanced dialysis system, the advos procedure can eliminate watersoluble and protein-bound substances, regulate the acid-base balance as well as fluid and temperature. in 2017, a national registry was established to collect data under "real-life" conditions of patients treated with advos without any trial-specific interventions (drks id: drks00017068). methods: data from 01/2017 to 02/2019 from 4 german hospitals (university hospitals in hamburg-eppendorf, mainz, essen, and klinikum weiden) were analyzed. clinical parameters, treatment settings and adverse events were documented. the 28-and 90-day mortality rates were compared with extrapolated rates based on the sofa score. results: 118 patients with a median age of 60 years (iqr 45-69), of whom 70 (59%) were male, were evaluated. patients had a median sofa score of 14 (iqr: 11-17) before the 1st advos treatment, which is associated with an expected mortality of 80%. the number of failing organs was 3 (iqr 2-4): cardiovascular (74%), lungs (57%), liver (47%), kidneys (74%), coagulation (69%) and cns (29%). 429 treatments with a median duration of 16 (iqr: 10-20) hours were evaluated. 87 were discontinued, of which 25 (6%) were due to a device error. 79 adverse events were documented, 13 were related to the device (all due to clotting and recovered without sequelae). significant removal of protein-bound (bilirubin: 11.2 vs 9.2 mg/dl) and water-soluble toxins (bun 32 vs 20 and creatinine 1.9 vs 1.4 mg/dl). in addition, improvement in acid-base balance was observed: ph (7.33 vs. 7.40), bicarbonate (21.3 vs. 25.5 mmol/l) and base excess (-4.5 vs. 1.0 mmol/l) ( table 1) . 28-and 90-day mortality rates were 60% and 65%, respectively. in a cohort of patients with multiple organ failure, we observed an improvement in the expected mortality rate, especially if the advos procedure was applied early. adverse events are comparable to other dialysis therapies in intensive care patients. introduction: acute kidney injury (aki) due to ischemia-reperfusion affects onethird of the patients in cardiac surgery. we investigated the potential role of cyclosporine (csa) to prevent postoperative aki and mitigate inflammatory response to extracorporeal circulation (ecc). methods: double-blind, randomized, placebo-controlled single-center study. patients (n=67) scheduled for elective cardiac surgery were randomized to 2,5 mg/kg csa or placebo before the surgery. the primary objective was to assess the role of csa to reduce the incidence of postoperative aki. the secondary objective was to study csa induced changes in the inflammatory response to ecc. results: all enrolled patients were analyzed. postoperative aki was more pronounced in the cyclosporine group compared to placebo. or=5.03 (1.76-15.74), 95% ci. the cytokine production in response to ecc was not affected by cyclosporine (figure 1) . in patients undergoing cardiac surgery, a single preoperative dose of csa does not prevent the postoperative decrease in renal function. csa does not alter cytokine release in response to extracorporeal circulation. elevated post-ecc levels of pro-inflammatory cytokine il-6 are associated with kidney dysfunction and may be predictive. new generation adsorbent such as oxiris r was introduced as novel technique in renal support for critically ill patients [1] . septic shock patients require decatecholaminization strategies emphasizing blood purification to remove catecholamine-producing mediators and evacuate overload fluid in interstitials. our 64-year-old female patient, admitted to icu after surgery with history of ovarium cancer. her septic shock was worsened with ards, hypercoagulable state and aki. vasopressors were set. patient was controlled with mode simv16,ps12,tv350 ml,peep7,fio270%. renal support was implemented by diuretic and cvvh started on the second day. at first,regular adsorbent was used, post-filter mode was set, and periodic fluid removal target was 50 ml/h. but after 24hours, no significant changes observed. oxiris r added and after 12 hours passed, requirements of vasopressors reduced, tidal volume increased, hemodynamic parameters stabilized, urine production increased. it was continued for 2 days and patient was recovered. our patient had fallen into inadequate cars stage in which not able to counter septic effects on vital organs (figure 1 ). renal would be primary target for filtration and monitoring tool. adsorbent consisted of an69 and polyethyleneimine was useful to purify blood from endotoxins conjoined with slower filtration. continuous yet cautious process in cvvh evacuate fluid and mediators while maintain steady hemodynamics. biomarkers could not be evaluated due to limited resources, but improving parameters could be signs that showed recovery process had already took place. advanced hemofiltration is a privilege. implementing and enhancing it with new generation adsorbent would increase survivors by extracting unnecessary fluids and eliminating catastrophic endotoxins and mediators. consent to publish: written informed consent for publication was obtained from the patient. analysis of retrospective cohort study data of 120 patients (pt) treated for dka at icu of kaunas clinics during 2014 -2019 has been carried out. serum kalemia, glycemia; hypokalemia, hypoglycemia episodes; rate of insulin interruption for hypo-and normoglycemia during ketoacidosis; use of nah 2 co 3 for ketoacidosis, and los in icu were analysed. spss 23.0 was used for statistic calculations. traits evaluated as significant at p < 0.05. at the beginning of dka treatment in totally hypokalemia (3.1 ± 0.3 mmol/l) was recorded in 64/120 pt (53.3 %). due to ignoring of blood ph (6.8 -7.3 (7.0 ± 0.1) kalemia was falsely misinterpreted as "normo-" or "hyper-" 3.5 -7.1 (5.1 ± 0.9 mmol/l) in 49/68 pt (72.1 %), thus disregarded so complicated by obvious hypokalemia additionally in 26/49 pt (53.1 %). in hypokalemia los in icu was 52.9 ± 29.7 vs 32.8 ± 18.6 h, p < 0.05. insulin use has caused hypoglycemia (1.2 -3.3 (2.5 ± 0.7 mmol/l)) in 22/120 pt (18.3 %), los in icu 63.2 ± 38.5 vs 38.9 ± 21.2 h, p < 0.05.insulin use was interrupted in case of normoand hypoglycemia with still persisting ketoacidosis in 39/120 pt (32.5 %), los in icu was found to be 56.5 ± 30.7 vs 37.0 ± 22.5 hr, p < 0.05. nah 2 co 3 was given for symptomatic treatment of ketoacidosis during first 10 h of dka in 33/120 pt (27.5 %) with stable hemodynamic: hco -3 buffer has increased (4.8 ± 3.3 -7.9 ± 3.1 mmol/l), p < 0.05, but it didn't control ketoacidosis, and los in icu was 55.2 ± 27.5.2 vs 39.1 ± 25.6 h, p < 0.05. hypokalemia, hypoglycemia, precocious interruption of insulin use were recorded as complications of dka treatment. all of them have prolonged los in icu. symptomatic treatment of ketoacidosis with nah 2 co 3 had no effect on it, and prolonged los in icu as well. a growing interest exists about co 2 derived parameters in shock management. central venous-arterial pco 2 difference (p cv-a co 2 ) is strictly related to cardiac output; central venous-arterial pco 2 difference to arterial-central venous o 2 content difference ratio, p cv-a co 2 / c a-cv o 2 , has been proposed as anaerobic metabolism when it's >1.4 mmhg/ml [1] . to evaluate p cv-a co 2 /c a-cv o 2 reliability in detecting anaerobic metabolism, we analyzed it in 7 consecutive patients affected by mala admitted to our icu, considering these patients as a prevalent anaerobic metabolism model. we calculated, by douglas formula, central venous-arterial co 2 content difference to arterial-central venous o 2 content difference ratio, c cv-ca co 2 /c a-ccv o 2 , as a respiratory quotient surrogate. we performed arterial and central venous blood gas analysis simultaneously at admission, we calculated p cv-a co 2 , p cv-a co 2 /c a-cv o 2 and c cv-a co 2 /c a-cv o 2 and we recorded scvo 2 . we verified relationship between p cv-a co 2 /c a-cv o 2 and scvo 2 and arterial ph, arterial lactates, sofa score at admission and c cv-a co 2 /c a-cv o 2 by linear regression analysis. pcv-aco2/ca-cvo2 greatly increases in mala (2.16 ± 0.84). pcv-aco2/ ca-cvo2 (fig.1) shows significant co-variation with ph (r2=0.618; p= 0.003) and sofa score at admission (r2=0.628; p=0.003). pcv-aco2/ ca-cvo2 has poor agreement with ccv-aco2/ca-cvo2 (r2=0.008) and disagrees with it in identifying anaerobic metabolism, in our series, in fact, ccv-aco2/ca-cvo2 is, in 3 patients, < 1 like an aerobic rq value. pcv-aco2/ca-cvo2 shows better agreement with ph, sofa score and lactate level than scvo2. in our series, p cv-a co 2 /c a-cv o 2 is good illness and acidosis severity marker, but it seems to be affected by ph value in accord with haldane effect [2] . p cv-a co 2 /c a-cv o 2 , in our study, doesn't seem to be a reliable anaerobic metabolism marker nor a rq surrogate. it is thought that early administration of basal insulin to patients with diabetic ketoacidosis (dka) may improve outcomes. small studies have shown trends towards decreases in time to closure of anion gap (tcag), rates of rebound hyperglycemia following discontinuation of intravenous (iv) insulin, rates of hypoglycemia, intensive care unit (icu) length of stay (los), and hospital los [1] [2] [3] [4] . this was a single-center, retrospective chart review of our institution's dka protocol between january 2010 and august 2019. patients that received early basal insulin within 24 hours of initiation of iv insulin and before closure of the anion gap (ag) were compared to those that did not receive early basal insulin. the primary outcome was median tcag. secondary efficacy outcomes include: time on iv insulin infusion, time to de-escalation of level of care, hospital los, and re-elevation of ag. secondary safety outcomes included incidences of hyperglycemia, hypoglycemia, and hypokalemia. a total of 334 patients were identified meeting inclusion and exclusion criteria. median tcag was longer in the experimental group (9 vs. 6 hours, p <0.01). incidence of re-elevation of ag and incidence of hyperglycemia were lower in the experimental group. other outcomes were similar (figure 1 ). early administration of basal insulin to patients with dka resulted in a longer tcag with a lower incidence of re-elevation of ag and hyperglycemia. early administration of basal insulin appears to be safe with respect to hypoglycemia and hypokalemia. glycaemic control continues to be a challenge in critically ill patients. stress induced hyperglycaemia has been associated with increased morbidity and mortality [1] . conversely, patients receiving intensive glucose control have a higher risk of death [2] . a quality improvement project was designed to develop a comprehensive insulin protocol that recognized pre-existing diabetes and reduced hypoglycaemia. data was collected prospectively in all adult patients admitted to the rah intensive care unit (icu) between october 2018 and august 2019 from the national icu audit database and electronic patient records. daily figures were collected for numbers of hypoglycaemic episodes (<4mmol/l), "in range" (4-10mmol/l) blood sugar measurements and patients with a pre-existing diagnosis of diabetes. data was collected and analysed using microsoft excel. results: 307 patients were identified; 56 patients (18.2%) had pre-existing diabetes. a total of 6908 blood sugar measurements were reviewed; 5268 (76.3%) were "in range" and 126 hypoglycaemic episodes (1.8%) occurred. there was no significant correlation between number of diabetic patients and measurements within range. of note, there was an increase in number of measurements per patient in the second half of the time period (11 vs 32). the development of this protocol has improved glycaemic control in our icu. there are considerably fewer episodes of hypoglycaemia and a large proportion of blood sugar measurements are in range. we hope to continue data collection and interrogate the prevalence of pre-existing diabetes further to reduce glycaemic variability. the optimal management of blood glucose levels for critically ill patients remains unclear. hypoglycemia, hyperglycemia and glycemic variability are associated with mortality. the time in targeted blood glucose range (tir) has been suggested to correlate with mortality depending on the status of antecedent glycemic control, but it has not been verified optimal tir and whether there is an optimal disease-specific tir. a retrospective observational study was performed at a single center. in the present study, we enrolled all critically ill patients admitted in intensive care unit from 1 january 2016 to 31 october. patients with diabetic ketoacidosis or hyperosmolar hyperglycemic syndrome and patients who had < 10 blood glucose readings were excluded. gathered information included, in part, demographics, comorbidities, severity of illness scores, diagnosis at admission, length of icu stay and hospital discharge status. the primary outcome was 28-day mortality. we analyzed to find the optimal tir for critically ill patients. several tirs were each tested for correlation with mortality. a total of 1,523 patients, 51.8% of whom had diabetes, were studied. tir 70 to 139 mg/dl (or, 0.33; 95%ci, 0.18-0.58), tir 70 to 179 mg/ dl (or, 0.33; 95%ci, 0.23-0.47) and tir 110 to 179 mg/dl (or, 0.28; 95%ci, 0.17-0.44) > 80 % was independently associated with mortality in critically ill patients respectively. the optimal tir did not differ depending on diagnosis at admission. in this retrospective evaluation, tir 110 to 179 mg/dl > 80 % was independently associated with mortality in critically ill patients, especially those with good antecedent glucose control. these findings have implications for the design of future trials of intensive insulin therapy. the prevalence of chronic dysglycemia (diabetes and prediabetes) in patients admitted to swedish intensive care units (icus) is unknown. we aimed to determine the prevalence of such chronic dysglycemia and asses its impact on blood glucose control and patient-centred outcomes in critically ill patients. in this retrospective, observational study, we obtained routine glycated hemoglobin a1c (hba1c) measured in patients admitted to four tertiary icus in sweden between march and august 2016. based on previous diabetes history and hba1c we determined the prevalence of chronic dysglycemia (prediabetes, undiagnosed diabetes and known diabetes). we compared indices of acute glycemic control in the icu and explored the association between chronic dysglycemia and icu-associated infections, mechanical ventilation, renal replacement therapy, vasopressor therapy, and mortality within 90 days. of 943 patients, 312 (33%) had chronic dysglycemia. of these 312 patients, 127 (41%) had prediabetes or undiagnosed diabetes and fig. 1 (abstract p259) . results 185 (59%) had a known diabetes diagnosis. during icu stay, patients with chronic dysglycemia had higher average blood glucose, spent less time in target glucose range, had greater glucose variability, and were more likely to develop hypoglycemia than patients without chronic dysglycemia. chronic dysglycemia was associated with greater need for renal replacement therapy (odds ratio 2.10, 95% ci 1.35-3.27) and increased 90-day mortality (hazard ratio 1.33, 95% ci 1.01-1.77) after adjustment for simplified acute physiology score 3. in contrast, chronic dysglycemia was not associated with mechanical ventilation, vasopressor therapy, or icu-associated infections. in four tertiary swedish icus, measurement of hba1c showed that 1/ 3 of patients had chronic dysglycemia (prediabetes or diabetes). chronic dysglycemia was associated with marked derangements in glycemic control during icu stay, greater need for renal replacement therapy and with increased mortality at 90 days. case report: modern antidiabetic therapie causes ketoacidosis am heiden, m emmerich krankenhaus bad oeynhausen, institut für anästhesie, bad oeynhausen, germany critical care 2020, 24(suppl 1):p263 the modern antidiabetic class of sglt2-inhibitors, that are known to reduce the risk for cardiac events [1] , are increasingly used in the last few years. a 68-year old male patient with diabetes mellitus suffered 10 days after colectomy surgery from abdominal pain and nausea. the patient had an antidiabetic therapy with empaglifozin that was paused until day 5 after surgery (nutrition start on day 5, weaning on day 6). methods: this is a case report of one male patient seen in the icu setting. daily blood values including arterial blood gases, vital parameters and clinical status of the patient were observed and evaluated. the blood gases showed this metabolic acidosis: ph 7.38; pco2 20.3 mmhg, bicarbonate 12 mmol/l, be -11.63 mmol/l, lactate 1.6 mmol/l, glucose 7 mmol/l. a ketonuria despite normal blood glucose values was noticed, so that the diagnosis of ketoacidosis was clear. after analyzing the possible causes we found out, that empaglifozin in times of catabolism and fasting can cause this severe symptomatic. we terminated the therapie with empaglifozin and under the treatment with insulin the symptoms disappeared within 3 days and the patient could be discharged from the icu on day 17 after surgery. after one episode of ketoacidosis the therapy with sglt2-inhibitors should lifelong never be started again. we recommend that intensivists should be aware of the modern sglt2-inhibitors because of the shown severe complications and the increased use of this medication. consent to publish: written informed consent for publication was obtained from the patient. while obesity confers an increased risk of death in the general population, numerous studies have reported an association between obesity and improved survival among critically ill patients. this contrary finding has been referred to as the obesity paradox. this retrospective study uses two causal inference approaches to address whether the survival of non-obese critically ill patients would have been improved if they had been obese. the study cohort comprises 6,557 adult critically ill patients hospitalized at the intensive care unit of the ghent university hospital between 2015 and 2017. obesity is defined as a body mass index of ≥30 kg/m 2 . two causal inference approaches are used to estimate the average treatment effect in the untreated (atu): a naive approach that uses traditional regression adjustment for confounding and that assumes missingness completely at random, and a robust approach that uses super learning within the targeted maximum likelihood estimation framework and that uses multivariate imputation of missing values under the assumption of missingness at random. obesity is present in 18.9% of patients. the in-hospital mortality is 14.6% in non-obese patients and 13.5% in obese patients. the marginal associational risk difference for in-hospital mortality between obese and non-obese patients is -1.06% (95% confidence interval (ci) -3.23% to 1.11%, p=0.337). the naive approach results in an atu of -2.48% (95% ci -4.80% to -0.15%, p=0.037), whereas the robust approach yields an atu of -0.59% (95% ci -2.77% to 1.60%, p=0.599). a robust causal inference approach that may handle confounding bias due to model misspecification and selection bias due to missing data mitigates the obesity paradox, whereas a naive approach results in even more paradoxical findings. the robust approach does not provide evidence that the survival of non-obese critically ill patients would have been improved if they had been obese. bowel management within an icu environment is often difficult. recent data collection from an intensive care unit at the rvi identified either loose stool or constipation on > 50% of patient days. it was postulated this could be improved with a more tightly controlled bowel management regimen. to test this hypothesis a step-wise bowel protocol was created and introduced. data was collected in the 4 month period following its implementation with the following aims: 1) assess effectiveness of the protocol 2) further observe the reasons for loose or constipated stool on an diarrhea is an important problem in each critically ill pateints [1] . we aimed to investigate the frequency and management of diarrhea in our icu. in this study 47 patient retrospectively reviewed, in our icu between 01.01.2017-03.01.2018. patients were divided into two group as diarrhea "positive" and "negative". patients with diarrhea had fluid or loose stools 3 or more times a day. each diarrhea period of the patients with diarrhea was examined separately and compared with the group without diarrhea. nutritional status, enteral product formulation, leukocyte, neutrophil, albumin values, gastric sparing, antibacterial and antimycotic use, los in hospital and in icu were compared. in diarrhea positive group, on the day of hospitalization, laxative and/or enema administration, toxin a in stool, nitrogen balance before and after diarrhea, enteral product change in diarrhea, probiotic, metronidazole or oral vancomycin use were examined. the incidence of diarrhea was 68.3%. the most common diagnosis of icu admision was respiratory failure (60-85%) in both groups. diarrhea occurred in two days after laxative and/or enema treatment. enteral nutrition was higher in both groups (≥90%). nasogastric tube feeding was significantly higher in the diarrhea group (p=0.041). there was no difference between nutritional product formulation and diarrhea development (p>0,1). antibacterial use was high in both groups (75%); however, teicoplanin use was significantly higher in the group diarrhea negative group (p=0.028). the los in icu, and hospital was higher in diarrhea group (p<0.001). no difference in mortality rates (p>0.5). many factors may cause diarrhea in icu, and diarrhea may adversely affect patient treatment and increase morbidity. we think that preventive methods are as important as the treatment of diarrhea. the use of parenteral glutamine is studied in number of rcts and systemic reviews (heyland d 2013, wischmeyer p 2014), while there is a lack of data about the use of enteral glutamine. the aim of our study was to determine the effect of enteral glutamine supplementation on the incidence of hospital infections and death. design: retrospective cohort study. inclusion criteria: males and females > 18 years of age, tbsa burned 20%-80%, nasogastric intubation.patients were divided in two groups: glutamine group (n=25) and control group (n=17). in the study group enteral glutamine was administered to the patients for 5 days after admission to the icu. baseline characteristics were well balanced between groups. no significant difference was found between groups on patients' age, sex, tbsa, need for mechanical ventilation and rate of inhalation injury. primary outcome was all-cause mortality. secondary outcome was rate of nosocomial infections (skin and skin structure infections (sssi), lower respiratory tract infections, urinary tract infections, bacteremia, sepsis). mortality rate was 6 (24%) and 7 (41%) in the glutamine group and the control group, respectively, p=0.40. rate of nosocomial infections was 14 (56%) in the glutamine group and 11 (65%) in the control group, respectively, р=0.81. rates of sssi, lower respiratory tract infections, urinary tract infections and sepsis did not differ significantly between the groups: 11 (44%) and 6 (35%), p=0.81; 6 (24%) and 7 (41%), р=0.40; 1 (4%) and 1 (6%), р=1.00; 6(24%) and 5 (29%), р= 0.97, respectively. rate of bacteremia was significantly different between the groups: 1 (4%) in the glutamine group and 5 (29%) in the control group, p=0.03. retrospective design is a significant limitation of our study. enteral glutamine supplementation may reduce the incidence of bacteremia in burn patients, but has no influence on the incidence of other nosocomial infections and mortality. further large clinical trials are needed. with outcomes were assessed with multivariable logistic regression and cox proportional hazard analyses, adjusted for baseline risk factors and randomization. in sensitivity analyses, models were further adjusted for key regulators of ketogenesis to assess whether any effect was direct or indirect. late pn increased plasma 3hb as compared with early pn, with maximal effect on day 2 (p<0.0001 for day 1 to 5 and for the "maximal effect" day in the 1142 patients). adjusted for baseline risk and randomization, plasma 3hb associated with a higher likelihood of earlier live weaning from mechanical ventilation (p=0.0002) and of earlier live picu discharge (p=0.004). as plasma 3hb replaced the effect of the randomization, the 3hb effect statistically explained these benefits of the randomization. further adjustment for key regulators of ketogenesis did not alter these findings. plasma 3hb did not independently associate with the risk of infections and mortality. withholding early pn increased ketogenesis in critically ill children, an effect that statistically mediated part of its clinical benefits. critical care patients are prone to frequent feeding interruptions for various reasons including feeding intolerance. these interruptions can lead to adverse outcomes. the aim of the study was to determine the reasons for and the duration of interruptions of enteral nutrition (en). single-center observational, cross-sectional study in a 19-bed mixed icu of a tertiary hospital. duration: 6 months. 50 patients, aged 65.4 years old (±14.6), that stayed in the icu > 48 hrs and were fed with en were included. anthropometric data, bmi, time of initiation of prescribed en, type of en formula, daily calories delivered were recorded. energy intake was calculated according to espen guidelines (25 kcal/ kg bw/day). the causes for and duration of interruption were reviewed from the patient's chart. apache ii and mnutric score was calculated for all patients. mnutric score ≤5 was used to diagnose malnutrition. all patients included in the study were endotracheally intubated. apache ii was 22.4 ±5.6. 58% of patients had increased risk of malnutrition. icu stay was 24.4 (8.0±32.0) days, and the in-hospital mortality was 24%. there were 318 episodes of en interruptions over a median icu stay of 24.4 days. median 2.5 interruptions/patient. the most common reason for en interruption was gastric residual volume monitoring followed by diagnostic and therapeutic procedures (figure 1 ). other reasons include surgery, intolerance and/or delayed feeding and extubation. the median lost feeding time was 5.4 hours/ day (3.7-7.4) for all causes, while the mean loss of total energy intake was 790 kcal/day (±321)/day. average body weight of the patients was 78 kg (±12). caloric deficit was calculated at 1950 kcal/day or 40% of the prescribed caloric goal. the results of this study showed that interruptions can lead to substantial caloric deficit, malnutrition and adverse events. an interruptionminimizing protocol could be useful in order to reduce the missing hours and to improve the clinical outcomes. relationship of goal-directed nutritional adequacy with clinical outcomes in critically ill patients pc tah there are controversies surrounding the effects of optimal nutritional intake on clinical outcomes in critically ill patients. this study aimed at investigating the relationship of goal-directed energy and protein adequacy on clinical outcomes which includes mortality, intensive care unit(icu) and hospital length of stay (los), and length of mechanical ventilation (lomv). this was a single centre prospective observational study. nutritional requirements were guided by indirect calorimetry and 24-h urinary urea.nutritional intake was recorded daily until death, discharge, or until day 14 of icu stay. clinical outcomes were collected from patient's hospital record. the relationship between the two groups (< 80% and ≥80% of overall nutritional requirement) with mortality outcomes was examined by using logistic regression with adjustment for potential confounders. terlipressin, despite being one of the main treatments for acute variceal bleeding, may lead to severe hyponatremia due to its antidiuretic activity.we aimed to identify risk factors for development of hyponatremia during terlipressin treatment. retrospective study of patients admitted to acute intermediate care unit for hypertensive upper gastrointestinal bleeding due to chronic liver disease who received terlipressin(december2011-decem-ber2018).hyponatremia was defined as a decrease in na serum levels ≥5meq and severe hyponatremia as >10meq within 3 days of treatment. we studied 191 patients, 84.3% male, mean age of 58.6 years (sd 10.8). alcohol-related liver disease was the most frequent etiology. hyponatremia occurred in 53 patients (27.7%). serum na δbetween -5 and -10meq and serum na δ>-10meq occurred in 20.4 and 7.3%, respectively (table 1) . severe hyponatremia occurred in 11 patients (5.8%) and symptoms were reported in two cases (status epilepticus and altered mental status). patients with higher baseline levels of na were more susceptible to terlipressin-induced hyponatremia and a longer length of stay was observed in patients with serum naδ>-10 meq (6.3 vs 4.4 days, p<0.022). the prevalence of hyponatremia in our study was lower than previously reported.higher serum na at admission and aih as etiology of cirrhosis were predictors of terlipressin-induced hyponatremia. neither the cumulative dose of terlipressin nor the duration of treatment appear to be related to the development of hyponatremia a δ48h-[na] >5 mmol/l was associated with larger hazards of mortality ( figure 1 ). an increase in serum sodium in the first 48 hours of icu admission is independently associated with a higher mortality in patients admitted with mild hyponatremia, normonatremia, and hypernatremia. based on our findings, it is possible that mild hyponatremia may be a protective mechanism in critical illness, which questions common practice of routinely correcting serum sodium when it is too low. introduction: acute liver failure (alf) represents a life-threatening organ dysfunction associated with increased mortality and liver transplantation represents the only definitive treatment. the aim of this study was to assess the effects of renal replacement therapy in combination with hemoadsorption in alf patients. twenty-nine patients with alf admitted to the intensive care unit (icu) of fundeni clinical institute were included in the study. after icu admission, 3 consecutive session of hemoadsorption in combination with continuous veno-venous hemodiafiltration were applied. number of organ dysfunctions and sirs criteria were recorded at icu admission. the following data were recorded before and after the 3 hemoadsorption therapies: glasgow coma scale, pao2/fio2, creatinine, 24-hours urine output, bilirubin, leucocyte and platelet count, heart rate, mean arterial pressure and vasopressor support, c-reactive protein and procalcitonine. clif-sofa score was calculated before and after the therapy. icu length of stay and 28-days outcome were noted. the mean age in the study group was 34±14 years. the median number of sirs criteria was 3 [2, 4] and the median number of organ dysfunctions was 3 [1, 6] . the use of hemoadsorption was associated with a decrease in creatinine (from 1.9±1.4 to 1.2±0.8 mg/dl, p= 0.02), bilirubin (from 14.2±12.6 to 9.2±9.1 mg/dl, p=0.05) and platelet count (96482±70913 / ul to 51275±24393 /ul, p=0.01). we also observed a decrease in clif-sofa score from 12.0±2.1 to 10.0±2.6 (p= 0.05). overall mortality was 37.9% (n=11). six patients (20.7%) underwent liver transplantation with 100% 28-days survival. the use of hemoadsorption in patients with alf is associated with improvement in liver and kidney functional tests and may represent a new therapy in bridging these patients to liver transplantation. introduction: impairment of intestinal mucosal barrier function is the initiating factor of sepsis. in order to explore the effect of lactic acid bacteria on intestinal barrier function impaired by sepsis, it is necessary to establish sepsis and lactic acid bacteria ecological models. however, how to construct these models is still unclear. co-cultures with a gradient of lactic acid bacteria and caco-2 cells were constructed. the symbiotic state was observed under an inverted microscope and lactate dehydrogenase (ldh) toxicity tests, transepithelial electrical resistance(teer) tests and western blots were used to determine effective concentrations of lactic acid bacteria in monolayer cell models. lipopolysaccharide (lps) was used to treat cells, and cell counting kit-8, quantitative reverse transcription pcr(rt-qpcr) and enzyme linked immunosorbent assays (elisa) were used to determine the appropriate concentration for sepsis models. the number of living cells decreased significantly when the moi(number of lactic acid bacteria/cell number) reached 8 ( figure 1 , panels 1a, b). the release of ldh indicated that damage to cells began to increase when the moi exceeded 10 (panels 2a, b). at an moi of 0.5, resistance values began to increase over time, whereas resistance values began to decrease when the moi reached 10 (panel 3). as the number of lactobacilli increased, the expression of tight junction protein increased and then decreased (panel 4a, b, c). in sepsis model experiments, the cell survival rate began to decrease once the concentration of lps exceeded 10^4 ng/ml (panel 5). rt-qpcr results showed that 10 2 ng/ml lps significantly increased inflammatory cytokines (panel 6), and elisa results consistently showed that tnf-α and il-6 increased significantly when lps concentrations reached 10 2 ng/ml (panel 7a, b). it is feasible to construct a cell monolayer model of lactic acid bacteria and lps. the appropriate moi of lactic acid bacteria is 0.5 and the optimal concentration of lps is 10 2 ng/ml. introduction: sepsis is associated with high mortality and morbidity. as the severity increases, physiological parameters such as ph changes are one of the most notable features in metabolic acidosis secondary to high lactate. currently there is no point of care test other than blood gas measurement that could detect these ph changes. this is challenging especially in prehospital environment. the aim of this study is to develop a novel rapid point of care testing using a sensor to detect ph change in blood. sensors were produced by screen printing graphene and silver electrodes and functionalizing the graphene working electrode with an active layer of melanin. a preclinical sensor model was produced by adding lactic acid to a citrated plasma sample thus altering its ph over a clinically relevant range. the ph sensors were exposed to modified plasma, recording any changes in the voltage. the relationship between the voltage potential and plasma ph was established using weighted least squares regression. a ph dependent change in the measured voltage, with respect to the ph of the solution, was observed with a sensitivity of -111.27 mv/ph +/-15.95 over a physiologically relevant ph range between ph7.1 and ph7.6. in this first phase proof of concept study a low cost, ph sensor was fabricated and demonstrated to be effective in measuring the ph of the plasma. this is the first time that such a sensor has been demonstrated and validated to work in this preclinical model of acidosis. the technology demonstrated here is a promising candidate for a point of care test whereby abnormal blood ph levels can be detected and monitored outside of a laboratory environment in a rapid manner. further studies are now underway to detect this change in whole blood. (figure 1) . over one year only a small proportion of patients (n=7, 4%) were classified as 'intermediate high' risk and potential candidates for reperfusion therapies. the revised national early warning score (news) with modified glasgow prognostic score (mgps) is superior to the news for predicting in-hospital mortality in elderly emergency patients t mitsunaga jikei university school of medicine, emergency medicine, tokyo, japan critical care 2020, 24(suppl 1):p286 the national early warning score (news) was developed in the ukto identify the risk of death. the previous study showed that the modified glasgow prognostic score (mgps) correlate with frailty in elderly patients [1] . the aim of this study is to evaluate the predict value of the revised news with mgps for in-hospital mortality (in 28 days) in elderly emergency patients. this study is secondary analysis and was carried out in jikei university kashiwa hospital, in japan, from 1 april 2017 to 31 march 2018. the acute medical patients aged 65 and older were included. the news was derived from seven physiological vital signs. the mgps was derived from c-reactive protein (crp) and albumin. discrimination was assessed by plotting the receiver operating characteristics (roc) curve and calculating the area under the roc curve (auc). the aucs for predicting in 28 days in-hospital mortality were 0.818 for revised news with mgps and 0.797 for the original news. the auc of the revised news with mgps was significantly higher than that of the original news for predicting in-hospital mortality (p < 0.001) (figure 1) . our single-centred study has demonstrated the utility of the revised news with mgps as a high predictor of acute phase in-hospital mortality in elderly emergency patients. the diagnostic performance of the five main emergency department (ed) triage systems has been shown to be poor in distinguishing acute coronary syndromes (acs) from mild severity diseases in chest pain patients. these ed triage systems are either clinically-based, being more sensitive or ecg-based, more specific [1] . the goal of the study was to evaluate if incorporation of cardiovascular risk factors (cvrf) into ecgbased triage could increase his diagnostic performance. cecidoc is a prospective, observational, single-center study in an academic hospital. all consecutive adult patients admitted for acute chest pain were included. we compared the ecg-based french triage system [2] to a modified system upgrading patients with a normal ecg but significant cardiovascular risk from a low acuity triage score (waiting period before medical assessment of max. 60 min.) to a high acuity triage score (waiting period before medical assessment of max. 20 min.). the final diagnosis was determined after a 30-day follow-up. we predefined as being adequate a high-acuity triage score (level 1 or 2) for acs and a low-acuity score (level 3, 4 or 5) for mild severity diseases. a total of 190 patients was enrolled over a 5-month period (age 56.8 ± 16.4; m/f ratio 1.7). triage scores of 35 patients (18.4%) with acs were compared to 103 patients (54.2%) with mild severity diseases. taking into account cvrf, the sensitivity of the triage system increased from 60 to 80% whereas the specificity decreased from 74 to 61%. area under the roc curve (auc) went from 0.69 to 0.72 (fig. 1) . for chest pain triage at ed, addition of cardiovascular risk factors into ecg-based triage increases his diagnostic performance. approximately 20% of patients presenting to hospital with an intentional overdose require admission to an intensive care unit (icu) [1] . there are currently no uk guidelines regarding the optimal use of ct head scans (cth) in this patient cohort [2, 3] . this study aims to determine whether we should be performing ct head scans in obtunded patients with suspected overdose requiring admission to intensive care. we performed a retrospective search of the icnarc database for plymouth university hospital trust, looking for patients admitted to the icu with overdose or self-poisoning as a primary diagnosis. 146 patients were identified and 86 of these patients required intubation due to obtundation(gcs<8). there were 59 males and 27 females with an average age of 38 years old. the median length of stay on the unit was 1 day. 52 of the patients has a past medical history of mental illness, and 53 overdosed on prescribed medications. the average gcs recorded on admission was 5. 52 of the 86 (64%) patients had a cth on admission, of which 5 were part of a trauma scan. 11 were known overdoses and 7 were suspected overdose as per the cth request form. the main rationale behind those requests were to exclude additional intracranial injury. none of those cth showed any signs of acute pathology (figure 1) . in this retrospective study, obtunded patients with suspected or known overdose with no history of apparent trauma or injury do not benefit from cth. in the absence of a history of trauma or focal neurological signs our conclusions are that cth provides limited value in the management of these patients. the audit was carried out to objectively investigate the problems associated with technique of folley catheterization in emergency department and 3 indoor units of internal medicine wards [1] . introduction: cellular and molecular mechanisms, epigenetic aspects of acute clozapine poisoning are studied insufficiently. the aim of this study was to identify morphological and epigenetic alteratons in brain neurons during acute exposure to clozapine combined wit ethanol. the experiments were carried out on male wistar rats weighting 200-250g (n=21). group i (control) received 0.9% nacl solution enterally; group iiclozapine 150 mg/kg in 0.9% nacl solution; group iiiclozapine 150 mg/kg in 40% ethyl alcohol. after 4 hours euthanasia was performed. autopsy included withdrawal of brain samples for histological examination (n = 21) and for determination of global dna methylation level (n = 21). the global dna methylation level (5-mc%) was determinated by fluorimetric method. inter-group comparisons were made by kruskal-wallis test. histological examination of paraffin sections of brains stained with hematoxylin and eosin was performed by light microscopy. in acute сlozapine poisoning and its combination with ethanol morphological changes in neurons of the cerebral cortex were detected. in acute сlozapine with alcohol poisoning an increase of global dna methylation level was observed. probably the identified changes have a common pathogenesis which will be clarified in our further studies. there is limited information available regarding the prevalence of adder bites and the complications of envenomation. nhs data suggests there are 100 adder bites annually in the uk with the last fatality in 1975 [1] . we performed an audit into adder bites in south west wales to identify the number attending our emergency departments, their management and clinical course as well as any environmental factors that predict increased likelihood of being bitten or the severity of the bite. a retrospective study of adder bites attending emergency departments in south west wales was undertaken (jan 2014 to aug 2019). measurements included were patient demographics, clinical presentation, type of treatment (conservative vs anti-venom) and outcome. results: 31 patients were included, age range 2-72 years ( figure 1 ). the majority of bites occurred in sand dunes (41.9%) and all bites were on extremities. anti-venom was administered to 45.2% (14/31) of patients. there was a significant positive association between the use of anti-venom and the length of hospital stay (r = 0.520; p=0.003) and a significant negative correlation between the anti-venom use and both diastolic and systolic blood pressure (p= 0.501 and 0.487 respectively p=0.01). all patients fully recovered. in this study, we demonstrated that with a full clinical assessment on presentation it is safe to decide whether anti-venom is required. the current guidelines are safe and effective in the treatment of adder bites. 98μmol/l, for pao2 < 9.9kpa and > 12.3 kpa, platelets < 165*10^9/l and > 327*10^9/l, and bilirubin > 12μmol/l. in our population of adult ed patients, the thresholds of vital values associated with increased 7-day mortality were very close to routinely used values, and most of the thresholds were included in the lowest urgency level in triage and risk-stratification scoring systems. the workload in the emergency room: direct assessment by the therapeutic intervention scoring system-76 and indirect assessment by the nasa task introduction: the number of emergency room admissions continues to increase each year, which increases the care workload of the emergency department staff, who should to use its theoretical and practical knowledge in order to provide quality care in difficult working conditions. the aim of our study was to assess the emergency room staff workload its impact on health workers and patients and to suggest an improvement strategy to decrease this workload. a prospective, monocentric cohort study with descriptive and analytic approach over one month (december 2018) conducted at the emergency department of an academic hospital. the workload endured by the emergency room staff was evaluated by the nasa task load index and on patients by the therapeutic intervention scoring system-76. there were 286 cumulative days of hospitalization in 67 consecutive patients admitted to the emergency room. the average age was 61 ±15 years. the average length of stay at the emergency room was about 103 ±48h. the average tiss-76 score was 31.7 ±14.9. factors associated with important care workload were: age ≥ 65 years, diabetes, more than 3 comorbidities, the use of intravenous antibiotics; the use of vasoactive drugs and the use of mechanical ventilation; a high tiss score was predictive of emergency room mortality. in the indirect assessment of the care workload, 41 medical and paramedical staff were interviewed, 73% of them were under 40 years old with a sex ratio of 0.58. a high level of mental and physical workload was expressed by ed staff with considerable level of frustration; the ed staff suggested mainly to improve the working conditions, communication and to redefine tasks "who does what". our study had shown a significant workload in the emergency room, a process to reduce this workload is being implemented medical simulation is a modern teaching tool increasingly used in specialties such as anesthesia, emergency medicine and obstetrics. however, it's not widely used in specialties like cardiology, althought cardiovascular emergencies are very frequent. the purpose of our study was to assess the effectiveness of simulation-based medical education in the management of cardiovascular emergencies among moroccan graduate students. we conducted a prospective, observational, multi-centrer study including the students of three moroccan universities from the 5 th to the 7 th year of medicine who underwent 6 phases: first a pre-test, then a theoretical and practical training on cardiovascular emergencies after which the students were separated in two groups, one undergoing the medical simulation training (group 1) and one who didn't (group 2), followed by a theoretical then a practical post-test on resusci anne and simman®. at last, the students were asked to answer a satisfaction survey. the reform procedure in the tunisian army consists in repairing the physical damage and deciding on the applicant's ability to continue working. terrorism increases the impact of the co-morbidity generated and the socio-economic consequences that result from it. the purpose of this work was to study the epidemiological, clinical and evolutionary profile of terrorist injuries, to specify the rates of consequent partial permanent disability (ppi) and the possibilities of returning to work. descriptive retrospective cross-sectional study of 177 reform files on military personnel injured during anti-terrorist operations from fig. 1 (abstract 296) . changes in total bcpr rate in family-and friends-witnessed ohca cases with dispatcher-assisted instruction during 20-week period after the day of disaster during three years january 2013 to september 2019. the data collection was carried out on the basis of a collection form. our 177 wounded were male, 96% of whom belonged to the army. the average age was 36 years and 3 months ± 8.869. half of our wounded were troopers. infantry and special forces were the most exposed military units. half of the accidents were recorded in the kasserine region (88 cases). chronic post-traumatic stress disorder (cptss) was found in 130 injured, followed by amputations in 18 injured. the after-effects were psychological in 32%, physical in 26% and mixed in 39% of our injured. the ppi rate ranged from 36% to 75% in 23.7% of injuries.. more than half of the injured had returned to their professional activity, 33% were put on reform for health reasons. our results showed that the esptc was the most recorded sequel, and that the ppi rate was significant in a quarter of our injuries. in our series, a third of our wounded were put on reform for health reasons. to state the importance of initial care and adequate and rigorous follow-up to recover a greater number of war wounded. introduction: the rapid response system (rrs) has been shown to decrease hospital mortality [1] . the japanese coalition for patient safety has set a major goal for hospitals to more widely implement the rrs. however, prevalence and actual circumstances of use in acute care hospitals (including small scale hospitals) in japan are as yet not well-known. web-based questionnaires were sent to acute care hospitals (of scale 75 beds-or-larger) of 17 prefectures in western japan. each participant hospital selected a certain department which answered the questionnaire. the rrs included the medical emergency team (met), the rapid response team (rrt), and the critical care outreach team (ccot). we investigated the presence and circumstances of in-hospital emergency calls, rrs and other systems, and then illuminated issues to be solved. our study suggests that delays in patient transfer to the icu after rrt activation in the wards were associated with slower physiological improvement.these findings support further and larger studies. blood and blood products use in intensive care unit m akcivan, s bozbay, o demirkiran istanbul university cerrahpasa, anesthesiology and intensive care, istanbul, turkey critical care 2020, 24(suppl 1):p304 blood and blood product (bp) transfusions are frequently used in intensive care units (icu) [1] . it is important to know transfusion epidemiology and the effect of adverse transfusion reactions and their effect on mortality and morbidity.we aimed to investigate the blood and bp transfusions in the icu. blood and bp transfusions in icu, between 2013-2017 were reviewed retrospectively. we evaluated each transfusion as a data and examined the pre-and post-transfusion laboratory values, demographic data, cause of icu admission and comorbidities. results: 284 patients who underwent transfusion in the icu, and 2188 transfusion data from these patients were included. the most frequent cause of hospitalizations were respiratory failure and sepsis. the rate of patients transfused in the five-year period decreased from 73.9% to 36.67%. the hemoglobin threshold before transfusion decreased from 8.34 g / dl to 7.91 g / dl. a total of 148 transfusion reactions were observed and the most common transfusion reaction was febrile non-hemolytic reaction. the most commonly transfused product was red blood cell suspension. transfusion reactions were found to be slightly higher in men than women in young age group(<65y) (p = 0.44 and p=0.021, respectively). transfusion reactions were found to be more frequent in emergency transfusions (p <0.01). the number of transfusions was significantly lower in patients with apache ii score <20 (p <0.01). the need for transfusion was found to be higher in patients with hematological malignancy (p <0.01). it was observed that as the mean number of transfusions increased the mortality is also increased (p <0.01). transfusion therapies are the treatments that are vital but have a serious mortality and morbidity risk. in particular, intensive care patients should be considered in detail because of their specific features. restrictive transfusion practices have positive results. association between anemia or red blood cell transfusion and outcome in oncologic surgical patients. figure 1a) . the association between rbc transfusion and adverse events also remained after adjustment (or 4.3 [2.2 -8.8] ; p < 0.001) ( figure 1b) . in oncologic surgical critically ill patients, there was an independent association between anemia (even moderate anemia) or rbc transfusion and patient outcomes. our findings highlight the need for further research to determine the optimal transfusion strategy in surgical oncologic patients. transfusion impaired skin blood flow when initially high e cavalcante dos santos, w mongkolpun, p bakos, al alves da cunha, c woitexen campos, jl vincent, j creteur, fs taccone erasme hospital, intensive care department, brussels, belgium critical care 2020, 24(suppl 1):p306 red blood cell transfusion (rbct) increases global oxygen delivery (do 2 ) and may improve microcirculation. however, the effects on blood flow have been found to be conflicting. we studied icu patients with stable hemodynamic status (mean arterial pressure (map) ≥ 65 mmhg for at least 6 hours) and without active bleeding, who received a rbct. skin blood flow (sbf) was determined (periflux system 5000, perimed, index finger; perfusion unit, pu) together with map, heart rate (hr), hemoglobin (hb), lactate levels and scvo 2 before and after rbct. sbf was measured before rbct (t0) and after (t1) for each 3 min. according to previous data indicating the lowest sbf value found in noninfected icu patients was 151 pu, all patients were analyzed according to the baseline sbf (i.e. <151 pu -low sbf vs. ≥151 puhigh sbf). the relative change of sbf (δsbf) was calculated after rbct and the responders were defined by the function of >10%. results: 63 icu patients were studied. rbct was associated with increases in map and scvo 2 but no change in sbf. at baseline, scvo 2 was lower in the responders than in the non-responders (p=0.04) and lower in patients with low sbf than in the high sbf (p=0.04). there was no difference in hb, map, and lactate, between the patients with low and high sbf. after rbct, map rose in the responders (p<0.01) and in the non-responders (p=0.03), sbf (p<0.01) rose in patients with low sbf, and sbf (p=0.02) decreased in patients with high sbf. there was a negative correlation between baseline scvo 2 (r= -0.363, p<0.01) or baseline sbf (r= -0.560, p<0.01) and the relative increase in sbf after rbct. rbct increases skin blood flow only when it is impaired at baseline. severe immune dysregulation is associated with adverse outcomes and is common in intensive care unit (icu) patients [1] . erythropoietin-stimulating agents (esas) have both anti-apoptotic and immune-modulating properties [2] . despite potential benefit, both the safety and efficacy of these agents remains unclear [3] . here we evaluate the impact of esas on morality at hospital discharge in critically unwell adult patients admitted to the icu. we conducted our search strategy in accordance with a predetermined protocol. the use of ffp is associated with an increased incidence of complications such as acute respiratory distress and infections, and the rate of complications increased with the quantities of ffp transfused [1] . pcc contain several important coagulation factors and it has been suggested that they could replace ffp. this has been shown mainly in case reports or series in which coagulation factor deficit was detected by using poc viscoelastic tests in trauma [2] or traditional hemostatic tests in obstetric patients [3] . multicenter observational study of the safety and efficacy of the prothrombin complex concentrate. a survey of anesthetists was conducted in 19 maternity hospitals at various levels of care in the russian federation. data has been collected and processed. as a result, 251 patients were analyzed. pph was determined as a volume of blood loss more than 500 ml during vaginal delivery or cs. the most significant risk factors for pph were: preeclampsia or arterial hypertension and a history of postpartum hemorrhage. 32.3% had no risk factors for pph. it was determined that the use of prothromplex 600 iu decreased the number of patients with transfusion ffp 12-15 ml/kg by 27.8% and increased the number of patients without transfusion by 25.9%, compared with patients without use of prothromplex 600 iu (figure 1 ). no complications were detected. the use of pcc safety and efficacy reduce use of ffp during pph. the full analysis included 45 patients on either hfc (n=22) or cryoprecipitate (n=23). the intraoperative and postoperative changes in etp and fibrinogen concentration are shown in table 1 . for fibtem a20 (intraoperatively) and fibrinogen concentration (intraoperatively and postoperatively), the mean numerical values appeared higher with hfc than cryoprecipitate. fxiii (hfc: 121.86%, 66.85%; cryoprecipitate: 115.55%, 68.68%, at baseline and 4hr after surgery start), fviii and vwf were maintained throughout surgery in both treatment groups. this was also the case for laboratory tests activated partial thromboplastin time, prothrombin time and platelet count. the forma-05 coagulation parameters analyses showed broad overlaps between hfc and cryoprecipitate, with satisfactory maintenance of the clot quality parameters, fxiii concentrations and thrombin generation parameters. the study group includes 118 men and 40 women with a mean age of 43,008 vs. 40.32 years (p=0.639) admitted with the diagnosis of multiple trauma. we found a directly proportional and highly significant statistical correlation between base excess and fibrinogen level diagnosed using the mcf/fibtem parameter(r=0.6382, p<0.0001)and an inverse proportional correlation between lactate level and fibrinogen level (r= -0.2164, p=0.0065). in the roc analysis that uses as a variable the level of base excess and as a criterion of classification the fibrinogen deficit (mcf/fibtem<12 mm) it can be observed that at a value of be<-7 mmol/l, we can diagnose a fibrinogen deficit with a sensitivity of 88.2% and a specificity of 80.6% (auc= 0.872,p< 0.0001). lactate appears to be inferior to the excess base (figure 1) , but still has a good diagnostic power, a value of 2.6 mmol/l has a sensitivity of 67.1% and a specificity of 75% (auc= 0.754,p<0.0001). the difference between the two roc curves (0.118) is statistically significant (p = 0.0028). both base excess and serum lactate can be used to diagnose fibrinogen deficiency with the mention that base excess appears to have a higher sensibility and specificity ability. based goal-directed algorithm. this approach requires further clinical validation. we conducted a retrospective study comparing transfusion strategies in patients with major trauma between 2013 and 2018. we retrieved demographic data and blood products administered from patients with at least one red-blood cell (rbc) transfusion. primary outcome was a reduction of rbc administration. secondary outcomes were mortality, icu length of stay and acute kidney injury. we included 141 patients admitted in the icu due to severe trauma (sapsii:41.5 ±21.9), and mainly after emergent surgery (68.8%). they featured a mean age of 45.3±19.3y, were predominantly male (76.6%) and 73% were in shock. in the first 24 hours of hospital admission a mean of 3.6±4.5 rbc units were administered. most patients received a fibrinogen-based protocol (fbp) (78%), with an average of 5±3g of fibrinogen and 1±3 fresh-frozen plasma (ffp) units, versus 3±4 g of fibrinogen and 6±4 ffp units in the ffp group. the fbp was associated with a decrease administration of rbcs in the first 24 hours (r = -2.6; p < 0.004), even after adjustment for severity (p=0.003) and for tranexamic acid use (p = 0.003). it was associated also with a decrease of platelet transfusion (p=0.004). fibrinogen-based protocol was not associated with a decrease in mortality, acute kidney injury or noradrenaline dose. treatment of tic in past years has progressively changed to a goaldirected fibrinogen-based approach. in our population, the use of fbp lead to a reduction of rbc administration in severe trauma patients. prospective, multicenter, randomized study comparing administration of clotting factor concentrates with a standard massive hemorrhage protocol in severely bleeding trauma patients the objective of this study was to assess the ability of the quantra® qstat® system (hemosonics) to detect coagulopathies in trauma patients. many level 1 trauma centers have adopted whole blood viscoelastic testing, such as rotational thromboelastometry (rotem®, fig. 1 (abstract 315) . study treatment plan instrumentation lab) for directing transfusion therapy in bleeding patients. the quantra qstat system is a cartridge-based point-of-care (poc) device that uses ultrasound to measure viscoelastic properties of whole blood. and provides measures of clot time, clot stiffness and a test of fibrinolytic function. methods: adult subjects were enrolled at two level 1 trauma centers which use a rotem based protocol to guide transfusion decisions. study protocols were approved by the site's ethics committee. for each subject, whole blood samples were drawn upon arrival to the emergency department and again, in some cases, after administration of blood products or antifibrinolytics. samples were analyzed on the quantra (at poc) in parallel to rotem delta (in lab). a total of 54 patients were analyzed. approximately 42% of samples had a low clot stiffness (cs) values suggestive of an hypocoagulable state. the low stiffness values could be attributed to either low platelet contribution (pcs), low fibrinogen contribution (fcs), or a combination ( figure 1) . additionally, 12% of samples showed evidence of hyperfibrinolysis based on the quantra clot stability to lysis parameter. samples analyzed on standard rotem assays showed a lower prevalence of low clot stiffness and fibrinolysis based on extem, fib-tem results. the correlation of cs and fcs vs equivalent rotem parameters was strong with r-values of 0.83 and 0.78, respectively. this first clinical experience with the quantra in trauma patients showed that the qstat cartridge detected coagulopathies associated with critical bleeding and may be useful for directing blood product transfusions in these patients. ability to perform testing at poc may provide additional clinical advantage. the objective of the study was to describe the conditions of use of fibryga® 1g, a new, highly purified, human fibrinogen (hf) recently granted a temporary import authorization for use in congenital and acquired fibrinogen deficiencies in france. observational, non-interventional, non-comparative, retrospective study conducted in 5 french hospital centres using fibryga®. data from patients with fibrinogen deficiency having received fibryga® from december 2017 to july 2019 were retrieved from their medical files. indications, modalities, efficacy and safety outcomes were recorded. indications encompassed non-surgical bleeding (nsb) either spontaneous or traumatic, including post-partum hemorrhage (pph), bleeding during surgery (sb) or administration to prevent bleeding during planned surgery. treatment success was defined as control of the bleeding or hemoglobin loss <20% for bleeding treatment and as absence of major perioperative hemorrhage for pre-surgical prevention. this analysis included 110 patients aged 56,7 ± 17.7 years and 60% were male. all presented an acquired fibrinogen deficiency requiring administration of hf. indications were nsb (n=45, 40.9%) including 15 (13.6%) pph, sb (n=31, 28.2%), and prevention of sb (n=34; 30,9%). cardiac surgeries were the main procedures associated with treatment and prevention of sb. mean total doses of fc were 2.95± 1.66g, 2.00±1.37g and 2.21±1.23g for nsb, sb and prevention of sb. success rates were 88.4% (95%ci 78.8-98.0%), 96.8% (95%ci 90.6-100%) and 91.2% (95%ci 81.6-100%) respectively. for pph, mean dose of hf was 2.53±0.74g with a success rate of 86.7% (95%ci 69.5-100%). overall, tolerance was good. fibrinogen concentrate fibryga® is mostly used for bleeding control. in one third of patients, hf was administered preventively to avoid bleeding during surgery. use of fibryga® was associated with favourable efficacy outcomes. functional testing for tranexamic acid effect duration using modified viscoelastometry t kammerer 1 , p groene 2 , s sappel 2 , p scheiermann 2 , st schaefer 2 1 ruhr-university bochum, institute of anaesthesiology, heart and diabetes center nrw, bad oeynhausen, germany; 2 ludwig-maximilans university, department of anaesthesiology, munich, germany critical care 2020, 24(suppl 1):p318 tranexamic acid (txa) is the gold standard to prevent or treat hyperfibrinolysis [1] . effective plasma concentrations are still under discussion [2] . in this prospective, observational trial using modified viscoelastometry we evaluated the time-course of the antifibrinolytic activity of txa in patients undergoing cardiac surgery. methods: 25 patients were included. modified viscoelastometry (tpa-test) was performed and txa-plasma-concentration, plasminogen-activatorinhibitor-1 (pai-1) and pai-antigen-plasma-concentrations were measured over 96h. additionally, in vitro dose-effect-curves from blood of healthy volunteers were performed. data presented as median with interquartile range (q1/q3). results: txa plasma-concentration was increased compared to baseline (t1:0 μg ml -1 ) at every time-point with a peak concentration 30min (t2) after application (p<0.0001; see fig.1a ). lysis was inhibited from 30min (lysistime tpa-test : p<0.01; lysisonsettime tpa-test :p<0.0001). maximumlysis tpa-test was decreased at t2 (t1:97% (96/97) vs. t2: 9% (6/11); p<0.0001). of note, after 24h some patients (n=17) had normalized lysis whereas others (n=8) had strong lysis inhibition (ml< 30%;p<0.05) up to 96h. high and low lysis groups differed regarding kidney function (cystatin c:1.64mg l -1 (1.42/2.02) vs. 1.28mg l -1 (1.01/ 1.52);p=0.002) and active pai-1 (93.05ng ml -1 (33.15/9100.0) vs. 16.13ng ml -1 (6.62/79.98);p=0.047). in-vitro, txa concentrations >10μg ml -1 were effective to inhibit fibrinolysis. in our trial, after 24h there was still completely blocked lysis in patients with moderate renal impairment. this could be critical with respect to postoperative thromboembolic events [3] . here modified viscoelastometry could be helpful to detect the individual fibrinolytic capacity. introduction: peri-operative coagulopathy correction based on viscoelastic hemostatic assays (vhas) and single-factor coagulation products has changed the paradigm of bleeding management in cardiac surgery [1] . in a retrospective study, we analysed patients with emergency surgery for thoracic acute aortic dissection (taad), before and after the introduction of fibrinogen concentrate in clinical practice. data were collected from paper and electronic records. the study was approved by the institutional ethical committee. 60 patients were included in the analysis, 19 operated in 2012, before fibrinogen concentrate was approved for human use, and 41 in 2018-2019. therapy was guided by a rotational thrombo-elastometry (rotem) algorithm. exclusion criteria were non-compliance with the institutional protocol and intra-operative death. we investigated allogeneic blood transfusion (abt), fibrinogen use, peri-operative bleeding (pob), surgical reexploration and post-operative complications (poc). the groups were similar in gender, age, body weight, additive euro-score and aortic cross-clamp time. fresh frozen plasma, cryoprecipitate and red blood cell transfusion were lower in the fibrinogen group, but not platelet transfusion (table). 48,7% of patients in the study group received fibrinogen concentrate and median dose was 2 g (iqr 2-3). day 1 postoperative chest tube drainage and surgical reexploration were significantly lower. there were no differences in stroke, renal replacement therapy, mechanical ventilation time and icu stay. in patients with taad surgery, rotem-guided algorithms which include fibrinogen concentrate are associated with less (pob), surgical re-exploration and abt. further research is needed to document the role of vhas and concentrated factors in reducing (poc). andexanet alfa (aa, portola pharmaceuticals, san francisco, ca) represents a modified factor xa agent which is approved antidote for apixaban and rivaroxaban. andexanet alfa may also neutralize the anti-xa effects of betrixaban and edoxaban. this study aims to compare the relative neutralization of these four anti-xa agents by andexanet alfa in different matrices. andexanet alfa was diluted at 10 mg/ml. apixaban (a), betrixaban (b), edoxaban (e) and rivaroxaban (r) were diluted in ph 8.4, 0.5 m tris buffer (tb), blood bank plasma (bbp) and in 5% albuminated buffer (ab) at 0.062 -1.0 ug/ml. anti-xa activities of all four agents were measured in three systems and the reversibility indices of aa were profiled. the reversibility index (ri 50 ) of anti-xa effects by aa was determined at 25 -100 ug/ml. each of the four agents produced varying degrees of inhibition of anti-xa at 0.062 -1.0 ug/ml, the ic 50 ranged 0.61 -1.53 ug/ml in bbp, 0.47 -1.28 ug/ml in ab and 0.49 -1.4 ug/ml in tb. andexanet alfa produced a concentration dependent reversal of all four anti-xa agents. in the bbp, the ri 50 values for a (192 ug/ml), b (32 ug/ml), e (152 ug/ml) and r (85 ug/ml). in the ab, the ri 50 values for a (140 ug/ml), b (46 ug/ml), e (176 ug/ml) and r (58 ug/ml). in the tb, the ri 50 values for a (154 ug/ml), b (79 ug/ml), e (>400 ug/ml) and r (110 ug/ml). each of the four anti-xa agents exhibit varying degrees of matrix independent anti-xa potencies in different systems, the collective order follows edoxaban > apixaban > betrixaban > rivaroxaban. andexanet alfa produced matrix dependent differential neutralization of the anti-xa effects of these agents. individualized dosing of andexanet alfa may be required to obtain desirable clinical results. the diagnostic and prognostic value of thromboelastogram (teg) in sepsis has not been determined. this study aimed to assess whether teg is an early predictor of coagulopathy [1, 2] and is associated with mortality in patients with sepsis. in total, 518 patients with sepsis on intensive care unit admission were prospectively evaluated. we measured teg and conventional coagulation tests(ccts)on preadmission and observed for development of 1, 3 days and 1, 3, 7days respectively. multivariable logistic regression was utilized to determine odds of icu/hospital mortality. the parameter of teg (maximum amplitude, reaction time; ma/r ratio) was calculated to evaluate sepsis-induced coagulopathy. the admission patients were divided into three groupsma/r0 group(ma/r= 5-14mm/min); ma/r1group(ma/r>14 mm/min)and ma/r2 group(ma/r<5mm/min). in our cohort of patients with severe sepsis, coagulopathy defined by ma/r ratio was associated with increased risk of icu/hospital mortality. introduction: blood sampling for coagulation assessment is often carried out in either arterial or venous samples in the intensive care unit (icu). there is controversy as to the accuracy of this method due to the inherent differences in physicochemical properties as well as the underlying effects of individual diseases in arterial and venous blood. clot microstructure has shown to be a new biomarker (fractal dimension-d f ) which encompasses the effects of diseases in all aspects of the coagulation system [1, 2] . in this study, we compared the effect of all these factors in venous and arterial blood to see if there is a difference in the clot microstructure and quality. 45 patients admitted to a tertiary intensive care unit and busy teaching hospital were recruited. arterial and venous blood was sampled from an arterial line and central venous catheter in situ from the same patient. standard markers of coagulation (pt, aptt, fibrinogen, full blood count), rotational thromboelastometry (rotem), whole blood impedance aggregometry and measured clot microstructure (d f ) were measured on both arterial and venous samples. no significant difference was observed in standard laboratory markers, rotem and platelet aggregation between arterial and venous blood. there were no differences in the fractal dimension (d f ) between the arterial and venous blood samples (d f 1.658 ± 0.10 vs 1.654 ± 0.08 respectively, p=0.830). samples from patients with critical illness give comparable results from either arterial or venous blood despite their underlying pathophysiological process or treatment. this confirms blood for coagulation testing can be taken from arterial or venous blood. clinicians in the emergency setting use a wide range of hemostatic markers to diagnose and monitor disease and treatment. current methods rely on the anticoagulant effect of citrate on whole blood prior to laboratory analysis. despite the well-recognized modulatory effects of citrate on hemostasis, the use of anticoagulated blood has clear analytical advantages, including repeat sampling and storage. however by altering the physiological state of the blood reproducibility and accuracy of the test is affected. recent studies have shown the potential of a novel functional biomarker of clot formation: fractal dimension (d f ), that may give an improved diagnostic accuracy. in this study we assessed the potential of this new biomarker in scientifically measuring the effects of recalcification of citrated samples. methods: 35 healthy volunteers were included. unadulterated and sodium citrate samples of blood were taken from each volunteer. citrated samples were recalcified using (1m cacl 2 ). in the study we compared unadulterated whole blood d f results to citrated d f results and repeated the citrated d f experiments 5 times for each sample over a 2 hour period to ascertain reproducibility. the d f of citrated blood was significantly lower than that of unadulterated blood (1.57±0.04 vs 1.69±0.04, p<0.001). the results of the citrate samples when tested 5 times over 2 hrs gave a coefficient of variation of 1.16%. for the first time we show that a functional biomarker of clot microstructure, d f , can precisely quantify and measure accurately the direct effect that the addition of the anticoagulant sodium citrate has on whole blood clot microstructure. the study also shows that the test is reproducible and has potential utility as a biomarker of acute disease in the emergency setting in citrated blood. this procedure now needs to be evaluated in a group of acute disease states. in this study, we analyzed the hematological abnormalities of dengue patients by thromboelastography (teg) at initial and 1-hour of fluid resuscitation. methods: this is a cross-sectional study evaluating teg readings of dengue patients with different severities presenting to the emergency department. laboratory confirmed dengue patient (positive ns1 antigen or igg/igm) was consecutively sampled. teg readings were taken at presentation and after 1-hour of fluid resuscitation. twenty dengue patients with varying severity had a median reaction time (r), α -angle, k time, maximum amplitude (ma) and lysis 30% (ly30) of 0.495 min, 68.74 ο , 3.58 min, 44.64 mm and 0.54% respectively. mean fibrinogen was normal before and after fluid infusion. there is a non-significant reduction in ma with prolongation of other teg parameters between different dengue severities. there is a statistically significant reduction of α-angle and ma between pre and post 1-hour fluid resuscitation (p=0.019 and p=0.040). normal fibrinogen with low ma, which signifies a weak clot strength, may indicate either a platelet reduction, platelet dysfunction or both. reduction in ma and α-angle post fluid resuscitation is an alarming finding. this is in contrast with previous teg studies although none of it used normal saline exclusively, studied initial fluid resuscitation in emergency department settings or studied a subject with dengue. a bigger study, especially in severe dengue is needed to validate our findings. agreement between the thromboelastography reaction time parameter using fresh and citrated whole blood during extracorporeal membrane oxygenation with teg®5000 and teg®6s m panigada, s de falco, n bottino, p properzi, g grasselli, a pesenti fondazione irccs ca´granda ospedale maggiore policlinico, intensive care unit, milano, italy critical care 2020, 24(suppl 1):p327 the r (reaction time) parameter of kaolin-activated thromboelastography (teg) may be used to assess the degree of heparinization of blood during ecmo. a teg analysis is usually performed on two types of samples: fresh (f) or citrated-recalcified (c) whole blood. teg®5000 can perform the analysis on c and f whole blood, the new teg®6s (haemonetics corp., ma, usa) only on c whole blood. aim of the study was to compare the response of r to heparin using the two types of samples and two teg devices methods: during a three months period at fondazione irccs ca' granda -policlinico of milan, teg was performed (using teg5000® and teg 6s® with and without heparinase, an enzyme that degrades heparin) on 13 consecutive ecmo patients (as part of the gatra study, nct03208270) and in 8 consecutive non-ecmo patients in whom a teg was requested for clinical purposes. bland altman analysis and lin's concordance correlation coefficient were used to assess agreement results: a total of 84 paired samples were taken (74 in-ecmo and 10 off-ecmo). ecmo patients received 19.2 (12.6-25.8) iu/kg/h of heparin. among non-ecmo patients, 5 of them did not receive any dose of heparin, two of them a very low prophylactic dose (1.6 and 2.9 iu/ kg/h, respectively), and one of them 13.1 iu/kg/h of heparin. using teg®5000, r was -21.0 (-65.5; 23.4) min shorter on c compared to f blood in patients receiving heparin (this difference disappeared using heparinase) and only -1.58 (-8.70; 5.54) min shorter in patients notreceiving heparin. r was -26.6 (-77.3; 24.2) min shorter using teg®6s (which performs the analysis only on c blood) than teg®5000 on f blood (figure 1) . when evaluating the effect of heparin using teg, clinicians should be aware that results obtained using citrated-recalcified or fresh whole blood are not interchangeable. using citrated-recalcified blood to perform teg might lead to underestimation of the effect of heparin trauma patients are at high risk for venous thromboembolism (vte). the 2002 east guidelines recommend low molecular weight heparin (lmwh) for vte prevention and antixa monitoring after initiation of the medication or after adjusting doses in certain populations [1] . studies have shown standard enoxaparin dosing of 30 mg every 12 hours may result in low antixa levels [2] . this study aims to evaluate the efficacy of a pharmacist-lead protocol for adjusting enoxaparin dosing based on antixa levels in trauma patients. this single center retrospective chart review included adult trauma patients admitted from 3/1/2018 to 9/20/2019. per protocol, patients with body mass index (bmi) ≤40 kg/m 2 were initiated on enoxaparin 30 mg twice daily, and patients with bmi > 40 kg/m 2 were initiated on enoxaparin 40 mg twice daily. peak antixa levels were drawn 4 to 6 hours after at least the third dose of enoxaparin with a goal therapeutic range of 0.2-0.4 iu/ml. the primary objective was time in days to goal peak antixa level. secondary objectives include vte occurrence, bleeding attributed to lmwh, and dosing regimens utilized. subgroups were analyzed based on body mass index (bmi). of 511 patients identified, 375 patients met inclusion criteria. median time to therapeutic antixa level was 3 days (iqr 2-6). of 337 patients fig. 1 (abstract 327) . agreement between teg®6s and r teg®5000 on citrated recalcified and fresh whole blood with bmi ≤ 40 kg/m 2 , 319 patients (94.7%) were dosed initially per protocol and 153/319 patients (48.0%) met goal antixa level at first check (table 1) . of 38 patients with bmi > 40 kg/m 2 , 24 patients (63.1%) were dosed initially per protocol and 8/24 patients (33.3%) met goal antixa level at first check. our results indicate the protocol is safe due to lack of bleeding attributed to enoxaparin, but less than 50% of patients achieved goal antixa level at first check. however, despite low rates of achieving goal antixa level, vte rates also remained low. introduction: most patients in the icu are given prophylactic anticoagulation with a fixed dose of 40 mg once daily of enoxaparin (clexane) if cct is normal and 20 mg if cct is low. studies on non icu patients have shown that afxa is below desired range for venous thromboembolism (vte) prevention. in the icu, many factors might influence afxa levels including weight, creatinine clearance (cct), shock and other medication. atxa activity was not yet reported in a big mixed icu population with variable morbidity. our study hypothesis is that enoxaparin is underdosed in most cases and routine afxa activity should be monitored in all icu patients. preventive enoxaparin (40 mg qd) was given to all patients unless therapeutic dose was needed or contraindication existed. levels of afxa activity were taken 4 hours after the 3 rd dose. therapeutic vte preventive effect was defined as afxa activity of 0.2-0.5. patient data was collected from medical files. the study is still ongoing, preliminary results were analyzed for 31 patients. 14 of 31 patients (45%) had afxa activity below normal (subtherapeutic). weight and cct were negatively correlated with afxa activity (figure 1 ). mean weight in the subtherapeutic afxa was significantly higher than the therapeutic group (83.2 vs. 72.6 respectively, p=0.031). cct in the subtherapeutic afxa was significantly higher than the therapeutic group (95.1 vs. 60.4 respectively, p= 0.003). the normal cct group (>50) had significantly more patients with subtherapeutic afxa (13 vs 8, p=0.017). in our icu, 45% of the patients receive insufficient vte prophylaxis. overweight patients and patients with normal cct should probably receive higher enoxaprin dose. afxa activity should be routinely monitored in icu patients. in this study we use a new bedside biomarker to test its ability to measure anticoagulation effects on patients who present with acute first time deep vein thrombosis (dvt). dvt requires oral anticoagulants to prevent progression to potentially fatal pulmonary embolism and recurrence. therapeutic efficacy monitoring of direct oral anticoagulants (doac) including rivaroxaban is problematic as no reliable test is currently available. advances in hemorheological techniques have created a functional coagulation biomarker at the gel point (gp) which allows quantitative assessment of: time to the gel point (t gp ), fractal dimension (d f ) and elasticity (g') [1, 2] . the prospective observational cohort study measured t gp , d f , g', standard coagulation and cellular markers in first time dvt patients at three sample points: pre-treatment and approximately 20 and 60 days following 15mg bd and 20mg od rivaroxaban respectively. strict inclusion and exclusion criteria applied. results: 40 dvt patients (mean age 64 years [sd±14.8]; 23 male, 17 female) and 177 non-dvt patients were well matched for age, gender and co-morbidities. mean t gp on admission was 267s (sd±63.3s) and 262.9s (sd±73.5s) for dvt and non-dvt respectively. doac therapy significantly increased t gp to 392.3s (sd±135.7s) after 20 days, and subsequently increased to 395.5s (sd±194.2s) at 60 days as shown in table 1 . d f , g' and standard hemostatic markers all remain within the normal range. conclusions: t gp demonstrates its utility in determining the anticoagulant effect of rivaroxaban. the significant difference in t gp between males and females needs further exploration. localized stasis as a result of transient provoking factors appears not to generate a systemic strength fig. 1 (abstract p329) . correlation of anti factor xa activity with patient cct and weight. anti fxa activity value below 0.2 (red line), was considered "non-effective prevention" introduction: trauma remains the leading cause of death all over the world. to better exploit the trauma care system, precise diagnosis of the injury site and prompt control of bleeding are essential. here, we created a nursing protocol for initial medical care for trauma. the aim of this study was to evaluate the impact of protocoled nursing care for trauma on measures of quality performance. this was a retrospective historical control study, consisted of consecutive severe trauma patients (injury severity score >16). people were divided into two groups: protocoled group (from april 2017 to march 2019) and control group (from april 2014 to march 2017). we set the primary endpoint as mortality for bleeding. the secondary endpoints included time allotted from arrival to start of ct scan and surgery, administration rate of several drugs (sedations, painkillers, preoperative antibiotics, and tranexamic acid). for the statistical analysis, continuous variables were expressed as median (interquartile range) and were compared by wilcoxon rank sum tests given a nonnormal distribution of the data. we included 152 patients in the study: 84 in the control group before the introduction of the protocol, 68 in the protocoled group. as a primary endpoint, the mortality for bleeding was similar between two groups (5% in the control group and 5% in the protocoled group). as a secondary endpoint, the time to ct initiation [group a 11 (7-16) min vs group b 7 (5-10) min; p <0.001], and emergency procedure [group a 41 (33-56) min vs group b (29-43); p <0.001] were shortened by the protocol introduction. furthermore, the administration rates of sedations, painkillers, preoperative antibiotics, and tranexamic acid were increased in the protocoled group compared with the control group. although the mortality as a patient-oriented outcome was not affected, improved quality of medical care by nursing protocol introduction may be suggested in this analysis. this single-institutional prospective study included 72 patients with uprf who were admitted to the trauma surgical intensive care unit (tsicu) and survived until discharge to home between 2012 and 2017. we evaluated the activities of daily living after the discharge using physical and mental component scores of sf-36® and defined physical dysfunction (pd) as physical function (pf-n) score of 40 or less. we divided the patients in the pd (n=34) and control (without pd, n=38) groups and compared the groups. the patients had experienced blunt injuries, including falls (38%) and pedestrian injuries (33%). the mean age was 59.9 years (men: 65.3%); the median injury severity score was 22 (interquartile range: 13-29); and the mean length of tsicu stay was 3.3 days. the average period from the injury until the survey was 34.1 months. there was no difference between the pd group and the control group in the patient characteristics, fracture type, pelvic fixation, and complications. at the time of the survey, the pd group had significantly more painful complaints than the control group (pd: 67.6%, c: 23.6%, p <0.01), and had more physical and mental problems. the sf-36®subscale score showed a significant positive correlation between physical function and body pain, mental health respectively. the percentage of those who were able to return to work was not different in both groups (pd:26.5%, c:47.4%). in the multivariate analysis of pd, only age (odds ratio: 1.039, 95% ci: 1.00-1.07, p = 0.03) was relevant. long-term pd was observed in 47% of patients with uprf. the elderly were particularly prominent, and there was an association between pain and mental health. cells (rbc) this can lead to inhibition of oxygen transport function and development of hypoxia. currently used methods for analyzing the state of rbc either do not have sufficient accuracy or require lengthy analysis and expensive equipment. the use of a simpler and more informative electrochemical approach to assessing the state of rbc is very promising. electrochemical measurements in rbc suspensions (~5 • 109 cells / l) were carried out in a special electrochemical cell [1] in the potentiodynamic mode in the potential range from -0.5 to +1.2 v using the ipc pro mf potentiostat (kronas, russia); optical measurements were performed using an eclipse ts100 inverted microscope (nikon, japan), a cfi s plan fluor elwd 60x / 0.70 lens (nikon, japan); rbc morphology was recorded in real time using a ds-fi1 digital camera (nikon, japan). when examining rbc of patients with severe multiple trauma a decrease in the ability of rbc to change their shape during electrochemical exposure was observed, indicating a decrease in deformability, which can lead to a disruption in the oxygen supply to tissues. at the same time, with the stabilization of the patient's condition a restoration of the ability of rbc to change morphology was detected which in turn could have a positive effect on the rheological characteristics of the blood (fig. 1) . the results of the analysis of red blood cells using electrochemical changes in their morphology can be used as an additional method for the diagnosis of critical conditions. severe trauma should be treated immediately. whole-body ct (wbct) is widely accepted to improve the accuracy of detecting injuries. however, it remains the problem of time-consuming. therefore, we focused on the scout image taken in advance of wbct. detecting major traumatic injuries from a single scout image would reduce the time to start treatment. a previous study suggested that even specialists could not easily find chest and pelvic injuries using wbct scout image alone. in this study, we aimed to develop and validate deep neural network (dnn) models detecting pneumo/hemothorax and pelvic fracture from wbct scouts. we retrospectively collected 2088 anonymous wbct scouts together with their clinical reports at the osaka general medical center between january 1, 2013, and december 31, 2017. we excluded incomplete, younger than 7 years old, postoperative, and poorly depicted images. the part of this dataset from january 1, 2017, until december 31, 2017, was used for validation and the rest for training dnn models. pneumo/hemothorax detection model and pelvic fracture detection model were trained respectively. accuracy, and areas under the receiver operating characteristic curves (aucs) were used to assess the models. the training dataset for pneumo/hemothorax contained 984 images (mean age 48 years; 30% female patients), and for pelvic fracture consisted of 783 images (48 years; 28%). the validation dataset for the former contained 258 images (54 years; 30%), and for the latter consisted of 186 images (55 years; 24%). the models achieved 59% accuracy and an auc of 0.57 for detecting pneumo/hemothorax, 72% and 0.62 for pelvic fracture. our results show that dnn models can potentially identify pneumo/ hemothorax and pelvic fracture from wbct scouts. increasing the number of samples, dnn model could accurately detect severe trauma injuries using wbct scout image. clinical information system (cis) is a computer system used in collecting, processing, and presenting data for patient care. it can reduce staff workload and errors; help in monitoring quality of care; track staff's compliance to care bundles; and provide data for research purpose. however, the transition from paper record format to electronic record involves changes in all kind of workflow in icu. therefore, an effective, efficient and evaluative rollout plan was required to minimize the risk that might arise from the new practice. methods: 1. small groups training were provided. a working station with different case scenarios were set up for practices. 2. individual tutorials were conducted to clarify questions. emphasis on patient care was always top priority. 3. contingency plans were available in case of server breakdown and power failure. downtime drills were conducted to prepare the staff in emergency situations. 4. step-by-step transition from paper record to electronic format was gradually carried out. a plan was discussed among cis team with clear dates and goals. 5. new items in cis were first reviewed and amended in team meeting until consensus was made; then were promulgated to all staffs during handover before implementation. fig. 1 (abstract p333) . the effect of therapy on the electrochemically induced change in the morphology of red blood cells in patients with combined trauma 6. staff compliance and outcomes were then monitored; further review and amendment would be possible if necessary. cis roll-out plan was smooth. all staffs were able to integrate cis into the daily routine. the contingency plans were well acknowledged. new items were followed as planned. ongoing enhancement in cis was put forward on nursing orders, handover summary, and integration with inpatient medication order entry (ipmoe) system. with emerging benefits cis brings along, our staff has more time to devote to direct patient care. human input in data interpretation and clinical judgment on top of cis play an irreplaceable role in patient care. the daily request for laboratory tests in intensive care units is a common practice. although common, this strategy is not supported, since more than 50% of the exams requested with this rationale may be within the normal range [1] . misconduct based on misleading results, anemia, delirium and unnecessary increase in costs may happen [2] . we have developed a strategy to reduce laboratory tests without clinical rationale. observational retrospective study, from july 2018 to june 2019. the number and type of laboratory orders requested, the epidemiological profile of hospitalized patients, the use of advanced supports, the average length of icu stay and the impact in outcomes such as mortality and hospital discharge at a private tertiary general hospital in the city of rio de janeiro / rj -brazil were analyzed. a strategy was implemented to reduce the request for exams considered unnecessary. approximately 1,300 patients underwent icu during this period. the epidemiological profile and severity of patients admitted to the unit were similar to those observed historically. there was a significant reduction (> 50%) in the request for laboratory tests and there was no negative impact on outcomes such as mortality, mean length of stay and no greater use of invasive resources. over the period evaluated, the estimated savings from reducing the need for unnecessary exams were approximately $ 150,000 per year. the rational use of resources in the icu should be increasingly prioritized and the request for routine laboratory tests reviewed. a strategy that avoids such waste, when properly implemented, enables proper care, reducing costs and ensuring quality without compromising safety. evaluating the medication reconciliation errors in 2 icus after implementing a hospital-wide integrated electronic health record system a rosillette, r shulman, y jani university college hospital, centre for medicines optimisation research and education, london, united kingdom critical care 2020, 24(suppl 1):p337 introduction: medication errors in intensive care unit (icu) are frequent [1] and can arise from a number of causes including transition of care. our aim was to investigate the impact of an integrated electronic health record system (ehrs) on medication reconciliation (mr) errors occurring at 2 critical steps: during the transition from an icu to the hospital ward and from the ward to hospital discharge. the objective was to examine the influence of icu admission on long-term medication. we performed a monocentric study in 2 icus of a university-affiliated hospital using drug chart and medical notes review to identify mr errors before, during and after icu admission. data were collected retrospectively from ehrs for 50 consecutive patients discharged from the icu between 1 june-31 july 2019, and who were newly initiated on specific drugs of interest. results: 129 drugs of interest were initiated in icu. many of these were continued after hospital discharge as shown in table 1 . there was appropriate discontinuation of all the antipsychotics newly initiated in icu. other than anticoagulants, there was no reason documented for continuation of the initiated drugs. the planned durations were documented more often after hospital discharge than icu discharge for the following drug classes (% of patients with a plan after icu discharge to the ward; % after home discharge): antibiotics (47.6%; 52.6%), and steroids (45.4%; 57.1%), but less so for analgesics (36.3%; 14.3%), insomnia (0.0%; 20.0%), and gastroprotective drugs (0.0%; 20.0%). our study has shown that medications initiated in the icu can be inadvertently continued at icu and hospital discharge due to failure in documenting indication or duration. systems are required to deprescribe icu only drugs at discharge or communicate a plan for ongoing treatment. introduction: the surviving sepsis campaign advocates the use of care bundles to guide the management of sepsis and septic shock [1] . our study aim was to assess compliance with a locally introduced sepsis pathway and to review intensive care unit admission outcomes. we carried out a prospective audit of patients admitted to the icu at royal surrey county hospital with a diagnosis of sepsis between 19/ 3/19 and 19/11/19, assessing compliance with local sepsis bundle delivery, outcome of icu admission and degree of associated organ dysfunction. results: 119 patients were identified, 71 male (59.7%), with a mean age of 65.7 (18-96). mean 1 st 24 hour sofa score on icu was 6.65 (2-15). 81% of patients required vasopressors, with 67% requiring noradrenaline >0.1mcg/kg/min, and 19% requiring an additional vasopressor/ inotrope. 36% required niv, 32% invasive ventilation and 15% rrt. icu mortality was 15%, in-hospital mortality 24%, mean icu stay 8 days (1-49), and mean length of hospital stay 28 days . in the presence of septic shock mortality was 47% with post-resuscitation lactate >4, versus 21% in patients with no vasopressor requirement or lactate <2 (p<0.05). the sepsis bundle was delivered in one hour to 61 patients (51%). where the bundle wasn't completed, antibiotics were delayed in 26% of cases and blood cultures weren't taken in 66%. where the bundle was fully delivered, unit mortality was 12% vs. 21% where it was not (p<0.05), but there was no significant difference in hospital mortality (26% vs. 30%, p>0.5) or rates of vasopressor requirement, niv, ippv or rrt. there is room for improvement in timely delivery of the sepsis bundle in our hospital and various measures are being instituted. though there was no significant difference in hospital mortality, icu mortality was significantly lower in patients when the bundle was fully delivered. surviving sepsis campaign recommends 3h and 6 h sepsis resuscitation bundle for sepsis. the study was done to assess the feasibility of the guideline and the compliance to sepsis-3 recommendations at an emergency department. prospective interventional study was conducted during one year. were involved in the study all sepsis cases with a qsofa ≥ 2. were assessed a composite of six components (measurement of serum lactate, obtaining blood culture before antibiotic administration and provision of broad-spectrum antibiotic before the end of h3 and provision of fluid bolus in hypotension, attainment of target central venous pressure assessed by cardiac ultrasonography, target lactate to normal level before the end of h6). time base line was the first medical contact at triage zone. secondary outcomes of study were the mortality rate and length of stay at intensive care unit (icu). were involved in the study, 128 patients (mean age 54±17 years, sex ration 2,3). pulmonary infections were the main cause of sepsis (37%) and urinary tracts infections (22%). at h3 components were achieved in 79% of cases [lactates (100%), blood culture (82%) and provision of antibiotics (79%)]. at h6 components were executed in 64% of cases (fluid provision achievement in 89%, ultrasonography assessment in 64% and normal lactate target achieved in 71%) (figure 1 ). the reliability-adjusted rate for completion of the 3 hours and 6 hours bundle was at 68%. patients compliant to composite bundle got the mortality benefit (odds ratios = 0.31, 95% [confidence interval, 0.11-0.72]). the study, however, did not show any benefits of mean intensive care unit (icu) length of stay. faisability of 3-6h bundle ratio was at 68%. it has shown a significant improvement in adaptation and mortality benefit without reducing mean hospital/icu length of stay. more adapted procedures are needed to improve results targeting full compliance of patients to the 3-6h bundle sepsis management. patterns and outcome of critical care admissions with sepsis in a resource limited setting m edirisooriya maddumage 1 , y gunasekara 2 , d priyankara 1 1 national hospital of sri lanka, medical intensive care unit, colombo 10, sri lanka; 2 sri jayawardenepura general hospital, department of critical care, nugegoda, sri lanka critical care 2020, 24(suppl 1):p340 introduction: paucity of epidemiological data is a major barrier in expansion of critical care services, especially in resource limited settings. we evaluated the patterns and the outcome of critically ill patients with sepsis admitted to a level 3 medical intensive care unit in sri lanka. a retrospective cohort study was performed to describe the characteristics and outcome of patients with sepsis, admitted to a medical intensive care unit. sepsis is defined according to sepsis 3 definition. we examined 360 critically ill patients admitted over a period of 6 months. sepsis was the commonest presentation, accounted for 63.6 % of all admissions. mean age was 49.2 ± 16.1 years. septic shock was present in 41.4% on admission. pneumonia (35.4%) was the commonest cause, while leptospirosis (17.9%) and meningoencephalitis (12.2%) accounted for fig. 1 (abstract p339) . sepsis 3-6h bundle components (% of goals achievment) second and third commonest causes of sepsis respectively. the sofa score on admission (8.8 ± 5.0 vs 7.5 ± 4.9, p< 0.025), occurrence of aki (62% vs 49.6%, p<0.02) and the length of icu stay (8.8 days vs 6.2 days, p <0.001), were significantly higher in sepsis than in patients without sepsis. icu mortality in sepsis (n=92) did not show a significant difference to nortality (n= 45) in those without sepsis (40% vs 35%, p= 0.5). patients with leptospirosis had a mean sofa score of 13.3, however the mortality (37.5% vs 40%, p = 0.75) was similar to others with sepsis. in contrast, mortality related to sepsis was significantly high ( 60%, p< 0.02) in the packground of immunosuppression (n= 35). respiratory failure secondary to pneumonia was the commonest cause of critical care admission with sepsis. sepsis related icu mortality was high in the background of immunosuppression. introduction: training in placement, and the subsequent safe confirmation of position, of a nasogastric (ng) tube, relies on clinicians completing an e-learning module at our trust. feeding through an incorrectly placed ng tube is a 'never event,' associated with significant morbidity and mortality [1] . analysis of these incidents reveal that the misinterpretation of chest radiographs, by medical staff, who had not received competency-based training, is the most frequent cause [2] . e-learning has revolutionized the delivery of medical education [3] , however, there are barriers to its use [4] . we hypothesized that, by taking e-learning content, and delivering it face-to-face, we would improve training rates, and thus patient safety. a questionnaire was completed by 50 critical care doctors, concerning their knowledge of the existence of the e-learning module, whether they had completed formal training in ng tube placement, and how confident they were, on confirming correct positioning, using a 5 point likert scale. all clinicians underwent training in the interpretation of ng placement, using chest radiographs. after the session they were asked to re-appraise how confident they felt. results were compared using paired t tests. confidence improved in all, rising from a pre-test average score of 3.74 (sd=0.92), to post-session 4.76 (sd=0.48), p=<0.0001. prior to the intervention, 63% of the doctors were aware of the trust guidelines, but only 17% had completed the training. after the session, 100% were aware of the guidelines, and 100% had completed the training (figure 1) . conclusions: e-learning is a useful tool, but has its limitations. by using course content, delivered with more traditional learning methods, we im-proved the number of appropriately trained clinicians, and thus the safe use of ng tubes in our unit. a systematic review of anticoagulation strategies for patients with atrial fibrillation in critical care a nelson, b johnston, a waite, i welters, g lemma university of liverpool, liverpool, united kingdom critical care 2020, 24(suppl 1):p342 there is a paucity of data assessing the impact on clinical outcomes of anticoagulation strategies for atrial fibrillation (af) in the critical care population. this review aims to assess the existing literature to evaluate the effectiveness of anticoagulation strategies used in critical care for atrial fibrillation. only 4 studies contained analysable data. anticoagulated patients had a lower mortality at 30 days and 365 days post admission to critical care, however there was an increased incidence of major bleeding events compared to the non-anticoagulated population. thromboembolic events were comparable in both cohorts. data from current literature is scarce and inferences regarding the effectiveness of anticoagulation in patients in critical care with af requires further investigation and research. every new admission to the icu prompts a handover from the referring department to the icu staff. this step in the patient pathway provides an opportunity for information to be lost and for patient care to be compromised. mortality rates in intensive care have fallen over the last twenty years, however, 20% of patients admitted to an icu will die during their admission [1] . communication errors contribute to approximately two-thirds of notable clinical incidents; over half of these are related to a handover [2] . nice have concluded that structured handovers can result in reduced mortality, reduced length of hospital stay and improvements in senior clinical staff and nurse satisfaction [3] . a checklist was created to review the information shared and to score the handover. this checklist was created with doctors and nurses and is relevant for handovers between all staff members. information was gathered prospectively by directly observing 17 handovers on the icu. there is a notable discrepancy in the quality of handovers of new patients ( figure 1 ). this is true of handovers between doctors, nurses and a combination of the two. it is also true of all staff grades. whilst a doctor may have reviewed the patient prior to their arrival, 41% (n=7) of patients weren't handed over to a doctor. the most commonly missed pieces of information were details of the patient's weight (96%, n=16), their height (100%, n=17), whether the patient has previously been admitted to an icu (78%, n=15) and whether the patient has any allergies (71%, n=12). the handover of new patients to the icu is often unstructured and important information is missed. this can be said for all staff members and grades, and for handovers from all hospital departments. post intensive care syndrome-family (pics-f) describes new or worsening psychological distress in family and caregivers after critical illness but remains poorly studied within specialist groups [1] . we aim to define the degree of pics-f within our tertiary referral cardiothoracic centre and map change over the course of 12 months. caregivers attended a 5-week multi-professional clinic alongside patients. peer support was facilitated through a café area and a caregiver group psychology session was offered with individual appointments if required. caregiver surveys were completed including: caregiver strain index; hospital anxiety and depression scale (hads); and insomnia severity index. patients also completed hads questionnaires. repeat surveys were completed at 3 and 12 months. results: over 5 cohorts, 23 caregivers attended, of which 17 were spouses (74%), 3 children (13%), and 3 others (13%), with 13 caregivers completing surveys at 12 months. patients' median apache 2 score was 17 (iqr 14-18.5) and median icu length of stay was 11 days (iqr 8-23.5). most admissions were from scheduled operations (56%). severe caregiver strain was present in 4/23 (17%) with changes to personal plans (35%) the most common sub category. hads demonstrated 13 caregivers (57%) with anxiety and 8 (35%) with depression. caregiver anxiety exceded that of patients', only reaching fig. 1 (abstract p343) . each handover was scored according to the information accurately given to icu staff similar levels at 12 months, while depression remained static ( figure 1 ). median number of nights with 'bothered' sleep was 5 (iqr 1-6.75) and 77% of caregivers expressed problems with sleep. conclusions: significant psychological morbidity in caregivers from our tertiary cardiothoracic centre is in keeping with the general icu population [2] . caregiver strain was reduced suggesting higher levels of resilience. future work should address mental wellbeing, particularly anxiety, to minimise the effects of pics-f. burnout syndrome is an illness that has increasingly affected health professionals. it is characterized by great emotional stress, physical and mental exhaustion and depersonalization of the individual. more serious cases can lead to job loss or even suicide. the described work identifies the burnout level of the multidisciplinary team through a specific questionnaireburnout syndrome is an illness that has increasingly affected health professionals. it is characterized by great emotional stress, physical and mental exhaustion and depersonalization of the individual. more serious cases can lead to job loss or even suicide. the described work identifies the burnout level of the multidisciplinary team through a specific questionnaire methods: application of a questionnaire suitable for the multidisciplinary group in november 2019. the same was answered by 85 professionals among physicians and nursing team. there was no identification of employees. after analysis of the results it is observed that 50% of the group presents initial burnout, 30% with the syndrome installed and about 5% with characteristics of greater severity. main factors found were: mental and physical exhaustion during the work day, the level of responsibility existing in the activity and the perception of disproportionate remuneration by work performed. all interviewees presented some degree of burnout or high risk to develop it. the most severe cases should be traced through occupational medicine and anti-stress measures with reorganization of work performance should be discussed in order to reduce the prevalence of this syndrome. introduction: burnout affecting the psychological and physical state of healthcare workers is recognized in the last 10 years. burnout has been shown to affect the quality of care. whilst some risk factors have been identified, there are gaps within the literature related to mental health and burnout. the aim of this study is to measure levels of burnout across 3 icu units in the metropolitan setting. to determine the level of burnout we used 2 surveys, the maslach burnout inventory human services survey (mbi-hss) and the centre for epidemiologic studies depression scale (ces-d). with the mbi-hss we analysed 3 different variables of burnout; exhaustion, cynicism and emotional exhaustion. basic demographic data and information regarding workout schedules were collected. we studied prevalence and contributing risk factors using and analysing the outcomes of the 2 self-scoring questionnaires. analysis was performed using descriptive statistical analysis. there were 90 respondents, 36% scored the threshold for depressive symptoms on the ces-d depression scale. interestingly, 40% (ci 25.4-57.7%) of those meeting the score for depressive symptoms identified as having frequent restless sleep compared with 11% (4.6-21.8 %) from those not meeting. gender did not affect depressive symptoms 35% of females and 37% of males met the threshold. with the mbi-hss for exhaustion the mean was 17.16 (sd 4.6) which is a high level of exhaustion, the second variable cynicism the mean score was 14.08 (sd 4.2), which was considered high. the final variable was emotional exhaustion the mean was 25.16 (sd 9.90), this is considered moderate levels of emotional exhaustion. fig. 1 (abstract p344) . hospital anxiety and depression scale (hads) scores for patients and caregivers at baseline, 3 months, and 12 months there was high prevalence of burnout in icu in all different categories as well as depressive symptoms. age and gender had no affect on burnout. interestingly, we identified that sleep and shift variables were linked to increased burnout. following the implementation of a fully integrated ehrs on 31 march 2019 at our university-affiliated hospital we conducted a prospective study in 2 icus by analysing pharmacists' contributions during 4 data collection periods of 5 days at 10, 15, 19 and 21 weeks post implementation. a pharmacists' contribution was defined as contacting the physician to make a recommendation in a change of therapy/ monitoring [1] . the 3 types of contribution were: a medication errorrectification of an error in the medication process; an optimizationproactive contribution that sought to enhance patient care, and a consult -reactive intervention in response to a request. a panel of experts composed of a senior pharmacist, a consultant, a nurse, and a pharmacy student assessed the impact of each contribution, scoring low impact, moderate impact or high impact. there were 160 pharmacist contributions recorded in the 4 periods. of these, 66 (41.2%) were medication errors, 93 (58.1%) were optimizations, and 1 (0.6%) was a consult ( table 1) . 37% of the contributions were assessed as having medium impact, 36% as high impact and 27% as low impact. in general, the consultant assessed fewer contributions as having high impact compared to other members of the panel, with 7 contributions assessed as high impact by the consultant versus 97 by the senior pharmacist. implementing an ehrs in combination with contributions of clinical pharmacists can prevent medication related issues. interestingly the types of incident did not change over time. introduction: most icu's are noisy and may adversely affect patients outcomes and staff performance [1] . who reports that the noise level in hospitals should not exceed 35 db at daylight and 30 db at night. the aim of this study is to evaluate the noise levels in intensive care unit, to apply awareness training to intensive care staff in terms of noise and to compare the noise levels before and after education. noise measurement areas are separated into 17 points including 12 patient bedsides, nurse desk, staff desk, wareroom, corridor and entrance of intensive care unite. measurements were performed 14 times per day. after 10 day, awareness training were given to staff in terms of harmful effects of noise. after the training, noise measurements were repeated during 10 days. after total 20 days the measurements were terminated. noise was measured with incubator analyzer (fluke model: bio-tek serial no:6050274). the mean noise values before and after the training were not statistically different from the mean average noise values (p>0.05). when the time of measurement were compared, the noise levels were higher between 10-16 hours to other measurements before and after the training statistically (p=0.001). seventeen different noise measurement areas were compared in terms of noise level, there was no statistically significant difference (p>0.05). the differences were examined at the same hours between before and after training. contrary to expectations, noise levels were found to be higher after training statistically (p<0.05). all of noise measurements were higher than the threshold values that who recommended. increased noise levels in critical care units may lead to harmful health effects for both patients and staff. our results suggest that much noise in the icu is largely attributable to environmental factors and behavior modifications due to education have not a meaningful effect. critical care medicine has focused on continuous, multidisciplinary care for patients with organ insufficiency in the face of lifethreatening illness. despite significant resource limitation low income countries carry a huge burden of critical illness. available data is insufficient to clearly show the burden and outcomes of intensive care units in these developing countries [1] . the objective of our study is to evaluate the morbidity and outcomes of patients admitted to the intensive care unit of a tertiary university hospital in hawassa, ethiopia. this was a prospective observational study. data was registered and analysed starting from patient admission to discharge during a 12 month period beginning september 2018. data regarding demographics, sources of admission, diagnosis, length-of-stay and outcomes were analysed. the total number of patients admitted to the icu was 218, with 71 patients dying over a one year period. the highest admission was from emergency medical unit, 36% and the lowest source was from pediatrics department, 8%. out of these, 69.8% were males. the mean age was 27 years (2-62). the most frequent aetiologies of morbidity in the admitted patients were traumatic brain injury (24.6%), acute respiratory distress syndrome (22.9%) and seizure disorder (8%). average median length of stay was 3.0 days (interquartile range: 1.0 -27.0). the overall mortality rate was 32.5%. the top four causes of death in the icu were respiratory illness at 24% followed by sepsis with multiorgan failure at 20%, trauma (16%) and central nervous system infection (12%). infection morbidity and mortality remains very high and needs institution of aggressive preventive strategies. the increase in frequency of trauma patients need to receive due attention. sepsis causes a high number of deaths, though overtaken by respiratory illnesses. improving the overall system of icu may achieve better outcomes in resource limited countries. introduction: icu mortality has been widely studied in the literature in relation to outcome index that primarily value organic failure [1] . however, early mortality, in the first 48 hours of admission has been little documented in the literature. the aim of this study is to analyze factors related to early mortality in icu. retrospective study at a second-level hospital. time of study was 12 months. patients who died in icu were included, patients were classified according timing of dead, including those who died within the first 48 hours of icu admission. the variables analyzed were age, sex, comorbidity, charlson index, apache ii, need for supportive treatments, more frequent admission diagnosis, origin and support treatment limitation decisions. the statistical study was carried out using the spss statistical program. 138 patients were included during the study period, 72 (52.2%) died within the first 48 hours of admission. no differences in the needs of support treatments were observed, more than 90% of patients received mechanical ventilation and vasoactive therapies. table 1 shows characteristics of patients. half of icu deaths occur within the first 48 hours of admission. severity at icu admisison was the main factor related with early mortality. severe stroke and coronary disease were the most frequent causes of early deaths in icu. in august 2018 the royal college of anaesthetists published guidelines on care of the critically ill woman in childbirth and enhanced maternal care [1] . approximately 11000 babies are born across the area covered by leicester university hospitals that includes two large maternity units and is part of the uk ecmo network. this audit sets out to assess current practice and form a basis for future planning, which will likely be representative to most major obstetric centres. a retrospective audit of all patients admitted to 'intensive care units' in leicester over a 12 month period following publication of the guidelines. the focus was on patients admitted to general adult intensive care and excludes all patients cared for in 'enhanced obstetric care' units. 9 simple standards were proposed relating to accessibility, resuscitation, follow up and multi-disciplinary learning. in total 49 women were identified with a broad range of diagnosis. the intensive care services are split across 3 hospitals and we found this led to a number of problems. the presence of trained staff to resuscitate a newborn were easily accessible, no steps to provide necessary equipment pre-emptively were present in any centre. none of our critical care units had a plan for perimortem section. on-going reviews by the obstetric and midwifery teams were very variable. contact with the infant and breastfeeding support was also poor. despite the large number of deliveries significant work needs to be done in order to come in line with the new national guidelines for critically ill woman in childbirth. clearly defined pathways around escalation of care, resuscitation of both the mother and baby, integrating care of the mother and the infant in the first few days of life, and multidisciplinary learning events are being produced de novo in response to these guidelines, some of which will be illustrated in the associated poster. interprofessional collaboration scale [3] . data were analyzed with ibm spss 25.0 results: it was found that cooperative attitudes with an average score of 49 to 75 are considered to be of average significance. interprofessional cooperation at an average score of 2,568 states that the level of cooperation is high and the quality of working life averages 125 to 150, suggesting that it is very good. as far as professional satisfaction is concerned, nurses are happy, content and satisfied with their work, despite workload and burnout conclusions: interprofessional cooperation at the icu of the general hospital of larissa is high, but satisfaction from wages, resources, working environment and conditions is low. in addition, the results showed that improvements in hospital communication between staff, has a positive impact on the quality of professional life (table 1) . contrasting with previous reports, decreased admissions per unit population in older and oldest age groups, and those with high comorbidity, suggest resource constraints may have influenced admission discussion and decision-making over the 10-year study period in wales. further investigation is warranted. icu discharge into weekends and public holidays: an observational study of mortality n mawhood, t campbell, s hollis-smith, k rooney bristol royal infirmary, general intensive care unit, bristol, united kingdom critical care 2020, 24(suppl 1):p355 introduction: up to a third of in-hospital deaths in icu patients occurs following ward stepdown [1] . discharge time seems to be associated with in-hospital prognosis, but meta-analyses have not shown a difference in weekday compared to weekend discharge [2, 3] . however, papers that examined discharge 'into' out-of-hours days, particularly on fridays, have found differences [3] . our aim was to assess whether discharge from icu 'into' out-of-hours (ooh -weekends and public holidays) is associated with in-hospital mortality or re-admission to icu, and whether these patients were seen on the wards ooh by medical staff. all adults discharged from the general icu to a ward at the bristol royal infirmary in december 2016-18 were included. in-hospital mortality rates were assessed for each day, with 'into weekdays' defined as sunday to thursday and 'into ooh' friday, saturday and the day before a public holiday. a subset of patients with data on readmission rate to icu was also examined. all available notes from patients discharged into ooh in 2018 were reviewed. the study included 1732 patients with a subset of 443 with readmission data. 117 sets of notes were reviewed from patients discharged into ooh (figure 1 ). the in-hospital mortality was significantly higher in patients discharged into ooh (5.1% vs 7.6%, p=0.012). within the subset, ooh was associated with in-hospital mortality or readmission to icu (6.8% vs 11.9%, p=0.044), though readmission rate alone was not (1.7% vs 2%, p=0.35). of patients discharged into ooh, once on a ward 64% were reviewed by a specialty doctor but 20.5% were not seen. this is the first study to examine icu discharge 'into' ooh days including public holidays. we found increased hospital mortality in ooh, similar to other studies [3] . up to a fifth of high-risk icu stepdown patients were not reviewed by a doctor on ooh days. exploring the experiences of potential donors' family members (fm) in a follow up clinic is crucial to analyze the effects of organ procurement (op) on the bereavement process, to gain insight on the reasons of family refusals (fr), and to improve family care during op. a mixed-method study involving fm at 3 and 12 months after patients' death was developed and approved by local ethics committee. fm of potential donors after brain (dbd) and cardiac death (dcd) treated in careggi teaching hospital, florence (italy) were eligible if adult and consenting. invitation letters were sent to the entitled 2 months after death and those who actively responded were involved in an encounter with a multidisciplinary group including a clinical psychologist, two nurses and two cultural anthropologists with expertise in op. organ replacement procedures such as ecmo (extracorporeal membrane oxygenation), lvad (left ventricular assist device) and dialysis are routinely used to treat multi-organ failure (mov). globally transplantation programs struggle with increasing organ shortage. patients (pts) with mov are a potential source for procurement. however, outcome data after kidney transplantation (ktx) from such donors are sparse. we retrospectively studied the cadaveric ktx at the charité berlin in 2018 and identified donors with ongoing organ replacement procedures. donor and recipient risk factors were assessed. overall patient and graft outcomes were analyzed at 12 months post-transplant. a total of 220 kidneys were transplanted. we identified 11 ktx from 7 donors with mov (6 following cardio-pulmonary resuscitation, 5 with acute renal failure -4 on dialysis) (figure 1 ). in 3 donors, a venoarterial ecmo was implanted during ecls-resuscitation. one donor needed a veno-venous ecmo due to ards, and 1 donor had a lvad implanted due to cardiac failure. the donor age was 41 ± 10.5 years (yrs). in addition, 6 donors had at least one cardiac risk factor. the kidney donor risk index averaged 0.94 (sd ± 0.14) and s-creatinine prior to ktx was 2.41 (sd ± 1.27 one way to expand the potential donor pool is donation after circulatory death (dcd), and a strategy to reduce the complications related to the ischemic time is the use of normothermic regional perfusion (nrp) with extracorporeal membranous oxygenation (ecmo) [1, 2] . we compare the use of standard nrp with an effective adsorption system inflammatory mediators (cytosorb®) in the regional normothermic reperfusion phase via regional ecmo, that involves a reduction in cellular oxidative damage, assessed as a reduction in levels of proinflammatory substances. we report a case series of 9 dcd-maastricht iiia category donors, treated in ecmo with nrp, to maintain circulation before organ retrieval, in association with cytosorb® in 5 patients. during perfusion, from starting nrp (t0), blood samples are collected 3 times, every 60 minutes (t1, t2, t3). during treatment with cytosorb®, lactate levels progressively decrease, ast and alt increase less than without cytosorb®, as sign of improvement in organs perfusion ( figure 1 ). nrp with cytosorb® might help to successfully limit irreversible organ damages and improve transplantation outcome [2] . development and implementation of uniform guidelines will be necessary to guarantee the clinical use of these donor pools. introduction: shock is a common complication of critical illness in patients in intensive care units (icus), who are undergoing major surgery. this condition is the most common cause of death in postsurgical icus. nowadays, there are different icu scoring systems for predicting the likelihood of mortality, such as apache or sofa. nevertheless, they are used rarely because they also depend on the reliability and predictions of physicians. in these sense, gene expression signatures can be used to evaluate the survival of patients with postsurgical shock. methods: mrna levels in the discovery cohort were evaluated by microarray to select the most differentially expressed genes (degs) between groups of those that survived and did not survive 30 days after their operation. selected degs were evaluated by quantitative real time polymerase chain reactions (qpcr) for the validation cohort to determine the reliability of the expression data and compare their predictive capacity to that of established risk scales. introduction: this study evaluates the prognostic ability of frailty and comorbidity scores in patients with septic shock. the 90-day mortality rate of individual medical conditions are also compared. the burden of comorbid illness and frailty is increasing in the critical care patient population [1] . outcomes from septic shock in patients with chronic ill-health is poorly understood. interstitial lung disease is a group of diseases associated with poor prognosis in the intensive care unit despite major improvement in respiratory care in the last decade. the aim of our study is to assess factors associated with hospital mortality in interstitial lung disease patients admitted in the intensive care unit and to investigate the long-term outcome of these patients. we performed a retrospective study in an intensive care unit of teaching hospital highly specialized in interstitial lung disease management between 2000 and 2014. a total of 196 interstitial lung disease patients were admitted in the intensive care unit during the study period. overall hospital mortality was 55%. two years after intensive care unit admission, 70/196 patients were still alive (36%). one hundred eight patients (55%) required invasive mechanical ventilation of whom 80% died in the hospital (figure 1 ). acute exacerbation of interstitial lung disease was associated with hospital mortality (or=5.4 [1.9-15.5] ), especially in case of acute exacerbation of idiopathic pulmonary fibrosis. multiorgan failure (invasive mechanical ventilation with vasopressor infusion and/or renal replacement therapy) was associated with very high hospital mortality (64/66; 97%). survival after intensive care unit stay of patients with interstitial lung disease is good enough for not denying them from invasive mechanical ventilation, except in case of acute exacerbation for idiopathic pulmonary fibrosis patients. if urgent lung transplantation or extracorporeal membrane oxygenation are ruled out, multiorgan failure should lead to consider withholding or withdrawal life support therapies. αgi is a malfunctioning of the gi tract in icu patients associated with prolonged mechanical ventilation, enteral feeding failure and high mortality risk. the wgap of esicm proposed a grading system for agi. four grades of severity were identified: agi grade i, a selflimiting condition; agi grade ii (gi dysfunction), interventions are required to restore gi function; agi grade iii (gi failure); agi grade iv, gi failure that is immediately life threatening. the aim was to evaluate the feasibility of using agi grades i and ii as predictors of malnutrition and 1-year mortality in critically ill patients methods: single-center retrospective cohort study in a tertiary university hospital (2015 -2017). agi grade iii and iv patients were excluded. αnthropometric data, gi symptoms (vomiting,diarrhea), feeding intolerance, gastric residual volumes and abdominal hypertension were recorded. daily prescribed caloric intake was calculated using a standard protocol and daily achievement of caloric intake was recorded. mnutric score was calculated for all patients. a score ≤5 was used to diagnose malnutrition. 200 patients (59% men, mean age 65 years) that stayed in the icu for >48 hours were included in the study. 52% were at high nutritional risk. 1-year mortality was 31%. the prevalence of agi ii was 14%. age, gender, bmi, mortality and energy intake did not differ significantly between patients with agi ii and those with agi i (table 1) . logistic the study aimed to assess the effects of icu admission on frailty and activities of daily living in the ≥80's population at 6-months. a prospective observational study with data used as a subset of the vip-2 trial [1] . research ethics committee approval from the mater misercordiae university hospital (mmuh). inclusion criteria -≥ 80 years of age and acute admission to icu from may to july 2018. data collected on 20 consecutive patients. frailty and activities of daily living (adl) were assessed using the clinical frailty score (cfs) and the katz index of independence in activities of daily living (katz). results: csf pre-admission frailty was present in 60% of patients, increasing to 93% at 6 months ( figure 1 ). 74% of survivors at 6-months had a cfs score increase by ≥ 1 point. pre-frail and frail cfs patients suffered an average 2-point deterioration in their instrumental activities of daily living (iadl). 60% of katz patients were fully functional preadmission, deteriorating to 13% at 6 months. 74% of patients declined by 1 adl at 6 months. 60% of the deceased were deemed fully functional initially. we demonstrate an association between an icu admission event and enduring functional decline at 6 months. icu admission resulted in patients acquiring on average 1.5 new iadl limitations despite their initial cfs. this is echoed in a study by iwasyna et al. who also showed similar deteriorations in iadl and cognitive impairment [2] . katz benefits may be best used in describing functional decline. 74% of patients developed at least one new limitation. however, the cfs takes into account iadl's and thus may be more sensitive in predicting the functional outcomes of an icu event at 6 months. frailty: an independent factor in predicting length of stay for critically ill t chandler, r sarkar, a bowman, p hayden medway maritime hospital, critical care, gillingham, united kingdom critical care 2020, 24(suppl 1):p366 frailty has attracted attention in the healthcare community in recent years, as it is associated with worse outcomes and increased healthcare costs [1] . our objective was to study the impact of frailty as recorded by clinical frailty scale(cfs) to prospectively evaluate the effect of frailty on hospital length of stay (los). a retrospective analysis of consecutively admitted critical care (cc) patients' data (jan'19-oct'19) was performed. electronic health records were used to collect demographics, cfs and clinical outcomes. statistical analysis was performed using stata. students t-test, simple and multiple (adjusted for age, disease severity/icnarc score) linear regression were used for comparison between groups and to see group effect. we excluded extreme outliers (los>50 days; n=13). frailty was defined as cfs>4. out of the 848 patients (male 58%), 554(66%) were emergency admissions, the rest elective (table 1) . 288(40%) were non-frail. the mean los were 15 days (d) ±12 and 10d±9 (p<0.0001) in the frail and non-frail patients respectively. for emergency patients, los were 16d(±12) and 10d(±10) for the groups, (p<0.0001). for elective patients; los were 12d(±10) and los 8d(±7), (p=0.01) for frail and nonfrail respectively. after adjusting, los was significantly higher in frail patients by 5 days (95%ci 3,7; p<0.0001), by 4 days (95%ci 1,6; p= 0.002) and by 5 days (95%ci 4,8; p<0.0001) for total cohort, elective and emergency admissions respectively. the los was 6 days higher in frail than non-frail (p<0.001) for cc survivors. frailty was associated with significantly increased los in this cohort, independent of age and illness severity. hospital capacity planning should take this into consideration when modelling bed allocation fig. 1 (abstract p365) . clinical frailty score 6-month trend robust clinical governance requires analysis of patient outcomes during an icu admission [1] . on one adult icu weekly mortality meetings are used for this purpose and aid multidisciplinary reflections on individual patient deaths. however, such reviews run the risk of being subjective and fail to acknowledge themes which may relate to preceding or subsequent deaths. this paper describes a new mortality review process in which: a) reviews are structured using the structured judgement review (sjr) framework [2] ; and b) themes are generated over an extended period of time to create longitudinal learning from death. the sjr framework has been developed by nhs improvement for the new medical examiner role, looking at inpatient deaths. we adapted this to better suit the icu creating a novel review structure. this involves explicit judgement comments being recorded, and the use of a scoring system to analyse the quality of care during the patient's stay with a focus on elements of care delivered on the icu. tabulation of this information allows analysis over time, identifying trends across all patients, and in specific subgroups. this framework has been rolled out at the st george's cardiothoracic icu weekly mortality meetings. themes that have emerged include parent team ownership, delayed palliative care referrals and inadequate documentation of mental capacity. this will continue as part of a three-month trial and following review of this trial may be extended to other critical care units in the trust. this system allows greater insight into patient deaths in a longitudinal fashion and facilitates local identification of problems at an early stage in a way that is not possible within the traditional mortality review format. the nature of the process means that key areas for change can be identified as a routine part of the clinical week. [1] . in this study, we evaluated three distinct machine-learning methods for predicting possible patient deterioration after surgery. the data was collected retrospectively from the catharina hospital in eindhoven. this dataset contained all the surgeries conducted in the hospital from 2013 up to 2017. the variables in this dataset were tested on their ability to differentiate between patients with a normal recovery versus patients with an unplanned icu admission after being admitted to the ward. the dataset contained 44 variables related to either the preoperative screening, surgery or recovery room. all variables were tested for statistical significance using a univariate logistic regression (lr), from which a subset of 34 statistically significant (p<0.05) variables was created. these variables were used to train three different types of models, namely, the lr, support vector machine (svm) and bayesian network (bn). the network structure of the bn was designed using expert knowledge and the probabilities were inferred using the data. the three models were validated using five-fold cross-validation, resulting in the following areas under the receiver operating characteristic curve: 0.82(0.79-0.86) for lr, 0.82(0.78-0.88) for svm and 0.65(0.63-0.68) for bn (fig. 1) . the results indicate that machine learning is a promising tool for early prediction of patient deterioration. the bn was included because it permits incorporating clinical domain knowledge into the learning process. however, its performance resulted inferior to the lr and svm. in future work, we will investigate alternative domainaware methods, and compare the performance with that of the clinical experts. intensive care unit (icu) admission decisions of patients with a malignancy can be difficult as clinicians have concerns about unfavourable outcomes, such as mortality [1] . a diagnosis of a malignancy is associated with an almost 6-fold increased likelihood of refusal of icu admission [1] . recent large long-term mortality studies of patients with a malignancy admitted to the icu are scarce. therefore, our aim was to compare mortality of patients with either a hematological or a solid malignancy to the general icu population, all with an unplanned icu admission. all adult patients registered in a national intensive care evaluation registry with an unplanned icu admission from 2008 to 2017 were included. subsequently, we divided these patients into 3 cohorts: cohort 1 (all patients with a hematological malignancy), cohort 2 (all patients with a solid malignancy), and cohort 3 (a general icu population without malignancy). as primary outcome, we used 1-year mortality, and as secondary outcome, icu and hospital mortality. we included 10,401 (2.2 %) patients in cohort 1, 35,920 (7.6%) patients in cohort 2 and 423,984 (90.2%) in cohort 3 ( table 1 ). the 1year mortality of patients of cohort 1, 2, and 3 was 60.1%, 46.2% and 28.3%, respectively (p<0.001). age, comorbidities, organ failure, and type of admission (i.e. surgical or medical) were positively associated with 1-year mortality in all cohorts (p <0.05). one-year mortality is higher in both patients with a hematological malignancy and patients with a solid malignancy compared to the general icu population. in addition, several factors were positively associated with 1-year mortality, i.e., age, comorbidities, medical icu admission, and organ failure. future research should focus on predictive modelling in order to identify patients with a malignancy that may benefit from icu admission. introduction: drug abuse is associated with immunosuppression in multiple mechanisms. despite that, the only study retrospectively reviewing drug abusers in the icu demonstrated less infections and better outcomes. we compared matched patient populations in order to fully understand whether drug abuse is a risk factor for infection and a predictor of poorer prognosis as is perceived by most physicians. we hypothesized that the drug abusers admitted to the icu will fare as good as or better than non-abuser icu patient populations. methods: this is a prospective study done between the years 2010-2012 on the entire patient population of the detroit medical center. after the drug abuse population was identified, controls were matched according to age and admission icu units. patients charts were reviewed and data regarding baseline demographics, infectious complication and outcome was extracted. data was retrospectively collected for 323 drug abusers and 305 matched controls. comorbidities and hospital admission diagnosis were significantly different between the two groups. disease severity scores were significantly higher in the drug abuser's patient group (dapg) on admission and during the icu stay. dapg had significantly more organ failure: more need for ventilation (30.5% vs 46.4% in the dapg (p<0.001)), more ards (1% vs 3.7%, p=0.03), more renal failure (33% vs 45.5%, p=0.002) and more need for renal replacement therapy (6.6% vs 11.2%, p<0.05) .they had longer hospital length of stay (los). there was no difference in icu or hospital mortality. multivariable modeling did not find drug abuse to be an independent risk factor for hospital mortality, icu mortality (hosp: or = 1.37, p = 0.3397; icu: or=1.43, pp = 0.07), but was a risk factor for a longer hospital los (me=1.46, p < 0.0001). drug abuse is not an independent risk factor for mortality or icu los. drug abusers should be evaluated like other patients based on baseline comorbidities and disease severity. this is a small audit which although it did not include general icu still reflects the need for encouraging clinicians and patients to speak freely regarding escalation plans. medical decsions is clinician led however this audit was carried by nursing staff as we have a duty to be advocate for our patients involvement in medical care [2] . a retrospective analysis of independent risk factors of late death in septic shock survivors c sivakorn 1 , c permpikul 2 , s tongyoo 2 (fig. 1) . the pap and katz scales seem to be adequate for predicting mortality of critically ill patients admitted to a medical icu. this finding may help in the elaboration of future icu mortality scoring systems, as well as in more rational use of resources. however, further multicenter studies are needed to better elucidate these results. adherence this last group was chosen because of its experience and specific training in the field of bioethics as a control group or reference. a total of 444 respondents participated in the study. 22.2% were emergency physicians, 14.8% intensivists, 11.2% emergency nursing, 6.2% icu nursing, 24.9% resident doctors, 13.8% medical students and 6.9% other professions. we observed variability in the responses observed not only between different groups of professionals but even within the same group reflecting the difficulty in decision making. variability was observed regarding decisions in end of life ethics conflicts. a high degree of similarity with the group of master in bioethics was observed in the responses issued by medicine students. the barriers and facilitators to framing goals of patient care (gopc) and factors motivating decision making is relatively unexplored [1, 2, 3] . a three part survey of physicians at an australian hospital in a culturally and linguistically diverse suburb ( table 1) . identification of levels of confidence and barriers and facilitators to gopc discussion and decision making was the main outcome measure. factors influencing decision-making was analysed through scenarios. results: 22 out of 96 eligible participants responded; 12 female, 10 male, clinical experience 4-31 years. level of confidence was ranked between "somewhat confident and very confident." all but one respondent had six months of icu experience. no differences in the level of confidence among physician groups. 14 barriers and 8 facilitators were identified; poor prognosis and patient or family request were most common facilitators; conflict between treating teams and the patient/surrogate and language barriers were most common barriers. factors driving gopc decision-making included clinical, value judgement, communication, prognostication, justice and avoidance. numerous barriers and facilitators were identified. factors driving decision making did not just consider clinical factors; conflict and we aimed to investigate physician-related factors contributing to individual variability in end-of-life (eol) decision-making in the intensive care unit (icu). qualitative study with semi-structured interviews with 19 specialists in critical care, (experience 2-32 years) from 5 swedish icus. data was analyzed in accordance to principles of thematic analyses. most of the respondents felt that the intensivist's personality played a major role in eol decisions (table 1) . individual variability was considered inevitable. views on acceptable outcome: respondents experienced that the possible outcome for patients was interpreted very differently and subjectively among colleagues, and what seemed an acceptable patient-outcome for one doctor, was not acceptable for another. values: most of the respondents were well aware that they might be affected by their own values and attitudes in the decision-making process. interestingly, several respondents mentioned that they thought that patients that were marginalized by society, especially drug-abusers could be at risk for receiving decisions to limit life sustaining treatments (lst) more often than others. none of the respondents thought that their own religious beliefs played any part in decision making. fear of criticism: among the less experienced respondents there was a clear sense of fear of making a questionable assessment of the patient's medical prognosis. there was a fear for criticism from colleagues that were not directly involved in the decision-making, and may have made another decision. this created a wish among younger respondents to defer or avoid participating in decision-making. physician-related, individual variability in eol decisions primarily consisted of differing views on acceptable outcome, values and fear of criticism. can (figure 1 ). within each quartile of sofa score, mortality was highest in patients with pneumonia and peritonitis and lowest in patients with cellulitis (see figure 1 ). the sepsis-3 consensus definition identified organ dysfunction as the hallmark feature of sepsis [1] . in developing sepsis-3, the sequential organ failure assessment (sofa) score was chosen for its prognostic value and relative ease of implementation clinically [2] . we propose an update based on epidemiologic data from two intensive care databases that more effectively captures organ dysfunction in the context of sepsis-3. using the mimic-iii (exploration) and e-icu (validation) databases, we extracted patients with suspicion of infection to form the study cohort. the predictive power of each sofa component was assessed using the area under the curve (auc) for in-hospital mortality. a logistic model with the lasso penalty was used to find an alternative statistically optimal score. results: by utilising alternate markers of organ dysfunction (e.g. lactate, ph, urea nitrogen) we demonstrated a significant improvement in auc for several versions of the new score, sofa2.0 ( figure 1 ). the sofa score can be updated to reflect current advances in clinical practice. using epidemiologic data, we have shown that substitution of existing components with more powerful measures of organ dysfunction may provide an improved score with greater predictive power. moreover, sofa 2.0 exhibits equivalent ease of implementation, but better reflects organ dysfunction in the context of sepsis-3. introduction: risk of acute organ failure (aof) in cancer patients(pts) on systemic cancer treatment isunknown. however, 5% of non-hematologic and 15% of hematologic cancer pts will need admission to intensive care unit (icu). ipop-sci-2017/01 is a prospective cohort study designed to ascertain the cumulative incidence of aof in adult cancer pts. single centre prospective cohort study with consecutive sampling of adult cancer pts admitted for unscheduled inpatient care while on, or up to 8 weeks after, systemic cancer treatment. primary endpoint was aof as defined by quick sofa. six months accrual expected an accrual of 400 pts to infera population risk aof with a standard error of 1%. between 08/2018 and 02/2019 10392 pts were on systemic anticancer treatment, 358 had unscheduled inpatient care and were eligible for inclusion and 285 were included. median age was 64 years, 51% were male, 52% had adjusted charlson comorbidity index (cci) &gt; 3 and hematologic cancers accounted for 22% of pts. the cumulative risk of aof on hospital admission was 35% (95%ci: 31-39); and of aof during hospital stay was 40% (95%ci: 35-44). aof was associated with older age, cci &gt; 3,hematologic malignancy, shorter median time from diagnosis and &gt; 1 prior line of therapy. on admission, 62% of pts were considered not eligible for artificial organ replacement therapy (noaort) and 34% of pts who developed aof while inhospital were judged noaort. overall, 17 (15%) of aof pts wereadmitted to icu, 31.5% for aort. median follow up 9.5 months (min 6; max 12). inpatient mortalitywas 18%, with icu mortality rate of 59%, with median cohort survival 4.5 months (95%ci: 3.5-5.4). on multivariate analysis, aof was an independent poor prognostic factor (hr 1.6; 95%ci 1.2-2.1). risk of aof in cancer pts admitted for unscheduled inpatient care while on systemictreatment is 35%, and risk of icu is 15%. aof in cancer pts was an independent poor prognostic factor. a severity-of-illness score in patients with tuberculosis requiring intensive care u lalla, e irusen, b allwood, j taljaard, c koegelenberg tygerberg academic hospital, internal medicine, division of pulmonology and icu, cape town, south africa critical care 2020, 24(suppl 1):p383 we previously retrospectively validated a 6-point severity-of-illness score aimed at identifying patients at risk of dying of tuberculosis (tb) in the intensive care unit (icu). parameters included septic shock, human immunodeficiency virus with cd 4 <200/mm 3 , renal dysfunction, ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao 2 :fio 2 ) <200mmhg, diffuse parenchymal infiltrates and no tb treatment on admission. the aim of this study was to validate and refine the severity-of-illness score in patients with tuberculosis requiring intensive care. we performed a prospective observational study with a planned post-hoc retrospective analysis, enrolling all adult patients with confirmed tb admitted to the medical intensive care unit from 1 february 2015 to 31 july 2018. descriptive statistics and chi-square or fisher's exact tests were performed on dichotomous categorical variables, and t-tests on continuous data. patients were categorized as hospital survivors or non-survivors. the 6-point score and the refined 4-point score were calculated from data obtained on icu admission. results: forty-one of 78 patients (52.6%) died. the 6-point scores of nonsurvivors were higher (3.5+/-1.3 vs 2.7+/-1.2; p=0.01). a score ≥3 vs. <3 was associated with increased mortality (64.0% vs. 32.1%; or 3.75; 95%ci, 1.25-10.01; p=0.01)( table 1) . post-hoc, a pao 2 :fio 2 < 200mmhg and no tb treatment on admission failed to predict mortality whereas any immunosuppression did. a revised 4-point score (septic shock, any immunosuppression, acute kidney injury and lack of lobar consolidation) demonstrated higher scores in non-survivors (2.8+/-1.1 vs. 1.6+/-1.1; p<0.001). a score ≥3 vs. ≤2 was associated with a higher mortality (78.4% vs. 29.3%; or 8.76; 95%ci, 3.12-24.59; p<0.001) ( table 1) . the 6-point severity-of-illness score identified patients at higher risk of death. we were able to derive and retrospectively validate a simplified 4-point score with a superior predictive power. chronic critical illness remains a scientific challenge, from its conceptualization to its impact on patient prognosis [1] . we evaluated the long-term evolution of icu survivors by identifying the real burden of prolonged critical illness on survival, quality of life and hospital readmissions. we conducted a prospective cohort in 16 brazilian hospitals including 1616 icu survivors with an icu stay > 72h. we compared the patients diagnosed with chronic critical illness with the other patients. telephone follow-up at 3 and 6 months. quality of life was measured by the sf-12 questionnaire. it was observed that 38% of patients had some definition of chronic critical illness. chronic critically ill patients had higher mortality at 6 months (p=0.012). this difference is mainly due to higher intrahospital mortality (p=0.0001). mortality after hospital discharge was similar between groups. there was no difference in hospital readmission rate at 6 months. various scores are developed to predict pulmonary complications such as ariscat for patients at-risk of postoperative pulmonary complication [1] and lips for patients at-risk of lung injury [2] . the aim of this study was to compare these scores with ours for predicting pulmonary complications in mechanically ventilated patients in sicu. this prospective observational study was conducted in sicu at a university hospital. adult patients admitted to sicu and required mechanical ventilation >24 hours were included. primary endpoint was the composite of pulmonary complications including pneumonia, ards, atelectasis, reintubation, and tracheostomy. multivariate analysis was performed to identify risk factors of pulmonary complications and the predictive score was developed. the roc analysis was performed to compare power of ariscat, lips and our newly developed score for predicting pulmonary complications. outcomes in intensive care units have been reported to be better in higher-volume units [1, 2] . we compared outcomes for high-risk patients between low and higher volume units. audit data from irish icus is analysed and reported by the intensive care national audit & research centre (icnarc) in london. icnarc report risk-adjusted mortality rates in all patients and in low-risk patients(predicted mortality rate <20%) for each unit, using the icnarch-2015 model to predict the risk of death. we used this data to calculate the proportion of high-risk patients(predicted mortality >20%) in each unit, the mortality rate for high-risk patients, the riskadjusted mortality rate and we compared the overall risk-adjusted mortality between low and high volume units. the median number of annual new-patient admissions among 18 participating units was 390; units below this were defined as lowvolume and those above as high-volume units. the proportion of all admissions to each unit who were high-risk ranged from 8% to 54%(mean 34%). unit mortality rates for high-risk patients ranged from 33% to 69%. the ratio of observed to expected mortality(standardized mortality ratio -smr) for high risk admissions in each unit ranged from 0.87 to 1.34(mean 1.07). in fig. 1 introduction: adl weakening is often seen after intensive care and called postintensive-care syndrome (pics). this is also seen in even outside icu and proposed to be called post-acute-care syndrome (pacs), especially in elderly patients. in patients with infection, sofa score is famous for predicting in-hospital mortality, but there are no tools for predicting adl weakening during admission. to search for risk factors for adl weakening during admission other than the age, we conducted a retrospective observational study. the subjects were surviving patients with infection, aged from 16 to 89 who were admitted to our department from april 1, 2018 to may 31, 2019. information of basic characteristics, laboratory data on admission and adjunctive therapies were extracted from our database. we use barthel index (bi) as adl evaluation, and the bi at discharge were evaluated by nurses. we stratified patients by bi at discharge of over 60 or not, and investigated factors that predicted it. we compared each factor between 2 groups, and perform a logistic regression analysis with those that had a significant effect clinically or statistically. despite improved outcomes of intensive care unit (icu) patients, sleep deprivation remains a major concern after icu discharge. multifaceted causes make it difficult to treat and understand [1] . not many studies have explored sleep deprivation beyond icu. this is evidenced by findings from a recent systematic review [2] which included 8 studies with only one study [3] reporting sleep deprivation beyond icu. the aim of this paper is to present findings of sleep deprivation beyond icu from a larger study that examined the experience of critical illness in icu and beyond in the context of daily sedation interruption. hermeneutic phenomenology was used to conduct the study. 12 participants aged 18 years and above who fulfilled the enrolment criteria were enrolled into the study. the cohort comprised 7 male and 5 female participants. in-depth face to face interviews at two weeks after discharge were conducted and repeated at six to eleven months. interviews were audio taped, transcribed and thematically analysed. significant statements were highlighted and categorized for emergent themes. six participants continued to experience sleep deprivation up to eleven months after icu. two cited dreams about icu, three could not explain why they continued to fail to sleep and one stated that he continued hearing icu alarms in the silence of the night. sleep deprivation continues beyond icu due to nightmares, delusional memories and unexplained reasons. further research is needed to establish causes of sleep deprivation and explore ways to promote sleep in critical illness survivors after icu discharge. frailty is being increasingly seen as an independent syndrome. frail patients now account for an increasing proportion of hospital and critical care admissions [1] . we aimed to compare frailty and mortality in our intensive care unit. clinical frailty score (cfs) was incorporated within the electronic health record (ehr) 2019. we performed this retrospective analysis on the data collected between jan'19 and oct'19. the predictor and outcome for this study were frailty and hospital mortality respectively. all demographic data, acute physiology score, critical care and hospital outcome data were automatically collected in the ehr and recorded. we used a cut off of cfs>3 and above to define non-frail and frail respectively. chi-squared test, simple and multiple logistic regression were used. adjustment was done for icnarc score and age. total number of patients was 848, of which 140 (16.5%) died in hospital. within the patients<65 years (n=392), 79 (20%) were recorded as frail or vulnerable. the number of elective and emergency admission were 292(34%) and 556(66%) respectively. in the frail and nonfrail, mortality rates were 30% and 9.5% (p<0.001) respectively, with odds ratio of 4.14 (95% ci 2.8, 6; p<0.001) ( age is a well-known risk factor for critical care (cc) outcome and is incorporated into many prognostic tools; however, this has been criticized for assumption of normal physiology for young at baseline. in recent years, frailty in cc prognostication has been of interest, with meta-analysis correlating worsening outcomes with increasing frailty [1] . in this study, we compared the effect of frailty versus age for determining hospital survival for critically ill patients. we conducted a prospective cohort in 16 brazilian hospitals including 1616 survivors of an icu stay > 72h. we compared chronic critically ill patients (icu stay> 10 days) and the other patients. we performed psychological and functional presential assessment in patients within 48 hours of icu discharge and by telephone at 3 and 6 months. the prevalence of chronic critically ill patients was 26%. regarding outcomes, chronic critically ill patients had a higher incidence of depressive symptoms than other patients in the immediate post-icu discharge (p = 0.004), as well as a higher incidence of muscle weakness (p <0.001). however, in subsequent evaluations, we found no difference between groups regarding psychological symptoms -depression, anxiety and post-traumatic stress. higher functional dependence was observed in critically ill patients, but without difference in the quality of life score, both in the physical (p = 0.87) and mental (p = 0.84) domains. chronic critically ill patients, when compared to patients with stay> 72h, have a higher incidence of depressive symptoms at icu discharge. this difference disappears in the follow up. chronic critically ill patients present higher levels of functional dependence but without repercussions on quality of life scores. introduction: activation of the inflammatory response after cardiac arrest (ca) is a welldocumented phenomenon that may lead to multi-organ failure and death. we hypothesized that white blood cell count (wbc), one marker of inflammation, is associated with one-year mortality in icu treated ca patients. we used a nationwide registry with data from five academic icus to identify adult ca patients treated between january 1 st 2003 and december 31 st 2013. we evaluated the association between the most abnormal wbc within 24 hours of hospital admission and one-year mortality. we accounted for baseline risk of death using multivariable logistic regression (adjusted for age, gender and 24h sequential organ failure assessment [sofa] score). a total of 5,543 patients were included in the analysis. of those patients 2,387 (43%) were alive one year after ca. we plotted wbc against baseline risk of death and through graphic examination of a locally weighted scatterplot smoothing (lowess) curve found the lowest risk of death to be associated with a wbc of 12 (e 9 /l) ( figure 1 mrps were identified by a specialist icu pharmacist during this programme and classified by their significance on a scale of one to four. logistic regression was used to determine if demographic factors were associated with the occurrence of a clinically significant mrp -a significance score of two or above (figure 1) . the adjusted model included age, icu los, hospital los, apache ii, number of days of renal replacement therapy, number of days of ventilation, the number of medications prescribed at icu discharge, and the who analgesia classification at ins:pire. there were increased odds of having a clinically significant mrp for hospital los (or results: 62·8% (n=115) of patients required at least one pharmacy intervention. the median number of interventions required per patient was one (iqr 0-2); the maximum number was six. 198 mrps were recorded in this cohort. the most common intervention was clarifying duration of treatment (n=44), followed by education (n=33), and correcting drug omissions (n=27). the bnf drug class most frequently associated with mrps was neurological (n=65), which comprises analgesics (n=45) and psychiatric medications (n=20) ( figure 1 ). this was followed by cardiovascular medications (n=40), gastrointestinal medications (n=34), nutritional medications (n=25), and others (n=34). many icu survivors experience mrps. the most common class of mrp was neurological, reflecting the high incidence of chronic pain and psychiatric illness in this population following discussion with icu staff, ward staff and fy1 doctors, a formal standardized handover system was introduced. this involved a verbal handover to the appropriate fy1 by an icu doctor and the patient drug chart to be rewritten in icu at the time of handover. the next change was to display posters on the wards to alert staff that the medical team are to be contacted when a patient comes to the ward from icu and to ensure the drug chart is completed. the baseline data showed a median time delay of 4 hours, with one patient waiting 14 hours for a drug chart. following the interventions the median time delay has decreased to 0 hours within 4 months as demonstrated in figure 1 . the changes have received positive feedback from icu staff, ward staff and fy1 doctors. the aim of reducing the time delay by 50% has been achieved with the median time delay now 0 hours. this has improved patient safety by significantly reduced delays in medications and through the introduction of a standardized handover. this has also provided an opportunity for junior doctors on the wards to seek clarification regarding medications and the clinical management plan for the patient. this has established a communication channel between icu and the wards making patient care safer and more effective. telemonitoring outside the icu is scarce. but with innovative wearables measuring respiratory and heart rate wirelessly, culture on intrahospital telemonitoring should definitely change. however, culture has been known to be one of the most crucial success factors in innovation, especially in health care. human design thinking is a promising tool in health care innovation but rarely used in a multidisciplinary team to initiate an innovation culture and stimulate sustainable collaboration. the aim of this study was to initiate a pilot project with a multidisciplinary team to start using wearables for early warning score (ews) on a clinical ward. human design thinking was used to write a value proposition on wearables in clinically admitted neutropenic hematologic patients in an academic center. a multidisciplinary team was performed to cover all disciplines involved in the technical, clinical and administrative parts of the project. a vendor was chosen based on its product specifications in relation to the present hospital monitoring infrastructure. in design thinking sessions, critical appraisal of multiple telemonitoring factors was performed by sub teams and a canvas projectplan was constructed. the project team was formed of registered nurses, physicians, itspecialists, electronic health record consultants; a critical care physician was appointed as project leader. the main critical factors were: unseamlessly transmitting of both heart and respiratory rates including appropriate movements filtering to the nurse's smartphones direct uploading into electronic health record with automated ews calculation nurse driven protocol on ews follow up. philips healthcare with their intellivue guardian wearable biosensor was the chosen vendor ( figure 1 ). design thinking in a multidisciplinary health care team could positively influence the innovation culture. scientific evaluation of this wearable will focus on both nurse's acceptance and data storage and is expected in the summer of 2020. severity, readmission and lengh of stay were lower in patients receiving discharges directly to home. it seems like a safe way to discharge low-risk short stay patients. it seems to save resources and reduce costs, as well as the need for hospital beds. however, futher estudies are needed to actualy evaluate this safety. forty-four cultures were analyzed with eplex ( figure 1 ). complete agreement with conventional diagnostics was observed in 38/44 cases. no false-positive results were observed, yielding a sensitivity and specificity of 90% and 100% respectively for target pathogens. time to result was, on average, 10.4 h faster with eplex compared to conventional diagnostics. antimicrobial therapy could have been optimized in 5 patients based on the eplex result, but treatment was only changed in one case (e.coli ctx-m+) receiving meropenem 8.5 h before the antibiogram was available. the eplex blood culture panels provide high accuracy and significantly faster results. the current implementation offers substantial potential value at a minimal cost, and is a feasible approach to 24-h/ 7 days blood culture diagnostics in many hospital settings. however, efforts to increase adherence are needed. the rapid increase of extended spectrum β-lactamases (esbl)-producing pathogens worldwide makes it difficult to choose appropriate antibiotics in patients with gram-negative bacterial infection. cica-beta reagent (kanto chemical, tokyo, japan) is a chromogenic test to detect beta-lactamases such as esbl from bacterial colonies. the purpose of the study was to reveal whether cica-beta reagent could detect esbl-producing pathogens directly from urine rather than bacterial colonies to make a rapid bedside diagnosis of the antibiotic susceptibility of gramnegative pathogens. we conducted a prospective observational study from july 2019 to october 2019. patients were eligible if they were performed urinary culture tests and gram negative pathogens were detected at least 1+ from their urine samples. the urine sample was centrifugated at 200 x g for 5min. the supernatant of sample was re-centrifugated at 1500 x g for 5min and the pellet was mixed with cica-beta reagent. the test was considered positive when the enzymatic reaction turned from yellow to red or orange. (fig.1) . the bundle approach could be an effective strategy to prevent hospital-acquisition of drug-resistant pathogens in icus. fig. 1 in the aspect-np trial, c/t was noninferior to mem for the treatment of habp/vabp. we evaluated outcomes from that study in the subgroup of pts failing current antibacterial therapy for habp/vabp at enrollment. methods: aspect-np was a randomized, controlled, double-blind, phase 3 trial in which mechanically ventilated pts with habp/vabp received 3 g c/t or 1 g mem every 8 h for 8-14 days. pts with >24 h of active gram-negative antibacterial therapy within 72 h prior to first dose of study therapy were excluded, except those pts failing current treatment (i.e. signs/symptoms of the current habp/vabp were persisting/worsening despite ≥48h of antibiotic treatment). primary and key secondary endpoints, respectively, were 28-day all-cause mortality (acm) and clinical response at test of cure (toc; 7-14 days after end of therapy) in the intent to treat (itt) population. pts failing current antibacterial therapy for habp/vabp were prospectively categorized as a clinically relevant subgroup. at baseline, failing current therapy for habp/vabp was reported in 53/362 (15%) c/t and 40/364 (11%) mem itt pts, mostly piperacillin/ tazobactam (34%), 3rd/4th-generation cephalosporins (31%), fluoroquinolones (29%), and aminoglycosides (12%). baseline demographic and clinical characteristics in this subgroup, including prior therapy regimen, were generally similar between treatment arms. there were greater proportions of patients with esbl+ enterobacterales (30%) and pseudomonas aeruginosa (25%) in the c/t arm than the mem arm (20% and 13%, respectively). lower 28-day acm was seen with c/t than mem, as evidenced by 95% confidence intervals for treatment differences that excluded zero ( figure 1 ); statistical significance cannot be assumed because subgroup analyses in this study were not corrected for multiplicity. conclusions: c/t was an effective treatment for habp/vabp pts who had failed initial therapy. catheter-related blood stream infection (crbsi) is common serious infections and associated with increased mortality in intensive care units (icu). one of the most important strategy to prevent crbsi is to minimize the duration of central venous catheterization. we built a medical team consisting of doctors, nurses and pharmacists in icu to discuss whether patients needed central venous catheter (cvc) in terms of monitoring hemodynamics and administering drugs, and recommend catheter removal to attending physicians every day in april 2019. the purpose of this study is to evaluate whether our team-based approach could shorten the total duration of catheterization and reduce crbsi. this was a retrospective historical control study conducted from april 2018 to october 2019 in the icu of a tertiary care hospital in japan. every patient admitted to the icu during the study period was eligible if they were inserted cvc. patients were divided into 2 groups: conventional (from april 2018 to march 2019) or intervention (from april 2019 to october 2019). we set the primary endpoint as onset of crbsi. the secondary endpoints included the duration of central venous catheterization, the length of icu stay and hospital mortality. crbsi was defined as bloodstream infection in patients with cvc, not related to another site. we included 428 patients: 259 in the conventional group and 169 in the intervention group. the reduced, though nonsignificant, tendency of crbsi was observed in the intervention group [hazard ratio, 0.341(95% confidence interval, 0.074-1.557; p = 0.213)]. the intervention group was significantly associated with reduced duration of central venous catheterization (5 days vs 7 days; p < 0.01). no difference was observed in the length of icu stay and in-hospital mortality between groups. the team-based approach to assess cvc necessity could shorten the duration of central venous catheterization and might reduce crbsi. introduction: empiric antibiotic therapy decisions are based upon a combined prediction of infecting pathogen and local antibiotic susceptibility, adapted to patients' characteristics. the objective of this study was to describe the pathogen predominance and to evaluate the probability of covering the most common gram-negative pathogens in icu patients with respiratory infections. methods: data were collected from multiple us and european hospitals as part of the smart surveillance program (2018). mic (mg/l) testing was performed by broth microdilution, with susceptibility defined as follows for p. aeruginosa & enterobacterales: ceftolozane/tazobactam results: 78 hospitals from 24 countries provided 3384 gram-negative respiratory isolates from patients located in an icu in the us (22%), eastern europe (40%) and western europe (38%) in 2018. the 4 most common pathogens isolated were p. aeruginosa (26%), k. pneumoniae (15%), e. coli (13%), and a. baumannii (10%). among enterobacterales, 30% (588/1955) were esbl positive. figure 1 provides the probability of covering the most common respiratory gram-negative pathogens from icu patients. co-resistance between commonly prescribed first line β-lactam antibiotics is common: when nonsusceptibility (ns) of one agent was present, susceptibility to other βlactams was generally <35%. ceftolozane/tazobactam provided the most reliable in vitro activity in both empiric and adjustment prescribing scenarios compared to other β-lactam antibiotics. ceftolozane/tazobactam ensured a wide coverage of the most common gram-negative respiratory pathogens demonstrating high susceptibility levels and provided the most reliable in vitro activity in both empiric and adjustment antibiotic prescribing scenarios. further studies are needed to define the clinical benefits that may translate from these findings. evaluation of compliance of icu staff for vap prevention strategies on the outcome of patients a kaur fortis hospital, critical care, mohali, india critical care 2020, 24(suppl 1):p426 ventilator-associated pneumonia is the most common nosocomial infection diagnosed in adult critical care units. it is associated with prolonged duration of mechanical ventilation, increased icu stay and increased mortality. it continues to be a major challenge to the critical care physicians despite advances in diagnostic and treatment modalities. the primary objective of the study was to determine the compliance of icu staff towards vap prevention bundle and secondary objective was to determine the incidence, risk factors and outcome of vap patients. single center, prospective, observational study carried out from february 2017 to july 2018. patients mechanically ventilated for more than 48 hours and satisfying the inclusion and exclusion criteria were enrolled in the study. vap was diagnosed using the cdc criteria and clinical pulmonary infection score. vap preventive strategies were employed and compliance of icu staff was assessed. a total of 1617 patients were admitted to icu over the set time period and out of them 483 patients were ventilated for more than 48 hours. among them only 166 patients fulfilled the inclusion and exclusion criteria and were enrolled in the present study. excellent compliance was observed in head end elevation, sedation vacation, stress ulcer prophylaxis, and heat moist exchanger filter use, good compliance in oral care and hand hygiene and moderate to poor compliance in subglottic suctioning. the incidence of vap was 24.7% with a vap rate of 30.87/1000 ventilator days. there was a significant correlation between primary diagnosis, hemodialysis, massive blood transfusion and development of vap (p<0.05)). mean duration of ventilation (p<0.001) and mortality (p<0.05) were highly significant in vap patients. conclusions: improvement in compliance towards vap bundle and reduction of risk factors can help decrease incidence of vap and related morbidity and mortality. preventive strategies are effective in reducing ventilation-associated pneumonia (vap) in adults [1, 2] . in paediatric population there are no data about vap prevention, so we introduced a new bundle (vap-p) based on the available evidence for adults. this was designed as a before-after study. we enrolled all patients admitted to 8-bed medical-surgical paediatric icu at gemelli hospital in rome, requiring mechanical ventilation for at least 48 hours. patients with pre-existing tracheostomy were excluded. vap-p has been introduced since 2018 in order to improve quality of assistance. our bundle consisted in twice a day oral hygiene with chlorhexidine swab, daily check of oral bacterial colonization and aspiration prevention. comparison was made with an historical group including patients admitted before vap-p introduction (since 2016 to 2017). all data about demographics, antimicrobial therapy, icu stay and treatments, were collected. results: 162 patients were included (82 after and 80 before vap-p introduction). 5(6%) events of vap were recorded in vap-p group compared to 16 (20%, p=0.01) vap-p group had less vap per days of mechanical ventilation (1/100 compared to 3.3/100 p=0.01). multivariate analysis yielded an or of 0.23 (95%ci 0.07-0.81) for vap incidence after bundle introduction. mortality rate was slightly reduced in vap-p group (2.4%vs 6.2% p=ns). patients who developed vap required more days on mechanical ventilation and had higher mortality rate (12 vs 5 days p<0.001 and 14%vs 3% p=0.047, respectively). our vap-p seems effective in reducing vap incidence in critically ill paediatric population. introduction: ceftolozane/tazobactam (c/t) is a new antibiotic against mdr gramnegative bacteria infections, whose target population are the critically ill patients. even though 2/1 g dose safety administered as a 3hour-infusion has been already assessed, these patients can be under renal replacement therapy (rrt) and suffer changes in their volume of distribution (vd) that may affect antibiotic concentrations. the objective was to determine concentration reached by 3g c/t (3hour infusion) in septic patients on rrt (cvvhdf) and interdose behavior. we have used rrt machine prismaflex with oxyris filter and m100. hplc-uv method was used for simultaneous quantification of c/t. study population consisted of three obese critically ill patients with sepsis, on cvvhdf while receiving 3g c/t every 8 hours. samples were taken of prefilter, post filter blood and effluent, 30 min before infusion and 1, 2 and 4 hours after the end of it. we found great interpatient variability with the lowest cconcentration values in the patient with more hemodynamic instability using oxyris filter. even though cmax was less than reported in healthy subjects, we found similar values of auc and t ½ in comparison with healthy population studies. cmax of t was also compromised in comparison with values reported in healthy subjects, but with higher auc and t ½. cvvhdf contributes to c/t clearance. m100 filter showed the least clearance and higher values of auc and t ½. extraction rate was similar in all patients and filters (figure 1) . cmax achieved may be impaired because of the varying vd caused by obesity and rrt, but not affecting the antibiotic characteristics and behaviour. we conclude that because of the variety of clinical conditions, c-concentration is compromised particularly in hemodynamically unstable patients. however, the small sample doesn´t let us extrapolate these results. the extended infusion seems to be adequate to achieve the interdose antibiotic concentration. the use of biomarkers in sepsis is useful for early diagnosis and prognosis. the desired marker should be sensitive, specific, fast and accurate. procalcitonin (pct) measurement is approved by the fda even its efficacy is still under question. the determination of alfatorquetenovirus (ttv) could be a useful marker [1] . we analyzed 55 samples from 23 patients admitted to icu with clinical suspicion of sepsis. analytical data of c-reactive protein (crp), neutrophils and procalcitonin were collected. the sofa and apache ii scales were calculated and patients stratified according to these values in good and poor prognosis. ttv quantitative determination was carried by using a quantitative crp 2 . we calculated area under the curve (auc) of ttv plasma levels as a function of time. the statistical analysis involved u-mann-whitney and spearman test, using chi 2 for qualitative variables. results showed a not significant (ns) inverse relationship between the ttv auc and the patient proinflammatory level. a tendency (ns) was found between poor prognosis and the pct median values and crp being higher in the poor prognosis.group. a trend showed lower ttv dna count related to worse prognosis. an inverse relationship was found between pct and crp values and the ttv copies /ml plasma, ns correlation in the case of pct. there was a clear trend between the neutrophils´expansion and the regression line slope, obtained between ttv loads in the first two study steps. fig. 1 (abstract p432) . patient pk/pd measurements value>0.05), suggesting that the adsorptive mechanism wasn't primarily mediated by plasma protein. ha330 was saturated after adsorption of a total of 280.28±12.33 mg of van. the adsorptive kinetics showed an exponential reduction of van mass that reached a plateau after 30 minutes of circulation. in our study, simulating in vivo conditions of hp using ha330 during sepsis, a rapid and clinically relevant removal of van has been shown. after 2 hours of hp, we suggest to assess van plasma concentration and a loading dose of van should be considered. however, not knowing the potential interactions with other drugs, further in vivo studies are warranted to confirm these findings. assessing the volume of blood taken for blood culture and culture positivitydo we need to take less blood? it is commonly accepted that larger blood culture (bc) volumes (bcv) increase the yield of true positive cultures, and optimally 20 cc of blood should be obtained per set (2 bottles). only scarce data exists on the matter of optimal bcv. it is unknown what is the minimal volume that is acceptable for bc. the objective of this study was to determine the association between bcv and the rate of positive bc. blood taken for cultures in bd bactec plus aerobic/f negative bottles was collected from icus and acute care floors at 8 hospitals at the dmc over 6 months. blood volume was estimated automatically from blood background signal data in the bd bactec fx instrument. cultures were analyzed for each bottle. data was summarized for every month as the average volume and number of cultures taken and rate of positive bc for every unit. units were classified according to unit type (icu, medicine, surgery, mixed, emergency department (ed), organ/bmt or "other" which did not fit the previous categories) and analyzed as a group. a total of 23795 cultures were taken in 84 units. there is a positive association between bv and positive bc rate for ed and "other" units (irr=1.27, p=0.006 for the ed, irr=5.00, p<0.001 for "other" unit). all other units had no association between bv and positive bc rate (figure 1 ). secondary analysis, excluding pediatric units, gave very similar results. when comparing bv between unit types, the ed and "other" unit had significantly lower bv (2.4 ml in the ed and 3.5 ml in "other" unit compared to 4.9 ml in the icu, 4.7 ml in surgery, 4.2 ml in mixed and 7.7 ml in bmt). the correlation between bv and positive bc rate is probably limited to units taking very low bv for cultures. units taking volumes above 4 ml show no improvement in positive bc rate when higher volumes are taken. better prospective studies should be done to further establish the minimal bcv needed and spare unnecessary blood loss to hospitalized patients without compromising bc yield. de-escalating antibiotics in sepsis with the use of t2mr in a 35bed greek university icu c vrettou, e douka, i papachatzakis, k sarri, e gavrielatou, e mizi, s zakynthinos 1st icu department, university of athens, evangelismos general hospital, icu, athens, greece critical care 2020, 24(suppl 1):p440 in septic patients, the early use of appropriate empiric antibiotic therapy reduces morbidity and mortality. de-escalation refers to narrowing the broad-spectrum antibiotics once the pathogen and sensitivities are known. t2 magnetic resonance (t2mr) is a novel method of detecting eskape pathogens. we aim at investigating if using t2mr technology can expedite de-escalation of broad spectrum antibiotics. this is a prospective observational study conducted in our 35-bed university icu. inclusion criteria were critically ill patients age>18 y.o., with newly diagnosed sepsis and clinical suspicion of eskape bloodstream infection. a sample for t2mr and a blood culture (bc) sample were collected simultaneously from the patients enrolled. the t2mr bacteria panel test was run according to the manufacturer's guidelines and the bcs were processed according to the hospital standard procedures. we recorded clinical data and administered antibiotics. results: 26 patients were included in the study. mean time to culture positivity was 84 hours while mean time to t2mr result was 3.5 hours. in 20 patients the results of t2mr were in concordance with the bcs. in the remaining 6 cases, the bcs were negative while the t2 mr detected one or more eskape pathogens. there were no false negative results. de-escalation in at least one drug was applied to 8 patients (30.8%). no escalation was applied to 15 patients (57.7%) and antibiotic escalation in 3 (11.5%). conclusions: t2mr provides a quicker detection time that could shorten the time to targeted therapy. in our population this corresponded to early (within 6-12h) antibiotic de-escalation in approximately 1/3 of the included patients. antibiotic stewardship in icu. a single experience l forcelledo 1 , e garcía-prieto 1 , l lópez-amor 1 , e salgado 1 , j fernández dominguez 2 , m alaguero 3 , e garcía-carús 4 the increasing antibiotic resistance in microorganisms urged interventions such as the antibiotic stewardship programs in icu focused on reducing the inappropriate use of antibiotics by improving the antibiotic selection, the dosage, administration route and length as well as improving clinical outcomes and reducing antibiotic resistance. retrospective study where antibiotic consumption was analysed and measured in days of therapy (dots) between 2015 and 2018 in a medical-surgical icu of a university hospital where a multimodal educational program was established. specific training in infectious diseases in critically ill patients, periodic clinical and formative sessions fig. 1 (abstract p439) . correlation of blood culture positivity rate with blood culture volume by unit type were performed for icu staff and specific leaders within the icu staff designated. results: 4128 patients were admitted to icu. there was a reduction of 20,5% in dots (figure 1 ), reduction in antimicrobial resistance rates (14,36 in 2015, 7,4 in 2018 [days of resistant microorganism/1000 patientdays]) without an impact in icu global mortality (19,7% in 2015, 19,4% in 2018). the resistant bacteria registered were acinetobacter baumannii, s. aureus mr, blee and carbapenemase-producing enterobacteriaceae, pseudomonas aeruginosa mr and clostridium difficile. the safe in antimicrobial consumption was 391500€ (70% reduction). the icu stay decreased from 8,2 days (2015) to 6,9 (2018) , with no variation in mean apache ii (17,8) . the bigger decrease in antibiotic consumption was in colistin related to the reduction in resistance bacteria, in special acinetobacter baumannii, in linezolid and in piperacilin/tazobactam, even more remarkable in 2018 due to shortage of supplies which meant an increase in meropenem. the application of an antibiotic stewardship program in icu succeeded in reducing antibiotic consumption, antibiotic resistance and costs without an impact in clinical outcomes like mortality or icu stay. clinical outcomes of isavuconazole versus voriconazole for the primary treatment of invasive aspergillosis: subset analysis of indian data from secure trial p kundu, s kamat, a mane pfizer limited, medical affairs, mumbai, india critical care 2020, 24(suppl 1):p442 the secure trial was designed to compare the safety and efficacy of isavuconazole (a) versus voriconazole (v) for primary treatment of invasive mould disease caused by aspergillus and other filamentous fungi. the present analysis is aimed at comparing the indian subset of patients with that of the overall trial population and to ascertain any similarity or difference in the primary efficacy endpoint and safety/tolerability in these two groups. in secure trial, 258 patients in one group received (i) & another 258 patients received (v). the indian subset had 29 patients. we have done a qualitative analysis as the sample size of the indian subset was small. non-inferiority of (i) to (v) in terms of all cause mortality from first dose to day 42 was assessed in overall patients. the treatment difference between (i) and (v) group in the indian subset of patients was analyzed. proportion of patients who had to discontinue treatment due to teaes was analyzed. the all-cause mortality in the overall trial population met noninferiority margin (table 1 ). in the indian subset, it was higher for (i) than (v). there was a lower incidence of ocular, hepatobiliary, skin & subcutaneous tissue disorders in the (i) treated patients (see table 1 ). in indian subset, the above adverse events were less in the (i) group, but statistical inference could not be done due to small sample size. however, similar trend of less number of patients discontinuing therapy due to teaes in the (i) treated patients was seen in the overall patients & the indian subset. the all-cause mortality in the indian subset was higher in the (i) patients. a trend similar to the overall population regarding safety parameters favoring (i) was seen in the indian patients. considering the significantly higher prevalence of ia in india, suitably powered study design is necessary to draw definitive conclusions on the non-inferior efficacy & better safety & tolerability of (i) over (v) in patients of ia. introduction: ventilator-associated pneumonia (vap) is one of the most frequent healthcare-associated infections, correlated with increased mortality,extended hospital stay and prolonged mechanical ventilation. considering the latest outbreak of multiresistant a. baumannii infections in the critically ill patients with vap, there is a growing concern regarding challenges of the antibiotherapy in these patients. although ceftazidim-avibactam is considered to have limited effects on a. baumannii, it is reported to have a synergic activity in combination with other antibiotics. we performed a retrospective, observational study which included 24 icu patients diagnosed with vap(cpis > 6). oxa 23 a. baumannii was isolated from the tracheal secretions using a rapid molecular diagnostic platform(unyvero a50 system). patients were divided in two groups according to the antibiotherapy:group a meropenem + colistin and group b meropenem + colistin + ceftazidim-avibactam.statistical analysis was performed using graphpad6 applying t-test and kaplan-meier curves, having the in-hospital mortality as primary outcome and days of mechanical ventilation and hospital stay as secondary outcomes. mean age(y.o) in group a was 46 and 52 in group b and in both groups mean charlson comorbidity index was 3 points. survival percent was higher in the group treated with ceftazidim-avibactam (67 % vs 57 %, p = 0.08)(fig. 1) . length of stay was significantly decreased in group b (26.5 days vs 43 days in group a, p = 0.046). number of days under mechanical ventilation was also decreased in the ceftazidim-avibactam group (19 vs 22) but the data was not statistically significant. in light of the important thread of multiresistant a. baumannii and the lack of therapeutic measures, the synergistic activity of ceftazidim-avibactam use in combination with other antibiotics may be a promising approach to lower the mortality and hospitalization in critically ill patients diagnosed with vap. impact of patient colonization on admission to intensive care on 28 and 90 days mortality g dabar 1 , c harmouch 2 , e nasser ayoub 3 , y habli 4 , g sleilaty 5 , j infections caused by multi resistant bacteria are a major health problem, especially in icus, and it may be associated with high mortality rates. colonization precedes infection in most instances; therefore it may be a marker of a poor outcome. we tried to determine the impact of colonization on mortality at 28 and 90 days in a population of patients admitted to one medical and one surgical icu in the same institution. medical records review over three years 2016-2018 of all patients admitted to one surgical et one medical icu at hotel dieu de france hospital staying more than 24h. colonization to resistant bacteria was defined as mrsa, esbl, mdr, and vre. all patient received a nasal and rectal screen on icu admission, in intubated patients tracheal aspirate was considered as colonization in the absence of clinical respiratory tract infection. demographics, apache, sofa, immunosupression, charleston comorbidity index, length of stay, mechanical ventilation, hospitalization and antibiotic use in the previous 3 month were collected. mortality at 28 and 90 days was assessed through medical records or phone call. pearson chi-square was calculated for the association of colonization and mortality at 28 and 90 days, and subsequently odd ratio was estimated. introduction: critically unwell patients have been observed to respond unpredictably to traditional intermittent dosing (id) schedules of vancomycin, likely due to the complex physiological derangements caused by critical illness. continuous infusion (ci) of vancomycin has been suggested to overcome such problems by allowing more regular therapeutic drug monitoring and subsequent effective dose titration [1] . this study conducted at a tertiary intensive care unit, reports our experience following implementation of a continuous vancomycin infusion protocol. prospective data was collected over two consecuative periods of three months, initially capturing plasma levels for id (target level of 15-20mg/l) followed by reviewing plasma concentration levels in a ci protocol (target level of 20-25 mg/l). patients recieving renal replacement therapy were excluded. a total of 22 intermittent vancomycin prescriptions were administered and dosing levels observed. in the three month ci period, 26 patients received ci vancomycin and levels subsequently checked. the ci protocol resulted in increased blood sampling (107 samples in ci group vs. 73 samples in id cohort). two non serious incidents were reported in the ci cohort relating to preparation of vancomycin. both groups had a comparable median time to therapeutic range (48 hours). however, ci vancomycin group had a greater proportion of first samples outside the desired therapeutic range (70%vs 36%) (figure 1 ). as the therapy continued, ci vancomycin demonstrated a greater propensity towards consistent therapeutic levels than that observed with id. 83% of patients on a ci regime achieve the desired target levels compared to 77% in the id cohort (fig. 1) . it was positive for single or multiple microbes in 9(26.5%) and 22(64.7%) samples respectively. single or multiple resistance genes were detected in 5(25%) and 20(80%) samples respectively. bfpcr was positive only for bacteria in 13(38.2%), virus in 2(5.9%) and for both in 16(47.1%) cases. influenza a was found in 10(29.4%) cases. the most common organisms in community and hospital acquired pneumonia were streptococcus pneumoniae (4/12) and a. baumannii (10/22) respectively. bacterial cultures were concordant with bfpcr in 11/11 (100%) of positive cases. decisions to change antibiotics could be taken earlier based on bfpcr (p< 0.001) than if were based solely on culturesboth in culture positive (9.7± 14.3 vs 50.03±6.0 hrs) and negative cases (14.7±14.9 vs 48.0+4.3 hrs) where antibiotics would have remained unchanged. based on bfpcr antibiotics were escalated in 17(50%) patients and teicoplanin (11/ 19) was most often stopped. bal bfpcr were obtained significantly earlier, identified more organisms and bacterial resistance than culture reports and lead to more frequent and earlier antibiotic changes. severe community-acquired pneumonia (scap) is a frequent cause of hospitalization and mortality. ceftaroline is efficacious for treatment of cap (port risk class iii or iv). most severe patients were excluded from the clinical trials, so the efficacy of ceftaroline in these kind of patients is unknown methods: this is a health record-based retrospective before-after study in a tertiary care hospital. all scap patients admitted in icu between november 2017 and february 2019 receiving ceftaroline were included. control group included patients with same inclusion criteria but receiving ceftriaxone. propensity scores to adjust for potential baseline differences between groups were performed. levofloxacin or azythromicin were administered in both groups. primary outcome was the change in sofa score over the first 96h and secondary were days of mechanical ventilation, respiratory failure at 96h, need of rescue antibiotics, length of stay and mortality results: there were 28 patients in ceftaroline group and 43 in ceftriaxone group. baseline characteristics were similar except from more intubated patients in ceftaroline group (figure 1 ). there were less respiratory failure at 96h in patients with ceftaroline treatment (-73.3% vs. -51.6%; p 0,015), but no differences in other organ failures, mortality, days of mechanical ventilation or los. there were more need of rescue antibiotics in ceftriaxone group (7.1% vs .46.5%; p 0,001). we found more streptococcus pneumoniae isolation in ceftaroline group (18 (64.2%) vs 11 (25.5%); p = 0.001); more empiric use of oseltamir (16 (57.1%) vs 11 (25.5%); p = 0.012), but no more influenzae infections (11 (39.2%) vs 8 (18.6%); p = 0.101). s. aureus was detected in 1 patient in ceftaroline group and in 5 in ceftriaxone group. introduction: acute respiratory failure (arf) due to pulmonary infections is a usual cause of intensive care unit (icu) admission. immigration patterns and iatrogenic immune-suppression have made tuberculosis (tb) a common disease in western europe. severe tb requiring icu care is rare. nevertheless, mortality associated with active tb and arf is poor [1] . adult patients with tb admitted to icu from 2014-2018 were identified retrospectively. diagnosis was based on: positive cultures of sputum, bronchial aspirates or bronchioalveolar lavage fluid. demographic characteristics, reasons for admission, hiv status, anti-tb treatment and mortality were recorded. total of 25 patients with tb were admitted to icu. mean apache ii score was 20,2±6,9. sixteen were male. mean age 49,1±14,7 years. eight (32%) were hiv-positive, 3 (12%) diabetes mellitus type 2, 3 (12%) chronic liver disease. six (24%) had other causes of immunesuppression. main causes for icu admission were arf due to nonmycobacterium tuberculosis pathogens in 64%, acute liver failure in 12%, septic shock due to non-respiratory cause in 8%. overall, 52% were on anti-tb treatment at time of admission. tb involved the lung parenchyma in all patients. pleural involvement was present in 12% and lymph node in 20%. extrapulmonary sites were present in 28%: urogenital, gastrointestinal, bone marrow. pathogens identified in over-infections: 16% gram positive coccus, 20% gram negative bacilli, 16% fungal, 4% mdr-pathogen. one patient hiv-positive suffered arf due to pneumocystis jiroveci. overall, 64% died during icu stay. besides its latent evolution, mortality of tb patients admitted to icu is extremely high. arf due to over-infection seems to be the main cause for icu admission and mortality. better preventive approach of these patients may improve their outcome. introduction: human african trypanosomiasis (hat) is rarely encountered by critical care clinicians, but is an important differential for fever in the returning tropical traveler. late disease is characterized by seizures, fever and multi-organ failure [1, 2] . we present an anonymized case presenting from an endemic area in zambia referred for tertiary critical care management. the patient was too obtunded to give informed consent and his relatives could not be contacted despite extensive efforts. a middle-aged man with no past medical history from rural zambia presented to a local clinical officer post with fever and arthralgia. he was treated twice with anti-malarial medication without resolution of symptoms. two months later he was admitted febrile and obtunded to a local hospital with worsening confusion. he was transferred 8 hours by ambulance to our facility in lusaka, which is the only public tertiary critical care unit in zambia results: gcs on arrival was e3m4v2 without localizing neurology. microbiology investigations were negative, including for toxoplasma, cryptococcus, hiv or malaria. the patient suffered a generalized seizure followed by a sustained gcs of 3 and was admitted to the icu for invasive ventilation and seizure control. peripheral blood smears demonstrated trypanosomes consistent with hat secondary to trypanosoma brucei rhodesiense. he was commenced on melarsoprol but rapidly deteriorated, with signs of melarsoprol-induced arsenic encephalopathy and subsequent tonsillar herniation. his death was confirmed by neurological criteria. conclusions: icu management of fulminant hat involves supportive neurocritical care plus melarsoprol, a toxic arsenic compound with common side effects of hepatotoxicity and dysrhythmia. arsenic encephalopathy occurs in 10% of late hat, with a fatality rate of 70% [1] . early diagnosis is associated with a 95% survival rate in developed world travelers repatriated from endemic areas [2] . lithium chloride to prevent endothelial damage by serum from septic shock patients (in vitro study) a kuzovlev 1 the aim of the study was to investigate into effectiveness of lithium chloride (licl) as agent that prevents damage to the monolayer of endothelial cells under the action of serum from multiple trauma patients with septic shock. methods: serum from 5 pts with septic shock (sepsis-3) and 5 healthy donors was withdrawn. monolayer of ea.hy926 endothelial cells were incubated for 3 hrs at 37°c with healthy person's serum and with septic patient's serum without licl and with it at concentrations of 0.01 mmol, 0.1 mmol, 1 mmol, 10 mmol. licl was added 1 hour before the change of serum. after incubation cells were washed and fixed with 2% paraform solution and permeabilized with 1% triton x-100 solution. fixed cells were stained with primary antibodies to vecadherin and then incubated with secondary antibodies conjugated with oregon green 488 fluorescent dye as well as with phalloid red and hoechst dye 33342. images were processed by fluorescence microscope and imagej 1.44p and metavue 4.6 programs. western blotting was used to detect antibodies to ve-cadherin, claudin and gsk-3beta. statistics included mann-whitney test and chi-square test. incubation of a monolayer of endothelial cells with 5% serum of septic shock patients led to loss of ve-cadherin contacts and decrease of claudine. preincubation with licl 0.01 mmol did not prevent dismantling of claudine, actin, ve-cadherins; 0.1 mmol licl prevented it (p>0.05), but at higher concentrations (1 mmol, 10 mmol) almost completely protected endothelial monolayer from destruction of intercellular contacts (p<0.05). serum had almost no effect on the phospho-gsk-3β level after 5 min, 15 min, 30 min and 1 hr, but caused a significant (60%) decrease in its level after 2 and 4 hrs. licl (1 mmol) caused a significant increase in phospho-gsk-3β already 15 mins and up to 4 hrs after exposure. licl prevents septic damage to the monolayer of endothelial cells in vitro in a gsk-3beta mediated way. introduction: the autonomic nervous system (ans) controls both heart rate and vascular tone, which are known to be impaired during septic shock (ss) . acute inflammation is presumed to increase arterial stiffness of large arteries in experimental studies [1] . the objectives of this work are to verify if standard ss resuscitation modulate mechanical vascular properties and to verify if alterations in these vascular properties and ans activity are correlated. a protocol of fecal peritonitis septic shock and standard resuscitation (fluids and noradrenaline) was applied on 6 pigs. the arterial blood pressure waveform was recorded in the central aorta and in the femoral and radial arteries. the characteristic arterial time constant tau was computed at the three arterial sites, based on the twoelement windkessel model [2] . the total arterial compliance (ac) and the total peripheral resistance (tpr) were also estimated. baroreflex sensitivity (brs), low frequency (lf, 0.04-0.15 hz) spectral power of diastolic blood pressure, and indices of heart rate variability (hrv) were computed to assess ans functionality. results: septic shock induced a severe vascular disarray, decoupling the usual pressure wave propagation from central to peripheral sites, as shown by the inversion of pulse pressure (pp) amplification, with a higher pp in the central aorta than in the peripheral arteries during shock. the time constant tau together with ac and tpr were independently decreased. a decrease in brs, lf power, and hrv describe an ans dysfunction. after the administration of fluids and noradrenaline, both vascular and autonomic dysfunction persisted and these were found to be significantly correlated. measures of mechanical vascular function and ans activity could represent an useful end-point to guide further clinical investigations and refine our understanding of ss mechanisms, especially under medical treatment. introduction: lipopolysaccharide (lps), is a component of gram-negative bacteria known for its activation of the host immune system. the phospholipid transfer protein (pltp) has previously been shown to promote the binding of lps to lipoproteins, to limit inflammation and to lower mortality following injections of lps or bacterial infection. the aim of the present study was to investigate the role of pltp and lipoproteins in the detoxification of lps from the peritoneal cavity. injection of lps intra-peritoneally (ip) (1mg/kg) to wild type (wt) and pltp knocked-out mice (pltp-ko) (n = 9 per group). mass concentration and activity of lps were quantitated by lcmsms analysis of 3-hydroxymyristate and lal bioassay, respectively. lipoprotein fractions in plasma were separated by ultracentrifugation (n=10 vs n =12). following intra-peritoneal injection, clearance of intra-abdominal lps was faster and plasma neutralization was more efficient in wt than in pltp-ko mice ( figure 1) . indeed, lps found in plasma of wt mice was proportionally less active, sustaining a higher capacity for wt mice to neutralize lps (figure 1b) . quantitative dosage of lps in portal blood, 15 minutes after ip injection, revealed that plasma lps associates rapidly with the lipoprotein fraction (hdl plus ldl), and in higher proportions as compared to pltp-ko mice (66 [62-72] % vs 50 [41-54] %, respectively; p < 0.01). in line with previous studies, these observations now indicate that, lps readily associates with lipoproteins in a neutralizing process pltp mediated. finally, even with a heavy lps load (25 mg/kg), the bulk of lps was still found in the lipoprotein fraction (80 [80-90] %), suggesting that lipoproteins plus pltp in wt mice have a high capacity to detoxify intraperitoneal lps. in a model of peritonitis, lipoproteins and pltp were found to constitute key playors for peritoneal clearance and neutralization of lps. it emerges as a key pathway for the resolution of the inflammatory response in peritonitis. introduction: autotaxin (atx, enpp2) is a secreted enzyme present in biological fluids that catalyses the production of lysophosphatidic acid (lpa). lpa is a bioactive phospholipid evoking various cellular responses in most cell types. upregulated atx levels have been reported in various chronic inflammatory diseases. given the established role of lpa in the inflammatory response, we investigated a possible role for the atx/lpa axis in lps-induced endotoxemia. methods: lps was injected intraperitoneally (20 mg/kg) in mice producing 50% atx levels (atx df/+ , heterozygous null mutant mice), in mice producing 20-30% reduced atx levels upon inducible inactivation (r26creer t2 /enpp2 n/n mice) and in mice expressing 150-200% increased atx levels (enpp2-tg mice). kaplan-meier survival analysis was performed. atx activity was measured using the toos activity assay. results: atx df/+ mice that produce almost 50% reduced serum atx levels show increased survival compared to their littermate controls. for the inducible inactivation of atx, enpp2 n/n targeted mice were crossed with the r26cre-er t2 mice and tamoxifen induction enabled temporal control of floxed gene expression. r26creer t2 /enpp2 n/n mice were more protected against lps-induced endotoxemia compared to control mice. enpp2-tg mice overexpressing autotaxin and showing a 2-fold increase in plasma levels do not display improved survival rates compared to control group. conclusions: atx participates in systemic inflammation, as reduced atx levels in circulation decrease lethality of mice from caused by lps. the excess amount of circulating atx does not exacerbate the systemic inflammatory response to lps. introduction: pneumonia (pn) is a prevalent and severe infectious lung disease. host genetics plays an essential role in the pathogenesis of infectious diseases including pn [1] . the aim of the study was to analyze the variability of genes associated with neutrophil activation in pneumonia. to identify differential expressed genes (degs) in communityacquired (cap) and hospital-acquired pneumonia (hap) dataset «genome-wide blood transcriptional profiling in critically ill patients -mars consortium» (gse65682) from gene expression omnibus was analyzed (logfc≥2.0, fdr-corrected p-value<0.05). degs associated with neutrophil activation were selected according to gene ontology go:0042119 («neutrophil activation»). with the use of gtex portal and blood eqtl browser, we searched for esnps (expression single nucleotide polymorphisms) in whole blood for neutrophil activation genes differentially expressed in cap/hap. these esnps were further analyzed for their association with pn via the global biobank engine (gbe). a total of 46 degs from gse65682 correspond to go:0042119 genes (43 up-and 3 down-regulated) of which 39 genes were common to cap and hap. functional enrichment of 46 degs based on disgenet detected top-5 diseases associated with these genes (fdr-corrected p-value<0.05): myeloid leukemia, chronic; sepsis; asthma; lung diseases; allergic asthma. for these 46 genes 1366 esnps common to gtex portal and blood eqtl browser were identified. more than half of all variants were located on the second chromosome and influenced the expression of tnfaip6 and il18rap genes. among all esnps we identified variants associated with pn in the gbe (table 1) . we identified genes related to neutrophil activation, genetic variability of which was associated with pneumonia. sepsis was induced in wild-type c57bl6 mice (n=41) and cse knockout mice (n=41) by i.p. injection of 10 8 cfu/mice mdr p. aeruginosa. similar experiments were repeated after cyclophosphamide induced neutropenia. survival was recorded for 7 days. mice were sacrificed for determination of bacterial load and myeloperoxidase (mpo) activity as a surrogate marker of myeloid cell recruitment. cytokines were measured in serum by legendplex inflammatory panel. total leukocytes from mice spleens, with or without pretreatment with the h 2 s donor gyy3147, were incubated with 1 x 10 4 cfu/ml mdr p. aeruginosa. bacterial clearance was recorded. we observed a significant decrease in survival of cse -/mice as compared to cse +/+ mice (12% vs. 47%; p: 0.025). this survival advantage was eliminated in neutropenic mice (17% for both groups, p: 0.873). cse -/mice had increased pathogen load in the liver (6.57 ± 0.13 vs 5.26 ± 0.50, p: 0.029) and lung (6.70 ± 0.17 vs 5.29 ± 0.55, p: 0.035). mpo activity was lower in cse -/mice in the liver (634 ± 71 vs 1029± 179, p: 0.048) and lung (7627 ± 585 vs 11121 ± 1468, p: 0.34). cse +/+ mice had increased serum levels of il-23 (121.13 ± 33.68 vs 31.41 ± 7.02 of cse -/-, p: 0.001); mcp-1 (4769.91 ± 908.83 vs 1940.37 ± 1062.65, p: 0.026) and gm-csf (22.91 ± 4.66 vs 8.11 ± 1.92, p: 0.004). phagocytic activity of leukocytes from cse -/mice was reduced compared to cse +/+ mice. this deficit was eliminated after gyy4137 pretreatment (fig. 1) . deficiency of host-derived h 2 s leads to increased susceptibility to mdr p. aeruginosa infection due to an inefficient neutrophil chemotaxis and neutrophil mediated phagocytosis. acknowledgement funded by the itn horizon 2020 marie-curie european sepsis academy introduction: neuroinflammation often develops in sepsis along with increasing permeability of the blood-brain barrier (bbb), which leads to septic encephalopathy [1] . the barrier is formed by tight junction structures between the cerebral endothelial cells [2] . we investigated the expression of tight junction proteins related to endothelial permeability in brain autopsy specimens in critically ill patients deceased with sepsis, and analyzed the relationship of bbb damage and measures systemic inflammation and systemic organ dysfunction. case series included all adult patients deceased with sepsis in the years 2007-2015 with brain specimens taken at autopsy available. specimens were categorized according to anatomical location (cerebrum, hippocampus, cerebellum). the immunohistochemical stainings were performed for occludin, zo-1 and claudin. patients were categorized as having bbb damage if there was no expression of occludin in the endothelium of cerebral microvessels. results: 38% (18/47) developed multiple organ failure before death. 74.5% (35/47) had septic shock. the deceased with bbb damage had higher sofa maximum scores (16 vs.14, p=0.04), and had more often procalcitonin levels above 10 (56 % vs.28 %, p= 0.045). bbb damage in cerebellum was more common in cases with c reactive protein above 100 mg/l as compared with crp less than 100 (69% vs. 31 %, p=0.025). absence of zo-1 expression in cerebral meningeal samples associated with bbb damage (17 % vs. 0 %, p=0.046). positive blood cultures (n = 22) were associated to absence of zo-1 expression in cerebellar glial cells (92 % vs. 44 %, p=0.018). in fatal sepsis, damaged bbb defined as loss of cerebral endothelial expression of occludin ( figure 1 ) is related with severe organ dysfunction and systemic inflammation. loss of zo-1 in endothelial cells associates with bbb damage, and sepsis contributes to zo-1 loss in cerebellar glial cells. oxylipins are oxidative breakdown products of cell membrane fatty acids. animal models have demonstrated that various vasoactive oxylipin pathways may be implicated in septic shock pathophysiology but these have been poorly studied in humans. oxylipin profiling was performed on serum samples collected on enrolment to the vanish (vasopressin vs. norepinephrine as initial therapy in septic shock) trial. samples were analysed with liquid chromatography-mass spectrometry. patients were followed up until 28 days. results: samples were collected from 154 of 409 (37.7%) patients on inclusion to the trial and 39 (25.3%) had died by 28 days. non-survivors were found to have higher levels of a number of oxylipins including: 14,15-dihydroxyeicosatrienoic acid (dhet) (p<0.01), 11,12-dhet (p=0.03), 15(s)-hydroxyeicosatetraenoic acid (p=0.02), 14-hydroxyoctadeca-pentaenoic acid (p=0.04) but lower levels of the precursor eicosapentaenoic acid (p=0.012). when corrected for multiple comparisons with the benjamini-hochberg test, only 14,15-dhet remained significant (p=0.025). although there was a difference in median 14,15-dhet levels between survivors and non-survivors, many values were below the level of detection (n=84/154 (54.5%)). as such, we also analysed 14-15-dhet as a binary variable (figure 1 ). patients with detectable 14,15-dhet were more likely to die (hr 2.4 [95% ci 1.2-4.6], p< 0.01) and have a higher median lactate (p =0.01) and total sofa score (p< 0.01) than those patients where baseline 14,15-dhet was undetectable. our study suggests the oxylipin 14,15-dhet may be associated with septic shock severity and 28-day mortality. these results are consistent with the known vasodilatory actions of this class of oxylipin. more work is needed to confirm its exact role in septic shock and whether this pathway is amenable to therapeutic intervention. introduction: activation of neutrophils is a mandatory stage and a sensitive marker of systemic inflammatory conditions that can lead to the development of multiorgan failure. the aim of the study was to investigate into the antiinflammatory effects of lithium chloride on human neutrophils in vitro. study was carried out on neutrophils isolated from the blood of 5 healthy donors. 50% of neutrophils were activated by 100 mkm fmlp, 50% -by 100 ng/ml lipopolysaccharide (lps); then their activity was evaluated by fluorescent antibodies to cd11b and cd66b degranulation markers. intact and activated neutrophils were treated with a solution of lithium chloride (9 mmol). immunoblotting was used to assess gsk3b activity in neutrophils. mann-whitney criterion and p<0.05 were used for statistics. results: lithium chloride 9 mmol decreased the level of expression of cd11b on intact neutrophils by 16% (p=0.07), cd66b by 15% (p=0.07). fmlp increased cd11b expression on neutrophils by 2.6 times (p=0.0007), cd66b by 2.5 times (p=0, 0022). addition of lithium chloride solution to fmlp activated neutrophils reduced the expression of cd11b (p= 0.0317) and cd66b (p=0.0079). lps increased cd11b and cd 66b expression by 2.1 times (p=0.0007, p=0.0022, respectively); addition of lithium chloride reduced the expression of cd11b (p=0,0317) and cd66b (p=0.0079) on neutrophils. fmlp led to a dephosphorylation of gsk-3b by 47% (p<0.05), lithium chloride increased its phosphorylation by 387% (p <0.05). adding lithium chloride to activated fmlp neutrophils restored the level of gsk-3b phosphorylation by 277% compared to controls (p<0.05). lithium chloride modulates the inflammatory activation of neutrophils by bacterial components through the phosphorylation of gsk3b in neutrophils. human host immune responses to lipopolysaccharide: a comparison study between in vivo endotoxemia model and ex vivo lipopolysaccharide stimulations using an immune profiling panel dm tawfik introduction: sepsis, a leading cause of mortality among critically-ill patients in the icu, recently recognized by the who as a global health burden. patients that suffer from sepsis exhibit an early hyper-inflammatory immune response which can lead to organ failure and death. in our study, we assessed the immune modulations in the human in vivo endotoxemia model and compared it to ex vivo lipopolysaccharides (lps) stimulation using 38 transcriptomic markers. methods: eight healthy volunteers were challenged with intravenous lps in vivo. in parallel, blood from another 8 volunteers was challenged with lps ex vivo. blood was collected before and after 4 hours of lps challenge and tested with the immune profiling panel (ipp) prototype using the filmarray® system. the use of ipp showed that markers from the innate immunity dominated the response to lps in vivo, mainly markers related to monocytes and neutrophils. comparing the two models, in vivo and ex vivo, revealed that most of the markers were modulated in a similar pattern (68%). some cytokine markers such as tnf, ifn-γ and il-1β were under-expressed ex vivo compared to in vivo. t-cell markers were either unchanged or up-modulated ex vivo, compared to a down-modulation in vivo. interestingly, markers related to neutrophils were expressed in opposite directions, which might be due to the presence of cell recruitment and feedback loops in vivo. the majority of ipp markers showed similar patterns of expression post-lps challenge in both models, except for several markers related to neutrophils and t-cells. the ipp tool was able to capture the early immune response in the human in vivo endotoxemia model, which is a translational model mimicking immune host response in septic patients. introduction: serum levels of tyrosine kinase receptor mer and its ligand gas6 predict mortality in septic patients in the intensive care unit. however, whether their early measurement at emergency department (ed) presentation also predicts mortality and organ failure still needs to be clarified. in this multicentre observational study, septic patients admitted to 5 italian eds were included [1] . at ed presentation blood samples were taken for routine biochemical analyses and serum mer and gas6 measurement. urinalyses, blood gas analyses and chest x-ray were routinely performed. mortality at 7 and 30 days, as well as the presence of organ damage such as acute kidney injury (aki), thrombocytopenia, pt-inr derangement and sepsis-induced coagulopathy (sic) were evaluated according to baseline levels of mer and gas6. in conclusion, neither mer nor gas6 are early predictors of mortality in septic patients at ed presentation. however, mer independently predicted the development of sic, thrombocytopenia and pt-inr derangement in this population. glycocalyx shedding correlates with positive fluid balance and respiratory failure in patients with septic shock n takeyama, y kajita, t terajima, h mori, t irahara, m tsuda, h kano aichi medical university, department of emergency and critical care medicine, aichi, japan critical care 2020, 24(suppl 1):p463 endothelial hyperpermeability would play a major role in septic shock related organ failure. the aim of this study is to clarify the relationship between glycocalyx shedding and respiratory failure, sofa score, plasma angiopoietin (ang)-2 level and patient survival. methods: plasma samples were collected from 30 septic shock patients from admission to icu discharge and 10 healthy volunteers. plasma syndecan (syn)-1 and ang-2 were measured and clinical data was also collected. septic shock patients were classified into 3 groups according to the time-course change of syn-1 levels. excess syn-1 (>400 ng/ml) during 0 to 3 days and remaining high following 4 to 7 days were assigned to group i. excess ang-2 during 0 to 3 days and decreased following 4 to 7 days were assigned to group ii. moderate increase (<400 ng/ml) during 0 to 7 days were assigned to group iii. results: plasma syn-1 levels are positively associated with increased ang-2 levels (r2=0.41, p= 0.005), suggesting that ang-2 is involved in endothelial hyperpermeability. fluid balance and ventilator-free days (vfd) are significantly increased in group i as compared with group iii. sofa score, apache ii and patient outcome does not show any differences between groups i, ii, and iii. the positive correlation between glycocalyx shedding and fluid balance indicates plasma syn-1 may be a valuable marker for endothelial hyperpermeability. the negative correlation between glycocalyx shedding and vfd indicates plasma syn-1 may be a valuable marker for respiratory failure. the plasma level of syn-1 for prognosis and organ failure excluding ards in patients with septic shock requires further investigation. serial procalcitonin measurements in the intensive care unit at hiroshima university hospital k hosokawa, s yamaga, m fujino, k ota, n shime hiroshima university hospital, department of emergency and critical care medicine, hiroshima, japan critical care 2020, 24(suppl 1):p464 introduction: serum procalcitonin (pct) is a promising biomarker for differentiating bacterial infections from other inflammatory states. moreover, including serial pct measurements in the management of acute respiratory infection reduces the duration of antibiotic therapy without increasing the mortality. however, limited real-world information is available regarding the use of pct in intensive care units (icus). we extracted and analysed data from january 1 to december 31, 2018 from all the orders and results of pct measurements in the icu (26 beds) at hiroshima university hospital. a total of 1,252 pct measurements from 409 icu patients were included. in 170 patients, pct was tested ≥3 times during a single icu stay. serial pct measurements showed a fade-out pattern (76 [45%] patients), a second day-peaked decrease pattern (35 [21%] patients), and a series of negative patterns (30 [18%] patients). compared to patients who demonstrated the fade-out pattern, those who demonstrated the second day-peaked decrease pattern had higher mortality rates (3% vs. 20%, p < 0.01). approximately one-third patients in the icu who had decreasing serial pct values demonstrated the second day-peaked decrease pattern. since this group of patients had poorer survival, further studies are needed to clarify the association between a late rise in pct levels and delayed therapeutic intervention. the research was performed on 200 full-term newborns; no clinical signs of bacterial infection were diagnosed. on the 1, 5, 20 days the plasmà concentration of il-1ß, il-6, il-8, tnf-α, g-csf, sfas, fgf, no was determined by capture elisa; cd3cd19, cd3cd4, cd3cd8, cd69, cd71, cd95, hla-dr, cd34, cd14, cd3cd56, lymphocytes in apoptosis -immunophenotype analysis. by applying the statistical cluster population analysis of the immunological criteria under study we have evaluated the feasibility of sepsis diagnostics at the admission to the intensive therapy unit. the diagnostic rule for sepsis has been formulated by applying the "decision tree" approach to the "r" statistic medium. the cluster analysis confirms the presence of two clusters (presence of absence of sepsis: these two components explain the 60.81% of the point variability). the diagnostic rule for the early diagnostics of sepsis is as follows: disease develops providing during the first 48 hours cd95≥16.8%, no≤9.6 mkmol/l or cd95≤16.8%, cd34≤0.2%, cd69≥4.12% or cd95≤16.8%, cd34≤0.2%, cd69≤4.12% and lymphocytes annexinv-fitc+pi-≥12.3%. 45 newborns featured the confirmed sepsis development. the accuracy of this diagnostics amounts to 95.41%; sensitivity to 97.06%; specificity to 94.67%; diagnostic false positive share to 5.33%; diagnostic false positive share to 2.94%; positive result accuracy to 89.19%; negative result accuracy to 98.61%. the aggregate determination of cd95, cd69, annexinv-fitc+ pi-, cd34 and the plasma concentration of no enables the pre-clinical diagnostics of sepsis development. efficacy of pancreatic stone protein in diagnosis of infection in adults: a systemic review and metaanalysis of raw patient data j prazak 1 , p egimann 2 , i irincheva 3 , mj llewelyn 4 , d stolz 5 , lg de guadiana-romualdo 6 , r graf 7 , t reding 7 , hj klein 8 , ya que 1 fig. 1 (abstract p469) . impact of 24h lactate and bio-adm values in patients with elevated lactate level at admission. the green curve in the left km-plot illustrates data from 75 patients with 5 events; the red curve 70 patients with 18 events. the green curve in the right km-plot illustrates data from 28 patients with 4 events; the red curve 96 patients with 48 events. of note, differences in numbers between admission (n=328) and 24h (n=269) is related to initial mortality introduction: adrenomedullin (am) is a peptide synthesized in vascular endothelial cells and cleared by the lungs. the use of am as an inflammatory biomarker and his predictive value has been studied in critically ill patients, but not yet in veno-venous extracorporeal membrane oxygenation (ecmo). the purpose of this study was to describe the plasmatic levels of am in patients supported with ecmo for acute respiratory failure methods: am (normal values <0.55 nmol/l) was measured at 5 time points: immediately before (t0), 24-h (t1) and 72-h after (t3) ecmo initiation and immediately before (t4) and 72-h (t5) after ecmo removal, in consecutive patients with severe respiratory failure supported with ecmo enrolled in the gatra study (nct03208270) at fondazione irccs ca' granda -policlinico of milan. data are reported as median (25 th -75 th percentile). statistical analysis was performed using logistic and random effects regression models (to account for repeated measurements within individuals) results: a total of 131 measurements were taken in 32 consecutive patients. am (nmol/l) decreased along the course of ecmo: t0=2.0 (1.5-6.4), t2=2.0 (1.5-6.4), t3=1.6 (1.1-3.1), t4=1.3 (0.8-2.0), t5=0.9 (0.6-2.1) (mean diff.= -0.65, 95%: ci -0.96, -0.35). am was lower in patients with viral compared to bacterial ards (mean diff.= -2.7, 95%ci -5.2, -0.2) (figure 1 ). am was higher in more severe patients (sofa>= 10, n=14) compared to less severe patients (sofa< 10, n=18): 5.7±4.8 vs 1.4±0.8 nmol/l, respectively p<0.001. basal values of am could not predict mortality at 28 days (or=0.8, 95%ci: 0.5-1.2) after conditioning for sofa score and respiratory failure etiology conclusions: am plasmatic values seem to be higher in more severe patients and in patients with bacterial ards. am decreased along the ecmo course but could not predict mortality in our group of patients fig. 1 (abstract p471) . plasmatic adrenomedullin during ecmo heparin binding protein (hbp) is released from activated neutrophils upon stimulation of b2 integrins. this pro-inflammatory effect generates the hypothesis that it can be a sepsis biomarker for patients admitted at the emergency department (ed) methods: the prompt study (clinicaltrials.gov nct03295825) took place at the ed of six greek hospitals. participants were admitted with suspected acute infection and at least one vital sign change. hbp was measured by an enzyme immunosorbent assay in plasma. sepsis was diagnosed by the sepsis-3 criteria. the primary study endpoint was the sensitivity for the diagnosis of sepsis. outcome prediction was the secondary endpoint. a total of 371 patients were enrolled; 166 had sepsis. the most common infections among patients without and with sepsis were upper respiratory tract infections in 30.2% and 1.2%; community-acquired pneumonia in 6.8% and 28.3%; and acute pyelonephritis in 9.3% and 28.3%. median hbp was 24.0 and 32.7 ng/ml respectively (p: 0.027). following analysis of the area under the curve (auc) it was found that the best discriminatory cut-off for sepsis was 19.8ng/ml. the comparative diagnostic performance of hbp versus qsofa score is shown in figure 1 . the odds ratio for sepsis with hbp above 19.80 ng/ml was 2.07 (p: 0.001). at the same cut-off point the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) for the prediction of early death after 72 hours was 100%, 35.7%, 4.1% and 100% respectively. hbp is more sensitive but less specific than qsofa for the diagnosis of sepsis in the ed. the rule-out prediction of early death seems the great merit. chronobiological and recurrence quantification analysis of temperature rhythmicity in critically ill patients introduction: rhythmicity and complexity of several circadian biomarkers, such as melatonin, cortisol and temperature have been found to be modified by critical illness. we examined the potential alterations of core body temperature (cbt) fluctuations and complexity in three groups (n=21): patients with septic shock upon icu admission (group a, n=10), patients who developed septic shock at icu hospitalization (group b, n= 6) and controls (group c, n=5). the hourly, average cbt was computed for 24 h upon icu admission and discharge in groups a and c, as well as during septic shock onset in group b. cosinor analysis of cbt curves was performed leading to the estimation of mesor (mean value), amplitude (the difference between peak and mean values) and acrophase (phase shift of maximum values in hours). complexity of cbt signals was evaluated with recurrence quantification analysis (rqa). no significant alterations in any circadian feature within groups were found, except for amplitude. controls exhibited increased entry cbt amplitude (0.45 ± 0.19) compared to groups a (0.28 ± 0.18, p < 0.05) and b (0.32 ± 0.13, p < 0.05). higher entry cbt amplitude in groups b and c was related with lower saps ii (r = -0.72 and -0.84, p < 0.003) and apache ii scores (r = -0.70 and -0.6, p < 0.003) respectively, reduced icu and hospital stay in group b (r = -0.62 and -0.64, p < 0.003) and entry sofa score in group c (r = -0.82, p < 0.003). recovery cbt time series appeared more periodic in relation with icu entry, for all groups. a more random cbt signals pattern upon results: among 23.011.601 individuals, 159.691 received inpatient treatment for sepsis. 41% had severe sepsis. 21% of sepsis and 28% of severe sepsis patients had an explicitly coded hai. the proportion of hai was higher in patients that received icu-treatment than in patients without icu-treatment (35% in icu/14% in non-icu sepsis, 37% in icu/17% in non-icu severe sepsis patients). tab. 1 shows the foci of explicitly coded hai. nosocomial pneumonia was the most common hai in all patient groups. clabsi occurred more frequently in icutreated patients; 21% were affected. cauti and c. diff infections were more common among non-icu-treated sepsis patients. more than one quarter of non-icu-treated sepsis patients had a c. diff infection. hai are common causes of sepsis and pose a significant healthcare burden. the proportion of patients affected and the distribution of foci differ between non-icu-and icu-treated sepsis patients with important implications for sepsis management within hospitals. impact of sepsis protocol triggered by ramathibodi early warning score (rews) in ipd sepsis on clinical outcomes s matupumanon 1 , y sutherasan 2 , d junhasawasdikul 2 , p theerawit 3 sepsis is now early identified and managed during triage in the emergency department. however, there is less focus on the effect of patients' management at the ward level. we aim to evaluate the impact of the implementation of the sepsis protocol on clinical outcomes in in-patients with new-onset sepsis. we conducted a prospective observational cohort study among adult medical patients admitted to the general wards in a university hospital. a 25-month pre-protocol period (august 2016 to august 2018) was assigned to a control group, and a 14-month protocol period (september 2018 to october 2019) was allocated to a protocol group. an in-patient sepsis protocol comprised nurse-initiated sepsis protocol by ramathibodi early warning score (rews)≥ 2 plus suspected infection, prompt antibiotic, lactate measurement, and fluid resuscitation was implemented. (table 1) . the implementation of in-hospital sepsis protocol was associated with significant improvement in patients' outcomes, namely lactate measurement, starting antibiotic within 1 hr, fluid management, and the shorter length of icu stay. icu routine nursing procedures interfere with cerebral hemodynamics in a prolonged porcine fecal peritonitis model sl liu 1 , dc casoni 2 , w z'graggen 3 , d bervini 3 , d berger 1 , sj jakob 1 routine nursing procedures (np) can interfere with blood pressure and cardiac output and may therefore alter cerebral hemodynamics in critical illness. this may be risk factor of sepsis-associated encephalopathy. methods: 20 sedated and mechanically ventilated pigs were randomized to fecal peritonitis or controls (n=10, each). after 8 hours of untreated peritonitis, the animals were resuscitated for 76 hours (resuscitation period). np [assessment of sedation (as), tracheal suctioning (ts), change in body position (cp), lung recruitment maneuver (rm)] were performed at baseline and 8h, 32h, 56h and 72h after start of rp. systemic and cerebral hemodynamics and o 2 saturations were recorded continuously. shock is the most common cause of death in the postsurgical icu, including septic shock and hypovolemic shock, reaching the 50-60% mortality in septic shock. the inadequate response of the immune system to the infection triggers a potent inflammatory cascade, where the c-reactive protein (crp) is an essential key in the amplification and maintenance of this cascade. the gene encoding to crp is located on the proximal long arm of human chromosome 1 (1q32). the gt polymorphism in the promoter sequence of crp gene (rs2794521) has been associated with invasive pneumococcal disease. thus, we analyze the relationship between rs2794521 polymorphism and the risk of developing septic shock in postsurgical patients. an observational, retrospective and single-center study was conducted on a sample of caucasian patients undergoing major abdominal surgery, of which one part developed septic shock and another part developed systemic inflammatory response syndrome, who were used as control. the rs2794521 polymorphism was analyzed by vasoactive medications are commonly used in sepsis treatment but may correlate with peripheral ischemia and the well-publicized complication of limb and digit loss. yet, the association between limb and digit threat and the intensity, duration, and pattern of vasopressor exposure are unknown. we studied adults (2010-2014) at 12 hospitals in an integrated health system who met criteria for sepsis-3. we identified the time to clinically apparent limb or digit threat using clinical adjudication among those with vasopressor-dependent sepsis (i.e. >1 hour of vasopressors at sepsis onset) who had a surgical evaluation within 28-days of sepsis onset. we defined daily vasopressor intensity as 0 to 4 vasopressors administered. then, we created a time-dependent model for threat with mortality as a competing risk with a weight function to estimates the varying contribution of vasopressors over time. we determined the subdistribution hazard (sh) ratio of threat for various patterns of vasopressor exposure and intensity, adjusted for age, baseline risk factors, and sequential organ failure assessment (sofa) score at sepsis onset. of 110,621 adults with sepsis, 13,147 (12%) were vasopressordependent (age, 66 [iqr, 56-77]; 7,040 [54%] males; max sofa score, 8 [sd 5] ). of these, 3,664 (28%) died and 117 (0.9%) had evaluations for limb or digit threat 4 [iqr, 1-8] days after sepsis onset. the model-based weight function showed the contribution of vasopressors to threat was stable over time ( fig 1a) . overall, a 1 unit increase in cumulative vasopressor exposure was associated with risk of threat (sh ratio, 2.60 [95%ci, 1.60-4.23], p<.001). for various patterns of vasopressor exposure, greater intensity associated with increased risk of threat ( fig 1b) . compared to constant exposure, an increasing and peak pattern associated with the greatest sh (fig 1c) . cumulative vasopressor exposure was associated with an increased risk-adjusted hazard of limb or digit threat following sepsis. fig. 1 (abstract p509) . relationship between vasopressor exposure and limb or digit threat following vasopressor-dependent sepsis. panel a demonstrates the estimated contribution of daily vasopressor intensity prior to surgical evaluation for limb or digit threat, with mortality as a competing risk. panel b and c explore the relationship between threat and both cumulative vasopressor exposure and the pattern of exposure following sepsis onset. (b) the maximum cumulative vasopressor exposure was associated with the highest risk of limb or digit threat (shr 17.5) when compared to reference exposure pattern (shr 1.0, reference). (c) increasing (shr 1.2) and peak (shr 1.2) patterns of cumulative exposure were associate with an increased sh of limb threat, while a decreasing pattern was associated with a lower risk (shr 0.8) when compared to constant intensity (shr 1.0, reference). abbreviations: shr: subdistribution hazard ratio proportion of encounters transitioning from phenotype at presentation within 24hrs, by arrival phenotype assignment and probability of membership. (c) tsne plots for α-type, ß-type, y-type, and ∂-type, with core (dark), marginal (light), and non-members (grey) in plots on the left and core, marginal, non members, and transitioning members (black) on the right fig. 1 (abstract p005). isolated microorganisms critical care references: 1. wertz et al. critical care explorations 1: e0058 the process investigators choosing wisely guidelines for the provision of intensive care services, version 2. ics structured patient handovers references: 1. care of the critically ill woman in childbirth the proqol manual: the professional quality of life scale:compassion satisfaction, burnout & compassion fatigue/secondary trauma scales references: 1. shimabukuro-vornhagen a et al. ca the code: professional standards of practice and behaviour for nurses, midwives and nursing associates p415 introduction: the aim of this study was to compare factors associated with the icu mortality for vap due to multidrug-resistant (mdr) klebsiella spp. in case of monobacterial (mo) vs polibacterial (po) origin. methods: retrospective data analysis of patients treated in icu with mdr klebsiella spp. strains as pathogens of vap during three year period was carried out. results: data of 71 patients were evaluated. mo vs po of mdr klebsiella spp. vap cases was found to be 35 (49.3 %) vs 36 (50.7%), p = 0.906. the icu mortality was 9/35 (25.7%) in mo, and 17/36 (47.2%) in po one, p = 0.060. statistical significant differences of survivors vs non-survivors in mo and po vap due to mdr klebsiella spp. were found in medians of neutrophilosis 78 p430 introduction: we study the population structure and resistome of mdr enterobacterales and pseudomonas aeruginosa isolates, c/t-susceptible or -resistant, recovered from low respiratory, intraabdominal and urinary tract infections of icu patients of 11 portuguese hospitals (step study results: in e. coli, two vim-2 producers were found (st131-b2-h30-o25:h4-ctx-m-15 and st88-c-h39-o22:h4) (c/t-mic=0.5/4-1/4 mg/l). a kpc-3-st5463-cladev-h160-o164:h56 (16/4 mg/l) was also detected. the most frequent esbl-e. coli clone was st131 cpr klebsiella pneumoniae (32 patients), candida spp. (21 patients). the comparison subgroup consisted of 217 patients with bacteremia caused by non-escape pathogens. we evaluated the days of mechanical ventilation, duration of antibiotic therapy (amt), icu length of stay (los), hospital los and mortality (table 1). results: mortality in patients with bacteremia caused by non-eskape pathogens was 23.5%, candida spp vancomycin mass removal over 120 minutes of hemoperfusion using ha330. bars refer to vancomycin mass (mg): blue (experiment 1) and red (experiment2) bars using blood while green (experiment 3) bar using balanced solution. yellow dashes are mean mass values of the three experiments (with standard deviations) and yellow line represents the reduction curve over time table 1 (abstract p438). results. * p-value versus non-eskape subgroup mechanical ventilation p453 translational value of the microbial profile in experimental sepsis studies sp tallósy 1 , a rutai 1 , l juhász 1 , mz poles 1 , k burián 2 , d érces 1 , a szabó 1 , m boros 1 invasive hemodynamic monitoring and blood gas analyses were performed on anesthetized animals between 18-24h of sepsis. the respiratory, cardiovascular, renal, hepatic and metabolic dysfunctions were evaluated with the species-specific sequential organ failure assessment (sssofa) score, the microbial profile was determined with selective media and maldi-tof ms in the initial inoculum and in the abdominal fluid taken 20h after sepsis induction. results: strong correlation was found between the initial dose of the inoculum (cfu) and the sssofa scores for organ dysfunction (rats: r = 0.656, p=0.0186; pigs: r=0.570, p = 0.0391) p466 introduction: pancreatic stone protein (psp) has shown promise as a biomarker of infection however, its diagnostic potential has not been systematically evaluated. we performed a systematic review and meta-analysis of available data on psp to evaluate its value for detecting infection in adults and determining a plasma or serum threshold value. methods: the pubmed and cochrane library database were searched for studies on psp in adult patients and their raw data were analyzed to estimate the best psp cut-off value that could detect infected patients using the youden's index. the cut-off sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were computed and compared to those for procalcitonin (pct) and c-reactive protein (crp). finally, we explored the potential value of a model combining all three biomarkers to detect infection. results: from a total of 44 potentially eligible published studies, 5 containing 631 patients were included in quantitative analysis. among them, 370 patients suffered from a clinically confirmed infection. the median appropriate statistical tests were used using spss 23. cd 64 was expressed as % age of neutrophils expressing positivity. results: sixty patients were analyzed. all parameters were compared between survivors and non survivors. demographics were comparable. most common source of sepsis was lungs and majority were admitted due to medical reason. non-survivors had significantly increased number of days with septic shock. at day 8 median values of all the biomarkers and the sofa score were significantly higher in the nonsurvivor group (p<0.05). there was a decreasing trend of all 3 biomarkers and sofa score amongst survivors. on multivariate logistic regression analysis, increased cd64 and crp levels between baseline and day 8, increased days with septic shock and increased sofa references: 1 introduction: we characterized the association of c-reactive protein (crp) with extracellular vesicles (evs) in plasma from sepsis patients and assessed a commercial crp adsorbent (pentrasorb, pentracor, hennigsdorf, germany) to deplete free and ev-associated crp. in addition, we characterized the potential pro-inflammatory effects of ev-bound crp on monocytes and endothelial cells monocytes and human umbilical vein endothelial cells (huvecs) were stimulated with isolated evs (20,000 g, 30 min) monocyte il-8 secretion was quantified by elisa; the activation of huvecs was assessed by their expression of icam-1 and e-selectin using confocal microscopy. results: septic plasma (n=30) contained 227.0±88.6 mg/l crp vs. 0.7±0.4 mg/ l for healthy controls (n=5). both, total evs and crp + evs were significantly elevated in septic plasma as incubation of septic plasma with pentrasorb resulted in depletion of free crp (247.2±72.6 mg/l before vs. 1.8±0.7 mg/l after adsorption) as well as in a significant reduction in crp evs from crp-depleted septic plasma induced significantly lower il-8 levels. huvec icam-1 or e-selectin expression, however, did not increase upon stimulation with septic evs. conclusions: treatment of septic plasma with pentrasorb efficiently removes free crp and detaches crp from the ev surface, resulting in reduced proinflammatory effects flow cytometry confirmed the association of monocytes with platelets and platelet-derived evs as well as the uptake of evs by monocytes. conclusions: storage of isolated monocytes induces a shift towards cd16 expressing proinflammatory monocytes, which seems to be mediated by residual platelets and platelet-derived evs. it remains to be clarified whether evs released from activated platelets can also trigger a shift towards proinflammatory, intermediate monocytes in vivo ethical approval was provided by ucl research ethics committee (5060/001). paired parametric analyses were performed and data displayed as mean +/-95% ci. results: plasma calprotectin concentration began to increase 1.5hours after endotoxin administration, was significantly higher than baseline by 2 hours (356.7ng/ml vs. 737ng/ml, p <0.01), peaked at 4 hours (mean 1373ng/ml, figure 1) and normalized by 24 hrs. calprotectin peaked earlier than comparator soluble mediators (procalcitonin 8hrs, crp, 24hrs) and exhibited 100% sensitivity; all participants demonstrating a minimum 2-fold increase from baseline (mean 3.84x). calprotectin displayed greater baseline variability (sd 147.9ng/ml) than either crp or procalcitonin. conclusions: our results indicate the potential of plasma calprotectin as a biomarker for bacterial infection. it increases earlier and peaks more rapidly than standard biomarkers. whilst higher baseline variability was observed p501 a multicenter randomized controlled study on landiolol for the treatment of sepsis-related tachyarrhythmia: subanalysis of the j-land 3s study o nishida kagoshima university graduate school of medical and dental sciences, department of emergency and intensive care medicine methods: we analyzed a retrospective cohort of electronic health records from adult sepsis patients at 12 upmc hospitals from 2010 to 2014. we defined sepsis-3 by i.) suspected infection (e.g., administration of antibiotics or body fluid culture) & ii.) organ dysfunction (e.g., 2 or more sofa points) in the first 6 hours of care. data were organized by hour and included vital signs, lab values, and treatments (e.g., total hourly iv fluids (ml) and norepinephrine equivalent dose). for each hour we describe, i.) available data elements, ii.) presence of sepsis-3, and iii by hour 6, most patients had vital signs (99%; n=70,559), basic labs (88%; n=62,719), fluid cultures (94%, n= 66,995), while serum lactate was completed in 24% (n=17,818) conclusions: early sepsis care patterns are variable. iv fluids were given during early hours, when uncertainty about sepsis was greatest, while vasopressors were administered after sepsis-3 elements were present. p504 effects of abdominal negative pressure treatment on splanchnic hemodynamics and liver and kidney function in a porcine fecal peritonitis model sl liu 1 department of intensive care medicine splanchnic hemodynamics and laboratory parameters were measured at baseline (bl, start of rp), and 24h, 48h and 72h after start of rp. two/three-way rm-anova or mixed-effects analysis, and student t tests were performed. results: npt in controls had no effect. after sepsis induction, mean arterial pressure (map) decreased by 15 (7-22) mmhg, cardiac output (co) by 1.3 (0.7-2.2) l/min, and arterial lactate increased by 0.2 (0.1-0.5) mmol/l. sepsis and resuscitation was associated with increasing hepatic and renal arterial flows (p≤0.002, both), and increasing prothrombin time npt in sepsis resulted in numerically less noradrenaline administration (0.3±0.3 ug/ min/kg in sepsis with npt vs. 0.8±0.7 ug/min/kg without npt, p= 0.310) and positive fluid balance (2.8±0.4 ml/h/kg with npt vs. 3.1± 0.4 ml/h/kg without, p=0.241). conclusions: in our experimental fecal peritonitis model, npt did neither impair splanchnic hemodynamics nor abdominal organ function. whether npt helps to reduce noradrenaline and volume administration in abdominal sepsis should be evaluated in further studies. p505 association between a c-reactive protein gene polymorphism (rs2794521) with the risk of develop septic shock in postsurgical patients of major abdominal surgery p martínez-paz 1 valladolid, spain; 2 hospital of medina del campo notably, the three groups received a comparable pro kg dose of acetaminophen. no difference was found between groups in term of toxic effects. patients carrying the cyp3a5p showed a more pronounced effect on body temperature in respect of wt and ugt1a1p °c respectively, but it does not reach statistical significance (fig.1b). only 50% of the patients reach a temperature <38°c at t2 and only 20% <37.5°c. conclusions: polymorphisms in enzymes involved in the metabolism of acetaminophen are relatively common. cyp3a5p seems to lead to higher peak plasmatic concentration and a slightly increased efficacy in fever control panel a: variations of acetaminophen plasmatic levels after 30 minutes (t1) and 2 hours (t2) after administration of an iv dose of 1g of paracetamol in wt patients and patients carrying mutation; panel b: body temperature variations in wt patients and patients carrying mutations clinical research, investigation, and systems modeling of acute illness (crisma) center, department of biostatistics we determined phenotype cohesiveness using probability of assignment at presentation, defining core members as ≥90% and marginal as <90% probability. we determined how members transitioned to other phenotypes over 24hrs using t-distributed stochastic neighbor embedding (tsne) plots and determined the odds (95%ci) of transition. results: we studied 37,198 adult sepsis encounters (median age 69 1c) the odds of ever transitioning from presenting phenotype increased significantly for marginal members vs publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the department of education of the basque government (piba 2019-57) and the university of the basque country upv/ehu (ppg17/65, giu 17/32) for their financial support. a great disaster affects the family-and friend-performance of bcpr by diminishing the willingness of family and friend bystanders to follow the instruction provided by dispatchers. the experimental method ifitem could be an alternative of fibtem in cases when internal coagulation pathways assessment is prioritized (i.e. heparinized patients on extracorporeal supports). patients undergoing limitation of life-sustaining therapy had lower karnofsky scale scores. therefore, this scale may be useful to guide end-of-life decisions in the future, but further studies with larger number of patients are needed. readmission after discharge home from critical care: a qualitative study c robinson 1 , f nicolson 1 , p mactavish 1 , t quasim 2 , jm mcpeake 1 1 nhs greater glasgow and clyde, nhs greater glasgow and clyde, glasgow, united kingdom; 2 university of glasgow, nhs greater glasgow and clyde, glasgow, united kingdom critical care 2020, 24(suppl 1):p392 readmissions to acute care occur in a high number of critically ill patients within 90 days of hospital discharge [1] . biomedical drivers such as frailty and pre-existing co-morbidities have been identified as drivers for readmission. however at present there is limited data on the influence of social problems on readmission. this study, using a grounded theory approach, sought to understand from a patient/caregiver perspective what the drivers for readmission to acute care were. ethical approval was granted from the west of scotland research ethics service (19/ws/0091). a grounded theory approach was used to explore from a patient and caregiver perspective what the drivers for readmission are [2] . using a clinical database, we identified those patients who had an icu admission ≥ 3 days who were readmitted to acute care within 90 days of hospital discharge. the researcher attended the ward and after discussion with the direct care team conducted a semi-structured interview with patient and/or caregiver. the interview was recorded and transcribed verbatim. the transcripts were analysed to generate initial codes, followed by the development categories and sub-categories. theoretical sampling was undertaken. results: 15 participants were interviewed.10(66.6%) were patients and 5 (33.3%) were caregivers. the themes that have emerged from the data were: pain and polypharmacy; lack of social support and/or isolation; strained relationships with primary care providers and information provision across the patient journey. subsequent theory development is underway to understand how this learning could help reduce readmissions in future. in conclusion, both social and biomedical drivers are likely to contribute to acute care readmission in this group. future interventional work is required in order to identify modifiable factors to reduce this burden for patients and the healthcare service. frailty has shown to have prognostic relevance for patients with critical illness. since a wide range of tools has been described to screen for frailty, we aimed to describe the association of two frailty screening tools, the clinical frailty scale (cfs) score and the modified frailty index (mfi) in critically ill patients. we performed a post-hoc analysis of a multicenter cohort of patients admitted to six canadian intensive care units (icu) between february 2010 and july 2011. frailty was identified using the clinical frailty scale (cfs) and the modified frailty index (mfi). concordance of the frailty screening tools was evaluated with partial spearman rank correlation and intraclass correlation (icc). discrimination and predictive ability of the tools for hospital mortality, 1-year mortality, hospital readmission and adverse events were compared using concordance statistic (c-statistic) and calibration plot adjusting for age, sex, sequential organ failure assessment (sofa) score and icu admission source, respectively. the cohort included 421 patients. prevalence of frailty was 32.8% (95% confidence interval [ci] 28.3%-37.5%) with the cfs and 39.2% (95% ci 34.5%-44.0%) with the mfi. concordance between the two tools was low [(icc of 0.37; 95% ci 0.29-0.45) and partial correlation coefficient of 0.38 (95% ci 0.29-0.47)], even after adjustment. hospital and 1-year mortality were greater for frail compared to non-frail patients using of both tools. similarly, both tools found frail patients were less likely to be living independently after hospital discharge, and more likely to be rehospitalized when compared to non-frail patients. while the cfs and mfi show low concordance, both showed good discrimination and predictive validity for hospital mortality. both tools identify a subgroup of patients more likely to have worse clinical outcomes. the post-intensive care syndrome (pics) is a myriad of physical, psychiatric and cognitive disorders secondary to critical illness, leading to a decreased quality of life and an important socioeconomic burden. this study aimed to identify if the conformity to a pics prevention bundle was able to reduce the incidence of the syndrome at icu discharge. all patients admitted to the icu from january 1 st to december 31 st 2018 were included. the conformity to each of the ten components of the pics prevention bundle was assessed daily, and the patients were evaluated for anxiety, depression, cognitive dysfunction, muscular weakness, mobility impairment and nutritional risk at icu discharge and at a 3-to-6-months follow-up consultation. the patient cohort was divided in terciles according to bundle conformity for the analysis. results: from the 1145 enrolled patients, 352 (31%) were evaluated at icu discharge, and 103 (29%) attended to the follow-up consultation. there was no difference in baseline characteristics between the cohorts. there was no correlation between the prevalence of pics at discharge and bundle conformity during icu stay (90% vs. 87% vs 87%, p 0.834), though there was a decrease in nutritional risk and days in mechanical ventilation (table 1) . after 3 to 6 months there was a reduction on the prevalence of any kind of pics, mobility impairment, muscular weakness and nutritional risk. the patients that developed pics were older and had a higher simplified acute physiology score iii at icu admission. a higher adhesion to a pics prevention bundle was not able to prevent the occurrence of the syndrome. post intensive care syndrome (pics) is well recognized following general icu care [1] . intensive care syndrome:promoting independence and return to employment (ins:pire) is a multidisciplinary complex intervention designed to address pics [2] . with a paucity of evidence on pics after cardiothoracic intensive care, we aim to evaluate pics and the feasibility of the ins:pire intervention in this population. those attending the clinic received 5 weeks of intervention including individual appointments with icm nurse, physician, pharmacist, and physiotherapist. a café area facilitated peer support alongside psychology group sessions. primary outcome was quality of life measured by eq-5d-5l. further surveys included: pain, mental health, and selfefficacy. questionnaires were taken at baseline, 3 and 12 months. results: over 5 cohorts, 27 patients attended, 67% male, median age 66 years (iqr 61-75), median apache 2 score of 17 (iqr 14-18.5), and median icu length of stay was 13 days (iqr 9-21). a total of 14 (53%) patients completed surveys at one year. scheduled admissions represented 56% of those attending. mean euroqol eq-vas score was 70/ 100 (sd +/-18) at baseline increasing to 78/100 (sd +/-16) by 1 year (table 1) . those with problems in at least one domain of eq-5d-5l fell from 92% at baseline to 73% at 1-year with the breakdown shown in table 1. severe problems were seen in 22% falling to 18% at 1 year. hads demonstrated an anxiety or depression rate of 44%. brief pain inventory identified 14 patients (52%) with ongoing chronic pain. mean self-efficacy was 32/40 (sd +/-6) at baseline and 34/40 (sd +/-5) at 1 year. cardiothoracic intensive care patients have ongoing and persistent features of pics with significant effects on health-related quality of life. further, the ins:pire multi-professional complex intervention is feasible within this specialist group. screening approach might be implemented whenever screening of the total icu population is not deemed feasible. influenza is an acute viral illness with a significant financial burden. point of care testing for influenza is available and has demonstrated accuracy [1, 2] , the current gap in knowledge is the question around the opportunity cost of influenza testing. if poct is financially a less costly test this could free up scarce resource. the study adopts a cost minimisation approach. the point of care test is the roche cobas® liat® machine which can detect flu a/b and is compared with the west of scotland specialist virology centre's established in house multiplex real time pcr assay.the model was developed using microsoft excel and has 2 arms comparing analysis of the above mentioned tests. the model estimates that the total cost of poct per patient tested is £3926.33 compared with £4053.92 for lab testing ( figure 1 ). this is a saving of £127.60 per patient when poct is used. the result swings in favour of the lab test when poct specificity falls to 95.72%. if the lab could provide the result of influenza testing within 12 hours the result would swing in favour of lab testing. zanamivir which will potentially be used increasingly in the intensive care setting can more than double the difference between the 2 tests in favour of poct. this research suggests that poct offers potential cost savings in the icu setting. this is the case as long as poct specificity is higher than a threshold of 97.52% and the lab take longer that 12 hours to return the result. the sensitivity analysis should allow for external validity given the usual variations in icu practice. the aim of the present study is to describe the demographic, clinical, microbiological aspects and the outcome of patients with intensive care unit-related (icu-related) bacteremia. moreover, we aimed to study the patient outcome in association with colistin susceptibility. retrospective, single-center study in a 16-bed icu for 12 months, from 1/10/2018 to 30/9/2019. icu-related bacteremia was defined as bacteremia in patients with icu stay >48 hours or icu readmission (first admission ≥ 1 month before). only the first episode of bacteremia was considered. the primary outcome was 28-day mortality. data regarding clinical, demographic and outcome characteristics were retrieved from the patient files. the hospital's ethics committee approved the present protocol. moreover, the patients with bacteremia due to colistin-resistant pathogens were compared with the patients affected by colistin sensitive microbes. forty episodes of gram-negative icu bacteremia were collected during the aforementioned period in 40 patients (61.5% male) with a mean age and apache ii of 60.6±16.8 years and 18±8.1, respectively. the event had taken place at an average of 13.7 days. the responsible isolates were resistant to carbapenems in 82.5% of the episodes. the majority of the events were due to a single isolate (85%). acinetobacter baumannii and klebsiella pneumoniae presented the majority of the implicated microbes (35% and 37.5%, respectively). the crude 28-day mortality was 30%. finally, we could not detect any difference in mortality between the colistin sensitive and the colistin-resistant pathogens ( figure 1 ). the present study denotes that, in a setting of extremely drugresistant pathogens with limited treatment options, gram-negative bacteremia in the icu is associated with increased mortality. image 1 : characterization of resistance mechanisms affecting ceftolozane/ tazobactam in enterobacterales and pseudomonas aeruginosa icu isolates using whole genome sequencing (step study) m hernández-garcia 1 , cc chaves 2 , jm melo-cristino 3 , ds silva 4 , ar vieira 5 , mp f. pinto 6 , jd diogo 7 , eg gonçalves 8 , jr romano 9 , rc cantón 1 1 hospital ramón y cajal-irycis, microbiology department, madrid, spain; 2 introduction: clostridium difficile infection (cdi) is the main cause of hospital acquired diarrhoea [1] . the aim of this study was to compare characteristics of cdi during yr 2011 and 2015. a retrospective observational study was carried out in lithuanian university of health sciences hospital -the largest teaching facility of tertiary care in country. according to department of infection control records, patients (pt) with (w.) diarrhoea and the first positive stool test for c.difficile toxin a/b were included. age, charlson comorbidity index (cci) score, profile of hospital department (medical (md), surgical or icu) where cdi was diagnosed, type of cdi (healthcare-associated (ha), hospital or community-acquired) and rate of risk factors (rf) have been estimated in both 2011 and 2015. ibm spss 23.0; pearson's chi-square, fisher's exact tests were used for statistics. p < 0.05 was statistically significant. results: in total 7 pt from 2011, 72 from 2015 were enrolled. in 2011 n=4 (57%) pt were ≥65 yr old, in 2015 -n=45 (63%), (p=0.045). in 2011 cci>5 was estimated in n=6 (86%) pt in comparison of n=46 (64%) in 2015, (p=0.025). in 2011 n=0 (0%) of cdi cases were ha, in 2015 -n=12 (17%), (p=0.01). in 2011 n=5 (71%) of cdi were diagnosed in md in comparison of n=60 (83%) in 2015, (p=0.01). in 2011 12 weeks prior to cdi n=5 (71%) pt have been admitted to hospitals, n=7 (100%) have been treated w. antibiotics, n=4 (50%) -w. ppis, n=5 (36%) -w. h2 antagonists, n=3 (43%) -w. immunosupressants in comparison of n=51 (71%), n=69 (96%), n=36 (57%), n=26 (71%) and n=21 (29%) in 2015, respectively, (p>0.05). overall rate of cdi cases among in-hospital patients increased tenfold by yr 2011 and 2015. in 2015, more elderly patients had cdi and severe comorbidities were less frequent in comparison with 2011. in 2015, more cases of cdi were hospital-acquired and have occured in medical departments. rate of risk factors of cdi remained unchanged.these results indicate a possible relationship between ttv dna count and immunological alteration. the ttv quantitative determination could be useful as a proinflammatory marker in sepsis, with some benefits: low cost, easy determination and good correlation with immune system functionalit. it will be necessary to perform a larger study to check our hypothesis and to establish a ttv level threshold that may allow to anticípate the disease prognosis. introduction: acute kidney injury (aki) is a serious complication in sepsis and associated with high morbidity and mortality. the combination antimicrobial regimens with vancomycin (vcm) and broad-spectrum betalactams (bsbl), such as piperacillin tazobactam and cefepime, have been identified as potentially nephrotoxic combinations, but existing studies have not provided sufficient evidence. the aim of this study was to evaluate detailed association between the combination antimicrobial therapy and the risk of aki in septic patients. this investigation was a post hoc analysis of 2 prospective nationwide cohorts enrolling consecutive adult patients with sepsis in intensive care units in japan. in this study, progression of aki was defined as one or more elevation of renal sub-score in sequential organ failure assessment score from day 1 to day 4. we regarded anti-pseudomonal penicillins, fourth generation cephalosporines, and carbapenems as bsbl. multivariable logistic regression analysis including a two-way interaction term (vcm x bsbl) was performed to assess the add-on effects of each antimicrobial agent on the progression of aki. the final study cohort comprised 1837 patients with sepsis. among them, 45 received vcm without bsbl, 1055 received bsbl without vcm, 249 received both vcm and bsbl, and 488 received other type of antimicrobials. the administration of vcm was associated with an increased risk of aki in patients with bsbl [odds ratio (or), 1.57 (0.96-2.57); p=0.072]. however, the tendency was not evident in patients without bsbl [or, 0.23 (0.03-1.56); p=0.133]. the interaction effect on the progression of aki between vcm and bsbl were statistically significant (p for interaction=0.038). the regression model including two-way interaction term suggested that the combination of vcm and bsbl might synergistically increase the risk of aki in patients with sepsis. increasing resistance to carbapenems due to carbapenemase productionone of main actual problems of antibacterial resistance in burn icu. production of several types of carbapenemases (kpc, ndm and oxa-48) is common in k. pneumoniae strains. carbapemenase production is a marker of extreme antibacterial resistance. the aim of our study was to investigate the epidemiology of nosocomial infections caused by producing kpc, ndm and oxa-48 k. pneumonia strains in burn icu. total of 26 patients with nosocomial infections caused by 26 carbapenem resistance strains of k. pneumoniae were included in the study, from whom 3 had lower respiratory tract infection, 23 had skin and skin structure infection. initial identification of isolates was performed in laboratory by automatic microbiological analyzer. for all of k. pneumoniae isolates presence of bla ndm , bla oxa-48 and bla kpcgenes were examined by pcr method. baseline characteristics of patients: me (iqr) of age -50 (39; 64) years, me (iqr) of tbsa -40 (29; 52) percent, me (iqr) of icu los -30 (21; 35) days. inhalation injury was diagnosed in 10 (38.4%) patients. total of 11 patients died, mortality rate was 42.3%. all patients were diagnosed with nosocomial infection caused by k. pneumoniae. from 26 k. pneumonia strains 1 (3.8%) were found to be producing kpc, 3 (11.5%)producing ndm and 20 (76.9%) -producing oxa48. only 5 (19.2%) carbapenem resistance k. pneumoniae isolates were not producing carbapenemases. from 20 patients infected by oxa48 producing k. pneumoniae 20 patients died, mortality rate was 40%. from 23 patients infected by oxa48 or ndm producing k. pneumoniae 10 patients died, mortality rate was 43.5%. from 5 patients infected by non-carbapenemase producing k. pneumonia no one died. carbapenemase producing strains are widely spread among carbapenem resistance strains of k. pneumoniae in burn icu. mortality of patients infected by producing oxa48 or ndm k. pneumoniae strains reaches 43.5%. the rationale for blood purification as adjunctive therapy during sepsis involved the capacity in removing endogenous and exogenous toxins, but currently no recommendations exists [1] . a critical point may be the potential interaction with antimicrobial therapy, which remains the mainstay of sepsis treatment. the aim of our study was to investigate the vancomycin (van) removal during blood purification using an in vitro model of hemoperfusion (hp) with ha330 cartridge (jafron, zhuhai city, china), most widely used in china and actually available in europe. this is an experimental study. three independent experiments were performed: we injected 250 mg of van in 500ml of whole blood from healthy donors (experiment 1 and 2) or in 500ml of balanced solution (experiment 3) in order to assess membrane saturation. a closed-circuit (blood flow of 250ml/min) simulating hp ran using ha330. samples were collected from arterial line at 0, 5, 10, 15, 30, 45, 60, 90, 120 minutes; van plasma concentrations were measured and removal was evaluated using mass balance analysis. differences in mass removal was assessed using kruskal-wallis test. results: figure 1 shows van mass at each timepoints. we observed no difference between in blood and in balanced solution experiments (p the aim of this study is to determine if routine bbv testing in the icu contributes to the discovery of undiagnosed bbv infections. icu patients may require renal replacement therapy (rrt). sharing rrt equipment carries a risk of bbv transmission, which mainly relates to hepatitis b (hbv), hepatitis c (hcv) and hiv. since 2012, all glasgow royal infirmary icu patients undergo routine bbv screening, with rrt machines allocated for patients with specific bbv statuses. routine bbv testing is beneficial to both the individual and society. hcv is a pertinent health issue in scotland. the scottish government aims to eliminate hcv by 2030 and is researching innovative and costeffective methods to identify undiagnosed infections. this single-centre retrospective observational study examined prospectively collected clinical data from 1069 icu admissions. proportions were compared using a two-proportion z-test and a logistic regression model was carried out to determine if deprivation quintile was independently associated with the seroprevalence of bbvs. the bbv seroprevalence in the cohort studied: 0.45% (hbv), 11.7% (hcv), 0.91% (hiv). the seroprevalence of hbv in the cohort studied was similar to that of scotland (p=0.11), but the seroprevalence of hcv (p<0.001) and hiv (p=0.01) were statistically significantly higher than that of scotland. due to the small number of reactive test results for hbv and hiv, the relationship between deprivation and bbv seroprevalence was explored for hcv only. the only independent variable associated with a reactive anti-hcv test result was "current or previous illicit drug use" (adjusted odds ratio of 40.2; 95% confidence interval of 21.1-76.4; p<0.001). this study shows that routine bbv testing in the icu is useful in discovering new bbv infections. this is the first observational study focusing on the value of routine bbv testing in an icu setting to our knowledge. continuous infusion vancomycin protocol is a safe, acceptable and effective alternative to intermittent dosing of vancomycin in critical care. ceftaroline is an efficacious treatment in patients with severe cap, admitted in icu. it relates to earlier resolution of respiratory failure and less rescue antibiotics. we need an adequately pragmatic trial to confirm our findings organ dysfunction in scrub typhus, incidence and risk factor a sarkar 1 , a guha 2 , r dey 3 [1, 2, 3, 4, 5] . its preads by bite of larval stageof thromboculid mites or chigger [1] . clinical features may include fever, headache, myalgia, lymphadenopathy, eschar, skinrash. it may also cause pneumonia, renal failure, shock, meningoencephalitis, multiple organ failure [1, 2] . our study aims to discuss the incidence of organ dysfunction in a comprehensive way taking the overall population of patients with identified scrub typhus infection. there is lack of data in eastern india regarding the incidence and risk factors of developing multiorgan dysfunction syndrome (mods) in scrub typhus. in this retrospective study we studied the incidence of various organ involvement and the risk factors associated with the development of mods in scrub typhus. we collected data from december 2016 to november 2019 in tertiary care hospital at kolkata. we have included all patients who are having fever, scrub typhus igm antibody positive, age more than 14 years. sofa score was used in evaluating patients with mods. exclusion criteria involves patient who are having coinfectional ong with scrub typhus. in a cohort (n=114), patients with multiorgan dysfunction syndrome was seen in 27 patients (23.68%), the mean age in group of patients with mods was 50.0+/-14.96 years (mean+/-sd). in group of patients with mods, fever duration in days was of 11+/-3.58 days (mean+/-sd), interval from treatment to defervescenc in days was 5.11+/-2.39 days (mean +/-sd). among patients with mods, hematologic involvement was seen in 7 patients (25.92%), hepatic involvement was seen in 19 patients (70.37%), renal involvement was seen in 17 patients (62.96%), neurologic involvement was seen in 24 patients (88%), respiratory involvement was seen in 14 patients (51.85%), cardiovascular was seen in 8 patients (29.63%), icu shifting was necessary in 20 patients (74.07%), mechanical intubation was needed in 14 patients (51.85%) in multiorgan dysfunction syndrome patients. hospital mortality in patients with mods was 3 patients (11.11%). no mortality was seen in patients without mods. other parameters were evaluated among patients with mods. they include eschar in 1 patient (3.7%), seizure in 7 patients (25.93%), hepatoslenomegaly in 26 patients (96.3%), leucopenia in 3 patients (11.11%), leucocytosis in 13 patients (48.14%), thromnbocytopenia in 7 patients (25.92%),decreased hemoglobin in 22 patients (81.48%), transaminitis in 19 patients (70.37%). the risk factors associated with the development of mods are platelet counts, bilirubin, transaminitis, glasgow coma scale, time interval from treatment to defervescence, hemoglobin, total leucocyte count and fever duration. scrub typhus is an important cause of acute febrile illness in this part of the country and is frequently associated with organ dysfunction. however, the overall mortality is low which is similar to other studies done before [2] . score at baseline were significant (p<0.05) predictors of mortality.highest area under the roc curve was obtained for number of days with septic shock (0.857) followed by increased cd64 between baseline and day 8 (0.798). though serial pct levels significantly increased amongst non-survivors, it did not predict mortality. serial level of biomarkers in icu patients may predict mortality. larger trials are needed to confirm the results. plasma strem-1 levels were retrospectively measured at day 1-2, 3-4 and 6-8 in 116 septic shock patients from the immunosepsis cohort (nct02803346), included between 01/2016 and 12/2018, using a validated elisa method. the associations between strem-1, mhla-dr, 28-day survival status, and occurrence of icu-acquired nosocomial infection (ni) were assessed. neither strem-1 nor mhla-dr levels at d1/2 were associated with the occurrence of icu-acquired ni. however, 28-day mortality was significantly higher in patients with d1-2 strem-1 value superior to the median (39.6% vs 11.3%, p=0.0103; median=539 pg/ml). a significant inverse correlation was found between mhla-dr at d6-8 and strem-1 at d1-2 (sp -0.378, p<0.0001) and at d6-8 (sp -0.382, p<0.0001). at d6-8, when stratifying patients based on strem-1 (400pg/ml) and mhla-dr (5000 ab/c), patients combining elevated strem-1 and low mhla-dr presented with significantly higher 28day mortality (47.6% vs 8.7%, p = 0.0003, chi-squared test) and ni incidence (31.8 vs 12%, p=0.044) compared with patients with low strem-1 / high mhla-dr. this study shows for the first time that trem-1 pathway activation is associated with septic shock-induced immunosuppression, as shown by an inverse correlation between strem-1 at baseline and mhla-dr expression at d6-8. persisting high strem-1 values and low mhla-dr expression in septic shock patients are significantly associated with higher rate of icu-acquired infection and mortality. introduction: sepsis mortality remains high [1] . the surviving sepsis campaign (ssc) recommends to guide resuscitation on normalization of lactate levels [2] , however this is debated [3] . we have shown that plasma levels of bio-adrenomedullin (bio-adm) were associated with patient outcome during sepsis [4] . we therefore aimed to evaluate the added value of bio-adm to lactate measurement in the adrenoss cohort. this is a post-hoc analysis of the adrenomedullin and outcome in severe sepsis and septic shock (adrenoss) cohort study. the adre-noss study is a prospective observational study conducted in twenty-four centers and included 583 septic patients [4] . we studied the relationship between the association of initial evolution of lactate plasma levels and bio-adm level at 24h and outcome in patients for whom both markers were available at admission and one day later ("24h"). bio-adm levels below 70 pg/ml were considered as low, and high if greater than 70 pg/ml [4] . in patients with high lactate levels (>2 mmol/l) at admission (n=328), lactate normalization (<2 mmol/l) at 24h was associated with better outcome than in patients with persistently high lactate at 24h (28day mortality 15.9% vs 41.9% respectively, hr 3.3 [2.0-5.3], p<0.001) ( figure 1 ). among patients with decreasing lactate, high and low bio-adm levels at 24h identified patients with different outcomes (28day mortality 7% vs 26% for low vs high bio-adm respectively, hr 4.4 [1.6-11.7], p<0.005). high and low bio-adm levels at 24h also differentiated outcome of patients with persistently elevated lactate (hr 4.5 [1.6-12.3], p<0.005). in patients with low initial lactate, neither lactate or bio-adm had no added prognostic. our data suggest that measurement of bio-adm in addition to lactate may help physicians to refine risk stratification and therefore to guide resuscitation during sepsis. the effect of fluid replacement in sepsis, severe sepsis and septic shock in first 24 hrs in clot quality and microstructure s pillai 1 , g davies 2 the inflammatory response in sepsis can lead to a spectrum of coagulation system defects [1] . sepsis and severe sepsis is associated with a hypercoagulable state where the clot microstructure is known to be a tight and highly elastic clot, which is potentially resistant to fibrinolysis ( figure 1 ). conversely, septic shock is associated with a hypocoagulable state where the clot microstructure is loose and structurally weak. the study aim to investigate the effect of fluid resuscitation and replacement in clot microstructure over 24 hours. methods: 100 patients (50 sepsis, 20 severe sepsis and 30 septic shock) were included in the study. all these patients received standard fluid replacement therapy with crystalloids. blood samples were collected at 0 hours, 4 hours and 24 hours. clot microstructure, standard markers of coagulation and inflammatory markers were measured. in sepsis group following fluid administration, the d f reduced initially and then remained stable (1.78-0 hours, 1.74-4 hours, 1.73-24 hours, normal d f range 1.73 ± 0.04). in severe sepsis group, the d f reduced initially, then increased (1.80-0 hours, 1.71-4 hours, 1.76-24 hours) and in septic shock, the df was very low to start with and there were only slight increase with fluid administration (1.66-0 hours, 1.68-4 hours, 1.67-24 hours). the hypercoagulable state and clot quality in both sepsis and severe sepsis group improved with fluid resuscitation, however despite an early improvement in clot quality, ongoing fluid resuscitation resulted in markedly reduced functional clot with very low clot strength and functionality. this study demonstrates that d f as a marker of clot quality and function may have potential in fluid and component replacement in critical illness and injury. this study analyses the prognostic ability of white blood cell count (wbc), neutrophil:lymphocyte ratio (nlr) and c-reactive protein (crp). hypo-and hyperimmune responses have been associated with increased mortality from septic shock [1] . patients with septic shock (sepsis 3.0) admitted to queen elizabeth hospital birmingham, between december 2017 and july 2019 were included. the primary outcome was 90-day mortality. data was tested for normality and presented as median (iqr) and analysed using a mann whitney u test. categorical data was presented as % and analysed using a chi-squared test. a p value of < 0.05 was used to determine significance. a multivariate binary logistic regression analysis was conducted using age, apache ii, charlson comorbidity index, performance status, and initial lactate as covariates. a hosmer lemeshow test of >0.05 indicated good fit. results: 474 patients were admitted with septic shock. the majority (61%) were male, with a median age of 64 (55-75) and a 90-day mortality of 37%. on day 1, wbc was lower in patients who died compared to patients who survived (9 [7] [8] [9] [10] [11] [12] [13] [14] [15] patients who died of septic shock had a lower wbc, nlr and crp response early on compared to survivors. this may represent early immunoparesis that allows infection to propagate unchecked. however, this was not independently associated with mortality when confounding factors were accounted for. a specific metabolite of mitochondriaitaconic acid is formed upon proinflammatory activation. the attempts of various researches to find the itaconic acid in peripherical blood of patients with sepsis were unsuccessful [1] . some phenylcarboxylic acids (phcas) are known to be microbial metabolites and sepsis biomarkers; they also affect the mitochondrial functions [2] . concentrations of phcas (phenyllactic, p-hydroxyphenylacetic, phydroxyphenyllactic acids) and mitochondrial metabolites (succinic, itaconic acids) in 48 serum samples from 8 patients on the 1 st day of diagnosis of sepsis and 35 serum samples from 22 patients with late stages of sepsis (sepsis-3) were measured by gas chromatographymass spectrometry; control group -20 donors. results: itaconic acid was found in low concentrations (0.5-2.3 μm) only at early stage of sepsis. the multiple increase in levels of phcas and mitochondrial metabolites were detected in patients with late stage of sepsis in comparison with early stage and donors, p<0.001. increased succinic acid (up to 100-1000 μm) concentration is the result of succinate dehydrogenase inhibition by microbial metabolism intermediates (phcas), which was confirmed by in vitro experiments in isolated mitochondria (fig.1) . itaconic acid may be a promising marker in early stage of sepsis, which needs to be proved. prediction of severe events in clinical sepsis is challenging. for such prediction we aimed to compare the novel biomarker calprotectin in plasma, with routine biomarkers. in a prospective study, blood samples were collected from consecutive patients who triggered the sepsis alert in the emergency department in our hospital. c-reactive protein (crp), procalcitonin, neutrophils, and lymphocytes were analysed according to routine practice. p-calprotectin was analysed using a specific particle enhanced turbidimetric assay (gentian diagnostics as). the composite endpoint, which was termed severe event, was defined as death or admission to the intensive care unit (icu)/high dependency unit (hdu) within 48 hours from arrival. the study included 367 patients with written informed consent, of whom 335 were considered to have infection (defined as obtained blood culture and subsequent antibiotic therapy for at least 4 days or until discharge or death), and 32 had no infection. seventy-four patients (22%) with infection developed a severe event. mean pcalprotectin was 2.99 mg/l (standard deviation (sd) 2.10) among patients with infection and 2.35 mg/l (sd 2.64) among patients without infection (p=0.02). in patients with infection mean p-calprotectin was 3.81 mg/l (sd 3.18) among those with and 2.75 mg/l (sd 2.50) among those without a severe event (p=0.006). analysis of area under the receiver-operating characteristic (roc) curve for prediction of severe events showed superiority for p-calprotectin compared with procalcitonin and neutrophil-lymphocyte-ratio, both regarding all sepsis alert cases and regarding the patients with infection (p< 0.05 for all comparisons), fig 1. in addition, there was a trend toward superior performance compared to crp (p=0.10 and 0.15). in sepsis alert patients, p-calprotectin was elevated in those who subsequently developed severe events. p-calprotectin was superior to traditional biomarkers for prediction of severe events. introduction: rapid diagnosis of acute infections and sepsis is critical in emergency departments (eds). current tests have slow turnaround times, low sensitivities, and/or signals from contaminant or commensal organisms. empirical antimicrobial treatment may result in severe adverse events and contributes to antimicrobial resistance. diagnostics to distinguish bacterial from viral infections and noninfectious etiologies support clinicians in efforts toward antimicrobial stewardship. in a prospective, non-interventional study in the eds of 6 sites in greece (prompt study nct03295825), we evaluated hostdx sepsis, a host response test for suspected acute infections and suspected sepsis. hostdx sepsis measures 29 human mrna targets and employs advanced machine learning to differentiate patients with bacterial and viral infections, and noninfectious etiologies. adult patients presenting with suspected acute infection and at least one vital sign change were enrolled. whole blood rna was quantified using nano-string ncounter. predicted probabilities of bacterial and viral infection were calculated (bvn-1 algorithm). patients were adjudicated in a retrospective chart review by 3 independent infectious disease specialists blinded to hostdx sepsis results. among 396 patients adjudicated as bacterial (56), viral (45), noninfected (1), or indeterminate (294) the area under the receiver operating characteristics (auroc) of hostdx sepsis for predicting bacterial vs. viral/non-infected patients was 0.92, and auroc for viral vs. bacterial/non-infected patients was 0.87 (fig.1) . our results indicate that hostdx sepsis distinguishes bacterial from viral infections and other etiologies with high accuracy. hostdx sepsis is currently developed as a rapid point-of-care device with a turnaround-time of less than 30 minutes. hostdx sepsis may therefore assist ed doctors in making appropriate treatment decisions earlier, towards the ultimate goal of antimicrobial stewardship. we studied the diagnostic value of a leukocyte deformability assay that rapidly quantifies the immune activation signatures of sepsis in an undifferentiated population of adults presenting to the ed. ed clinicians must balance the benefits of early intervention against the risks of indiscriminate use of resource-intensive interventions. there are no currently available rapid diagnostics with acceptable performance to achieve this balance. we prospectively enrolled adult patients within 5 hours of presentation with signs of suspicion of infection in two eds in the usa. edta-anticoagulated blood was drawn and analyzed using deformability cytometry [1] . procalcitonin (pct) levels were also measured. patients were retrospectively adjudicated for sepsis-3 by physician committee using the entire medical record. diagnostic performance characteristics and receiver operating curves were used to examine the diagnostic performance of the assay as well as pct. of the 190 patients enrolled, 17.4% were adjudicated as septic. the leukocyte deformability assay demonstrated 91% sensitivity, 68% specificity, and 97% negative predictive value for a single cutoff. the auc was 0.86 ( figure 1 ). pct with a cutoff of 0.5 ng/ml had 55% sensitivity, 87% specificity, and 90% negative predictive value. the auc for pct (as continuous variable) was 0.8. the leukocyte deformability assay of immune activation signatures demonstrated superior diagnostic performance for sepsis when compared to pct. the assay's diagnostic performance and rapid turnaround time of 5 minutes may positively impact patient outcomes while minimizing indiscriminate use of valuable resources in the ed. it is already known in literature that high levels of midregional proadrenomedullin (mrproadm) are related with organ disfunction in infections despite of source and pathogens [1] . similarly, microcirculatory impairment has been reported in sepsis. we examine the correlation between microcirculatory disfunction and mrproadm as a sign of early organ failure. we included 20 consecutive adult patients with suspected infection, sepsis or septic shock admitted to our intensive care unit (icu) as first hospital admission with an expected icu stay of > 24hours. mrproadm was measured daily during the first five consecutive days and sublingual microcirculation was assessed with incident dark field (idf) technology at t1, t2, and t5. we collected information on saps ii, apache scores, and sofa score for each timepoint. results: ten patients had septic shock, 5 sepsis and 5 infection. three patients died during icu stay. a mrproadm clearance of 20% or more between t1 and t2 was found associated with the improvement of mfi (mann-whitney u test, median increase 12.35% versus 2.23%, p= 0.005) (figure 1) . a mrproadm >1.42 nmol/l at the icu admission was associated with a worse sofa score at all the timepoint. moreover, mrproadm levels at admission was found significantly related with icu mortality (auc 0.941 [0.819-1]; p=0.017). mrproadm shown no relation with absolute value of mfi. the study shows a good correlation between the clearance of the biomarker and the improvement in mfi. moreover, our results support previous findings on the prognostic value of mrproadm in terms of sofa and icu-mortality. clinical performance of a rapid sepsis test on a near-patient molecular testing platform r brandon 1 , j kirk 2 , t yager 2 , s cermelli 2 , r davis 2 , d sampson 2 , p sillekens 3 , i keuleers 3 , t vanhoey 3 1 immunexpress, seattle, united states; 2 immunexpress, immunexpress, seattle, united states; 3 biocartis nv, biocartis, mechelen, belgium critical care 2020, 24(suppl 1):p481 the purpose of this study was to clinically validate a new, rapid version of the septicyte™ assay on a near-patient testing platform (biocartis idylla™). septicyte™ lab is the first-in-class sepsis diagnostic to gain fda-clearance but has a complex workflow and a turnaround time (tat) of~6 hours. the assay in idylla™ cartridge format is called septicyte™ rapid. septicyte™ lab was translated to the biocartis idylla™ near-patient testing platform and analytically validated. for this study, 0.9ml of peripheral blood paxgene tm solution from previously collected patient samples was pipetted directly into the cartridge and inserted into the idylla™ reader. patients were part of an independent cohort (n=200) from intensive care units located in the usa and europe. septicyte™ rapid results were reported as a septiscore™ between 0 and 10 with higher scores representing higher probability of sepsis. assay performance determined included technician hands-on-time (hot), assay tat, failure rates, and area under roc curve based on comparison to retrospective physician diagnosis. average hot was 2 minutes, and average tat was 65 minutes. clinical samples could be processed immediately with septicyte™ rapid and did not require 2 hour pre-incubation of paxgene blood, greatly improving tat. correlation of septiscore™ values between lab and rapid, based upon a subset of samples run on both platforms, was very high (r 2 >0.97). estimated roc auc performance for discriminating sepsis from non-infectious systemic inflammation (nisi/sirs) was similar to that previously reported for septicyte™ lab. this is the first demonstration of a validated, fully-integrated, rapid, reproducible, near-patient, immune-response sepsis diagnostic, providing actionable results~1 hr, to differentiate sepsis from non-infectious systemic inflammation / sirs. accuracy of septicyte™ for diagnosis of sepsis across a broad range of patients r brandon 1 , k navalkar 2 , d sampson 2 , r davis 2 , t yager 2 1 immunexpress, seattle, united states; 2 immunexpress, immunexpress, seattle, united states critical care 2020, 24(suppl 1):p482 the purpose of the study was to demonstrate sepsis diagnostic performance of the biomarkers of septicyte™ in subjects other than critically ill adults, and in hospital locations other than icu. septicyte™ lab was the first immune-response sepsis diagnostic assay to gain fda-clearance (k163260) and, as part of gaining this clearance, clinical validation was performed on adult patients admitted to intensive care (icu) only [1] . we therefore performed an in silico analysis across a broad range of patients using the septicyte™ host immune response biomarkers and algorithm. peripheral blood gene expression data, including public and private datasets, were chosen based on quality, annotation, and clinical context for the intended use of septicyte™. multiple comparisons were performed within datasets to better understand the diagnostic performance in certain cohorts including healthy subjects. diagnostic performance was determined using area under curve (auc). results: table 1 shows some characteristics of the selected datasets and patients, including number of datasets (n=22) and comparisons (n=55), number of cases (n=2234) and controls (n=2089) used in comparisons, patient category and hospital location. septicyte™ aucs for the three groups of adults, adult / pediatric and pediatric / neonates were 0.88, 0.85, and 0.87 respectively, which is similar to that previously reported (0.82 -0.89) [1] . these results suggest that the septicyte™ signature has diagnostic utility beyond adults suspected of sepsis and admitted to icu. this signature has now been translated to the near-patient testing platform biocartis idylla™ (as septicyte™ rapid) which promises rapid (~1 hour) diagnosis of sepsis in a broad patient population following further validation. introduction: especially extracorporeal cardio pulmonary bypass (cpb) is known to induce severe inflammation. postoperative inflammation is associated with a sepsis like syndrome including endothelial barrier disruption, volume depletion and hypotension. sphingosine-1-phosphate (s1p) is a signaling lipid regulating permeability and vascular tone. in septic humans decreased serum-s1p levels could be identified as marker for sepsis severity. we addressed three main issues: (1) are serum-s1p levels affected by cardiac surgery? (2) are potential alterations of serum-s1p levels related to changes of acute-phase proteins, s1p sources or carrier? (3) is the invasiveness of the surgery a factor that may influence serum-s1p levels? methods: 46 elective major cardiac surgery patients were prospectively enrolled in this study. serum samples were drawn pre-, post-procedure and on day 1 and day 4 after surgery. we analyzed s1pand its potential sources: red blood cells (rbc) and platelets. we further quantified levels of other inflammatory markers and documented other clinical parameters. median serum-s1p levels in all patients before the procedure were 0.77 (iqr 0.61-0.99) nmol/ml. serum-s1p levels decrease after surgery, whereas all other inflammatory markers increase. serum-s1p levels dropped by 58% in the on-pump and 31% in the off-pump group. changes of serum-s1p levels are associated with s1p sources and carriers: albumin, hdl and vwf:ag activity. patients with a full recovery of their serum-s1p levels after surgery compared to their individual baseline presented with a lower sofa score (p>0.05) and shorter icu stay (p<0.05). serum-s1p levels are disrupted by open heart surgery and levels might be negatively affected by endothelial injury or loss of s1p sources. low serum-s1p levels may contribute to prolonged icu stay and worse clinical status. future studies may investigate the beneficial effects of s1p administration during cardiac surgery. the aim of study is to measure and correlate the expression of ncd64, mhla-dr, pct (procalcitonin) and qcrp (quantitative creactive protein) to predict development of sepsis and its outcome. in this tertiary centre based longitudinal cohort study, a total 110 patients were enrolled in whom sepsis was suspected on the basis of clinical diagnosis and supported by lab investigations. they were divided into two groups sepsis/case and non-sepsis/control. disease severity in icu was assessed by sequential organ failure score (sofa). blood samples for routine lab investigations and biomarkers were taken at the time of admission in icu before administration of first dose of antibiotics at time d 0 /d 1. assessment of biomarkers was done simultaneously with tlc at d 0 /d 1 , d 3 and during follow up of patients till their final outcome. there was no significant (p>0.05) mean change in pct, qcrp, sofa, ncd64, mhla-dr from day 1 to day 3, however, mean change was higher among cases than controls.on comparison of mhla-dr between the groups across time periods, mhla-dr was significantly (p=0.0001) lower among septic patients than controls at both day 1 and day 3. all biomarker correctly predicted cases among different percentage of patients with different sensitivity and specificity. there was no significant (p>0.05) association of mortality with the study biomarkers except for pct. in our study, diagnostic value of pct in differentiating sepsis from non-sepsis was similar to ncd64 among all biomarkers studied. no advantage of ncd64 or mhla-dr was found over pct in diagnosis and correlation with disease progression and mortality. introduction: aqp4 is a water channel protein contributing to astrocyte and immune cells migration, blood-brain barrier maintenance and cell survival [1] [2] . aqp4 genetic variants represent biomarkers associating with outcome after traumatic brain injury and intracerebral hemorrhage [3] [4] . linking aqp4 genetic polymorphism to the course of sepsis has not been studied. methods: study cohort included 124 icu patients diagnosed according to sepsis-3 consensus. aqp4 rs11661256 polymorphism was studied by analyzing pcr products in a 2% agarose gel using an aqp4 specific polynucleotide tetraprimer set. data were analyzed by log rank test (medcalc 18.11.3), and odds ratios/hazard ratios were computed. statistical significance was determined by fisher test (ft) or mann-whitney test. results: 23 of 124 sepsis patients had the minor mutation a for snp rs11661256 located within the regulatory 3' region of the aqp4 gene. septic shock occurred more frequently in homozygotic carriers of aqp4 c allele vs. patients with aa or ca genotype: or=3.75 (95%ci: 1.47-9.56), p=0.006 (ft). lethality in septic shock patients, n= 85, significantly increased compared to sepsis patients with no shock, n=39 (82% vs. 20%, p=0.001, ft). maximum sofa values were significantly lower in patients with minor allele a compared to cc carriers of (9.6 vs. 12.0, respectively, p=0.008). in post-surgery group of patients, carriers of ac or aa genotypes had significantly increased survival compared to patients with cc genotypes: chi-square=5.804; hr=0.455 (95%ci: 0.24 -0.863) for lethality; p=0.016 (figure 1) . association of minor allele a of aqp4 snp rs11661256 with survival in sepsis patients seems secondary to linking the snp to decreased development of multiorgan failure and septic shock that contribute to mortality. validation of presepsin as a biomarker of sepsis in comparison to procalcitonin, il-6 and il-8 v chantziara 1 , f kaminari 1 , c sklavou 1 , s fortis 2 , p kogionou 2 , s perez 2 , a efthymiou 1 1 saint savvas hospital, icu, athens, greece; 2 saint savvas hospital, cancer immunology and immunotherapy center, athens, greece critical care 2020, 24(suppl 1):p486 sepsis is an everyday challenge for the intensivist and biomarkers are useful tools for identification and treatment of this syndrome. we sought to validate presepsin as a biomarker of sepsis in comparison to pct(procalcitonin) and interleukins (il-6,il-8). we enrolled 25 patients,18 men and 7 women average age 58 (39.5-74) years old, apache ii 16 (13.5-20.5), saps ii 48(40.5-58.5), sofa 8 (6.5-9). 11 patients were septic on admission (according to surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016), 9 had a septic episode during their hospitalization in the icu while 5 patients never endured sepsis. we measured presepsin, procalcitonin, il-6, il-8 during sepsis and on remission. results: all septic patients had increased values of presepsin, pct, il-6 and il-8 during sepsis with a cutoff value for presepsin 800pg/ml, while the values of these biomarkers were significantly decreased during remission or in comparison to non-septic patients(presepsin p = 0.002, pct p≤ 0.001, il-6 p≤ 0.001, il-8 p= 0.004. all patients who were not septic survived while among septic patients 8 died (40% mortality). presepsin correlated significantly with pct, il-6 and il-8 (p<0.05). presepsin is a valid biomarker of sepsis and correlates significantly with all the other values of pct, il-6 and il-8. clinical sepsis phenotypes are proposed at hospital presentation. these phenotypes, biomarker profiles, and outcomes are not yet reproduced in prospective data. even less is known about the biologic mechanism the drives these distinct groups. thus, we sought to validate clinical phenotypes and to determine markers of innate immunity, coagulation, tolerance and tissue damage in a prospective cohort. we prospectively studied patients with sepsis-3 criteria within 6 hours of presentation at 12 hospitals in pennsylvania (2018-2019) using automated electronic alerts. using 29 clinical variables, we predicted phenotypes (α, β, γ, δ) for each patient using euclidean distance anchored to published seneca phenotype centroids. discarded blood was analyzed in a subset (n=160) for markers of innate immunity (e.g. il-6, il-10), coagulation (e.g antithrombin iii, eselectin), tolerance (e.g. ho-1, igfbp7), and tissue damage (e.g. serum lactate, bicarbonate) results: among 549 patients, α-type was present in 146 (27%), β-type in 140 (25%), γ-type in 168 (31%) and δ-type in 95 (17%, figure 1a ). on average, β-type was older and more comorbid (mean 73, sd 9 yrs; mean elixhauser 4.6, sd 2.2) with renal dysfunction (median creatinine 1.8 [iqr 1.1 -2.9] mg/dl, p<0.01 all). the δ-type had more acidosis (mean hco3 -20.1, sd 4.7 meq/l), higher serum lactate (median 1.8 [iqr 1.0-3.5] mmol/l, p <0.01 both) and inpatient mortality (13%, figure 1b) . the γand δ-type had greater markers of innate immunity and abnormal coagulation (e.g il-6, icam p<0.01 both), while markers of increased tissue damage (lactate) and poor tolerance (ho-1) were present in δ-type, compared to α-type (figure 1c) . the distribution and characteristics of clinical sepsis phenotypes were reproduced in a prospective validation cohort. similar to the seneca study, distinct biomarker profiles of tissue damage, innate immunity and poor tolerance were present for the δ-type. the effect that neoadjuvant chemotherapy and hyperthermic intraperitoneal chemotherapy (hipec) may have in the postoperative kinetics of biomarkers remains unknow. some studies demonstrate that neoadjuvant chemotherapy and hipec do not invalidate the use of inflammatory markers in postoperative patient monitoring, but none have compared biomarkers kinetics between patients who underwent hipec or only cytoreduction surgery. our main purpose was to identify a difference pattern in c-reactive protein (crp). we conducted a single-center observational study from january 2015 to november 2019, including all patients who underwent cytoreductive surgery with or without hipec. crp was measured daily until seven post-operative day. we compared patients with and without hipec. a total of 19 patients were included, 15 were female. mean age was 63 yrs (44-76). no clinical and demographical differences were observed between groups. no documented infection was found. after surgery crp increased markedly in both groups. crp time-course from the day of surgery onwards was significantly different in hipec patients (9.78 ± 3.95 mg/dl vs 14.80 ± 5.63 mg/dl; p=0.035). multiple comparisons between hipec and non hipec patients were performed and crp concentration was significantly different on the 5th and 7th pod (figure 1 ). no differences were found in other biomarkers (leucocytes and platelets) neither in body temperature. after a major elective surgical insult crp levels markedly increase independently of hipec. serum crp time-course showed a higher pattern in hipec patients despite no infection detected. decreased thrombin generation potential is associated with increased thrombin generation markers in sepsis associated coagulopathy d hoppensteadt 1 , f siddiqui 1 , e bontekoe 1 , r laddu 1 , r matthew 2 , e brailovsky 3 , j fareed. introduction: sepsis associated coagulopathy (sac) is commonly seen in patients which leads to dysfunctional hemostasis in which uncontrolled protease generation results in the consumption of clotting factors. the purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sac patients and demonstrate their relevance to thrombin generation markers. baseline citrated blood samples were prospectively collected from 49 patients with sac at the university of utah clinic. citrated normal controls (n=50) were obtained from george king biomedical (overland park, ks). thrombin generation studies were carried out using a flourogenic substrate method. tat and f1.2 were measured using elisa methods (seimens, indianapolis, in) . functional antithrombin levels were measured using a chromogenic substrate method. the peak thrombin levels and auc levels were lower in the sac patients in comparison to higher levels observed in the normal plasma ( table 1 ). the sac group showed much longer lag time in comparison to the normal group. wide variations in the results were observed in these parameters in the sac group. the f1.2 and tat levels in the sac group were much higher in comparison to the normal. the functional antithrombin levels were decreased in the sac group. these results validate that thrombin generation markers such as f1.2 and tat are elevated in patients with sac. however, thrombin generation parameters are significantly decreased in this group in comparison to normal. this may be due to the consumption of prothrombin due to the activation of the coagulation system. thus, persistent thrombin generation with simultaneous consumption of clotting factors such as prothrombin contributes to the consumption coagulopathy observed in sepsis patients. introduction: procalcitonin (pct) is used in the icu as an inflammatory marker to monitor bacterial infections and guide antibiotic therapy. whether pct can predict bacteremia and therefore could prevent expenses attached to bloodcultures is unknown . we investigated whether pct can predict the outcome of blood cultures in the icu and reduce expences. a single centre observational cohort study was performed in a dutch community teaching hospital . adult patients who were staying in the icu and were suspected of bacteremia were included. simultaneously with drawing of blood cultures, samples for pct measurement were obtained. expenses for pct measurement and bloodcultures were calculated. in the study period of one year, a total of 120 patients were included. three patients were excluded because of incomplete data. out of the 117 included patients, ten patients had positive blood cultures. there was a significant difference in pct levels between patients who had positive bloodcultures versus patients with negative bloodcultures (8.01 ng/ml vs 0.71 ng/ml) ( figure 1 ). the negative predictive value for negative blood cultures is 97% when pct is below 2ng/ml, there was no difference in crp levels between the two groups (148 mg/l vs 179 mg/l, p= 0.83).a set of negative blood cultures in our centre costs 35 euros. positive blood cultures however costs significantly more depending on the micro-organisms found. pct only costs 8.50 euros per measurement. so when blood cultures are omitted when the pct level is below 2ng/ml, a cost reduction of 38% can be achieved. a pct value below 2 ng/ml is a good predictor of a negative blood cultures in icu patients suspected of bacteremia. pct guided bloodculture management in these patients could lead to a significant cost reduction introduction: level of cfdna in plasma is a promising prognostic candidate biomarker in critical illness [1] . oxidized cfdna (ocfdna) have not been studied as a biomarker although its functional role in cellular stress have attracted attention of researches [2] . the goal of our study was to assess the early prognostic value of plasma cfdna/ocfdna for sepsis in a nicu setting. the cohort included 115 nicu patients diagnosed with stroke, intracerebral hemorrhage (ich), anoxia, encephalopathy. cfdna was isolated from day 1 plasma and stained with picogreen. oxidized dna was determined using dna immunoblotting with anti-8-oxo-desoxiguanosine antibodies. genotyping of allelic variants of the tlr9 rs352162 gene was performed using a pcr and designed allele-specific tetraprimers followed by electrophoretic separation of the products statistics was performed by the fisher test and mann-whitney test. results: sepsis was diagnosed by sepsis-3 criteria in 35 patients (30.4%). average nisu staying was 8,8±11,1 days. circulating dna plasma levels on day 1 predicted the future sepsis development (figure 1 ): or for cfdna was 7.54 (95%ci: 3.03-18.76), p<0.001; or for ocfdna was 5.57 (95%ci: 1.64-18.97), p=0.008. power of both performed tests with alpha=0.05: 1.0. log rank test demonstrated better predictive value of cfdna vs. ocfdna (figure) . concentrations of cfdna, but not ocfdna, on day 1 significantly positively correlated with maximum sofa values during hospitalization, day 3 and pre-outcome leukocyte count and neutrophil-to-lymphocyte ratios in a limited cohort of nisu patients with tlr9 rs352162 cc genotype and not in other patients with genotype tlr9 ct+tt. increased level of plasma cfdna better then ocfdna predicts sepsis development in nisu. further studies are warranted to clarify the fig. 1 (abstract p490) . pct values in patients with positive blood cultures and patients with negative blood cultures possible utility of tlr9 rs352162 polymorphism determining for sepsis risk stratification early on nisu admittance. admission was related with higher severity of illness and extension of icu stay for all groups. reduced cbt fluctuations upon icu admission was found to more severely ill patients with worse clinical outcomes, while the more periodic cbt patterns were correlated with high cbt rhythmicity and better outcome. the impact of sex on sepsis incidence and mortality have been elucidated in previous studies, and sex is increasingly recognized as one key factor in sepsis [1] . some studies indicate that women have better immunologic responses to infections [2] . later investigations assume this advantage is linked to immune modulating genes located on the x-chromosome [3] . the purpose of this study is to reveal sex differences in incidence of and mortality of sepsis in a large population-based cohort. methods: 64049 adult participants in the hunt2 study (1995-97) were followed from inclusion through end of 2011. incident bloodstream infections (bsi) from all local and regional hospitals in nord-trøndelag county were identified through linkage with the mid-norway sepsis register, which includes prospectively registered information on bsi used as a specific indicator of sepsis. we estimated age-adjusted cumulative incidence of first-time bsi and compared the risk of a first-time bsi and bsi mortality in men and women using age-adjusted cox proportional hazard regression. during a median follow-up of 14.8 years 1840 individuals experienced at least one episode of bsi, and 396 died within 30 days after a bsi. cumulative incidence and cumulative mortality curves are shown in fig. 1a introduction:the proportion of hospital-acquired infections (hai) among sepsis patients is unknown in germany. systematic differences in hai foci between sepsis patients with and without icu treatment are insufficiently described. retrospective cohort study based on nationwide health claims data of the german statutory health insurance aok. incident inpatient sepsis cases were identified in 2013/2014 among insured persons >15y without preceding sepsis in 24 months prior to index hospitalization. sepsis was defined according to explicit sepsis icd-10-codes (incl. severe sepsis/septic shock). hai were defined based on specific icd-10-codes for surgical site infection, catheterintroduction: elevated renin is associated with an increased risk of death in patients with vasodilatory shock (vs). recent data show that patients with vs and elevated renin levels have improved survival when treated with angiotensin ii (ang ii) + standard care (sc) vs placebo + sc. patients with acute respiratory distress syndrome (ards) can develop angiotensin-converting enzyme (ace) defects that can lead to elevated renin levels and insufficient endogenous ang ii production. we hypothesized that patients with severe ards and elevated renin shock would have improved survival when treated with ang ii + sc vs placebo + sc. in the randomized, placebo-controlled, double-blind athos-3 study, 321 patients with severe vs receiving >0.2 μg/kg/min of norepinephrine or the equivalent were randomized to intravenous ang ii (n= 163) or placebo (n=158). in a post hoc analysis, we assessed the subset of patients with elevated renin (defined as a renin level greater than the median value of the overall athos-3 population) and ards (defined by a pao2/fio2 ratio <300) at the time of randomization. survival to 28 days was compared between the ang ii group (n=41) and the placebo group (n=61). in patients with elevated renin and ards, baseline age, acute physiology and chronic health evaluation ii score, and blood pressure were similar in the ang ii and placebo groups. the median serum renin level was 459.5 pg/ml (iqr: 285.8-1036.0) compared to the normal range for serum renin: 5-58 pg/ml. a significantly higher proportion of patients receiving ang ii survived to day 28 compared to those in the placebo group (51% vs 31%; p=0.01). elevated renin identified patients with vs and ards who were most likely to gain a survival benefit from ang ii. elevated renin is likely caused by an ace defect and may describe an important subset of patients with a biotype that responds well to ang ii therapy. introduction: elevated renin levels have been shown to be associated with an increased risk of death and more severe acute kidney injury (aki) in patients with vasodilatory shock (vs). recent data show that patients with vs and elevated renin levels have improved survival when treated with angiotensin ii (ang ii) + standard care (sc) vs placebo (pbo) + sc. we hypothesized that vs patients with severe aki and elevated renin levels would have improved survival and enhanced renal recovery with ang ii treatment. in the randomized, pbo-controlled, double-blind athos-3 study, 321 patients with severe vs received >0.2 μg/kg/min of norepinephrine or the equivalent and were randomized to intravenous ang ii + sc (n=163) or pbo + sc (n=158). in a post hoc analysis, we assessed the subset of patients with elevated renin (defined as a renin level greater than the median value of the overall athos-3 population) and severe aki (defined as those with aki requiring renal replacement therapy [rrt] at baseline). survival and renal recovery were assessed in patients treated with ang ii + sc (n=45) and pbo + sc (n=60). in patients with elevated renin and severe aki, baseline age, acute physiology and chronic health evaluation ii score, and blood pressure were similar between ang ii + sc vs pbo + sc. the median baseline serum renin level in the whole group was 352.5 pg/ml (iqr: 115.9-785.4; normal range for serum renin: 5-58 pg/ml). a significantly higher proportion of patients receiving ang ii + sc vs pbo + sc survived to day 28 (43% vs 22%, respectively; p=0.03). ang ii recipients also had a higher rate of discontinuation from rrt by day 7 (43% vs 12%; p=0.04). in this study, elevated-renin shock patients with aki treated with ang ii + sc gained a survival benefit and earlier discontinuation from rrt compared to those receiving pbo + sc. elevated renin is likely caused by an angiotensin-converting enzyme defect and may identify those patients with a biotype that responds well to ang ii therapy. most clinical trials conclude the ineffective use of anticoagulation for sepsis-induced coagulopathy [1] . however, post hoc analyses of randomized control trials report positive results [2] , suggesting anticoagulation is effective in specific populations exhibiting coagulopathy. further, anticoagulants should be administered in the early phase [3] ; however, methods for precisely predicting the progression of sepsis-induced coagulopathy are not established. this study aimed to create and evaluate a prediction model of coagulopathy progression using machine-learning techniques. we performed a subgroup analysis of data from a retrospective cohort study involving adult septic patients in 40 japanese institutions from january 2011 to december 2013 and used the japanese association for acute medicine disseminated intravascular coagulation (dic) score as a dic severity index test. the predictive ability of δdic ([dic score on day 3] -[dic score on day 1]) was evaluated using various statistical methods. using variables available at the outset, we compared the predictive ability of random forest (rf) and support vector machine (svm) with that of multiple linear regression analysis. a total of 1110 adults with sepsis were included in the analysis. the root mean square error in δdic score for the multiple linear regression analysis model was 2.1168 compared with values of 1.6508 and 1.9394 for rf and svm, respectively. thus, the rf method predicted the progression of sepsis-induced coagulopathy more accurately than multiple linear regression analysis. conclusions: rf, a machine-learning technique, was superior to multiple linear regression analysis in predicting the progression of sepsis-induced coagulopathy. this prediction model might enable us to use anticoagulation in an early phase. this study examined the efficacy and safety of landiolol, an ultrashort-acting β1-blocker, for treating sepsis-related tachyarrhythmia, according to patient background characteristics. the j-land 3s study (japiccti-173767) was conducted in patients with sepsis, diagnosed according to the sepsis-3 criteria, and tachyarrhythmia (atrial fibrillation, atrial flutter, or sinus tachyarrhythmia). the patients had a mean heart rate of ≥100 beats/min and required catecholamine administration to maintain a mean blood pressure of ≥65 mmhg. the efficacy endpoint was the percentage of patients whose heart rate could be controlled within 60-94 beats/min at 24 h of registration. the safety endpoint was the incidence of adverse events within 168 h of registration. subgroup analyses of efficacy and safety were performed after stratifying the patients according to various patient background characteristics. a total of 151 patients were randomized, 76 to landiolol and 75 to the control group. the efficacy endpoint, percentage of patients with a heart rate of 60-94 beats/min at 24 h of registration, was significantly higher in the landiolol group (54.7% vs 33.3%; mantel-haenszel test: p = 0.0031). the incidence of adverse events was 63.6% and 59.5% in the landiolol and control groups, respectively, and there was no difference between the two groups. most adverse events were related to sepsis or septic shock. the subgroup analyses showed that no patient background characteristic clearly affected the efficacy and safety of landiolol. landiolol is a well tolerated and effective therapeutic agent for controlling heart rate in patients with sepsis-related tachyarrhythmias; its safety and efficacy were not affected by the patient background characteristics investigated. tissue oxygenation monitoring in sepsis r marinova, at temelkov umhat alexandrovska, anesthesiology and intensive care, sofia, bulgaria critical care 2020, 24(suppl 1):p502 near-infrared spectroscopy (nirs) was proposed as a concept in the end of 20th century. this method offers noninvasive monitoring of oxy-and deoxyhemoglobin in tissues.nirs could be measured on the thenar or forehead within few santimeters of the skin. it was first applied as a monitoring in cardiovascular surgery. patients with sepsis have changes in the microcirculation which are important target for therapy. invasive monitoring of oxygen delivery and consumption has been used in patients with sepsis but as every invasive technique such a monitoring hides risks. nirs offers a noninvasive method for tissue oxygenation monitoring (sto2) and could be useful in patients with sepsis and septic shock. the aim of the study is to compare noninvasive tissue oxygenation monitoring with hemodinamic monitoring and lactate values in patients with sepsis methods:the study includes 19 critically ill patients in icu of umhat alexandrovska, sofia. 10 of the patients fullfil the criteria for septic state. the other 9 patients do not have sepsis. in both group of patients are measured tissue oxygenation with invios monitor, mean arterial pressure, oxygen saturation in mixed venous blood and lactate values during 72 h after icu admission. patients with sepsis are reported with significantly lower values of tissue oxygenation, compared to patients without sepsis. the values of tissue oxygenation correlate well with the mixed venous blood oxygenation, mean arterial pressure and lactate values but not significantly with apache scores. conclusions: nirs when used for tissue oxygenation monitoring correlates well with the hemodinamic monitoring and lacate values in patients with sepsis and could be used as an noninvasive monitoring for guiding teurapeutic strategies. tissue oxygenation monitoring has no linear correlation with the severity of illness in patients with sepsis and could not be reccomended as a guidance in the early ressuscitating stage of sepsis. further investiganions in these field are needed.the sequenom´s massarray platform and a recessive inheritance model was selected (cc vs tt/ct). the possible association between the cc recessive form of the rs279451 polymorphism and the septic shock risk was analyzed, demonstrating a statistically significant relationship (p=0.02) between both conditions. among patients who developed septic shock, 79.2% presented a recessive inheritance pattern while 54.5% showed the ct/tt genotype. on the other hand, those patients with the recessive form of the rs279451 polymorphism were selected and a statistical analysis was performed comparing those patients who developed septic shock from those who did not develop it, obtaining a statistically significant relationship (p=0.036) between the presence of the recessive form of polymorphism and the likelihood of developing septic shock. the recessive form of rs279451 polymorphism is a risk factor for septic shock in post-operative patients of major abdominal surgery. introduction: sepsis remains one of the major causes of morbidity with mortality rates as high as 50 % worldwide, representing significant clinical challenge to confront highly intangible therapeutic needs. rnabased structures are emerging as versatile tools encompassing a variety of functions capable to bypass the current protein-and cellbased therapies. rna aptamers act as disease-associated protein antagonists. here, the effects of an aptamer, apta-1, were evaluated in animal models that mimic systemic inflammation in humans. high dose of lps endotoxin was used to induce systemic inflammation in mice and in non-human primate animal models. apta-1 was administered intravenously in two doses post lps infection. animals were monitored and blood samples collected up to 72 hours after apta-1 administration. healthy-and lps-only treated animals served as control groups. complex analyses of clinical parameters, hematology, serum biochemistry, inflammation and tissue damage markers were performed. results: apta-1 increased survival of endotoxin challenged animals up to 80% in a dose-dependent manner and exerted profound effects on wellbeing and recovery of healthy eating habits. administration of apta-1 led to delayed coagulation and enhanced fibrinolysis; maintained the complement cascade activated while preventing it from further amplification. expression of pro-inflammatory cytokines was reduced while anti-inflammatory increased. endogenous pro-inflammatory molecules (damps), secreted from injured cells, were preserved at healthy level in animals treated with apta-1. systemic inflammation and sepsis lead to severe dysregulation of several arms/axis of innate immune response. our studies showed that apta-1 affects various components of this system and restores the organism's control over its dysregulated immune response. thus, apta-1 might be a promising potential therapeutic candidate to treat life-threatening conditions such sepsis. several preclinical studies demonstrated beneficial effects for methane (ch 4 ) administration in various inflammatory conditions. our aim was to investigate the consequences of post-treatment with inhaled ch 4 in a clinically relevant intra-abdominal sepsis model. anesthetized minipigs were subjected to fecal peritonitis (0.6 g/kg, 5-9x10 6 cfu i.p.; n=22) or sham-operation (sterile saline i.p; n=5). invasive hemodynamic monitoring with blood gas analyses was started between 16-24 hours, organ dysfunction parameters (pao 2 /fio 2 ratio; mean arterial pressure; lactate, bilirubin, creatinine; urine output and platelet counts) were determined according to a modified porcinespecific sequential organ failure assessment (ps-sofa) score system, the perfusion rate (pr) of sublingual microcirculation was measured by incident dark field illumination imaging. the animals were divided into non-treated septic or septic shock groups (n=6-6) and ch 4treated septic or septic shock (n=5-5) subgroups, ch 4 inhalation started from the 18th hr (2.2% ch 4 in normoxic air; 500 ml/min). despite the standardized induction, heterogeneous severity of organ damage was evolved. in septic and septic shock groups the median values of ps-sofa score reached 5 (4.75-5.65) and 13 (11.75-14), respectively. septic shock was characterized by significant elevations of creatinine and bilirubin levels, while the platelet count decreased (from 332 to 76 *10 9 /l). inhalation of ch 4 increased the sublingual pr by 22% in the septic group, the creatinine and bilirubin levels were decreased by 28% and 80%, respectively. ch 4 post-treatment significantly decreased the ps-sofa score (to 1; 0.5-2.75) and resulted in lower values in septic shock group (to 10; 9.5-12.4). methane post-treatment effectively influences sepsis-related end organ dysfunction. up to a severity threshold it may be a promising additional organ protective tool. evaluation of sepsis awareness among various groups in turkey: a survey study s erel, o ermis, ö nadastepe, l karabıyık gazi university school of medicine, anesthesiology and intensive care, ankara, turkey critical care 2020, 24(suppl 1):p510 introduction: sepsis is a common life-threatening condition in critically ill patients [1] . public awareness is important for early recognition of sepsis and improvement of outcomes [2] . we aimed to evaluate sepsis awareness among different groups of people. methods: prospective paper-based surveys were issued between 1st july and 1st august 2019 to patients, the relatives of the patiens, hospital staff and general public who gave consent to participate in the study. the questionnaire included ten questions about demographic informations, occupational informations of hospital stuff and sepsis awareness. a total of 588 participated in the survey. of these participants, 87 (14.3%) were patients, 50 (8.5%) were relatives of patients, 134 (22.8%) were physicians, 125 (21.3%) were medical students, 49 (8.3%) were nurses, 51 (8.7%) were other hospital stuff and 92 (%15.6) were other people. of these participants, 425 (72.3%) had heard of the word "sepsis". 206 (35.0%) responded correctly regarding the definition of sepsis. 325 (55.3%) of the participants heard the word "sepsis" during their education, but only 53 (9%) heard it through the media. in the groups of high school graduates, university graduates and postgraduates, the rate of hearing the word sepsis and correctly identifying sepsis is significantly higher than the primary school graduates or illiterate groups. (p<0.05). physicians, nurses and medical students were heard of the word "sepsis" significantly more than other groups (p<0.005). physicians and medical students responded more accurately to the definition of sepsis than other groups (p<0.05). public awareness of sepsis is limited compared to healthcare workers. increasing public knowledge of sepsis through education and through media may contribute to raising public awareness and improving outcomes. the association between clinical phenotype cohesiveness and sepsis transitions after presentation jn kennedy 1 , eb brant 1 , km demerle 2 , ch chang 3 , s wang 4 , dc angus 1 , cw seymour 5 1 key: cord-026031-hnf5vayd authors: ford, richard b.; mazzaferro, elisa m. title: emergency care date: 2009-05-21 journal: kirk and bistner's handbook of veterinary procedures and emergency treatment doi: 10.1016/b0-72-160138-3/50002-3 sha: doc_id: 26031 cord_uid: hnf5vayd nan in the event that you suspect peritonitis and have a negative tap with abdominal paracentesis, a diagnostic peritoneal lavage can be performed. to perform abdominal paracentesis, follow this procedure: 1. place the patient in left lateral recumbency and clip a 4-to 6-inch square with the umbilicus in the center. 2. aseptically scrub the clipped area with antimicrobial scrub solution. 3. wearing gloves, insert a 22-or 20-gauge needle or over-the-needle catheter in four quadrants: cranial and to the right, cranial and to the left, caudal and to the right, and caudal and to the left of the umbilicus. as you insert the needle or catheter, gently twist the needle to push any abdominal organs away from the tip of the needle. local anesthesia typically is not required for this procedure, although a light sedative or analgesic may be necessary if severe abdominal pain is present. in some cases, fluid will flow freely from one or more of the needles. if not, gently aspirate with a 3-to 6-ml syringe or aspirate with the patient in a standing position. avoid changing positions with needles in place because iatrogenic puncture of intraabdominal organs may occur. 4. save any fluid collected in sterile red-and lavender-topped tubes for cytologic and biochemical analyses and bacterial culture. monitor hemorrhagic fluid carefully for the presence of clots. normally, hemorrhagic effusions rapidly become defibrinated and do not clot. clot formation can occur in the presence of ongoing active hemorrhage or may be due to the iatrogenic puncture of organs such as the spleen or liver. if abdominal paracentesis is negative, a diagnostic peritoneal lavage can be performed. peritoneal dialysis kits are commercially available but are fairly expensive and often impractical. to perform a diagnostic peritoneal lavage, follow this procedure: 1. clip and aseptically scrub the ventral abdomen as described previously. 2. wearing sterile gloves, cut multiple side ports in a 16-or 18-gauge over-the needle catheter. use care to not cut more than 50% of the circumference of the catheter, or else the catheter will become weakened and potentially can break off in the patient's abdomen. 3. insert the catheter into the peritoneal cavity caudal and to the right of the umbilicus, directing the catheter dorsally and caudally. 4. infuse 10 to 20 ml of sterile lactated ringer's solution or 0.9% saline solution that has been warmed to the patient's body temperature. during the instillation of fluid into the peritoneal cavity, watch closely for signs of respiratory distress because an increase in intraabdominal pressure can impair diaphragmatic excursions and respiratory function. 5. remove the catheter. 6. in ambulatory patients, walk the patient around while massaging the abdomen to distribute the fluid throughout the abdominal cavity. in nonambulatory patients, gently roll the patient from side to side. 7. next, aseptically scrub the patient's ventral abdomen again, and perform an abdominal paracentesis as described previously. save collected fluid for culture and cytologic analyses; however, biochemical analyses may be artifactually decreased because of dilution. remember that you likely will retrieve only a small portion of the fluid that you instilled. during the early stage of repair, granulation tissue, some exudate, and minor epithelialization is observed. place a nonadherent bandage with some antibacterial properties (petroleum or nitrofurazone-impregnated gauze) or absorbent material (foam sponge, hydrogel, or hydrocolloid dressing) in direct contact with the wound to minimize disruption of the granulation tissue bed. next, place an absorbent intermediate layer, followed by a porous outer layer, as previously described. granulation tissue can grow through gauze mesh or adhere to foam sponges and can be ripped away at the time of bandage removal. hemorrhage and disruption of the granulation tissue bed can occur. later in the repair process, granulation tissue can exude sanguineous drainage and have some epithelialization. a late nonadherent bandage is required. the contact layer should be some form of nonadherent dressing, foam sponge, hydrogel, or hydrocolloid substance. the intermediate layer and outer layers should be absorbent material and porous tape, respectively. with nonadherent dressings, wounds with viscous exudates may not be absorbed well. this may be advantageous and enhance epithelialization, provided that complications do not occur. infection, exuberant granulation tissue, or adherence of absorbent materials to the wound may occur and delay the healing process. moist healing is a newer concept of wound management in which wound exudates are allowed to stay in contact with the wound. in the absence of infection a moist wound heals faster and has enzymatic activity as a result of macrophage and polymorphonuclear cell breakdown. enzymatic degradation or "autolytic debridement" of the wound occurs. moist wounds tend to promote neutrophil and macrophage chemotaxis and bacterial phagocytosis better than use of wet-to-dry bandages. a potential complication and disadvantage of moist healing, however, is the development of bacterial colonization, folliculitis, and trauma to wound edges that can occur because of the continuously moist environment. use surfactant-type solutions (constant clens; kendall, mansfield, massachusetts) for initial wound cleansing and debridement. use occlusive dressings for rapid enzymatic debridement with bactericidal properties to aid in wound healing. bandage wet necrotic wounds with a dressing premoistened with hypertonic saline (curasalt [kendall] , 20% saline) to clean and debride the wounds. hypertonic saline functions to desiccate necrotic tissue and bacteria to debride the infected wound. remove and replace the hypertonic saline bandage every 24 to 48 hours. next, place gauze impregnated with antibacterial agents (kerlix amd [kendall] ) over the wound in the bandage layer to act as a barrier to bacterial colonization. if the wound is initially dry or has minimal exudate and is not obviously contaminated or infected, place amorphous gels of water, glycerin, and a polymer (curafil [kendall] ) over the wound to promote moisture and proteolytic healing. discontinue moisture gels such as curafil once the dry wound has become moist. finally, the final stage of moist healing helps to promote the development of a healthy granulation tissue bed. use calcium alginate dressings (curasorb or curasorb zn with zinc [kendall] ) in noninfected wounds with a moderate amount of drainage. alginate gels promote rapid development of a granulation tissue bed and epithelialization. foam dressings also can be applied to exudative wounds after a healthy granulation bed has formed. change foam dressings at least once every 4 to 7 days. for closed wounds without any drainage, such as a laceration that has been repaired surgically, a simple bandage with a nonadherent contact layer (telfa pad [kendall] , for example), intermediate layer of absorbent material, and an outer porous layer (elastikon, vetrap) can 1 be placed to prevent wound contamination during healing. the nonadherent pad will not stick to the wound and cause patient discomfort. because there usually is minimal drainage from the wound, the function of the intermediate layer is more protective than absorptive. any small amount will be absorbed into the intermediate layer of the bandage. it is important in any bandage to place the tape strips or "stirrups" on the patient's limb and then overlap in the bandage, to prevent the bandage from slipping. place the intermediate and tertiary layers loosely around the limb, starting distally and working proximally, with some overlap with each consecutive layer. this method prevents excessive pressure and potential to impair venous drainage. leave the toenails of the third and fourth digits exposed, whenever possible, to allow daily examination of the bandage to determine whether the bandage is impairing venous drainage. if the bandage is too tight and constricting or impeding vascular flow, the toes will become swollen and spread apart. when placed and maintained properly (e.g., the bandage does not get wet), there usually are relatively few complications observed with this type of bandage. in some cases, it is necessary to cover a wound in which a penrose drain has been placed to allow drainage. in many cases, there is a considerable amount of drainage from the drain and underlying soft tissues. the function of the bandage is to help obliterate dead space created by the wound itself, absorb the fluid that drains from the wound and that will contaminate the environment, and prevent external wicking of material from the external environment into the wound. when the bandage is removed, the clinician can examine the amount and type of material that has drained from the wound in order to determine when the drain should be removed. when placing a bandage over a draining wound, the contact layer should be a commercially available nonadherent dressing and several layers of absorbent wide-mesh gauze placed directly over the drain at the distal end of the incision. overlay the layers of gauze with a thick layer of absorbent intermediate dressing to absorb fluid that drains from the wound. if the gauze and intermediate layers are not thick or absorbent enough, there is a potential for the drainage fluid to reach the outer layer of the bandage and provide a source of wicking of bacteria from the external environment into the wound, leading to infection. some wounds such as lacerations have minor bleeding or hemorrhage that require an immediate bandage until definitive care can be provided. to create a pressure bandage, place a nonadherent dressing immediately in contact with the wound, followed by a thick layer of absorbent material, topped by a layer of elastic bandage material such as elastikon or vetrap. unlike the bandage for a closed wound, the top tertiary outer layer should be wrapped with some tension and even pressure around the limb, starting from the distal extremity (toes) and working proximally. the pressure bandage serves to control hemorrhage but should not be left on for long periods. pressure bandages that have been left on for too long can impair nerve function and lead to tissue necrosis and slough. therefore, pressure bandages should be used in the hospital only, so that the patient can be observed closely. if hemorrhage through the bandage occurs, place another bandage over the first until the wound can be repaired definitively. removal of the first bandage will only disrupt any clot that has formed and cause additional hemorrhage to occur. fractures require immediate immobilization to prevent additional patient discomfort and further trauma to the soft tissues of the affected limb. as with all bandages, a contact layer, intermediate layer, and outer layer should be used. place the contact layer in accordance 1 with any type of wound present. the intermediate layer should be thick absorbent material, followed by a top layer of elastic bandage material. an example is to place a telfa pad over a wound in an open distal radius-ulna fracture, followed by a thick layer of cotton gauze cast padding, followed by an elastic layer of kling (johnson & johnson medical, arlington, texas) , pulling each layer tightly over the previous layer with some overlap until the resultant bandage can be "thumped" with the clinician's thumb and forefinger and sound like a ripe watermelon. the bandage should be smooth with consecutive layers of even pressure on the limb, starting distally and working proximally. leave the toenails of the third and fourth digits exposed to monitor for impaired venous drainage that would suggest that the bandage is too tight and needs to be replaced. finally, place a top layer of vetrap or elastikon over the intermediary layer to protect it from becoming contaminated. if the bandage is used with a compound or open fracture, drainage may be impaired and actually lead to enhanced risk of wound infection. bandages placed for initial fracture immobilization are temporary until definitive fracture repair can be performed once the patient's cardiovascular and respiratory status are stable. wounds with exuberant granulation tissue must be handled carefully so as to not disrupt the healing process but to keep an overabundance of tissue from forming that will impair epithelialization. to bandage a wound with exuberant granulation tissue, place a corticosteroid-containing ointment on the wound, followed by a nonadherent contact layer. the corticosteroid will help control the exuberant growth of granulation tissue. next, carefully wrap an absorbent material over the contact layer, followed by careful placement of and overlay of elastic bandage material to place some pressure on the wound. leave the toenails of the third and fourth digits exposed so that circulation can be monitored several times daily. bandages that are too tight must be removed immediately to prevent damage to neuronal tissue and impaired vascularization, tissue necrosis, and slough. because wound drainage may be impaired, there is a risk of infection. gaping wounds or those that have undermined in between layers of subcutaneous tissue and fascia should be bandaged with a pressure bandage to help obliterate dead space and prevent seroma formation. an example of a wound that may require this type of bandage is removal of an infiltrative lipoma on the lateral or ventral thorax. use caution when placing pressure bandages around the thorax or cervical region because bandages placed too tightly may impair adequate ventilation. to place a pressure bandage and obliterate dead space, place a nonadherent contact layer over the wound. usually, a drain is placed in the wound, so place a large amount of wide-mesh gauze at the distal end of the drain to absorb any wound exudate or drainage. place several layers of absorbent material over the site to further absorb any drainage. place a layer of elastic cotton such as kling carefully but firmly over the dead space to cause enough pressure to control drainage. place at least two fingers in between the animal's thorax and the bandage to ensure that the bandage is not too tight. in many cases, the bandage should be placed once the animal has recovered from surgery and is able to stand. if the bandage is placed while the animal is still anesthetized and recumbent, there is a tendency for the bandage to be too tight. finally, the tertiary layer should be an elastic material such as elastikon or vetrap. many wounds require a pressure relief bandage to prevent contact with the external environment. wounds that may require pressure relief for healing include decubitus ulcers, pressure bandage or cast ulcers, impending ulcer areas (such as the ileum or ischium of recumbent or cachexic patients), and surgical repair sites of ulcerated areas. pressure relief bandages can be of two basic varieties: modified doughnut bandage and doughnut-shaped bandage. to create a cup or clamshell splint, follow this procedure (figures 1-7 to 1-11): 1. place a nonadherent contact layer directly over the wound. 2. place stirrups of tape in contact with the skin of the dog, to be placed over the intermediate layer and prevent the bandage from slipping. 3. place a fairly thick layer of absorbent intermediate bandage material over the contact layer such that the bandage is well-padded. pull the tape stirrups and secure them to the intermediate layer. 4. place a length of cast material that has been rolled to the appropriate length, such that the cast material is cupped around the patient's paw, and lies adjacent to the caudal aspect of the limb to the level of the carpus or tarsus. in the case of a clamshell splint, place a layer of cast material on the cranial and caudal aspect of the paw and conform it in place. 5. take the length of cast padding and soak it in warm water after it has been rolled to the appropriate length. wring out the pad, and secure/conform it to the caudal (or cranial and caudal, in the case of a clamshell splint) aspect of the distal limb and paw. 6. secure the cast material in place with a layer of elastic cotton gauze (kling). 7. secure the bandage in place with a snug layer of elastikon or vetrap. short or long splints made of cast material can be incorporated into a soft padded bandage to provide extra support of a limb above and below a fracture site. for a caudal or lateral splint to be effective, it must be incorporated for at least one joint above any fracture site to prevent a fulcrum effect and further disruption or damage to underlying soft tissue structures. a short lateral or caudal splint is used for fractures and luxations of the distal metacarpus, metatarsus, carpus, and tarsus. to place a short lateral or caudal splint, follow this procedure: 1. secure a contact layer as determined by the presence or absence of any wound in the area. 2. place tape stirrups on the distal extremity to be secured later to the intermediate bandage layer and to prevent slipping of the bandage distally. 3. place layers of roll cotton from the toes to the level of the mid tibia/fibula or mid radius/ulna. place the layers with even tension, with some overlap of each consecutive layer, moving distally to proximally on the limb. 4. secure the short caudal or lateral splint and conform it to the distal extremity to the level of the toes and proximally to the level of the mid tibia/fibula or mid radius/ulna. 5. secure the lateral or caudal splint to the limb with another outer layer of elastic cotton (kling). 6. cover the entire bandage and splint with an outer tertiary layer of vetrap or elastikon. make sure that the toenails of the third and fourth digits remain visible to allow daily evaluation of circulation. long lateral or caudal splints are used to immobilize fractures of the tibia/fibula and radius/ulna. the splints are fashioned as directed for short splints but extend proximally to the level of the axilla and inguinal regions to immobilize above the fracture site. â�¢ packed cell volume drops rapidly to less than 20% in the dog and less than 12% to 15% in the cat â�¢ acute loss of more than 30% of blood volume (30 ml/kg in dog, 20 ml/kg in cat) â�¢ clinical signs of lethargy, collapse, hypotension, tachycardia, tachypnea (acute or chronic blood loss) â�¢ ongoing hemorrhage is present â�¢ poor response to crystalloid and colloid infusion â�¢ life-threatening hemorrhage caused by thrombocytopenia or thrombocytopathia â�¢ surgical intervention is necessary in a patient with severe thrombocytopenia or thrombocytopathia plasma support â�¢ life-threatening hemorrhage with decreased coagulation factor activity â�¢ severe inflammation (pancreatitis, systemic inflammatory response syndrome) â�¢ replenish antithrombin (disseminated intravascular coagulation, protein-losing enteropathy or nephropathy) â�¢ surgery is necessary in a patient with decreased coagulation factor activity â�¢ severe hypoproteinemia is present; to partially replenish albumin, globulin, and clotting factors type a cats typically possess weak anti-b antibodies of igg and igm subtypes. transfusion of type b blood into a type a cat will result in milder clinical signs of reaction and a markedly decreased survival half-life of the infused rbcs to just 2 days. because type ab cats possess both moieties on their cell surface, they lack naturally occurring alloantibodies; transfusion of type a blood into a type ab cat can be performed safely if a type ab donor is not available. the life span of an rbc from a type-specific transfusion into a cat is approximately 33 days. . indications for fresh whole blood transfusion include disorders of hemostasis and coagulopathies including disseminated intravascular coagulation, von willebrand's disease, and hemophilia. fresh whole blood and platelet-rich plasma also can be administered in cases of severe thrombocytopenia and thrombocytopathia. stored whole blood and packed rbcs can be administered in patients with anemia. if pcv drops to below 10% or if rapid hemorrhage causes the pcv to drop below 20% in the dog or less than 12% to 1 *indicates that this must be done for each donor being tested. minor crossmatch* 2. obtain a crossmatch segment from blood bank refrigerator for each donor to be crossmatched, or use an edta tube of donor's blood. make sure tubes are labeled prop-erly. 3. collect 2 ml of blood from recipient and place in an edta tube. centrifuge blood for 5 minutes. 4. extract blood from donor tubing. centrifuge blood for 5 minutes. use a separate pipette for each transfer because cross-contamination can occur. 5. pipette plasma off of donor and recipient cells and place in tubes labeled dp and rp, respectively. 6. place 125 âµl of donor and recipient cells in tubes labeled dr and rr, respectively. 7. add 2.5 ml 0.9% sodium chloride solution from wash bottle to each red blood cell (rbc) tube, using some force to cause cells to mix. 8. centrifuge rbc suspension for 2 minutes. 9. discard supernatant and resuspend rbcs with 0.9% sodium chloride from wash bottle. 10 . repeat steps 8 and 9 for a total of three washes. 11. place 2 drops of donor rbc suspension and 2 drops of recipient plasma in tube labeled ma (this is the major crossmatch). 12. place 2 drops of donor plasma and 2 drops recipient rbc suspension in tube labeled mi (this is the minor crossmatch). 13. prepare control tubes by placing 2 drops donor plasma with 2 drops donor rbc suspension (this is the donor control); and place 2 drops recipient plasma with 2 drops recipient rbc suspension (this is the recipient control). 14. incubate major and minor crossmatches and control tubes at room temperature for 15 minutes. 15. centrifuge all tubes for 1 minute. 16. read tubes using an agglutination viewer. 17. check for agglutination and/or hemolysis. 18. score agglutination with the following scoring scale: 4+ one solid clump of cells 3+ several large clumps of cells 2+ medium-sized clumps of cells with a clear background 1+ hemolysis, no clumping of cells neg = negative for hemolysis; negative for clumping of red blood cells fresh whole blood coagulopathy with active hemorrhage (disseminated intravascular coagulation, thrombocytopenia; massive acute hemorrhage; no stored blood available) stored whole blood massive acute or ongoing hemorrhage; hypovolemic shock caused by hemorrhage that is unresponsive to conventional crystalloid and colloid fluid therapy; unavailability of equipment required to prepare blood components packed red blood cells nonregenerative anemia, immune-mediated hemolytic anemia, correction of anemia before surgery, acute or chronic blood loss fresh frozen plasma factor depletion associated with active hemorrhage (congenital: von willebrand's factor, hemophilia a, hemophilia b; acquired: vitamin k antagonist, rodenticide intoxication, dic); acute or chronic hypoproteinemia (burns, wound exudates, body cavity effusion; hepatic, renal, or gastrointestinal loss); colostrum replacement in neonates frozen plasma acute plasma or protein loss; chronic hypoproteinemia; (contains stable colostrum replacement in neonates; hemophilia b and clotting factors) selected clotting factor deficiencies platelet-rich plasma* thrombocytopenia with active hemorrhage (immune-mediated thrombocytopenia, dic); platelet function abnormality (congenital: thrombasthenia in bassett hounds; acquired: nsaids, other drugs) cryoprecipitate congenital factor deficiencies (routine or before surgery): (concentration of factor hemophilia a, hemophilia b, von willebrand's disease, viii, von willebrand's hypofibrinogenemia; acquired factor deficiencies factor, and fibrinogen) *must be purchased because logistically one cannot obtain enough blood simultaneously to provide a significant amount of platelets; platelets infused have a very short (<2 hours) half-life. dic, disseminated intravascular coagulation; nsaids, nonsteroidal antiinflammatory drugs. universal donor (e.g., should be administered whenever possible. because there is no universal donor in the cat and because cats possess naturally occurring alloantibodies, all cat blood should be typed and crossmatched before any transfusion. if fresh whole blood is not available, a hemoglobin-based oxygen carrier (oxyglobin, 2 to 7 ml/kg iv) can be administered until blood products become available. table 1 -4 indicates blood component dose and administration rates. blood products should be warmed slowly to 37â°c before administering them to the patient. blood warmer units are available for use in veterinary medicine to facilitate rapid transfusion without decreasing patient body temperature (thermal angel; enstill medical technologies, inc., dallas, texas). red blood cell and plasma products should be administered in a blood administration set containing a 170-âµm in-line filter. smaller in-line filters (20 âµm) also can be used in cases in which extremely small volumes are to be administered. blood products should be administered over a period of 4 hours, whenever possible, according to guidelines set by the american association of blood banks. the volume of blood components required to achieve a specific increment in the patient's pcv depends largely on whether whole blood or packed rbcs are transfused and whole blood 20 ml/kg will increase max rate: 22 ml/kg/ max: 22 ml/kg/ volume by 10% 24 hours hour packed red 10 ml/kg will increase critically ill blood cells volume by 10% patients (e.g., cardiac failure or renal failure): 3-4 ml/kg/hour fresh frozen 10 ml/kg body mass (repeat 4-10 ml/minute or use rates as for plasma in 2-3 days or in 3-5 days whole blood (infuse within 4-6 hours) or until bleeding stops); monitor act, aptt, and pt before and 1 hour after transfusion cryoprecipitate general: 1 unit/10 kg/12 hours 4-10 ml/minute or use rates as for whole or until bleeding stops blood (infuse within 4-6 hours) hemophilia a: 12-20 units factor viii/kg; 1 unit of cryoprecipitate contains approximately 125 units of factor viii platelet-rich 1 unit/10 kg (1 unit of 2 ml/minute plasma platelet-rich plasma will check platelet count before and 1 hour increase platelet count after transfusion 1 hour after transfusion by 10,000/âµl) whether there is ongoing hemorrhage or rbc destruction. because the pcv of packed rbcs is unusually high (80% for greyhound blood), a smaller total volume is required than whole blood to achieve a comparable increase in the patient's pcv. in general, 10 ml/kg of packed rbcs or 20 ml/kg whole blood will raise the recipient's pcv by 10%. the "rule of ones" states that 1 ml per 1 lb of whole blood will raise the pcv by 1%. if the patient's pcv does not raise by the amount anticipated by the foregoing calculation(s), causes of ongoing hemorrhage or destruction should be considered. the goal of red blood component therapy is to raise the pcv to 25% to 30% in dogs and 15% to 20% in cats. if an animal is hypovolemic and whole blood is administered, the fluid is redistributed into the extravascular compartment within 24 hours of transfusion. this will result in a secondary rise in the pcv 24 hours after the transfusion in addition to the initial rise 1 to 2 hours after the rbc transfusion is complete. the volume of plasma transfused depends largely on the patient's need. in general, plasma transfusion should not exceed more than 22 ml/kg during a 24-hour period for normovolemic animals. thaw plasma at room temperature, or place it in a ziplock freezer bag and run under cool (not warm) water until thawed. then administer the plasma through a blood administration set that contains an in-line blood filter or through a standard driptype administration set with a detachable in-line blood administration filter. the average rate of plasma infusion in a normovolemic patient should not exceed 22 ml/kg/hour. in acute need situations, plasma can be delivered at rates up to 5 to 6 ml/kg/minute. for patients with cardiac insufficiency or other circulatory problems, plasma infusion rates should not exceed 5 ml/kg/hour. plasma or other blood products should not be mixed with or used in the same infusion line as calcium-containing fluids, including lactated ringer's solution, calcium chloride, or calcium gluconate. the safest fluid to mix with any blood product is 0.9% sodium chloride. administer fresh frozen plasma, frozen plasma, and cryoprecipitate at a volume of 10 ml/kg until bleeding is controlled or source of ongoing albumin loss ceases. the goal of plasma transfusion therapy is to raise the albumin to a minimum of 2.0 g/dl or until bleeding stops as in the case of coagulopathies. monitor the patient to ensure that bleeding has stopped, coagulation profiles (act, aptt, and pt) have normalized, hypovolemia has stabilized, and/or total protein is normalizing, which are indications for discontinuing ongoing transfusion therapy. plasma cryoprecipitate can be purchased or manufactured through the partial thawing and then centrifugation of fresh frozen plasma. cryoprecipitate contains concentrated quantities of vwf, factor viii, and fibrinogen and is indicated in severe forms of von willebrand's disease and hemophilia a (factor viii deficiency). platelet-rich plasma must be purchased from a commercial source. one unit of fresh whole blood contains 2000 to 5000 platelets. the viability of the platelets contained in the fresh whole blood is short-lived, just 1 to 2 hours after transfusion into the recipient. because platelet-rich plasma is difficult to obtain, animals with severe thrombocytopenia or thrombocytopathia should be treated with immunomodulating therapies and the administration of fresh frozen plasma. in dogs, blood and plasma transfusions can be administered intravenously or intraosseously. the cephalic, lateral saphenous, medial saphenous, and jugular veins are used most commonly. fill the recipient set so that the blood in the drip chamber covers the filter (normal 170-âµm filter). with small amounts of blood (50 ml) or critically ill patients, use a 40-âµm filter. avoid latex filters for plasma and cryoprecipitate administration. blood can 30 1 emergency care be administered at variable rates, but the routine figure of 4 to 5 ml/minute often is used. normovolemic animals can receive blood at 22 ml/kg/day. dogs in heart failure should receive infusions at no more than 4 ml/kg/hour. volume is given as needed. to calculate the approximate volume of blood needed to raise hematocrit levels, use the following formula for the dog: anticoagulated blood volume (ml) = body mass (kg) ã� 90 ã� pcv desired â�� pcv of recipient pcv of donor in anticoagulant an alternative formula is the following: 2.2 ã� recipient body mass (kg) ã� 30 (dog) ã� pcv desired â�� pcv of recipient pcv of donor in anticoagulant surgical emergencies and shock may require several times this volume within a short period. if greater than 25% of the patient's blood volume is lost, supplementation with colloids, crystalloids, and blood products is indicated for fluid replacement. one volume of whole blood achieves the same increase in plasma as two to three volumes of plasma. if the patient's blood type is unknown and type a-negative whole blood is not available, any dog blood can be administered to a dog in acute need if the dog has never had a transfusion before. if mismatched blood is given, the patient will become sensitized, and after 5 days, destruction of the donor rbcs will begin. in addition, any subsequent mismatched transfusions may cause an immediate reaction (usually mild) and rapid destruction of the transfused rbcs. the clinical signs of a transfusion reaction typically only are seen when type a blood is administered to a type a-negative recipient that has been sensitized previously. incompatible blood transfusions to breeding females can result in isoimmunization and in hemolytic disease in the puppies. the a-negative bitch that receives a transfusion with a-positive and that produces a litter from an a-positive stud can have puppies with neonatal isoerythrolysis. cats with severe anemia in need of a blood transfusion are typically extremely depressed, lethargic, and anorexic. the stress of restraint and handling can push these critically ill patients over the edge and cause them to die. extreme gentleness and care are mandatory in restraint and handling. the critically ill cat should be cradled in a towel or blanket. supplemental flow-by or mask oxygen should be administered, whenever possible, although it may not be clinically helpful until oxygen-carrying capacity is replenished with infusion of rbcs or hemoglobin. blood can be administered by way of cephalic, medial saphenous, or the jugular vein. intramedullary infusion is also possible, if vascular access cannot be accomplished. the average 2-to 4-kg cat can accept 40 to 60 ml of whole blood injected intravenously over a period of 30 to 60 minutes. administer filtered blood at a rate of 5 to 10 ml/kg/hour. the following formula can be used to estimate the volume of blood required for transfusion in a cat: anticoagulated blood volume (ml) = body mass (kg) ã� 70 ã� pcv desired â�� pcv of recipient pcv of donor in anticoagulant the exact overall incidence and clinical significance of transfusion reactions in veterinary medicine are unknown. several studies have been performed that document the incidence of transfusion reactions in dogs and cats. overall, the incidence of transfusion reactions in dogs and cats is 2.5% and 2%, respectively. transfusion reactions can be immune-mediated and non-immune-mediated and can happen immediately or can be delayed until after a transfusion. acute reactions usually occur within minutes to hours of the onset of transfusion but may occur up to 48 hours after the transfusion has been stopped. acute immunologic reactions include hemolysis and acute hypersensitivity including rbcs, platelets, and leukocytes. signs of a delayed immunologic reaction include hemolysis, purpura, immunosuppression, and neonatal isoerythrolysis. acute nonimmunologic reactions include donor cell hemolysis before onset of transfusion, circulatory volume overload, bacterial contamination, citrate toxicity with clinical signs of hypocalcemia, coagulopathies, hyperammonemia, hypothermia, air embolism, acidosis, and pulmonary microembolism. delayed nonimmunologic reactions include the transmission and development of infectious diseases and hemosiderosis. clinical signs of a transfusion reaction typically depend on the amount of blood transfused, the type and amount of antibody involved in the reaction, and whether the recipient has had previous sensitization. monitoring the patient carefully during the transfusion period is essential in recognizing early signs of a transfusion reaction, including those that may become life threatening. a general guideline for patient monitoring is first to start the transfusion slowly during the first 15 minutes. monitor temperature, pulse, and respiration every 15 minutes for the first hour, 1 hour after the end of the transfusion, and every 12 hours minimally thereafter. also obtain a pcv immediately before the transfusion, 1 hour after the transfusion has been stopped, and every 12 hours thereafter. monitor coagulation parameters such as an act and platelet count at least daily in patients requiring transfusion therapy. the most common documented clinical signs of a transfusion reaction include pyrexia, urticaria, salivation/ptyalism, nausea, chills, and vomiting. other clinical signs of a transfusion reaction may include tachycardia, tremors, collapse, dyspnea, weakness, hypotension, collapse, and seizures. severe intravascular hemolytic reactions may occur within minutes of the start of the transfusion, causing hemoglobinemia, hemoglobinuria, disseminated intravascular coagulation, and clinical signs of shock. extravascular hemolytic reactions typically occur later and will result in hyperbilirubinemia and bilirubinuria. pretreatment of patients to help decrease the risk of a transfusion reaction remains controversial, and in most cases, pretreatment with glucocorticoids and antihistamines is ineffective at preventing intravascular hemolysis and other reactions should they occur. the most important component of preventing a transfusion reaction is to screen each recipient carefully and process the donor component therapy carefully before the administration of any blood products. treatment of a transfusion reaction depends on its severity. in all cases, stop the transfusion immediately when clinical signs of a reaction occur. in most cases, discontinuation of the transfusion and administration of drugs to stop the hypersensitivity reaction will be sufficient. once the medications have taken effect, restart the transfusion slowly and monitor the patient carefully for further signs of reaction. in more severe cases in which a patient's cardiovascular or respiratory system become compromised and hypotension, tachycardia, or tachypnea occurs, immediately discontinue the transfusion and administer diphenhydramine (1 mg/kg im), dexamethasone-sodium phosphate (0.25 to 0.5 mg/kg iv), and epinephrine to the patient. the patient should have a urinary catheter and central venous catheter placed for measurement of urine output and central venous pressures. aggressive fluid therapy may be necessary to avoid renal insufficiency or renal damage associated with severe intravascular hemolysis. overhydration with subsequent pulmonary edema generally can be managed with supplemental oxygen administration and intravenous or intramuscular administration of furosemide (2 to 4 mg/kg). plasma products with or without heparin can be administered for disseminated intravascular coagulation. the hbocs can be stored at room temperature and have a relatively long shelf life compared with red blood component products. the hbocs function to carry oxygen through the blood and can diffuse oxygen past areas of poor tissue perfusion. an additional characteristic of hbocs is as a potent colloid, serving to maintain fluid within the vascular space. for this reason, hbocs must be used with caution in euvolemic patients and patients with cardiovascular insufficiency. central venous pressure (cvp) measures the hydrostatic pressure in the anterior vena cava and is influenced by vascular fluid volume, vascular tone, function of the right side of the heart, and changes in intrathoracic pressure during the respiratory cycle. the cvp is not a true measure of blood volume but is used to gauge fluid therapy as a method of determining how effectively the heart can pump the fluid that is being delivered to it. thus the cvp reflects the interaction of the vascular fluid volume, vascular tone, and cardiac function. measure cvp in any patient with acute circulatory failure, large volume fluid diuresis (i.e., toxin or oliguric or anuric renal failure), fluid in-and-out monitoring, and cardiac dysfunction. the placement of central venous catheters and thus cvp measurements is contraindicated in patients with known coagulopathies including hypercoagulable states. to perform cvp monitoring, place a central venous catheter in the right or left jugular vein. in cats and small dogs, however, a long catheter placed in the lateral or medial saphenous vein can be used for trends in cvp monitoring. first, assemble the equipment necessary for jugular catheter (see vascular access techniques for how to place a jugular or saphenous long catheter) and cvp monitoring (box 1-7). after placing the jugular catheter, take a lateral thoracic radiograph to ensure that the tip of the catheter sits just outside of the right atrium for proper cvp measurements (see to establish an intravenous catheter for cvp, follow this procedure: 1. assemble the cvp setup such that the male end of a length of sterile intravenous catheter extension tubing is inserted into the t port of the jugular or medial/lateral saphenous catheter. make sure to flush the length of tubing with sterile saline before connecting it to the patient to avoid iatrogenic air embolism. 2. next, insert the male end of a three-way stopcock into the female end of the extension tubing. 3. attach a 20-ml syringe filled with heparinized sterile 0.9% saline to one of the female ports of the three-way stopcock and either a manometer or a second length of intravenous extension tubing attached to a metric ruler. 4. lay the patient in lateral or sternal recumbancy. 5. turn the stopcock off to the manometer/ruler and on to the patient. infuse a small amount of heparinized saline through the catheter to flush the catheter. 6. next, turn the stopcock off to the patient and on to the manometer. gently flush the manometer or length of extension tubing with heparinized saline from the syringe. use care not to agitate the fluid and create air bubbles within the line or manometer that will artifactually change the cvp measured. 7. next, lower the 0 cm point on the manometer or ruler to the level of the patient's manubrium (if the patient is in lateral recumbancy) or the point of the elbow (if the patient is in sternal recumbancy). 8. turn the stopcock off to the syringe, and allow the fluid column to equilibrate with the patient's intravascular volume. once the fluid column stops falling and the level rises and falls with the patient's heartbeat, measure the number adjacent to the bottom of the meniscus of the fluid column. this is the cvp in centimeters of water (see figure 1 -4). 9. repeat the measurement several times with the patient in the same position to make sure that none of the values has been increased or decreased artifactually in error. alternately, attach the central catheter to a pressure transducer and perform electronic monitoring of cvp. there is no absolute value for normal cvp. the normal cvp for small animal patients is 0 to 5 cm h 2 o. values less than zero are associated with absolute or relative hypovolemia. values of 5 to 10 cm h 2 o are borderline hypervolemia, and values greater than 10 cm h 2 o suggest intravascular volume overload. values greater than 15 cm h 2 o may be correlated with congestive heart failure and the development of pulmonary edema. in individual patients, the trend in change in cvp is more important than absolute values. as a rule of thumb, when using cvp measurements to gauge fluid therapy and avoid vascular and pulmonary overload, the cvp should not increase by more than 5 cm h 2 o in any 24-hour period. if an abrupt increase in cvp is found, repeat the measurement to make sure that the elevated value was not obtained in error. if the value truly has increased dramatically, temporarily discontinue fluid therapy and consider administration of a diuretic. delaforcade am, rozanski ea: central venous pressure and arterial blood pressure measurements, vet clin north am small anim pract 31 (6) the diagnosis of intracellular fluid deficit is difficult and is based more on the presence of hypernatremia or hyperosmolality than on clinical signs. an intracellular fluid deficit is expected when free water loss by insensible losses and vomiting, diarrhea, or urine is not matched by free water intake. consideration of the location of the patient's fluid deficit, history of vomiting and diarrhea, no visible clinical signs of deficit 4% dry mucous membranes, mild skin tenting 5% increased skin tenting, dry mucous membranes, mild tachycardia, normal pulse* 7% increased skin tenting, dry mucous membranes, tachycardia, weak pulse pressure 10% increased skin tenting, dry corneas, dry mucous membranes, 12% elevated or decreased heart rate, poor pulse quality, altered level of consciousness* the respiratory system further contributes to acid-base status by changes in the elimination of carbon dioxide. hyperventilation decreases the blood pco 2 and causes a respiratory alkalosis. hypoventilation increases the blood pco 2 and causes a respiratory acidosis. depending on the altitude, the pco 2 in dogs can range from 32 to 44 mm hg. in cats, normal is 28 to 32 mm hg. venous pco 2 values are 33 to 50 mm hg in dogs and 33 to 45 mm hg in cats. use a systematic approach whenever attempting to interpret a patient's acid-base status. ideally, obtain an arterial blood sample so that you can monitor the patient's oxygenation and ventilation. once an arterial blood sample has been obtained, follow these steps: 1. determine whether the blood sample is arterial or venous by looking at the oxygen saturation (sao 2 ). the sao 2 should be greater than 90% if the sample is truly arterial, although it can be as low as 80% if a patient has severe hypoxemia. 2. consider the patient's ph. if the ph is outside of the normal range, an acid-base disturbance is present. if the ph is within the normal range, an acid-base disturbance may or may not be present. if the ph is low, the patient is acidotic. if the ph is high, the patient is alkalotic. 3. next, look at the base excess or deficit. if the base excess is increased, the patient has higher than normal bicarbonate. if there is a base deficit, the patient may have a low bicarbonate or increase in unmeasured anions (e.g., lactic acid or ketoacids). 4. next, look at the bicarbonate. if the ph is low and the bicarbonate is low, the patient has a metabolic acidosis. if the ph is high and the bicarbonate is elevated, the patient has a metabolic alkalosis. 5. next, look at the paco 2 . if the patient's ph is low and the paco 2 is elevated, the patient has a respiratory acidosis. if the patient's ph is high and the paco 2 is low, the patient has a respiratory alkalosis. 6. finally, if you are interested in the patient's oxygenation, look at the pao 2 . normal pao 2 is greater than 80 mm hg. the metabolic acidosis early in renal failure may be hyperchloremic and later may convert to typical increased anion gap acidosis. 7. next, you must determine whether the disorders present are primary disorders or an expected compensation for disorders in the opposing system. for example, is the patient retaining bicarbonate (metabolic alkalosis) because of carbon dioxide retention (respiratory acidosis)? use the chart in table 1 -6 to evaluate whether the appropriate degree of compensation is occurring. if the adaptive response falls within the expected range, a simple acid-base disorder is present. if the response falls outside of the expected range, a mixed acid-base disorder is likely present. 8. finally, you must determine whether the patient's acid-base disturbance is compatible with the history and physical examination findings. if the acid-base disturbance does not fit with the patient's history and physical examination abnormalities, question the results of the blood gas analyses and possibly repeat them. the most desirable method of assessing the acid-base status of an animal is with a blood gas analyzer. arterial samples are preferred over venous samples, with heparin used as an anticoagulant (table 1-7) . potassium primarily is located in the intracellular fluid compartment. serum potassium is regulated by the actions of the sodium-potassium-adenosinetriphosphatase pump on cellular membranes, including those of the renal tubular epithelium. inorganic metabolic acidosis artifactually can raise serum potassium levels because of redistribution of extracellular potassium in exchange for intracellular hydrogen ion movement in an attempt to correct serum ph. metabolic acidosis potassium is one of the major players in the maintenance of resting membrane potentials of excitable tissue, including neurons and cardiac myocytes. changes in serum potassium can affect cardiac conduction adversely. hyperkalemia lowers the resting membrane potential and makes cardiac cells, particularly those of the atria, more susceptible to depolarization. characteristic signs of severe hyperkalemia that can be observed on an ecg rhythm strip include an absence of p waves, widened qrs complexes, and tall tented or spiked t waves. further increases in serum potassium can be associated with bradycardia, ventricular fibrillation, and cardiac asystole (death). treatment of hyperkalemia consists of administration of insulin (0.25 to 0.5 units/kg, iv regular insulin) and dextrose (1 g dextrose per unit of insulin administered, followed by 2.5% dextrose iv cri to prevent hypoglycemia), calcium (2 to 10 ml of 10% calcium gluconate administered iv slowly to effect), or sodium bicarbonate (1 meq/kg, iv slowly). insulin plus dextrose and bicarbonate therapy help drive the potassium intracellularly, whereas calcium antagonizes the effect of hyperkalemia on the myocardial cells. all of the treatments work within minutes, although the effects are relatively short-lived (20 minutes to 1 hour) unless the cause of the hyperkalemia is identified and treated appropriately (box 1-10). dilution of serum potassium also results from restoring intravascular fluid volume and correcting metabolic acidosis, in most cases. treatment with a fluid that does not contain potassium (preferably 0.9% sodium chloride) is recommended. hypokalemia elevates the resting membrane potential and results in cellular hyperpolarization. hypokalemia may be associated with ventricular dysrhythmias, but the ecg changes are not as characteristic as those observed with hyperkalemia. causes of hypokalemia include renal losses, anorexia, gastrointestinal loss (vomiting, diarrhea), intravenous fluid diuresis, loop diuretics, and postobstructive diuresis (box 1-11). if the serum potassium concentration is known, potassium supplementation in the form of potassium chloride or potassium phosphate can be added to the patient's intravenous fluids. correct serum potassium levels less than 3.0 meq/l or greater than 6.0 meq/l. potassium rates should not exceed 0.5 meq/kg/hour (table 1 -8) . metabolic acidosis from bicarbonate depletion often corrects itself with volume restoration in most small animal patients. patients with moderate to severe metabolic acidosis may benefit from bicarbonate supplementation therapy. the metabolic contribution to acid-base balance is identified by measuring the total carbon dioxide concentration or calculating the bicarbonate concentration. if these measurements are not available, the degree of expected metabolic acidosis can be estimated subjectively by the severity of underlying disease that often contributes to metabolic acidosis: hypovolemic or traumatic shock, septic shock, diabetic ketoacidosis, or oliguric/anuric renal failure. if the metabolic acidosis is estimated to be mild, moderate, or severe, add sodium bicarbonate at 1, 3, and 5 meq/kg body mass, respectively. patients with diabetic ketoacidosis may not require bicarbonate administration once volume replacement and perfusion is restored, and the ketoacids are metabolized to bicarbonate. if the bicarbonate measurement of base deficit is known, the following formula can be used as a gauge for bicarbonate supplementation: base deficit ã� 0.3 = body mass (kg) = meq bicarbonate to administer osmolality osmolality is measured by freezing point depression or a vapor pressure osmometer, or it may be calculated by the following formula: mosm/kg = 2[(na + ) + (k + )] + bun/2.8 + glucose/18 where sodium and potassium are measured in milliequivalents, and bun and glucose are measured in milligrams per deciliter. osmolalities less than 260 mosm/kg or greater 38 1 emergency care than 360 mosm/kg are serious enough to warrant therapy. the difference between the measured osmolality and the calculated osmolality (the osmolal gap) should be less than 10 mosm/kg. if the osmolal gap is greater than 20 mosm/kg, consider the presence of unmeasured anions such as ethylene glycol metabolites. the volume of extracellular fluid is determined by the total body sodium content, whereas the osmolality and sodium concentration are determined by water balance. serum sodium concentration is an indication of the amount of sodium relative to water in the extracellular fluid and provides no direct information about the total body sodium content. unlikely to cause hyperkalemia in presence of normal renal function unless iatrogenic (e.g., continuous infusion of potassium-containing fluids at an excessively rapid rate) acute mineral acidosis (e.g., hydrochloric acid or ammonium chloride) insulin deficiency (e.g., diabetic ketoacidosis) acute tumor lysis syndrome reperfusion of extremities after aortic thromboembolism in cats with cardiomyopathy hyperkalemic periodic paralysis (one case report in a pit bull) mild hyperkalemia after exercise in dogs with induced hypothyroidism infusion of lysine or arginine in total parenteral nutrition solutions nonspecific î²-blockers (e.g., propranolol)* cardiac glycosides (e.g., digoxin)* urethral obstruction ruptured bladder anuric or oliguric renal failure hypoadrenocorticism selected gastrointestinal disease (e.g., trichuriasis, salmonellosis, or perforated duodenal ulcer) late pregnancy in greyhound dogs (mechanism unknown but affected dogs had gastrointestinal fluid loss) chylothorax with repeated pleural fluid drainage hyporeninemic hypoaldosteronism â�  angiotensin-converting enzyme inhibitors (e.g., enalapril)* angiotensin receptor blockers (e.g., losartan)* cyclosporine and tacrolimus* potassium-sparing diuretics (e.g., spironolactone, amiloride, and triamterene)* nonsteroidal antiinflammatory drugs* heparin* trimethoprim* from dibartola sp: fluid, electrolyte and acid-base disorders in small animal practice, st louis, 2005, saunders. *likely to cause hyperkalemia only in conjunction with other contributing factors (e.g., other drugs, decreased renal function, or concurrent administration of potassium supplements). â�  not well documented in veterinary medicine. if refractory hypokalemia is present, supplement magnesium at 0.75 meq/kg/day for 24 hours. alone unlikely to cause hypokalemia unless diet is aberrant administration of potassium-free (e.g., 0.9% sodium chloride or 5% dextrose in water) or potassium-deficient fluids (e.g., lactated ringer's solution over several days) bentonite clay ingestion (e.g., cat litter) alkalemia insulin/glucose-containing fluids catecholamines hypothermia hypokalemic periodic paralysis (burmese cats) albuterol overdosage patients with hyponatremia or hypernatremia may have decreased, normal, or increased total body sodium content (boxes 1-12 and [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] ). an increased serum sodium concentration implies hyperosmolality, whereas a decrease in serum sodium concentration usually, but not always, implies hypoosmolality. the severity of clinical signs of hypernatremia and hyponatremia is related primarily to the rapidity of the onset of the change rather than to the magnitude of the associated plasma hyperosmolality or hypoosmolality. clinical signs of neurologic disturbances include disorientation, ataxia, and seizures, and coma may occur at serum sodium concentrations less than 120 meq/l or greater than 170 meq/l in dogs. therapy of hypernatremia or hyponatremia with fluid containing low or higher concentrations of sodium should proceed with caution, for rapid changes (decreases or increases) of serum sodium and osmolality can cause rapid changes in the intracellular and extracellular fluid flux, leading to intracellular dehydration or edema, even though the serum sodium has not been returned to normal. a rule of thumb is to not raise or lower the serum sodium by more than 15 meq/l during any one 24-hour period. restoration of the serum sodium concentration over a period of 48 to 72 hours is better. in almost all circumstances, an animal will correct its sodium balance with simple fluid restoration. if severe hypernatremia exists that suggests a free water deficit, however, the free water deficit should be calculated from the following formula: hypernatremia can be corrected slowly with 0.45% sodium chloride plus 2.5% dextrose, 5% dextrose in water, or lactated ringer's solution (sodium content: 130 meq/l). correct hyponatremia initially with 0.9% sodium chloride. sodium is balanced predominantly by chloride and bicarbonate. the difference between these concentrations, (na , has been called the anion gap. the normal anion gap is between 12 and 25 meq/l. when the anion gap exceeds 25, consider the possibility of an accumulation of unmeasured anions (e.g., lactate, ketoacids, phosphate, sulfate, ethylene glycol metabolites, and salicylate). abnormalities in the anion gap may be helpful in determining the cause of metabolic acidosis (boxes 1-14 and 1-15). the colloid oncotic pressure of blood is associated primarily with large-molecular-weight colloidal substances in circulation. the major player in maintaining intravascular and interstitial oncotic pressure, the water-retaining property of each fluid compartment, is albumin. albumin contributes roughly 80% to the colloidal oncotic pressure of blood. the majority of albumin is located within the interstitial space. hypoalbuminemia can result from increased loss in the form of protein-losing enteropathy or nephropathy and wound exudates, or it may be due to lack of hepatic albumin synthesis. serum albumin pools are in a constant flux with interstitial albumin. once interstitial albumin pools become depleted from replenishing serum albumin, serum albumin levels can continue to decrease, which can lead to a decrease in colloidal oncotic pressure. serum albumin less than 2.0 g/dl has been associated with inadequate intravascular fluid retention and the development of peripheral edema and third spacing of fluid. oncotic pressure can be restored with the use of artificial or synthetic colloids or natural colloids (see colloids). maintenance fluid requirements have been extrapolated from the formulas used to calculate a patient's daily metabolic energy requirements because it takes 1 ml of water to metabolize 1 kcal of energy (table 1 -9) . the patient's daily metabolic water (fluid) requirements can be calculated by the following formula: administration of an isotonic crystalloid fluid for maintenance requirements often can produce iatrogenic hypokalemia. in most cases, supplemental potassium must be added to prevent hypokalemia resulting from inappetance, kalliuresis, and supplementation with isotonic crystalloid fluids. the most reliable method of determining the degree of fluid deficit is by weighing the animal and calculating acute weight loss. acute weight loss in a patient with volume loss in the form of vomiting, feces, wound exudates, and urine is due to fluid loss and not loss of muscle or fat. lean body mass normally is not gained or lost rapidly enough to cause major changes in body weight. one milliliter of water weighs approximately 1 g. this fact allows calculation of the patient's fluid deficit, if ongoing losses can be measured. when a patient first presents, however, the body weight before a fluid deficit has occurred rarely is known. instead, one must rely on subjective measures of dehydration to estimate the patient's percent dehydration and to calculate the volume of fluid required to rehydrate the patient over the next 24 hours. to calculate the volume deficit, use the following formula: body mass (kg) ã� (% dehydration) ã� 1000 = fluid deficit (ml) the patient's fluid deficit must be added to the daily maintenance fluid requirements and administered over a 24-hour period. ongoing losses can be determined by measuring urine output, weighing the patient at least 2 to 3 times a day, and measuring the volume or weight of vomitus or diarrhea. a crystalloid fluid contains crystals of salts with a composition similar to that of the extracellular fluid space and can be used to maintain daily fluid requirements and replace fluid deficits or ongoing fluid losses (table 110) . metabolic, acid-base, and electrolyte imbalances also can be treated with isotonic fluids with or without supplemental electrolytes and buffers. depending on the patient's clinical condition, choose the specific isotonic crystalloid fluid to replace and maintain the patient's acid-base and electrolyte status ( table 1-11) . crystalloid fluids are readily available, are relatively inexpensive, and can be administered safely in large volumes to patients with no preexisting cardiac or renal disease or cerebral edema. following infusion, approximately 80% of the volume of a crystalloid fluid infused will redistribute to the interstitial fluid compartment. as such, crystalloid fluids alone are ineffective for ongoing intravascular volume depletion when given as a bolus. the crystalloid fluid bolus must be followed by a constant rate infusion, taking into consideration the patient's daily maintenance fluid requirements and ongoing fluid losses. administration of a large volume of crystalloid fluids can cause dilutional anemia and coagulopathies. *30 ã� bw kg + 70 = kcal/day = ml/day. note: this formula will slightly underestimate the requirements for patients that are less than 2 kg and will slightly overestimate the requirements for patients greater than 70 kg. retain fluid in the vascular space, the volume of crystalloid fluid infused (maintenance + deficit + ongoing losses) should be decreased by 25% to 50% to avoid vascular volume overload. two major classes of colloids exist: natural and synthetic. natural colloids (whole blood, packed rbcs, plasma) are discussed elsewhere in this text. concentrated human albumin is a natural purified colloid that recently has become more popular in the treatment of advanced hypoalbuminemia and hypoproteinemia and will be discussed here. synthetic colloids are starch polymers and include dextrans and hetastarch. concentrated human albumin is available as a 5% or 25% solution. the 5% solution has an osmolality similar to that of serum (308 mosm/l), whereas the 25% solution is hyperoncotic (1500 mosm/l). a 25% albumin solution draws fluid from the interstitial space into the intravascular space. concentrated albumin solutions often are used to restore circulating volume when synthetic colloids are not available. albumin not only is important at maintaining the colloidal oncotic pressure of blood but also serves as a valuable free-radical scavenger and carrier of drugs and hormones necessary for normal tissue function and healing. albumin levels less than 2.0 g/dl have been associated with increased morbidity and mortality. concentrated human albumin solutions can be administered as an effective method of restoring interstitial and serum albumin concentrations in situations of acute and chronic hypoalbuminemia. albumin (25%) is available in 50-and 100-ml vials and is more cost-efficient as an albumin replacement than procurement and administration of fresh frozen plasma. recommended albumin infusion rates are 2 to 5 ml/kg over 4 hours, after pretreatment with diphenhydramine. although concentrated human albumin is structurally similar to canine albumin, closely monitor the patient for signs of allergic reaction during and after the infusion. dextran-70 is a synthetic high-molecular-weight polysaccharide (sucrose polymer) with a molecular weight of 70,000 d. particles less than 50,000 d, are cleared rapidly by the kidneys, whereas larger particles are cleared more slowly by the hepatic reticuloendothelial system. dextran-70 can coat platelets and inhibit platelet function and so must be used with caution in patients with known coagulopathies. the total daily dosage should not exceed 40 ml/kg/day. hetastarch (hydroxyethyl starch) is a large-molecular-weight amylopectin polymer, has molecules with a molecular weight that exceeds 100,000 d, and has an average half-life of 24 to 36 hours in circulation. hetastarch can bind with vwf and cause prolongation of the act and aptt; however, it does not cause a coagulopathy. recommended rates of hetastarch infusion are 5-to 10-ml incremental boluses for the treatment of hypotension and 20 to 30 ml/kg/day as a constant rate infusion for maintenance of colloidal oncotic pressure. many are the acceptable ways to administer the fluids prescribed for each patient based on the degree of dehydration, estimation of ongoing losses, ability to tolerate oral fluid, and metabolic, acid-base, and electrolyte derangements. administer the fluids in a manner that is best for the patient and most appropriate for the practice. to determine the rate of intravenous fluid infusion, take the total volume of fluids that have been prescribed and divide the total volume by the total number of hours in a day that intravenous fluids can be delivered safely and monitored. the safest and most accurate way to deliver intravenous fluids, particularly in extremely small animals or those with congestive heart failure, is through an intravenous fluid pump. fluid should not be administered intravenously if the patient cannot be monitored to make sure that the fluids are being delivered at a safe rate and that the fluid line has not become disconnected. supplement fluids over as many hours as possible to allow the patient as much time as possible to redistribute and fully utilize the fluids administered. fluids administered too quickly can cause a diuresis to occur, such that the majority of the fluids administered will be excreted in the urine. if time is limited or if extra time is needed for safe administration of fluids, consider using a combination of intravenously and subcutaneously 46 1 emergency care administered fluids. intravenous is the preferred route of administration of fluids in any patient with dehydration and hypovolemia. as intravascular volume depletion occurs, reflex peripheral vasoconstriction occurs to restore core perfusion. the subcutaneous tissue are not perfused well and therefore fluids administered subcutaneously will not be absorbed well into the interstitial and intravascular spaces. subcutaneously administered fluids can be absorbed slowly and delivered effectively in the management of mild interstitial dehydration and in the treatment of renal insufficiency. subcutaneously administered fluids should never take the place of intravenously administered fluids in a hypovolemic patient or one with severe interstitial dehydration. intramedullary (intraosseous) infusion works well in small patients in which vascular access cannot be established. shock doses of fluids and other substances, including blood products, can be administered under pressure through an intraosseous cannula. because of the inherent discomfort and risk of osteomyelitis with intraosseous infusion, establish vascular access as soon as possible. the safest and most efficient method of intravenous fluid infusion is through a fluid pump. in cases in which a fluid pump is unavailable, infusion by gravity feed is the next option. infusion sets from various manufacturers have calibrated drip chambers such that a specific number of drops will equal 1 ml of fluid. fluid rates can be calculated based on the number of drops that fall into the drip chamber per minute: fluid volume to be infused (ml) = ml/hour number of hours available many pediatric drip sets deliver 60 drops/ml, such that milliliters/hour equals drops/ minute. carefully record fluid orders so that the volume to be administered is recorded as milliliters/hour, milliliters/day, and drops/minute. this will allow personnel to detect major discrepancies and calculation errors more readily. the volume actually delivered should be recorded in the record by nursing personnel. all additives should be listed clearly on the bottle on a piece of adhesive tape or a special label manufactured for this purpose. a strip of adhesive tape also can be attached to the bottle and marked appropriately to provide a quick visualization of the estimate of volume delivered. includes a large-bore flexible orogastric lavage tube, permanent marker or white tape, lubricating jelly, warm water, two large buckets, a roll of 2-inch white tape, and a manual lavage pump. to perform the orogastric lavage, follow this procedure: 1. place all animals under general anesthesia with a cuffed endotracheal tube in place to protect the airway and prevent aspiration of gastric contents into the lungs. 2. place a roll of 2-inch white tape into the animal's mouth, and secure the tape around the muzzle. you will insert the tube through the hole in the center of the roll of tape. 3. next, place the distal end of the tube at the level of the last rib, directly adjacent to the animal's thorax and abdomen. measure the length of the tube from the most distal end to the point where it comes out of the mouth, and label this location on the tube with a permanent marker or piece of white tape. 4. lubricate the distal portion of the tube, and gently insert it through the roll of tape in the animal's mouth. 5. gently push the tube down the esophagus. palpate the tube within the esophagus. two tubes should be palpable, the orogastric tube, and the patient's trachea. push the tube down into the stomach. you can verify location by blowing into the proximal end of the tube and simultaneously auscultating the stomach for borborygmi. 6. insert the manual pump to the proximal end of the tube, and instill the warm water. alternate instilling water with removal of fluid and gastric debris by gravity. repeat the process until the efflux fluid is clear of any debris. 7. save fluid from the gastric efflux fluid for toxicologic analyses. hackett tb: emergency approach to intoxications, clin tech small anim pract 15 (2):82-87, 2000. hypoxia, or inadequate tissue oxygenation, is the primary reason for supplemental oxygen therapy. major causes of hypoxia include hypoventilation, ventilation-perfusion mismatch, physiologic or right-to-left cardiac shunt, diffusion impairment, and decreased fraction of inspired oxygen (table 1-12) . inadequate tissue perfusion caused by low cardiac output or vascular obstruction also can result in circulatory hypoxia. finally, histiocytic hypoxia results from inability of cells to use oxygen that is delivered to them. this form of hypoxia can be observed with various toxin ingestions (bromethalin, cyanide) and in septic shock. a patient's oxygenation status can be monitored invasively by drawing of arterial blood gas samples or noninvasively through pulse oximetry, in most cases (see acid-base physiology and pulse oximetry). inspired air at sea level has a po 2 of 150 mm hg. as the air travels through the upper respiratory system to the level of the alveolus, the po 2 drops to 100 mm hg. tissue oxygen saturation in a normal healthy animal is 95 mm hg. after oxygen has been delivered to the tissues, the oxygen left in the venous system (pvo 2 ) is approximately 40 mm hg. normally, oxygen diffuses across the alveolar capillary membrane and binds reversibly with hemoglobin in rbcs. a small amount of oxygen is carried in an unbound diffusible form in the plasma. when an animal has an adequate amount of hemoglobin and hemoglobin becomes fully saturated while breathing room air, supplemental oxygen administration will only increase the sao 2 a small amount. the unbound form of oxygen dissolved in plasma will increase. if, however, inadequate hemoglobin saturation is obtained by breathing room air, as in a case of pneumonia or pulmonary edema, for example, breathing a higher fraction of inspired oxygen (fio 2 ) will improve bound and unbound hemoglobin levels. the formula for calculating oxygen content of arterial blood is as follows: where cao 2 is the arterial oxygen content, 1.34 is the amount of oxygen that can be carried by hemoglobin (hb), sao 2 is the hemoglobin saturation, and 0.003 ã� pao 2 is the amount of oxygen dissolved (unbound) in plasma. dissolved oxygen actually contributes little to the total amount of oxygen carried in the arterial blood, and the majority depends on the amount or availability of hemoglobin and the ability of the body (ph and respiratory status) to saturate the hemoglobin at the level of the alveoli. oxygen therapy is indicated whenever hypoxia is present. the underlying cause of the hypoxia also must be identified and treated, for chronic, lifelong oxygen therapy is rarely feasible in veterinary patients. if hemoglobin levels are low due to anemia, oxygen supplementation must occur along with rbc transfusions to increase hemoglobin mass. whenever possible, use arterial blood gas analyses or pulse oximetry to gauge a patient's response to oxygen therapy and to determine when an animal can be weaned from supplemental oxygen. the goal of oxygen therapy is to increase the amount of oxygen bound to hemoglobin in arterial blood. oxygen supplementation can be by hood, oxygen cage or tent, nasal or nasopharyngeal catheter, or tracheal tube. in rare cases, administration of oxygen with mechanical ventilation may be indicated. administration of supplemental oxygen to patients with chronic hypoxia is sometimes necessary but also dangerous. with chronic hypoxia the patient develops a chronic respiratory acidosis (elevated paco 2 ) and depends almost entirely on the hypoxic ventilatory drive to breathe. administration of supplemental oxygen increases pao 2 and may inhibit the central respiratory drive, leading to hypoventilation and possibly respiratory arrest. therefore, closely monitor animals with chronic hypoxia that are treated with supplemental oxygen. oxygen hoods can be purchased from commercial sources or can be manufactured in the hospital using a rigid elizabethan collar, tape, and plastic wrap. to make an oxygen hood, place several lengths of plastic wrap over the front of the elizabethan collar and tape them in place. leave the ventral third of the collar open to allow moisture and heat to dissipate and carbon dioxide to be eliminated. place a length of flexible oxygen tubing under the patient's collar into the front of the hood, and run humidified oxygen at a rate of 50 to 100 ml/kg/minute. animals may become overheated with an oxygen hood in place. carefully monitor the patient's temperature so that iatrogenic hyperthermia does not occur. commercially available plexiglass oxygen cages can be purchased from a variety of manufacturers. the best units include a mechanical thermostatically controlled compressor cooling unit, a circulatory fan, nebulizers or humidifiers to moisten the air, and a carbon dioxide absorber. alternately, a pediatric (infant) incubator can be purchased from hospital supply sources, and humidified oxygen can be run into the cage at 2 to 10 l/minute (depending on the size of the cage). high flow rates may be required to eliminate nitrogen and carbon dioxide from the cage. in most cases, the fio 2 inside the cage reaches 40% to 50% using this technique. disadvantages of using an oxygen cage are high consumption/ use of oxygen, rapid decrease in the fio 2 within the cage whenever the cage must be opened for patient treatments, lack of immediate access to the patient, and potential for iatrogenic hyperthermia. one of the most common methods for oxygen supplementation in dogs is nasal or nasopharyngeal oxygen catheters: 1. to place a nasal or nasopharyngeal catheter, obtain a red rubber catheter (8f to 12f, depending on the size of the patient). a. for nasal oxygen supplementation, measure the distal tip of the catheter from the medial canthus of the eye to the tip of the nose. b. for nasopharyngeal oxygen supplementation, measure the catheter from the ramus of the mandible to the tip of the nose. 2. mark the tube length at the tip of the nose with a permanent marker. 3. instill topical anesthetic such as proparacaine (0.5%) or lidocaine (2%) into the nostril before placement. 4. place a stay suture adjacent to (lateral aspect) the nostril while the topical anesthetic is taking effect. 5. lubricate the tip of the tube with sterile lubricant. 6. gently insert the tube into the ventral medial aspect of the nostril to the level made with the permanent marker. if you are inserting the tube into the nasopharynx, push the nasal meatus dorsally while simultaneously pushing the lateral aspect of the nostril medially to direct the tube into the ventral nasal meatus and avoid the cribriform plate. 7. once the tube has been inserted to the appropriate length, hold the tube in place with your fingers adjacent to the nostril, and suture the tube to the stay suture. if the tube is removed, you can cut the suture around the tube and leave the stay suture in place for later use, if necessary. 8. suture or staple the rest of the tube dorsally over the nose and in between the eyes to the top of the head, or laterally along the zygomatic arch. 9. attach the tube to a length of flexible oxygen tubing, and provide humidified oxygen at 50 to 100 ml/kg/minute. 10. secure an elizabethan collar around the patient's head to prevent the patient from scratching at the tube and removing it. the rule of 60s states that if a patient's pao 2 is less than 60 mm hg, or if the paco 2 is 60 mm hg, mechanical ventilation should be considered. for mechanical ventilation, anesthetize the patient and intubate the patient with an endotracheal tube. alternately, a temporary tracheostomy can be performed and the patient can be maintained on a plane of light to heavy sedation and ventilated through the tracheostomy site. this method, a noninvasive means of determining oxygenation is through the use of pulse oximetry. a pulse oximeter uses different wavelengths of light to distinguish characteristic differences in the properties of the different molecules in a fluid or gas mixture, in this case, oxygenated (oxyhemoglobin) and deoxygenated hemoglobin (deoxyhemoglobin) in pulsatile blood. the process is termed pulse oximetry. oxyhemoglobin and deoxyhemoglobin are different molecules that absorb and reflect different wavelengths of light. oxyhemoglobin absorbs light in the infrared spectrum, allowing wavelengths of light in the red spectrum to transmit through it. conversely, deoxyhemoglobin absorbs wavelengths of the red spectrum and allows wavelengths in the infrared spectrum to transmit through the molecule. the spectrophotometer in the pulse oximeter transmits light in the red (660 nanometers) and infrared (920 nanometers) spectra. the different wavelengths of light are transmitted across a pulsatile vascular bed and are detected by a photodetector on the other side. the photodetector processes the amount of light of varying wavelengths that reaches it, then transmits an electrical current to a processor that calculates the difference in the amount of light originally transmitted and the amount of light of similar wavelength that actually reaches the photodetector. the difference in each reflects the amount of light absorbed in the pulsatile blood and can be used to calculate the amount or ratio of oxyhemoglobin to deoxyhemoglobin in circulation, or the functional hemoglobin saturation by the formula: where hbo 2 is oxygenated hemoglobin, and hb is deoxygenated hemoglobin. four molecules of oxygen reversibly bind to hemoglobin for transport to the tissues. carbon monoxide similarly binds to hemoglobin and forms carboxyhemoglobin, a molecule that is detected similarly as oxygenated hemoglobin. thus sao 2 as detected by a pulse oximeter is not reliable if carboxyhemoglobin is present. in most cases, pulse oximetry or sao 2 corresponds reliably to the oxyhemoglobin dissociation curve. oxygen saturation greater than 90% corresponds to a pao 2 greater than 60 mm hg. above this value, large changes in pao 2 are reflected in relatively small changes in sao 2 , making pulse oximetry a relatively insensitive method of determining oxygenation status when pao 2 is normal. because pulse oximetry measures oxygenated versus nonoxygenated hemoglobin in pulsatile blood flow, it is fairly unreliable when severe vasoconstriction, hypothermia, shivering or trembling, or excessive patient movement are present. additionally, increased ambient lighting and the presence of methemoglobin or carboxyhemoglobin also can cause artifactual changes in the sao 2 , and thus the measurement is not reliable or accurate. most pulse oximeters also display a waveform and the patient's heart rate. if the photodetector does not detect a good quality signal, the waveform will not be normal, and the heart rate displayed on the monitor will not correlate with the patient's actual heart rate. the efficiency of ventilation is evaluated using the paco 2 value on an arterial blood gas sample. alternatively, a noninvasive method to determine end-tidal carbon dioxide is through use of a capnograph. the science of capnometry uses a spectrophotometer to measure carbon dioxide levels in exhaled gas. the capnometer is placed in the expiratory limb of an anesthetic circuit. a sample of exhaled gas is aliquoted from the breath, and an infrared light source is passed across the sample. a photodetector on the other side of the sample flow measures the amount or concentration of carbon dioxide in the sample of expired gas. the calculated value is displayed as end-tidal carbon dioxide. this value also can be displayed as a waveform. when placed in graphic form, a waveform known as a capnograph is displayed throughout the ventilatory cycle. normally, at the onset of exhalation, the gas exhaled into the expiratory limb of the tubing comes from the upper airway or physiologic dead space and contains relatively little carbon dioxide. as exhalation continues, a steep uphill slope occurs as more carbon dioxide is exhaled from the bronchial tree. near the end of exhalation, the capnogram reaches a plateau, which most accurately reflects the carbon dioxide level at the level of the alveolus. because carbon dioxide diffuses across the alveolar basement membrane so rapidly, this reflects arterial carbon dioxide levels. if a plateau is not reached and notching of the waveform occurs, check the system for leaks. if the baseline waveform does not reach zero, the patient may be rebreathing carbon dioxide or may be tachypneic, causing physiologic positive end-expiratory pressure. the soda-sorb in the system should be replaced if it has expired. conversely, low end-tidal carbon dioxide may be associated with a decrease in perfusion or blood flow. decreased perfusion can be associated with low end-tidal carbon dioxide values, particularly during cardiopulmonary cerebral resuscitation. end-tidal carbon dioxide levels are one of the most accurate predictors of the efficacy of cardiopulmonary cerebral resuscitation and patient outcome. additionally, the difference between arterial carbon dioxide levels (paco 2 ) and end-tidal carbon dioxide can be used to calculate dead-space ventilation. increases in the difference also occur with poor lung perfusion and pulmonary diffusion impairment. thoracocentesis refers to the aspiration of fluid or air from within the pleural space. thoracocentesis may be diagnostic to determine whether air or fluid is present and to characterize the nature of the fluid obtained. thoracocentesis also can be therapeutic when removing large volumes of air or fluid to allow pulmonary reexpansion and correction of hypoxemia and orthopnea. to perform thoracocentesis, follow this procedure: 1. first, assemble the equipment necessary (box 1-16). 2. next, clip a 10-cm square in the center of the patient's thorax on both sides. 3. aseptically scrub the clipped area. 4. ideally, thoracocentesis should be performed within the seventh to ninth intercostal space. rather than count rib spaces in an emergent situation, visualize the thoracic cage as a box, and the clipped area as a box within the box. you will insert your needle or catheter in the center of the box and then direct the bevel of the needle dorsally or ventrally to penetrate pockets of fluid or air present. 5. attach the needle or catheter hub to the length of intravenous extension tubing. attach the female port of the intravenous extension tubing to the male port of the three-way stopcock. attach the male port of the 60-ml syringe to one of the female ports of the three-way stopcock. the apparatus is now assembled for use. 6. insert the needle through the intercostal space such that the bevel of the needle initially is directed downward. 7. next, push down on the hub of the needle such that the needle becomes parallel with the thoracic wall. by moving the hub of the needle in a clockwise or counterclockwise manner, the bevel of the needle will move within the thoracic cavity to penetrate pockets of air or fluid. in general, air is located dorsally and fluid is located more ventrally, although this does not always occur. 8. aspirate air or fluid. save any fluid obtained for cytologic and biochemical analyses and bacterial culture and susceptibility testing. in cases of pneumothorax, if the thoracocentesis needs to be repeated more than 3 times, consider using a thoracostomy tube. place a thoracostomy tube in cases of pneumothorax whenever negative suction cannot be obtained or repeated accumulation of air requires multiple thoracocentesis procedures. thoracostomy tubes also can be placed to drain rapidly accumulating pleural effusion and for the medical management of pyothorax. before attempting thoracostomy tube placement, make sure that all necessary supplies are assembled (box 1-17; table 1-13) . to place a thoracostomy tube, follow this procedure: 1. lay the patient in lateral recumbency. 2. clip the patient's entire lateral thorax. 3. aseptically scrub the lateral thorax. 4. palpate the tenth intercostal space. 5. have an assistant pull the patient's skin cranially and ventrally toward the point of the elbow. this will facilitate creating a subcutaneous tunnel around the thoracostomy tube. 6. draw up 2 mg/kg 2% lidocaine (1 mg/kg for cats) along with a small amount of sodium bicarbonate to take away some of the sting. 7. insert the needle at the dorsal aspect of the tenth intercostal space and to the seventh intercostal space. inject the lidocaine into the seventh intercostal space at the point where the trocarized thoracic drainage catheter will penetrate into the thoracic cavity. slowly infuse the lidocaine as you withdraw the needle to create an anesthetized tunnel through which to insert the catheter. 8. while the local anesthetic is taking effect, remove the trocar from the catheter and cut the proximal end of the catheter with a mayo scissors to facilitate adaptation with the christmas tree adapter. 9. attach the christmas tree adapter to the three-way stopcock and the three-way stopcock to a length of intravenous extension tubing and the 60-ml syringe so that the apparatus can be attached immediately to the thoracostomy tube after placement. 10. aseptically scrub the lateral thorax a second time and then drape it with sterile huck towels secured with towel clamps. 11. wearing sterile gloves, make a small stab incision at the dorsal aspect of the tenth intercostal space. 12. insert the trocar back into the thoracostomy drainage tube. insert the trocar and tube into the incision. tunnel the tube cranially for approximately 3 intercostal spaces while an assistant simultaneously pulls the skin cranially and ventrally toward the point of the elbow. 13. at the seventh intercostal space, direct the trocar and catheter perpendicular to the thorax. grasp the catheter apparatus at the base adjacent to the thorax to prevent the trocar from going too far into the thorax. 14. place the palm of your dominant hand over the end of the trocar, and push the trocar and catheter into the thoracic cavity, throwing your weight into the placement in a swift motion, not by banging the butt of your hand on the end of the stylette. for small individuals, standing on a stool, or kneeling over the patient on the triage table can create leverage and make this process easier. the tube will enter the thorax with a pop. 15. gently push the catheter off of the stylette, and remove the stylette. 16. immediately attach the christmas tree adapter and have an assistant start to withdraw air or fluid while you secure the tube in place. 17. first, place a horizontal mattress suture around the tube to cinch the skin securely to the tube. use care to not penetrate the tube with your needle and suture. 18. next, place a purse-string suture around the tube at the tube entrance site. leave the ends of the suture long, so that you can create a finger-trap suture to the tube, holding the tube in place. 19. place a large square of antimicrobial-impregnated adhesive tape over the tube for further security and sterility. 20. if antimicrobial adhesive is not available, place a gauze pad 4 ã� 4 inches square over the tube, and then wrap the tube to the thorax with cotton roll gauze and elastikon adhesive tape. 21. draw the location of the tube on the bandage to prevent cutting it with subsequent bandage changes. an alternate technique to use if a trocar thoracic drainage catheter is not available is the following: 1. prepare the lateral thorax and infuse local lidocaine anesthetic as listed before. 2. make a small stab incision with a no. 10 scalpel blade, as listed before. 3. obtain the appropriately sized red rubber catheter and cut multiple side ports in the distal end of the catheter, taking care to not cut more than 50% of the circumference of the diameter of the tube. 4. insert a rigid, long urinary catheter into the red rubber catheter to make the catheter more rigid during insertion into the pleural space. 5. grasp the distal end of the catheter(s) in the teeth of a large carmalt. tunnel a metzenbaum scissors under the skin to the seventh intercostal space and make a puncture through the intercostal space. 6. remove the metzenbaum scissors, and then tunnel the carmalt and red rubber tube under the skin to the hole created in the seventh intercostal space with the metzenbaum scissors. 7. insert the tips of the carmalt and the red rubber catheter through the hole, and then open the teeth of the carmalt. 8. push the red rubber catheter cranially into the pleural cavity. 9. remove the carmalt and the rigid urinary catheter, and immediately attach the suction apparatus. secure the red rubber catheter in place as listed before. placement of a temporary tracheostomy can be lifesaving to relieve upper respiratory tract obstruction, to facilitate removal of airway secretions, to decrease dead space ventilation, to provide a route of inhalant anesthesia during maxillofacial surgery, and to facilitate mechanical ventilation. in an emergent situation in which asphyxiation is imminent and endotracheal intubation is not possible, any cutting instrument placed into the trachea distal to the point of obstruction can be used. to perform a slash tracheostomy, quickly clip the fur and scrub the skin over the third tracheal ring. make a small cut in the trachea with a no. 11 scalpel blade, and insert a firm tube, such as a syringe casing. alternately, insertion of a 22-gauge needle attached to intravenous extension tubing and adapted with a 1-ml syringe case to attach to a humidified oxygen source also temporarily can relieve obstruction until a temporary tracheostomy can be performed. in less emergent situations, place the patient under general anesthesia and intubate the patient. assemble all the equipment necessary before starting the temporary tracheostomy procedure (box 1-18). to perform a tracheostomy, follow this procedure: 1. place the patient in dorsal recumbency. 2. clip the ventral cervical region from the level of the ramus of the mandible caudally to the thoracic inlet and dorsally to midline. 3. aseptically scrub the clipped area, and then drape with sterile huck towels secured with towel clamps. 4. make a 3-cm ventral midline skin incision over the third to sixth tracheal rings, perpendicular to the trachea. 5. bluntly dissect through the sternohyoid muscles to the level of the trachea. 6. carefully pick up the fascia overlying the trachea and cut it away with a metzenbaum scissors. 7. place two stay sutures through/around adjacent tracheal rings. 8. incise in between trachea rings with a no. 11 scalpel blade. take care to not cut more than 50% of the circumference of the trachea. 9. using the stay sutures, pull the edges of the tracheal incision apart, and insert the tracheostomy tube. the shiley tube contains an internal obturator to facilitate placement into the tracheal lumen. remove the obturator, and then insert the inner cannula, which can be removed for cleaning as needed. 10. once the tube is in place, secure the tube around the neck with a length of sterile umbilical tape. postoperative care of the tracheostomy tube is as important as the procedure itself. because the tracheostomy tube essentially bypasses the protective effects of the upper respiratory system, one of the most important aspects of tracheostomy tube care and maintenance is to maintain sterility at all times. any oxygen source should be humidified with sterile water or saline to prevent drying of the respiratory mucosa. if supplemental oxygen is not required, instill 2 to 3 ml of sterile saline every 1 to 2 hours to moisten the mucosa. wearing sterile gloves, remove the internal tube and place it in a sterile bowl filled with sterile hydrogen peroxide and to be cleaned every 4 hours (or more frequently as necessary). if a shiley tube is not available, apply suction to the internal lumen of the tracheostomy tube every 1 to 2 hours (or more frequently as needed) with a sterile 12f red rubber catheter attached to a vacuum pump to remove any mucus or other debris that potentially could plug the tube. unless the patient demonstrates clinical signs of fever or infection, the prophylactic use of antibiotics is discouraged because of the risk of causing a resistant infection. after the temporary tracheostomy is no longer necessary, remove the tube and sutures, and leave the wound to heal by second intention. primary closure of the wounds could predispose the patient to subcutaneous emphysema and infection. baker gd: trans-tracheal oxygen therapy in dogs with severe respiratory compromise due to tick (i. holocyclus) toxicity, aust vet pract 34 (2) urohydropulsion is a therapeutic procedure for removal of uroliths from the urethra of the male dog. the technique works best if the animal is heavily sedated or is placed under general anesthesia (figure 1-12) . to perform urohydropulsion, follow this procedure: 1. place the animal in lateral recumbency. 2. clip the fur from the distal portion of the prepuce. 3. aseptically scrub the prepuce and flush the prepuce with 12 to 20 ml of antimicrobial flush solution. 4. have an assistant who is wearing gloves retract the penis from the prepuce. 5. while wearing sterile gloves, lubricate the tip of a rigid urinary catheter as for urethral catheterization. 6. gently insert the tip of the catheter into the urethra until you meet the resistance of the obstruction. 7. pinch the tip of the penis around the catheter. 8. have an assistant insert a gloved lubricated finger into the patient's rectum and press ventrally on the floor of the rectum to obstruct the pelvic urethra. 9. attach a 60-ml syringe filled with sterile saline into proximal tip of the catheter. 10. quickly inject fluid into the catheter and alternate compression and relaxation on the pelvic urethra such that the urethra dilates and suddenly releases the pressure, causing dislodgement of the stone. small stones may be ejected from the tip of the urethra, whereas larger stones may be retropulsed back into the urinary bladder to be removed surgically at a later time. the type of catheter that you choose for vascular access depends largely on the size and species of the patient, the fragility of the vessels to be catheterized, the proposed length of time that the catheter will be in place, the type and viscosity of the fluid or drug to be administered, the rate of fluid flow desired, and whether multiple repeated blood samples will be required (table 1-14) . a variety of over-the-needle, through-the-needle, and over-the-wire catheters are available for placement in a variety of vessels, including the jugular, cephalic, accessory cephalic, medial saphenous, lateral saphenous, dorsal pedal artery, and femoral artery. one of the most important aspects of proper catheter placement and maintenance is to maintain cleanliness at all times. the patient's urine, feces, saliva, and vomit are common sources of contamination of the catheter site. before placing a peripheral or central catheter in any patient, consider the patient's physical status including whether vomiting, diarrhea, excessive urination, or seizures. in a patient with an oral mass that is drooling excessively or a patient that is vomiting, peripheral cephalic catheterization may not be the most appropriate, to prevent contamination. conversely, in a patient with excessive urination or diarrhea, a lateral or medial saphenous catheter is likely to become contaminated quickly. whenever one places or handles a catheter or intravenous infusion line, the person should wash the hands carefully and wear gloves to prevent contamination of the intravenous catheter and fluid lines. one of the most common sources of catheter contamination in veterinary hospitals is through caretakers' hands. in emergent situations, placement of a catheter may be necessary under less than ideal circumstances. remove those catheters as soon as the patient is more stable, and place a second catheter using aseptic techniques. in general, once the location of the catheter has been decided, set up all equipment necessary for catheter placement before starting to handle and restrain the patient. lists the equipment needed for most types of catheter placement. after setting up all of the supplies needed, clip the fur over the site of catheter placement. make sure to clip all excess fur and long feathers away from the catheter site, to prevent contamination. for catheter placement in limbs, clip the fur circumferentially around the site of catheter placement to facilitate adherence of the tape to the limb and to facilitate catheter removal with minimal discomfort at a later date. next, aseptically scrub the catheter site with an antimicrobial scrub solution such as hibiclens. the site is now ready for catheter insertion. consider using a central venous catheter whenever multiple repeated blood samples will need to be collected from a patient during the hospital stay. central venous catheters also can be used for cvp measurement, administration of hyperoncotic solutions such as parenteral nutrition, and administration of crystalloid and colloid fluids, anesthesia, and other injectable drugs (figures 1-13 and 1-14) . to place a jugular central venous catheter, place the patient in lateral recumbancy and extend the head and neck such that the jugular furrow is straight. clip the fur from the ramus of the mandible caudally to the thoracic inlet and dorsally and ventrally to midline. wipe the clipped area with gauze 4 ã� 4-inch squares to remove any loose fur and other debris. aseptically scrub the clipped area with an antimicrobial cleanser. venocaths (abbott laboratories) are a through-the-needle catheter that is contained within a sterile sleeve for placement. alternately, other over-the-wire central venous catheters can be placed by the seldinger technique. sterility must be maintained at all times, regardless of the type of catheter placed. wearing sterile gloves, drape the site of catheter placement with sterile drapes, and occlude the jugular vein at the level of the thoracic inlet. pull the clear ring and wings of emergency diagnostic and therapeutic procedures 59 1 figure 1 -13: lateral thoracic radiograph of a central venous catheter. note that the tip of the catheter is inserted in its proper location, just outside of the right atrium. the catheter cover down toward the catheter itself to expose the needle. remove the guard off of the needle. lift the skin over the proposed site of catheter insertion and insert the needle under the skin, with the bevel of the needle facing up. next, reocclude the vessel and pull the skin tight over the vessel to prevent movement of the vessel as you attempt to insert the needle. in some cases, it may be difficult actually to see the vessel in obese patients. if you cannot visualize or palpate the needle, gently bounce the needle over the vessel with the bevel up. the vessel will bounce in place slightly, allowing a brief moment of visualization to facilitate catheter placement. once the vessel has been isolated and visualized, insert the needle into the vessel at a 15-to 30-degree angle. watch closely for a flash of blood in the catheter. when blood is observed, insert the needle a small distance farther, and then push the catheter and stylette into the vessel for the entire length, until the catheter and stylette can be secured in the catheter hub. if the catheter cannot be inserted fully into the vessel for its entire length, the tip of the needle may not be within the entire lumen, the catheter may be directed perivascularly, and the catheter may be caught at the thoracic flexure and may be moving into one of the tributaries that feeds the forelimb. extend the patient's head and neck, and lift the forelimb up to help facilitate placement. do not force the catheter in because the catheter potentially can form a knot and will need to be removed surgically. remove the needle from the vessel, and have an assistant place several 4 ã� 4-inch gauze squares over the site of catheter placement with some pressure to control hemorrhage. secure the catheter hub into the needle guard, and remove the stylette from the catheter. immediately insert a 3-to 6-ml syringe of heparinized saline and flush the catheter and draw back. if you are in the correct place, you will be able to draw blood from the catheter. to secure the catheter in place, tear a length of 1-inch white tape that will wrap around the patient's neck. pull a small length of the catheter out of the jugular vein to make a semicircle. the semicircle should be approximately 1 /2 inch in diameter. let the length of catheter lie on the skin, and then place 4 ã� 4-inch gauze squares impregnated with antimicrobial ointment over the site of catheter insertion. secure the proximal end of white tape around the white and blue pieces of the catheter, and wrap the tape around the patient's neck so that the tape adheres to the skin and fur. repeat the process by securing the gauze to the skin with two additional lengths of white tape, starting to secure the gauze in place by first wrapping the tape dorsally over the patient's neck, rather than under the patient's neck. in between each piece of tape and bandage layer, make sure that the catheter flushes and draws back freely, or else occlusion can occur. gently wrap layers of cotton roll gauze, kling, and elastikon or vetrap over the catheter. secure a male adapter or t port that has been flushed with heparinized saline, and then label the catheter with the size and length of catheter, date of catheter placement, and initials of the person who placed the catheter. the catheter is ready for use. monitor the catheter site daily for erythema, drainage, vessel thickening, or pain upon infusion. if any of these signs occur, or if the patient develops a fever of unknown origin, remove the catheter, culture the catheter tip aseptically, and replace the catheter in a different location. as long as the catheter is functional without complications, the catheter can remain in place. central catheters also can be placed via the seldinger or over-the-wire technique. a number of companies manufacture kits that contain the supplies necessary for over-the-wire catheter placement. each kit minimally should contain an over-the-needle catheter to place into the vessel, a long wire to insert through the original catheter placed, a vascular dilator to dilate the hole in the vessel created by the first catheter, and a long catheter to place into the vessel over the wire. additional accessories can include a paper drape, sterile gauze, a scalpel blade, local anesthetic, 22-gauge needles, and 3-or 6-ml syringes. restrain the patient and prepare the jugular furrow aseptically as for the percutaneous through-the-needle catheter placement. the person placing the catheter should wear sterile gloves throughout the process to maintain sterility. pick up the skin over the site of catheter placement, and insert a small bleb of local anesthetic through the skin. the local anesthetic should not be injected into the underlying vessel (figure 1-15) . make a small nick into the skin through the local anesthetic with a no. 10 or no. 11 scalpel blade. use care to avoid lacerating the underlying vessel. next, occlude the jugular vein as previously described, and insert the over-the-needle catheter into the vessel. watch for a flash of blood in the catheter hub. remove the stylette from the catheter. next, insert the long wire into the catheter and into the vessel (figures 1-16 and 1-17) . never let go of the wire. remove the catheter, and place the vascular dilator over the wire and into the vessel (figure 1-18) . gently twist to place the dilator into the vessel a short distance, creating a larger hole in the vessel. the vessel will bleed more after creating a larger hole. remove the vascular dilator, and leave the wire in place within the vessel. insert the long catheter over the wire into the vessel (figure 1-19) . push the catheter into the vessel to the catheter hub (figure 1-20) . slowly thread the wire through a proximal port in the catheter. once the catheter is in place, remove the wire, and suture the catheter in place to the skin with nonabsorbable suture. cover the catheter site with sterile gauze and antimicrobial ointment, cotton roll bandaging material, gauze, and kling or vetrap. flush the catheter with heparinized saline solution, and then use the catheter for infusion of parenteral nutrition, blood products, crystalloid and colloid fluids, medications, and frequent blood sample collection. examine the catheter site daily for evidence of infection or thrombophlebitis. the catheter can remain in place as long as it functions and no complications occur. place the patient in sternal recumbency as for cephalic venipuncture. clip the antebrachium circumferentially, and wipe the area clean of any loose fur and debris (figure 1-21) . aseptically scrub the clipped area, and have an assistant occlude the cephalic vein at the crook of the elbow. the person placing the catheter should grasp the distal carpus with the nondominant hand and insert the over-the-needle catheter into the vessel at a 15-to 30-degree angle ( figure 1-22) . watch for a flash of blood in the catheter hub, and then gently push the catheter off of the stylette (figure 1-23) . have the assistant occlude the vessel over the catheter to prevent backflow. flush the catheter with heparinized saline solution. make sure that the skin and catheter hub are clean and dry to ensure that the tape adheres to the catheter hub and skin. secure a length of 1 /2-inch white tape tightly around the catheter and then around the limb. make sure that the catheter hub does not "spin" in the tape, or else the catheter will fall out. next, secure a second length of 1-inch adhesive tape under the catheter and around the limb and catheter hub (figure 1-24 ). this piece of tape helps to stabilize the catheter in place. finally, place a flushed t port or male adapter in the catheter hub and secure to the limb with white tape. make sure that the tape is adhered to the skin securely, but not so tightly as to impede venous outflow (figure 1-25) . the catheter site can be covered with a cotton ball impregnated with antimicrobial ointment and layers of bandage material. label all catheters with the date of placement, the type and gauge of catheter inserted, and the initials of the person who placed the catheter. the femoral artery can be catheterized for placement of an indwelling arterial catheter. indwelling arterial catheters can be used for continuous invasive arterial blood pressure monitoring and for procurement of arterial blood samples. place the patient in lateral recumbancy, and tape the down leg in an extended position. clip the fur over the femoral artery and aseptically scrub the clipped area. palpate the femoral artery as it courses distally on the medial surface of the femur and anterior to the pectineus muscle. make a small nick incision over the proposed site of catheter placement using the bevel of an 18-gauge needle. place a long over-the-needle catheter through the nick in the skin and direct it toward the palpable pulse. place the tip of the catheter so that the needle tip rests in the subcutaneous tissue between the artery and the palpating index finger. advance the needle steeply at a 30-degree angle to secure the superficial wall of the vessel and then the deep wall of the vessel. the spontaneous flow of blood in the catheter hub ensures that the catheter is 1 figure 1 -25: catheter is taped in place with a t-port. situated in the lumen of the artery. feed the catheter off of the stylette, and cover the hub with a catheter cap. flush the catheter with sterile heparinized saline solution, and then secure it in place. some persons simply tape the catheter in place with pieces of 1 /2-and 1-inch adhesive tape. others use a "butterfly" piece of tape around the catheter hub and suture or glue the tape to the adjacent skin for added security. the dorsal pedal artery commonly is used for catheter placement. to place a dorsal pedal arterial catheter, place the patient in lateral recumbency. clip the fur over the dorsal pedal artery, and then aseptically scrub the clipped area. tape the distal limb so that the leg is twisted slightly medially for better exposure of the vessel, or the person placing the arterial catheter can manipulate the limb into the appropriate position. palpate the dorsal pedal pulse as it courses dorsally over the tarsus. place an over-the-needle catheter percutaneously at a 15-to 30-degree angle, threading the tip of the needle carefully toward the pulse. advance the needle in short, blunt movements, and watch the catheter hub closely for a flash of pulsating blood that signifies penetration into the lumen of the artery. then thread the catheter off of the stylette, and cover the catheter hub with a catheter cap. secure the catheter in place with lengths of 1 /2-and 1-inch adhesive tape as with any other intravenous catheter, and then flush it with heparinized saline solution every 2 to 4 hours. any vessel that can be catheterized percutaneously also can be catheterized with surgical cutdown. restrain the patient and clip and aseptically scrub the limb or jugular vein as for a percutaneous catheterization procedure. block the area for catheter placement with a local anesthetic before cutting the skin over the vessel with a no. 11 scalpel blade. while wearing sterile gloves, pick up the skin and incise the skin over the vessel. direct the sharp edge of the blade upward to avoid lacerating the underlying vessel. using blunt dissection, push the underlying subcutaneous fat and perivascular fascia away from the vessel with a mosquito hemostat. make sure that all tissue is removed from the vessel. using the mosquito hemostat, place two stay sutures of absorbable suture under the vessel. elevate the vessel until it is parallel with the incision, and gently insert the catheter and stylette into the vessel. secure the stay sutures loosely around the catheter. suture the skin over the catheter site with nonabsorbable suture, and then tape and bandage the catheter in place as for percutaneous placement. remove catheters placed surgically as soon as possible and exchange them for a percutaneously placed catheter to avoid infection and thrombophlebitis. the most important aspect of catheter maintenance is to maintain cleanliness and sterility at all times. an indwelling catheter can remain in place for as long as it is functional and no complications occur. change the bandage whenever it becomes wet or soiled to prevent wicking of bacteria and debris from the environment into the vessel. check the bandages and catheter sites at least once a day for signs of thrombophlebitis: erythema, vessel hardening or ropiness, pain upon injection or infusion, and discharge. also closely examine the tissue around and proximal and distal to the catheter. swelling of the paw can signify that the catheter tape and bandage are too tight and are occluding venous outflow. swelling above the catheter site is characteristic of perivascular leakage of fluid and may signify that the catheter is no longer within the lumen of the vessel. remove the catheter if it is no longer functional, if there is pain or resistance upon infusion, if there is unexplained fever or leukocytosis, or if there is evidence of cellulitis, thrombophlebitis, or catheter-related bacteremia or septicemia. aseptically culture the tip of the indwelling catheter for bacteria. animals should wear elizabethan collars or other forms of restraint if they lick or chew at the catheter or bandage. catheter patency may be maintained with constant fluid infusion or by intermittent flushing with heparinized saline (1000 units of unfractionated heparin per 250 to 500 ml of saline) every 6 hours. flush arterial catheters more frequently (every 2 hours). disconnect intravenous connections only when absolutely necessary. wear gloves whenever handling the catheter or connections. label all fluid lines and elevate them off of the floor to prevent contamination. date each fluid line and replace it once every 24 to 36 hours. if an intravenous catheter cannot be placed because of small patient size, hypovolemia, hypothermia, or severe hypotension, needles can be placed into the marrow cavity of the femur, humerus, and tibia for intraosseous infusion of fluids, drugs, and blood products. this technique is particularly useful in small kittens and puppies and in exotic species. contraindications to intraosseous infusion is in avian species (which have air in their bones), fractures, and sepsis, because osteomyelitis can develop. an intraosseous catheter is relatively easy to place and maintain but can cause patient discomfort and so should be changed to an intravenous catheter as soon as vascular access becomes possible. to place an intraosseous catheter, clip and aseptically scrub the fur over the proposed site of catheter placement. the easiest place for intraosseous placement is in the intertrochanteric fossa of the femur. inject a small amount of a local anesthetic through the skin and into the periosteum where the trocar or needle will be inserted. place the patient in lateral recumbency, and grasp the leg in between your fingers, with the stifle braced against the palm of your hand. push the stifle toward the abdomen (medially) to abduct the proximal femur away from the body. this will shift the sciatic nerve out of the way of catheter placement. insert the tip of the needle through the skin and into the intertrochanteric fossa. gently push with a simultaneous twisting motion, pushing the needle parallel with the shaft of the femur, toward your palm. you may feel a pop or decreased resistance as the needle enters the marrow cavity. gently flush the needle with heparinized saline. if the needle is plugged with bone debris, remove the needle and replace it with a fresh needle of the same type and size in the hole that you have created. a spinal needle with an internal stylette also can be placed. the stylette will prevent the needle from becoming clogged with bone debris during insertion. secure the hub of the needle with a butterfly length of white adhesive tape and then suture it to the skin to keep the catheter in place. the catheter is now ready for use. the patient should wear an elizabethan collar to prevent disruption or removal of the catheter. the intraosseous catheter can be maintained as any peripheral catheter, with frequent flushing and daily evaluation of the catheter site. the definition of pain has been debated philosophically over the ages and has changed as knowledge has increased. pain is defined as an unpleasant sensory or emotional experience associated with actual or perceived tissue damage. until recognition of a noxious stimulus occurs in the cerebral cortex, no response or adaptation results. rational management of pain requires an understanding of the underlying mechanisms involved in pain and an appreciation of how analgesic agents interact to disrupt pain mechanisms. multiple factors and causes produce pain in human beings and domestic animal species. the causes of pain, psychological and physical, may derive from many different mechanisms within emergency medicine, among them trauma, infectious disease, neglect, environmental stress, surgery, and acute decompensation of chronic medical conditions. the two major classes of pain are acute and chronic pain. box 1-20 gives specific categories and causes of pain. the pain sensing and response system can be divided into the following categories: nociceptors, which detect and filter the intensity of the noxious stimuli; primary afferent nerves, which transmit impulses to the central nervous system (cns); ascending tracts, which are part of the dorsal horn and the spinal cord that conveys stimuli to higher centers in the brain; higher centers, which are involved in pain discrimination, some memory, and motor control; and modulating or descending systems, which are a means of processing, memorizing, and modifying incoming impulses. current analgesic therapies may inhibit afferent nociceptive transmission within the brain and spinal cord; directly interrupt neural impulse conduction through the dorsal horn, primary afferent nerves, or dorsal root ganglion; or prevent the nociceptor sensitization that accompanies initial pain and inflammation. the physiologic aspects of pain are believed to be produced by the transmission, transduction, and integration of initial nerve endings, peripheral neuronal input, and ascending afferent nerves via the thalamus to the cerebral cortex. ascending afferent nerves to the limbic system are believed to be responsible for the emotional aspects of pain. there are several classification schemes for different types of pain. acute pain, such as that which results from trauma, surgery, or infectious agents, is abrupt in onset, relatively short in duration, and may be alleviated easily by analgesics. in contrast, chronic pain is a long-standing physical disorder or emotional distress that is slow in onset and difficult to treat. both types of pain can be classified further based on site of origin. somatic pain arises from superficial skin, subcutaneous tissue, body wall, or appendages. visceral pain arises from abdominal or thoracic viscera and primarily is associated with serosal irritation. analgesia, then, is the loss of pain without the loss of consciousness. this is in contrast to anesthesia, which is the loss of sensation in the whole body or a part of the body with the loss of consciousness or at least depression of the cns. untreated pain causes immediate changes in the neurohormonal axis, which in turn causes restlessness, agitation, increased heart and respiratory rates, fever, and blood pressure fluctuations, all of which are detrimental to the healing of the animal. a catabolic state is created as a result of increased secretion of catabolic hormones and decreased secretion of anabolic hormones. the net effect the majority of neurohormonal changes produce is an increase in the secretion of catabolic hormones. hyperglycemia is produced and may persist because of production of glucagon and relative lack of insulin. lipolytic activity is stimulated by cortisol, catecholamines, and growth hormone. cardiorespiratory effects of pain include increased cardiac output, vasoconstriction, hypoxemia, and hyperventilation. protein catabolism is a common occurrence and major concern regarding healing. pain associated with inflammation causes increase in tissue and blood levels of prostaglandins and cytokines, both of which promote protein catabolism indirectly by increasing the energy expenditure of the body. powerful evidence indicates that local anesthetic, sympathetic agonist, and opioid neural blockade may produce a modification of the responses to these physiologic changes. variable reduction in plasma cortisol, growth hormone, antidiuretic hormone, î²-endorphin, aldosterone, epinephrine, norepinephrine, and renin is based on the anesthetic technique and the drugs selected. prophylactic administration of analgesics blunts the response before it occurs; analgesics administered following perception or pain are not as effective, and higher doses are generally necessary to achieve an equivalent level of analgesia. effective pain control can be achieved only when the signs of pain can be assessed effectively, reliably, and regularly. the experience of pain is unique to each individual, which makes pain assessment difficult, especially in traumatized and critical patients. most attempts to assess clinical pain use behavioral observations and interactive variables in addition to assessment of physiologic responses such as heart rate and respiratory rate, blood pressure, and temperature. but many factors can influence the processing and outward projection of pain, including altered environments, species differences, withinspecies variations (age, breed, sex), and the type, severity, and chronicity of pain. within-species differences (age, breed, and sex) further complicate the pain assessment. most notable is that different breeds of dogs act differently when confronted with pain or fear. labrador retrievers tend to be stoic, whereas greyhounds and teacup breeds tend to react with a heightened state of arousal around even the simplest of procedures (e.g., subcutaneous injections and nail trims). the individual character and temperament of the animal further influences its response. pediatric and neonatal animals seem to have a lower threshold for pain and anxiety than older animals. in any species, the duration and type of pain make it more (acute) or less (chronic) likely to be expressed or exhibited outwardly. unfamiliarity with normal behaviors typical of a particular species or breed makes recognition of their painful behaviors and responses impossible. the definition and recognition of pain in an individual animal is challenging. because of all the differences discussed, there is no straight line from insult, albeit actual or perceived, to degree of pain experienced. nor is there a formula for treating "x" type of pain with "y" type of analgesic. a goal of analgesia is to treat all animals with analgesic drugs and modalities as preemptively as possible and using a multimodal approach. use analgesic treatment as a tool for diagnosis of pain in the event that recognition of these phenomena is difficult for the patient. in other words, with countless drugs and treatment modalities available, analgesic administration should never be withheld in an animal, even if pain is questionable. it is important to remember that no behavior or physiologic variable in and of itself is pathognomonic for pain. interactive and unprovoked (noninteractive) behavior assessments and trending of physiologic data are useful to determine the pain in an individual animal. this is known as pain scoring. baseline observations, especially those observations from someone who has known the animal well, can be helpful to serial behavior and pain assessments. pain scoring systems have been developed and are reviewed elsewhere; the purposes of these systems are to evaluate and to help guide diagnostic and analgesic treatments (table 1 -15) . regardless of the scale or method used to assess pain, the caregiver must recognize the limitations of the scale. if in doubt of whether pain is present or not, analgesic therapy should be used as a diagnostic tool. classic behaviors associated with pain in dogs and cats include abnormal postures, gaits, movements, and behaviors (boxes 1-21 and . stoicism is the apparent apathy and pain: assessment, prevention, and management 71 indifference in the presence of pain and is perhaps the no. 1 sign of ineffective pain relief or persistent pain in many animals, because so many display apathy and classically normal physiologic parameters even in the face of severe distress, overt suffering, or blatant trauma and illness. the absence of normal behaviors is also a clinical sign of pain, even when abnormal behaviors are not observed. acute pain results in many of the aforementioned behavioral and physiologic signs, but chronic pain in small animals is an entirely different and distinct entity. chronic pain is often present in the absence of obvious tissue pathology and changes in physical demeanor. again, the severity of the pain may not correlate with the severity of any pathologic condition that may or may not be present. chronic pain, especially if insidious in onset (cancer, dental, or degenerative pain), may well go unnoticed in dogs and cats, even by family members or intermittent caregivers. inappetance, lack of activity, panting in a species classically designed to be nose breathers, decreased interest in surroundings, different activity patterns, and abnormal postures are just a few signs of chronic pain in cats and dogs. cats are a species that in particular are exemplary in their abilities to hide chronic pain. they will exhibit marked familial withdrawal, finding secluded areas where they may remain for days to weeks when they experience acute and chronic pain. when deciding on a pain management protocol for a patient, always perform a thorough physical examination and include a pain score assessment before injury and pain has occurred, whenever possible. form a problem list to guide your choice of anesthesia and analgesia. for example, using a nonsteroidal antiinflammatory drug (nsaid) in an animal with renal failure would not be wise. remember to account for current medications that the patient may be taking that may augment or interfere with the analgesic or anesthetic drugs. use multimodal techniques and regional therapy and drugs to target pain at different sites before it occurs. once a strategy is decided upon, frequently reassess the patient and tailor the protocol to meet each patient's response and needs. drug therapy (in particular, opioids with or without î± 2 -agonists) is a cornerstone for acute pain treatment and surgical preemptive pain prevention. however, local anesthetics delivered epidurally, via perineural or plexus injection, intraarticular or trigger point injection, are also effective analgesics for acute and chronic forms of pain and inflammation. the nsaids that classically have been reserved for treatment of more chronic or persistent pain states now are being used regularly for treatment of acute and perioperative pain once blood pressure, coagulation, and gastrointestinal parameters have been normalized. an opioid is any natural or synthetic drug that is derived from the poppy, which interacts with opiate receptors identified on cell membranes. the drugs from this class constitute the most effective means of controlling acute, perioperative, and chronic pain in human and veterinary medicine (table 1 -16) . their physiologic effects result from the interaction with one or more of at least five endogenous opioid receptors (âµ, ï�, î´, îµ, and îº). âµ-receptor agonists are noted for their ability to produce profound analgesia with mild sedation. these drugs diminish "wind-up," the hyperexcitable state resulting from an afferent volley of nociceptive impulses. they elevate the pain threshold and are used preemptively to prevent acute pain. as a class, opioids cause cns depression with their intense analgesia. dose-related respiratory depression reflects diminished response to carbon dioxide levels. cardiac depression is secondary only to bradycardia and is more likely with certain opioids such as morphine and oxymorphone. narcotics produce few if any clinically significant cardiovascular effects in dogs and cats; they are considered cardiac soothing or sparing. because opioids increase intracranial and intraocular pressure, use them more cautiously in patients with severe cranial trauma and or ocular lesions. opioids directly stimulate the chemoreceptor trigger zone and may cause nausea and vomiting. most opioids depress the cough reflex via a central mechanism; this may be helpful in patients recovering from endotracheal intubation irritation. a key characteristic of opioids that makes them desirable for use in emergency and critical care situations is their reversibility. antagonists block or reverse the effect of agonists by combining with receptors and producing minimal or no effects. administer all reversal agents, such as naloxone and naltrexone, slowly if given intravenously and to effect. î± î± 2 -agonists as a class of drugs, î± 2 -agonists warrant special attention because most members of the group possess potent analgesic power at doses that are capable of causing sedation, cns depression, cardiovascular depression, and even general anesthetic states. originally developed for antihypertensive use, î± 2 -agonists quickly have attained sedative analgesic status in veterinary medicine (table 1 -17) . like the opioids, î± 2 -agonists produce their effects by aggravating î±-adrenergic receptors in the cns and periphery. 1 emergency care among them cyclooxygenase-1 (cox-1), the major constitutive enzyme primarily involved in normal physiologic functions, and cox-2, the enzyme responsible for most of the hyperalgesia and pain responses experienced after tissue injury or trauma. some nsaids inhibit cyclooxygenase and lipoxygenase activity. most of the currently available oral and parenteral nsaids for small animal medicine and surgery target the cyclooxygenase pathways predominantly, although one (tepoxalin) is thought to inhibit both pathways. inhibition of cox-1 and cox-2 can inhibit the protective effects and impair platelet aggregation and lead to gastrointestinal ulceration. there are definite contraindications and relative contraindications for the use of nsaids. nonsteroidal antiinflammatory drugs should not be administered to patients with renal or hepatic insufficiency, dehydration, hypotension or conditions that are associated with low circulating volume (congestive heart failure, unregulated anesthesia, shock), or evidence of ulcerative gastrointestinal disease. trauma patients should be stabilized completely regarding vascular volume, tone, and pressure before the use of nsaids. patients receiving concurrent administration of other nsaids or corticosteroids, or those considered to be cushingoid, should be evaluated carefully for an adequate "washout" period (time of clearance of drug from the system) before use of an nsaid or before switching nsaids. patients with coagulopathies, particularly those that are caused by platelet number or function defects or those caused by factor deficiencies, and patients with severe, uncontrolled asthma or other bronchial disease are probably not the patients in which to use nsaids. other advice is that nsaids not be administered to pregnant patients or to females attempting to become pregnant because cox-2 induction is necessary for ovulation and subsequent implantation of the embryo. the administration of nsaids should be considered only in the well-hydrated, normotensive dog or cat with normal renal or hepatic function, with no hemostatic abnormalities, and no concurrent steroid administration. nonsteroidal antiinflammatory drugs can be used in many settings of acute and chronic pain and inflammation. among these are the use in well-stabilized musculoskeletal trauma and surgical pain, osteoarthritis management, meningitis, mastitis, animal bite and other wound healing, mammary or transitional cell carcinoma, epithelial (dental, oral, urethral) inflammation, ophthalmologic procedures, and dermatologic or otic disease. whereas opioids seem to have an immediate analgesic effect when administered, most nsaids will take up to 30 minutes for their effect to be recognized. as such, most perioperative or acute nsaids use is part of a balanced pain management scheme, one that uses narcotics and local anesthetic techniques. nonsteroidal antiinflammatory drugs are devoid of many of the side effects of narcotic administration; namely, decreased gastrointestinal motility, altered sensorium, nausea/vomition, and sedation. nonsteroidal antiinflammatory drugs are also devoid of many of the side effects of steroid administration; namely, suppression of the pituitary adrenal axis. the toxic effects of salicylates in cats are well documented. cats are susceptible because of slow clearance and dose-dependent elimination because of deficient glucuronidation in this species. because of this, the dose and the dosing interval of most commonly used nsaids need to be altered in order for these drugs to be used. cats that have been given canine doses of nsaids (twice daily or even once daily repetitively) may show hyperthermia, hemorrhagic or ulcerative gastritis, kidney and liver injury, hyperthermia, respiratory alkalosis, and metabolic acidosis. acute and chronic toxicities of nsaids have been reported in cats, especially after repeat once daily dosing. ketoprofen, flunixin, aspirin, carprofen, and meloxicam have been administered safely to cats, although like most antibiotics and other medications, they are not approved and licensed for use in cats. an important note, though, is that dosing intervals ranging from 48 to 96 hours have been used, and antithrombotic effects often can be achieved at much lower doses than those required to treat fevers and inflammation. i recommend the use of no loading doses, minimum 48-hour dosing intervals, and assurance of adequate circulating blood volume, blood pressure, and renal function. because many of the nsaids are used off-label in cats, it is imperative that the clinician carefully calculate the dose, modify the dosing interval, and communicate this information to the client before dispensing the drug. even drugs that come in liquid form (meloxicam), if administered to cats via box-labeled directions used for dogs, will be given in near toxic doses. to worsen the misunderstanding about dosages for cats, drops from manufacturer's bottles often are calibrated drops; when these same liquids are transferred into pharmacy syringes for drop administration, the calibration of course is lost, and the animal potentially is overdosed. a more accurate method of dispensing and administering oral nsaids in cats is to calculate the dose in milligrams and determine the exact number of milliliters to administer, rather than use the drop method. ketamine classically was considered a dissociative anesthetic, but it also has potent activity as an n-methyl-d-aspartate (nmda) receptor antagonist. this receptor located in the cns mediates windup and central sensitization (a pathway from acute to chronic pain). blockade of this receptor with microdoses of ketamine results in the ability to provide body surface, somatic, and skin analgesia with potentially lower doses of opioids and î±-agonists. loading doses of 0.5 to 2 mg/kg are used intravenously with continuous rate infusions of 2 to 20 âµg/kg/minute. in and of itself, this drug possesses little to no analgesic ability and indeed in high doses alone often can aggravate, sensitize, or excite the animal in subacute or acute pain. amantadine is another nmda blocker that has been used for its antiviral and parkinson's stabilizing effects. amantadine has been used for neuropathic pain in human beings but is only available in an oral form. suggested starting doses for cats and dogs range from 3 to 10 mg/kg po daily. when the drug is given orally and intravenously, patients are unlikely to develop behavioral or cardiorespiratory effects with ketamine or amantadine. tramadol is an analgesic that possesses weak opioid âµ-agonist activity and norepinephrine and serotonin reuptake inhibition. tramadol is useful for mild to moderate pain in small animals. although the parent compound has very weak opioid activity, the metabolites have excellent binding affinity for the âµ-receptor. tramadol has been used for perisurgical pain control when given orally in cats and dogs at a dose of 1 to 10 mg/kg po sid to bid. cats appear to require only once daily dosing. regardless of its affinity for the opioid receptors, the true mechanism of action of tramadol in companion animals remains largely unknown. gabapentin is a synthetic analog of î³-aminobutyric acid (gaba). originally introduced as an antiepileptic drug, the mechanism of action of gabapentin remains somewhat unclear in veterinary medicine. the drug is among a number of commonly used antiepileptic medications used to treat central pain in human beings. the rationale for use is the ability of the drugs to suppress discharge in pathologically altered neurons. gabapentin does this through calcium channel modulation without binding to glutamate receptors. chronic, burning, neuropathic, and lancinating pain in small animals responds well to 1 to 10 mg/kg po daily. local anesthetic agents are the major class used as a peripheral-acting analgesic ( table 1 -19) . local anesthetics block the transmission of pain impulses at the peripheral nerve nociceptor regions. local anesthetics may be used to block peripheral nerves or inhibit nerve "zones" using regional techniques. although all local anesthetics are capable of providing pain relief, agents with a longer duration of action are preferred for pain management purposes. bupivacaine is an example of a long-acting local anesthetic drug that is used along with lidocaine for long-acting pain relief. a single dose of bupivacaine injected at a local site will provide local anesthesia and analgesia for 6 to 10 hours. when lidocaine is administered as an intravenous constant rate infusion (50 to 75 âµg/kg/minute in dogs, 1 to 10 âµg/kg/minute in cats) is effective in the treatment of chronic neuropathic pain and periosteal and peritoneal pain (e.g., pancreatitis). mexiletine, an oral sodium channel blocker, can be used as an alternative to injectable lidocaine for provision of background analgesia. many drugs (table 1 -20) are used in combination with opioids, î± 2 -agonists, and ketamine to provide anxiolysis and sedation. injection of local anesthetic solution into the connective tissue surrounding a particular nerve produces loss of sensation (sensory blockade) and/or paralysis (motor nerve blockade) in the region supplied by the nerve. local anesthetics also may be administered epidurally, intrathoracically, intraperitoneally, and intraarticularly. lidocaine and bupivacaine are the most commonly administered local anesthetics. lidocaine provides for quick, short-acting sensory and motor impairment. bupivacaine provides for later-onset, longerlasting desensitization without motor impairment. combinations of the two agents diluted with saline are used frequently to provide for quick-onset analgesia that lasts between 4 and 6 hours in most patients. adding narcotic and/or î± 2 agent often maximizes the analgesia and increases the pain-free interval to 8 to 18 hours. epinephrine and preservative-free solutions are recommended. precision placement of anesthetic close to nerves, roots, or plexuses is improved with the use of a stimulating nerve locator. cats seem to be more sensitive to the effects of local anesthetics; as such the lower ends of most dosing ranges are used for blockades in this species. unlike most instances of general anesthesia, during which the animal is rendered unconscious and nerve transmission is decreased by virtue of cns depression, local and regional techniques block the initiation of noxious signals, thereby effectively preventing pain from entering the cns. this is an effective means of not only preventing initial pain but also reducing the changes that take place in the dorsal horn of the spinal cord, spinothalamic tracts, limbic and reticular activating centers, and cortex. frequently, the neurohormonal response that is stimulated in pain and stress is blunted as well. overall, the patient has fewer local and systemic adverse effects of pain, disease processes are minimized, chronic pain states are unlikely, and outcome is improved. regional techniques are best used as part of an analgesic regimen that consists of their continuous administration, narcotics, î±-agonists, anxiolytics, and good nursing. lidocaine can be added to sterile lubricant in a one-to-one concentration to provide decreased sensation for urinary catheterization, nasal catheter insertion, minor road burn analgesia, and pyotraumatic dermatitis analgesia. proparacaine is a topical anesthetic useful for corneal or scleral injuries. local anesthetics can be used to infiltrate areas of damage or surgery by using long-term continuous drainage catheters and small, portable infusion pumps. this is an effective means of providing days of analgesia for massive surgical or traumatic soft tissue injury. even without the catheter, incisional or regional soft tissue blocking using a combination of 1 to 2 mg/kg lidocaine and 0.5 to 2 mg/kg bupivacaine diluted with equal volume of saline and 1:9 with sodium bicarbonate is effective for infiltrating large areas of injury. administration of local anesthetic drugs around the infraorbital, maxillary, ophthalmic mental, and alveolar nerves can provide excellent analgesia for dental, orofacial, and ophthalmic trauma and surgical procedures. each nerve may be desensitized by injecting 0.1 to 0.3 ml of a 2% lidocaine hydrochloride solution and 0.1 to 0.3 ml of 0.5% bupivacaine solution using a 1.2-to 2.5-cm, 22-to 25-gauge needle. precise placement perineurally versus intraneurally (neuroma formation common) is enhanced by using catheters in the foramen versus needle administration. always perform aspiration before administration to rule out intravascular injection of agents. this block is used to provide analgesia for thoracic, lower cervical, cranial abdominal, and diaphragmatic pain. following aseptic preparation, place a small through-the-needle (20-to 22-gauge) catheter in the thoracic cavity between the seventh and ninth intercostal space on the midlateral aspect of the thorax. aseptically mix a 0.5 to 1 mg/kg lidocaine and a 0.2 to 0.5 mg/kg bupivacaine dose with volume of saline equal to the volume of bupivacaine, and slowly inject it over a period of 2 to 5 minutes following aspiration to ensure that no intravascular injection occurs. depending on where the lesion is, position the patient to allow the intrapleural infusion to "coat" the area. most effective is positioning the patient in dorsal recumbency for several minutes following the block to make sure local anesthetic occupies the paravertebral gutters and hence the spinal nerve roots. the block should be repeated every 3 hours in dogs and every 8 to 12 hours in cats. secure the catheter to the skin surface for repetitive administration. administration of local anesthetic around the brachial plexus provides excellent analgesia for forelimb surgery, particularly that distal to the shoulder, and amputations. nerve locator-guided techniques are much more accurate and successful than blind placement of local anesthetic; however, even the latter is useful. to administer a brachial plexus blockade, follow this procedure: 1. aseptically prepare a small area of skin over the point of the shoulder. 2. insert a 22-gauge, 1 1 /2-to 3-inch spinal needle medial to the shoulder joint, axial to the lesser tubercle, and advance it caudally, medial to the body of the scapula, and toward the costochondral junction of the first rib. aspirate first before injection to make sure that intravenous injection does not occur. 3. inject one third of the volume of local anesthetic mix, and then slowly withdraw the needle and fan dorsally and ventrally while infusing the remaining fluid. 4. local anesthetic doses are similar to those for intrapleural blockade. epidural analgesia refers to the injection of an opioid, a phencyclidine, an î±-agonist, or an nsaid into the epidural space. epidural anesthesia refers to the injection of a local anesthetic. in most patients a combination of the two is used. epidural analgesia and anesthesia are used for a variety of acute and chronic surgical pain or traumatically induced pain in the pelvis, tail, perineum, hind limbs, abdomen, and thorax (table 1 -21) . procedures in which epidural analgesia and anesthesia are useful include forelimb and hind limb amputation, tail or perineal procedures, cesarean sections, diaphragmatic hernia repair, pancreatitis, peritonitis, and intervertebral disk disease. epidural blocks performed using opioids or bupivacaine will not result in hind limb paresis or decreased urinary or anal tone (incontinence), unlike lidocaine or mepivicaine epidural blocks. morphine is one of the most useful opioids for administration in the epidural space because of its slow systemic absorption. epidural catheters used for the instillation of drugs through constant rate infusion or intermittent injection can be placed in dogs and cats. routinely placed at the lumbosacral junction, these catheters are used with cocktails including preservative-free morphine, bupivacaine, medetomidine, and ketamine. extremely effective for preventing windup pain in the peritoneal cavity or caudal half of the body, the catheters may be maintained if placed aseptically for 7 to 14 days. to provide epidural analgesia or anesthesia, follow this procedure: 1. position the animal in lateral or sternal recumbency. 2. clip and aseptically scrub over the lumbosacral site. 3. palpate the craniodorsal-most extent of the wings of the ileum bilaterally and draw an imaginary line through them to envision the spine of l7 located immediately behind the imaginary line. 4. advance a 20-to 22-gauge, 1 1 /2-to 3-inch spinal or epidural needle through the skin just caudal to the spine of l7. 5. the needle will lose resistance as it is introduced into the epidural space. drop saline into the hub of the needle, and the saline will be pulled into the epidural space as the needle enters. discrete intercostal nerve blocks can provide effective analgesia for traumatic or postsurgical pain. identify the area of the injury, and infiltrate three segments on either side of the injury with analgesic. to perform an intercostal nerve block, follow this procedure: 1. clip and aseptically scrub the dorsal and ventral third of the chest wall. 2. palpate the intercostal space as far dorsally as possible. 3. use a 25-gauge, 0.625-inch needle at the caudolateral aspect of the affected rib segments and those cranial and caudal. 4. direct the tip of the needle caudally such that the tip of the needle "drops" off of the caudal rib. (this places the needle tip in proximity to the neuromuscular bundle that contains the intercostal nerve that runs in a groove on the caudomedial surface of the rib.) 5. aspirate to confirm that the drug will not go intravenously. 6. inject while slowly withdrawing the needle. inject 0.5 to 1.0 ml at each site, depending on the size of the animal. gaynor js, an acute condition in the abdomen is defined as the sudden onset of abdominal discomfort or pain caused by a variety of conditions involving intraabdominal organs. many animals have the primary complaint of lethargy, anorexia, ptyalism, vomiting, retching, diarrhea, hematochezia, crying out, moaning, or abnormal postures. abnormal postures can include generalized rigidity, walking tenderly or as if "on eggshells," or a prayer position in which the front limbs are lowered to the ground while the hind end remains standing. in some cases, it may be difficult initially to distinguish between true abdominal pain or referred pain from intervertebral disk disease. rapid progression and decompensation of the patient's cardiovascular status can lead to stupor, coma, and death in the most extreme cases, making rapid assessment, treatment, and definitive care extremely challenging. often the patient's signalment and history can increase the index of suspicion for a particular disease process. a thorough history often is overlooked or postponed in the initial stages of resuscitation of the patient with acute abdominal pain. often, asking the same question in a variety of methods can elicit an answer from the client that may lead to the source of the problem and the reason for acute abdominal pain. important questions to ask the client include the following: â�¢ what is your chief complaint or reason that you brought your animal in on emergency? â�¢ when did the signs first start, or when was your animal last normal? â�¢ do you think that the signs have been the same, better, or getting worse? â�¢ does your animal have any ongoing or past medical problems? â�¢ have similar signs occurred in the past? â�¢ does your animal have access to any known toxins, or does he or she run loose unattended? as with any other emergency, the clinician must follow the abcs of therapy, treating the most life-threatening problems first. first, perform a perfunctory physical examination. examination of the abdomen ideally should be performed last, in case inciting a painful stimulus precludes you from evaluating other organ systems more thoroughly. briefly observe the patient from a distance. are there any abnormal postures? is there respiratory distress? is the animal ambulatory, and if so, do you observe any gait abnormalities? do you observe any ptyalism or attempts to vomit? auscultate the patient's thorax for crackles that may signify aspiration pneumonia resulting from vomiting. examine the patient's mucous membrane color and capillary refill time, heart rate, heart rhythm, and pulse quality. many patients in pain have tachycardia that may or may not be accompanied by dysrhythmias. if a patient's heart rate is inappropriately bradycardic, consider hypoadrenocorticism, whipworm infestation, or urinary obstruction or trauma as a cause of hyperkalemia. assess the patient's hydration status by evaluating skin turgor, mucous membrane dryness, and whether the eyes appear sunken in their orbits. a brief neurologic examination should consist of whether the patient is actively having a seizure, or whether mental dullness, stupor, coma, or nystagmus are present. posture and spinal reflexes can assist in making a diagnosis of intervertebral disk disease versus abdominal pain. perform a rectal examination to evaluate for the presence of hematochezia or melena. finally, examination of the abdomen should proceed first with superficial and then deeper palpation. visually inspect the abdomen for the presence of external masses, bruising, or penetrating injuries. reddish discoloration of the periumbilical area often is associated with the presence of intraabdominal hemorrhage. it may be necessary to shave the fur to inspect the skin and underlying structures visually for bruising and ecchymoses. auscultate the abdomen for the presence or absence of borborygmi to characterize gut sounds. next, perform percussion and ballottement to evaluate for the presence of a gas-distended viscus or peritoneal effusion. finally, perform first superficial and then deep palpation of all quadrants of the abdomen, noting abnormal enlargement, masses, or whether focal pain is elicited in any one area. once the physical examination has been performed, implement initial therapy in the form of analgesia, fluid resuscitation, and antibiotics. treatment for any patient with an acute condition in the abdomen and shock is to treat the underlying cause, maintain tissue oxygen delivery, and prevent end-organ damage and failure. a more complete description of shock and oxygen delivery is given in the section on shock. 1 emergency care the administration of analgesic agents to any patient with acute abdominal pain is one of the most important therapies in the initial stages of case management. many patients with acute abdominal pain are clinically dehydrated or are in hypovolemic shock because of hemorrhage. careful titration of intravenous crystalloid and colloid fluids including blood products is necessary based on the patient's perfusion parameters including heart rate, capillary refill time, blood pressure, urine output, and pcv. fluid therapy also should be based on the most likely differential diagnoses, with specific fluid types administered according to the primary disease process. in dogs, a shock volume of fluids is calculated based on the total blood volume of 90 ml/kg/hour. in cats, shock fluid rate is based on plasma volume of 44 ml/kg/hour. in most cases, any crystalloid fluid can be administered at an initial volume of one fourth of a calculated shock dose and then titrated according to whether the patient's cardiovascular status responds favorably or not. in cases of an acute condition in the abdomen from known or suspected hypoadrenocorticism, severe whipworm infestation, or urinary tract obstruction or rupture, 0.9% sodium chloride fluid without added potassium is the fluid of choice. when hemorrhage is present, the administration of whole blood or packed rbcs may be indicated if the patient has clinical signs of anemia and shows clinical signs of lethargy, tachypnea, and weakness. fresh frozen plasma is indicated in cases of hemorrhage resulting from vitamin k antagonist rodenticide intoxication or hepatic failure or in cases of suspected disseminated intravascular coagulation (dic). a more thorough description of fluid therapy is given under the sections on shock and fluid therapy. the empiric use of broad-spectrum antibiotics is warranted in cases of suspected sepsis or peritonitis as a cause of acute abdominal pain. ampicillin sulbactam (22 mg/kg iv q6-8h) and enrofloxacin (10 mg/kg once daily) are the combination treatment of choice to cover gram-negative, gram-positive, aerobic, and anaerobic infections. alternative therapies include a second-generation cephalosporin such as cefotetan (30 mg/kg iv tid) or cefoxitin (22 mg/kg iv tid) or added anaerobic coverage with metronidazole (10 to 20 mg/kg iv tid). tissue oxygen delivery depends on a number of factors, including arterial oxygen content and cardiac output. if an animal has had vomiting and subsequent aspiration pneumonitis, treatment of hypoxemia with supplemental oxygen in the form of nasal, nasopharyngeal, hood, or transtracheal oxygen administration is important (see oxygen supplementation under emergency diagnostic and therapeutic procedures). perform a complete blood count in all cases of acute abdominal pain to determine if lifethreatening infection or coagulopathy including dic is present. in cases of sepsis, infection, or severe nonseptic inflammation, the white blood cell count may be normal, elevated, or low. examine a peripheral blood smear for the presence of toxic neutrophils, eosinophils, atypical lymphocytes, nucleated rbcs, platelet estimate, anisocytosis, and blood parasites. a falling pcv in the face of rbc transfusion suggests ongoing hemorrhage. perform a biochemistry panel to evaluate organ system function. azotemia with elevated bun and creatinine may be associated with prerenal dehydration, impaired renal function, or postrenal obstruction or leakage. the bun also can be elevated when gastrointestinal hemorrhage is present. serum amylase may be elevated with decreased renal function or in cases of pancreatitis. a normal serum amylase, however, does not rule out pancreatitis as a source of abdominal pain. serum lipase may be elevated with gastrointestinal inflammation or pancreatitis. like amylase, a normal serum lipase does not rule out pancreatitis. total bilirubin, alkaline phosphatase, and alanine transaminase may be elevated with primary cholestatic or hepatocellular diseases or may be due to extrahepatic causes including sepsis. obtain a urinalysis via cystocentesis whenever possible, except in cases of suspected pyometra or transitional cell carcinoma. azotemia in the presence of a nonconcentrated (isosthenuric or hyposthenuric) urine suggests primary renal disease. secondary causes of apparent renal azotemia and lack of concentrating ability also occur in cases of hypoadrenocorticism and gram-negative sepsis. renal tubular casts may be present in cases of acute renal ischemia or toxic insult to the kidneys. bacteriuria and pyuria may be present with infection and inflammation. when a urinalysis is obtained via free catch or urethral catheterization, the presence of bacteriuria or pyuria also may be associated with pyometra, vaginitis, or prostatitis/prostatic abscess. serum lactate is a biochemical indicator of decreased organ perfusion, decreased oxygen delivery or extraction, and end-organ anaerobic glycolysis. elevated serum lactate greater than 6 mmol/l has been associated with increased morbidity and need for gastric resection in cases of gdv and increased patient morbidity and mortality in other disease processes. rising serum lactate in the face of adequate fluid resuscitation is a negative prognostic sign. obtain abdominal radiographs as one of the first diagnostic tests when deciding whether to pursue medical or surgical management. the presence of gdv, linear foreign body, pneumoperitoneum, pyometra, or splenic torsion warrants immediate surgical intervention. if a loss of abdominal detail occurs because of peritoneal effusion, perform additional diagnostic tests including abdominal paracentesis (abdominocentesis) and abdominal ultrasound to determine the cause of the peritoneal effusion. abdominal ultrasonography is often useful in place of or in addition to abdominal radiographs. the sensitivity of abdominal ultrasonography is largely operator dependent. indications for immediate surgical intervention include loss of blood flow to an organ, linear bunching or placation of the intestinal tract, intussusception, pancreatic phlegmon or abscess, a fluid-filled uterus suggestive of pyometra, gastrointestinal obstruction, intraluminal gastrointestinal foreign body, dilated bile duct, or gallbladder mucocele, or gas within the wall of the stomach or gallbladder (emphysematous cholecystitis). the presence of peritoneal fluid alone does not warrant immediate surgical intervention without cytologic and biochemical evaluation of the fluid present. see also abdominal paracentesis and diagnostic peritoneal lavage. abdominal paracentesis (abdominocentesis) often is the deciding factor in whether to perform immediate surgery. abdominocentesis is a sensitive technique for detecting peritoneal effusion when more than 6 ml/kg of fluid is present within the abdominal cavity. abdominal effusion collected should be saved for bacterial culture and evaluated biochemically and cytologically based on your index of suspicion of the primary disease process. if creatinine, urea nitrogen (bun) or potassium is elevated compared with that of serum, uroabdomen is present. elevated abdominal fluid lipase or amylase compared with serum supports a diagnosis of pancreatitis. elevated lactate compared with serum lactate or an abdominal fluid glucose less than 50 mg/dl is highly sensitive and specific for bacterial/ septic peritonitis. the presence of bile pigment or bacteria is supportive of bile and septic peritonitis, respectively. free fibers in abdominal fluid along with clinical signs of abdominal pain strongly support gastrointestinal perforation, and immediate surgical exploration is required. text continued on p. 93 the following are clinical conditions, patient signalment, common history, physical examination, and characteristic findings of various diagnostic tests. a blank column next to a condition indicates no specific signalment, history, physical examination, or diagnostic test characteristic for a particular disease process. lack of contiguity of body wall surgical ( medical unless perforation present present c-shaped abnormal gas pattern with plication on radiographs surgical (immediate) dilation of bowel cranial to foreign object, radiopaque object in surgical (immediate) stomach or intestines, hypochloremic metabolic acidosis on bloodwork if pyloric outflow obstruction is present elevated or decreased wbc; foreign material, wbcs and medical unless perforation bacteria on abdominal fluid, elevated lactate and decreased present glucose on abdominal fluid target shaped soft tissue density on abdominal u/s, soft tissue surgical (immediate): density with gas dilation cranially on abdominal radiographs medical management of primary cause colonic distension with hard feces on radiographs medical increased or decreased wbc, septic abdominal effusion surgical (immediate) elevated t bili, alt, alk phos, and wbc hypoechoic hepatic medical after biopsy parenchyma on ultasound hepatomegaly elevated t bili, alt, alk phos, and wbc hyperechoic foci in surgical (immediate) gallbladder or sludge on u/s, free gas in wall of gall bladder abdominal effusion, bile pigment in effusion surgical (immediate) elevated t bili, alk phos, alt surgical (immediate) elevated or decreased wbc, elevated t bili, alk phos and surgical (immediate) alt, free gas in hepatic parenchyma on rads, hypoechoic mass with hyperechoic material in hepatic parenchyma on u/s heteroechoic liver with hyperechoic center on ultrasound surgical (immediate) mixed echogenic mass on ultrasound, soft tissue mass surgical (immediate or density on radiographs, elevated alk phos, alt, delayed) t bili, hypoglycemia pain-cont'd elevated t bili, alk phos, alt, amylase and/or lipase, elevated medical in most cases or decreased wbc, hypocalcemia, focal loss of detail in right unless abscess or cranial quadrant on radiographs hypo-to hyperechoic phlegmon is present pancreas with hyperechoic peri-pancreatic fat on ultrasound, abdominal and/or pleural effusion on radiographs and ultrasound pancreatic soft tissue mass effect on radiographs and surgical if mass identified, ultrasound, elevated amylase and lipase, hypoglycemia, otherwise medical elevated serum insulin management of hypoglycemia splenomegaly on radiographs, hyperechoic spleen with no surgical (immediate) blood flow on ultrasound soft tissue mass effect and loss of abdominal detail on surgical (immediate) radiographs, cavitated mass with abdominal effusion on u/s hyperechoic spleen with no blood flow on abdominal u/s, surgical (immediate) abdominal effusion, thrombocytopenia loss of abdominal detail on radiographs, peritoneal effusion medical unless refractory on u/s, hemoabdomen on abdominocentesis hypotension diagnosis based primarily on clinical signs medical fracture of the os penis on radiographs largely medical unless urethral tear diagnosis based primarily on clinical signs medical, although prepuce may need to be incised to allow replacement of penis into sheath prostatomegaly on radiographs and ultrasound hypoechoic medical prostate on u/s, pyuria and bacteriuria and u/a prostatomegaly on radiographs and ultrasound hypo-to surgical (delayed) hyperechoic prostate on u/s, bacteriuria and pyuria on u/a prostatomegaly on radiographs and ultrasound, prostatic medical/surgical mineralization on radiographs and ultrasound hypoechoic kidneys on u/s, pyuria on u/a, elevated wbc, medical azotemia pyuria, bacteriuria on u/a medical pyelectasia in abdominal u/s, azotemia surgical (immediate) renomegaly on radiographs, azotemia renal mass on u/s, renomegaly on radiographs surgical (immediate) renal mass on u/s, azotemia, lack of renal blood flow surgical (delayed) on u/s calculi in renal pelvis on radiographs and ultrasound, azotemia medical unless both kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, medical unless both azotemia kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, surgical (delayed until fluid or soft tissue density on u/s, azotemia electrolyte stabilization) diagnosis largely based on physical examination medical unless cannot pass findings urethral catheter azotemia, no peritoneal effusion, lack of urine output or surgical (delayed until outflow with ureteral catheterization, double contrast electrolyte stabilization) cystourethrogram indicated transitional cellular casts on u/a, hematuria, mass effect or surgical and medical thickened irregular urethra on ultrasound or management cystourethrogram hypoechoic swollen testicle on testicular ultrasound surgical (immediate) fluid or gas-filled tubular structure on abdominal ultrasound or surgical (immediate) abdominal radiographs soft tissue tubular structure on radiographs, fluid-filled uterus surgical ( in the event of a negative abdominocentesis, but peritoneal effusion or bile or gastrointestinal perforation are suspected, perform a diagnostic peritoneal lavage. peritoneal dialysis kits are commercially available but are often expensive and impractical (see p. 6). animals that have acute abdominal pain can be divided into three broad categories, depending on the primary cause of pain and the initial definitive treatment (table 1-24) . some diseases warrant a nonsurgical, medical approach to case management. other conditions require immediate surgery following rapid stabilization. other conditions initially can be managed medically until the patient is hemodynamically more stable and then may or may not require surgical intervention at a later time. specific management of each disease entity is listed under its own subheading. box 1-23 lists specific indications for exploratory laparotomy. the best means to explore the abdominal cavity accurately and thoroughly is to open the abdomen on midline from the level of the xyphoid process caudally to the pubis for full exposure and then to evaluate all organs in every quadrant in a systematic manner. address specific problems such as gastric or splenic torsion, enteroplication, and foreign body removal, and then copiously lavage the abdomen with warmed sterile saline solution. suction the saline solution thoroughly from the peritoneal cavity so as to not impair macrophage function. in cases of septic peritonitis, the abdomen may be left open, or a drain may be placed for further suction and lavage. the routine use of antibiotics in irrigation solutions is contraindicated because the antibiotics can irritate the peritoneum and delay healing. when the abdominal cavity is left open, secure sterile laparotomy towels and water-impermeable dressings over the abdominal wound with umbilical tape, and then change these daily or as strike-through occurs. open abdomen cases are often effusive and require meticulous evaluation and management of electrolyte imbalances and hypoalbuminemia. the abdomen can be closed and/or the abdominal drain removed when the volume of the effusion decreases, when bacteria are no longer present, and when the neutrophils become more healthy in appearance. bischoff mg: radiographic techniques and interpretation of the acute abdomen, clin tech small anim pract 18 (1) anaphylactic shock occurs as an immediate hypersensitivity reaction to a variety of inciting stimuli (box 1-24). in animals, the most naturally occurring anaphylactic reaction results from wasp or bee stings. most other reactions occur as a result of an abnormal sensitivity to items used in making medical diagnoses or treatment. during an anaphylactic reaction, activation of c5a and the complement system results in vascular smooth muscle dilation and the release of a cascade of inflammatory mediators, including histamine, slow-reacting substance of anaphylaxis, serotonin, heparin, acetylcholine, and bradykinin. clinical signs associated with anaphylaxis differ between dogs and cats. in dogs, clinical signs may include restlessness, vomiting, diarrhea, hematochezia, circulatory collapse, coma, and death. in cats, clinical signs often are associated with respiratory system abnormalities. clinical signs may include ptyalism, pruritus, vomiting, incoordination, bronchoconstriction, pulmonary edema and hemorrhage, laryngeal edema, collapse, and death. the most important steps to remember in any emergency is to follow the abcs of airway, breathing, and circulation. first, establish an airway through endotracheal intubation or emergency tracheostomy, if necessary. concurrently, an assistant should establish vascular or intraosseous access to administer drugs and fluids (box 1-25). the patient should be hospitalized until complete resolution of clinical signs. after initial stabilization and treatment, it is important to maintain vascular access and continue intravenous fluid therapy until the patient is no longer hypotensive, and vomiting and diarrhea have resolved. in cases of fulminant pulmonary hemorrhage and edema, administer supplemental oxygen until the patient is no longer hypoxemic or orthopneic on room air. normalize and maintain blood pressure using positive inotropes (dobutamine, 3-10 âµg/kg/ minute cri) or pressors (dopamine, 3 to 10 âµg/kg/minute iv cri; see shock). if bloodtinged vomitus or diarrhea has been observed, administer antibiotics to decrease the risk of bacterial translocation and sepsis (cefoxitin, 22 mg/kg iv tid; metronidazole, 10 mg/kg iv tid). also consider using gastroprotectant drugs (famotidine, 0.5 to 1.0 mg/kg iv; ranitidine, 0.5 to 2.0 mg/kg po, iv, im bid; sucralfate, 0.25 to 1.0 g po tid; omeprazole, 0.7 to 1.0 mg/kg po sid). a second and less serious form of allergic reaction is manifested as angioneurotic edema and urticaria. in most cases, clinical signs develop within 20 minutes of an inciting allergen. although this type of reaction causes patient discomfort, it rarely poses a life-threatening problem. most animals have mild to severe swelling of the maxilla and periorbital regions. the facial edema also may be accompanied by mild to severe generalized urticaria. some animals may paw at their face, rub at their eyes, or have vomiting or diarrhea. the treatment for angioneurotic edema involves suppressing the immune response by administration of short-acting glucocorticoid drugs and blocking the actions of histamine by the synergistic use of histamine 1 and histamine 2 receptor blockers (box 1-26). in some cases, the inciting cause is a known recent vaccination or insect sting. many times, however, the inciting cause is not known and is likely an exposure to a stinging insect or arachnid. differential diagnoses for acute facial swelling and/or urticaria include acetaminophen toxicity (cats), anterior caval syndrome, lymphadenitis, vasculitis, hypoalbuminemia, and contact dermatitis. observe animals that have presented for angioneurotic edema for a minimum of 20 to 30 minutes after injection of the short-acting glucocorticoids and antihistamines. monitor blood pressure to make sure that the patient does not have concurrent anaphylaxis and hypotension. after partial or complete resolution of clinical signs, the animal can be discharged to its owner for observation. in dogs, mild vomiting or diarrhea may occur within 1 to 2 days after this type of reaction. wherever possible, exposure to the inciting allergen should be avoided. â�¢ administer short-acting glucocorticoid: complications observed while a patient is under anesthesia can be divided into two broad categories: (1) those related to equipment malfunction or human error and (2) the patient's physiologic response to the cardiorespiratory effects of the anesthetic drugs. careful observation of the patient and familiarity with anesthetic equipment, drug protocols, and monitoring equipment is necessary for the safest anesthesia to occur. despite this, however, anesthetic-related complications are frequent and need to be recognized and treated appropriately. many anesthetic drugs have a dose-dependent depressive effect on the respiratory system and cause a decrease in respiratory rate and tidal volume, leading to hypoventilation. respiratory rate alone is not a reliable indicator of the patient's oxygenation and ventilatory status. the respiratory tidal volume can be measured with a wright's respirometer. perform pulse oximetry and capnography as noninvasive measures of the patient's oxygenation and ventilation. ventilation can be impaired as a result of anesthetic drugs, patient position, pneumothorax, pleural effusion (chylothorax, hemothorax, pyothorax), equipment malfunction, rebreathing of carbon dioxide, thoracic wall injury, or alveolar fluid (pulmonary edema, hemorrhage, or pneumonia). problems such as a diaphragmatic hernia, gdv, or gravid uterus can impede diaphragmatic excursions once the patient is placed on its back and can lead to impaired ventilation. the work of breathing also may be increased because of increased resistance of the anesthesia circuit and increased dead space ventilation. this is particularly important in small toy breeds. clinical signs of inadequate ventilation and respiratory complications include abnormal respiratory pattern, sudden changes in heart rate, cardiac dysrhythmias, cyanosis, and cardiopulmonary arrest. end-tidal carbon dioxide, or capnography, gives a graphic display of adequacy of ventilation. rapid decreases in end-tidal carbon dioxide can be caused by disconnection or obstruction of the patient's endotracheal tube or poor perfusion, namely, cardiopulmonary arrest (see capnometry [end-tidal carbon dioxide monitoring]). postoperatively, hypoventilation can occur because of the residual effects of the anesthetic drugs, hypothermia, overventilation during intraoperative support, surgical techniques that compromise ventilation (thoracotomy, cervical disk surgery, atlantooccipital stabilization), postoperative bandaging of the abdomen or thorax, ventilatory muscle fatigue, or injury to the cns. cardiac output is a function of heart rate and stroke volume. factors that influence stroke volume include vascular and cardiac preload, cardiac afterload, and cardiac contractility. the patient's cardiac output can be affected adversely by the negative inotropic and chronotropic and vasodilatory effects of anesthetic drugs, all leading to hypotension. 96 1 emergency care bradycardia, tachycardia, cardiac dysrhythmias, and vascular dilation can lead to hypotension and inadequate organ perfusion. table 1 -25 lists the normal heart rate and blood pressure in dogs and cats. bradycardia is defined as a heart rate below normal values. many anesthetic drugs can cause bradycardia. causes of bradycardia include the use of narcotics or î± 2 -agonist drugs, deep plane of anesthesia, increased vagal tone, hypothermia, and hypoxia. table 1 -26 lists the causes of bradycardia and the necessary immediate action or treatment. tachycardia is defined as a heart rate above normal values. common causes of tachycardia include vasodilation, drugs, inadequate anesthetic depth and perceived pain, hypercapnia, hypoxemia, hypotension, shock, or hyperthermia. table 1 -27 lists the causes and immediate action or treatment for tachycardia. hypotension is defined as physiologically low blood pressure (mean arterial pressure less than 65 mm hg). a mean arterial blood pressure less than 60 mm hg can result in inadequate tissue perfusion and oxygen delivery. the coronary arteries are perfused during diastole. inadequate diastolic blood pressure, less than 40 mm hg, can cause decreased coronary artery perfusion and myocardial hypoxemia that can predispose the heart to dysrhythmias. causes of perianesthetic hypotension include peripheral vasodilation by anesthetic drugs, bradycardia or tachyarrhythmias, hypothermia, inadequate cardiac preload from vasodilation or hemorrhage, decreased venous return from patient position or surgical manipulation of viscera, and decreased cardiac contractility. electrocardiogram monitoring is useful for the early detection of cardiac dysrhythmias during the perianesthetic period. clinical signs of cardiac dysrhythmias include irregular pulse rate or pressure, abnormal or irregular heart sounds, pallor, cyanosis, hypotension, and an abnormal ecg tracing. remember that the single best method of detecting cardiac 98 1 emergency care vagolytic drugs atropine allow time for the drug to wear off. glycopyrrolate allow time for the drug to wear off. sympathomimetic drugs epinephrine allow time for the drug to wear off; administer a î²-blocker; turn off infusion. isoproterenol administer a î²-blocker. turn off infusion; administer a î²-blocker. allow time for drug to wear off. inadequate anesthetic depth increase anesthetic depth. hypercapnia increase ventilation (assisted ventilation). hypoxemia increase gas flow and oxygenation. hypotension decrease anesthetic depth; administer an intravenous crystalloid or colloid bolus, positive inotrope drug, positive chronotrope drug, or pressor. hyperthermia apply ambient or active cooling measures; administer dantrolene sodium if malignant hyperthermia is suspected. hypothermia provide ambient rewarming. hypocalcemia * administer calcium chloride (10 mg/kg iv) or calcium gluconate (23 mg/kg). decrease vaporizer setting/anesthetic depth. reverse with opioids or a 2 -agonists. vasodilation administer an intravenous crystalloid bolus (10 ml/kg). administer an intravenous colloid bolus (5 ml/kg). administer a pressor (epinephrine, phenylephrine dysrhythmias is with your fingertips (palpate a pulse or apex heartbeat) and ears (auscultate the heart). confirm the dysrhythmia by auscultating the heart rate and rhythm, identify the p waves and the qrs complexes, and evaluate the relationship between the p waves and qrs complexes. is there a p wave for every qrs, and a qrs for every p wave? during anesthesia, fluid, acid-base, and electrolyte imbalances can predispose the patient to dysrhythmias. sympathetic and parasympathetic stimulation, including the time of intubation, can predispose the patient to dysrhythmias. if the patient's plane of anesthesia is too light, perception of pain can cause catecholamine release, sensitizing the myocardium to ectopic beats. atrioventricular blockade can be induced with the administration of î± 2 -agonist medications, including xylazine and medetomidine. thiobarbiturates (thiopental) can induce ventricular ectopy and bigeminy. although these dysrhythmias may not be harmful in the awake patient, anesthetized patients are at a particular risk of dysrhythmia-induced hypotension. carefully monitor and treat all dysrhythmias (see cardiac dysrhythmias). box 1-27 lists steps to take to prevent perianesthetic dysrhythmias. awakening during anesthesia can occur and can be caused by equipment failure and simply, although no one likes to admit it, human error. table 1 -29 lists causes of arousal during anesthesia and appropriate immediate actions. awaken patient, and administer dantrolene arousal (e.g., malignant hyperthermia) sodium. â�¢ stabilize acid-base and electrolyte balance before anesthetic induction, whenever possible. â�¢ rehydrate patient before anesthetic induction. â�¢ select anesthetic agents appropriate for the particular patient. â�¢ be aware of the effects of the drugs on the myocardium. â�¢ ensure adequate anesthetic depth and oxygenation before anesthetic induction. â�¢ ensure ventilatory support during anesthesia. â�¢ monitor heart rate, rhythm, blood pressure, pulse oximetry, and capnometry during anesthesia. â�¢ ensure adequate anesthetic depth before surgical stimulation. â�¢ avoid surgical manipulation to the heart or great vessels, whenever possible. â�¢ avoid changes in perianesthetic depth. â�¢ avoid hypothermia. delayed recovery can be caused by a number of factors, including excessive anesthetic depth, hypothermia, residual action of narcotics or tranquilizers, delayed metabolism of anesthetic drugs, hypoglycemia, hypocalcemia, hemorrhage, and breed or animal predisposition. careful monitoring of the patient's blood pressure, acid-base and electrolyte status, anesthetic depth, pcv, and vascular volume intraoperatively and taking care with supportive measures to prevent abnormalities can hasten anesthetic recovery and avoid postoperative complications. gaynor the presentation of a patient with a bleeding disorder often is a diagnostic challenge for the veterinary practitioner (boxes 1-28 and 1-29). in general, abnormal bleeding can be caused by five major categories: (1) vascular trauma, (2) circulating inhibitors of coagulation heparin fibrin degradation products development of spontaneous deep hematomas, unusually prolonged bleeding after traumatic injury, bleeding at multiple sites throughout the body involving multiple organ systems, delayed onset of severe hemorrhage after bleeding, and an inability on the practitioner's part to find an organic cause of bleeding. the signalment, history, clinical signs, and results of coagulation often can aid in making a rapid diagnosis of the primary cause of the disorder and in the selection of appropriate case management. when taking a history, ask the following important questions: â�¢ what is the nature of the bleeding? â�¢ what sites are affected? â�¢ how long has the bleeding been going on? â�¢ has your animal had any previous or similar episodes? â�¢ is there any possibility of any toxin exposure? â�¢ if so, when and how much did your animal consume? â�¢ is there any possibility of trauma? â�¢ does your animal run loose outdoors unattended? â�¢ have you ever traveled, and if so, where? â�¢ has your animal been on any medications recently or currently? â�¢ has your animal been vaccinated recently? â�¢ have any known relatives of your animal had any bleeding disorders? â�¢ are there any other abnormal signs that you have seen? abnormalities found on physical examination may aid in determining whether the hemorrhage is localized or generalized (i.e., bleeding from a venipuncture site versus bleeding diathesis). note whether the clinical signs are associated with a platelet problem and superficial hemorrhage or whether deep bleeding can be associated with abnormalities of the coagulation cascade. also, make an attempt to identify any concurrent illness that can predispose the patient to a bleeding disorder (i.e., pancreatitis, snakebite, sepsis, immunemediated hemolytic anemia, or severe trauma and crush or burn injury). abnormalities associated with coagulopathies include petechiae and ecchymoses, epistaxis, gingival bleeding, hematuria, hemarthrosis, melena, and hemorrhagic cavity (pleural and peritoneal or retroperitoneal) effusions. disseminated intravascular coagulation is a complex syndrome that results from the inappropriate activation of the clotting cascade, leading to disruption of the normal balance between thrombosis and fibrinolysis. the formation of diffuse microthrombi with concurrent consumption of platelets and activated clotting factors leads to end-organ thrombosis with various degrees of clinical hemorrhage. in animals, dic always results from some other pathologic process, including various forms of neoplasia, crush and heat-induced injury, sepsis, inflammation, and immune-mediated disorders (box 1-30). the pathophysiologic mechanisms involved in dic include vascular endothelial damage, activation and consumption of platelets, release of tissue procoagulants, and consumption of endogenous anticoagulants. because dic always results from some other disease process, diagnosis of dic is based on a number of criteria when evaluating various coagulation tests, peripheral blood smears, platelet count, and end products of thrombosis and fibrinolysis. there is no one definitive criterion for the diagnosis of dic (box 1-31). thrombocytopenia occurs as platelets are consumed during thrombosis. it is important to remember that trends in decline in platelet numbers are just as important as thrombocytopenia when making the diagnosis. in some cases the platelet count still may be within the normal reference range but has significantly decreased in the last 24 hours. early in dic the procoagulant cascade dominates, with hypercoagulability. activated clotting time, aptt, and pt may be rapid and shorter than normal. in most cases, we do not recognize the hypercoagulable state in our critically ill patients. later in dic, as platelets and activated clotting factors become consumed, the act, aptt, and pt become prolonged. antithrombin, a natural anticoagulant, also becomes consumed, and antithrombin levels decline. antithrombin levels can be measured at commercial laboratories and in some large veterinary institutions. the end products of thrombosis and subsequent fibrinolysis also can be measured. fibrinogen levels may decline, although this test is not sensitive or specific for dic. fibrin degradation (split) products also become elevated. fibrin degradation products are normally cleared by the liver, and these also become elevated in cases of hepatic failure because of lack of clearance. more recently, cageside d-dimer tests have become available to measure the breakdown product of cross-linked fibrin as a more sensitive and specific monitor of dic. management of dic first involves treating the primary underlying cause. by the time dic becomes evident, rapid and aggressive treatment is necessary. if you are suspicious of dic in any patient with a disease known to incite dic, then ideally, you should begin treatment before the hemostatic abnormalities start to occur for the best possible prognosis. treatment involves replacement of clotting factors and antithrombin and prevention of further clot formation. to replenish clotting factors and antithrombin, administer fresh whole blood or fresh frozen plasma. heparin requires antithrombin as a cofactor to inactivate thrombin and other activated coagulation factors. administer heparin (50 to 100 units/kg sq q6-8h of unfractionated heparin; or fractionated enoxaparin [lovenox], 1 mg/kg sq bid). aspirin (5 mg/kg po bid in dogs; every third day in cats) also can be administered to prevent platelet adhesion. management of dic also involves the rule of twenty monitoring and case management to maintain end-organ perfusion and oxygen delivery (see the rule of 20). hemophilia a is a sex-liked recessive trait that is carried by females and manifested in males. female hemophiliacs can occur when a hemophiliac male is bred with a carrier female. hemophilia a has been reported in cats and a number of dog breeds, including miniature schnauzer, saint bernard, miniature poodle, shetland sheepdog, english and irish setters, labrador retriever, german shepherd, collie, weimaraner, greyhound, chihuahua, english bulldog, samoyed, and vizsla. mild to moderate internal or external bleeding can occur. clinical signs of umbilical cord bleeding can become apparent in some animals shortly after weaning. gingival hemorrhage, hemarthrosis, gastrointestinal hemorrhage, and hematomas may occur. clotting profiles in animals with factor viii deficiency include prolonged aptt and act. the pt and buccal mucosa bleeding time are normal. affected animals have low factor viii activity but normal to high levels of factor viii-related antigen. carrier females can be detected by low (30% to 60% of normal) factor viii activity and normal to elevated levels of factor vii-related antigen. von willebrand's disease is a deficiency or defect in von willebrand's protein. a number of variants of the disease have been described: von willebrand's disease type i is associated with a defect in factor viir/protein concentration, and von willebrand's disease type ii is associated with a defect in viiir:vwf. type i von willebrand's disease is most common in veterinary medicine. von willebrand's disease has been identified in more than 29 breeds of dogs, with an incidence that varies from 10% to 60% depending on the breed of origin. affected breeds include doberman pinchers, german shepherd dogs, scottish terriers and standard manchester terriers, golden retrievers, chesapeake bay retrievers, miniature schnauzers, and pembroke welsh corgis. two forms of genetic expression occur: (1) autosomal recessive disease in which homozygous von willebrand's disease individuals have a bleeding disorder, whereas heterozygous individuals carry the trait but are clinically normal. the second variant of genetic expression involves an autosomal dominant disease with incomplete expression such that heterozygous individuals are affected carriers and homozygous individuals are severely affected. von willebrand's disease has high morbidity, but fortunately a low mortality. dogs with 30% or less than normal vwf tend to hemorrhage. platelet counts are normal, but bleeding times can be prolonged. the aptt can be slightly prolonged when factor viii is less than 50% of normal. routine screening tests are nondiagnostic for this disease, although in a predisposed breed with a normal platelet count, a prolonged buccal mucosa bleeding time strongly supports a diagnosis of von willebrand's disease. documentation of clinical bleeding with low or undetectable levels of factor viii antigen or platelet-related activities of vwf support a diagnosis of von willebrand's disease. recessive animals have zero vwf:antigen (a subunit of factor iii); heterozygotes have 15% to 60% of normal. in the incompletely dominant form, levels of vwf antigen are reduced (less than 7% to 60%). clinical signs in affected animals include epistaxis, hematuria, diarrhea with melena, penile bleeding, lameness, hemarthrosis, hematoma formation, and excessive bleeding with routine procedures such as nail trimming, ear cropping, tail docking, surgical procedures (spay, neuter), and lacerations. estrous and postpartum bleeding may be prolonged. a dna test to detect carriers of the vwf gene is available through vetgen (ann arbor, michigan) and michigan state university. patients with von willebrand's disease should avoid drugs known to affect platelet function adversely (sulfonamide, ampicillin, chloramphenicol, antihistamines, theophylline, phenothiazine tranquilizers, heparin, and estrogen). hemophilia b is an x-linked recessive trait that occurs with less frequency that hemophilia a. the disease has been reported in scottish terriers, shetland and old english sheepdogs, saint bernards, cocker spaniels, alaskan malamutes, labrador retrievers, bichon frises, airdale terriers, and british shorthair cats. carrier females have low (40% to 60% of normal) factor ix activity. clinical signs are more severe than for hemophilia a. congenital deficiencies of factor vii have been reported as an autosomal, incompletely dominant characteristic in beagles. heterozygotes have 50% factor vii deficiency. bleeding tends to be mild. the pt is prolonged in affected individuals. factor x deficiency has been documented in cocker spaniels and resembles fading-puppy syndrome in newborn dogs. internal or umbilical bleeding can occur, and affected dogs typically die. bleeding may be mild in adult dogs. in severe cases, factor x levels are reduced to 20% of normal; in mild cases, factor x levels are 20% to 70% of normal. factor xii deficiency has been documented as an inherited autosomal recessive trait in domestic cats. heterozygotes can be detected because they have a partial deficiency (50% of normal) of factor xii. homozygote cats have less than 2% factor xii activity. deficiency of hageman factor usually does not result in bleeding or other disorders. factor xi deficiency is an autosomal disease that has been documented in kerry blue terriers, great pyrenees, and english springer spaniels. in affected individuals, protracted bleeding may be observed. homozygotes have low factor xi activity (< 20% of normal), and heterozygotes have 40% to 60% of normal. the management of congenital defects of hemostasis typically involves replenishing the clotting factor that is present. usually, this can be accomplished in the form of fresh frozen plasma transfusion (20 ml/kg). if anemia is present because of severe hemorrhage, fresh whole blood or packed rbcs also can be administered. recent research has investigated the use of recombinant gene therapy in the treatment of specific factor deficiencies in dogs; however, the therapy is not yet available for use in clinical practice. in cases of von willebrand's disease, administration of fresh frozen plasma (10 to 20 ml/kg) or cryoprecipitate (1 unit/10 kg body mass) provides vwf, factor viii, and fibrinogen. doses can be repeated until hemorrhage ceases. 1-desamino-8-d-arginine vasopressin (ddavp) also can be administered (1 âµg/kg sc or iv diluted in 0.9% saline given over 10 to 20 minutes) to the donor and patient to increase the release of stored vwf from endothelial cells. a fresh whole blood transfusion can be obtained from the donor and immediately administered to the patient, or spun down and the fresh plasma administered if rbcs are not needed. administer a dose of ddavp to any affected dog before initiating any elective surgical procedures. a supply of fresh frozen plasma and rbcs should be on hand, should uncontrolled hemorrhage occur. platelets are essential to normal blood coagulation. after a vessel is damaged, release of vasoactive amines causes vasoconstriction and sluggish flow of blood in an attempt to squelch hemorrhage. platelets become activated by platelet activating factor, and attach to the damaged vascular endothelium. normal platelet adhesion depends on mediators such as calcium, fibrinogen, vwf:antigen, and a portion of factor viii. after adhesion, the platelets undergo primary aggregation and release a variety of chemical mediators including adenosine diphosphate, prostaglandins, serotonin, epinephrine, thromboplastin, and thromboxane a that promote secondary aggregation and contraction. platelet abnormalities can include decreased platelet production (thrombocytopenia), decreased platelet function (thrombocytopathia), increased platelet destruction, increased platelet consumption, and platelet sequestration. thrombocytopathia refers to platelet function abnormalities. alterations in platelet function can affect platelet adhesion, aggregation, or release of vasoactive substances that help form a stable clot (box 1-32). in von willebrand's disease there is a deficiency in vwf:antigen that results in altered platelet adhesion. vascular purpuras are reported and have been seen in collagen abnormalities such as ehlers-danlos syndrome, which can be inherited as an autosomal dominant trait with complete penetrance and has been recognized in german shepherd dogs, dachshunds, saint bernards, and labrador retrievers. thrombasthenic thrombopathia is a hereditary autosomal dominant abnormality that has been described in otterhounds, foxhounds and scottish terriers. in this condition, platelets do not aggregate normally in response to adenosine diphosphate and thrombin stimulation. evaluation of platelet function is based on a total platelet count, buccal mucosa bleeding time, and thromboelastography. platelet function defects (thrombocytopenia and thrombocytopathia) can affect both sexes. clinical signs can resemble von willebrand's disease. in most cases, buccal mucosa bleeding time will be prolonged, but platelet count and clotting tests will be normal. platelet count can be decreased because of problems with production, increased consumption, sequestration, or destruction. causes of accelerated platelet destruction are typically immune-mediated autoantibodies, drug antibodies, infection, and isoimmune destruction. consumption and sequestration usually are caused by dic, vasculitis, microangiopathic hemolytic anemia, severe vascular injury, hemolytic uremic syndrome, and gram-negative septicemia. primary thrombocytopenia with no known cause has been called idiopathic thrombocytic purpura. in approximately 80% of the cases, thrombocytopenia is associated with immune-mediated destruction caused by immune-mediated hemolytic anemia, systemic lupus erythematosus, rheumatoid arthritis, dic, and diseases that affect the bone marrow. in systemic lupus erythematosus, 20% to 30% of the affected dogs have concurrent idiopathic thrombocytic purpura. when immune-mediated hemolytic anemia and idiopathic thrombocytic purpura are present in the same patient, the disease is called evans syndrome. pf-3 is a non-complement-fixing antibody that is produced in the spleen and affects peripheral and bone marrow platelets and megakaryocytes. antibodies directed against platelets are usually of the igg subtype in animals. antiplatelet antibodies can be measured by a pf-3 release test. platelet counts with immune-mediated destruction typically are less than 50,000 platelets/âµl. infectious causes of thrombocytopenia include ehrlichia canis, anaplasma phagocytophilum (formerly, ehrlichia equi), and rickettsia rickettsii (rocky mountain spotted fever). primary immune-mediated thrombocytopenia has an unknown cause and most frequently is seen in middle-to older-aged female dogs. breed predispositions include cocker spaniels, german shepherd dogs, poodles (toy, miniature, standard), and old english sheepdogs. thrombocytopenia usually is manifested as petechiae, ecchymoses of skin and mucous membranes, hyphema, gingival and conjunctival bleeding, hematuria, melena, and epistaxis. to make a diagnosis of idiopathic thrombocytic purpura, measure the severity of thrombocytopenia (< 50,000 platelets/âµl), analyze the peripheral blood smear for evidence of platelet fragmentation or microthrombocytosis, normal to increased numbers of megakaryocytes in the bone marrow, detection of antiplatelet antibody, increased platelet counts after starting glucocorticoid therapy, and elimination of other causes of thrombocytopenia. if tick-borne illnesses are suspected, antibody titers for e. canis, a. phagocytophilum (formerly e. equi), and r. rickettsii should be performed. treatment of immune-mediated thrombocytopenia involves suppression of the immune system to stop the immune-mediated destruction and to stimulate platelet release from the bone marrow. traditionally, the gold standard to suppress the immune system is to use glucocorticoids (prednisone or prednisolone, 2 to 4 mg/kg po bid divided, or dexamethasone, 0.1 to 0.3 mg/kg iv or po q12h). more recently human serum immunoglobulin (igg) also has been used (0.2 to 0.5 g/kg iv in saline over 8 hours; pretreat with 1 mg/kg diphenhydramine 15 minutes before starting infusion). vincristine (0.5 mg/m 2 iv once) can stimulate the release of platelets from the bone marrow if megakaryocytic precursors are present; however, the platelets released may be immature and potentially nonfunctional. treatment with fresh whole blood or packed rbcs is appropriate if anemia is present; however, unless specific platelet-rich plasma has been purchased from a blood bank, fresh whole blood contains relatively few platelets, which are shortlived (2 hours) and will not effectively raise the platelet count at all. finally, long-term therapy is usually in the form of azathioprine (2 mg/kg po once daily, tapered to 1 mg/kg daily to every other day after 1 week) and cyclosporine (10 to 25 mg/kg po divided). if a tickborne illness is suspected, administer doxycycline (5 to 10 mg/kg po bid) for 4 weeks or if titers come back negative. thrombocytopenia also can occur in the cat. causes for thrombocytopenia in cats include infections (29%), neoplasia (20%), cardiac disease (7%), primary immune-mediated disease (2%), and unknown causes (20%). in one study of cats with feline leukemia and myeloproliferative disease, 44% of cases had thrombocytopenia. warfarin and coumarin derivatives are the major class of rodenticides used in the united states. vitamin k antagonist rodenticides inhibit the epoxidase reaction and deplete active vitamin k, causing a depletion of vitamin k-dependent coagulation factors (ii, vii, ix, x) within 24 hours to 1 week of ingestion, depending on the ingested dose. affected animals can spontaneously hemorrhage anywhere in the body. clinical signs can include hemoptysis, respiratory difficulty, cough, gingival bleeding, epistaxis, hematuria, hyphema, conjunctival bleeding, petechiae and ecchymoses, cavity hemorrhage (pleural, peritoneal, retroperitoneal) with acute weakness, lethargy or collapse, hemarthrosis with lameness, deep muscle bleeds, and intracranial or spinal cord hemorrhage. diagnosis of vitamin k antagonism includes prolonged pt. a pivka (protein induced by vitamin k absence or antagonism) test also can be performed, if possible. treatment of vitamin k antagonist rodenticide intoxication and other causes of vitamin k deficiency involves supplementation with vitamin k 1 (phytonadione, 5 mg/kg sq once with 25-gauge needle in multiple sites, and then 2.5 mg/kg po bid to tid for 30 days). never administer injections of vitamin k intramuscularly, because of the risk of causing deep muscle hematomas, or intravenously, because of the risk of anaphylaxis. the pt should be rechecked 2 days after the last vitamin k capsule is administered, for some of the secondgeneration warfarin derivates are fat-soluble, and treatment may be required for an additional 2 weeks. act, activated clotting time; aptt, activated partial thromboplastin time; bmbt, buccal mucosa bleeding time; fdp, fibrin degradation products; n, normal; pt, prothrombin time. thermal burns are fortunately a relatively infrequent occurrence in veterinary patients. box 1-33 lists various causes of malicious and accidental burns. the location of the burn is also important in assessing its severity and potential to lose function. burns on the perineum, feet, face, and ears are considered to be the most severe because of loss of function and severe pain. often the severity of thermal injury is difficult to assess in animals because hair coat potentially can mask clinical signs and because the thermal injury can continue after the animal has been removed from the heat source. the skin cools slowly and warms slowly, considerations that become important when initiating therapy for burns. the severity of thermal injury is associated with the temperature to which the animal is exposed, the duration of contact, and the ability of the tissue to dissipate heat. the tissue closest to the heat source undergoes necrosis and has decreased blood flow. the severity of thermal burn injury is associated directly with the temperature to which the animal is exposed, the percentage of total body surface area affected, the thickness of injured tissue, and whether underlying complications with other body systems occur. prognosis largely depends on the total body surface area affected (table 1-31) . superficial partial thickness, or first-degree, burns offer the most favorable prognosis. the affected epidermis initially appears erythematous and then quickly desquamates within 3 to 6 days. in most cases, fur grows back without leaving a scar. deep partial thickness, or second-degree, burns involve the epidermis and dermis and are associated with subcutaneous edema, inflammation, and pain. deep partial thickness burns heal from deeper adnexal tissues and from the wound edges and are associated with an increased chance of scarring and depigmentation. the most severe type is known as full thickness, or third-degree, burns, in which thermal injury destroys the entire thickness of the skin and forms an eschar. thrombosis of superficial and deeper skin vasculature and gangrene occurs. treatment involves sequential wound debridement. healing occurs by second intention and reepithelialization or by wound reconstruction. in most cases, scarring is extensive in affected areas. burns greater than 20% of total body surface area will have systemic effects, including impaired cardiovascular function, pulmonary dysfunction, and impaired immune function. burned tissue, with capillary damage, has increased permeability. the release of inflammatory cytokines, oxygen-derived free radical species, prostaglandins, leukotrienes, 108 1 emergency care histamine, serotonin, and kinins results in increased vascular permeability and leakage of plasma proteins into the interstitium and extravascular space. at the time of presentation, first examine the patient and ascertain whether airway obstruction, impaired ventilatory function, circulatory shock, or pain are present. if necessary, establish an airway with endotracheal intubation or emergency tracheostomy. next, cool the burned area(s) with topical cool water. use care to avoid overcooling and iatrogenic hypothermia. the best approach is to cool only one portion of the patient's body at a time, then dry, and repeat the process for all affected areas to avoid overcooling and iatrogenic hypothermia. establish vascular access and administer appropriate and judicious analgesic drugs and intravenous fluid therapy. whenever possible, avoid placing a catheter through an area of burned or damaged skin. in the early stages of burn injury, shock doses of intravenous crystalloid fluids usually are not required. later, however, as severe tissue exudation occurs, protein and fluid losses can become extensive, requiring aggressive crystalloid and colloid support to treat hypovolemia and hypoproteinemia. flush the eyes with sterile saline and examine behind the third eyelids for any particulate matter. stain the corneas to make sure that superficial corneal burns are not present. treat superficial corneal burns with triple antibiotic ophthalmic ointment. next, assess the total body surface area affected, as this will gauge prognosis. depending on the extent of the damage, decide whether the burn is superficial and local therapy is indicated or whether more severe injuries exist that may involve systemic therapy or possibly euthanasia. in most cases the diagnoses of thermal burns are based on a clinical history of being in a house fire, clothes dryer, or under a heating lamp. too frequently, however, thermal burns become apparent days after an elective surgical procedure in which the patient was placed on a faulty heating pad rather than a circulating warm water or warm air blanket. superficial burns appear as singed fur with desquamating, easily epilated hair. this condition also can resemble a superficial or deeper dermatophytosis if history is unknown. other differential diagnoses include immune-mediated vasculitis or erythema multiforme. unless the superficial dermis is blistered, it may be difficult to distinguish between a thermal burn, chemical burn, or electrical burn if the trauma went unnoticed. management of burn injury largely depends on the depth of injury and the total body surface area affected. partial thickness burns and those affecting less than 15% of the total body surface area will require support in the form of antibiotic ointment and systemic analgesic drugs. burns affecting greater than 15% of total body surface area or deep thickness burns require more aggressive therapy. central venous catheters can be placed to administer crystalloid and colloid fluids, parenteral nutrition if necessary, antibiotics, and analgesic drugs. monitor perfusion parameters closely, including heart rate, blood pressure, capillary refill time, and urine output. respiratory function can be impaired because of concurrent smoke inhalation, thermal damage to the upper airways and alveoli, and carboxyhemoglobin or methemoglobin intoxication. respiratory function also can be impaired because of burn injury to the skin around the thoracic cage. thoracic radiographs may reveal patchy interstitial to alveolar infiltrates associated with pulmonary edema, pneumonia, and atelectasis. bronchoscopy often reveals edema, inflammation, particulate matter, and ulceration of the tracheobronchial tree. in some cases, upper airway inflammation is so severe that an emergency tracheostomy must be performed to treat airway obstruction. administer supplemental humidified oxygen at 50 to 100 ml/kg/minute via endotracheal tube, tracheostomy, nasal or intratracheal tube, or hood oxygen if respiratory function and hypoxemia are present. perform blood work including a hematocrit, albumin, bun, creatinine, and glucose at the time of presentation. monitor serum electrolytes, albumin, and colloid oncotic pressure closely because derangements can be severe as burns become exudative. the goal of fluid therapy in the burn patient is to establish and maintain intravascular and interstitial fluid volume, normalize electrolyte and acid-base status, and maintain serum albumin and oncotic pressure. in the first 24 hours following burn injury, direct fluid therapy to maintaining the patient's metabolic fluid requirements. crystalloid fluids in the form of normosol-r, plasmalyte-m, or lactated ringer's solution can be administered according to the patient's electrolyte and acid-base status (see fluid therapy). monitor urine output, and keep it at 1 to 2 ml/kg/hour. avoid overhydration in the early stages of burn injury. in affected burn patients, calculate the amount of fluid that should be administered over a 24-hour period from the formula 1 â�� 4 ml/kg ã� percent total body surface area. administer half of this calculated dose over the first 8 hours and then the remaining half over the next 16 hours. in cats, administer only 50% to 75% of this calculated volume. to administer this volume and also avoid fluid overload is often difficult in critically ill patients with pulmonary involvement associated with smoke inhalation injury. avoid colloids in the first 6 hours after burn injury. monitor the patient closely for serous nasal discharge, chemosis, and rales that may signify pulmonary edema. as burns become exudative, weigh the patient at least twice daily. infused fluid should equal fluid output in the form of urine and wound exudates. acute weight loss signifies acute fluid loss and that crystalloid fluid infusion should be more aggressive. ideally, keep the patient's serum albumin equal to or greater than 2.0 g/dl and total protein between 4.0 and 6.5 g/dl using a combination of fresh frozen plasma or concentrated human albumin. adjunct colloidal support can be provided with synthetic colloids including hetastarch or hbocs. keep serum potassium within 3.5 to 4.5 meq/l using potassium chloride or potassium phosphate supplementation. if potassium supplementation exceeds 80 to 100 meq/l and the patient continues to have severe refractory hypokalemia, administer magnesium chloride (0.75 meq/kg/day) to enhance potassium retention. if anemia occurs, administer packed rbcs or whole blood (see blood component therapy). lavage wounds daily with lactated ringer's solution or 0.9% sodium chloride solution. place wet-to-dry bandages or bandages soaked in silver sulfadiazine or nitrofurazone ointment over the wounds. depending on the thickness of the burn, epilation and eschar formation and separation may take 2 to 10 days. at each bandage change, debride devitalized tissue to normal tissue. perform staged partial or total escharectomy, and leave the wound to heal by second intention or by reconstruction using skin advancement flaps or grafts. maintain meticulous sterility at all times, given that burn patients are at high risk for infection. administer broad-spectrum antibiotics including cefazolin and enrofloxacin. perform wound culture if a resistant bacterial infection is suspected. the most common cause of electrical injury is associated with an animal chewing on low-voltage alternating current electrical cords in the household. damage is caused by the current flowing through the path of least resistance, causing heat and thrombosis of vessels and neurons. in some cases, the owner witnesses the event. in other cases, the owner presents the patient because of vague nonspecific signs, and characteristic abnormalities on physical examination support a diagnosis of electrocution. burns on the face, paws, commissures of the mouth, tongue, and soft palate may be present. electrocution causes a massive release of catecholamines and can predispose the patient to noncardiogenic pulmonary edema within 36 hours of the incident. clinical signs may be isolated to the pulmonary system, including orthopnea, pulmonary crackles, and cyanosis. assess the patient's lips, tongue, soft palate, gingivae, and commissures of the mouth. early after electrocution, the wound may appear small and white, black, or yellow. later, the wound may become larger as tissue sloughs because of damaged vascular supply. assess the patient's respiratory status. auscultate the lungs to determine whether pulmonary crackles 110 1 emergency care are present. if the patient is stable, thoracic radiographs may demonstrate an interstitial to alveolar lung pattern in the dorsocaudal lung fields. measure the patient's heart rate, blood pressure, oxygenation as determined by pulse oximetry or arterial blood gas and urine output. immediate treatment consists of judicious use of analgesics for the burn injury, antibiotics (cefazolin, 22 mg/kg q8h; cephalexin, 22 mg/kg q8h), and humidified supplemental oxygen (50 to 100 ml/kg/minute). direct fluid therapy at providing the patient's metabolic fluid requirements. because of the risk of development of noncardiogenic pulmonary edema, avoid overzealous administration of crystalloid fluids. differential diagnoses for the patient with electrical burn injury and electrocution include chemical or thermal burn, immune-mediated glossitis, cardiogenic pulmonary edema, and pneumonia. management of the patient with electrical burn injury and electrocution primarily involves the administration of analgesic agents, supplemental humidified oxygen, and topical treatment of electrical burns. the noncardiogenic pulmonary edema is typically unresponsive to diuretics (i.e., furosemide), bronchodilators (i.e., aminophylline), and splanchnic vascular dilators (i.e., low-dose morphine). the use of glucocorticoids has no proven benefit and may impair respiratory immune function and is therefore contraindicated. oral burns may require debridement and advancement flaps if large defects or oronasal fistulas develop. if oral injury is severe, place an esophagostomy or percutaneous gastrostomy tube to ensure adequate nutrition during the healing process. if an animal survives the initial electrocution, prognosis is generally favorable with aggressive supportive care. chemical burns are associated with a number of inciting causes, including oxidizing agents, reducing agents, corrosive chemicals, protoplasmic poisons, desiccants, and vesicants. the treatment for chemical burns differs slightly from that for thermal burns, so it remains important to investigate the cause of the burn when providing initial treatment, whenever possible. at the scene, advise the owner to wrap the patient in a clean towel for transport. chilling can be avoided by then wrapping the patient in a second or third blanket. placement of ointments by well-doers should be avoided. encourage immediate transport to the nearest triage facility. the first and foremost consideration when treating a patient with chemical burn is to remove the animal from the inciting cause or offending agent. make no attempt to neutralize alkaline or acid substances because the procedure potentially could cause an exothermic reaction, leading to thermal injury in addition to the chemical injury. remove collars or leashes that may act as tourniquets or constricting devices. flush affected areas with copious amounts of cool water for several minutes, not cooling more than 10% to 20% of the body at any one time to prevent iatrogenic hypothermia. support breathing by extending the patient's head and neck. carefully clip the fur over affected areas for further evaluation of the extent of the injury. lavage exposed eyes with sterile saline, and stain the cornea to evaluate for any corneal burns. debride any wounds carefully, knowing that the full extent of the wound may not manifest itself for several days. then cover the wounds with antibiotic burn ointment such as silver sulfadiazine and an occlusive dressing. without a history of exposure, the differential diagnosis for any chemical burn includes thermal burn, necrotizing vasculitis, erythema multiforme, or superficial or deep pyoderma. contact local or national animal poison control regarding whether to attempt neutralization. perform daily bandage changes with staged debridement as the full extent of the wound manifests itself. place antimicrobial ointment and silver sulfadiazine ointment over the wound to prevent infection. the routine use of antibiotics may promote the development of a resistant bacterial infection. first-generation cephalosporin can be administered. if a more serious infection develops, perform culture and susceptibility testing to direct appropriate antibiotic therapy. the wound can heal by second intention or may require reconstructive repair for definitive closure. the primary cause of radiation injury in small animal patients is radiation therapy for neoplastic conditions. the goal of radiation therapy is to kill neoplastic cells. an unfortunate side effect is damage to adjacent normal tissue that results in necrosis, fibrosis, and impaired circulation to the affected area. radiation burns result in dermatitis, mucositis, impaired surgical wound healing, and chronic nonhealing wounds. in many cases, the degree of secondary radiation injury to normal tissue can be prevented or decreased with careful radiation planning and mapping of the radiation field, such that radiation exposure to normal tissue is limited to the smallest extent possible. with the advent of three-dimensional imaging modalities such as computed tomography (ct) and magnetic resonance imaging (mri), this has become more routine in veterinary oncology to date. radiation injury can be early and appear at the later stage of the course of radiation therapy. late effects can be delayed and occur 6 months to years after treatment. the degree of radiation injury is categorized based on the depth of tissue affected. first-degree changes cause cutaneous erythema. second-degree changes cause superficial desquamation. thirddegree changes cause deeper moist desquamation, and fourth-degree changes are associated with complete dermal destruction and ulceration. during the early stages of radiation injury, affected tissues may appear erythematous and edematous. wound exudates may be moist, or the skin may appear dry and scaly with desquamation or ulceration. later, the area may scar and depigment or may have induration, atrophy, telangiectasia, keratosis, and decreased adnexal structures. treatment for radiation dermatitis is to irrigate the area with warmed saline and to protect the area from self-mutilation. no-bite, or elizabethan, collars or loose clothing can be used to protect the area for patient-induced injury. mucositis can be treated with topical green tea baths and the administration of an oral solution of l-glutamine powder (4 g/m 2 ). local irrigation of xylocaine or lidocaine viscous jelly can be used in dogs but should be avoided in cats because of the risk of inducing hemolytic anemia and neurotoxicity. topical and systemic antibiotics (cephalexin, 22 mg/kg po tid) also can be administered. avoid antibiotics that can be sensitized by radiation (i.e., metronidazole). because most radiation burns are associated with a known exposure to radiation therapy, the cause of the patient's injury usually is known. if an animal presents to you with a scar, however, differential diagnoses may include nasal planum solar dermatitis, pemphigus foliaceus, discoid lupus, superficial necrolytic dermatitis, superficial or deep pyoderma, chemical burn, or thermal burn. treatment of radiation injury involves making the patient as comfortable as possible with analgesic drugs, prevention of self-mutilation, and staged debridement techniques. wounds can heal by second intention or may require reconstructive surgery. distress syndrome (ards), and anesthetic agents. the acute onset of bradycardia, change in mucous membrane color and capillary refill time, change in respiratory pattern, and change in mentation are signs of possible deterioration and impending cardiopulmonary arrest. the diagnosis of cardiopulmonary arrest is based on the absence of effective ventilation, severe cyanosis, absence of a palpable pulse or apex heartbeat, absence of heart sounds, and ecg evidence of asystole or other nonperfusing rhythm such as electricalmechanical dissociation (aka pulseless electrical activity) or ventricular fibrillation. the goals of cpcr are to obtain airway access, provide artificial ventilation and supplemental oxygen, implement cardiac compressions and cardiovascular support, recognize and treat dysrhythmias and arrhythmias, and provide stabilization and treatment for cardiovascular, pulmonary, and cerebral function in the event of a successful resuscitation. even with aggressive treatment and management, the overall success of cpcr is less than 5% in critically ill or traumatized patients and 20% to 30% in anesthetized patients. basic life support involves rapid intubation to gain airway access, artificial ventilation, and cardiac compressions to promote blood flow and delivery of oxygen to the brain and other important tissues (figure 1-26 ). perform the abcs or cabs of cpcr, where a is airway, b is breathing, and c is compression and circulation. recently, the paradigm has shifted to cabs. while a team member is grabbing an endotracheal tube, clearing the airway of foreign debris, and establishing airway access through endotracheal intubation, a second person starts external cardiac compressions to deliver oxygen that is in the bloodstream to the vital organs. the patient should be positioned in dorsal (> 7 kg) or lateral (< 7 kg) recumbency for external cardiac compressions. approximately 80 to 120 external compressions should be performed over the patient's sternum. a team member should palpate for a peripheral pulse to determine whether cardiac compressions are actually effective. if a peripheral pulse cannot be palpated for every chest compression, change the patient's position and have a larger individual perform compressions, or initiate open-chest cardiac resuscitation. once the patient is intubated, tie in the endotracheal tube and attach it to an oxygen source (anesthetic machine or mechanical ventilator or ambu bag) for artificial ventilation. the oxygen flow rate should be 150 ml/kg/minute. give two long breaths, and then 12 to 16 breaths per minute. simultaneous ventilation with thoracic compression increases the pressure difference in the thorax and allows more forward flow of oxygenated blood through the great vessels into the periphery. if possible, a third team member can initiate interposed abdominal compressions, compressing the abdomen when the thoracic cage is relaxed, to improve forward flow. if only one person is available to perform the thoracic compressions and ventilation, give two breaths for every 15 compressions (i.e., 15 thoracic compressions followed by two long breaths, and then start thoracic compressions again). the jen chung maneuver can be performed by placing a 25-to 22-gauge hypodermic needle through the skin of the nasal philtrum and twisting the needle into the periosteum to stimulate respirations. this maneuver appears to work better in cats than dogs at return to spontaneous respiration. advanced life support during cpcr involves ecg, pulse oximetry and capnometry monitoring, administration of drugs, and the administration of intravenous fluids (in select cases). most of the drugs used during cpcr can be administered directly into the lungs from the endotracheal tube (intratracheal tube). therefore, only in select instances is it necessary to establish vascular or intraosseous access during cpcr (figure 1-27) . if an animal experiences cardiopulmonary arrest because of extreme hemorrhage or hypovolemia, inappropriate vasodilation caused by sepsis or systemic inflammation, or vasodilation resulting from anesthesia, the administration of shock volumes (90 ml/kg/hour in dogs and 44 ml/kg/hour in cats) is appropriate. if a patient is euvolemic and experiences cardiopulmonary arrest, however, an increase in circulating fluid volume actually can impair coronary artery perfusion by increasing diastolic arterial blood pressure and is asystole is one of the most common rhythm disturbances that causes cardiac arrest in small animal patients. one of the most important things to do when the ecg looks like asystole is to make sure that the ecg monitor is working properly and that all ecg leads are attached properly to the patient. if asystole is truly present, reverse any opiate, î± 2 -agonist, or benzodiazepine drugs with their appropriate reversal agents. lowdose epinephrine (0.02 to 0.04 mg/kg diluted with 5 ml sterile saline) can be administered directly into the endotracheal tube via a rigid or red rubber catheter. if vascular access is available, epinephrine (0.02 to 0.04 mg/kg) can be administered intravenously. no drug should ever be administered directly into the heart by intracardiac injection. unless the heart is in the veterinarian's hand during open-chest cpcr, intracardiac injection is risky and potentially could lacerate a coronary artery or cause the myocardium to become more irritable and refractory to other therapies, if a drug is delivered into the myocardium and not into the ventricle. for these reasons, intracardiac injections are contraindicated. administer atropine (0.4 mg/kg iv, io, or 0.4 mg/kg it) immediately after the epinephrine. atropine, a vagolytic drug, serves to decrease tonic vagal inhibition of the sinoatrial and atrioventricular node and increase heart rate. administer atropine and epinephrine every 2 to 5 minutes during asystole while cardiac compressions, interposed abdominal compressions, and artificial ventilation are continued. although discontinuation of thoracic compressions can decrease the chance of success during cpcr, you must intermittently evaluate the ecg monitor for any rhythm change that may require different drug therapies. if the cardiac arrest was not witnessed or more than 2 to 5 minutes have passed without successful return to a perfusing rhythm, perform open-chest cpcr, if the client wishes. administer sodium bicarbonate (1 to 2 meq/kg iv) every 10 to 15 minutes during cpcr. sodium bicarbonate is the only drug used in cpcr that should not be administered intratracheally because of inactivation of pulmonary surfactant. electrical-mechanical dissociation also is known as pulseless electrical activity and is an electrical rhythm that may look wide and bizarre and irregular with no associated mechanical contraction of the ventricles. the rhythm can appear different from patient to patient. electrical-mechanical dissociation is one of the more common nonperfusing rhythms observed during cardiopulmonary arrest in small animal patients (figure 1-28) . when electrical-mechanical dissociation is identified, first confirm the rhythm and proceed with cpcr as previously described. electrical-mechanical dissociation is thought to be associated with high doses of endogenous endorphins and high vagal tone. the treatment of choice for electrical-mechanical dissociation is high-dose atropine (4 mg/kg iv, it [10 times the normal dose]) and naloxone hydrochloride (0.03 mg/kg iv, io, it). administer epinephrine (0.02 to 0.04 mg/kg diluted in 5 ml sterile 0.9% saline it). if the rhythm does not change within 2 minutes, consider open-chest cardiac massage. ventricular fibrillation can be coarse (figure 1-29) . patients with coarse ventricular fibrillation are easier to defibrillate than those with fine defibrillation. if ventricular fibrillation is identified, initiate cpcr as described previously (figure 1-30) . if an electrical defibrillator is available, administer 5 j/kg of direct current externally. when a patient in cardiopulmonary arrest is attached to ecg leads, it is important to use contact electrode paste, water-soluble gel such as ky jelly, or water, rather than any form of alcohol. electrical defibrillation of a patient who has alcohol on the ecg leads can lead to fire and thermal burns. reverse any opioid, î± 2 -agonist, and phenothiazine drugs that have been administered to the patient. if fine ventricular fibrillation is identified, administer epinephrine 1 figure 1 -28: electrical-mechanical dissociation (emd), also known as pulseless electrical activity (pea). the complexes often appear wide and bizarre without a palpable apex beat or functional contraction of the heart. this is just one example of emd, as many shapes and complexes may be observed. organized according to whether an electrical defibrillator is available. after each intervention step, the ecg should be reevaluated and the next step initiated if v-fib is still seen. if a new arrhythmia develops, the appropriate therapy for that rhythm should be inititated. if a sinus rhythm is seen with a palpable apex beat, postresuscitation measures should be implemented. perform open-chest cpcr immediately if a pathologic condition exists that prevents enough of a change in intrathoracic pressure that closed-chest cpcr will not be effective in promoting forward blood flow (box . to perform open-chest cpcr, place the patient in right lateral recumbency. clip a wide strip of fur over the left fifth to seventh intercostal space and quickly aseptically scrub over the clipped area. using a no. 10 scalpel blade, incise over the fifth intercostal space through the skin and subcutaneous tissue to the level of the intercostal muscles. with a mayo scissors, make a blunt stab incision through the intercostal muscles in the left sixth intercostal space. make sure that the person who is breathing for the patient deflates the lungs as you make the stab incision to avoid iatrogenic lung puncture. after the stab incision, open the tips of the mayo scissors and quickly open the muscle dorsally and ventrally to the sternum with a sliding motion. avoid the internal thoracic artery at the sternum and the intercostal arteries at the caudal aspect of each rib. cut the rib adjacent to the sternum and push it behind the rib in front of and at the caudal aspect of the incision to allow more room and better visualization if a rib spreading retractor is not available. visualize the heart in the pericardial sac. visualize the phrenic nerve, and incise the pericardium just ventral to the phrenic nerve. make sure to not cut the phrenic nerve. grasp the heart in your hand(s) and gently squeeze it from apex to base, allowing time for the ventricle to fill before the next "contraction." if the heart does not seem to be filling, administer fluids intravenously or directly into the right atrium. the descending aorta can be cross-clamped with a rummel tourniquet or red rubber catheter to improve perfusion to the brain and heart. postresuscitation care and monitoring (prolonged life support) postresuscitation care involves careful monitoring and management of the adverse effects of hypoxia and reperfusion injury on the brain and other vital organs. the first 4 hours after an arrest are most critical, because this is the time period in which an animal is most likely to rearrest unless the underlying cause of the initial arrest has been determine and treated (table 1 -32) . until an animal is adequately ventilating on its own, artificial ventilation by manual bagging or attaching the patient to a mechanical ventilator with supplemental oxygen must continue. the efficacy of oxygenation and ventilation can be monitored using a wright's respirometer, pulse oximetry, capnometry, and arterial blood . once an animal is extubated, administer supplemental oxygen (50 to 100 ml/ kg/minute) (see oxygen supplementation). the brain is sensitive to ischemia and reperfusion injury. the effects of cellular hypoxia and reperfusion include the development of oxygen-derived free radical species that contribute to cerebral edema. administer mannitol (0.5 to 1 g/kg iv over 5 to 10 minutes), followed by furosemide (1 mg/kg iv) 20 minutes later, to all patients that have experienced cardiopulmonary arrest and have had successful resuscitation. mannitol and furosemide work synergistically to decrease cerebral edema formation and scavenge oxygen-derived free radical species. the combination of cardiac arrest, myocardial ischemia and acidosis, and external or internal cardiac compressions often make the myocardium irritable and predisposed to dysrhythmias following successful cpcr. start lidocaine (1 to 2 mg/kg iv, followed by 50 to 100 âµg/kg/minute iv cri) in all patients following successful resuscitative efforts. monitor the ecg continuously for the presence of cardiac dysrhythmias and recurrence of nonperfusing rhythms. perform direct or indirect blood pressure monitoring. if a patient's systolic blood pressure is less than 80 mm hg, diastolic pressure is less than 40 mm hg, or mean arterial blood pressure is less than 60 mm hg, administer positive inotropic drugs (dobutamine, 1 to 20 âµg/kg/minute) and pressor agents (epinephrine, 0.02 to 0.04 mg/kg iv, io, it) to improve cardiac contractility, cardiac output, and core organ perfusion. the kidneys are sensitive to decreased perfusion and cellular hypoxia. place a urinary catheter and monitor urine output. in a euvolemic patient, normal urine output should be no less than 1 to 2 ml/kg/hour. if urine output is low, administer low-dose dopamine (3 to 5 âµg/kg/minute iv cri) in an attempt to dilate afferent renal vessels and improve renal perfusion. maintain acid-base and electrolyte status within normal reference ranges. monitor serum lactate as a rough indicator of organ perfusion and cellular oxygen extraction. the presence of elevated or rising serum lactate in the face of aggressive cardiorespiratory and cerebral support makes prognosis less favorable. cole sg, otto cm, hughes d: cardiopulmonary cerebral resuscitation: a clinical practice review part i, j vet emerg crit care 12 (4) immediate action depends largely on recognition of the primary or secondary cause of the dysrhythmia and treating the dysrhythmia and underlying cause. diagnosis of cardiac dysrhythmias is based on physical examination findings of abnormal thoracic/cardiac auscultation, the presence of abnormal pulse rhythm and quality, and recognition of ecg abnormalities. the ecg is critical to the accurate diagnosis of dysrhythmias. ventricular dysrhythmias arise from ectopic foci in the ventricles that cause the wave of depolarization to spread from cell to cell rather than spread through fast-conducting tissue. this causes the qrs complex to appear wide and bizarre, unless the ectopic focus originates close to the atrioventricular node high in the ventricle. other ecg features of ventricular dysrhythmias include a t wave polarity that is opposite to the qrs complex and nonrelated p waves. ventricular dysrhythmias may manifest as isolated ventricular premature complexes, couplets, or triplets; bigeminy; or ventricular tachycardia. relatively slow ventricular tachycardia is known as an idioventricular rhythm and is not as hemodynamically significant as faster ventricular tachycardia. idioventricular rhythm usually is less than 130 beats per minute and may alternate spontaneously with sinus arrhythmias (figures 1-31 to . supraventricular dysrhythmias arise from ectopic foci in the atria and are commonly associated with atrial dilatation and structural heart disease such as advanced acquired or congenital heart disease, cardiomyopathies, cardiac neoplasia, or advanced heartworm disease. occasionally, supraventricular dysrhythmias may be associated with respiratory or other systemic illness. sustained supraventricular tachycardia in the absence of underlying structural heart or systemic disease is disturbing and should alert the clinician that an accessory pathway conduction disturbance may be present, particularly in labrador retrievers. supraventricular dysrhythmias can manifest as isolated premature complexes (atrial premature complexes or contractions), sustained or paroxysmal supraventricular tachycardia (atrial tachycardia), or atrial fibrillation or flutter. in the dog, atrial fibrillation most commonly is associated with dilative cardiomyopathy. rarely and primarily in giant breed dogs, lone atrial fibrillation can occur with no underlying heart disease. atrial fibrillation and the resultant sustained elevation in ventricular rate are presumed to progress to dilative cardiomyopathy in such breeds. by comparison, atrial fibrillation is relatively uncommon in cats because of the small size of their atria but is associated most commonly with hypertrophic and restrictive cardiomyopathy. the ecg is critical to the diagnosis of a supraventricular dysrhythmia. the ecg usually demonstrates a normal appearance to the qrs complex unless aberrant conduction occurs in the ventricles, in which case the qrs can be wide but still originate from above the atrioventricular node. in most cases of a supraventricular dysrhythmia, some evidence of atrial activity including p waves, atrial flutter, or atrial fibrillation is apparent. in some cases, it may be difficult to diagnose the exact rhythm without slowing the rate down mechanically or through pharmacologic intervention. once a rhythm diagnosis is made, appropriate treatment strategies can be implemented (figures 1-35 and 1-36 ). treatment of ventricular dysrhythmias largely depends on the number of ectopic foci discharging, the rate and character of the dysrhythmia, and whether the presence of the abnormal beats is of adverse hemodynamic consequence, including risk of sudden death. many ventricular dysrhythmias, including slow idioventricular rhythms, ventricular bigeminy, or intermittent ventricular premature complexes, do not warrant antiarrhythmic therapy unless the patient is hypotensive and the dysrhythmia is thought to be contributing to the hypotension. in such cases, correction of the underlying disease process including hypoxia, pain, or anxiety often alleviates or decreases the incidence of the dysrhythmia. more serious ventricular dysrhythmias that warrant antiarrhythmic therapy (table 1 -33) include sustained ventricular tachycardia (>160 beats/minute in dogs; >220 beats/minute in cats), multifocal ventricular premature complexes originating from more than one place in the ventricles, and the presence of r-on-t phenomena where the t wave of the preceding complex is superimposed on the qrs of the next complex with no return to isoelectric shelf in between complexes. treat these ventricular dysrhythmias immediately and aggressively. in dogs, the mainstay of emergency treatment for ventricular dysrhythmias is lidocaine therapy. administer lidocaine (1 to 2 mg/kg iv bolus) over a period of 5 minutes to prevent the adverse side effects of seizures or vomiting. the bolus can be repeated an additional 3 times (total dose 8 mg/kg) over 15 minutes, or the patient can be placed on a constant rate infusion (50 to 100 âµg/kg/minute) if control of ventricular tachycardia is accomplished. also correct the patient's magnesium and potassium deficiencies to maximize the success of lidocaine therapy in the treatment of ventricular tachycardia. procainamide (4 mg/kg iv slowly over 3 to 5 minutes) also can be used to control ventricular tachycardia. if procainamide is successful at controlling ventricular tachycardia, administer it as a constant rate infusion (25 to 40 âµg/kg/minute). side effects of procainamide include vomiting, diarrhea, and hypotension. chronic oral therapy may or may not be necessary in the treatment of acute ventricular tachycardia. the decision to continue antiarrhythmic therapy depends on the underlying disease process and the expectation of persistent arrhythmogenesis of the underlying disease process. oral antiarrhythmic therapy is warranted in cases in which a serious ventricular dysrhythmia is recognized but the animal does not require hospitalization, such as the syncopal boxer with intermittent ventricular dysrhythmias and no evidence of structural heart disease. it deserves emphasis that asymptomatic, low-grade ventricular dysrhythmias probably do not require treatment. if maintenance therapy for ventricular dysrhythmias is needed, use an oral drug based on the underlying disease process, clinical familiarity, class of drug, dosing frequency, owner compliance, concurrent medications, cost, and potential adverse side effects. in the cat the mainstay of antiarrhythmic therapy is the use of a î²-adrenergic antagonist. in the acute management of ventricular dysrhythmias in cases of hypertrophic, restrictive, or unclassified cardiomyopathies, consider using injectable esmolol (0.05 to 1.0 mg/kg iv slowly to effect) or propranolol (0.02 to 0.06 mg/kg iv slowly to effect), particularly if the dysrhythmia results from hyperthyroidism. for chronic oral ventricular antiarrhythmic therapy in cats, propranolol (2.5 to 5.0 mg po per cat q8h) or atenolol (6.25 to 12.5 mg po per cat q12-24h) can be used. the decision to treat supraventricular dysrhythmias depends on the ventricular rate and the hemodynamic consequences of the dysrhythmia. for intermittent isolated atrial 124 1 emergency care procainamide 10-20 mg/kg po q6-8h tocainide* 10-20 mg/kg po q8h sotalol 40-120 mg per dog q12h (start low, then titrate up to effect) mexiletine 5-8 mg/kg po q8h atenolol 0.25-1.0 mg/kg po q12-24h (start low, titrate upward to effect) *do not use for longer than 2 weeks because of idiosyncratic blindness. premature contractions, couplets, and triplets, usually no treatment is required. when the ventricular rate exceeds 180 beats/minute, diastolic filling time is shortened, causing the heart to not fill adequately. the consequence is decreased cardiac output and decreased coronary artery perfusion. the goal of therapy is rhythm control or, in most cases, rate control. in cases of atrial fibrillation and congestive heart failure, conversion to a normal sinus rhythm rarely can be achieved, although electrocardioversion or pharmacoconversion can be attempted. in the dog a vagal maneuver can be attempted by pressing on the eyeballs or massaging the carotid body. for sustained supraventricular tachycardia, diltiazem (0.25 mg/kg iv), esmolol (0.05 to 0.1, titrated upward to a cumulative dose of 0.5 mg/kg iv), or propranolol (0.04 to 0.1 mg/kg iv slowly to effect) can be administered in an attempt to slow the ventricular rate in emergent situations. administer oral diltiazem (0.5 mg/kg po q8h), diltiazem (dilacor-xr) (1.5 to 6 mg/kg po q12-24h), propranolol (0.1 to 0.2 mg/kg tid, titrated up to a maximum of 0.5 mg/kg po q8h), atenolol (0.25 to 1 mg/kg q12-24h), or digoxin (0.005 to 0.01 mg/kg bid or 0.22 mg/m 2 for dogs greater than 15 kg). in the cat a vagal maneuver can be attempted by ocular or carotid massage. (diltiazem [dilacor] 30 to 60 po q12-24h), propranolol (2.5 to 10 mg/kg q12-24h), or atenolol (6.25 mg q12-24h) also can be administered. if structural heart disease is present, treat pulmonary edema and start angiotensin-converting enzyme inhibitor therapy. table 1 -34 summarizes the drugs used in the management of supraventricular dysrhythmias. severe bradycardia often results from systemic disease, drug therapy, anesthetic agents, or hypothermia and thus rarely requires specific therapy except to treat or reverse the underlying mechanisms promoting bradycardia. hemodynamically significant bradyarrhythmias that must be treated include atrial standstill, atrioventricular block, and sick sinus syndrome. atrial standstill most commonly is associated with hyperkalemia and is seen most often in urinary obstruction, renal failure, urinary trauma with uroabdomen, and hypoadrenocorticism. characteristic ecg abnormalities observed in atrial standstill are an absence of p waves, widened qrs complexes, and tall spiked t waves (figure 1-37 ). the treatment for hyperkalemia-induced atrial standstill is to correct the underlying cause and to drive potassium intracellularly and protect the myocardium from the adverse effects of hyperkalemia. regular insulin (0.25 to 0.5 units/kg iv) followed by dextrose (1 g/unit insulin iv, followed by 2.5% dextrose cri to prevent hypoglycemia) or sodium bicarbonate (1 meq/kg iv) can be administered to drive potassium intracellularly. calcium gluconate (0.5 ml/kg of 20% solution iv over 5 minutes) also can be administered as a cardioprotective drug until the cause of hyperkalemia has been identified and resolved. also administer sodium chloride fluids (0.9% sodium chloride iv) to promote kaliuresis. less commonly, atrial standstill is associated with atrial cardiomyopathy or silent atrium syndrome. persistent atrial standstill has been recognized without electrolyte abnormalities in the english springer spaniel and the siamese cat. short-term therapy for persistent atrial standstill includes atropine (0.04 mg/kg sq) until definitive treatment by implantation of a cardiac pacemaker can be performed. complete or third-degree atrioventricular block or high-grade symptomatic seconddegree atrioventricular block can be hemodynamically significant when ventricular rates are less than 60 beats/minute in the dog. classic clinical signs include weakness, exercise intolerance, lethargy, anorexia, syncope, and occasionally seizures. advanced atrioventricular block usually is caused by advanced idiopathic degeneration of the atrioventricular node. less commonly, atrioventricular block has been associated with digoxin toxicity, magnesium oversupplementation, cardiomyopathy, endocarditis, or infectious myocarditis (lyme disease). an accurate diagnosis is made based on the ecg findings of nonconducted p waves with ventricular escape beats. first-and second-degree atrioventricular block may not be hemodynamically significant and therefore may not require therapy. initially treat third-degree (complete) or symptomatic high-grade second-degree atrioventricular block (<60 beats/minute) with atropine (0.04 mg/kg sq or im). perform a follow-up ecg in 15 to 20 minutes. atropine is rarely successful in treating complete atrioventricular block. also attempt treatment with isoproterenol (0.04 to 0.08 âµg/kg/minute iv cri or 0.4 mg in 250 ml 5% dextrose in water iv slowly), a pure î²-agonist. definitive treatment requires permanent pacemaker implantation. consultation with a veterinary cardiologist who implants pacemakers is suggested. never attempt to convert or treat the observed ventricular escape beats with lidocaine ( figure 1-38) . sick sinus syndrome most commonly is recognized in the miniature schnauzer, although any dog can be affected. sick sinus syndrome usually results from idiopathic degeneration of the sinus node in the dog. in the cat, sinus node degeneration usually is associated with cardiomyopathy. dysfunction of the sinus node may manifest as marked bradycardia with periods of sinus arrest followed by junctional or ventricular escape complexes. a variant of sick sinus syndrome is the presence of severe bradycardia followed by periods of supraventricular tachycardia, often termed bradycardia-tachycardia syndrome. the most common clinical signs are syncope, exercise intolerance, and lethargy. in cats, hypertrophic cardiomyopathy is the most common form of acquired cardiac disease observed. congestive heart failure resulting from hypertrophic cardiomyopathy can occur in animals as young as 6 to 10 months of age. hypertrophic cardiomyopathy is characterized by stiff, noncompliant ventricles that do not relax during diastole, causing an increase in left atrial pressures and left atrial enlargement. other cardiomyopathies, including unclassified, restrictive, and dilated, are less common but also can occur in the cat. cats often develop acute exacerbation of clinical signs because of stress or arterial embolization. the rapid diagnosis of chf often is made on owner history, signalment, and physical examination findings (box 1-36). typical physical examination findings include a cardiac murmur or gallop dysrhythmia, abnormal breath sounds, respiratory difficulty and orthopnea, tachycardia, weak pulse quality, cool peripheral extremities, and pale or cyanotic mucous membrane. initiate immediate treatment based on physical examination findings and index of suspicion. in some cases, it is difficult to distinguish between chf and feline lower airway disease (asthma) without performing thoracic radiographs. let the animal rest and become stabilized before attempting any stressful procedures, including thoracic radiographs. immediate treatment consists of administering supplemental oxygen, decreasing circulating fluid volume with furosemide, dilating pulmonary and splanchnic capacitance vessels with topical nitroglycerine and morphine, and alleviating patient anxiety and stress (box 1-37). primary differential diagnoses are made based primarily on the patient's breed, age, clinical signs, history, and physical examination abnormalities. the most common differential diagnoses in a patient with chf are cardiac abnormalities and respiratory disease (chronic bronchitis [asthma], pulmonary hypertension, cor pulmonale, neoplasia). postpone diagnostic tests in any patient with suspected chf until the immediate treatments have taken effect and the patient is cardiovascularly more stable. in most cases, lateral and dorsoventral thoracic radiographs are one of the most important diagnostic tools in helping make a diagnosis of chf. increased perihilar interstitial to alveolar infiltrates are characteristic of pulmonary edema. left atrial enlargement may be observed as a "backpack" sign at the caudal cardiac waist. cardiomegaly of the right or left side also may 128 be present in cases of valvular insufficiency. in cats, increased sternal contact and a classic valentine-shaped heart may be observed in cases of hypertrophic cardiomyopathy. perform a vertebral heart score (sum) to measure cardiac size and determine whether cardiomegaly is present (box 1-38). also obtain arterial blood pressure and ecg readings to determine whether hypotension and dysrhythmias are present. atrial fibrillation, ventricular premature contractions, and supraventricular tachycardia are common rhythm disturbances that can affect cardiac output adversely and influence treatment choices. the echocardiogram is a useful noninvasive and nonstressful method to determine the degree of cardiac disease present. the echocardiogram is largely user-dependent. the quality of the study is based on the experience of the operator and the quality of the ultrasound machine. echocardiography can be a useful tool in making a diagnosis of pericardial effusion, dilated or hypertrophic cardiomyopathy, cardiac neoplasia, and endocarditis. the medical management of chf is designed to improve cardiac output and relieve clinical signs. the immediate goal of therapy is to reduce abnormal fluid accumulation and provide adequate cardiac output by increasing contractility, decreasing preload and ventricular afterload, and/or normalizing cardiac dysrhythmias. strict cage rest is of utmost importance when managing a patient with chf. after initial administration of furosemide, morphine, oxygen, and nitroglycerine paste, clinical signs of respiratory distress should show improvement within 30 minutes. if no improvement is observed, administer repeated doses of furosemide. reevaluate severe cases that are refractory to this standard treatment protocol. vasodilation should be the next step in the management of refractory cases, provided that a normal blood pressure is present. sodium nitroprusside is a potent balanced vasodilator that should be administered (1 to 10 âµg/kg/minute iv cri), taking care to monitor blood pressure continuously because severe vasodilation and hypotension can occur. the goal of nitroprusside therapy is to maintain a mean arterial blood pressure of 60 mm hg. sodium nitroprusside should not be considered in cases of refractory chf with severe hypotension. for more long-term management of chf, the use of angiotensin-converting enzyme (ace) inhibitors including enalapril (0.5 mg/kg po q12-24h), benazepril (0.5 mg/kg po q24h), and lisinopril (0.5 mg/kg po q24h) have become the mainstay of therapy to reduce sodium and fluid retention and decrease afterload. start angiotensin-converting enzyme inhibition as soon as a patient is able to tolerate oral medications. dobutamine (2.5 to 10 âµg/kg/minute cri diluted in 5% dextrose in water) can be administered to improve cardiac contractility, particularly in cases of dilated cardiomyopathy. at low doses, dobutamine, primarily a î²-adrenergic agonist, will improve cardiac output with minimal effects on heart rate. dobutamine must be given as a constant rate infusion with careful, continuous ecg monitoring. despite minimal effects on heart rate, emergency management of specific conditions 129 the vertebral heart sum can be calculated by performing the following steps: 1. measure the long axis of the heart from the apex to the carina on the lateral view and mark the distance on a sheet of paper. 2. measure the length of the long axis of the heart in terms of vertebral bodies, starting by counting caudally from the fourth thoracic vertebra; count the number of vertebrae that are covered by the length of the long axis of the heart. 3. measure the short axis of the heart at the caudal vena cava, perpendicular to the long axis of the heart. 4. count the number of thoracic vertebrae covered by the short axis of the heart, starting at t4. 5. add the two numbers together to yield the vertebral heart sum; a vertebral heart sum greater than 10.5 is consistent with cardiomegaly. sinus tachycardia or ventricular dysrhythmias may develop during infusion. cats are more sensitive to the effects of dobutamine than dogs. monitor carefully for seizures and facial twitching. digoxin is a cardiac glycoside that acts as a positive inotrope and negative chronotrope in the long-term management of chf. digoxin has a long (24 hours in dogs, and 60 hours in cats) half-life and so has minimal use in the emergency management of chf. in chronic management of chf resulting from dilated cardiomyopathy or advanced mitral disease, however, digoxin is extremely useful. oral digitalization protocols have been developed but are risky in that dysrhythmias and severe gastrointestinal side effects can occur. cats with chf often have fulminant pulmonary edema, pleural effusion, arterial thromboembolism, or some combination of all three. if the pleural effusion is significant, perform therapeutic thoracocentesis to relieve pulmonary atelectasis and improve oxygenation. once the diagnosis and initial management of chf has been made, formulate a plan for continued management and monitoring. tailor the therapeutic plan to the patient based on the cause of the chf, the presence of concurrent diseases, and response to therapy. an important and often overlooked part of the successful emergency management of chf is the open communication with the owner regarding the owner's emotional and financial commitment for immediate and long-term management to ensure appropriate quality of life for each patient. pathophysiology and treatment, vet j 162 (3) caval syndrome resulting from severe heartworm disease is caused by the rapid maturation of a large quantity of adult worms in the right atrium and cranial and caudal venae cavae. most cases of caval syndrome occur in regions of the world where heartworm disease is highly endemic and dogs spend a large portion of time living outdoors. caval syndrome is recognized by the following clinical signs and results of biochemical analyses: acute renal and hepatic failure, enlarged right atrium and posterior vena cava, ascites, hemoglobinuria, anemia, acute collapse, respiratory distress, dic, jugular pulses, circulating microfilariae, and sometimes tricuspid insufficiency. immediate action in cases of caval syndrome in dogs involves immediate stabilization of the cardiovascular and respiratory systems with supplemental oxygen, furosemide (4 mg/kg iv), and careful crystalloid fluid infusion. diagnosis of caval syndrome is based on clinical signs of cardiogenic shock with right ventricular heart failure, intravascular hemolysis, and renal and hepatic failure. thoracic radiographs reveal cardiomegaly of the right side and enlarged tortuous pulmonary arteries. a right axis deviation may be seen on ecg tracings. clinicopathologic changes observed include azotemia, inflammatory leukogram, regenerative anemia, eosinophilia, elevated hepatocellular enzyme activities, hemoglobinuria, and proteinuria. circulating microfilariae may be observed on peripheral blood smears or in the buffy coat of microhematocrit tubes. heart worm antigen tests will be strongly positive. echocardiographic changes include visualization of a large number of heartworms in the right atrium, pulmonary arteries, and vena cava, tricuspid insufficiency, and right atrial and ventricular enlargement. treatment involves surgical removal of as many of the adult heartworms as possible from the right jugular vein and right atrium. glucocorticosteroids are recommended to decrease inflammation and microangiopathic disease associated with heartworm infection. for more long-term management, administer adulticide therapy several weeks following surgery, followed by routine microfilaricide therapy and then prophylaxis. calvert pericardial effusion often develops as a consequence of neoplasia in the older dog and cat. the most common types of neoplasia that affect the heart and pericardium include hemangiosarcoma, chemodectoma, mesothelioma, and metastatic neoplasia. more rarely, other causes of pericardial effusion include benign idiopathic pericardial effusion, coagulopathy, left atrial rupture in dogs with chronic mitral valvular insufficiency, infection, or pericardial cysts. regardless of the cause of the effusion, the development of pericardial tamponade adversely affects cardiac output. cardiac output is a function of heart rate and stroke volume. stroke volume depends on cardiac preload. the presence of pericardial effusion can impede venous return to the heart and thus adversely affect preload. in addition, as preload decreases, heart rate reflexively increases in an attempt to maintain normal cardiac output. as heart rate increases more than 160 beats/minute, diastolic filling is impaired further, and cardiac output further declines. animals with pericardial effusion often demonstrate the classic signs of hypovolemic or cardiogenic shock: anorexia, weakness, lethargy, cyanosis, cool peripheral extremities, tachycardia, weak thready pulses, hypotension, and collapse. physical examination abnormalities may include muffled heart sounds, thready femoral pulses, pulsus paradoxus, jugular venous distention, weakness, tachycardia, cyanosis, and tachypnea. electrocardiogram findings may include low amplitude qrs complexes (<0.5 mv), sinus tachycardia, ventricular dysrhythmias, or electrical alternans (figure 1-39) . thoracic radiographs often demonstrate a globoid cardiac silhouette, although the cardiac silhouette rarely may appear normal with concurrent clinical signs of cardiogenic shock in cases of acute hemorrhage. in such cases the removal of even small amounts of pericardial effusion by pericardiocentesis can increase cardiac output exponentially and alleviate clinical signs (table 1-35) . unless an animal is dying before your eyes, ideally perform an echocardiogram to attempt to determine whether a right atrial, right auricular, or heart base mass is present before pericardiocentesis. before attempting pericardiocentesis, assemble all of the required supplies (box 1-39) . to perform pericardiocentesis, follow this procedure: 1. place the patient in sternal or lateral recumbency. 2. attach ecg leads to monitor the patient for dysrhythmias during the procedure. 3. clip a 6-cm square caudal to the right elbow over the fifth to seventh intercostal space. 4. aseptically scrub the clipped area, and infuse 1 to 2 mg/kg of 2% lidocaine mixed with a small amount of sodium bicarbonate just dorsal to the sternum at the sixth intercostal space. bury the needle to the hub, and inject the lidocaine as you withdraw the needle. 5. while the local anesthetic is taking effect, assemble the intravenous extension tubing, three-way stopcock, and 60-ml syringe. 6. wearing sterile gloves, make a small nick incision in the skin to decrease drag on the needle and catheter during insertion. 7. slowly insert the needle and catheter, watching for a flash of blood in the hub of the needle, and simultaneously watching for cardiac dysrhythmias on the ecg monitor. 8. once a flash of blood is observed in the hub of the needle, advance the catheter off of the stylette further into the pericardial sac, and remove the stylette. 9. attach the length of intravenous extension tubing to the catheter, and have an assistant withdraw the fluid slowly. 10. place a small amount of fluid in a red-topped tube, and watch for clots. clot formation could signify that you have penetrated the right ventricle inadvertently or that active hemorrhage is occurring. withdraw as much of the fluid as possible, and then remove the catheter. monitor the patient closely for fluid reaccumulation and recurrence of clinical signs of cardiogenic shock. less rd, bright jm, orton ec: intrapericardial cyst causing cardiac tamponade in a cat, j am anim hosp assoc 36 (2) foreign bodies within the ear canal (e.g., foxtails) can present as emergencies because of acute inflammation and pressure necrosis of the tissue of the external auditory meatus causing pain and discomfort. clinical signs may be limited to incessant head shaking or scratching of the ear canal. complete examination of the ear canal and removal of any foreign body often requires administration of a short-acting anesthetic agent. once the animal has been restrained sufficiently and placed under anesthesia, carefully examine the ear canal and remove any foreign material with an alligator forceps. stimulation of the ear canal can cause awakening after removal of all debris and detritus, gently wipe the internal and external ear canal with a sterile gauze. place a topical antimicrobial-antifungal-steroid ointment such as otomax in the ear every 8 to 12 hours. if pain and discomfort is severe, systemically effective opioids or nsaids may be required. otitis externa is a common emergency that causes excessive head shaking, scratching, and purulent malodorous aural discharge. clean the ear canal with an irrigating solution such as epiotic and wipe it clean of debris. perform a complete aural examination to determine whether a foreign body or tumor is present and whether the tympanic membrane is intact. heat-fix any discharge and examine it cytologically for bacteria and fungal organisms. following careful cleansing, instill a topical antibiotic-antifungal-steroid ointment. in severe cases in which the ear canal has scarred and closed down with chronicity, consider administering systemically effective antibiotics (cephalexin, 22 mg/kg po tid) and antifungal agents (ketoconazole, 10 mg/kg po q12h) instead of topical therapy. systemically effective steroids (prednisone or prednisolone, 0.5 mg/kg po q12h) may be indicated in cases of severe inflammation to decrease pruritus and patient discomfort. presentation of a patient with otitis interna often is characterized by torticollis, head tilt, nystagmus, circling to the affected side, or rolling. fever, pain, vomiting, and severe depression may accompany clinical signs. most cases of severe otitis interna are accompanied by severe otitis media. both conditions must be treated simultaneously. the most common causes of otitis interna are staphylococcus aureus, pseudomonas, escherichia coli, or proteus spp. otitis interna can develop by infection spreading across the tympanic membrane, through the eustachian tubes, or by hematogenous spread from the blood supply to the middle ear. in most cases of otitis media, the tympanic membrane is ruptured. perform a culture and susceptibility test of the debris behind the tympanic membrane and within the aural canal. carefully clean the external ear canal. medicate with a topical combination antibiotic, antifungal, and antibiotic ointment. administer high-dose antibiotics (cephalexin, 22 mg/kg po q8h, or enrofloxacin, 10 to 20 mg/kg po q24h). if the tympanic membrane is not ruptured but appears swollen and erythematous, a myringotomy may need to be performed. if clinical signs of otitis media persist despite topical and systemic therapy, radiographic or ct/mri examination of the tympanic bullae may be required. chronic shaking of the head and ears or aural trauma (bite wounds) causes disruption of the blood vessels and leads to the development of unilateral or bilateral aural hematomas. aural hematomas are clinically significant because they cause patient discomfort and are often due to the presence of some other underlying problem such as otitis externa, atopy, or aural foreign bodies. acute swelling of the external ear pinna with fluid is characteristic of an aural hematoma. in some cases, swelling can be so severe that the hematoma breaks open, bathing the patient and external living environment in blood. when a patient has an aural hematoma, investigate the underlying cause. perform a complete aural examination to determine whether an aural foreign body, otitis externa, or atopy are present. carefully examine and gently clean the inner ear canal. treat underlying causes. management of an aural hematoma involves draining the hemorrhagic fluid from the aural tissue and tacking the skin down in multiple places to prevent reaccumulation of fluid until the secondary cause is resolved. many techniques have been described to surgically tack down the skin overlying the hematoma. after the animal has been placed under general anesthesia, lance the hematoma down the middle with a scalpel blade and remove the fluid and blood clot. tack down the skin with multiple through-and-through interrupted or mattress sutures through the ear. some clinicians prefer to suture through and attach a sponge or length of x-ray film to the front and back of the ear for stabilization and support. more recently, a laser can be used to drill holes in the hematoma and tack the skin down in multiple areas. compress the ear against the head with a compression bandage, whenever possible, for 5 to 7 days after the initial surgery, and then recheck the ear. the patient must wear an elizabethan collar until the surgical wound and hematoma heal to prevent selfmutilation. also systemically treat underlying causative factors such as otitis externa with antibiotics, antifungals, and steroids as indicated. investigate and treat other underlying causes such as hypothyroidism or allergies. bass electrocution usually is observed in young animals after they have chewed on an electric cord. other causes of electrocution include use of defective electrical equipment or being struck by lightning. electric current passing through the body can produce severe dysrhythmias, including supraventricular or ventricular tachycardia and first-and thirddegree atrioventricular block. the electric current also can produce tissue destruction from heat and electrothermal burns. electrocution also commonly results in noncardiogenic pulmonary edema caused by massive catecholamine release and increase in pulmonary vascular pressures during the event. ventricular fibrillation can occur, although that depends on the intensity and path of the electrical current and duration of contact. clinical signs of electrocution include acute onset of respiratory distress with moist rales, and localized necrosis or thermal burns of the lips and tongue. often the skin at the commissures of the mouth appears white or yellow and firm to the touch. muscle fasciculations, loss of consciousness, and ventricular fibrillation may occur. thoracic radiographs often reveal an increased interstitial to alveolar lung pattern in the dorsocaudal lung fields. noncardiogenic pulmonary edema can develop up to 24 to 36 hours after the initial incident. the first 24 hours are most critical for the patient, and then prognosis improves. the most important aspect in the treatment of the patient with noncardiogenic pulmonary edema is to minimize stress and to provide supplemental oxygen, with positive pressure ventilation, when necessary. although treatment with vasodilators (low-dose morphine) and diuretics (furosemide) can be attempted, noncardiogenic pulmonary edema is typically resistant to vasodilator and diuretic therapy. positive inotropes and pressor drugs may be necessary to treat shock and hypotension. opioid drugs (morphine, hydromorphone, oxymorphone) may be useful in controlling anxiety until the pulmonary edema resolves. administer broad-spectrum antibiotics (cefazolin; amoxicillin and clavulanic acid [clavamox]) to treat thermal burns. use analgesic drugs to control patient discomfort. if thermal burns are extensive and prohibit adequate food intake, place a feeding tube as soon as the patient's cardiovascular and respiratory function are stable and the patient can tolerate anesthesia. prolapse of the uterus occurs in the immediate postparturient period in the bitch and queen. excessive straining during or after parturition causes the uterus to prolapse caudally through the vagina and vulva. immediate intervention is necessary. examine the bitch or queen for a retained fetus. treatment consists of general anesthesia to replace the prolapsed tissue. if the uterus is edematous, physical replacement may be difficult or impossible. application of a hypertonic solution such as hypertonic (7%) saline or dextrose (50%) to the exposed endometrium can help shrink the tissue. that, combined with gentle massage to stimulate uterine contraction and involution and lubrication with sterile lubricating jelly, can aid in replacement of the organ into its proper place. to ensure proper placement in the abdominal cavity and to prevent recurrence, perform an exploratory laparotomy and hysteropexy. postoperatively, administer oxytocin (5 to 20 units im) to cause uterine contraction. if the uterus contracts, it is usually not necessary to suture the vulva. administer antibiotics postoperatively. recurrence is uncommon, even with subsequent pregnancies. if the tissue is damaged or too edematous to replace or if the tissue is devitalized, traumatized or necrotic, perform an ovariohysterectomy. in some instances, replacement of the damaged tissue is not necessary before removal. pyometra occurs in dogs and cats. the disease process occurs as a result of infection overlying cystic endometrial hyperplasia under the constant influence of progesterone. during the 2-month luteal phase after estrus or following copulation, artificial insemination, or administration of hormones (particularly estradiol or progesterone), the myometrium becomes relaxed and favors a quiescent environment for bacterial proliferation. clinical signs of pyometra are associated with the presence of bacterial endotoxin and sepsis. early, affected animals become lethargic and anorectic. polyuria with secondary polydipsia is often present because of the influence of bacterial endotoxin on renal tubular concentration. if the cervix is open, purulent or mucoid vaginal discharge may be observed. later in the course of pyometra, vomiting, diarrhea, and progressive debilitation resulting from sepsis occur. diagnosis is based on clinical signs in an intact queen or bitch and radiographic or ultrasonographic evidence of a fluid-filled tubular density in the ventrocaudal abdomen, adjacent to the urinary bladder (figures 1-40 and 1-41) . treatment of open and closed pyometra is correction of fluid and electrolyte abnormalities, administration of broad-spectrum antibiotics, and ovariohysterectomy. close pyometra is a life-threatening septic condition. open pyometra also can become life-threatening and so should be treated aggressively. in closed pyometra, conservative medical therapy is not advised. administration of prostaglandins and oxytocin do not reliably cause the cervix 136 1 to open and can result in ascending infection from the uterus into the abdomen or uterine rupture, both of which can result in severe peritonitis. for animals with an open pyometra, ovariohysterectomy is the most reliable treatment for chronic cystic endometrial hyperplasia. although less successful than ovariohysterectomy, medical therapy may be attempted in breeding bitches as an alternative to surgery. the most widely used medical therapy in the breeding queen and bitch is administration of prostaglandin f 2î± . this drug has not been approved for use in the queen or bitch in the united states. to proceed with medical management of pyometra, first determine the size of the uterus. start the patient on antibiotic therapy (ampicillin, 22 mg/kg iv q6h, or enrofloxacin, 10 mg/kg po q24h). administer the prostaglandin f 2î± (250 âµg/kg sq q24h) for 2 to 7 days until the size of the uterus approaches normal. measure serum progesterone concentrations if the bitch is in diestrus. as the corpus luteum degrades under the influence of prostaglandin f 2î± , serum progesterone levels will decline. prostaglandin f 2î± is an abortifacient and thus should not be administered to the pregnant bitch or queen. clinical signs of a reaction to prostaglandin f 2î± can occur within 5 to 60 minutes in the bitch and can last for as long as 20 minutes. clinical signs of a reaction include restlessness, hypersalivation, panting, vomiting, defecation, abdominal pain, fever, and vocalization. in a very ill animal, death can occur. the efficacy of prostaglandin f 2î± is limited and may require more than one treatment. the bitch should be bred on the next heat cycle and then spayed because progressive cystic endometrial hyperplasia will continue to occur. acute metritis is an acute bacterial infection of the uterus that typically occurs within 1 to 2 weeks after parturition. the most common organism observed in metritis is e. coli ascending from the vulva and vaginal vault. sepsis can progress rapidly. clinical signs of acute metritis include inability to nurse puppies, anorexia, lethargy, foul-smelling purulentsanguineous vaginal discharge, vomiting, or acute collapse. physical examination may reveal fever, dehydration, and a turgid distended uterus. septic inflammation will be observed on vaginal cytologic examination. an enlarged uterus can be observed with abdominal radiographs and ultrasonography. treatment of acute metritis is directed at restoring hydration status with intravenous fluids and treating the infection with antibiotics. because the primary cause of metritis is e. coli infection, start enrofloxacin (10 mg/kg iv or po once daily) therapy. as soon as the patient's cardiovascular status is stable enough for anesthesia, perform an ovariohysterectomy. if the patient is not critical and is a valuable breeding bitch, medical therapy can be attempted. medical management of acute bacterial metritis includes administration of oxytocin (5 to 10 units q3h for three treatments) or administration of prostaglandin f 2î± (250 âµg/kg/day for 2 to 5 days) to evacuate the uterine exudate and increase uterine blood flow. either drug should be used concurrently with antibiotics. rupture of the gravid uterus is rare in cats and dogs but has been reported. uterine rupture may occur as a consequence of parturition or result from blunt abdominal trauma. feti expelled into the abdominal cavity may be resorbed but more commonly cause the development of peritonitis. if fetal circulation is not disrupted, the fetus actually may live to term. uterine rupture is an acute surgical emergency. an ovariohysterectomy with removal of the extrauterine puppies and membranes is recommended. if only one horn of the uterus is affected, a unilateral ovariohysterectomy can be performed to salvage the remaining unaffected puppies and preserve the breeding potential for the valuable bitch. if uterine rupture occurs because of pyometra, peritonitis is likely, and copious peritoneal lavage should be performed at the time of surgery. the patient should be placed on 7 to 14 days of antibiotic therapy (amoxicillin or amoxicillin and clavulanic acid [clavamox] with enrofloxacin). vaginal prolapse occurs from excessive proliferation and hyperplasia of vaginal tissue while under the influence of estrogen during proestrus (figure 1-42) . the hyperplastic tissue usually recedes during diestrus but reappears with subsequent heat cycles. vaginal prolapse can be confused with vaginal neoplasia. the former condition occurs primarily in younger animals, whereas the latter condition occurs primarily in older animals. treatment for vaginal hyperplasia or prolapse generally is not required if the tissue remains within the vagina. the proliferation can lead to dysuria or anuria, however. in some cases, the tissue becomes 138 1 emergency care dried out and devitalized or becomes traumatized by the animal. such extreme cases warrant immediate surgical intervention. the treatment for vaginal prolapse consists of ovariohysterectomy to remove the influence of estrogen, placement of an indwelling urinary catheter if the patient is dysuric, and protection of the hyperplastic tissue until it recedes on its own. although surgical resection of the hyperplastic tissue has been recommended, excessive hemorrhage after removal can occur, and so the procedure should not be attempted. the patient should wear an elizabethan collar at all times to prevent selfmutilation. administer broad-spectrum antibiotics for a minimum of 7 to 14 days or until the hyperplastic tissue recedes. keep the tissue clean with saline solution. dystocia, or difficult birth, can occur in the dog and cat but is more common in the dog. a diagnosis of dystocia is made based on the time of onset of visible labor and the time in which the last puppy or no puppy has been born, the intensity and timing of contractions, the timing of when the amniotic membranes first appear, the condition of the bitch, and the timing of gestation. causes of dystocia can be maternal or fetal and include primary or secondary uterine inertia, narrowing of the pelvic canal, hypocalcemia, psychological disturbances, or uterine torsion. maternal-fetal disproportion, or large fetus size in relation to the bitch or queen, also can result in dystocia (box 1-40). obtain an abdominal radiograph for all cases of suspected dystocia at the time of presentation to determine the size of the fetus, presentation of the fetus (both anterior or posterior presentation can be normal in the bitch or queen, but fetal malpositioning can cause dystocia), and whether there is radiographic evidence of a uterine rupture or torsion. if maternal-fetal disproportion, uterine torsion, or uterine rupture is observed, take the patient immediately to surgery. if the puppies or kittens are in a normal position for birth, medical management can be attempted. clip the perineum and aseptically scrub it. wearing sterile gloves, insert a lubricated finger into the vagina and palpate the cervix. massage (or "feather") the dorsal wall of the vagina to stimulate contractions. place an intravenous catheter, and administer oxytocin (2 to 20 units im), repeating up to 3 times at 30-minute intervals. in some cases, hypoglycemia or hypocalcemia can contribute to uterine inertia. administration of a calciumcontaining solution (lactated ringer's solution) with 2.5% dextrose is advised. alternately, administer 10% calcium gluconate (100 mg/5 kg iv slowly). if labor has not progressed after 1 hour, immediately perform a cesarean section. uterine torsion is an uncommon emergency seen in the gravid and nongravid uterus and has been reported in dogs and cats. the onset of clinical signs of abdominal pain and straining as if to whelp/queen or defecate is usually acute and constitutes a surgical emergency. in some cases, there may have been a history of delivery of a live or dead fetus. vaginal discharge may or may not be present. radiographs or ultrasound examination reveal a fluid-filled or air-filled tubular density in the ventral abdomen. treatment consists of placing an intravenous catheter, stabilizing the patient's cardiovascular status with intravenous fluids and sometimes blood products, and performing an immediate ovariohysterectomy. if there are viable feti, the uterus should be delivered en mass and the puppies or kittens delivered. the expulsion of one or more fetus before term is known as spontaneous abortion. in dogs and cats, it is possible to expel or abort one or more fetuses and still carry viable fetuses to term and deliver normally. clinical signs of spontaneous abortion include vaginal discharge and abdominal contractions. in some cases, the fetus is found, or there may be evidence of fetal membranes or remnants. causes of spontaneous abortion in dogs include brucella canis, herpesvirus, coronavirus, and toxoplasmosis. in cats, herpesvirus, coronavirus, and feline leukemia virus can cause spontaneous abortion. in both species, trauma, hormonal factors, environmental pathogens, drugs, and fetal factors also can result in spontaneous abortion. the safest method of pregnancy termination in the bitch or queen is by performing an ovariohysterectomy. oral diethylstilbesterol is not an effective mechanism of pregnancy termination in the bitch. a so-called mismating shot, an injection of estradiol cypionate (0.02 mg/lb im) is effective at causing termination of an early pregnancy but can be associated with severe side effects, including bone marrow suppression and pyometra. estradiol cypionate is not approved for use in the bitch or queen and is not recommended. prostaglandin f 2î± is a natural abortifacient in the bitch if treatment is started within 5 days of cytologic evidence of diestrus (noncornified epithelium on a vaginal smear). the prostaglandin f 2î± causes lysis of the corpora lutea and a rapid decline in progesterone concentration. the prostaglandin f 2î± is administered for a total of eight injections (250 âµg/kg q12h for 4 days), along with atropine (100 to 500 âµg/kg sq). side effects can occur within 5 to 40 minutes of injection and include restlessness, panting, salivation, abdominal pain, urination, vomiting, and diarrhea. walking the patient for 20 to 30 minutes after each treatment sometimes decreases the intensity of the reactions. bitches in the first half of the pregnancy often resorb the embryos. if prostaglandin f 2î± is administered in the second half of the pregnancy, the fetuses are aborted within 5 to 7 days of treatment. measure serum progesterone concentrations at the end of treatment to ensure complete lysis of the corpus luteum. prostaglandin f 2î± is not approved for pregnancy termination in the bitch. in cats, prostaglandin f 2î± can terminate pregnancy after day 4 of gestation. prostaglandin f 2î± should be used only in healthy queens (100 to 250 âµg/kg sq q24h for 2 days). side effects in the queen are similar to those observed in the bitch but typically have a shorter duration (2 to 20 minutes). prostaglandin f 2î± is not approved for use in cats in the united states. the use of prostaglandin f 2î± does not preclude breeding and pregnancy at a later date. biddle d, macintire dk: obstetrical emergencies, clin tech small anim pract 15 (2) in the dog and cat the majority of injuries to the scrotum are associated with animal fights or shearing and abrasive injuries sustained in accidents involving automobiles. scrotal injuries should be categorized as superficial or penetrating. treatment of superficial injuries to the scrotum includes cleaning the wound with dilute antimicrobial cleanser and drying it. administer antiinflammatory doses of steroids (prednisolone, 0.5 to 1.0 mg/kg po q12-24h) or nsaids (carprofen, 2.2 mg/kg po q12h in dogs) for the first several days after scrotal injury to prevent or treat edema. administer topical antibiotic ointment until the wound heals. in most cases, place an elizabethan collar to prevent self-mutilation. prognosis is generally favorable; however, semen quality may be affected for months after injury because of scrotal swelling and increased scrotal temperature. penetrating injuries to the scrotum are more serious and are associated with severe swelling and infection. surgically explore and debride penetrating scrotal wounds. administer systemically effective antibiotics and analgesics. in extreme cases, particularly those that involve the testicle, consider castration and scrotal ablation. scrotal dermatitis is common in intact male dogs and can be associated with direct physical injury, self-infliction from licking, chemical irritation, burns, or contact dermatitis. in affected animals, the scrotum can become extremely inflamed, swollen, and painful. if left untreated, pyogranulomatous dermatitis can develop. make an attempt to determine whether an underlying systemic illness is present that could predispose the animal to scrotal dermatitis. widespread vasculitis with scrotal edema, pain, fever, and dermatitis has been associated with rickettsia rickettsii (rocky mountain spotted fever) infection. brucella canis also has been associated with scrotal irritation and dermatitis. if scrotal dermatitis follows from an infectious cause, empiric use of glucocorticosteroids potentially can make the condition worse by suppressing immune function. empiric treatment with antibiotics also potentially can confound making an accurate diagnosis. treatment of scrotal dermatitis is to eliminate predisposing causes, if possible. place an elizabethan collar at all times to prevent self-mutilation. bathe the scrotum with a mild antimicrobial soap and dry it to remove any offending chemical irritants. topical medications including tar shampoo, tetracaine, neomycin, and petroleum can cause further irritation and are contraindicated. use oral or parenteral administration of glucocorticosteroids or nsaids to control discomfort and inflammation. scrotal hernias occur when the contents of the abdomen (intestines, fat, mesentery, omentum) protrude through the inguinal ring into the scrotal sac. like inguinal hernias, scrotal definitive therapy for a scrotal hernia involves exploratory laparotomy and surgical reduction of the contents of the hernia, surgical correction of the rent in the inguinal ring, and castration. trauma to the epididymis or testicle can cause testicular pain and swelling of one or both testes. treat penetrating trauma to the testicle by castration to prevent infection and selfmutilation. administer oral antibiotics (amoxicillin or amoxicillin-clavulanate) for 7 to 10 days after the injury. nonpenetrating injuries to the scrotum and testicle rarely may cause acute testicular hemorrhage or hydrocele formation. palpation of the affected area often reveals a peritesticular, soft, compliant area. treatment consists of cool compresses on the scrotum and testicle and administration of antiinflammatory doses of glucocorticosteroids or nsaids. if the swelling does not resolve spontaneously in 5 to 7 days, consider surgical exploration and drainage. increased scrotal temperature and testicular inflammation can affect semen quality for months after the initial incident. testicular torsion, or torsion of the spermatic cord, causes rotation of the testicle, ultimately causing obstruction to venous drainage. testicular torsion often is associated with a neoplastic mass of a retained testicle within the abdomen but also can be observed with nonneoplastic testes located within the scrotum. the predominant clinical signs are pain, stiff stilted gait, and the presence of an abnormally swollen testicle (if located within the scrotum). if an intraabdominal testicular torsion is present, pain, lethargy, anorexia, and vomiting can occur (see acute condition in the abdomen). an intraabdominal mass may be palpable. perform an abdominal or testicular ultrasound, preferably with color flow doppler to evaluate perfusion to the testicle. treatment involves surgical removal of the involved testes. bacterial infections of the testicle or epididymis most commonly are caused by ascending infections of the normal bacterial flora of the prepuce or urethra. common inhabitants include escherichia coli, staphylococcus aureus, streptococcus spp., and mycobacterium canis. brucella canis and r. rickettsii are also capable of causing orchitis and epididymitis in the dog. clinical signs of orchitis or epididymitis include testicular enlargement, stiff stilted gait, and reluctance to walk. physical examination often reveals a fever and self-induced trauma to the scrotum from licking or chewing at the inflamed area. collect a semen sample by ejaculation, and culture it to identify the causative organism. alternately, collect samples by needle aspiration of the affected organ(s) and test serologically for b. canis. treatment of infectious orchitis involves a minimum of 3 to 4 weeks of specific antimicrobial therapy, based on culture and susceptibility testing, whenever possible. if a bacterial culture cannot be obtained, initiate fluoroquinolone therapy (enrofloxacin, 10 mg/kg po q24h). doxycycline (5 mg/kg po bid for 7 days) has been shown to suppress but not eradicate b. canis infection. testicular inflammation and increased temperature can affect sperm quality for months after infection. the most common causes of acute prostatitis are associated with acute bacterial infection (e. coli, proteus spp., pseudomonas spp., and mycoplasma spp.). less common causes include fungal infection (blastomyces dermatitidis) or anaerobic bacterial infection. acute prostatitis is characterized by fever, caudal abdominal pain, lethargy, anorexia, blood in the ejaculate, hematuria, dyschezia, and occasionally stranguria or dysuria. the patient often appears painful and depressed and may be dehydrated on physical examination. symmetric or asymmetric prostatomegaly and prostate pain may be evident on rectal palpation. in severely affected dogs, clinical signs of tachycardia, hyperemic or injected mucous membranes, bounding pulses, lethargy, dehydration, and fever may be present because of sepsis. death can occur within 2 days if a prostatic abscess ruptures. diagnosis of acute prostatitis is confirmed based on the presenting clinical signs, neutrophilic leukocytosis (with or without a left shift), and positive urine culture results. prostatic samples may be obtained from the prostatic portion of the ejaculate, prostatic massage, urethral discharge, urine, or (less commonly) prostatic aspirate. although semen samples can yield positive bacterial cultures, dogs with acute prostatitis are often unwilling to ejaculate. radiography may reveal an enlarged prostate, but this alone does not confirm the diagnosis of prostatitis. an abdominal ultrasound often reveals prostatic abscessation and allows for the collection of samples from the affected area(s) via prostatic aspirate. aspiration of the affected tissue potentially can wick infection into periprostatic tracks. cytologic examination of the patient's ejaculate or prostatic wash from a dog with acute prostatitis reveals numerous inflammatory cells and may contain bacterial organisms. the treatment of a patient with acute prostatitis is directed at correcting dysuria and constipation associated with prostatic enlargement. enrofloxaxin (10 mg/kg po sid) can penetrate the inflamed prostatic tissue and is effective in treating gram-negative and mycoplasma spp. infections. ciprofloxacin does not appear to penetrate prostatic tissue as readily. alternatives to enrofloxacin therapy are trimethoprim-sulfamethoxazole (30 mg/kg po q12h) or chloramphenicol (25-50 mg/kg po q8h) for a minimum of 2 to 3 weeks. castration is recommended because benign prostatic hyperplasia may be a predisposing factor in the development of acute prostatitis. do not perform castration until the patient has been on antibiotic therapy for a minimum of 7 days, to prevent the surgical complication of schirrous cords. finasteride (proscar, 1 mg/kg po q24h), an antiandrogen 5î±-reductase inhibitor, may help reduce the size of prostatic tissue until the effects of castration are observed. if a prostatic abscess is present, perform marsupialization, surgical drainage, or ultrasonographic drainage. surgical therapy is associated with a large incidence of complications, including incontinence, chronic drainage from fistulas and stomas, septic shock, and death. fracture of the os penis is an uncommon condition encountered in male dogs. os penis fractures can occur with minimal soft tissue damage but cause hematuria and dysuria. on physical examination, urethral obstruction and crepitus in the penis are found. a lateral abdominal radiograph is usually sufficient to document the fracture. treatment consists of conservative therapy, in most cases, and consists primarily of analgesia administration. if the urethra also is damaged, place a urethral catheter for 5 to 7 days to allow the urethral mucosa to heal. fractures of the os penis that are comminuted or severe enough to cause urethral obstruction require open reduction and fixation, partial penile amputation, or antescrotal (prescrotal) urethrostomy. lacerations of the penis cause significant bleeding because of the extensive vascular supply to the penis. dogs and cats tend to lick penile lacerations and prevent adequate clot formation. sedation or general anesthesia often is required to evaluate and treat the laceration. after sedation or general anesthesia, place a urinary catheter and examine the penis under a stream of cold water. small lacerations can be managed with cold compresses and one to several absorbable sutures. extensive suturing usually is not required. prevent erection by isolating the patient from females in estrus or allowing excitement or excessive activity. place an elizabethan collar to prevent self-mutilation. initiate systemic antibiotic therapy to prevent infection. the inability to withdraw the penis into the prepuce in male dogs or cats is known as paraphimosis. paraphimosis usually develops following an erection in young male dogs and in 144 1 emergency care older dogs after coitus. mucosal edema, hemorrhage, self-mutilation, and necrosis requiring penile amputation can occur if left untreated. treatment consists of applying cold water to the penis and reducing edema with application of an osmotic substance such as sugar. examine the base of the penis for hair rings that can prevent retraction of the penis into the prepuce. rinse the penis carefully with cold water and lubricate it with sterile lubricant and replace it into the prepuce. if the penis cannot be reduced easily into the prepuce, anesthetize the patient and make a small incision at the lateral aspect of the preputial opening. replace the penis and close the incision with absorbable suture. place a purse-string suture and leave it in place for several days to prevent recurrence. instill topical antimicrobial ointment with steroids into the prepuce several times a day. in severe cases, a urinary catheter may need to be placed to prevent urethral obstruction, until penile swelling and edema resolve. place an elizabethan collar to prevent excessive licking during the healing process. prolapse of the distal urethra is a condition usually confined to intact male english bulldogs, although isolated incidences also have been reported in yorkshire and boston terriers. the exact cause of this condition is unknown but usually is associated with a condition that causes increased intraabdominal pressure or urethral straining, including sexual excitement, coughing, vomiting, obstructed airway or brachycephalic airway syndrome, urethral calculi, genitourinary tract infection, and masturbation. the urethral prolapse usually appears as a mushroom-tip congested, irritated mass at the end of the penis that may or may not bleed (figure 1-44) . in some cases, bleeding occurs or worsens with sexual excitement. clinical signs associated with the prolapsed urethra include excessive licking of the prepuce, stranguria, and preputial bleeding. once the mass is observed, other differential diagnoses include transmissible venereal tumor, urethral polyp, trauma, urethritis, and neoplasia. in most cases, however, the prolapse occurs in intact young dogs, making neoplastic conditions less likely. treatment for urethral prolapse should occur at the time of diagnosis to prevent selfinduced trauma and infection. immediate therapy includes manual reduction of the prolapsed tissue and placement of a purse-string suture around an indwelling urinary catheter. the purse-string suture can remain in place for up to 5 days until definitive repair. until the time of surgery, place an elizabethan collar on the patient to prevent self-mutilation. several forms of surgical correction have been described. in some cases, surgical resection of the prolapsed tissue with apposition of the urethral and penile mucosa can be attempted. more recently, a technique involving placement of several mattress sutures to reduce and secure the prolapsed tissue has been described. recurrence of prolapse can occur with either technique, particularly if the inciting event recurs. because there may be a genetic predisposition in this breed and because the prolapse can recur with sexual excitement, neutering should strongly be recommended. local freezing or frostbite most commonly affects the peripheral tissues of the ears, tail, paws, and genitalia that are sparsely covered with fur, are poorly vascularized, and may have been traumatized previously by cold. clinical signs of frostbite are paleness and appearance of a blanched pink to white discoloration to the skin. the skin also may appear black and necrotic. immediate treatment consists of slowly rewarming the affected area with moist heat at 29.5â° c (85â°f) or by immersion in warm water baths. analgesics may be required to alleviate patient discomfort. carefully dry the injured areas and protect them from further trauma. the use of prophylactic antibiotics is controversial because it can promote resistant bacterial infection. use of antibiotics should be based on the presence of infection. treatments that are ineffective and may be harmful include rubbing the affected areas, pressure bandages, and ointments. corticosteroids can decrease cellular immunity and promote infection and are therefore contraindicated. many frostbitten areas that appear nonviable can regain function gradually. use care when removing areas of necrotic tissue. affected areas may take several days to a week before fully manifesting areas of demarcation between healthy viable and necrotic nonviable tissue. chilling of the entire body from exposure or immersion in extremely cold water results in a decrease in core body temperature and physiologic processes that become irreversible when the body temperature falls below 24â°c (75â°f). mild hypothermia can be 32â°to 37â°c, moderate hypothermia from 28â°to 32â°c, and severe hypothermia below 28â°c. the duration of exposure and the general condition of the animal influences its ability to survive. clinical signs and consequences associated with hypothermia include shivering, vasoconstriction, mental depression, hypotension, sinus bradycardia, hypoventilation with decreased respiratory rate, increased blood viscosity, muscle stiffness, atrial and ventricular irritability, decreased level of consciousness, decreased oxygen consumption, metabolic (lactic) acidosis, respiratory acidosis, and coagulopathies including dic. if the animal is breathing, administer warm, humidified oxygen at 4 to 10 breaths per minute. if the animal is not breathing or is severely hypoventilating, endotracheal intubation with mechanical ventilation may be necessary. place an intravenous catheter and infuse warmed crystalloid fluids. if the blood glucose is less than 60 mg/dl, add supplemental dextrose (2.5%) to the crystalloid fluids. monitor the core body temperature and ecg closely. rewarming should occur in the form of external circulating warm water blankets, radiant heat, and circulating warm air blankets (bair hugger). never use a heating pad, to avoid iatrogenic thermal burn injury. severe hypothermia may require core rewarming in the form of intraperitoneal fluids (10 to 20 ml/kg of lactated ringer's solution warmed to 39.4â°c [103â°f]). place a temporary peritoneal dialysis catheter, and repeat the dialysis every 30 minutes until the patient's body temperature reaches 36.6â°to 37.7â°c (98â°to 100â°f). the body temperature should rise slowly, ideally no more than 1â°f per hour. because the response of the body to drugs is unpredictable, avoid administering drugs whenever possible, until the body temperature returns to normal. complications observed during rewarming include dic, cardiac dysrhythmias including cardiac arrest, pneumonia, pulmonary edema, cns edema, ards, and renal failure. heat stroke and heat-induced illness in dogs can be associated with excessive exertion, exposure to high environmental temperatures, stress, and other factors that cause an inability to dissipate heat. brachycephalic breeds, obesity, laryngeal paralysis, and older animals with cardiovascular disease can be particularly affected. hyperthermia is defined as a rectal temperature of 41â°to 43â°c (105â°to 110â°f). clinical signs of hyperthermia include congested hyperemic mucous membranes, tachycardia, and panting. more severe clinical signs include collapse (heat prostration), ataxia, vomiting, diarrhea, hypersalivation, muscle tremors, loss of consciousness, and seizures. heat-induced illness can affect all major organ systems in the body because of denaturation of cellular proteins and enzyme activities, inappropriate shunting of blood, hypotension, decreased oxygen delivery, and lactic acidosis. cardiac dysrhythmias, interstitial and intracellular dehydration, intravascular hypovolemia, central nervous dysfunction, slough of gastrointestinal mucosa, oliguria, and coagulopathies can be seen as organ function declines. excessive panting can result in respiratory alkalosis. poor tissue perfusion results in a metabolic acidosis. loss of water in excess of solutes such as sodium and chloride can lead to a free water deficit and severe hypernatremia. a marked increase in pcv occurs because of the free water loss. severe abnormalities in electrolytes and ph can lead to cerebral edema and death. treatment goals for the patient with heat-induced illness are to lower the core body temperature and support cardiovascular, respiratory, renal, gastrointestinal, neurologic, and hepatic functions. at the scene the veterinarian or caretaker can spray the animal with tepid (not cold) water. immersion in cold water or ice baths is absolutely contraindicated. cold water and ice will cause extreme peripheral vasoconstriction, inhibiting the patient's ability to dissipate heat through conductive and convective cooling mechanisms. as a result, core body temperature will continue to rise despite the good intentions of well-doers at the scene. animals that present to the veterinarian that have been cooled to the point of hypothermia have a worse prognosis. once the animal has presented to the veterinarian, the goal is to cool the animal's body temperature with towels soaked in tepid water, cool intravenous fluids, and fans until the temperature has decreased to 103â°f. organ system monitoring and support is based on the severity and duration of the heat stroke and the ability of the body to compensate and respond to treatment. management of the patient with heat-induced illness involves prompt aggressive cooling without being overzealous and creating iatrogenic hypothermia. administer cool intravenous crystalloid fluids to replenish volume and interstitial hydration and correct the patient's acid-base and electrolyte abnormalities. management consists of rule of twenty monitoring (see rule of 20), taking care to evaluate, restore, and maintain a normal cardiac rhythm, blood pressure, urine output, and mentation. administer antibiotics if there are any signs of gastrointestinal bleeding that will predispose the patient to bacterial translocation. monitor baseline chemistry tests including a complete blood count, biochemical panel, platelet count, coagulation tests, and urinalysis. treat coagulopathies including dic aggressively and promptly (see also disseminated intravascular coagulation). severe changes in mentation including stupor or coma worsen a patient's prognosis. following initial therapy, monitor the patient for a minimum of 24 to 48 hours for secondary organ damage, including renal failure, myoglobinuria, cerebral edema, and dic. dogs that are going to die of heat-induced illness usually die within the first 24 hours. animals that survive longer than 24 hours have a more favorable prognosis. immediate treatment consists of cooling the patient with cooling measures as for hyperthermia and heat-induced illness (see the previous discussion), and eliminating the cause (i.e., exertion, anesthesia, or neuromuscular blockers such as succinylcholine). if the patient is under general anesthesia, hyperventilate the patient to help eliminate carbon dioxide and respiratory acidosis. administer dantrolene sodium (1 to 2 mg/kg iv) to stabilize the sarcoplasmic reticulum and decrease its permeability to calcium. animals with malignant hyperthermia should avoid any predisposing factors, including exertion, hyperthermia, and anesthesia. after an episode of malignant hyperthermia, administer crystalloid fluids intravenously to aid in the elimination of myoglobin. monitor renal function closely for myoglobinuria and pigment damage to the renal tubular epithelium. monitor and correct acid-base and electrolyte changes. walters jm: hyperthermia. in wingfield we, editor: the veterinary icu book, jackson, wyo, 2001, teton newmedia. sometimes it is difficult to assess whether an animal has been bitten by a poisonous or nonpoisonous snake. in colorado, the bull snake closely resembles the prairie rattlesnake. both snakes make similar noise and can be alarming if noticed on a hike or in the backyard. whenever possible, identify the offending reptile but never risk being bitten. know what types of venomous creatures are in the geographic area of the practice. if an animal has been bitten by a nonpoisonous snake, usually the bite marks are small with multiple small tooth punctures, and the bite is relatively nonpainful. usually local reaction is negligible. however, large boas or pythons also can inflict large crushing injuries that can cause severe trauma, including bony fractures. treatment for a nonpoisonous snakebite involves clipping the bite wound and carefully cleaning the area with antimicrobial scrub solution. broad-spectrum antibiotics (e.g., amoxicillin-clavulanate, 16.25 mg/kg po q12h) are indicated because of the extensive bacterial flora in the mouths of snakes. monitor all snakebite victims for a minimum of 8 hours after the incident, particularly when the species of the offending reptile is in question. if clinical signs of envenomation occur, modify the patient's treatment appropriately and aggressively. the two major groups of venomous snakes in north america are the pit viper and the coral snake. all venomous snakes are dangerous. the severity of any given bite depends on the toxicity of the venom, the amount of venom injected, the site of envenomation, the size of the animal bitten, and the time from bite/envenomation to seeking appropriate medical intervention. the majority of reptile envenomations in the united states are inflicted by pit vipers, including the water moccasin (cottonmouth), copperhead, and numerous species of rattlesnakes. pit vipers are characterized by a deep pit located between the eye and nostril, elliptic pupils, and retractable front fangs (figure 1-45) . localized clinical signs of pit viper envenomation may include the presence of bleeding puncture wounds, local edema close to puncture wounds, immediate severe pain or collapse, edema, petechiae, and ecchymosis with subsequent tissue necrosis. systemic signs of pit viper envenomation may include hypotension, shock, coagulopathies, lethargy, weakness, muscle fasciculations, lymphangitis, rhabdomyolysis, and neurologic signs including respiratory depression and seizures. neurologic signs largely are associated with envenomation emergency management of specific conditions 149 by the mojave and canebrake rattlesnakes, although a potent neurotoxin, mojave toxin a, also has been identified in other subspecies of rattlesnake. clinical signs of envenomation may take several hours to appear. hospitalize all suspected victims and monitor them for a minimum of 24 hours. the severity of envenomation cannot be judged solely on the basis of local tissue reaction. first aid measures by animal caretakers do little to prevent further envenomation. the most important aspect of initiating therapy is to transport the animal to the nearest veterinary emergency facility. to determine whether an animal has been envenomated by a pit viper, examine a peripheral blood smear for the presence of echinocytes. echinocytes will appear within 15 minutes of envenomation and may disappear within 48 hours. other treatment should be initiated as rapidly and aggressively as possible, although controversy exists whether some therapies are warranted. the mainstay of therapy is to improve tissue perfusion with intravenous crystalloid fluids, prevent pain with judicious use of analgesic drugs, and when necessary, reverse or negate the effects of the venom with antivenin. because pit viper venom consists of multiple fractions, treat each envenomation as a complex poisoning. obtain vascular access and administer intravenous crystalloid fluids (one fourth of a calculated shock dose) according to the patient's perfusion parameters of heart rate, blood pressure, and capillary refill time (see also shock and fluid therapy). opioid analgesics are potent and should be administered at the time of presentation. (see also pharmacologic means to analgesia: major analgesics). diphenhydramine (0.5 to 1 mg/kg im or iv) also can be administered to decrease the effects of histamine. famotidine, a histamine 1 receptor antagonist, also can be administered (0.5 to 1 mg/kg iv) to work synergistically with diphenhydramine. although antihistamines have no effect on the venom per se, they may have an effect on the tissue reaction to the venom and may prevent an adverse reaction to antivenin. the use of glucocorticosteroids is controversial. glucocorticosteroids (dexamethasone sodium phosphate [dex-sp], 0.25 to 0.5 mg/kg iv) may stabilize cellular membranes and inhibit phospholipase, an active component of some pit viper toxins. polyvalent antivenin is necessary in many cases of pit viper envenomation, except in most cases of prairie rattlesnake (crotalus viridis viridis) envenomation in colorado. a recent study demonstrated no difference in outcome with or without the use of antivenin in cases of prairie rattlesnake envenomation. clinically, however, patients that receive antivenin are more comfortable and leave the hospital sooner than those that do not receive antivenin. the exact dose of antivenin is unknown in small animal patients. administer a dose of at least 1 vial of antivenin to neutralize circulating venom. mix antivenin with a swirling, rather than a shaking motion, to prevent foaming. mix the antivenin with a 250-ml bag of 0.9% saline, and then administer it slowly over a period of 4 hours. pretreat animals with diphenhydramine (0.5 to 1 mg/kg im) before the administration of antivenin, and then monitor the animal closely for clinical signs of angioneurotic edema, urticaria, tachyarrhythmias, vomiting, diarrhea, and weakness during the infusion. administration of antivenin into the bite site is relatively contraindicated and ineffective because uptake is delayed, and systemic effects are the more life-threatening. management of pit viper envenomation largely involves maintenance of normal tissue perfusion with intravenous fluids, decreasing patient discomfort with analgesia, and negating circulating venom with antivenin. hydrotherapy to the affected bite site with tepid water is often soothing to the patient. the empiric use of antibiotics is controversial but is recommended because of the favorable environment created by a snakebite (i.e., impregnation of superficial gram-positive bacteria and gram-negative bacteria from the mouth of the snake into a site of edematous necrotic tissue). administer amoxicillin-clavulanate (16.25 mg/kg po q12h, or cephalexin, 22 mg/kg po q8h). also consider administration of nsaids (carprofen, 2.2 mg/kg po q12h). monitor the patient closely for signs of local tissue necrosis and the development of thrombocytopenia and coagulopathies including dic (see management of disseminated intravascular coagulation). treat coagulopathies aggressively to prevent end-organ damage. coral snakes are characterized by brightly colored bands encircling the body, with red and black separated by yellow. "red on black, friend of jack; red on yellow, kill a fellow." types of coral snakes include the eastern coral, texas coral, and sonoran coral snakes. clinical signs of coral snake envenomation may include small puncture wounds, transient initial pain, muscle fasciculations, weakness, difficulty swallowing/dysphagia, ascending lower motor neuron paralysis, miotic pinpoint pupils, bulbar paralysis, respiratory collapse, and severe hemolysis. clinical signs may be delayed for as long as 18 hours after the initial bite. immediate treatment with antivenin is necessary in cases of coral snake envenomation before the clinical signs become apparent, whenever possible. support respiration during paralysis with mechanical ventilation. secure the patient's airway with a cuffed endotracheal tube to prevent aspiration pneumonia. clinical signs will progress rapidly once they develop. rapid administration with antivenin is the mainstay of therapy in suspected coral snake envenomation. respiratory and cardiovascular support should occur with mechanical ventilation and intravenous crystalloid fluids. keep the patient warm and dry in a quiet place. turn the patient every 4 to 6 hours to prevent atelectasis and decubitus ulcer formation. maintain cleanliness using a urinary catheter and closed urinary collection system. perform passive range of motion and deep muscle massage to prevent disuse atrophy of limb muscles and function. treat aspiration pneumonia aggressively with broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h, with enrofloxacin, 10 mg/kg iv q24h, and then change to oral once tolerated and the patient is able to swallow) for 2 weeks past the resolution of radiographic signs of pneumonia, intravenous fluids, and nebulization with sterile saline and coupage chest physiotherapy. several weeks may elapse before a complete recovery. the adult black widow spider (latrodectus spp.) can be recognized by a red to orange hourglass-shaped marking on the underside of a globous, shiny, black abdomen. the immature female can be recognized by a colorful pattern of red, brown, and beige on the dorsal surface of the abdomen. adult and immature females are equally capable of envenomation. the male is unable to penetrate the skin because of its small size. black widow spiders are found throughout the united states and canada. black widow spider venom is neurotoxic and acts presynaptically, releasing large amounts of acetylcholine and norepinephrine. there appears to be a seasonal variation in the potency of the venom, lowest in the spring and highest in the fall. in dogs, envenomation results in hyperesthesia, muscle fasciculations, and hypertension. muscle rigidity without tenderness is characteristic. affected animals may demonstrate clinical signs of acute abdominal pain. tonic-clonic convulsions may occur but are rare. in cats, paralytic signs predominate and appear early as a ascending lower motor neuron paralysis. increased salivation, vomiting, and diarrhea may occur. serum biochemistry profiles often reveal significant elevations in creatine kinase and hypocalcemia. myoglobinemia and myoglobinuria can occur because of extreme muscle damage. management of black widow spider envenomation should be aggressive in the cat and dog, particularly when the exposure is known. in many cases, however, the diagnosis is made based on clinical signs, biochemical abnormalities, and lack of other apparent cause. antivenin (one vial) is available and should be administered after pretreatment with diphenhydramine. if antivenin is unavailable, administer a slow infusion of calcium-containing fluid such as lactated ringer's solution with calcium gluconate while carefully monitoring the patient's ecg. the small brown nonaggressive spider is characterized by a violin-shaped marking on the cephalothorax. the neck of the violin points toward the abdomen. brown spiders are found primarily in the southern half of the united states but have been documented as far north as michigan. the venom of the brown spider has a potent dermatonecrolytic effect and starts with a classic bull's-eye lesion. the lesion then develops into an indolent ulcer into dependent tissues promoted by complement fixation and influx of neutrophils into the affected area. the ulcer can take months to heal and often leaves a disfiguring scar. systemic reactions are rare but can include hemolysis, fever, thrombocytopenia, weakness, and joint pain. fatalities are possible. immediate management of an animal with brown spider envenomation is difficult because there is no specific antidote and because clinical signs may be delayed until necrosis of the skin and underlying tissues becomes apparent through the patient's fur 7 to 14 days after the initial bite. dapsone has been recommended at a dose of 1 mg/kg for 14 days. surgical excision of the ulcer may be helpful if performed in the early stages of wound appearance. glucocorticosteroids may be of some benefit if used within 48 hours of the bite. the ulcer should be left to heal by second intention. deep ulcers should be treated with antibiotics. bufo toad species (b. marinus, aka cane toad, marine toad, giant toad; and the colorado river toad or sonoran desert toad b. alvarius) can be associated with severe cardiac and neurotoxicity if an animal licks its skin. the severity of toxicity depends largely on the size of the dog. toxins in the cane toad, b. marinus, include catecholamines and vasoactive substances (epinephrine, norepinephrine, serotonin, dopamine) and bufo toxins (bufagins, bufotoxin, and bufotenine), the mechanism of which is similar to cardiac glycosides. clinical signs can range from ptyalism, weakness, ataxia, extensor rigidity, opisthotonus, and collapse to seizures. clinical signs associated with b. alvarius toxicity are limited largely to cardiac dysrhythmias, ataxia, and salivation. the animal should have its mouth rinsed out thoroughly with tap water even before presentation to the veterinarian. if the animal is unconscious or actively seizing and cannot protect its airway, flushing the mouth is contraindicated. once an animal presents to the veterinarian, the veterinarian should place an intravenous catheter and monitor the patient's ecg and blood pressure. attempt seizure control with diazepam (0.5 mg/kg iv) or pentobarbital (2 to 8 mg/kg iv to effect). ventricular dysrhythmias can be controlled first with esmolol (0.1 mg/kg). if esmolol is ineffective, administer a longer-acting parenteral î²-antagonist such as propranolol (0.05 mg/kg iv). ventricular tachycardia also can be treated with lidocaine (1 to 2 mg/kg iv, followed by 50 to 100 âµg/kg/minute iv cri). case management largely depends on supportive care and treating clinical signs as they occur. monitor baseline acid-base and electrolyte balance because severe metabolic acidosis may occur that should be treated with intravenous fluids and sodium bicarbonate (0.25 to 1 meq/kg iv). monitor ecg, blood pressure, and mentation changes closely. control seizures and cardiac dysrhythmias. eubig pa: bufo species intoxication: big toad, big problem, vet med 96 (8) lizards of the family hemodermatidae are the only two poisonous lizards in the world. they are found in the southwestern united states and mexico. the venom glands are located on either side of the lower jaw. because these lizards are typically lethargic and nonaggressive, bite wounds are rare. the lizards have grooved teeth that introduce the venom with a chewing motion as the lizard holds tenaciously to the victim. the majority of affected dogs are bitten on the upper lip, which is very painful. there are no proven first aid measures for bites from gila monsters or mexican bearded lizards. the lizard can be disengaged by inserting a prying instrument in between the jaws 1 and pushing at the back of the mouth. the teeth of the lizard are brittle and break off in the wound. topical irrigation with lidocaine and probing with a needle will aid in finding and removing the teeth from the victim. bite wounds will bleed excessively. irrigate wounds with sterile saline or lactated ringer's solution, and place compression on the affected area until bleeding ceases. monitor the patient for hypotension. establish intravenous access, and administer intravenous fluids according to the patient's perfusion parameters. antibiotic therapy is indicated because of the bacteria in the lizard's mouth. because no antidote is available, treatment is supportive according to patient signs. the majority of musculoskeletal emergencies are the result of external trauma, most commonly from motor vehicle accidents. blunt trauma invokes injury to multiple organ systems as a rule, rather than an exception. because of this, massive musculoskeletal injuries are assigned a relatively low priority during the initial triage and treatment of a traumatized animal. perform a rapid primary survey and institute any lifesaving emergency therapies. adhere to a crash plan or the abcs of resuscitation (see initial emergency examination, management, and triage). although musculoskeletal injuries are assigned a relatively lower priority, the degree of recovery from these injuries and financial obligation for fracture repair sometimes becomes a critical factor in a client's decision whether to pursue further therapy. one of the most important deciding factors is the long-term prognosis for the patient to have a good quality of life following fracture repair. the initial management of musculoskeletal injuries is important in ensuring the best chance for maximal recovery with minimal complications after definitive surgical fracture repair. this is particularly important for open fractures, spinal cord compromise, multiple fractures, open joints, articular fractures, physeal fractures, and concomitant ligamentous or neurologic compromise (box 1-41). immediately after the initial primary survey of a patient, perform a more thorough examination, including an orthopedic examination. multiple injuries often are observed in the patient that falls from height (e.g., "high-rise syndrome"), motor vehicle accidents, gunshot wounds, and encounters with other animals (e.g., "big-dog-little-dog"). address the most life threatening injuries, and palliate musculoskeletal injuries until more definitive repair can be attempted when the patient is more stable. in animals with the history of potential for multiple injuries, search thoroughly and meticulously for areas of injury to the spinal column, extremities, and for small puncture wounds. helpful signs that can provide a clue as to an underlying injury include swelling, bruising, abnormal motion, and crepitus (caused by subcutaneous emphysema or bony fracture). if the patient is alert, look for areas of tenderness or pain. in unconscious or depressed patients, reexamine the patient after the patient becomes more mentally alert. injuries often are missed during the initial examination in obtunded patients because of the early response and attenuation of pain. unconscious or immobile patients must have radiographic examination of the spinal column following stabilization and support. palpate the skull carefully for obvious depressions or crepitus that may be associated with a skull fracture. localization of the injury can be determined by motion in abnormal locations, swelling caused by hemorrhage or edema, pain during gentle movement or palpation, deformity, angular change, or a significant increase or decrease in normal range of motion of bones and joints. perform a rectal examination in all cases to palpate for pelvic fractures and displacement. once the diagnosis of a fracture or luxation has been confirmed, look for any evidence of skin lacerations or punctures near the fracture site. in long-haired breeds, clipping the fur near the fracture site often is necessary to perform a thorough examination of the area. if any wounds are found, the fracture is classified as an open fracture until proven otherwise. in some cases, the open fracture is obvious, with a large section of bone fragment protruding through the skin. in other cases, the puncture wound may be subtle, with only a small amount of blood or pinpoint hole in the skin surface. characteristics observed with open fractures include bone penetration, fat droplets or marrow elements in blood coming from the wound, subcutaneous emphysema on radiographs, and lacerations in the area of a fracture. protect the patient from further injury or contamination of wounds. excessive palpation to intentionally produce crepitus is inappropriate because it causes severe patient discomfort and has the potential to cause severe soft tissue and neurologic injury at the fracture site. sedation and analgesia aids in making the examination more comfortable for the patient and allows localization of the injury and comparison with the opposite extremity. higher-quality radiographs can be performed to determine the extent of the injury when the animal is sedated adequately and pain is controlled. sedate the patient judiciously with analgesic drugs. opioid drugs work well for orthopedic pain, produce minimal cardiorespiratory depression, and can be reversed with naloxone if necessary. handle the fracture site gently to avoid causing further pain and soft tissue injury at the fracture site. rough or careless handling of a fracture site can cause a closed fracture to penetrate through the skin and become an open fracture. cover open fractures immediately to prevent contamination of the fracture with nosocomial infection from the hospital. administer a first-generation cephalosporin (cephalexin, 22 mg/kg po q8h, or cefazolin, 22 mg/kg iv q8h). the bandage also serves to control hemorrhage and prevent desiccation of the bones and surrounding soft tissue structures. leave the initial bandages in place until the patient's cardiorespiratory status has been determined to be stable and more definitive wound management can occur in a clean, preferably sterile location. examine the neurologic status and cardiovascular status of the limb before and after treatment. determine the vascular status of the limb by checking the color and temperature of the limb, the state of distal pulses, and the degree of bleeding from a cut nail bed. in patients with severe cardiovascular compromise and hypotension caused by hemorrhagic shock, the viability of the limb may be in question until the cardiovascular status and blood pressure are normalized. reduction of the fracture or straightening of gross deformities may return normal vascularity to the limb. when checking neurologic status, examine for motor and sensory function to the limb. swelling may increase pressure on the nerves as they run through osteofascial compartments, resulting in decreased sensory or motor function, or neurapraxia. diminished function often returns to normal once the swelling subsides. serial physical examinations in the patient and response to initial stabilization therapy can lead to a higher index of suspicion that more occult injuries are present, such as a diaphragmatic hernia, perforated bowel, lacerated liver or spleen, or uroabdomen. to prevent ongoing trauma, reduce any fracture and then stabilize the site above and below the fracture. a modified robert jones splint or bandage often works well for fractures emergency management of specific conditions 155 involving the distal extremities. fractures of the humerus or femur are difficult to immobilize without the use of spica or over-the-hip coaptation splints to prevent mobility. inappropriate bandaging of humerus or femur fractures can result in a fulcrum effect and worsen the soft tissue and neurologic injuries. further displacement of vertebral bodies or luxations can cause cord compression or laceration such that return to function becomes impossible. immediately place any patient with a suspected spinal injury on a flat surface, and tape down the animal to prevent further movement until the spine has been cleared by a minimum or two orthogonal radiographic views (lateral and ventrodorsal views performed as a cross-table x-ray technique). wounds associated with musculoskeletal trauma are common and include injury to the bones, joints, tendons, and surrounding musculature (box 1-42). major problems associated with these cases are the presence of soft tissue trauma that makes wound closure hazardous or impossible, because of the risk of infection. chronic deep infection of traumatized wounds can cause delayed healing and sequestrum to develop, particularly if there is avascular bone or cartilage within the wound. in the early management of an open fracture, the areas should be splinted without pulling any exposed bone back into the soft tissue. the wound should not be probed or soaked, as nosocomial bacteria and other external contaminants can be introduced into the wound, leading to severe infection. because of the risk of actually causing infection, probing, flushing, or replacing tissues back into the wound should be performed at the time of formal debridement when the patient is physiologically stable. immediate bactericidal antibiotic therapy with a first-generation cephalosporin should be started immediately to obtain adequate concentrations of antibiotics at the fracture site. the duration of antibiotic therapy should ideally be limited to 2-3 days to prevent the risk of superinfection. treatment of open musculoskeletal injury involves three considerations: initial inspection and wound debridement, stabilization and repair, and wound bandaging. 156 1 emergency care when associated with a fracture, wound is created from the inside out by penetration of bone fragments through the skin or from a low-energy gunshot. simple or comminuted fracture pattern good stability of the two main bone segments treatment and prognosis are good and similar to those of a closed injury if wound is debrided and stabilized within 6 to 8 hours. when associated with a fracture, wound is created from the outside in. major deep injury with considerable soft tissue stripping from bone and muscle damage simple or comminuted fracture pattern prognosis is good if wound is debrided within 6 hours of injury and provided rigid stabilization with a bone plate or external fixator. results from major external force severe damage and necrosis of skin, subcutaneous tissue, muscle, nerve, bone, tendon, and arteries soft tissue damage may vary from crush injury to shearing injury associated with bite wounds or low-speed automobile accidents. requires immediate and delayed sequential debridement and rigid external fixation can require prolonged healing times guarded prognosis initial inspection and wound debridement include the following steps: 1. after the patient's cardiovascular status has been stabilized and it has been determined that it can withstand anesthesia, place the animal under general anesthesia and remove the temporary splint. 2. keeping the wound covered, shave the surrounding fur. 3. remove the covering and then place sterile lubricant jelly over the wound. shave the fur to the edges of the wound margin. 4. wash away any entrapped fur and the lubricant jelly. 5. complete an antiseptic scrub of the surrounding skin. 6. if the wound is a small puncture (e.g., gunshot pellets or bites), probe the wound with a sterile hemostat. do a thorough debridement if tissues deep to the hole are cavitated. if not deep, create a hole for drainage. 7. flush the wound with a physiologic solution (lactated ringer's solution is preferred). 8. debride the wound from outward to inward. cut away damaged areas of skin and deeper tissues to open up underlying cavitations and tissue injury. 9. continuously irrigate with warm physiologic solution (lactated ringer's solution is preferred). the stream must be strong enough to flush debris out of the bottom of the wound. to accomplish this, attach a 20-gauge needle to a 35-ml syringe (will deliver 7 psi). excise any obviously devitalized tissue. 10. do not remove any bone fragments that are firmly attached to soft tissue. do not cut into healthy soft tissue to find bullet or bone fragments, unless the bullet can cause injury to joints or nerve tissue. 11. do a primary repair of tendons and nerves if the wound is type i and recent (within 8 hours of the initial injury). if the wound is too severe or if there is obvious infection, tag the ends of the tendons and nerves for later repair. it is best to stabilize and repair open fractures as soon as the patient's cardiovascular and respiratory status can tolerate general anesthesia, provided that adequate stabilization is possible. if this is not possible because of the level of experience of the surgeon or the lack of necessary equipment, it is best to perform wound management and place a temporary splint until definitive repair can be performed. wound bandaging is discussed in the section on bandaging techniques. structural injuries to the joints are common and can involve both ligaments and articular cartilage injuries. cartilage does not heal well; therefore, injuries involving articular cartilage can lead to a significant loss of function and degenerative joint disease (osteoarthritis). cartilage injuries that are superficial evoke a short-lived enzymatic and metabolic response that does not stimulate enough cellular growth to repair the defect. superficial lesions remain as defects but do not progress to chondromalacia or osteoarthritis. deep cartilage lacerations that extend to subchondral bone produce an exuberant healing response from the cells of the underlying cartilage. in many cases, this material undergoes degeneration and leads to osteoarthritis. impact injuries to surface cartilage can cause chondrocyte and underlying bone injury. these lesions rapidly progress to osteoarthritis; however, they may be totally or partially reversible. treatment of grade i injuries requires short-term coaptation splints and has a good prognosis. grade ii injuries require surgical treatment with a suture stent and consistent postoperative coaptation splints to heal and maintain good function. healing of grade iii injuries often is a problem, and suture stents or surgical reapproximation may be indicated. failure to immobilize joints that are frequently flexed (elbow and stifle) can result in late complications of ligament repair. ligamentous injuries of joints, particularly the collateral ligaments of the stifle, elbow, and hock, and carpal hyperextension injuries are commonly missed and may require surgical fixation, including arthrodesis (box 1-43). fractures in immature animals differ from those in adults in that young puppies and kittens have a great ability to remodel bone. remodeling is dependent on the age of the patient and the location of the fracture. the younger the puppy or kitten and the closer the fracture to the epiphysis or growth plate, the greater the potential for remodeling and the development of angular limb deformities. remodeling occurs more effectively in longlimbed breeds of dogs than in short-limbed breeds. fractures through the growth plate of immature animals may potentially cause angular limb deformities, joint dislocations or incongruity, and osteoarthritis. this form of injury is commonly observed in the distal ulnar growth plate and the proximal and distal radial growth plates. high-rise syndrome in cats is seen in cats that fall from a height usually greater than 30 feet. it occurs most frequently in high-rise buildings in urban areas where cats lie on window ledges and suddenly fall out the window. the most common lesions observed in cats that fall from heights are thoracic injuries (rib and sternal fractures, pneumothorax, and pulmonary contusions) and facial and oral trauma (lip avulsions, mandibular symphyseal fractures, fractures of the hard palate, and maxillary fractures). limb and spinal cord fractures and luxations, radius and ulna fractures, abdominal trauma, urinary tract trauma, and diaphragmatic hernias are also common. the injuries sustained are often found in combination, rather than as an isolated injury of one area of the body. follow the mnemonic a crash plan when managing a cat suffering from high-rise syndrome, treating the animal immediately for shock. following cardiovascular and respiratory stabilization, evaluate thoracic and abdominal radiographs, including those of the spine. evaluate the bladder closely, making sure that the cat is able to urinate effectively. examine the hard palate, maxilla, and mandibular symphysis for fractures. palpate the pelvis and carefully manipulate all limbs to examine for fractures or ligamentous injuries. finally, perform a complete neurologic examination. patients that fall less than five stories often have a more guarded prognosis than patients that fall from higher levels. sometimes the owner witnesses the ingestion of a foreign body during play, such as throwing a stick or fetching a ball. cats tend to play with string or thread that becomes caught around the base of the tongue. in many cases, however, ingestion of the foreign object is not witnessed, and diagnosis is made based on clinical signs and physical examination. foreign bodies lodged in the oral cavity often cause irritation and discomfort, including difficulty breathing and difficulty swallowing. often, an animal paws at its mouth in an attempt to dislodge a stick or bones wedged across the roof of the mouth. irritation, inability to close the mouth, and blockage of the orpharynx can result in excessive drooling. the saliva may appear blood-tinged due to concurrent soft tissue trauma (figs 1-46 and 1-47) . obstruction of the glottis by a foreign body (e.g., tennis ball or toy) can result in cyanosis secondary to an obstructed airway and hypoxemia. in many cases, the object is small enough to enter the larynx but too large to be expelled. if a foreign object is lodged in the mouth for more than several days, halitosis and purulent discharge may be present. many animals are anxious at the time of presentation and may require sedation or a light plane of anesthesia to remove the foreign object. the animal may bite personnel and may have bitten the owner during his or her attempt to remove the object from the mouth en route to the hospital. propofol (47 mg/kg iv) or a combination of propofol with diazepam (0.5-1 mg/kg iv) is an excellent combination for a light plane of anesthesia. exercise caution when anesthetizing a patient with a ball lodged in the airway, as further compromise of respiratory function may occur and cause worsening of the hypoxemia. before inducing anesthesia, assemble all supplies necessary to remove the object. make sure that rigid towel clamps, sponge forceps, and bone forceps are on hand, because the foreign object is often very slippery with saliva. hemostats and carmalts may slip and not be useful in the removal of the foreign object. place a peripheral intravenous catheter to secure vascular access prior to anesthetic induction. have available the supplies necessary for an emergency tracheostomy, if the foreign object cannot be removed by usual methods. induce a light plane of anesthesia and then grasp the object with the sponge forceps or towel clamps, and extract. monitor the cardiorespiratory status of the animal at all times during the extraction process. if you are unable to remove the object, and if severe respiratory distress, including cyanosis, bradycardia, or ventricular dysrhythmias, develop, perform a tracheostomy distal to the site of obstruction. once the foreign body has been removed, administer supplemental flow-by oxygen until the animal awakens. if laryngeal edema or stridor on inspiration is present, administer a dose of dexamethasone sodium phosphate (0.25 mg/kg iv, im, sq) to decrease inflammation. the patient should be carefully monitored for 24 hours, because noncardiogenic pulmonary edema can develop secondary to airway obstruction. esophageal foreign bodies pose a serious medical emergency. it is helpful if the owner witnessed ingestion of the object and noted rapid onset of clinical signs. in many cases, however, ingestion is not witnessed, and the diagnosis must be made based on clinical signs, thoracic radiographs, and results of a barium swallow. the most common clinical signs are excessive salivation with drooling, gulping, and regurgitation after eating. many animals will make repeated swallowing motions. some animals exhibit a rigid "sawhorse" stance, with reluctance to move immediately after foreign body ingestion and esophageal entrapment. after completing a physical examination, evaluate cervical and thoracic radiographs to determine the location of the esophageal obstruction. esophageal foreign objects are lodged most commonly at the base of the heart, the carina, or just orad to the lower esophageal sphincter. if the object has been lodged for several days, pleural effusion and pneumomediastinum may be present secondary to esophageal perforation. endoscopy is useful for both diagnosis and removal of the foreign object; however, it is invasive and requires general anesthesia ( fig. 1-48) . remove foreign objects lodged in the esophagus with a rigid or flexible endoscope after the patient has been placed under general anesthesia. evaluate the integrity of the esophagus both before and after removal of the material because focal perforation or pressure necrosis can be present. necrosis of the mucosa and submucosa of the esophagus often leads to stricture formation or perforation. attempt to retrieve the object with a flexible fiberoptic endoscope if available. rigid tube endoscopy can also be performed. in many cases, smooth objects that cannot be easily grasped can be pushed into the stomach and allowed to dissolve or may be removed by gastrotomy. if the foreign body is firmly lodged in the esophagus and cannot be pulled or pushed into the stomach, or if perforation has already occurred, the prognosis for return to function without strictures is not favorable. in such cases, referral to a surgical specialist is recommended for esophagostomy or esophageal resection. after removal of the object, carefully examine the esophagus and then administer gastroprotectant agents (famotidine, 0.5 mg/kg po bid; sucralfate slurry, 0.5-1.0 g/dog) for a minimum of 5 to 7 days. to rest the esophagus, the patient should receive nothing per os (npo) for 24 to 48 hours. if esophageal irritation or erosion is moderate to severe, a percutaneous gastrotomy tube should be placed for feeding until the esophagus heals. perform repeat endoscopy every 7 days to evaluate the healing process and to determine whether stricture formation is occurring. persistent vomiting immediately or soon after eating is often associated with a gastric foreign body. in some cases, the owner knows that the patient has ingested a foreign body of some kind. in other cases, continued vomiting despite lack of response to conservative treatment (npo, antiemetics, gastroprotectant drugs) prompts further diagnostic procedures, including abdominal radiographs and bloodwork. obstruction to gastric outflow and vomiting of hydrochloric acid often cause a hypochloremic metabolic acidosis. radiopaque gastric foreign bodies may be observed on plain films. radiolucent cloth material may require a barium series to delineate the shape and location of the foreign body ( fig. 1-49) . treatment consists of removal with flexible endoscopy or a simple gastrotomy. most animals with uncomplicated gastric foreign bodies are relatively healthy, but any metabolic and electrolyte abnormalities should be corrected prior to anesthesia and surgery. small intestinal obstruction can be caused by foreign bodies, tumors, intussusception, volvulus, or strangulation within hernias. regardless of the cause, clinical signs of small intestinal obstruction depend on the location and degree of obstruction, and whether the bowel has perforated. clinical signs associated with a high small intestinal obstruction are usually more severe and more rapid in onset compared with partial or complete obstruction of the jejunum or ileum. complete obstructions that allow no fluid or chyme to pass are worse than partial obstructions, which can cause intermittent clinical signs interspersed with periods of normality (table 1 -36). the most common clinical signs associated with a complete small intestinal obstruction are anorexia, vomiting, lethargy, depression, dehydration, and sometimes abdominal pain. early clinical signs may be limited to anorexia and depression, making a diagnosis challenging unless the owner has a suspicion that the animal ingested some kind of foreign object. obstructions cranial to the common bile duct and pancreatic papillae lead to vomiting of gastric contents, namely hydrochloric acid, and a hypochloremic metabolic alkalosis. obstructions caudal to the common bile duct and pancreatic papillae result in loss of other electrolytes and sometimes mixed acid-base disorders. eventually, all animals with small intestinal obstruction vomit and have fluid loss into dilated segments of bowel, leading to dehydration and electrolyte abnormalities. increased luminal pressure causes decreased lymphatic drainage and bowel edema. the bowel wall eventually becomes ischemic and may rupture. linear foreign bodies should be suspected in any vomiting patient, particularly cats. string or thread often is looped around the base of the tongue and can be visualized in many cases by a thorough oral examination. to look properly under the tongue, grasp the top of the animal's head with one hand, and pull the lower jaw open with the index finger of the opposite hand while pushing up the thumb simultaneously on the tongue in between the intermandibular space. thread and string can be observed lying along the ventral aspect of the tongue. in some cases, if a linear foreign body is lodged very caudally, it cannot be visualized without heavy sedation or anesthesia. linear foreign bodies eventually cause bowel obstruction and perforation of the intestines along the mesenteric border. the foreign material (e.g., string, thread, cloth, pantyhose) becomes lodged proximally, and the intestines become plicated as the body attempts to push the material caudally through the intestines ( fig. 1-50) . continued peristalsis eventually causes a sawing motion of the material and perforation of the mesenteric border of the intestines. once peritonitis occurs, the prognosis is less favorable unless prompt and aggressive treatment is initiated. reevaluate any patient that does not respond to conservative symptomatic therapy, performing a complete blood count, serum biochemical panel (including electrolytes), and abdominal radiographs. intestinal masses may be palpable on physical examination and are often associated with signs of discomfort or pain when palpating over the mass. radiography and abdominal ultrasound are the most useful diagnostic aids. plain radiographs may be diagnostic when the foreign object is radiodense or there is characteristic dilation or plication of bowel loops. as a rule of thumb, the width of a loop of small bowel should be no larger than twice the width of a rib. diagnosis of small intestinal obstruction or ileus can be based on the appearance of stacking loops of dilated bowel. comparison of the width of the bowel with the width of a rib is often performed. with mild dilation, the bowel width is three to four times the rib width; with extensive dilation, five to six times the rib width ( fig.1-51) . in cases of linear foreign bodies, c-areas (comma-shaped areas) of gas trapped in the plicated bowel will appear stacked on one another. blunt, wedge-shaped areas of gas or square linear areas of gas adjacent to a distended bowel loop are characteristic of a foreign body lodged in the intestine. contrast radiography is indicated when confirmation of the suspected diagnosis is necessary and ultrasonography is not available. contrast material may outline the object or abruptly stop orad to the obstruction. the definitive treatment of any type of small intestinal foreign body is surgical removal. linear foreign bodies sometimes pass, but they should never be left untreated in a patient that is demonstrating clinical signs of inappetence, vomiting, lethargy, and dehydration. the timing of surgery is critical because the risk of intestinal perforation increases with time. prior to surgery, correct any acid-base and electrolyte abnormalities with intravenous fluid therapy. administer broad-spectrum antibiotics. perform an enterotomy or intestinal resection and anastomosis as soon as possible once the patient's acid-base and electrolyte status have been corrected. clinical signs of a foreign body in the large bowel are usually nonexistent. in most cases, if a foreign object has passed successfully through the small bowel, it will pass through the large bowel without incident unless bowel perforation and peritonitis occur. penetrating foreign bodies such as needles often cause localized or generalized peritonitis, abdominal pain, and fever. hematochezia may be present if the foreign object causes abrasion of the rectal mucosa. symptomatic patients should have abdominal radiographs performed. colonoscopy or exploratory laparotomy should be performed if survey radiographs are suggestive of a large intestinal obstruction or perforation. in most cases, large intestinal foreign bodies will pass without incident. surgery is required to treat perforations, peritonitis, or abscesses. 164 1 emergency care 1 figure 1 -51: after 60 minutes, the barium has stopped moving and has reached a blunt, intraluminal intestinal foreign body. note that barium appears wedge-shaped or square at the site of the foreign body. foreign bodies in the rectum and anus often are the result of ingestion of bones, wood material, needles, and thread, or malicious external insertion. often the material can pass through the entire gastrointestinal tract and then get stuck in the anal ring. clinical signs include hematochezia and dyschezia with straining to defecate. diagnosis is made by visual examination of the item in the anus, or by careful digital palpation after heavy sedation or short-acting general anesthesia. radiography is helpful in locating needles that have penetrated the rectum and lodged in the perirectal or perinatal tissues. treatment consists of careful removal of the needle digitally or surgically. intussusception is the acute invagination of one segment of bowel (the intussusceptum) into another (the intussuscipiens). the proximal segment always invaginates into the distal segment of bowel. intussusception most commonly occurs in puppies and kittens less than 1 year of age but can occur in an animal of any age with hypermotility of the small bowel, gastrointestinal parasites, and severe viral or bacterial enteritis. intussusception occurs primarily in the small bowel in the jejunum, ileum, and ileocolic junction. clinical signs include vomiting, abdominal discomfort, and hemorrhagic diarrhea. usually, hemorrhagic diarrhea is the first noticeable sign, and in puppies, may be due to parvoviral enteritis, with secondary intussusception. usually, the obstruction is partial with mild clinical signs. more serious clinical signs develop as the obstruction becomes more complete. differential diagnoses include hemorrhagic gastroenteritis, parvoviral enteritis, gastrointestinal parasites, intestinal foreign body, bacterial enteritis, and other causes of vomiting and diarrhea. the diagnosis of intussusception is often made based on palpation of a sausage-shaped firm, tubular structure in the abdomen accompanied by clinical signs and abdominal pain. plain radiographs may demonstrate segmental or generalized dilated segments of bowel, depending on the duration of the problem. ultrasonographs of the palpable mass resemble the layers of an onion, with hyperechoic intestinal walls separated by less echogenic edema. treatment consists of correction of the patient's acid-base and electrolyte abnormalities with intravenous fluids and surgical reduction or removal of the intussusception with resection and anastomosis. although enteroplication has been suggested, the technique has fallen out of favor because of the increased risk of later obstruction. the primary cause of intestinal inflammation and hypermotility must be identified and corrected. gastric dilatation can occur with or without volvulus in the dog. gastric dilatationvolvulus (gdv) occurs primarily in large-and giant-breed dogs with deep chests, such as the great dane, labrador retriever, saint bernard, german shepherd dog, gordon and irish setters, standard poodle, bernese mountain dog, and bassett hound. the risk of gdv increases with age; however, it can be seen in dogs as young as 4 months. deep, narrow-chested breeds are more likely to develop gdv than dogs with broader chests. the overall mortality for surgically treated gastric dilatation-volvulus ranges from 10% to 18%, with most deaths occurring in patients that required splenectomy and partial gastrectomy. clinical signs of gdv include abdominal distention, unproductive vomiting or retching, lethargy, weakness, sometimes straining to defecate, and collapse. the owner may think that the animal is vomiting productively because of the white foamy froth (saliva) that is not able to pass into the twisted stomach. in some cases, there is a history of the dog's being fed a large meal or consuming a large quantity of water prior to the onset of clinical signs. instruct the owner of any patient with a predisposition for and clinical signs of gdv to transport the animal to the nearest veterinary facility immediately. physical examination often reveals a distended abdomen with a tympanic area on auscultation. in dogs with very deep chests, it may be difficult to appreciate abdominal distention if the stomach is tucked up under the rib cage. depending on the stage of shock, the patient may have sinus tachycardia with bounding pulses, cardiac dysrhythmias with pulse deficits, or bradycardia. the mucous membranes may appear red and injected or pale with a prolonged capillary refill time. the patient may appear anxious and attempt to retch unproductively. if the patient is nonambulatory at the time of presentation, the prognosis is more guarded. the definitive diagnosis of gdv is based on clinical signs, physical examination findings, and radiographic appearance of gas distention of the gastric fundus with dorsocranial displacement of the pylorus and duodenum (the so-called "double-bubble" or "popeye arm" sign) ( fig.1-52) . in simple gastric dilatation without volvulus, there is gas distention of the stomach with anatomy appearing normal on radiography. with "food bloat," or gastric distention from overconsumption of food, ingesta is visible in the distended stomach ( fig. 1-53) . as soon as a patient presents with a possible gdv, place a large-bore intravenous catheter in the cephalic vein(s) and assess the patient's ecg, blood pressure, heart rate, capillary refill time, and respiratory function. obtain blood samples for a complete blood count, serum biochemistry profile, immediate lactate measurement, and coagulation tests before taking any radiographs. rapidly infuse a colloid (hetastarch or oxyglobin, 5 ml/kg iv bolus) along with shock volumes of a crystalloid fluid (up to 90 ml/kg/hour) (see section on shock). monitor perfusion parameters (heart rate, blood pressure, capillary refill time, and ecg) and titrate fluid therapy according to the patient's response. the use of short-acting glucocorticosteroids is controversial. glucocorticosteroids may help stabilize cellular membranes and decrease the mechanisms of ischemia-reperfusion injury, but no detailed studies have proved them to be beneficial versus not using glucocorticosteroids in the patient with gdv. attempt gastric decompression, either with placement of an orogastric tube or by trocharization. to place an orogastric tube, position the distal end of the tube at the level of the patient's last rib ( fig. 1-54 ) and place it adjacent to the animal's thorax; then put a piece of tape around the tube where it comes out of the mouth, once it is in place. put a roll of 2-inch tape in the patient's mouth behind the canine teeth and then secure the roll in place by taping the mouth closed around the roll of tape. lubricate the tube with lubricating jelly and slowly insert the tube through the center of the roll of tape into the stomach. the passing of the tube does not rule out volvulus. in some cases, the front legs of the patient need to be elevated, and the caudal aspect of the patient lowered (front legs standing on a table with back legs on the ground) to allow gravity to pull the stomach down to allow the tube to pass. once the tube has been passed, air within the stomach is relieved, and the stomach can be lavaged. the presence of gastric mucosa or blood in the efflux from the tube makes the prognosis more guarded. if an orogastric tube cannot be passed, clip and aseptically scrub the patient's lateral abdomen and then insert 16-gauge over-the-needle catheter. "pinging" the animal's side with simultaneous auscultation allows determination of the location that is most tympanic-that is, the proper location for catheter insertion. once intravenous fluids have been started in the animal, take a right lateral abdominal radiograph to document gdv. if no volvulus is present, the owner may elect for more conservative care, and the animal should be monitored in the hospital for a minimum of 24 hours. because some cases of gdv intermittently twist and untwist, the owner should be cautioned that although the stomach is not twisted at that moment, a volvulus can occur at any time. if radiographs demonstrate food bloat, induce emesis (apomorphine, 0.04 mg/kg iv) or perform orogastric lavage under general anesthesia. documentation of gastric dilatation-volvulus constitutes a surgical emergency. 1 figure 1 -53: example of "food bloat" with severe gastric distention caused by overconsump-following diagnosis of gdv, continue administration of intravenous fluids. serum lactate measurements greater than 6.0 mmol/l are associated with an increased risk of gastric necrosis, requirement for partial gastrectomy, and increased mortality. administer fresh frozen plasma (20 ml/kg) to patients with thrombocytopenia or prolonged pt, activated partial thromboplastin time (aptt), or activated clotting time (act). cardiac dysrhythmias, particularly ventricular dysrhythmias, are common in cases of gdv and are thought to occur secondary to ischemia and proinflammatory cytokines released during volvulus and reperfusion. lidocaine (1-2 mg/kg followed by 50 mcg/kg/minute iv cri) can be used to treat cardiac dysrhythmias preemptively that are associated with ischemia-reperfusion injury, or administration can be started when ventricular dysrhythmias are present. correct any electrolyte abnormalities, including hypokalemia and hypomagnesemia. the use of nonsteroidal antiinflammatory drugs (flunixin meglumine, carprofen, ketoprofen) that can potentially decrease renal perfusion and predispose to gastric ulcers is absolutely contraindicated. administer analgesic drugs (fentanyl, 2 âµ/kg iv bolus, followed by 3-20 âµ/kg/hour iv cri; or hydromorphone, 0.1 mg/kg iv) before anesthetic induction. after carrying out a balanced anesthesia protocol, the patient should be taken immediately to surgery for gastric derotation and gastropexy. postoperatively, assess the patient's ecg, blood pressure, platelet count, coagulation parameters, and gastric function (see section on rule of twenty). if no resection is required, the animal can be given small amounts of water beginning 12 hours after surgery. depending on the severity of the patient's condition, small amounts of a bland diet can be offered 12 to 24 hours postoperatively. continute supportive care with analgesia and crystalloid fluids until the patient is able to tolerate oral analgesic drugs (tramadol, 1-3 mg/kg po q8-12h). once the patient is ambulatory and able to eat and drink on its own, it can be released from the hospital; instruct the owner to feed the animal multiple small meals throughout the day for the first week. when the intestines twist around the root of the mesentery, a small intestinal or mesenteric volvulus occurs. the problem is most common in the young german shepherd dog, although it has been observed in other large and giant breeds. predisposing factors include pancreatic atrophy, gastrointestinal disease, trauma, and splenectomy. clinical signs of mesenteric volvulus include vomiting, hemorrhagic diarrhea, bowel distention, acute onset of clinical signs of shock, abdominal pain, brick-red mucous membranes (septicemia), and sudden death. diagnosis is based on an index of suspicion and the presence of clinical signs in a predisposed breed. plain radiographs often reveal grossly distended loops of bowel in a palisade gas pattern. in some dogs, multiple, tear-drop-shaped, gas-filled loops appear to rise from a focal point in the abdomen. usually, massive distention of the entire small bowel is observed ( fig. 1-55) . the presence of pneumoperitoneum or lack of abdominal detail secondary to the presence of abdominal fluid is characteristic of bowel perforation and peritonitis. in a patient with mesenteric volvulus, immediate aggressive action is necessary for the animal to have any chance of survival. treatment consists of massive volumes of iv crystalloid and colloid fluids (see section on iv therapy), broad-spectrum antibiotics (ampicillin, 22 mg/kg iv qid, with enrofloxacin, 10 mg/kg iv once daily), and surgical correction of the bowel. because of the massive release of proinflammatory cytokines, bacterial translocation, and ischemia, treatment for shock is of paramount importance (see sections on rule of twenty and shock). prognosis for any patient with mesenteric volvulus is poor. obstipation (obstructive constipation) is most common in the older cat. in cases of simple constipation, rehydrating the animal with intravenous fluids and stool softeners is often volvulus. this consistutes an immediate surgical emergency, and the prognosis is often poor. this condition is most common in young german shepherd dogs, but can be observed in any breed. sufficient for it to regain the ability to have a bowel movement. obstipation, however, is caused by adynamic ileus of the large bowel that eventually leads to megacolon. affected cats usually are anorectic, lethargic, and extremely dehydrated. treatment consists of rehydration with intravenous crystalloid fluids, correction of electrolyte abnormalities, enemas, and promotility agents such as cisapride (0.5 mg/kg po q8-24h). the use of phosphate enemas in cats is absolutely contraindicated because of the risk of causing acute, fatal hyperphosphatemia. in many cases, the patient should be placed under general anesthesia and manual deobstipation is performed with warm water soapy enemas and a gloved finger to relieve and disimpact the rectum. stool softeners such as lactulose and docusate stool sofener (dss) may also be used. predisposing causes of obstipation such as narrowing of the pelvic canal, perineal hernia, and tumors should be ruled out. adenocarcinoma is the most common neoplasm of the gastrointestinal tract that causes partial to complete obstruction. adenocarcinomas tend to be annular and constricting, and they may cause progressive obstruction of the lumen of the small or large bowel. siamese cats tend to have adenocarcinomas in the small intestine, whereas in dogs, the tumor tends to occur in the large intestine. clinical signs of adenocarcinoma are both acute and chronic and consist of anorexia, weight loss, and progressive vomiting that occur over weeks to months. effusion may be present if metastasis to peritoneal surfaces has occurred. diagnosis is based on clinical signs and physical examination findings of a palpable abdominal mass, radiographic evidence of an abdominal mass and small or large intestinal obstruction, or ultrasonographic evidence of an intestinal mass. treatment consists of surgical resection of the affected bowel segment. the prognosis for long-term survival (10-12 months) is good if the mass is completely resected and if other clinical signs of cachexia or metastasis are observed at the time of diagnosis. median survival is 15 to 30 weeks if metastasis to lymph nodes, liver, or the peritoneum are absent at the time of diagnosis. in dogs, the prognosis is more guarded. leiomyoma and leiomyosarcoma are tumors that can cause partial or complete obstruction of the bowel. clinical signs are often referred to progressive anemia, including weakness, lethargy, inappetence, and melena. hypoglycemia can be observed as a paraneoplastic syndrome, or due to sepsis and peritonitis secondary to bowel perforation. leiomyomas are most commonly observed at the ceco-colic junction or in the cecum. surgical resection and anastomosis is usually curative, and has a favorable prognosis. incarceration of a loop of bowel into congenital or acquired defects in the body wall can cause small bowel obstruction. pregnant females and young animals with congenital hernias are most at risk. rarely, older animals with perineal hernias and animals of any age with traumatic hernias can be affected. clinical signs are consistent with a small intestinal obstruction: anorexia, vomiting, lethargy, abdominal pain, and weakness. diagnosis is often made based on physical examination of a reducible or nonreducible mass in the body wall. hernias whose contents are reducible are usually asymptomatic. treatment consists of supportive care and rehydration, administration of broad-spectrum antibiotics, and surgical correction of the body wall hernia. in some cases, intestinal resection and anastomosis of the affected area is necessary when bowel ischemia occurs. the potential for bowel perforation should be suspected whenever there is any penetrating injury (knife, gunshot wound, bite wound, stick impalement) of the abdomen. injuries that result in bowel ischemia and rupture can also occur secondary to nonpenetrating blunt 170 1 emergency care trauma or shear forces (e.g., big dog-little dog/cat). perforation of the stomach and small and large intestines can occur with use of nonsteroidal antiinflammatory drugs. diagnosis of bowel perforation first depends on the alertness to the possibility that the bowel may have been perforated or penetrated. as a general rule, all penetrating injuries of the abdomen should be investigated by exploratory laparotomy. diagnostic peritoneal lavage (dpl) can be performed; however, early after penetrating injury of the bowel, dpl may be negative or nondiagnostic until peritonitis develops. whenever any patient with blunt or penetrating abdominal trauma does not respond to initial fluid therapy, or responds and then deteriorates, the index of suspicion for bowel injury should be raised. the findings of pneumoperitoneum on abdominal radiographs or of intracellular bacteria, extracellular bacteria, bile pigment, bowel contents, and cloudy appearance of fluid obtained by abdominocentesis or diagnostic peritoneal lavage fluid (see sections on abdominocentesis and diagnostic peritoneal lavage) warrant immediate surgical exploration. treatment largely consists of stabilizing the patient's cardiovascular and electrolyte status with intravenous fluids, administration of broad-spectrum antibiotics, and definitive surgical exploration and repair of injured structures. prolapse of the rectum is observed most frequently secondary to parasitism and gastrointestinal viral infections in young puppies and kittens with chronic diarrhea. older animals with rectal prolapse often have an underlying problem such as a tumor or mucosal lesion that causes straining and dyschezia. the diagnosis of a rectal prolapse is made based on physical examination findings. the diagnosis of rectal prolapse is sometimes difficult to distinguish from small intestinal intussusception. in rare cases, the intussusception can invaginate through the large bowel, rectum, and anus. the two entities are distinguished from one another by inserting a lubricated thermometer or blunt probe into the cul-de-sac formed by the junction of the prolapsed mucosa and mucocutaneous junction at the anal ring. inability to insert the probe or thermometer indicates that the rectal mucosa is prolapsed. passage of the probe signifies that the prolapsed segment is actually the intussusceptum. treatment can be performed easily if the prolapse is acute and the rectal mucosa is not too irritated or edematous. the presence of severely necrotic tissue warrants surgical intervention. to reduce an acute rectal prolapse, after placing the patient under general anesthesia, lubricate the prolapsed tissue and gently push it back into the rectum, using a lubricated syringe or syringe casing. apply a loose purse-string suture, leaving it in place for a minimum of 48 hours. de-worm the patient and administer stool softeners. if a rectal prolapse cannot be reduced, or if the tissue is nonviable, surgical intervention is warranted. in patients in which viable tissue does not stay reduced with a purse-string suture, a colopexy can be performed during a laparotomy. first, place tension on the colon to reduce the prolapse, and then suture the colon to the peritoneum of the lateral abdominal wall with two to three rows of 2-0 or 3-0 monofilament suture material. if the prolapsed tissue is nonviable, it must be amputated. place four stay sutures at 90-degree intervals through the wall of the prolapse at the mucocutaneous junction. resect the prolapse distal to the stay sutures and then reestablish the rectal continuity by suturing the seromuscular layers together in one circumferential line and the mucosal layers together in the other. replace the suture incision into the anal canal. following surgery, de-worm the patient and administer a stool softener and analgesic drugs. avoid using thermometers or other probes in the immediate postoperative period because they may disrupt suture lines. acute gastritis may be associated with a variety of clinical conditions, including oral hemorrhage, ingestion of highly fermentable nondigestable foods or garbage, toxins, foreign bodies, renal or hepatic failure, inflammatory bowel disease, and bacterial and viral infections. diarrhea often accompanies or follows acute gastritis. hemorrhagic gastroenteritis often occurs as a shock-like syndrome with a rapidly rising hematocrit level. clinical signs of gastritis include depression, lethargy, anterior abdominal pain, excessive water consumption, vomiting, and dehydration. differential diagnosis of acute gastritis includes pancreatitis, hepatic or renal failure, gastrointestinal obstruction, and toxicities (box 1-44). the diagnosis is often a diagnosis of exclusion of other causes (see preceding text). a careful and thorough examination of the vomitus may be helpful in arriving at a diagnosis. a complete blood count, serum biochemistry profile including amylase and lipase, parvovirus test (in young puppies), fecal flotation and cytology, abdominal radiographs (plain and/or contrast studies), and abdominal ultrasound may be warranted to rule out other causes of acute vomiting. while diagnostic tests are being performed, treatment consists of withholding all food and water for a minimum of 24 hours. after calculating the patient's degree of dehydration, administer a balanced crystalloid fluid to normalize acid-base and electrolyte status. control vomiting with antiemetics such as metoclopramide, prochlorperazine, chlorpromazine, dolasetron, and ondansetron (table 1-37). if vomiting is accompanied by diarrhea, administer broad-spectrum antibiotics (cefazolin, 22 mg/kg iv q8h, with metronidazole, 10 mg/kg iv q8h; or ampicillin, 22 mg/kg iv q6h, with enrofloxacin, 10 mg/kg iv q24h) to decrease the risk of bacterial translocation and bacteremia/septicemia. although antacids (famotidine, ranitidine, cimetidine) do not have a direct antiemetic effect, their use can decrease gastric acidity and esophageal irritation during vomiting. if gastritis is secondary to uremia or nonsteroidal antiinflammatory drug use, administer gastroprotectant and antiemetic drugs (ranitidine, 1 mg/kg po q12h; sucralfate, 0.25-1 g/dog po q8h; or omeprazole (0.5-1 mg/kg po q24h) to decrease acid secretion and coat areas of gastric ulceration (table 1 -37) . once food and water can be tolerated, the patient can be placed on an oral diet and medications, and intravenous fluids can be discontinued. do not use until a gastrointestinal obstruction has been ruled out. hemorrhagic gastroenteritis (hge) is an acute onset of severe hemorrhagic vomiting and diarrhea most commonly observed in young small-breed dogs (e.g., poodles, miniature dachshunds, miniature schnauzers) 2 to 4 years of age. clinical signs develop rapidly and include vomiting and fetid diarrhea with hemorrhage, often strawberry jam-like in appearance. the hematocrit can rise from 55% to 75%. often, the animal is extremely hypovolemic but has no apparent signs of abdominal pain. there is no known cause of hge, although clostridium perfringens, escherichia coli, campylobacter, and viral infections have been suggested but not consistently confirmed. other differential diagnoses of of hematemesis and hemorrhagic diarrhea include coronavirus, parvovirus, vascular stasis, sepsis, hepatic cirrhosis with portal hypertension, and other causes of severe shock. immediate treatment consists of placement of a large-bore intravenous catheter and replenishment of intravascular fluid volume with crystalloid fluids (up to 90 ml/kg/hour), while carefully monitoring the patient's hematocrit and total protein. administer broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h, and enrofloxacin 10 mg/kg iv q24h) because of the high risk of bacterial translocation and sepsis. control vomiting with antiemetic drugs. monitor the patient's platelet count and coagulation tests for impending disseminated intravascular coagulation (dic), and administer fresh frozen plasma and heparin, as needed (see section on disseminated intravascular coagulation). when vomiting has ceased for 24 hours, offer the animal small amounts of water, and then a bland diet (e.g., boiled chicken and rice or boiled ground beef and rice mixed with low-fat cottage cheese). pancreatitis occurs most frequently in dogs but can occur in cats as well. in dogs, the onset of pancreatitis is sometimes preceded by ingestion of a fatty meal or the administration of drugs (e.g., potassium bromide or glucocorticoids). glucocorticoids can increase the viscosity of pancreatic secretions and induce ductal proliferation, resulting in narrowing and obstruction of the lumen of the pancreatic duct. pancreatitis can also occur following blunt or penetrating abdominal trauma, high duodenal obstruction causing outflow obstruction of the pancreatic papilla, pancreatic ischemia, duodenal reflux, biliary disease, and hyperadrenocorticism. in cats, acute necrotizing pancreatitis is associated with anorexia, lethargy, hyperglycemia, icterus, and sometimes acute death. chronic pancreatitis is more common in cats and results in intermittent vomiting, anorexia, weight loss, and lethargy. predisposing causes of chronic pancreatitis in cats include pancreatic flukes, viral infection, hepatic lipidosis, drugs, organophosphate toxicity, and toxoplasmosis. clinical signs of acute pancreatitis include sudden severe vomiting, abdominal pain, and lethargy. depending on the severity of pancreatic inflammation, depression, hypotension, and systemic inflammatory response syndrome (sirs) may be present. subacute cases may have minimal clinical signs. severe pancreatic edema can result in vascular changes and ischemia that perpetuates severe inflammation. hypovolemic shock and dic can also decrease pancreatic perfusion. severe pancreatic edema, autolysis, and ischemia lead to pancreatic necrosis. duodenal irritation is manifested as both vomiting and diarrhea. pain may be localized to the right upper abdominal quadrant or may be generalized if peripancreatic saponification occurs. differential diagnosis of pancreatitis is the same as for any other cause of vomiting. complications that occur in patients with severe pancreatitis include dehydration, acidbase and electrolyte abnormalities, hyperlipemia, hypotension, and localized peritonitis. hepatic necrosis, lipidosis, congestion, and abnormal architecture can develop. inflammatory mediators (bradykinin, phospholipase a, elastase, myocardial depressant factor, and bacterial endotoxins) stimulate the inflammatory cascade and can lead to sirs, with severe hypotension, clotting system activation, and dic. electrolyte imbalances and hypovolemia secondary to vomiting all can lead to multiple organ dysfunction syndrome (mods), and ultimately, death. if a patient survives an episode of acute pancreatitis, long-term sequelae can include diabetes mellitus. monitor patients with recurrent pancreatitis for clinical signs of polyuria, polydipsia, polyphagia, hyperglycemia, and glucosuria. the diagnosis of pancreatitis is based on the presence of clinical signs (which may be absent in cats), laboratory findings, and ultrasonographic evidence of pancreatic edema and increased peripancreatic echogenicity. serum biochemistry analyses can sometimes support a diagnosis of pancreatitis; however, serum amylase and lipase are often unreliable indicators of pancreatitis, depending on the chronicity of the process in the individual patient. both serum amylase and lipase are excreted in the urine. impaired renal clearance/ function can cause artifactual elevations of serum amylase and lipase in the absence of pancreatic inflammation. furthermore, serum lipase levels can be elevated as a result of gastrointestinal obstruction (e.g., foreign body). early in the course of the disease, levels can be two to six times normal, but they may decrease to within normal ranges at the time of presentation to the veterinarian. the transient nature of amylase elevation makes this test difficult to interpret, and it is not highly sensitive if a normal value is found. lipase levels also increase later in the course of the disease. amylase and lipase should be tested concurrently with the rest of the biochemistry profile. other changes often observed are elevations in bun and creatinine levels secondary to dehydration and prerenal azotemia, hyperglycemia, and hyperlipemia. hypocalcemia can occur secondary to peripancreatic fat saponification, and its presence warrants a more negative prognosis. a more specific measure is pancreatic lipase immunoreactivity, which becomes elevated in dogs and cats with pancreatitis. this test, combined with ultrasonographic or computed tomography evidence of pancreatitis, is the most sensitive and specific test available for making an accurate diagnosis. however, because the results of this test take time to obtain, animals must be treated in the meantime. abdominal effusion or fluid from diagnostic peritoneal lavage can be compared with serum amylase and lipase activity. abdominal lipase and amylase concentrations in the fluid greater than that in the peripheral blood are characteristic of chemical peritonitis associated with pancreatitis. wbc counts greater than 1000 cells/mm 3 , the presence of bacteria, toxic neutrophils, glucose levels less than 50 mg/dl, or lactate levels greater than that of serum are characteristic of septic peritonitis, and immediate exploratory laparotomy is warranted. if a biopsy sample obtained during laparotomy does not demonstrate inflammation, but this does not rule out pancreatitis, because disease can be focal in nature and yet cause severe clinical signs. abdominal radiographs may sometimes reveal a loss of abdominal detail or a ground glass appearance in the right upper quadrant. pancreatic edema and duodenal irritation can displace the gastric axis toward the left, toward the left with dorsomedial displacement of the proximal duodenum (the so-called "backwards 7" or "shepherd's crook" sign). ultrasonography and ct are more sensitive in making a diagnosis of pancreatitis. treatment of pancreatitis is largely supportive in nature and is designed to correct hypovolemia and electrolyte imbalances, prevent or reverse shock, maintain vital organ perfusion, alleviate discomfort and pain, and prevent vomiting (see section on rule of twenty). when treating pancreatitis in dogs, all food and water should be restricted. however, food should not be withheld from cats with chronic pancreatitis. give fresh frozen plasma to replenish alpha-2-macroglobulins. administer antiemetics such as chlorpromazine (use with caution in a hypovolemic or hypotensive patient), dolasetron, ondansetron, or metoclopramide to prevent or control vomiting. analgesic drugs can be provided in the form of constant rate infusion (fentanyl, 3-7 âµ/kg/hour iv cri, and lidocaine, 30-50 âµ/kg/minute iv cri), intrapleural injection (lidocaine, 1-2 mg/kg q8h), or intermittent parenteral injections (morphine, 0.25-1 mg/kg sq, im; hydromorphone, 0.1 mg/kg im or sq). because the pancreas must be rested, consider using parenteral nutrition. acute hepatic failure may be associated with toxins, adverse reaction to prescription medication, and bacterial or viral infections. the most frequent clinical signs observed in a patient with acute hepatic failure are anorexia, lethargy, vomiting, icterus, bleeding, and cns depression or seizures (associated with hepatic encephalopathy). differential diagnosis and causes of acute hepatic failure are listed in box 1-45. diagnosis of acute hepatic failure is based on clinical signs and biochemical evidence of hepatocellular (ast, alt) and cholestatic (alk phos, t bili, ggt) enzyme elevations. ultrasonography may be helpful in distinguishing the architecture of the liver, but unless a mass or abscess is present, cannot provide a specific diagnosis of the cause of the hepatic damage. management of the patient with acute hepatic failure includes correction of dehydration and acid-base and electrolyte abnormalities, as shown in the following list: â�¢ hypoalbuminemia: plasma or concentrated albumin. plasma also is an excellent source of clotting factors that can become depleted. â�¢ clotting abnormalities: vitamin k 1 (2.5 mg/kg sq or po q8-12h) to â�¢ severe anemia: fresh or stored blood â�¢ gastric hemorrhage: gastroprotectant drugs (omeprazole, ranitidine, famotidine, cimetidine, sucralfate) â�¢ hypoglycemia: dextrose supplementation (2.5%-5%) â�¢ hepatic failure, particularly when hypoglycemia is present: broad-spectrum antibiotics (ampicillin 22 mg/kg iv q6h; with enrofloxacin, 5 mg/kg iv q24h) â�¢ hepatic encephalopathy: lactulose or betadine enemas â�¢ cerebral edema: mannitol (0.5-1.0 g/kg iv over 10 to 15 minutes) followed by furosemide (1 mg/kg iv 20 minutes later). deterioration of clinical signs may signify the development of cerebral edema. applewhite aa, cornell kk, selcer ba: diagnosis and treatment of intussusception in dogs. comp cont educ pract vet 24 (2) often, systemic hypertension is diagnosed when the animal is seen by the veterinarian because of some other clinical sign, such as acute blindness, retinal detachment, hyphema, epistaxis, and cns signs following intracranial hemorrhage. diagnosis of systemic hypertension is often difficult in the absence of clinical signs and without performing invasive or noninvasive blood pressure monitoring. normal blood pressure (bp) measurements in dogs and cats are listed in table 1-38. hypertension is defined as a consistent elevation in systolic bp >200 mm hg, consistent diastolic bp >110 mm hg, and consistent mean arterial blood pressure >130 mm hg. the effects of systemic hypertension include left ventricular hypertrophy, cerebrovascular accident, renal vascular injury, optic nerve edema, hyphema, retinal vascular tortuosity, retinal hemorrhage, retinal detachment, vomiting, neurologic defects, coma, and excessive bleeding from cut surfaces. 176 1 emergency care dog 100-160 80-120 90-120 cat 120-150 70-130 100-150 patients with systemic hypertension should have a thorough diagnostic work-up to determine the underlying cause. although uncommon, hypertensive emergencies can occur with pheochromocytoma, acute renal failure, and acute glomerulonephritis. sodium nitroprusside (1-10 âµ/kg/minute iv cri) or diltiazem (0.3-0.5 mg/kg iv given slowly over 10 minutes, followed by 15 âµ/kg/minute) can be used to treat systemic hypertension. with the use of sodium nitroprusside or diltiazem, monitor carefully for hypotension. diagnosis is based on consistent elevations in systolic, diastolic, and/or mean arterial bp. because many of the clinical signs associated with systemic hypertension involve hemorrhage into some closed cavity, other causes of hemorrhage, such as vasculitis, thrombocytopenia, thrombocytopathia, and hepatic or renal failure, should be investigated (see section on coagulation disorders). diagnostic testing is based on clinical signs and index of suspicion for an underlying disease and may include a complete blood count; urinalysis; urine protein:creatinine ratio; acth stimulation test; thoracic and abdominal radiographs; thoracic and abdominal ultrasound; tick serology; brain ct or mri; and assays of serum electrolytes, aldosterone concentration, t4, endogenous tsh, plasma catecholamine, and growth hormone. management of systemic hypertension involves treatment of the primary underlying disorder, whenever possible. long-term adjunctive management includes sodium restriction in the form of cooked or prescription diets to decrease fluid retention. obese animals should be placed on dietary restrictions and undergo a weight reduction program. thiazide and loop diuretics may be used to decrease sodium retention and circulating blood volume. alpha-and beta-adrenergic blockers may be used, but they are largely ineffective as monotherapeutic agents for treating hypertension. calcium channel blockers and angiotensin-converting enzyme (ace) inhibitors are the mainstay of therapy in the treatment of hypertension in dogs and cats ( diabetic ketoacidosis (dka) is a potentially fatal and terminal consequence of unregulated insulin deficiency and possible glucagon excess. in the absence of insulin, unregulated lipolysis results in the beta-hydroxylation of fatty acids by abnormal hepatic metabolism. as a result, ketoacids-namely, acetoacetic acid, beta-hydroxybutyric acid, and acetoneare produced. early in the course of the disease, patients exhibit clinical signs associated with diabetes mellitus: weight loss, polyuria, polyphagia, and polydipsia. later, as ketoacids stimulate the chemoreceptor trigger zone, vomiting and dehydration occur, with resulting hypovolemia, hypotension, severe depression, abdominal pain, oliguria, and coma. at the time of presentation, often a strong odor of ketones (acetone) is present on the patient's breath. physical examination often reveals dehydration, severe depression or coma, and hypovolemic shock. in extreme cases, the patient exhibits a slow, deep kussmaul respiratory pattern in an attempt to blow off excess co 2 to compensate for the metabolic acidosis. a serum biochemistry profile and complete blood count often reveal prerenal azotemia, severe hyperglycemia (blood glucose >400 mg/dl), hyperosmolarity (>330 mosm/kg), lipemia, hypernatremia (sodium >145 meq/l), elevated hepatocellular and cholestatic enzyme activities, high anion gap, and metabolic acidosis. although a whole body potassium deficit is usually present, the serum potassium may appear artifactually elevated in response to metabolic acidosis. with severe metabolic acidosis, potassium moves extracellularly in exchange for a hydrogen ion. phosphorus too moves intracellularly in response to acidosis, and serum phosphorus is usually decreased. hypophosphatemia >2 mg/dl can result in intravascular hemolysis. urinalysis often reveals 4+ glucosuria, ketonuria, and a specific gravity of 1.030 or greater. the urine of all diabetic animals should be cultured to rule out a urinary tract infection or pyelonephritis. treatment of a patient with dka presents a therapeutic challenge. treatment is aimed at providing adequate insulin to normalize cellular glucose metabolism, correcting acidbase and electrolyte imbalances, rehydration and restoration of perfusion, correcting acidosis, providing carbohydrate sources for utilization during insulin administration, and identifying any precipitating cause of the dka. obtain blood samples for a complete blood count, and serum biochemistry electrolyte profiles. whenever possible, insert a central venous catheter for fluid infusion and procurement of repeat blood samples. calculate the patient's dehydration deficit and maintenance fluid requirements and give appropriate fluid and electrolytes over a period of 24 hours. it is advisable to rehydrate patients with severe hyperosmolarity for a minimum of 6 hours before starting insulin administration. use a balanced electrolyte solution (e.g., plasmalyte-m, normosol-r, lactated ringer's solution) or 0.9% saline solution for maintenance and rehydration. balanced electrolyte solutions contain small amounts of potassium and bicarbonate precursors that aid in the treatment of metabolic acidosis. treat animals with severe metabolic acidosis with an hco 3 â�� >11 meq/l or a ph <7.1 with supplemental bicarbonate (0.25-0.5 meq/kg). add supplemental dextrose to the patient's fluids as a carbohydrate source during insulin infusion. both insulin and carbohydrates are necessary for the proper metabolism of ketone bodies in patients with dka. the rate and type of fluid and amount of dextrose supplementation will change according to the patient's blood glucose concentration. serum potassium will drop rapidly as the metabolic acidosis is corrected with fluid and insulin administration. measure serum potassium every 8 hours, if possible, and supplement accordingly (see section on fluid therapy for chart of potassium supplementation). if the patient's potassium requirement exceeds 100 meq/l, or if the rate of potassium infusion approaches 0.5 meq/ kg/hour in the face of continued hypokalemia, magnesium should be supplemented. magnesium is required as a cofactor for many enzymatic processes and for normal function of the na,k-atpase pump. hypomagnesemia is a common electrolyte disturbance in many forms of critical illness. replenishing magnesium (mgcl 2 , 0.75 meq/kg/day iv cri) often helps to correct the refractory hypokalemia observed in patients with dka. patients with hypophosphatemia that approaches 2.0 mmol/l should receive potassium phosphate (0.01-0.03 mmol/kg/hour iv cri). when providing potassium phosphate supplementation, be aware of the additional potassium added to the patient's fluids, so as to not exceed recommended rates of potassium infusion. to determine the amount of potassium chloride (kcl) to add along with potassium phosphate (kpo 4 ), use the following formula: meq k + derived from kcl = total meq of k + to be administered over 24 hours â�� meq in which k + is derived from kpo 4 clinical signs of severe hypophosphatemia include muscle weakness, rhabdomyolysis, intravascular hemolysis, and decreased cerebral function that can lead to depression, stupor, seizures, or coma. regular insulin can be administered either im or as a constant rate infusion in the treatment of patients with dka. subcutaneous insulin should not be administered. because of the severe dehydration present in most patients with dka, subcutaneous insulin is poorly absorbed and is not effective until hydration has been restored. in the low-dose intravenous method, place regular insulin (1.1 units/kg for a cat, and 2.2 units/kg for a dog) in 250 ml of 0.9% saline solution. run 50 ml of this mixture through the intravenous line to allow the insulin to adsorb to the plastic tubing. administer the patient's insulin fluid rate according to blood glucose levels ( table 1 -40) . adjust the patient's total fluid volume according to changes in the insulin fluid rate as necessary. in many cases, multiple bags of fluids are necessary because they must be changed when fluctuations in blood glucose concentrations occur in response to therapy. infusion of the insulin mixture should be in a separate intravenous catheter. to replenish hydration, use a second intravenous line for the more rapid infusion of non-insulin-containing fluids. to administer the regular insulin im, first give 0.22 unit/kg im and then re-check the patient's blood glucose every hour. additional injections of regular insulin (0.11 unit/kg other fluid type (ml/hour) >250 10 0.9% nacl 200-250 7 0.45% nacl + 2.5% dextrose 150-200 5 0.45% nacl + 2.5% dextrose 100-150 5 0.45% nacl + 2.5% dextrose <100 0 0.45% nacl + 5% dextrose im) should be administered based on the patient's response to subsequent injections. once the patient's blood glucose falls to 200 to 250 mg/dl, add 2.5% to 5% dextrose to the fluids to maintain the blood glucose concentration at 200 to 300 mg/dl. continue intramuscular injection of regular insulin (0.1-0.4 unit/kg q4-6h) until the patient is rehydrated, no longer vomiting, and able to tolerate oral fluids and food without vomiting. even in patients with intramuscular regular insulin therapy, a central venous catheter should be placed for frequent blood sample collection. as the patient begins to respond to therapy, monitor electrolytes, glucose, and acid-base status carefully. hypokalemia, hypophosphatemia, and hypomagnesemia can occur. when the patient's hydration and acid-base status has normalized and the patient is able to tolerate oral food and water, a longer-acting insulin can be administered as for treatment of a patient with uncomplicated diabetes. extreme hyperosmolarity can result in a coma, if uncorrected. in patients with diabetes mellitus, hyperglycemia and hypernatremia secondary to osmotic diuresis and free water loss can lead to severe hyperosmolarity. in dogs, normal serum osmolality is <300 mosm/l of serum. hyperosmolarity is expected when serum osmolality is >340 mosm/l. if equipment for determining serum osmolarity is not available, osmolarity can be calculated by the following formula: osm/l = 2(na + k) + (glucose/18) + (bun/2.8) patients with severe dehydration, hyperglycemia, hypernatremia, and azotemia may experience cerebral edema without ketonemia. treatment is directed solely at rehydrating the patient and slowly reducing blood glucose levels using a hypotonic solution such as 0.45% nacl + 2.5% dextrose or 5% dextrose in water (d 5 w). after the initial rehydration period, administer potassium supplementation conservatively. red blood cells and the brain absolutely depend on the oxidation of glucose for energy. hypoglycemia can be caused by various systemic abnormalities that can be related to intestinal malabsorption of nutrients, impaired hepatic glycogenolysis or gluconeogenesis, and inadequate peripheral utilization of glucose. clinical signs of hypoglycemia are extremely variable and can include weakness, tremors, nervousness, polyphagia, ataxia, tachycardia, muscle twitching, incoordination, visual disturbances, and generalized seizures. clinical signs typically occur when serum glucose levels are <60 mg/dl. the combination of the clinical signs listed previously, documentation of low serum glucose, and alleviation of clinical signs upon glucose administration is known as whipple's triad. whenever a patient presents with hypoglycemia, consider the following important factors: the age of onset, the nature of the hypoglycemic episode (transient, persisent, or recurrent) , and the pattern based on the patient's history . treatment of hypoglycemia is directed at providing glucose supplementation and determining any underlying cause. administer supplemental dextrose (25%-50% dextrose, 2-5 ml/kg iv; or 10% dextrose, 20 ml/kg po) as quickly as possible. do not attempt oral glucose supplementation in any patient having a seizure or if the airway cannot be protected. administer intravenous fluids (e.g., normosol-r, lactated ringer's solution, 0.9% saline solution) with 2.5%-5% supplemental dextrose until the patient is eating and able to maintain euglycemia without supplementation. in some cases (e.g., insulinoma), eating or administration of supplemental dextrose can promote insulin secretion and exacerbate clinical signs and hypoglycemia. in cases of refractory hypoglycemia secondary to iatrogenic insulin overdose, glucagon (50 mg/kg iv bolus, then 10-40 ng/kg/minute iv cri) can also be administered along with supplemental dextrose. to make a glucagon infusion of 1000 ng/ml, reconstitute 1 ml (1 mg/ml) of glucagon according to the manufacturer's instructions and add this amount to 1000 ml of 0.9% saline solution. 1 emergency care the diagnosis of eclampsia (puerperal tetany) is often made on the basis of history and clinical signs. clinical signs can become evident when total calcium decreases to <8.0 mg/dl in dogs and <7.0 mg/dl in cats. the disease is often observed in small, excitable dogs, and stress may play a complicating role in the etiology. in most bitches, the disease manifests itself 1 to 3 weeks after parturition. in some cases, however, clinical signs can develop before parturition occurs. hypophosphatemia may accompany hypocalcemia. clinical signs of hypocalcemia include muscle tremors or fasciculations, panting, restlessness, aggression, hypersensitivity, disorientation, muscle cramping, hyperthermia, stiff gait, seizures, tachycardia, a prolonged qt interval on ecg, polydipsia, polyuria, and respiratory arrest. treatment of eclampsia consists of slow, cautious calcium supplementation (10% calcium gluconate, 0.15 mg/kg iv over 30 minutes). severe refractory tetanus can be controlled with intravenous diazepam. supportive care includes intravenous fluid administration and cooling (see section on hyperthermia and heat-induced illness). instruct the owner to give the patient oral calcium supplements (e.g., 1 to 2 tablets of tums bid-tid) after discharge from the hospital. also instruct the owner about how to wean the puppies, allowing the bitch to dry up, in order to prevent recurrence. recurrence with subsequent pregnancies is common, particularly in patients that receive calcium supplementation during gestation (table 1-41) . hypercalcemia can occur from a variety of causes. the gosh darn it mnemonic can be used to remember the various causes of hypercalcemia in small animal patients (box 1-47) . the gastrointestinal, renal, and nervous systems are most commonly affected, particularly when serum total calcium rises above 16.0 mg/dl. clinical signs of severe hypercalcemia include muscle weakness, vomiting, seizures, and coma. ecg abnormalities include prolonged pr interval, rapid qt interval, and ventricular fibrillation. the most serious clinical signs are often seen when hypercalcemia is observed in combination with hyperphosphatemia or hypokalemia. pay special attention to the "calcium ã� phosphorus product." if this product exceeds 70, dystrophic calcification can occur, leading to renal failure. renal complications include polyuria, polydipsia, dehydration, and loss of renal tubular concentrating ability. renal blood flow and the glomerular filtration rate (gfr) are impaired when serum total calcium exceeds 20 mg/dl. the extent, location, and number of renal tubular injuries are the main factors in determining whether renal damage secondary to hypercalcemia is reversible or irreversible. emergency therapy of hypercalcemia is warranted when severe renal compromise, cardiac dysfunction, or neurologic abnormalities are present, or if no clinical signs occur but the calcium ã� phosphorus product exceeds 70. the treatment of choice is correction of the underlying cause of hypercalcemia, whenever possible. in some cases, the results of diagnostic tests take time, and emergency therapy should be initiated immediately, before a definitive cause of the hypercalcemia is found. emergency management of hypercalcemia consists of reduction of serum calcium levels. administer intravenous fluids (0.9% saline solution) to expand extracellular fluid volume and promote calciuresis. to promote diuresis, initial intravenous fluid rates should approach two to three times maintenance levels (120-180 ml/kg/day). potassium supplementation may be required to prevent iatrogenic hypokalemia. administration of a loop diuretic such as furosemide (2-5 mg/kg iv) will promote calcium excretion. calcitonin (4 iu/kg im q12h for cats and 8 iu/kg im q24h for dogs) can be administered to decrease serum calcium levels. in severe refractory hypercalcemia secondary to cholecalciferol toxicity, more aggressive calcitonin therapy (4-7 iu/kg sq q6-8h) can be attempted. side effects of calcitonin treatment include vomiting and diarrhea. alternatively, bisphosphonates (pamidronate, 1.02-2.0 mg/kg iv) are useful in rapidly reducing serum calcium concentrations. glucocorticosteroids reduce calcium release from the bone, decrease intestinal absorption of calcium, and promote renal calcium excretion. administer glucocorticosteroids only after the underlying cause of hypercalcemia has been determined and appropriate therapy started. because many forms of neoplasia can result in hypercalcemia as a paraneoplastic syndrome, empiric use of glucocorticosteroids can induce multiple drug resistance, making the tumor refractory to the effects of chemotherapeutic agents. hypoadrenocorticism is most commonly observed in young to middle-aged female dogs, but it can occur in animals of any age, gender, and breed. clinical signs, which are referable to deficiency in glucocorticoid (cortisol) and mineralocorticoid (aldosterone) hormones, may develop slowly over time, leading to a waxing and waning course; acute clinical signs occur when >90% of the adrenal functional reserve has been destroyed. in such cases, complete adrenocortical collapse can result in an addisonian crisis. lack of aldosterone causes a lack of renal sodium and water retention, and impaired potassium excretion. the most significant clinical signs associated with hypoadrenocorticism are depression, lethargy, weakness, anorexia, shaking, shivering, vomiting, diarrhea, weight loss, abdominal pain, weakness, hypotension, dehydration, and inappropriate bradycardia (box 1-48) . the diagnosis of hypoadrenocorticism is made based on the patient's clinical signs in combination with electrolyte abnormalities that include hyperkalemia, hyponatremia, and hypochloremia. serum sodium concentration (115-130 meq/l) is often greatly reduced, and serum potassium is elevated (>6.0 meq/l). a sodium:potassium ratio of <27 is characteristic of hypoadrenocorticism, although not exactly pathognomonic. electrocardiographic changes associated with hyperkalemia include inappropriate bradycardia, absence of p waves, elevated spiked t waves, and widened qrs complexes. other more variable bloodwork abnormalities include a lack of a stress leukogram, eosinophilia, hypoglycemia, hyperphosphatemia, hypercalcemia, azotemia, and hypocholesterolemia. a definitive diagnosis of hypoadrenocorticism is based on an adrenocorticotropic hormone (acth) stimulation test. in patients with hypoadrenocorticism, baseline cortisol levels are usually low, with a lack of appropriate cortisol release after administration of acth analogue. rarely, animals with "atypical" hypoadrenocorticism lose glucocorticoid secreting ability from the zona fasciculata, but retain mineralocorticoid secretory ability from the zona glomerulosa. atypical addisonian patients have normal serum electrolytes but still have clinical signs of vomiting, diarrhea, weakness, lethargy, inappetence, muscle wasting, and weight loss. the diagnosis is more difficult in such cases because of the presence of normal electrolytes. an acth stimulation test should be considered, particularly in predisposed breeds. treatment of hypoadrenocorticism includes placement of a large-bore intravenous catheter, infusion of intravenous crystalloid fluids (0.9% saline solution), and replenishment of glucocorticoid and mineralocorticoid hormones. administer dexamethasone or dexamethasone-sodium phosphate (0.5-1.0 mg/kg iv). dexamethasone will not interfere with the acth stimulation test, unlike other longer-acting steroids (e.g., prednisolone, methylprednisolone sodium succinate, triamcinolone). depending on the severity of the patient's condition, consider monitoring using the rule of twenty. administer antiemetics and gastroprotectant drugs to treat nausea, vomiting, and hematemesis. give the patient broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h) if hematochezia or hemorrhagic diarrhea is present. if severe gastrointestinal blood loss occurs, whole blood, packed red blood cells, or fresh frozen plasma may be required. control hypoglycemia with 2.5%-5.0% dextrose. use sodium bicarbonate, regular insulin with dextrose, or calcium gluconate to correct severe hyperkalemia with atrial standstill (see section on atrial standstill). chronic therapy for hypoadrenocorticism consists of mineralocorticoid and glucocorticosteroids supplementation for the rest of the animal's life. mineralocorticoid supplementation can be in the form of desoxycorticosterone pivalate (docp) (2.2 mg/kg im) or fludrocortisone acetate (0.1 mg/2.5-5 kg body weight daily). fludrocortisone acetate possesses both mineralocorticoid and glucocorticoid activities and can be used as the sole daily treatment of hypoadrenocorticism. (because fludrocortisone is poorly absorbed in some dogs, it may not completely normalize electrolyte abnormalities in these animals.) docp is primarily a mineralocorticoid. give supplemental glucocorticosteroids in the form of prednis(ol)one (1-0.25 mg/kg/day). in dogs, iatrogenic hypoadrenocorticism can be caused by abrupt discontinuation of glucocorticosteroid treatment. long-term glucocorticosteroid supplementation can downregulate the pituitary gland's excretion of endogenous acth and the zona fasciculata's ability to excrete cortisol. however, the zona glomerulosa's ability to secrete aldosterone does not appear to be affected. clinical signs of iatrogenic hypoadrenocorticism include inability to compensate for stress, weakness, lethargy, vomiting, diarrhea, and collapse. treatment of iatrogenic hypoadrenocorticism is the same as for naturally occurring disease. following immediate emergency treatment, the patient should be weaned slowly from exogenous glucocorticosteroid supplementation. severe hyperthyroidism can manifest as a medical emergency as a result of hypermetabolism. clinical signs in affected cats with severe thyrotoxicosis include fever, severe tachycardia (heart rate >240 bpm), vomiting, hypertension, congestive heart failure with pulmonary edema, and fulminant collapse. clinical signs typically are manifested as an end-stage of chronic debilitation associated with hyperthyroidism and are often preceded by polyphagia, weight loss, cardiac murmur, polyuria/polydipsia (pu/pd), vomiting, and diarrhea. treatment of thyrotoxicosis includes antagonizing the adrenergic activity by administration of a beta-adrenergic blocker (esmolol, (25-50 âµ/kg/minute, or propranolol, 0.02 mg/ kg/hour). administration of glucocorticosteroids (dexamethasone, 1 mg/kg) may inhibit the conversion of thyroxine (t 4 ) to the active form triiodothyronine (t 3 ) and decrease peripheral tissue responsiveness to t 3 , effectively blocking its effects. correct hypoglycemia with supplemental dextrose (2.5%). use care to avoid overhydration in a patient with cardiac failure or insufficiency. start the patient on methimazole as quickly as possible and consider the use of radioactive iodine therapy. to maintain cerebral perfusion pressure, blood pressure must be normalized. if other concurrent injuries are suspected (e.g., pulmonary contusions), administer synthetic colloid fluids (dextran-70, 5-10 ml/kg iv, or hetastarch, 5-10 ml/kg iv) to normalize blood pressure. although the use of colloids is controversial because of their potential to leak into the calvarium, the benefits of reestablishing cerebral perfusion far outweigh the risks of their use. hypertonic saline (7.5% nacl, 3-5 ml/kg iv) can also be administered over 10 to 15 minutes to expand intravascular volume. maintain blood glucose within normal reference ranges whenever possible, because hyperglycemia is a negative prognostic indicator in cases of head trauma. if tremors or seizures cause hyperthermia or increased metabolism, active cooling of the patient is warranted (see sections on hyperthermia and heat-induced injury). all patients with head trauma should receive care and monitoring based on the rule of twenty (see section on rule of twenty). examine the patient's level of consciousness, response to various stimuli, pupil size and reactivity to light, physiologic nystagmus, and cranial nerve deficits. in dogs, damage to the midbrain often produces coma and decerebrate rigidity. initial consciousness followed by a unconsciousness or stupor usually involves an injury to the brainstem. brainstem lesions can be caused by compressive skull fractures, extradural or subdural hematomas, or herniation through the foramen magnum from cerebral edema (box 1-49) . the patient's pupil size and response to light can be used to localize a diagnosis and give a rough prognosis for severity of disease and possibility for return to function. pupils can be normal in size, mydriatic, or miotic. whenever a pupil appears miotic, direct ocular 186 1 emergency care unconscious with no response to noxious stimuli injury with uveitis or secondary miosis due to brachial plexus injury should be ruled out. the eyes should always be examined to rule out ocular trauma. in a patient with head trauma, a change from dilated to constricted to normal pupil size is suggestive of improvement in clinical function. bilateral mydriatic pupils that are unresponsive to light in an unconscious animal are a grave prognostic sign and usually indicate an irreversible severe midbrain contusion. bilateral miotic pupils with normal nystagmus and ocular movements are associated with diffuse cerebral or diencephalic lesions. miotic pupils that become mydriatic indicate a progressive midbrain lesion with a poor prognosis. unilateral, slowly progressive pupillary abnormalities in the absence of direct ocular injury are characteristic of brainstem compression or herniation caused by progressive brain swelling. asymmetric pupils are seen in patients with rostral brainstem lesions and can change rapidly. unresponsive pupils that are seen in the midposition occur with brainstem lesions that extend into the medulla and are a grave sign. visual deficits are common with intracranial injury. lesions that are less severe and limited to the cerebrum produce contralateral menace deficits with normal pupillary light response. bilateral cerebral edema can cause blindness with a normal response to light if the midbrain is not disturbed. a patient that is severely depressed and recumbent may not respond to menacing gestures, even when visual pathways are intact. ocular, optic tract, optic nerve, or optic chiasm lesions can interfere with vision and the pupillary light response. brainstem contusion and cerebral edema may produce blindness and dilated unresponsive pupils due to disturbance of the oculomotor area. examine all cranial nerves carefully. cranial nerve abnormalities can indicate direct contusion or laceration of the neurons in the brainstem or where they exit the skull. cranial nerves that are initially normal then later lose function indicate a progressively expanding lesion. when specific cranial nerve deficits are present, the prognosis is considered guarded. clinical signs such as rolling to one side, torticollis, head tilt, and abnormal nystagmus are usually associated with petrosal bone or cerebellomedullary lesions that produce vestibular neuron dysfunction. fractures of the petrosal temporal bone often cause hemorrhage and cerebrospinal fluid (csf) leak from the external ear canal. if the lesion is limited to the membranous labyrinth, the loss of balance will be toward the injured side and the quick phase of the nystagmus will be toward the injured side. normal physiologic nystagmus requires that the pathway is between the peripheral vestibular neurons and the pontomedullary vestibular nuclei to the nuclei of the cranial nerves that innervate the extraocular muscles (iii, iv, vi). severe brainstem lesions disrupt this pathway. disruption of the pathway is manifested as an inability to produce normal physiologic nystagmus by moving the patient's head from side to side. in patients with severe central nervous system depression, this reflex may not be observed. next, assess postural changes and motor function abilities. a loss of the normal oculocephalic ("dolls-eye") reflex is an early sign of brainstem hemorrhage and a late sign of brainstem compression and herniation. any intracranial injury may be accompanied by a concurrent cervical spinal cord injury. handle animals with such injuries with extreme care to avoid causing further damage. whenever there is uncertainty whether a spinal cord lesion exists, strap the patient down to a flat surface and obtain radiographs of the spine. at least two orthogonal views may be required to see fractures; however, do not manipulate the patient until radiography has been completed. crosstable views, in which the bucky is turned perpendicular to the patient's spine, with a radiograph plate secured behind the patient, may be required to minimize patient motion. in patients with cerebral lesions, hemiparesis usually resolves within 1 to 3 days. evaluation of cranial nerve function at frequent intervals may reveal an initial injury or a progressively expanding lesion in the brain. signs of vestibular disorientation, marked head tilt, and abnormal nystagmus occur with contusions of the membranous labyrinth and fracture of the petrous temporal bone. hemorrhage and cerebrospinal fluid otorrhea may be visible from the external ear canal. rolling movements indicate an injury to the cerebellar-medullary vestibular system. respiratory dysfunction and abnormal respiratory patterns are sometimes observed with severe head injury. lesions of the diencephalon produce cheyne-stokes respirations, in which the patient takes progressively larger and larger breaths, pauses, then takes progressively smaller and smaller breaths. mesencephalic lesions cause hyperventilation and can result in respiratory alkalosis. medullary lesions result in a choppy, irregular respiratory pattern. clinical signs of respiratory dysfunction in the absence of primary respiratory damage indicate a guarded prognosis. after injury, seizures may be associated with intracranial hemorrhage, trauma, or an expanding intracranial mass lesion. immediately begin medical therapy to control the seizure. administer diazepam (0.5 mg/kg iv or 0.1-0.5 mg/kg/hour iv cri) to treat seizures. if diazepam is not effective in combination with other treatments to control intracranial edema, consider giving pentobarbital . loading doses of phenobarbital (16-20 mg/kg iv divided into 4 or 5 doses, given every 20 to 30 minutes) may be beneficial in preventing further seizures. severe refractory seizures or decreased mentation may be associated with cerebral edema and increased intracranial pressure. mannitol, an osmotic diuretic, is effective at reducing cerebral edema (0.5-1.0 g/kg iv over 10 to 15 minutes). mannitol also acts as a free radical scavenger that can inhibit the effects of cerebral ischemia-reperfusion injury. mannitol works synergistically with furosemide (1 mg/kg iv given 20 minutes after the mannitol infusion). corticosteroids have not been demonstrated to be beneficial in the treatment of head trauma and may induce hyperglycemia. hyperglycemia has been shown to be a negative prognostic indicator in cases of head trauma. also, glucocorticoids can suppress immune system function and impair wound healing. because of the known risks and lack of known benefits of glucocorticosteroids, their use in treatment of head trauma is contraindicated. the prognosis for any patient with severe head trauma is guarded. management of head trauma patients may include intense nursing care for a period of weeks to months, depending on the presence and extent of concurrent injuries. if progressive loss of consciousness occurs, surgery for decompression of compressive skull injuries should be considered. the most common injury associated with head trauma in small animals is a contusion with hemorrhage in the midbrain and pons. subdural or extradural hemorrhage with space-occupying blood clots is uncommon. diagnostic tests of head trauma may include skull radiographs, ct, and mri of the brain. special studies can help detect edema and hemorrhage in the brain and brainstem, and aid in making an accurate diagnosis and prognosis. a cerebrospinal fluid tap is contraindicated in patients with head trauma because of the risk of causing a rapid decrease in intracranial pressure and brainstem herniation. if a compressive skull fracture is present, the patient should be stabilized for surgery to remove the compression. surgery to alleviate increased intracranial pressure is rarely performed in veterinary medicine because of the poor prognosis and results. in some cases, when a lesion can be localized to one area, 1-to 2-cm burr holes can be placed through the skull over the affected area of the cerebrum, exposing the underlying brain tissue. blood clots can be removed through the holes. the bone flap may or may not be replaced, depending on the surgeon's preference and the degree of brain swelling. spinal cord injuries may be associated with trauma, disk rupture, fractures, and dislocation of the spinal column. proceed with caution when moving a patient with suspected spinal cord injury. avoid flexion, extension, and torsion of the vertebral column. all animals that are unconscious following a traumatic event should be considered to have cervical or thoracolumbar spinal injury until proved otherwise by radiography, ct, or mri. the animal should be moved onto a flat surface (e.g., board, door, window, picture frame) and taped down to prevent motion and further displacement of vertebrae. sedation with analgesics or tranquilizers may be necessary to keep the animal immobile and to minimize patient motion. whenever possible, avoid the use of narcotics in patients with head trauma because of the risk of increasing intracranial pressure. as in other emergencies, the abcs 188 1 emergency care should be evaluated, and the patient treated for shock, hemorrhage, and respiratory compromise. once the cardiovascular and respiratory systems have been evaluated and stabilized, a more thorough neurologic examination can be performed. protrusion of an intervertebral disk indicates that the disk is bulging into the vertebral canal as a result of dorsal shifting of the nuclear pulposus disk material. disk extrusion refers to the rupture of the outer disk membrane and extrusion of the nuclear material into the vertebral column. in dogs and cats, there are 36 intervertebral disks that potentially can cause a problem. chondrodystrophic breeds of dogs are predisposed to endochondral ossification and include the dachshund, shih tzu, french bulldog, bassett hound, welsh corgis, american spaniel, beagle, lhasa apso, and pekingese. initial examination of the patient with suspected intervertebral disk disease includes identifying the neuroanatomic location of the lesion based on clinical signs and neurologic deficits and then establishing a prognosis. the neurologic examination should be carried out without excessive manipulation of the animal. the presence of pain, edema, hemorrhage, or a visible deformity may localize an area of vertebral injury. once an area of suspected lesion is localized based on physical examination findings, take radiographs to establish a diagnosis and to institute therapy. in most cases, the animal must receive a short-acting anesthestic for proper radiographic technique and to prevent further injury. lateral and crosstable ventrodorsal (vd) or dorsoventral (dv) radiographs require less manipulation of the animal compared with traditional vd and dv projections. myelography is often required to delineate the location of the herniated disk material. prognosis in spinal cord injury depends on the extent of the injury and the reversibility of the damage. perception of noxious stimuli, or the presence of "deep pain," by the animal when the stimulus is applied caudal to the level of the lesion is a good sign. to apply a noxious stimulus, apply firm pressure to a toe on one of the rear limbs using a thick hemostat or a pair of pliers. flexion or withdrawl of the limb is simply a local spinal reflex, and should not be perceived as a positive response to or patient perception of the noxious stimulus. turning of the head, vocalization, dilation of the pupils, change in respiratory rate or character, or attempts to bite are behaviors that are more consistent with perception of the noxious stimulus. absence of perception of the noxious stimulus ("loss of deep pain") is a very poor prognosis for return to function. focal lesions are usually associated with vertebral fractures and displacement of the vertebral canal. focal lesions in one or more of the spinal cord segments from t 3 to t 4 can cause complete dysfunction of the injured tissue as a result of concussion, contusion, or laceration. the degree of structural damage cannot be determined from the neurologic signs alone. transverse focal lesions result in paraplegia, with intact pelvic limb spinal reflexes and analgesia of the limbs and body caudal to the lesion. clinical signs in patients with spinal injury are summarized in table 1 -43. carefully evaluate the cardiovascular and respiratory status of patients with spinal injuries. immediately address specific injuries such as pneumothorax, pulmonary contusions, hypovolemic shock, and open wounds. if there is palpable or radiographic evidence of a vertebral lesion causing compressive injury, surgery is the treatment of choice unless the displacement has compromised most or all of the vertebral canal. displacements through 50% to 100% of the vertebral canal are associated with a poor prognosis, particularly if deep pain is absent caudal to the lesion. in the absence of a radiographic lesion and in the presence of continued neurologic deficits, an mri or ct scan or myelography is warranted to localize a potentially correctable lesion. surgical exploration can be considered: with the objectives of providing spinal cord decompression by hemilaminectomy or laminectomy with removal of disk material or blood clots, realign and stabilize the vertebral column, and perform a meningotomy, if necessary. place the patient on a backboard or other rigid surface, taped down for transport and sedated, to be transported to a surgical specialist. the presence of worsening or ascending clinical signs may signify ascending-descending myelomalacia and is characteristic of a very poor prognosis.in acute spinal trauma, the use of glucocorticoids has been the mainstay of therapy; however, controversy exists about whether they actually offer any benefit. traditional glucocorticosteroid therapy is listed in box 1-50. more recently, the use of propylene glycol has proved to be beneficial in the treatment of acute traumatic herniated disk. high-dose glucocorticoids should only be used for the first 48 hours after initial injury. side effects of glucocorticosteroid therapy include gastric and intestinal ulceration. the prophylactic use of gastroprotectant drugs will not prevent gastrointestinal ulcer formation; however, if signs of gastrointestinal ulcer are present, institute gastroprotectant therapy. management of the patient with spinal cord injury includes aggressive nursing care and physical therapy. many patients with spinal cord injury have little to no control over bladder function, which results in chronic dribbling or retention of urine and overdistention of the urinary bladder with overflow incontinence. urinary bladder retention can lead to urinary tract infection, bladder atony, and overflow incontinence. manual expression of the bladder several times a day may be enough to keep the bladder empty. alternatively, place a urinary catheter to maintain patient cleanliness and to keep the bladder decompressed. (see section 5 on urinary catheterization). paralytic ileus and fecal retention are frequent complications of spinal cord injury. to help prevent constipation, provide highly digestable foods and maintain the patient's hydration with oral and intravenous fluids. mild enemas or stool softeners can also be used to treat fecal retention. to prevent decubital ulcer formation, turn the patient every 4 to 6 hours, and use clean, dry, soft padded bedding. apply deep muscle massage and passive range of motion exercises to prevent disuse atrophy of the muscles and dependent edema. the radial nerve innervates the extensor muscles of the elbow, carpus, and digits. the radial nerve also supplies sensory innervation to the distal craniolateral surface of the forearm and the dorsal surface of the forepaw. injuries to the radial nerve at the level of the elbow 190 1 emergency care cranial to c6 spastic tetraplegia or tetraparesis hyperreflexive all four limbs severe injury can result in death from respiratory failure. c6-t2 tetraparesis or tetraplegia depressed thoracic limb spinal reflexes (lower motor neuron) hyperreflexive pelvic limbs (upper motor neuron) t1-t3 horner' syndrome (prolapsed nictitans, enophthalmos, and miosis) t3-l3 schiff-sherrington syndrome (extensor rigidity of thoracic limbs, flaccid paralysis with atonia, areflexia, and analgesia of pelvic limbs) result in an inability to extend the carpus and digits. as a result, the animal walks and bears weight on the dorsal surface of the paw. there is also loss of cutaneous sensation, which leads to paw injury. injuries to the radial nerve above the elbow (in the shoulder area) results in an inability to extend the elbow and bear weight on the affected limb. it can take weeks before the full extent of the injury and any return to function are manifested. the animal may need to be placed in a carpal flexion sling or have eventual amputation if distal limb injury or self-mutilation occurs. the sciatic nerve primarily innervates the caudal thigh muscles that flex the stifle and extend the hip. the tibial branch of the sciatic nerve innervates the caudal leg muscles that extend the tarsus and flex the digits. the tibial nerve provides the sole cutaneous sensory innervation to the plantar aspect of the paw and digits. the peroneal branch of the sciatic nerve provides the sole sensory cutaneous innervation to the dorsal surface of the paw ( table 1 -44) . sciatic nerve injury may occur with pelvic fractures, particularly those that involve the body of the ileum at the greater ischiatic notch, or with sacroiliac luxations that contuse the l6 and l7 spinal nerves that pass ventral to the sacrum to contribute to the sciatic nerve. with sciatic nerve injury, there is decreased stifle flexion and overflexion of the hock (tibial nerve), and the animal walks on the dorsal surface of the paw (peroneal nerve). clinical signs of tibial or peroneal damage are seen with femur fractures or with inadvertent injection of drugs into the caudal thigh muscles. the femoral nerve innervates the extensor muscles of the stifle. the saphenous branch of the femoral nerve provides the sole cutaneous innervation to an area on the medial distal thigh, the leg, and the paw. the femoral nerve is protected by muscles and is rarely injured in pelvic fractures. clinical signs of femoral nerve injury are inability to support weight on the pelvic limb, absence of a patellar reflex, and analgesia in the area of cutaneous innervation. coma is complete loss of consciousness, with no response to noxious stimuli. in some animals that present in a coma or stuporous state, the immediate cause will be apparent. in other cases, however, a careful and thorough diagnostic work-up must be performed. a coma scale devised to assist in the clinical evaluation of the comatose patient is shown in table 1 -45. whenever an animal presents in a comatose state, immediately secure the emergency management of specific conditions 191 c6-t2 nerve roots radial nerve paralysis musculocutaneous nerve inability to flex the elbow axillary or thoracodorsal dropped elbow nerve median and ulnar nerves loss of cutaneous sensation on the caudal surface of the forearm and palmar and lateral surfaces of the paw; inability to flex the carpus and digits c8-t1 nerve roots radial, median, or ulnar nerve injury c6-c7 nerve roots musculocutaneous, suprascapular, and axillary injury c7-t3 horner's syndrome (miosis, enophthalmos, and prolapsed nictitans) airway by placing an endotracheal tube (see section on endotracheal intubation). if necessary, provide respiratory assistance, or at a minimum, supplemental oxygen. control existing hemorrhage and treat shock, if present. take a careful and thorough history from the owner. make careful note of any seizure, trauma, or toxin exposure, and whether prior episodes of coma have ever occurred. perform a careful physical examination, taking note of the patient's temperature, pulse, and respiration. an elevated temperature may suggest the presence of systemic infection, such as pneumonia or hepatitis, or a brain lesion with loss of hypothalamic thermoregulatory control. very high temperatures associated with shock and coma are often observed in animals with heat stroke (see section on heat stroke and heat-induced illness). circulatory collapse or barbiturate overdose can produce coma and hypothermia. abnormal respiratory patterns also may be observed in a comatose patient. hypoventilation may occur with elevated intracranial pressure or barbiturate overdose. rapid respiratory rate may be associated with pneumonia, metabolic acidosis (dka, uremia), or brainstem injury. examine the skin for any bruises or external trauma. examine the mucous membranes and make note of color and capillary refill time. icterus with petechiae or ecchymotic hemorrhage in a comatose patient may be associated with end-stage hepatic failure and hepatic encephalopathy. smell the patient's breath for the odor of ketones that may signify dka or end-stage hepatic failure. motor activity 6 normal gait, normal spinal reflexes hemiparesis, tetraparesis, or decerebrate activity 5 recumbent, intermittent extensor rigidity 4 recumbent, constant extensor rigidity 3 recumbent, constant extensor rigidity with opisthotonus 2 recumbent, hypotonia of muscles, depressed or absent spinal reflexes 1 normal papillary reflexes and oculocephalic reflexes 6 slow pupillary light reflexes and normal to reduced oculocephalic reflexes 5 bilateral unresponsive miosis with normal to reduced oculocephalic reflexes 4 pinpoint pupils with reduced to absent oculocephalic reflexes 3 unilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes 2 bilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes 1 occasional periods of alertness and responsive to environment 6 depression of delirium, capable of responding to environment but response 5 may be inappropriate semicomatose, responsive to visual stimuli 4 semicomatose, responsive to auditory stimuli 3 semicomatose, responsive only to repeated noxious stimuli 2 comatose, unresponsive to repeated noxious stimuli 1 *neurologic function is assessed for each of the three categories and a grade of 1 to 6 is assigned according to the descriptions for each grade. the total score is the sum of the three category scores. this scale is designed to assist the clinician in evaluating the neurologic status of the craniocerebral trauma patient. as a guideline and according to clinical impressions, a consistent total score of 3 to 8 represents a grave prognosis, 9 to 14 a poor to guarded prognosis, and 15 to 18 a good prognosis. (modified from the glasgow coma scale used in humans.) from shores a: craniocerebral trauma. in kirk rw, ed: current veterinary therapy x. small animal practice. philadelphia, wb saunders, 1989, p 849. finally, conduct a complete neurologic evaluation. the presence of asymmetric neurologic signs may suggest an intracranial mass lesion (e.g., hemorrhage, neoplasia, injury). usually, toxicities or metabolic disturbances (e.g., dka, hepatic encephalopathy) cause symmetric clinical signs of neurologic dysfunction, with cerebral signs predominating. in hepatic encephalopathy, pupils are usually normal in size and responsive to light. in toxicities, the pupils are abnormal in size and may be unresponsive to light. obtain a complete blood count, serum biochemistry profile, urinalysis, and specific tests for glucosuria and ketonuria. findings of a drastically elevated blood glucose with glucosuria, ketonuria, and high specific gravity are characteristic of dka. fever and uremic encephalopathy are characterized by severe azotemia with a low urine specific gravity. if barbiturate intoxication is suspected, save urine for later toxin analysis. evaluate urine sediment for calcium oxalate crystalluria that may indicate ethylene glycol toxicity. calculate plasma osmolality (see following section) to check for nonketotic hyperosmolar diabetes mellitus. elevated blood ammonia levels may be associated with hepatic encephalopathy. in uncontrolled diabetes mellitus, hyperosmolarity can result in clinical signs of disorientation, prostration, and coma. plasma osmolarity can be calculated from the formula: mosm/l = 2(na + k) + (glucose/18) + (bun/2.8) clinical signs of hyperosmolarity can occur when the plasma osmolarity exceeds 340 mosm/l. treatment of dka or nonketotic hyperosmolar syndrome is aimed at reducing ketoacid production, stimulating carbohydrate utilization, and impeding peripheral release of fatty acids. the treatment of choice is rehydration and provision of supplemental regular insulin and a carbohydrate source (see section on diabetic ketoacidosis). during ketosis, insulin resistance may be present. slow rehydration with 0.9% saline solution or other balanced crystalloid fluids (e.g., normosol-r, plasmalyte-m, lactated ringer's solution), should occur, with the goal of rehydration over 24 to 48 hours. too rapid rehydration can result in cerebral edema and exacerbation of clinical signs. hepatic encephalopathy (he) is characterized by an abnormal mental state associated with severe hepatic insufficiency. the most common cause of he is congenital or acquired c o m a portosystemic shunts. acute hepatic destruction can also be caused by toxins, drugs, or infectious causes. the treatment of he is considered a medical emergency (table 1 -46) . absorption of ammonia and other nitrogenous substances from the gastrointestinal tract is thought to be one of the complicating factors in he. prevent absorption of ammonia and other nitrogenous substances from the gastrointestinal tract by restricting dietary protein to 15% to 20% for dogs, and to 30% to 35% (on a dry matter basis) for cats. dietary protein should be from a nonanimal plant source (e.g., soybean) whenever possible. caloric requirements are met with lipids and carbohydrates. also prescribe cleansing enemas to rid the colon of residual material, and antibiotic therapy to reduce gastrointestinal tract bacteria. neomycin (15 mg/kg q6h) can be administered as a retention enema. metronidazole (7.5 mg/kg po, q8-12h) or amoxicillin-clavulanate (16.25 mg po q12h) can also be administered. administer lactulose (2.5-5.0 ml q8h for cats; 2.5-15 ml q8h for dogs) to trap ammonia in the colon to prevent absorption (table 1 -46) . administer lactulose orally to an alert animal, or as a retention enema to a comatose animal. if lactulose is not available, betadine retention enemas will change colonic ph and prevent ammonia absorption. a side effect of lactulose administration (po) is soft to diarrheic stool. a seizure is a transient disturbance of brain function that is sudden in onset, ceases spontaneously, and has a tendency to recur, depending on the cause. most seizures are generalized and result in a loss of consciousness and severe involuntary contraction of the skeletal muscles, resulting in tonic-clonic limb activity and opisthotonus. mastication, salivation, urination, and defecation are common. partial (petit mal) seizures range from limited limb activity, facial muscle twitching, and episodic behavioral abnormalities to brief loss of consciousness. similar clinical signs also can occur with syncopal episodes. conduct a careful cardiac examination in any patient with a history of petit mal seizures. seizures of any form constitute a medical emergency, particularly when they occur in clusters, or as status epilepticus. most seizures are of short duration and may have subsided by the time the animal is presented for treatment. whenever a seizure occurs, however, it is important that the animal does not inadvertently injure itself or a bystander. it is important to evaluate whether the patient has a coexisting disease that can predispose it to seizures, such as hepatic failure, uremia, diabetes mellitus, hypoglycemia, toxin exposure, insulin-secreting tumors, and thiamine deficiency. many toxins are responsible for clinical signs of tremors or seizures (see section on poisons and toxins). treatment of a primary disease entity can help control seizures, in some cases, provided that the underlying cause is investigated and treated. status epilepticus, a state of continuous uncontrolled seizure activity, is a medical emergency. when an animal is in a state of status epilepticus, immediately place a lateral or medial saphenous intravenous catheter and administer diazepam (0.5 mg/kg iv) to help control the seizure. in most cases, the seizure must be controlled before a diagnostic workup is attempted. whenever possible, however, blood samples should be collected before administration of any anticonvulsant agent because of the risk of incorrect test results. for example, the propylene glycol carrier in diazepam can cause a false-positive ethylene glycol test using an in-house testing kit. whenever possible, check blood glucose levels, particularly in young puppies or kittens, to evaluate and treat hypoglycemia as a cause of seizures. if hypoglycemia exists, administer 25% dextrose (1 g/kg iv). if diazepam partially controls the status epilepticus, administer a constant rate infusion (0.1 mg/kg/hour in 5% dextrose in water). diazepam is sensitive to light, and the bag and infusion line must be covered to prevent degradation of the drug. if diazepam fails to control status epilepticus, give pentobarbital (3-25 mg/kg iv to effect). the animal's airway should be intubated and protected while the patient is kept in the drug-induced coma. protracted cases of seizures may require mannitol and furosemide therapy to treat cerebral edema. administer intravenous fluids (balanced crystalloid at maintenance doses [see section on intravenous fluid therapy]). the patient should be turned every 4 to 6 hours to 194 1 emergency care prevent atelectasis. insert a urinary catheter for cleanliness, and place the animal on soft dry padded bedding to prevent decubital ulcer formation. depending on the length of time that the patient is rendered unconscious, apply passive range of motion exercises and deep muscle massage to prevent disuse atrophy of the muscles and dependent or disuse edema. monitor the patient's oxygenation and ventilation status by arterial blood gas measurement or pulse oximetry and capnometry (see section 5 on blood gas, pulse oximetry, and capnometry). administer supplemental oxygen to any patient that is hypoxemic secondary to hypoventilation or other causes. severe refractory seizures can result in the development of neurogenic pulmonary edema. lubricate the animal's eyes every 4 hours to prevent drying out and corneal abrasions. depending on the cause of the seizure, administer phenobarbital at a loading dose of 16 to 20 mg/kg iv given in four to five injections, every 20 to 30 minutes; make sure that the patient is rousable in between injections). seizures in cats often are associated with structural brain disease. the occurrence of partial focal seizures is unequivocally associated with a focal cerebral lesion and acquired structural brain disease. an initial high frequency of seizures is also a strong indication that structural brain disease is present. seizure activity in cats may occur as mild generalized seizures or complex partial seizures and may be associated with systemic disorders such as feline infectious peritonitis virus, toxoplasmosis, cryptococcus infection, lymphosarcoma, meningiomas, ischemic encephalopathy, and thiamine deficiency. thiamine deficiency in the cat can be a medical emergency characterized by dilated pupils, ataxic gait, cerebellar tremor, abnormal oculocephalic reflex, and seizures. treatment consists of administration of thiamine (50 mg/day) for three days. steffen f, grasmueck s: propofol for treatment of refractory seizures in dogs and a cat with intracranial disorders. j small anim pract 41 (11) (1997) (1998) (1999) . j am vet med assoc 218 (7): [1124] [1125] [1126] [1127] [1128] [1129] 2001 . an ocular emergency is any serious condition that causes or threatens to cause severe pain, deformity, or loss of vision. treat ocular emergencies immediately, within 1 to several hours after the emergency, whenever possible (box 1-51, 1-52). to assess the location and degree of ocular injury, perform a complete ocular examination. in some cases, short-acting sedation or general anesthesia in conjunction with topical local anesthetic may be necessary to perform the examination, because of patient discomfort and blepharospasm. the equipment listed in box 1-53 may be necessary and may be invaluable in making an accurate diagnosis. to perform a systematic and thorough ocular examination, first obtain a history from the owner. has there been any prior incident of ocular disease? is there any history of trauma or known chemical irritant or exposure? did the owner attempt any irrigation or medical techniques prior to presentation? when was the problem first noticed? has it changed at all since the owner noticed the problem? after a history has been obtained, examine the patient's eyes for discharge, blepharospasm, or photophobia. if any discharge is present, note its color and consistency. do not attempt to force the eyelids open if the patient is in extreme discomfort. administer a short-acting sedative and topical local anesthetic such as 0.5% proparacaine. note the position of the globe within its orbit. if the eye is exophthalmic, strabismus and protrusion of the third eyelid are often visible. exposure keratitis may be present. in cases of retrobulbar or zygomatic salivary gland inflammation, the patient will resist opening the mouth and exhibit signs of discomfort or pain. note any swelling, contusions, abrasions, or lacerations of the eyelids. note whether the lids are able to close completely and cover the cornea. if a laceration of the lid is present, determine the depth of the laceration. palpate the orbit for fractures, swelling, pain, crepitus, and cellulitis. examine the cornea and sclera for penetrating injury or foreign material. the use of lid retractors or small forceps can be very helpful in these cases. if a wound appears to penetrate completely into the globe, look for loss of uveal tissue, lens, or vitreous. do not put any pressure on the globe, because intraocular herniation may result. examine the conjunctiva for hemorrhage, chemosis, lacerations, and foreign bodies. examine the superior and inferior conjunctival cul-de-sacs for foreign material. in such cases, placement of a topical anesthetic and use of a moistened cotton swab is invaluable to sweep the conjunctival fornix to pick up foreign bodies. use a small, fine-tipped forceps to retract the third eyelid away from the globe and examine behind the third eyelid for foreign bodies. next, examine the cornea for opacities, ulcers, foreign bodies, abrasions, or lacerations. place a small amount of fluroescein stain mixed with sterile water or saline on the dorsal sclera. close the eye to disperse the stain over the surface of the cornea, then flush gently with sterile saline irrigation. examine the cornea again for any defects. a linear defect perpendicular to the long axis of the eye should alert the clinician to investigate the conjunctiva for dystechia. record the pupil size, shape, and response to light (both direct and consensual). examine the anterior chamber and note its depth and whether hyphema or aqueous flare are present. is the lens clear and is it in the normal position? lens luxation can cause the lens tissue to touch the cornea and cause acute corneal edema. measure intraocular pressure with a schiotz tonometer or tonopen. finally, dilate the pupil and examine the posterior chamber using a direct or indirect ophthalmoscope to look for intraocular hemorrhage, retinal hemorrhage, retinal detachment, tortuous retinal vessels, optic neuritis, and inflammation. the basic surgical instruments listed in box 1-54 may be useful in the treatment of ocular lacerations and other ophthalmic injuries: bite wounds and automobile trauma commonly cause lacerations and abrasions of the lid margins. the lids can be considered to be two-layer structures, with the anterior composed of the skin and orbicularis muscle and the posterior layer composed of the tarsus and conjunctiva. the openings of the meibomian glands in the lid margin form the approximate line separating the lids into anterior and posterior segments. splitting the lid into these two segments facilitates the use of sliding skin flaps to close wound defects, if necessary. clean and thoroughly but gently irrigate the wound with sterile saline solution before attempting any lid laceration repair. use sterile saline solution to irrigate the wound and conjunctiva. a 1% povidone-iodine scrub can be used on the skin, taking care to avoid getting any scrub material in the soft tissues of the eye. drape the eye with an adhesive ocular drape, if possible, to prevent further wound contamination. trim the ragged wound edges, but be very conservative with tissue debridement. leave as much tissue as possible to insure proper wound contracture with minimal lid deformity. close a small lid wound with a figure-of-eight or two-layered simple interrupted suture of absorbable suture material or nylon in the skin. the lid margins must be absolutely apposed to prevent postoperative lid notching. direct blunt trauma to the eye can cause severe ecchymosis because of the excellent vascular supply of the eyelids. other associated ocular injuries such as orbital hemorrhage, proptosis, and corneal laceration may also occur. trauma, allergic reactions, inflammation of the sebaceous glands (hordeolum), thrombocytopenia, and vitamin k antagonist rodenticide intoxication can all cause ecchymoses of the lids. treat eyelid ecchymoses initially with cool compresses, followed by warm compresses. resorption of blood can occur from 3 to 10 days after the initial insult. ocular allergies respond well to topical application (dexamethasone ophthalmic ointment q6-8h) and systemic administration of glucocorticosteroids, along with cool compresses. in order to fully assess the conjunctiva for abnormalities, it may be necessary to carefully dissect it away from the underlying sclera. when performing this dissection, do not place undue pressure on the globe because of the risk of herniation of the intraocular contents through a scleral wound. repair large conjunctival lacerations with 6-0 absorbable sutures, using an interrupted or continuous pattern. carefully approximate the margins of the conjunctiva to prevent formation of inclusion cysts. when large areas of the conjunctiva have been damaged, advancement flaps may be required to close the defect. subconjunctival hemorrhage is a common sequela of head trauma, and it may also be observed in various coagulopathies. by itself, it is not a serious problem but may signify severe underlying intraocular damage. a complete ocular examination is indicated. other causes of subconjunctival hemorrhage include thrombocytopenia, autoimmune hemolytic anemia, hemophilia, leptospirosis, vitamin k antagonist rodenticide intoxication, severe systemic infection or inflammation, and prolonged labor (dystocia). uncomplicated subconjunctival hemorrhage usually clears on its own within 14 days. if the conjunctiva is exposed because of swelling and hemorrhage, administer a topical protective triple antibiotic ophthalmic ointment every 6 to 8 hours until the conjunctival hemorrhage resolves. toxic, acid, and alkaline chemical injuries to the eye can sometimes occur. the severity of the injury caused by ocular burns depends on the concentration, type, and ph of the chemical and on the duration of exposure. weak acids do not penetrate biologic tissue very well. the hydrogen ion precipitates the protein upon contact and therefore provides some protection to the corneal stroma and intraocular contents. precipitation of corneal proteins produces a ground-glass appearance in the cornea. alkaline solutions and very strong acids penetrate tissues rapidly, causing saponification of the plasma membrane, denaturation of collagen, and vascular thrombosis within the conjunctiva, episclera, and anterior uvea. severe pain, blepharospasm, and photophobia are produced by exposure of free nerve endings in the corneal epithelium and conjunctiva. severe alkaline burns cause an increase in intraocular pressure. intraocular prostaglandins are released, and the intraocular aqueous ph increases, producing changes in the blood-aqueous barrier and secondary uveitis. uveitis with anterior synechia formation, eventual chronic glaucoma, phthisis, secondary cataract, and corneal perforation can occur. healing of the corneal epithelium is usually accomplished by neovascularization and sliding and increased mitosis of the corneal epithelium. severe stromal burns within the cornea heal by degradation and removal of necrotic debris, followed by replacement of the collagen matrix and corneal epithelial cells. the release of collagenase, endopeptidase, and cathepsins from polymorphonuclear cells serves to cause further corneal breakdown. in severe cases, only pmns may be present, and fibroblasts may never invade the corneal stroma. all chemical burns should be washed copiously with any clean aqueous solution available. if any sticky paste or powder is adherent to the conjunctival sac, remove it with moist cotton swabs and irrigation. begin mydriasis and cycloplegia by topical application of 1% atropine ophthalmic drops or ointment. start antibiotic therapy with triple antibiotic ophthalmic ointment or gentocin ointment every 6 to 8 hours. treat secondary glaucomas with topical carbonic anhydrase inhibitors. to avoid fibrinous adhesions and symblepharon formation, keep the conjunctival cul-de-sacs free of proteinaceous exudate that can form adhesions. analgesics are required for pain. oral nonsteroidal antiinflammatory agents such as carprofen, ketoprofen, meloxicam, or aspirin are recommended. persistent epithelial erosions may require a conjunctival flap left in place for 3 to 4 weeks or placement of a topical collagen shield (contact lens). topical antibiotics, mydriatics, and lubricants (lacrilube or puralube ointment) should also be used. strong acid or alkali burns can result in severe corneal stromal loss. in the past, topical n-acetylcysteine (10% mucomyst) has been recommended. this treatment is very painful. other treatments are also available, such as ethylenediaminetetraacetic acid (edta) (0.2 m solution) and patient serum to inhibit mammalian collagenase activity. to prepare patient serum, obtain 10 to 12 ml of whole blood from the patient. spin it down in a serum separator tube after a clot forms and then place the serum in a red-topped tube on the patient's cage. (the contents of the tube are viable for 4 days without refrigeration.) apply the serum topically to the affected eye every 1 to 2 hours. avoid using topical steroids because they inhibit fibroblast formation and corneal healing. in severe cases, if conjunctival swelling and chemosis also are present, antiinflammatory doses of oral steroids can be administered short-term. oral steroids and nonsteroidal antiinflammatory drugs should never be administered to the patient concurrently, because of the risk of gastrointestinal ulcer and perforation. corneal abrasions are associated with severe pain, blepharospasm, lacrimation, and photophobia. animals with such intense pain are often difficult to examine until analgesia has been administered. topical use of proparacaine (0.5% proparacaine hydrochloride) is usually sufficient to permit relaxation of the eyelids so that the eye can be examined. using a focal source of illumination and an eye loupe, examine the cornea, inferior and superior conjunctival fornixes, and medial aspect of the nictitans for foreign bodies. place a sterile drop of saline on a fluorescein-impregnated strip and touch the superior conjunctiva once to allow the stain to spread onto the surface of the eye. irrigate the eye to remove excess stain and then examine the corneal surface for any areas of stain uptake. if an area of the cornea persistently remains green, there is damage to the corneal epithelium in that area. initial treatment consists of application of a topical mydriatic (1 drop of 1% atropine in affected eye q12h) to prevent anterior synechiae and improve cycloplegia. triple antibiotic ointment is the treatment of choice (a 1 /4-inch strip in the affected eye q8h) until the ulcer heals. in some cases, nonhealing ulcers (e.g., boxer ulcer, indolent ulcer) form in which the epithelial growth does not adhere to the underlying cornea. gently debride the loose edges 1 of the ulcer/erosion with a cotton swab and topical anesthesia. more severe cases in which only minimal healing has occurred after 7 days of treatment require grid keratectomy, in which a 25-gauge needle is used to gently scratch the surface of the abrasion or ulcer in the form of a grid to promote neovascularization. apply a topical anesthetic before performing the procedure. a collagen contact lens also may be required to promote wound healing. all corneal abrasions should be reevaluated in 48 hours, and then every 4 to 7 days thereafter until they have healed. acute infectious keratitis secondary to bacterial infection is characterized by mucopurulent ocular discharge, rapidly progressing epithelial and corneal stromal loss, inflammatory cellular infiltrates into the corneal stroma, and secondary uveitis, often with hypopyon formation. confirmation of infectious keratitis is based on corneal scrapings and a positive gram stain. initial treatment for bacterial keratitis consists of systemic antibiotics and topical ciprofloxacin (0.3% eyedrops or ointment). penetrating injuries through the cornea may result in prolapse of intraocular contents. frequently, pieces of uveal tissue or fibrin effectively but temporarily seal the defect and permit the anterior chamber to re-form. avoid manipulation of these wounds until the animal has been anesthetized, as struggling or excitement can promote loss or dislodgement of the temporary seal and cause the intraocular contents to be extruded. superficial corneal lacerations need not be sutured and can be treated the same as a superficial corneal ulcer or abrasion. if the laceration penetrates more than 50% the thickness of the cornea, or extends more than 3 to 4 mm, it should be sutured. when placing sutures in the cornea, it is helpful to use magnification. referral to a veterinary ophthalmologist is advised. if a veterinary ophthalmologist is not available, use 7-0 or 8-0 silk, collagen, or nylon sutures on a micropoint spatula-type needle. use a simple interrupted suture pattern and leave the sutures in place for a minimum of 3 weeks. because many corneal lacerations are jagged and corneal edema forms, most of the wound edges cannot be tightly juxtaposed. in such cases, pull a conjunctival flap across the wound to prevent leakage of aqueous fluid. never suture through the full thickness of the cornea; rather, the suture should pass through the mid-third of the cornea. following closure of the corneal wound, the anterior chamber must be re-formed to prevent anterior synechia formation with secondary glaucoma. taking care to avoid iris injury, use a 25-or 26-gauge needle to insert sterile saline at the limbus. any defect in the suture line will be apparent because of leakage of the fluid from the site and should be repaired. incarceration of uveal tissue in corneal wounds is a difficult surgical problem. persistent incarceration of uveal tissue can result in development of a chronic wick in the cornea, a shallow anterior chamber, chronic irritation, edema, vascularization of the cornea, and intraocular infection that can lead to panophthalmitis. referral to a veterinary ophthalmologist is strongly recommended. the most common foreign bodies associated with ocular injuries in small animals are birdshot, bb pellets, and glass. the site of intraocular penetration of the foreign bodies may be obscured by the eyelids. a foreign body entering the eye may penetrate the cornea and fall into the anterior chamber or become lodged in the iris. foreign bodies may occasionally penetrate the lens capsule, producing cataracts. some metallic high-speed foreign bodies may penetrate the cornea, iris, and lens to lodge in the posterior wall of the eye or vitreous chamber. direct visualization of a foreign body is the best means of localization. examination of the eye with an indirect ophthalmoscope or biomicroscope (if available) is invaluable for locating foreign bodies. indirect visualization of the ocular foreign body can also be achieved through radiographic techniques. three separate views should be obtained to determine the plane of location of the foreign object. ct or mri may prove useful, although scatter from the foreign body may make it difficult to directly visualize with these techniques. ocular ultrasound is perhaps the most useful and refined radiographic technique for locating intraocular foreign bodies. before removing any foreign body from the eye, the risk and surgical danger of removing it must be weighed against the risks of leaving it in place. metallic foreign bodies in the anterior chamber are much easier to remove than nonmagnetic ones. attempted removal of foreign objects from the vitreous chamber of the eye has consistently produced poor results. for the best chance of recovery, ocular foreign bodies should be removed by a veterinary ophthalmologist whenever possible. blunt trauma to the globe can result in luxation or subluxation of the lens. the subluxated lens may move anteriorly and make the anterior chamber more shallow. trembling of the iris (iridodonesis) may be noticed when the lens is subluxated. in complete luxation, the lens may fall totally into the anterior chamber and obstruct aqueous outflow, causing secondary glaucoma. alternatively, the lens may be lost into the vitreous cavity. luxation of the lens is almost always associated with rupture of the hyaloid membrane and herniation of the vitreous through the pupillary space. emergency surgery for lens luxation is required if the lens is entirely within the anterior chamber or incarcerated within the pupil, causing a secondary pupillary block glaucoma. acute elevation in intraocular pressure can cause vision loss within 48 hours; thus, lens removal should be accomplished as quickly as possible. referral to a veterinary ophthalmologist is recommended. severe trauma to the globe or a direct blow to the head can result in retinal or vitreous hemorrhage. there may be large areas of subretinal or intraretinal hemorrhage. subretinal hemorrhage assumes a discrete globular form, and the blood appears reddish-blue in color. the retina is detached at the site of hemorrhage. superficial retinal hemorrhage may assume a flame-shaped appearance, and preretinal or vitreous hemorrhage assumes a bright-red amorphous appearance, obliterating the underlying retinal architecture. retinal and vitreous hemorrhage secondary to trauma usually resorbs spontaneously over a 2-to 3-week period. unfortunately, vitreous hemorrhage, as it organizes, can produce vitreous traction bands that eventually produce retinal detachment. expulsive choroid hemorrhage can occur at the time of injury and usually leads to retinal detachment, severe visual impairment, and total loss of vision. treatment of vitreal and retinal hemorrhage includes rest and correction of factors that may predispose to intraocular hemorrhage. more complicated cases may require vitrectomy performed by a veterinary ophthalmologist. hyphema refers to blood in the anterior chamber of the eye. the most common traumatic cause of hyphema is an automobile accident. hyphema may also present because of penetrating ocular wounds and coagulopathies. blood within the eye may come from the anterior or posterior uveal tract. trauma to the eye may result in iridodialysis or a tearing of the iris at its root, permitting excessive bleeding from the iris and ciliary body. usually, simple hyphema resolves spontaneously in 7 to 10 days and does not cause vision loss. loss of vision following bleeding into the anterior chamber is associated with secondary ocular injuries such as glaucoma, traumatic iritis, cataract, retinal detachment, endophthalmitis, and corneal scarring. treatment of hyphema must be individualized, but there are severe general principles of treatment. first, stop ongoing hemorrhage and prevent further bleeding whenever possible. this may involve correction of the underlying cause, if a coagulopathy is present. next, aid in the elimination of blood from the anterior chamber, control secondary glaucoma, and treat associated injuries, including traumatic iritis. finally, detect and treat any late complications of glaucoma. in most cases of traumatic hyphema, little can be done to arrest or prevent ongoing hemorrhage. it is best to restrict the animal's activity and prohibit exertion. rebleeding can occur within 5 days, and intraocular pressure must be monitored closely. after 5 to 7 days, the blood in the anterior chamber will change color from a bright red to bluish-black ("eight-ball hemorrhage"). if total hyphema persists and intraocular pressure rises despite therapy, surgical intervention by a veterinary ophthalmologist may be necessary. the primary route of escape of rbcs from the anterior chamber is via the anterior drainage angle. iris absorption and phagocytosis play a minor role in the removal of blood from the anterior chamber. because of the associated traumatic iritis in hyphema, topical administration of a glucocorticoid (1% dexamethasone drops or 1% prednisolone drops) is advised to control anterior chamber inflammation. a cycloplegic agent (1% atropine) should also be used. the formation of fibrin in the anterior chamber of the eye secondary to hemorrhage can produce adhesions of the iris and secondary glaucoma (see section on glaucoma secondary to hyphema) by blocking the trabecular network. hyphema secondary to retinal detachment (collie ectasia syndrome) and end-stage glaucoma are extremely difficult to treat medically and have a poor prognosis. proptosis of the globe is common secondary to trauma, particularly in brachycephalic breeds. proptosis of the globe in dolichocephalic breeds requires a greater degree of initiating contusion than the brachycephalic breeds because the orbits are so much deeper. therefore, secondary damage to the eye and cns associated with proptosis of the globe may be greater in the collie or greyhound than in the pug. when proptosis occurs, carefully evaluate the cardiovascular system for evidence of hypovolemic or hemorrhagic shock. examine the respiratory and neurologic systems. be sure to establish an airway and treat shock, if present. control hemorrhage and stabilize the cardiovascular system before attempting to replace the globe within its orbit or perform enucleation. during the initial management of the cardiovascular and respiratory systems, the eye should be covered with an ophthalmic grade ointment or sponges soaked in sterile saline to prevent the globe from drying out. proptosis of the globe can be associated with serious intraocular problems including iritis, chorioretinitis, retinal detachment, lens luxation, and avulsion of the optic nerve. stain the surface of the eye with fluorescein to look for topical abrasions or ulcers. carefully examine the sclera, cornea, and conjunctiva for penetrating injuries that may allow aqueous leakage. evaluate the size, location, and response to light of the pupil. a reactive pupil is better than a mydriatic fixed pupil. topical administration of a mydriatic (atropine 1%) to prevent persistent miosis and synechia formation is indicated, along with topical and oral antibiotics and oral analgesic therapy. reposition the proptosed globe with the patient under general anesthesia. make a lateral canthotomy incision to widen the palpebral fissure. lavage the globe with sterile saline irrigation to remove any external debris. place a copious amount of triple antibiotic ophthalmic ointment on the surface of the eye and then gently press the globe into the orbit using the flat side of a scalpel handle or a moistened sterile surgical sponge. do not probe the retro-orbital space with a needle or attempt to reduce intraocular pressure by paracentesis. when the globe is replaced in the orbit, close the lateral canthotomy incision with simple interrupted sutures. place three non-penetrating mattress sutures in the lid margins but do not draw them together. tighten the lid sutures through small pieces of a red rubber catheter or length of intravenous extension tubing to prevent the sutures from causing lid necrosis. leave the medial canthus of the eye open in order to allow topical treatment. postoperative treatment is directed at preventing further iritis and preventing infection. administer systemic broad-spectrum antibiotics (clavamox, 16.25 mg/kg po bid) and analgesic drugs. apply topical triple antibiotic ophthalmic ointment ( 1 /4 inch in affected eye q6-8h) and atropine (1% in affected eye q12h) to prevent infection, cycloplegia, and anterior synechiae. antiinflammatory doses of systemic steroids can also be added to the treatment 202 if severe periorbital inflammation is present. systemic steroids should never be used in conjunction with nonsteroidal antiinflammatory drugs, because of the risk of gastrointestinal ulceration and perforation. the sutures should remain in place for a minimum of 3 weeks. after this time, remove the sutures and inspect the globe. if proptosis recurs, repeat the treatment. following proptosis, strabismus is common secondary to periorbital muscle injury. even after extensive treatment, vision in the eye may still be lost. nonvisual eyes can remain in place, but phthisis may develop. carbonic anhydrase inhibitors such as acetazolamide and dichlorphenamide decrease aqueous secretion and may effectively reduce intraocular pressure if the trabecular outflow is still functioning at 40% of its capacity. an eye with a poorly functional trabecular outflow system will respond poorly to therapy with carbonic anhydrase inhibitors. osmotic agents such as mannitol or glycerol may be helpful in controlling glaucoma secondary to hyphema. reduction in vitreous chamber size can make the anterior chamber deeper and may allow increased aqueous outflow. evacuation of blood or blood clots from the anterior chamber is not advisable unless the glaucoma cannot be controlled medically or there is no indication after a prolonged period of time that blood is being resorbed. tissue plasminogen activator (t-pa) has proved to be useful in may be helpful in lysing blood clots and preventing excessive fibrin formation. the t-pa is reconstituted to make a solution of 250 âµ/ml, which is then frozen at â��70â°c in 0.5-ml aliquots. the thawed, warmed reconstituted t-pa is injected into the anterior chamber. blind probing of the anterior chamber of the eye and surgical intervention in an attempt to remove blood clots can cause serious complications such as rebleeding, lens luxation, iris damage, and damage to the corneal epithelium, and therefore is not advised. acute glaucoma is a rise in intraocular pressure that is not compatible with normal vision. glaucoma may present as early acute congestive or noncongestive glaucoma, or as end-stage disease. cardinal signs of glaucoma are a sudden onset of pain, photophobia, lacrimation, deep episcleral vascular engorgement, edematous insensitive cornea, shallow anterior chamber depth, dilated unresponsive pupil, loss of visual acuity, and buphthalmia. intraocular pressure usually exceeds 40 mm hg but may be normal or only slightly increased if glaucoma is secondary to anterior uveitis. most forms of clinical glaucoma in dogs are secondary to some other intraocular problem. primary glaucoma is recognized in some breeds, including the bassett hound, cocker spaniel, samoyed, bouvier des flandres, and some terrier breeds either from goniodysgenesis or a predisposition to lens luxation. other common causes of acute glaucoma are anterior uveitis and intumescent lens secondary to rapid cataract development, particularly in dogs with diabetes mellitus. treatment involves investigation of the underlying cause of the sudden rise in intraocular pressure and rapid reduction in intraocular pressure. permanent visual impairment is often associated with chronically buphthalmic globes or the presence of rippling or striae formation on the cornea. referral to a veterinary ophthalmologist is recommended. if the eye is still visual and not buphthalmic, the prognosis is favorable, depending on the cause of the acute glaucoma. treatment to reduce intraocular pressure consists of improving aqueous outflow, reducing intraocular volume with osmotic agents, and reducing aqueous formation (table 1 -47). the use of topical mydriatic agents in acute glaucoma is contraindicated because of the risk of making lens luxation or anterior uveitis worse. referral to a veterinary ophthalmologist for emergency surgery is indicated in cases of iris bombe, intumescent lens, or lens subluxation. administer osmotic agents to reduce the size of the vitreous body and the amount of aqueous. osmotic agents create an osmotic gradient between the intraocular fluids and the emergency management of specific conditions 203 vascular bed, thus allowing osmotic removal of fluid independent of the aqueous inflow and outflow systems. if no other treatments are available, oral glycerol (50%, 0.6 ml/kg or 1.4 g/kg) can be used to effectively reduce intraocular pressure. an adverse side effect of oral glycerol treatment is protracted vomiting. do not use glycerol in a diabetic patient. mannitol (1-2 g/kg iv over 1 hour) also effectively reduces intraocular pressure but does not cause vomiting. carbonic anhydrase inhibitors can be used to reduce intraocular volume by reducing aqueous production. oral administration of dichlorphenamide, methazolamide, and acetazolamide (2-4 mg/kg) is usually not very effective alone in reducing aqueous volume and intraocular pressure and also can cause metabolic acidosis. topical carbonic anhydrase inhibitors appear to be more effective (dorzolamide, trusopt) when used in conjunction with topical beta-blockers (timolol, 0.25% or 0.5% solution q8h). the most effective treatment for acute pressure reduction is use of a topical prostaglandin inhibitor (latanaprost). usually just one or two drops effectively reduces intraocular pressure in the emergency stages, until the patient can be referred to a veterinary ophthalmologist the following day. many clinical conditions that are presented as emergencies may be due in part or wholly to the presence of a neoplasm. paraneoplastic signs are summarized in table 1 -48. prompt identification of the neoplasia combined with knowledge of treatment, expected response to therapy, and long-term prognosis can aid owners and practitioners in making appropriate treatment decisions. hemorrhage or effusion can occur in any body cavity as a result of the presence of benign or malignant tumors. tumors secrete anticoagulants to allow angiogenesis to grow unchecked. hemorrhage often occurs as a result of rupture of a neoplasm or invasion of a neoplasm into a major vascular structure. effusion may be the result of direct fluid production by the mass or may be due to obstruction of lymphatic or venous flow. hemorrhagic effusions in the abdominal cavity occur most commonly with neoplastic masses of the spleen or liver. the most common causes are hemangiosarcoma and hepatocellular carcinoma. clinical signs associated with acute abdominal hemorrhage, regardless of the cause, are related to hypovolemic shock and decreased perfusion and include pale mucous membranes, tachycardia, anemia, lethargy, and acute collapse. treatment for abdominal hemorrhage includes placement of a large-bore peripheral cephalic catheter and starting one fourth of a shock dose (90 ml/kg/hour for dogs, and 44 ml/kg/hour for cats) of intravenous crystalloid fluids, taking care to carefully monitor perfusion parameters of heart rate, capillary refill time, mucous membrane color, and blood pressure. administer intravenous colloids such as dextran-70, hetastarch, and oxyglobin (5-10 ml/kg iv bolus) to restore intravascular volume and normotension. treat severe anemia with whole blood or packed rbcs to improve oxygen-carrying capacity and oxygen delivery (see sections on transfusion medicine and treatment of shock). confirm the presence of hemoabdomen abdominocentesis (see section on abdominocentesis). the presence of nonclotting hemorrhagic effusion is consistent with free blood. packed cell volume of the fluid is usually the same or higher than that of the peripheral blood. an abdominal compression bandage can be placed while further diagnostics are being performed. in cases of acute hemoabdomen, obtain right lateral, left lateral, and ventrodorsal or dorsoventral thoracic radiographs to help rule out obvious metastasis. monitor the patient's ecg and correct dysrhythmias as necessary (see section on cardiac dysrhythmias). surgery is indicated once the patient is stabilized. in some cases, hemorrhage is so severe that the patient should be taken immediately to surgery. when recommending surgery for a hemorrhaging intraabdominal mass, it is important to discuss likely diagnoses and long-term prognosis with the owner. hemangiosarcoma usually involves the spleen or liver or both. the presence of free abdominal hemorrhage is associated with a malignant tumor in 80% of cases. even when free abdominal hemorrhage is not present, the tumor is malignant in 50% of cases. approximately 66% (two thirds) of masses in the spleen are malignant (hemangiosarcoma, lymphoma, mast cell tumor, malignant fibrous histiocytoma, leiomyosarcoma, fibrosarcoma), and approximately one third are benign (hematoma, hemangioma). hepatocellular carcinoma usually affects one liver lobe (usually the left), and surgery is the treatment of choice. with complete surgical excision, median survival in dogs is longer than 300 days. if diffuse disease is observed at the time of surgery, the prognosis is poor. nonhemorrhagic effusions are associated with mesothelioma, lymphoma, carcinomatosis, or any mass that causes vascular or lymphatic obstruction. clinical signs of respiratory distress and abdominal distention with nonhemorrhagic effusions are usually slowly progressive in onset and not as severe as those observed with hemorrhage. treatment is usually aimed at identification of the underlying cause. obtain a fluid sample via thoracocentesis or abdominocentesis. to obtain further cells for cytologic evaluation, aspirate fluid from the thoracic or abdominal mass with ultrasound guidance. cytologic evaluation of the fluid will often elucidate the causative tumor type. an abdominal ultrasound can determine the degree of metastasis. perform therapeutic abdominocentesis or thoracocentesis if the effusion is causing respiratory difficulty. rapid re-accumulation of the fluid potentially can cause hypoproteinemia and hypovolemic shock. mesothelioma is a rare tumor most commonly observed in urban environments. in humans, mesothelioma has been associated with exposure to asbestos. it is sometimes difficult to differentiate between reactive mesothelial cells and malignant mesothelial cells. treatment is aimed at controlling the neoplastic effusion. intracavitary cisplatin has been demonstrated to slow rates of fluid re-accumulation, but is largely a palliative therapy. lymphoma is another tumor type that can cause thoracic or abdominal effusion. cytologic evaluation of the fluid usually reveals abundant lymphoblasts. treatment with multiagent chemotherapy protocols, with or without adjunctive radiation therapy, can prevent tumor remission and stop fluid accumulation. carcinomatosis occurs as a result of diffuse seeding of the abdominal cavity with malignant carcinomas and has a poor prognosis. carcinomatosis may occur de novo or from 1 metastasis of a primary tumor. treatment consists of fluid removal when respiratory difficulty occurs, with or without intracavitary cisplatin as a palliative measure. cisplatin should never be used in cats due to fatal acute pulmonary edema. clinical signs of hemorrhagic thoracic effusion include acute respiratory distress, anemia, hypovolemic or cardiogenic shock, and collapse. hemorrhagic thoracic effusions are rare in association with neoplastic effusions. a notable exception is intrathoracic hemorrhage in young dogs with osteosarcoma of the rib. hemorrhage can result when a primary lung tumor erodes through a vessel. hemangiosarcoma of the lungs or right auricular area can also result in hemorrhagic thoracic effusion. in many cases, hemorrhage may be confined to the pericardial sac with a right auricular mass, causing a globoid cardiac silhouette on thoracic radiographs. treatment consists of pericardiocentesis (see section on pericardial effusion and pericardiocentesis) and placement of a pericardial window, or the mass may be removed if it is in the right auricular appendage and resectable. although surgery can resolve clinical signs of right-sided heart failure, metastatic disease often develops soon afterward. nonhemorrhagic thoracic effusion is more common than hemorrhagic thoracic effusion, and is caused most commonly by mesothelioma, lymphoma, carcinomatosis, and thymoma. clinical signs develop gradually and include respiratory difficulty, cyanosis, and cough. supplemental oxygen should be administered. in many cases, thoracocentesis can be therapeutic and diagnostic. obtain thoracic radiographs both before and after thoracocentesis to determine whether a mass effect is present. following identification of a cause, definitive therapy can be instituted. mesotheliomas are rare and are associated with diffuse serosal disease. they are more common in dogs than in cats. effusions caused by mesotheliomas can affect the pleural or pericardial cavities. treatment is directed at removing effusion fluid and controlling reaccumulation with use of intracavitary platinum compounds, carboplatin, and cisplatin can be used in dogs. (cisplatin and carboplatin should never be used in cats.) chemical or physical pleurodesis may be helpful in controlling reaccumulation of fluid, but it is very painful in small animal patients. thoracic effusion secondary to lymphoma often is associated with an anterior mediastinal mass. t-cell lymphoma is the most common type of mediastinal mass observed in dogs. b-cell lymphoma is associated with a decreased response to chemotherapy and shorter survival times. treatment consists of combination chemotherapy with or without radiation therapy to decrease mass size. carcinomatosis is a diffuse disease of the pleural cavity that often is a result of metastasis from a primary pulmonary carcinoma or mammary adenocarcinoma. treatment is similar to that for mesothelioma and is aimed at controlling the effusion and delaying its recurrence. thymomas have been documented in both dogs and cats. dogs most commonly present with a cough, while cats present with clinical signs of respiratory distress and a restrictive respiratory pattern associated with the presence of pleural effusion. an anterior mediastinal mass is often observed on thoracic radiographs. in some cases, the pleural effusion must be drained via thoracocentesis before a mass is visible. ultrasound-guided aspiration and cytologic evaluation of the mass reveal a malignant epithelial tumor with small lymphocytes and mast cells. prognosis is good if the tumor can be completely excised. treatment consists of surgical removal with or without presurgical radiation therapy to shrink the mass. paraneoplastic syndromes of myasthenia gravis have been documented in dogs with thymomas. if megaesophagus or aspiration pneumonia is present, the prognosis is more guarded because of the high rate of complications. obstructive lesions affecting the urinary tract can be extramural (intra-abdominal, pelvic, or retroperitoneal) or intramural (urethral, bladder, or urethral wall) . transitional cell 1 carcinoma is the most common type of bladder tumor observed in dogs. prostatic adenocarcinoma, or neoplasia of the sublumbar lymph nodes (lymphoma, adenocarcinoma from apocrine gland adenocarcinoma), also can cause urethral obstruction. treatment is aimed at relieving the obstruction and then attempting to identify the cause of the disease. to alleviate the obstruction, pass a urinary catheter whenever possible. perform cystocentesis only as a last resort because of the risk of seeding the peritoneal cavity with tumor cells if transitional cell carcinoma is the cause of the obstruction. institute supportive therapy including intravenous fluids and correction of electrolyte abnormalities. plain radiographs may reveal a mass lesion or may not be helpful without double contrast cystography. abdominal ultrasound is more sensitive in identifying a mass lesion in the urinary bladder. masses in the pelvic urethra are difficult to visualize with ultrasonography. double contrast cystourethrography is preferred. once the patient is stabilized, biopsy or surgery is indicated to identify the cause of the mass and attempt resection. urine tests for transitional cell carcinoma are available for identification of transitional cell carcinoma in the dog. complete surgical excision of transitional cell carcinoma or removal of benign tumors of the urinary bladder yields a favorable prognosis. poorer prognosis is seen with incomplete excision. many transitional cell carcinomas are located in the trigone region of the bladder and cannot be completely excised. the nonsteroidal antiinflammatory drug piroxicam is helpful in alleviating clinical signs for a reported 7-month median survival. in some dogs, cisplatin and carboplatin may delay recurrence of transitional cell carcinoma. tumors of the prostate gland are always malignant and occur with equal frequency in castrated and uncastrated male dogs. diagnosis of prostatic tumors is based on ultrasonographic evidence of a mass effect or prostatomegaly and on transrectal or transabdominal aspiration or biopsy. surgery, chemotherapy, and radiation therapy generally are unrewarding over the long term, although palliative radiation therapy may relieve clinical signs for 2 to 6 months. luminal tumors of the gastrointestinal tract typically cause obstruction, with slowly progressive clinical signs including vomiting, inappetence, and weight loss, or with acute severe protracted vomiting. extraluminal obstructive lesions usually arise from adhesions, or strangulation may occur, resulting in obstruction. perforation of the mass through the gastric or intestinal wall can cause peritonitis. treatment consists of initial stabilization and rehydration, evaluation for evidence of metastasis, and surgical resection of the affected area in cases of adenocarcinoma, leiomyoma, leiomyosarcoma, and obstructive or perforated lymphoma. gastric and intestinal adenocarcinoma are the most common gastrointestinal tumors observed in dogs. affected animals typically have a history of anorexia, weight loss, and vomiting. obtain an abdominal ultrasound before performing any surgery. fine needle aspirates of the mass and adjacent lymph nodes are usually diagnostic and can determine whether there is local metastasis. many tumors are not resectable, and metastasis occurs in approximately 70% of cases. dogs with smaller tumors that can be resected typically have longer survival times. leiomyosarcomas occur in the intestines of dogs, and carry a more favorable prognosis than adenocarcinoma if the mass can be completely resected. with complete resection, the average survival time is longer than 1 year. the paraneoplastic syndrome of hypoglycemia has been observed with this tumor type. gastrointestinal lymphoma is the most common tumor of the gastrointestinal tract observed in cats. in comparison, it is relatively rare in dogs. unless there is complete obstruction or perforation of the gastrointestinal tract, surgical treatment for gastrointestinal lymphoma is not indicated. rather, multiple chemotherapy drugs are used in combination to achieve remission and resolution of the clinical signs of anorexia, weight loss, and vomiting. treatment responses unfortunately are poor. mast cell tumors of the gastrointestinal tract typically are manifested as gastrointestinal ulceration and hemorrhage in up to 83% of patients. the gastrointestinal hemorrhage that occurs with mast cell tumors results from increased acid secretion as a result of histamine receptor stimulation. treatment consists of histamine or proton pump inhibition (ranitidine, famotidine, cimetidine, or omeprazole). bowel perforation is a rare complication. many chemotherapy agents exert their effects on rapidly dividing normal and neoplastic cells. normal tissues that are commonly affected include the bone marrow, gastrointestinal tract, skin and hair follicles, and reproductive organs. some drugs have unique organspecific toxicities that must be monitored. knowledge and recognition of the expected type and onset of complications can alleviate their severity by rapid treatment, when complications occur (see table 1 -48) . neutropenia is the most common bone marrow toxicity observed secondary to chemotherapy in small animal patients (table 1 -49) . in most cases, the neutropenia is dose-dependent. the nadir, or lowest neutrophil count, is typically observed 5 to 10 days after chemotherapy treatment. once the nadir occurs, bone marrow recovery is observed, with an increase in circulating neutrophils within 36 to 72 hours (table 1 -49) . treatment of myelosuppression is largely supportive to treat or prevent sepsis. prophylactic antibiotics are recommended in the afebrile patient with a neutrophil count <2000/âµl. acceptable antibiotics include trimethoprim-sulfa and amoxicillin-clavulanate. granulocyte-colony stimulating factor (g-csf) (e.g., neupogen) is a recombinant human product that stimulates the release of neutrophils from the bone marrow, and its use shortens the recovery time following myelosuppressive drug therapy. disadvantages of g-csf include antibody production in response to the drug within 4 weeks of use and its high cost. to prevent ongoing neutropenia, subsequent chemotherapy dosages should be decreased by 25%, and the interval in between treatments increased. whenever possible, overlap of myelosuppressive drugs should be avoided. acute gastrointestinal toxicity can occur within 6 to 12 hours after administration of cisplatin and actinomycin d. in many cases, pretreatment with the antiemetics metoclopramide, butorphanol, chlorpromazine, dolasetron or ondansetron can prevent chemotherapyinduced nausea and vomiting. vomiting can also occur as a delayed side effect 3 to 5 days after treatment with doxorubicin (adriamycin), actinomycin d, methotrexate, and cytoxan. in delayed reactions, vomiting and diarrhea are caused by damage to intestinal crypt cells. treatment consists of administration of antiemetics, intravenous fluids, and a bland highly digestible diet. doxorubicin also can cause hemorrhagic colitis within 5 to 7 days of administration. treatment includes a bland diet, metronidazole, and tylosin tartrate (tylan powder). 1 emergency care mild to none not observed vincristine (low-dose), l-asparaginase, glucocorticosteroids moderate 7-10 days melphalan, cisplatin, mitoxantrone, actinomycin d severe 7-10 days doxorubicin, cyclophosphamide, vinblastine 1 paralytic ileus can be observed 2 to 5 days after administration of vincristine. this side effect is more common in humans than animals and can be treated with metoclopramide once a gastrointestinal obstruction has been ruled out. cardiotoxicity doxorubicin (adriamycin) causes a dose-dependent dilative cardiomyopathy when the cumulative dose reaches 100 to 150 mg/m 2 . in many cases, however, clinical signs do not occur until the cumulative dose is 240 mg/m 2 . the myocardial lesions are irreversible. treatment of cardiac dysrhythmias is dependent on the type of dysrhythmia (see section on treatment of dysrhythmias). discontinue doxorubicin and administer diuretics and positive inotropic therapy for dilative cardiomyopathy in order to delay the progression of congestive heart failure (see sections on treatment of congestive heart failure). if abnormalities are shown on electrocardiography performed before beginning therapy, substitute liposome-encapsulated doxorubicin or mitoxantrone substituted in the chemotherapy protocol. cardioprotectant drugs such as vitamin e, selenium, and n-acetyl cysteine have shown some promise in the prevention of doxorubicin-induced cardiotoxicity. cyclophosphamide can cause a sterile hemorrhagic cystitis. damage to the urinary bladder mucosa and vessels is caused by the toxic metabolite acrolein. clinical signs of sterile hemorrhagic cystitis include a history of cyclophosphamide administration, stranguria, hematuria, and pollakiuria. treatment for sterile hemorrhagic cystitis is discontinuation of the drug, treatment of any underlying urinary tract infection with antibiotic therapy based on susceptibility testing, and intravesicle drug administration. in extremely refractory cases, surgical debridement and cauterization of the bladder mucosa may be necessary. prevention of sterile hemorrhagic cystitis includes emptying the bladder frequently and administering the drug in the morning. concurrent administration of prednisone can induce polyuria and polydipsia. if sterile hemorrhagic cystitis occurs, chlorambucil can be substituted as a chemotherapeutic agent. anaphylactic reactions have been observed with the administration of l-asparaginase, adriamycin, etoposide, and paclitaxel. the risk of anaphylaxis increases with repeated administration, although in some animals anaphylaxis will occur on the first exposure to the drug. treatment consists of administration of epinephrine, diphenhydramine, famotidine, and glucocorticosteroids, as with any other life-threatening allergic reaction (see section on treatment of allergic reactions). to decrease the risk of an adverse reaction, give diphenhydramine (2.2 mg/kg im) 15 to 30 minutes before drug administration. slowing the rate of intravenous infusion also can decrease the chance of an anaphylactic reaction. cisplatin can cause a fatal irreversible pulmonary edema in cats, even at low dosages. 5-fluorouracil (5-fu) can cause a severe neurotoxicity in cats that results in ataxia and seizures. never use cisplatin or 5-fu in cats. poisoning cases benefit from a rapid, organized approach. key points in this approach are giving appropriate advice over the telephone, being able to access information sources, and providing appropriate treatment. there are only a few classes of poisons that account for the majority of toxicities reported in dogs and cats. every veterinarian should develop a familiarity with the clinical management of rodenticide and insecticide toxicity and be prepared with antidotes on hand. beyond the most common toxins, the spectrum of possibilities is endless, and the veterinarian must rely on appropriate information resources. it is important to have available a comprehensive source of pharmaceutical and plant identification resources. remarkably, considering the myriad of potentially toxic substances to which an animal can be exposed, relatively few specific antidotes are commonly used in veterinary medicine. because of the lack of specific antidotes, the veterinarian must treat each toxicity with general methods of poison management, applying basic critical care in the treatment of specific clinical signs associated with the poison exposure or toxicity. the adage "treat the patient, not the poison" often comes into play when the exact toxic substance is unknown, or has no specific antidote. before an animal arrives, the staff should be prepared to ask specific questions over the phone, and provide initial advice for clients, particularly if the animal lives some distance from the hospital (box 1-55.) it is important to have access to a database of information on toxic substances. thousands of potentially toxic substances are available on the market today. the american society for the prevention of cruelty to animals (aspca) animal poison control center provides direct access to veterinary toxicologists 24 hours a day, 365 days a year. for additional information, call the nearest veterinary school or emergency center (box 1-56). also, see section 6 for a table of emergency hotlines. check your local telephone book for a poison control center listing under emergency numbers, usually found on the front cover. although these numbers are for human poisonings, they have access to extensive poison and toxin databases and can potentially provide useful information for veterinarians, particularly regarding antidotal substances suitable for out of the ordinary toxins and human medications. information on the toxic ingredients in thousands of medications, insecticides, pesticides, and other registered commercial products has been confidentially placed by the government in these poison control centers. as new products are marketed, information regarding toxin ingredients is forwarded to the centers. various e-mail discussion lists can serve as an informative resource for practitioners, but access generally requires an initial subscription and may have the disadvantage of delayed 1 *do not keep the client on the telephone for too long. lengthy histories can be performed once the animal is at your hospital and you have started to initiate treatment. â�  hair dressing products sometimes have hydrogen peroxide as a 30% w/v; this concentration is not suitable for induction of emesis. is your animal breathing or does it have respiratory difficulty? what is the color of the gums or tongue? is your animal able to walk? is there any vomiting, diarrhea, trembling, or seizures? does it appear lethargic or hyperactive? what is the substance that your animal ingested (was exposed to)? did you witness the ingestion or exposure? how much did the animal consume? how long ago was the exposure? was the substance swallowed, or is it on the animal's skin or eyes? how is the patient acting? how long has the animal been acting that way? or when was the last time you saw your animal act normally? 2. first aid instructions for the client: induce vomiting at home and save the vomitus. never induce vomiting if the patient is depressed, appears comatose, or is actively seizing. if the animal has ingested a caustic substance (strong alkali or acids) or a petroleum-based product (kerosene or turpentine), never recommend induction of emesis. hydrogen peroxide (3% w/v â�  ) 5 ml = 1 tsp/10 lb of body weight can repeat once if no vomiting occurs after 10 minutes 3. remind the owner to bring a sample of the toxin and the vomitus in with the patient. 4. advise the owner to transport the patient as rapidly as possible to the nearest veterinary hospital. 1 response times. they are useful for ideas on standard and long-term therapy, but not emergency stabilization. an exception to this is the veterinary interactive network (vin), which posts message board communications. previous communications from veterinarians who treated a case with the same poison/toxin can be accessed with a subscription. many manufacturers operate an information service about their products. if the product label or name is available, check for a telephone number that may route you to a specialist. there are six essential steps in treating toxicities: 1. performing a physical examination 2. stabilizing the patient's vital signs 3. taking a thorough history 4. preventing continued absorption of the toxin 5. administering specific antidotes when available 6. facilitating clearance or metabolism of the absorbed toxin it is most important to provide symptomatic and supportive care both during and following emergency treatment. immediately on presentation, perform a brief but thorough physical examination. obtain a minimum database as well as serum, urine, or orogastric lavage samples for later toxicologic analyses. it is important at this time to systematically evaluate the patient's physical status, focusing particularly on the toxins most common to a particular geographic location and the organ systems most commonly affected by toxins in veterinary medicinenamely, the neurologic and gastrointestinal tracts. a checklist is useful when performing a complete physical examination (box 1-57). the minimium database includes a urine sample, packed cell volume, total protein, serum urea, and serum glucose. the information obtained from these simple cage-side tests is useful for determining dehydration, hemoconcentration, azotemia (renal or prerenal), and hypo-or hyperglycemia. when appropriate, obtain samples for serum biochemistry profiles, serum electrolytes, blood gases, serum osmolality, a complete hemogram, and coagulation profiles. samples of serum, urine, and any vomitus or orogastric lavage contents should be collected and saved for later toxicologic analyses as required later. stabilization of vital signs includes four major goals of treatment: maintain respiration, maintain cardiovascular function, control cns excitation, and control body temperature. in any patient with clinical signs of respiratory distress or respiratory dysfunction, supplemental oxygen should be administered via flow-by, oxygen hood, oxygen cage, nasal, nasopharyngeal, or transtracheal oxygen sources. ventilatory assistance may be necessary. irritant or corrosive substances can cause damage to the oropharyngeal mucosa to such an extent that airway obstruction occurs. when necessary, a temporary tracheostomy should be performed. arterial blood gases, pulse oximetry, and capnometry may be required to monitor oxygenation and ventilation. at the time of presentation, immediately place an intravenous catheter for administration of intravenous fluids, inotropes, antiarrhythmics, and antidotes, if necessary. the initial fluid of choice is a balanced crystalloid solution such as normosol-r, plasmalyte-m, or lactated ringer's solution. fluid therapy can later be changed based on the patient's acidbase and electrolyte status. some toxins can cause severe dysrhythmias and hyper-or hypotension. monitor blood pressure and perform ecg and correct any abnormalities according to standard therapy (see sections on hypotension and cardiac dysrhythmias). what is the pupil size? what is the pupil reactivity to light? is the ocular examination normal? what is the sensitivity to light or sound? nose: is it moist, dry, bubbling, or frothy, or caked with dirt? throat: are there any characteristic odors on the breath? are there any traces of foreign material on the tongue or in the crevices of the teeth or gums? are there petechiae or ecchymosis on the gums or bleeding from the gumline? what is the mucous membrane color? is it normal and pink, or dark red (injected), pale, or icteric? what is the capillary refill time? is it fast, normal, or slow? what is the patient's heart rate? are there any pulse deficits or dysrhythmias auscultated? what is the patient's blood pressure? what is the quality of the femoral pulse? is it synchronous with the heart rate, or are there dropped pulses? is the pulse bounding, normal, thready, or not palpable? what is the patient's electrocardiogram? what is the patient's respiratory rate? what is the patient's respiratory character? is it normal, fast, shallow, or labored? what do you hear on thoracic auscultation? do you hear harsh airway sounds or pulmonary crackles? what is the patient's rectal temperature? is there excessive salivation? is there evidence of vomiting or diarrhea? is abdominal palpation painful? do the intestinal loops feel normal, or are they fluid-filled or gas-filled? what is the color and consistency of the feces? is there a palpable urinary bladder? is there urine production? what is the color of the urine? peripheral lymph nodes should be normal in poisonings. some toxins cause hemolysis, methemoglobinemia, heinz body anemia, and coagulopathies. whole blood, fresh frozen plasma, packed rbcs, or hemoglobin-based oxygen carriers should be available and used if necessary. treat methemoglobinemia with a combination of ascorbic acid and n-acetylcysteine. many toxins affect the cns, producing clinical signs of excitation and/or seizures. diazepam is the drug of choice for most but not all seizures and tremors. if an animal has cns excitation secondary to the ingestion of selective norepinephrine reuptake inhibitors, avoid using diazepam, as it can potentially exacerbate clinical signs. muscle relaxants such as guaifenesin or methocarbamol may be required to control muscle spasm and tremors associated with some toxicities. consider animals that are in status epilepticus because of toxin exposure at high risk. such patients may not require the full dose of anesthetics or sedatives for seizure control. give phenobarbital (16-20 mg/kg iv) or pentobarbital (3-25 mg/kg iv to effect) for longer-term management of seizures. core body temperature can easily increase or decrease secondary to increased muscle activity or coma. animals may present as hypo-or hyperthermic, depending on the toxin ingested and the stage of toxicity. manage hypothermia with circulating hot water or hot air blankets, or place bubble wrap or saran wrap around the animal's peripheral extremities. manage hyperthermia by placing lukewarm wet towels on the patient until the rectal temperature has decreased to 39.5â°c (103â°f). (see section on of hyperthermia and heat-induced illness). if sedatives or anesthetics have been used, initial hyperthermia may initially resolve due to hypothalamic loss of thermoregulatory control, cool water bathing should not be performed. when the patient is first presented to the veterinarian, have the owner complete a toxicologic history form (figure 1-56) while the animal is being initially assessed and vital signs are being stabilized. when initial stabilization of vital signs has been accomplished, the veterinarian can discuss the patient's history with the owner. in urgent situations, the veterinarian should obtain a brief history as an initial procedure (box 1-58). knowing when the animal was last seen as normal provides a time frame in which the toxic substance was most likely accessed, allowing differential diagnoses to be ranked in some order of probability by rate of onset. in eliciting a history from the owner about the animal's access to poisons, it is important not to take anything for granted. many owners do not realize how poisonous some substances can be, such as insecticide products, garbage, cleaning chemicals, and over-the-counter drugs commonly used by humans. many owners will deny that an animal could have ingested anything that might be toxic, not wanting to believe that the source of the toxin is within their household or property, particularly if recreational drug exposure is suspected. it is useful to phrase questions in a neutral fashion-for example, "is such-and-such present on the premises?" rather than "could the dog have eaten such-and-such?" if recreational drug exposure is suspected, another way to question the owners is to ask whether they have had any guests in their house recently that may have had such-and-such (e.g., marijuana, cocaine, methamphetamine). this approach serves to minimize the suggestion of any bias or preconceptions. when questioning an owner about recent events, it is useful to realize and acknowledge that disruption in the household routine is a distinct factor in the occurrence accidents, including poisonings. examples of such disruptive events include moving from the house, family member is ill or in the hospital, and renovations or recent construction. while these events are occurring, the safeguards followed by a normally careful owner may be disrupted. often, doors or gates may be left open, animals may be outside instead of inside (or vice versa), and inexperienced people may be pet-sitters. once owners are made aware of the importance of assessing such risks, they are often able to provide insight into otherwise baffling circumstances. various methods can be used to remove toxins from the gastrointestinal tract, including emesis, orogastric lavage, cathartics, and enemas. adsorbents, ion exchange resins, or 1 precipitating or chelating agents may be used. removal of a toxic substance from the body surface may be necessary, depending on the toxin.the use of both emesis and orogastric lavage is less and less frequent in human medicine because of the risk of aspiration pneumonia and doubts about their efficacy. currently, management of poisonings in human medicine relies heavily on the use of activated charcoal combined with sorbitol as a cathartic, when appropriate, and supportive critical care. it should be emphasized, however, that the majority of poisonings in humans are due to drug overdoses (illicit or otherwise) (which have a relatively small volume and rapid absorption), for which this treatment is appropriate. furthermore, adoption of the approach rests on the availability of a hospital intensive care infrastructure, which is not always available in veterinary practice. induce emesis if the animal's physiology and neurologic status are stable (i.e., does not have respiratory depression or is not actively seizing, obtunded, unable to swallow or protect its airway). do not administer the same emetic more than twice. if the emetic doesn't work after two doses, give a different emetic or perform orogastric lavage under general anesthesia. emetics are strictly contraindicated for toxicity from petroleum-based products and corrosives because of the risk of aspiration pneumonia and further esophageal damage. emetics may also be of little value if poisons with antiemetic properties have been ingested, such as benzodiazepines, tricyclic antidepressants, and marijuana (table 1-50) . various emetics traditionally have been recommended for use in veterinary medicine. many have fallen out of favor because of the risk of causing adverse consequences and side effects. apomorphine (0.04 mg/kg iv or in the conjunctival sac) remains the standard but is less useful in certain situations in which the poison causes cns excitation or stimulation. it is ineffective in cats. other emetics include xylazine and hydrogen peroxide. do not use table salt because of the risk of severe oropharyngeal irritation and hypernatremia. do not use mustard powder or dishwashing liquid detergent because of the risk of severe oropharyngeal, esophageal, and gastric irritation. orogastric lavage is described in detail in the section on emergency procedures gastric lavage is contraindicated in treatment of toxicity from petroleum-based compounds and acid/alkali ingestion. the procedure can be messy but is very effective if performed within 1 to 2 hours of ingestion of the poison. to prevent aspiration, the patient should be placed under general anesthesia. keep the animal's head lowered during the procedure to prevent aspiration of stomach contents into the trachea. it is sometimes helpful to put the animal in both right and left lateral recumbency to allow complete emptying of gastric contents. repeat the procedure until the fluid runs clear from the stomach. in some cases in which solid material has been ingested, this process can take a long time, so be prepared with a large volume of warm water. following successful evacuation and lavage, administer a slurry of activated charcoal through the orogastric tube before removing it. keep the endotracheal tube cuffed and in place until the animal is semi-conscious, is starting the fight the tube, and is visibly able to swallow and protect its airway. â�¢ when was the animal last seen as normal? â�¢ what clinical signs developed? â�¢ how fast did the clinical signs develop? â�¢ when was the onset of clinical signs? â�¢ what is the animal's activity level? â�¢ does the animal have access to any poisonous substances? â�¢ this includes known toxins or chemicals, over-the-counter or prescription medications (including the owner's), and recreational drugs. enemas are useful to facilitate the action of cathartics and in cases in which the poison is a solid material (e.g., compost, snail bait, garbage) (box 1-59). it is best to use just lukewarm water. commercially available phosphate enema solutions can cause severe electrolyte disturbances (hyperphosphatemia, hyponatremia, hypocalcemia, and hypomagnesemia) and acid-base abnormalities (metabolic acidosis); therefore, they are absolutely contraindicated in small animal patients. use nonsterile nonspermicidal water-soluble lubricants (k-y jelly) old intravenous fluid bag enema bag 60-to 120-ml syringe fluid warm water, with or without hand or liquid dish soap the fluid volume required depends on the size of the animal and the state of its lower gastrointestinal tract. as with orogastric lavage, continue the procedure until the water runs clear. if difficulty is encountered emptying the lower gastrointestinal tract, repeat the enema in 1 or 2 hours, rather than be overzealous on the first attempt. cathartics are useful for hastening gastrointestinal elimination of toxins, and they are particularly useful for elimination of most solid toxicants (e.g., compost, garbage, snail baits). cathartics can be used in conjunction with activated charcoal. do not use magnesium-based cathartics in patients with cns depression, because hypermagnesemia can worsen this disorder and also cause cardiac rhythm disturbances (table 1-51) . activated charcoal (1-4 ml/kg) is the safest and to date the most effective adsorbent for the treatment of ingested toxins. activated charcoal can be administered after emesis or orogastric lavage or can be administered as the sole treatment. various preparations are available on the market, including dry powder, compressed tablets, granules, liquid suspensions, and concentrated paste preparations. commercially available products are relatively inexpensive and should be used whenever possible for ease of administration. vegetableorigin activated charcoal is the most efficient adsorbent and binds compounds with weak, nonionic bonds. some preparations are combined with sorbitol to provide simultaneous administration of an adsorbent and a cathartic; this combination has been shown to be most efficacious. repeated administration of activated charcoal every 4 to 6 hours has been shown to be beneficial in the management of a toxin that undergoes enterohepatic recirculation. administration of an oily cathartic or mixing the activated charcoal with food only serves to reduce the absorptive surface of the activated charcoal and therefore is not recommended. in general, substances that are very soluble and are rapidly absorbed are not well adsorbed by activated charcoal, including alkalis, nitrates, mineral acids, ethanol, methanol, ferrous sulfate, ammonia, and cyanide. kaolin and bentonite are clays that have been used as adsorbents. both are usually less effective than activated charcoal. however, they are reported to be better adsorbents than activated charcoal for the herbicide paraquat. ion exchange resins can ionically bind certain drugs or toxins. cholestyramine is one such resin, commonly used in human medicine to bind intestinal bile acids and thereby decrease cholesterol absorption. its application in toxicology extends to the absorption of fat-soluble toxins such as organochlorine and certain acidic compounds such as digitalis. ion exchange resins also have been used to delay or reduce the absorption of phenylbutazone, warfarin, chlorothiazide, tetracycline, phenobarbital, and thyroid preparations. precipitating, chelating, and diluting agents precipitating, chelating, and diluting agents are used primarily in the management of heavy metal intoxications, such as alkaloids or oxalates. they work by binding preferentially to the metal ion and creating a more soluble complex that is amenable to renal excretion. those chelating agents in common usage are calcium edta, deferoxamine, and d-penicillamine. calcium edta and deferoxamine should both be on hand in the veterinary hospital because they are necessary to treat zinc and iron toxicity, respectively, both of which have a short window of opportunity for therapeutic intervention. d-penicillamine has a wide application for a number of metal toxicities but tends to be used for long-term chronic therapy because it can be administered orally. various agents used for nonspecific dilution of toxins, including milk of magnesia and egg whites, although old-fashioned, still have wide application in many cases in which low-grade irritants have been ingested. bathing the animal is an important aspect of treatment for topical exposures to toxins such as insecticidal products, petroleum-based products, and aromatic oils. bathing an animal is not an innocuous procedure. to avoid hypothermia and shock, use warm water at all times. actively dry the animal to further minimize the risk of hypothermia. when bathing the animal, use rubber gloves and a plastic apron to avoid exposure to noxious agents. in most cases, a mild dishwashing soap is appropriate. medicated or antibacterial shampoos are less appropriate in this situation. for petroleum-based products in particular, dawn dishwashing liquid that "cuts the grease" works well to remove the oils. if dawn is not available, mechanics' hand cleaners or coconut oil-based soaps can be used instead. as a general principle, best results are obtained by barely wetting the patient's fur until the detergent is worked well into the fur, keeping the amount of water to a minimum until ready for the rinse. oil-based paint is best removed by clipping rather than by attempting removal with solvents, because solvents are also toxic. to remove powder products, brush and vacuum the animal before bathing it to eliminate further toxic exposure. with caustic alkaline or acidic products, the primary treatment is to dilute and flush the skin with warm water; do not attempt neutralization. neutralization can cause an exothermic reaction that causes further damage to the underlying tissues. eliminating poison from the eyes for ocular exposures, irrigate the eyes for a minimum of 20 to 30 minutes with warm (body temperature) tap water or warmed 0.9% sterile saline solution. the use of neutralizing substances is not recommended because of the risk of causing further ocular damage. following adequate irrigation, treat chemical burns of the eyes with lubricating ointments and possibly a temporary tarsorrhaphy. atropine may be indicated as a cycloplegic agent. systemic nonsteroidal antiinflammatory drugs can be used to control patient discomfort. daily follow-up examinations are required because epithelial damage may be delayed, especially with alkali burns, and it is difficult to predict the final extent of ocular damage. topical glucocorticosteroids are contraindicated if the corneal epithelium is not intact. if severe conjunctival swelling is present with a corneal ulcer, parenteral glucocorticosteroids can be administered to help alleviate inflammation, but nonsteroidal antiinflammatory drugs should not be used simultaneously due to the risk of gastrointestinal ulceration or perforation. whenever possible, administer specific antidotes to negate the effects of the toxin and prevent conversion of the substance to the toxic metabolite. three categories of agents are used in the management of poisonings. the first category is specific antidotes. unfortunately, few specific antidotes are available for use in veterinary medicine. some "classic" toxins and antidotes are now considered to be rare, such as curare and physostigmine, thallium and prussian blue, and fluoride and calcium borogluconate. these and a few others have been omitted from the table. the second, broader category of antidotes includes those drugs used in the symptomatic management of clinical signs, which are part of our routine veterinary stock. drugs such as atropine, sedatives, steroids, antiarrhythmics, and beta-blockers fall into this category. the third category comprises nonspecific decontaminants such as activated charcoal, cathartics, and emetics. these were discussed previously. many patients benefit from efforts to enhance clearance or metabolism of the absorbed toxins. some specific therapies have been developed for this purpose, including 4-methylpyrazole for ethylene glycol toxicity and specific antibodies such as digibind (digoxin immune fab [ovine]) for digitalis toxicity. other strategies are aimed at promoting renal excretion. renal excretion strategies include diuresis, ion trapping, and peritoneal dialysis or hemodialysis (see section on peritoneal dialysis). diuresis and ion trapping are applicable to a large number of toxins and are discussed here in more detail. other toxins respond to urine acidification and urine alkalinization. enhancing renal excretion of substances is most useful for those organic substances that are present in significant concentrations in the plasma. substances that are non-ionic and lipid-soluble, such as certain herbicides, are likely to be less affected by attempts to promote rapid renal elimination. before starting diuresis or ion trapping, intravenous fluid therapy should be adequate as determined by normal central venous pressure, urine output, and mean arterial blood pressure. if any of these values are less than normal, use other measures to ensure adequate renal perfusion, including but not limited to a constant rate infusion of dopamine. simple fluid diuresis can influence the excretion of certain substances. the use of mannitol as an osmotic diuretic may reduce the passive reabsorption of some toxic substances in the proximal renal convoluted tubule by reducing water reabsorption. dextrose (50%) can be used as an osmotic diuretic. furosemide can be used to promote diuresis, but again, there is no substitute for intravenous fluid therapy. the use of mannitol, dextrose, and furosemide is contraindicated in hypotensive or hypovolemic patients. take care to avoid causing dehydration with any diuretic; central venous pressure monitoring is strongly recommended. ion trapping is based on the principle that ionized substances do not cross renal tubular membranes easily, and are not well reabsorbed. if the urinary ph can be changed so that the toxin's chemical equilibrium shifts to its ionized form, then that toxin can be "trapped" in the urine and excreted. alkaline urine favors the ionization of acidic compounds, and acidic urine favors the ionization of alkaline compounds. those toxins that are amenable to ion trapping are mostly weak acids and weak bases. ammonium chloride can be used to promote urinary acidification. contraindications to the use of ammonium chloride include a preexisting metabolic acidosis, hepatic or renal insufficiency, and hemolysis or rhabdomyolysis leading to hemoglobinuria or myoglobinuria. signs of ammonia intoxication include cns depression and coma. when performing urine acidification, frequently check the serum potassium concentration and urine ph. urine alkalinization can be performed with use of sodium bicarbonate. contraindications to the use of sodium bicarbonate include metabolic alkalosis (particularly with concurrent use of furosemide), hypocalcemia, and hypokalemia. as with urine acidification, monitor the serum potassium concentration and urine ph frequently. the major steps in management of poisonings discussed here must be accompanied by application of the fundamentals of critical care. respiratory and cardiovascular support have been discussed previously. renal and gastrointestinal function and analgesia are particularly important in the management of the poisoning patient. maintenance of renal perfusion is a priority in the poisoning patient. fluid, electrolyte, and acid-base balance must be controlled and be accurate. poisoning patients are at particularly high risk for renal damage and acute renal failure, whether by primary toxic insult to the renal parenchyma or by acute or prolonged renal hypoperfusion. for this reason, a protocol that aims at preventing oliguria and ensuing renal failure is one of the therapeutic strategies that should be routinely employed. this protocol is described in box 1-60. gastrointestinal protectant drugs may be indicated for the management of those poisons that are gastrointestinal irritants or ulcerogenic. commonly used gastroprotectant drugs include cimetidine, ranitidine, famotidine, omeprazole, sucralfate, and misoprostol. antiemetics may be used to suppress intractable vomiting. metoclopramide is commonly used, and it is the drug of choice for centrally mediated nausea. antiemetics that work by different mechanisms can be used in combination as necessary. examples are dopamine 2-receptor antagonists such as prochlorperazine, 5-hydroxytryptamine antagonists such as ondansetron and dolasetron, and h-1 receptor antagonists such as diphenhydramine and meclizine. analgesics are more appropriate to treat poisonings than once thought. common effects of poisons including severe gastroenteritis and topical burns or ulcerations may warrant the use of analgesics. longer-acting analgesics such as morphine, hydromorphone, and buprenorphine are particularly useful. nutritional support may be necessary in the form of enteral or parenteral feeding in patients that have esophageal or gastric damage or that need to be sedated for long periods of time. endoscopy may be useful in assessing the degree of esophageal and gastric damage, particularly after ingestion of caustic substances. introduction: acetaminophen (paracetamol) is the active ingredient in tylenol and many over-thecounter cold products. acetaminophen is converted to n-acetyl-p-benzoquinonimine in the liver, a toxic substance that can cause oxidative injury of red blood cells and hepatocytes. clinical signs of acetaminophen toxicity include respiratory distress from lack of oxygen-carrying capacity, cyanosis, methemoglobinemia (chocolate-brown appearance of the blood and mucous membranes), lethargy, vomiting, and facial and paw swelling (cats). the toxic dose of acetaminophen is >100 mg/kg for dogs, and 50 mg/kg for cats. treatment of acetaminophen toxicity includes induction of emesis or orogastric lavage if the substance has been ingested within 30 minutes. activated charcoal should also be administered. in cases of severe anemia, give supplemental oxygen along with a packed rbc transfusion. administer intravenous fluids to maintain renal and hepatic perfusion. n-acetylcysteine, vitamin c, and cimetidine are the treatments of choice for methemoglobinemia in patients with acetaminophen toxicity. introduction: hydrochloric, nitric, and phosphoric acids cause chemical burns through contact with the skin and/or eyes. localized superficial coagulative necrosis occurs upon contact. usually, the patient's skin is painful to the touch or the animal may lick or chew at an irritated area that is not visible under the haircoat. if the chemical is swallowed, do not induce emesis or perform orogastric lavage, because of the risk of worsening esophageal irritation. rinse the patient's skin and eyes with warm water or warm saline for a minimum of 1 /2 hour. use analgesics and treat corneal ulcers (see section on corneal ulcers) as required. do not attempt chemical neutralization, because of the risk of causing an exothermic reaction and worsening tissue injury. aflatoxin (aspergillus flavus) is found in moldy feed grains. clinical signs of toxicity occur after ingestion and include vomiting, diarrhea, and acute hepatitis; abortion may occur in pregnant bitches. treatment of suspected aflatoxin ingestion consists of gastric decontamination, administration of activated charcoal, intravenous fluids, and hepatic supportive care (s-adenosyl methionine [same], milk thistle). drinking (ethanol), rubbing (isopropyl), and methyl (methanol) alcohols can be harmful if ingested (4.1 to 8.0 g/kg po). all cause disruption of neuronal membrane structure, impaired motor coordination, cns excitation followed by depression, and stupor that can lead to cardiac and respiratory arrest, depending on the amount ingested. affected animals may appear excited and then ataxic and lethargic. contact or inhalant injury can occur, causing dermal irritation and cutaneous hyperemia. methanol also can cause hepatotoxicity. 1 and diarrhea result from muscarinic overload. nicotinic overload produces muscle tremors. toxicity can result in seizures, coma, and death. 1 and cause severe irritation and corrosion of the mucous membranes and skin. some compounds also can cause clinical signs similar to those observed with anticholinesterase compounds, including muscle tremors, seizures, paralysis, and coma. methemoglobinemia can occur. 1 signs of ethylene glycol intoxication and renal impairment or failure, a negative test for the presence of calcium oxalate crystalluria means that there is no more ethylene glycol in the patient's serum because it has all been metabolized. cats are very sensitive to the toxic effects of ethylene glycol. in many cases, cat may have ingested a toxic dose, but because the sensitivity of the assay is low, test results will be negative. lack of treatment can result in death. there are three phases of ethylene glycol intoxication. in the first 1 to12 hours after ingestion (stage i), the patient may appear lethargic, disoriented, and ataxic. in stage ii (12 to 24 hours following ingestion), the patient improves and appears clinically normal. in stage iii (24 to 72 hours following ingestion), the patient demonstrates clinical signs of renal failure (polyuria and polydipsia) that progress to uremic renal failure (vomiting, lethargy, oral ulceration). finally, seizures, coma, and death occur. crosses, old english sheepdogs, and some terriers. clinical signs of ivermectin toxicity include vomiting, ataxia, hypersalivation, agitation, tremors, hyperactivity, hyperthermia, hypoventilation, coma, seizures, signs of circulatory shock, bradycardia, and death. clinical signs often occur within 2 to 24 hours after ingestion or iatrogenic overdose. blood ivermectin levels can be measured, but diagnosis is often made based on clinical signs and knowledge of exposure in predisposed breeds. there is no known antidote. the clinical course can be prolonged for weeks to months before recovery occurs. to treat known exposure, induce emesis or perform orogastric lavage if the substance was ingested was within 1 hour of presentation and the patient is not symptomatic. administer activated charcoal. control seizures with phenobarbital, pentobarbital, or propofol administered as intermittent boluses or as a constant rate infusion. diazepam, which potentially can worsen central nervous stimulation, is contraindicated. administer intravenous fluids to maintain perfusion and hydration, and treat hyperthermia. supportive care may be necessary, including supplemental oxygen (or mechanical ventilation, if necessary), frequent turning of the patient and passive range-of-motion exercises, placement of a urinary catheter to maintain patient cleanliness and monitor urine output, lubrication of the eyes, and parenteral nutrition (see section on rule of twenty). specific antidotes used to treat ivermectin toxicity include physostigmine and picrotoxin. physostigmine therapy was beneficial in some patients for a short period; picrotoxin caused severe violent seizures and therefore should be avoided. introduction d-limonene and linalool are components of citrus oil extracts used in some flea control products. the toxic dose is unknown, but cats appear to be very sensitive to exposure. clinical signs of toxicity include hypersalivation, muscle tremors, ataxia, and hypothermia. treatment of d-limonene and linalool exposure includes treatment of hypothermia, administration of activated charcoal to prevent further absorption, and careful, thorough bathing to prevent further dermal exposure. lead is ubiquitous, and is found in some paints, car batteries, fishing equipment/ sinkers, and plumbing materials. lead can be toxic at doses of 3 mg/kg. if more than than 10-25 mg/kg of lead is ingested, death can occur. lead causes toxicity by inhibiting sulfur-containing enzymes, leading to increased rbc fragility, and cns damage. clinical signs of hyperexcitability, dementia, vocalization, seizures, and lower motor neuron polyneuropathy can occur. affected animals may appear blind, or vomiting, anorexia, and constipation or diarrhea may occur. if lead toxicity is suspected, blood and urine lead levels can be measured. treatment of lead toxicity is supportive and is directed at treatment of clinical signs. control seizures with diazepam or phenobarbital. if cerebral edema is present, administer mannitol (0.5-1.0 g/kg iv), followed by furosemide (1 mg/kg iv 20 minutes after mannitol). sodium or magnesium sulfate should be administered as a cathartic. initiate chelation therapy with dimercaprol, penicillamine, or calcium edta. if a lead object is identified in the gastrointestinal tract on radiographs, remove the object using endoscopy or exploratory laparotomy. 1 hyperthermia, that occurs within 15 -30 minutes of ingestion. diarrhea and convulsions can develop. if hyperthermia is severe, renal failure secondary to myoglobinuria and disseminated intravascular coagulation can result. delayed hepatic failure has been described days after initial recovery. if metaldehyde toxicosis is suspected, analysis of urine, serum, and stomach contents is warranted. to treat metaldehyde toxicity, procure and maintain a patent airway and control cns excitation and muscle tremors. if an animal has just ingested the metaldehyde and is not symptomatic, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by administration of one dose of activated charcoal. administer intravenous fluids to control hyperthermia, prevent dehydration, and correct acid-base and electrolyte abnormalities. methocarbamol is the treatment of choice to control muscle tremors. diazepam can be used to control seizures if they occur. introduction mushroom ingestion most commonly causes activation of the autonomic nervous system, resulting in tremors, agitation, restlessness, hyperexcitability, and seizures. in some cases slud (salivation, lacrimation, urination, and defecation) is seen. some mushrooms (amanita spp.) also can cause hepatocellular toxicity. clinical signs include vomiting, anorexia, lethargy, and progressive icterus. 1 hemoglobinuria and pigment damage of the renal tubular epithelium. heinz bodies may be observed on cytologic evaluation of the peripheral blood smear. 1 paint in a sorbitol or glycerol carrier. when large quantities of these osmotically active sugars are ingested, osmotic shifts of fluid cause a sudden onset of neurologic or gastrointestinal signs, including ataxia, seizures, and osmotic diarrhea caused by massive fluid shifts into the gastrointestinal tract. the loss of water in excess of solute can result in hypernatremia, a free water deficit, and increased serum osmolality. following orogastric lavage, treatment of ingestion includes administering warm water enemas to help speed the movement of the paintballs through the gastrointestinal tract. do not administer activated charcoal (usually in a propylene glycol carrier), because the compound's cathartic action will pull more fluid into the gastrointestinal tract. baseline electrolytes should be obtained and then carefully monitored. if severe hypernatremia develops, administer hypotonic solutions such as 0.45% nacl + 2.5% dextrose or 5% dextrose in water after calculating the patient's free water deficit. because of the large volume of fluid loss, intravenous fluid rates may seem excessive but are necessary to normalize acid-base, electrolyte, and hydration status. in most cases, these patients can survive if the problem is recognized promptly and corrected with careful electrolyte monitoring, aggressive decontamination strategies, and intravenous fluid support. introduction paraquat, a dipyridyl compound, is the active ingredient in some herbicides. the ld 50 of paraquat is 25-50 mg/kg. paraquat initially causes cns excitation. it also causes production of oxygen-derived free radical species in the lungs, that can lead to the development of acute respiratory distress syndrome. initial clinical signs include vomiting, diarrhea, and seizures. within 2 to 3 days, clinical signs associated with severe respiratory distress and acute respiratory distress syndrome (ards) can develop, leading to death. chronic effects include pulmonary fibrosis, if the patient survives the initial toxicity period. the prognosis for paraquat toxicity is generally unfavorable. to treat paraquat ingestion, remove the toxin from the gastrointestinal tract as rapidly as possible after ingestion. there are no known antidotes. if the compound was ingested within the past hour and the animal is able to protect its airway, induce emesis. otherwise, perform orogastric lavage. activated charcoal is not as effective as clay or bentonite adsorbents for removing this particular toxin. early in the course of paraquat toxicity, oxygen therapy is contraindicated because of the risk of producing oxygen-derived free radical species. later, oxygen therapy, including mechanical ventilation, is necessary if ards develops. experimentally, free radical scavengers (n-acetyl cysteine, vitamin c, vitamin e, same) have been shown to be useful in preventing damage caused by oxygen-derived free radical species. hemoperfusion may be useful in eliminating the toxin, if it is performed early in the course of toxicity. pennyroyal oil is an herbal flea control compound that contains menthofuran as its toxic compound. menthofuran is hepatotoxic and may cause gastrointestinal hemorrhage and coagulopathies. to treat toxicity, administer a cathartic and activated charcoal and antiemetic and gastroprotectant drugs, and thoroughly bathe the animal to prevent further dermal exposure. petroleum distillates: see fuels phenobarbital: see barbiturates phenylcyclidine (angel dust) introduction phenylcyclidine (angel dust) is an illicit recreational drug that causes both cns depression and excitation, decreased cardiac output, and hypotension. to treat phenylcyclidine toxicity, place an intravenous catheter, and administer intravenous fluids and antiarrhythmic drugs to maintain organ perfusion. administer supplemental oxygen, and administer diazepam to control seizures. urine alkalinization can help eliminate the compound. phenylephrine is an î±-adrenergic agonist in many over-the-counter decongestant preparations. clinical signs of intoxication include mydriasis, tachypnea, agitation, hyperactivity, and abnormal flybiting and staring behavior. tachycardia, bradycardia, hypertension, hyperthermia, and seizures can occur. to treat phenylephrine toxicity, place an intravenous catheter and give intravenous fluids to maintain hydration, promote diuresis, and treat hyperthermia. administer prazosin or sodium nitroprusside to treat hypertension, antiarrhythmic drugs as necessary, and diazepam to control seizures. phenylpropanolamine has both î±and î²-adrenergic agonist effects, and is used primarily in the treatment of urinary incontinence in dogs. the drug was taken off of the market for use in humans because of the risk of stroke. clinical signs of phenylpropanolamine intoxication include hyperactivity, hyperthermia, mydriasis, tachyarrhythmias or bradycardia, hypertension, agitation, and seizures. to treat toxicity, administer prazosin or nitroprusside to control hypertension, a betablocker (esmolol, propranolol, atenolol) to control tachyarrhythmias, diazepam to control seizures, and intravenous fluids to maintain hydration and promote diuresis. urine acidification may aid in facilitating excretion. if bradycardia occurs, do not use atropine. pseudoephedrine is an î±and î²-adrenergic agonist that is a component of many over-thecounter decongestants and is used in the manufacture of crystal methamphetamine. clinical signs of toxicity include severe restlessness, tremors, mydriasis, agitation, hyperthermia, tachyarrhythmias or bradycardia, hypertension, and seizures. to treat toxicity, administer activated charcoal, intravenous fluids to promote diuresis and treat hyperthermia, chlorpromazine to combat î±-adrenergic effects, a beta-blocker (propranolol, esmolol, atenolol) to treat î²-adrenergic effects, and cyproheptadine (per rectum) to combat serotoninergic effects. piperazine is a gaba agonist, and causes cervical and truncal ataxia, tremors, seizures, coma, and death. salt used for thawing ice commonly contains calcium chloride, a compound that has a moderate toxic potential. calcium chloride produces strong local irritation and can cause gastroenteritis and gastrointestinal ulcers if ingested. respiratory emergencies consist of any problem that impairs delivery of oxygen to the level of the alveoli or diffusion of oxygen across the alveolar capillary membrane into the pulmonary capillary network. decreased respiratory rate or tidal volume can result in hypoxia and buildup of carbon dioxide, or hypercarbia, leading to respiratory acidosis. conditions most frequently encountered result in airflow obstruction, prevention of normal lung expansion, interference with pulmonary gas exchange (ventilation-perfusion mismatch), and alterations of pulmonary circulation. evaluation of the patient with respiratory distress is often challenging, because the most minimal stress can cause rapid deterioration, or even death in critical cases. careful observation of the patient from a distance often allows the clinician to determine the severity of respiratory distress and localize the lesion based on the patient's respiratory pattern and effort. animals in respiratory distress often have a rapid respiratory rate (>30 breaths per minute). as respiratory distress progresses, the patient may appear anxious and start openmouth breathing. the animal often develops an orthopneic posture, characterized by neck extension, open-mouthed breathing, and elbows abducted or pulled away from the body. cyanosis of the mucous membranes often indicates extreme decompensation. clinical signs of respiratory distress can develop acutely, or from decompensation of a more chronic problem that was preceded by a cough, noisy respirations, or exercise intolerance. localization of the cause of respiratory distress is essential to successful case management. in any patient with clinical signs of respiratory distress, the differential diagnosis should include primary pulmonary parenchymal disease, airway disease, thoracic cage disorders, congestive heart failure, dyshemoglobinemias (carbon monoxide, methemoglobin), and anemia. careful observation of the patient's respiratory pattern can aid in making a diagnosis of upper airway disease/obstruction, primary pulmonary parenchymal disease, pleural space disease, and abnormalities of the thoracic cage. it is often helpful to rest a hand on the patient and breathe along with the patient's effort, to confirm the periods of inhalation and exhalation. the pharynx, larynx, and extrathoracic trachea comprise the upper airway. obstructive lesions are associated with a marked inspiratory wheeze or stridor and slow deep inspiratory effort. auscultation of the larynx and trachea may reveal more subtle obstructions of normal air flow. stridor can usually be auscultated without the use of a stethoscope. lung sounds are usually normal. the neck should be carefully palpated for a mass lesion, tracheal collapse, and subcutaneous emphysema. subcutaneous emphysema suggests tracheal damage or collapse secondary to severe trauma. in some cases, there is a history of voice, or bark, change secondary to laryngeal dysfunction. differential diagnosis is usually based on the patient's signalment, history, and index of suspicion of a particular disease process. differential diagnoses of upper airway obstruction are listed in box 1-61. diseases of the pleural space often are associated with a restrictive respiratory pattern. inspiratory efforts are short, rapid, and shallow, and there is often a marked abdominal push. the pattern has been referred to as a choppy "dysynchronous" respiratory pattern. depending on the disease present, lung sounds may be muffled ventrally and enhanced dorsally. percussion of the thorax reveals decreased resonance if fluid is present. increased resonance is present with pneumothorax. decreased compressibility of the anterior thorax may be present with an anterior mediastinal mass lesion, particularly in cats and ferrets. a pneumothorax or diaphragmatic hernia is commonly associated with evidence of trauma, with or without rib fractures. respiratory distress due to hemothorax may be exacerbated by anemia. differential diagnoses for patients with evidence of pleural cavity disease include pneumothorax, diaphragmatic hernia, neoplasia, and various types of pleural effusion. primary pulmonary parenchymal disease can involve the intrathoracic airways, alveoli, interstitial space, and pulmonary vasculature. a rapid, shallow, restrictive respiratory pattern may be observed with a marked push on exhalation, particularly with obstructive airway disease such as chronic bronchitis (asthma) in cats. crackles or wheezes are heard on thoracic auscultation. differential diagnoses for pulmonary parenchymal disease include cardiogenic and noncardiogenic pulmonary edema, pneumonia, feline bronchitis (asthma), pulmonary contusion, aspiration pneumonitis, pulmonary thromboembolism, neoplasia, infection (bacterial, fungal, protozoal, viral) , and/or chronic bronchitis. other abnormal respiratory patterns may be evident, and warrant further consideration. tachypnea present in the absence of other signs of respiratory distress can be a normal response to nonrespiratory problems, including pain, hyperthermia, and stress. a restrictive respiratory pattern with minimal thoracic excursions can be associated with diseases of neuromuscular function, including ascending polyradiculoneuritis, botulism, and tick paralysis. if adequate ventilation cannot be maintained by the patient, mechanical ventilation may be indicated. kussmaul respiration manifests as very slow, very deep respirations when a metabolic acidosis is present. this type of respiratory pattern typically is observed in patients with severe diabetic ketoacidosis and renal failure in a compensatory attempt to blow off carbon dioxide. cheyne-stokes respiration is usually observed with a defect in the central respiratory control center. the classic pattern of cheyne-stokes respiration is normal or hyperventilation followed by a period of apnea or hypoventilation. in cases of lower cervical cord damage or damage to the central respiratory control center in the cns, the diaphragm alone may assume most of the ventilatory movement. with diaphragmatic fatigue, severe hypoventilation and resultant hypoxemia may require mechanical ventilation. immediate management of any patient in respiratory distress is to minimize stress at all costs. relatively benign procedures such as radiography or intravenous catheter placement can be fatal in patients with severe respiratory compromise. stabilization should always precede further diagnostic evaluation. in some cases, sedation may be required before performing any diagnostics, to prevent further stress. all patients should receive some form of supplemental oxygen, either by mask, cage, or flow-by techniques. in cases in which a severe pneumothorax or pleural effusion is suspected, perform therapeutic and diagnostic thoracocentesis bilaterally to allow lung re-expansion and alleviate respiratory distress, whenever possible. if thoracocentesis alone is not effective at maintaining lung re-expansion, place a thoracostomy tube (particularly in cases of tension pneumothorax). if hypovolemic/ hemorrhagic shock is present, initiate treatment while stabilizing the respiratory system (see section on shock). if an animal is suspected of having an upper airway obstruction, reestablish airflow. in cases of laryngeal paralysis, tracheal collapse, and brachycephalic airway syndrome, sedation is often very useful in alleviating the distress of airway obstruction. in cases of laryngeal collapse, however, sedation may make the condition worse. if laryngeal edema is severe, administer a dose of short-acting glucocorticosteroids (dexamethasone sodium phosphate) to decrease laryngeal inflammation and edema. if a foreign body is lodged in the pharynx, perform the heimlich maneuver by thrusting bluntly several times on the patient's sternum. objects such as balls or bones may be small enough to enter the larynx but too large to be expelled, and will require rapid-acting general anesthesia to facilitate dislodgement and removal. if the obstruction cannot be removed, bypassing the obstruction with an endotracheal tube or temporary tracheostomy should be considered. in an emergency, a temporary transtracheal oxygen catheter can quickly be placed in the following manner. connect a 20-or 22-gauge needle to a length of intravenous extension tubing and a 3-ml syringe. place the male connector of the syringe into the female portion of the extension tubing. cut off the syringe plunger and connect the resulting blunt end to a length of flexible tubing attached to a humidified oxygen source. run the oxygen at 10 l/minute to provide adequate oxygenation until a tracheostomy can be performed. (see sections on oxygen supplementation and tracheostomy). once the animal's condition has been stabilized, specific diagnostic tests, including arterial blood gas analyses, thoracic radiographs, and/or transtracheal wash, can be performed, depending on the patient's condition and needs. specific therapies for management of upper airway obstruction, pleural space disease, and pulmonary disease are discussed next. upper airway obstruction can occur as a result of intraluminal or extraluminal mass lesions or foreign bodies in the oropharynx (abscess, neoplasia), laryngeal paralysis, trauma, and anatomic abnormalities. clinical signs of an upper airway obstruction are associated with an animal's extreme efforts to inhale air past the obstruction. marked negative pressure occurs in the extrathoracic airways and can cause worsening of clinical signs. mucosal edema and inflammation further worsen the obstruction. therapy for upper airway obstruction is aimed at breaking the cycle of anxiety and respiratory distress. administer the anxiolytic tranquilizer acepromazine (0.02-0.05 mg/kg iv, im, sq) to decrease patient anxiety. many animals develop hyperthermia from increased respiratory effort and extreme anxiety. implement cooling measures in the form of cool intravenous fluids and wet towels soaked in tepid water placed over the animal (see section on hyperthermia). administer supplemental oxygen in a manner that is least stressful for the animal. short-acting glucocorticosteroids can also be administered (dexamethasone sodium phosphate, 0.25 mg/kg iv, sq, im) to decrease edema and inflammation. if the airway obstruction is severe and there is no response to initial measures to alleviate anxiety and decrease inflammation, establish control of ventilation by placement of an endotracheal tube (see section on endotracheal intubation), tracheal oxygen catheter, or temporary tracheostomy. to obtain airway control, administer a rapid-acting anesthetic (propofol, 4-7 mg/kg iv to effect), and intubate with a temporary tracheostomy. an intratracheal oxygen catheter can be placed with sedation and/or a local anesthetic (see technique for transtracheal wash). laryngeal paralysis is a congenital or acquired condition that occurs primarily in largebreed dogs secondary to denervation of the arytenoid cartilages by the recurrent laryngeal nerve. congenital laryngeal paralysis occurs in the bouvier des flandres, siberian husky, and bull terrier. acquired laryngeal paralysis occurs in labrador retrievers, saint bernards, and irish setters. acquired laryngeal paralysis can be idiopathic, acquired secondary to trauma to the recurrent laryngeal nerve, or can be a component of systemic neuromuscular disease. although rare, this condition also occurs in cats. with dysfunction of the recurrent laryngeal nerve, the intrinsic laryngeal muscles atrophy and degenerate. as a result, the vocal folds and arytenoid cartilage move in a paramedian position within the airway and fail to abduct during inhalation, causing airway obstruction. laryngeal paralysis can be partial or complete, unilateral or bilateral. in many cases, a change in bark is noted prior to the development of clinical signs of respiratory distress or exercise intolerance. when a patient presents with severe inspiratory stridor (with or without hyperthermia) initiate stabilization with anxiolytic tranquilizers, supplemental oxygen, and cooling measures. once the patient's condition has been stabilized, definitive measures to accurately document and assess the patient's airway should be considered. place the patient under very heavy sedation with short-acting barbiturates or propofol (4-7 mg/kg iv) and observe the arytenoid cartilages closely in all phases of respiration. administer just enough drug to allow careful examination without getting bitten. if the arytenoid cartilages do not abduct during inhalation, administer dopram (doxapram hydrochloride, 1-5 mg/kg iv) to stimulate respiration. absent or paradoxical laryngeal motion (closed during inspiration and open during exhalation) is characteristic of laryngeal paralysis. correction of the defect involves documentation and treatment of any underlying disorder and surgical repair of the area to open the airway. partial laryngectomy, arytenoid lateralization ("tie-back" surgery), or removal of the vocal folds has been used with some success. aspiration pneumonitis is common following these procedures. brachycephalic airway syndrome is associated with a series of anatomic abnormalities that collectively increase resistance to airflow. affected animals typically have stenotic nares, an elongated soft palate, and a hypoplastic trachea. components of the syndrome can occur alone or in combination. in severe cases, laryngeal saccular edema and eversion, and eventual pharyngeal collapse, can occur secondary to the severe increase in intrathoracic airway pressure required to overcome the resistance of the upper airways. specific airway anomalies can be identified with general anesthesia and laryngoscopy. severe respiratory distress should be treated as discussed previously. treatment requires surgical correction of the anatomic abnormalities. in animals with laryngeal collapse, surgical correction may not be possible, and a permanent tracheostomy may be required. because an elongated soft palate and stenotic nares can be identified before the onset of clinical signs, surgical correction to improve airflow when the animal is young may decrease the negative intra-thoracic pressure necessary to move air past these obstructions. the chronic consequences of everted laryngeal saccules and laryngeal collapse potentially can be prevented. tracheal collapse is common in middle-aged and older toy and small-breed dogs. the owner typically reports a chronic cough that is readily induced by excitement or palpation of the trachea. the cough often sounds like a "goose honk." diagnostic confirmation is obtained by lateral radiography or fluoroscopy of the cervical and thoracic trachea during all phases of respiration. acute decompensation is uncommon but does occur, particularly with excitement, exercise, and increased environmental temperatures or ambient humidity. therapy of the patient with acute respiratory distress secondary to tracheal collapse includes sedation, administration of supplemental oxygen, and provision of cooling measures to treat hyperthermia. cough suppressants (hydrocodone bitartrate-homatropine methylbromide, 0.25 mg/kg po q8-12h, or butorphanol, 0.5 mg/kg po q6-12h) are useful. tracheal collapse is a dynamic process that usually involves both the upper and lower airways. because of this, bypassing the obstruction is often difficult. tracheal stents have been 256 1 emergency care 1 used with limited success in combination with treatment of chronic lower airway disease. crush or bite injuries to the neck can result in fractures or avulsion of the laryngeal or tracheal cartilages. bypassing the obstructed area may be necessary until the patient is stable and can undergo surgical correction of the injury. if there is avulsion of the cranial trachea, it may be difficult to intubate the patient. a long, rigid urinary catheter can be inserted past the area of avulsion into the distal segment, and an endotracheal tube passed over the rigid catheter, to establish a secure airway. neck injury can also result in damage to the recurrent laryngeal nerve and laryngeal paralysis. foreign bodies can lodge in the nasal cavity, pharynx, larynx, and distal trachea. signs of foreign bodies in the nares include acute sneezing and pawing at or rubbing the muzzle on the ground. if the object is not removed, sneezing continues and a chronic nasal discharge develops. respiratory distress is uncommon, but the foreign body is severely irritating. pharyngeal and tracheal foreign bodies can cause severe obstruction to airflow and respiratory distress. diagnosis of a foreign body is based on the patient history, physical examination findings, and thoracic or cervical radiographs. smaller foreign bodies lodged in the distal airways may not be apparent radiographically but can cause pulmonary atelectasis. foreign bodies of the nose or pharynx can often be removed with an alligator forceps with the patient under anesthesia. if removal is not possible with a forceps, flushing the nasal cavity from cranial to caudal (pack the back of the mouth with gauze to prevent aspiration) can sometimes dislodge the foreign material into the gauze packing. rhinoscopy may be necessary. if an endoscope is not available, an otoscope can be used. foreign objects lodged in the trachea can be small and function like a ball valve during inhalation and exhalation, causing episodic hypoxia and collapse. when attempting to remove these objects, suspend the patient with its head down. remove the object with an alligator forceps, using a laryngoscope to aid in visualization. foreign bodies lodged in the trachea or bronchi require removal with endoscopic assistance. nasopharyngeal polyps (in cats, tumors, obstructive laryngitis, granulomas, abscesses, and cysts) can cause upper airway obstruction. clinical signs are usually gradual in onset. the lesions can be identified through careful laryngoscopic examination performed with the patient under general anesthesia. the nasopharynx above the soft palpate should always be included in the examination. pedunculated masses and cysts are excised at the time of evaluation. biopsy of diffusely infiltrative masses is indicated for histologic examination and prognosis. it is impossible to distinguish obstructive laryngitis from neoplasia based on gross appearance alone. whenever possible, material should be collected from abscesses and granulomas for cytologic evaluation and bacterial culture. extraluminal masses impinge on and slowly compress the upper airways, resulting in slow progression of clinical signs. masses are usually identified by palpation of the neck. enlarged mandibular lymph nodes, thyroid tumors, and other neoplasms may be present. diagnosis is usually based on a combination of radiography and ultrasonography. ct and/or mri are helpful in identifying the full extent and invasiveness of the lesion. definitive diagnosis is made with a fine-needle aspirate or biopsy. many thyroid tumors bleed excessively. the inside of each side of the hemithorax is covered in parietal pleura. the lung lobes are covered in visceral pleura. the two surfaces are in close contact with each other, and are contiguous at the hilum under normal circumstances. pneumothorax refers to free air within the pleural space, accumulating in between the parietal and visceral pleura. the term pleural effusion refers to fluid accumulation in that area but does not reflect the amount or type of fluid present. the mediastinal reflections of the pleura typically are thin in dogs and cats, and usually, but not always, connect. bilateral involvement of pneumothorax or pleural effusion is common. both pneumothorax and pleural effusion compromise the lungs' ability to expand and result in hypoxia and respiratory distress. pneumothorax can be classified as open versus closed, simple versus complicated, and tension. an open pneumothorax communicates with the external environment through a rent in the thoracic wall. a closed pneumothorax results from tears in the visceral pleura but does not communicate with the outside. a tension pneumothorax occurs as a result of a tear in the lung or chest wall that creates a flap valve, such that air is allowed to leave the lung and accumulate in the pleural space during inhalation, and closes to seal off exit of air from the pleural space during exhalation. tension pneumothorax can cause rapid decline in cardiopulmonary status and death if not recognized and treated immediately. a simple pneumothorax is one that can be controlled with a simple thoracocentesis. complicated pneumothorax involves repeated accumulation of air, requiring placement of a thoracic drainage catheter. in many cases, pneumothorax develops as a result of trauma. spontaneous pneumothorax occurs with rupture of cavitary lesions of the lung that may be congenital or acquired as a result of prior trauma, heartworm disease, airway disease (emphysema), paragonimiasis, neoplasia, or lung abscess. pneumothorax also rarely occurs as a result of esophageal tears or esophageal foreign bodies. rapid circulatory and respiratory compromise following traumatic pneumothorax can develop as a result of open or tension pneumothorax, rib fractures, airway obstruction, pulmonary contusions, hemothorax, cardiac dysrhythmias, cardiac tamponade, and hypovolemic shock. any patient that is rapidly decompensating after a traumatic episode must be quickly assessed, and emergency therapy initiated (see section on immediate management of trauma, a crash plan). diagnosis of pneumothorax is usually made based on a history of trauma, a rapid, shallow, restrictive respiratory pattern, and muffled heart and lung sounds on thoracic auscultation. the clinical signs and history alone should prompt the clinician to perform a bilateral diagnostic and therapeutic thoracocentesis before taking thoracic radiographs (see section on thoracocentesis). the stress of handling the patient for radiography can be deadly in severe cases of pneumothorax. although the mediastinum on both sides of the thorax connects, it is necessary to perform thoracocentesis on both sides to ensure maximal removal of free air in the pleural space and allow maximal lung expansion. if negative pressure cannot be obtained, or if the patient rapidly reaccumulates air, place a thoracostomy tube connected to continuous suction. (see section on thoracostomy tube placement). treat all penetrating wounds to the thorax as open sucking chest wounds unless proved otherwise. to "close" an open sucking chest wound, clip the fur around the wound as quickly as possible, and place sterile lubricant jelly or antimicrobial ointment circumferentially around the wound. cut a sterile glove to provide a covering. place the covering over the wound, making sure to cover all of the sterile lubricant, thus creating a seal to close the wound temporarily from the external environment. evaluate the patient's thorax via thoracocentesis while placing a thoracostomy tube. once the patient is stable, the open chest wound can be surgically explored, lavaged, and definitively corrected. all animals with open chest wounds should receive antibiotics (first-generation cephalosporin) to prevent infection. following stabilization, radiographs can be taken and evaluated. pneumothorax is confirmed by evidence of elevation of the cardiac silhouette above the sternum, increased density of the pulmonary parenchymal tissue, free air in between the parietal and visceral 1 pleura (making the outline of the lungs visible), and absence of pulmonary vascular structures in the periphery. parenchymal lesions within the lungs are best identified after as much air as possible has been removed from the thorax. obtain left and right lateral and ventrodorsal or dorsoventral views. a standing lateral view may reveal air-or fluid-filled cavitary masses. if underlying pulmonary disease is suspected as a cause of spontaneous pneumothorax, a transtracheal wash, fecal flotation, and heartworm test may be indicated. treatment of pneumothorax includes immediate bilateral thoracocentesis, covering of any open chest wounds, administration of supplemental oxygen, and placement of a thoracostomy tube if negative pressure cannot be obtained or if air rapidly reaccumulates. serial radiography, ct, or mri should be performed in dogs with spontaneous pneumothorax, because the condition can be associated with generalized pulmonary parenchymal disease. strict cage rest is required until air stops accumulating and the thoracostomy tube can be removed. the patient's chest tube should be aspirated every 4 hours after discontinuing continuous suction. if no air reaccumulates after 24 hours, the chest tube can be removed. exercise restriction is indicated for a minimum of 1 week. if bullae or mass lesions are present, exploratory thoracotomy should be considered as a diagnostic and potentially therapeutic option for long-term management in prevention of recurrence. pleural fluid cytologic analysis is indicated for all patients with pleural effusion before administration of antibiotics. the general term pleural effusion means a collection of fluid in the space between the parietal and visceral pleura but does not indicate what kind or how much fluid is present. clinical signs associated with pleural effusion depend on how much fluid is present, and how rapidly the fluid has accumulated. clinical signs associated with pleural effusion include respiratory distress, reluctance to lie down, labored breathing with an abdominal component on exhalation, cough, and lethargy. auscultation of the thorax may reveal muffled heart and lung sounds ventrally and increased lung sounds dorsally, although pockets of fluid may be present, depending on the chronicity of the effusion. percussion of the thorax may reveal decreased resonance. in stable patients, the presence of pleural effusion can be confirmed radiographically. radiographic confirmation of the pleural effusion should include right and left lateral and dorsoventral or ventrodorsal views. a handling or standing lateral view should be obtained if an anterior mediastinal mass is suspected. the standing lateral view will allow the fluid to collect in the costophrenic recess. in patients with respiratory distress, muffled heart and lung sounds, and suspicion of pleural effusion, thoracocentesis should be performed immediately. thoracocentesis can be both therapeutic and diagnostic. radiography is contraindicated because the procedure can cause undue stress and exacerbation of clinical signs in an unstable patient. pleural effusion can cause severe respiratory distress, and can be the result of a number of factors that must be considered when implementing an appropriate treatment plan. pathology of the pleura is almost always a secondary process except for primary bacterial pleuritis and pleural mesotheliomas. causes of pleural effusion in the cat and dog include pyothorax, feline infectious peritonitis, congestive heart failure, chylothorax, heartworm disease, hemothorax, hypoalbuminemia, lung lobe torsions, neoplasia, diaphragmatic hernia, and pancreatitis (box 1-62). in stable animals, diagnosis of pleural effusion can be made based â�¢ imbalance of transpleural or hydrostatic or protein osmotic forces â�¢ change in membrane permeability â�¢ decrease in rate of fluid reabsorption â�¢ combination of foregoing mechanisms on thoracic radiography or ultrasound. thoracic radiographs can show whether the pleural effusion is unilateral or bilateral. effusions in dogs and cats are usually bilateral. the lung parenchyma and the cardiac silhouette cannot be fully evaluated until most of the fluid has been evacuated from the pleural cavity. following thoracocentesis, radiography should be performed with left and right lateral and ventrodorsal or dorsoventral views. in cases of suspected heart failure, echocardiography also is necessary. pleural fluid cytologic analysis is indicated for all patients with pleural effusion. collect specimens before administering antibiotics, whenever possible, because treatment with antibiotics can make a septic condition (pyothorax) appear nonseptic. the remainder of the diagnostic workup and treatment is based on the type of fluid present (table 1 -52). the fluid may be a transudate, nonseptic exudate, septic exudate, chylous, hemorrhagic, or neoplastic. ultrasonographic evaluation of the thorax can be helpful in identifying intrathoracic masses, diaphragmatic hernias, lung lobe torsions, and cardiac abnormalities. unlike radiography, ultrasonography is facilitated by the presence of fluid in the pleural space. pyothorax refers to a septic effusion of the pleural cavity. the infection is generally the result of a combination of aerobic and anaerobic bacteria. rarely, fungal organisms are present. the source of the underlying organisms is rarely identified, particularly in cats, but can be caused by penetrating wounds through the chest wall, esophagus, migrating foreign bodies (especially grass awns), or primary lung infections. the most common organisms associated with pyothorax in the cat are pasteurella, bacteroides, and fusobacterium. fever is often present in addition to clinical signs of pleural effusion. septic shock is ununcommon. diagnosis of pyothorax is made based on cytologic analysis and the demonstration of intracellular and extracellular bacteria, toxin neutrophils and macrophages, and sometimes the presence of sulfur granules. gram stains of the fluid can assist in the initial identification of some organisms. bacterial cultures are indicated for bacteria identification and antibiotic susceptibility testing. administration of antibiotics before cytologic evaluation can cause a septic effusion to appear nonseptic. emergency treatment for pyothorax involves placement of an intravenous catheter, intravenous fluids to treat hypovolemic shock, and broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h, and enrofloxacin, 10 mg/kg iv q24h). chloramphenicol also is an appropriate antibiotic to use for penetration into pockets of fluid. administration of a beta-lactam antibiotic (ampicillin or amoxicillin) with a beta-lactamase inhibitor (amoxicillin clavulanate or ampicillin sulbactam) is helpful in achieving better coverage of bacteroides spp. treatment of pyothorax differs in the cat and dog. in the cat, placement of one or two thoracic drainage catheters is recommended to allow continuous drainage of the intrathoracic abscess. inadequate drainage can result in treatment failure. fluid should be evaluated and the pleural cavity lavaged with 10 ml/kg of warmed 0.9% saline or lactated ringer's solution every 8 hours. approximately 75% of the infused volume should be recovered after each lavage. in dogs, or in cats with refractory pyothorax, perform an exploratory thoracotomy to remove any nidus of infection. rarely a foreign body is visible that can be removed at the time of surgery, but this finding is rare. antibiotics are indicated for a minimum of 6 to 8 weeks after removal of the thoracostomy tube. early diagnosis and aggressive treatment result in a good prognosis in the majority of patients with pyothorax. in cats, clinical signs of ptyalism and hypothermia at the time of presentation worsen the prognosis. chylothorax refers to the abnormal accumulation of chyle (lymphatic fluid) in the pleural cavity. the cisterna chili is the dilated collection pool of lymphatic ducts in the abdomen that accumulate chyle prior to entry into the thoracic duct located within the thoracic cavity. the thoracic duct enters the thorax at the aortic hiatus. numerous tributaries or collateral ducts exist. the functions of the lymphatic vessels collectively serve to deliver triglycerides and fat-soluble vitamins into the peripheral vascular circulation. damage of the thoracic duct or lymphatic system or obstruction to lymphatic flow can result in the development of chylous effusion in the pleural or peritoneal space. it is difficult to identify chylous effusions based on their milky appearance alone. to identify a chylous effusion versus a pseudochylous effusion, the triglyceride and cholesterol levels of the fluid must be compared with those of peripheral blood. chylous effusions have a higher triglyceride and lower cholesterol levels than peripheral blood. pseudochylous effusions have a higher cholesterol and lower triglyceride levels than peripheral blood. disease processes that can result in chylous effusions are listed in the box 1-63. clinical signs associated with chylous effusion are typical of any pleural effusion and of the disease process that caused the effusion. weight loss may be evident, depending on the chronicity of the process. the diagnosis is made based on thoracocentesis, cytology, and biochemical evaluation of the fluid (i.e., triglyceride and cholesterol levels). the fluid often appears milky or bloodtinged but can be clear if the patient has significant anorexia. typical cytologic characteristics are listed in table 1 -52. lymphangiography can be used to confirm trauma to the thoracic duct, but this is usually not necessary unless surgical ligation is going to be attempted. the diagnostic evaluation must also attempt to identify an underlying cause. therapy for chylothorax is difficult and primarily involves documentation and treatment of the underlying cause. if an underlying cause is not found, treatment is largely supportive and consists of intermittent thoracocentesis to drain the fluid as it accumulates and causes respiratory dysfunction, nutritional support, and maintenance of fluid balance. a variety of surgical techniques, including ligation of the thoracic duct, pleural-peritoneal shunts, and pleurodesis, have been attempted but have had limited success. most recently, the combination of thoracic duct ligation with subtotal pericardectomy has been shown to improve surgical success rates in the treatment of chylothorax. rutin, a bioflavinoid, has been used with limited success in the treatment of idiopathic chylothorax in cats. prognosis in many cases of chylothorax is guarded. extensive hemorrhage into the pleural cavity can cause fulminant respiratory distress due to sudden hypovolemia and anemia and interference with lung expansion. hemothorax typically is associated with trauma, systemic coagulopathy, lung lobe torsions, and erosive lesions within the thorax (usually neoplasia). diagnosis of hemothorax involves obtaining a fluid sample via thoracocentesis. hemorrhagic effusion must be differentiated from systemic blood inadvertently collected during the thoracocentesis procedure. unless the hemorrhage is peracute, fluid in cases of hemothorax is rapidly defibrinated and will not clot, has a packed cell volume less than that of venous blood, contains rbcs and macrophages. hemorrhagic effusions also usually contain a disproportionately higher number of white blood cells compared with peripheral blood. hemothorax commonly is the sole clinical sign observed in animals with vitamin k antagonist rodenticide intoxication and systemic coagulopathy. whenever an animal presents with signs of a hemorrhagic pleural effusion, perform coagulation testing immediately to determine whether a coagulopathy exists. the prothrombin time test is fast and can be performed as a cage-side test (see section on coagulopathy). therapy for hemorrhagic pleural effusions should address the blood and fluid loss. administer intravenous crystalloid fluids and rbc products (see section on transfusion therapy). when necessary, administer coagulation factors in the form of fresh whole blood or fresh frozen plasma, along with vitamin k 1 (5 mg/kg sq in multiple sites with a 25-gauge needle). if severe respiratory distress is present, evacuate the blood within the pleural space via thoracocentesis until clinical signs of respiratory distress resolve. fluid that remains aids in the recovery of the patient, because rbcs and proteins eventually will be reabsorbed. autotransfusion can be performed to salvage blood and reinfuse it into the anemic patient. in cases of neoplastic or traumatic uncontrollable hemorrhagic effusions, surgical exploration of the thorax is warranted. diaphragmatic hernia, or a rent in the diaphragm, can result in the protrusion of abdominal organs into the thoracic cavity and impair pulmonary expansion. organs that are commonly herniated into the thorax include the liver, stomach, and small intestines. diaphragmatic hernia usually is secondary to trauma but can occur as a congenital anomaly. in cases of trauma, rib fractures, pulmonary contusions, traumatic myocarditis, hemothorax, and shock are also often present concurrently with diaphragmatic hernia. respiratory distress can be caused by any one or a combination of the above lesions. animals with prior or chronic diaphragmatic hernias may have minimal clinical signs despite the presence of abdominal organs within the thorax. clinical signs of acute or severe diaphragmatic hernia include respiratory distress, cyanosis, and shock. a diagnosis of diaphragmatic hernia is made based on the patient's history (traumatic event), clinical signs, and radiographs. in some cases, ultrasonography or contrast peritoneography is necessary to confirm the diagnosis. contrast radiographs may show the presence of the stomach or intestines within the thorax following oral administration of barium. never administer barium directly into the peritoneal cavity or in cases of suspected gastrointestinal rupture. treatment of a patient with a diaphragmatic hernia includes cardiovascular and respiratory system stabilization before attempting surgical repair of the diaphragm. if the stomach is within the thorax, or if the patient's respiratory distress cannot be alleviated with medical management alone, immediate surgery is necessary. if the respiratory distress is minimal and the stomach is not located within the thorax, surgery can be postponed until the patient is a more stable anesthetic candidate. at the time of surgery, the abdominal organs are replaced into the abdominal cavity, and the rent in the diaphragm is closed. air must be evacuated from the thorax following closure of the diaphragm. if chronic diaphragmatic hernia is repaired, the complication of reexpansion pulmonary edema can occur. cardiac injury is a common complication secondary to blunt thoracic trauma. in most cases, cardiac injury is manifested as arrhythmias, including multiple premature ventricular contractions, ventricular tachycardia, st segment depression or elevation secondary to myocardial hypoxemia, and atrial fibrillation (see section on cardiac emergencies). myocardial infarction and cardiac failure can occur. careful and repeated assessments of the patient's blood pressure and ecg tracing should be a part of any diagnostic work-up for a patient that has sustained blunt thoracic trauma. rib fractures are associated with localized pain and painful respiratory movements. radiographs are helpful to confirm the diagnosis. careful palpation may reveal crepitus and instability of the fractured ribs. common problems associated with rib fractures 264 1 emergency care include pulmonary contusions, pericardial laceration, traumatic myocarditis, diaphragmatic hernia, and splenic laceration or rupture. a flail segment results from rib fractures of more than three adjacent ribs that produce a "floating segment" of the chest wall. the flail segment moves paradoxically with respiration-that is, it moves inward during inhalation and outward during exhalation. respiratory distress is associated with the pain caused by the fractures and the presence of traumatic underlying pulmonary pathology. therapy for rib fractures and flail chest includes administration of supplemental oxygen, treatment of pneumothorax or diaphragmatic hernia, and administration of systemic and local anesthesia to alleviate the discomfort associated with the fractures. although controversial, positioning the patient with the flail segment up may reduce pain and improve ventilation. avoid the use of chest wraps, which do nothing to stabilize the flail segment and can further impair respiratory excursions. following administration of a systemic analgesic, administer a local anesthetic at the dorsocaudal and ventrocaudal segment of each fractured rib, and in one rib in front of and behind the flail segment. often, pulmonary function will improve once the pain associated with rib fractures has been adequately treated. in rare cases in which the flail segment involves five or more ribs, surgical stabilization may be necessary. single rib fractures or smaller flail segments are allowed to heal on their own. feline bronchitis has a variety of names (bronchial asthma, asthma, acute bronchitis, allergic bronchitis, chronic asthmatic bronchitis, feline lower airway disease) and refers to the acute onset of respiratory distress secondary to narrowing of the bronchi. cats may present with an acute onset of severe restrictive respiratory pattern associated with lower airway obstruction. acute bronchitis in cats typically has an inflammatory component in the lower airways, resulting in acute bronchoconstriction, excessive mucus production, and inflammatory exudates. in cats with chronic bronchitis, there may be damage of the bronchial epithelium and fibrosis of the airways. these patients often have a history if intermittent exacerbation of clinical signs, intermittent cough, and periods of normality throughout the year. because there appears to be an allergic or inflammatory component in feline bronchitis, clinical signs can be acutely exacerbated by stress and the presence of aerosolized particles such as perfume, smoke, and carpet powders. causes of feline bronchitis include heartworm disease, parasitic infestation (lungworms), and (rarely) bacterial infection. on presentation, the patient should be placed in an oxygen cage and allowed to rest while being observed from a distance. postpone performing stressful diagnostic procedures until the patient's respiratory status has been stabilized. after careful thoracic auscultation, administer a short-acting bronchodilator (terbutaline, 0.01 mg/kg sq or im) along with a glucocorticosteroid (dexamethasone sodium phosphate 1 mg/kg im, sq, iv) to alleviate immediate bronchospasm and airway inflammation. clinical signs of feline bronchitis are characterized by a short, rapid respiratory pattern with prolonged expiration with an abdominal push. wheezes may be heard on thoracic auscultation. in some cases, no abnormalities are found on auscultation, but become acutely worse when the patient is stimulated to cough by tracheal palpation. radiographs may reveal a hyperinflated lung field with bronchial markings and caudal displacement of the diaphragm. in some cases, consolidation of the right middle lung lobe is present. a complete blood count and serum biochemistry profile can be performed, but results usually are unrewarding. in endemic areas, a heartworm test is warranted. fecal examination 1 by flotation and the baermann technique is helpful in ruling out lungworms and other parasites. bronchoalveolar lavage or transtracheal wash is useful for cytologic and bacterial examination. long-term management of feline bronchitis includes isolation from environmental exposure to potential allergens (litter dust, perfumes, smoke, incense, carpet powders) and treatment of bronchoconstriction and inflammation with a combination of oral and inhaled glucocorticosteroids and bronchodilators (table 1 -53). antibiotic therapy is contraindicated unless a pure culture of a pathogen is documented. oral therapy with steroids and bronchodilators should be used for a minimum of 4 weeks after an acute exacerbation and then gradually decreased to the lowest dose possible to alleviate clinical signs. metered dose inhalers are now available (aerokat.com) for administration of inhaled bronchodilators and steroids. fluticasone (flovent, 100 mcg/puff ) can be administered initially every 12 hours for 1 week and then decreased to once daily, in most cases. inhaled glucocorticosteroids are not absorbed systemically, and therefore patients do not develop the adverse side effects sometimes documented with oral glucocorticosteroid administration. because it takes time for glucocorticosteroids to reach peak effects in the lungs, administration of inhaled glucocorticosteroids should overlap with oral prednisolone administration for 5 to 7 days. treatment of pulmonary contusions is supportive. administer supplemental oxygen in a manner that is least stressful for the animal. arterial blood gas analysis or pulse oximetry can determine the degree of hypoxemia and monitor the response to therapy. intravenous fluids should be administered with caution to avoid exacerbating pulmonary hemorrhage or fluid accumulation in the alveoli. treat other conditions associated with the traumatic event. possible complications of pulmonary contusions are rare but include bacterial infection, abscessation, lung lobe consolidation, and the development of cavitary lesions. the routine use of antibiotics or steroids in cases of pulmonary contusions is contraindicated unless external wounds are present. empiric antibiotic use without evidence of external injury or known infection can potentially increase the risk of a resistant bacterial infection. steroids have been shown to decrease pulmonary alveolar macrophage function and impair wound healing and are contraindicated. aspiration pneumonia can occur in animals as a result of abnormal laryngeal or pharyngeal protective mechanisms or can be secondary to vomiting during states of altered mentation, including anesthesia, recovery from anesthesia, and sleep. megaesophagus, systemic polyneuropathy, myasthenia gravis, and localized oropharyngeal defects such as cleft palate can increase the risk of developing aspiration pneumonitis. iatrogenic causes of aspiration pneumonia include improper placement of nasogastric feeding tubes, overly aggressive force-feeding, and oral administration of drugs. aspiration of contents into the airways can cause mechanical airway obstruction, bronchoconstriction, chemical damage to the alveoli, and infection. severe inflammation and airway edema are common. pulmonary hemorrhage and necrosis can occur. diagnosis of aspiration pneumonia is based on clinical signs of pulmonary parenchymal disease, a history consistent with vomiting or other predisposing causes, and thoracic radiographs demonstrating a bronchointerstitial to alveolar pulmonary infiltrate. the most common site is the right middle lung lobe, although the pneumonia can occur anywhere, depending on the position of the patient at the time of aspiration. a transtracheal wash or bronchoalveolar lavage is useful for bacterial culture and susceptibility testing. treatment of aspiration pneumonia includes antibiotic therapy for the infection, administration of supplemental oxygen, and loosening the debris in the airways. administer intravenous fluids to maintain hydration. nebulization with sterile saline and chest physiotherapy (coupage) should be performed at least every 8 hours. antibiotics to consider in the treatment of aspiration pneumonia include ampicillin/enrofloxacin, amoxicillinclavulanate, ampicillin-sulbactam, trimethoprim sulfa, and chloramphenicol. the use of glucocorticosteroids is absolutely contraindicated. continue antibiotic therapy for a minimum of 2 weeks after the resolution of radiographic signs of pneumonia. pulmonary edema arises from the accumulation of fluid in the pulmonary interstitial alveolar spaces, and airways. ventilation-perfusion abnormalities result in hypoxia. pulmonary edema can be caused by increased pulmonary vasculature hydrostatic pressure, decreased pulmonary oncotic pressure, obstruction of lymphatic drainage, or increased capillary permeability. multiple factors can occur simultaneously. the most common cause of edema is increased pulmonary hydrostatic pressure resulting from left-sided congestive heart failure. decreased plasma oncotic pressure with albumin <1.5 g/dl can also result in accumulation of fluid in the pulmonary parenchyma. overzealous intravenous crystalloid fluid administration can result in dilution of serum oncotic pressure and vascular overload. obstruction of lymphatic drainage is usually caused by neoplasia. other causes of pulmonary edema include pulmonary thromboembolic disease, severe upper airway obstruction (noncardiogenic pulmonary edema), seizures, and head trauma. increased capillary permeability is associated with a variety of diseases that cause severe inflammation (systemic inflammatory response syndrome). the resultant pulmonary edema contains a high amount of protein and is known as acute respiratory 1 distress syndrome (ards). ards can be associated with pulmonary or extrapulmonary causes, including direct lung injury from trauma, aspiration pneumonia, sepsis, pancreatitis, smoke inhalation, oxygen toxicity, electrocution, and immune-mediated hemolytic anemia with disseminated intravascular coagulation. diagnosis of pulmonary edema is made based on clinical signs of respiratory distress and the presence of crackles on thoracic auscultation. in severe cases, cyanosis and fulminant blood-tinged frothy edema fluid may be present in the mouth and nostrils. immediate management includes administration of furosemide (4-8 mg/kg iv, im) and supplemental oxygen. sedation with low-dose morphine sulfate (0.025-0.1 mg/kg iv) is helpful in dilating the splanchnic capacitance vasculature and relieving anxiety for the patient. if fluid overload is suspected secondary to intravenous fluid administration, fluids should be discontinued. severely hypoalbuminemic patients should receive concentrated human albumin (2 ml/kg of a 25% solution) or fresh frozen plasma. furosemide as a constant rate infusion (0.66-0.1 mg/kg/hour) also can dilate the pulmonary vasculature and decrease fluid accumulation in cases of ards. following initial stabilization of the patient, thoracic radiographs and an echocardiogram should be assessed to determine cardiac side, pulmonary vascular size, and cardiac contractility. further diagnostic testing may be required to determine other underlying causes of pulmonary edema. heart failure is managed with vasodilators, diuretics, oxygen, and sometimes positive inotropes. treatment ultimately consists of administration of supplemental oxygen, minimal stress and patient handling, and judicious use of diuretics. in cases of cardiogenic pulmonary edema, administer furosemide (4-8 mg/kg iv, im) every 30 to 60 minutes until the patient loses 7% of its body weight. positive inotropic and antiarrhythmic therapy may be necessary to improve cardiac contractility and control dysrhythmias. the clinician should determine whether the cause of the pulmonary edema is secondary to congestive heart failure with pulmonary vascular overload, volume overload, hypoalbuminemia, or increased permeability (ards). pulmonary edema secondary to ards typically is refractory to supplemental oxygen and diuretic therapy. in many cases, mechanical ventilation should be considered. a diagnosis of pulmonary thromboembolism (pte) is difficult to make and is based on clinical signs of respiratory distress consistent with pte, lack of other causes of hypoxemia, a high index of suspicion in susceptible animals, the presence of a condition associated with pte, and radiographic findings. virchow's triad consists of vascular endothelial injury, sluggish blood flow with increased vascular stasis, and a hypercoagulable state as predisposing factors for thromboembolic disease. clinical conditions that predispose an animal to pte include hyperadrenocorticism, disseminated intravascular coagulation (dic), catheterization of blood vessels, bacterial endocarditis, protein-losing nephropathy or enteropathy, hyperviscosity syndromes, heat-induced illness, pancreatitis, diabetes mellitus, inflammatory bowel disease, and immune-mediated hemolytic anemia. definitive diagnosis requires angiography or a lung perfusion scan. clinical signs associated with pte include an acute onset of tachypnea, tachycardia, orthopnea, and cyanosis. if the embolism is large, the patient may respond poorly to supplemental oxygen administration. pulmonary hypertension can cause a split second heart sound on cardiac auscultation. in some cases, a normal thoracic radiograph is present in the face of severe respiratory distress. this is a classic finding in cases of pte. potential radiographic abnormalities include dilated, tortuous, or blunted pulmonary arteries; wedge-shaped opacities in the lungs distal to an obstructed artery; and interstitial to alveolar infiltrates. the right heart may be enlarged. echocardiography can show right heart enlargement, tricuspid regurgitation, pulmonary hypertension, and evidence of underlying cardiac disease, possibly with clots in the atria. measurement of antithrombin (at) and d-dimer levels can be useful in the identification of hypercoagulable states, including dic. treatment of any patient with at deficiency or dic includes replenishment of at and clotting factors in the form of fresh frozen plasma. treatment of pte includes therapy for cardiovascular shock, oxygen supplementation, and thrombolytic therapy (see section on thromboembolic therapy). for short-term treatment, administer heparin (heparin sodium, 200-300 units/kg sq once, followed by 100 units/kg q8h of unfractionated heparin; or fractionated heparin). thrombolytic therapy may include tissue plasminogen activator, streptokinase, or urokinase. long-term therapy with low molecular weight heparin or warfarin may be required to prevent further thromboembolic events. ideally, management should include treatment and elimination of the underlying disease. smoke inhalation commonly occurs when an animal is trapped in a burning building. the most severe respiratory complications of smoke inhalation are seen in animals that are close enough to the flames to also sustain burn injuries (see section on burn injury). at the scene, many animals are unconscious from the effects of hypoxia, hypercapnia, carbon monoxide intoxication, and hydrogen cyanide gases that accumulate in a fire. carbon monoxide produces hypoxia by avidly binding to and displacing oxygen binding to hemoglobin, resulting in severe impairment of oxygen-carrying capacity. the percentage of carboxyhemoglobin in peripheral blood depends on the amount or carbon monoxide in inhaled gases and the length of time of exposure. clinical signs of carbon monoxide intoxication include cyanosis, nausea, vomiting, collapse, respiratory failure, loss of consciousness, and death. smoke inhalation of superheated particles also causes damage to the upper airways and respiratory tree. the larynx can become severely edematous and obstruct inspiration. emergency endotracheal intubation, tracheal oxygen, or tracheostomy tube may be required in the initial resuscitation of the patient, depending on the extent of airway edema. inhalation of noxious gases and particles can cause damage to the terminal respiratory bronchioles. specific noxious gases that can cause alveolar damage include combustible particles from plastic, rubber, and other synthetic products. pulmonary edema, bacterial infection, and ards can result. in any case of smoke inhalation, the first and foremost treatment is to get the animal away from the source of the flames and smoke and administer supplemental oxygen at the scene. at the time of presentation, carefully examine the animal's eyes, mouth, and oropharynx suction soot and debris from the mouth and upper airways. evaluate the patient's respiratory rate, rhythm, and pulmonary sounds. arterial blood gases should be analyzed with co-oximetry to evaluate the pao 2 and carboxyhemoglobin concentrations. evaluation of sao 2 by pulse oximetry is not accurate in cases of smoke inhalation, as the pao 2 may appear normal, even when large quantities of carboxyhemoglobin are present. radiographs are helpful in determining the extent of pulmonary involvement, although radiographic signs may lag behind the appearance of clinical respiratory abnormalities by 16 to 24 hours. bronchoscopy and bronchoalveolar lavage provide a more thorough and accurate evaluation of the respiratory tree; however, these procedures should be performed only in patients whose cardiovascular and respiratory status is stable. management of the patient with smoke inhalation includes maintaining a patent airway, administration of supplemental oxygen, correction of hypoxemia and acid-base abnormalities, preventing infection, and treating thermal burns (see section on burn injury). if severe laryngeal edema is present, a temporary tracheostomy may be necessary to allow adequate oxygenation and ventilation. glucocorticosteroids should not be empirically used in the treatment of smoke inhalation, because of the risk of decreasing pulmonary alveolar macrophage function and increasing the potential for infection. in cases of severe laryngeal edema, however, glucocorticosteroids may be necessary to decrease edema and inflammation. the use of empiric antibiotics is contraindicated unless clinical signs of deterioration and bacterial pneumonia develop. epistaxis can be caused by facial trauma, a foreign body, bacterial or fungal rhinitis, neoplasia, coagulopathies, and systemic hypertension. acute, severe bilateral hemorrhage without wounds have been classified in several ways according their degree of tissue integrity, etiologic force, degree of contamination and duration, and degree of contamination and infection (table 1 -54) . there are also unique causes of wounds such as burns, psychogenic dermatoses, frostbite, decubital ulcers, and snake bite. the animal should be transported to the nearest veterinary facility for definitive care. the wound should be covered or packed with dry gauze or clean linen to protect the wound, and to prevent further hemorrhage and contamination. if an open fracture is present, the limb should be splinted without placing the exposed bone back into the wound. replacing the exposed bone fragment back through the skin wound can cause further damage to underlying soft tissue structures and increase the degree of contamination of deeper tissues. if a spinal fracture is suspected, the patient should be transported on a stable flat surface to prevent further spinal mobilization and neurologic injury. at the time of presentation, first refer to the abcs of trauma, taking care to evaluate and stabilize the patient's cardiovascular and respiratory status. after a complete physical examination and history, ancillary diagnostic techniques can be performed if the patient is hemodynamically stable (see section on triage, assessment, and treatment of emergencies). initially, every patient with superficial wound should receive some degree of analgesia and an injection of a first-generation cephalosporin, preferably within 3 hours of the injury. evaluate the wound after the patient's cardiovascular and respiratory status have been stabilized. always cover an open wound before taking an animal to the hospital to prevent a nosocomial infection. evaluate limb wounds for neural, vascular, and orthopedic abnormalities. carefully examine the structures deep to the superficial wounds. when there has been a delay in assessment of the wound, obtain samples for culture and antimicrobial susceptibility testing. if the wound is older and obviously infected, a gram stain can help guide appropriate antimicrobial therapy pending results of culture 1 and susceptibility testing. place a support bandage saturated with a water-soluble antibiotic ointment or nonirritating antimicrobial solution (e.g., 0.05% chlorhexidine, if bone or joint tissue is not exposed) around the wound. in addition to a first-generation cephalosporin, other appropriate antibiotic choices include amoxicillin-clavulanate, trimethoprim-sulfadiazine, amoxicillin, and ampicillin. if gram-negative flora are present, administer enrofloxacin. administer the antibiotics of choice for a minimum of 7 days unless a change of antibiotic therapy is indicated. at the time of wound cleansing or definitive wound repair, the patient should be placed under general anesthesia with endotracheal intubation, unless the procedure will be brief (i.e., less than 10 minutes). in such cases, a short-acting anesthetic combination open lacerations or skin loss closed crushing injuries and contusions etiologic force abrasion loss of epidermis and portions of dermis, usually caused by shearing between two compressive surfaces avulsion tearing of tissue from its attachment because of forces similar to those causing abrasion but of a greater magnitude incision wound created by a sharp object; wound edges are smooth and there is minimal trauma in the surrounding tissues laceration irregular wound caused by tearing of tissue with variable damage to the superficial and underlying tissue puncture penetrating wound caused by a missile or sharp object; superficial damage may be minimal; damage to deeper structures may be considerable; contamination by fur and bacteria with subsequent infection is common class i 0-6 hours with minimal contamination class ii 6-12 hours with significant contamination class iii >12 hours with gross contamination (analgesia + propofol, analgesia + ketamine/diazepam) can be administered to effect. heavy sedation with infiltration of a local anesthetic may also be appropriate for very small wounds, depending on the location of the wound and temperament of the patient. protect the wound by packing it with sterile gauze sponges soaked in sterile saline, or with watersoluble lubricating gel such as k-y jelly. clip the fur surrounding the wound, moving from the inner edge of the wound outward, to help prevent wound contamination with fur or other debris. scrub the wound and surrounding skin with an antimicrobial soap and solution such as dilute chlorhexidine until the area is free of all gross debris. gross debris within the wound itself can be flushed using a 30-ml syringe filled with sterile saline or lactated ringer's solution and an 18-gauge needle. pressure-lavage systems are also available for use, if desired. grossly contaminated wounds can be rinsed first with warm tap water to eliminate gross contamination, and then prepared as just described. debride the wound, removing skin and other soft tissue that is not obviously viable. obviously viable and questionable tissue should remain, and the wound left open for frequent reassessment on a daily basis. remove any dark or white segments of skin. questionable skin edges may or not regain viability and should be left in place for 48 hours, so the wound can fully reveal itself. excise grossly contaminated areas of fat and underlying fascia. blood vessels that are actively bleeding should be ligated to control hemorrhage, if collateral circulation is present. if nerve bundles are ligated cleanly in a clean wound, the nerve edges should be reapposed and anastomosed. if gross contamination is present, however, definitive neurologic repair should be delayed until healthy tissue is present. excise contaminated muscle until healthy bleeding tissue is present. anastamoe tendon lacerations if the wound is clean and not grossly contaminated. if gross contamination is present, the tendon can be temporarily anastomosed and a splint placed on the limb until definitive repair of healthy tissue is possible. thoroughly lavage open wounds to a joint with sterile saline or lactated ringer's solution. infusion of chlorhexidine or povidone-iodine solution into the joint can cause a decrease in cartilage repair and is contraindicated. smooth sharp edges and remove any obvious fragments. whenever possible, the joint capsule and ligaments should be partially or completely closed. after removing bullets and metal fragments, the subcutaneous tissue and skin should be left open to heal by second intention, or should be partially closed with a drain. the joint should then be immobilized. injuries and exposed bone should be carefully lavaged, taking care to remove any gross debris without pushing the debris further into the bone and wound. the bone should be covered with a moist dressing and stabilized until definitive fracture repair can be made. this type of injury typically is seen with shearing injuries of the distal extremities caused by interaction with slow-moving vehicles. perform wet-to-dry or enzymatic debridement until a healthy granulation bed is present. if large areas of contamination are present (e.g., necrotizing fasciitis), en bloc debridement may be necessary. en bloc debridement consists of complete excision of badly infected wounds without entering the wound cavity, to prevent systemic infection. this technique should be used only if there is sufficient skin and soft tissue to allow later closure and it can be performed without damaging any major nerves, tendons, or blood vessels. open wounds often are managed by second intention healing, delayed primary closure, or secondary closure. see section on wound management and bandaging for a more complete discussion on the use of various bandaging materials in the treatment of open wounds. if an animal is presented very shortly after a wound has occurred and there is minimal contamination and trauma, the wound can be closed after induction of anesthesia and 1 careful preparation of the wound and surrounding tissues. close any dead space under the skin with absorbable suture material in an interrupted suture pattern. avoid incising major blood vessels or nerves. close the subcutaneous tissues with absorbable suture material in an interrupted or continuous suture pattern. take care that there is not too much tension on the wound, or else surgical dehiscence will occur with patient movement. close the skin with nonabsorbable suture or surgical staples (2-0 to 4-0) . if there is any doubt at the time of repair about tissue status or inability to close all dead space, place a passive drain (penrose drain) so that the proximal end of the drain is anchored in the proximal aspect of the wound with a suture(s). leave the ends long so that the suture can be accurately identified at the time of drain removal. pass the suture through the skin, through the drain, and out the other side of the skin. place the rest of the drain into the wound and then secure it at the most ventral portion of the wound or exit hole in the most dependent area of the body, to allow drainage and prevent seroma formation. close the subcutaneous tissue over the drain before skin closure. during wound closure, be sure to not incorporate the subcutaneous or skin sutures into the drain, or it will not be possible to remove the drain without reopening the wound. bandage the area to prevent contamination. the drain can be removed once drainage is minimal (usually 3 to 5 days). active drains can be constructed or purchased; their use is indicated in wounds that are free of material that can plug the drain. to construct a small suction drain, remove the female portion or catheter hub at the end of a butterfly catheter. fenestrate the tubing so that there are multiple side holes, taking care to avoid making the holes larger than 50% of the circumference of the tubing. place the tubing into the wound via a small stab incision distal to the wound. use a purse-string suture around the tubing to facilitate a tight seal and prevent the tubing from exiting the wound. following wound closure, insert the butterfly needle into a 5-to 10-ml evacuated blood collection tube to allow fluid to drain into the tube. incorporate the tube into the bandage, and replace it when it becomes full. alternatively, the butterfly portion of the system can be removed and the tube fenestrated as described previously. place the tube into the wound and suture it in place to create a tight seal. secure the catheter hub to a syringe in which the plunger has been drawn back slightly to create suction. insert a metal pin or 16-to 18-gauge needle through the plunger at the top of the barrel to hold it at the desired level. incorporate the suction apparatus into the bandage and replace it when it becomes full. delayed primary closure should be considered when there is heavy contamination, purulent exudate, residual necrotic debris, skin tension, edema and erythema, and lymphangitis. delayed primary closure usually is made 3 to 5 days after the initial wound infliction and open wound management has been performed. once healthy tissue is observed, the skin edges should be debrided and the wound closed as with primary closure. secondary wound closure should be considered when infection and tissue trauma necessitate open wound management for more than 5 days. secondary wound closure is performed after the development of a healthy granulation bed. this technique also is useful when a wound has dehisced and has formed granulation tissue. if the wound edges can be manipulated into apposition and if epithelialization has not begun, the wound can be cleansed and the wound edges apposed and sutured. this is known as early secondary closure. late secondary closure should be performed whenever there is a considerable amount of granulation tissue, the edges of the wound cannot be manipulated into position, and epithelialization has already started. in such cases, the wound should be cleaned, and the skin edges debrided to remove the epithelium. the remaining wound edges are then sutured over the granulation tissue ( shock is defined as a state of inadequate circulating volume and inability to meet cellular oxygen demands. there are three types of shock: hypovolemic, cardiogenic, and septic. early recognition of the type of shock present is crucial in the successful clinical management of shock syndrome. tissue oxygen delivery is based on cardiac output and arterial oxygen concentration. knowledge of the components of normal oxygen delivery is essential to the treatment of shock in the critical patient. improper handling of animal during further tissue and neurologic damage may occur transport (e.g., improper limb or spine immobilization). inadequate assessment of animal's animal's condition may worsen or animal may general condition or wounded tissues succumb; tissue injuries may be overlooked. inadequate wound protection during further wound contamination may occur at assessment, resuscitation, or veterinary facility. stabilization procedures inadequate wound protection while further wound contamination with fur and preparing the surrounding area debris may occur. insufficient wound lavage wound infection may occur. hydrogen peroxide wound lavage lavage offers little bactericidal activity and contributes to irritation of tissues and delayed healing. lavage has short residual activity and absorption with large wound. overly aggressive initial layered debridement may result in the removal of viable debridement tissue. en bloc debridement debridement results in removal of large amounts of tissue and a large defect for closure. use of drains potential exists for bacteria to ascend along the drain, for drain removal by the animal or breakage of the drain, and for possible tissue emphysema with air being sucked under the skin with patient movement. tube-type drains drains may cause postoperative discomfort; fenestrations may become occluded to stop intraluminal drainage. deeply placed sutures in the presence drain may be incorporated into the repair and of a drain prevent drain removal. active drains high negative pressure may cause tissue injury; highly productive wounds may necessitate changing the evacuated blood tubes several times a day with constructed drains. oxygen delivery (do 2 ) = cardiac output (q) ã� arterial oxygen content (cao 2 ) where q = heart rate ã� stroke volume. stroke volume is affected by preload, afterload, and cardiac contractility. where hb = hemoglobin concentration, sao 2 = oxygen saturation, and pao 2 = arterial partial pressure of oxygen in mm hg. thus, factors that can adversely affect oxygen delivery include inadequate preload or loss of circulating volume, severe peripheral vasoconstriction and increased afterload, depressed cardiac contractility, tachycardia and decreased diastolic filling, cardiac dysrhythmias, inadequate circulating hemoglobin, and inadequate oxygen saturation of hemoglobin. during septic shock, enzymatic dysfunction and decreased cellular uptake and utilization of oxygen also contribute to anaerobic glycolysis. an inadequate circulating volume may develop secondary to maldistribution of available blood volume (traumatic, septic, and cardiogenic origin) or as a result of absolute hypovolemia (whole blood or loss of extracellular fluid). normally, the animal compensates by (1) splenic and vascular constriction to translocated blood from venous capacitance vessels to central arterial circulation, (2) arteriolar constriction to help maintain diastolic blood pressure and tissue perfusion, and (3) an increase in heart rate to help maintain cardiac output. arteriolar vasoconstrictions support perfusion to the brain and heart at the expense of other visceral organs. if vasoconstriction is severe enough to interfere with delivery of adequate tissue oxygen for a sufficient period of time, the animal may die. hypovolemic shock can result from acute hemorrhage or from severe fluid loss from vomiting, diarrhea, or third spacing of fluids. early in shock, baroreceptors in the carotid body and aortic arch sense a decrease in wall stretch from a decrease in circulating fluid volume. tonic inhibition of sympathetic tone via vagal stimulation is diminished, and heart rate and contractility increase and peripheral vessels constrict to compensate for the decrease in cardiac output. the compensatory mechanisms protect and support blood supply to the brain and heart at the expense of peripheral organ perfusion. this is called early compensatory shock. early compensatory shock is characterized by tachycardia, normal to fast capillary refill time, tachypnea, and normothermia. as shock progresses, the body loses its ability to compensate for ongoing fluid losses. early decompensatory shock is characterized by tachycardia, tachypnea, delayed capillary refill time, normotension to hypotension, and a fall in body temperature. end-stage decompensatory shock is characterized by bradycardia, markedly prolonged capillary refill time, hypothermia, and hypotension. aggressive treatment is necessary for any hope of a favorable outcome. septic shock should be considered in any patient with a known infection, recent instrumentation that could potentially introduce infection (indwelling intravenous or urinary catheter, surgery or penetrating injury), disorders or medical therapy that can compromise immune function (diabetes mellitus, immunodeficiency virus, parvovirus or feline panleukopenia virus infection, stress, malnutrition, glucocorticoids, chemotherapy). the presence of bacteria, viruses or rickettsiae, protozoa, or fungal organisms in the blood constitutes septicemia. septic shock is characterized by the presence of sepsis and refractory hypotension that is unresponsive to standard aggressive fluid therapy and inotropic or pressor support. septic shock and other causes of inflammation can lead to systemic inflammatory response syndrome (sirs). in animals, the presence of two or more of the criteria in table 1 -56 in the presence of suspected inflammation or sepsis constitutes sirs (table 1 -56). clinical signs associated with sepsis may be vague and nonspecific, including weakness, lethargy, vomiting, and diarrhea. cough and pulmonary crackles may be associated with pneumonia. decreased lung sounds may be associated with pyothorax. abdominal pain and fluid may be associated with septic peritonitis. vaginal discharge may or may not be present in patients with pyometra. diagnostic tests should include a white blood cell count, serum biochemical profile, coagulation tests, thoracic and abdominal radiographs, and urinalysis. the white blood cell count in a septic patient that is appropriately responding to the infection will be elevated with a left-shifted neutrophilia and leukocytosis. a degenerative left shift, in which leukopenia with elevated band neutrophils suggests an overwhelming infection. biochemical analyses may demonstrate hypoglycemia and nonspecific hepatocellular and cholestatic enzyme elevations. in the most severe cases, metabolic (lactic) acidosis, coagulopathies, and end-organ failure, including anuria and ards, may be present. cardiogenic shock occurs as a result of cardiac output inadequate to meet cellular oxygen demands. cardiogenic shock is associated with primary cardiomyopathies, cardiac dysrhythmias, pericardial fluid, and pericardial fibrosis. abnormalities seen on physical examination often are similar to those seen in other categories of shock, but they can also include cardiac murmurs, dysrhythmias, pulmonary rales, bloody frothy pulmonary edema fluid from the nares or mouth, orthopnea, and cyanosis. it is important to distinguish the primary cause of shock before implementing treatment (table 1-57) , whenever possible, because treatment for a suspected ruptured hemangiosarcoma differs markedly from the treatment for end-stage dilatative cardiomyopathy. the patient's clinical signs may be similar and include a peritoneal fluid wave, but the treatment for hypovolemia can dramatically worsen the congestive heart failure secondary to dilatative cardiomyopathy. when a patient presents with some form of shock, immediate vascular access is of paramount importance. place a large-bore peripheral or central venous catheter for the infusion of crystalloid or colloid fluids, blood component therapy, and drugs. monitor the patient's cardiopulmonary status (by ecg), blood pressure, oxygen saturation (as determined by pulse oximetry or arterial blood gas analyses), hematocrit, bun, and glucose. ancillary diagnostics, including thoracic and abdominal radiography, urinalysis, serum biochemistry profile, coagulation tests, complete blood count, abdominal ultrasound, and echocardiography, should be performed as determined by the individual patient's needs and the type of shock. the following list, called the "rule of twenty," is a guideline for case management of the shock patient. consideration of each aspect of the rule of twenty on a daily basis ensures temperature <100â°f or >103.5â°f <100â°f or >103.5â°f heart rate >120 beats/minute in dogs <140 or >250 beats/minute in cats respiratory rate >20 breaths/minute or paco 2 >40 breaths/minute or paco 2 <32 mm hg <32 mm hg white blood cell >18,000 cells/âµl 19,000 cells/âµl count or <4000 cells/âµl o r <5000 cells/ml or >10% bands or >10% bands 1 that major organ systems are not overlooked. the list also provides a means to integrate and relate changes in different organ systems functions with one another.* the treatment of hypovolemic and septic shock requires the placement of large-bore intravenous catheters in peripheral and central veins. if vascular access cannot be obtained percutaneously or by cutdown methods, intraosseous catheterization should be considered. once vascular access is achieved, rapidly administer large volumes of crystalloid or colloid fluids. as a rule of thumb, administer 1 /4 of a calculated shock dose of fluids-that is, 1 /4 ã� (90 ml/kg/hour) in dogs and 1 /4 ã� (44 ml/kg/hour) in cats) of a balanced crystalloid fluid ( normosol-r, plasmalyte-m, lactated ringer's solution, or 0.9% sterile saline). reassess the patient's perfusion parameters (heart rate, capillary refill time, blood pressure, urine output) on a continual basis to direct further fluid therapy. synthetic colloid fluids (hetastarch, dextran 70, or oxyglobin) can also be administered in the initial resuscitation from shock. a guideline is to administer 5 to 10 ml/kg of hetastarch or dextran as a bolus over 10 to 15 minutes and then reassess perfusion parameters. hypertonic saline (0.7% nacl, 4 ml/kg) can be used in cases of hemorrhagic shock to temporarily restore intravascular fluid volume by drawing fluid from the interstitial space. because this type of fluid resuscitation is short-lived, hypertonic saline should always be used with another crystalloid or colloid fluid, and it should not be used in patients with interstitial dehydration. if hemorrhagic shock is present, the goal should be to return a patient's blood pressure to normal (not supraphysiologic) levels (i.e., systolic pressure 90-100 mm hg, diastolic pressure >40 mm hg, and mean arterial pressure â�¥60 mm hg) to avoid iatrogenically causing clots to fall off and hemorrhage to re-start. in critically ill patients, fluid loss can be measured in the form of urine, vomit, diarrhea, body cavity effusions, and wound exudates. additionally, insensible losses (those that cannot be readily measured from sweat, panting, and cellular metabolism) constitute 20 ml/kg/ day. measurement of fluid "ins and outs" in conjunction with the patient's central venous pressure, hematocrit, albumin, and colloid oncotic pressure can help guide fluid therapy (see also section on fluid therapy). maintenance of normotension is necessary for adequate oxygen delivery to meet cellular energy demands. blood pressure can be measured using direct arterial catheterization, or through indirect means such as doppler plesthymography or oscillometric methods. the systolic pressure should remain at or greater than 90-100 mm hg at all times. the diastolic pressure is very important, too, as it constitutes two thirds of the mean arterial pressure; it must be greater than 40 mm hg for coronary artery perfusion. the mean arterial pressure should be greater than 60 mm hg for adequate tissue perfusion. if fluid resuscitation and pain management are not adequate in restoring blood pressure to normal, vasoactive drugs including positive inotropes and pressors should be considered (table 1 -58). in cases of cardiogenic shock, vasodilator drugs (table 1 -59) can be used to decrease vascular resistance and afterload. low-dose morphine (0.05 mg/kg, iv, im) dilates splanchnic vessels and helps reduce pulmonary edema. furosemide (1 mg/kg/hour) also can dilate pulmonary vasculature and potentially reduce edema fluid formation in cases of ards. cardiac output is a function of both heart rate and stroke volume. stroke volume or (the amount of blood that the ventricle pumps in 1 minute) is affected by preload, afterload, and contractility. during hypovolemic shock, there is a fall in cardiac preload due to a decrease in circulating blood volume. during septic and cardiogenic shock, there is a decrease in contractility secondary to inherent defects of the myocardium or due to the negative inotropic effects of inflammatory cytokines such as tnf-alpha, myocardial depressant factor, il-1, and il-10 released during sepsis and systemic inflammation. afterload also may be increased because of the compensatory mechanisms and neurohumoral activation of the renin-angiotensin-aldosterone axis in hypovolemic or cardiogenic shock. as heart rate increases to compensate for a decline in cardiac output, myocardial oxygen demand increases and diastolic filling time becomes shorter. because the coronary arteries are perfused during diastole, coronary perfusion can be impaired, and myocardial lactic acidosis can develop, causing a further decline in contractility. in addition to lactic acidosis, acid-base and electrolyte abnormalities, inflammatory cytokines, direct bruising of the myocardium from trauma, and areas of ischemia can further predispose the patient to ventricular or atrial dysrhythmias. cardiac dysrhythmias should be controlled whenever possible. treatment of bradycardia should be directed at treating the underlying cause. administer anticholinergic drugs such as atropine (0.04 mg/kg im) or glycopyrrolate (0.02 mg/kg im) as necessary. in cases of third-degree or complete atrioventricular (av) block, administer a pure betaagonist such as isoproterenol (0.04-0.08 âµg/kg/minute iv cri, or 0.4 mg in 250 ml of 5% dextrose in water iv slowly). perform passive rewarming if the patient is hypothermic. receptor activity dosage (iv) dopamine da 1 , da 2 , î± +++ , 5-25 âµg/kg/minute (blood pressure support)* î² +++ 1-5 âµg/kg/minute (renal afferent diuresis) dobutamine î± + , î² +++ 3-20 âµg/kg/minute* (blood pressure support, positive inotrope) norepinephrine î± +++ , î² + 0.05-0.3 mg/kg/minute; 0.01-0.02 mg/kg phenylephrine î± +++ , î² 0 0.05-0.2 mg/kg epinephrine î± +++ , î² +++ 0.02-0.5 mg/kg, 0.05-0.2 mg/kg/minute +++, strong receptor activity; 0, no receptor activity; +, weak receptor activity. *monitor for tachyarrhythmias at higher doses. correct any underlying electrolyte abnormalities such as hyperkalemia and hypo-and hypermagnesemia. treat ventricular dysrhythmias such as multifocal premature ventricular contractions (pvcs), sustained ventricular tachycardia >160 beats per minute, and r on t phenomenon (the t wave of the preceding beat occurs superimposed on the qrs complex of the next beat, and there is no return to isoelectric shelf), or if runs of ventricular tachycardia cause a drop in blood pressure. intravenous lidocaine and procainamide are the first drugs of choice for ventricular dysrhythmias. supraventricular tachycardia can impair cardiac output by impairing diastolic filling time. control supraventricular dysrhythmias with calcium channel blockers, beta-adrenergic blockers, or quinidine (table 1-60) . (disorientation); is 1 minute; 2 minutes) light sensitive and must be covered in foil and not kept for longer than 4 hours 1 albumin can decrease as a result of loss from the gastrointestinal tract, urinary system, and wound exudates, or into body cavity effusions. albumin synthesis can decrease during various forms of shock due to a preferential increase in hepatic acute phase protein synthesis. serum albumin contributes 80% of the colloid oncotic pressure of blood, in addition to its important roles as a free radical scavenger at sites of inflammation and as a drug and hormone carrier. albumin levels <2.0 g/dl have been associated with an increase in morbidity and mortality in human and veterinary patients. administer fresh frozen plasma (20 ml/kg) or concentrated human albumin (2 ml/kg of 25% solution) to maintain serum albumin â�¥2.0 g/dl. additional oncotic support can be in the form of synthetic colloids, as indicated. colloid oncotic pressure within the intravascular and interstitial spaces contributes to fluid flux. oncotic pressure can be measured with a colloid osmometer. normal oncotic pressure is 15 mm hg. in cases of sepsis and sirs, increased vascular permeability increases the tendency for leakage of fluids into the interstitial spaces. colloids that can be administered until the source of albumin loss resolves include the synthetic colloids hetastarch and dextran 70 (20-30 ml/kg/day), synthetic hemoglobin-based oxygen carriers (oxyglobin, 3-7 ml/kg/day), concentrated human albumin (25% albumin, 2 ml/kg), and plasma (20 ml/kg). oxygenation and ventilation can be evaluated by arterial blood gas analysis or by the noninvasive means of pulse oximetry and capnometry (see sections on pulse oximetry and capnometry). oxygen delivery can be impaired in cases of hypovolemic shock because of hemorrhage and anemia, and thus a decrease in functional capacity to carry oxygen, and is not to be used for more than 2 weeks due to idiosyncratic blindness. in cases of cardiogenic shock as a result of impaired ability to saturate hemoglobin due to pulmonary edema in the lungs, or decrease in cardiac output. in septic shock, decreases in cardiac output due to inflammatory cytokines and a decrease in cellular oxygen extraction can lead to lactic acidosis. increased cellular metabolism and decreases in respiratory function can lead to respiratory acidosis as co 2 increases. administer supplemental oxygen as flow-by, nasal or nasopharyngeal catheter, oxygen hood, or oxygen cage. supplemental oxygen should be humidified, and delivered at 50-100 ml/kg/minute. if oxygenation and ventilation are so impaired that the pao 2 remains <60 mm hg with the patient on supplemental oxygen, a paco 2 >60 mm hg, or severe respiratory fatigue, develops, and mechanical ventilation should be considered. glucose is a necessary fuel source for red blood cells and neuronal tissues, and serum glucose should be maintained within normal reference ranges. glucose supplementation can be administered as 2.5-5% solutions in crystalloid fluids, or in parenteral and enteral nutrition products. arterial and venous ph can be measured by performing blood gas analyses. decrease in tissue perfusion, impaired oxygen delivery, and decreased oxygen extraction in the various forms of shock can lead to anaerobic metabolism and metabolic acidosis. in most cases, improving tissue perfusion and oxygen delivery with crystalloid and colloid fluids, supplemental oxygen, and inotropic drugs will help normalize metabolic acidosis. serial measurements of serum lactate (normal, <2.5 mmol/l) can be used as a guide to evaluate the tissue response to fluid resuscitative efforts. serum electrolytes often become severely deranged in shock states. serum potassium, magnesium, sodium, chloride, and total and ionized calcium should be maintained within normal reference ranges. if metabolic acidosis is severe, sodium bicarbonate can be administered by calculating the formula base deficit ã� 0.3 ã� body weight in kg = meq bicarbonate to administer because iatrogenic metabolic alkalosis can occur, a conservative approach is to administer 1 /4 of the calculated dose and then recheck the patient's ph and bicarbonate levels. if the base excess is unknown, sodium bicarbonate can be administered in incremental doses of 1 meq/kg until the ph is above 7.2. complications associated with bicarbonate therapy include iatrogenic hypocalcemia, metabolic alkalosis, paradoxical cerebrospinal fluid acidosis, hypotension, restlessness, and death. massive trauma, neoplasia, sepsis, and systemic inflammation can all lead to coagulation abnormalities, including disseminated intravascular coagulation (dic). cage-side coagulation monitors are available for daily measurement of prothrombin time (pt), activated partial thromboplastin time (aptt), and platelet counts. fibrin degradation products (fibrin split products) become elevated in dic, trauma, hepatic disease, and surgery. coagulation proteins (clotting factors) and antithrombin often are lost with other proteins in hypoproteinemia or are consumed when microclots are formed and then dissolved. antithrombin levels can be measured by commercial laboratories. antithrombin and clotting factors can be replenished in the form of fresh frozen plasma transfusions. a more sensitive and specific test for dic is the detection of d-dimers, which can be measured by commercial laboratories. treatment for dic involves treatment and resolution of the underlying disease and administration of antithrombin and clotting factors in the form of fresh frozen plasma (20 ml/kg) and heparin (unfractionated, 50-100 units/kg sq tid; fractionated [lovenox], 1 mg/kg sq bid). monitor the patient for changes in mental status, including stupor, coma, decreased ability to swallow and protect the airway, and seizures. elevation of the patient's head can help to protect the airway and decrease the risk of increased intracranial pressure. serum glucose should be maintained within normal levels to prevent hypoglycemia-induced seizures. one of the major components of oxygen delivery is the binding to hemoglobin. packed cell volume must be kept above 20-30% for adequate cellular oxygen delivery. acid-base status can adversely affect oxygen offloading at the tissue level if metabolic or respiratory alkalosis is present. oxygen-carrying capacity and hemoglobin levels can be increased with administration of rbc component therapy or with hemoglobin-based oxygen carriers. monitoring of renal function includes daily measurement of bun, creatinine, and urine output. normal urine output in a hydrated euvolemic patient is 1-2 ml/kg/hour. fluid ins and outs should be measured in cases of suspected oliguria or anuria. in patients with oliguria or anuria, furosemide can be administered as a bolus (4-8 mg/kg) or by constant rate infusion (cri)(0.66-1 mg/kg/hour). mannitol should also be administered (0.5-1 g/kg over 10 to 15 minutes). dopamine (1-5 âµg/kg/minute cri) can be administered to dilate renal afferent vessels and improve urine output. the patient's white blood cell count may be elevated, normal, or decreased, depending on the type of shock. the decision to administer antibiotics should be made on a daily basis. superficial or deep staphylococcus or streptococcus infection usually can be treated with a first-generation cephalosporin (cefazolin, 22 mg/kg iv tid). if a known source of infection is present, administer a broad-spectrum antibiotic (cefoxitin, 22 mg/kg iv tid; ampicillin, 22 mg/kg qid, or enrofloxacin, 5-10 mg/kg once daily) pending results of culture and susceptibility testing. if broader anaerobic coverage is required, metronidazole (10 mg/kg iv tid) should be considered. gentamicin (3-5 mg/kg iv once daily) is a good choice for gram-negative sepsis, provided that the patient is well hydrated and has normal renal function. ideally, patients receiving any aminoglycoside antibiotic should have a daily urinalysis to check for renal tubular casts that signify renal damage. in dogs, the gut is the shock organ. impaired gastrointestinal motility and vomiting should aggressively be treated with antiemetics and promotility drugs (dolasetron, 0.6 mg/kg iv once daily, and metoclopramide, 1-2 mg/kg/day iv cri). metoclopramide is contraindicated in cases of suspected gastrointestinal obstruction. histamine-receptor blockers such as famotidine (0.5 mg/kg bid iv) and ranitidine (0.5 to 2 mg/kg iv bid, tid) or proton-pump inhibitors (omeprazole, 0.5-1 mg/kg po once daily) can be administered for esophagitis. administer sucralfate (0.25-1 g po tid) to treat gastric ulceration. if the gastrointestinal barrier function is diminished due to poor perfusion, infection, or inflammation, administer broad-spectrum antibiotics such as ampicillin (22 mg/kg iv qid) to prevent gastrointestinal bacterial translocation. the course of drug therapy should be reviewd daily and the patient should be monitored for potential drug interactions. for example, metoclopramide and dopamine, working at the same receptor, can effectively negate the effects of each other. cimetidine, a cytochrome p450 enzyme inhibitor, can decrease the metabolism of some drugs. drugs that are avidly protein-bound may have an increase in unbound fraction with concurrent hypoalbuminemia or when hypoalbuminemia is present. decreased renal function may impair the renal clearance of some drugs, requiring increased dosing interval or decreased dose. nutrition is of utmost importance in any critically ill patient. patients with septic shock may become hypermetabolic and require supraphysiologic nutrient caloric requirements, while others may actually become hypometabolic. enteral nutrition is preferred, whenever possible, because enterocytes undergo atrophy without luminal nutrient stimulation. a variety of enteral feeding tubes can be placed, depending on what portion of the gut is functional, to provide enteral nutrition in an inappetent patient. loss of gastrointestinal mucosal barrier function may predispose the patients to the development of bacterial translocation and may contribute to sepsis. if enteral nutrition is impossible because of protracted vomiting or gastrointestinal resection, glucose, lipid, and amino acid products are available that can be administered parenterally to meet nutrient needs until the gastrointestinal tract is functioning and the patient can be transitioned to enteral nutrition. assessment of pain in animals in shock can be challenging. pain can result in the release of catecholamines and glucocounterregulatory hormones that can impair nutrient assimilation and lead to negative nitrogen balance, impaired wound healing, and immunocompromise. in any animal determined to be in pain, analgesic drugs should be administered to control pain and discomfort at all times. opioids are cardiovascularly friendly, and their effects can easily be reversed with naloxone if adverse effects such as hypotension and hypoventilation occur. if the patient is nonambulatory, rotate the animal from side to side every 4 to 6 hours to prevent lung atelectasis. passive range-of-motion exercises and deep muscle massage should be performed to increase tissue perfusion, decrease dependent edema, and prevent disuse atrophy. animals should be kept completely dry on soft, padded bedding to prevent the development of decubital ulcers. all bandages, wound sites, and catheter sites should be checked daily for the presence of swelling, erythema, and pain. soiled bandages should be changed to prevent strike-through and contamination of the underlying catheter or wound. hospitalization can be a stressful experience for patient and client alike. allowing brief visits and walks outside in the fresh air can improve a patient's temperament and decrease stress. the preemptive use of analgesic drugs on a regular schedule (not prn) should be used to prevent pain before it occurs. pain decreases the patient's ability to sleep. lack of sleep can promote further stress and impaired wound healing. the use of glucocorticosteroids and antiprostaglandins in shock therapy remains a topic of wide controversy. although the use of these agents potentially may stabilize membranes, decrease the absorption of endotoxin, and decrease prostaglandin release, the routine use of glucocorticosteroids and antiprostaglandins can decrease renal perfusion and gastrointestinal blood flow, promoting gastrointestinal ulceration and impaired renal function. the administration of supraphysiologic levels of glucocorticosteroids in patients in any type of shock can increase sodium and water retention, depress cellular immune function, and impair wound healing. in clinical studies of small animal patients, the routine use of glucocorticosteroids and antiprostaglandins has not demonstrated definite improved survival. the risks of therapy do outweigh the anecdotal reported benefits, and therefore the empiric use of glucocorticosteroids and antiprostaglandins in any shock patient is urinary tract emergencies azotemia azotemia occurs when 75% or more of the nephrons are nonfunctional. the magnitude of the azotemia alone cannot be used to determine whether the azotemia is prerenal, renal, or postrenal in origin, or whether the disease process is acute or chronic, reversible or irreversible, progressive or nonprogressive. before beginning treatment for azotemia, the location or cause of the azotemia must be identified. take a thorough history and then perform a physical examination. obtain blood and urine samples before initiating fluid therapy, for accurate assessment of the location of the azotemia. for example, an azotemic animal with a history of vomiting and diarrhea that appears clinically dehydrated on physical examination, normally should have a concentrated urine specific gravity (>1.045) reflecting the attempt to conserve fluid. if this level is found, the azotemia is much less likely to be renal in origin, and the azotemia will likely resolve after rehydration. if, however, the urine specific gravity is isosthenuric or hyposthenuric (1.007-1.015) in the presence of azotemia and dehydration, primary intrinsic renal insufficiency is likely present. if the azotemia resolves with fluid therapy, the patient has prerenal and primary renal disease. if the azotemia does not resolve after rehydration, the patient has prerenal and primary renal failure. dogs with hypoadrenocorticism can have both prerenal and primary renal disease secondary to the lack of mineralocorticoid (aldosterone) influence on the renal collecting duct and renal interstitial medullary gradient. medullary washout can occur, causing isosthenuric urine in the presence of dehydration from vomiting and diarrhea. the patient often has azotemia due to fluid loss (dehydration and urinary loss) and gastric or intestinal hemorrhage (elevated bun). the prerenal component will resolve with treatment with glucocorticoids and crystalloid fluids, but the renal component may take several weeks to resolve, until the medullary concentration gradient is reestablished with the treatment and influence of mineralocorticoids. drugs such as corticosteroids and diuretics can influence renal tubular uptake and excretion of fluid, and cause a prerenal azotemia and isosthenuric urine in the absence of primary renal disease. treatment of azotemia includes calculation of the patient's dehydration estimate and maintenance fluid volumes, and administering that volume over the course of 24 hours. identify and treat underlying causes of prerenal azotemia (shock, vomiting, diarrhea). monitor urine output closely. once a patient is euvolemic, oliguria is defined as urine output <1-2 ml/kg/hour. urine output should return to normal in patients with prerenal azotemia as rehydration occurs. if a patient remains oliguric after rehydration, consider the possibility of oliguric acute intrinsic renal failure, and administer additional fluid therapy based on the patient's urine output, body weight, central venous pressure, and response to other medical therapies. prerenal azotemia is caused by conditions that decrease renal perfusion, including hypovolemic shock, severe dehydration, hypoadrenocorticism, congestive heart failure, cardiac tamponade, cardiac dysrhythmias, and hypotension. once renal perfusion is restored, the kidneys can resume normal function. glomerular filtration rate decreases when the mean arterial blood pressure falls to less than 80 mm hg in a patient with normal renal autoregulation. renal autoregulation can be impaired in some diseases. passive reabsorption of urea from the renal tubules can occur during states of low tubular flow (dehydration, hypotension) even if glomerular filtration is not decreased. if renal hypoperfusion is not quickly restored, the condition can progress from prerenal disease to acute intrinsic renal failure. prerenal and renal azotemia can coexist in animals with primary renal disease, as a result of vomiting and ongoing polyuria in the absence of any oral fluid intake. the treatment of prerenal azotemia consists of rehydration, antiemetic therapy, and treatment of the underlying cause of vomiting, diarrhea, or third spacing of fluids. acute intrinsic renal failure is characterized by an abrupt decline in renal function to the extent that azotemia and an inability to regulate solute and fluid balance. patients with acute intrinsic renal failure may be oliguric or polyuric, depending on the cause and state of renal failure. in small animals, the most common causes of acute intrinsic renal failure are renal ischemia and toxins. there are three phases of acute intrinsic renal failure: induction, maintenance, and recovery. during the induction phase, some insult (ischemia or toxin) to the kidneys occurs, leading to a defective concentrating mechanism, decreased renal clearance of nitrogenous waste (azotemia), and polyuria or oliguria. if treatment is initiated during the induction phase, progression to the maintenance phase potentially can be stopped. as the induction phase progresses, there is worsening of the urine-concentrating ability and azotemia. renal tubular epithelial cells and renal tubular casts can be seen on examination of the urine sediment. glucosuria may be present. the maintenance phase of acute intrinsic renal failure occurs after a critical amount of irreversible nephron injury. correction of the azotemia and removal of the cause of the problem do not result in return to normal function. in patients with oliguria, the extent of nephron damage is greater than that observed in patients with polyuria. the maintenance phase may last for several weeks to months. recovery of renal function may or may not occur, depending on the extent of injury. the most serious complications (overhydration and hyperkalemia) are observed in patients with oliguria. the recovery phase occurs with sufficient healing of damaged nephrons. azotemia may resolve, but concentrating defects may remain. if the patient was oliguric in the maintenance phase, a marked diuresis develops during the recovery phase that may be accompanied by fluid and electrolyte losses. this phase may last for weeks to months. treatment of acute intrinsic renal failure consists of determining the cause and ruling out obstruction or uroabdomen whenever possible. a careful history can sometimes determine whether there has been exposure to nephrotoxic drugs, chemicals, or food items. if ingestion or exposure to a toxic drug, chemical, or food occurred recently (within 2 to 4 hours), induce emesis with apomorphine (0.04 mg/kg iv). next, administer activated charcoal either orally or via stomach tube, to prevent further absorption of the toxin. obtain blood and urine samples for toxicologic analysis (e.g., ethylene glycol) and to determine whether azotemia or abnormalities in the urine sediment exist. (see section on ethylene glycol, grapes and raisins, and nonsteroidal antiinflammatory drugs). obtain a complete blood count, biochemical profile, and urinalysis to determine the presence of signs of chronic renal failure, including polyuria, polydipsia, and nonregenerative anemia. radiographs and abdominal ultrasound can help in determining the chronicity of renal failure. normal renal size is 2.5-3.5 times the length of l2 in dogs and 2.4-3.0 times the length of l2 in cats. monitor the patient's body weight at least twice a day to avoid overhydration. also monitor urine output; normal output is 1-2 ml/kg/hour. in cases of polyuric renal failure, massive fluid and electrolyte losses can occur. place a urinary catheter for patient cleanliness and to facilitate urine quantitation. measure fluid ins and outs (see section on fluid therapy). after the patient has been rehydrated, the amount of fluids administered should equal maintenance and insensible needs plus the volume of urine produced each day. if a urinary catheter cannot be placed or maintained, serial body weight measurements and central venous pressure should be used to monitor the patient's fluid balance and prevent overhydration. if the patient is oliguric (urine output <1-2 ml/kg/hour), pharmacologic intervention is necessary to increase urine output. first, administer furosemide (2-4 mg/kg or 0.66 mg/kg/hour iv cri). repeat bolus doses of furosemide if there is no response to initial treatment. if necessary, administer low-dose dopamine (3-5 âµg/kg/minute iv cri) to increase renal afferent dilatation and renal perfusion. dopamine and furosemide may be synergistic if administered together. if dopamine and furosemide therapy is ineffective, administer mannitol (0.25-0.5 g/kg iv) once only. if polyuria is present, management is 1 simplified because of the decreased risk of overhydration. if oliguria cannot be reversed, monitor the central venous pessure, body weight, and respiratory rate and effort, auscultate for crackles, and examine the patient carefully for signs of chemosis and the presence of serous nasal discharge. correct hyperkalemia with sodium bicarbonate (0.25-1.0 meq/kg iv) or with insulin (0.25 units/kg) plus dextrose (1 g/unit of insulin iv, followed by 2.5% dextrose iv cri). treat severe metabolic acidosis (ph <7.2 or hco 3 â�� <12 meq/l) with sodium bicarbonate. if anuria develops or oliguria is irreversible despite this therapy, begin peritoneal dialysis. obtain a renal biopsy to establish a diagnosis and prognosis (see section on renal biopsy). administer gastroprotectant drugs and antiemetics to control nausea and vomiting. if possible, avoid the use of nephrotoxic drugs and general anesthesia. initiate nutritional support in the form of an enteral feeding tube or parenteral nutrition as early as possible. once the patient enters the recovery phase, diuresis may occur that can lead to dehydration and electrolyte imbalances (hyponatremia, hypokalemia). dehydration and electrolyte imbalances can be treated with parenteral fluid and electrolyte supplementation. postrenal azotemia is primarily caused by urethral obstruction or leakage from the urinary tract into the abdomen (uroabdomen). complete urinary tract obstruction and uroabdomen are both ultimately fatal within 3 to 5 days if left untreated. in dogs, the most common causes of urethral obstruction are urinary (urethral) calculi or tumors of the urinary bladder or urethra. in male cats, feline urologic syndrome (fus) is the most common cause of urethral obstruction, although there has been an increased incidence of urethral calculi observed in recent years. a ruptured urinary bladder is the most common cause of uroabdomen and is usually secondary to blunt trauma. clinical signs of urinary tract obstruction include dysuria, hematuria, inability to urinate or initiate an adequate stream of urine, and a distended painful urinary bladder. late in the course of obstructive disease, clinical signs referable to uremia and azotemia (vomiting, oral ulcers, hematemesis, dehydration, lethargy, and anorexia) occur. the initial goal of treatment of urinary tract obstruction is to relieve the obstruction. in male dogs, a lubricated catheter can be inserted past the area of obstruction with the animal under heavy sedation or general anesthesia (see section on urohydropulsion). depending on the chronicity of the obstruction, serum electrolytes should be measured;an ecg should be obtained before administering any anesthetic drugs, because of the cardiotoxic effects of hyperkalemia (see section on atrial standstill). correct fluid, electrolyte, and acid-base abnormalities. if a urinary catheter cannot be placed, perform cystocentesis only as a last resort, because of the risk of urinary bladder rupture. definitive treatment includes identification and treatment of the underlying cause (tumor versus urinary calculi). in most cases, surgical intervention is necessary. if an unresectable tumor is present, a low-profile permanent cystostomy tube can be placed, if the owner desires. administration of piroxicam (feldene, 0.3 mg/kg po q24-48h) with or without chemotherapy may shrink the tumor mass and delay the progression of clinical signs. a complete discussion of this disorder is beyond the scope of this text (see additional reading for other sources of information). feline lower urinary tract disease can cause urethral obstruction, particularly in male cats. clinical signs include stranguria, dribbling of small amounts of urine, lethargy, inappetence, and vomiting. often, owners call with the primary complaint of constipation, because the cat is making frequent trips to the litterbox and straining. cases with a duration of obstruction <36 hours are considered uncomplicated; those with a duration >36 hours are complicated. treatment of urethral obstruction includes stabilizing and normalizing the patient's electrolyte status, induction of sedation or general anesthesia, and relieving the obstruction. obtain blood samples for analysis of electrolyte abnormalities. treat hyperkalemia (k + > 6.0 meq/l) with sodium bicarbonate (0.25-1.0 meq/kg iv), regular insulin (0.25 unit/ kg iv) plus dextrose (1 g//unit of insulin iv), followed by 2.5% dextrose iv cri to prevent hypoglycemia; or calcium gluconate (0.2 ml/kg 10% iv slowly). administer non-potassiumcontaining intravenous fluids in 0.9% saline solution. obtain an ecg to detect atrial standstill (see section on atrial standstill). in some cases, a urethral plug is visible at the tip of the penis. the urethral plug can sometimes be manually extracted or massaged from the penis, and the obstruction temporarily relieved. in such cases, it is still necessary to pass a urethral catheter to flush sediment from the urethra and urinary bladder. unless a patient is obtunded, administer an anesthetic such as ketamine, atropine, or propofol (4-7 mg/kg iv) with diazepam iv for patient comfort and muscle relaxation. once the patient is under anesthesia or heavily sedated, urinary catheterization should be performed. in some cases, it will be difficult to advance the catheter. lubricate a closedended tomcat catheter and pass the tip into the distal urethra. fill a 12-ml syringe with sterile saline and sterile lubricant and connect the syringe to the hub of the catheter. pulse the fluid into the catheter as you gently move the catheter tip back and forth against the urethral obstruction. when the catheter has been passed into the urinary bladder, obtain a urine sample for urinalysis. drain the bladder and flush with sterile saline solution until the urine efflux appears clear. remove the tomcat catheter and insert a 3-5 fr red rubber tube or argyle infant feeding catheter into the urethra for urine collection and quantitation. secure the urinary catheter to prepuce with a butterfly strip of 1-inch adhesive tape secured around the catheter and then sutured to either side of the prepuce. the catheter should be connected to a closed urinary collection system for cleanliness and to reduce the risk of ascending bacterial infection. an elizabethan collar should be placed at all times to prevent the patient from damaging or removing the catheter. when the urethral obstruction has been relieved and the catheter placed, continue intravenous fluid diuresis to alleviate postrenal azotemia. monitor the urine for bacteria and other sediment. in some cases, postobstructive diuresis can be severe. carefully monitor fluid ins and outs, along with body weight, to maintain adequate hydration and perfusion. remove the urinary catheter can be removed after 24 to 48 hours. palpate the bladder frequently to make sure that the patient is voiding normally and to detect the recurrence of obstruction. in patients with severe penile or urethral trauma or edema, administer a short-acting steroid (dexamethasone sodium phosphate, 0.25 mg/kg iv, im, sq). at the time of initial diagnosis and again at the time of discharge, the clients need to be instructed about the long-term management of feline lower urinary tract disease at home, and informed of the risks and consequences of recurrence. uroabdomen can occur from trauma or leakage from the kidneys, ureter, or urinary bladder. clinical signs of uroabdomen (azotemia, uremia, hyperkalemia) can also occur secondary to third spacing of urine and leakage into muscular tissue from a ruptured urethra. in most cases, urinary bladder trauma and rupture are secondary to blunt trauma. abdominocentesis should be performed in any animal with suspected blunt abdominal trauma, and any fluid obtained should be analyzed for creatinine or potassium and compared with the patient's serum levels. an abdominal effusion that has a low packed cell volume and a potassium or creatinine level greater than that of the patient's serum is consistent with the diagnosis of uroabdomen. uroabdomen is not a surgical emergency. however, medical management consists of placement of a temporary abdominal drainage catheter into the abdomen, to facilitate removal of urine from the peritoneal cavity. to place the catheter, position the patient in dorsal or lateral recumbency, shave the ventral abdomen, as for any exploratory laparotomy. aseptically scrub the clipped area, and instill a local anesthestic (lidocaine, 1-2 mg/kg) caudal and to the right of the umbilicus, through the skin, subcutaneous tissues, and rectus 290 1 emergency care clinical differentiation of acute necrotizing from chronic nonsuppurative pancreatitis in cats: 63 cases acute pancreatitis in dogs mesenteric volvulus in the dog: a retrospective study of 12 cases incidence and prognostic value of low plasma ionized calcium concentration in cats with pancreatitis: 46 cases (1996-1998) review of feline pancreatitis. part 2: clinical signs, diagnosis and treatment gastric dilatation-volvulus syndrome in dogs diagnostic approach to acute pancreatitis pathophysiology of organ failure in severe acute pancreatitis in dogs washabau rj: gastrointestinal motility disorders and gastrointestinal prokinetic therapy watson pt: exocrine pancreatic insufficiency as an end-stage of pancreatitis in 4 dogs clinical signs, underlying cause, and outcome in cats with seizures: 17 cases fibrocartilaginous embolism in 75 dogs: clinical findings and factors influencing the recovery rate kirk's current veterinary therapy xiii intervertebral disc extrusion in six cats medical management of acute spinal cord disease risk factors for recurrence of clinical signs associated with thoracolumbar intervertebral disk herniation in dogs: 229 cases intervertebral disk disease in 10 cats long-term functional outcome of dogs with severe injuries of the thoracolumbar spinal cord: 87 cases canine status epilepticus: a retrospective study of 50 cases risk factors for development of status epilepticus in dogs with idiopathic epilepsy and effects of status epilepticus on outcome and survival time: 32 cases (1990-1996) skills laboratory part i: performing a neurologic examination skills laboratory part ii: interpreting the results of the neurologic examination accuracy of localization of cervical intervertebral disk extrusion or protrusion using survey radiography in dogs medical and surgical management of the glaucoma patient the feline glaucomas: 82 cases (1995-1999) the canine glaucomas traumatic ocular protrusion in dogs and cats: 84 cases traumatic glaucoma in a dog ocular and orbital porcupine quills in the dog: a review and case series hyphema: pathophysiologic considerations. comp cont educ pract vet van der woerdt a: the treatment of acute glaucoma in dogs and cats administer crystalloid intravenous fluids at maintenance rates using a balanced electrolyte solution perform urinary catheterization and collection to monitor urine output monitor serum urea nitrogen and creatinine every 12 hours treat oliguria, defined as a drop in urine output to less than 1 ml/kg/hour ml/kg) bolus start dopamine at 3 to 5 âµg/kg/minute if no response to crystalloid/colloid bolus occurs within 30 minutes consider mannitol (0.5 to 1 g/kg iv) administration if no response to dopamine occurs within 30 minutes consider furosemide (4 to 8 mg/kg iv, or 0.66 to 1 mg/kg/hour iv cri) if no response to dopamine or mannitol occurs in 30 to 60 minutes if no response to furosemide, peritoneal dialysis or hemodialysis is indicated immediately, particularly if anuria is present administered with caution, because of the risk of exacerbating increased capillary permeability and causing pulmonary edema. animal patients. chlorphenoxy derivatives exert their toxic effects by an unknown mechanism, and cause clinical signs of gastroenteritis and muscle rigidity severe anemia should be treated with packed rbcs or hemoglobin-based oxygen carriers handbook of small animal toxicology and poisonings macadamia nut toxicosis in dogs the recognition and treatment of the intermediate syndrome of organophosphate poisoning in a dog acute renal failure in four dogs after raisin or grape ingestion pleural effusion in cats pulmonary function, ventilator management, and outcome of dogs with thoracic trauma and pulmonary contusions: 10 cases (1994-1998) acute lung injury and acute respiratory distress syndrome smoke exposure in cats: 22 cases (1986-1997) smoke exposure in dogs: 27 cases (1988-1997) thoracic duct ligation and pericardectomy for treatment of idiopathic chylothorax use of intraluminal nitinol stents in the treatment of tracheal collapse in a dog clinical approach to epistaxis the veterinary icu book. teton newmedia radiographic diagnosis of diaphragmatic hernia: review of 60 cases in dogs and cats tracheal collapse: diagnosis and medical and surgical management acute respiratory distress syndrome brachycephalic syndrome in dogs outcome and postoperative complications in dogs undergoing surgical treatment of laryngeal paralysis: 140 cases (1985-1998) full recovery following delayed neurologic signs after smoke inhalation in a dog aspiration pneumonitis the veterinary icu book. teton newmedia allergic airway disease canine pleural and mediastinal effusion, a retrospective study of 81 cases suggested strategies for ventilatory management in veterinary patients with acute respiratory distress syndrome laryngeal and tracheal disorders the veterinary icu book. teton newmedia medical and surgical treatment of pyothorax in dogs: 26 cases traumatic diaphragmatic hernia in cats: 34 cases canine pyothorax: clinical presentation, diagnosis, and treatment canine pyothorax: pleural anatomy and pathophysiology treatment of chronic pleural effusion with pleuroperitoneal shunt in dogs: 14 cases (1985-1999) effects of doxapram hydrochloride on laryngeal function of normal dogs and dogs with naturally occurring laryngeal paralysis an overview of positive pressure ventilation risk factors, prognostic indicators, and outcome of pyothorax in cats: 80 cases (1986-1999) use of percutaneous arterial embolization for the treatment of intractable epistaxis in 3 dogs systemic inflammatory response syndrome, sepsis, and multiple organ dysfunction cardiogenic shock and cardiac arrest hemostatic changes in dogs with naturally occurring sepsis multiple organ dysfunction syndrome in humans and dogs increased lactate concentrations in ill and injured dogs the role of albumin in health and disease pathophysiologic characteristics of hypovolemic shock usefulness of systemic inflammatory response syndrome criteria as an index for prognosis judgement current principles and application of d-dimer analysis in small animal practice choosing fluids in traumatic hypovolemic shock: the role of crystalloids, colloids and hypertonic saline colloid and crystalloid resuscitation thromboembolic disease: predispositions and management marks sl: systemic arterial thromboembolism retrospective study of streptokinase administration in 46 cats with arterial thromboembolism feline arterial thromboembolism: an update arterial thromboembolism in cats: acute crises in 127 cases (1992-2001) and long-term management with low-dose aspirin in 24 cases cut multiple holes in the side of a 14-16 fr red rubber tube or thoracic drainage catheter, using care not to make the cut wider than 50% of the circumference of the tube. insert the catheter into the abdominal cavity in a dorsal caudal direction. make sure that all incisions within the abdomen. secure the tube by placing a pursestring suture around the tube entrance site in the abdominal musculature with absorbable suture material. close the dead space in the subcutaneous tissues with absorbable suture. close the skin around the tube with another purse-string suture secured using a finger-trap technique. connect the tube to a closed urinary collection system and bandage the catheter to the abdomen. the tube can remain in place until the patient retrospective evaluation of acute renal failure in dogs uroabdomen in dogs and cats drug-induced nephrotoxicity: recognition and prevention peritoneal dialysis in emergency and critical care acute renal failure caused by lily ingestion in six cats early diagnosis of renal disease and renal failure acute renal failure in four dogs after raisin or grape ingestion disorders of the feline lower urinary tract the use of a low-profile cystostomy tube to relieve urethral obstruction in a dog renal biopsy: methods and interpretation feline idiopathic cystitis: current understanding of pathophysiology and management today's problem when did you first notice that something was wrong with your pet? when was the last time you noticed your pet act normally? what was the first abnormal sign noticed? what other conditions have developed and what are they? how soon did other signs develop? have the signs become better or worse since you first saw them? what is the name of the product? do you have the container with you today? is it a liquid concentrate, dilute spray, or solid? how long ago do you think that your pet was exposed to the poison? where do you think it happened? do you have any over-the-counter or prescription medications that your animal may have had access to? did you give any medications to your animal? is there any possibility of recreational drug exposure?your pet's recent activity did your pet eat this morning or last night? what is he/she normally fed? is there a chance that your pet may have gotten into the garbage? have you fed table scraps or anything new recently? if so, what? has your pet been off your property in the last 24-48 hours? does your pet run loose unattended? has your pet had any antiflea/tick medication within the last week?your pet's environment is your animal kept inside or outside of the house? is your pet kept in a fenced-in yard or allowed to run loose unattended? does your pet have access to neighboring properties (even for a short time)? where has your pet been in the last 24 hours? has your pet traveled outside of your immediate geographic location? if so, when? has your pet been to rural areas in the last week? has there been any gardening work recently? does your pet have access to a compost pile? any fertilizers or weed killer used in the last week? any construction work or renovation recently? any mouse or rat poison in your house, yard, or garage? any cleaning products used inside or outside the house within the last 48 hours? if so, which? have you changed your radiator fluid or does a car leak antifreeze? induce and maintain a patent airway and stabilize the patient's cardiovascular and respiratory status. control cns excitation with diazepam, if necessary, and control the patient's body temperature (both hypo-and hyperthermia) . induce vomiting if the patient is alert and can protect its airway; otherwise, perform orogastric lavage with the patient under general anesthesia with a cuffed endotracheal tube in place. alcohols do not bind well with activated charcoal. treat dermal exposure by bathing the area with warm water. introduction: if ingested, sodium or potassium hydroxide can cause severe contact dermatitis or irritation of the gastrointestinal tract. esophageal burns and full-thickness coagulative necrosis can occur. if an animal ingests a caustic alkali substance, feed the animal four egg whites mixed with 1 quart of warmed water. perform endoscopy within 24 hours to evaluate the extent of injury and to place a feeding tube, in severe cases. do not induce emesis , and do not perform orogastric lavage, because of the risk of worsening esophageal irritation. in cases of contact exposure to the skin or eyes, rinse the exposed area with warm water baths for at least 30 minutes. administer gastroprotectant, antiemetic, and analgesic drugs as necessary. avoid neutralization, which can cause a hyperthermic reaction and worsen injury to the skin and gastrointestinal tract. amitraz is the active ingredient in ascaricides and anti-tick and anti-mite products such as mitaban and taktic. the toxic dose is 10 to20 mg/kg. amitraz exerts its toxic effects by causing î±-adrenergic stimulation, and causes clinical signs similar to those observed with administration of xylazine: bradycardia, cns depression, ataxia, hypotension, hyperglycemia, hypothermia, cyanotic mucous membranes, polyuria, mydriasis, and emesis. a coma can develop. treatment of amitraz intoxication includes cardiovascular support with intravenous crystalloid fluids and induction of emesis in asymptomatic animals. if clinical signs are present, orogastric lavage may be required. many toxic compounds are impregnated in a collar form. if the patient has ingested a collar and does not vomit it, it should be removed using endoscopy or gastrotomy. administer activated charcoal to prevent or delay absorption of the toxic compound. yohimbine or atepamizole, both î±-adrenergic antagonists, are the treatment(s) of choice to reverse the clinical signs of toxicity. avoid the use of atropine, because it can potentially increase the viscosity of respiratory secretions and cause gastrointestinal ileus, thus promoting increased absorption of the toxic compound. ammonium hydroxide, or cleaning ammonia, can be caustic at high concentrations (see alkalis/caustics) and cause severe injury to the respiratory system if inhaled. pulmonary edema or pneumonia can occur, resulting in respiratory distress. ingestion of ammonia can cause severe irritation to the gastrointestinal tract and cause vomiting and esophageal injury. if ammonia is ingested, administer a dilute solution of egg white.administer gastroprotectant, antiemetic, and analgesic drugs as necessary. if pneumonia or pulmonary edema occurs secondary to aspiration of ammonia into the airways and alveolar spaces, treatment is largely supportive with supplemental oxygen administration, antibiotics, fluid therapy, and mechanical ventilation as necessary. diuretics may or may not be useful in the treatment of pulmonary edema secondary to ammonia inhalation. amphetamines cause cns excitation due to neurosynaptic stimulation, resulting in hypersensitivity to noise and motion, agitation, tremors, vomiting, diarrhea, and seizures. clinical signs of amphetamine toxicity include muscle tremors, tachyarrhythmias, mydriasis, ptyalism, and hyperthermia. amphetamines are rapidly absorbed from the gastrointestinal tract. treatment includes administration of intravenous fluids to maintain hydration and renal perfusion and correction of hyperthermia. administer sedative drugs such as chlorpromazine to control agitation and tremors, and diazepam to control seizures. urinary acidification can promote excretion and prevent reabsorption from the urinary bladder. in severe cases, treat cerebral edema with a combination of mannitol followed by furosemide to control increased intracranial pressure.antifreeze: see ethylene glycol antihistamines introduction antihistamines (loratadine, diphenhydramine, doxylamine, clemastine, meclizine, dimenhydrinate, chlorpheniramine, cyclizine, terfenadine, hydroxyzine) are available as over-thecounter and prescription allergy and anti-motion sickness products. clinical signs of antihistamine toxicity include restlessness, nausea, vomiting, agitation, seizures, hyperthermia, and tachyarrhythmias. treatment of antihistamine intoxication is largely symptomatic and supportive, as there is no known antidote. if ingestion is recent (within 1 to 2 hours) and the patient is not actively seizing and can protect its airway, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. monitor the patient's heart rate, rhythm, and blood pressure. treat cardiac arrhythmias, if present, with appropriate therapies (see section on cardiac dysrhythmias). administer cooling measures and intravenous fluids to treat hyperthermia. a constant rate infusion of guaifenasin can be used to control muscle tremors. introduction î±-naphthylthiourea (antu) is manufactured as a white or blue-gray powder. the toxic dose in dogs is 10-40 mg/kg, and in cats is 75-100 mg/kg. younger dogs appear to be more resistant to its toxic effects. antu usually causes profound emesis and increased capillary permeability that eventually leads to pulmonary edema. treatment of antu toxicity includes respiratory support. mechanical ventilation may be required in severe cases of pulmonary edema. if an animal does not vomit, orogastric lavage should be performed. administer gastrointestinal protectant, antiemetic, and analgesic drugs. cardiovascular support in the form of intravenous crystalloids should be arsenic introduction inorganic arsenic (arsenic trioxide, sodium arsenite, sodium arsenate) is the active ingredient in many herbicides, defoliants, and insecticides, including ant killers. the toxic dose of sodium arsenate is 100-150 mg/kg; that of sodium arsenite is 1-25 mg/kg. sodium arsenite is less toxic, although cats are very susceptible. arsenic compounds interfere with cellular respiration by combining with sulfhydryl enzymes. clinical signs of toxicity include severe gastroenteritis, muscle weakness, capillary damage, hypotension, renal failure, seizures, and death. in many cases, clinical signs are acute in onset. treatment of arsenic toxicity involves procuring and maintaining a patent airway. administer intravenous crystalloid fluids to correct hypotension and hypovolemia, and normalize acidbase and electrolyte balance. if no clinical signs are present and if the compound was ingested within 2 hours, induce emesis. if clinical signs are present, perform orogastric lavage followed by administration of activated charcoal. if dermal exposure has occurred, throughly bathe the animal to prevent further absorption. dimercaprol (bal, 3-4 mg/kg im q8h) can be administered as a chelating agent. n-acetylcysteine (mucomyst) (for cats, 140-240 mg/kg po iv, then 70 mg/kg po iv q6h for 3 days; for dogs, 280 mg/kg po or iv, then 140 mg/kg po iv q4h for 3 days) has been shown to decrease arsenic toxicity in rats. aspirin causes inhibition of the production of prostaglandins, a high anion gap metabolic acidosis, gastrointestinal ulceration, hypophosphatemia, and decreased platelet aggregation when ingested in high quantities (>50 mg/kg/24 hours in dogs; >25 mg/kg/24 hours in cats). clinical signs of aspirin toxicity include tachypnea, vomiting, anorexia, lethargy, hematemesis, and melena. treatment of aspirin toxicity is largely supportive. if the ingestion was recent (within the last hour), induce emesis or perform orogastric lavage followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration and correct acid-base abnormalities. administer synthetic prostaglandin analogues (misoprostol), gastroprotectant drugs, and antiemetics. alkalinization of the urine can enhance excretion. introduction baclofen is a gaba agonist centrally acting muscle relaxant. clinical signs of toxicity include vomiting, ataxia, vocalization, disorientation, seizures, hypoventilation, coma, and apnea. clinical signs can occur at doses as low as 1.3 mg/kg. treatment of baclofen ingestion includes induction of emesis if the animal is asymptomatic. otherwise, perform orogastric lavage. emesis or orogastric lavage should be followed by administration of activated charcoal. perform intravenous crystalloid fluid diuresis to promote elimination of the toxin, maintain renal perfusion, and normalize body temperature. supplemental oxygen or mechanical ventilation may be required for hypoventilation or apnea. if seizures occur, avoid the use of diazepam, which is a gaba agonist and can potentially worsen clinical signs. control seizures with intravenous introduction î²-adrenergic agonists, including terbutaline, albuterol (salbutamol), and metaproterenol, are commonly used in inhaled form for the treatment of asthma. animals commonly are exposed to the compounds after chewing on their owners' inhalers. clinical signs of î²-adrenergic stimulation include tachycardia, muscle tremors, and agitation. severe hypokalemia can occur. treatment of î²-adrenergic agonist intoxication includes treatment with beta-blockers (propranolol, esmolol, atenolol), intravenous fluids, and intravenous potassium supplementation. diazepam or acepromazine may be administered for sedation and muscle relaxation. introduction barbiturates such as phenobarbital are gaba agonists and induce cns depression. clinical signs of barbiturate overdose or toxicity include weakness, lethargy, hypotension, hypoventilation, stupor, coma, and death. treatment of barbiturate toxicity includes maintenance and support of the cardiovascular and respiratory systems. if clinical signs are absent and the patient can protect its airway, induce emesis followed by repeated doses of activated charcoal. perform orogastric lavage if emesis is contraindicated. administer supplemental oxygen if hypoventilation occurs. some animals may require mechanical ventilation. administer intravenous fluids to control perfusion and blood pressure. positive inotropic drugs may be required if dosedependent decrease in cardiac output and blood pressure occurs. alkalinization of the urine and peritoneal dialysis can be performed to enhance excretion and elimination. hemodialysis should be considered in severe cases, if available. automotive and dry cell batteries contain sulfuric acid that can be irritating on contact with the eyes, skin, and gastrointestinal tract. button batteries, which contain sodium or potassium hydroxide, cause contact irritation if chewed. to treat exposure, rinse the eyes and skin with copious amounts of warm tap water or sterile saline solution for a minimum of 30 minutes. if ingestion occurred, administer gastroprotectant and antiemetic drugs. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of aspiration pneumonia and worsening esophageal irritation. no attempt should be made at performing neutralization because of the risk of causing an exothermic reaction and worsening tissue damage. administer analgesics to control discomfort. benzoyl peroxide is the active ingredient in many over-the-counter acne preparations. ingestion can result in production of hydrogen peroxide, gastroenteritis, and gastric dilatation. topical exposure can cause dermal irritation and blistering. if an animal has ingested benzoyl peroxide, do not induce emesis, because of the risk of worsening esophageal irritation. instead, perform orogastric lavage. administer gastroprotectant and antiemetic medications and closely observe the patient observed for signs of gastric dilatation.bismuth subsalicylate (pepto-bismol): see aspirin bleach, chlorine (sodium hypochlorite) introduction sodium hypochlorite is available in dilute (3%-6%) or concentrated (50% industrial strength or swimming pool) solutions for a variety of purposes. sodium hypochlorite can cause severe contact irritation and tissue destruction, depending on the concentration. affected animals may have a bleached haircoat. treatment of exposure includes dilution with copious amounts of warm water or saline baths and ocular lavage. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of causing further esophageal irritation. to treat ingestion, give the animal milk or large amounts of water, in combination with gastroprotectant and antiemetic drugs, to dilute the contents in the stomach. administration of sodium bicarbonate or milk of magnesia is no longer recommended. nonchlorine bleaches (sodium peroxide or sodium perborate) have a moderate toxic potential if ingested. sodium peroxide can cause gastric distention. sodium perborate can cause severe gastric irritation, with vomiting and diarrhea; renal damage and cns excitation followed by depression can occur, depending on the amount ingested. to treat dermal or ocular exposure, rinse the skin or eyes with copious amounts of warm tap water or sterile saline for a minimum of 30 minutes; treat ocular injuries as necessary, if corneal burns have occurred. if the bleach has been ingested, do induce emesis and perform orogastric lavage. administer milk of magnesia (2-3 ml/kg). boric acid is the active ingredient in many ant and roach killers. the toxic ingredient (in amounts of 1-3 g/kg) can cause clinical signs in dogs by an unknown mechanism. clinical signs include vomiting (blue-green vomitus), blue-green stools, renal damage, and cns excitation and depression. treatment of boric acid or borate ingestion includes gastric decontamination with induction of emesis or orogastric lavage, followed by administration of a cathartic to hasten elimination. activated charcoal is not useful to treat ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. clostridium botulinum endospores can be found in carrion, food, garbage, and the environment. ingestion of endospores and c. botulinum endotoxin rarely can cause generalized neuromuscular blockade of spinal and cranial nerves, resulting in miosis, anisocoria, lower motor neuron weakness, and paralysis. respiratory paralysis, megaesophagus, and aspiration pneumonia can occur. clinical signs usually develop within 6 days of ingestion. differential diagnosis includes acute polyradiculoneuritis (coonhound paralysis), bromethalin intoxication, and tick paralysis. treatment of botulism is largely supportive; although an antitoxin exists, it often is of no benefit. treatment may include administration of intravenous fluids, frequent turning of the patient and passive range-of-motion exercises to prevent disuse muscle atrophy, and supplemental oxygen administration or mechanical ventilation. administer amoxicillin, ampicillin, or metronidazole. recovery may be prolonged, up to 3 to 4 weeks in some cases. bromethalin is the active ingredient in some brands of mouse and rat poisons. it usually is packaged as 0.01% bromethalin in green or tan pellets, and packaged in 16 -42.5 g place packs. the toxic dose for dogs is 116.7 g/kg, and for cats 3 g/kg. bromethalin causes toxicity by uncoupling of oxidative phosphorylation. an acute syndrome of vomiting, tremors, extensor rigidity, and seizures occurs within 24 hours of ingestion of high doses. delayed clinical signs occur within 3 to 7 days of ingestion of a lower dose and include posterior paresis progressing to ascending paralysis, cns depression, and coma. treatment of known bromethalin ingestion includes induction of emesis or orogastric lavage, and repeated doses of activated charcoal every 4 to 6 hours for 3 days, because bromethalin undergoes enterohepatic recirculation. supportive care includes intravenous fluids, anticonvulsants, muscle relaxants (methocarbamol up to 220 mg/kg/day iv to effect), frequent turning of the patient, and passive range-of-motion exercises. supplemental oxygen and /or mechanical ventilation may be required in patients with coma and severe hypoventilation. administer mannitol (0.5-1 g/kg) in conjunction with furosemide (1 mg/kg iv) if cerebral edema is suspected. the majority of caffeine toxicities occur in dogs that ingest coffee beans. caffeine causes phosphodiesterase inhibition, and can cause cardiac tachyarrhythmias, cns stimulation (hyperexcitability and seizures), diuresis, gastric ulcers, vomiting, and diarrhea. muscle tremors and seizures can occur, resulting in severe hyperthermia. treatment of caffeine toxicity is largely symptomatic and supportive, as there is no known antidote. if clinical signs are not apparent and the patient is able to protect its airway, induce emesis. alternatively, orogastric lavage can be performed, followed by administration of activated charcoal. administer diazepam to control seizures. administer betaadrenergic blockers (e.g., esmolol, propranolol, atenolol) to control tachyarrhythmias. give intravenous fluids to maintain hydration and correct hyperthermia. the patient should be walked frequently or have a urinary catheter placed to prevent reabsorption of the toxin from the urinary bladder. carbamate compounds are found in agricultural and home insecticide products. examples of carbamates include carbofuran, aldicarb, propoxur, carbaryl, and methiocarb. the toxic dose of each compound varies. carbamate compounds function by causing acetylcholinesterase inhibition. toxic amounts cause cns excitation, muscarinic acetylcholine overload, and slud (salivation, lacrimation, urination, and defecation). miosis, vomiting, treatment of carbamate intoxication includes maintaining an airway and, if necessary, artificial ventilation. administer intravenous crystalloid fluids to control the patient's hydration, blood pressure, and temperature. cooling measures may be warranted. induce emesis if the substance was ingested within 60 minutes and the animal is asymptomatic. give repeated doses of activated charcoal if the animal can swallow and protect its airway. control seizures with diazepam (0.5 mg/kg iv). bathe the patient thoroughly. atropine (0.2 mg/kg iv) is useful in controlling some of the muscarinic signs associated with the toxicity. pralidoxime hydrochloride (2-pam) is not useful in cases of carbamate intoxication. control muscle tremors with methocarbamol (up to 220 mg/kg iv) or guaifenesin. in humans, ingestion or inhalation of 3-5 ml of carbon tetrachloride can be fatal. clinical signs of carbon tetrachloride toxicity include vomiting and diarrhea, then progressive respiratory and central nervous system depression. ventricular dysrhythmias and hepatorenal damage ensue. the prognosis is grave. treatment of carbon tetrachloride inhalation includes procurement and maintenance of a patent airway with supplemental oxygen, and cardiovascular support. to treat ingestion, administer activated charcoal, and give intravenous fluids to maintain hydration and support renal function. chlorinated hydrocarbons include ddt, methoxychlor, lindane, dieldrin, aldrin, chlordane, chlordecone, perthane, toxaphene, heptachlor, mirex, and endosulfan. the toxic dose of each compound varies. chlorinated hydrocarbons exert their toxic effects by an unknown mechanism, and can be absorbed through the skin and the gastrointestinal tract. clinical signs are similar to those observed in organophosphate toxicity: cns excitation, seizures, slud, (salivation, lacrimation, urination, defecation), excessive bronchial secretions, vomiting, diarrhea, muscle tremors, and respiratory paralysis. secondary toxicity from toxic metabolites can cause renal and hepatic failure. chronic exposure may cause anorexia, vomiting, weight loss, tremors, seizures, and hepatic failure. the clinical course can be prolonged in small animal patients. treatment of chlorinated hydrocarbon toxicity is largely supportive in nature, as there is no known antidote. procure and maintain the patient's airway. normalize the body temperature to prevent hyperthermia. if the substance was just ingested and the patient is not demonstrating any clinical signs, induce emesis. if the patient is symptomatic, perform orogastric lavage followed by activated charcoal administration. bathe the patient thoroughly in cases of topical exposure. administer intravenous crystalloid fluids to maintain hydration. these compounds do not appear to be amenable to fluid diuresis. introduction: chlorphenoxy derivatives are found in 2,4-d, 2,4,5-t, mcpa, mcpp, and silvex. the ld 50 of 2,4-d is 100 mg/kg; however, the toxic dose appears to be much lower in small treatment treatment of chlorphenoxy derivative toxicity is largely supportive in nature, as there is no known antidote. secure the patient's airway and administer supplemental oxygen, as necessary. control cns excitation with diazepam (0.5 mg/kg iv). intravenous crystalloid fluid diuresis and urinary alkalinization can promote elimination. administer gastroprotectant and antiemetic drugs, as needed. the toxic effects of chocolate are related to theobromine. various types of chocolate have different concentrations of theobromine and thus can cause clinical signs of toxicity with ingestion of varying amounts of chocolate, depending on the type. the toxic dose of theobromine is 100-150 mg/kg in dogs. milk chocolate contains 44 mg/oz (154 mg/100 g) of chocolate, and has a low toxic potential. semisweet chocolate contains 150 mg/oz (528 mg/100 g), and baking chocolate contains 390 mg/oz (1365 mg/100 g). semisweet and baking chocolate, being the most concentrated, have a moderate to severe toxic potential, even in large dogs.clinical signs of theobromine intoxication are associated with phosphodiesterase inhibition and include cns stimulation (tremors, anxiety, seizures), myocardial stimulation (tachycardia and tachyarrhythmias), diuresis, and (at very high doses) gastrointestinal ulceration. with treatment, the condition of most dogs returns to normal within 12 to 24 hours (t1 /2 = 17.5 hours in dogs). potential side effects include gastroenteritis and pancreatitis due to the fat content of the chocolate. treatment of chocolate toxicity includes obtaining and maintaining a protected airway (if necessary), intravenous fluid diuresis, induction of emesis or orogastric lavage followed by administration of repeated doses of activated charcoal, and placement of a urinary catheter to prevent reabsorption of the toxin from the urinary bladder. cholecalciferol rodenticide ingestion can lead to increased intestinal and renal reabsorption of calcium, causing an increase in serum calcium and dystrophic mineralization of the kidneys and liver at 2-3 mg/kg. clinical signs include lethargy, anorexia, vomiting, constipation, and renal pain within 2 to 3 days of ingestion. seizures, muscle twitching, and central nervous system depression may be observed at very high doses. as renal failure progresses, polyuria, polydipsia, vomiting/hematemesis, uremic oral ulcers, and melena may be observed. if the compound was ingested recently (within 2 to 4 hours) induce emesis or perform orogastric lavage, followed by administration of activated charcoal. check the patient's serum calcium once daily for three days following ingestion. if clinical signs of toxicity or hypercalcemia are present, decrease serum calcium with loop diuretics (furosemide, 2-5 mg/kg po or iv q12h) and glucocorticosteroids (prednisone or prednisolone, 2-3 mg/kg po bid) to promote renal calcium excretion. in severe cases, salmon calcitonin (4-6 iu/kg sc q2-12h in dogs) or bisphosphonate compounds may be required. correct acid-base abnormalities with intravenous crystalloid fluid diuresis and sodium bicarbonate, if necessary. (see section on hypercalcemia.) denture cleaners contain sodium perborate as the active compound. sodium perborate can cause severe direct irritation of the mucous membranes and may also act as a cns depressant. clinical signs are similar to those seen if bleach or boric acid compound is ingested, namely vomiting, diarrhea, cns excitation then depression, and renal failure. treatment for ingestion of denture cleaner includes gastric decontamination along with induction of emesis or orogastric lavage and administration of a cathartic to hasten elimination. activated charcoal is not useful for treatment of ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. deodorants are usually composed of aluminum chloride and aluminum chlorohydrate. both have a moderate potential for toxicity. ingestion of deodorant compounds can cause oral irritation or necrosis, gastroenteritis, and nephrosis. treatment of deodorant ingestion includes orogastric lavage, and administration of antiemetic and gastroprotectant drugs. introduction anionic detergents include sulfonated or phosphorylated forms of benzene. dishwashing liquid is an example of an anionic detergent that can be toxic at doses of 1 -5 g/kg. anionic detergents cause significant mucosal damage and edema, gastrointestinal irritation, cns depression, seizures, and possible hemolysis. ocular exposure can cause corneal ulcers and edema. treatment of anionic detergent exposure is largely symptomatic, as there is no known antidote. to treat topical toxicity, flush the patient's eyes and skin with warmed tap water or 0.9% saline solution for a minimum of 30 minutes, taking care to avoid hypothermia. to treat ingestion, feed the patient milk and large amounts of water to dilute the toxin. do not induce emesis, because of the risk of worsening esophageal irritation. to dilute the toxin, perform orogastric lavage, followed by administration of activated charcoal. closely monitor the patient's respiratory status, because oropharyngeal edema can be severe. if necessary, perform endotracheal intubation in cases of airway obstruction. monitor the patient for signs of intravascular hemolysis. administer intravenous crystalloid fluids to maintain hydration until the patient is able to tolerate oral fluids. cationic detergents and disinfectants include quaternary ammonia compounds, isopropyl alcohol, and isopropanol. quaternary ammonia compounds have a serious toxic potential treatment treatment of cationic detergent exposure includes careful bathing and ocular rinsing of the patient for a minimum of 30 minutes, taking care to avoid hypotension. secure the patient's airway and monitor the patient's respiratory status. administer supplemental oxygen, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation. give milk or large amounts of water orally, as tolerated by the patient, to dilute the toxin. nonionic detergents include alkyl and aryl polyether sulfates, alcohols, and sulfonates; alkyl phenol; polyethylene glycol; and phenol compounds. phenols are particularly toxic in cats and puppies. clinical signs of exposure include severe gastroenteritis and topical irritation. some compounds can be metabolized to glycolic and oxalic acid, causing renal damage similar to that observed with ethylene glycol toxicity. topical and ocular exposure should be treated with careful bathing or ocular irrigation for at least 30 minutes. administer activated charcoal to prevent absorption of the compound. as tolerated, give dilute milk or straight tap water orally to dilute the compound. administer antiemetic and gastroprotectant drugs to control vomiting and decrease gastrointestinal irritation. administer intravenous crystalloid fluids to maintain hydration and decrease the potential for renal tubular damage. monitor the patient's acid-base and electrolyte status and correct any abnormalities with appropriate intravenous fluid therapy. introduction diclone (phigone) is a dipyridyl compound that is a cns depressant. the ld 50 in rats is 25-50 mg/kg. dichlone reacts with thiol enzymes to cause methemoglobinemia and hepatorenal damage. to treat dichlone ingestion, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. procure and maintain a patent airway. perform intravenous fluid diuresis to maintain renal perfusion. n-acetylcysteine may be useful in the treatment of methemoglobinemia. diethyltoluamide (deet) is the active ingredient in many insect repellants (e.g., off, cutters, hartz blockade). the mechanism of action of deet is not fully understood, but it acts as a lipophilic neurotoxin within 5 to 10 minutes of exposure. cats appear to be particularly sensitive to deet. a lethal dermal dose is 1.8 g/kg; if ingested, the lethal dose is much less. the toxic dose of dermal exposure in dogs is 7 g/kg. clinical signs of toxicity include aimless gazing, hypersalivation, chewing motions, and muscle tremors that progress to seizures. recumbency and death can occur within 30 minutes of exposure at high doses. treatment of deet toxicity is largely supportive, as there are no known antidotes. procure and maintain a patent airway and perform mechanical ventilation, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to control hydration and treat hypotension, as necessary. treat seizures with diazepam (0.5 mg/kg iv) or phenobarbital. because of the rapid onset of clinical signs, induction of emesis is contraindicated. perform orogastric lavage if the compound was ingested within the last 2 hours. administer multiple repeated doses of activated charcoal. cooling measures should be implemented to control hyperthermia. if dermal exposure has occurred, bathe the patient thoroughly to avoid further exposure and absorption. diquat is a dipyridyl compound that is the active ingredient in some herbicide compounds. the ld 50 of diquat is 25-50 mg/kg. like paraquat, diquat induces its toxic effects by causing the production of oxygen-derived free radical species. clinical signs of diquat intoxication include anorexia, vomiting, diarrhea, and acute renal failure. massive dehydration and electrolyte imbalances can occur as a result of fluid loss into the gastrointestinal tract. treatment of diquat intoxication is similar to that for paraquat ingestion. if the animal had ingested diquat within 1 hour of presentation, induce emesis. in clinical cases, orogastric lavage may be required. both emesis and orogastric lavage should be followed by administration of kaolin or bentonite as an adsorbent, rather than activated charcoal. place an intravenous catheter and administer crystalloid fluids to restore volume status and maintain renal perfusion. monitor urine output. if oliguria or anuria occurs, treatment with mannitol, furosemide, and dopamine may be considered. ecstasy (3,4-methylenedioxymethylamphetamine; mdma) is a recreational drug used by humans. ecstasy causes release of serotonin. clinical signs of intoxication are related to the serotonin syndrome (excitation, hyperthermia, tremors, and hypertension), and seizures may be observed. a urine drug screening test can be used to detect the presence of mdma. treatment of ecstasy intoxication is largely supportive, as there is no known antidote. administer intravenous fluids to maintain hydration, correct acid-base status, and treat hyperthermia. serotonin antagonist drugs (cyproheptadine) can be dissolved and administered per rectum to alleviate clinical signs. intravenous propranolol has additional antiserotonin effects. administer diazepam (0.5-2 mg/kg iv) to control seizures. if cerebral edema is suspected, administer mannitol, followed by furosemide. ethylene glycol is most commonly found in antifreeze solutions but is also in some paints, photography developer solutions, and windshield wiper fluid. ethylene glycol in itself is only minimally toxic. however, when it is metabolized to glycolate, glyoxal, glyoxylate, and oxalate, the metabolites cause an increased anion gap metabolic acidosis and precipitation of calcium oxalate crystals in the renal tubules, renal failure, and (ultimately) death.the toxic dose in dogs is 6.6 ml/kg, and in cats is 1.5 ml/kg. the toxin is absorbed quite readily from the gastrointestinal tract and can be detected in the patient's serum within an hour of ingestion. colorimetric tests that can be performed in most veterinary hospitals can detect larger quantities of ethylene glycol in the patient's serum. in a dog with clinical treatment begin treatment of known ethylene glycol ingestion immediately. induce emesis or perform orogastric lavage and adminiser repeated doses of activated charcoal. place an intravenous catheter and perform crystalloid fluid diuresis with a known antidote. the treatment of choice for dogs is administration of 4-methylpyrrazole (4-mp), which directly inhibits alcohol dehydrogenase, thus preventing the conversion of ethylene glycol to its toxic metabolites. the dose for dogs is 20 mg/kg initially, followed by 15 mg/kg at 12 and 24 hours and 5 mg/kg at 36 hours. 4-mp has been used experimentally at 6.25 times the recommended dose for dogs. in cats, treatment with 4-mp is effective if it is administered within the first 3 hours of ingestion.cats will demonstrate signs of sedation and hypothermia with this treatment. if 4-mp is not available, administer ethanol (600 mg/kg iv loading dose, followed by 100 mg/ kg/hour), or as a 20% solution (for dogs, 5.5 ml/kg iv q4h for five treatments, then q 6h for five more treatments; for cats, 5 ml/kg q8h for four treatments). grain alcohol (190 proof) contains approximately 715 mg/ml of ethanol. antiemetics and gastroprotective agents should be considered. urinary alkalinization and peritoneal dialysis may enhance the elimination of ethylene glycol and its metabolites. many fertilizers are on the market, and may be composed of urea or ammonium salts, phosphates, nitrates, potash, and metal salts. fertilizers have a moderate toxic potential, depending on the type and amount ingested. clinical signs of fertilizer ingestion include vomiting, diarrhea, metabolic acidosis, and diuresis. nitrates or nitrites can cause formation of methemoglobin and chocolate-brown blood. electrolyte disturbances include hyperkalemia, hyperphosphatemia, hyperammonemia, and hyperosmolality. treatment of fertilizer ingestion includes cardiovascular support, and administration of milk or a mixture of egg whites and water, followed by induction of emesis or orogastric lavage. correct electrolyte abnormalities as they occur (see section on hyperkalemia). administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to control hydration and maintain blood pressure. n-acetylcysteine may be useful if methemoglobinemia is present. fipronil is the active ingredient in frontline, a flea control product. fipronil exerts its effects by gaba antagonism and can cause cns excitation. treatment of fiprinol toxicity includes treatment of cns excitation, treatment of hyperthermia by cooling measures, and administration of activated charcoal. fire extinguisher fluid contains chlorobromomethane or methyl bromide, both of which have a serious toxic potential. dermal or ocular irritation can occur. if ingested, the compounds can be converted to methanol, and cause high anion gap metabolic acidosis, cns excitation and depression, aspiration pneumonitis, and hepatorenal damage. to treat ocular or dermal exposure to fire extinguisher fluids, flush the eyes or skin with warmed tap water or 0.9% saline solution for a minimum of 30 minutes. do not induce emesis or perform orogastric lavage to treat ingestion, because of the risk of causing severe aspiration pneumonitis. gastroprotectant and antiemetic drugs may be used, if indicated. administer intravenous fluids to maintain hydration and renal perfusion. supplemental oxygen or mechanical ventilation may be required in severe cases of aspiration pneumonitis. fireplace colors contain salts of heavy metals-namely, copper rubidium, cesium, lead, arsenic, antimony, barium, selenium, and zinc, all of which have moderate toxic potential, depending on the amount ingested and the size of the patient. clinical signs are largely associated with gastrointestinal irritation (vomiting, diarrhea, anorexia). zinc toxicity can cause intravascular hemolysis and hepatorenal damage. to treat ingestion of fireplace colors, administer cathartics and activated charcoal and gastroprotectant and antiemetic drugs. place an intravenous catheter for intravenous crystalloid fluid administration to maintain hydration and renal perfusion. specific chelating agents may be useful in hastening elimination of the heavy metals. fireworks contain oxidizing agents (nitrates and chlorates) and metals (mercury, copper, strontium, barium, and phosphorus). ingestion of fireworks can cause hemorrhagic gastroenteritis and methemoglobinemia. to treat firework ingestion, induce emesis or perform orogastric lavage and administer activated charcoal. administer specific chelating drugs if the amount and type of metal are known, and administer gastroprotectant and antiemetic drugs. if methemoglobinemia occurs, administer n-acetylcysteine; a blood transfusion may be necessary. introduction fuels such as barbecue lighter fluid, gasoline, kerosene, and oils (mineral, fuel, lubricating) are petroleum distillate products that have a low toxic potential if ingested but can cause severe aspiration pneumonitis if as little as 1 ml is inhaled into the tracheobronchial tree. cns depression, mucosal damage, hepatorenal insufficiency, seizures, and corneal irritation can occur. if fuels are ingested, administer gastroprotectant and antiemetics drugs. do not induce emesis or perform orogastric lavage, because of the risk of aspiration pneumonia. to treat topical exposure, rinse the skin and eyes copiously with warm tap water or 0.9% saline solution. administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to maintain hydration and treat acid-base and electrolyte abnormalities. children's glue contains polyvinyl acetate, which has a very low toxic potential. if inhaled, the compound can cause pneumonitis. treatment of polyvinyl acetate should be performed as clinical signs of pneumonitis (increased respiratory effort, cough, lethargy, respiratory distress) occur. introduction superglue contains methyl-2-cyanoacrylate, a compound that can cause severe dermal irritation on contact. do not induce emesis. do not bathe the animal, and do not apply other compounds (acetone, turpentine) in an attempt to remove the glue from the skin. the fur can be shaved, using care to avoid damaging the underlying skin. the affected area should be allowed to exfoliate naturally. glyophosate is a herbicide found in roundup and kleenup. if applied properly, the product has a very low toxic potential. clinical signs of toxicity include dermal and gastric irritation, including dermal erythema, anorexia, and vomiting. cns depression can occur. treatment includes thorough bathing in cases of dermal exposure, and induction of emesis or orogastric lavage followed by administration of activated charcoal. administer antiemetic and gastroprotectant drugs as necessary. administer intravenous crystalloid fluids to prevent dehydration secondary to vomiting. even small amounts of grapes and raisins can be toxic to dogs. the mechanism of toxicity remains unknown. clinical signs occur within 24 hours of ingestion of raisins or grapes, and include vomiting, anorexia, lethargy, and diarrhea (often with visible raisins or grapes in the fecal matter). within 48 hours, dogs demonstrate signs of acute renal failure (polyuria, polydipsia, vomiting) that can progress to anuria. to treat known ingestion of raisins or grapes, induce emesis or perform orogastric lavage, followed by repeated doses of activated charcoal. if clinical signs of vomiting and diarrhea are present, administer intravenous fluids and monitor urine output. aggressive intravenous fluid therapy, in conjunction with maintenance of renal perfusion, is necessary. in cases of anuric renal failure, dopamine, furosemide, and mannitol can be useful in increasing urine output. peritoneal or hemodialysis may be necessary in cases of severe oliguric or anuric renal failure. calcium channel blockers such as amlodipine and diltiazem can be used to treat systemic hypertension. supportive care includes treatment of hyperkalemia, and administration of gastroprotectant and antiemetic drugs and (if the animal is eating) phosphate binders. aromatic hydrocarbons include phenols, cresols, toluene, and naphthalene. all have a moderate toxic potential if ingested. toxicities associated with ingestion of aromatic hydrocarbons include cns depression, hepatorenal damage, muscle tremors, pneumonia, methemoglobinemia, and intravascular hemolysis. if an aromatic hydrocarbon is ingested, do not induce emesis, because of the risk of aspiration pneumonia. a dilute milk solution or water can be administered to dilute the compound. perform orogastric lavage. carefully monitor the patient's respiratory and cardiovascular status. administer supplemental oxygen if aspiration pneumonia is present. to treat topical exposure, thoroughly rinse the eyes and skin with copious amounts of warm tap water or 0.9% saline solution. imidacloprid is the compound used in the flea product advantage. clinical signs of toxicity are related to nicotinic cholinergic stimulation, causing neuromuscular excitation followed by collapse. the compound may induce respiratory paralysis. to treat imidacloprid toxicity, procure and maintain a patent airway with supplemental oxygen administration. control cns excitation with diazepam, phenobarbital, or propofol. administer enemas to hasten gastrointestinal elimination, and administer activated charcoal. bathe the animal thoroughly to prevent further dermal absorption. closely monitor the patient's oxygenation and ventilation status. if severe hypoventilation or respiratory paralysis occurs, initiate mechanical ventilation. iron and iron salts can cause severe gastroenteritis, myocardial toxicity, and hepatic damage if high enough doses are ingested. lawn fertilizers are a common source of iron salts. treatment of ingestion of iron and iron salts includes cardiovascular support in the form of intravenous fluids and antiarrhythmic drugs, as needed. induce emesis or perform orogastric lavage for gastric decontamination. a cathartic can be administered to promote elimination from the gastrointestinal tract. antiemetic and gastroprotectant drugs should be administered to prevent nausea and vomiting. in some cases, radiographs can aid in making a diagnosis of whether the compound was actually ingested. iron toxicity can be treated with the chelating agent deferoxamine. ivermectin is a gaba agonist that is used in commercial heartworm prevention and antihelminthic compounds and can be toxic in predisposed breeds, including collies, collie loperamide is an opioid derivative that is used to treat diarrhea. clinical signs of loperamide intoxication include constipation, ataxia, nausea, and sedation. induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. naloxone may be beneficial in the temporary reversal of ataxia and sedation. ingestion of macadamia nuts can cause clinical signs of vomiting, ataxia, and ascending paralysis in dogs. the toxic principle in macadamia nuts is unknown. there is no known antidote. treatment consists of supportive care, including administration of intravenous fluids and antiemetics and placement of a urinary catheter for patient cleanliness. clinical signs resolve in most cases within 72 hours. marijuana is a hallucinogen that can cause cns depression, ataxia, mydriasis, increased sensitivity to motion or sound, salivation, and tremors. along with these findings, a classic clinical sign is the sudden onset of dribbling urine. urine can be tested with drug test kits for tetrahydrocannabinoid (thc), the toxic compound in marijuana. there is no known antidote for marijuana toxicity; therefore, treatment is largely symptomatic. place an intravenous catheter and administer intravenous fluids to support hydration. administer atropine if severe bradycardia exists. induction of emesis can be attempted but because of the antiemetic effects of thc, is usually unsuccessful. orogastric lavage can be performed, followed by repeated doses of activated charcoal. clinical signs usually resolve within 12 to 16 hours. introduction "strike anywhere" matches, safety matches, and the striking surface of matchbook covers contain iron phosphorus or potassium chlorate. both compounds have a low toxic potential but can cause clinical signs of gastroenteritis and methemoglobinemia if large quantities are ingested. treatment of match and matchbook ingestion includes gastric decontamination with induction of emesis or orogastric lavage and administration of activated charcoal and a cathartic. if methemoglobinemia occurs, administer n-acetylcysteine, intravenous fluids, and supplemental oxygen. metaldehyde is the active ingredient in most brands of snail bait. the exact mechanism of toxicity is unknown but may involve inhibition of gaba channels. clinical signs associated with metaldehyde toxicity include severe muscle tremors, cns excitation, and treatment treatment of mushroom toxicity is largely supportive. if the mushroom was ingested within the last 2 hours, induce emesis or perform orogastric lavage and then administer activated charcoal. symptomatic treatment includes intravenous fluids to promote diuresis and treat hyperthermia and skeletal muscle relaxants to control tremors and seizures (methocarbamol, diazepam). if amanita ingestion is suspected, administer hepatoprotectant agents including milk thistle. mycotoxins from penicillium spp. are found in moldy foods, cream cheese, and nuts. clinical signs of intoxication include tremors, agitation, hyperesthesia, and seizures. if tremorigenic mycotoxin toxicity is suspected, a sample of the patient's serum and gastric contents or vomitus can be submitted to the michigan state university veterinary toxicology laboratory for tremorigen assay. there is no known antidote. perform orogastric lavage, followed by administration of activated charcoal. control tremors and seizures with methocarbamol, diazepam, phenobarbital, or pentobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. in cases in which cerebral edema is suspected secondary to severe refractory seizures, administer intravenous mannitol and furosemide. naphthalene is the active ingredient in mothballs and has a high toxic potential. clinical signs associated with naphthalene toxicity include vomiting, methemoglobinemia, cns stimulation, seizures, and hepatic toxicity. a complete blood count often reveals heinz bodies and anemia. do not induce emesis if naphthalene ingestion is suspected. if the ingestion was within 1 hour of presentation, perform orogastric lavage. control seizures with diazepam or phenobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. n-acetylcysteine can play a role in the treatment of methemoglobinemia. a packed rbc transfusion may be necessary if anemia is severe. observe the patient for clinical signs associated with hepatitis. nicotine toxicity occurs in animals as the result of ingestion of cigarettes, nicotine-containing gum, and some insecticides. nicotine stimulates autonomic ganglia at low doses, and blocks autonomic ganglia and the neuromuscular junction at high doses. absorption after ingestion is rapid. clinical signs include hyperexcitability and slud (salivation, lacrimation, urination, and defecation). muscle tremors, respiratory muscle fatigue or hypoventilation, tachyarrhythmias, seizures, coma, and death can occur. if the patient presents within 1 hour of ingestion and has no clinical signs, induce emesis, followed by administration of repeated doses of activated charcoal. in patients with clinical signs of toxicity, perform orogastric lavage. administer intravenous fluids to maintain hydration and promote diuresis, and treat hyperthermia. administer atropine to treat cholinergic symptoms. urinary acidification can promote nicotine excretion. nonsteroidal antiinflammatory drugs (nsaids) include ibuprofen, ketoprofen, carprofen, diclofenac, naproxen, celecoxib, valdecoxib, rofecoxib, and deracoxib. nsaids cause inhibition of prostaglandin synthesis, leading to gastrointestinal ulceration, renal failure and hepatotoxicity. ibuprofen toxicity has been associated with seizures in dogs, cats, and ferrets. the toxic dose varies with the specific compound ingested. to treat nsaid toxicity, induce emesis or perform orogastric lavage, followed by administration of multiple repeated doses of activated charcoal. place an intravenous catheter for crystalloid fluid diuresis to maintain renal perfusion. administer the synthetic prostaglandin analogue misoprostol to help maintain gastric and renal perfusion. control seizures, if present, with intravenous diazepam. administer gastroprotectant and antiemetic drugs to control vomiting and gastrointestinal hemorrhage. continue intravenous fluid diuresis for a minimum of 48 hours, with frequent monitoring of the patient's bun and creatinine. when the bun and creatinine levels are normal or have plateaued for 24 hours, slowly decrease fluid diuresis 25% per day until maintenance levels are restored. onions, garlic, and chives contain sulfoxide compounds that can cause oxidative damage of rbcs, leading to heinz body anemia, methemoglobinemia, and intravascular hemolysis. clinical signs of toxicity include weakness, lethargy, tachypnea, tachycardia, and pale mucous membranes. vomiting and diarrhea can occur. intravascular hemolysis can cause treatment treatment of onion, chive, and garlic toxicity includes administration of intravenous fluid diuresis, and induction of emesis or orogastric lavage, followed by administration of activated charcoal and a cathartic. in cases of severe anemia, packed rbc transfusion or administration of a hemoglobin-based oxygen carrier should be considered. opiate drugs include heroin, morphine, oxymorphone, fentanyl, meperidine, and codeine. opiate compounds bind to specific opioid receptors throughout the body and produce clinical signs of miosis or mydriasis (cats), and cns excitation, followed by ataxia and cns depression, leading to stupor and coma. hypoventilation, bradycardia, hypoxia, and cyanosis can occur. to treat known overdose or ingestion of an opiate compound, induce emesis (in asymptomatic animals) or perform orogastric lavage, followed by administration of activated charcoal. administer intravenous fluids and supplemental oxygen to support the cardiovascular and respiratory systems. mechanical ventilation may be necessary until hypoventilation resolves. administer repeated doses of naloxone as a specific antidote to reverse clinical signs of narcosis and hypoventilation. if seizures are present (meperidine toxicity), administer diazepam. organophosphate compounds traditionally are used in flea control products and insecticides. common examples of organophosphates include chlorpyrifos, coumaphos, diazinon, dichlorvos, and malathion. the toxic dose varies, depending on the particular compound and individual animal sensitivity. organophosphate toxicity causes acetylcholinesterase inhibition, resulting in clinical signs of cns stimulation, including tremors and seizures. muscarinic acetylcholine overload causes the classic slud signs of salivation, lacrimation, urination, and defecation. miosis, excessive bronchial secretions, muscle tremors, and respiratory paralysis can occur. an intermediate syndrome of generalized weakness, hypoventilation, and eventual paralysis with ventral cervical ventroflexion that may require mechanical ventilation has been described. if organophosphate toxicity is suspected, whole-blood acetylcholinesterase activity can be measured and will be low. treatment of toxicity includes careful and thorough bathing in cases of dermal exposure and, if the substance was ingested, gastric decontamination with induction of emesis or orogastric lavage, followed by administration of activated charcoal, and administration of the antidote pralidoxime hydrochloride . atropine can help control the muscarinic clinical signs. supportive care in the form of cooling measures, intravenous crystalloid fluids, and supplemental oxygen or mechanical ventilation may be required, depending on the severity of clinical signs. introduction ingestion of large amounts of paintballs can cause neurologic signs, electrolyte abnormalities, and occasionally death. paintballs are gelatin capsules that contain multiple colors of if ingestion was recent and if no clinical signs of toxicity are present, induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal. there is no known antidote. treatment includes supportive care in the form of intravenous fluids and administration of phenobarbital or methocarbamol to control seizures and tremors. diazepam, a gaba agonist, is contraindicated, because it can potentially worsen clinical signs. urine acidification may hasten elimination. clinical signs can last from 3 to 5 days. pyrethrin and pyrethroid compounds are extracted from chrysanthemums, and include allethrin, decamethrin, tralomethrin, fenpropanthrin, pallethrin, sumethrin, permethrin, tetramethrin, cyfluthrin, and resemethrin. the oral toxicity is fairly low; however, the compounds can be significantly harmful if inhaled or applied to the skin. pyrethrin and pyrethroid compounds cause depolarization and blockade of nerve membrane potentials, causing clinical signs of tremors, seizures, respiratory distress, and paralysis. contact dermatitis can occur. to distinguish between pyrethrin/pyrethroid toxicity and organophosphate toxicity, acetylcholinesterase levels should be obtained; they will be normal if pyrethrins are the cause of the animal's clinical signs. treatment of toxicity is supportive, as there is no known antidote. carefully bathe the animal in lukewarm water to prevent further oral and dermal exposure. both hyperthermia and hypothermia can worsen clinical signs. administer activated charcoal to decrease enterohepatic recirculation. atropine may control clinical signs of excessive salivation. to control muscle tremors, administer methocarbamol to effect. administer diazepam or phenobarbital to control seizures, as necessary. rotenone is used as a common garden and delousing insecticide. fish and birds are very susceptible to rotenone toxicity. rotenone inhibits mitochondrial electron transport. clinical signs of tissue irritation and hypoglycemia can occur after topical or oral exposure. if the compound is inhaled, cns depression and seizures can occur. to treat toxicity, perform orogastric lavage, followed by administration of a cathartic and activated charcoal. bathe the animal carefully to prevent further dermal exposure and further ingestion. administer diazepam or phenobarbital to control seizures. the prognosis generally is guarded. treatment of ingestion includes dilution with milk, water, or egg whites. perform orogastric lavage, followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration. administer antiemetic and gastroprotectant drugs to treat gastroenteritis and vomiting.shampoos, nonmedicated: see detergents, nonionic shampoos, selenium sulfide introduction selenium sulfide shampoos (e.g., selsun blue) have a low toxic potential, and primarily cause gastroenteritis. treatment of ingestion includes dilution with water, milk, or egg whites and administration of activated charcoal. carefully and thoroughly rinse the skin and eyes to prevent further exposure. administer antiemetic and gastroprotectant drugs in cases of severe gastroenteritis. zinc-based (zinc pyridinethione) anti-dandruff shampoos have a serious toxic potential if ingested or if ocular exposure occurs. gastrointestinal irritation, retinal detachment, progressive blindness, and exudative chorioretinitis can occur. treatment of ingestion includes gastric decontamination. induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal.to treat ocular exposure, thoroughly rinse the patient's eyes for a minimum of 30 minutes. carefully monitor the animal for clinical signs of blindness. implement intravenous fluid to maintain hydration and renal perfusion in cases of severe gastroenteritis. silver polish contains the alkali substance sodium carbonate and cyanide salts, and has a serious toxic potential. ingestion results in rapid onset of vomiting and possibly cyanide toxicity. to treat ingestion, monitor and maintain the patient's respiration and cardiovascular status and administer intravenous crystalloid fluids. induce emesis, followed by administration of activated charcoal. administer sodium nitrite or sodium thiosulfate iv for cyanide toxicity. bath soap (bar soap) usually has low toxic potential and causes mild gastroenteritis with vomiting if ingested. to treat ingestion, include dilution with water, administration of intravenous fluids to maintain hydration, and administration of antiemetic and gastroprotectant drugs to treat gastroenteritis. sodium fluoroacetate is a colorless, odorless, tasteless compound that causes uncoupling of oxidative phosphorylation. the toxic dose in dogs and cats is 0.05-1.0 mg/kg. clinical signs of toxicity include cns excitation, seizures, and coma secondary to cerebral edema. the prognosis is guarded. to treat toxicity, procure and maintain a patent airway, monitor and stabilize the cardiovascular status, and control hyperthermia. perform orogastric lavage, followed by administration of activated charcoal. if clinical signs are not present at the time of presentation, induce emesis. administer intravenous fluids and supplemental oxygen, as necessary. strattera (atomoxetine hydrochloride) is a selective norepinephrine reuptake inhibitor used in the treatment of attention deficit hyperactivity disorder (adhd) in humans. peak serum concentrations occur in dogs within 3 to 4 hours of ingestion, with a peak half-life at 4 to 5 hours following ingestion. clinical signs of toxicity include cardiac tachyarrhythmias, hypertension, disorientation, agitation, trembling, tremors, and hyperthermia. treatment of intoxication is largely symptomatic and supportive in nature. first, induce emesis if the patient is conscious and has an intact gag reflex. orogastric lavage can also be performed. administer one dose of activated charcoal to prevent further absorption of the compound from the gastrointestinal tract. identify cardiac dysrhythmias and treat accordingly. control hypertension with sodium nitroprusside or diltiazem as a constant rate infusion. administer acepromazine or chlorpromazine to control agitation. do not use diazepam, because it can potentially worsen clinical signs. administer intravenous fluids to maintain hydration and promote diuresis. strychnine is the active ingredient in pesticides used to control rodents and other vermin. the toxic dose in dogs is 0.75 mg/kg, and in cats is 2 mg/kg. strychnine antagonizes spinal inhibitory neurotransmitters and causes severe muscle tremors, muscle rigidity, and seizures. clinical signs are stimulated or exacerbated by noise, touch, light, and sound. mydriasis, hyperthermia, and respiratory paralysis can occur. if strychnine toxicity is suspected, gastric contents should be collected and saved for analysis. if the animal is asymptomatic at the time of presentation, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by the administration of activated charcoal. administer intravenous crystalloid fluids to support the cardiovascular system, aid in cooling measures, and improve renal diuresis. treat cns stimulation with methocarbamol, diazepam, or phenobarbital. the animal should have cotton packed in its ears to prevent noise stimulation, and should be placed in a quiet, dark room. treatment of ingestion includes dilution with milk of magnesia or water, administration of antiemetic and gastroprotectant drugs, and administration of intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation.sunscreen: see zinc and zinc oxide suntan lotion: see shampoos, zinc-based, and alcohols tar: see fuels tea tree oil (melaleuca oil) introduction tea tree (melaleuca) oil is an herbal-origin flea-control product. the toxic principles in tea tree oil are monoterpenes, which produce clinical signs of neuromuscular weakness, and ataxia. treatment of tea tree oil toxicity includes administration of cathartics and activated charcoal to prevent further absorption. carefully bathe the animal to prevent further dermal exposure. tetanus spores from clostridium tetani organisms are ubiquitous in the soil and feces, particularly in barnyards. cases have been reported in dogs after tooth eruption and after abdominal surgeries performed with cold sterilization packs. anaerobic wound infections can contain tetanus spores. the neurotoxin from c. tetani inhibits spinal inhibitory neurons, causing motor neuron excitation. extensor muscle rigidity ("sawhorse stance"), erect ears, and risus sardonicus (a sardonic grin) are characteristic features of tetanus. administer tetanus antitoxin if toxin has not already been bound in the cns. to eliminate the source of the toxin (e.g., abscess), open and debride all wounds. intravenous administration of ampicillin or penicillin g is the treatment of choice for tetanus. supportive care in the form of skeletal muscle relaxants, intravenous fluids and parenteral nutrition, and nursing care to prevent decubitus ulcer formation is required. in extreme cases, mechanical ventilation may be necessary. triazene compounds include atrazine, prometone, and monuron (telvar). the toxic mechanism of triazene compounds is unknown. clinical signs of toxicity include salivation, ataxia, hyporeflexia, contact dermatitis, hepatorenal damage, muscle spasms, respiratory difficulty, and death. treatment of triazene exposure includes cardiovascular and renal support in the form of intravenous crystalloid fluids, inotropic drugs, and antiarrhythmic agents, as necessary. if the exposure is recent, induce emesis. perform orogastric lavage in animals that cannot protect the airway. emesis and orogastric lavage should be followed by the administration of activated charcoal and a cathartic. carefully bathe the patient to prevent further dermal absorption. a variety of tricyclic antidepressants are available for use in both humans and animals, including amitriptyline, amoxapine, desipramine, doxepine, fluoxetine (prozac), fluvoxamine (luvox), imipramine, nortriptyline, paroxetine (paxil), protriptyline, sertraline (zoloft), and trimipramine. selective serotonin reuptake inhibitors (ssris) are rapidly absorbed from the digestive tract, with peak serum concentrations occurring 2 to 8 hours after ingestion. the elimination half-life for each drug differs in dogs, but typically last 16 to 24 hours. ssris inhibit the reuptake of serotonin, causing serotonin to accumulate in the brain. this can cause "serotonin syndrome," characterized by trembling, seizures, hyperthermia, ptyalism or hypersalivation, cramping or abdominal pain, vomiting, and diarrhea. other clinical signs of ssri intoxication include depression, tremors, bradycardia, tachyarrhythmias, and anorexia. any animal that has ingested an ssri should be promptly treated and carefully observed for at least 72 hours for side effects. the treatment of suspected ssri intoxication involves gastric decontamination if the patient is not depressed and has an intact gag reflex. perform orogastric lavage and administer activated charcoal to prevent further toxin absorption and hasten elimination from the gastrointestinal tract. treat other clinical signs symptomatically. administer intravenous diazepam to control seizures. treat tachyarrhythmias according to type. administer methocarbamol to control muscle tremors. cyproheptadine (1 mg/kg), a serotonin antagonist, can be dissolved in water and administered per rectum. vitamin k antagonist rodenticides, which are commonly found in pelleted or block form, inhibit the activation of the vitamin k-dependent coagulation factors ii, vii, ix, and x. clinical signs of hemorrhage occur within 2 to 7 days of exposure. hemorrhage can occur anywhere in the body, and can be manifested as petechiation of the skin or mucous membranes, hemorrhagic sclera, epistaxis, pulmonary parenchymal or pleural hemorrhage, gastrointestinal hemorrhage, pericardial hemorrhage, hematuria, retroperitoneal hemorrhage, hemarthrosis, and central nervous system hemorrhage. clinical signs include respiratory distress, cough, bleeding from the gums or into the eyes, ataxia, paresis, paralysis, seizures, hematuria, joint swelling, lameness, lethargy, weakness, inappetence, and collapse.diagnosis is made based on clinical signs and a prolonged activated clotting time, or prothrombin time. the pivka (proteins induced by vitamin k antagonism) test may be helpful but usually cannot be performed in-house. slight thrombocytopenia may be present secondary to hemorrhage; however, blood levels usually do not reach the critical level of <50,000 platelets/âµl to cause clinical signs of hemorrhage. in some cases, severe stressinduced hyperglycemia and glucosuria may be present but resolves within 24 hours. if the rodenticide was ingested within the last 2 hours, induce emesis. alternatively, orogastric lavage can be performed in an uncooperative patient. both emesis and orogastric lavage should be followed by administration of activated charcoal. the stomach contents can be submitted for analysis. following successful treatment, administer oral vitamin k for 30 days after the exposure; or a check prothrombin time 2 days after gastric decontamination. if the prothrombin time is prolonged, administer fresh frozen plasma and vitamin k.if the prothrombin time is normal, gastric decontamination was successful, and no further treatment is necessary.if an animal presents with clinical signs of intoxication, administer activated clotting factors in the form of fresh frozen plasma (20 ml/kg), and vitamin k 1 (5 mg/kg sq in multiple sites with a 24-gauge needle). packed rbcs or fresh whole blood may be required if the patient is also anemic. supportive care in the form of supplemental oxygen may be necessary in cases of pulmonary or pleural hemorrhage. following initial therapy and discharge, the patient should receive vitamin k 1 (2.5 mg/kg po q8-2h for 30 days), and prothrombin time should be checked 2 days after the last vitamin k capsule is administered. in some cases, depending on the type of anticoagulant ingested, an additional 2 weeks of vitamin k1 therapy may be required. xylitol is a sugar alcohol that, when ingested by humans, does not cause a significant increase in blood glucose, and therefore does not stimulate insulin release from the human pancreas. in dogs, however, xylitol causes a massive rapid and dose-dependent release of insulin from pancreatic beta-cells. following insulin release, clinically significant hypoglycemia can develop, followed by signs of vomiting, weakness, ataxia, mental depression, hypokalemia, hypoglycemic seizures, and coma. clinical signs associated with xylitol ingestion can be seen within 30 minutes of ingestion and can last for more than 12 hours, even with aggressive treatment. known xylitol ingestion should be treated as for other toxin ingestion. if no neurologic abnormalities exist at the time the patient is seen, induce emesis, followed by administration of activated charcoal. it remains unknown at this time whether activated charcoal actually delays or prevents the absorption of xylitol from the canine gastrointestinal tract. if clinical signs have already developed, perform orogastric lavage and gastric decontamination. blood glucose concentrations should be analyzed and maintained with supplemental dextrose as a constant rate infusion (2.5%-5%) until normoglycemia can be maintained with multiple frequent small meals. hypokalemia may develop because it is driven intracellularly by the actions of insulin. treat hypokalemia with supplemental potassium chloride by infusion, not to exceed 0.5 meq/kg/hour. pennies minted in the u.s. after 1982 contain large amounts of zinc rather than copper. other sources of zinc include zinc oxide ointment and hardware such as that found in metal bird cages. zinc toxicity causes intravascular hemolysis, anemia, gastroenteritis, and renal failure. if zinc toxicity is suspected, take an abdominal radiograph to document the presence of the metal in the stomach or intestines. (if zinc-containing ointment was ingested, this will not be visible on radiographs.) induce emesis or perform orogastric lavage, depending on the size of the object ingested. often, small objects such as pennies can be retrieved using endoscopy or surgical gastrotomy/enterotomy. always take an additional radiograph after the removal procedure to ensure that all objects have been successfully removed. administer intravenous fluids to maintain renal perfusion and promote fluid diuresis. administer gastroprotectant and antiemetic drugs. chelation therapy with succimer, calcium edta, dimercaprol, or penicillamine may be necessary. do not administer pulmonary contusions are a common sequela of blunt traumatic injury. a contusion basically is a bruise characterized by edema, hemorrhage, and vascular injury. contusions may be present at the time of presentation or can develop over the first 24 hours after injury. a diagnosis of pulmonary contusion can be made based on auscultation of pulmonary crackles, presence of respiratory distress, and the presence of patchy interstitial to alveolar infiltrates on thoracic radiographs. radiographic signs can lag behind the development of clinical signs of respiratory distress and hypoxemia by 24 hours. in most cases, cage rest is sufficient to temporarily diminish blood loss. sedation (acepromazine, 0.02-0.05 mg/kg iv, im, sq) may be helpful in alleviating anxiety and decreasing blood pressure. the hypotensive effects of acepromazine are potentially harmful if severe blood loss has occurred. if evidence of hypovolemia is present (see section on hypovolemic shock), intravenous fluid resuscitation should be administered. rapid assessment of clotting ability, with a platelet count estimate and clotting profile (act or aptt and pt), should be performed. if epistaxis secondary to vitamin k antagonist rodenticide intoxication is suspected, administer vitamin k 1 and fresh frozen plasma or fresh whole blood.persistent hemorrhage from a nasal disorder can be treated with dilute epinephrine (1:100,000) into the nasal cavity with the nose pointed toward the ceiling to promote vasoconstriction. if this fails, the animal can be anesthetized, and the nasal cavity packed with gauze, and the caudal oropharynx and external nares covered with umbilical tape to control hemorrhage. a rhinoscopy should be performed to determine the cause of ongoing hemorrhage. continued excessive hemorrhage can be controlled with ligation of the carotid artery on the side of the hemorrhage, or with percutaneous arterial embolization. systemic thromboembolism is most commonly recognized in cats with cardiomyopathies (hypertrophic, restrictive, unclassified, and dilatative) but can also occur in dogs with hyperadrenocorticism, disseminated intravascular coagulation (dic), systemic inflammatory response syndrome (sirs), protein-losing enteropathy and nephropathy, and tumors affecting the aorta and vena cava. thrombosis occurs through a complex series of mechanisms when the components of virchow's triad (hypercoaguable state, sluggish blood flow, and vascular endothelial injury or damage) are present. in cats, blood flow through a severely stretched left atrium is a predisposing factor to the development of clots and thromboembolism.the most common site of embolism is the aortic bifurcation, or "saddle thrombus." other, less common locations of thromboembolism include the forelimbs, kidneys, gastrointestinal tract, and cerebrum. diagnosis usually is made based on clinical signs of cool extremities, the presence of a cardiac murmur or gallop rhythm, auscultation of pulmonary crackles resulting from pulmonary edema, acute pain or paralysis of one or more peripheral extremities, respiratory distress, and pain and lack of a palpable pulse in affected limbs. the affected nailbeds and paw pads are cyanotic, and nails do not bleed when cut with a nail clipper.client education is one of the most important aspects of emergency management of the patient with thromboembolic disease. concurrent congestive heart failure (chf) occurs in 40% to 60% of cats with arterial thromboembolism. more than 70% of cats are euthanized during the initial thromboembolic event because of the poor long-term prognosis and the high risk of recurrence within days to months after the initial event, even with aggressive therapy. although the long-term prognosis varies from 2 months to 2 years after initial diagnosis and treatment, in the majority of cats thromboembolic disease recurs within 9 months. rectal temperature hypothermia and bradycardia on presentation are negative prognostic indicators.immediate treatment of a patient with chf and thromboembolic disease involves management of the chf with furosemide, oxygen, and vasodilators (nitroglycerine paste, morphine, nitroprusside). additional management includes analgesia (butorphanol, 0.1-0.4 mg/kg iv, im) and prevention of further clot formation. aspirin (10 mg/kg po q48h) is beneficial bcause of its antiplatelet effects. heparin works in conjunction with antithrombin to prevent further clot formation (100-200 units/kg iv, followed by 250-300 units/kg sq q8h in cats, and 100-200 units/kg sq q8h in dogs). acepromazine can cause peripheral vasodilation and decreased afterload but also can promote hypotension in a patient with concurrent chf. acepromazine (0.01-00.02 mg/kg sq) should be used with extreme caution, if at all.thrombolytic therapy can also be attempted, but in most cases is not without risk, and may be cost-prohibitive for many clients. streptokinase (90,000 units iv over 30 minutes and then 45,000 units/hour iv cri for 3 hours) was administered with some success in cats; however, many died of hyperkalemia or other complications during the infusion. tissue plasminogen activator (0.25-1 mg /kg/hour iv cri, up to 10 mg/kg total dose, to effect) has been used with some success but is cost-prohibitive for most clients. side effects of thrombolytic therapy include hyperkalemia with reperfusion and hemorrhage.in cats, the primary cause of arterial thromboembolism is cardiomyopathy. once an animal is determined to be stable enough for diagnostic procedures, lateral and dv thoracic radiographs and an echocardiogram should be performed. ultrasound of the distal aorta and renal arteries should also be performed to determine the location of the clot and help establish the prognosis.other diagnostic procedures to evaluate the presence and cause of thromboembolism include a complete blood count, serum biochemistry profile, urinalysis (to rule out proteinlosing nephropathy), urine protein:creatinine ratio, antithrombin levels, acth stimulation test (to rule out hyperadrenocorticism), heartworm antigen test (in dogs), thyroid profile (to rule out hyperthyroidism in cats, and hypothyroidism in dogs), thoracic radiographs, arterial blood gas analyses, coagulation tests, and coombs' test. selective and nonselective angiography can also be performed to determine the exact location of the thrombus.long-term management of thromboembolism involves management of the underlying disease process and preventing further clot formation. begin therapy with heparin until the aptt becomes prolonged 1.5 times; then administer warfarin (0.06-0.09 mg/kg/day). monitoring therapy based on prothrombin time and the international normalized ratio (inr, 2.0-4.0) is recommended. low-dose aspirin (5-10 mg/kg q48h) also has been recommended. physical therapy with warm water bathing, deep muscle massage, and passive range-of-motion exercises should be performed until the patient regains motor function. future therapy may involve the use of platelet receptor antagonists to prevent platelet activation and adhesion. key: cord-023364-ut56gczm authors: nan title: education day monday: plenary session 1 monday: parallel sessions date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00651.x sha: doc_id: 23364 cord_uid: ut56gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023346-8sqbqjm1 authors: nan title: monday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00652.x sha: doc_id: 23346 cord_uid: 8sqbqjm1 nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023354-f2ciho6o authors: nan title: tuesday plenary session 3 tuesday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00654.x sha: doc_id: 23354 cord_uid: f2ciho6o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023095-4dannjjm authors: nan title: research abstract program of the 2011 acvim forum denver, colorado, june 15–18, 2011 date: 2011-05-03 journal: j vet intern med doi: 10.1111/j.1939-1676.2011.0726.x sha: doc_id: 23095 cord_uid: 4dannjjm nan clinics ãã also see infectious disease abstracts id-1 -id-14 (thursday, june 16, 2:15 pm -6:15 pm) ãã also see pharmacology abstracts p-1 -p-5 (thursday, june 16, 5:00 pm -6:15 pm) ãã also see gasteroenterology abstracts gi-1 june 17, 8 hypertrophic cardiomyopathy (hcm) is the most commonly observed myocardial disease in cats. beta-blockers and calcium channel inhibitors are frequently administered drugs to cats with preclinical hcm despite the fact that neither drug category has been proven to slow disease progression or improve survival. ivabradine (procorolan s , servier, france) is a novel negative chronotropic agent used in the treatment of ischemic heart disease in people. little is known about its efficacy and safety in cats. the purpose of this study was to determine the short-term effects of ivabradine on heart rate (hr), blood pressure, left ventricular (lv) systolic and diastolic function, left atrial (la) performance, and clinical tolerance in healthy cats after repeated oral doses. ten healthy laboratory cats were involved in the present study. physical examination, systolic blood pressure measurement, and transthoracic echocardiography were performed in all cats at baseline and after oral administration (4 weeks each) of ivabradine (0.3 mg/kg, q12 h) and atenolol (6.25 mg/cat, q12h; 1.0-1.7 mg/kg) in a prospective, double-blind, randomized, active-control, fully crossed study. a-priori non-inferiority margins for the effects of ivabradine compared to atenolol were set at 50% (f 5 0.5) based on predicted clinical relevance, observer measurement variability, and in agreement with fda guidelines. variables were compared by use of 2-way repeated measures anova. ivabradine was clinically well tolerated with no adverse events observed. hr (ivabradine, po0.001; atenolol, po0.001; ivabradine vs. atenolol, p 5 0.721) and rate-pressure product (ivabradine, p o 0.001; atenolol, p 5 0.001; ivabradine vs. atenolol, p 5 0.847) were not different between treatments. at the dosages used, ivabradine demonstrated more favorable effects than atenolol on echocardiographic indices of left ventricular (lv) systolic and diastolic function and left atrial performance. ivabradine is non-inferior to atenolol with regard to effects on hr, rate-pressure product, lv function, la performance, and clinical tolerance. clinical studies in cats with hcm are needed to validate these findings and further assess safety. the aim of this study was to compare outcome from cpa in dogs following initial administration of either epinephrine or vasopressin during cardiopulmonary resuscitation (cpr). dogs having cpa in the er or icu of a university hospital were randomized to receive either iv epinephrine (0.01-0.02 mg/kg) or vasopressin (0.5-1.0 u/kg) in a blinded fashion immediately following establishment of iv access and again three minutes later. a standardized cpr protocol was followed. other vasopressors were not permitted during the six minute study period, at the end of the study period additional cpr interventions were at the discretion of the managing clinician. the primary end point was return of spontaneous circulation (rosc) within the study period; secondary end points included rosc at any point, survival to 20 minutes, and survival to one hour. sixty dogs completed the study, 31 received epinephrine and 29 received vasopressin. rosc within six minutes was 53% (13 vasopressin, 19 epinephrine p 5 0.2), rosc at any time was 60% (15 vasopressin, 21 epinephrine p 5 0.2). survival to 20 minutes was 32% (6 vasopressin, 13 epinephrine p 5 0.077), survival to one hour was 18% (2 vasopressin, 9 epinephrine p 5 0.027). five dogs survived to 24 hours, one survived to hospital discharge. of animals dying after rosc, 13/35 were euthanized and 22/35 rearrested. no advantage of routine substitution of vasopressin for epinephrine was seen for rosc, a small survival advantage at one hour was seen in the group receiving epinephrine. the study also demon-strated that prospective clinical cpr research in animals is both possible and practical. three dogs were evaluated in 3 phases. phase-1: single-dose diltxr at approximately 6 mg/kg po. phase-2: same dose q12h for 4.5 days. phase-3: after a 14 day wash-out the single-dose protocol was repeated using cut tablets to assess affect on extended release properties. blood pressure (bp), 6-lead ecg, echocardiogram, and 24h-ambulatory ecg were performed at baseline, and conclusion of phase-2. blood samples and bp was obtained 0, 1, 2, 4, 8, 12, and 24h after final dosing. peak median plasma diltiazem concentrations (mcg/ml) measured by hplc for each phase were 0.0979, 0.016, and 0.07 respectively. diltiazem concentrations were below the limit of detection in the majority of samples in phase-2. median diltiazem concentration reached purported therapeutic concentrations (0.05-0.2 mcg/ml) by 2 h post-pill in phase-1 and 1h post-pill in phase-3. therapeutic concentrations were maintained for 24 h in phase-1, but only 2h in phase-3. median bp (mmhg) was 142.7 at baseline and 126.0 at peak concentration in phase-1. median heart rate (bpm) was 127.1 at baseline. 24h-ambulatory ecg analysis revealed the median hourly heart rate was 101.5 at baseline and 102 during phase-1. median heart rate at peak concentration in phase-1 was 127.5. lack of detectable plasma diltiazem during phase-2 may be due to up-regulation of drug metabolism via p-glycoprotein (abcb1-1) mutations. ongoing data collection and analysis will include mutation testing. adiponectin (adpn) is a cytokine produced by fat cells which has been shown to be correlated with adverse cardiac conditions in humans. in the heart, adiponectin activates several pro-survival reactions, including the ampk pathway and cox2 receptors, which protect the heart following ischemic injury. recent studies have shown that higher levels of adpn influence cardiac remodeling signaling, inhibiting protein synthesis and suppressing pathological cardiac growth. in humans, adpn plasma levels rise with decreased activity of the sympathetic nervous system and b-adrenergic agonists inhibit adpn at the level of gene expression. in contrast c-reactive protein (crp), a marker of systemic inflammation is elevated in humans with congestive heart failure (chf) and correlates to the severity of disease. first, we hypothesized that dogs with chf would have reduced adpn and elevated crp compared to normal dogs and that cytokine concentrations would predict severity of chf. second, we hypothesized that adpn receptor-1 (r1) and adpn protein would be elevated in the myocardium of chf dogs reflecting a compensatory process. we collected serum from 32 dogs (18 healthy and 18 chf). circulating adiponectin and crp levels were quantified using a mouse/rat adiponectin elisa and a canine crp elisa. we found lower mean crp concentrations in normal dogs (3.5 ae 2.3 mg/ ml) than dogs with chf (17.9 ae 17.1 mg/ml), however, the results were not statistically significant due to the large variability seen among the chf dogs (p 5 0.07). we found greater mean adpn concentrations in normal dogs (11.46 ae 3.3 mg/ml) than chf dogs (9.2 ae 3.2 mg/ml) (p 5 0.05). in general, the greater the severity of the heart failure, the lower the level of serum adpn. when the 2 tests the purpose of this study was to determine if there are any clinically important differences between the approaches (including devices) used in non invasive transvascular (interventional) closure of patent ductus arteriosus (pda) in dogs in our institution. initial and follow up records from all dogs (n 5 112) that underwent attempted transvascular pda occlusion from january 2006-december 2009 were examined. dogs were placed into 4 groups depending on the device and route of vascular access (transvenous or transarterial). group 1: amplatz s canine ductal occluder (acdo) (transarterial) -36 dogs; group 2: gianturco s or mreye flipper s detachable embolization (flipper) coil (transarterial) -38 dogs; group 3: amplatzer s vascular plug (avp) (transarterial) -23 dogs; group 4: flipper coil (transvenous) -15 dogs. statistical comparisons were made using the kruskal-wallis test with mann-whitney tests to compare pairs of groups when significance was detected. p o 0.05 was considered significant. there was no significant difference in ages between the 4 groups. there was a significant difference in body weight between groups with dogs receiving a coil either transarterially or transvenously (groups 2 and 4) being significantly smaller than dogs receiving an acdo or avp. this was by design since the acdo and avp cannot be used in small dogs. overall, the success rate of the total procedure (including vascular access and satisfactory pda occlusion) was high (92%) with success rates being comparable between groups (87-97%). there was a significant difference in complication rate between groups (p o 0.0001) with the acdo group having a markedly lower complication rate than the remaining groups (3% for acdo versus 24-33% for the other groups). total fluoroscopy time ranged from 3-78 minutes (median 8 minutes). fluoroscopy time for the transvenous method was significantly longer (median 13 minutes; range 10-78 minutes) than in the remaining groups (median 6 minutes; range 3-33 minutes) (p o 0.0001). number of dogs with residual flow immediately following the procedure and 24 hrs later was significantly less in the acdo group than in the remaining groups (2 dogs from group 2 and 3 from group 3 had moderate persistent flow while 1 dog from group 2 and 1 from group 4 had severe persistent flow 24 hours after the procedure). the acdo appears superior in ease of use, complication rate, completeness of occlusion and fluoroscopy time than other devices. the remaining limiting factor with this device is patient size. until a smaller acdo device is marketed, coils remain the only choice for interventional closure in very small dogs ( o 2.5kg). previously presented at the university of california davis, house officers seminar day. subvalvular aortic stenosis (sas) is one of the most commonly reported canine congenital heart defects and is inherited in newfoundland dogs and human beings. the golden retriever and rottweiler are breeds over-represented in dogs with subvalvular aortic stenosis; however, a genetic cause of this disease in these breeds has not been described. we performed genome wide association analysis in both normal and sas affected rottweilers and golden retrievers to identify chromosomal regions of interest that could implicate a causative mutation by high density single nucleotide polymorphism (snp) array. 48 (24 unaffected/24 affected) adult golden retrievers and 48 (20 unaffected/28 affected) adult rottweilers were included in this study. criteria for affected included a subcostal continuous-wave doppler aortic velocity ! 2.5 m/s and presence of a left basilar systolic ejection murmur; criteria for unaffected included a doppler aortic velocity 1.8 m/s. dna samples were obtained from anticoagulated blood. genotypes were obtained using high density (173,662) snp arrays, and genome wide association with sas was evaluated for each breed. significance cut-off was set at p 5 5 â 10 à5 , and all snps meeting this criterion were plotted within each breed and compared across breeds using plink. affected golden retriever data implicate the most significant region of genetic variation on chromosome 21 at location 27384260 (p 5 1.15 â 10 à5 ; odds ratio 7.58) with 11 other significant surrounding snps . affected rottweiler data also implicate the most significant region of genetic variation on chromosome 21 at location 27895300 (p 5 8.98 â 10 à6 ; odds ratio 23.39) with 3 other significant surrounding snps . other regions of statistical significance were on chromosomes 4 and 7 in the golden retriever and 11 and 38 in the rottweiler. genome wide association with subvalvular aortic stenosis in the golden retriever and rottweiler implicate overlapping chromosomal regions of interest for causative mutations on chromosome 21. the different secondary chromosomal regions of interest (chr 4, 7 in golden retrievers and 11, 38 in rottweilers) supports the known familial nature of this disease within different breeds and may suggest the presence of multiple mutations or breed specific disease modifiers. these data highlight the need for candidate gene evaluation on chromosome 21 in golden retrievers and rottweilers with sas. heart valves share developmental signaling pathways with bone and cartilage. degenerative aortic valve disease in humans is characterized by valve stenosis and calcification. recent evidence suggests that degenerative aortic valves are undergoing pathologic processes that mimic osteogenesis. degenerative mitral valves in dogs and humans are characterized by valve regurgitation, and rarely undergo calcification. we tested the hypothesis that canine and human degenerative mitral valves might be undergoing pathologic processes that mimic chondrogenesis. to test this hypothesis, expression of bone morphogenic protein 2 (bmp2), a chondrogenic growth factor; sox 9, a chondrogenic transcription factor; aggrecan, a proteoglycan abundant in cartilage; and type ii collagen were evaluated utilizing immunohistochemistry. normal canine mitral valves, different stages of canine degenerative mitral valves (early, intermediate, and late), and late-stage human degenerative mitral valves were studied. canine and human degenerative mitral valves showed focal areas that co-expressed all four markers of chondrogenic signaling and phenotype. valve interstitial cells and surrounding extracellular matrix in these focal areas adopted a morphologic appearance reminiscent of cartilage. focal chondrogenesis was present in all stages of canine degenerative mitral valves, but not normal canine mitral valves. focal areas of chondrogenesis did not coincide with nodular areas of glycosaminoglycan accumulation on the leaflet edge, but rather seemed to occur at points of chordae attachment to leaflets. in conclusion, canine and human degenerative mitral valves undergo pathologic processes that mimic chondrogenesis. this finding suggests that mitral valve degeneration may be recapitulating developmental signaling pathways shared by heart valves and cartilage. the triggering events for chondrogenesis in mitral valves remain unknown; as does the reason why aortic and mitral valves appear to be undergoing different pathologic processes. the fact that humans exhibit degeneration of both the aortic and mitral valve, and that dogs commonly exhibit only the latter could eventually provide insight into both processes. arrhythmogenic right ventricular cardiomyopathy (arvc) is a familial cardiomyopathy characterized by right ventricular fibrofatty infiltration and ventricular ectopy of left bundle branch block morphology (vpc) . a deletion in the striatin gene has been associated with arvc in at least some boxer families. syncope and sudden death (sd) occur in some affected dogs, although many affected dogs survive for years. the objective of this study was to define clinical characteristics of arvc in boxers that experienced sd, and compare them to those of a contemporaneous group of arvc boxers that had not died suddenly (nsd). data for both groups were collected from adult boxers enrolled in a long term prospective study of arvc in which echocardiograms and 24 hour ambulatory ecg (aecg) are evaluated annually. aecg are quantitated for vpc numbers and arrhythmia grade (1-4). arvc diagnosis requires at least 100 vpcs/ 24 hours in the absence of other disease. forty three adult boxers that entered the study had died suddenly at the time of analysis (sd defined as the absence of observed clinical signs within 24 hours prior to an unexpected and unexplained death). striatin genotype was available for 28 of the 43 sd dogs (17 heterozygotes, 11 homozygotes); 19 were female (9 intact) and 24 were male (14 intact). sd occurred at a mean age of 8 years (range, 1-12); 27 sd dogs (63%) had no prior history of syncope. twelve sd dogs (28%) were on antiarrhythmics at the time of death (metop-p0oooooooooooprolol (1), sotalol (4), amiodarone (1), procainamide (2), mexiletine & atenolol (3), atenolol (1)). eleven sd dogs (25%) had decreased myocardial systolic function defined by a shortening fraction (%fs) o 25% (range 11-45, mean 5 27) on the most recent echocardiogram prior to sd. median vpcs/24 hours on annual aecg was 7,700 (range 106-91,000) with a median arrhythmia grade of 4 (range 2-4). twenty one contemporaneously entered arvc boxers that had survived to at least the median age of the sd group with nsd were available for comparison; 15/21 were genotyped (10 heterozygous, 1 homozygous, 4 negative), 13 were female (3 intact) and 8 male (2 intact). twelve nsd dogs (57%) had no prior history of syncope. median nsd group age was 10 years (range, 8-13); 11/21 (52%) were on an antiarrhythmics (sotalol (9), mexiletine & sotalol (1), mexiletine & atenolol (1)). one nsd dog had decreased %fs (nsd group %fs range 20-42, mean 5 32). the nsd median number of vpcs was 5,342 (range 622-62,622), median arrhythmia grade was 4 (range 2-4). striatin genotype was significantly associated with sd. no significant differences were found between groups with respect to vpc numbers or arrhythmia grade. shortening fraction was significantly lower in the sd group (p o 0.01). sd in arvc appears to be associated with the presence of the striatin mutation and reduced % fs, it does not appear to be associated with number of vpcs or arrhythmia grade. coughing in the small breed dog may be related to cardiac causes associated with myxomatous mitral valve degeneration (mmvd) including pulmonary edema and compression of the mainstem bronchus by a severely enlarged left atrium, or due to respiratory causes such as tracheal and/or bronchial collapse or chronic bronchitis. the purpose of this study was to evaluate the association between left atrial enlargement and large airway collapse in dogs with mmvd and chronic cough. we hypothesized that airway collapse was independent of degree of left atrial enlargement. twelve dogs with mmvd and a chronic cough in the absence of congestive heart failure were prospectively evaluated with thoracic and cervical radiography, echocardiography, fluoroscopy, bronchoscopy and bronchoalveolar lavage (bal). group 1 dogs (n 5 8) had moderate to severe left atrial enlargement based on an echocardiographically calculated left atrial:aortic surface area [la:ao(a)] 4 6. group 2 dogs (n 5 4) had no to mild left atrial enlargement [la:ao(a) 6]. the site and severity of airway collapse was graded on bronchoscopy and bal cytology was assessed for evidence of inflammation or infection. the occurrence of bronchoscopic abnormalities was compared between groups using fisher's exact test. p o 0.05 was considered significant. age and body weight did not differ between groups. left atrial size was interpreted radiographically as moderately to severely enlarged in 7 of 8 dogs in group 1 and as moderately enlarged in 2 of 4 dogs in group 2. fluoroscopy revealed variable degrees of airway collapse during normal respiration and induced cough in both groups. radiography and fluoroscopy were not accurate in identifying site and degree of collapse in either group when compared to bronchoscopy. cervical tracheal collapse was identified during bronchoscopy in both group 1 (2 of 8) and group 2 (3 of 4) dogs but was subjectively less severe in group 1 dogs. bronchial collapse 4 50% was evident at multiple sites in both groups of dogs with no difference between groups. all dogs had suppurative and/or lymphocytic inflammation on airway cytology. infection was not present in either group of dogs, although non-specific light bacterial growth was detected in 6 of 8 group 1 dogs and 1 of 4 group 2 dogs (p 5 0.22). preliminary results failed to identify an association between left atrial enlargement and airway collapse in dogs with mmvd but did suggest that airway inflammation is common in affected dogs. further studies are needed to identify factors contributing to airway collapse in dogs with and without mmvd. atenolol is often used empirically in cats with asymptomatic hcm, even though clinical and experimental evidence of efficacy is lacking. cardiac biomarkers play a critical role in the early detection of subclinical cardiac disease, in the prediction of long-term prognosis, and in monitoring the response to therapy in humans. we hypothesized that serum concentrations of the biomarkers, nterminal pro-brain natriuretic peptide (nt-probnp) and cardiac troponin i (ctni), would improve following the chronic administration of atenolol po to asymptomatic cats with hcm. six maine coon or maine coon cross cats with severe hcm from the research colony at ucdavis were administered atenolol (12.5 mg po twice a day) for 30 days. no cat had severe left ventricular dynamic outflow tract obstruction due to systolic anterior motion of the mitral valve. the concentrations of nt-probnp and ctni were assayed prior to drug administration and on the last day of drug administration. there was no statistically significant difference identified in nt-probnp [median before: 394 pmol/l (range: 71-1500 pmol/l), median after: 439 pmol/l (range: 24-1500 pmol/l); p 5 0.63] or ctni [median before: 0.24 ng/ml (range:0.10-0.97 ng/ml), median after: 0.28 ng/ml (range: 0.09-1.0 ng/ml); p 5 0.69] concentrations before and after drug administration using the wilcoxon matched pairs test. the ctni finding suggests that atenolol does not reduce chronic myocyte death in cats with hcm. the lack of improvement in nt-probnp suggests that atenolol does not improve myocardial wall stress in cats with hcm. a clinical trial is warranted to confirm or refute the findings from this study. therefore, leptin-gene expression was investigated in blood samples of dogs with congestive heart failure (chf; n 5 8) in comparison to dogs presented for cardiac screening (n 5 8) without abnormalities. additionally myocardial samples (interventricular septum, right and left atrium and ventricle) of 10 dogs with no cardiac abnormalities (controls), seven dogs with acquired and three with congenital cardiac diseases were investigated using quantitative rt-pcr. leptin blood levels were significantly higher in dogs with chf in comparison to dogs without diseases (p 5 0.013). there was an association with gender with higher myocardial leptin levels in female dogs with cardiac diseases (p 5 0.001). differences between cardiac regions were present (p o 0.05) and cardiac diseases resulted in an increase in atrial leptin levels in both sexes (p 5 0.006). interestingly, a significant reduction of myocardial leptin was present in dogs with congenital cardiac diseases (p 5 0.016), whereas acquired cardiac diseases resulted in an increase in leptin (p 5 0.035) in comparison to controls. these results suggest that the heart might be a target of leptin action in the dog and myocardial leptin production might play a role in regulating cardiac function in an auto-and paracrine manner. predicting risk of chf in asymptomatic dogs with mitral valve disease (mvd) is challenging. we examined ability of nt-probnp to identify asymptomatic dogs at high risk for chf. dogs with isachc-1b (la:ao 4 1.6) were prospectively recruited; dogs with current or previous chf or diuretic therapy were excluded. physical examination, radiography, echocardiography, and nt-probnp were performed at 2-9 mo intervals for 66 dogs (median follow-up 5 276 d, range, 17-1334 d). thirty-one patients reached a study endpoint of radiographic pulmonary edema; 35 remained asymptomatic. parameters from the visit immediately previous to onset of chf (future-chf) or prior to the most recent visit without chf (remain-asympt) were analyzed. median nt-probnp of future-chf (3001pmol/lpmol/l, iqr 2255-3001) was significantly different from remain-asympt (1600 pmol/l, 984-2863; p 5 0.0004). median time to chf of future-chf was 86d (iqr, . groups also differed in median la:ao (future-chf 2.20 [2.00-2.50]; remain-asympt 1.96 [1.85-2.13], p 5 0.0014); vhs (future]; remain-asympt 11.3 [10.75-12.0], p 5 0.003); and lvidd:ao ]; remain-asympt 2.33 [2.15-2.64], p 5 0.0001). roc analysis to predict if chf would occur prior to the next visit yielded auc 5 0.753 (95%ci, 0.632-0.851). sensitivity was 90.3% or 77.4% and specificity 48.6% or 68.6% for nt-probnp 41490 pmol/l or 42150 pmol/l, respectively. mean increase in nt-probnp between penultimate visit and two visits prior to endpoint: future-chf 5 1440.1 pmol/l vs. remain-asympt 5 110.5 pmol/l. within 6 mo, 4.5%, 10.7%, 17.4%, and 36.7% of dogs with nt-probnp o 1000, , and 43000 pmol/l developed chf. nt-probnp and heart size helped assess risk of chf in asymptomatic mvd. increasing the assay's upper limit of detection would likely improve utility of nt-probnp. piiinp is a serum biomarker of collagen biosynthesis and is described as a marker of myocardial fibrosis in human patients. we hypothesised that piiinp concentrations would vary according to the degree of remodelling demonstrable in dogs with naturallyoccurring myxomatous mitral valve disease (mmvd). serum piiinp concentrations (mg/ml) were measured in dogs with mmvd and healthy controls using a validated commerciallyavailable radioimmunoassay. results are reported as (mean ae sd). non-normally distributed variables were logarithmically transformed. comparisons of continuous variables were made between groups using t-tests and one-way anovas with tukey's post-hoc comparisons. univariable analyses were used to evaluate associations between piiinp and clinical characteristics (age, breed [cavalier king charles spaniel (ckcs) yes/ no], sex, weight, heart rate [measured from ecg], treatment with acei [yes/ no], treatment with diuretics [yes/ no] and echocardiographic measurements [la/ao, lvedd/ lvfwd, lveddn, lvesdn]). multivariable analysis was initially performed with all dogs included and then repeated excluding all dogs receiving therapy. dogs with mmvd were divided into those with no cardiomegaly (nc) (la/ao o 1.5 and lveddn o 1.8), those with cardiomegaly (la/ao ! 1.5 and/ or lveddn ! 1.8) but no clinical signs (c) and those dogs with cardiomegaly requiring treatment for congestive heart failure (chf). one hundred and fifty-four dogs with mmvd and 23 control dogs were studied. there was no difference in age (p 5 0.870) or weight (p 5 0.606) between the mmvd and control groups. there was a significant difference in serum piiinp (p 5 0.034) between normal (11.2 ae 3.66), nc (11.6 ae 4.57), c (10.1 ae 3.44) and chf (9.4 ae 3.06) groups. post-hoc comparisons demonstrated a difference between nc and chf groups (p 5 0.038). there was no difference in serum piiinp between genders (p 5 0.228). in the univariable analysis ckcs (yes/ no) (p 5 0.016) was positively associated with serum piiinp. age (p o 0.0001), log (la/ao) (p 5 0.002) and lveddn (p 5 0.002) were negatively associated with serum piiinp. in the multivariable model including all dogs, lveddn (p o 0.0001, b 5 à4.04 (95%ci 5 à6.11 to à1.97)), age (p 5 0.006, b 5 à0.28 (95%ci 5 à0.47 to à0.08)) and ckcs (yes/ no) (p 5 0.003, b 5 2.01 (95%ci 5 0.67 to 3.34)) were independently associated with serum piiinp. in the multivariable model including only dogs not receiving therapy (n 5 141), lveddn (p 5 0.006, b 5 à4.30 (95%ci 5 à7.32 to à1.29)), age (p 5 0.011, b 5 à0.31 (95%ci 5 à0.55 to à0.07)) and ckcs (yes/ no) (p 5 0.034, b 5 1.75 (95%ci 5 0.13 to 3.37)) were independently associated with serum piiinp. in conclusion, serum piiinp decreases with age and with increasing lveddn. ckcs have higher serum piiinp measurements independent of age and lveddn, which may reflect a difference in collagen turnover in this breed. left atrial (la) chamber dilation and congestive heart failure (chf) are common consequences of cardiac conditions in cats. in some cases chf is manifest as right-sided chf (r-chf) or pleural effusion, in other cases chf manifests as left-sided chf (l-chf) or pulmonary edema. it is not always readily apparent as to which cats will develop what form of chf. a general impression has been that la enlargement is associated with the average burden of elevated filling pressures, but little attention has been paid to the function of the la chamber itself. since chf is classically preceded by abnormal atrial chamber dilation and alterations in atrial chamber function, we want to understand how these changes may help us manage or predict chf in the cat. we hypothesized that cats manifesting r-chf have la failure with the la acting primarily as a conduit, resulting in greater pulmonary hypertension, whereas l-chf cats maintain some booster pump and reservoir function. we measured la maximum and minimum areas from right parasternal long axis four-chamber views on 2d echo, and la m-mode dimensions at maximum, minimum, and beginning of atrial contraction. la area change, fractional shortening, active emptying fraction, and expansion index were calculated from these measurements. right ventricular internal diastolic diameter was also measured on m-mode views. preliminary data revealed that maximum left atrial size is not significantly different between r-chf and l-chf cats on 2d or m-mode measurements due to high variability. however, total left atrial fractional shortening is significantly reduced in r-chf cats (11.32% ae 4.9) compared to l-chf cats (19.7% ae 5.1)(p 5 0.001), and r-chf cats have reduced left atrial active emptying fraction (6.12% ae 3.5) as compared to l-chf cats (13.7% ae 3)(p o 0.001). left atrial expansion ability is poorer in r-chf cats (13.11% ae 6.7) than in l-chf cats (25.0% ae 8)(p 5 0.002). these findings may suggest that atrial stiffness and poorer atrial function is associated with a greater degree of pulmonary venous and thus secondary pulmonary arterial hypertension resulting in pleural effusion (r-chf). right ventricular diameter on m-mode was increased in r-chf cats (4.8 mm ae 2.1) when compared to l-chf cats (2.89 mm ae 0.8)(p 5 0.002) and normal cats (2.74 mm ae 0.8)(p 5 0.002), which may also be evidence for a greater degree of pulmonary arterial hypertension in these cats. episodic weakness and syncope are common in boxer dogs. reported causes include rapid ventricular tachycardia (vt) and exertion-excitement triggered neurally-mediated bradycardia (nmb) .the purpose of this retrospective study is to describe the features of presumed nmb in boxers. to be included in the study, each dog must have been overtly healthy with a history of exertion-excitement triggered syncope or presyncope; had a normal echocardiogram (ec); had absence of vt and fewer than 500 ventricular premature complexes (vpc) on an initial and subsequent 24 hour holter recordings; and been alive and overtly healthy for at least six months following the initial evaluation. a total of 27 boxers were identified. sixteen were male and 11 were female. most (90%) dogs were either less than 4 or more than 7 years of age. most dogs had multiple, but infrequent, episodes and heart rhythm was documented at the time of an episode in only 8 (30%) and only once (bradycardia) on the first holter recording. owners were instructed to attempt to precipitate episodes. bradycardia related episodes were subsequently recorded in 5: during the 2nd (1), 3rd (2) or 4th (1) day of 120 hour holter recordings and during the 5th day of an event recording (1). collapse and bradycardia were documented during auscultation in 2 additional dogs. the heart rate during syncope was never documented in 19 (70%) dogs. a presumptive diagnosis of nmb was based on the absence of initial and follow-up of ec abnormalities and the presence of no or few vpc during extended ecg monitoring. multiple holter recordings (48-168 hours) were performed in 8 of 19 (42%) dogs and event monitoring of 5 days (1) and 7 days (1) was performed in 2 additional dogs. in conclusion, documentation of the heart rhythm during episodes of collapse was difficult, accomplished in only 30% and was unlikely during the first holter recording. in boxers with suspected nmb, extended ecg monitoring and implantable loop recorders may be best for hr documentation. arrhythmogenic right ventricular cardiomyopathy (arvc) is an inherited myocardial disease with high prevalence in the boxer dog population, and is associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of myocardium with fatty or fibro-fatty tissue. though several genes have been linked to the disease both in humans and in boxers, the etiology of arvc is still unclear. several mechanisms for the development of arvc have been suggested, including dysfunction of the canonical wnt pathway, which results in an arvc phenotype in the mouse. the canonical wnt pathway has been linked to many cellular functions, including growth and differentiation of adipocytes. with the recent discovery that the gene encoding striatin, a protein involved in wnt signaling, may be involved in the development of boxer arvc, we hypothesized that changes in the wnt pathway may also play a role in the etiology. here, we show changes in the localization and decreased amount of proteins affiliated with the canonical wnt pathway. afflicted boxers were identified by 24-hour holter monitoring and histopathological examination of the heart. samples from the right ventricle rv) of 15 arvc afflicted boxers, and 5 unafflicted dogs (1 beagle, 2 mongrels, and 2 german shepherds) were collected, fixed in 10% formalin, processed, treated with antibodies recognizingcatenin, striatin, and calnexin, and examined using confocal microscopy. western blots were performed on 3 unafflicted rv samples, and 2 arvc afflicted rv samples. frozen tissue samples were homogenized in laemmli buffer, and 10 mg of protein was loaded into each well of a 8-16% gradient gel. -catenin, an integral modulator of the wnt pathway, and striatin were colocalized with the endoplasmic reticulum (er) marker, calnexin. in the unafflicted animals, -catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern, with no detectable localization in the er. in contrast, in the 15 arvc boxers, bothcatenin and striatin were colocalized with calnexin in an er pattern. in the afflicted samples, -catenin and striatin were not visualized to the intercalated disc and intracellular space, respectively. western blots of striatin and -catenin revealed no changes in the amount of protein. interestingly, a western blot for the wnt protein revealed a decrease in the amount of protein in arvc samples, compared to unafflicted samples. our preliminary data suggest that disturbances of the canonical wnt pathway may play an etiological role in the development of arvc in the boxer dog. there are numerous benefits of omega-3 fatty acid supplementation in human heart disease, including reduction in arrhythmias, decreased incidence of sudden death, and improved survival in heart failure. antithrombotic effects of omega-3 fatty acids have been demonstrated in people, which may have particular benefit in cats given their risk of thromboembolic complications with cardiac disease. benefits also have been found in canine heart disease, and reduced serum concentrations of the omega-3 fatty acids, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) have been found in dogs with congestive heart failure (chf) secondary to dcm. to the authors' knowledge, no studies to date have investigated fatty acid concentrations in cats with cardiomyopathy. the purpose of this study was to measure serum fatty acid concentrations in normal cats and cats with cardiomyopathy. serum fatty acid concentrations were measured in normal cats and cats with cardiomyopathy using gas chromatography. cats with cardiomyopathy and at least mild left atrial (la) enlargement (la to aortic ratio 4 1.5 on two-dimensional echocardiography from a right parasternal short axis view) were candidates for study. normal cats had a normal history, physical examination, echocardiogram, packed cell volume, total solids and platelet count. cats with evidence of other systemic disease or those receiving anticoagulants were excluded from the study. normally distributed and skewed data were compared between the cardiomyopathy and control groups with independent t tests or mann whitney u tests, respectively. statistical significance was set at p o 0.01. thirty cats with cardiomyopathy (23 neutered males and 7 neutered females) and 27 healthy controls (13 neutered males and 14 neutered females) were enrolled. median age was 8 yr (range, 2-16 yr) in the cardiomyopathy group and 5 yr (range, 1-10 yr) in the control group (p 5 .009). cats in the cardiomyopathy group were classified in the international small animal cardiac health council stage 1b (n 5 10), 2 (n 5 8), 3a (n 5 3) and 3b (n 5 9). compared to control cats, cardiomyopathic cats had higher concentrations of palmitic acid (p 5 .002) and dha (p o .001), and lower concentrations of linoleic acid (p 5 .005). among cats with cardiomyopathy, there was no significant correlation between any serum fatty acid concentration and left atrial size or age. these findings warrant further investigation into the role of fatty acids in cats with cardiac disease. platelet mapping is an application of thromboelastography that relies on the generation of at least three tracings: ma thrombin (maximum platelet activity),ma fibrin (fibrin activity only), an-dma aa or adp (platelet activity not inhibited by arachidonic acid or adp receptor antagonists, respectively). using these three tracings, the % inhibition of platelets can be calculated. the purpose of this study was to evaluate the platelet mapping assay in normal cats and assess platelet inhibition in cats receiving clopidogrel. employee-owned cats with normal history, physical exam, echocardiogram, thromboelastography, packed cell volume, total solids and platelet count were eligible. clopidogrel (18.75 mg po q24h) was administered for 10 days with platelet mapping performed on days 0 and 10. platelet mapping values were compared using a paired t test, with significance set at p o 0.05. seven cats (4 fs, 3 cm, aged 2-10 years) were enrolled. compared to day 0, ma adp (p o .001) and ma fibrin (p o .001) were lower on day 10. the latter unexpected result prompted measurement of fibrinogen concentrations at day 0 and 10 in the last 5 of these 7 cats. fibrinogen was not different from day 0 to 10 in these 5 cats. these results suggest that platelet mapping may be a simple, outpatient clinical tool to measure antiplatelet activity in cats receiving clopidogrel. this clopidogrel dose resulted in significant platelet inhibition as measured by ma adp in all cats. further studies correlating antiplatelet effects measured by platelet mapping with clinical outcomes in cats with cardiomyopathy are warranted. this study investigated the hemodynamic effects of application of an itd in a canine model of cardiopulmonary arrest. 8 laboratory beagles which were part of a separate terminal study were anesthetized and instrumented for continuous measurement and recording of right atrial pressure, arterial pressure and carotid blood flow. following euthanasia, cpr was performed for one minute, then a pause of one minute followed by a second one minute period at a compression rate of 100-120/minute, ventilation with 100% oxygen was delivered at eight breaths/min and 15 ml/kg tidal volume. cpr was performed with the itd in place (itd-cpr) and without the itd (s-cpr) for one period each in a randomized fashion with the rescuer blinded to its application. baseline, s-cpr and itd-cpr data were assessed for normality, a kruskal-wallis one-way anova on ranks was used (baseline v cpr). when appropriate a pairwise multiple comparison procedure (dunn's method) was used. percentage of baseline s-cpr v itd-cpr was assessed using the student t-test. the right atrial diastolic pressure was significantly more negative with the itd attached than without (p 5 0.02), the coronary perfusion pressure and carotid blood flow were significantly higher during cpr with the itd than standard cpr (p 5 0.015, p 5 0.017). no significant differences in diastolic, mean or systolic arterial pressure or end tidal co 2 were seen. application of the itd resulted in significantly improved hemodynamics during cpr in dogs. clinical evaluation of the device is warranted to examine whether this translates into improved success in resuscitation and survival. left ventricular (lv) systolic dysfunction is a common problem in dogs and can be due to a variety of etiologies. one potential etiology for systolic dysfunction is persistent or paroxysmal tachyarrhythmias, leading to tachycardia-induced cardiomyopathy (ticm). in humans, ticm carries a relatively good prognosis in that remodeling may be reversible with normalization of heart rate. differentiating between primary and secondary tachyarrhythmias in dogs with systolic dysfunction is critical for prognostic purposes as primary tachyarrhythmias may be associated with a better outcome. the goal of our study was to describe a population of dogs with ticm and to determine if treatment of arrhythmias was associated with reversible cardiac remodeling as indicated by standard echocardiographic parameters. medical records of dogs referred to the cardiology service of ksu vmth from 2008 to 2010 were reviewed. ticm was defined as the presence of severe tachyarrhythmias that were reversible with treatment, systolic dysfunction or ventricular enlargement that improved with treatment of the arrhythmia, or dogs with severe tachyarrhythmias and systolic dysfunction of breeds with that are atypical for idiopathic dilated cardiomyopathy. exclusion criteria were dogs with congenital heart disease, severe mitral regurgitation, and endocarditis. transthoracic echocardiography, thoracic radiographs and electrocardiography (ecg) were performed in all dogs. ventricular enlargement and systolic dysfunction were defined according to standard echocardiographic parameters. arrhythmias were confirmed with a holter monitor in 7 dogs. a total of 12 dogs were included in the study. mean age was 7 years (range 2-12 years) with 8 males (4 intact, 4 castrated) and 4 females (3 spayed, 1 intact). four dogs had pulmonary venous congestion or pulmonary edema and two dogs had ascites. at initial presentation, the meanaesd values were as follows: heart rate 219ae75 bpm, m-mode fractional shortening (fs) 17.29ae9.05%, ejection fraction (ef) using the area-length method 30.69ae13.45%, and left atrial to aortic root ratio (la/ao) 1.92ae0.37. initial meanaesd m-mode derived lv internal dimensions corrected for body weight were as follows: diastolic 1.92ae0.37 and systolic 1.49ae0.38. at least one of the following tachyarrhythmias were identified in each dog: atrial fibrillation (4), supraventricular tachycardia (5), junctional tachycardia (2), and ventricular arrhythmias (3). ten dogs were available for follow up. seven dogs improved in at least one of the following parameters: resolution of tachyarrhythmia (3), improved systolic function (3) antidiuretic hormone (adh) has been shown to be elevated in humans with congestive heart failure (chf). recently, antidiuretic hormone antagonists were successful during investigational treatment of refractory congestive heart failure in humans. adh levels have been only modestly investigated in dogs with cardiac disease, primarily due to the technical difficulty in measuring adh levels via radioimmunoassay. based on the homologous structure of canine and human adh, we aimed first to determine the feasibility of measuring adh in dog plasma using a human elisa kit, and secondly to investigate the level of adh in dogs with congestive heart failure due to acquired cardiac disease. elisa assay kit validation was performed using six healthy dogs with normal clinical and echocardiographic examinations. pooled canine plasma was spiked with synthetic adh and intra-assay precision, dilutional parallelism, and linearity were assessed. to address the second aim of the study, samples were collected from normal dogs and dogs with heart failure due to one of two types of acquired cardiac disease: chronic degenerative valve disease (cdvd) or dilated cardiomyopathy (dcm). patients underwent clinical, radiographic, and echocardiographic examination to confirm diagnosis, assess severity of disease, and determine presence of pulmonary edema. whole blood was collected into edta tubes containing protease inhibitors, cold centrifuged, and plasma was stored at à801 until analysis. following ether extraction, plasma adh in each sample was measured in duplicate using a human elisa kit. statistical analysis included a d-agostino and pearson test for normality; group results were compared using a nonparametric mann-whitney test. adh was measured in canine plasma using the human elisa kit with acceptable intra-assay precision, linearity, and dilutional parallelism. intra-assay coefficient of variation was 11%. twenty-four dogs were recruited for the second phase of the study. six normal dogs and twelve dogs with radiographic evidence of pulmonary edema due to either cdvd or dcm were selected for participation. the remaining six dogs were excluded due to lack of definitive radiographic evidence of congestive heart failure. median adh values were 3.67 ae .38 pg/ml for the normal group (n 5 6) and 6.155 ae .94 pg/ml for the heart failure group (n 5 12). median adh values for the two groups were statistically different (p 5 0.0057). our preliminary results indicate that measuring canine adh using a human elisa kit is feasible and provides results with an acceptable coefficient of variation. we also showed that dogs with congestive heart failure due to cdvd and dcm have elevated adh levels in comparison to normal dogs. our findings motivate further investigation to assess the degree of plasma adh level elevation and the possible use of adh antagonists as an adjunct treatment for refractory congestive heart failure in dogs. aortic thromboembolism (ate) occurs in both cats and dogs. whereas ate in cats is strongly associated with structural heart disease and typically has an acute catastrophic presentation; the pathogenesis and presentation of ate in dogs is less well known or understood. further, an effective antithrombotic strategy for ate in dogs has not been reported. medical records of dogs diagnosed with ate between 2003 and 2010 were examined retrospectively. diagnosis of ate was based on ultrasonography, doppler flow studies, and diminished or absent femoral pulses. dogs were treated with various acute and chronic antithrombotic therapies. the severity of ambulatory dysfunction was graded as none, mild, moderate, severe, or non-ambulatory at presentation and after therapy. a cohort of dogs in this study received a standardized protocol of chronic warfarin therapy with or without antiplatelet drugs. target international normalized ratio for warfarin therapy was 2 to 3. twenty-six dogs were diagnosed with ate. all had an apparent mural aortic thrombus caudal to the renal arteries with most having evidence of embolization to the iliac and femoral arteries. none had structural heart disease at diagnosis. twenty dogs (77%) were still ambulatory at diagnosis. the median duration of ambulatory dysfunction prior to presentation was 7.8 weeks (range 1 day -52 weeks). a majority of dogs (58%) had no concurrent conditions at diagnosis. nine dogs (33%) had protein-losing nephropathy. four dogs (15%) were hypothyroid. fourteen dogs were treated with a standard warfarin protocol for a median period of 22.9 months (range 0.5-53 months). eight dogs were treated concurrently with aspirin, 2 dogs were treated concurrently with clopidogrel, and 4 dogs were treated with warfarin only. ambulatory function improved between 1 and 3 grades in dogs treated with chronic warfarin. the median period until clinical improvement was 13.9 days (range 2-49 days). two dogs treated with chronic warfarin therapy had documented resolution of ate, and 5 dogs had complete resolution of ambulatory dysfunction. none of the 14 dogs treated with chronic warfarin became nonambulatory, died, or underwent euthanasia because of ate. the median period of freedom from an adverse event was 24.2 months. no serious hemorrhagic events were reported. four dogs were treated with tpa. three of these had an unfavorable outcome. two dogs were ambulatory before tpa and become non-ambulatory after treatment. two dogs underwent surgical thrombectomy. one had a favorable outcome until ate recurred 14 months after surgery. in conclusion, the pathogenesis of ate in dogs is not associated with structural heart disease or left atrial thrombus formation. the presentation tends to be for chronic ambulatory dysfunction. most dogs are still ambulatory at presentation. warfarin, with or without concurrent antiplatelet therapy, is an effective antithrombotic treatment strategy for dogs with ate. information is known. through previous work, investigators have encountered norfolk terriers (nt) with echocardiographically apparent dmvd in the absence of a heart murmur. in order to more fully understand dmvd in this breed of dog, we sought to characterize findings from the physical and echocardiographic exam, biochemical, biomarker, and nutritional profile, and select environmental variables from a cohort of apparently healthy nt. overtly healthy nt ! 6yrs old were recruited by 3 different veterinary hospitals and underwent historical, physical, ecg, and 2d/color-flow doppler echocardiographic exam. anterior mitral valve length, maximal thickness, area, and prolapse were measured from 2d images. presence of dmvd was defined as thickened, prolapsing, or flail mitral valve leaflets in the presence of color flow doppler evidence of mitral regurgitation. blood samples were obtained for serum biochemistry and serotonin, plasma nt-probnp, amino acid profile, c-reactive protein, and cardiac troponin-i. forty-eight dogs were entered into the study (median age, 8yrs iqr [7-10]; gender, 29f, 19m; median bcs, ). of the 48 dogs, 23 (48%) had murmurs, 2 (4%) had mid-systolic clicks, 11 (23%) had ecg p-pulmonale, and 41 (85%) were deemed to have echocardiographic evidence of dmvd, including 18 nt without a murmur. seven (15%), 28 (58%), and 13 (27%) dogs were classified as isachc 0, 1a, and 1b, respectively. mean indexed echocardiographic mitral leaflet length (p o 0.0001), thickness (p 5 0.019), prolapse (p 5 0.0005), and la:aod (p 5 0.01) were significantly different between isachc 1a/b vs 0. between isachc 1a/1b and 0, there were no differences in serum amino acids, c-reactive protein, troponin, diet, or environmental factors; however 6 different amino acids (ala, gly, phe, pro, trp, tyr) were significantly higher in isachc 1b vs. 1a. median serum serotonin was increased in dogs with 1a/b vs. 0 (p 5 0.025). dogs whose diets contained some canned food (p 5 0.12) and dogs residing in suburban environments (p 5 0.03) had higher serotonin concentrations. nt-probnp tended (p 5 0.07) to be higher in isachc 1a/1b vs. 0. dmvd appears to be relatively common in nt and echocardiographic changes consistent with mild dmvd can be seen in dogs without a heart murmur. the results of this study establish a foundation of useful information upon which additional prospective studies can be developed. left ventricle (lv) evaluation is one of the most important contributions of echocardiography in the assessment of cardiac function. however, lv analysis can be made from images obtained by different modes and views of the heart. the aim of this study was to compare lv measurements, shortening fraction (sf) and ejection fraction (ef) obtained from four methods: m mode in short-axis and in long-axis, bidimensional mode in short-axis and in long-axis views of the heart. forty normal adult german shepherds were selected. echocardiographic study of lv of each animal was performed by the four methods described above. ancova test was used to examine the effects of axis, mode, weight and gender over lv measurements. isolated effect of the axis was observed for lv end-diastolic diameter (lvedd), with greater values obtained from short-axis views. there was isolated effect of mode over ef and sf, with greater measurements derived from bidimensional mode methods. weight correlated with all linear lv measures at least in one method, but not with ef and sf. weight had positive effect over lv endsystolic diameter and lv end-diastole posterior wall thickness in all methods, except from m mode in short axis in the last one. gender had isolated effect over lvedd, males showing greater values than females in bidimensional mode in short and long axis. the combined effect of axis, gender and weight was identified in interventricular septal end-diastolic thickness. we concluded that normal reference values obtained by different echocardiographic modes and planes should not be used interchangeably. abstract c-29 assessment of left ventricular diastolic func-tion by color tissue doppler imaging echo-cardiography in maine coon cats tested for mypbc-ap31 mutation hypertrophic cardiomyopathy (hcm), characterized by increased cardiac mass and diastolic dysfunction, is the most common feline heart disease. myocardial analysis by color tissue doppler imaging (tdi) is more sensitive than conventional echocardiography. this study evaluated diastolic dysfunction in various stages of feline hcm. maine coon cats (n 5 57) were screened for the mybpc-a31p mutation and examined with both echocardiography and tdi. then, were phenotypically classified in: normal (n 5 45), suspects for hcm (n 5 7) and hcm group (n 5 5); and genotypically classified in: negative (n 5 28), heterozygous (n 5 26) and homozygous group (n 5 3). myocardial velocities, measured in the basal and mild ventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw) and in radial segment of left ventricular wall, was compared among different groups. a significant decreased (p 5 0,01) longitudinal em/am at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. a significant increased (p 5 0,01) longitudinal e/em at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. and a significant decreased (p 5 0,02) longitudinal sm at the basal segment of the lvw was observed in heterozygous cats compared with negative cats, both without hypertrophy. there was a significant positive correlation between summated early and late diastolic velocities (emam) and heart rate (p o 0,001); and a positive correlation between sm and em velocities and heart rate (p o 0,01). the mybpc-a31p mutation is not consistently associated with ventricular hypertrophy and negatives cats can also develop hcm. the tdi alone is not able to identify cats with mutation before myocardial hypertrophy. diastolic dysfunction occurs in many cats with hypertrophic cardiomyopathy (hcm) but less is known about systolic function in various stages of hcm. myocardial strain analysis by tissue doppler imaging is a noninvasive echocardiographic method to assess systolic function. this study evaluated systolic function in various stages of feline hcm. maine coon cats (n 5 57) were screened for the mybpc-a31p mutation an examined with both echocardiography and strain. then, were phenotypically classified in: normal (n 5 45), suspects for hcm (n 5 7) and hcm group (n 5 5); and genotypically classified in: negative (n 5 28), heterozygous (n 5 26) and homozygous group (n 5 3). peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw), left ventricular anterior wall (lvaw), left ventricular posterior wall (lvpw) and radial segment of left ventricular wall, was compared among different groups. whereas conventional echocardiography demonstrated an apparently normal contractile state based on fractional shortening, myocardial strain (at mildventricular segment of ivs) in hcm cats was significantly decreased compared with normal group (p 5 0,01). myocardial strain (at basal segment of lvaw) also was significantly decreased in heterozygous cats compared with negative group (p 5 0,00); and was significantly decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy (p 5 0,019). there was a significant negative correlation between strain values and wall thickness (p o 0,05). this method allows detection of abnormal systolic deformation in maine coons cats with hcm mutation despite apparently normal systolic function. the abnormal systolic deformation already can be present in heterozygous cats without hypertrophy and increased with progressive ventricular hypertrophy. recently, multiple advanced resting electrocardiographic (ecg) techniques have been applied in humans for detection of cardiac autonomic and repolarisation function. this has improved the diagnostic and/or prognostic value of short-time ecg in detection of common human cardiac diseases even before onset of symptoms or changes in the standard ecg. therefore, this study investigates, if advanced ecg can predict the severity of mitral regurgitation (mr) in dogs with myxomatous mitral valve disease (mmvd) and thereby improve the diagnostic value of ecg. the study included 77 privately owned cavalier king charles spaniels (ckcss) (age 6.0 ae 2.7 years; 30 males and 47 females). all dogs were examined by echocardiography and a short-time (3-5 min) high-fidelity 12-lead ecg, with the dog in a resting position and in sinus rhythm. dogs were divided into 5 groups according to the degree of mr estimated as the percentage of the left atrium area using color doppler mapping (0%; 0% o jet 15%; 15% o jet 50%; jet 4 50%; jet 5 100% and with clinical signs of congestive heart failure). ecg recordings were evaluated via custom software programs to calculate 76 different parameters, including heart rate variability (hrv), qt variability (qtv), t-wave complexity, wave morphology and 3-d ecg. one-way anova determined 21 ecg parameters, which were significantly different (p o 0.05) between the 5 dog groups. principal component factor analysis identified a 5factor model with 83.2% explained variability. qrs dipolar voltage and two repolarization indices of qtv increased significantly with mr severity, whereas total power of the frequency spectrum of rr interval and the standard deviation of qtv decreased significantly with mr severity. for the 5 selected parameters the prediction of mr jet value was tested by multiple linear regression. a correlation coefficient (r) of 0.65 indicated that the prediction value was significant (p o 0.01). if age was included in the multiple linear regression the prediction value was further increased (r 5 0.80). our results indicate that for a cut-off criteria of mr ! 50% jet the five selected ecg parameters could predict the severity of mr caused by mmvd in ckcss with sinus rhythm with sensitivity 65% (78% with age inclusion) and specificity 98% (92% with age inclusion) (p o 0.05). nt-probnp concentration is increased in canine patients with heart disease. relatively little is known about the stimuli for release of nt-probnp in dogs. physical activity independent of cardiac disease and the stress of being in the hospital could influence nt-probnp release and affect diagnosis and management of patients. we hypothesized that nt-probnp concentration in healthy dogs would not exceed the normal reference value (900 pmol/l) following a period of exercise. the goal of this study was to examine whether physical activity could elevate plasma nt-probnp and cause false positive results in healthy dogs. the study population included healthy dogs 41 yr of age without heart murmur or known systemic disease, and normal 2d/color flow echocardiographic exam. plasma samples for nt-probnp were obtained before, immediately after, and 1 hour after a standardized 5-minute submaximal exercise regimen. the study included 14 dogs with a median age of 5.3yrs and included 6 females and 8 males. there was no statistical difference in median plasma nt-probnp concentration across the three time points (baseline median, 617 [iqr, immediately post, ; p 5 0.11). the average coefficient of variation of nt-probnp concentration across the exercise regimen was 23.0 ae 18.9%. in 1 of 14 dogs (7.1%), nt-probnp increased from 790 to 1054 pmol/l immediately after exercise. the results of this study demonstrate that submaximal exercise does not significantly change median nt-probnp concentration and the incidence of false positive results is low. further studies should investigate effects of exercise on nt-probnp concentrations in dogs with heart disease. obesity is an increasing problem in veterinary medicine. obese human patients are shown to present lower levels of natriuretic peptides, regardless of an increased volume and pressure load, what raises the possibility that the natriuretic response is impaired in these individuals. considering the controversial findings in obese humans, and the lack of studies reported in dogs, this study proposed the evaluation of nt-proanp concentration in obese dogs. nt-proanp concentration was determined prospectively in 39 obese dogs (27 females; 12 males; 6-108 months) and in 23 non-obese dogs (controls; 13 females; 10 males; 12-108 months) from a veterinary hospital population. obesity was determined by body condition score [2 (4/9); 21 (5/9); 1 (6/9); 3 (7/9); 19 (8/9); 18 (9/9)]. dogs were excluded if they had any primary cardiac disease, renal insufficiency, endocrine disease, or if they were receiving diuretics, vasodilators, antiepileptic drugs or corticosteroids. commercial kits were used (vetsign s canine cardio screen nt-pro-anp vc3167 -guildhay/biomedica). mann whitney test was used for group comparison. results are presented as median; interval; p25 and p75). nt-proanp was significantly lower in obese dogs [413.56fmol/ml (0.0-1287.14); p25 5 228.673; p75 5 595.205] than in controls [647.98fmol/ml (34.99-1596.82 ); p25 5 490.785; p75 5 957.055]; (p 5 0.004). results were similar to what has been found in obese humans. lower levels of natriuretic peptides are also seen in obese heart failure patients. this study provides important information regarding nt-proanp concentration in obese dogs, which should be better explored characterizing the behavior of natriuretic peptides after weight loss, and also in obese dogs with primary heart disease. left-to-right shunting patent ductus arteriosus (pda) is one of the most common canine congenital cardiovascular defects. human studies have shown that bnp and nt-probnp concentrations are elevated in patients with pda, and can be used to detect hemodynamically significant pda. the purpose of this study was to measure nt-probnp concentrations in dogs with pda, and to assess whether additional indicators of hemodynamics correlate with ntprobnp. we hypothesized that nt-probnp will serve as a simple non-invasive marker of hemodynamic significance in dogs with pda prior to and following transcatheter ductal occlusion. nt-probnp was measured in 30 client-owned dogs with echocardiographically normal hearts. ten dogs with pda were initially evaluated with thoracic radiographs, transthoracic and transesophageal echocardiography, pulmonary capillary wedge pressure (pcwp) and nt-probnp. nt-probnp and echocardiography were repeated at 1 day and 3 months following ductal occlusion. pcwp was repeated at 3 months. baseline nt-probnp was significantly higher in pda dogs compared to control (1877 ae 2081 pmol/l (mean ae sd), 651 ae 301; p o 0.0025). at 1 day and 3 months following ductal occlusion, nt-probnp was 1347 ae 1256 and 466 ae 380, respectively. the following decreased significantly from baseline: pcwp (11.8 ae 4.7 to 6.4 ae 3.2 mmhg; p 5 0.018), and indexed left ventricular internal dimensions in diastole (2.26 ae 0.47 to 1.67 ae 0.19; p 5 0.006) but not significantly in systole (1.40 ae 0.31 to 1.20 ae 0.19; p 5 0.13). nt-probnp is elevated in dogs with pda and transductal closure is associated with a reduction in nt-probnp, pcwp and left ventricular size. cardiac biomarkers, particularly nt-probnp, are becoming more commonly used in dogs and cats as part of a diagnostic work up. multiple studies already have documented the correlation of this peptide with cardiac disease status and potential clinical implications. in a portion of these reports the manner in which samples were handled was placement of whole blood into an edta tube, followed by centrifugation and decanting of the supernatant that was ultimately stored at à201c or à801c prior to shipment, either with or without protease inhibition. our objective was to compare the nt-probnp concentrations in feline plasma collected using the previously reported methods to the california animal hospital (cah) collection method using tubes containing a protease inhibitor. this study compared nt-probnp concentrations using the protease inhibitor tubes vs. edta tubes from 18 privately owned feline patients, with confirmed cardiac disease, and 6 control feline patients. for all study participants, we performed a full history and physical examination, a hematology and chemistry panel, thoracic radiographs, ecg, and echocardiogram. in each study participant, at least 4 ml's of whole blood was drawn from a peripheral vein, and transferred to a plastic edta tube. the sample was centrifuged within 1 hour after collection. 1 ml of plasma was then transferred to a tube containing a protease inhibitor, which was stored at 41c until being shipped within 24 hours of collection. the remaining plasma was placed into 2 separate microtubes, which did not contain a protease inhibitor. one microtube was then stored and shipped as previous studies have reported (à201c, styrofoam container, shipment within 24 hours), and the second microtube was frozen at à801c. all samples were shipped, received and analyzed within 24 hours of collection. results of this study showed that no difference was found between the 2 frozen sample methods (663 pmol/l and 650 pmol/l p 5 0.40). it was determined that both frozen methods had lower nt-probnp levels (655 and 646 pmol/l) when compared to plasma samples shipped in protease inhibitor tubes (756 and 794 pmol/l). the findings of this trial demonstrate that the nt-probnp levels are significantly different between samples placed in edta tubes vs. contain protease inhibitor (p 5 0.008 and p 5 0.0006). utilizing protease inhibitor tubes allows more accurate measurement of plasma nt-probnp. as for its relevance for future research and publications, authors should take care to investigate the manner in which blood samples were handled and the conclusion/results of these studies should be taken in light of the methodologies used in collecting, storing, shipping and analyzing the samples. degenerative mitral valve disease (dmvd) is one of the most common heart disease and is present approximately 75% of the canine heart disease. although the high prevalence exists in small dogs, the underlying molecular mechanism of its pathophysiology is rarely known. dmvd is functionally and pathologically similar in humans and dogs, thus, there will be a common pathogenesis in human and dogs with naturally occurring dmvd. serotonin and serotonin-related mechanisms have been implicated as a cause of valvular disease in human and animals, including spontaneous dmvd in dogs. increased circulating 5ht concentration as a potential source of heightened 5ht signaling is demonstrated in small dogs with dmvd. the aim of this study was to investigate whether serum 5ht concentrations were associated with severity of naturally occurring dmvd in small dogs and to investigate potential associations of dog characteristics on serum 5ht concentrations in our study population. forty-eight dogs were included in this study and were classified into control and dmvd groups according to the results of physical and echocardiographic examinations. based on the la:ao ratio, dogs with dmvd were classified as follow: control (la:ao ratio 1.5 and no mr), mild (la:ao ratio 1.5 and mr), moderate (1.5 o la:ao ratio 1.8 and mr), severe (la:ao ratio 41.8 and mr). serum serotonin concentrations were measured by elisa. an overall significant difference (p o .05) was found among 4 groups and 5ht concentrations (control, 72.38 ng/ml [51.34-95.11 dmvd, ). significantly higher 5ht concentrations were observed in dogs with moderate (p o .05) and severe (p o .05) dmvd, compared with concentration in control group. additionally, 5ht concentration in dogs with moderate dmvd were significantly higher (p o .05) than concentration in dogs with mild dmvd. also, dogs with severe dmvd had significantly higher 5ht concentration than dogs with mild (p o .05) and moderate (p o .05) dmvd. there was no significant association of age, platelet, and lvidd, on serum 5ht concentration, however, weak correlation between serum 5ht increased significantly and la:ao ratio (r 2 5 .211, p o .05) was observed. the results of this study indicate that serum 5ht concentrations were higher with increasing severity of spontaneous dmvd, which may be the potential cause to advance the progression of dmvd. further studies should be performed to reveal the role of 5ht in inducing and accelerating spontaneous dmvd and to investigate if lowering serum 5ht concentration could alter the progression of dmvd. the objective of this prospective study was to evaluate the utility of cardiac troponin i (ctni) in differentiating between underlying etiologies of pericardial effusion in the canine patient. patients were prospectively recruited at time of diagnosis of novel pericardial effusion. serum samples were collected prior to pericardiocentesis. patients were evaluated by echocardiography and classified with the diagnosis of hemangiosarcoma (hsa), heart base tumor (hbt), or unknown etiology at the initial evaluation based on established characteristic echocardiographic findings. idiopathic pericardial effusion (ipe) was defined by histopathology, echonegative for a mass lesion with no recurrence of pericardial effusion 46 months, or symptom free 412 months from time of enrollment. patients were excluded from analysis if a diagnosis could not be established based on above criteria or concurrent moderate azotemia (creatinine 43.0 mg/dl) was present at time of sample collection. serum samples were frozen and analyzed in batches within 60 days of collection by a ctni assay with a 0.2ng/ml lower limit sensitivity. sixty-three patients were recruited over a one year period with 15 patients excluded due to lack of diagnosis (13) or azotemia (2). median ctni levels of dogs with hsa (n 5 24), hbt (n 5 19), and ipe (n 5 5) were 14.8 ng/dl (interquartile range (iqr) o 0.2-11.3), 0.23 ng/dl (iqr o 0.2-0.29), and o 0.2 ng/dl (iqr o 0.2-o 0.2) respectively. concentrations of ctni differed significantly between dogs with hsa and hbt (p 5 0.001) and ipe (p 5 0.0029). there was no difference between ctni concentrations between hbt and ipe dogs (p 5 0.911). receiver operating curve analysis to determine the optimal cutoff for differentiation of dogs affected with hsa and both hbt and ipe revealed a significant (p 5 o 0.001) area under the curve (0.79). a cut-off point of ctni of 40.78 yielded a sensitivity of 67% (95% ci, 45-84%) and specificity of 95% (95% ci, 79-99%). utilizing a higher cut-off point of 43.0 yielded a lower sensitivity of 50% (95% ci, 29-71%), but a higher specificity of 100% (95% ci, 86-100%) which may have more clinical utility given the disparity in prognoses of the etiologies compared. in conclusion, this study supports the diagnostic utility of ctni concentrations to delineate between patients with hsa and other etiologies of pericardial effusion, but does not reliably differentiate between dogs with ipe and other neoplastic etiologies. the pathogenesis of degenerative mitral valve disease (dmvd) in dogs remains to be fully elucidated. the high sheer stress caused by mitral regurgitation damages the endothelial surface of the valve, and a previous study demonstrated increased transcription of intercellular adhesion molecule-1 (icam-1) and e-selectin in affected mitral leaflet tissue. we hypothesized that this may be responsible for platelet recruitment and adhesion, and initiation of a proliferative cascade, resulting in further myxomatous changes. the goal of this study was to compare plasma levels of icam-1 and e-selectin in healthy dogs and those with dmvd. the study population included dogs with echocardiographic evidence of dmvd and healthy control dogs 41 year old with no heart murmur or known systemic diseases. dmvd dogs underwent 2d/color-flow doppler echocardiographic exam. blood samples were obtained for plasma icam-1 and e-selectin analysis using commercially available elisa kits. the study included 34 dogs, of which 20 had dmvd and 14 were normal. the dmvd group had a median age of 9.5yrs ) and included 6 females and 14 males. two (10%), 13 (65%), 2 (10%) and 3 (15%) dogs were classified as isachc 1a, 1b, 2 and 3a, respectively. of the control dogs, median age was 4.5yrs [2-6.5], with 5 females and 9 males. there was no statistical difference in plasma e-selectin between control dogs (median 2.71 [2.05-7.66]) and those with dmvd (2.46 [1.54-3.55]); p 5 0.35. plasma icam-1 concentrations were higher in dmvd dogs (1.58 [1.20-10.58 ]) than controls (median 1.31 [1.11-1.65], but this difference did not reach statistical significance (p 5 0.22). linear regression analysis showed no significant correlation between icam-1 or e-selectin and serum serotonin level, nt-probnp or echocardiographic measures of dmvd severity (la:ao, lvidd:ao, lvids:ao). the results of this study demonstrate no significant difference in circulating adhesion molecules icam-1 and e-selectin in dogs with dmvd as compared with healthy controls. further studies investigating adhesion molecules within the mitral valve tissue itself are likely needed if icam-1 and e-selectin play a role in the pathophysiology of dmvd. the rate of glucose utilization in the heart is greater than in other tissues, and impaired glucose uptake may play a major role in the pathogenesis of heart failure (hf). glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (gluts), which includes glut-4, the major cardiac isoform, and glut-12, a recently discovered isoform, the role of which is unknown in the heart. in addition, despite the wellknown regional differences in myocardial structure and function, potential regional patterns in glucose transport have not been investigated. thus, we hypothesized that glut-4 and -12 protein and gene expression would be chamber specific in healthy dogs and during chronic hf. using a canine model of tachypacing induced chronic hf, glut protein and messenger rna in both ventricles and atria were investigated by immunoblotting and real time pcr. in control dogs, glut-4, but not glut-12, protein expression were significantly higher in the atria compared to the ventricles, with the highest content in the right atrium (ra, p o 0.001). glut-4 and -12 mrna were homogeneously expressed in all the cardiac chambers. during chronic hf, glut -4 and -12 expression was highest in the left ventricle (lv, by 2.5 and 4.2 fold, respectively, p o 0.01), with a concomitant increase in glut-4 and -12 mrna (p o 0.001). glut -4, but not glut-12, was decreased in ra during chronic hf (p 5 0.001). our data suggest that glut-4 protein was differentially expressed across the cardiac chambers in the healthy heart. during chronic hf, lv was the primary site dependent on both glut4and glut12-mediated glucose transport, which was transcriptionally regulated. in addition, the paradoxical decrease in glut4 content in ra may induce perturbations in atrial energy production during chronic hf. some obese dogs are suspected to have cardiac disease because they have enlargement of the heart on thoracic radiograph. it has been reported in cats that the fat increases the cardiac silhouette, while echocardiograms revealed normal cardiac dimensions. the purpose of this study was to determine whether obesity overestimates cardiac dimension in radiographs compared to echocardiographic findings in dogs. twenty three obese dogs and 20 controls were included based on a 1-9 body condition scoring (bcs). computerized radiography was obtained and vhs measurement was performed as previously described. echocardiographic measurements were interpreted based on reference values according to lean body weight regression equations. results for echo and vhs measurements were classified in scores as normal, mild, moderate or severe increase. student's t test was used for comparison of vhs between groups. mann-whitney rank sum test was used to assess echocardiographic scores between groups. spearman rank order correlation was used to assess relationships between any pairs of variables between echo and vhs scores, echo vs bcs and vhs vs bcs. groups were similar regarding age [obese (69ae24); control (62ae27); p 5 0.368], breeds and gender distribution. obese dogs had significantly higher vhs and echo scores compared with controls [vhs: (10.58ae0.69) vs (9.77ae0.54); p o 0.001; echo score: range (1-4) vs (1-2); p 4 0.05]. there were no relationships between any pair of variables analyzed. these results show that there are changes both in echo and radiographic appearance of the heart in obese dogs, but vhs overestimates cardiac silhouette compared to echo, probably related to pericardial fat accumulation. heart rate variability (hrv) is an indirect measurement of the autonomic modulation of heart rate (hr). reduced hrv measured from short-time electrocardiography is seen in dogs with heart failure (hf) secondary to myxomatous mitral valve disease (mmvd). however, hrv is suggested to increase with disease severity at early stages of mmvd. the aims of this study were 1) to associate hr and hrv with severity of mmvd in cavalier king charles spaniels (ckcs) and 2) to compare hr and hrv between ckcs and other dog breeds in a group of dogs in hf secondary to mmvd. one-hundred dogs were examined by echocardiography and 24hour electrocardiography. the dogs were divided into five groups: 1) ckcs with no/minimal mitral regurgitation (mr) (mr jet 15% of the left atrial area using color doppler mapping) and no murmur, 2) ckcs with mild mr (20% o jet 50%), 3) ckcs with moderate/ severe mr (jet450%) and no clinical signs of hf, 4) ckcs in hf (hf defined as left atrium to aortic root ratio (la/ao) 41.5, clinical signs of hf and furosemide responsiveness) and 5) non-ckcs in hf. dogs in hf were allowed hf therapy. both hr and hrv were analyzed over a 24-hour period, while hrv were also analysed over a 6-hour nightly period. analyses of variance were performed with hr or hrv as response variables and the explanatory variables dog group and echocardiographic indices of mmvd were included separately. all p-values were bonferroni corrected. minimum-and mean hr were significantly higher in ckcs with moderate/severe mr and in hf compared to ckcs with no/ minimal and mild mr (all p o 0.001). seven out of 26 hrv variables were significantly decreased in ckcs with moderate/ severe mr and in hf compared to ckcs with no/minimal and mild mr (all p o 0.02). another 10 hrv variables showed the same groupwise differences (all p o 0.02), except that the difference between ckcs with mild mr and ckcs with moderate/severe mr did not reach statistical significance. mminimum hr, mean hr and the hrv variables (7 and 10) differing between dog groups, also consistently decreased with increasing mr, la/ao and the proximal isovelocity surface area in ckcs. non-ckcs in hf had a lower minimum hr compared to ckcs in hf (p 5 0.03) and a higher triangular index measured in both periods (all p o 0.04). in conclusion, hr increased and most hrv variables decreased with increasing severity of mmvd in ckcs, even prior to the development of hf. other breeds in hf secondary to mmvd had lower minimum hr, but higher triangular index compared to ckcs in hf. although the cells in the specialized conduction system in the heart are capable of initiating their own impulse, the rate in which those impulses are generated can be influenced by autonomic nervous system. different types of respiratory patterns can stimulate autonomic nervous system in different manners. thus, non-sedated rabbits were studied during forced respiration aiming to evaluate the influence of this breathing pattern on heart rate. twenty male, one-year-old healthy new zealand rabbits were enrolled in the study. animals were set in right lateral recumbency and maintained that way by physical contention. chemical sedation was not used. partial nasal obstruction by digital compression was applied to those rabbits for five seconds, eliciting a forced inhaling and exhaling against semi closed nostrils. heart rate was obtained by measurement of two consecutives rr intervals in the computerized electrocardiography, recorded continuously prior and during the maneuver. heart rate before the intervention was 251 ae 34 bpm (mean ae standard deviation). all rabbits submitted to the maneuver showed a dramatic reduction in this parameter. after nasal partial obstruction, heart rate was 142 ae 32 bpm. data was submitted to statistical analysis by paired student's t test and a significant difference between the heart rate before and after the maneuver was observed (p o 0.0001). although the exactly mechanism involved in this response was not elucidated, the presented data support the applicability of this maneuver as an efficient method for non-pharmacological heart rate reduction in rabbits. obesity can affect cardiac function due to effects on cardiac rhythm, ventricular volume and blood pressure. the purpose of this study was to determine the effects of obesity and overweight on noninvasive systemic blood pressure and doppler echocardiographic parameters in cats without others causes of cardiac hypertrophy. the study groups comprised fifteen obese cats with mean body score index (bsi) of 8,8, seven overweight cats (bsi 5 6,3) and seven cats with ideal bsi (4,9). the blood pressure was measured by doppler method and the doppler echocardiography was performed in conscious animals. the statistical analysis was performed by analysis of variance followed by tukey's test and pearson's correlation. the blood pressure values of the obese cats were superior (159,12 ae 11,22 mmhg, p o 0,0001) than in overweight (134,45 ae 13,81 mmhg) and normal cats (136,90 ae 13,17 mmhg) and 57% of the obese cats had blood pressure higher than 160 mmhg. there were observed differences on the ratio of early (e) and late (a) left ventricular filling velocity (p 5 0,008) of obese animals (e/a 5 1,07 ae 0,39) compared to overweight (1,68 ae 0,37) and normal cats (1,43 ae 0,24). seven obese cats (50%) had inversion of e/a compatible with diastolic dysfunction and there were negative correlation (r 5 à0,453, p 5 0,026) between the e/a ratio and blood pressure values. other differences observed were increases in left ventricular septum in diastole (p 5 0,002) and in free wall in diastole (p 5 0,023) and systole (p 5 0,042) of the obese animals compared to overweight and normal cats. these results demonstrate the possibility of cardiovascular effects related to obesity in cats, such as systemic arterial hypertension and secondary diastolic dysfunction. diuretic therapy reduces preload, and relieves congestion secondary to cardiac dysfunction. torsemide (torasemide) is a loop diuretic with longer duration of action, less diuretic resistance, and adjunctive aldosterone antagonism as compared to furosemide. we hypothesized that torsemide was no less effective than furosemide at diuresis, control of clinical signs, and maintenance of quality of life in dogs with congestive heart failure. a double-blinded, randomized, crossover clinical trial was performed in 7 dogs with stable heart failure receiving bid furosemide and adjunctive medications. dogs were randomized to their current furosemide dose or torsemide (calculated as 1/10 of the daily furosemide dose divided into bid dosing). crossover occurred at day 7 and the study ended on day 14. clinical, laboratory, radiographic, and owner-perceived quality of life variables were evaluated on days 0, 7 and 14. no dog developed recurrent heart failure during the study. average furosemide dose on day 0 was 5.13 mg/kg/day (range, 2.8-9.6). following torsemide treatment, blood urea nitrogen (p 5 0.0028), albumin (p 5 0.0287), and albumin:globulin ratio (p 5 0.0012) were significantly increased, and urine specific gravity (p 5 0.0062) and chloride (p 5 0.0051) were significantly decreased as compared to baseline and/or furosemide dosing (one-way anova with bonferroni correction). no differences in qol were found. results indicate that torsemide is equivalent to furosemide at controlling clinical heart failure in dogs, and might in fact, achieve greater diuresis vs. furosemide. larger clinical trials evaluating furosemide resistance and/or torsemide as a first-line loop diuretic for congestive heart failure in dogs with heart failure are warranted. the purpose of this study was to investigate the feasibility of speckle tracking echocardiography (ste) in healthy cats and to determine whether or not it can detect myocardial dysfunction in cats with diseased heart. radial and circumferential strain and strain rate were measured by ste using left ventricle short-axis view in clinically healthy cats. eighteen cats with hcm whose lv thickness at end-diastole with 6 mm or more were evaluated with ste analysis, and compared with healthy cats. index of left ventricular synchrony (trs-sd) was also assessed in cats with hcm, and compared to healthy subjects. ste resulted in technically adequate images in 100% of the cats. fusions of early and late diastolic (e and a) wave in strain rate were seen in 3 of 16 cats. percent errors in analysis with or without simultaneous ecg monitoring were 5.3-14.7% in all parameters. inter-and intraobserver variability of ste parameters in healthy cats was minimal (4.1-15.6%) except for the systolic circumferential strain rate. sedation using buprenorphine and acepromazine did not affect any ste parameter. e wave in radial and circumferential strain rate of hcm cats was significantly decreased compared with healthy cats. no significant difference was seen in trs-sd. ste analysis was considered clinically feasible to assess cardiac function in cats, and could detect myocardial dysfunction in cats with hcm. further study is warranted to investigate to assess whether or not ste can differentiate the etiology of left ventricular concentric hypertrophy since it is clinically important. carvedilol, a 3rd generation non-selective beta-blocker with ancillary alpha 1 -blocking and antioxidant properties may have therapeutic implications for multiple diseases in cats; however, pharmacokinetics and bioavailability of commercially prepared oral carvedilol has not been determined. hplc for carvedilol measurement in feline plasma was validated and standardization curves created. the pharmacokinetics (pk) of carvedilol was evaluated in 5 apparently healthy male neutered adult cats (average weight of 5 kg) following single dose intravenous (iv) of 0.5 mg/kg and single dose oral administration of 1.6 to 2.0 mg/kg. concentrations of the active parent compound, carvedilol, were detected in plasma using hplc analysis. lower limit of quantification was 5 ng/ml. the mean peak concentration after iv administration of carvedilol was 8639 ng/ml (range, 901 to 8648), elimination half-life was 2.9 hours (range, 2.0 to 5.4), and clearance was 0.35 l/hr/kg. the volume of distribution was 1.18 l/hr. after a single oral administration of carvedilol, the time to peak plasma concentration was 60 minutes (range, 30 to 90 minutes) and the mean residual time was 4.8 hours. the half life was 4.44 hours. maximal concentration 294 ng/ ml and the mean bioavailability was 15.7% with a median of 9.97% (range, 4.7% to 46%). these data demonstrate a low bioavailability of oral carvedilol and a wide variation in cats. all cats tolerated the oral dose of carvedilol with no major adverse effects. also, a mean residual time of 4.8 hours would suggest that a more frequent dosing schedule may be required to maintain therapeutic plasma levels. pharmacodynamic studies investigating beta-adrenergic blockade duration may provide a more accurate dosing interval of carvedilol. abstract c-49 effects of sildenafil citrate on dogs with ei-senmenger's syndrome. k nakamura, m yamasaki, h ohta, m takiguchi. department of veterinary clinical sciences, graduate school of veterinary medicine, hokkaido university, sapporo, hokkaido, japan. sildenafil has shown to be effective for dogs with pulmonary hypertension; however, its efficacy for dogs with eisenmenger's syndrome (es) and secondary erythrocytosis has not yet been determined. the objective of this study is to determine the effect of sildenafil for dogs with eisenmeger's syndrome and secondary erythrocytosis. this was a prospective, single arm, open-label study. five clinical dogs with es and secondary erythrocytosis were included. new york heart association (nyha) functional class, pcv, and pulmonary artery acceleration time to ejection time ratio (pa at : et) were evaluated before and after sildenafil therapy (0.5 mg/kg, bid). nyha functional class was significantly improved after 1 (median 2; range 1-2, p 5 0.031) and 3 months (median 2; range 1-2, p 5 0.031) of sildenafil therapy, compared with the baseline (median 3, range 2-3). pcv was significantly decreased after 1 month (62.4 ae 4.7%, p 5 0.015) and 3 months (59.9 ae 3.6%, p 5 0.01) of therapy, compared with the baseline (68.0 ae 5.6%). at : et was significantly increased after 1 month of therapy (0.39 ae 0.06, p 5 0.013) from the baseline (0.28 ae 0.04). sildenafil resolved the clinical signs and secondary erythrocytosis in dogs with es. sildenafil therapy could be the treatment of choice for dogs with es. sepsis is the number one cause of mortality in neonatal foals. the role of the raas and hpaa in systemic inflammation and response to stress is well documented in critically ill human neonates, but limited information exists in foals. we hypothesized that in septic foals the raas and hpaa will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality. blood samples were collected on admission from 60 septic (sepsis score 4 12), 102 sick non-septic (sns), and 18 healthy foals of o 7 days of age. blood concentrations of corticotropin-releasing hormone (crh), adrenocorticotropin (acth), cortisol, aldosterone, angiotensin-ii (ang-ii), arginine vasopressin (avp) and plasma renin activity were determined by radioimmunoassays. acth, cortisol, aldosterone, ang-ii and avp concentrations were higher while crh was lower in septic and sns foals compared to healthy foals (p o 0.05). septic non-survivor foals had higher concentrations of aldosterone, cortisol, acth and avp and lower concentrations of ang-ii and crh than survivors. avp was associated with ang-ii in septic, and with acth in septic and sns foals (p o 0.05). there was no difference in renin activity and ang-ii concentrations among foal groups. septic foals had a higher acth:aldosterone ratio than healthy foals (p o 0.001). this study shows that in response to sepsis there is raas and hpaa activation in critically ill foals. we propose that in sick foals avp is more important than crh in regulating acth secretion. the increased acth:aldosterone ratio further supports relative adrenal insufficiency in septic foals. this prospective, cohort study aimed to characterize alterations in coagulation and blood-derived inflammatory biomarkers in adult horses that developed diarrhea during hospitalization. physical and hematological parameters were evaluated at times 0 (onset of diarrhea), 6, 12, 24 and 48h, then every 48 h until resolution of diarrhea or death. each hematological analysis included a complete blood count (cbc), thromboelastography (teg), partial-thromboplastin-time (ptt), prothrombin-time (pt), plasma concentrations of lactate, tumor necrosis factor alpha (tnf-a), interleukin (il)-1, il-6, il-10 and nt-proc-type-natriuretic peptide (pcnp). horses were categorized into three groups based on the duration of diarrhea and evidence of systemic inflammation. group 0: diarrhea o 6 h without systemic inflammation (si); group 1 -diarrhea ! 6 h without si; group 2-diarrhea with si. assessment of vital parameters and cbc established a diagnosis of si as previously described (levy, 2003) . descriptive and univariate outcome analyses were based on data normality. 19 horses were enrolled, of which 16 (84.2%) survived to discharge. the mean age was 13.61/-5.3 years. eight horses (42.1%) were categorized as group-0, 2 (10.5%) as group-1 and 9 (47.4%) as group-2. two horses developed thrombophlebitis. based on the results of teg, 6/19 (31.6%) were normocoagulable, 7/19(36.8%) were hypocoagulable and 6/19 (31.6%) were hypercoagulable, at one or more time points. of these, 7/9 (77.8%) group-2 horses were coagulopathic. additionally, group-2 horses had a significantly lower ma than group-0 horses at baseline (43.6 ae 16.7 vs. 61.9 ae 7.2) and 6h (45.9 ae 15.9 vs. 65.5 ae 5.8). biomarker analyses are pending. in conclusion, si was associated with coagulation disorders in horses with hospital acquired diarrhea. clostridium difficile and clostridium perfringens are commonly associated with colitis and diarrhea in equines but asymptomatic carriers exist. reported carrier rates of toxigenic c. difficile and c. perfringens strains in feces range between 0-25% and 0-30% respectively. toxigenic c. difficile has also been isolated from the small intestine of diseased foals and is implicated as etiologic agent of duodenitis/proximal jejunitis in adult horses however scarce information is available on prevalence in gastrointestinal compartments other than feces in healthy horses, and it is unclear whether fecal samples are good predictors of the status of proximal intestinal sites. the objectives of this study were to investigate the presence of c. difficile and c. perfringens in various intestinal compartments of healthy adult horses and to molecularly characterize isolates. intestinal contents were collected from the stomach, duodenum jejunum, ileum, cecum, right dorsal and left ventral colon, small colon and rectum of 10 euthanized horses free of apparent gastrointestinal disease. enrichment culture was performed for c. difficile and c. perfringens and c. difficile isolates were further characterized via toxin gene detection and ribotyping. c. difficile was isolated from 9/90 (10%) samples from 5/10 (50%) horses. between zero and three sites were positive per horse, and multiple sites were positive in three horses. isolates were recovered from duodenum (n 5 1), right dorsal colon (n 5 3), small colon (n 5 1) and rectum (n 5 4). in one horse, the rectal sample was negative but c. difficile was isolated from a proximal site, all other horses were positive on the rectal sample if a more proximal compartment was positive. in three horses multiple compartments were positive however different strains were always present within the same horse (n 5 2). all isolates possessed genes encoding toxins a and b. five isolates were ribotype 078 and also possessed genes encoding the binary toxin. the other isolates were ribotype 001 and were negative for the genes encoding the binary toxin. despite using a method with a detection level as low as 9 cfu/g of feces, no c. perfringens was recovered. rectal samples were a good predictor of overall c. difficile carrier status (4/5 horses), however rectal samples were not always representative for the ribotype carried in more proximal compartments. the presence of variable strains within the same horse suggests transient passage of the bacterium through the gastrointestinal system rather than actual colonization although further study testing multiple colonies per site is needed. the predominance of ribotype 078 is consistent with recent emergence of this strain in this region, as earlier studies found other strains (027, 001) to be more prevalent and a variety of ribotypes were typically recovered from horses. interestingly ribotype 078 has recently emerged as a hypervirulent strain in humans in our area. clostridium difficile, clostridium perfringens and salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. additionally, antimicrobial resistance is a pressing concern. commensal e.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. the objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for e. coli. fecal samples were collected from 25 adult horses from five farms on a monthly basis over the course of one year. selective cultures were performed for c. difficile, c. perfringens, salmonella and e. coli. c. difficile isolates were characterized via toxin gene pcr and ribotyping. broth microdilution was performed to assess antimicrobial susceptibility profiles of e. coli. clostridium difficile was isolated from 15/275 (5.45%) samples from 11/25 (44%) horses. four horses were positive on more than one occasion, three were positive in two consecutive months. different ribotypes were found in two of the latter horses. most isolates were ribotype 078 (n 5 6) with ribotype 001 (n 5 5) and ribotype c (n 5 4) also identified. ribotypes 078 and c possessed genes encoding toxins a, b and binary toxin, while ribotype 001 only possessed toxin a and b genes. despite a detection threshold of 9 cfu/g feces, c. perfringens was not detected in any samples, nor was salmonella. e coli was isolated from 117/225 (52%) samples. resistance to !1 antimicrobial was present in only 19/117 (16.9%) isolates. multidrug resistance (! 3 antibiotics) was present in 5/117 (4%). most commonly, isolates were resistant to sulfisoxazole (17/ 117) and trimethoprim sulfamethoxazole (16/117). the overall detection rate for toxigenic c. difficile in fecal samples of healthy horses was 5.4% which is consistent with previous studies. the cumulative prevalence of 44% was striking but only one horse shed the same strain for more than one month, indicating c. difficile shedding is a transient and dynamic state. the predominant isolation of ribotype 078 is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. the low prevalence of c. perfringens and salmonella is in agreement with some other studies. the low prevalence of antibiotic resistance in commensal e. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria. type 1 polysaccharide storage myopathy (pssm) in horses is associated with a dominant missense mutation (r309h) in the skeletal muscle glycogen synthase gene (gys1). since disease severity varies between affected horses, we hypothesised that some clinical variability could be accounted for by the underlying genotype. 107 belgian / percheron horses were genotyped using a validated restriction fragment length polymorphism assay enabling grouping of horses as homozygotes (hh), heterozygotes(hr) or normal (rr). subsequently, semimembranosis muscle samples were biopsied from each of six matched sedentary horses from each group; one sample was formalin-fixed and one fresh frozen. sections were stained using haematoxylin and eosin, periodic acid schiff 1/diastase. anti-dystrophin, nnos and myosin heavy chain immunohistochemistry was performed to examine sarcolemmal intregrity, there were significant differences in resting ck activity (p 5 0.023) (median hh 5 364u/l interquartile range(ir) 332-764; hr 5 301u/l ir222-377; rr 5 260u/l ir216-320) and ast activity (p o 0.001) (ast mean hh 5 502u/l sd116; hr 5 357u/l sd92; rr 5 311u/l sd64) and muscle pathology between the 3 groups, with severity increasing rr o hr o hh. there were significantly more type 2a (p 5 0.04) and fewer type 2x fibres (p 5 0.02) in homozygotes (2a 55% sd 10.2; 2x 32% sd 18) compared with the other groups (hr 2a 38% sd 10.9, 2x 44% sd 10.8; rr: 2a 36.5% sd 12.4 2x 57% sd 11.5). more type 2a fibres contained polysaccharide inclusions in homozygotes (30% sd 11.1) than in heterozygotes (10.6% sd 6.9) (p o 0.001). both dystrophin and nnos expression was normally localised to the sarcolemma in pathologically normal and vacuolated fibres from mutant horses. in conclusion, sedentary homozygotes have more severe skeletal muscle pathology and higher resting plasma ck and ast activities than heterozygotes, and pssm1 is associated with a fibre type shift towards type 2a. although subsarcolemmal vacuolation likely disrupts the contractile apparatus's attachment to the sarcolemma, the latter's integrity appeared intact. the recumbent horse presents a logistic, diagnostic, and therapeutic challenge to the equine practitioner. there is currently very little data available on the prognosis and outcome of horses that are recumbent. therefore, the purpose of this study was to investigate the outcome of hospitalized horses that had been recumbent in the field or in the hospital and the factors affecting their survival. records of horses admitted to the school of veterinary medicine, university of california davis from january of 1995 to december of 2010 with a history of recumbency or horses that became recumbent while hospitalized were evaluated. a horse was defined as recumbent if it was unable to stand on its own. the medical record was examined for the following criteria: history pertaining to the current illness including treatment by the rdvm, breed, age, weight, date of presentation, physical and neurological examination findings, cbc and biochemical profile results, initial drugs administered on arrival, time spent recumbent, time spent in a sling, diagnosis, and hospitalization costs. statistical analysis correlating factors associated with survival was performed using logistic regression. overall there were 112 non survivors and 49 survivors. factors that favored survival included early initiation of treatment in the field by the rdvm, horses that tolerated a sling and spent more time in a sling, increased duration and costs of hospitalization, horses that were recumbent post anesthesia, and those recumbent due to disease of the musculoskeletal system. factors that increased likelihood of non survival included horses that were ataxic on presentation, horses with increased bun, horses that spent more time recumbent, those that did not tolerate a sling, and horses diagnosed with botulism and spinal cord disease. in conclusion, this retrospective study demonstrated that both the cause of recumbency and the ability of horses to tolerate a sling had a direct effect on survival. abstract e-7 plasma peak and trough gentamicin concentra-tions in hospitalized horses receiving once daily gentamicin. jr read 1 , pa wilkins 2 , rd nolen-walston 1 . 1 university of pennsylvania, new bolton center, kennett square, pa. 2 university of illinois, champaign-urbana, il. gentamicin is often used to provide gram negative antimicrobial coverage in horses at 6.6 mg/kg iv every 24 hours. therapeutic drug monitoring in our hospital suggests larger doses are required in many clinical cases to achieve the desired concentration (8-10â minimum inhibitory concentration) for common bacterial isolates (peak target range 32-40 mg/ml). the aim of this study was to determine the correlation between gentamicin dose and plasma concentration in hospitalized horses receiving gentamicin treatment in order to identify an optimum dose range for this population. review of records (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) identified 71 horses ! 3 months old receiving once-daily gentamicin with peak and trough assays performed (n 5 107 sets). spearman rank correlation coefficient analysis revealed a weak (r 5 0.289) but statistically significant correlation (p 5 0.003) between gentamicin dose and peak plasma concentration. horses receiving 7.7-9.7 and 4 9.7 mg/kg gentamicin (groups 2 and 3) had higher median peaks (31 mg/ml) than horses receiving 5.6-7.6 mg/kg (group 1; 25.7 mg/ml). higher doses were more likely to result in peaks 4 32 mg/ml (42 and 41%, groups 2 and 3 respectively) than horses receiving 5.6-7.6 mg/kg (group 1; 16%). all 22 hour post-gentamicin administration trough values were o 2 mg/ml. no correlation was found between dose and change in plasma creatinine during treatment, nor dose and trough level. these data suggest that gentamicin dosage in horses should be individually determined by therapeutic monitoring. additionally, these data support an initial dose of 7.7-9.7 mg/kg iv every 24 hours in order to achieve desired peak concentration and an appropriately low trough concentration. heaves is a common respiratory inflammatory disease, characterized by a pulmonary neutrophilia. this disease is also characterized by an activation of circulating neutrophils after antigen challenge but their specific role in heaves is not well understood. also, there are anecdotal studies concerning heaves-affected horse to be more susceptible to infection. however, to our knowledge, the antibacterial host defense role mediated by circulating neutrophils was not investigated in heaves-affected horses. the objective of this study was to compare phagocytosis activity and bacterial killing by circulating blood neutrophils of heaves-affected and control horses. peripheral neutrophils were isolated from heaves-affected (n 5 6) and control (n 5 6) horses using a density gradient technique. the killing capacity was assessed by incubating neutrophils with streptococcus equi spp equi and spp zooepidemicus. after 1 h of bacterianeutrophil coculture, total viable bacterial cells were measured by quantitative plating. the phagocytosis was evaluated by flow cytometry using fluorescent beads and gfp-transformed streptococcus suis suilysin-negative mutant strain. circulating neutrophils from heaves-affected horses showed a significant decrease in their killing capacity toward s. zooepidemicus (p 5 0.046). a reduced, although not significant (p 5 0.1), killing capacity of s. equi by these neutrophils was also observed. the phagocytosis activity was not different between groups. this impairment of blood neutrophil bactericidal activity in heaves-affected horses could contribute to an increase susceptibility to infection. obesity is a common disorder of the horse, with current prevalence estimated at 30%. in people, obesity is associated with dyslipidemia, insulin resistance, mitochondrial dysfunction and downregulation of lipid and glucose metabolic pathways. in the horse, obesity is similarly associated with insulin resistance and alterations in lipid profiles; however, metabolic regulatory gene expression profiles have not been fully characterized. we hypothesized that obese horses have decreased expression of metabolic regulatory genes and decreased mitochondrial content in skeletal muscle compared with non-obese horses. sixteen light breed horses, 2-27 years of age were included. body condition score (n 5 16) and neck circumference (n 5 9) were recorded. post-mortem biopsy samples of the semi-membranosus muscle were obtained. dna and rna were isolated. relative expression of the metabolic genes, peroxisome proliferator activated receptor g (pparg), pparg coactivator-1a (pgc-1a), fatty acid translocase (fat) and estrogen related receptor a (erra) was determined by quantitative polymerase chain reaction (qpcr). mitochondrial content was assessed by determining mitochondrial dna/nuclear dna ratio by qpcr, using nadh-dehydrogenase subunit 2 and cytochrome oxidase subunit 2 as mitochondrial genes and beta actin as the nuclear reference gene. non-normal data was log transformed for analysis and a pearson coefficient of correlation was calculated for relative gene expression, body condition score and neck circumference. a value of p o 0.05 was considered significant. body condition score was strongly correlated with neck circumference (n 5 9, r 5 0.72, p 5 0.03). relative expression of erra and glut-4 increased with body condition score (erra: n 5 16, r 5 0.66, p 5 0.005; glut-4: n 5 16, r 5 0.50, p 5 0.05). copy number of the mitochondrial genes (nadh-dh and cox-2) was not related to body condition score or metabolic gene expression. expression of glut-4, erra, pparg, and fat were strongly correlated to each other, but not pgc-1a. there was a strong trend towards correlation between pparg and pgc-1a in horses with body condition score 4 6 (n 5 6, r 5 0.77, p 5 0.07). in this study, there was no change in mitochondrial content in obese horses. assessment of mitochondrial function in obese horses and horses with ems is under way. the strong correlation between pparg and pgc-1a observed only in horses with high body condition scores suggests this pathway is activated with obesity. the role of pparg and pgc-1a in equine obesity should be further investigated to determine their potential as therapeutic targets. upregulation of erra and glut-4 in horses with increasing body condition score is unexpected, and may indicate a compensatory response to dysfunction of a downstream pathway. further studies to better define the role of metabolic regulatory gene expression in obese horses and those with ems are ongoing. previously presented at the 7th annual harold hamm diabetes center research retreat, oklahoma city, ok. inflammatory bowel disease is a cause of weight loss, decreased performance, and colic in horses. this condition is difficult to diagnose and clinicians rely upon absorption tests to document malabsorption. the purpose of this study was to compare glucose and xylose absorption tests in normal horses and determine the repeatability of these procedures. eight horses received 500 mg/kg dextrose or d-xylose powder mixed as a 10% solution in water or water alone via nasogastric intubation on three different occasions within the same week for three consecutive weeks (9 tests/horse). a crossover design was employed and the order of treatments was randomized. blood samples were collected at time 5 0, 30, 60, 90, 120, 150, and 180 min. data were analyzed by repeated measures anova and t-tests. results showed that the xylose response over time differed significantly from the glucose response over time (test â time; p o 0.001). mean time to maximum concentration differed (p o 0.001) between tests (glucose 90 min; xylose 60 min). within-horse area under the curve, maximum concentration, and time to maximum concentration values for dextrose and xylose did not differ significantly when tests were repeated. results indicate that glucose and xylose absorption tests are repeatable within the same horse, but plotted curves differ between tests, with peak concentrations occurring at a later time point for the glucose absorption test. we conclude that both tests provide repeatable measures of intestinal absorption, but glucose and xylose appear to differ in their rates of absorption and clearance. the purpose of this study was to examine the records of a population of thoroughbreds with cervical vertebral malformation (cvm) and to determine which factors have an effect on these horses achieving athletic function. this was a retrospective case study of 119 thoroughbreds with cvm treated medically from 2002 to 2010. forty-one were euthanized after diagnosis, while the remaining 78 were discharged for treatment. racing records were reviewed to determine which horses raced after treatment. horses were separated into groups based on whether or not they raced. medical records were reviewed, and results of neurologic examination, radiographic and laboratory findings, treatments, and outcome were assessed and compared between groups. twenty-one of 78 horses treated medically (27%) improved enough to race. median neurologic grade between groups was significantly different (p o 0.0001), with a hind limb grade of 2.0 (range 1-3) for the raced group and 2.5 (range 0.5-4) for the unraced. intravertebral sagittal ratios measured from standing lateral cervical radiographs were equivocal between groups. radiographs of all horses were examined for kyphosis, dorsal over-riding arch, caudal epiphysitis, degenerative joint disease, cystic bone lesions, and cranial stenosis of the vertebral canal. horses with kyphosis (p 5 0.0178), degenerative joint disease (p 5 0.0497), or cranial stenosis (p 5 0.0357) at any site were less likely to return to racing. racing prognosis for horses with cvm treated conservatively is equivalent to that of those treated surgically as reported by rush moore et al (javma, 1993) . radiographic changes and neurologic grade may help serve as indicators for whether a horse will respond to conservative therapy. since pain assessment is vital for management of colic, a valid, reliable and feasible tool for assessing the severity of acute abdominal pain in horses is urgently needed. our aim was to construct and validate a behavior-based pain scale by methodology utilized in construction of pain scales in non-verbal humans. the project consisted of four stages. firstly, behaviors to include in a scale were empirically identified. thirty equine clinicians noted behaviors in each of 23 random film clips of horses with colic using a checklist. nine behaviors (e.g. rolling, pawing, and flank watching) demonstrated good inter-observer agreement without bias (multi-rater kappas: 0.5-0.95). secondly, the clinical judgment of experts was utilized to identify and to weight behaviors. six expert clinicians independently expressed opinions as to which of 46 behaviors to include and the severity of pain they indicate. two contending scales (equine acute abdominal pain scales (eaaps) 1 & 2) were constructed based on both the empirical and the judgmental approaches. each included 12 identical behaviors with a 1-5 point score range; eaaps-2 with gradations to some of the behaviors and eaaps-1 without. in the third stage, blood cortisol and lactate levels and heart rate were shown to only approximate pain since they correlate poorly with degree of pain as assessed by visual analog scale (vas) in 32 horses with colic and 8 controls (spearman rho; lactate 0.359; cortisol 0.214; heart rate 0.261). finally, reliability and validity of the pain scales were evaluated including constructs of pain; face validity, convergent and discriminate validity and extreme groups. thirty of 40 films of horses with colic were randomly presented to 44 expert equine clinicians internationally who were randomly allocated into three groups to score pain; one group by both vas and numerical rating scale (nrs)), and two groups, each by one of the two eaaps scales. inter-observer reliability of both eaaps scales was excellent (intraclass correlation 0.8). intra-observer reliability based on scores given for identical films demonstrated; 87% and 56% agreement, kappa 0.9 and 0.35, and spearman's rho 5 0.97 and 0.58 for eaaps-1 and 2, respectively. both scales varied by 1 score between observations. face validity; each group reported their scale to be valid (67% & 81%). convergent validity; the scales compared favorably with vas/nrs scores (spearman's rho: 0.84-0.89). discriminate validity; correlation to heart rate, lactate and cortisol levels was predictably low (rho 5 0.2-0.5). extreme group validity; colic horses scored significantly higher than control horses; scores of 0.6-0.7 in controls versus 2.6-2.7 in cases. in conclusion, methodology established in human medicine but novel in veterinary medicine was used to construct and validate two clinically feasible equine abdominal pain severity scales that showed excellent reliability and validity. further refinement of the eaaps scale is advised prior to introduction into clinical practice. aortic valve prolapse (avp) is a common echocardiographic finding in horses, but when compared with mitral valve prolapse in dogs, little is known about the natural progression of this condition. previously published data has shown that echocardiographic identification of avp in horses is reliable, diagnostic criteria have been established and that development occurs with training. the aims of this study were to evaluate the different rna and protein expressions of smooth muscle actin (sma), transforming growth factor-b 1 (tgf), nitric oxide synthase (nos) and the concentrations of elastin and collagen in normal, prolapsing and diseased cusps to evaluate what structural changes may predispose them to prolapse. valve cusps were harvested and processed from a group of 176 horses at a commercial abattoir following disease classification using echocardiography. horses were aged 13.7 ae 6.7years, weighing 518 ae 51 kg and with a median body condition score of 4/9. cusps were collected in rnalater s and stored at à801c prior to processing. cdna was produced from half a valve using a standard protocoland qrt-pcr performed to assess relative rna expression of sma, tgfb 1 , endothelial (enos) and inducible nos (inos) and compared with the housekeeping gene 18s. a quarter of cusp was processed using an adapted commercial protocol to evaluate protein expression of sma, tgf b 1 , enos and compared to vimentin. specific antibody binding was assessed with western blotting and protein expression evaluated using dot blots. the remaining quarter cusp was used to measure soluble collagen and elastin concentrations using commercial assays 3 . statistical analyses included one way anova with post-hoc bonferoni, paired student's t-test, linear and logistic regression. there was no effect of gender or age on any of the measurements. valves from animals with avp had lower expression of sma and elastin compared to normal and diseased valves, increased expression of tgfb 1 and enos, whereas inos expression was greater than normal valves (table 1) . collagen content of valves from horses with avp was increased compared to normal but lower than horses with valve disease. prolapsing cusps appear to be a different phenotype from diseased cusps. further studies will help to elucidate the significance of these findings in vivo. a clear association between heart rate (hr) and body mass has been observed across a wide range of mammalian species. furthermore, it is well known that electrocardiographic (ecg) time intervals vary with heart rate and body mass. within the equine species, small breeds are generally thought to have higher heart rates than large breeds. however, despite the large differences in size among different equine breeds, there is little information about normal heart rates and normal ecg time intervals in horses and ponies of different body size. similarly, the relationship between hr and body mass in dogs of various breeds and sizes is still under debate. the goal of this study was to investigate the relationship between heart rate and ecg time intervals to body mass in apparently healthy horses and ponies and to calculate normal ranges for different weight groups. 250 adult horses and ponies at an age of 5.5 (1-30) y [median (range)] and a body weight of 479 (46-1120) kg were included in the study. all animals were considered clinically healthy based on history and physical examination. a standard base-apex ecg was recorded at a speed of 25 (n 5 4) or 50 mm/s (n 5 246) using a multiparameter monitor (datascope passport). during the procedure, the horses were unsedated, standing quiet in a box stall. mean hr over 15 sec was determined for each recording. the following ecg time intervals were measured in triplicate and averaged for further analyses: pq interval, qrs duration, qt interval, and difference between qt and qrs (qt-qrs duration). the relationship between hr, ecg time intervals, and body mass was assessed using linear regression analyses. normal ranges (2.5% to 97.5% percentile) were calculated for 5 different weight groups. the level of significance was p 5 0.05. heart rate was inversely related to body mass (p o 0.0001, r 2 5 0.122). the pq interval (p o 0.0001, r 2 5 0.413), qrs duration (p o 0.0001, r 2 5 0.147), qt interval (p o 0.0001, r 2 5 0.089), and qt-qrs duration (p o 0.0001, r 2 5 0.028) were directly related to body mass. normal ranges for hr, pq, qrs, and qt within the different weight groups were 36-64 bpm, 107-230 ms, 60-127 ms, 360-510 ms (o200 kg); 28-68 bpm, 162-319 ms, 74-155 ms, 362-610 ms (200-399 kg); 28-60 bpm, 211-423 ms, 89-147 ms, 390-581 ms (400-599 kg); 28-54 bpm, 220-380 ms, 87-150 ms, 367-587 ms (600-799 kg); and 24-52 bpm, 240-463 ms, 87-140 ms, 437-533 ms (4 799 kg). we conclude that in healthy horses there is a significant but weak relationship between body mass and hr and between body mass and ecg time intervals, respectively. this study therefore supports the hypothesis that within the equine species, small breeds have faster heart rates and shorter ecg time intervals than large breeds. therefore, body mass has to be considered when comparing hr and ecg time intervals to normal ranges in horses. horses with pituitary pars intermedia dysfunction (ppid) often have elevated plasma acth concentrations. however, ppidaffected horses rarely have resting serum cortisol levels above the reference range or adrenal hyperplasia. we hypothesized that this apparent dissociation between plasma acth levels and adrenal response in horses with ppid is due to the secretion of acth that is less biologically active than that from normal horses. to test our hypothesis, a bioassay to evaluate acth activity was developed. adrenocortical explants were harvested aseptically from normal horses at euthanasia and stimulated with plasma from healthy (n 5 9) and ppid-affected horses (n 5 11). the assay was performed three times with explants obtained from different horses. cortisol secreted by the explants and plasma acth levels were measured with commercially available radioimmunoassays. cortisol secretion stimulated by each sample was standardized to the respective explant protein concentration. cortisol data was normalized for acth concentration in each plasma sample and expressed as a cortisol:protein:acth ratio. ratios from horses with ppid and normal horses were compared by unpaired t-test. horses with ppid had significantly lower cortisol:protein:acth ratios compared to normal horses. (assay 1: 0.06 ae 0.04 vs. 0.33 ae 0.11, p o 0.001; assay 2: 0.06 ae 0.04 vs. 0.30 ae 0.10, p o 0.001; assay 3: 0.07 ae 0.06 vs. 0.20 ae 0.13, p o 0.01). these results suggest that plasma acth from ppid horses is less biologically active than plasma acth from normal horses. our findings give further insight into the pathophysiology of ppid and may aid in the development of novel diagnostic testing protocols. an online survey was conducted to determine perceived needs of potential employers of new acvim-laim diplomates. the survey was designed as the first step in determining what is needed for success in the various sectors of practice employing acvim-laim diplomates. demographic and background data were collected using questions and drop-down menus on the first page. the survey evaluated 189 skills or concepts in 26 areas of veterinary practice. participants answered 4 questions about each skill or concept using drop-down ranked lists. those participants that had completed an acvim-laim training program were asked 3 additional questions about whether they were taught the skill or concept during their own residency. data were collated and descriptive statistics calculated. the mean scores or frequencies of use for each skills or concepts were ranked to determine which of the skills or concepts were most important for an entry-level diplomate. eighty-eight individuals participated in the survey with 86 respondents being acvim diplomates, 1 respondent was not board-certified and 1 respondent was an act diplomate. nineteen respondents were diplomates of acvim and an additional specialty. eighty-three respondents had completed an acvim residency. the majority of respondents were in academia (65%) with 30% being in private practice. equine specialists prevailed (53%) followed by mixed large animal (28%) and then food animal only specialists (13%). the distribution of years post-residency was slightly skewed toward younger diplomates, but overall there was a good distribution of diplomates across years of experience. most respondents stated that they did not make hiring decisions in their practice. competency in disciplines other than internal medicine was expected with ultrasonography and radiology being the most desirable followed by theriogenology and lameness. surgical skills, both equine abdominal (10%) and food animal general surgery (11%) were considered important by some respondents. thirty-six per cent of respondents thought that a new diplomate should expect to make o $50,000 per annum, while only 13% of respondents thought that a new diplomate should expect to make ! $90,000 per annum. not all respondents answered questions on all skills or concepts. the mean number of skills or concepts evaluated was 86 (sd 5 75) with only 18 respondents answering all 189. all skills or concepts evaluated were found to be at least somewhat important, were estimated to be used at least occasionally, were recommended for inclusion in training programs as core or elective, and some level of knowledge was expected. at least some of the respondents were taught each of the skills or concepts during their residency, practiced the skill or concept at least occasionally during their residency, and some degree of competency was expected at the time of completion of their residency. these data will provide a framework for designing future laim residency programs. abstract this study evaluated pharmacokinetics and clinical safety of an oral paste formulation of a commercially available cox1-sparing nsaid in clinically healthy pony foals in a randomized controlled clinical trial. values for complete blood count, serum chemistry profile, urinalysis, pharmacokinetic assay, and gastric endoscopy were evaluated in eighteen shetland pony foals treated with firocoxib (0.1 mg/kg, po, q 24 h) or placebo for 14 days. foals were divided into 3 treatment groups. group 1 and 2 foals received firocoxib while a 3rd group was administered an oral placebo. gastric endoscopy was performed on group 1 and 3 foals prior to treatment and on days 7 and 14 to monitor for the presence of gastric ulcers. group 2 and 3 foals had blood and urine samples taken sequentially for pharmacokinetic analysis, cbc, serum chemistry evaluation, and urinalysis. physical examinations were performed prior to treatment and daily for 17 days. data were analyzed using anova and paired t-tests (p o 0.05). none of the foals presented adverse clinical effects. there were no significant changes in cbc, biochemical profiles within groups, or differences between groups. pretreatment gastric endoscopy scores were not significantly different from evaluations at 7 and 14 days. firocoxib was quickly absorbed with an observed maximum concentration at 2 hr, the first sampling interval, for the majority of animals. firocoxib plasma concentrations decreased in a log-linear manner after reaching the maximum concentration and steady state concentrations were achieved by the 7th dose. based on the sampling times after the final and 14th dose, an average half life of 1.3 days was estimated. administration of firocoxib did not cause any adverse effects on gastrointestinal, or hematological or serum biochemical variables, appears to have been well tolerated, and follows a predictable pharmacokinetic pattern in 4-6 week old foals. equine herpesvirus 1 (ehv-1) is highly prevalent in most horse populations. horses are routinely vaccinated against ehv-1, and neutralizing antibodies have helped to prevent disease. however, the usda has recently classified ehv-1 myeloencephalopathy (ehm) as an emerging disease, in response to the apparent increase in incidence, morbidity, and mortality of ehm that suggests a change in virulence of the virus. it has been reported that cellular immune mechanisms, in particular cytotoxic t-cells (ctls), are important in controlling ehv-1 viremia. interferon-alpha (ifn-a) has a key function in innate immune regulation by inducing the differentiation and maturation of ctls. here, we investigated the influence of abortogenic (racl11, ny03) and neuropathogenic (ab4) ehv-1 virus strains on ifn-a, il-4 and il-10 secretion in equine pbmc. equine pbmc were infected with racl11, ny03 or ab4 ehv-1 strains or kept in medium for 24 hours. ifn-a, il-10 and il-4 secretion was detected in the supernatants by a fluorescent bead-based cytokine assay. the production of ifn-a increased with increasing viral doses and similarly for all three ehv-1 strains. the production of the antiinflammatory cytokine il-10 was significantly decreased after ab4 infection compared to racl11 and ny03 strains at viral infection doses of moi 0.3-1. at high doses (moi 3), il-10 production was suppressed by all three ehv-1 strains. the results suggested that abortogenic and neuropathogenic ehv-1 strains equally induce antiviral ifn-a production in equine pbmc. they also illustrated the differences in the ability of ehv-1 strains to modulate anti-inflammatory il-10. neuropathogenic ab4 strain had an increased potential to down-regulate il-10 production suggesting specific viral mechanisms that interfere with the control of inflammation in the host. the variations in innate il-10 secretion might influence the development of protective immunity and might offer an explanation why neuropathogenic ab4 induces more severe disease, including myeloencephalopathy, than abortogenic ehv-1 strains. previously presented at a conference of research workers in animal disease. rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. treatment protocols are long, expensive and do not always produce good results. rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. recent studies are based on the use of the virulence associated protein a (vapa) which has been described as an important inducer of immunity against r. equi. the present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of s. enterica typhimurium expressing vapa antigen (test group) or s. enterica typhimurium without the vapa gene (control group), previous to and following experimental challenge. two experimental phases were established according to the immunization route: intranasal or oral vaccination up to 12 hrs following birth and at 14 days of age. the experimental and control groups were challenged on day 28 with a virulent stain of r. equi. clinical examination, cbc and image complementary exams were used to evaluate the development of clinical signs. immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation assays, isolation of r. equi and cytological profiles of tbw. clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group (2/3) and was due to r. equi pneumonia. the test group produced a more intense iggb response when compared to controls however no statistical difference was observed. lymphoproliferation and th1 cytokine expression were higher in the test group. in contrast, controls produced an il-4 response. local iga was significantly higher in animals immunized with salmonella carrying vapa. immunization protocols produced no severe toxic effects. the vaccination of neonatal foals with s. enterica typhimurium expressing vapa was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental r.equi infection. this study was conducted to test the hypothesis that the 32 kd protein, myristolated alanine-rich c-kinase substrate (marcks), is involved in equine neutrophil migration and adhesion. in other species, marcks phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. to investigate marcks phosphorylation in horses, neutrophils were isolated from whole blood and stimulated with platelet activating factor (paf), leukotriene b 4 (ltb 4 ) or phorbol myristate acetate (pma). western blot was performed using specific phospho-marcks and total marcks primary antibodies. these results determined marcks phosphorylation is maximal 30 seconds following stimulation and that dephosphorylation occurs within 3 minutes. to investigate the requirement for marcks in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with mans (a cell permeant peptide identical to the n-terminal 24 amino acids of marcks), rns (a control peptide) or vehicle control (vc) prior to a migration assay toward known neutrophil chemoattractants (ltb 4 or paf). pre-treatment of equine neutrophils with mans significantly inhibited migration while rns pre-treatment had no effect. to investigate marcks requirement in equine neutrophil adhesion, mans, rns or vc treated cells were stimulated to adhere to immulon 2 plates coated with 5% fbs. pre-treatment of equine neutrophils with mans significantly inhibited adhesion while rns pre-treatment had no effect. inhibition of marcks using a cell permeant peptide identical to the protein's n-terminus significantly inhibited equine neutrophil adhesion and migration. these results indicate that marcks is an important regulator of equine neutrophil chemotaxis and represents a potential target for anti-inflammatory therapy. amongst other tests, a thorough neurologic examination of horses may include walking with the head elevated and during blindfolding, in order to help differentiate normal from abnormal and to help with neuroanatomically localising any lesion(s) i.e. in the ataxic horse. consensus amongst equine neurologists suggests that gait abnormalities associated with these specific tests are often exacerbated in horses with underlying proprioceptive deficits however the effect of these tests on temporal gait characteristics in normal horses has not previously been assessed quantitatively. we hypothesized that head elevation or blindfolding, in comparison with walking in a straight line would result in a compensatory decrease in lateral (left front-on to left hind-on and right front-on to right hind-on) and diagonal coupling intervals (left front-on to right hind-on and right front-on to left hind-on) in normal horses. four thoroughbreds without any history or clinical signs suggestive of neurological disease (age range 3 to 5 years) were included in the study. retroreflective markers were applied to the withers, to the sacrum and to left and right tuber coxae; for each limb, lateral fetlock markers and dorsal and lateral hoof wall markers were used. a minimum of 3 trials each with 2-4 walk strides for each task were analysed as horses walked across an 8-force-plate runway i surrounded by a 12-camera kinematic system. ii force-plate data were processed with semi-automated custom written matlab iii scripts. data were analysed with a mixed model using the statistical software r. there was a significant fixed effect of normal walk on a straight line and head elevation on left and right lateral coupling intervals (p o 0.0001) and of the left and right diagonal coupling intervals (p o 0.0001). there was no significant effect of blindfolding on neither lateral nor diagonal coupling intervals. the random effect of horse had no influence on the coupling intervals. the decrease of the lateral coupling intervals indicates a tendency towards a pacing gait during head elevation. we conclude that there is a significant change in temporal gait characteristics of non-neurologic horses when the head is elevated but not during blindfolding compared to normal walking. current results suggest that pacing and increased variation in foot-placement during head elevation should be interpreted with caution however further work is required to determine whether the change differs between horses with neurological disease and non-neurologic disease. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder associated with a mutation in cyclophillin b that leads to impaired collagen folding, aberrant wound repair, and corneal abnormalities. it affects young quarter horses, appaloosa, and paints. herda shows similarities to the human hereditary connective tissue syndrome ehlers danlos (eds). many eds patients suffer from joint pain and osteoarthritis (oa) as adults. the similarity between eds and herda raises the question whether horses suffering from herda develop oa. in oa, excess production of inflammatory mediators such as prostaglandin e2 (pge 2 ) activate enzymes that degrade cartilage as well as impede wound healing. the present study examined articular cartilage from yearling horses afflicted with herda. we hypothesized that chondrocytes from these horses are continually activated to produce inflammatory mediators. to test this hypothesis, articular cartilage from carpal and tarsal joints of herda horses were evaluated using histology. pge 2 production by chondrocyte cultures was measured by elisa and analyzed by one-way anova, tukey post-hoc test, p o 0.05 significance. we also determined the antiinflammatory effects of an avocado/soybean unsaponifiables (asu), glucosamine (glu), and chondroitin sulfate (cs) mixture (ingredients found in cosequin s asu) and phenylbutazone (pbz) on chondrocytes. cosequin s asu and pbz are used alone or in combination for the management of oa. chondrocyte cultures were incubated for 24 hrs with control media alone, a clinically relevant concentration of pbz (4 mg/ml), or the combination of asu (nmx1000 s , 8.3 mg/ml) 1 glu (fchg49 s , 11mg/ml) 1 cs (trh122 s , 20 mg/ml). articular cartilage from joints of five herda-afflicted horses showed gross and histologic evidence of osteoarthritic lesions. chondrocyte cultures from cartilage of horses suffering from herda spontaneously produced greater pge 2 than chondrocytes from normal horses (41000-fold). pbz significantly decreased pge 2 production by $90% (p o 0.001). the combination of asu1-glu1cs also significantly reduced pge 2 production by $60% (p o 0.001). the present study supports anecdotal findings that horses suffering from herda are likely to develop oa. the inhibition of pge 2 synthesis by asu1glu1cs suggests that this combination may be beneficial for the management of oa in herda. research supported by nutramax laboratories, inc. equine inflammatory airway disease (iad) is a common condition often treated empirically with corticosteroids. gene expression analysis in the bronchoalveolar lavage fluid (balf) may help understand the effects of corticosteroids in iad. the first part of the study aimed at identifying reference genes in the balf of iad horses treated with corticosteroids. the second part of the study investigated the effects of dexamethasone and fluticasone propionate treatments on the mrna expression of il-1b, il-4, il-8 and il-17. the expression stability of seven candidate reference genes was determined in balf taken pre-and post-treatment with dexamethasone and fluticasone propionate in horses with iad. primers' efficiencies were calculated using linregpcr. normfinder, genorm and qbaseplus softwares were used to rank the genes according to their stability. gapdh was the most stably expressed gene whereas b2m was the least stable gene. in addition, genorm analysis revealed that the number of genes required for optimal normalization was four (gapdh, sdha, hprt, rpl32). in the second part of the study the mrna expression of il-1b, il-4, il-8 and il-17 was measured in balf samples from seven iad horses treated in a randomized cross-over design with dexamethasone (0.05 mg/kg sid, 15 days) or inhaled fluticasone propionate (3000 mcg bid with aerohippus, 15 days). the balf samples were taken at baseline and after each treatment period. there was no significant effect of the corticosteroids treatment on the mrna expression of il-1b, il-4 and il-8 in the balf. the mrna expression of il-17 was suppressed by dexamethasone and fluticasone propionate treatments. pneumonia is observed in horses after long distance transportation in association with confinement of horses' head position leading to a reduction in tracheal mucociliary clearance time (tmct). we hypothesize that clenbuterol, a beta-2 agonist shown to increase tmct in the horse, will ameliorate the affect of a fixed head position on large airway contamination and inflammation in a long-distance shipping model. six adult horses were enrolled in a cross-over design prospective study. horses were housed with their heads in a fixed position for 48 hours to simulate long distance transport, and treated with clenbuterol (0.8 ug/kg po q12 h) or a placebo starting 12 hours before simulated shipping. tmct was measured using a charcoal clearance technique. data were collected at baseline and 48 hours, and included tmct, tracheal wash cytology and quantitative culture, rectal temperature, cbc, fibrinogen, and serum tnfa, il-10 and il-2 levels. there was a 3-week washout between study arms, and each horse served as its own control. the data was analyzed using regression analysis and wilcoxon rank-sum tests. no statistically significant difference was seen between treatment and placebo groups for any of the variables investigated. tmct did not differ after treatment (1.71 ae 0.64 cm/min) versus placebo (1.55 ae 0.82 cm/ min; p 4 0.10), and intratracheal bacterial counts were similar for treatment (105 â 10 3 ae 42 â 103 cfu; p 4 0.10) and placebo (98 â 10 3 ae 41 â 103 cfu) groups. a reduction of tracheal b hemolytic streptococcus. spp. after clenbuterol versus placebo was also nonsignificant (0% versus 33%; p 4 0.10). in conclusion, treatment with clenbuterol does not appear to combat the deleterious effects of this long-term shipping model. breathing cold air during strenuous exercise is associated with airway inflammation. under these conditions, warming and humidification of inspired air occurs in the lower respiratory tract resulting in mucosal cooling, desiccation, and hyperosmolarity. the purpose of this research was to test the hypothesis that airway hypertonicity causes inflammatory cell migration and alterations in cytokine expression associated with exercise induced airway inflammation. horses (n 5 9) were examined in a randomized crossover design after exposure to hypertonic aerosols (5 minute nebulization with solutions of either isotonic or hypertonic mannitol). airway leukocytes were harvested 5 and 24 hours post aerosol challenge via bronchoaveolar lavage, and were used to determine total and differential nucleated cell count and expression of cytokinespecific mrna. hypertonic aerosol challenge resulted in an increase in total number of cells 5 hr after challenge, characterized by increased macrophage (p 5 0.04) and neutrophil (p 5 0.03) concentrations, but there was no effect on airway leukocyte concentrations 24 hours after nebulization. no significant changes in the relative quantity of mrna for airway cytokines were noted at either time point. these data demonstrate that transient airway hypertonicity can cause airway leukocyte influx and may be responsible for the airway inflammation commonly found in athletes that exercise in cold conditions. however, our data do not support the hypothesis that hypertonicity is the sole initiating cause of changes in cytokine expression secondary to cold weather exercise. it is likely that factors such as airway temperature, shear stress or epithelial damage also play a role in this phenomenon. we studied the importance of abdominal sonograms in neonatal foals suffering from gastrointestinal conditions. we hypothesized that there would be a subgroup of neonates with sonographically detectable pneumatosis intestinalis (pi) as a reflection of a necrotizing component of the disease. records of foals 7 days of age hospitalized between 2005 and 2009 with signs of gastrointestinal disease were evaluated (n 5 89). the association of sonographic, clinical, pathological and clinicopathological signs with outcome and severity of disease was determined. pneumatosis intestinalis was imaged in 19 foals. twenty-seven foals were classified as having necrotizing gastrointestinal disease based on the presence of gastrointestinal signs (colic, diarrhea, gastric reflux or abdominal distension) and pi detected sonographically (19), surgical (2) or pathological (6) evidence of gastrointestinal necrosis. there was a difference between overall survival rate (58%) and survival rate in foals with necrotizing disease (33%, p 5 0.005) or foals with pi detected sonographically (37%, p 5 0.02). pneumatosis intestinalis was the only sonographic finding associated with outcome. sonographic abnormalities in peritoneal fluid, stomach, duodenum, jejunum, cecum, umbilicus or the presence of meconium were associated (p o 0.05) with surrogates of severity of disease (hospitalization cost or days of hospitalization). hypoproteinemia was associated with pi (p 5 0.02). the presence of blood in the feces, reflux and abdominal distension was associated with necrotizing gastrointestinal disease (p o 0.05). abdominal sonograms have prognostic value in neonatal gastrointestinal disease. pneumatosis intestinalis was a common sonographic sign that worsened the prognosis. the therapeutic implications of detecting a necrotizing component of the gastrointestinal disease deserve further study. the interaction of insulin and the microvascular endothelial insulin receptor (irc) plays an important role in the normal and insulin resistant (ir) individual. while endothelial irc signaling is normally vasodilatory, this effect is well-documented to reverse in the ir individual, resulting in vasoconstriction. although vascular dysfunction has been reported in sepsis-associated equine laminitis, the role of the laminar microvasculature in endocrinopathic laminitis remains poorly characterized. the purpose of this study was to characterize the pattern of irc expression in digital laminae in ponies subjected to a dietary carbohydrate challenge that mimicked abrupt exposure to pasture rich in nonstructural carbohydrates (nsc). mixed-breed ponies (body weight 270.9 1/-74.4 kg) received a diet of hay chop (nsc $6% on a dm basis) for 4 weeks prior to initiation of the experimental feeding protocol. following conditioning, ponies either remained on the control diet (n 5 11) or received the same diet supplemented with sweet feed and oligofructose (total diet $42% nsc; n 5 11) for a period of 7 days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including dorsal digital laminae, were collected immediately following euthanasia. the samples were formalin-fixed for 48 hours, transferred to 70% ethanol, and paraffin-embedded. laminar sections were stained immunohistochemically for irc using a commercially-available antibody (abcam); the number of irc (1) cells was quantified in 40x light microscopy fields (n 5 10) for each section. the total number of irc (1) cells was greater in the laminae of challenged ponies than control ponies (p 5 0.0096), and there was a significant correlation between the change in serum basal insulin concentration and number of laminar irc (1) endothelial cells (r 5 0.74; p o 0.05). while the number of irc (1) endothelial cells was significantly greater in the dermal laminae of challenged ponies (p 5 0.0095), there was no difference in the number of interstitial irc (1) cells (p 5 0.82). no epithelial irc (1) cells were observed in any laminar section, and irc (1) cells were conspicuously absent from the deep dermal tissue (including vessels). up-regulation of irc expression in the laminar vasculature occurs acutely in response to dietary carbohydrate challenge and accompanies hyperinsulinemia in ponies. the dramatic increase in endothelial irc expression in the laminar microvasculature in nutritionally challenged ponies, with no apparent epithelial irc present, suggests that hyperinsulinemia associated with exposure to increased dietary nsc may induce laminar injury by causing a similar vasoconstriction in ir equids as described in the microvasculature of ir humans. glucose transport from the blood stream into cells, the limiting step in whole-body glucose utilization, is regulated by a family of glucose transporter (glut) proteins in insulin-sensitive (i.e., muscle and adipose) tissues. we previously demonstrated that glut4, the major isoform, is a key factor in the pathogenesis of equine insulin resistance (ir). while it has been recently demonstrated that glut12 (a newly discovered isoform) increases insulin-stimulated glucose transport in human muscle, its role in other tissues, particularly in the setting of ir, is not well characterized in any species. in addition, as160 has recently emerged as a key downstream signaling molecule regulating translocation of glut to the cell surface, the rate-limiting step in glucose uptake. we hypothesized that glut12 content would be differentially expressed across tissues and that ir would induce alteration in glucose transport by affecting active cell surface glut12. biopsies of skeletal muscle, and subcutaneous and visceral adipose tissue were collected from light-breed horses, characterized as either insulin sensitive or compensated ir, based on the results of an insulin-modified frequently-sampled intravenous glucose tolerance test (n 5 5/group). we specifically quantified active cell-surface glut12 in these biopsies, using an innovative exofacial bismannose photolabeled assay, which has not been previously applied to glut12. total glut12 protein expression was measured by western blots, as well as total and phosphorylated (indicating activation of) as160. glut12 was expressed in all the depots with a significant regional effect. total glut12 protein content was increased (p o 0.05) in visceral (omental and mesenteric) compared to subcutaneous (nuchal ligament and tailhead) adipose tissue and skeletal muscle of healthy horses. ir did not induce alterations in active cell-surface and total glut12 content nor in total and phosphorylated as160 in any of the tissues evaluated. our data suggests that glut12 is abundant in visceral adipose tissue and is therefore likely to play a substantial role in the regulation of glucose transport. however, neither glut12 translocation nor as160 activation are impaired in insulin-sensitive tissues of ir horses. it is concluded that, in contrast with glut4, glut12 does not appear to contribute to glucose transport alterations during naturally-occurring equine ir. insulin resistance (ir), characterized by exaggerated glycemic or insulinemic responses to glucose challenge, is a key metabolic disturbance in horses that develop obesity-associated laminitis. in addition to obesity, diet and age have been demonstrated to affect tissue sensitivity to insulin in other species but these factors have received limited investigation in horses. we hypothesized that there would be greater glycemic and insulinemic responses to a sweet feed meal in aged horses, as compared to adult horses, as well as in horses adapted to a forage-only diet. three diets, grass hay only, grass hay plus sweet feed (starch and sugar-rich, ss), and grass hay plus a fat and fiber (ff) feed, were fed to 17 mares, 8 adult (5-12 yr) and 9 aged (4 19 yr), for a 6-week adaptation period in a randomized design. glycemic and insulinemic responses to a standardized meal of sweet feed (4 g/kg bw offered for 1 hour) were determined for 6 hours from the onset of feeding. peak glucose and insulin concentrations and areas under the glucose or insulin vs. time curves (auc-g, mg/ dl/360 min, and auc-i, mu/ml/360 min) were determined and data were analyzed by one-and two-factor repeated measures anova. there were no differences between age groups in glycemic responses to any of the diets. however, in aged horses peak glucose concentration (p o 0.03) and auc-g (p o 0.01) were greater after adaptation to the forage-only diet, as compared to the other two diets. in contrast, aged horses had a greater peak insulin concentration (p o 0.05) and auc-i (p o 0.03) than adult horses on all diets but no differences in peak insulin concentration or auc-i was found between diets within age groups. as hypothesized, the insulin response, but not the glycemic response, to a sweet feed meal was greater in aged horses, regardless of background diet. further, the glycemic response was greatest after adaptation to a forage-only diet, but this finding was only significant in aged horses. morbidity, mortality, and economic loss to the equine industry. in obese humans and rodent models of nutritional obesity, systemic insulin resistance and hyperinsulinemia are followed temporally in a majority of individuals by decreased glucose tolerance, pancreatic bcell failure, and type ii diabetes mellitus. in stark contrast to humans, obese horses and ponies chronically remain in what is termed a ''prediabetic'' state in human ir, characterized by hyperinsulinemic euglycemia. few data exist describing the biology of the equine endocrine pancreas in the chronically ir animal that may both: 1) explain this unique equine endocrine physiology and 2) characterize the animal at-risk for hyperinsulinemia-associated laminitis. the purpose of the study reported here was to characterize the morphology and physiology of the equine endocrine pancreas in response to a dietary carbohydrate challenge. twenty-two mixedbreed ponies (body weight 266.6 ae 170.5 kg) were conditioned to a diet of chopped hay (nsc $6% on dm basis) for 4 weeks; following conditioning, ponies either remained on the control diet (n 5 11), or received the same hay supplemented with sweet feed and oligofructose (total diet $42% nsc; n 5 11) for 7 days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including pancreas, were collected immediately following euthanasia. the samples were formalin-fixed for 48 hours, transferred to 70% ethanol, and paraffin-embedded. immunohistochemistry was performed on pancreas sections using a commerciallyavailable anti-insulin antibody (abcam), and measurements of islet surface area and b-cell surface area were performed (n 5 10 islets per tissue section) using a commercially-available computer software program (image j). there was a trend for greater total islet surface area in pancreatic tissue from ponies fed the high nsc diet when compared to the ponies on the hay diet (p 5 0.068); however, no difference was noted in b-cell surface area between diet treatments (p 5 0.12). the change in serum insulin concentration was significantly greater in the high nsc-fed ponies than in controls (403.8 1/-317.1 miu/l vs. 1.00 1/à 4.03 miu/l; p 5 0.002); however, this variable was not correlated with total islet surface area (r 5 0.32; p 5 0.17) or b-cell surface area (r 5 0.25; p 5 0.3). due to the relatively modest changes in pancreatic islet surface area that accompany marked increases in serum insulin concentrations in ponies fed a high nsc diet, it is important to assess both b-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in this model. humans that suffer from obesity show exaggerated inflammatory responses and this may be relevant to the association between increased adiposity and laminitis in horses with equine metabolic syndrome (ems). this study was performed to test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and those affected by ems. six healthy adult mares and 6 horses with ems received an intravenous infusion of lipopolysaccharide (lps; 20 ng/kg in 60 ml sterile saline) or saline alone. a crossover design was employed with a 7-day washout period. physical examinations were performed hourly for 9h and whole blood was collected at 30, 60, 90, 120, 180, and 240 min for assessment of inflammatory cytokine gene expression. a liver biopsy was performed between 240 and 360 min postinfusion. data were analyzed using mixed model anova. mean rectal temperature, heart rate, and respiratory rate increased following lps infusion (treatment â time; p o 0.001), with higher heart (group â treatment; p 5 0.087) and respiratory rates (group; p 5 0.017) detected in ems horses. lipopolysaccharide infusion significantly increased whole blood gene expression of tumor necrosis factor a (tnfa), interleukin (il)-1b (p o 0.001), il-6 (p o 0.001), il-8 (p o 0.001), and il-10 (p 5 0.002), and hepatic gene expression of il-6 (p o 0.001), il-8 (p o 0.001), and il-10 (p 5 0.016). inflammatory gene expression did not differ significantly between groups, so our hypothesis was not supported. heart rates tended to be higher when lps was administered to horses with ems. elevated serum concentration of cardiac troponin i (ctni) is a biomarker for myocardial damage in horses. preferred times to test blood for ctni levels following athletic performance or other events that may cause myocardial injury are not yet established and would be affected by time of release from the myocytes, location of release within the myocytes, duration of release and half-life of ctni in the horse. this information would be necessary to more accurately and reliably test horses for myocardial injury. the aim of this study was to determine the elimination half-life (t1/2) of equine ctni. to establish the t1/2 of equine ctni in horses, ctni was recombinantly expressed in e.coli. two healthy ponies received intravenous injections of recombinant equine ctni and plasma ctni concentrations were measured with a point-of-care ctni analyzer at multiple time points after injection. standard pharmacokinetic analysis was performed to establish the elimination half-life of ctni. for comparative purposes, data were subjected to pharmacokinetic models describing a single versus biphasic elimination profile. elimination of recombinant equine ctni following intravenous administration exhibits a short half-life. establishing the t1/2 of troponin provides critical information in understanding the clinical application of this cardiac biomarker in clinical practice. this study describes a true biological ctni t1/2, which has not been documented in any species thus far. stall-side assessment of this cardiac biomarker in horses should enhance the ability of clinicians to detect myocardial damage and aid in the management and treatment of horses with cardiac disease. the objective of the study was to evaluate the between-pony, within-pony, between-analyser and within-analyser variation of flow-mediated vasodilation (fmd) measurement in healthy ponies, to investigate the hypothesis that fmd occurs in healthy ponies. six healthy, native breed, unrelated pony mares of varying weight (236-406 kg), body condition score (3/9-7/9) and age (14-25 years) were used. the median artery was occluded for 5 minutes. twodimensional (2d) ultrasonographic images of the artery were recorded for 30 seconds prior to and for 2 minutes after occlusion. the peak luminal diameter was compared to baseline diameter to calculate the relative percentage increase in luminal size (fmd). images were obtained from six ponies on one occasion and from one pony on six occasions. analysis of images was performed by two independent analysers and by one analyser twice. the mean (sd) fmd in 6 ponies was 12.57% (4.28%) and in 1 pony (6 occasions) was 7.30% (2.11%). coefficients of variation were 34.09% and 28.84% respectively. agreement between analysers was fair (icc 5 0.47) and within analyser was poor (icc 5 0.30). fmd is used to assess endothelial function in humans and has recently been assessed for its use in canine subjects. fmd occurs and measurement is feasible in ponies. fmd could be used to assess endothelial function, in the context of laminitis or other cardiovascular diseases. current state-of-the-art technique for measuring blood pressure (bp) in the horse is invasive and involves cannulation of the facial artery. indirect techniques, such as oscillometry, have proven useful in the anaesthetised horse, but have not become routine in the standing horse. monitoring bp can be indicated for the diagnosis and treatment of the hypotensive patient (ie. caused by endotoxemia, hypovolemia, systemic inflammatory response syndrome and cardiac failure) or the hypertensive patient (ie. due to equine metabolic syndrome or pain). the objective of this study was therefore to a) describe the methodology for application of oscillometric bp using a cuff applied to the tail in the standing horse and b) and to determine accuracy and precision of this method applied to the normotensive standing horse. the oscillometric method is simple to apply in a clinical setting. a pneumatic cuff is snugly applied to the unclipped tail-base with the cuff bladder centered over the middle coccygeal artery. the tail circumference must match the manufacturers description of the cuffs diameter range. the oscillometric apparatus inflates the cuff and obtain systolic, diastolic and mean arterial bp (sap, dap and map). at least 2 consecutive measurements must be obtained. a correction of 0.7 mmhg/cm vertical distance between cuff and heart level is added to the measurement to correct for hydrostatic pressure difference. for determination of accuracy and precision of indirect sap, dap and map, eight healthy horses (age 3 to 16 years), was equipped with an intra-arterial catheter ii in the facial artery and a commercial tail-cuff oscillometric apparatus. i measurements were recorded every 2 minutes for 20 minutes. the data were analysed with the statistical software r using a mixed model with repeated measurements and a bland-altman analysis corrected for repeated measurements. oscillometric bp was accurate and precise for map (mean bias, lower confidence level, upper confidence level, variation in difference, all mmhg) (à0.3, à18.5, 19.1, 33.2, respectively) in the conscious horse but not for sap (à1.5, à19.3, 16.3, 38.2, respectively) and dap (0.05, 15.9, 16.0, 49, respectively) . there was no significant contribution to the statistical model of either horse or measurement number. all horses tolerated the tail-cuff well and the method was simple to apply. only map could be measured with acceptable accuracy and precision in the normotensive standing horse using the described oscillometric method. reference intervals for thyroid hormone (th) concentrations have not been established for donkeys. therefore, clinicians must use reference ranges from horses, potentially leading to misdiagnosis of thyroid diseases. we hypothesized that th concentrations are different between donkeys and horses. the purposes of this study were: a) to compare th concentrations between donkeys and horses and, b) to determine whether the age may influence th concentrations. thirty-eight healthy donkeys (8.5 ae 0.8 years), mixed breeds, and 20 healthy andalusian horses (6.4 ae 0.5 years) were used. donkeys were divided into three groups: o 5 years (n 5 13), 5-10 years (n 5 12), and 411 years (n 5 13). serum concentrations of total triiodothyronine (tt3), free triiodothyronine (ft3), total thyroxine (tt4), free thyroxine (ft4), reverse triiodothyronine (rt3) and thyroid-stimulating hormone (tsh) were quantified by radioimmunoassay. all blood samples were collected the same day. neither horses nor donkeys had received any treatment for 30 days before sampling and both farms had similar production conditions. total t3, ft3, ft4 and tt4 concentrations were higher (p o 0.01) in donkeys than horses. in contrast, no statistical differences were found for rt3 and tsh concentrations. young donkeys ( o 5 years) had higher ft4, tt4 and rt3 concentrations compared to other donkey groups (p o 0.05). old donkeys (411 years) had lower tt3 and ft3 concentrations than both younger donkeys groups (p o 0.05). this study shows that there are differences in th concentrations between donkeys and horses, raising awareness on the possibility of misdiagnosis of thyroid gland dysfunction when using values from horses, being necessary to determine exclusive reference intervals for donkeys. ovariectomy is associated with alterations of responses to many hormones, not just those associated with reproductive function. in humans and rats, ovariectomy leads to insulin resistance, increased adiposity and altered fat mobilization. the effects of ovariectomy on energy metabolism have not been reported in horses. ovariectomized mares have been shown to respond normally to an acth stimulation test, but the response to suppression of the hypothalamo-pituitary-adrenal axis has not been previously described. the aim of this study was to evaluate the effect of ovariectomy on insulin response in mares and to determine if mares exhibit alterations in response to dexamethasone administration after ovariectomy. six healthy mares underwent an intravenous glucose tolerance test (ivgtt), an insulin sensitivity test (ist) and a dexamethasone suppression test (dst) before and 5 weeks after bilateral ovariectomy. body weight, cortisol values at baseline, 15 and 24 hours after dexamethasone injection and acth values at baseline, 15 and 24 hours after dexamethasone injection, basal insulin/glucose ratio, time to reach a 60% decrease in blood glucose in the ist, time to reach baseline glucose concentration in the ivgtt and area under the curves plotting blood glucose and time to injection of glucose or insulin were compared before and after ovariectomy using a paired t-test or an anova for repeated measures. significance level was p o 0.05. average body weight was decreased after surgery (6kg ). the injection of dexamethasone resulted in a serum cortisol concentration of less than 1 mg/dl in all mares before ovariectomy, whereas after ovariectomy, dexamethasone injection resulted in a serum cortisol concentration of less than 1 mg/dl in 5 out of 6 mares. in all cases, acth concentration was within the reference range (9-35 pg/ml) before and after ovariectomy. however, acth concentrations at t 0 and at t 15 were significantly higher after ovariectomy. each mare had a normal ivgtt, both before and after ovariectomy. additionally, no significant differences were observed in basal blood glucose (84 ae 12 mg/dl before and 85 ae 3 mg/dl after) or in the time to reach glucose baseline (108 ae 66 min before and 99 ae 51 min after). serum basal insulin concentration and insulin/glucose ratio was not significantly different before or after ovariectomy (22.0 ae 7.9 miu/ml and 17.5 ae 8.9 miu/ml and 0.26 ae 0.09 and 0.20 ae 0.10, respectively), nor was the average time to reach a 60% decrease in blood glucose after insulin injection (30 ae 7 min and 25 ae 9 min, respectively). these findings suggest that, as reported in other species, the shortterm effect of ovariectomy may modify dexamethasone response in mares and that, contrary to other species, it may not modify insulin response. equine gastric ulcer syndrome (egus) is a common medical problem in horses. the high prevalence of gastric ulcers, vague clinical signs and negative effect on performance make it a significant clinical and economic problem within the horse industry. current pharmaceutical treatments are expensive and alter the acidic environment of the stomach. berries and pulp from the seabuckthorn plant (hippophae rhamnoides) are a rich source of vitamins, trace minerals, amino acids, antioxidants, and other bioactive substances and have been used successfully to treat stomach ulcers in man and rats. the purpose of this study was to evaluate the efficacy of a commercially sold, liquid extract of seabuckthorn berries (seabuck tm sbt gastro-plus) for treatment and prevention of gastric ulcers in horses. eight thoroughbred and thoroughbred-cross horses (3-10 years of age, 5 geldings & 3 mares, 380-600 kg) were used in a blinded two-period cross-over study. treatments consisted of control (untreated) and treatment (seabuck tm sbt gastro-plus) twice daily mixed with the grain meal. horses were treated for 5 weeks followed by a 1 week alternating feed-deprivation period to induce or worsen existing ulcers. gastroscopies were performed on all horses on day 0, week 5, and week 6 (at the end of the alternating feed-deprivation period). gastric juice was aspirated and ph was measured. during gastroscopy, gastric ulcer scores were assigned to each stomach based on lesion number and severity. horses acted as their own controls, and between each treatment period the horses had a 2-week washout period. data was analyzed by anova for repeated measures via the glm procedure (sas inst. inc., cary, nc). when significant differences (p o 0.05) were observed, a post-hoc tukey's test was used to determine differences. non-glandular gastric ulcer scores significantly increased in all control and sbt-treated horses from week 5 to week 6, after the feed-deprivation phase of the study. there was no significant difference in the non-glandular gastric number (p 5 0.84) and nonglandular gastric severity (p 5 0.51) scores in sbt-treated horses compared to non-treated controls. glandular ulcer number (p 5 0.02) and glandular ulcer severity (p 5 0.02) was significantly lower in the sbt-treated horses compared to the control horses. there was no significant difference in the ph (p 5 0.06) in sbt-treated horses compared to non-treated controls. thus, seabuck tm sbt gastro-plus, mixed in the feed twice daily, may be efficacious in controlling the severity of glandular ulcers in horses during stress, without increasing stomach ph. the availability of rapid and accurate quantitative fibrinogen measurements may be useful for evaluation of hospitalized equine patients. the abaxis vspro analyzer was evaluated for precision using two levels of human fibrinogen controls (300 mg/dl and 150 mg/dl), four different vspro machines, and two different lots of cartridges, assessed over 5 subsequent days. the coefficients of variation of the assay ranged from 4% (300 mg/dl) to 8% (150 mg/ dl). we subsequently evaluated the abaxis vspro fibrinogen assay compared to fibrinogen concentration measured using the beckman coulter acl-1000 in 50 equine samples of varying fibrinogen concentrations obtained from horses with gastrointestinal disease. all samples were measured in citrated plasma. fibrinogen samples measured on the acl-1000 ranged from 226 to 959 mg/dl (median 501 mg/dl). vspro samples were run in duplicate, and the mean compared to the acl values. pearson correlation coefficient analysis generated an r value of 0.949 (p o 0.001). duplicate measurements on the vspro were strongly correlated to each other with an r value of 0.9690 (p o 0.001). bland-altman analysis of these samples for the vspro compared to the acl-1000 noted a bias of à84 ae 57 mg/dl the results of this study indicate that the vspro benchtop fibrinogen analyzer provides accurate and precise fibrinogen data compared to the acl-1000 reference analyzer. the immune response of foals to r. equi is incompletely understood and believed to be responsible for clinical disease caused by this pulmonary pathogen. in a recent study foals receiving a large inoculum exhibited th2 skewing with pneumonia and a small inoculum exhibited th1 skewing without clinical disease. we hypothesized that cytokine/chemokine production by pulmonary alveolar macrophages, in vitro, would increase with the infective dose and that the magnitude of the response would differ between foals and adults. alveolar macrophages were obtained by bronchoalevolar lavage from 7 healthy mares and their 5-week-old foals. macrophage cultures were infected with r. equi (337011 or 33701-) at a multiplicity of infection (moi) of 1 or 100. total rna was harvested 4 and 24 hours post-infection, reverse transcribed and used as template for quantita-tive pcr. the ddct method was used to calculate relative gene transcripts for il-6, il-12p40, tnfa and cxcl10. cellular infections at moi 100 resulted in significantly higher expression of il-6, il-12p40 and tnfa mrna transcripts compared to moi 1. however, the dose-effect was reversed for cxcl10 with significantly lower expression at the higher moi. there was no difference in magnitude of cytokine/chemokine responses by the alveolar macrophages between adults and foals. dose-dependent responses of alveolar macrophages may represent a novel mechanism by which r. equi could modulate immune responses and therefore disease. significant down-regulation of cxcl10 mrna transcripts associated with a higher dose is of particular interest as this chemokine plays a role in development of protective th1 responses. the intent of this study was to develop likelihood ratios (lrs) for infection attributable to corynebacterium pseudotuberculosis in horses based on synergistic hemolysis inhibition (shi) test titers. medical records for horses presented to the uc davis veterinary teaching hospital with serum submitted for shi titer determination were evaluated and 171 cases met study inclusion criteria. these cases were grouped based on evidence of internal and/or external infection attributable to c. pseudotuberculosis and likelihood ratios with 95% confidence intervals determined. results showed increasing lrs indicating increasing odds for any form of active disease as titer increased with all cases considered. lrs for internal infection were 4 1 for titers ! 1280 overall and for titers 4 160 with external abscess cases excluded. no difference from 1 (and therefore no significant change in pre-test to post-test odds) was seen in any lrs for internal disease when only cases with external disease were examined (external and internal disease vs. external only). overall, the shi test results showed usefulness in determining internal c. pseudotuberculosis infection in horses with no evidence of external abscessation. overall, however, higher titers were more indicative of active external or internal disease than internal disease specifically in contrast to previous reports. the shi test was unable to distinguish internal infection when external abscesses were present. salmonella enterica is a zoonotic pathogen that has tremendous impact on many different animal production and management systems. rapid detection of s. enterica in fecal samples may facilitate effective infection control practices. current detection methods require 24-48 hours (polymerase chain reaction or pcr) or 48-72 hours (enriched aerobic culture) to obtain results. alternatives have been developed, lateral flow antigen detection systems (lfads), which are currently marketed for salmonella detection related to food safety microbiology. the objective of this study was to evaluate two commercially available rapid salmonella detection systems in equine feces. fecal samples collected from repeatedly culture-negative horses were inoculated with known concentrations of salmonella enterica serotype typhimurium (five uninoculated control samples, and 5 samples of each 10-fold dilution [1.9 â 10 0 -1.9 â 10 4 cfu/gram of feces]). all samples were aerobically cultured using a standard enrichment technique. in a blinded fashion, samples were tested using two different lfads as well as plated on agar media for confirmatory testing. at 24 hours of incubation, using bacterial culture as the reference method, test 1 was correctly identify 70% of samples ( bacterial contamination of stalls with salmonella sp. is a serious problem in equine hospitals. salmonella sp. exposure to horses in the facility can result in nosocomial infections which results in temporary facility closure, until the organism is eradicated. hospital closure can result in loss of revenue, damage to reputation and interference with patient care. the purpose of this study was to evaluate three stall cleaning methods on eradication of salmonella sp. at an equine veterinary teaching hospital (vth). horses admitted to the vth were assigned to salmonella sp.negative stalls within areas of the vth during the study period (september 2009 -january 2010 . when the horses were discharged stalls were randomly assigned to one of three cleaning methods (pressure-washing only [pw] , pressure washing and hand scrubbing [pws] , or hand scrubbing only [s]) in a single period, non cross-over design. all stalls were stripped of bedding and surfaces sprayed with tap water and cleaned with a disinfectant quaternary-ammonia solution (super hdq neutral, spartan chemical co., inc, maumee, oh). the pressure-washing system (psc cleaning systems, inc., toronto, canada) used, provided a pressure of 3000 psi and a temperature range of 1851-2151f. following cleaning, each stall was allowed to air dry and within 48 hours, stall surfaces were sampled using three 4 00 â 4 00 sponges moistened with sterile saline. the person collecting the samples was masked to the method of cleaning. sponges were submitted to the louisiana animal disease diagnostic laboratory (laddl) for culture of salmonella sp. a chi-squared analysis was used to determine significant differences (limit p o 0.05) between cleaning methods and salmonella sp. isolation. during the study period, 112 stalls (pw [n 5 29]; pws [n 5 50]; s [n 5 33] were included. all stalls had negative environmental salmonella sp. cultures prior to beginning the study. for pw cleaned stalls, 6/23 (20.7%) were salmonella sp.-positive, for pws cleaned stalls, 12/38 (24%) were salmonella sp.-positive, and for s cleaned stalls, 4/29 (12.1%) were salmonella sp.-positive. although, there were fewer salmonella sp.-positive stalls (12.1%) in the handscrubbed stalls, cleaning method did not significantly (p 5 0.4057) affect the isolation of salmonella sp. from the stall environment. in conclusion, power washing alone, power washing and hand scrubbing, and hand scrubbing alone, using a quaternary-ammonia solution did not significantly affect environmental isolation of salmonella sp. from stalls surfaces in the vth during this study. the objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against streptococcus equi subsp. zooepidemicus (s. zooepidemicus) and rhodococcus equi isolates. a single dose of gamithromycin (6 mg/kg of body weight) was administered intramuscularly. concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (pelf), bronchoalveolar lavage (bal) cells, and blood neutrophils were determined using hplc with tandem mass spectrometry detection. the minimum inhibitory concentration of gamithromycin required to inhibit growth of 90% of r. equi and s. zooepidemicus isolates (mic 90 ) was determined. additionally, the activity of gamithromycin against intracellular r. equi was measured. mean peak gamithromycin concentrations were significantly higher in blood neutrophils (8.35 ae 1.77 g/ml) and bal cells (8.91 ae 1.65 g/ml) compared to pelf (2.15 ae 2.78 g/ml) and plasma (0.33 ae 0.12 g/ml). mean terminal half-lives in neutrophils (78.6 h), bal cells (70.3 h), and pelf (63.6 h) were significantly longer than that of plasma (39.1 h). the mic 90 of s. zooepidemicus isolates was 0.125 g/ml. the mic 90 of gamithromycin for macrolide-resistant r. equi isolates (128 g/ml) was significantly higher than that of macrolide-susceptible isolates (1.0g/ ml). the activity of gamithromycin against intracellular r. equi was similar to that of azithromycin and erythromycin. intramuscular administration of gamithromycin at a dosage of 6 mg/kg would maintain pelf concentrations above the mic 90 for s. zooepidemicus and phagocytic cell concentrations above the mic 90 for r. equi for approximately 7 days. eight western stock yearling horses were infected with ehv-1 (ab4) by nasopharyngeal instillation. venous blood samples for collection of plasma were collected in na-citrate tubes on the day prior to infection (d -1) and on d4 through d11. in addition, clinical data, nasal swabs and peripheral blood mononuclear cells (pbmc) for detection of viremia were collected on the day before infection (d -1) and on d1 through d14 post-infection. d-dimer concentrations were determined in citrated plasma samples using a latex agglutination test (minutex d-dimer, biopool, ireland). viral load in pbmc was determined using quantitative pcr. all horses showed bi-phasic fevers typical for ehv-1 infections. one horse developed acute ehm on d10 and was euthanized after samples were collected. in all horses d-dimers were undetectable on d -1 and on d4, 5 and 11. in contrast, all horses had increased ddimer concentrations for 3 to 5 consecutive days starting on day 6 post-infection. d-dimer concentrations in 2 horses increased to 1000 ug/ml and one of these horses was the horse with acute ehm. interestingly, mean increased d-dimer concentrations showed timely overlap with the mean fever curve and, delayed by 1 day, with the mean viremia curve. because plasma samples for d-dimer measurements were not collected during the first 3 days post-infection, which are typically associated with a primary fever, conclusion on the association of d-dimers with fever of viremia await analysis of a second study currently conducted in our laboratory. in conclusion our data indicates that during ehv-1 infection with neuropathogenic strains activation of the coagulation cascade and production of cross-linked fibrin is wide-spread; not limited to horses with clinical signs of ehm, and can be expected between days 6 and 10 post-infection. lawsonia intracellularis is an emerging pathogen in horses and the causative agent in equine proliferative enteropathy (epe). the goal of this study was to evaluate the exposure of pre-weanling foals and broodmares to lawsonia intracelluaris on several farms in louisiana with a history of epe and compare the results to several farms with no known clinical cases of epe in foals. an additional goal of the study was to identify whether a relationship exists between lawsonia intracelluaris and other gastrointestinal pathogens in foals. whole blood and fecal samples were collected from 66 mares and 68 foals from four breeding farms in louisiana. farms a and b had no known clinical cases of epe, while farms c and d had previous know cases of epe in 2009. serum samples were examined for the presence of antibodies against lawsonia intracellularis using an immunoperoxidase monolayer assay (ipma). dna was extracted from fecal samples using a commercial dna kit and molecular detection of lawsonia intracelluaris was assayed using real-time pcr. fecal ova were determined using quantitative sucrose floatation. the presence of fecal clostridium difficile toxin was measured using a commercial enzyme linked immunosorbent assay (elisa). three of the 4 farms examined had foals and mares with exposure to l. intracellularis as evidenced by serum antibodies against the organism. of the total population sampled, 6 foals (8.8%) and 14 mares (21.2%) had evidence of antibodies to l. intracellularis based on serology. three foals (4.4%) tested positive for l. intracellularis organism by fecal pcr, and all of these foals were located on farm c. of these, one of the foals was seronegative, while the other two were seropositive. farm c also had the highest percentage of mares (28.6%) serologically positive for l. intracellularis, while farm a had the highest percentage of foals (14.3%) with antibody titers against l intracellaris. farm c also had the only pairs (n 5 3) of serologically positive mares with seropositive foals. while farm a and b had seropositive mares and/or foals, none of the foals were positive for l. intracellularis fecal shedding by pcr. all serum and fecal samples were negative for evidence of l. intracellaris on farm d. ten foals (14%) had fecal egg counts greater than 200 egg per gram and 2 foals (3%) were positive for c. difficile toxin. this study demonstrated evidence of natural exposure to l intracellularis on farms both with and without a history of epe in louisiana. further, this study failed to establish a relationship between l intracellularis and other gastrointestinal pathogens. the objective of this study was to examine the clinical, hematological, biochemical, and outcome data from equids infected with anaplasma phagocytophilum presented to a primary care field setting in southeastern pennsylvania. computerized medical records from 19 febrile equids with confirmed anaplasma phagocytophilum infection were reviewed. confirmation of anaplasma phagocytophilum was defined by the presence of granular inclusion bodies seen within leukocytes or eosinophils on microscopic blood smear evaluation and/or a positive polymerase chain reaction (pcr) for anaplasma phagocytophilum. 18 horses and 1 donkey presented with a mean fever of 104.41f and mean fever duration of 39 hours. the mean age at presentation was 9.6 years and the mean pack cell volume was 30.8%. 15/19 cases were diagnosed in the months of may to december. equids ages 5 to 15 years had significantly lower platelet counts. 16/18 cases were positive on blood smear for inclusion bodies and 6/6 cases were positive for anaplasma phagocytophilum on pcr. treatments included intravenous oxytetracycline, oral doxycycline, or both. mean treatment duration was 4.8 days and mean treatment cost was $621. 17/19 cases were normothermic within 48 hours. the treatment used in the two remaining cases was changed from oral doxycycline to intravenous oxtetracycline and was successful. this is the first case series of equine granulocytic anaplasmosis in the mid-atlantic states. all cases were examined and treated in the field. in order to make a definitive diagnosis, some cases required pcr. treatment failures were documented with the use of oral doxycycline alone. 100% of the cases survived. a high incidence of clinical and possibly genetic abnormalities has been reported amongst friesian horses including dwarfism, hydrocephalus, dissecting aortic aneurism and esophageal dysfunction. the purpose of the current study was to develop a new electromyography (emg) method to assess neurophysiological function of the esophagus especially for friesian horses. five friesian horses with esophageal dysfunction were included (ranging in age from 0.5-24 years and comprising 4 mares and a stallion) and two friesian control horses (a 10-and 12-year-old gelding). all five horses with esophageal dysfunction had a history of recurrent esophageal obstruction and were examined histopathologically post-mortem. barium contrast radiography was used as the gold standard to distinguish the diseased from the control horses. an endoscopically-guided percutaneous needle emg procedure (viking quest r ; software version 11.0) was performed just caudal to the larynx and just cranial to the thoracic inlet (to monitor striated and smooth muscle, respectively) to visualize esophageal motility. esophageal contractility in both control horses was predominantly reflected by interference patterns associated with longer duration and lower amplitude in smooth muscle compared to striated muscle. mean (ae sd) values were 35.1 ae 19.4 ms and 167.7 ae 96.7 mv (n 5 19 readings) and 10.8 ae 14.3 ms and 305.8 ae 233.7 mv (n 5 24 readings), respectively. in diseased horses, aperistalsis in smooth muscle was the most remarkable finding suggesting a loss of inhibitory neurogenic input resulting in aperistalsis and thus esophageal dysfunction. preliminary findings suggest that endoscopically-guided percutaneous needle emg might become a valuable method in elucidating the pathophysiology of dysfunction of esophageal motility especially in friesian horses. lymphoma affects horses of all ages. unlike in humans, no etiologic agent has been discovered. a 9 year old thoroughbred/warmblood cross mare presented with signs of upper and lower respiratory disease and was subsequently diagnosed with lymphoma and equine multinodular pulmonary fibrosis (empf) and was positive for equine herpes virus 5 (ehv-5) in both the pulmonary tissue and the lymph nodes. retrospective polymerase chain reaction (pcr) testing of six lymphoma cases found that 5 of 6 of the cases were positive on pcr for ehv-5 (83.3%, p 5 0.0045, rr 5.55). electron microscopy was performed on one sample and herpes virus particles were identified. of the samples in which immunohistochemistry was performed (3 of 6), only t-cell rich b-cell lymphoma was identified. samples of mesenteric or submandibular lymph nodes from 20 clinically healthy horses were submitted for ehv-5 pcr analysis; 15% were positive. gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as kaposi's sarcoma and burkitt's lymphoma. equine herpesvirus 5, also a gamma herpesvirus, is found in association with equine lymphoma; although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia remains unknown. pathologic events reported to occur in the digital laminae in early stages of sepsis-related equine laminitis include leukocyte extravasa-tion into the laminar interstitium, pro-inflammatory cytokine expression, and epithelial stress. while these events have been documented early in the disease process at both a developmental stage and at the onset of obel grade 1 (og1) lameness in the carbohydrate overload (cho) model of laminitis, the later events occurring at the onset of obel grade 3 lameness(og3, time point at which structural failure of the laminae usually occurs) have not been determined. we hypothesized that the inflammatory events described above are sustained through og 3 lameness, likely playing an injurious role culminating in laminar failure. our objectives were to determine pro-inflammatory gene expression, leukocyte extravasation, and epithelial stress at og 3 induced using the cho model. archived laminar tissue samples (snap frozen and paraffin embedded sections) were used from a previous cho study at louisiana state university (control group [n 5 6, water], cho group [n 5 7, corn starch]. calprotectin (cp) immunohistochemistry (ihc) was used to assess both laminar myeloid leukocyte numbers and epithelial stress; rt-qpcr was used to assess inflammatory gene expression. minimal inflammatory changes were present at og3 compared to published values at og1 stage in the cho lameness model including decreased mrna concentrations of cytokines (i.e. 20-fold increase in il-6 at og3 vs. 4 2000-fold increase at og1, no increase in il-1b at og3 vs. 11-fold increase at og1), chemokines (no change in mcp-1at og3 vs. 4 30 fold increase at og1, 8-fold increase in il-8 at og3 vs. 95fold increase at og1) and adhesion molecules (no change in e-selectin at og3 vs. 10-fold increase at og1). laminar leukocyte emigration was also decreased at the onset of og 3 lameness compared to previously reported leukocyte infiltration at og 1. interestingly cox-2, underwent a greater increase at og3 (approx. 50-fold) compared to that reported at og1 lameness (35-fold). finally, epithelial stress at og3 evidenced by cp ihc did not follow the uniform widespread distribution reported at og1 lameness, but instead was present in focal areas in which secondary epidermal laminae on either side of a common primary dermal vascular supply demonstrated increased cp signal. overall, laminar inflammation appears to be subsiding at og3 lameness, with epithelial stress possibly more dependent on vascular dysregulation instead of inflammatory events. the sustained increase in cox-2, central to the induced production of vasoactive prostanoids in disease processes, may play a role in vascular dysregulation. this study was conducted to characterize clinical, laboratory and postmortem findings associated with oleander toxicosis in equids and to determine factors predictive of survival in these cases. retrospective analysis of medical records from our veterinary medical teaching hospital from january 1, 1995 to july 15, 2010 was completed. records of equids demonstrating detectable oleandrin in serum, plasma, urine or gastrointestinal fluid samples or detectable serum digoxin in the absence of pharmaceutical cardiac glycoside administration were included. descriptive statistics were used to evaluate the history, physical examination, and laboratory and postmortem data of affected individuals. logistic regression analysis was used to detect physical examination and laboratory factors significantly associated with survival. thirty equids met inclusion criteria of the study. three of 30 subjects were dead on arrival or died immediately upon arrival (10%). of the remaining 27 equids, 85% presented with gastrointestinal abnormalities, 70% were azotemic and 48% had cardiac arrhythmias. mortality was 50% for all subjects and 44% for those treated. the predominant cause for non-survival was cardiac dysfunction. factors significantly associated with survival included relatively decreased hematocrit and serum glucose, relatively increased serum chloride, absence of cardiac arrhythmias, and increased duration of hospitalization. equids with oleander toxicosis frequently present with gastrointestinal upset and may develop cardiac and renal disturbances. patients with cardiac arrhythmias and relatively increased hematocrit and serum glucose and decreased serum chloride are significantly less likely to survive. oleander intoxication is a differential diagnosis for colic in endemic areas, particularly with concurrent azotemia or cardiac dysrhythmia. the quantitative physicochemical approach emphasizes the importance of strong ions (na, k, cl, lactate), pco 2 , and the plasma protein concentrations in determining plasma ph. serum concentrations of strong ions, proteins, and total co 2 are reported on modern biochemical profiles. we hypothesized that the results of serum biochemical analysis can be used for acid-base interpretation in horses. the objective was to determine whether blood ph, anion gap, and strong ion gap could be quantitatively estimated and clinically used based on the results of serum or plasma biochemical analysis. 100 horses (70 adults and 30 foals) presented to the isolation unit of our veterinary teaching hospital for suspected infectious diseases were prospectively enrolled. a venous serum sample was analyzed using a hitachi 911 or copas 6000 c501 automated machine. measured parameters included strong ion difference (sid 5 {na1k}-{cl1lac-tate}), total protein concentration (tp), and total co 2 (tco 2 ), with lactate being measured by blood gas analyzer. a second venous blood sample was collected into a na-heparin blood gas syringe and analyzed for ph (ph m ), pco 2 and concentrations of na, k, cl, and lactate using a radiometer 800 flex blood gas analyzer; sid was calculated from the measured values, and total solids (ts) were estimated using refractometry. serum/ plasma ph (ph calc ) was calculated using stewart's 8 factor equation from the results of serum or plasma biochemical analysis, assuming pco 2 5 40 mmhg for serum and pco 2 accurate for plasma. anion gap (ag) was calculated as: ag 5 (na1k)-(cl1tco 2 ). strong ion gap (sig) was calculated as: sig 5 0.21x[total protein, g/l]/ (1110 {pka-ph} )-ag. linear regression analysis was used to compare ph calc to ph m, as well as ag and sig to blood lactate concentrations. measured ph ranged from 7.03 to 7.48 (7.37 ae 0.07). measured sid from serum biochemistry (sid sb ) ranged from 22.3 to 51.4 meq/l (36.2 ae 4.7 meq/l) and sid from blood gas analyzer (sid bg ) from 14.3 to 46.6 meq/l (33.3 ae 5.4 meq/l; r 2 5 0.59; sid bg 5 0.919 â sid sb ). sid sb and sid bg showed small variability in measurements. tp ranged from 18 to 88 g/l (51.0 ae 13.9 g/l) and ts from 20-84 (55.2 ae 13.8 g/l; r 2 5 0.77; ts 5 1.071 â tp). using sid sb and tco 2 values with constant pco 2 , ph calc was poorly associated with ph m (r 2 5 0.27; ph calc 5 0.48 1 3.89). in contrast, using sid bg with accurate pco 2 , ph calc was closely associated with ph m (r 2 5 0.54; phcalc 5 0.98 1 0.15) and the equation was not different from the line of identity. anion gap and sig (meq/l) calculated were significantly linearly correlated with lactate concentrations (mmol/l); ag 5 0.93 â [lactate] 1 8.3 (r 2 5 0.39), and sig 5 à0.91 â [lactate] 1 0.5 (r 2 5 0.41). we conclude that ph calc using sid sb , tco 2 and constant pco 2 values is not accurate. however, variability of measured biochemical parameters between machines was small, permitting use of serum biochemistry for clinical metabolic acid-base abnormalities interpretations of patients. these results reemphasize the importance of strong electrolytes and proteins in acid-base balance. metalloproteinases (mmps) are critically important in remodeling processes and in wound healing. however, excessive activation of mmps by pro-inflammatory mediators including cytokines, prostaglandin e2, and nitric oxide lead to tissue breakdown. this is observed in osteoarthritis (oa) which is characterized by erosive lesions in articular cartilage. in hereditary equine regional dermal asthenia (herda), afflicted horses exhibit collagen abnormalities and can have associated chronic inflammation and aberrant wound repair. herda affects horses with quarter horse bloodlines and is similar to the human hereditary connective tissue syndrome ehlers danlos (eds). many adult eds patients suffer from joint pain and oa. we hypothesized that chondrocytes from articular cartilage of herda horses have increased activity of mmps. to test this hypothesis, chondrocytes were retrieved from articular cartilage of homozygous herda carpal and hock joints. chondrocytes from normal horses were also obtained for comparison. chondrocytes were seeded at 5 x 10 6 /ml into 6-well plates and incubated at 371c, 5% co 2 for up to seven days. activity of secreted mmps was determined by zymography using equal amounts of proteins for loading. secreted mmps were analyzed by western blot. zymography showed that normal chondrocytes secreted two major bands with gelatinolytic activity observed at 92 and 72 kda suggestive of the latent form of mmp-2 and mmp-9, respectively. less intense bands of gelatinolytic activity were observed at about 82 and 62 kda suggestive of the active form of mmp-2 and mmp-9. another band of activity was also seen at 240 kda which is suggestive of a dimer of mmp-9 that has been reported when mmps are in excess of tissue inhibitors of metalloproteinases (timps). chondrocyte cultures from homozygous herda cartilage showed a similar profile but with decreased activity by 90% at 92 kda and 10-50% increased activity at 72 kda compared to normal chondrocytes. western blot analysis detected mmp-2 and mmp-9 immunoreactivity in chondrocyte culture media of herda-afflicted and normal horses. the present study demonstrates for the first time that horses suffering from herda have increased mmp activity which may predispose them to the development of lesions in articular cartilage. research supported by nutramax laboratories, inc. equine polysaccharide storage myopathy (pssm) type 1 is a dominantly inherited glycogenosis caused by a mutation in the gene coding for skeletal muscle glycogen synthase type 1 (gys-1). the disease has been reported to affect the haflinger breed but so far its prevalence is unknown. aim of this preliminary study was to estimate the occurrence of the gys-1 mutation in austrian haflingers and establish which of the seven haflinger sire lines appear mostly affected. gys-1 genotyping of 50 randomly chosen haflingers was performed with a validated restriction fragment length polymorphism assay. resting and post-exercise muscle enzyme activities (creatine kinase (ck), aspartate aminotransferase (ast), lacate dehydrogenase (ldh)) and blood lactate concentrations were compared between horses with and without the mutation. among the 50 horses 9 were heterozygous (hr) carrier of the mutation. no homozygotes (hh) were identified. all horses with the gys-1 mutation were descendents of the a-or w-sire lines. the estimated hr prevalence was 18% (95% ci: 8.6-32.4%). ck activity after exercise (p 5 0.022) was significantly higher in hr horses compared with horses not carrying the mutation (rr). ast activity was significantly higher in the hr group at rest and after exercise (p o 0.001). there was no statistically significant difference in resting ck, resting and post exercise ldh activity or blood lactate between hr and rr. results suggest that the prevalence of hr in the austrian haflinger population is higher than in the overall quarter horse population and might be as high as 30%, similar to some draft horse breeds. further research is needed to establish the prevalence within the different breeding lines. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder affecting quarter horse lineages. 1 although a mutation in the gene encoding cyclophilin b has been genetically linked to herda, its causal association with the disease is not yet documented. 2 previously, we demonstrated reductions in ultimate tensile strength (uts), modulus of elasticity, and energy to failure (toughness) of skin from many corporal regions of herda animals. 3 given the presumed relationship between her-da and abnormal collagen structure, and the predominance of type i collagen in skin, we hypothesized that altered biomechanical properties would be detected in tendons which are rich in type i collagen. to evaluate this hypothesis we compared the uts, modulus of elasticity, and energy to failure of forelimb deep digital flexor tendons (dft) from six herda horses to six age-matched controls. isolated dft was secured and pulled to failure on an instron s 1011 universal testing instrument using purpose-built cryogenic clamps. analysis of variance was executed using sas 9.2 proc glimmix program (sas institute, 2009). p-values 0.05 were identified as significant. uts and modulus of elasticity were significantly lower in herda dft when compared with controls (p o 0.0001); energy to failure did not differ between groups. these findings document abnormal biomechanics in herda tendon, leading us to postulate that lower uts and modulus of elasticity associated with the herda defect could convey a competitive advantage in the athletic disciplines in which this defect has segregated. (references on request). a proprietary herbal biocontamination product (bios) approved for cosmetic use in france, inhibits proliferation of medically relevant bacteria, mold, and viruses. these properties make bios potentially useful as a topical wound medication, prompting us to compare bios to silver sulfadiazine (ssd) in a distal extremity wound healing model in horses. 1 using general anesthesia, two 6.25 cm 2 wounds were aseptically created on the dorsomedial aspect of all limbs. for the duration of the study, two contralateral limbs were randomly chosen to be bandaged; the other two limbs were un-bandaged -with one limb of each group being treated with 10% bios and the other with ssd. for each limb the most proximal wound served as an untreated control. every 48 hours wounds were evaluated, digitally photographed, and perimeter and area determined using morphometric software (imagej, nih). analysis of variance did not identify significant differences between ssd or bios treatment for wound perimeter (p 5 0.76) or area (p 5 0.95). at individual time points the effect of bandaging was significant when area was evaluated (p 5 0.019) and trended toward significance for perimeter (p 5 0.084) comparisons, substantiating published reports that bandaging modifies wound healing. difference in perimeter and area between control and treatment were highly significant (p o 0.0001), substantiating the importance of topical treatment. over the study duration, effects of bandaging (p o 0.0001) and topical treatment (perimeter p o 0.0001; area p 5 0.0016) continued to be highly significant. bios performance in the equine distal extremity wound model was equivalent to ssd. both bandaging and topical treatment significantly impacted wound healing. this effect was compounded when both variables were evaluated over time. radiolabeled leukocytes are the only scintigraphic method currently available for identifying sites of infection and/or inflammation in horses; however the clinical applicability of this technique is limited by expense and poor efficacy. this pilot study compares the accumulation of 99m tc-labeled igg, peg-liposomes and leukocytes in an equine muscle abscess model. three mixed breed adult horses had 2 â 10 6 cfu s. equi zooepidemicus inoculated into the right semitendonosis to create an abscess. peg-liposomes were prepared via the film hydration method and labeled using 200 mci 99m tc-hexamethyl-propylene-amine-oxime ( 99m tc-hmpao). autologous leukocytes were obtained from 120 ml whole blood and labelled using 200 mci 99m tc-hmpao. commercial equine polyclonal igg was conjugated with the chelator hydrazinonicotinamide (hynic) and labelled with 200 mci 99m tc. radiopharmaceutical administration was initiated 24 hours after inoculation. horses 1 and 2 received 5 mg 99m tc-igg, 2.7 mmol/kg 99m tc-liposomes and 99m tc-leukocytes, with a 48 hour interval between each radiopharmaceutical. horse 3 received only 99m tc-leukocytes. scintigraphic examinations were performed at 8 and 21 hours post injection (p.i.) with each radiopharmaceutical. after the final study, horses were euthanized and tissue samples collected. the percentage of injected dose per kilogram of tissue (%id/kg) was calculated for the region of the abscess, normal muscle and multiple organs. scintigraphic examinations demonstrated increased radiopharmaceutical in the region of the abscess with all three techniques at both time-points. at 8 hours p.i. abscess-to-background ratio was highest using 99m tc-igg (3.7 ae 0.2). at 21 hours p.i. abscess to background ratio was highest using 99m tc-liposomes (5.9 ae 2). tissue biodistribution data revealed abscess to muscle ratios of 36 ( 99m tc-igg), 24 ( 99m tc-liposomes), and 4.1 ( 99m tc-leukocytes). this preliminary data demonstrates that 99m tc-liposomes, 99m tc-igg and 99m tc-leukocytes exhibit long circulating characteristics and accumulate at inflammatory/infectious foci after intravenous injection in horses. 99m tc-igg and 99m tc-liposomes appear to be superior to 99m tc-labelled leukocytes in this model. due to its low cost and ease of preparation, 99m tc-igg has great potential for clinical use where identification of infectious or inflammatory foci is necessary. digital hypothermia is used clinically to decrease the incidence of sepsis-related equine laminitis, a disease causing structural failure of digital laminae resulting in crippling lameness. due to the fact that hypothermia was recently reported to effectively decrease laminar expression of inflammatory molecules including pro-inflammatory cytokines, chemokines and cox-2 in equine laminitis, our laboratory is investigating the effect of hypothermia on central upstream signaling cascades which may induce expression of these diverse inflammatory molecules. the p38 mapk pathway has recently been reported to be a central component of inflammatory signaling in multiple diseases including human sepsis, and is currently being assessed as a therapeutic target. we thus hypothesized that 1) p38 mapk is upregulated and activated in affected laminae in equine laminitis and 2) digital hypothermia inhibits inflammatory mediator expression by blocking p38 mapk phosphorylation (indicator of p38 mapk activation). western hybridizations using both a total p38 mapk and a phospho-p38 mapk antibody were performed on archived pooled laminar samples from black walnut extract (bwe) model (10 control, 2 developmental (dev) groups [1.5h & 3h post bwe administration] and the onset of obel grade 1 lameness (og1) [n 5 5 each]) and carbohydrate overload (cho) models (con [n 5 8], dev [n 5 6], og1[n 5 6]) of laminitis, and individual laminar samples from two groups of horses from a digital hypothermia (dh) study. in the dh study, one forelimb of each horse was kept at approximately 41c in ice water and the other at ambient temperature following administration of 10 g/kg oligofructose (of). dorsal laminae were harvested for snap freezing at either 24 hours after of administration (dev, n 5 7) or at the onset of lameness (og1, n 5 6) using protein extracted from treated and untreated digital laminae of each horse. increased laminar concentrations of phospho-p38 mapk were present in the developmental periods (1.5h and 3h) in the bwe model, and in both the dev and og1 periods in the cho laminitis models. however, digital hypothermia had no effect on laminar phospho-p38 mapk concentrations. thus, p38 mapk is activated in affected laminae in multiple models of laminitis, but does not appear to be the central signaling cascade through which hypothermia works to block the expression of inflammatory molecules. therefore, p38 mapk is not likely to be a viable therapeutic target as a sole source for blocking the multiple inflammatory signaling mechanisms inhibited by local hypothermia. abstract e-60 does cefquinome penetrate the blood brain barrier in the normal horse? hollis ar 1 duggan ve 2 and corley ktt 3 . 1 scott dunn's equine clinic, berkshire, uk; 2 university college dublin, dublin, ireland; 3 anglesey lodge equine hospital, the curragh, ireland. meningitis is a rare but serious condition that occurs in both foals and adult horses. there is currently a restricted choice of antimicrobials that are both safe to use in horses and penetrate the blood brain barrier. cefquinome is a fourth generation cephalosporin that has activity against streptococcus, the most commonly reported causative organism in adult horse meningitis. therefore, if cefquinome were to achieve therapeutic concentrations in cerebrospinal fluid following routine administration, this would be an exciting advance for the treatment of meningitis in the horse. 5 mature, healthy horses were used on 2 separate occasions, seven days apart, in a crossover design. each horse was administered either cefquinome (1 mg/kg) or saline (equivalent volume). cerebrospinal fluid was collected via atlanto-occipital puncture under general anaesthesia 1 and 4 hours after administration of cefquinome or saline placebo. blood samples were collected prior to, and 1 and 4 hours after administration of cefquinome or placebo. all samples were analysed for the presence of cefquinome by a laboratory masked to treatments administered. cefquinome was detectable in the cerebrospinal fluid in all horses 4 hours after intravenous administration, and in 2 horses 1 hour after administration. cefquinome penetrates the blood-brain barrier and it is therefore a potential treatment for equine meningitis. further investigation of the pharmacokinetics and pharmacodynamics of cefquinome in the cerebrospinal fluid is warranted to establish the optimum intravenous dose. the purpose of this study was to determine if enrofloxacin alters the pharmacokinetics of firocoxib in the horse. firocoxib is a coxibclass nonsteroidal anti-inflammatory drug (nsaid) approved for use in horses to control pain and inflammation associated with osteoarthritis. dosages of firocoxib are species dependent, with the recommended dose for horses being 0.1 mg/kg as an oral paste every 24 h. the main elimination pathway of firocoxib is hepatic; however the effects of concurrent administration of drugs that may inhibit its metabolism have not been evaluated. enrofloxacin is a synthetic antibacterial agent from the flouroquinolone group developed for veterinary use. it is primarily used for gastrointestinal, urogenital, skin and respiratory tract infections in various animals. a well acknowledged problem associated with flouroquinolone usage is their effect on the metabolism of other drugs. co-administration of multiple drugs can result in unpredictable therapeutic outcomes. often it is either diminished therapeutic efficacy or increased toxicity of one or more of the administered drugs. various pharmacokinetic interactions between antimicrobials and nsaids have been described. six healthy, adult mares were administered 0.1 mg/kg of firocoxib orally. samples were collected by direct venipuncture of the jugular vein at 0 (control), 15, 30, and 60 min, 2, 4, 8, 12, 16, 24, 48, 72 , and 96 h after administration. after a 20 day washout period the six horses were pretreated 3 days with enrofloxacin 5 mg/kg intravenously every 24 h then on the fourth day given 0.1 mg/kg of firocoxib orally. samples were collected at 0 (control), 15, 30, and 60 min, 2, 4, 8, 12, 16, 24, 48, 72 , and 96 h after administration. all samples were stored at à801c until analysis using a validated hplc method. the t1/2, c max , t max , auc 0-24 and auc 0-f after firocoxib administration were 30. 66 angiotensin converting enzyme (ace) inhibitors improve survival and quality of life in humans and small animals with cardiovascular and renal disease. there is limited information regarding their effects in horses. the purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ace inhibition in horses. six healthy horses were administered quinapril at 120 mg iv, 120 mg po or 240 mg po in a 3-way crossover design. blood was collected at predetermined times for measurement of quinapril and quinaprilat concentrations using high pressure liquid chromatography, as well as ace concentrations using a radioenzymatic assay. normally distributed data were analyzed with one way repeated measures analysis of variance (rm-anova) and non-normally distributed data were analyzed using friedman rm_anova on ranks. significance was set at p o 0.05. no adverse effects were observed during the study period. plasma quinapril concentrations were low and rapidly declined after iv administration. quinaprilat concentrations were below the limit of quantification (0.1 mg/ml). ace activity was significantly decreased from baseline at 0.5 and 1 hour after iv dosing and at all timepoints after oral dosing. maximum % ace inhibition was 72, 53 and 47% with the iv, high and low oral doses, respectively. these results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption and results in inhibition of ace in healthy horses. controlled studies in clinically affected horses are indicated. this study determined the pharmacokinetic profile of firocoxib in healthy neonatal foals. foals are more sensitive to the side effects of nsaid, primarily due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. firocoxib, a novel, second generation nsaid, is reported to have reduced side effects due to cox-2 selectivity. the pharmacokinetic profile of firocoxib in neonates has not been established. we hypothesized that firocoxib given po to neonatal foals would achieve therapeutic concentrations in plasma. seven healthy foals of mixed gender were administered 0.1 mg/kg firocoxib po q24h for nine consecutive days, commencing at 36h old. blood was collected for firocoxib analysis at 0 (dose #1 only), 0.25, 0.5, 1, 2, 4, 8, 16 and 24h after doses #1, 5 and 9. for all other doses (2, 3, 4, 6, 7 and 8) blood was collected immediately prior to the next dose (24h trough). elimination samples were collected after dose #9. plasma was stored at à801c until analysis. physical examinations were performed on foals daily and body weight obtained every two days during the sampling period. analysis of plasma samples by liquid chromatography-mass spectrometry revealed firocoxib was rapidly absorbed. after the initial dose, a maximum plasma concentration was reached in 30 min, minimal accumulation after repeat dosing occurred and steady state was obtained after approximately four doses. after the final dose, plasma drug concentration decreased in a linear manner with an estimated terminal t1/2 of 11h. seventy-two hours after the final dose, firocoxib was not detectable (o 2ng/ml). erythrocytosis is reportedly a rare finding associated with hepatocellular carcinoma in horses. the purpose of this study was to determine the relative frequency of erythrocytosis and the clinicopathologic abnormalities and hepatic histopathology associated with erythrocytosis in horses with liver disease. ninety-seven horses aged !1 year with clinicopathologic or clinical signs of liver disease, a complete blood count (cbc), and hepatic histopathology were included. information on cbc, biochemical variables, and hepatic histopathology was collected from records. data from horses with erythrocytosis (packed cell volume 4 45%) were compared to those without using the mann-whitney rank sum test with significance set at p o 0.05. there were no differences between groups in white blood cell count, gamma-glutamyl transferase, sorbitol dehydrogenase, aspartate aminotransferase, and alkaline phosphatase activities, total protein, albumin, globulin, blood urea nitrogen, or glucose concentrations. fibrosis (64%), biliary hyperplasia (56%), inflammatory infiltrate (50%), megalocytosis (25%), degeneration (25%), necrosis (25%), cholestasis (25%), anisocytosis and anisokaryosis (11%), and lipidosis (3%) were observed in livers of horses with erythrocytosis. neoplasia (3%) was observed rarely. this study reports a high frequency of erythrocytosis in horses with liver disease. erythrocytosis is associated with higher total bilirubin and serum bile acids concentrations. common histopathologic changes include fibrosis, biliary hyperplasia, and inflammatory infiltrate. hepatic neoplasia was rare. this study was performed to determine if horses diagnosed with equine proliferative enteropathy (epe) from lawsonia intracellularis (li) infection had long term effects from disease based on their sale price as yearlings and race earnings. a retrospective review of medical records of thoroughbred horses that were treated for lawsonia intracellularis infection between january 1, 2002 and january 31, 2008 at hagyard equine medical institute in lexington, kentucky was performed. three criteria were used for inclusion in this study. first, each horse had presumptively been diagnosed with li based on physical examination findings such as ventral edema, diarrhea, lethargy, or poor body condition. second, horses had hypoalbuminemia of less than 2.5 mg/dl (normal reference range: 3.4-4.5 mg/dl). third, each horse had a positive fecal polymerase chain reaction (pcr) for li, a positive serum immunoperoxidase monolayer assay (ipma), or both. an ipma titer greater than or equal to 60 was considered positive for disease. 116 horses met the initial criteria. 36 of the 116 horses sold at public auction as yearlings. the sale price of these horses was compared to the average sale price of all yearlings by the same sire as the affected horse (control group). 30 of the 116 horses raced in the united states. their monetary earnings from racing were compared to the average monetary earnings of all progeny by the same sire as the affected horse (control group). earnings of horses that were between 3 and 7 years of age (23/30 horses) at the conclusion of the study were compared to the lifetime average earnings of the stallion's progeny. earnings from horses that were two years of age (7/30) at the end of the study were compared to the two year old average earnings of the stallion's progeny. monetary earnings from all races prior to december 31, 2008 were included in the study. 12 horses both sold at public auction and raced. as well as being included in the total number of horses that sold and raced, their sale records and monetary earnings were compared to the averages from their respective sire as a separate group. this retrospective study indicated that yearling horses previously infected with li do not sell for as much at public auction as their herdmates, but their monetary earnings from racing are not significantly different from other horses. these results should assist practitioners in guiding owners in determing if treatment of horses with epe is appropriate and it may aid in reassuring owners that despite the poor condition of the horse during and shortly after the course of disease, horse may still have future athletic potential. this abstract was presented at the aaep in december 2009. bronchopneumonia caused by streptococccus equi subsp. zooepidemicus (s. zooepidemicus) is one of the most important causes of morbidity in weanling foals. ceftiofur crystalline free acid (ccfa) is a long acting third-generation cephalosporin antimicrobial recently approved for the treatment of bronchopneumonia associated with s. zooepidemicus in adult horses. the objective of the present study was to determine the disposition of ccfa in plasma and pulmonary epithelial lining fluid (pelf) of weanling foals. six healthy 4-to 5month-old weanling foals were administered a single intramuscular injection of ccfa at a dose of 6.6 mg/kg of body weight. concentrations of desfuroylceftiofur acetamide (dca) and related metabolites were measured by use of ultra-high performance liquid chromatography and tandem mass spectrometry. following im administration, median time to maximum plasma and pelf concentrations was 24 h (12-48 h) . mean (ae sd) peak dca concentration in plasma (1.44 ae 0.46 mg/ml) was significantly higher than that in pelf (0.46 ae 0.03 mg/ml). terminal half-life of dca in plasma (74.8 ae 20.9 h) was not significantly different from that of pelf (58.5 ae 11.4 h). time above the therapeutic target of 0.2 mg/ml was significantly longer in plasma (185 ae 20 h) than in pelf (107 ae 31 h). based on the results of the present study, intramuscular administration of ccfa at a dose of 6.6 mg/kg would be appropriate for the treatment of bronchopneumonia caused by s. zooepidemicus and other susceptible pathogens in weanling foals. fgf-23 is secreted by osteocytes and osteoblasts in response to hyperphosphatemia. fgf-23 enhances phosphaturia and is postulated to have a central role in the development of secondary renal hyperparathyroidism. hyperthyroid cats have elevated plasma phosphate and parathyroid hormone concentrations, which may in part be associated with underlying chronic kidney disease (ckd). the aim of this study was to determine if plasma fgf-23 concentrations were associated with the presence of underlying ckd in hyperthyroid cats, and to investigate the changes in plasma fgf-23 concentrations that occur following treatment of hth. hyperthyroid cats were recruited from two london-based first opinion practices between 1999 and 2009. cats that were azotemic at diagnosis were excluded. hth was treated with anti-thyroid medication alone or in combination with thyroidectomy. cats were included in the study if they had a plasma total thyroxine concentration o 40 nmol/l documented for a six month period following commencement of treatment. cats were classified as having azotemic ckd if they developed renal azotemia within six months of establishment of euthyroidism. otherwise cats were deemed to have normal renal function. stored edta plasma samples were assayed for fgf-23 using a recently validated elisa. the mann-whitney u test and the wilcoxon signed rank test were used to compare between the groups and assess the response to treatment respectively. results are reported as median [25 th , 75 th percentiles]. correlations were made using spearman's correlation coefficient. thirty one cats with hth (14 azotemic and 17 non-azotemic) were included in the study. plasma phosphate concentrations decreased following treatment in cats that did not develop azotemia (4.84 [3.91, 5.64] mg/dl vs. 3.91 [3.38, 4.37] mg/dl; n 5 13, p 5 0.01) whereas plasma phosphate concentrations did not change significantly following treatment in cats that did develop azotemia (4.28 [3.81, 5.64] mg/ dl vs. 4.22 [3.10, 5.33] mg/dl; n 5 14, p 5 0.158). plasma fgf-23 concentrations were significantly higher in cats that developed azotemia than cats that did not at both pre treatment (211.7 [176.4, 356 .3] pg/ml vs. 148.3 [118.8, 274 .9] pg/ml; p 5 0.039) and post treatment (514.0 [250.2, 800.0] pg/ml vs. 195.1 [160.7, 287 .3] pg/ml; p 5 0.001) timepoints. plasma fgf-23 concentrations increased following treatment in both azotemic (p 5 0.004) and non-azotemic groups (p 5 0.025). plasma fgf-23 concentrations and plasma phosphate concentrations were not correlated at baseline (r s 5 0.189, p 5 0.335) or following treatment (r s 5 0.136, p 5 0.472). plasma fgf-23 concentrations were higher in pre-azotemic cats than non-azotemic cats and increased following treatment of hth. the reason that fgf-23 concentrations increased following treatment, particularly in the face of decreasing plasma phosphate concentrations in cats that remain non-azotemic, is unclear but may be related to the decline in glomerular filtration rate. hyperthyroidism is a disorder resulting from the excessive production and secretion of t4 and t3 by the thyroid gland. although the disorder and its pathological lesions have been well studied and described the cause remains illusive. whole blood and solid tissue samples from non-diseased, severe disease and mild disease cats based on t4 levels and thyroid histology were used in this study. whole blood samples from 29 non-disease cats, 28 severe disease cats and 17 mild disease cats as well as solid thyroid tissue samples from 30 non-disease cats, 31 severe disease cats and 27 mild disease cats were collected and processed. the resulting total rna samples were used for genechip analysis using our custom feline gene chip designed by affymetrix. data analysis was performed using the partek s gs software for gene expression data. the robust multichip average algorithm was used for background adjustment, normalization, and probe-level summarization of the raw data. anova analysis was performed to find significant differentially expressed genes with a minimal false discovery rate control of 0.1 and a fold change of 1.3 in each direction. during the mild disease state, pathways associated with dna damage and apoptosis are most prominent. at later stages when the histopathological disease is more severe in addition to the aforementioned pathways others associated with tgf-beta signaling, cell adhesion and extracellular matrix remodeling take more prominence. the analysis of this unique data set generated from the use of our proprietary genechip revealed molecular mechanisms that are associated with the transition from non-disease, to mild disease to severe disease, in the thyroid tissue as well as the blood. these mechanisms could provide insights into the causes of the disease and identify potential new therapeutic and diagnostic targets. although it is well established that concurrent chronic kidney disease (ckd) develops in about 30% of hyperthyroid cats, no one has reported the use of the iris staging system for ckd before and after treatment of these hyperthyroid cats. the purpose of this study was to compare the effects of treatment in hyperthyroid cats with known stage 1 and 2 ckd in order to determine the effects of restoring euthyroidism or inducing hypothyroidism has on the iris stage in these cats. we evaluated 36 hyperthyroid cats (median age, 14 years) in this study. one day prior to treatment, serum t 4 concentration, serum chemistry analysis, complete urinalysis, and urine protein-to-creatinine ratio (upc) were measured. all cats were again evaluated with the same parameters again 3 months after treatment with 131 i. prior to treatment, 26 (72%) of the 36 cats had no evidence of azotemia (serum creatinine o 1.6 mg/dl), whereas 10 cats (28%) had stage 2 ckd (serum creatinine, 1.6-2.8 mg/dl). in the 36 cats, iris staging revealed proteinuria in 33 cats (92%), 21 with borderline proteinuria (upc, 0.2-0.4) and 12 with overt proteinuria (upc 4 0.4). hyperthyroidism was cured in all 36 cats (median post-t4, 1.3 mg/dl). all cats had a good response to treatment; there were no signs of ckd except for polyuria and polydipsia in some cats. a significant (p o 0.001) increase in median values for both serum urea nitrogen (26 mg/dl to 31 mg/dl)) and creatinine (1.1 to 1.7 mg/dl) occurred after treatment. nine of the 26 cats (34.6%) classified as nonazotemic or iris stage 1 prior to 131 i progressed to stage 2 ckd after 131 i. all 10 cats with stage 2 ckd before treatment remained azotemic after 131 i, with 5 cats remaining in stage 2 ckd, and 4 cats progressing to stage 3 ckd (serum creatinine, 3.1-3.7 mg/dl). there was a significant inverse relationship (p 5 0.002) between pretreatment urine specific gravity (usg) and post-treatment serum creatinine in the 36 cats. of the 19 cats with post-treatment serum creatinine values 4 1.5 mg/dl (stage 2 to 3 ckd), 15 (79%) had pretreatment usg of o 1.040. in contrast, in the 17 cats with post-treatment serum creatinine values o 1.5 mg/dl, only 3 (18%) had pretreatment usg of o 1.040. a significant (p o 0.001) decrease in median upc from 0.3 to 0.1 occurred after treatment, but there was no relationship between degree of proteinuria and iris stage in these cats. two cats developed iatrogenic hypothyroidism after 131 i, diagnosed by finding low serum t 4 and high ctsh concentrations. both hypothyroid cats had progressed from stage 1 before treatment to stage 2 and 3 ckd, respectively, after 131 i; after thyroxine replacement, serum creatinine decreased to near pretreatment concentrations in both cats. conclusions: 1) iris stage 2 ckd is common in untreated hyperthyroid cats. 2) progression to next higher iris stage is common after treatment, but most cats with remain relatively asymptomatic for ckd. 3) usg may be helpful in predicting which cat's iris stage will progress after 131 i. 4) iatrogenic hypothyroidism worsens azotemia, an effect that appears reversible with replacement therapy. home blood glucose monitoring (hbgm) of diabetic pets is likely to result in superior glycaemic control, minimizing episodes and impact of dangerous hypoglycaemia and reducing costs. nevertheless, it has proven difficult to objectively establish a clear benefit of hbgm using biological parameters (clinical signs, blood glucose, fructosamine). the current study aimed to assess the impact of hbgm on owner perceived quality of life (qol) aspects of diabetes mellitus (dm) treatment, using the recently validated psychometric tool diaqol-pet. owners of insulin treated diabetic cats were recruited to complete the 29-item tool, evaluating areas affecting the cat's and owner's qol, including: worry about pet's dm, hypoglycaemia, costs, owner's desire for autonomous control over the pet's dm, etc. item-weighted-impact-scores (iwis), reflecting frequency and importance ratings of each item, were calculated, as well as averageweighted-impact-scores (awis; average iwis of all items), as an overall measure of diabetes dependent qol. frequencies, iwis and awis were compared between owners practising hbgm and those who did not using mann whitney u test (significance p o 0.05). two hundred and eleven owners of insulin treated diabetic cats completed the diaqol-pet; 161 owners practised hbgm, whereas the remaining 50 did not practise any form of home monitoring (including urine glucose). iwis for 'excessive drinking' and 'owner wanting more control' were significantly different between the hbgm-group (mean1/-standard deviation: à2.011/à2.4 and à5.071/à4.8) and the non-hbgm-group (à3.361/à3.8 and à1.861/à3.2). there was no significant difference between the groups with regards to the iwis for other items, including 'worry about hypoglycaemia' or 'worry about pet's dm'. polydipsia was reported significantly more frequently in the non-hbgm-group and this was the reason for the difference between groups in this item's iwis as it was considered of equal importance. frequency and iwis of reported occurrence of hypoglycaemia signs were not significantly different. awis for both groups was not significantly different (hbgm: à1.851/à1.3; non-hbgm: à1.871/à1.1). the current study suggests that hbgm is predominantly practised by owners who desire more autonomous control over their cat's dm. the frequency of polydipsia was lower in the hbgm-group perhaps suggesting superior control. however, hbgm did not detectably affect the impact of the majority of qol-items, nor the frequency of hypoglycaemic episodes. overall diabetes dependent qol of diabetic cat and owner, as measured per diaqol-pet, was unaffected by hbgm. these data argue for the use of hbgm in selected pet-owner combinations rather than as part of a practice's standard dm management protocol, although further studies are indicated. insulin resistance is associated with impaired activation of the insulin signaling pathway in peripheral tissues such as skeletal muscle, visceral and subcutaneous (sc) adipose tissue. high plasma glucose, fatty acid and endotoxin levels are three major causes of insulin resistance in feline and human obesity and in type 2 diabetes mellitus. however, the mechanisms by which these factors influence insulin action are still unclear. therefore, our aim was to investigate the tissue-specific expression of crucial mediators of insulin action such as the insulin-receptor substrate 1 (irs1), the serine/threonine protein kinase b (pkb/akt) and of the principal insulin-dependent glucose transporter protein (glut4) in feline models of hyperglycemia, hyperlipidemia and subacute endotoxemia. healthy cats were infused through the jugular vein with glucose (n 5 5), lipids (n 5 6) or lipopolysaccharide (lps; n 5 5) for 10 days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: 25-30 mmol/l; triglycerides: 3-7 mmol/l) or to induce a systemic low-grade inflammation (lps; rectal temperature: 39.2-40.51c), respectively. healthy control cats were infused with saline (n 5 10). on day 10, specimens were collected from skeletal muscles, visceral and sc fat and processed for irs1 mrna expression, total and phosphorylated pkb/akt and glut4 protein expression. gene transcripts of irs1 were not different between the groups. compared to controls, skeletal muscle pkb/akt phosphorylation was 91% lower in cats infused with glucose (p o 0.01); lipid-infused cats showed a trend for a decrease in pkb/akt phosphorylation (31% lower than saline) and had decreased glut4 expression (p o 0.05) in muscle. total (p o 0.01) and phosphorylated (p o 0.05) pkb/akt protein expression were decreased in the sc adipose tissue of lps-infused cats compared to controls. in these cats, phosphorylation of pkb/akt protein was also decreased in visceral fat (p o 0.01). sustained hyperglycemia and, to a lesser extent, hyperlipidemia impaired insulin signaling and glucose transport pathways primarily in skeletal muscle; endotoxemia reduced insulin sensitivity mainly in adipose tissues. thus, the development of insulin resistance in response to hyperglycemia, hyperlipidemia or endotoxemia might be affected by tissue-specific mechanisms in cats. separately used, single photon emission computed tomography (spect) and computed tomography (ct) both lack sensitivity and are additionally hampered by a poor anatomical location capacity and a lack of specificity, respectively. these drawbacks suggest an interest in the fusion of images obtained by the 2 techniques. the aim of this study is to test spect/ct fusion performance in dogs with insulinoma. inclusion criteria were: 1/ a biological diagnosis of insulinoma; 2/ an examination by high resolution ct scan and 111 in-pentetreotide spect followed by spect/ct fusion; 3/ a surgical or post mortem examination completed by histopathological analysis. spect examination showing abnormal foci and ct scan showing pancreas, lymph nodes (ln) or liver abnormalities were considered positive. in case of double positivity, presence (imp1) or absence (imp-) of superimposition of abnormal images was noted. ten dogs were included. in 2/10 dogs, superimposition of abnormalities couldn't be tested. ct scan detected 3 abnormal images [2 pancreatic nodules (pn), 1 enlarged ln (eln)] while spect failed to show any abnormal uptake. both dogs became euglycemic after removal of pn and ln designed by ct scan. in 6/ 10, all abnormal images were classified as imp1 [6 pn, 1 eln and 1 diffuse hepatic infiltration (dhi)]. surgery performed on 5/6 resulted in euglycemia in 4; 1 dog remained hypoglycemic after partial removal of 1 pn. pn localization and dhi were confirmed after necropsy in the 6 th dog. in 2/10 dogs imp1 and imp-images were both recorded. in 1 dog, a dhi was classified as imp1 but pn localization was imp-: localized in the left lobe by ct scan and in the corpus by spect, the latest localization being confirmed after necropsy. in the other dog pn localization was imp1 but a diffuse spect signal superimposing to the liver considered as normal on ct scan was noted. hepatic biopsy confirmed spect results. this study confirms an imperfect sensitivity of both ct scan and spect. it confirms that ct scan can be associated with unspecific abnormal images. subject to a confirmation on a larger cohort of dogs, it indicates that imp1 images provide specific detection and accurate localization of canine insulinomas' primary lesions and metastasis. the majority of dogs with primary hypoadrenocorticism (ph) reveal clinical and laboratory abnormalities of gluco-and mineralocorticoid deficiency. in some of them sodium and potassium levels are normal, a phenomenon currently called atypical addison's. it has been postulated that in those cases adrenal destruction is confined to the zona fasciculata/reticularis, resulting in isolated glucocorticoid deficiency. however, there are no histological studies confirming a normal zona glomerulosa and in most reported cases diagnosis was based solely on low post-acth cortisol levels. the aim of the study was to evaluate aldosterone (aldo) levels in dogs with ph with and without electrolyte abnormalities. seventy dogs with newly diagnosed ph were included. aldo concentrations (ria, coat-a-count s , siemens) were measured before and 60 min after administration of 250 mg synthetic acth (synacthen s , novartis) iv. results were compared to those of 19 healthy dogs and 17 dogs with diseases mimicking ph. to confirm that peak concentrations were not missed aldo was additionally measured 15, 30 and 45 min after acth in 19 dogs (5 with ph, 14 with ph mimicking diseases). results were analysed by means of non-parametric statistical methods (p o 0.05). post-acth aldo was significantly lower in dogs with ph (0-253 pg/ml, median 0 pg/ml) than in healthy dogs (46-602 pg/ml, median 187 pg/ml) and in dogs with ph mimicking diseases (0-639 pg/ml, median 155 pg/ml). low post-acth aldo was found in 67/70 dogs with ph, in 64/67 of them levels were below the detection limit of the assay. normal sodium and potassium levels were found in 5/67 dogs (7%), 6/67 dogs (9%) had hyponatremia and normal potassium, 56/67 dogs (84%) had hyponatremia and hyperkalemia. electrolyte abnormalities ranged from mild to severe. there was no correlation between post-acth aldo and sodium and a weak correlation between post-acth aldo and potassium (r 5 à0.28). aldo concentrations were not different 30, 45 and 60 min after acth. the results demonstrate that aldo levels are low in most dogs with ph independent of the degree of electrolyte abnormalities. this implicates that all three zones of the adrenal cortex are compromised and that there are mechanisms which allow maintenance of a normal electrolyte balance without aldo. definitive diagnosis of canine hypoadrenocorticism (ha) is based on inadequate cortisol secretion following adrenocorticotropic hormone (acth) administration. an abnormal serum sodium to potassium (na:k) ratio can be used to determine whether an acth stimulation test is warranted. the aim of this study was to examine the utility of combining the na:k ratio with white blood cell counts to determine whether an acth stimulation test is warranted. a retrospective review of medical records of dogs examined between 2005 and 2009 was performed. 53 dogs diagnosed with ha and 110 control dogs, in which a diagnosis of ha was excluded during the study period, were included. inclusion criteria for all 163 dogs were hospitalization with intravenous fluid therapy, a complete blood count, and serum na and k measurements at the time of initial examination. dogs were included in the ha group if they also had pre and post acth stimulation serum cortisol concentrations 1.0 mg/dl. dogs were included in the control group if they had resting or post acth stimulation serum cortisol concentration 4 2.0 mg/dl. exclusion criteria were recent administration of glucocorticoids, prior treatment of hyperadrenocorticism, or serum cortisol concentration 4 1.0 mg/dl but 2.0 mg/dl. continuous variables were compared between groups using the mann-whitney u test. receiver operating characteristic (roc) curves were produced to assess the sensitivity and specificity of detecting ha with various cutoffs for each variable. data is presented with 95% confidence intervals (ci) and statistical significance was defined as p o 0.05. the na:k ratio, neutrophil count and neutrophil:lymphocyte ratio were significantly lower in dogs with ha than in dogs without ha (p o 0.01 for each). lymphocyte and eosinophil counts were significantly higher in dogs with ha compared to dogs without ha (p o 0.001 for each). the areas under the curve by roc analysis were largest for na:k ratio (0.87, ci:0.81-0.92) and lymphocyte count (0.85, ci:0.78-0.90). a na:k ratio 39.6 was 100% sensitive (ci:93-100%) but only 15% specific (ci:9-23%) for detecting ha. a lymphocyte count ! 0.77 x10 3 cells/ml was 100% sensitive (ci:93-100%) and 35% specific (ci:27-45%). conversely a na:k ratio 20.1 was 51% sensitive (ci:37-65%) but 100% specific (ci:97-100%) and a lymphocyte count ! 6.1 â 10 3 cells/ml was 9% sensitive (ci:3-20%) but 100% specific (ci:97-100%). a na:k ratio 35.5 was 96% sensitive (ci:87-100%) and 35% specific (ci:26-44%) for detection of ha and a lymphocyte count ! 0.79 â 10 3 cells/ml was 98% sensitive (ci:90-100%) and 36% specific (ci:27-46%) for detection of ha. a combination of this na:k ratio ( 35.5) and lymphocyte count (! 0.79 â 10 3 cells/ml) was 96% sensitive (ci:87-100%) and 55% specific (ci:47-66%) for detection of ha. these results indicate that the combination of lymphocyte count and na:k ratio results in a better screening test for ha than the use of the na:k ratio alone. pheochromocytoma is a malignant, catecholamine-producing, adrenomedullary tumor. clinical signs resulting from excessive catecholamine secretion are typically non-specific, making differentiation from other adrenal tumors a challenge. elevated plasma concentrations of the catecholamine breakdown products metanephrine (mn) and normetanephrine (nmn) are used to identify pheochromocytoma in humans. this study tested the hypothesis that plasma metanephrine concentrations are greater in dogs with pheochromocytoma than in dogs with other adrenal neoplasms, healthy dogs and dogs with non-adrenal illness. edta plasma was collected from healthy dogs and unwell, hospitalized dogs with non-adrenal illness, pheochromocytoma and cortical tumors between april 2007 and october 2010. samples were stored at à801c before measurement of free mn and nmn concentrations using high pressure liquid chromatography at the central laboratory for clinical chemistry at the university of groningen (33 samples) or the mayo clinic, rochester, minnesota (7 samples). kruskal-wallis tests followed by dunn's multiple comparison analysis were used to compare results between groups. significance was set at p o 0.05. results are reported as median [range] . eight dogs with pheochromocytoma, 5 healthy dogs, 15 dogs with non-adrenal illness and 12 dogs with cortical tumors were sampled. pheochromocytoma was diagnosed histologically (7 dogs) or cytologically (1 dog). cortical tumors were diagnosed histologically (9 dogs) or by response to trilostane treatment after obtaining consistent endocrine test results (3 dogs occult hyperadrenocorticism (hac) has been theorized to exist in which excess adrenal sex hormone secretion induces the clinical signs and laboratory changes associated with classic hac. however, the ability of sex hormones to cause such alterations has never been closely evaluated. if sex hormones can cause a syndrome similar to classic hac, they should be able to induce expression of classic glucocorticoid-induced genes. the purpose of the study was to determine if in vitro expression of the gene for corticosteroid-induced alp (cialp) could be induced by clinically relevant concentrations of cortisol and sex hormones believed to cause occult hac. canine hepatocytes were purchased from a commercial source (cellzdirect or invitro) in 6-well plates. upon arrival (3-4 plates per shipment), the cells were allowed to recover in general media per supplier recommendations. after 4 hrs, media was changed to william's e media (-l-glutamine) containing concentrations of cortisol or sex hormones that have been documented in the literature in dogs with hac or with purported occult hac. each plate was treated with a different hormone (cortisol, 17-hydroxyprogesterone [17ohp], progesterone, estradiol or androstenedione), and each well contained a different concentration (starting with no hormone added as a negative control) to evaluate a dose response. media was changed daily. after 5 days of hormone exposure, rna was extracted. reverse transcription was performed and the product used for quantitative pcr for cialp and beta-actin (roche lightcycler) using a gene-specific fluorescent probe for detection. standard curves were created for each gene. all samples and standards were run in duplicate. using the lightcycler software (vers 4.0), cialp expression was normalized to that of beta-actin. fold change in expression was determined relative to the negative control. each sex hormone was used to treat 3 plates; one plate in each shipment was treated with cortisol as a positive control. for cortisol, a dose response was seen in expression of the cialp gene. compared to no cortisol, 10, 50, 100, 150, 250 and 500 nmol cortisol increased expression 2.8, 4.6, 7.1. 9.3, 9.9 and 9.8 fold, respectively. a 2-fold increase is considered significant (j.vandesompele et al genome biol 2002). expression of cialp was not significantly induced in response to any concentration of 17ohp (10 nm maximum), progesterone (5 nm maximum), estradiol (max 400 pm maximum) or androstenedione (100 nm maximum). we conclude that in vitro these sex hormones do not induce expression of the cialp gene which is classically induced by cortisol in vivo; indeed, elevated serum cialp activity is a hallmark of classic hac. thus, the ability of the sex hormones to induce the gene in vivo must be questioned and evaluated. measurement of sex hormones has been advocated as an adjunct means for diagnosing typical hyperadrenocorticism (hac), i.e. disease due to excess cortisol secretion, as well as for diagnosis of atypical hac, i.e. disease due to excess adrenal sex hormone secretion. however, measurements in either setting have not been widely studied. therefore, our objectives were: 1. to determine the sensitivity of 17-hydroxy-progesterone (17ohp) and estradiol concentrations pre-and post-acth for diagnosis of typical hac. 2. to determine the specificity of 17ohp and estradiol concentrations preand post-acth for diagnosis of occult hac. dogs that had pdh (n 5 12), dogs that were suspected to have hac but proven not to (had non-adrenal illness [nai, n 5 89]) or dogs that were healthy (n 5 20, used to establish reference ranges [rr]) were enrolled. acth stimulation tests were performed (5 mcg/kg cosyntropin iv); blood samples were drawn pre and 60min post; 17ohp and estradiol were measured by previously validated radioimmunoassays. a kruskal-wallis rank sum test was used to compare values between the groups. significance was set at p o 0.05. for basal and acth-stimulated 17ohp concentrations, the rr were determined to be 0.03-0.6 ng/ml (mean ae 2 s.d.; range 0.03-0.55) and 0.3-2.2 ng/ml (range 0.31-1.95), respectively. in pdh dogs, 3 and 7 had basal and post-acth 17ohp concentrations above the rr, respectively; in the nai group, 23 and 25 dogs had concentrations above the rr, respectively. thus, the sensitivity of basal and post-acth 17ohp measurement for diagnosis of hac is 25% and 58%, respectively. specificity of diagnosis is 72% and 74%, respectively. post-acth 17ohp concentration was significantly different between groups. for basal and stimulated estradiol concentrations, the rr were determined to be 81-180 pg/ml (range 89-195) and 71-170 pg/ml (range 74-151), respectively. for both basal and stimulated estradiol, 4 pdh dogs (n 5 9) had concentrations above the rr; for those with nai (n 5 80), 14 and 15 had concentrations above the rr, respectively. thus, the sensitivity of estradiol measurement for diagnosis of hac is 44% for both pre-and post-acth. specificity of estradiol for diagnosis for hac is 83% and 81% for pre-and post-acth, respectively. overall, 23 dogs with nai had at least one elevated estradiol concentration (total specificity 70%). post-acth estradiol concentration was not significantly different between groups. we conclude that use of 17ohp and estradiol concentrations for diagnosis of hac can be problematic. sensitivity and specificity are relatively low, potentially leading to misdiagnoses. diabetes mellitus is one of the most common feline endocrinopathies and is considered to have a similar pathophysiological basis to human type 2 diabetes. several studies have identified risk factors for development of diabetes mellitus in cats, which include age, obesity, inappropriate diet and physical inactivity. however, to date, no specific genetic risk factors have been identified. genome-wide association studies in humans have identified several genes that predispose to obesity and/or diabetes mellitus, one of which is the melanocortin receptor 4 (mc4r) gene. the aim of the current study was to identify polymorphisms (snps) in the feline mc4r gene and to use these to perform a case:control study to determine whether these candidate gene snps were associated with diabetes mellitus in cats. genomic dna from 10 cats (6 domestic short hair [dsh], 4 burmese) was initially analysed by pcr and direct sequencing using felmc4r-specific primers, which identified a missense mutation (mc4r:c.92 c 4 t) in the region encoding the extracellular domain of the receptor protein in dsh cats only. one hundred and nineteen dsh cats were subsequently recruited into the case:control study. fifty nine cats were obese diabetic (29 male, 30 female), mean age 11.8 years (range 6-18y); mean weight 6.68 kg (range 5.15-10 kg). sixty lean cats were used as controls (30 male, 30 female), mean age 13.81 years (range 9-19y), mean weight 3.99 kg (range 2.56-5.68 kg). the t to c base change alters a restriction site in the sequence recognized by the enzyme bstoi, such that dna from cats with the mutant (c) allele can be cut, whereas that from the wild-type (t) allele cannot. primers were designed that flanked the mutation to allow pcr amplification of this region of mc4r from genomic dna obtained from edta blood. the pcr products were purified and subject to restriction fragment length polymorphism (rflp) analysis. bstoi digestion products were then analysed by agarose gel electrophoresis. of the 59 diabetic cats, 32 (54%) were homozygous for the mutation (cc), compared to 21 (35%) of 60 control cats. statistical analysis (two tailed fisher's square test) revealed that this difference between groups was statistically significant (p 5 0.0431). in conclusion, this pilot study has identified a missense mutation in the coding sequence of mc4r. this could be an important predisposing factor for development of diabetes and/or obesity in dsh cats. polymorphisms in a similar region of human mc4r predispose to obesity, which in turn is a major risk factor for type 2 diabetes. hyperadrenocorticism (hac) is one of the most common endocrine disorders of dogs. the two most effective medical treatments are trilostane (vetoryl s ) and mitotane (lysodren s ). previous studies evaluating the effect of treatment on aldosterone secretion measured the hormone at 60 min post-acth administration. however, the optimal sampling time would be at the time of maximal secretion, which occurs 30 minutes after the 5 mg/kg dose commonly used for the test (carlson et al, jvim, 2010). thus, the true effect of either medication on aldosterone secretory capacity is unknown. our objectives were: 1) to assess and compare the effect of treatment with trilostane and mitotane in dogs with pituitarydependent hac (pdh) on aldosterone secretory reserve at 30 min post-acth stimulation and 2) to determine if changes in aldosterone concentration at that time correlate with changes in serum sodium and potassium concentrations. forty-six dogs being treated for pdh with either mitotane (n 5 30) or trilostane (n 5 16) have been enrolled. the dogs could be treated for any length of time. all had acth stimulation tests performed (5 mcg/kg cosyntropin iv); blood samples were drawn before and at 30 and 60 min post-acth for monitoring of cortisol and aldosterone concentration using previously validated radioimmunoassays. ten historical normal controls were also included. serum sodium and potassium concentrations were measured in the basal samples. a kruskal-wallis rank sum test was used to compare values between normal dogs and those treated with mitotane or trilostane. linear regression analysis was used to determine if a correlation existed between electrolyte and aldosterone concentrations or between cortisol and aldosterone concentrations. significance was set at the p o 0.05 level. acth-stimulated aldosterone concentrations in mitotane-treated but not trilostane-treated dogs were significantly lower than that in normal dogs at both the 30 and 60 min time points. no difference was detected between aldosterone concentrations at 30 and 60 min after acth injection in either treatment group. a positive correlation existed between the 60-min cortisol and 30-min aldosterone concentrations in the trilostane-treated group (r 5 0.813), i.e. the peak post-acth concentration for each hormone, but not in dogs treated with mitotane. basal serum sodium and potassium concentrations were not correlated with the basal aldosterone concentration in either treatment group. in conclusion, treatment with mitotane resulted in decreased aldosterone secretory reserve, but this did not correlate with hyperkalemia or hyponatremia. measurement of aldosterone concentrations is not predictive of electrolyte concentrations. previously presented at the auburn university phi zeta research emphasis day, november 10, 2010. antioxidant depletion is documented in humans with hyperthyroidism, and is reversible with treatment. in addition, antioxidant depletion has been shown to increase the risk of methimazole toxicity in rats. the primary aim of this study was to determine whether deficiencies in glutathione (gsh), ascorbate (aa), or vitamin e, along with increases in urinary 8-isoprostanes, were present in hyperthyroid cats, and were reversible after radioiodine treatment. a secondary aim was to determine whether antioxidant abnormalities were associated with a prior history of methimazole toxicity. ongoing prospective, controlled, observational study. otherwise healthy client-owned hyperthyroid cats presenting for radioiodine therapy (n 5 26 to date) and healthy age-matched controls (n 5 32 to date) were recruited. all cats were screened with cbc, biochemical panel, urinalysis, and t4, as well as red blood cell (rbc) gsh, plasma aa, plasma vitamin e, and urinary 8-isoprostanes. hyperthyroid cats were re-evaluated 2 months after radioiodine treatment. unlike in humans, median blood antioxidants were not significantly different in hyperthyroid cats (gsh 1.5 mm; aa 11.4 mm, and vitamin e, 19 g/ml) compared to controls (gsh 1.4 mm; aa 12.5 mm, and vitamin e, 17 g/ml). results for urinary isoprostanes are pending, and associations with methimazole toxicity will be investigated after full recruitment. rbc gsh concentrations did increase significantly (to 1.6 mm; p 5 0.019) after radioiodine treatment. however, this modest change is unlikely to be clinically significant. preliminary data do not indicate clinically significant blood gsh, ascorbate, or vitamin e deficiencies in hyperthyroid cats. with appropriate insulin therapy and a low carbohydrate diet, up to 90% of newly diagnosed diabetic cats are eventually able to maintain euglycemia without insulin administration, and these cats are considered to have achieved remission. there are currently no published data reporting the glucose tolerance status of cats classified as being in remission, and it is unknown whether these cats are truly in diabetic remission, or should be classified as non-insulin dependent diabetics, or having impaired glucose tolerance, and/or impaired fasting blood glucose. the aim of this study was to determine fasting blood glucose concentrations and glucose tolerance status of cats in remission. the study was a prospective study in a feline-only clinic. for inclusion, diabetic cats had to have achieved remission through insulin therapy, and insulin withheld for a minimum of two weeks. five diabetic cats in remission and five matched non-diabetic cats were enrolled in the study. blood samples were obtained via the ear vein but where the cat's temperament precluded this, from the jugular.glucose concentration was measured using a meter calibrated for feline blood (abbott alphatrak). a simplified glucose tolerance test was performed after food was withheld for 24 hours. a 22g catheter was placed in a cephalic vein three hours before the gtt was commenced, to minimize the effects of stress on blood glucose concentration. blood glucose concentration was measured at time 0 and then a 1 g/kg dose of glucose was administered slowly via the intravenous catheter. further blood glucose measurements were made at 2 hours and then hourly until glucose had returned to o 117 mg/dl (o 6.5 mmol/l). in the control group, all cats had a fasting blood glucose below 117 mg/dl, and following glucose administration, glucose had returned to o 117 mg/dl by 3 hours. fasting blood glucose in the remission group was o 126 mg/dl (7 mmol/l) in all cats except one, which had fasting blood glucose of 135 mg/dl (7.5 mmol/l). following glucose administration, all five cats in remission had blood glucose above 117 mg/dl (6.5 mmol/l) at three hours, four were o 117 mg/dl at four hours, and one returned to o 117 mg/dl at five hours. the cat with impaired fasting glucose subsequently became diabetic after steroid administration. the results of this study show that these cats, while no longer diabetic, have mildly impaired glucose tolerance compared to nondiabetic cats, and a minority have impaired fasting glucose. the objective of this study was to determine the role of iodine restriction in the nutritional management of cats with naturally occurring hyperthyroidism. five domestic shorthair cats ranging in age from 8-17 years were confirmed to have hyperthyroidism based on persistently increased serum total thyroxine concentrations (tt4), palpable thyroid nodule and weight loss. serum tt4 concentrations ranged from 55-146 nmol/l (reference range 10-55 nmol/l). the cats were then fed a low iodine containing food (0.47 ppm iodine dmb, as measured by epiboron neutron atomic activation). serum tt4 concentrations were measured every 3 weeks. biochemistry parameters were also evaluated at weeks 0, 6 and 9. at 9 weeks, serum tt4 concentrations had decreased in all cats with 4 of 5 cats (80%) being euthyroid (mean 48 nmol/l; range 41-54 nmol/l). the remaining hyperthyroid cat had an initial serum tt4 of 146 nmol/l, which decreased to 83 nmol/l after being fed the iodine-restricted food. mean decrease in tt4 for all 5 cats was 26 nmol/l (range 8-63 nmol/l). renal parameters remained stable in all 5 cats. these 5 cats along with 4 additional newly diagnosed hyperthyroid cats were transitioned to a similar food that contained less iodine (0.28 ppm dmb). baseline serum tt4 concentrations in the 4 new cats ranged from 55-73 nmol/l. serum tt4 and other biochemical parameters were monitored every 3 weeks for 9 weeks, and then every 4 weeks for an additional 8 weeks. with the 0.28 ppm iodine food the four new cats became euthyroid with a mean tt4 concentration of 41 nmol/ (range 29-50nmol/l). the 4 euthyroid cats from the earlier feeding study had a further decrease in tt4 concentration (mean tt4 5 39 nmol/l, range 38-54nmol/l). the single non-euthyroid cat from the first study had a serum tt4 concentration of 61 nmol/l, a decrease from the baseline concentration of 83 nmol/l. the average decrease in serum tt4 for all 9 cats was 20 nmol/l (range 2-35 nmol/l). finally, 8 of the 9 cats were fed a third iodine-restricted food (0.17 ppm dmb) along with one other newly diagnosed hyperthyroid cat (79 nmol/l serum tt4) and evaluated every 4 weeks. all 9 cats in this evaluation were euthyroid (mean tt4 33 nmol/l; range 23-50 nmol/ l). this result included the cat whose serum tt4 remained in the hyperthyroid range in the first two evaluations. the average decrease in tt4 was 13 nmol/l (range 0-43 nmol/l). biochemical features of renal function remained stable and no other biochemical abnormalities were observed. in summary, the results of these three feeding studies demonstrate that feline hyperthyroidism can be managed effectively with dietary iodine restriction. we have shown previously that restriction of dietary iodine (i) is a safe and effective method for decreasing serum thyroxine concentrations (tt4) in cats with hyperthyroidism. the objective of this study was to determine the maximum level of iodine in a nutritionally balanced feline mature adult food required to maintain normal serum tt4 concentrations in hyperthyroid cats currently being controlled on a food containing 0.15 ppm i (dmb) as measured by epiboron neutron atomic activation. all cats were previously diagnosed at least 14 months prior to the start of the study and their tt4 concentrations were maintained in the normal range by dietary iodine restriction for a minimum of 10 months (range 10 months-3 years). serum tt4 concentrations ranged from 9-42 nmol/l (reference range 10-55 nmol/l) at the beginning of the study. the cats were divided into two groups each containing 9 cats. groups were similar in age and gender distribution (mean age 5 13.8 years, range 12-18 years). one group (group a) was placed on a food that was formulated for mature adult cats containing 0.39 ppm i (dmb). the other group (group b) was placed on a similar food that differed only in that it contained 0.47 ppm i (dmb). blood was collected from all cats every three weeks and analyzed for serum tt4 concentration. biochemistry parameters were also evaluated at weeks 0, 6 and 9. all group a cats exhibited increases in serum tt4 concentration (mean increase of 25 nmol/l above baseline, range 5-48 nmol/l). seven of the cats remained in the euthyroid range (mean serum tt4 5 36 nmol/l, range-27-54 nmol/l). two cats exceeded the upper limit of the reference range (59 and 76 nmol/l respectively). the cats in group b also exhibited increases in serum tt4 concentration but to a greater degree than the cats in group a (mean increase 39 nmol/l, range 20-60nmol/l). four cats remained in the euthyroid range (mean serum tt4 5 41, range 29-49 nmol/l). the five remaining cats all exceeded the upper limit of the reference range (mean serum tt4 5 76 nmol/l, range-59-99 nmol/l). all cats returned to a euthyroid state within 1 month of being returned to a diet containing 0.17 ppm i (dmb). it was determined that serum tt4 concentrations are not ideally controlled in the normal range in hyperthyroid cats fed a food containing ! 0.39 ppm i (dmb). hyperthyroidism is a common disease in old cats. excessive production of thyroid hormones is the hallmark of the disease. three main treatments for feline hyperthyroidism include radioactive iodine, thyroidectomy, and antithyroid drugs such as methimazole. previously we have shown that limiting dietary iodine to or below 0.27 ppm induces euthyroidism in cats with hyperthyroidism compared with a similar diet containing 0.42 ppm iodine. the objective of this study was to test whether dietary iodine at 0.32 ppm would induce euthyroidism in cats with naturally occurring hyperthyroidism. fourteen cats with hyperthyroidism confirmed by serum tt 4 and ft 4 measurements were stratified into two groups based on gender and age. one group (control: 4 males and 3 females, age ranged from 11 to 15 years) was given a positive control dry cat food (0.17 ppm iodine) while the other group (test: 3 males and 4 females, age ranged from 12 to 17 years) was fed a commercial dry cat food (1.9 ppm iodine) for at least 6 weeks before the study. afterwards (week 0), the control cats continued to receive the same food while cats in the test group were given a test food (0.32 ppm iodine) for additional 12 weeks. all cats had free access to their food and deionized water during the study. blood samples were collected during weeks 0, 3, 6, and 12 of the study. the control cats maintained euthyroidism during the study. the test food significantly reduced serum tt 4 (72 ae 12, 43 ae 9 ã , 42 ae 9 ã , 40 ae 6 ã nmol/l in weeks 0, 3, 6 and 12, respectively; ã : p o 0.05 compared with week 0, dunnett's t test). it also significantly reduced ft 4 at the end of the study (17 ae 2 vs. 23ae 2 pmol, week 12 vs. week 0; dunnett's t test, p o 0.05). serum ft 4 was within the reference range (10-55 pmol/l) in cats in both groups. serum tt 3 , ft 3 , and tsh were not affected by the test food and were within the reference ranges (tt 3 : 0.6-1.4 nmol/l, ft 3 : 1.5-6 pmol/l, and tsh: 0-21 mu/l) in cats of both groups during the study. this study demonstrates that dietary iodine at or below 0.32 ppm provides an effective and inexpensive therapy for cats with naturally occurring hyperthyroidism. radioactive iodine ( 131 i) is a widely used treatment for feline hyperthyroidism. prior to 131 i administration, many cats receive methimazole therapy. it has been suggested that recent withdrawal of methimazole prior to 131 i may increase the risk of hypothyroidism, inhibit the response to therapy, or have no effect. to further address this question, a retrospective medical records search was performed to identify hyperthyroid cats that received 131 i therapy after methimazole treatment. inclusion criteria included documentation of the time interval between discontinuation of methimazole and 131 i administration, and measurement of thyroxine (t 4 ) at 7-14 days after 131 i. cats were divided into 2 groups: those receiving 131 i within 1 day of stopping methimazole, and those receiving 131 i treatment 5 or more days after stopping methimazole. sixty cats met the inclusion criteria. forty received 131 i within 1 day of stopping methimazole. of those, 20 (50%) had a low t4 (o 1.2 mcg/dl), 17 (42.5%) had a normal t4 (1.2-4.8 mcg/dl), and 3 (7.5%) had an elevated t4 (4 4.8 mcg/dl) at 7-14 days after 131 i therapy. fourteen cats received 131 i 5 or more days after stopping methimazole: 8 (57%) had a low t 4 , 5 (36%) had a normal t 4 , and 1 (7%) had an elevated t 4 at 7-14 days after 131 i therapy. the results were compared with a fisher's exact test and there was no difference between the groups (p 5 0.76). these findings indicate that stopping methimazole therapy within 1 day of 131 i therapy does not inhibit the response to therapy. pharmacokinetic studies evaluating synthetic insulin analogs such as glargine necessitate the ability to measure the blood concentrations of glargine without cross-reactivity to endogenous insulin. although the cross-reactivity between endogenous human insulin assays and synthetic analogs is often known for commerciallyavailable assays, the degree of cross-reactivity of human insulin assays with feline insulin is not. the purpose of this study was to evaluate the cross-reactivity of feline insulin with a commerciallyavailable human insulin elisa with known cross reactivity to several synthetic analogs. pre-and post-prandial blood samples were collected from four healthy cats immediately prior to and approximately 15 minutes following a meal, for a total of 8 samples. dextrose was added to the meals given to two of the cats. blood samples were immediately centrifuged and the serum was collected, aliquoted, and stored at à201c until analysis. serum insulin levels were determined in parallel with commercially-available feline insulin and human insulin elisas. the elisas were run in duplicate and according to the manufacturer's instructions. concentrations of serum insulin measured by the feline insulin elisa ranged from 12.7 ng/l to 4 700 ng/l. despite the wide range of concentrations of feline insulin, all 8 samples evaluated with the human insulin elisa yielded absorbance readings equal to or lower than the absorbance of the negative control, indicating no crossreactivity between the evaluated human insulin assay and feline insulin. since this assay is reported to cross-react significantly with glargine, it is a great candidate for determination of serum glargine concentrations in cats. the aim of this prospective, controlled study was to compare the efficacy of two trilostane protocols for treatment of canine pituitary-dependent hyperadrenocorticism (pdh). among the 28 client-owned dogs diagnosed with pdh, only the dogs weighing o 5 kg were selected (n 5 16). group a (n 5 9; low-dose treatment group) and group b (n 5 7; high-dose treatment group) received 0.8 ae 0.3 mg of trilostane/kg orally every 12 hours and 30 mg of trilostane/ body orally every 24 hours, respectively. all of the dogs were reassessed at 2, 4, 12, and 24 weeks after the initiation of treatment. the improvement in post-acth stimulation serum cortisol concentration, as well as clinical signs in group a, required more time than group b; however, 2 of 7 dogs in group b had clinical signs and abnormal laboratory findings consistent with hypoadrenocorticism after treatment for 20 weeks. twenty-four weeks later, all of the dogs of both groups improved the abnormal clinical findings. the present study suggests that twice daily, low-dose administration of trilostane is effective in the management of canine pdh and may be safe without the potential adverse effects of once daily, high-dose treatment. however, because this study involved only a small number of dogs, a population-based control study will be needed to clarify the efficacy of low-compared to high-dose trilostane treatment. cobalamin is essential for a variety of metabolic processes in many tissues and organs, and has effects on cell growth and peripheral and central nervous system function. chronic distal small intestinal disease in humans, cats, and dogs has been shown to cause cobalamin deficiency. an immunoassay for the measurement of serum cobalamin concentration in these species is being used in routine practice for the diagnosis of cobalamin deficiency. in pigs, the role of cobalamin has not yet been extensively investigated. thus, the aim of this study was to analytically validate an immunoassay, labeled for use in humans, for the measurement of cobalamin in porcine serum samples and secondly to determine serum cobalamin concentrations in weaned pigs. for the analytical validation of the assay, serum cobalamin concentrations were measured using the commercially available immulite s 2000 cobalamin immunoassay (siemens healthcare diagnostics ltd., deerfield, il, usa) in 30 surplus porcine serum samples from a variety of studies. validation of the assay consisted of determination of dilutional parallelism, spiking recovery, and intra-and inter-assay variability. additional surplus serum samples from 27 piglets from four litters at a texas a&m university farm were obtained. each piglet had been bled twice, the first at weaning (21 days of age) and the second one 12 days later. to investigate results in comparison between age groups, serum cobalamin concentrations were compared using a wilcoxon matched pairs test. significance was set at p o 0.05. observed to expected ratios (o/e) for serial dilutions ranged from 87.3 to 124.9% (mean ae sd: 104.2 ae 14.5%) for four different serum samples at dilutions of 1:1, 1:2, and 1:4, and from 96.8 to 118.3% (mean ae sd: 107.6 ae 15.2%) for one serum sample at dilutions of 1:2, 1:4, and 1:8. o/e for spiking recovery ranged from 87.4 to 116.7% (mean ae sd: 102.0 ae 7.5%) for five different porcine serum samples that had been spiked with each other in a 1:1 dilution. intraassay coefficients of variation (%cv) for five different serum samples were 4.3, 5.7, 4.3, 3.7, and 6.1%. inter-assay %cvs for five different serum samples were 4.9, 7.2, 9.6, 3.5, and 7.6%. serum cobalamin concentration was significantly lower in piglets post weaning (median: 242 ng/l) compared to those at the time of weaning (median: 324 ng/l; p 5 0.009). the immulite s 2000 cobalamin immunoassay labeled for use in humans is linear, accurate, precise, and reproducible for measurement of serum cobalamin concentrations in pigs. this study also showed that piglets that differ in age by only 12 days have significantly different serum cobalamin concentrations. further investigations of cobalamin concentrations in both sows and piglets at different stages of weaning are warranted. primigravid dairy heifers can be infected with mastitis pathogens during the periparturient period. the prevalence of intramammary infection (imi) ranges from 30-75% of quarters pre-partum and 12-45% at parturition. some pre-partum infections self-cure before parturition, however a number of these imis persist into early lactation. these imis may impact milk production and quality and may serve as a reservoir for contagious pathogens. no study has specifically investigated the risk of an imi persisting from the prepartum period into early lactation. the objectives of this study were to describe the prevalence of mastitis pathogens in heifers on a grazing dairy before and after parturition and calculate the relative risk (rr) and attributable fraction of population (afp) for the association between a post-partum and pre-partum imi. two-hundred-ninety-four heifers were systematically assigned to 1 of 3 groups: g1) pre-partum secretions from all mammary quarters (n 5 98), g2) no pre-partum secretions collected (n 5 98) and g3) pre-partum secretions from two diagonal quarters (n 5 98). group assignments were designed to assess whether pre-partum sampling increased the likelihood of imi at calving. mammary quarter secretions were collected for bacterial culture approximately 2 weeks prior to expected calving date. quarter milk samples were collected for bacterial culture once weekly during the 1 st 3-weeks of lactation. bacterial isolates were classified as staphylococci, non-agalactiae streptococci and gram-negatives. mammary quarter samples yielding 2 different bacteria were classified as mixed infections and those yielding ! 3 bacterial types were classified as contaminated. bacterial isolates were speciated using gene sequencing methods and strain-typed using pulse-field-gel-electrophorysis to evaluate the relatedness of bacteria isolated from pre-and post-partum samples from the same mammary quarter. relative risk and afp were calculated using 2 â 2 tables. forty-five percent of mammary quarters had a pre-partum imi. during the 1st 3 weeks of lactation the mean prevalence of imi was 23.3% of quarters. staphylococci were most frequently isolated bacteria from pre-partum secretions and milk with s. chromogenes and s. aureus being the most common species. using data from 228 mammary quarters, the rr and afp for the association between a post-partum and pre-partum imi were 11 and 77%, 43 and 86%, and 12 and 68% for all staphylococci, s. aureus only and cns only imis, respectively. mammary quarters sampled pre-partum were no more likely to have a post-partum imi than those not sampled (chisquare, p ! 0.27). these data demonstrate that pre-partum imis persist into early lactation and that pre-partum secretion cultures may be a useful, not only in predicting imi at calving, but also in assessing risk of introducing new contagious mastitis pathogens, e.g., s. aureus, into the lactating herd. despite concerns about antimicrobial resistance and clostridium difficile in food animals, there has been little study of the prevalence or mechanisms of resistance. this study evaluated the impact of tetracycline treatment on c. difficile shedding in veal calves and the impact on resistance. calves arriving on 1 veal farm received oral oxytetracycline for 5 days as per farm protocols. calves were sampled at arrival and 6 days later. selective culture for c. difficile was performed. isolates were ribotyped, and tested for tetracycline susceptibility and the presence of tetracycline resistance genes. multivariable logistic regression models were used to determine the relationship between tetracycline resistance and the presence of tetracycline resistance genes. clostridium difficile was isolated from 32% (56/174) and 51% (88/ 172) calves, at the first and second samples, respectively. the percentage of tetracycline resistant isolates increased from 79% to 93%. isolates from the second sample were 3 times more likely to be tetracycline resistant (p 5 0.016) and 5 times more likely to possess tet(m) (p 5 0.004). tet(m) was detected in 13% (7/53) and 43% (39/ 91), tet(o) in 23% (12/53) and 19% (17/91) and tet(w) in 2% (1/53) and 1% (1/91) of isolates from first and second samples, respectively. tet(l), tet(k) and tet(s) were not detected. 54 resistant isolates were not carrying any of the genes investigated. routine tetracycline use may have had an impact on both the prevalence of c. difficile, as well as the strain distribution and resistance patterns. this is the first report of presence of tet ( the objectives of this study were to 1) estimate the prevalence of antimicrobial resistance in the study population and 2) to investigate the associations between exposures to antimicrobial drugs and antimicrobial resistance in fecal non-type specific e. coli (ntsec) recovered from individual feedlot cattle. two-stage random sampling was used to identify cattle for enrollment at 4 western canadian feedlots. a fecal sample was collected per rectum from each individual at arrival and in the middle of the feeding period when cattle were rehandled as part of standard feedlot protocol. from samples collected at this second time point, a total of 2,133 ntsec isolates were tested for susceptibility to antimicrobial drugs by disk diffusion. parenteral and in-feed exposures to antimicrobial drugs were recorded for each individual enrolled in the study. the least square means estimates and 95% confidence intervals for the prevalence of resistance at each time point were modeled using poisson regression. multivariable logistic regression was used to investigate associations between antimicrobial resistance and exposure to antimicrobial drugs. regression models were adjusted for clustering of observations among individuals and pens. the most common resistances identified in arrival samples were sulfisoxazole (7.5%; 95%ci: 6.1-9.2), streptomycin (7.7%; 95%ci: 6.3-9.5) and tetracycline (20.0%; 95%ci: 17.7-22.6). at the second sampling point, resistance prevalence was 25.6% (95%ci: 23.5-28.0) for sulfisoxazole, 25.0% (95%ci: 22.8-27.3) for streptomycin, and 72.7% (95%ci: 70.5-75.1) for tetracycline. logistic regression modeling identified weak associations of exposures to tetracycline and macrolide classes of drugs with antimicrobial resistance at the second time point. abstract fa-5 premature/dysmature syndrome in cria: a ret-rospective study of 63 cases (1999) (2000) (2001) (2002) (2003) (2004) (2005) . c. gerspach, d. anderson. the ohio state university, columbus oh. prematurity is widely acknowledged as risk factor for subsequent morbidity and mortality in llama and alpaca cria. a review of medical records for premature cria alive at the time of admission to the veterinary teaching hospital between 1999 and 2005 was performed to determine risk factors of prematurity and to report the outcome and related conditions or diseases in affected cria. medical records for 63 premature or dysmature cria were included in this study. of these cria, 51 were alpaca and 12 llama, 36 were female and 27 were male. reasons for referral were prematurity, failure of passive immunity, dyspnoea, weakness and failure to gain weight. cria were presented at a mean age of 1.4 days and were premature by a mean estimated time of 19.5 days. overall survival rate was 82.5%, with all llama cria surviving. a multivariate logistic regression model was used to identify risk factors associated with not surviving. cria receiving camelid colostrum had a significant better outcome than cria receiving no colostrum or colostrum from different species. dyspnea and tachypnea was associated with a poor outcome. all cria that were able to nurse, without assistance prior to referral, survived. clinical pathology parameters most commonly associated with death were hyperphosphatemia and acidosis. enrofloxacin is approved for the treatment of swine respiratory disease, however there are no published studies describing the pharmacokinetics of enrofloxacin at the approved dose and route in pigs (7.5 mg/kg subcutaneously). furthermore no studies have assessed the unbound concentrations of enrofloxacin at its site of action, the extracellular tissue fluid. therefore the objective of this study was to use an in-vivo ultrafiltration method to measure the active fraction of enrofloxacin, and the metabolite ciprofloxacin, at 3 tissue sites relevant to pigs, and to compare these concentrations with plasma concentrations collected at similar time points. six healthy pigs were used in this study. pigs were recently weaned and weighed an average 16.3 kg. on the day before the experiment, pigs were anesthetized for the placement of jugular vein sampling catheters and interstitial fluid collection probes. three ultrafiltration probes were placed in each pig in a subcutaneous site near the right shoulder, an intramuscular site along the epaxial muscles, and in the pleural space of the chest cavity. each pig received an injection of enrofloxacin (baytril 100, bayer animal health) at a dose of 7.5 mg/ kg subcutaneously behind the left ear. plasma and interstitial fluid samples were collected at pre-determined time points, and enrofloxacin and ciprofloxacin concentrations were measured using hplc with fluorescence detection. protein binding was determined with a microcentrifugation system. pharmacokinetic data was analyzed using a one compartment model. the analysis of plasma and isf showed that only a small fraction of ciprofloxacin was produced in these pigs, therefore ciprofloxacin concentrations were not used in pharmacokinetic measurements. the plasma half-life (t 1/2 ), volume of distribution, clearance, and peak concentration (c max ) for enrofloxacin was 25.9 hr (ae 6.2), 6.29 l/kg (ae 1.23), 0.168 l/kg/hr (ae 0.076), and 1.07 mg/ml (ae 0.28), respectively. the concentrations from each of three tissues were not different in each pig. when pharmacokinetic values from all tissues were combined for the isf, the t 1/2 was 23.6 hr (ae 4.1) and the c max was 1.26 mg/ml (ae 0.10). the enrofloxacin plasma protein binding was 31.1% (ae 3.28) and 37.13% (ae 16.54) at a high and low concentration, respectively. this study has demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. the half-life of enrofloxacin is longer in tissues and plasma than has been reported in previous studies. the high tissue concentrations and long half-life produce an auc/mic ratio sufficient for the pathogens that cause respiratory infections in pigs. ceftiofur crystalline free acid (ccfa), a long-acting ceftiofur formulation labeled for use in cattle, pigs, and horses for treatment of respiratory disease has been used for treatment of ovine respiratory infections in clinical practice. pharmacokinetic data, however, do not exist for ccfa administered subcutaneously in sheep. the present pharmacokinetic study evaluated the single dose subcutaneous administration of ccfa in sheep (n 5 9) at 6.6 mg/kg body weight. concentrations of ceftiofur free acid equivalents (cfae) in plasma were measured by high performance liquid chromatography for 14 days following drug administration. pharmacokinetics of subcutaneous ccfa in sheep were best described using a single compartment model with the following average (ae sd) parameters: area under the concentration time curve 0! 1 (206.6 hãug/ml ae 24.8), observed maximum plasma concentration (2.4 ug/ ml ae 0.5), and observed time of maximum plasma concentration (23.1 h ae 10.1). no significant adverse drug reactions were observed. adequate cfae plasma concentrations were attained to effectively treat respiratory tract pathogens associated with pneumonia in sheep. the purpose of this study was to assess, using thoracic ultrasonography, the prevalence of lung lesions in pre-weaned dairy calves. subsequent aims were to describe ultrasonographic changes within the lung, clinical respiratory score, and treatment of respiratory disease. a longitudinal study was performed using female dairy calves from 6 commercial dairy farms in new york state. calves were enrolled based on age. thoracic ultrasound and clinical respiratory scoring were performed on each calf at 2 time points. a standard 5 mhz linear ultrasound probe was utilized to evaluate intercostal spaces 1 through 11 of each hemi-thorax with the calf in lateral recumbency (us1) or standing (us2). lesion appearance, size, and location were recorded. respiratory score (rs) was assigned based on a previously published protocol incorporating fever, nasal discharge, cough, ocular discharge and ear droop, with a higher numerical score corresponding to more severe disease. abnormal lung on ultrasound was defined as one or more areas of !1 cm width or depth of non-aerated lung. farm records were evaluated to identify treated calves. calves were treated for respiratory disease at the farm manager's discretion, not based upon ultrasound findings or rs. non-parametric methods were used to evaluate the data. ninety-one calves were enrolled into the study, with 6 lost to follow-up. an average of 4 minutes was spent performing the rs and ultrasound on each calf. the median ages at first (us1) and second (us2) examination were 13 (interquartile range 12-15) and 46 (interquartile range 44-47) days, respectively. the majority of calves had a low rs (o 5) and only 3.2% of calves had a rs high enough to warrant treatment based on previous recommendations (rs!5). the prevalence of calves that had abnormal lungs on ultrasound but a low rs (o 5) was 5.5% (us1) and 16.5% (us2). the prevalence of calves that had abnormal lungs on ultrasound and a high rs (! 5) was 0% (us1) and 3.5% (us2). of the calves that had abnormal lungs on ultrasound but a low rs, 13% were treated with antimicrobials within 7 days of examination. none of the calves with high rs and abnormal lungs on ultrasound were treated with antibiotics within 7 days of examination. this study demonstrates a high prevalence of abnormal lungs, as detected by thoracic ultrasonography, without significant clinical signs in pre-weaned dairy calves. the relatively low treatment rate in these calves may suggest an area of opportunity for improvement in calf health, welfare, and herd longevity. further studies and follow up are needed to elucidate the significance of these findings and whether or not treatment is indicated. literature regarding diseases causing lameness in beef cattle is limited. this retrospective study was undertaken to examine beef cattle presented for lameness. medical records of beef cattle having a lameness examination done during the period 2007 to 2010 were reviewed and descriptive statistics generated. lameness was classified based on clinical diagnosis. the medical records of 270 beef cattle were reviewed of which 63.2% were male and 36.8% were female. beef cattle presented for lameness most often during the summer months (34%) and least during autumn (18%). causes of lameness were categorized as infectious (44.6%) or non-infectious (55.4%) and infectious lameness subcategorized as either a primary disorder or a secondary infection. all cases of a primary infectious disorder were interdigital phlegmon. secondary infections diseases included sole abscess (25.9%), septic arthritis (11.1%), tenosynovitis (2.8%), and pedal osteitis (1.3%). non-infectious lameness included proximal limb lameness (19.6%), foot trauma (14.6%), hoof horn cracks (9.5%), hoof defects (2.5%), interdigital fibromas (1.9%), overgrown hooves (1.9%), sole bruise (1.3%), subclinical laminitis (1.3%), white line disease (0.9%), osteoarthritis (0.9%), heel erosion (0.3%), sole ulcers (0.3%), and sole hemorrhage (0.3%). the most frequently affected claw was the lateral digit of the hind limb (36.4%), followed by the medial digit of the front limb (27.1%), lateral digit of the front limb (23.6%), and the medial digit of the hind limb (12.9%). the findings of this study suggest significant differences in the frequency of disease causing lameness in beef cattle compared to published reports for dairy cattle. in people, endoscopic ultrasound (eus) has become the technique of choice for assessing pancreatic disease and eus-guided fineneedle aspiration (eus fna) has proven a useful and safe modality for characterizing pancreatic lesions. reported complications include infections, bleeding and acute pancreatitis. in dogs, laparoscopic-assisted pancreatic biopsy has been suggested to be a safe procedure, however eus and eus fna have not been evaluated in dogs so far. thus the aim of the present study was to assess the practicability and safety of eus examination of the abdominal cavity as well as pancreatic eus fna in healthy dogs. this study was approved by the cantonal committee for the authorization of animal experimentation, zurich, switzerland. the study population consisted of 14 healthy beagle dogs with a median bodyweight of 13.4 kg (10.9-15.7). eus was performed with an olympus gf-uc140p-echoendoscope and fna were performed using 19 g needles (cook echotipultra). after completion of the eus-examination of the abdominal cavity from the stomach (liver, gallbladder, bile ducts, kidneys, adrenals, pancreas), the scope was advanced into the duodenum and eus fna of the pancreas was performed. fna tissue acquisition was made applying negative pressure and 6 to 8 needle passes were made. all dogs received 30 mg/kg metimazole im after eus fna and were re-checked ultrasonographically 20 minutes post eus fna. postoperative activity was assessed using a standardized scoring system. a cbc, serum biochemistry, urinalysis and spec cpl s were measured before, as well as 24 and 48 h after eus fna. the eus examination was complete in 13/14 dogs, the pancreas could not be visualized in 1 dog. the pancreas was hypo-(4/13) to isoechoic (9/13) to the surrounding mesenterium in all cases. in 3/13 dogs parts of the pancreas presented hyperechoic. the mean measured thickness was 0.88 cm. the pancreas was aspirated in 12 dogs using a transgastric approach (3) or transduodenal approach (9). duodenal transmural puncture was not accomplished in 1 dog where a re-sterilized needle was used. a minimal amount of peripancreatic fluid was observed in 1/12 dogs after eus fna. all dogs recovered uneventfully and required no further analgesia. all laboratory results including the spec cpl s measurements were within reference ranges on all three time points. cytologically, conglomerates of exocrine pancreatic cells were seen in 8/12 cases, duodenal villous epithelial cells were seen in 11/12 cases. in 1 dog the aspirated pancreatic material was sufficient for a histological assessment. the 8 aspirates with exocrine pancreatic cells on cytology were obtained by transgastric (4) and transduodenal (4) aspirations. in conclusion, (1) eus examination of the abdomen is feasible in medium-sized dogs, (2) the healthy canine pancreas can be difficult to visualize completely, and (3) eus-guided pancreatic fna using a 19 g needle is a safe procedure in healthy dogs. studies evaluating its use in dogs with pancreatic disease are warranted to assess its clinical utility. miniature schnauzers have a high prevalence of idiopathic hyperlipidemia, which is characterized by an increased serum triglyceride (tg) concentration, with or without an increased serum cholesterol (chol) concentration. a common initial therapeutic approach for the management of hyperlipidemia is the use of a low-fat diet. also, it is believed that low-fat diets may be beneficial in the treatment of pancreatitis in dogs. however, the efficacy of this approach has not been evaluated for either condition. the aim of the present study was to evaluate the effect of a commercially available low-fat diet on serum concentrations of tg, chol, and canine pancreatic lipase immunoreactivity (cpli; measured as spec cpl s ) in apparently healthy miniature schnauzers with hypertriglyceridemia. blood samples were collected from 15 apparently healthy miniature schnauzers with hypertriglyceridemia (serum triglyceride concentrations 4 108 mg/dl). common causes of secondary hyperlipidemia were excluded based on historical information, physical examination findings, and the measurement of serum glucose, total t4, and free t4 (by ed) concentrations. the owners of the dogs were asked to switch their dog to the study diet (royal canin gastrointestinal low fat s ; fat content: 18.6 g/1,000 kcal) and have a second blood sample collected 8 weeks after their dog had been on the new diet. all blood samples were collected after food had been withheld for 15 hours. serum tg, chol, and spec cpl concentrations were measured both before and after the diet change. results were compared between the two time-points using the wilcoxon signed rank and fisher's exact tests. serum tg concentrations were significantly higher before (median: 432 mg/dl) than after the diet change (median: 178 mg/dl; p 5 0.003). the proportion of dogs with hypertriglyceridemia was significantly higher before (15/15) than after the diet change (10/15; p 5 0.042). also, the proportion of dogs with serum tg 4 500 mg/dl was significantly higher before (6/15) than after the diet change (0/15; p 5 0.016). serum chol concentrations were significantly higher before (median: 296 mg/dl) than after the diet change (median: 258 mg/dl; p 5 0.004). the proportion of dogs with hypercholesterolemia was significantly higher before (8/15) than after the diet change (0/15; p 5 0.006). finally, the difference in serum spec cpl concentrations before (median: 88 mg/l) and after the diet change (median: 56 mg/l) approached but did not reach significance (p 5 0.052). also, the proportion of dogs with high serum spec cpl concentrations before (4/15) and after the diet change (0/15) was different, but this difference was not significant (p 5 0.099). in summary, a commercially available low-fat diet was effective in reducing serum tg and chol concentrations in miniature schnauzers with hypertriglyceridemia. toll-like receptor 5 (tlr5) is an extracellular pattern recognition receptor which recognizes flagellin present in motile bacteria. we have previously demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the tlr5 gene (g22a, c100t and t1844c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). recently, we have confirmed that two of these tlr5 snps (c100t and t1844c) are significantly associated with ibd in other canine breeds. to further substantiate the role of tlr5 in canine ibd functional analysis of these polymorphisms would be needed. therefore the aim of this study was to determine the functional significance of the tlr5 snps by transfecting wild-type and mutant receptors in to human embryonic kidney cells (hek) and carrying out nuclear factorkappa b (nf-kb) luciferase assay and il-8 elisa. the tlr5 gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) as determined by the case-control analysis in gsds with ibd were cloned into plasmids expressing yellow-fluorescent protein (yfp). these were then stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were then stimulated with various ligands (0.1 mg/ml flagellin, 0.01 mg/ml flagellin, 1 mg/ml lps, 1 mg/ml pam3csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk) according to the manufacturer's recommendations. the supernatants were harvested and used in an il-8 elisa (r&d systems). human tlr5 transfected hek cells (invivogen) served as positive controls in all experiments. independent t-test was used to determine the significance of relative luciferase activity and il-8 concentration between wild-type and mutated tlr5 cells. although there was no significant difference between the wild-type and mutated receptor when they were stimulated with 0.01 mg/ml of flagellin (p 5 0.14), there was a significant increase when the cells with mutated tlr5 were stimulated with 0.1 mg/ml of flagellin compared to the cells expressing wild-type tlr5 (p 5 0.027). similarly, there was a significant increase in il-8 concentration in the supernatants in the cells with the mutated tlr5 receptor when stimulated with 0.1 mg/ml flagellin compared to the wild-type (p 5 0.025-one-tailed, 0.05-two-tailed) but not with 0.01 mg/ml flagellin (p 5 0.26). we show for the first time that polymorphisms associated with ibd are functionally hyper-responsive to flagellin compared to the wild-type receptor. this suggests that tlr5 may play a role in canine ibd and that blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. however, further in-vivo functional analysis of tlr5, especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions. tlr5 has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (ibd). similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the canine tlr5 gene (g22a, c100t and t1844c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). therefore the aim of this study was to determine the functional significance of the tlr5 snps in the breed of gsds. the tlr5 gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) were stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were stimulated with various tlr ligands (0.1 mg/ ml flagellin, 0.01 mg/ml flagellin, 1 mg/ml lps, 1 mg/ml pam3csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk). the supernatants were harvested and used in an il-8 elisa (r&d systems). peripheral whole blood from dogs carrying the wild type and mutant tlr5 genes was cultured and stimulated with tlr ligands as above. canine tnf-alpha was measured in the supernatant by commercially available elisa (r&d systems). t-test was used to determine differences of relative luciferase activity, il-8 concentration and tnf-alpha concentration between wild-type and mutated tlr5 cells. there was a significant increase in nf-kb activity when the cells with mutated tlr5 were stimulated with 0.1 mg/ml of flagellin compared to the cells expressing wild-type tlr5 (p 5 0.027), which correlated with il-8 expression in the supernatant (p 5 0.26). similarly, in the whole blood assay the tlr5 risk haplotype for ibd in gsds (acc) was significantly hyperresponsive to flagellin at a concentration of 0.1 mg/ml compared to the tlr5 wild-type haplotype (gtt) (p 5 0.001). we show for the first time that polymorphisms associated with canine ibd in gsds are functionally hyper-responsive to flagellin compared to the wild-type receptor. blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. proton pump inhibitors (ppi) are widely used in human and also veterinary medicine. side-effects of ppi treatment reported in people are atrophic gastritis, gastric and esophageal cancer, and rebound hyperacidity following cessation of treatment, which has been speculated to be due to a sustained increased in circulating gastrin concentration. moreover, long-term ppi treatment has been associated with an increased risk for osteoporosis in people. little is known about the effect of ppi treatment on serum gastrin concentration or calcium metabolism in dogs. eight healthy adult research dogs (4 males and 4 females) were enrolled into the study. the dogs received an average dose of 1.1 mg/ kg of omeprazole orally twice daily for 15 days. blood samples were collected prior to initiating the treatment and every 3 days during the 15 days of treatment and during the 15 days after discontinuation of treatment for determination of serum gastrin, ionized calcium, pth, and 25 oh vitamin d3. gastric fluid was collected via gastroscopy after an overnight fast for measurement of gastric ph prior to, during, and after the omeprazole treatment period. normally distributed data were compared with a repeated measures anova and post hoc dunnett's test. data that were not normally distributed were compared with a friedman's test and a post-hoc dunn's test. gastric fluid ph was significantly higher (p o 0.01) at the end of the treatment period (median: 7.4; range: 4.2-8.1) when compared to pretreatment values (median: 1.7; range: 1.0-6.8). serum gastrin concentrations increased significantly from a median baseline of 10.0 ng/l (range: 10.0-27.0) to a maximum median of 379.5 ng/l (range: 49.9-566.0) at day 9 of treatment (p o 0.01). serum gastrin remained significantly increased above baseline values from day 6 to day 15 of the treatment, but was not different from pre-treatment values 3 days after the end of the treatment. omeprazole treatment had no effect on ionized calcium or pth for the duration of the study. marginal, but significant changes of 25 oh vitamin d3 were observed at day 15 (end of the treatment period -increased by 13.8%) and day 21 (6 days after the end of the treatment -decreased by 14.7%). this study shows that treatment with omeprazole for 2 weeks results in a profound and sustained increase in serum gastrin concentration in dogs. this effect is rapidly reversible after cessation of the treatment. no effect on calcium metabolism was observed. however, this study documents only the effect of short-term treatment and it is possible that the effects of long-term administration are different. omeprazole treatment has been associated with small intestinal bacterial overgrowth and a higher risk for infectious enteropathies in humans. using a semi-quantitative sequencing approach, we have previously shown that omeprazole treatment may lead to alterations in both duodenal and gastric bacterial populations in healthy dogs (acvim 2010). however, a sequencing approach can only estimate relative proportions of genomic bacterial targets. therefore, significant changes in the total number of bacteria could not be evaluated. the aim of this study was to quantify gastric and duodenal bacterial populations in dogs undergoing omeprazole treatment. eight 9 month-old healthy research dogs (4 males and 4 females) were enrolled. the dogs received an average dose of 1.1 mg/kg of omeprazole orally twice a day for 15 days. endoscopic gastric and duodenal biopsies were harvested 30 and 15 days before starting omeprazole treatment, on the last day of treatment (day 15), and 15 days after the end of treatment (day 30). all biopsies were fixed in 10% formalin for 24 hours, processed, and embedded in paraffin blocks. fluorescent in situ hybridization was used to quantify mucosa-associated bacteria using fluorescently-labeled probes targeting the 16s ribosomal rna. statistical analysis aimed to compare changes in helicobacter spp. in gastric biopsies and total bacteria in both gastric and duodenal biopsies using the glimmix and npar1way procedures in sas s 9.2. bacteria were counted in 1,174 and 989 microscopic fields (63â) obtained from 155 and 132 gastric and duodenal biopsies, respectively. in the stomach, omeprazole treatment led to a decrease in helicobacter spp. (log of average counts ae standard error: 1.98 ae 0.14 at day 15) when compared to the counts 30 (2.43 ae 0.13, p0.0001) and 15 (2.40 ae 0.13, p0.0001) days before treatment. after completion of omeprazole treatment, helicobacter spp. increased and returned to baseline counts (2.45 ae 0.13 at day 30, p0.0001 vs day 15). also, in the stomach, non-helicobacter spp. bacteria were observed more often during omeprazole treatment (median: 3, range: 0-20) than on days 30 (median: 0, range: 0-3) and 15 (median: 1, range: 0-6) before and 15 days after (median: 0, range: 0-2) omeprazole treatment; however, statistical comparison across time points did not reach significance. in the duodenum, while the median number of bacteria for all time points was zero, non-parametric comparison of median scores (number of points above median) revealed significantly higher numbers of bacteria during omeprazole treatment (p 5 0.0063). our results suggest that omeprazole treatment for 2 weeks leads to a lower abundance of helicobacter spp. organisms in the stomach of healthy dogs. also, this transient decrease in helicobacter spp. was accompanied by a higher abundance of other bacteria in both the stomach and the proximal duodenum. the smartpill ph.p s capsule (the smartpill corporation) is a wireless motility capsule that measures ph, pressure, and temperature as it passes through the gastrointestinal (gi) tract. analysis of this data allows the calculation of gastric emptying time (get), small and large bowel transit time (slbtt), and total gi transit time (tgtt). this study evaluated the variability associated with repeated measurement of gi transit times and the effect of oral administration of ranitidine (zantac s ) on gi transit times in dogs using this system. it was hypothesized that ranitidine would reduce gi transit times. six privately owned healthy adult dogs weighing between 25.0 kg and 41.0 kg were used. on 3 occasions each dog was fed a standard meal followed by oral administration of a capsule. data were recorded until the capsule had passed in the dog's feces. on a 4th occasion each dog was given 75 mg of ranitidine po q12 hrs starting 48 hrs prior to testing. the dogs were then fed the test meal and the capsule was administered as above. ranitidine was given until the capsule had passed in the dog's feces. proprietary smartpill software was used to calculate get, slbtt, tgtt, and the median gastric ph (mgph). mean intra-individual and inter-individual coefficients of variation (cv%) were calculated for get, slbtt, and ttt for the first 3 time points. transit times and gastric ph recorded at all 4 time points were compared using a repeated measures anova. where significant differences were identified, post-hoc testing was performed using a bonferroni's multiple comparisons test. significance was set at p o 0.05. a sharp rise in ph indicating exit of the capsule from the stomach was identified in each experiment. mean (ae sd) get, slbtt, and tgtt without ranitidine were 775 ae 144, 1563 ae 614, and 2338 ae 577 min, respectively. mean get, slbtt, and tgtt during treatment with ranitidine were 719 ae 11, 1442 ae 885, and 2155 ae 897 min, respectively. mean intra-individual cv% before ranitidine for get, slbtt, and tgtt were 12.9, 29.7, and 17.8%, respectively. mean inter-individual cv% before treatment with ranitidine for get, slbtt, and ttt were 16.1, 40.5, and 24.7%, respectively. no significant differences in get, slbtt, or tgtt were found at any of the 4 time points. the mean mgph during treatment with ranitidine (ph 2.85) was significantly higher than at all other time points (overall mean ph for the 3 time points: 1.73; p o 0.05). the smartpill system is an easy to use, ambulatory, non-invasive, non-radioactive method for assessing gi transit times in medium to large breed dogs. measurements of gi transit times, especially slbtt, were subject to considerable intra-individual and interindividual variation. no significant effect of oral ranitidine on gi motility was identified in this group of dogs. however, as expected, oral ranitidine caused a significant increase in gastric ph. the intestinal microbiota has been implicated in the pathogenesis of various gastrointestinal disorders in both humans and dogs. recent metagenomic data suggest that specific bacterial groups, including bacteria within the clostridium clusters iv and xiva (i.e., faecalibacterium spp., ruminococcaceae, and lachnospiraceae) and bifidobacterium spp. are decreased, while proteobacteria are increased in dogs with clinical signs of gastrointestinal disease. the objective of this study was to establish quantitative polymerase chain reaction (qpcr) assays for these specific bacterial groups and evaluate their abundance in healthy dogs and dogs with clinical signs of gastrointestinal disease. fecal samples were collected from 21 healthy dogs (14 females and 7 males) and 20 dogs with clinical signs of gastrointestinal disease (11 females and 9 males). novel quantitative pcr assays were established for faecalibacterium spp., ruminococcaceae, and lachnospiraceae by aligning respective group specific sequences against canine specific sequences obtained from 16s rrna gene clone libraries and sequences available from the ribosomal database project. primers for bifidobacterium spp. and proteobacteria were selected from previously published studies. the specificity of the qpcr assays was confirmed by sequencing of obtained qpcr amplicons. the bacterial dna abundance in fecal samples was compared between healthy dogs and dogs with clinical signs of gastrointestinal disease using a mann-whitney u test. significance was set at p o 0.05. a significantly lower abundance of faecalibacterium spp. (p o 0.01) and ruminococcaceae (p 5 0.02) was observed in dogs with clinical signs of gastrointestinal disease when compared to healthy dogs. proteobacteria were more abundant in dogs with clinical signs of gastrointestinal disease, but this difference did not reach statistical significance (p 5 0.07). there was no significant difference in the abundance of lachnospiraceae (p 5 0.23) and bifidobacterium spp. (p 5 0.77) between both groups. in conclusion, we established novel qpcr assays for faecalibacterium spp., ruminococcaceae, and lachnospiraceae. we observed significant decreases in the abundance of faecalibacterium spp. and ruminococcaceae in dogs with clinical signs of gastrointestinal disease. these bacterial groups are considered major short-chain fatty acid producers and studies are warranted to determine if a decrease in these bacterial groups is associated with decreases in short chain fatty acid production. further studies are also needed to determine if these bacterial shifts are associated with specific gastrointestinal disorders. the pathogenesis of chronic enteropathies (ce) in dogs likely involves complex interaction between the mucosal immune system and the intestinal microbiota. while the application of bacterial 16s rdna sequence-based analysis has shown an association between altered microbial composition and duodenal inflammation in dogs, relatively little is known about alterations in non-invasive mucosal and luminal bacteria seen with diseases involving the ileum and colon. the present study sought to evaluate the relationship of enteric bacteria to type and severity of mucosal inflammation affecting the ileum and colon of dogs with ce. eleven client-owned dogs with ce involving both the small and large intestines were prospectively enrolled. ce was diagnosed on the basis of a history of chronic gastrointestinal signs, exclusion of identifiable underlying disorders, and histopathologic evidence of intestinal inflammation. mucosal bacteria were detected in formalinfixed ileal and colonic tissue sections with fluorescence in situ hybridization (fish) using 16s rdna-targeted probes directed against all bacteria, enterobacteriaceae, e. coli, eubacterium rectale-clostridium coccoides group, bacteroides/prevotella, and helicobacter spp. sections were examined by epifluorescence microscopy and the number of bacteria and their spatial distribution (luminal, superficial mucus, epithelial adherent, within mucosa) was determined in ten 60x fields of each section. microbial composition in ce dogs was compared to the ileal/colonic microbiota of 7 healthy control (hc) dogs using a mixed effect anova model. p values o 0.05 were considered significant. the final diagnoses for dogs with ce included ibd (n 5 8) and lymphosarcoma (n 5 3). when compared to hc dogs, dogs with ce showed regional (ileum versus colon) imbalances in microbiota composition characterized by selective enrichment of mucosa-associated populations. evaluation of colonic biopsies in dogs with ce showed that the total number of bacteria (p o 0.04), clostridium (p o 0.005), enterobacteriaceae (p o 0.04) and e. coli (p o 0.03) were increased in the adherent mucus regions of dogs with ibd as compared to hc dogs. total bacteria (p o 0.05) and e. coli (p o 0.02) were also more numerous in dogs with lsa versus hc and ibd dogs (p o 0.04 for e. coli). ileal biopsies from ce dogs similarly showed variable dysbiosis with increased total bacteria (p o 0.05) but decreased helicobacter spp (p o 0.001) and bacteroides (p o 0.02) observed within inflamed intestines as compared to hc tissues. the spatial distribution of these bacteria was also appreciably different from hc dogs, with higher numbers of bacteria generally found within the adherent mucus compartment as compared to other ileal regions. our data demonstrate that dogs with ce affecting the ileum and colon have altered microbiota composition that may be a cause or consequence of mucosal inflammation. recognition of these microbiota imbalances may provide new opportunities for therapeutic intervention. trichomonads have been rarely reported in the feces of dogs and their pathogenicity remains uncertain. although pentatrichomonas hominis (ph) is considered to be a commensal that may overgrow in dogs with other causes of diarrhea, little is known regarding the history, clinical presentation or prevalence of concurrent gi infections in dogs with trichomonosis. the aim of this study was to determine whether dogs with diarrhea and trichomonosis could be distinguished from dogs having diarrhea without trichomonosis on the basis of clinical signs or presence of concurrent enteric infections. fecal samples from 39 dogs were submitted to ncsu from 2007-2010 for trichomonas spp. pcr testing. dna was extracted using a zr fecal dna mini-prep kit and absence of pcr inhibitors verified by amplification of bacterial 16s rdna. pcr for ph and tritrichomonas foetus (tf) was performed as well as real-time pcr assays for 9 possible concurrent enteric infectious agents. obtainable medical records were reviewed. all submitted fecal samples were submitted from dogs with diarrhea that was variably described as soft, mucoid, hemorrhagic, or watery. mean age of the dogs was 2.33 years (median 1.9; range: 2.5-120 months) and represented a total of 24 breeds. ph, tf, or concurrent ph and tf were diagnosed in 18, 1, and 1 dogs respectively (group a). the remaining 19 dogs were negative for ph and tf by pcr no dogs were identified as infected with canine distemper virus or parvovirus. five samples from each group had insufficient quantity or quality of dna for concurrent infectious disease testing. in this large study of canine trichomonosis, no differences in age, clinical signs, or prevalence and identity of concurrent enteric infection between diarrheic dogs with or without ph were identified. thus, these findings do not appear to support a primary pathogenic role for ph as a causative agent of diarrhea in dogs. gastrointestinal motility disorders are a common clinical problem in domestic animals. many of the g.i. motility disorders have been treated previously with 5-ht 4 agonists although limited availability of drugs in this classification have stimulated interest in the use of new (and old) drug therapies. the dopaminergic antagonists are a group of drugs with well-known anti-emetic effects at central dopamine d 2 receptors, and putative gastrointestinal prokinetic effects at peripheral d receptors. domperidone has been shown, for example, to reverse gastric relaxation induced by dopamine infusion in the dog. similar studies have not been reported in the cat or rabbit, two species at risk for distal gastrointestinal motility disorders. our aim was to study the effects, mechanisms, and sites of action of domperidone in feline colonic and rabbit gastrointestinal smooth muscle contraction. portions of stomach (fundus and antrum), intestine (duodenum and ileum), cecum (rabbits only), and colon (ascending and descending) were obtained from healthy cats and rabbits from 9-12 months of age. longitudinal and circular smooth muscle strips from each site were suspended in physiologic (hepes) buffer solution, attached to isometric force transducers, and set to optimal muscle length (l o ) using acetylcholine (ach; 10 à4 m). muscle strips were treated with domperidone (d; 10 à8 to 10 à4 m) in the presence or absence of ach (10 à8 to 10 à4 m), and maximal force output (p max ) was normalized for cross-sectional area (n 5 â 10 4 newtons/m 2 ). domperidone (d) had a minor direct effect of inducing feline and rabbit gastric, cecal, and colonic smooth muscle contraction. direct effects were similar whether in the longitudinal or circular muscle orientation. the direct effect of domperidone was dose-dependent and maximal (feline colon p max 5 0.15-0.22 n; rabbit colon p max 5 0.10-0.19 n) at a dose of 10 à4 m. domperidone had a much greater indirect effect in augmenting cholinergic (ach; 10 à4 m) contractions in feline and rabbit gastric, cecal, and colonic smooth muscle. domperidone-augmented cholinergic contractions were 157-200% (feline colon p max 5 1.54 ae 0.24 n ach only; feline colon p max 5 2.03 ae 0.23n ach 1 d) of baseline cholinergic contractions. domperidone contractions were of a similar magnitude to those induced by cisapride. domperidone effects were similar in mucosaintact and mucosa-dissected preparations. domperidone contractions were unaffected by prazosin (a 1 receptor antagonist), yohimbine (a 2 receptor antagonist), or terbutaline (b 2 receptor agonist), but were somewhat attenuated by dopamine (d 2 receptor agonist) and a non-specific cholinergic antagonist (atropine). in vitro studies show for the first time that domperidone has minor direct and major indirect effects in augmenting cholinergic contractions of feline and rabbit gastrointestinal (stomach, cecum and colon) smooth muscle. as recognition of acute and chronic pain in dogs has increased, so too has the use of non-steroidal anti-inflammatory drugs (nsaids) often in conjunction with tramadol. in people and rats, co-administration increases the risk of perforation and gastric injury over nsaids alone. using an ex vivo model of acid injury in canine gastric mucosa, we examined the effects of indomethacin and tramadol on gastric permeability and concentrations of gastroprotective prostaglandin e 2 (pge 2 ). mucosa from the gastric antrum was harvested from 5 shelter dogs immediately after euthanasia, and mounted on ussing chambers. the tissues were equilibrated for 30-minutes prior to addition of acidic ringer's solution (ph, 1.2). after 45-minutes of injury, the acid was replaced with neutral ringer's and the tissues were treated with indomethacin, tramadol or both. tissues were maintained for 210minutes total, during which time permeability was assessed electrically. prostanoid concentrations were quantified using a commercially available elisa. western blots were performed for cox-1 and à2. recovery of gastric barrier function after acid injury was inhibited by co-administration of tramadol and indomethacin ( figure 1 ) but not by tramadol or indomethacin alone (data not shown). prostaglandin e 2 increased with acid injury. the increase in pge 2 was inhibited by co-administration of indomethacin and tramadol (in pg/ml: acid injury 681.25 ae 324.88, indo 1 tramadol 149.11 ae 35.24). there was no significant effect of treatment on cox-1 or à2 expression. co-administration of tramadol with a non-selective nsaid inhibits the return of gastric mucosal barrier function after acid injury in canine tissue, suggesting that caution is required in prescribing concurrent use of these drugs in dogs at risk for gastric ulcers. these drugs may exert this effect by decreasing levels of gastroprotective prostanoids. further study is needed to understand the mechanism of this drug interaction. an increased intestinal permeability (ip) has been suggested to be both cause and consequence of gastrointestinal (gi) disease, such as inflammatory bowel and celiac disease, in people. a novel tight junction regulator, larazotide acetate (alba therapeutics, baltimore, md) has been shown to significantly decrease ip in rats and in humans with celiac disease. the purpose of this study was to determine if larazotide acetate reduces ip in soft coated wheaten terriers (scwt) and norwegian lundehunds (nl) with chronic gi disease and ameliorates clinical signs. four nl (2 females, 2 males; median age: 2.5 yrs, range: 1.5-4.5 yrs) and 9 scwt (7 females, 2 males; median age: 5.0 yrs, range: 1.5-9.0 yrs) were enrolled based on presence of clinical signs of gi disease and hypoalbuminemia, increased fecal alpha 1proteinase inhibitor (a 1 -pi) concentrations, and/or hypocobalaminemia. scwt with protein-losing nephropathy were excluded. dogs were fed q12 hrs and received 0.5 mg (4 nl and 2 scwt) or 2.0 mg (7 scwt) of larazotide acetate po before each meal for 90 days. prior to start of treatment (day 1) and at the end (day 90), ip was evaluated by calculating the lactulose/rhamnose (l/r)-ratio in serum samples obtained at 30, 60, 90, and 120 min after oral dosing. also, 3 consecutive fecal samples each were collected prior to day 1 and day 90 for n-methylhistamine (nmh) measurement. pre-and post-treatment data were compared using a wilcoxon signed rank test. the 0.5 mg vs. 2.0 mg dose groups were compared using a mann-whitney u test. statistical significance was set at p o 0.05. l/r-ratios (medians) for the 60 min sampling time point were significantly lower on day 90 (0.046) than on day 1 (0.080; p 5 0.018). dogs treated with 2.0 mg q12 hrs had significantly lower 60 min l/r ratios on day 90 than dogs treated with 0.5 mg (0.033 vs. 0.094; p 5 0.014). no difference was found between breeds. fecal nmh concentrations were not different between time points, treatment groups, or breeds. fecal a 1 -pi concentrations were available for 11 of the 13 dogs and were significantly higher on day 90 compared to day 1 (p 5 0.033). no differences were found between pre-and post-treatment serum albumin or cobalamin concentrations. weight gain was seen in all 4 nl. resolution of diarrhea, vomiting, hyporexia, as well as an increased activity was seen in 1 scwt. another scwt had resolution of diarrhea and a decrease in pruritus. no changes in clinical signs were reported in the remaining 7 scwt. this study indicates that larazotide acetate might be able to reduce ip in dogs. this effect may be dose-dependent. however, not all dogs showed an improvement in clinical signs, suggesting that factors other than increased ip might have been responsible for the clinical signs in these dogs. breed-related effects cannot be ruled out, and further studies are warranted to determine the efficacy of larazotide acetate in dogs of other breeds with gi disease. to analyze different biochemical markers, calculate clinical activity scores, and assess survival in dogs with ple and compare them with those in dogs with food-responsive diarrhea (frd) without protein loss. 29 dogs with ple and 18 dogs with frd, referred to the university of bern, ch, were enrolled. selection criteria included a history of chronic diarrhea (4 3 weeks), exclusion of identifiable underlying causes, and histopathologic evidence of intestinal inflammation, but not neoplasia. underlying disorders were excluded based on cbc, chemistry profile, urinalysis, fecal analysis, trypsinlike immunoreactivity, cobalamin, folate, and transabdominal ultrasound. also, canine pancreatic lipase immunoreactivity (spec cpl s ), c-reactive protein (crp), calprotectin and alpha 1 -proteinase inhibitor (a 1 -pi) were measured in serum from 18 dogs and compared with 18 dogs with frd without ple. all dogs were scored using the canine ibd (cibdai) and the canine chronic enteropathy (cce) clinical activity index (ccecai). total protein, albumin (5-28.1 g/l), and total calcium (1.21-2.32 mmol/l) were decreased in all 29 dogs. cobalamin was decreased in all but 3 dogs ( o 100-490 ng/l). spec cpl was mildly increased in 3/18 dogs with ple and normal in 15/18 ple and all frd dogs. crp was normal in 5/18 dogs with ple (16/18 frd), mildly increased in 7/18 (1/18 frd), and moderately increased in 6/18 ple dogs (1/18 frd). calprotectin was slightly higher in dogs with ple, but all ple and frd dogs yielded values in the normal range. serum a 1 -pi was significantly lower in dogs with ple than in those with frd (p o 0.001), with 13/18 ple dogs below the reference range (1/18 frd). cibdai ranged from 4 to 16 and ccecai from 6 to 19. at the end of the study, 12/29 dogs were still alive with survival times between 26 and 2544 days. 17/29 dogs died with a median survival of 96 days (range 2-874 days). dogs with mildly increased crp died earlier than dogs with a normal or moderately increased crp (p 5 0.011), whereas albumin, calcium, spec cpl, calprotectin, cibdai, and ccecai had no significant impact on outcome and survival. in conclusion, dogs with ple have a significantly lower a1-pi in the serum than dogs with frd. furthermore, most dogs with ple have an increased crp and a decreased cobalamin. a mild increase in crp appears to be a poor prognostic factor. while hypoalbuminemia is a common finding associated with chronic enteropathies, its impact on survival in this population is poorly defined. the aim of this study was to compare dogs with chronic enteropathies on the basis of their serum albumin concentration at the time of presentation. we hypothesized that dogs with a protein losing enteropathy (ple) have a significantly shorter survival time compared to dogs with chronic enteropathies which are not hypoalbuminemic (controls). information obtained from the medical records included signalment, duration and characteristics of clinical signs, physical examination findings, clinicopathologic data and survival time. one hundred seventeen cases fit the inclusion criteria; 68 in the ple group and 49 controls. there was no statistical significance between groups for age (p 5 0.12), weight (p 5 0.17), weight loss (p 5 0.59) and body condition score (p 5 0.072). compared to control dogs, ple dogs had decreased serum concentrations of cobalamin (p 5 0.002), total calcium (p o 0.0001), globulin (p o 0.0001), cholesterol (p o 0.0001) and ionized calcium (p o 0.001). survival analysis revealed a significantly decreased survival time for ple dogs (p 5 0.0008); median survival was 701 days for ple dogs and 4 3,500 days for controls. while the ple group did not survive as long, survival was not directly associated with severity of hypoalbuminemia; patients with albumin concentration o 1.3 g/dl survived longer than those with mild hypoalbuminemia (1.6-1.9 g/dl). this study supports the observation that chronic enteropathy patients have decreased survival time when presented with hypoalbuminemia; however this study suggests the severity of hypoalbuminemia is not a reliable indicator of survival. cobalamin (vitamin b 12 ) deficiency in the chinese shar pei (shar pei) is suspected to be hereditary. inherited causes of cobalamin deficiency have been reported in humans and may affect absorption, transport, or cellular processing of cobalamin. based on human and veterinary studies, an increased serum methylmalonic acid (mma) concentration has been suggested to reflect cobalamin deficiency at the cellular level. in this context, it has been shown in humans that mma concentrations are higher in patients with genetic disorders affecting intracellular processing than in patients with genetic defects affecting gastrointestinal processing and extracellular transport of cobalamin. therefore, the aim of this study was to evaluate serum mma concentrations in shar peis and dogs of six other breeds with cobalamin deficiency. from 2008 in conclusion, serum cobalamin deficient shar peis had a 10 times higher median serum mma concentration compared to cobalamin deficient dogs of six other dog breeds. further studies are needed to investigate the intracellular processing of cobalamin in shar peis with cobalamin deficiency. chinese shar peis (shar peis) have a high prevalence of cobalamin deficiency. two other conditions reported frequently in this breed are shar pei fever and cutaneous mucinosis. shar pei fever is an autoimmune disorder causing periodic flare-ups and is associated with increased serum concentrations of c-reactive protein (crp), a nonspecific marker of inflammation. cutaneous mucinosis is characterized by excessive deposition of mucin in the dermis. also, hyaluronic acid (ha), the main component of mucin, was shown to be significantly higher in serum from shar peis with cutaneous mucinosis than in healthy controls. to date, a possible association between shar pei fever and/or cutaneous mucinosis on one side and cobalamin deficiency on the other has not been investigated in shar peis. thus, the aim of this study was to compare serum concentrations of ha (an indicator of cutaneous mucinosis) and inflammatory markers (crp, calprotectin, and s100a12), assumed to be increased in episodes of shar pei fever, in shar peis with and without cobalamin deficiency. serum samples from 40 shar peis, collected from 2008 to 2010, were analyzed. serum ha and crp (reference interval (ri): 0.0-7.6 mg/l) were quantified by using commercial elisa kits (echelon biosciences, salt lake city, ut, usa and tridelta, maynooth, ireland; respectively). serum calgranulin concentrations were measured using an in-house elisa (calprotectin; ri: 0.9-11.9 mg/l) and ria (s100a12; ri: 33.0-233.0 mg/l), respectively. mann-whitney u tests were used to compare serum ha, crp, calprotectin, and s100a12 concentrations between shar peis with and without cobalamin deficiency. significance was set at p o 0.05. fourteen shar peis were severely cobalamin deficient, defined by an undetectable serum cobalamin concentration ( o 150 ng/l). in the remaining 26 dogs, serum cobalamin concentrations were within the reference interval (251-908 ng/l). serum concentrations of ha, crp, calprotectin, and s100a12 were not significantly different between cobalamin deficient shar peis (medians: 649.9 ng/ml, 4. . fifty percent of cobalamin deficient shar peis had serum calprotectin concentrations above the upper limit of the reference interval, and 43% had serum s100a12 concentrations above the suggested upper reference limit. in this study, serum concentrations of ha, crp, and the calgranulins did not differ between cobalamin deficient shar peis and shar peis with a normal serum cobalamin concentration. this finding leads us to speculate that increased ha and/or inflammatory markers are not associated with cobalamin deficiency in shar peis. further studies are needed to investigate serum cobalamin concentrations in patients with shar pei fever or cutaneous mucinosis. cobalamin deficiency (cd) has been associated with gastrointestinal and pancreatic disease in dogs. hereditary cd has been demonstrated in giant schnauzers and single case reports have suggested congenital cd in the border collie (bc) breed. clinicopathologic findings of cd vary and can be unspecific as cobalamin acts as a co-factor for a multitude of enzymatic reactions. the two most important reactions concern the conversion of methylmalonyl-coa to succinyl-coa and the re-methylation of homocysteine (hcy). these two metabolites increase when cobalamin is lacking and act as markers for cobalamin availability on a cellular level. preliminary data from dogs suggested that measurement of methylmalonic acid (mma) may be a better diagnostic test for cd than serum cobalamin concentration. therefore the goals of the study were (1) to establish reference values for serum cobalamin, urine mma and plasma hcy in healthy pet dogs, (2) to screen a larger bc population from switzerland for cd, and (3) to perform genomic analyses on bc with cd. for determination of reference values 35 healthy pet dogs were used. serum cobalamin was measured using an automated chemiluminescence assay (immulite 2000), urine mma was determined using gas chromatography and expressed as a ratio to urine creatinine and plasma hcy was measured using high pressure liquid chromatography and fluorimetric detection. to calculate reference ranges the 10th and 90th percentile were used. data were analyzed using non-parametric tests. reference ranges for cobalamin, hcy, and mma were: cobalamin 279.2-972.8 ng/l; urine mma 2-4.65 mmol/mol creatinine; and plasma hcy 4.73-18.34 mmol/l. the screened bc population comprised 113 purebred dogs and 4 bc (median 11.5 months; range 8-41) suffering from congenital cd could be identified. clinical signs differed and consisted of tiredness (4), stunted growth (4), anemia (3), dysphagia (2) and persistent fever (1). median (ranges) results for healthy bc and bc with cd were: for cobalamin 592 (142-1855) and 72.00 (30-139) ng/l; for urine mma 2 (2-360) and 4148 (1800-6665) mmol/mol creatinine; for hcy 8.5 (2.8-22.4) and 41.00 (40-86.6) mmol/l. strikingly, healthy bc with cobalamin concentrations well within the reference range had significantly higher urine mma concentrations compared to control dogs. under the assumption that the four affected bc are inbred to a single founder animal, first results of genotyping on the 170 k illumina canine_hd snp chip suggest that mutations in the cubn and amn gene can be excluded to cause the observed cd in these dogs. we conclude that cd is a rare familial disease in bc with variable clinical signs. to define the genomic region responsible for cd further genetic analysis is in progress. it remains to be determined why some bc have high urine mma concentrations despite a serum cobalamin concentration within the reference range. calprotectin is a protein complex that plays an important role in the innate immune response. preliminary data suggest that canine calprotectin (ccp) is a useful marker for the detection of inflammation in dogs. recently, a radioimmunoassay for the measurement of ccp has been developed and analytically validated, but this test requires the use of a radioactive tracer. therefore, the aim of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (elisa) for the quantification of ccp in serum and fecal specimens from dogs. canine calprotectin (ccp) was purified, antiserum against purified ccp was raised in rabbits, monospecific antibodies were purified by affinity chromatography, and a sandwich-elisa was developed. purified antibodies were used for capturing and, after coupling with horseradish peroxidase (hrp), for reporting. a hrp substrate was used for color development. the assay was analytically validated by determination of analytical sensitivity and specificity, dilutional parallelism, spiking recovery, and intra-and inter-assay variability. control intervals for serum and fecal ccp were established from 110 and 52 healthy pet dogs, respectively, using the central 95 th percentile. sensitivity of the assay for serum samples assayed in a 1:400 dilution and for fecal extracts assayed in a 1:4,000 dilution was 0.3 mg/l and 3.2 mg/g, respectively. over a wide range of the assay, there was no cross-reactivity with cs100a12, the closest structural analogue of ccp available. observed to expected ratios (o/e) for serial dilutions ranged from 83.2-118.5% (mean ae standard deviation [sd]: 101.3 ae 10.0%) for four different serum samples, and from 81.7-129.1% (mean ae sd: 101.8 ae 14.0%) for five different fecal extracts. o/e for spiking recovery ranged from 87.8-130.4% (mean ae sd: 100.6 ae 6.5%) for four different serum samples and 6 different spiking concentrations, and from 95.9-152.0% (mean ae sd: 104.6 ae 11.6%) for 4 different fecal extracts and 6 different spiking concentrations. intra-assay coefficients of variation (cv) for 4 different serum samples were 7.8, 5.0, 7.4, and 12.7%, and 10.0, 6.1, 6.2, and 7.0% for 4 different fecal extracts. inter-assay cv for 4 different serum samples were 17. 2, 8.1, 9.9, and 12.6%, and 12.3, 8.3, 7.2, and 9 .6% for 4 different fecal extracts. the control intervals for serum and fecal ccp were established as 0.9-11.9 mg/l and 3.2-65.4 mg/g, respectively. we conclude that this new elisa for the measurement of ccp is analytically sensitive, linear, accurate, precise, and reproducible, and does not cross-react with canine s100a12. further studies evaluating the clinical usefulness of measuring serum and/or fecal ccp are currently under way. the syndrome of hemorrhagic gastroenteritis (hge) is characterized by a peracute onset of hemorrhagic diarrhea, vomiting, depression, and anorexia, and can be associated with a high mortality if untreated. the etiology of hge is unknown, but it is speculated that an abnormal response to bacterial endotoxins, bacteria, or dietary components may play a role. hge is characterized by an increased vascular/mucosal permeability, thought to represent a type i-hypersensitivity reaction, whereas inflammation and necrosis appear to be rare. however, markers of gastrointestinal (gi) inflammation and changes in the intestinal microbiota have not been studied extensively in dogs with hge. therefore, the aim of this study was to evaluate fecal canine calprotectin (cp) and s100a12 (a12), a 1 -proteinase inhibitor (a 1 -pi, a marker of gi protein loss), and bacterial groups that have previously been shown to be decreased (i.e., faecalibacterium spp., ruminococcaceae, bifidobacterium spp.) or increased (i.e., proteobacteria) in fecal samples from dogs with hge. fecal samples from 3 consecutive days were collected from 7 dogs with hge. fecal cp, a12, and a 1 -pi concentrations were measured by in-house immunoassays. bacterial dna was extracted from each fecal sample and was analyzed for faecalibacterium spp., proteobacteria, rumino-coccaceae, and bifidobacterium spp. using quantitative pcr assays. concentrations of fecal cp, a12, and a 1 -pi, and the abundance of bacterial dna were compared using a friedman test with dunn's post-hoc tests. significance was set at p o 0.05. at the time of diagnosis (day 1), fecal cp, a12, and a 1 -pi were above the suggested reference intervals in 6, 6, and 5 of the 7 dogs, respectively. until day 3, this number decreased to 2, 1, and 4, respectively. decreases in concentrations were significant between days 2 and 3 for a12 (p 5 0.016), and between days 1 and 3 for a 1 -pi (p 5 0.012), but not for cp despite a trend (p 5 0.085). no differences in the abundance of faecalibacterium spp. (p 5 0.085), bifidobacterium spp. (p 5 0.192), or proteobacteria (p 5 0.305) were observed. however, the abundance of rumino-coccaceae was significantly lower on day 3 when compared to day 2 (p 5 0.008). in this study, fecal markers of inflammation and gi protein loss were increased in dogs with hge. although the number of patients was small, following initiation of treatment, two of the markers decreased significantly. these results suggest a loss of protein into the gi tract at the onset of hge. the lack of significant increases of faecalibacterium spp., bifidobacterium spp., and ruminococcaceae, and decreases in proteobacteria may suggest gi dysbiosis. further longitudinal studies are needed and are currently under way to evaluate gi dysbiosis in canine hge patients. the most recent antiemetic approved for use in dogs is maropitant citrate (cerenia s , pfizer animal health). maropitant is a selective nk1 receptor antagonist that acts by blocking the binding of substance-p within the emetic center and chemoreceptor trigger zone. label dosage recommendations for maropitant citrate are 1 mg/ kg sc or 2 mg/kg orally once daily for up to 5 consecutive days (acute emesis) and 8 mg/kg orally once daily for up to 2 consecutive days (motion sickness). the study objective was to determine when steady-state is reached and the pharmacokinetics of maropitant administered at label oral dosages once daily for 14 consecutive days. two groups of eight healthy beagles were administered maropitant citrate at 2 or 8 mg/kg orally once daily for 14 days. concentrations of maropitant and its metabolite were measured in plasma using a lc-ms/ms assay. pharmacokinetic parameters were estimated using non-compartmental pharmacokinetic techniques and a modeling approach was used to estimate steady-state. the accumulation ratio for maropitant was 2.46 (auc0-24) and 2.03 (cmax) for the 2 mg/kg dose; and 4.81 (auc0-24) and 2.77 (cmax) for the 8 mg/kg dose after 14 days. the model estimate for the number of doses required to reach 90% of steady-state was 4.30 for 2 mg/kg and 8.09 for 8 mg/kg. three dogs experienced a single episode of vomiting. dosing maropitant citrate beyond the label duration was well tolerated by healthy dogs. steady-state was reached after approximately 4 doses for daily 2 mg/kg and 8 doses for daily 8 mg/kg oral dosing. previously presented at the veterinary cancer society, november 2010. cobalamin (vitamin b 12 ) is involved in a variety of metabolic processes. altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders, such as exocrine pancreatic insufficiency (epi) or severe and longstanding ileal disease. this study was conducted to identify breeds with a higher proportion of a decreased serum cobalamin concentration that were submitted to the gastrointestinal laboratory. the study was also aimed at investigating serum trypsin-like immunoreactivity (tli) concentrations that were diagnostic for epi in the dogs with a decreased serum cobalamin concentration. except for csp, breeds identified here, have not previously been identified to have a higher rate of a decreased serum cobalamin concentration. also, a possible association between an undetectable serum cobalamin and a decreased serum tli in ai needs to be further investigated. calprotectin (cp) is a widely used marker for the diagnosis and monitoring of gastrointestinal (gi) inflammation in humans. studies in humans usually report fecal cp concentrations based on a single stool sample although considerable day-to-day variability of fecal cp was found in patients with gi disease and in healthy controls. intra-individual variation of canine cp (ccp) was also substantial in a small number of healthy dogs but has not been determined in dogs with chronic gi disease. thus, the aim of this study was to compare the day-to-day variation of fecal ccp in dogs with chronic gi disease before and during treatment to that in healthy dogs. we hypothesized that fecal ccp would be less variable in patients with chronic gi disease than in healthy controls, and thus collection of a single fecal sample would be sufficient. fecal samples from 3 consecutive days were prospectively collected from 15 dogs (group a; median age: 6.1 years) referred for diagnostic work-up of chronic signs of gi disease, from 8 dogs (group b; median age: 5.6 years) with stable gi disease while being treated, and from 44 healthy adult dogs (group c; mean age: 5.4 years). fecal samples were extracted and ccp was measured by an in-house immunoassay. mean ccp, standard deviation, coefficient of variation (cv), and difference between maximum and minimum ccp for the 3-day sample collection period were calculated for each dog and were compared among groups using a kruskal-wallis test. fecal ccp ranged from 2.9-102.7 mg/g (median: 17.6 mg/g) in dogs with gi disease (group a), from 2.9-265.1 mg/g (median: 18.4 mg/g) in dogs of group b, and from 2.9-93.1 mg/g (median: 7.9 mg/g) in healthy controls (group c). cvs were 0-121.3% in group a (median: 20.0%), 2.0-89.4% in group b (median: 47.4%), and 0-145.2% in group c (median: 40.4%), respectively. patients in group a appeared to have less variable fecal ccp than dogs in group b and c, but this difference was not significant (p 5 0.326). the difference between maximum and minimum ccp for the 3-day sample collection ranged from 0-31.1 mg/g in group a (median: 7.2 mg/g), from 0.1-240.0 mg/g in group b (median: 12.5 mg/g), and from 0-57.4 mg/g in group c (median: 14.8 mg/g), and were not significantly different between any of the groups (p 5 0.530). in this study, considerable day-to-day variation of fecal ccp was found in dogs with chronic gi disease (regardless of treatment) and was comparable to that in healthy dogs. results of this study suggest that for evaluating fecal ccp in dogs with clinical signs of gi disease, three consecutive fecal samples rather than a single fecal sample should be analyzed. because we did not intend to evaluate the clinical usefulness of fecal ccp as a marker of gi disease in dogs, disease severity, quality, and location differed among dogs in groups a and b. the diagnostic utility of fecal ccp in dogs with gi disease is currently being investigated. it has been suggested that diagnosis of clostridium perfringens related enteropathy should be based on the detection of the c. perfringens enterotoxin gene (cpe-gene) by pcr and/or c. perfringens enterotoxin (cpe) by elisa in feces. however, the prevalence of the cpe-gene and cpe in dogs and especially cats with gastrointestinal disease has not yet been reported. also, there is limited information about the stability of cpe in fecal samples at various storage conditions. the aim of this study was to evaluate the prevalence of the cpe-gene and cpe and the stability of cpe in fecal samples from dogs and cats. to evaluate the prevalence of the cpe-gene, a total of 481 fecal samples from dogs and cats with clinical signs of gastrointestinal disease (273 dogs and 208 cats) and 109 fecal samples from those without such signs (80 dogs and 29 cats) were examined using pcr. to evaluate the prevalence of cpe, a total of 90 fecal samples from dogs and cats with clinical signs of gastrointestinal disease (31 dogs and 59 cats) and 11 dogs without such signs were evaluated using a commercially available elisa kit (techlab, blacksburg, va). the results were analyzed using a fisher's exact test. significance was set at p o 0.05. to evaluate the stability of cpe, 8 fecal samples from dogs and 2 from cats with clinical signs of gastrointestinal disease that were positive for cpe were examined. also, 5 cpe negative samples from dogs were evaluated as negative controls. each sample was subdivided into 8 aliquots and evaluated on day 0; on days 2, 5, and 10 after being stored at room temperature (rt) or 41c; and on day 10 after being stored at à201c. the prevalence of the cpe-gene was not significantly different between dogs with signs of gastrointestinal disease (99/273; 36.3%) and dogs without (27/80; 33.8%; p 5 0.79). also, the prevalence of the cpe-gene in cats with signs of gastrointestinal disease (80/208; 38.5%) was not significantly different compared to cats without (6/ 29; 20.7%; p 5 0.06). pcr and elisa results were available for 90 samples. of the 65 pcr positive samples, only 6 (9.2%) were elisa positive. of the 35 pcr negative samples, only 1 (2.9%) was elisa positive. the prevalence of cpe was not significantly different between dogs with clinical signs of gastrointestinal disease (3/31; 9.7%) and those without (1/11; 9.0%; p 5 1.0). the prevalence of cpe in cats with signs of gastrointestinal disease was 4/59 (6.8%), but no samples from cats without such signs were available. when evaluating the stability of cpe, results for all aliquots were consistent with the initial result, except for one sample (on day 5, stored at rt, which was initially cpe positive). these results indicate that only a small proportion of samples that are pcr positive for the cpe-gene are also positive for cpe. studies are warranted to further compare the prevalence of cpe among animals with gastrointestinal disease and those without. furthermore, the results indicate that cpe is relatively stable in fecal samples at various storage temperatures. clostridium perfringens has been implicated as a cause of diarrhea in dogs. the main study objective was to compare two culture methods for the identification of c. perfringens. a secondary objective was to evaluate c. perfringens toxin genes a, b, b 2 , e, ı and cpe from canine isolates using a multiplex pcr and determine their prevalence in a group of normal and diarrheic dogs. fecal samples were collected from clinically normal (nd, n 5 105) and diarrheic dogs (dd, n 5 54) at a primary care veterinary facility. isolation of c. perfringens was performed using direct inoculation of feces onto 5% sheep blood agar (sba) as well as enrichment of stool in bhi broth followed by inoculation onto sba. isolates were tested by multiplex pcr for the presence of a, b, b 2 , e, ı and cpe genes. c. perfringens was isolated from 84% (88/105) of nd fecal samples using direct culture and 87.6% (92/105) with bhi enrichment (p 5 0.79). in the dd, corresponding isolation rates were 90.7% and 93.8% (p 5 0.45). all isolates possessed a toxin gene. b, b 2, e, ı and cpe toxin genes were identified in 4.4%, 1.1%, 3.3%, 1.1% and 14.4% of nd isolates, respectively. in the dd group, b and b 2 were identified in 5%, e and ı were not identified and the cpe gene in 16.9% of isolates. bhi enrichment did not significantly increase the yield of c. perfringens compared to sba but increased time and cost involved. c. perfringens (p 5 0.64) and c. perfringens toxin genes were present in equal proportions in nd and dd groups (p ! 0.15). culture of c. perfringens and pcr for toxin genes are of limited diagnostic utility due to the high prevalence of c.perfringens in normal dogs and the lack of apparent difference in toxin gene distribution between normal and diarrheic dogs. endoscopic biopsies are a relatively convenient, non-invasive test for feline infiltrative intestinal disorders. commonly, only the duodenum is examined due to cost, risks and time required to prepare the colon using lavage solutions, cathartics and/or enemas. the purpose of this study was to evaluate the consistency between endoscopic biopsies of the duodenum and ileum in cats. endoscopic biopsies from 70 cats which had duodenal and ileal tissue specimens were evaluated retrospectively. all slides were randomized and reviewed by a single pathologist (jm) for quality, number of biopsies, and diagnosis according to wsava standards. no information regarding history, clinical signs, endoscopic findings, or previous histological diagnosis was made available to the pathologist. statistical comparison of the diagnosis of sc-lsa and ibd by intestinal location was conducted using fisher's exact test (p o 0.05 significant). 18 of 70 cats (25.7%) were diagnosed with sc-lsa in the duodenum and/or ileum. of these 18 cats, 7 (38.9%) were diagnosed with only duodenal sc-lsa, 8 (44.4%) were diagnosed with only ileal sc-lsa, and 3 (16.7%) had sc-lsa in both duodenum and ileum. in 8 cats with only ileal sc-lsa, 3 had severe ibd in duodenal biopsies, possibly consistent with early sc-lsa. 5 of these 8 had duodenal biopsies without evidence of sc-lsa. our results suggest there is a population of cats in which diagnosis of sc-lsa may only be found by evaluating ileal biopsies. clinicians should consider performing both upper and lower gi endoscopic biopsies in cats with suspected infiltrative small bowel disease. periodontitis is one of the most common diseases in cats and is mainly due to the presence of plaque and calculus. in this study, we investigated putative correlations between dental tartar and gingivitis and also between gingivitis and subgingival bacteria in cats. twelve cats (median age: 5 years; range: 1-10 years; 6 dsh and 6 persians; 8 females and 4 males) were enrolled. dental tartar was obtained during scaling for a dental prophylactic procedure. all cats were negative for felv and fiv infection as assessed by a commercial elisa test (snap s fiv/felv combo test). severity of gingivitis (scores: 0-3; 0 5 normal, 1 5 mild, 2 5 moderate, and 3 5 severe) and dental tartar (scores: 0-3) were scored in each cat. endodontic paper points were applied for collecting a bacterial sample from the subgingival area and transferred to thioglycollate transporting media for bacterial culture. the relationship between gingivitis and tartar thickness scores was analyzed by spearman correlation. a student's t-test was used to compare the mean differences (gingivitis and tartar thickness scores) between upper and lower teeth. the association between severity of gingivitis and bacterial type was tested by chi square test. the spearman correlation coefficient for the average gingivitis score and the average tartar thickness score was 0.91 (p o 0.05). interestingly, the average tartar thickness scores from the upper jaw were significantly higher than those from the lower jaw (p o 0.05). the highest scores were found for the molar teeth in all cats. bacterial culture revealed 28.9% anaerobic bacteria species (i.e., bacteroides spp., peptostreptococcus anaerobius, and eubacterium aerofaciens) and 71.1% aerobic bacteria species (i.e., pasteurella multocida, streptococcus spp., enterococcus spp., staphylococcus spp., bacillus cereus, escherichia coli, and pseudomonas aeruginosa). anaerobic bacteria were found mostly in cats with higher gingivitis scores (2-3; chi square: p o 0.05), while pasteurella multocida was found mostly in cats with lower gingivitis scores (0-1; chi square: p o 0.05). antimicrobial sensitivity testing indicated that all of the anaerobic bacteria were sensitive to clindamycin, chloramphenicol, metronidazole, cefoxitin, or tetracycline, 90% were sensitive to erythromycin, and 80% were sensitive to penicillin. the most abundant aerobic bacterial species, pasteurella multocida, was sensitive to cefoxitin in all cases in which it had been cultured. these results suggest that anaerobic bacteria may be associated in the pathogenesis of severe gingivitis. these data warrant further studies of the prophylactic use of antibiotics in cats undergoing dental prophylactic procedures. inflammatory bowel disease is the most common cause of vomiting and diarrhea in dogs. although it can occur in any canine breed, certain breeds are more susceptible. we have previously shown that polymorphisms in the tlr4 and tlr5 gene are significantly associated with inflammatory bowel disease (ibd) in the german shepherd dog (gsd), a breed at risk of developing this disease. it would be useful to determine if these polymorphisms are significant in other canine breeds as this may allow the development of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. therefore the aim of this study was to investigate whether polymorphisms in canine tlr 4 and tlr 5 genes are associated with ibd in other non-gsd canine breeds. four non-synonymous snps in the tlr4 gene; t23c, g1039a, a1572t and g1807a and three non-synonymous snps in the tlr5 gene; g22a, c100t and t1844c previously identified in a mutational analysis in gsds with ibd were evaluated in a case-control study using a snapshot multiplex reaction. sequencing information from 85 unrelated dogs with ibd consisting of 38 different non-gsd breeds from the uk were compared to a breed-matched control group consisting of 162 unrelated dogs from patients treated for noninflammatory disease at the royal veterinary college, london, uk. as in the gsd ibd population the two tlr5 snps; c100t and t1844c were found to be significantly protective for ibd in other breeds included in this study (p 5 0.023 and p 5 0.0195 respectively). this study confirms the protective effects of the two tlr5 snps (c100t and t1884c) in other canine breeds with ibd. this highlights the importance of tlr5 in the pathogenesis of canine ibd and may represent common pathological pathways of ibd in different canine breeds due to the high degree of haplotype sharing seen among breeds. this may allow for the future expansion of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. further functional studies looking at the role of tlr5 in the pathogenesis of canine ibd are needed to confirm these findings. toll-like receptor 5 (tlr5) is an extracellular pattern recognition receptor belonging to the innate immune system. we have recently shown that three non-synonymous single nucleotide polymorphisms (snps) in the tlr5 gene (g22a, c100t and t1844c) are significantly associated with inflammatory bowel disease (ibd) in german shepherd dogs. in addition, we confirmed that two of these tlr5 snps (c100t and t1844c) were significantly associated with ibd in a population consisting of 38 different dog breeds. in order to determine if other novel snps exist in the tlr5 gene in addition to the ones identified in the gsd population, mutational analysis was carried out in seven boxer dogs with ibd. polymerase chain reaction was carried out to amplify the tlr5 coding region in the seven dogs with ibd. sequencing was carried out using sequence based typing with the abi prism sequencing kit (applied biosystems, uk) and analyzed using an abi3100 automated sequencer (pe applied biosystems). sequencing information from seven boxer dogs with ibd from the uk were compared to the reference sequence published on the ensemble webserver (www. ensembl.org/canis_familiaris). in addition to the three snps identified previously in the tlr5 gene, a novel non-synonymous snp; t443c was identified in the boxer dog population with ibd. this snp has never been reported before and was present as the homozygote genotype in three dogs with ibd and in one dog as the heterozygote genotype. using the simple modular architecture research tool (smart) web server (http:// smart.embl.de/) we were able to map the t443c snp to the leucine rich repeat domain of the tlr5 protein. the leucine rich repeat domain is involved with ligand binding and therefore a change in the amino acid in this region may affect function, especially as the t443c snp results in a change in the amino acid from non-polar to polar. our study further confirms the role of tlr5 in the pathogenesis of canine ibd. our results suggest that in addition to shared risk polymorphisms amongst breeds, individual breeds may harbor unique snps arising after breed formation which may further affect their susceptibility to this disease. however, a case-control study would be needed in the boxer dog to confirm the significance of the tlr5 t443c snp and further functional data would be needed to elucidate the exact role of this polymorphism in canine ibd. an automated power driver device (oncontrol, vidacare) has recently become available for bone marrow aspiration (bma) and bone marrow biopsy (bmb) in humans. the purpose of our study was to compare this automated technique to the traditional manual technique for bone marrow sampling in cats. twelve healthy research cats were anesthetized using a standardized protocol on 2 different occasions, 2 days apart, to have bmas and bmbs performed by the same operator. on day 1, half of the cats were randomized to have a bma performed at both the proximal humerus and the iliac crest, and a bmb performed at the iliac wing, using the oncontrol device (15-gauge needle for bma; 11-gauge needle for bmb). the other half of the cats had the same procedures performed using a manual technique (15-gauge illinois needle for bma; 11-gauge jamshidi needle for bmb). on day 3, each cat had bma performed at the opposite humerus and iliac crest, and a bmb performed at the proximal humerus using the opposite technique from day 1. for each procedure, the operator was given a maximum of 3 attempts to successfully collect a sample. the rate of success, as well as the number of attempts were recorded. the ''ease of use'' of the device was rated by the operator on a 5-point scale after each procedure. using previously determined criteria, the macroscopic and microscopic qualities of the bma and bmb samples were assessed by a board-certified pathologist, blinded to the technique used. the level of pain experienced by each cat was evaluated 6, 12, 18, 24, 36 and 48 hours following each set of procedures, using a previously validated pain scoring system. two sample t-tests were used to compare the automated technique to the manual technique and to compare the humerus to the iliac crest site for bmas and the humerus to the iliac wing site for bmbs. for all procedures, at all sites, the ''ease of use'' was better for the automated technique than for the manual technique (p o 0.05). the duration of the procedure and the number of attempts to collect a sample were significantly lower with the automated technique for bma at the proximal humerus (p o 0.05). there was no significant difference in the level of pain at any time point following each set of procedures with either technique. performing bma at the proximal humerus was associated with a higher rate of success (p o 0.05), a lower number of attempts (p o 0.05), a shorter duration of the procedure (p o 0.05), a higher-rated ''ease of use'' of the technique (p o 0.05), and a better quality sample (p o 0.05) when compared to sampling from the iliac crest, in conclusion, we found the automated bone marrow sampling technique suitable for use in adult cats. this technique was easier to use than the manual technique for both bma and bmb, and reduced the duration of the procedure and the number of attempts for successful bma at the proximal humerus. performing bma at the proximal humerus was faster, easier and allowed collection of better quality samples than at the iliac crest, independently of the technique used. the fractious nature of some feline patients sometimes makes sedation or general anesthesia necessary for routine procedures such as blood collection for hematologic analyses. it has been anecdotally reported that sedation or general anesthesia could induce variations in hematologic parameters in cats, making it important for the clinician to be able to anticipate potential changes on hematologic parameters that could result from chemical restraint. this study evaluated the effects of a standardizecd anesthetic protocol using ketamine (10 mg/kg, iv), midazolam (0.5 mg/kg, iv) and buprenorphine (10 mg/kg, im) on the hematologic parameters of 12 healthy adult research cats. each cat had blood samples collected before and after induction of anesthesia on 2 different occasions, 2 days apart. in total, 24 pairs of complete blood counts were obtained. analyses were performed at a certified veterinary laboratory. paired sample t-tests were used to determine whether there were any statistical differences between hematologic parameters before and after induction of general anesthesia, for each cat, on 2 different occasions. compared to preanesthetic values there was a significant decrease in red blood cell count, hemoglobin concentration, hematocrit, lymphocyte count and plasma total protein concentration after induction of anesthesia. there was no significant difference in the segmented or band neutrophil, eosinophil, basophil, monocyte and platelet counts between the samples taken before and after induction of anesthesia. on average, there was a 23.7% decrease in the red blood cell count (9.06 â 10 12 /l to 6.91 â 10 12 /l) (p o 0.0001), a 23% decrease in hemoglobin concentration (133.88 g/l to 103.08 g/ l) (p o 0.0001), a 24.4% decrease in the hematocrit (0.41 l/l to 0.31 l/l) (p o 0.0001), a 25.3% decrease in the lymphocyte count (3.68 â 10 9 /l to 2.77 â 10 9 /l) (p 5 0.0023), and a 12.1% decrease in the plasma total protein concentration (84.79 g/l to 74.54 g/l) (p o 0.0001) when samples taken before and after induction of anesthesia were compared. if only the hematocrit was considered as a marker of anemia, 29% of the samples from these 12 healthy cats, taken while they were under general anesthesia, would have been misinterpreted as belonging to anemic patients (hematocrit o 0.285 l/l), using the reference interval established in our laboratory. none of the cats would have been considered anemic before induction of general anesthesia. in practice, the decrease in lymphocyte count following anesthesia is unlikely to be of clinical relevance, as all the samples except 2 had a lymphocyte count that was within the reference interval for cats established by our laboratory. this study suggests that complete blood counts performed on blood taken under general anesthesia with this combination of anesthetic drugs in cats should be interpreted cautiously in order not to make a false diagnosis of anemia. the mechanism responsible for the decrease in circulating red blood cell mass following anesthesia induction in cats is unknown and requires further investigation. rivaroxaban is an oral inhibitor of activated coagulation factor x (xa). it is expected to have similar coagulation effects as low molecular weight heparin, without the need for injection, making it an attractive alternative for long-term anticoagulant therapy in cats. citrated blood obtained from five healthy adult cats was exposed in vitro to varying concentrations of rivaroxaban, followed by coagulation testing. the rivaroxaban was extracted from commercially available tablets (xarelto s ) and dissolved in dmso prior to addition to the blood. tests performed included kaolin-activated thrombelastography (teg), prothrombin time (pt), dilute pt (dpt), activated partial thromboplastin time (aptt), and anti-factor xa (axa) activity. dose-dependent prolongations were seen in all coagulation parameters. similar to human data, therapeutic axa levels (between 0.5-1.0 axa units) were achieved at in vitro concentrations between 160 and 220 mg/l. at 220 mg/l, dpt measurements were clinically prolonged in all cats (29.2 ae 4 sec vs. 18.5 ae 0.8 sec, p 5 0.148), while aptt values were only mildly prolonged from baseline (21.9 ae 5 sec vs. 15.6 ae 2 sec, p 5 0.07). significant prolongations were seen in dpt at 500 (60.4 ae 42 ec, p 5 0.005). teg r time did not prolong from baseline values until concentrations of 2000 mg/l were reached (16.0 ae 9 min compared to 3.1 ae 0.7 min, p 5 0.006). rivaroxaban has similar coagulation effects in the cat as in other species and may play a role in feline thromboprophylaxis. kaolinactivated teg does not appear to be sensitive to low concentrations of rivaroxaban in the cat. anticoagulated blood is required for platelet function studies. sodium citrate, a calcium chelater, is the most commonly used anticoagulant to measure coagulation parameters including platelet aggregation but it may have a negative effect on platelet responsiveness. dogs are generally considered moderate responders to collagen on platelet aggregation and are notorious for being poor or inconsistent responders to adp-induced platelet aggregation using citrated whole blood. hirudin, a selective thrombin inhibitor, can also be used as an anticoagulant for coagulation assays and is the anticoagulant of choice for certain assays including the multiplate s platelet function analyzer. ten adult healthy dogs were used to compare whole blood platelet aggregation between citrated and hirudinated blood samples. venous blood was collected atraumatically from the external jugular vein directly into tubes containing 3.2% trisodium citrate or hirudin. whole blood platelet aggregation was performed (whole-blood lumi-aggregometer, chrono-log corporation, havertown, pa, usa) with collagen (5 mg/ml) and adp (10 mm) as agonists. maximal platelet aggregation (ohms) was recorded. there was a significant increase in collagen-induced platelet aggregation from the hirudinated samples compared to the citrated samples (31.2 ae 6.1 vs. 17.2 ae 8.6 o, p o 0.0005). there was also a significant increase in adp-induced platelet aggregation from the hirudinated samples compared to the citrated samples (15.6 ae 6.0 vs. 2.6 ae 2.6 o, p 5 0.0001). the results of this study show a significant difference in platelet responsiveness between citrated and hirudinated whole blood using the chrono-log impedance aggregometer. while both collagen and adp-induced platelet aggregation was attenuated from citrated blood samples, this was most notable for adpinduced aggregation where almost all samples had no objective measurable platelet aggregation. it is suggested from this data that future whole blood platelet aggregation studies performed on the chrono-log impedance aggregometer should use hirudinated blood samples although new reference limits would need to be established. low-molecular-weight heparin (lmwh) is now used to prevent thrombotic complications in dogs. a functional assay such as the calibrated automated thrombogram (cat) may provide a new approach for monitoring lmwh therapy. we hypothesized that cat would detect decreased endogenous thrombin potential (etp) in healthy dogs receiving lmwh (fragmin s ). twenty-four healthy adult beagles were included in this study and divided equally in four groups. one dose of 50 u/kg, 100 u/kg or 150 u/kg of lmwh were given subcutaneously to healthy dogs and compared to a control group. platelet poor plasma (ppp) was collected over a 24 hour period. using a repeated-measure linear model, effect of lmwh on etp was time and dose dependent with a significant interaction (p o 0.0001). compared to control dogs, significant differences were obtained for group 50 u/kg at t60 (p 5 0.037), for group 100 u/kg at t15 (p 5 0.013) and between t30-t240 minutes (p o 0.0001) respectively, and for group 150 u/ at t15 (p 5 0.011), between t30-t300 minutes (p o 0.0001) respectively and at t360 (p 5 0.004 the cat assay can be employed to measure the effects of lmwh at different doses in healthy dogs, resulting in significant time and dose-dependent decreases in etp and warrants further investigation as a tool for monitoring lmwh therapy in dogs. the purpose of this study was to determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation parameters in healthy dogs, with an emphasis on thromboelastography (teg). this was a prospective, randomized, blinded study utilizing fourteen dogs determined to be healthy based on normal physical examination, complete blood count, biochemistry, urinalysis, and fecal floatation. dogs were evenly divided into either prednisone plus aspirin (pa) or prednisone plus placebo (pp) groups. baseline values for teg parameters (r, k, angle, ma, ly30, ly60, g, ci) were measured twice two days apart, and thrombin-antithrombin complexes (tat), and traditional coagulation parameters (prothrombin time, activated partial thromboplastin time, d-dimer, antithrombin (at), fibrinogen) were measured once. each dog received 2 mg/kg/ day of prednisone, and either 0.5 mg/kg/day of aspirin (pa group) or placebo (pp group) for 14 days. a complete blood count, biochemistry profile, teg, tat, and traditional coagulation parameters were then repeated on each dog. day to day variation was calculated for the teg parameters using the two baseline measurements. the change from baseline between and within each group were compared using t-tests, or wilcoxon 2 sample test where appropriate, for teg, tat, traditional coagulation parameters, and hematocrit. day to day variation in teg was acceptable ( 10%) for ma, g, and angle, unacceptable (4 10%) for r, k, ly30 and ly60, and not meaningful for ci. within both groups, ma, g, ci and fibrinogen significantly increased from baseline (p o 0.05). within both groups, ly30 and at significantly decreased from baseline (p o 0.05). for the pp group, ly60 significantly decreased from baseline (p 5 0.03), and approached significance for the pa group (p 5 0.0504). all other within group changes from baseline were not statistically significant (p-values 4 0.05). for all parameters, there was no difference between groups for change from baseline (p values 4 0.05). day to day variation in some teg parameters is high and may preclude their clinical utility. prednisone causes hypercoagulability in healthy dogs based on increased g, ma, and ci. the addition of ultra-low dose aspirin to prednisone has no effect on the parameters measured in this study. 'aspirin resistance' has been identified in people and dogs that develop thrombi despite low dose aspirin therapy. variability in platelet cyclooxygenase (cox) isoform expression is one proposed mechanism for aspirin resistance in people. two isoforms (cox-1 and cox-2) have been identified in canine platelets. high (antiinflammatory) dose aspirin inhibits platelet function and alters expression of both cox isoforms in most dogs. this study evaluated the effects of low dose aspirin on platelet function and cox expression in normal dogs. twenty-five healthy client-owned dogs were evaluated before and at two time points (days 3 and 10) during aspirin therapy (1 mg/kg po sid). platelet response to aspirin (siemens pfa-100 s ; collagen/ epinephrine cartridges), was stratified into one of three groups [aspirin responders (9 dogs), non-responders (8 dogs), or inconsistent responders (8 dogs)]. flow cytometry identified platelet cox-1 and cox-2 expression. an elisa was used to measure urine 11-dehydro-thromboxane b 2 (11-dtxb 2 ). there were no significant differences between groups for cox-1, cox-2 or 11-dtxb 2 at any time point. when all dogs were considered as a single group, there was a significant increase (p o 0.0001ã) in cox-1 and cox-2 mean fluorescent intensity (mfi) from baseline to day 10, 70.1% ae 38.0 (mean ae sd) and 70.8% ae 71.2, respectively. there was a significant decrease in mean urine 11-dtxb 2 :creatinine on day 3 and 10 by 23.4% (p 5 0.0044 ã ) and 45% (p o 0.0001 ã ). as with our previous high dose studies, cox-1 expression was increased with aspirin exposure. however, there was a significant increase in cox-2 expression with low dose aspirin in contrast to the decrease seen at higher doses. our study suggests that levels of platelet cox-1 and cox-2 expression do not influence aspirin response in dogs. although thromboxane levels decreased in most (23 of 25) dogs on low dose aspirin, platelet function was consistently affected in only 36% of dogs, suggesting that differences in response to thromboxane may play a role in the variable affects of low dose aspirin on canine platelet function. delayed postoperative bleeding is common in retired racing greyhounds (rrgs), despite normal results of routine hemostasis assays. the excessive postoperative bleeding in the rrgs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. providing a method to prevent or minimize the severity of postoperative bleeding in rrgs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed. epsilon aminocaproic acid (eaca) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms. the objective of this double-blinded, prospective, randomized study was to evaluate the effects of eaca versus placebo on the prevalence of bleeding in rrgs, and to investigate its mechanism of action by using thromboelastography (teg). we compared the effects of eaca and placebo in 100 rrgs that underwent elective ovariohysterectomy or orchiectomy at the veterinary medical center, the ohio state university during 2 years. the main endpoint was bleeding (prevalence and severity); minor endpoints included most teg parameters. thirty percent (15/50) of the rrgs in the placebo group had delayed postoperative bleeding starting 36 to 48 hours after surgery, compared to 10% (5/50) in the eaca group (p 5 0.0124). on the teg parameters, the r time (clot formation time) was significantly different between treatment groups (p 5 0.0321). the postoperative administration of eaca significantly decreased the prevalence of postoperative bleeding in rrgs. thromboembolism associated with protein losing nephropathy (pln) has been long recognized as a serious and unpredictable complication in dogs, however its prevalence remains unknown. in humans, surrogate indicators are frequently used to assess thromboembolic risk. this study aimed to investigate the prevalence of hypercoagulability in pln dogs based on thromboelastography (teg), and to determine whether hypercoagulability in these patients could be predicted by clinical assessments that identify systemic hypertension (systolic blood pressure 4 160 mmhg), hypoalbuminemia (serum albumin o 2.7 mg/dl), antithrombin activity (o 70%), and degree of proteinuria (urine protein:creatinine ratio [upc] ! 2). between march 2009-september 2010, twenty-seven dogs were identified with pln at the animal medical center. the prevalence of hypercoagulability based on a teg g-value 4 9.6 was 83.3%. there was no statistically significant relationship, either categorically or continuously, in univariate as well as multivariate analyses of all variables. univariate logistic regression (odds ratio; lower and upper confidence limit; p value) for hypertension was à1.18; 0.201, 6.99; 0.851; for albumin -1.37; 0.228, 8.26; 0.728; and for antithrombin activity -0.73; 0.12, 4.39; 0.728. thus, in this patient population, in the absence of teg, prediction of hypercoagulability using abnormalities in commonly measured clinicopathologic variables was not helpful. however, given the documented high prevalence of hypercoagulability in patients with pln, early institution of prophylactic anti-platelet or anticoagulant therapies should be considered. thromboelastography (teg) is a test of global hemostasis. due to the effects of extrinsic factors on whole blood coagulation, sample collection method (scm) may influence results. the purpose was to determine if scm influenced teg using kaolin-activated citrated whole blood (wb). healthy dogs with normal platelet counts were prospectively enrolled. three wb samples were obtained from each dog at least 48 hours apart from alternating jugular veins in a randomized order of three methods: 1) vacutainer s into citrated tube (vac), 2) citrated syringe with transfer into plain tube (cit), or 3) plain syringe with transfer into citrated tube (plain). draw time was recorded in seconds. kaolin-activated teg was performed, with measurement of reaction time (r), clot formation time (k), maximum amplitude (ma), and alpha angle. eleven dogs were enrolled. there were no significant differences in teg indices between vac samples and either cit or plain samples. cit samples had a significantly higher k value (p 5 0.004) and a lower alpha angle (p 5 0.004) compared to plain samples. draw times ranged from 2-10 seconds. a longer draw time was significantly correlated (p 5 0.046; r 5 à0.35) with a shorter r time. higher platelet count was significantly correlated (p 5 0.001; r 5 0.575) with a higher ma. scm did not have a significant effect on teg parameters when comparing vac samples to either cit or plain samples. minimizing sample collection time and trauma during venipuncture may be important in minimizing hypercoagulable changes in teg indices. liquid plasma (lp) is defined as either plasma collected and refrigerated immediately after collection or fresh frozen plasma (ffp) that is thawed and stored refrigerated until use. stability studies in people have shown that adequate clotting factor activity is preserved for at least 14 days. lp is transfused in human level i trauma centers to critically ill people requiring rapid infusion of clotting factors as the time required to defrost ffp is considered prohibitive. the use of lp has not been described in veterinary critical care. the purposes of this study were to 1) determine the length of time required in a water bath for a unit of canine ffp to thaw and 2) describe the use of lp in a busy university emergency room (er). for part 1: six units (250 ml) of canine ffp were individually thawed in a 371c water bath. the duration of time (in minutes) to thaw was recorded. for part 2: the transfusion log was reviewed for dogs receiving lp in the last 6 months. the indications and outcome were recorded. the mean time ae sd thaw time was 34.7 ae 1.38 minutes. ten units of lp were transfused to 7 critically ill or injured dogs during the study time. indications for lp transfusion included hypovolemic shock due to intra-abdominal hemorrhage in 6 dogs (2 traumatic, 4 non-traumatic) and rapid correction of hemorrhage following parenteral tissue plasminogen activator administration in 1 dog. lp volume transfused ranged from 11.1 to 50.5 ml/kg. no transfusion reactions were identified. effect on coagulation was not consistently evaluated. time required to thaw a unit of ffp is greater than 30 minutes which could be detrimental in a bleeding, coagulopathic dog. lp was transfused without incident to critically ill and injured dogs and represents a potential new addition to the armamentarium of treatments in a veterinary er setting. further investigation of canine lp is warranted including evaluation of in vitro factor stability and in vivo efficacy in correcting coagulopathy. immune mediated thrombocytopenia (imt) is associated with increased morbidity and mortality. large prospective research studies in dog platelet antibodies and clinical utilization of platelet immunoglobulin assays are limited. potential explanations include limited availability and low specificity due to nonspecific binding. the focus of this study is to evaluate optimized direct and indirect platelet surface associated immunoglobulin (psaig) and staining with anti-cd61 antibodies (cd61ab) for the utilization in classifying thrombocytopenic dogs. one hundred clinically ill and 30 apparently healthy dogs were prospectively evaluated. data collected included a history of hemorrhage, physical examination evidence of bleeding, complete blood count, and measurement of psaig and cd61ab. the psaig assay utilized polyvalent antibodies with correction for non-specific binding by subtraction of background fluorescence with control antiserum. thrombocytopenia was defined as o 164,000/ml and all dogs were clinically classified into 1 of 4 groups (g): g1 imt, n 5 45, g2 thrombocytopenia from non-immune mediated diseases, n 5 37, g3 ill with normal platelet counts, n 5 18, g4 healthy dogs, n 5 30. median platelet counts, by groups, were g1, 20,000; g2, 69,000; g3, 324,500; and g4, 212,000/ml. for the direct and indirect psaigs in dogs with itp (g1), more dogs (n 5 6 and n 5 9) with clinical evidence of bleeding had antibodies compared to those who were not bleeding (n 5 3 and n 5 6). considering only direct psaig the sensitivity and specificity was 13% and 95%, respectively for the diagnosis of imt. for indirect psaig the sensitivity and specificity was 27% and 97%, respectively, for the diagnosis of imt. when considering both direct and indirect psaig together, the sensitivity was 33% with a specificity of 93%. in g1 interference from high control antiserum background staining was noted in 26.7% of dogs and resulted in a negative direct psaig classification. minimal background interference was noted in g2, g3, or g4. the percentage of platelets stained with cd61ab was significantly less in g1 (median 38, p 5 0.0001) vs. g2 (median 74, p 5 0.007) vs. g3 (median 95.1, p 5 0.857) and g4 (median 95.6). these findings indicate the optimized platelet surface associated immunoglobulin assay has a high specificity, however poor sensitivity, for the diagnosis of imt. the decreased cd61 staining in g1 (imt group) may reflect decreased surface gpiiia expression, blocking by anti-gpiiia antibodies or other proteins, clearance by macrophages, or increased non-platelet debris and has potential applications in the diagnosis and treatment of imt. greyhounds have lower serum concentrations of a-globulin than other breeds, explained by negligible levels of haptoglobin (hp) measured using different methods (colorimetric, immunoturbidimetric and protein electrophoresis). the purpose of the present study was to characterize the hp gene in greyhounds. we isolated dna and rna from blood samples of akc-registered and retired racing greyhounds (akcg, rrg), and a german shepherd dog (gsd). we sequenced the hp exons and splice sites, and conducted array comparative genomic hybridization to identify associated dna structural variation (custom 1m agilent oligonucleotide array). additionally, we tested for the presence of one or multiple haplotypes spanning hp in greyhounds using a high density snp array (180k illumina hd). sequencing results of hp in both dna and cdna revealed three synonymous snps in the racing greyhound. we did not identify structural variation overlapping or near the hp gene. notably, we detected that the rrg and akcg do not appear to share a specific haplotype spanning hp. despite having low or undetectable serum concentrations of hp, we did find that rrg hp mrna is expressed and lacks amino acid variation. this suggests that the clinical absence of the hp is attributable to post-transcriptional hp effects or to an unknown physiological interaction. finally, given the existence of distinct rrg and akcg haplotypes spanning hp, it is important to characterize serum levels of hp in akcg in follow on studies. we reported that hemoglobin in retired racing greyhounds (rrg) has higher oxygen carrying properties and affinity than other breeds. surprisingly, very little is known about canine hemoglobin genetics. the purpose of this study was to characterize genetics of canine beta globins. using computational blast analysis of the dog genome, we identified five beta globin genes in a single locus: two human hbelike followed by three hbb-like genes. we isolated dna and rna from blood of rrgs, akc registered greyhounds (akcg), and german shepherd dog (gsd). all beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom 1m agilent array). additionally, we determined the number of common haplotypes that span this locus in rrgs and akcgs using high density snp array (180k illumina hd). expression and sequence analysis of cdna showed all five beta globin genes are actively expressed in adults. canhbb1 and 2 were created by relatively recent segmental duplication and have identical protein sequence. canhbb1/2 are abundantly expressed in adults; canhbb3 is expressed at greatly reduced levels. sequencing results revealed one rare non-synonymous single nucleotide polymorphism (snp) in hbe1 of rrgs, but no variation that could explain their abnormal hemoglobin. we did not detect structural variation overlapping or near the beta globin locus. notably, rrg and akcg do not share haplotypes spanning the beta globin locus. this is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults. sampling of the bone marrow in the dog from the costochondral (cc) junction can be performed with minimal to no sedation and readily available equipment but is not in widespread use in the united states. the aim of this study was to compare the number of attempts needed to successfully obtain a sample, the time needed for the procedure, and the sample quality between aspirates obtained from the cc junction and more traditional sites (humerus or femur) in healthy dogs when performed by novice and seasoned practitioners. samples were obtained from healthy anesthetized laboratory reared adult dogs after undergoing terminal endoscopic surgery. paired samples from separate dogs were obtained by each practitioner using either a 22 gauge needle and 6 cc syringe at the cc junction or an 18 gauge rosenthal needle and 12 cc syringe from either the proximal humerus or femur (clinician preference). three small animal veterinary interns, one experienced technician and one boarded internist were monitored for number of attempts to success and length of time needed for success of each procedure. slides were prepared by a single investigator and read by a blinded clinical pathologist. data were compared using the paired t-test for normally distributed data and wilcoxen signed rank test for non-gaussian distributions. five pairs of samples from three dogs were evaluated. two dogs had two pairs drawn from opposite limbs and ribs. mean number of attempts to success for traditional sampling sites (1.4 1/à0.54) and time to success (8.6 minutes 1/à5.2) did not differ significantly from attempts (2.01/-0.70, p 5 0.25) or time (2.7 1/-0.98, 0.06) needed when aspirating from the cc junction. subjectively, samples were of similar quality with regards to cellularity and number of particles present when compared within practitioners. myeloid: erythroid ratio and percentage of lymphocytes were also not significantly different between sites (m:e ratio p 5 0.37, lymphocyte % p 5 0.07) and were within normal limits. while there were no significant differences between the two sites in terms of number of attempts or time to success, it should be noted that the ''seasoned'' practitioners had never performed an aspirate at the cc site and had an increased number of attempts compared to the traditional sites. if the number of attempts needed for success decreases with experience, it is likely the time required would decrease as well. both subjectively and objectively, there were no significant differences in quality or cell populations between the two sampling sites in healthy dogs. based on this data, bone marrow aspiration from the cc junction appears to be equivalent to more traditional sampling sites in healthy dogs. larger studies in clinically ill dogs should be performed before routinely using the site in the clinical setting. recent research on iron homeostasis has elucidated the tightly controlled intestinal uptake of iron. hepcidin, the major hormone limiting iron absorption and release from macrophages, is downregulated by matriptase-2, a transmembrane serine protease (tmprss6) produced by the liver. while iron deficiency is commonly caused by chronic blood loss anemia and rarely dietary deficiency or intestinal disorders in dogs and other species, we report here the clinical to molecular investigations of a dog with iron-refractory iron deficiency anemia (irida) caused by a matriptase-2 deficiency homologous to a recently described autosomal recessive disorder in humans. the proband, a spayed female cocker spaniel without any clinical signs except for recent occasional idiopathic seizures, exhibited a lifelong history of microcytosis and hypochromasia but not anemia. there was no evidence of any blood loss and the dog was receiving an appropriate meat-based diet. mean values of complete blood cell counts, performed from 0.5-5 years of age, were: hematocrit 41% (normal reference range 39-56); rbc count 9.6 â 10 6 /ml (5.8-8.5 x 10 6 ); mcv 43 fl (62-72); mchc 31 g/dl (33-36). serum iron parameters revealed severe iron deficiency with serum iron 34 mg/dl (88-238); total iron binding capacity 378 mg/dl (246-450); serum iron saturation o 9% (15-50%), and ferritin 410 ng/dl (80-800). prolonged courses of oral ferrous sulfate supplementation and several short courses of intramuscular (dextran) injections and intravenous iron infusions did not result in improvement of any red cell or serum iron parameters. however, this dog was never anemic and the partial seizures could not be directly related to the iron deficiency status. no family members were available for further studies. genomic dna was extracted from the proband's edta blood and the exons of the tmprss6 gene were amplified with flanking primers and then sequenced. in comparison to the normal canine tmprss6 sequence and that of a sequenced control dog we found a homozygous missense muation, r723h, toward the c-terminal end of the protein in the proband's gene. in conclusion, the severe microcytosis and hypochromasia, low serum iron parameters and lack of a response to oral and parenteral iron therapy led to the diagnosis of irida. the missense mutation in the matriptase-2 at position 723 from an arginine, which is conserved across all species currently deposited in the genbank, to a histidine is likely the disease-causing mutation. this is the first report of an irida in the dog with features very similar to those observed in humans. dogs with naturally-occurring irida may be helpful in developing and assessing novel therapies. accidental ingestion of copper-coated zinc pennies minted after 1982 is the most common causes of zinc toxicity anemia in the dog. zinc toxicity anemia may also be seen with ingestion of zinc from other sources as ingestion of metallic foreign material other than pennies, medicines containing zinc, and zinc supplements. the purpose of this study was to determine if there is a weight below which dogs are more susceptible to zinc toxicity anemia secondary to metallic foreign body ingestion. records of dogs presented to the internal medicine service at the veterinary medical center of long island for metallic foreign body ingestion were reviewed for signalment, weight, presenting pcv, and type of metallic foreign body ingested. eighteen dogs met the inclusion criteria and were compared. of the 18 dogs, there were 15 cases of coin ingestion (83%), with 11 (73%) involving ingestion of 1 or more pennies. the other 3 cases involved ingestion of a metallic object (1), decorative garland (1), and bb pellets (1).of the 14 dogs exposed to zinc, 13 (93%) were less than 27 pounds (12.3 kgs). of those 13 cases 11 (85%) had ingested one or more pennies. eleven out of the 14 (79%) zinc exposure dogs were anemic at presentation. the average weight of the 11 dogs was 17.5 pounds (8 kg). this study showed that dogs less than 27 pounds appear to be more susceptible to developing anemia secondary to zinc toxicosis, with the majority of cases due to ingestion of pennies minted after 1982. zinc toxicity anemia secondary to penny ingestion is more commonly seen in small dogs. we suspect larger dogs are able to pass pennies through the pyloric sphincter and thus not develop clinical signs. although thrombocytopenia is common in hospitalized dogs, canine cryopreserved platelet concentrate (pc) is used infrequently. data suggest in vitro efficacy of pc and when administered to research dogs, but efficacy is unknown in clinical patients. study objectives were to determine clinical characteristics of dogs receiving pc as well as safety and efficacy of pc in thrombocytopenic dogs. medical records were evaluated retrospectively to identify dogs that received pc. information evaluated included patient characteristics, platelet count, acute transfusion reactions, and survival. twenty six dogs met study criteria. dogs receiving pc ranged in age from 1-13 years (mean 7.8 years) and 17/26 (65.4%) were spayed or intact females. hemorrhage was reported in 20/26 dogs (76.9%) prior to pc transfusion. platelet counts prior to transfusion ranged from 0 to 77 â 10 3 /ul (mean 29.9 1/à43.9 â 10 3 /ul). change in platelet count was measured in 23 dogs and the mean change was17.1 1/à 45.5 â 10 3 /ul. dose of pc administered ranged from 4.8 to 40 ml/kg with a mean of 14.5 1/à 8.8 ml/kg. no acute adverse reactions were reported. there was no correlation between transfusion dosage and platelet count change post transfusion. survival to discharge occurred in 15/26 (57.7%) of dogs. the only variable correlated with survival was age with survivors being younger than non-survivors (6.4 years-old ae 3.9 vs. 10.1 years-old ae 3.1.; p 5 0.02). efficacy of cryopreserved pc transfusions for improving clinical outcome in dogs with thrombocytopenia is yet to be determined; however, pc is well tolerated in clinical patients. fresh frozen plasma (ffp) is used to treat coagulopathies in dogs. current transfusion guidelines recommend that ffp be administered within 4 hours of thawing to avoid decreasing clotting factor function and bacterial contamination. the purpose of this study was to assess clotting factor activity and bacterial contamination of ffp that had been thawed and refrigerated for 5 days. blood was collected from 10 client-owned healthy dogs with no known history of coagulopathy or of administration of drugs affecting coagulation. plasma was separated from whole blood and frozen (à201c) within 30 minutes of collection. thawed plasma was maintained at 41c (1/à21c). aerobic and anaerobic bacterial culture, prothrombin time (pt), activated partial thromboplastin time (ptt), and factor ii, vii, ix, and x analyses were tested at time of whole blood collection, ffp thaw, 24 hours post-thaw, 72 hours post-thaw, and 120 hours post-thaw. there were no statistically significant differences in pt and ptt at any of the measured time points. statistically significant differences occurred between initial measurements of factors ii, vii, ix, and x and subsequent time points, but there was no difference in activity levels of the factors once ffp was thawed. one bacterial colony was grown from each of two samples from post-thaw plasma. thawed plasma protocols do not significantly decrease the function of factors ii, vii, ix, and x or prolong pt and ptt. bacterial contamination of the plasma supply seems unlikely, but strict aseptic technique should be used when obtaining plasma for patient use. erythrocyte pyruvate kinase (pk) deficiency is the first and most common erythroenzymopathy described in dogs, cats, and humans. the pk enzyme plays a crucial role in the erythrocyte energy metabolism and its absence causes severe hemolytic anemia, often misdiagnosed as autoimmune hemolytic anemia. the disease is inherited as an autosomal recessive trait and affected dogs also develop osteosclerosis. in dogs, the enzymatic diagnosis is complicated by the anomalous expression of malfunctioning m 2 -pk expression, but breed-specific r-pk mutation tests have been reported for basenjis, west highland white terriers (whwt), and beagles. we report here on a survey of canine pk deficiency studied at the penngen laboratory. a biased group of samples were received for screening from dog breeds with known mutations as well as from dogs with chronic, prednisone-and antibiotic-resistant hemolytic anemia and their relatives. edta blood samples and/or cheek swabs as well as medical record information were received and genomic dna and/ or enzyme activity testing were performed. among the 237 whwts 7% and 37% were found to be homozygous deficient dogs or carriers, respectively, with a mutant allele frequency of 0.26. the average age at the time of diagnosis was 1.5 years ranging from 2 months to 5 years of age; some samples came from europe and south america. of the 67 beagles studied, 36% were affected and 3% were carriers (mutant allele frequency 0.37). the average age at the time of diagnosis was 2 years ranging from 7 months to 9 years. surprisingly, very few samples from basenjis were received for screening, and none showed the mutant allele. while pk-deficient basenjis lived o 5 years, whwt and beagles often show milder signs and can reach 9 years of age. several dogs from other breeds were also examined because of chronic regenerative anemia and none had any of the known mutations seen in the other breeds. however, based upon pk enzyme activity studies, chihuahua, dachshund, miniature poodle, spitz, eskimo toy, and labrador retriever dogs were found to be affected; they also had osteosclerosis and at least one labrador retriever developed severe hemochromatosis (hepatic iron 37,300 ppm; normal o 1,200 ppm, analyzed on a dry weight basis). moreover, sequencing of the r-pk cdna from a pk-deficient labrador retriever revealed a new nonsense mutation in exon 6. in conclusion, pk deficiency appears to be a common cause for hemolytic anemia in certain breeds, and mutation testing makes screening simple. pk deficiency should also be considered in dogs of other breeds which may require the more cumbersome enzyme testing. studies to identify new mutations will confirm and simplify the diagnosis. supported in part by nih grant rr02512. immune-mediated hemolytic anemia (imha) is a common hematological condition observed in dogs. the diagnosis is based on clinical history, presenting signs and hematological evidence of imha such as regenerative anemia, leucocytosis and presence of spherocytes. the definitive diagnostic procedure is the coomb's test (direct antiglobulin test, dat) which is known to be highly specific but lacks diagnostic sensitivity. direct flow cytometric assay (fca) for igg, igm or c3 coated red blood cells (rbcs) detection might be more sensitive and thus could be introduced as an alternative diagnostic tool. to investigate the usefulness of fca for imha diagnosis, evaluation of igg, igm or c3 coated rbcs was performed from 15 dogs presented at the veterinary hospital at usp that fulfilled clinical and hematological criteria for imha. thirty eight healthy dogs were included as controls. dat was performed with polyvalent and monovalent anti-dog sera with twofold serial dilutions of each one, incubated with 2% rbcs suspension at 371c and 41c. for fca, 2% rbcs from anemic and healthy dogs were incubated with fitc anti-dog igg, anti-dog igm and anti-dog c3 and submitted to flow cytometry evaluation. specific software and mann whitney u test were used for data analysis. five dogs showed positive results for dat with polyvalent coombs reagent at 41c (titer 64 to 4096) and 371c (titer 64 to 2048) but only three of them had agglutination titer for anti-igg at 4 0 c (1024 to 8192) and 371c (512 to 4096). no positive results were observed for anti-igm and anti-c3 dat. by fc, percentage of igg, igm and c3 coated rbcs in normal and anemic dogs were, respectively, 1,18% and 17,11% (p o 0,001); 1,15% and 21,29% (p o 0,001); 0,66% and 6,99% (p o 0,001). igg coated rbcs percentage were higher in dogs showing dat positive results (min. 33,94%; max. 99,96%; median 54,25%). direct flow cytometric erythrocyte immunofluorescence assay is more sensitive than dat for detection of antibodies coated rbc in anemic dogs and may provide quantitative data useful for laboratorial diagnosis of imzha. bone marrow aspirates from cats are typically obtained from the ilium, humerus or femur, but may be difficult to obtain and/or of poor quality. in this study the feasibility, safety, and nature of sternal aspiration in cats was investigated. under general anesthesia, bone marrow aspirates were obtained in a randomized order by a single investigator from the sternum and ilium of 10 healthy cats weighing 3.4-8.4 kg, with body condition scores of 5-9 (on a scale of 1-9). for sternal aspirates, cats were positioned in sternal recumbency and a 1-inch, 22-23 ga hypodermic needle attached to a 12cc syringe was inserted into the cranial manubrium and directed caudally along the long axis of the sternum. aspirates were also obtained from the right iliac crest using an 18 ga illinois needle attached to a 12cc syringe. difficulty of site localization, needle insertion and advancement, and specimen aspiration, were scored from 1 (easiest) to 5 (hardest). bone marrow smears were prepared by one investigator and reviewed by a pathologist blinded to aspiration site and cat. sample quality was scored from 0 (no marrow particles) to 5 (excellent) based on the number of wellsmeared marrow particles on the slide. particle cellularity was scored from 1 (4 75% fat) to 3 (o 25% fat). post-procedure, cats were treated with tramadol (4-5 mg/kg, po, q12h) for 3 days, and assessed for post-biopsy pain (colorado state university feline acute pain scale, range 0 [no pain] -4 [maximum]) and site swelling (range 0 [none] -3 [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. pneumothorax was not identified. it was significantly easier to perform sternal than iliac aspiration, but the quality of the sample was significantly better for iliac than for sternal aspirates. because of limitations due to sample quality, bone marrow morphology in sternal samples could not be compared to iliac samples in all cats. for samples that could be compared, cellularity was identical for sternal and iliac samples from 1 cat but underestimated in the sternal sample from another cat. myeloid:erythroid ratios and lymphocyte numbers were the same for sternal and iliac samples in 2 and 3 cats, respectively. megakaryocyte numbers were the same in one sample, less in sternal samples compared to iliac samples from 2 cats, and overestimated in the sternal sample from 1 cat. bone marrow cell morphology was normal in all acceptable samples. it was concluded that sternal aspiration of bone marrow using a 22-23 ga hypodermic needle is 1) easier to perform than iliac aspiration; 2) safe; but 3) provides samples of lower quality than iliac aspiration in cats. the diameter of 11-13ga jamshidi-type needles makes bone marrow core biopsy difficult in cats. in this study, biopsies of the left humeral head were taken under anesthesia using a 1-inch, 15ga needle (ez-io s intraosseous infusion system, vidacare) from 10 healthy cats weighing 3.4-8.4 kg with body condition scores of 5-9 (on a scale of 1-9). humeral biopsies were compared to biopsies taken from the left iliac crest using a 2-inch, 13ga jamshidi needle. biopsies were performed in randomized order by one investigator. biopsy was repeated to a maximum of 3 attempts until a specimen ! 5 mm long was obtained. difficulty of site localization, needle insertion and needle advancement were scored from 1 (easiest) to 5 (hardest). specimens were wrapped in tissue paper and placed in davidson's fixative for 15 min and then transferred to formalin. biopsy sections were reviewed by a pathologist blinded to biopsy site and cat. biopsy length on the slide was measured, and biopsy quality was scored from 0 (no hematopoietic tissue) to 5 (! 5 intertrabecular spaces free of artifact). post-procedure, cats were treated with tramadol (4-5 mg/kg, po, q12h) for 3 days, and assessed for postbiopsy pain (colorado state university feline acute pain scale, range 0 [no pain] -4 [maximum]) and swelling of biopsy sites (range 0 [none] -3 [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. there were no significant differences between 15ga and 13ga biopsies except for post-biopsy swelling, and there were no significant effects of body weight and body condition. six (60%) of 15 ga and 5 (50%) of 13ga biopsies were considered acceptable specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoiesis and cell morphology. comparison of acceptable 15 ga to 13 ga biopsy specimens from 4 cats showed no significant differences for cell density and lymphocytes/plasma cells, while cellularity, assessed as high in 2 of the 13 ga biopsies, was assessed as medium in corresponding 15ga biopsies; and megakaryocytes, assessed as 4-9/low-power field in one 13ga biopsy, were assessed as 3/low-power field in the 15 ga biopsy. myeloid:erythroid ratios were greater in 15 ga biopsies compared to 13 ga biopsies in 2 cats, and less in the 15 ga biopsy in one cat. discordant results between biopsies were not related to differences in quality. in conclusion, 15 ga bone marrow biopsy of the humerus was as likely to yield a specimen of acceptable quality as was 13 ga biopsy of the ilium, and resulted in less post-biopsy swelling. reports on canine acute liver failure (alf) include individual or small case series of animals with a specific diagnosis. the aim of this study was to describe the clinical course, outcome and etiology of alf in dogs presenting to a referral hospital. medical records (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) were reviewed for a diagnosis of alf (elevated serum bilirubin or icterus with concurrent coagulopathy or hepatic encephalopathy (he)). fifty cases were identified representing 22 breeds: 7 labradors retrievers, 6 golden retrievers, 3 german shepherds, and 3 cocker spaniels. median age was 6 years (1 m to humerus, 15 ga 5.1 ae 0.9 5.6 ae 0.6 2.1 ae 0.5 6.0 ae 0.7 0 0 ã (2.2-9.3) (3-9) (0-4) (3-10) ilium, 13 ga 6.1 ae 0.6 9.0 ae 0.7 1.9 ae 0.6 7.5 ae 0.8 0.2 ae 0.1 0.9 ae 0.1 ã (4.5-9.5) (6-12) (0-5) (5-12) (0-1) (0-1) 13 yrs). presenting signs included anorexia (31/50), vomiting (26/50), polydipsia (9/50) and neurologic signs (6/50 granulomatous hepatitis (gh) is a histopathological diagnosis characterized by focal aggregations of activated macrophages mixed with other inflammatory cells that is usually part of a systemic disease process (wsava). published case reports describe many potential infectious causes, but only one retrospective study involving nine dogs with gh has detailed clinically relevant findings. the aims of this study were to describe the clinical and clinicopathologic findings in dogs with a histopathological diagnosis of gh, and to identify infectious agents using differential staining techniques, pcr, and fluorescence in-situ hybridization (fish) in archival paraffin-embedded tissues from dogs with gh. medical records of dogs with a histopathological diagnosis of gh (n 5 22) were reviewed and signalment, historical toxin exposure or evidence of other systemic diseases, clinical signs, physical exam findings, clinicopathologic test results, imaging findings, concurrent diagnoses, treatments administered, and case outcome, when available, were extracted and summarized. twelve archival formalin-fixed, paraffin-embedded hepatic tissue samples were available for special staining and molecular diagnostic testing. two of these samples had sufficient tissue for only pcr. the mean age of dogs with gh was 7 years (median 6.5 years; range 2 to 17 years) and included 12 males and 10 females representing 14 different breeds. common presenting complaints included inappetance or anorexia (n 5 12), weight loss (n 5 11), lethargy (n 5 9), fever (n 5 8), and vomiting (n 5 8). high mixed liver enzyme activity (14/22) was the most common clinicopathologic abnormality. leukemia was diagnosed in one dog and copper-associated hepatopathy in 6 dogs. no infectious agents were identified using differential staining techniques. bartonella species dna was not pcr amplified from the extracted archival tissue. furthermore, no bacteria were identified by means of fish using a universal eubacterial probe. these data suggest a possible role for copper accumulation in the genesis of gh in dogs and support further evaluation of dogs with gh for evidence of copper-associated hepatopathy. future studies should include detailed environmental histories, the collection of adequate sample volumes for quantification of hepatic copper content and the examination of frozen tissues using novel molecular diagnostic platforms. hepatocyte copper and iron accumulation contribute to cell loss, inflammation, and fibrosis. the purpose of this study was to compare copper and iron accumulation in feline liver samples with different disease processes. liver biopsies (n 5 104) submitted between july 1, 2007 and june 30, 2009 were evaluated using wsava guidelines and categorized as non-hepatic/normal, congenital, inflammatory/infectious, neoplastic, and other. copper (by rubeanic acid) and iron (by prussian blue) accumulation were graded by increasing amounts (0-3) and location (centrilobular 5 cl, midzonal 5 mz, periportal 5 pp, random 5 r). associations between metal scores and diagnosis category were assessed using the kruskal-wallis test. histologic diagnoses were non-hepatic/normal (n 5 12), congenital (n 5 6), neoplastic (n 5 16), infectious/inflammatory (n 5 39), and other (n 5 31). ninety-two samples were negative for copper; remaining samples were graded 1 (n 5 5), 2 (n 5 6), and 3 (n 5 1). histologic diagnoses (pattern) for positive samples were congenital (1cl), infectious/inflammatory (7: 2cl, 1mz, 2pp, 2r), neoplastic (2pp), and other (2cl). iron staining was negative in 18 samples; remaining samples were graded 1 (n 5 38), 2 (n 5 40), and 3 (n 5 8). distribution was primarily cl (n 5 38) or r (n 5 33), though mz (n 5 13) and pp (n 5 2) distribution occurred. there were no significant differences by kruskal-wallis analysis for amount or location of hepatocellular iron or copper for the different disease categories. in this study, copper accumulation was rare, had variable distribution and occurred primarily in samples with inflammatory/ infectious disease. in contrast, iron accumulation was common and did not correlate with disease category. further prospective evaluation of copper and iron accumulation in feline liver disease and association with outcome may be warranted. chronic hepatitis (ch) in dogs is a progressive condition without clearly defined treatment. glucocorticoids are commonly used to stop progressive inflammation and fibrosis but are associated with significant side effects including a steroid hepatopathy that complicates enzyme monitoring. cyclosporine is proposed as an alternative therapy, but there are no published reports of its use for canine ch. patient records at the csu veterinary teaching hospital were searched for histologically confirmed cases of ch treated with cyclosporine. data were compiled on cyclosporine dosing, concurrent medications, clinical course and biochemical parameters. 13 patients over a 50-month period were identified. serum alanine aminotransferase (alt) decreased by an average of 71% in 12 dogs. the alt normalized completely in 6 of 10 dogs treated for 4 60 days. in 5 of 6 dogs on 4 9 mg/kg/day, the alt also normalized. five of the 6 patients that exhibited clinical signs prior to treatment showed measurable improvement (weight gain, fewer gastrointestinal signs). eight patients had hyperbilirubinemia or ascites prior to treatment; these resolved in 7. post-treatment histopathology, available in one patient, revealed decreased severity of ch. five patients exhibited adverse effects including gastrointestinal signs (3), gingival hyperplasia (1), and papillomatosis (1). cyclosporine was discontinued in 2 dogs with gastrointestinal signs. cyclosporine was an effective therapy for many cases of ch and should be considered for patients who are refractory to or cannot tolerate glucocorticoids. prospective clinical trials with histological documentation are needed to better define cyclosporine's effectiveness in ch. insertion of the veress needle and establishment of pneumoperitoneum is associated with 22 to 57% of all laparoscopic complications in humans. the purpose of this study was to determine the accuracy of interpretation of tissue impedance measurements for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in 20 cadaverous dogs euthanized for reasons unrelated to the study. placement order was randomized. a third individual evaluated impedance measurements using a handheld device (sensormed, knoxville tn) to determine correct versus incorrect needle placement. veress needle locations were marked using contrasting colors of india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified 29/33 correct and 7/ 7 incorrect placements, respectively. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. agreement was moderate (kappa 0.50, p 5 0.01) for placements by operator 1 and very high (kappa 5 0.88, p o 0.01) for placements by operator 2. results for tissue impedance measurement interpretation are superior to published data for currently available tests. impedance measurements accurately detected all incorrect needle placements. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether it increases operator detection of inappropriate veress needle placement and decreases installment phase complication rates. delayed detection of intestinal perforation during veress needle insertion is associated with high mortality. the purpose of this study was to evaluate the accuracy of tissue impedance measurement interpretation for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in 24 cadaverous cats. placement order was randomized. a third individual evaluated impedance measurements (sensormed, knoxville, tn) to determine placement location. needle locations were marked using india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified 36/38 correct and 2/10 incorrect placements. all 8 undetected incorrect placements were located within the retroperitoneal fat pad. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. correlation was absent (kappa à0.15, p 5 0.34) for placements by operator 1 and substantial (kappa 0.78, p o 0.01) for operator 2. there was no association between correct or incorrect placement and operator on chi-squared analysis. failure of impedance measurements to identify placement in the retroperitoneal fat pad resulted in poor accuracy and discordant kappa statistics. small cat size limited the number of appropriate placement sites, perhaps resulting in excessively dorsal placement. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether impedance measurements increase detection of inappropriate veress needle placements or decrease installment phase complication rates. best clinicopathologic tests detecting portosystemic shunting (pss) in dogs remains controversial. this retrospective study examined performance of single random "fasting" and paired serum bile acids (sba; pre-and 2-hr post-feeding) in a large population of non-icteric dogs with confirmed pss (abdominal ultrasound, colorectal scintigraphy, radiographic or spiral-ct portography, laparotomy, or necropsy). sba were measured by enzymatic colorimetric method with normal o 25 mmol/l. dogs meeting inclusion criteria (n 5 568) included 467 portosystemic vascular anomalies (psva; 368 extrahepatic [e-psva], 99 intrahepatic [i-psva]), and 101 acquired pss (apss). signalment and laboratory parameters were recorded. non-parametric statistical analyses were used, two-tailed p o 0.05 applied with bonferroni corrections. median age and weight of 74 breeds were 1.2 (0.2-12) yrs and 6.6 (0.2-48) kg, with equal gender distribution. random "fasting" sba detected 90% psva and 81% apss, whereas post-feeding sba detected 100% psva and 98% apss. low protein-c (o 70% activity) occurred in 96% psva and 74% apss. low mcv and creatinine occurred in 82% and 94% of psva dogs, respectively; other tests were less helpful. in apss, post-feeding sba was superior. compared to apss, psva had significantly (p 0.002) lower mcv, cholesterol, bun, creatinine, glucose, and protein-c. compared to e-psva, i-psva had significantly (p 0.009) lower post-feeding sba, mcv, albumin, urine specific gravity, and protein-c but higher cholesterol and glucose. post-feeding sba reflect physiologically provoked bile acid challenge and should be the preferred sba test in non-icteric dogs for pss detection. protein-c assists in identifying psva but its utility in apss may be complicated by concurrent coagulopathies and inflammation. this study compared outcomes of treatment with adjunctive nonsteroidal anti-inflammatory drugs (nsaids) or anti-inflammatory glucocorticoids in dogs with severe pulmonary blastomycosis. medical records were reviewed for dogs diagnosed with blastomycosis at the university of illinois veterinary teaching hospital between 1992 and 2007. dogs with a presenting pao 2 of 80 mmhg, and clinical or radiographic signs of respiratory blastomycosis were included. all dogs were treated with either itraconazole, fluconazole, amphotericin b, or a combination of these. group 1 (g1) dogs were treated with nsaids and group 2 (g2) dogs were treated with glucocorticoids as anti-inflammatory adjunctive therapy. the following comparisons were made: days of oxygen supplementation, days in hospital, survival to discharge, and long term patient survival. mann-whitney u tests and chi-squared tests were performed on continuous and categorical data, respectively. p o 0.05 was considered significant. sixty-eight dogs fit the inclusion criteria. g1 consisted of 31 dogs and g2 consisted of 37 dogs. the two groups were found to be similar in weight, age, and sex distribution. there was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and patient survival. there does not appear to be a difference between the clinical course or patient outcomes between groups of dogs with severe pulmonary blastomycosis treated with nsaids or anti-inflammatory glucocorticoids. further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ards and additional respiratory complications. diagnosis of feline histoplasma capsulatum infection traditionally relies upon identification of organisms in circulating monocytes or affected organs. in recent years, an antigen assay (aa) was developed for the diagnosis of disseminated histoplasmosis in human patients, but there is little information describing this test in cats. the goal of this study was to determine the sensitivity and specificity of h. capsulatum aa in cats with clinical disorders suggestive of histoplasmosis. urine and serum h. capsulatum aa results for feline patients from 3 veterinary hospitals were evaluated. medical records were reviewed for confirmatory evidence of histoplasmosis (based on cytological or histopathological findings) or an appropriately supported alternate diagnosis. aa results were available for 78 cats; initial testing was performed on 72 urine samples, 6 serum samples, and 1 unspecified sample. of these cats, 17/78 had a definitive diagnosis of histoplasmosis based on organism identification, and 10 had a definitive alternate diagnosis (e.g., neoplasia, other infection) based on necropsy findings (n 5 5) or other clinical data (n 5 5). an additional 16 cats had a clinical alternate diagnosis with no cytological or histopathological evidence of histoplasmosis in the affected body system(s). the remaining cats had unverified histoplasmosis (n 5 9) or an open diagnosis (n 5 26). of the 17 cats with confirmed histoplasmosis, 16 were positive on initial urine aa. one cat (with rectal involvement) was negative, indicating a test sensitivity of 94%. one cat was positive on urine aa but negative on serum aa. all of the 26 cats with definitive or clinical alternate diagnoses had negative results on the aa, suggesting an excellent specificity (100%). however, this result should be interpreted with caution, as the possibility of primary or concurrent histoplasmosis was only definitively excluded in the 5 patients who underwent necropsy examination. these findings suggest that the aa for h. capsulatum is a reliable diagnostic tool in this species. a positive result appears to reliably support the presence of infection, but a small percentage of infected cats may be negative on aa. in addition, tests performed on urine may be more sensitive that those performed on serum. the purpose of this study was to evaluate the sensitivity and specificity of an aspergillus galactomannan antigen enzyme immunoassay (ga-eia) for the diagnosis of canine systemic aspergillosis. serum and urine samples were collected from sick dogs at 2 hospitals (ucd and tamu). group 1 dogs were diagnosed with systemic aspergillosis using culture (sterile site) or microscopy and culture (non-sterile site). group 2 dogs had clinical findings suggestive of aspergillosis but an alternate diagnosis was established. group 3 dogs were not suspected to have aspergillosis. samples were tested using the ga-eia and results expressed as a galactomannan index (gmi). gmis 4 0.5 were considered positive. comparisons were performed using the mann-whitney test. there were 10 dogs in group 1, 22 in group 2, and 23 in group 3. serum was collected from all dogs, and urine from 6, 9, and 10 dogs, respectively. serum gmis did not differ from urine gmis across groups. serum gmis of group 1 dogs were higher than those of group 2 and group 3 dogs (p o 0.0001). results from dogs in group 2 did not differ from those in group 3 (p 5 0.43). two dogs in group 1 tested negative, but had localized pulmonary infections. one dog in group 2, which had paecilomycosis, tested positive. two dogs in group 3 tested positive. one was being treated with plasmalyte. the other had a cutaneous opportunistic mycosis. these data support the utility of this assay to aid in the diagnosis of systemic aspergillosis in dogs. anaplasma phagocytophilum, an ixodes tick transmitted rickettsial bacterium has a wide mammalian host range that is not commonly reported in cats. clinical signs in humans, dogs and cats are often vague and include lethargy, anorexia and malaise. the purpose of this retrospective study was to describe the clinical signs, laboratory data and response to treatment in cats that tested positive for a. phagocytophilum on a commercially available pcr of peripheral blood (fastpanel tm ). this study describes and reports the appearance of intracellular morulae in feline neutrophils contributing to the diagnosis of a. phagocytophilum. the a. phagocytophilum real-time pcr (rt pcr) assay consists of four multiplexed primer systems designed to detect a total of three distinct genes. amplicons were confirmed as a. phagocytophilum by dna sequencing. clinicopathologic data was obtained by review of medical records and interview of primary veterinarians. complete blood counts were available from 13/15 cats and 10/13 blood smears were reviewed. the cats included in this study were all positive for a. phagocytophilum by real-time pcr. the cats ranged from 4 months to 13 years of age with an average age of 3.7 years. fifteen of 15 cats had a history of tick exposure and lived in the northeastern region of the us, an ixodes endemic area. all cats presented with lethargy, 13/15 were anorexic and 14/15 had a fever (temperature 4 103 o f). other clinical findings included hepatomegaly, splenomegaly, ataxia and ocular changes of conjunctivitis and elevation of the nictitating membrane. hematologic findings included leukopenia (1/13), neutropenia (1/13) and lymphopenia (4/13). thrombocytopenia was not noted in any case. morulae were seen within neutrophils in 2/13 cases. all cases in this report responded to treatment with doxycycline. this is the first report of the identification of morulae within neutrophils via peripheral blood smear review in cats confirmed by rt pcr to be infected with anaplasma phagocytophilum in north america. infection with anaplasma phagocytophilum should be considered in a clinically ill cat with tick exposure, living in an ixodes endemic area that presents to a veterinarian for lethargy, anorexia and fever. the spectrum of disease manifestations and the accompanying clinicopathological abnormalities indicative of bartonellosis in dogs have not been thoroughly characterized. the objective of this unmatched case-control study was to compare signalment, clinical and pathologic findings in clinically-ill dogs suspected of a tick-borne disease that were negative for bartonella sp. dna (controls) as were the dogs diagnosed with bartonellosis by pcr amplification, dna sequencing and the bapgm (bartonella alpha proteobacteria growth medium) enrichment culture approach. both groups were tested under the same laboratory conditions and in the same time frame. medical records were reviewed for information regarding signalment, medical history, physical examination findings, clinicopathological abnormalities, microbiological data and treatment. the study population consisted of 47 bartonella-infected dogs and 93 non-infected dogs. healthy dogs with no historical illnesses, such as blood donors, were excluded. the following species were amplified: b. henselae (n 5 28, 59.6%), b. vinsonii subsp. berkhoffii (n 5 20, 42.6%), b. koehlerae (n 5 3, 6.4%), b. volans-like (n 5 3, 6.4%), b. bovis (n 5 1, 2.1%). nineteen (40.4%) bartonella-infected dogs were febrile and lethargic and ten (21.3%) had neurological signs. laboratory abnormalities for both groups are summarized below (number of affected dogs provided in parenthesis): multivariate logistic regression using confounding factors was performed to establish potential associations between specific variables and bartonella sp. infection. there were no differences in signalment, age, sex, body weight and duration of clinical signs between the two groups. compared to the control population, infection with the genus bartonella was associated with a diagnosis of endocarditis (p 5 0.0196, or 5 7.95, 95%ci 5 1.39-51.11) and hypoglobulinemia (p 5 0.0057, or 5 5.30, 95% ci 5 1.62-18.55). controls were more likely to have joint effusion (p 5 0.0059, or 5 5.89, 95% ci 5 1.62-27.22) and azotemia (p 5 0.0353, or 5 2.93, 95%ci 5 1.07-8.86) than were the bartonella sp. infected dogs. bartonella was detected in dogs with signs such as fever, anemia, thrombocytopenia, hyperglobulinemia and proteinuria that are typically associated with tick-borne diseases. when endocarditis or hypoglobulinemia are detected, testing for bartonella should be prioritized. likewise, the detection of bartonella should prompt further testing for endocarditis, if not already investigated. surveillance studies in other species depend on detection of antibodies to the highly conserved influenza a nucleoprotein (np); however, no such antibody detection assay is approved for canine use in the u.s. the purpose of this study was to determine the diagnostic accuracy of a commercial blocking elisa used for avian species in detecting influenza a np antibody in dogs. since the blocking elisa is not a species-specific or viral subtype-specific format, we hypothesized that it would detect np antibodies in dogs infected by influenza a virus. serum samples from uninfected dogs (n 5 204) and dogs naturally infected with canine influenza h3n8 (n 5 150) were tested using the idexx flockchek blocking elisa for influenza a np antibody according to manufacturer instructions. the sample/negative control (s/n) absorbance ratios for infected dogs ranged from 0.12 to 0.67 compared to 0.53 to 1.40 for uninfected dogs. a receiver operating characteristic (roc) curve analysis determined optimum diagnostic sensitivity (99.3%) and specificity (99.0%) at a s/n cutoff ratio of 0.647. using this cutoff ratio, the overall diagnostic accuracy was 99.2%. coefficients of variation for intra-assay (4.7%) and inter-assay (6.1%) testing demonstrated good repeatability with canine sera. the excellent diagnostic accuracy of the commercial blocking elisa makes it a suitable tool for large-scale surveillance of influenza a virus exposure in dogs. upper respiratory disease (urd) can affect a majority of cats in shelters and is one of the leading reasons for euthanasia of otherwise adoptable cats. the purpose of this study was to determine prevalence and risk factors for upper respiratory pathogens in four different models for managing unowned cats: short-term animal shelters (shel), long-term sanctuaries (sanc), home-based foster care (fost), and trap-neuter-return (tnr) programs. conjunctival and oropharyngeal swabs were collected from 543 cats, half of which had clinical signs of urd, and tested for feline herpesvirus (fhv), feline calicivirus (fcv), chlamydiophila felis, bordetella bronchiseptica (bb) and mycoplasma felis by real-time pcr. management model, vaccination, sex, age, body condition, and clinical signs were evaluated as risk factors for infection. a majority of cats in all management models carried one or more organisms capable of causing urd. in many cases, prevalence was similar in cats with or without clinical signs. unlike diseases that can be controlled by segregation of symptomatic animals, the lack of strong correlation between the presence of pathogens with the presence of clinical signs suggests that feline urd control should be managed by vaccination before or at the time of intake ,biosecurity protocols that presume all cats may be shedding pathogens, and minimizing stressful conditions that contribute to disease susceptibility. depending on geographical location, sex, age and environment, 2-40% of cats worldwide are infected with the feline immunodeficiency virus (fiv). knowledge of the fiv status of cats is important to limit the spread of disease and to institute appropriate health management. however, like all lentiviruses, fiv is highly variable in nucleotide sequence, and viral load in cats is variable during different disease stages. detection of antibodies is the most widely employed diagnostic approach, but does not distinguish fiv-infected from fiv-vaccinated cats. in this study, samples from 30 fiv-seronegative cats, 30 fivseropositive cats, and 30 fiv-historically seronegative but vaccinated cats, were analyzed by a commercial quantitative pcr (qpcr) assay and virus isolation. replicate blood samples were coded, and then submitted for 1) qpcr (idexx); and 2) mononuclear cell isolation with 7-day culture and viral p24 antigen detection by elisa. for the p24 antigen elisa, cutoff absorbance values were established from analysis of 10 fiv-negative samples. fiv infection status was pre-determined based on 2 antibody-elisa results and vaccination history. results indicated that qpcr had a sensitivity of 76% for samples from fiv-seropositive cats, and a specificity of 100% and 94.1% for samples from fiv-seronegative and fiv-vaccinated cats, respectively. at a cutoff value of 3 standard deviations above the mean absorbance for p24 antigen elisa, results from fiv-negative samples yielded a sensitivity of 69.2% for samples from fiv-seropositive cats, and a specificity of 77.1% and 64.7% for samples from fiv-seronegative and fiv-vaccinated cats, respectively. conclusions from this study are 1) the commercial fiv qpcr assay has high specificity but limited sensitivity for diagnosis of fiv infection; 2) 7-day virus culture has limited sensitivity and specificity. hence, detection of antibodies remains the most reliable test for diagnosis of fiv infection, but qpcr may be suitable to rule out infection. oral disease is an important clinical problem in feline medicine and includes common painful conditions such as oropharyngeal inflammation (formerly known as gingivostomatitis) and tooth resorptive lesions. a number of infectious agents have been associated with private veterinary clinics in the u.s. were recruited to test feline patients presenting with oral disease. presenting cases included cats with plaque, calculus, gingivitis, stomatitis, periodontal disease, tooth resorptive lesions and other oral diseases as defined by the practitioner. all cats were tested using a commercially available point-of-care elisa test (idexx snap combo). confirmatory tests were not performed as part of the study. seroprevalence was calculated as the percentage of positive tests in the study population for each virus. a total of 11,262 cats were tested. seroprevalence for felv was 6.8% and for fiv was 7.1%. of these, 119 cats (1.0%) were infected with both viruses. seroprevalence was higher in cats with inflammatory oral disease than in cats characterized with other types of oral disease. of 7,805 cats with gingivitis, seroprevalence for felv was 7.4% and for fiv was 7.9%, with 1.1% of cats co-infected. of 1,953 cats with stomatitis, seroprevalence for felv was 12.2% and for fiv was 13.9%, with 2.2% of cats co-infected. the seroprevalence for felv and fiv reported in this population of cats with oral disease was higher than in a recent large study where samples from u.s. cats not specifically selected for oral disease were tested (felv 2.3%, fiv 2.5%). results of this study indicate that further investigation of the role of retroviruses in cats with oropharyngeal inflammation is warranted. reliable tests and preventive vaccines and medications for feline retroviral and heartworm (hw) infections are available, but compliance with protocols to reduce transmission is unknown. no largescale longitudinal studies evaluating prevalence over time have been reported. the purpose of this study was to determine the prevalence and risk factors for infection compared with a similar study completed for the first time 5 years previously. veterinary clinics and animal shelters in the us and canada submitted results of testing using a point-of-care elisa for felv antigen, fiv antibody, and hw antigen (idexx snap triple) and risk factor information for cats tested during march-september 2010. bivariable and multivariable analyses were used to evaluate risk factors for infections. a total of 62,301 cats were tested. only 16% of owned cats were prescribed hw preventive. risk of retroviral infections was increased by outdoor access, adulthood, and male gender. the most important risk factor associated with all 3 infections was clinical disease; in particular, respiratory and oral diseases and abscesses or bite wounds. multivariate analysis revealed differences among geodivisions and across infection types. feline retroviral and heartworm infections are easily prevented, but difficult to treat. despite availability of effective management protocols, compliance remains inadequate to reduce the prevalence of these infections. improved use of preventive care and testing to identify and segregate contagious cats, particularly those at high-risk, is required to reduce the morbidity of these preventable infections. infectious disease outbreaks are common in animal shelters and are frequently managed by depopulation when risk-assessment tools are not available. during a canine distemper virus (cdv) and parvovirus (cpv) outbreak in sheltered dogs, we used a cdv/cpv point-of-care antibody titer elisa, a cdv quantitative rt-pcr test, and a cpv fecal antigen test as risk assessment tools to guide release of exposed dogs from quarantine and euthanasia of diseased dogs. serum samples (for antibody titers) and swabs of the conjunctiva and upper respiratory tract (for cdv pcr) were collected from 111 asymptomatic dogs starting on day 4 of the outbreak. dogs with positive cdv pcr tests were retested every 2 weeks until euthanized for progressive disease or released following recovery from infection. dogs with clinical signs of parvoviral infection were tested using a cpv fecal antigen test. for dogs ! 4 months old, protective antibody titers correlated with resistance to clinical disease, but 10% of dogs shed cdv. lack of protective cdv antibody titers correlated with susceptibility to clinical infection, but most dogs recovered. risk assessment and outcome in 60 dogs !4 months of age feline herpesvirus 1 (fhv-1) is a common ocular and respiratory pathogen of cats that can have clinical illness exacerbated by stress. cyclosporine (csa) is commonly used for the treatment of a number of inflammatory diseases in cats and can induce immune suppression. a small number of cats administered csa to block renal transplant rejection have developed clinical signs of upper respiratory tract disease that may have been from activated fhv-1. in this study, young adult cats experimentally inoculated with fhv-1 several months previously were divided into three groups and administered methylprednisolone acetate (8 cats, 5 mg/kg, im, day 0 and day 21), csa (8 cats, 7.0 mg/kg, po, daily for 42 days), or a placebo (7 cats, corn syrup; 0.075 ml/kg, po, daily for 42 days). each cat was assigned a daily individual clinical score by a trained, masked observer using a standardized score sheet during the initial pre-treatment time period (day -14 to day 0) and throughout the 42 day treatment period. each individual clinical score (conjunctivitis, blepharospasm, ocular discharge, sneezing, nasal discharge, nasal congestion, and body temperature scores), the total clinical score (sum of all parameters), the total ocular score (sum of conjunctivitis, blepharospasm, ocular discharge), and total respiratory score (sum of ocular discharge, sneezing, nasal discharge, nasal congestion) were analyzed using sas proc glimmix with 'treatment', 'time', and the two-way interaction 'treatment by time' all as fixed effects. statistical significance was defined as p o 0.05. on day 42 of the study, all of the csa treated cats had detectable concentrations of csa in serum (mean 5 406.1 ng/ml; standard deviation 5 291.8 ng/ml; median 5 388.5 ng/ml). when group mean values for clinical signs were compared over time as described, significant differences in individual clinical score measurements, in total score, total ocular score, or total respiratory score were not detected over time among any of the treatment groups. while clinical signs of activated fhv-1 occurred in some cats administered methylprednisolone or csa, disease was mild and self-limited in most cats and there were no significant csa sideeffects. these results suggest that the csa protocol described here is unlikely to reactivate latent fhv-1 infection and cause significant clinical illness. the purpose of this study was to determine the prevalence and risk factors for enteropathogens in four different models for managing unowned cats: short-term shelter, long-term sanctuary, home-based foster care, and trap-neuter-return (tnr) programs. fecal samples were collected from 482 cats, half with diarrhea (d) and half with normal feces (n), and tested for a panel of feline and zoonotic enteropathogens by polymerase chain reaction, antigen, and fecal flotation. risk factors for infection evaluated include management practices, fecal consistency, and signalment. a majority of cats had at least one enteropathogen of feline or zoonotic importance, regardless of management model or preventive healthcare protocol. for most enteropathogens, the presence or absence of diarrhea did not correlate with infection, the exceptions being t. foetus in sanctuary cats and fcov in foster cats. prevalence of specific enteropathogens varied between management models, reflecting differences in preventive healthcare and housing conditions. management protocols for unowned cats were inadequate for elimination of infections present at the time of intake and for prevention of transmission of enteropathogens among shelter cats. improved compliance with effective vaccination, deworming, sanitation, and housing protocols is needed to reduce zoonotic and feline health risks. several allergic diseases of cats, including atopy and gingivostomatitis, can be resistant to glucocorticoids but responsive to cyclosporine. toxoplasma gondii infection occurs in approximately 30% of cats and the effect cyclosporine therapy has on the t. gondii oocyst shedding period is unknown. the objective of this study was to determine whether administration of cyclosporine before or after t. gondii infection influences the oocyst shedding period. the young adult cats were t. gondii seronegative when administered 1,000 t. gondii tissue cysts orally on day 42. group 1 cats (n 5 10) were never administered cyclosporine; group 2 cats (n 5 10) were administered cyclosporine (7.5 mg/kg, po) daily on days 84-126; and group 3 cats (n 5 10) were administered cyclosporine (7.5 mg/kg, po) daily from days 0-126. available feces from individual cages were collected daily and fecal flotation by sugar centrifugation was performed for 84 days after t. gondii inoculation. group 3 shed oocysts for a significantly shorter period than groups 1 or 2 and had a significantly lower oocyst shedding scores than groups 1 and 2 on days 5-11 after t. gondii inoculation. group 2 cats had completed the oocyst shedding period prior to being administered cyclosporine and repeat oocyst shedding was not detected during administration of the drug. administration of cyclosporine prior to t. gondii infection lessened oocyst shedding which is likely from the anti-t. gondii effects of the drug. administration of cyclosporine using this protocol is unlikely to induce repeat t. gondii oocyst shedding in client-owned cats. ã 5 group with diarrhea significantly different than group with normal feces p o 0.05 known about its metabolic pathways or mechanism of pathogenicity and whole genome sequencing of feline hemoplasmas has not yet been reported. the aim of this study was to completely sequence the genome of m. haemofelis to further characterise this important pathogen. mycoplasma haemofelis genomic dna was purified and subjected to whole shotgun roche 454 sequencing. gaps were closed using targeted pcr and amplicon sequencing. ribosomal genes and potential open reading frames (orfs) were predicted in silico. putative orfs were annotated and orthologous groups identified. analysis showed a circular genome of 1.15 mbp with a gc content of 38.9%. thirty-one transfer rnas (trnas) were identified, accounting for all amino acids, including a tryptophan trna for the opal codon (uga). of the 1,545 putative proteins identified, 328 (21.2%) matched to proteins from other bacterial species. in common with the pneumoniae group of mycoplasmas, the closest phylogenetic relatives of the hemoplasmas, genes involved in carbohydrate metabolism were limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source for m. haemofelis. the majority of the pentose phosphate pathway genes present in other cultivatable mycoplasmas appear to be incomplete or absent in m. haemofelis, suggesting an alternative mechanism for sourcing purine and pyramidine bases such as scavenging from the host. a gene encoding a glyceraldehyde-3-phosphate dehydrogenase homolog of the immunogenic msg1 protein of mycoplasma suis was present. of the uncharacterized hypothetical proteins, 1,115 were arranged in series of orthologous repeats, or comprised fragments there-of, encoding putative proteins of approximately 200 amino acids. the predicted motifs of the majority of these putative proteins were consistent with these proteins being presented on the cell surface; an n' terminal signal peptide or transmembrane region followed by a non-cytoplasmic tail. these data have provided valuable information as to why this pathogen remains highly fastidious; it lacks some of the metabolic pathways found in cultivatable mycoplasmas. we have also identified a homolog of a known m. suis immunogenic protein, and identified a potential mechanism for host immune system evasion by way of highly repetitive, putatively surface-expressed hypothetical proteins with variable sequences. canine leptospirosis has been recognized as a re-emerging disease in the u.s. over the past 20 years, and several serosurveys of the prevalence of leptospiral antibodies in dogs have been published during that time. the role of cats in the epidemiology of leptospirosis has received little attention. serosurveys of cats for exposure to or infection with leptospires have been published from other geographic areas, but none for cats in the u.s. in the past four decades. the new england states have been found to have a high incidence of canine leptospirosis. the purpose of this pilot study was to determine the prevalence of leptospiral antibodies in a population of feral cats in central massachusetts. blood was collected from 63 sexually intact feral cats presented to a spay and neuter program. microagglutination titers to leptospira serovars autumnalis, hardjo, bratislava, icterohaemorrhagiae, canicola, pomona, and grippotyphosa were determined. three of 63 cats (4.8%) had a positive titer to one or more serovars, with autumnalis being the most common. these results are consistent with previously published prevalence rates in feral cats. further studies are required to determine the role of leptosporosis in clinical disease in the domestic cat. since years the rivalta's test is routinely used in several european countries as a tool to diagnose feline infectious peritonitis (fip) in cats with effusion. it is inexpensive and easy to perform in private practice. there is, however, only little information about mode of action or its diagnostic value. the objectives of this study were to evaluate sensitivity, specificity, positive (ppv) and negative predic-tive values of the rivalta's test to diagnosis of fip and to examine if there is a correlation with any effusion or blood parameters. medical records of 782 cats with effusion in which the rivalta's test was performed between 1999 and 2010 were reviewed concerning diagnosis, blood and effusion parameters, and survival time. effusion and blood parameters were compared between rivalta-positive and -negative effusions using the mann whitney u test. prevalence of fip in cats with effusion was 31.7%. the rivalta's test showed a sensitivity of 91.3%, a specificity of 66.6%, a ppv of 55.9%, and a npv of 94.3% for the diagnosis of fip. the ppv improved, when cats with lymphoma or bacterial infection were excluded (ppv 69.2%) and also, when only cats younger than 2 years (ppv 87.5%) or 1 year (ppv 90.8%) of age were included. the most important significantly different parameters between rivalta-positive and -negative effusions were specific gravity as well as cholesterol, triglyceride, and glucose concentration in the effusion. the rivalta's test in general is a useful tool to diagnose fip, but its sensitivity and specificity are not as high as previously assumed. if the rivalta's test, however, is performed in young cats or if certain diseases have been ruled-out, its diagnostic value is high. effusion total protein is not highly correlated with test outcome. therefore, it is still unclear, which components in the effusion of cats with fip lead to a positive rivalta's test. canine parvovirus (cpv) and canine distemper virus (cdv) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. our study aimed to determine the antibody protection status of dogs at the time of admission into an animal shelter (pre-vaccination) and over the following 2 weeks after vaccination. serum samples were obtained from 57 incoming shelter dogs aged 4 months and older with no known history of vaccination. immediately following serum collection, the dogs were vaccinated against cpv and cdv using a modified live vaccine (mlv). cpv and cdv antibody protection status was determined using synbiotics titerchek. dogs with unprotective serum antibody levels against cpv and/or cdv were retested at 6-8 days post-vaccination and again at 13-15 days post-vaccination, if antibody levels were still unprotective against cpv and /or cdv. at the conclusion of the study, stored duplicate sera were submitted for batch 'gold standard' testing to determine canine distemper virus serum neutralization and canine parvovirus hemagglutination inhibition antibody titers. based on the synbiotics titerchek results, 43/57 dogs (75.4%) were protected against cpv and 21/57 (36.8%) were protected against cdv at intake. older incoming dogs were more likely to be protected against cpv (p o 0.0001) and cdv (p 5 0.0174). dogs that were spayed/neutered were more likely to be protected against cpv on intake than intact animals, although this result was not statistically significant (p 5 0.0627). the number of dogs with protective titers against cpv/cdv was increased at 6-8 days post-mlv (cpv -48/56, 85.7%; cdv -31/52, 59.6%) and further increased at 13-15 days post-mlv (cpv -54/54, 100.0%; cdv -47/48, 97.9%). we conclude that incoming shelter dogs often do not have protective antibody titers against cpv and cdv, but older shelter dogs are more likely to be protected against cpv. based on this population, we further conclude that a large percentage of dogs develop protective antibody titers to cpv and cdv within 1 to 2 weeks when vaccinated with a mlv. mycoplasma spp. are common inhabitants of the feline oral cavity and so likely contaminate many cat bite abscesses. mycoplasma spp. are cell-wall deficient and so do not respond to beta-lactam class antibiotics, the class most commonly use for the treatment of cat bite abscesses. the objectives of this study was to determine whether mycoplasma spp. are common contaminants of cat bite abscesses and are associated with beta-lactam resistant clinical disease. privately owned cats with clinical evidence of an acute abscess suspected to be from a cat bite were included in the study. participants were given a free aerobic and anaerobic culture as well as mycoplasma spp. culture and polymerase chain reaction using mycoplasma genus specific primers. mycoplasma spp. amplicons were sequenced to determine the species. all cats were initially treated with appropriate wound management, were administered an antibiotic in the beta lactam class (amoxicillin-clavulanate or cefovicin), and were rechecked in person or by phone 7 days after beginning treatment. of the 26 cats entered into the study to date, mycoplasma spp. were amplified from 4 cats (15.4%). of the 2 positive samples with adequate dna for sequencing, one was consistent with m. felis and the other was consistent with m. equigenitalium. of the 26 cats, 25 responded by day 7 to the initial treatment, including 3 of the 4 mycoplasma spp. positive cats. the cat that failed initial treatment was positive for m. equigenitalium on both day 0 and day 7 and ultimately responded to administration of a fluoroquinolone. the results suggest that while mycoplasma spp. commonly contaminate cat bite abscesses, routine wound management and antibiotic therapy is adequate for control. however, as mycoplasma spp. infections do not respond to beta lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class. molecular diagnostic assays are frequently used in clinical practice to aid in the diagnosis of suspected infectious respiratory diseases in dogs. however, most currently available assays cannot distinguish strains of the organisms used in vaccines from naturally occurring strains. our prior studies demonstrated that previously immune adult dogs are unlikely to shed nucleic acids of vaccine strains of adenovirus 2, parainfluenza, or bordetella bronchiseptica. however, whether this is true for puppies is unknown. puppies (n 5 8) at a breeding facility were moved into area without other dogs at 6 weeks of age. swabs of the nasal and pharyngeal mucosa were collected prior to vaccination and on days 0, 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 after vaccination with an intranasal adenovirus 2, parainfluenza, and b. bronchiseptica vaccine (intratrac 3, schering plough). the swabs were shipped on cold packs by overnight express for dna/rna extraction and assay in the fastpanel tm pcr canine respiratory disease profile at antech diagnostics. all puppies were negative for the infectious agents prior to vaccination. after vaccination, positive assay results for parainfluenza and b. bronchiseptica were first detected on day 1 and on day 2 for adenovirus 2. by day 3, dna or rna of the agents were amplified from all puppies from both sample sites and most samples were positive for all 3 agents through day 10. by day 24, only one dog was still positive for b. bronchiseptica. the results indicate that intranasal administration of adenovirus 2, parainfluenza, or bordetella bronchiseptica vaccines commonly leads to positive molecular diagnostic assay results for a short time period after primary vaccination. these findings should be considered when assessing the results of these assays in client-owned puppies with respiratory disease. antimicrobial resistance in escherichia coli is an increasing concern in both human and veterinary hospitals' patients. the choice drug for treatment in dogs is enrofloxacin, a second generation fluoroquinolone (fq) whose activity reflects, in part, ciprofloxacin. among the difficulties in effective e. coli treatment is rapid detection of fq resistance. the purpose of this study was to determine the specificity and sensitivity of a fret based assay for the rapid detection of urinary tract infections caused by fq associated multi-drug resistant e.coli. 306 clinical e. coli isolated from canine urine and 120 clinical veterinary urine samples being examined for e. coli were subjected to susceptibility testing for 14 drugs representing 6 drug classes. pure isolates were designated ndr (no drug resistance), sdr (single drug resistance) and mdr (multi-drug resistant) (n 5 101 mdr, 116 sdr and 89 ndr). minimum inhibitory concentration (mic) for enrofloxacin ranged from 0.03 mg/ml to 512 mg/ml, with high mic generally associated with mdr. extracted dna from culture and from urine were subjected to fret-pcr targeting single nucleotide polymorphisms in gyra. the resulting product was sequenced to detect other polymorphisms. further, to determine the level of detection, microbial free canine urine was inoculated with 10 6 to 10 1 cfu/ml of 7 isolates characterized by variable susceptibility to enrofloxacin (mic enro 5 0.03, 0.06, 0.15, 1, 64, 128, 256 mg/ml). of 306 pure isolates, 50 were confirmed positive for enrofloxacin resistance (mic enro 4 4 mg/ml), 43 of which were positively identified by the fret-pcr assay giving a sensitivity of 86.00%. only 1 isolate that was resistant was not detected (specificity of 96.66%). however, of the isolates expressing high level resistance (mic 4 8 x breakpoint [64 mcg/ml]), and mdr (n 5 34), sensitivity 5 97.06%. of the 120 urine samples 27 contained e. coli 7 of which determined to be fq-resistant by the assay. colony dilutions of e. coli confirmed the assay able to detect enrofloxacin resistance at as low as 101 cfu/ml. the relationship between cfus and the peak of the -(d/dt) fluorescence of the melting curve was r2 5 0.988. these results conclude that the assay is capable of detecting not only the presence of escherichia coli in clinical samples, but also detecting severity of fluroquinolone resistance and infection. the fluoroquinolones (fqs) are a key class of synthetic antimicrobial agents with an established history in both humans and companion animals of efficacy for treatment of urinary tract infections (utis) caused by e. coli, and fluoroquinolones are common therapy. among the commonly used fqs in dogs and cats are the 2nd generation drugs, enrofloxacin, marbofloxacin, orbifloxacin (all veterinary approved) and the human drug ciprofloxacin; no 3rd and 4th generation fq is routinely used. the purpose of this study was to assess the in vitro activity of different generation fqs toward e.coli uropathogens whose phenotype ranges from no resistance to multidrug resistance. a total number of 51 canine uropathogenic canine or feline e.coli isolates had been subjected to susceptibility testing to 6 drugs classes (15 drugs) and phenotyped as to resistance: none (ndr, n 5 12), single (sdr, n 5 15), or multiple, mdr (resistance to 2-6 drug classes; n 5 24). mdr included isolates susceptible (enr s -mdr, n 5 12) or resistant (enr r -mdr) to enrofloxacin. the minimum inhibition concentrations (mics) for 11 quinolones (1-1st generation, 3-2nd generation, 4-3rd generation and 3-4th generation) were determined for these isolates using broth microdilution methods according to clsi guidelines (e. coli atcc s 25922 served as a negative control). mic statistics were generated for each drug among phenotypes. the results showed that companion animal e. coli expressing ndr or sdr are largely susceptible to 2nd to 4th generation fqs. however, isolates expressing resistance to 1st or 2nd generation quinolone also express high level resistance based on the mic 90 to 3rd and 4th generation fqs. the overall potency (mic) for the 11 drugs for isolates not expressing enr resistance (that is, ndr, sdr and enr s -mdr) is gat canine leproid granuloma (clg) was first reported in brazil in 1990. over the past 20 years, 37 cases of clg were diagnosed in sa˜o paulo, brazil, and clinical and epidemiological findings were similar to those reported in australia. all dogs presented with one or more, uni or bilateral, ulcerated or not, papular, nodular or tumoral lesions, mainly observed in the dorsal surface of the ear, site usually more affected. in general, the lesions are painless and confined to the subcutis and skin, and it does not involve regional lymph nodes, nerves or internal organs, and systemic clinical signs frequently are absent. short-coated breeds show a marked predisposition for this disease. the definitive diagnosis of clg was obtained by histological examination of skin biopsies that were stained with acid fast (ziehl-neelsen) and diffquik s . thirty one (83.7%) of the dogs were purebred; in this study the breed pattern comprised 19 (61.3%) boxers, 2 (6.5%) german shepherd and labrador retriever, 1 (3.2%) dobermann, 1 (3.2%) brazilian terrier, 1 (3.2%) golden retriever, 1 (3.2%) bulldog, 1 (2.8%) american pitbull, 1 (3.2%) mastiff, 1 (3.2%) fila brasileiro and 1 (3.2%) cocker spaniel, 6 (16.3%) were of unknown breed. nineteen (51.4%) of the thirty seven dogs were males. twenty (54.1%) dogs were 4-6 years old. in most cases, dogs presented with unilateral or bilateral ear lesions, but rarely thoracic, foot and caudal lesions. the animals were successfully treated by use of rifampicin orally (''the brazilian protocol'') or enrofloxacin orally and topical rifamicin. anaplasma phagocytophilum is being recognized more frequently in dogs in endemic areas. currently, most suspected cases are evaluated for a. phagocytophilum antibodies by immunofluorescence assay (ifa) or elisa. since a. phagocytophilum is an acute disease, detection by antibody measurement may be negative on initial evaluation. it is possible that a. phagocytophilum dna can be amplified from blood or synovial fluid prior to seroconversion. wild caught ixodes scapularis adult ticks from rhode island were allowed to feed on 18 young adult (2-3 years), mixed sex beagles for up to 7 days. blood (weekly for 12 weeks), serum (weekly for 12 weeks), and synovial fluid (radiocarpal joint; alternating arthrocentesis weekly for 6 weeks) were collected prior to tick attachment and then weekly after tick attachment. joint fluid cytology was performed and total dna was extracted from blood and synovial fluid and assayed in a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii. serum was assayed for a. phagocytophilum antibodies by ifa. time to first positive results for serology and pcr were compared by paired student's t test. none of the beagles developed clinical evidence of disease, and no major changes in synovial fluid cytology were detected over time. of the 18 beagles, 15 were positive for a. phagocytophilum dna in blood or synovial fluid or ifa antibodies in at least one sample after tick attachment. antibody titers appeared in 14 of 15 dogs from weeks 2 to 12 (median to 1 st positive 5 3 weeks ae 1). titer magnitude ranged from 1:40 to 1:10,240. anaplasma phagocytophilum dna was amplified from the blood of 14 of 15 dogs with positive test results ranging from 1 to 12 weeks (median to 1 st positive 5 2 weeks ae 1.5). anaplasma phagocytophilum dna was amplified from synovial fluid from 8 of 15 dogs between weeks 1 to 4 (median to 1 st positive 5 4 weeks ae 1). of the 8 dogs, 7 were pcr positive for only one week and 1 dog was pcr positive for two consecutive weeks. of the 15 dogs, 13 were positive for a. phagocytophilum in both blood and joints by dna analysis. anaplasma phagocytophilum dna was amplified from blood more quickly than seroconversion was detected by ifa antibody titer (t 5 à3.4, p o 0.01) or dna was amplified from synovial fluid (t 5 5.4, p o 0.001). anaplasma phagocytophilum dna can be amplified from the blood prior to development of detectable antibody titers by ifa. amplification of a. phagocytophilum dna from synovial fluid does not occur in all dogs, appears to be transient in most dogs, and a negative test result does not preclude a diagnosis of a. phagocytophilum infection. canine granulocytic anaplasmosis and granulocytic ehrlichiosis are tick-transmitted infections caused by anaplasma phagocytophilum (aph) and ehrlichia ewingii (eew), respectively. both organisms induce an acute clinical disease, frequently accompanied by fever, polyarthropathy and thrombocytopenia. however, aph and eew have different tick vectors, i.e. ixodes scapularis and ambylomma americanum, respectively, with different, but overlapping geographic distributions. in addition, infection outcome may be affected by other regional ticktransmitted pathogens, such as borrelia burgdorferi (mn) or ehrlichia chaffeensis (ar). therefore, we compared serology and pcr results derived from dogs examined at two private practices located in highly endemic areas for either aph or eew. serum collected between april-december, 2005 from minnesota dogs (n 5 420) was tested by snap s 4dx s and whole blood was tested by aph pcr. serum collected from arkansas dogs (n 5 708) for 1 year beginning in august 2009 was tested using microtiter plate elisas for antibodies to eew, e. canis, and e. chaffeensis (ech) while whole blood was tested by ehrlichia pcr. comparisons were evaluated using chi square (ã) and binomial (w) tests with an alpha of 5%. the above results indicated that dogs are frequently exposed to both aph and bb in mn, whereas ar dogs are often exposed to eew, but less frequently to ech. antibodies to e. canis peptides were found infrequently in both mn and ar with only 10 seroreactive dogs detected in both locations. active eew infection, as determined by pcr, was four times more frequent in ar pet dog seroreactors as compared to active aph infections among aph seroreactors. although both organisms induce acute disease, the number of aph and eew pcr positive dogs that were also seropositive was relatively high suggesting that both organisms induce persistent infections or that dogs are frequently re-infected, despite the presence of a measurable humoral immune response. additional studies are needed to determine regional infection profiles in other areas that are endemic for these pathogens. anaplasma phagocytophilum and ehrlichia canis are two of the most common vector borne disease agents that infect dogs and cats. while pcr assays that amplify the dna of these agents from blood are currently available, there is minimal information concerning the performance of these assays in different commercial laboratories that utilize different techniques. the purpose of this study was to compare the e. canis and a. phagocytophilum results of two different laboratories on the same samples collected from client-owned animals. veterinarians in 3 states (az, md, ct) were recruited to participate in the study based on high prevalence rates for e. canis or a. phagocytophilum infection. blood in edta was collected from dogs or cats with fever, thrombocytopenia, or clinical evidence of polyarthritis and an equal volume of the same blood sample was simultaneously shipped on cold packs by overnight express to colorado state and to antech diagnostics. standard operating procedures at each laboratory were followed for total dna extraction and amplification of gapdh as the dna control. at colorado state university, a previously published pcr assay that amplifies the dna of ehrlichia spp., anaplasma spp., neorickettsia spp., and wolbachia was performed on each sample with positive amplicons sequenced to determine the species. at antech diagnostics, a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii was performed. in the study to date, samples from 35 animals (30 dogs and 5 cats) have been assayed at both laboratories. dna of a. phagocytophilum (2 cats and 2 dogs) and e. canis (1 dog) were amplified at both laboratories with a percentage agreement between laboratories of 100%. the results to date suggest that the assay results of the two laboratories for a. phagocytophilum and e. canis are comparable. ehrlichiosis and bartonellosis are zoonotic diseases caused by extremely small, obligate intracellular bacteria that require a mammalian reservoir and a blood sucking arthropod vector. human ehrlichiosis is present in peru, with a seroprevalence as high as 23% in the highlands. bartonella species in humans were also identified in peru since 1905 (b. bacilliformis). recently, a new species (b. rochalimae) was isolated from an american woman who became febrile after travelling to peru. dogs can become infected with the same ehrlichia species, and the majority of bartonella species that affect human beings. the role of dogs as reservoirs for human infections has not been clearly established, but exposure and/or infection in dogs has been used to monitor human exposure to tick-borne disease (tbd), since they share the same environment. the objective of this study was to determine the serological and molecular prevalence of anaplasmosis, ehrlichiosis and bartonellosis in rural dogs in the highlands of peru. a total of 122 healthy adult dogs were enrolled in this study from four communities in the central highlands of peru: ondores, pachacayo, san juan de pachayo, and canchayllo. edta-blood samples were collected from 108 dogs, whereas serum samples were available from 110 dogs. serum samples were tested for ehrlichia canis, anaplasma, borrelia burgdorferi and dirofilaria immitis infections using a qualitative dot-elisa (snap s 4dx). the edta-blood samples were screened by conventional pcr for the groel gene of the genus anaplasma and ehrlichia, and for the intergenic transcribed spacer of the genus bartonella. speciation was conducted by nucleotide sequencing. bartonella genus dna was detected from seven of the 108 dogs (6.5%) and ehrlichia canis dna was detected and sequenced from one dog (0.9%). four of the bartonella positive samples were identified by dna sequencing as b. rochalimae (genbank accession numbers hq185696 and hq185695). the other three bartonella positive samples were identified as b. vinsonii subspecies berkhoffii, the causative agent of endocarditis in dogs and humans. no dog was infected with anaplasma species by dna amplification, but one dog was seroreactive for this genus (0.9%). no specific antibodies against ehrlichia canis and borrelia burgdorferi and no antigens of dirofilaria immitis were detected. this study expands the current knowledge about tbd in peru and describes for the first time the infection of b. rochalimae in dogs in peru. the results suggest that dogs may play an important role in the epidemiology of this infection in humans, since they can be asymptomatic but bacteremic. bartonella spp. dna is commonly amplified from the blood of cats exposed to ctenocephalides felis. in previous work, it was shown that cats administered imidacloprid and experimentally exposed to b. henselae infected cats and c. felis did not become pcr positive for b. henselae whereas untreated cats all developed infection. the purpose of this study was to determine if administration of imidacloprid to clientowned cats likely to be exposed to bartonella spp. and c. felis in the field lessens prevalence of bartonella spp. infection. veterinary students in tennessee and florida that owned cats that spent at least 20 days per month outside and that were willing to apply imidacloprid to their cats monthly for six months were recruited for the study. blood for bartonella spp. pcr assay was collected from the cats seven months after starting imidacloprid administration and assayed at colorado state university. to serve as a control group that was unlikely to have been administered flea control products in the previous 6 months, blood was collected from feral cats during tnr programs in each of the two cities and assayed for bartonella spp. dna. the bartonella spp. dna prevalence rates between the groups were compared by chi square analysis with significance defined as p o 0.05. the overall prevalence rates for bartonella spp. dna in the blood of veterinary student cats (7.4%) and the feral cats (39.1%) were significantly different (p o 0.0001). the distribution of results is shown in table 1 . the results suggest that florida feral cats were more commonly exposed to c. felis than tennessee feral cats. while the cats in the groups were not exactly matched, the student cats were allowed outdoors for approximately 20 days per month and lived in the same cities as the feral cats, so c. felis exposure rates were likely similar. as previously shown in experimentally-exposed cats, the use of imidacloprid monthly may influence transmission rates of bartonella spp. amongst naturally-exposed cats. in an endemic area for leishmaniosis and filariosis, coinfection can occur and immunomodulation produced by wolbachia might influence the clinical signs and progression of both diseases. the aims of the present study were 1) to determine the prevalence of wolbachia in dogs infected with dirofilaria immitis (di) and other filarial nematodes, 2) to evaluate the level of coinfection of leishmaniosis and filariosis by molecular assays and 3) to evaluate any associations between leishmania infantum (li) infection, filariosis with or without wolbachia and clinical presentation and outcome. statistical differences between groups were tested for significance by the fisher exact test using spss v.14.0 software (significance: p-value o 0.05). one-hundred and eighteen owned dogs from southeastern spain presenting for clinical evaluation were included in the study. criteria the results of this study highlight the increased sensitivity of pcr for diagnosis of filariosis, confirm the presence of wolbachia in dogs from the mediterranean basin, show the increased severity of hwd when li-filaria coinfection is present and suggest that wolbachia could play a protective role for leishmaniosis. wolbachia antigens can stimulate a th1-type immune response, as has been previously described. however other factors (as treatment with doxycicline) might be responsible for the lower prevalence of wolbachia among filaremic dogs infected with li and further studies must be done to clarify this interaction. the purpose of the present study was investigate the occurrence of leishmaniasis in 200 cats in the municipality of arac¸atuba, sa˜o paulo, brazil, an endemic area for canine visceral leishmaniasis. animals were evaluated by direct parasitological examination of lymphoid organs and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). thirteen (6.5%) out of 200 cats studied were diagnosed with visceral leishmaniasis; eight (4%) by parasitological diagnosis through cytological examination of lymphoid organs, six (3%) were considered positive by elisa and one (0.5%) by ifat. only two (15.38%) out of the thirteen infected cats had clinical signs, characterized by the presence of crusty lesions on the dorsal cervical region and hepatosplenomegaly. regarding age five cats (38.5%) had between six months and two years, being the others older than 2 years (61.5%). only one cat (7.7%) was positive for the three employed methods. pcr confirmed leishmania sp infection in nine (69.2%) cats, of which six were diagnosed previously by cytological examination, two by elisa and one by the three techniques employed. since its first description in 1912 feline leishmaniosis has been reported in several countries. the purpose of this study was to assess the prevalence of leishmania chagasi infection in cats showing dermatologic lesions from an endemic area for visceral leishmaniasis in brazil. animals were evaluated by direct parasitological examination of lymphoid organs, immunohistochemical technique for detection of amastigotes in lesioned skin and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). twenty seven (49.1%) out of the 55 cats studied were diagnosed with visceral leishmaniosis. twelve (44.4%) were positive by parasitological diagnosis; amastigote forms of leishmania sp were identified in lymphoid organs from 10/55 (18.2%) infected cats, and immunohistochemical technique allowed the identification of nine (16.4%) positive animals. the seroprevalence of leishmaniosis was 25.4% (14/55) by elisa and 10.9% (6/55) by ifat. fiv specific antibodies were found in 6/55 cats (10.9%), of which 5/6 (83.3%) had leishmaniosis. real time pcr confirmed leishmania chagasi infection in three cats. based on the evidence of the high occurrence of leishmaniosis in cats in this study, this disease should be included in the differential diagnosis of skin diseases of felines living in endemic areas. blastomyces dermatiditis is a dimorphic fungus that commonly affects large-breed hunting dogs. a recent advancement in diagnosis has come with the advent of a urine antigen screening test that has both high sensitivity and moderately high specificity. therapy for the disease involves use of antifungal agents, usually itraconazole, and length of treatment is based chiefly on resolution of clinical and radiologic signs. with the new urine antigen test, however, a noninvasive route of monitoring treatment progress is available and could be an adjunct device utilized to determine treatment efficacy and may even reveal a need for prolonged treatment. therefore, the purpose of this study was to determine if monitoring the blastomyces urine antigen test and comparing to pulmonary radiographic signs would elucidate the necessity for prolonged antifungal therapy, even after resolution of radiologic signs. to this end, a retrospective case review was performed that identified a series of client-owned animals with naturally occurring blastomycosis. the inclusion criteria were radiographic pulmonary parenchymal signs consistent with fungal disease and urine antigenconfirmed blastomycosis with repeated testing of both radiographs and urine antigen quantification as monitoring parameters until negative results achieved in each. ideally, intervals between testing dates would be between two and five months. radiographs were considered negative if all radiographic changes had resolved or if repeated radiographs separated by at least one month were considered static after documented improvement had occurred from original diagnostic radiographs (suspected scarring). urine antigen testing was considered negative if concentrations were less than 1.0 enzyme immunoassay units, a reference interval set by the testing laboratory. preliminary data analysis reveals resolution of radiographic signs of blastomycosis occurred earlier in many of the cases presented than did attaining a negative urine antigen concentration. ceasing treatment 1 month after radiographic resolution of signs as has been recommended in the past might have resulted in premature discontinuation of therapy in many of the cases. monitoring of urine antigen concentrations may be of additional clinical use for determining when cessation of treatment should occur in cases of blastomycosis. persistent elevation of urine antigen concentrations after radiographic resolution of infection may account for apparent recrudescence of blastomycosis after suspected clinical resolution. giardia spp. and cryptosporidium spp. are both known to cause infections in dogs and humans in the united states. nevertheless, prevalence rates for dual infection in dogs had not been widely reported. in this study, fecal samples from dogs housed in a northern colorado animal shelter (n 5 121), dogs owned by veterinary students in northern colorado (n 5 132), and dogs from the pine ridge reservation in south dakota (n 5 84) were collected. each sample was assayed with a commercially available fluorescent antibody assay that detects giardia spp. cysts and cryptosporidium spp. oocysts. those samples that were positive for giardia spp. or cryptosporidium spp. with adequate dna available for sequencing were genotyped by the glutamate dehydrogenase [gdh] and by the heat shock protein-70 [hsp-70] genes, respectively. overall, 45 (13.3%) of the dogs had current evidence of a protozoal infection ( table 1 ). the dogs from pine ridge reservation had the highest prevalence rates for giardia infection and also for dual infections. from the student dogs, sequencing was successful for the three giardia isolates (assemblage d from 2 dogs; assemblage c from one dog) and one cryptosporidium isolate (c. canis). from the reservation dogs, sequencing was successful for nine giardia isolates (assemblage d from 4 dogs; assemblage c from 5 dogs) and one cryptosporidium isolate (c. canis). cryptosporidium and giardia co-infections are commonly detected in dogs; in this study dual infections were more common than cryptosporidium infections alone. further studies will be required to determine the clinical importance of this finding. although the giardia and cryptosporidium isolates that were sequenced were the dog specific assemblages/genotypes, more samples should be analyzed in order to assess the potential for zoonotic transmission of either parasite. the current study was conducted to determine the prevalence of intestinal parasites in dogs visiting the veterinary teaching hospital, chiang mai university, northern thailand. fecal samples (n 5 301) were collected and submitted by owners between august 2009 to february 2010. demographic and geographic data were recorded. intestinal parasitic infection was diagnosed by both microscopic examination after zinc sulfate centrifugation flotation and commercially available ifa for giardia spp. and cryptosporidium spp. polymerase chain reaction and dna sequencing were performed on all giardia and cryptosporidium positive samples to provide genotyic information. overall prevalence of intestinal parasitic infection in dogs in chiang mai was 38.9%. the most prevalent parasite was giardia spp. (24.9%) followed by ancylostoma spp. (12.0%), cryptosporidium spp. (7.6%), cystoisospora spp. (6.0%), toxocara canis (2.7%), trichuris vulpis (2.0%), coccidian-like (1.7%), toxascaris leonina (0.7%), and strongyloides spp. (0.7%). the prevalence of having at least one parasite in dogs o 1 year, 1-7 years, and 4 7 years were 49.3%, 39.8%, and 27.4%, respectively. of these infected dogs, 59.0%, 34.2%, 5.1%, and 1.7% were infected with one, two, three, and four organisms, respectively. available dna sequences from giardia spp. positive samples were shown to be dog specific. only one adequate dna sequence was available for cryptosporidium spp., which was shown to be c. canis. the findings suggested that intestinal parasitic infection was common in dogs in chiang mai, thailand. dogs could be potential source for zoonotic intestinal parasitic infection since dogs in this area are allowed for free roaming. regular deworming program is indicated to prevent not only transmission among dogs but also to human. a retrospective study was conducted on 135 parasite positive fecal specimens consisting of 90 canine, 29 feline, 14 equine and 2 from other host species, comparing recovery of eggs, protozoan cysts and coccidian oocysts using 2 standardized methods of parasite concentration: the formalin/ethyl acetate (f/ea) sedimentation concentration and the commercial fecalyzer (flotation) kit procedures. specimens were processed by each technique either according to manufacturer's instructions or according to standard laboratory procedures. formalin/ethyl acetate concentrations used at a ratio of 10 ml normal saline to 4 ml ethyl acetate for extraction of lipophilic material from pelleted stool samples, previously fixed in sodium acetate/acetic/acid/formalin (saf) solution. flotations with the fecalyzer kit were performed with concentrated zinc sulfate solution (s. g. 1.22) . the range of parasites recovered from these specimens included flagellate cysts (40 total), coccidian oocysts (28 total), ova and larvae of nematodes (80 total), and ova of trematodes (12 total) , and cestodes (16 total). recovery rates by fecalyzer flotation were good for protozoan cysts, coccidian oocysts and nematode eggs and larvae, but very poor for cestode and trematode eggs. formalin/ethyl acetate concentration showed excellent recovery of all parasites and consistently outperformed fecalyzer in recovery rates. recoveries by f/ea concentrations were higher by 27.5% for giardia, by 10.7% for coccidia and by 10.0% for nematode eggs and larvae. with the exception of coccidian oocysts, based on z-test analyses, recovery rates were significantly higher, at a confidence level of at least 95%, for all parasites, using formalin/ethyl acetate sedimentation concentration. although capc recommends the use of flotation with centrifugation methods for standard fecal ova and parasite examination for veterinary patients, sedimentation concentration methods are widely and effectively used in human diagnostic parasitology laboratories. these results provide good evidence for the use of f/ea concentration as a preferred method to flotation procedures for stool ova and parasite examinations in veterinary laboratories. cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases. cyclosporine inhibits calcineurin-dependent pathways of t-cell activation and the resultant cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. little work has been done comparing the effects of these agents on cytokine production in dogs. our study assessed these effects by measuring t-cell cytokine production using flow cytometry, and cytokine gene expression using quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) in activated canine t-cells treated with cyclosporine and dexamethasone. for flow cytometric assays, peripheral blood mononuclear cells were separated using density gradients and cultured for 12 hours in the presence of cyclosporine (5, 25, or 100 ng/ml), dexamethasone (10 à7 , 10 à6 , 10 à5 m), or cyclosporine plus dexamethasone. for qrt-pcr, whole blood was cultured for 5 hours with the same drugs at the same concentrations, and rna was then extracted from leukocytes. expression of cytokines il-2 and ifn-g was analyzed in pma/ionomycinactivated t-cells by flow cytometry, and gene expression for il-2 and ifn-g in activated t-cell populations was assessed via qrt-pcr. flow cytometry and qrt-pcr both demonstrated inhibition of il-2 and ifn-g that was generally dose-dependent in response to both cyclosporine and dexamethasone. flow cytometry results from the average of samples collected from 3 different dogs are shown in figure a . similar results were achieved using qrt-pcr ( figure b ). suppression of il-2 and ifn-g in activated t-cells has potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine t-cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. idiopathic eosinophilic diseases are described in several breeds, but are over represented in rottweilers. the immunopathogenesis of idiopathic eosinophilic disorders is poorly characterised. studies in people highlight the importance of cytokines, particularly interleukin-5 (il-5), in mediating eosinophil maturation, differentiation, egress from the bone marrow, migration and polyclonal expansion. eotaxin-2 and eotaxin-3 also appear important for induction of chemotaxis and release of reactive oxygen species from eosinophils. the aim of the current study was to establish whether definable differences in specific cytokines associated with mediation of eosinophil production and survival are present between healthy rottweilers, non-rottweilers and rottweilers with non-parasitic eosinophilia. secondly, by evaluating cytokine profiles the study aimed to improve understanding of the pathophysiology of eosinophilia therefore assisting development of potential molecular treatment options. quantitative real-time reverse transcriptase polymerase chain reaction (qrt-pcr) assays were used to quantify messenger rna (mrna) encoding cytokines il-4, il-5, il-10, il-23p19, il-12p35, il-12p40, il-18, interferon gamma (ifn-g) and chemokines eotaxin-2 and eotaxin-3 from peripheral blood mononuclear cell (pmbc) samples obtained from healthy non-rottweiler dogs with normal eosinophil counts (n 5 5) and rottweilers with normal (n 5 6), mildly increased (n 5 7) and high (n 5 3) eosinophil counts. quantification of serum ifn-g was also performed using a commercially available canine-specific elisa. all samples were positive for housekeeping genes and all cytokines could be quantified with the exception of eotaxin-2 and -3. results were normalised using three stably expressed housekeeper genes (rpl13a, sdha and ywaz) and a relative copy number was calculated for each sample with the sample with the fewest copies given a value of 1. no significant differences were found between groups but there was a tendency for ifn-g mrna expression to be lower in the rottweilers with moderate to severe eosinophilia versus control dogs (p 5 0.062). this trend was not seen in the concentration of serum ifn-g quantified by elisa as there were no significant differences between normal and diseased animals. in conclusion, there were no significant differences in cytokine mrna profiles between normal dogs and rottweilers with varying degrees of eosinophilia. additional studies including larger numbers of affected dogs are warranted before any accurate conclusions can be made. the presence of large amount of antibody on erythrocyte membrane can accelerate red blood cell (rbc) removal process by the mononuclear phagocyte system. an antigenic stimulus such as the one promoted by vaccines, for example, can induce hypersensitivity reactions and may accelerate rbc destruction. the study objective was to evaluate the erythrocytic membrane potential in inducing lymphocyte proliferative response of recently immunized dogs. healthy adult dogs (n 5 17) were immunized with multiple antigens (commercial vaccine with eight antigens: distemper virus, parvovirus, coronavirus, parainfluenza virus, adenovirus, infectious hepatitis virus and leptospire; and anti-rabies). blood samples from each animal were collected into edta tubes in two moments: pre (immediately before vaccination) and pos (28 to 35 days after vaccination). mononuclear cells were separated by gradient, marked with cfse-fitc and cultured. the stimuli for lymphocyte proliferation used were autologous erythrocytic membrane (aem) and concanavalin a (cona). aem was obtained by hypotonic lysis and tested in two concentrations (m1: 0.1ug/100ul; m2: 0.2 ug/100ul). the proliferation assay was evaluated by flow cytometry and analyzed with specific software. the proliferation index (pi) was calculated dividing the fluorescence intensity of the basal sample by the stimulated one. statistical analysis was performed using paired t-test for parametric samples and wilcoxon test for non-parametric samples (a 5 0.05). the for the tested concentrations, autologous erythrocytic membrane does not constitute a stimulus for lymphocyte proliferation in vitro, either before or after vaccination procedure. additionally, there was no evidence of self-reagent lymphocytes to erythrocyte membrane after vaccination. e. coli is a common cause of canine urinary tract infection. current treatment emphasizes eradication of established infection rather than infection prevention but increased antibiotic resistance necessitates strategies to prevent infection. proanthocyanidins found in cranberry juice inhibit e. coli attachment to human uroepithelial cells, impairing bacterial adherence and colonization. we hypothesized that purified cranberry extract (ce) inhibits bacterial adhesion to canine uroepithelial cells. five healthy female dogs received an oral ce supplement (vetoquinol; 100 mg ce/tablet) according to body weight for 30 days. voided urine collected from each dog before (pre) and after (30-day) completion of the protocol was membrane filtered (22 mm) and stored frozen (-20c). bacterial adhesion was determined using an in vitro assay. briefly, urine samples were incubated with an uropathogenic e. coli strain that had been subcultured to promote fimbriae expression. urine samples containing e. coli were next incubated in 96-well plates containing methanol-fixed madin-darby canine kidney (mdck) cells for 1-hr (35c) to permit bacterial attachment. after incubation, plates were washed to remove nonadherent bacteria and fresh media added. plates were incubated (35c) for 4-hr to grow attached bacteria to detection level. bacterial concentration in each well was determined using a spectrophotometer (650 nm). results were analyzed using the chi-square test. ce significantly reduced bacterial adhesion by 30% (n 5 5; p 0.5) in 30-day urine samples compared with pre samples. the results show that ce supplementation can reduce adhesion of uropathogenic e. coli to canine uroepithelium and suggests one mechanism by which ce might improve urinary tract health. the purpose of this study was to determine prevalence of 4 urovirulence factors (uvfs) and antimicrobial resistance in canine uropathogenic e. coli (upec) and to evaluate associations between uvfs and antimicrobial resistance. two hundred and twenty-one upec isolates from samples collected from 184 different canine patients submitted to the university of tennessee microbiology laboratory in 2007 were evaluated. a multiplex pcr assay was used to detect cnf, hlyd, sfa/foc, and papgiii in dna lysate. in vitro susceptibility was evaluated and if the isolate was resistant to any antimicrobial in a class, it was considered resistant to that class. of the 221 samples, the number of uvf expressed per isolate was: 0 5 127/221 (57%), 1 5 27/221 (12%), 2 5 4/221 (2%), 3-22/221 (10%), and 4 5 41/221 (19%). expression of uvf was sfa (33%), hly (24%), cnf (24%), and pap (19%). presence of 4 uvfs was associated with less resistance (p o 0.0001). the combination of hly, cnf, and sfa was associated with less resistance (p o 0.0001). when sfa was present alone, resistance was less (p o 0.0001). average resistance to antimicrobial class by number of uvfexpressed was: 0 uvf 5 4.1 ae 3.3 classes, 1 uvf 5 1.2 ae 1.1 classes, 2 uvf 5 0.0 ae 0.0 classes, 3 uvf 5 0.8 ae 1.6 classes, and 4 uvf 5 0.5 ae 1.2 classes. urovirulence factors were present in a moderate number of upec and correlated negatively with resistance. neither individual nor combinations of uvfs were associated with increased resistance. obesity is associated with several comorbidities in dogs including pancreatitis, osteoarthritis, oral disease, neoplasia, and lower urinary tract disease. investigator observations led to the hypothesis that morbidly obese dogs are more likely to have asymptomatic bacterial urinary tract infections (abuti) than overweight and moderately obese dogs. therefore, a pilot study was conducted to screen for abuti in obese dogs. urinalysis with urine culture and dual energy x-ray absorptiometry (dxa) were performed on fortythree dogs with body fat (bf) percentages ranging from 36 to 56%. following dxa, subjects were categorized as obese (o)(bf 5 35-44%, n 5 17) and morbidly obese (mo)(bf 4 45%, n 5 26). no dogs had owner-reported symptoms indicative of uti. the prevalence of abuti in o dogs was 6% (n 5 1) and 31% (n 5 8) in mo dogs. the dog in the o group with abuti was close to being mo with a bf equaling 44.3%. of the nine dogs with positive cultures, 4 were neutered males and 5 were spayed females. the prevalence ratio of abuti in mo dogs was 7.5, indicating dogs with 45% or greater bf are 7.5 times more likely to have the condition then dogs o 45% bf. the results of this pilot study coincide with other surveillance data describing an increased prevalence of lower urinary tract disease in obese dogs. in conclusion, dogs with body fat percentages greater than 45% are at risk for abuti, and veterinarians should consider screening all morbidly obese patients for urinary tract infections. calcium carbonate (cac) is recommended to decrease phosphate intake in chronic kidney disease. however, its effect is poorly documented in dogs. our objectives were to assess within-day, postprandial and cac effects on phosphatemia variations in healthy dogs. phosphatemia was measured every 2 hours for 24 hours in eight adult healthy beagle dogs in i) fasted condition and ii) a 2 â 2 crossover design. one group received cac mixed with maintenance diet (0.8% phosphorus), while the second group received the diet alone. after a 1-week wash-out period, groups were switched. a general linear model was used to test the period, sequence, treatment, dog and time effects on phosphatemia and the area under the phosphatemia versus time curve (auc 0-24 ). a significant (p o 0.001) circadian variation existed in fasted dogs. the maximum difference (mean: à1.9 mg/dl; 95% c.i.: à2.4 mg/dl; à1.4 mg/dl) was observed between 8 a.m. and midnight. the auc 0-24 with cac (5936 ae 533 mg.min/dl) was mildly but significantly lower (p 5 0.027) than without cac (6239 ae 631 mg.min/dl). however, it was similar to the auc 0-24 in fasted conditions. feeding, with and without cac, has minor effect on phosphatemia. however, circadian variation of fasted phosphatemia might affect its interpretation. gfr measurement permits diagnosis of kidney injury prior to development of azotemia, and is the gold standard for kidney function assessment. accurate and rapid (o 60 min) gfr measurement has been performed in rats by simultaneous transcutaneous assay of two intravascular fluorescently-labeled markers. a recently developed analyzer assays fluorescence via a fiberoptic cable introduced through a peripheral catheter, and thus should also allow rapid gfr determination in larger species. the purpose of this study was to determine correlation and agreement between fluorescent ratiometry (fr) and iohexol plasma clearance (ipc) in dogs over a range of gfrs. acute kidney injury (aki) was induced in 5 female hound-type dogs (10 mg/kg gentamicin iv q8h), and fr and ipc gfr were simulta-neously determined on days 0, 3, 6 and 9. a 9-sample, 5-hr protocol was used for ipc; fr was determined following bolus injection of a dextran conjugate mixture (2-sulfohexamine rhodamine-carboxymethyl 150 kd dextran, 5-aminofluorescein-carboxymethyl 5 kd dextran) with fluorescence measured over 60 min. gfr was calculated using 2-compartment model concentration-vs.-time curves for both techniques. correlation was determined via spearman's rho; agreement was analyzed via bland-altman plots. ipc gfr and serum creatinine confirmed progressive aki in all dogs. correlation between fr and ipc was 0.91 (p o 0.001). bland-altman plots confirmed good agreement between techniques with slight underestimation of gfr by fr across most observed values. these results suggest fr is suitable for gfr determination in dogs with aki. importantly, the portable analyzer allowed for point-of-care gfr determination in o 60 min using a peripheral vein. previously presented at the american society of nephrology renal week (related but not identical abstract). dogs with protein-losing nephropathy (pln) are at risk of thromboembolic disease, but the mechanism of hypercoagulability and the population of dogs at risk are unknown. the purpose of this study was to characterize thromboelastography (teg) in dogs with pln. twenty-eight client-owned dogs with pln (urine protein:creatinine ratio (upc) 4 2.0) and 8 control dogs were enrolled. teg parameters, antithrombin activity, serum biochemical profiles, and upc were measured. teg analyses were run in duplicate with kaolin activation; reaction time (r), clot formation time (k), maximal amplitude (ma), and g (global clot strength) were analyzed. a wilcoxon sum rank test was used to evaluate differences between groups. twelve pln dogs (42.8%) were azotemic. nineteen pln dogs (67.8%) were hypoalbuminemic [serum albumin (salb) o 3.0 g/dl]; 11 had salb o 2.5 g/dl. dogs with pln had higher k (p o 0.01), ma (p o 0.005) and g (p o 0.005) than controls. r was similar between the two groups. pln dogs with salb o 2.5 g/dl had higher g (p o 0.05) values than dogs with salb 4 2.5 g/dl; however, even pln dogs with normal salb (4 3.0 g/dl) had significantly higher g values than controls (p o 0.005). no significant relationship between upc and g, salb and g, antithrombin and g, or salb and antithrombin was noted using linear regression analysis. these results indicate that antithrombin, salb, and upc cannot be used alone to predict hypercoagulability as assessed by teg in dogs with pln. a comprehensive evaluation of the coagulation system in individual patients may be necessary to predict the point at which to initiate anti-thrombotic therapy. cystinuria is a hereditary renal tubular reabsorption defect of cystine, ornithine, lysine and arginine (collectively, cola). the low solubility of cystine in acidic urine predisposes to the formation of uroliths. type i cystinuria in newfoundland and labrador retriever dogs is an autosomal recessive trait caused by mutations in the slc3a1 gene, whereas in other breeds, the cause of cystinuria has not yet been determined. we report here on the clinical, biochemical and molecular features of cystinuria in irish terriers. urine and edta blood were collected from 222 irish terriers from europe and australia. a nitroprusside screening test was used to identify increased cystine in urine. urinary amino acid concentrations were determined by high-pressure liquid chromatography. cystinuric dogs were defined as having cystine calculi, a positive nitroprusside result, urinary cystine (4 179 mmol/g creatinine) and/ or a cola concentration of 4 700 mmol/g creatinine. all 83 females tested nitroprusside negative and had normal urinary cystine (o 150 mmol/g creatinine) and cola (o 500 mmol/g creatinine) concentrations. the 10 intact males that formed calculi as adults exhibited cystine concentrations ranging from 323-1580 and cola from 1029-4302 mmol/g creatinine. an additional 41 males had similarly high cola values with cystine levels from 0-1580 mmol/g creatinine. among the affecteds tested, 75% were nitroprusside positive. the negative nitroprusside results and/or low urinary cystine levels of affecteds may be due to precipitation of cystine in acidic urine. sequencing the coding regions of the slc3a1 and slc7a9 genes from edta blood identified no mutations. the mode of inheritance remains undetermined. however, castration appears to lower the urinary cystine and cola concentrations and to prevent cystine calculi formation, while diet changes have lesser effects. in conclusion, non-type i cystinuria in irish terriers (and several other breeds like mastiffs and scottish deerhounds) is a unique form characterized by increased aminoaciduria only in males, with lower cystine and cola excretion and fewer and later urolith formation compared to type i cystinuria. castrating cystinuric irish terriers lowers their cystine and cola excretion and thus their risk for calculi formation. cats and dogs that are diagnosed with acute kidney injury (aki) and resultant uremia that is not responsive to standard medical therapy are likely to benefit from renal replacement therapies, such as intermittent hemodialysis (ihd). the purpose of this study was to evaluate the long-term outcome of patients with aki treated with ihd, and to establish whether renal function, as determined by serum or plasma creatinine concentrations, is associated with longterm survival. medical records of 20 cats and 35 dogs that were diagnosed with aki, treated with ihd, and survived longer than 30 days following the last ihd treatment were retrospectively analyzed. standard methods of survival analysis using kaplan-meier product limit curves and the log-rank test were performed. for all-cause mortality, the median survival time was 1823 days (95% confidence interval: 841, 4667) for cats and 1049 days (95% confidence interval: 893, 1931) for dogs. when only renal-related causes of death were taken into account, the median survival time was not reached for cats or dogs. survival time for all-cause mortality was inversely associated with the lowest creatinine concentration within the 30 to 90 day period following the last ihd treatment (p o 0.0011 for cats, p o 0.0104 for dogs). this study demonstrates that veterinary patients that are diagnosed with aki, treated with ihd, and survive greater than 30 days after the last ihd treatment have a good longterm prognosis and frequently die from causes that are unrelated to renal impairment. renal fine-needle aspiration (r-fna) is oftentimes attempted during evaluation of dogs and cats with renomegaly, mass lesions, or suspected infiltrative processes. diagnostic utility of fna is dependent upon the organ being sampled; additionally, in some organs, certain diagnostic imaging findings are associated with improved concordance of fna with final diagnosis. objectives of this study were to evaluate the diagnostic utility of r-fna and determine whether concordance with final diagnosis is associated with specific clinicopathologic or diagnostic imaging findings. we hypothesized that r-fna is most useful in patients with diagnostic imaging results suggestive of renal neoplasia (i.e. masses or suspected infiltrative processes). dogs and cats that had undergone r-fna from jan 1, 1998 to dec 31, 2008 were identified by database search. patient signalment, serum creatinine and blood urea nitrogen concentration, urine specific gravity, dipstick protein, r-fna result, and final diagnosis were recorded. patients were excluded if abdominal radiographs or sonographic images were not available for review, or if diagnostic test results were insufficient for determination of final diagnosis. a single coauthor blinded to final diagnoses interpreted all abdominal images using a pre-set list of descriptors and grading criteria. radiographic kidney shape, margin distortion, and ventrodorsal kidney-to-l2 ratio were evaluated. sonographic kidney margin distortion, cortical echogenicity, and corticomedullary junction distinction were described, and presence of nodules or masses, peri-renal effusion, or a peripheral sonolucent rim was noted. concordance of r-fna and final diagnosis was determined, and the chi-squared or fisher's exact test were used to determine association of concordance with the above variables; p o 0.05 was considered significant. 37 dogs and 41 cats (78 animals) met all inclusion criteria. r-fna results were concordant with the final diagnosis in 43 (55.1%) patients, discordant in 12 (15.4%) patients, and inadequate for cytologic interpretation in 23 (29.5%) patients. neoplasia or fip were the final diagnoses in 19 of 43 (44.2%) and 3 of 43 (7.0%) patients with concordant results, respectively. renal lymphoma (p 5 0.196), renal carcinoma (p 5 0.364), and renal neoplasia in general (p 5 0.451) were not associated with a higher likelihood of r-fna and final diagnosis concordance. there was no association noted between likelihood of r-fna and final diagnosis concordance when patients were stratified by species, serum creatinine or blood urea nitrogen concentration, urine specific gravity, dipstick proteinuria, or any diagnostic imaging variables. this study failed to identify concurrent clinicopathologic or diagnostic imaging findings that enhanced the diagnostic utility of r-fna. future studies should use standardized criteria to prospectively identify patients in which r-fna will be performed, evaluate additional variables that may be associated with increased r-fna diagnostic utility, and directly compare the utility of r-fna with that of other diagnostic techniques. feline lower urinary tract disease (flutd) is a disease with increasing prevalence in private practices and veterinary teaching hospitals. although several underlying causes can cause the obstructive form in male cats, the idiopathic form (feline interstitial cystitis) often is diagnosed as underlying reason in cats o 10 years. the goal of this retrospective study was to identify possible predisposing factors in order to optimize the therapy of these patients. as a study group, 40 cats hospitalized with obstructive flutd at the veterinary university of vienna were examined during a 2 year period (2008) (2009) (2010) . as a control group 40 cats presented for other reasons were randomly chosen during the same time period. the data were examined concerning the signalment and history. furthermore, the long-term outcome was evaluated with a questionnaire. based on assumptions a student's t-test or a chi-square test was used. there were no significant differences in age and breed. the body weight was significant higher in the flutd group than in the control group (p o 0.01). we could observe a significant risk for the disease of a weight of 4 5 kg (p o 0.01). there were significant less cat toilets in the flutd group compared to the control group (p o 0.05). furthermore we could observe that in the households of flutd cats there was significant less than one toilet per cat (p o 0.01) and more cats diseased on flutd lived strictly indoor than outdoor (p 5 0.05).there were no significant differences at the time of hospitalization in age, breed, number of cats per household or season of the year between the two groups. in summary, we could observe that cats over 5 kg body weight kept indoor with less than one toilet per cat have a significant higher possibility to be affected by obstructive flutd. further studies with an extensive history of animal husbandry are needed to identify risks predispoing cats to this frequent and cost-intensive disease. although purine uroliths (ammonium urate, sodium urate, xanthine, uric acid, etc.) represent the third most common stone type in cats, purine uroliths have the highest rate of recurrence (13% in 22 months). in dogs, mutation of the urate transporter (slc2a9) and portovascular anomalies are common risk factors. however the underlying cause(s) for purine urolith formation in cats is unknown. the purpose of this study was to test the hypothesis that hyperuricosuria without alterations in liver function is common in cats with urate uroliths. urine concentrations of purine metabolites were measured by high-performance liquid chromatography in 5 cats with ammonium uroliths (cases), 5 clinically healthy, breed and gender matched cats (negative controls), and 2 cats with naturally occurring xanthine uroliths (positive controls). prior to urine collection, all cats were fed a standard maintenance food (protein 5 7 g/100kcal) for 4 weeks. urinary xanthine, uric acid, and allantoin concentrations and concentration to creatinine ratios were calculated and compared between groups. also, serum pre-and post-prandial bile acid concentrations were measured. when compared to control cats, urinary uric acid concentration was significantly higher in case cats (p 5 0.002). xanthine was not detected in the urine of cases or negative controls. a significant difference in fasted and post-prandial serum bile acid concentrations was not detected in cases or controls (p 5 0.197, 0.212).hyperuricosuria without increased concentrations of urinary xanthine or allantoin appears to be a risk factor for ammonium urate urolith formation in cats. an association between portovascular shunts and purine urolithiasis was not observed in this population of cats. studies indicate that proteinuria is predictive, on a population basis, of those cats at risk of developing azotemia. seldi-tof-ms is a sensitive, high-throughput, proteomic technique utilising chromatographic surfaces to facilitate separation and detection of proteins and peptides within biological fluids such as urine. individual low molecular weight (lmw) urinary proteins have been considered as potential biomarkers for renal damage but provide only a limited representation of the urinary proteome; seldi-tof-ms may provide a more global assessment. normotensive, non-azotemic geriatric cats (4 9 years) were recruited prospectively from two first-opinion clinics for routine health screening. at entry cats received a full physical examination, plasma biochemistry, evaluation of total t4 concentration and urinalysis including urine protein to creatinine ratio. re-examination was offered at 6 and 12 months. cats were divided into two groups based on clinical status at the 12 month re-examination (azotemic; creatinine concentration ! 2.0 mg/dl and non-azotemic). optimisation studies were performed to facilitate the automated preparation (biomek 3000) of cm10 (weak cation exchange) arrays for seldi-tof-ms analysis (ciphergen enterprise 4000) of urine samples from cats at entry to the study. results are reported as median [25 th , 75 th percentile]. mann whitney u-test and wilcoxon signed rank test were used to compare variables between groups and between timepoints, respectively. ciphergen express (3.06) software was used to analyse spectral data and a mann whitney u-test was used to identify clusters which differed significantly between groups (p o 0.05) at entry to the study. twenty non-azotemic cats were recruited, of which 10 cats developed azotemia by 12 months. no significant differences in age, body weight, biochemical or urinalysis variables were identified between groups at entry to the study. as might be expected creatinine increased significantly (1.73 mg/dl [1.58, 1.77], 2.17 [2.00, 3.23], p 5 0.002) between study entry and 12 months in the cats that developed azotaemia and there was a commensurate increase in phosphate concentration (3.81 mg/dl [3.60, 4.22], 4.65[3.94, 6 .48], p 5 0.019). creatinine and phosphorus did not change significantly over time in the cats that did not develop azotaemia. seven clusters with m/z values of 2822, 10 033, 10 151, 10 234, 11 635, 11 700 were found to differ significantly between groups at entry to the study. the low protein concentration of feline urine makes the use of proteomic techniques challenging. however, this pilot study indicates that seldi-tof-ms can be utilised to examine the feline urinary proteome and that differences in low molecular weight protein patterns may be useful to differentiate those cats which are at risk of the development of azotemia. further work is necessary to identify these proteins/peptides. fibroblastic growth factor 23 (fgf-23) is a phosphotonin with an important physiological role in the regulation of phosphorous and vitamin d metabolism, and may therefore play a part in the development of renal secondary hyperparathyroidism. previous studies in cats have shown parathyroid hormone (pth) to be elevated prior to the development of azotemia. the study objectives were to explore the hypothesis that fgf-23 is a mediator of the development of renal secondary hyperparathyroidism in the nonazotemic stages of feline ckd. healthy, non-azotemic (plasma creatinine concentrations (cr) o 2.0 mg/dl) geriatric cats were recruited into the study prospectively and followed for 12 months. at the study end point cats were categorised into the following 3 groups: group 1 (n 5 15)-cr 1.58 mg/dl, group 2 (n 5 33)-cr !1.58 mg/dl but did not meet the criteria for group 3 and group 3 (n 5 14)-cr 4 2.0 mg/dl in association with reduced urine concentrating ability (usg o 1.035) or demonstration of persistent azotemia (cr 4 2.0 mg/dl). plasma samples were subjected to routine biochemical analysis, intact pth, calcitriol and intact fgf-23 assay. variables were compared between the 3 groups at the baseline time point. gfr was measured in an additional group of 19 cats (11 non-azotemic, 4 iris stage ii, 4 iris stage iii) using a corrected slope-intercept iohexol clearance method. relationships were explored using linear regression analysis and determining the coefficient of determination (r 2 ). results are presented as median [range] . at the baseline time point fgf-23 concentrations were significantly higher in group 2 (208.1[51.4-814.6 ], p 5 0.001) and group 3 (237.6[127.4-908.1], p 5 0.001) compared to group 1 (126.2[69.4-505.2] ). weak positive relationships were identified between fgf-23 and pth (r 2 5 0.126, p 5 0.005, n 5 62) and fgf-23 and cr (r 2 5 0.077, p 5 0.029, n 5 62). however, the positive relationships between fgf-23 and phosphate (r 2 5 0.016, p 5 0.323, n 5 62) and fgf-23 and calcitriol (r 2 5 0.085, p 5 0.212, n 5 20) were not significant. the additional group of cats in which gfr measurement was performed there was an inverse relationship between fgf-23 and gfr (r 2 5 0.208, p 5 0.040). in conclusion, fgf-23 was elevated in cats prior to the development of azotemia. the role of fgf-23 in the development of feline renal secondary hyperparathyroidism remains to be determined and should be explored through interventional studies. however, considering the relationship between fgf-23 and gfr, it cannot be excluded that the phosphotonin is simply a marker of reduced filtration. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to the progression of this disease. the aim of this study was to evaluate urinary vascular endothelial growth factor (vegf) as a marker of renal hypoxia. cats were recruited through geriatric clinics held at two first opinion london practices. vegf was measured in stored samples using a canine elisa kit validated for use on feline urine and indexed to creatinine concentration to yield a vegf to creatinine ratio (vcr). two studies were undertaken -firstly a cross-sectional analysis of clinical variables associated with vcr in cats with ckd. diagnosis of ckd was based on concurrent findings of plasma creatinine ! 2 mg/dl and usg 1.035, with persistence of azotemia for ! 2 weeks. only patients receiving no medical therapies were included. normotensive and (pre-treatment) hypertensive cats were included, but borderline cases (mean systolic blood pressure 160-170 mmhg on the date of sampling) were not. hyperthyroid cats were also excluded from this cross-sectional study. associations between vcr and clinical data were initially assessed using the spearman's coefficient and mann whitney test. linear regression was then used for multivariate analysis. the second study used samples from a trial in which hypertensive cats that had been treated with amlodipine for at least 3 months were entered into a randomised cross-over study where they received placebo or benazepril (0.5 to 1 mg/kg daily) for 12 weeks in turn. vcr on placebo was compared with that on benazepril using the wilcoxon signed ranks test. cats with well controlled hyperthyroidism were included in this intervention study. results are reported as median [25th, 75th percentile]. vcr was higher (49.5[33.3, 74.1] vs. 36.1[25.6, 42 .1] fg/g, p 5 0.010) in untreated hypertensives (n 5 30) than normotensives (n 5 63). vcr was correlated with pcv (r 5 à0.236, p 5 0.024, n 5 92), upc (r 5 0.444, p o 0.001, n 5 93), plasma phosphate (r 5 0.286, p 5 0.005, n 5 93), and usg (r 5 à0.284, p 5 0.006, n 5 93), but not plasma creatinine concentration. in the best multivariate model, pcv was associated with vcr independently of upc (r 2 5 0.435, n 5 92). vcr was significantly reduced by benazepril therapy (65.3 [43.1, 92 .7] fg/g) compared with placebo (76.0[48.0, 116.7] fg/g; p 5 0.031, n 5 17) with a reduction seen in 76% of cases. these results suggest urinary vegf excretion is associated with proteinuria in cats with ckd and might be a marker of renal hypoxia induced by low pcv. ace inhibitor therapy might reduce urinary vegf excretion because angiotensin ii causes constriction on efferent arterioles resulting in tubular hypoxia. fgf-23 is a phosphaturic hormone. fgf-23 concentrations increase with declining renal function in humans. the objectives of this study were to validate a method for fgf-23 quantification in feline plasma and to assess the association between fgf-23 concentration and plasma creatinine or phosphate concentration in cats with chronic kidney disease (ckd). non-azotemic and azotemic (plasma creatinine concentration (cr) 4 2.0 mg/dl) geriatric (4 9yrs) cats were recruited into the cross-sectional study from two london first opinion practices. cats were excluded from the study if they were fed a phosphate restricted diet, or had evidence of concurrent disease. the cats were categorized, using a modified iris staging system, into the following four groups: group 1 (cr 1.6 mg/dl), group 2 (cr 2.0-2.8 mg/dl), group 3 (cr 2.9-5.0 mg/dl), group 4 (cr 4 5.0 mg/dl). groups 2 and 3 were further subdivided based on the iris targets for plasma phosphate concentration (po 4 ): group 2a (po 4 4.5 mg/dl), group 2b (po 4 4 4.5 mg/dl), group 3a (po 4 5 mg/dl), group 3b (po 4 4 5 mg/dl). fgf-23 concentrations were measured in feline edta plasma using a human intact fgf-23 elisa, validated by intraand inter-assay variability and assessment of dilutional parallelism. comparisons between groups were made using the kruskal-wallis test and mann-whitney u test, with statistical significance defined as p o 0.05. bonferroni correction was applied where appropriate (statistical significance then determined as p o 0.008). results are reported as median [25th, 75th percentiles]. fgf-23 concentrations ! 800pg/ml (upper limit of quantification) were assigned the value of 800pg/ml. intra-and inter-assay variability of fgf-23 measurements were o 10.0% and dilutional parallelism between feline samples and the calibration curve were demonstrated. plasma fgf-23 concentrations increased with increasing creatinine concentrations (group 1: 158 [115, 274] , n 5 20, group 2: 354 [239, 473] , n 5 20, group 3: 800 [425, 800], n 5 23, group 4: 800 [800, 800], n 5 14). fgf-23 measurements were significantly different between all groups (p 5 0.005 to o 0.001) except between groups 2 and 3 (p 5 0.01). fgf-23 concentrations were significantly higher in cats with higher plasma phosphate concentrations (group 2a: 329 [237, 423] , n 5 16 vs. group 2b: 576 [374, 793], n 5 4; p 5 0.047) and (group 3a: 432 [167, 800] , n 5 10 vs. group 3b: 800 [625, 800], n 5 13; p 5 0.028). in conclusion, fgf-23 concentrations were higher in cats with more severe ckd or higher plasma phosphate concentrations as would be predicted from its known biological actions. further work is warranted to explore the role of fgf-23 in the development of renal secondary hyperparathyroidism by measuring parathyroid hormone (pth) and calcitriol in cats at different stages of ckd. progressive non-cardiogenic edema and lung dysfunction are common complications of acute kidney injury (aki) in people. pulmonary abnormalities have not been systematically reviewed in dogs with renal azotemia, but anecdotal reports of dogs with aki and concurrent non-cardiogenic pulmonary edema are suggestive of uremic pneumonopathy (up), a centrally-distributed pulmonary edema syndrome associated with kidney disease in people. we therefore hypothesized that pulmonary-associated clinical signs or thoracic radiograph abnormalities are more common in dogs with renal azotemia than in non-azotemic dogs, and that this association is more likely in dogs with aki than dogs with chronic renal failure (crf). our study objectives were 1) to describe thoracic radiograph and lung histopathologic abnormalities in dogs with renal azotemia, 2) to compare the occurrence of these findings in dogs with aki, crf, or non-systemic illness, and 3) to determine if these abnormalities are associated with shorter survival times. records of dogs with renal azotemia evaluated from 1/1/2000 to 8/20/2010 were reviewed; dogs which could be classified as having aki or crf and which had complete thoracic radiograph studies available for review were included. dogs with primary intracranial disease and normal serum creatinine and a complete thoracic radiograph study were selected as controls. signalment, weight, presence of pulmonary-related clinical signs, azotemia duration and severity at time of radiography, and leptospirosis antibody titer were noted. alveolar, bronchial, interstitial, or nodular lesions were described using a 4-point scale, and lung tissue collected at time of necropsy was reviewed; both the radiologist and pathologist were blinded to final diagnoses. significance was p o 0.05 for all analyses. the final study population included 54 aki, 50 crf, and 63 control dogs. crf dogs were older (p o 0.001) than aki and control dogs. pulmonary-related clinical signs were more commonly diagnosed at first evaluation in aki dogs (29/53 dogs, 54.7%) than in crf (13/50, 26.0%; p 5 0.003) or control dogs (9/63, 14.3%; p o 0.001). presence of an alveolar pattern was the only radiographic finding which differed amongst groups (more common in aki [n 5 8, 14.8%, p 5 0.047] and crf [n 5 8, 16%, p 5 0.028] dogs than in control dogs [n 5 2, 3.2%]). there was no association between presence of an alveolar pattern and any other variable. alveolar mineralization was the most common lesion in aki dogs (5/8 dogs; 62.5%), with concurrent alveolar space concretions or mineralization of vessels or bronchioles noted in 1 dog each. necropsies had not been performed in any of the crf dogs, but mineralization was not seen in lung tissues from any control dogs (n 5 9). neither pulmonary-associated clinical signs nor alveolar pattern were associated with median number of days from discharge until death in dogs with aki (p 5 0.220 and 0.468, respectively) or crf (p 5 0.280 and 0.253, respectively). in this group of dogs, presence and type of radiographic pulmonary abnormalities were associated with renal azotemia but not with median time until death. the association between and clinical relevance of alveolar mineralization in aki dogs were not determined, but both the radiographic and histopathologic abnormalities reported here differ from up in people. chronic kidney disease (ckd) is a common cause of morbidity and mortality in cats. the purpose of this study was to investigate the effects of chinese rhubarb (rheum officinale) supplementation on the progression of feline ckd. cats with stable iris stage ii or iii ckd and without comorbidity were included in the study. cats were divided into 3 treatment groups and administered rhubarb extract (group 1, rubenal s , vetoquinol, 75 mg tablet po q 12 h), benazepril as a positive control (group 2, 0.5 mg/kg po q 24 h), or both (group 3). cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. body weight, laboratory data, and blood pressure were recorded every 3 months for up to 34 months. variables between groups at enrollment and within groups over visits were compared with anova and repeated measures ano-va, respectively. a treatment by visit interaction term was included in all repeated measures models. significance was set at p 0.05. except for body weight there was no significant differences between treatment groups at enrollment. there was no significant change in body weight, hematocrit (hct), upc, or creatinine over time as compared to baseline within any group. there was no significant difference between groups over time in regards to change in weight, hct, upc, or creatinine. the treatment by time interaction was non-significant in all models. although there was no benefit associated with combination treatment, the results for rhubarb treatment alone were not different from benazepril treatment. azodyl, an encapsulated, enteric-coated probiotic/prebiotic nutraceutical, is marketed for reduction of azotemia (bun & creatinine) in dogs and cats. cat owners often sprinkle contents onto cat food to facilitate administration. however, exposure to air and stomach acid are thought to inactivate the lyophilized bacteria within the product. therefore, we examined the ability of foodsprinkled azodyl to reduce azotemia in cats with ckd. 10 cats with ckd were enrolled in the study and randomized receive azodyl or placebo. owners were provided with 3-4 capsules of azodyl prior to enrollment to ensure compliance with administration. 2 baseline blood samples were obtained 1 month apart, and then 1 & 2 months after beginning therapy. clinicians and owners were masked as to medication assignment. we hypothesized that a 30% decrease in bun and/or creat in the azodyl group would be significant, and set a 5 0.2. in order to maximize the probability of detecting a difference, we determined the % change as being the difference between the maximal baseline analyte concentration and minimal therapeutic concentration. we compared the % change between groups by mann-whitney u test. bun and creatinine did not differ between groups. based on these results, azodyl, applied by sprinkling onto food fails to reduce azotemia in cats with ckd. whether intact capsule administration reduces azotemia in cats with ckd remains unknown. lower urinary tract disease (lutd) occurs commonly in cats, and idiopathic cystitis (fic) and urolithiasis account for over 80% of cases in cats less than 10 years of age. although several strategies have been recommended, a common recommendation is to induce dilute urine resulting in more frequent urination and to dilute calculogenic constituents. in addition to conventional therapy using modified diets, traditional chinese and western herbs have been recommended, although only one, chorieto, has published data. we evaluated 3 commonly used herbal treatments recommended for use in cats with lutd including (1) san ren tang, (2) wei ling tang, and (3) alisma. we hypothesized that these 3 chinese herbal preparations would induce increased urine volume and decreased urine saturation for calcium oxalate and struvite. six healthy, spayed female, adult cats were evaluated in a placebocontrolled, randomized, cross-over design study. cats were randomized to 1 of 4 treatments including placebo (p), san ren tang (srt), wei ling tang (wlt), or alisma (a). treatment was for 2 weeks each with a 1 week washout period between treatments. at end of each treatment period, a 24-hour urine sample was collected using modified litter boxes. urine volume and biochemistries were measured, and urine saturation for struvite and calcium oxalate was estimated using equil 1.5b. analysis of variance (anova) was used to analyze data statistically if distributed normally and kruskal-wallis was used to analyze data statistically if data were not distributed normally. a p o 0.05 was considered significant. body weights were not different between treatments. no differences were found in 24-hour urinary analyte excretions, 24-hour urine volume, urine ph, or 24-hour urinary saturation for calcium oxalate or struvite between treatments (table) . urolithiasis is a multifactorial disease, frequent and recurrent in dogs in the worldwide, in which breed, sex, age, diet, some anatomical abnormalities, urinary tract infection, urine ph and some geographical and hereditary features in the populations studied have been implicated as risk factors. the effective long-term management of urolithiasis depends on identification and control of the pathophysiological mechanisms involved, which, in turn, depend on accurate knowledge of the mineral composition of the uroliths. the aim of this study was to determine for first occasion the main epidemiological data of canine urolithiasis in mexico. this study was developed with 491 dogs with urolithiasis from 25 of the 33 states of the country. chemical composition of the uroliths was determined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and x-ray microanalysis. urolithiasis affected nearly the same number of males and females; with ages ranging from two months to 15 years with a median age of 5 years. adult animals were the most affected. breeds more affected were schnauzer miniature, poodle, dalmatian, yorkshire terrier, scottish terrier, chihuahua and bichon frisee´. uroliths were found in the lower urinary tract in 97.74% of the cases. mineral composition of the uroliths was: struvite 49.69%, followed by calcium oxalate 25.46%, purines 7.13%, silicate 6.72%, others 0.20%, mixed 8.15% and compound uroliths 2.44%. struvite uroliths affected females in most cases, whereas calcium oxalate, purines and silicate uroliths, were mainly observed in males. our results are similar to studies developed in other countries and continents, though we found a higher frequency of uroliths containing silicate, either pure, mixed or compounds uroliths (10.79%); in mexico city the frequency reached 15%. this high frequency may be due to high consumption of silicate in home-made food or in the groundwater derived from aquifers. acknowledgments: this work has been partially supported by a project of waltham foundation in mexico and the consejo nacional de ciencia y tecnologı´a (conacyt) of mexico. voiding urohydropropulsion is a non-invasive method for removing small urocystoliths from the dog, most commonly used in females due to the relatively wider and shorter urethra. this procedure is typically performed under general anesthesia to allow complete relaxation of the urethra, however, anesthesia results in longer procedure times and difficult endotracheal tube stabilization due to the vertical positioning of animals, especially in larger dogs. the aim of this study was to devise a novel injectable sedation protocol for urohydropropulsion when cystoscopy was not concurrently required. an intravenous catheter was placed, and a combination of medetomidine (10 to 15 mg/kg iv) and hydromorphone (0.025 to 0.05 mg/kg iv) was administered, with the addition of ketamine (2 mg/ kg iv) in fractious animals; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this protocol was considered in cardiovascularly healthy, non-diabetic dogs without evidence of urinary obstruction. monitoring equipment included electrocardiography, blood pressure measurement, and pulse oximetry, and supplemental flowby oxygen was provided. two dogs received the proposed sedation protocol in order to perform urohydropropulsion. dog one was a 3 year old female spayed shih tzu cross, and dog 2 was a 2 year old female spayed standard poodle. ultrasonography revealed a moderate number of urocystoliths present in both dogs, measuring up to 1 mm in dog 1 and 2.3 mm in dog 2. urohydropropulsion was performed and resulted in retrieval of 15 urocystoliths in dog 1, and approximately 20 urocystoliths in dog 2. repeat ultrasonography revealed no uroliths present after urohydropropulsion in both dogs. the time from administration of sedation to administration of reversal agent was 6 minutes for dog 1, and 8.5 minutes for dog 2. records were obtained from 3 dogs that had traditional general anesthetic protocols for urohydropropulsion with cystoscopy for confirmation of urocystolith removal, performed within the last 2 years, and the average anesthetic time was 64 minutes. subsequent to the use of medetomine-based sedation protocols for the above dogs, cystoscopy was performed in a 9 year old neutered male golden retriever with prostatomegaly. medetomidine (15 ug/kg iv) and butorphanol (0.2 mg/kg iv) were administered; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this sedation allowed adequate immobilization for cystoscopy of the urethra and urinary bladder, and endoscopic biopsying of the prostatic urethra and urinary bladder. the time from administration of sedation to administration of reversal agent was 15 minutes for this dog. in conclusion, a novel sedative protocol for urohydropropulsion is proposed which allows for an appropriate level of sedation along with a short procedure time and rapid recovery. this sedation protocol may also be useful for certain cystoscopic procedures. analysis may be delayed for a variety of reasons, including the need for sample batching within the laboratory or shipping to an outsourced location. therefore, it is important to know how storage of the sample may affect enzyme activity. we hypothesized that urinary nag and ggt activity would be affected differently in samples stored by refrigeration vs. freezing. thirty-four canine urine samples submitted to the clinical pathology laboratory at kansas state university were included. samples were collected from clinical patients with a variety of medical/surgical disorders and were selected based on the day of the week and a minimum volume of 10 ml. a complete urinalysis was performed on each sample; however there were no exclusion criteria based on urinalysis results. nag and ggt activity in the urine supernatant was assessed by colorimetric assay. aliquots of each supernatant were refrigerated for 5 days and frozen at à201c for 5 and 30 days at which time enzyme activity was re-assessed. compared to baseline values, enzyme activity for both nag and ggt were stable after 5 days of refrigeration, however there were significant (p o 0.01) declines in ggt and nag activity when urine supernatants were frozen for 5 and 30 days. treatment for canine urinary tract infections (uti) typically consists of 7-14 days of antimicrobial drugs in primary care veterinary practice. compliance with this drug regimen can be difficult for some clients. enrofloxacin is a veterinary approved fluoroquinolone antimicrobial and is useful for treatment of canine uti. fluoroquinolones are often used in human medicine to treat uncomplicated utis in women and can be prescribed for as little as 3 days. the primary objective of this study was to determine if dogs with naturally occurring uncomplicated uti have equivalent microbiologic cure with a high dose short duration protocol of enrofloxacin, compared to a standard antimicrobial protocol. client-owned adult dogs with naturally occurring, uncomplicated uti were prospectively enrolled in a multi-center clinical trial and assigned to 1 of 2 groups in a randomized blinded manner. group 1 received treatment with 18-20 mg/kg oral enrofloxacin once daily for 3 consecutive days. group 2 dogs were treated with 13.75-25 mg/kg oral amoxicillin-clavulante twice daily for 14 days. both groups had urinalyses and urine cultures submitted on day 0, 10, and 21. at the time of this interim analysis, thirty-six dogs have completed the trial. bacteriological cure was achieved in 15 dogs (83%) treated with enrofloxacin and 14 dogs (78%) treated with amoxicillinclavulante, respectively. these data suggest that the high-dose, short-duration enrofloxacin protocol was equally effective to the standard protocol in treating uncomplicated canine uti in the sample patient population. and may represent a viable alternative therapeutic regimen for similar patients. azotemia is frequent in dogs with dmvd (nicolle et al; jvim 2007; 21:943-949) and could result from renal hemodynamic alterations. renal resistive index (ri) allows assessment of renal vascular resistance. the aim of this prospective study was to assess ri in dogs with different dmvd stages. fifty-five dogs with dvmd were used (isachc class 1 (n 5 28), 2 (n 5 19), and 3 (n 5 8)). physical examination, renal ultrasonography and echo-doppler examinations were performed in awake dogs by trained observers. plasma creatinine, urea and nt-probnp were measured. statistical analyses were performed using a general linear model. whereas ri of renal and arcuate arteries were unaffected by isachc class, left interlobar ri increased (p o 0.001) from 0.62 ae 0.05 (mean ae sd) in class 1 to 0.76 ae 0.08 in class 3. left interlobar ri was also higher (p o 0.001) in azotemic (0.74 ae 0.008) than in non azotemic (0.62 ae 0.005) dogs. similar findings were observed for right interlobar ri. a positive effect of nt-probnp (p 5 0.002), urea (p o 0.001), creatinine (p 5 0.002), urea-to-creatinine ratio (p o 0.001), left atrium-to-aorta ratio (p o 0.001), regurgitation fraction (p 5 0.011), systolic pulmonary arterial pressure (p o 0.001) and shortening fraction (p 5 0.028) on ri was also observed. in conclusion, interlobar ri increases with the severity of dmvd and azotemia. a cause-effect relationship remains however to be established. antibodies against alpha-enolase are associated with immunemediated nephritis in people. it was previously shown that vaccinated cats commonly develop antibodies against alpha-enolase. the purpose of this study was to assess for associations between alphaenolase antibodies and azotemia in privately-owned cats. clinically stable privately owned cats ! 10 years of age, with and without azotemia (creatinine 4 2 mg/dl), and with an available vaccine history for ! 5 years were recruited for the study. sera were assayed for creatinine concentrations and alpha-enolase antibodies by use of previously validated techniques. results from cats with and without azotemia were compared by student's 2-tailed t test or fisher's exact test with significance defined as p o 0.05. median ages were 15 years (range: 10-18) and 12 years (range: 10-15) for cats with (n 5 35) and without azotemia (n 5 27), respectively. there was no significant difference in vaccine events (number, type, or route of administration) between groups. azotemic cats (34.3%) were more likely than normal cats (12.5%) to be positive for antibodies against alpha-enolase (p 5 0.016). in addition, alpha-enolase antibody concentrations were greater (p 5 0.041) in azotemic cats (mean % elisa 5 62.5%) than cats with normal creatinine concentrations (mean %elisa 5 47.2%). results of this study suggest that antibodies against alpha-enolase in cats may be associated with renal disease. additional prospective evaluation in a larger number of cats is indicated. aki is used in human medicine as a predictor of mortality based on the akin (acute kidney injury network) scoring system which utilizes relative increases in creatinine to determine stage. with this scheme, mortality has been shown to increase as the stage of kidney injury (indicated by akin score) increases. accordingly, we hypothesized that this system would improve predicting prognosis in dogs and cats. we retrospectively evaluated 1088 dogs and 856 cats (2008) (2009) ) that had ! 2 creatinine measurements within 7 days, and whose first creatinine was o 1.6 mg/dl. patients were categorized as: level 0 (no aki); level 1 (second creatinine value o 1.6 mg/dl, but creatinine increased ! 0.3 mg/dl); or level 3 (second creatinine 4 1.6 mg/dl with a creatinine increase ! 0.3 mg/dl). thirty and 90 day survival for each level was compared to level 0. adjusted odds ratio (or) in dogs for 30 day survival was 1.3 for level 1 (ci 95%, 0.8-2.2) and 3.2 (ci 95%, 1.8-5.5) for level 2; or for 90 day survival was 1.3 for level 1 (ci 95%, 0.8-2.2) and 3.7 (ci 95%, 2.1-6.5) for level 2. for cats, or at 30 days was 1.5 (ci 95%, 0.5-4.6) for level 1 and 3.1 (ci 95%, 1.5-6.7) for level 2; or for 90 day survival was 0.9 (ci 95%, 0.3-2.8) for level 1 and 4.1 (ci 95%, 1.8-9.3) for level 2. thus, detecting increasing stage of aki helps predict mortality in dogs and cats. abstract n/u-27 feline urate urolithiasis: 143 cases (2000 -2008 . j dear 1 , r shiraki 2 , a ruby 2 , j westropp 3 . 1 william r pritchard veterinary medical teaching hospital, university of california, davis, ca, 2 gerald v. ling urinary stone analysis laboratory, university of california, davis, ca and 3 the department of veterinary medicine and epidemiology, university of california, davis, ca. feline urate urolithiasis accounts for 10% of the feline stones our laboratory analyzes each year; little information is known about this disease, particularly the incidence of those cats with hepatopathies. the objective of the study was to characterize the signalment, clinicopathologic data, and diagnostic imaging of cats with this disease as well as the salts of uric acid present. a retrospective analysis of feline urate uroliths submitted to the stone lab between january 2000-december 2008 were included. from these data, primary veterinarians were solicited to submit records. furthermore, all records from cats with urate uroliths from the vmth were analyzed separately. 143 records were received from the primary care veterinarians. sixteen cases were identified from the vmth. median values for the cbc and chemistry panels available were within the reference ranges provided, with only a few outliers present. of the 78 cats with radiographic reports, 70 (90%) had visible evidence of uroliths. two external cases had confirmed pss; five cases from the vmth had a pss. cats with urate uroliths and pss were younger than cats without a documented hepatopathy (2 years vs. 7 years). the siamese breed was overrepresented. all stones were ammonium hydrogen urate. the pathogensis of urate uroliths in cats is poorly understood. most cats were not completely evaluated for pss, however, there were few clinicopathologic parameters which indicated hepatopathies were present. further studies are warranted to evaluate genetics and purine metabolism in cats with urate uroliths to help tailor proper management and breeding strategies. 3-indoxyl and p-cresyl sulfate (is, and cs, respectively), small protein-bound molecules derived from gastrointestinal protein metabolism, are among the most important uremic solutes affecting morbidity and mortality in human chronic kidney disease (ckd). in the blood stream, these compounds are predominantly bound to protein, but their debilitating effects on prognosis and quality of life in ckd appear to be driven by the free fraction. the objectives of the present study were to assess the normal, physiological levels of is and cs in healthy cats and to evaluate the correlation of the respective free and protein-bound levels. blood samples were taken from 105 clinically healthy adult cats enrolled at five participating veterinary practices in germany. after centrifugation, the serum was deep frozen until transport on dry ice to the analytical laboratory. serum creatinine and urea levels were quantified by vettest s (idexx laboratories, inc.). total and free is and cs, respectively, were quantified by turbulent flow chromatography coupled with a tandem mass spectrometry detector. statistical analysis of the results comprised i) a descriptive report of the median with upper and lower bounds of the 95% confidence interval for reference values of is and cs, ii) a calculation of various pearson correlation coefficients r, also tested with reference to the null hypothesis of no relationship, and iii) wilcoxon-mann-whitney utest for an estimation of the effect of hemolysis on serum is or cs levels. six animals with serum creatinine or urea levels outside the reference range were excluded from the calculation of reference values. median levels of is in cat serum were 1.19 mg/l with upper and lower bound 95% confidence intervals at 1.46 and 0.99 mg/l, respectively. the corresponding median levels of cs were 2.11 mg/l (median) and 2.46 vs 1.57 mg/l (upper vs lower bound levels, respectively). these values showed a low, non-significant correlation with serum creatinine or urea levels. however, is and cs serum levels were moderately correlated (total levels r 5 0.4808, p o 0001). their respective free levels constituted about 5% of the total serum levels (r ! 0.8977, p o 0.001). non-hemolytic samples tended to yield lower values than hemolytic samples. due to the low number of hemolytic samples (n 5 14) , the group difference could, however, not be statistically confirmed. the results indicate that it is sufficient to determine total levels of either is or cs in serum while studying the effects of therapeutic or dietetic interventions on the evolution of these parameters in feline ckd. reference values are provided for orientation towards clinically relevant changes. disrupted urothelial differentiation has been implicated in the pathogenesis of feline idiopathic cystitis (fic). studies of cultured human urothelium have shown that abnormalities in urothelial differentiation and repair may be mediated by persistent 15-hydroxy-prostaglandin dehydrogenase (pgdh) activity and subsequent metabolism of cytoprotective prostaglandins. the goal of this study was to confirm persistent pgdh expression in fic bladders compared to desmoplakin i1ii expression, a marker of urothelial differentiation. urinary bladder biopsy specimens were obtained by cystotomy from 9 symptomatic cats with chronic fic. cats with a history of another major disease, previous cystotomy, or recent treatment with corticosteroids, nsaids, antihistamines, antidepressants, or glycosaminoglycans were excluded. urinary bladder tissue specimens were also obtained from 10 untreated clinically normal specific-pathogen-free cats. tissue specimens were fixed in buffered 10% formalin and embedded in paraffin. tissue sections were deparaffinized and subjected to citrate buffer microwave antigen retrieval. tissues were stained for pgdh using a rabbit anti-pgdh antibody, an isotype negative control or goat anti-desmoplakin i1ii and developed using the avidin-biotin peroxidase complex method. all fic (9/9) and normal (10/10) cat bladder samples showed similar staining of urothelial cytoplasm for pgdh. however, desmoplakin i1ii staining, found on the luminal cell surface in 4/4 normal tissues, was disrupted in 6/6 fic bladder samples. desmoplakin i1ii staining confirmed altered urothelial differentiation in fic cats. however, pgdh expression remained intact in fic samples. we hypothesize that pgdh expression in fic may contribute to its pathophysiology due to breakdown of prostaglandins essential for urothelial healing. additional studies will explore this hypothesis. the university of tennessee college of veterinary medicine's picture archiving and communication system was searched over a 9 month period for cats that had undergone both abdominal radiographs and ultrasound during the same visit. one hundred and three cats were identified (age range o 1 to 18yrs; median 11yrs). kidney size was determined based on radiographic and ultrasound findings. of the included cats, 41.8% had two normal sized kidneys, 18.4% had one small and one normal, 15.5% had one large and one normal, 11.7% had two small, 8.7% had two large, and 3.9% had one small and one large kidney. the presence of mineralization, uroliths and hydronephrosis was also noted. medical records were reviewed for clinical chemistry data and historical information concerning previous urinary disease. no significant differences were found between kidney size and renal function, kidney size and the presence of uroliths, renal mineralization and function or the presence of uroliths and function. the presence of uroliths was significantly associated with hydronephrosis. of the 30 cats with at least one large kidney, 9 (30%) had hydronephrosis. of the 23 cats with current or previously diagnosed uroliths, urinary tract infections or other uropathies, 10 (43.5%) had at least one small kidney. small kidneys were commonly found in older cats, however, this correlation was not statistically significant. based on these findings, small kidneys are more likely to be the result of urinary disease as opposed to being either congenital or due to aging. this study aimed to evaluate ife, which has been advocated for treatment of lipid-soluble drug intoxication, in the treatment of clinically-occurring canine ivermectin toxicosis. one australian shepherd and two miniature australian shepherds were included. all three dogs were homozygous for the mdr-1 gene mutation. two dogs roamed on horse ranches where ivermectin-based deworming products had recently been used. ivermectin was administered to the third dog (165 mg/kg po). all three dogs exhibited tremors, ptyalism, and cns depression, which progressed over several hours to stupor in two dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. a 20% formulation of ife (liposyn ii, hospira) was administered as a bolus (1.5 ml/kg) followed by a slow iv infusion (7.5-15 ml/kg over 30 minutes). no change was observed in the neurologic status of any patient. lipemia visible upon blood sampling persisted for 36 hours in one dog. no other adverse effects were noted. serum ivermectin levels confirmed ivermectin exposure in each case. in this study, ife administration did not result in clinical benefit in cases of ivermectin toxicosis. brain ivermectin concentrations in mdr1 mutant/mutant genotype dogs may be too high to be overcome by ife. additionally, these dogs may lack p-glycoprotein-mediated biliary clearance mechanisms needed for optimal ife function. further investigation is needed to determine the utility and optimal dosing of ife in canine toxicoses, to characterize its safety, and to determine how mdr-1 status may alter the efficacy of ife in treatment of canine ivermectin intoxication. rufinamide is a recently approved antiepileptic drug used for the treatment of seizure disorders in human patients. rufinamide is administered at a dose of 45 mg/kg divided twice daily to achieve therapeutic concentrations of 15 mg/ml. the objective of this study was to determine the pharmacokinetic properties and short-term adverse effects of single-dose oral rufinamide in healthy dogs in preparation for a possible clinical trial evaluating the efficacy of rufinamide in the treatment of canine epilepsy. six healthy adult dogs were included. the pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of 20.0 mg/kg (range 18.6-20.8 mg/kg), extrapolated from the dose used in human patients. dogs were monitored by repeat physical examinations, electrocardiograms and blood pressure assessments during the course of the study. plasma rufinamide concentrations were determined using high-performance liquid chromatography. pharmacokinetic data were analyzed using winnonlin version 1.0. no adverse effects were observed. the mean terminal half-life was 9.86 1/à 4.77 hours. the mean maximum plasma concentration was 19.561/à5.82 mg/ml and the mean time to maximum plasma concentration was 9.33 1/à 4.68 hours. mean clearance was 1.448 1/à 0.703 l/hr. auc inf was 410.72 1/à 175.88 mgãh/ml. results of this study suggest that rufinamide given orally at 20 mg/ kg twice daily in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted. the aims of this study were to investigate the abg for (i) the prevalence of skull abnormalities; (ii) the prevalence of sm; (iii) an association between lateral ventricular size, cerebellar size and sm; and (iv) associations between sm, skull abnormalities, csf pleocytosis and clinical signs. seventy-six abgs, recruited as part of a larger epidemiological and genetic study, underwent brain and spinal mri evaluation (3.0t general electric signa hdx, milwaukee, wi). all dogs were evaluated neurologically, recording deficits and the presence of spinal pain. sequences acquired included t2w, t1w pre-and postcontrast, and t2w flair, sagittal and transverse. cervical spinal cord central canal (cc) and or syrinx size and its percent area of spinal cord was measured using osirix s . the presence of chari-like malformation (cm) was assessed by recording the presence of caudal cerebellar deviation and/or foramenal vermal herniation. lateral ventricle and cerebellar volume was expressed as a percent of the cerebrum and intracranial volume2qa respectively. forty-five dogs underwent atlanto-occipital cerebrospinal fluid tap at the time of mri and the white blood cell (wbc) count was recorded. student's t-tests were used to compare the measured variables between groups with and without skull abnormalities, spinal pain and neurological signs. the mean age of the 30 males (24 intact) and the 46 females (34 intact) was 50.4 months (range 8-135; median 44 months). neurological deficits and neck pain were noted in 21 (27%) and 15 (19.7%) of dogs respectively; 5 dogs (6.57%) exhibited both. cerebellar deviation and vermal herniation were present in 37 (48.68%) and 46 (60.52%) dogs respectively; twenty-three dogs (30.26%) had both. mean height of the cc was 2.3 mm (0-7.2 mm). forty (52.63%) ccs were greater than 2 mm in height; the mean length of these lesions was 2.03 vertebrae (0.5-7). mean csf wbc count was 4.97/ml (0-39). syrinx height and extent were significantly higher in dogs with neurological signs (size p 5 0.01; extent p 5 0.0004). there were no significant differences in syrinx sizes and extent in dogs with or without skull abnormalities or spinal pain. there were no associations of syrinx height or extent with csf wbc count or age of dog. intact females had a significantly lower syrinx extent than intact males (p 5 0.009). there were no significant differences in presence of spinal pain or neurological signs between dogs with or without skull abnormalities. there was a significant negative association of ventricular percentage and cerebellar percentage (p o 0.0001). there was a significant association of ventricular percentage with syrinx percentage (p 5 0.0015) and height (p 5 0.0007). this study suggests that sm and cm are prevalent in abgs. syrinx size and extent are associated with neurological signs and ventriculomegaly is associated with both small cerebellar size and large syrinx size. however, sm may not be associated with cm as defined by cerebellar herniation and deviation and is not associated with csf inflammation. the power tissue resection device (ptrd) is a hand-piece comprised of an outer cannula with motor driven vacuum-assisted inner cutting blade. this device was designed and is marketed for human neurosurgical brain/spinal cord tumor resection. the purpose of this study is to describe the use of the ptrd for intervertebral disc fenestration and to compare the effectiveness of manual fenestration to that of the ptrd. fifteen cadaveric lumbar spines were randomly placed into three study groups: group 1 was the control group on which no fenestrations were performed, group 2 was the manual fenestration group and group 3 was the ptrd fenestration group. the effectiveness of fenestration via both manual and ptrd was assessed by calculating the ratio of remaining nuclear weight post fenestration to total nuclear volume. discs with lower ratios were more effectively fenestrated. results showed a smaller ratio of post fenestration remaining nuclear weight to nuclear volume following fenestration with the ptrd (0.23 ae 0.09) as compared to manual fenestration (0.30 ae 0.10). these results did not show statistical significance. when fenestrated samples were compared to control samples (0.39 ae 0.07), there was a statistically significant reduction in ratios. in conclusion, the ptrd is easy to use and is as effective as the manual technique for canine intervertebral disc fenestration. according to the human who classification gliomatosis cerebri (gc) is a rare astrocytic tumor affecting at least three lobes of the brain with extensive infiltration, but relative preservation of brain architecture. gc has not been reported to occur as a hereditary disease, neither in man nor in animals. here, we report the temporally clustered occurrence of gc in a family of bearded collies. a 7 years old female bearded collie with forebrain signs was presented. differentials included inflammatory/ infectious, metabolic/ toxic, and neoplastic diseases. within a time period of 12 months, 3 offspring of this bitch were presented with similar clinical signs. two dogs were full siblings (2 males). the remaining female dog originated from a match with a different male dog. mri was performed in all 4 dogs and revealed a diffuse and extensive intra-axial lesion with moderate mass effect and midline shift. the ill defined lesion showed mainly a white matter distribution with hyperintense signal in t2-w and flair images and iso-to hypointense signal in t1-w images without contrast enhancement. the lesion was bilateral in all cases, continued along the white matter extending partially into the gray matter with contact to the brain surface. neuropathology revealed a diffuse and extensive infiltration of the brain and spinal cord by a neoplastic glial cell population involving white and gray matter of both hemispheres, thalamus, brainstem and cerebellum in all 4 dogs. based on the cell morphology and immunoexpression of glial fibrillary astrocytic protein by neoplastic cells diagnosis of gc was made. this is the first report of familial occurrence of gc, which is likely the result of a germ-line mutation. several human hereditary cancer syndromes are associated with cns tumors including amongst others the li-fraumeni cancer family syndrome (p53 mutation), neurofibromatosis (type 1 and 2) (neurofibromin, merlin mutation), and tuberous sclerosis (hamartin, tuberin mutation). furthermore, familial clustering of human gliomas unassociated to the known inherited cancer syndromes has been described. in the dog, hereditary cns tumors are not known. the exact mode of inheritance and putative gene mutations of gc in this bearded collie family are currently under investigation. preliminary results are consistent with a monogenic autosomal dominant mode of inheritance, although a recessive inheritance cannot be completely ruled out at this time. mutations in the tp53 gene were not found following amplification and sequencing of exons 5-8 in 2 affected dogs. previously presented at the ecvn annual meeting in cambridge, uk. the gm 2 gangliosidoses are characterized by a deficiency of bhexosaminidase. there are two isoforms: hex a composed of an a and b subunit encoded by hexa and hexb genes respectively and hex b with two b subunits. hex a requires an activator encoded by gm2a. two japanese chin dogs with confirmed gm 2 gangliosidosis showed elevated total hexosaminidase and normal hexosaminidase a activity, a pattern associated with the ab variant in humans and consistent with prior reports in the breed. this study was performed to identify the mutation responsible using resequencing with an applied biosystems 3730xl dna analyzer as previously described (awano 2009). mutations in gm2a cause the ab variant in humans, but resequencing gm2a revealed no mutation that could account for the disease. resequencing hexa and hexb revealed a c.967g 4 a mutation in hexa which was homozygous in both affected dogs. sixty-five normal japanese chin dogs were screened for the mutant allele; 60 were homozygous for the ancestral allele and 5 heterozygous. this mutation predicts a p.323e 4 k substitution affecting one of two primary active-site amino acids that participate in the hydrolysis of gm 2 ganglioside. substitution of a lysine residue at this site is likely to eliminate subunit a enzymatic activity. the apparently normal levels of hexosaminidase a activity in affected dog samples may be a result of b subunit overexpression. human hex b possesses low levels activity against the artificial substrate used to assess hex a activity, but specificity of activity of the canine enzyme is not known. previously presented at the american society for neurochemistry: additional data in this abstract. phenytoin (pht) is the intravenous drug of choice in humans for seizure emergencies following benzodiazapines. iv fosphenytoin (fos) is a pht pro-drug which causes less administration related adverse events. while the short half-life of pht is not suitable for chronic oral therapy in dogs, iv fos has not been studied. two dogs received 15 mg/kg phenytoin equivalent (pe) and two dogs received 25 mg/kg pe of fosphenytoin intravenously at a rate of 50 mg pe/min. blood for plasma levels were drawn at 10 time-points over 12 hours; total and unbound drug levels were measured by hplc. vital signs including ekg, blood pressure, and neurological examination were monitored. the half-life of metabolism of fos to pht was $10 min, with 4 80% of fos metabolized to pht by 30 minutes. eighty to 84% of pht was protein-bound during the first 15 minutes after dosing, compared to 90-95% in humans. the elimination half-life for total pht ranged from 2.8-3.5 hours and for unbound pht ranged from 1.9-5.4 hours. dogs receiving 15 mg/kg pe intravenously achieved unbound pht plasma maximum concentrations of 2.2-2.4ug/ml at 5 minutes, consistent with human loading dose levels. adverse events observed in some dogs included vomiting, mild ataxia, and short lived tremors, the severity of which appeared dose dependent. all dogs were clinically normal within 30 minutes of all doses. a 15 mg/kg pe dose of iv fos appears adequate for production of pht levels predicted to be effective for the treatment of canine seizure emergencies. further studies in clinical canine patients are warranted. acquired myasthenia gravis (mg) is caused by antibodymediated inactivation of the acetylcholine receptor on the neuromuscular endplate causing focal, regional or generalized muscle weakness. many medical treatments have been reported; however, responses to therapy and outcomes are unpredictable and death often results from aspiration pneumonia. therapeutic apheresis is an extracorporeal procedure that separates blood into its components for removal or specific alteration prior to return to the patient. therapeutic plasma exchange (tpe) is an apheresis treatment in which plasma (containing pathologic antibodies) is removed and exchanged with donor plasma. tpe is used routinely to treat mg in human patients with severe disease or disease unresponsive to conventional therapy. we report the successful use of tpe to treat 2 large breed dogs with confirmed mg (aceytlcholine receptor antibody concentration: 3.05 and 3.32 nm/l, respectively; normal concentration: o 0.6 nm/ l) that was severe and not adequately responsive to traditional therapies. both dogs were non-ambulatory, recumbent, and demonstrated megaesophagus and aspiration pneumonia. three tpe treatments (1 plasma exchange each) were performed over 5 and 7 days, respectively, in each dog without complication. both dogs became ambulatory within 3 days of starting tpe treatment with subsequent resolution of regurgitation and megaesophagus. pyridostigmine was continued during tpe sessions and discontinued in both dogs within 3-6 months. both dogs remain asymptomatic and have had no recurrence of mg during 16 and 4 months of follow-up, respectively. tpe is a viable treatment option for dogs with mg that have severe disease, life-threatening complications or that remain unresponsive to traditional therapies. tpe may alleviate clinical signs more rapidly, and improve long-term outcomes when compared to historical experiences in patients with comparable disease. clinical findings, clinicopathologic data, imaging features, and treatment of canine spinal meningiomas have been described in the veterinary literature, but histological characteristics and tumor grading have less commonly been reported. the aims of this retrospective case series were to describe the clinical, imaging, and histologic features of seven canine spinal meningiomas including a cervical spinal cystic meningioma that had imaging and intraoperative features of a subarachnoid cyst. medical records from dogs with a histopathological diagnosis of spinal cord meningioma presented to the veterinary teaching hospital between 2006 and 2010 were reviewed. signalment, presenting clinical signs, physical and neurologic examination, clinicopathologic data, surgery reports and available images were reviewed. all meningiomas were histologically classified and graded following the international who human classification for cns tumors. seven dogs were included, 4 males and 3 females. median age at presentation was 8.7 years (range, 3.5-11.4 years), and median weight was 35 kg (range, 8-45 kg) . median time between onset of clinical signs and diagnosis was 108 days (range, 45 days -1 year). cerebrospinal fluid (csf) analysis was performed in 4 dogs, showing increased protein concentration in 2 cases, and being normal in the other 2. spinal radiographs revealed vertebral canal widening in one case. myelography (4/7) showed intradural/extramedullary lesions in three cases, one of them consistent with a csf-filled subarachnoid cavity, and an extradural lesion in one case. magnetic resonance imaging (mri) was performed in all cases and revealed mild to marked hyperintensity on t2w and precontrast t1w images and homogeneous contrast enhancing (ce) intradural/extramedullary masses (4 cervical and 2 thoracic) in six cases, with one of these showing an additional intramedullary ce pattern. a dural tail was identified in two dogs. one dog had a fluid-filled subarachnoid enlargement located dorsally to the spinal cord. this lesion was hyperintense on t2w, hypointense on t1w and flair images, and did not enhance. it was diagnosed as a spinal subarachnoid cyst, but the histopathological study of the surgically resected mass revealed a grade i cystic meningioma. five other cases underwent cytoreductive surgery, two transitional meningiomas (grade i) that survived 3 (alive at the time of writing) and 7 months; and three anaplastic meningiomas (grade iii) that survived 10-16.6 months before neurological deterioration and euthanasia. another anaplastic meningioma was euthanized right after diagnosis. there are few reports grading canine spinal meningiomas, with most being grade i or ii. of the few grade iii tumors reported, only one had been treated surgically and was euthanized 90 days later because of neurological deterioration. we report four grade iii (anaplastic) meningiomas, three of which surgically treated and with longer survival times. finally, cystic meningioma should be considered in the differential diagnosis of cases with imaging features consistent with arachnoid cyst because of their similar appearance, making histopathological analysis essential for a definitive diagnosis. head trauma is a common veterinary emergency, but few prognostic indicators have been studied in dogs, making it challenging for clinicians to counsel clients about the odds of recovery. a recent meta-analysis showed that higher plasma glucose, lower plasma ph and lower hemoglobin at admission were associated with increased risk of death in human head trauma. the goal of this retrospective study was to investigate the association between admission point of care blood gas parameters and survival to discharge in dogs with head trauma. fifty one dogs presenting to the cornell university hospital for animals with head trauma from 2007 to 2010 that had a blood gas analysis done within 1 hour of presentation were eligible for inclusion. parameters assessed included glucose, base excess (be), anion gap (ag), ph, hemoglobin, and sodium. biochemical data were found to be normally distributed using the kolmogorov-smirnov test. t-tests or welch tests were used to compare parameters between survivors (s,n 5 42) and non-survivors (ns, n 5 9). of glucose, be, ag, ph, hemoglobin, and sodium, only mean glucose (s 5 131 mg/dl, ns 5 171.4 mg/dl, p 5 0.029) was significantly different between groups, although there was a trend for a difference in mean be (s 5 à3.6, ns 5 à8,5, p 5 0.055). logistic regression analysis showed that of the parameters, only be was independently associated with outcome (odds ratio 0.79, 95% ci 5 0.63-0.98, p 5 0.036). these results suggest that two easily measured biochemical parameters (glucose and be) may yield useful prognostic information in dogs with head trauma, but further studies are needed to further elucidate these findings. type i intervertebral disc disease (ivdd) commonly affects chondrodystrophic dogs. neurological recovery and outcome following surgical decompression may be unpredictable due to suspected ischemic neuronal injury. hyperlactatemia has been associated with spinal cord injury in humans and experimental animals. the purpose of the study was 1) to determine the relationship between serum and csf lactate levels and 2) to compare lactate levels with neurological outcome following decompressive surgery in dogs with ivdd. healthy, chondrodystrophic dogs diagnosed with ivdd localized to the t3-l3 spinal cord were included. serum lactate levels were obtained at: anaesthetic induction, skin incision, muscle dissection, and extubation. in patients with hyperlacatemia at extubation, additional samples were obtained. csf was analyzed for lactate concentration. neurological status was recorded at presentation and multiple times during the recovery period. 31 dogs were included in the study (3-12 years old). 22/31 dogs had normal lactate levels throughout the study. 9/31 dogs had serum hyperlactatemia prior to anaesthetic induction; 6/9 dogs returned to normal during anaesthesia and 3/9 dogs had continued hyperlactatemia until the end of the observation period. neurological status of the dogs varied similarly between all groups. in 12/14 dogs where csf lactate levels were measured, initial serum levels were lower than csf lactate levels; in 5/14 dogs where csf and serum were collected simultaneously, serum lactate concentration was consistently lower than csf lactate. no association between presenting neurological status or neurological outcome and serum or csf lactate concentration was made. neither serum nor csf lactate concentration is useful for predicting neurological outcome in dogs with ivdd. chiari-like malformation (cm) has been associated with syringomyelia (sm) in cavalier king charles spaniel (ckcs) and is postulated to result from a mismatch between the volume of the caudal cranial fossa and the brain parenchyma contained within. the objective of this study was to assess the role of cerebellar volume in caudal cranial fossa overcrowding and syringomyelia. three dimensional models were created using t2-weighted transverse magnetic resonance images in the commercial software package mimics s . volumes of cerebellar parenchyma were analyzed as percentages of caudal cranial fossa volume (cerebellar caudal cranial fossa percentage) and total brain parenchyma volume (cerebellar brain percentage). data was assessed for normality and the appropriate statistical test was used to compare means/medians between groups. forty-five small breed dogs (sb), 58 ckcs and 31 labradors (ld) were compared. as sm is thought to be a late onset disease process, two subgroups were formed for comparison: 21 ckcs younger than 2 years with sm (group 1) and 13 ckcs older than 5 years without sm (group 2). ckcs had a larger cerebellar caudal cranial fossa percentage than the other groups .76%] vs. sb 47.81% [40.36-62.91%] and ld 41.32% [32.59-52.95%]; p o 0.001). the cerebellar brain percentage was also larger in ckcs compared to the other groups (ckcs 8.90% [6.62-11.46%] vs. sb 7.37% [5.25-11.34%] and ld 7.23% [6.36-9.54%]; p o 0.001). group 1 had a significantly larger cerebellar caudal cranial fossa percentage than group 2 (53.71% ae 1.27 vs. 49.31% ae 2.35, p 5 0.001) and a significantly larger cerebellar brain percentage (9.45% ae 0.43 vs. 8.58% ae 0.55, p 5 0.021). our findings show that the ckcs has a relatively larger cerebellum than small breed dogs and labradors and there is an association between increased cerebellar volume and sm in ckcs. chiari-like malformation (cm) is nearly omnipresent in the cavalier king charles spaniel (ckcs) breed. the mis-match of the caudal cranial fossa and the parenchyma within is thought to lead to syringomyelia (sm). there is currently a lack of information if the morphological changes seen in ckcs with cm are progressive or non-progressive. in this retrospective study we used established measurements of cerebral volumes, foramen magnum height and cerebellar herniation length to assess if there is a significant difference between subsequent magnetic resonance (mr) imaging of the brain of the same dog. electronic patient records were reviewed for ckcs with cm which had two separate mri scans, which were a minimum of 3 months apart. ckcs with diseases affecting measurements were excluded. for the volumetric measurements three-dimensional models were created using t2-weighted transverse mr images in the medical imaging software (mimics v12.0, materialise n.v, 2008) . volumes of the caudal cranial fossa parenchyma were analyzed as percentages of caudal cranial fossa volume and caudal cranial fossa volume was analyzed as a percentage of total cranial cavity volume. the volume of the ventricular system was recorded as a percentage of total parenchymal volume. data was assessed for normality and the appropriate statistical test was used to compare means/medians. twelve ckcs were included with a median scan interval of 9.5 months (3-83 months). the size of the foramen magnum increased significantly between the first and second scan (1.52 ae 0.08cm vs. 1.59 ae 0.09cm; p 5 0.03), as did the length of cerebellar herniation (0.17 ae 0.05cm vs. 0.22 ae 0.09cm; p 5 0.02) and the caudal cranial fossa percentage (13.44% [9.9-15.28%] vs. 13.96% [9.9-15.48%]; p 5 0.02). there was no significant difference noted between the two time points in any of the other volumetric measurements ( this work could suggest that overcrowding of the caudal cranial fossa in conjunction with the movements of cerebrospinal fluid and cerebellar tissue secondary to pulse pressures created during the cardiac cycle causes pressures on the occipital bone. this leads to a resorption of the bone and therefore an increase in caudal cranial fossa and foramen magnum size allowing cerebellar herniation length to increase. the cord dorsum potential (cdp) is a stationary potential arising in dorsal horn interneurons after stimulation of sensory nerves. cdps have been recorded in normal anesthetized dogs previously, and normal latency values have been determined for tibial and radial nerves. this study was undertaken to determine whether cdps could be reliably recorded from the caudal nerves in normal dogs, thus allowing electrophysiological assessment of the cauda equina, and whether neuromuscular blockade improved recording quality. ten adult dogs weighing from 23.2 to 32.0 kg were anesthetized and cord dorsum recordings were compared before and after administration of atracurium. recording needles were placed onto the dorsal lamina at intervertebral sites from l7/s1 to l2/3. stimulations were made on the lateral aspect of the caudal vertebrae approximately 5-8 cm from the tail base. recordings from 500 stimulations were averaged. cdps were recorded successfully in all dogs. onset latency varied from 2.2 to 4.7 ms. the cdp was largest when recorded closest to the site of entry of the stimulated nerve into the cord, as determined by post-mortem examination immediately after testing in 6 dogs. administration of atracurium did decrease muscle artifact, and in some cases helped isolate the origin of the cdp. these data show that cdps can be readily assessed from the caudal nerves of anesthetized dogs, with or without atracurium. cord dorsum potentials from caudal nerves may add important information about the integrity of the cauda equina in dogs with suspected degenerative lumbosacral stenosis. canine intracranial glial tumors and many human brain tumors express heat shock proteins (hsps) associated with their degree of malignancy. the up-regulation of hsps during tumor cell growth helps keep tumor proteins stable and therefore makes them a reasonable target for therapy. ki67 expression and ec have been strong indicators of cell proliferation and dedifferentiation, respectively.the aims of this study were to determine (i) if canine meningiomas express hsp 27 and/or hsp 72; (ii) whether the expression of the hsps was associated with ki67 and/or e-cadherin (ec) expression; and (iii) whether peritumoral edema was associated with hsp, ki67 and/or ec expression. forty-one formalin-fixed, paraffin-embedded canine intracranial meningiomas underwent immunohistochemical staining using anti-hsp 27, or 72 antibodies. these tumor samples were also immunohistochemically stained for ki67 and ec expression. canine mammary carcinoma and squamous cell carcinoma tissues served as the control samples, as both have previously been shown to express hsps. skin was used as control for ki67 and ec. four non-overlapping high power fields of each stained sample were selected and cell staining was analyzed using a semi-quantitative method for hsps and ki67; a qualitative assessment was used for ec. all analyses were performed using sas v 9.2 (cary, nc). descriptive statistics of staining percentages were calculated for all tumors tested. simple pearson's correlation was used to test for correlations of ec area with hsp areas and ki-67 percent positive cells and of ec intensity with hsp intensities and ki-67 percent positive cells. all hypothesis tests were 2sided and the significance level was a 5 0.05. thirteen meningiomas had mr images quantitatively evaluated for peritumoral edema using t2flair sequences. the edema index (ei) was evaluated for an association with hsp 27, hsp 72, ec and ki67 expression. hsp 27 was expressed in 36% (mean 8.7% of cells; range 0-58%), hsp 72 in 52% (mean 5.2% of cells; range 0-26%) and ec in 68% of meningiomas. there was no association demonstrated between either hsp expression variable and ec or ki-67 expression. there was also no association between the ec expression variables and ki-67. however, there was a significant negative association between hsp 72 extent (p 5 0.03) and area (p 5 0.04) with ei. in conclusion, hsp 27 and 72 expression was demonstrated in canine intracranial meningiomas but was not associated with ki-67 or ec expression. this study suggests that hsps may not have a significant role in the maintenance of canine meningiomas and so do not represent a novel treatment target for this group of tumors unlike canine glial cell tumors. however, hsp 72 may be involved in the pathogenesis of peritumoral edema in meningiomas and warrants further investigation. an extended release (xr) formulation of levetiracetam, a second generation antiepileptic drug, was recently approved for human use on a once daily basis. although levetiracetam is clinically effective for seizure control in dogs, it requires a three times daily administration. the potential benefits of the xr formulation include reduced daily dosing leading to improved compliance and relatively constant plasma concentrations. the aim of this study was to compare the pharmacokinetics of levetiracetam xr tablets with immediate release (ir) tablets following single dosing in dogs. five clinically and neurologically normal mixed breed dogs were used in a cross-over design. all dogs (mean body weight 25.9kg; range 23.2-30.5) had normal hematology, serum chemistry and urinalyses. following a 12 hour fast, each dog was administered oral ir levetiracetam (500 mg; mean dose 19.3 mg/kg; range 16.4-21.6). heparinized blood for drug analysis was taken from each dog prior to administration and 0.25, 0.5, 0.75, 1, 2, 4 and 8 hours after. blood was immediately centrifuged and supernatant plasma was stored at à801c until analysis. after a 4 day wash-out period, each dog was administered 500 mg oral xr levetiracetam and blood samples were taken at identical timings. plasma samples were thawed at room temperature before preparation by solid phase extraction for hplc analysis. reverse phase chromatographic separation was performed. levetiracetam and an internal standard were detected using ultraviolet spectroscopy at 205 nm. concentrations of levetiracetam were determined by peak area comparison to the internal standard. mean data were fit to a one compartment pharmacokinetic model with first order elimination and absorption and included a lag-phase for xr formulation. no adverse clinical effects were noted in any of the dogs. the auc associated with xr was 230 hr ug/ml, a 5.14 fold increase over that with ir (44.8 hr ug/ml). the absorption half-life was 3.2 hr with xr and 0.41 hr with ir, a 7.75 fold difference. the elimination halflife was 3.13 hr with xr and 2.19hr with ir, a 1.43 fold difference. the tmax associated with xr 5.01 hr and 1.22 hr with ir, a 4.21 fold difference. the cmax associated with xr was 18.5 mg/ml and 9.62 mg/ml with ir, a 1.92 fold difference. the plasma concentration of ir levetiracetam was not detectable at 8hr after administration whereas it was greater than 10 mg/ml at 8hr after xr administration. based on the auc data, there is an approximately 5 fold increase in bioavailability of the xr compared to the ir formulation. the cmax was approximately 2 times greater following xr administration and a high plasma level in excess of the suggested canine therapeutic concentration (5 mg/ml) for at least 8 hours. although specific dosing recommendations cannot be made from this data, the favorable pharmacokinetics of xr over ir suggests that single, daily administration could be efficacious. thoracic and lumbar vertebrae are frequently affected by fractures and or luxations in dogs following trauma. surgical repair is part of the emergency treatment described for this disorder but does not guarantee improvement of the associated clinical signs. multiple surgical repair techniques have been described but have not been compared in terms of their success and the factors associated with a positive outcome. the aims of this study were to retrospectively evaluate the effect of 3 different types of vertebral repair, injury type and injury location on outcome in dogs with thoracolumbar (tl) and lumbosacral (ls) spinal trauma. medical records were searched for dogs with radiographic evidence of a tl or ls vertebral fracture and or luxation (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) ; signalment, body weight and duration of disease were recorded. dogs were retrospectively scored neurologically (0-5; normal to plegic with absent pain perception) on admission and at re-evaluation following surgery. lesion location was classed as t3-l3 and l4-s3; dogs were evaluated as one group and as two separate groups with respect to outcome. a subset of lesions were classed as cord compression or not based on advanced imaging. three repair techniques were evaluated (i) pins and polymethylmethacrylate (pmma); (ii) screws and pmma; and (iii) spinal stapling. regression analysis was applied to test for an association between the type of surgery and a successful outcome (non-painful and ambulatory). simple bivariate analyses were performed to investigate for variables predictive of a successful outcome. fifty-nine dogs were included. twenty-eight dogs were classed as t3-l3 and 31 were l4-s3. there were 55 dogs with fractures and 43 with luxations; 23 dogs had both. thirty-one of 35 dogs evaluated had spinal cord compression. ten dogs were repaired with im pins and pmma, 18 dogs with screws and pmma and 31 dogs with spinal stapling. overall, there was a 78.7% success rate; there was no significant difference in outcome between the anatomic sites (p 5 0.5). all dogs initially graded as 1-3 pre-operation were classed as a successful outcome after at least one week following surgery; 79% of dogs initially graded as 4 (plegic with pain perception) were classed as successful recovery. one dog (12.5%) initially as graded as 5 (plegic with no pain perception) had a successful outcome. a low admission score was statistically predictive of a successful outcome (p o 0.0001). surgery type was not associated with a successful recovery (p 5 0.13). signalment, body weight, location of injury, injury type (fracture, luxation or both), presence of compression, and duration of disease did not predict outcome. from this study, the successful recovery of dogs following surgical fixation is high and is only dependent on the neurological score at the time of admission. the choice of surgical technique does not seem to influence outcome although a prospective study comparing two surgery types is warranted to further investigate this issue the results of which can be confounded by surgeon experience and variable follow-up. cranial thoracic intervertebral disc disease (ivdd) is extremely rare due to the presence of the intercapital ligament, although anecdotic data suggest german shepherd dogs (gsd) can share some predisposition for this disorder. the objective of the study was to retrospectively evaluate through mri if cranial thoracic ivdd is significantly more common in gsd compare to other large breed dogs. a search was done through database of the ontario veterinary college. any gsd were a spinal mri including t1-t10 spine was performed was recruited. a group of large-breed non-gsd was used as a control. in the midsaggital t2wi plane, three variables were assessed and graded for each intervertebral disc space t1-t9: spinal cord compression (scc), disc degeneration (dd), and herniation. wilcoxon sign rank test was used to assess if scores were different between groups. exact conditional logistic regression was used to determine whether any intervertebral disc space was a risk factor. 22 gsd and 46 large breed non-gsds were recruited. the gsd group had significantly higher scores than the non-gsd for scc, and herniation. regarding the individual intervertebral discs, in the gsd group t2-3, t3-4, t4-5 discs had significantly an increased risk for scc, and t3-4 for herniation. the results of this study show that gsd have a higher risk of cranial thoracic disc ivdd than other large breed dogs. that risk was higher in discs t2-t3, t3-4, and t4-5, particularly in t3-4. genetic and/or conformational factors, such as weakness of the intercapital ligament, may predispose gsd to this lesion. diskospondylitis is a common disease of the canine spine; however, few reports of mr imaging findings in dogs are available. the purpose of this study was to describe the signalment, clinical and mr imaging features in affected dogs. twenty-three dogs with a diagnosis of diskospondylitis based on clinical signs, mr imaging, and urine, blood, csf and/or intervertebral disk cultures were included. large breed dogs (4 25kg) accounted for 21of the cases. the mean age was 6.8 years with males and females equally represented. most dogs (15/23) were ambulatory with varying degrees of pain and paresis. mr imaging characteristics of 27 sites were reviewed. on t2w images, vertebral endplates were of mixed signal intensity (16/27) while the vertebral body was hypointense (11/27). the intervertebral disk space was hyperintense on t2w (16/27) and stir (14/15) images and mixed signal intensity (7/13) on t1w images. paravertebral soft tissue hyperintensities were noted on 10/27 t2w and 12/16 stir images. contrast enhancement occurred at 12/19 endplates and 15/19 intervertebral disk spaces. only 4/18 vertebral bodies and 7/18 parvertebral soft tissues contrast enhanced. intramedullary spinal cord t2w hyperintensity was noted at 10/23 sites. spinal cord or cauda equina compression occurred at 22/27 sites. based on the spearman correlation coefficient, a significant direct correlation was found between the degree of spinal cord or cauda equina compression and the patient's neurologic status (p 5 0.0011). the incidence and severity of spinal cord compression in canine diskospondylitis may have prognostic value and may have been previously underestimated using other imaging modalities. hemilaminectomy and pediculectomy are both well described and commonly utilized techniques to access the spinal canal. these procedures are most often performed to approach a compressive lesion, such as intervertebral disc disease and neoplasia, the goal being adequate visualization of the spinal canal and access to the offending lesion. a proposed benefit of pediculectomy is preservation of the articular facets and thus better maintaining stability of the vertebral column, but at the cost of reduced access to the spinal canal. the purpose of this study was to describe standardized anatomical limits of each technique and report any observed differences that could be considered during presurgical planning. ten canine cadavers had both procedures performed on opposite sides to access the t11-12, t13-l1, and l2-3 spinal canal. measurements were obtained after performing a computed tomography study of the spine and recorded from the transverse slice most representative of the defect. the surgical technique, vertebral site, and side of vertebral column were compared with the mean spinal canal and defect height using a covariate model. dorsal and ventral remnant lamina heights were also compared. the height of the defect relative to the spinal canal was 76-87% with hemilaminectomy and 58-75% with pediculectomy. the observed difference in defect height of 12-18% (p o 0.0001) and varied with spinal canal height. dorsal remnant lamina height was 0.9-2.4% of spinal canal height with hemilaminectomy and 7-19% with pediculectomy. ventral remnant lamina height ranged from 1-2% and 0.6-2.3%, respectively, though the difference was not statistically significant. while a larger defect is expected with a hemilaminectomy procedure, our results demonstrate that this difference increases with increasing spinal canal height. interestingly, the proportion of exposed spinal canal decreases with increasing canal height for both procedures. the difference in defect height between techniques was due to greater removal of the dorsal spinal canal, possibly making the hemilaminectomy technique better suited for more dorsal lesions, while no statistically difference in access to the ventral canal is observed. no effect of vertebral site was detected. of note was the involvement of articular facets in half of the pediculectomy defects, involving an average of 22% of the articular facet height. this result questions the suggested benefit for the vertebral stability, but further biomechanical studies would be required. low level laser therapy (lllt) is a treatment used in human and veterinary medicine for a variety of clinical syndromes. some uses in human medicine include acute pain associated with osteoarthritis, rheumatoid arthritis, tendonitis, tmj disorders, chronic joint disorders, and wound healing. research is currently on-going to determine the adequate wavelengths to promote effective treatment results with lllt in these conditions. it is purported that lllt acts via the mitochondria to increase cellular metabolism promoting wound healing and a decrease in pain and inflammation. in this study, we hypothesized that dogs treated with lllt in conjunction with hemilaminectomy would display quicker recovery times regardless of the presence or absence of deep pain sensation. seventeen dogs (9 dachshunds, 2 chihuahuas, 2 french bulldogs, 2 lhasa ahpsos, and 1 each of a pembroke welsch corgi, and a miniature poodle) were selected and divided into two groups. the dogs ranged in age from 2 to 11 years old, weighed between 7 and 33 pounds, and underwent hemilaminectamies after acute onset of paraplegia secondary to intervertebral disc disease (surgically confirmed). one group received laser treatments on days 1 through 4 of hospitalization. the second group did not receive lllt, but followed the same peri-operative medication protocol. the laser used in this study was an erchonia laser model pl5000 (635nm). the hertz setting was similar for each patient using the previously established protocol for intervertebral disc disease (ivdd) with pulse rate ranging from 9hz to 1151 hz. all dogs received advanced imaging pre-operatively with myelogram or mri. results of the study revealed that treatment with lllt of 635nm wavelength did not shorten or improve recovery times for dogs with acute onset paraplegia secondary to ivdd after hemilaminectomy procedures. dogs that showed recovery to ambulation at the two week recheck were consistently dogs that were deep pain positive on presentation. a lengthened recovery time or no recovery was seen in the majority of those dogs with absent deep pain on presentation as has been revealed historically in past studies. lllt did not appear to have an effect on this result. however, there are few data describing normal glucose uptake of the canine brain for comparison with suspected or confirmed disease. thus the purpose of this study was to assess the normal distribution of fdg uptake of canine brain structures using a high-resolution research tomography-pet and 7 t-magnetic resonance imaging (mri) fusion system. fdg-pet and t2-weighted mr imaging of the brain were performed on 4 healthy laboratory beagle dogs. acquired pet and mr images were automatically co-registered by the image analysis software. on mr images, regions of interest (roi) were manually drawn over 48 intracranial structures, including 6 gross structures (whole brain, telencephalon, diencephalon, mesencephalon, dorsal metencephalon, ventral metencephalon and myelencephalon). a standard uptake value (suv) and relative suv ratio (rsuv 5 suv of roi/suv of whole brain) were calculated for each roi. 7 t-mr images compensated the low anatomical resolution of pet qj;by proving good spatial and contrast resolution for the identification of the clinically relevant brain anatomy. among gross structures, mesencephalon and ventral metencephalon had the highest (suv: 4.17 ae 0.23; rsuv: 1.12 ae 0.03) and the lowest (suv: 3.34 ae 0.35; rsuv: 0.90 ae 0.06) fdg uptake respectively. when suvs were calculated on 42 detailed regions, rostral colliculus and corpus callosum had the highest (suv: 6.00 ae 0.16; rsuv: 1.62 ae 0.05) and the lowest (suv: 2.81 ae 0.12; rsuv: 0.76 ae 0.06) value respectively. these data acquired from normal dog brain will be used in clinical neurology to investigate various intracranial diseases such as inflammation, neoplasm and behavioral disorders. degenerative lumbosacral stenosis (dlss) is a multifactorial condition affecting predominantly large breed dogs. the combination of stenosis and compressive neuropathy cause lumbar pain, lameness and neurologic dysfunction. previous reports describe urinary and fecal incontinence in severely affected dogs. the objectives of this retrospective case series were to describe the clinical signs associated with dysuria and eventual diagnosis of dlss in dogs, and to describe factors associated with regained micturition following prompt diagnosis and treatment. medical records from the university of georgia and the university of missouri between 1995 and 2009 of 11 dogs were reviewed. inclusion required observation of dysuria, urine retention, absence of structural lower urinary tract disease and concurrent presumptive diagnosis of dlss. dysuria was defined as inability to initiate or sustain a urine stream. urine residual volume was evaluated postvoiding. dysuria was further evaluated using urethral contrast studies, urodynamic testing (urethral profilometry (4) and cystometry (4)), ultrasonography (5), and urine culture (8). presumptive diagnosis of dlss was based on imaging using plain radiography and epidurography (8), computed tomography (1) or magnetic resonance imaging (2). breeds represented included the german shepherd dog (n 5 3), golden retriever (n 5 2), burnese mountain dog (n 5 2), and 1 each labrador retriever, weimaraner, rottweiler and mixed-breed. all dogs were male. 8 were intact at onset of clinical signs. median body weight was 38.5 kg (range 29.5-46) and median age was 5 years (range 2-10). median duration of clinical signs prior to admission was 2 months (range 0.25-12). other pertinent presenting clinical signs included dyschezia (2), fecal incontinence (4), general proprioceptive ataxia (2), weakness (2), and difficulty rising (1). physical examination findings included pelvic limb muscle atrophy (2) and prostatomegaly (1). abnormal neurologic examination findings included postural reaction deficits (6), hyporeflexia (4), decreased tail tone (3) and lumbosacral hyperesthesia (6). neurologic examination was normal in 3 dogs. dorsal laminectomy was performed and diagnosis confirmed in 9 dogs; recovery was monitored for a median of 5.5 months (range 0.25-9). three of the 9 dogs (33%) regained normal micturition within 0.25-1.5 months of surgery. though not statistically significant, dogs that regained micturition tended to have a shorter duration of clinical signs (median 0.25 months, range 0.25-2) versus dogs that remained dysuric (median 5 months, range 2-12). two of the 3 dogs that regained micturition were neutered at the onset of clinical signs, but only 1of 6 dogs that remained dysuric was neutered. signs improved in all dogs with postural reaction deficits and decreased tail tone. hyperesthesia resolved in 5 of 6 dogs (83%) and fecal continence returned in 2 of 4 dogs (50%). these findings suggest that following prompt diagnosis and surgical decompression, normal micturition could be regained in dlss affected dogs presenting with signs of dysuria. glycogen storage disease type ia (gsdia; von gierke disease) is an inherited metabolic disorder resulting from a deficiency of glucose 6-phosphatase-a (g6pase). previous reports indicate that clinical manifestations of gsdia occur only in individuals with homozygous expression of a p.i121l mutation. heterozygote dogs (het) have been previously reported to exhibit an overall normal outward phenotype. the purpose of this report is to briefly describe some differences that have been observed between het and homozygous wild type (wt) dogs. a colony of dogs at the university of florida contains a mix of affected, wt, and het individuals. in the course of studies designed to determine the effectiveness of gene therapy for correction of gsdia in dogs, both wt and het dogs have been utilized as controls. available information about body weights, clinical pathology tests, fasting studies, and liver biopsies was retrieved from records for both wt and het dogs and compared. although birth weights are similar, het dogs have a slower average rate of weight gain than wt dogs and this difference is especially prominent during the first few months of life (figure 1) . in contrast to affected dogs, both wt and het dogs are able to maintain normal blood glucose concentrations for up to 10-12 hours of fasting, however, after longer fasts of 12-17 hours, het dogs have lower glucose and higher lactate concentrations (table 2 ). in addition, liver biopsy samples from het dogs had greater apparent levels of glycogen suggested by pas staining than did samples from wt dogs, and this correlated with the results of proton magnetic resonance spectroscopy which demonstrated 2.9 times greater glycogen content in a liver biopsy sample from a het dog compared to a sample from a wt dog. together, these findings suggest that the level of g6pase activity in heterozygote dogs does not provide a completely normal physiological, biochemical, or histological phenotype as previously reported. the glucokinase gene (gck) encodes an enzyme involved in cellular glucose-sensing mechanisms in pancreatic beta cells and hepatocytes. gck mrna is present in feline pancreas but the gene is not expressed in feline liver. hepatic gck expression is abundant in omnivores so its absence may reflect an evolutionary adaptation of strict carnivores, like feline species. we hypothesized speciesspecific features in the gck hepatic promoter may underlie the gene expression pattern observed in cats. the putative feline gck (fgck) promoter region was located using bioinformatic software to identify homology with human gck (hgck). genomic dna from a dsh cat was subjected to direct sequencing using a series of pcr reactions with speciesspecific primers. dna clones thus obtained were aligned to generate the feline sequence. direct sequencing yielded 8.9 kb of genomic dna sequence with high homology with sequences (acbe01359231, acbe01359221) archived in the feline genome project. the feline sequence had six regions homologous with non-coding regions of hgck; four of these conserved regions are upstream of the putative fgck start. a 0.8 kb segment immediately upstream of feline hepatic exon 1 is not present in hgck. the 0.8 kb insert is the reverse complement of a conserved sequence located downstream of exon 1 in feline and human sequences. in conclusion, the putative hepatic promoter of fgck shares extensive homology with the hgck promoter but contains a 0.8 kb insert not found in hgck. functional studies are needed to confirm the role of the unique insert in regulation of fgck gene expression. deuterium oxide (d 2 o) dilution has been proposed for quantifying body water content, but remains difficult to perform routinely. the objective of this study was to assess if the volume of distribution (vd) of creatinine could be proposed as an alternative in dogs for such a measurement. creatinine and d 2 o vd were measured before (c) and after induction (o) of obesity (by giving an hypercaloric diet (6100 kcal/ kg) for 6 months) in six healthy adult beagle dogs. creatinine (40 mg/kg) and d 2 o (200 mg/kg) were simultaneously injected by bolus iv. blood was collected before administration and then at 5, 10, 30, 60, 120, 180, 240, 360, and 480 min post-injection (creatinine), and 10, 30, 60, 90, 120, 150, 180 and 240 min (d 2 o) . plasma concentrations of both markers were determined. vd was calculated using pharmacokinetic equations. the body weight increased from 10.7 ae 0.6 (c) (mean ae sd) to 15.8 ae 1.0 kg (o). d 2 o vd decreased from 636 ae 18 (c) to 468 ae 19 (o) ml/kg. similarly, creatinine vd decreased from 624 ae 31 (c) to 420 ae 25 (o) ml/kg. the individual difference between creatinine and d 2 o vd (expressed in % of d 2 o vd) ranged from à9.5 to 1.0% (c) and from à17.3 to à7.1% (o). in conclusion, creatinine vd provides a good estimate of d 2 o vd in both normal and obese conditions. a 16 wk double blinded study was conducted comparing the affect of two foods on mobility in dogs. all work was approved by an iacuc. 53 healthy beagle dogs (7-15 years old, mixed gender) were used. 33 affected (a) and 20 non-affected (na) dogs were identified based on orthopedic examination and radiography as having or not having evidence of naturally occurring joint pathology (presence of osteophytes, dysplasia, effusion, pain on manipulation etc) in one or more joints. a and na dogs were evenly distributed between two locations. foods had nutrient profiles adequate for maintenance according to the 2009 aafco official publication. the test food contained greater amounts of methionine, manganese, carnitine, vit. e,, vit. c, alpha linolenic acid (ala), and eicosapentaenoic (epa) acid: the food provided 388 mg n3 fatty acids and 847 mg n6 fatty acids per 100kcal. all dogs were fed the control food for 4 wks followed by a 12 wk feeding period where 17 a and 10 na dogs consumed the test food and 16 a and 10 na dogs the control. blood and urine were collected at weeks 0, 4, 8 and 12 and analyzed for serum fatty acids and urine thromboxane:creatinine ratios were determined. evaluators in this study were different than those making the original diagnosis and so were blinded as to treatment and diagnosis. orthopedic exams were performed by two veterinary surgeons at each site on weeks 0, 4, 8 and 12. the same two evaluators examined the same dogs throughout. the data was evaluated for the difference between a and na dogs and between foods with age, gender and location as covariates. body weight, disease status, age and gender were blocked. analysis included anova repeated measures mixed procedure (sas version 9.0) to determine treatment effects over time.serum epa was greater and arachidonic acid lower at weeks 4, 8 and 12 in the test food fed dogs (p o 0.05). urine thromboxane:creatinine ratios were decreased in the a dogs fed the test food compared to the a dogs fed the control food at 12 wks (p o 0.05). lameness score was significantly improved (p o 0.05) within and between groups of dogs fed the test food. a significantly greater proportion of a dogs fed the test food had improvement in total het (n511) blood glucose (mg/dl) 78 (ae17) 64 (ae13) blood lactate (mmol/l) 1.1 (ae0.5) 1.7 (ae1.1) joint score, lameness, functional disability and overall assessment score at 12 wks compared to a dogs fed the control food. 69% of a dogs had an improved overall assessment score on the test food after 4 wks and at 12 wks compared to 40% at 4 wks and 27% at 12 wks of a dogs consuming the control food. this study shows that a food with moderate amounts of added linolenic acid and epa can have a positive impact on systemic inflammation and mobility in 4-12 weeks. a similar abstract will be presented at the orthopedic research society meeting in january 2011 to an audience largely of orthopedic researchers interested in human orthopedics. fat is an important dietary component, serving both as a source of energy and as a supplier of essential fatty acids (fa). medium-chain triglycerides (mct) contain intermediate length fa that do not rely on l-carnitine for transport across the inner mitochondrial membrane, bypassing this rate-limiting step in fa oxidation. longchain (n-3) polyunsaturated fatty acids (pufa) from fish oil (fo), and in particular eicosanoids derived from eicosapentaenoic acid (epa), may protect against excessive inflammatory reactions, which may be exacerbated by eicosanoids derived from (n-6) arachidonic acid (aa). this study investigated the effects of adding mct:fo and l-carnitine to a control diet (prescription diet s k/d s ) on lean body mass, and serum fa and metabolites. forty healthy beagles (3.1 to 14.8 y) were fed one of three foods (n 5 13 to 14 dogs each) for 6 mo. the study protocol was reviewed and approved by iacuc, hill's pet nutrition, inc. all foods were complete, balanced, and sufficient for maintenance of adult dogs; and had similar concentrations of moisture, protein, and fat (approx. 7.4%, 14.0%, 18.1%, respectively). composition of serum fa was determined by gas chromatography of fa methyl esters. metabolomic profiles of serum samples were determined from extracted supernatants that were split and run on gc/ms and lc/ ms/ms platforms, for identification and relative quantification of small metabolites. body composition was determined by dual energy x-ray absorptiometry. serum concentrations of lauric and myristic fa increased; epa and dha increased in a dose-dependent manner; and aa decreased in dogs fed treatment food 3 (proc-mixed procedure in sas; all p 0.05) when compared to dogs fed treatment foods 1 or 2. serum concentrations of acetylcarnitine and succinylcarnitine increased, indicating lcarnitine incorporation, in dogs fed treatment foods 2 and 3. thus, a diet enriched with mct:fo significantly altered serum fa composition, enriching (n-3) pufa and lowering aa concentrations. there was no change in lean body mass for any of the diets compared to baseline values, and no difference between treatments, showing that all three treatment foods met protein requirements. ten owned dogs, obese for more than 12 months (body condition score [bcs] of 9; fat mass [fm] 5 45.7 ae 1.51%) were studied. these dogs had their weight reduced by 20% (bcs 5 8; fm 5 33.5 ae 1.92%; p o 0.001) being designated weight reduced (wr) group and then were fed to maintain constant body weight during 150 days (bcs 5 8.2; p 5 0.6), designated maintenance (main) group. a control (ct) group of 10 beagles was also included (bcs 5 4.5; fm 5 18.3 ae 1.38%; p o 0.01). in all groups the glucose postprandial response test was performed after 12 hours of fasting. blood samples were taken prefeeding and after 5, 10, 15, 30, 45, 60, 120, 180, 240, 300 and 360 minutes of the consumption of cooked rice enough to the ingestion of 6g of starch/kg body weight. tnf-a and il-6 were dosed in milliplex tm map panel, insulin and leptin by radioimmunoassay. statistical analysis included paired or non-paired t-tests and wilcoxon (p o 0.05). the regimen normalized meal glucose response, the area under the curve (auc) of glucose for wr was lower than for obese (p o 0.05) and similar to main and ct (p 4 0.05). insulin secretion did not normalize immediately, as obese and wr exhibited similar auc of insulin and higher values than for ct (p o 0.05). main, however, presented similar auc of insulin than ct, with lower values than obese and wr (p o 0.01), suggesting that dogs require some time to adapt their metabolism. leptin, tnf-a, and il-6 presented significant reductions after weight loss (p o 0.05), without differences between wr, main and ct (p 4 0.05), suggesting an improvement of the pro-inflammatory state consequent to obesity. studying food base excess (be) modification, methionine intoxication was described. in a basal kibble dog diet (be 5 305 meq/kg; 1.98 g/kg of s) two dosages of ammonium sulphate and methionine was added, resulting in diets with be of 135 meq/kg (4.26 g/kg of s) and à51 meq/kg (6.65 g/kg of s), or be of 142 meq/kg (4.68 g/kg of s) and 4 meq/kg (7.49 g/kg of s), respectively. a 2 â 2 factorial plus a control diet design, resulting in five treatments, and 32 adult health beagle dogs were used, in a completed randomized design with six dogs per diet. a 5-d adaptation phase followed 3-d of total urine collection (in bottles with 100 mg of thymol). urine were pooled by dog and analyzed for density, volume and ph. food macroelements were determined by standards methods (aoac, 1995) and used for be calculation. dog's acid-basic status was studied by blood gas analysis of venous blood, at 8:00h (pre feeding) and 6 hours after meal. a dose-dependent reduction of urinary ph was verified for both compounds (p o 0.01). blood bicarbonate (r 5 0.98; p o 0.01), and blood base excess (r 5 0.75; p o 0.01) were highly correlated with food be. acidemia and reduced blood be were verified in diets with be close to zero (higher dose of both compounds, or 4 6.6 g/kg of s), resulting in daily or each other day vomiting episodes in the dogs. ataxia, seizures, and vomiting were previously describe in dogs fed 47 g/kg of methionine, but our results suggest that a much lower value (27.7 g/kg) was toxic and that the safe upper limit should be between this value and 15.7 g/kg (the lower evaluated dose). in people with chronic kidney disease and heart failure, obesity is associated with longer survival times. this association, called the "obesity paradox," also has been recognized in dogs and cats with heart failure. excess weight appears to modulate the serious deleterious effects of muscle loss in these diseases. the purpose of this study was to determine the effects of body condition and body weight changes in dogs with naturally-occurring chronic kidney disease (ckd). dogs diagnosed with ! iris stage ii ckd between 2008 and 2009 at iowa state university and tufts cummings school of veterinary medicine were eligible for the study. dogs o 1 year of age and those with acute renal failure or suspected congenital renal diseases were excluded. medical records were reviewed using a standardized data form, and data were collected for initial body weight and body condition score (bcs, 1-9 scale), clinicopathologic values, changes in body weight and bcs, comorbidities, and treatments. dogs were classified as underweight (bcs 5 1-3), moderate weight (bcs 5 4-6), or overweight (bcs 5 7-9). a change in body weight was defined as 4 0.2 kg. survival times were determined for all dogs that were discharged from the hospital and lived 4 1 day. associations between survival and bcs or body weight changes were analyzed using cox proportional hazards models. one hundred two dogs were enrolled in the study. at the time of diagnosis, 21 dogs were classified as iris stage ii, 57 dogs were stage iii and 24 dogs were stage iv. median body weight at baseline was 18.7 kg (range, 2.4-50.1 kg). for dogs with body condition scores recorded (n 5 74), 13 were underweight (18%), 51 were moderate (69%), and 10 were overweight (14%). for dogs that had at least two body weights recorded over the course of their disease, 22 gained weight, 46 lost weight, and 10 had no change in weight. changes in body weight were not associated with survival; however, bcs at the time of diagnosis was significantly associated with survival. dogs classified as underweight had a significantly shorter survival time compared to both moderate (p o 0.001) and overweight dogs (p o 0.001). these results suggest that body condition is an important consideration in dogs with acquired chronic kidney disease. further studies are warranted to evaluate the relationship between obesity and longer survival in dogs with ckd. protein restriction is the cornerstone of dietary management of kidney disease. the national research council recommends 20% crude protein and the american association of feed control officials (aafco) recommends a minimum of 26% crude protein for maintenance for healthy adult cats. protein requirement is unknown for adult cats with kidney disease. most commercially produced cat foods for adult maintenance contains 30% or more crude protein on a dry matter basis. a typical therapeutic food for cats with kidney disease contains about 28% crude protein. the objective of the present study was to investigate whether dietary crude protein at 28.5% would be adequate for the maintenance for adult cats with impaired kidney function. seven adult cats, 3 female and 4 male, with age ranging from 5 to 13.7 years old (mean: 8.1 years) were used in the study. all cats had elevated serum creatinine concentration (4 1.6 mg/dl, range: 1.61-2.10 mg/dl) and reduced glomerula filtration rate (mean: 32% reduction; range: 12-60% reduction) during the study. they did not have other systematic diseases, e.g., hyperthyroidism, at the beginning of the study. cats were fed an expanded dry food made with ingredients commonly used in commercial dry cat foods. the food contained 28.5% crude protein (chemical analysis) and 4366 kcal/kg (calculated) on a dry matter basis, or 65.3 g protein/1000 kcal. each essential amino acid in the food was at least 130% of that recommended by aafco. other nutrients in the food also exceeded aafco's recommendations for maintenance for adult cats. cats were fed the food for 30 weeks. lean body mass (dual x-ray absorptionmetry; hologic, hologic, inc, ma) and serum albumin concentration were measured periodically to monitor protein status of cats. the average lean body mass (mean ae sd) was 3.63 ae 0.82 kg, 3.72 ae 0.85 kg, 3.74 ae 0.84 kg, and 3.75 ae 0.89 kg in weeks 3, 11, 17, and 30 of the study, respectively. paired t-test did not detect statistical difference (p 4 0.05) when comparing the lean body mass in weeks 3 versus weeks 11, 17, and 30, respectively. serum albumin concentration were within the normal reference range during the study (mean ae sd: 3.04 ae 0.35%, 2.80 ae 0.35%, 3.04 ae 0.32%, and 3.07 ae 0.40% in weeks 3, 11, 17, and 30, respectively) . these data show that 28.5% dietary crude protein in a dry food with 4366 kcal/kg on a dry matter basis, or 65.3 g protein/1000 kcal, is adequate for maintenance for cats with impaired kidney function. in humans, several disease conditions exist that involve abnormal patterns of polyunsaturated fatty acids and similar abnormalities may be present in companion animals. indeed there have been reports of decreased plasma arachidonic acid and reduced delta-6 desaturase activities in dogs with atopy and other skin disorders. the present study investigated serum fatty acid profiles in dogs and cats presented to the texas a&m university veterinary teaching hospital, clinical pathology laboratory over the past one year period. results were compared with normative data generated among dogs and cats from earlier feeding studies. sera used were residual samples submitted to the laboratory for other diagnostic procedures and stored frozen for no more than 2 months after collection. the samples were grouped according to presenting disorders involving liver, kidney, digestive, and cardiac diseases. total lipids were extracted using chloroform:methanol (2:1 v/v) and fatty acid methyl esters were prepared for capillary gas chromatography. relative percentage distribution of individual serum fatty acids for each animal were then compared with average normative serum phospholipid fatty acid values (dogs, n 5 44; cats, n 5 29) by calculating the ratio of the value in the diseased individual to the normal mean value and used as an index of normalcy. normalcy ratios were then plotted on a logarithmic scale with normal at 1.0. the ratio was then compared to changes greater than 3, 2 and 1 standard deviations of the normal mean values. in this way a graphical presentation of resultant values was obtained. although the animals had been fed various commercial diets and some home-prepared foods, a number of noteworthy patterns emerged from this analysis. dogs showed increased linoleic acid, decreased arachidonic acid, increased total monounsaturated and decreased saturated fatty acids at p o 0.001; oleic acid was increased at p o 0.01. remarkably, these findings were similar for all canine disease categories evaluated (n 5 30, heart; n 5 17, kidney; n 5 28, liver, and n 5 28 digestive disorders). in cats, a slight decrease in arachidonic acid and large decrease in 22:0 was observed but only in heart disorders. by contrast, modest elevations of arachidonate were observed in kidney, liver, and digestive disease groups but at p o 0.01. sample sizes of the feline sera were considerably smaller (range of 4-13 per group). a limitation of this analysis is that variability of normal data may exist depending on diet fed making comparisons less reliable however, these preliminary data suggest that metabolic diseases of dogs may depress plasma arachidonic acid independent of diet fed suggesting either reduced conversion from linoleic acid or increased utilization of arachidonate for eicosanoid production during times of metabolic stress. conversely, in cats, increases in arachidonic acid may be associated with diet arachidonate or other mechanisms. additional studies to verify these findings are warranted. the objective of this study was to determine whether or not lalanyl-l-glutamine (ala-gln) supplementation in dogs with parvoviral enteritis improves the survival rate and ameliorates clinical signs without side effects. this randomized, double-blinded, placebo-controlled clinical trial included 39 client-owned dogs. the dogs were randomly assigned into two groups and administered ala-gln solution (dipeptiven; 0.4 g/kg) or an equivalent volume of placebo orally twice a day. all of the dogs (ala-gln group [n 5 20] and placebo group [n 5 19]) received standard treatment while hospitalized and were monitored daily according to a clinical scoring system and diagnostic evaluation for 11 days. among the 39 dogs, 17 (ala-gln-treated group [n 5 8] and placebo group [n 5 9]) were vaccinated and 22 (ala-gln-treated group [n 5 12] and placebo group [n 5 10]) were not vaccinated. the population consisted of 29 purebreds and 10 mixed breed dogs, with a mean age of 12.2 ae 2.2 weeks. the survival data were compared statistically by means of a log-rank test for the kaplan-meier survival curves. the clinical scores of ala-gln-treated dogs improved significantly relative to the placebo group. there was a significant difference between the two groups in the survival distribution (p 5 0.038); specifically, 3 of the ala-gln-treated dogs (15.0%) died, whereas 8 of the dogs in the placebo group (42.1%) died. no side effects were associated with the administration of ala-gln. these results suggest that the oral administration of ala-gln is effective in improving clinical signs and survival rate in dogs with parvoviral enteritis. bleeding disorders, thrombocytopenia and alterations in platelet function have been documented in humans receiving lipid-containing parenteral nutrition formulations. despite a lack of evidence in the veterinary literature, it is believed that parenteral lipids are contraindicated in critical illness when the development of bleeding disorders is likely. the objective of this study was to determine if there is an in vitro effect on platelet function and thromboelastography (teg) in normal dogs with varying concentrations of a 20% soybean oil emulsion (intralipid s ). twelve clinically healthy dogs were used for this study. whole blood platelet aggregation, using adp and collagen agonists, was measured using multiple electrode aggregometry in hirudinated blood with final lipid concentrations of 0, 1, 10, and 30 mg/ml. the teg parameters r, k, a-angle, and maximum amplitude (ma) were evaluated from citrated whole blood with equivalent final lipid concentrations as platelet aggregation. there was no significant difference between groups with collageninduced platelet aggregation. there was a significant increase in the area under the curve (auc) with adp-induced aggregation at a lipid concentration of 30 mg/ml (p 5 0.027). the ma was significantly reduced at both the 10 mg/ml (p o 0.001) and 30 mg/ml (p o 0.001) lipid concentration. there was no statistical difference between groups evaluating the other teg parameters. while platelet aggregation appeared enhanced at the highest concentration evaluated, this concentration is not clinically relevant. the reduction in ma seems discordant but both fibrinogen and platelets contribute to the ma. therefore the higher lipid concentrations may be interfering with fibrinogen kinetics or fibrinogenplatelet interaction. in vivo studies are indicated to determine if any of these changes are clinically significant. rosiglitazone is a peroxisome proliferator-activated receptor gamma (pparg) agonist and an fda-approved anti-diabetic agent in humans that has been investigated for its ability to reduce tumor cell growth. specifically, the combination of rosiglitazone and carboplatin has demonstrated enhanced tumor control. the purpose of this study was to determine the peak plasma concentrations and side effect profile of rosiglitazone after oral administration in dogs with spontaneously occurring cancer. all dogs received carboplatin intravenously concurrently with oral rosiglitazone. ten cancer-bearing dogs with normal pre-treatment hepatic and renal function were enrolled. complete pre-treatment hematological and biochemical parameters were available in ten dogs and post-treatment parameters in nine dogs. peak plasma concentrations varied with dose and ranged from 150.3-959.4 ng/ml and occurred between 30 minutes and 4 hours post administration and rapidly declined after the peak. the dose limiting toxicity was hepatic at a dose of 9 mg/m 2 . there was one grade iii, two grade i alt, and one grade iii ast elevations noted. no changes in total bilirubin, alkaline phosphatase, or ggt values were noted. blood glucose values remained within normal limits. mild, self-limiting gastrointestinal and hematologic toxicities were observed when rosiglitazone was administered in combination with carboplatin. based on this study, the recommended dose of rosiglitazone in cancer-bearing dogs with normal hepatic function is 6 mg/m 2 orally once daily. side effects of the combination appear similar to side effects noted with carboplatin alone. further study is needed to determine efficacy of this combination and if more frequent dosing is required to maintain plasma concentrations. carboplatin has shown little activity as a single agent for the treatment of canine transitional cell carcinoma (tcc). however, gemcitabine has shown synergism with carboplatin in human cell lines. the purpose of this study was to evaluate the activity of gemcitabine against canine tcc cell lines alone or in combination with carboplatin. we hypothesized that gemcitabine in combination with carboplatin would have synergistic effects in vitro. the results of this study could provide a rationale for treatment of canine tcc with the combination of these drugs. tcc cell lines tcc-kiss, tcc-knapp-js, tcc-axa, tcc-hxc, and tcc-sh were treated with gemcitabine, carboplatin, or the combination. cell proliferation was assessed using cyquant assay, cell cycle was evaluated using propidium iodide staining, and apoptosis was assessed by measuring caspase-3/7 activation. synergy was quantified by combination index analysis using compusyn software. treatment of canine tcc cell lines with carboplatin or gemcitabine decreased cell proliferation, induced cell cycle arrest, and apoptosis. when tcc cell lines were treated with gemcitabine and carboplatin in combination at a therapeutically relevant concentration (gemcitabine o 100um, carboplatin o 250um), a significant decrease in cell proliferation was observed compared to gemcitabine or carboplatin alone, and the drug combination was synergistic in 3 of 5 cell lines, and additive in the remaining 2 lines. gemcitabine exhibits biologic activity against canine tcc cell lines and carboplatin combined with gemcitabine exhibits synergistic activity at biologically relevant concentrations. our results support further evaluation of these drugs in dogs with tcc to determine the clinical efficacy of this combination. metronomic chemotherapy has been shown in murine models and humans to improve tumor control by inhibiting tumor angiogenesis and suppressing regulatory t cells (treg). treg are a subset of t lymphocytes demonstrated to be increased in humans and dogs with cancer and are thought to suppress cellular immune responses against tumors. the purpose of this study was to determine whether metronomic cyclophosphamide therapy depletes treg and/or exhibits antiangiogenic activity in dogs with soft tissue sarcoma. client owned dogs with histologically confirmed grade i or ii soft tissue sarcoma were administered cyclophosphamide at 12.5 mg/m 2 or 15 mg/m 2 orally once daily for 28 days. whole blood and tumor biopsies were obtained on days 0, 14, and 28. flow cytometric analysis of blood was performed to assess changes in t lymphocyte subsets, including cd4 1 and cd8 1 cells as well as cd4 1 foxp3 1 treg. tumor microvessel density (mvd) was assessed by performing immunohistochemistry for cd146. five dogs were enrolled in the 12.5 mg/m 2 /day dose cohort and six dogs were enrolled in the 15.0 mg/m 2 /day dose cohort. in patients that received cyclophosphamide at 12.5 mg/m 2 /day, the mean number of treg decreased from day 0 to 28 but there was no change in the mean percentage of treg or mvd. for patients that received 15.0 mg/m 2 /day, both the mean number and percent of treg as well as mvd decreased over the 28 day time period. cyclophosphamide at 15.0 mg/m 2 /day or greater selectively depletes treg and inhibits angiogenesis in dogs with soft tissue sarcoma. arsenic trioxide (ato) is used to treat leukemias, multiple myeloma, and relapsed lymphoid malignancies in humans; its use has not been explored in veterinary oncology. prior therapy with glucocorticoids decreases likelihood and duration of remission for dogs with lymphoma treated with chemotherapy. we hypothesized that ato will re-sensitize glucocorticoid-resistant canine lymphoma cells to glucocorticoid-induced apoptotic death. the osw canine lymphoma cell line was cultured with 200 um dexamethasone. remaining viable dividing cells were considered resistant. resistant cells were exposed to 0.01 um and 0.02 um of ato without dexamethasone, after which cells were washed and re-exposed to 200 um dexamethasone. after 24, 48 and 72 hours of dexamethasone exposure, cells were counted using trypan blue stain. apoptosis was assessed by tunel assays on cytospin preparations collected at 24, 48, and 72 hours from ato-exposed and control groups. statistical analysis was performed using 1 way anova and tukey's test. the proportion of dead cells increased over time in both 0.01 um and 0.02 um ato exposed groups. the proportion of dead cells was greater for 0.01 um ato (p o 0.001) and 0.02 um (p o 0.001) groups compared to control. apoptosis increased with increasing ato concentration and duration of dexamethasone exposure compared to control. these results support the effectiveness of ato at re-sensitizing glucocorticoid-resistant canine lymphoma cells to apoptotic death following re-exposure to glucocorticoids. ongoing gene expression studies aim to elucidate this mechanism. additional studies to determine if this effect is seen with other chemotherapeutic agents are warranted. lymphoma is the most common hematopoietic tumor of dogs. protein disturbances may be associated with this disease including monoclonal gammopathies in a low percentage of cases. serum protein electrophoresis (spe) is routinely used to aid diagnosis of various canine diseases including lymphoma when total protein concentration is elevated. the purpose of this study was to compare spe changes in lymphoma patients without elevated total proteins with a population of healthy dogs. agarose gel electrophoresis was performed on residual serum from 17 healthy control dogs and 21 untreated dogs with multicentric lymphoma (stage iii -v) after measuring total protein (tp) using the biuret method. densitometric traces of the protein bands were obtained using computer software (totallab 100) and the albumin, alpha-1, alpha-2, beta-2 and gamma globulin subfractions were identified by visual inspection. the total protein concentration, the number of subfractions and the relative and absolute protein subfraction concentrations were then compared statistically between the two populations. in lymphoma dogs, tp, absolute albumin, beta-2 and gamma globulin concentrations and both relative and absolute concentrations of the alpha-1 globulins were significantly lower however relative and absolute alpha-2 globulin concentrations were significantly elevated. no monoclonal gammopathies were identified in any of the dogs and not every patient with lymphoma had the above changes in their electrophoretogram. this study has demonstrated that significant changes occur in the albumin and globulin fractions of canine lymphoma patients despite no obvious increase in tp. further investigation is required to identify the proteins responsible for these changes. it is well known that immunophenotype has a prognostic value for the outcome of canine lymphoma, with t-cell lymphomas having a worse prognosis than b-cell lymphomas. the recent advent of flowcytometric techniques allowed easy detection of many different markers on lymphoma cells and therefore, not only distinguish between t and b cells, but also estimate possible aberration on immunophenotype. in human oncology, although some controversy persists, it seems that non-hodgkins lymphoma and acute leukemia carrying aberrations have a worse prognosis. the aim of this study was to evaluate the role of immunophenotype aberration in canine high-grade lymphoma considering outcome and time span to achieve complete response under chemotherapy. samples of bone marrow, blood and lymph node suspensions from twentythree dogs were evaluated with flow-cytometry. eleven dogs had aberrant expression of neoplastic lymphocytes and twelve were non-aberrant. the most common aberrations found were: positivity to cd34, biphenotypes, double expression of tantigens (cd41, cd81), diminished expression of cd45. all dogs were treated with a chop-based protocol. there was a significant difference for the time to achieve response to chemotherapy (partial or complete). 12/12 non aberrant lymphomas went into cr or pr after the first treatment (l-asparaginase), while aberrant lympho-mas needed more than 2 treatment to reach cr or pr. there was a trend for a prolonged disease free interval with non-aberrant versus aberrant, although it was not statistically significant. aberration of immunophenotype may be a prognostic factor for canine lymphomas, but further studies with larger groups are needed. class ii major histocompatibility expression is a significant and independent predictor of prognosis in human b cell lymphoma. low class ii mhc is consistently associated with poorer outcome. the mechanism underlying this relationship is not clear, but one hypothesis is that high class ii mhc allows for better antigen presentation and tumor-specific immune responses. in the this study, we investigated whether that class ii mhc expression in canine b cell lymphoma was associated with remission and survival times. a total of 160 patients were categorized by level of class ii mhc,expression of cd34 and cell size for on neoplastic b cells. multivariable cox-proportional hazard analysis was used investigate this research question using a randomly selected subset of the data, and the predictive ability of this model was validated on the remaining 1/3 of patient data. results suggested that low class ii mhc expression was associated with decreased times to relapse and death as is seen in human b cell lymphoma, and that large neoplastic cells were associated with decreased survival time. cd34 expression was not associated with patient outcomes. these findings have implications for the use of dogs to model human lymphomas, for the study of tumor vaccines, and for prediction of mortality in dogs with b cell lymphoma with a high level of specificity. one of the reasons for the failure of canine lymphoma treatment is related to the resistance of tumor cells against chemotherapy drugs. the major form of this resistance is provide by multidrug resistance abc transporters. abc transporters proteins comprise a large superfamily of transmembrane proteins, atp-dependent, that extrude a large variety of drugs from the cells. multidrug resistance phenotype in cancer cells is associated with overexpression of these transmembrane proteins. abcg2, also known as bcrp, is a 655 residue half-transporter protein that protect hematopoetic stem cells against toxic compounds. the aim of this study was to investigate the expression of bcrp (abcg2) in canine multicentric lymphoma. samples were collected by fine needle aspiration of an enlarged lymph nodes, from 25 dogs with multicentric lymphoma (stage iii to v) at diagnosis, and 8 normal lymph nodes (control). dogs that were previously treated with prednisone or chemotherapy were excluded from the study. quantitative rt-pcr was used to measure the mrna expression level of bcrp and flnb expression as a endogenous reference canine gene. a widely range expression value for abcg2 expression was found for canine multicentric lymphoma. high gene expression was observed in 52% (13/25) canine lymphoma, but 48% of dogs had a lower expression when compared with normal lymph node. gene expression was not associated with clinical staging, complete or partial remission, relapse and survival time. in conclusion, abcg2 was expressed in canine lymph node and canine multicentric lymphoma at the diagnosis, and it was not correlated with clinical response. osteosarcoma (osa), the most frequent primary malignant bone tumor of dogs, is both locally aggressive and highly metastatic. prognostic factors for canine osa include tumor location, distant metastatic disease, and serum alkaline phosphatase (alp) concentration. an increased serum alp concentration is associated with poor prognosis; however the mechanisms underlying this phenomenon are currently unclear. during normal bone development alp may be used as a marker for osteoblasts. additionally, alp is a downstream target of activated canonical wnt/b-catenin signaling. therefore, we hypothesized that increased serum alp would be associated with increased expression of b-catenin in canine osa. the goals of this study were: (1) characterize and compare cellular alp expression in osa tissue from patients with normal and high serum alp; and (2) assess b-catenin expression in those same patient populations. we used frozen osa samples collected from patients with either high alp (n 5 3) or normal alp (n 5 3). total rna was isolated from the frozen tissue, converted to cdna, and analyzed using quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) with either target gene alp (aim 1), or target gene b-catenin (aim 2). additionally, b-catenin expression was analyzed by western blot. qpcr data for bcatenin and alp expression were normalized to 18s, and relative expression was calculated by the ddct method. the relative expression of cellular alp was higher in high serum alp samples compared to normal serum alp samples: 44.74 ae 22.04 (mean relative expression ae standard deviation; p o 0.001). further, the relative expression of b-catenin was also increased; b-catenin expression of high serum alp samples relative to low serum alp samples was 24.57 ae 11.41 (p o 0.001), which is also seen by western blot. this study begins to clarify the mechanism behind high serum alp in canine osa, and suggests the wnt signaling pathway may be active in this population of patients. further work will focus on elucidating the role active wnt signaling plays in the biology of osa. in the future, serum alp status of osa patients may help identify patients that would benefit from therapies targeting this pathway. accurate assessment of abdominal lymph node status is of vital importance for appropriate treatment planning and determining prognosis in dogs with apocrine gland adenocarcinoma of the anal sac (agaas). pretreatment knowledge of lymph node status is helpful for determining prognosis and planning the optimal extent of lymphadenectomy. in addition, pretreatment knowledge of lymph node status may help in selecting patients who might benefit from adjuvant chemotherapy and radiation therapy. abdominal ultrasound is currently the most commonly employed test to screen for abdominal lymphadenopathy in dogs with agaas. imaging studies in people indicate that magnetic resonance imaging ( to determine and compare the plasma concentration of cyclophosphamide and its metabolite 4-ohcp, within the plasma of lymphoma bearing dogs being treated with either oral or intravenous cyclophosphamide. in this prospective study, patients were randomly assigned to either receive oral or intravenous cyclophosphamide, at a dose of 250 mg/m2. based on a priori power calculation eight patients per treatment group were enrolled. plasma was obtained at times 0, 15, 30, 60 minutes, and then at 2, 4, 6, 8, 24 hours post administration for evaluation of 4-ohcp concentrations by liquid chromatography-dual mass spectrometry (lc/ms/ms). average values were obtained for both cyclophosphamide and 4-ohcp concentrations within the plasma of both groups. the following values were obtained, half life (hl), time to maximum concentration (tmax), maximum concentration (cmax), and area under the curve (auc). the mann-whitney statistical test was used to compare the groups. the auc for cyclophosphamide was statistically significant (p o 0.05) when compared between the two groups. the auc for 4-ohcp was not statistically significant between the groups. the difference between cmax for cyclophosphamide and 4-ohcp was statistically significantly (p o 0.05) between the groups. although the auc for cyclophosphamide was statistically significant between the two groups, the auc for the active metabolite 4-ohcp was not different when administered intravenously or orally. thus drug exposure to the active metabolite of cyclophosphamide is the same when administered intravenously or orally. previously the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g3 power driver by a wide spectrum of novice participants in feline cadavers were evaluated. novice users' mean io catheter insertion time using the ez-io g3 driver was also compared to the mean iv catheter insertion time in normovolemic feline and canine patients presented to the western college of veterinary medicine (wcvm) small animal hospital. novice users included 40 wcvm personnel (8 technicians, 11 veterinary students, 5 interns, 8 residents, 8 clinicians). after watching a 5-minute ez-io g3 training video, each participant inserted 3 io catheters using the ez-io g3 driver. site (proximal humerus or trochanteric fossa of the femur) and side of cat (right or left) were randomized for each attempt for each participant. a 15 gauge x 15 mm long needle and a 15 gauge x 25 mm needle were used for io catheter insertion in the humerus and femur, respectively. participants then graded the ''ease of use'' of the ez-io g3 device on a visual analog scale (vas) that was converted to a 5-point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by wcvm small animal hospital personnel (6 technicians, 16 veterinary students, 1 intern, 1 resident, 2 clinicians) were then timed and compared to the mean io catheter insertion time in feline cadavers by study participants using the ez-io g3 device. the io catheter was inserted correctly on every attempt by 70% (28/40) of participants. no difference was found between participant groups for mean io catheter placement confirmation percentage (p 5 0.72). percentage of io catheter ''slippage off the bone'' at the time of placement did not vary across participant groups (p 5 0.12). mean io catheter insertion times were all less than 10 seconds and did not differ significantly as a function of attempt number (p 5 0.93) or as a function of participant group (p 5 0.78). participants rated the ez-io g3's ''ease of use'' favorably and subjective scores did not differ across participant groups with varying levels of clinical experience (p 4 0.2). compared to the mean insertion time for iv catheterization (188 sec), mean io catheter insertion by participants using the ez-io g3 (8 sec) was significantly faster (p 5 0.001). regardless of their level of clinical experience, participants rated the ez-io g3 device favorably in terms of its ''ease of use'' and their willingness to use the device in the future. regardless of their level of clinical experience, study participants successfully placed io catheters using the ez-io g3 device and did so significantly faster than the reported iv catheter insertion time in normovolemic feline and canine patients in the wcvm small animal hospital. intraosseous catheterization using the ez-io g3 has the potential to provide very rapid vascular access and is a skill that can be easily learned. previously presented at the western college of veterinary medicine undergraduate poster competition. multicavitary effusion is a common cause of presentation for dogs to emergency medical centers. the goal of this study was to identify common underlying causes of multicavitary effusion as well as determine their relative importance. a retrospective analysis of 43 cases of multicavitary effusion admitted to the icu of a tertiary referral center (ontario veterinary college) from 2007 to 2010 was performed. twenty-three different breeds, with golden and labrador retrievers (25.6% and 14%, respectively) being most commonly seen, were included in the study. ages ranged from 2 to 14 years with a median age of 9 years and a mean of 8.1 years. 69.8% of cases were males (30/43 cases). most common presenting signs included lethargy (48.8%), anorexia (32.6%), vomiting (21%) and dyspnea (21%). cavitary effusion was detected by either ultrasonography (pericardial, pleural or abdominal) or radiographs (pleural). bicavitary effusion was present in 28 cases (65.1%) whereas 15 cases (34.9%) had tricavitary effusion. neoplasia was found to be the most common underlying cause overall (51.2%), with hemangiosarcoma being the leading type (40.9% of neoplasia cases), followed by congestive heart failure (9.3%), gastrointestinal lymphangectasia (4.7%), peritonitis/pancreatitis (4.7%), cirrhotic liver disease (2.3%) and acute renal failure (2.3%). in 11 cases (25.6%), no underlying cause could be found. of these, 4 (9.3% of all cases) were diagnosed as having idiopathic pericardial effusion. taken together, these findings suggest a strong association between multicavitary effusion and diseases carrying a guarded prognosis in dogs. infection control practices in veterinary clinics and hospitals are becoming increasingly important, with rising client expectations, growing concern about the spread of antimicrobial-resistant pathogens, and the potential for zoonotic transmission of disease. surgical patients are at increased risk of developing infections, and can serve as sources of these pathogens for other animals and people with whom they have contact within and outside the clinic. taking all reasonable precautions to reduce the risk of surgical site infections, beginning with preoperative preparation of the surgeon and patient, is therefore an important part of any infection control program. while guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these guidelines in veterinary practices. the objectives of this pilot study were to describe a range of preoperative hand scrub and surgical site preparation practices in veterinary clinics, and to determine if there were any areas that consistently require improvement. observation of preparation practices was performed in each of ten clinics over 9-14 days using 2-3 small wireless surveillance cameras. data was coded for 148 surgical patients, and 31 surgeons performing a total of 190 hand scrubs. patient hair removal was most commonly performed after induction of the animal (129/140, 92%) and using clippers (114/137, 83%) . steps in surgical site aseptic preparation ranged from 1-4. contact time with soap ranged from 18-369s (mean 82s, median 53s), and with alcohol from 3-220s (mean 41s, median 30.5s). application of alcohol or antiseptic using a ''cleanest to dirtiest'' pattern was infrequent (29/66 (44%) and 11/83 (13%), respectively). potential contamination of the surgical site occurred most frequently when the animal was moved to the surgery table after initial preparation (40/58, 69%). preoperative alcohol hand rub was used in 2/10 facilities, but soap and water hand scrub was still more commonly used even at these clinics. proximal-to-distal scrubbing was noted in 95/142 (67%) of soap and water scrubs. contact time during surgeon hand preparation ranged from 7-529s (mean 144s, median 124s) for soap and water and from 4-123s (mean 34s, median 25s) for alcohol-based hand rub. approximately 75% of the variation in contact time was due to inter-surgeon variation. no significant changes in practices were identified over the course of the observation period. some preoperative preparation practices were fairly consistent between clinics in this study, while others varied considerably. contact times with preparatory solutions were often far shorter than recommended, and there was a high frequency of non-sterile contact with the surgical site during movement of patients to the surgical suite. the camera system used to perform this study did not have a significant time-dependent effect on the behavior of participants, and could be useful for performing similar field-based observational studies in the future. this prospective randomized study compared the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g3 power driver to manual io catheterization in feline cadavers. the io catheter insertion time in cadavers using the ez-io g3 device was also compared to iv catheter insertion time in normovolemic feline and canine patients. after a purposely limited training period, a preclinical veterinary student was timed and video-recorded as she performed 20 io catheter placements in feline cadavers (10 io insertions by placing an illinois needle manually and 10 io insertions using the ez-io g3). order of technique (manual or ez-io g3), site of io placement (proximal humerus or trochanteric fossa of the femur), and side of cat (right or left) were randomized for each attempt. when using the ez-io g3, a 15 gauge x 15 mm long needle and a 15 gauge x 25 mm needle were used for io catheter insertion in the humerus and femur, respectively. after each attempt, the student graded the ''ease of use'' of each technique on a visual analog scale (vas) that was converted to a 5-point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by western college of veterinary medicine (wcvm) small animal hospital personnel (6 technicians, 16 veterinary students, 1 intern, 1 resident, 2 clinicians) were then timed and compared to the student's mean io catheter insertion time using the ez-io g3.median io catheter insertion times for the 2 techniques were significantly different (manual io technique 24 sec; ez-io g3 5 sec) (p o 0.001); the manual method took 48 seconds longer (95% confidence interval of 28 to 67 seconds) than the ez-io g3 method. insertion time was more variable for the manual technique than for the ez-io g3. percentage of catheter ''slippage off the bone'' and extravasation around the inserted catheter were significantly higher for placement of the manual io catheter compared with placement of the ez-io g3 catheter (p o 0.001). student's subjective ratings were more favorable and more consistent for the ez-io g3 technique compared to the manual technique for io catheter insertion. compared to the mean insertion time for iv catheterization in the wcvm small animal hospital, io catheter insertion by the student using the ez-io g3 was significantly faster (iv catheter 188 sec; ez-io g3 io catheter 7 sec) (p o 0.001). intraosseous catheter insertion using the ez-io g3 can be said to be significantly faster, less traumatic, more user-friendly, and as effective as io catheter placement using the manual technique. vascular access via io catheter insertion using the ez-io g3 device may be suggested to be faster than iv catheter insertion. previously presented at the western college of veterinary medicine undergraduate poster competition. computed tomography (ct) has been widely investigated and applied as a means for non-invasive quantitative bone mineral determination in human medicine. the aim of this study was to assess age-related changes and anatomic variation in bone mineral density (bmd) using quantitative ct in normal cats. seventeen normal cats were included in this study and divided into the following 3 age groups: o 1 year (n 5 4); 2-5 years (n 5 10); and 4 6 years (n 5 3). a computed tomographic scan of each vertebra from the 12 th thoracic to the 7 th lumbar spine, and the pelvis, was performed with a bone-density phantom (50, 100, and 150 mg/cm 3 , calcium hydroxyapatite, cirs phantom s ). on the central transverse section, the elliptical region of interest (roi) was drawn to measure the mean hounsfield unit value. those values were converted to equivalent bmd by use of the bone-density phantom and linear regression analysis (r 2 4 0.95). the mean bmd value of the thoracic vertebrae (651.3 ae 100.4 mg/cm 3 ) was significantly higher than of the lumbar vertebrae (520 ae 119.5 mg/cm 3 ). the maximum bmd occurred at the t12, t13, and l1 levels in all age groups. there was a statistically significant difference in the mean bmd value among the 3 age groups at the t12 (p o 0.001), t13 (p o 0.001), and l4 levels (p 5 0.013), respectively. in addition, there was no significant difference between the mean bmd value of the left and right iliac bodies (485.1 ae 140.4 mg/cm 3 and 482.1 ae 148.2 mg/cm 3 , respectively). the present study suggests that age-related changes and anatomic variation in bmd values should be considered when assessing bmd using quantitative ct in cats with bone disorders. dynamic contrast-enhanced computed tomography (dce-ct) is a rapid and widely available method of cerebral perfusion imaging. however, there is no established reference value of cerebral blood flow (cbf) measured by dce-ct according to a dog's age. the purpose of this study was to identify the correlation between regional cbf and aging in clinically normal dogs using dce-ct. fourteen dogs with no evidence of hemodynamic disorders and central nervous system dysfunction were included in this study. dogs were assigned to the following 3 age groups: o 1 year (group 1); 3-6 years (group 2); and o 10 years (group 3). dce-ct scans were performed at the level of the third ventricle and mesencephalic aqueduct. cbf in the gray and white matter was calculated using stroketool-ct s software. the overall mean ae standard deviation quantitative estimate for regional cbf in clinically normal dogs was 67.8 ae 12.9 ml/min/ 100 g, 44.7 ae 4.1 ml/min/100 g, and 31.0 ae 3.4 ml/min/100 g in groups 1, 2, and 3, respectively. there was no significant regional cbf difference between the right and left sides of the brain in each group. also, a statistically significant difference in the regional cbf was observed between groups 2 and 3 (p o 0.001). thus, aging affects the regional cbf in normal dogs and the values should be considered assessing the results of dce-ct. according to several clinical behavior guidelines, ''toileting'' type inappropriate urination (i.e. large amounts of urine deposited in horizontal surfaces) can arise in cats suffering from a medical problem (typically lower urinary tract disease). by contrast, ''spraying'' type behaviour (i.e. possibly smaller amounts of urine deposited on vertical areas) is more typically associated with anxiety brought about by a threat to local resources, arising from either a change in the physical environment or threat to these resources from another cat. however, there is some evidence that ''sprayers'' may also be presented with a medical problem, which might be linked to the disease (e.g. painful voiding associated with crystalluria may lead to a standing posture being adopted and small amounts eliminated at a given time). this might be associated with an apprehensive state or simply a co-morbid state. as part of a larger research project aimed at investigating behavioral and physical aspects of cats presented with inappropriate urination, owners of 14 ''spraying'' and 18 ''toileting'' cats with appropriate control subjects from the same households were recruited throughout local media coverage and the internet. the case-control dyads were brought by the owners to the veterinary hospital of the university of sa˜o paulo, at the same time, for a medical work-up (i.e. physical examination, complete blood count, biochemical profile, urinalysis, urine culture and abdominal ultrasound). no significant differences between the ''sprayers'' and ''toileters'' regarding the occurrence of medical problems were found. both groups had a similar proportion of cats affected by medical illnesses (sprayers: 35.7%, toileters: 44.4%; chi 2 , p 5 0.618), directly or indirectly relating to the urinary system (e.g. diabetes, chronic kidney disease). in both groups, control cats also had a relatively high occurrence of medical concerns (21.4% and 27.8%, respectively for each control group). these results emphasize the importance of careful medical evaluation of cats presented for a urinary housesoiling problem. the relatively high prevalence of medical concerns among apparently healthy cats in multi-cat households may have arisen, at least in part, as a result of an inability/failure of owners to monitor individuals, thus allowing some early signs to pass unnoticed. the way in which medical and behavioral elements are linked (if at all) remain unknown but deserve further investigation. considered as a semi-social species, domestic cats appear to be highly sensitive to the effects of social stress, especially when living in high density populations. cats are capable of adapting to live ingroup; nonetheless, they do not appreciate living in close proximity with others as result of an environment lacking of great opportunities of escaping and hiding. this study aimed at testing the following hypotheses: (a) owners' perceived quality of life affects cats' global levels of stress; (b) cats' global levels of stress are influenced by cats' personality; (c) cats' living style (single housing versus large group housing) does affect stress levels in cats. to our knowledge, this is the first study investigating stress levels of domestic owned cats, under natural conditions, throughout measurement of faecal glucocorticoids metabolites concentration, and taking into consideration cat personality, cat living style and owner's subjective life quality. in this study, adrenocortical activity, as a valuable physiological indicator of emotional stress, was evaluated throughout the measurement of faecal glucocorticoids metabolites in fourteen single and sixteen in-group housed cats. cat personality as well as owners life quality was evaluated by self reported questionnaires given to the owners to answer. significant differences in mean glucocorticoids metabolites concentrations (mgcm) between the two populations (i.e. single versus in-group cats) were not detected (random effect model, p 5 0.178). however, when mgcm were taken as a function of cat personality, there were differences regarding single catstimid cats showed higher levels in comparison to easy-going (random effect model, p 5 0.018) and bossy (random effect model, p 5 0.020) cats. as to owner subjective life quality, a direct association between the scores given by the owners to the social dimension and mgcm was found for single cats only (i.e. the better the owner felt itself social wise the higher the mgcm of the cat; random effect model, p 5 0.002). social stratification may compensate the stress resulting from spatial restriction in large in-group living cats. other underexplored factors such as feline personality and owner life style seem to play an equally important role in domestic cats' day to day levels of stress, especially in the cats kept as single pets. in dogs, raas activation is a major feature of congestive heart failure (chf). benazepril (fortekor s ) is a potent ace inhibitor with well-documented effectiveness in canine chf. although ace activity (ace a ) has been used in preclinical studies as a surrogate marker of efficacy, some authors have reported a poor correlation between plasma ace a and changes in angiotensin ii (aii) or aldosterone (al). the purpose of this study was to investigate the effect of benazepril on canine plasma renin activity/concentration (pra/prc), angiotensin i (ai), aii, al, and fractional excretion of potassium (ufek), sodium (ufena) and aldosterone (ufeal). sixteen beagle dogs were fed a low-sodium diet and dosed with placebo or benazepril tablets (10 mg po, q24h) for 5 days. blood and urine samples were collected on day 1 (d1) and day 5 (d5) over 24-hour periods. data were analyzed by repeated measures anova of baseline corrected values, and anova of auc 24hours . compared with placebo, benazepril induced a significant increase in pra and ai at d1 (p-value [pra] :0.001, p-value [ai] :0.002) and d5 (p-value [pra] :0.001, p-value [ai] o 0.001). no differences in prc were noticed. based on auc 24hours, aii levels were 34% lower in the benazepril group at d5 (p-value [aii] :0.01). ufeal and al decreased by up to 27% and 24% at d1 and d5, respectively, though differences did not reach statistical significance. benazepril markedly influences raas dynamics in dogs. decreased exposure to aii and al are likely to be the key events required to counteract pathological remodeling of the heart in chf. this study compared two intravenous anesthetic agents, alfaxalone (alf) (alfaxan s , jurox pty. ltd.) and propofol (ppf) (rapinovet s , schering plough animal health) and their effects on spontaneous ventilation after induction of anesthesia in dogs at various doses. this randomized, crossover, dose-escalation study used six dogs in weight and gender-matched pairs (3m-3f). for each drug, each dog was dosed incrementally at 1, 2, 5, 10 and 20 times the labeled anesthetic induction dose rate (alf 2 mg/kg, ppf 6.5 mg/kg) or until a dose was reached that rendered the dog apneic. a minimum of three days was allowed between doses. for each dose administration, the entire calculated dose was delivered constantly over 1 min. the primary variable was apnea, defined as an absence of spontaneous ventilation for 1 minute. apneic dogs were manually ventilated with oxygen until they resumed adequate spontaneous ventilation. once the apneic dose was determined for an individual dog for one drug, the dog began incremental doses with the alternate drug. for each anesthetic episode times were recorded from completion of induction dose to; removal of endotracheal tube, dog lifting head, dog attaining sternal recumbency and dog standing. pulse rate, respiratory rate, spo 2 and etco 2 were each measured every 5 min. within-dog comparisons were made using the paired student's t-test. for both alf and ppf all 6 dogs respired voluntarily at the labeled (1 x) dose. for ppf at 2 and 5 x doses, 4 and 0 dogs respired voluntarily respectively. for alf at 2, 5 and 10 x doses, all 6, 4 and 1 dog respired voluntarily respectively. for all six dogs to become apneic required 5 x dose of ppf and 20 x dose of alf. the mean no observable adverse effect dose (noael) expressed as a multiple of the labeled dose was higher for alf (4.8 x) than for ppf (1.7 x) (p 5 0.035). there were no significant differences between times to extubation, head lift or attaining sternal recumbency after alf and ppf at 1, 2 and 5 x doses. at the 2 x dose, dogs took longer to stand after alf (29.0 ae 7.0 min) than ppf (25.0 ae 8.3 min). we concluded that based on anaesthetic duration, the manufacturer's labeled dose rates of 2 mg/kg for alf and 6.5 mg/kg for ppf were equivalent. however, based on the dose escalation, the number of dogs becoming apneic at each dose-multiple is consistent with ppf having a narrower safety margin, i.e., ppf caused more respiratory depression than alf. parenteral levetiracetam (lev) has been shown to rapidly attain therapeutic levels in dogs when given iv or im, and has been used offlabel for the treatment of seizure emergencies. the purpose of this study was to determine the safety and pharmacokinetics of subcutaneously administered levetiracetam in healthy dogs. potential application of these results would be use of sq lev instead of or in addition to rectal diazepam for the treatment of cluster seizures at home. lev was administered sq between the shoulder blades to 4 healthy, purpose-bred hound dogs, at a dose of 60 mg/kg (undiluted). blood samples were collected at 15, 120 and 420 minutes after lev administration via jugular venipuncture. plasma lev concentrations were measured by high pressure liquid chromatography. none of the dogs became sedated, nor was there pain evident on palpation of the injection site. mean (standard deviation) lev concentration was 65.2 (29.5), 114.5 (10.5) and 84.9 (20.6) mg/ml at 15, 120 and 420 minutes, respectively. administration of sq lev was well tolerated, and exceeded the suggested therapeutic range (5-45 mg/ml) within 15 minutes of administration, and remained above the range for at least 7 hours. these data indicate that sq lev administration may be an alternative for the at-home treatment of cluster seizures in dogs, and prospective studies in epileptic dogs are warranted. the purpose of this study was to assess the effects of cyp inhibitors (ketoconazole, chloramphenicol, fluoxetine, trimethoprim, cimetidine, and medetomidine) in varying combinations on the bioavailability of oral methadone in healthy greyhound dogs. the iacuc approved this study. cyp inhibitors were administered po for 48 hours prior to methadone administration. methadone hydrochloride was administered po at a targeted dose of 1 mg/kg. blood was obtained for the determination of methadone plasma concentrations by mass spectrometry. the area under the curve (auc) of methadone for each treatment group was compared statistically to the auc of methadone administered without inhibitors using the mann-whitney rank sum test. significant increases (p o 0.001) in the methadone auc occurred in all treatment groups which included chloramphenicol, including chloramphenicol as the only inhibitor. the magnitude of increase was at least 50 fold. mean concentrations of methadone exceeded 10 ng/ml for at least 10 hours in all groups administered concurrent chloramphenicol. no significant increases in the auc occurred in any of the groups which did not include chloramphenicol. in conclusion, chloramphenicol significantly inhibits the metabolism of methadone in greyhound dogs. as a result, the oral bioavailability of methadone is significantly increased and plasma concentrations are achieved that are reported to be effective in humans for 10-36 hours after a single oral administration. doxycycline hyclate is used frequently in small animals, horses and exotic animals for treatment of a wide variety of infections. because doxycycline hyclate tablets may not be suitable for oral administration in some animals, particularly horses and cats, it has been compounded into liquid suspensions. the commercially available doxycycline calcium 10 mg/ml oral suspension, vibramycin s (pfizer) is not suitable for use in animals due to its low concentration and flavoring that animals find unpalatable. because of the known inherent instability of doxycline in aqueous vehicles under storage, this study was conducted to determine the potency of two formulations stored in dark and light conditions. a high pressure liquid chromatography (hplc) assay with uv absorption at 350 nm was developed for analyzing doxycycline in formulations, in comparison to a reference standard from the united states pharmacopeia (usp). doxycycline hyclate 100 mg tablets were first tested for potency. the tablets were then crushed and mixed with a pharmaceutical vehicle to make two concentrations: 33.3 mg/ml and 166.7 mg/ml. the vehicle used was a 50:50 mixture of a vehicle for oral solution (ora-sweet, usp-nf) and vehicle for oral suspension (ora-plus, usp-nf). the suspensions were prepared in replicates of 3. each replicate was divided, with one aliquot stored at room temperature in lighted conditions, and the other aliquot stored at room temperature in the dark. doxycycline was extracted from the formulations and measured by hplc at day 0, 1, 4, 7, 14, 21, and 28 . each replicate was tested and the potency reported as the percent doxycycline relative to the usp reference standard. on day 0, 1, 4, and 7, the potency of each formulation was within 90-110% of the reference standard (range 93.4-109%). this value is within the accepted range cited in usp o 795 4 on pharmaceutical compounding-non-sterile preparations. however, starting at day 14, the potency declined dramatically and remained low for the tests performed on day 21 and 28. the potency on day 14, 21, and 28 was below 20% of the reference standard (range 14-18%). there was also a noticeable change in the quality of the formulation starting on day 14, and a change in the color of the formulation to a dark brown. these results indicate that when doxycycline hyclate tablets are compounded as a suspension in an aqueous vehicle as described in this study, at 33.3 and 166.7 mg/ml under the storage conditions used in this study, potency of the formulation cannot be assured beyond 7 days. we recommend a beyond-use-day (bud) of 7 days for formulations prepared and stored at room temperature in light or dark conditions. therapeutic options for multidrug resistance (mdr) escherichia coli urinary tract infections (uti) are limited. fosfomycin (fos) tromethamine is an oral, broad-spectrum, cell-wall active, bactericidal drug approved for treatment of uncomplicated uti in humans. the purpose of this study was to determine time dependency of fos and the disposition of fos tromethamine in dogs. using a randomized, double crossover design, 12 client-owned dogs received fos sodium iv (40 mg/kg) and fos tromethamine (po, 80 mg/kg) either with (n 5 6) or without food (n 5 6). serum and urine were collected for 24 hr; fos was quantitated with a bioassay (atcc e. coli 25922, serum or atcc proteus vulgaris 13315, urine). in-vitro killing curves (cell counts through 24 hours) were performed at 0 (control),0.5,1,2,8,16 and 32 x mic for mdr e. coli canine fos susceptible (e-test s ) uropathogens. killing curves indicated fos to be time dependent. after iv administration, clearance (mlãkg/hr), volume of distribution (l/kg), elimination half-life (hl; hr) and mean residence time (mrt, hr) were (mean ae sd): 210 ae 104, 0.23 ae 0.15, 0.36 ae 0.19, 1.14 ae 0.35 and 1.7 ae 0.4, respectively. for po, c max , hl and mrt were 66 ae 21, 2.5 ae 1.09 and 5.1 ae 1.7, respectively. serum fos exceeded the mic 90 reported for multidrug resistant (mdr) e. coli (1.5 mg/ml) for 7hr (iv; 2.5 mg/ml) and 12hr (po, 9 mg/ml diminazene is an aromatic diamidine, anti-protozoal drug that has shown promise in a small number of cases of cytauxzoonosis. in a noncontrolled case series, 5 of 6 cats with clinical cytauxzoonosis given 3 mg/ kg of diminazene aceturate survived infection. dosage frequency was two intramuscular injections given one week apart. commercial formulations contain the diminazene diaceturate salt. the active base is diminazene with the salt consisting of two aceturate molecules. currently there is no data available on the pharmacokinetics of either diminazene compound in cats. the objective of this study was to determine the pharmacokinetics of diminazene diaceturate in healthy cats. four purpose bred cats with normal physical examination, cbc, chemistry and urinalysis were used. a powdered commercial drug formulation (veriben s , ceva sanet animale) was freshly reconstituted with sterile water to a concentration of 7 mg/ml prior to administration and sterile filtered solution. heparinized blood samples were collected just before (hour 0) or 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, 120 , and 168 hours after intramuscular administration of 3 mg/kg (1.68 mg/kg of diminazene base) diminazene diaceturate. the plasma was separated by centrifugation within 30 minutes of collection and frozen (à801c) until analysis. concentrations of diminazene were measured by hplc analysis using uv absorption and ion-pairing conditions. the pharmacokinetic profile was analyzed using a simple one-compartment model. in these cats, diminazene had a mean terminal half life (t 1/2 ) of 1.70 (1/-0.29) hrs and mean peak plasma concentration (c max ) 0.51 (1/à 0.11) mg/ml. the mean residence time (mrt) of diminazene was 2.45 hrs (1/à0.42). systemic clearance (cl/f) was 1.38 (1/à 0.26) l/kg/hr. the volume of distribution per fraction absorbed (vd/f) was 3.36 (1/à 0.72) l/kg. a single intramuscular dose of diminazene diaceturate was well tolerated by all 4 cats. without knowing the concentration required to inhibit or kill cytauxzoon felis, it is not yet possible to make suggestions regarding optimum dosing schedules for this drug. additional toxicology data and studies to assess clinical efficacy for the treatment of cytauxzoonosis are indicated before routine clinical use can be considered. meloxicam has been shown to accumulate in areas of inflammation in both the rat and human. the objective of this study was to investigate the concentration of meloxicam in synovial fluid of inflamed joints versus that of non-inflamed joints in dogs. eight male dogs were treated with 0.2 mg/kg of meloxicam on day one and 0.1 mg/kg of meloxicam on day two. all treatments were administered orally. on day three reversible acute synovitis was induced in one stifle by aseptic, intra-articular administration of 1 ml sodium urate crystal suspension (10 mg/ml). in four dogs synovitis was induced in the l stifle and in four dogs the same procedure was used in the r stifle. in each dog the stifle without induction of synovitis served as the ''normal'' joint sample. a synovial fluid sample was collected from both the r and l stifle of each dog. sample collection occurred eight hours after administration of sodium urate and twenty four hours after the last administration of meloxicam. synovial meloxicam concentration was analysed using high performance liquid chromatography-mass spectrometry (hplc/ ms-ms). the concentration of meloxicam in the inflamed versus non-inflamed joint in each dog was compared using the paired t-test. the results indicate that meloxicam preferentially accumulates in inflamed joints in the dog as meloxicam concentrations are statistically significantly higher in inflamed joints than in non-inflamed joints. no national surveillance system exists for monitoring emergent resistance in companion animals. however, e. coli resistance is an increasing therapeutic and public health concern in these in dogs and cats. the purpose of this study was to describe current resistance patterns of canine and feline pathogenic e. coli throughout the united states and identify risk factors of antimicrobial resistance. isolates (n 5 1512) of clinical e. coli collected from dogs or cats from may 2008 through may 2010 located in 6 different regions. susceptibility was determined to 15 drugs (6 drug classes) by broth microdilution methods. pharmacodyamaic statistics were described regionally. phenotypes were determined and type of resistance was based on the number of drug classes to which resistance was expressed: none (ndr), single (sdr) and multi (mdr). the majority of isolates were from urinary tract (71.5%) and dogs (75.7%). the proportion of resistance type for each drug was: ndr (17.5%), sdr (56.4%) and mdr (26.12%). the proportion of mdr was greatest in the southwest (25.29%) and least in the northwest (9.19%) (p o 0.05). for all regions, the proportion of resistance was: cephalothin (cph, 65.2%) 4 amoxicillin-clavulanic acid (amx, 59.4%), ampicillin (amp, 52.9%), tricarcillin-clavulanic acid (tcx, 21.8%), doxycyline (dxy, 15.9%) 4 cefoxitin (cfx, 15.2%), cefpodoxime (cpx, 13.7%), chloramphenicol (chp, 13.2%), enrofloxacin (enr, 13.0%) , ciprofloxacin (cif,12.3%), trimethoprim-sulfamethoxazole (tmx, 10.7%), ceftazidime(cfz, 10.0%), gentamicin (gtm, 10.0%), cefotaxime (cft, 9.2%) 4 meropenem (1.1%) (p o 0.05). the mic 90 exceeded the resistant breakpoint for amp, amx, cpx, cph, cif, cfx, dxy and enr whereas mic 50 did not surpass the susceptible breakpoint. beta-lactams (96.37%) was the most and aminoglycosides the least (0.12%) sdr. the drug class most frequently involved in mdr was beta-lactams (97.72%) and least, gen (30.13%). resistance differs regionally, being greatest in the southwest. cph is the most and meropenam is the drug least associated with resistance; these patterns are consistent with current drugs used by veterinarians. the fluoroquinolones (fqs) are common choices for treatment of e. coli urinary tract infections (utis) in animals and humans. 2nd generation drugs approved in animals include enrofloxacin (enr), marbofloxacin (mar), orbifloxacin (orb); human drugs include ciprofloxacin (cip). 3rd and 4th generation fq for humans include moxifloxacin (mox), gatifloxacin (gat) and ofloxacin, (ofl]), its lisoform levofloxacin (lev). for animals, pradofloxacin (pra) is approved for use in europe. the purpose of this study was to assess the in vitro activity of 1st (naladixic acid [nal] through 4th generation fqs (n 5 11) toward dog or cat e.coli uropathogens (n 5 51). isolates were subjected to susceptibility testing to 6 drugs classes (15 drugs). isolate phenotypes included no (ndr; n 5 12), single (sdr; n 5 15) or multidrug (to more than 2 drug classes; mdr; n 5 24) resistance (including enr resistant [enr r -mdr; n 5 12] or enr susceptible (enr s -mdr, n 5 12). the minimum inhibition concentrations (mics) were determined for each isolates using broth microdilution (e. coli atcc s 25922 served as a negative control). mic statistics were generated for each drug among phenotypes. the overall potency (mic 90 ) for all enr susceptible isolates (ndr, sdr and enr s -mdr) was gat 4 pra, mox, mar, lev, cip 4 sar, orb, ofl 4 enr 4 nal. each e. coli isolate expressing ndr or sdr was susceptible to all fq. however, isolates expressing resistance to 1st or 2nd generation fq were also resistance to later generation drugs. glucocorticoids (gc) are standard therapy for allergic asthma but do not reverse the underlying type i hypersensitivity. allergenspecific immunotherapy (asit), a process of ''desensitization'', is potentially curative but requires identification of offending allergens. the purpose of this study was to determine if oral or inhaled gc administered at routinely used dosages would interfere with allergen identification. we hypothesized that oral but not inhaled gc would interfere with accurate identification of allergen-specific ige using skin and serum testing in experimentally asthmatic cats. asthma was induced in eighteen cats using bermuda grass allergen (bga). cats (n 5 6/group) were randomized to receive oral gc (10 mg prednisolone q 24 hr po), inhaled gc (600 ug budesonide q 24hr) or placebo (gelatin capsule q 24hr po) for one month. intradermal skin testing (idst) and bga-specific ige amounts were measured prior to, during (weeks one and four) and every two weeks after treatment until both tests were positive. a paired t test was used to compare serum ige among groups pre-and post-treatment (p o 0.05 significant). idst reactivity was eliminated in 4/6 cats on oral gc, 3/6 on inhaled gc, and 1/6 placebo-treated cats. within two weeks after stopping treatment, idst was again positive in all cats. contrary to our hypothesis, serum ige reactivity to bga was not significantly diminished by any treatment. in conclusion, a two week withdrawal from gcs is adequate for idst identification of allergen but no withdrawal is required prior to serum ige testing to identify the sensitizing allergens. previously in people, increasing severity of asthma is associated with low serum concentrations of 25-hydroxyvitamin d (25-oh-d). 25-oh-d is thought to ameliorate lower airway inflammation primarily by decreasing the production of pro-inflammatory mediators, and by increasing the production of the anti-inflammatory cytokine il-10. in people, serum 25-oh-d concentration is associated with sunlight exposure as well as dietary intake. cats do not rely on sunlight for vitamin d synthesis; all vitamin d comes from dietary intake. cats have a naturally occurring lower airway disease syndrome (lad) that shares many features with human asthma. the goal of this study was to evaluate serum 25-oh-d concentrations in cats with lad. cats with naturally developing lad were enrolled. criteria for a diagnosis of lad included a history of cough, wheeze or respiratory distress, radiographic evidence of a bronchial pattern and hyperinflation, negative heartworm antigen and antibody test, and a resolution of clinical signs in response to glucocorticoids. dietary history was obtained. 25-oh-d concentrations were determined on serum samples by a commercial laboratory. twelve cats with lad were enrolled. all cats ate commercial cat food. the median 25-oh-d concentration was 112 nmol/l with a range of 65-176 nmol/l which is within the reported reference range of 65-170 nmol/l. in contrast to human asthma, lower airway disease in cats is not associated with low serum concentrations of 25-oh-d. interstitial lung diseases (ild) are uncommon in dogs, with the most commonly recognized ild idiopathic pulmonary fibrosis (''westie fibrosis''). in human medicine, ild represent a large umbrella of pulmonary diseases, with ipf only a subset. other, more treatable, ilds are also identified, and may respond to either the removal of a stimulus (hypersensitivity) or steroid therapy. the goal of this report is to describe the clinical course, including outcome, computed tomography and histopathology of dogs affected with an ild. the computed tomography (ct) log was reviewed for dogs that underwent thoracic ct scanning for evaluation of respiratory signs, and had changes consistent with ild as the primary abnormality, including the presence of diffuse disease in all lobes, and at least 2 of the following: reticulation, ground glass opacity, consolidation, or traction bronchiectasis. survival time from ct date was calculated. the presence of moderate pulmonary hypertension [phtn] (4 50 mmhg) as estimated by tricuspid regurgitant jet, was also reported and survival times were compared with a mann-whitney rank sum with p o 0.05 considered significant. thirteen dogs were identified. terriers and chihuahuas were the most commonly affected breeds. two dogs were adolescents, the remaining dogs ranged from 7-16 years, with a median of 11 years. histopathology results (n 5 5), including moderate to severe interstitial fibrosis (3) alveolar proteinosis with fibrosis (1), and interstitial eosinophilic pneumonia (1). one had suspected cryptogenic organizing pneumonia and had a good response to glucocorticoids. eight dogs died of respiratory failure, with a median post ct survival time of 42 days (range 2-365), two dogs died of non-pulmonary disease, 2 dogs had severe lower respiratory infections as puppies with persistent respiratory signs, and both are still alive at 4 2 years since diagnosis, 1 terrier is alive at 19 months and 1 was lost to follow up. 5 dogs had phtn, with a median survival of 60 days (42-590), while the 5 dogs without had a survival of 180 days (range 21-730), [p 5 0.3]. interstitial lung disease in dogs is not just idiopathic pulmonary fibrosis. following respiratory infection, young dogs may develop an ild with a relatively indolent course and rare ild is steroid responsive. ct is useful to identify ild but further research correlated with echocardiography and histopathology is advised to use it to prognosticate. idiopathic pulmonary fibrosis (ipf) is an interstitial pulmonary disease, mainly described in west highland white terriers (whwt). identification of molecular pathways important in the pathogenesis of ipf would improve our understanding of this disease and may help identify therapeutic targets. the aim of the present study was to investigate gene expression in lungs of whwt with ipf using oligonucleotide microarray. total rna was extracted from post-mortem pulmonary samples from five whwt with ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. genes with a minimum of a two-fold difference in expression between the two groups were selected for further analysis. the most significant biological functions for these genes were identified using ingenuity pathways analysis. more than 1000 genes were identified as having greater than twofold difference in expression. the significant biological functions associated with these genes were related to cellular movement, cellular proliferation and apoptosis. most notable among these were genes encoding the leukocyte chemotactic proteins: ccl2 (fold change 14.93), ccl17 (12.74) and il8 (14.32); the proteins involved in fibroblast migration; and the matrix metalloproteinases (mmps) involved in matrix degradation: mmp7 (à17.55), mmp9 (-3.42), mmp1 (à2.76). this study has identified genes which may be important in pathogenesis of ipf, e.g. proteins involved in leukocytes chemotaxis, fibroblast recruitment and activation, regulation of apoptosis, and extracellular-matrix turn-over. however, real-time quantitative rt-pcr studies are needed to confirm these results before any definitive conclusions can be drawn. idiopathic pulmonary fibrosis (ipf) is an interstitial disease, mainly described in west highland white terriers (whwt). defini-tive diagnosis ultimately relies on lung histopathology. identification of specific biomarkers would be very helpful. expression microarray is a powerful screening tool to study local gene expression in a disease state. the aim of the present study was to measure gene expression profiles in lungs of whwt with ipf to identify potential blood or bronchoalveolar lavage fluid (balf) biomarkers. total rna was extracted from post-mortem pulmonary samples from five whwt with histopathologically confirmed ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. ipa-biomarkers analysis (ingenuity system) was used to filter and prioritize biomarkers candidates using the three following criteria: a minimum of a two-fold difference in expression between ipf and ctrl; expression of the gene in lung tissue; possible detection of the protein in blood or in balf. fifty-four molecules met all the criteria. based on difference in expression, promising proteins included ccl7 (fold change 17.8), a3-actinin (15.7), ccl2 (14.9), serum amyloid a1 (14.4), il8 (14.3), plunc (à25.1), mmp7 (à17.6). some are well-known biomarkers of ipf in humans either for diagnosis (mmp7, il8) or prognosis (ccl2). these results provide novel potential biomarkers of canine ipf. measurement of these proteins in blood and balf of healthy dogs, dogs with ipf and with other respiratory diseases is needed to assess their use as biomarkers of canine ipf. heliox is a mixture of helium and oxygen that has been used therapeutically in human medicine for treatment of airway obstruction. helium's low density and other physical properties have been shown to reduce the work of breathing by limiting turbulence. the purpose of this study was, therefore, to evaluate respiratory parameters in response to inhaled heliox in dogs with meso-and brachycephalic conformation. eleven healthy dogs were recruited, five were mesocephalic and six were brachycephalic. flow-volume loops were collected using commercial software (buxcor) while breathing 70:30 helium: oxygen (heliox) and 70:30 nitrogen:oxygen (nitrox) in a randomized order via a low dead-space face mask. due to the intrinsic gas properties, gas flow rates and volumes were corrected in-vitro by a conversion factor for the effect of helium on the pneumotachograph. respiratory rate, tidal volume (ml), minute ventilation (l), inspiratory time (ti), expiratory time (te), peak inspiratory flow (pif) and peak expiratory flow (pef) were recorded while breathing heliox or nitrox. values were compared using a paired sample t-test, with p o 0.05 considered significant. all dogs cooperated with testing. there was no significant difference in respiratory rate, tidal volume, minute ventilation, inspiratory or expiratory times, or peak inspiratory flow. peak expiratory flow was significantly higher (p 5 0.01) while breathing heliox than when breathing nitrox in brachycephalics but not in mesocephalics (p 5 0.22). heliox is well-tolerated in healthy dogs and results in an increased expiratory flow rate in brachycephalic dogs. further investigation of heliox is warranted in dogs with airway obstruction. of this prospective multicentric study is to assess the effects that surgical correction has on the severity of clinical signs and levels of acute phase proteins (c-reactive protein [crp] , haptoglobin [hp]) and cardiac troponin i (ctni). thirty three brachycephalic dogs with boas were included and evaluated before and, approximately two months, after surgical correction. the most common components of boas found were elongated soft palate (33/33; 100%), stenotic nares (31/33; 94%) and everted laryngeal saccules (13/33; 39.4%). staphylectomy was performed by means of two different surgical techniques: laser (n 5 12) or electrical scalpel (n 5 21). there were significant differences between dogs depending on the surgical technique used, with a higher reduction of respiratory signs (p o 0.002) and a better postsurgical improvement (p o 0.015) with the use of laser. the levels of crp, hp and ctni were categorized into normal or elevated. before surgical treatment three (9.1%), six (18.2%) and thirteen (39.4%) dogs had elevated values of crp, hp and ctni, respectively. two months after surgical correction, five (15.1%), eleven (33.3%) and fourteen (42.4%) dogs had elevated values of crp, hp and ctni, respectively. there were no statistical differences between values of crp and ctni before and after surgical correction but the levels of hp increased significantly after surgical treatment (p o 0.008), probably due to postsurgical treatment with corticosteroids. as previously suggested by others, there was a statistically significant reduction of respiratory and gastrointestinal signs in dogs with boas submitted to surgical correction (p o 0,001). according to the results obtained in the present study, the determination of crp, hp and ctni before and two months after surgical treatment do not have a prognostic value in dogs with boas. even though, near half of the dogs studied had elevated levels of ctni (39.4%) that persisted after surgical treatment (42.4%), suggesting some degree of myocardial damage is present. further studies are needed considering the influence of breed and age. to the authors' knowledge, this is the first description of crp, hp and ctni determination in dogs with boas. overweight and obesity are common conditions that lead to alterations in respiratory mechanics, airway resistance, pattern of breathing and gas exchange in humans. the objective of the present study was to investigate if there are significant differences on respiratory parameters and arterial gas analysis of obese and overweight cats, in conscious state and under general anesthesia. twenty nine adult cats were arranged in three groups: obese (n 5 15), overweight (n 5 7) and with ideal body score index (bsi) (n 5 7). mean of bsi in the groups were: 8,8 (obese), 6,3 (overweight) and 4,9 (ideal bsi). cats did not had respiratory, cardiac or others systemic diseases. the respiratory parameters were evaluated with a ventilometer equipment coupled to facemasks in conscious cats and directly to the endotracheal tube in anesthetized cats under spontaneous respiration. the anesthesia was performed with propofol (3 ae 1,2 ml/kg) and the cats were maintained in the same anesthetic plan. the three groups were compared by analysis of variance followed by tukey's test and conscious and anesthetized cats were compared by student's t test, with a 5% significance level. there were not observed differences on the respiratory parameters evaluated on ventilometry (tidal volume, expiratory and inspiratory times and peak pressures, respiratory rate and partial pressure of end tidal co 2 (petco 2 )) and on arterial gas parameters (pao 2 e paco 2 ) in the three groups. the pao 2 of cats with ideal bsi was 88,1 ae 13,5 mmhg, although was not significantly different (p 5 0,06) from overweight (72,0 ae 11,9 mmhg) and obese cats (72,6 ae 14,6 mmhg). comparison of anesthetized to conscious cats, it was detected decreases in tidal volume, expiratory and inspiratory times and peak pressures and increase in petco 2 in respiratory rate in the anesthetized cats. only petco 2 , inspiratory time and respiratory rate in overweight cats did not differ in anesthetized cats. these results suggest that obesity and overweight did not result in impairment of respiratory function in cats and propofol induced respiratory depression. osteosarcoma (osa) is the most common bone tumor in dogs, however, little is known regarding the mechanisms underlying malignant transformation in these tumors. breeds such as rottweilers and greyhounds are at higher risk for developing osa, suggesting that heritable factors play a role in this disease. mirnas have tumor/tissue specific roles in regulating gene expression and dysregulated mirna expression is found frequently in cancer. we hypothesize that canine osa is characterized by a unique mirna expression profile(s) with dysregulation of some mirnas being associated with specific breeds. mirna expression profiling of primary osa tumors from 6 greyhounds and 6 rottweilers was performed using the nanostring technologies ncounter mirna expression assay kit, interrogating the mirna expression profile of 651 human mirnas, 168 of whose mature sequences are 100% conserved between human and dog. 17 mirnas were differentially expressed in greyhound versus rottweiler tumors (p o 0.05), suggesting that breed-specific dysregulation of mirnas may contribute to the development and progression of spontaneous osa. hierarchical clustering revealed distinct mirna expression signatures in greyhound osa tumors as compared to rottweilers. based on these preliminary results, we are evaluating a larger cohort of osa tumor samples including greyhounds, rottweilers, golden retrievers, and a mixed population of other breeds. statistical analysis will be performed to determine the association of mirna transcript levels with specific breeds and overall outcome. characterization of mirna expression in canine osa will facilitate our understanding the biology of this disease and has the potential to identify targets for therapeutic intervention. originally combination therapies using drugs with documented single-agent activity and lack of overlapping toxicities could potentially improve outcome. the hypothesis intended to be tested is that palladia s can be safely administered concurrently with a standard weekly protocol of vinblastine (vbl), at dosages known to have activity against mast cell tumors. dogs with histologically confirmed measurable mast cell tumors were evaluated for eligibility to enter a standard phase i dose-finding trial (313 cohort), at a starting dose of 2.3 mg/m2 iv vbl (weekly for a total of 4 treatments) and 2.25 mg/kg po palladia s eod, concurrently. dose escalation of palladia s was scheduled in 0.25 mg/kg increments until mtd was established or fda label dose completed (3.25 mg/kg). safety evaluation was performed weekly throughout the 4 week study period. dose-limiting toxicities were described following established vcog-ctcae(v1.0) criteria. while antitumor response is not a primary endpoint of phase i trials, activity was documented prior to vbl treatments 2-4, and monthly thereafter, based on recist criteria. nine dogs have been enrolled; cohort 3 is filled and approaching completion of the evaluation period. hematologic dose limiting toxicity led to 2 de-escalations of vbl. the current safe combination appears to include vbl at 1.6 mg/m2 every other week and palladia s at 2.25 mg/kg eod. response was seen in all but one dog. without head to head trials comparing efficacy of bi-weekly vbl combined with palladia s and vbl alone, choice of therapy should remain at the clinician's discretion. originally prostate specific membrane antigen (psma) is a transmembrane protein expressed by tumor-associated neovasculature, but not normal blood vessels. based upon its selective expression in endothelial cells associated with cancer, psma may serve as a conserved angiogenic target shared by macroscopic solid tumors of various histologies. to investigate the feasibility of targeting a homogenous population of psma-expressing endothelial cells as a novel anticancer strategy, we have investigated psma expression in several canine hemangiosarcoma (chsa) cell lines, and subsequently developed self-assembling nanoparticles containing diagnostic (near infrared dyes) and therapeutic (doxorubicin) cargo which selectively bind to psma by means of the a10 aptamer, a commercially-available oligonucleotide. the expression of psma by chsa cells was confirmed transcriptionally and translationally by real time pcr and immunohistochemistry, respectively. selective binding and endocytosis of a10 decorated nanoparticles was studied by fluorescent microscopy. the ability of a10 decorated nanoparticles encapsulating doxorubicin to exert in vitro cytotoxic effects in chsa cells was assessed by colony forming assays. using a chsa xenograft murine tumor model, clinically-relevant anticancer effects of a10 decorated nanoparticles encapsulating doxorubicin were tested. all chsa cell lines expressed psma mrna and protein. a10 decorated nanoparticles were selectively endocytosed by psma-expressing cells, and when these nanoparticles encapsulated doxorubicin, significant cytotoxic effects were exerted in vitro. finally, a10 decorated nanoparticles encapsulating doxorubicin significantly reduced the size of macroscopic chsa tumor burdens in transplanted mice. diagnostic and therapeutic nanoparticles can be targeted to psma-expressing endothelial cells, and chsa provides a comparative model for the future study of nanoparticle therapeutics. canine transitional cell carcinoma (tcc) is the most common tumor of the urinary tract, and is similar to human invasive tcc in histopathologic characteristics, molecular features, sites of metastasis, and response to medical therapy. prevalence is increasing, and novel therapies and strategies are needed to effectively treat this aggressive form of cancer in both species. personalized medicine techniques intend to improve treatment outcome by using patient tumor profiling to identify potential and individualized therapeutic targets. a genomic algorithm has been developed termed ''coexpression extrapolation'', or coxen, that aims to use expression microarray data to predict drug activity in patient tcc samples. the utility of this predictive methodology has been established in other types of cancer in vitro, however its clinical utility has not yet been determined. validation studies of coxen in 10 canine tcc cell lines were conducted. the goal was to determine the value of coxen in predicting baseline sensitivity of canine tcc to 5 chemotherapy agents (gemcitabine, mitoxantrone, carboplatin, vinblastine and cisplatin) that would then be used in a proposed clinical trial. additionally, expression data from 25 canine treatment-naı¨ve primary tumor samples were generated on an affymetrix array platform (canine genome v 2.0). both the expression data and tcc cell line data (antiproliferative effects, 50% growth inhibition or gc 50 ) were used to establish a canine specific predictive coxen algorithm. coxen scores for canine tcc cell-line drug activity were then analyzed. scores predicted the activity of cisplatin, gemcitibine, and mitoxantrone in all 10 cell lines, and of carboplatin in 6 cell lines. because all of the cell lines were sensitive to vinblastine (gi 50 o0.05 mm), the coxen score was not predictive of its potency. interestingly, coxen fails to predict vinblastine response in human tcc cell line data as well. in concurrent work, comparative genomic studies to define and compare the gene expression signatures of tcc in dogs and humans provides further evidence that canine tcc is a valuable genomic model of the human disease. current studies involve testing the chemo-predictivity of this derived canine coxen algorithm in additional canine tcc cell lines. canine tcc offers an excellent model for in vitro and in vivo studies of the coxen approach. this preclinical work will be used to guide the feasibility of future coxen clinical trials in dogs and humans with tcc. a small molecule complex (aminoact) isolated from bovine milk is a natural peptide mixture with multi-kinase inhibitory effects against epidermal growth factor receptor (egfr) and insulin-like growth factor receptor-1 (igfr-1). ingestion of aminoact in people with cancer results in lower serum tnf-alpha, an increase in antioxidant superoxide dismutase (sod) enzyme activity, and subjects' blood serum causes apopsotis in cancer cell lines. this study was designed to first assess safety and secondly the efficacy of three dosage levels of ax-3 in sustaining progression free survival (pfs) for dogs with refractory advanced and/or metastatic cancer. the prospective, open label study included dogs of different breeds with naturally occurring histologically confirmed malignancies. the first 13 dogs received aminoact at 1g/m 2 ; the second group of 7 dogs subsequently received the same dosage 1 350 mg of aminoact; and the third group of dogs subsequently received 2g/ m 2 . each dog was treated orally daily for six weeks along with 550 mg betaine hcl, that aids in peptide absorption. all patients were evaluated for toxicity using the vcog-ctcae and efficacy using the recist criteria via assessment of clinical parameters, blood work and client questionnaires. no toxicity other than mild, transient (grade i) nausea was noted, nor were there any changes in hemograms or biochemical profiles in any patient. dogs with tumors that were confirmed as responders (4 50% reduction in size) include pulmonary adenocarcinoma, mast cell tumor, trichoepithelioma and soft tissue sarcoma. it appears in limited studies that the response rate may be more durable at higher dosages. the response to aminoact is dose dependent and only transient mild toxicity was observed, which suggest the maximum effective dosage has not been reached. further clinical studies will be valuable in determining the effective dosage and response duration. treating cancer in dogs with aminoact offers a unique opportunity as a model for human cancer biology and translational cancer therapeutics. stereotactic radiation therapy (srt) combines patient immobilization, image guidance, and intensity modulated delivery to achieve ablative radiation doses within the tumor, while preferentially sparing surrounding normal tissues. the purpose of this study was to evaluate the efficacy of srt as a means of achieving local tumor control for canine nasal tumors. retrospective analysis was performed on dogs with a nasal tumor confirmed by histopathology and computed tomography, no previous surgical or radiation therapy, at least six months of follow-up, and completion of three fractions of srt at csu.srt was administered via the varian trilogy linear accelerator once daily for three consecutive days. the varian eclipse treatment planwas reviewed to determine the planned target volume (ptv) and dose to 95% of the ptv. kaplan-meir survival analysis was performed for disease free interval (dfi) and overall survival (os). sixteen patients with nasal tumors (8 adenocarcinoma/carcinomas, 2 squamous cell carcinomas, 3 chondrosarcomas, 2 osteosarcomas, and 1 undifferentiated sarcoma) were treated with srt. a median dose of 29.1gy was administered to 95% ptv with a median ptv of 75.3cc. srt was well tolerated by the normal tissues with minimal, manageable side effects. to date, the median dfi is 270 days, while the median os is 367 days. based upon the initial clinical experience, stereotactic radiation therapy is an emerging modality in the management of canine nasal tumors. canine leptospirosis can vary from subclinical infection to illness that ranges from mild to severe, including death, depending on the susceptibility of the dog, virulence of the organism, and route and degree of infection. the objective of this study was to evaluate the ability of a canine leptospira bacterin to prevent infection and disease following challenge with virulent leptospira canicola, l. pomona, l. grippotyphosa, or l. icterohaemorrhagiae. groups of 8week-old beagles were vaccinated (day 0) and boosted (day 21) with placebo (n 5 10) or the 4-way bacterin (n ! 20) and subsequently challenged with each serovar. the results demonstrated that blood and various tissue samples from placebo-recipients became reliably infected, and the dogs developed typical clinical signs of leptospirosis including loss of appetite, ocular congestion, depression, dehydration, jaundice, hematuria, melena, vomiting, petechiae, and death. in addition, placebo-recipients developed kidney and liver dysfunction. in contrast, some vaccine-recipients became infected, but the organisms were cleared quickly from the blood. vaccinated dogs failed to develop severe clinical disease requiring medical intervention, and no animals died (p ! 0.001). a few of the vaccinated dogs developed clinical abnormalities, but the clinical signs remained mild and were self-limiting (p o 0.0001 for each serovar). administration of the bacterin also prevented thrombocytopenia ( ciprofloxacin, a synthetic fluoroquinolone antimicrobic, is not fda-approved for veterinary use. however, due to recent availability of less expensive generic formulations, extra-label use of ciprofloxacin by veterinarians appears more common. although ciprofloxacin crystalluria and uroliths have been reported in humans, we are unaware of any published reports in dogs. this is surprising since mean urine ciprofloxacin concentration (0.36 mg/ml) in dogs following a modest iv dose (13 mg/kg) was 3 times higher than the solubility of ciprofloxacin in water (0.12 mg/ml). to identify the occurrence of ciprofloxacin uroliths in dogs, records from the minnesota urolith center were reviewed. between january 2001 and december 2009, ciprofloxacin was identified in uroliths from 58 dogs; uroliths were composed of 100% ciprofloxacin in 10, mixed uroliths containing ciprofloxacin were identified in 6, a shell of ciprofloxacin was observed in 21, and ciprofloxacin surface crystals were identified in 21. based on an experimental study in which 83% of human volunteers consuming 1000 mg of ciprofloxacin with nahco3 exhibited ciprofloxacin crystalluria (urine ph 47.3), while no volunteers consuming 1000 mg of ciprofloxacin and nh4cl to acidify urine formed crystals; we postulated that ciprofloxacin uroliths could be dissolved in acidic urine. to test this hypothesis, canine uroliths composed of 100% ciprofloxacin from a single source (6-yr-old male, english bulldog receiving 24 mg/kg of ciprofloxacin po, q12 hr to manage superficial pyoderma; turbulent flow chromatography/tandem mass spectrometry detected 517 mg of ciprofloxacin/g of urolith) were incubated in urine at selected ph's and monitored for dissolution. urine obtained from multiple dogs not receiving fluoroquinolones, was pooled and divided into 5 aliquots. aliquots were adjusted with hcl or naoh to a ph of 3, 5, 6, 7, or 8. aliquots were capped and preserved by refrigeration; ph was monitored and readjusted weekly. ten uroliths of approximately equal weight were randomly assigned to individual flasks containing 10 mls of urine. flasks were constantly agitated and maintained at 381c. every 24 hours, urine was discarded and replaced with 10 mls of urine of identical ph until stone dissolution was complete. ciprofloxacin urolith dissolution times at each urine ph are reported below. ciprofloxacin uroliths are a newly recognized disease and a potential adverse effect of ciprofloxacin administration in dogs. in vitro dissolution of ciprofloxacin uroliths was achieved in canine urine, supporting the premise that in vivo dissolution is possible. urolith dissolution times were shortest at lower and higher ph's, which is consistent with the pka (6.0 and 8.8) of this amphiprotic antimicrobic (more soluble at ph below the acidic pka and above the alkaline pka). foods designed to promote struvite urolith dissolution may be designed for short term feeding facilitating rapid dissolution or may be formulated with a more moderate target urine ph to allow for dissolution and then life-long maintenance feeding minimizing recurrence. the purpose of this study was to compare the efficacy and rate of dissolution of a maintenance food with a struvite dissolution food. sixteen client-owned adult cats (13 fs, 3 mc) with naturally occurring struvite urocystoliths (mineral composition based on history, radiographs, urinalysis, urine culture and physical examination) were randomized to either a dry maintenance food (test) or a dry food known to dissolve struvite uroliths (control). the clinical care team and owner were blinded to treatment assignment. the test food was formulated to provide 0.06% mg (dm), 0.65% p, 35% protein, and a calculated target urine ph value (uph) of 6.2-6.4. the control food was formulated to provide 0.06% mg (dm), 0.77% p, 35% protein, and a targeted urine ph of 5.9-6.1. owners were advised to feed the assigned diet exclusively in an amount to maintain body condition. after diet assignment radiographs were performed at eight weekly intervals until there was no evidence of uroliths or until there was evidence that the uroliths were the same size or larger. a physical examination, complete blood count, serum chemistry profile, urinalysis and urine culture were repeated at the conclusion of the study. statistical analysis was by anova. all uroliths dissolved in all cats and both foods were palatable. radiographs of cats fed the control food indicated the uroliths dissolved in a significantly shorter time (mean ae std dev of 1.6 ae 0.7 weeks) compared to cats consuming the test food (mean 3.9 ae 2 weeks; po0.05).). cats in the control group finished the study at 1 (n 5 4), 2 (n 5 3) and 3 weeks. cats in the test group finished the study 1, 2, 3 (n 5 2), 4, 5, 6, and 7 weeks. all the minnesota urolith center occasionally receives uroliths for analysis that are immersed in formalin. results of quantitative analysis of these uroliths revealed that some submitted in formalin consisted of newberyite (magnesium hydrogen phosphate trihydrate). because newberyite is uncommonly found in uroliths formed by cats and dogs, we hypothesized that this mineral was an in vitro artifact caused by exposure of struvite (magnesium ammonium phosphate hexahydrate) to formalin. the purpose of this study was to determine if formalin alters the mineral composition of uroliths. urolith submissions containing stones of either 100% struvite (n 5 5 dogs and 5 cats), 100% calcium oxalate (n 5 5 dogs and 5 cats), 100% calcium phosphate apatite (n 5 5 dogs and 5 cats), 100% cystine (n 5 5 dogs and 5 cats), 100% ammonium urate (n 5 5 dogs and 5 cats), and 100% silica (n 5 5 dogs) preserved by only air drying were tested. one urolith from each submission was quantitatively analyzed by polarized light microscopy or infrared spectroscopy. a subsequent urolith from the same submission was immersed in 1 ml of 10% buffered formalin for 48 hours at room temperature. uroliths were then air dried for 30 minutes and the analysis was repeated. after exposure to formalin, portions of all 10 struvite uroliths were transformed into newberyite. three (1 dog and 2 cats) of 10 ammonium urate uroliths were completely dissolved. newberyite was not detected in any of the remaining uroliths. likewise quantitative mineral analysis of non-struvite uroliths remained unchanged. to avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis. we previously reported that transfusion to normal dogs of autologous erythrocyte concentrates (prbcs) that had been stored for 21 days causes a profound inflammatory response (2x increase in leucocyte count and fibrinogen, 60x increase in c-reactive protein). we speculated that inflammation was due to cytokines produced during the storage period, and hypothesized that transfusion of fresh (f) prbcs would elicit less inflammation than would stored (s) prbcs. a whole blood unit was collected from healthy dogs (n 5 10) for prbcs on day 0, then again on day 32. on day 35 dogs received an autologous transfusion of prbcs stored for either 35 days (s, n 5 5) or 3 days (f, n 5 5). cbcs and in-tem thromboelastometry (ct:coagulation time, cft:clot formation time, a:alpha, mcf:maximum clot firmness) were evaluated on blood samples collected at 0 (pre) and 5,9,24,48, and 72 hours after transfusion. fresh prbcs did not elicit any change in leucocytes, platelets, or thromboelastometry. stored prbcs elicited a degenerative left shift (5 hr) followed by a regenerative left shift (9-48 hr), thrombocytopenia (36% decrease at 5 hr), and marked hypocoagulability characterized by prolonged ct (5,9,24 hr) and cft (5,9 hr), and decreased a (5,9 hr) and mcf (5,9, 24 hr). data are mean(sd). a: p o 0.05 between groups f and s by t test. b: p o 0.05 compared to ''0'' by rm anova. transfusion of autologous stored prbcs elicits a greater inflammatory response than fresh prbcs, and results in hypocoagulability on thromboelastometry. clopidogrel is a potent antiplatelet drug that is gaining popularity in veterinary medicine for antithrombotic therapy. the parent molecule is an inactive prodrug that must be converted by hepatic isozymes to an active metabolite. the majority of the parent molecule is directed to the formation of inactive metabolites with only an extremely small proportion of parent molecule directed to the formation of the active metabolite. there are multiple hepatic isozymes responsible for the formation of the active metabolite. a non-specific hepatic isozyme inducer such as rifampin could increase the formation of the active metabolite of clopidogrel thereby increasing the pharmacodynamic response which may allow a reduced drug dose to achieve a clinical effect. we have previously presented data supporting the increased pharmacodynamic response of clopdiogrel after rifampin therapy. the goal of this study was to demonstrate an increased pharmacokinetic response of clopidogrel after rifampin induction of hepatic isozymes. six healthy, purpose-bred dogs were used for this study. the pharmacokinetics of clopidogrel were determined by measuring the parent molecule, primary inactive metabolite and active metabolite through lc/ms/ms. the pharmacodynamics of clopidogrel were determined by measuring collagen-induced whole blood aggregation. blood samples were collected prior to clopidogrel administration (baseline), after 7 days of 2 mg/kg clopidogrel po q 24 hrs, and after 7 days of 2 mg/kg clopidogrel po q 24 hrs 1 10 mg/ kg po q 12 hrs rifampin. given the absence of a known standard for the active metabolite, only a semi-quantitative assessment of active metabolite concentration can be made. there was no identifiable active metabolite peak noted at baseline or after clopidogrel treatment. however, with clopidogrel and rifampin combined administration there was an active metabolite peak identified in all dogs with a mean area of 41.7 ae 23.5. the development of the active metabolite peak was associated with an increase in the pharmacodyamic response of clopidogrel in the dogs. this is the first study in any species to document the increased formation of the active metabolite of clopidogrel in response to a strong, non-specific hepatic isozyme inducer. this increased pharmacokinetic response was associated with an increased pharmacodynamic response of clopidogrel. this data provides supportive evidence to develop therapeutic protocols to improve the pharmacodynamic response to clopidogrel in dogs that may reduce dosing requirements or correct subtherapeutic pharmacodynamic response. critical illness-related corticosteroid insufficiency (circi) has been identified in humans, foals, dogs and cats with lower-thanexpected circulating cortisol concentrations, and/or by a blunted cortisol response to acth stimulation. our purpose was to determine if circi exists in critically ill horses. endogenous plasma acth and serum cortisol concentrations, and cortisol at t 5 0 and t 5 30 min after 0.1 mg/kg cosyntropin, were measured by radioimmunoassay from horses with colic or systemic illness on admission, and days 2, 4 and 6 of hospitilization. horses were divided into mild, moderate, or severe illness groups based on clinicopathologic data. inappropriately low cortisol was defined as endogenous cortisol o mean-1sd achieved after administration of 0.1 mg/kg cosyntropin to normal horses ( o 269 nmol/l). inadequate delta cortisol was defined as o mean delta cortisol in normal horses after 0.1 mg/kg cosyntropin ( o 159 nmol/l). cortisol, acth and delta cortisol were compared using anova between groups, with p o 0.05 considered significant. fifty-eight horses classified as having mild (11), moderate (30) and severe (17) disease at admission had survival rates of 100%, 97% and 35% respectively. admission acth and cortisol concentrations were highest in severely ill horses (93 ae 198pg/ml, 361 ae 137 nmol/l) compared to moderate (31 ae 36, 279 ae 137) and mildly ill horses (18.0 ae 12.0, 237 ae 133). admission cortisol concentrations were higher overall in severely ill horses (p 5 0.016), but were low in 24% (4/17). admission delta cortisol was low in 85% (11/13) of severely ill horses, and was associated with marked adrenal hemorrhage in non-survivors. severely ill horses have high cortisol and acth, but low cortisol and delta cortisol may indicate circi secondary to adrenal hemorrhage. equine pituitary pars intermedia dysfunction (ppid) is a common endocrinopathy of aged horses that results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. factors that initiate spontaneous dopaminergic neurodegenerative disease remain elusive, however accumulation of misfolded a-synuclein protein and dysfunctional protein clearance have been implicated. misfolded protein accumulation occurs due to increased protein production or decreased clearance of damaged macromolecules through the process of autophagy. while have previously demonstrated that horses with ppid have increased asynuclein in the periventricular neurons compared to controls, it remains unknown whether the protein accumulates due to increased production or decreased clearance. we hypothesized that autophagy is decreased in the pituitary neurointermediate lobe from horses with ppid compared to controls. neurointermediate lobe pituitary tissue was from collected from horses with ppid (n 5 12) and healthy horses (n 5 37, 2-35 years). realtime pcr was used to determine the relative expression of autophagy genes (mtor, beclin1, atg12, atg7, atg5, pink, lamp2) and a-synuclein relative gene expression from horses with ppid were compared to healthy horses by t-test following log transformation. a pearson coefficient of correlation was calculated comparing a-synuclein expression with autophagy gene expression. the expression of a-synuclein, autophagy-related genes (atg12, beclin, lamp2), and mtor was greater in horses with ppid than in healthy horses. age was not correlated to a-synuclein or autophagy gene expression. there was a significant positive correlation between expression of a-synuclein and beclin1, atg12, atg7, atg5, and pink, but not mtor expression. accumulation of a-synuclein protein in horses with ppid may result from increased a-synuclein expression. autophagy genes are upregulated in horses with ppid, suggesting a compensatory response, although these findings need to be confirmed by demonstrating an increased functional response. asynuclein expression was positively correlated to expression of autophagy genes except mtor, suggesting a-synuclein may stimulate autophagy in an mtor independent manner. acvim forum session 86a efficacy of delayed antiviral therapy against ehv-1 challenge. lk maxwell 1 , ll gilliam 1 , n pusterla 2 , r carmichael 1 , rw eberle 1 , jw ritchey 1 , tc holbrook 1 , t gull 1 , gb rezabek 1 , d mcfarlane 1 , cg macallister 1 . 1 oklahoma state university, stillwater, ok. 2 university of california, davis, ca. equine herpes virus type-1 (ehv-1) outbreaks are often not recognized until exposed horses are at immediate risk for developing equine herpes myeloencephalopathy (ehm). the objective of this study was to determine whether delayed therapy with the antiviral drugs valacyclovir or ganciclovir could protect those horses most at risk for ehm. eighteen aged ( 4 20 years) mares were randomized to treatment: no therapy (control), oral valacyclovir therapy, or intravenous ganciclovir therapy. drug administration was initiated at the onset of the second febrile phase, between days 4-6 after ehv-1 inoculation (pi), and continued for one week. neurological examinations were performed prior to the study and for three weeks pi. one horse was excluded from the study for failure to become febrile. body temperature was significantly lower in the ganciclovirtherapy horses as compared to control horses on days 6-8 pi (p o 0.05), whereas valacyclovir-therapy horses did not differ from control horses. viremia in whole blood, as determined by pcr, was also lower in the ganciclovir-therapy horses on days 7-10 pi and on day 7 pi in the valacyclovir-therapy horses (p o 0.05). although antiviral drug administration did not reduce the risk of ataxia (p 5 0.06) or nasal shedding, ganciclovir therapy did decrease the severity of ataxia (p o 0.05) as compared to valacyclovir-therapy and control horses, where 0/6, 2/5, and 4/6 horses, respectively, developed at least a two grade change in ataxia. in summary, ganciclovir administration provided better protection against ehm than did valacyclovir when therapy was initiated just prior to the onset of neurological disease. equine vaccination is amongst the most important method of prophylaxis against equine influenza virus (eiv), a pathogen in which continuous antigenic drift can lead to vaccine failure. a 6month duration of immunity (doi) challenge infection study was conducted using commercial inactivated vaccines containing different strains of a/equine/2/influenza virus's, including innovator tm , containing kentucky/97 (pfizer animal health, new york, ny), and calvenza, containing a combination of ohio/2003, kentucky/ 95, and newmarket93 (boehringer ingelheim vetmedica, st. joseph, ms) . the challenge virus strain was colorado/07, the most contemporary challenge strain currently in use. the study design was a blinded, randomized challenge trial. three groups of 10 yearling ponies, with no history or serological evidence of eiv infection were established. each group received one of three treatments: vaccination with innovator tm ; vaccination with calvenza tm ; or injection with a saline placebo. each treatment was administered 3 times, at intervals of 1 month between the first two treatments, and 3 months between the second and third treatments. all ponies were challenged by nasal nebulization of 5x10 7 eid 50 influenza virus a/eq/2/colorado/07 6 months after the third treatment. clinical signs of disease, including rectal temperature, nasal discharge, anorexia, coughing, and depression, were recorded daily for 2 days prior to challenge infection, and 14 days post-challenge. nasal shedding of eiv was measured on the same days, using a realtime pcr test procedure. eiv-specific antibody responses were measured by elisa. differences between groups were analyzed by non-parametric repeated measures anova, and differences were declared significant when p o 0.05. all control group ponies demonstrated clinical signs of disease consistent with eiv infection post-challenge infection, including pyrexia, nasal discharge, inappetance and partial anorexia. these signs were significantly lower in both vaccine groups; mean body temperature was elevated ( 4101.51f) for 8 days in controls, but only 2 days in vaccine groups. nasal shedding of eiv was detected in all ponies in all groups: over the duration of the study the calvenza group shed significantly less virus than innovator and control. over time antibody titers were significantly higher in the calvenza than the innovator group, and both were significantly greater than controls. this study demonstrated that both current commercial inactivated eiv vaccines have a duration of clinical protection of at least 6 months after a highly pathogenic challenge with a recent eiv isolate. both antibody responses and virological protection differed between the vaccines. formulation difference between the vaccines, including the eiv antigens employed, may have contributed to this performance difference. degenerative myelopathy (dm) may be homologous to a form of amyotrophic lateral sclerosis in humans which has excitotoxic and immunologic pathogeneses described. the aims of this study were to determine (i) presence or absence of abnormalities in concentrations of csf amino acid (aa) neurotransmitters (glutamate, glycine and gàaminobutyric acid (gaba)) and cytokines in dogs with dm and if present (ii) investigate associations with disease severity. twenty-two dogs histopathologically confirmed for dm and 21 dogs with suspected dm based on thorough diagnostic investigations and 42 clinically normal age-matched control dogs were included in the study. the neurological severity of the dm dogs was graded (1-4) using an established scale. csf was evaluated for presence of glutamate, glycine and gaba by high performance liquid chromatography and for gm-csf, ifn-g, il-2, il-4, il-6, il-7, il-8, il-10, il-15, il-18, ip-10, kc (keratinocyte chemoattractant), mcp-1 (monocyte chemotactic protein-1) and tnf-a using a commercially available, canine multiplex immunoassay (millipore, billerica, ma). all data analyses were performed using sas v 9.2 (cary, nc). analyte levels were compared between dm confirmed, dm suspected and control dogs by an analysis of variance (anova). spearman correlation was used to test for correlations of analyte levels and neurological grades. all hypothesis tests were 2-sided with a 5 0.05. there were no significant differences between individual csf analytes in dm confirmed and dm suspected dogs. glutamate levels were not significantly different between dm affected (mean 5 0.09 mg/ ml; range 5 0.01-0.29; sd 5 0.069) and control dogs (mean 5 0.07 mg/ ml; range 5 0.01-0.29; sd 5 0.056). control dogs (mean 5 0.86 mg/ml; range 5 0.08-1.16; sd 5 0.213) had significantly higher levels of gaba (p o 0.0001) than dm dogs (mean 5 0.12 mg/ml; range 5 0.07-0.79; sd 5 0.12). control dogs (mean 5 2.14 mg/ml; range 5 0.01-3.83; sd 5 0.76) also had significantly higher glycine concentrations (p o 0.0001) than dm dogs (mean 5 0.45 mg/ml; range 5 0.01-0.98; sd 5 0.24). dm-affected dogs also had significantly higher levels of il-2 (p 5 0.03), kc (p o 0.0001) and mcp-1 (p 5 0.005) than control dogs. neurotransmitter levels were not significantly associated with neurological grade. kc levels were significantly higher in the least affected dogs (p 5 0.0015). there were no associations with disease severity and analyte concentrations. dm affected dogs have an imbalance of csf aa concentrations creating a relatively excitotoxic environment. reports in human als confirm an imbalance between csf excitatory and inhibitory aas suggesting a pathogenic role for excitotoxicity in als. it also appears that dm affected dogs have increases in csf cytokines and chemokines suggestive of an immunologic component to the pathogenesis as is similar to als. further prospective analysis of dm is warranted to evaluate the role of treatment on csf variables. the pathogenesis of neuropathic pain (np) and syringomyelia (sm) in association with chiari-like malformation (clm) in dogs has focused on the anatomical anomalies and secondary cerebrospinal fluid (csf) flow abnormalities. neuropathic pain in humans has been associated with abnormalities of neurotransmitters such as glutamate and serotonin as well as immunologic mechanisms. the aim of this study was to investigate the csf neurotransmitter and cytokine levels in brussels griffon dogs (bgs) with clm, sm and np. as part of an mri study investigating the prevalence of sm in bgs, atlanto-occipital csf was acquired from 46 dogs and stored at -80c until analysis. all dogs underwent a neurologic exam prior to mri; osirix s software was used to measure sm and the presence of cerebellar herniation and deviation were recorded. deproteinized csf samples were analysed for presence of serotonin (ng/ml), glutamate, glycine and gaba (mg/ml) by high performance liquid chromatography. all csf samples were evaluated simultaneously for gm-csf, ifn-g, il-2, il-4, il-6, il-7, il-8, il-10, il-15, il-18, ip-10, kc, mcp-1 and tnf-a. a commercially available, canine multiplex immunoassay (millipore, billerica, ma) was used for the cytokine analysis (pg/ml). student's t-tests were used to compare the means of neurotransmitter and cytokine values between groups with and without skull abnormalities or spinal pain. simple pearson's correlation was used to test for correlations of neurotransmitter and cytokine values with syrinx dimensions and correlations of neurotransmitter with cytokine values. all hypothesis tests were 2-sided and the significance level was a 5 0.05. np was detected in 8 dogs (17%); sm was present 24 dogs (52%); and cm was detected in 24 dogs (52%). ifn-g levels were significantly lower in dogs with np than without (p 5 0.036). there were significant positive correlations between syrinx size and il-8 (p 5 0.017), kc (p 5 0.025) and mcp-1 (p 5 0.003). there were significant negative correlations between ifn-g and syrinx height (p 5 0.025) and extent (p 5 0.042). there was a significant negative correlation between il-2 and syrinx height (p 5 0.042). neurotransmitter levels were not associated with skull abnormalities or spinal pain, but there was a positive correlation of glycine with il-2 (p 5 0.004) and mcp-1 with glutamate (p 5 0.0147) and serotonin (p 5 0.0059). the size of the syrinx in bgs with sm is associated with several cytokine elevations but only a decrease of ifn-g was associated with np. based on this study it does not appear that excitotoxicity plays a role in either sm development or np. further work is justified on the role of the immune system in cm, sm and np. current knowledge about the conservative management of disk associated cervical spondylomyelopathy (da-csm) is rather limited and mainly based on retrospectively retrieved data. the goals of this study were to prospectively evaluate the evolution of clinical signs in dogs treated conservatively for da-csm. additionally, several potential prognostic parameters and the correlation of initial clinical signs with magnetic resonance imaging (mri) and transcranial magnetic stimulation (tms) were investigated. twenty-one dogs were included. after neurological evaluation, neurological status was graded from 0 (5 normal) to 6 (5 tetraplegia). all animals underwent low-field mri and tms with measurement of onset latencies and peak-to-peak amplitudes from the extensor carpi radialis and cranial tibial muscles. from the mr images, the following dimensions were calculated: remaining spinal cord area; compression ratio; vertebral occupying ratio of the spinal cord; canal height to body height ratio (cbr); canal height to body length ratio (cblr); and the canal compromise ratio. intraparenchymal intensity (isi) changes were graded from 0 to 3. all dogs were reevaluated by the same person after 1, 3, 6, 12, and 24 months. eight of 21 dogs (38%) experienced a positive clinical evolution with improvement of clinical signs or stabilization of mild clinical signs. all dogs with a negative clinical evolution 1 month after diagnosis experienced a further progression of clinical signs resulting in a poor outcome. the opposite was true for all dogs with a positive clinical evolution after 1 month. outcome was further significantly associated by the remaining spinal cord area and the vertebral canal compromise ratio. prognosis was not significantly affected by clinical presentation or tms. progression of clinical signs, in unsuccessfully treated dogs, was generally characterized by a rapid and dramatic deterioration of neurological status. there were no significant correlations between clinical presentation, mri and tms. two dogs underwent necropsy and histopathological examination. this revealed in both cases chronic wallerian degeneration and segmental myelomalacia. the results of this study suggest that conservative treatment of da-csm is associated with a rather guarded prognosis. clinical evolution 1 month after diagnosis and selected mri parameters can be considered as prognostic indicators. the lack of correlation between clinical presentation and outcome, medical imaging and electrophysiological evaluation is disturbing and warrants further investigation. a mri-guided stereotactic brain biopsy system has not been clinically evaluated in dogs. the purpose of this study was to determine the ability of the brainsight tm system to obtain histologically diagnostic samples and access the impact of this procedure on neurologic status for 72 hours after the biopsy. five dogs with mri definable lesions in the brain have been enrolled. breeds included a pitbull mix, pembroke welsh corgi, french bulldog, border terrier and west highland white terrier. age ranged from 5-11 years. weight ranged from 8.5-18.8 kg.dogs presented with seizures (n 5 5), ambulatory paresis(n 5 4), unilateral blindness(n 5 2) and head tilt(n 5 1). one dog had a normal neurologic exam. lesions chosen for biopsy were in the olfactory and/or frontal lobes (n 5 3), parietal lobe(n 5 1), and pyriform lobe(n 5 1). lesions were between 14-22 mm in diameter. all lesions were well-circumscribed and contrast enhancing except for one. histologic diagnosis of meningioma(n 5 3) and granulomatous meningoencephalitis(n 5 1) were made. the poorly-circumscribed, non-contrast enhancing frontal mass yielded non-specific necrosis. following biopsy, three dogs returned to pre-biopsy neurologic status within 12 hours. the french bulldog took 48 hours to return to previous neurologic status due to brachycephalic syndrome that required oxygen support. one dog had acute respiratory arrest 16 hours post-biopsy. necropsy is pending. these results suggest that this mri-guided biopsy system can provide an accurate histologic diagnosis of brain lesions. biopsies of poorly-circumscribed and non-contrast enhancing brain lesions may be less diagnostic. further evaluation is on-going to determine the true diagnostic yield and complication rate of this procedure. concurrent malformations of the craniocervical junction are commonly identified in humans with chiari type i malformation. recent evidence suggests such craniocervical junction abnormalities (cjas) also occur in dogs suspected of having chiari-like malformation (clm). the purpose of this study was to objectively describe morphometric features of the craniocervical junction region of dogs with suspected clm and to investigate for associations between these features and the occurrence of other malformations in this region. magnetic resonance (mr) and computed tomographic (ct) images from 274 dogs with clm were evaluated. three regions of neural tissue compression were assessed: cerebellar compression (cc); ventral compression at the c1/c2 articulation, termed ''medullary kinking'' (mk); and dorsal compression (dc) at the c1/c2 articulation. a compression index (ci) was calculated for all abnormal regions for each dog. multiple logistic regression analysis was performed (p o 0.05) to ascertain whether ci values for the different regions of compression were associated with the incidence of other craniocervical junction abnormalities. 68% of dogs had mk and 38% of dogs had dc. 28% of dogs also had evidence of atlanto-occipital overlapping (aoo medical infrared imaging (mii) is a non-invasive diagnostic imaging technique that measures skin surface temperature and generates thermal pattern maps based on predetermined color scales. because skin temperature, dependent on regional perfusion, is under direct control of the sympathetic nervous system, mii provides information about the function of the autonomic nervous system. because of recent advances in technology and lack of sedation needed to image patients, mii has potential use as a screening test for a variety of conditions that may result in autonomic dysregulation like chiari-like malformation in dogs (clm). the purposes of this study were to establish a mii protocol for dogs suspected of having clm, to identify thermal imaging patterns for various regions of interest (roi), to evaluate changes in thermal patterns and compare the results to those of mri findings, considered the standard for diagnosing clm in dogs. one hundred and five cavalier king charles spaniel dogs with clinical signs attributable to clm and confirmed clm with mri were evaluated with a complete blood count and chemistry profile, examination by a board certified surgeon/neurologist, multidetector ct scan of the craniocervical junction, whole body mri and mii. the protocol for thermal imaging included cranial and caudal views of the body, full lateral right and left body views, dorsal views of the head and body, and right and left lateral views of the head. thermal patterns were assessed with custom image recognition software. after each dog was imaged awake, general anesthesia was administered and the dogs re-imaged using the same protocol. mri findings in dogs with severe or moderate cerebellar compression and cerebellar herniation were compared with mii results. the top of head and front of head roi were 89.2% and 97.3% successful in identifying dogs with clm. based on these preliminary findings, mii may be a viable screening tool to detect clm in dogs. medical infrared imaging (mii) is an imaging technique that measures skin surface temperature derived from cutaneous perfusion and generates thermal pattern maps based on color scales. mii has been used as a test for a variety of conditions that cause autonomic dysregulation resulting in altered cutaneous perfusion. acute thoracolumbar intervertebral disk disease (tlivdd) is common in dogs. the purpose of this study was to: 1) determine the success of mii in identifying dogs with tlivdd, 2) compare the mii localization with mri results and surgical findings 3) determine if the mii pattern returns to that of normal dogs following decompression surgery. 72 small breed chondodystrophic dogs with tlivdd confirmed with mri and 14 dogs with no tlivdd were evaluated with mri and mii. regions correlating with the intervertebral disk spaces were analyzed for average temperatures and thermographic patterns. thermal patterns were assessed with computer recognition pattern analysis (crpa) software. 21 dogs were re-evaluated 8 weeks after surgery using the same protocol. when analyzing temperature averages over a region, no significant difference was found between control and affected dogs. crpa was 90% successful in differentiating normal from affected dogs. crpa was 97% successful in identifying the intervertebral disk space when compared with mri and surgical findings. based on these findings, mii may be a viable screening tool to detect tlivdd in dogs. microglia physiologically shows regional topographical differences in immunophenotype and function within the central nervous system indicating the endowment for a prompt response to pathological stimuli such as trauma. spinal cord injuries (sci) consist of a primary injury encompassing the mechanical impact and the ''secondary wave'' of injury occurring minutes to weeks later and comprising various consecutive effects such as increased production of free radicals, excessive release of excitatory neurotransmitters and inflammatory reactions. activated microglia has the potential to perform some of these reactions, their contribution to the secondary wave is therefore controversially discussed. it has to be considered a double-edged sword as both, beneficial and deleterious effects have been attributed to these cells. the purpose of the presented study was to assess microglial involvement, particularly in the ''secondary wave'' following sci. microglia from 15 dogs with sci was isolated and characterized ex vivo in terms of morphology, immunophenotype, and function by flow cytometry. the results were compared to region-specific findings obtained from healthy control dogs (n 5 30). the histopathological exam confirmed the diagnosis of sci in the cervical (n 5 5) and thoracolumbar (n 5 10) spinal cord, and revealed a significant activation of microglia/ macrophages and upregulation of myelinophagia in dogs with sci 5 days or longer prior to euthanasia. microglial ex vivo examination showed significantly increased expressions of b7-1, b7-2, mhc ii, cd1c, icam-1, cd14, cd44, and cd45, and significantly enhanced phagocytosis and generation of reactive oxygen species (ros) in sci compared to healthy controls. microglial cells seem to be highly activated following sci with an immunophenotype indicating their active role in co-stimulation of t cells, in leukocyte adhesion and aggregation, and in lipid and glycolipid presentation. microglial phagocytosis might play a pivotal role in removal of injured or damaged cells and initialize subsequent healing processes. however, as ros can be directly neurotoxic an enhanced microglial generation might lead to bystander damage of the traumatized spinal cord and might therefore add to the deleterious effects of the secondary wave. modulating the microglial response in sci might be a valuable novel therapeutic strategy alleviating further damage to the spinal cord. thymidine kinase (tk) is a soluble biomarker present in s-phase of a salvage pathway for dna synthesis, and can be measured in serum. tk activity correlates with stage, prognosis, and relapse in canine and human lymphoma. we previously reported the results of a pilot study evaluating tk activity in archived canine osteosarcoma, transitional cell carcinoma, and hemangiosarcoma (hsa) sera, and found elevated tk activity in 80% of canine hsa sera evaluated. the purpose of this study was to prospectively evaluate serum tk activity in a large number of dogs presenting to emergency clinics with hemoabdomen and a splenic mass, to determine if tk activity could be used as a noninvasive means to distinguish hsa versus benign conditions in this population. dogs presenting with hemoabdomen and a splenic mass identified on ultrasound examination were studied. serum was collected prior to anesthesia, euthanasia or surgical intervention and frozen until batch analysis. tissue from all patients was evaluated histologically by a single pathologist. sera from age-matched normal dogs comprised a control population. an elisa using azidothymidine as a tk1 substrate was used. comparisons between groups were made using 2-tailed student t-tests, and receiver-operator characteristic (roc) curves were generated. sixty-two patients and 39 normal controls were studied. there were 35 dogs with hsa, 10 dogs with other splenic neoplasia, and 17 dogs with benign diseases. using a training set of 24 normal dogs, a cutoff of 6.55 u/l was established from the roc curve. tk activity was significantly higher (p o 0.0001) in dogs with hsa than in the validation set of 15 normal dogs (mean1/àsd 5 17.711/à4.5 and 2.011/à0.6 respectively), but not between dogs with hsa and benign splenic disease (mean1/àsd 5 7.021/à3.7, p 5 0.13). using a cutoff of 6.55 u/l, tk activity demonstrated a sensitivity of 0.54, specificity of 0.76, positive predictive value of 0.83 and negative predictive value of 0.45 for distinguishing hsa versus benign splenic disease. when interval thresholds of o 1.55 and 47.95 u/l were used together, diagnostic utility was markedly increased for distinguishing both hsa versus normal and hsa versus benign disease. in conclusion, serum tk evaluation may assist in detection of canine hsa, and may also discriminate between benign disease and hsa in dogs with hemoabdomen and a splenic mass. t cell chronic lymphocytic leukemia (cll) is a heterogeneous disease that affects a number of dog breeds. cll patients have variable disease outcomes. the objectives of this study were to use gene expression profiling of cd8 t cell leukemias with variable outcomes in order to identify markers that can be used in routine diagnostic tests to distinguish good from poor prognosis disease, and to identify potential targets for novel therapy. gene expression profiling of 12 cd8 t cell leukemias (7 good, 5 poor prognosis) was conducted. samples from 6 normal dogs were also profiled. several differentially expressed genes were found including cd9, cd94, and cd 25. these were selected for further study using flow cytometry to determine expression of protein on the cell surface. seventy nine cases of cd81 t cell leukemia were screened for cd 9 expression. forty seven had associated outcome information. based on analysis to date, cd9 expression as assessed by flow cytometry does not appear to provide prognostic information. a monoclonal antibody to cd25 was recently made available. to date 33 patients with cd8 t cell leukemia have been profiled. cd25 is variably expressed on t cell leukemias compared to normal cd8 t cells. cd25 is the receptor for interleukin 2. cyclosporin, a commonly used immunosuppressive drug, inhibits il-2 production, and has been used to treat a subset of t cell leukemias in people. thus, the finding that cd25 is up regulated on t cell leukemias compared with normal t cells suggests a possible new therapeutic avenue. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of dogs. there is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (ibd). similarly, compositional changes of the intestinal bacterial ecosystem have been associated with ibd in humans. the aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders using a next generation sequencing technique. fecal samples were obtained from healthy dogs (n 5 31), dogs with acute uncomplicated diarrhea (n 5 7), dogs with acute hemorrhagic diarrhea (ahd; n 5 13), and dogs with active (n 5 8) and therapeutically controlled ibd (n 5 10). the bacterial composition was analyzed by massive parallel 16s rrna gene 454-pyrosequencing. differences between groups were analyzed using mann-whitney u tests and kruskal-wallis tests followed by dunn's multiple comparison tests. statistical significance was set at p o 0.05. significant differences in the proportions of several bacterial groups were identified between healthy and diseased dogs. dogs with gastrointestinal disease had significantly higher proportions of proteobacteria (p o 0.01). proportions of firmicutes were lower in diseased dogs, but this difference did not reach significance (p 5 0.06). within the firmicutes the most notable findings were decreases in bacterial groups belonging to clostridium clusters iv and xiva (i.e., ruminococcus, dorea, and faecalibacterium spp.; p o 0.01 for all). dogs with ahd had the most profound changes of the microbiota, followed by dogs with acute uncomplicated diarrhea, and dogs with active ibd. faecalibacterium spp. was the bacterial group most prominently depleted in dogs with active ibd, but was not significantly different between healthy dogs and dogs with therapeutically controlled ibd (p 5 0.66). results of this study revealed bacterial dysbiosis in fecal samples of dogs with various gi disorders. bacterial changes were more profound in dogs with severe disease, but were not identified in dogs with therapeutically controlled ibd, suggesting that the microbiota is stable in non-active disease. the bacterial groups identified are considered to be important short chain fatty acid producers and may serve as candidates for the diagnosis or therapeutic monitoring of gi disease. future studies are necessary to determine if these microbial changes correlate with functional changes in the intestinal microbiota. ciprofloxacin oral tablets, available in a generic formulation for people, are widely used for treatment in dogs. oral absorption data for ciprofloxacin in dogs has been variable, and too limited to guide accurate dosing. subsequently, published doses for dogs in veterinary formularies have varied from 5 to 25 mg/kg. this study was undertaken to explore the factors that may affect oral absorption of generic ciprofloxacin in dogs, and to derive a pharmacokinetic-based dose for treating susceptible bacteria. six healthy adult beagle dogs were used for the study (11.2 kg mean weight). after placing jugular vein catheters for collecting blood samples, these dogs were administered either a single oral dose of ciprofloxacin (250 mg tablet; mean dose 23 mg/kg), or an intravenous (iv) dose (10 mg/kg; 2 mg/ml solution). a randomized crossover design was used with a washout time between treatments. blood was collected for plasma drug analysis for 24 hours. ciprofloxacin concentration in plasma was analyzed using high pressure liquid chromatography (hplc) and pharmacokinetics analyzed using a computer program. oral absorption was also evaluated via deconvolution analysis. the oral dose was well-tolerated, but the iv dose produced transient vomiting and depression in some dogs. after the oral dose, the peak plasma concentration (c max ) was 4.4 mg/ ml (cv 55.9%), terminal half-life (t1/2) 2.6 hr (cv 10.8%), auc 22.5 mg á hr/ml (cv 62.3%), and systemic absorption (f) 63.7% (cv 41.6%). after the iv dose, the t1/2 was 3.7 hr (cv 52.3%), systemic clearance 0.587 l/kg/hr (cv 33.9%), and volume of distribution 2.39 l/kg (cv 23.7%). after examining the pharmacokinetic results from the oral dose, it was apparent that oral ciprofloxacin was absorbed well in some dogs (approximately 80%), but poorly in others (approximately 30%). to explore the factors that may have affected oral absorption, two high absorbers and two low absorbers were administered an additional oral dose as a 10 mg/ml solution (250 mg total dose) via gastric tube. after administration of the oral solution, the plasma concentrations were more uniform and consistent among dogs. absorption of the oral solution of ciprofloxacin was 71.0% (cv 7%) with a t1/2 of 3.1 (cv 18.6%) hr and c max of 4.67 mg/ml (cv 17.6%). therefore, it appears that inconsistent oral absorption of ciprofloxacin in some dogs may be formulation-dependent, and affected by tablet dissolution in the canine small intestine. doses were calculated using the data for oral tablets in these dogs. the pharmacokinetic-pharmacodynamic (pk-pd) target was an auc/ mic ratio of 100. because of the wide range in oral absorption of tablets, a dose to reach the pk-pd target ranged from canine distemper (cd) is a highly contagious, acute or subacute systemic viral disease of dogs and other carnivores which can be controlled efficiently by the use of modified live-virus (mlv) vaccines. however, mlv strains do cross-react with molecular diagnostic tests and cause significant confusion for clinicians. the purpose of this study was to use quantitative real-time pcr viral load information to differentiate between vaccine virus used in mlv vaccines and wildtype infections in dogs. a real-time pcr test for cd virus (cdv) based on the p gene for phosphoprotein was used to determine viral loads in vaccinated and wildtype infected animals. a total of 158 respiratory mucosal swab samples from mlv vaccinated and asymptomatic dogs were obtained within the first 3 weeks after mlv vaccination. based on the viral load in vaccinated animals, a cutoff value was established for the differentiation of dogs with clinical signs of respiratory distress and presumably infected with a wildtype strain of cdv. two hundred clinical cases with known clinical and vaccination histories were analyzed to validate the cutoff value. the cdv real-time pcr proved to be of high analytical and diagnostic sensitivity: a standard curve was established using known numbers of cdv molecules to allow absolute quantitative cdv viral load data. the limit of detection was in the single molecule range while the limit of quantitation was established at around 10 molecules per pcr reaction. a comparison to ifa showed real-time pcr to be 30% more sensitive. the cdv viral load in vaccinated animals averaged 26,738 viral particles per swab. a cutoff value of 107,903 viral particles was calculated by adding 3 standard deviations to the average value. this cutoff value correctly detected 95.2% of the vaccinated samples. acutely infected dogs with cdv compatible clinical signs have high viral loads normally several logs higher than the cutoff value. in dogs with clinical distress, recent cdv mlv vaccination but viral loads below the cutoff value, other infectious agents were detected by using a panel of real-time pcr tests. testing additional infectious agents in clinical settings is important in order to explain clinical signs when viral loads below cutoff values indicate that cdv is not the cause of clinical signs. in conclusion, quantitative real-time pcr is a sensitive, rapid and reliable test regardless of recent vaccination. the use of a cutoff value will be of significant help to discriminate between vaccine interference and wildtype infection in clinical settings. feline ureteral obstructions are a common urinary dilemma and traditional therapy is associated with substantial morbidity/mortality. feline nephrostomy tubes are reported as being effective when pelvic drainage is required. the biggest limitation is externalized drainage, requiring careful management to prevent infection/dislodgement. the development of an indwelling ureteral bypass using a combination locking-loop nephrostomy/cystostomy tube was modified from humans, resulting in permanent indwelling drainage, reduced complications, and improved quality of life. the objective is to describe the technical and clinical outcome using a novel device called a subcutaneous ureteral bypass (sub) in cats with ureteral obstructions. fifteen cats (16 kidneys) had a sub placed for: ureterolithiasis (9), ureteral stricture (1/à stones) (6), and ureteral stent rejection (2). the median pre-and post-procedure creatinine was 6 mg/dl (range: 2.8-13) and 2.5 mg/dl (range: 2.4-9), respectively. the median pelvis diameter pre and post-procedure were 15 (range: 7-28) and 5 mm (range 3.4-6), respectively. six french tubes were placed in 14, and 5 fr. in 2. the bypass remained indwelling for a median of 4240 days (range 64-4450). there were 3 major complications resulting in nephrostomy tube dislodgement (2) and port leakage (1) 5 days after surgery. one patient with severe coagulopathy developed a clot which resolved with tpa infusion through the port. no sub got occluded/obstructed long-term. overall, the use of a sub for cats with ureteral obstructions can be considered a functional option when other therapies have failed or are contraindicated, but shtime. oxidative stress is considered central to the pathogenesis of many systemic diseases. in humans, biomarkers of oxidative stress, antioxidant depletion and lipid peroxidation, have been correlated with disease severity and associated with poor clinical outcomes. therapeutic antioxidant supplementation with nac in glutathione (gsh)-deficient patients has shown clinical benefits, including repletion of intracellular gsh levels. we have shown that clinically ill dogs are gsh-deficient, and that gsh deficiency correlates with mortality, but it is not clear whether there are direct benefits of antioxidant intervention in these patients. the purpose of this randomized, investigator-blinded, placebo-controlled, prospective study was to evaluate the effect of nac to normalize blood antioxidants (rbc reduced gsh (rbc gsh), plasma cysteine (cys), serum vitamin e (vit e), and whole blood selenium (se)), reduce lipid peroxidation (urine isoprostane/creatinine ratio (u i/ c)), and improve illness scores (spi2) and outcome (survival to discharge) in clinically ill dogs. clinically ill client-owned dogs, admitted to the uw veterinary medical teaching hospital that did not receive blood transfusions, tpn, vitamins, or antioxidants were eligible for the study. dogs enrolled in the study were randomized to receive iv infusions q. 6 h. of either nac (1 â 140 mg/kg and 6 â 70 mg/kg) or equal volumes of 5% dextrose (placebo) over 48 hours. at the time of enrollment, and 2 hours following the final 48 hour infusion, blood and urine were collected to quantify rbc gsh, cys, vit e, and se concentrations; u i/c ratios; and calculate spi2 scores. rbc gsh and cys concentrations were quantified by hplc. commercially available hplc, atomic absorption spectroscopy, and eia were used to quantify vit e, se, and u i/c ratios, respectively. nonparametric statistical analyses were used, with results reported as medians and p o 0.05 considered significant. sixty-one ill dogs were randomized to either nac (n 5 30) or placebo (n 5 31). overall this group of ill dogs had significantly decreased rbc gsh (1.50 vs. 1.91 mm; p 5 0.0013), vit e (27 vs. 56 mg/ml; p 5 0.0002), and se (0.37 vs. 0.55 mg/ml; p 5 0.0308) levels and elevated u i/c ratios (969 vs. 398 pg/mg; p 5 0.0005) in comparison to healthy control dogs. dogs in the placebo group showed a significant further decrease in rbc gsh over the next 48 hours (1.48 to 1.42; p 5 0.035). nac supplementation significantly increased plasma cys levels (9.1 to 15.1 mm; p o 0.0001), and prevented a further decline in rbc gsh (1.55 to 1.58 mm; p 5 0.174). however, serum vit e (30 vs. 28 mg/ml), se (0.40 vs. 0.35 mg/ ml), u i/c ratios (946 vs. 806 pg/mg), spi2 scores (0.74 vs. 0.75), and outcome (81% vs. 76%) were not significantly different between the nac and placebo groups after treatment. the results of this study further support that clinically ill dogs experience oxidative stress, and suggest that antioxidant supplementation with nac within the first 48 hours of hospitalization prevents further rbc gsh depletion. further studies are necessary to investigate whether longer duration or combined antioxidant supplementation normalizes the redox state and impacts long-term outcome. diabetes mellitus in cats is very similar to type ii diabetes in humans, preceded by a period of insulin resistance. evaluating insulin resistance in a cat is a time consuming, expensive, and difficult procedure. there is a need for a simple biomarker based test predictive of insulin resistance. there is a biomarker based assay predicative of insulin resistance in humans. the purpose of this study was to evaluate the utility of this assay in overweight cats and show improvement in insulin sensitivity following weight loss and weight maintenance. the insulin resistance assay is based on the quantitative analysis of 5 metabolites (2-hydroxybuterate, creatine, palmitate, decanoylcarnitine, and oleoyl-lpc). a proprietary algorithm (metabolon, inc, durham nc) was used to generate a predictive rd (rate of disposal) value (normal range in cats 6.5-10.5). individuals with an rd value less than 6 will have a greater than 50% chance of being insulin resistant and an rd value less than 3 will have a greater than 90% chance of being insulin resistant. initial studies demonstrated that the rd values indicating insulin resistance in cats correlated with age, obesity and severity of diabetes as determined by histopathology and blood glucose levels. in a feeding study of 40 cats (4 39% vs. o 25% body fat) rd values improved from 6.19 ae 1.23 to 6.8411.38 (p 5 0.029). during weight maintenance, 19% body fat for 4 months, further improvement was observed (rd, 8.30 1 1.08 (p 5 9.2e-12)). these results demonstrate that long term weight maintenance following weight loss is critical for increasing insulin sensitivity in cats. the use of monoclonal antibodies and antibody fragments to directly target tumor antigens and neutralize their growth factors has shown promising results in human clinical trials. however, these targeted approaches have not been possible in dogs since specific tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. to overcome these obstacles, we have generated canine antibody phage display libraries from canine splenocytes. these libraries consist of single chain variable fragments (scfv) comprised of canine variable heavy (vh) and variable light (vl) immunoglobulin chains displayed on the surface of bacteriophage (fig. 1) . the antigen specificity within these libraries is diverse and recapitulates the antigen-experienced immunoglobulin repertoire of the dog. we can now use simple panning techniques to isolate scfv of canine origin that bind to either known targets or unknown targets which can then be identified using standard molecular techniques. canine hsa is a highly aggressive malignancy of vascular endothelial cells that affects large breed dogs. although there are no confirmed immunological targets for hsa, serum levels of vascular endothelial growth factor (vegf) are elevated in these patients and, as in many human cancers, vegf may represent an important therapeutic target for neutralization. we used simple panning techniques to screen canine scfv libraries generated from the spleens of dogs with hsa against canine vegf and successfully isolated 3 scfv clones that bind and neutralize canine vegf in vitro. these scfvs are now being taken into a murine model of canine hsa to determine whether they can inhibit tumor growth and metastases. in addition, we have panned the same antigen-experienced scfv phage display libraries against allogeneic primary canine hsa cells of low passage number to isolate canine-derived antibody fragments that can target malignant endothelial cell surface molecules. early results demonstrate enrichment of scfv phage libraries for malignant endothelial cell binders. these scfv can be readily linked to chemotherapeutic agents or other toxins and used to deliver high doses directly to the malignant cell. this novel approach aims to reduce side effects of systemic chemotherapy and augment therapeutic response. calcitriol, (vitamin d 3 ), has antineoplastic activity and acts synergistically to potentiate the antitumor activity of a diverse array of chemotherapeutics. ccnu, vinblastine, corticosteroids, and tyrosine kinase inhibitors, are used to treat canine mast cell tumors (mct). vitamin d receptor is expressed in the majority of canine mcts, suggesting a role for calcitriol in the management of dogs with these tumors. the purpose of our study was to examine the in vitro effects of calcitriol in combination with ccnu, vinblastine, imatinib, or toceranib on canine mastocytoma c2 cells. also, we evaluated the antitumor activity of dn101, a highly concentrated oral formulation of calcitriol, as single-agent treatment in dogs with naturally occurring mcts. c2 cells were incubated with serial dilutions of calcitriol (0.1-25 nm). twenty-four hours later, cells were then treated with vehicle control or serial dilutions of ccnu (2.5-20 um), vinblastine (1.25-10 nm), imatinib (1.67-12.5 nm), or toceranib (3.13-25 nm). cell viability was assessed with an mtt assay after 48 hours and data was used to derive a combination index (ci: values o 1, 1, 41 indicate synergism, additivity, antagonism, respectively). in the phase ii clinical trial, dogs were eligible if they had at least 1 measurable, histologically confirmed, mct. calcitriol was administered orally. recist criteria were used to assess tumor response. calcitriol, ccnu, vinblastine, imatinib, and toceranib each suppressed c2 cell viability in a dose-dependent manner. ci values o 1 were obtained for calcitriol (0.1-6.25 nm) combined with ccnu (5 and 10um), vinblastine (2.5 and 5 nm), imatinib (1.67-12.5 nm) and toceranib (0.1-12.5 nm). due to the occurrence of toxicity (vomiting, anorexia, hypercalcemia), the phase ii trial was terminated early; only 10 of 20 planned patients were treated. one dog with a metastatic muzzle mct had a complete response that lasted 89 days. three dogs achieved partial response lasting from 74-90 days. in summary, our in vitro data demonstrate that calcitriol combined with ccnu, vinblastine, imatinib or toceranib has synergistic effects on c2 mastocytoma cells. antitumor responses were observed in dogs with spontaneously occurring mcts treated orally with single-agent calcitriol, but the frequency of adverse effects was high. together these results suggest calcitriol combination therapies might have significant clinical utility in the treatment of canine mcts but refinement of the calcitriol-dosing regimen must be done. cyclosporine is a potent immunosuppressive agent used to treat many canine inflammatory and immune-mediated diseases. cyclosporine has gained popularity as an immunosuppressive agent because of a favorable toxicity profile compared to many other immunosuppressive agents. optimal dosing regimens for cyclosporine in the dog remain unclear, primarily because standard methods that monitor effectiveness of immunosuppression have not been established. pharmacokinetic testing is currently used during treatment with oral cyclosporine to adjust doses based on measurement of blood drug levels. individual patients, however, often demonstrate marked variations in blood drug levels while on similar oral doses of cyclosporine, and can also demonstrate different clinical responses even at comparable drug levels, making correlation of blood cyclosporine levels and degree of disease control extremely difficult. pharmacodynamic testing offers an alternative method for regulating cyclosporine dosing by objectively measuring the effects of cyclosporine on t-cells, the drug's main cellular target in the body. our acvim foundation-funded research has focused on developing and evaluating a comprehensive panel of biomarkers of immunosuppression that can be utilized for pharmacodynamic monitoring during treatment with cyclosporine and other immunosuppressive agents that affect t-cell function. we have completed several studies using flow cytometry to evaluate activated t-cell expression of surface molecules (cd25 & cd95) and cytokines (il-2, ifn-g & il-4) as potential biomarkers. our first study was an in vitro study evaluating expression of surface molecules and cytokines in canine t-cells exposed to varying concentrations of cyclosporine. this study established consistent drug-associated suppression of the cytokines il-2, ifn-g and il-4. our second study was an in vivo study in normal dogs evaluating the effects of two doses of oral cyclosporine, a high dose considered to be reliably immunosuppressive (starting dose 10 mg/kg bid, titrated upwards as needed to attain trough drug blood levels of at least 600 ng/ml) and a lower dose used to treat atopy (5 mg/kg sid), on t-cell expression of these three cytokines. significant suppression of il-2 and ifn-g expression was seen at the high cyclosporine dose, while at the lower dose only ifn-g expression was suppressed. because tcell expression of il-4 was not significantly suppressed at the high cyclosporine dose, il-4 was not evaluated at the lower drug dose. because of specialized sample handling requirements, flow cytometry is not as practitioner friendly as other assays (such as pcr) for routine use in pharmacodynamic testing. we have therefore conducted an in vitro study comparing the effects of cyclosporine on activated t-cell expression of il-2 and ifn-g using flow cytometry and qrt-pcr, and demonstrated dose dependent and comparable suppression of il-2 and ifn-g using either methodology. we are currently evaluating, using qrt-pcr, the effects of oral cyclosporine on t-cell expression of il-2 and ifn-g in normal dogs prior to moving on to pharmacodynamic trials in our clinic patients. effect of hypothyroidism on reproduction in bitches. dl panciera 1 , bj purswell 2 , ka kolster 2 , sr werre 3 . departments of 1 small animal clinical sciences, 2 large animal clinical sciences, and 3 laboratory for study design and data analysis, virginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va. numerous reproductive abnormalities, including irregular interestrous period, anestrus, and infertility have been attributed to hypothyroidism. we previously documented reduced fertility and lower birth weight and increased periparturient mortality in pups born to bitches with experimentally-induced hypothyroidism for a median duration of 56 weeks. the purpose of this study was to evaluate reproductive function in these same bitches after hypothyroidism was treated with a replacement dose of levothyroxine. twelve multiparous bitches were studied. hypothyroidism was induced in 6 dogs by administration of 1 mci/kg 131 i. hypothyroidism was confirmed by finding serum t4 concentrations before and 4 hours after iv administration of human recombinant tsh that were o 5 nmol/l. levothyroxine (0.02 mg/kd q 24 h) was administered to all hypothyroid bitches. six bitches served as euthyroid, untreated controls. dogs were evaluated daily for signs of estrus and were bred by 1 of 2 males when serum progesterone was !5 ng/ml. interestrous interval, gestation length, strength and duration of contractions during whelping, time between pups, number of live pups and stillbirths, viability of pups at birth, weight of pups, and periparturient mortality were recorded. the student's t-test and anova were used to compare differences between control and hypothyroid bitches for continuous, normally distributed data. the wilcoxon rank sum test was used to analyze data between groups that was not normally distributed. the mean duration of hypothyroidism prior to levothyroxine administration was 102 ae 8.1 weeks. breeding took place after levothyroxine treatment for 46 ae 12.6 weeks in the hypothyroid group. all 6 dogs in the hypothyroid group and 5/6 control dogs were pregnant, while 4/8 hypothyroid and all 6 control bitches became pregnant prior to levothyroxine administration. no difference in interestrus interval or gestation length was noted between groups. during whelping, no difference in strength of contractions, contraction duration, interval between pups, or viability scores of pups was found between groups. litter size, birth weight and peirparturient mortality were similar between groups. levothyroxine administration reverses the detrimental effects of hypothyroidism on fertility and neonatal health. racing sled dogs have a high prevalence of exercise-induced gastric erosions/ulcers, with reports ranging from 50-67% of dogs running at least 100 miles in a day or less. omeprazole reduces the severity of, but does not completely prevent, gastritis under racing conditions, and can be difficult to administer under these conditions. famotidine can be administered in food, but has only demonstrated efficacy under less intense training conditions. the purpose of these studies was to evaluate different acid suppression strategies under racing conditions for the prevention of exercise-induced gastritis. experiment #1 was a randomized placebo-controlled study using 36 sled dogs (3-8 years) competing in a 330 mile race over 50-60 h. treatment groups were famotidine (approx 1 mg/kg qd) or no treatment, beginning 2 days prior to the start of the race and proceeding until gastroscopy was performed 24h after the race. experiment #2 was a randomized positive-control study using 52 sled dogs (2-8 years) running a mock race of 300 miles in 50h. dogs were divided into omeprazole (approx 1 mg/kg qd, administered 30 min prior to a meal) or famotidine (approx 2 mg/kg bid) groups beginning 2 days prior to the exercise challenge and continuing for 24h after completion. gastroscopy was performed immediately prior to the start of dosing and 24h after completion of the exercise. in all cases, mucosal appearance during gastroscopy was blindly scored using previously described scoring system. famotidine (1 mg/kg qd) reduced the prevalence of clinicallyrelevant, exercise-induced gastric lesions compared to no treatment (7/16 vs 11/16, p 5 0.031). compared to famotidine at 2 mg/kg bid, omeprazole significantly decreased the severity (0.4 vs 1.2, p 5 0.0002) and prevalence (2/23 vs 7/21, p 5 0.049) of gastric lesions. although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, neither the recommended dose nor the higher dose were considered acceptable in the prevention of exerciseinduced gastritis as between 33-44% of the dogs receiving famotidine had clinically significant lesions. a previous study examining omeprazole under racing conditions, but without careful administration on an empty stomach, resulted in a 22% prevalence of clinically significant gastric lesions. however, the bioavailability of omeprazole is reduced in the presence of food, and when the daily administration of the drug is carefully scheduled to coincide with an empty stomach, the resulting prevalence of clinically significant lesions induced by racing-intensity exercise is reduced to just over 10%. the conclusions of these studies are that omeprazole is superior to famotidine in preventing gastritis in racing sled dogs during competition. routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing alaskan sled dogs. mares may be an important source of environmental contamination with rhodococcus equi on breeding farms. attempts to reduce fecal shedding of r equi by the mare and the effects of the mare's fecal r equi concentration on airborne concentrations in the foaling stall have not been previously reported. twenty-one arabian mares were treated daily with either oral gallium nitrate or placebo in a randomized double-blind study. fecal samples were collected at day 320 of gestation (time 1), the week before foaling (time 2), and the week after foaling (time 3). airborne concentration of r equi were measured in the stall within 6 hours post foaling using a microbial air sampling system into which standard (100-mm) culture plates with a media selective for r. equi have been loaded. concentration of total r equi were determined by morphological characteristics. the concentration of virulent r equi was determined using a modified colony immunoblot method. concentrations of total and virulent r equi were compared among mares to examine effects of treatment, time, and treatment by time interaction. there were significant (p o 0.05) effects of treatment that depended on time of sample collection. at sample times 1 and 2 there were no significant differences between groups in the fecal concentration of virulent r equi. at time 3 concentrations of virulent r equi were significantly lower among mares in the treatment group (p o 0.05) compared to control. effects of time depended significantly on groups: for the control group, there were no significant effects of time. for the treatment group, concentrations tended to decrease over time, and concentrations at time 3 were significantly (p o 0.05) lower than those at time 1. no other differences among times for concentrations in the treatment group were statistically significant. there were no significant effects of treatment, sample time, or their interaction on the concentration of total r equi between groups; however, the pattern for these data was similar to that observed for the virulent isolates. no significant differences were determined between treatment groups for airborne concentrations of virulent or total r equi. treatment of mares with oral gallium nitrate significantly reduced the fecal concentrations of virulent r equi over time, but had no impact on the airborne concentration of r equi shortly after foaling. the purpose of this study was to evaluate the protein profile of bronchoalveolar lavage fluid (balf) in horses affected with recurrent airway obstruction (rao) and in control horses using proteomics and western blot techniques. rao-affected (n 5 5) and control horses (n 5 6) were subjected to an experimental exposure trial; when the rao-affected horses showed clinical signs of disease, balf was collected from all horses. balf was also collected from client-owned rao-affected horses (n 5 15) with naturally-occurring clinical signs of disease and client-owned control horses (n 5 12) from the same environments. the balf from the experimental exposure trial horses was subjected to trypsin digestion and proteomics analysis with mass spectrometry (ms). peaks detected with ms were identified using tandem ms analysis and database searches. western blot was used to confirm the identity and expression levels of two proteins identified using proteomics techniques in the balf of all horses. data from ms experiments were analyzed with the student's t-test to compare peak intensity between rao-affected and control horses. western blot band density data was analyzed with the kruskal-wallis anova for comparison between groups of horses. significance level was set at p o 0.05. with ms proteomic analysis of the balf from the experimental exposure trial horses, 2049 total peaks (peptides) were identified. of these peaks, 100 were differentially expressed between the rao-affected (24 over-expressed) and control horses (76 over-expressed). identifications were made for 250 balf proteins. transferrin and secretoglobin were chosen for validation with western blot. proteomics indicated that secretoglobin was not differentially expressed between the experimental exposure trial group; this was confirmed with western blot analysis. western blot also showed that clientowned rao-affected horses had lower secretoglobin expression than client-owned control horses and control horses before experimental exposure. according to the proteomics data, transferrin was over-expressed in control horses after experimental exposure compared to rao-affected horses. while the western blot analysis did not show a statistically significant difference in this comparison, transferrin was significantly over-expressed in control horses before experimental exposure compared to client-owned rao-affected horses. in addition, both secretoglobin and transferrin band densities on western blot were negatively correlated with airway obstruction and neutrophilic pulmonary inflammation. this study demonstrates that proteomics techniques can be used in the investigation of equine balf proteins. the proteins identified as differentially expressed between rao-affected and control horses in this study including, but not limited to, secretoglobin and transferrin should undergo further evaluation for their use as biomarkers of rao, and as potential targets of new therapeutic agents for rao. cardiotoxic effects of rattlesnake venom in the horse are not well defined. the first aim of this study was to document cardiac damage in naturally envenomated horses. twenty horses with clinical diagnosis of snake bite were included. a snake venom elisa was utilized to confirm envenomation when possible. serum and plasma were collected at selected intervals. plasma was assayed for cardiac troponin i (ctni) using a flurometric assay (stratus cs s , dade behring). holter monitors (zymed s , philips) were placed at admission, 1 week and 1 month post presentation. echocardiography was performed on available horses 5-7 months after envenomation. the second aim of this study was to investigate potential mechanisms of the cardiac damage. serum samples were assayed for tnfalpha using a commercial assay (endogen). antibody titers to crotalus atrox venom were measured at admission, 1 week and 1 month after natural envenomation and compared to titers in vaccinated horses (crotalus atrox toxoid, red rock biologics). a significant number of horses showed elevations in ctni (p o 0.05) at one or more time point indicating myocardial damage. holter readings revealed the presence of arrhythmias or persistent tachycardia in 19 horses. five of twenty horses were available for echocardiography; no abnormalities were noted. horses with increased ctni tended to have greater tnfalpha concentrations compared with horses without increased ctni. peak venom titers in bitten horses were significantly higher than peak titers in vaccinated horses (p o 0.05). rattlesnake envenomation was associated with evidence of cardiac damage in a significant proportion of bitten horses. further studies are needed to determine the cause as well as mechanisms to treat and/or prevent its occurrence. little is known about the gastric mucosal flora in healthy horses and its role in gastric disease has not been critically examined. our laboratory previously reported that a diverse microbial flora with a predominance of streptococcus spp. and lactobacillus spp. exists in healthy horses using fluorescence in situ hybridization (fish). the present study sought to further characterize the gastric mucosal flora of healthy horses using massive parallel 16srrna bacterial tag encoded flx-titanium amplicon pyrosequencing (btefap). biopsies of the squamous, glandular, antral and any ulcerated mucosa were obtained from 4 healthy horses via gastroscopy after a 12-hour fast and 2 horses immediately post-mortem. dna was extracted from the mucosal biopsies and btefap and data processing was performed. hierarchical cluster analysis based on relative abundance data on the genus level were performed to look for trends in bacterial diversity among the individual horses. pyrosequencing yielded between 4,500 and 13,000 reads per horse with 9238, 16587, 16587 reads in the antrum, squamous and glandular regions, respectively. the microbiome segregated into two distinct clusters: cluster 1 comprised of 2 horses that were stabled, fed hay and sampled at post-mortem and cluster 2 consisted of 4 horses that were pastured on grass, fed hay and biopsied gastroscopically after a 12-hour fast. samples from different antomic regions clustered by horse rather than region. despite being very similar at the higher taxonomic level (phyla) differences in the distribution of bacteria were seen at the genus and species level. the dominant bacteria in cluster 1 horses were firmicutes (483% reads/sample) consisting of mainly streptococcus spp., lactobacillus jensenii, l. fornicalis and sarcina maxima. cluster 2 had more diversity with a predominance of proteobacteria, bacteroidetes and firmicutes and 51 genera identified such as streptococcus spp., moraxella spp., actinobacillus spp., and others. though the relative abundance of the individual taxonomic groups was significantly different between individual horses, no significant differences in the overall diversity could be found (as assesed by shanon weaver, ace and choa i diversity indices). helicobacter spp. sequences were not identified in any sample (out of 58,891 reads). the ulcerated mucosa from horse 3 (group 1) had lower diversity and higher numbers of bacteria predominated by lactobacillus equigenerosi. this data shows that the equine gastric mucosa harbors an abundant and diverse microbiome which is unique to each individual and differs by sampling method, fasting prior to sampling and diet. seasonal pasture myopathy (spm; atypical myopathy [am] in europe), typified by nonexertional rhabdomyolysis, occurs in pastured horses during autumn or spring. clinical signs rapidly progress from muscular weakness to recumbency and frequently death. extensive myonecrosis and intramyofiber lipid storage occur in highly oxidative respiratory and postural muscles. recently, a defect of lipid metabolism called madd has been identified in european horses with am. this report documents the first cases of equine madd in the united states. six midwestern us horses suspected of having spm in the spring or fall of 2009 were evaluated for madd by urine organic acids, plasma acylcarnitines and/or muscle carnitine and histopathology. five horses had clinical signs and clinicopathologic data consistent with severe rhabdomyolysis. one horse was found dead on pasture after 2 days of rear limb stiffness and inappetance. urinary organic acid profiles revealed markedly elevated ethylmalonic and methylsuccinic acids, butyrylglycine, isovalerylglycine, and hexanoylglycine, consistent with equine madd. plasma acylcarnitine profiles from 2 horses had marked elevations of short chain acylcarnitines, while the third horse and only survivor had minor elevations of short chain acylcarnitines. affected muscle showed extensive degeneration with intramyofiber lipid accumulation, a marked decrease in free carnitine, and high levels of carnitine esters. spm appears to be a highly fatal emerging disease of pastured horses in the us characterized by weakness, colic-like signs and myoglobinuria. the disease is associated with a defect in muscular lipid metabolism that can be diagnosed by performing lipid staining of muscle samples and urine organic acid profiles. candidatus mycoplasma haemolamae (cmhl) is a common red blood cell parasite of new world camelids. the high degree of parasitemia that develops in an infected splenectomized animal allows for the efficient collection of parasitic dna. this dna can then be used in the development of genetically-derived tools such as pcr and in-situ hybridization. thus, one splenectomized animal can replace many immunologically intact animals within a research setting. the purpose of this study was to track the natural progression of cmhl parasitemia and associated clinical signs in a splenectomized alpaca. an intact, 9-month-old, 39.1 kg male alpaca was used in this study. he had tested positive via pcr for cmhl on three different occasions, although no organisms were seen on peripheral blood smears. the alpaca was placed under general anesthesia and a ventral midline incision was made. the spleen was located, the vessels ligated, and the organ removed. buprenorphine and flunixin meglumine were given for 2 and 4 days after surgery respectively. body weight, attitude, rectal temperature, blood glucose, and pcv were recorded daily. in addition, a peripheral blood smear was examined daily and the percent of red blood cells that were infected with mycoplasma organisms was determined. the alpaca was not parasitemic prior to surgery. one percent of the rbc's contained mycoplasma on days 2 and 3 after splenectomy. parasitic bloom developed on day 4 with 85% of the red blood cells infected, and over 70% containing 3 or more organisms. the alpaca was treated with 20 mg/kg oxytetracycline i.v. on day 4. on postoperative day 5 no parasites were seen in the peripheral blood. the peripheral blood remained free of parasites for 11 days. on the morning of the 12 th day, 3% of the peripheral red blood cells contained mycoplasma. by late that afternoon, 75% of the observed rbc's contained 3-4 organisms. the alpaca again received oxytetracycline. there were no more parasites observed from that time until the alpaca was euthanized 5 days later. the alpaca lost 3.7 kg between days à1 and 12 after surgery. his weight fluctuated between 34.9 and 35.4 kg for the remainder of the study period. blood glucose ranged between 87 and 163 mg/dl there was no major change in pcv (range 21-29%), a finding that was expected as the spleen was not available to remove infected red blood cells. body temperature ranged between 38.1 and 39 degrees celsius except for days 4 and 16 when more than 70% of red blood cells contained parasites. on those days rectal temperature reached 39.4 and 39.1 degrees respectively. this study confirmed that a non-parasitemic, yet pcr positive alpaca did indeed harbor cmhl. the time from splenectomy to parasitic bloom was shorter, and the length of oxytetracycline suppression longer than has been observed in other species. gastro-intestinal (gi) disease frequently results in increased wall thickness in many species. identification of changes in gi wall thickness using ultrasound has proved to be a useful diagnostic tool and is widely used in human patients, small animals and horses. although gi motility has been evaluated in cattle, normal reference ranges for wall thickness has not been reported in ruminants. the aims of this study were to report normal values for wall thickness of various gi structures and to assess the repeatability of this technique in adult dairy cows, sheep and goats. eight healthy adult holstein friesian (hf) cattle (656 ae 11 kg), eight jersey (j) cattle (458 ae 56 kg), thirteen adult sheep (79 ae 11 kg) and eleven adult goats (43.5 ae 8 kg) were recruited and examined on three consecutive days. ultrasonographic images were optimised for the structure of interest. structures were identified based upon appearance and anatomical position. a minimum of three cineloops were obtained of the abdominal organs per intercostal space (ics) and three along the ventral midline in each ics; images were analysed offline. data were analysed using anova and post-hoc bonferoni, student's ttest and intra-class correlation coefficients. each structure was measured per ics per species; if no differences were noted for structures in different ics, then measurements were pooled. no differences were noted between hf and j cattle so data were pooled. data are displayed in table 1. good repeatability (icc40.91) was obtained for all measurements and no differences were noted between animals of the same species or between days. these measurements for assessment of normal gi thickness are repeatable and may allow valuable additional information to be gained from ruminants with gi disease. ocular infections with the infectious bovine keratoconjunctivitis (ibk) agent moraxella bovis (m. bovis) are associated with significant economic loss in the cattle industry.although antibiotic therapy is the treatment of choice for ibk, treatment failures are common and current vaccines are not optimally effective mainly due to antigenic variation. as a result, our laboratory has been actively investigating the therapeutic potential of bdellovibrio bacteriovorus 109j (b. bacteriovorus); a predatory bacterium capable of attacking and inducing lysis of gram-negative bacteria, as a new treatment for ibk. we have previously shown that b. bacteriovorus can reduce the number of m. bovis attached to bovine epithelial cells in an in vitro model of ibk and that b. bacteriovorus can be trained to kill m. bovis as effectively as e. coli using serial passages. in this study, we hypothesized that b. bacteriovorus can remain viable in bovine tears without its prey for up to 24 hours. this hypothesis was addressed by incubating inocula of active b. bacteriovorus in its preferred media peptone yeast extract (pye) and comparing b. bacteriovorus viability in bovine tears or phosphate buffered saline (pbs) at time 0, 2, 4, 12, and 24 hours. using a plaque assay to quantify the mean amount of plaque forming units (pfus) of b. bacteriovorus exposed to each treatment, it was determined that viability of b. bacteriovorus over time was comparable between treatment groups. overall, the results supported that b. bacteriovorus can remain viable in tears for up to 24 hours in the absence of prey bacteria. further studies are needed to determine the therapeutic potential of b. bacteriovorus in an in vivo model of ibk. correction of the measured ionized calcium concentration (cca 21 ) to a ph 5 7.40 is routinely applied in experimental studies in order to assist in the interpretation of measured values relative to a reference range. the equation most commonly used for ph correction in bovine plasma is: cca 21 ph 5 7.40 5 cca 21 â10 (-0.24â{7.40 -ph}) . the validity of this equation for bovine plasma is unknown. accordingly, our first objective was to characterize the in vitro relationship between cca 21 and ph for bovine plasma. feeding rations with a low dietary cation-anion difference (dcad) during late gestation mitigates periparturient hypocalcemia in dairy cows, particularly when chloride containing acidodgenic salts are fed. the mechanism for this beneficial effect remains unclear. our second objective was to determine whether hyperchloremia displaces calcium from binding sites to albumin, thereby increasing cca 21 . the in vitro relationship between plasma log(cca 21 ) and ph in was investigated using lithium heparin anticoagulated blood from 10 healthy holstein-friesian calves. plasma was harvested and tonometered with co 2 at 371c over a ph range of 7.10-7.70. plasma chloride concentration (ccl -) was altered by equivolume dilution of plasma with 3 electrolyte solutions of varying ccl -(97 ae 1, 110 ae 1, and 123 ae 1 meq/l; mean ae sd). the slope of the linear regression equation relating log(cca 21 ) to ph for 112 tonometered plasma samples from the 10 calves was -0.24 ae 0.05 at normal values for cca 21 (2.63 ae 0.09 meq/l), albumin concentration (30.9 ae 2.0 g/l), and ccl -(99.1 ae 2.2 meq/l). the experimentally-determined value for the slope for bovine plasma was identical to that determined previously for human plasma. the formula for correcting cca 21 in bovine plasma for change in ph from 7.40 is therefore: cca 21 ph 5 7.40 5 cca 21 â 10 (à 0.24 â {7.40-ph}) . this equation is only valid at normal concentrations of albumin and chloride in plasma. equivolume dilution of plasma by electrolyte solutions of varying cclindicated that cca 21 ph 5 7.40 increased by 0.007 meq for every 1 meq/l increase in ccl -. in other words, plasma cca 21 at a given ph increases directly in response to an increase in plasma ccl -, presumably because the additional chloride displaces calcium that is electrostatically bound to albumin. furthermore, the increase in cca 21 is independent to the change in plasma ph induced by an increase in ccland decrease in plasma strong ion difference. our finding that hyperchloremia directly increases plasma cca 21 provides an additional mechanism by which ingestion of high chloride (acidogenic) rations prevents the clinical signs of periparturient paresis. our finding is consistent with the results of other studies that indicate acidogenic salts that contain chloride as the predominant anion (ie, nh 4 cl, cacl 2 ) are more effective in increasing cca 21 than equimolar quantities of acidogenic salts such as mgso 4 . coagulase negative staphylococci (cns) are among the most common bacteria isolated from the bovine mammary gland. historically, these bacteria were lumped together as minor mastitis pathogens. modern molecular techniques have allowed accurate speciation and fingerprinting of the cns species. these methodologies have recently been applied to the study of cns in bovine mastitis. the aim of the studies presented here was to evaluate the role of individual cns species on milk somatic cell count (scc) and duration of intramammary infection (imi). in the first study, mammary quarter foremilk samples were aseptically collected from all lactating cattle ($180 head) at the university of missouri dairy research center once monthly for 17 months for bacterial culture and milk scc. staphylococcal isolates were speciated by sequencing the rpob gene and strain-typed using pulsed-field gel electrophoresis (pfge). using species and fingerprint data along with published definitions for staphylococcal imi, 91 cns imis were identified. overall, 11 species of cns were identified with staphylococcus chromogenes, s. cohnii, s. epidermidis, and s. simulans being most prevalent. duration of imi and scc data were analyzed using regression models accounting for repeated measures. mean milk scc and duration of imi were found to differ between cns species (p o 0.05). although most imis were of short duration (1 month), staphylococcus capitis and s. chromogenes imis had longer mean durations of infection than 3 or more of the other species isolated. mean sccs were under 350,000 cells/ml in most cases. however, staphylococcus simulans and s. xylosus imis were more inflammatory (mean 4 600,000 cells/ml) and had a higher mean scc than s. cohnii, s. epidermidis, and s. haemolyticus. to examine the relationship between cns imi and milk scc in a larger population of cattle, cns isolates from the canadian bovine mastitis research network (cbmrn) culture collection were obtained for speciation. speciation and fingerprinting were performed as above. isolates were from subclinical imi from before and after the dry period and from subclinical imi during lactation. data associated with each isolate were obtained from the cbmrn database. nine-hundred-thirty-eight isolates from 696 mammary quarters in 89 herds were successfully speciated. twenty-two different species of cns were identified. staphylococcus chromogenes was the most frequent species identified accounting for 40% of the infections. three species, s. chromogenes, s. xylosus, and s. simulans accounted for 475% of all infections. data were analyzed using a linear hierarchical repeated measures mixed model. differences in mean scc were found between some cns species and culture negative control quarters and also between different species of cns (p o 0.05). overall, our data demonstrate potential differences in pathogenicity between strains of cns that cause bovine mastitis. passive transfer of maternally derived antibodies via ingestion of good-quality colostrum within the first 24 hours of life is crucial for the health and future productivity of dairy calves. however, infectious diseases can be transmitted via colostrum feeding, which may require use of a colostrum replacement product or pasteurization to decrease disease transmission. while pasteurization of colostrum is effective for sterilization, heating during pasteurization can alter the viscosity of colostrum, destroys important nutritional biomolecules, and has been shown to decrease colostral igg concentrations. the purpose of this study was to investigate the effect of high pressure processing (hpp) on the viscosity, igg concentration, and bacterial contamination of bovine colostrum. first milking colostrum samples were collected from 18 cows from 3 different farms, and 50 ml aliquots of each sample were pooled for analysis. pooled colostrum was processed in triplicate using an isostatic press at 400 mpa (60,000 psig) for 0, 5, 15, 30, and 45 minutes. samples were tested for the effects of hpp on the viscosity, bacterial load (cfu/ml), and igg concentration. there was a significant decrease (p o 0.05) in bacterial load at each time point when compared to time 0. no significant difference in igg concentration was found between any time points. subjectively, the colostrum viscosity appeared to increase with the processing time, though the rheologic assessment has not been completed at this time. hpp appears to be an effective method to decrease bacterial contamination of colostrum while maintaining appropriate igg concentrations. minimizing the processing time or pressure may be necessary to maintain an acceptable viscosity of the colostrum. based on these results, additional studies are justified in order to determine the optimum combination of processing time and pressure and the effect of hpp on specific bovine pathogens. the heme-associated iron-binding apoprotein lactoferrin (lf) is known for its, anti-inflammatory, anti-parasitic, antimicrobial and bactericidal effects. lactoferrin demonstrates ubiquity throughout mammalian host biological fluids: saliva; tears; mammary secretions, as well as at mucosal surfaces. it is also released from immune cells under pathogenic stimulation. the purpose of this study which has been approved by western university's institutional animal care and use committee is to further characterize the mechanisms through which lf modulates inflammation in the face of bacterial endotoxin. it was hypothesized that lf would inhibit p38 phosphorylation. numerous studies speak to the ability of lf to alter leukocyte function, inhibit cytokine production, and bind lipopolysaccharide (lps); mechanisms through which it is believed to achieve its anti-inflammatory effects. recently, investigators demonstrated its ability to interact with host dna while others describe regulation of granulocyte adhesion and motility; elucidating its roles in the apoptotic signaling. in earlier studies, dawes me, et al. demonstrated lactoferrin's ability to limit the expression of inducible cyclooxygenase-2 and the gelatinase, matrix metalloproteinase -9 by lps-induced macrophages. the generation of these inflammatory mediators is modulated by pro-inflammatory cytokines such as interleukin-1 b (il-1 b) and tumor necrosis factor-alpha (tnf-a), the production of both being dependent on signaling through the p38 mitogenactivated protein kinase (mapk) pathway. peripheral mononuclear cells (5 â 10 6 )isolated from buffy coat cells of healthy neonatal to 4-month old holstein calves were cultured in the presence and absence of lf (200 ng/ml), lps (1 mg/ml), anisomycin (25 mg/ml), a known p38 activator -the positive control, and 50 mm of sb203580, a known p38 inhibitor -the negative control. sample lysates obtained post culture was subjected to immunoprecipitation and kinase reactions. reactions were terminated under reducing conditions and evaluated using western immunoblotting. phosphorylation of activated transcription factor-2 (atf-2) by phosphorylated p38 served as the marker of investigation. immunologically reactive atf-2 expression by lps and anisomycin-treated cells was compatible with a prominent band at 40 kd. evidence of lf-induced inhibition of lps-induced p-38 activation was observed in lanes representative of co-cultures of lf 1 lps; lf 1 anisomycin; and anisomycin 1 sb203580, which was demonstrated by decreased immunological reactivity at 40kd. the findings here, suggest that lf interferes with lps-induced p-38 activation of transcription factor atf-2, in vitro. this serves as additional proof of its potential use in attenuating the systemic effects of lps. six (6) clinically normal, purpose-bred cats of similar age and body condition were imaged with [ 18 f] fluorodeoxyglucose ([ 18 f]fdg) and [ 18 f]ftha by using dynamic cardiac-gated fused pet/ct for kinetic assessment of myocardial glucose and fatty acid uptake and metabolism, respectively. kinetic tracer uptake within the myocardium was achieved by initiating image data acquisition simultaneously with tracer injection. pet data were acquired over a 1 hour period with the heart in the center of the scanner field of view. regions of interest were drawn in the left ventricular wall and thoracic aorta for the purpose of measuring the kinetics of tracer redistribution. serial blood samples were also taken during pet imaging for comparison with image data. the equilibrium biodistribution of both tracers was documented 1 hour post-injection in a whole body pet/ct image. standard echocardiographic examination of cardiac structures was also performed. both radiotracers remained in the plasma fraction; however, [ 18 f]ftha was cleared from the more rapidly than [ 18 f]fdg (t 1/2 $ 2 and $ 20 min, respectively). the tracers were readily visualized within the feline myocardium in dynamic pet images and analysis of the blood pool clearance from the kinetic image data agreed with blood sampling data. myocardial uptake of each tracer was best described by a double exponential analysis and was rapid but variable among animals (range 1-30 bq/cc/min), although blood glucose levels were similar in all cats during image acquisition. physiologic [ 18 f]fdg was observed in the brain, salivary tissue, gastrointestinal tract, renal pelves and urinary bladder, with [ 18 f]ftha seen in the myocardium, liver and renal cortex. all cats were normotensive with normal echocardiographic parameters. this study demonstrates the utility of kinetic imaging using the left ventricle (lv) shape has been suggested to change from elliptical to more globular in response to chronic volume overload. real-time three-dimensional echocardiography (rt3de) offers new modalities for lv assessment. the aim of the study was to investigate left ventricular changes in shape and volume occurring in response to different severities of naturally acquired myxomatous mitral valve disease (mmvd) in dogs using rt3de. privately owned dogs were classified by standard echocardiography into: healthy (20), mild (20), moderate (8) and severe mmvd (17). a lv cast was obtained using semi-automated endocardial border tracking from rt3de dataset, from which global and regional (automatically acquired basal, mid, and apical segments based on lv long-axis dimension) end-diastolic (edv) and endsystolic volumes (esv), lv long-axis dimension and rt3de sphericity index, were derived. global and regional edv and esv increased significantly with increasing mmvd severity, assessed by mmvd group-wise comparisons and linear regression analyses using left atrial to aortic root ratio, and lv end-diastolic and end-systolic dimensions. all three segments contributed to the overall increased global volumes, but the mid edv segment was strongest associated with increasing lv end diastolic dimension (p 5 0.048). furthermore, lv long axis distance and lv sphericity index increased with increasing mmvd severity. the basal and apical edv segments were strongest associated with sphericity index (p o 0.0001). in conclusion, this rt3de study showed that increased lv edv, primarily in the mid segment, leads to rounding of lv apical and basal segments in response to increasing mmvd severity in dogs. 84 dogs from shelters in florida with naturally acquired di infection were euthanized and necropsied. all adult di in each dog were sexed using morphological features. total worm burdens and numbers of males and females were recorded. no other information was available for any dog. all data, raw and transformed, were examined visually and descriptively. raw numerical data were further examined by a paired t-test; log-odds transformed data were examined by logistic regression. we also conducted a binomial distribution goodness of fit analysis assuming a null hypothesis of a m:f 5 1.0. worm intensities ranged from 1 to 143 di per dog. eight dogs had unisex infections: 7/8 had all-female infections. dogs with lowintensity dual-sex infections were more likely to have greater numbers of female di. overall, sex ratios were equal (paired t-test, p 5 0.7). however, logistic regression demonstrated that the probability of being female is strongly affected by the total worm intensity, with lower intensities increasing the probability of having a predominance of female worms. our data show that di sex ratios in naturally-infected dogs equal 1 when examining the entire dog population, but deviate to favor female worms at low worm intensities. these data could impact adulticide treatment strategies. the reasons for sex ratio distortion in di are unknown. we evaluated cardiac reverse remodeling after mitral valve repair under cardiopulmonary bypass (cpb) for mitral regurgitation in small breed dogs. fifty dogs (body weight 1.8-9.3 kg, age 5-14 years) with mitral regurgitation were treated between august 2006 and november 2009. the cardiac murmur was grade 4/6-6/6. the preoperative chest x-rays showed cardiac enlargement (vertebral heart scale (vhs) 11.0-13.1). echocardiography showed severe mitral regurgitation and left atrium enlargement (la/ao 2.0-4.2). after inducing anesthesia, a thoracotomy was performed in the fifth intercostal space. cpb was started by using a cpb circuit connected to carotid artery and jugular vein catheters. after inducing cardiac arrest, the left atrium was sectioned and chordae tendineae rupture confirmed. the chordae tendineae were replaced with expanded polytetrafluoroethylene. a mitral annulus plasty was also done, and the left atrium was closed. after de-clamping for restarting the heart, the chest was closed. heart rate decreased from 118-164 bpm to 75-138 bpm. the grade of cardiac murmur was reduced to 0/6-3/6 three months postoperatively, and the heart shadow was reduced (vhs 9.8-11.5) in the chest x-rays. echocardiography confirmed the marked reduction in mitral regurgitation and the left atrial dimensions (la/ao 1.2-2.2). mitral valve repair reduced enlarged cardiac size by reduction of regurgitant rate. pulmonary arterial hypertension (pah) is a well recognized condition in dogs leading to considerable morbidity and mortality. the majority of therapeutics has focused on endothelial dysfunction causing reduced production of vasodilators, such as nitric oxide and prostacyclin, coupled with overproduction of vasoconstrictors, such as endothelin-1. more recently, it has been shown that the mitochondria play an important role in the development of pah as oxygen sensors and regulators of cellular proliferation. in pah, pulmonary artery smooth muscle cells undergo a metabolic shift from oxidative phosphorylation in the mitochondria to glycolysis in the cytoplasm as the major energy source and this leads to suppression of apoptosis and increased proliferation. dichloroacetate (dca) inhibits pyruvate dehydrogenase kinase to activate pyruvate dehydrogenase which catalyzes the rate limiting step for entry of pyruvate into the krebs cycle, thus increasing mitochondrial respiration. in three different rat models of pah, dca has been shown prevent and reverse pah by normalizing molecular pathology, stimulating apoptosis of pulmonary artery smooth muscle cells, and reducing pulmonary artery hypertrophy. dca has known toxic effects, including reversible hepatotoxicity and peripheral neuropathy, and has not been studied in any species with naturally occurring pah. the objective of this open label pilot study is to evaluate the therapeutic and toxic effects of dca in naturally occurring canine pah. three dogs with pah diagnosed by doppler echocardiography and no correctable underlying cause are enrolled in the study. dogs are orally administered 25 mg/kg of dca divided daily for 2 weeks, and then 12.5 mg/kg of dca divided daily for the remainder of the study. at baseline, 2, 4, 8, and 12 weeks, an echocardiogram, cbc, serum chemistry profile, urinalysis, nt-probnp, blood uric acid, blood lactate, noninvasive blood pressure, nerve conduction study, and trough dca level (12 hr post-dose) are obtained. the measured echocardiographic parameters include peak and mean tricuspid regurgitant flow velocity and pressure gradient, peak and enddiastolic pulmonary regurgitant flow velocity and pressure gradient, pulmonary valve flow velocity acceleration time and ejection time, pulmonary valve flow velocity time integral, right ventricular myocardial performance index, tricuspid annular plane systolic excursion, and systolic tricuspid annular tissue velocity. variables are inspected for normalcy and equality of variances and a two-sided paired t-test is used to compare the variables before and after treatment at each evaluation time. the basis for the role of the mitochondria in pah and the results of this pilot study will be presented to determine if dca warrants further study as a therapy for dogs with pah. study produced the strongest associations between the ncl phenotype and cfa2 markers. all 19 ncl-affected tibetan terriers were homozygous for the same haplotype which extended for 22 consecutive snps spanning 0.95 mb. none of the 20 annotated genes within this target region had previously been associated with human or rodent ncl. we used dna from ncl-affected tibetan terriers to resequence the coding regions and intron-exon borders of several genes harbored within the target region and found a single base pair deletion, c.1623delg, in exon 16 of positional candidate atp13a2. this deletion produces a frame shift and a predicted premature termination codon. we genotyped all 454 tibetan terrier dna samples in our collection and found all 45 ncl-affected tibetan terriers to be homozygous for the c.1623delg allele. eleven additional c.1623delg homozygotes were either less than 5 years old, or lost to follow up. there were no known cases of ncl in the remaining 398 tibetan terriers which were either heterozygous (n 5 149) or homozygous for the ancestral allele (n 5 249). atp13a2 is a member of group of ion transport genes and has been associated with lysosomes. mutations in human atp13a2 cause kufor-rakeb syndrome (krs), a rare neurodegenerative disorder with clinical features that include parkinsonism plus spasticity, supranuclear upgaze paresis, and dementia. post-mortem findings in krs have not been reported. we conclude that ncl in tibetan terriers is caused by a mutation in atp13a2. our results suggest that krs may be a form of adult onset ncl in humans. niemann-pick type c (npc) disease is a progressive neurological disorder characterized by dementia and ataxia, hepatic and pulmonary disease, and death typically within the first or second decade. despite the identification of causative mutations, the pathogenesis is not clear and therapies to successfully treat npc disease have been ineffective to date. the recent use of intravenously administered 2-hydroxypropylbeta-cyclodextrin (hpbcd), an fda-designated orphan drug (may 2010), in a small number of children with npc disease is based on favorable treatment outcome data in subcutaneously treated mouse and cat models. to rigorously evaluate the mechanistic, pharmacologic, and toxicity issues associated with hpbcd therapy in npc disease, we have utilized the spontaneous feline npc model harboring a missense mutation in npc1 (pc955s), orthologous to the most common mutation in juvenile-onset patients. the feline npc model has clinical, neuropathological and biochemical abnormalities similar to those present in juvenile-onset patients making this model homologous to the most common disease form seen in human patients. we identified that intrathecal administration of hpbcd ameliorated all clinical aspects of neurological disease at least up to 24 weeks of age (an age when untreated cats die) but had no effect on hepatic disease. we identified that while subcutaneous therapy with hpbcd at all doses ameliorated liver disease, only 8000 mg/kg substantially affected neurological disease but also resulted in early death due to pulmonary toxicity. finally, we identified a dose-related toxic effect of hpbcd on hearing function that had not been described in any other species. leukodystrophies are disorders of myelin synthesis and maintenance that affect cns myelin. they are subdivided as leukodystrophies, hypomyelinating disorders and spongy degenerations. although infrequently seen, several forms have been described in various dog breeds. we present a novel form of complex leukodystrophy consisting of hypomyelination and spongy degeneration that presents primarily with hind end tremors in border terrier puppies. three border terriers from two different litters (and lineages) are described here that presented with a history of shaking movements. the youngest dog was a 3-week old male. it was the only dog affected in the litter. the other two dogs were 6-week old female littermates. there were two unaffected males in the same litter. physical examination revealed no abnormalities. on neurological examination, the affected dogs displayed severe hind end tremors, with a characteristic swinging side-to-side movement (best described as ''rumpshaker''). the tremors also involved the head and thoracic limbs but to a lesser degree, and disappeared when the dogs were asleep or at rest. severe cerebellar ataxia was observed when the dogs ambulated. proprioceptive positioning was delayed in the pelvic limbs of all 3 dogs. spinal reflexes and nociception appeared normal. necropsy was performed in all 3 puppies. no macroscopic changes were observed. histologic evaluation of the cns revealed spongy degeneration and hypomyelination in all funiculi of the cervical and thoracic spinal cord. white matter of the frontal, temporal and parietal cortices had mild multifocal spongy degeneration and hypomyelination, whereas white matter of the cerebellum, medulla and pons showed severe diffuse spongy degeneration and hypomyelination with gliosis. the combination of reduced myelin formation combined with spongiform white matter changes in the absence of microglial responses suggest a complex pathogenesis affecting both oligodendrocytes' capacity to synthesize myelin and the stability of the myelin that was formed. the number of oligodendrocytes and axons appeared subjectively normal indicating a primarily hypomyelinating process. the clinical and pathological features of this disease have not been described in any other canine leukodystrophy. the primary and most striking clinical feature is the presence of severe tremors in the hind end, causing the ''rumpshaker'' pheynotype. genetic studies are underway to determine if the disease is inherited and the inheritance mode. a syndrome of border collie collapse (bcc) appears to be common in dogs used for working stock. this syndrome has also been called malignant hyperthermia, heat intolerance, exerciseinduced collapse and ''wobbles''. a presumptive diagnosis of bcc can only be made by eliminating other causes of exercise intolerance and weakness. the purpose of this study was to describe the clinical features of collapse in affected dogs and determine if there were characteristic clinical or laboratory features at rest or after exercise that could aid in diagnosis. seven adult border collies with a history of collapse during sheep herding (affected) and 5 adult border collies regularly used for sheep herding but showing no signs of exercise intolerance (normal) were evaluated before and after participating in a videotaped 10 minute exercise protocol consisting ofa series of continuous short outruns and fetches of three sheep in an outdoor pen. exercise was halted at 10 minutes or earlier if there were signs of gait or mentation abnormalities. pre-exercise evaluation included physical examination, orthopedic and neurological exam. pre and immediate post exercise rectal temperature, pulse and respiration, patellar reflexes, ecg, cbc, serum biochemistry profile, cortisol, arterial blood gas and plasma lactate and pyruvate concentrations were measured. clinical parameters (gait, temperature, reflexes) and lactate and pyruvate concentrations were evaluated at intervals up to 120 minutes after exercise. additional testing in affected dogs included measurement of acetylcholine receptor antibodies (achrab) and dna testing for dynamin-associated exercise induced collapse (deic) and the ryanodine receptor mutation associated with canine malignant hyperthermia(mh). one week after exercise affected dogs had thoracic radiographs and echocardiography performed and were anesthetized for emg and muscle biopsies. there were no significant differences in temperature, pulse, respiration, or any laboratory parameter at any time point between normal and affected dogs. no arrhythmias were detected. affected dogs were negative for the dna mutations tested and for achr ab. thoracic radiographs, echocardiograms, emgs and muscle biopsies were normal. the 5 normal dogs had no alterations in mentation or gait during or after exercise. three of the affected dogs had exercise halted early (6 min-9 min) because of altered gait or mentation. all 7 of the affected dogs were abnormal in the 15 minutes following exercise. abnormalities seen in all dogs included disorientation, dull mentation, swaying, falling to the side, exaggerated lifting of limbs each step, choppy gait, delayed limb protraction, scuffing of rear and/or forelegs, and crossing legs when turning. all dogs returned to normal by 30 minutes. bcc appears to be an episodic nervous system disorder that can be triggered by exercise. genetic testing excluded deic and the described canine mh mutation. common causes of exercise intolerance were eliminated, but the cause of collapse in bcc was not determined and no clinical or biochemical marker to aid diagnosis was established. equine cushing's disease (ecd) is common in older horses. the purpose of this study was to determine the frequency of diagnosis, identify prognostic factors and assess owner satisfaction with treatment. the study was a retrospective cohort design evaluating equine accessions reported to the veterinary medical data base (vmdb) and the ohio state university from 1993-2004. proportional accessions, annual incidence and demographic characteristics of horses with ecd were compared with all accessions in the vmdb. medical records for a subset of horses were extracted and owners contacted to obtain long-term follow up information. two hundred seventeen new cases of ecd were reported to the vmdb. incidence increased from 0.25/1,000 in 1993 to 3.72/1,000 in 2002. eighty-one percent of horses were ! 15 years of age. average delay from onset of signs to diagnosis was 180 days (range 1 to 1,824 days). hirsutism (84%) and laminitis (50%) were the most common clinical signs. improvement in one or more signs 2 months after diagnosis was reported by 9/22 (41%) of horse owners. none of the clinical or laboratory data were associated with survival and, 50% of horses were alive, 4.6 years after diagnosis. 17/20 (85%) of horses were euthanatized and 13/17 (76%) were euthanatized due to conditions associated with ecd. twenty-eight of 29 (97%) of horse owners said they would treat a second horse for ecd. ecd is becoming a more frequent diagnosis. fifty percent of horses survived 4.5 years after diagnosis and owners were satisfied with the horse's quality of life. supported by centers of excellence in livestock diseases and human health, college of veterinary medicine, university of tennessee. the role of the hypothalamic-pituitary-adrenal (hpa) axis in sepsis has been a subject of a great deal of research. the role that the somatogenic axis plays in sepsis is less well understood and how these two axes interact during critical illness is not clear. the purpose of this study was to assess inter-relationships of adrenocorticotropin (acth), cortisol, and insulin-like growth factor-i (igf-i), in septic and non-septic term foals. blood samples were obtained from term septic foals less than 7 days of age (n 5 20) admitted to texas a&m university veterinary medical teaching hospital or mid-atlantic equine hospital. the foals were classified as septic by a sepsis score ! 11 and/ or a positive blood culture. non-septic term foals less than 7 days of age (n 5 8) and having a sepsis score o 11 and a negative blood culture, were obtained from texas a&m university veterinary medical teaching hospital and mid-atlantic equine hospital. plasma and serum were processed from whole blood collected by jugular venipuncture upon admission, at 24 hours post admission and at 5 days post admission or at the time of discharge. plasma concentrations of acth, and serum concentrations of cortisol and igf-i were determined by specific rias. data were analyzed using linear mixed-effects modeling with foal modeled as a random effect and day of admission modeled as an ordered categorical variable; post-hoc testing of pair-wise comparisons was made using the method of sidak. significance was set at p o 0.05, and analyses were performed using s-plus software (tibco, inc., seattle, wa). plasma concentrations of acth were not significantly different between septic and non-septic foals whereas septic foals had greater serum cortisol (37 ae 8 ng/ml vs 25 ae 7 ng/ml) but lower serum igf-i (116 ae 14 ng/ml vs 152 ae 15 ng/ml) relative to non-septic foals pooled overall sampling times. the positive association of the peripheral blood concentrations of acth and cortisol depended on disease status of the foals. specifically, cortisol and acth were positively correlated for the septic foals (p 5 0.027) but not significantly correlated in the non-septic foals. peripheral concentrations of acth and igf-i were not significantly correlated whether data were pooled overall or stratified by sepsis status. however, peripheral concentrations of cortisol and igf-i were negatively associated (p 5 0.019); disease status did not influence this association, although it appeared to be a stronger association for the septic than the non-septic foals. the negative correlation between serum concentrations of the adrenal axis steroid cortisol and the somatogenic axis peptide igf-i may reflect interactions of these homeorhetic hormones. further studies of these and other metabolic hormones in a greater number of foals are warranted to better understand how these factors contribute to survival or non-survival of critically ill foals. botulism is a potentially fatal paralytic disorder which definitive diagnosis is difficult. the purpose of this study was to investigate if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of suspected botulism in foals. four healthy foals were used for its comparison with 3 foals with suspected botulism. controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. the common peroneal nerve was chosen for its superficial location and easy access. stimulating electrodes were placed along the common peroneal nerve. for recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies (1 to 50 hz). amplitude, area under the curve and percentages of decrement or increment for each m wave over subsequent potentials for each set of stimuli were analyzed. baseline m waves were decreased in affected foals compared to controls. a decremental response was seen at all frequencies in control foals. decremental responses were also observed in affected foals at low frequencies. however, an incremental response in amplitude and area under the curve was seen in all affected foals at 50 hz. reduced baseline m waves with incremental responses at high rates are supportive of a presynaptic neuromuscular disorder which botulism was the most likely cause in these foals. repetitive nerve stimulation is a safe, simple, fast, and non-invasive technique that can aid in the diagnosis of suspected botulism in foals. this study examined the frequency with which dogs are exposed to e. chaffeensis and e. ewingii relative to e. canis, which is transmitted by the more ubiquitously distributed brown dog tick (rhipicephalus sanguineus). a total of 6,512 canine serum samples, ranging from 182 to 614 from each of the 14 participating institutions, collected at random from clinical accessions, diagnostic laboratories and/or shelters were evaluated. all serum samples were tested by three microtiter plate elisas using species-specific peptides for antibodies to e. canis, e. chaffeensis and e. ewingii. zip code information for sample origin was provided by the collaborator and was used to assess seroprevalence by region. comparisons were evaluated using the chi-square test. seroreactivity for at least 2 of 3 ehrlichia spp was found in samples from every institution both mississippi and oklahoma had greater than a 7% samples from ohio had the lowest aggregate seroprevalence (1.0%) with only 4 dogs e. canis seropositive, one e. ewingii seropositive and no e. chaffeensis seroreactors. the geospatial pattern of e. chaffeensis and e. ewingii seropositive samples was similar to that previously reported based on modeling seroreactivity to e. chaffeensis in white-tailed deer as well as the distribution of human monocytic ehrlichiosis (hme) cases reported by the cdc. this study provides the first large scale regional documentation of canine exposure to these three ehrlichia spp., highlighting where infections most commonly occur and thus identifying areas where heightened awareness about these emerging vector urinary incontinence (ui) occurs in approximately 20% of spayed female dogs. the most common cause is urethral sphincter mechanism incompetency (usmi). pharmacological agents are effective, however, not all dogs respond, and dogs may become refractory to treatment over time. urethral bulking, where a compound is injected submucosally in the urethra, has been used in women and in female dogs with urinary incontinence. new synthetic compounds have been used in human medicine; the most promising is polydimethylsiloxane (pdms), which has been shown to be more effective than glutaraldehyde cross-linked collagen. the purpose of this descriptive clinical trial is to evaluate the safety and effectiveness of pdms urethral bulking agent (pdms uba) in client-owned, spayed female dogs with naturally-occurring ui due to usmi.twenty-two, spayed female dogs were included. dogs had a median age of 6 years (2 to 11 years). eighteen dog breeds were represented, and dogs weighed a median of 29.9 kg (7.3 to 62.7 kg). average length of time of ui was 2.5 ae 2.3 years; 18/22 dogs had been treated medically, of which 2/18 were continent, 14/18 were improved, and 2/18 had no improvement. dogs were deemed healthy based on results of physical examination, complete blood cell counts, plasma biochemical analysis, and urinalysis; urine cultures were negative.dogs were anesthetized, positioned in dorsal recumbency, and cystoscopy performed using a 2.7 mm, 0-or 30-degree, 18 cm rigid cystoscope. urethral bulking was performed with pdms uba. on average, 2.5 ae 0.9 ml were injected in 3 to 5 locations approximately 1 to 1.5 cm distal to the trigone submucosally in the proximal urethra. good coaptation was achieved in all dogs. the procedure took on average 15.9 ae 4.3 minutes. one dog experienced urethral obstruction after the procedure; a foley catheter was inserted for approximately 12 hours and removed at which time she urinated normally and was continent. three dogs experienced an acute allergic reaction characterized by blepharedema and urticaria treated successfully with diphenhydramine. dogs were discharged on day of procedure except for the one dog that experienced urethral obstruction. all dogs were treated with meloxicam (0.1 mg/kg po q24h for 3 days).owners were contacted on day after discharge and 21/22 dogs were continent; 1/22 dogs was improved. dogs were re-evaluated 1 week after discharge and 21/22 dogs were continent and 1/22 dogs polyneuropathy in large breed dogs is a relatively common clinical problem for which the genetic basis is generally unknown. the first cases of polyneuropathy in the leonberger breed (leonberger polyneuropathy or lpn) were identified in 1999 by one of the authors (gds) and a report published in 2003 (musclenerve 27:471-477) . in this report a spontaneous, distal and symmetrical polyneuropathy with onset between 1 to 9 years of age was described and characterized clinically, electrophysiologically, histologically and morphometrically. there were striking similarities between lpn and the charcot-marie-tooth group of human inherited sensory and motor polyneuropathies, which have many known genetic mutations.a genome-wide case-control association study for lpn was performed with 53 cases and 42 controls on high-density 170k canine snp arrays and revealed a significantly associated region on cfa 16 (p raw 5 2.36 â 10 à10 p genome 5 9.99 â 10 à5 ). a clear association of an approximately 1 mb cfa16 haplotype with cases (p 5 1.71 â 10 à8 ) was observed, particularly with those cases that were affected more severely and at a younger age (p 5 2.55 â 10 à11 ). a positional candidate gene, arhgef10, which has previously been associated with peripheral nerve abnormalities in humans, was sequenced, revealing a deletion that results in a frame shift and premature stop codon. of all leonbergers with young onset lpn (before 4 years), 48.5% (32 of 66) have two copies of this deletion, and, of all young onset leonbergers that are nerve biopsy positive for lpn, 59.4% (19 of 32) have two copies of this deletion. importantly, nearly all dogs carrying two copies of the deletion (32 of 34 or 94.1%) are affected with lpn by the age of 4 years.the leonberger breed was generated from crossing several breeds, including the st. bernard, and a polyneuropathy clinically and histologically similar to lpn occurs in this breed. to determine if the arhgef10 mutation was associated with polyneuropathy in the st. bernard, dna was extracted from archived frozen muscle biopsy specimens from clinical cases (n 5 3). the identical arhgef10 startle disease or hyperekplexia is caused by defects in mammalian glycinergic neurotransmission resulting in an exaggerated startle reflex and extensor hypertonia triggered by noise or touch. in humans and animals, startle disease is typically caused by mutations in one of three genes (glra1, glrb, and slc6a5) encoding postsynaptic glycine receptor subunits (a1 and b) or a presynaptic glycine transporter (glyt2). a litter of seven irish wolfhounds was recently identified in which two puppies developed muscle stiffness and tremor beginning at 5-7 days of age post-partum. signs were dramatic when the puppies were handled and resolved when the puppies were relaxed or sleeping. both puppies were euthanized due to ongoing stiffness, tremor and breathing difficulties. necropsies were performed, but no microscopic pathological abnormalities were identified in the peripheral or central nervous system.based on the clinical signs, exons from the three candidate genes were amplified from genomic dna isolated using pcr and directly sequenced. no deleterious polymorphisms were identified in either glra1 or glrb. however, difficulties were experienced in amplifying slc6a5 exons 2 and 3 from affected animals, although control samples were positive, suggesting that the pcr primer designs and conditions were not at fault. further pcrs revealed that the reason for this anomaly was the presence of a homozygous 4.2 kb deletion encompassing exons 2 and 3 of the glyt2 gene in both affected animals. this deletion is predicted to result in the loss of part of the large cytoplasmic n-terminus that is vital for trafficking of glyt2 to synaptic sites, and a loss of all subsequent transmembrane domains via a frameshift. this genetic lesion was confirmed by defining the deletion breakpoint, southern blotting and multiplex ligationdependent probe amplification (mlpa). this analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, suggesting recessive inheritance of this disorder. wider testing of related animals has identified a total of 18 carriers of the slc6a5 deletion and enabled the identification of non-carrier animals to guide future breeding strategies. insulin resistance (ir), obesity, and type 2 diabetes affect glucagon-like peptide 1 (glp-1) concentrations in humans and rodents, but this incretin hormone has not been examined in horses. we therefore hypothesized that glp-1 concentrations would change in horses as obesity and ir were induced or exacerbated by overfeeding. six horses previously diagnosed with equine metabolic syndrome were provided with twice the amount of digestible energy required for maintenance as sweet feed and hay for 8 weeks. intravenous and oral glucose tolerance tests (ogtts) were performed at 0 and 8 weeks. effects of time and period (0 and 8 weeks) were assessed by repeated measures anova.mean body weight increased from 438 ae 61 kg (range, 381 to 533 kg) to 464 ae 61 kg (range, 394 to 550 kg) over 8 weeks, with individual horse weight gain varying from 2 to 10%. mean body condition score increased (p 5 0.006) from 6 ae 2 (range, 4 to 8.5) to 8 ae 1 (range, 7 to 9). three horses developed mild laminitis. glucagon-like peptide 1 concentrations increased over time during ogtts (p 5 0.023), but the period â time effect was not significant (p 5 0.141). area under the glp-1 curve remained unaffected by weight gain, whereas area under the insulin curve increased (p 5 0.003) over time, indicating a reduction in insulin sensitivity. obesity and ir were induced or exacerbated when horses previously diagnosed with ems were overfed, but glp-1 concentrations did not change as a result. hypertonic saline solution (7.2%) (hss) is an intravenous fluid used for the emergency treatment of intravascular volume deficits. the use of this fluid in horses with severe dehydration is controversial. the purpose of this study was to compare the use of hss and isotonic saline solution (0.9%) (iss) for the emergency treatment of endurance horses.endurance horses eliminated from competition and requiring intravenous fluid therapy were eligible for enrollment in the study. twenty-two horses were randomly assigned to receive 4 ml/kg of either hss or iss along with 5 l lactate ringer's solution (lrs). following this bolus, all horses were treated with an additional 10l of lrs. blood and urine samples were collected before, during and after treatment. data was compared using two-way anova with repeated measures.as compared to iss, hss horses showed a greater decrease in pcv (p 5 0.04), total protein (p 5 0.01), albumin (p 5 0.01), and globulin (p 5 0.02). hss horses showed a greater increase in sodium and chloride (p o 0.001) as compared to iss horses. horses receiving hss had a shorter time to urination (p 5 0.03) and lower specific gravity (p o 0.001) than those receiving iss.results of this study indicate that hss may provide faster restoration of intravascular volume deficits than iss in endurance horses receiving emergency medical treatment. more profound electrolyte changes should be expected with hss however. b 2 -adrenergic receptor agonists have been shown to increase erythrocyte carbonic anhydrase activity, which may stimulate the jacobs-stewart cycle and increase pulmonary circulation transvascular fluid fluxes during exercise. increase in pulmonary transvascular fluid fluxes (j v-a ) and consequent increase in the pulmonary interstitial fluid would be detrimental for alveolar o 2 exchange during the fast erythrocyte transition time across the pulmonary capillaries. therefore, we hypothesised that treatment with inhaled b 2 -adrenergic receptor agonist will increase j v-a and the alveolar-arterial po 2 difference (aado 2 ) during exercise.six stb horses were exercised on a high-speed treadmill at 80% vo 2 peak until fatigue. horses were randomly assigned to treatment with salbutamol (sal: 500mcg) or placebo (control: con) inhalation via aeromask ã 60 min prior to exercise, with cross over treatment used at the repeated exercise test (8 days later). arterial and mixedvenous blood, as well as co 2 elimination and o 2 uptake, were sampled simultaneously at rest, during exercise at 60 sec intervals until fatigue, and into recovery. blood gases were analyzed. aado 2 was calculated using the inspired po 2 (149 mmhg), and blood partial pressure of o 2 and co 2 . blood volume (%) changes across the lung were calculated from changes in hemoglobin and hematocrit values in venous and arterial blood. cardiac output (q) was calculated using the fick equation. j v-a was calculated using q and blood volume changes across the lung. variables were analyzed using two-way repeated-measures anova (p o 0.05).the duration of exercise to fatigue was 4.3 ae 0.3 min and 4.4 ae 0.4 min in both con and sal, respectively. at rest sal had no effect on j v-a , oxygen consumption (vo 2 ), blood oxygen saturation (so 2 ) or aado 2 (p40.05). at the onset of exercise j v-a increased in con and sal (p o 0.0001) and at fatigue reached 10.0 ae 2.4 l/min and 10.0 ae 1.6 l/min, respectively. treatment with sal had no effect on j v-a during exercise (p 5 0.9). at the onset of exercise so 2 and vo 2 increased in con and sal (p o 0.0001). treatment with sal had no effect on so 2 or vo 2 during exercise (p40.05). aado 2 increased during exercise in con and sal (p o 0.0001) and at fatigue reached 19.4 ae 2.3 mmhg and 18.1 ae 2.4 mmhg, respectively. treatment with sal had no effect on aado 2 during exercise (p 5 0.3).inhaled b 2 -adrenergic receptor agonist salbutamol at the dose of 500mcg given 60 min before exercise did not affect the duration of exercise to fatigue, j v-a , vo 2 , so 2 or aado 2 . therefore, it had no detrimental effect on alveolar-capillary diffusion distance and the ventilation/perfusion mismatch in exercising horses. inflammatory airway disease (iad) and recurrent airway obstruction (rao) represent two classes of equine lung inflammatory diseases that may share some similar immunologic mechanisms. there is evidence that th2 cytokines and il-17 play some role in rao. iad is a common condition in horses, but its pathophysiology is still not understood. the aim of the present study was therefore to determine the mrna expression of th1, th2 and th17 inflammatory cytokines, to understand the immunological mechanisms of iad.the mrna expression of ten inflammatory cytokines and chemokines was measured in the bronchoalveolar fluid (balf) of seventeen horses with iad and compared with ten control horses. the horses were selected based on 1-their clinical signs, 2-the inflammatory cells count in the balf, 3-their physical examination and 4-their medical history. the mrna expression of il-5, il-1b, il-6, il-8 and il-10 was significantly up-regulated in balf from horses with iad.furthermore, the balf samples were subdivided in two groups based on the differential cells count 1-balf with increased mast cells (iad-mast) and 2-balf with increased neutrophils (iad-neutro). il-4 was significantly down-regulated in the iad-neutro group compared to the iad-mast group. il-17, il-5 and il-8 were significantly up-regulated in the iad-neutro group compared to the iad-mast group.the present study shows that iad in horses is characterized by a th2 and a th17 mrna inflammatory expression profile and that different immunological mechanisms are involved in mast cells or neutrophils accumulation in the balf of horses with iad. b 2 -adrenoreceptor (b 2 -ar) agonists are a class of medications that promote smooth muscle relaxation and bronchodilation in horses and humans with airway disease. activated human peripheral blood lymphocytes (pbls) also respond to b 2 -ar agonist stimulation by attenuating the production of cytokines associated with the pathogenesis of asthma and recurrent airway obstruction (rao). the aim of this study was to develop an in vitro technique for measuring the response of equine pbls to stimulation with salbutamol, a b 2 -ar agonist. this method was then used to compare the response of pbls from rao-affected and non-affected horses to b 2 -ar agonist stimulation. pbls from 4 rao and 4 nonaffected horses were cultured (4x10 6 /ml) in rpmi complete media with concanavalin a (cona, 2ug/10 6 cells) for 0, 1, or 2 days then stimulated with salbutamol (30 minutes). using flow cytometric techniques, response was measured by detecting protein kinase a phosphorylation of vasodilator stimulated phosphoprotein (vasp). results were verified by western blot analysis. activated pbls were then incubated with cona for one day were pre-incubated with b 2 or b-adrenoreceptor antagonist (ici 118,551, sigma s ; atenolol, sigma s ) for 15 minutes, followed by 30 minutes salbutamol (500 nm) stimulation. results were analyzed by anova or ancova and differences were considered significant when p o 0.05.response to b-antagonist was only observed in activated pbls (pre-cultured with con a) and was greater in cells from rao horses as compared to cells from non-affected horses. the addition of b-antagonist attenuated the response of pbls to salbutamol while the addition of a b 1 -antagonist had no effect. these findings indicate that activated pbls from rao-affected horses have a greater response to salbutamol as compared to pbls from non-affected horses, and this response is mediated mainly through the b 2 -ar.human b 2 -ar are known be polymorphic and this polymorphism results in a variable response to b 2 -agonist binding that affects long term outcome in human asthmatics. further studies are required to determine if the difference in response of pbls from rao affected as compared to non-affected horses is due to genetic polymorphism in the equine b 2 -ar, and whether this difference is associated with a propensity for horses to develop equine rao. key: cord-015021-pol2qm74 authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: 1994 journal: intensive care med doi: 10.1007/bf02258437 sha: doc_id: 15021 cord_uid: pol2qm74 nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on 2-5 march 1994. when, in the mid-1980s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in 1988 provided stateof-the-art information and consensus on factors involved in injury and sepsis. in 1991, the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until 1995 or 1996. however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il-6 to il-11, il-12 and il-13 and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in 1994, to be dealt with at this symposium and congress? (1) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. (2) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. (3) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. (4) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. (5) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. (6) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of 1994 and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past 25 years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~2ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha-2macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro2-iarg]5]aprotinin (bay 4620) or the b2 kinin antagonist, hoe 140 on the hypotensive response to dxs. in the first study, anesthetized miniature pigs (5 pigs/group, randomly assigned) were given one of the following treatment protocols: 1) dxs (5 mg/kg), 2-5) dxs plus bay 4620 (45, 90, 180, or 360 rag), or 6) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c3adesarg and fibrin mom)mer were all increased. bay 4620 produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c3adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay 4620. llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs (3/group) were given either dxs alone (2 mg/kg) or dxs 10 minutes after a bolus injection of hoe 140 (30 #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe 140 pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse 20, d-80336 munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f6r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion (0,01 mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to 50 u/1. in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to 200 u/1. in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs (1). when very small plasma kailikrein activities (0,3 u/kg bodyweight) were given intravenously a 50% decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c1-inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin-2 (5 x 106 iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of 100, 250 or 500 u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion (100-125 u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was 90 % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c1-inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; 2thrombosis research center, temple university, penn., usa; 3oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c6b7 which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c3b/c and c4b/c, and a significant reduction was observed in 5 animals that had received a lethal dose of e. coli together with mab c687 (treatment group), compared to 4 animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-=2antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c3a, c3, and c5a were measured in 30 patients from an internal intensive care unit. 20 patients were clinically septic defined by the criteria of bone et al.(l) . the remaining 10 patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l0 days period. during the first 3 days blood samples were drawn every 8 h, on day 4-6 every 12 h and the last 4 days once daily. mean plasma concentrations of c3a within the first 32 h after clinical onset of sepsis were 1019 + 618 pg/ml, whereas non-septic-patients exhibited mean values of only 595 +_ 312 p_g/m/. c3 levels were lower for septic-patients (840 + 480 lag/ml) than for non-septic-patients (1340 _+ 770 lag/ml). the most profound difference between both groups was found, when the c3a/c3 ratio was compared (1.84 + 2.28 for septic-patients and 0.4 _+_ 0.18 for the control group). no significant differences between both patient groups were observed in c5a plasma levels (5.9 + 2.1 ng/ml in septic-patients vs. 5.6 _+ 1.5 ng/ml in control patients). in 13 of 20 cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n=9) and non-survivors (n=l 1) within the septic-group revealed, that the c3a/c3 ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c3a and c3 in only 20-25 minutes (2) and the significant difference of the c3ajc3 ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of 65 amino acids with 6 cysteine residues (mr 7000 daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw 023, behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of 0.5 p.g/kgxh thromboplastin for 7 hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to 27.0 % and 33,0 % respectively, and by an increase of fibrin monomers from 13.2 to > 85.0 ~tg/ml. we administered rec. hirudin to rabbits in 3 different concentrations (0.5, 1.0 and 2.0 mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even 7 hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin (0.5, 1.0 and 2.0 mg/kg i.v. bolus) as late as 2 hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, 35001 marburg, germany $3 novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r-020) and its effect on sepns-lnduced organ injury in rat atsuo murata 1, hitoshi toda 1, ken'ichi uda 1, hidewaki nakagawa 1 , takesada mori 1, hideaki morishita 2, tom yamakawa 2, jiro hirese 2, atsushi ni~ 2, nariaki matsuura 3 1osaka university medical school, osaka, 2mochida pharmaceutical co. ltd. tokyo, 3wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which 2 kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je5505 which lacks the membrane lipoprotein. the recombinant second domain (r-020) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r-020 was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r-020 significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai.,1990) . the cathepsin l-like and cathepsin b-like activity were measured in serum of 42 patients with chronic bronchitis (14 -with obstructive, 28 -with nonobstructive bronchitis),acute bronchitis (15) and 35 healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai.,1991) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls (2-4 fold) .after treatment there was a tendency to normalization of indices,but the increase was about 30-40% more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis -2-4-times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/31-6 diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and 4 amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd14 and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il-1/3, ifn-a, il-6, il-8, il-10, paf, pge, ltb 4 and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur (1,2), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. 1987 gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/3 selectively t cells to produce lymphokines including i1-2 and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/38 + t cells accumulate within 90-120 min mrna for the ii-2, i1-4, tnfai~ and ifn-3,. parallel in time high concentrations of bloodborne i1-2 and tnf are noted. anti tnf t~//3 neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within 12-18 h distinct levels of tolerance to seb become discernable on ligand reactive v/38 t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd2, cd4, cd8 accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als0 sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c3h/hej mice. the absence of sensitization to lps in lps d c57bl/10sccr mice was identified as being due to their inability to produce interferon-7 (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn7 is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c57bl/10sccr mice concerns only the ifn7 response to bacteria, since the response to the t-cell mitogen con a and to cd3 monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c57bl/10sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il-6 levels, whereas treatment with aztreonam resulted in progressively increasing levels of il-6 and a significant increase in mortality. the increased mortality in pbp-3 specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd1 mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as 100-to lo00-fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its 0 side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf-1 dgaln-treated mice with e. coli olll:b4 resulted in an lds0 of approximately 2.5 x 104 cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection (3 fold vs 8 fold respectively, p< .01). parallel studies with endotoxin hyporesponsive c3h/hej mice indicate significantly greater levels of protection with imipenem (> 80 fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since 1893, when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe 1972; pollack 1979) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: 1 ) enhancing of opsonization and phagocytosis(antibactericidai activity) 2) synergistic effects with [3-1actam antibiotics 3) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis 4) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c3 receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il-6 release in mice (baumgartner 1990, corriveau and danner 1993) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger 1990 (berger ,1993 , in animal models (stephan 1985 , berger 1993 and also in prospective, randomized, controlled clinical trials (schedel 1991 , poynton 1992 , behre 1992 . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg2ak) has been selected and chimerized into a human igglk (sdz 219-800). in elisa and in immunoblots on purified lps both sdz 219-800 and wni 222-5 show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz 219-800 and wni 222-5 have biological activity as they inhibit the lal assay and the secretion of monokines (il-6 and tnf) by mouse and human macrophages. moreover, sdz 219-800 and wni 222-5 inhibit the release of il-6 and tnf in vivo. in vivo sdz 219-800 as well as wni 222-5 neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni 222-5 as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch4002 basel, $chweiz $7 presentation of lps to cd14 by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd14 (mcd14). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd14 but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd14 (scd14). these complexes of lps with scd14 are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd14, either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd14. these results show that the delivery of lps to scd14 is catalysed by lbp, i.e., lbp is not included with the lps-scd14 complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd14. the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm37696, ai32021, ai15136, gm08172, and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm-12, 10666 n. torrey pines rd. la jolla, ca usa 92037. modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d-23845 borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i1_-1, and il-6, which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound 406) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin-2 and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a 45% amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd14 receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd14 receptors. a recombinant protein (rbpi23), corresponding to the amino terminal 23kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi23 will have to successfully compete with relatively high serum levels of lbp (5-60 ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi23 and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of 2.6 nm for rbpi23 and 58 nm for rlbp. in a competition assay format rbpi23 was approximately 75-fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi23 has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi23 in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi23 at 0.2 ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a 100-fold weight excess of rlbp over rbpi23. and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions 17-45, 65-99 and 142-169 . a single synthetic peptide (85-99) was bactericidal. these results suggest that rbpi23 contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi23 analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in 80 patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first 3 days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p55 and p75 receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p75 stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only 40% of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il-1[3-inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered (300kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in 5% plasma +/-bpi (500 ng/ml). pbmc were incubated for 24 hours at 37°c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n=7, mear~+sem, *p<0.05 vs control). 0 control 0.1_+0 + bpi 0.1+0 5% plas. 0.2_+0 + bpi 0.2_+0 pmp (ng/ml) lps (ng/ml) 60 600 3000 1 10 3.2_+1 8.0_+1 16.3_+3 1.2_+. 2 5.2+.6 0.2_+.1 3.3_+.9" 13_+3" 0.1_+0" 0.1_+0" 2.2_+. 5 5.7+1 14_+2 5_+.6* 14_+2" 0.6+. 4" 5.3+1 15-+2 0.1-+0" 0.5_+.3" cba, c57bl/6, balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection 59-75% of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats (250-300g) were studied [no operation (n=39) , sham operation (n=40), and bile duct ligation for 21 days (bdl)(n=50)]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii 30 rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n=8), sham op(n=8) or bdl(n=i4). blood was taken at this time (to) and 21 days later(t20 at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp<0.001, mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t21 [p<0,001,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp<0.05, paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than 9.8 pglml. polymyxin b was administered at a dose of 12,500 u every 6 hours. plasma endotoxin rapidly decreased to the normal range in 40 of the 42 patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin 6 decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. 19-1 uchimaru, morioka 020, japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, 12 n o r w e g i a n b r e d landrace pigs (17-30 kg) of either sex, 6 pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a 50/50 air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e 0.6 m g / k g h a n d d i a z e p a m 2.4 m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a 5f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( 1 0 m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n 2.5 x l09 cfu of e colt intraperitoneally as a bolus in 100ml saline, the a n t i b o d y g r o u p received in a d d i t i o n 5 m g / k g e5 a n t i e n d o t o x i n i n t r a v e n o u s l y over 1 h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for 6 hours. results : a t 5 a n d 6 hours, the o x y g e n c o n s u m p t i o n increased by 30 % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of 30 % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po2. in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h-6720 szeged~ hunbary 46 involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome 3 heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit6 des toxines microbiennes and "unit6 d'immuno-allergie, institut pasteur, 28, rue du docteur roux -75015 paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il-1 d and ~, tnf ~ and il-6 in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il-6 and il-8. however, the most preponderant cytokine was tnf~, which peaked at i0 ng/ml after stimulation with i0 ~g eta. comparable amounts of tnfd (ca 4 ng) were induced by 0.i ~g eta and 0.i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon 7 in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i,3-glucan (ram 120000) was injected twice 48 and 24 h before the shock i.v. in a dose of 50 ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli 0127 bs, 5 mg/kg. aiiofcmg pretreated ruts survived during first 24haher ¢ndotoxine, while in controi shock group the lethality was 90%. the concentration of ~col)terin in serum was significantly elevated 24 hafterthc second cmginjection (appare~tly 80 % if compare with the control rats), but didu't chartged 60 rain and 1 s0 rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i20 and 180 min after endotoxine onset ( 8i % trod 72~, respectively of initial level) than in the control shock group (64 % and 52% at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after 3 h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at 40 %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; 2. modify records; 3. erase records; 4. searches; 5. reports; 6. configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of 34 areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd9) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over 65 years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of 31 antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of 37 organisms and the chance of identifyin 3 organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group (40 cases), the total effective rate was 87,5%, in the contral group (combined administration of gentamycin and dexamethasone, 25 cases) the total effective rate was 84%. however the obviously effective rate in sa group 70% was significantly higher than in contral group 44% (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o5). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and 13-glucuronidase (13-gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c3h/hen mice at i and 24 h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n=6/grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at 24 h the thymic cell yield from clp mice was significantly decreased (sham:3.6!0.2 x l06 vs clp:0.3i0.1 x 106* cells; *,p<0.05), the % of cells apoptotic by facs analysis increased from 1.7i0.1% in sham to 8.7±3.2%* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from 1982 to 1992 31 patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch 16 ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of 6 years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). 21 patients (80%) out of 26 patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all 5 patients with chronic septic knee joints showed poor results. additionally, in 3 of these 5 patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco-2 cell line) is grown on a 3 micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco-2 monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco-2 system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco-2 monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. 2) caco-2 cells are actively involved in the transcellular passage of bacteria across the caco-2 monolayer, since inhibition of caco-2 microtubule or microfilament function decreases the magnitude of st. 3) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco-2 cell monolayer. 4) caco-2 cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco-2 bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco-2 cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers (1.3 x 108 colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about 99.9% within the first 15 minutes and are totaiy cleared within 90 minutes. 57% offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about 100 cfu of viable e.eoli per ml blood could still be counted 3 hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with 25% respectively 11% of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at 24 (n~ 7), 4g (n ~ 10), and 96 (n~ 10) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker -25 % at 24 hour~ with return to 51% of gut leugth (similar to ~m) at 96 hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at 2,; hours with return to sham control at 96 hours (9 log cfu/g veraus 6 log cfu/g ia ileum). translocation to mesenteric nodes -71, 90, and 30 percent at 24, 48, and 9fi hours compared to 49b in sham controls (n=l of 26). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il-6, soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs (40 mmhg mean arterial pressure: map for 2-4 h terminated at an accumulated oxygen debt of 180-200 ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of 30-35 mmhg 90-180 rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at 90 min and were still markedly increased at 150 rain postshock. plasma tnf, il-6, and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at 30 min following resuscitation in rats subjected to hs for 90 min, similarly at 3 h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at 120 min, while in baboons higher levels up to 300 pg/ml were found at the end of shock and 1 h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the 48-hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a2 (txa2) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky-046, a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup753, an a-11 receptor antagonist as a posthum treatment modality was evaluated. treatment with dup753 prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup753 from 86% to 29% (p<0.05, fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf3-dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il-2 and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus 9101,6500 lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci2mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. 90 male swiss (icr) mice were used weighing 21-25 g elimination of liver and spleen macrophages was accomplished by intravenous injection of 200/11 suspension of ci2mdp-liposome suspension. control mice received an i.v. injection with 200 pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either 01 mg/g, 0.5 mg/g or 1.0 mg/g body weight (n=8 in each group), signs of st and bacterial translocation bt were measured 24 hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n=6 in each group) were tested to verify that neither paraffin nor pbs or ci2mdpqiposome suspension alone induced bt or st. furthermore survival over a 12-day period was assessed in a control and macrophage-depleted group with a dose of 1d mg/g z. results. neither the paraffin nor ci2mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: 11/32 vs. 1/32 at 0.1 mg/g zymosan (p~o.01); 19132 vs. 10132 at 05 mg/g zymosan (p~q05); 23/32 vs. 11/32 st 1.0 mg/g zymosan (p~o.o1).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of 0.5 mg/g zymosan (3.14 -+ 0.69 vs. 7.13 -+ 3, p~o.01) and 1.0 mg/g zymosan (4.38 -+2.50 vs. 11.25 -+ 2.5, p<_.o.01) . the 12 day mortality after 1.0 mg/g zymosan was 27% in the control group and 0% in the macrophage depleted group (p=o.05). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n=3) and two experimental (n=6 each). a portal vein catheter was placed and remained in situ 48 hrs. a femoral artery and vein catheter were in place 12 hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of 6.5 hrs., given 2 more hours of anesthesia, and studied for further 48 hrs. 40% of the estimated blood volume was withdrawn from each experimental animal over 30 min. these animals were kept in shock for 6.5 hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for 48 hrs. samples from portal blood for cultures and measurement of leukotriene b4 (ltb4), prostaglandin e2 (pge2), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to 48 hrs. after 48 hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb4, pge2 and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha-1a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c3hb-iej mice). five days post-transfusion the animals were gavaged with lxl09 viable escherichia coil and randomized into 3 groups (n=22/group) to receive a sham burn (sb group) or a 20% thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies (6 mg/kg) (ha-1a group) or a 20% burn injury and administration of equal volume of sterile saline (c group). all 3 groups were resuscitated by intraperitoneal injection of 0.3 ml of saline. the animal survival rate was observed for 10 days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n=8/group) were gavaged with 109 viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine 0xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da0 declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da0 goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline 24.5 and 24 hr prior to sacrifice, group ih treated mice receiving ip of utinastatin 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group ilk sham4reated mice receiving ip of saline 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group iv: saline control mice receiving ip twice during 30 rain intervals,group v: mice received ip of utinasmtin, and 30 min later they were received ip of endotoxin (5mg/kg), group vi: mice received ip of saline,and 30 min later they received ip of endotoxin (5mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for 3 days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu 520-21,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: 31 polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (>7 points at 2 days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range 0.6 -0.8 mind) was studied after i.v. injection of 100 mbq 99rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results 86.0 148+ 239+ liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r=0.88) and between l/m and ela (r=0.71). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il-6 release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol 50 patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and 5, 30, 90, 180 min, . the plasma concentrations (,pc) of 6-keto-pgft,, txb: and-ki-12-pgf ~ (stable metabolites of pgi2, txa2 and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il-6 levels by elisa. data are given as mean+sem (* p< 0.05 placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed 180 minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t5 0.16+0.02e[0.32+0.06] eu/ml). il-6 pc demonstrated an increase continuously from t4 to t7 (t7 115+12 [91 + 15] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of 6-keto-pgf~,, pc up to 6h after mt in untreated patients with a peak of 2450*[60] ng/1 at tl. also txb: and kh2pge 2 pc showed a considerabe increase up to 6h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il-6 release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into 6 groups. group 1 received standard laboratory chow feeding ad lib. in group 2 a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group 3 50% of the calculated daily required calory intake (drci) (307/kcal/kg) was given by iv-tpn (28% glucose, 4,25% amino acids) and 50% by limited chow administration. groups 4 and 5 received 70% and 90% of the drci by i.v. tpn and 30% and 10% respectively by chow feeding. group 6 received iv-tpn only. after 7 days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. 4 5,5+0,9 4 12 23,9 -2,3 16 ,7 2/12 63+11 125~1 "7,7-+0,7 6,7-+1 5 12 24~6 -3,5 67 8/12 241+~243 170+163 8_+0,7 7+1,1 6 18 25, 5 -4 88,8 16118 2061-+3224 2211+4015 8,4~0,7 8,1-+1~1 conclusions: the administration of 30% of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. 3 previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r2lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal (90 %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for 15 hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for 6 days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and 17 % in animals subjected to 90 % bepatectomy and lreatment with fermented oatmeal, while 80-90 % and 34-50 %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < 0.05 vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < 0.01) in salinetreated animals following 90 % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in 90 % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. 190_+20" 16+_3" 11.7" data=mean_+sd, * stats anova p<0.05 vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<.05) in lps gut translocation compared to control and l+co2. this correlated with a significant increase in peritoneal inflammatory responses (o2-,tnf) above that of the control and l+co2 groups, while mac-1 and cr3 opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin 9, ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in 32 patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), 6-keto-prostaglansin, leueotriene c4, interleukin 6 and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of 32 patients showed a significant increase of endotoxin plasma levels during colonoscopy (p=0.003), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after 24 hours. ~hromboxane b o levels also increased after five min. from 89 to 376 pgyml peaking at 30 min. with 1332 pg/ml. 6-keto-prostaglandin,leucotriene c 4, ii-6 and crp remained unchanged. a control group of i0 volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b 2 levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into 4 groups were subjected to 30, 60 and 120 minutes of 20%, 30% and 40% of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p 0.05). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p 0.05). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with 5 known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: 1.long term(8-12 days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. 2.in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. 9athol. 73. 161. 1992.) . there was a increasing infiltration of the mucosa c111m~nating at the 3d to 4th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. 45 ~in isehemia was followed by a 4 hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at 5 m_in, i, 2~ 3, and 4 hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the 4th hour in the arterial blood .and at the 3d and 4th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the 2d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for 20 and 30 minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion 2, 4 and 6 hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr51). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats 6 hours after reperfusion in ischemia-20 rain group and 4 and 6 hours after reperfusion in the ischemia-40 rain group. delayed small intestinal transit time was seen from 2 hours and on after intestinal ischemia for both 20 and 40 rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < 0.01). the distribution of radioactivity in segments 4 and 5 in the group with repeffusion 6 hours after intestinal iscbemia for 20 rain was significantly higher than that noted in the group with repeffusion 6 hours after intestinal ischemia for 40 min (p < 0.01). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy (90 %) in the rat. water-soluble ehec was administered orally 1 and 12 hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a 80-100 % translocation rate to mlns or blood 2 and 4 hours after operation, while only 0-1% of rats subjected to sham operation or animals with 90 % hepatectomy and pre-treatment with ehec (p < 0.01). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal 2-hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following 1hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s-221 85 lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca 2÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca 2+ concentration, [ca2*]i, and ca 2+ fluxes were measured by the use of fluorescent ca 2+ chelating dyes (fura-2 or indo-1) and 45 ca respectively. to assess sepsis-related changes in ca 2+ dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca2+ i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response (100% ¢) in the liver, and a blunted insulin mediated sugar utilization (60% 4) and increased proteolysis (50% ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca2+]i by 35-40% was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm 32288). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c3b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca +2 from intracellular stores. this protein kinase dependent process of [ca+2] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca +2 signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn-7 (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute 01 biochemistry, albert-ludwigs-university, hermann-herder-str. 7, d-79104 freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a 50 and 65 kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: 5"-gggpunnf'ypycc-3") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il-1b, tnf, [l-6, il-8, gm-csf etc), cell adhesion molecules (icam-1, elam-1, vcam-1), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p65. upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a 58/60 kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g2 cells showed that lbp transcripts could be induced 10 to 100 ford within 24 hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter 1.5 kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il-6 responsive elements hstf-hsp 70.5 and h-apf-1 rs as well as the transcription factor hnf-5 and the glucocrticoid-responsive elements oct-2-d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g2 cells by il-6, il-1 and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd14.transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd14, a 55 kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd14, which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd14 may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd14 (cho/cd14). stimulation of cho/cdi4 with as little as 1 ng of lps/ml resulted in the release of 3h-arachidonic acid (3h-20:4) into the culture supernatant. in addition to 3h-20:4 release, we examined lps-imiuced activation (transl0cation) of the transcription factor nf-~b. similar to lps-induced 3h-20:4 release, these responses were observed only in cho cells lransfected with cd14 closely resembling the same response in the marine macrophage cell line raw 264.7. maximum nf-~cb expression occurred at 30 minutes. in contrast to lps-induced 3h-20:4 release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd14, suggesting that nf-r,b activation begins early after lps binding to cd14 and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd14, lps-induced tk activity in cho/cd14 was not observed. although the tk inhibitor herbimycin inhibited the release of 3h-20:4 in cho/cd14 and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd14 cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd14-related lps signal transducer in cho/cd14 is not a tymsine ldnase. dimished cardiac inotropic function and response to 13-adrenergic receptor (13-ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin (0:2 gm/kg). to stimulate various components of the transmembrane signal cascade of 15-ar was isoproterenol (i3-ar), acetylcholine (m2ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia (22°c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that 1 h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c3b and cdb was given to rats at a dose of 12 mg/kg by intravenous bolus, 15 min before reperfusion of the ischemic gut. a control group was given pbs buffer. at 4 h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri (2.49±0.22 to 1.73_+0.17,p<0.05) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced (0.05 ± 0.01 to 0.02 ± 0.01 u/g). c3 was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced (11.3±1.4 x i0 3 to 3.9±0.8 x 103, p<o.05). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic3b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia (4hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j2sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within 60 minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc 0, interleukin-i (1l-i) and il-6 plasma levels increased significantly, reaching maximum levels after 2 hours ofreperfusion. polyclonal antibodies to tnfet and 1l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by 41% and 74% respectively and mpo was reduced by 72% and 83%. antml-1 yielded a protection in muscle and lung vascular permeability of 24.7°,4 and 52.4% respectively whereas muscle mpo was reduced by 46.4% and lung mpo by 25.9°,4. these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using 125mabeled antmcam-i, constititive expression of icam-1 was evident only in lung but not in muscle. during reperfusion icam-1 expression increased by 98.2%. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by 73.9°,4 and 74.8% respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following 90 min of ischemia (mi) and 4.5 h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within 10 min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb1.3 to p-selectin in a feline model of mi+r. pb 1.3 (1 mg/kg) administered 80 rain post-occlusion (10 min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp1.6) for p-selectin (15 + 3 vs 33 + 5% of area-at-risk, p<0.01). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb 1.3 compared to nbp1.6 (67 + 6 vs 11 + 3, p<0.01). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb1.3 treated cats (64 + 7 vs 136 + 12 pmns/mm z, p<0.01). also, normal coronary endothelium activated with thrombin (2 u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p<0.01) blocked by pb1.3, but not by nbp1.6. furthermore, p-selectin is markedly upregulated 10-20 min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested 2, 4, 8, 16, 24, 48, 72, 120 and 168h after transplantation (n=6/time point) . snap frozen sections of organs were stained for cd-4 (w3/25), cd-8 (ox8), t-lymphecytes (tl) (ox-19), macrophages (ed1, ed2), neutrophils (pmn) (mom), icam-i (iap29), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w3/25, ox8, ox19, ed1, ed2 and mom, staining for icam1, lain and fib was assessed visually using a scale (ve=0-10). as early as 2h after transplantation, fibrinogen and laminin expression increased in i, peaking 8h after transplantation (ve= 4.5; nc: ve=i.5) . at 16 and 24h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i.5) but rose again at 48h, peaking at 72h (ve=4) and remaining elevated thereafter (ve=3). the high expression at 8h correlated well with the maximal pmn infiltration at that time (cf=6) in i returning to baseline at 24h (cf=0.5) and thereafter. the second peak at 72h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il-6, tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the 60minute interval (il-lb: 6,1__+2,6 to 161,1__+98,5 pg/ml; il-6:30.24-7,7 to 274,2__+75,8 pg/ml; tnf-a: 544,2__+363,6 to 1651,0+25,7 pg/ml; p<0.05). immediately after the beginning of reperfusion, soluble icam-1 and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at 60 minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam-1, lfa-1 or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n=30/group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within 30 rain, ischemic hearts were stored in cold ringer's solution for 3 hours before revascularization. representative grafts were removed after 12. 24, 72hrs, 10 and 100 days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts (5 vs 1 rain). after 12 hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked (3 vs 1 c/f in non-ischemic grafts), whereas edl+macrophages (9 vs 2 c/f) and tnfe expression peaked by 24hrs. by 72 hrs t-lymphocytes began to enter ischemic myocardium and icam-1 was moderately increased. after 10 days cellular infiltration had returned to baseline, and no differences were seen among both groups after 100 days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il-1 for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd18 treatment in the same model. in other species similar controversial results with anti-cd18 therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a 3-fold increase in neutrophil numbers associated with dispersed lullg tissues 2 hours after lps treatment, while macrophage levels increased by 4-fold at the 24 hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin-8 (il-8). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by 4 hours, while mip-i levels were maximal at 2 hours (2,5 ng/ml), with a second peak at 24 hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a 4-fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from 24 hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by 2 hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip-1. in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by 62-integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of 62-integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib4 and dreg 200, baseline values were determined immediatley after drawing blood. in addition cells were incubated 45 min at 37°c to allow for spontaneous regulation of adhesion molecules. 55 blood specimens from 8 septic patients were obtained during the course of their illness. control values were determined in 12 healthy volunteers. results: baseline expression of 62-integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g2-integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to 2000 pg/ml tnfc~ or 1000nm fmlp (but not at 400pg/ml tnfc~ or 200nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied 23 patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n =7, bacteremia = 6); group b : sepsis syndrome without septic shock (n = 16, bacteremia = 9). serial plasma samples obtained on day 0, 1, 3, 6, 10 and 14, were assayed using elisas method (british biotechnology), normal control levels of soluble icam-1 and e-selectin, obtained from 11 healthy volunteers, were respectively 205 ± 19.4 ng/ml and 45 ± 5.7 ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis,1989) . compared to control mean values, initial levels of groups a and b were significantly higher for icam-1 (p < 10 -4) and e-selectin (p < 10-3). comparisons of group a and [] (* = p<0.05; ** = p<0.00t) soluble icam-1 levels of group a enhanced significantly (p<0.05) during the first 24 hours, and a sustained high levels was of bad prognosis (25% of survivors at day 6). the evolution of soluble icam-1 and e-selectin levels were significantly correlated with murray's score (spearman test : p <0.001). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam-1 and e-selectin are correlated with endothelial lung damage, and loam-1 seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il-6 and il-8, govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il-1, have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam-1. such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: 1) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, 2) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and 3) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room 2600; 702 barnhill drive; indianapolis, in 46202 usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd18 monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)18 chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd18 is expressed on all leukocytes, whereas cd 11 b/cd 18 and cd 11 c/cd 1.8 are restricted to cells of myeloid origin. cd i 1/ cd 18 interacts with intracellular adhesion molecule-1 (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd18 antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd18, however, would affect all leukocytes, as would antibodies to cdlla/cdi8. targeting cdllb/cd18 would affect cells of the myeloid lineage only, which could prove to be beneficial. cd11b/cd18 is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd1 lb/cd18 may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd 11 b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups 0a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b0hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt0gs r.he, t focus on replacing slslic ~sd ~nd fuc0s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein8 t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex2aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ 0u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c0.an does sle:, s=h compounds are m2ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the2~peutics for i~fi~anmat0ry disease. lr:rng maimai models of acute lung lu3ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi 8 or its endothelial ligaud, intercellular adhesion molecule-1 (icam-1, cd54). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two 5 x 30-mm zones were produced by applying brass probes heated to 100°c to the animals' backs for 30 sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for 72 hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd 18 r 15.7 prior to burn injury (pre-r 15.7); animals given r 15.7 30 min after burn injury (post-r 15.7); animals given a monoclonal antibody to icam-i, r 6.5 prior to burn (pre-r 6.5); and animals given the r 6.5 30 min postburn injury (post-r 6.5). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies 30 min after injury was as effective as preburn administration in preserving blood flow. at 72 hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to 34.7 _+ 12% of baseline (p < 0.05 by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy-1787, a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but 2 patients who died during the first 24 hours were excluded, as this was part of the protocol. cy-1787 was administered as a single intravenous bolus of 0.1 mg/kg (n=3), 0.33 mg/kg (n=3) or i mg/kg (n=3) mg/kg. the antibody was well tolerated. none of the 9 patients died during the 28 day follow-up period. organ failure was assessed for 5 organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially 2.7 ± 1.2, decreased to 0.2 ± 0.4 at the end of the study (p<o.o01). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd3 complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin-2 (il-2) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh2/sh3 domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb-2 and shc, via direct binding of the shc sh2 domain to the tyrosine-phosphorylated chain of the tcr/cd3 complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/3 t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~//3 t cell population further segregates into cd4 t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd8 t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a//3 t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of 7/$ t cells. moreover, these studies show that the relative contribution of cd8 or cd4 t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl-2 and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl-2 does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il-1, il-6 and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format 2-d page, autoradiography of 35s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at 3 and 24 hrs. following clp. serum il-6 levels were determined at 3,12, and 24 hours by the 7 tdi bioassay. by 12 hrs. 100% of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant 286% elevation (p < 0.05) in serum il-6 levels at 12 hrs. there was no difference in il-6 at 3 and 24 hrs. when compared to the control groups. 2d page studies examined splenic macrophage total protein distribution and biosynthetic activity at 3 and 24 hrs. post-clp. at 3 hrs. macrophages from clp rats showed a substantial decrease in total protein pattern (353 protein spots vs 503 protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< 40 kd) was observed. at 24 hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< 40 kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early (3 hrs.) staining with cd14 and cd16 mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd14 antigen (cd14++) and a minor population with weak expression of cd14 and expression of the cd16 antigen lcd14+/ cd16+ cells). in healthy control donors(n=35) the latter cells account for 45+22 cells/mm 3 and 9%+5% of the monocytes. in sepsis patients, the cd]4+/cd16+ cells can become a major population with more than 50% of all monocytes in 3 of 18 patients and with more than 500 cells/mm 3 in 4 of 18 cases. there was no correlation of cd14+/ cd16+ cells to any clinical parameter except for cd14+/cd16+ percentage and body temperature (p= 0.013). the cd14++ monocytes showed a substanttial decrease in cd14 antigen density in 9 of 11 patients. three-color-if shows that the cd14+/ cd16+ cells in sepsis patients when compared with the cd14++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd33 antigens, consistent with a more mature nature of the cd14+/cd16+ cells. levels of il-6 were increased in sepsis patients: 3 of 5 patients with high numbers of cdi4+/cd16+ cells ( 200/mm 3) had high levels of il-6 (250u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il-6. human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl 953) and production of the cytokines il-lb, il-6, tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl 953 to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il-6 production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il-6 and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl 953 were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il-6-production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps 1) on mo mediator release with a second endotoxin stimulus (lps2). methods: in vitro cultures of marine peritoneal exudate mo were exposed to 2 hr. of hypoxic or normoxic conditions, then incubated 22 hr under identical normoxic conditions _+ 10 ng/ml of lps 1 pretreatment. during the final 24 hours all m~l were exposed to a range of lps 2 concentrations. m~i supernatants were assayed for tnf, il-1, il-6, pge2, nitric oxide (no), and superoxide release. results: lps 1 markedly inhibited mo tnf release by lps 2 but hypoxia had no effect on lps 2-triggered tnf release. hypoxia increased mo il-6 production in the absence of lps 1, but inhibited lps 1 augmentation seen under normoxic conditions. pretreatment with lps 1 increased no production from lps 2 under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps1 signifcantly augmented mo release of il-i and pge 2, but hypoxia had no significant effect (data not shown) superoxide production was increased by lps 1 under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. (1) . h materials and methods. different groups of icu patients, postoperative (n=12), sepsis (n=12), septick shock (n=13), and healthy control (n=10), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf75 pl oflps (10ng/ml) to 675 id of whole blood in presence of various concentrations of ptx (0, 10 "5, 10 -4, 10 "3, i0"2m). after 5 hrs incubation at 37°c, the tubes were centrifuged and supematants frozen at -80°c. tnfa and/l6 were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h6000. the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and 11,6, corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being 100%, 1) stimulation by lps. the control group is much more stimulated (>21000% for il6, >8000% for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (>1500% for il6, >600% for tnfa ). the lowest stimulation was observed in patients with septic shock (median = 168%), some patients being not stimulated at all. 2)effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at 10 -3 m (reduction to less than '¼ of the median values), and is almost complete at 10" 2 m. the septic shock group has a decreased sensitivity to ptx. il6 production exhibits a lesser reduction at 10 -3 m (~ 'a to ½ of the median values), further increased at 10 -2 m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at 10 -3 m and the inhibition is nearly complete at 10 -2 m. surprisingly, there is a lesser, but significant suppressive action of ptx on il6, not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. (1) lymphokine research (1989) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp-1 was induced with phorbol ester (48 h) and activated for 4 h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il-1 and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received 3 ml of either saline or blood from brown norway donors,iv. the animals were killed 3 or 7 days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day 3; 44-+8 vs 99+7, p<0.001). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure (93+9, p<0.001 vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from 90% in ileum on day 3 to 20%, p<0.001). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb 910 i, 6500 hb nijmegen, the netherlands. leukemia cell line, teip-1. robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. 29425. adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa2, an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa 2 may alter monocyte adhesiveness. we selected the thp-1 cell line, a human monocytic leukemia cell line to further investigate the effect of txa 2 on adhesion. we tested the hypothesis that txa 2 alters lpsinduced adhesion of thp-1 cells and that txa 2 exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa 2 mimetic i-bop (0.11.tm,) or txa 2 receptor antagonists bms180291 and l657925 (10~m). cells were allowed to adhere for 24 hours and adherent protein/well was determined. lps-induced a significant (p<0.05;n=7) increase in adherence of thp-1 cells (basal, 2.4+0.85gg protein/well; lps, 20.4+_6.0p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between 0.5 -0.6 mm, and is reduced in septic patients up to 80 % (0.1 -0.2 mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from 500 ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than 90 %. subsequently cells were cultured in phenolred-free rpmi 1640 medium with various concentrations of glutamine (0.05, 0.1, 0.2, 0.3, 0.6, 1, 2 mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between 70 and 90 %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam-1/cd54 on monocytes in a dose-dependent manner. the receptor for fc'/rucd64 as well as the receptors for complement cr3/cdllb and cr4/cdllc were down-regulated. cr1/cd35 which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi4, the receptor for transferrin cd71 and fc'friii/cd16 were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than 300 patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up 2-3 times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> 5 days) of low hla-dr expression (< 30%) predicts fatal outcome (mortality > 85%). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [7 of 33 patients recovered from "immunoparalysis" following repeated plasmapheres; 16 of them survived (46 %). 7 patients recovered temporarely and 9 patients did not respond (all 16 died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was 4 of 38 (11%; p<0,01). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study 1: in vivo stimulation:cd-1 mice (n=35) were randomized to receive taurolidine (200mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after 24 hours and were assessed for pm0 function [superoxide anion generation (o2-), nitric oxide (no), tumor necrosis factor (tnf), fc/cr3-mediated phagocytic function (phago) study 2: control pm0 were harvested and cultured in vitro with taurine (1.0mg/ml for 10 hrs), after which time they were assayed for 0 2-and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m0. release of the inflammatory mediators 0 2-and tnf, and fc/cr3-mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & 2, and nitric oxide. but injury and inflammation als0 cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a 21 amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi 1640 +5% fetal calf serum and stimulated with i0 ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced 87 ! 6 fm of et/106 cells (vs. unstimulated controls of <25). this calculates to 15-30% of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal 5, germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< 30 % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il-1 receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i0, which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il-10 message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il-10 message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l0, which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il-10 production and the effects of il-10 on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med 17:511, 1993) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il-6 production. macrophages were obtained from peritoneal exudate of thioglycollate treated c3heb/fej mice and cultured in rpmi 1640 medium containing 10% fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b4) for 6 hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for 18 hours. the amount of il-6 in the supernatant was measured by il-6 dependent cell line (b9 and 7td1) proliferation assay. il-6 was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il-6 production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il-6 during the initial incubation with lps and subsequent desensitization. il-6, like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il-6 shared the similar potentiational pathway, but differed by the fact that only il-6 was inhibited. (supported by r37 ai23447 and po1 a54474.) department of microbiology, molecular genetics and immunology and the cancer center, 1000 wahl east, university of kansas medical center, kansas city, ks 66160-7832. korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, 150 ~g/ml,30 min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice 1-14 days after (peak at 2nd day for i.v. and 5th day for i.p. mode of administration of the same dose of 5 mg/100 g b.w.).the preliminary injection of cryelan (5 mg/100 g, 24 or 48 h before) to mice with acute cold stress (-10 ° c, 1 h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al.,1990) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium (3-4 fold); bcg possessed similar effect.cmg in the same concentrations (50 /~g/ml) increased release of these enzymes only saightly (1.6 times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren,1989) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the 5th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased 2-5 fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, 6, 8, prostaglandin e2, and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et-4 (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. 0.5 x 10ucells/ ml in rpmi 1640 + 10% fcs were incubated i0 min., 6 & 24 hrs. with 10 -8 m et-i, 10 -8 m vic or i0 ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps 1 ) for 24 hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin-1 (il-i) in response to a subsequent endotoxin stimulus (lps2). in this study we examined the kinetics of lps 1 inhibition of tnf and augmentation ofil-1. methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or 10 ng/ml of lps1 for intervals of 0 to 24 hours. culture medium was then replaced with 0, 10 or 1000 ng/ml of lps2 for 24 hrs. tnf and il-1 in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin-6 (il-6), interleukin-i (il-i) and prostaglandin e2 (pge2). we showed that preexposure to lps (lps 1) alters the response of murine m~i to subsequent lps stimulation (lps2). we hypothesized that in vitro cytokine release by lps2 in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n=8), cultured in vitro 24 hrs, then pretreated 24 hr _+ lps 1-cultures were then stimulated with lps 2 and mediators in mo supernatant measured: tnf, il-i, and il-6 by specific bioassays, pge2 by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps2= 1000 ng/mh the table shows that all mediators were increased, in the absence of lps 1. pretreatment with lps 1 resulted in complete inhibition of lps2-triggered tnf release. in contrast, lps 1 significantly increased mo secretion of il-1, il-6 and pge 2 (data not shown). serum cytokine levels were as follows: tnf 407_+21, il-i 241+208, and il-6 8.0-+2.5 ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps2-triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps 1. provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing 210-290g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd18, cdllb, icam-1 and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd18, and icam-1, but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi8 on activated neutrophils and icam-1 on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il-6 and il-8 as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps 4 hrs following endotoxin infusion followed at 24 hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p2 integrln (cdlla, b,c/cd18) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~2 integrlns, but not cd14, is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (>30~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng-10p.g/ml) or f'mlp (10 "1 1 to 10"6m) et 37oc for 30 mln, in 1~; human ab serum, the expression of the 132 ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase (3 fold) in the expression of cd11b on pmns from burned patients compared to an 8 and 14 fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd11 b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd1 ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = 0 and total occlusion of blood flow to the lower extremities was maintained for g0 mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h202 production was determined by a previously described 2'7'-dichlorofluorescein flowcytametric assay. 185 _+ 53 amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < 0.01 vs. baseline; 1"p < 0.05 vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at 15 min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h20 2 production was increased by 2.3-fold above baseline values by 1 hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by 2 hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at 90 rain. post-ischemia, however after release 01 the ligature a significant reactive hyperemia was observed by 15 mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from 14 burned patients (mean age 42,1 +_ 17.7 years, mean burn size 39.8% _+ 22.2%) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age 34 years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to 370c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at 0°c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure 1 displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and 1,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la5 that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ ,2s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated 20 blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of 166.7 u/ml (recommended by manufacturer is the range of 10-200 o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns (107.2% of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin-6 (il-6) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il-6 and tgf-61 in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il-6 and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il-6 and tgf-i~ 1 on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll-6 up to 104u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf-61. in conclusion, these data suggest a contribution of il-6 in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at 24 h followed by return of this parameter to normal levels at 48 h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at 3 h. this hi~her index persisted until the end of experiments (5 days). the early (3-5h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at 24 h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes (1). there are reports of nentrophil dysfunction in inflammatory disorders of the skin (2), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score 21±5) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously (2) . in parallel, granulocytes from 20 healthy individuals were tested. two granulocyte functians were studied in vitro: 1. migration of the ceils in a boyden chamber through a filter matrix following stimulation with 5 different receptor dependent stimuli (c5a, intefleukin-8, platelet-activating-factor, leukotrien b4, fmlp). 2. release of 13glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of 13-glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure (10/11) and nonsurvivors (8/11), whereas 8/10 patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. 85: 194-198, 1985 klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i00 ~g/ml) on 8 granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load (8% of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in 12 h of the recovery and cytosol proteolytic activity (ph 7.4; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was 2-3 -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by 6 h of the recovery (the comparison was done with the sedentary rats). in 12 h cytosol proteolytic activity decreased and then increased again by 24 h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate (70-80%) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il1,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from 85 polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s3mdrome. the presence of interleukin (il)-8 in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il-8 in animal models can attenuate lung injury. collectively, evidence to date suggests that il-8 attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il-8 is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il-8 can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, 1251.albumi n leak was quantitated by measuring the 1251 counts which crossed the monolayer, using a gamma counter. il-8 (lpg/ml) was incubated in the culture medium with 1251.albumi n for 21 hrs. the il-8 dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to 11.-8 addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt8 expression and primes pmn's for subsequent 02generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd 18 expression and pmn priming via the [3 adrenergic signal transduction pathway. methods: normal human pmns were primed with paf (10ng/ml for 5 min) or pre-treated with 10-5m of isoproterenol (i) or forskoklin (f) for 5 rain and then primed with paf. cd18 expression was measured by flow cytometry (fig.l) and 02-generation in response to 10-rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig.2 holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf109203x on cytokine-and endotoxin induced expression of intercellular adhesion molecule 1 (icam-1) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo0-and lipopolysaccharide (lps)-induced icam-1 expression on human endothelioma celts (eahy926) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf109203x significantly reduced icam-1 expression induced by interferon-y (ifn-?) and interleukin-1 (il-1). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il-1 induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il-1 driven icam-1 expression on eahy926 cells as well as the il-1 induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody (195f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf109203x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b 4 (ltb4, 5x10 "6 tool/l) or intravenous injection of interleuldn-2 (il-2, 5-106 iu/kg b.w.) and lipopolysaccharide (lps, 15 mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of 3 mg/kg b.w., superoxid-dismutase (sod, 10.000 iu/kg b.w.) was able to decrease the ltb 4 effect, and the immuumodulating drug leflunomide (hwa 486) exerted inhibitory effects on il-2-induced permeability at a dose of 3 mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb 4 or il-2 induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam-1 in neutrophil migration. blocking antibodies to human pecam-1, in which the antibodies are crossreactive with rat pecam-1, were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam-1/ integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi8 adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs (15-25 kg) were subjected to 40% arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at 24hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added (0,1,5,10 i.tg/ml) ex vivo. after 3hr incubation at 22-24°c, %cd18 (+) pmns were determined with fitc-ib4 and flow cytometry from mean channel fluorescence histograms. 49±8 # p<0.05 vs baseline * p<0.05 vs sham $p<0.05 vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd18 expression was depressed for at ]east 24hr after trauma relative to sham but by 72hr, was enhanced, relative to sham, and because fentanyl anesthesia at 72hr had a greater effect on cd18 expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps (50ng/ml), tnf(10ng/ml), and lps/tnf(25ng/10ng/ml). cultures of the human ec tine(ecv-304) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for 6, 12, 18, and 24hrs. ec necrosis was assessed by a 51cr release cytotoxicity assay. pmn activation was assessed by cd1 lb receptor expression and respiratory burst activity 6hr 0_+0 1.7-+ 1 0-+0 3.2_+ 1 0_+0 4.4_+ 1 0_+0 2.3_+0.9 12hr 0+0 3.24_3 0_+0 9.3_+3 0_+0 11_+3" 0+_0 12+--2.1" lghr 04-0 0.9_+2 0+_0 284-12" o:fo 25,12" 0~0 30+-9.5* 24hr 0_+0 4.14-4 0-+0 33+_3* 0_+0 30_+9* 0_+0 32_+10" data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p<0.05 vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received 3 ml/100g bw normal saline. at 5 hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, 2xi0 -7 m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at 5 hr post-clp. after pre-contraction of blood vessels in the isolated gut with 5xl04 m ne, vascular responses to ach (5x10 6 m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, 5xl0 -7 m), were determined. total vascular resistance (tvr, mmhg/mi/min/100g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n=6/group) in the aortic rings were: ach lxl0 i~s, st-in ~ ~ significantly at 5 hr post-clp (i.e., increased *p(o 05 vs. sham; n-4 per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin-1 on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. 200-230g) shook was induced by fractionated withdrawl of arterial blood within 5 rain and maintained for 1 h (map at 40 mm hg, cardiac output 50 % of baseline). rats were adequately resuscitated with 60% of shed blood and twice the volume in ringer's solution additionally. following 5 h of reperfusinn (map >100 mm hg, co >120% of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, 5mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n=6) received the il-lra as pretreatment beginning 5 min prior to shock induction. in the group t (n=6) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of 5 mg/kg and was followed by continuons administration of 5 mg/kg/h. the control group c (n=4) received equal volumes in nac10,9%, the sham-operated group s (n=4) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: 9,1% +1,1%; c: 42,1% _+5,4%) was significantly reduced by pretreatment or treatment with il-lra (p: 16,9% -+1,9%; p<0.001, t: 28,8% -+3,6%, p<0.01, anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: 31993iam3/s +40971um3/s, p: 32768llm3/s +4445}am3/s, t: 326211ams/s -+2998 lam3/s, s: 390201am3/s -+39041am3/s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il-1. pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to 6 retool/1 asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc (1 g/kg bw bolus, followed by 0.2 g/ kg-h infusion) on the endotoxin-induced (0.5 ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p<0.0005 at 6 mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p<0.025 at 6 mmol/l asc) and reduced the adhesion dose-dependently (p<0.0005 at 6 mmol/l asc); ec damage was also reduced (p<0.0005 at 6 retool/1 asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p<0.05), decreased the protein clearance (p<0.05) as well as the zymosan-induced rom production (p<0.03), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi8 on neutrophils and lymphocytes. methods: from blood samples (n=5) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after 0,5, 15,30,60 and 120 minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi8 and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at 15min was observed in wbc-cultures (n/r: 0min 91/9, 15min 76/24, 30min 68/32, 60min 52/48, 120min 49/51). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to 30min followed by a decrease to normal values at 60min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl8-expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c5-delicient mice in a model of zymesan indt~ed mof. materials and methods. 25 c5-deficient b2d10/oid and 25 c5-sufficient b2d10/new mice were used in this study. on day 0 all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of 1 mg/g body weight. between day 0 and 12, biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score=4). on day 12 all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x100) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day 0-4) and a late phase (day 8-12). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c5 deficient mice (all p<0.005). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was 2/25 (8%) in c5 deficient mice and 8/25 (32%) jn c5 sufficient mice (p=0.049), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c5 appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c5 could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c57bl/6 mice received zymosan (1 mg/g body weight) intraperitoneally. groups of 14 animals were killed after 3, 6, 8, 18 and 24h and subsequently at each day until day 12. plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin 1-ct, and -6 (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c 0 were measured in plasma and culture fluid by means of a ria (detection limit 0.1 ng/ml). interleukin-6 (il~) levels were assayed using the b9 hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day 7, they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within 3h after zymosan injection and disappeared within 8h. from day 8 onwards, tnf levels started to rise again. plasma il-6 behaved almost similarly in the acute phase, but in the mof phase plasma il-6 remained low. no circulating il-16 could be detected at any time point. macrophage lps-stimulated production of il-lcq il-11~ and tnf--c~ was suppressed immediately after zymosan injection. production of il-16 and tnf-~ was normalized within 6h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day 6 did production of il-i~ reach control values. il-16 production was higher than control values from day 3 onwards. il6 production was similar to that of ili-il the production of tnf-ct was strongly elevated between days 1 and 6 and again during days 10 to 12. the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il-16 or il-6. also, the ability of macrophages to produce excessive amounts of il-16 and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~1-acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with 5 antisense dna's (as) complementary to the 5' region of the il-6 gpl30 receptor. the antisense were 5'-agtttagggatgagg-3' (asl), 5'-atcttcatcttctgaat-3' (as2), 5'-aagtgaatgattaaaacact-3' (as3), 5'-aaacctttataggcg-3' (as4), and 5'-cgttctacaactgcaacgt-3' (as5). using hepg2, the biological effects of these antisense complexes on the high affinity il-6 receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for 24 h. underivatized asl was less effective and the complex, asgp-pll-as2, had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il-6 (i00 units) asgp-pll-asi alone showed a dose dependent (0.i-0.4 ~m) inhibition of 3ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem 91120, israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l0 female merino-sheep were included (mean weight: 30kg). day 0: hemorrhagic shock (mean arterial pressure (map) 50mmhg for 2 hrs.), closed femoral nailing (ao-technique), day 1-5: bolusinjection of endotoxin (et) (0,75~tg/kgbw) und zymosan-activated plasma (zap) (20ml) every 12hrs., day 6-10: observation. bronchoalveolar lavage (bal): day 1, 6, 10. the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm-5), pap -putm.art.pressure (mmhg), pap2 -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov1), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p<0005) results: baseline day 4 day 6 day 8 day 10 heart: co 4,96_+0,32 5,33_+0,36 5,4_+0,2 7,30_+0,77* 7,98_+0,66* svr1520_+134 1395+141 1403_+89 1119+_122" 1089+-91" lung: pap 17,0_-20,7 19,6_+1,6" 22,0+-1,2"25,8+2,0" 28,8+-1,7' pap2 103,1+1,5101,5+-5,7 97,3_+4,7 91,9+-5,6 89,8+_3,9* liver: bill 2, 94_+0, 344, 82_+1, 34' 5, 13_+0, 68'6, 13_+0, 7" 7, 19_+0, 91" kidney:crcl 86, 5+_30, 9 109, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] 4 74, 7_+10, 868, 8+14, 1 53, 1+17, 6 histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge2 is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il-6 and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg3 responses, t cell proliferation, and il-2 synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il-4 and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by 2d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after 30-45 rain even without cyclooxygenase inhibition although pgi2 levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated 26 patients undergoing major abdominal surgery as compared to 26 ibuprofen (400 rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time 5, 15, 30, 45, 90, 180, 360 , 1440 rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of 6-keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of 6-k-pgf~,tp~, peaked 5 minutes after m.t. (2274_+284, ibu: 102_+12, to: 60+i ng/l) and declined monotonously over 6 h (203 +_42, ibu: 84_+ 12 ng/1). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after 45 rain (66_+23, ibu: 18+3, to: 16-+2/~u/ml); aldosteronc also presented a maximum after 45 rain (110 + 12, ibu: 66-+ 15, to: 56 +-7 pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum 6 h after m.t. (26+_1, ibu: 24-+3, to: 10+_1 ~g/1). adh pc peaked 5 min after m.t. (71+7, ibu: 20-+6, to: 5+_1 pg/ml) and showed analogously to 6-k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated 45 min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, 89070 elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. 20 rats of them received sma clipping and others wee used as control. control and treated rats at 2, 4, 6, 8 hours were perfused and fixed. the brain were sectioned at 20 pm and stained by abc method using c-fos antibody. plasma endotoxin level of 5 rats were measured at 0, 2, 4, 6, 8 hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of 0, 2,4, 6, 8 hours after the treatment were 6.48 ± 5.57, 15.0 _-4-4.37, 10.0 _+ 4.18, 14.4 ± 3.52 and 58.4 ± 28 .6 pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi2). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied 52 patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed 5, 15, 30, 45, . tile plasma concentrations (pc) of 6-keto-pgft, (stable metabolite of pgi2) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < 0.05 placebo vs. [ibuprofen] [117] mmhg (*p< 0.0i). these changes were accompanied by a marked increase of 6-keto-pgf~ pc up to 180 rain after mt in arterial and mixed venous blood of untreated patients with a peak of 2450*[60] ng/l tl (*p< 0.00ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao2 while 6-keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for 6-keto-pgft~ was not detectable. a splanchnic vascular source for pgi2 release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e 43, 89075 ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day 1, two groups of n=36 8 week old aj mice were subjected to either a 20% scold burn (ti), or were untreated (c) n=18. on day 10, 18 mice (ti+clp) and 24 mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days 11, 12 and 13 after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il-2 and adherent splenocytes (as) were cultured with lps for il-1, tnf, il-6 and pge2. results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi2) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi 2 during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. (109/ml) at rates increased incrementally from 0.2 to 4.0 mi/kg/hr over 4 hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n=6); pga (n=5), graded bacteremta plus anti-pgiz antibody, 50 ml/hr iv, beginning at 2 hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po2, mmhg, cardiac index (ci), l/min/m2, oxygen delivery index (do2i) and consumption index (vozi), ml/min/m2, and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg6-keto f1 alpha ; in the first instance~ peak values of lt ~4 after i~ hrs post infarction were 6 times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b4 led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets4, ~ev252024, ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical (121 patients) and experimental material(128 rats and 74 dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period (1-7 days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin (15 microgram per kilogram of body mass intrap'eritoneally within 5 days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [2] . peep can affect rv function by reducing preload and ejection fraction (ef) [3] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this 34-year-old patient (apache-ii:24) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [4] . the therapeutic procedure included volume replacement, vasopressors (dopamine 5, dobutamine 3 gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf-30, od=6 mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id=8 ram, bennett 7200a, pressure controlled mode, pm~x=25 mbar, peep=8 mbar, i:e=i:i, fio2=1.0). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [1 ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new 55,000 dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [2] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included 10 rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to 15 min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing 150 mg% glucose & 5 gm% bovine albumin and incubated in dubnoff shaking water bath for 60min at 37°c the results revealed that there was an enhancement in release of free fatty acids (ffa) (240%) and lactate (163%) and in glucose uptake (160%) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition (200 ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by 67%. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a23187, but not with a direct vasodilator (nano2). this failure to relax to ach and a23187 reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by 2 weeks. exogenous no donors (e.g., c87-3754 or spm-5185), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor (30 p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at 7 days. rat carotid artery rings relaxed only 17 + 5% and 15 + 5% to 10 gm ach in vehicle treated rats and in inactive no donor treated rats respectively 7 days following injury compared with 67 + 6% in no donor rats (p<0.001). relaxation to 100 gm nan02 was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name (1 mm) was markedly reduced by injury, but was protected by no donors (p<0.01). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il-1, ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin-1 and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) and the cytochrome p450 (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to 35 mmhg for 3 hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, 100 nm) was reduced by 80% compared to control sma segments (p<0.01) while relaxation to sodium nitrite (100 gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p<0.0l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p<0.01), attenuated mesenteric mpo (p<0.05) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm-5185 (schwarz pharma), following regional myocardial ischemia (1 hour) and reperfusion (4.5 hours) . treatment with spm-5185 (500 rim) significantly reduced myocardial necrosis by 70% (p<0.01) compared to an no-deficient analog of spm-5185, spm-5267. furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p<0.01) reduced following treatment with spm-5185 compared to spm-5267 (1.6 + 0.5 vs. 3.8 + 0.3 u/100 mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps (0 mg/kg, n=15:8 mg/kg, n=21 ; or 16 mg/kg, n=30) and treated with l-nma (0, 1,2, 4,10 mg/kg/hr) for 22 hours following a 0, 10, or40 mg/kg loading dose. animals were resuscitated with iv ringers solution (10 ml/kg/hr). hemodynamic data were collected at 0, 2, 6, 10, 14, and 22 hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p=0.07), and systemic vascular resistance index (p=0.ol) and decreased cardiac index (p=0.05) and oxygen delivery index (p=0.01). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p<0.05), but this differential effect on the venous and pulmonary circulation occurred, >6 hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied (1, 2, 4, or 10 mg/kg/h) in either the low (8 mg/kg) or high dose (16 mg/kg) lps-challenge groups. a nonsignificant (p>0.2) trend toward a beneficial effect on survival ol low dose l-nma (1 mg/kg/h) in animals given the 8 mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study (10 mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p<0.05). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad 2. recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [1]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no (18 and 36 ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad2 [2] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation (5-20 ppm) consistently lowered the pap and augmented the pao2/f.o 2 for up to 53 days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below 1.5%. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [3] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa02 and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately 2-3 ppm, the improvement of oxygenation had a maximum at 10 ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately 100 ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad2, thus allowing reduction of the f.o 2. no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd-3-phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il-1, ifnj and lipopolysacchafide (lps). 24 hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after 3 more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of 1,3-diphosphoglycerate to glyceraldehyde-3-phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about 5 times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by 49_+3%. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only 32_+12%. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il-1, tnf, ifn, and lps for 16 hours. 20, 60, 120 and 200 ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no2); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n=4.) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont 14.1 + 0.3 32 + 4.0# stim 13.4 + 0.6 139 + 12 stim+2o tzm nmma 12.7 + 1.1 88 + 5.0# stim+60 ~m nmma 9.9_..+ 0.4* 68 + 4.5# stim+120 pm nmma 7.6 + 0.6* 57 + 3.0# stim+200 )lm nmma 5.0 + 0.5* 37 + 2.5# anova 0,0002 0.0001 (* p < 0.05 versus stim, # p < 0.01 versus stim; anova with neuman-keuls) gsh in cont+120 i~m l-nmma was equivalent to that of cont (13.3 vs.14.1). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c57bl/6 mice and lew rats underwent a 50% and 70% ph, respectively. sc were prepared 1-6 days after ph and plated at 1 to 2 x 10ecells per well. after 20 h incubation at 37°c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient 3 days aider liver partial resection (1.4 nmol nitrite/106 cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: 1) encletoxin and l-name, 2) endotoxin, 3) naci and l-name, 4) nach preliminary results from groups 1 (n=7) and 2 (n=8) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin (1.7 gg/kg/h) was given as a continous portal infusion over the entire observation period of 8 hrs. l-name (25 mg/kg) was given as a bolus after 3 hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by 25%* and hepatic artery flow (hal e) by 45%*, while l-name caused a further and lasting reduction in flow (pvf 78%, haf 90%)*. transhepatic (portal-hepatic vein) vascular resistance increased to 3 times baseline value during endotoxemia while l-name caused a further marked increase in resistance to 12 times initial value. portal oxygen saturation (so2) decreased by 60%* during endotoxemia. l-name caused a reduction in portal so2 by 87%*. arterial so2 was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a 27% reduction in cardiac output (co). the addition of l-name reduced co by a total of 71%*. *: p < 0.05. conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; 2 mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; 20 mg kg -i s.c., at 16 h prior to lps) or with an il-i receptor antagonist (il-ira; 16 mg/kg bolus and 2.4 mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at 90-180 min after lps. for instance, endotoxaemia for 180 min resulted in a fall in map from 114-+6 (control) to 79-+4 mmhg (p<0.01; n=8). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at 180 min when compared to lps-control: 118-+3 mmeg (n=5) and 103-+7 mmhg (n=5), respectively (p<0.01). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, 20 min, 3x10 -4 m). pretreatment of rats with tnfab or il-ira significantly (p<0.05) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at 180 min after lps there was an induction of calcium-independent nos activity in the lung (5.14-+0.57 pmol citrulline/mg/min, n=8), which was attenuated by tnfab and !l-ira by 37-+6% and 46-+6%, respectively (n=5; p<0.05). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with 109 live e. coli, which consistently increased cardiac output 15-20% above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name,1 mg/kg iv), prevented this increase and elevated map by 25-30%. in the first 3 groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other 3 groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are 60 rains after e. cob. n=5-7/group. * p<0.05 vs bl by remanova and § p<0.05 vs e. coli alone by anova. ec+l-name 11-+2 -57_+10" § -45_+4* § -89_+3* § -33+5* -20+5* § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 5'untranslated region (utr) of ferritin mrna and 3'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k562 myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of 1re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j774) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k562 cells, where nos was stimulated by increasing the availability of the essential nos cofactor 5,6,7,8-tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn-5or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of 7 pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group 1, no, 50 ppm, was added to the inhaled gas from the start of dialysis. in group 2 no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group 1, from 2 minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats (250-300g, n=4-5/group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received 3 rrd/100g bw normal saline. the animals were killed at 10, 20, or 35 h post-clp (10 h post-clp=early sepsis; 20-35 h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, 10 9 to 10 -5 m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, 300/~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at 10 and 20 h after clp. in contrast, l-nmma administration produces an 18% increase (p<0.05) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at 35 h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a 68-years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of 200 mg of l-nmma, a continuous infusion was installed (0.05 mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose (62 to 134 mmhg}, heart rate stayed stable (124 + 4 b/rain), systemic vascular resistance increased (225 to 354 dyne.sec/cm5), cardiac output decreased (17 to 15.2 l/rain), and cardiac index declined (9.94 to 8.63 l/min/m2}. before and after 90 minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after 140 matutes. neopterine increased from 16.51 to 32.55 ng/ml, tumour-necrosis-factor-a increased from 24.16 to 36.61 pg/ml and intedeukin-6 increased from 61.9 to 98.2 pg/ml. immunoglobulines a, g, and m (3.2 to 2.9, 8.1 to 7.6, 1.1 to 0.9 g/i), complement factor c-3c and c-4 (0.54 to 0.50, 0.22 to 0.23 g/i), alpha-l-antitrypsine (3.86 to 3.83 g/i), c-reactive-protein (111.1 to 105.6 rag/i), interleukin-1 (0 pg/ml) and soluble interleukin-2 (2128 to 1983 units/ml) did not change significantly. pulmonary-shuntvolume decreased from 54.1% to 35.4% within one hour and to 36.6% after 140 minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse 100, 8091 zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. 1986. microbiol.immuno130:425) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc 50 ,um, ifn-,/250 u/ml, lps 1 fig/m/, ngmonomethyl-l--arginine (mmla) 2ram. nitrite (no2-) content of the supematants was determined by a standard griess reaction, and h202 release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x-100, and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with 0.5 ml of a suspension of 5.10 ~" s.typhimurium, strain c5, and injected with aminoguanidine (ag) 1 mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from 60% in the control group to 20% in the treated group, 10 days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom 78% to 57%, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no 2-accumulation, 4.7 nmole/well v.s. 21 nmole/well. conversely h202 production is enhanced from 09 nmole/well up to 2,33 nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, 77% in iron exposed macrophages v.s, 68% in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity (57% v.s. 68%) whereas the addition of afc to these macrophagas restores an elevated (72%) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h20 2 release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit4 de microbiologie. bp 87. 38702 la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at 37°in tcm-199/5% fcs, with or without additional rat serum. after 20 h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of 240 u cobra venom factor/kg b.w. at days 0 and 2. significantly higher (p<o.o1) lps-stimulated no-generation was found in spleen cells prepared at day 4 (13.0 4-1.3 nmol nitrite/106 cells, n=6) compared to spleen cells from non-treated animals (3.6 ± 1.3 nmol, n=6) complement activation was effective also in vitro. when incubated in 25% zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli (10qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at 4 hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l929 bioassay. survival rate (%) qrql/,p n 8 hr 24 hr 4 dav control (normal saline 0.1ml/body i.p.) 10 100 50 40 n10 (l-name 10mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to 60 min of partial liver ischemia (70%) and 30 min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i0 mg/kg). sod, when used, was given as mn-containing preparation at a dose of 6000 u. i.v. i0 min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : 0.93±0.11"#/ 0.52±0.08/ 0.48±0.05. alt (u/2) • 171±40~#/ 477±173/ 506±182. gldh (u/l): 5.2±1.2e#/ 10.7±4.2#/ 24.3±9.1. in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous 140~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [1] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [2] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no2-chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of 0.07-0.13 parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non 0.089 0.0422 0.0721 0.0311,2 parts per million (ppm) no, mean, n = 10; ~ p < 0.05 (t-test) between smokers and non-smokers; 2 p < 0.05 (t-test) between in-and expiration. about 50% of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than 95% {o.043 ppm before intubation vs. 0.003 ppm after intubation, ~, < 0.01), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [3] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi 1, yoshihiro inoue 1, masao yoshida 2 1 critical care and emergency center, and 2department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il-2 contribute to the occurrence of septic shock (circ shock 38:264; . the present study investigated the in vitro effects of lps, tnf-o~, il-2 and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il-2, tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il-2 and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il-2 and tnf-c( enhanced lps-facilitated no production to a greater extent than il-2 or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. 19-1 uchimaru, morioka 020. japan. t. yolk 1,2, i. ioannidis 1, w.j. kox 2 and h. de groot 1 objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: 3-morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin-1 (5mm), which releases both no. and o2-., caused a time-dependent cytoreduction of 60% and 80% after 3 and 6 hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes 02--to form h202 and 02. in contrast, the addition of catalase (cat), which removes h202 to form h20 and 02, diminished the cytotoxic effect ot sin-1 to 30%, equivalent to high concentrations of snp (20mm), which solely releases no.. in addition, the amount of h202 formed by 5mm sin-1 equaled the amount ot enzymaticany produced h202 by 5mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration (5mm) alone were not toxic to endothelial cells, the combination caused an 80% reduction of viability, comparable to the effect observed with sin-1 (5mm) alone. moreover, toxicity mediated by high concentrations of snp (20mm and 50ram) was reduced by 20% under hypoxic conditions indicating synergism with no-and 02. conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with 02-. however, toxicity increases dramatically in the presence of h202. this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no2 and no3 levels were measul*d sliectrophotometricallymonocyte cultures were done for 24 itrs with or without lps+ifn-t stimulation mid no2 and no3 levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no2 levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. 2)both no2 levels in the supematmlts of monocytes cultured with lps+ifn-t mid no2, no3 levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. 2)based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j774 with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [200u/ml]. after 18 hours of incubation no-release was determined as nitrite (no2) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d609, that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no2-production (p<o,001). although we observed no toxic effect on cell viability, no2-production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo0nm] released 20% less no 2 in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [30}jm] and bim [ioijm] reduced the no2-formation by 50% and 85%, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d609 cells pretreated with d609 [301~g/mt] released 96% less no 2 than stimulated controls the inhibitory effect on no 2production decreased the later the inhibitore were added after stimulation; e.g. bim added 6 and 12 hours after stimulation reduced no2-production only by 50% and 12%, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr 12, 91054 erlangen interleukin-1 (il-1) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il-1ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the 5' utr of the human il-1ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable (75%) gc rich region. the translation of the type i il-1r appears under a significant suppression and may limit the biological activities of il-1 by low level of expression. on the other hand, the type ii il-1r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il-1 activity; inhibition of il-1 converting enzyme which cleaves pro-il-l[3, anti-il-1ri, the il-i receptor antagonist (il-1ra) and soluble (extracellular) il-1ri and il-1rii. il-1ra is structurally related to il-1 and binds at 4°c to the il-1ri with near equal affinity as that of bona fide il-i; however, il-1ra does not transduce a signal. il-1ra has been given to humans in pharmacologic levels (mean plasma levels of 30gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by 50% and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il-1ra are in 100-fold molar excess to those of il-i [3. it is unclear whether these endogenous levels of il-1ra are sufficient to block il-1 activity in disease. a single injection of ]l-6 or ifna in humans does not induce il-1 but rather high levels of il-1ra (10ng/ml). prof.dr.l.a. aarden interleukin 6 (il6) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il6 is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il6 has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il6-anti-il6 complexes, suggesting that plasma il6 measurements represent a gross underestimation of il6 production in the patient. until now no natural antagonists of il6 have been described. the biological activities of il6 results from binding to a lowaffinity il6-receptor (il6r). the il6/il6r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp130. studies on the structure-function relation of il6 revealed that disruption of the gpl30-interaction site on il6 gives rise to a mutant il6 that, by virtue of its intact binding to the il6r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin-8 (il-8), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il-8 and the ability of il-8 to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il-8 during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il-8 was found to be a potent angiogenic factor at concentrations ranging from 2-40ng/ml. sequential fluorescein angiograms demonstrated that il-8 induced neovascularization by 14 days, which regressed by 6 weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il-8. in addition, an il-8 antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il-8 has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th0, thl and th2 cd4 ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il-10 are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il-10 also directly inhibits t-cell proli/eration induced by cross]inked anti-cd3 or anti-cd2 mabs (e.g. in the absence of professional apc), by specifically inhibiting il-2 gene transcription and il-2 protein production. il-10 also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il-6, and tnf-a and the chemokines il-8 and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il-10 enhances the production of the il-1 receptor antagonist, a cytokine with anti-inflammatory activity. il-l0 produced by monocytes downregulates class ii mhc expression and il-10 production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il-10 is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il-10 also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il 8) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il 8 preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd4 and cd8 memory t cells. ip-10 chemoattracts only preactivated (not resting) cd4 or cd8 t cells. hip-l~ preferentially chemoattracts gd8 t calla, while hip-18 more readily attracts the cd4 t call subset. the 8 chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd3 activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla-4. these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h 8 integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln 30', peaked by 1 hr and was completed in 3 hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf4 and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin-12 (il-12) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of 35kd and 40 kd. il-12 was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il-12. phagocytic cells produce il-12 in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il-12 exert biological activity are t and nk cells. the major direct biological function of il-12 on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il-12, produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il-12 to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il-12, other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il-12 has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type 1 (th-i) response and suppressing a th-2 response. the presence of il-12 during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il-2, and inhibits the generation of th-2 cells producing il-4. il-12 induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il-12, however, il-12 needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il-12-mediated inhibition of il-4 producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il-12 for thl cells and an inhibitory effect of ifn-y on th2 cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il-12 on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il-12 appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. 1993, interleukin-12 and its role in the generation of t e 1 cells, imm~ol. on peripheral blood monocyte/macrophages, il-13 behaves like il-4 in a variety of assays, and shows both similar and contrasting, activties with either il-10 or ifn-y. il-13 reduces inflammatory monokine and il-10 production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, 1993, febs lett. 328, 268) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi4 expression. il-13 produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il-4. it inhibits niv-i production (montaner et el, 1993, i. exp. mad. 178, 743) , and interferes with hiv-induced cell fusion. the activities of il-13 and il-4 differ on t-ly•ocyte and large granular lymphocyte population. in particular il-13 does not exhibit the suppressive effects of il-4 on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il-4 and il-l3 share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. 1993, embo j.7. 2663-2670), explaining both thai common and divergent activities. the levels of il-13~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il-4 mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t8 cytotoxic lymphocytes express markedly higher levels of il-13~rna. in vivo, il-13 may thus be contributing, significantly to some of the activites previously ascribed to il-4 il-10. recently, we cloned the human cdna encoding interleukin-13 (il-13). human il-13 is a non-glycosylated protein of 132 aa with a molecular mass (mr) of 10 kd and has biological activities on monocytes and b cells, il-13, like il-4, strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd23 (fc~rii). furthermore, il-13, like il-4 and il~10, inhibited the production of pro-inflammatory cytokines il-1, ]l-6, tnfo~ and chemokines miplc~ and tl-8 by lps activated monocytes. both il-13 and il-4 enhanced the production of il-lra by monocytes. in contrast, il-13 lacked the t cell stimulating activities of il-4. the responses of monocytes to il-13 and il-4 were not additive or synergistic, supporting the hypothesis that il-13 and il-4 receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il-13, il~4 and il-10 have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il-13 is unique in that it can modify inflammatory reactions without stimulating (as does il-4) or inhibiting (as does il-10) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of 390 amino acids that is inactive until processed to a disulfide linked homodimeric molecule of 1t2 amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf-131 has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf-13 isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[3 family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il-6, pge2 and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il-10 and il-12, have been characterized as critical in regulation of mo tnfa and il-6, as well as in induction of t lymphocyte proliferation and ifn 7 production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score >35) were analyzed for their il-10 levels, their in vitro proliferation to mitogen (pha) and their response after il-12 addition. since il-10 produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il-10 levels. increased patient il-10 production might also be resulting from the high levels of tnfa a known stimulator of il-10. conversely, since il-12 augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il-12 deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il-10 found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il-10 production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il-10 were explored. increases in tgf[3 may have partially contributed to the patients' depressed il-10 level, but elevated pge2 had no effect. addition of il-12 to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il-6, pge2) and a dearth of other monokines (il-12, il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells (13mmc) may result in the production of different cytokines including the interleukins (il) 3, 4, 5, and 6 as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il-1, 1l-6, tnf-c 0 as well as various mast cell growth factors (il-3, il-4, il-9, il-10, ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl06 bmmc/ml; ll.um ionomycin; 24-48h). the actixdties of bmmc supernatants were assessed in specific biological (il-3, il-4 il-6, 1l-9) and/or elisa assays (il-5, il-9). here we show that homogeneous populations of bmmc (>97%alcian blue+/safranln-; in vitro age: 4 weeks) generated in the presence of recombinant (r) rail-3 from normal balb/c mice produced modest amounts of 1l-6 and low or undetectable levels of il-3, -5, and -9 after induction with lp.m ionomycin only. however, a dramatic increase (5-to 10-fold) of these cytokine activities was noted, when in addition to ionomycin also human (11) rll-la was provided during the induction period. this il-1 effect was dose dependent with a maximgm at 2-10 u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with 20 ng/ml hrll-1 receptor antagonist abolished the action of 2 u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll-6 or rmtnf-~. both mrll-10 and hrll-10 substantially enhanced ionomycin-induced 1l-6 production of bmmc in the absence or presence of il-1. il-10 significantly enhanced il-6 and il-9 production while decreasing il-3 activities to abont 50-90% of control levels, when il-i0 was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t0 antibody (ascites 1:200) abrogated the effects of mrll-10. other mast cell-active cy~okines (]1,-3, il-4, 1l-9, ngf, or kl) added to ionomycia-or 1l-1/ionomycin-treated bmmc had no major effects on cytokine production. il-1 and il-i0 did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of 10"6m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il-1/ll-10induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il-9 inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il-9 previously recognized as a product of th2type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il-10, and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of 17 different httman cytokines, ilia, ill [3, illra, il2, il3, il4, il5, il6, ils, ill0, gm-csf, tnfa, ifn 7 and tgf[31.3, was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with 4 % paraformaldehyde and permeabilized by 0.1% saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[31_3, could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il-6 in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n=10) were incubated at 37°c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after 0,30,60,120,240 and 360 minutes plasma levels of tnf-alpha, il-i beta and il-6 were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from 0,5 pg/ml at 0min to 14pg/ml, 236pg/ml, 343pg/ml after 60, 120 and 240 min. il-ibeta (0,3pg/ml, 0,6pg/ml, 1,5pg/ml, 81pg/ml and 169pg/ml after 0,60,120,240 and 360min) and il-6 (0,2pg/ml, l,lpg/ml, 4,1pg/ml, 58pg/ml and 72,9pg/ml after 0,60,120,240 and 360min) plasma levels were increased 60min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il-6 to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr.9, 89070 ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf-131) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf-131 (0.1-1.0 ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf-131 (>5.0 ng/ml) potentiate production of the immunedepressive prostaglandin e 2. other investigators have shown that tgf-]31 can cause the appearance of cd16 (fc immunoglobulin receptor) on monocytes exposed to 10 ng/ml of tgf-[~i for 24 hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd14 is depressed in septic patients and therefore we hypothesized that tgf-131 could account for the down-regulation of cd14 observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[31 for 2 and 24 hours at 37°c and examined ceils for cd14 and cd16 expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at 37°c alone. one displaying high density of cd14 had increased fluorescence over the homogeneous expression of cd14 in cells maintained at 4°c (baseline). the other population displayed decreased cd14 expression relative to the baseline cells. tgf-i~i (10-50 ng/ml) caused a shift of ceils from the high density into the low density cd14 population. this trend was observed within 2 hours of incubation and was complete by 24 hours. we observed a net decrease in cd14 expression 0f32% for all subjects studied (p<0.05 vs controls). phorbol myristate acetate (100 ng/ml) also caused down-regulation of cd14 to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd16 after incubation with tgf-131 (10 ng/ml) for 24 hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd14 by tgf-131 deplete their surface expression of cd14 while generating cd16. this down-regulation of cd14 by tgf-131 correlates with our clinical observations of lower cd14 expression on monocytes obtained from septic patients. for over 25 years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw264.7 and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw 264.7 cells, mif secretion was induced by as little as 10 pg/ml of lps (e.coli 011 l:b4), peaked at 1 ng/ml, but was not detectable at lps concentrations >1 txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn 7. production of mif by lps-stimulated (l ng/ml) macrophages peaked at 12 hr. expression ofmif mrna and tnf mrna by lps-stimulated raw 264.7 macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations (1 pg/ml to 1 p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately 2-fold after lps induction (100 pg/ml). mif secretion also was induced by tnfoc (1 ng/ml) and ifn? (10 iu/ml), but not by il-113 and il-6 (up to 10 ng/ml). lps and ifn 7 had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif (1 gg/ml) were found to secrete bioactive tnf~ (>750 pg/ml by l929 cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il-4, il-10, and tgf8, have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il-1, il-6, il-8, tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il-8 by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at 37°c for 24h in the absence or presence of lipopolysaccharide (lps) or tnfa. il-8 release was measured in the supernatants using a specific elisa. among tested cytokines, il-10 was the most efficient inhibitor of il-8 production by lps-activated pmn. il-4 was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il-8 production. northern analysis are under investigation to precise the level of the il-4-and il-10-induced inhibition of il-8 production by pmn. our data illustrate that il-10 and il-4 possess the capacity to down regulate the production of il-8 by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il-6 superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. 40 female, wistar (175-200 gm) rats received either 250 or 25 pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with 10 mg/kg ibuprofen lp prior to 250 pg/kg bw cntf. rats treated with 250 ,ug/kg bw cntf lost 6.8_+0.4 gms bw as compared to freely-fed controls which gained 17.0_+3.1 gms (p<o.001 by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (-5.8_+0.7). animals pair fed to the high dose cntf gained body weight (10.2-+3.5), although weight gain was less than seen in freely-fed controls. rats treated with 25 pg/kg bw had reduced body weight gain (+7.8+_4.4 gin). food intake was also reduced by 29% in 250 pg/kg cntf (from 23.7+_0.7 gm/day to 16.8_+0.6 gm/day; p<o.05) and was also unaffected by prior ibuprofen treatment (18.4+-0.6 gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight (8.6+0,1 gms vs. 9.4_+0.3 gins), total protein (54.5+-2.9 gms/l vs 44.8+-3.6 gins/l) and albumin (29.0+1.5 gms/l vs. 26.0+_1.6 gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il-1 and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box 100286, gainesville, fl. 32610. of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il-1, have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il-1 are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp130 signal transd,uction protein, including il-6 and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il-6 and cntf also induced hepatic acute-phase proteins. thus il-6 and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il-1-and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of 10 to 1000 pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound 506) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound 406) or the 1phosphono-oxyethyl analogue pe-4, structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd4 ÷-as welt as cd8 ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between 1/500 to 1/1000 cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il-2 and ifnf, but not for il-4 or il-5 (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd4 ÷ as well as cd8 + t cells express cd25, the high affinity il-2 receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il-2 receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t,1 cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)-8 when stimulated with calcium ionophore (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was re/eased into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il-8 in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il-8 concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il-8 in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by 20-30% within 14 days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. 15 versus 2 kin/day). they developed elevated plasma corticosterone levels (30_+5gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il-1 a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il-6 and tnf activity in plasma was not affected. after administration of 1001.tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il-6 activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin 6 (il-6). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il-1) and those of their specific antagonists interleukin-1 receptor antagonist (il-1 ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from 20 patients with acute myocardial infarction at presentation in the coronary unit, every three hours for 24 hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il-1 and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within 24 hr whereas stnfr and 1l-lra peaked at 3-6 hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin-6 (il-6) is a multifunctionai cytokine known to be involved in important homeostatic processes. il-6 levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il-6 binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il-6 receptor (sll-6r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil-6r and il-6 are measured using elisa's. both elisa's are tested for interference of sil-6r mid il-6. the effect of sll-6r in the il-6 bio-assay (b9) is tested. results: baseline levels are (mean + sd): 76.6 -+ 19.3 ng/ml in serum, 3.7 -+ 1.3 ng/ml in urine, 1.64 _+ 0.4 ng/ml in csf and 11.6 + 3.3 ng/ml in sf. there is no interference of sll-6r in the il-6 elisa or bio-assay and no interference of 1l-6 in the sil-6r elisa. conclusions: in all investigated biological fluids sll-6r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin 1 (il 1) and to turnout necrosis factor ~x (tnfc0. methods. bum patients (n=24, 25->75 % total body surface area) were studied weekly up to 42 days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il 1~-and tnfcz-binding t(cd 5) lymphocytes was assessed by flow cytometry analysis. levels of il 1 receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p<0.05) increased up to 3 weeks post-bum (means 3.5+1 in non-surviving and 2.6+ 0.6 u/ml in surviving patients vs < 1u/ml in the control). within 2 weeks of bum, the percentage oft ceils expressing receptors for tnfa and il 1[~ constitutively was elevated (by 10-15 fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range 2.5-40x10 j pg/ml vs <0.2x10 j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range 20-30x10 -3 pg/ml vs 4-8x10j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl 1~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of 1l lra may affect il 1-regulated biological responses including specific immune reactions. while studies suggest that il-10 is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il-10 contributes to depressed t-cell responses seen following hem; and 2) how other agents, known to play a role in hem, effect il-10 release. to study this, c3h/hen mice were bled to and maintained at a map of 35 mmhg for 1 h and then adequately resuscitated. mice were killed 2 h post-hem to obtain splenic t-cells (nylon-wool purified). il-10's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il-10 to markedly improve the t-cell proliferative response [2.5 #g the marked increase in capacity of t-cells from hem mice to produce il-10 was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il-6 are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il-10 release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il-10 down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il-10 has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n=40) were randomized to 4 groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received 20% etoh or ns daily for 14 days by gavage. a 30% full thickness bum was induced 4 hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed 4 days post bum. splenic lymphocytes were cultured for 5 days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma 2.5ug/ml, 10ug/ml). splenocyte production of il-10, interferon-gamma, il-2, pge2 were measured, and lymphocyte proliferative response examined. results: il-10 production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of 11.-10 in a dose-dependent manner, indicating nitric oxide may modulate il-10 and interferon-gamma production in thermal injury. il-10 production is normal in etoh-burn animals. conclusion: il-10 and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il-10 production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin-1 (i1-1), interleukin-6 (i1-6), the interleukin-1 receptor antagonist (i1-1ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn 0, 1, 2, 4, 6, 12, 24 and 48 hours following operation. a portion of the results are shown (mean -+ sem). 135+37 1738-+843 145_+24 one and two factor anova; *p<0.0002, #p<0.0001, §p<0.0009, ¶p<0.0014, for differences between groups i1-1 was not detected at any time point. both ii-ira and i1-6 increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for 24 hours; the b-ira increase was a thousand fold greater than the rise in i1-6 levels. i1-6 levels increased up to 24 hours after surgery. crp levels reflected maximum ii-ira and i1-6 levels (r2=.676, p<0.0001 and r2=.282, p<0.04 respectively). high ii-1ra and i1-6 levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i1-1ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin 8, ireland. change of il-6 and soluble il-6 receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il-6 plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il-6 receptor (sll-6r) is considered to be agonistic to il-6, unlike other soluble type receptors of cytokines. here we measured il-6 and sll-6r levels in the serum and drain fluid from surgical field in order to investigate the changes of il-6 and sll-6r after surgery and their origins. [materials and methods] serum and drain fluid samples from 21 cases (6 of esophagectomy and 15 of gastrectomy ) were serially collected before and after surgery. il-6 and sll-6r levels were measured by elisa. [results] (1) serum il-6 : all cases reached the maximum level on pod-l, more precisely 3-6 hours after operation. (2) il-6 in the drain : maximal il-6 levels in the drain were recognized 3-6 hours after operation, at almost the same time as serum il-6. furthermore the il-6 values in the drain were much higher, about 100 times, than those in serum. (3) sll-6r in the serum : all cases reached minimum levels 3-12 hours after operation and recovered to the preoperative levels a few days later (decrease ratio : 43.9+5.6~,, range : 9-84~'). (4) sll-6r in the drain : sll-6r levels in the drain showed almost the same value and change as serum sll-6r. [conclusions] (1) il-6 is produced from the cells gathering around operative fields whereas sll-6r is considered to be produced in the cells which do not gather around the operative fields. (2) there may be a mechanism that down-regulates sll-6r in the early stage of surgery. [objectives] il-6 plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il-6 concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il-6. [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into 6 groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il-6 was measured by elisa and il-6 mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] (1) serum il-6 levels reached the maximum concentration on the 1st postoperative day in all patients. (2) the il-6 peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r=0.554, p<0.01, r=0.427, p<0.01, respectively). (3) the peak concentration of serum il-6 in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p<0.05), despite a similar degree of surgical trauma. (4) peak 1l-6 concentration observed in a patient who underwent esophagectomy was about 100 fold greater in the drain fluid of thorax than in the peripheral blood. (5) il-6 mrna was demonstrated in leukocytes from thoracic and abdominal exudate at 6, 24 and 48 hours after surgery. in contrast, il-6 mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il-6 is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over 60 years old show a different serum level cytokine pattern than pts under 60 after a standard surgical procedure considered as a "medium strength trauma". patients and methods: 33 pts(22 females 11 males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a:17 pts under 60 years(mean age:49.5+-7)gr.b:16 pts over 60 years(mean age:65.5_+5). all pts underwent cholecystectomy and cholangiography. 6 pts in gr.a and 7 pts in gr. b underwent common duct exploration. 1 spbintercctomy was performed in each group. on the day of surgery (pre) and on the 1st and 7th postoperative day(leo, 7po) : percentages of cd19, cd5, cd4, cd8 and cd16 cells we measured by means of flow cytometry using moab. and levels of il-1, il-4, il-6 and tnf "in vivo" by elisa using moab. results: ere: cd16% was 5.3_+3 in gr.a and 7. 5 objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin 8 (il-8) levels to determine a role of g-csf and il-8 in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il-8 levels were measured before and after surgery in 12 patients with esophageal cancer (ec), and 12 patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p<0.01) and peripheral pmn chemiluminescence (p<0.05) of patients with ec were significantly decreased compared to those of patients with gc at 4 and 8 hours after surgery. serum g-csf levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4 and 8 hours after surgery. serum il-8 levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4, 8 and 24 hours after surgery. significant inverse correlations (p<0.0l) between peripheral pmn count and serum g-csf and il-8 levels were seen at 4 hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il-8, may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin-6 (il-6) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system (1, 2, 3). in our study we monitored il-6 and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il-6 and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) <8 and ct abnormatities or gcs <8 over 6 hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over 24 hours csf and serum. il-6 and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described (4) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il-6 and il-6 antibody. astrocyte migration was tested in a chemstaxis chamber. results 17 head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il-6 were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il-6 could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il-6 antibodies, purified il-6 exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il-6 was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o1 il-6 on astrocyte functions, il-6 promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il-6 stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il-6 represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of 30-35 mmhg for 180 pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. 15 animals received a bolus dose (20 mg/kg) of tnf mab (celltech, berkshire, uk) at 15 min after resuscitation (tn3). the control group (n = 15) was treated similar to the tn3 group but received ringer's lactate (con). results: at 180 min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph (7.16 + 0.04), hco3-(16.3 ___ 2.0 mm), and base excess (-12.5 + 1.9 ram) values with pco2 (47.9 + 7.7 mmhg) and po2 (136.2 + 20.1 mmhg) in the tn3 with no difference to the con group. immediately after resuscitation (230 min) plasma endotoxin levels were found to be increased in both groups (48.8 + 73.3 in tn3 vs 30.1 _ 25.7 pg/ml in con group) . prior to the treatment with tnf mab (230 min) there was also no difference between plasma tnf levels of the two groups (42.1 + 60.7 in tn3 vs 60 + 141.8 pg/ml in con group). treatment with the tnf mab at 65 rain post-hs improved the 48hour survival rate to 73.3 % as compared to 26.7 % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy 2 animals in the tn3 vs 11 in the con group. no bleeding in the kidneys was found in the tn3 but in 4 rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = 11) was prevented in animals which died in the tn3 (n = 4) group ((9.3 +_ 2.4 vs 6.3 +_ 1.0 g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. 55-+8 n=ll*$ 15-+5 n=7 78_+22 n=5* * p<0.05 vs baseline :~p<0.05 no anesthesia vs anesthesia thus 1) tnf production increased 2-3 fold by 48-72 hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; 2) anticd18 had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd18 does not involve tnf suppression; 3) fentanyl anesthesia at 72 hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from 26 to 72 yeas of age. the percentages of bum ranged from 40% -90%. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of30pgjml -150pg/ml. peaks in the tnfa values were above 90pg/ml and were also associated with a peak in the stnfr levels. these levels began at <10,000pghnl within the in,st 6 ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl5 there is a significant corresponding peak in il-trn (>40~8/ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin-1 beta 0l-ii3), intefleukin-6 (il-6) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until 48 hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the 48 hours. patients' ages ranged from 17 to 86 years. iss varied from 9 to 50 and transfusion requirement from 0 to 14 units. five patients died after the study period. ]1,-6 was raised in 9/10 patients (max level 12,500 pg/ml) but was unrelated to condition or outcome. 5/8 showed a rise in il-1b (max level 90 pg/ml) which was negatively correlated to iss (i=-0.789, p<0.02). tnfa was raised in 2/10 (max level 95 pg/ml). peak tnfc~ was positively correlated with iss (1=0.676, p<0.05) and haemorrhage (i=0.602 but p<0.5). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak 11,-6 and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [1] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [2] . radioligand binding studies with ~261odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc2) for 12 hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./(1 + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o0o (mm h¢i): _k, (nm}: n (molecules/cell): 130 -150 1.83 ± 0.15 2600 _+ 500 55 -70 1.27 ± 0.15 3600 + 500 25 -35 0,41 ± 0.13 4800 -+ 500 10-15 0.18 + 0.07 5900 -+ 300 presented are calculated values on the idealized curve + 95 % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [3] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il-6 and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c,10 see, °0v bsa, ld 50) and (80 c, 30 see, 20 ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il-6, 1-3 hr following injury and of try activity 6hr after scald, followed by elevated levels of il-i and il-6 at 12hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted 24 hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and 3 postburn. il-6 increase peaked 3-12 hr after scalding and levels remained elevated 3-5 days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il-6 activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska 17,11002 belgrade~yu. asadullah k (1), woiciechowsky c (2), liebenthai c (1), doecke wd (1), volk hd (1), vogel s (2), v. baehr r (1); depts. of med. immunology (1) and neurosurgery (2) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, 30 patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l0 patients undergoing neurosurgery from the first preoperative day until at least day 6 after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)-6 levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il-6( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il-6 and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit (180-200 ml/kg) hypotension phase (mean arterial pressure = map of 35-40 mmhg for 2-4 hours followed by adequate resuscitation). rats subjected to hs (map of 30-35 mmhg for 90 rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin (1 ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin (100 ng/ml) for 2 hrs at 37 °c. results: at 90 min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group (956 _+ 585 vs 276 + 199 pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats (12 + 4 post-hs vs 164 + 89 ng tnf/ml pre-hs) and baboons (8 ± 16 post-hs vs 233 ± 188 pg tnf/ml pre-hs). in contrast, the il-6 productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs (224 ± 106 pre-vs 1545 ±_ 1254 pg il-6/ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h01mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., 1992, new biol. 4: 147 ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin-1 (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et-1/et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et-1 on the release of histamine, serotonin, and leukotriene c 4 (ltc4) by primary mouse bmmc (in vitro age: 4 weeks) caltured with different recombinant mttrine cytokines (interleukin 3 (il-3) and/or kit ligand (kl) in the presence or absence of il4) for two weeks prior to activation. et-1 (5x10 -8 to lxl0 -6 m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to 20% and 16% of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il-3 and il-4. only when cultured in the additional presence of il-4 rather than in medium containing kl or kl plus il-3 alone, bmmc released histamine and serotoaln upon challenge with et-1.furthermore, et-1 stimulated ltc 4 biosynthesis up to 4-fold in bmmc cultured in the presence of il-4. in contrast, the peptide was without major effect on ltc4 biosynthesis in bmmc culturd without il-4. the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il-4 prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l-4. et-1 (10"6m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of 10 -8 m. crude peritoneal cells (containing 2-3% serosal mast cells) obtained from normal balb/c-mice released 87% histamine upon challenge with et-1 (10 -6 m; 20 rain). taken together, these resu/ts demonstrate that et-1 can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc 4 production in mast cells. il-4 appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a2 (type ii pla2) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla2 and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea2, endotoxin, tnf-c~, il-6 and il-8 were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla2 and tnf-a in these patients were 218.3 ± 179.9 ng/ml and 98.5 ± 92.1 pg/ml, respectively, the two parameters being significantly correlated (r = 0.6460, p = 0.0001 ). there were also a significant correlation between the level of type ii pla2 an il-6 (r = 0.4262, p = 0.0188). there was no significant correlation between the level of type ii pla2 and il-8. these in vivo findings suggest that tnf-a may be involved in the production of type ii pla2. a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)-4 down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. 1991, j. immunol. 147: 4224) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. 1993, cancer res. 53: 3638) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. 1991, embo j 10: 4121) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il-4, tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: 4 weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il-3 the additional presence of rmull-4 for 3 or 14 days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack-2) nor kl-mediated proliferation or chemotaxis. in contrast, il-4 syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes0. pre-incubation of bmmc with rhutgfl~ 1 (5.0, 1.0, or 0.1 ng/ml) for 24h had no effect on kit receptor expression, although tgffil at concentrations of 0.5 -5.0 ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg 1 were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il-3, 1i,-4, il-9, and/or il-10). moreover, pre-incuhation (24h) of bmmc with tgf-gl (>500 pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il-3-or kl-induced chemetaxis. when bmmc were preancuhated with rmukl (200 ng/ml) for 1, 3. or 5 days, a transient down-modulation of kit receptors with a maximum effect on day 1 was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il-4 nor tgfi31 affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi 164 cells from tnf-mediated cytolysis. material and methods: wehi 164 subclone 13 ceils (5 x 105), that are highly sensitive to the cytolytic activity of tnf, were grown on 96-well culture plates in rpm11640 medium. after 24 hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn (20-29bp) were added (0.1, 1 and 10 pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after 18 hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at 590 nm. results: wehi 164 ceils incubated with tnf (1-8ng/ml) were completely lysed after 18 hours (0% abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with 2.5ng/ml tnf and 11jm odn showed still 30% of the absorbance observed in control ceils without tnf (100% abs). the protective effect of odn started at 0.1pm, reached a maximum at 1,um, and diminished at 101jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at 8ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e 12, d-91054 erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il-1), in the sepsis model of lipopolysaccharide (lps, 5mg/kg i.p.) induced hepatitis in galactosamine (gain, 18rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at 3 hr and 8 hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, 50mg/kg i.p.), 30 rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il-1 levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, 2 x 100mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, 2 x 200 unit/mouse i.v) r6spectively. pretreatment with al and sod (24 and 1 hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il-1 into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee4 b'~ th~ recombinant mmlse tnf-o' (0.3 ~/rno~e j.p.) ~d oi~lps 1~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il-1 release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients (62+ 14 years) in severe sepsis (sepsis score > 15 according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma (59+19 years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. 50 pl of whole blood were incubated either with 450/4 of a standard medium or with 400 pl of a standard medium and 50 pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours plasma concentrations of tnf-a, il-la, il-16 and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if-3,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients (6 males, 4 females, mean age 62+_14) with severe sepsis (sepsis score > 15, elebute and stoner} were drawn on day 1,3,6,8, and 14 of disease. blood was immediately placed on ice and processed within 1 hour. 50 pl of whole blood were incubated with 450 pl rpmi-medium supplemented with antibiotics and l-glutamlne or with 400 pl of rpmi-medium and 50 pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours samples were centrifuged and plasma was harvested and stored at -30 ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < 0,05) for circulating mean arterial tnf-a concentration (237 pg/ml _+ 34 sem} and central venous tnf-a (203 pg/ml +_ 29 sem). upon in vitro stimulation there was also a significant difference (p < 0,002) between released arterial tnf-~' {746 pg/ml _+ 88 sem) and venous tnf-a (637 pg/ml +_ 76 semi. conclusions: these results are difficult to interprete but could reflect the influence of pao 2 and sao 2 on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il-1), tumor necrosis factor-a (tnf-c 0 and interleukin-8 (il-8) the multifunctional cytokine intedeukin-6 (il-6) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il-6-concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il-6 may be a potential substrate of these preteases. the serine preteases elastase (ec 3.4.21.37 ) and cathepsin g (ec 3.4.21.20) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin-6 (il-6) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il-6 leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin-6. furthermore the loss of the biological activity of both, recombinant and natural human il-6, was demonstrated by determination of the capacity of protease-treated il-6 to stimulate hybddoma growth (7td1 bioassay). these data suggest a possible downregulation of pathologically elevated il-6 levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il-1, il-6, 11_-8 -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of 39 patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il-1, 11_-6 and il-8 were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to 12 days, for a total of four tests performed (to, tl, t2, t3). i1.-1 was not detected in any samples. the serological profiles of the two cytokines 11.-6 and 11_-8 were similar; augmented levels were found at study entry and throughout the observation period, peaking at t3 and decreasing at t4. however, in patients with sepsis, il-6 and 11_-8 concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il-6 and il-8 release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il-6 and il-8 production appears more significantly elevated, suggesting a role of il-6 and 11_-8 in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc 0 and interleukin 6 (il-6) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after 4 hours and every 24 hour. tnfct and il-6 levels were measured using the bioassays with cell lines wehi-164 ci13 and 7td1, respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il-6 was found in all the patients' plasma samples and five patients' (55.5%) ultrafiltrate samples. the il-6 blood level ranged from 17.4 to 3061.7 u/ml (mean 518.2, sd 686.8). the il-6 level in positive ultrafiltrate samples ranged from 21.0 to 1150.2 u/ml (mean 252.2, sd 401.8). conclusions: our preliminary results suggest that il-6 is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il-1 and il-6 and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd-1 mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first 4h with a peak 2h after clp at a significantly lower level than after lps. il-1 was measurable in serum only after 24h, significantly increased in spleen and liver after 4 and 8h and in mesenteric lymphonodes from 8 to 24h after clp compared with shammice. il-6 was significantly increased in serum throughout the first 16h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if 7 to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= 3 ld50) to the sublethal (= 1/3 ld50) of three different lps (e.coli oiii:b4 and 026:b6; p.aeruginosa r261) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i0 h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps 026:b6 induced as much tnf~ as the sublethal dose of lps 0111:b4; furthermore, lps r261, whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r261 2) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to 8 h post lps administration moreover, the treatment with 6 mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. 3) circulating and organ levels of if 7 are proportional to the dose and degree of toxicity of all the administered lps even if lps r261 was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if 7. to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i00 mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if 7 production. together with the total ablation of if7, the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin-1 (il-1) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd-1 mice were randomized to 15 mg/kg e.coli 0111:b4 lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at 0, 30', lh, 2.5h, 4h, 6h, 12h, and 24h (3 mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit 100 pg/ml) and expressed as ng/g weight + sem (lowest detectable amount 1 ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [0~-32p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present (6.2 + 1.6 ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced 3-fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was 19.2 + 0.6 ng/gwt at lh, reached maximal levels at 2.5h (31.8 -+ 0.5 ng/gw-i) and returned to baseline at 24h. saline controls maintained their constitutive il-i~ levels. sb had 2fold more il-1 ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r2=0.9). separate mice were pretreated with saline i.p. orthe il-1 receptor antagonist (irap, 30 mg/kg bolus i.p.) and were challenged 20 rain later with 1.5 mg/kg lps i.p. or saline i.p. specific blockade of il-1 by irap decreased intestinal secretion at 4h and 6h post-lps challenge (p<_. 0.05, student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content (0.05% of total pituitary protein) and peak serum mif levels (80-340 ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps (15 mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone (85% vs. 35%, p = 0.003). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps (17.5 mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from 50% to 100% (p = 0.0004). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by 38.0 _+ 9.5% in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin-1 (il-1) antibodies and il-1 receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il-1 in the serum does not correlate well with outcome. we hypothesized that this may be because il-1 acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il-1 levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n=5 in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. 1990, j. inununol. 145: 3762) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after 1 and 2 hours of lps injection (100 btg/ 0.5 ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels 30 iron after injection amounting to only 12 to 25% of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in 92% mortality in w/w v versus 8% mortality in +/+ mice 2.5 days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin-10 (il-10) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli 055:b5, 100 gg ip) into balb/c mice induces the rapid release of circulating il-10 (17±9 u/ml at 90 min). blocking endogenous il-10 using monocional antibody (jes5-2a5, 2 mg, 2h before lps) resulted in a massive increase in tnf production (107±47 in lps+anti-il-10 treated mice vs 6±2 ng/ml in lps alone, p<0.05, n=4 to 5 mice per group) and an enhanced lps-induccd lethality (53% vs 7% in anti-il-10+lps or lps alone respectively, p=0.01, n=15 mice per group). irrelevant igg1 rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il-10 during human septicemia. plasma samples were obtained from 65 patients with gramnegative (gns, n=22) or gram-positive septicemia (gps, n=43) and from 20 healthy volunteers. among these patients, 17 suffered from septic shock at the time of sampling. il-10 levels were measured by elisa (detection limit: i 1 pghrd). we found that 35 patients (54%) had increased il-10 plasma levels (range 12 to 2740 pg/nd). patients with gps had il-10 levels similar to the ones observed in gns (median: 12 vs 17.5 pg/m, respectively). patients with septic shock had higher il-10 values (median: 58 pg/ml) than septicemic patients without shock (11 pg/ml, p=0.002). no il-10 was detected in plasma from healthy volunteers. we conclude that il-10 is produced daring human septicemia. our experimental data suggest that il-10 might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, 808 route de lennik, 1070 brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache 1i and sss). patients and melhods: 20 septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i (60kda) were measured by enzyme immuneassays (norms1 values = 13+8 pg/ml and 1.06_+0a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were 93+64 pg/ml and 11.1+5.5 ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p<0.001). mean tnf concentrations for each patient were significantly greater in non survivors (112+52 vs 64_+74 pg/ml p<0.05); stnfr-i levels also were greater in this group, but the difference was not statistically significant (12.6+5.2 vs 8.8_+5.5 ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = 0.65 p<0.005). mean tnf levels were significantly correlated with apache ii (r = 0.49 p<0.05) and sss (r = 0.54 p<o.01); stnfr-i levels were likewise correlated with both scores (r = 0.80 p<0.001 and r = 0.76 p<0.001 respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in 33 septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il-6). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of 100 pg/ml and 1 ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in 11 and 2 out of the 33 patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il-6. multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il-6. peak plasma levels of both lif and il-6 correlated inversely with patients survival duration. cox's analysis for plasma levels of lif >480 pg/ml yelded a hazard ratio of [exp (1.86)=6.44]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value 480 pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human 55 kda tnf receptor (stnfri) and the 75 kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: 20 points). serial blood samples were obtained within 12 h after diagnosis of sirs, every 6 hrs for the first 48 hrs and every 12 hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o01) compared to healthy controls (normal value: tnfrh 1,5 ng/ml; tnfrii: 2,3 ng/ml). the serum concentration of tnfri (14,1 ng/ml) as well as tnfrii (18,2 ng/ml) were significantly higher in nonsurvivors (n=6) compared to survivors (8,6 and 9,5 ng/ml; n=8) at the onset of sirs and during the course of the inflammation response (p<0,05). a positive correlation exists between both receptors (r-0,57; p < 0,01 ). increasing levels and maximal values of stnfri (59 ng/ml) and i1 (82 ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in 10 out of 14 patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and 11 are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. 11-2 is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first 24 hours after administration of il-2. in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il-6 and il-8 using immuno assays. in these plasma samples we also measured phospholipase a 2 activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il-2 therapy the cytokines tnf, il-6, and il-8 are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il-2. objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin-6 (il-6) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il-6 by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of 5 healthy volunteers and cultured at a concentration of 2.5 • 10 ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after 24 hours. using elisa, tnf-c~, il-ib and il-6 concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il-11] and il-6 by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il-6 in ng/ml as mean _+ standard error. ca/ml 0.0 1. (2) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity (3,8x10-9m) as the fab-fragment (2,7x10-9m) but bind the antigen with a eightfold lower avidity compared to the parental mab (4,8x10-10). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl-60 and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l 929, the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a 15-fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at 370c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies (1) reveales that the glucocorticoid antagonist ru 38486, nufepristone, sensitizes both endotoxin responder and nonresponder, c3h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru 38486 sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru 38486 on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and 2 hrs following lps injection, ru 38486 significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru 38486 induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru 38486. however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru 38486 treatment. these studies suggest that ru 38486 aggravates lps lethality via factors other than tnf in endotoxin non-responder, c3h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of 14 different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi3 response in 10-16% of the pbmc after 48-96 hours of cell stimulation. in contrast, il2 and tnfc~ containing cells was only detected in 1-3% of the pbmc. the induction ofil3, il4, il5, and ill0, was weak or completely absent. thus, the response indicates activation of th1 cells. however, illc~, illl3, illra and il8, were consistently found and gradually produced, peaking at 24 hems in approximately 5-8% of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of 30rag 2,4,6-trinitrobenzenesulphunic acid in 0.5ml 50% ethanol (n=12). control animals received an isovohlmetric instillation of normal saline (n=12). faecal pellets were collected before induction of colitis (day 0) and throughout the experiment. after 8 days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day 4 -#p=0 02 alld the total ftnf measured during the study correlatcs with the cms oll day 8 -p=0.04 there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice (20-25g) were primed intraperitoneauy with either lipopolysaccharide (lps) 10ug]anlmal or saline. five days later, the animals were subjected to hypoxia (8%02) for 160, followed by 20 of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at 20, 40, and 60 and assayed for tumor necrosis factor (tnf), prostaglandin e2 (pge2) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia (21%o2 disease 5, berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl 60. we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of 24h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il-1 receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps (10ng/ml) supplemented with different concentrations of mps (0, 500pg/ml, 50mg/ml). after 24hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by 3 cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il-1 ra, and tnfbp-i (tnfsrp55). il-la, il-1 ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to 5.4+1.6%(50mg/ml) by mps in a dose dependent fashion, whereas about 50% decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased 12 times more in the supernatant of lps-stimulated pbmc with 500gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional 13-adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that 13-ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected 3 times with !3-ar agohist isoproterenol (0.5 mg/kg i.m. and 0.5 mg/kg s.c.) 4, 2 and 0 hours prior to a lethal dose of lps injection (25 mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. 1) . these data suggest that 13-ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at 37 °c in rotating polystyrene tubes. samples were taken before addition of 1 x 103 ng/l e. col± endotoxin and after 2 hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin-6 (il-6) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of 4mg/ml or 8 mg/ml or with the monoclonal lipid a igm antibody ha-1a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il-6 values were found.in the plasma. cytokine values at 2 hours: tnf-ec 1689 pg/ml (mean) and 1l-6 1123 pg/ml (mean). preincubalion with 8 mg/ml standard immune globulins reduced il-6 values by 49 % (mean) while there were no effects on tnf-c~ values at 2 hours. a slight reduction in il-6 values was also found with the lower concentration, 4mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il-6 nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha-1a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced 1l-6 release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha-1a had no effect on the il-6 or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto 40% of the estimated blood volume was withdrawn over 80-40 rain, to reduce bp to 40-50mmttg and co by 2/8 mad withheld for 60 rain. shed blood (60%) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of 120%. zymoeen activated plasma (0,36gg/ml) was given 10 rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at 72h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r6, rat igg1, 8mgkg i.e. n=7) or emine (nffi6), 60 rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p<0.05) higher in saline compared to r6-treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto 72h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p<0.05) higher in ealine ve r6 animals at 48 and 72h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p<0.05) attenuated in r6 treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin-1 (il-1 ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il-1 in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, 24 sprague-dawley rats were studied. anesthetized rats were divided in 3 groups, implanted with alzet r minipumps and randomized to receive the following treatment for 14 days:i: 25 gg/kgbw/min of il-lcc; ii: 25 ~tg/kgbw/min of il-1 receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a 30% bsa, full thickness scald burn and the wounds then infected with 10 + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first 8 days and improved food intake between days 5 and 8. we conclude that blockade of il-1 after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il-6. ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd14 antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il-6 producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx (200 mg/day) in septic patients resulted in tnf and il-6 productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( 5ml/kg/day ). the prevention of lpsinduced in vitro tnf and il-6 production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam-1 in sera of septic patients was significantly higher (800-1200 ng/ml) than in normal individuals (50-150 ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy6rgyi medical university, institute of microbiology, szeged, hungary h-6720 ddm t. 10. e~i~im~/tii, septic sh0~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit3' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il-6 concentrations. methodology female wistar rats (wt 160-200g) (n-6 per group) were prefed with 2% tanrine, 3.33% glyciuc ( in tap water) or tap water (tw) for 15 days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received 0.9% saline subcutaneously (3 mls/100g) post-procedure. animals were venosected by direct cardiac puncture at 6 or 18 hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at -70°c was completed within 2 hours. tnf and il-6 were measured using wehi and b9 bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il-6 estimation revealed a statistically significant reduction at 6 hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin 4 (il-4), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c3h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after 5 days systemic blood was harvested to determine the circulating levels of il4 and tnf. a second group was treated with thy for 5 days and then received a gavage wide lxl09 escherichia coli and a 20% burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for 5 days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had 8 mice/group. in separate groups (n=20/group), receiving the same treatments, mortality was observed for 10 days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since 1977 that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this 02 consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av02 difference. it was mclean in a study reported in 1967 that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in 1992 shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon 14 labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by 25 percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by 25 percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately 120 percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are 5,000 neutrophils per cubic millimeter of blood, a 70kg man would have ~1.75xi0 u circulating neutrophils. when activated ixl0 ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate -1.75xi0 nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of -4.2ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m6 is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m6 and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least 96 h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m6 ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il-1, il-6, and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m6 from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m6 populations; it decreases splenic m6 but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon-7 offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin-3, which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il-6 and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il-3 and il-3+m-csf treatments increased the production of tnf in burn 4 day macrophages and progenitor cells compared to normal cells. il-3+m-csf+il-10 increased the production of il-6 by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il-3+m-csf increased the mrna for tnf in the 4-day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il-6/~ actin mrna indicated that il-3, il-3+m-csf, and m-csf increased the il-6 ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~2servojf of appr 200 m ~ separating 10 i~ eucayotic ceils of the hosl ttom l0 ~4 bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if1 i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every 24 hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (.31 u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= 1%' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h2-receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h2-receptors by h2-agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,-2 decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il-2, 3 and 5, and shared mscrophags funclion with loss nr f and c 3b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we8 g~ven in doses of ~37.5 to ~2,500 u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m0zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~4 animals, interferon ~amma was a2so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to 22,s00 uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< 0,05). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c0, interleukin-16 (il-ii3), interleukin-6 (il-6), and interleukin-8 (il-8) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin-4 (il-4), interleukin-10 (il=10), transforming growth factor-6 (tgf-j3), and the newly sequenced interleukin-13 (il-i3). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u3. in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii3 when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il-1b and tnf-a. the il-1 receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p55) and type ii (p75). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il-4, il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il-4 and 1l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, 2) early decreases in total t and helper t cells in the peripheral bloo~ 3} lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and 4) severe and prolongei deficiency of interleukin-2 (il-2) prcductlon by peripheral blood mononuclear cells (pbmo). this il-2 deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il-2 deficiency end shown that the defect lies post-transcrlptionally since mrna for il-2 is also depressed following thermal injury. we have also investigated the potential of a defect in ii-2 receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il-2r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd4+) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: 1) occurrence of activation-induced cell death (aicd), 2) activation-related phenotypic and functional_ diversity in the pool of peripheral cd4+ t cells. initially, (up to 7 days post-bum), peripheral blood lympliocytes from severely injured (> 35% total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l 2 (cd 25) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd4+/cd45ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il-2 secretion and downregulating excessive il-6 and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either 2.4 g/kg of e in a 20% solution by garage (ge), or 1.25 g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a 30% body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el1sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p55 and p75 were both 2-3 fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p55 and p75 during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. decker, d., sch6ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages 0-ii according to me sherry), were 35-55 years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn 24 h before surgery, immediately before incision (after anaesthesia), 2 h and 24 h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in 18 laparoscopic and 15 open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were 1. the interleuldn-2 receptor and im soluble form (cd25/scd25); 2. the activation antigen fd-1 and its soluble form (cd30/scd30), a member of the nerve-growth-factor receptor family; 3. the cd45ro epitope which characterizes t memory ceils; 4. the trausferrin receptor cd71; 5. the soluble adhesion molecule icam-1; and 6. the cytokines interieukin-6 and interleuldn-8. on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to 24 h before surgery. 2 h after incision the tra index in open cholecystectomy showed a distinct 22-37 fold increase, whereas in laparoseopic surgery a mere 2-3 fold increase was noted. 24 h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from 19 patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was 44.7% (range 24%-84%) of total body surface and mean age was 38 years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd14 and most strongly positive for cd45. this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd4 and cd8 positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd20+) was unchanged and nk (cd16+) cells were decreased by ahnos[ 50% compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd4ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd29 were unchanged. cd25 (il2 receptor) and cd69 (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days (13) t, 3, 5, 7 and 10 post injury. we analyzed the cumulative data for interleukin-1 beta (il-i[3), il-2, il-8 as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d5 post-trauma and recovery thereafter. *p < 0.05, ** p < 0.01 vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il-8 and 11,-2, suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r=0.612). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss>30, n=16). i1-6, 11-8, g-csf, fpa, and c3a -levels differed significantly (p<0.005) between the iss-groups (>-< iss 30) at the time of admission, whereas on day 3 tnfa, c3a, 11-6, and ealpi showed significant differences. beyond the first week, major differences were restricted to pge 2 and c3a. the formation of two groups with respect to later multiple organ failure (mof < 3; mof > 2 n= 14) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd14 (monocytes), cd3/cd25 (i1-2r + t-cells), and cd29/cd4 (th 2calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving 20% or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma (5% tbsa). following a 20% tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a 5% tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of 5% tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with 20% and 5% tbsa thermal injuries, respectively. a 20% tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a 5% tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm46899 and the office of navy research n00014-92-j-1612. division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut 84132. post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. 50 cd-1 mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, 150 cd-1 mice were randomised into control (c), l or s groups and peritoneal cells studied at 24 hours for bacterial phagocytosis and killi:~g, superoxide (02-) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd-11b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac-1 was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac-1 expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in 9,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. 1-3 mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a 37,c chamber for recording by real-time video confocal microscopy. the cells were subjected to 4 msec, 150 v/era, 1 a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was 0,2.c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with 1 mm mg%. experiments were repeated with 4 mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. 60 patients (34 males 26 females,mean age: 59.5_+5) 30 with gallstone disease and 30 with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least 6 months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the 1st and 7th postoperative days (pre, lpo, 7po) percentages of cd19, cd5, cd4, cds, cdi6 were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il-1, il-2, il-4, il-6, tnf) were measured in 10 pts. of each group by means of elisa using moab. _r. esults:variations of il-2 and il-4 were not s.s.. il-6 increased but differences between groups were not statistically significant (s.s). il-i decreased on 1po and increased on 7po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p<0.05).tnf decreased in the l.g. and increased in the th.g. on the 1po, the difference was s.s(p<0.05); on 7po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on 7po, except for %cd19 cell that increased on lpo and decreased on 7po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< 0.0i), but differences between them were not s.s. in contrast, igg decreased on 1po (p<0.01) and increased on 7po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to 3 years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, 70% nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia,4 and 24 hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound 4 or 24 hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd3) and helper-t-cells (cd4) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd8) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd19) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd3+hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd3+cd16+leu19-pos) was increased just after anaesthesia being decreased at g and 24 hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples 4 hours after the operation with recovery at 24 hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at 4 hours (p<o.01) and 24 hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in 40 trauma patients on hospital admission and 3x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age 36-+ 16 years (33 m, 9 f), mean injury severity score (iss) 26+ 10, sepsis (19 patients), sepsis syndrome (9 patients), ards (4 patients), renal failure (5 patients). ige increased in 28 patients (70 %). the mean increase was 527 ng/ml (range 0 to 7362 ng/ml; 95% ci was 160 to 895 ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p=0.021) and renal failure (p<0.001). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p>0.05). plasma ige increase was not related to severity of injury by iss score (p =0.42). the mean day to highest ige was 9.1-+6.5. the day sepsis was first observed preceded the day of highest ige by 4.2+6.6 days. there was a significant association between the day of sepsis onset and the day of highest ige (p=0.018). eight of nine patients with sepsis syndrome had > 100% increase in plasma ige from admission. one patient's ige levels were normal (20-100 ng/ml) for 24 days and then increased to 7300 ng/ml over the next 12 days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il-4, which concurrently regulate production of ige and il-1 receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during 24 hrs. adult male wistar rats (250+_23 gin) were submitted to hs (sbp 40 tort) and after t hr (shock i hr) and 24 hrs (shock 24 hrs) hsl (nac17.5%, 3.5 ml/kg) treatment, 1131 e. coli (i 131) was injected into the portal vein 10 ~tci (n_>12). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p<0.05, ~" p<0.001 and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung 1131 uptake was increased and liver and spleen 1131 uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after 1 hr of hs hsl reatment, diminishing by 24 hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after 1 hr hs hsl treatment, returning to normal values by 24 hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of 30 rabbits, labelled with 111indium-tropolene and reinjected intravenously into the rabbits, i0 rabbits received an infusion of escherichia coli endotoxin 2 ~g/kg, while i0 rabbits were subjected to a major sham operation and i0 rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and 2, 4 and 6 hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd2, cd3, cdlla/b, cdis, cd20, cd44, mhcii and cd4/cd8 ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd4/cd8 ratio while the cd3 positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to 129% of initial values following endotoxaemia and to 125% following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd4 cells compared to cd8 positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin-2(il-2) and interleukin-2 reeeptor(il-2r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at 0, i, 2, 4, 6 days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il-2 stimulated by concanavalin a(con a): the sera from the rabbits 2 days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, 30kd,milipore; centricon-10,10kd,amicon), that are less than 10kd, between i0 and 30kd, and more than 30kd. each group of the substances also was tested for the expression of il-2 and il-2r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il-2 by the con a-stimulated splenocyte in mice; 2) the sera from the rabbits 2 days after injury had more profound suppression than other injured sera; 3) there was a marked band at about 10kd in sera from the rabbits 2 days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; 4) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il-2 and il-2r than other groups substances, of which molecular weights are more than 10kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; 2) there is kind of substance, of which molecular weight is about 10kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; 3} the substance can depress the expression of mrna of both il-2 and il-2r. research institute of surgery daping, chongqing, 630042 p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m(3 results in decreased antigen presentation, induces tgf-13 and pge2 while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il-6. consequently, here we investigated rnonokine production in trauma patients (n=10) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t0) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days 0-3 post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the 4-8 days post injury period became higher in alcoholic trauma patients. furthermore, over 9 days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml 0-3 days post-injury 9 days post injury stimulus ale. pt. pt 9.3 11.9 5.6 4.0 immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol (25-150mm) exposure resulted in a significant down-regulation of tnfc~ (p<0.001) and il-6 (p<0.01) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd14+ receptor, the proinflammatory mediator il-6, the macrophage activating factor ifn-5,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days 1, 3, 5, 7 and 10 after mechanical trauma of 9 and after bum trauma of 5 patients (mean age 38~17 years; mean iss 34±2 pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il-6 and ifn-7 in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly (16+5 u/ml) compared to controls (58+6 u/ml) as well as mechanical trauma (59+12 u/ml). il-6 showed a significant suppression following mechanical trauma (662+58 u/ml) vs control (1266+91 u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than 85% of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l-6 and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il-1 agonist) in eutrophic rats subjected to either 20% tbsa cutaneous scald injury (bi), muscle scald injury of equivalent 20% tbsa (mbi), sham muscle bum (resection of skin only, up to 20% tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation (15 mukg i.p.). separate rats were infused with 15 mg/kg e.coli 0111:b4 lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first 24h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit 150 pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount 1.5 ng/gwt). il-lo~ was constitutively present only in the skin (102 + 16.4 ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at 2.5h (in the liver, bi = 16.5 _+ 6.2 ng/gwt, sbi = 1.7 + 0.1 ng/gwt, p _< 0.05; in the lung, bi = 10.3 + 1.3 ng/gwt, sbi = 1.9 + 0.8 ng/gwt, p -< 0.002). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint 0 already (2 min real-time). these levels increased in parallel until 30 min and became eventually different by 1 log at 1 -2.5h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il-10~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il-1 c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma 02114. j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c3h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of 40 mmi-ig for 60 rain. the animals were then resuscitated with either 4 times (x) the shed blood vohune as lactated ringer's solution (lrs) or 2x lrs + lx 6% lies. sham mice were neither hemorrhaged nor resuscitated. at 2 or 24 hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il-6 ( alternatively pmqb showed no differences in il-1 release between all groups at 2 and 24h, while sm~ from the lrs + hen group showed a depression at 24h. tnf production by pm~ was depressed in all groups at 2h and remained so in the lrs + hes group at 24h. sm~b showed decreased tnf release values in both hem groups at 2 and 24h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il-6) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in 39 patients with multiple trauma (median injury severity score = 20,5). blood samples.were collected shortly after injury and after 0,5, 1, 3, s and l0 days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p<0,0s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury (0-3h) at 0,425 eki/ml (median), decreased thereafter to 0,04 eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day 3 revealing the lfighest level to la followed by pa (= 80% of la-sac), ec (= 65% of la-sac) and vc (= 40% of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day 5). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day 5 and 10. however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r>0,5; p<0,01) most often between antibodies of igm-elass (73%) followed by igaclass (40%) and lgg class (2%]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin-6 (ii-6) and c-reactive protein (crp) were measured in 30 patients. i0 female and 20 male, ageing from 30 to 73 with a median age of 59. blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, 2 and 4 hours after the operation and then every morning until the 5th postoperative day. blood was drawn into heparinized tubes (i0 iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. 11-6 levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p 0.01) in endotoxin plasma occurred during surgery, culminating in a peak (median value of 0.795 eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the 5th day after surgery, whereas those of interleukin-6 rose at the end of the operation and were at their highest 4 hours later (median value of 217.5 pg/ml). crp levels were also high postoperatively with a median value of 114 mg/l, and were markedly raised on day 2 (191 mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and 11-6 during the operation was establisthed (p 0.05), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of 11-6, and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or 11-6 plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map 35±5mm hg) for 60 minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at 24 and 72 hours following hs for quantitation of slga by ria. animals were sacrificed at 24 hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in 5 groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled 10 and 30 days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day 10 in the control 29.3 + 3.2, in group 1 58.1 +2.0, in group ii 47.1 + 1.6, in group iii 50.8+2.0, in group iv 48.5_+2.0, in group v 37.4_+4.9x10(6). the evident increase in bmc yield in all groups continued until day 30. increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from 1987 through 1991, and who had serum cholesterol assayed during the first 24 hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< 0.05 level. results: 2909 trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first 24 hrs. 24 patients had cholesterol levels less than 50 mg/dl; 8 of these (33.3%) died, 3 (12.5%) developed ards, 5 (20.8%) developed acute renal failure, and 8 (33.3%) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was 5 times greater and risk of multi-organ failure 3 times greater in this group than in those with a normal serum cholesterol (>if0 mg/dl; 1820 patients; p<0.05). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared 35 patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n=8) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n=9) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi 2) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f2~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p<0.001) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa 2 which was more pronounced in c (p<0.05) and most in b (p<0.01). similar results were found for pge~ and pgf2=. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p<0.05 vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated 45 patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first 24 hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first 24 hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r=0.71, p<o.o001) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt 32.9 + 6.4 tt 28,7 +_ 9,5 n.s. pla-ca(s) mt 11.7 +_ 2.6 tt8.3 _+ 2.6 n.s. goris mt 3.5 4-0.7 tt3.6 _+ 1.3 n.s. iss mt 35 4-3 tt21 4-3 p<0.01 we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim6nez f j, l6pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha-1 antitrypsin (aat), alpha-2 macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of 33 patients, 27 males and 6 females (mean age: 43 years), with compound fractures. 57 % of them were located in tibia and fibula. intercurrent infection developed in 4 patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days 0, 3, 10, 17, 24, 31 and 38. a control group of 30 healthy volunteers was also evaluated. aat and cp showed increased values from day 3 which kept elevated untill the end of the study. amg elevated from day 24. when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il-1, il-6, tnf, pge 2 and tgf-b have all been implicated in these immune system changes. in vitro, pge 2 causes many of the immune system changes following hs, yet the time course of pge 2 release has not been well deliniated. the aim of this study was to determine the time course of pge 2 release by peritoneal and splenic macrophages following hs. c3h/hen mice were bled to a mean bp of 35+5 mm hg for 60 minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at 2, 12, 24, 48, and 72 hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for 24 hours. the supernatants were collected and frozen until assayed. pge 2 was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge 2 release by peritoneal macrophages was significantly elevated at 24, 48 and 72 hours over control. it was maximal at 48 hours, but by 72 hours it had started to decline. splenic macrophage pge2 release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge 2 is significantly elevated at 24 hours following hs, and reaches a peak at 48 hours. pge 2 release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge 2 following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in 12 patients (9 male, 3 female, mean age 31.42), admitted to our icu following serious trauma (mean iss=34) and in 10 control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was 9.25 (range 5-13). 5 patients didn't develop organ failure(nof-group), 4 patients survived, developing mof (s,mof-group), 3 patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured 48h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days 5-6. n and crp levels showed a parallel release peaks on the days 6-7 and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values 24-48h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all 10 control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats (300 g) divided in 4 groups (observation time 120 hrs): a: mild hemorrhage by sequential blood sampling (0.9 ml each)(0, 2, 4, 6, 24, 48, 72, 96, 120 hrs). b: a with hemoperitoneum (hp)(2 ml/100g), c: hemorrhagic shock: blood withdrawal of 3 ml/100g over 30 min and resuscitation with ringer's lactate (2 x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); 59fe labeling of erythrocytas (application of 1 ci 59fe i.a. into a donor rat, 5 days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at 24 and 48 hrs, respectively (212+30 vs. 248+35; 185+_28 vs. 255+_22). hp reduced this increase (209+_17 vs. 177+21; 194+25 vs. 197+-48, p<.05). i-ip significantly increased hemoglobin between 24 to 96 hrs in mild (12.1+_.8 vs. 14.2+.7) and severe hemorrhage (10.3+.6 vs. 13.1+.6). in mild and severe hemorrhage comparable maximal resorption (59fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at 24 hrs (mild: 318+_71 vs. severe: 364+-114 counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: 10/17; hp: 10/13). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at 96 hrs a significant higher transferrin level than with hp alone (3.2+_.5 vs. 2.23 + .2). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during 30 days after cct. antigenic challenge was made on day i, 5, 15 or 30. number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day 5 after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day 5 and reached a maximum when antigen was presented on day 15 after trauma (pfc number 2.4 times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed 30 days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day 15 after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a 21 amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, 6 & 8, prostaglandin e 2 (pge2) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (>20%.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output >30 cc's per hour had et levels drawn on admission, at i, 6 12, 24, and 48 hrs. et levels were measured by radioimmunoassay. mean levels were elevated at 205±28 pg/ml at all time points versus levels in healthy controls of 39±9. in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin 9 these processes since it eradicates the influence of infectious factc~rs. 32 dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il-6, saa and neopterin, the percentage of cd14+ cells, the in-vitro il-2 synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of 27 patients with cholecystolithiasis undergoing either laparascopic (16) or open (i 1) cholecystectomy on consecutive perioperative days -1, 1, 2 and 3. there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients (67 versus 145 minutes, 5 versus 20 days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i 40 + 1 lng/ml, d1 362 + 43ng/ml) versus lap group (d-1 61 + 19ng/ml, d1 457 + 45ng/ml), while in serum il-6 levels we saw a less steep increment in the lsc group (7-fold from d-1 to d 1) compared to the lap group (10-fold from d-1 to d 1). the analysis of cd14+ receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d -1:24.000 + 3.330 cpm, d 1:17.300 + 3.000 cpm) compared to the lsc group (d -h 22.500 + 4.200 cpm, d h 26.000 + 2.900 cpm). in both groups il-2 synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd14 receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors (13-19 and 25 kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il-2 mrna and il-2 production was detected. the nucleotide sequences of the human il-2 gene regulatory region bounding by the splenic nuclear proteins were determined between +39-141 b.p. the il-2 trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il-2 production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il-2 gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania 195 non-pregnant patients from rural areas were followed. of preoperative patients 10% had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied 120 patients of which 16% had a parasitaemia on admission and 6% had postoperative malaria. half of the surgical patients came from rural areas, whilst only 14% of those with malaria lived in the city (with much less exposure and immunity). 57% underwent major surgery and 43% minor. bloodtransfusions (4% with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about 10% of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that 16% was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co2 derived from [i-14c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol 0.12 ~g/ml, glucagon 80 #g/m1, epinephrine 20 ~g/ml, insulin 18 t~u/ml; traumaeortisol 0.40 ~g/ml, glucagon 500 /*g/ml, epinephrine 420~g/ml, insulin 36 ~ij/ml. analysis of twenty subjects showed a reduction of 207° ~mp shunt activity by lymphoeytes and a ]3% reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < 0.05. lymphocyte glucose uptake was also reduced by trauma hormones p~0.05. it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence 0f.neutr0phils. there~bre, neutr0pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery 0f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln-8 (i1..-8) and mo~tocyte chemotacde pop, de 1 (x¢cp-1) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from 12 ipf and 15 sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii.4 arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i (20.1 ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf (41.8 ng!mg) in comparises to 1.1 normal individuals (4.24 ng/mg) and to 15 patients w~th obreic bronentis (cb) (~, 16 rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp-1 e.qu/va]ent) was strongly increased in sareoidosis (86.03 ngjmg) as well as ~n 12f pag,nts (54.47 ng/mg). norra.al indlvidu~s and cb patiants hzd a 7. or 3-fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il-8 ievei~ (15.5 and 26.0 rig/rag, respe~veiy; nomzls: 2.14 rig/rag; cb: 4.23 ng/mg) mad nvatropmi ohemotax~ (60.25 ~'~d 49.68 nnmg, res!z~ztiveiy; aormals: 0.25 ng,'mg; cb: 1l06 ngmg). these data suggest that increased ievels of born mcp.1 ~d il-8 may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ (5 fold vs. lps or paf alone), ll-lg (80 fold), kc (60 fold) and il-6. taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day 0, 12hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays 1-5 and further observation and measurements at days 6-10. from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight 30 kg) neutrophil counts (10e6 pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood (10e6 cpm/250,000 pmn), and of blood-and bal-isolated pmn (10e6 cpm/25,000 pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca 2+ second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in 12 polytranmatized patients (mean injury severity score = 32) arterial and venous blood samples during 15 days. daily evaluation of horowitz-quotiant (po2/fio2), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day 6 and 7 (day 0: 36+.4; day 7: 39.8+.4). plasma lactate was significantly increased at day l (36+2) and day 7 (16.2+2). hurowitz-quotient (day 0: 344+43) was low at day 4 (199+19) and day 7 to 10 (178+25)(p<.05). at day 7 a substantial rise in venous pmnl-superoxide production (day 5: 1.9+_.45, day 7: 3.3+.7, day 9: 1.72+_.8), oecured with significant increase in plasma lipid peroxidation (day 1:0.6+0.2 ; day 7: 1.3+0.2). pivin~-myeloperoxidase activity was high at day 2 (2.3+--.5) and then continuously declined (day 7: 1.3+.3). plasma antiexidant activity (glutathione pemxidase) was reduced by 34% at day 7 (day 1: 5.3+.3; day 4: 4.4+_.2; day 7: 3.5+.2). whereas basal ca 2+ concentration remained unchanged (day 4: 68+_17, day 7: 68+_15), fmlp-stimulated cytosolic ca 2+ mobilization increased at day 7 (day 4: 457+89, day 7: 11084410, day 9: 670+262). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca 2+ mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, 66421 homburg/saar 1donnelly sc, 1haslett c, 1dransfield i, 2robertson ce, 3grant is, 4carter c, 4ross ja, 5tedder tf. 1dept's of respiratory medicine, 2accident & emergency, 3intensive care, 4surgery, university of edinburgh, scotland and 5dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n=19), perforated bowel (n=12), and multiple trauma (n=51)), of whom 14 progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o.0001) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to 24 hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity (632 _+ 231 pmol/i vs 1666 _+ 187; p -0.027), but not the release of neutrophil related mediators (2.20 _+ 0.56 ng/i vs 1.80 _+ 0.17; p=0.63). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb (1.4 + 0.8 % vs 25.0 _+ 14.3; p =0.04). although not significant, nac prevented also the increase in elastase levels in lavage fluid (19.0 + 18.5 pmol/i vs 172.7 + 101.4; p = 0.12). it is concluded that nac is able to protect against the inactivation of a 1-proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion (30 rain) and repeffusion (120 min). sprague dawley rats were randomized into: control (n=i 1); ischaemia repeffusion (ir) (n=12); and ir treated with gtn (2ug/kg/min) during repeffusion (n=10). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n=6 per group). control 573_+t02"* 992_+125 579_+54** bal pmn/mm 3 36l+-126 3"219+1087# 820_~221 *p<0.05, #p<0.02 v control/gtn;**p<0.01 v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from 9.4+0.76 to 13.9_+1.92 mcf after 40 minutes repeffusion (p<.01). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin 9, ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, 500 #g in 0.25 ml saline). three groups (n=10 each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh (100 ~tg in 0.2 ml saline) at 0, 2, and 4 hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f2,29=6.2, p<.003), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<.05). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<.01), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia (75 mg/kg, i.p.) in female balb/c mice (16-20g) that were then randomly assigned (15-17 mice per group) to receive at time 0 and 3 hr later: control saline thjectioas (i.p.), injections of ~-msh (100 ~tg), gentamicin sulfate (i0 mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in 14 patients (3 female, 59+4 years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and 60 min after declamping. arterial pla 2 concentrations were higher than venous (113+32 vs. 71+30). while cytosolic basal pmnl ca 2+ concenlxation was not altered at the end of ischemia (v: 52.3+8; a: 42_+6) and during reperfusion (v: 60+8; a: 69+12), fmlp-stimulated cytosolic ca 2+ mobilization showed a significant arterial increase as compared to venous (1652_+601 vs. 638+85, p < .05). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by 1) pulmonary membrane damage possibly due to pmnl-degranulation, 2) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and 3) a significant arterial increase of pmnl cytosolic ca 2+ mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca 2+ messenger system by inflammatory mediators (e,g. ii-8, tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h202 and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p2 cell) is not only an important target, but as recently shown it is also capable of producing h202. the aim of this study was to investigate the influence of endotoxin on h202 release of p2 cells. we obtained p2 cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h202 release of fresh isolated cells and on cells cultured for 2 days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for 16 hours with 1 p.g/ml lps and tested the conditioned medium (mcm) on cultured p2 cells. p2 cells 5 h ex vivo were _> 70 % pure, _> 95 % vital and had an basal h202 release of 64.5 _+ 1.8 nmol h202 per hour and million cells. adding 1 p.g/ml lps to the incubation medium the h202 release decreases slightly but significantly to 54.2 _+ 2.3 nmol. adding 0.3 p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h202 release increased 3-fold to 148.3_+ 26 nmol. pma induced h202 release decreased to 128.3 + 41.5 nmol after addition of 1 p.g/ml lps. after 2 culture days the p2 cells were _> 98 % pure and showed a pma inducible h202 release of 174.8 _+ 9.9 nmol addition of 1 p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h202 release up to 216.2 _+ 2.4 nmol. addition of mcm to cultured p2 cells increases pma-stimulated h202 release to 223.3 +_ 10.7 nmol. the release decreased to 191.9 _+ 2.4 nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > 96 % in all experiments. the results show that lps has an direct effect on p2 cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h202 release of p2 cells could be important for generating internal oxidative stress in p2 cells before external oxygen radicals exceed. the produced h202 did not necessarily damage p2 ceils, but it can effect surfactant metabolism, especially when extracellular h202 release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled 23 patients with severe lung injury after sepsis (17) or polytrauma (6) and obtained multiple blood samples (14 days) for endotoxin, tumor necrosis factor e (tnfa), interleukin 18 (il-18) and interleukin 6 (il-6) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l8 and il-6 were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, 0.1 ng/ml) for 2 hours at 370 c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao2/fiao2 quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < 200 (0.288 ± 0.038 eu/ml, mean values ± sem) versus the patients with a sli > 200 (0.175 ± 0.023 eu/ml, p<o.01). the basal plasma concentration of ll-6 also correlates with the sli index (p<o.o01). the delta tnfe and delta il-6 values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n:11), the deceased patients (n=12) showed higher endotoxin level, a reduced tnfa and il-6 releasing capacity of the blood and higher basal il-6 levels. we can conclude that endotoxemia, high il-6 plasma level and a decreased capacity of the blood to release tnfa an il-6 under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il-8 in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado2) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($4 or $5) of the right and left lung through a bronchoscope on days l, 3, and 5 days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado2 unexceptionally deteriorated postoperatively and the mean aado2 in 17 patients reached the maximum (mean ± sem; 372 _+ 23 mmhg) on the 3rd postoperative day. the mean concentrations of il-8, ne and neutrophil count in balf also increased postoperatively and reached their maximum level (2137 + 618 pg/ml, 1603 -+ 493 gg/l, and 1561 ± 477 x 103 cells/ml, respectively) on the 3rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il-8 and ne levels in balf (r=0.82, p=0.0001). also, significant correlations between aado2 and the neutropbil count, and the concentration of ne or 1l-8 were observed. in contrast to il-8, neither il-6, il-113 nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il-8 increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes (8 mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats 24 h prior to pma exposure (25 pg/kg, intratracheally) and treated rats were killed 3 h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, 24 h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp-140,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n-24) weighing 5.d-5.5 kg oaselinepao>11 kpa and mean pulmonary arterial pressure (mpap)<i.5 kpa were included in this study. anaesthesia was induced with thiopental 20-25 mg/kg bw and "blind" jntubation was performed. a 5f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma 25 ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose 100 mg iv or metylprednisolone 150 mg iv 15 min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap(2.0-+o.~6 kpe3 ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp-140 or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients (3 sepsis, 4 trauma) with an fie 2 of 0.59+0.(36, a peep of 8.50±0.99, and a murray score of 2.83 ±0.18 were included in this study. 15 healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) 0.74~: 0.46* 0.37 ± 0.16 *= <0.01, **=p<0.001 condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io2), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of 99 serum samples were collected from 39 patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n=12) and non-septic (n=27) patients. patients with ards (n=10, 52.0+4.85ng/ml) and with respiratory disease other than ards (n=12, 49.6+-5.35ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n=17, 27.4_+4.86ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio 2 ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using 6-hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after 6 hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase (30.i-49.6%) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej 24a 15-276 ~ia~ystok, poland, tel/fax +48 85 619742. in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) 5-1ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. 41:757, 1992) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n-3-and n-6 -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf (5 um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of 4-series lts and 5-hete (total sum of 5-1ipoxygenade products rd. 7 and rd. 27 pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of 5-1ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. 67 pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. 308 pmofml). eicosapentaeooic acid (10 um) elicited exclusive generation of 5-series lts and 5-hepe (total sum rd. 82 pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived 5-1ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. 224 pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. 545 pmol/ml). we conclude that the paf-evoked 5-1ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n-3-versus n-6enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr-51, incubated with either pmn or mnc for 18 hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin 32 (cx32) which forms channels between cells capable of pas.~ing any molecule smaller than 1000 d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > 20 adjacent hepatocytes within 60 seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx32 is markedly decreased while the secondary gap junction protein cx26 is either unchanged or even increased. while cx 26 readily effects electrical coupling, molecules > 350 d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of 30 % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $94 although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., 2 and 5 h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci2, peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after 60 minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for 1, 4 and 24 hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after 60 minutes of hepatic ischemia and 24 hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to 20 min of hepatic ischemia followed by reperfusion (rp) and injection of 0.5 mg/kg salmonella enteritidis endotoxin (et) at 30 min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from 870+122 u/l (i/rp only) to 3900+470 u/l at 4h rp. inhibition of kupffer cells (kc) with gadolinium chloride (7 mg/kg) attenuated liver injury in this model by 70%, however, monoclonal antibodies (cl26, wt3) directed against adhesion molecules (132 integrins, cd18) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time (467+52 pmns/50 hpf; baseline: 13+3). isolation of kc and neutrophils from the postischemic liver indicated a 10-fold increase of the spontaneous superoxide formation only in the kc fractions [3.85+0.68 nmol o2-/h/10%elts (kc2); 6.82_+0.92 (kca) ] at 4h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments (4h), the antibody wt3 (4 mg/kg) attenuated liver injury by 90% at 24 h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es-06091 and gm-42957) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was 10 ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type 1 (scri), 25 mg/kg iv after vascular occlusion (n=8) or by depleting the complement system using cobra venom factor (cvf), 0.5 mg im, 24 and 6 hours before ischaemia (n=10). non-ischaemic rats (n=8) and ischaemic non-treated rats (n=15) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during 45 minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c3 and c9 were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p<0.001) and haemoglobin saturation (p<0.001). c3 and c9 were present in the endothelium of the ischaemic control group. no deposits of c3 or c9 were found in the cvf group. few c3 and no c9 were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, 23 control and 38 cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to 50 + 5 mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by 25%.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [500 mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < .05) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < 0.01); fhm (19 < .01); and liver blood flow (p < 0.01) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < 0.05) in cirrhotic rots. tnf levels were higher (p < 0.05) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin 32 (cx32) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx32 to 18% of sham controls. immunostaining of liver sections using anti-cx32 revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles 6 hrs after animals were treated with lps, suggesting the internalization of cx32 without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at 6hr post injection 3.6% ofpixels exceeded threshold, compared to 16.2% in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased 6hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca 2÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca 2÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats (250g, n>_6/group; pentobarbital 50 mg/kg) with hemorrhage for 60 rain at 40 mm hg. reperfusion by ringer's lactate (2 x maximal bleed out/60 min) and 60% of citrated shed blood. tnfcz-moab (tn3, ceutech, 20 mg/kg in 0.9% nac1) infused during flrst 5 min of reperfusion. at baseline, end of ischemia and 60 min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " 45 hepatocyte incubation (30 mg w.w./ml) with caci 2 (0.05 2+ 2+ 2+ mbq/ml) for 60 rain (ca influx [slope, 1/mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, 100 nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca 2+ (ca2+ex) [nmol ca2+/mg protein]; ca 2+ membrane flux [nmol ca2+/mg protein'min]). during incubation, aliquots (100 ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca2+ex and membrane ca 2+ flux were significantly increased at both, the end of ischemia (14.3+.9; 0.41+.05) and reperfusion (16.4+.9; 0.50+.2), as compared to sham-operated animals (7.6+_.5; 0.09+.02)(19<.05 ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca2+ex (10+.8) and membrane ca 2+ flux (0.17+.03)(p<.01). fast ca 2+ influx was significantly increased by epinephrine in hepatecytes from sham-operated rats (0.23+.03 vs. epi: 0.52+.1, p< .05). this hormone effect was not observed in isehemia (0.47+.05, epi: 0.6!-_.2) or reperfusion (untreated: 0.13+.06, epi: 0.07+.01; tnft~-moab: 0.26_+.04, epi: 0.33+.07). conclusion. the present study clearly demonstrated hepato-cellular ca 2+ overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca 2+ fluxes and hormone ca 2+ mobilization suggests disturbances of membrane ca 2+ transport mechanisms, e.g. through ca2+-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca 2+ buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing 20-28 kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. 6.5 fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive 40% of tbs a third degree burn. eighteen hours after burn. 100 gg/kg e. coli lps was intravenously administered over 30 rain. ali animals were studied for additional 24 hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc13) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc13 and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc13 pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc13-indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc13. inositol(l, 4, 5) triphosphate (ip3) has been proposed as a second messenger for calcium mobilization. the addition of ip3 at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip3 binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip3 receptor binding was performed with tridium label ip3. the results shewed that the ip3 binding was significantly depressed by 40-47% (p<0.05) during late sepsis (18 hrs after clp), but not in early sepsis (9 hrs after clp). the ip3 binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p<0.05), but not on smooth subfraction. since ip3 binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip3 binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h202) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h#09 a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h202 was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was 62 consecutive patients with liver cirrhosis admitted to the department of surgery over a 1 year period from january to december 1992 were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c3, c4) were measured and functional assay for complement (ch50) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c3, c4 and the functional chs0 complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch50) showed marked impairment in child's c patients (p<0.000001) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p=0.0177) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o5 per 0.5 ml medium fi2+dmem (1:1) with 10% fetal calf serum for 2 days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by 15-50% (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than 2.5 times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., 1983) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., 1970 (kudryavtseva et al., ,1974 the ability of 02 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in 02 consumption supported by nad+-iinked substrates, but showed almost no change in 02 consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state 3 oxidation of site i substrates and with the increase in state 4 oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca2+ release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca2+ cycling was prevented, either by chelating ca 2+ with egta or by inhibiting ca2+ re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca2÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally 3 mmol/kg and 10 mmollkg, respectively, and lps (2 mg/kg) given 1 h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase 6 h after lps injection. the degree of hydrogen peroxide (h202) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [014]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h202 synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: 1.evaluation nfthe course of the ees due to the biochemic changes 2.determination of the influence of the ees on the activity of lysosomal enzymes in liver cell 3.deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on 30 dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into 5 groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: 1.activity of acid phosphatase in the venous blood serum. 2-6.total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after 4 hours of the experiment). conclusions 1. essential increase of activity of lysosomal enzymes was observed in the ees. 2. increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia (45 min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. 260: g355, 1991) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. 15:277, 1991) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. 4: 3355, 1990 ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats (230-250 g) were subjected to 60 rain of hemorrhage (mean arterial pressure 40 mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from 2.3+0.2 txmol/g to 0.24_+0.08 (60 min shock) indicating severe ischemia, and recovered to 1.04--0.2 at 90 min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by 120% compared to baseline levels (0.69!-_0.31 i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was 0.32+0.05 nmol o2-/min/10ecells (kc2) and 0.51+0.11 (kc3) in controls and did not change significantly after shock and 90 rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after 60 min of hemorrhagic shock and up to 90 rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after 3 weeks animals were sacrificed and livers were "perfusion fixed" with 3% glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after 120 rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as 4-fold of basal levels at the end of 120 min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: (1) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda (2) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. (3) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan 407, r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing 180-220 m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps(236.gg.10)0.7rag/kg intraperitoneally. at the end ,:,f l,gand 5 hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at 3-5 hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier 1 hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a0c in wlstar rats was developed by tigatlng the common bite duct and injecting d.3mt solution of e. goli % b, (g×10' efm/mt) into the bite duct. the mortality rate was 40~ at 48h after aoc. at 6, 12, 24, 48h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at 12h after aoc. at 48h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p<8.01), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a0c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at 48h after a0c (p<0. og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho0ensis of hepatic dysfunction and m0f following h06. " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il-1, il-2, 1l-6, i58, tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il-2 and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: (1) the same immurzoassay (elisa) yielded comparable results in different laboratories; (2) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; (3) assays for soluble receptors yielded less discrepant results; (4) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately 50% for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p55 and tnf-r p75. these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin-2 and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta-2-microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, 1 ) increasing neopterin levels predict rejection episodes in allograft recipients 2) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv-1 infection 3) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease 4) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv-1 infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ 1-proteinase inhibitor (a 1pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a 1pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage 20, 80336 m0nchen, germany procalaitonin (proct) a 116 aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as 3 hours after the andoto:edn release and readied maximal level with a 200 to 80b-fold increase 24 h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii-6, that reached peak levels at 2 h and 3 h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about 80 000 daltons is unable to cross the kidney and the c_n9 barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau 0.1ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat3vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp0site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i00 to 10 000 fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over 500 patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost 20 years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp70) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin 1 and interleukin 6 (ill, il6). class1 acute phase genes are mainly controlled by ill and by combinations of ill plus il6; class2 genes mainly by ii,6. the human c-reactive protein(crp), serum amyloid a(saa) and complement c3 genes will be discussed as examples of class1 genes, and rat c~2 macroglobulin as a prototypical class2 gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class1 genes use the transcription factor nf-il6 or c/ebp as the key factor to mediate cytokine responsiveness; class2 genes use a different, so far unidentified factor to achieve responsiveness to il6. ongoing efforts to purify this factor, called the il6-response factor (il6-rf), from rat liver nuclear protein extracts will be described. the il6-rf apparently mediates the effects of il6 in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il6 as nf-il6. the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly 50 kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over 2 mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 70's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex3~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of 109 critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to 109 uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt 10 build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf0sioi0 to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than 18 mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to 40% and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than 80% when sepsis is present or when at least 3 organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of 16 to 18 ml/100g/min are reflected in isoelectric eeg, and values about t5ml/100g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about 10 ml/100g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding 6 seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds 10 min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm 30, 12200 berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e 2 synthesizing m~ and a simultaneous decrease of functionally competent cd3+ and cd4+ lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin-2 (il-2) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il-2, most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper-1 (thl) secreting principally il-2 and ?-ifn and t-helper-2 (th2) acting principally in inducing antibody formation with a secretion of il-4, il-5, il-10 and tnf-[3 has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u3, tnf-c~, il-6, il-8) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: 70% of all pmn's in this compartment show downregulated or non existent ~-integrins within 72 hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within 7 days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr3+ receptors (cdll/cd18 complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+2) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered 1o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge2 by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in-2 (il-2) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge2. administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il-2 production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il-2 after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen 24 -48 hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc02 gradients. supranorma[ 02 delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. 1. myocardial depression with a drop in 02 delivery (do~) 2. redistribution of blood flow between the different organ systems 3. inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ 02 supply may therefore be inadequately matched with cellular 02 needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april 1989 to august 1993 89 patients, aged from 15 months to 60 years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia 13 patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other 76 patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in 23 out of these 76 patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between 40 -55 s and an activated clotting time between 120 -150 s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four 4 days. in the conventionally treated group 87% survived and in the additionally with ecmo treated group 58% survived, representing a 75% survival rate in patients whose risk of death is considered more than 50%. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po 2 in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro6hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po 2 by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med 1994) . in contrast, mean skeletal muscle po 2 was found to be abnormally high in patients with sepsis (48 _+ 10,4 mmhg, n=39) compared to patients with limited infection (28, 4 + 4, 7 mmhg, p<0.001, n=16) , to patients with cardiogenic shock (22,3 + 6,2 mmhg, p<0.001, n=ls) and to healthy subjects (33,3 _+ 8,1 mmhg, p<0.001, n=10). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po 2 was higher (44,1 _+ 10,3 mmhg) on days with severe sepsis than on days with intermediate state (30,1 _+ 8 mmhg) and than on days without sepsis (26, 8 _+ 5, i mmhg) . thus, a decrease of abnormally high skeletal muscle po 2 was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po 2 with the decrease of apache ii score or elebute score as well as interleukin-6 serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il-8 and c5a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on 110 consecutive pat. after elective open-heart surgery, we had determined ele daily for at least 3 pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n =515 patient-days, both ele and sirs showed significant (p<0.0001) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: 0.59 vs. 0.32; icu treatment days: 0,51 vs. 0.32; days on mechanical ventilation: 0,54 vs. 0.32; days with vasopressor requirement: 0.59 vs. 0.33; number of microbiological findings: 0.44 vs. 0.29), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of 0.87 for ele (ele >_12 on >_2 days, accuracy: 94%) compared to 0.42 for sirs (sirs present on >3 days, accuracy: 67%). accordingly, ele roc curve areas for both overall (0.92) as well as sepsis-related prognostic evaluation (0.99) were significantly (p<0,05) larger compared to sirs (0.79 and 0.86, resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate (46% and 53%, compared to 7% and 1%, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. 15, d-81377 munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score 1, while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation 2. four of the nine patients died. apache iii scores at 72 h were lower in survivors (s) than in non-survivors (ns), (51-+13 vs 111-+1 5 p<0.05), while apache iii scores at admission were not significant different between s and ns (82-+13 vs 95-+13). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an 3 fold increase of tnf-a bioactivity with[r~ 72 hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il-1[3 and il-6 immunoactivity. tnf was measured by wehi bioactivity, il-1[~ and il-6 immunoactivity were determined by elisa. the sensitivity was 35 pg/ml for tnf, 30 pg/ml for il-ll3 and 35 pg/ml for il-6, respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day 3, p<0.05, the pattern of cytokine production by pbmc upon lps stimulation over the first 72h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin-6 (il-6), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, 40 polytraumatized patients were prospectively studied (mean age 42y, 71% male, iss 28). serum and edta-plasma samples were taken in 12h intervalls until the patient left the icu. il-6, tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, 1987) with at least 4 of 7 clinical signs: (1) tachycar-dia> 100/min, (2) temperature >38,9°c, (3) blood pressure < 100mmhg, (4) mechanical ventilation, (5) leukocytosis > 15.000 /ml, (6) thrombocytopenia < 100.000/ml and (7) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day 2 after trauma. results: 21 of 40 patients developed a systemic sepsis (52.5%), and 5 died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il-6 (510 vs. 195 ng/l; p=0.029), crp (160 vs. 121 mg/l; p=0.035) and tnf (32 vs. 15 ng/l; p=0.045) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il-6, tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n=26) was determined by the injury severity score (iss). lidocaine is given at a dose of 1 mg/kgbw over 2 rain. i.v. and is metabolized in the liver by a cytochrome p-450 mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the 1 ~t and the 11 ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin-6 (il-6), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~5, stnfr75) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il-6, tnfot, stnfr55 , stnfr75 ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il-6 was detected in an biological assay using the/l-6 dependent 13-cell line 1313/29. detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (>10 mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with (143±33 mg/l) or without (129a:39) multiple organ failure (mof) nor in survivors (133±41) or non-survivors (147:t:44). in contrast, 1l-6 was elevated in patients wilh mof (mean 740 pg/ml, range 0-8900 pg/ml). il-6 levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il-6 and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r=0.7). patients could be divided into two groups by values for stnfr~5 and stnfr75: the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il-6 correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f0r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in 135 consecutive patients with multiple injuries (iss 40.6) the inflammatory response to trauma was prospectively studied. the patients were divided into 4 groups according to their iss points. additionally, the alterations after 79 secondary operations (>24 hr) were determined (msteosynthesis of the femur (n=28), pelvic girdle (n=ll) and spine (n=8), facial reconstruction (n=13), smaller osteosynthesis (n=13) and others (n=6)). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every 6 h, beginning on admission of the patient to the emergency room for a period of 48 hr, and in the operative patients on the morning of the operation, at the end of the procedure and every 6 hr during the first two days. results: lactate, neutrophil elastase, heart rate, po2/fio2-ratio, and other parameters discriminated significantly between the 4 injury severity groups during the first 48 hr (kruskal-wallis-test, p<.05). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<.05) between the 5 types of operations for lactate, heart rate, po2/fio2-ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss 1 to 24) or moderate (iss 25 to 40) accidental trauma for neutrophil elastuse, heart rate, po2/fio2-ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~65 years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a 1-year period, 538 patients were consecutively admitted into the 8icu; and during a 2-year period, 487 patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o1), sepsis (p<o.o01), and mortality rate (27% vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o01). of the total group of 1,025 patients, there were 406 (40%) patients 65 years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o02). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients (29% vs. 11% in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of 2.48 (95% confidence limits, 1.97-3.12), age did not increased [odds ratio 1.05 (i.01i.i0)], while admission to the sicu reduced risk of death by a factor of 0.15 (0.08-0.30). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan 1117, 1081 ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il-6,1l-8,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n=5) and non-survival group (n=4).tn these patients,ll-6,1l-8,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were 12 points in survival septic group, l l points in non-survival septic group and 10 points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p<0.05. results: il-8 levels in non-survival septic group were higher than in both survival septic and los group.ll-6 levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/3ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha-2-maeroglobulin, c 1estemse inhibitor, antithrombin ill and alpha-2-antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for 7 days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in 256 patients and continued postoperatively in 78 patients, 70 % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication 1-3 days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by 1-3 days. dept. of surgery m'alm6 general hospital, s-2 i4 01 malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of 17 patients admitted with burns covering more than 70 per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: 7 out of 17 burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were 0.512--1.127 eu/ml, significantly higher than that of 10 patients without mof (0.216-0.553 eu/ml) on 3, 14, 21 and 28 days postburn (p< 0.05--0.01). significantly more incidence of positive endotoxin test (>_ 0.120 eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p<0.05). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t4 patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache 1i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp-140, and gp53) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs1 expression) was significantly elevated (p<0.05) and correlated well with severity of disease (f0.597). no significant change in surface expression ofthrombospondin, gmp-140 or gp53 was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (!0<0.05) that correlated well with severity of mof (gmp-140, r=0.611; thrombospondin, r=0.643).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a 10-year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period 1983-1992 i66 patients with abdominal sepsis were treated at the icu at our university hospital. 60 patients were women and 106 men with a mean age of 64 (17-101) years. in 74 cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for 40 (-273) days out of which 7 (-178) in the intensive care unit. 46 out of 166 patients (28 %) died in median after 18 (1-194) days. the primary cause of mortality was multiple organ failure (38/46; 83 %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin (83 %) and in 60 % cultures grew multiple bacteria. 134 patients bad organ failure and 64 multiple organ failure. 29/166 patients (17 %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about 1/5 of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january 1985 to september 1993 56 patients, mean age 24+4 years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for 7.3_+5.7 days. diagnosis of sepsis was confirmed in 6 cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, 2-patients with sepsis, the control group consisted of 20 patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c3,c4), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c3(mg/dl) c4(mg/dl) k 203+-31 971+47 205_+21 134+12 30+-3 1 154-+29" 568-+48* 142_+24" 81-+14' 17_+4" 2 102+_21'* 420-+30** 96-+21"* 67-+10"* 11_+3" in a prospective study we investigated serum of 12 severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n =24) was investigated as. well as serum of patients undergoing elective herniotomy (n=12) 24 hours before (op i) and 24 hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain 08:k87:h19. 200/11 suspension with a final concentration of 250 -400 cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co 74.8 _+ 6.3 opi 77.8 _+ 6.4 opii 68.2 _+ 7.7* tri 38.4 _+ 9.1"* trii 57.6 + 8.9** (*:p< 0.05; **:p<o.01) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi 24h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i0 % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied 62 patients with sls (group i: age 61.1 ~ 10.8 years); they were compared to 52 control patients (group 2: age 58 ± 14.3 years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone (200 mg/24 hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in 48 pts. preoperatively pts in group 1 revealed a significantly lower cardiac index (2.5 ± 1 i/min/m 2) compared to group 2 (3.1 ± 1.2 i/min/m2; p < .02) higher left atrial pressure (group i: 17.8 ~ 8.1 mmhg; group 2: 13.1 ~ 6.1 mmhg; p < .005) and lower ejection fraction (group i: 57.9 z 16.3 %; group 2 65.2 ± 10.9 %; p < .03). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: 15,700 ± 8,200/ui; group 2: 10,200 ~ 4,400/ui; p < .0005) and core temperature 12 hours pop was higher (group i: 38.3 7.6 °c; group 2: 37.6 ~ 8.4 °c; p < .004). serum lactate was already elevated on arrival on the icu pop (group i: 5,0 z 1.9 mmol/l, group 2: 2.2 ~ 0.9 mmol/l) but dropped within 24 hours (p < .0005). icu stay (group i: 3.7 ! 4.3; group 2: 1.6 ± 1.9 days; p < 0.001) and mortality over 3 months (group i: 19.1 %; group 2: 3.1 %; p < .005) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. 33 patients who were admitted to the intensive care units between january and july '92 with polytrauma, sepsis or those requiring ventilatory support for more than 48 hours were selected for the study. the patients were randomized into 3 groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored 4 hourly and the patients underwent an upper gi endoscopy 48 hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the 33 patients selected for the study, 14 had undergone cardiac surgery, 6 had polytrauma and 13 had sepsis. the change in average pre and post drug gastric ph was +1.1 in the omeprazole group, +0.5 in the ranitidine group and -).9 in the placebo group (p < 0.05). endoscopic evidence of stress ulcers was seen in 62% of patients not on prophylaxis, 18.1% of those on omeprazole and 54.5% of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, 82% of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < 0.01). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin-6 (il-6) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn-2 receptors (sil-2r). the aim of this study was to evaluate: the significance oftnf c~, il-6 and sll-2r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il-6 and sll-2r. we examined 19 patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within 24 hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, 11,-6 and sil-2r were measured in 19 septic patients and in 20 adult healthy controls. tnf ct, il-6 and sil-2r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il-6 and sil-2r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll-2r serum levels were significantly (p<0.005, student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ]1,-6 levels. we conclude that only the increased serum sil-2r levels were predictive of bacterial sepsis within 24 hours after the first septic signs. tnf c~ and il-6 levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of 200 cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has 5 headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed 35 children with light and 45 children with severe course(l-3 class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch50, level of c 1-c5, factors b told d, c3a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group (60%-1 st and ioo%-2 nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c4, c3 components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~3) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr6der j, braun j, rennekampff ha, schr6der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in 16 trauma patients with an injury severity score (iss) > 20 (mean iss = 32; mean age 41 years) lymphocyte and neutrophil phenotypes cd 3 (t-cells), cd 4 (t-helper cells), cd 8 (t-suppressor cells), ratio cd 4/cd 8, cd 11b (receptor for cr 3) and cd 16 (fcriii) were measured on day 1,2,3,5,7 and 10 post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd 14) and il 2-receptors on t-helper cells (cd 25/cd 41 were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd 14 were significantly lower an day 2,5,7 and 10 in patients who developed mods (p<0,05) compared to patients without mods and a healthy control (p<o,o01). the expression of il 2receptors on cd 4 was significantly different only on day 2. no differences could be measured in lymphocyte phenotypes cd 3, 4, 8 and cd 4/cd 8-ratio as well as in expression of cd 11b and cd 16 on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > 280/zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the 1025 consecutively admitted patients to a surgical and a medical intensive care unit during 1 -ye0r period, 136 (13%) had arf. arf mortality was 52%. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o,02). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p<0.02), the number of osf before was higher compared to after arf in both groups of patients (p<0.001). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p<0.03). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p<0.002). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml 1rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl12 cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama 1993;270:1z33-1~41) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of 58,737 recent admissions to 107 hospitals in the united states and western europe, we identified 1,195 patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their 2g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors (82% of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original 1,195 patients with a receiver d_perator characteristics (roc) area of 0.76 and in two independent databases, the first independent database contained 295 hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc=0.80); in the second, the 302 placebo patients withtn the recently completed phase ill e~aluatlan of il-1ra (rocffi0.76). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low 1% to a very hlglt 99%. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are 2 ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il (1) -to use a model for risk of death either from a score (apache ii, lii, (2) saps ii) or directly (mpm ii system (3)) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are 2 methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system (4)) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm 1i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii 0) before any therapy. 2 -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. 3 -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: (1) toxicity models with injection of bacterial endotoxin or exotoxins; (2) live or killed bacterial challenge models; (3) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: (1) hemodynamic parameters; (2) modulation of host-derived inflammatory mediators; (3) resolution of end organ injury; or (4) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least 10 study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than 70 years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in 41%-66% of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao 2 and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y0126ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of 24 mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field (1). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the 24 mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a 1pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ 1pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to 140 %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa 1pi, tat), cytokines (il-8, il-6), and soluble intercellular adh6sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il-1, il-6 and tnfe) as well as breakdown products of complement proteins (e.g. c5a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, 1990) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd3 mab wig was able to down-regulate production of il-2, il-10, ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin-1 receptor antagonist (il-lra) but not of il-1; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il-6), some only by solid-phase detection systems (anti-ll-8, anti-ifn% anti-tnfc 0. infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il-6 in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il-6, and il-8. self limitiation of this process might be due to demonstrable instant release of il-1 ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( 1 ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il-1, il-6, tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. (2) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c3hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi 17,hokkaido,japan $120 470 immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h2mon departmen~ of surgery, san frandsco general hospital, urdversr 3, of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of 1c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi4 is down-regu/ated, along wl~ cd 32 and cd64. thus responsiveness to lipopolysacchmdde (128) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc42 is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e2. in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge2. ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s!0otlse. by examining m0n0cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e5 monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il-6 was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than 500 pg/ml (normal: less than 4 pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il-6 level and the number of organs showing failure (r=0.883). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p<0.05). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i-1-1, kumamoto 860, japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock (1). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated 12 patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of 100 mg/ 30 rain, followed by a continuous infusion (10 mggh). with hemodynamic improvement (dopamine < 2 gg/kg/min) the infusion rate nfhc was tapered over a period of 3 days. phospbolipase a 2 (pla2), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after 4 days dopamine requirements decreased to less than 2 gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla 2 activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day 5 and 7) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla2, and finally by the relapse of septic shock in four patients on days 7,8 9, and 1o. a second infusion of hc again resulted in shock reversal and in a decrease in pla2, crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il-16, il-6, and il-8 is attenuated by-100 rag hc prior to endotoxin challenge in humans (2, 3) . cytokines like tnf, il-16, il-6, and il-8 are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla 2 synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: 20 patients with an iss nf more than 28 points who could be treated with at iii administration within 180 rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of 140% by using the following calculation: at iii to administer = (140% -at iii measured) x body weight. at iii was given every six hours for a period nf48 hours and on the 4th day. patients were followed up throughout their stay in the icu but at least 14 days. in this abstract the data of the first 10 patients are included. results: the patients' median injury severity score was 41 points with a median age of 42 years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received 18,100 units of the inhibitor on average during the first 4 days. with our regimen, the at iii levels could be kept between 125% and 140% of normal, whereas control patients an at hi concentration of 40% to 60% was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to 206% of normal. white blood count, pmn--elastase, interleukin 6 and 8 and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii1 group (8.5 vs. 15.8 days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of 140%, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, 45122 essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. 20 patients with severe multiple trauma (7 treatment [at-ill] --13 controls [c]) were investigated. study cdteda were age > 16 and < 65 years, injury severity (iss) > 30 points and glasgow-coma-scale > 8 points; randomization and treatment has to be started within 6 hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp-0127, a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp-0127 in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. 24h later bk was injected ia and the pain score ranked from 0 (no responses) to 5 (vocalization). cp-0127 at 3.6 umoles/kg completely inhibited the pain responses for a period of 2.5 -3h. cp-0127 at 3.6 umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a 3-4h period by an intraplantar injection of 0.1% carrageenan. the abdominal constriction response 1o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp-0127. when 50 ul of 0.5% formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp-0127 significantly inhibited both the first (0-5min) and second (15-30 min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at 55°c for 15 sec the increase in paw volume was significantly reduced by pretreatment with cp-0127, 3.6 umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (-78°c for 10 sec) to the dural surface following a craniectomy. cp-0127 at 3.6 umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls 24 h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp-0127, may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi23, a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats (230-250 g.) received a 70% liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either 0.2 mg/kg/hr rbpi23 (phx-bpi, n=8; sh-bpi, n=7) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n=8; sh-con, n-8). various parameters were measured 4 h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi23. blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il-6 levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd14 or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at 90 -120 rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi21 (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of 30-35 mmhg for 180 rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. rbplg. 1 was administered at a total dose of 5 mg/kg i.v. (2.0 mg/kg at the16-eginning followed by two doses of 1.5 mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi21. results: imrffe?diately after resuscitation (230 min) the detected plasma endotoxin levels in the control group (mean = 61, range = 0-120 pg/ml) were almost neutralized by rbpi21 treatment (mean = 13, range = 0-53 pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points 230 and 320 min of experiment (means: 65 and 51 in bpi vs 49, 65 pg/ml in the control group). the 48-hour survival rate was improved from 6/16 (37.5 %) in the control to 11/16 (69 %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi21 might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. 42 rabbits weighed 900 + 200 grams divided into two groups. group i included 22 tabbits were treated with allopurinol 80 mg/kg for seven days before induction of haemorrhage. group ii as a control included 20 rabbits. all rabbits were subjected to 25% arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for 10 days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was 10 out of 22 while in group ii it was two out of 20. our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h2 receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, 26 pigs were first anesthetized, then injured with 100 joules of energy to the posterior thigh, then hemorrhaged 30-40% of their blood volume. after i hr of shock, all the shed blood plus 2x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine (1.5 mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after 72 hrs, a septic challenge was administered (15 bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for 180 min. baseline values and units were cardiac index 102 _+ 9 ml/min/kg (ci), arterial po2 228 + 11 mmhg(pao2), base excess 6.5 -+ 1 meq (be), physiologic dead space fraction 23 +_ 4% (pds), and tnf 0.4 + 0.1 units/ml. baseline gastric ph was 3.9 -+ 0.4 and 4.8 _+ 0.5 in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao2* be* gastric* pds* peak* 120 rain 60 rain 120 rain ph 120 min tnf ranitidine 167_+17 118_+15 -4.4±1. bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin-6 (ii-6). this study was designed to clarify the role of lps and 11-6 in the metabolic response to bum injury. twenty-five burn patients (49 -+ 17%; 16 + 15% ft bsa burn; 32 _+ 16 years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i1-6 were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b (5,000 u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic (3.1 +_ 5.6 pg/ml; normal <40 pg/ml). i1-6 did demonstrate a significant correlation with cole temperature (tr~ = 37.6 + 0.21ogi1-6, p=0.04) and with nitrogen excretion (nou t = -5.2 4-0.091ogi1-6 + 0.14tr, p=0.01). administration of polymixin b had no effect on metabolic rate, temperature or i1-6 levels but did reduce nitrogen excretion resulting in more positive nitrogen balance (0.t grn/day vs. -0.1 gm/day, p=0.04). although bum injury does not produce an obligatory endotoxemia, i1-6 does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within 5 rain and shock state was hold for 1 h at a map of 40 mm hg (cardiac output of 50 %). following adequate resuscitation with 60% of shed blood and twice of this volume as ringer's solntion, animals recovered to map >1130 mm hg and co >120%. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at 1, 3, or 5 hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [2 mg/kg, celltech, uk) , pentoxffylline (ptx, 50 mg/kg, hoechst, d), and a paf antagonist (web 2086, boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n=6-8 group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence 32-35 of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from 95,7 to about 20 pglml, but did not abolish it. the inhibition of the lps(0.1 ug/ml)-induced tnf production occured already in presence of 0,1 pg peptide/ml and peaked at the concentration of 0,01 ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp-3 and tp-5), thymocartin did not enhanced the pwm-induced pge 2 production in pbmc cultures up to a concentration of 1 ng/ml, inclusive. the selection of thymocartin (tp-4) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about 90% registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than 260 patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: 20 patients (pts) formed control group a. 20 group b pts received 3x50 mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d5) intravenously to block prostaglandin e2induced suppression of cmi response as well as 50 mg thymopentin (tp-5) subcutaneously preop., on d2 and d4 to augment cmi reactivity. in vitro investigations preoperatively, on dl and d7 included measurements of the percentage of cd4+ t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)-2 as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il-6 as one cytokine primarily produced by th2-cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d7 were used as in vivo tests for thl-and th2-induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd4+ th cells, il-2/il-6 ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p<0.05 vs.bl). in group b pts no change in cd4+ th cells, il-2/il-6 ratio, or dth skin response was observed (p<0.05 vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: 1.these results clearly indicate a significant suppression of th1 induced cmi response, while th2 induced response remains normal following ohs. 2.these alterations may gain clinical significance whenever a postoperative infection requires a th1 response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. 3 the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing 250-300 g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il-2. they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in 11o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from 1 to 4. results.it was found that in 100% of infants with 3-4 classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then 2 times, ratio th/ts was reduced to 1.5 or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c3a subcomponent were increased but the level of c2, c3,, c4 and c5 components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with 2 point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i-3 mcg/kg per day and 2 mg/kg per day accordingly during a week in children with 3 -4 classes el heaviness. aiter 11-14 days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of (1-3)-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c3h/hen or c3h/hej mice, 6 to 12 wkolds of both sexes were used through experiments. highly purified (1-3 )-{5-d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b-139. induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c3h/hen and c3h/hej mice, whereas lps only induced such activities in c3h/hen mice. most effective dose of bgc was 10 ug/ml. 2) bgc-dependent proliferative response of c3h/hen splenic cells was not detected under the culture condition used. 3) induction of tnf~ prodction was apparently observed in bgc-stimulated c3h/hej pd4 cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr94 and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~4 activation, intracellular signaling pathways of p~4 by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il-2 in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il-2. under anesthesia, 8-weekold male c3h-hej mice were subjected to a 20% scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of 105 organisms of p. aeruginosa 24 hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells (2x105) intraperitonelly after septic challenge. the mice were scored for survival daily. on day 7 after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il-1 and il-6 in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, 2 positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l 6 were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il 6 (n=6) were raised to 45.9_+17.4 and 3866_+1045 pg/ml, while the levels in lak treated mice were reduced to 4.23_+2.37 and 437_+110 pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l6 has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included 20 operated patients, 10 of which received 100mg indomethacin before and 1,2 and 3 days after surgery (group a) and 10 patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd4), suppressor (cd8), nk-cells and interleukin-1 and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day 0 and 1, 3 and 7 days after operation. in group a we detected a significant (p<0.05) increase in cd4/cd8 ratio on day 3 while no differences were ~oted in nk-cells or cytokine production in both groups (table 1) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of 15 mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. 100 female cd-1 mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p<0.05 vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p<0.05). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p<0.05). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin 9, ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h2-antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. 21-aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these 21aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi0)(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p<0.004) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi0 and plasma cholesterol (p<0.0005) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, 21-aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, 5x10 u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. 4-hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the 21-aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in 41 neurosurgical centers and included 1023 patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and 8 groups of tirilazad in escalating dosage (0.6 -6 mg/kg/day). the clinical outcome was compared for these 4 groups and cerebral a~giography was performed in 75 % of patients on day 7 -11 follovang sah, to study the effect 0f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint 6 months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the 6 rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z0 patients sufferin s from severe ap (c-reactlve protein >120 me/l) we determined on day 1 and day 8 after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i0 patients were randomly treated with 600 ug/day of se for 8 days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only 1/3 of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains 85% hes and 15% chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation 0fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats (190-215 g, n>6; pentobarbital anesthesia 50 mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate (1) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and (2) whether dfo-hes could prevent this tissue damage. methods: in 67 rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to 2 groups: one group was treated with 3 ml dfo-hes (100 mg/kg iv), the other rats received solely 3 ml of the carrier starch solution. 30, 60, 120, and 240 min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> 1.84 eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p<0.05) in the lies group already 30 min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p<0.02). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after 60 min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p<0.02) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca2+ levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose (150mg%) and bovine albumine (5gm%) for 60min at 37°c.it composed of 3 groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo&200 mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to 15 min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ 200mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were 267% in group ab and 767% in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a2, leukotriene b4 and leukotriene c4 are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after 0,5,15,30 and 60 minutes plasma levels of thromboxane b2 (txb2), leukotriene b4 (ltb4) and leukotriene c4 (ltc4) were determined via elisa technique. another volunteer had taken 1000mg aspirin one day before taking the blood sample (no7). results: txb2 plasma levels increased from 14pg/ml at 0min to 144pg/ml, 350pg/ml, 370pg/ml and 660pg/ml at 5,15,30 and 60min (p<0,01) . ltb4 and ltc4 plasma levels showed an increase during the first few minutes (ltb4: 0min: llpg/ml,5min: 87pg/ml; ltc4: 0min: 23pg/ml, 5min: 97pg/ml (p<0,05)) followed by a decrease to normal values at 15min. in the sample no7 the cyclooxigenase-pathway was completely inhibited, the txb2 plasma-levels did not alter at all, whereas ltb4 and ltc4-plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il-2 secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il-2 productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e2 secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c3lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early 20th century, however the protein was not isolaled until 1977. the human gene was identified and cloned in 1983, which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a 3-5 day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet 1993; 342:1328-33) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p<0.05) in patients receiving allogeneic blood (n=36) or operated without blood transfusions (n=17). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n=24). in contrast, specific igg-levels increased sigmficantly (p<0.05) in patients receiving allogeneie blood (from 1.6 + 4.4 to 9.7 _+ 18.4 ie/ml) whereas in patients receiving autologous blood a smaller increase (from 1.9 + 4.1 to 4.0 + 6.7; p=0.068) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l 1.4 + 12.0 to 28.5 + 11.4 u/ml) compared to those patients with autologous blood (8.7 + 12.5 to 17.1 + 23.0). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin-1 (il-i), interleukin-6 (il-6) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during 4-6 hours after surgery in 10 patients undergoing arthroplasty (9 hips and 1 knee). 7kf, il-1, it-6, thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il-6 concentrations ~ere analysed at the beginnlqg and end of the 4-6 hour blood retrieval period. in a separate study (5 hip arthroplasties) f~f, il-i and il-6 ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il-6 coocemtratiems rose from a median value of 0 to 116 pg/ml, p<o.01, during the blood ret.rieval period. in c was detectable in 4 patients, range:15-50 pg/ml. il-i was not detectable in the patients. in retrieved blood tag was >200 mg/ml in all samples (ref:<4.1 mg/ml) and at was 0. 16-0.51 units/ml (ref:o.80-1.20) . the il-6 concentrations in retrieved blood was >1000 pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n=5), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: 11-277 pg/ml) and il-i (range:5-125 pg/ml) ~re present in all samples but ii-6 (range:o-25 pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: 159 patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients (52.8%) were perioperatively transfused. thirty-six (42.8%) patients were given autologous blood , while 48 (57.2%) received homologous blood. no patients received both autologous and homologous blood. twenty eight (17.9%) patients developed postoperative infections. non transfused patients had a 9.3 % infection rate , those receiving autologous blood had a 13.9 % infection rate, whi]e in the homologous blood group the infection rate was 33.3% (p < 0.001). univariate analysis showed that infections were significantly related to operative blood loss (p<0.01), length of operation (p<0.05) blood transfusion (p<0.05) and attending surgeon (p<0.05) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, 2 month old rats were transfused intravenously with syngeneic heparinized venous blood (3x2ml, every other day), and 3, 7 and 14 days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a 10:9:0.9:0.1 mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of 0.3 microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai1y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over 24-48 hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of 1-2-weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il-6. we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium-99m (99mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a 25% topload of leh (200 mg of phospholipid, 2.5 g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at 20 hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, 8% at 30 minutes and 15% by 2 hours. the spleen accumulated activity at a slower rate, 3% at 30 minutes and 7% at 2 hours. at 20 hours, autopsy biodistribution studies revealed that approximately 42.6% of the dose is in the blood pool, 15.4% in liver, 18.1% in spleen, 3.2% in lungs, 2.4% in muscle and 1.6% in urine, with trace levels in kidney, brain and heart (<1 °/o). in a hypovolemic model, rats were 10% or 50% exchange transfused with 99mtc-leh. in the 10% exchange model, 99mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at 20 hours. with the 50% exchange, 50% of the leh was in circulation at 20 hours. the interaction of leh with platelets labeled with indium-111 was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next 30 minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha (2) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens (3) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine (4) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength (5) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t 5-7 years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. 526 o]~ga-3 pufa there continues to much interest in the application of the 0mega-3 pufa in clinical nutrition. the basic principle has been that the 0mega-3 pufa will displace arachidunic acid and result in a decrease in eic0san0id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega-3 incorporation with continuou~ enteral feeding both in control and endotoxic animals within 3 days. this includes the liver, spleen, circulating and alveolar marc0phages and the lung. this incorporation resuls in significant changes in the eicosan0id production including pgf 2 and ket0-pgflalpha. there is improvement in the cardio-vascular reep0nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit0gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of 0mega-3 pufa. clinical studies using short term entsral nutrition with 0mega-3 either alone or with other enteral supplements in a number of clinical settings have shown significant 0mesa-3 incorporation and decreased eicosan0id production. these positive results must be discussed with the additional evidence that long term omega-3 supplementation decrease eic0san0id production but als0 induce a state of immune suppression that is capable of increasing transplant sunvival. these 10ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr0hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega 3 fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) 14 gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf-1 mice aged 8-12 wks received 32% tbsa fullthickness dorsal burns & were resuscitated with 2 cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast 24 hrs, chow 24 hrs). at 48 hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p<0.01, ** p<0.05 compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of 326 adult icu patients who required enteral feeding for > 7 days. patients entered the study within 48 hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least 60 ml/hr by 96 hr after event. results. both groups tolerated administration of formula well. for patients fed > 7 days, the median los was 25% shorter (p=o.ol) for the--supplemented group (21 days) compared to the conventional group (28 days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o05). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia (19 vs. 27 days) and skin/soft tissue infection (19 vs. 37 days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > 7 days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into 3 groups ( n= 9 each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega 3 fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within 12 hours following surgery (10 ml/hour). it was progressively increased reaching a full regimen on day 4. on hospital admission and on post-operative day 1 and 8, the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day 1, 4, 8. the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day 1. comparing post-operative day 1 versus day 8 a significant increase of prealbumin (p<0.02) and rbp (p<0.005) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p<0.001) and a significant recovery in delayed hypersensitivity response (p< 0.005) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent (18% in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = 41% vs vivonex ten = 35%), mean mof score (immun-aid = l.b vs vivonex ten = 3.6), or mortality (immun-aid 0% vs vivonex ten = 6%) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> 25), patients receiving immun-aid appeared to have fewer septic complications (33% vs 50%) and their mean mof was significantly lower (1.8 _+ 0.7 vs 5.8 + 1.7, p = 0.05, student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from 10 patients in septic shock and 17 heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in 3 icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for 10 minutes. pmn (p{o,05) and monocytes (p<o,01) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the 49%-bcaa vs 16%-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o01 for all). clearances of bcaa increased with increasing bcaa dose (p<o.o01); arg and gln clearances were directly related to the bcaa clearances (rz=o.65, p<o.o0i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation (7gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to 14 days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with 3x103 t.spiralis larvae. thirty to fifty days later rats received 7gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by 3 dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o5 mucosal mast cells and their subsequent degranulation. mast cell-derived 5-ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with 3xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with 10ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for 24 hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l929 bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group (60% vs 0%). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m-6 polyunsaturated fatty acids (pufa) from safflower oil (so) or (o-3 pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at 30% in the diets was adjusted to provide 10% each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for 5 days, lipopolysaccharide (lps, 9mg/100g) was injected through the tail vein. the percent of animals surviving in the mo group was 67% (14/21), and did not differ significantly compared to 57% (12/21) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so (1.2+3.0 ng/ml) and those fed mo (1.8+1.1) did not differ significantly. the levels of prostaglandin(pg)e2, 6-keto-pgflc~ and tumor necrosis factor-ct (tnf-c 0 were determined 90 min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and 3h after the lps administration (lps+). the data (mean_+sd; n=6) are as follows. group these data indicate that a marked reduction in the plasma levels of poe2 and 6-keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a 2.8% reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after 5 days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf-1 outhred female mice (12-15 g) were fed purina #5002 chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the 21 d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from 0 to 8 days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a-5 desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at 15wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for 3 weeks. the incorporation of dihomo-y-linolealc acid (dgla: 20:3 o)-6) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between 1-3% compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps (10p.g/kg body wt), the plasma levels of both pge2, and txb2, were significantly decreased (p<0.02) by nearly 50% for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a-5 desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a 2 (pla2) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was 65% (13/20) which was markedly higher (p<0.01) compared to only 20% (4/20) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly 50% reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla 2 and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c57bl/6 mice that have been stressed by immobilization. the proteins have been labelled with 3ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of 3ss-methionine reveal about 440 protein spots, from which a small fraction -about 20are altered in both qualitative and quantitative manner. the major changes are in the range of 25 to 60.000 d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery 6 days and 1 days preoperatively and on the ist and 6th postoperative days (study-days i, 2, 3, and 4) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived 6 days preoperatively an enteral nutrition enriched with omega-3 fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a23187 (7.3 pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb5 (mean values 6.6 ng/l x i~ cells) compared to the placebo group (2.6 ng) at study-day 2 [p, 0.01). in contrast, the capacity of neutrophils to produce ltb4 was not significantly different in both patients populations at study-days 2 and 3. the omega-oxidation of ltb4 to its biologically less active metabolites oh-ltb4 and cooh-ltb4 was not significantly different in both groups. however, neutrophils from group f patients generate more ltc4 at study-day 2 (p(0/5). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a23187 exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega-3 fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega-3 fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega-3 fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with 18 male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for 10 days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution (250 kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn (0,5 g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide (0,15 g n derived from ala-gin =30% gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o,01). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p<0,01). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p<0,01). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o1~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai1y erodes both fimetianal and stm~ tissue. wiu3_b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej2ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed 5ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. 235als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~2po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh 24 iu daily three days before the experiment (gh-3), another group treated with gh 24 ie at the start of the surgical procedure (gh-1) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u-14c-glutamine. substrate fluxes were measured one (lh) and five (5b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh-3:30.8+4.2 mg/kg, gh-i: 37.6+_.5.8 mg/kg, con: 55.4+-3.1 mg/kg, p=0.004). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh-3:0.9 + 0.8gmol/min at lh and -2.4+1.4 in.tool/rain at 5h, gh-i: -3.4+1.5 i.tmol/min at lh and -1.4+1.2 pmol/min at 5h, con: -8.7+_2.8g mol/min at lh and -11.6+3.4/amol/min at 5h, p=0.005, p=0.006), whereas the absolute uptake of glutamine in hindleg was unchanged (p=0.84). glucose uptake in the hindleg was decreased (p=0.0006). net glutamine uptake in liver was attenuated (p=0.03 at 5h), whereas net glutamate release from liver was increased (p=0.005). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. 48 metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of 0,075; 0,15 or 0,3 i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from 20.9 calories per kg body weight to 26.6 calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus 12.7 to plus 8.5 g n / 7 days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in 3 patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in 30 patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either 81u of rhgh bid (sc) or no alternative medication. the study period lasted 14 days. from daily 24 hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days 7 and 14 to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf1 and igf2, igfbp3 were measured daily during the study period, on day 3 a 24 hour profile of gh was.performed. samples were collected each 20 minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first 4 study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf1 differed significantly between the groups for the whole study period, but not igf2 and igfbp-3. the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first 4 days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, 29 candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n=14, age range 25-59 yr, bmi 23.9+5.7 kg/m 2) or for 14 days additional gh (2x8 i.u.s.c.) (gh group, n=14, 21-59 yr, 21.8+2.2 kg/m2). metabolic studies (calorimetry and lsc-leucine infusion) were performed 5 (test a) and 11 days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the 2-week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was 117+10; 111+15 (co) and 110+7; 100±12 (gh) pmol/kg ffm/h (n.s.). protein breakdown was 134±16; 131+30 (co) and 156+14; 178+30 (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was 119±9; 127+16 (co) and 139±11; 177+20 (gh) pmol/kg ffm/h (p<0.05 for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days 1-4: gh 52+3 vs co 66±7 g; p<0.05). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf-1 levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf-1. this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: 38 patients (both sexes, age: 40-75 years, bmi 17-30 kg/m 2) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received 80 ug rhlgf-1/kg body weight/twice daily or placebo during 5 treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition (3g chng, o.6g aa/kg) troughout the whole study period. cumulated nitrogen balance, 3-methyl-histidin exretion in urine as well as serum-igf-1 levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to 3 groups (n=10/gronp): gh-high (4+8mg/kg/day), gh-iow (0a8mg/kg/day) or control (saline). following subcutaneous treatment (every 8 hours for 6 days) with gh or saline, mice were challenged i.p. with e.coli (lxl08/body). survival rate was recorded every 2 hours. in the next experiment, gh-high and control animals were sacrificed 4 hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for 24 hours with lipopolymccharide (10p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf-1) and the insulin like growth factor binding protein (igfbp-3) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf-1 and igfbp-3. materials and methods: 21 patients sheduled for esophagus resection were investigated once they had given written informed consent. 8 patients showed acute alcohol abuse and were treated with clonidine postoperatively• 13 patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf-1 and igfbp-3 were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf-1 and igfbp-3 levels showed no differences between the groups. discussion: igf-1 and igfbp-3 plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n=10, 24mg/kg/day), low dose igf-i (n=10, 2.4mg/kg/day) or saline (11=10) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx108 e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for 24 hours with lipopolysaccharide (10).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l929 bioassay. results igf-i improved the survival rate at a dose of 24mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than 500/~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated 36 patients (32 scn, 4 cyclic neutropenia) with r-methug-csf (filgrastim). thirty of 32 patients with scn and all 4 cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than 1000/~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between 0.8 and 120 ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to 6 years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in 12 patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. these levels were 1000-fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately 2-4-fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day 15 of gestation (parturition in rodents occurs on approximately day 21) survived a lethal challenge of group b-13 hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res 31:574, 1992) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for 7 days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood 76:1788 , 1990 cairo, pediatr res 27:612, 1990) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. 42 infants <72 hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm (26-30 wk), preterm (31-35 wk), and term (>36 wk) and randomized to receive either a placebo or 1, 5, and 10 gg/kg/qd or 5 and 10 p.g/kg/bid of rhg-csf infused by pump over 1 hour for 3 consecutive days. cbcs were obtained at 2, 6, 24, 48, 72 and 96 hrs. tibial bone marrow aspirations were performed 72 hrs after study entry and differential counts and cfu-gm pools were determined. c3bi expression was determined at 0 and 24 hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at 48, 72 and 96 hrs following administration of both 5 and 10 ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred 72 hrs after 5 and 10 ~tg/kg/d (397 -116%) (p<0.01) and (621% -+ 200%) (p<0.001), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool (18 _+ 4% vs. 62 + 6%) (p<0.01) (placebo vs. 10 ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo (53.5 _+ 8.6 vs. 87 -+ 15) (colonies/l(p cells/plate). c3bi expression was significantly increased 24 hrs after 10 bg/kg/d of rhg-csf (233 + 81% vs. 83 +-26%) (p<0.012). peak serum g-csf levels occurred at 2 hrs and were dosedependent. the half-life of rhg-cse was 4.44 + 0.4 hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. 31 of 42 patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research.41:240, 1993) . methods: awake 2 ylo beagles had serial cardiopulmonary and laboratory studies before and for up to 10 days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until 3 days after clot placement, animals received high (n=17) or low dose (n=17) g-csf or protein control (n=20) subcutaneously. results: survival in high dose g-csf animals (14/17) was significantly improved compared to low dose (10117) and controls (12120) (p<0.04 wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day 1 after clot, p<0.001) and mean arterial pressure (day 2, p<0,02) compared to low dose and controls. high dose rcg-csf increased (p<0.001) peripheral neutrophil numbers both before and after clot implantation (2 hours to 4 days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p<0.0001) rise (day 2) and fall (day 4) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin (2 to 8h, p<0.002) and tumor necrosis factor (tnf, 2h, p<0.02) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given 480 uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and 20 or 44 hours later. lps-inducible tnf, il-1, stnf-r p75 and il-lra were assessed in the supernatant of whole blood incubations stimulated with 10 ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about 50% 20 hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p75 which was indetectable in blood incubations from untreated donors increased dramatically 44 hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as 10 /.tg/kg iv every 12 h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin 21 and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either 1 or 7 days before or 4 hours after cp. results. mortality was reduced from 90% to about 50% with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin 21 and lactate were attenuated the first 24 hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin 21 and lactate are suppressed the first 24 hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels 2h after lps administration. gm-csf pretreatment (50 ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin-6 (il-6) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il-6-tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include 60 icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in 24 hs. continuous infusion for 15 days. safetyandefyieacy will be assessed till to day 30. the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > 16 (score 16 corresponds to expected mortality rate of 38%).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than 30, were included in the study. filgrastim was given by daily continuous intravenous infusion for 7 days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > 4 apache 1i score points on day 4 after onset of treatment. results: none of the 10 patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to 10.110 + 2.000/~tl (mean + sem) and reached a level of 29.280 +_ 5.810/tal at day 6 after onset offilgrastim therapy. the apache ii score initally was 14 + 1.57 (mean + sem) and as an indicator of filgrastim response a decrease of 5 points ~dthin 4 days oceured in 7 out ot 10 patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= 41) or colorectal (n= 47) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il-2, which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to 48 hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of 900 pg/ml (preoperative < 70 pg/ml) on day 1 and day 3 to 5 after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < 0,01) correlated with infectious complications in patients whh gastric cancer (n= 17/22). secondly patients could be arranged into two groups according to an anergic (n= 25) or normergi¢ (n = 63) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= 11/41) or colorectal (n= 14/47) cancers. interestingly we found a significant correlation (p < 0,01) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= 30) within the pre-and postoperative period resulted in reduced mean values (about 50%) for ifn-ff release (preoperative means llo0 pg/nfl), if compared to patients with normergic dth (n= 63, preoperative means 1960 pg/ml). similar, but less significant results were obtained for il-2 secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il-2) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h 2 02 production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s0orl0o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe2-gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)(50m$/k9 bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h 20 2 producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after 2 weeks was significantly decreased by administratlon of rh~-csf(p<0,01).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached 57.1%. nextly,treatn%ent wlth rhg-csf(s0 ~ $/k9 body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p<0.01). iv~oreover,functions of neutrophlis which were phagocytic activity and h2 02 p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~(50 ~ 9/ks b ody wt) (p<0.(15). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in 17 renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < 500/mm 3) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < 1000/ram 3) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of 2 to 5 mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within 1 to 4 days. in most of them, spectacular correction was observed within 24 hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed (2 cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below 5 mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, 161, rue de s~vres, hopital necker, 75743 paris, france. changes in serum g-csf and il-6 after surgical intervention hitoshi toda 1, atsuo murata 1, hidewaki nakagawa 1 , takesada mori 1, nariaki matsuura 2 1osaka university medical school, osaka, 2wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin 6 (il-6) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il-6 and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l-6 was less than 2 pg/ml and g-csf less than 4 pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il-6 levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il-6 (x) was y=3.28x+6.16 (r=0.786, n=86, p<0.01 ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il-6 was 50 pg/ml and g-csf 69 pg/ml respectively. the estimated value of g-csf was 171 pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a 20kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). 10 postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than 30 were treated with filgrastim (0.5 -1 l.tg/kg/day) for prophylaxis of sepsis on 7 days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to 14 days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced (10 -7 reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of 797% +-570% (217% -1826%) compared to pretreatment values of 100%. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within 48 hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in 38 neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p<0.05). before initiation of csf treatment urinary neopterin was similar in both groups of patients (283+/-38 and 267+/-40 lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day 4 the mean value was about 40% above the basal level (p<0.05). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than 2 fold induction was noted and the difference between g-csf and gm-csf was highly significant (p<0.01). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp-1 human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r37 a123447 and pot ca54474. john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, 20850. at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i.8], dmpg[0.2], cholesterol [1.5] , and monophosphoryl lipid a [20-200 gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected 10-11 days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing 0, 20 or 200 gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds0 for control (liposomes with no lipid a) mice was 3x10 bacteria; ld50 for mice vaccinated with 20p.g was 24x103 (8-fold increase in resistance) and with 200~tg was 48x103 bacteria (16-laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld50 for control (liposomes with no lipid a) mice was 30 ng lps; the ld50 for 20gg lipid a was 70rig lps (2-fold increase in resistance) and for 2001xg was 570ng lps (19-fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld50 of franciseua in normal mice or mice inoculated with liposomes without lipid a was 20-40 bacteria. in contrast, mice vaccinated with liposomal lipid a (200ggl survived challenges as high as 30,000 bacteria, (3 logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers 24 hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il-6, and il-8 responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd14. to evaluate if blocking lps-cd14 interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi4 monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a 24 hour period. to evaluate the effect of treatment with anti-cd14 mab, animals were given either nothing, an isotype control or anti-cd14 mab (5mg/kg) 30 rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il-113,1i,-6, il-8 and il-10, and liver enzymes during a 24 hour period revealed that treatment with anti-cd14 mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd14 mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd14 interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi23). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a 23 kd n-terminal fragment (rbpi;3) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi23 bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi23 has equivalent antibacterial activity to bpi against rough gnb but is up to 30x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi23 compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi23 block the cd14-dependent lpsinduced synthesis of the cytokines tnf, il-1, el-6 and il-8 in vitro. rbpi23 has also been shown to inhibit the lps-induced synthesis of reactive 02 metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi23 increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi23 also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi23 was not toxic to rats after 10 daily consecutive i.v. doses of 10 mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi23 have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd14 as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd14 is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd14) of myeloid cells or as a soluble serum protein (scd14) lacking the gpi-anchor. binding of lps to mcd 14 triggers cell activation while binding of lps-scd14 complexes to cells such as endothelial or epithelial cells that normally do not express mcd14 activates these cells. these pathways are shown in schematic form below. 4 ~di mcd14 plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: 15 mg/kg ---0.1 mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to 4h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il-6, or il-8, but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p80 tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of 10-10 m and has a t1/2 of • 63h in normal humans. methods: 18 normal volunteers were randomized to receive either tnfr:fc vehicle (n=6), or low dose (ld) tnfr:fc (10 mg/m 2 iv, n=6), or high dose (hd) tnfr:fc (60 mg/m 2 iv, n=6) infused over 30 rain prior to endotoxin (e) (escherichia coil 0:113; 4 ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point (4-5h) in subjects given tnfr:fc compared to vehicle (3h) (p=.004). at 3h, 5h (post fluid load ), and 8h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=.0001 ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<.03) and later leukocytosis (p<0.05). peak tnf bioactivity in subjects given vehicle was 154 + 44 pg/ml and <10 pg/ml in the ld or hd tnfr:fc groups (p<.001). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=.0001) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<.001). decreased levels of il-8 (p=.00t), granulocyte colony stimulating factor (p=.03), and il-1 receptor antagonist (p=.001) were found after ld or hd vs vehicle. il-6 levels were lower after ld vs vehicle or hd (p=.006). il-10 levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the 5' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 3'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥1ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c1 inhibitor complexes (1). in this in vitro plasma model we also found that methylpredoisolone alone in doses of 1 mg/ml induced contact activation with the formation of kallikrein-c1 inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade (2) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when 103 ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with 20 gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et (79 %) and interleukin-6 (63 % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c1-1nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c3-1nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c1-1nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c1-1nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type-1 in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type-1 (cr-1) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c3/c5 convertase formation. we have recently purified the human recombinant cr-1 in a truncated form (brl55730, tp-io, scr-1) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: 1) endotoxin-induced ards in paf primed animals; 2) 11.-2induced ards; 3) thermal injury (30% body surface)-induced ards; 4) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr-1, 1-25 mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il-2), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr-1 in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that (1) ptx suppresses lps-induced tnf release from activated macrophages; (2) ptx prevents tnf-induced pmn activation; (3) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; (4) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse 15, d-81377 m0nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il-6 most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to 4 hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il-6 and il-8. moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt3 first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons (14.5 +.7 kg), were randomized to a control group receiving 101° cfu/kg of intravenously administered e. co/i (n=5) and a treatment group (n =5), pretreated with .1 mg/kg of paf receptor antagonist ro24-4736 two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving 4 ng/kg of intravenously administered endotoxin (lps) (n=5) and a treatment group (n=5) pretreated with 10 mg of ro24-4736 eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for 24 hours. results: baboons pretreated with the paf antagonist had improved oxygenation (96 + 11 mm hg v 59 -+ 6 in controls, p < 0.05) and creatinine clearance 12 hours post e.coli (39.8 + 23.4 ml/min v 0.1 _+ 0.1 in controls, p < 0.05). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at 1 hour post lps. this was in concert with a diminished peak cortisol (668 +_ 107 mmot/l v 959 +-159 mmol/l in controls, p < 0.05), epinephrine secretion (1057 + 165 nmol/l v 2029 + 431 nmol/l in controls, p < 0.05). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il-1 and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il-1ra, a naturally occurring il-1 antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha-1a and e5. future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa2 production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa2, and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn 52021, have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il-1 and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)i 8 chain covalently linked to one of three different alpha chains (cd11 a, cd1 lb, cd1 lc) and exist on the cell surface as three distinct heterodimers. cd1 l a/cdi 8 is expressed on all leukocytes, whereas cd1 l b/ cd18 and cd1 lc/cd18 are restricted to cells of myeloid origin. cd1 i/cd18 interacts with intracellular adhesion molecule-1 (icam-1), its ligand on endothelial cells. icam-1 is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = 27) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria1 endotoxin) during the first 3 days after inciosion in the study. the other randomized group (n = 28) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of <6 wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one (4%) of 27 patients who received immunoglobulln. by comparison, nine (32%) of 28 control group patients died during this period (p <. 01 ). within the first 48 hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; 2) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_20 (meaning a mortality risk >70%) were accepted into this protocol. patients were randomized to receive 0.4 g/kg of ivig or placebo on day 0 (when they reached sepsis score>20), repeated on day +1 and +5. at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: 38% vs, 67% respectively; p< 0,05). in conclusion, the results of our study in patients with severe surgical sepsis were the following: 1) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', 2) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg 8(1993)61-83) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in 163 patients with sepsis and septic shock (infection 19~1991)216-227), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score 20-35) and severity of sepsis (elebute sepsis score 12-27) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo (0.i % albumin) or polyglobin n" -12 ml (0.6g)/kg on day 0 and 6ml (0.3 g)/kg on day i. with an anticipatedpopulation size of 800 patients the study should comply with the statlstical requirements (estimated mortality: 30%, with a 33 % reduction in 28-day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november 1993, more than 460 patients had been included; patient enrollment will be finished in 1994. previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il-1, increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in 99 patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled 893 patients at 63 academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) 1.0 or 2.0 mg/kg/hr by continuous intravenous infusion for 72 hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over 28 days utilizing a linear dose-response model, assuming a log-normal distribution. results: 563 patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< 0.04 endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn 52021 (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d28) and tolerance of bn 52021 (2 iv infusion of 120 mg x 2/day over 4 days) in severe sepsis has enrolled 262 pts. the 28day mortality rate was 51% for the placebo group and 42% for the bn 52021 group (p = .17). the efficacy of bn 52021 was greater in pts with gram-negative sepsis: the 28-day mortality rate was 57% for the placebo group and 33% for the bn 52021 group (p = .01). bn 52021 also reduced mortality among pts with gram-negative septic shock (mortality was 65% for placebo vs 37% for bn 52021; p = .01). using statistical adjusments for pronostic factors, the relative risk of death of the bn 52021 group was 0.61 (0.34 -1.08, 95% confidence interval; p = .09). this risk corresponds to an adjusted reduction in mortality of 39% for pts receiving bn 52021. no differences in mortality rates were found between the placebo and the bn 52021 groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn 52021 groups. bn 52021 is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~3lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for 28-day mortality (jamb. 1993; 270:12,33-1241) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin-1 receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x2 = 7.6, p [] 0.02, log.normal) for all 893 patients in the trial• survival benefit began st approximately 24% baseline risk of 28-day mortality. for the $80 patients with a predicted risk > 24%, there was a 22% reduction (p=0,00$ log normal). when we examined the variation in patients above and below the 24% risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < 24% risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > 24% risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after 14 days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp-0127, in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp-0127 on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to 28 days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population (95.2%) of pop. low-risk pat. (ap< 19; mortality: 1%) and the small groups of pop. pat. at risk lap= 19-23) and high risk lap_24) with a significantly higher mortality (14% and 76%, mainly due to sepsis). subsequently, among 1341 consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n =41): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day 1:8 ml/kg, day 2:4 ml/kg). second study period (n=25): iggma (pentaglobin r, biotest, dreieich, frg, 5 ml/kg on days 1 to 3). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n=21, high-risk: n-21), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n=26: p<o.05; iggma, n=13: p: 0.08). in this group, ig led to higher (p<0.05) response rates, defined as an ap score decrease ->7 within four days (igg: 54%, iggma: 62%; con.: 19%), and reduction in mortality (igg: 46%, iggma: 46%; con.: 76%), statistically significant (p<0.05) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, 21 aminoacids et (et 21) in rat abdominal sepsis. methods. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et 21 were determined with amersham tm endothelin rias 4, 8 and 12 h after sepsis induction. experimental groups: 1. cp control, 2. volume replacement (vr); 0,9% saline 10 ml/kg/h continous iv infusion started after 2 h, 3. antibiotic; imipenem 40 mg/kg iv after 2 h, 4. e5®; 10 mg/kg iv after 2 h, 5. vr + imipenem + es® after 2 h. results. high concentrations of both big et and et 21 could be demonstrated after 2 h and lasting for 12 h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e5® significantly reduced et 21 both 4, 8 and 12 h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et 21 was the same as for e5® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e5® reduces circulating lps and tnf which is the probable mechanism of the suppressed et 21 synthesis. the unaltered big et fraction after e5® treatment indicates conversion of big et to et 21 as the site of action responsible for reduced et 21. conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et 21. e5® anti-lps antibody significantly reduces plasma et 21 while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e-75 in congestive heart failure has been demonstrated 03aumann, 1989). therefore, we investigated the effect of bu-e-75 in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [20] [21] [22] [23] [24] [25] were anesthesized with fentanyl/dormicum, ventilated (n20:o 2 = 2:1) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study (4 animals) to evaluate the effective concentration of bue-75 in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of 10 ~g lps/kgkg/h (055:b5, fa. difco). the hypodynamic state was defined as a decrease of cardiac output by 30 % of steady state levels. a wedge pressure of 10-12 mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) 3 groups with 6 animals per group were studied. the groups were treated as follows: group i, lps and 0,9 % nac1; group ii, lps and bu-e-75 (100 #g/kgkg/h); group iii, famotidine (h2-blocker) pretreatment (1 mg/kgkg), lps and bu-e-75. results: the pilot study in healthy pigs revealed, that bu-e-75 had positive inotropic effects. these effects were inhibited by the h 2antagonist famotidin. bu-e-75 however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do2) were not significantly influenced by bu-e-75 application (group i versus group ii). bu-e-75 did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e-75 led to a further significant decrease of lvsw (p < 0,05). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h2-agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. 29 male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci 0.9% (control without endotoxin, n=10). group e : endotoxin 120 tjg kg "1 bolus i.v. + naci 0.9% (control with endotoxin, n=10). group c : endotoxin 120 pg kg -1 bolus i.v. + cll 400 u kg -1 bolus + 400 u kg "1 4h "~ i,v. (treatment group, n=9). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m1) and after 120 (m2) and 240 rain. (m3). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p<0.05) between group e (lung 180, liver 159) and c (lung 89, liver 0) (group k : lung 11, liver 0). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july 1992 to june 1993, 15 patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of 66.7 years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of 2mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the 1st, 3rd, 5th and 7th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are 2 main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk 2 receptor is constitutive. the bk 1 receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk 1 receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk 2 (bradycor [cp-0127]),bk~ (cp-0298) and bkz/bk 1 (cp-0364) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk 1 agonist) in the lps-treated (10ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk 2 agonists increased with time and at time 210min appeared maximally induced. constant iv infusions of cp-0127 blocked bk 2 but not bk~ and cp-0298 bk~ but not bk 2 responses. cp-0364,cp-0127+cp-0298 and antril+cp-0364 blocked both bk 2 and bk~ responses. antril alone had no effect on bk 2 or bk~ responses. within 30-60 min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk2,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk 1 receptors. in vitro studies demonstrated that beth bradycor and cp-0364 (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk 1 receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp-0364 is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september 1988 and july 1993 400 olt's in 368 patients were performed at our department. the actuarial 1-year patient survival is 90 %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from 48 to 72 hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss -30), mean temp. was 101.4f, leukocytosis was 16k, pan2 was 87, fin2 was .47, and peep was 5.1 at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t00 am and a thickness of 4nm. the ami-fungal efficacy is 2-4 times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than 100 nm with a mutina ld50 of > 175 mg/kg. it has been used in dosages up to 6 mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a 83% response rate for invasive candida infections to 41% response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was 8% among placebo treated patients compared to 3% for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from 16% to 0% ill placebo and ambisome treated patients respectively (p<0.01). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study 40 kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of 100 mg/kg at day 0,7,14,35,56 and 77 after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop0sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in 143 consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of 1200 elisa units during 3 months. cmv infection occurred in 21/65 ~eronegetlve and in 40/81 seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower (3/34] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f18t29) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection (11121 v8 10/40), but no difference in incidence of disease (11165 vs 10/81 ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from 21 patients with different types of leucemia after autologous and allogenzc bmt 19 had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in 62% of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity 10-14 days after intranasal inoculation of influenza virus a (h3n2-3) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest 1.00 consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years 1990 and 1993. in the same period of time 732 pri~ total hlp replacen~nts were performed under i4entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than 1%. in the selected i00 cases the septic rate was 4% indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of 45 patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c3) and fourth (c4) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate (1"= -0.766, p < 0.001). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet 36 patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c3, c4, crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il-1ra, il-2, il-2r, il-6r, tnf-ct, tnf-~r (p75) and icam-1. in contrast, pmn-elastase, il-6 and il-8 closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd4+/cd8+ ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of 30 % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il-6 and il-8, however, closely correlate to the clinical course and may prove valuable for follow-up. the cd4+/cd8+ ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of 30 % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and (2) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of 20 mu/ml xanthine oxidase (xod), 500 mm hypoxanthine (hx), 50 mm fec13 and 75 mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il-8. cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h202 is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& 2) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, 3) the trant~ of vim.©n~e traits between ~renr~a.,dsms and 4) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky 40292 inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il-1 receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h6pital erasme, route de lennik 808, 1070 bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin 8) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il-8 by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il 8 and monocyte chemotactic activity when cultured. these activies were significantly increased (2-fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il-6 in 19 patients with bacteriologic proven abdominal infection (intraabdominal abscess 10, diffuse peritonitis 4, pancreatic necrosis 2, pancreatic abscess 3) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a 6 to 10 hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il-6 determination were drawn preoperatively, intraoperatively, and until 2h postoperatively in regular intervals (min 7/pat), results: preoperative apache ii was 12 in median (rain 4, max 25). 10 patients fulfilled the criteria of sirs. 4 of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p=0,000 i, spearman coefficient). preoperative cardiovascular (systol. rr<90 mmhg) and respiratory (pao2<75mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p=0,000 i, wilcoxon; pulmonal: p=0,0003) and il-6 (cardial: p=0,2; pulmonal: p=0.0001) overall, lps, tnfa and il-6 values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i 0, mean 182 pg/ml) than in those who did not fulfill these criteria (group b; n=9, mean 24 pg/ml; p=0,00 i, wilcoxon). il-6 was significantly higher in group a (mean 2169 pg/ml) than in group b (mean 191 pg/ml; p=0,0o i wilcoxon). conclusion: perioperative tnfa and il-6 were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel 18-20, 1090 wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in 30 lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= 12) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after 6 hours. stastistieal calculations were performed on a personal computer with the spss programm vers. 4.01 (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla2-activities [endotoxin range from 0.064 eu/i, sd=0.19 to 102.38 eu/1, sd-145.4 (p < 0 0001), ifn-¥ levels, range from 147.9 pg/ml, sd-141.6, to 4591 pg/ml, sd=6541 (p < 0.0001), circulating pla2-activities range from 116.2, sd=45.4 to 185.4 u/1, sd=105.4 (p < 0.01) and biopterin range from 54.4 nmol/l sd=17.2 to 127.8 nmol/l, sd=76.8 (p < 0.001)]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = 0.72, p < 0.0001) and bioptefin synthesis (rv= 0.82, p < 0.0001). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = 0 73 p < . .. p • , . 0.0001) and to the pla2-actwtues (rp = 0.50, p < 0.005). the biopterin synthes~s correlates slightly with the pla2-actn,ities (rp= :0.38; p < 0.05). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of 87 %. anaerobes were found in 53.4%, anaerobes were isolated in 22.8%. there were aerobic and anaerobic associations in 12.1% and microflora was not found in 11.6% of the cases. express method of anaerobes discovering let to receive information on 1 -3 days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of 166 patients with abdominal sepsis treated at the surgical intensive care unit during a 10-year period, 72 (43 %) had an age of 70 years or more. 28 were women and 44 were men, a sex distribution not differing with patients younger than 70 years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was 29 (-183) days including in median 10 days in the icu. figures in patients less than 70 years of age were comparable (34 (-293) days out of which in median 10 days in the icu). apache ii and sss-scores did not significantly differ (14.2 vs 13 and 23.3 vs 26.5, respectively), between the groups. neither did the incidence of organ failure differ (57/73 vs 77/94). however, the incidence of multiple organ failure was significantly lower in elderly patients (21/72 vs 42/94 (p < 0.001)). the mortality rate, however, did not differ between the groups (21/72 vs 26/94). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. 96 adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n--48 in each group). at 3, 6, 12, 24, 48 and 72 hours (n=6/timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of :25i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was 33 % both at 48 and 72 hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from 6 hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from 12 hours and on and in the proximal and distal small intestine from 24 hours and on. different types of alterations in capillary permeability seem to appear: 1) a temporary short increase e.g. in the proximal small intestine and spleen; 2) a temporary longer increase e.g. in the colon and kidneys; 3) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p 2 integrins (cd1 l a, b,c/cd18), and the fc'?r (cd16, cd32, and cd64), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score >10) or thermal injury (>30~ total body surface area, 15% full thickness), and healthy controls. the expression of cd11 b and c and to a lesser degree cdi 1 a was significantly reduced on pmns. the expression of cd16 and cd32 but not cd64 was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of 132 integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, 2~1 8ethesda ave, cincinnalt, oh, usa, 45267-0s5b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet1, dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj 07107 a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e2) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi0atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in 44 recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il-1. the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =.28, mean b state = .57, mean ~ state = .61). the accuraay of this estimate was prospectively analyzed in this group of 44 m~itlple patients of whom 72% had sepsis and 36% had ssptlo ards. the 22 survivors had a mean p death of 0.39 and the 22 deaths had a mean p death of 0.63. the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb6 was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of 80 patients who were enrolled into one of four treatment groups: (1) 0.1 mg/kg of anti-tnf mab, (2) 1.0 mg/kg, (3) 10 mg/kg or (4) 1.0 mg/kg at study entry and the second dose 24 hours later. the small sample size in each group (n=20) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin-6 may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first 48 hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x1351) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in 31 hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of 15 mg/kg tnf~ mab, 7.5 mg/kg tnf~ mab or placebo (0.25% human albumin).patients received standard aggressive medical/surgical care during the 28 day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced 28 day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day 28 post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca2, was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca2. as a single infusion or in combination with ha-1a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage 1 was an open label trial in which 4 groups of 5 patients each with the clinical diagnosis of sepsis received ascending doses of ca2 (0.1, 1, 5, 10 mg/kg). stage 2 was a randomized, double blind study in which patients received a single dose of ha-1a (100 mg) and placebo or one of 3 doses of ca2 (1,5,10 mg/kg). stage 3 was a randomized, double blind study in which patients received a single dose of placebo or one of 3 doses of ca2 (0.1, 1, 10 mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il-1, and il-6 levels.a total of 141 patients were enrolled from 13 clinical sites (20 in stage 1, 60 in stage 2 and 61 in stage 3). primary analyses were performed on patients in stage 1 and 3. there were 65 patients who received ca2 exclusively and 16 patients received placebo. administration of ca2 was well tolerated at doses up to 10 mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in 61% (30/49) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca2. the mean half-life was -70 hrs (58-96 hrs). a dose related decrease in tnf concentration was observed 1 hr post infusion of ca2. tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak 195f. it is a f(ab')2 fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. 122 patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak 195f or placebo were administered (0,1; 0,3 and i mg/kg) over a perid of 72 hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il 6, il 8 and tnf), and outcome. at this time only an interim analysis of 60 patients is available i indicating that mak 195f in all 3 dosage groups resulted in a decrease in il 6. this contrasted to a further in crease of il 6 in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all 122 patients in the study will be presented and discussed.$152 s154 staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a 13 atom-chain spacer. the detoxification capacity was 150 ug[et/ml column material. the biocompatbility resulted in 90~ platelet recovery. the column contained 7 ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine 1.000 iu/h in the inflowline after bolus injection of 5.000 iu. hp was performed on 8 pigs at a rate of 50 ml/min by means of a roller-pump until the animals succumbed (h). 8 animals served as controls (c). serum et levels rose from 3.40 pg/ml to 74,9 pg/ml after 5 hours in the c and from 2.77 pg/ml only to 28 pg/ml in the h group after 7 hours whieh was highly significant. survival time could be extrended from 216 to 313 min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b(2rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from 1989 to 1991 78 consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in 21°,4 and by septic-toxic event in 79%. all patients required mechanical ventilation (fio2>0, 5) . 86°4 showed hyperdynamic shock. 85% had renal and 53% hepatic failure. medium appache ii score amounted to 29,8 points. cvvh was performed in postdilution mode with a polyamide membrane (fh 66) and high volume exchange (50 l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in 71%, hemodynamic in 68% and renal in 66% of the cases. 42% of the patients survived and were discharged from hospital. 10 of 45 non-survivors (58%) died because of fatal mof within 24h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web 2086), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin (6p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n=6), or after pretreatment with web 2086 (5xl0-gm, n=6), or diclofenac (10#g/ml, n-6). furthermore, the application of specific antitoxin (mg/ml, n=6) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, 7.5 mg/ml) (n=6) and the combination of immunogiobulins, web 2086 and diclofenac (n=6). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at 30 rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of 2.5 times higher than the control, starting after 1 min and a delayed onset of edema formation resulting in a mean weight gain of 20 g after 120 min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about 10 times that of controls in the toxin treated lungs. pretreatment with web 2086 or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web 2086 and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st 899 (4-n,n,n trimethylammonium-(r)-3-isovaleroyloxy-butanoic acid z-3-(5-chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of 3h-paf (ki=7.5x10 -8 m) to rabbit platelets. the present study shows that pretreatment of c57bl/6 mice with st 899, administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli 055:b5 injected at 30 mg/kg intraperitoneally). very interestingly, st 899 administered at the same doses as above (i.e. 1.25, 2.5, and 5 mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st 899 (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia (42.5°c) induces heat shock protein ('asp) 72 and increases smvival 2-4 fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp-72 by sodium arsenite improves survival in a rat sepsis model (abstract a95 am. rev. resp. dis. vol. 147, 1993) . the effects of heat are complex and in addition to formation of lisp-72 include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp-72 in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite (10 mg/kg) and ethanol (400 ~1 of 39% ethanol) caused marked induction of hsp-72 in lung, gut, kidney, and liver, which was comparable to heat-induced hsp-72. female nd4 mice weighing 22-25 gms were pretreated with arsenite or alcohol 8 hours prior to challenge with escherichia coli endotoxin (-ld 80) and survival was compared to control mice. results: survival at 24 hrs. for arsenite treated and alcohol treated mice was 96% and 88% respectively and was statistically different from the 40% survival for control mice. (p<0.01) (n=25 mice per group). however, at 7 days post endotoxin, there were no differences in survival in the 3 groups, i.e., ~ 4% survival for all 3 groups. in contrast, the protective effect of hyperthermia remains present at 7 days, i.e., ~ 70% survival vs 20% survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp-72 formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered 4x per day from the day be ~ fore the operation until the 7th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day 7 postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in 3/93 after 137 pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from 10.6 % in the placebo-group to 3.5 % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from 33.3 % in the placebo-group to 16.4 % in the treatment-group (p ~ 0.0281;fisher's exact test). in march 1994 final results with more than 200 patients will be presented for the first time. (= po2 <80 mm hg, b s-creatinin >2 mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only 2 of 14 deaths occurred during the 1st week, all other deaths occurred late (after 4-12 weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. 11--6) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only 15% of deaths today occur eady in the course of the disease, whereas this percentage varied between 40-70% just 10 years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for 20 to 25 years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in 24 patients with acute pancreatitis (14 males, 10 females; median age: 56 years, range: 32-81 years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type 1 (slcam-1) were determined using immunoassays (henning, bender, t cell sciences). 13/14 had increased concentrations of stnf-r compared to the 75th percentile obtained in healthy controls (>4.2 ng/ml), and 9/24 patients had increased neopterin (> 8.7 nmol/i), 9/24 presented with elevated slcam-1 (>440 u/i). all patients with increased neopterin also had increased stnf-r, 4 patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = 0.728, p < 0.001 ). weak associations between age and stnf-r (rs=0.435, p=o.05) or neopterin (rs=0.456, p = 0.044) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as 1970 ( 1,2) and characterized by increased serum or urine amylase levels (in about 33% of patients) in the 5 fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the 198ffs, 3 studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery (3, 4, 5) . in a recent study (6), evidence of pancreatic cellular injury was found in 80 out of 300 patients undergoing cardiac surgery, with 22 out of these 80 patients presenting abdominal signs or symptoms and 3 developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied 300 patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), 8 hours alter surgery and in the folfowing days alter surgery (days 1, 2, 3, 4 and 8). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day 1. we measured abnormal levels of trypsin and pancteatic iso-amylase in 30% of patients and observed 2 simultaneous releases of these 2 enzymes, the fi,'st one in the 24 hours after surgery and the second more intense from day 4 and pa~icularly on day 8 after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about 2% of our patients : pancrealic abnormalities were revealed by scan in 7 patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic 1so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu 7 diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion (7). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin-6(il-6) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii-6(elisa), transferrin and crp (nephelometry) were analysed daily along the first i0 days of hospitalisation by 28 patients with acute pancreatitis admitted to our hospital from 4/1991 to 4/1993 . it was judged whether the patients have either interstitial (aip) (n=9) or necrotizing (anp) (n=lg) pancreatitis. 5 patients with anp have died during the course of pancreatitis (mortality=17.5%). results: 1-severity o~ pancreatitis: signifcant differences (p<o.05, mann-whitney test) could be calculated between aip and anp for endotoxin, il-6, transferrin and crp during the i0 days of monitoring. 2-mortality: except of il-6 on days 2, 3, 4 and 5 after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. 3-organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il-6 and crp along the i0 days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il-6 and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from 10/92 -8/93, 23 patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n=12) and n (n=l 1) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in 13 patients. the etiology of ap was gallstones in 9, alcohol abuse in 7, hyperlipidemia in 2 and other or unknown causes in 5 patients.five of 11 patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least 3 days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il-6 concentration in blood reached up to 2600pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of 600 pg/ml was correct in 20 patients (87%, sensitivity (se): 82%= specificity (sp): 91%, (z:0,001). high levels of il-6 over 500 pg/ml correlated well with the prognosis ifri in the first week (accuracy: 87%, se: 80%, sp: 100%, c~: 0,001). the blood levels of il-8 reached a maximum of 1381 pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of 1l,-8 blood levels indicated correctly the severity of ap in 18 out of 23 patients using a cut offtevel of 200 pglml (accuracy: 78%, se: 82%, sp: 75%, ~: 0,01). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il-6 and il-8. in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il-6, and to a lesser extent of [l-8, can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: 18 opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe0 tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n=6), around the pancreatic duct (group b, n=6) or around both ducts (group c, n=6). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a 99mtcalbumin colloid with a diameter of 25 nm, was measured before, 2,5 and 6 days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day 6, the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day 6. the opossums in groups a & 8 had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day 5 in group a & b (p<o,05) and at day 2 it~ group c (p<0,ol). at day 6, res function in group c was significantly worse than that in group a & b (p<0,01). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day 2 (p<0.005), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b6) in lewis rats weighing 220-250g. there were five groups: group a (n=14) were untreated controls; group b (n=7) were given isotonic saline intraperitoneally and observed for 48 hours; group c (n=7) 2 ml x 108 e. coli intraperitoneally and observed for 48 hours; group d (n=7) were given 4 ml x 108 e. coli and observed for 48 hours; group e (n=7) 4 ml x i08 e. coli and observed for 24 hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin 2 receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser 11). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p=0,003) and spleen (p=0,0001 )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p<0,0001 ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel 18-20, 1090 wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in 60 children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar(26-1ocalized jo-diffuse, 9-~eneralized) and 5-hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen-4, the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, 5-com:lissural ileus). 2he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; 720) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit 2 can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial 24 hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and 4) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding 4000/co 2 hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate 3-6 wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. 80 grafts were cryo'd in 10% dmso, quick-thawed & sutured onto 2x2 cm excised full-thickness dorsal wounds on athymic mice. 40 "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained >60% cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in 10-10,000x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through 40 days of followup. histologic exams on days 6-40 showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than 80% total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we1] as ready availability of urdform lis~ue~. on dec~mt~r 14, 1993, the fda assumed control of as1 tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, "13ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as 3 years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki-67 (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since 1984 when galllco et el reported theh'use in two brothers with greater than 90% total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day 0), the full thickness wounds were covered with epigard, a synthetic dressing until day 10. after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from 30 patients at the beginning of surgical treatment (day 0) and after 3, 6, and 10 days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf-6, double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.(1) pharmatec gmbh, frankfurt (2) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group 1, cells migrating through skin: 75+4% t lymphocytes (cd3), 6+3% langerhans and dendritic cells (cdla, hla dr, s100), 41+9% cd4, 18+6% cd8, no b cells (cd22, 23), 74% cd45r0 (memory cells), 6+3% il2r. approximately 90% cells possessed cdlla and 18 antigens. cd1 lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group 2, cells in skin: langerhans cells were found only in epidermis, cd3,4 and 8, cd45r0, rb, ila/18 cells around venules, cd68 (macrophages) uniformly dispersed, no il2r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group 3, one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il2r cells, perivenular infiltrates of cd68, 45r0 but less cd3 cells, high density of cdlla/18 cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht3 bioassay). the hollow lipid microcylinders (50 microns in length and i micron in diameter) show an initial burst (5-10 ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed 1, 3, and 5 days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd3 and cd4, mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code 6900, naval research laboratory, washington, dc. 20375-5348. expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet 1992) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from 200ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of 30 ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. 22 patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to 50 go of the origional size (t50%) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from 5 juvenile human bum patients, immediately after wound debridemerit mad on day 8 after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*'1~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of 6 psoriatics and 6 healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx105 t-lymphocytes were then incubated alone or in coculture with 2x104 irradiated autologous lymphocytes in culture medium containing 10 -7 mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce11-surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t2si-hsa over a 30 rain period, the chemotac~c peptide imlp (0.1. 30ng) and bradykinin were used to induce cell.dependent and cell-5ndependent leakage respectively, the antibodies used were 6.5e (cdis), nri85 (cdlla) and antibody 198 (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien (15.~. ffi 2.1p.l to 54.4 z b.bttl and 16.4 ,-1.8~ to 59.8 z 6.71d respectively. 6.5e (0.0072-2.4mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at 0.24mk/kg, the plasma leakage was completely inhibited, antibody nr185 produced similar results, treatment with antibody 198 did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . 22 patients without infectious coz~lications and 2 group -53 patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection 23 patients of the 2 group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i-3 days after injury revealed a considerable decrease of lymphocytes, ed3",$d4 ~ and cd8 cells amo~it in the blood, cd4 /cd8 ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the 7-10 days in pateents of+the i group. but stable ~eple$ion of cd3 and cd4 cells amount, lower cd4 /cd8 ratio and indexes of leukocyte migration inhibition test in patients of the 2 group were observed~ besides that, these persons showed higher cd19cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in 80% of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd4 tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. 6, 194175, st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p2 integrlns (cd11a,b,c/cd18) and the fw receptors (cd16, and cd32) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~2 integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z 10) or thermal injury (>30~; total body surface area, >15~ full thickness) and incubated /n v/tro with gm or g-csf. the j32 integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd1 lb, cd16, and cd18 while gm bur not c-csf induced an increase in the percentage expressing cdi 1 a, cd1 lc, and cd32, gm-csf and to a lesser extent g-csf induced an increase in the density (1,5 fold) of the ~2 integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, 231 bethesda ave, cincinnati, oh, usa, 45267-0558. funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra5 loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in 6% and in association with bacterial infection(l 1%), viral infection(3*/.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a.1 of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e1-ebiary, a. torres et al, 1993 reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of 415 olt in adult patients 100 olt were performed for hbsag positive liver disease (cirrhosis n=86, fulminant liver failure n=8, retransplantation n=6) in 94 pat. all pat. received 10.000 u hig in the anhepatic phase and 2.000 u/per day for the first week. a small group of 9 pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above 100 uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are 100% in pat. who were transplanted for fulminant hepatitis, 96% in pat. with cirrhosis and long term prophylaxis, and 78% ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was 100% under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in 28% in pat, negative for hbv dna. in hbv dna positive pat. infection rate was 56%, the total rate of reinfection for all pat. with long term prophylaxis was 36%the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢11 capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il-8, ltb4, c5a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi2, poe, lt's), complement components (c3a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il,1, il-6 or tnf), and immunosuppressivc agents (poe2, il-10). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, 325-9th ave za-16, seattle, wa 98104 usa key: cord-005814-ak5pq312 authors: nan title: 8th european congress of intensive care medicine athens greece, october 18–22, 1995 abstracts date: 1995 journal: intensive care med doi: 10.1007/bf02426401 sha: doc_id: 5814 cord_uid: ak5pq312 nan objectives: evaluate the levels of tnf, il-6 and pai-i in different moments of the ards and the possible relationships among them. methods: 23 septic patients with ards were studied. also significant differences for: tnf, pai-i and il-6 in septic patients and both evaluations of ards with control gropup; pai-1 between septics and 2nd evaluation in ards, and between the ist and 2nd evaluation in ards; il-6 between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il-6, with clinical signs, are suggestive of infection; 2) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; 3) due to its own kynetics, il-6 takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, 30 patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution (10% hes, mean molecular weight (mw) 200,000 daltons, degree of substitution (ds) 0.5) or human albumin 20% (ha) for volume therapy for 5 days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule-1 [selam -i] , intercellular adhesion molecule-1 [sicam -i] , vascular cell adhesion molecule-1 [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next 5 days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from 89• to 106• during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from 626• to 1,329• sgmp-140 increased significatly only in the ha-group (483• to 683• only pao2/fio 2 was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august 1994 all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the 8 month period ,2059 patients were hospitalized on our intensive care unit. 230 central venous catheters were examined for microbiological culture. 118 specimens appeared to be possitive (51%). 13 patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was 13/118 (11%). the incidence of sepsis due to the presence of all central venous lines was 13/230 (6%). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n=5), escherichia colt (n=7), others (n=6). in the epic study the percentage for sepsis on the icu was 17.6% for the netherlands and 17.8% for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of 59 admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl 0 and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than 0,5 ]09]1). results: five patients (8,5%) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( 20.9+4.6 and 16.5+3.0, respectively, p<0,01, mann-whitney u test). no patient with a saps greater than 20 survived. mortality among the 27 patients with saps from 9 to 20 was 81,5%o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only 56 hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=-0,52, p=0.001). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than 20, failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of 52 neutropenic(wbc <0,5 109/1)patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of 8 patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in 4 from 8 patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within 40 minutes up to 1,5 hours after administration of antipyretics. three patients received 0,5 g of metamisol and one 0,5 g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level (35.4+0.4~ accompanied'by hypotension. but in 8-12 hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required 1200+350 ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by 3 other methods used in icu patients. methods: we prospectively studied 12 adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution 0"d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and 95% limits of agreement (l of a = d_+2sd .15 td-fick -2.36 -8.06 to 3.34 fick-echo 0.60 -5.92 to 7.12 there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n 12; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years 120 patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il-1, il-2, il-3, tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from 23.4% to 12.5% in patients with abscessed pneumonia and lung abscesses and from 50% to 13% if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il-2. intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in 81% and mortality was decreased from 46% to 19%. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of 1) rbct ; 2) number of units transfused; and 3) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from 1992 to 1994 and excluded patients who died in the first 5 days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first 5 days after onset of sepsis. overall 30-day mortality was 22%. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco2 (pico2) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified 5 patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico2 > 50 mmhg. pentaspan| (500 ml) was infused in 30 rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico2 were performed before and repeated 30 miff and 2 hr after pentaspun| infusion. we calculated the pico2 -arterial pco'2 difference (pico2-paco2) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was 73 :1:13 mmhg and lactate 1.2:1:0.6 mmol/l. pentaspan| increased ci by 22% (p<0.05) but did not change pico2 ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco2 in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co2 in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between 12 and 24 hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in 5 patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in 5 patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion 2, nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: 1. moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem 1995: 41/6. 2. moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med 1993; 329: 2002 -2012 ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita180 mg/kg (tb), or varied doses of ec, followed 2 hours later by bolus 30 mg/kg iv lps. 7-day survival is shown below: group: s tb ec(0.1gmikgi ec(0.sgm/kg) ec(i.2gm/kg) alive (n) t 0 0 3 9 ~ total (n) 10 s s 7 10 "signiflcant;y different from all other groups, p<0.0s 5/5 rats given lps followed 2 hours later by ec (1.2 gm/kg) also died. additional rats were treated with s (n=10) or 1 2 gm/kg ec (n=10) followed by 30 mg/kg lps, then sacrificed at 4 hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl0~/mm3), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, 0-4 scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = 0 hz impedance (z; {dyn.sec.cm "5 }); zl = first harmonic z; zc = characteristic z; z1 ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < 0.05 between fio2 0.4 and 0.12, fio2 0.4 and fio2 0. 4&no -0.46_+0.1 -0.26_+0.1 # -0.24+0.1 m -0.85+0.1 * -0.85+0.1 * -0.74+0.1 * -0.88_+0.1 * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa (26+1 rnmhg versus 16+1 mmhg; p < 0.01). hypoxia increased (ppa-ppao) at all levels of q by an average of 13 mmi-ig in minipigs and 2 mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude 1 ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and 2 ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: 1) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. 2) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august 1993 to january 1995, thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from 1 to 40 mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as 40 mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from 70.9 mm hg + 3.4 to 109.0 _+ 4.3 s.e.m., p=0.0004, systemic vascular resistance (430 + 57 to766 + 93 dyne.sec/ cm s, p=.002), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output (8.1 _+ 0.8 to 7.0 _+ 0.9 i/min p=.003) and increased pulmonary artery pressure (35.6 _+ 2.1 to 43.5 _+ 2.0 mm hg; p=.004) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of 13 patients survived for more than 28 days, 4 patients died of cancer complications (all 5 patients had maintained blood pressure for 24 h on nma) and 4 patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and 1 patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as 40 mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included 18 mongrel dogs (25• pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of 3 mg/kg of e. coli endotoxin, the dogs were randomized to receive a 2ml/kg infusion in 30 rain of a hypertonic (2895 mosm]l) solution iucludiug either a mixture of glucose 50 % with 750 u insulin and 2000 meq kcl/l (glk-group 1) or hydroxyethyl starch 7.5 % in naci 8.4 % (hes-group 2). in each dog, a 0.9 % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and 30 miu, 60 rain, 120 rain, and 240 nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table 1) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. 1, 75 for group 2, 105 for group 3) were drawn and immediately analysed at 37~ using the abl500 radiometer for po2, pco2 and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the ps0 at ph=7.40, pco2=40 mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps0"in vivo" (i.e. the ps0 at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in 36 patients aged from 19 to 72 with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of 25nm(mmi) & 280nm (mm2), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic2) and also some clinical and biochemical findings on the 1,3,5 day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from 12.6~1.4 to 2.6+.5 on the 5-th day after the operation. true decrease of the level mm2 from .704~.09 to .402+.08 un & optimal density and increase of distribution index from .96 to 1.09 are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from 8.4+.6g/i to i0.8+.7g/i on the 5-th day after the operation simultaneously with the decrease of cic2 from 806.9~60 to 624.7~54.8(p .05) were founded. some stages of the investigation true increase of lymphocytes from 9.0+2.6% to 15.0+1.8% was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=.5-.7 in p<.05) was discovered and it also showed positive dynamics of the course of the disease. a 34 year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died 24 hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin 6 (il 6) and 8 (il 8), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il 1 ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : 6 patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n=3), ards (n=2) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a 30 rain resting period (baseline), then 30 min after dopamine (2 mcg/kg/min) infusion, and finally 30 rain after the end of dopamine infusion (recovery gmbf 185 _+ 23 (perfusion units) gmbf 0 ~a% vs baseline) * p < 0.05 vs "baseline" and "recovery". conclusions : 1) despite a slight increase in co (+13%), the dramatical increase in gmbf (+87%) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. 2) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and 7,8-dihydroneopterin (nh2) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n (20 pm), nh2 (20 ilm), lipopolysaccharide (lps, 5 ~g/ml), and interferone-~/(ifn-~/, 100 u/ml) for 9 h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn-7 seem to have a more than additive effect nh2 did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in 64 patients, 30 with sepsis, 22 with severe sepsis and 12 with septic shock, as well as in 14 healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in 30 anesthetized rats, catheters were placed in the femoral vein and artery. 23 rats were given an iv bolus of lipopolysaccharide (lps, 5 mg/kg), at baseline (to). after 1 h (t1), rats received at random an infusion of either can (20 mg/kg/h; can group, n=l 1) or an equivalent volume of 0.9% naci (2cc/kg/h; nac1 group, n=12), giyen over 4 h (t1-t5). a third group (sham group, n=7) received 0.9% nac1 in place of lps, and then was treated like the nac1 group. mean blood pressure (mbp), blood lactate and nitrates (no3) were measured each h. glucose, creatinine and asat were also measured in 18 rats (n=6 in each group). the can 98_+9* 304+52"t 3.2+1.1"~ 3.6+_1.4"t 138+46" 48+10" *p<0.05 can vs naci ?p<0.05 vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no3, confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc1tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), 11 patients with non septic shock (nss), 14 patients with bacterial infection without shock (1nf) and 8 icu patients without shock and without infection (control) were compared for proct levels at dayl, 2, 3, 7, 14 . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p=0.0002), infection (3.7+_3 vs 61_+25,p=0.0007), shock (13_+10 vs 60+.-27,p=0 .002), death at day28 (12_+7 vs 96_+44,p=0.003). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day 1 proct levels (19=0.003) in patients with shock, dayl proct levels are correlated with saps, infection and death at day28, but not with arterial lactate levels (p=0.37), white blood calls (p=0.2) or fever (p=0.1). proct levels remain higher i~i septic shock patients at day 1, 2 and 3( figure) . i c0edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in 10 fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin (10 gg/kg). the blood flow to the mucosal and the muscular layer was measured in 3 regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using 8 adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map (95.8_+3.0 vs 53.9-+2.4, p<0.01), and phi (7.29!-_0.02 vs 7.13_+0.01, p<0.01), with a constant co (133_+4 vs 144_+8, p=0.28) and qst, aa (21.5_+0.8 vs 22.8_+0.9, p=0.09). the results of regional pertusion are presented in the table. (flow in ml/rain 9 100g of tissue; mean _+ sem ; * p<0.001 vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a 50% increase in flow. acute increase in wbc count (from a mean of lo.oo01mm a to 42 o00/mm~), between the 3rd and the 7th day of therapy. there was a decline of the wbc count to an average of about 25.0001mm a after decreasing the daily dose of the medication to 200 mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c3 complement between 0.26 to 0.29 g/i. normal values 0.5 to 0.9 g/i there was no change in c4 values. conclusions : an early increase in wbc count was observed (3rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c3 fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin-1 receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of 71 evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, 2 h, 3 h, 4 h, 8 h, and 12 h after the administration of 1 mg/kg (n=20) or 2 mg/kg (n=22) of i1-1 ra or the corresponding placebo (n =29). 58/71 patients (82 %) were treated with adrenergie agents and 66/71 (93 %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man8 and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l0beck from 1992 to 1994. results: within the observation period s. was isolated from the stool cultures of 748 patients. in 13 patients (g m, 4 f, median age 52 yrs) s. could be detected in blood cultures (9 s. enteritidis, 4 s. typhimurium). in addition, in 10 of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all 13 patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least 4 up to 62 days, 4 of the 13 patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids (3), immunosuppressive drugs (2), chronic alcoholism (2), malignancies (2), none (4). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < 2 % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. 30 %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) 65 + 9 58 + 14 death (n) 22 18 duration of shock (h) 27 + 37 54 + 44 noradrenaline (rag/h) 5,9 _+ 6 2 + 5 temperature (~ 38,2 + 2 38,1 + 1 pvr (dynxsecxcm -5) 1195 + 408 572 + 418 co (ljmin) 4,2 _+ 1,6 9,9 + 3,6 lactate (mmol/l) 7 + 5,9 9,2 + 9 interleukin-6 (pg/ml) 829 _+ 798 1331 + 888 interleukin-1 (pg/ml) 9,3 _+ 9,4 9,8 + 7,3 tnf-alpha (pg/ml) 23,5 + 31,7 166 + 209 neopterin (nmol/l) 43,2 + 43,7 218 + 193 crp (rag/l) 131 _+ 97 233 +_ 138 pro-ct (ng/ml) 22,6 + 50,5 77,9 + 160 there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within 24 h did not. however, in late cardiogenic shock (>24h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine (16.7 mg/kg base in 4 h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from 02/1993 to 12/94, pts with sfm (who 1990) treated with iv quinine for less than 6 h were included prospectively. blood samples were collected at o, 6, 12, 18, 24, 36 and 48 h viability was assessed by culturing parasitized red blood cells in the presence of 3h-hypoxanthine, and radioactivity was determined at 42 h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/1)xlo00/icso for quinine (nmo]/]) was called the parasiticida/ index. results:5 pts were included, 42• saps1 18.6-+4.9. the initial parasitemia was 2t.4+7.2%. complications of malaria were coma (4 pts), shock (3 pts), renal failure (2 pts) and acute lung injury (2 pts). all strains were sensitive to quinine (icso 174--67 nmol/1). in 2 pts who were not given a loading dose, parasite viability increased by 63 and 157%, with concomitantly low quinine levels (22 and 19 #mow] at 12 h); 1 pt died. in 3 pts that received a loading dose (serum quinine at 12 h = 33.1--2.0 ~mol/]) a marked decrease of parasite viability (by 73+_10% at 12 h) was shown. viability was inversely correlated with plasma quinine (r=.677, p-.o11) and parasiticidal index (r=.678, p-.o1). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in 3077 consecutive admission to a university hospital icu in 1992 -93 and the icu and hospital survival of the bacteremia patients. during these years 73 patients had 176 positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of 65.8 % (vs. 92,7 % in all icu patients) and six month survival of 50.7 % (vs. 85.8 % in all icu patients). the most common bacteria were enterobacteriaceae (27,3 %), staphylococcus aureus ( 18,8 %) , coagulase negative staphylococci ( 14.2 %), pseudomonas ( 14.8 %) and slieptococci (9.1%). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from 03-1991 to 12-1993 , records of consecutivepts with septic shock (crit care med 1992, 20: 864-74) admitted to the icu were reviewed retrospectively. results: among 76 pts with septic shock admitted during the study period, 28 had hiv infection-26 of whom had aids-(gr. i) and 46 were hiv-negative (gr. ill. ten gr. ii pts (21%) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in 89% gr. i and 83% gr. ii pts in 8 gr . i pts (29%) a multivariate analysis demonstrated that hiv infection and sap5 i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i 0 levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il-10 and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il-6, the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied 94 critically ill patients (63 men, 31 women; 18-86 years old) in three diferents groups. group i: 23 patients without evidence of infection, group i1:34 patients with sepsis and 37 with septic shock (group iii). we measured plasma il-lo, tnf and il-6 levels in the first 12 hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il-10 were higher in group iii (median, 51 pg/ml; range, 5-6000 pg/ml) than in group ii (median, 10 pg/ml; range, 2-970 pg/ml; p <.001) and group i (median, 5 pg/ml; range, 2-133 pg/ml; p <.001). median il-10 concentrations did not differ among patients who survived (median 7 pg/ml; range, 2-6000 pg/ml) and those who died during the overall follow-up period (28 days) (median, 15; range, 5-5400 pg/ml); but patients who died in short-term (< 24 hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, 1200 pg/ml; range, 51-5400 pg/ml). in patients with bacteriemia (34%), levels of il-10 were higher (median, 51 pg/ml; range, 2-6000 pg/ml) than in those with negative blood culture (median, 8,5 pg/ml; range 2-5.400 pg/ml; p< .001). there was a good correlation between plasma il-io concentration and levels of tnf (r= .59; p < .001) and il-6 (r= .60; p < .001). the correlation between levels of il-10 and the apache ii score was significant only in the septic shock group (r= 0.48; p <.005). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il-10 levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital -59037 lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : 6 patients (age : 65• years ; saps ii : 46• were prospectively investigated (septic shock : bone criteria). following parameters were collected during 12 hours : systemic hemodynamic parameters (swan ganz catheter 93a434h -ref1 computer -baxter lab.), calculated systemic oxygen transport (do2), oxygen consumption (vo2) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco 2 (pco2ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter 2024, cold z021 computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp-140) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < 0.05) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam-1 concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il-6, il-8 and increased expressior of adhession molecules (selectin e, p, icam-1) measure of icam-1 and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: 20 male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi02=0.5). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/1o0 g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx (0.25 b6, difco) in saline at 1 mg/kg or h. both interventions produced 50% reduction in map within 3 min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:(1)baseline, (2) mid-point of map reduction; (3) nadir of hypotension, (4)midpoint of map recovery, & (5) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o4o:oc4., objectives : evaluate the relationship between il-10, a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il 6 and ils in severe sepsis and septic shock. methods : twenty-eight icu patients (19 m, 9 f, mean age 54 + 17 y) were studied as soon as they developped a severe sepsis (n = 16) or a septic shock episode (n= 12) as defined by a conference consensus in 1992 (1). tnf~, il 6, il s and il-10 plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il-10 plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < 0.05) between il-10 and tnf a (r= 0.6), il-10 and il~ (r = 0.73) and il-10 and il s (r = 0.65) as well as between il-10 and apache n score (r= 0.52). patients who died (n = 13) had il-10 levels higher than patients who survived but this difference was not statistically significant (114 pg/ml vs 34.5 pg/ml; p>0.05). conclusions : during severe sepsis and sepsis shock, il-10 seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : (1) crit care med 1992;20:864-74. objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and 24 hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin (0.250 mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least 30 mmhg and/or a decrease in the necessary vasoactive medication of at least 50% within 24 hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than 500 nmol/l. results: 15 of 23 patients were found to respond to steroid medication, 8 did not. mean cortisol levels before and after corticotropin were 534 • 366 and 737 • 396 nmol/l in the responder group (rg) and 583 • and 907 • 301 nmol/l in the non responder group (nrg). in the rg 9 out of 15 (60%) were found to have an adrenal insufficiency, in the nrg 3 out of 8 (37%). in the rg 2-weeks mortality was 6.7% (l out of 15), the overall mortality 33% (5 out of 15). mortality in the nrg was 62% (5 out of 8) (p <0.01) and 75% (6 out of 8) (p <0.005) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam-1 and s-tnt levels continuously starting at the beginning of sepsis. methods: 19 patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within 24 hrs following the beginning of sepsis. blood was drawn every 4 hrs in the first ;~4 hrs, after 48 hrs, followed once per day for 7 days. s-tnt, icam-1, elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) 0.6 (0.0-1.66), 3.12 (2.4-12), 6.29 (0.0-15.3) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. .45 (0.06-7.6) 0.0003 13 patients had s-tnt levels >0.2pg/l. 11 of these died, whereas only 2 of 6 patients died with s-tnt values <0.2 pg/l (p=0.0296). all patients that died had elevated sjcam-1 levels (232 ilg/l:cut-off ) whereas in the survivor group only 50% had elevated icam-1 levels (p=0,043). conclusions: increased slcam-1 and s-tnt levels were found during early sepsis in the majority of patients, a high sicam-1 and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on 20 critically ill postoperative septic patients (their multiple organ failure score was 8-9/with icg monitor. in 9 cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during 3-72 hours in every 30 minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on 300 patients using previously defined criteria. methods: 300 normotensive patients with fever (temperature >38.0 ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of 7 days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients (69%) had or developed sirs within the first three days, 16 patients (5%) did not. sepsis was present in 25% at inclusion. in patients with sirs, 76% did not progress to sepsis or septic shock, 24% progressed to sepsis (mean interval 2.55 • 1.97 days), and 1 patient (<1%) directly progressed from sirs to septic shock. in patients with sepsis, 17% progressed to septic shock (mean interval 2.08 • 1.56 days). sepsis was preceded by sirs in 40%. septic shock was preceded by sepsis in 92% and by sirs in 8%. conclusions: 94% of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. 20 patients with double pneumonia were subjected to the evaluation. sh in the concentration of 600 mg/l in the volume of 400-800 m1/24 hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even 1 hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence 0.5-5 times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, '02, h202) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by 15% in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for 28 days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. 1987 , goris et al arch. surg. 1985 , kuaus et al ann. surg. 1985 colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca1 in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il-6 with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c4xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for 4-6 hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, 1o00 ng/ml lps was required to increase cgmp levels, whereas the presence of 10 % fbs shifted the lps concentration curve i00 times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (>80 ~ 30 min) but not removal of small molecules (<10,000 d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first 120 min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin 1-~ (il-i). 10% fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed >72 hours following admission to our icu, during a 10 year period ( january 83 through january 94) were reviewed. 30 patients (pts) were included, mean age 68,1y (sd 13,1), saps 2 35,9 (sd 11,1), mac cabe (1 and 2) 53%, mortality directly due to sepsis 30%. 16 pts had mrsa bacteremia and 14 methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age (71,8+ 4,8 vs 63,9+ 1,8) , saps2 (33,6+6,6 vs 38,2+14,1), use of vancomycin (94% vs 71%), mechanical ventilation (94% vs 100%), number of days (d) before the drawing of the first positive blood culture (median 20 d, range 7-150 d vs median 30 d, range 7-120 d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) (25 % vs 0% p<0,001), central venous catheter infection due to staph.aureus (62,5% vs 14% p<0,01), but previous use of antibiotics was not significantly different (37,5% vs 21%). the outcome of the bacteremic pts was not statistically different: saps 2 at the first day of bacteremia (33,6+_.7,2 vs 40,7+14,5), severe sepsis and septic shock (31% vs 28%), persistence of the bacteremia (43% vs 78%), mortality directly due to bacteremia (25% vs 45%). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio11992; 13:587-593) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of 62 cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of 5 dead cases had been analized. results: in 45 cases (72,6%) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in 36 cases (58%) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in 21 cases (34%) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of 5 dead cases (8%) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. 4owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. 3bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs (30 kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a 30 minute intravenous infusion )f escherichia colt (serotype 34h4113, sigma, st. louis, mo) was begun ~t a rate of 150 pg/kg. an infusion of either saline at 1.7 ml/kg/min (group ; n=5) or saline and dobutamine at a rate of 5 pg/kg/min (group ii; n=5) vas begun 30 mn after the end of the endotoxin infusion. tesults : to td0 1120 t180 ~ fl0w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: 20 patients with sepsis (criteria according to accp/sccm, 1992) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output<250 ml/8h, cr>2.5 mg/dl and bun>60 mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on 24, 48 and 72 hours. cytokines tnfa, i1-1 and ii-6 were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and 24 h solute mass transfer of tnf and i1-6 were calculated (v24 h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included 8 pts and group b 12 pts with mean+sd age (65 _+19 vs 64_+9, ns) and apache scores(24_+2 vs 24-+2.2, ns). the mean ta-+ sd was 3.6+2 vs 10-+6, p<0.05. the mean_+se morbidity period of group b vs group c was 20_+4 vs 8_+0.8 p<0.05 . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was 0.2 and for i1-6 was 0.25. the solute mass tranfer was 6-fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, 02 delivery (do2) and 02 consumption (c02). objectives: the main purpose of this article is to determine the relationship betwee~ 02 delivery an 02 consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d02 and v02. we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after 24 hours. high levels of do2 and v02 may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january 1989 and december 1994. specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of 372 patient there were 324 (87,09~) adults and 48 (12,9(3~) children. almost two thirds of the patients (238 -63,97~) were males. the predominantly cause (68,759~) of children's burns was scalding b~y hot liquids and flame burns ~63,97~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa (64"1796 in adult patients) and staphyloccocus epidermidis (78,57% in children). in only five patients (1,349~ 1 the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in 6,72% of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was 6,98~, but the mortality rate caused of sepsis was low (i 34%) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do 2 (vigilance ~ baxter), v02 (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t1) na infusion (0, 1 to 0, 19 hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp02) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in 19 patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il6 and il8 were determined every 8 hours for 3 days and on day 7 after fulfilling the criteria of ss and severe sepsis. results : in 9 pts, sepsis was caused by a gram (-) microorganism, in 6 pts by a gram (+) and in 4 pts no microorganism was identified. there were 12 survivors (63%) (s) and 7 non-survivors (37%) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il-6 were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl (546c88) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70mmhg) or the requirement for a noradrenaline (na) infusion >_ 0.i ]tg/kg/min with a map _< 90mmhg. cardiovascular support was limited to na _+ dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t = 1); and at the end of treatment (t = 8) with 546c88. conclusions: 546c88 can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. 546c88 is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t1); vl, dobu and na. results: in 5 patients with treated ss, 16 tests were performed (vl n=8; dobu n=4; na n=4 method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70 mmhg) or the requirement for a noradrenaline (na) infusion ~> 0.1 ~g/kg/min with a map _< 90mmhg. cardiovascular support was limited to na + dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either i, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at 1 h from the start of treatment (t = 1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. 546c88 is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc1 (546c88) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 [xg/kg/ min with a map _< 90 mmirlg. cardiovascular support was limited to na + dobutamine. 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = 0); at the end of treatment (t = 8); and 12 h after treatment (t = 20) with 546c88. results. -median values (* assessment made at 8 h or when 546c88 discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to 546c88. the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of 546c88 in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc1 (546cg8) given by continuous infusion for 8 h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 ~tg/kg/min with a map _< 90 mmhg. cardiovascular support was limited to na • dobutamine. 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. plasma was collected from each patient over a 24 h period and analysed for 546c88. pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/2 -plasma elimination halflife). conclusion: the pharmacokinetics of 546c88 in patients with septic shock are dose-independent at infusion rates up to 2.5 mg/kg/h. at higher rates, clearance of 546c88 decreases without any marked change in volume of distribution. 546c88 metabolism may be partially saturable at dose-rates above 2.5 mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc1 (546c88) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd-1 mice (25-35 g) were instrumented under gaseous anaesthesia (isofluorane, 2%) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after 24 h recovery, endotoxin administration (e. col• 026:b6, 6-12.5 mgkg -1 i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from 101 + 5 to 59 + 4 mmhg (n=5, p<0.05) at 12 h, with a survival rate at 24 h, 48 h and 72 h of 80%, 40% and 20% respectively. 546c88 administered as a 24 h continuous infusion (3 mgkg-th -t i.v., n=5), 4 h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from 105 + 2 to 70 + 3 mmhg (n=5) at 12 h, with an improved survival rate at 24 h, 48 h and 72 h of 100%, 100% and 60% respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using 546c88 represents a novel and promising treatment for septic shock. cultures of e.coli (19,5%) and candida(8, 3%) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia (23,7%cultures) and common e.coli(18,6%)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in 1994. objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of 12 months all surgical patients on icu were registrated prospectively, more than 270 parameters for each of them were documented'daily in a paradox file. results (see table 1): 20 of 286 patients fulfilled the criterion of a septic shock (r. bone, 1991 ) , 11 of them died at the 2 lth day, while the surviving group of patients stayed almost 51 days at icu. obiectives: to compare the effects of 6 and 10% pentastarch solutions to a human albumin solution on oxygen delivery (do2) in septic patients. methods: this stud}, included 41 septic patients with fever (t > 38~ tachycardia flqr > 90/rain), tachypnea (rr > 20/min) or mechanical ventilation, leukocytosis (wbc>12000/mm 3) or leukopcnla (wbc<400()/mm 3) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < 12 mmhg. patients were randomized to receive 400 ml of 4% albunun (n:i4), hydroxyethyl starch (hes -mw200/d.s. 0.5) 6% (n:14) or t0% (n=i3); 33 patients were also treated with adrenergic agents. results cardiac index (c1) increased significantly only in 10% lies (table) hemoglobin (hb) decreased significantly at 40 min in the same group. there was not significant change in oxygen delivery ( do2 ). baseline ci alb 3.7::1.3 (l'min/m 2) hes 6% 3.9=0.9 hes 10% 3. polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of 25 patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after 3 days ( day 1 = onset of septic shock ) in 68% of the patients pci was demonstrable. after 14 days 80% of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. 2. pci develops early in the course of illness ( 85% after 5 days ). 3. pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: 1) can serial measurements of vapco2 and avph also reflect the onset of tissue hypoxia during endotoxemia? 2) are whole body changes in vapco2 and avph associated with parallel changes in regional circulation? methods: in 12 anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo2). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n=6), group 2 (n--6) received 2 mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do2. results: systemic do2crit was higher (12.1_+2.2 vs 7.9+2.6 ml/kg.min, p <0.05) and critical oxygen extraction ratio (o2ererit) was lower (45.1+_9.7 vs 74.1+_9.1%, p<0.05) in the endotoxie than in the control group. meslenteric and femoral do2crit were higher in the endotoxic than in the control group (8.2_+2.5 vs 4.1_+0.5 mlll00 g tissue.min and 8.3_+2.3 vs 4.6_+0.9 ml/min, respectively, both p<0.05). regional o2ercrit were lower in we endotoxic than in the control group (mesenteric: 37.1_+15.4 vs 71.i_+7.4 %, renal: 30.7+ 24.6 vs 53.9-+28.7 % and femorah 48.1-+9.2 vs 75.3_+6.9 %, all p<0.05). vapco2 and avph followed a similar pattern in both groups, increasing slightly before do2crit was reached, but dramatically wh,en do2 fell below do2crit. in the presence like in the absence of endotoxin, systemic and regional do2crit calculated from vo2, from vapco2, or from avph were similar; do2crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do2crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco2 and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. 2) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco2 and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: 14 anesthetized dogs received endotoxin (2 mg/kg iv), followed 30 min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo2) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n=7) maintained low arterial pressure and systemic vascular resistance. the 7 dogs receiving a continuous infusion of 1,2,4 ~ug/kg.min of sin-1 starting 30 min following initial fluid resuscitation (each dose for 1 h), had a higher cardiac index (0.23-+0.03 vs 0.16_+0.08 l/min.kg, p<0.05) and lower systemic and pulmonary vascular resistance than the control group (1006-+170 vs 1460-+445 and 114-+38 vs 244-+84 dyne.sec/cm5, respectively, both p<0.05). arterial pressure and pulmonary artery pressure were not influenced. sin-1 significantly increased the mesenteric blood flow from 63-+17 to 148+49 % of baseline levels and renal blood flow from 67-+25 to 96-+24 % (both p<0.05) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo2, oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin-1 increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl0w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv-30g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) -6 il-8, and soluble (s) e-selectin were measured in both tcv-309 and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv 309 versus placebo (takeda chemical industries ltd., osaka, japan). tcv-309 was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock (1.0 mg/kg body weight intravenously over 2 hrs, twice a day for one week). day 28 mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of 29 patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining 28 patients 12 patients were randomised to receive tcv-309 and 16 to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il-6 and il-8, considered to reflect severity of disease, tended to be higher in tcv treated patients: 24.59. vs 2.37 ng/ml for il-6 (p.06) and 0.34 vs 0.04 ng/ml for il-8 (p.07) in tcv-309 and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day 7 i.e. the end of tcv treatment (mortality: 2 out of 12 in the tcv group and 7 out of 16 patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il-6 and plasma il-8 levels tended to decrease more rapidly in tcv treated patients compared to controls (p=.17 and p=.09, for il-6 and /l-8 respectively) conclusions: tcv-309 is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv-309 may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged 20-81y.) with verified septicemia had been included in this study. beginning from day 1 oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days 1-7. 4 patients, 2f and 2m died @u= ring their stay at the icu, 3 of them in less than 48h~urs: after admission. at the begin surviving patients showed olab values between 75-119%, non-survivors between 68-104%, com= pared to neopterin values of 38-75 m~ol/l in survivors and 20-250 ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in 10 consecutive children on day 1 after uncomplicated open heart surgery was compared to gtt in 10 children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o,4g/kg in a 10% aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within 480 minutes in 7/10. after closed heart surgery gtt was delayed 138(+-80) min vs. 56(+-20) compared to controls, but transit was achieved in all aptients within 285 rain. mean dopamine dose was 2,7mcg/kgmin after cpb and 4,8meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge13', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only 16 newborns, b.w. 2000 -4300 g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were 51 +12, and aado2 79,6 -+7,6 kpa. results. 4 newborns (25%) died shortly alter" admission or during transport without possibity to further is 5 newborns (31,3%) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in 6 newborns (37,9%) and 3 of them survived the mortality rate of group mentioned was 44%. conclusion. the fact that in the czech republic (10 mil. population with natality rate of about 100.000 newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: 8urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, 11-6, ltb 4 and thromboxane b 2release in whole blood: each of 1.5 ml hepariaised blood of healthy adult volunteers was iucubated lbr 2 hears with eillier ill ug/ml lps, 1 mg/ml b&,ine smfactant (alveulhct/themae), 1 rag/nil artificial surfactant (exosurf/wellcome), 10 og lps + i mg/ml bovine surfactant or i0 ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. '111e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of 1.5 ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr 2 hours with eilher 10 aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or 10 ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of 11-6 but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with 1.1~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b2) and the lipooxygenase (lencotrieae b4) pathways. conclusion: we cooclude: 1) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. 2) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. 3) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope2, tcpco 2 or san 2 leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo 2 and 1 lb content, capillary flow rate and oxygen uptake rate. aim of the study: 1 ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. 2) to investigate the possibility of obtainiug infonnations about the local 02nptake and the venous as well as arterial 02 satnration. patients and methods:we investigaled 16 non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope2, tcpco 2 and san 2 were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded 1000 spectres within 2 minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo 2 vahms were sorted by a software package. on the assumption that the low values correspond to the tibo 2 value of the veaons ends of the capillaries and the high values to those of the arterial ends we 2) it is possible to gel infonnations about the local o2-uptake and arterial 02 saturation ia the peripheral tissue by photoslg'ctroscopy. 3) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from(27-32)ws.continnous positive air way pressure was applied to six cases at peep level from (3-6)cm h2o through nasal pronge,(group i),the other 8 cases were managed as routine,(group ii).blood gases, tcpo2,tcco2,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among (83.3%)of cases,while (16.7%)of cases need imv.the cases of group ii need imv among (75%)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel 2, the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht 2department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all (10) pediatric intensive care units (icus) in the netherlands (6 tertairy and 4 non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality 0aenee, standardized mortality rate). efficiency was determined by 2 objective criteria (mortality risk > 1% or administration of at least 1 icu-dependent therapy)'. 1063 consecutive admissions aged < 18 years old were included. overall, observed and expected mortality were in good agreement (p > 0.5). between hospitals, crude mortality showed wide variations (mean 7.1%, range 1-10%). however, in each center, observed and expected mortality were similar (mean ratio 0.99, range 0.8-1.5). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was 72%, however large variations between units were found (range: 22-95 %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the 14-bed neonatal intensive care unit of a university hospital material and method: a total of 209 consecutive outbem newborns admitted to nlod from 01,10.1991 to 31.,19.1992 were enrolled to the study. babies who died or were discharged from the unit within 48 hours of treatment were excluded from the study, mean birth weight was 2040g (+/-820r), mean gestatational age was 35 weeks (+/-3.5 wks), mean age at the admission was 50 h (+/-110hi. multiple (~_2j organ system failure occurred jn 38.3% of babies at the admission./~tertal lactates were measure/at the admission, among 22 -26 hour and 46 -50 hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was 68.4%, mean length of nicu stay for survivors was 17.8 days (+/-15.1 day). we found high lactate levels at the admission in 82.8% babies (~9.2% with levels above 5.0 retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of 23 pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of 5 years was recorded in 9 patients (39.1%), 6 patients (26.1%) reported a 2-3 year remission and 8 patients (34.8%) had a remission lasting less than 12 months before they became pregnant. results: seven patients (30.4%) developed medium attacks in the second half of pregnancy, four patients (17.4%) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first 16-18 weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used 132adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during 1-2 weeks. a significant portion of pregnant women with b.a. (78%) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight (2800-2500 gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about 25% of newborns weighing below 1500 g who are born before 32 weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in 9 animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i0-ml and 25-ml pump volume. the right sided pumps are 10 % smaller for biventricular use. between february 1994 and may 1995 we implanted this assist system in 6 children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in 3 infant. patient n.l: 4 kg, 7 lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl 4 mcg/kg/h, hypocapnia of 27 mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at 10 ppm, and the gas aoalyser for no and no2 (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/3 of systemic was obtained with a dramalic improvement. no was continued at 5 ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu 5 days after. patient n.2 : 4.5 kg, 6 monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was 76% wilh a pap at 75% of systemic. two hours of c01wenlional therapy failed 1o improve ihc patient and no administration was slarled at 10 ppm. so2 dramatically incrcased to 95%, but the pap dropped only to 50% of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at 5 ppm in the following 6 days. she was extubaled 8 days after surgery and discharged from the icu 20 days after. patient n.3:12 kg, 3 3'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than 7 mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after 1 h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after 4 d;b's and disclmrged from the icu after 15 days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case 1: severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy 13 month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day 4 of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after 17 days (420 hours) -the patient was extubated 7 weeks later. case 2: tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a 4 month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, 5 days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced 4 days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis (4 days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further 3 days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died 9 months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: 1) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. 2) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to 23 near term neonates with severe persistent pphn, oxygenation index > 25 and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the 23 patients studied responded to the initial trial of 1no (20ppm x 20 minutes), as defined by a greater than 20 percent improvement in pad2 as well as a fall in the el to < 40. these 21 patients were continued on ino therapy, with 3 patterns of response emerging: pattern 1 babies (n=8) continued to show a sustained response to ino and were successfully weaned from it within 5 days -all survived. pattern 2 babies (n=9) failed to sustain their response to ino over 24 hours, as definded by a rise in the el > 40. six survived, five with ecmo. pattern 3 babies (n=3) had a sustained dependence on ino for 3 -6 weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern 3) not only required ino for longer periods of time than did the sustained responders (pattern 1), but also required significantly higher doses of ino we report on the air transport of 32 paediatric intensive care patients. these transports fall into three categories: 1) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = 12). one patient was transferred on ind. mean transfer time 2.2 hours (se +0.6hrs). 2) long distance international transport using chartered aircraft (n = 11). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time 4.4 hours (se +0.4hrs) 3) long distance international transport using commercial aircraft (n = 9). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time 14 hours (se _+1 .fhrs, longest 24 hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; 1) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication 2) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. 3) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials (2 hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_.110 children,asa i-if,median age 6 years,range 9-13 who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( 25 children,age 0 -3 yrs,ketamine 2,5 mg/kg, diazepam 3-6 mg total dose ) or per os ( 85 children,ketamine 5-7 mg/kg,diazepam 0,3 mg/kg or midazclam 0,4 -0,5 mg/kg ) _resu_l_t_s. 20 -25 minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted 30 -60 minutes and allowed adequate needle emg and nerve conduction investigation. 11 children recieved additional 0,6 -1,0 vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad2 by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was 88 neonates admitted during the years 1990 to 1992, with gestational age _< 32 weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo2) <0.22), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo 2 before the administration of the 1st dose of surfeclant, delivery, apgar score at 5min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x 2 and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was 31.8%. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo 2 before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every 2 weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: 1.92, 95% c1:1.14, 3.22). for every 0.02 on average decrease of a/apo 2 before surfactant administration the risk of p-ivh in the neonates was 1.27 times higher (95% ci: 1.02, 1.58). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: 0.10, 95% ci: 0.01, 0.82). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than 24 hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of 103 (51 boys, 52 girls) accidental ingestions of foreign bodies in children (mean age : 2.8 years, range : 7 months-10 years). no child had ingested more than 1 foreign object. the majority of the ingested foreign bodies were : coins (n : 44), toy parts (n : 11 ), jewellery (n : 3), batteries (n : 16), "sharp" materials such as needles and pins (n : 21), "large" amounts of food (n : 8). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. 28/103 patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only 4 children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : 3 hours, range 20 min. -28 hours). chest and/or abdominal x-ray was performed as first-line investigation ( 93/103 objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in 6 children. in 87/103 the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from 0.5 to 13 years. methods: protocol n1 consisted of intravenous administration of diphtheria antitoxic serum, prednisolone (2 mg/kg bw/day), plasmapheresis and supportive care. protocol n2 included the use of antitoxic serum against the background of high-dose dexasone (2-3 mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n1 consisted of 17 children with malignant diphtheria, 11 of them with severe malignant diphtheria (grade 2 and 3). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all 11 patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the 11 children in the group died seven. the children of the second group were treated according to the protocol n2. out of total of 22 patients of this group. 11 patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of 22 children admitted to the trial 21 survived, one child with malignant diphtheria of grade 3 and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m611er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. 31-35, 23538 l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a 18 months period 10 non-neonatal pediatric ards patients (age 1-38 months) in a "pre-ecmo"-situation (oi 40 over 4 h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia (5), sepsis (2), immunosuppression (1), near drowning (1), neurogenous ards (1). a total of 20-120 mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. 4 hrs. if there was no improvement of the oi by at least 10, further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of 1 mg/kg in 2 doses. patients with no benefit (oi remained unchanged or increased within the max. 2-4 hrs) were taken on ecmo. results: nine patients improved within 4 hours after surfactant therapy: the oi decreased from a level of 41 (mean, range 22-100) before our treatment to a level of 16 (mean, range 6-60) thereafter. in 6 patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within 3-10 days. the other 3 patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than 3 organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after 10 days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other 3 death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient 1: in a boy with down's syndrome an avsd had been surgically corrected at 11 month of age. at 5,6 yr of age a catheter examination revealed a pulmonary vascular resistance of 70 % of systemic vascular resistance in room air and at an fin2 of 1.0. prostacyclin (0.5 mcg/ml) was administered with a jet nebulizer at an fin2 of 0.21. pvr declined to 0.4 systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion (5 ng/kg rain) had to be stopped after 5 minutes because of systemic arterial hypotension. patient 2: a 8 month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing 60 % oxygen via face mask pao2 was 37 mmhg, arterial ph was 7.35. systolic arterial pressure was 85 mmhg, a rv-ra gradient of 100 mmhg was measured by cw-doppler. while fio2 was maintained aerosolized prostacyclin was administered over 30 minutes. rv-ra gradient was 70 mmhg, systemic blood pressure 75 mmhg, pao2 58 mmhg. two hours later nitric oxide (20 ppm) was inhaled at an fio2 of (3,6. rv-ra gradient declined from 100 to 65 mmhg, systemic systolic blood pressure remained stable at 105 mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient 2 the poor response to inhaled pgi1 compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years (1993-94), 51 infants weighing less than 1000gr. admitted to our referral unit. thirty four of them (67%) survived, (28% of infants weighing 500-700g and 78% of infants weighing 701-1000gr survived) for the years 1986-87-88 the survival of these infants was 53% and for the years 1976-77-78, 14% (p<0.01). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors (1) to neonatal deaths (2) shows: gestational age: 27.6 w (1) to 26.4 w (2) (s). birth weight: 923.5g (1) to 724.7 (2) (s). apgar score: 7,90 (1) to 7.56 (2) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of 8 weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants 32.3",,(1) to 95% (2) (s) use of surfactant: 70% (1) to 95% (2). bpd observed in 62% of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in 20% of the mothers. in conclusion: 1) a great improvement in the survival rate observed in these infants the last 20 years in our unit. 2) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated 19 healthy preterms less then 2 weeks postnatal age (gestational age 315 +_ 2.1 wks; x + sd, weight 1653 +_ 370 g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair .0.4 + twall 0.6). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress (15rain). ii) insulation increased by a cap (30 min), iii) 30 min after its removal, iiii) increased operant temperature by 1.6 + 0.5~ (60min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded (6) or diagnosticated an upper pouch fistula (1). in patients who suspected "h" fistula (2) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july 1992 14 newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in 1 case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible 3-4 french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients (30 primary anastomosis, 2 resections of tef, 6 oesophagocoloplasty and 1 died with gastrooesofagostomy). the late mortality 7.7% (3) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il-1, il-6) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, 18 children (9 months-16 years) were admitted in our picu in 1994 with the following diagnosis: bacteriemia (4) sepsis (4) and septic shock (10) according to jacob's r f criteria. measurements: il-1, il-6, tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, 12, 24, 48 hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il-2, il-6 and tne high levels of tnf and il-6 are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd (77%). in 10 patients pd lasted less then one week and there were no complications; in 3 patients it lasted 13 -29 days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. 5 patients (38%) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles (5%, 10%, 50~ 90%, 95%) of the most important pulmonary parameters were determined non-parametrically in 6 weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of 300-600 healthy newborns ageing between 1-28 days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised 100% scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on 14 routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first 24 hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. 1988; 16:1110 -1116 . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of 33 pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during 1 year period. as the validation study started on july 94, data collection is still on going and validation analyses will be carried out on july 95. up to now 23 centers recruited 1116 cases. at present, characteristics of the sample recruited are the following: most of the patients were male (62%); the mean age is 3 years with 30% of patiens having less than 30 days; more than half were medical cases (59%) admitted from emergency room or from hospital floor (51%); 52% cases were admitted with an organ failure while 48% to be intensively monitored. icu-mortality was l 1%. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from 1528 consecutive paediatdc icu admissions over a 6 year pedod. a proportional weight (pwt) was calculated as a percentage from the 50th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes 0nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors (6.8kg and 8.3kg medians p = 0 63) a lower pwt (85% and 90% medians p = 0.6"0. the incidence of malnutdtion , in our icu population was 33%. the mortality of manoudshed patients was' significantly increased (p = 0.001), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from 0.72 to 0.79). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample 0.82, az validation sample 0.81). the modified formula is: legit = -2.864 +0.134*prism score -0.006*age + 0.463*weight category, where the probability of mortality is exp(iog/t)/1 + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to 40 ppm inhaled no in 12 patients(9 m, 3 f, median age 4.5 months, range 1day to 17years) with signs of increased pulmonary vascular resistance, there were 5 pre and 7 postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a 20% reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + 12 mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders(7/12) were characterised by left to right shunts or pulmonary venous hypertension(4/7). patient#11 was weaned from ecmo with inhaled no. patient#2, without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient#1 had a pulmonary hypertensive crisis which responded to no. all non-responders(5/12) had evidence of anatomic obstruction to pulmonary blood flow (#7,10,12)or a low pvr(#8) on subsequent cardiac catheterisation. in patient #3, lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these 18 years, the incidence of sdra was 1.3% of the total of admissions. the most common etiology was meningococcic septic shock. since 1989, there is a decrease of its incidence. (from 24% to 11%) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was 2,7 years (61% males, 39% females), total mortality by sdra was 59% and there is an increase up to 72% since 1989 mean time of stay of the dead was 4,3 days and 12,4 days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de1 roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: 60 cases of severe traumatism in children admitted to our icu in the period from january 1990 to june 1990 were reviewed. age of patient ranged from 4 months to 9 years, 65% were males. in our series, 53% of cases suffered traumatism due to a traffic collision and 38% had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in 76% of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: 75% of our patients suffered cranioencephalic traumadsm (ct); in 53 % it was an isolated picture and in 22 % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in 16 % of cases. 10% of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was 3.3%. in 8.3% important sequelae remained. all of these patients presented tepas on admission equal or lower than 4. 16% of those with traumatises had slight sequelae. 71.6% of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation 20% of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a 14 year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only 30% in spite of a ventilation of 70% of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of 20 ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of 10 ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m 40 to 32 nun h20 and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to 70ca/of the total pulmonary perfusion. after 3 days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a 14 year old girl. results: a total of 11 children with ards were treated with bovine surfactant (alveofact| 9 cases were evalable. the median age was 1.3 years (range 2 weeks to 4,9 years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of 22 clays (range 3-68 days) after first symptoms of ards witha median doseof 84 mg/ kg (range 42-133mg/kg). in 9 patients 28 doses of surfactant were applied. during the hour before therapy, the median pao2 / fio2 -ratio was 70-65. within 30 min. after application of exogenous surfactant the pao2 / fio2 -ratio increased to 90 with successive decrease over a period of 8 hours to 75. accordingly, an increase in pao2 and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. 2 of 9 treated patients survived (3 of the 11, respectively). 13 of the 28 surfactant doses were applied in the 2 surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of 8-12 hours. the effect often could repeatedly reproduced, in one case after 11 applications. the increase in oxygenation often allowed the reduction of fio2 and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency 22 days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with 3 patients detected antenatally and another diagnosed at 2 months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a 10 year period 13 patients under 14 y of age fullfilled diagnostic criteria; 3 were detected prenatally (25-34 weeks) and one was diagnosed at age 2 mo. the 3 fetuses had intermitent svt during gestation. all 3 of them had pjrt in the first month of life at rates between 180 and 240 bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in 2 to 20 days. one was in sinus rhytm until age 4y; he then showed persistent pjrt over 70% of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over 90% of the day (follow-up 2ran and 4 y). the 2 mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction 15%) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to 185; rfca was performed at 3 m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient (15 to 35 see) recovery of sinus rhytm. ff,s162 clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients (5%) died. these were 4 meningitis, 2 head injury, 2 sub-arachnoid bleeds, 1 status epileptieus, 1 leukaemie, 1 drowning, and 1 multiple trauma. calculated from the a 2 admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from 0-100%. of the 12 deaths, i 1 were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p 43% & 73%) survived. the median length of stay was 2 days (range 1-34 days). 98 patlents(50%) received ventilatn~ support and 10 patienta(4%) were transferred to specialist units (5 neurosciences, 3 liver, 1 cardiac, 1 bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths (9 of 12) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, 8m and 2y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was 215 and 225 bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of 34~ there was a gradual decrease in heart rate in the following hours (130-150bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in 48 to 72 hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september 1 to december 31, 1994 a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n=9) were treated with et. in total 15 newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than 24 hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was 164.3 ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of 26 newborns, treated for culture-positive sepsis in the same icu during the first eight months in 1994. students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: 1 death of 15 cases during the study period, compared to 9 deaths among the 26 controls (p<0.05). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o.05) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl 7 years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant 6 months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a 17 years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for 7 days. a significant increase of granulocytes was ever noted already after the second administration until 24 hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, 18 months old, submitted to a neurosurgical interventiol after 2 days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after 2 days an intrathecal administration of vancomyein 40 m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january '95 till april '95, all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied (19 females, 8 males)i out of which 7 doctors, 7 residents and 13 nurses. it was considered sign!ficant as mild bo, a score between 3-4 and as severe, the score of bo >. 4. results: 10 subjects (37%) resulted positive for bo, out of which 8 were females (80%) and 2 were males (20%). the subjects with mild bo were 7/10:1 was a doctor, 3 residents and 3 nurses. the subjects with severe bo were 3/10, out of which 1 resident and 2 nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses (5 subjects), as well as residents (4 subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized 99mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of 9~tc-dtpa from the lung. methods: three groups of 5 white sealand rabbits (bw 3.5 kg) were anesthetized, tracheotomized and paralyzed. 2 groups were ventilated for six hours at pressure regulated volume control, set to deliver 10 ml/kg with a frequency of 30/rain, i/e ratio = 1:2 and peep = 3 cm hzo using a modified servo 300 ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either 0/25 or 20/25 [no (ppm) / fioz]. after six hours of ventilation in these 2 groups and immediately after anesthesia in group 3 (control), 99~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for 20 minutes, monoexponential curves were fitted to the data and the clearance half-time (t 89 was calculated. the t~/2 mean • sd of the different groups were: t~a (mean 4-sd) h"e,i witl~ arf 1:14 di.ff:erent kinds, aged .q-ore 2 mon't.hes to [4 gears o11:1 (bodi weight .~rom 4.,5 to 45 kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se...14~ 2.ards'-8; :~,;,,arf o~ ::entral genes:i s .-3,0,~ :inc lud ing men ingeenceph 1 it :is-3~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.."5). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba'i~ier was. about 4. tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to 25 dayns.,~ <6.-:in 351, 6"t2-irl lo;and>12 davs'-in 6'l~atierr~{s i'i"ai3s:ltiol~ o4; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to 16-17 ml~ar, fi02 ~ecreased to 0,4. lind less with 5a02=901/,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th 2f'f'v, teoph :i. :1. l:i.r~ (is-24.mg/kg/day), g lucecdr t icostei~oids (2 .... :~;mg/kg/day), when r exceeded in 2,2-.];,4 times normal va i tea the e aqes/,'!:l"oln 2~j,, ite :i.~;::.!;, 8 ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom !1.20 2'.' 26i', 8 to !..';51,0-106, 1 mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i.2e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl 9 of: 1. health children (asa i, ii) facing single uncomplicated surgical elective procedures 2. various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones 3. unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods 2-50 ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure >50% systemic pressure, critically depressed right, ventricular function or an oxygenation index >10. assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in 29 situations (19 patients, age 3 days-71,1 years), ino was applied 0-628 h postoperatively. oxygenation was improved in 13 situations from 114_+51 to 171+53 mmhg pc2; pulmonary pressure was reduced in 17 situations from 70-*22% to 34_+17% of systemic pressure. in 7 situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume +39-*12%, cardiac output +20-*11%). in 8 situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n=4], multi-organ-failure [n=4]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under 12 months of life belonging to asa group 1. they all had been anaesthetised in 1985.95 in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in 2350 cases we tbund: lafingospasm during induction in anaesthesia (0,5%), bronchospasm during induction in anaesthesia (0,22%), impaired intubation (0,1~ postanaesthetic laringospasm (0,1%), supposed aspiration (0,04%),postanaesthetic inspiratory stridor (0,05%), postinductional inngoedema (0,03%), death after 4 months in consequative of infection pneumonie (0,12%), none of these disorders was correlated the prematurity, 3 infants suffered of post anaesthetic apnea, 2 of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi 2 lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi 2 could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb 100 ) were investigated dudng mechanical ventilation over a pedod of 24 hours. 20 minipigs with hemorrhagic shock were intubated and ventilated for 5 days in an animal intensive care unit (icu). after 24 hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after 24 hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of 24 hours 3 of 4 types of filters showed an increase in weight of 10 + 6% and airway pressure. bactedal celonisation 0ccured in nearly all fillers (93 of 100) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: 21 patients in respiratory failure, age range 3 weeks to 15 years in a total of 29 patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from 4 hours to 8 days. indications for use ef the device were: 1) to facilitate weaning from ippv 2) prevent reintubation of patients following unsuccessful extubation, and 3) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao2:fio2 ratio after cnp and eov-nb (p <0.0001, and p=0.01 respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco2 both with eov-nb and cnp (p=0.02 and p=0.01 respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p<0.0001) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao2:fio2, the fall in paco2 and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b 7 the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all 52 indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: 70 women with ~h-gestosis (i group) and 48 somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was 94% in the ist group and 90% in the iind group; gaseous metabolism -91% and 86%, respectively; central hemodynamics was 87~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, (3 males and 2 females; mean age 41.6• were enrolled in the study. all patients presented criteria for pneumonia with pao2/fio2 ratio ~ 100 and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = 16 em h20; peep = 5 emh20; trigger = -lemh20) continuously admires" tered ( 10' before fob fio2 = .7; during fob, fio2 =1 and for 90' alter fob, fio2 = 0.7). pao2/fio2 ratio as well as 02 saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis (2), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of 113 consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a 12 months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first 24 hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: 113 mechanically ventilated patients, 76 male and 37 female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia (2), ards (2) and septic shock (1); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: 43 consecutive patients with severe ards (murray-score > 2,5; pao2/ fit 2 < 160 mmhg; 29 male, 14 female, mean age 62 years) were conventionally ventilated (pcv, peep 6-16 mbar, i:e=i:i, ppeak < 30 mbar). if after 24 hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every 12 hours. beside ultimate survival, parameters investigated were aado2, pao2/fio2, and venous admixture (qs/qt). results: during the first 12 hours in prone position 39 of 43 patients showed a significant decrease in qs/qt (25.3% vs. 17.8%) and aado 2 (235 vs. 187 mmhg), and an increase in pao2/fio2 (151 vs. 201 mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than 48 hours before first application of prone position. after an average of 6 position changes (2 to 16) 28 of 43 patients could be weaned from the ventilator. 22 patient could leave tile hospital. i11 the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co2, and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the 40% of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < 20 mbar, ppeak < 30 mbar) pulmonary function does not improve within 12 -24 hours prone position should be applied. when after 2 -3 position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed 100% 02 for 60 h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged 16,g • 10 days in patients with ardsvs 1,7 _+ l,4 days in control . mean staylcuwas lg _+ 10,g days in the ards group vs 4,9 • 2,7 days in control group postoperative mortality rate was 53% in ards patients vs 5,7% in those without respiratory failure. 1-ards incidence in liver transplantation is low (11,2% in our sene) but it causes high mortality (53%) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (<15kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio2 of 0.6, peep of 5 cm h20, imv of 6 bpm, it of 50%, and tv of 16 ml/kg), sheep weighing 58.9 ~ 8.3) kg had lung injury induced by instilling 2 ml/kg of 0.05n hc1 into the trachea. ten minutes after injury, sheep with pao2<100 ton" were randomized to continue gas ventilation (control, n=9) or to institute page (n=9). page was instituted by instilling 1.6 l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, 10 minutes after injury, 30 minutes after injury, and then every 30 minutes for 4 hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age 71_+2y;fev~ 0.98_+0.12l) and 11 ards patients (age 57_+5; lis 2.8_+0.1) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao2, svo2, hb and methb were measured (abl 500,osm3). mean intratracheal concentrations of no and no2 were continuously monitored using a chemiluminescence analyzer (nox 2000) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no 20 ppm, no 30 ppm and basal2 . after a steady state of 20 rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a 20% increment in pao 2. results: papm was similar in both groups and decreased significantly after no (ards, basal 33.6_+9.7 mmhg, no 29.7 +6.7 mmhg, p <0.01) (copd, basal 27.8_+6.3 mmhg, no-10 24.4_+5.3 nrmhg, p<0.01). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio 2 189_+53 mmhg) vs ards group (paojfio 2 100_+40 mmhg)(p<0.01). after no-10, pao2 increased (69_+20 mmhg to 97_+40 mmhg, p<0.01) and qs/qt decreased (37+11% to 31_+10%, p<0.01) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao2/fio2 was not correlated with basal paoflfio2. no responders were 8/11 (73 %) in ards group and 2/10 (20%) in copd group (p<0.05). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis 95/1390. objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest 1994; 105:219-223) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co2 elimination in different clinical conditions. methods: we have studied 37 adult patients (8 controls, 11 acute respiratory failure, 9 severe ards and 9 copd) mechalfically ventilated (servo 300 and 900c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco2/vt] =facoz, vco2 vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of 5% variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at 3 levels of vt (0.3, 0.5 and 0.8 l) and four levels of peep (0, 5, 10 and 15 cmh20 objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied 3 groups of subjects. n: 8 normal subjects scheduled for minor surgery; arf: 9 critically ill patients with mild acute respiratory failure; ards: 8 patients with early ards (< 72 h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne (0.5l) a peep level of 0,5,10 and 15 cmh20 was applied in all patients. we calculated ers (cmh20/l), rmin, c~rrs (cmh20/l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co2 slope beyond vae (sipco2 in contrast to synthetic surfactant natural suffactants (alveofact|174 are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe 35, 6020 innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days (1, 2). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated 7 ventilated patients with severe ards (entry criterias: murray score > 2,5) in the ct scann of the university hospital. after a stabilisation period of 30 minutes in supine position a thoracic ct scan slice 1 cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) 60 degree of left lateral position, b) 60 degree of right lateral position. each lateral position was held for 20 minutes. at the end of each of these periods a thoracic ct scan slice 1 cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined 3 compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) (3). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: 45%; l: 65%) as well as a significant decrease of the noninflated compartment (s: 34%, l: 12%) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: 45%, l: 26%) whereas the noninflated compartment increased significantly (s: 34%, l: 51%). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within 20 minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who required no inhalation with a dose of 5 ppm for at least 5 days. entry criteria for no injaalafioa were murray score >i 2.5 aud pat/fie 2 < 125 nun hg with peep >~ 8 em i~o for at least 24 hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox 4000, see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least 20 % and was stopped when the change in pao2/fio ~ was below 20% or when the patient presented a paojf02 > 150 mm hg a~er 30 minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao2/fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was 60.1 +_ 10.2 years and mean lung injury score was 3.3 • 0.2. mortality was 60 % (6/10), metahemoglobinemia 1.1 • 0.2 %, and no2 concentrations zero. paojf~o 2 always improved significantly al~er 5 ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > 2.5, and a pao2/f~o 2 < 100 with peep ~8 cm h20 for at least 24 h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after 60 minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were 6 men and 3 women with a mean age of 54.2 years (21-71) and mortality was 55 % (5/9). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied 14 patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to 35 emh20 with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr 1975; 11:709-28) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a 100 ml v r with a respiratory rate of 20/min and i/e ratio 1:4; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who were treated with no (5 ppm). the onta'y criteria for no inhalation were murray score >/2.5 and paojfo z < 125 mm fig and peep >/8 cm i~o for at least 24 hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between 6 and 10 ml&g, with constant inspiratory flow, respiratory rate was 15-25/rain, and i/e ratio between 1:2 to 1:3. all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox 4000, see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo 2 above 20 % was observed after the inhalation of 5 ppm no (group r) . when the cha~age in paojfi0 z was below 20 % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ 10%) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. 37 patients (age:24-62 years; 22 males) with no serious complications al~er mitral or multiple valves replacements and with more than 15 hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head #2; differential pressure transducer pm-131-tc "statham" w amplifier "kistler 7251") and flow-volume recording of forced expiration (fleish head #4) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on 2 (2 nay) and 3 (3 day) days. airways specific conductance (sg aw) has been calculated as a mean of 7-10 consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at 50 and 25% of vc on 3-4 f-v curves (mef .~0, mef 25) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of 51 patients (72 • 10 years) presenting with acute exacerbation of copd (fevi: 610 • 396 ml, paco2:62 • 17, ph: 7.33 • 0.08) and nonmvasively ventilated (pressure support through a full-face mask) between april 1990 and may 1994 twenty-seven (53%) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while 24 (47%) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to 35 respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score (15 • 3 vs 11 • 3, p = 0.0003), creatinine serum concentration (122 • 45 vs 86 • 25 gm/l, p = 0.005), blood urea nitrogen (bun : 12 • 6 vs 8 9 3 mm/l, p = 0.009), age (75 • 9 vs 69 • 10, p = 0.01) were higher and encephalopathy (71 vs 30%, p = 0.005) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = 4, p = 0.001) older than 65 years (or = 4, p = 0.04) and presenting with bun >_ 10 mmyl (or = 3, p = 0.01) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco2, pao2, fev1) were unable to predict tile failure of niv. conclusion : copd patients older than 65 years, presenting with acute exacerbation, encephalopathy and bun > 10 ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december 1994 we studied 30 pa_ timnts,25 males and 5 females(mean age 68+/-5);18 of the se had emphysema,lo chronic bronchitis,2 dilatative car diomyopatia,with tracheostomy and emphysema.mean pac02 at admission in icu was 95+/-8mmhg,while when weaningbegan, 60+/-5.mean autopeep was 8 cmh20(4-12).all patients were ventilated in crpv as long as four hours to calculate st8 tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep 7cmh20 objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within 12 h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond 12 hours after trauma), is more insidious. the clinical and radiographic picture has to clear within 24 to 48 hours. (1) methods: all headtrauma patients admitted from january 1 to december 31, 1993 in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao2-fio2 ratio < 250. results: 151 neurotrauma patients were admitted during 1993.94 patients (63%) presented with severe head injury (gcs<8), 42 patients (27.8%) with moderate (gcs 8-12) and 15 patients (9.9%) with minor head injury (gcs 12-15). overall mortulity was 19.2% early (within 12 h. after trauma) and delayed onset respiratory incidents were distinguished, counting for 29 (19.2%), respectively 27 patients (17.8%), 7 patients (4.6%) had early and late respiratory complications. early respiratory insufficiency was caused in 9 patients (25.0%) by aspiration, in 11 patients (30.1%) by lung contusion, in 1 patient (2.7%) by fat embolism and in 15 patients (41%) by npe. in the late onset group 31 patients (91.2%) presented with pneumonia, 1 (3.0%) with fat embolism and 2 (5.8%) with npe. the npe group, 17 patients, presented as follows: 15 patients (88.2%) developed early npe, and 2 (11.8%) delayed onset npe. 9 patients (53%) died within the first days after admission, showing high mortality. gcs was less than 8 in 16 patients (94.1%), indicating severity of head injuries. conclusions: high incidence of arf with various etiology (41,7~ was found in this population. in about 10% of all admitted hcadtrauma patients (26,9% of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of 0 em h20 with demand-flow continuous positive airway pressure (cpap) 5 cm h20. after a minimum of 30 minutes on the above sehiogs: gastric intramucosai pc'o2, abg, and a p0.1 were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for 24 hours for any reason. results: twenty patients met criteria and were studied over one month period in october 1994. six of the twenty patients (30%) failed weaning. the mean and standard deviation is outlined in failure 7.01+/-0.10 6.8+/-3.9 74.3+/-43.3 87.5+/-43.6 comparison between roc areas shows phi and p0.1 to each show a statistically significant difference from an area of 0.5 (p<o.05). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #'2. results. 9 of the 21 newborns referred to us for ecmo-therapy developed barotrauma, 6 of the 9 required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates 36, d-35392 giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in 16 ards patients mechanically ventilated for 24 -96 h (mean murray lung injury score [1] 2.75 + 0.05). patients were randomized to receive either first no and then pgi2 (n=8), or vice versa (n=8), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi2 were compared to pro-and past-challenge values. no (mean dose 17.8 + 2.7 ppm) increased the mean oxygenation index (pao2/fio2) from 115 + 12 to 144 + 15 mmi-ig (p < 0.01) and reduced the shunt-flow from 33.1 + 3.6 to 26.6 5:4.5 % (p < 0.05). aernselized pgi2 (mean dose 7.5 + 2.5" ng/kg rain) augmented pao2/fio2 from 114 + 12 to 135 + 12 mmhg (p < 0.01), and decreased shunt from 33.5 + 3.8 to 26.0 + 3.9 % (p < 0.05). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in 10 patients, both no and pgi2 caused an increase in pao2/fio2 by at least 10 rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from 34.8 5:2.2 to 33.0 5:1.8 mmhg in response to no, and from 35.0 5:2.2 to 31.9 5:1.7 mmhg (p < 0.05) in response to pgi2. cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co2-removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since 1989, 47 patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf4, pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: 1) in mean, platelets dropped from 305/nl to 76/nl within 60 h after onset of ela. 2) tat levels were already elevated before ela start (55 ug/]) and demonstrated an additional peak 2 h after onset of ela (up to 240 ug/[). 3) in parallel, there was a transient peak of pai-i levels (from 5 to 75 au/ml} 2 h after onset of ela. 4) in mean, fibrinogen levels dropped from 470 to 310 mg/100ml within 24 h after onset of ela. 5) in mean, cl-inhibitor levels dropped significantly from 132% to 115% after onset of ela and reached the initial levels within 48 h. 6) after membrane change, there were significant changes for c1-inhibitor, fibrin-d-dimers and tt. 7) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: 22 patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for 20 seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep (5-10-15-10-5 cm h20) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was -30 _+ 3 mi (x _+ se) or a -5.2 _+ 0.6 % error. increasing peep from 5 to 15 cm h20 induced a 511 + 93 ml change in eelv. a difference of 18 + 12 ml (3.5 % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was 187 _+ 69 ml/120 min (fig.i) . validation of calibration showed a mean error of 1 -+ 1.78 % after 120 rain. the new rip had a higher drift when cold (cold 28 + 10 ml/30 min vs warm 3 _+ 1 ml/30 min), whereas the old rip was thermally stable (cold -3 + 3 ml/30 rain vs warm i + 1 ml/30 min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo 2) values is higher than suspected. objectives: determine the incidence, the significance and the pro9nosis value of those differences,if they exist,in severe head injured-patients. material and methods: 35 severe head-injured patients, coma glasgow scale (gcs) 8, with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than 12 hours, were studied. data from bilateral sjo 2 were obtained simultaneously, every 6 hours. discrepancies were considered significant, when differences in sjo 2 were > 10%. no chan9es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: 30 men and 5 women were studied, aged 32• yrs. at arrival at hospital, gcs were < 5 in 20 and ) 5 in to. the incidence of high icp() 20 mmhg) were 7sz at the entry. the mean therapy index level required to control lop was 4~l all patients required vasopressor therapy to maintain upp over ds mmhg. in 20 patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo 2. in the others 15, sj02 were intermittently controhed.the mean time of monitoring were d.8• days. ten patients died within this period. a total of 1.240 blood samples were analized. at arrival, sjo 2 discrepancies were found in 22 patients, b2%. at 48 hours, the incidence were lower, 18/35, 51.4%. at 4th day, were h/29, 38z and at day 7, when the catheters were retired, ii[25, 44z showed discrepancies. the ct showed new injuries in g4z of patients with differences > 10~ in sd02 values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo 2 values and sjo 2 differences. conclusions: the incidence of discrepancies between sjo 2 was higher than expected in severe head-injured patients. these situation could reflect disturbances between 02 demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied 8 healthy, non-atop• nonsmoking subjects (6m/2f, 24+4 yr), which were pre-treated with s (300,ug) or placebo, with a randomized, double-blind, crossover, design, before paf (24,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every 4 min over a 30 min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc99m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from 5410 2 1125 to 33022934x109/l), and pmn(from 29752693to 1222 _+ 767 x109/l), and increased aapo 2(from 2.1 _+9.5 to 14.7+ 12.2 mmhg, p<o.05) and rrs(from 3.0 2 0.6 to 3.7 2 0.7 cmh20.s.i q ) (p < 0.05 each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r =0.83, p <0.05). by contrast, after s, paf did not induce any change in white ceil count (from 596321766 to 6147+1721 x109/i), pmn count (from 354321628 to 2953 2 1793 x109/i), aapo 2 (from 7.3 2 10 to 5.6 + 8.6 mmhg), and rrs (from 2.9 2 0.5 to 2.9 2 0.7 cmh20.s.i 1). compared with placebo, after s there was a lower ~lln-pmn lung activity (2.020.4 vs 1.320.7 cts/mci/pixel, p<o.01 ), lower intrapulmonary pmn retention (15.627.7 vs 11.1 24.8 %, p=o,09), and a faster washout (0.1420.08 vs 0.1720.007 s ~, p=0,4). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)>0.15-0.20 has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied 10 failed weaning trials in 5 copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco2 and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco2 >20mmhg from baseline accompanied by a fall in ph<7.35; a respiratory frequency (f) >30/min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi2 and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning 0f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo4 pneumoeytes. sections of generation 5, 10 and 15 from bronchial tree were free of pathological changes. conclusion: alter 8h inhalation of p~ji2 no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation 3. objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = 15) trauma patients with acute lung injury (ali), group if (n = 15) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett 7200 ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using 0.375 % marcain. the following parameters were analysed: pao2, sao2, paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao 2 and sao2, decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t8 pts. with ari (pao2:8,24@1,49 kpa. paco2: 5,01i-0,77kpa, ph:7 42~:0,59, hco3: 26,3:~8,10 mmol/1, sao2 : 90,4~-7,42%) who were hospitally treated due to pneumonia(9 pts.),emboly of the pulmonary artery(3 pts.) and severe attack of bronchial asthma (6 pts). among tham there were 12(66,7%) males and 6(33,3%) females, average age 51,5~:16,1 years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during 24 hours (sodium-na,potassium-k, chlorine-c1, calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l 4o,94 the object of our investigation was a group of 21 pts with massive pneumonias, 14 males (66.6%), 7 females (33.3%),mean age 55 yrs.thirteen (62%) of them were smokers,8(38%) nonsmokers. only 1 pt (4.7%) had pre-existing chronic respiratory disease, and 20 (95.2%) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in 15 pts(71.4%),physicaly ~onchial breathing in 19 i~s (90.4%),white cell count onder 10 x 109 /l in 18 pts(85.7%). radiographicly, bilateral massive homogeneous shadows were found in 7 pts (33.3%), onilateral in 12 pts(57.1%),pleural effusion in 2 pts (9.52%). abnormal renal function was found in 14 pts (66.6%). sputum culture was positive in 8 pts (38%): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in 4, 2, 2 cases respectively. all patients had remarcable hypoxernia (pao2 range from 4,75 to 8,1 kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while 2 pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf6 intrapulmonary shunt) were measured at controlled flow. in 8 dogs (treatment), data were collected at baseline, during long injury (obtained 90 rain after intravenous administration of oa 0.06 ml/kg), and again after bos (10 mg/kg intravenously). in 5 dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < 0.05 vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar 02 tension is measured by the application of the alveolar air equation in which the arterial pco 2 is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of 0.8. the purpose of this study was to estimate the effective alveolar 02 tension (pao2eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in 14 normal subjects and 27 patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po 2 (pao2(i)), measured from the alveolar air equation in which paco 2 was substituted by the effective alveolar pco 2 (paco2eff) and with the alveolar po 2 measured from the simplified alveolar air equation (pa02). this study is complemantary to previous work for the estimation of paco2eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response 02 and co 2 analyzer. the method is a computerised calculation of the effective alveolar po2quite similar to that of paco2eff, obtained from the simultaneously recorded at the mouth expiratory flow, 02 and co 2 concentration versus time curves. results: the results showed a mean difference (pao2eff-pa02(i)) of -0.061 kpa in normal subjects and -0,711 in patients. the mean of the difference (pao2eff-paq 2) and (pad2(i]-pao z) was much greater than 0.281 in all subjects. the limits of agreement for the difference (paozeff-pa02(i))were -0.691 to 0.568 kpa in normal subjects and -2.040 to 0.596 in patients, while those for the differences (pao2eff-pad 2) and (pao2(i)-pad 2) were very large ( > -1.5 to > 1.7) in all subjects. conclusions: the effective alveolar po 2 is very close to the ideal one in normal subjects, tn patients pao2eff may excessively deviate from pa02(i) due to the observed significant difference between the alveolar/tidal volume ratio for o 2 and that for co 2. the alveolar po 2 measured from the simplified alveolar air equation (pao 2) differed substantially from pao2eff and pad2(i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood 02-carrying capacity and increased affinity of hemoglobin for 02 are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa02>lo0 mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i1. erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value 98• mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value 21.6+15.2 mu/ml in group i, 36.8• mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i1. we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, (1.0<g-i1<2.5); and group iii comprises 5 patients with severe ards (all score > 2.5). broncoalveolar lavage (bal) was obtained after infusion of normal saline at 37~ to intubated patients and cooled immediately. cells were removed after mild centrifugation (350 x g, 30 min, 4oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with 3h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of 20% acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: 60 pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below 1l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than 2l]s. we have investigated 10 patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was 1.7l/s _+0.3l/s. the maximal deliverable flow of 2l/s was obtained in 3 of 10 patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than 2l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no2) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl (100 ~g apob/ml) was incubated with cu 2+ (2,5 ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from 10 to 500 ~m. oxidation of ldl was monitored continuously with conjugated diene formation (234 nm) and 4 hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to 1 hour renal isehaemia following by 2 hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (<0.05 nmol/g tissue in sop versus 0.6 nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a 10-year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in 130 patients (22%). at the hospital admission, pac02 was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years 1990 through 1994, 244 patients were admitted to hospital in acute asthma episode. there were 11 (4,5%) pts with complications; mean age of 27 yrs; 6 females (54%). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was 14 yrs (range from 2 months to 17 yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from 1 to 7 days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during 1993 a total of 52 patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco2 greater than 6,7 kpa and/or an altered state of consciousness. mean age was 56,2-d:16,2 yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was 28~4/min and puts rate 118.d: 12 bts/imn. arterial blood gases revealed a pao2 of 6,95~1,33 kpa, paco2 of 7,87• kpa and ph of 7,274-+-0,132. area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai1 patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every 6 hours the first day and every 12 hours the second and third day. at the end of 72 hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total 116 patients, 57 were finally included in the so group and 59 in the po group.patients in so group and po group had comparable ages (p<.01).peep in both groups was comparable (=.06) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at 48 h.(p<.05) and 72 h. (p<.03).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at 48 h. (p<.05) with respect to initial values.fifteen cases of ards (26.3%) in the so group and twenty five cases (42.3%) in the po group were identified. the time of onset of ards was 35_+ 14 hours in the so group and 11 + 4 b hours in the po group.the final outcome was very similar th both groups : mortality of 36% in the so group versus 37% in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi2) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score >3) recived inhaled pgi 2 at 15-20 ng.kg.min "1 using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after 30 rain of pgi inhalation. results: short-terro p~i 2 inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao2) increased from 101 to 166 mmhg in patient 1 and from 87 to 108 in patient 2, the ratio pao to the fraction of inspired oxygen increased from 1262 to 207 (patient 1) and from 124 to 154 (patient 2). venous admixture decreased from 36% to 29% and from 34% to 27% in patient 1 and 2 respectively. mean pulmonary artery pressure decreased slightly from 25 to 23 mmhg in patient 1 and from 41 to 37 mmhg in patient 2. no effects on systemic haemodynamics were observed in any patient. conclusions: pgi 2 inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated 67 ards-patients (age 41 yr (16-75) mean, range) during 1991-94. the lowest pao2/fio2-ratio was 74 (29-140), the worst murray score 3.0 (2.3-4.0), icu-stay 41 (1-121) days and hospital mortality 40%. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments (1-4). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is 25 years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) 65 000 lowering cholesterol using iovastatin 51 000 treating hypertension using nifedipine 32 900 heart transplantation 28 000 intensive care of ards-patients 3300 conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients (5). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of 38.5 % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= 1) to analyse the effect of different levels of peep (0, 10 and 20omh20) on evlw during hpe; 2) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in 3 groups of dogs by inflating a foley catheter in left auricular to a pressure of 24-26 r~uhg. peep levels of 0, i0 or 20 m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over 30 kg) were randomly divided in three gmups immediately alter infusing via a central vein 0.1 ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n=4) : vt: 10-15 ml/kg; zeep. group 2:(n=4) : vt: 10-15 ml/kg; peep: 10 cm h20. group 3:(n=4) : vt: 5-10 ml/kg; peep: 10 emil20. (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi>40 kg/m 2) we partitioned the total respiratory system mechanics into its lung (1) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap 1989; 67: 2556) . at three different levels of peep (0, 5, 10 cmh20 ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in 1993-94, selecting adult patients with arf defined as: intubation and pao2/fio2<250 mmhg plus an fio2>0.5 or peep>5 cm i120. results. 146 patients met the arf criteria: 40 of them (27.4%) underwent prone positioning (p+). prone position use began in the early phase of arf (3.5• days from the beginning, range 1-32, median 2).25 out of 40 p+ pts were treated with controlled ventilation (cppv or pcv), while 14 were on assisted ventilation (simv+ps) and 1 on spontaneous breathing (cpap). only 2 pts were awake when turned prone, while 11 pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght 4.7• h, range 0.5-12 h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least 12.5 mmhg increase in pao2/fio2:35/40 patients (87.5 %.) were responders when first pruned. the pao2/fio 2 changes induced by prone position are reported in the figure. pao2/fio 2 increased when patients were pruned (*p<0.001) and remained higher than baseline values when returning supine(*p<0.001). paco 2 remained unchanged. prone positioning was used at least twice in 21/40 ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis 2.7+.5) submitted to mechanical ventilation randomly received 8 inhaled no doses in increasing or decreasing sequence: 0.5, 1, 5, 10, 20, 50 and 100 ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after 25 min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_2 cmh20) and fit2 (.56+.14). the changes in vt and fit2 due to the no (1000 ppm in n2) injection in the ventilator external circuit were compensated for. results .34 the dose of 0.5 ppm, ineffective on papm, significantly improved oxygenation. the increase of pat2 and the decrease of q'va/q' and papm were nearly maximal at 5-10 ppm. no deterioration of arterial oxygenation was observed at no doses as high as 100 ppm. co2 exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around 10 ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [1] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no (2-130 ppb) in hospital central-supplied medical air. [2] we have realised nitric oxide measurements in ten healthy volunteers, (4 smokers and 6 non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat0ry limb of the ventilator. unfiltered central supply medical air contained :20 -50 ppb of no and 10 -30 ppb of no2, whereas central supplied oxygen was no/no 2 free. samples were taken after equilibration periods of 5 minutes, with increasing fit2 levels of 0.21, 0.50 and 1.0 for subsequent 5 minutes periods; paired values were recorded every 30 s. the mean no value was 4.57 ppb (sd 2.51) and n o significant differences were found for different fit2 levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [2] and that, previously reported, differences between smokers and non-smokers are not always striking [3] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either 10rag (n=9) or 5rag (n=7) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed 30, 60, and 120 rain after the start of nebulization. results: compared with 10mg regimen, 5mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace2 did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of 10ng of salbutamol is not more effective than 5rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of 107 neutropenic patients (wbc < 1000/mm 3 and/or pmn < 500/ram3), admitted to the icu between 1986 and 1990, were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the 1 st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients (53.3%) had a h~ematologic malignancy, and 50 (46.7%) a solid tumor. fifty-nine of the 107 patients died (55.1%); the mortality rate did not differ between both groups (61.4 and 48% respectively, p = 0.16). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < 10 -4) and cardiovascular (p < 10 -3) failures, and the number of organ system failures (p < 10 -4) are associated to the risk of death. the saps (p < 10 -3) and saps ii scores (p < 10 -4) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = 10-4); neither the features of the underlying malignancy (p > 0.8), nor the duration of neutropenia before admission in icu (p = 0.83), nor the severity scores figs ii: p = 0.068) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co 9 removal. generally, this has been used for short periods of time (<24h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used (70% of the patient's cardiac output), sufficient to achieve both co 2 removal and oxygenation. satisfactory gas-exchanged was accomplished (pco2=50-60 mmhg) with nearly total lung rest for a prolonged period (60h). however, the long ecmo duration was associated with two severe complica-ti0ns:1) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and 2) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, 1981: 36; 135) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt2), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects (5 male, 31 -+5yr) and 13 copd patients (4 male, 61 -+10yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial 10-15 min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a5 min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was <10%. lung function tests were: in normals (mean-sd), fevl%pred = 100• fevl/fvc%=81-+3% , and in copd patients, fev~%pred=53__.20 and fevi/fvc%=51 --.12%. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay 0s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in 75,0~ os patients; inconsiderable improvement -in 15,9~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e 68,7h and 20,5~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means 0s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease 3ust afte~ the course o~ t~eatment (29,3h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s 6 months in 84,5~ os patients, till one yea~ in 69~9~). in 12,5~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in 73,5~ os patients a period os ~emission lasted s ha]s a yea~. 42,9~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: 19 mechanically ventilated patients (5 copd, 6 ards, 8 other pulmonary diseases) were studied in two phases: 1) during the acute phase of respiratory failure; 2) during recovery 5-73 days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with 3-8 cmh20) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase 1) or for two hours when they could sustain sb indefinitely (phase 2). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. 1. during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. 2. inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit 10 pg/ml)in balf (lavage of 3 x 50 ml in the right middle lobe). results: lif was not detected in the balf of 13 healthy control patients and in only one (34 pg/ml) out of 26 patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal 4 h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of 10 out of 12 patients with full-blown ards (196 + 80, mean + sem, range 10-985 pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r:0.70, p= 0.01), albumin ( r:0.75, p=0.008) and protein level (r:0.74, p=0.006). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter0genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, 1995 ; 23 : 481-485) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : 10 mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which 50% of a tcg~-labelled test meal was eliminated from the stomach (t 1/2). baseline data (do) were recorded after enteral nutrition reached 1500 to 2000 ml daily. scintigraphic measurements were repeated 4 days after cisapride (10 ml orally, q.i.d) had been added to this regimen (d4). patients were considered cisapride responders when gastric clearance improved by more than 50% from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be 31 + 15 min. in healthy volunteers (crit care med, 1995 ; 23 : 481-485) . five patients responded to cisapride (t 1/2 : 81 + 31 rain vs. 26 + 10 min at do and d4, respectively) and five did not (t 1/2 : 36 + 18 min vs. 33 _+ 11 rain at do and d4, respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> 150 ml) gastric residues, vomiting (> 3 times/day and abdominal distension) which disappeared in 4 of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove 500 36, czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within 24 hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval 25 patients were enrolled in the study enteral feeding was begun within 24 hours of injury in 14 trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of 11 patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in 38 patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ 1 l short bowel syndrome) and group b (intensive care, 27 surgical patients). all patients likely to require pig for a period of 10 days (group a) or 7 days (group b) were studied. the pn regimens administered were the following : combination with 50 g of mct/lct fat emulsion end 9,6 g of nitrogen, in 1 liter end glucose requirements were met by imfizsion of l liter of glucose 20-30 % via a "y " connection. lipid thus provided 3040 % of the non introgen calories. total daily calorie intake was 1540 to ] 940 kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many 1cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: 36 consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf-1, gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within 8 hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md 1, chiolero r md 1, pannatier a phd 2, berger l 2, cayeux c 1, voirol p 2, hurni m md 3. 1 surgical icu, 2 pharmacy, and 3 cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, 24 patients, aged 63_+8 years (mean_+sd) were assigned to 2 groups (anaesthesia: fentanyl 20 gg/kg + midazolam): group 1 (n=10): reference group, with normal hemodynamic status, easy recovery. group 2 ('n=14): patients in low output syndrome, cardiac index < 2.5 i/m2 on day 1 (d1) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol 1 g, was given intragastrically on d1 + d3: plasma levels measured (h.p.l.c), at administration (to), t30-60-90-120-180-240 and 480 rain. hemodynamic status assessed with pulmonary artery catheter. 5 healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d1 in all patients (no difference between groups). on d3, peak paracetamol level was significantly lower in group 2 (low cardiac output): in group 2 the area under the curve on d1 and d3 were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d1 in all patients, and reverted to normal between d2 and d3 in case of normal cardiac function, but not in case of low output syndrome. the decrease on d1 can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [992 till november, 1994 there was carried out the randomized study of 155 patients with general purulent peritonitis; among them 70 persons constituted the control group and 85 -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega-3 fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: 390 patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of 30.8 (std. dev. -+ 22.9). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving 5 or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of 20 consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg2, hospal,uk, lactate concentration 40 retool/l). date obtained 1 hr before and 4 -6 hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow 100ml/mlu, dialysate solution flow i 1/hr), and approximate daily lactate flux into the patient was calculated to be as high as 920 mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels (3.80+0.67 axtd 2.88+0.68,respectively; p<0.01,paired t-test). when patiente with increased basal lactete (~-7) were compared to paliente with normal basal values (n=13), no difference in laotete increase was fmmd (p=0.22, manova). patiente with severe liver dysfunction (2 points in mop scomlg, n=9) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste (0 or 1 point in mof scoring, n=ll), rite values being 4.04 + 1.17 and 1.84 + 0.23, respectively (p<0.05, student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p=0.86, manova). in 11 pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t2=o.ol; p--o.81) or oxygen consumption (reversed fie, k) (r2-q).o7;p--0.66) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of 20 polytraumafized patients, split in two groups of ten patients each, with mean ages of 35 _+ 17 an 45 -+ 20 years. due to their condition, all patients required p.n. for at least 9 days. both groups were subjected to isocalorie and isonitrogenous solutions (45 ci/kg/ day and 0.24 g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a 10 % concentration and solution b 23 %. blood samples determinations during days 0, 3, 6, 9 after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < 0.05) and prealbumin (p < 0.05). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < 0.05). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p<0.05) and prealbumin (p < 0.005) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < 0.01) when considering the interaction of both the type of diet and the time course of pn. conclusions: 1. solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. 2. no significant differences on plasma proteins were found between the two solutions having 10 % or 23 % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids 50 g/i; glucose/xylito180 g/40 g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids 50 g/i; glucose 120 g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on 31 patients with severe brain injury. there was no difference in body mass index bmi (group a: 25.9 +/-2.7 kg/m 2 and group b: 25.1 +/-2.4 kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started 24 -48 hours after trauma or surgery with carbohydrates and aminoacids. fat was added 24 h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for 4 days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for 4 days in both groups. serum tipase steadily rose to 202 lull in group a and 320.2 lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a 2-fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design 3 groups (n=7 each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct-20%), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct 50/50-20%) and group iii placebo (control), during 12 h (2 mg/kg/min each). we measured before, at the end of 12 h infusion, and 12 h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, 6-keto pgf~,, and ltb4). at the end of the fat emulsion, groups (i and il) to 1,10• to 0,51 • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly (12.5% and 19%) (p<0.05). eicosanoids were increased at baseline compared to reference values (p<0,05). a decrease (p<o,05l in the arterio-venous difference of the vasodilatador prostanoid (6-keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at 2 mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of 2/1 in group i (n=6) , and a carbohydrate/fat ratio of 1/2 in group i] (n = 6) during 8 consecutive days. indirect calorimetry was set at baseline and 24 hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day 8 of enteral feeding. urine was collected during 24 hours and was used to measure 24h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose 6-phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after 24 h of nutrition patients in group i showed an increase in carbohydrate oxidation (from 17.8% to 55.4%, p=0.06), and in group ii a decrease in protein oxidation (from 27% to 21%, p=0.03). after 8 days of enteral feeding a tendency to increase in glycogen concentration (group i, p=0 .06; and group ii,p=0.1) was observed. while patients in group i showed a tendency (p=0.06) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p =0.01 ). in group i, the pao2/fio 2 ratio increased after 8 days of treatment (from 235.9 to 284.5; p=0.03), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in 10 healthy humans (control) and in 30 patients admitted to the intensive care unit (icu) at days 1, 3 and 5. the apache ii of the patients studied was 15_+4. there was an increase of uer in the critically ill patients compared with controls (o.26_+0.1 vs 0.036_+0.01) (p<0.001). in contrast, no changes in uer between days 1, 3 and 5 were observed (0.26 -+ 0. . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r=0.89) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl00/av). patients were divided for days of en according to the presence (group 1) or absence (group 2) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p<0.05 for statistical significance. (vr%) 53 61 58 89 67 63 65 72 62 60 64 59 67 7461 group2 (vr%) 85 90 90 94 94 93 92 90 92 91 92 94 90 92 tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: 1) abdominal distension (abd), 2) increase of gastdc residuals (igr), 3) vomits (vom), 4) diet regurgitation (reg), 5) diarrhea (dia) and 6) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co2 production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co2 output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t00gr glucose/24h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only 100 gr/24h glucose was provided, the mean values of vco2 output were 134_+_25 and 140!-_26 ml/min respectively. there was no difference in vco2 output between with low carbohydrates and high fat regimen and standard regimen, mean values were 16t .5-+27 and 163_+26 respectively.the high calories regimen and tpn resulted in higher vco2 output, mean values 184_+25 and 211• respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco2 output, in the contrary, high calories intake as well as tpn increased vco2 output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b6raud j.j. m6tabolic intensive care unit and nephrology department. lapeyronie hospital. 34059 montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january 92 to january 95. 33 patients (pts) (male:23, female:lo; mean age: 57+4 y.o.) were admitted with rh defined as cpk>1000 iu/1. etiologies were: traumatic and ischaemic 24, infectious 4, toxic 4, excess activity 1. factors studied were: simplified acute physiologic score (saps: 10.3+1.1), organ systemic failure (osf: 1.02_-!-0.2), diagnosis delay (d: 33+_5h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a 24 hours period. arf was observed in 25 pts. in non-arf and arf groups respectively, saps (5.5_+0.5 vs 11.8+1.3), deshydratation (0 vs 11), sepsis (0 vs 12), phosphorus (1.03+0.16 vs 2.21-+0.21), calcium (2.1+0.07 vs 1.8_+0.07), ward score (4_+0.65 vs 11.8+0.8) were significantly different. however, no significance was observed in uar (310-+89 vs 210-+35) and ci (26_+7 vs 34_+3). 16 patients required hemodialysis (hd) (85:2 sessions) and 9 remained dialysis free. only osf (1.1_+0.1 vs 1.9-+0.23), ward score (9.2_-/-0.95 vs 13.25_+0.92) and ci (29+_3 vs 41-+3) appeared significantly higher in pts requiring hd. 5 pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over 7) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x 0.043. tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, 89070 uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a 4 hr infusion were investigated. methods: after approval by the local ethic committee 14 healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using 6,6-d2glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor (0.14 pg/kg/min) and dob (6 pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ 8~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p<0.01, anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a /3-adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial 30 patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi6ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet (15 patients) or an isonitrogenous isocaloric parenteral support (tpn) (15 patients) (total energy: 2880 kcal, nitrogen: 14.5 g, carbohydrates: 380 g, fat: 112 g, n/non protein calories: 1/175) at least for 10 days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day 3 in the tpn group and by day 5 in the enteral group (66.6% of the enterally fed patients and 80% of the tpn patients maintained in positive balance the day 10 of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p=0.003). thyroxine-binding prealbnmin rose significantly in both groups as well (p=0.019 and 0.004 respectively). statistically significant rises in lymphocyte counts (p=0.003 and 0.001 respectively), in levels of c 3 (p=0.009 and 0.01)1 respectively), iga (p=0.002), igg (p=0.004 and 0.003 respectively) and igm (p=0.004) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group (73.3%) and the tpn group (60%). by the end of the study the incidence of negative responsiveness was 40.0% and 26.6% respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in 73.3% of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (>250 ml/24 hours) fasting gastric aspirates. methods : antral (4 sites separated each other from 1.5 cm), duodenal (1 site) and jejunal (1 site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder (7758a recorder, hewlett-packard) then anal)7,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), 2) presence of bursts (> 2min) of nonpropagated phasic pressure and 3) presence of sustained (>30 min) uncnardinate pressure activity. 11 patients with a volume of gastric aspirates of 731 • 506 (sd) [median 5001 ml/24 hrs were investigated for 538 4-271 [median 4551 minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n=4) or migrated abnormally (retrograde propagation : n=4; retrograde and stationnary : n=2) in 10 pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in 9 pts and sustained uncoordinate pressure activity was found in 2 pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n=2}. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included 20 patients with hypokalemia in whom rapid potassium replacement (20 meq kci in 1 h) was performed: 14 patients received mg sulfate (6 g in 3 hours) and 6 patients received a corresponding saline infusion. measurements were made at time 0, +1, +3 and +6 hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time 1 and 3 h (p < 0.05 -students-newman-keuls). (table 1 ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the3" authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients 1. aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a 2 months period and requiring artificial ventilation and enteral nutrition for a period of at least 3 days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin (200 rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. 15 patients (male/female : 13/2: mean age : 56.9 + 22.2 years; mean apacbell score : 23.t • 7.0) wore enrolled into the study.the procedure was performed within the 2 dab,s following institution of mechanical ventilation. jejunal access was obtained in all 15 patients without resort to enduscopy in 10,81 • 7.31 min.(total duration of the procedure). mean duration of fluoroscopy was 3.54 + 2.97 rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow 80% of normal values in 3 or more of the 6 above criteria were considered undernourished (und).statistical analysis was performed using %2 analysis.statistical significance was established at p<o.o5. results: a total of 129 patients(47 female, 82 male) -with a mortality rate (~) of 31,8% -were studied, aged 56-+8 years and a median ]enght of stay of 22 days. 1 -nourished (nou) at ad and at dis -22; ~ 0%; 2 -nou at ad and und at dis -17; ) 41,2% ; 3 -und at ad and und at dis -73; ~ 45.2%; 4 -und at ad and nou at dis -17; ~ 5,9%. we observed a p< 0.05 when we compared groups 1 and 2 (p=0.0012), and groups 1 and 3 (p=0.00026). variation in nutritional status and longht of stay: 1-nou at ad and nou at dis = > median lenght of stay 18 days; 2 und at ad and und at dis = > median lengbt of stay 22days; nutritional status and age at admission: 1-age > = 60 years : nou (13) , und (53) 2-age < 60 years: nou (26), und (37) nutritional status and age at discharge: 3-age > = 60 years : nou (12) , und (54) 4-age < 60 years: nou (36), und (27) we observed a p<o.05 when we compared groups 1 and 2 (p=o.01) and groups 3 and 4 (p =0.004) conclusion: such study permits to conclude that most of our patients (69.8%), staying in icu for more than 6 days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf-1 levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol2jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: 9 icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient 6 baseline hlood samples for circulating 1gf-i were obtained over a 24 hour period, prior to il!iection of 40 p.g/kg of rhlgf-1 subcutaneously. a further l4 samples were taken fi'om each subject over the next 48 hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv2 were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age 35(29-73) years, apache ii score 18(12-34), and length of icu stay 26(10-92) days. baseiine circulating igf-1 levels were low 28( < 20-166) ng/ml reaching a peak of only 84( < 20-246) ng/ml. 2 of the 9 patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining 7 patients tmax was at 3(2-6) hours, clearance was 89(31-452)0 ml/min, vd was 0.29(0.12-0.73) l/kg, and tia 9.2(4.2-30.1) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients (0.29 vs 0.33 l/kg), but clearance was greater (89 vs 25 ml/min), tmax earlier (3 vs 5 hours) and t~a shorter (9.2 vs i0.8 homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january 1991 to march 1994, 65 patients underwent surgery for acute bacterial endocarditis: 36 had native valve endocarditis and 29 had prosthetic valve endocarditis. there were 48 men and 17 women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was 1902+1450cc and in the aprotinin was 850:t:80 (p < 0,0001). the mortality in icu was 10,7%, the staphyloccocical endoearditis mortality was 18,2% and the no-staphyloccocical endocarditis mortality was 2,6% (p<0,05). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality (24%). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: 65 patients (ventilation period > 5 days) were randomized and allocated to the sdd group (n=32) or the control group (n=33). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of 50 rag polymi~ 80 mg tobramycin and 500 mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: 625 smears were examined in the sdd group. mrsa strains were detected in 66 samples (10.5%) from 7 patients, and in 5 patients they were detected for a period of up to 4 weeks. the positive smears were districted as follows: tracheal 11/117 (9.4%), nasal 28/199 (14.0%), pharyngeal 15/111 (13.5%) and rectal 121198 (6.1%). severe mrsa-induced infections were observed in 2 patients (infection rate 28.6% of the colonized sdd patients). 560 smears were examined in the control group. ivlrsa swains were r in 15 samples (2.6%) from 6 patients, but only repeatedly over a period of up to 10 days in 3 patients. the po~tive snmars were distributed as follows: traclmal 1/114 (0.8%), nasal 8/174 (4.6%), pharyngeal 4/98 (4.0%) and rectal 2/174 (1.1%). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p1p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip 4.5g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: 2151 hospitalized patients have been documented in this multicenter trial during a 2 year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > 38~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip 4.5g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n=926), followed by intraabdominal (n=765) and skin and soft tissue infections (n=460). 61% of the 926 lrtis wvre nosocomial acquired and in 75% the treatment was conducted as monotherapy. in 53% the lrti was treated with taz/pip b.i.d, and in 45% t.i.d. pseudomonas spp. (n=138) and staph..aureus (n=134) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip 4.5g b.i.d, and t.i.d, were 89% and 81% respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to 4.5g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of 9 cases of severe generalized tetanus (sgt), treated in our icu the last 7 years. we review 9 cases of sgt (6m, 3f), mean age 66.7 years. in 5 eases the entry site of c.tetanus was a skin laceration, in 1 case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first 6 cases incubation period was short (3-11days) and so was the period of onset (1-6 days). all patients needed mechanical ventilation (range 15-58 days), initally through an orotracheal tube,and later through a tracheostomy, performed 6• days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to 6 weeks in the most severe cases. significant autonomic nervous system dysfunction was present in 3 cases occurring 5-12 days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in 5 cases. nosocomial infections occurred in 7 eases, with sepsis and mof in one. average stay in the icu was 18-62 days. one patient died with severe septic complications and one was discharged with severe 9 disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, 12 disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, 6 out of 14 examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi6, intensive care unit, kb firule split spin~ideva 1! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo4 patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a 24-hour period.criteria for the suspected bacteremia were body temperature above 38 c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial 5 ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in 76 (73%) patients hemocultures was negative at both sites,whereas in the remaining 28 (27%) they were positive.for twentyone (26~176 of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in 7 (6.7%) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than 4 days in 81 patients and for more than 7 days in 23 patients.from 7 patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other 6 had the catheter from 6-1o days. we neither found significant differences in hemodynamic dates : objectives: 1, to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). 2. to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included 20 consecutive ventilated patients (15 male and 5 female, 56_ + 16 yr; apache ii score 19-+7) over a period of 3 months. et aspirated samples with and without saline were taken daily from day 0 of intubation until pathogen~ were presented in counts of _> 105 per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between 3 to 65 days. bacterial transfer between staff and patients was noted in 80% of patients until day 5 of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was 45% with an overall mortality rate of 30%. acinetobacter anitratas (no 15), staphylococcus aureus (no.15), klebsiella pna~moniae (no.9) and pscudomonas aeruginosa (no.4) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. 2. there is a close relationship between the microflora of personnel, patients and equipment. 3. bacteria transfer was noted both to and from patients. 4. strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to 20% of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from 30~ to 70%. enteric gram-negative bacilli have been implicated in 75% to 85% of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for 27% to 40% of these pneumonias. importantly, epidemics of/3-1actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than 95% of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and 5;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from 37 to 40 ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i0 patients) or equal volumes of human albumin 20% (control group; 10 patients). wig (sandoglobulin) was administered in a total dose of 1 g/kg divided in a four time regimen on days 1, 2, 3 and 6 post-admission. three blood collections were performe& before the first dose (day 0) and 24 hours after the third and the fourth dose (days 4 and 7 respectively). complement, lgg fractions, the sba at 37 ~ and at 40 o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day 0 (743+_130 vs 898• ns and 394+103 vs 472+101, ns), whereas they were significantly higher (p<0 05) in the 1v1g group on days 4 (1700_+_274 vs 799+197, p<0 05) and 7 (1740_+227 vs 864+i64, p<0.05). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas (37 ~ c) at 30 rain in ivig group vs control group were: -53_+32 vs -56• ns for day 0, 9_+46 vs -54_+46 p<005 for day 4 and 7_+34 vs -54+47 p<005 for day 7. the mean (+sd) sbas (40 ~ c) at 30 rain presented a significant improvement over those of 37 ~ c but for the control group remained negative a~d were respectively as following: -~8• vs -26+33, ns for day 0, 22+_39 vs -29_+35, p<0.05 for day 4 and 24_+31 vs -27_+36, p<0.05 for day 7. the increase of temperature induced a 3-fold improvement of sba in iv1g group and 2-fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p<0 05) in the ivig group than in the control (2 vs 7, and 440 vs 1900, respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune 168, d-53115 bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as 3rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from 1990 the'overall resistance in intensive care units towards oftoxacin was 22 %, whereas in other hospital wards the percentage of resistance was 5.3 %, in out-patients, however, only 2.$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than 60 fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid•177 in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of 2617 postoperative adults pts of mean age 61.1+8.9 years old, with a mean icu stay of 1.6_+0.6 days, following an open heart surgery from july 1993 to april 1995, 54 pts (2%) remained in icu for more than 10 days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole (800 mg/day) (10 patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of 54 postoperative pts with prolonged jcu stay, 11 pts (20.3%) developed systemic candid•177 usually after the 20th postoperative day. they were 8 males and 3 females of mean age 64+_7.4 years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group (119 min vs 84, and 64 vs 49 min). all these pts received inotropes per• (mean value=2.3). during their icu stay, 9 pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of 50 days. the median icu and hospital stay was 58 and 60 days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: 1. a significant percentage of patients who remained in the postoperative icu for more than 10 days developed systemic candidiasis. 2. all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. 3. fluconazole seems to be a very good alternative to amphotericin-b. 4. fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may 1982 to february 1995. results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a 24-hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the 4th to 7th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis (100%), dry mouth (100 %), dysfagia (100 %), asthenia (55 %), palpebral ptosis (55 %), accomodation paralisis (66%) and urinary retention (55%). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was 10 days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an 18-month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group 2 for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high (70-100%). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in 35 patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and 6 month outcome. 18 patients required mv; 10 were immunocompromised (ic) including 8 hiv. tb was pleuropulmonary in 24, disseminated in 9 and meningeal in 2. results: mr was 31% in icu, 34% in hospital and 47% at 6 month. 12/16 (75%) <0.001 mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po2/fio2 ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high (47% at 6 month). need for mv increased mortality (72% vs 18%). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january 1985 tit march 1995. in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total 0f42 pts were studied, 26 males; mean age 55,8 _+ 16 (16-81); apache ii score 16,6 + 7,9; glasgow coma scale (gcs) at admission 12,5 _+ 1,7; 17 (40%) pts suffer from cronic pathology; 5 (12%) pts diabetes mellitus (dm), 4 (9,5 %) pts had had a previous cranial traumatism. in 22 cases the source of infection was otic and also in 22 (52 %) episodes of pm there were bacteriemia. in 21 out of 26 (80%) pts that ct was performed no radiologic abnormalities were shown, 3 of them presented cerebral oedema and 1 pts a cerebral abscess. twenty-eight percent presented seixures, 14% hemiparesia, 46,3% respiratory failure, 17,5% shock, i5% renal failure, 5,1% multiple organ failure (mof). as for treatment refers 5,5% pts recieved only penicillin, 69,4% pts only tcg, 11,1% pts tcg followed by penicillin and 8,3% pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and 25,6% vasoaetive drugs. the mean icu stay was 7,5 5:6 days (1-28). twelve (28,5 %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the 48 and 72 hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at 48 and 72 hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of 30 patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of 2(} immunologycal parameters has been offered:t-calls total/cd3+/;t-helper/cd4+/;t-supressor/cd8+/ th/ts ratio;b-cells/cd19+/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd3+mean=37.62%/14.3%-47.9%/and cd4+mean=22.11%/9% -28.8%/;inverted th/ts ratio ,mean=o.72/0.37-0,90/;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population 9 we prospectively studied 245 pts, 9.4% (23/245-group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t4 and t8 type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) 9 the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in 2.7%, tv in 6.0%, av in 2% and pv in 0.9~ and was multiple in 2.0%. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations (39% tr, pe 33%, mortality 32%). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within 7th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type 2,enterotoxin a and toxic shock syndrome toxin-1 .eight of 1 6 patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from 1992.futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from 1992. these countermeasures for prevention of nosocomial infections after 1992 significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr6sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a 36 year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao2:46mmhg with fio2:1.0, pco2:27 mmhg, ph:7.4, hco3:20mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung4/4). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was 8.7001mm8, platelet: 40.0001ram3, hematesrlt:30%, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high (1/800, negative<ill00) and iglm antibodies also very high (elisa, a=1.492, negative<0.150) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period (1990-94): group a, 19 patients hiv+, and group b, 152 patients hiv-. apache ii was identical in the 2 groups (p=ns). group a required more often mechanical ventilation (p=0,o07), had a higher p(a-a)o2 (p=0,004) and metabolic acidosis was more frequent (p=0,001). regarding laboratorial parameters group a had a lower no. of linfocytes (p=0,02), a higher ldh (p=0,04) and a more marked hypoalbuminemia (p=o,03). mortality was higer in group a (52,6%) than in group b (29,6%), (p=0,04). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p=0,02). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o2, metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, 3 gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, 2 gr tid) plus netilmicin (netromycin, 150 mg bid) (group b). a total of 926 patients (pts) (767 males and 159 females, of mean age 60.6+8.7 years old) were included in the study over a period of one year; 424 in group a and 502 in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was 5.8%; 5.4% in group a and 6.1% in group b (p=ns). 34 pts (4.7%) developed infection following cabg, 17 pts (7.9%) following valve replacement and 6 pts (17.6%) after other cardiac surgery. they were 43 males (5.6%) and 11 females (6.9%). endocarditis has occurred 0.4 % in group a and 0.2 % in group b. severe wound infection was recorded in 0.4% in group a and in 0.8% in group b. one case of sepsis (0.2%) in group a and in group b (0.2%). respiratory infection occurred in 11 pts of group a (2.6%) and in 11 pts of group b (2.2%). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in 5 (1.1%) in group a and 6 (1.1%) pts in group b. 3 pts (0.6%) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the 8 patients with lpp admitted in our icu during the last 8 years (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) . they represented 4.6% of cap. seven patients were males and 1 female, with mean age 46.7+12.1 years. tiss was 24.1+10.9 and apache ii 21.0+5.2. all, but 1 patient, were under mechanical yen tilation (mv) during a mean period of 11.7• (min-l, max-44) days. two pneumonias occurred beyond the season, while 4 patients had an epidemiological history. only 1 patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in 5, renal failure in 4 (due to rhabdomy~ lysis in 3). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in 3 patients. mortality rate was 50%. deceased patients had initially higher apache ii, (a-a) 02, and lower natriemia. comparing lpp with the other cap (n=84), both submitted to mv, mortality rate was similar (57,1% versus 54.7%). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > 10 cfu 10 3 cfu/ml. broncho-alv~lat lavage (bal) ~= 104 c'fu/rnl or positive blood culture. 10 were excluded for rapture of treatment ; 63 were analysed (shift with oral antibiotic8 : 3 ; prohibited antibiotics associations : 5 ; resistant germ : 2). clinical data : age 60,6 • 18,7 ; saps 12 • 2,86 ; mac cabe i : 76,2% -ii : 22,2 % -iii : 1,6. 63,5% of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in 35 (55,6%), copd 9 (14,3%), aspiration pneumonia 19 (30,2%). 75 germs were isolated (psb 67, bal 1, blood culture 7) : s. pneumoniac 28 (37,3%), h. influeazae 14 (18,7%), sttep~:occns 10 (13,3%), saar6ns 10 (13,3%), enterobaetdrindr 5 (6,7%), mosexella catarrhalis 2 (2,7%), othem 6. 71/75 (94,7%) were sensitive to freatment. the ltentment was 100 mg/kg/d of ampiclllin and 50 mg/kg/d of sulbactam in continuous iv adminisu'ation during at least 10 days. clinical eff~ienev : success 46 (73%), failures 17 (27%) with superinfeetion 7, worsening or relapse 3, dead 5, side effects 2. there was no difference between etiologies : primiti~;e~ 74,3%, copd 77,8%, aspiration pneamoniae 68,4%. the bacteriological effieieacy was evaluated only for 41 patients with eradication 30 (73,2%), eradication but super~ection 6 (14,6%) : with pseadomoaas a&ogiuosa 2, eater~ac~ 3 ; beeteriological failure 5 (12,2%). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > 48h were randomized to received topical application oga cl suspension (2%) qshrs, a cl gel (1%) q6hrs or a placebo. in addition all vpts received a nasal and a op spray (2%) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n=8 n=10 n=9 n=10 5/8 6/10 7/9* 3/10 3/8 4/10 2/9* 7/10 1/8 2/10 3/9 4/10 0/8 1/10 2/9 2/10 i *p = 0,03byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q6hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r6enmmtion, * service des admissiens-urgeuces centre hospitalier g-6ndral -02 321 saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a 9 beds unit. the prospective study includes all the patients comn~ in from 01/01/1993 to 30/03/1995. the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than 2 days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of 3 nosoeomjal inthrtions: i abcess and 2 bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed 2 different periods: period 1( 1/01/93 to 31/10/93 ) and period 2 (01/11/93 to 30/03/95 ) for all 1 .e peripheral catheters inserted in the i.c.u. period 1 44,9 % 46,2 % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every 72 hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo2) to measurements of the mechanical work of breathing (web) and p0.1. methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation (6-75 days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto 15, 10, 5, 0 mbar far 20 rain periods each. all patients had a peep between 5-8 mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display (7200ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p0.1 was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo2 and vco2 were measured using the pb 7250 metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb 7200ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p<0.05, compared to ips 0 mbar. results: the values for breathing pattern, web, p0.1, vo2 and vco2 are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: 13 episodes in 12 consecutive patients (mean age: 57.6 years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, 7 patients had nosocomial pneumonia, 6 had urinary tract infection [2 with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations (103 -10 s cfu/ml) in 2 occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over 1994. mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p<0.05 results: 140 of the total 178 admissions were fully evaluable. 38 of them (27%) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every 48h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for >48h were compared: group1= hme replaced every day, n= 61 episodes of mv in 61 patients; group 2 = hme changed every 48h, n=68 episodes in 64 patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~103 cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every 48h. results: the groups were similar in terms of age, indication for and overall duration of mv (10+_8.6 vs 10+_9days, p=0.9), and severity of illness (saps: 16---4.9 vs 16.4+_5.5, p=0.6). the maximal values for peak airway pressure were identical in both groups (33.4-+7.8 vs 33.7• cmh20, p=0.9). obstruction of the tracheal tube was observed in only one instance in a group 1 patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same (6/61 vs 8/68, p=0.7), as was duration of mv before the occurence of pneumonia (9• vs 10.5+_4.7, p=0.6) and overall mortality rate (17161 vs 17168, p=0.7). conclusions: the clinical efficiency of this hme does not seem altered after 2 days of use. indeed, replacing this hme every 48h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed 13 patients (pts) admitted to our icu from july 1989 to december 1993 with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv (2 cases), herpes zoster varicellae (1), measle (1), rabies (1), unidentified (8). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts (6/12) had an abnormal mri. among them, 2 pts made a complete recovery, in comparison with 5/6 pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in 3 cases (with poor outcome in one) and was improved in one. spect was found abnormal in 10/11 pts (among them, 4 pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in 56% of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied 102 pts, 68 # (66,7%), 34 (33,3%). hean age:48,3• years (16-82). mean stay in icu:13,3• days (8-75). they were divided in 2 groups: a(44 pts) who did not suffer from pneumonia and b (58 pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) 3 times: on days o, 4 and 7 of the pneumonia for group b and respectively for g~oup a. conclusions: 1) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts<65 years (p<o,01). 2) the frequency of isolation of kp in icu pts during 1985-92 was 25-30%, but now (1992-95) kp becomes more and more rare (12-15%) and is isolated especially in elderly pts. 3) its sensibility to antibiotics remains always the same. 4) the prognosis of np in our study was independent from age (p<o,1), sex, presence of bacteremia (p<0,1) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at 1 and 4 hours after the iv (bolus) or the et (nebulized) administration of 80 mg of gm in seven criticcally ill patients (3 with established renal failure). safety was defined as peak and trough plasma gm levels _< than 8 and 2 ijg/ml respectively and adequancy as gm levels in bronchial secretions _> 0,5 ijg/ml. results: gentamicin was administered by the et and iv routes in 18 and 7 separate sessions respectively. a total of 107 samples were assayed, 69 in bronchial secretions (bs) and 38 in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route (3,26 + 2,86 vs 2,1 _+ 2,1 pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in 100% of patients after et administration, compared to 66% after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route (1,56 + 1,95 vs 5,56 • 1,96 pg/ml respectively, p _< 0.01). the et administration resulted in toxic bronchia~ gm levels in 47% of the specimens. 66% of these samples were from patients with renal failure, however toxic blood levels were reached in only 12% of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from 4 groups of patients were tested. group i: 13 patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of 30 patients with a np without anaerobic bacteria, group ill: a control group of 27 patients with dental stumps but without pulmonary infection, group iv: a control group of 30 healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i (63• was statistically higher (p=o.o05) than in the 3 control groups (respectively: 29+25, 32_+25, 31_+23). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t >38.2 ~ c) in all adult patients admitted for >-48 hours in the icu during a two month period. these patients were studied for 14 consecutive days. and werc classified in 3 groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of 208 patients studied, 74 dec'eloped fever (35 6%). including (after complete evaluation) 39 d, 15 p and 20 u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u (38.7•176 versus 38.5•176 and 39.2-~0.9~ versus 38.64-0.4, respectively p= 0.05 and p= 0.003). most common sources of infection in d were the lungs in 25 patients (64%) and urina .ry tract in 4 (10%). 14 of these patients had positive blood cultures (36%). the overall mortality was 27% (23% in d, 40% in p and 25% in u. differences ns). antibiotics were given in 100% of d, 73% of p and 15% of u (3 patients). in p there was a non significant lower mortality." in patients who received antibiotics (3/11 (27%) versus 3/4 (75%) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: 126 patieet with a stay of more than 7 days in inteaasive care were screened for candida infection. 70 patients were treated with antifungal therapy due to either an increased antigen titre of -> 1:8 or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of 1:640 or higher. results: the median length of stay was 37 (ranging from 8 to 132) days, the mean apache ii score on admission was 18 (+_ 5.8 sd) points. of 126 patients 31 patients died (24.6%). in the group treated with antifungnls (71 patients) 19 patients died (26.7 %). although of the 126 patients only 51 (40.4 %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher (37.2% vs. 14.6%, p < 0.05 [chi-square-test]). in 34 patients an antigen concentration-> 1:16 was measured. seroconversion was found in 41 patients. the most common fungus was candida albicans (66.4 %). furtberm0re, candida glabrata was found in 21.1%. most of the patients were treated with 2 x 200 mg fluconazole (66 patients). in 38 patients therapy was changed to amphotericin b/flucytosine. in 5 patients therapy was started with amphotericine b and flucytosine. in 40 patients a threefold decrease of candida antigen titre was found. 27 patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least 24 hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied 47 patients (36 males and 11 females, age 49 + 23) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at 1500 rpm for 15 min revealed: i) more than 20 leukocytes per optic field, ii) squamous epithelial cell less than 1 percent and iii) one or more microorganisms per optic field on 1000 magnification. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 8 patients had vap and 39 patients did not. in 5 cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was 15,7 -+ 5,5, while in patients without vap was 17,9 + 6,4. there was a significantly higher incidence of vap in patients who had i) coma (gcs <8) and ii) been receiving neuromuscular blockade (p<0.05) . the sensitivity of the gram stain for vap diagnosis was 75%, the specificity 89,5%, the positive predictive value 60%, and the negative predictive value 94,6%. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october 1992 and february 1995 all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the 153 psb performed for suspected mvp (132 nosocomial and 21 community-acquired pneumonia), 81 yielded at least one micro-organism (positive psb : 53%). 63 of positive psb demonstrated only aerobic bacteria and 18 (23%) yielded with anaerobes. in 14 out 18 patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia (17/76 positive psb). 27 strains of anaerobes were isolated. prevotella species represent 19 out these 27 strains (70%) the most frequent anaerobic species were prevotella oralis (6) p. intermedia (5) and p. buccae (4). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied 64 multi-trauma patients (mean age 58 + 19 years, apache ii 16.5 + 6), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than 105 and 103 cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 14 patients had vap, and 50 patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)<8 (x2=4.15, p<0.05), the administration of neuromuscular blockade (x2=7.9, p<0.05), the duration of mechanical ventilation to be greater than 5 days (x2=5.5, p<0.05), the flail chest (x2=4.1, p<0.05), the parenteral nutrition (x2=5.6, p<0.05), the ards (x2=3.9, p<0.05), the abbreviated injury scale (ais) of more than 4 for thorax (:,:2=5.9, p<0.05), the pneumothorax (x2=5.1, p<0.05) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: 4.8, p<0.001), flail chest (f:3.0, p=0.003), and gcs (< 8) (f:2.1, p= 0.039). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (<8), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of 160 cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of 2 or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= 0,2042 + 0,0243x~ +0,2931x 2 +0,1504x3 , were x i-apache-ii score points, x2-evidence of septic shock, x3-endocrinopathy. the explanatory power of this quation was evidenced by roc of 0.88, se (v 0 -0.033 introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in 26 patients during the first week after trauma or the onset of sepsis. patients: group 1: (n = 11) multiple trauma iss > 30, group 2: (n = 15): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin4cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z-021, pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. 1+4.4 110&-_11 7(4-9) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of 52 granulocytopenie(wbc<0,5xl09) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in 28.8% of patients before ss and in all patients after ss. only 3 patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only 5 frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte8 in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in 257 patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto2) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was 0.8 mt-m "2, and le,~ and ie'i~ w~ 2.4 units in sic. we managed to maintain op of 1.0-1.7 ml.m "2 and ieto2 of 1.9-2.6 units in hst. 41 patients from 135 with mosf survived in sic and 96 patients from 122 survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab9 2zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and 28 day survival were also recorded. biochemical data were compared with results from 20 healthy subjects. results: xanthine oxidase activity was 6.30 + 1.59 units/i in patients (mean :t: sem) and 0.74 + 0.12 units/i in controls (p<o.01, mann whitney u test), and was highest in the 6 patients who survived (p<0.05). malondialdehyde concentrations were elevated (0.81 • 0.11 prnol/i compared with 0.29 • 0.05 pmol/i in controls, p<0.001). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco2) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p7) methods the pco 2 electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography 70% of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on 35-40% and general peripheral resistance increased on 80-100%). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was 10-15 mg/kg during 4-6 days. as a result of such therapy was also the oliguric stage shortening (ac n 1464083).in 9,6% of the cases unsatisfactory datas of central hemodynamic,combined with high (more than 15 cm h20) venous pressure,lung oedema.then nanipruss (0,5-1,5mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton (0,25-0,5 mg/kg) and dopamine (5-10 mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during 5-7 days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in 4 different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of 76 surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; 1 .ruptured aneurysm of the abdominal aorta (raaa) n =20 2.elective vascular surgery (evs) n =20 3.abdominal sepsis n -17 4.trauma n = 8 5.others ( e.g. malignancy, obstetric emergencies) n-11 seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients 72% , group 1 75% , group 2 70% , group 3 88% , group 4 38 % , group 5 75 % . mortality increased with the number of failing organ systems; mortality for all patients with > 4 failing organsysterns was 80% the only exception being the subgroup of trauma patients where mortality under these circumstances was 5o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > 4 failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first 30 day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( 60% ~ 40 % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between 10 and 19, and the function of 2,3 or 4 organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol 97 patients (18-60 years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: 1) to increase the effect on microbic agents and the efficacy of treatment in combined infections; 2) to lessen the possibility of the exciters'resistence to antibiotics; 3) to prevent the development of superinfection: 4) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was 19 (10-25). when indicated, 9 patients had blood volume measurements simultaneously based on the techniques of, i) dilution of 5~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co2 removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was 6310ml (4710-7959ml) compared with 4690ml(3755-5778ml) with slcr labelled red cells (r=0.9). regression analysis produced an intercept at 769ml. the slope of the regression line was 0.62 (0.33-0.9, 95 % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock (30-35 mmhg for 180 rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of 2.0 mg/kg or 20.0 mg/kg at the end of resuscitation. results: infusion of 2.0 mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the 48 h survival rate was significantly improved from 26.7% in the control group to 68.8% in the treatment group (p<0.05). in contrast, the high dose of 20.0 mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at 2 h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest-1993) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: 733 patients have been hospitalized in our icu from january 1992 to may 1. 994, 198 (27%) with mosf. among mosf pati~ts, 57 (29%) have had hepatic dysfunction defined according to hebert (bilirubin ~ 60 ttmop1, chest 1993). thirty six of these 57 patients acquired hepatic dysfunction after admission in the icu. these 36 patients were compared with 36 mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< 0.05 are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao2/fio2, arterial lactate, do2 were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in 15 % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard8 that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to 0amag/~5 factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on 13 consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f 60 tm [fresenius] , ct 190 tm [baxter] , and filtra116 tm [hospal]) were assessed. each filter was changed intentionally after a 24 hours" use. together the data of 54 filters were evaluated, each at three different times (immediately after its onset [0 hi, after 8 h, and after 24 h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p<0,05) showing the f 60 tm membrane excelling filtra116mand ct 190 tm the more. the loss of inuline clearance (3 mi/min/m 2) after 24 h, however, was insignificant for all 3 filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after 8 h (p<0,05), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a 24 hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on 100 patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was 60 years (y), 57 patients (57 %0) were <65 y, 43 patients (43%) were >= 65 y. the incidence of dialysed arf in our mixed intensive care departement is 3%/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively 70%, 44 % and 35 %. treatment: cavh (cd) or cvvh (cd) was used in 40 patients (40%), hemedialysis (hd) was used in 60 patients (60 %). data: mean apache 2 scores were the same for cd and hd (27 for both groups), patients treated with continuous dialysis techniques had significantly (p<o,05) more need for ventilation (100 % vs 70 %), elevated bilirubin (82 % vs 57 %) and a higher vasopresser need (89 % vs 46 %@ than patients treated with hemedialysis. there was a trend towards more sepsis (83 % vs 62 %; p=0.06) mad coaguiation disorders (50 % vs 27 %; p=0.07) in cd. taere were sign/ficantly more younger patients (<65 y) treated with cd (70 % vs 30 %, p<0.os). mean apache 2 score was significantly lower in patiants<65 y than as patienta>=65 y (26 vs 30; p<0.05). patients<65 y had significantly (i}<0.05) more coagulation disorders (53 % vs 17 %) and elevated bilirabin (81% vs 52 %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd (43 % vs 15~ p<0.05). patiants>=65 y had a comparable sr vs patients<65 y (3") */e vs 28 %; p----a.s.). tha global survival rate (sr) was 32 % (32 patients). conclusions : diaiysed arf has a well known lowsurvival rate (32 %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache 2 score couldn't illustrate that. patient~65 y with arf have the same survival rate as patients<65 y: although patients >=-65 y have a higher apache 2 score they have less organ faille. the avacbe 2 score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < 7.32 initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: 94 patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of 64.9%. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo2 vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: 15 patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo2. sao2, diuresis, pha, and hemoglobin were repeatedly registered during the first 6 hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: 176 measurements of co were obtained, ranging 2. objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo2) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do2/vo > its co> relation to other parameters of do2/vo 2 disturbance, and with postoperative complications and clinical course. methods: 20 patients (16 male, 4 female) undergoing cardiac surgical procedures were included (16 myocardiai revascularizations, 3 valvular substitutions, 1 constrictive pericarditis). mean age was 63 + 12 years, mean weight 70 _+ 10 kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period (90', 120', 240", 360' and 18 h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo2), diuresis and arterial ph (pha) were also recorded. results: phi values ranged 7.20 to 7.65 (mean 7.40 (0.8); the mean values of clinical evolution were: extubation time, 20 _+ 12 hr.; discharge from postoperative care unit, 88 4-50 hr.; and hospital total postoperative time, 10 _+2.2 days. complications registered were: 2 perioperative acute myocardial infarctions, 2 cases of respiratory insufficiency, 1 occlusion of coronary bypass, an 1 ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > 0.05) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo 2 disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo2, pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: (1) gutidrrez g: lancet 1992; 339:195-202. (2) landow i: acta anaesthesiol scand 1994; 38: 629-639. the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in 34 critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p<0.05. according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb<8 and b: >sg/dl: i: 1ac<2.8 and ii: >2.smm. in either group blood transfusion results in zt significant increase in hb (a: 7.5_+0.4 to 9.4+0.8 g/dl; b: 9.(~0.8 tt, 10.5+0.09 g/dl; i: 8.0-+1.0 to 10.2-+0.6 jdl; i1:8.4-+0.9 to 10.1+0.7 g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: 467-+86 to 581-+158; b: 521+125 to 577+137; 1:512 -+1 13 to 599-+141; i1:516-+123 to 632-+214), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i68-+148 to 158-+38; b: 180-+162 to 175-+52; i: 171-+38 tu 179-+35; 11: 199-+60 to 219+_71); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of >7.5-+0.4 g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in 0.1% of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a 48-years-old worn an with a breast cancer, diagnosed in 1989, initial staging t3nim0, treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of 678 mg/m2 until august 1994. the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of 100 mg paclitaxel, given over 1 hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to 20%. she died 20 days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selectin (selam-1). sicam-1, svcam-1, selam-1, and inflammatory cytokines were determined. methods: sicam-1, svcam-1, and selam-1 were determined by elisa. tnf-a, il-6, and il-8 were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam-1 and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam-1 level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il-8, but the level of il-6. discussion and conclusion: the slcam-1 and svcam-1 levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il-8 also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam-1, elam-1, and vcam-1, appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut-175, reduced release of inflammatory cytokines (tnf, il-lg, il-6), activation of complement (c3a, c4a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut-175 on soluble adhesion molecules following cpb surgery. 20 patients undergoing cpb surgery were divided into two groups, group a consisted of 10 patients who received 1omg of fut-175 in priming solution, followed by a continuous infusion at 2mg/kg/hr during cpb in addition to initial heparin dose of 3mg/kg. group b, a control group, included 10 patients who were injected with heparin only. the plasma slcam-1, selam-1, and svcam-1 concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam-1 and slcam-1 reached their peaks on 3 hours after cpb and on pod 1 respectively in both groups, but they remained lower in group a (selam-i: 32.1+11.5 vs. 38.6• ng/ml, p<0.05, slcam-i: 327• vs. 483• ng/ml, p<0.05), svcam-1, in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut-175 reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, 1l-2, il-6, il-8, s-cd-14 and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of 10 critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: 100-130 ml/h, filtration rate 500 ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock (4), the so called hepatorenal s~aldrome (3) and a severe pancreatitis (3). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha (5 cases), scd 14 (9 cases), il-2 (l case) and il-6 (4 cases), hemofiltrates contained no activities. only il-8 was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il-1 and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il-2, il-6 could not be eliminated in cvvh with a filtration rate to 500 ml/h. in contrast to findings of other investigators with a higher filtration rate (> 1000 ml/h), we found no significant concentrations of tnf-alpha and il 6 in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (>1000 ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~4ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed 19 times using nafamostat mesilate as an anticoagulant in 5 hours with 12 l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~1-proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = 25 consecutive pts treated with hd; n 2 = 25 consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the 5 first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = 22 _+ 7; mean number of organ system failure (osf) = 1.5 -+ 1; mean blood pressure (mbp) = 75 • 22 mmhg (first day of application of hd). cvvhf-group : apachen score : 25 + 6; osf = 3 -+ 1; mbp = 57 + 20 mmhg (first day of application of cwhf discussion : during the 5 first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) 20 patients with mof were treated with cvvh in the period between january in 1992 to march in 1995. the mean (• age of our patient population was 52,1• years, being 65% male and 35% female the whole patient population was with mof iust at the moment the technique was accomplished; 75% was in mechanical ventilation, 90% needed vasopressor support and 65% required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was 17,84~:6,97 (range 5-28) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine 1, st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta 2 and dopaminergic receptor agonist. the specific beta 2 adrenoceptor antagonist ici 118551 has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs (25-30kg) divided into 2 groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the 2 groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and 200 pg/kg ici 118551 bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at 10-12mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici 118551 blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b2 antagonist blocked any protective effect of endogenous beta 2 adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr0chloride, a beta 2 and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta 2 adrenoceptor antagonist ici 118551 was infused to block any protective effect of dopexamine. nine anaesthetised pigs (25-30kg) were randomised into 3 groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and 5 ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition 200 pg/kg ici 118527 bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at 10-12mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. 30.5_+4.1, dopexamine 13.5+2.9", blocker 31.5+3.7. data expressed as mean + sd. significance p<0.05. * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the 62 antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta 2 adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) (7 patients) with bilirubin >3.5 mg/dl and lung injury score <1.8, it) mof-ards (9 patients) with respective values <2.0 and >2, iii) 5 patients with head injury with respective values <2 and <1, served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index >300 ml/min/m2 prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was 7.2_+0.8 vs 6.9_+0.3 (ns) and 6.3_+0.6 respectively, of the paop was 8.7+_1.6 vs 10+:3 (ns) and 8.2+3.1 respectively and of the cvp was 12.+.2.3 vs 11.6+2.3 (ns) and 5.8 respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was 6.8+9.5 vs 1.20+0.7 (13<0.05) and 0.8_+0.2 respectively and lung injury score was 1. objectives: oxygen delivery (do2) and oxygen consumption (vo2) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo 2 determination by either indirect calorimetry (vo2m) or tick calculation (vo2c). the purpose of this study was to investigate the changes in vo2m and vo2c following do 2 increase. methods: the relatives of 24 critically ill patients (mean age 63 years, mean apache ii 24, mean mof-score 9) gave their written informed consent to participate in this institutionally approved, prospective study. do 2 was increased by fluid loading (hydroxyethylstarch 10%: mean volmne 750 ml, mean duration of infusion 80 min) and catecholamine support (dobutamine: mean dose 14,3 ~g/kg/min). changes in vo2m and v02c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor 7250 integrated in the ventilator 7200 (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in 25 patients with peritonitis of different origins (18 nonsurvivors and 7 survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol (1099 +_ 4,83 nmol/ i), aldosterone (0,895 • 0,687 nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=-0,539, p < 0,02 between cortisol and pao2; r = 0,817, p < 0,001 between cortisol and d (a-v) 02; olso renin -cao 2 r=-0,824, p < 0,001, renin d ~,vl o2 r = 0,626, p < 0,001). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b 2 and p6fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : 14 consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of 2 pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next 24 hrs fzom 516 + 140 ml/24 hrs to 817 + 229 ml/24 hrs (ns). data expres,uxl as mean + sem. three patients (21%) became polyuric with urine output >100 ml/hr within 3 days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was 5.09 + 0.84 days. serum potassium levels were well controlled. day 5 or immediate pre-dialysis levels were 4.48 + 0.19 mmol/l compared with pre-lreatment 4.67 + 0.2 mmol/l overall mortality in this series was 4/14 (28%). duration of acute dialysis in survivors with renal recovery was 16.8 +_ 1.82 days. 2 patients (14%) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: 13 patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co2 in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do2 and vo2 calculations and temperature) and determined the respiratory mechanics (compliance and pao2 /fie2 relatinship) before starting the procedure, after 10 minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler 30 and 60 minutes of zero fluid balance bemofiltration and after 120 minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for 10 minutes without filtered fluid production and after 60 minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in 5 dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate (1800 mosm/l), was injected in the dose of 10.o ml/ kg into v. cephalica antebrachii after 24 hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc02 p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco 2 and phi obtained simultaneously by gastric tonometry. material and method : in a patient (55 y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co 2 and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : 19 sets of data were obtained and are expressed in mean + sd. gastric pco z was 46,5+18 compared to 40,4+3.5 mmhg for portal pco 2. phi was 7.32+._0,17 vs 7.34+._o,04 for portal ph. no correlation was found for these 2 parameters. p (gast-a) c02 was 6.4+15 mm hg vs 2+1.6 mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = 0,61) [figure] but no correlation with p (gast-a) c02. obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange (1). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: 10 chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator 2 dilution method, with indocyanine green and the stable isotope h20 as tracers, was used to measure evlw (2). arterial bloodgases and endtidal co2 were registered. evlw data was compared to a group of 18 renal healthy patients (0). dcp n evlw, ml -pao2, mmhg h~o +, nmol/l control group 0 18 243 -2--61 l 10 332 _+ 91"* 13 -+ 3 38 _+4 crfgroup ii 10 269 -+ 84~ 11 +-2ns 34-+2"(" iii 10 283 +-78t 11 _+3ns 34-+2t ** p < 0.01 dcp i from dcp 0, t p < 0.01 dcp li or i1 from dcp i, :~ p < 0.001 dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco2 (5.3+0.6 kpa) and etco2 (5.2+0.8 kpa) were unchanged while h3 o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao2. the reduction in h30 + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao2 in bicarbonate dialysis. 1. prezant lung 1990; 168: 1-14.2. wallin j appl physio11994; 76: 1868-75. a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value 7.32, can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered 20 patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h2-bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every 30 minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below 7.32. results: among the 20 patients, 9 had a drop of phi below 7.32 during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other 6 patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi=7.25 and the second 6.97. the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the 20th day after operation and died on the 25th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other 4 patients with a fall of phi had a lowest phi=7.24. 7.18. 7.26. 7.28 respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the 25th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the 5th pest-operative day and a dehiscence of the surgical wound on the 8th day: the third one, operated on for a sigmoid carcinoma, had melena in 41h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these 4 patients were discharged alive from the hospital. the other 11 patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of 3 hours duraticn. all l:atients nm.'-~ms_(~lly va~2ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e 15 rain after the ~, ~ne at the middle, ~ne 15 rain before lhe erd ard one 30 rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re (0ve)emm 9 to 7.5 ~ ~, 2) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n 13 to 11 ~mg hg, 3) i~09 from 33 to 30.5 mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o.05) of: i) s~o~ from 67 to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc02 in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: 16 patients (15 males and 1 female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. 14 patients were operated on eleetively, 1 had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a 67 month period (1/89 -7/94), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to 12,489 deliveries occurring during that time period for a .22% rate. the following procedural indications for icu transfer occured: 4 insertions of endotracheal tubes, 2 arterial catheterizations, 1 temporary tracheostumy, and 1 venous catheter. the following medical diagnoses were found: 5 hypertensive/eclamptic episodes, 2 hypotensive episodes, 1 pulmonary embolism, 1 mitral stenosis, and 1 coagulation deficiency. there were four episodes of respiratory failure (14%). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for 7 months in a multidisciplinary 18-bed icu. methods: data from 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995 were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm0, mpm24 and odin. data from 2453 days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = 90%) were evaluated from data collected during the day of admission (saps ii, rpm0, odin) or after 24 hrs (mpm24). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary 18-bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995. data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied 176 men and 94 women, with a mean age of 55 years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from 6 clinical variables and 6 laboratory items for 200 patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i1. thresholds were calculated with the youden's test (se+sp-1). results : 23 patients were admitted in the intensive care unit (icu)and 177 patients in medical units; 23 patients died. the saps ii was higher in patients which died than the survivors (31,0+11,4 vs. 1,9+9,3, p<0,0001 ; respectively), the best threshold was 24, se was de 86%, sp was 92%, the ac was 920/0. thus for patients admitted in icu than in medical unit (27,0:1:13,5 vs. 12,5+9,9 , p<0,0001; respectively); the best threshold was 21, se was 69%, the sp was 78%, the ac was 77%. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a 16 bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, 52 hiv positive and 350 hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure (71% versus 49%, p < 0.05) and septic shock (39% versus 15%, p , 0:05) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. 1, n= 112) and january-march 1994 (period 2, n= 127) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache 11 scores. the icu and hospital mortalities decreased from 33.8% to 27.6% (p=ns) and 38.2% and 34.2% (p=ns) respectively in period 2. the mean icu and hospital stay was also decreased from 4.6 __+ 7.1 to 3.6 + 4.0 days (p=ns) and 19.3 __+ 357 to 17.2 • 22.2 days (p=ns) respectively. the improved micu length of stay for survivors in period 2 was statistically significant (4.2 • 4.2 days vs 2.8 • 2.8 days = 0.015). there was no significant differences in the vse of peritoneal dialysis (5.4% vs 6.3%) and mechanical ventilation (55.4% vs 49.6%). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from 0% in both to 23.6% and 5.5% respectively in period 2. conclusion: the duration of critical illness was reduced in period 2. hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, 59 ald patients (37 males; 22 females; median age 46 years, median apache ii score 19; hospital mortality 68%)with severe complications (30 [50%] with bleeding oesophageal varices, 16 [27%] with hepatorenel failure, 5 [9%] with respiratory failure, 3 [5%] with septic shock, and 5 [9%] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians 12 and 20, p<0.0003), 2 or more organ systems in failure in the first 24 hours of icu admission was associated with a 91% (21123) mortality. mortality was also related to child's grading and the numbers of organs supported. of the 29 patients who received more than one form of organ support only one survived. organs supported mortality a (n=2) 0% none 3/13; 23% b (n=6) 33.3% one 8/17; 47% c (n=51) 74.5% two or more 28/29; 97% the icu costs for the 59 patients were only s 440 but s 687 (75%, 216 patient days) was utilised by the 40 patients who died, giving an effective cost per survivor (ecps) of s 496. although the cost of treating the 23 patients with 2 or more organ systems in failure on the day of admission was s 510 (36% of the budget) the ecps was s 755. conclusion patients with ald who have 2 or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which 107 surgical patients were randomly assigned to either a control group (n = 549 to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = 53) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than 600 ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at 1993/94 prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality 28 days postoperatively. results: in the protocol group, there was a 75 % reduction in mortality 28 days postoperatively (p = 0.015), a significant reduction in the number of complications (p = 0.008) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a 94.3 % survival rate 28 days postoperatively was s 9,894, resulting in an average cost of s 10,488 per surviving protocol patient. the average cost of a control patient was s 11,331 to achieve a 77.8 % survival rate 28 days postoperatively, resulting in an average cost of s 14,568 per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s 1,436 per patient together with a 75 % reduction in mortality at 28 days postoperatively. hence, the cost per surviving protocol patient was s 4079.8 less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet 1988; 2: 1392-94; madl et al. lancet 1993; 341: 855-58 ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: 90 recordings of standard bilateral sensory evoked potentials were performed in 25 patients with acute liver failure. findings were focused on cortical n70 peak -a stable negative deflection occuring 70 • 9 ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, 8 patients were referred to emergency liver transplantation, and 8 patients died. in all 9 patients who recovered spontaneously, n70 peak was detectable between 74 and 162 ms. all 8 patients who were referred to liver transplantation and 7 of 8 patients who died, developed a loss of a prior detectable n70 peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year 1993 were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \2-test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows 6.0.1 was used for statistical calculations. results: in-unit mortality: 184/1853 patients (9.9%) --oth 20.0% > as 10.6%> ni 9.1% > ed 6.8%; p = 0.01. in-hospital mortality: 365/1853 patients (19.7%) --oth 30.9% > ni 20.4% > as 19.6% > ed 16.2 %; p = 0,05. mean survival time in days after discharge: as 620 < ni 682 < oth 708 < ed 777; p = 0.66. conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january 1990 asd ]lay 1993 data from 1,425 patients over 14 years of aqe who retained in the icu for mare than 24 hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s 17.2% and in-hospitul mortality 22.7%. patients who died showed a higher spas score then survivors (15,3 ~ i0,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection (1~ 7.18; 95% el, 1.9 to 27.1), pmo~othor~x related to mocasnical ventilation (@ 6.28; 95% cl, 1.7 to 22.3) and delay in the insertion of a fln~-quidod catheter (ii~ 5.49; 95% ic, i.i to 26.9). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~5, j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~4i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january 1990 and april 1994, all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within 24 hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. 0eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi3tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of 815 intnmtsd patients amre stndied. self-extu~atien occurred in 54 (6.6%) patients and 25.6% required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe (52.1 v_s 60.4 years, p = 0.~02), ~verity of illness (11.4 ~ 13.1 spas score, p = 0.02), dia~isstia category (4s.6% v_s 66.7% of patients with res~iratury conditiono, p =0,002} and mean length of stay (10,9 ~ 20,7 days~ p = 0.05) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a 0.4 iir for mortality (95% ci, 0.i to 0.9) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber 1993 all patients who fulfilled criteria to be almittod to the ic0 who for waste~r reason retained in tile observation unit for more than 24 hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of 1,425 patients who suffered no delay is admission to icii over a period of 3 years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~9&ults: a total of 50 patients remained in the observation nit for more than 24 hours with a del w in igd admission of 55.8 _+ 25.1 hoers. assisted mechanical ventilation was requited in 22% of patients and only monitericatien in 46%. itsse patients were cspared with 112 ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn 16% as compared with 17.5% for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = 0.779; 95% ci, 0,2 to 2,5). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for 24 borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: 150 patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was 9 for survivors and t6 for non-survivors (p < 0.001). the mortality ratio was 1.1 and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent (233) of icu bed days were occupied by 4% of patients (non-sarvivors) which resulted in cancellation of 60 cardiac sot#cat sessions in 6 momhs. conclusions: this study concludes that apache 1t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a 30 day period. 2) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december 1990 through to april 1992, patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of 7 organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until 30 days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score=7, maximum 30 day off scorn=210, sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at 0.05 (two-sided), with n/group = 2[(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as 30 to 50 patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from 40% to 30% (25% relative risk reduction) where the n/group=356. scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. 2. non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" 3. autonomy: in every particular medical attendance, the patient has ability to decide by himself. 4. justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. (1-84) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p =0.51 ). the mean aiii score when considering all admissions was 59,9 (8-153) . the initial score between survivors and non survivors showed ststistical difference (48.6 vs 92.5) respectively (p < 0.0001). univariate logistic regresion analysis demostrated a 90% increment in death probability for every 10 points augmentation in the aiii score with a sensitlbity of 94.9% and specificity of 62.7%, the roc curve showed that the best cut off point for death prediction was 75 points with a sensitivity of 75.6% and specificity of 79.9%. if a patient is classified as high risk (> 75) the bayesian analysis showed a 52.8 probability of death and for one class(fed as low risk (<75) a death probability < 10%. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > 75 points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama 1994, 272: 619] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, 160 co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. 1986, 14: 777-782] and then a 5 step verbal scale (pqolv): best, good, fair, poor, worst. of the 160 studied patients, at the first interview, 116 were able to use both scales, but 44 (27.5%) understood only the verbal one. at the second interview, 8 patients were not able to answer, 113 used both scales and 39 only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, 42 pts (1.6%) died in icu. they were 33 males (1.5%) and 9 females (1.6%). their mean age was 66 (+7) years and mean ef was 0.38 (+0.1). nineteen pts (45%) had low (<0.35) preoperative ef. mortality was 0.9% in the coronary artery bypass grafting (cabg) group (n=2014) and 2.8% in the valve replacement (vr) group (n=359). in the cabg +vr group, mortality was 8.4% (n=95), and 3.3% in the remaining pts (n=149). cardiogenic shock was the sole cause of death in 24 pts (57%), septic shock in 6 pts, whereas sepsis in combination with ards in 4 pts, sepsis and stroke in two pts. in addition, 6 pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p<0.001) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: 1. although cardiogenic shock is the main cause of death (57%)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. 2. patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. 3. although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of 27 patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after 12 and up to 24 hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from 12/89 to 4/95 364 consecutive pts with ami were treated by primary ptca in the wuppertal heart center 9 27 pts (7,4%) were admitted to our hospital > 12 hours and < 24 hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was 62 years (49-78). 23 pts were male, four female. 37% had an anterior wall myocardial infarction, 63% suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in 70.4%, multi-vessel-disease in 29.6%. average time of onset of pain to recanalisation was 929 min (720-1440). angiography revealed timi-flow 0 in 85.2% of the pts, timi-flow i in 11.1%, timi-flow ii in 3.7%. average follow-up (fu) period was 12 months (4-28 months). timi iii lv-ef ~ 30-day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality 92.6% 58%/63% 7.4% 7.4% 3.7% 0% early mortality occured in the two pts, who were in cardiogenic shock at admission 9 no pt required emergency coronary artery bypass grafting.restenosis >50% was seen in 37% of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about 90% (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of 128 icu's representative of the italian icu system 9 methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the 3004 patients recruited during one month period, a total of 1813 patients were included in this study. for the purpose of the comparison of the two scores, patients with less than 18 years, or having cardiac surgery or staying in the icu less than 24 hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$01~: saps ii fits the data equally well compared to the older version (goodness-of-fit p=0.29 and 0 9 in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve 0.81 and 0.73, respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p--0.47) and discrimination (receiver operating characteristics area=0.80) were observed. c0nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: 1) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); 2) assess the efficiency by means of a resource use index (rui); 3) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. 1270 critical from 17 ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was 1.15 and it ranged from 0.58 to 2.05 in the 17 icus. the overall rui was 1 and it ranged l~ont 0.61 to 1.51. there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine 1993;21:1432-42): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on 1621 random admissions to 8 specialty icus with 90 beds (range 8-14) between february i and december 3 l, 1994. post-operative solid organ transplant recipients were excluded. units included 2 general medical, 2 general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active 1cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from 36.9-55.5; predicted risk of hospital death ranged from 8.5-21.1%. standardized mortality ratios ranged from 0.40 to 1.24 with 4 icus performing significantly better and 1 performing worse than predicted (p<0,05). los ratios and icu readmission rates ranged from 0.95 to 1.09 (ns) and 2.1 to 13.2% respectively. patients predicted at low risk of requiring active icu treatment ranged from 6,6 to 45.8% conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day 1 -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals (0-5; 6-10; 11-15; 16-20; > 20) , we compared tcu mortality within 6 age intervals (< 40; 41-50; 51-60; 61-70; 71-80; > 80 years -10, 11-15, 16-20) . in these groups mortality may be twice higher in the > 60 years patients than in the _< 60 years. mortality does not vary with age in low (physio a n = 0-5) and high (physio a n = > 20) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear (1994), 216 patients were treated at the unit, 45 of them died, and 16 ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only 142 patients were evaluated on the fu. the, were classified into the follov4ng three groups: acute medical, elective surge d 9 and acute and emergency postoperative. the patients were seen at 3, 6 and 12 months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~4ous jobs. apache 11 scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that 19/142 patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and 20% showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only 7% had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii0 i mpmp ii24), apach ii end gaprik were evaluated o~ 456 patients with acute myocardial infurction admitted to 17 intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii24cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii24, saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii24, and apache-saps were adapted and reduced to 5 (mpm ii24cor) end to 4 variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to 5 or 4 variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included 343 consecutive intensive therapy unit (itu) admissions with an itu stay>24 hrs in a 18 month prospective study (exclusion criteria: burn injury and age <16 yrs). data were couectsd applying the criteria described by the developers [1, 2] . the definition of coma (mpm24ii) was modified and the best assessment within 24 in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was 58 yrs (ranse:16-92) with a male:female ratio of 1.6:1. the actual hospital mortality was 26.8%, mean predicted death rates were 22.8% (mpmz4ii) and 15.2% (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p<0.000l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion 50%: apache ]/i 77.1%, mpm24ii 71.4%). the area under the roc curve was 0.75 (ap iii) and 0.66 (mpm24ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~2-59, mpm24b ;(2-52, lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -<50% with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks >40%0 ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of 715 adult patients consecutively admitted to a medical-surgical itu over a period of 18 months. patients with burn injury, age < 16 yrs and itu stay < 4 hrs were excluded. using a eomputerlsed database, we routinely recorded 24 hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;(2) statistics and calibration curves [1 ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the 453 male and 262 female patients was 59 yrs (range: 16-92 yrs). of these patients, 64% were medical admissions, 17% were admired after emergency and 21% after elective surgery. the observed hospital mortality was 25.4%, the overall mean predicted death rate was 16.8%. mean predicted risks were siguifieanfiy greater for nonsurvivors (38.0 %o) than for survivors (9.6%, p<0.00l, t-test). apache iii showed good calibration (z2-~59, lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was 89.3% (decision criterion: 50%). the area under the roc curve was 87.6% confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than 24 houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected 5092 patients, in two months in summer in 1992 all over italy. regional data were echieved in a three years entlection (1990-i992) in lombardia' icus from archidia group (arehivio diagnostieo), including 10065 patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr162 observed patients account for 23.2% and 22% of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data (2.3%) and extremely low mortality in regional data (0.6%). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: 25.7% and 22.3%; 8.a.p.s. score: 10.6 and 13; iength of stay: %7 and 9). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively 47% vs. 57%, medical patients were 34% vs. 31% and traumatised were 19% vs. 13%. 92% of "observed patients" in national study and 93% in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than 90% did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients (19% vs. 13%); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during 1994. the authors collected data of patients outcome and predictor variables. overall mortality rate was 39,3%. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death (90%). average length of stay was 6.6 days ~. 55% patients died in the first ten hosiptal days and only 18% after 30 days. age was directly correlated with death 50% of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose (55%) and natrium (71%) were in optimal physiological values. serum proteins (72%) and haemoglobin (50%) levels were inversely related to death. multivariate showed that alveolo-arterio difference in 02 content was the most informative of all mortality predictors (mean value 22,4 mmhg in 90% patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. 1985; 13: 818-29) and the sepsis score of elebute and stoner (br. h surg. 1983; 70: 29-31) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past 8 years for patients in our icu was calculated = 62% (prior probability). the outcome of 230 patients who were admitted to our icu for > 72 hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died (21.6 i 7.2 v~s 25.6 • 6.5 and 10.9 • 5 v's 15.2 • 5.9 respectively p < 0.001). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at 0, 12 and 24 h while in the intervention group phi measurements were made 4 hourly for 24 h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure >70 mmhg, systolic arterial pressure >90 mmhg, urine output >0.5/ml/kg, haemoglobin >8 g/dl, blood glucose < 12 mmol/1, arterial oxygen saturation >94% and correction of uncompensated respiratory acidosis. if the phi was < 7.35 after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at 5 then 10 mcg/kg/h, titrated against phi. this additional therapy was continued until 24 h after entry into the study. in each year patients were subdivided in two series with random selection, so that the 1st series contained abeat 2/3 and the 2nd 1/3 of the patients. the 1st series of all the years constituted the devdoping data set and the 2nd series the validation data set. with data of the 1st series (642 patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die 1st, 5th, 10th and 15th day, calculating for each lime the apache ii score (for a total of 1444 records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january 1994 and february 1995,185 anonymous questionnaires were sent to 20 intensive care units, all over greece. results : 107 questionnaires (57,7%) were replied and returned back. of them 58,9% were answered by male and 41,1% by female. the doctors replied in the following rate :81,2% aged up to 34,80% aged between 3544 and 94,4% aged over 45. 36 questions were answered and were divided into 3 main topics, as following: 1. admission criteria: limited bed availability was the main cause for refusing admission in 54,5% of icu's. 54,5% evaluated each case's viability and only 10,3% used some prognostic score system. 21,5% of icu's accepted all cases and a significant percentage (64%) gave in to pressure coming from their colleagues (72,7% female and 58,7% male). 2. informing the patient/relatives: only 6,5% was willing to tell the whole truth, while 39,2% had given selective information.. in the case of iatrogenic incident, 58,9% withheld it, because either they feared legal implications (34,6%), or lost of trust (46,7%). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of 82,3%, while only 55,1% found informed consent necessary for the proposed treatment procedure. 3. withdrawal of therapy/dnr orders/organ donation: 80,4% were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in 34,6% such a decis~n was unanimous, while the percentage of those carrying it out was 69,1% (72,2% female, 63,9% male). in case of brain stem death 87,8% (67,3% female, 85,7% male) withdrew any life support. 67,3% would like therapy withdrawal to be legally established, while only 12,1% would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only 11,2%. 83,2% considered organ donation to be a necessary proposal, while 10,3% refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it (3,7%), or because they doubted the procedures' objectivity (4,7%). note: in greece, icu beds are less than 1% from the total number of hospital beds available. only a percentage of 35-50% of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of 25 consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n=14) and mitral valve replacement (mvr) (n=t 1). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at 1 hour and at 6 hours after operation. results: there were no significant hemodynamic changes during the first 6 hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t9 trauma and 26 sepsis patients randomly received either 0.25 mg/h (group i, n=15) or 0.5 mg/h (group 2, n=15) of enalaprilat i.v. or saline solution (control, n=15) as placebo for 5 days. plasma levels of endothelin-1 (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next 5 days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion (0.25mg/h: from 53.1• to 22.1• pg/ml; 0.50mg/h: 62.1• to 17.9• whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p<0.01) and decreased after 0.50mg/h of enalaprilat. et remained almostunchanged in group 2, whereas et increased significantly in the control patients (from 4.9• to 10.t• on the 5th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p<0.01), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied 8 adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, 15 min after the patient was placed in a trp and again 15 min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo 2 monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo 2 by monitoring partial concentration of o2and co 2 ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed 15 min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco 2 without a similar trend in vo 2 values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after 15 min in trp. evaluation of vo 2 from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after 15 rain. since vo 2 did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo 2 and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied 10 adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for 3 hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo 20ximetrix, abbott) allowing continuous svo 2 monitoring was 5sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for 3 hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for 3 hours and/or successful weaning (s-w). (j physiol 1995; 78." 696-701) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and 3% co2 inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, 25 year, performed three incremental exercise tests in a randomized order: 1) inspiring air without vd (air control, ac); 2) inspiring air with vd of 920 ml (avd); 3) inspiring 3% co2; 21% oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to 90% peak hr. results: ventilation (vi) was significantly greater (p<0.0001) during the avd and co2 tests than during the ac test at the same work rates. end-tidal co2 (petco2) and estimated arterial co2 (paco2) were significantly greater (p<0.01) at 150 w and 200 w. oxygen saturation was significantly lower (p<0.05) during the avd test than during the ac and 3% co2 exerdse. at matched hrequivalent to 90% peak hr, vi was significantly greater (p<0.01) during the avd and 3% co2 tests than during the ac exerdse (123 l, 121 l, and 91/). conclusion: we conclude that the increase in xri and petco2 due to vd loading and 3% co2 inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu (46 men and 12 women; mean age 50.6 4-16.02 years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was 3.01 4-2.13 hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt (0, 3, 6 and 9 hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (<15 hours). table i list the variation of plasma eoneenlrations of tbars (mean 4-sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe 0 time cuncentzatiens reveal a difference p<o.01 between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~).05) at 3,6 and 9 hours fi-om de begin~g of'it. comparison ofthe 3,6 and 9 hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in 51 healthy subjects (group i) , 43 patients with several risk factors of ischaemic cardiopathy (group if) and 99 icu patients with possible aim (87 were confirmed ami (group ma) and 12 were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i11. electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h 4, s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< 0.001) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: 58 pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i (29 pts) -p:ithout d and group ii (29 pts) -d ~as administered 0.15 mg/kg iv bolus 12 hours prior to anesthesia and 0.15 mg/kg/h upon the end of operation and for at least 12 hours post-operatively. iv was substituted by oval administration of 2x120 mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during 90 minutes starting 5 minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of 1o0 fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period (4 hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), 60 % survived, the remainder died of incomplete recovery. in vi with bvad, only 40 % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in 67 patients by comparing paopee, paoptmc, and paopnadir obtained within the first 3 seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in 38 patients (group a) but present in 29 others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : 11 +_ 2 ; gr b : 11 _+ 3 cmh20 ). results : l. gr a : paopee (12 _+ 6 mmhg) differed (p < 10 -3) from paopnadir (8 • 6 mmhg) and paoptmc (8 _+ 6 mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves (14 "2-6, 12 + 6, 10 + 6 mmhg respectively). 2. good correlatkm (r (i.99 ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). 3. lung compliance (v t/bpah,) correlated well with i t in both groups (r -0.92), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in 16 consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (>50 ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) 4 hours after operation, e) 22 hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline 22 hours after operation. methods: 10 patients with preoperative lvef<40% undergoing coronary artery bypass grafting were studied. after surgery, a 3f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z-02t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m2), intrathoracic bood volume (itbv,ml/m2), pulmonary blood volume (pbv,ml/m2) and extravascular lung water (evlw,ml/kg). with a 7f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while 02 extraction ratio (o2exr,%) and oxygen delivery (do2,ml/min/m2) was calculed. peak inspiratory pressure (pawp,cmh20) and mean airway pressure (mawp,cmh20) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after 60 minutes (to) of volume controlled standard ratio ventilation (vc), and after 60 minutes (ti) of stabilisation period of pcirv (67% inspiratory time, 0 % pause). vt,ve and total peep were held constant in every mode of ventilation. 1+_0.4" *'p < 0,05 versus to conclusions: these data show that pcirv 2:1 is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo2. paw eight patients (12 melee end 16 females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so2/co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo2 were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o.05) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do2 and tissue oxygenation (pro2) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do2 and pro2. objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight 17+2 kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio 2 0.3). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao2) and mixed venous (pvo2) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, 300 pm microspheres (sephadex g50 coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to 45 mmhg initially. 45 rain after embolisation when haemodynamics were in a steady state (control 1), inhaled no was administered. further measurements were taken 5 min after 40 ppm no (no 40), 5 min after 80 ppm no (no 80), and 10 min after discontinuation of no administration (control 2). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ 9oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in 1994 five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given 100rag of actij.yse as ~ 2hrs infusion, followed by heparin~single 5000j. ihjaction snd iefusion ie doses 300-400j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im 3.24. hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao 2 and sao 2 in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed 45% improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation (1ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv (1). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: 13 normothermic who were stable (< 20 % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after 30 rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit2 to insure both normal abg and a peak airway pressure < 35 cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi.3,5) with synchfjv runs interposed (hfjvz4). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > 40% ; n=10 and < 40%; n=4) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than 50x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate 3h-bpap. trace mnou~ of ~fi-bpap (4gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of 1.5pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g0-a:3%, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to 21• reflecting a 85• inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to 52:l:10%, representing a 50-a:15% inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as 6.8x10 "4 sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi2) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi2 and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from 1664 + 75 to 1054 -+ 93dyn*s*cm 4 (p<0.001) and pulmonary artery pressure from 63.3 + 3.1 to 528 + 3.4 mmhg (p < 0.05), cardiac output increased from 2.66 + 0.11 to 3.57 _+ 0.16 i/rain (p < 0.001), mixed venous oxygen saturation from 49.6 _+ 2.2 to 63.3 + 2.8 % (p < 0.001) and arterial oxygen saturation from 87.9 + 2.6 to 93.6 _+ 2.2 % (mean _+ sem of 7 trials in 3 patients). 5-month iloprost nebulisation in one patient (100 gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi2 or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selecfin (selam-1) levels in 27 patients with acute myocardial infarction admitted within 6 hours after onset. peripheral venous plasma-samples were collected at the time of admission, 12, 24, 36, 48, and 72 hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into 3 groups as follows: group 1; killip's class (k) 1 and 2 without thrombolytie therapy, group 2; k 1 and 2 with thrombolytic therapy and group 3; k 3 and 4. both plasma sicam-1 and svcam-1 concentrations in group 2 and 3 were elevated rapidly and significantly and maintained at a high level during the first 3 days. plasma selam-1 level did not change in any of the groups. these results suggest that the adhesion molecules icam-1 and vcam-1 may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no27no3-(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were 15 patients admitted within 3 hours after onset. venous blood samples were collected at 3-hour intervals on the first day, 6-bour intervals on the 2nd day and 12-hour intervals on the 3rd day and 4 th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about 24 hours and 2-3 days after onset (basal level; 32.8_+4.9, first peak; 42.0!-_7.0, second peak;47.0+7.6 ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of 60 hours after onset were significantly correlated with maximal plasma creatine kinase level (r=0.83, p<0.01). the levels of plasma nox in the early stage of myocardial infarction (from admission to the 4th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range 36-85) fullfilling the high risk criteria of shoemaker (colectomy 13, gastrectomy 10, pancreaticoduodenectomy 4, others 6). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci >2.5 l/min/m 2, pcwp >10 mmhg, hb >100 g/l, sat2 >.94. objectives: evaluate the acute effects of 0,08 mg ipratropium bromide and 0,2 mg fenoterol (ibf) inhaled dose on pulmonary function in 17 nonsmocers (nb:m) and 14 smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and 12 healthy subjects. methods: pulmonary function tests were performed < 3h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred 4 puffs-ipratropium bromide (0,08rag) and fenoterol (0,2 rag). for 0,5 h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in 1 second(eevt) was 8,1% (p<0,00t) improvement and 3,9% (p<0,ob), forced expiratory flow betwen 25% and 75% of the forced vital capacity (fef25.75) was 30,8% (p<0,001) and 23,6% (p<0,001) and maxamal voluntary ventilation (mw) was 21,7% (p<0,05) and 16,2% (p<o,05) improvement. the mean changes in normal subjects were mild (fev 1 increased by 3,4%,fef 25-75 by 14,5% and mvv by 6,3%). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group 1 (n=13) received both imas as grafts and group 2 (n=12) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao2/fio2 ratio was measured in all patients in the icu, at 30,120,240 minutes on mechanical ventilation (mv), at 30, 120, 240 minutes after extubation (ex) and on postoperative day (pod) 5. in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group 1 had significantly longer bypass (101_+23 vs 70+15. p<0,001) and aortic cross clamp time (45+-19 vs 28+7, p=0,009). pao2/fi02 ratio was significantly higher in group 1 only at 30 minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group 2 (13+_3 vs 9_+2, p=0,004), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group 2 and one case of pulmonary infection in each group. after extubation 30 m[n 120 min 240min 30min* 120rain 240min pod 5 1' 342_+87 302_+92 369+87 278-+67 2664-79 294-+102 376_+76 2 319_+105 324_+89 320_+76 223-+52 257_+94 249_+82 400-+43 mean_+sd, *p=o, o03 conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in 10 adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after 4 minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in 8 patients without previous pulmonary disease undergoing elective cardiac surgery. there were 6 males and 2 females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were 69_.27 and 39• min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb-4 h after 7 h after stemotomy closed chest cpb cpb estrs (cmh20/l) 17.7+4.9 19.94-5.6" 22.5_+5.8* 22.64-4.3* est cw " 6.7_+4.1 6.3• 6.61+4.09 6.5-+4.1 est l " 10.9--4.3 13.54-5.4" 15.9-_4.9" 16.1_+5.1" x + sd. * p < 0.05: anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first 4 hours and stabilizes 7 hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied 220 pts, 23% (50/220) developed dcm during the follow-up study. among this group 26% (13/50 pts) were in class i and ii and 64% (37/50 pts) in class iii and iv of the nyha classification. we divided this population in a acei+ (22 pts) and acei-(28 pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t4 and t8 type cell population and its t4/t8 ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography (3-de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new 3-de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of 16 idcm pts, mean age 58_+11 yrs, 70% male gender, using a combined 3-de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated 3-de software. computer processing and analysis of the tee images led to a 3-de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using 3-de derived indices. mean values of 3-de lvesv=149-+20 ml, lvedv=197_+32 ml, lvstv=48_+25 ml and lvef=24-+5% were obtained. for all these 3-de parameters, good correlations were observed with 2-de lv measurements obtained through biplane tee analysis of the lv cavity (r>.70; p<.01) as well as regional analysis of sequential 3-de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new 3-de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of 23 dcm patients, mean age 57_+11 yrs, 74% male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ 62% pts; group slvd-38% pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. 3-de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. 19+5 305-_8 0.02 simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with 250 (fl250) and 500 (fl 500) ml of dextl'an 40 (rhemacrodex| in 12 patients free of previous cardiopulmonary disease (66 • 3 yrs) with acf (ci < 2.5 l/rain/m2) due to angiographicalty proven mpe (miller score > 21) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = 0.83) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial 02 content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep (0-5-10-15 cmh20) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in 20 mechanically ventilated arf patients (lis = 2.6 ~-0.45 sd). according to the response to peep 15, two groups of patients were defined: group a (9 pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep5, rvef decreased at peep10 (~0.8%)~ rvesvi increased only at peep15 (+21.5%) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep5, 'rvesvi decreased only at peep15 (-21.4%), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the 4 levels& peep were studied. rvedvi and ci were significantly correlated in 10 out of:l 1 patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in 5 out of 9 patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in 8 patients of group b and in 2 patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is 73.2 + 8.56 nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only 52 • 6.71 nun hg is tolerated (p < 0.01). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after 15 minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at 5, 10 and 15 minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after 5 minutes of reperfusion there was a highly significant increase (p < 0.001) in the mda values (mean =2.00 /zmols/l). these returned to basal levels after 10 and 15 minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used 10 "large white" pigs. mean weight was 22 kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated 7 centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends 20 patients studied as two groups of 10 individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered 400 rag/8 hours of vitamin e (tocopherol acetate) 72 hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and 15 nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a 55-year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of 100 mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of 200.000 u/hr for 24 hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a 38 years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed (250.000 ui of streptokinase on days 23, 25, 27, 29). fluid drainage was improved and cardiac output was also improved (day 23 : 2.53 1.min "i, day 27 : 3.58 l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after 37 days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload (1,2) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of 4 successive breaths of increasing magnitude (5, 10, 15, and 20 ml/kg). a line of best fit is drawn between the 4 minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh20). in 14 mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of 7 ml/kg of plasma expander. the initial mean downslope of the rsvt was -.40 + .40 mmhg/cmh20. following volume loading the downslope decreased to -.23 + .44 (ns). at the same time, cardiac output (co) increased by .96 + 1.2 l/min (p<.02), end-diastolic area (determined by tee) increased from 18.5 + 6.9 to 20.3 + 7.1 cm2 (ns), and paop increased from 12 + 8 to 17 + 9 mmhg ( p < .001). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = .81) and the eda (r = .70). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion ~ 0.1 9g/kg/ rain with a map --< 90 mmhg. cardiovascular support was limited to na + dobutamine (db). 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t -1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to 8 h. 546c88 is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope 2hzo in aorta (q2v~2o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h20 (9.4 ml), 2h20 (0.6 ml) and icg (10 mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and 2h20 in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= 18 boluses) over group i (n=18 boluses) (1.3<-0.3 vs. 1.0+-0.1, p=0.04). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz (2) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope 2h20 for the estimation of extravascular lung water (evlw2hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg (10 rag) and 2h20 (0.6 ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh20 was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q2n2o, t202o) (1). data analysis: evlwg~av was reference for evlwzhzo calculated as q2hzo times the difference in mean transit time between t2nzo and rico (atm2n). as reference for atzn2o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for 2h20 was calculated as the sum of central blood volume and evlwg=v. 54 boluses were administrated in a group (i) of 6 anaesthetized pulmonary healthy sheep while q was altered. another 18 boluses were administrated in a group (ii) of 6 anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. 2 results: for 72 boluses h20 parameters were not significantly different from their respective reference parameter: at2vao 5.3+_3.5 s vs. atg~, 5.4+3.2 s, evlwzh2o 332-+195 ml vs. evlwg~,~ 338+169 ml. in group i the ratio between 2hzo parameters and respective reference parameters (n=54) were independent of qlco from 1.4 to 7.1 l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg (10 mg) in water (10 ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( 19 men, 2 women ) age from 18 to 56 were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in 24% (5pts), without high pvr-in 43% opts) and with left ventricular failure-in 33% (7pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than 90min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i8oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in 2 cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed-39~-pat~e~ts (273 males and 118 woman), average 59,8 years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture (0 ~ 45 ~ 75~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from 7.1 +__ 3.6 mm to 17.71 +_ 5.5 ram (p = 0.009) at 45 ~ tilt angle, and from 8.4 + 3.8 mm to 19.3 + 5.8 mm (p = 0.009) at 75" tilt angle. the tidal volume increased from 211 + 78 to 373 + 99 rut a145* tilt angle, and from 229 + 78 to 447 + 143 ml at 75* tilt angle (p = 0.009). two patients could not bear the horizontal position (0' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion (9.2 + 4.9 mm to 15.5 + 7.3 ram). after an overnight respiratory assistance, pao2 increased from 66.4 +_. 8.7 to 73 + 10.1 mmhg (17 = 0.015), sao2 increased from 91.1 + 2.5 % to 91.9 +_. 3 % (p = 0.015), and paco2 decreased from 52 + 6.4 to 46.4 +_. 4 mmhg (p = 0.015) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity 1 and 5 l/min) vs pressure triggering (-lcmh20) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in 8 stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized 15 min. runs using a bird 8400 st ventilator at zero peep (zeep). trigger values for pressure (-lcmh20) and flow (1 l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv (10.6+6.8 cmh:0 x sec) and a/c (10.1+2.5) with pressure triggering, as respect to psv (8.9+7.5, p<0.02) and a/c (5.8+4.4, p<0.001) with flow triggering (1 l!m). no differences were observed between 1 and 5 l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being 5.5+1.5 cmh20 (psv flow trigger) vs 8.1+1.5 (psv pressure trigger) and 4.4+_1.3 (a/c flow trigger) vs'f~5+l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs 1 . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung 2. we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in 6 anaesthetized and paralyzed piglets we applied short periods (30 s) of one-sided ventilation (10 breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied 1-to-r and r-to-i cross-talk at ventilatory rates of 10, 15 and 20 bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during 10 bpm the r-to-i crosstalk was 23 _+ 4.7 % (mean +__ sd) of the tidal volume to the right lung and the 1-to-r crosstalk 31 _ 6.3 % of the left tidal volume. both values increased at 20 bpm to 30 _ 4.1% (p < 0.05) and 39 _ 7.7 % (p < 0.01) respectively. the values at 15 bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of 13 administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = 8) or as a slow infusion (n = 5) in 10 infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every 3 minutes with a lung function cart (sensormedics 2600) without interrupting ventilation. 3 infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > 400 %, maximum r > 700 % of baseline value. retreatment was followed by an increase in r in all 3 patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i31ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores >3) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l.0 and a peep level <15 cme=o} was not able to get either a p.ojf=o= ratio >55 or a s.o= >85%. patients were sedated, paralyzed, and a ventilator (serve 900c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at 1.0 and peep removed. continuous gas flow (250• ml/kg] was humidified and jet delivered through a tube (7 ram id, 18 ml capacity, 0.09 ml/cm h=o compllancel ended in a nozzle (0.8 mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded 15 minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo2) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin2 and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin2. further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies 2 -25 hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies 2 -25 hz with the relative inspiratory time (ti) 0.2 -0.8. the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats (400-430 g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature 37~ the arterial pressure was monitored. after traeheotomy the metal cannula (2mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = 0.1 kpa, ti = 0.5. the dead space of ventilator including canula was 0.45 ml. the initial frequency was 2hz and 10 rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : 2 hz-->4 hz-->8 hz-->4 hz-->16 hz-->4 hz--~25 hz-->4 hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco2 = 40 + 2 mm hg. the arterial po2 was always above 70 mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt1 = 5.91 --+ 0.30 ml/kg for ft = 2 hz, vt 2 = 4.31 +0.12 mukg for fz =4 hz, vt3 =3.30 +_0.27 ml/kg forf3 = 8 hz, vm4=2.61+0.08ml/kg forf4=16hz andvmt= 2.18 + 0.13 mukg for fs = 25 hz (presented as mean values _+ s.d., n = 6 ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = 37. 5 * f-e.30 . conclusions: the described system allowing ventilation in a wide frequency range 2 -25 hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr 1448-3 and gacr nr 305. s122 intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on 6 ventilated patients (puritan bennett 7200ac with metabolic monitor pb 7250 set to measure end tidal co2). all measurements were repeated with the patient stabilised at 5cm. 10cm and 15cm peep. inclusion criteria were: 1) haemedynamic stab(l( .ty for 1 hr; 2) pulmonad" anon" flotation catheter in situ: 3) volume control ventilation with plateau of 0.5s: 4) fio2~ > 11.6 to maintain pao~. > 10 kpa with 5em peep: 5) qs/ot > 20%; 6) pao2/fio2 ratio <150. measured variab!es included: r162 minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co2; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p<0.01), compliance (p<t).05) and o,'gcn deliver, index (do2, p<0.02) occurred with increasing peep (table 1) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april 1995 were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of 59 men and 46 women were studied (n= 105). the mean distance of ett tip to carina was 4.1 cm. using the reference markings, 29 cases (27.6%) had the ett tip < 3 cm from the carina and 5 cases (4.8%) > 7 cm. one case resulted in an endobronchial intubation. the mean height of all patients were 167 cm (153-187) for males and 155 cm (140-170) for females. of the patients with ett tip < 3 cm from carina, the mean height was 163 cm and 151 cm respectively. ~2onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases (32.4%). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp-100 pulmonary monitor were recorded 24 hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre 33.4 _+ 11.8 to post 28.6 _+ 9.2, p < 0.05). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group 1 included eight patients who were completely weaned from ventilator; group 2 included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups (1.1 _+ 1.6 vs 2.7 + 1.4 in peepi; 47.3 _+ 36.9 vs 28.8 _+ 16.5 in cld, p < 0.05). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group 2 (3%5 % in group 1 vs 66.6 % in group 2). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first 6 hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were 25 survivors and 23 deaths in this series (mortality 48%).using arterialpco 2 values measured 2 hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co 2 retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was 90%. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was 97%. arterial co 2 accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po2, pc02) and the ph (abg) was examined in 20 stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for 9 and ps plus intermitted mantatory ventilation in ii. [n each patient, 6 speciments of abg were measured at 10 min intervals during a 1-11 study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was 3.45 percent for po2, 3.53 percent for pco2 and 2.27 percent for hco3. the average sd for ph was 0.008, the corresponding c for systolic bp, diastolic bp, cf, tv, rf were 4.35, 5.21, 3.68, 2.51, 4.18 percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in 496 patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in 104; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in 73 patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, 102 del, vo2 by improving the rheologic properties of blood and plasma, normalizes 02 ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by 1.3-1.4 limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co2 which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo2) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in 20 critically ill patients treated with mechanical ventilation for at least 48h, who fulfilled at least 3 of 4 standard weaning criteria (vt>5ml/kg; respiratory frecuency (f) <35; pimax > 20 cm h20; pao2/fio2 > 150). baseline measurements: t, vt, p0.1, pimax, f/vt, p0.1*(f/vt), p0.1/pimax. study protocol: measurement of vo2, vco 2 (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after 30 and 120 minutes of spontaneous ventilation with cpap 5 cm h20. the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, 16 were extubatad, and 2 of them had to be reintubated before 48h, being considered also weaning failures. during cmv, vo2/kg was 4.07 + 0.2 ml/kg/min, and 5.09 _+ 0.4 mlo-2/kg/min after 30' on cpap 5 cm h20 (p < 0,01 ). 14 of 15 patients (93%) with 4 standard criteria were extubated, while only 2 of 5 (40%) with 3 criteria (p<0,01). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa (0.06-0.09 ml/kg) in anesthetized, paralyzed, and intubated dogs (n=10) during volume controlled ventilation: rate=12/min, peep=5 cmh20, ti/ttot=0.3, fio2=0.6, vt=15 ml/kg. animals were rotated during the oa infusion and the following 90 minute stabilization period to assure uniform injury. in the supine position, peep was set 1-2 cmh20 above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of 35 cmh20: animals were ventilated in either the prone (n=5) or supine (n=5) position for four hours. pulmonary artery occlusion pressure was maintained constant (4-6 mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale 0-3). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at 2(10 to 10(10 cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two 30 rain periods of chfo (sequences : 6(10 and 901) c/rain : group l, n = 1 l: 900 and 600 c/rain : group 2, n = 8). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu2 and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa02 is not significantly modified during chfo at 600 or 900 c/rain. paco2 is slightly decreased at 600 c/rain (p = 0.(16). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. 2). there is no more effect of chfo for patieets who are at a flu2 higher than 0.50 (n = 9). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < 0.ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co 2 was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co 2 (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = 10): two levels of assist were studied; flow assist (fa) of 2 cmh20/i/sec and volume assist (va) of 2 cmh20/i (assist 1), and fa of 2 cmh20/i/sec and va of 4 cmh20/i (assist 2). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in 5 subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist 2) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco2 of 55 mmhg v t was increased significantly by 0.4+0.1 i and 0.7+0.2 i in protocol a with assist 1 end 2, respectively, and by 0.5_+0.1 i in protocol b with assist 2 (p<0.05). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ (50 mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco2 (59 mmhg), pdi and pmus at the end of neural inspiration decreased by 18.8-+8.3% and 11.7+15.7%, respectively, from control values. neither change was significant (p>0.05). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from 30 to 90 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in 8 subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol 8) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps (3-4 breaths each) from 30 to 70 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and 10 during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p<0.05); at 30 i/min ttot, expressed as % of that at 70 i/rain, was 110.2+_1.3% during sleep and 127.8+_3.9% during wakefulness. during wakefulness, at 30 i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was 60% of that at 70 i/min (p<0.05). the corresponding value during sleep, was 86% (p>0.05). in four sleeping subjects the increase in v~ was sustained for 1.5-2 min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only 3-4 breaths. we conclude that 1) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and 2) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively 14 asa i pediatrics patients (2 to 10 yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel 210 (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of 10 mm. id and 1.5 m. long (children < 30 kg), and a corrugated dar set of 15 mm. id and 1.5 m. long (children > 30 kg) were respectively used for ccl an cc2 values of 9.3 and 9.5 ml/cm h20. a vtof 10 mlg/kg and respiratory frequency was adjusted for an end-tidal co2 (etpco2) between 30 35 mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was 38.8 % and reached as high as 64.1%. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as 69.6 %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago-2) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically 111 patients. the ventilatory setting is pressure support mode. the pressure level and fit2 were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: 0'(immediately before h/d), 15', 30', 60', 120', 180', 240'. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp-100). the mean value of the breaths 5 minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po2 and pco> however, a potential problem in mechanically ventilated patients is that the 10 l mixing box (mb-10l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb-10l on the v c were compared with those produced by a new 1 l mixing box (mb-1 l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb-10l was substantially higher (vc= 7.4*ppeak) than that provoked by the new mb-1l (vc= 1.4*ppeak). at a ppeak = 50 cm h20 ~ the v c were 377 ml (mb-10l) and 67 m{ (mb-1l), respectively (p< 0.001). in a group of 6 subjects (4m/2f, 57_+6 years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb-1 l and mb-10l fell on the identity line. it is concluded that the new mb-1l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied 15 polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age 52 (37-71) yrs . they all were connected to servo ventilators siemens 900c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo2, swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with 5 cm h20 cpap for at least one hour. we turned them to psv with 0 inspiratory assistance (psv 0 cm h20) and after 60 rain we applied psv 10 cm h20, and after 60 min psv 20 cm h20 . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< 0.001 ) . respiratory rate (rr) decreased significantly (p<0.01) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do2) decreased ns, oxygen consumption (vo2) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo2 and pvo2 values in a group of patients, following open heart surgery. methods: upon transfer to icu, 67 patients (54 males and 13 females) of mean age 63_-+6 years, were randomly assigned to receive 0 (n=22), 5 (n=24), or 10 cm of peep (n=21). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of 10 ml/kg. the fraction of inspired oxygen (fio2) was adjusted to keep a pao2 around 100 mmhg. mixed venous po2 and svo2 were measured at 30 min, 4 and 8 hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at 4 hours post-extubation. differences at each study time were analysed by anova. results: mean svo2 and pvo2 values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo2 and pvo2) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two 7l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co 2 sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from 70 to 500ml, at rr from 10 to 120 bpm, while closely monitoring cpap airway pressure swings. at v v of 400 and 500ml the rr was limited to 60 bpm. for comparison we explored aps of a one 16l balloon cpap system, the cpap mode of the puritan bennett 7200, and siemens 300 ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one 7l balloon system was linear over a range from 1.0 to 3.3l, with a cpl. of 4.0 l/em h20.the cpl. of the 16 l balloon (0.5 l/em h20) was linear between a volume of 13 and 14.5 l. apf of the weighted balloon system was under 1 em h20 at all v r (except at a v r of 500ml aps was 1.5em h20), while the apf in the 16l balloon was up to 3 em h20. apf witli human volunteers with the two commercially available ventilators in the cpap mode was about 7 cm h20; while under identical conditions apf in the 16l balloon system was 1.5 emhzo; and in the two 7l balloon system was below lcm h20. conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < 25 crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co2 (etco2) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < 25 cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco2-etco2 variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) (224 + 46 ml vs 377 + 766 ml, p<0 001) two individual behaviours were observed on tl in pl: four patients presented low etco: (range 18 -31 mmhg)and normal pacoz (range 38 -42 mmhg), while three patients had normal etco2 (range 35 -45 mmhg) with high pac02 (range 44 -61 mmhg). one hour before stopping ilv (t2), vt, etc02 and paco2 were the same in each lung. the pao2/fio: ratio improved in all patients from the beginning ofllv cst of pl was 526 + 30 % of the normal lungs' cst on ti and improved to 97.6 + 27 % ofnl's cst on t2 (p<0.005 vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco2 and of the differential paco2-etco2 gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med 20:$41, 1994) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in 5 intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from 20 to 80% of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav 0%), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of 596 adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an 18 month period. we compared assist/controlled mandatory ventilation (s-cmv, 123 patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, 431 patients) and biphasic positive airway pressure ventilation (bipap, 42 patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation (10.1 h, p< 0.05) than patients treated with s-imv/-psv (14.7 h) and s-cmv (13.2 hi. with s-cmv 39.9% of the patients required single or multiple doses of midazolam but only 13.5% in the s-imv-/psv group and 9.5% in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group (8.8 mg) than in the s-imv/psv group (6.6 mg, p<0.05) and in the bipap group (4.3 mg, p<0.05). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p<0.05). after extubation the paco2 patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~2 +pst, rs (bates) [1] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r 2 is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h 1 -h 2 log t (hildebrandt) [2] where h~ and h 2 are parameters depending on lung volume, and initial time is 1 s for analytical reasons. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of 200 hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. 1 was 0,912 -+ 0.168, and using eq. 2 was 0.884 + 0.045. the values ofz~ (eq. 1), however, decreased with increasing the tidal volume (vt) according to the following equation: "~2 = 1.52 -0.65 v t, similary, the values of h~ and h 2 increased with increasing v t according to the following functions: h~ = 4.4 + 13 v i and h 2 = 1.15 + 1.88 v t. conclusions: the behaviour of "% of eq. 1 suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range (6-14 ml/kg) in ventilated patients. infect this model predicts that "c 2 is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r 2 obtained by fitting an exponential for data of eq. 2, is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r 1 2 1 wh t lsms ira tlmeand r given by:pv~c.~ = 2d~( -'e-~ )[ ] ere " ' p" tory " 2 and "r 2 are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c 2 and r 2 which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z 2 during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z2 + e~t.r= [2] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~2 by analyzing the time-course of ptr according to eq 2 and next determining r 2 according to eq. 1, using the expedmental values of p,i=~, ~ and ti, as well as "~2 obtained with eq. 2. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of 200 hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. 2 was 0.912. with v t of 7 ml/kg, the mean values (+ sd) of ':2 and r 2 of the 8 subjects amounted to 1.128 • 0.100 s and 3.990 • 0.890 cmh20 i "~ s. with the traditional multi breath method the corresponding values were 0.711 + 0.257 s and 4.445 _+ 1.474 cmh20 i "1 s, respectively. with the t-test the difference between new and traditional "~2 was statistically significant, between new and traditional r2 was not significant. conclusions: with the single breath method it is possible to compute ':2 and r 2. the mean values of r 2 with v t of 7 nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term 02 therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during 1993-94. a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. 1) post tb respiratow failure 6(26%) 2) neuromuscular diseases, 3(21%) 3} undiagnosed sas 3{13%) 4) cope) 9(39%) (3 patients had a overlap syndrom). the programme consists of : 1) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). 2) group and individual respiratory therapy 3) mobilization 4) nutritional support 5) educational classes for the members of the family. three from the patients passed away (during the year), 11 are under nippv during night with or without 02 supply, 13 pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in 28 decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in 28 decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao2/pao2) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients (64 + 6 yrs) suffering from ards (lung injury score > 2.5) for 48 hours or less entered into the study. irv (volume controlled, decelerating flow, 20 % inspiratory pause, lie = 2/1) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= 1/2). these two modes were applied for 6 hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume (8.0 • 0.7 ml/kg), respiratory rate (20 • 0"bpm) mad fit2 (63 • 2 %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after 1, 2, 4 and 6 hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support 0ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a 70-year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of 10 mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of 10 mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above 1000 ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * 1(30. rr162 18 of 50 patients examined showed moderate to severe desynchronization. in 17 of these 18 patients a (re)activation of the hbr was found. the strength of hbr amounted to 134 + 51%. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below 1000 ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined 20 patients after open-heart surgery and 50 patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in 13 of 37 intubated patients, 6 of which showed cs. in 4 of these 6 patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining 2 patients. in 1 of these 2 patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k2.w. the constant tube coefficients k~ and k2 are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k2 are changed rendering the p~,ch calculations inaccurate. therefore, k1 and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to 30%. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [1] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [2] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be intergil cano a, san pedro jm ~, sandar d, herntndez .1, carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: 1) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and 2) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than 40 mmhg or an absolute decrease in systolic blood pressure below to 90 mhg within 1 hour of intabatian. 14 patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h (8), cardiac arrest (5) .1 there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < 0.1). high paco 2 before lrasheal intubatian (87.4 4-9 mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < 0.01) in patients who developed h. paw. 3) thexe was a good relatienship between h and high arterial paco 2 before traqueal intahatian and its fast "washing" with mechanical ventilation. 4) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco 2 changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit (1). application of ips has been shown to partly compensate this added work (1). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd (2). therefore, atc has been developed as a new form of assisted spontaneous breathing (3), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls 4000, dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls 4000 was connected to an artificial trachea (at, 10 cm long, 22 mm id). the at was intubated with three different tubes of 7.0, 8.0, 9.0 mm id and connected to an evita 2 ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of 5 and 10 cm i420 and atc all with a peep of 10 cm h20. the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/1 are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<.05, **-p<01, ns = non significant, f versus s neveltheless, in 5/26 patients, invasive ventilation was necessary in mean 12.6_+12 hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps1 & 2, clinical signs (rr, spo2, heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : 1.a low value of pac02 on admission until the patients were intubated. 2. an increased level of cpk in relation with an acute myocardial infarction (4/5 cases in the failure group, vs 3/21 cases in the success group, x~(with continuity correction) : p<.05). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco2 (<35mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators (1, 2). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) (3, 4). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls 4000, driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls 4000 was connected to an artificial trachea (at), intubated with an 8.0 nun et]', end connected to one of four ventilators (servo 900c and servo 300, siemens,-elema, sweden; evita2, driiges, liibeck, frg; pb 7200ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips 5 and 10 mbar; trigger set at maximal sensitivity, peep always 10 mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied 31 paralyzed cppv ventilated ali patients (lung injury score =2.25• that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve 900c), first during cppv and then within the 24th psv hour. airway pressure and flow signals were recorded (cpi00 bicore) for subsequent analysis. an airway pressure plateau was defined as a 0 flow tracing in which airway pressure was stable for at least 0.25 sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; 2) during psv inspiratory plateaus are longer than the expiratory ones; 3) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = 6 patients). we selected 17 ali patients (lis=2.5• in vcv mode who had available a chest ct scan performed within 4 days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao2 >80 mmhg, irrespective of fie2 (peep <15 cmh20). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate <40 bpm, an increase in fie2 lower than 0.2 compared to vcv, a pace2 increase <20% of vcv value and hemodynamic stability were maintained during the next 48 hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with 10 ventilated patients, 7 after head injury and 3 with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep (0-5-15 cm h20). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: 7.3 + 2.5 for peep 0, 7.4 + 2.7 for peep 5 and 6.9 + 1.7 l/rain for peep 15. simultaneously the bioimpedance device recorded a significant increase in cardiac output from 4.4 + 1.3 for peep 0 to 6.0 + 1.9 l/mi for peep 15. (p < 0,05). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of 12 patients who had been hypercapnic (paco2>50mmhg) for at least 3 days (range 3 to 60 days) before the end of cv. the psv trial was started as soon as pao2 was > 80 mmhg, irrespective of fie2 and with peep < 15 cmh20 and the psv level had to be < (pplateau-peep) as measured during cv. pace2, pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: 05) . 2) weaning is more difficult in pts with head injury(p<o,05) and iss>30 (p<o,ol), in elderly(p<o,05) and in pts who need fi02>0,6 (p<o,05) and peep>io cm h20 (p<o,05). 3) pts with iss>30 need longer duration of mv (p<o,ol). 4) the mortality rate is higher in pts with iss>30 (p<o,o01), with head injury (p<o,ol) and in elderly (p<o,05). 5) paradoxally, compliance was found to be higher in pts>60 years than in pts<60 years (p<o,05). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in 4 patients (individual analysis for each of the eight lungs) at 4 peepe levels: peepe = 0 cmh20 (zeep) and peepe amounting to 50%, 100% and 150% of the peep• at zeep (9.5 • 4 cmh20). tidal volume was kept constant (8 • 1.4 ml/kg). each lung section was divided into 3 zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. 1st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: 4 pts with acute (a) (paco2= 39• mmhg, pao2/pao2= 0.4• and 4 pts with chronic (c)(paco2= 51• mmhg, pao2/pao2= 0.3:50.1) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh20*s/min), which is an index of oxygen muscle consumption. measurements were collected, after 15 rain of stabilization, at 5 and 15 cmh20 ps and at two different peep levels (0 and 8 cndd20 conclusions: during psv: 1) breathing pattern and ptp are different between a and c; 2) in both groups peep does not modify breathing pattern; 3) peep reduces ptp in c but not in a; 4) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p0.1 and rr. changes in p0.1 were remarkable, homogeneous and highly significant (p<.002), while changes in rr were less pronounced, inhomogeneous and less significant (p<.02). it seems therefore that p0.1 is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. 9 sets of measurements were obtained in each of 7 ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to 15% of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of 160 consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of 3 measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=.87.ctotlsf+l.14, r2=.982), and with cqs (cqs =.94.ctotlsf +1.19, r2=.984). the average difference between ctotlsf and cqs, yet, was much lower (+.36+1.79 ml/cmh20) than the one between ctotlsf and cdyn (+2.43+2.40 ml/cmh20). rtotlsf correlated much better with rmax (rmax=.88*rtotlso+.98, r2=.914) than with rmin (rmin =.58.rtotlsf +. 1.71, r2=.804). the average difference between rtotlsf and rmin was +4.48+3.48 cmh20/1/s, while the one between rtotlsf and rmax was +.81+2.03, confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. 9 sets of measurements were obtained in 7 mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these 3 reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all 3 reference measurements, rcdyn (ve/v'epeak= 1.07orcdyn-.18, r2=.939), rcstat (ve/v'epeak = .97 9 rcstat -. 12, r2= .929), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator 900c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period 1985-1995 we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao2/fio 2 < 100, despite maximal ventilator settings: peep > 8 cm hz0 , fit 2 > 0.6. a total of 43 patients matched these criteria, 27 males and 16 females with a median age of 44 (17-72) years. seventeen suffered from severe trauma. chfjv was started following a median period of 3 (1-22) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit 2 0.8, pao2/fio 2 78, peep 12 cm h20 peak airway pressure (pap) 48 cm h20. chfjv consisted of high frequency jet ventilation with a frequency of 100 to 300 breaths/minute, driving pressure of 1.8 to 3.5 arm, and inspiration time of 20 to 30 percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l0 to 20 breaths/minute and tidal volumes of 100 to 400 ml. results: following two days of chfjv 31 of 43 patients showed an improvement of ventilatory parameters; peep could be reduced to < 8 cm h20 in 14 patients, the pap was decreased with > 5 cm h:o in 20 patients, fio 2 could be reduced to < 0.6 in 27 patients and finally the median pao2/fio 2 ratio changed from 78 to 133. during chfjv 23 patients died, 4 of respiratory failure and 19 due to multiple organ failure, 6 died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was 6 days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm 1995; 151(4) :a432). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with 4 groups to ventilate 40 nonperfused, lavaged rat lungs with small vt (5 to 6 ml/kg) for 2 hours and studied the effect on compliance and lung injury. group 1: peep>ping group 2: peep<pin f with an si, inflation to 30 on h20 over 30 sec, to recruit volume; group 3: peep<pinf without si; group 4: control group, lungs were inflated at peep<pin f, but not ventilated. results: in group 2 and group 4 static compliance did not change after ventilation (table) . in group 3 static compliance fell significantly. group 1 showed significant changes in the pv curve, which were less severe than in group 3. results from morphological examination are pending but preliminary results showed less lung injury in group 2, compared to group 3. conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy (1) minimizes lung injury as well as, or better than using peep above pint" and (2) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ 2 _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles 42 eeg examinations with fourier spectral analysis' of 37 patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co2 and blood gases during weaning. results: 13 patients had invariable eeg activity or short 13-waves period (till one hour). the weaning of this patients was fast arid sucsessful. other 24 patients have had a decreasing of a-activity, an appearence of 13-waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u 296, hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett 7 200) but not others (siemens serve 300). we studied the two types of systems in two groups of 8 patients mechanically assisted with pressure support ventilation (15 + 6 cmh20). in the first group (pb 7 200) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt (139 + 40 cmh20.s/min -1 vs 158 + 32, p< 0.05). by contrast no significant difference appeared in the second group (serve 300), as predicted by the bench study despite marked interindividual differences (134 + 55 cmh20.s/min -1 vs 160 + 61, p = 0.1). we conclude that 1) rigorously performed bench studies can predict in vivo effects, 2) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs0, lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: 16 (8 arf and 8 ards) sedated paralysed patients were studied using a serve 900c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to 1500-2000 ml or a maximum paw of 50 cm h20 (alfi). the quasistatic elastic pressure (pel,qs0 was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v0 from 50 up to 1500-2000 ml were followed by a 3 s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~111i~: in ards, mean cst was 27.9 + 3.5 and cqst 29.7 + 3.9 ml/cm h20 (us), lipst 5.2 + 5.0 and lipqst 7.0 + 4.6 cm h20 (us), uipst 23.1 + 10.8 and uipqst 26.0 + 5~4 cm h20 (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, !983). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of 15 patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every 48 hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > 103 colony forming units / ml. results: for the 15 patients studied, the mean saps score (+ sd) was 16+_2, the initial pao2/fio2 ratio was 100-&-_35, the duration of mechanical ventilation (mv) was 19+9 days. the mean delay before the onset of the first np was 8.6+5.6 days (5-12), and the mean pao2/fio2 ratio was 110+-28. respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in 80% of the patients studied. alteration of gas exchange was present in 8 of the 15 patients (7 np) . a new pulmonary infiltrate was present in only 1 np (10%). an increase of fever was noted in 6 patients, an increase of leukocytosis > 20% in 8 patients, an increase of volume and purulence of sputum in 3 of the 10 patients with np. the degree ofgazometric worsening (pao2/fio2 before np minus pao2/fio2 during np) during the first episode of np was 44+17 mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was 1 in 1/10 patients, 2 (5/10), 3 (2/10) or 4 (2/10). two patients only had a pulmonary colonization (ptc: < 1102 cfu / ml) before the first episode of np. the incidence of np is high (53%) during severe ards. the first episode occurs in average:at the 9 th day, and is the cause of a severe hypoxemia (pao2/fio2 110) . the onset of a np may contribute to the high mortality rate observed in our patients (93%). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of 0.27 • j/l, a mean value of inefflency, ~ of 0.25 ~=0.05 and a mean value of resistance, 7~, of 4.40 • cm h20 s/1. the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ----3.90 • 0.66 cm h20 s/l. during methacholine provocation~ ae rises more than five-fold up to 1.48 • j/l~ doubles to 0.54 • and t~ increases to a maximum of 22 • cm h20 s/l, with armax : 15.1 • 7.0 cm h20 s/1. the variation in rk becomes more pronounced with higher doses of methacholine. methods: 10 ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with 20 breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group 1 patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo 2, and aado 2 decreased while paco 2 increased and tda vo ume and co remained unchanged. on the contrary, in group 2 v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo 2 and aado 2 increased with constant co, the pressure support level needed to achieve the target rate was much lower in group 1 than in group 2 (19,8-+1.3 vs 29.4_+2.0). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, 20-40 years)breathed spontaneously through an uncut ett of 7.5 mm id via a mouthpiece. the ett was connected with a prototype ventilator evita 2 modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, (1) asb (10 mbarover 5 mbar peep), (2) atcin, (3) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+1), ,,equal" (0) or ,,worse" (-1). inspiration and expiration were investigated separately by presenting 120 mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within 4 hours after cardiac arrest and repeated 3, 6, 9, 12, 18 and 24 hours later. results: we studied 10 patients, 6 females, mean age 64,1+13,7 years. the pi decreased s!gnificantly between th~ first and the last measurement from 1.86 _+ 1.02 to 1.05 + 0.22 (p = 0.03). cerebral oxygen extraction ratio decreased also from 0.39+ 0.13 to 0.24 + 0.11 (.p = 0.015). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels 24 hours later, rasp. 2.95 pmol/ml (median; 25th 2.48-75th 5.43) and 7.5 pmol/ml (median; 25th 6.2-75th 14.00) (p = 0.02). eighteen and 24 hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = 0.64, p = 0.05 and r = 0.76, p = 0.01). we.also found 12 hours after the first measurement a significant correlation between pi and cgmp levels ( r = 0.69, p = 0.03). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next 24 hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology (1,2). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged 34-48 years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model 7219d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m1 segment of the middle cerebral artery (mca). results: a total of 12 phases of vf were investigated. duration of vf ranged from 6 to 26 seconds, with cbfvmc a (mean_+sd, cm sec -1) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: 42 icu nurses were asked to inflate a testlung model 10 times with a specific air volume (600 ml, 800,ml or 1000 ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated 600 ml (250 ml). the mean inflated volume for this group was 843 ml. when the icu nurses had to inflate 800 ml the mean absolute volume difference was 181 ml with a mean inflated volume of 913 ml. inflating 1000 ml produced an absolute volume difference of 131 ml with an mean inflated volume of 1042 ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the 42 ml and 66 ml for all specific air volumes. 42% of the last 5 inflations with feedback were significantly smaller than 50 ml from the specific air volume (p < 0.05). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when 600 ml and the smallest when inflating 1000 ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in 11 domestic pigs. following 4 rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for 8 min. electrical defibrillation was then attempted and 6 of 11 animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of 1.13 ml/kg to 0.74 ml/kg (p<0.05), and left ventricular stroke work index (lvswi) from 1.57 to 0.77 mmhg,ml/kg (p<0.05). both svi and lvswi remained depressed for another 3 hours. these decreases were associated with increases in heart rate from 145 bpm to 185 bpm (p<0.05). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k6ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d-13353 bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach 30 -40% depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from 2 -31 min, mean 8 min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after 30 -60 min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report 5 cases of prehospital cpr with primary asystolia terminated after 45 -60 rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after 2 -5 min and cpr efforts were immediately resumed, nevertheless, 3 of the patients died at the scene, while 2 could be hospitalized with stable circulation. one of them died 3 hours after admission to the icu, the other survived for 3 weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least 5 rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [1]. we reported our results on such monitoring in 1969 [f.coaobbeb,r.fe6enb~-kap~monorm~,1969,n7,p.28-39] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po2,pco2 (mm rg),so2,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po2+pco2) in mm hg in ap blood. these data were derived from:i)86 subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc2 and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [2j. in control group it is equal to 1,91• mm hg. tests on sgp neither exclude nor substitute conventional (pc2 and pco2) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: 1) using towel clips to reapproximate skin only, 2)i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, 3) using loosely tied retention sutures for partial closure, 4) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since 1989 we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of 27 patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from 19 to 51. all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died 4t days later of multiple organ failure. there were no complications related to the closure technique in any of the 27 patients. conclusions; 1. selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first 24 hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from 12/1993 through 3/1995, 2140 pts underwent open heart surgery at our hospital. of th~se, 23 pts (1%) (age 65_+9 yrs) had a cardiac arrest during the first 24 hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) (17 pts), valve replacement (vr) (3 pts), cabg and vr (2 pts) and aortic aneurysm (1 pt). the preoperative ejection fraction was 44_+12% whereas bypass and aortic cross-clamp time were 127+70 and 72+42 rain, respectively. prior to arrest, they had a cardiac index of 2.23_+ 0.5 l/min/m 2 and were receiving 1.3+1 inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation (10pts) and bradyarrythmia (9 pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in 12 pts. eighteen pts (78%) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t2 pts, 52%), tamponade (3 pts, 13%), rupture of the proximal vein gra& anastomosis (1 pt, 4%), graft occlusion (2 pts, 9%); no cause was found in 5 pts (21%). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (-1% incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, 2 buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of 25 doctors and 25 nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between 0-10. after the first screening we held a 2 x 2 hours training. 8 doctors and 8 nurses were trained for the endotracheal intubation (group it1, 1t2) , 8 doctors and 9 nurses were trained to use the laryngeal mask (group lm1, lm2) . all respondent were trained to use the bag-valve-mask device. 1 day, 1 month and 3 month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. 3 x 6 hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r01e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and 100% responded the course "very useful". 73.6% of doctors and 79.4% of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s 144 objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in 22 patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies (45.5 %), penetrating abdominal wounds with injuries of mesenterial vessels (22.8 %), injuries of the liver (9.1%), blunt abdominal trauma with lien ruption (22.8 %). in 27.3 % patients had the previous somatic pathology. blood loss volume was 1500-4500 ml, & the reihfuside blood volume was 500-2000 ml, consisting 30-70 % of blood loss. it was needn't to fransuse donor blood in 18.2 % in further but 300-2500 ml of contanined erythrocytes were frasfused for supporting of hb concentration on the 80 g/l (8 g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just 40 % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of 4500 ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened 14 days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than 10 days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ 52 functional indices of cardiorespiratory function were evaluated. results: 74 pregnant women with ~h-gestosis were examined before and after i/v infusion of i00 ml of 0.0001% clophelin solution, 0.005 mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by 17%, of the heart rate by io.5%, of the oardial output index by 9.5%. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for 214 patients. the mean iss value was 27.65 + 17.36 while the median value was 21. minor injuries were present in 90 (42 %) patients with iss less than 21, while 124 (58 %) patients with iss more than 22 had severe injuries. increased mortality of the older patients was noted in the range 21-30. all patients older than 50 died while 20 % of patients below 50 yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above 50 yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during 4 months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp-1). results : fifteen patients were admitted in icu and 46 in a medical unit. fifteen patients died. a group of 35 patients had a lactatemia up to 2 mmol.l" 1. in this group of patients, 3 had acidocetosis, 3 had asthma, 3 had cerebral vascular ischemia, 3 had neoplasia, 2 had cardiogenic shock, 1 was epileptic, 8 had congestive heart failure, 6 had acute respiratory failure, 2 had septicaemia, 2 had hyperosmolar status finally 3 had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( 5.5• vs. 2.3+1.0, p<o.oool, respectively), the cut point of lactatemia was 3.7 mmoll" 1 se was 60%, sp was 91% and the accuracy was 83%. on the other hand lactatemia was higher in patients admitted in icu than in medical unit (4.5+3.6 vs. 2.0• p<o.01, respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over4 months. setting: a 16-bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < 4 x 109, neulropaenia < 2 x 109, and lymphopaenia < 1.5 x 10 ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days 0, 5, and 10. the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < 0.05) and hollow visceral intra-abdominal injury (p < 0.001). eight (27%) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < 0.001) and significant differences were found in absolute neutrophil counts (p < 0.02) and differential percentages (p < 0.01) on days 5 and 10. no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that 58.1% traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: 1. besides a physician specialized (vii --+ vii2 -~ viii gradus) in urgent medicine, it's necessary to introduce two (2) medical technicians instead of one as is existing now (1 ~ 2). 2.the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). 3.the technicians have been specially educated for urgent cases (iv ~ v ~ vi). 4. such a new team structure should be more capable in professional sense. 5.we can see an importance of team-work in ects. 6. such teams should also be more economic in comparison with existing one. 7. we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since 1983 we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis (164 dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc14, dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at 5-10 mg of cells per i kg of patients weight. dialysis was performed during 1-2 hrs. 168 patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: 40.0 ml of hemosorbent and 30.0 ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of 80-120 ml/min, during 90-120 min. average volume of hemoperfusion made 3.51. conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam (0.2mg/kg),thiopentone (4mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone (24mg/ kg/die),mannitol (0.smg/kg) and diphenylydantoin (20mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c0nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi0surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in 20 patients (14 m.,6 f.,61 + 10 y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is 3,5 -5,0 mmol/l. although more than 90 % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in 65 % of patients (13120) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from 1st of january 1991 till 31st of december 1994. results; in this four years period there were 250 abdominal injures. there were 110 blunt injures. among them in 43 patients peritoneal lavage was applied. results were positive in 28 patients and negative in 11 patients. false positive finding were in 2 patients and false negative in two patients. in patients with positive lavage there were more than 90,000/mm3 red blood cells, more than 1,000/mm3 white blood cells and the level of amylase was up to 8 u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than 40,000/mm3 red blood cells, less than 500/mm3 white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se6en md, z.milo~evi6 md, *s.srdi6 md, k.sar6ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the 1st of january 1991 till 31st of december 1994, are presented. results: among 92 operated patients there were 31 females (33.70%) and 61 males (66.30%). there were 52 patients (56.53%) with gastric bleeding and 40 patients (43.47%) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in 23 patients (44.24%), ulcer suture in 12 patients (23.07%), billroth i in 9 patients (17.31%) and billroth ii in 8 patients (15.38%). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in 34 patients (85%), duodenotomy with ulcer suture was done in three patients (7.5%) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj 0b0ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in 60 patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in 34(57%) patients, while in 26 (43%) it appeared unaffected. cpk-mb of 3% and more in comparison to total cpkwas 6.4+4,2% in 34 patients, and 1,9+0,6% in 26 pts (p<0.01).c0 of 5.8+1.17 l/min in patients with cardiac contusion, measured 16 hrs after admission at the latest, was significantly lower than in group of 26 patients with co of 4.8+0.86 l/min (p<0.01). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in 28 patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was 23 % lower than in the blood inflowing. 24 hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was 18 % lower compared to the vb and did not change for 24 hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent 5 years 150 patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. 4 (2.7 %) underwent urgent surgery, of them 2 (1.33 %) with cardiogenic shock and 2 (1.33 %) with massive pulmonary embolism. 146 patients (97.3 %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in 133 cases (91.1%), in 13 (8.9 %) patients subsequent pulmonary embolectomy was performed. mortality rate was 2 (1.36 %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and 4 (100 %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in 89 % patients. pe mortality rate is repoted to be 4 %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr4),free thyroxine(ft4),total tri-iodothyronine(tr3),free tri-iodothyronine (ff3) levels were measured within 24 hr.of intensive care unit admission(first day) in 17 multiple tratmmtized male patients(injury severity score higher than 17). in fifth day the same hormone levels were repeated. tsh,et4,ft4,tr3 and ft3 levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt4,fr4,et3 and ft3 in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm (1), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose 20 rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose 1 mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( 10,5+1,5 mmol& norm -4,9+0,3 mmol/i ), whereas rats without glucagon had urea content 30,3 +1,8 mmol/1 ( p < 0,001 ). osmolarity of blood plasma in rats having glucagon injections was 301,5 + 2,9 mosm/kg h20, without the drug -317,0 + 3,5 mosm/kg h20 ( p < 0,01), ldh activity -27,7 + 2.9 and 51,0 + 9,6 ( p < 0,01) mcmol cec-1 accordingly. lethality in the group of rats without glucagon was 70% /n=50/, with glucagon -0 (n = 70 ). in patients with hypovolemic shock in 15 minutes after glucagon administration stroke volume increased to 71% (sv), miocardium contractility -to 70% (mc), peripheral resistance of vessels ( vr ) decreased to 10%. in 1-1,5 hrs sv increased to 100%, mc-to 126%, pvr -decreased to 116%. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing,1993 ), 172 p. n, vasina (t) ,n.sebbota hph (2). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k3r-key, [7maine (2), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in 85 patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first 8 we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i9~26 mmol/l down to 89#15 mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal 37 z, there of 2i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in 25% and morphologically in 80-90% of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last 5 years 27 patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of 27 patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june 30 1994 protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were 30 patients with acute myocardial infarction treated before hospital by the cardiological groups. 100 patients were treated with the early infusion of nitroglycerin and 200 without. the results of treatment were: in the group with infusion: mortality in the first hours 5 % the full disappear of pain 90 % in the group without infusions mortality in the first hours 8 % the full disappear of pain 82 % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used 1 mg prazosin tablets sub lingua and measured blood pressure 4 ones during 2 hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was 69. the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in 90 % of patients. the general condition of patients became better in 10-15 min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose 1 mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: 16 patients, aged 34_+9 years (mean _+ sd), and burnt 37_+11% of body surface, were studied from day 1 (d1) to d20. serum samples were collected at do, serum and urine from d1 to d7, and on dio, d15, d20. cutaneous exudates were extracted from the textiles surrounding the patients, collected over 24h periods from d1 to d7, using bidistilled acidified water. mg supplements (8.2 to 123 mmol/24h) were prescribed from d1 to dio according to the severity of hypomg (serum mg < 0.7 mmol/i). results: mg serum levels decreased below reference ranges in 12 patients between d1 and d4, and normalised thereafter during supplementation. urinary mg excretion was increased in 11 patients, mean excretion being 4.8_+1.3 mmol/24h until d20. mean daily mg cutaneous losses, were 16 mmol/24h, i.e. 112 mmo; in 7 days. large inter-& intra-patient variability: the daily exudative losses ranged from 0 to 83 mmol/24h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t1/2,id50). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for 25 seconds with 75~ hot water to achieve a 30% tbs partial-thickness burn. blood samples were taken prior and after surgery as well as 15, 45 minutes, 4, 8, 12 and every further 12 hours after thermal trauma. two control groups (each n=6) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n=4) received c 1-inhibitor concentrate at an initial dose of 100 u/kg body weight either immediately or 12 hours after thermal trauma (n-2), followed by three further applications (of reduced amounts) every 12 hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c 1 -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin-6 (il-6) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of 12 multiply injured ( 238 (73) 148 (46) 154 (57) il-6 (nis) 156 (81) 88 (54) 65 (39) il-6 (nin) 117 (68) 150 (76) 157 (04) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il-6 is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no2) and nitrates (no3). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included 16 patients admitted to the intensive care unit of the burn center of brussels (age 35:1-18 years; burned surfaee area 374-19 %), and 6 control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age 64 4-18 years). the patients in both groups were fed enterally with 30 to 40 kcal/kg/day of a standard solution. in the burn group, daring the study period, 6 patients were mechanically ventilated, 4 required vasopressor (dopamine) therapy, and 5 received antibiotics; 13 patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no2/no3 determinations (griess reaction) daily from day 1 to day 5 (n=ll) or to discharge (day 3, n=2; day 4, n=3) in the burn group, and once the control group. in the burn group, no2/no3 levels were elevated at day 1 (70_+ 12 #moles/i), reached a maximal value on day 2 (874-26 p.moles/l) and progressively decreased to day 5 (fig). these values were higher than in the control group (5.6:t:7.6 #moles/l, all differences p<0.01). however, no correlation was found between the plasma no2/no3 levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no2/no3 levels than those who did not (177 + 131 vs 83 4-48 #moles/i, ns). the present study indicates that burn 120 noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no2/no3 plasma levels, which are ao so consistent with an enhanced no pro-4o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics (1). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury (2). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa 1, 4 male, 2 female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a 25 gauge needle was inserted into the lumbar subarachnoid space at level l3/l4 and connected to a trancducer (mx860| medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco2 constant. after a resting period of 10 minutes basline measurements were performed including csfp, cvp, map, hr, etco2 (cardiocap 2| datex). then the volunteers were given urapidil 0.2mg/kg over a period of 1 minute. 5 minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p<0.05 significant; values: mean_+sd, si = mmhg. 10-2_1" 74_+5* 64_+5* -3_+1" 35_+2 csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map (2). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after 75 min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( 6 h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for 75 min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to 6 h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of 75-min ischemia, reaching peak at 0.5 h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at 6 h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied 13 patients with severe head injury (glasgow coma score < 8) admitted to our icu between fobmaly 1990 and december 1993. icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of 13 patients were additionally icpc monitored by a fiber optic transducer (comma 420r~), and seven of 13 patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = 0.84 (p < 0.001) with a regres~on line y = 0.75 + 0.93x. four of ~hx lcpc -icpv patients had r > 0.85 when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = 0.47 (p < 0.001) with a regression line y = 7.2 + 0.43x. corralalmu eoetfieiont was inwer than 0.5 in all seven patients. corrdation eoelfieients for levals of icpv > 20 man hg, > 25 mm hg and > 30 tuna hg with icpe showed r = 0.89, r = 0.91 and r = 0.97 respectively; and with icpe r = 0.25, r = 0.13 and r = 0.09. the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors (1,2). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam (2 -5 mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of 1 mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam (2-8 mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, 79 patients aged over 60 years suffering from head injury, with glasgow coma scale (gcs) of 8 or less, were studied retrospectively. twenty-six patients (32.9%) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three (67.1%) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs (6.57 vs. 6.56) and median age (67.5 vs. 67). the incidence of brain pathology in the two groups was the following: epidural haematoma 7.69% vs. 11.32%, acute subdural! haematoma 30.7% vs. 30.19%, intracerebral haematoma 19.23% vs. 5.66%, subarachnoid haemorrhage 38.46% vs. 39.62%, diffuse haemorrhage 11.54% vs. 13.21%, contusion 26.92% vs. 49.06% and non-visible pathology (normal ct) 3.85% vs. 1.89%. unilateral pupilary dilatation was found to be 15.38% in group a and 18,87% in group b. the mortality during hospitalisation in the i.c.u. was almost the same: 50% iu group a and 47.2% in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a 12 month period (1993 -9r the icp was monitored in 23 p~,tients (14 male, 9 female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was 36.9 years (4 -83). the glasgow coma scale at admission was 6.9 (3 -15). in all cases the device was placed wfihln the first 10 hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was 7. 2 days (1 -19) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c0ntra!s by ct-scan. in 1 case an intracranlai hemorrhage was detected and drained. the overall survival rate was 78.3 %. 113 showed a complete resolutl0n, in other 33.3 % psychological residuals like decreased mentatlon, in 17.4 % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and 11.1% of the injured remained in a comatous status. in 87 % of our cases the measured values correlated with clinical course and management. in 2 cases (8.6 %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality 1. identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age 37.6 _+ 19.6, injury severity score 28.3 +10.9) admitted to icu were studied. incidence of arf was 31.4 % (48/153). arf was defined as persisteat plasma creatinine >2mg/dl with or without oligoanuria 2. arf was defined as early when occurring within the first 96 hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in 31 patients while larf developed in 17 patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis (76.6% for both) were observed in larf group, when compared to earf (25% and 23% respectively). abdominal trauma was more frequent in earf group (32% vs 18%). the gs for earf and larf were respectively 8_+4.4 and 9_+4.15 while in the group who not developed arf (narf) the gs was 10.5• conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher (91.7 versus 64.5) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc (1992) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied 146 consequitive patients (75 in group i) and (71 in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was 9min. in group i we recorded r.o.s.c. (return of spontaneous circulation) 17,5%, death 73%, continuation of cpr efforts 9,5%. while in group ii, 21%, 69%, and 9,9% respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost 0 times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: 12 patients, aged 40_+16 yrs (mean_+sd), with burns covering 49+18 % of body surface were studied from day 1 (d1) to d30 post-injury, were randomised in 2 groups (g): g1-control receiving recommended te supplies + placebo; g2 -receiving in addition large supplements of cu, se and zn from d1 to d8. enteral nutrition was started within 12 hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d10 and d20. infectious episodes and micro-organisms were monitored until d30. results: the patients' characteristics were similar g1 & g2. the total leukocyte counts were higher in g2 between d10 and d20, due to increased neutrophils (significant from d13 to d15). total cd3+ and cdlg+ cells did not differ, whereas cd14+ (monocytes) were significantly increased at d20. proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g2 with a mean of 2.0_+ 0.9 infections during the first 30 days versus 3.3_+ 0.8 in the control group (p < 0.03). conclusions: the large te supplements for 8 days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of 20% body surface area iiib-iv burn in 335 rats. a burn eschar was excised on the 3rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of 78 patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the 14th day after burn infliction the activity of cathepsin d in injm'ed muscles is 6 times lower under an adsorptive dressing than under a gauze bandage (control) (p<0,05), the activity of trypsin-like proteases is 1.5 -3.4 times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is 25-35% and 7-20%, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first 6 hours icu data and icu admission had to occur within 12h of traumatic insult. over the last 2 years, 150 pts with severe head injury (gcs<8) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of 4.8h after trauma, range between 2-9h). in a total of 85 pts (=56.6%), jugular bulb desaturatiens (<55%) were noticed during this early 6h period. in 24 pts (=16%), jugular bulb saturations higher than 75% were observed, whereas 41 pts (=27.4%) revealed no abnormal sjo 2 data (55-75%) during these first 6h. concerning the periods with too low jugular bulb saturations (n:85), we found the following correlation ; in 49 pts (=57.6%) cerebral perfusion pressure (cpp) was below 70 mmng, in 36 pts (=42.3%) paco~ was below 30 mmhg and finally in 6 pts (=7%) we found primary intracranial hypertension. for the high jugular saturations (n:24) we found a primary intracraniaf hypertension in f0 pts (=41%), and a pace 2 level above 40 mmhg in 6 pts (=25%). in all patients we could restore jugular bulb saturation within normal range (55-75%) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the 2 major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above 90 mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo2 should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury (1). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last 18 months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp>20 mmhg) and cerebral hyperaemia (sjo~>75%) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of 98 head injured pts received treatment for intracranial hypertension over the last 18 months. six of them met the criteria set for the administration of indomethacine. in 2 pts, no decrease in icp or in sjo 2 was observed and both pts died due to therapy refractory intracranial hypertension. in the other 4 pts, a significant fall in icp and in sjo 2 was observed shortly after indomethacine administration. in 2 pts we observed a catastrophic fall of sjo= even below 55%, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the 4 pts, icp remained under control without further administration of indomethadne, but he died 3 days later in multiple organ failure. the other 3 pts, needed multiple indomethacine administrations (for 1 pt even during 4 consecutive days) to finally control icp. in all 3 pts, icp was finally controlled, but only 1 pt survived. both other pts died from systemic causes (multiple organ failure in 1 pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in 86 consecutive pts, suffering from severe head injury (gcs<8). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo2 information. pts revealing ich and sjo=values above 75%, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than 75%, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=>55%). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed 86 pts with ich, with an overall mortality of 13.7% due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer 16 pts, csf drainage alone was sufficient to control ice of the remaining 70 pts, 38 pts received mannitol and 32 pts were hyperventilated as second approach. in the third line, 14 pts were managed with barbiturates, 12 with mannitol and 10 pts with hyperventilation. finally, barbiturates were used as the final rescue in 14 pts. these results reveal a less frequent use of mannitol as only 50 pts received mannitol, compared to the 70 pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as 32 lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier (14 pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo2-guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po2, icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in 6 piglets (14 days old, 3,3-5 kg) of either sex we produced a moderate brain injury (1,5 arm., 20 msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and 5 min. after brain trauma and after 3, 5 and 24 hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po 2 by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to 130 mm hg being highly significant (before mean i0 mm hg, range 4-12 mm hg) could be observed: mean arterial blood pressure rose from appr. 85 mm hg to ii0 mm hg for 3-5 min. in two animals this was followed by an ischemic period lasting 15 min. accordingly icp values gradually returned to starting measures within 3 hours; in the ischemic animals they remained at a level of about 30 mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within 24 hours. cerebral blood flow (ldf) fell from mean values being i00 before trauma to appr. zero and recovered to around 50. brain po 2 started at mean values of 20 mm hg (range 15-30 mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of 15 mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po 2 measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: 1) suction of gastric contents from trachea during intubation, 2) presense of a predisposing factor, e.g. coma. 8) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale 3, because of cerebral injury, and patients who died within 3 days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were 96 and the non trauma patients 49. from the trauma patients, 77 had cerebral injury and 19 were polytreumatized without cerebral damage. from the non trauma patients, 13 had malignant neoplasms, 14 neurological diseases in terminal stage, 7 old age, 10 drug overdose, and 5 several diseases. eighty seven from 96 trauma patients (91%) and 45 from 49 non trauma patients (92%) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the 2 groups (p>0,1). the severity of complications was also proportional in the 2 groups. eighteen deaths were recorded in the trauma patients (mortality 19%). only 7 deaths correlated directly or indirectly with the aspiration (7%). in non trauma patients, 32 deaths were recorded (71%). twelve deaths were recorded in 13 patients with neoplasms, 12 deaths in 14 patients with neurological diseases, 6 deaths in 7 aged patients, 1 death in 10 drug overdose patients, and 1 death in 5 patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< 0,001). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p<0,001). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in 2 groups. the patients of group a (15 patients) received fluconazole 200 rag/day for 15 days. and the patients of group 8 (20 patients) amfotericin-b 50 rag/day for also 15 days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from 15 of the group a (60%), and 18 from 20 of the group b (90%), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p<0,05). all the patients before treatment had positive for fungi blood cultures. after 10 days of treatment, 3 patients of group a and none of group b had positive cultures. eight patients (from 13 who had positive cultures of bronchial secretions before treatment) of group a. and 5 (from 17) of group 8. had positive cuttures of bronchial secretions after 10 days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p<0,1 and p>0,05): ten patients (from 12) of group a and 7 patients (from 16) of group b had positive urine cultures, after 10 days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p<0,05). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in 2 patients, chills and fever during infusion in 3 patients, and hypokalemia in 12 patients). three patients of group a and 1 patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of 15 days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into 4 groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the 4 groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were 85. all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, 19 in group b, 20 in group c. and 18 in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the 4 groups as to number of complications ( twenty four patients had chest complications in group a, 16in group b, 17 in group c, and 16 in group d. p>0,1}. the duration of mechanical ventilation was not statistically different among the 4 groups (the mean duration was 15,9 days in group a, 16,8 in group b, 16,5 in group c, and 17,5 in group d, p>0,1). there was also no statistical difference in mortality among the groups (six patients died in group a. 4 in group b, 4 in group c, and 5 in group d, p>0,1 ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in 129 trauma patients admitting between december 1, 1992 and july 31,1993 in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, 29 of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in 44 (44%) of survivors and in 26 (86%) of non survivors .we noted low fncidence 5 of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least 3 times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.(3.4 vs 2.4, 4.1 vs 2.8meq/l, p<0.01)the duration of hyperlactemia also was higher in the patients with organ complications. (2.2 vs 1.0 days, p<0.01). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.(4.0 vs 2.8,4.6 vs 3.4meq/l, p<0.01) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s155 current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj02, kjdo 2 and ceo 2 in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l 8 at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and 8jo 2 monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous 4 hours, normal x ray chest and within the first 48 hours from injury, were considered candidates. peep value where increased fro~ zero to 15 cmh20, in three different steps, each one with 20 min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo 2, in the case of hemodynamic deterioration, during the study, ppc 6ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. 40 patients were candidates, 15 of them were exc;uded due to haemodynamic instability, k total of 25 completed all steps and were e~amined, 22 men and 3 women, age 32!7.9 yrs. at hospite; arrival, glasgow were < 5 in 18 patients, and >5 in the rest 7. 15 patients 20 mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied 36 patients(16 females), aged (mean-..+-sd) 56.2_ 17.3yrs, which were consecutively (from august 1994 to march 1995) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was 17.4-+6.8 (range 4-31). the following data were evaluated: 1) number of medical departments, where health care was provided until final admission to the icu, 2) ambulance transportation conditions, 3) catheters and tubes inserted before admission, 4) vital signs upon admission 5) information provided by referring physician (scored on a 1 to 5 scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), 6) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu (1:22.2%, 2:47.2%, 3:27.7%, 4:4%). thirty/36 (83.4%) patients were escorted by a physician. twenty-six/36 (72.2%) were transported on oxyge n, fio2(mean__.sd): 46-+3%, pao2: 78.6-+35.2mmhg. five of the remaining 10, for whom no oxygen was provided, had pao2: 46.2-+7mmhg. twelve/36 (33.3%) were intubated and ventilated during transportation. thirtyfour/36 had a peripheral venous line, 5/36 had an arterial line, 13/36 had a nasogastdc tube, 20/36 had a urinary catheter. eleven/36 were sedated and 2/36 were paralysed. three/36 were on inotropes. vital signs upon admission were: arterial blood pressure, systolic 100.6-+44mmhg, diastolic 57-+23mmhg, heart rate 104-+22 bpm, temperature 36.3 -+2cc. patient information score was 217--. 1.7. the actual state upon admission was found substantially different, as compared to the description of the referring physician, in 28/36(77.7%) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since 78.8% of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from 18 to 84 years. dosing ranged from 25mglkg to 300mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over 150 patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from 1st june 1994 to 14 july 1994, all the calls for emergency care were analysed using a questionnaire of 114 items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : 996 calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively 2% and 100%. the performance of the model is 88 %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may 1991 to april 1995, 4 patients were admitted to our icu with bci (2m,2f, age 29+ 1 3). a history of direct trauma was present in 2 patients. admission gcs was 15 in all patients, and was associated with hemiparesis in 3 of them; the last became paretic 48 hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan (2-4 days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the 3 groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table 3x2 in z2 test. res.cl~s: of 43 patients 34 were submitted to thrombolysts and 3 died. the higher incidence of bracb, ar~lhmias (ii degree gg p t39e 1 and 2 av block. 11i degree av block. avsb 6.141 <ii.05 sinus arrest) that requited the insertion of avsc 11.121 <cl005 temporar~ pace-maker was recorded in group a b vs c 0.004 ns in 64% of the cas6~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v4r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and 7,5%nacl/6%dextran 70. methods:in 40 polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q2) prevalid.replacement of the volume loss 5egan in institutions of general medicine,frequently by rl and 69dextran 70 or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution 8dministered ~o the injured wer~2375-2550ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the 7,5%nacl/6%dextran 7@(groupii)(4-sml/kg observed to the final surgical management,had statistically higher map(increase of 58% in relation to admission), cvp(average increase of 8,25cmh~o),diuresis(averagely 35omi after 30min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q8%,average increase c~ of cvp 2,5cmh20 and average diuresis 20oml after 3omin). total quantity of the administered solutions was significantly smaller in the groupii(2800ml:4925ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within 32hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl:20ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of 7,5%nacl/6%dextran 70 lasted 5 minutes. w.scherzer(l~,k.lechner(1),r.simon(2). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin 9 !(2)neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson,1975) ,in general anaesthesia (bennet,1985) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger,1992) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro4 vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter(1980) ,basal stimulation (bienstein, 1991) , bobath-therapy(1966) and facilitation of a physiological moving pattern(pnf)according to knott(1968) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s157 objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: 21 patients (mean age: 69) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n=5), acute congestive heart failure (n=4) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of 10rrd ice-cold glucose 5% non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, 159 paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of 6 months. the tt is a eompetitve immunoassay which gives qualitative results within i0 minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study 25 patients were enrolled, in 19 of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of 0.94 and specificity of 0.94. conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure >210 rorohg and diastolic blood pressure > 110 nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received 5 rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below 180 mmi-ig and diastolic blood pressure below 95 mmi-ig within 75 minutes after start of treatment. results: 35 patients were included in our study, 20 (57%) patients responded successfully to treatment. mean arterial pressure decreased in responders by 36.5 mmhg and in non-respenders by 12.7 mmhg (p<0.001). responders and non-respenders differed signii'icantly concerning pra (p=0.001), ace (p=0.003) and ang ii (p=0.04). 0.003 0.04 the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r=0.43; ang ii: r=0.66). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july 1992 to june 1993, files of patients consulting to the ed for smp have been retrospectively analyzed. results: 727 patients, 482 women and 245 men, 33.3 + 12 years old (range 15-92) have been admitted for 804 episodes of smp (4% of all consultations) whose 77 relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively 42.6%, 9.1% and 2,9% of patients. the interval of time between the ingestion and emergency consultation was noted for 43% of smp (332 + 532 min, ranges 15-4320). the involved products name was known in totality in 89% of cases with an average number by episode of 1.7 + 1 drugs (ranges 1-8). the most often, 1 (52%) or 2 (21%) different products were interfered. the nonbarbiturate psychotropic drugs accounted for 76.7% of the products (benzodiazepines 67%, antidepressants 9.5%, neuroleptics 8%, carbamates 5.8%, imidazopyridines 5.1%, cyclqpyrrol0nes 2.7%). analgesics and nonsteroidal antiinflammatories represented 6.8% of all drugs, anticonvulsants 3.4%, cardiovascular drugs 2%, antiinfective agents 1.9%, drugs against cough 0.86%, muscle relaxants 0.86% and antihistamines h1 0.5%. the benzodiaz6pines were present in 531 episodes, alone in 316 episodes. in 36.5% of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in 32.5% of cases, activated charcoal in 16.7% of cases, flumazenil in 16.9% of cases, naloxone and acetylcysteine in 3.4% of cases; orotracheal intubation was performed in 12 patients. admission in hospital was effective for 280 patients, in medical ward (n = 156), psychiatry (n = 63) or icu (n = 62); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz6pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a 18 bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately 3.500 m2 is based on the principle of three identical 6 bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate (13). method: a plaque of 64 electrodes, positioned on the left anterior ventricular wall of 9 anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused (0.5 mg/kg/min iv) during 75 rain; at t60, dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least 30% of its value at to was demanded before the administration of b. results: 1 dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively (13<0.05) compared to values control fro)except qrs at t65. the b (7 + 6 ml/kg; ranges 2.8 and 20.5 ml/kg) modified no studied dements outside of the ( }rs. to ti5 t30 t45 t60 t65 t75 a 2,1 + 0,6 2,1 + 0,5 2,1 + 0,4 2,1 + 0,4 2,1 + 0,7 2,1 + 0,7 2,1 +-0,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase 0 of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval0ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to 205 doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from 1 (--full agreement) to 5 (=disagreement). overall, 64"/. of the active emergency physicians participated in the survey. results: 61.1% of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only 13% would not hesitate to go into action by dark. 14.8 % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only 29% would do it. 52.8% of the doctors would rather stop flying. the most common reasons for 9,uitting were wish of family and fear of an accident. 47.2% conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. 14 crash landings within less than 10 years with 15 deadcases and 17 badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in 37 patients (age 21-68 years). the indication for intubation was elective surgery in 21 patients (asa i-ii) and emergency intubation in 16 patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the 16 emergency patients was dyspnea in 8, cardiac arrest in 2, trauma in,2 and unconsciousness due to drug overdose or seizures in 4 patients. intubation was facilitated with medication in 12 patients. results: of the elective 21 patients, 19 (91%) were successfully intubated. six patients (29 %) needed two attempts before successful intubation. the duration of intubation exceeded 30 seconds in 8 patients (38 %). of the emergency patients, 14 (88%) were successfully intubated. six patients (38%) needed two attempts, and the duration of intubation was more than 30 seconds in 9 patients (56 %). in 54 % of all 37 patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in 11 patients (30 %). oesophageal intubation occurred in 2 patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < 50 x e9/i. in each treatment the same type (av-400, fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between 200 and 250 s. the initial enoxaparin dose was 80 rag every 8:th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels 24.3 and 25.2 mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin (35.7 +31.6 h versus 22.3+26 h, ns). the mean aptt-levcl during heparin treatment was 79s and during enoxaparin treatment 54s (ref. 24 -34s). the mean daily dose of heparin was 422 nag, that of enoxaparin lg7mg. the mean anti-xa activities were 0.40 u/mi and 0.47 u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load 17 data obtained through direct measurement from the monitors and the lab, the program calculates 18 parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e8 of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of 10065 pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma 24 hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for 8059 pts during aperiod of three .y~m , treama pts were 1156 04.3%), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g 2=-8a7 p>0.25; roc = 0,94 ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in 6 dogs 1-~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made 29.8+1.9 ml/kg). the treatment was begun after 7.u+o.2 hrs of h. "sl", created on the basis of-sorblt and natrium lactate (1800 mosm/l) was injected into v. femofalls at the dose of io.0 ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to 30.0 mm hg and -0.6+0.2 cm h20 (p4.o01), while heart rate (hr)-increased to 190.0+9.6 per min (p<.o01). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by 42.2% (p<.i) and 6.4% (p~.05), while fibrin degradation products (fdp) enlarged by 215.6% (p~ .001). after 30-40 min of treatment termination abp and cvp increased to 98.3+6.1 mmhg and 4.1+o.2 cm h20 (p<.o01), and ~[r diminished to t80.0+6.3 per min (p>.5). at the same time the indtces of pic and pf enlarged by 36.4% and 2.6% (p>.i), while fdp diminished by 8.2% (p>.i). one of 6 dogs survived. life duration of the other 5 dogs was 3.9+1.2 hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied 64 icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo2 through opljcal fiber and the cerebral flux with tcd. methods: since january 1990 until june 1994, we collected 152 ht admitted to the icu, and 64 of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ 8, with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo 2 and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< 8, sjo2 is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , (16m,6f) , (m. age: 39+4 years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was >25 in all studied patients. tbe group a (9 m, 41") had no tremor and/or fever on admisskm, while em group b (tin, 211 the above criteria were ix)sitive. bhx~d samplings were taken 2-9 hours after accident and 10-25 rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, 25 healthy people were examined. _resu!_ts-il-lct, il-ii~, il-2 and tnf-tt levels were similar to control group levels in both groups a and b. i!,-6 and g-csf levels were found increased in both groups (p<0jxjl), while il-6 levels were statistically significant comparing to group a. in con_tin_skin, during immediate post4raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than 11,.6, cannot be detected,whereas 11.-6 was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep 5% burn, cornplicated by bloodshed the of blood grows at 6-7 times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to 30%, and 77% in 3 hours. in 3 hours after the trauma the-process is accompanied b3! 59,6% lower inhibitory activity of blood, where as at the peak of the trauma it was 14,5~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and 5-fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at 2 times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at 2 times; [15emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing 2-3,5 kgr and at age of 4-6 months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and 0.1 ml (this volume assuring a normal incranial pressure after kaufman 1985) of autologous blood was injected into a depth of 3 mm via a needle of 0.36 mm bore. the wound was closed and the animals were left to recover. nimodipine, of 2,1 mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into 10% formalin solution. tissue sections of 5 ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was 260 --. 26 ~t while that of group b was 76 + 12 ~t (p<0,01) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: 20 pts with 20-40% bsa second to third degree flame burns (respiratory tact injury excluded) 19 to 61 yrs of age were randomised to study (n=10) and control (n=10) groups. burn shock was treated with hypertonic saline -bicarbonate solutions (250 mmol/l na +) 2ml/kg/%bsa for the first 24 hours and 1 ml/kg/%bsa for second day. analgesia in control group for the first 48 hours was provided by regular 6 hourly intramuscular administration of 20 mg of morphine sulphate and 500 mg of analgesic -antipyretic analgin with 10 mg of diphenhydramine (dimedrol). from the 3rd day regular administration of morphine was finished. in the study group 100 ixg of clonidine was added 8-hourly for 72 hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, 1 -5), sleeping time, spo2, hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of 300 ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first 3 days was 3.1 -3.3 vs 1.3 -1.7 in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first 24 hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population 7 pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only 1 pt showed sign of aws. mean vas score was in 2.4 -2.9 range for first 3 postburn days. 7 pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo 2 values in study group were in 95 -98 % range, among controls 90 -95 %; vomiting was absent in. cionidine group vs 4 cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs (22-+4kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter-30 polysulphone membrane) and remained stable for one hour (pao~= 98.8• peco2=34-+8mmhg, ph=7.37-+0.07, bp=137-+12mmhg and pap=15-+2mmhg). all was induced two hours after oleic acid infusion (0.07ml/kgr) into the pulmonary artery (poo~=46.6_+6 -p<0.001, paco~-50.2_+10 -p<0.05, ph=7.10-+0.10 -p<0.01, bp=158-+25 -p=ns, and pap=29_+5 -p<0.01). fio 2 70% for the next 30 minutes did not significantly altered the b3ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter (2l/min) while filtrate and gas were removed from the outlet port (bypass flow 220 ml/min) b) through a thin intratracheal catheter positioned 2cm above the carina (4l/min). the fio 2 given through the ventilator readjusted to 21%. results replacement fluids/filtrate during the next four hours were not exceed 0.7l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.=88. objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied 3 randomised clinical groups of patients each containing 24 patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding 1 500 ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the 1-st and on the 7-th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c5 and c4 fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the 1-st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the 1-st day, while on the 7-th and on the 30-th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, 3 g. sofiiski str.,1606 sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a 18-year old male patient with extremely massive intraabdominal haemorrhage ( 7 300 m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of 6 600 ml of autologous blood using an original autotransfusion system developed by us ( pat. no 95311/ 11.10.1991) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first 3 days after the trauma in 20 patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret 1000 cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over 18. results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the 2-nd and the 18-th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the 24 supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in 6h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity (160+3 pg/ml compared to 50+4.9 pg/ml in control) were found od day 3 following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages 3h following trauma (60-70% compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase (25%, 3h following trauma and 60% and 50% on days 1 and 3 after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days 1 and 3 following trauma, and initially but persistently high levels of plasma tnf alpha in da rats (4-5 fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least 100 people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, 75080, iso 9001, yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca 5-8t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least 50 wounded and sik people. in federal republic yugoslavia, it was proposed 24 such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < 30% may be more dangerous than short-term levels of 45-50%. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a 15 year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was 8/15 and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of 10% was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef 20%). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf 80 %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a 44 year old male ingested 2500 mg doxepin (sinequanr), a tca and 3500 mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs 6/15), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of 140 b.p.m., an arterial blood pressure of 90/70 mmh4g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively 410 gg/l (toxic level 5130 #g/l) and 3900 i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed 1 1/2 h later. nahco3 (250 meq/24 h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. 8 h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for 2 days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of 85 patients treated" during a two year period (1-3-1992 to 1-3-1995) in our i.c.u who meeted the following criteria: (1) coma score _< 8 gcs within the first 24 hours, (2) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes 1-6), (3) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ 155 meq/l). survival was defined up to the 30th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = 0.95). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august 1994 by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in 1992 [1] the pre-existing equipment of the volunteers was completed with type a ambulances and 1 special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide 24 hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, 300 consecutive emergency interventions in the time period from novembre 1, 1994 to april 30, 1995. the emergency physicians treated 131 male (44 %) and 169 female (56%) patients, 15 patients died before hospital admission and 75 patients (25%) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period 1 year earlier (november 1993 to april 1994) without attending physician the volunteer rescuers transferred all 257 first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of 19 patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate 3 ml/min in the recirculatory mode. its duration was 180 -240 min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products 80 -300 times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e3tabli~mczr 3bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, 29 yejin-l)a-l)ao, wuhan 430080 fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to 90 far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving (91.6~), the successful rate of saving tra ma (93.~), the suscessfut rate of saving shock (98.~) and the successful rate of cardioputmonary resuscitation (52.4~) daring the three years after t he network operated with these before (86.6~), (91]. 4~), (92. ~) and (4ft. 5~), the successful rates after operating were remrk~iy higher ( p<i).o5 ). conctusions~ the above results showed estabiishmnt and mena0emont of region at medical first-aid network had very si~nificont action during mergentty s avin9 the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med 1995; s. nouira. chest 1995) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among 600 consecutive victims of se presenting to the emergency department, 135 were managed using sav (group sav+). these patients were matched to 135 envenomated patients who did not receive sav (group savchest injuries are the cause of death in 25% of trauma fatalities and a major contributing factor in an additional 50%. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january 1 1993 to december 31 1994, all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao2/fio2 < 200) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: 52 severe trauma patients were considered. 8 of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais<3). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. 44 patients with severe thoracic trauma (ais>=3) were admitted into the study. the mean iss was 36.2 (16-75). thirty-six patients required artificial ventilation for at least 24 hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in 7 cases pneumothorax was diagnosed an the initial cxr 14 more patients had a pnx which was identified only on the ct. in 4 cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, 54% (24/44) presented a pnx that required the insertion of a thoracic drainage. only one third (7/21) of the pneumothorax could be recognised on the initial cxr, while other 3 were decompressed before performing the cxr. as many as 58% of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in 4 cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a 20% pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli6, 0.~osid, m.zesevid, m.risovid, d.stepi6, d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod:30 a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given 5-5 prcpofol bolus doses (o,5 ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion 6 ~ ~ropafol. as sdflitianal were given fan-i~l (o,2 n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro4o l.patients in gr~4o 2 received i~ (an initial bolus dose of 35 rg slowly intcavax~ 8rd 25 mg as infusion over ~0 rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj 10-15 rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, 30 snd 60 rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min 9-~a4~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than 1 hour map < 60 mmhg or use of vasoactive agents or blood lactates > 5 mmol/1 were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at 3.6, 12, 24, 48 hrs. an immunoradiometric assay was used to determine plasma concentrations of il6 (n<0.03ng/ml) and tnfc~ (n<5pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n<0.1eu/ml)(1 endotoxine unit= 100pg). results : 9 severe multiple trauma patients (age = 42_+18 yrs, iss = 40-!-_15, saps = 19+'~, mean-+sd) were studied. they received 15+6 packed red cells during the first 24h. mean duration of collapsus before inclusion was 5.1_+2.9 hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il6 level and saps was observed. in the early post-injury period phi, sao2, svo2, vo2 were significantly associated with ;il6 release (p<0.05 at ho, h3, h6). later a significant correlation existed between lactates and ii6 (h6, h24). a peak of tnf was detected at 24 and 48 hrs. it was associated with low phi and low arterial ph of the early post-injury period (p<0.05 iat ho, h3, h6 ,h12, h24) and with high lactate levels of later period (_>h12). only the late release of endotoxins (i{48) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il6 in the early phase of trauma . i16 and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in 30 sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect 1000 brain wave monitor from january 1 to june 1, 1995. results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo2) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in 5 sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett 7200ae ventilator with integrated 7250 metabolic monitor. inclusion criteria were; 1) stable cardiorespirator s" status; 2) systemic and pulmonary artery catheters already in situ; 3) inspired oxygen < 80%. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, 150 ug/kg, followed by an infusion of 80 ug/kg/h to maintain the train-of-four ratio at 0. a further four sets of measured and calculated variables were obtained at 20 min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo2fick) , and measured oxygen consumption (vo2gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: 18 patients (12 women and 6 men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than 0.5 and normal coronary circulation. for postoperation sedation propofol was administered in 0.5 mg/kg i.v. bolus, followed by a 2.2 mg/kgth perfusion. all data were registered before administering propofol and after 20 minutes, the patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about 56 years old, and the average period of aortic cross-clamp was 77.50 min. the adminstering of propofol caused a decrease in the coronary blood flow (-9%), great curonary vein flow (-23%), myocardial oxygen consumption (-14%), regional myocardial oxygen constanption (-11%), myocardial oxygen extraction (-6%), regional myocardial ooxygen extraction (-10%), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+16%) as well as in the great cardiac vein (+32%). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (-23%), diastolic (-20%) and mean (-25%) arterial pressure, systemic vascular resistance (-20%), and cardiac output (-8%). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of 0.4% "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: 16 patients (12 women and 4 men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than 0.5 and normal coronary anatomy, were selected. after the surgical operation, 0.4 "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and 20 minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was 57 83 -+ 8.87 years. during surgical operation the period of aortic cross-clamp was 78.56 _+ 32.09 rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (-26%), coronary vascular resistance (-29%), regional coronary vascular resistance (-29 %), regional myocardial oxygen consumption (-7%), regional myocardial oxygen extraction (-6%) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+16%) and in the great cardiac vein (+32%). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (-23%), diastolic (-25 %), mean (-25 % ) arterial pressure, and systemic vascular resistances (-28%) decreased, while cardiac output did not. discussion: the administering of 0.4% "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: 78 anaesthesias have been conducted in patients aged 15 75 years (28 females, 50 males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of 100 % 02 insnffladon through mask. operations lasted from 0.5-1.5 h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao2, t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics (0.01 mg/kg) and h2-blockers (0.3 mg/kg) on the operational table was sumplemented by administration of 0.5-1.0 mg/kg of lidocaine, 1.50.0 mkg/kg of clonidine, 0.5-1.0 mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: 0.05-0.1 mg/kg of droperidoi, 0.l-0.15 mglkg of diazepam and analgesia: 2-3 mkg/kg of phentanyl, 1.0--1.5 mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was 5-15 ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the 30-th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of 70-80 per rain. adequate external breathing: decrease (rr) by 2-3 per rain., with sao2 increase from 9446 % to 98-100 %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by 10-t5 % to the end of the operation with the initial moderate hyperglykhaemia of up to 10 mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state (3-48) and analgesia lasted 6-8 b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a 64 yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt 0.44/qtc 0.49 sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days 8 and 16 he received haloperidol, 10-44 mg/d (cumulative dose 209 rag) for agitation and delirium. icu day 11: qt 0.46/qtc 0.57 sec. icu day 12: for better control of delirium, trazodone " 50 mg q hs was added. icu day 15: he developed frequent nonsustained ventdcular ectopy. icu day 16: qt 0.70/qtc 0.74 sec, pha 7.48, paco2 50 mm hg, pao2 72 mm hg, k 4.9 meq/l, mg 2.0 meq/l. later in icu day 16 the patient had 3 brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day 17: qt 0.42/qtc 0.53 sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to 1000 mg/day. over past decade, 9 cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental (104 m), propofol (4p_g/ml), the solvent of propoful (intralipid), fentanyl (30ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm 30 minutes, pmn 15 minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp (1o 7 m) was measured in a two chamber 24 well system for 3 hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from 5 independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e 1 ). 77% conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january 10 to february 8, 1994, data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of 138 icus, 128 of which enrolled at least one case. the total sample included 2932 patients. overall, 60.7% of patients analyzed (t780/2932) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these 1780 patients in 5014 days in icu. although over 38 drugs were reported to be used, 10 pharmacological principles accounted alone for 89% of all prescriptions. opioids were actually used in 33% of prescriptions; propofol in 24% and benzodiazepine in 18.3%. as regards the way of administration, intravenous administration was applied in 74% of cases and, followed by intramuscular in 17.3%. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in 19% of patients and neuromuscular blockade agents (nmba) in 23%. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in 1994, 162 selected patients (pts) (>70y, 62f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon 1cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at 8h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao2 >75 mmhg with flu 2 <0.4; d) stable ph>7.35 with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (>1.(i ml/kg/h); f) mediastinal bfeeding<100ml/h for at least 2h; g) normothermia (core temp>36~ and no shivering). subsequent reintubation was for: 1) rr>35/min; 2) spontancx)us ventilation for 30 rain with paco2>50 mmhg; 3) pao2<50 mmhg with fio2>0.4; 4) ph>7.45; 5) heart rate>]20 bm; and/or 6) non mental alertness; and 7) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after 24h was considered: 1) spontaneous breathing without any forrn of mechanical assistance; 2) stability in haemodynamics; and 3) elimination of fever threat. results: 122 pts (75%) were extubated at 8h without complication; 29 other pts (18%) at 8h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by 48h. only 11 pts (7%) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation (75%=8h and 18%=24h), exempting pts with developed post-operative complications (7%=extubation<72h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p 30h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first 15-20 hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than 10 b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia 0b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged 25-61. group a (23 patients) received baprenorphine hydrochloride (norphin) at dosages of #.52• mg/kg. group b (30 patients) received tramadel hydrochloride (t~aasl) st dosages of 1.50z0.38 mg/kg. before the procedure diazepam was administrated i.v. (0.24!0.03 mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by 17.7% on the onset of the procedure while sap and sv decreased by 8.%% and 9.6%, respectively (p<0.05). however, there were no reliable co chsnges. spoz ~educed by @.2% (p<0.05) and remained lower than the initial one after the procedure was oyez. when administrating tramsl 50 min. after ste~ting the procedure sap and svr increased by ~1.2% and 7.3% respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o.05). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, 2, 3, 4) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of 5 up to 20 microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by 50%. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate 1-7 rag/h) and antipyrine (1000rag p.o.) were infused to ci with (10 patients -group 1) and without (10 patientsgroup 2) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group 1 chypermetabolizer") as compared to group 2 chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac12) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups (20 pts a and 20 pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl 7 mg• kg < in 60", pancuronium 0.1 mg x kg -1 and propofol 2 mg • kg -1 in 60". a blind investigator administered via another venous way at same speed (60") saline in patients of group a and 10 mg x kg -1 cac12 in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), 2' after anesthesia induction (t1) and 2" after tracheal intubation (t2). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t2. this trend was statistical significant (p < 0.05) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < 0.05) dramatically dropping at t2, while in group b it showed a negligible decrease at t1 (p = ns) and value similar to baseline at t2. svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac12 simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac12 were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to 20 primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was 2 hours more than in control and made 4 hours, though their average labor duration and in control was the same and made 12 hours. 98 % of the examined women experienced independant labor, as for the control group -only 68 %. supplementary methods of anesthesia were required in 73 % in control,that increased medicamentous depression of the fetus, but for the examined group -only in 16 %. the examinees's newborns experienced favourable terminations, in control 3 newborns were in state of light rate asphixia. analgetic effect was complete in control just in i5 %, and of examinees -in 82 %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for 42 % and in control only for 24 %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on 17 patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of 20 mg. than we were increased the dose to 80 mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had 13-rhythm with slow 0and a-activity. an c~-activity index have been lower than 5%. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to 35%. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts (13-14 points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and 15 min after each treatment. clonidine (i-4 ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of 4 gg/mouse, tfl increased 2.37-fold compared to time zero. l-arginine increased tfl at a high concentration (i00 gg/mouse). l-name suppressed tfl 1.2-fold compared to time zero at a concentration of 50 gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: 30 adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium (0,5 mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and 5 min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that 1,2 mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of 16 female swine (20-22 kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n=8): induction with intravenous sodium thiopental in a dose of 5 mg/kg, group b (n=8): propophol in a dose of 2 mg/kg for induction'and 10 mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with 4-5 it of cold (4oc) university of wisconsin solution for a period of 5 hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c-9263,1.3mg/ml) was infused via portal vein and the liver was incubated at 37oc for 45 rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (-20oc). results: histology was completed in 8/16 experiments (4 in group a and 4 in group b). mononuclear infiltration (halothane type injury) was found in all specimens (8/8). focal zonal necrosis and acidophilic bodies were found in specimens from group a (2/4 and 1/4 respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b (1/4). smears of hepatocytes -beth formalin fixed and cryopreserved at -20oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: 256 patients were treated from jan. 1994 till dec. 1994 results: in 157 patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of 20 patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic (4 ml 0.5~ bupivacaine) when analgesia was first demanded; second group received 2 ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received 2 m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : 51 consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature <35~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius 3000 a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within 10 minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, 1986 ; i : 307-310 objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in 1993 during a period of 5 months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of 18.5% on the short stay unit and 42.8% on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in 1994 that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: 1) there is no indication of a reduction in patient days with pressure sores (short stay unit: 14%, long stay unit: 55%); 2) there is a reduction in severity of pressure sores (grade iv decreased from 2.4 % to 0.7 %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from 17.8 to 19.8 and the mean frequency of nursing patients on alternate sides decreased from 1.4 to 0.6 times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads (4-6 times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch 22 are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' 6x7 with the so called bookend method and are connected to a low vacuum suction system (1-2 kph) results: we studied the frequency of dressings changes on 12 patients in the itu. in total they had 363 days with an open abdomen. during this period there were 63 dressing changes. this means one dressing change every 6 days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo 900 c: siemens), ventilation modes 0ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (-76 cm h20) was produced with a tv of 600 ml and a frequency of 6 /min in the ippv mode with trigger level on -3 cm h20 and a peep level of + 10 cm h20. the servo 900 c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set (27 cm h20), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( 1-4 cm h20 ) and the ippv mode the largest (16-27 cm h20 ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q1, n = 43, 13 items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by 38 persons (88 %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q 2, n = 38, 12 items). at the issue, iger was created. results : q1 : 30 responders (70%). law's misinformation : 33 to 70 %, disagreement for limitation of intensive cares : 40 %, hope to improve collective decisions and creation of iger : 88% q 2 : 29 responders (76%) : satisfied by the training sessions : 65%, non satisfied : 31%, want to continue the project : 68 %, refuse : 10 %. iger was constitute by physicians, nurses, secretary, dietetic (n = 15). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: 1) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. 2) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: 48 mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the 1st, 2nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in 23 patients (13 hmef and 10 hme) 3 h and 24 h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, 10 (42%) ventilator tubing yielded microorganisms in, at least, one sample as compared to 7 (29%) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups (25% vs 17%, p:ns, for hmef and hme respectively). among the 16 bacterial species isolated from ventilator tubing in the hmef group, 7 (44%) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group (6/15, 40%). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air (97% • vs 96% • 3.%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after 3h of mechanical ventilation in the hme group as compared to hme group (28.5% • vs 26.5% • 2%; p=0.03). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in 85~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in 2~ end endocrine pancreatic tumors in 3%. in the intensive oere unit, these patients wore screened by noninwsive monitoring in 85~ of cases: and invasive monitoring was applied in 15% of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of 24 patient submitted to wchf during the last year. we used a total of 32 filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x 2 test to compare the results. results" from 80 nurses of staff 5 refused to flu the quetionary, and 8 were not available. of the 67 remaining, 71%were v~men and 29% men. the mean age were 31.51% had an svable contract and 499( eventual, the mean professional experience were of 10 years and 44% worked in the unit since more than 6 years. the 88% answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: 1) to inform about patient mood. 2) to inform about happenings from the last visit. 3) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients (29 male and 22 female, aged 69+2,5 and 73,912,4 years respectively, ~+sym) being on 02 therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without 02. during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa02 and sa02 were signif~canthy decreased in the case of oxygen discontinuation (p<0,00i). paco2 was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p<0,01) . heart rate increased in all phases of the transport when 02 administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s6~ment depression was progressively increasing and became very high at the end of transport in the case of 02 therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao2 and sa02, increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results:92 patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the 1 group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was 23,3+-1,78. the duration of coma was from 30 rain. to 48 hour,average 11,9+-4,sh.ln the 2 group (the deads) the depth of come was 13,8+-0,86.the artificial lung ventilation was used in all patients:in the 1 group 2,64+-0,92 days,in the 2~ 6,1 +-1,1 days.apallish syndrome developed in 5 cases,in 5 patients diagnozed <<brain death~. the 4th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( 1 group) and slow (2 group) activity. the relative power of delta-and theta-ranges was about 77-87 per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the 1 group decreased on 19,5% and 44,3z, in the 2 -on 45,9% and (;3,3%.general peripheral resistance (gpr) increased in the 1 group on 40,5%,in the 2 -on (;9,4%.in the 1 group brain blood flow was increased, in the 2 -decreased on 37,6n.in the 1 group there was the normotonic type of reg-curve,in the 2 -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on 107 and 121n) and vascular ones (an increase of serotonin concentration on 90 and 110%, hi stamine content on 116 and 180%) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the 1 group on 59%,in the 2 -69%.the level of middle molecules (mm) increased in the 1 group on 79,2%, in the 2 -on 120,8%,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was 0,891. immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: 1. protective inhibition of brain and reduction of its energy requirements. 2. recovery of cell and vascular membrane fuoctions.3.recovery of blood circulatiom4.oxygenation of nervous tissue and drainage of brain disintegration products.in 22 patientshbo carried out.5.active methods of detoxication (homo-and enterosorption).6.correction of cerebral metabolism.7. dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. (67). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co (36), asystolia (8), eclampsia (5), acute renal failure (5), control group (13). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in 8 monopolar channels. the estimation of eeg patterns by zhirmunskaya method (1984) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) 2,6,10,20 hz. we studied also the figures of absolute and relative power in such diapason : delta (1-4 hz), theta (5-7 hz), alpha (8-12 hz), betal(13-25hz), beta2(25 hz and more). results: all eeo changes were divided into following types: 1 -organized (3,4 groups); iii -asynehronic (8 group); iv -disorganized with prevalence of alpha-waves (12,13,14 groups); v -disorganized with prevalence of delta-and theta activity (16,17,18;19,20 groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied 20 pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf-800,with effective area-800 cm,the volume of extracorporal contour-60 ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. (2). post-operative cerebral neoplasm (1), post-operative subdural hematoma (1). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the 30 minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the 30 minutes p0st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) 30 (14) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of 25 cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: 25 patients were included in this study: 9 men and 16 women. the mean age was 64 years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was 5 h. 32 m. +/-7 h. 10 m. hypertension was found in 19 patients (76 %), ischemic heart disease in 4 patients (16 %), hypercholesterolemia in 10 patients (40 %), hypertrigliceridemia in 3 patients (12 %), smoking in 2 patients (8 %), atrial fibrillation in 1 patient (4 %), history of epilepsy in 1 patient (4 %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in 4 patients (16 %), a single hypodense lesion in 4 patients (16 %). in 11 patients electroencephalogram was normal (44 %), in 8 patients there were widespread nonspecific electrical changes (32 %), in 6 patients there were focal nonspecific eeg abnormalities (24 %). conclusion: in our study tga was more common in women (64 %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. 94 march 95 thirty-seven patients with intracranial aneurysms were operated on within 48 hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n=12) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in 23~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_.7 bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin2~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: 1) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, 2)the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and 3) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co 2 vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. 1991; 35:113-122) . we wondered whether co 2 vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: 6 patients (5m/1f, age 32-65yrs, glasgow 4-8) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco 2 25, 30, 35, and 40 mmhg by increasing pico 2 with mechanical ventilation kept constant. normal co 2 vasoreactivity was defined as an increase in cbf of at least i ml/min.100 g per mmhg paco 2. results: 2 patients had normal co 2 vasoreactivity bilaterally, 2 patients had altered co 2 vasoreactivity at both sides, and 2 patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the 6 patients left cbf was in mean !7 ml/min.100g lower than right cbf (figure methods: following institutional approval 4 piglets (body weight 25:tl .5) were anaesthetized by 2% fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs-3 abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo 2 determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at 25 mmhg was achieved by appropriate balloon inflation for 10 rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio2=l on respirator (o). samples were obtained at 0 time (1), 10' min at hypoperfusion (2) arid at reperfijsion at 1' (3), 3' (4) and 10' (5). pao2, pjo 2 and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of 500~tm hydrogen peroxide (h202). resul'~s: the mean pao 2 was 34 mmt-ig for group i and methods: we retrospectively reviewed all 411 upper gi-endoscopies, performed in the period january 1992-july 1994 in 301 patients (199 men and 102 women) admitted at the 4 icu's of our hospital. results: it concerned 129 surgical, 103 medical, 50 eardiological and 19 neurological patients with a mean age of 57.9 yrs (range: 14-91). in 86%, the endoscopy was performed at the icu and in 14% at the endoscopy department. in 56% of the cases, the endoscopy was primarily diagnostic, of which 70% was performed for localization of upper gi blood loss. in 44 % the endoscopy was primarily thempentic, of which 89 % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees (31%), duodenal ulcer (20%), oesophagitis (13%), gastric ulcer (11%), others (13%) and none (10%). as coincidental findings were noted: cesophagitis (37%), gastritis (16%), gastric deer (14%), duodenal ulcer (9%), duodenitis (8%), oesophageal ulcer (7%) and others (8%). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis (1). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: 32 patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above 2 mmoltl, measurement was repeated every 4 hours for 12 hours or until normaiisation (blood lactate level less than 2 mmol/1). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was 12,5 % and mortafity was 5,5 % in a group of patients with blood lactate level less than 2,5 mmol/l (n = 18). frequency of complications (62,5 %) was significantly increased in a group of patients with blood lactate levels 2,5-4 mmol/l (n = 8), mortality was 12,5 %. mortality (60 %) and frequency of complications (80 %) were significantly increased in a group of patients with blood lactate levels above 4 mmol/l (n = 5). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo2) after major liver surgery, as shvo 2 is considered an indirect parameter for splanchthc and hepatic blood flow. methods: 30 consecutive patients were included in this study after liver resections for primary or secondary liver tumors. 5 patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo 2 was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in 26 out of 30 attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein (12.9 _+ %5 rain vs. 3.7 + 2.5 rain; p < 0.001). the procedure requires a total of between 45 and 75 minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo 2 showed a span from 27.4% to 90.0% with a mean of 67.0% -+ 10.8%. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without (53.4% • 5.3% vs. 68.7% • 10.1%; p < 0.001). (b) a negative correlation between shvo 2 immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = -0.58; p < 0.05). this correlation could also be seen for the first 12 post-operative hours (r = -0.42; p < 0.01). (c) a negative correlation between shvo2 and the difference between arterial and hepatic venous lactate levels was found (r = -0.39; p < 0.02). conclusions: the routine measurement of shvo 2 appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo 2 findings cannot be given yet. methods: in 5 cases after major liver resection, in which abnormally low readings of shvo 2 suggested an impaired hepatic blood flow, pgi 2 was applied at a dose rate of 5 ng/kg/min. as shvo 2 can be considered an indirect parameter for hepatic blood flow, the effect of pgi 2 infusion on shvo 2 was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo 2 for all 5 patients .was 54.1% (47-9 -47-3). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi 2 lead to a sustained increase of shvo z to an average of 67.1% (65.6 -69,1 ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi 2 infusion could be observed. both patients died 32 and 45 days respectively after operation in protracted liver failure. side effects of pgi 2 included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to 10% as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of 5 ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h2-receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/2 could have hidden smaller differences between c and r as has been described in volunteers. between april 22, 1990 and april 19, 1993 , 102 patients with severe acute pancreatitis were admitted to 16 participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ 3) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and 52 were assigned to the control group. there were 18 deaths in the control group (35%), compared with 11 deaths (22%) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = 0.048). this difference was mainly caused by a reduction of late mortality (> 2 weeks) due to significant reduction of gram-negative panreatic infection (p = 0.003). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < 0.05). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients (6%) and transient gramnegative pancreatic infection was seen in one (2%). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p-450. active c10 provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline (0,85-0,9% naci solution) in electrochemical set e~io-4 (russin,moscow). methods: 1. the nacio in concentration 600 ragfl (400-800 ml/24h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis 2-3/t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. 2. after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i000 mg/t in 10:1 ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca 35 g/l), the concanlration of medium molecular oligopeptides (mm 0,2) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: 1. the intravenous administration of nac10 excels detoxicative effect of hemosortion by 17-20% provides effictive presentation of protein components and blood cells and improves the transport function of albumin by 37%. 2. the return of exfused plasma after its purification ieco was 70-80%. only the remaning 20-30% of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of 26 mos 12 pts, 8 m and 4 f, mean age 64+6.6 yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin 9 days (mean 6.25). the mean peak level of ast, alt and ldh was 4340 iu/l (range 2105 to 7500), 3453 iu/l (range 1685 to 5150) and 2868 iu/l (range 1440 to 6960) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of 1.95 mg/dl (range 1.1 to 2.7), while altered coagulation paran-,ete's (pt> 1.5 times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt (8.3%). renal impairment (cr>2.0 mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure (50%) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range 3-10pg/kg/min) and dopamine (range 7-25 pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired (66.6%), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure (75%). it is concluded that: 1) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. 2) clinically significant coagulopathy is uncommon complication of ih. 3) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in 15 patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, 4, 12 and 24 hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: 52 pts, 34 ~(65,4%), 18 (34,6%) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age:54,3• years. hean stay in icu:29,2• days. 38 pts were operated, 15 of them twice. hean value of ranson's scale:4,4• (2-7). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: 5 patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf -33 pts. b)early ards with typical xrf -5 pts(1 died), c)early arf with xrf(atelectasis,infiltrates)-15 pts(9 died). d)late ards with typical xrf-32 pts(31 died), e)pleural effusions in various combinations with the above patterns -38 pts. overall mortality rate: 41/52 = 78,8%. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. 2)even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. 3)deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. 4)delay of surgical therapy is related to worse prognosis(p<o,05) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o,05). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of 12 months all surgical patients on icu were registrated prospectively (n = 1572). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. 5,2 m!ll. of single patient informatioas. results: 1300 of 1572 patients didn't suffer from liver disease or have any operation on liver or bile tract. 1201 of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by 99 patients. at a ,,cut-off" of 4rag% (total-bilirubine) the sensitivity was 0,7, specivity 0,6, positive predicive level 0,8, negative predictive level 0,5 for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface 1.2 cm z, 02 flow=3 itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight 790 gr (min 610, max 870 gr) were obtained and after a mean cold ischemia time of 35 min were perfused fo} a mean of 7.5 h (min 4.5, max 9 h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of 16 (12-25) mmhg at 38~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t8) sampling: po2, 4" + +4" pco2, ph, hco3-, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from 7.45 at t o to 7.168 at t 8 while mean output po 2 fell from 527 at t o to 10 at t 8. mean output ast values increased from 361 at t o to >2500 at t 8 while mean output alp values increased from 3.66 at t o to 197 at t 8. mean output k + values increased from 3.93 at t o to >8 at t 8. histology revealed lesions of ischemic necrosis, more prominent after t 6. conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between 1982 and 1993 the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years 205 organs were donated from 458 potential donors i139 single, 66 multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from 28 (1982) ,to 61 (1992) with a concomitant drastic reduction of donations from 64% in 1982 to 26% in 1992; amounting to a net unchanged number of donations over the last 10 years (1982 = 18; 1992 = 17) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from 0% in 1986 to 90% in 1993 despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho2) provides online information about hepatic-splanchnic oxygen supply-demand ratio [1]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [2] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi2, flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho2) in human liver transplantation. methods: with institutional approval and informed consent, 14 consecutive patients, mean age 50-2-_10 years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of 0.1 gg/kg/min and pgi2 at 5 ng/kg/min were initiated for a 45 rain period. each sequence was followed by baseline therapy for 45 rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p<0,05. results: ng at 0.1 gg/kg/min induced a decrease of mean arterial pressure (map) (84_49 [baseline] vs. 75+9 mmhg) and pulmonary artery wedge pressure (pcwp) (8j:2 [baseline] vs. 65:1 mmhg). cardiac index (ci) (5-41 vs. 4+1 l/rain/m2), oxygen delivery index (do2i) (655-+108 vs. 618+123 mgnfin) and svho2 (74_~12 vs. 69-l-_19%) were decreased (p<0.05). pgi2 at 5 ng/kg/min induced a reduction in map (73• nm~. _g) and pcwp (6+1 mmhg). ci (6_+1 l/rain/m2), do2i (7555:135 ml/min) and svhoz (81+6%) were increased (!o<0.05). 9 vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. 9 pgi2 increased systemic oxygen delivery in parallel with svho2, suggesting a corresponding improvement of hepatic-splanchnic okygenation. 9 thus, if vasedilator therapy is indicated in th6 15orient receiving liver grafting, pgi2 appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi2 in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, 1994 until now 56 donors were involved into this prospective study. every other donor was treated with 30 mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group 1 -steroid pretreatment goup (y~=35), and group 2 -control group (n=37). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the 0-5th, 14th and 30th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group 2 had slightly lower ereatinin level (819 -+ 244 g.,non vs.923 -+ 254gmol/1) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first 5 postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group 2. the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august 1993 and september 1994, 81 patients with 85 transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n=10) included microbiologic evidence and more than 2 secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n=37) referred as control groups. results: 1-year patient survival was 88.9% (72/81): 5 patients died due to serious infections, while 4 died for other reasons. mean bilimbin, stnf-rii-, ifn-7-, il-4-, il-8-, il-10-, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: 28310_+788 pg/ml vs 20452• pg/ml; ifn-7:466_+57 pg/ml vs 4.4-+1.8 pg/ml; il-4:214-+35 pg/ml vs 148-+29 pg/ml; il-8:667-+48 pg/ml vs 251_+26 pg/ml; il-10:149_+52 pg/ml vs 52• pg/ml; laminin: 3010-+312 ng/ml vs 1263-+ 117 ng/ml; neopterin: 247_+37 nmol/1 vs 96_+19 nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: 455-+31 mg/l vs 685• mg/1. conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: 81 patients with 85 transplants were monitored for various cytokines on a daily basis between august 1993 and september 1994. rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, 28 patients (34.6%) developed rejection; 14 patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further 14 patients required additional treatment with fk506 or okt3 (steroid-resistant rejection). 4 patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn-7, il-ib, il-2r, il-4, il-6, il-8, hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: 18.2-+4.1 mg/dl vs 11.2-+1.7 rag/all; tnf-rii: 23374-+798 pg/ml vs 18246_+679 pg/ml; il-8:1024+-192 pg/ml vs 275-+67 pg/ml; neopterin 148_+37 nmol/1 vs 49-+21 nmol/1; hyaluronic acid: 290_+63 ~tg/l vs 223_+28 ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: 437_+34 mg/l vs 671_+55 mg/1. ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il-8, hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of 8 consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals (1500 beds) between december 1992 and may 1994. exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the 8 patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms 15 and 35 days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal 32 days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that 35 % is critical value for cpv loss and for cev it is 65 %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to 85 % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june 1991 and april 1995 39 patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to 10 days (mean 2.4 days) we reported 22 of these patients as possible organ recipients to eurotransplant. all of these 22 patients were transplanted within 48 hours, 16 (73 %) of them even within 24 hours. the principal aetiologies were hepatitis b (7), hepatitis c (1), nanb hepatitis (5), mushroom poisoning (amanita phalloides 1). after transplantation 2 patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was 82 %, 5 patients died within 3 months after transplantation due to various reasons. 17 patients were not referred for liver transplantation. 10 of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning (4), paracetamol intoxication (4) and fulminant hepatitis a (1). 7 patients suffering from fulminant hepatitis (6) or intoxication (1) were excluded from emergency liver transplantation for various contraindications. 6 of these 7 patients (86 %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within 48 hours. but 9 the good survival rate in the transplantation group (82 %) 9 the 100 % recovery rate in the group, where there was no transplant-indication in our opinion 9 and the fatal outcome (86 % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t3), increased reverse t3 (rt3) with normal levels of tetraiodothyronine (2"4) and tsh, this lacking in "1"3 levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: 1.to study thyroidal pattern in brain death patients potential organ donors. 2.to avoid organ impairment by administration of t3. 3.to study the hemodynamic and hormonal changes after the administration of t3 in these patients. material and methods:population: 22 brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group 1) and unstable (group 2). group 2 received a bolus of 0.25p.gr/kg. and a perfusion at a dose of 2-3.5 p.gr]h of t3. hormonal assays: total t3 (tt3), total 2"4 (tt4), tsh. fxee t3 (ft3), free 1"4 (ft4) and rt3 were determine at the moment of clinical brain death (0 hrs) and in group two these assays were repeted at hours 4, 8 and 12. results: 22 patients (17 male) with a mean age of 33 years (range 17 to 71yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were 15 patients in group 1 (68,1%) and 7 patients in group 2 (31,8%). hormonal pattern: at the moment of brain death tt3 was normal in 21 cases (95,4%) and decreased in i (4,6%); tt4 was normal in 9 patients (40,9%) and decreased in 13 (59,1%); ft3 was normal in 3 cases (i3,6%), decreased in 19 (86,4%); fl'4 was normal in 19 patients (86,4%) , decreased in 3 (13,6%) .rt3 was normal in 17 cases (77,2%) and increased in 5 cases (22,8%). there were no statistically significant differences in hormonal pattern between the two groups. only t3 levels at hours 0, 4 and 8 were significant in group 2. in the 19 cases with ft3 decreased, the tt3 was normal in 18 (84%) and decreased in 1 (16%), tt4 was decreased in 11 (57,8%) and normal in 8 (42,1%), tsh was decreased in 1i (57,8%), normal in 7 (36,8%) and increased in i(5,2%) and ft4 decreased in 3 (15,7%) and normal in 16 (84,2%) and rt3 was normal in 14 (73,68%) and increased in 5 (26,3%). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t3. conclusions: 1. the hormonal pattern most often find in brain death patients was: normal tt3, decreased tt4, normal tsh, decreased ft3, normal fr4 and normal rt3. 2 . there were discrepancies in the values of ft3 and tt3 3. there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace2) could be detected by mra. methods: following institutional approval and informed consent, 10 mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace2: 30, 50, 40 mmhg; arterial blood gas probes; avl). mra was performed with a 1.5 tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight-3d-angiography (tr: 39 ms; te: 7 ms; fl: 20 deg; slab: 56 mm) for vessel diameter detection and a flash-2d-gradient-echo-sequence (tr: 28 ms; te: 5 ms; fl: 30 dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest 0toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace2 correlated with changing signal intensities, whereby under hyperventilation a decrease of 23,7% (p 0.01) and under hypoventilation an increase of 28.4% (p 0.01) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace2 dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of -3.5% (p _< 0.01) and in peripheral diameter of -22.2% (p _< 0,001). a change from normo-to hypoventilation produced an increase in proximal diameter of +6.1% (p < 0.05) and of +21.3% (p -< 0.001) in peripheral diameter. conclusions: pace2 related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co2-reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: 1) bronchoscopic potency of airways and air bronchogram sign 2} resolution time of atelectasis and broncoscopic potency of airways. 3) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were 23, men 19 women 4, seventeen patients had atelectasis of whole lung, 3 of upper lobe, and 3 of lower lobe. ten patients had atelectasis in right and 13 in left lung. eight from 28 patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [6 from 8 patients with air bronchogram sign (75%) and 11 from 15 without air bronchogram sign (73%), had bronchoscopic potency of airways, p>0.1], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency 2,29 days and in bronchoscopically closed bronchi 2,33 days, p>0,1). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign 2,25 days, and without air bronchogram sign 2,33 days. p>0). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer (4.0 mhz) and a linear transducer (7.0 mhz) using acuson 128xp/10 c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. 1) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. 2) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. 3) . from march 1994 to april 1995 we did 55 examinations by cs in 34 icu patients (20 male, 14 female; age from 29-87). patients examinations pulmonary embolism 10 16 pneumonia 7 16 pleural effusion 13 19 us-guided thoracic punctions were performed in 7 patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an 83 % concordance. results of the tee classification were : class 0 : n = 21 (34 %) ; class 00 : n = 13 (21%) ; class 1: n = 7 (12 %) ; class 2 : n = 8 (13 %) class 3 : n = 12 (20 %). therapeutic implications of tee in class 3 patients were : cardiac surgery in 5 patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in 8 patients with endocarditis and prescription of anticoagulant therapy in 2 patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in 79 patients admitted to the icu, an ultrasound-guided method (ultrasound group : 37 patients) with an external landmark guided technique (control group : 42 patients). all jugular vein accesses were performed by young interns with an experience of < 5 procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in 10 patients (24 p.cent) in the control group (p < 0.01). average access time was longer in the control group (235 • 408 sec. vs 95 • 174 see. ; p = 0.06) and puncture of the carotid artery occurred in 5 patients in each group (p = 0.83). 32 patients (86 p.cent) in the ultrasound group and 23 patients (55 p.cent) ia the control group (p < 0.05) were cannulated in 3 rain. or less. the cannula was therefore unabie to be inserted within 3 minutes in 19 patients in the control group, with failure of eannulation in 10 of these patients (53 p.cent). failure was due to thrombosis (n = 1), small calibre of the internal jugular vein (< 4 ram) (n = 5), abnormal vascular relations (n = 3) or cervical irridation (n = 1). among the 10 primary failures of cannulation, an internal jugular vein catheter was able to be inserted in 4 cases by an experienced physician on the side initially selected and with ultrasound guidance in 2 cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining 4 cases. jugular cannulation was obtained at the first attempt in 26 p.cent in the control group and 43 p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within 3 minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of 3-4%. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from 40-50%. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases (4 male, i female, 55-74 years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in 3 pts, a massive pe in 2 pts. one patient (pt) had a cardiac arrest. in all 5 cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in 2 cases. all pts were treated with 100 mg rt-pa, 3 pts in a front-loaded regimen over 90 minutes, 1 pt over 120 minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after 1 to 15 hours. all pts survived and were put on coumadine, 1 pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein 10-20 cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a 7.5 mhz transducer (ultrasound depth 4-5 cm) fitted with a needle guide and a 6 cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in 46 patients 55 catheters were introduced.in 6 cases more than one (2-4) attempt was made and in 3 patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > 4-5 cm), during the 55 procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of 9 days (range 1-22) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first 20 patients 3 cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following 26 patients duplex scanning performed after removal of the catheter yielded another 3 cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation 10-20 cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary 8-bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from 137 consecutive patients admitted to the icu from 1/10/1994 to 30/3/1995 were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a 12 month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over 3 years evaluate the necessary efforts and expenses to implement a cis in the routine of a 16-bed stcu. methods: in june 1992 a commercially available, unix-based cis was installed on a 16-bed surgical icu. the goal was a paperless documentation at the bedside. after more than 2 years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, 1-3 trained health care professionals for configuration, 1 system administrator. on a single icu (12-20 beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after 3 years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an 18 month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo2). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab 1989;9:717). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo2), at ips (sipso2) and at mid jugular vein (5th cervical vertebra) (smj 02) bilaterally. results: we found significant differences between right sjbo 2 and both right sipso 2 (p= 0.007) and right smjo 2 ( p= 0,017) and between left sjbo 2 and both left sipso 2 (p= 0.01) and left smjo 2 (p= 0.017) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). 163 patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : 8channel electroencephalograph, 4-channel roencephalograph, ad-converter (16 analog inputs, 12 bit resolution, 60 k hz), ibm 486 dx2, software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal 4.0), expert programs for estimation objective and humoral patient status (written in clipper 5.0) and statistics. there were used following programme-language instruments : borland c++ 3.0, nantucket clipper 5.01, ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: 1. direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. 2. indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner 50% of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to 146 centers and we received 84 responses (57.5 %). there was at least one uncological (i.e. with > 50 % of cancer patients) icu in 59 (% % an 18-year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at 155/minute and a temperature of 40~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was 1300/mm ~ with 0 neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv02 (59%) (n 70-80%) and cardiac index (2,37) (n 2,5-3). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n=9,2-21pmo/l) went from 143,6 to 16,6 after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i0 days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: 1087 patients were included in a prospective study from june 92 to november 94. 220 patients presented arf defined by creatinin serum leve(s greater than 150 pmol/l and previous normal levels. patients were divided in three groups. group i (control) : 156 patients with arf who did not receive substitutive techniques. group ih 21 patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii1:43 patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was 19.7 and mean length of tcu stay was 6.7 days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics (1) cardiovascular drugs (2), gastrointestinal system drugs (3), enteral and parenteral nutrition (4), respiratory system drugs (5), sedative, analgesics and paralysing agents (6), parenteral solutions with electrolytes, vitamins and trace elements (7), anti-inflammatory agents (8), protein substitutes and immunomodulation agents (9), anticoagulative agents (10). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was 30.530 drs ($122). total tc/patient was 295.195 drs ($1.108.7). ii) partitioning total tc per group of drugs reveals : (1) 43%, (2) 2.7%, (3) 2.7%, (4) 9.2%, (5) 0.3%, (6) 8.6%, (7) 9.3%, (8) 1.3%, (9) 15.8%, (10) 2.5%. t11) concerning antibiotics which consist the major cost component, group a and group b contributed by 29.1% and 13.9% to the total icu tc respectively. group b were administered to 13.9% of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso2 cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. 2-to determinate the variations of the rso2 during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso2 ) and rso2 were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam (50 mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental (4 mg/kg),fentanyl (3 mcgr/kg) and atracnrium (0,5 mg/kg) have been used for induction of anesthesia. co2te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane (0,5 -1,5%) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each 5 minutes: blood pressure, cardiac frequency, pso2, co2te and rso2. the rso2 cerebral variate in relation with: the anesthetic induction, blood ~ressure, co2te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso2 cerebral was in relation with the :ross-clampping carotid ( minimal value: 52 ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than 8000 anesthesia in chelyabinsk emergency hospital every year. to 80% patients of it emergency anesthesia is applied. more than 900 patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than 5% of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes 21 parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of 12 parameters simultaneously. results: 187 pathients extremly needed peryoperative monitoring of hemodinamics. from these 187 patients more 40% had stroke volume (sv) less than 30 ml, 18n -co less than 2.1/mim/m2, 25% -ejection fraction (ef) less than 65n and 32% -puls bioimpedans microvessels (pbm) less than 10 morn. 100 patient had intensive care in special department. 42 out of 187 died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of 12 basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do2), oxygen consumption (vo2), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do2]. methods: 18 patients (15 males and 3 females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo2, do2, ci, (a-v)do2 were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a 24-hour study period. a total number of 52 measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p<0.05 value was considered as significant. results: mean values were phi=7.19+0.1, do2=984+313, vo2=181+71, c.1= 3.7+ 1.2, (a-v)do2 = 4.47+1.2. no correlation was found between phi and do2, phi and vo2, phi and c.i, phi and (a-v)do2. on the contrary in 14 patients phi remained below 7.30 for more than 24 hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high (85%). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, 0berlingen) in combination with a standard gastroscope. measurements were performed on 9 ventilated, traumatized patients (ages 17 -75 years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of 9 cm 2 was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc0sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o 2 affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~0) and standard p~0 (ps0st) 22 consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in 11 patients (mean age 73-*7 y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at 37 ~ c using the abl500 radiometer for po2, pco: and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the p~0 at ph=7.40, pco:=40mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps0"in vivo" (i.e. the pso at the patient's value of ph, pco2 and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was 6.1 -* 1.2 houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo2 and oxygen extraction ratio (oer), r=0.94,p=0.00000. the improvement of ci (1.41 -* 0.47 to 2.55 + 0.5 l/min/m 2, p<0.0000001) induced a significant increase in svo~ (0.495 -* 0.131 to 0.636 • 0.060 %, p<.0001). a significant decrease in p50 (32.5 • 6.7 to 27.9 • 2.5 mmhg, p<0.05) without any significant change in p~0st (29.1 • 2.2 to 28.7 • 2.3 mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo2 occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: (1)personal data (2)patient's pathology (3) laboratory and~ monitor lug data (4)drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. (5)reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month (6) access to de questionnaired apache ii to load up new data. (7) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: (1)detailed drugs interactions, including toxic elements (2)diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy (3) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and (4)perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = 2 days, los = 3-4 days, los = 5-7 da~, los = 8-14 days, los >15 day~. area under the carve (auc) was ud~ninted for each 8ro~. s~ing: 25 imlimlcus. patents: of 10~65 pts comec~ively admired ~ a period of three yeet~ (1990) (1991) (1992) , a total of 8059 was i~leded in this study. pts without saps, p~ yolmger them 18 yearn, p~ with los shorter ~ 24 hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g2: 9.24, p > 0.25; auc = 0.79i-0.01) and in validation ~t (g.o.g g2: 8.95, p > 0.50 ; auc = 0.78..+0.01). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being 0.90i-0.01 in pts with los = 2 days el 0.59~.02 ia tm with los > 15 da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: 1.-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with 15 items grouped in 3 sub-scales: physiological functions (4 items); functional capacity (8 items) and subjective aspects (3 items). qol is classified in 4 levels: normality (0 points); slight deterioration (1-4 points);moderate deterioration (5-9 points); significant deterioration (>i0 points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first 24 hours as evaluated by tiss, and related factors. results: the sample was 9,291 patients, sge 57.91~17.46 years. severity by apache ii system was 15.59• points. the tiss score was 19.87• points, distributed as follows: i (<i0 points): 14%; ii (i0 -19 points): 44%; iii (20 -39 points): 36%; iv (>39 points):5%.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = 0.46, p < 0.001), and a very weak but negative correlation between tiss and age (r = -0.048, p < 0.001), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v4.5 for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of 421 items classified in 15 categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and 300 patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances (1.850), b) all generic names of medications available in our country for each substance, c) incompatibilities (2.300 cases) and d) interactions with other substances (50.000 cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among 113 critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on 113 consexa~e ~ilically ill paliffas, subdivided in 3 series following the diastases at the admission: medical pafiea~ (29 s and 23 ns), surgical patients (19 s and 22 ns), a~d poliwauntas ( 14 s and 6 ns). follow up was done at 20 d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and 18 laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at 7 fin~es (t): at fiae ~draission (t0), at 121x~ars from t0 (t1), at 24 (f2), 48 (y3), 72 (t4), % (t5) and 120 horus from t0 cf6). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < 0.05 w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the 3 series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty1 hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t2 has a sensitivi .ly of 100~ ,and a ~ecificity of 27.8%: sv1>32.89 nffmin/n~, map>92 mmhg, pmap<27 nmalqg cvp<i0 nunhg will4 nmah& lvswi>34 g m/m 2, sxo2>67~ do21>515 mlhnin/m2, o2er<31%. for lx~dical l~tienls at t5 the following criteric~a has a ser~tivi.ty of 100% and a ~zificity of 36.8~ cvp<7.5 mn~g, sao2>97%, s,g)2>74~ vo2i<133 ml/nfin/m 2, o2er<25%, shunt<19% survlvops' data of the 3 series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox3rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. 2hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n0[ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh0~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure 1) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure 2 ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied 20 patients ventilated with a siemens 900c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of 10 inflations (100, 200,... 1000 ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of 3 sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves ((31, c2, c3 the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g01ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of 80 paired act/p't-/" values was analysed in 40 patients (28 m.,12 f.,66 + 12 y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of 20.000 u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding 5 ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within 30 minutes. results ac-i -~ pi-f~ cact/~pi7 r = 0,62) cvc samples 132 88 +34 +22 . v.femoralis samples 1"28 84 +29 +24 p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing 5 ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january 1991 through august 1994, we studied filter surviva[ and hemorrhagic complications during 240 filter periods in 78 critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: 141 filters (59%) were disconnected because of coagulation. mean survival of multiflow an69 filters was twofold shorter compared to survival of fh66 gambm filters. a total of 48 hemorrhagic complications occurred of which three patients died at aptt values of respectively 39, 48 and 56 seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased 25% (relative risk 0.76, 95%c1 0.68-0.85) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased 50% (relative risk 1.50, 95%ci 1.38-1.64) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between 35 and 45 seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied 6 patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a 48hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure 40mmhg on admission and 48hours later were respectively 864.2 + 249.5mmhg, 270.0 +_ 92.0 mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and 48hours later were respectively, pas; 96.5_+20.4, 152+18.7mmhg, ad; 1.21_+1.02, 0.07_+0.04 ng/ml, nor; 1.60_+1.48, 0.65+0.39 ng/ml, pra; 26.6_+37.8, 2.5_+2.9ng/ml/hr. the ev values correlated significantly with ad (r=0.47, p=0.006, n=33), nor (r=0.47, p=0.005, n=33), pra (r=0.38, p=0.032, n=33). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p=0.00079) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf 60, 1,2 m 2, fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of 10 patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s-2403. the reaction is allowed to proceed at,. room temperature for 15 minutes before discontinuation. the absorbance at 405 nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied 4 patients, who were admitted to our neurosurgical intensive care unit between january and march 1995 with head injury and no other associated pathology (age 20-60 yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way 16 gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) 6-10 hours after trauma (t 1) and t 2 hours later (t2 the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd(1).tbe n'mimum effect occurs after 3 hours from the fast administration and is still present after 24 hours. ~-979 patients, mean age 62---11 yrs., weight 68---10 kg were studied. 321 (32.8%) were male and 658 (67.2%) female. 858 onderwent 713 hip prosthesis (7 previously plate and screw removal) 145 hip revim'un (10 stem, 33 cop and 102 stem + cop), 121 tutal knee prosthesis, in the 1st anaesthesidogy depl from 14-1992 to 30-6-1994. as for antithromboembolic ptephylam, apart from hemodihitiun 668 pts were with treated indobufen 0ndo), 199 with heparin ealdum caeca) and 112 with low mo!lecular weight hepam (lwr,91). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p=0.000l) in the padeats treated with indobufen (4.256) compared 7.'ith heca (14.5%). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p=0.000l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro0.0001) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec,7.a (13.7-+1.4 days) and lmwh (13.5+-1a days) compared with indo(ll.8_+1a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt797 recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from -100 a +50 mmhg, which activates a peristaltic pump connected m drainage robes. the bt797 recovery display shows hourly bleeding in the first 8 hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at -10 mmhg and then modified according to bleeding/minute, g bt797 recovery also has an alarm that sounds automatically if.' blood loss is more than 300 ml/hour; air is in the circuit; the batteries are running low. materials and methods: 191 pts were studied (53 m and 138 ~), aged 63.5-+11.lyears, basal hemoglobin 13.1-+ 13(range 7.8-16.6)g/all, treated from 1st january, 1992 to mst december, 1994 in the 1st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision (132pts), cup revision (~ipts), stem revision (13 pts), total knee revision (2 pts). the average dumtion of the operations was 173-+58 min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on 57 pts. and integrated (epidural analgesia + light general) on the remaining t34. anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in 131(68.6%)pts., calalc heparin in 35(18.3%)pts., low molecular weight heparin in 25(13.1%)pts.; 1 pt did not give a predepoalt of blood, 4 gave 1 unit, 45 pts 2 units, 110 pts 2 units, 31 pts 4 units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was 420-+345 ml, average postop salvage was 420-+265 mi the average intra+postop 909+-460 ml. average postop loss was 677-+359 ml. the global incidence of postop complications was: h~natomata 5.2%, dvt 1.1%, pulmonary thromboembolism 1,196, myocardiac ischemia 0.5%, acute myocardic infarction 0.5%, respiratory deflciecy 2.6%, arrhythmia 2%, cystitis 0.5% there were nn complications in 86.4% of pts. postop bleeding over 300 ml in under 60 minutes (with bleeding alarm activation) occurred in 30 pts (15.7%). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p=0.034) and with a significant decrease (p~0.003) in the pruthrombic activity detected about 8 hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in 16% of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the 30 patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references 1)borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. 60 patients with sah were studied. in 17 of them sah was due to closed eraniocerebral injury and in the rema ining 43 resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in 3 patients with closed craniocerebral injury and in 14 with vas. cular malformation despite the lack of clinical symptoms the tests in 5 posttraumatic patients and in 6 patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period (85-94) were reviewed. diagnosis of dic was based on the association of fibrinogen < 2 g/1 -platelets <150 109/1 -fpd > 40 ~tg/ml in the 24 hours of the admission. results : 40 patients -mean age 29+6 y -saps 8+_5 -gestanional age 35_+5weeks -the two first conditions associated with d.i.c. were placental abruption (35 %) and preeclampsia or eclampsia (22,5 %). bleeding episode was present in 22 pts (55 %) and surgical treatment has always been necessary. 26 pts (65 %) were given packed red ceils (12+10 u) and fresh frozen plasma (9+8 u). 6 patients were given platelets packs. heparin was never administered. 6 pts required mechanical ventilation and two patients hemodialysis. all the 40 patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t24 h 69~2 % -f at t36 h 2,60+1,5 g/i). but platelets count remained low (plat. at t48 h 80 +42109/1) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, 40 patients with a dic score (1988, japan) more than 5 points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i-2 mg/kg/h for 7 days or more) treated group and no foy group, each of 20 patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, 3, 5 and 7 days after the admission. at-iii at 1500 units for 3 days was administered if the at-iii at admission was less than 70%. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ 70%; group b, foy (+) and the at-iii < 70%" group c, foy (-) and the at-iii 70%; group d, foy (~) anffthe at-iii <70%, each of 7 patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day 3 to 7 were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were 57%, 43%, 71% and 57% in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. 20 icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: 55 adults, mean age 54 years and 11 children, mean age 18 months (2 me.-5 yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and 2/3 of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed 60 see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, 1993, 460 . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction 1, to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group 1 (control} 5 dogs and group 2 (no inhaled) 6 dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of 02 and n~ (f[q2 0,16) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation (80 ppm) in group 2 was started 15 rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan2) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and 5, 15, 30 and 45 rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao21fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < 0,05. : no produced an increase in arterial oxigenation (pao2/fio~ ratio) and reduced pap before pe induction in group 2. after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations 9 method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to 100% 02) spills automatically into the circle, balancing o5 uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. -10 (25%) underwent conventional surgical therapeutics. 9" (90%) with resection of tracheal stenosis with end-to-end anastomosis(rts). i (10%) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. -29 (72,5%) have received endoscopic laser ablation with or without calibration tubes. 17 of them (58,6%) are receiving continued endotracheal treatment until now. 12 (41,4%) have sp wihout continued treatment. -i (2,5%) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas (21% oxxygen, 5 % carbon dioxide, 84 % nitrogen); gaseous no (20ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after 270 minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, 30, 90, 150, 210, 270, 330, 390 , and 450 minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at 96 + 3,2 % (percentage of baseline value, mean -+ sd) until 270 min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after 150 minutes ( 96,4 9 +3,5% vs90_+3,1%(p<0.05);210min. 95,9-+4,5%vs84,5_+2,2%(p< 0.05); 270 min. 82,7 _+ 2,5 % ( p < 0.05). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at 81,4 +_ 4,5 % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to 92,6 _+ 5,4 %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp-140, cd62p) and glycoproteiu 53 (gp53, lysosomal protein, cd63) are expressed only after platelet activation and degranulation. activation was quantified in thrombin (0.4 u/ml) and adp (0.1 ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n=7), who had no drugs for at least 14 days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i (0.1 mm) (3-morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd41a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd62p (fitcconjugated), and cd63 (fitc). only cd41a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was 1.6 + 0.7 %, and increased to 75.2 _+ 12.2 % after thrembin-activation, and to 26.7 + 5.3 % in adp-stimulated samples. addition of sin-1 attenuated p-selectin expression to 34.0 -4-193 % in thrombin (p<.001, two-tailed paired t-test), and 5.2 + 2.2 % (p<.001) in adpactivated platelets. basal gp53 expression was 1.7 _+ 0.5 % and increased to 63 .0 + 6.4 % in thrombin, and to 7.7 _+ 3.4 % in adp-stimulated samples. with sin-l, gp53 expression decreased to 346 _+ 10.4 % (p<.001) in thrombin, and 3.0 5:1.4 (p .001) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a 53 year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on 10th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died 19 days after exposure. the metal he had been working with was a 10% cadmium alleation. blood cadmilam concentration 15 days after exposure was 0.34 mcg cd/g cr, and urine cadmium concentration was 16.9 mcg/l. on postmortem examination, tissue cadmium concentrations were: blood 175 ng/ml, liver 823 ng/g, kidney 3571 ng/g and lung 1143 ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : 8 patients were studied. on admission, plc ranged from 4.8 to 53 mmol/l, and bcc from 12.7 to 256 gmol/l. mean systolic blood pressure was 80 • 56 mm hg, mean arterial ph 7.34 • 0.14, mean anion gap was 29.7 + 7.7 mmol/l and mean pao 2 32.2 • 27.0 kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = 0.008), anion gap (p = 0.008) and bcc (p = 0.016) but not with heart rate, pao2, paco 2 and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> 7 retool/1 was a sensitive and specific indicator of a blood cyanide concentration > 40 ~tmol/1. sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a 63-year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received 10 g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury 12 days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were 53 mmol/l and 256 gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r=0.976, p<0.0001). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r2=0.968) with a blood half-life of 1.14 h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r2=0.986) with a blood half-life of 3.94 h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > 20 %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a 5 g dose of hoco (10 g in case of cardiovascular collapse) was administered intravenously over 15 min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were 28 females and 22 males. the mean blood cn concentration was 83 • 73 pmol/1. the mean blood carbon monoxide was 3.2• 2.1 mmol/1. nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < 40 ~mol/1), there was no significant change in arterial blood pressure. in the 33 cn-intoxicated patients (blood cn > 40 gmol/1) a significant increase in blood pressure was observed both immediately (p < 0.001) and 1 hour later (p < 0.001) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ 1] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a 800-beduniversity hospital from 1989 through 1994. the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal 40ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between +130 and 270* 798+310 915+453 5609• the best correlation with severity grade was found with qrs-and qtc-duration (p=0.0001), the tca-dose (p=0.0003) and hf (p= 0.027); tqrs did not correlate. 2 patients died (5.7%). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal 40ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative 3,4-methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a 25 year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken 40 tablets of "eve" (mdea, approx. 4 g) and 12 g of "smack" (heroin) by oral route approximately 2 h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was 1400 ng/ml (lethal range 950-2000 ng/ml). trace amounts of cocaine and substantial amounts of heroin (115 ngtml; mean value in heroin-related deaths: 190 ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day 4 and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around 10 %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients (20 females, 1 male) with documented aip were seen over a 10-year period in the university hospital. 1 patient was in clinical remission and 20 were with the acute attack of aip, among them 4 with respiratory paralysis were required artificial lung ventilation and 4 -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on 15 patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range 1-2 mkg per/1 kgr creatinine) fill from 188 mkg on admission 140.8 mkg, then on 3-5 day raise to 193 mkg and then during treatment with normosong and prasmapheresis lowest level was 32.9 mgk. fatalities occured in two females during attacks with proforma cerebral involvement and 13 patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in 6 anaesthetized and ventilated (fio2 0.4) minipigs and 6 dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above 35 mmhg. results: flow (03 and ppa matched pvz data (mean-+sem) are shown in the table. [zo = 0 hz impedance (z; {dyn.sec_em-5}); zl = first harmonic z; zc = characteristic z; z1 phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < 0.05 between dog and minipig, and before v~. after embolisation respectively]. before case report: a 53-yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt=800 ml, rr=12/mm, fio2=l, the patient had ph=7.28, pao2=57 mmhg and paco2=45.1 systemic bp was 130/80 mmhg and hr=80 b/min with low dose epinephrine (0.12 g/kg/min) a thrombolytic infusion (rtpa: 100mg/2h) through a peripheral vein was started tee imaging and clinical status 3 hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from 46/30 mmhg to 36/25 mmhg, etco2 increased from 26 to 30 mmhg and sao2 improved from 89% to 96% three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also 1~, properties. case report.an obese 75 },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine 0.3/die was used for prophylaxis of thrombotic disease, on day 8th at 11.00 a.m. she started to be hypoxic (sa02 90%), tach3,tardic l1 l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to 6r mm/hg, the central venous pressure (cvp) raised [rom lb to 27 mm/hg and the end tidal co2 was 7mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase 750 x i(i 3 u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp 54/32 mm/hg, cvs 23 mm/hg, spo2 90%) despite a cominnus infusion of dobutamine 20 fag/kg/min and adrenaline 0.5 ~tg/kg/min. at this stage a bolus of aoradrenaline 20 ,g was given followed by a cnntinous infusion of 0.05 !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was 149/77 mmhg, the cvp 24 mm/hg, the sao2 100% and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou16 be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i 14 pte patients (pts), 63 males (average age 47,7 yrs) and .q females (average age 53,2 yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in 94,7% of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain (79%), dyspnea (64%), hemoptysis (49%), cough (39%) with association of two or more symptoms in 93%. chest xrays findings were abnormal in 92% with diaphragmal elevation (74,2~ lung opaeilies (69,5%), atelectasis (48,5%), plemal effusion (35,2%), main pulmonary brancah asimetry (22,8~ oligemia (19%), heart shadow changes (10,4%) and pulmonary arteries "cut off' (6,6%). the association of two or more abnormalities was found in 92,1% while normal chest x-rot was found in 8~ of the cases. hypoxemia with pao2<10,4 kpa was found in 64,4% followed with hypocapnia and respiratory alealosis in 34,6% in 27,8% of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i0,5%, ecg changes-st q3t3 type in "~1,6%. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet 2:669,1971) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared 100 patients arrived in our intensive care unit (i.c.u.) in.1980: (group a) with 100 arrived in 1994: (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and 24 hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of 11,4 +/-1,8 (hb) and 34 +/-2 (hct) in group a while in group b mean value are 9,7 4-/-1,2 (hb) and 29 +/-2(hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate 25 patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took 400 ml of patients' blood and separated it within 15 min with rotation frequensy of 2000/rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made 3-4 procedures during 3-4 days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from 140 + 3.2 g/l to 120 _+ 2.3 g/1 and from 44 + 2.1% to 35 _+ 1.8 % respectively). the time of blood coagulation by lee-white has increased by 2-2.5 times (up to 10-12 rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from 480.7 + 34.6 ml/min to 625.4 _+ 35.4 ml/min (p < 0.05); b) the regional brain blood flow from 52.2 _+ 2.8 ml/min to 87.1 + 6.2 ml/min (p < 0.01); c) the brain blood flow in internal carotid arteries from 166.1 _+ 12.2 ml/min to 206.3 + 14.6 ml/min (p < 0.05). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of 199 consecutive patients, 100 on verapamil, 99 on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil (10 mg over 2 minutes) was followed by a rapid loading infusion (0.375 mg/min) for 30 minutes and finally a maintenance infusion (0.125 rag/rain) for 72 hours. results: a mean plasma level of verapamil of 150 ng/ml was obtained only after more than 24 hours. atrial fibrillation occurred in five out of 78 patients who tolerated the verapamil infusion, and in 15 out of 99 patients on placebo (p = 0.08). verapamil infusion was not tolerated in 22 patients because of hypotension or a heart rate of less than 50/min, within 6 hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion (132 + 22) compared to placebo (147 + 20). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to 0.62 + 0.03 g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to 0.40 _+ 0.04 g/l, monofibrin soluble complex concentration to 0.08-+ 0.04 g/l, blood plasmin level was enhanced to 34.0 + 0.2 mmol/1, plasminogen proactivator level was also enhanced to 153.0 + 0.60 ram, plateletes aggregation has decreased to 52 %. after plasmopheresis aggregation was decreased in 1.6 times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in 1.7 times, and plasminogtnt activator level in 4.6 times. at the same time we have observed increase in total antifibrinalitic activity of blood in 1.3 times. activity of activators plasmine and plasminogene proactivators has decreased in 1.2 times and in the same time activity of activation inhibitors and antiplasmines has increased in 7 times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc0) monitoring based on thermodilution technique in 35 critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique (10ml of ice-cold saline solution) was used to compare cc0 with intermittent bolus cardiac output (ic0) measurements. the following subgroups were prospectively studied: i. heart rate (hr) >120 beats/min, 2. cardiac output >10 i/min 3. cardiac output <4.5 i/min, 4. rectal temperature >39.0~ and 5. pa catheter was inserted for more than 4 days. results: a total of 404 pairs of ic0 and cc0 measurements were obtained from the 35 patients. bias (ico measurement minus cc0 measurement) of all measurements were 0.03• i/min and the 95% confidence limits (mean difference• were -1.01/1.06 i/min. also in the subgroups, cc0 measurement agreed closely with ico measurement (c0 >10 i/min: bias=0.16• i/min; co <4.5 i/min: bias=-0.17• i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc0 measurement with ic0 measurement neither (>39~ bias=0.09• i/min). conclusions: monitoring of cc0 using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january 1994, this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since 1994, 30 patients aged 54 to 77 years (mean 66 years) underwent cas without ecc. the mortality in programmed surgery was 0%. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: 2 units per patient, b) need of mechanical ~entilation: i3,6 hours, c) postoperative bleeding: 900 cc, d) days at icui 2,5. we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments 4 per patient (p<0,0001), b) need of mechanical ventilation: 29 hours (p<0,0001), c) postoperative bleeding: 1300 cc (p<0,004), d) days at icu: 4 (p<0,001), e) programmed surgery mortality: 7% (p<0,05). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography (3 and l0 days after beginning of the treatment), stress-echocardiography and stress ecg (8-10-th day after the onset of mi). enalapril was included into the treatment of 42 pts with mi (study group), with normal or increased blood pressure, from the 1-st day of the disease. the data were compared with 30 pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in 6 pts of the study group and 8 of control group, the arrhythmia episodes during stress test -in 5 and 8 pts and episodes of silent nocturnal isehemia -in 7 and 12 pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh %0 stress test. in 10 pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by 25%; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t0ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included 60 patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef<45%). besides cesf, the ejection fraction was calculated before and after administration of 25 mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) 32.12 • 2.51 ef (simpson) 35 . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: 41 pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! 6 13 ! 1 [ gedv, itbv and pdr showed a significant decrease dufing the first 12-24h after the operation, cfi and rvef si~canfly improved after 48k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at 48h. comparison of cold/explorer m~ements sb0wed good correlations. discussion: concerning m0~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati0n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included 12 adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least 0.5~ (max 60 rain apartl the patients were separated in two groups according to their cardiac function: 4 patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below 45% and 8 patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction (02er), the q2er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o2er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied 34 mechanically ventilated icu patients (64• years) in stable hemodynamic status (no change in vasoactive treatment for at least 2 hours), separated in 4 groups: group 1 = deep sedation, cardiac dysfunction required dobutamine (n=5)r group 2= deep sedation (barbiturates), unaltered cardiac function (h=lo), group 3= moderate sedation, altered cardiac function (h=7) and group 4= moderate sedation, unaltered cardiac function (n=12). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline (2x) and twice (q.10 min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of 59 hypertensive surgical patients. patients were allotted to 3 groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)<0.6, 29 patients; eukinetic (ef>0.5),i6 patients and hyperkinetic (ef>0.6),i4 patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p<0.001) while systolic arterial pressure (sap) (p<0.05) and peripheral resistance (pr) (p<0.01) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p<0.00t) stroke volume (sv) (p<0.0l) and co (p<0.001) were increased while pr(p<0.0t), sap(p<0.001) and diastolic arterial pressure(p<0.05) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p<0.01) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p<0.0 i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : 6 palients, 4 post cardiac surgery, 2 uremic (3men, 3 women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a 8fr polyurethane catheter with end and multiple side holes over 18 ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes(30-70ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht=15% ,protein 5,1gr/dl) in all cases. in first 24 hours the mean volume of fluid removed was 550ml (350to 720ml). in that period echo showed no residual fluid. the catheter remained within the pericardium 1 to 3 days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by 2-d echo guided perieardiocentesis. a 8-fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this 2-d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first 24 hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after 5 rain, 30 min, and finally with one hour rate, within first 24 hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate 3hbenzoyl-phe-ala-pro (bpap; 30p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi(1-m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as 0.5xf v. results: similar values of %m (69.6+3.8 vs 68.9• and v (1.29• vs 1.25• were observed in both and one lung respectively. a~k~ decreased from 4185• ml/min (both ltmgs) to 2122:~175 (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the 50% reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: 37 patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients (8 females), with a mean age of 59+8 (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin 24 hours postoperatively. square wave stimuli of 0.1 msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding 9.75 msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, 7.69• msec for the left nerve and 7.98• mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients (7.93• msec) , whereas left pnct was normal in 45 patients (7.86• msec) and abnormal in 7 patients (incidence 13.5%). in 6 patients the left phrenic nerve was inexcitable and in 1 patient left pnct was prolonged (10.50 msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction (45• vs 53• p=0.03). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper 2 to 3 cm of lima were preserved (p=0.001). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo 2 shifts. methods: we studied 26 males (554-7 yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature 27-28 c we set pump flow at 2.1 i.m~2.min -1. when map was higher than 85 mmhg we calculated vo 2 by using fick equation. then we infused sodium nitropruaside (sn) to control map at 55-65 mmhg for 10 min and we calculated vq 2. without changing the sn infusion rate we set q at 2.5 i.m'2.min "1. ten min later we measured vo 2. we took vo 2 changes into consideration if greater than 15%. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo 2 response to sn infusion during low and high q we classified pts in four groups (table). i. vo 2 increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo 2 increases with sn but the addition of high q results in systemic shunt. iii. vo 2 increase during high q proves that vasodilatation can turn flow insufficient. iv. vo 2 does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo 2 monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > 2.8 l/min/m 2, 8 _< paop < 18 mm hg,and svri __< 30 mmhg/ll/min/m 2 decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: 44 aaar patients and 41 pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > 24 hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > 0.03 sec or cpk-mb > 5%. results are presented as means _4-. sd. opt was achieved in 32 of 41 (78%) and in 36 of 44 (82%) in the pvs and aaar group, respectively. events did nat differ between groups 9 of 41 (21,9%) and 12 of 44 (27,2%) in the pvs and aaar group, respectively (p>o.05). mort was 0 of 41 (0%) and 1 of 44 (2.2%) in the pvs and aaar group, respectively (p > 0.05), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, 8 of 9 (89%) patients who developed events in the pvs group had a ci < 2.8 in contrast to 4 of 12 (33%)in the aaar group. the positive and negative predictive value were 89% and 97% in the pvs and 50% and 75% in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; 2. pvs patients who have net achieved opt are at extraordinary risk of perioperative events; 3. preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< 0.6; ratio >_ 0.8 may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade (1). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in 100 consecutive patients (pts) after uncomplicated cardiac surgery (78 coronary artery bypass grafts; 22 valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : 3.2 _+ 0.7 1/minlm~; laotate: 144 + 68 rag/i; cvp range : 4-17 rnmhg; pewp range : 2-16 mmhg. mean cvp/pcwp ratio before extubation is 0.94 (95 % confidence imerval : 0.86-1.02) and after extubation, 0.93 (95 % confidence interval : 0.83 -.1.03), (ns, paired t-test). in 25 % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = -0.04) and after extubation (r = -0.09) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = 0.08) and after extubation (r = -0.13), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< 0.6) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as 1 within a large range of cvp (4-17 mmhg) and pcwp (2-16 mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : (1) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from 50 to 120 minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. 1 audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period 1 june to 31 december 1994, 79 patients with chest paros were treated within a 0eriod varying from 30 minutes to 6 hours from onset of symtoms. ages ranged from 33 to 86 (average 64,4), oelng 49 males and 30 females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l0 m.. 204 potentia ami patients were then admitted to the coronary unit, 103 [)atients, under age 75 with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was 15 minutes (12. 17), -he total median delay was 58 minutes ~40-i h.45min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). 2 objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to 8-i 0 minutes and treatment delays to less than i 5 minutes results. following rmplementatlon of this protocol in january 1995, 30 fts were glven, with an over all average delay of 24 minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a 59% reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati0n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly0c agents to 73 patients with suspect of ami from the begin!ng of september 1994 until the end of february 1995. in this time there are two different periods, during the first 4 months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from 28 to 90) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to 28 -30 ~ c. renal dysfunction was defined as a 20 % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x2 when appropriate. data are expressed as mean +__sd. p value less than 0.5 was considered statistically significant. results: thirtytwo patients (13,5 %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod1 and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p<0,05). mortality rate was statistically different in patients with or without renal dysfunction(~,2,5 % and 0 %, respectively, p=0,001). incidence of acute renal failure following renal dysfunction was 6,2 % (2 patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature (1). however renal dysfunction appeared significantly associated with a poor prognosis. reference: 1-settergren g, ohqvist g current opinion in anaesthesiology 1994, 7:59-64 r 66; 159, 200 tzelepis, g. 112, 127, 142 late complications were observed in 14% of cannulations: local infection in 2 (i,7%), catheter displacement by the patient in 4 cases (3,5%), catheter displacement during nursing care in 5 (4,4%) and malfunction in 5 cases (4,4%). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, 33 selected patients (65_+9 years) undergoing peripheral vascular surgery (n=17) or carotid endarterectomy (n=16) were investigated. patients included had either documented cad (n=18) or two or more (n=15) dsk factors (age >65 years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia >240 mg/dl). 12-lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and 20 h, 48 h, 72 h, and 84 h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression >0.2 mv and/or new t inversion). in addition results: 22 of the patients investigated developed ecg-documented pmi, 86% occurdng in the immediate postoperative phase. troponin i levels >1.6 ng/ml were found in 19 of these 22 patients thus, comparing a cardiac troponin i cut-off level of 1 ng/ml with intermittent 12-lead ecg recordings, we found a sensitivity of 86% and a specificity of 91% methods: demographic, clinical and ecg data were analyzed. 53.8% of patients were male; 46.2% female. cad was the most common underlying cardiac disease (85.7%) and 58.4% underwent open heart surgery. 69% received proeainamide for supraventricular and 31% for ven~cular arrhythmias. 27% received a loading dose. maintenance was provided by iv route in 36.8% and by po in 63.2% (40.8%sr end 22.4% ir). 40.4% of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was 54 • 16 years, for group b 64 • 11 years, and for group c 59 • 13 years. a history of previous myocardial infarction was present in 6 pts of group a, in 3 of group b and in 54 of group c. three pts in group a, 5 in group b and 15 in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was 5 days (2 -19) for group a we used a continuous fixed intravenous a infusion at a dose of 140 the sn was 100% in groups a and b, 98% in c, and sp 100% for group a, (fixed defects included) and 50% for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, 2 pts -group b, and 8 pts -group c), transient hypotension (1 pt -group c), headache (5 pts, group c), dyspnea (1 pt -group a), while st depression was seen in 2 pts of group b and in 2 pts in group c. the rate of a infusion was decreased to 70 7/kgr/min in one group b pt due to development of chest pain s228 five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch 133 bertschat, e 141 betbes6 75 blanch, l1 177 del nogal saez 93 e1-meneza 220 nolla, j. 98 nolla-salas 24 pilz~ u puig de la bellacasa e 38 scarpa, n. 77 217 van de wetering objectives: only 50% of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines (2) able to induce pronounced conduction block (3). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted 18 g catheters in 24 gbs-and 22 cidp -patients under sterile conditions. some of them had not responded very well to established therapies. 30-40 ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed (200 -300 ml) over one week. results: the 24 gbs patients improved after 19 days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after 16 days (median). patients not at all treated before (16/24) responded better than patients that had been pretreated (8/24) with plasmaexchange or intravenous immunoglobulines. 18/22 cidp patients drew benefit from treatment, 10 stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower (1/3) than those for plasmaexchange or intravenous immunoglobulines. references:(1)wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve (1995) . (2) clinical observations were made in 24 patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok-01 apparatus, the exposure within 20 minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in 550 bed university hospital, covering 345.000 inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march 31th 1993, were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score >10, at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant (7th, 25~ and 60th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from 285 patients ( apache ii score: 12.82) admitted in the icu, 16 (5.6%) enter the study protocol. seven (2,45%) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was 22.6 vs 16.6. the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, 5/9 and mof, 6/11. conclusions: 1. -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. 2. -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(>60 years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, 103 patients were evaluated; 21(23%)had pancreatitis of unknown etiology . results:(1)patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity(48% vs 22%),mortality(24% vs 8%),and longer hospital stays than p~tierf~ with pancreatitis of known cause.(2)the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.(3)curlously,no difference existed in associated medical conditions between patierts withknown and ur6~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in 240 cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for >3 days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a 36 hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were 1.6% ie at ph 7.3 _+0.12 (95% limits) and between old & new catheters were 2.2%, ie ph 7j3 _+0.16 (95% limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis8. therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in 173 patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of <9. according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n=37); ih analgesiajsedation plus pirenzepine 60 mg/day (n=54); .[ih anatgcsia/sedalkm plus sncraltate 6 x [ g/day (n=47); iv: analgesidsedatkm plus pirenzcpine 60 mghlay plus sucralfate 6 x 1 e/day (n=35). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p <0.05. 28/173 patients (16.2 %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb (5.61 + 1.65 vs 6.12 -+ 1.65). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = 0.89) has been shown. the frequency of gisb among the groups is as follows: h 18.9 %; lh 18.5 %; llh 17.0 %; iv: 8.6 % (ch1 -~ =1.94; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" =5.86; p=0.1186) and reached an overall-value of 75.1%.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a 24-h esophageal ph-metry was performed in 40 patients in recumbent position at 30 ~ who had a ngt for more than 10 days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied 5 cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than 4) (ger-t) more than 3%, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale=0-3) was noted. two ph-metrles repeated on 48 h and on 7 days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than 4 (tph<4). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: 23 out of 40 patients presented a ger-t>3%. eleven out of 23 group i group (n=6) 1i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise (0,50 and 100 w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: 0.18 +/-0.38 i/min, vtc: -33 +/-108 ml and frc: 0.028 +/-0.048 liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol 1994; 76: 2832-2839. objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co 2 vasoreactivity in brain injury (stroke 1992;23:962-6), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: 17 mechanically ventilated patients (age 30-81 yrs, glasgow 3-10) in coma due to acute brain injury were investigated during stepwise changes in paco 2 (25, 30, 35, and 40 mmhg) by increasing inspired pco 2. middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of 10% n20 through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco2. objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: 93 patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at 80 mm hg. 1. ringer's solution, 2. 3.5% gelatine solution, 3. 6% hydroxyaethylstarch (mean m.w. 70.000). we evaluated the following parameters within intervals of 30 min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in 17 consecutive patients (6 sepsis/sirs, 2 acute heart failure, 3 ards, 6 transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z-021 system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of 1ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f -139, df = 99, p < 0.001, (figure) ). aitbv was correlated to ac1 in a linear fashion (r = 0.76, f = 134, df = 99, p < 0.o01). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in 5 anaesthetized pigs using a centrifugal pump with a 14 f arterial catheter and a 16 f left atrial aspiration line. animals were supported for two hours at a mean flow of 3.1 liter (3'680 rpm), a mean hematocrit of 29% and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), 10 minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in 9 patients with ph of various causes: 2 primary; 1 post atrial septal defect closure (asd); 2 chronic thromboembolic (te); 1 anorexigen; 1 iv drugs; 2 collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate 3h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me0. we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to 9 control humans (mean %met = 71.4% _+ 11.3% s.d.), the mean %met in ph patients was 54.3% _+ 14%. the %met in ph patients correlated inversely with cardiac output (r=0.74), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph (1663 + 536 ml/min) as compared to controls (4225 _+ 1018 ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from 20% to less than 1%. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in 32 patients with (raaa).a short acth stimulation test (synacthen test; 250 ug 1-24 acth iv) was performed at 0800 hrs within 24 hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > 0.55 umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the 32 patients investigated 6 died and 26 survived. mean cort basal in nonsurvivors was significantly (p<0.o04) higher than in survivors; 1.03 (range 0.72-1.29) vs 0.69 (range 0.24-1,14). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; 1.30 (range 0.96-2.25) vs 1.00 (range 0.57-1.53). while 8 patients showed a cort basal < 0.55, no cort stim <0.55 was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; 70 vs75 and 19 vs 21. conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: 131 pts. aged 68+14 years, admitted to our ccu from 1992 to 1994 with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters (7f, 85cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into 3 groups. in group a we included 54 pts with anterior ami, in group b, 30 pts with inferior ami, and in group c, 47 pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p<0.05 was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were 8+3 mmhg and 14_+7 mmhg respectively. despite the signifanf correlation (p<0.01) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, 19 pts (35%) presented cvp>8 mrnhg and 16 of these (84%) had pwp_>15mmhg. in group 3, the cvp was 11_+8 mmhg and the pwp, 13_+6 mmhg. significant correlation (p<0.05) between the two variables also existed, however it was impossible to predict the pwp value. 6 pts (20%) had cvp>8 mmhg but only 2 of these (33%) had pwp>15 mmhg, similar was the relation between cvp and pwp in group c (p<0.01). cvp averaged 12+6 mmhg, and pwp, 17_+8 mmhg. 33 pts (70%) had cvp>8 mmhg and 25 from these (76%) presented pwp>15 mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as 6-1~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated 35 pts (age 48+12 yeats nyha class: 12 ii, 15 hi, 8 iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo2 max determination, a 6-mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was 23.5 (18-32) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every 24 hours until somatic death. we we detected severe diffuse hypokinesia (ef<50%) in 2 patients and mild hypokinesia in 3 others (ef 50-60%). systolic function was strictly normal in only 2 patients. corrected qt interval (qtc) in ecg was 54.6_+5.5 msec (normal range 38-43 msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref-1; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc (62 + 13 95.8 + 18.4, p < 0.01) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from 58.5 + 8.8 to 79.9 + 8.6 (p < 0.01); redv was reduced from 227.7 -+ 76 to 139.14 _+ 36.4 (p < 0.05); resv was reduced from 142 • 68.7 to 82 + 35.3 (p < 0.05). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group (40.07 + 9.7|• 4.7) compared with the c"group (39 • 10.9($ 38.3 • 8.5) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: 20 elective cardiac surgery patients. interventions: dobutamine was infused at 4 ug/kg/min for 3 hours immediately after admission to the icu. hemodynamics were measured every 30 minute periods until 3 hours and again 2 hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline 90 objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied 21 patients 58.9-+ t4.4 years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< 80mmhg, urine output< 20 ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to 9 pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the 15th post infarction day and 6 months later. results: three patients refused iabp treatment and 0/3 survived on the 15th day. on the 15th day 3/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p <0.01). on the 6th month 2/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p<0.01). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from 27 to 85; 89 females and 211 males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n=179), valvular replacement (vr, n=98), combination of both (vr-cabg, n=15), or others (n=6) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first 48 hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x2 with continuity correction when appropriate. results: seventy4hree patients (24.5%) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p<0.001). type of surgery, preoperative ef, and nyha classification are the first 3 significant factors related to inotropic support (p<0.005). most patients operated on for double-vr or vr=cabg required inotropic support (57 and 60%, respectively). postoperative mortality was higher in patients receiving inotropic support (8,2% vs 0,9% 'overall mortality, p=0.003). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing 12 morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do2i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every 30-60 seconds. cardiac 3 output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa-2 1 sing do2i to at least 600 ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do21 was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached 18 mmhg. vasoactive or inotropic drugs were infused if the desired do21 was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics 4. materials: 15 asa group ill-iv patients with a mean age of 75 years (range 60-92) and a mean weight of 67 kg (range 36-93)) scheduled for major abdominal surgery were included. results: 2 patients were excluded because do2i could not be raised at all. mean do2i was increased from 488 ml/min/m 2 (range 384-610) to 688 ml/min/m 2 (range 490-967). mean volume of preoperativdy infused colloid was 954 ml (range 0-2000). during surgery 1230 ml (range 5002300) of colloid was infused. mean length of surgery was 150 minutes (range 60-300). mean blood loss was 920 ml (range 04800). expected mortality and morbidity rates ("possum") were 56% and 92%, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of 8 % and 54%, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do21 in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in 16 patients (pts) with severe left heart faille (left veutrieul0x filling pressure (lvfp) >25 mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in 11 furosemide (f) + d0butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in 6 of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other 5 pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n=5): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela 12 . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am1) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: 42 consecutive patiens with ami, from october 1, 1994 to february 28, 1995, admitted within 6 hours alter onset of symptoms, undergone successfull systemic thrombolysis. 21 of them were anterior (group a) and 21 inferior (group b) . noradrenaline plasma levels at 0 (na1), 60 (na2) and 240 (na3) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns 0.01 ns means 4-sem (normal limit for our laboratory: na < 37/0 pg/ml; ck < 170 u/i ) conclusions: 1. the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. 2. the decrease in na is more evidence in amis with anterior location. 3. this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. 3. there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: 38 patients scheduled for multiple cardiac valve replacement in 1994 were divided into two groups: group i (10 patients) -without pp; group ii (28 patients) -pp was performed preoperatively. the first pp was made ten days and the second -3 days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing (17-18~ and absence of dmso. this made it possible to preserve 90 % functionally active platelets during 20 days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the 1st p. o. d. was significantly greatest in the group i (725 _+ 97 ml) and all the patients required transfusion of the donor blood (480 + 112 ml) whereas the blood loss in group ii was 489 +_ 75 ml and olny 12 patients required the donor blood. the number of platelets on the 1st p.o.d, was 107 _+ 12. 109/l (group i) and 153 + 19. 109/l (group ii), p < 0.05.conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the 567 patients of the swiss centers) and in the 36381 patients randomized in the isis-3 trail mortality rate in women was 14.8% (1421/9600) compared to 9.1% (2417/26480) in men; in switzerland: in-hospital mortality for women was 15.5% (20/129), for men 7.1% (31/438).the table shows the results of isis-3 in terms of odds ratios and their 95% confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. (27) and bubble (13) oxygenators a, ere used. anaesthesia was balanced and pts were extubated 12 to 18 hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia (1), after induction of anaesthesia (i[), 5 mins after cpb (iii) 2 (iv) and 6 (v) hrs afiter cpb, 30 rains after extubation (vi), 24 hrs after cpb (v[1) and on the 2nd, 3rd, 5th, 8th and 13tb postoperative day (pd) (viii, 1 x, x, xi, xi1, respectively). analysis of data was performed by two-way analysis of variance, p < 0.05 being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the 13th pd. qs/qt reached maximum at 30 rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over 9ubbie type oxygenators. the aim of this study was to evaluate the influence of 3x3'genator type on pulmonary shunt (as/at) after coronary surgery. methods: 40 patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a1 (n = 27) with a membrane o~genator and a2 (n = 13) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), 30 mins after extubation (11), 24 hrs alter cpb (111) and on the 2nd, 3rd, 5th, 8th and 13th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < 0.05 being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at 30 rains after extubation (ii) in a2 group. the difl'ereuce between the two groups was no more significant from 24 hrs after cpb (iii) to the end of the investigated period. ! i * p < a.0s betw~n ~o~ conclusions: membrane ox3'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation (12 to 18 hrs after cpb). the difference in qs/qt disappeared 24 his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence (20 to 61% ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent (10-65% are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in 74 patients on admission to the icu, using a nova 8 electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs 50 ). normal range of img++ was found between 0.45-0.55 mmot/l (healthy volunteers). results: for the entire population, we found a 61% prevalence (45/74) of hypomagnesemia (figure 1) . among the surgical patients, the prevalence was highest after cardiac surgery (85%) and after thoracic surgery (80%) and was lowest after neurosurgery (8%). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure (100% for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i-5%. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from 11/93 to 11/94 1146 consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was 1% (12). in all cases diagnosis was proven by a positive hipa. 2/12 patients died. in 3/12 hatt could be confirmed before severe thromboembolic complications occured. 4/12 patients developed a deep vein thrombosis (dvt), 2/12 dvt and pulmonary embolism (pe), 2/12 dvt, pe and arterial thrombosis (at) and 1/12 a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of 1146 pts. is 1%. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality (2/12). computed tom0graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of 73 patients with cardiogenic shock, as defined by an initial ct below 2.5 l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n=26), heart failure (n=7), valvulopathy (n=2) or recent cardiac surgery (n=38). after exclusion of 10 patients with concurrently suspected/documented infection, this study included 63 patients, of whom 23 (36.5%) survived. treatment of shock included dopamine (n=43), dobutamine (n=56), norepinephrine (n=18) and epinephrine (n=23). 39 patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about 20-25 mm hg ayter single drug administration observed only in 4 patients. ezap monotherapy accomplished during one week with 20 mg daily dose. hypotensive effect observed in 5 patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of 25 mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in 3 addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of 40 mg. this new drug combination permits to lower ap in 23 patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra(78,7%)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in 59% patients with ah and low pra. methods: in 35 patients with cardiogenic shock due to ischemic heart disease (n=26), heart failure (n=7) and valvulopathy (n=2), hemod31aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least 3 times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n=30), dopamine (n=24), norepinephrine (n=i2) and epinephrine (n=7 objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing 15-20 kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h 4 it of heparinised r/l solution at 4~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing 30-35 kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was 600 ml. temperature was maintained at 38~ and portal vein pressure at 16 (12-20) mmhg. the flow was 0.5-0.7 ml/gr of graft liver mass per minute. observation period was 8 hours (t8). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t0=124mmhg , t8=118mmhg), hr (t0=177, t8=201), rap (t0=11mmhg , t8=19mmhg), pap (t0=26mmhg, t8=31mmhg), pcwp (t0=14mmhg, t8=15~mhg), svr (t0=1940dyn'sec/cm '5, t8=2190dyn'seclcm~ pvr (t0=206dyn.sec/cm o, t8= 354 dyn.sec/cm ,'~), co (t0=4.631t/min, t8=3.61t/min), do 2 (t0=662ml/min, t8=261.6 ml/min), vo 2 (t0=118ml/min, t8=111ml/min), o2er (t0=17.8%, t8=42.5% ), ph (to= 7.48, t8=7.39 ), po 2 (t0=292mmhg, t8=371mmhg), pco 2 (t0=28mmhg, t8=30 mmhg), pvo 2 (t0=47mmhg, t8=36mmhg), svo 2 (t0=84%, t8=66%), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. 02 consumption, co 2 production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: 24 paced pts (mean age 55+-13 yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every 6 months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a 41 year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to 39.5 ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity 34%), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a 49-year-old woman presented a 3 year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was 60 mm hg, pulse 140 beats/mir~ute, hematocrit 0.59, seln~n protein 46 9/i, and se~um albumin 23 q/l. an leg-kappa pa[apfotein was demostrated (7.82 g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, 1.000 units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the 48 bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in 30 critically ill patients 16 men and 14 women ages from 32-74 who had sepsis -arf 10 congestive heart failure 8 postoperative multiple organ failure 8 and polytrauma 4.this method was applied to these patients from 24 to 168 hours. 20 % of the patients recovered completely their kidney function, 50 % improved their kidney function and 30 % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to 2 groups: in group i, (n=48) airf was inducted by bilateral renal artery clamping for 60 rain. group h (n=48) rats underwent a sham procedure. in each group, rats were dialyzed twice (4th and 8th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an69) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day 5 was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): 0.78_+0.54; cupro: 0.84_+0.9; hemo: 0.82_+0.28; an69: 0.77_+0.31). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day 5 (p<0.001), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: 150 patients who underwent first cadaver kidney transplantation in our unit between january and december in 1994 were involved. the recipients were divided into 3 groups: group i." non functioning graft (n=27); group ii: delayed graft function (n=59), group ili: good graft function (n=64). the grouping criteria were: a/haemodialysis in the fii~t 5 postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the 1st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. (0.36) and croup ii. and iii. (0.31 and 0.30, p< 0.05). intmo!0emtive significant differences were found between the different groups in systolic blood pressure (group i. 110 hgrmn, group ii. 140 hgnnn, group iii. 165 hgmm, p<0.05), mean arterial pressure (group i. 66 hgmm, vs. group ii. 83 hgnun p<0.05, vs. group iii. 116 hgmm p<0.001), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. 39 inin. vs. group ii. 43 rain. p< 0.05, vs. group i. 49 rain. p< 0.00!). the rejection rate was higher in the first 5 days in the patients with non-functioning grafts (group i. 53% and group ii. 24% vs. group iii. 12 %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems. key: cord-010119-t1x9gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober 7‐10, 2017 date: 2017-09-04 journal: transfusion doi: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna1 blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to 3 months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by 3 months. urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months. of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple zikv related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* 1 , yuelong guo 2 , ritchard g. cable 3 , joseph e. kiss 4 , michael paul busch 5 , grier page 2 , stacy endres-dighe 2 , steve kleinman 6 , simone glynn 7 , alan mast 8 and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) 9 . 1 american red cross, 2 rti international, 3 american red cross blood services, 4 blood systems inc., 5 blood systems research institute, 6 university of british columbia, 7 nih/ nhlbi, 8 blood research institute, 9 nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over 12,600 donors were enrolled from 4 us blood centers for ferritin testing. the study population was enriched for racial minorities [1600 african-american (aa), 1600 asian (as), 1000 hispanic (hisp)] and for "super donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral). the minority donors and the remaining 6800 non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml). results/findings: across all subjects, 19% had ais and 42% had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed %20% decreased risk for ais compared to nhw, while hisp donors had 25% higher risk. daily use of exogenous iron reduced risk for lf and ais by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection). regular use of antacids was associated with a 20% or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean (6sd) *p < 0.05 compared to batf31/1, 200ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d1 and d14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on d14 vs 100% on d1 of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at 2h posttransfusion. at 24h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* 1 , amanda l richards 2 and krystalyn e hudson 2 . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod 1 otii 1 mice are predisposed to have autoreactive cd4 1 t cells. study design/method: four cohorts of hodxotii f1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoab production. peripheral rbcs were stained with anti-complement (c3) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd71 and ter119 to assess for the presence of rbc progenitors. statistical analysis between hod 1 otii 1 autoab 1 vs. hod 1 otii 1 autoabvs. hod -otii 1 was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd4 1 t cells were not deleted in the thymus of hod 1 otii 1 mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod 1 otii 1 . however, as they aged, 15-50% of hod 1 otii 1 were positive for rbc autoab by 6 months. thereafter, $50% of the autoab 1 mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in 3 of the 4 cohorts, 60-100% of autoab 1 mice were female. hod 1 otii 1 autoab 1 mice also had enlarged spleens compared to hod 1 otii 1 autoaband hod -otii 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd71 hi ter119 inter ) were observed in the spleens of hod 1 otii 1 autoab 1 mice but not in hod 1 otii 1 autoaband hod -otii 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03). moreover, autoab and c3 deposition were found (0.1-2% and 3-10%, resp.) on ter119 1 rbcs in all of the hod 1 otii 1 autoab 1 mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b5-a01a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards 1 , christopher a tormey 2 and krystalyn e hudson* 1 . background/case studies: red blood cell (rbc) alloimmunization occurs in up to 10% of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c57bl/6 (b6) mice were treated with pbs, or anti-ly6g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at 18-24 hours post-transfusion. anti-hod alloantibody generation was assessed 14 days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n55), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n55). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls (3/3 experiments, p<0.01). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio1 leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l1), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b9-a03b cxcr5 1 pd1 1 and ccr7 1 expressions characterize responders to rbc immunization benoît vingert* 1,2,3 , marie tamagne 1,2,3 , sadaf pakdaman 1,2,3 , anoosha habibi 2,3,4 , philippe bierling 1,2,3,4,5 , rachid djoudi 1 and france pirenne 1,2,3,5 . 1 efs ile de france, 2 laboratory of excellence gr-ex, 3 imrb u955 -eq2, 4 ap-hp, 5 universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd4 1 t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd4 1 t cells have a th17 profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd4 1 t cells with a cxcr5 1 pd1 1 phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr5 1 pd1 hi profile, with a differentiated expression of ccr7. ccr7 is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr6 and cxcr3 can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr5 1 pd1 1 lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr5 1 pd1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510). the analysis was also performed in non-transfused healthy controls (n512). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr5 1 pd1 hi subpopulation expression was identical between transfused groups and controls. ccr6 and cxcr3 expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr7 expression was very strong independently of the expression of pd1. in the aim to determine the help of the circulating cxcr5 1 pd1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr5 1 pd1 1 subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr5 1 pd1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr7 on circulating cxcr5 1 pd1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr5 1 pd1 1 profile and the ccr7 1 expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd4 t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* 1 , ashley bennett 1 , kathryn girard-pierce 1 , connie arthur 1 , amanda mener 1 , patty zerra 1 , christopher a tormey 2 , jeanne hendrickson 3 and sean stowell 4 . 1 emory university, 2 yale-new haven hospital, 3 yale university, 4 emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd4 t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd4 t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b6 recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod 1 kel). to examine the role of cd4 t cells, pic/kel primed b6 recipients were cd4 t cell depleted prior to transfusion. in addition, b6 recipients were adoptively transferred with cd4 t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd4 t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < 0.001); pic/kel primed recipients transfused with (hod 1 kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd4 t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < 0.0001) and transfer of cd4 t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < 0.001). conclusion: these results demonstrate that cd4 t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd8 t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd8 t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd8 t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h2kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c57bl/6) mice or oti mice, whose cd8 t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h2kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes 48 hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5. results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is 28-35% versus >60% in wt recipients at 24 hours (p<0.05), whereas transfusion of wt platelets into either oti or wt recipients is approximately 60% at 24 hours after transfusion. adoptive transfer of oti cd8 t cells into wt mice recapitulates the effect, with significant mova platelet clearance at 24 hours compared to wt platelet clearance (p<0.05). conclusion: this work extends the ability of cd8 t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv1r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv1r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv1r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c57bl/6 mouse blood and treated with uv1r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv1r treated wbc-rich prp, or uv1r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c57bl/6 donor blood. a second transfusion of untreated wbc-rich c57bl/6 prp was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv1r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) wbcs, but not those given uv1r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c57bl/6 wbcs were reduced in recipients of either uv1r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv1r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv1r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line. however, a recent investigation of so 2 levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so 2 distribution (mean 45.9%617.5% [yoshida et al. 2017; blood transfusion 15, 172] . the present study was undertaken to determine the distribution of so 2 in lr-rcc produced at a medium-size blood center using a novel non-invasive so 2 probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so 2 levels. study design/method: the so 2 from 977 units of lr-rcc were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection. so 2 was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a3u11; pendar technologies, cambridge ma). in addition to so 2 , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n54) from human volunteers were stored in as-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (so 2 ranging from <3 to >95%) prior to uhplc-ms metabolomics analyses in presence of 13 c, 15 n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of lr-rcc procured and sampled invasively within 24 hours after blood collection [yoshida ibid]. the shape of the so 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (iqr) of 31.4%-61.9%. male donors showed higher so 2 compared to female donors (p<0.04). no correlations were observed between so 2 levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so 2 levels ameliorate the energy and oxidative metabolic lesion. lower so 2 levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp 1 ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so 2 levels was observed from lr-rcc manufactured at a blood center using 8-hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a 3-fold increase in the absolute lymphocyte count (ko 7.59 6 4.63 x10 9 /l vs. wt 2.90 6 1.32 x10 9 /l, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko 6.00 6 0.29 fl vs. wt 5.24 6 0.56 fl, p50.0140). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd81foxp31 regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd8 1 foxp3 1 tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd8 1 foxp3 1 tregs derived exosomes and their functions involved in cd8 1 tregs mediated immune-modulation were seldom reported. study design/method: cd8 1 t cells were freshly purified from pbmcs, cultured with anti-cd3/cd28 antibody packaged beads and il-2, and then polarized with tgf-b and rapamycin into cd8 1 foxp3 1 treg cells. the harvest cells were co-cultured with cd3/cd28 beads stimulating cd41cd25effector cells in the transwell plate. the supernatant derived from cd81 tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd63, cd81, tsg101 and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir-155, let-7b, let-7d were measured by qpcr. the precipitated exosomes were further purified by cd63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd8 1 treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the cd8 1 treg mediated immune modulation. a total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by nta). cd63 and cd81 were expressed on these background/case studies: regulatory t cells (tregs), containing cd4 1 and cd8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd4 1 foxp3 1 regulatory t cells (ntregs) in inflammation conditions (including instability of foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd81 regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd8 1 treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd8 1 regulatory t cells in inflammation and transfusion. study design/method: human cd8 1 tregs were induced with tgf-b1 and rapamycin from cd8 1 t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd8 1 tregs when encountering with inflammation were test by foxp3 expression, th1 and th17 cells conversion in inflammations conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6) on day3, day6 and day9. in vivo, cd8 1 tregs were transfused into cia mice and then their survival in mice and foxp3 express were evaluated to reveal the stability of cd8 1 tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd8 1 tregs when induced factor tgf-b1 and rapamycin were removed by testing the foxp3 expression on day3, day6 and day9. results/finding: ex vivo induced human cd8 1 treg were foxp3 1 (90.40 6 1.40%) and did not secret il17a (both in supernatant and % of cells). foxp3 express in cd8 1 tregs were maintained after induced factor tgf-b1 and rapamycin were removed on day3, day6 and day9. in vitro, foxp3, il2 and ifn-c expression has no significant difference when compared with controlled tregs on day3, day6 and day9 and did not secret il17a when encounter with inflammation conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6). in vivo, cd81 treg cells were transfused into cia mice on the peak of disease onset (35 days after the first collagen immunization, has inflammation condition in vivo) to test cd8 1 tregs survival. cd8 1 tregs were found in cia mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd8 1 tregs are stable in inflammation and transfusion and can maintain foxp3 expression when induced factor were removed, these make cd8 1 treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s6k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd83/cd80/cd86 expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p70s6k and its downstreanm proteins, especially the protein s6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s6 related protein translation inhibition. xiaoyun fu* 1,2 , mikayla anderson 1 and james c zimring 1,2 . 1 bloodworksnw research institute, 2 university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in 405 leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day 43 (one day past their expiration). 35 bioactive lipids including 10 common fatty acids, 10 oxylipins, and 15 lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in 405 stored rbc units. for example, arachidonic acid (aa) ranges from 0.5 -10.7 mm, linoleic acid (la) (1.4-28 mm), dihomo-c-linolenic acid (dgla) (0.1-0.8 mm), eicosapentaenoic acid (epa) (0.03-3.1 mm), docosahexaenoic acid (dha) (0.2-3.0 mm), and alpha-linolenic acid (ala) (0.06-2.3 mm). ten oxylipins including hetes, hodes, and dihomes, and 15 lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way anova testing (fdr<0.05). the ab rh1 blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh1). the fold increase of o rh-/o rh1 among pufas ranges from 1.3 to 2.1, suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among 405 stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna-223 targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r 2 rnaprobe 5 0.96 and r 2 dnaprobe 5 0.98) with mirna-223 concentration, down to a 10-nm limit of detection. hybridization assays in 1% human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in 1% human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. 6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 total atp,lm/ghb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 extracellular lactate,mm 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 extracellular glucose,mm 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 extracellular na 1 ,mm 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 extracellular k 1 ,mm 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso100201600009c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n56) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, p50.030, chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 . in vitro quality of single dose amotosalen/uva treated platelets in 35% plasma/65% pas-3 after 5 days of storage crystal stanley 1 , marguerite kelher 2 , nero evero 1 , melissa vongoetz 3 , betsy donnelly 3 and anna erickson* 3 . 1 belle bonfils memorial blood center, 2 university of colorado, 3 cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas-3 to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct02298842) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas-3 and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% pas-3, collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, 7.5 6 0.6 310 11 platelets in 602 6 52 ml, were collected on the trima apheresis platform in 35% plasma/65% pas-3. a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n56, were 302 6 26ml (t) and 300 6 27ml (c) with doses of 3.8 6 0.3 3 10 11 (t) and 3.7 6 0.3 3 10 11 (c). all pc were stored under the same conditions and evaluated on day 5 and day 7 for physical/metabolic characteristics. results/finding: on days 5 and 7 all t and c pc had ph228c !6.2. the dose recovery for t was 87%63%. on day 5, t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table 1) . conclusion: trima pc in 35% plasma/65% pas-3 treated with the inter-cept blood system for platelets using the sv set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan 1,2 , yang yu 1 , li-ping sun 1 , shu-fang wang 1 , rui wang 1 , lei-ying zhang 1 and deqing wang* 1 . 1 the department of blood transfusion, the pla general hospital, 2 the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: 1 five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35. we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in 17% final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. 2 levels of k and lactic acid (la) were tested using automatic biochemical analyzer.3 k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. 4 treated hips-cms with d35 ssrbcs, d35 k and cell culture media for 48h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: 1 d0 ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d14 ssrbcs stop beating, but beating patterns restored at 48h. hips-cms treated with d35 ssrbcs stop beating, and beating patterns did not restored at 48h. 2 levels of k and la in ssrbcs changed most obviously. 3 only d35 k solution made hips-cms stop beating and can restore in 48h; d0 k, d14 k and la solution did not influence the beating pattern in at the end of the treatment for 24h, hips-cms treated with d35 ssrbcs show obvious shrinkage. at the end of the treatments for 48h, cells treated with d35 k and d35 ssrbcs both show obvious shrinkage, the shrinkage in d35 ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. 5 gene expression array results show a total of 140 genes were differentially expressed in d35 ssrbcs group compared with naive group. there was no consistent separation within the d35 k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. 2 in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. 3 further study should be applied to signal pathways on ssrbcs induced cytotoxicity. 4 large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as-3 as compared to sagm. the presence of citrate in as-3 seems to be necessary to prevent hemolysis of thawed cells. during storage in as-3, atp and 2,3-dpg levels rapidly decline. recently developed additive solutions like pag3m and as-7 have shown to better maintain 2,3-dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag3c in which the mannitol of pag3m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at 2-68c in pag3c. study design/method: leukoreduced rcc (n56) in pag3c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68c. on day 8, rccs were glycerolized using acp215 (haemonetics v r , braintree, ma) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808c. after thawing and deglycerolization using acp215, rcc were resuspended in pag3c. during storage at 2-68c, stability (hemolysis), atp and 2,3-dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n58) resuspended in or sagm (n54). results/finding: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels at day 8 as compared to storage in sagm, resp. 9.1 6 7.6 mmol/g hb and 1.9 6 0.7 mmol/g hb. hemolysis during post-thaw storage in pag3c remained below 0.8% for 35 days and was comparable with storage in as-3. in sagm, hemolysis remained below 0.8% for 7 days. during the first 2 weeks of post-thaw storage in pag3c, both atp and 2,3-dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag3c showed significantly higher atp and 2,3-dpg levels compared to as-3 or sagm. while in sagm and as-3, 2,3-dpg levels were undetectable after 7 days post-thaw storage, in pag3c, 2,3-dpg levels only decreased to 6.1 lmol/g hb after 35 days of storage. conclusion: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels. as compared to as-3, post-thaw storage in pag3c showed comparable hemolysis while atp and 2,3-dpg levels were much better maintained. based on a maximum allowed hemolysis of 0.8% and an atp content of >2.7mmol/g hb, thawed rcc can be stored at 2-68c for 35 days in pag3c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold (48c, 4c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only 2 apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n54-5) in 65% iso using a trima or in 65% int using an amicus and stored for 15 days at rt and 4c. samples were tested on day 1 (baseline, bl), 5, 10, and 15 of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at 40% hct. labeled blood was perfused through microfluidic channels (fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate. images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p<0.05. results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day 5 of storage compared to bl (bl: 11.6 6 1.7%, rt: 4.9 6 1.2%; p<0.005). 4c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, 4c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day 5 (p50.03) and compared to 4c-int by day 10 (p<0.01). conclusion: our work suggests that 4c storage of plts collected with a trima ap system in iso for up to 15 days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at 4c. these results are surprising since both 4c-int and 4c-iso have been shown to express similar levels of cd62p, pac-1, and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at 4c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table 1. comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th17 cells and increase in cd4 regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th1,th2,tfh and tfr cd4 subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* 1 , sarah kloss 1 , sara crew 1 and sandra j nance 2 . 1 american red cross, 2 american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to 28 unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only 510k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from 40 plasma and serum clinical specimens. group 1 contained a single hpa alloantibody specificity with or without hla antibodies (n526). group 2 included 5 specimens with hla antibodies alone and group 3 consisted of 9 patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa-1, hpa-2, hpa-3, hpa-4, hpa-5, gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is 100% concordance observed for hpa-1a, hpa-1b and hpa-5b antibodies. the pak-plus assay had difficulty discriminating hpa-5b from hpa-5a antibody when hpa-5a antibody was present (3 false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa-5a when compared to hpa-5b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for pakplus and <2.0 adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb231 cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac6 specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut1 overexpression on cell motility. results/findings: fut1 overexpression increases both ley expression and cell migration, while fut1 knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut1 overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac6. as tumor promoter, hdac6 becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac6 function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at 48c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n53) and stored at 48c for up to 10 days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and 30 minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd62p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means6sem, and paired student's t-tests were used to determine statistical significance (p<0.05). results/finding: on day 10, p-selectin levels were significantly higher in pre than bl (p50.03). mirasol treatment caused a significant increase in pac-1 expression compared to pre (pre: 10.5 6 3.1%, post: 28.1 6 4.7%; p50.04), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after 10 day storage at 48c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, 48c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day 10 4c-stored pas plts followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* 1 , arthur p. bode 1 , anne s hale 2 , michael stanton 3 , mark johnson 4 and g. michael fitzpatrick 3 . 1 cellphire, 2 bodevet, inc, 3 cellphire, 4 background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to 33, 10, and 3.3% of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to 33% of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of 10% and 33% of the tcpc produced a significant decrease in blood loss. the lcps at 10% and 33% tcpc were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to 33% of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of 10% and 33% of the tcpc reduced blood loss. these results suggest a starting dose above 3.3% tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as 10% tcpc had similar efficacy signals as 33% tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso100201300021c. the study on pcr-ssp technique for the genotyping of cd36 329-330del.ac mutation and the genetic polymorphism of cd36 329-330del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd36 (platelet glycoprotein iv, scarb3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd36 is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd36 deficiency in china. cd36 gene mutation is the main reason that leads to cd36 deficiency. cd36 329-330del.ac (frameshift at aa110) mutation is one of the cd36 mutations that causes cd36 deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in 28 pediatric patients who received both doses of the mmr vaccine at 12 and 18 months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are !90% for all mmr components. results/finding: table 1 shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years). thirteen patients (46%) were chronically transfused at the time of serology. twenty-three patients (82%) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to 6 months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper 1,2 , franklyn cladis 2 , richard saladino 2 , darrell triulzi 3 , barbara a gaines 2 and mark yazer* 1 . 1 university of pittsburgh, 2 children's hospital of pittsburgh of upmc, 3 institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (<50) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately 11 months, 15 trauma patients received wb: 7 group o and 8 group a recipients, 53% male, median (iqr) age was 11 (4.5-14) and 73% blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of 36 (22-51) and 47% mortality rate. the median (iqr) quantity of wb transfused to group o recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean 6 standard deviation haptoglobin concentrations for non-group o recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group o recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514). the mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to 30 ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) 41 with anti-human igg only, and a 31 positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a 60 minute 378c incubation, followed by 3 automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer 4096 and the breast milk showed anti-d with a titer between 16 and 64. the patient had a consistent plasma anti-d titer of 8. the patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that 30% of c1 scd patients from the west indies and west and central africa are partial c and at (30%) risk for alloimmunization to the c antigen through transfusion of c1 rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce(733g,1006t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce(4-7)-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of 255 patients with genotype/rh phenotype data available, 78 (30.6%) were c antigen positive serologically. the allele frequency of rhce*ce(733g,1006t) was 0.071. in total, 15 (5.9%) patients possessed rhce*ce(733g, 1006t) in the absence of conventional c gene in trans. of the 78 c antigen positive patients, 15 individuals (19.2%) were predicted to be partial c based on four molecular profiles [rhce*ce(733g, 1006t)/rhce*ce:12; rhce*ce(733g, 1006t)/rh*ce:1; rhce*ce(733g, 1006t)/rh*ce(733g):1; rhce*ce(733g, 1006t)/rh*ce(733g, 1006t):1]. in these 15 partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after 60 transfusion exposures (57 c-antigen negative units; mean: 4, range: 0-36), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce(733g, 1006t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* 1 , megan l nguyen 1 , melanie c proctor 1 , david e krysztof 1 , gregory a foster 1 , erin k sash 1 , sandy s dickson 1 , joua yang 1 , jeffrey m linnen 2 , kui gao 3 , jaye p brodsky 4 and susan l stramer 1 . 1 american red cross, 2 grifols diagnostic solutions inc., 3 grifols diagnostic solutions, inc, 4 quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and 4 suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on 6/20/16 (fl, ga, sc, ms, al). following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on 12/12/16. travel history questions were discontinued on 1/23/17. confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 mps. no reactive donations were identified by mp-nat. of the 1,895,142 id-nat donations, 72 were initial reactive (ir) of which 8 (11%) confirmed positive (cp) by subsequent testing (cp rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%). five (62%) cp donations were id-nat repeat reactive (rr); 3 (38%) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and 3 in fl, 2 of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from 2 to 73 days prior to donation. two donors with a travel risk reported clinical symptoms; 6 cp donors (75%) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than 40 copies (c)/ml to about 8ê 5 c/ml. at the time of writing, the longest period of detection in rbcs was 91 days vs. 17 days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from 1 ir and all rr donors, ranging from 12 to 2000 c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat1 samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of 6 were prepared by diluting nat1 plasma 1:6 and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm1/high vl; igm1/low vl. results/finding: of 52,942 donations collected april 3-december 31, 352 were reactive for zikv rna. igm-index donations had higher vls (mean 1.1 x 10 6 vs 8.3 x 10 4 iu/ml) and higher proportions of simulated mp-detectable results (93% vs 23%) than igm1 donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm1 donations increased (table 1) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the 2016 pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r 6800/8800 systems lisa lee pate* 1 , phillip c williamson 2 , michael paul busch 3 , susan rossmann 4 , scott jones 5 , ann butcher 1 , john duncan 1 , jean stanley 1 and susan a galel 1 . 1 roche molecular systems, inc., 2 creative testing solutions, 3 blood systems research institute, 4 gulf coast regional blood center -sugar land, 5 qualtex laboratories background/case studies: in february 2016, the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march 30, 2016 and testing of puerto rico donations began on april 3, 2016. as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august 2016, the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april 3, 2016 -february 28, 2017 using the investigational cobasv r zika for use on the cobas v r 6800/8800 systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a 1:6 dilution to simulate mini-pool testing. results/findings: a total of 1,776,190 blood donations were screened using cobasv r zika. of 56 ir donations, 12 were repeat reactive (rr), 39 non-rr and 5 had no repeat testing. of the 12 rr donations, 7 were positive by altnat; 3 of these were igm positive. all 4 altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the 5 rr donors that were not igm positive on index, 3 enrolled in follow-up and all seroconverted. of 39 non-rr donations, 38 were altnat negative and 1 is pending supplemental testing. 8/38 donors were igm positive on index. 30 donors were igm negative on index; 15/30 enrolled in follow-up; 14 remained igm negative and 1 was 1gm inconclusive. of 5 donations without repeat testing results, 2 met criteria for positive (1 was altnat positive, igm negative and 1 altnat negative, igm positive). 1 donation is pending additional testing. altogether, 22/56 ir donations met the criteria for true positive on the index donation. 9/22 (41%) true positive donations were reactive when retested in a simulated minipool. 16/22 were igm positive. conclusion: 0.001% of the 1,776,190 donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* 1 , marc germain 2 , gilles delage 3 , maria esther lopes 1 and yves gr egoire 3 . 1 hemorio, 2 hemaquebec, 3 h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia (2013) , and in brazil (2015/2016), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since 80% of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january 1 st , 2016 through november, 26 th , 2016, from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for wb and 1.75 for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x (1 -proportion of refused donors) x (1proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain 1:13,598, for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from 6 donations). the residual volume of mp plasma, 0.35 -0.45 ml, is routinely discarded. beginning in april 2016 each blood center saved $67 mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april 3-15) 3 mp6 were combined into pools of 18 donations; thereafter mp6 were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first 6 months of samples is complete for 6,292 mp, comprised of 37,752 donations collected from april 3 to october 9, 2016. a total of 77 pools were positive, with 76 detected between april-june 2016. the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over 0.6% of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over 0.4% of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in 2016, zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* 1 , sze sze chua 1 , mars stone 2 , michael paul busch 2 and ai leen ang 1 . 1 health sciences authority, blood services group, 2 blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on 26 august 2016. the numbers rose rapidly to 386 cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since 1 october 2016. zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the who international standard for zikv and 25 replicates of negative controls prepared by bsri. probit analysis was performed to determine the 50% and 95% limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a 14 member blinded zikv reference panel from the usa-fda. results/finding: a total of 63,144 donations were screened from 1 october 2016 to 31 march 2017, with 1 false positive case and 1 zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml. zika igm was negative in the index donation sample but present in the 10-day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be 2.1 copies/ml at 50% lod and 10.0 copies/ml at 95% lod. the procleix zikv assay detected rna in 6 out of 9 patient samples and provided 85.7% agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with 1 confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types 1, 2 and 3 express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to 21 reactivity on initial gel testing. if genotyping demonstrated weak d types 1, 2 or 3, the intent was to manage the patient as rhd-positive. if weak d types 1, 2 or 3 were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in 2 to 4 weeks. results/finding: rhdgenotyping was performed on 22 patient samples over 15 months. of these 22 patient samples, 13 (59%) were weak d types 1 or 2. the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type 1 required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types 1 and 2 patients have not received transfusions at this institution since they were genotyped. four of 4 obstetric weak d types 1 and 2 patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this 15 month study period 13 serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using 3 fda approved anti-d reagents. when reactivity with all 3 reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over 80 rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of 8 months we tested 509 rhd-negative blood donors. there were 3 (0.6%) partial-d, 1 weak d (0.2%), and 3 (0.6%) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs5-46_42deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these 8 donors showed that 6 rhd-negative recipients received rbcs from 4 of these donors. five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, 5 grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which 63% of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d1" with 31 reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october 2016 to march 2017, we performed routine d typing (neo, immucor) on 1875 obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of 31 using at least 1 antibody. solid phase and manual testing used the series 4 and series 5 reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh1) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all 13 samples. two of 13 (15.4%) were d1 with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs1-29c, rs2301153; ivs3 1 117c, rs28521909; and ivs3 1 124a, rs28562109). two (15.4%) were d1 and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four (30.8%) patients had rhd alleles with known potential to make anti-d (rhd*dol2, rhd*dar1.2, and 2 with weak d type 4.0). one had weak d type 96, which has uncertain susceptibility to alloimmunization and one was weak d type 1, which has not yet been associated with anti-d. interestingly, two (15.4%) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type 2 is a variant of the rhd protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c.1154 g>c which is the first nucleotide of the exon 9 of the rhd gene and thus could be implicated in exon 9 skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v6 (agilent) and the nextseq500 platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type 2 rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by beadchip technology. interestingly, 4 out of 10 carry the c.1154-31c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last 4 patients, one has been previously characterized as rhd weak type 2 carrying the c.1154g>c (p.gly385ala). independently, sanger sequencing on 50 unrelated rhd weak type 2 samples pinpoint to a linkage disequilibrium between c.1154g>c (exac, maf 5 0.001145) and the c.1154-31c>t (exac, maf 5 0.2496). in silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon 9, mutant rhd c.1154g>c, mutant rhd c.1154-31c>t and double rhd mutants c.1154g>c plus c.1154-31c>t, we showed no influence on skipping of exon 9 due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type 2 between ala385 (transmembrane helix 12) and val183 (transmembrane helix 6) hampering membrane insertion. conclusion: the c.1154-31c>t variation is always associated in cis with the missense mutation c.1154g>c on the allele rhd weak type 2. the c.1154-31c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has 10 transmembrane (tm) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p.280. we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and 2010-2016 aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, 2012) . results/finding: seven snmvs located within 1 amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all 3 at the exofacial ends (p.a93t, p.w240r, p.v333d) are jk-weak; the two jkneg exceptions p.g298e and p.g299e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, 13 snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v10m, p.e44k, p.v76i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the 13 jk-neg variants are within 19 aa (p.270-p.299) of jk a/b at p.280. none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n289s and p.s291p are adjacent to p.288f and p.292l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among 13 jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc14a1 gene, which encodes the urea transporter ut-b1. the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in 1965. in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc14a1 gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within 1 aa from tm a-helix end v76i, a93t, w171r, w240r, g298e, g299e, v333d* cytoplasmic n-terminal v10m, g40s, e44k, l45p in membrane tm and urea-pore a-helices r64w, r64q, g65d, i117t, a183v, l246r, a248t ‡, a270a §, l272f, n289s, s291p, t319m 2/10 * second nucleotide variant in this allele is synonymous (p.p196p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a 5 mbp region in 19q13.11-13.2 with an lod score of 9.6. using deep sequencing, we identified a potential deleterious mutation in the znf850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain. the identical del84-znf850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf850 locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf850del84. none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc14a1gene, is a urea transporter that has been associated with renal function, we found that people with the znf850del84 in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf850, prevalent in southern spain due to a founder mutation, leads to ut-b1 dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-(2-ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis(2-ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at 30 days and 1 year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the 12 pools included 5 group a, 6 group o and 1 group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than -208c within 8 hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day 0 (pool), day 30, and 1 year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day 0, day 30, and 1 year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp 5 2.9 ppm; mehp 5 0.3 ppm; deht 5 0.9 ppm; and meht 5 0.2 ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than 80% of its initial value. plasma stored in deht bags had an average plasticizer content 90% lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate rbcs from platelet-rich plasma (prp). prp was diluted 3-fold in pipes-saline with 1.4mm pge1 and centrifuged at 1900 g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of 34-40% and 150,000-250,000 platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted 1:1 (spdp50%) with plasma from a patient with type 3 vw disease (t3vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was 20% (spdp/ffp > 0.8). results/finding: six batches of spdp/ffp were evaluated using 17 subjects. there was no statistical difference between the spdp/ffp pairs (p50.7558). the mean ratio of spdp/ffp was 1.21 with a 95% ci of 0.84 -1.57. comparing spdp vs. spdp50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was 20% greater than in samples reconstituted with ffp. the lower limit of the 95 th % ci is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%. even with 50% dilution with t3vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* 1 , qiyong peter liu 2 , grantham c. peltier 1 , ryan c. carney 2 , ashley s. taylor 1 , colby s. mcintosh 1 , james a. bynum 1 and andrew p cap 1 . 1 u.s. army institute of surgical research, 2 velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: (1) ffp; (2) ffp with 70mm glycine; (3) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; (4) spdp pretreated with glycine-hcl (20mm); and (5) spdp pretreated with glycine-hcl:glycine (20mm:50mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood (40% hct with 200 platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < .05). fibrin polymerization density was slightly diminished in rspdp vs. ffp (0.879 vs. 0.742 o.d., p < .01), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < 0.001). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < .01) and an additional twofold in pretreated spdps vs. rspdp (p < .05). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < 0.01). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* 1 , anita tuip-de boer 1 , ruqayyah almizraq 2 , jason p. acker 3 , philip j. norris 4 , jennifer a muszynski 5 and nicole juffermans 1 . 1 academic medical center, 2 university of alberta, 3 canadian blood services, 4 blood systems research institute, 5 nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from 8 donors (blood type a or b). supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and ev analysis. a549 type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to 25% stretch using a cellstretcher. control cells were not stretched. after 24 hours, il-8 and il-6 production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il-6 and il-8 production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p<0.05) . incubation of stretched cells with stored wbf products resulted in higher il-8 production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry). monocytes were co-cultured in media plus 20% rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in 5 replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean 6 sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table 1) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin-8 production was higher after exposure to fresh wbf (248 6 115 % control, p 5 0.02) or wbd at expiry (292 6 111 % control, p 5 0.0005). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* 1 , alessandro tocchio 1 , anita howell 2 , kaushik sridhar 1 , jason p. acker 3 and utkan demirci 1 . 1 stanford university, 2 canadian blood services, centre for innovation, 3 background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from 24 volunteers with four different age and sex categories (male, 18-40 years; male, >60 years; female, 18-40 years; female, >60 years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young (1.098 g/ml) and older female donors (1.109 g/ml) (p < 0.01). moreover, rbcs from young males (1.096 g/ml) were significantly less dense compared to rbcs profiled from older female donors (1.109 g/ml) (p < 0.05). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il-6 (pg/ml) il-8 (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: 120 icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of 426 rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p<0.001). substantial correlations were also found between orp and free hemoglobin (p<0.05) and orp and free heme (p<0.05). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than 35 days compared to rbcs stored for 7 days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of 1 to 42 days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to 7 days storage duration -reference group), medium age (at least 1 rbc of 8-35 days storage), and oldest (at least 1 rbc greater than 35 days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for 7 days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days. results/finding: 24,726 patients receiving 90,530 rbcs were included in the analysis. exposure to rbcs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (hr 5 0.91; 95% ci: 0.72, 1.14; p 5 0.400). exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (hr 5 0.90; 95% ci: 0.73, 1.10; p 5 0.295). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than 35 days compared to exclusive exposure to rbcs stored 7 days or less was not significant (hr 0.90; 95% ci 0.72, 1.14; p 5 0.381). the confidence intervals around the hazard ratios for the other 7-day intervals all include 1. similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to rbcs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for 1-3 days (hr 0.82; 95% ci 0.37, 1.83; p 5 0.635). the confidence intervals around the hazard ratios for the other 3-day intervals all include 1. conclusion: individuals exposed to rbcs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for 7 days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* 1 , cynthia walser 1 , tatsuro yoshida 2 , andrew dunham 2 and pedro cabrales 1 . 1 university of california san diego, 2 new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o 2 ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o 2 carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o 2 saturation <10%) stored rbcs, or anaerobic/hypercapnic (o 2 saturation <10% and pco 2 (@378c) $70mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as-3 after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions. rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with prbc stored for either 1 or 3 weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. 1(11%) neg patient showed short term response and 6(67%) patients showed progressive disease. at the neg group standard eval 1(11%) patient showed response and 3(33%) had progressive disease. 1(11%) neg patient had long term response compared to 11(21%) pos patients. at the pos short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease. at the pos group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease. overall, 28(53%) pos patients responded compared to 2(22%) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd38 neutralizing substance could play a role in treatment response. alternatively, reduced cd38 expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate 24% replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of 0.2m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* 1 , laurie sutor 1,2 , germ an leparc 3 , marjorie doty 3 and william crews 1 . 1 carter bloodcare, 2 ut southwestern medical center, 3 oneblood background/case studies: anti-cd38 drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with 0.2m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a 28 day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and 0.2m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual (18 th edition). each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. (25) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than 11 with the untreated or dtt-treated cells during the study. conclusion: long term storage of 0.2m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque-10771), pooled, suspended in cryopreservation media (20% dmso; 1:1) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips (378c, 5% co 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o1) rbcs sensitized with either anti-d (positive control), anti-scianna-2 (sc2) or anti-anwj or lipopolysaccharide stimulated for 2 h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/100 monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed 96.2 6 1% viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il-1b, il-6, il-8, mip-a (p < 0.01), mip-b and gro (p < 0.05) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc2-and anwj-sensitised rbcs resulted in a pi of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-d sensitized rbcs (pi: 72 6 8.7%). a weak (11) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in 41 iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi>5%). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in 5 patients, involving 3 antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all 5 patients had 3-41 positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients 1 and 2 typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c1 by hea precise-type. ega-treated rbcs gave 31 reactions with the same anti-c reagent. patient 1 rbcs gave variable reactivity (vw-11) with bio-rad seraclone and ortho bioclone anti-c. patient 2 rbcs gave 11 reactivity with all 3 anti-c reagents when incubated for the maximum incubation time allowed. patient 3 rbcs were jk(a1) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat1rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients 4 and 5 tested s1 with bio-rad seraclone anti-s (3-41), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat1 rbcs but not all manufacturers include reagent limitations regarding testing of dat1 rbcs. we describe 2 cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and 3 cases with false positive tests with anti-s (n52) and anti-jk a (n51) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with 3-41 positive dat and supports testing to dissociate igg from rbcs strongly dat1 before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/2opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r :5 prob(ab|al-loexp), so that prob(ab) 5 prob(ab|alloexp)*prob(alloexp) and 0 r 1; rewriting prob(alloexp) 5 prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) 5 nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) 5 r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past 12 months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in 2 states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for state b, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a 13-and 3-fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from 13 to 567 (state a) and 151 to 1,496 (state b), which annualized, represents a potential gain of 2,216 (state a) and 4,035 (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female. there were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to 10.5 ml/kg or 15% of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit. variable volume scales [vvs] can be programmed to vary unit volume (up to 550 ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by 10 ml at ebvs <3.5l in donors !23 yo, but increase by 5-40 ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the 18 mos. before a 6 mo. phased implementation of the vvs, and the subsequent 24 mos. multivariable analysis [mva] by 6-mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods 1 & 2, continued during impl and post-impl periods 1 & 2, returning to the baseline rate in post-impl periods 3 & 4 (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods 3 & 4. the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of 3.8 ml during post-impl periods 1 & 2 from the temporally matched baseline & pre-impl period 1. conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* 1 and yves gr egoire 2 . 1 hema-quebec, 2 h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, 2017. the vaccine is produced with live and attenuated yfv, which can circulate for at least 4 weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a 4 week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview. the deferral rate was 26.9%. at the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations. the deferral rate was 27.3%. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates. that represents a 177.34% increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017. conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of !3.0x10 11 is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: 1,000 apheresis collections from 4 centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (s), 6.3x1011 11 for double (d), and 9.5x10 11 for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for s and 6.7x10 11 for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as 99%) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc 2rbc plt/p plt plt/rbc/p plt/rbc 2plt 2plt/rbc 2plt/p #donations 47990 63 1775 10832 968 476 67 1150 142 10252 citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 study design/method: a randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model. cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of 68 aes: 40 were treatment emergent (teae), of which 8 were treatment-related (6 thrombosomes and 2 control). all teaes were mild or moderate in severity. in cohorts 4 and 5, 3/4 thrombosomes subjects had treatment related adverse events. one cohort 4 subject developed an upper respiratory infection and elevated wbcs within 8 hours post infusion, which resolved by 24 hours, and an elevated d-dimer at 24 hours post infusion, which resolved by day 7. this subject also had an elevation of prothrombin fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by day 14. one cohort 5 subject developed non-specific t-wave changes at 1 and 2 hours following her 2nd infusion that resolved by day 21 without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort 5 subject developed an igg platelet autoantibody on days 7-21, which was undetectable on days 42-60; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days 7-14, and negative on days 21-60. background/case studies: cryopreservation of platelets (plts) could extend the shelf life from 5-7 days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with 5% dmso and stored at 2808c. after thawing, the unit was reconstituted in thawed ffp spiked with either 500 lm puromycin (pm) or 250 nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd62p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd62p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage. immunoblot analyses of the plts showed a 2-and 4-fold increase in pm incorporation after 4 and 24 hours of storage, respectively. massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac1, rap1 and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac1, rap1 and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in 2015, the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of 3 days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r 4-496 cooler with 2 units of ffp, 2 units of rbcs, and 1 unit of whole blood. three to 5ml of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding) were measured by coulter counter, 2 channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c49-a03h molecular sieving: beyond genotyping ghazala hashmi 1 , reinhard klemm 2 and michael seul* 1,2 . 1 biomolecular analytics, 2 immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi2005 http://bit.ly/2ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of 30 rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, 4*4*96 samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("sca") patients (tb1 in cas-tro2002, http://bit.ly/2oplxhr, excluding le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells. in another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from sca patients, a yield exceeding 2.5x. sieving alone typically fills 65-75% of requests of moderate complexity ( 5 ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez 1 , monica kalvelage 2 , ghazala hashmi* 1 and michael seul 1 . 1 biomolecular analytics, 2 lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou2013 http://bit. ly/2ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the rhce locus) that encode 401 mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag2" (e.g. e2,c2,e2,c2) to specific combinations of "ag2" (e.g. c2e2k2fya2 and c2e2jsa2) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, 96 pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the 124 c2 samples, 24 that were also v2 and vs2 and, among the e2 samples, 12 that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select 6 specific "ee" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b (8 pools), co a|b (8) and others. conclusion: molecularsieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c2, e2 and jsa2. these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam4 (landsteiner-wiener) and ackr1 (duffy). for longer genes, such as abo of >20 kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of >21 kb each was used for all physically confirmed 48 ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least 4 clades representing clusters of 5 to 11 alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt1 genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors1) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors1. a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos1(b3galnt1) cells, and cell-surface expression of fors1 antigen was immunologically monitored with a monoclonal anti-fors1 antibody. results/findings: we found that met69thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon 3 or 4 of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors1 antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met69thr/ ser or exon 3/4 deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt1 genes is reminiscent of common ancestral origin of alpha 1,3-gal(nac) transferase genes. the finding that at can synthesize fors1 implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of 29,308 cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period 7/1/2008 -4/1/2017. abo genotyping targeting specific snps for groups a, a2, b, o1, and o2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two (0.21%) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in 53% the predicted abo phenotype was a rh neg (table 1a ). the predominant donor race was caucasian (65%). four cbu with abo discrepancy were also evaluated by genotyping (table 1b) . in 3 of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r 6800/8800 systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: 30,695 fresh and 20,029 frozen edta plasma samples from american red cross donors, collected from february 2015-2016, were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r 8800 system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of 50,724 valid results, a total of 3 donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a 65-year old male in indiana, a 21-year old male in california, and a 55-year old female in kentucky. all 3 donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna (1440 iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as 3a, the california donation genotype 3b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was 100% (95% exact ci: 99.993% to 100%). conclusion: based on the 3 confirmed-positive donations of 50,724 tested, the hev prevalence was 0.006% (95% exact ci: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% ci, 1:588-1:100,000). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report 13 months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using 95% confidence intervals. this analysis contains data from 9/1/15-9/30/16. results/findings: among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 cp results for hbv, hcv and hiv with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv 8.3 vs 4.2; hcv 23.5 vs 15.2; hiv 3.9 vs 1.0). in general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when 3-month periods were compared. conclusion: data from 4 major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* 1 , melanie c proctor 2 , deanna self 1 , monique portugal 1 , adrian gurrola 1 , laura tonnetti 2 , sonia bakkour 3 , cheryl lobo 4 , michael paul busch 3 , susan l stramer 2 and jeffrey m linnen 1 . 1 grifols diagnostic solutions inc., 2 american red cross, 3 blood systems research institute, 4 new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to 16 donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from august 25 th 2016 to april 7 th 2017 in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of 16. results/finding: the procleix babesia assay detected all four babesia species with a 95% lod ranging from 7.10-13.51 copies/ml. the preliminary 95% lod in parasites/ml ranged from 0.64-3.61 p/ml for b. microti (n59), from 0.92-1.52 p/ml for b. duncani (n52), and from 0.62-4.95 p/ml for b. divergens (n52). of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%ci: 99.972-99.997%). of the confirmed positive specimens, 8 were reactive by both ifa and pcr, 5 by ifa only and 1 by pcr only. all confirmed positive samples were reactive in lysate pools of 16. donors of reactive donations resided in ct (11), nj (1), nh (1) and me (1) for an overall incidence of 1:2,305, and 1:1,433 in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* 1 , whitney r steele 1 , ed p notari 1 , james haynes 1 , roger y dodd 2 and susan l stramer 1 . 1 american red cross, 2 american red cross (retired) background/case studies: from 2004 -2012 , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from 2008-2015. study design/methods: prevalence was calculated in 2-year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of 18.5, 7.4 and 9.1 days for hbv, hcv and hiv, respectively. linear regressions were calculated with p<0.05 (*) as significant. results/findings: from 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% first-time (ft), 81.4% caucasian). there were significant decreases in donation prevalence for hbv and hcv (p50.014 and 0.044), but no significant decrease in hiv during the 8 years (see table for f and r 2 values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p50.026 and 0.042). prevalent ft donors were significantly more likely to be male (68.3% -hbv, 59.8% -hcv, 79.7% -hiv; p<0.001). incidence for all agents declined (significant only for hbv; p50.035). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) . hcv incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011). overall, incident donors were more likely to be caucasian males (p<0.01). rrs for all 3 agents decreased over time with rrs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the 8-year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in 2015, mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as-5 rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with 200lm amustaline, and incubated for 18hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero76 cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, >6.9 log 10 , or >6.2 log 10 pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was >6.2 log 10 , or >5.5 log 10 pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts-13 inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts13 activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts13 deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts13 activity of <5% and high inhibitor (1.4-8). mean age of cohort 22.8 years (range 17-64). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /l, range 9 -27 x 10 9 /l) and low a-ipc (mean 1.5 x 10 9 /l, range 0.5 -3.6 x 10 9 /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient). tpe continued until platelet count reached 150 x 10 9 /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in a-ipc from baseline (mean 11.1 x 10 9 /l, range 2.2 -25.3 x 10 9 /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean 217.6 x 10 9 /l, range 200 -294 x 10 9 /l) and a-ipc (mean 19.4 x 10 9 /l, range 13 -28.5 x 10 9 /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /l (range 14 176 x 10 9 /l) and mean a-ipc 3.2 x 10 9 /l (range 0.7 -6.6 x 10 9 /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of 20.8 days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts13 inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* 1 , michelle n stram 1 , joan sevcik 2 , alesia kaplan 2,3 and joseph e. kiss 2,3 . 1 department of pathology, university of pittsburgh medical center, 2 blood systems inc., 3 university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january 1, 2013 to november 1, 2016 was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version 14 (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the 96 ttp patients identified, 22 were excluded due to missing temporal data for important variables. the majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients (20%) had a prior history of ttp and 26% had severe adamts13 deficiency on admission. the median time from tpe request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table 1) . treatment was not started within an 8-hour window in 13 patients; the median time to cv access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001). two of these patients had a prior history of ttp and only four patients had severe adamts-13 deficiency. the majority (more than 70%) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus 4-8 hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi-48a transfusion 2017 vol. 57 supplement s3 hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, 2006 through january, 2017, we performed cytapheresis (cy) treatments (txs) for 123 pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. 84 pts (68%) had cml-at and received 319 leukapheresis (lp) txs; 39 pts (32%) had et and received 124 thrombocytapheresis (tc) txs. cml-at pts presented with median wbc 398 x 10 9 /l (range 193-689 x 10 9 /l), of which 63% had blast percent >75% or blast count >100 x 10 9 /l. median age was 42 years (8-79 years); 62% were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. 22% of cml-at pts had no sxs of lks; 40% pts had sxs of either cns or pulm lks (1 sxs), and 38% pts had sxs of both cns and pulm lks (2 sxs). et pts presented with median platelet (plt) count of: 1738 x 10 9 /l (642-3510 x 10 9 /l)and 71% pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was 66 years (31-89 years); 58% pts were male. results/finding: all pts received a course of cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) <100 x 10 9 /l for cml-at pts, and plt ct <450 x 10 9 /l for symptomatic et pts and <750 x 10 9 /l for asymptomatic et pts. cml-at pts received median of 3 lp txs (mean 3.9 txs/pt; range 2-8 txs). et pts underwent median of 2 tc txs (mean 3.4 txs/pt; 1-7 txs). outcomes were evaluated by percentage of pts who: 1) reached wbc (or plt ct) tx goal, and 2) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved >50% reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened. in et cohort, 85% improved, 14% stabilized, and 1% were unchanged. for cml-at pts, median final wbc ct 5 96 x 10 9 /l (range 66-307 x 10 9 /l); 94% pts received ind chemo. for et pts, median final plt ct 5 705 x 10 9 /l (263-1087 x 10 9 /l); 95% pts had resolution of thrombotic 49a transfusion 2017 vol. 57 supplement s3 symptoms. 4% of cml-at pts and 0% of et pts expired within 1-4 days after course of cy tx. of 3 expired pts, 2 pts had both blast crisis and sxs of cns/ pulm lks; 1 pt had intracranial hemorrhage or cva; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20. covariates included were fluid types, age and gender. odds ratios (or) and 95% confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < 0.05. results/finding: during the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20. the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, or (ci): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, or (ci):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, or (ci): 0. 24 (0.06, 0.95)]. age had a significant effect on having a hypotensive event [p50.04, or (ci):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a 10 year period and compared it to published literature. study design/method: we conducted a 10-year retrospective study of ta procedures performed and aes were classified according to criteria described in table 1 . during the study period, ta were performed using cobe spectra (software versions 4.7 and 6.1) and since 2013 the spectra optia apheresis system (version 8.0). literature search was conducted for data published on aes associated with ta. four studies from us and 13 non-us studies (canada, europe and japan) were analyzed. trend for ae rates from 2007-16 was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was 6.9% (396 of 5,684 procedures) during 10 year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher (8.5%, p<0.00001) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe aes with a spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively. there were no fatalities during the study period. majority of aes were grade i (60%) and grade ii (28%): 32/5684 (0.6%) procedures were not completed due to aes. comparison of aes [6.9% (396/5,684)] to both european [11.2% (n513, 12, 256/ 109, 842) ] and other us studies [13.6% (n54, 860/6,324)] showed a statistically significant difference (p<0.0001). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table 1) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within 2 hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* 1 , jordy jurgens 1 , jacoline buchner-doeven 1 , joris roelofs 1 , philip spinella 2 , jennifer a muszynski 3 , carel goslings 1 and nicole juffermans 1 . 1 academic medical center, 2 washington university school of medicine, 3 nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing. plasma was filtered through a 0.22um filter. rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57ml/kg. hemorrhage continued until a mean arterial pressure of 40mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of 17ml/kg of blood products in a 1:1:1 ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april 2013, which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january 2008 and july 2015 (n53535). because of missing data on patient characteristics, 257 patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january 2008 to march 2013, n51987) and after (april 2013 to july 2015, n51291) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively). however, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table 1 ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy16. length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of 4-factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: 4-factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in 2015. marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh5 rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p5.005) and ptt (p5.05) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use. 20 cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were 5.5 times (p5.0072) more with cell saver or anh, and 5.3 (p5.0130) times more with cpb. post-pcc thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every 3 days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days. the transfusion service medical director reviews the case and gives final approval. we observed only 1 patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight (38) patients were in-patients continuously until delivery. five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was 17 days (range 0-63). six (6) patients delivered within 3 days of approval. after approval, the mean number of additional specimens per patient was 2.1 (range, 0-9). no patient required transfusion prior to delivery. five patients received transfusion of at least 1 rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each. since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* 1,2 , jan m. binnekade 1 , benjamin nota 2 , pieter r tuinman 3 , kirsten van de groep 4 , olaf l cremer 4 , janneke horn 1 , marcus j schultz 1 , robin van bruggen 2 and nicole p juffermans 1 . 1 academic medical center, 2 sanquin research and landsteiner laboratory, 3 vu university medical center, 4 university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in 2 tertiary icus in the netherlands comparing 30 patients who developed ai during icu stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/l, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/l, p<0.001). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells4life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the cd341 fraction post separation and freeze thaw (table 1) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table 1) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd451cd611) and early projenitor cells expressing oct4 and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: 1. routine recovery of the wcf at levels higher than current methods, independent of volume. 2. higher percentage recoveries of all cell types tested than can be achieved with existing methods. 3. markedly higher post-thaw recovery of viable nucleated cells than any current methodology. 4. almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd341 target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd341 cell target of 4.0x10 6 /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd341 yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf 1 plerixafor (g1pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< 0.05 considered significant. results/finding: 110 no alc and 159 alc collections occurred among the 50 patients. fenwal amicus was used for 91% of the no alc and 99% of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was 5 hodgkin's and 45 non-hodgkin's lymphoma (no alc); 7 hodgkin's and 43 non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc 49.3, lymph 2.0x10 9 /l) than alc (wbc 39.1, lymph 1.2x10 9 /l). equivalent whole blood (corrected for ac) was processed for no alc (16.4l) and alc (17.1l). for alc group, extra collections beyond cd341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%. significantly more patients were mobilized with g1pl in no alc group (n581) than alc group (n560) and 42 collections in alc group had mobilization discontinued after cd341 cell target reached. there was no significant difference in g (13.2x10 9 lymph) compared to g1pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph). days to wbc engraftment (13.5 no alc vs 13.0 alc) and platelet engraftment (13.0 no alc vs 12.0 alc) were not significantly different. median number of collections for no alc (2) and alc (3) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the 0.5x10 9 lymph/kg or even the 0.3x10 9 lymph/kg targets. implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/kg from 40% to 54%. only 12% had <0.3x10 9 lymph/ kg. discontinuation of mobilization once cd341 cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* 1,2 and nicolas pineault 2,3 . 1 canadian blood services, 2 university ottawa, 3 canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost 48 hours at room temperature (rt) as long as units are cryopreserved by 48-hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd451 cells and cd341 cell (n53). primary nsg mice were transplanted with a ucb cell dose that contained a total of 7,500 annexinv neg viable cd341 cells. the latter was done to avoid any bias towards one group or another. short term platelets (190 vs. 140 hplt/ml, p50.06) and leucocytes (1.2% vs. 0.2% hcd451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant. strikingly, the frequency of human cd451 bm cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2. conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to 97%, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of 1sec). total lymphocyte recovery was 43% and monocyte concentration was reduced 76%. furthermore, in a two-pass process platelets were reduced by 75%. in a 12-fold parallel system we tested rbc separation from plasma and achieved 90% separation at 72ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past 15 years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from 9 clinical protocols from 2008-2016. an infusion reaction was defined as any symptom from the time of nk cell infusion up to 4 hours afterwards. a severe reaction was defined as any symptom with grade 3 or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r 3.3.1. two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of 127 nk cell infusions. there were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42). infusion rate (ml/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively). incubation of nk cell product overnight in il-2 vs il-15 had similar reaction rates for those with any symptom (88% had reaction with il-2, 86% had reaction with il-15, p50.94) and those with severe reaction (28% had severe reaction with il-2, 24% had severe reaction with il-15, p50.80). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan 1 , maryanne c herzig* 1 , barbara a christy 1 , james a. bynum 2 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at -80. mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias. on day 0, mscs were washed, resuspended in pbmc media and incubated with or without 150,000 freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, 0-5 lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by 72 h, with >6 fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was 10.0%. inter-assay variation of msc preps run under identical conditions was 7.5%. inhibition of pbmc proliferation was graded from 0-100% over the range msc concentrations therefore an ec50 of msc cell number resulting in 50% suppression of pbmc could be determined for each msc prep. this ec50 however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the us blood supply. differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged 16-49 were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the 2015/16 academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of 4265 donors contributed 6219 donations. donors were evenly split by gender, 66% were ft donors, and 87% were 16-18yo. ft and rpt 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo. odds for lf were 4 to 6 times greater in the younger donors, and for ais were 3-to 4fold higher. preliminary statistical models indicate 16yo donors may have greater risk for lf than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector. testing was performed on successful 16-18 y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < 20 ng/ml in females (f) and < 30 ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations (12 months for f and 6 months for m) and counseled to take 18-28 mg of elemental iron daily for 60 days. for m and f, a ferritin < 12 ng/ml indicated absent iron stores (ais) and < 26 ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! 20 ng/ml in f and ! 30 ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior 24 months. an appreciable number of donors with no rbc donations in the prior 24 months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* 1 , joan williams 1 , michelle humphries 1 , nancy haubert 1 , ben reynolds 1 , michael phillips 1 , randall spizman 1 , ralph r vassallo 2 , hany kamel 2 , sally caglioti 1 , german leparc 1,3 and phillip c williamson 1 . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. 1 new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. 2 study design/methods: over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the beckman coulter au680 instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. 3 results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016. study design/method: abnormal risk factor levels were defined as hba1c ! 5.7%, sbp/dbp ! 120/80 mm hg and tc !170mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females). of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as !13.5 g/dl for men and !12.5 g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from 52 blood services worldwide and complete data were available for 25 blood services. deferral percentages for low hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with 53% lower hb deferral rates in men (95% confidence interval [ci] 11% to 75%) and 61% lower rates in women (95%ci 15% to 82%). iron supplementation was associated to 57% lower hb deferral rates among women (95%ci 22% to 76%) but there was no evidence of such an effect among men (p50.680). each one-week increase in minimum donation intervals resulted in 8% lower hb deferral rates among women (95%ci 1% to 14%) but not among men (p50.454). at the 5% level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: 9.15% of all candidates for wb donation were deferred in continental france in 2015. deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from 20 to 30 weeks. analysis (table) identified 3 main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the 5 previous years. conclusion: the 3 main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, 2 ng/kg). blood was sampled every 2 hours up to 8 hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from 1.4e108 (iqr 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (iqr 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: 200 cases of taco and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from may 2015 until july 2016. taco incidence was 1 case per 100 patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march 2016 fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from 5 to 7 days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july 2008 and to routinely extend ap outdate to day 7 since february 2016. this study reports a 103 month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july 2008-january 2016, ap underwent rt on day 4. day 6 and 7 units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day 8 had a second rt performed. from february 2016-january 2017, ap underwent rt on day 5 with routine outdate extension to 7 days by performing a second rt on day 6 and a third rt on day 7, as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type 1) or repeat rt positive with negative confirmatory culture (type 2). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july 2008, 20,010 ap were entered into inventory. of these, 11,840 (59%) were transfused prior to rt testing. the remaining 8170 (41%) underwent rt on day 4 or day 5. of these 43 (0.5%) were rt positive (29 type 1 fp, returned to inventory; 14 type 2 fp, discarded), leaving a total available inventory of 8156 units tested by rt. of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate. a total of 1561 (8% of original inventory) were transfused on day 6 or day 7. of these, 768 underwent a second rt on day 6 (2 rt positives; 1 fp type one and 1 fp type 2) and 233 underwent a third rt on day 7 (no positive results). a total of 964 (5% of original inventory) outdated on day 7. of these, 754 underwent a second rt on day 8 (no positive results). conclusion: to date we have performed 9925 rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted 13% to only 5%. a total of 1522 ap have been tested twice by rt (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed fp by repeat testing or culture. a total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every 24 hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with 8 to 10 million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in 2007. contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/ml. each parasite concentration in wb was tested x2. an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at 48c for up to 42 days; platelets were stored at 228c (rt) under agitation for 5 days and plasma was frozen at -208c. aliquots for culture were removed weekly from rbcs, daily from platelets and after 30 days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at 278c for detection of live parasites for up to 16 weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at 48c, rbcs from all units spiked with 10,000 parasites/ml were positive for up to 21 days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with 1000 parasites/ml were positive for up to 7 days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with 10,000 and 1000 parasites/ml were positive up to 5 days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at 48c for up to 3 weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* 1 , marion lanteri 2 and larry corash 1 . 1 cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom (2006 -2015 ), french (2006 -2015 , swiss (2011 -2015 ), and belgium(2009 -2015 hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately 1.35 million dlvbc-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/uva-treated pc were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored >2.83 million conventional, non-dlvbc-screened pc and recorded 58 str and 9 fatalities. concurrently, zero definite and 2 possible str were reported with 607,871 amotosalen/uva-treated pc, significantly fewer than with conventional pc (table 1 ) (20.5 str per million vs. 0.0 per million, p<0.001). one definite, 1 possible, 7 undetermined/indeterminate non-fatal str and 5 contaminated "near miss" pc were reported with 1.35 million dlvbc-screened pc between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 str per million vs. 16.3 per million, p <0.05). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october 2016 for all platelets received at our institution. at time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, 10 ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at 35c for three days. results/finding: a total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in october 2016 and march 2017 respectively). over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us16.83 per product tested. the cost per averted case was $us79,707. conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january 2009 and december 2016. the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were bcptr (0.55% of transfusion reactions). of the 18 bcptr, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma. recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51). an organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%). blood pressure (bp) decreased in 22%, increased in 17%; 61% of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie 1 , jenna lebedev 1 , linda kapp 1 , xiaohong wang 1 , meghan delaney 2 , lay see er 1 and james c zimring* 3 . 1 bloodworksnw research institute, 2 bloodworks nw, 3 university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg1-igg4), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least 29 natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all 29 known variants. study design/methods: the heavy and light chain variable regions of an anti-k1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k11 rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg2, igg3, and igg4 had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg1 or for any of the monoclonal ahgs tested. monoclonal anti-igg3 had a blindspot for igg3-04, due to the shorter hinge region on igg3-04. no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* 1 , yves dominique pastore 1 and maryse st-louis 2 . 1 chu sainte-justine, 2 hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since 2008, our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of 2014, 205 scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-c, anti-e (2), anti-hrb, anti-fya, anti-jka, anti-jkb (2), anti-s, anti-m, anti-sc2, anti-leb (2). seventeen patients (8.3%) were either d2 or partial d. rhce results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. as for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed. fy(a2b2) phenotype was found in 182 (89%) patients. a total of 2606 genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos 1 , emilia sippert 1 , mayra dorigan de macedo 1 , sheila fatima perecin menegati 1 and lilian castilho* 1,2 . 1 hemocentro unicamp, 2 university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa-308a, il1b-511t cytokine polymorphisms, rhag 808g>a and hla-drb1*15 alleles may predict a good responder phenotype (sippert et al, transfusion 2017) and that rhag 808a and hla-drb*15 alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included 96 non-alloimmunized patients with scd, homozygous for hbs, receiving a range of 5-289 rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa-308g>a, il1b-511c>t) and the rhag 808g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for rh variant alleles. from those, 6 had rhag 808a and/or hla-drb*15 alleles and at least one cytokine polymorphism (tnfa-308a or ilb1-511t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other 15 patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and 2 were confirmed by sequencing. the third sample was found to be rhce*cevs.01,rhce*cebi on sequencing (predicted phenotype v1,vs1). the 3 samples were typed as v1 (or ce s ) and vs1 (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[712g]in 2 samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c1 by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising 80% of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies (20% of patients) bound young and old rbcs with no apparent prejudice. band-3 is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from 22 patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band-3 tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing 5 type i and 5 type ii patients, we found that type i is characterized by 5 percollv r fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by 3-4 percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band-3 suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* 1 , burak bahar 1 , jeanne hendrickson 2 , krystalyn e hudson 3 and christopher a tormey 1 . 1 yale-new haven hospital, 2 yale university, 3 background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam30 abstract algorithm, e value51x10 -6 , word size5 6, gap costs: existence59 exten-sion51). search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of 162 alloimmunized patients reviewed, 105 were culture-positive. of these, 76% of the anti-c/c group (13 of 17 patients) and 16% of the anti-k group (7 of 43 patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from 0 -11.1%. overall, 21.9% (23 of 105 patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of >80% sequence identity. while 27.6% (29 of 105) patients reviewed had positive cultures for klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed 109 tegs performed on 76 patients undergoing cv surgeries at our institution from jan 1 to dec 31, 2016. no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the 56 tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (r>10 min, k>3 min, a<53 degrees, ma<50 mm) and "hypercoagulable" (r<5 min, k<1 min, a>72 degrees, ma>70 mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of 56 tegs analyzed, 37 patients (66%) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients (8% vs. 32%, p50.02). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused (32% vs. 11%, p50.07). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused (100% versus 33%, p50.06). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, 670 olts were performed. a total of 345 patients was submitted to cs. the median age was 51 years (range 10-78 yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in 31,6% of the patients. the average meld score was 29,6 6 9,4 and it was slightly higher in the cs group (31,3 vs 27,9, p<0,001) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml. allogeneic blood transfusion was required in 71,8% patients in the cs group, compared to 46,7% patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group (2,4 units vs 3,39 units, p<0,001 background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july 2015-december 2016 was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp (27 and 15 products, respectively) however, obp wastage occurred more frequently in the 18 month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products). this is skewed by one month when 20 products were wasted due to expiration of product on the floor. cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p50.176). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with 5 units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d1) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d(table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* 1 , edward smith 2 , thomas brown 2 , foeks jeremy 2 , metcalf suzanne 2 , james johnson 2 , peter davis 2 , karafa sw badjie 1 and abba zubair 1 . 1 department of laboratory medicine and pathology, transfusion medicine, mayo clinic, 2 department of anesthesia, mayo clinic background/case studies: our institution performs an average of 398 solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a 29 y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused 2 o(1) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused 10 more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the 2 o(1) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in 1% of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o. female patient should not have received o(1) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o(1) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a 50 y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o(1) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic pfd surgeries range from 0.2-1.6% and 0.3-1.4%, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may 2015 -may 2016 in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p<0.05. results/finding: we identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative t&s ordered. two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine (90.8%) of the 65 patients required a second abo/rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table 1) . no abo/rh discrepancies were identified. one patient received 1 unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n527) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs-1000i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units. this was performed twice for a total of 10 units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%). in units between 21 and 42 days (n510), the estimated mean residual k1 was 1.89 meq (range 0.61 to 2.21). in the two mock mtp trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births. uc collections were divided into 3 segments to test 3 conditions. segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of 21 days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the 2 remaining tissue segments were soaked in (ab/am) saline solution for 1 hr and 24 hrs at 48c, respectively. tissue segments were frozen in cryo bags with a proprietary 10% dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process 3 or 4 times the total blood volume (bv) of the patient, up to a maximum of 25 liters (l) per procedure, to obtain peripheral blood cd341 stem cells. as a consequence, a patient often would need to spend 6 hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd341 pre-count and cd341 yield, normalized per liter of blood processed, was derived utilizing the patient's cd341 pre-count, the patient's weight in kilograms (kg), and the target cd341 dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd341 stem cells. the initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 l. the range was 4.9 l -21.6 l. the target dose was achieved in all patients. our previous practice for these 8 patients would have required, assuming a standard 4 bv procedure, processing an average of up to 28 l per patient, with a range of 20-62 l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd341 yield. the result was a high correlation between these two ratios (r 2 5 0.92), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r 2 5 0.92, confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd341 mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june 2015 and march 2016. patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and primary amyloidosis (n51) and were mobilized according to standard protocols. collection cd341 cellularity target was established ! 2x10e6/kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd341 count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v4.0. results/finding: the media (range) general collection parameters were: cd341 (day 5) 27.50/ml (4.5-157.5/ml), blood volume processed 23204ml (9718-39618ml) and 4.96 (2-7.30) exchanged volemias. seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p50.071]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1]. two patients could not undergo hematopoietic stem cell transplantation due low cd341 cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd34) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with 20% fetal bovine serum. bmsc and amsc at passage 2-5 were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par4) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within 30min and 2hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: 334 6 35 seconds, versus low, medium and high doses of amsc (145 6 2, 111 6 6, and 75 6 12 seconds), and bmsc (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (cft, p<0.05) and increased alpha angle (p<0.05) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par4. no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* 1 , neil bagamasbad 2 , reynold dilag 2 , melissa nasser 2 , nicole bauer 2 , jennifer wheeler 3 and mary berg 1 . 1 department of pathology, university of colorado -anschutz medical campus, 2 department of medicine, division of hematology, university of colorado hospital, 3 scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from 120 collection procedures using the mnc protocol and 173 collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd34)-positive (cd341) throughput, cd341 collection efficiency (ce%), platelet loss 71a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included 14 and 22 allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd341 throughput was significantly higher in the cmnc group than the mnc group. the cd341 ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded 20 ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient #1, originally typed as an a2, had 1 bone marrow donor and 2 cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient #1 is now typing as type o. patient #2 was originally typed as a2 and received a bone marrow transplant from a type b donor. patient #2 is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient #1 and patient #2 indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with 20% fetal bovine serum under either normoxia (20% o 2 ) or hypoxia (3.5% o 2 ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd90/cd29 and cd45 were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc (1.5 3 10 5 /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by 15%, but depressed the growth of amsc by 30% at day 5 in comparison to normoxia. both bmsc and amsc equally expressed cd90 and cd29 but not cd45 under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: 468 6 64 (control), versus 170 6 13 (bmsc), and 195 6 60 (amsc) seconds) by natem. hypoxia also significantly shortened ct (165 6 20 (bmsc), 169 6 50 (amsc) seconds, p<0.05 as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd341 cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd341 cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd341 cells. study design/method: cryopreserved cd341 cells from 2 healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef1-alpha-yfp lentivirus (2.5% concentration) and media (x-vivo-10, human serum albumin(hsa), 100 ng/ml each of cytokines (scf, tpo and flt3-l) over 2 days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of 5% dmso, 6% pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd34 1 cd38 -cd45ra -cd90 1 cd49f 1 cells) phenotyping and cfu assays were done following first thaw (pt1), post-transduction (ptxn) and second cryopreservation-thaw (pt2). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd34%, cfus were similar before and after pt2. hscs ranged from 824 to 1655 cells/10 6 cd341 cells in the pt2-tr arm compared to a range of 286 to 1416 /10 6 cd341 cells after pt1. viability, % cd341 and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt2 (table) . conclusion: dec of mpb human cd341 cells decreases tnc recovery, but has minimal effects on cd341 cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd34% in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd341 cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% dmso at final concentration. pbsc were cryopreserved by direct immersion on -808c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /l and platelet count >20 x10 9 /l after 07 days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd341 was below 10 x 10 6 cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd341 on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd341 on the day of the collection versus collected cd341 per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd341 was calculated. final laboratory count of cd341 per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss 23 software. results/findings: among patients collecting hpc for autologous transplantation, 69,23% needed only one day of hpc harvesting, while 25,64% needed two days and 5,13% needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was 49 1-2,91%. after comparing predicted values with cd341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% cd90, cd105, cd73 and 2% cd14, cd19, cd34, cd45, hla-dr) study design/method: umbilical cord tissue (n510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline. fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence. all cells were tested on an msc flow panel at passage 2 just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of 8 days (fresh 5 7.8, frozen 5 8.1), and 13 days (total) for the msc's to reach passage 1 (fresh 5 12.6, frozen 13.4). all cells were ready for flow analysis in approximately 3 weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all). flow cytometry showed average !95% for positive markers and 2% negative markers. there was no statistical difference between fresh and frozen flow result (p > 0.05). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with 10% concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a 378 c water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-aad marker through flow cytometric analysis. results/finding: ucb storage period was 7.24 years (mean) and cell recovery was 86.31% (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11). post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for 5 years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln 2 vapor in a dmso-based cryoprotectant for 5yrs. (5.49 6 0.431; n54). units were rapidly thawed and rinsed in dpbs, then 25 pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a 5x5 grid pattern in msc-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged. cells were isolated on the 14 th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate. cells were positive for the msc markers cd73, cd90, and cd105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers cd34/45 (1.1 6 0.7%). passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical 75a transfusion 2017 vol. 57 supplement s3 research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd34 yield prediction algorithm ines bojanic* 1 , nelly besson 2 , ivana vidovic 1 and branka golubic cepulic 1 . 1 department of transfusion medicine and transplantation biology, university hospital centre zagreb, 2 terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd341cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version 11) in adult and pediatric lvl. a prediction algorithm for cd341cell yield was also tested. study design/method: we evaluated retrospectively 67 lvl performed in 46 adult patients, and 14 lvl in 11 pediatric patients treated in uhc zagreb from march 2016 till september 2016. mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio 1:24). in patients weighting 25kg (n59), a rbc prime was performed. cd34, lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd341cell count and cd341cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd34 values to real cd34 yield. results are presented as median (iqr). results/finding: in both groups, cd34, ly and mo ces were high. target cd34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd341cells and cd341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting cd34 yield could be predicted based on precd341cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd34 yield and observed cd34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)l of blood in 20 adult procedures, and 5.9(3.5-7.8)l in 7 pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd34, ly and mo ce1 were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* 1 , aniko barta 2 , arpad batai 2 , zoltan csukly 2 , zita farkas 2 , laszlo gopcsa 2 , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg. clinical response was assessed 28 days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all 12 patients had received 13 cycles of msctreatment (4 dose per cycle). the median age was 47 years old (19-56) with a male/female ratio of 1:2. distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; t-cell lymphoma: 1. nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median 63rd day (7-455). the involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases. the median time of msc's first infusion was 274 days after the stem cell transplantation (hsct) and 165 (19-1974) days after the first episode of gvhd. 4 of the 13 cycles of msc-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with 83% overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is 2 million cd341 cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd341 cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal. the ideal minimum cd341 cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from 55 patients to evaluate the predictive value of the cd341 cells/ml level. data was collected over 6 months from every patient who underwent a stem cell collection. four patients were allogenic donors and 51 were autologous donors. the patients' weight, diagnosis, and pre-procedure cd341 cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd341 cells collected were recorded. the collection efficiency and the cd341 cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd341 cells/ml and post-procedure cd341 cells/kg (r50.95). any patient who had a pre-procedure cd341 cells/ml count of 29 or greater had a collection of at least 2 million cells/kg. any patient who had a pre-procedure cd341 cells/ml count of 16 or less collected less than 2 conclusion: the pre-procedure cd341 cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd341 cells/kg level. to confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure cd341 cells/ml count of at least 29 should be obtained. for any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 cd341 cells/ml should be conducted. heidi elmoazzen 1 , antonio giulivi 1 , michael halpenny* 1 , lisa martin 1 , donna perron 1 , chris bredeson 2 , lin yang 1 , locksley mcgann 1 , paul birch 1 and jason p. acker 1 . 1 canadian blood services, 2 ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso (5% final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd34, viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in 5% dmso and 1.7% hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of 12.6 days for anc500 with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current 5% dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using 5% dmso and 1.7% hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from 5% to 40%. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost. in 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg. this retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses. study design/method: patients weighing >100 kg with crcl >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd341/cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd34 in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %cd34 level than any of the other groups, reaching statistical significance when comparing the %cd34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs). hispanic donors show statistically similar %cd34 pre-apheresis levels over all age groups. moreover, the hispanic older age group (>540yrs) had a statistically higher %cd34 pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of 121 sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd34 level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd34 level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc (234 x 10 6 cell / ml) and three low wbc (114 x 10 6 cells / ml) concentrations, each at high (505 ml), low (265 ml) and median (355 ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax 2 (pericell protocol, cs.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and 7-aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting 7-aad viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the 6 mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-aad viability of 2 6 2 [0, 11] % from the input product. the method was found to have a cv of 2.0%. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating 78a transfusion clinical assessment. in the first phase, 2 cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd34 7-aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw. the second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than 20% within 1 hour, while cd341 cell viability remained stable up to 3 hours post thaw. small aggregates appeared after 1 hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075). platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207). infusion rates were slightly higher for the pump group. for control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the 278 zikv ineligible cbus was: caucasian 52%, asian 9%, black/aa 20%, and multi-race 21%. racial distribution of all clinical cbu donors was caucasian 49%, asian 15%, black/aa 20%, and multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: 78% of all ineligible cbus and 21% of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring 2,3-dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a 10 ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n 5 20), which were stored in sagm for 22 days, to act as untreated controls. the remainder of each unit ($270 ml) underwent treatment with the rejuvenation solution (50ml, 60minutes at 37 o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p 1 , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of 22 day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp 215 (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros 60. pc were centrifuged at 1250g in a sorvall rc3c1 centrifuge (sorvall, usa) for 10 min. the combination cryoprotectant dmso1dextran (cryosure dex40, germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at 37 degrees c (barkey plasmatherm) for 10 min. cpc osmolality was measured with an osmomat 030 osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru 169287 u1). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso1dextran (cryosure dex40) , as a cryoprotectant, to obtain a final concentration of 5% dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m3 were instrumental in automating that phase. pc to be frozen had an osmolality of no less than 1500 mosm/l. prp and ppp were frozen at a cooling rate of 1-38c/min and stored at -85 0 in the chest freezer for up to 24 months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru 167874 u1). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to 380 mosm/l. freeze-thaw recovery of platelets was 80% or more of the original population. defrosted pc were stored at 20-24 0 with continuous gentle stirring from a helmer platelet agitator for no longer than 4 hours before transfusion. it took no more than 30 min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over 20 min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day 4 and day 5 evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day 4 for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day 5. a total of 60 lrap units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5. the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were 59 true negatives (tn) and 1 false positive (fp) on day 1 when tested by bact/alert, with 60 tns on day 4. bactx testing results showed 50 tns on day 4 and 10 tns on day 5. testing using the pgd kit showed 50 tns on day 4; and 8 tns and 2 fps on day 5. fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and 30 minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within 20 minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in 100% plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for 1 year jos lorinser 1 , pieter f van der meer 1 , hans van der heiden 2 and dirk de korte* 1 . 1 department of product and process development, sanquin blood bank, 2 mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers (140 ml) is unknown. if these products can be stored at -188c it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188c or <-25 to -358c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from 500 ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <-258c for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 ll) containers, which were refrozen and stored at either -188c or <-258c. during storage at 3 months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <-808c. growth factors tested were pdgf-aa&ab/bb, tgf-ß1/2/3, vegf, 80a transfusion 2017 vol. 57 supplement s3 egf, fgf2. the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß1 were the most abundant growth factors, on average 35, resp. 40 ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average 11 ng/ml. tgf-ß2, egf and vegf were detected at relatively low values, resp. 3 ng/ml, 0.5 ng/ml and 0.3 ng/ml. average levels of fgf2 and tgf-ß3 were close to detection limit (< 0.2 ng/ml). the controls stored at <-808c showed for all growth factors close to 100% of the initial values in samples at t50 (moment of filling mini containers). for serum stored at <-258c for up to 12 months, most factors showed less than 2 % decrease, except for pdgf-aa and tgf-ß2, showing 6% resp. 3% lower values. for serum stored at -188c the values for tgf-ß1, egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß2 showed a decrease of resp. 9, 17 and 3%. conclusion: human serum eye drops can be stored in the new micro dose device at -188c (3-star household freezers) or <-258c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at -188c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year. ruqayyah almizraq* 1 , heather inglis 2 , phillip norris 2,3 , jennifer a muszynski 4 , nicole juffermans 5 , jelena holovati 1 and jason p. acker 1,6 . 1 university of alberta, 2 blood systems research institute, 3 university of california, san francisco, 4 nationwide children's hospital, 5 academic medical center, 6 canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n58 per method). residual platelets and white blood cells (wbcs) were measured on day 5 using flow cytometer (fc). on storage day 5 and 42, number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day 5, apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p<0.01, wbd p<0.05) and wbf (vs: apheresis p<0.0001, wbd p<0.01) methods. while rcf units yielded the lowest count of platelet-evs (cd41a1) on day 5 and 42, the highest number of platelet-evs were in apheresis (day 5) and in wbd (day 42). similarly, there was significant difference among methods in the number of wbc-evs (cd31, cd141, cd161, cd191, cd66b1) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day 42 vs day 5 in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< 200 nm) was greater than large evs (! 200 nm) in all of the products on day 5 and 42, and the highest level of evs < 200 nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p<0.05). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at 48 centigrade maryanne c herzig* 1 , crystal lafleur 2 , chriselda g fedyk 1 , sherrill j. slichter 3 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research, 3 university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at 48c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a2 (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection. units were stored for 12 days without agitation. units stored for 10, 15 or 22 days were agitated during storage with a model 400 hybridization incubator at 48c set for end over end rotation at 2-3 rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at -808c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd40l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai-1) as another fibrinolytic measure; and complement activation markers c3a, c4d, c5a and c5b-9. data was analyzed by one way repeated measure anova. results/finding: only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation. these levels did not meet fda requirements of 5.5 x 10 10 platelets per wb unit. subsequently, wb was agitated and platelet recovery was 71-76%. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t0 (day of collection) and t10, 12, 15, or 22 measurements. significant elevations of pai-1 and scd40l indicate activation of platelets and inhibition of fibrinolysis (p<0.001). activated complement peptides c3a, c5a, and c4d were all elevated over time (p<0.001) while sc5d-9 was not. however, only c3a and c4d levels at t22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at 48c for 10-22 days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc5d-9 reported, wb showed elevation of c3a, 5a and c4d and not sc5d-9. complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* 1 and christian todd 2 . 1 cerus corporation, 2 community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 rcf 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 wbd 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350ml, 6.6x10 11 in 400ml, 6.8x10 11 in 400ml, and 7.0x10 11 in 400ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: 64% of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was 1.34. conclusion: it is possible to treat 64% of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* 1 , andrey skripchenko 1 , fei xu 1 , ying li 1 , stephen j wagner 2 , pamela h whitley 3 and jaroslav g vostal 1 . 1 fda/cber/ obrr/dbcd/lch, 2 american red cross holland laboratory, 3 american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature (4-6 o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts (11 hrs ct: 1 hr 37 o c [tc]). autologous apheresis plts stored for 7-days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled plts were also infused into mice (n590). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts <5% were considered background. results/finding: the mean recoveries of infused plts were 51.2 6 16.7% for rt, 37.7 6 12.3% for tc and 23.1 6 8.8% for ct in humans. in mice, mean recoveries of the same plts were 24.9 6 10.3% for rt, 19.1 6 9.8% for ct and 16.2 6 6.9 for ct (mean6sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $74% and ct was $45% of rt. in mice tc was $76% and ct was $64% of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to 100% for rt plts. human tc plts had 26% auc while ct plts had 11% auc compared to rt plts in humans. in comparison, the same tc plts had 39% auc and ct plts had 26% auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are 2.4 and 1.5, respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* 1 and geeta paranjape 1,2 . 1 coastal bend blood center, 2 carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g5 with the compomaster net software for data management. implementation was planned for a november 2014 go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g5s and compomaster in june 2014. training and validations were successfully completed and a full launch occurred mid-march 2015. device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december 2014. validation was completed and signed off in march of 2015. manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g5 system. data points were collected from 210 units bi and 302 units ai. results/finding: upon initial implementation, staff training and use, the compomat g5 was found to be easy. plt weight spread was reduced from an average of 22gm to an average of 15 gm. actual plt weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit. plt count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by 31.8% after implementation of the compomat g5 and our plt concentrations increased on average by 5%. we were able to consistently produce a smaller volume plt (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell 1 , angela hill 1 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: 13 abo/rh matched lr sagm rccs were pooled and split to produce 6 large (354 ml) and 6 small (244 ml) rccs. the rccs were stored to 14 d and glycerolized manually by mixing 400 ml of glycerol with the rcc in a 2000 ml freezing bag. units were frozen at -658c for ! 72 h before being removed from frozen storage and thawed in a 378c water bath. 3 large rccs and 3 small rccs were deglycerolized using the organization's current procedure on the cobe 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose. the remaining rccs were transferred into a 1l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 ml centrifuge bowl on the acp-215 with re-suspension in as-3. rbc quality was tested at 24 6 2 h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p50.006, acp215: p50.007) and lower cell recovery (cobe: p50.002, acp215: p<0.001) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume rccs. large cobe 2991 rccs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large acp-215 rccs. however, all cobe 2991 rccs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did acp-215 rccs (0.31 6 0.02 %). cobe 2991 rccs failed to meet regulatory hemolysis standards of 0.8%. conclusion: addition of a 400 ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp-215 cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using 0.9% saline and centrifugation and the semi-automated washing method (sam) using the cobe 2991blood cell processor. study design/method: in this study, 20 units of single donor platelets were evaluated (10 washed using the mm and 10 washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas 6000. results/finding: table 1 shows that the average platelet recovery for the sam (92%) was significantly higher compared to the mm (82%). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took 10-15 minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 ml of 47 to 68% plasma and 32-53% pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 ml. study design/methods: apheresis pcs (amicus v r ) were collected in 35% plasma and 65% pas-3. one study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 ml) in 65% pas/35% plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 ml) using either single or pooled donations. input pcs (n520) were treated with the intercept ts set by the end of day 1 post collection; the incubation time in the compound adsorption device (cad) container ranged from 4 to 16 hours and the intercept treated pcs were stored in 3 containers (n560). day 5 and 7 post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas-3 treated in the intercept ts set demonstrated acceptable in vitro function (table 1 ). all intercept treated pcs had ph(228c) !6.2. platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing 100% plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n56). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n56). numerous in-vitro quality markers (plt concentration, atp, po2, pco2, ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days 1, 3, 5 and 7 for apheresis pcs, and on days 2, 3, 5 and 7 for wb-derived pcs. two flow cytometry assays were used to evaluate cd62p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion 2017 vol. 57 supplement s3 results/finding: platelet recovery was 92 6 5% and 81 6 10% for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells (3% 6 1 (test), vs. 1.7% 6 0.5 (ctl) on day 5) and a higher rate of cd62p expression than control pc units (58% 6 7 (test), vs. 23% 6 6 (ctl)) on day 5). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* 1 , lorraine blagg 2 , christi e marshall 1 , herman woodson 1 , sean erony 1 , krishna patel 1 and eric gehrie 3 . 1 the johns hopkins hospital, 2 johns hopkins hospital transfusion medicine dept, 3 johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. 340 intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day 4. as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day 3. the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of 340 lrap were tested. 335 lraps initially tested negative by bactx, while 5 lraps initially tested positive by bactx. all 5 initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was 98.5%. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only 340 platelet units. the expected rate of bacterial contamination of platelets is less than 1 per 2000 units. the 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as-1 and cp2d/as-3 rbc alan d. gray* 1 , matt landrigan 2 , pamela whitley 3 , michael wellington 3 , sherrie sawyer 3 , shalene hanley 3 , emily rondeau 4 , louise herschel 4 , neeta rugg 5 , patricia a.r. brunker 3 , shawnagay nestheide 5 , jose cancelas-perez 5 , larry dumont 6 and zbigniew m. szczepiorkowski 7 . and 2,3-dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for >24 hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood (530-550 ml) was collected and processed at 3 sites into leukocyte-reduced rbc (a total of n563 cpd/ as-1 and n564 cp2d/as-3). 50 ml of rejuvenation solution (citra labs) was added to each rbc on day 35 (d-35), incubated for 60 minutes with agitation at 378c water bath (helmer dh4), washed (haemonetics acp215), and stored in as-3 at 1-6 oc for 7 days (d-36 through d-42). in vitro recovery (%) was calculated and hemolysis, atp, and 2,3-dpg were determined on day 0, d-35, d-35 after rejuvenation and washing (postrjv), d-36, d-38, d-40, and d-42. all units were cultured on d-35 postrjv and on d-42, and then concentrated by centrifugation on d-42. results/finding: in vitro rbc recoveries were 95.7% and 95.5% (as-1 and as-3, respectively) and no bacterial growth was observed. hemolysis on d-42 was maintained <1% in 58/63 (92%) as-1 units and 63/64 (98.4%) as-3 units. all as-1 and as-3 units (100%) had hemolysis <1% following concentration by centrifugation. morphology score was reduced to 77% (as-1) and 74% (as-3) by d-35, restored after rejuvenation (91%, 92%, respectively) and maintained through d-42 (>90%). atp was restored and maintained above fresh levels after rejuvenation. 2,3-dpg was restored above fresh levels and was maintained !80% of fresh levels through d-38. all values were significantly different compared to d-35 except as noted (p<0.001, paired ttest) ( table 1) . conclusion: rejuvenation of stored rbc restores atp and 2,3-dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d-42 when compared to nonrejuvenated rbc on d-35. this study is funded by zimmer biomet. storage >24 hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante 1 , jason p. acker* 1,2 and jelena holovati 1 . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during 42-day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and 2,3-dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome 1 rejuvesol-treated (l1r). the prbcs were incubated for 1 h at 378c with hepes-nacl (sham), liposomes (dopc:chol, 7:3 mol%, 2 mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and 2,3-dpg at day 42 hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): l (0.53 6 0.01%, p50.042), r (0.43 6 0.02%, p50.004), l1r (0.48 6 0.06%, p50.020). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r (0.55 6 0.01, p50.010) and l1r (0.55 6 0.01, p50.010) treatments compared to s (0.53 6 0.01) but not l (0.53 6 0.01, p50.936). rbc rigidity (kei) increased in all treatments compared to sham (1.19 6 0.07): l (1.28 6 0.06, p50.025), r (1.44 6 0.17, p50.010) and r1l (1.44 6 0.06, p50.004). aggregation amplitude was significantly increased by r treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004). atp levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g hb): l (2.00 6 0.21 mmol/g hb, p50.010), r (4.70 6 1.20 mmol/g hb, p50.004), l1r (5.00 6 1.56 mmol/g hb, p50.004). the levels of 2,3-dpg were no longer detectable in s and l treatments at day 42. the combined treatment was comparable to r (2.38 6 3.26 mmol/g hb vs. 2.62 6 2.20 mmol/g hb, p50.868). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l1r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer 1 , eric ducas 1 , patricia landry 1 , nathalie dussault 1 , jacques bernier 1 , danny brouard* 1 and anne maltais 2 . 1 h ema-qu ebec, 2 institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < 108c) of one to six 500-ml whole blood units (wbu) within 8h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between 18c and 108c for 24h under extreme external conditions (-308c to 408c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for 24h at 20-248c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, 500-ml wb bags were filled with 555 ml saline 0.9% at t 5 308c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (-308c) and summer (408c) conditions. shipping boxes were filled with either one or six bags (n5 2). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under 108c in 4.55 6 0.62h and maintain their internal temperature between 18c and 108c for 24h with final values ranging between 6.38c and 9.38c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the 108c threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than -138c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* 1 , christopher c. c silliman 2 , beth shaz 1 , marguerite kelher 2 and claudia s. cohn 3 . 1 new york blood center, 2 bonfils blood center, 3 department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either 100% donor plasma (n550) or 65% pas-3 / 35% donor plasma (n550). within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd40 ligand (scd40l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c (3/50 products) compared to plasma platelets (2/50 products); however, the hla-antibody screen-positive supernatants of pas-86a transfusion 2017 vol. 57 supplement s3 abstract c platelets had fewer hla specificities (2 specificities) compared to those of the plasma platelets (18 specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd40l levels were increased in the supernatant of pas-c compared to plasma platelets (table 1) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd40l and not bioactive lipids. although scd40l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd40l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below 5x10 6 wbc in us and 1x10 6 wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n52) and leukoreduced (lr) rbc units (n52) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of 1000 wbc/ul. the spiked samples of 5, 12.5, 5, 25, 50 and 100 wbc/ul were prepared from the source sample of 1000 wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations (0, 12.5, 5, 25, 50, 100 wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 wbc/ul). 10 tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of 20 samples (plt and rbc) were run on both analyzers, repeated for 5 days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt50.996, slope50.972), (r-rbc50.999, slope50.992). the %diff-plt at 5, 25, 50 wbc/ul were 7.8, 4.7 and 10, respectively. the %diff-rbc at 5, 25, 50 wbc/ul were 10.8, 3.2 and 14.7, respectively. the average total testing time was similar on both instruments; 89 min for the facsvia and 92 min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated 62% (56 of 89 min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of 56 minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* 1 and mickey koh 2 . 1 department of laboratory medicine and pathology, university of minnesota, 2 st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of 62 completed and partially completed surveys were received. results/findings: responses came from 13 countries, but the majority of responses came from the united states (us). of the respondents, 35% reported aprp use in their hospital. aprp was used predominantly for outpatients, though >40% of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by 1-5 mds; however, 3 hospitals had >10 mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures (97%); however, 3 respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the 3 hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, 2017) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n56) under blood bank conditions at day 3 (d3), at day 42 (d42), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from 2.1-8.8% at d3 to 8.3-68.1% at d42. rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell 1 , angela hill 1 , tracey turner 2 , april xu 2 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units (314 6 15) than in rcf units (275 6 16), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: 12 small rcf (252-263 ml) and 12 large wbf (322-353 ml) rccs were stored for 21 d before being glycerolized and frozen at -658c for !72 h. large rccs whose red cell mass exceeded the capacity of the 275 ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a 378c water bath, deglycerolized and re-suspended in as-3. rccs were stored 14 d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant k 1 and na 1 concentration, deformability (ei max ), and higher (p<0.001) recovery than did large wbf units. no significant differences in hemolysis, atp, 2,3-dpg, p50, rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table 1) , however 8 of 12 large wbf units had rbc recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small rcf units had hemoglobin values < 35 g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. 0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 elongation index (30pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 this study is funded by zimmer biomet. (hasan 1994) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo 2 ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for 42 days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two (52) rbc units (leukocyte-reduced), cpd/as-1 or cp2d/as-3, on day 0, day 42, and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o 2 /g hb) and total releasable oxygen (tro) of the unit (ml o 2 ). orc was determined by assessing the change in % o 2 saturation from 100 mm hg po 2 (e.g., lung) to 40 mm hg po 2 (e.g., venous blood) multiplied by 1.34 ml o 2 /g hb (li 2016). a simulated baseline pretransfusion vo 2 of 115 ml o 2 /min was estimated using the day 0 orc and assuming a 7 g/dl transfusion trigger with a cardiac output of 5 l/min and 5 l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day 42 restored orc and tro to levels greater than day 0 ( table 1) . orc of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than rbc on day 0 and day 42, respectively (p<0.001). vo 2 increased after a simulated single unit transfusion of rbc (day 0, day 42, and pw) by 19.3%, 8.9%, and 28.8% over the pre transfusion vo 2 , respectively (p<0.001). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release 3.3 times the volume of o 2 compared to standard, untreated rbcs stored for 42 days. inferior oxygen delivery to tissues (vo 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (bennett-guerrero 2017). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* 1 , jay srinivasan 1 , gustaaf de ridder 2 , alan d. gray 3 , matt landrigan 4 , keaton charles stoner 5 , angela crabtree 6 , jessica poisson 7 and ian welsby 8 . 1 duke university school of medicine, 2 duke health pathology, 3 citra labs, a zimmer biomet company, 4 zimmer biomet, 5 duke university, 6 department of pathology, durham veterans affairs medical center, 7 duke university hospital, 8 duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted 2,3-diphosphoglycerate (2,3-dpg). the loss of 2,3-dpg increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation). decreased p50 may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and 2,3-dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at 378c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a1, leukoreduced prbc stored in as-1 were obtained from our local blood center. after 3 days of storage, units were divided into 4 separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution (50ml) was added to the cr group, and all groups were then stored for another 12 days at 1-68c. on day 15 of storage, the sr group was incubated for 1 hour at 378c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p50 was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps1) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a 5 0.05. results/finding: significant differences in p50 were noticed between all groups (table 1) . ei, ps1, and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p50 that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k 1 , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc (300ml) units stored in as-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 ml of multifunctional polymer bead, at a flow rate of 20 ml/min. supernatants were analyzed for k 1 removal as well as free hb, antibodies and cytokines (27-plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k 1 ] from 54.2 6 5.0 to 1.98 6 1.3 meq/l; equivalent to an 84.6% reduction. free hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/ml. antibodies, specifically igg, iga, and igm decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/ml (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/ml (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/ml (31.5%), respectively. inflammatory cytokines were significantly reduced, specifically: ip-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/ml (87.2%), mip-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/ml (80.7%), and pdgf from 1348.3 6 291.9 to 77.91 6 22 pg/ml (94.2%). filtration had no significant impact on cell surface markers of rbc viability (<0.1% decrease) or sensitivity to osmotic changes. values listed represent mean 6 sem (p < 0.01 for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k 1 as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k 1 along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency (1.5%) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia 2120 (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on 10000 permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and 2,3-dpg profiles to fresh levels. the objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was 97.2 6 2.2%. hemolysis (%) was similar on day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%). percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24hr (0.51 6 0.19%). average atp and 2,3-dpg were restored above the average fresh values. the morphology score decreased $25% by day 42, which was restored to near fresh values following rejuvenation and washing and storage 24hr (93.7% and 95.1%, respectively). rbc oxygen affinity, as assessed by p50, was restored above fresh values. all values were significantly different compared to day 42 (p<0.001, paired t-test) ( table 1) . conclusion: rbc morphology was restored to near fresh and average atp, 2,3-dpg, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and 2,3-dpg were maintained during storage 24hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d9tetrahydrocannabinol (thc) and 11-oh-d9-tetrahydrocannabinol (11-oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and 11-oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and 11-oh-thc to produce samples for lc-ms/ms analysis. lc used a c18 column. post-column detection by ms/ms used positive ion electrospray with q1:q3 ion pairs of m/z 5 605.3:225.3 (internal standard (is), d3-thc), m/ z 5 602.2:225.2 (thc), and m/z 5 618.3:256.1 (11-oh-thc). quantitative results for thc and 11-oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ ml for both thc and 11-oh-thc. limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/ml for thc and 7 ng/ml for 11-oh-thc. results/finding: a total of 424 donor plasma samples were tested for thc and 11-oh-thc. no samples tested positive for either thc or 11-oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than 1% among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of thc and/or 11-oh-thc in plasma. conclusion: testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* 1 , ruqayyah almizraq 2 , daniel millar 3 and jason p. acker 4 . 1 university of british columbia, 2 university of alberta, 3 lightintegra technology inc., 4 canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days 7, 21, and 42 of storage. one rcf rcc was tested on days 1, 5, 14, 21, and 43 and six 10 ml aliquots were stored in parallel and tested on days 14, 21, and 43. all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the 6 aliquots were consistent at each time point but statistically higher than in the original rcc on and after day 21 of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow 100% screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n 5 60) and paired ffp aliquots were stored for 31-33 days at 2-68c and -188c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! 80% levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c1 esterase and alpha 1-proteniase inhibitors). the level of factor xiii in odp was slightly lower, about 70% of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i1ii and ddimer) and complement (c3a and c5a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion 2017 vol. 57 supplement s3 abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to 62% in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to 15 minutes. in addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from 12-15 minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n56) were prepared from one 12-15 minutes bc and 60 ml of autologous plasma in a 600 ml pvc-dehp container. as a reference, spc from donations with collection times of <12 minutes were prepared (n55). in addition, pc were prepared from 5 bc, of which at least 4 bc were from 12-15 minutes donations (n55). after pooling of the bc, 300 ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for 8 days at 22 6 28c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean6 sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume (67 6 5 vs. 66 6 16 ml) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day 8, ph(378c): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day 6 and/or 8 in 2/5 pc (possibly because sometimes ab0 incompatibility was accepted). on day 8, plt showed low cd62p expression (17.1 6 1.8%) and phosphatidylserine exposure (annexin v binding, 8.9 6 1.9%). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage. to substantiate that the exclusion of 12-15 minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* 1 , jessie miller 1 , ranee marie wannarka-farlinger 1 , sandra bryant 1 , scott a hammel 1 , sherry stern 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as-3 rbcs, 20 irradiated and 21 non-irradiated, were selected for the study. the units varied in age, ranging from 2 to 42 days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< 0.05) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p50.02). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after 5 days of storage pei lun karen lim* 1 , erma sofia sumardi 1 , isamar eduardo ancheta 1 , susan lim 2 , christina yip 1 , lip kun tan 2 and shir ying lee 3 . 1 national university hospital singapore, 2 national university hospital, 3 national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within 8 hours of processing and stored at -188c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for 5 days and kept at 1 to 68c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than 50 iu/dl. study design/method: randomly selected units of ffp (n510) were measured for fviii concentration based on clotting assay (stav r -deficient 92a transfusion 2017 vol. 57 supplement s3 viii diagnostica stago). fviii levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at 30 to 378c for 35 minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration (0 hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at 1 to 68c for 5 days for subsequent testing. results/finding: results obtained were listed in table 1 . units 7 to 9 were not tested for fviii at post thaw-24 hour due to operational issues. the overall fviii concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour. after further storage of tp post thaw-24 hour and -72 hour, residual fviii level remain to be above 50 iu/dl except unit 10 which had a lower initial fviii concentration. at post thaw-120 hour, 7 out of 10 units tested had residual fviii activity within the pre-set standard of 50 iu/ dl. the average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least 50 iu/dl of fviii. typically patients with factor levels below 30 iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi 1 , ayman mohamad sabri 1 , ali abdullah alajeafi 1 , ashwaq hasan alhekri 1 , saleem bin mahfouz 1 , ali hasan alkhodari 1 , rawya saeed shealy 1 , marcus picard-maureau* 2 and hussain bana almalki 1 . 1 king abdulaziz hospital and oncology center, 2 cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) prp pc in 100% plasma over a 7 day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of 4 leucoreduced test pcs were assessed at day 7 of storage and compared to leucoreduced control pc at day 5 of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day 0; the process was completed by day 1 post-collection. samples were taken daily for quality analysis from test and control pc until day 5 and day 7, respectively. for bacterial spiking, additional pc were spiked with each receiving 4 ml of 1 mcfarland ($ 1.2x10 9 cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8. the average platelet loss in the control units at day 5 was 9.5% 61.4. the average ph of the test units at day 7 was 6.64 60.04 and in the same range as the control pc, ph 5 6.89 60.09. glucose concentration in test pc at day 7 (13.8 63.0 mmol/l) was lower than in the day 5 control units (18.32 61.06 mmol/l). lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* 1 , jay srinivasan 2 , jessica poisson 3 and ian welsby 4 . 1 duke university, 2 duke university school of medicine, 3 duke university hospital, 4 duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf (4.44 mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial (0á2 lm) filtration. cryoprecipitate mini-pools (400 6 20 ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di(2-ethylhexyl) phthalate (dehp)] adsorption device and a 0á2 lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table 1) . kit ensured bacterial sterility (table 3 ) and most importantly, final product was free of hbv, hcv and hiv (table 2) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane a 2 from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n518) were prepared from a nsaid-bc and 60 ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for 8 days at 22 6 28c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n55) were investigated as a reference. values are expressed as mean6sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume (69 6 4 vs. 66 6 16 ml) and plt content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups. on day 8, both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day 8 was significant higher in a subset of donors who had used ibuprofen (n55). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day 1 in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, 2014] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type 2 diabetes (t2d). because of the strong rise of people with t2d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t2d, but accepted as donor. study design/method: twelve whole blood donors with t2d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for 8 days at 22 6 28c and sampled on day 1, 4 or 5 and 8. the diabetic marker hba1c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 3 'good' (ph day8 >6.6) and 3 'poor' (ph day8 <6.3) storing spc were selected and analysed in more detail. results/finding: donors were of age 57 6 10 year and primarily men (75%). donors with t2d had a higher mean bmi (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher hba1c than controls. the spc of both groups had the same volume (70 6 5 vs 726 2 ml) and plt content (71 6 9 vs 73 6 11x10 9 ) but on day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mm, p<0.05). on day 8, the average in vitro quality was comparable in both groups (data not shown). when combining 94a transfusion 2017 vol. 57 supplement s3 the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc-1) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01). conclusion: bc from donors with t2d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t2d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of 30 minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of 2017 detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table 1). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to 60 minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* 1,2 , katharine a downes 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts13 are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: 32 x 10 9 /l, platelets: 62 x 10 9 /l, bun: 77mg/dl, creatinine: 2.4mg/dl, lactate dehydrogenase 1940 u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to 44 x 10 9 /l, adamts13 sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation (74 x 10 9 /l and a-ipc of 4.7 x 10 9 /l). two consecutive tpe were completed which resulted in a platelet count decrease to 54 x 10 9 /l and a-ipc of 5.1 x 10 9 /l. a-ipc ratio was 1.1 below the ratio of 3 which has been reported for ttp patients. similarly a-ipc count was not below 5 x 10 9 /l threshold reported in setting of ttp with severe adamts13 deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o157:h7 toxin. testing of c3, c4, factor h, factor h autoantibody, factor i and factor b were normal. adamts13 activity was 93%. patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /l, with resolution of her renal failure: bun: 42 mg/dl, creatinine: 0.65 mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts13 testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts13 activity. many patients with severe autoantibody-mediated adamts13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts13 activity <5%) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value 2-3 days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column 1), increased peak a-ipc value (results shown as percent increase, column 2), delayed a-ipc peak, and delayed plt recovery (table 1) . moreover, recurrent episodes required more procedures compared to initial presentation (table 1) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february 2016 to march 2017 on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the 2 nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs1 separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version 5.0 after taking consent from the donors. the target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered. data was analyzed by stata 14. within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the 98 donors, 35 repeated the plateletpheresis within a week (group i) and 63 underwent 2 nd plateletpheresis within 8-30 days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025). though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at 2 nd plateletpheresis in group ii donors. there were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000). plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type 1 receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type-1 receptor antibody (at1rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at1rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at1r ab are reviewed. results/findings: case 1: the patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38. three years post-transplant, her creatinine (cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed banff criteria grade 2 amr, grade 2a t-cell mediated rejection (tcmr) and grade 3 interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at1rab was identified at >40 u/ ml (high: >17 u/ml, intermediate: 12-17 u/ml, negative: <12 u/ml). she received 6 tpe treatments every other day and started losartan. after a course of tpe, at1rab decreased to 32 u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to 1.9 ml/dl. in one month, her at1rab increased again to >40 u/ml, therefore, she received 3 more tpe treatments with a decrease in her at1rab to 16 u/ml. although at1rab level increased slightly to 20 u/ml after 3 months, her cr has been stable at 1.3-1.6 ml/dl. case 2: the patient is a 25-year-old mean 1/-se -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96a female with malignant hypertension who received a deceased donor kidney transplant at age 24. her cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at1rab level at 18 u/ml. she received 6 tpe procedures every other day and at1rab decreased to 8 u/ml with a decrease of cr to 1.98 mg/dl and improved arteriopathy in histology. because her at1rab level slightly increased to 12 u/ml over the next 2 weeks, she started weekly tpe treatment. after 5 weekly tpe, tpe treatment was stopped because her at1rab level remained relatively unchanged. her cr has been stable at around 1.5 ml/dl to date. conclusion: we present 2 kidney transplant recipients who received tpe treatments for high at1rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at1rab levels; however, weekly tpe had no effect on reducing at1rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a 36 year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < 10%], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a 33 year old man (patient b) with a medical history of hypothyroidism (on synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (ef of 20-25%) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within 12 hours of the procedure completion. their total t4, t3 and free t4 levels trended to normal or near normal range within 24 hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* 1 , laura martínez molina 2 , cristina muniesa montserrat 2 , octavio servitje bedate 2 , silvia cosano navarro 1 and maria isabel gonz alez medina 1 . 1 banc de sang i teixits, 2 dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since 30 years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category 1. since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of 10 patients diagnosed with ss and compare them in their first evaluation once the 20 th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from 1.5 to 2 total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in 2 where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in 7/10 patients) and with online system (therakos) just in 3. main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : 3835 u/l) requiring transfusions, mild thrombocytopenia (144 x 10 9 /l), acute kidney injury (bun 175 mg/dl, creatinine 2.51 mg/dl). by the third hospitalization day hgb improved to 10 g/dl, however with worsening thrombocytopenia (16 x 10 9 /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days 6-10). platelet count and a-ipc improved to 52 x 10 9 /l and 6.6 x 10 9 /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count (30 x 10 9 /l) and a-ipc 1.98 x 10 9 /l. these dynamics did not resemble those which had been described for ttp patients with adamts13 deficiency. adamts13 obtained prior to tpe initiation was resulted at this time and was 67%. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc (2.64 x 10 9 /l) that preceded platelet count increase to 80 x 10 9 /l three days later when patient was discharged. other laboratory values at this time were ldh of 635 u/l, hgb: 11.2 g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts13 deficiency eiman hussein* 1 and jun teruya 2 . 1 department of clinical pathology, cairo university, 2 texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts13. since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts13, the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts13. the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january 2008 through march 2017 were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts13 activity of less than 10%. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts13 deficiency. eight patients (25%) were associated with suspected bacterial infection. four of the 8 patients (50%) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in 3 patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure. study design/method: in october 2015 and june 2016, two children with possible leukemia were submitted to tl procedure. they were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with 285 ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin (750 ml of acd-a and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1. a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 . the formula "collection pump flow 5 0,0003 x inlet flow x preapheresis wbc count" was used with the goal of removing up to 3 x 10 9 leukocytes/ml. a single leukapheresis procedure was performed with 2 total blood volume processed per patient. immediately after the 2-hour procedures, wbc count were 74.000 and 92.000 wbc/mm3, and 12-hour post tl, wbc count were respectively 45.000 and 70.000/mm3. net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since 2006. we report the data from the year 2016. study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of 13,302 apheresis procedures were performed at 37 hospitals. therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (pbsc) collection (23.9%), allogeneic pbsc collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%). cobe spectra (37.4%) and amicus (16.8%) were the most widely distributed instruments. centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis. detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out). spectra optia (42.7%) and cobe spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 ffp (11.1%). most of the procedures were performed for 1 plasma volume (72.4%). acd (88.4%) and heparin (11.5%) were used for anticoagulation. central venous catheter (91.9%) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for abo compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%). adverse reactions were observed in 8.5% of the procedures. allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported. therapeutic effect was achieved in 86.5% of the patients. our apheresis registry has been well run for 10 years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride >1000-2000 mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of 41 pts who were diagnosed with hp from january, 2009 through january, 2017, and referred for immunotherapy evaluation. 27/41 (66%) pts received conventional therapy (ct) and pe (pe group), and 14/41 (34%) pts received ct alone (ct group). mean age was 36 years (range 16-79), and 56% were female. baseline mean triglyceride level (normal <150 mg/dl) for pe group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for ct group. baseline mean lipase level (normal <393 u/l) for pe group was 1,798 u/l (797-2,745) versus 923 u/l (472-1,796) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications. 24/27 (89%) of pe group and 11/14 (79%) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. 20/27 (75%) of pe group and 6/14 (43%) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of 2.85 pe treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 u/l (2.5-3.5 x upper limit of normal). mean triglyceride levels after 2 pe txs were 1,976 mg/dl (627-3,968) for pe group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing ct were 1,576 mg/dl (487-2,873) for ct group (mean decrease 50%). while the pe group achieved a greater mean decrease in triglyceride levels after 2 pe txs (compared to the ct group after 48 hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa 2016 guidelines, in a patient unresponsive to optimal medical management. asfa 2016 guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed 39 tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* 1,2 , metha apiwattanakul 3 , sompis santipong 2 , jutaluk jaipian 2 , jettawan siriaksorn 2 and ponlapat rojnuckarin 1 . 1 chulalongkorn university, 2 king chulalongkorn memorial hospital, 3 prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june 2016 through february 2017 were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a 20% solution. before using, it was diluted to a 4% albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of 156 tpes in 38 patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was 3,000 (range 1,750-4,200) ml. although the corrected calcium level was low (<8 mg/dl) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in 2 patients. the first patient had 2 events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was 1.9% (3/ 156). in 2014, the incidence of tpe adverse effects was 1.6% (2/125) when commercial albumin was used. the difference was not statistically different (p 5 1.000). median serum albumin levels pre-tpe and post-tpe were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dl. the increase in serum albumin after tpe was statistically significant (p<0.001). eighty-two percent of pre-tpe serum albumin levels were lower than 4.0 g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether 60 outpatients who underwent a total of 100 la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). based on differences in the study protocols la was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was 1.4x10 10 wbc consisting of 1.1x10 10 mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg4-related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category 1 indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to 57,000 iu/mls (ref. range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref. range < 20) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were 4610, 2890 and 1320 mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was 1.74. peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref. range 1.6 -1.9). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to 2.4 cp. serum igg, igm and iga levels decreased to 2040, 1510 and 672 mg/dl respectively. her rf had decreased to 19,900 iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than 3 cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan 2013 -dec 2016). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with 5% human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between 4 to 35 years age (m: f; 1:2) underwent 62 tpe procedures with an average of 5.6 per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only. three (27%) out of the seven tested, were positive for aqp4-igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven. adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2). follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after 3 months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from 6.0 to 6.4 for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version 6.0. the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to 1/16/2016, plt collections were performed on nine trima accel machines operating with version 6.0. upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016. the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05. results/findings: following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version 6.4 of the trima accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0. both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4. conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version 6.4 software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is 30 weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of 500 ml 5% albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using 500 ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of 500 ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a 45 years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at 20 years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of 4980 mg/dl, lipase 92 u/l, glucose 250 mg/dl, bicarbonate 24 mmol/l, anion gap 12. ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega 3 fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day 3 after admission. results/finding: the patient tg decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day 6 after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below 1000 mg/dl at 20 days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from 2009 to 2013, a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/l. and the upper limit was set to be 244 lg/ l in male and 158 lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times. the high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%). the mean ferritin was 82.0 lg/l in male (95% ci: 80.7-83.2 lg/l) and 66.5 lg/l in female (95% ci: 60.9-72.0 lg/l). the result of anova indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05). the average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/l in younger and elder 50 y/o male and 18 and 23 lg/l in female. and then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/l in the first period to 4.1 lg/l in the third period (1 period5150$160 days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* 1 , mary townsend 2 and lizabeth rosenbaum 3 . 1 university of new mexico hospital, 2 blood systems, inc., 3 blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately 10 minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within 24 hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* 1 , william korzun 1 , teresa nadder 1 , susan roseff 2 and elizabeth ripley 1 . 1 virginia commonwealth university, 2 virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for 1% of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of 142 subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location. in 2015, basic trainees at site a were scheduled at day 57 of 70. in january 2016, the blood drive date changed to day 60 of 70. the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in 2016. these observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* 1 , eva alonso 1 , oscar bascuñana 1 , monica romero 1 , teresa vich 1 , elena castaño 1 , laura carbonell 1 , eva palomas 1 , saray almerge 1 , francesc carpio 1 and xavier curia 2 . 1 banc de sang i teixits, 2 institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january 2015 to december 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t5 due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t5 and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t5 excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t5 but his hemoglobin levels were lower than our selection criteria. of the 204 donors selected for donation 16 (7.8%) had sci lower than t5 and t6. adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t5 have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of 2013, automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an 11 month period from january 2013 to november 2013. the same information was assembled for the automated bp process for the 11 month period of january 2014 to november 2014. the automated bp process implemented in mid-december 2013; so the december data for both 2013 and 2014 has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < 0.05. results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p50.006). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < 0.03) with the automated bp while and reactions remained non-significantly lower (p 5 0.086). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05. results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under 12.5. statistically more visits with hgb less than 12.5 g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from 1 january to 31 march 2017. after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study. 85% of donors (n55678) accepted automatic appointment booking, whereas some donors (n51002) were not comfortable with it. 77% of those who declined still preferred walk-ins (n5771) based on their own time schedule, the rest decided that variable situations (n5112), donation frequency (n569) and choice of preferred donation locations (n550) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was 19%. a comparison was made and found that this study shown a significant increase of acceptance rate by 66%. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has 4 collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. 4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105a transfusion (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%). mean donor sbp, dbp, and pulse were 125 6 14.7 mmhg, 75.1 6 9.6 mmhg, and 75.9 6 11.2 bpm, respectively. screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female). no differences were observed in the 45-64 year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females. developing blood donor educational materials gay wehrli* 1 , susan rossmann 2 , louis m. katz 3 and dan a waxman 4 . 1 university of virginia health system, 2 gulf coast regional blood center -sugar land, 3 americas blood centers, 4 indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an 8 th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, 3.5 page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table 1. results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has 4 major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from 2011 to 2016 after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016. moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to 24 times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately 1.3 million doses of ap transfused within 1.375 billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since 2006. firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than 180 days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by 7.46 times from 5550 to 41420 and the doses of ap increased by 7.41 times from 7363 to 54553 within 10 years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into 5 groups: those who donated ap once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively. it was found that the number of permanent ap donors who donated ap more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of ap (29.2%) from 2006 to 2016. conclusion: aps increased at a rapid and steady pace in wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer <26 ng/ml and zpp levels >100 umol/mol heme) at 3 hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all 3 hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp 87.8% (r50.937) at first and 86.5% (r50.93) at second donations. at first donation when compared to fs hb, only 10.4% (r50.323) of variation could be explained by variation in fs zpp, 12.3 % (r50.35) by ven zpp and 9.4% (r50.307) by ven fer. at second donation, when compared to fs hb, only 9% (r50.30) of variation could be explained by variation in fs zpp, 14.4% (r50.38) by ven zpp and 20.1% (r50.448) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p<0.001) suggesting strong evidence against correlation. 55% (181) responded to the survey of which 4% (13) reported not feeling well after donation. it should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/ml but the zpp levels were less than 100 umol/mol heme. of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/ml nor ven or fs zpp levels above 100 umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from 12.5 to 13.0 gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from 59 blood centers over two intervals, july-dec. 2015 and july-dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male5m, female5f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab 17.0, chicago il). p <.05 was considered significant. results/findings: data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 wb donations and 272,094 and 319,161 ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for m ap from 1.8 to 3.5% (p<.001). the mean "by center" deferral rates (table) were similar to that and significant (p<.001). mean by center hb deferral rates among f donations during the two intervals were 11.6 and 11.9% (p50.241) for wb, 11.8 and 13.0% (p5.041) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none. for bp, 8 provided detail (high vs. low), 28 summary and 4 none. p deferrals increased in the successive intervals among f wb donors from a center mean of 0.57 to 1.49% (p5.018) and for m wb donors from 0.78 to 1.16% (p5.006). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba1c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba1c levels among those with rbs >180 mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from 1 st march 2017 to 31 st march 2017. total of 1,861 blood donors were tested for rbs. those with rbs > 180 mg/dl were further tested for hba1c by gold standard hplc method using variant ii biorad. blood donors with >180 mg/dl rbs and hba1c > 6.5% were advised to consult a physician for further evaluation. results/findings: of the 1,861 donors tested, 44 (2.36%) donors showed a rbs of > 180 mg/dl. forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years). of these, 14 (31.81%) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining 30, 8 (26.66%) of them had a family history of dm. of these 30 donors, 8 donors did not give a consent for testing for hba1c. among the 22 donors tested for hba1c levels, 16 (72.72%) had hba1c > 6.5%. all the 16 donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is 0.87% (16 of 1839 donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* 1 , hwee huang tan 1 and ai leen ang 2 . 1 health sciences authority blood services group, 2 health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last may 2016. the current minimum acceptable hemoglobin for male donors in singapore is 12.5 g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above. study design/method: during a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin 12.5-12.9) group b (whole blood with hemoglobin !13, group c (apheresis with hemoglobin 12.5-12.9) and group d (apheresis with hemoglobin !13). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below 30 ug/l is considered low and levels below <12 ug/l are considered having absent iron stores. results/findings: 55.1% of donors in the study have ferritin levels below 30 ug/l. there were more donors with low ferritin in group a compared to group b, 80% and 53% respectively (p<0.05). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, 49% and 30% respectively (p50.001876). ferritin results for the 4 groups can be seen in table 1. conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to 13.0 g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among 600 students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of 54% male and 46% female students in the age group of 18-28 years. only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment. 42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood. 80 % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. (21); miscellaneous effects were reported in 23 courses. side effects led to interruption of supplementation in 55 instances. ferritin levels (mgt6sd) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/l in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets. ferritin levels<26mg/l were found in 4,8% of participants and 14.7% of controls. deferral for low hemoglobin was below 1% in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21cfr630.10 and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in 2001, an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of 50 -75 ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are c282y homozygotes. without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors. the mean current age is 59.7 years, 65% male, 96% caucasian. the majority of hh donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate. over the last 5 years, hh donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over 16 years. since 5/23/16, with an increase in male hgb deferral threshold to 13g/dl, there has been only 1 hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm-200 non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm-200 non-invasive occlusion spectroscopy device. over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the nbm-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the 200 blood donors for the performance of hb measurement on the sysmex hematology analyzer within 1-3 hours of collecting the venous samples. results/finding: the sd of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl. the hb measurements obtained from the nbm-200 and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be 0.978 g/dl. the precision of the nbm-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl. conclusion: the operators found the nbm-200 easy to install, maintain, and operate with minimal training. the nbm-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder 1 , ravi reddy 1 , dhuly chowdhury 2 , don brambilla 2 and edward l. murphy* 3 . 1 sanbs, 2 rti international, 3 ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in 2014 and followed them for one year. within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used 4-point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. 2015) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included 2,902 first-time black donors with median age 23 and female predominance (59%). within one year, 1,786 donors (62%) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) 5 1.16, 95% ci 1.06-1.28), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or51.11, 95% ci 1.00-1.23). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or50.83, 95% ci 0.72-0.96) and "i wasn't treated well by the <blood center> staff" (or50.85, 95% ci 0.74-0.97). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or51.19, 95% ci 1.03-1.37). a secondary analysis treating the likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard 1 , ramya ghantasala 1 , obhijit d hazarika 1 , nicole leonard 1 , cori a polonski 1 , zachary b wunrow 1 , michelle heleba 2 , jan k carney 1 and mark k fung* 1 . 1 university of vermont larner college of medicine, 2 american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of 292 surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" (27.5 %) and "traveling is a time for me to relax." (30.6 %). of the respondents who travel in the summer, very few reported donating while traveling (3.4 %). summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%). willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%). of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv6, and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(il-4) and il7. others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a compomatev r g5. plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb1 loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers (13m [29%]: 32 [71%] f); median age 42 years (range 21-70) donated a unit (500 ml) blood from which buffy coats (average volume 56 ml) were processed. the buffy coat process was previously validated on 20 wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2. all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for 34 days. results/findings: there were 158 completed responses, of which 73 (46%) indicated that their hospital had an msbos and 85 (54%) did not. the majority of hospitals without an msbos were academic centers (36/85, 42%) from oceania (26/85, 31%) or europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 rbcs (21/85, 25%) per year. 15/85 (18%) are going to implement an msbos in 2017. of those with an msbos, the majority 23/73 (32%) were from north america. the majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 rbc units per year (21/73, 29%) offering a wide range of surgical services. on average there were 207 6 577 procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching rbc units for 28%, and for 4% of procedures a different recommendation was made. most (32/73, 44%) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of msbos' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents (30%) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in 2015 was approximately $15.8m. in nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $1.2m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee 1. development of evidence based transfusion triggers. 2. education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3. clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to "1" unit instead of "2" units. 4. updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015. conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* 1 , anna w rains 2 and christopher t clark 3 . 1 university of tennessee graduate school of medicine, 2 univeristy of tennessee medical center, 3 univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc 3000 system. by using the blood volume values, and assigning a value of 1.5 ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in 2016, there was a total of 3,331 infants delivered at our facility. out of all the deliveries, 487 (14%) infants were transferred to the nicu. of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion. of the 487 infants transferred to the nicu, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%. in those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* 1 , shannon davis 2 , suzan new 2 , vaishali patel 2 and oren guttman 2 . 1 university of texas southwestern medical center, 2 ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of 79 randomized controlled trials (8, conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* 1 and stephanie rogers 2 . 1 dignity health st joseph's medical center, 2 dignity health background/case studies: over 12 million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across 39 hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels >5 7.0 g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of 7.0 g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of 7.0 g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to 21 hospitals including 16 cme presentations, online physician and nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb >5 7.0 g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy2015 to fytd2017, there was a 26% reduction in prbc units transfused to patients with hgb >5 7.0 g/dl, starting at a baseline of 67% down to 41%. this represents an fy17 annualized savings of $9.732m, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels >5 7.0 g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm5-0.258) reduced rbc units and two studies decreased the percentage of patients transfused (or5 0.700). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or 50.264) and rbc units transfused (sdm5-0.553). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a-6 method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was 7g/dl/21% or 8g/dl/24%. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately 42% of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was 590 1 24 units/ month. after the first 5 months of buc activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (p50.003 by t-test). non-surgical rbc use now represents approximately 29% of the total rbc use hospital wide a 13% reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital -30 month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital (4001 beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first 30 months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating 115a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance (2q14 vs. 1q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3q15 vs.1q17) vital sign compliance (39% vs.71%). jwp increased from 27 to 225 (04/16-03/17). cost savings were realized by decreased utilization and implementation of bpa. (table 2016 -1q17) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately 30-40 trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used. it also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november 2016 implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level 1 trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains 2-4 units of group o rhd negative, 4 units of o rhd positive, and 4 units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in 2015, the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < 10 k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june 2015-october 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a 17-month period, 1,270 cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table 01 below. overall, 532 (42%) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." (325), "active bleeding" (303), "platelet count of . . ." (173), and "downtrend" (92), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, 618 (49%) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years 2014-2016. study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by 207 km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of 5,5g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> 9 g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of 32 complete responses (16%). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dl (56%), platelet count of 20-50,000 (38%), and inr of greater than 2.0 (69%). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per 1,000 inhabitants may vary 3 folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of 18 key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%. we estimate that 384,704 patients would be eligible for pbm strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (daly) relative to the current clinical practice. a decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate. in this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: 237 adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted <1250ml of rbcs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used. if 1250-2000ml rbcs were predicted to be transfused, >2000ml were used 25% of the time. if predicted usage was >2000ml, 53% of the time it exceeded 2000ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for >1250ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (jan to mar 2017). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of 494 units of blood were requested. 154 units were crossmatched, of which 138 units were sent to the operating theatre (ot). only 33.3% of blood issued to ot were transfused (n546) while the rest were unutilized. the observed ct ratio was 3.35. conclusion: although only 31% of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study 35 patients who received tranexamic acid (txa) (study group) and 31 patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa 1gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < 7.0 g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were 1732 unique alert encounters. of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at 4 academic hospitals with data-derived msbos. study design/method: the 4 hospitals were in 2 groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if 5-24% of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if !25% of the patients had been transfused. data were collected at each center over a 1 month period between january to march 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers results/finding: between these 4 centers there were a total of 1599 cases analyzed. some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%). there were 1362 t&s ordered for these cases, of which 5 were positive for antibodies on the day of surgery. of all the t&s ordered, 52% were ordered in accord with the msbos recommendation, 26% were ordered when the msbos did not recommend one, and in 0.2% a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (hgb) 8-8.9). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of 29% among obstetric patients. study design/method: we studied a sample of anemic (hgb<10.0 g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks. mean hgb before referral was 7.5 g/dl and most were already taking oral iron therapy. a total of 169 women were hiv positive with mean cd41 lymphocytes counts of 394 cells/ul; 29 (12%) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and 156 (92%) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women. there was concurrent chronic disease (n52), infection (n52), vitamin b12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a 2 month old boy presented to our institution after a 1 month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige (15270 ku/l; rr: 0-2.9). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp3, while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp31 cd41 lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 mtps. results/finding: in 2016, mtp was activated 28 times. in 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of rbc allocation/issue. this left 11 cases to evaluate. the median time to plasma availability was 29 minutes (range 4 minutes -61 minutes). the mean plasma wastage for mtp activations was 32% (range 0-100%). of the 9 cha replies, 3 were using thawed plasma and their wastage was </5 5%. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes. our implementation began with thawing a single unit of plasma for "level 1 neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated 5 cases of gtx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with 40gy. results/finding s: granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85). in general donors reported no significant adverse reactions. from 56 donations, only in 4 was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table 1 . adverse reactions for patients were minor and not frequent (4 in 66 gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* 1 , elena kurnikova 1 and pavel trakhtman 2 . 1 russian institute for paediatric haematology, oncology and immunology, 2 instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period 04/2012-02/2016. donors underwent granulocyte mobilization with g-csf in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58. the mean increment of wbc after stimulation was 22,6x10 9/l (9,2-41,9 x10 9/l; f-23,2 x10 9/l; m-21.9 x10 9/l); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of wbc was respectively 22,2 x10 9/l (9,2-41,9x10 9/l) and 24,02x10 9/l (12,6-35 x10 9/l). the mean volume of treated blood was 566, a50,05) . the mean number of total wbc in the product was 38,43x10 9/l (13.38-77x10 9/ l; f-34,8x10 9/l, m-42,7x10 9/l). only three donors (1,96%) had apheresisrelated adverse effects the analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients. the mean number of transfusions was 6.9 (1-60). each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of wbc on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg). wbc increment was able to estimate after 144 transfusions. wbc increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/l (0,01-11,38 x10 9/l). efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%. in the cases of severe sepsis, the efficacy of transfusions was 76.2%. the frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-hla antibodies formation 2,7% (n 5 1) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp150 hemolytic disease of the newborn due to anti-rh17 keegan barry-holson* 1 and jennifer andrews 2 . 1 stanford university, dept of pathology, 2 stanford university, dept of pathology & pediatrics background/case studies: anti-rh17 is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh17 alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g3p1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dl), severe hyperbilirubinemia (total bilirubin (t bili) 9.0 mg/dl), reticulocytosis (8%) and a positive direct antiglobulin test (igg 21). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of 16.7mg/dl on day 8 of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh17 was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh17 sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d1 c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh17 antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* 1 , cyril jacquot 1 , valli criss 1 , philippe p pary 1 , jay greenberg 1 , naomi lc luban 1 and edward cc wong 2 . 1 children's national medical center, 2 quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as 4-11%. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of 1.6 g/dl/4.9%. wbc counts (19 x 10 9 /l) were mildly elevated and platelet counts (410 x 10 9 /l) were within normal limits. her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dl, direct 1.4 mg/dl), normal ldh (318 u/l), and undetectable haptoglobin (<7 mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol. dat was 41 positive for anti-igg and negative for c3 despite a positive cold antibody screen. the patient weighed 6.9 kg with an estimated total blood volume of 620 ml. she initially received simple transfusions totaling 20 ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with 463 ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of 40%, utilizing the central venous catheter. no adverse events took place over the course of the 2 hour exchange. her one hour post-exchange hb was 9.5 g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21). after initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of 11 g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels (3168 mg/dl). at a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a 24 or 16 gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the 24g catheter, the mean change in hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p<0.0001). during rapid manual transfusion with a 24g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in hct versus time: r50.58, p50.23). correlations between time and hemolysis were similar, but insignificant using 24g with washed rbcs and the 16g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in 99.9% of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge3. study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-d and an anti-ge3 titer of 256. she was d-and ge:-2,-3, 4 by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge3. the birth hemoglobin (hgb) was 12.6 g/dl, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dl; the infant was discharged. on day 7 of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb 7.6 g/dl, retic 2.6%, and bili 6.6 mg/dl. ge3-blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh1. obstetrics had to authorize maternal blood donation due to her hgb of 10.9 g/dl. maternal blood collection and rbc washing was expedited and the infant received 40ml of maternal rbcs within 24 hours, at which time his hgb was 6.1 g/dl. post-transfusion hgb was 10.8 g/dl. one week later, the infant was symptomatic with hgb 7.1 g/dl, retic 1.0%, bili 2.1 mg/dl. a 2 nd aliquot of 60ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb 7.8 g/dl, retic 0.7%, anti-ge3 titer 8, and needed another transfusion. the maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion. at 6 weeks, the infant's anti-ge3 titer was 2, hgb 9.2 g/dl, retic 1.7%; no transfusion was necessary. at 8 weeks of life, hgb was 10.2 g/dl, retic was 3.3%, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge3 hdfn. molecular analysis revealed that the mother was homozygous ge3-negative ge*01.-03, the father had homozygous wild type ge*01, and the infant was heterozygous ge*01/ge*01.-03. conclusion: the infant had hdfn due to antibodies to the high prevalence ge3 antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge3. hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in 2016, 14, 247 rbc orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was 9.85 g/dl as measured in the 72 hours prior to rbc order placement. in 2016, 3105 ffp orders occurred and the top three patient groups were: 46% in neonates in the nicu, 28% in congenital heart disease patients and 13% in pediatric intensive care patients. average inr of every patient was 2.09 as measured in the 72 hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day 5 bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after 4 months, pr-plt represented 30% of our platelet inventory (average daily plt inventory: 45 units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among 16 transfused neonates (0-4 m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of 60 pediatric patients receiving prbc transfusion over a 12 month period were retrospectively reviewed. a total of 44 patients were identified as receiving allogeneic prbc transfusion. 16 patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was 10.6 g/dl with post-transfusion hgb rising to 14.5 g/dl. the mean prbc volume transfused was 46.3 ml using a dose of 15ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of 10-15 ml/kg in a 2 kg patient, for instance, would translate into 3 full prbc units (about 1000 ml) in an average size adult. the current standard dose of 10-15 ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dl and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dl with a direct fraction of 0.43 mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable. the patient was started on a 5-day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c3. an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her 8-day hospital course, the patient received 2 rbc transfusions on the day of admission and several rbc transfusions thereafter (see table 1 ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day 6. her hemoglobin rose to 8.4 g/dl on hospital day 7 and increased to 9.5 g/dl on hospital day 8. at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dl on day 57 after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is 3 or 41. platelet and leukocyte immunohematology, testing and genetics table 1 . of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2). the matching rate of hla-dpb1 in our study is 13%. conclusion: the matching rate of hla-dpb1 in 10/10 hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb1 and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china (81401732) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts13. therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts13 with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a 56 year old male with a 7-year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts13 <5% and inhibitor of 3.6. on day of admission platelet count was 95 x 10 9 /l which decreased within five days to 14 x 10 9 /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were 1 x 10 9 /l and 14 x 10 9 /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to 3.2 x 10 9 /l achieving the ratio of 3 previously shown to be diagnostic of ttp. on day 5 his a-ipc and platelet counts had improved to 7.5 x 10 9 /l and 218 x 10 9 /l respectively. absence of anti-pf4 antibodies ruled out heparin-induced thrombocytopenia at this time. on day 6 he had an unexpected decrease in both a-ipc and platelet count to 4.8 x 10 9 /l and 132 x 10 9 /l respectively, worsening by day 8 to 1.7 x 10 9 /l and 40 x 10 9 /l respectively despite daily tpe. patient received 25 additional tpes that failed to improve a-ipc or platelets which on day 32 were 0.4 x 10 9 /l and 13 x 10 9 /l respectively. a-ipc had remained at this level for 16 days suggesting that the observed decrease was irreversible. adamts13 activity remained <5% low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa-1a, hpa-3a, and hpa-5a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa-1a, hpa-3a, and hpa-5a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p2 and gi9, specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa-1aa, hpa-3ab and hpa-5aa) were collected and reacted with anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex100. the hpa-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa-1a, hpa-3a, hpa-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa-1a, anti-hpa-3a, anti-hpa-5a. furthermore, because the platelet was hpa-5aa, the hpa-5b serum did not react with the coupled beads with mfi was comparable to negative control (286.59 vs 127.25). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa-1a was 1/128 (0.78iu/ml) and 1/64 (1.56iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa-1a, hpa-3a and hpa-5a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 january 2014 to 31 december 2016. clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c3, c4bp, thbd, dgke, cfhr3, cfhr1, cfhr4 and cfhr5) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and vus in 35% (47/134). 20% (27/134) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and 9 (33%) had vus. 31% (42/134) of patients had primary ahus; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had vus. 48% (65/134) of patients had secondary tma; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had vus. in patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of ahus and 28% (5/18) had recurrent disease. patients with primary ahus had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl80 and sysmex xn9000 compared with a flow cytometric method farshid ezligini 1 , kjersti roen eriksen 1 , annette vetlesen 1 , thomas larsen titze 1 and geir hetland* 1,2 . 1 oslo university hospital, 2 university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion 2012). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and 33 buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl80 (horiba abx, montpelier, france) and xn9000 sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd41a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x10 9 /l 6 sd were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by sysmex toa, pentraxl80, and the gallios flow cytometer, respectively. sysmex count was the very lowest 129a transfusion 2017 vol. 57 supplement s3 abstract (31.4% less than for flow cytometry), but all plt counts were significantly different (p<0.001), although least so (7.4%) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl80 seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing 1 , arishma lata 1 , roland russnak 2 , zachary antovich 2 , heather dunckley 1 and thierry viard* 2 . 1 new zealand blood service, 2 linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant snps located within hpa genes (hpa-1 through hpa-11, and hpa 15). genomic dna purified from 48 blood samples, previously genotyped for hpa-1,-2,-3,-4,-5 and -15 by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were 100% concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late 2016 and to date has tested 749 dna samples from 400 blood donors (349 donors were tested in duplicate). concordance between the sample replicates was 100%. there were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa-3 (4.7%) and hpa-5 (1.2%) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd36 antigen deficiency expression in jiangsu chinese han population qing chen* 1 , jianyu xiao 1 and chengyin huang 2 . 1 jiangsu province blood center, 2 jiangsu province blood center background/case studies: cd36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in asians and 2.4% of african americans, respectively. however, there is little information on the molecular basis of individuals with cd36 deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd36 expression levels and to determine the molecular basis of cd36 deficiency on the platelet surface of the han population in jiangsu region. cd36 expression levels on platelets were detected by flow cytometry among 243 blood donors in jiangsu region. donors without cd36 antigen expression on their platelet surface were further to be determined the expression of cd36 antigen on their peripheral blood monocyte cells. the coding exons of cd36 gene and adjacent introns were amplified and sequenced in cd36 deficient individuals. results/finding: among these 243 blood donors, cd36-deficient and cd36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively. the frequencies of type i and type ii cd36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively. among 237 individual with platelet cd36 expression, according to mean fluorescence intensity (mfi) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of cd36, respectively, and their mfis were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (p<0.05), respectively. the type i cd36 deficiency individual were heterozygous for 1200-13a>g and 430-14c>g, respectively. among type ii cd36 deficiency individuals, two harbored a t insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a t>c exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a a insertion before the 17th bp of the start codon atg in the promoter region; one were heterozygous for 748 1 2t>c and 1006 1 2t>g, respectively. conclusion: platelet cd36 surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd36 deficiency was higher than that in type i. the study findings indicated that the frequency of cd36 deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd36-deficient individuals widely varies among ethnic groups, with 3% to 11% in japanese, 8% in sub-saharan africans, 2.4% in african americans, and 0.3% in caucasians. although some studies of cd36 deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd36 expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the eastern china. the expression of cd36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd36 and peanti-cd41). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd36-nagtive phenotype. for those donors with cd36-negative platelets, cd36 antigen expression on monocytes was analyzed further to distinguish between cd36 type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china (81570170) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd36 phenotypes using the (mean 1 3sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd36 deficiency on platelet, in which one sample was cd36 negative both on platelet and monocyte. the frequency of cd36 type i and type ii deficiency in the eastern chinese donors was 0.08% and 3.3%, respectively. the average mfi of cd36 deficiency samples was significantly lower than cd36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, p< 0.0001). conclusion: the frequency of platelet cd36 deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd36 antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd36 antibody. background/case studies: cd36 (gpiv, chromosome 7q11.2) is an 88 kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd36 deficiency (cd36-n) is observed in 3-10% of africans (t1264g) & is classified as either type i (cd36-n plt, cd36-n mono) or type ii (cd36-n plt, cd361 mono). an acquired type ii cd36-n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type 1 cd36-n individuals can develop anti-cd36 alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd36 in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd36 phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd36 staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd36 dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an 80 year-old, group o1 african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin 4.4 g/dl & plt count 5k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < 5. hla antibody testing was negative (class i panel reactive antibody (pra)50%). the patient was plt-xm-incompatible with most donors (10/14). a trial of 4 group o, plt-xm-compatible plts was unsuccessful (cci 1). subsequent testing for plt-specific alloantibodies identified anti-cd36. fc-phenotyping showed no cd36 on patient's mono or plt, consistent with type i cd36-n. preliminary dna results show that the patient is heterozygous for t1264g. because cd36-n apheresis plt were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: all showed normal cd36 expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day 16) demonstrated new class i alloantibodies (pra 5 55%) in response to transfusion (21 apheresis plts, 5 rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd36-n & severe plt refractoriness in the setting of new mds, and 7q-chromosomal abnormalities. the absence of cd36 on plt & mono support congenital type 1 cd36-n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel 1 , kristopher fernandez* 1 , eric senaldi 2 , pascal george 2 , michael seul 1 and ghazala hashmi 1 . 1 biomolecular analytics, 2 central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made ! 6 donations in the previous 12 months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for hla class 1 (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/2pdplf8) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a *26:82 chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are 16,429 hla alleles documented according to the imgt / hla sequence database in janury2017, and more than 80% of them were identified in the last 10 years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a *26:82 allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn72d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove1 nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 c-a (codon225 acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a 13 year-old pregnant woman typed as ab1, who delivered a baby affected by severe hdfn. the newborn was typed as b1 and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted 41 with the four monoclonal anti-d used (igm clones p3x61 and rum 1 and the blends clones th281ms26 and d1751d415) and were typed as c-c1e-e1. an anti-d was identified in her serum. molecular analysis showed the 410c>t and 455a>c in exon 3, the snp 509t>c changes in exon 4 and the 667t>g nucleotide change in exon 5. the set of snps found is similar to the molecular background of dol3, except for 455a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of 1089 immuno hematological tests (465 abo/d grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 dat) and 20 crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in 99.2% of the abo/d tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the dat tests (n510). there were 4 discrepancies (2 abo/d for the same patient, 1 for antibody screening and 1 antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min). detection threshold of the d antibody was assessed at 2.5 ng/ml (0.0125 ui/ml) whereas the french recommendations are 20 ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso 15189 accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a 31 year old g5p3 presented during her fifth pregnancy with anti-kpb with an initial titer measured of 64. by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams (1.7 moms) peaking at 27 weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at 35 weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was 3.4 mg/dl with 13.6 g/dl hemoglobin. the baby typed as o positive, kp (b1) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at 6 weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by 8 weeks of age. his hemoglobin recovered to 9.0 g/dl with an indirect bilirubin of 1.4 mg/dl at 9 weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c3d-specific dat may be too insensitive to detect low, but significant levels of c3d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a "1111" reaction corresponds to about 500 molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c3d have been published. however, these are mainly designed to quantify the fraction of rbcs with c3d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c3d. study design/method: ten microliters (ul) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-c3d) mouse monoclonal anti-human anti-c3d (abcam, clone 7c10) were added to 5 ul of a 2.5% rbc suspension. after incubation for 60 minutes at 4c, samples were washed x3, and 25 ul of 1:10 diluted anti-mouse-f(ab)2-pe (ro480, dako) were added. after incubation at 4c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro480 was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c3d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a1 rbcs stained with 10 levels (2fold dilution, 1:1 -1:512) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c3d, edta-stabilized samples from 4 healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c3d (values ranging from 0 to 3,393 abc) with level of 0-serum dilution (used to sensitize a1 rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001). compared with dc-screening 1, the sensitivity of the flow cytometric assay was superior. it detected c3d sensitization at least 4 dilution steps further. the median normal level of rbc-bound c3d was 11 abc (range 7-20 abc, n54). the assay enabled demonstration of specific c3d-sensitization in the patient; the level of rbc-bound c3d in the sample was significantly elevated (1,907 abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c3d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt1 gene. in humans, the abo and gbgt1 genetic loci are located on chromosome 9q34, and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors1) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt1 genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt1 genes have been constructed of 88 vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k1 antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion 2017 vol. 57 supplement s3 the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c1, e-, e1 and k1-. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d1, c-, e-, partial c1, partial e1. the probable rhce genotype, rhce*ce-jal/rhce*ce733g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant woman and her newborn carolina bonet bub* 1 , maria giselda aravechia 1 , thiago costa 1 , marilia sirianni 1 , eduardo bastos 1 , leandro santos 1 , lilian castilho 2 and jos e kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp background/case studies: rhd*weak d type 2 is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion 2007) the c.1154g>c change (p.g385a), which characterizes the rhd*weak d type 2 allele is a splicing variant that induces skipping of the whole rhd exon 9. we report an altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d1 w c-c1e1e1. the samples showed weak hemagglutination reactions (11/21) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon 9 in both dna samples. sequencing showed the c.1154g>c change and the intronic c.1154-8t>a and c.1154-31t>c substitutions, which are associated to the rhd*weak d type 2 allele. conclusion: our results showed that c.1154g>c associated with c.1154-8t>a and c.1154-31t>c variations had probably a functional impact on splicing inducing exclusion of exon 9 in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type 2. background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than 50% of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with 1,057 patients and 3 studies from other regions (brazil, egypt and france) with 641 patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was 0 to 26 years and for the other countries 0 to 20 years. available data from 5 us studies included a total of 91 alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% ci) vs. 9.4% for non-us studies (7.3-11.8, 95% ci) (p50.0008) and 134a transfusion 2017 vol. 57 supplement s3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001). similarly, the number of alloantibodies per 100 transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort (0á68 vs. 0.33, p50á0005). average number of rbc units transfused per patient in the us was also higher compared to data from france (77 vs. 45, p50.0001). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* 1 , melissa grohotolsky 2 , lisa deblass 1 , bala carver 1 and kip kuttner 2 . 1 health network laboratories, 2 miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: 23 year old white mennonite female g1,p0 presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated 11 reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was 2 at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at 2. although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a 15 year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g1 (r g r) cells. both rhce*c and rhd genes encode ser103 which determines g expression. rare rhd variant antigens lacking ser103 are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e1c1e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r 2 r 2 , r'r, r g r and rr cells. this patient is predicted to be r 2 r 2 (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is 135a transfusion 2017 vol. 57 supplement s3 excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* 1 , christa voliva 1 , kathy fletcher 1 , heather vaught 2 and tracie ingle 1 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than 7 days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were >21 dat positive, 2 were weakly dat positive and 16 were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman 6.0-8.1ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: 5 bb, 14 bg and 1 bp. the ph of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a ph of 8.1 (35%). sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%). four eluates with different results are summarized in table 1 . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results (3) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in 1900 led to the discovery of human blood groups. in the abo system >200 alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor (38 units over 17 years) who is actually a w . study design/methods: donations were tested with the pk7300 instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns 1,2 and 4 and exons 6 and 7. specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table 1 . tests with anti-a, -a1, -b anti-a,b were negative as were the a2 cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table 2 . the significant changes were found in exons 6 and 7. in exon 6 there was a nucleotide (nt) deletion of 261g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly117ala. mutations in exon 7 included a nt substitution causing a pro156-leu change and a nt deletion 1061c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a2 cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o.01.01/abo*aw.02] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a1 and b cells. this abo discrepancy was caused by the presence of anti-a1 in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a2 cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer >1:1000. (schmidt, nacarrow et al. 1959) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. 136a transfusion 2017 vol. 57 supplement s3 extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to 96 blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to 15 days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: 92 edta blood tubes collected from random blood donors were used to extract dna from 200 microliters of whole blood on day 5, 12 and 15 days post collection. blood samples were stored at 2-8c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a260/a280 using a nanodrop 2000 (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result50) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the 92 samples, 20 samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from 10.8 to 62.6 ng/ul. all readings with the exception of 1 (10.8ng/ ul) had concentrations >5 15ng/ul. interestingly, the one that was <15ng/ ul on day 5, yielded >515ng/ul on day 12 and 15 post collection. over the next 3 months, 67 sets of 92 samples were extracted and tested by hea. eighty-three (1.3%) failed extraction and 82 (1.3%) failed hea. none of the samples that failed extraction were 12 or 15 days post collection; none of those that failed hea were 15 days post collection; 3.7% were >10 <15 days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to 15 days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas 1 , carlos villa 1 , rachel davis-rauser* 1 , helen carpenter 1 and vrunda patel 2 . 1 university of pennsylvania, 2 hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd38 monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in 2015. communications suggest all patients receiving therapy would have a positive antibody screen because cd38 is a common antigen expressed on red blood cells. currently, 154 patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for 1 hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a 28 y/o g1p0 at approximately 23 weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [1, 2] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m1n-s1s1 phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [3] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays 137a transfusion 2017 vol. 57 supplement s3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in 67 dna samples from chronically transfused patients with scd, in 65 patients with thalassemia and in 3000 dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in 3 levels: (1) rh and k matching; (2) extended matching and (3) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for 100% of our thalassemic and scd patients at level 1, 90% for scd patients and 70% for patients with thalassemia at level 2 and 30% for patients with scd and 90% for patients with thalassemia at level 3. the patients were transfused with a median of 36.4 rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with c e k matching and <1% with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of 164 clinically significant antibodies were detected using sprca technique, as well as 9 warm autoantibodies and 97 nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being 100% specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than liss (93). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd38 on rbcs patricia a arndt* 1 , anthony salazar 2 and regina m. leger 1 . 1 american red cross blood services, 2 long beach memorial medical center background/case studies: monoclonal anti-cd38, e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd38 on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd38 antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or 2aminoethylisothiouronium bromide (aet). chapuy et al described (2015) and validated (2016) 2), and 6% aet (ph 8.0) as per the aabb technical manual, 17th ed. these treated and untreated rbcs were stored in alsevers at 4c and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from 8 total dara patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (pe)labeled anti-cd38. rbcs were also tested on days 1 and 5 or 8 with a serum containing anti-k by peg iat. results/findings: the 0.2m dtt in ph 8.0 pbs had a final ph of 7.3 and the ph of the commercial 0.2m dtt was 6.5. results are in table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8. rbcs treated with 0.2m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd38 by flow cytometry for up to 8 days after treatment. rbcs treated with 0.01m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening (10-30%) of reactivity with pe anti-cd38. background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery 1997 of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon 5 and 10) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr5) in gestational week 25. the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of 1618 pregnant women were included. nip rhd was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%). compared to the postnatal rhd type, 9/987 (0.1%) of nip rhd results were false positive (fp) and 4/582 (0.7%) were false negative. in 5/49 (10%) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p50,71). the fraction of ccfdna was calculated for 150 randomly selected nip rhd true positive cases. median ccfdna ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r2 50,012; p50.49). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the 25th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an 83 year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only 1 in 20,000 donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, 4 lan-rbc units were transfused over 4 days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (hb) 5.3 g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of 10.2 g/dl was maintained for 4 days. the antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive dat (igg 21, c3 11) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from 10.9 g/dl on day 5 to 7.4 g/dl by day 8 with no bleeding identified, and increase in total bilirubin and ldh (peak 2.4 mg/dl and 304 u/l on day 7) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day 8 with good response (hb 8.1 g/dl). the patient remained stable and was discharged to a skilled nursing facility 6 days later. conclusion: transfusion of lan1 rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* 1 , andrea gerner 1 , lynne stewart 1 , carol sostok 1 , mollie bell 2 and gregory r halverson 2 . 1 st. elizabeth healthcare, 2 hoxworth blood center background/case studies: anti-f was first described in 1953 by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the rh blood group system (isbt rh6). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a 56 year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused 1 unit o-rbcs. two weeks later the patient received an additional o-rbc. within 4 days the hgb had decreased from 8.3 to 7.1 g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused 3 r 1 r 1 (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c1e1f1 phenotype. the rh phenotype and as was repeated on a sample collected 18 days later. the c typing was micro positive, mixed field only after 5 minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post 24 hour hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl. the last transfusion of 3 units increased by only 1.2 g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 r 1 r 1 units was nearly complete. in a case from 1989, ohto and kariyone (transf. 1989; vol29, no. 3) reported a 51 cr ásurvival study of f1 rbcs in a patient with anti-f. they showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd38 drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to 9 days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with 0.2m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table 1 ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to 9 days following the dtt treatment of rrbc. reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table 1 for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to 9 days. this suggests that dtttreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd38 therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd38 that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with 0.2m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract 2-5c, and observed for hemolysis (none was seen) for up to 21 days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted 2-41 with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in 4 patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than 6 months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* 1 , monique scott 2 , garcia curtis 2 , ellice wong 2 , alexa j siddon 1 and christopher a tormey 1 . 1 yale-new haven hospital, 2 va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately 25% to 50% of persons of african descent is characterized by neutrophil count of <1.5x10 9 /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p<0.05. results/finding: subjects who were clinically identified as having probable ben included 7 patients (mean age 48.7; all self-identified as african-american; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as african american; (50/50 male). all of the cases (100%) diagnosed with ben had fy(a-b-) phenotype. mean anc (1.95x10 3 /ul) and wbc counts (4.04x10 3 /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p50.0008 and 0.001, respectively) compared with controls (mean anc 5 5.46x10 3 /ul ; mean wbc count 5 8.14x10 3 /ul). there was no significant difference in mean platelet counts (161x10 3 /ul vs 213x10 3 /ul; p50.2301) or mean hemoglobin levels (12.4 g/dl vs 11.7 g/dl; p50.6031) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c3d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih-1000 had 100% concordance for all blood grouping assays. for ahg assays, the ih-1000 detected an anti-jka1e, anti-fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih-1000 identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih-1000 with anti-igg,-c3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih-1000 analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih-1000 is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in 99.9% of samples tested. there were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti d and 1 positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp201 evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* 1 , kathleen bensing 1 , michael schanen 1 , cindy piefer 1 , randall w. velliquette 2 , christine lomas-francis 2 and connie m. westhoff 2 . 1 immunohematology reference laboratory, versiti/bloodcenter of wisconsin, 2 immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new 7/0 7/0 7/0 cecf 1/1 2/0 2/0 rhce*ce or rhce*ce compound heterozygotes ce254g 1 ce733g or ce48c,733g or ces or ceti 6/0 6/0 6/0 ce733g 1 ce48c,712g or 48c,733g 4/0 4/0 4/0 ce733g 1 ces or cemo or ceek or ceek(var) or cern 8/0 8/0 8/0 ce48c,733g 1 ce48c,712g or cemo or ceti 2/0 2/0 2/0 ce48c,712g/ce254g/733g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi 7/0 7/0 7/0 total 106/8 110/4 113/2 142a transfusion 2017 vol. 57 supplement s3 reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd9/4 and rd12/2, and a licensed comparator anti-e (p3gd5121ms63), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n 5 42) or edta blood from donors (n 5 72) and were tested using a manual tube method or on a pk7300 automated platform. a score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table 1) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r 1 r 1 , r 2 r 2 , r 1 r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: 40 were rhce*ce that were in trans to rhce*ce; 16 were various rhce*ce plus rhce*ce48c compound heterozygotes; 31 were rhce*ce or rhce*ce homozygotes; 27 were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for 6 rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with 1 rhce*cear/rhce*ce48c compound heterozygote, and with 1 rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and 1 of 2 rhce*cecf homozygotes were detected using the comparator reagent. rd9/4 and rd12/2 failed with 4 and 1 e variants, respectively (table 1) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd9/4 clone. none of the reagents detected e antigen variant expressed on 1 example of rhce*cehar/rhce*ce. conclusion: rd9/4 and rd12/2 anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems. we used the illumina's hiseq 2000/2500 system to perform next generation sequencing first on sureselect-enriched genes from 16 dna reference samples with average target design coverage of 97.5%, and then on haloplex-enriched genes from 32 dna reference samples with average target design coverage above 97.0%. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads (80.54%) were mapped to the target regions relative to the sureselect reads (29.23%). the mean sequence coverage depth of the targeted bases was around 200x for sureselect method and 300x for haloplex method. some exons, such as rhd exons 4 and 8, 10, rhce exon 10, ermap exons 5 and 12, cd55 exons 10 and 11, cr1 gene (most exons) and gypb exon 5, are consistently covered with less than 10x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on 38 blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than 90% concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes. evidence rhce*cehar does not encode for rh34 (hr b ) antigen debra j bailey* 1 , trina horn 2 , paul mansfield 2 , najmi qazi 1 , pamela nickle 2 , jessica keller 2 , margaret a keller 2 and jan r hamilton 1 . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in 1996 and has a phenotype of c2e2c1e1 w f1 w , g2, hr 0 1 w , hr2, hr s 2, rh:33, rh:50 with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh:234 (hr b 2) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c.254c>g and rhd c.1136c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d1c1e2c1e1. her plasma contained an alloanti-s and an antibody that reacted strongly with all random e2k2s2 reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d22 and dc2 red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e2s2k2 red cells homozygous for the rhd*diiia-ce(4-7)-d, rhce*-ce48c,733g,1006t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce48c,733g,1006t/ rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr1 (2 of 2 sources) and hr b 2 (2 of 3 sources) phenotype. conclusion: the rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t haplotype is one of the rh haplotypes expressed by the original hr b 2 individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between 1/1/2006 and 3/ 31/2017. all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately 11 years, 81 patients had htla established at least once by titration studies. serological investigations on a total of 118 samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on 20 samples was successful in rule out in 60% of cases. in an additional 12 patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only 40% of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for 71% of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c.1136c>t (p.thr379met). the dau0 allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-d alloimmunization for 18 dau family alleles. we investigated two samples with the c.1136c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample 1 was from a 17 yo multiracial female. her rbcs reacted 11 s at immediate spin (is) and 31 in iat with immucor gammaclone and series 4 and 5, and mi1 at is and 41 in iat with ortho bioclone anti-d. rbcs did not react with 2 of 12 (lhm 174/102 & 57/17) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c.1136c>t characteristic of dau. rhd sequencing confirmed c.1136c>t and identified two adjacent changes, c.787g>t and c.788g>t (c.787_788delinstt), in exon 5 encoding p.gly263leu. sample 2 rbcs reacted 1w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series 4 and 5, and quotient albaclone blend and alpha anti-d. papain treated rbcs were 11s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c.1136c>t. sequencing confirmed c.1136c>t and found a new c.761c>t change (p.ser254-leu) in exon 5. the c.761t has not been reported, but c.761g encodes a stop codon (p.ser254stop) in japanese (vox sang 2015, 109:359). conclusion: we report two new alleles: rhd with c.787_788delinstt (p.gly263leu) and rhd with c.761c>t (p.ser254leu), both also carrying the c.1136c>t (p.thr379met) characteristic of the african dau cluster. d antigen associated with p.263leu is a partial d antigen with a novel epitope pattern. the p.254leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by 5/7 commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to 21. the number and diversity of alleles in the dau cluster supports that the c.1136c>t change is a major ancestral african background allele (wagner et al, blood 2002,100:306). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september 2016 to present day we got 130 samples of repeated blood donors who are known to be d negative, c positive and/or e negative from 15 blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon 4, exon 7 and exon 10 in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227g>a and c.1222t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the 130 sample, we identified 71 cases (54.6%) of total rhd deletion, 18 cases (13.8%) of rhd-ce-d hybrid, and 41 cases (31.5%) of rhd variant. 39 of rhd variant were determined to be asian type del with c.1227g>a variation. 2 cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was 10 % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b 3 phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b 3 phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from 30 taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n552) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon 6 and exon 7 of the abo gene were amplified and sequenced. the abo*b3.03 allele was confirmed by pcr-rflp analysis. results/finding: among 52 subjects with b 3 or ab 3 phenotypes, 47 were genotyped as abo*b3.03. the abo*b3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage (37.81 6 6.62) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other 5 subjects with b 3 or ab 3 subjects, genotyped as abo*b3.06(n51), abo*bw.03(n51), abo*bw.11(n51), abo*bw.12(n51) and abo*bw.29(n51), displayed flow patterns differed from the abo*b3.03 group. the abo*bw.03, abo*bw.11 and abo*b3.06 subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (<10% in abo*bw.03 and abo*bw.11 subjects and <20% in abo*b3.06 subject). both abo*bw.12 and abo*bw.29 displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b3.03 genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout 2016, the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of 398 patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n54), anti-jka (n53), anti-k (n52), anti-jkb (n51), both anti-e and anti-c (n51) (see table 1 ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr1 alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr1 gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr1 gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study 60 volunteers in the gambela region (nct01282021). the whole ackr1 gene was amplified in one reaction covering 12,125 base pairs (bp). this primary amplicon was re-amplified using nested primers covering 5782 nucleotides. nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using ncbi refseq ng_011626.2. the sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-utr, 53 bp of 3'-utr and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbsnp and nhlbi esp databases. results/finding: among the 60 samples, a total of 15 snps, including one novel snp in 5'-utr were observed. 4 snps occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-utr and 2 in the intron. all 60 individuals carried the snp indicative of the common fy:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the gata box mutation. no splice site mutation was detected. as 46 individuals were observed as being homozygous or heterozygous for 1 snp, we could unambiguously assign 8 distinct alleles. in the remaining 14 individuals with 2 or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than 5.5 kb of the ackr1 gene and identified at least 8 different alleles. the present study found that the vast majority of alleles (117/120) in the gambela population as defined by 15 snps, were similar to the clinically relevant fy*02n.01 allele, which in turn is defined by only 2 snps at positions c.1-67t>c and c.125g>a. out of the remaining 3 alleles, 2 were similar to fy*02 with the fy(b1) phenotype and 1 was similar to fy*02w.01 with the fy x phenotype. the high frequency of fy*02n.01 (95%) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique (95%-100%). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce(4-7)-d is the most common hybrid and is found in african blacks. it arose by conversion of exons 4-7 of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample 1 (male) and sample 2 (multiracial female), both c1c2e2e1 (presumed r1r1), presented with weaker than expected d typing; 11 is and 31/41 at iat. rhd beadchip identified the common african rhd*diiia-ce(4-7)-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c.733c>g and c.1006g>t (heterozygous) was also detected. as rhce*ce with 733g and 1006t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons 2 and 3 replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce(4-7)-d were found in both samples. sample 3 (scd male), d1c2e2c1e1, by rh beadchip had one conventional rhd and rhd*diii type 8, and rhce*ce733g/ces. as rhd*diiia type 8 has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce(48c) exons 1 and 2 had replaced those exons in the common hybrid rhd*diiia-ce(4-7)-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky926711and ky926710. we report two different and novel complex rh rearrangements: two samples thought to be r1r1 had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia(2-3)-ce. in kind, a sample genotyped as diii type 8 rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce48c(1-2)-diiia(3)-ces(4-7)-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples 2 and 3 have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r1 haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a 81-year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at 81g/l. her pregnancy history was not provided. she had received 5 units of packed red blood cells (rbcs) in the past including 1 unit within the last 3 months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r 1 r 1 , r 2 r 2 , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse 29 polymorphisms which determine 37 antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a1) and lu(b1). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of 36 antigens encoded by the kel gene, is organized into 19 exons. there are approximately 30 kel alleles associated with a kell null phenotype (k 0 ) in which no kell antigens are expressed, and 12 alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a 53 year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k1 kp(a-b1) js(a-b1). kel-cdna sequence analysis was performed and detected a single transcript species with c.578c, c.841c, 1790t, and missing the sequences corresponding to exons 11, 12 and 13. amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel*02 allele. this donor was presumed to have a k 0 phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel*02m.12. here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table 1 ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a1 and a2 cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c.261 deleted g, characteristic of o alleles, c.467t, characteristic of a2 and some uncommon o alleles, and c.703a and c.1096a, characteristic of b alleles. genomic sequencing of exons 6 and 7 confirmed the presence of an o allele, abo*o09 261del g, 318t, 467t), and the presence of a b allele (297g, 526g, 657t, 703a, 796a, 803c, and 930a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , diana gazito 2 , afonso cortez 2 , lilian castilho 4 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the 17-bp deletion in smim1 in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of 400 blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31. dna was isolated from peripheral blood and smim1 was sequenced. results/finding: from 400 donor samples studied, 4 were serologically vel negative by gel-iat but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31. genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the a allele rs1175550 in homozygous status. from the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the a allele of rs1175550 and 14 (66.7%) had the a allele of rs6673829. in contrast, in the 16 samples with stronger reactions we found the a allele of rs1175550 in 5 (31.25%) samples and the a allele of rs6673829 in 3 (18.75%) samples. conclusion: the molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides. this study reinforces the association of the a allele of rs1175550 with reduction of vel expression and suggests the involvement of a new rs6673829 change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as 0 (rh negative), or !31 on the neo or !21 on the echo (rh positive) for both series 4 and series 5 anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series 4 and series 5 anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: 80 patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in 51 of 80 (63.7%) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in 16 of 80 (20%) of samples. 67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. 40 of 80 (50%) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but <31 on the galileoneo or positive but <21 on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , rosangela person 2 , lilian castilho 4 , afonso cortez 2 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of 108 samples were included in the study, being 69 previously genotyped as rhd*dar1.2, 37 rhd*dar3.1 and 2 rhd*dau6. the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from 0 -99 corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon 10. rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon 3 was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that 10 of 108 samples (5 dar1.2, 4 dar3.1 and 1 dau6) had 2 rhd genes, were phenotyped as c1e-c1e1 and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these 10 samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in 2 dar1.2 samples showed the rhce*-cear/ce s genotype, in 2 dar3.1 samples the rhce*cevs.02/ce s genotype and in the dau6 sample the rhce*ce s /ce genotype. table 1 describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized 5f9 antibody (hu5f9-g4) that binds human cd47 has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu5f9-g4 (anti-cd47) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in phase 1b/2 trial of hu5f9 g4 in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a 48 year old male with refractory diffuse large b cell lymphoma enrolled in hu5f9-g4 clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd47 therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing (41 with anti-a, non-reactive with anti-b) and the reverse typing (31 with both a 1 cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase (11 to 41), at liss-37c (11 to 31), at liss-polyspecific ahg (m1), and at peg-anti-igg (m1 to 11). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and 37c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at 37 o c ahg phase. the abo typing of the second case performed after anti-cd47 administration showed a discrepancy between the forward (41 with anti-a) and the reverse (41 with both a 1 and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is (21), at 37c in liss (21), and liss-polyspecific ahg (m1). the dat and autocontrol were negative. his genotype was determined to be a1/o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd47 therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, 37c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd38 interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected 31 reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o.01.01/ o.01.01, consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n523) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt1 and a4galt. papain-treated patient rbcs were used to screen donor plasmas (n578) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with 3 polyclonal anti-a,b and a monoclonal anti-b (clone g1/2) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors1 or nor antigens. the patient was le(a-b1) and thus a secretor. a positive crossmatch was seen with 47% of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type 1 or 2) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type 1 chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $15 years earlier. the reactions are likely due to uptake of recipient-derived ble b (type 1) antigen (isbt no. 007006), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b1). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of 2017. shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017. reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?" results. assuming 30 seconds of technologist time per "?", we estimate that 13.6 hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table 1) . in 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells. all cases had mts-gel tm antibody identification panels performed, 25 of 26 also had a mts-gel tm ficin panel. the yield for the 51 panels performed was two routine panels with weak reactivity against hla1 cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; 13 were negative. one patient newly demonstrated anti-jka. fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2. conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in 16/28 patients. further testing was performed in 9/16. eight were tested for the presence of antibodies at 188c and confirmed in 8/8. rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict 378c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* 1 , sandra nance 2 , david moolten 3 and p. dayand borge 3 . (2):47-53). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this 2.5 year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing 0.6% bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed 50k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* 1 , anna burgos 1 , virginia lew 2 , sunitha vege 1 , susan veneman 3 , christopher j gresens 3 , jonathan hughes 3 and connie m. westhoff 1 . 1 immunohematology and genomics laboratory, new york blood center, 2 blood centers of the pacific, 3 bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least 100 rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m1n-individuals (meyer et al. br j haematol. 2016; 174:624-36) . the st a allele, also described as gyp*401, is a hybrid gypb-gypatranscript with the crossover in intron 3. we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a 24 years old pregnant, african american female g1p0 was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a 1 antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a 3 like phenotype. genetic testing did not support the serological findings of a 3 subgroup and a new abo allele, abo*784c that has never been reported in correlation with an a 3 like subgroup was detected study design/method: the patient rbcs were typed with anti-a 1 (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a 1 antibody work up was performed using three different lots of a 1 cells and three lots of a 2 cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and 48c for 30min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at 35 weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a 3 b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo*784c found in the mother. the previously reported abo*784a allele encoded an aspartic acid to asparagine change at position p.256 consistent with an a weak phenotype. also, at least five other alleles encoding an a 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a3/ aweak phenotype is encoded by the variant allele abo*784c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated biorobot m48 robot using the magattract dna mini m48 extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of 38.1ng/ml and 1.83 respectively. in the second phase of the study (n 5 39), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (a260/280) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was 100% concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano 1 , izaskun apraiz 1 , maría azcarate 2 , miguel angel vesga 2 , montserrat rubia 2 , mercedes piedrabuena 2 , fernando puente 3 , barbera veldhuisen 4 , ellen van der schoot 4 and m onica l opez* 1 . 1 progenika biopharma, a grifols company, 2 centro vasco de transfusi on y tejidos humanos, 3 banco de sangre y tejidos de arag on, 4 sanquin blood supply research background/case studies: it is well established that weak d 1, 2 and 3 phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type 1, rhd*weak d type 2, rhd*weak d type 3, rhd deletion, rhd*pseudogene and rhd*diiia-ce(3-7)-d and itgb3 gene: hpa1a and hpa1b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa-1 blood group typing. study design/method: a cohort of 1000 previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications 2009/108/ce for a ivd product of list a (!10% clinical samples, >2% neonatal specimens and !2% weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n5160, 16%). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa-1 predicted phenotype were used for comparison. transfusion results/finding: no system failure, 100% call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a 100% concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was 100%. the following id rhd xt predicted phenotype results were obtained: d negative (n5361), no amplification variant (n515), weak d type 1 (n522), weak d type 1 heterozygous (n51), weak d type 2 (n532), weak d type 2 heterozygous (n51), weak d type 3 (n534), weak d type 3 heterozygous (n51), weak d types 1, 2 or 3 not detected (n5533). regarding hpa-1 blood group, the predicted phenotype results obtained by id rhd xt were 100% concordant with bds results: hpa-1a positive (n5157) and hpa-1a negative (n56), hpa-1b positive (n546) and hpa-1b negative (n5117). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types (100% specificity and 100% sensitivity for d antigen, hpa-1a and hpa-1b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with 11 rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value <50.05 (clinical analysis) or <5x10 -8 (gwa) was considered statistically significant. results/finding: of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100). in multivariable logistic regression models, age (or 4.2, p50.009, for age 501 compared to 0-4), gender (or 1.3, p50.04, for female compared to male), transfusion history (or 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (or 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or 4.5, p<0.0001) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from 32 newborns less than 2,000 gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on 32 sample pairs. dat test was negative on 30 sample pairs and two were positive. there was 100% concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on 32 placental blood samples and 29 heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns. o-(7.4%), ab1(6.8%), b-(2.7%), and a-(0.6%). among the tested donors, 89.2% were d positive with r1r being the most common rh phenotype. in the kell blood group system, 4.5% of the donors were k positive, while the k antigen was found to be 99.4%. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a1b1) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a1b1) and m1n-s1s1 at 47% and 22.6% respectively. the le a 1 and le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while lu b -phenotype was found in 3.3% of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a1b1) and lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the m1n-s-s-and m-n1s-s-phenotypes were not found. the frequency of the p1 antigen was found to be at 78.9% similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph 7.2 and the supernatant was discharged. a dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with 10ll of washed red cells. the cell suspensions were incubated for 30 min at 378c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional 15 min at 228c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for 515-548nm. events were recorded at a frequency of 1000 cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* 1 , mark yazer 2 , nancy m. dunbar 3 and biomedical excellence for safer transfusion (best) collaborative 1 . 1 university of vermont medical center, 2 university of pittsburgh, 3 dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of 50. study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a1 and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a 4-year and 5-year period, respectively. one center provided plasma and wb testing data for a 1-year period. results/findings: in total there were 7106 group a plasma units tested and 654 (9.2%) had a high titer anti-b. the range of high titer group a plasma units between these three centers was 7.4%-12.5%. of the 1778 wb units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-a, 61/1778 (3.4%) had a high titer anti-b, and 106/1778 (6%) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with 0.2m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd38 on reagent rbcs and render them free from plasma anti-cd38 drug interference. procedures for the preparation of 0.2m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on 0.2m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of 0.2m dtt, was adjusted to ph7.16, ph 7.56, ph 7.96 using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n53) were treated with the 0.2m dtt solutions in parallel by mixing 1:4 ratio of packed rbcs to 0.2m dtt solution followed by incubation at 378c. for up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each ph level. the reduction in average scores between different phs were also calculated at every 5 minutes to measure the impact of 0.2m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of dtt treatment at ph 7.96; 15 minutes at ph7.56 and 20 minutes at ph7.16. complete loss of k expression was seen after 25 minutes of dtt treatment at ph7.96; 35 minutes at ph7.56 and 35 minutes at ph 7.16. the reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other. the reductions in average scores were seen between 15 to 30 minutes range of dtt treatment time when ph 7.16 was raised to ph7.56; 15 to 30minutes range when ph7.56 was raised to ph7.96; and 15 to 30 minutes range when ph7.16 was raised to ph7.96. conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to 15 minutes and/or beyond 35 minutes of incubation. the ph of the 0.2m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of 0.2m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between july 1, 2013 and june 30, 2014. these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent. the three most commonly encountered antibodies were anti-d (n 5 7639) 63.1%, anti-m (n 5 1288) 0.6%, and anti-e (n 5 1227) with a frequency of 10.1%. a total of 455 (3.9%) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with 182 instances (40.0%) followed by anti-e and anti-c with 79 (17.4%), and anti-c, anti-e with 26 (5.7%). of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with 7474 or 61.8% of the total. the west had 2111 (17.4%), the midwest 1538 (12.7%) and the northeast with 979 (8.1%). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p 5 0.17). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* 1 , lindsy rich 1 , sherry stern 1 , sharon wangen 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous 4 days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was 1 to 2 days. results/finding: of the 30 ntd specimens from the immucor neo, 8 resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present. the other 19 samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the 23 absc specimens that were resulted out as positive on the immucor neo, 11 specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: 3-41 with ms24 (n515), 1-31 s with ms23 (n59), no reaction with ms273, dgc02, p3x255 (n514). 14 samples tested with a polyclonal anti-c showed a 1-31 reactivity. 3 d1c1e1c1e1 cases tested with a polyclonal and monoclonal anti-e (ms16, ms21, ms62, ms63) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c.286g>a mutation in exon 2, predicted to encode the p.gly96ser substitution. for 2 apparent r 1 r 2 donors, a f-negative type allowed the prediction of a rhce*ce286a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce286a allele (c and e in cis) in all samples. 3 d1c1e1c1e1 individuals were reactive 11 s with the original source of anti-rh55, slightly weaker when compared to rhce*ce286a/rhce*ce rbc samples available from our cryobank (21). conclusion: our results confirm that the c.286g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh55). the locr reactivity appears to be rather similar when coded by rhce*ce286a or rhce*ce286a alleles. this was quite an unexpected finding, since the p.gly96ser substitution is close to the critical amino-acid for c/c expression (p.pro103ser). none of our 15 cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce286a was reported to code for a partial c (rh:-26), we consider that rhce*ce286a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly96ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes 1, 2 or 3. the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d 1, 2, or 3 genotypes. study design/methods: between 9/2015 and 2/2017 50 samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types 1, 2, or 3 genotypes, but 156a transfusion 2017 vol. 57 supplement s3 had evidence of rhd genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons 1-10 to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided. 34 samples (68%) were found to be weak d type 1, 2, or 3 (24, 6, and 4 samples, respectively). 5 samples (10%) appear to be genetically rhd negative. genetic sequencing was performed on 11 samples; 9 had rhd genetic variants that were not weak d types 1, 2, or 3 (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples (4%) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types 1-3. of the 11 samples that had evidence of an rhd gene and did not carry the known weak d types 1-3 polymorphisms, 9 (82%) of were found to have other rhd variants, and 2 (18%) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types 1-3 variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel 2006, curr opin hematol13:476) , that individuals with weak d types 1, 2, and 3 are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report 15 months experience with rhd genotyping on 352 samples referred with discrepant or weak d typing investigated from january 2016 to april 2017. study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for 153 samples (53.3% caucasian, 32.2% african american/african, 6.6% multiracial, 4.6% hispanic, 2% asian, and 1.3% other). results/finding: rhd genotyping identified weak d types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial d phenotypes in 168/ 352 (47.7%) (table) . uncommon or rare weak d alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%). the partial d alleles found were diverse, but the largest number included partial rhd*d 4.0 (n562) and *dar ( conclusion: in a multiracial cohort of 352 individuals with weaker than expected d typing 44% were due to weak d types 1, 2, or 3 and would not be considered at risk of clinical significant anti-d, but for 56% there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp242 rhd*07.02 allele causes discrepant genotyping results for rhce small c sabine scholz* 1 , sandra schneider 1 , sabrina k€ onig 1 , susanne helmig 1 and vicky van sandt 2 . 1 inno-train diagnostik gmbh, 2 rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c (307c) and c (307t) is caused by the snp on position 307 on the rhce gene. the rhd*07.02 allele (also known as rhd cat vii type 2) carries the snp 307t>c on the rhd gene and additionally the snp 329t>c. this rhd*07.02 allele has been described to partially express rhc on the d polypeptide (faas, transfusion, 2001) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, 81938) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles (307t>c, 329t>c) confirming a rhd*07.02 and one rhd*01 allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series 4 and 5 anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series 4 and series 5, and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c.463a>g change in exon 3 encoding an amino acid change p.met155val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c.463a>g (p.met155val) change in exon 3. several snps, deletions, and insertions have been reported with changes in exon 3. phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c.463a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* 1 , juan merayo-rodriguez 2 , christopher lough 1 and nancy eckert 3 . 1 lifesouth community blood centers, 2 life south community blood centers, 3 lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is 100% in most populations and greater than 99% in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, 57 year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found 251 eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to 7.1/21. the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient 1 was a 29 yo female, c2e2c1e1, whose rbcs reacted 11 by echo and 31 by neo with anti-d4, and '?' with anti-d5. testing with d4 and d5 by tube gave 21 and 11 w on initial spin (is) respectively and 41 by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and 1 w with immucor gammaclone anti-d, and all were 21 at iat. rbcs did not react with two (lhm 174/102 & 57/17) of 12 anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c.780c>a change encoding p.his260gln. patient 2 was a 20 yo pregnant female, c2e2c1e1, whose rbc were 1 w at is and 31 at iat with immucor series 4 and 5 and gammaclone, and moderately reactive, 21 is and 41 iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm 174/102 & 170/45) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon 2 gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.ile60leu and ser68asn. conclusion: we found two previously reported rare alleles: rhd with a c.780c>a (p.his260gln), previously found in france (lefloch et al. genbank ku363612), and rhd*dar with part of exon 2 replaced by rhce, reported in sub-sahara africa (granier et al. transfusion 53:3009) designated rhd*dar(ce2:v505v-s68n) with an allele frequency of 0.002 to 0.016. blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu5f9-g4 is a human monoclonal igg4 antibody recognizing cd47 that is in clinical trials to treat hematologic or solid malignancies. cd47 is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd47 is thought to enhance phagocytosis and promote anti-tumor responses. cd47 is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd47 drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n57) from 2 patients were tested over the course of 1 month treatment. plasma was tested at immediate spin (is) and by iat with r2r2, rr, d--, rh mod and rh null rbcs, as cd47 expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg4) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd47 was observed in plasma as soon as 1 hour post infusion. plasma reacted 31 to 41 at is and 41 with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker (31 and 21) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was 31. in contrast, iat reactivity using gamma-clone anti-igg was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd47 titer was 1 at is and peg iat with gamma-clone anti-igg, but was ! 256 with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n54) were 31 reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after 4x allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu5f9-g4 anti-cd47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd47 expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu5f9-g4 is igg4. reactivity was observed in all phases and with all test methods. cd47 is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd47 reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg4, can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd47 on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat1 rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in 1985. in august 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh1k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:-3 etc. had been excluded, k-phenotyping revealed a k 0 -phenotype. a total of 38 silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k 0 -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel*02n.19 with c.2023t encoding p.675ter (reported in an individual from austria in 2007). there are two known k 0 -patients in our country, both homozygous for c.2023t. the daughter was a c.2023t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k 0 -donors are available in our country. with the help of the isbt rare donor working party, a k 0 o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* 1 , kshitij srivastava 2 , houda romdhane 3 , saloua jemni yacoub 4 and willy albert flegel 1 . 1 nih, 2 dtm/cc/nih, 3 regional blood transfusion center sousse, 4 regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, 159a transfusion 2017 vol. 57 supplement s3 since 2009. the tunisian population has the largest known prevalence of weak d type 4.0 alleles, occurring in 1 of 105 rh haplotypes, compared to 1 in 6,060 or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of 13,431 random blood donors were serologically screened for the d antigen using 3 routine techniques. samples with weak reactivity were tested with a panel of 6 monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak d phenotype. among them, 60 carried an allele of the weak d type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak d type 4.0 allele. only 1 sample each was found for the weak d types 1, 3 and 100 and the dvii, while 3 samples showed the consensus rhd sequence. no mutation in any of the 10 rhd exons was detected in another 3 samples. the molecular analysis of the rhce gene showed that 59 out of 67 samples with serologic weak d phenotype (88.06%) had a variant rhce allele and the most common associations were: weak d type 4.0 linked to rhce*cevs.04.01; weak d type 4.2.2 with cear; and weak d type 4.1 to rhce*cevs.02, while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost 90% of the weak d phenotypes in tunisia were caused by alleles of the weak d type 4 cluster, of which 88% represented the weak d type 4.0 allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type 4.0 in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type 4.0 phenotype. there is a possibility that the rhce*cevs.04.01 allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* 1 , heather simmons 1 , christine lomas-francis 2 , gayane shakarian 2 , sunitha vege 2 , lauren hutelmyer 3 , sandra nance 4 , jessica poisson 1 , nicholas bandarenko 1 and connie m. westhoff 2 . 1 duke university hospital, 2 immunohematology and genomics laboratory, new york blood center, 3 arc pennjersey, 4 american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a1, 2 year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with 0.2m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c2, k2, fy(a2),s2]. reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x peg alloadsorption. the adsorbed plasma reacted with 0.2m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k2, k2, js(b2), kp(a2b2) and sc:21,23. her plasma reacted with k o , mcleod, sc:21,22 rbc samples and dtt-treated sc:21 rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused 4 aliquots of crossmatch incompatible kp(b2), s2 rbcs. her post-transfusion dat was 21 with anti-igg, 11 with anti-c3d. the eluate reacted with all rbc samples except 1 kp(b2) sample. she tolerated additional aliquots from 4 phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k2, k1, kp(a2b1), js(a2b1) and sc:1,22, discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c.1481a>t (p.glu494val) (kel*02.10) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a2). sc sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional sc*01, predicting sc:1,22,3. kel and sc results on the mother were kel*02/kel*02.10, heterozygous for the sc change 5'-2g>a, and her rbcs typed k2k1 kp(a2b1), sc31, ula1, consistent with dna predictions. plasma collected 7 months later was nonreactive at rt and in peg iat. her rbcs were dat2 and now typed k1, kp(a2b1), ul(a1) sc11 and sc31,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel*02.10 homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc:21,23 rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b1). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a 55-year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron 5 polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs5-1a)genotype, associated with a jkb null phenotype. anti jk3 was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused 64 old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a1) 41 and jk(b1) 21. genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk3. however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk3 or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms 160a transfusion 2017 vol. 57 supplement s3 (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a1b-); kp(a1b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on 3 separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon 2 revealed a 287g>a mutation, fy01*n.04, known to silence fya. sequencing of kel exons 1-19 exposed a silent polymorphism in exon 8, 846g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy01*n.04 mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly 100% of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, 64 self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of african descent. only 1 was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient 1. study design/methods: patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age. anti-b changed from undetectable/weak to strong at the age of 7 years. patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was 0/11. both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table 1 . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient 1 with 0-11 reactions up to 7 years of age. thereafter, abo typing showed mainly strong anti-b. patient 2 had 0/11 anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for 2 children on long-term tpn. patient 1 had absent/weak anti-b since birth up to 7 years of age, then developed strong anti-b with no change in feeding regiment and medications. patient 2 had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used 28 times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* 1 and elizabeth hart 2 . 1 brigham and women's faulkner hospital, 2 university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title 42, cfr part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in 2016. a total of 232 samples were evaluated. each abid was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and (2) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of 83 abids were performed on new patient samples. of the new abid samples, 29 (35%) had microscopic absc results. for the previously known antibody patients, there were 35 which accounted for 15% of the total abids performed. when reviewing the total abid workups, a total of 64 (28%) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the 29 new antibody samples were: conclusion: a total of 86% of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than 99% in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only 10 cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received 10 rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only (1-21), and negative with anti-c3b, c3d reagent. the antibody showed a peak gel-igg iat titer of 32. results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w1), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody 12 anti-p 1 4 anti-m 2 anti-sd a 6 anti-le b 1 anti-jk a 1 anti-k 1 anti-e 1 anti-c 1 results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42. further investigation was conducted to determine the molecular identity of the «others». out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon 5 or both exons 4 and 5. a surprising amount of 38 samples were discovered to be normal rhd. conclusion: along with sandler et al. (2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types 1, 2, 3 and 42 obtained in serological weak d, 45 years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was 0,6%. no trali happened in the period. prophylaxis were used in 98% of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred 3 times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, 358 rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p <0.05). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) (0.27% vs 0.11%, p <0.001) or aes with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes (0.028% vs 0.024%, p 5 0.614) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than 1 week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd10 codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found 11 patients from 2011-2016, who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those 10 cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as 1 out of 100 transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was 8.7 g/dl but declined to 7.3 g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days 2 and 3 with poor responses. on day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dl to 1.8 mg/dl (reference 0.8-1.3 mg/dl), and lactate dehydrogenase was above reportable linearity, >2500 u/l (reference 122-222 u/l). testing revealed additional anti-e, anti-jkb, dat c31, plasma free hb 64.4 mg/dl (reference 1-15.2 mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b1), one of which was also e1. one volume tpe was performed to remove free hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at 3.7 mg/dl on day 13, decreased to 2.3 mg/dl before discharge on day results/findings: twenty three cases were identified, of which 20 had medical records available for analysis. ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (cad). the implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 ml (range 20-280 ml). the mean time to onset of chest pain was 92.15 minutes (sd 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion. chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%). a post-transfusion chest x-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia. three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a 6 months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts 13. based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase. check cells were found to be 21. these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o1 to a1) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a 63-year-old o1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for olt (donor o1). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving 46 units of o1 rbcs and 26 units of o1 plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a1 plasma were transfused to wash out anti-a antibody prior to transfusion of a1 rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant. the patient's total estimated blood loss was >20l. he received a total of 71 units of rbcs (including 23 a1), 63 units of plasma (including 37 a1), 6 units of cryoprecipitate, and 9 units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o1. on postoperative day (pod) 1, a ts showed predominantly a1 rbcs with trace o1 rbcs, as well as very low anti-a igm and igg titers (table 1) . he received two additional o1 rbc units (1 each on pod 4 and pod 12) with increasing o1 rbcs on ts and rising anti-a titers. his blood type was unequivocally o1 by pod 13. the patient showed recovery of liver synthetic function on pod 1 (factor 5 activity 5 58%) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod 13, the patient had reverted to o1 with recovery of anti-a titers. at 3 months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* 1 , alfred mingo 1 , maria isabel gonzalez 1 , antoni mena 2 and juan pedro benitez 2 . 1 bst, 2 at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h1) and an oncology center (h2). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of 2016 transfusion activity in both h1 and h2 shows 7970 pse and 12572 bca, out of 13163 and 20676 respectively, since the tss deployment in 2015. retrospective analysis and classification of 6700 security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h1, nm related to pse accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%). the nm detected in bcas were 12.1% of all and mostly (74.52%) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet. for h2, nm related to pse accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%). the nm detected in bcas accounts for 24.48% of all and in 69.08% occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are 28.02% of all and in 65.61% of them the blood components were assigned to another patient. (1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available elisa. comparison of adequate response to ppv23, defined as ! 2 mcg/ml for >7 serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6). of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv23 in the previous 10 years; 9/44 also reported previous history 13-valent sp conjugate vaccine within the last 5 years. baseline pre-vaccination titers (n572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13). in the group with pre-and post-vaccination (n519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016). patient harm events recorded within the veritas system from january to july 2016 were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of 114 tres per month and 1300 per year were found over five years. 81% of tres are associated with pre-bb activities, 10% occur within bb, and 9% are post-bb events. sample collection and handling represent 80% of total tres. most tres (96%) were reported by bb staff, 4% were reported by non-bb staff. patient harm analysis revealed an average of four level 0 (near miss), three level 1 (no known harm), and 0.3 level 2 (patient harm) per month. no deaths related to tre were detected over the seven month january to july 2016 period. patient harm was associated with tres occurring in the bb (17%) and post-bb (83%). these events were reported externally (78%) and by bb staff (22%). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january 1, 2013 and march 28, 2017 was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by 5 and added to the number of individual whole blood plts; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was 31. the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029). there were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the prcs and 25gypcs. we did not find significant differences between prcs and 25gypcs in cci1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in cc24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.) transfusions. there were no significant differences between prcs and 25gypcs also in ma1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) pc transfusions. reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after prpc transfusions and in 51 (84%) of 61 cases after 25gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006. it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented. it was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient. results/finding: we identified 67 canadian tm experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%). for a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium-51 ( 51 cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n514) were divided into two 150ml aliquots, which were labeled with selected concentrations of s-nhsbiotin (3 and 30 lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of 38.4 6 1.6%). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately 2 million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that 561nm excitation of phycoerythrin (pe)-sa and high laser power (150mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with 3lg/ml of biotin resulted in $50,000 mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, 95% ci) of 1 in 380,000 (0.0003%). the lld95 for rbc labeled with biotin at 30lg/ ml was $ 1 in 1 million (0.0001%). biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes (150ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (hboc-201) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: (1) wb, (2) wb110% hboc volume (model of two units in an adult), (3) wb110% fdp, and (4) wb110% hboc110% fdp. samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4. susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with hboc and/or fdp, with or without prior 25% plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb1hboc, and wb1hboc1fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb1hboc in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc (10%) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even 75% hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc1plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in 8/2013 and 2/2015. we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of 81 patients, 24 were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery (43%). pcc was given for warfarin reversal in 31% of cases. a subset of patients received plasma within 2 hours prior to pcc (40%) or 24 hours after (47%). pcc was most frequently ordered in the or/perioperative service (25%). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in 2016, several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) simplify and expedite the process; 2) improve communication and expectations to decrease tat; 3) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood (4 units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of 61 er episodes were received. the average tat from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for er orders and physician documentation was 100% (61/61), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february 2016 -february 2017. mt was defined as the transfusion of ! 10 rbc units in a 24-hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at 30 days. results/findings: thirty mts occurred during a one year period. a total of 192,441 blood products were transfused during that time period. gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age. surgical patients accounted for 26/30 (86.7%) mts, and 4/30 (13.3%) were critical care patients. tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30). mtps were activated in only 8/30 (26.7%) cases. thirty-day survival was seen in 25/30 (83.3%) patients. only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage (3/ 5) or perisplenic hematoma (1/5). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a 70-year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* 1,2 , rebecca ross 2 , christopher a tormey 1,2,3 and amit gokhale 1,2 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: daratumumab (dara) is a igg1 monoclonal antibody therapy that specifically targets cd38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd38 expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, 54 subjects were identified for analysis. their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of 0% (0/54) prior to administration of dara. of these patients, 22 were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the 22 patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of 2015 for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september 1, 2015 through april 7, 2017 were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in 2016, 2,523 unique women receiving a total of 5,889 units of red cells (rc) in 2,906 transfusion episodes were identified. their median age was 45 (range 11 -60). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in 46 (1.6%) and 283 (9.7%). 635 (21.9%) transfusion episodes were associated with the use of 3 units or more rc. as a result, 1385 (47.7%) episodes resulted in a post transfusion hb ! 9g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively. upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to 386 (16.0%) women. conclusion: in the present study, it is observed that 49.7% transfusion episodes were given at hb ! 7g/dl. a substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! 9g/dl). for iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes (0.8-1.3x10 10 per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with 25 gy and transfused over 3-4 hours within 24 hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* 1 , lee grabner 1 , brenda herdman 2 , robert fallis 1 , amin kabani 3 and charles musuka 3 . 1 canadian blood services, 2 kenora rainy-river regional laboratory program, 3 diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over 50 years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and 38% were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dl; p50.94); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units (9.9 vs 9.8 g/dl; 1.2 vs. 1.1 g/dl; both p50.02). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients (1.3 g/dl vs. 1.0 g/dl; p<0.001). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than 1% of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered 33 times in total, affecting 15 patients and 21 users. stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver icu (6 times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin 25% iv solution, human albumin 5% iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* 1,2 , mahmut akgul 1,2 , hollie m reeves 1,2 , robert w maitta 1,2 , marcie pokorny 2 , anne capetillo 2 and katharine a downes 1,2 . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a 7-month retrospective study (january-july 2016) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were 301 adult (>18 years) orders reviewed by tms out of which 299 were approved. of the 299 approved orders, 136 (45.5%) were in agreement with tms's estimated dose. of 163 (54.5%) orders that were not in agreement with the tms's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated. seventeen of 299 orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001). seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e. 2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was 2.36 and posttransfusion was 1.91. only 22% of patients had their inr corrected to 1.5, while 28% had no change, or had increased inr. (table) . the majority (67%) of patients received 2 units of plasma. the mean plasma dose was 6 ml/kg. there were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the icu. two patients experienced bleeding during ir procedures (tips) and 1 developed a hematoma (tunneled central line). the median of inr correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is 1.9. randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. 3 of the 111 patients experienced bleeding complications indicating that inr of 1.9 may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately 4% of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up 45 % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population (40%) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately 85% of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july 1, 2014 as follows: 2 units of group a plasma and 3 units of group ab plasma is provided for the massive transfusion protocol (mtp) along with 5 units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed 235 mtps at our institution between july 2014 and march 2017. twenty patients (8.5 %) were transfused with incompatible group a plasma (5 group ab and 15 group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining 15 patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april 2017, our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in 2013, bonfils immunohematology reference lab (irl) sent out approximately 245 special platelets for patients with hla antibodies. by 2016, hla platelet orders increased dramatically and the irl sent out over 650 special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over 10,000 donors in the database with historical hla typing. however, only approximately 3500 of those donors actively donate. in the denver area, one of the most common hla types is a1 a2 b7 b8. only 81 of the 10,000 donors have this type (0.81%). therefore, to fill an hla platelet order request for a common hla type, only 28 donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a9 a11 b17 b35, there is only 1 out of 10,000 donors (0.01%) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a1 for example, all of the a1 positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in 2012, approximately 27% of these special order platelets were pra matched and the remaining 73% were hla matched by donor recruitment. by 2017, approximately 59% of special platelets sent are pra matched. this change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains 6 packed red blood cells (prbcs), 4 fresh frozen plasma (ffp) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august 2013 to march 2017 mtps were activated at ucm, of which 251 orders could be traced to the origin: 118 on inpatient floor (including icus), 58 in the operating rooms, 46 in the emergency department, 25 in labor and delivery, and 4 in other procedure rooms. of the 2207 prbcs that were issued, 1406 were transfused (64% utilized); of the 1446 units of ffp that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized). since march 2016, the time of first product issue after the initiation of an mtp has also been tracked. of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less. another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes. only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($60-70%). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* 1 , shailesh macwan 1 , arline stein 1 , jane fischman 1 , nancy nikolis 1 , matthew bank 1 , lennart logdberg 1 , alexander indrikovs 2 , sherry shariatmadar 1 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177a transfusion 2017 vol. 57 supplement s3 ongoing bleeding. our mtp was officially implemented in 2013 in preparation for an initial verification as a level 1 trauma center by acs. our mtp has the following packages: 1st pack has a ratio of 4:4:1 (rbcs, plasma & platelets) and subsequent packs a ratio of 6:6:1. our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march 2016 to add cryo and pcc at a defined point in the mtp (cryo is included in the 3rd pack and pcc in the 4th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had 8 patients who received >20 rbc in 2015 and 2016. mtp had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt >16 seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of 200 mg/dl (table 1) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of >20 rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective 10-year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of 63 published cases (31 reports) of ta-pls, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants. all 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the 4 reports of stem cell ta-pls, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ ta-pls, 1 actively screened for antibodies. these screens detected 5 cases of stem cell ta-pls before hemolysis became apparent and 2 cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* 1 , rosario mallari 2 , marc de asis 2 , elaine shu 3 , jonathan hughes 4 and tho pham 1,3 . 1 stanford university, 2 stanford health care, 3 stanford blood center, 4 bloodsource background/case studies: mur antigen is present in 7-10% of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. 41 year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received 1-2 rbc units every 1-2 weeks to maintain a hemoglobin (hb) level of 8 g/dl. the patient remained stable for 5 months when his hb level acutely dropped to 6.6 g/dl. the antibody screen remained negative for an additional 2 months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for 30 of the 33 rbc units he received. 3 units were from an asian donor, and a unit transfused 13 days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where asians comprise approximately 30% of the population. results/finding: 6.6% of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for 5245 of 37933 (13.8%) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over 10% of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* 1 , princess maynie 1 , carol chandler 1 , shelia garret 1 and pampee young 2 . 1 vanderbilt, 2 vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [3] [4] [5] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of 26 samples have been analyzed (table) , 17 rh and 9 non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being 2.77 (range 1-7) times higher. the average fold change for rhd/c/e antibody titers were 3.2, whereas the average fold change for non rh titers was 1.03 (range 1-2). the range for anti d titers was particularly variable, 2-7, whereas for c/e, it was 1-3. the overall reproducibility/precision of the automated analyzer was $90%. to correlate the 178a transfusion 2017 vol. 57 supplement s3 increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $3 times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of 250. units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of a1 and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with 15 min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, 32 group a, and 16 group b. results/finding: of the 100 whole blood edta samples tested, 26 (25 group o and 1group b) exceeded a high titer threshold of 250. when the pas samples of these 26 donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of 250 when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from 2 main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year 2016 was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the 27(59%) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the 2 mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house plt. the other 13 hospitals had wastage rates ranging between 9-54%. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* 1,2 , christopher j gresens 3 , anne capetillo 2 , hollie m reeves 1,2 and katharine a downes 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center, 3 bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of <0.1%. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm hg to 205/89 mm hg) followed by hematuria (500 ml). chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of 256 (igm) and 1,024 (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* 1 , leila patricia de sousa fontenele 1 , isabel nagle reis 1 , carolina bonet bub 1 , araci sakashita 1 , raffael zamper 1 , cristiane nakazawa 1 , tatiane almeida omura paula 1 , patricia silva batista 1 , marcio dias almeida 1 , fernanda loureiro de andrade orsi 2 and jose mauro kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of 671 cases of olt performed between 2011 and 2015 in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or 1,726 -95% ci: 1,147-2,597, p:0,009), absence of hcc (or 0,295 -95% ci:0,199-0,437, p:0,0), cirrhosis of any cause (or 4,161 -95% ci 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or 5,236 95%ic 2,212-12,394) and retransplantation due to primary non function of the graft (or 5,791 95%ci 1,33-4,25,206, p: 0,019) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a1/b in group o platelet products. charles k. childers* 1 , mark destree 2 , ashley rose 2 and theresa nester 3,4 . 1 madigan army medical center, 2 bloodworks northwest, 3 bloodworks nw, 4 dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a1 or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a1/b in group o platelet products is presented from a large regional blood center collected over 10-12 months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in 2 ml edta sample tubes. a single 1:150 dilution of plasma was prepared using a hamilton microlab 600 series dilutor using 2235.0 ml saline diluent and 15.0 ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a1 or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a1 cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a1 or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group o apheresis platelets will have a high titer, most commonly with anti-a1. less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate. after 10 months of testing pspp units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for pspp units. rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to 19% (from 24% on the previous day). his hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of 8 in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c3 in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of 24% while reducing the number of a rbcs in his circulation by approximately 70%. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the 2016 calendar year from 9 hospitals. an additional hospital* provided data for august-december 2016. rbc transfusions in patients <1 year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/5 70 years was also determined. results/findings: see table 1. the fraction of all transfused rbcs that were oneg ranged from 5-14% (row f). the percentage of oneg rbcs transfused to oneg patients ranged from 37-89% (row g); thus, non-oneg patients received 11-63% of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients (68%-100%; row i). overall use of oneg rbcs could have been reduced by 10%-39% if opos units had been given to all oneg patients >/ 5 70 years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* 1 , nehad mohammed 2 , marwa aly 2 and nashwa fahmy 2 . 1 national blood transfusion services, 2 nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center. they were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). 29 oncology patients, 32 chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june 2016. screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified 136 articles for data abstraction, where 48 articles were transfusion guidelines. there were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (table 1) . review b identified 3 retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or 9.4, 95% ci 5.02-17.60), although heterogeneity was high (i 2 597%). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december 2016 and february 2017 was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the 3 months, 1723 units of platelets were transfused to 238 recipients. over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69. the overall distribution of products used was 58% plasma, 24% pr, 7% pas f and 11% pas c. thirty percent of patients (n572) received all of their products on a single day. single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively. the distribution by product type was 56% plasma, 25% pr, 13% pas c and 4% pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (p5 1.00). the distribution by service was different for the groups receiving multiple units. for single units the distribution was 44% hematologic malignancy, 22% infusion clinic (nos), 13% solid tumor medicine, 11% surgery, and 9% pediatrics. for those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of 0.022. conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* 1,2 , yvemarie b.o. somsen 2 , maike e. van hezel 2 , marleen straat 2 , robin van bruggen 1 and nicole p juffermans 2 . 1 sanquin research and landsteiner laboratory, 2 academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains 220 mg of iron and 25% of the rbcs are cleared by macrophages within 1 hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in 52 icu patients who received one rbc transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il-6 levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/l, p50.69). also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/l, p50.74) and il-6 levels (35.0 vs. 25.5 pg/ml, p50.09). hepcidin levels increased in these icu patients after rbc transfusion (223 vs 332 ng/ml, p50.01). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il-6 or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016. in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016. during this time pas c, pas f and pr went from 13% to 40% of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month. the intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively. the 5 year average was 5.66. the slope of the graph for all 5 points was y5 -0.004 15.672. the two sample t-test showed that the plt/recipient/month from 2012 to 2016 was not statistically different with a p value of 0.81. conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab 17 statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 k/ul. the analysis revealed that the mean of 9.35 k/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 k/ul (p<50.001) results/findings: 123 (median 4 range [1-43]) hla matched leucoreduced irradiated sda platelets were transfused to 17 (6m/11f) patients, median age 60 years (range 27-83). 15/17 (88%) patients showing broad alloimmunization to hla class i/class ii antigens. 2/17(12%) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority 16/17 (94%) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of hsct. 63 (51%) platelets were abo identical-platelet increment median 7 k/ul (range -14 to 61), 53 (43%) were abo compatible -platelet increment median of 2k/ul (range -10 to 46) and 7(6%) were abo incompatible with platelet increments median 8k/ul ( range -10 to 32). platelet counts were performed within 24 hours in 73 (57%) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* 1 , renee leblanc 2 , dongfu xie 2 , alice cabe 1 and yanyun wu 2 . 1 overlake hospital, 2 bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. 1 these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and 68 % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than 0.25% of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a 32-year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dl (baseline 9-10 g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october 2015 to march 2017. patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were 139 rbc products transfused to 40 waiha patients. twenty-three (57.5%) patients received at least 1 incompatible unit. the mean age was 51.4 years (range 4-93 yrs) with 50% women. ethnic composition was 55% african-american, 40% caucasian, and 5% patients of mixed/other ethnicity. one hundred fourteen (82%) of these products were released as li products and 25 (18%) were compatible. ninetythree (81.6%) of the li product transfusions had a post-tfn hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn hct change of <3% (p50.0092, v 2 (1), exact methods). the mean hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the li group (p50.82, t-test, 2-tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for 12 units. units that were 31 incompatible had a lower mean post-tfn hct rise compared to all other li units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected 3% per unit more frequently than it was for waiha patients who received compatible products (81.6% vs. 56%). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first 4-hours from mt onset) was calculated with 95% and 99.8% control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were 5482 mt cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's). number of mt cases per hospital ranged from 5 to 721. patient median age was 65 years (iqr 49, 76), 62% were male and 73% required admission to intensive care. the most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit. cb that required !10 rbcs within 24-hours of mt onset occurred in 40% of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of 5 bcs and 280 ml of platelet additive solution iii (pasiii) were used to produce pcs (n55). pcs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28c for 4-7 days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of 300 mm hg was applied. using clamps, a flow velocity of 90-120 ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted 10-30%, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /l) and number (>250x10 9 /unit). simulated transfusion had no effect on the percentages of cd62p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* 1,2 , joan sevcik 2 and joseph e. kiss 1,2 . 1 university of pittsburgh, 2 blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, 2 ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for 3 patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution 1:50 is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control). results/finding: all 3 patients were ab blood type. patient 1, a 57 year old female with recurrent adamts13 deficient ttp, received 2 courses of tpe (total 12 tpe procedures) for relapse and exacerbation. ten out of 12 procedures were performed with ab and a plasma (average 916 ml of a plasma or 24% of total plasma volume for 10 tpe procedures). patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of 12 tpe procedures. four out of 12 procedures were performed with ab and a plasma (average 1210.5 ml of a plasma or 48% of total plasma volume for 4 tpe procedures). patient 3, a 33 year old female with adamts13 deficient ttp who served as a control, received a total of 10 procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between 3 patients. the trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3. all 3 patients had negative dat. only patient 3 received 2 rbc transfusions. all 3 patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to 48% of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd36 negative platelet unit is not available for a patient with anti-cd36 antibodies sameer khatri* 1 , charles harmon 1 , brian r curtis 2 and chisa yamada 1 . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd36) is one of the identified plt surface ags and deficiency is rare, but found in asians (3-11%), sub-saharan africans (7-8%) and also in some people from mediterranean descent. two types of cd36 deficiency have been described. type 1 deficiency is the complete lack of cd36 on both plts and monocyte-macrophages whereas type 2 deficiency lacks cd36 on plts with variable expression (12-99%) on monocytemacrophages. transfusing plts in a patient with cd36 deficiency is challenging given the rarity of cd36 negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd36 negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than 20 units of apheresis plt units over a 2 week period without any significant increase in plt count. cross-match compatible plt unit found in 1 of 32 units and hla matched units were tried without success. at that point, a cd36 ab was identified in the serum and the patient's type 1 cd36 deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was 95% due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd-36 negative (but blood type different and hla 187a transfusion 2017 vol. 57 supplement s3 unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd36 non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every 2 weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his hla class i pra has decreased to 37%. he is currently receiving 2 apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to 1.0 k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd36 abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct 2015 utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec 2016, 101,854 blood donations were screened for b microti by immunetics elisa. of those, 267 (0.26%) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%. no patient babesia transmission has been reported since implementing this test, but we only had 4 documented babesia ttd cases from 2007-2017. donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations. lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to 80% were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/ml of plasma, a median value of 80.5 copies/ml of plasma, ranging from 0 to 1.87x10 3 copies/ml. pre-transplant viral loads were similar. for 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/ml of plasma, a median value of 88 copies/ml of plasma, ranging from 0 to 1.18x10 5 copies/ml. post-transplant viral loads were remarkably different. for 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/ml of plasma, a median value of 1.25x10 5 copies/ml of plasma, ranging from 0 to 4.6x10 7 copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt (17) 0.2 post-ivig: all plt (41) 5.5 post-ivig: cd36-negative plt from relative (1) 0.8 post-ivig: single donor apheresis (23) 4.3 post-ivig: cross-match compatible (15) 6.1 post-ivig: flow cross-match compatible plt (2) 12.6 188a transfusion 2017 vol. 57 supplement s3 detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv (1/2/3/4), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of 1 tcid 50 /ml for denv-1, denv-3 and chikv and of 10 tcid 50 /ml for denv-2, denv-4 and zikv. finally, the first results obtained on 110 denv(1), 69 zikv(1) and 50 chikv(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step. pools of 4 or 8 samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $100 cfu/ml of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc 12924, were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta 3d and virtuo the organism was recovered 100% . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc 12924. however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of 4ml lrap demonstrated 100% recovery when loaded onto the virtuo and 3d ( table 1) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus 1-4, and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in 2014, a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of 150 mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as-5 rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final 200 mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on vero76 cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, >5.1 log 10 or log 10 /ml of rrv was achieved, with >5.5 log 10 or >5.2 log 10 /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; 255-325 ml), large volume (lv; 300-390 ml) and dual storage containers (ds; 300-420 ml) designed to treat platelet doses between 2.9 and 8.0x10 11 . the new triple storage (ts) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 ml, generating either 2 or 3 doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or 100% plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in 47% plasma/53% pas or 100% plasma with a final volume of $650 ml and a dose of 9-12 3 10 11 platelets. these conditions represent inactivation using the lowest amotosalen concentration (135 mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log 10 titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of 6-10 logs in respiratory secretions of mers patients, and with lower genomic titers of 4-5 logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log 10 reduction/ml (log 10 /ml) 47%plasma/ 53% pas e .coli 6.0 <-1.0 >6.0 e. cloacae 6.4 <-1.0 >6.4 k. pneumoniae 6.6 <20.1 >6.5 s. aureus 6.7 <-1.0 >6.7 blue tongue virus 4.9 <-1.0 >4.9 bovine viral diarrhea virus 4.6 <-1.0 >4.6 adenovirus-5 1 3.9 <-0.6 >3.9 100%plasma k. pneumoniae 1 6.5 -0.5 >6.5 s. aureus 1 6.2 <-0.7 >6.2 adenovirus-5 1 4.5 <-0.6 >4.5 1 n53 190a transfusion 2017 vol. 57 supplement s3 abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer. no viral replication was observed after 9 days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* 1 , jen-wei chen 1 , chi-ling chen 2 , sheng-nan lu 3 and pei-jer chen 2 . 1 department of research, head office, taiwan blood services foundation, 2 graduate institute of clinical medicine, college of medicine, national taiwan university, 3 division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since 1992) and 8-sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since 2013) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively. age-standardized prevalence and its 95% confidence interval (95% ci) were calculated with adjustment of who world standard population 2000-2025. for the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its 95% confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% ci: 14.8-15.7) to 4.0 per 1,000 (95% ci: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% ci: 25.6-28.4) to 7.7 per 1,000 (95% ci: 6.9-8.5). the agestandardized prevalence of anti-hcv was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03). a total of 1,036 hcv rna positive cases, 1.9% of them were anti-hcv negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of hcv rna was 1.8 per 1,000 (95% ci: 1.7-1.9) and 5.0 per 1,000 (95% ci: 4.3-5.7), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend<0.0001). in the incidence study, a total of 68 new hcv rna positive cases, 23.5% of them were anti-hcv negative, found from 1,202,165 donors followed for 2,415,668 person-years. the incidence of hcv rna was 2.8 per 100,000 person-years (95% ci: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37). conclusion: the prevalence of hcv infection has been dramatically decreased by 71.5% during 1999-2013. it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* 1 , sahar muhmmad 2 and dalia el dewi 2 . 1 national blood transfusion services, 2 azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is 70-12 days, hiv from 22 to 11 days, and hbv from 25-30 days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from 2012 to 2015, the total number of donor samples to be screened is 178685, the age of the donors ranged from 18 to 50 years, and they were of both sexes (m: f 5 3:1).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v2) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of 75 nat yield donations among 178685 (0.04%) seronegative donors. among these 75 nat yields cases, 53 (0.03%) were reactive for hbv, 20 (0.011%) were reactive for hcv and 2 (0.001%) were reactive for hiv-1. we stratified the age of the donors into 3 groups; group a (18 -28 years), group b (29 -39 years) and group c (40 -50 years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p 5 0.0089; with 95% confidence interval (ci) 5 0.0085 -0.0520 & p 5 0.0247; with 95% ci 5 0.0025 -0.0534 respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p 5 0.0224; with 95% ci 5 0.0032 -0.0413). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p 5 0.0335; with 95% ci 5 0.0015 -0.0352). nat-hcv; did not differ significantly between the three groups (p 5 0.3222; with 95% ci 5-0.0089 to 0.0161 between groups a and b & p 5 0.1340; with 95% ci 5 -0.0055 to 0.0270 between groups a and c & p 5 0.4277; with 95% ci 5 -0.0080 to 0.0215 between groups b and c). nat-hiv; did not also differ significantly between the three groups (p 5 0.3801; with 95% ci 5-0.0077 to 0.0077 between groups a and b & p 5 0.3172; with 95% ci 5 -0.0056 to 0.0077 between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% ci 5 0.0136 to 0.0507). nat hbv was significantly higher in males (p 5 0.002; with 95% ci5 0.0103 -0.0413), but the prevalence of either hcv or hiv did not differ significantly between males and females (p 5 0.3835; with 95% ci 5 -0.0077 -0.0145 & p 5 0.2751; with 95% ci 5 -0.0044 -0.0066; respectively). conclusion: in this study the nat yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients. hence, in effect the nat yield becomes 3 times that is, 225 in 178685. saving 225 recipients from tti out of 178685 (0.13%) is indeed very significant. results/finding: of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at 1:128 titer. the screening elia s/co of this donor was 0.2782. both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (table 2 ). in the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of 4 years of additional nat testing at blood bank, dmch, ludhiana from september 2012 to december 2016. results/finding: results 1.73% (2041 of 118021) units were initially nat reactive. these units were further tested, of which 90.98% were discriminated (70 hiv, 1051 hcv, 726 hbv and 10 co-infections). the remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated. overall, nat yield rate was one in 837, whereas virus-specific nat yield rates were one in 59,010 for hiv, one in 1873 for hcv, one in 1639 for hbv and one in 29,505 for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections. assuming 100 % component preparation it saved 423 transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu 1,2 , wei mao 3 , tao he 3 , yashan yang 1,2 , zhan gao 1,2 , chunhong zhang 3 , hongmei liao 3 , jingxing wang 1,2 and miao he* 1,2 . 1 institute of blood transfusion, chinese academy of medical sciences & peking union medical college, 2 sichuan blood safety and blood substitute international science and technology cooperation base, 3 chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from 5,000 voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a 250pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch37 human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as 1e -5 . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: 1.23 gb raw data with 2,450,046 reads were generated in the dna library. meanwhile, 1.98gb raw data with 3,967,242 reads were generated in the rna library. after cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (table 1) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table 1) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in 2015 most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of 1 february 2016 to 1 february 2017. thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method. results/finding: over the period 260 platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* 1 , germ an leparc 2 , phillip c williamson 1 , lani palmer 1 , ben reynolds 1 , maria noedel 1 and lindsey houghton 1 . 1 creative testing solutions, 2 oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february 16, 2016 recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march 1, 2016. with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february 16, 2016, the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in 2015, who reported $212 million new cases worldwide, resulting in >400,000 deaths. malaria prevalence is highest in sub-saharan africa, home to 90% of all infections accounting for 92% of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of 0.2 mm amustaline and 2 mm glutathione (gsh) and a 24h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to 7 days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with 5% fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching >1% parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with 7 day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from 5 to 7 days using an fda cleared rapid test (rt). in february 2016, our hospital based transfusion service implemented the use of rt on day 5, 6 and 7 to routinely extend ap shelf life to 7 days. prior to this, we tested aps by rt on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets. study design/methods: data were obtained for two 12-month study periods: october 2014-september 2015 (pre-implementation) and february 2016-january 2017 (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table 1 . the number of ap transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91). the number of rts performed increased by 130%. the percentage of transfused units tested at least once by rt prior to transfusion increased by 21% (p<0.0001). the outdate rate decreased from 5% to 2% (p<0.0001). ad-hoc ordering decreased from 21% to 9% (p<0.0001). conclusion: use of an approved rt for routine ap outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* 1 , vito scalia 1 , carla osiowy 2 , michael carpenter 2 , anton andonov 2 and margaret fearon 1 . 1 canadian blood services, 2 public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in 2011 the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of 6 units. hcv nat was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march 2016 all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at -20 o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the 5' ntr-e1 and ns5b regions. sanger sequencing of these regions represents approximately 15% of the genome. results/findings: all confirmed positive donations were whole blood donations. there were 42 hbv positive donations. of these, 37 had tested hbv nat positive. genotypes were 8 type a, 6 b, 4 c, 17 d and 2 e. there were 5 samples hbv nat negative but hbsag positive (2 were anti-hbc reactive). of these, 4 could not be sequenced and one was genotype a (also anti-hbc reactive). there were 30 samples considered hcv positive. of these, 17 samples were hcv nat positive. genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a. there were also 13 samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first 8 months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert 3d microbial detection system (bta 3d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert 3d (bta 3d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels (1-20 cfu/ml) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta 3d and the other into virtuo and incubated until signaled positive by the instruments or for up to 7 days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally 98 bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august 2013. this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a 100% voluntary donor program since 2011 and is the only center in the country that has achieved this goal. results/findings: a total of 264,343blood donations were studied from august 2013 to december 2016. in the rbdc, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for hiv (ic95% 8-34:100,000); 14 per 100,000 for hbv (ic95% 7-29:100,000) and 18 per 100,000 for hcv (ic95% 8-34:100,000). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for hiv (ic95% 77-103:100,000); 70 per 100,000 for hbv (ic95% 59-83:100,000) and 78 per 100,000 for hcv (ic95% 66-91:100,000). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of 1: 66,086 for hbv; 1: 132,172 for hiv and 1: 264,343 for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s201 system. hbv s region was also sequenced. results/finding: 234 obi were found in the 230,000 donations. in the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20iu/ml. the mean viral loads was 1.85 6 0.41 (log10) iu/ml in other 87 samples,while the mean viral loads with hbsag1/hbv dna1 samples was 2.38 6 0.83 (log10) iu/ml. 60 samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype (69.0%) and the other was hbv c genotype(31.0%). compared the samples with hbsag1/hbv dna1 ,we found two obi samples carrying with 318t>c mutation, which could cause an amino acid s55f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag1/hbv dna1, and some unique variation was identified in the obi individuals. 195a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat 15 th days, 1month, 3 months& 6 months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may 2015, we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may 2015 to dec 2016 was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, 68547 seronegative donations were screened by nat and a total of 20 hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections. most of the hbv dna cases (about 80.0%) reached senior high school education. the average hbsag dna positive rate was 0.029% (20/ 68547). incidence among apheresis donors in this period for hbsag dna were 2.91/10000. these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf24 with 5 day stability at 228c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control (225 610 ml) and test components (625 ml 625 ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within 8 hr and wbd pf24 within 24 hr. cryo was manufactured according to site sops and frozen at -308c (test 62 6 2 ml, control 22 6 2 ml ). test and control cryos were thawed at 378c, and characterized immediately post thaw (t50), and after 5 d storage at 228c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over 5d at 228c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through 5d storage at 228c. conclusion: pr cryo can be processed from 3 plasma sources, including pf24, and stored at rt for 5 days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , lynne fleischmann 1 , mirjana sarac 3 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics, 3 abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects 6-8 million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over 20 days using a panel of positive and negative samples. sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples. results/finding: precision was 7% cv or less for positive samples over 20 days. the overall specificity in a blood donor population was 99.99% (7620/ 7621). sensitivity was 100.00% for 407 presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , lynn martin 1 , daniela kaleve 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over 20 days. sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the who standard, 23 hbsag mutants, and 94 hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.98% (7998/8000). sensitivity was 100% for 511 presumed positive samples. sensitivity was 100% for all genotypes. 100% of the mutants were detected vs 83% for the comparator assay. seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the alinity s assay and 154 reactive samples detected by the comparator assay. analytical sensitivity ranged from 0.015 to 0.016 iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including 3 hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , kevin callear 1 , susan sullivan 1 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types 1 and 2 (anti-hiv-1/2). blood centers require very high throughput anti-hiv-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen was evaluated on the abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv-1, hiv-2 and hiv group o antibodies and hiv-1 p24 antigen. seroconversion sensitivity was evaluated with 41 commercial seroconversion panels. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.96% (8082/8085). sensitivity was 100% for 813 presumed antibody positive samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 135 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , joyce siregar 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who 1st international standard. seroconversion sensitivity was evaluated using 10 commercial seroconversion panels. results/finding: precision was less than 6% cv for positive samples over 20 days. the blood donor specificity was 99.93% (6946/6951). sensitivity was 100% for 500 samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from 0.57 to 0.62 iu/ml. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 134 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* 1 , anton vanweert 2 , ed bakker 2 , mark paradowski 1 , jane bryant 1 , tuan bui 1 , joyce siregar 1 , george chen 1 , george schlauder 3 and gregg williams 1 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over 20 days using htlv i and htlv ii positive samples. specificity was evaluated using 8,001 blood donor specimens from europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 500 preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot 2.4. results/finding: imprecision was less than 7.0% for positive samples over 20 days. clinical sensitivity was 100.00% (500/500) on preselected htlv i and htlv ii positive samples. the specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez 1 , michel garcia* 2 , fernando palomino 3 and guillermo orjuela-falla 1 . 1 national blood bank colombian red cross, 2 universidad del rosario, 3 fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, 387 anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than 1.0; abbott architect i2000sr) underwent supplemental testing by immunoblot (either chiron riba hcv 3.0 sia or hcv blot 3.0 test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: 1-4.99, 5-9.99, >10. band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%). in samples with indeterminate results, ns3_2 was the most frequent band detected (52,9%). in contrast, the most frequent band in the group of positive results was core (93,2%). only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group (5-9.99) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (>5). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns3_1 and ns3_2 cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* 1 , ivanka mihaljevic 2 , manuela miletic 2 , miljana stojic vidovic 2 , irena jukic 2 , jane bryant 1 , mark paradowski 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 croatian institute of transfusion medicine, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over 20 days using positive samples. specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 514 preselected positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than 6.0% cv for positive samples over 20 days. clinical sensitivity was 100.00% (514/514) on preselected syphilis positive samples. the specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* 1 , ed bakker 2 , anton vanweert 2 , jane bryant 1 , mark paradowski 1 , tuan bui 1 , lynn martin 1 , george chen 1 , gregg williams 1 and george schlauder 3 . 1 abbott laboratories, 2 sanguin diagnostics, 3 background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over 20 days evaluating positive samples. sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels. specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than 7.0% cv for positive samples over 20 days. overall clinical sensitivity was 100% on 501 preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying 5 more bleeds than the comparator assay. the specificity was 99.99% (8,111/8,112) for blood donor specimens and 98. 98% (194/196) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of 2015, was declared as the public health emergency of international concern by who in feb 2016. in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where 2.8% of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, 73.8% of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately 1:5 to 1:6. thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of 1lm and assayed after illumination with visible light from both sides for 5, 15, and 30min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was 4.5 log 50% tissue culture infectious dose (tcid 50 )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial 5min of treatment whereby ct-value jumped from 18.25 (control) to 25.50 (mbpt for 5min) (table 1) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* 1 , margaret fearon 2 , susan l stramer 3 , megan l nguyen 3 , france bernier 1 , sheila o'brien 2 , vito scalia 2 , sakina smith 3 , yves gr egoire 1 and boris hogema 4 . 1 h ema-qu ebec, 2 canadian blood services, 3 american red cross, 4 sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in 14,000 canadian blood donors in 2013. in a subset of 4,000 donor samples the seroprevalence was 5.9%. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of 250 iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately 50,000 canadian whole blood donors including 30,000 from canadian blood services (cbs) and 20,000 from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all 199a transfusion 2017 vol. 57 supplement s3 donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test (95% lod 18.6 iu/ml, 95% ci 15.9-25.6) for use on the cobas v r 6800/8800 system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed. recipients will be traced in the event of any products transfused in the previous 6 months. results/finding: as of april 10, 2017, 9 of 39,834 (19,395 cbs, 20,439 hq) tested samples with valid results have been found hev-nat reactive: 8 donors have been confirmed by further testing to date. confirmation is pending in 1 donor. of the 9 donors, 7 were from quebec, and one each from nova scotia and alberta (7 male, 2 female). ages ranged from 21 to 70 years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water. one donor had no identifiable risk factor. viral loads ranged from 3 to 151 iu/ml, of which 2 were <10, 3 were 10-50, and 3 were >50 iu/ml; 2 were anti-hev igm positive and 4 anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around 1/4400. the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august 2014. both thin and thick glass stained blood smears of 417 blood samples with giemsa was examined using microscope. results/finding: of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p>0.05), majority of the blood donors that tested positive belonged to blood group o (64.71%). the prevalence of malaria parasite in the study was 4.1%. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert 3d (bta 3d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life (3, 4 and 5 days after collection). study design/method: pooled lrap were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection. the seeded lrap were inoculated into bpa and bpn bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each. seeded bottles were loaded into a virtuo and a bta 3d and incubated until declared positive or negative (up to 7 days). additionally, bpa and bpn bottles inoculated with 4 ml of unseeded lrap were tested on the virtuo and the bta 3d (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of 99.9% for the virtuo and 99.5% for the bta 3d. the virtuo demonstrated an average improved ttd of 3.2 hours, when compared to the bta 3d in the presence of 4 ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within 5 day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for 120 days barring continued zikv testing and nonreactive results. a total of 2,485 donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus 17d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn 1 , felicia santa maria 1 , yvette girard 1 , peter bringmann 1 , marion lanteri* 2 and adonis stassinopoulos 2 . 1 microbiology department, cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in 2015. the rapidly increasing number of infections in brazil, with hundreds of fatalities since december 2016, is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a 2 weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate 17d-yfv using amotosalen (s-59) and uva light prt of platelet components (pc). study design/method: pc in 65%pas (n53) or 100% plasma (n51) were spiked with high titers of 17d-yfv and treated with s-59/uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero76 cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were 4.71 6 0.7 log 10 pfu/ml for pc in 65% plasma and 5.19 log 10 pfu/ml for pc in 100% plasma while titers in post-prt samples were <-0.7 6 0.0 log 10 pfu/ml for pc in 65% plasma and <-0.7 log 10 pfu/ml for pc in 100% plasma. inactivation to the limit of detection of >5.41 6 0.7 log 10 or inactivation of >4.71 6 0.7 log 10 pfu/ml was achieved for pc in 65% plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately 1000 ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples (1 hiv-1 antibody and 1 hiv-1 p24 antigen, and 1 htlv-i antibody and 1 htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were 0.00 for hcv, hbc, syphilis, cmv igg, and chagas, 0.01 for hiv ag/ab and htlv i/ii, and 0.03 for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for hiv ag/ab combo; 0.90% (htlv i antibody sample) and 0.32% (htlv ii antibody sample); -1.43% for anti-hcv, -2.52% for chagas, -0.71% for hbsag, -0.37% for anti-hbc, -1.62% for syphilis, and -0.59% for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/ml. robustness of the abbott prism methods to biotin interference c fischer 1 , r schneider 2 , w leonard 2 , m cobb 3 , g schlauder 3 , g williams 3 , m zuske 2 m janulis* 2 . 1 transfusion medicine, abbott diagnostics, wiesbaden, germany, 2 add diagnostics, 3 transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (cfus)/bag (i.e. 0.03 to 0.3 cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 cfu/ml over the 5 days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day 2 after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, 24 hours (day 1) after collection a sampling volume of spiked platelets (0.1-1 cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of 25 bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate (7.5%). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by 2030 in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann 1 , frank tolksdorf 2 , wiebke handke 1 , thomas h. m€ uller 1 and axel seltsam* 1 . 1 german red cross blood service nstob, 2 maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from 5 buffy coats using the additive solution ssp1 (macopharma) with a residual plasma content of 35%. for inactivation kinetics, pcs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 cfu/ml. bacteria were allowed to grow for 6 h in the pcs at 22 6 28c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log 10 reduction factors ranged from 6 to 7 for enterobacter cloacae (6.3 6 0.6, pei-b-p-43), pseudomonas fluorescens (7.1 6 0.4, pei-b-p-77), staphylococcus aureus (6.6 6 0.4, pei-b-p-63), and streptococcus bovis (7.0 6 0.3, pei-b-p-61). pcs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12). treated pcs remained sterile during storage for 7 days, while bacteria in non-treated pcs grew to high titers of 10 6 -10 8 cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of 7 days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july 1, 2008 to july 30, 2013 was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version 16 software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than 0.05 were considered significant. results/finding: a total of 173, 207 consecutive blood donors were screened between 2008 and 2013. the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively. the hiv-hbv co-infection was higher among blood donors 135(41.79%) followed by hbv-hcv co-infection whish accounts about 103(31.89%). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of 26-35. in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a 14 month old infant patricia davenport* 1 , geeta paranjape 1 and laurie sutor 1,2 . 1 carter bloodcare, 2 ut southwestern medical center background/case studies: in 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ecmo). over this time 113 blood products were transfused. about 10 days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified 27 donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed 3 years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october 2014 and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an 81 y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of 13 units of red blood cells. approximately 4 weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 20's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of 1:256. the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* 1 , andrea j linscott 2 and donny dumani 3 . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from 2011-2015, 8% of 173 transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day 1 of hospitalization showed no growth after five days. on day 3, the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the 318 ml unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient. all 26 ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was 37.18c. within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38c and subsequently reached a maximum of 39.58c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at 4-68c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus 3 standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run. inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively. the hbsag assay detection limit was 0.065 iu/ ml for source plasma and 0.120 iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than 5% for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than 14%. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all 6 donors. denv antibodies were negative in 9 donations and equivocal in 1. our initial reactive rate is higher than that reported to date for the procleix zikv tma of 1 per 23, 342 [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since 2014. over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june 2016, bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, 300 blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation #1: updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation #2: implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors. on average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year. if both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average likert scale rating of 4.1 (very good). 89% (63/71) of hybrid group students improved their post-test scores and had an average likert scale rating of 4.2 (very good). 89% (90/101) of online only students improved their post-test scores and had an average likert scale rating of 3.0 (good). the average changes in scores were statistically significant within all training groups (p value < 0.0001). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in 2016. all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july 2016 through january 2017 were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $68,000 of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* 1 , david lancaster 2 , dianne geary 2 , robert scott 1 , anh thu nguyen 1 , adam garcia 2 , raina shankar 1 , leslie buchanan 1 and tho pham 2 . 1 stanford health care, 2 stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (t:s), (2) the number of products ordered as stat, and (3) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. 2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period. these differences did not reach statistical significance (p50.28). we also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28). an estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to 0.175 fte and $18,200 per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over 360 hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* 1 , yulia lin 2 , troy thompson 1 , allison collins 1 and sheena scheuermann 1 . 1 ontario regional blood coordinating network, 2 sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the ptqip and 43% of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october 2016 a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in 2017. publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* 1 , rana hajjeh 1 and cees th. smit sibinga 2 . 1 world health organization regional office for the eastern mediterranean, 2 international quality management (iqm) consulting background/case studies: more than 76 million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may 2016 in tunisia. results/finding: we found 24 publications on disaster from five countries in the region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries (54.5%) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. (63, 85, and 108 for 2014-2016) . the number of collections per registered trt donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016. conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016. it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of 2010. after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march 2017 to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a 77% response rate (n533). of these, 78.8% have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. 87.9% of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although 93.9% of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only 48% of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs1 to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in 2015, apheresis red cells (arc) represented 4.7 % of total red cell collections at our center. hae mcs1 ln8150 was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff. validation of the 12 alyx began 06/01/16 and took approximately 45 days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs1 machines were removed from service 07/08/16. alyx go-live occurred 07/13/16. additional operator training continued through september 2016. results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs1 and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $19.21 each providing an estimated annual savings of $239,000. conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october 2016-march 2017. background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at 125g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! 130 g/l) and for females (minimum interdonation interval increased from 56 to 84 days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october 2016, changes in rebooking of donation appointments in december 2016 and culminating with eprogesa criteria changes on march 5, 2017. both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by 100. percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were 12.6% in september, 12.0 % in october/november, and 9.9 % from december to march, 2017. conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to 130 g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories. whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%). less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%). adverse donor reactions were infrequent (0.33% of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in 100% plasma. the corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of 1867 x 10 3 / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* 1 and steve cihura 2 . 1 bonfils blood center, 2 bbc / bsi background/case studies: in 2012, the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective hgb/hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested 50 donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february 2013 with a targeted implementation date of july 2013. after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately 15%. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015. during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in 2016, the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may 2016. conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* 1 , ayumi araki 1 , hiromi sanyoshi 1 , hiromi kanai 1 , hiroya kikuchi 2 , katsushi tsukada 2 and kazuhide mure 2 . 1 hokkaido red cross blood center, 2 japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from 354 individuals who donated platelets during the 6-month period between february and august 2015, and data from the 30 donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the 30 donors in the vvr group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-vvr group. at a maximum dbf threshold of 45%, sensitivity for discriminating between vvr and non-vvr donors was 93.3% and accuracy was 94.4%. when 45% dbf was used as the alert level, alerts were issued for 44 donors, including 25 in the vvr group. therefore sensitivity for predicting vvr was 83.3% and specificity was 94.1%. mean time from alert to diagnosis in the vvr group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%. some of the vvr could not be predicted even the value of maximum dbf exceeded 45%. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* 1,2 , salam abdus 3 and shabrina shah 3 . 1 inova blood donor services, 2 inova fairfax medical campus, 3 inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of 10 scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every 4 to 8 weeks from december 2013 to march 2017. blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of 10 patients, 3 females and 7 males, who underwent a total of 178 rbcx from october 2013 to march 2017, using an average number of 7 rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for 8 patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the 178 rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component 3 ( c3) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c3 decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at 12 weeks among men and 16 weeks among women, while c3 significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women. conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women. donating 400 ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low 40% split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below 3 percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october 2015. after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september 2016 for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in 2015 and 2016, a total number 1,097 rns and 965 rns completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively. the overall mean score in 2015 was 6.24 points (range 0 to 8). the mean score in 2016 was 6.57 points (range 2 to 8). the percentage of rns having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016. table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at .05 level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f(3,260) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute powerpoint presentation on a fundamental tm topic, after which 2 other members had 2 months to review and edit. therefore, each member created 1 and reviewed 2 presentations (three total steps). during each step, members wrote 2 multiple-choice questions for those particular topics. in the end, each topic would have 6 quiz questions to assess learning. at completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: 1) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; 2) pre and post-lecture 20 question validated examination (best collaborative) to assess learning; 3) resident in-service examination trends specific to tm. results/finding: six presentations were developed as 6 of the 7 abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables 1 and 2. abo leaders pre-test data could not be obtained for institution b, and 3 trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsõ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a 10 question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017. during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than 350 healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10). nps varies between 83 and 98. according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased 2011-2016 from 8.4 to 9.0. conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* 1 , anne eder 2 , beth a. dy 1 and mary o'neill 1 . 1 american red cross, 2 georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. 2015, a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to 2,300 hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the 12 months before and after launching the educational outreach. results/finding: the web based course was completed by more than 700 participants; 117 were physicians. based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good. the blood center physicians gave over 200 presentations to hospital customers. reporting of suspected strs in 2016 increased by 23% compared to the prior year. the increased reporting came from 2 specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required 30-45 minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, 2014.) subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in 2016 by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june 2016, with a new equipment module assigned each month for the following five months. the series concluded in december 2016 with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of 3.5 on a 4-point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment. level 2: learning the average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of 1.647 and a t-stat value of 5.641. level 4: results while equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance. conclusion: our level 1 and 2 evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october 2013, a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n5151) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was 35%. of those, 96% endorse that fbc creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: 41 students of undergraduate semester 3 and 59 students of semester 4 participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with 86% of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, 12/7/16 from 9-11am. there were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, 4/20/17 from 7-11am. there were 14 attendees, including 2 regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and 11 surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) 50% as patients with fcr 50% may benefit from delaying the procedure for performance in the future. validation process included (1) a deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| 15%. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table 1 background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, 2016 with the group capacity 220a transfusion 2017 vol. 57 supplement s3 of up to 35 people the number of medical specialists who have attended the training is nearly 450. results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of 35 total technologists, 31 technologists took the pre esd survey and 25 technologists took the one year post esd implementation survey. table 1 shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were 14 unplanned sop deviations; in the year after esd implementation there were only 5 deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from 0600 to 1600. the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in 100% plasma is broken up in to two days. on day 0 platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for 12-24 hours. on day 1 products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day 0 and day 1. in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: 13 of 18 employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was 21. for the two month training period the daily average rose to 23. conclusion: our "flip the switch" training plan for implementing prt platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly 100% of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as 90% of an activity. in 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks. in 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a 2016 cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter 2015 (q4/15) with last quarter 2016 (q4/16). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q4/15 1756 blood bags were used compared to 1998 in q4/16, demonstrating an activity "13.78%. price negotiation resulted in 12.58% readjustment. both indicated an estimated cost "28.10% with a possible impact of over us$ 35,000. we have identified a real cost #2.31% in q4/16, representing an overall #14.89% and us$ 3,716.72 (r$12,235.16) savings. conclusion: economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih-1000 tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17, statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in 2006. in the 2015-2018 nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on 60% individual and 40% for the other four levels (10% for each). the bonus (%) is calculated based on the iis as follows; category a: 100% (iis >575%), category b: 75% (iis: 50 -<75%), category c: 50% (iis: 25-<50%) and category d: 0% (iis < 25%). on the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%. the iis were 76% to 81%. the number of staff in each bonus categories were 11, 92% (category a) and 1, 8% (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* 1 , deborah r fludd 1 and sandra j nance 2 . background/case studies: rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in 2016, there were 65,801 active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the 6398 (9.7% of total active donors) returned contact cards alerting ardp of changes in calendar year 2016, 355 (5.5%) were donors moving from one ardp facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes. other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ardp. in 2016, 5390 new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted. conclusion: with nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* 1 , barbara j bachman 2 , mike leamy 2 , susan olson 2 and candace williams 2 . 1 university of florida college of medicine, 2 bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih-1000 (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two (22) runs of one to six (6) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing 153,000 types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. regardless of quality or speed metrics evaluated, the ih-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < 0.001). ih-1000 process steps and time studies addressed in the table below did not account for the ih-1000 reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih-1000 (77% reduction, a difference of 43 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* 1 , kimberly monnin 1 , barbara j bachman 2 , kyla warren 2 , susan olson 2 and candace williams 2 . 1 clinical pathology labs, 2 bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih-1000 tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately 211,500 type & screens (t&s). the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < 0.001). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih-1000 (difference of 120 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih-1000 tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih-1000 and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih-1000 with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17. statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/findings: using automated result verification, only 0.93% out of 6,339 samples evaluated for abo/rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for abo/rh testing. for 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past 2 years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by 1% annually and peaked at 16% in mid 2016. to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an 8week period in late 2016. hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than 10 day shelf life remaining. units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was 100% of the 56 hospitals surveyed. mean percent o neg rbc gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively. mean percent o neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior. during the 3-month survey period o neg rbc monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior. for the 3 months after the survey the average o neg rbc return rate further increased to 28.9% while mean percent o neg rbc net sales trended slightly upward to 14.1%. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* 1 , jeremie gachelin 1 , veronique ollivier 2 , thibaut mutin 1 , xavier telot 1 , benoit ho tin noe 2 and sandra sanfilippo 1 . 1 aenitis technologies, 2 hôpital bichat, inserm u1148 background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from 14 donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in 2005, orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in 2007, when we moved from this equipment to atreus 2c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in 2011 we changed to atreus 3c (terumo bct) and finally in 2013, we moved to reveos system (terumo bct). since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in 2009, bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during 2008 and 2016. conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about 30% of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november 2015, the hb has significantly improved the turnaround time of rbc issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately 750 rbc per month out of the window for non-surgical patients. this has been reduced to approximately 300 rbc per month, a 60% average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the 131 st rescue squadron (131 rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the 131 rqs master sergeant in january 2016. we asked what 131 rqs's order and delivery expectations were. he said sporadic use and the blood order would be 2 rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the 131 rqs needs. staff was trained based on data from january 2016 meeting. we contacted the 131 rqs in september 2016 to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the 131 rqs association with a civilian blood center. based on his field experiences, he changed the blood order from 2 to 6 rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and 131 rqs performed a mock run on october 31, and we felt prepared for any future events. results/finding: on november 11, 2016, the 131 rqs was deployed to a civilian aeromedical evacuation. we anticipated a 6 rbc order. the actual order was 7 rbcs and 4 ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the 131 rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at 4:30am by the 131 rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november 28 th , we established a maximum blood order of 10 rbc and 4 liquid plasma, noting future orders may request fewer products, yet meet the preferred 2 rbc; 1 plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table 1 ). the highest mean fib concentration was 535 mg/donor unit; lowest mean fib concentration was 264 mg/donor unit. all sites had a mean fib concentration at least 100 mg/donor unit above the fda minimum requirement of 150 mg/donor unit. fifteen of 17 blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; 12 manufactured pooled donor cryoprecipitate. most froze plasma in a -188c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than 10 hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of 345 mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached >68c in 75 minutes as shown in table 1 . the control thermometer recorded temperatures maintained at 1-68c for the entire 12 hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68c for more than 45 minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: 100% conventional (c-pc), 100% pr-pc, and mix of 75% c-pc/25% pr-pc. the model predicts a modest ($4%) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january 2011. a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january 2015. a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in 2016, zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within 4 weeks (sept. phase 1) and nationwide within 12 weeks (nov. phase 2). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools (16-donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab 1 had 86% and lab 2 had 77% of clients requiring universal zkv testing. we evaluated a 12-month test result upload performance period to determine the impact of zkv test implementation. results/finding: during 2016, lab 1 upload time performance ranged from 92% to 94.2% from january to july; upload time performance fell between august through november, returning to 94.2% performance in december. lab 2 upload time performance ranged from 91.4% to 95.3% january to august. performance fell september through december 83.3% -88.5%. lab 1 experienced a low of 75% upload time performance during phase 1 when there was a rapid implementation; 69% clients required zkv nat. improved performance was observed during phase 2, with a 16% increase in zkv clients. for lab 2: phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of clients implementing zkv nat. performance was 87.1% in phase 2, when an additional 43.3% of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* 1 and colleen a. aronson 2 . 1 advocate lutheran general hospital, 2 acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services. the 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the 6 sites which implemented the verax pgd test perform testing on all day 4 and day 5 platelets to be issued for transfusion. this abstract summarizes the data collected for the first 5 weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice. inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets. in march that number dropped to 29.9%. it is expected that this number will level off at some percentage at or below 29.0% with further data collection. in february 25.9% of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, 5 of the 6 sites performing testing are level 1 trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from 400 ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n57). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd62p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of hsr was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 . the ph, hsr, and the cd62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%. there is significant change for wbc after filtering (p<0.01). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1. conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb 18469-2012) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of 500ml and 1000ml. two users then evaluated the ss tubing segment types with 500ml or 1000ml samples for a total of 10 data points. samples were collected into the attached 1ml or 3ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of 10 ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by 22%. the platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 fte to 6.2 fte, lowering fte by 11%. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* 1 , jaclyn mckay 1 , jennifer curnes 1 and rowena punzalan 1,2 . 1 bloodcenter of wisconsin, 2 children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a 3 month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: (1) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, (2) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection. this tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion). in particular, during the 4 th -quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the 2017-the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after 7-day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from 5 to 7 days in the usa using an fda cleared rapid test (rt). in august 2016, our hospital based transfusion service began using a rt on day 6 and 7 to routinely extend ap shelf life to 7 days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-day ap. study design/methods: data were obtained for two study periods: september 2015-february 2016 (pre-implementation) and september 2016-february 2017 (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from 5-day to 7-day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001). ad-hoc ordering was not statistically different between study periods (p50.10). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was 4892 units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively. the mean number of discarded rbc units of the five years of the study exceeds 50% of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below 1000 mg/dl, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including 4892 rbcs, 8546 plasma products and 24 apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dl to 1000 mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* 1,2 , rebecca ross 3 , debra mraz 3 , anne baker 3 , zenna neal 3 , melanie champion 3 and edward l. snyder 2,3 . 1 johns hopkins university school of medicine, 2 yale university, 3 yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first 4 months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, 777 pr-sdp were transfused at our hospital (out of a total of 3286 platelets transfused). after 4 months of scale-up, pr-sdp were approximately 30% of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in pr-sdp (<1 ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september 2017, the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from 10 donors was collected in k 3 edta tubes. plt concentrations were determined at the qc department using the coulter act 5 diff hematology analyzer (beckman coulter). sample tubes were stored at 20-248c and measured at 24, 48 and 72 hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd (250 ml; n58) or dpd units (500 ml; n54). plt pools were stored at 20-248c under mild agitation for seven days except for dpd, which were split in two 250-ml bags after 18 6 1 h. samples were taken on days 1 and 7. ph, po 2 and pco 2 , hypotonic shock response (hsr), extent of shape change (esc), cd62p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at 24h (247 6 32 pltx10 9 /l), 48h (247 6 27 pltx10 9 /l) and 72h (247 6 32 pltx10 9 /l) postdonation. dpd can be stored in the same collection bag for 24h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits (1141-1526 pltx10 9 / l) before splitting. on day 1, lactate and pco 2 concentrations increased, and po 2 decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march 2017. there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* 1 , colleen vincent 2 and patricia kopko 1 . 1 university of california -san diego, 2 american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment. 1 blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (bmt) ward (week 6). an e-mail communication explained the change to all physicians and nurses. in phase 1, we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase 2, we expanded usage to include the inpatient bmt ward. in phase 3, we lifted all restrictions so prp could be used throughout the health system, with the goal to reach 100% prp within 6 months. results/finding: in phase 1 (weeks 1-6), we requested 31 prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of 23 prp weekly (range 9-33), and prp constituted 44% of platelet transfusions in the cancer center. in week 2, excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase 1. in phase 2 (weeks 7-8), we formally expanded issuing of prp to include the inpatient bmt ward and requested 91 prp products weekly. our blood supplier provided an average of 57 prp weekly (range 44-69), and prp constituted 53% of platelet transfusions in the phased-in areas. in phase 3 (weeks 9-10), we began issuing prp throughout the health system. our supplier provided an average of 70 prp weekly (range 61-78), and prp constituted 43% of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises 21 hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, 2 ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a 48 hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table 1 ) resulting in a cost savings of $50k. an additional cost savings of approximately $25k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as 237a transfusion 2017 vol. 57 supplement s3 we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* 1 , james r stubbs 1 , scott a hammel 1 and manish gandhi 2 . 1 mayo clinic, 2 mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing 100% pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of 100 apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0. these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe-2100d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcl with the exception being the wbc failure with a count of 0.29. further monitoring of the wbc counts discovered a result of 0.04 which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of 0.03 will be tested on the adam to confirm a leukoreduced product. we also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0. we decided to increase the yields requiring post processing samples to include the 3.4. conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method. results/finding: the average number of platelets transfused from 9/16-12/ 16 was 18.2 u/d with a standard deviation of 5.3 u/d; the predicted amount was 13.7 u/d. the difference between the predicted amount and the number of units used was -4.5 u/d. 79% days (23d/month) were under-predicted (average: 6 u/d). 17% of days (10) were under-predicted by !10 u (average: 12 u; max: 15 u (4x)). the average number of platelets used from 1/17-4/17 was 17.5 u/d with a standard deviation of 4.4 u/d; the predicted amount was 18.3 u/d. the difference between the predicted and units used was a 10.8 u/d. 38% days (11d/ month) were under-predicted (average: 3.5 u/d). one day (1%) over this period was under-predicted !10 u (11 u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 u a 3.5 u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* 1 , maria noedel 1 , nancy haubert 1 , kenneth hudson 1 , larry morgan 1 , robert shaw 1 , tracy fickett 1 , jamie jue 1 , valerie winkelman 1 , sally caglioti 1 , german leparc 2,3 and phillip c williamson 1 . background/case studies: on 08/26/16, fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within 4 weeks; nationwide in 12 weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on 1 of 2 manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for 44 clients within 4 weeks, and an additional 21 clients within 12 weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* 1 , hala samuel boules 1 , fatemah saleh al matroud 1 , rabab hussien ali dashti 1 , hanan alawadhi 2 and reem al radwan 3 . 1 kuwait central blood bank, 2 kuwait central blood bank, 3 kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of 3000 rbcs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016. half of them (1500 units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb (60 units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is 100% efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march 2017. results/findings: a total of 339 products were tested. fifteen units (4%) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture. of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%). there were 166 platelets transfused (51%) and 158 expired after day 7 (49%). the cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436. if we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50. we averaged 40 expired platelet products per month (range 6-67) before verax testing and 26 (range 9-40) after implementation. conclusion: using verax point-of-care testing saved 166 platelet products from discard. the cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products. the average discard rate per month went from 40 to 26 after verax implementation. extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december 2016 were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for 2016 data, 129 units were investigated without the use of secure texting. of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: 1) product not released after procedure/ or when patient stabilized (42) 2) product returned outside of appropriate temperature range (40) 3) clinician unaware product was assigned (36). thus far in 2017, wastage records have identified 31 units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, 11 responses thanked the transfusion service for the information, and in 8 instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: 99.9% dmso, plas-a, 25% hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) 99.9%dmso: plas-a : 25%hsa51:2:2. plas-a and hsa are kept at room temperature (20-258c, rt) and refrigerated at 48c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point 18.58c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total 14 tests. at least 10 minutes cooling after dmso, before adding the next reagent. see table: (1) after directly adding 99.9% dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. (2) in tube, autologous plasma first, dmso next, powder-like precipitates. (3) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. (4) & (5) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at 48c. (6)&(7) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the 48c group is slightly milder/slower than rt group. so hsa should not be added first. (8)&(9) trace of hsa(<1ml) was mixed into the plas-a bag (500ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%. with an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* 1 , russell thorsen 1 , rosaline ma 1 , antonio g insigne 1 , amy decourten 1 , florence panganiban 1 , patricia mckean 1 , cyril jacquot 2 , sara bakhtary 1 and ashok nambiar 1 . 1 ucsf health, 2 children's national medical center background/case studies: plasma (ffp, pf24, pf24rt24) stored at 1-6c outdates 24 hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to 5 days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf24 for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk 1 and richard gammon* 2 . 1 oneblood, 2 oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average 2-5 minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp-215 using the low glycerol (40%) freezing method and frozen at -658c within six days of collection. thawing occurred in a 328c water bath in the following order: 7 o positive and 1 o negative on 3 january 2017; 7 o positive and 1 o negative on 7 february 2017; and 8 o negative on 22 february 2017. deglycerolization occurred on site using the acp-215 with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $240-280 and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $400 per unit to produce and distribute. drbcs have a shorter shelf life, 14 days versus the 211 days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp-215's and deglycerolize four units at a time. in january and february 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of 10 years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c8000 izekial butler* 1 , karen leighton 1 , scott jones 1 and rachel beddard 2 . 1 qualtex laboratories, 2 biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c8000 instrument. precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from 1 iu/ml to 60 iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 iu/ml. stability of samples was determined by testing samples stored at 2-8 0 c and -20 0 c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142. so, precision was acceptable since the %cv for all samples tested was 5%. the mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value. the linearity of the assay was acceptable with a r2 ! 99.0%. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 c and up to 1 month at -20 0 c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 c and stored up to one month at -20 0 c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, 2017) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april 1, 2014 to march 31, 2015. local research ethics board approval was obtained. results/finding: 40 patients received ivig for itp at smh over the study period for a total of 76 unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between 30-50 x 109/l. 6 patients received ivig for a likely diagnosis itp while 245a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. 18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg. of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to 32 staff members in early february of 2017. twenty-two technologists responded for a 69% response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* 1 , neil bangs 1 and kimberly sanford 2 . 1 vcu health system, 2 virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required. results/finding: a total of 3144 t&s orders were initiated from the ed in this time period. 2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of tsd and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of tsd. a total of 2119 (67.3%) patients required a second sample. of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of tsd and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within 24 hours of tsd amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* 1 , debra berry 1 , yunchuan delores mo 2 and gay wehrli 1 . 1 university of virginia health system, 2 children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* 1 , elizabeth halperin 2 , sharon breining 2 and mona papari 3 . 1 acl laboratories/ advocate hospitals, 2 advocate health care, 3 itxm background/case studies: a large midwest hospital system with 5 level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and 3 months of data were evaluated that contained 29 events. results/finding: there was an equal number of events that were initiated in the ed and the or (12). male patients were involved 69% of the time and 31% of time the patients expired. trauma of some type was the majority of the cause but 13.8% of the cases involved gi bleed and only 6.9% were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average 7.1 with the post hgb average of 9.7. ratios of 1:1 for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of 4:1. it was found that the rbc: plasma was 1.9:1, rbc: plt was 5.9:1 and rbc to cryo was 7.4:1. use of txa was only 24.1% and cacl was utilized in 58.6% of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* 1 , jody a barna 1 , donald e ulinski 1 and nancy m. dunbar 2 . 1 dartmouth hitchcock medical center, 2 dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july 2016, we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior 24 hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august 2015 and november 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of august 2016 and november 2016 (post-implementation). the tat for allergy alert entry for both periods is shown in table 1 . we observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067). using the new process, nearly all of the alerts were entered into the emr within 72 hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* 1 , shailesh macwan 1 , nancy nikolis 1 , arline stein 1 , janelle richardson 1 , manju bagu 1 , lennart logdberg 1 , alexander indrikovs 2 , vishesh chhibber 1 and sherry shariatmadar 1 . 1 north shore university hospital, 2 northwell health background/case studies: our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (cvs) in 2016, an increase of 117% after the healthcare system cvs integration in 2015. transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after 2 reported events in q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: 1. open discussions and collaboration between blood bank and cvs nursing teams 2. mapping the process using flowcharts for additional blood orders from cvs. 3. identify bottlenecks and brainstorm solutions. 4. a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. 5. the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. 6. follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* 1 , tracie ingle 1 and heather vaught 2 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter 12-6 and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with 8-11 o, d negative red blood cells over 30 days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series 4, anti-d series 5, a1 cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table 1 summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for 75% and 20% of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february 2014-june 2015 as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june 2015-october 2016. results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table 1) . this led to a reduction in observed overall sli (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and odr (0.9% vs 0.5%). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. (6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january 2014 and december 2016 thirty-five (35) notifications were sent to physicians using epic letters and of those, fourteen (14) responded to the epic notification and five (5) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining 21 cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* 1 , denden benabdessadek 2 , annu george 2 and alexandra jimenez 2 . 1 westchester medical center, 2 new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of 941 orders were reviewed. approximately, 30% of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (t&s) per day] blood banks 1 , respectively. our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was 30% longer and had a larger standard deviation (s.d.) than the published trial result of 32.2 6 4.5. transfusion 2017 vol. 57 supplement s3 abstract during phase i visionv r 1 performed 263 panels. during phase ii visionv r 1 performed 351 of the 361 visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* 1 , john roback 2 , ronald arkin 1 , michael bartlett 1 , robert geiger 1 and jaxk reeves 1 . 1 university of georgia, 2 emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february 2016 guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august 2016 guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over 4,000 emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a 10% target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of 317 participants responded to the survey (7.94% response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0. overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on 3 run cycles. ct was comprised of 3 metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table 1 provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc (2:50), xm (32:23), cb (13:18) and dat (10:00). by implementing the future state, an average $1.3 min. lt and vt is saved on each sample loaded for ts equating to a 73% labor reduction over the current state. a 19% improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a 59% lt reduction. on average, a 38 min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on 100 patient samples for abo/d typing and antibody screening; of which at least 10 had a positive antibody screen. out of the 10, 5 had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which 17 were d(-) and 25 were d(1). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance). all 10 samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave 100% sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over 3 months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* 1 , craig fletcher 2 and peter millward 1 . 1 beaumont hospital, 2 beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october 2014 and quality data was reviewed from august 2013 to december 2016. the collected data was then analyzed using descriptive statistical methods. results/finding: data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation. wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) . importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* 1 , lisa marie button 2 , lori scanlan-hanson 2 , karen koch 2 , janet finley 2 , deepi goyal 2 and camille van buskirk 3 . 1 mayo clinic-rochester, 2 mayo clinic, 3 mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in 2014 to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in 2013 and 2014 were 63/1187 (events/ed transfusions 2013-2014). study design/method: electronic ordering for the ed was implemented march 31 st 2015. any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg. 252a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for 2 years (april 2015 -march 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ed but not charted in the patient's medical record. in 2014, 18/536 (3.36%) transfusions were not charted. however, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option 1 (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option 2 (verbal) for verbal orders and coolers set up; and option 3 (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by 68% with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* 1 , steven zibrat 1 , geoffrey wool 2 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in 2015, blood bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was 0.006%. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september 2016. results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of 0.128%, three times the national average of 0.043%. under the new system, rejected blood bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease. implementation of the new process produced a net savings of $55.8k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. 253a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured 19 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-78 days (mean 19 days, sd 20). in the postimplementation period, we cultured 22 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-12 days (mean 7 days, sd 2; p50.0082). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* 1 , marcus holme 1 , johnathan bakst 1 , gunta musa 1 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an 80 minute turnaround time (tat). in april of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased 104%. tat analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month. average monthly tys samples performed is 2758. these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was 12/20/ 2016. results/finding: the average number of outliers decreased 61% from 57 per month to 22. further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017. conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from 0-65 days with a mean of 13.64 days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of 0-7 days with a mean of 2 days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* 1 and randy levine 2 . 1 northwell health, 2 lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%). post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated. again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* 1 , charlotte van dyke 2 , dina garza van hoose 2 and rachel beddard 1 . 1 biobridge global, 2 south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics 8150s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january 1, 2016 to april 19, 2017, 2,097 total collections were flagged for additional qc by our trima accels and haemonetics 8150 instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in 2015, the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: 1) risk assessment, 2) quality control plan and 3) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* 1 , rachel jug 1 , kimberly ingersoll 1 , nicholas bandarenko 2 , nicole guinn 3 and jessica poisson 2 . 1 duke health pathology, 2 duke university hospital, 3 duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced 6:6:1 transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july 2015-december 2016. data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* 1 , nancy nikolis 1 , linda benison 1 , ruthmire thelusca 1 , renee liberty 1 , sherry shariatmadar 1 , alexander indrikovs 2 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: our blood bank (bb) processes approximately 60,000 specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june 2016, a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed 24/7. the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of 2016. the implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from 75% to 96%, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* 1,2 , morgan rockwell 2 , joseph hagan 1 , jun teruya 1,2 and shiu-ki hui 2 . 1 texas children's hospital, 2 baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within 24 hours pre and post transfusion were evaluated. patients (0-18 years) receiving prophylactic ptx from july to december 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < 10ml/kg for patients < 35kg and one apheresis unit (au) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg. a significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001). conclusion: ptx threshold above ir for both groups were 31 ( 35 kg) and 48% (> 35 kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than 10% of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only 2.1% of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated 3220 (805x4) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, 7 columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by 29% with the communication category average rpn having the greatest reduction of 72%. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* 1 , karen king 1 and joseph sweeney 2 . 1 rhode island hospital, 2 lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level 1 trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was 80 6 32 minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different (30 minutes for the provuev r and 28 for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over 90 minutes are shown in the table. the results show a reduction in tat by 14 minutes with a 20% reduction of tat greater than 90 minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* 1,2 , lorna orengo 3 , monique scott 3 and christopher a tormey 1,2,3 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged 8.2%. reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, abstract 4) duplicate sample, and 5) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table 1) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s 201 system christopher shahan* 1 , christina dejesus 1 , mosi mccall 1 , fallon hampton 1 , tangi herring 1 , judy davis 1 , anjali patel 1 , sonya gomillion 1 and bonnie maltby 2 . 1 qualtex laboratories, 2 qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s 201 system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system. study design/method: the number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016. a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the 5 whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s 201 system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system. after counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%. patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: 265 patient charts were reviewed. 91 were excluded for bleeding and cardiovascular instability. 106/174 (60.9%) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dl vs 7.3 g/dl, p<0.0001). inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dl vs 9.4 g/dl, p<0.0001). moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/l vs 15.7 nmol/l, p50.029). 29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. 7/64 providers were responsible for 32.3% of all inappropriate transfusions. 1/68 appropriately-transfused patients experienced an fnhtr. 3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* 1 , willem martin smid 2 and ashley john duits 1 . 1 red cross blood bank foundation, 2 sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of 8 worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* 1 , dee dee cassidy 1 , jed b gorlin 1,2 and nancy l van buren 1,2 . 1 hennepin county medical center, 2 innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july 2016. data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january 2015-june 2016, and post change included august 2016-december 2016. july 2016 data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of 99 rbc/month in coolers. post change this dropped to 62 rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of 54 rbc/month were returned (55%). post change, the average rbc/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products. each rbc dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month. it was also noted that the average rbc/month transfused was 44 for baseline and 57 post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over 250 patent-applications have been filed related to "transfusion medicine" and over 400 related to "transfusion alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an 1/4 inch color cmos sensor, providing effectively 1.0 mp, a 3.6 mm lens, ir-cut, day/night minimum illumination 0.1 lux/f 1 and 808 viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a 24 hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood 260a transfusion 2017 vol. 57 supplement s3 abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h.264, video frame rate (fps) 1-30/s, refresh rate 50 hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/802.11/b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p2p is provided. typical 5v power-supply, sized 165x125x101mm and weighing 370 g. client software is required. the ir range is 10-15 m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of 10 m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a 32 gb sd-card. pan/tilt-horizontal 355 o and pan/tilt-vertical 90 o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo 2 , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than 6 3 %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to 4 participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot (30 cfu), multishot 550 cfu or highdose 10k organism preparations at a low level (< 50 cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at 36 c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < 1 days and the fungal organisms in < 2 days. the overall agreement was 99.8 % in 698 bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* 1 , diane schafer 2 , debra brown 1 , jesse cox 3 , scott koepsell 3 and sara shunkwiler 3 . 1 nebraska medicine, 2 the nebraska medical center, 3 university of nebraska medical center background/case studies: anticipating the implementation of the new (30 th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late '90s and in early 2000, intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since 2010 have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january 1, 2005 to june 30, 2016. data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: 57% were female and 7% were less than 34 weeks of gestational age. none had co-existing g6pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn (41%) and rhesus-hdn (59%). antibodies most often implicated in rh-hdn were anti-d (22/57), anti-d and anti-c (22/57) and anti-c (5/57). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg). neonates received one to four ivig administrations. table 1 shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, 14 required an et for rh-hdn and 3 for abo-hdn. forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with abo-hdn and 37 with rh-hdn. the mean number of transfusions was three (range:1 to 7). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over 90% case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, 30 coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than 1 hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, 2 georgia institute of technology ap72 reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with 2 o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add 2 type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to 1a) all adult males (am), 1b) women of non-childbearing age (wncba), and 1c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to 2a) women of childbearing age (wcba) and 2b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed 3%) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap73 saving apheresis platelets through use of verax point of care testing jennifer rhamy* 1 and rebecca wride 2 . 1 st. mary's regional blood donor center, 2 st. mary's regional medical center background/case studies: our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson 2 and jun teruya 1,2 . 1 texas children's hospital, 2 baylor college of medicine ap127 vision titers --easier or problematic? (table 1) . results/findings: post intercept, t had volumes of 261-320 ml, with 98 6 4% hemoglobin (hb) recovery. t had 10-fold less extracellular protein than c. after 35 days of storage t had higher atp and na 1 than c while lactate and hemolysis were lower. hct, ph, k 1 and glucose were equivalent between t and c on d35. d35 hemolysis for t was 0.08-0.31%, while for c it was 0.10-0.57%. t and c atp was >2mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table 1) . hematocrit (hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 ph (378c) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 total atp (mmol/g hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 k1 (mm) 1.5 6 0.3* 5.7 6 2.5 53. total tested total plts issued feb 87 121 64 48 24 185 181 mar 226 516 342 276 133 858 757 totals 352 730 471 375 174 1201 table: 2. resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 dpmo to 6.3 per day or 3,539 dpmo. this is a statistically significant difference since the p-value calculated was 0.002. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately 4,400 specimens per month. since 1998, the requirement of having a second blood type on record was met by:1. utilizing the historical blood type and the current specimen, or 2. having second type performed on same specimen by different technologist, and 3. each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards 29 th edition, #5.16.2.2 a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april 2016, the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding:1. there was a minor adjustment period with more phone calls made to blood bank to explain the process. 2. there was minimal impact on turn around times for release of components. 3. aborh retype workload decreased from 1500 to 950 (35% to 20% of t&s volume) per month. 4. unnecessary blood draws minimized, improving patient experience. 5. no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table 1 demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%). the cost analysis is summarized in table 2 . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated 41% decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after 23 samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc.15 samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another 8 samples were measured with fc in ucc maribor. results/finding: 15 samples (6 rbc, 3 plt, 3 ffp-all leucocyte depleted and 3 non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from 44,10 -173,21; plt from 86,60 -100,00; ffp from 86,60 -173,21; and for non-leucocyte depleted ffp from 0,69 -8,85 (table 1) . 8 samples (2 rbc, 2 plt, 2 ffp -all leucocyte depleted and 2 nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from 91, 56; plt from 78, 21, ffp from 66, 21 ; and for non-leucocyte depleted ffp from 11,17 -15,91 (table 2) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january 2017 we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control. key: cord-010092-uftc8inx authors: nan title: abstract of 29th regional congress of the isbt date: 2019-06-07 journal: vox sang doi: 10.1111/vox.12792 sha: doc_id: 10092 cord_uid: uftc8inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner 0 s fate illustrates the brain drain beginning in the early 30 s escalating in 1939 with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early 80 s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in 1995. as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu28. levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of 3% r&d intensity was first met in 2014 and is 2018 the sixth highest among oecd countries and the second highest in the eu28. austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon 2020 and the preceding 7th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd19 targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd19 directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd19car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about 30% of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to 75% of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in 4% of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il-1rap is expressed by the leukemic but not by the normal cd34 + /cd38-hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il-1rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il-1rap mab (#a3c3 clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with 3rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp9 (inducible caspase 9) and a monitoring/selection cell surface marker δcd19. we demonstrated in-vitro and in an in-vivo xenograft murine model that il-1rap car t cells can be activated in the presence of il-1rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a 4-year period does not affect transduction efficiency of cml patient t-cells by il-1rap car vector and that autologous cart-cells are able to target il-1rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il-1rap expression on a tissue macroarray comprising 30 normal human tissues (3 donors) , with #a3c3, detected various il-1rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a3c3 flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il-1rap. as expected, monocyte subpopulation is targeted by autologous il-1rap cart cells (ratio e: t = 1:1), but at a lower level that il-1rap cml cell line. in-vivo investigation of specific toxicities of autologous il-1rap cart-cells against hsc and/or immune cells on a human-cd34 + cord blood cell engrafted/nog murine model, but also by an in-vitro cd34 + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd34 + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp9/ rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than 90% of cart-cells, after exposure to ap1903. in conclusion, based on cml model, we demonstrated that il-1rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from 20 to 30% for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about 20% when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of 5% for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of 2 h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within 15 min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at 4°c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an 1/1/1 ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the 6th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and 24 h organ failure. this trial will be recruiting 200 patients in 6 french trauma centers and is planned to be initiated second half of 2019. local/neighbours day: innovation in germany 1c-03-01 mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct1: eudract number: 2011-005441-13; ortho-ct2: eudract number: 2012-002010-39; maxillo1: eudract number: 2012-003139-50) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. 2018 introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using 50 ml stirred flasks. cells were differentiated into mks using tpo, scf and il-3 in apel2 medium for a period of 22 days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il-2rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an 8-fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of 18.7 ae 6.8 9 10 7 in microcarrier-assisted bioreactors in comparison to 4.9 ae 1.3 9 10 7 mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il-2rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than 12 years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september 30, 2016 . it has travelled billions of kilometers, just to study a small (4 km diameter), black boulder named 67p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd19 expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. 1d-04-03 cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb2) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from 450-1200 ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between 3-28 days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation (450/470 ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard 450 ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline 28 days after blood donation, but endurance performance was normalized already after 14 days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, 4 -5 weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" (1970) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby 2004) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease 2a-02-01 immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be 29-36% and 53-72%, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be 8.4-14%, 12.5-27.7%, 3.3-3.5%, respectively, and the alloimmunization rates were 17.6%, 14.3-30%, 7.1%, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of 2-6% is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from 7% to 76%, the highest figures being established when an abo/rh1-only matching policy is implemented. in a meta-analysis of 24 publications, the overall alloimmunization rates were around 20%. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a 61% increased risk of producing additional antibodies versus 30% in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to 70% of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; 1) which antibody specificities are clinically relevant 2) which antigens are most immunogenic 3) what is the availability of specific antigen typings in the donor population 4) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over 50 years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to 15-30 min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first 72 h of life, and late thrombocytopenia, occurring after the first 72 h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet-2/matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of 50 9 10 9 /l had a significantly higher rate of death or major bleeding within 28 days after randomization than those who received platelet transfusions at a platelet-count threshold of 25 9 10 9 /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from 1% to 8%, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> 60 years or < 3 years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within 12 h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: 1) using normal pediatric values published in the nelson textbook of pediatrics; and 2) using patients as their own controls and comparing pre-and post-transfusion values with either a 10% or 20% difference threshold. we monitored for taco up to 24 h post-transfusion. a total of 136 patients were included. taco incidence varied from 1.5% to 76%, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of 96 samples. from november 2017 to january 2018, a total of 10,141 blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas 6800 platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than 5.00e+04 iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was 7.05e+03 iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence 1:1,268) , whereas 17 hev rna positive donations were identified by id-nat (incidence 1:597); all id-nat only positive donations had vl < 25 iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately 50 % higher if id-nat was used. however, vl were below 25 iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last 25 years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b 2 ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost 20 years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p1 and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p 1 and p 2 phenotypes. the system (isbt no. 003) currently includes three different antigens, p1, p k and nor. the p1 antigen was discovered already in 1927 by landsteiner and levine while p k and nor were described in 1951 and 1982, respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p1/p k carbohydrate structures. anti-p1 is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p1 have been reported to react at 37°c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p 1 k /p 2 k . the rbc of the fetus as well as of the newborn express low amounts of p1, p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p1. recently it was clarified that the same a4galtencoded galactosyltransferase synthesizes both the p1, p k and nor antigens and in addition the p 1 and p 2 phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p 1 allele in the 5'-regulatory region of a4galt, which enhance transcription of the gene. it has been debated whether the p k and p1 antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p1 antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p1, has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata1 nor klf1 represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf1 gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd44) and p1, among others. since the first description of klf1 variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf1 table of blood group alleles. other than klf1, a mutated gata1 gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata1 allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in 1995, when the disruption of a gata motif in the ackr1 gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata1 binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata1 binding to a control element located 3.7 kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata1 binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p1 antigen has been known for a long time to be determined by the a4galt gene but the molecular basis underlying the common p1/p2 phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon 2a, which showed a very good correlation with p1 antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx1 tf in the expression of p1 antigen, by selective binding to a regulatory site present in p1 but not in p2 alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p1 and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. 2c-06-03 clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of 1) guiding targeted rh prophylaxis in non-immunized rhd negative women and 2) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in 6-7 european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of 99.9%, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified 837 studies, but only 10 was eligible for full text review and 5 met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. 2d-07-02 applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in 1976. during the 1994 genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from 1995, the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs 4, 5 and 6. today, rwanda has an ambitious vision to put all 12 million citizens within 30 minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in 2016, the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in 15 -45 minutes depending on a hospital's location. the average duration was between 2 -4 hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to 25 health facilities throughout the country and are set to reach 90% of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of 3.1 hours per delivery and a total of 10,115 hours of lost time on road pick up they could instead dedicate to patient care. by march 2019, over 11,000 deliveries have been made, with 30% of those being emergency deliveries. a total of more than 19,500 blood units have been delivered. in february 2018, zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to 100 km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. 2d-07-03 scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna-1b, fcgriiib and hna-2 have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna-1a, -1b, -1c, -1d, hna-2, hna-3a, -3b, hna-4a, -4b and hna-5a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna-3a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna-1a, -1b, hna-2 and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna-1, -2, -4, -5 and hla class i antibodies are clearly detectable in the gift while hna-3 antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna-3 with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna-3, can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna-2 in the gift because the molecular reason for the hna-2-null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd177*787a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. 2d-08-02 norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa-1, -2, -3, -5 and -15), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa-4, -6 to -11 are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd109 and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the 19th international platelet immunology workshop of isbt (2018) . the investigations also include measurement of the anti-hpa-1a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa-1 typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. 2d-08-03 molecular basis of hna-2 expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen 2 (hna-2) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd177 (also known as nb1). hna-2 is absent on the neutrophil surface of 3-5% of the healthy individuals that divided the population to hna-2 positive and hna-2 null individuals. exposure of hna-2 null individuals to hna-2 positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna-2 and consequently the production of iso-antibodies. the hna-2 iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd177 on a neutrophil surface of hna-2 positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna-2 positive and negative subsets. the cd177 gene contains 9 exons encoding a protein of 437 amino acids. the lack of hna-2 (in hna-2 null individuals) is associated with the presence of a missense mutation, cd177*c.787a>t in exon 7 of cd177 gene inducing a premature stop codon in codon 263. this mutation alone or in combination with cd177*c.997delg has been introduced as the main reason for the absence of cd177 in hna-2 null individuals. a pseudogene (cd177p1) highly homologous to exons 4-9 of cd177 gene is located downstream of the cd177 gene. conversion of exon 7 of cd177p1 into cd177 gene is responsible for the generation of cd177*c.787a>t missense mutation. in addition, the heterozygosity or homozygosity of cd177*c.787a>t is accounted for regulation of hna-2 negative and positive neutrophils subpopulations. genotyping has revealed the hna-2 null individuals, heterozygous for cd177*c.787a>t mutation without cd177*c.997delg, indicating the presence of a complementary mechanism regulating cd177 expression. newly in hna-2 null individuals and individuals with atypical cd177 expression a cd177*1291g>a polymorphism in combination with cd177*787a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd177 expression on the neutrophil surface. this presentation will summarize recent findings on cd177 expression and highlights the potential genotyping methods for genetic assessment cd177 expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, 100 transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding 1) blood product ordered, 2) whether the patient experienced a reaction, and 3) the start and end times of the transfusion. for each of these fields across all 100 sampled notes, the claritynlp tool reproduced these data points with 100 percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than 1 h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and 24-h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid-1980s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:-1,2,3,4, (5),6,7. genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel*02.03/ 02.06 (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 5'and 3'-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than 1000 human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked 30 years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" 3a-s02-03 background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns1 proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at 650 nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of 10 -10 2 tcid 50 /ml for zikv and denv (serotypes 1/2/3/4) after a detection phase of around 5 min. the first results obtained on 16 zikv (+) clinical samples previously tested by commercial real-time pcr (ct < 36, altona) showed an 88 % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than 10 min (sampling + detection) instead of 2 h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during 2016, with up to~2% of blood donors reactive for zikv rna in id-nat testing at the peak in june 2016. aims: perform a serosurvey for anti-zikv igg using six panels of 500 donor specimens each collected in march 2015, at the beginning, peak and end of the 2016 epidemic, and from march 2017 and april 2018. methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns1 antigen from bio-techne to characterize zikv seroprevalence in the 6 9 500 cross-sectional sample sets (anonymized with selected demographic information). results: 500 pr donor samples collected in april 2015 were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative (3 positive, 3 equivocal) despite the high dengue virus seroprevalence (>90%) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected 1-3 months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > 99%, and an area under the curve (auc) of 0.999, demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional 5 sample sets (2500 samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in 6,636 female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, 17b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo-3am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp9 (a selective trpc6 activator). results: sex hormone intake by menstrual status was higher in premenopausal women (25.3%) than in postmenopausal women (7.4%). female hormone intake was significantly (all p < 0.0001) associated with reduced storage hemolysis in all females (0.32 ae 0.16% versus 0.35 ae 0.29% in controls), enhanced susceptibility to oxidative hemolysis (37.9 ae 9.3% versus 35.8 ae 9.9% in controls), and reduced osmotic hemolysis in postmenopausal women (23.1 ae 10.2% versus 26.8 ae 12.0% in controls). in vitro, supraphysiological levels of progesterone (10 or 20 lmol/l), but not 17b-estradiol or testosterone, inhibited spontaneous or hyp9-induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after 30 day cold storage (0.95 ae 0.18% versus 1.85 ae 0.35%, progesterone 10 lmol/l versus solvent control (dimethyl sulfoxide, 0.1%), p < 0.0001). co-incubations (2.5 h, 37°c) of rbcs in the presence of progesterone and a trpc6 activator (hyp9, 25 lmol/l) suggested that progesterone protected against hyp9-induced hemolysis (1.45 ae 0.13% and 1.01 ae 0.09% versus 2.63 ae 0.19%; hyp9 + progesterone at 10 or 20 lmol/l versus hyp9 alone, p < 0.05 by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc6 channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. 3a-s05-01 international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid-1970s, the red cross was active in the national blood programs in approximately 95% of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly 50% of all worldwide donations take place (home to only 17% of the population). who data shows that the median annual blood donation rate in high-income countries is 3.21% of the population compared to 0.46 % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last 30 years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their 2018 global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by 25 % in lebanon; vnrbds by 71% annually in kirgizstan, and from practically zero to 3'588 in south sudan. the importance of rc societies was also underlined in the 2018 published global mapping of gap, which showed that 36 (19%) of them provide level a (full blood service), 75 (39%) are level b (systematic blood donor recruitment) and 47 (25%) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ 132420/d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in 500 pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: (1) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. (2) corresponding educational materials for students and teachers were developed and distributed. (3) blood donation clubs were established in 500 pilot schools. (4) trainings were conducted for 688 personnel of moh and trc on blood donation regarding their responsibilities. (5) cascade trainings were conducted for 3218 personnel of transfusion centers and 4399 school principals in 81 provinces. (6) information seminars were delivered to 251.475 students and 15.739 teachers and family members of students during school campaigns. (7) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). (8) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. (9) media spots were produced and broadcasted 3.851 times in 33 different tv and radio channels. (10) billboard posters and brochures were prepared and distributed to 81 provinces for raising public awareness. (11) advertisements about the project and the importance of vnrd were displayed 386 times on national and local newspapers, 1.117 times on online news, and broadcasted on 6 national tv channels. (12) during the campaigns, 28.310 units of whole blood were collected in pilot schools. (13) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. (14) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to 32.07 % and 35.99 % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from 73.66% to 82.54 in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite 70% of pakistan's population being under 29 years, only 10% of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p0 checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with 3-10 walk-in donors and an average of more than 20 telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june 2019. the statistics generated since january 2019 are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to 31 patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since 01.01.2013, whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from 01.01.2013 until 01.03.2019, an extended determination of rbc antigens was performed in 352 subjects presenting for blood donation. twenty-nine rare donors (8%) were identified and included in the rare donor file: 25 fy(a-b-), 1 lu (a-b-), 1 lu(b-), 1 fy(a-b-) and s-, 1 ccddee (r'r'). overall, these 29 donors provided 105 rbc units (range . to date, all donors are still active and 14 are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately 100. -chf, resulting in a total financial effort of around 35,200.-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: 1.) avoidance of transfusion transmittable infections; 2.) quality of the blood product with a strong focus on component therapy; 3.) prevention of severe transfusion reactions; 4.) avoidance of clerical errors; 3.) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. 1.) sufficient availability of blood and proper utilisation; 2.) avoidance of transfusion transmittable infections; 3.) avoidance of clerical errors; 4.) prevention of severe transfusion reactions; 5.) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at -20°c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from 51 samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with 2654 probes covering exons of 64 genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, 51 samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > 2509, with 94% of target at a quality of q30. optimal sample plexity for a standard run was determined to be 14 samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted 99.5% of snp based red cell genotypes. script predictions were 100% concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna2 genotypes defined by cd177 could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p1pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to 14 samples can be reliably sequenced in a single run. our script correctly predicts over 99% of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type 600,000 uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > 1:20,000 identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = 7,871) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was 99.9% for 95,022 comparisons; 29 of the 89 discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across 48 antigens for which serology was available, genotyping provided a 3.6-fold increase in the number of typing results available per donor (47.9 vs 13.2). furthermore, genotyping provided data on an additional 224 clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from 3,146 patient referrals with > 3 alloantibodies between 2014 and 2018, 772 unique alloantibody profiles were identified. we found that there was a 2.6-fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the 4,721 nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from 293 to 246, representing an additional 164 patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. 3c-s06-04 biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near 1500 wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including 2 transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by 4 dna samples with targeted sequencing on illumina miseq, and specific variants were verified by 40 dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, 1) m, n: insufficient sequencing reads, 2) c, c: identical rhce exon 2 with rhd exon 2 for c allele, 3) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, 13 systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a2, has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. 8:14750, 2017) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine 27 (suppl.2) :42, 2017). here we have used it to analyse and document bel-a2 blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a2 cell-line. methods: bel-a2 cells (day 196) were cultured in expansion medium and genomic dna (gdna) was isolated from 1 9 10 6 cells on day 5. for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for 41 genes encoding all 36 known blood group systems and 12 further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a2 genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c4a/c4b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a2 did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr1, cdan1 and tmx4, these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a2 genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, 2017) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a2 blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from 16 countries were enrolled (67% in north america, 17% in europe, 7% in oceania, 5% in asia, and 4% in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = 360) differed significantly on a regional basis (p = 0.04) and ranged from 12 (8-41) x10 9 cells/l in the middle east to 45 (20-66) x10 9 cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = 0.08), nor did the tpc prior to therapeutic transfusions (n = 189) differ on either a regional (p = 0.16) or national (p = 0.57) basis. for children supported by ecls (n = 90), there were no regional (p = 0.06) or national (p = 0.40) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = 0.02) and national (0.04) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [28 (16-81) x10 9 cells/l]. for children with an underlying oncologic diagnosis (n = 233), no differences were seen in the tpc for prophylactic transfusions (n = 175) on a regional (p = 0.19) or national (p = 0.20) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional (0.86) or national (p = 0.33) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < 0.001) and national basis (p < 0.001). the median (iqr) dose based on volume ranged from 8.4 (5.0-11.2) ml/kg in north america to 12.6 (9.8-16.0) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < 0.001) and national (p < 0.001) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, 2015) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ 10 9 10 11 /l and a platelet dose of 1.1 9 10 11 per square meter (sm) of body-surface area (bsa) in inpatient and 2.2 9 10 11 /sm in outpatient setting. aims: in january 2018 we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of 1.1 9 10 11 /sm and outpatients a dose per transfusion of 2.2 9 10 11 /sm. platelets were transfused when the count was ≤ 10 9 10 11 /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january 2018 to december 2018 a total of 9839 platelet pediatric aliquots were transfused: 5534 (56.2%) were obtained from apheresis platelet concentrates and 4305 (43.8%) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots (7505-76.3%) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, 6365 aliquots were transfused in inpatient setting: 1675 (17%) in the hematology unit, 2772 (28.2%) in the oncology unit and 1918 (19.5%) in hsct unit. a total of 1140 (11.5%) aliquots were transfused in outpatient setting: 840 (8.5%) to patients affected by hematological malignancies and 300 (3%) to patients with solid tumors. five major bleeding events (who grade ≥ 3) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade 3 bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = 3, 1.2%) developed intracranial bleeding (who grade 4). the platelet count at the time of the event was 22 9 10 9 /l, 25 9 10 9 /l and 8 9 10 9 /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade 4 bleeding has been observed in inpatients setting only (1.2% vs 1% of plado trial, sj slichter, nejm, 2010) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ 3) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell 3000 elite (best theratronics, canada) at a dose of 25 gray. all rbcs were used for transfusion for 14 days from the harvest day. 70 pediatric patients with various oncological and hematological diseases were randomized to 2 groups of 35 members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. 3-5 days after the transfusion, the direct antiglobulin test (dat) was performed and after 14-21 days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = 0.2), as well as the concentration of potassium (p = 0.44) and haptoglobin (p = 0.25) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = 1). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = 0.39). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for 14 days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama 2018) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over 5 years duration from 2012 to 2016. methods: using 5 years data (2012) (2013) (2014) (2015) (2016) perioperative transfusions decreased steadily per year from 6.4% in 2012 to 5.5% (14% cumulative decline) in 2016 for children of all ages (or 0.966; 95% ci 0.957-0.976; p trend < 0.001). the cumulative change in elective procedures was 9.2% versus 27.2% decrease in urgent/emergent procedures (p trend < 0.001). summary/conclusions: in this large prospective registry study of > 350,000 children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across 5 years from 2012 through 2016 with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk 3c-s08-01 transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of 2-20% of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ 100 iu/ml and being id-nat (procleix-ultrio plus tm [95% lod: 3 iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < 6 iu/ml (n = 44) or nat repeat reactive (rr) with vl < 6 iu/ml (n = 32). french samples initially tested nat nr/nrr with procleix-ultrio (lod 95%: 11 iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: 6 iu/ml]). hbv dna was purified from 5 to 12 ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in 79% of all samples with undetectable or vl < 6 iu/ml. hbv genotypes were a1 (33.3%), a2 (18.4%), a3 (3.3%), b (3.3%), c (1.7%), d (25%), and e (15%). all samples were anti-hbc positive and 73% of ultrio-negative samples tested positive with ultrio plus. unusual 1-2 nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = 16) or deletions (n = 4) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of 30 samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. 3c-s08-03 background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since 1998, hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of 15 and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i1000 analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas 8800 platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p22cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in 69 blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, 59/69 (85.5%) donors were anti-hbc positive, and 10 (14.5%) negative. 18 donors had both anti-hbc and anti-hbe reactivities. a group of 11 young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and 9 patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g600 automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is 2 logu/ml and the lower limit of quantification (loq) is > 3 logu/ml, due to nonlinearity results between 2 and 3 logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss 19.0.0. results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: 3.0 logu/ml, range 2.0-4.9), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in 34/69 obi donors (49.3%), with a mean value of 2.21 logu/ml (range 2.0-3.20). hbcrag could be measured only in 2 obi donors (3.0 and 3.2 logu/ml), being below the loq of the test in the majority of obi (32/34). considering the presence of anti-hbc, hbcrag was detected in 29/59 (49.1%) anti-hbc+ and in 5/10 (50%) anti-hbc-obi, with no significant difference in their mean levels (2.21 ae 0.32 vs 2.14 ae 0.26; p = 0.44). interestingly, the presence of anti-hbe (18/62) was independently associated with higher hbcrag levels (2.38 ae 0.42 vs 2.14 ae 0.22; p = 0.03). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. 3c-s08-05 hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss 20.0 statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < 0.05 was considered statistically significant. results: 1. proliferation characteristics of t cells. the proliferation of cd4 + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group (3.0%, 3.3% vs. 1.7%), with significant difference (3.0% vs. 1.7%, p = 0.016, 3.3% vs 1.7%, p < 0.001). 2. the frequency of specific ifn-c secreted t cells. the response intensity of the obi group (25 sfc/10 6 pbmcs) and chb group (25 sfc/10 6 pbmcs) was higher than that of the hc group (5 sfc/10 6 pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group (64.0%). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term (15-20 years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under 10 themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = 2.11; 95% ci: 1.03-4.31). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for 72,000 participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = 150,000), were calculated for all dbds participants. 12,903 female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of 10 depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile 1 contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = 1 from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between -0.42 and 0.50 (mean 0.01). a total of 12,523 female donors were classified as "not tired" and 380 (2.9%) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: 0.00; not tired mean prs: 0.01). an age-adjusted logistic regression model found this to be insignificant (or: 0.74, 95% ci: 0.33-1.66), p = 0.47). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile 6) had or = 1.02 (p = 0.93) of being tired when compared to those in quantile 1 (or set as 1). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv-1 subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv-1 infected blood donors. methods: from 2016-2018, 177 blood donations collected from 24 blood centers, covering almost the whole of china, were confirmed as hiv-1 positive by national centers for clinical laboratories using abbott realtime hiv-1 assay or cobas taq-man hiv-1 test, version 2.0. then hiv-1 gag (973 bp, hxb2: 1074-2044), pol genes (1454 bp, hxb2: 2068-3521) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv-1 subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv-1 subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among 177 donations, gag and pr-rt regions of 160 samples were sequenced successfully. the distribution of hiv-1 genotype was as follows: crf_07bc = 66 (38.8%), crf_01ae = 58 (36.3%), b = 9 (5.6%), crf_08bc = 2 (1.3%), crf55_01b = 4 (2.5%), crf59_01b = 1 (0.6%), crf65_cpx = 1 (0.6%), crf67_01b = 2 (1.3%), crf79_0107 = 1 (0.6 %), crf85_bc = 1 (0.6 %), urf_0107 = 6 (3.8%) and urf = 9 (5.6%). 26 of 160 hiv-1 isolates were identified to have drms. there were 4 (15.4%, 4/26) protease inhibitors (pi) accessory drms, 3 pi major drms and 20 (76.9%, 20/26) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf01_ae and crf07_bc (61.5%, 16/26). 3 of 4 pi accessory drms were q58e. the pi major drms included m46l, m46i and n88s. n88s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v179d/e is main nnrti drm (70.0%, 14/20). a combination of v179d and k103r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k103n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf07_bc (38.8%), crf01_ae (36.3%). besides, other rare crfs and several urf_0107 and urfs were also found in these hiv-1 isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in 16.3% donors in the study, which may result in resistance to pis and nnrtis, especially the hiv-1 variants with n88s mutation in pr gene and k103n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv-1 in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv-1 among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in 100% plasma were biotinylated at day 1 and day 7 of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, 50 ml of pcs was washed twice and incubated with 5 mg/l biotin, dissolved in phosphate buffered saline-pas-e (1:9), for 30 min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd62p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in 65% pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, 98.4% ae 0.9% of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd62p expression was increased in biotinylated platelets 48.4% iqr(41.7-56.2%) compared to the control samples 12.3% iqr(9.5-12.7%) on day 1 of storage. however, biotinylated platelets were not more activated compared to sham samples 50% iqr(41.7-56.2%). thus only the procedural steps led to increased cd62p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day 7, a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd62p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd62p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs4 insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs4, these insulators are small-sized (119-284 bp vs 1.2 kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a1, one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il-3-dependent 32d cells, which upon transduction with oncogenic vectors become il-3-independent, leading to transformation. 32d cells were transduced with sin-lvs: the b-globin-τνs9.3.55-, the insulated b-globin-a1-tns9.3.55and the oncogenic sffv-gfp-vector. transduced cells were expanded in 10% il-3 and transduction efficiency was determined by vector copy number (vcn). transduced 32d cells were seeded in methylcellulose with 10% or 0-1% il-3 to detect the il-3-independent and potentially transformed clones. the il-3-independent clones were further expanded in 10% il-3 and infused in partially myeloablated and il-3-treated c3h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a1 insulator did not negatively affect vector titers (τνs9.3.55, a1-tns9.3.55, sffv-gfp: 2.8, 1.8, 2.5x10^8 iu/ml, respectively). 32d cells were successfully transduced with all vectors (%vcn positive colonies: 40-100%) and expanded up to 400-fold. the a1-insulator decreased the number of il-3-independent colonies by 78-93% over the uninsulated vectors. the uninsulated vector-transduced, il-3-independent colonies, were greatly expanded in culture with 10% il-3 over the a1-transduced colonies (sffv, τνs9.3.55, a1-tns9.3.55: 48, 40, 10 fold change, respectively). il-3 independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il-3-independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a1 insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a1 may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including 46 missense variants reported in rhd exons 6 and 7, was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published 3d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c.1065c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c.1154g>t (gly385val; d-negative) disrupts totally normal splicing, while c.1154g>c (gly385ala; weak d) and c.1154g>a (gly385asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a 3d model suggests that gly385asp, but not gly385ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons 6 and 7 by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in 15/46 (32.6%) variants, which correlates well with the quepasa-like prediction (sensibility = 0.93, specificity = 0.94). additionally, while normal exon inclusion is affected by c.1012c>g (weak d type 70), the associated leu338val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in 2014, participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of 6.210 compensated and 68.205 non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either 25 eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after 24, 36, 48 and 72 months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups (51.9%), refreshments (50.0%) and free (testing) laboratory parameters (38.3%) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by 28.6% and 25.3%, respectively. the lowest acceptance was for release from work (17.8%), complementary items (13.9%) and additional cash reimbursement (11.8%). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received 25 eur as a monetary reimbursement, the proportion of regular donors after 48 months (19.9%) was significantly higher than among comparable non-compensated donors (9.2%). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice (1-5 prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand 1) how deferral reasons and donor experience jointly affect donor lapse, and 2) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between 2013 and 2015 (n = 343,564). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included 25% new donors, 41% novice donors, 30% experienced donors, and 4% reactivated donors. deferral reasons included travel, hb, medical short-term (<28 days duration), medical long-term (>28 days duration), and miscellaneous. next, we interviewed 31 temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, 2012) to identify key topics and underlying themes. results: of target donations, 11% were deferred, mostly for travel (40%), medical short-term (23%), and hb (19%). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < 0.75, p's<0.001). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: 8.4 mmol/l (135 g/l) for men and 7.8 mmol/l (125 g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at 8 weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us 35% -75% of deferrals are due to low hb, especially in women (editorial, transfusion, 2012) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from 2007 to 2016. we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of 1000 new entrant donors (500 males and 500 females). prior information from the clinical literature (boulton, vox sanguinis 2004) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of 58 days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of 100 to 419 days, while the estimated recovery time for females varies between 54 to 503 days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of 23% reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, 2019) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (<30 years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: 330 ml water drink, 500 ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within 400 days after the index donation and we analysed their return for a donation attempt within 421 days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the 8199 episode participants who had received an invitation, 6538 (79.7%) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or 1.2, 95% ci 1.06-1.4 and 1.3, 1.12-1.5 respectively). return was slightly lower in women (or 0.88, ) and lower in first-time donors (or 0.86, 0.77-0.96) than after a 2 nd -4 th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or 0.47, 95% ci 0.37-0.60 and or 0.53, 0.46-0.61 respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, 79.7% returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > 50 years contributed 41% of all donations made in 2017. however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's 45 and up study baseline data collected between 2006 and 2009 to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up 22,058 active whole blood donors for 200,403 eligible person-years (average age at recruitment 56.4 years, 56.3% female, average follow up 9.1 years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the 2 years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made 1066 (95% ci 1056-1075), 987 (981-993), 900 (891-909), and 710 (690-730) donations per 1000 person-years, respectively. iron-deficiency was identified in 8.9% of donors in the study (n = 1964, 95% ci 8.5-9.3) . sixty percent of those with iron deficiency (n = 1,175, 95% ci 57.6-62.0) visited their general practitioner (gp) within 60 days of the identification of irondeficiency, and 48.4% (95% ci 45.5-51.3) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = 0.004). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting 25% of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to 30% of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp01-02 the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: 1) optimising the patient's own blood, 2) minimising blood loss and 3) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. 3d-s11-01 department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c3bc/c3d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c5 to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd163 and cd91-mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase-1 (ho-1) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho-1 has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. 3d-s11-02 understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. 3d-s11-03 cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il-12p70-producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il-12 production. in diseases characterized by local th1 inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band 3. other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd47. a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by 90%. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. 3d-s12-02 treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, 60,000 children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately 20% patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is 88% in paediatric and 65% in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss6 inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since 1987, include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with 204 national thalassemia associations in 62 countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive 2004/33/ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive 2005/61/ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to 15 days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers 95% of total blood supply. data on ttis in 3,067 patients with thalassaemia and scd-thalassaemia in 2010-2017 are analysed. results: tti prevalence in thalassaemia syndromes was: hbv 1.8% (occult type 1.3%), hcv 54%, hiv 0.3%, htlv 0.8%, wnv 0.5% and hev 3%. most frequent adverse reactions in 2015-17 were allergic (incidence 1:854), non-haemolytic febrile reactions 1:2,631, "other" 1:5,883, alloimmunisation 1:6,667, taco 1:100,013, tad 1:50,006, tt-hev 1:100,013. hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in 2010-2017 show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs10421768, rs117345431, and rs142126068 with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of 102 samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs10421768, rs117345431, and rs142126068. statistical analysis was performed on ibm*spss* statistic 23 using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.-582a>g variant (p = 0.02). for rs117345431 statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = 0.058). all samples were homozygous for allele t of rs142126068. summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to 20 years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by 2030 might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to 12 months in july 2016. since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s1. deferral of msm during the 4 months preceding the donation; s2. deferral of msm who have had more than one sexual partner in the 4 months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july 2016-december 2017 which is the baseline rr with the current 12month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤180 days) since all hiv-1 antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s1, from msm blood donors with the current deferral policy (12 months) and for s2, from monogamous msm of the general population. results: from july 2016 to december 2017, 8/28 (29%) hiv-1 positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at 0.16 in 1 million donations [95% ci: 0.04-0.34], or 1 in 6,380,000 donations. for s1, the number of additional msm donors was estimated at 733 and the number of additional hiv positive donations at 0.09, resulting in an hiv rr of 0.16 in 1 million donations [95% ci: 0.04-0.35] or 1 in 6,300,000 donations. for s2, the number of additional msm donors was estimated at 3,103 and the number of additional hiv positive donations at 4.92, resulting in an hiv rr of 0.23 in 1 million donations [95% ci: 0.05-0.56] or 1 in 4,300,000 donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by 1.5, the risk would be 1 in 6,225,000 donations for s1, and 1 in 3,000,000 for s2. summary/conclusions: for both scenarios, the hiv rr remains very low. for s1 (4-month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s2 (no more than one sexual partner, 4 months), the risk is 1.5 higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s1, since the risk could be 2 times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. 3d-s13-03 background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering 8 of 9 provinces in south africa. controls were frequency matched at a 3:1 ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < 1.5. eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the 6 months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november 2014 to january 2018, we enrolled 323 incident hiv cases and 877 controls; 202 (62.5%) cases and 544 (62%) controls were ≤ 29 years old. there were significantly more female cases 230 (71.2%) than female controls 439 (50.1%) (p < 0.0001). significant hiv risk factors (all p < 0.0001) reported within the 6-months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from 2000 to 2016, among male blood donors (mbds) found hiv-1 positive at blood donation screening, 31% did not disclose any risk factor for hiv infection during post-donation interviews, while 38% reported having sex with men (msm), and 24% and 7 % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv-1 infection in mbds, we performed an hiv-1 genetic network analysis, including hiv-1 positive mbds and patients included in the french primary hiv infection anrs co6 primo cohort (pc). methods: 284 mbds, who donated blood between 2000 and 2016, and 1340 pcs, included between 1998 and 2014, were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: <6 months). a partial transmission network was computed based on tamura-nei 93 nucleotidic distance (threshold for hiv-1 s/t b = 1.5%; for non-b s/ t = 0.8%) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv-1 strains from 81 mbds and 126 pcs were linked into 54 clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger (30 vs. 36 year-old; p < 0.01) and were more likely to have a recent infection (48% vs. 34%; p = 0.03). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < 0.001) and to have the same risk factor for hiv infection (p < 0.001) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: 38% vs. 49%, hts: 24% vs. 21%, other: 7% vs. 6% and unknown: 31 vs. 24%. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to 18% (31/176) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv-1 associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in 2002. monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in 2013 and 2015. aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every 2-3 years. results: in the uk 2002 -2017, 254 htlv-infected donors were identified. prevalence among new donors was steady around 5/100 000 donations. prevalence among repeat donors peaked in 2002 (2.7/100 000 donations), with most in previously untested. from 2004 to 2016, prevalence of 0.07 per 100,000 donations (average of one positive/year) was recorded. in 2017, prevalence among new donors increased to 9.9/100,000 donations (17 positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women (182/254, 72%), uk-born (125/254;49%) and htlv-1 infections (228/254;90%). mean age was 43 years. almost all positive donations were from previously untested donors (240/254), with seroconversion within a year of previous donation confirmed for only 5 of the 14 previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of 114 htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over 800-person years follow-up, none had developed atll or ham. summary/conclusions: over 16 years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in 2017 nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the 60°and 70°n latitude. the length of the country is 1150 km and width 550 km. by surface area it is the fifth largest country in eu. the population of the country is 5.5 million resulting in the lowest population density in eu (15.8 inhabitants/km3). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as 10-20% of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since 1948. frc bs collects annually about 200 000 whole blood units of which 50 % are collected in 10 fixed sites and 50 % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by 8 am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the 1930s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in 2007 and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice (4 courses) -pbm: general (7 courses) -pbm: medical (6 courses) -pbm: acute care and surgical (4 courses) -pbm: obstetrics and maternity (3 courses) -pbm: neonates and paediatrics (7 courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from 1 july 2007 to 31 january 2019: -489,600 people registered as learners -1,072,299 courses were completed -these learners came from 182 countries, with 11,141 (2.3%) of them from outside of australia. analysis by profession shows that: -82.4% are nurses and/or midwives -11.2% are medical -6.4% are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = 2,885) from 1 april 2015 to 31 january 2018 shows that these courses have a positive impact, with 88.7% of respondents stating they gained additional knowledge, 65.2% able to make changes to clinical practice, and 88.2% reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with 93.5% gaining knowledge, 76.4% able to change their clinical practice and 91.9% believing this will improve patient outcomes. analysis of red cell usage in australia shows that since 2012 there has been a 21.1% reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in 2012 and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. 3d-s14-03 prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a 12-month period (january 2018-december 2018). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > 50,000/ll, ≥2 doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy1) were compared to subsequent years (pgy > 1). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $500 for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < 0.05 considered statistically significant. results: there were 1446 platelet doses requiring approval with 670 (46%) routed to the pgy1 group and 776 (54%) to the pgy > 1 group. there were 847 (59%) ordered doses that were in compliance with hospital transfusion policy and 599 (41%) that were not in compliance with hospital policy. of the 847 appropriately ordered doses, the pgy1 group declined release of 7 necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > 1 group. when paged by the blood bank, pgy1 physicians approved product release not in compliance with policy for 191/670 (29%) doses while pgy > 1 physicians approved not indicated products for 79/776 (10%) of doses (p < 0.01). products not indicated by hospital policy were held from release by pgy1 physicians for 113/670 (17%) doses and 216/776 (28%) doses by pgy > 1 physicians (p < 0.01). the ordered doses not in compliance with hospital policy had an estimated cost of $299,500. of this cost, there was a calculated $164,500 savings of products not released due to prospective auditing. there was an additional potential savings of $135,000 for products not indicated but released ($95,500 from the pgy1 and $39,500 from the pgy > 1 group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > 1 group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy1 group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy1 physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. 3d-s14-04 what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in 2015 we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in 2017 and 2018 were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of 188 adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty (10.6 %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected 39 (20.7 %) events and formal internal or external reviews detected one event. seven (3.7 %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of 16 wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of 22 events related to blood ordering, two of seven errors in testing, six of 17 events where incorrect blood had been transfused, and eight of 64 events related to donor selection. in 80 reports human error was listed as the cause of the event and 27 of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average 1.2 g/l (0.9%) higher than from the venous blood samples. the range of the difference was -21 -+22 g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in 1.4 % (12/845) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + 9 g/l. 92 % of the hemoglobin results from the finger prick were in the range ae10 g/l compared to the venous hemoglobin results. 63 % of the results from the finger prick were between ae5 g/l (the precision of the device) compared to venous hemoglobin results. in 13 cases the difference between finger prick and venous measurements was outside 2 standard deviations from the mean i.e. 2.5 % from the bottom (n = 9) or top (n = 4) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about 8% (men) to 81% (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of 56 days, a donor needs to absorb 4.5 mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of 3-4 mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every 5th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < 15 lg/ l, or ≥ 15 and ≤ 30 lg/l (for 12 and 6 months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from 2020 onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. 3d-s15-02 superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (<7.8 mmol/l [<12.57 g/dl] for women and < 8.4 mmol/l [<13.54 g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since 2010 with more than 110,000 participants. extensive genotyping has been performed on approximately 72,000 dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than 150,000 icelandic individuals (an independent discovery cohort), we constructed 9 different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of 2017 was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at 5, 10, 25, 40, 60, 75, 90 , and 95 th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was 6.9 donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the 40-60 prs percentile group, donors below the 5 th percentile had lower (-0.23 hb mmol/l (95% ci: -0.25; -0.21)) and donors above the 95 th percentile higher (+0.19 hb mmol/l (95% ci: 0.18; 0.21) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the 40-60 prs percentile stratum as reference, donors below the 5 th percentile and donors above the 95 th percentile had odds ratios of deferral of or = 2.58 (95% ci: 2.27; 2.93) and or = 0.46 (95% ci: 0.39; 0.54), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, 2012) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles (58 features, 3,162 donations from 1,025 repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < 20 ng/ml for women and < 30 ng/ml for men), or absent-iron donation (ferritin < 12 ng/ml for men and women). we trained multiple machine learning models on 2,543 of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of 620 donations, which were not used to train or select the model. we then used our model to generate risk estimates for these 620 test donors as a function of days since their last donation, which varied from 56 days to 250 days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes 250 days after the last donation was lower than the risk 56 days after the last donation for most donors (risk of hemoglobin deferral decreased for 84% of donors; risk for low-iron donation decreased for 61%; and risk for absent-iron donation decreased for 94%). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, 2011) and from the danish blood donor study (rigas, transfusion, 2014) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for 15-24 month follow-up of donation frequency and iron status. a brief checklist of 8 food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of 2425 enrolled donors, 1406 were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was 7. 2, 13.1, and 22 .0 mg of heme iron weekly. these values are equivalent to approximately 3, 6, and 9 servings of beef per week, or alternately twice as many servings of chicken or pork. across 2236 follow-up visits with iron outcomes assayed, almost 33% of donor visits were associated with intermediate iron depletion (serum ferritin < 26 ng/ml) and 8.5% with complete depletion of iron stores, representing serum ferritin < 12 ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than 2fold higher risk for complete iron depletion during all follow-up visits (rr 2.40, 95% ci 1.51, 3.81, compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately 250 mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included 2,552 donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = 292). hb levels were measured in edta whole blood samples using a hematology analyzer (xt-2000, sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci8200, abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, 2,260 (1,186 female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) (95% confidence interval (95% ci)) in men and women respectively: 0.147 (0.069 to 0.225) and 0.094 (0.013 to 0.175) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (-0.015 (-0.026 to -0.004) and -0.018 (-0.032 to -0.005) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: 0.073 (0.046 to 0.107) and 0.065 (0.031 to 0.100) lg/l for heme and -0.003 (-0.008 to 0.000) and -0.007 (-0.013 to -0.002) for non-heme). more mvpa was negatively associated with hb levels in men only (-0.004 (-0.008 to -0.001)), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd20 moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. 4a-s16-02 thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple 1 , r aslam 2 , e speck 2 , j rebetz 1 and r kapur 1 1 lund university, lund, sweden 2 st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last 10 years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately 30% of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp4, amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood 2010) . methods: platelet glycoprotein (gp) iiia (cd61) knockout (ko) mice were immunized with cd61 + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra (1, 10 and 20 ug/kg) had significantly higher platelet counts by 72 h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after 2 weeks and were fully rescued to control levels after 3 weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: 112 itp sera were investigated in this study. 35 (31%) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): 3.23, range: 1.76-13.61, p = 0.0001). in addition, 23 (21%) sera caused higher ecl binding to test plts (ecl-fi: 2.31, range: 1.54-5.7, p = 0.0001). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between 2014 and 2018. the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from 64 patients (52% women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was 63 years (range 22-89 years). the mean hg level at diagnosis was 68.60 g/l. dat results were positive mostly with igg+c3d (59%) or igg (31%). most patients had warm aiha (70%). other types of aiha diagnosed were mixed aiha (15%), cad (11%), pch (1.5%) and dat negative aiha (1.5%). in 6 cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as 1st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of 80% of patients were transfused at a mean hemoglobin level of 67.88 g/l. during this period we detected 136 dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november 2017 to march 2018. completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented 42% (58/137) of english acute trusts. median number of adults with aiha diagnosed annually was 4-6. in the preceding 5 years, 31% (18/58) recalled at least one patient who had died due to aiha. although 7% (4/57) undertook a bone marrow biopsy in all patients, 93% required additional features, mainly: neoplasia, age over 60 or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, 59% (34/58) would not organise confirmatory tests, either because it was considered unnecessary (29/34), or because clinicians were unsure how to access tests (5/34). when determining aiha subtype, 29% (17/58) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with 12 considering this unnecessary and 5 unsure how to access tests. in 4 clinical scenarios of patients with aiha and dat positive to c3d ae igg ae cold associated symptoms, up to 87% (47/54) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone 1 mg/kg given before judging the patient refractory and reducing the dose was 3.5 weeks (sd 1.70, range 1-19 weeks). second line treatment of choice was rituximab for 82% (45/55) of respondents and splenectomy for 5%. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for 88% (49/56) but single agent steroid for 9%. we also explored the potential for future audit and research. 64% (37/58) of respondents were able to identify patients with aiha who previously required transfusion. 96% (55/57) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c3d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although 44% (25/57) would continue prednisolone at 1 mg/kg beyond the recommended 21 days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. 4a-s17-02 low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < 8.0 g/ dl, 5 mmol/l) vs. high-trigger (hemoglobin < 9.7 g/dl, 6 mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed (1) severe adverse transfusion reaction, (2) acute myocardial infarction, (3) stroke, (4) new-onset renal replacement therapy, (5) vascular reoperation, and (6) amputation of the lower limb. results: the primary outcome, mean hemoglobin within 15 days of surgery, was significantly lower in the low-trigger group: 9.46 g/dl vs. 10.33 g/dl in the hightrigger group (mean difference 0.87 g/dl; p = 0.022, longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november 1, 2017, standard double-unit rbc transfusion was indicated with a hb threshold ≤ 8.1 g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to 7.3 g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ 8.1 g/dl and ≤ 9.7 g/dl applied. we evaluated rbc blood product utilization over a 6 month period starting december 1, 2016 (liberal protocol) and december 1, 2017 (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ 7 days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, 137 patients were admitted who in total received 164 therapy cycles, including 56 acute myeloid leukemia (aml) induction cycles and 69 autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in 303/402 (75.4%) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from 29/112 (29.0%) to 81/111(73.0%) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units 4.0 (interquartile range (iqr) 2.0-8.0) during the liberal versus 2.5 (iqr 0.0-9.0) during the restrictive period (p = 0.36)). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: 0.25 (iqr 0.11-0.33) versus 0.15 (iqr 0.00-0.32) units per day during the liberal versus restrictive period, respectively (p = 0.06). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from 2.0 (iqr 0.0-2.0) to 0.0 (iqr 0.0-1.5) units (p = 0.06), corresponding to a reduction from 0.13 (iqr 0.00-0.21) to 0.0 (iqr 0.0-0.13) (p = 0.01) units per neutropenic day. moreover, 14/38 (36.8%) patients during the liberal versus 21/31 (67.7%) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. 4a-s17-04 assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain 1 , n marwaha 2 and s sachdev 2 1 transfusion medicine, aiims, new delhi 2 transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from 350 ml and 450 ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of 900 prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group 100 collections were done in double 350 ml, triple 450 ml and quadruple 450 ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) 9 volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = 0.007). hemoglobin concentration of prbc ranged from 14.2-29.6 g/dl; mean hb was 21.02 ae 2.90 g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = 0.0001) and rtvd (p = 0.008). the hb content of prbc units prepared from 450 ml collection ranged from 35.19-87.36 g and from 350 ml collection ranged from 30.77-65.78 g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = 0.730, p = 0.0001).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from 155 ml to 370 ml (including both 350 and 450 ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. 4a-s17-05 nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a 639 bedded tertiary care oncology centre with approximately 18,000 to 20,000 rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of 4413 rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < 0.05. results: total 4413 rbc transfusion events for 2012 patients were analyzed. there were 1877 (43%) events in 628 patients of medical oncology and 2536 (57%) in 1384 patients of surgical oncology. maximum transfusions were received by patients in age group of 41 to 60 years (47%). total 83% of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < 0.05. inappropriateness was more 53%(396/735) and significant in daycare setup (p < 0.05). anemia was the most common indication of rbc transfusion observed in 90% of events (3971/4413). total 62% rbc transfusions were given as planned and 38% as urgent transfusions. most common adverse transfusion event observed was allergic reaction in 0.3% of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. 4a-s18-01 paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed 0.38% of donors were positive for babesia dna or antibodies (moritz, nejm, 2016) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â 6800/8800 systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the 4 babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october 2017 under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of 6 (mp6), plus 3 idt replicates with cobas â babesia. reactive index donations were also tested with 2 validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, 256,802 valid donations have been screened with cobas â babesia, and 15 (0.006%) were reactive. 13 of 15 (87%) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. 1 of 13 (8%) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and 1 (8%) was collected in a state where babesia is not considered endemic (iowa). 11 of 13 (85%) confirmed positive donations were collected in states with high endemicity. 8 of 13 (62%) confirmed babesia-positive donations were detected in late fall or winter. all 13 (100%) confirmed babesia-positive donations were reactive in mp6. serology results are available for 12 of 13 confirmed-positive donations: at index, 6 of 12 confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; 3 were positive for both igg and igm. 3 of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of 99.999% (256,787/256,789; 95% exact ci: 99.997%>100%). summary/conclusions: the cobas â babesia test successfully identified 13 babesiapositive donations, including 3 confirmed-positive donations with no igm or igg reactivity. 2 donations were collected in states considered low-or non-endemic for babesia. 8 confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a 2013 study of~14,000 donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november 2018, 50,752 blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of 14,758 tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the 50,752 donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of 14,758 samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october 1, all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only 1 tma-confirmed-positive donation of 50,752 tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. 4a-s18-04 background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september 2015 and july 2017, 520 blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the 520 blood donors (mean age 38.55 ae 11.14; range 18-65 years old), 38.1% were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence (55.1%) followed by the north (37.2%) and the south (25.3%). blood donors living in rural areas had a significantly higher seroprevalence (p = 0.001) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence (60.2%) found in the age group of 46-55 years old (multiple logistic regression [mlr]: or = 7.68; ci: 4.08-14.46; p < 0.001), and decreased with educational level (p < 0.001). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = 0.02). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = 2.72; ci: 1.27-5.86; p = 0.001). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. 4a-s18-05 who is syphilis testing excluding? c reynolds 1 , c pearson 2 , k davison 2 and s brailsford 1 1 nhsbt/phe epidemiology unit, nhs blood and transplant 2 nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the 1940s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from 12 to 3 months in november 2017 and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between 2009 and 2018. methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within 12 months for regular donors. results: between 2009 and 2018 there were 153 recent syphilis cases, 121 hiv and 32 acute hbv infections identified by donation screening. recent syphilis rates per 100,000 donations increased from 0.66 to 1.64 whereas hiv decreased from 1.04 to 0.19 with less than 5 positive donations in 2018. acute hbv rates rose slightly from 0.28 to 0.38 in 2018. males outweighed females accounting for 71.9%, 63.6% and 59.4% of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below 25 years old. of the male donors with recent syphilis, 58.2% reported sex between men and women (sbmw), 19.1% sex between men (sbm) and 22.7% did not report a risk. this contrasted with hiv where 45.5% of male donors reported sbm, just 2.6% not reporting a risk. overall 19, 32 and 3 males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in 2018, 26 donors with recent syphilis aged 23-65 years (median 36 years) were excluded from the donor pool, including 3 non-compliant to the sbm deferral. there were fewer than 5 hiv cases identified in 2018, all over 40 years old, all compliant, reporting sbmw. of the 6 hbv acute cases in 2018, 5 were male, all but one in the 45 and over age-group. summary/conclusions: over the 10 year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a 3 month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated 113 million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last 10 years. plasma donors are bled up to 104 times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in 2014, the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into 6 categories (16 subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in 2018, with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between 1/1/2017 to 9/30/2018. severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade 2 or higher were individually evaluated by a physician for grading accuracy. results: in 1,511,758 needle-in collections, 31,320 dae were graded for severity. the majority (16,143, 51.5%) were vasovagal reactions (vvr), followed by 8,570 apheresis-related (27.4%), 6,572 needle-related (21.0%) and 35 allergic (0.1%) events. the majority of dae were grade 1 accounting for 98.6% of all dae, followed by grade 2 (1.2%), and grade 3 (0.1%). there were 2 grade 4 and no grade 5 dae. among the vvr, 98.1%, 1.6%, 0.2% and 0.01% were grade 1, 2, 3, and 4 respectively. grade 3 vvrs included 14 concussions, 11 fractures, 1 dental injury, and 2 pre-faint and 8 fainting events requiring hospitalization for work-up. two grade 4 vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only 6 and 5 grade 2 reactions respectively, and no grade 3 or 4 events. needle-related dae included 98.3% grade 1, 1.6% grade 2, 0.1% grade 3, and no grade 4 events. of the six grade 3 needle-related dae, 4 were nerve irritations lasting > 6 months, and 2 were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since 2006. aims: we analysed the data collected in 2006-2016 in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from 2008 the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in 2015. reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per 1000 donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was 7, iqr 2-8). the total number of country years (cy) was 138, covering 155 million donations. the overall complication rate was 6.3/1000 donations and the median country rate was 3.2 complications/1000 donations (iqr 1.1-10.1). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall 4.6/ 1000 donations, median country rate 3.1/1000 donations (iqr 0.6-7.7). rare and apheresis-related types of complications such as generalized allergic reaction (0.10 per 100,000, 40 cy), and major blood vessel injury (category available since 2015; overall 0.12 per 100,000, 6 cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total 89 cy, 101.6 million wbd and 26.3 million aphereses, total 128 million donations). for these hvs the median rate of vasovagal reactions was 3.4/1000 wbd (iqr 1.0-9.1) and 1.5/1000 apheresis procedures (1.0-4.2) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was 0.39/1000 wbd (iqr 0.31-1.2) vs 4.2/1000 aphereses (0.69-5.6); rates of arm pain and/or nerve injury (not separated in 2006-2013) also tended to be higher: median 0.09/1000 with wbd, iqr 0.03-0.34, vs 0.39/1000 with apheresis, 0.05-0.57. summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. 52000 registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in 8000 control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each 4 th (women) or 8 th (men) donation, and with yearly testing of young adult blood donors below 25 years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that 20% of female and 9% of male applicants cannot be registered because of low hb (125 and 135 mg/l respectively). adding ferritin testing, a preferred cut-off level of 30 lg/ml (male reference level), would result in additionally 24% female and 1.3% male applicant donor loss. as this would threat the blood demand, cut-off was set to 15 lg/ml for women, above the female reference 10 lg/ml, with an acceptable 6% loss of female applicant donors. for registered blood donors, 4000 mg of extra iron tablets were offered at low ferritin (10-29 lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above 30 lg/ml. donors with ferritin below 10 lg/ml (in 0.6% applicant donors, 0.8% registered donors) or above 600 lg/ml (0.5% applicant donors, 0.1% registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from 3 to 5 months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from 50% to 70% approved hb at return. the team of nurses processing ferritin and blood count results (1½ nurse fulltime weekdays) reacted to approximately 40 donor results daily, representing 20% of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november 2017 a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at 30 and 15 ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors 46% (44% of 16,433 male donors, and 48% of 13,525 female donors) have ferritin levels below 30 ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of 43 ng/ml. in contrast, we found that only 27% of new female donors (n = 13,283) and 1.7% of new male donors (n = 6,334) have a ferritin levels below 30 ng/ml. the average ferritin level in new donors was 148 ng/ml for males and 60 ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between 1.5 and 2.0 was observed in female donors and between 3.2 and 4.4 in male donors. both ratios increased with donor age. at the end of december 2018 2884 donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by 13 ng/ml per year whereas average ferritin levels in male donors increased by 34 ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from 1.5 to 2.0 in female and from 3.2 to 4.4 in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~7000 different rare diseases and the genes for half have been identified. approximately 3.5 million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts 2.2 years. the main aims of the 100,000 genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in 2013 and dna samples from 100,000 nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the 100,000 genomes project comprising phenotype and genotype data from 13,037 individuals recruited at 83 hospitals using approved eligibility criteria for 15 rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over 165 million unique variants in the 10,258 genetically independent samples, with 47% of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in 2,000 unique diagnostic-grade genes for the 15 domains. over 1,000 reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from 0.5% to 55%, while the proportion of novel causal variants ranged between 25% and 73%. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying >30 novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the 100,000 genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped 7588 donors from england and the netherlands with this new array and observed a 99.92% concordance when comparing 92,387 blood centredetermined antigen typing results with genotype-determined ones. for the 48 red cell and hpa antigens that were available for 7,473 donors, the array typing provided a 3.6-fold increase in typing results per donor (13.2 vs 47.9) and 38 rare donors were identified. using the genotyping data we identified 2.6 times more compatible units among this cohort of donors when blood demand was modelled using referral data from 3,146 english patients with more than three red cell alloantibodies. in conclusion the 100,000 genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another 500,000 dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early 1990s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found 4 amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons 266 and 268 are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than 250 abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a1,3-gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. 1: our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); 2: identification of novel abo alleles by others; 3: more snp data from genome sequences and potential problems for abo genotyping; 4: findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; 5: a1,3-gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and 6: the potential causes of generation of abo polymorphism and of species variations of the gbgt1 gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. 4c-s20-01 a large deletion spanning xg xg and gyg2 gyg2 constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd99. the cd99 gene resides within pseudoautosomal region 1 on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first 3 exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly 11% of women and 34% of men are xg(aà). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs311103c variant disrupts a gata motif between xg and cd99. this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from 13 archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the 1000 genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg2. database searches indicated that the most likely deletion was the infrequent genomic structural variant esv2662319 reported in the 1000 genomes project. further analyses with a short (714 bp) and a long (3555 bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately 114 kb and corresponded well with esv2662319. this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the 13 cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific 714-bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr6b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the 1000 genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg2 genes accounts for the xg null phenotype underlying the majority (10 of 11) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c.143c>t, p.thr48met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c.230c>t (p.thr77met) causing partial exon skipping and designated gybp*03n.01 (gypb*ny) and c.270 + 5g>t, an intron change causing complete skipping of exon 5, designated gypb*03n.03 (gypb*p2). aims: samples from three individuals, a previously transfused african american sickle cell patient (p1), a blood donor of unknown ethnicity (p2), and an african american patient (p3) (lapadat r. 2014 aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p2 were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p1 and p2. p1 was also tested by gypb*s/s as-pcr, exon 5 pcr-rflp for c.270 + 5g>t and as-pcr for c.230c>t. p3 was tested for gypb*s/s and c.230 c>t and c.270 + 5g>t changes by a real-time pcr-fluorogenic 5' nuclease taqman chemistry. for all, gypb exons 1-6 were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c.143t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c.230c>t and c.270 + 5g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sàu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c.143c/t, c.230c/t and c.270 + 5g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c.59t/g and c.60a/g (p.20leu/trp), c.67a/t (p.23thr/ser), c.71a/g and c.72g/t (p.24glu/gly), c.143c/t (s/s), c.208g/t (p.70val/leu), c.230c/t (p.77thr/met), and c.270 + 5g/t. all samples were also c.251g/g (p.84ser) and heterozygous for several previously recognized silent changes in exon 2, c.87t/c, c.96t/c and c.102a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sàs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c.230t or c.270 + 5t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c.270 + 5t change is on a gypb*03n.02 [gyp*he(ny)] background. c.230c>t (rs79492560) and c.270 + 5g>t (rs139511876) have a frequency of 0.0053 respectively 0.032 in the african population (exac). although we identified 3 samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of 25 antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin-5a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of 15 exons located on chromosome 19q13.2. a number of rare lutheran phenotypes have been previously recorded in israel, including lu:-6,9 observed among iranian jews, lu:-20 in one thalassemia patient and one case of lu:-21. in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with 2-aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:-5, lu:-6, lu:-8, lu:-13, lu:-21, lu:-22, and lu:-23 cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: -1,2,8,12,13,-19,21 . bcam sequence analysis confirmed the patient to be lu*02, lu*18 and revealed a novel homozygous mutation c.1351a>c in exon 11, encoding p.lys451gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c.1351a>c (p.lys451gln) in exon 11 of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises 25 antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome 19. currently, isbt lists 20 high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: -1, 2, 3, 5, 6, 8, 13, 20, 21 . bcam sequencing revealed a novel homozygous mutation c.121g>a in exon 2, encoding p.val41met in the lu glycoprotein. the c.121g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of 3.98 9 10 -6 and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val41met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema7a exon-specific primers and sequenced. results: the proband was a 32-year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at 27 weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted 1 + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge2 was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge2 and anti-ch1 could be ruled out. the serum was nonreactive with two jmh:-1 and positive with two jmh:-3 samples. the patient was found to be jmh1 positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg4. overall, these results were consistent with a probable jmh variant and prompted us to perform sema7a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon 7, c.709g>a (p.asp237asn, rs140707085, maf < 0.01, sift score = 1); a common synonymous change in exon 12, c.1545a>g (p.gln515gln, rs741761, maf = 0.5); a rare non-synonymous change in exon 14, c.1865g>a (p.arg622his, rs140128092, maf < 0.01, sift score = 0.36). the analysis of surface accessibility of asp 237 and arg 622 using the 3d structure of sema7a (rcsb pdb-3nvq https://www.rcsb.org/structure/3nvq) showed that only arg 622 was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg4 antibody showing a similar pattern of reactivity, and with the same three changes in sema7a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg622his substitution in sema7a. we propose to provisionally assign the name jmh7 for this antigen. interestingly, our two unrelated jmh:-7 individuals were from north african ancestry. background: the abo system was discovered almost 120 years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca3(fuca2)gal and gala3(fuca2)gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, 1985) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca3(fuca2)galb3galnaca3(fuca2)galb4glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a3-specific gh110 family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, 40% of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px2 but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a 1 b individual with the p1 k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p 1 k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen 500-litre reference preparation and confirmatory preparation from 24 freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bà human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a 1 b p 1 k plasma contained anti-px2 and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px2 was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex-4hexnac-hex-4hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type 2 hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala3(fuca2)galb4glcnacb3galb4glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 1990's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p2y12 antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. 4c-s21-02 university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between 0.05 -15% of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day 100 (33%) compared with those who had normal elpi at baseline (7%) (p = 0.00034). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < 50 ml plasma). until, in 2011, we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in 2017, we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under 50 years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (>24 days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (<10 days storage) red cells from ever-pregnant donors (3-year cumulative incidence of death 15.4% versus 4.8%). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. 7.2% versus 4.7%). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy (6-9 months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s-303) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (>5.3 log reduction) and plasmodium falciparum (>7.5 log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase 1 clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase 1 clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase 1 clinical trial in africa, 20 clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade 2) within the first 24 hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within 59 (ae3) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment 24 hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase 3 clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase 1 clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's (2014) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every 4 weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from 2 months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions 1 and 2, donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition 3) or the revised email (condition 4) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every 4 weeks, and a prompt to forward-book appointments. results: the final sample (n = 6788) comprised 3859 women (57%) and 2929 men (43%) aged 18-70 (mean = 32). after two months 37.2% of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition 4 (revised email + phone call), or = 1.305, ci = 1.128-1.510, and bau. donors assigned to the two telephone conditions (condition 3 and 4) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c282y mutation), but also carriers of other hfe mutations (p.c282y/p.h63d or homozygous h63d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< 200 ng/ml in females, < 300 ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis (2rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ 18-60 years, total blood volume ≥ 5 l, bmi < 35 kg/m 2 , hb ≥ 140 g/l, elevated sf levels and no end organ damage due to iron overload. 2rbcaph (360 ml rbc) were scheduled every 14 days and wbph (450 ml) every 7 days until sf was < 100 ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up 8 weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: 30 subjects (5 females; mean age 47 years) were randomized to wbph (n = 16; 1 female) or 2rbcaph (n = 14; 4 females). hfe mutations were p.c282/ p.c282y in 17 subjects, p.c282y/p.h63d in 9, and p.h63d/p.h63d in 4. at baseline, mean hb was 149 g/l (sd 7.8) and median sf was 504 ng/ml (iqr 406-620 ng/ml). 222 procedures (wbph n = 146, 2rbcaph n = 76) were completed; 9 were interrupted (local hematoma, insufficient flow); 35 (16 wbph, 19 2rbcaph) were postponed because of low hb and 15 for non medical reasons. there were 2 drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < 130 g/l in males, <120 g/l in females) occurred after 15 visits in 8 wbph subjects and after 5 visits in 3 2rbcaph subjects. fatigue was reported after 37 phlebotomies and 31 aphereses. only 5 participants (17%) completed the study per protocol. 136 blood components (94 rbc concentrates and 42 plasma units) for transfusion were obtained. overall, a median of 7.5 wbph (iqr 6.2-9.8) was needed to reach sf < 100 ng/ml, corresponding to 1.8 times of 2rbcaph (median 4.0, iqr 3.0-5.8) (p = 0.0001). analyzing separately p.c282/p.c282y and p.c282y/p.h63d carriers, the relation wbph to 2rbcaph was 1.6 and 1.8, respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = 0.0001 and 0.007, respectively). summary/conclusions: 2rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately 200 ml red blood cells (rbc) and 200-240 mg iron are lost with wb donation. double unit rbc (2rbc) collections of 360 ml (ca. 40 ml less than the rbc amount of two wb donations) lead to a loss of about 400 mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every 6 months for 2rbc and once every 3 months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and 2rbc at our institution. methods: we included 294 wb and 151 2rbc donors (n = 445) who donated with the maximal allowed donation frequency over 48 months between 2008 and 2013, yielding 4,704 wb and 1,208 2rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were 135 g/l for wb and 140 g/l for 2rbc donation. with 2rbc apheresis 360 ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was 53 (wb) and 48 years (2rbc), respectively. at the first donation, mean hb was 153 g/l (sd 13) in wb and 159 g/l (sd 8) in 2rbc donors; mean sf was 44 (sd 52) and 73 lg/l (sd 56), respectively. on average, hb and sf were higher in 2rbc donors (5.1 g/l and 26 lg/l, respectively; p < 0.001). there were 137 subjects with sf < 30 lg/l in wb and 19 in 2rbc group, and 85 with sf < 50 lg/l (but > 30 lg/l) and 40, respectively. in 2rbc donors, between the first and the last donation, mean hb declined from 159 g/l to 157 g/l (p < 0.05) and mean sf from 73 lg/l to 66 lg/l (ns). in wb donors, mean hb dropped from 153 g/l to 152 g/l (p < 0.05) and sf from 44 lg/l to 35 lg/l (p < 0.001). similar results were found when adjusting for age and season. hb values dropped from baseline until the 11 th donation for wb donors and until the 4 th donation for 2rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the 4 th donation in both wb and 2rbc donors (37 lg/l and 60 lg/l) and increased thereafter in 2rbc donors. in wb donors, sf followed a parabolic trend that peaked at the 10 th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and 2rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15% or 30% mfg (gelafundin 4%, b. braun melsungen ag) or hes (hespan 6%/450/0.7, b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd11b, cd62l and cd66b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < 0.001 respectively), whereas only the higher concentrations (15% and 30%) of mfg showed lower relative migration distances (p < 0.001 respectively). track straightness was reduced with both sedimentation agents at 15% and 30% to the same extent (p < 0.001 respectively). hes resulted in lower cd11b expression (p = 0.028) and higher cd62l expression (p = 0.007) compared to the controls, whereas the differences for cd66b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, 2001) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to 12 plateletpheresis donations per year, with a minimum 4 weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between 01/01/2014 and 08/31/2018 from the french civilian blood donors' base and then selected a cohort of donors with at least 24 donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. 8 measures for each donor. results: the cohort includes 2,186 donors (384 women and 1,802 men). mean platelet counts fluctuate between 276.4 and 278.6 platelets/ml. analysis of variance does not show any statistically significant difference (f = 0.462), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the 24 donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than 20,000 described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the 1950s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb1, -dqb1), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb3, -drb4, -drb5, -dqa1, -dpa1). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa1a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa1a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa 1a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa-1a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n162a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd61 (c17) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa 1a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n162a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, 166 plasma samples with anti-hpa1a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, 2016) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n162a-fccriiia correlated with the level of fucosylation of the hpa1a antibodies, as measured by mass-spectrometry (r = à0.524; p < 0.0001). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n162a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa 1a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa1a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa-1a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa-1a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa-1a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa-1a screening program in poland. aims: our aim was to assess the frequency anti-hpa-1a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa-1a screening of 24 244 pregnant women in 8-40 gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa-1a negative/hpa-1b/1b women were tested for hla drb3*01:01 and for anti-hpa-1a antibodies by maipa (followed up at week 17-20, 28, 32, 38-40 and 6 weeks after delivery). if anti-hpa-1a were detected, quantitative maipa was performed. all hpa-1a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa-1a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: 554 hpa-1a negative women were identified (2.3%). anti-hpa-1a was antibodies were detected by maipa in 44 women (two delivered tweens). in addition, anti-hpa-1a antibodies were later detected by paklx in further 3 women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was 47 (8.5%). they delivered 49 babies; 35 were boys. three women were treated by ivig: two by 4 and 8 injections since 33 th and 26 th gw respectively. the anti-hpa-1a concentration in the 1 st one was 0.4; 0.1; 5.88 iu/ml in 17, 28, 32 gw respectively and in the 2 nd <0.05 iu/ml in all examined samples. the decision on treatment was based on the low plt count~50 g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the 3 rd treated woman entered the program in 35 gw (anti-hpa-1a concentration was high 22.64 iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in 5/49 newborns: in 3 with anti-hpa-1a detected in paklx only and in 2 with antibody concentration in maipa -1 st : 6.86/12.91/ 23.36 at 28/32/38 th gw respectively; 2 nd : 8.85/17.58 at 32/38 th gw respectively. ich was observed in all of them; plt count was < 50x10 9 in four, 56 9 10 9 / in one. summary/conclusions: 1/ the severe thrombocytopenia due to anti-hpa-1a alloimmunisation in our prospective study occurred in 2/10 000 pregnancies 2/ the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative 3/ the hpa-1a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in 2014 that the diagnosis of alloimmunization was carried in only 33% of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by 3 different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all 2017 and 2018 data were collected using the laboratory information management system. results: 544 patient files were analyzed. no incompatibility is demonstrated in hpa-1 to -9, -11 and -15 systems in 19.3% (n = 105). hpa-1 and / or 3 and / or 5 incompatibilities were found in 271 cases (49.8%), hpa-2 and / or 15 in 87 cases (16%). platelet alloimmunization was globally confirmed in only 10.6% of the cases. 58 platelet alloabs were identified regardless of clinical manifestations: 24 anti-hpa-1a (41.4%), 20 anti-hpa-5b (34.5%), 8 anti-cd109 (13.8%), 2 anti-hpa-5a and anti-hpa-15b (3.4% respectively) and 1 anti-hpa-1b and anti-cd36 (1.7% respectively). 40 alloabs were found in the context of neonatal thrombocytopenia, 6 in ich and 10 in fdiu, and 1 in a follow-up of pregnancy. even if no anti-hpa-3 alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = 55, n = 32 and n = 17 on 114 cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa-3 and hpa-5 incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa-3 and hpa-15 systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa-1a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the 2% hpa-1a negative women. at present, hpa-1a typing is mostly done by genotyping. for costeffective implementation of anti-hpa-1a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa-1a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa-1a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg1 anti-hpa-1a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, 20 ll of the uppermost plasma of 3 -6 days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < 0.500 in the hpa-1a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, 506 consecutive samples were tested. in phase ii, the hpa-1a elisa was performed in another 62,171 consecutive samples, with confirmatory q-pcr in 1,825. the two phases combined, samples from in total 1,585 hpa-1a negative and 823 hpa-1a positive pregnant women were genotyped. the assay reached a 100% sensitivity with a cut-off od between 0.075 and 0.200, leading to a specificity of 99.6%. summary/conclusions: a quick, low-cost and reliable assay for hpa-1a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa1a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of 1047 pregnant women in 2002 found that 30.4% of irish women were cmv seropositive in comparison to 56% from western europe and 92% eastern europe and 97% from africa. an internal study carried out by the irish blood transfusion service (ibts) in 2010 indicated the rate of cmv seropositivity in irish blood donors was 21.97%. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in 2018 the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing 48 confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = 54). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing 6127 donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be 100%. the seroconversion sensitivity reported 42 out of 54 samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be 1.12 iu/ml. the validation reported 65 discordant results from 6127 donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. 60 discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified 27 discordant results as positive, 12 as negative and 21 as indeterminate. 5 discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be 99.80%. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity (99.80%) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of 6127 donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: 1) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, 2) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, 2014) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: 1. to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa 2. to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: 900 negative new donor samples and 100 confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was 0.56%, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than 750.000 blood and plasma donations are tested, on average 3.000 samples per day. for more than 10 years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july 2018, serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from 6 months before and 5 months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june 2018 (prism) and august to december 2018 (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: 1) hbsag p 0.01 % (42/390.736) versus a 0.03 % (87/322.394); 2) hiv p 0.07 % (262/390.735) versus a 0.06 % (201/322.470); 3) anti-hcv p 0.11 % (427/390.737) versus a 0.03 % (109/322.476). the rate of anti-hbc reactive samples was not significantly different between prism (0.39 %) and alinity s (0.42%). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s (0.39 %) and prism assays (0.34 %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to 0.16 %, which was significantly lower than for the three prism assays (0.33%). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by 0.17 % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on 2,748 blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag 6.0, enzygnost anti-hcv 4.0, and enzygnost hiv integral 4 assays and on the pk7300 with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i1000 system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas 8800 system) were available. results: based on the results from testing 2,748 blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around 90% of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e 801 analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e 801 analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e 801 analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using 30 preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar (99.81-100.00% versus 99.79-99.98%; n ≥ 5195). in specificity analyses, there were 14 discrepant results for hiv testing, 27 for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay (83.33%; 95% ci 65. 28-94.36) was higher than the prism hiv o plus assay (76.67%; 95% ci 57.72-90.07), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv (85.19%; 95% ci 67.33-95.97), hbsag (70.00%; 95% ci 50.60-85.27), anti-hbc (100.00%; 95% ci 85.75-100.00), and syphilis (100.00%; 95% ci 88.43-100.00); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e 801 analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e801 (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june 2018, close to 10,000 aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = 10%) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of 7,389 plasma and 2,242 serum samples, 41 test results representing 37 individual donations were found rr on one or both instruments. two samples were confirmed positive (1 9 hbsag, 1 9 hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e801): hbs ag: 0.04/0.03 % in a total of 9590/9589 samples tested; hcv ab: 0.01/0.09 % in 9620/9585, p < 10%; hiv ag/ab: 0.03/0.09% in 9362/9329, p < 10%; syphilis ab: 0.1/0.07% in 2971/2987; hbc: 0/0% in 1531/1549. no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd34 + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type 1 (hiv-1), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd8 + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd8 + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered 50 years of soviet occupation. we held hands in a 600 km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june 1 st , 2004. the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in 1991, the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in 1992. a lot of advice came from finnish colleagues. in 1997, it was decided to move towards non-paid voluntary donations. the process took 6 years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than 15 years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in 2006-2015. it resulted in 51% of the donations being unpaid. the second program initiated in 2016 is still ongoing, aiming towards 100% non-remunerated donations by 2020. by the end of 2018, they had reached 99.2%. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: 13.8 ae 0.01 vs 15.5 ae 0.01 g/dl; p < 0.000). percentage of females with low hb < 12.5 g/dl were 7.1%, 4.9%, 5.2%, 6.7% and 4.2%, percentage of males with hb < 13.5 g/dl were 0.5%, 0.6%, 0.7%, 1.7% and 1.2% for the age groups 1-5 respectively. ferritin values were higher in males compared to females (median; 25 th -75 th %>tile: 51;31-79 vs 157;106-231 lg/l; p < 0.000) and in older age groups compared to younger age groups (median; range in age groups 1-5 in females: 40;3-702, 52;6-619, 60;6-480, 60;8-725, 98; 10-604 and in males: 99;8-661, 161;18-1080, 206;15-1090, 220;26-1430, 221;33-981 respectively) . percentage of females with ferritin ≤ 15 lg/l were 8.6%, 3.8%, 3.7%, 6.7% and 2.0%, while percentage of males with ferritin ≤ 15 lg/l were 0.2%, 0.0%, 0.1%, 0.0% and 0.0% for the age groups 1-5 respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: 7.2 ae 0.02 vs 6.6 ae 0.03; p < 0.000). percentage of females with wbc > 12x10 9 /l were 1.8%, 1.3%, 1.7%, 1.4% and 0.3%, while percentage of males with wbc > 12x10 9 /l were 1.4%, 0.9%, 0.5%, 1.2% and 0.9% for the age groups 1-5 respectively. none had wbc < 2x10 9 /l. platelet counts (plt) were higher in females compared to males (meanaesem: 257 ae 0.6 vs 222 ae 0.6; p < 0.000).percentage of females with plt < 150x10 9 /l were 1.0%, 1.5%, 2.2%, 2.4% and 1.0%, while percentage of males with plt < 150x10 9 /l were 2.6%, 3.6%, 2.7%, 2.4% and 1.6% for the age groups 1-5 respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with 75% younger than 30 years of age and the female/male ratio was 58/42. of the 16 583 first time donors with data on ferritin available, 14% had low ferritin (≤ 30 lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb (6%<12.5 g/dl) and low iron stores (23%≤30 lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period (2017-2018), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november 2017 was assessed by comparing results in two study years. results: a total of 491 pdi were recorded: 266 in 2017 (1/396 donations) and 225 in 2018 (1/452 donations) with the following distribution: nonsexual risk as tattoo and piercing (17.5%), surgical procedures (17.1%), travel history (16.1%), infections/ contact (15.3%), other medical reasons (13.4%), endoscopy/invasive diagnostic procedures (12.2%), malignancy (4.3%), autoimmune diseases (3.7%) and sexual risks (0.4%). majority (76.4%) were late pdi, revealed on the future donation(s): 58.7% on the first next donation, 28.9% on the second and 12.3% after more than 2 subsequent donations. the mean age of blood donors associated with pdi was 40 ae 12 years (median 39 years), while the mean age of all donors in 2017/2018 was 38 years (median 37 years). of all pdi, 81.1% were related to male donors (84% in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > 0.05). the median number of all donations preceding pdi was 6 for female donors and 19 for male donors. implementation of education leaflet for blood donors resulted in 15.4% reduction of pdi in 2018 compared with 2017 (p > 0.05). the effect is more pronounced (p < 0.05) when comparing second and first half of 2018 (-25.6%). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from 21.1% in 2017 to 26.7% in 2018, which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. 45 % of omani populations are àa/àa gene carriers, 44% àa/aa, and 11% of the population are aa/aa. around 10 % of omani nationals carry the gene for hbs, and 2 -3 % carry the gene for b-thalassaemia. recent statistics show that there are around 400 patients with thalassaemia major and 3000 with scd in oman. the other rbc abnormality that is common in oman is g6pd deficiency which is found in 28 % of males and 12 % of females. omanis are known to have the highest frequency of a thalassaemia and g6pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get 40 new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: 418 patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the 418 scd patients, 49% of the patients were male and 51% female, mean age was 17 years, in the range of 1-72 years. 38 % of the scd cases were positive for the alloantibodies, 72% were female and 28% were male, the age range was from 6-68 years. 78% of the positive were scd, 19% s trait and 3% were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. 51% of the patients were with single alloantibody, 30 % of them with two antibodies, 10 % with three antibodies, 7 % with four antibodies and 2 % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority 23%, followed by anti-d 13%, anti-k 17%, anti-c 14%, anti-c 8%, anti-jk a 5%, anti-jk b 5%, anti-fy a 5%, anti-e 3%, anti-s 3%, antis 1%, anti-kp a 0.7%, anti-fy b 0.3% and igm being 2%. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and 19% of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in 2018, a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ 1, 2-5, 6-9 and ≥10), previous pregnancy, number of transfused units (2, 3-5 and 6-10 and > 10), and years after last transfusion (<1, 1-2, 2-5, >5y) with presence of alloantibodies results: 226 patients (100 males and 126 females, median age 17 years, range 1.7-66) were included. the median number of transfusions was 3 (range 2-40). the median years after last transfusion was 2 (range 2 weeks-55.5 years). in 56 patients, anti-rbc antibodies were detected. in 14 of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least 34 patients (15.0%) alloimmunization was demonstrated or suspected; in 11 patients the alloantibodies were 'enzyme only'. besides, the 36 alloantibodies of known specificity (8 anti-d, 2 anti-d+c, 16 anti-e, 1 anti-c, 3 anti-e, 1 anti-k, 1 anti-s, 1 anti-le a , 1 anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients (3 had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated (2 dau-alleles and 1 diii type 4 or diva-2). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the 34 patients. immunobiology -red cell alloimmunity 65 fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion (12 patients had dark urine). adverse reactions were associated with the number of units received (or 1.71 (95% ci, 1.25-2.33; p = 0.001), but not with the presence of antibodies (p > 0.7) summary/conclusions: in at least 15% of multi-transfused patients with scd alloimmunization could be demonstrated, mainly (80%) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of 12 patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately 1% of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman /3 rd pregnancy, 1 st delivery, 2 abortions in 1 st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd1) by biorad and anti-d duo igm+igg, clone: th28 + ms26 by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons 5,7,10) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon 1 to 9 revealed presence of a nucleotide deletion in position c.147dela, which is specific for allele rhd*01el.04. this nucleotide change results in the amino acid change p.val50leufs*5 causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the 2000 level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd*01el.04 allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt 00023736 and rvo-vfn 64165. 5a-s29-05 ea scharberg 1 , s rothenberger 1 , a st€ urtzel 1 , n gillhuber 1 , s seyboth 1 , e richter 1 , g rink 2 and p bugert 2 1 institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden 2 institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di6) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc4a1*c.1643c>t (p.pro548leu; isbt allele name: di*02.06) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of 125,742 sequenced genomes (allele frequency: 0.000004). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di6 in 1,700 blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in 3,652 patients and in 964 blood donors was performed in the gel technique (biorad ahg id-cards) using a 3 cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di*02.06 allele in 1,700 blood donors revealed no single positive individual. within the first 2 weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell 84 patients with anti-rb a were found. it was 2.3% of 3,652 patients tested in 21 laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in 18 of 964 randomly tested blood donors (1.9%) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di*02.06 allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around 1% of patients and donors. 5a-s30-01 national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since 1935. real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in 2003 in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in 2002, who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in 2017, national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since 2016, reporters to shot have been asked to score(0-10) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between 2010-2017(inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between 2016-2017 was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between 2010-2017; majority (56/67, 83.6%) were red cell transfusions but aboi plasma (9/67) and platelet transfusions (2/67) were also seen. most errors occurred in the clinical area (45/67, 67%), and could have been detected at point of administration. in 21(31%) cases, the error could not have be detected at the point of administration with a primary laboratory error in 10/21(48%) incidents. reviewing data from hfit for cases in 2016-2017 (13 aboi cases), the total score for staff culpability was 100, compared to a total score of 99 for all the other three organisational and system factors. this disparity is most obvious for the 4 aboi red cell cases, all of which scored the maximum 10 for staff culpability, i.e. 40/40 compared to 8/120 as the combined total score given to the other factors. in the preceding years (2010 to 2015), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by 37 cases that included an outcome of the local case review and 14 (37.8%) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with 2/56(4%) that resulted in death, 14/ 56(25%) major morbidity and 40/56(71%) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between 2015-2018 to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = 27 were possibly related to treatment, n = 2 trars were probable, and n = 2 were definitely related to treatment; n = 37 trars were grade 1, n = 4 were grade 2, and none were grade 4. in recipients of conventional wb, there were n = 13 (1.12%) ars, n = 32 (2.75%) fnhtrs, n = 1 (0.09%) taco, n = 0 trali, and n = 16 (1.38%) unclassified transfusion reactions. of the confirmed trars, n = 55 were possibly related to treatment, n = 1 trar was probable, and n = 8 were definitely related to treatment; n = 54 trars were grade 1, n = 1 was grade 2 and n = 1 was grade 4. there were 21 mirasoltreated wb transfusions in pregnant women and 2 trars (9.5%), both grade 1 and probably related. there were 80 transfusions of mirasol-treated wb and 84 transfusions of conventional wb in patients < 18 years old resulting in n = 7 (8.33%) trars in recipients of mirasol-treated wb and n = 10 (11.90%) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of 2181 wb transfusions in routine use in ghana, there were 4.02% trars in recipients of mirasol-treated wb and 5.59% in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january 2008 to october 2018 at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified 310 patients who received fludarabine (n = 52, 17%), bendamustine (n = 29, 9%), cladribine (n = 17, 5%), dacarbazine for hl (n = 164, 53%) and alemtuzumab (n = 48, 15%). the median age of patients was 46 years (range 8-92) and 171 (55%) were male. median follow-up was 30 months (range 0-132). post-exposure, 42 patients (14%) received transfusions with 33% correctly receiving irradiated rbcs. the remaining 28, all from haematology/oncology, received a total of 192 unirradiated rbcs. in 8 patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of 14.5 months (range 0-75) from first rbc transfusion. after medication administration, 99 patients had g&h requests after a median of 3 months (range 0-129). only 23% of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only 20% asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march 2017. for audited patients, these were written from 38 days prior to 104 days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february 2018 to february 2019. ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after 15 minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version 23.0. results: a total of 500 transfusions forms were analyzed. over all compliance rate was 18%. out of 500, 115(23%) forms were available in source notes and of 115, 88 (76%) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = 0.000). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion(3%) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion(89%). a total of 02(0.4%) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was 2 hours and 30 minutes for red blood cells and for platelets it was 1 hour and 13 minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after 6 hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. 5a-s31-01 as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately 15 cases per 100,000 but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of 2-4 weeks (range, 5 days to 5 months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and 130 (4.6%) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of 8.7 9 10 3 , 8.0 9 10 3 and 7.2 9 10 3 , respectively. mean cci decreased in a linear fashion between day ≤ 2 and day 7 pcs (9.0 9 10 3 , 9.1 9 10 3 and 8.5 9 10 3 at ≤ 2 days; 6.7 9 10 3 , 5.8 9 10 3 and 4.9 9 10 3 at 7 days, respectively), although the number of pc transfused on day 7 to autohsct patients was small (n = 71). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late 1990s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from 4 bcs (35% plasma in additive solution ssp+) were spiked with virus suspension (10% v/v). pcs (n = 2, 375 ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) j/cm 2 )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid 50 ) by endpoint titration in microtitre plate assays on vero 76 cells (atcc â crl-1587 tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of 6.2 (bag no. 1) and 6.5 (bag no. 2) log 10 tcid 50 /ml was received in the pcs. at a uvc dose of 0.10 j/cm 2 and higher niv was inactivated down to the detection limit of the system (1.9 log 10 tcid 50 / ml), resulting in log 10 reduction factors of ≥4.3 (bag no. 1) and ≥4.6 (bag no. 2). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. 364 ae 20.6 9 10e11 platelets/unit, p < 0.001), whereas the platelet content of apheresis pc did not change (305 ae 9.9 vs. 300, ae16.1, p = 0.57). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. 1478 first-time donors has attended to the study in 18 regional blood centres in 29 cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = 14). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = 3.55, close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: 0.47, 0.28. 0.19, 0.17, and 0.5, respectively). donation anxiety was the negative direct predictor of intention (-0.05). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy (0.90 and 0.08). paraphernalia anxiety was the negative indirect predictor of intention (-0.03). descriptive norm did not show any significance. our model accounted for 75.3% of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from 16 countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp1 coordination, wp2 dissemination, and wp3 evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp4 inventory of donor selection & protection practices; -wp5 development of risk-based guidelines for donor selection and protection; -wp6 development of a standard donor health questionnaire (dhq); -wp7 training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september 2017 and will complete in spring 2020. wp4 has completed its work in october, wp5 will complete its work in june 2019, and wp6 and wp7 have recently commenced. results: with the use of the deliverables created by wp4, we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp5's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from 16 countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to 1) provide a comprehensive inventory of current systems for selection and protection of donors and donations, 2) critically review them and 3) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of 163 experts (40 blood; 40 plasma; 27 tissues; 47 stem cells; 9 art) and 39 (24%) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of 24 stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years 2014-2017, both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by 77%), arterial puncture (77%) and nerve damage (54%). vasovagal reactions were also frequently included (75%); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma (86%), citrate reactions (86%) and arm pains and nerve damage (both 57%, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl-03-01 modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg1/2) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a 2 c 2 ). we have previously identified bcl11a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas9 editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas9:sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + 58 bcl11a erythroid enhancer results in highly penetrant disruption of gata1 binding motif, reduction of bcl11a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl11a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl11a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m101 was developed in the medical device named hemo 2 life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on 60 patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo 2 life â and grafted on recipients. in 2018, a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo 2 life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m101 making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since 2012; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at 9-11 march 2018. aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from 25 countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations 4 different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: 1. should have university degree preferably in marketing and business administration field. 2. should have a certificate and/or professional experience in public relation 3. should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team 4. should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter 5. should be a permanent staff 6. should have basic salary and performance bonus might be given 7. is eligible to monitor and modify mobile team working period at blood drive 8. should participate the mobile blood drive which he/she has organized 9. should participate the group who will create promotional materials for national blood service 10. number at each blood establishment should be defined based on annual blood collection such as 5 staffs for 50,000 whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga 1 and j emmanuel 2 background: africa is a large continent with 55 independent states and a total population of 1,275,710,034 (february 2018) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the 2 who regional offices; eastern mediterranean regional office (emro) supporting 8 arabic speaking countries and the african regional office (afro) responsible for 47 sub-saharan countries. population distribution is approximately 40.6% urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise 3 level accreditation program. in 2016 emro held a consensus meeting developing a "strategic framework for blood safety and availability for 2016-2025" with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in 2001 all 54 member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least 15% of the national fiscal budget. in 2013 ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha63.12 on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's 2016 global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the 2016 report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of 4 staff on the morning (am) shift (from 8:00 to 17:30) and 5 staff on the afternoon (pm) shift (from 13:00 to 22:00) on weekdays and 3 staff on the am shift and 5 staff on the pm shift on weekends. bdt lab has a staff strength of 15 to be rostered for the 2 work shifts. each staff is on a five-day work week and has to work 11 pm shift and 9 am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving 5 staff on am shift and 4 staff on pm shift was conducted. the total number of pm shift per month was reduced from 124 to 112 using the re-defined process. the 10% reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having 2760 licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of 70,000 blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of 6 months. after overcoming all the challenges we minimised hard copies to 6 registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by 20%. waiting time for attendant decreased by 10%. traceability of all the units became 100%.supervision of the activities being carried out was 90% accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by 50% thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p-007 ct smit sibinga 1 , y abdella 2 and f konings 2 1 iqm consulting, zuidhorn, netherlands 2 who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in 2013 blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only 9/22 countries are put in force by governments [1960 (egypt) till 2017 (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in 2008-2017 including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in 21 regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from 170 in 2008 to 133 in 2017 in favor of mobile collections, which increased from 8,672 to 13,189, the former is still the number one location for donating blood. on average 47.36% of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar (583,908 in 2008 and 588,184 in 2017); over 99% of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from 237,658 in 2008 to 143,038 in 2017). the total number of donations increased from 1,076,655 in 2008 to 1,249,655 in 2017; most frequent were whole blood collections (from 1,016,411 in 2008 to 1,171,302 in 2017) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs (996,678-1,154,239 units per year), fresh frozen plasma -ffp (1,090,369-1,289,021 units) and platelet concentrates -pcs (81,692-129,143 units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs (52-57.6% in respective years irradiated, 75.18-92.07% leukocyte-depleted), than to rbcs (4.46-8.46% irradiated, 7.65-20.7% leukocyte-depleted). in 2010, the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in 2017 approximately 11.5% of pcs and 8% ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland 80% of hospitals depend on blood for the treatment of patients; over 1.5 mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. 1 st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the 20 year-period in two stages. goals at 1 st stage: 1. data digitalization; scanning of paper documents. 2. development of a uniform template for collecting digital data from various sources. 3. standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. 4. selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) , b) digital stored in two file types (.doc and .xls, for the years 2005-2010 and 2011-2017, respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: 1. 1344 pages of paper documents were scanned. 2. models developed for data from 3 different sources: a. paper-data were rewritten and ascribed to its model; outcome -3 tables, 272 columns, 10,400 rows. b. .doc and .xls. filesdata were ascribed to 2 other models; outcome -6 tables, 1656 columns, 788 rows. 3. the 3 models were merged into 1 analytical table to create a 588 mb database (comparable to approx. 784 min of music). 4. the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. 5. selection of data for analysis at 2 nd stage. summary/conclusions: the 1 st stage provided a set of selected data for analysis in the 2 nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far 8 workshops were organized and each year around 30 countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. 6. supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from %96 whole blood (at 1996) to 5%. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management 7. around 11,000 blood bankers attended national courses, 7,000 attended national congresses, 18,000 attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in 2016 an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages 4.3% of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages 122.8% (2018) based on a 5-day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated (2016/2017) to be at 3.2% of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october 2018, an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level 6. prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the 12 months prior to emr implementation was compared with 3 months post. ntr numbers reduced from 4220/month to 2019/month (52% reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of 27/mth to 6/mth. wbit numbers have increased slightly: before having median 1 (range 0-2), after with median 2 (range 0-3). although it was hoped that wbit incidence may reduce with the new emr, 4 of the post implementation 6 wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa 1 , k teo 2 , s tsai 1 , p heng 1 , r sagun 1 and m wong 1 1 laboratory medicine 2 khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a 761-bed general and acute care hospital, opened in 2010, serving more than 800,000 people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase 1: an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase 2: the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase 3: elimination of the witness step for blood collection. specimen collection and rejection data from 2016 to 2018 was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january 2016 and march 2017, before the implementation of the e-t&s phase 1, the average rejection rate for blood bank specimens was 0.16% and 1.14% for identification and clerical errors respectively. during phases 1 and 2 of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february 2018, with the implementation of the final phase of the e-t&s system the specimen rejection rate was 0.38% and 0.12% for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after 15 min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on 30 patients admitted to the outpatient clinic for 45 red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january 2017-december 2017) 76 patients, 40 females and 36 males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was 12.0 months (interquantile range-iqr 26). the median length of stay was 16 days (iqr 30). in total 400 blood components were transfused in which of 227 red blood cell concentrates, 114 apheresis platelet concentrates, 6 granulocyte concentrates, 51 fresh frozen plasmas, and 1 cryoprecipitate and 1 whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < 5%, 5-10%, 11-15% and > 15%. most of the patients (63.2%) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were 5478.76 euros (iqr = 11280.02) and 130.57 euros (iqr = 354.86), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: 0.674, p < 0.01). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: 0.247, p = 0.032) there was also a significant weak positive correlation between the age and transfusion associated cost (p = 0.048, r: 0.227). a significant difference was found between the patients with and without hematological malignancies (p < 0.01) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < 0.05). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately 55.7% of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january 2016 to december 2018 were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total 177,370 ordered prc unit, 166,910 (94.1%) were subjected to pretransfusion testing and 63.1% (105,260) of ordering prc unit which are pretransfusion testing were transfused. this means that 5.9% (10,460) of ordering prc unit were not subjected to pretransfusion test. this showed savings of 1,098,300,000 rupiah. c/t ratio was 1.6 which demonstrate a good ordering pattern. however, 16.4% (27,451) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of 2,882,355,000 rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of 19 questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, 2014 to february, 2015 . results: the overall mean percentage of 'correct' responses for 19 questions in the baseline study was 61.75%, whereas post sensitization it was 77.21%. the mean percentage increase in general awareness related questions was 21.49%, 13.95% for storage of blood/blood components related questions, 17.37% for pre-transfusion checks and bedside transfusion practices related questions, 21.33% for testing and blood component preparation related questions and 27.27% for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < 0.001). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in 2014 to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online 24/7, complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during 2018, tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from 3/5 international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been 2,376 paediatric and neonatal courses completed from 6 march 2018 to 28 february 2019 with 89.6% of learners being nurses and/or midwives. analysis of course evaluation data (n = 33) showed that these courses: -provide knowledge (96.9%) -improve patient safety and outcomes (84.9%) -result in change to clinical practice (69.9%) -are relevant to clinical practice (70.9%) -are easy to use (67.7%) -are readily accessible (58.1%). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was 100%. the 2 nd group participants had an average seniority of 9 years . more than half of them (64%) had seniority of less than 10 years. only 12% had more than 20 years of experience. the rate of correct answers for all items combined was 44.4% for students versus 42.5% for practicing nurses. the theoretical knowledge part was more mastered in the 1 st group than that of practicing nurses (44.8% vs 33.4% of correct answers). on the other hand, the control of the transfusion act was better in 2 nd group (44% vs 50.5%). the overall "dangerous" response rate was 47% for students and 41.7% for practicing nurses. false practical knowledge was more common in group 1 (59.5% vs. 41.5%). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive 2004/33/ec on blood donor selection criteria is 15 years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than 24 stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive 2004/33/ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in 2018, we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive 2004/33/ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified 64 risks considered to be significant, distributed between donors and recipients. for 35/64 (55%) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for 9/20 criteria concerning general health and medication, 12/22 for transfusion transmissible infections, 10/13 for high-risk behaviour and travel, and 4/9 for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive 2004/33/ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of 5 possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since 2007, showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after 10 years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all 27 states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since 2007, when the assessment covered 109 blood services, marpsh reached 1218 blood services in 2017 (57% of the blood services registered) what corresponded to almost 100% of the inspection cover in this year. over this period (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from 26% to 9%. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over 10 years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = 2), training coordinator (n = 1), medical doctor for donor selection (n = 2), hemovigilance officer (n = 1), laboratory technicians performing donor sampling, blood qualification and production (n = 7), biomedical scientists (n = 3), microbiologist (n = 1), clinical biologist (n = 1), nurse (n = 3). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is >8 h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: 19.3 million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab0, rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version 4.2; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than 8800 blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over 1000 blood donors from more than 20 non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of 600 migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of 1.6%. amongst 509 hla-typed potential stem cell donors, we found 28 (5.5%) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund 2014-2020 (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of 1 year starting from june 2017 to may 2018 at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ 16) that were admitted within 24 h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period 885 patients (83.5% males) were enrolled. mean iss scores was 21.89 . mean time to hospital admission after injury was 9:03 (iqr 3.38-13:48) hours. mean time to first rbc transfusion following admission was 2:09 (iqr 0:27-2:45) hours. approximately 49.3% (436) patients were in shock (sbp < 90 mm hg &/or pulse rate > 110/min). whereas, 160 (18.1%) patients were coagulopathic (pt ≥ 1.5 times of normal). during initial 24 h of admission, these patients were transfused with 2929 (69.7%) rbc, 1986 (51.8%) ffp, 2327 (42.9%) rdp and 384 (81.5%) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ 5 units/4 h) was given to 190 (21.4%) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of 9% per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on 78 patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in 2016. pc consumption was examined from january 2016 to december 2018. the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched (10-8/10) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of 78 patients aged from 3 to 74 years were analyzed; 48 (62%) were male and 30 (38%) female; 35 (45%) were cmv-negative and 43 (55%) were cmv-positive. out of a total of 78 transplants, 14 (17.9%) were haploidentical and 64 (82.1%) hla-matched. according to the stem cell source, bm was transplanted in 22 cases (28.2%), and pbsc in 56 cases (71.8%). two patients also received a cd34 + stem cell boost. our analysis showed that, with a mean follow-up of 652 days, the number of pc transfused to our patients treated by hsct ranged from 0 to 383 units, with an average of 37 and a median of 15, illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = 0.034) higher number of pc transfused for patients treated with a haploidentical (89) versus hla-matched (26) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused (2016: 20% (371/1813), 2017: 19% (322/1742)). moreover, approximately 8 products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october 2017 to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july 2014 and december 2016 to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ 25% of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ 25%: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took 2.5 min using the pneumatic tube system (maximum of 2 units per tube) and 4.5 min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december 2017. one year following implementation of the change in policy 940 rbc units were issued to the or (a 54% reduction); 38% (361) were transfused, compared to 19% in 2017. wastage rates decreased from 8 products a month to 1 per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, 34% of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from 1 may 2018. the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired 5-days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july 2018 set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of 704 daily blood bank statements which were submitted between may and december 2018 from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was 130.4% (sd +/-101.6), the available stock was 142.3%: (sd +/-118.8) and the demand versus supply was at 95.6% (sd +/-11.3).the overall bsms was 122.8%; (sd +/-77.2) for the study period. gweru and masvingo nearly supplied all the demanded blood with 99.2%, overall bsms of 118.1% and 99.7%, overall bsms of 144.3% respectively. bulawayo supplied 98.3% of the blood demanded with an overall bsms of 99.1%. mutare supplied 97.8% with a bsms of 180.1% and harare 85.6% and a bsms of 78.0%. there were monthly variations but the service could supply above 90% of the blood demand. in the month of may the service met 91.6% of the demand and a bsms of 87.8%. in november and december it supplied 92.6%, bsms of 100.8% and 92.8%, bsms 131.8% respectively. august also had a below average supply of 93%, bsms -98.2%. june, october and september recorded above the average values; 97.3%, bsms of 126.8% and 98.7%, with a bsms of 117.2% respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in 2008. subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in 2016 and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where 4 or more red cell units are transfused in <4 h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: 65 from 10 treating units including cardiothoracic surgery (18) hepatobiliary/gastrointestinal/colorectal surgery (24), vascular surgery (6), neurosurgery (4), orthopaedic surgery (3), endocrine (2) and "other" (encompassing general surgery, urology, general medicine and oncology -8). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. 18 of 65 reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at à40°c, and irradiation. biolog-id tags are passive hf (13.56 mhz) tags. they are compliant with is0 15693, iso 18000-3 and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, 2010). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt 128 format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with 450 ml water. centrifugation at 4,500 rpm for 10 min. 270 tags processed, 15 tags per kit affixed at different positions. -shock-freezing at à80°c: shock-freezer (angelantoni, sf40), 50 units processed, reading immediately after removal from shock freezer. water, 30 tags irradiated at 30 gy and 30 tags at 50 gy results: all biolog-id tags were encoded and read with a 100% success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at à40°c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an 18-year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh34). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd*01/rhd*03n.01 and rhce*cevs.01/ rhce*cevs.03. aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion 2013 53(2s):174a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier 1 donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier 2 donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier 3 donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: 102 tier 1, 357 tier 2 and 100 tier 3 donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier 2 unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from 7 to 1.9 g/dl. at that time, she was transfused the only compatible units available -2 of the rare -dphenotype and her hb increased to 3.8 g/dl and eventually to 7 g/dl. the tier 2 rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform (1) assuring the patient identification with barcode techniques; (2) designing a structured order entry; (3) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; (4) automatically reminding physicians the happening of reaction and suggesting relevant test; (5) building a complete traceability log system; and (6) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected (0% after implementation, p < 0.01), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency 30-min blood crossmatch (98.1% after implementation, p < 0.05). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply (0% after implementation, p < 0.01). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow (2004 moscow ( -2011 . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during 7 days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in 7 days after were compared. results: in 2004-2011, 5 terrible ta occurred in moscow: 141 people died and more than 629 were injured. with the explosion in subway in 02/2004 42 people died, 250 were injured. the number of d-rbcus increased by 10% on ta-day, and by 30% during next 7 days. dbdn in the 1st day after ta increased 6,7 times, and in the next 7 days -1,6 times. second explosion in subway in 08/2004 resulted in 10 died, 50 injured. the number of d-rbcus increased by 80% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 1,1 times, and in the next 7 days -2,2 times. in 2006 (explosion on market) resulted in 14 died, 61 injured. d-rbcus delivery increased by 20% on ta-day, and by 10% during 7 days. dbdn in the 1st day increased 2,0 times, but decreased to 0,6 times during the next week. with subway explosion in 2010 40 people died, 88 were injured. the number of d-rbcus increased by 10% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 6,5 times, and in the next 7 days -1,6 times. with the explosion in airport in 2011 35 people died, 180 were injured. rbcus delivery increased by 50% on ta-day, and by 40% during next 7 days. dbdn in the 1st day after ta increased 6,1 times, and in the next 7 days -2,0 times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for 7 days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of 70,000 blood units. 2000 patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. 1000 patients were taken as control and the other 1000 were taken as cases. 2482 blood units of healthy donors were chosen (2442 were males and 40 were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to 1000 patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of 6 months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in 5 cases (0.5%) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common (99.44%) followed by d (95.45%), c (89.65%), c (53.1%) and e (17.65%). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of 12.5 million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab0, rhesus and kell systems were analyzed retrospectively in 352362 blood donors for 14 years (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab0, rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih-1000" (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) 35.8%, 0 (i) blood group 34.3%, b (iii) 21.3%, ab (iv) 8.6% (n = 352362). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was 82.8% and 17.2%, respectively. donor's phenotype detection was routinely conducted from the 2013 year, therefore the number of donors was 152883. the most common phenotype among donors ccdee (32.61%), the second in frequency ccdee (19.33%), the third in frequency rhesus negative phenotype ccddee (15.42%) in the studied population. the ccdee and ccdee phenotypes were 14.04% and 12.17%, respectively. the most rare are ccdee (2.65%), ccdee (1.86%), ccddee (1.54%). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of 0.37% (n = 152883). cw antigen was tested in 104230 donors and was detected in 5.28%. cw is most commonly found in donors with ccddee phenotypes (2.36%), ccdee (2.03%) and ccdee (0.79%), with other variants of the data phenotype, the antigen was detected in 0.1% of the examined (n = 104230). antigen k was detected in 6.8% of donors, in 93.2% of this antigen is absent (n = 352362). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in 2014 the number of outdated units at the hospital side dropped considerably. results: since 2008 when the issued number of red blood cell units (rbc) was 5170 the amount of issued units rose to 5678 in 2011 and then dropped more or less steadily to 4850 in 2018. in this period the hospitals' programmes rose for 10% in all areas. number of donated units declined from 6330 in 2011 to 4820 in 2018. after reorganization in 2009 the number of outdated units fell from 3% of stocked units to 1.5%. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from 397 to 41 in 2018. for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens 4 times a day; at 10 a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high (99%). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of 78% blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve 100% voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january 2013 to january 2017 were collected from donor management software. the data includes socio demographic data. data has been process with spss version -17 results: during 4 years study period, total of 276,290 blood donation happened from both mobile blood collection and in-house blood collection. out of 276,290 collection, 48351 (17.5%) are from 18-24 age group; 106924 (38.7%) are from 25-31 age group 53324 (19.3%) are from 32-38 age group; 34536 (12.5%) are from group 39-45 age group; 23761 (8.6%) are from age group 46-52 and 9394 (3.4%) from age group above 53 respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal 18-38 years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in 2018 to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by 80 of the 81 blood banks authorized nationwide between january and december 2018. results: sihevi-ins© received information of 854,462 accepted donors, 33% of them with more than one donation in the same blood bank in a period of 12 months. a total of 72 abo or rhd discrepancies were identified in 69 people, who made donations in 20 blood banks (estimated risk: one discrepancy per 11,876 accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of 6,086 ae 3,785 units, representing 3.6% of the national collect). the remaining blood banks are distributors (average collection: 31,389 ae 25,704 units per year, representing 44% of the national collect). 75% of blood group typing discrepancies (n = 36) were related to the abo group. the most common discrepancy was between a typing group and ab typing group (67%) . in 25% of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = 10) or b type (n = 4). rhd typing discrepancies account for 25% (n = 18) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of 4417 edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, 1 anti-s, 1 anti-jka and 6 anti-k. in one case ih-1000 failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples (0.045%) false-positive result were observed both on ih-1000 system and neo iris and in two cases (0.09%)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih-1000 system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from 2017 to 2018 in taiwan. aims: the proficiency testing (pt) held semiannually from 2017 to 2018 were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into 4 groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of 11 kinds of routine work categories of 841 blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the 11 categories were as follows: 1. characteristics of institution 2. the equipment of blood bank 3. the kinds of tube in blood bank 4. the present kinds of blood bank tests 5. abo and rh type tests 6. the cross-match tests 7. the irregular antibody tests 8. hemovigilance system 9. other blood bank tests 10. massive transfusion protocol 11. quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of 3rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum 7 days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since 2001 year with 844 collections of stem cells (686 in patients and 159 collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt 128 standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, 2017 to dec, 2018) qc data from archi-tect i2000sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of 6532 data points (3523 data points in 2017 & 3009 data points in 2018) were obtained for 10 different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% (5.70-12.05) were within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2018. five out of six positive quality control levels cv% (5.72-15.80) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2017 except syphilis tp positive control (15.8%). all four negative quality control levels showed the sd values within 0.009-0.41 in 2017 and 0.009-0.41 in 2018 respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: 1. to assess the existing transfusion practices in the institute with specific quality indicators 2. to introduce corrective reforms to improve the existing practice 3. to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, 295 transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within 30 min of release from the blood bank (iii) close observation of transfusions for the first 15 min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within 30 min of issue. transfusion practices were once again monitored by following up 306 transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. 57.3% transfusions were verified in phase i while 73.6% were verified in phase ii (p < 0.001). 69.5% transfusions were started within half an hour of issue while in the second phase, it rose to 83.1% (p < 0.001). 47.8% transfusions were observed in the first 15 min in phase i and 88.6% were observed in the second phase (p < 0.001). in phase i, 54.6% transfusions were completed within right time while the same in phase ii was 73.9% (p < 0.001). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a1 & b) antibodies were selected. the titers of 126 samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, 12 of these samples were tested in the 2 automated systems for 5 times (120 datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured (3 expert technicians) in 2 different sample workload (1 and 10 samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, 2 different sample workload (1 and 10 samples testing) were assessed in manual method (3 expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ 1 titer: 73% ≤0.5 titer). the results proved that both instruments were equivalent in performing titration (differences ≤ 1 titer; 85% ≤0.5 titer). the precision results showed no difference between titers obtained through the 100% of the runs performed with the grifols analyzers (differences ≤ 1 titer: 75% ≤0.5 titer). the manual hands-on in automated system was reduced in a 15% compared to manual method for 1 sample. when the number of samples was increased (10 samples), the difference in hands-on in was even more reduced (65%). in addition, the walk-away was 46% higher in automated system compared to manual method. furthermore, when the number of samples was increased (10 samples), the walk-away difference was increased even more (78%). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least 15% the hands-on, increasing at least 46% the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by 30%>50% of users and "easy" and by 50-80% of users; 90%>100% of the users considered "sufficient" the design to ease the interaction; and 60%>80% of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), 40% of users considered tasks "very easy" or "easy"; 40% of users considered "sufficient" the design to ease the interaction; and 40% of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), 100% of users considered tasks considered "very easy" or "easy"; 90% of users never found any situation of not knowing how to proceed; and 100% of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno (1 and 2) and correspondent two mp-readers lyra (1 and 2) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a1, b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of 4 + 2 tubes of whole blood and 2 tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno 1/lyra 1 system. ward to ward. methods: a prospective observational pilot study was done for around 140 prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to 90 min, with highest time taken 795 min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be 10 min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to 70 min, maximum being 310 min (jan 2019), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p-059 hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: 204 healthy blood donors of greek origin (18-24 years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < 0.05. results: b-thal-het represented 9% of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch (11% p = 0.039, 26% p = 0.008 and 30% p = 0.006, respectively) and b) increased rbc count (20%, p = 0.042) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = 0.053) and serum total protein concentration slightly reduced (p = 0.054) in the same group. a trend for higher plasma antioxidant capacity (p = 0.052) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by 13%, p = 0.022) and hemolysis (by 41%, p = 0.001) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = 10 per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > 0.05). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of 23) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding 100,000 per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years 2010-2017 obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from 400 in 2010 to 716 in 2017 (+79%). in the group of male donors this trend remained rather stable with a slight decrease: from 181 in 2011 to 151 in 2017 (à16.6%). in the group of female donors the growth was more prominent: from 219 in 2010 to 565 in 2017 (+158%). in terms of the recorded confirmed hcv infections a downward trend can be observed: from 63 in 2010 to 16 in 2017 (à74.6%). in the group of male donors from 43 in 2019 to 12 in 2017 (à72%), in the group of female donors from 20 in 2010 to 4 in 2017 (à80%). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last 6 months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a 6 month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, 18.3% of donors who received a reminder message attempted to return compared to 12.8% of donors in the control group (p < 0.05). looking at each time point, donors who received the message 1 week before their deferral ended were 64% more likely to attempt to return compared to the control group (p < 0.05). the 1 week prior reminder message was particularly effective with males, with 30.3% attempting to return to donate, compared with 25.2% of females (p < 0.05). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide 100% voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of 1000 interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: 1. provide 100% voluntary donation for safe blood. 2. establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with 60 questions in total was divided into 6 sections out of which 14 questions on blood practices were answered by all 1000 interviewees. 416 people answered 9 questions on the blood transfusion service. 5 questions on blood knowledge were answered by 270 people. 5 questions on the knowledge of the blood transfusion were answered by 220 people, 17 questions on blood donation were answered by 420 people and 10 questions on the communication channels were answered by 500 people. results: out of 1000 interviewees, 19% have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, 40% did not donate, but expressed the readiness to donate in the future, 10% have donated voluntarily only once, 24% were family donors, 3% regular volunteer donors, and 4% have donated voluntarily several times and did not want to donate anymore. from those who have donated, 59% have donated for one of their relatives, 28% have donated for thalassemic children, 10% have donated to benefit free check-up and 3% have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, 57% have answered yes, 22% no and 21% were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around 60% of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with 3 adults of 10 overweight and 1 of 10 obese in 2017 with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a 41 item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < 25 kg/m 2 , overweight with a bmi ≥ 25 and < 30 kg/m 2 , and obesity with bmi ≥ 30 kg/m 2 . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least 3 donations in the last 2 years, and were eligible to donate. results: of the 2468 blood donors enrolled between july 2017 and january 2018, 1390 were regular repeat donors, 1102 did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and 288 accepted, of which 83 were deferred from blood donation and were excluded from the analysis. among the 205 included participants 68.3% (n = 140) were male, age ranged from 19-61 years with a mean age of 39.8 ae 11.1 sd and 31.7% (n = 65) were female age ranged from 20-62 years with a mean age of 37.7 ae 11.0 sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found 50.7% in men and 16.9% in women. our survey showed that 84.4% of the participants evaluated their health as "good", without gender difference (men, 86.4% vs women, 80.0%). besides, 14.6% reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged 18-25 attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics 22.0. categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < 0.05 was considered statistically significant. results: a total of 600 students were surveyed (300 squ, 300 non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, 73% of the squ and 25% of non-squ students reported the university as the main source for information (p < 0.001), while 56% of squ and 45% of non-squ students reported that the social media was the main source respectively (p = 0.048). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = 0.868). about 84% of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation (44.3% vs 29.7%, p < 0.001). squ students reported greater influence of peers (84% vs 60.7%, p < 0.001), personal knowledge (82% vs 74.7%, p = 0.029) and personal experience (79.3% vs 69%, p = 0.005) when compared to non-squ students. they also reported more feeling of commitment to the society (90.3% vs 78%, p < 0.001). squ students reported lower influence of parents (53% vs 65%, p = 0.005), lower rates of fear from needles (18% vs 32%, p < 0.001) and lower rates of fear from blood (16% vs 29%, p < 0.001). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about 15% of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately 85% of the expatriate population (and 71% of the emirate's total population) are asian, chiefly indian (51%) and pakistani (16%). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in 2019. the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of 1080 donors were surveyed and asked about the languages which they can read and understand and 1860 responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = 760; 41%), arabic (n = 272; 14.6%), hindi (n = 268; 14.4%) and malayalam (n = 233; 12.3%). the donors come from different countries, most common 591 (54.7%) donors are indian and 73 (6.8%) are from uae. it was found that 45% donors can read and understand only one language. majority 884 (81.9%) donors can read and understand either of the official languages arabic or english. however, 196 (18.1%) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, 1290 responses were obtained. the most common mode of communication were sms and telephone (85% together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as 18.1% donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type 2 diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a1c (hba1c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period 2015-2017 and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was 83.7%, plasmapheresis donors -13.2%, blood cell apheresis, including plateletapheresis, donors -3.1%. for the period 2015-2017, the percentage of repeated and regular whole blood and apheresis donors increased from 66.2% to 72.2%. the percentage of first-time donors ranged from 27.8% to 33.8%. the largest proportion of plasmapheresis donors was observed in the volga fd (22.0%). about 28.9% of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from 1.9% to 2.3%. the largest percentage of plateletapheresis donors was observed in the central fd (2.9%), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to 69.9% in 2017. actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between 20 to 23 years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january 2016 to december 2018. the data were retrieved from the official reports of each mobile blood donation. results: a total of 828 young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is 25.6%. the main causes of deferral are low haemoglobin (hb) level (20.8%) followed by low blood pressure (16.0%), upper respiratory tract infection (11.3%) and sleep less than 5 h (9.4%). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than 5 h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is 12.7% and in non-pregnant women is 30.2%. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was 454913. repeated blood donors was 44.8% of total population and had the highest number of referrals, followed by first and lapsed donors with 31.2% and 24% respectively. in terms of gender distribution, 6.2% were female and 93.8% were male. the highest rate of hemoglobin level less than 12.5 g/dl was found in first-time donors with 51.6% and the lowest prevalence was observed in lapsed donors, followed by repeated donors with 24.7%. 44.8% of the repeated blood donors have hemoglobin higher than 16.5. there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit 50,000 blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of 10% (500,000) of the finnish population. (11.58%), dental examination 10(3.32%) and medication history 6(1.98%). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were 5(1.65%) and 8(2.64%) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years (2016-2018). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold (120 g/l in women, 130 g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies (15.9% versus 3.4%) or in continental france (2.9% and 0.8%). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march 11 and march 28, 2019. results: this study ferridon has been approved by ethical research committee. nine thousand (9000) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, 150 donations should be included in the french west indies and 120 in reunion island. additionally, 1500 whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than 26 ng/ml and iron overload is defined by ferritin higher than 300 ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may 2019 to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around 10 million people, only 3.8% are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of 95% in 2018, instead of 80% in 2016, which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . the mean interval between donations is shorter for former regular donors (4.9 months, p < 0.01) whereas donors with an interval of 9 to 12 months are more likely to be regular (aor 16; 95% ci 1.6-164). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least 60 9 10 9 cells per dose), and in kazakhstanin therapeutic doses (at least 200 9 10 9 cells per dose). in 2017, the estimated consumption of platelets in kazakhstan exceeded the russian indicator by 28.0%. 86% of platelets in kazakhstan and 69.9% in russia are harvested by the apheresis method. inactivation of pathogens is performed in 92% of platelets in kazakhstan and in 15.4% in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in 2017 exceeded the russian indicator by 141.4%. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in 10% of plasma in kazakhstan and in 3.6% in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. 0.7% of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p-091 finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: 1. no bacteria growth near the puncture spot (result 0 cfu) in ≥ 90% of the samples. 2. total amount of bacteria on average is at most 2 cfu/25 cm 2 . at most 10% of samples are allowed to have 2-10 cfu/25 cm 2 . methods: microbiological samples were taken with contact plates from 30 voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from 20 to above 500 cfu/25 cm 2 in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean (0 cfu/25 cm 2 ) in 93.3% of the samples and 86.7% of the samples had 0 or only 1 cfu/25 cm 2 . the average number of bacteria after disinfection was 0,6 cfu/25 cm 2 and the maximum number was 4 cfu/25 cm 2 . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the 2018 meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb201 + (hemocue ab) point-of-care (poc) device. donors with hb < 125 g/l for females or < 135 g/l for males or with a decrease > 20 g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = 305) and control (n = 331) groups, the proportion of male donors (27% and 26%) and the mean initial skin-prick hb (122 g/l and 123 g/l) were similar. significantly less donors were deferred from donation in the study group (40%) than in the control group (51%; chi-square test p = 0.004). the mean difference in venous hb with the poc device versus the hematology analyzer was à3 g/l (range à12 to + 6 g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only 1 g/l below the limit of donation eligibility (124 g/l for a female and 134 g/l for a male). interestingly, a further 33 donors (27% of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above 60 years of age. this age group has been reported to use up to 50% the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged 60 years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ 60 years from blood centres of four countries. the estimated proportion of blood donors aged ≥ 60 years old for each country was 0.81% in south korea (sk); 2.57% in hong kong (hk), <3.18% indonesia (indo) and 9% in japan (jap). the minimum hb criteria varied between each country and ranged from 11.5-12.5 g/dl for women and 12.5-13.0 g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe-2100d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are 6 medication questions on the donor history questionnaire (dhq), including any medication use in the last 3 days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in 2017 were included in the analysis. donors' answers to each of the 6 medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were 975,067 donation attempts with a completed dhq. overall, 34% of donors answered yes to medications in the last 3 days, 8% to vaccination, and less than 0.1% to others (38% any). slightly more were female (42 vs 36%) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from 24% of 17-19 year olds to 57% aged 60 + (p < 0.0001 for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel 7 and tomes software j schreier 1 , a davison 1 , j gambarte 2 , y l opez 2 , c calonge 2 and e herranz 2 1 terumo bct, lakewood, united states 2 centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than 4000 apheresis platelet collections were completed in 2018, of which almost 3000 were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel 7 was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel 7 devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel 7 with tomes compared to trima accel version 6. methods: this was a retrospective study analyzing 1372 apheresis procedures on trima accel version 6 during the control period from 2 january 2018 to 10 september 2018 compared to 541 apheresis procedures on trima accel 7 during the test period from 11 september 2018 to 28 december 2018. this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a 2-proportions test, whereas donor demographic data were analyzed using a 2-sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = 3.0 or 3.5 9 10 11 ) or double (target platelet yield = 6.0 9 10 11 ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = 5148 ml, test = 5124 ml, p = 0.545), hematocrit (control = 43.7%, test = 43.6%, p = 0.233), or platelet pre-count (control = 249 9 10 3 / ll, test = 253 9 10 3 /ll, p = 0.091). females represented 21% of donors in the control arm compared to 23% of donors in the test arm. platelet split rate (platelet products per procedure) increased from 1.15 with trima accel version 6 to 1.18 with trima accel 7; procedure time decreased from 55.7 min to 53.3 min for single collections and from 63.0 min to 60.9 min for double collections with trima accel 7 (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from 62.4% to 84.8% (p < 0.001) and the rate of completed apheresis procedures increased from 89.8% to 92.4% (p = 0.083) with trima accel 7. manual transcription of data during the procedure was discontinued with the implementation of trima accel 7 with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel 7 significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the 19 th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec 2017, distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of 36 respondents, 24 bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering 2,888,390 plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per 10,000 plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to 16% of the estimated total blood volume but maximally 750 ml, including anticoagulant. respondents had differing practices and scale of collection program 15 be were aligned or lower, and 9 be had higher collection volumes. altogether 14 reported the immediate vvr with loc rate, which mainly was lower than 10/10000 collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by 16 respondents. the retention rate banded in the range of 50%>80% (mean = 60%, min = 25%, interquartile range = 14%, max = 90%). the association between maximum allowed yearly plasma collection (25 l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide 0 s limits for collection volume of maximum of 750 ml per donation and 25 l per year per donor. methods: serum ferritin concentrations were established from sera stored at à30°c from repetitive platelet donors between 2014 and 2016, using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn 1800 â . sixteen samples were obtained from women (age: 35.3 ae 12.4 years, range: 18-61) and 35 samples from men (age: 36.2 ae 9.4 years, range 20-61), corresponding to 55.2% and 37.2% of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, 43.8% of women and 40% of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was à38.4 ae 52.2 ng/ml when the donations had an interval less than three months, vs 3.3 ae 13.0 ng/ml when the time between donations was higher (p = 0.046). in men the change in the ferritin levels was à38.5 ae 61.2 ng/ml with donation times less than three months vs © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 à3.3 ae 38.9 ng/ml with prolonged donation times (p = 0.042). in women, the change in platelet count was à2 ae 46.2 9 10 3 /ul, when the donations had an interval less than three months vs à22 ae 45.3 9 10 3 /ul when the time between donations was greater (p = 0.43). in men, the delta of the platelet count was à12 ae 37.0 9 10 3 /ul in donation times less than three months vs à13 ae 26.6 with higher donation times (p = 0.93). no correlation was found between the concentrations of serum ferritin and the platelet count (r = 0.05, q = 0.75 for males, and r = 0.34, q = 0.22 for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version 7 (ta7) by comparing its routine performance with that of the previous software version 6.06 (ta6). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta6 and ta7 sequentially. data were collected from december 2017 to april 2018 on ta6 and from june to july 2018 on ta7. simple and double doses of platelets (respectively 5.0 9 10 11 , 5.5 9 10 11 and 8.0 9 10 11 , 9.0 9 10 11 ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from 1.06 (ta6) to 1 (ta7). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety (590) collections with ta6 and 607 with ta7 were recorded, with 92% and 95% complete procedures respectively. mean duration of procedures was 70 min on ta6 against 72 min on ta7, p < 0.05. the mean alerts number per procedure on ta6 was 4.1 against 0.5 on ta7, p < 0.05 whereas the maximum alerts number per procedure was 57 and 12 respectively. on ta7, 76% procedures did not require operator's intervention against 40% on ta6,. with ta7 the inlet flowrate was automatically adjusted in 97.2% procedures. the inlet flowrate was increased in response to access pressure in 45.6% of procedures, for 3% of the procedures the inlet flowrate was decreased and for 48.6% of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta7 autoflow system. summary/conclusions: ta7 with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel 7 (ta7) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta7 leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta7 software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version 6 (ta6) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta6 from 4th january 2016 to 10th october 2017 were compared to 995 procedures, started on ta7 from 18th october 2017 to 31 st december 2018. procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta6 vs. ta7 respectively) were comparable and were characterized by: tbv -5207 vs. 5344 ml; platelet count pre-procedure -252 9 10³/ll vs. 252 9 10³/ll; hematocrit pre-procedure -44% vs. 44%. gender distribution was 11% female with ta6 vs. 2% with ta7. venous access pressure alerts were significantly improved by ta7 with an average of 0.2 alerts per procedure as compared to 2.0 with ta6, i.e. 92% decrease. this decrease went down to 91% if only male procedures were analyzed. the maximum number of pressure alerts went down by 84% from 90 alerts in one particular run in the ta6 cohort to 14 alerts in one ta7 procedure. procedure time for single platelet products was reduced from 51 to 49 min and for double platelet products from 59 to 54 min (ta6 and ta7 respectively). donor qualification possible was 52% of procedures yielding single products and 48% of procedures yielding double products with ta6. the percentage of procedures qualifying for doubles increased to 91% with ta7. in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from 1.48 to 1.91 from ta6 to ta7, respectively. in fact, the observed split rate rose modestly from 1.01 to 1.05, as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta7 decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta7 has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel 7 to trima accel version 6 august 11 2017 to october 17 2017 or trima accel 7 during the test period from november 28 2017 to january 9 2018. this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: 63 donors completed the survey during the control period whereas 86 donors completed the survey during the test period. the mean number of previous donations for the control period was 3.0 (min 1 max 6) and for the test period was 3.2 (min 0 max 7). there were no first time donors during the control period and 20 first time donors during the test period. 91% of donors rated their overall donation experience as good on trima accel 7 compared to 90% on trima accel version 6. zero (0) donor rated their experience on either trima accel device as poor. 100% of donors who responded to the question said they would donate on trima accel 7 again. summary/conclusions: no significant difference was observed in donor experience between trima accel version 6 and trima accel 7 as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in 2014, a higher number of the platelets was consumed compare to 2011 by 7.8%. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in 2018, 635 (8.6%) of the 7,371 patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is 14,292, which is 668 higher (by 4.9%) comparing to 2017. platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + 9000 (haemonetics), upp and trima accel (terumo bct) version 6.0 protocols. methods: the data for 4868 protocols of platelet donations performed in 2018 were analyzed: 2773 on trima accel and 2095 on msc + 9000. all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was 32 years old, height -175 cm, weight -76 kg, platelet count before donation -254 9 10 9 /l, hematocrit -42%. detailed data are presented in table 1 . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than 0.05 was considered as significant. results: the data obtained showed significant difference (p < 0.01) between average number of platelets collected on trima accel (6.3 ae 1.06 9 10 11 /l) and on mcs + 9000 (5.09 ae 0.78 9 10 11 /l). while cost of consumables are comparable, trima accel demonstrated 23.7% higher efficiency. procedure duration also was comparable and averaged within 94 min for both devices. detailed data are presented in table 2 . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during 2018 and reached 56, 7% in total (2017-19.4%). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from 7.64% up to 43.7%. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by 23.7% compared to mcs + 9000 while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from 2010 till 2019. all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ 3.0 9 10 11 equal to 12 random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was 150.000/ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was 273.000/ll, with range from 182.000/ll to 397.000/ll. male were 77% of the donors and females were 23%. the single procedure usually took 45-70 min. the median platelet count collected was 4.0 9 10 11 , range 2-6.5 9 10 11 . the median processed blood volume was 3215 ml and median used acd-a was 352 ml. mean total volume of collected product was 312 ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso4) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso4 solutions with specific weight (y) 1.053 can to detect value of hemoglobin above or same with 12.5 gr/dl. but, cuso4 solution specific weight 1.053 can not to detect and elimination value of hemoglobin above 17 gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso4) with a specific weight (y) 1062 to detect value of hemoglobin below 17gr/ dl and determine value of hemoglobin above 17 gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above 17gr/dl with the test of 35 samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso4 solution with specific weight 1.062 in to detect and elimination value of hemoglobin donors above 17 gr/dl. methods: used the method cyanmethemoglobin and cuso4 (y) 1.062 determination value of hemoglobin donor. test results were analyzed with spss software version 23 using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso4 solution with specific weight (y): 1.062 in detecting and eliminating hemoglobin value donors above 17gr/dl. from data processing using spss with the wilcoxon test p value 0.02. summary/conclusions: it was found that the cuso4 solution (y): 1062 can detect hemoglobins value above 17gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version 23 with the wilcoxon test p value < 0.05. it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, 8194 whole blood donors (20,864 whole-blood donations) were included in the zinc study, 63% being female donors. previous zpp showed a statistically significant association (p < 0.001) with hb levels in females, but the size of the association was quite small (regression coefficient, b = à0.17, 95% confidence interval à0.22 to à0.11). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was 66 years until the end of year 2013 and since beginning of 2014 donors between 66 and 70 years have been able to donate blood. blood donation after the age 66 is possible if the donor has donated within the last 24 months. the upper age limit was raised up based on adverse event data from frcbs donors up to 66 years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from 2013 with upper age limit of 71 years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than 66 years have more adverse events compared to other donors. the most common donor adverse event for donors over 66 years, haematoma, was registered 211 times (0.05%). in the other age groups haematoma was registered 4408 times (0.56%) and the difference between the oldest age group compared to all other donors was statistically not significant (chi2, p = 0.012). vvrs with loc were registered 21 times (0.05%), vvrs without loc 25 times (0.06), and the total number of all daes was 293 (0.65%) in the age group 66 years or older. the respective numbers in the other age group were: 1152, 0.15%; 11055, 1.41%; 17550 (2.23%). the number of vvrs with and without loc, and total number of all daes in the age group 66 years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi2, p < 0.0001). summary/conclusions: donors over the age of 66 years have less donor adverse events than other age groups. decision to raise the age limit from 66 to 71 seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in 2016-2018 years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last 3 years (2016-2018 inclusive) were reviewed to look for reports of dvt post donation results: a total of 135 saeds were reported in uk from approximately 5.8 million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for 2% of the saeds reported and rate of dvt of 1 in 1.9 million donations collected. -case 1: a regular male whole blood donor in his early 30s reported worsening arm pain 12 days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case 2: a female donor in her mid-40s gave her sixth whole blood donation without event. 11 days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case 3: a female donor in her 20s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february 2013 to march 2015. the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study 6,955 whole blood donors were included, during the period of 2 years, 105 (1.50%) adverse donor reactions were reported. majority 89 (84.76%) of adverse donor reactions were mild in nature such as, sweating; 27 (25.72%), light headedness; 19(18.09%), nausea and vomiting; 15(14.28), allergy and bruises; 11(10.47%), sore arm; 9(8.58%) and hematoma; 8(7.62%) while 16 (15.24%) were severe adverse reactions similarly, anaphylaxis; 11(10.49%), loss of consciousness; 3(2.85%) and convulsive syncope; 2(1.90%). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with 18-23 years old at higher risk compared to 24-55 years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th1/th2 cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, 2008; ellertsen & hetland, clin mol allergy, 2009). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for 7 weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm (82%), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v 1-induced basophil activation in whole blood determined by cd63 expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct03198455, clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v 1 and anti-t3, were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for 2 months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in 20/9/2014. the data presented in this abstract is till 28/2/ 2019. data is collected from total of 114475 blood donors (86.67% male donors and 13.33% female donors). repeat donors accounted for 59.34% against 40.66% of first time donors. of the total number of donor adverse events recorded, 2.73% (2633) reported for male donors and 6.97% (1063) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group 18-24 years (male 5.45% and female 13%). among age group 25-40 years, (male 2.4% and female 4.02%), whereas in age group 41-65 years, (male 1.52% and female 2.11%) data analysis of total 6226 reported and registered donor adverse events, are categorized as hyperventilation (864), sweating (1458), dizziness (pre-syncopal 3160), loss of consciousness (416), vomiting (108), convulsions (220), hematomas with re-bleed (255), nerve irritation (8) and off-site reactions (333). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january 1 st 2016 to december 31 st 2018 and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january 1 st 2016 to december 31 st 2018 was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into 8 categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < 0.05 was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, 193,228 donors were registered from january 1 st 2016 to december 31 st 2018 and 41,037 (21.24%) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from 24.07% (15302/63563) in 2016 to 20.79% (13180/63394)in 2017 and further to 18.74% (12419/66271) in 2018 (p < 0.05). the specific deferral rate due to low hb also significantly (p decreased during these three years (86/1000 in 2016, 68/1000 in 2017, 58/1000 in 2018), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june (6/1000) and then increasing with a peak in october (19/1000) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of 4/1000 in june and increasing to 10/1000 in october (p < 0.05). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels 2017 thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of 28 days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from 2015 to 2018 on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the 7 days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in 2015-2016 and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in 2015-2018, 16,400 donation attempts led to onsite deferral for wnv risk; 87% at whole blood donation attempts, 13% at new donor examinations. in total 19,624 offsite deferrals could not be traced directly to a donation, but based on the next donation more than 90% were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from 920 in august 2015 to 1711 in august 2018. this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred (92% versus 95%); for wnv deferred new donors the return rate is 75% (versus 87% for no deferral). thus wnv deferral resulted in approximately 400-500 extra lapsing donors during these 2 years. however wnv deferred donors, that are older (odds ratio (or) 1.03; 95% confidence interval (95% ci) 1.02-1.03), of male sex (or 1.16; 95% ci 1.03-1.31) and whole blood donors as opposed to new donors (or 2.01; 95% ci 1.70-2.38) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since 2012 with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given 14 days of one tablet of iron supplementation (100 mg elemental iron) since 2012. an electronic questionnaire was sent to blood donors in 2017 to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: 3212 donors (male to female was 1:1.9) replied to the questionnaire. of them, 594 received iron supplementation (male to female was 1:1.6). most of the respondents (94%) had one or more donations in the preceding 12 months. of the 594 donors received iron tablets, only 246 (41%) took all; 69 (12%) took more than 50% but not all; 130 (22%) took some but less than 50% and 149 (25%) did not take any. gastrointestinal upset was reported in 113 (25%) donors and constipation seen in 193 (43%) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than 50%) to those who did not (took less than 50%), significantly more donors in the former knew vitamin c could enhance iron absorption (p < 0.05). on the other hand, no difference was seen when they were asked if 1) iron can only be absorbed from meat; 2) tea and coffee consumed during meal can enhance iron absorption; 3) everyone can take iron supplementation on their own; and 4) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by 27% for whole blood donors and 22% for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with 36 whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor 10,000 study in 2015. we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may 2015 and december 2017. participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer 2018. we included the 1435 participants (596 men and 480 premenopausal and 359 postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = 31), good (n = 483), very good (n = 684) or excellent (n = 237) health tended to report improved (58%), similar (63%), similar (55%) or poorer (55%) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal 51%, post-menopausal women 38%), or = 1.37 95% ci 1.02-1.85). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in 2018. methods: the data of volunteers from january to december 2018 were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were 9081 blood donors in 2018, 69 (0.76%) of whom had adverse reactions and 9 causes were induced, among which mental stress was the most common factor that accounted for 78.26% (54 cases). there was no significant difference in the incidence of adverse reactions between men and female (p > 0.05). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < 0.01). when it comes to age, the incidence was different and the 18-25 age group was the highest (1.61%). among different blood group donations, there was no significant difference (p > 0.05). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least 8 weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to 24 times per year (if spaced more than 14 days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥4 whole blood donations or 16 plasmapheresis or plateletpheresis donations, and female donors with ≥3 whole blood donations or 12 component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past 2 years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ 2.07. the repeat donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of 53 male and 57 female repeat donors were recruited, and 30 each male and female ft-ra donors were recruited to the control group. after 4week iron supplementation, among male donors, the prevalence of: low hb level (<13.0 g/dl) decreased from 24.5% to 1.9%; low ferritin level (<15.0 ng/ml) decreased from 58.5% to 3.8%; high tibc level (>380 lg/dl) decreased from 66.0% to 37.5%; low transferrin saturation (<20.0%) decreased from 58.5% to 35.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 28.3% to 2.3%. among female donors, the percentage of: low hb level (<12.0 g/dl) decreased from 43.9% to 9.8%; low ferritin level (<15.0 ng/ml) decreased from 78.9% to 11.8%; high tibc level (>380 lg/dl) decreased from 68.4% to 24.5%; low transferrin saturation level (<20.0%) decreased from 70.2% to 22.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 94.7% to 44.4%. in total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. a total of 38 male (71.7%) and 36 female (70.8%) blood donors were administered iron supplementations for 28 days. 89 participants (85.6%) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, 4-week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than 3 mg/l has been independently associated with a 60% excess risk in incident of coronary heart disease (chd) as compared with levels less than 1 mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci4100 (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl 180, erba mannheim). data was analysed using stata version 15 (stata corp) statistical software. results: in total of 313 blood donors, 278(90.8%) were males and 28(9.2%) were females, the mean age was 32.3 ae 9.3 years. two hundred and thirty four (74.8%) were first time donors and 79(25.2%) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors (0.95 ae 3.7 vs 0.35 ae 1.7 mg/l, p = 0.06) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors (87.56 ae 23.9 vs 46.44 ae 31.4 ng/ml, p = 0.02). interestingly, levels of serum hs-crp were significantly higher in male than female population (0.52 ae 2.4 vs 0.17 ae 0.2 mg/l, p < 0.03) and smokers than non-smokers (1.4 ae 4.6 mg/l vs 0.4 ae 1.9 mg/l, p = 0.02). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes 250 mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = 13.5 g/dl for male and > or = 12.5 g/dl for female • inter donation interval = 56 days • 5 donations/year for male and 3/year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: 1056 first time and regular donors accepted to enroll in this study. only 38 female donors (3.6%) participated to this study. 11.3% of the participants were first time donor. 21% of male and 26% of female frequent donors are iron deficient 173 out of 1018 male blood donors were iron deficient (17%) with serum ferritin < 30 ng/ml. 98.8% were repeat donors. 7 out of 38 female donors were iron deficient (18.4%) with serum ferritin < 13 ng/ ml, all were repeat donors. 19.2% of repeat donors were iron deficient 3/180 of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a 12-month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years 2014-2018 in the group donors of aged 18-24. we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years 2014-2018 the total number of 1904 adverse reactions among the donors was recorded which is 0.39% of the total number of collected donations. 50% of the adverse reactions occurred in the group of donors aged 18-24. vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled 50.2% of all adverse reactions. in the group of donors ages 18-24 it totalled 27% of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled 29.8%, in the analysed group of donors 15.96%. vascular reactions (bruises) totalled 10.2% of all adverse reaction, in the analysed group 5.6%. the remaining adverse reactions totalled 9.8%. summary/conclusions: 1. vasovagal reactions (with and without fainting) were proved to be 2 most common adverse reactions in the group of donors aged 18-24 i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. 2. it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. 3. it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" (2 nd day) and "old" (42 nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd235a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd235a, cd44, cd47, cd55, cd59, and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0.5 lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units (201 ae 139 mvs per 1 ll), as compared to the microvesicles in the "fresh" (67 ae 53 mvs per 1 ll). at day 2, the microvesicles had elevated expression levels of cd47 and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd55 and cd59 molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of 42-day vesicles. the phagocytosis index was significantly higher (28.44%) for the microvesicles isolated from 42-day stored rbcs than for microvesicles from the 2background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of 5-7 days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to 7 days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in 30% plasma/70% ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into 3 units (n = 6 of each) on day 2 and stored either at room temperature (rt; 20-24°c with agitation) for 5 days, cold stored for 21 days (2-6°c no agitation) or cryopreserved (à80°c with the addition of 5-6% dimethyl sulfoxide) for 33-109 days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at à80°c. the mean size and concentration of microparticles were measured using nanosight ns300 nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < 0.05 was considered significant. results: at day 2, sheep pcs had a microparticle concentration of 3.77 9 10 10 ae0.84 9 10 10 microparticles/ml with a mean size of 156.6 ae16.7 nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration (4.39 9 10 10 ae0.81 9 10 10 microparticles/ ml; p = 0.0087) and the mean size (170.9ae 20.5 nm; p = 0.0008) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day 21 was comparable to room temperature pcs stored for 5 days (165.4 ae17.5 nm vs. 163.6 ae20.9 nm; p = 0.4081 and 4.11 9 10 10 ae 0.54 9 10 10 microparticles/ml vs. 3.82 9 10 10 ae0.71 9 10 10 microparticles/ml; p = 0.16 respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the 21 days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and 2,3-dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p50: the oxygen tension (po2) at which 50% of the hemoglobin is saturated with o2. an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po2. this relationship is dependent on temperature, ph, pco2 and 2,3-dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p50 is at a po2 of about 26 mm hg. not much is known about p50 of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. 2010;50:2386-92). methods: rbcs were prepared in sagm (n = 3) or pagggm (n = 3). pagggm is designed to better maintain both atp and 2,3-dpg during storage. rbcs were stored at 2-6°c and sampled on day 1, 35 and 56 for (internal) ph, atp, 2,3-dpg and p50. p50 was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment (2%) using n2 gas. p50 was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. 2,3-dpg content of sagm-rbcs decreased during storage and was below the detection limit after day 14. 2,3-dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day 7 on. at day 56, pagggm-rbcs still contained 2.3-dpg (2.2 lmol/g hb). p50 values decreased during storage from 26 mmhg at day 1 to 20 mmhg at day 56 for sagm-rbc and from 31 mmhg to 26 mmhg for pagggm-rbc. p50 values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p50 decreased in all rbcs. the p50 was higher for the pagggm-rbcs during the whole storage period. the higher p50 in pagggm-rbcs seems to correlate with the higher 2,3-dpg content in these cells. background: in belgium 100% of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of 3.0 9 10 11 per platelet concentrates (pc). therefore routine pools are produced with 6 buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from 6 to 5 bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with 5 bc and 250 ml platelets additive solution (pas) instead of 280 ml for 6 bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ 3.0 9 10 11 platelets with a ratio plasma/pas between 32 to 47% required for pi. after validation, deploy a dual pooling strategy (5 or 6 bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce 44 ml bc with 40% haematocrit (htc) and > 90% platelets recovery with average platelets content of 1 9 10 11 . 5 random bc are pooled with 250 ml or 6 bc are pooled with 280 ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl 80; horiba). results: during the study 45594 bc were processed into 8225 pools (3756 (45.7%) with 5 bc and 4469 (54.3%) with 6 bc). before tacsi separation, bc mixture with pas-e had volumes of 421 ae 9 ml (5 bc) and 491 ae 8 ml (6 bc) with respectively htc of 19 ae 1% and 20 ae 2%. the plasma/ pas ratio was 37 ae 1% in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was 3.8 9 10 11 ae 0.5 with 5 bc and 4.7 9 10 11 ae 0.5 with 6 bc (average ae standard deviation). pools below the limit of < 3.0 9 10 11 were 139/3756 (3.7%) with 5 bc and 1/4469 (0.02%) with 6 bc. the platelets concentrations (10 3 /ll) were 1395 ae 167 (5 bc) and 1491 ae 155 (6 bc). platelets recovery was 85% ae5 for 5 bc and 86% ae 6 for 6 bc. summary/conclusions: 45594 bc could theoretically produce 7599 pools of 6 bc or 9118 pools of 5 bc. this means a maximum potential gain of + 20% pc. in practice during shortage periods we switched from 6 to 5 bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods (626 pc, +8%). the disadvantage of pooling randomly 5 bc is that 139 pools contained less than 3.0 9 10 11 platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the 5 bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica 2018, pmid: 30115655). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and 4°c in the presence or in the absence of caspase-3 inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: 13 ae 1% vs. 5 ae 1% p = 0.018). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: 6.13 ae 1.89 vs. 18.53 ae 3.64, p = 0.038). accordingly, a decrease of the procaspase-3 level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase-3 inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: 74 ae 3% vs. 22 ae 3%, caspase 3 inhibitor vs. ionomycin, p = 0.005). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o 2 , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o 2 (so 2 )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within 24 h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for 3 h on an orbital shaker (50 rpm) at 22°cae2 and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at 4°c. a g-irradiation treatment (25 gy, gammacell 3000 elan, theratronics) was applied at day 2 or 14 and the rccs (expiry dates at day 16 or day 28, respectively) were stored until day 43. hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so 2 values were of 63.7%ae18.4 (n = 12) in control and of 20.8%ae9.8 (n = 12) in treated bags, and reached 90.8%ae9.1 and 6.6%ae5.9 at day 43, respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around 70 mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day 2 and the values stayed equivalent through end of storage (at day 16, hemolysis (control) = 0.37%ae0.24, hemolysis (treated) = 0.34%ae 0.15, p-value > 0.9999). when irradiated at day 14, hemolysis was lower (p-value = 0.033) in treated rccs at the end of storage (day 28, 0.67%ae0.16) compared to control (1.06%ae0.33). seven days post-irradiation, two-third of the control rccs were above the limit of 0.8% whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o 2 content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day 2 and was an advantage when irradiated at day 14. importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o 2 depleted rbc. in summary, the reduction of o 2 level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, 2017] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as 0.5 ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a 3d printed support. the accuracy of flow rate measurements between 0.5 ml/min and 1.5 ml/min was evaluated as well as the optimal measurement time. for 4 different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under 8% was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under 3% for a measurement time of 2s. the mean error is still under 5% for a measurement time of 0.5s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags 2, 3 and 4. hematocrit estimation for bag 1 diverges from reference for values above 6%. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under 8% without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood (450 ml) was drawn into compoflow blood bags containing cpd and sag-m from 10 healthy controls and 10 newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing 20 ml prp (range 270-320 9 10 9 platelets/l). platelet quality tests were performed on days 0, 1, 3, 5 and 7 of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd41, cd42b, and cd62p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd40l and scd62p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included 7 males and 3 females. the mean age was significantly lower in the control group, 35 years (22-55 years), than in the hh group, 55.7 years (29-77 years) (p = 0.004) while ferritin levels were significantly higher in hh patients (median 847.5, range 498-1889) than in controls (median 45.8 ng/ml, range 9.28-97 ng/ml) (p < 0.001). in the hh group, 5 each had the c282y/c282y and c282y/h63d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < 0.05) with the exception of glucose (higher in hh patients on all time points, p < 0.05). platelet aggregation and the expression of activation markers (cd62p and cd42b) on platelets and in the supernatant (scd62p and cd40l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd42b expression decreased and cd62p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = 16) were collected using a trima accel platform in 40% plasma/60% pas (ssp+). after resting for 1 h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (<6 h; t0), and the others remained within the shipper, without agitation. the second component was removed at 6 h post-collection (t6), and the third was removed at 23.5 h post-collection (rounded up to 24 h; t24). platelets were tested on day 1, 5 and 7 post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < 0.01 was considered significant. results: platelets held without agitation for 24 h consumed significantly more glucose than those removed at 6 h or immediately upon arrival (p < 0.0001), even on day 1 post-collection. this was accompanied by increased lactate production (p < 0.0001), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t24 platelets (p < 0.0001), and on average it was 0.4 ph units lower than in platelets held in the shipper for 6 h or less. however, the ph remained above 6.9 in all components. mean platelet volume was also reduced in t24 platelets (p < 0.0001), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd62p and microparticle generation were significantly higher in the t24 platelets throughout the storage period (all p < 0.0001), suggesting platelet activation. release of scd62p was also increased in t24 platelets (p = 0.0006), whereas extended storage in a shipper did not affect release of rantes (p = 0.4488). adp-induced activation of glycoprotein iib/iiia, measured by pac-1 binding, was decreased in t24 platelets (p < 0.0001), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = 0.0001) and adp (p = 0.003) were significantly lower in t24 platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for 24 h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to 5 days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after 5 days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, 9, 2018) . our new target is to extend this research in 2 extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from 5 to 7 days. methods: in this study, platelets were collected from 11 normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of 450 ae 10 ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc 1200). samples were drawn aseptically with a needless access coupler (cair-lgl) on days 1, 5, and 7. platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day 5 and day 7, maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days 5 and 7 of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of 7 days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at 4°c or room temperature (rt) over 10 days for 4 apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with 5% or 10% dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either 5% or 10% dmso. results: for pcs standard quality assurance tests did not show a major difference between 4°c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day 1-7, p < 0.001 and day 10, p < 0.02). for red cells, we found by rt-dc no impact of gamma irradiation with 30 gy over the entire storage period of 42 days assessing 3 different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation (0.052 for 5% dmso versus 0.034 for the control without dmso; p < 0.00004). however, this did not differ to high extent whether 5% or 10% dmso were used for cryopreservation (0.035 and 0.041, respectively; p < 0.02). hpsc viability was lower after cryopreservation using 10% dmso in comparison to using 5% dmso. overall, blood cell regeneration is comparable between 5% and 10% dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight (16 h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day 0 was separated into plasma, bc and red cell concentrates either at day 0 or at day 1. bcs were then stored until the pooling step at 20°c without agitation and pcs were prepared at day 1 by pooling 5 isogroup bcs. seven "16 h-pcs" were prepared from bcs stored for 16 h (whole blood separation at day 0) and six "90 min-pcs" from bcs stored for 90 min (whole blood separation at day 1). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd42) and of activated platelets (marked with cd62 the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: 32 dd-bc-pc were prepared with 8 bc and 280 ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a 600 ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within 1 h of preparation and after storage for 8 h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po 2 , pco 2 , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = 32) was on average 6.9 ae 0.3.10 11 in a volume of 393 ae 6 ml. the mean of plasma ratio was 42% [min: 39.3max: 43.9]. all pc contain <1.10 6 wbc [min: <lq; max: 0.38]. po 2 decreased from 166 ae 12 mmhg after separation (1 h) to 44 ae 18 mmhg after 8 h, ph (+22°c) and pco 2 was stable during the same time period. glucose decreased from 9.0 ae 0.3 mmol/l (1 h) to 8.7 ae 0.3 mmol/l (8 h) while lactate increased from 6.07 ae 0.46 mmol/l (1 h) to 6.57 ae 0.54 mmol/l (8 h). the vpm was stable with 7.6 ae 0.2 at 1 h and 8 h. ldh increased from 120.4 ae 8.6 u/l at 1 h and 125.8 ae 10.3 u/l at 8 h as p-selectin 45.7 ae 7.6 ng/ml at 1 h and 57.7 ae 7.0 ng/ ml. all units have maintained swirling. the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in 2018 the ibts collected 130,183 whole blood units and performed 9,654 platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september 2017, the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years 2016 to 2018 were analyzed. in 2016, the ibts prepared 5,609 orbisac platelet pools of which 5,477 were qc tested. in 2017 5,031 orbisac and 1,625 tacsi platelet pools were prepared and tested. in 2018 6,783 tacsi platelet pools were prepared of which 4671 were qc tested. qc data comprised pc volume, platelet content (10 9 /unit), residual leucocyte content (10 6 /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the 10,508 o-pcs tested in 2016 and 2017, mean pc volume was 292 ae 9 ml. initially the t-pcs mean volume was 273 ae 13 ml but more recently the volumes were increased by addition of a larger volume of additive solution to 303 ae 13 ml. platelet content (10 9 /unit) was significantly improved in t-pcs (384 ae 47) compared to o-pcs (356 ae 50) [p < 0.01] as well as residual leucocyte content (10 6 /unit) which was lower in t-pcs (0.05 ae 0.04) compared to o-pcs (0.1 ae 0.2) [p < 0.01]. ph at expiry was lower in t-pcs (7.32 ae 0.07) compared to 0-pcs (7.38 ae 0.06) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately 48 t-pcs per hour compared to a lower throughput of 20 o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a 10% increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga 1 , f puente 1 , a aranda 1 , j domingo 1 , l call en 1 and a bah 2 1 banco de sangre y tejidos de arag on, aragon, spain 2 terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes 44,000 whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november 2012 and routine use started in july 2013. in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november 2012 to improve transfusion safety. in november 2015, the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in 2012, 369 whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from 2013 to 2018, qc data of rccs (n = 3025), plasma (n = 660) and pcs (n = 660) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from 2015 to 2018 were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: 296 ae 21 ml vs 251 ae 19 ml; hb: 59.9 ae 6.2 g/ unit vs 49.2 ae 7.8 g/unit; hct: 59.5 ae 2.7% vs 56.1 ae 6.3%). control plasma units had higher volume (280 ae 17 ml) and lower residual platelet content (9 ae 7 10 9 /l) compared to reveos plasma units (volume: 249 ae 23 ml; residual platelet content 19 ae 13 10 9 /l). for pcs, volume was higher for units processed with reveos compared to control (350 ae 20 ml vs 310 ae 10), but no statistical difference was observed in platelet content (reveos: 361 ae 60 9 10 9 /unit; control: 386 ae 80.10 9 /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the 5 years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from 4% to 0% between 2013 and 2018 and platelet content in pcs increased from 370 9 10 9 /unit (first six months in routine) to 390 9 10 9 /unit. plasma volume increased from 249 ml during validation to 267 ml in 2018. survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from 10% in 2015 to 59.7% in 2018 with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations (450 ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm (100 ml) and then filtered by gravity in average 2-3 h after blood collection to obtain leukoreduced rbc. average temperature during filtration was 26°c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = 27) were performed. rbc characteristics post-filtration and separation were as follows: volume (266.8 ae 17.1 ml), hematocrit (57.8 ae 2.1%) hemoglobin (19.8 ae 1.1 g/dl) and hemoglobin per unit (53.0 ae 5.4 g); residual platelets (11.7 ae 2.2 x10³/ll); residual wbcs (0 à 0.004 9 10³/ll). the mean filtration time was 19 ae 3 min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between 2 and 8 h from collection using the 3c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day (42 nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate 4 whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in 2018. the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to 8 h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, 1005 therapeutic units of lpcs were produced after overnight hold: 38 pools of 4 ipus and 966 of 5 ipus were made using platelet pooling set (terumo bct), using 250 ml ssp+ solution (macopharma). results: 42 therapeutic units of lpc were analyzed. the mean volume of lpc was 387 ae 13 ml, platelet content was 3.67 ae 0.46 x10 11 , and white blood cells (wbc) count was 0.15 ae 0.13 x10 6 , 100% all lpcs had wbc below 1 9 10 6 . ph measurements were made on the fifth day of storage, the ph was determined in 15 units of lpc and was 7.2 ae 0.04. summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for 5 days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term (10 days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about 250 ml) were obtained from a healthy blood donors of type o blood group and divided into a 30 ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd62p, gpiba (cd42b), annexin v), degree of reactive oxygen species and sialidase activity on the 7th, 10th and 12th day, and phagocytosis using the hepg2 cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the 2nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day 12 and sialidase activity was maintained lower than that of control group on day 12. the degree of phagocytosis by the hepg2 cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for 2 h and 24 h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos 3c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of 2 h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within 8 h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately 4 to 6 ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool (4 units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately 19 units of whole blood using the reveos system and approximately 4 units of pooled platelet products. a total of 19 units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the 19 units, 10 units have been processed on the reveos device using the fresh procedure (within 8 h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a 22-min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn 2000 cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than 1 million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = 4) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax 20 (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including 40 units in 450 ml quadruple bags (450q) with/without integrated leucofilter and 18 units in 350 ml quadruple bags (350q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into 2 groups, mixed at 2 revolutions per minute (rpm) for 2 min (n = 30) and 30 min (n = 28), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both 450q and 350q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for 2 or 30 min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for 30 min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as 3, 5 or 10 min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year 2000, universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs2 for rcc in comparison with sepacell tm r-s11 and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over 40,000 whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from 5 (routine in italy) to 4. the 4-bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the 4-bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from 4-bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory (2.0 9 10 11 cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february 2018 to february 2019. study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version 23.0 results: a total of 2880 blood products were available at our institute including 960 each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was 3.5%. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. 102(10%). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with 02 common causes, including the expiry of unused products 60(59%) followed by broken bags 30(29%). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early 2000s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of 1 liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of 2010-2017. results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than 200 blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are 18 blood centers in the republicone for each of the 18 administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since 2012, the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in 100% of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group 0 rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis 30 units of pooled platelet concentrate derived from buffy coats and 30 units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl 80 analyser. 1. pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: 4.12 9 10 11 /unit ae 0.50 the number of platelets after the reconstitution: 3.40 9 10 11 /unit ae 0.50 the loss in the number of platelets: 17.5% 2. platelet concentrates derived from apheresis the number of platelets before the reconstitution: 3.68 9 10 11 /unit ae 0.55 the number of platelets after the reconstitution: 2.91 9 10 11 /unit ae 0.49 the loss in the number of platelets: 20.7% summary/conclusions: 1. reconstitution results in the loss of 17.5% of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. 2. the average number of platelets after the reconstitution is 3.4 9 10 11 /unit, which fulfils the quality requirements. 3. reconstitution results in the loss of 20.7% of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. 4. the average number of platelets after the reconstitution is 2.9 9 10 11 /unit, which slightly deviates from the quality requirements. 5. the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf4 was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf4 antibody on the column. hereafter, pf4 was purified with concentration tubes having 5 kda molecular weight cut-off and dialyzed via dialysis tubing with 3.5 kda cut-off. pf4 concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf4 specificity. molecular weight of obtained pf4 was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf4 had 30 kda molecular weight which is corresponding with a tetramer pf4. in addition, elisa showed that the pf4 qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf4. summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf4. nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf4 is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf4. unused human pcs and specific anti-pf4 antibody can considered as an alternative to separate the pf4 particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first 2 weeks of storage at 4°c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from 7 days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: 34 units of whole blood were leucodepleted within 24 h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for 7 days at 4 ae 2°c. on the 8 th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of 35 days at 4 ae 2°c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, 2.3 dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units (0.23 ae 0.2.10e6/u)) while maintaining 80% of the initial platelet content (85 ae 20.10e9/u). mean volume (464 ae 24 ml), hemoglobin content (54 ae 6 g/u) and hematocrit (36 ae 3%) was within conformance range. after 7 days of storage, 50% of the initial platelet content at collection is maintained and mean hemolysis is 0.27 ae 0.15%. rcc processing at day 8 was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume (266 ae 20 ml), hematocrit (56 ae 3%), hemolysis (0.18 ae 0.19%) were in conformance with mandatory standards at day 8. after 7 days of storage (i.e. day 14 after collection), rcc quality parameters were maintained with however an increase in hemolysis (0.34 ae 0.36%) which was above usual values routinely observed with rcc produced from day 1 processed units. 3 rcc out of 34 were above the maximum limit of 0.8% at day 14 after collection such increased hemolysis was more pronounced at day 28 after collection (0.70 ae 0.45%) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold (20% of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after 7 days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within 21 days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland 1,2 , f mahmood 3 , l nissen-meyer 1 and i nentwich 1 1 immunology and transfusion medicine, oslo university hospital 2 immunology, university of oslo, oslo 3 immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, 2005) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were 1) to assess sensitization profiles against an array of 55 allergens in 100 randomly selected blood donors and 2) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from 100 blood donors randomly selected on one donation day outside the pollen season. we used 300 ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for 55 analytes (euroline atopy screen, euroimmun, germany), which comprised 28 food allergen extracts and single allergens, 24 inhalant allergen extracts, 2 insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of 44 serum samples for ige against 55 allergens in less than 3 h. results are expressed as east (enzymoimmunoassay) classes: class 0 (no sensitization), 1-2 (weak sensitization), 3 (moderate sensitization), 4-5 (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class 5none detected. class 4, sesame seed: 1. class 3, apple 1; hazelnut 2. class 2, apple 1; almond 2; hazelnut 1; sesame seed 1; soybean 1; cow milk alpha-lactalbumin 3. class 1: 26 donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class 5, 4 and 3 none, class 2, wasp venom 7; honey bee 2. lower classes (0-1) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor (1%) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to 5 days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = 53) were collected, stored at 22°c with agitation, and tested at day 1, day 5 and 7 post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd61 + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a 2 (txa 2 ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin-1 (rca-1; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to 4-fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to 4-fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only 27% of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa 2 was also higher in the arachidonic acid responders (p = 0.027). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day 5 was weakly associated with donor age (p = 0.009, r 2 =0.2613) but not bmi (p = 0.9143, r 2 <0.001), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than 10%) are b-thal-het that meet the criteria for blood donation (hb > 13.5 g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from 10 healthy non-smoker donors (5 b-thal-het carriers and 5 controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca 2+ and proteasomal activities were determined. for statistical analysis, significance was accepted at p < 0.05. samples from the red cell units were collected aseptically, processed (dual centrifugation at 3,000 g for 15 min) and stored at à80°c. processed samples were thawed, and then analysed using the nanosight ns300 nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d2, red cell units contained an average of 8.1 9 10 9 ae 4.0 9 10 9 mvs/ ml. the mean size of these mvs was 112.8 ae 10.5 nm and the mode size was 79.9 ae 10.3 nm. the concentration of mvs increased gradually throughout storage (p = 0.0276), reaching a maximum at d42 of 32.4 9 10 9 ae 1.8 9 10 9 mvs/ml. both the mean (p < 0.0001) and mode (p < 0.0001) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d2 vs. d7: p < 0.0001 for both mean and mode). by d42, mean and mode size of mvs was 176.1 ae 4.8 nm and 171.8 ae 5.6 nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than 400 nm in red cell units. both the concentration and size of mvs present in red cell units increased during the 42 days of routine storage. the concentration of these mvs was approximately 100-fold higher than we had previously detected using flow cytometry (aung, pathology, 2017) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than 24 h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six 14 day old leukocyte depleted red blood cells (ld-rbc) and six 4 day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated (25 gy). a volume of 350 ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for 6.5 min at 2888 9 g at 4°c (hettich roto silenta 630 rs) before removing the supernatant using compomat g5 extractor. wash procedure was repeated twice using 450 ml sagm solution, and after removal of the last supernatant, 100 ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after 24 h, 7 days and 14 days storage at 4 ae 2°c. results: all washed ld-rbcs met european specification for haematocrit (0.50-0.70) and all but one for hb content (≥ 40 g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than 0.8%) 14 days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration 14 days after washing and irradiation did not exceed those levels found at the end of shelf life (day 35) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than 0.05 mg/dl (0.5 mg/l). our iga method 0 s lower limit for detection is 0.05 mg/l and all results were below this level. total albumin were well below finnish specification (<250 mg/unit). background: room temperature has been the standard storage condition for platelets since the 1970s, when it was shown that this improved in vivo survival compared to when stored at 4°c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at 4-9°c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from 5 buffy coats and 300 ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for 23 days at 4-9°c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d1-d16: 7.05-6.77 with platelet count 400 9 10 9 /u and glucose still at 2 mm at least until 16 days of storage. platelet activation maintained acceptable levels with cd62p expression < 30%, while ps exposure increased rapidly; >20% after 6 days of storage. aggregation tests showed functional platelets until 13 days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at 4-9°c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at 4-9°c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets 0 rhd negative were frozen in 5-6% dmso and stored at à80°c for 6 months. cpp were thawed at 37°c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was 24%. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount 194 9 10 9 /unit, concentration 0.73 9 10 9 /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value 3 in fp and on value 1 in cpp. the average plasma content in fp was 31% compare to 3.22% in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low (0-1:2). cpp had faster clot initiation (rotem clotting time (ct) in cpp 69.2 s, fp 81.8 s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp 39.8 mm, fp 53.2 mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of 24 ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, 250 ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept 1 h on bench in room temperature followed by 1 h on agitator at 22 ae 2°c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of 420 ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of 7 days. results: during validation of the method, 12 pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po2, pco2 and lactate on day 2, 5 and 7 of storage. the platelet count was 2.27 ae 15 9 10 11 per unit on day 2. the ph value was 6.9 on day 2, 7.1 on day 5, and 6.9 on day 7. the glucose concentration decreased from 5.8 to 3.5 and 1.6 mmol/l on day 2, 5 and 7, respectively. the mean po2 level was 25.3, 25.0 and 28.7 kpa while the mean pco2 was 3.8, 1.7 and 1.8 kpa and the lactate concentration was 5.9, 9.6 and 12.9 mmol/l on day 2, 5 and 7, respectively. since routine implementation of the method in april 2017, regular quality controls showed an average of platelet count of 2.63 ae 34 9 10 11 (n = 161) with a volume of 204 ae 7 ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of 7 ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from 11 published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested 4-10 ml sample volume per culture bottle. the 11 studies classified results based on aabb bulletin 04-07 definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta 3d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of 217,932 platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day 3, 4, or ≥ 6, and represented 8.9%, 44%, and 47% of the units tested, respectively. true positives were detected in 174 platelet units representing 0.08% of the total platelets tested. the majority were reported from platelets tested on day ≥ 6 with a total of 128. data showed the bta 3d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤27 h with the majority detected in ≤24 h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in 5 of the 11 studies for platelets tested at 3 days and ≥ 6 days with a total false positive rate of 0.27% (range of 0-1.1%). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, 1,136 culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at 3 to 8 days post collection provided evidence that the bta 3d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert 3d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day 3 and up to day 7 when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts (0.5-2 mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with 2 mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~2 nm, medium 30 nm and long~100 nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at 0.5-1.0 mg/ml hsa coated particles with 0.57 ae 0.36 and 0.54 ae 0.39 pg/platelet, respectively. however, the 1.0 mg/ml hsa coating resulted in~100-fold higher binding affinity to platelets than particles coated with 0.5 mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of 80, 220, and 360 pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day 3, by the increased expression of cd36. the percentage of cd36-positive cells among the population of large platelets did not change during storage. on the day 3, increased expression of cd42b and cd62p was detected, but only on large platelets. small and medium-sized platelets had increased cd62p expression only on day 5. the expression of cd42a on total platelets increased significantly on day 3, and stayed unchanged until day 5. the same pattern of cd42a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd41-positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the 5-day storage period. increased annexin and pf4 concentrations were detected on day 5. the concentration of b-tg increased on day 3 of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february 2009, the transfusion service in our hospital has managed 3 autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of 3 types of autologous blood components in a single institution over an approximately 9-year period. methods: between february 2009 and december 2017, we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of 9,193 patients (9% of whom received operations) received blood transfusion, of which 5,080 (55%) received autologous blood transfusion alone, 2,101 (23%) both autologous and allogeneic blood transfusions, and 2,012 (22%) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was 78%. pacs units were transfused to 5,830 patients (60%), pad units to 2,845 patients (30%), and anh units to 982 patients (10%), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was 98.6%. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were 12 and 0.1%, respectively, of which the most common was febrile non-hemolytic transfusion reaction at 9 (75%), followed by allergic reactions at 2 (17%), and hypotensive transfusion reaction at 1 (8%). the severity of adverse reactions to pad transfusion was grade 1 (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was 0.55%, that to frozen pad units was 0.05%, and that to autologous ffp units was 0.10%. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was 78%, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp215 (haemonetics corp.), is approximately 1%. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: 3-40%) for up to 24 h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for 48 h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in 100% plasma were washed on day one after blood collection using the automated closed-system cell processor acp215 (n = 6). wpc, which were rested 1 h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at 20-24°c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was 110.9 ae 1.9 (910 10 /l) and the volumes of the control and test units after the division were 120 ae 11 and 123 ae 12 (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than 6.85 during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco 2 , po 2 , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd62p expression were significantly higher in the test units than in the control units on days three (52.6% ae 6.0 vs 40.9 ae 5.7, p < 0.01); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd42b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a 2-month field trial over 10,000 data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < 0.0001) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ 0.3246). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > 90% of ld components have less than 1 9 10 6 rwbc/unit. for ld failures (n = 14) there was a good correlation (r 2 =0.9848) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around 6 and 8 rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < 0.0001) in rrbc contamination, with lower levels in apheresis platelets (median = 15 rbc/ll, n = 1820) compared to those produced from buffy coats (median = 783 rbc/ll, n = 77) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately 2-3 h for 60-90 samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was 60 to 220 nm (without oxidation). the heights (h) of dips were 4 to 10 nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only 10% to 15% of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from 8 donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn 2+ (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to 600 nm. the young's modulus e = 10 ae 5 kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn 2+ ) significantly increased the absolute value of the young's modulus of rbc membranes up 2-3 times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from 200 nm to 600 nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january 2016 to december 2018, in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january 1st 2016 to december 31st 2018. results: a total of 54,599 whole blood units and 3,166 apheresis platelets were collected during the study period. of the 66,289 blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of 2,891 (4.4%) blood components were discarded, of those 75.4% due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in 2016 was 5.6%, in 2017 was 3.8% and 0.2% in 2018. similar tendency was shown in the pooled platelets for 2 consecutive years with 4.8% (2016) and 3.8% (2017), but with an increase in 2018 (7.7%). the rate of apheresis platelets had a more variable behaviour from 2016 to 2018 with rates of 1.3%, 0.6%, and 0.9% respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from 1 buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, 31 apheresis pcs and 66 pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba1c from red cells. triglyceride levels > 2.5 mmol/l and hba1c levels > 42 mmol/mol were defined as high. results: twenty two percent (21/97) of the donors were marked as 'rapid acidifiers' and 71% (15/21) of these donors had health issues. 'rapid acidifiers' were of age 57, 31-69 (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type 2 diabetes, indicated by high cholesterol and/or triglycerides, high hba1c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' (29%) did not have high triglyceride or hba1c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, 2012). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than 10 visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, 2016). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis (1246.8 ae 90.1/ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from à600 mv to +1100 mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse 80i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + 500 mv and + 800 mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than 10%) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from 4 healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph 7.4. we used ultraviolet (uv) irradiation of blood or nano 2 as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico 2800, usa) to measure the absorption and scattering of light (0.5 nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo2,l c hbo2 l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno2 -,l c methbno2 -l+ e methbno,l c methbno l+k+s/k 4 l (1), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano 2 action (up to 90%). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r 2 = 0.98). incubation of rbcs with cytoflavin leads to reduction of methb to hbo 2 . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr5, the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr5 leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl2 and mgcl2) in ph 7.2 containing 2 mm edta and 4%(w/w) dextran 40 without additive amounts of triton x100 was the most optimized buffering system for lcr5 filter back flushing. total cell content was also determined as 6.28*10 8 granulocytes, 4.27*10 8 lymphocytes and 0.8*10 8 monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis (2019) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of 5.2 g ig (70% intravenous -privigenand 30% subcutaneous -hizentra) and 26 g albumin (alburex) per kg plasma fractionated, corresponding to 998.000 g ig and 4.992.000 g albumin; based on present demand, this represents 97% and 100% self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted 6.000 g fibrinogen (riastap) and 6.000.000 iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of 5.000.000 iu at and 3.000.000 iu pcc, capable of ensuring self-sufficiency for naip regions until 2020. summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in 2017 in italy 923,944 litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, 151 of the 176 italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of 3 parts: a) "surrogate" bags (check-bags) of 250 and 700 ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period 2016-2018, at the pievesestina be of emilia romagna region 210,871 plasma units were frozen so as to allow complete freezing within 60 0 to a temperature below à30°c, in 8,349 freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be 108 tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of 8,349 freezing sessions carried out at the pievesestina be, 27 (0.3%) were detected to fail to reach à30°c at the core of the check-bags within 60 0 . of the latter, in most cases (70%) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (<24 units), ii) labelling time (<8 0 ), iii) optimal storage t (à40°c vs. à30°c), iv) optimal time between two openings of storage sites (>30 0 ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january 2018 -december 2018) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check (6 months) and data with double check (6 months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of 6818 from the first six months a 38 mismatch was found which equals 5573 dpmo and 4.1 sigma metric. and in a sample size of 5854 from the second six months a 25 mismatch was found which equals 4277 dpmo and 4.2 sigma metric. out of the whole 12672 pooled units 63 were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a 4.1 sigma metrics obtained from the first 6 months, were shifted to a sigma metrics of 4.2 in the second 6 months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from 150 ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (à20°c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced 7 mc; 1 mc as the control, 3 mc were lyophilized with excipient and 3 mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at 30 days at room temperature (30-32°c) and blood bank refrigerator temperature (2-8°c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher (9.8 iu/ml) than without excipient (4.5 iu/ml) and the storage at blood bank refrigerator (2-8°c) is better than at room temperature (30-32°c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature (2-8°c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from 2016 to 2017. they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of 27 patients were included (12 patients as control group) and (15 patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph 2.5 after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was 6.99 d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of 4.2 million habitants. annually, it collects about 100,000 whole blood and 3,500 apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for 7 days at 22°c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of 4 bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in 33% plasma and 65% pas and mcd were subsequently evaluated also in 38% of plasma and 62% pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days 1, 5 and 7. bacteria sterility test was performed at day 7 for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = 10) and ad pcs (n = 8) stored in 33% plasma showed at day 7 an average ph ≥ 6.9; swirl ≥ 2.5 and yield = 3.3 9 10 11 . their untreated counterparts showed average values for ph ≥ 7:0, swirl ≥ 2.9 and yield 3.3-3.4 9 10 11 . mcd stored in 33% plasma (n = 6) that underwent prt showed at day 7 average values for ph = 6.7, swirl = 1.2 and yield = 3.05. control mcd showed average values for ph = 7.0, swirl = 2.8 and yield = 3.1 9 10 11 . mcd stored in 38% plasma (n = 8) that underwent prt showed average values for ph = 6.6., swirl = 1 and yield = 3.1. their untreated counterparts had average ph = 7.1, swirl = 2.9 and yield = 3.2. total protein content in pcs derived from wbd (n = 12), ad (n = 15) and mcd (n = 27) was 22 g/l, 20 g/l and 20 g/l, respectively. while the coefficient of variation of wbd and ad ranged from 3% to 4%, plasma products 129 respectively, the one of mcd reached 14%. all prt-pcs were negative for bacterial growth at day 7. summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day 7 and passed the requirements of the eu guidelines (19 th edition). quality of mcd units met eu criteria at day 5; swirl decreased significantly at day 7 which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~25% of the annual produced buffy coat platelet concentrates (bcp) (~40.000 bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~25% pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period (2017) . this cost was offset by substituting a semi-automated production method for bcp, which was used in 2017 to produce 28.7% of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by 89.2%. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in 2018 by 0.19%. the extension of max. storage time from 5 to 7 days for 24% of the bcp-units that were pr resulted in decreasing our overall outdating rates by 29% (versus 2017). this reduction in outdating rates reduced our opc in 2018 by 2.2%. in 2018 we gamma-irradiated 25.9% fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~25% pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day 14 of storage then stored for a further 14 days. rcs were also irradiated on day 5 of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into 4 arms on day 1 of storage, with 10 units in each arm. all units received an irradiation dose of 25.9-48.3 gy. two arms remained as standard volume rcc and were either cor x-irradiated on day 14 of storage. the other two arms were both split into 6 rcs on day 4 of storage before being irradiated on day 5 (early arm) or day 14 (late arm) of storage. for each replicate in these arms, 3 splits were c-irradiated and 3 splits x-irradiated. all arms were tested a day prior to irradiation and 1, 7 and 14 days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and 2,3 dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, 1 split was tested on each testing day post-irradiation. a 2-way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (>75% of units with < 0.8% haemolysis) and 100% of units had atp levels above the recommended minimum for acceptable post-transfusion survival (2.3 lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz 1 , c coello de portugal 2 , m morales 3 , f solano 4 , c perez parrillas 5 , a rodriguez hidalgo 5 , t diaz rueda 5 and m flores 5 1 direccion, regional transfusion center toledo-guadalajara 2 transfusion service, toledo hospital complex, toledo 3 transfusion service, general university hospital of guadalajara, guadalajara 4 transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera 5 regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves 3 general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of 943,000 inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since 2008, rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from 5 to 7 days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last 8 years divided into four periods ( results: pc were predominantly obtained from whole blood collections with 85% of bc platelets/15% of apheresis platelets. 77% of the available bc were used in production for period a and 88% for periods b, c and d. after wastes of approximately 1.9%, the distribution of pc was stable for the 4 periods studied. 7075 pc were distributed for period a, 7047 pc for b, 7467 pc for c and 7083 pc for d. the % of pi platelets with 7-day shelf life available in the 3 hospitals was limited to 13% during period a. it was then increased to 14.1%, 20.9% and 26% for periods b, c and d respectively. the percentage of wastes was stable at 0.2-0.3% but the discards due to expiry went down from 24.24% (period a) to stabilize at 10.9% in periods b and c and 10.3% in period d. in the 3 general hospitals the expiry went down from 20% to 5.89%(hct), 29.4% to 14.5% (ughg) and 33.36% to 17.68%(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with 7-day shelf life allowed reducing discards for expiry from 24.24% to 10.3% in the last period analyzed at rtc and the 3 major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in 100% donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day 5. the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh-800, beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of 2-3. the average volume per unit of the pre-inactivation was 288 ml (278-302 ml) and post inactivation was 269 ml (254-284 ml), with average volume loss during inactivation was 36 ml (24-43 ml), corresponding to 13% (8-15%). the average platelet yield per unit pre-inactivation was 3.7 9 10 11 (3.0-4.4 9 10 11 ) and post inactivation 3.2 9 10 11 (2.8-3.9 9 10 11 ) with an average platelet loss of 13% (3-20%) . the average rbc contamination per unit (0.01-0.02 9 10 9 rbc/ml). the culture tests were negative, the average ph at day 1 was 7.4 (7.1-7.6), average ph at day 4/5 was 7.3 (7. 2-7.4 ). the average residual amotosalen concentration post treatment was 0.30 lm (0.23-0.35 lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below 2 lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of 106 independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of 2 ml pre-inactivation and another sample post-inactivation (16 h after pi). the platelet viability of each sample was evaluated by demonstrating the cd62p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of 106 independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd62p marker, was 18%, while the percentage of functional platelets post inactivation was 19%, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a 95% confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, 2016" in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than 10%. for the intermediate precision, the coefficient of variation was less than 15%, but for the pcs after treatment, this result rose up to 21%. the r² value for the linearity was 97.7%. the detection limit corresponded to a result of 6 edel and the loq (equal to 10xsd) is 19 edel. concerning roc curves, the men apcs threshold was 58.5 edel compared to women apcs with 62.5 edel. the threshold for bcpc was 54 edel. all of these results had 100% of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, 404 apcs (182 women and 222 men) and 263 bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, 14.0% of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was 89 edel. our study showed a threshold with 100% specificity and 90% sensitivity at 58.5 edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at 62.5 edel compared to 66.5 edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold (54 edel) was lower than abonnenc threshold which was at 59.8 edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a 3-ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: 1) complete treatment, 2) plasmas with mb without illumination, 3) plasmas without mb with illumination and 4) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol 1 where n = 20, were realized during the viro-inactivation process, t1 corresponds to a dosage of plasmas before treatment, t2 the plasma after the mb dry tablet passage, t3 is the time after illumination and t4 corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than 50%, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series 1 and 3, the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series 2 was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t4 (238 ae 16 edel at t3 and 209 ae 17 edel at t4). however, in the absence of mb (series 3 and 4), the results at t1 and t4 were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis (2019) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ 2.00 g/l was 94% (>70% required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were 75% and 90%, respectively. residual platelets were < 25 9 10 9 /l, leucocytes < 1 9 10 4 /l and red blood cells < 4 9 10 9 /l. all units had a protein content > 50 g/l. residual amotosalen was below 2 lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c3a and c5a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at 37°c was consistently short (6-7 min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine 8-10 to 6-7 min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included 5 experiments. for each experiment 5 male-donor, abo-compatible whole-blood derived plasma units (≥ 270 ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the 5-unit pools were split into 2 equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into 3 (≥200 ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from 5 single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, 2018) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ 18 lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, 2017) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ 18 lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade (0 to 70 lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to 2 different fluorochromes: phycoerythrin (pe) or brilliant violet (bv421). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and 4 populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): 0 (mfi 0.4), 4 (gmp mfi 12; non gmp mfi 9.5), 18 (gmp mfi 44; non gmp mfi 42), 70 (gmp mfi 174; non gmp mfi 180). streptavidin-bv421 brighter than streptavidin-pe is a promising tool because it amplifies by 1.7 the signal of fluorescence and allows a good differentiation of the 4 populations of rbc treated with only 0, 1, 4 and 18 lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ 18 lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases 2 (timp2) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, 2016) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = 9) were centrifuged within 3 h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = 9) were used as adult counterpart. platelet concentration was adjusted to 7-10 9 10 11 / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at à30°c/37°c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of 50,000 au spot density was defined as cut-off. data were analyzed by graphpad prism 8 using two-way anova. results: semi-quantitative evaluation of 105 analytes per array revealed significant differences. in plasma samples and platelets 63 and 48 analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (>200,000 au spot density) compared to adult plasma (6/63 vs. 3/63). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor 15 (gdf 15), platelet derived growth factor aa (pdgf-aa) and serpin e1 (p < 0.0001). more highly prevalent proteins were detected in npl (10/48) compared to apl (1/48), and 20 proteins were significantly elevated including vascular cell adhesion molecule-1 (vcam-1), platelet factor 4 (pf4/cxcl4), epidermal growth factor and lipocalin-2 (p < 0.0001). in adult samples only 10 proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < 0.05 to p < 0.0001). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, 2018) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, 1999) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are 1) to describe a procedure to label injectable human platelets with 2 densities of biotin, 2) to evaluate the impact of biotinylation on platelet functions, 3) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with 1.2 and 10 lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap (60 lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid (1 mm), amplitude of aggregation is 81.7 ae 2.5% (bio 0); 82.6 ae 2.3% (bio 1.2 lg/ ml); 79.2 ae 1.6% (bio 10 lg/ml). using collagen (2.5 lg/ml), amplitude of aggregation is 65.6 ae 4.2% (bio 0); 61.4 ae 4.0% (bio 1.2 lg/ml) 40.8 ae 6.6% (bio 10 lg/ml). the 2 biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than 48 h. after 4 h, the mean fluorescence intensities are 0.13 ae 0.02 for unlabeled circulating mouse platelets, 1.01 ae 0.03 and 8.2 ae 0.15 for circulating human biopl covered respectively with 1.2 and 10 lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, 10 ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, 9 ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to 30%. auto seds were administered in the form of 20% eye drops. results: auto seds have been applied to 17 ophthalmological patients (10 men and 7 women), previously resistant to standard therapy. in total 44 treatments were performed (each lasted 20 days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were 71.3 and 19.6 respectively). also, objective progress was present in 83% of all patients (p < 0.001). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. 1 platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of 2,3-dpg while reducing storage lesions stemming from oxidative stress. 2, 3 on the other hand, effects of steady hypoxia (pco2~10-20 mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for 3-week storage at 1-6°c. methods: 11 units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into 70 ml cp2d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o2-free bag. 5 ml of wb were collected from each unit at day 1, weeks 1, 2, 3. plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < 0.05. results: h-plt counts declined to~60% by the o2-reduction process, while similar decline was observed after 1 week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac-1 showed large increase after 1 week, then remained steady (h << c). plt activation by trap or adp declined modestly over 3 weeks (~15%) while h-plt showed additional~10% reduction for all time points. collagen activation for c-plt increased after 1 week (74%) and gradually increased to 100% after 3 weeks (~20% reduction with h compared to c). plt-derived mv (cd61 and cd61/annexin v) increased~4-fold over storage time; day 0 mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd42a) in wb supernatant increased 17-fold after 3 weeks for c, while h suppressed increase to 7-fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over 3-week period when stored at 1-6°c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated 500 ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted 1:1 by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, 19 patients were enrolled, 4 of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): 1) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); 2) autologous serum eye drops (sed). the creation of both types of platelet gel started in 2014 but only in 2017 we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from 2018, is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. 1) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between 800-1200 10 3 /ll and negative blood cultures, required by the italian law; the units are frozen at à80°c and last 5-year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. 2) the ophthalmologist's patients, with dry eye syndrome, donate 120 ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution (30%) are divided in 2 boxes containing 30 single-dose vials each: they are stored at à80°c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc 10231 strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. 10% calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of 1 9 10 3 cfu/ml and 1 9 10 4 cfu/ml were inoculated on sabouraud dextrose agar at the 1 st , 2 nd , 4 th , 8 th and 16 th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after 18-24 h of incubation at 30°c. chemokines and kinocidins (platelet factor-4, interleukin-8 and thymosin-b4) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp-10 3 group showed that the antifungal activity was still going on at the 8 th hour. the difference in colony production between the two groups at 8 th hour was statistically significant (p < 0.05). it was observed that the antifungal activity continued at the 4 th hour, decreased at the 8 th hour in the group prp-10 4 group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp-10 3 group. although there was an increase in il-8 levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb4 values results from the antifungal activity on the advancing hours in the prp groups. whereas pf-4 did not act an antifungal activity on prp-10 3 and prp-10 4 . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that 4% of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions (883 ml of red cells) in the preceding 10-day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of 225 ml, the hemoglobin decreased from 8.6 to 5.8. anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant (68% reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as 7-10 units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over 27,000 responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over 3,500 samples from 75 blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered (0.06%); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for 3-5 days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for 20 h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected 0.5 ml of ipm (2 mg/kg) intravenously between 15 to 30 seconds into each 5 health mice, weighing 17-22 grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during 24 h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after 24 h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother 0 s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the 1980s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than 6 years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since 2 years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab0-identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: 31 patients were identified receiving allogeneic sed, 15 patients had been treated autologous previously. in total, allogeneic sed have been produced 54 times since june 2017. indications were ocular gvhd (n = 15.48%), neurotrophic keratopathy (n = 9.29%), mucous membrane pemphigoid (n = 2.7%), sj€ ogren syndrome (n = 1.3%) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = 4.13%). contraindications for autologous donation were underlying disease (n = 19.61%), poor venous access (n = 12.38%), low haemoglobin (n = 8.25%), low body weight (n = 8.25%), very young age (n = 7.22%), circulatory disturbances (n = 3.10%) and lack of response to auto-sed (n = 2.7%). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = 2.4%), high donor haemoglobin (n = 2.4%) and unspecific positive serological findings (anti-hbs n = 2.4%). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a 8 year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood (20 ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for 1 h at 37°c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into 0.3 ml segments (capsules)and the artificial tears applied to the left eye 8 9 daily. results: ulcer healing was reported after 4 weeks of therapy with artificial tears. the dosage was reduced to 4 9 daily. no recurrence of corneal ulceration was observed after subsequent 8 weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > 80% of ab+/rnadonations since 2016 testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ 1.5), longstanding (ab+/rna+, lag odn > 1.5) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit 0.02 lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during 2017, 1671 donors tested hiv-positive of whom 1315 had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was 9.3% (n = 122) with efavirenz most frequently detected (115), followed by lopinavir (5) and nevirapine (2) . potential elite controller cases had the highest proportion of detectable arv (68/80; 85%) (p < 0.0001) followed by longstanding (37/741; 4.9%) and recent (17/366; 4.6%) infections. none of 82 acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older (35 to 64 years) hiv-positive donors (49/305; 16.1%) were significantly more likely to test positive for arv than younger (15 to 34 years) donors (73/1010; 7.2%) (p < 0.0001). arv use was more frequent among first time (101/ 716; 14.1%) than in lapsed (13/277; 4.7%) or repeat (8/322; 2.5%) donors (p < 0.0001). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites (10.4% vs 5.0%; p = 0.0051). summary/conclusions: the 9.3% prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the 4.7% prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year 2018. methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of 11 yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than 160.000 samples annually from whole blood and apheresis donations, testing on average around 600 samples per day. for the last 5 years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of 2019. although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the 4 mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to 2.000 unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: 19/33 anti-hcv, 19/21 hiv ag/ab and 04/22 hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: 1) hbsag aly 100. 00% (1994 00% ( / 1994 00% ( ) vs arc 99.95% (1993 00% ( /1994 ; 2) hiv aly and arc 99.95% (1992/1993); 3) anti-hcv aly 99. 90% (1994 90% ( /1996 90% ( ) vs arc 99.75% (1991 90% ( /1996 . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of 2 years from january 2016 to december 2017. blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. (78) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of 0.09%, thereby total reactivity being 2.39%. 55% (22/40) of these were hcv reactive & 45% (18/40) for hbv. the nat yield was 1 in 1086 and the viral loads of nat reactives ranged from 1-9 x10 4 iu/ml for hcv & all the hbv yields had an extremely viral load of < 06 iu/ml. 12/40 nat reactive showed sero-conversion after 4-8 months with follow-up eclia screening, and 7 of these were hcv reactive and 4 hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. (1/190,298) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect 283 infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e801 equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv-1/2, hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv-1/2 or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than 900 plasma samples collected in 7 sub-saharan africa countries (2011) (2012) and the amazon region of brazil (2016) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv-1 (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b19, chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv-1 genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in 2014 during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i2000sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m2000 realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a (5.78 log iu/ml), b (5.90 log iu/ml), c (5.62 log iu/ml), crf01 (5.61 log iu/ml), crf02 (6.19 log iu/ml), and urf (6.33 log iu/ml), as well as single donor high viral titer hcv genotypes 1a (6.84 log iu/ml), 1b (6.73 log iu/ml), 3a (6.64 log iu/ml), 2 (6.10 log iu/ml), 2q (6.65 log iu/ ml), and 4t (6.07 log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately 1 ml of residual sample volume, was then tested using the 0.6 ml abbott realtime hiv assay (lod 40 copies/ml) or 0.5 ml abbott realtime hcv assay (lod 12 iu/ml) and an hcv ag immunoassay (lod 1.24 fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = 24 per platform per virus schema) run on alinity s, alinity i, and architect i2000sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< 0.20 s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< 0.20 s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i2000sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in 2015 per 2.083.914 donations, 54% % of confirmed positive hiv, 87% of hcv, and 93% of hbv cases has been reported among first blood donors. in the end of 2015 a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after 3 months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in 2015, about 0.156% of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in 2017 was 0.082%, which shows a significant decreasing the ttis prevalence among blood donation from 2015 to 2017. the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in 2017 compared with the 2015. prevalence of hiv among donations reduce from 0.0032% in 2015 to 0.0024% in 2017, for hcv and hbv the same results have been experienced, respectively from 0.040% and 0.113% in 2015 reduce to 0.026% and 0.053% in 2017. it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from 80.19% in 2015 to 86.97% in 2017. it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from 0.0156% in 2015 to .082% in 20117. abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 61 samples were tested (19 for hcvab, 24 for havab igm and 18 for havab igg) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hcvab ranged from 0 to 4.474%. 5 samples were tested for havab igm in a total of 55 essays and the %cv ranged from 0 to 11.475%. havab igg was tested in 3 samples during 33 essays and the %cv ranged from 0 to 4.861%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 85 samples were tested (10 for hbsag, 20 for hbsab, 18 for hbcab, 14 for hbeag and 23 for hbeab) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 30 essays we found the %cv hbsag ranged from 0-4.375%. 3 samples were tested for hbsab in a total of 33 essays and the % cv ranged from 0-5.196%. hbcab was tested in 3 samples during 32 essays and the %cv ranged from 0-2.811%. using 3 samples and a total of 28 essays we found the %cv hbeag ranged from 4.176-5.147%. 4 samples were tested for hbeab in a total of 29 essays and the %cv ranged from 0-4.444%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 38 samples were tested (12 for hivag/ab, 14 for syphilis and 12 for htlv i/ii) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hivag/ab ranged from 0 to 3.05%. 2 samples were tested for syphilis in a total of 22 essays and the %cv ranged from 0 to 1.481%. htlv i/ii was tested in 3 samples during 28 essays and the %cv ranged from 3.342 to 7.5%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were 1.27%, 0.20% and 0.50% respectively. consecutive positive results for hbv were 9.20% (63/685), for hcv were 6.0% (16/266) and nil for hiv. there was no sample carry over in this. out of 79 consecutive reactive donors 69 were donated for same patients and 32 were related with infected patient which were statistically significant (p < 0.0001). summary/conclusions: among all tti reactive donors 7.4% (79/1061) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study 32 reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < 0.0001). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june 2018. a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using 100 positive and 100 negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were 65% and 70% (hightop), 67% and 85% (rightsign), 62% and 73% (wondfo), 70% and 80% (accu-chek), 68% and 77% (fastep), 73% and 85% (abon), 77% and 83% (immumed), 80% and 90% (insta-answer), 67% and 81% (biocheck) and 72% and 83% (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were 69% and 80% (hightop), 76% and 83% (rightsign), 69% and 81% (wondfo), 78% and 79% (accu check), 68% and 68% (fastep), 63% and 73% (abon), 71% and 70% (immu-med), 79% and 68% (insta answer), 62% and 66% (biochek) and 69% and 78% (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be 100%, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , 1940 and 1945 , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. ,906 donors were routinely tested for hbv dna by using cobastaqscreen mpx-1 and mpx-2 (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: 348 hbsag-/dna+ donors (1:1,462) including 294 confirmed obi were identified (1:1,731 prevalence). among obi donors, 160 (54.5%) tested anti-hbc+/anti-hbs-, 108 (36.7%) were anti-hbc+/anti-hbs+, 25 (8.5%) were anti-hbc-/anti-hbs+, and 1 anti-hbc-/anti-hbs-primary obi (0.3%). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: 21 years [range: 18-57 years]) than those with anti-hbc+/anti-hbs+ (mean: 44 years [range: 19-60 years]) and anti-hbc+/anti-hbs-(median: 45 years [range: 21-55 years]) profiles (p < 0.0001). hbv vaccination was documented for 18 (72%) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: <10-155 iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = 1/20) and genotype c (n = 19/20). preliminary analysis of core protein (n = 16) and bcp (n = 10) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for 15/ 25 anti-hbc-/anti-hbs+ donors (2-6 samples/donor; range: 2-56 months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between 12 and 1,000 iu/l. no significant difference in hla-a, -b (except hla-b*46 more frequently detected in anti-hbc negative obi), and -drb1*. summary/conclusions: overall, the 8.5% prevalence of anti-hbs-only in hbv dna positive obi carriers (1:20,355 of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in 15/25 donors by the over 50 months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after 1986 is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since 2000. hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december 31, 2018, allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between 2012 and 2018. background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about 130,000 blood donors are submitted to serological screening tests (hbv, hcv, hiv-1/2, chagas disease, syphilis and htlv-1/2) and nat for hiv, hcv and hbv per year. approximately 2% of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was 0.65% in 2016. aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly 74,000 donations from may 2016 to december 2016. hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity 95% lod 300 iu/ml for hbv). reagent samples (n = 407) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity 10-20 ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno2pheno [hbv] 2.0. socio-demographic and epidemiological data were also analyzed. financial support: fapesp 2017/16658-6. results: among the 407 hbv reactive samples, 377 were reactive for anti-hbc only (92.6%), 10 for hbsag only (2.5%) and 20 were reactive for both markers and hbv dna (4.9%). routine testing and id-pcr-hbv identified 20 (0.027%) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from 1.08e+01 to 2.45e+09 cop/ml (median, 2.12e+08cop/ml). hbv sub-genotypes a1, a2, c1, d3 and f1 were found in 70%, 5%, 5%, 15%, and 5% of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in 30% (6/20), with the following main substitutions 100c (3x), 120r, 123n and 144g. the mean age of donors with active hbv infection was 40 years, mostly donors were males (80%), mixed (40%) or white (40%) and had concluded high school (45%). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a1 and mutations associated with escape were found in 30% of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period 15-30 november 2018, all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of 620 edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in 24 (3.87%) samples, among which 7 (19%) were also anti-hbe(+), while anti-hbs was found > 200 m iu/ml in 15 (62.5%), between 100 and 200 miu/ml in 3 (12.5%), and < 100 miu/ml in 6 (25%). among anti-hbcore positive donors, 4/24 were foreigners (16.6%) and 20 were greeks, while foreigners consisted 6,29% (39/620) of donors examined. so, anti-hbcore was found positive in 10,25% of foreigners (4/39, all from countries with high prevalence for hepatitis b infection) and in 3,44% of greeks (20/581). in total, 285 (45.96%) samples had anti-hbs > 10 miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors (45.96%) were immunized, 261 by vaccination and 24 (3.87%) by natural infection. the incidence of natural infection was significantly higher in foreigners (10.25% versus 3,44%). if not all anti-hbcore+ donors, 25% with anti-hbs < 100 iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < 100 or < 200, or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately 2 million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in 20 samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than 20 km to each other. pcr was positive in 15 samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and 11 of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the 1a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with 1b subtype originated from the same village, and their difference was only 2 nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to 2 or 3 primary human sources. during 2011 and 2014, a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from 1993. however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march 2014 to december 2017, there were 2083 blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from 15 chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, 1178 of 2083 donors in the study who could be classified into two categories for hcv status: 201(17.1%) true positive and 974 (82.9%) false positive. a total of 905 of 2083 donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors (82.9%) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at 6.6% and 0.9% respectively. between january and december 2016, in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming 8.1%. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: 235 hcv seropositive blood samples from 3 bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that 45/50 seropositive blood (90%) was rna pcr negative. in december 2018, 156/249 (63%) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between 1.00-1.99 (1.00 is the cut-off indicating a positive sample). data from the re-testing of 235 seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december 2018. preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november 2017, the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of 3 months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a 3-month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for 2010-2018 (2018: preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within 12-months, and/or microbiological and clinical evidence of recent infection. for donors positive in 2018, compliance with the 3-month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from 2010 to 2018 among new donors, annual hiv prevalence decreased significantly by an average of 11.3% each year (p = 0.02) to 1.49/100,000 donations in 2018; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of 17.3% each year (p = 0.03) to 0.23/100,000-person years (pyrs) in 2018 (based on 2 seroconverters). there was no significant trend in hbv incidence over the study period, however between 2017 and 2018 incidence increased from 0.35/100,000 pyrs to 0.84/ 100,000/pyrs (based on 3 and 7 seroconverters respectively). with the information available to date, none of the 2018 seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in 2018 although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the 1980s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a 5-year deferral was implemented in 2013, reduced to 12-months in 2016; a 3-month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a 3-month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a 12-month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a 3-month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current 12-month deferral, calculated as hiv positive donors with a previous negative within 12 months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a 3-month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the 2.5th and 97.5th percentiles over the simulation (95% ci). results: incidence, per 100,000 donations, was estimated to be 0.15, 0.13 and 0.27 for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the 3month deferral "most likely" scenario, hiv residual risk was predicted to be 1 in 32.6 million donations (95% ci: 1 in 217,692 million to 1 in 8.3 million). for the "optimistic" scenario, hiv residual risk was estimated to be 1 in 34.3 million donations (95% ci: 1 in 257,692 million to 1 in 9.1 million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be 1 in 16.0 million donations (95% ci: 1 in 34,091 million to 1 in 5.7 million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every 30 years for the "most likely" scenario, every 32 years for the "optimistic" scenario and every 15 years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a 3-month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above 25%). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september 2018, a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated 22 times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang 9 urumqi blood center, urumqi, china 10 rti international, rockville 11 national heart, lung, and blood institute, bethesda 12 stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in 2013-2016 participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv-1/2 on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately 129 days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the 2013-2016 estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least 2 donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of 1,276,544 whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between 2013 and 2016, including 648,607 donations from first-time donors and 627, 937 donations from repeat donors. hiv prevalence among first-time donors was 68.04 per 100,000 donors (95% ci, 61.68-74.40). hiv ir was estimated to be 37.93 per 100,000 person-years (95% ci, 30.62-46.97) among first-time donors and 20.55 per 100,000 person-years (95% ci, 16.95-24.91) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than 25 years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over 10 years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during 2009 to 2018 at thai national blood centre. results: a total of 6,408,024 voluntary blood donations over 10 years, the repeated reactive results on hiv serological screening were 5,978 (0.093%) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for 6 months and tested by using the different three principles of hiv serological testing. a total of 5,978 hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was 1,130 (35.7%) cases, 742 (23.5%) cases and 1,292 (40.8%) cases respectively. out of 1,130 hiv positive results, we found that 1,105 (97.8%) cases were positive with all hiv serological testing for the first-time follow-up and 25 (2.2%) cases were tested and become to positive results after follow-up more than one time. in 742 cases of inconclusive results, 539 (72.6%) cases were reactive only 1 or 2 testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, 203 (27.4%) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, 1,292 negative results cases showed 762 (59.0%) cases were negative results after follow-up over 6 months while 530 (41.0%) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over 10 years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, 241 donors were found (80% men); of these people 256 donations were obtained (97% whole blood). 166 donors (69%) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average 174 ae 83 days to be reported in sivigila. 25 donors (10.4%) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. 20 donors (60% men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to 6 months they were reactive by sivigila (93% of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for 28 recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around 10-100 cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = 4-12 per species) was inoculated with approximately 10 cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for 22 to 24 h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with 3 random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to 3.5 9 10 3 cfu/ml in bcs and up to 0.9 9 10 3 cfu/ml in the corresponding pcs right after preparation. in total, 33 out of 48 pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., 6/6 pcs tested positive after spiking with streptococcus pyogenes, while only 1/8 pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male (69 yo and 65 yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day 4 platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after 18 h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit 1: a. baumanii tntc and s. saprophyticus with 8.5 9 10 4 cfu/ml unit 2: a. baumanii 6.6 9 10 7 cfu/ml and s. saprophyticus 2.2 9 10 6 cfu/ml blood cultures collected from both patients became positive within 8 h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a-105 and yersinia enterocolitica a-176) were distributed from the paul-ehrlich-institut to 17 laboratories in 10 countries for enumeration, identification and growth measurement in a 7-day interval for a total of 42 days after low-count spiking of rbc bags (10-25 colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a-105 and y. enterocolitica a-176 achieved >10 6 cfu/ml at day 14 and 10 9 cfu/ml at day 21. growth of l. monocytogenes was lower reaching a maximum concentration of >10 6 cfu/ml at day 35. in 9 out of 17 laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert 3d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber (10 or 16 ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc 29523), streptococcus agalactiae (atcc 13813), escherichia coli (atcc 25922), clostridium perfringens (atcc 13124). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert 3d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range 4 9 100-2.8 9 103 cfu/ml) and tested on bact/alert with a 1 ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two 10 ml ssk, which were held for a period of 6 h at 20-25°c. the process was repeated with a 24-h period. once elapsed, 8 ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of 7 days (36ae0.5°c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of 0.6-log10 after 6 h (2.0 9 102 to 8.9 9 102 cfu/ml) and 2.0-log10 after 24 h (3.1 9 101 cfu/ml to 3.4 9 103 cfu/ml). s. agalactiae increased by 0.7-log10 after 6 h (2.1 9 102 cfu/ml to 1.1 9 103 cfu/ml) and 0.09-log10 after 24 h (1.0 9 102 cfu/ml to 1.2 9 102 cfu/ml). c perfringens increased by 0.6-log10 after 6 h (1.3 9 102 cfu/ml to 5.0 9 102 cfu/ml) and 2.7-log10 after 24 h (4.0 9 100 cfu/ml and 2.2 9 103 cfu/ml). for e. coli, the concentration after 6 h was reduced by 0.28-log10 (2.8 9 103 cfu/ml and 1.5 9 103 cfu/ml), however this was possibly a result of inherent counting errors. at 24 h, an increase in growth by 2.7-log10 (1.5 9 102 to 7.9 9 104 cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to 24 h does not have a negative effect on bacterial viability and detection using the bact/alert 3d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in 100% plasma or amicus tm for 65% intersol pas/35% plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken 7 h after successful and complete transfusion of an apheresis intercept-treated pc in 65% pas/35% plasma, from a patient with a suspected septic reaction that occurred 2 h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a 7 day shelf-life. methods: a double apheresis pc collected in 65% pas/35% plasma was split into three equal components, yielding approximately 250 ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~6 log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at 3, 5 and 7 days of storage. the samples were analyzed by plating on lb plates (100 ll-10 ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were 5.9-6.0 cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after 3, 5 and 7 days of storage, corresponding to > 5.9 log 10 inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after 7 days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between 0,3%>17,5%, up to 5000 times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december 2018, a total of 2411 blood products were sampled, of which 979 in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), 662 in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and 770 in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, 16 ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, 4 ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at 35°c for 7 days. cultures were checked daily for signs of growth. in addition, 2 ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated 48 h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of 1.6% (39/2411) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, 2.7% (26/979) of blood products were contaminated, in hpkll 1.5% (10/662) and in hslk 0.4% (3/770) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = 0.00047). there was no significant difference between the other sites (p = 0.051 and p = 0.12). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. (46.7%) and bacillus spp. (33.3%). the remaining 20% of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than 10³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at 10³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than 2000 blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate (1.6%) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (<10³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the 18 to 65-year-old general population registered at the nphc. methods: altogether, 474,017 18 to 65-year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between 2015 and 2017. reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ 1:8 and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the 18 to 65-year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk 1,2 across 2015-2017. statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < 0.05. results: anti-tp reactivity was confirmed in 167 blood donors. aeid was proved in 85 cases with 36 ft and 49 rt donors. in that period, 1696 syc were notified. both in aeid and syc, the age group of 25-34 years with approximately 35% and 36% of individuals was the dominant. the proportion of men was 74% and 77% (p = 0.5) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher (0.020%; 0.023%; 0.032%, p < 0.0001) than that of rt donors (0.007%; 0.009%; 0.009%) and the proportion of syc (0.008%; 0.009%; 0.010%) in the general population. pp of aeid showed a roughly homogenous distribution in the 7 regions (0.011%-0.025%), however, pp of syc had a significant (0.058%; p < 0.0001) central hungary dominance in relation to the other regions (0.008%-0.016%). comparing the pp of aeid to syc, central hungary indicated a significant difference (0.025% vs. 0.058%, p < 0.0001), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised 4135 donations, including 112 bm (2.70%), 3787 pbsc (91.60%) and 236 cb (5.70%) donations processed in our laboratory in the years 1996-2016. bm was collected in operating theatre-conditions, pbsc with cell separators -cs-3000 (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and 35°c) using a variety of culture media. pbsc and bm were tested using 2 samples with trypcase-soy broth (tsb-t) and 1 with schaedler + vit k3 (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/10/anaerobic/f (becton-dickinson). results: in the 1996-2016 period 42 contaminated products were found: 25 pbsc (0.66% of all tested pbsc units) and 17 cb (7.20% of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at 0,1%. in 2010, three (3) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis (61.36%). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around 12 million new cases of syphilis in the world, more than 90% occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between 2015 and 2017 we observed in our institution a significant increase of 24% in positivity of syphilis among blood donors from 0.62% in 2015 to 0.73% in 2016 and 0.77% in 2017 (p < 0.0000001). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in 2017 was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp 2017/23028-9. results: among 123,851 samples screened in 2017, 958 (0.77%) presented a positive result for cmia -syphilis. of these, 626 (65.4%) were included in the study. a total of 106 samples (17%) showed vdrl+/igm+; 96 (15%) vdrl+/igmand 28 (4.5%) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in 28 (4.5%) samples that presented positive results for elisa igm and vdrl negative with 21 (3.35%) positive results, 1 (0.15%) undetermined and 6 (0.95%) negative. none of the 626 samples showed the presence of treponema dna by real-time pcr. the prevalence was 0.77%, the incidence was 0.1% in the year 2017, and the incidence in relation to the total positivity was 20.4%. both, prevalence and incidence were higher in men, white, not married, aging 18-29 years and high school educational level. we observed a 6% a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of 1.5% htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in 2017 (0.77%) with an incidence of 20.4% of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found 22 (3.5%) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to 5 days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to 7 days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; 22°c) or refrigerated (cs; 4°c). aims: the aims of this study were to 1) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and 2) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to 60 rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg4 + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days 0, 1, 3, and 5 by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first 3-4 days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day 4. bacterial numbers remained unchanged in refrigerated aliquots through day 5. rt storage resulted in significantly (p < 0.05) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < 0.05) delay in bacterial growth at day 1. while growth of e. coli and p. aeruginosa recovered by day 2, growth of a. baumannii was significantly (p < 0.05) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < 0.05) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day 3, indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a 20-ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle (10 ml each) within 48 h after collection. the bottles were then placed in the bact/ alert system and incubated for at least 7 days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a 2-ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a 2-ml and a 20-ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september 2008 and march 2018, after maintaining in liquid nitrogen for 1 to 17 years, 205 pbpc products collected from 45 patients, which was preserved in a volume between 35 and 60 ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these 205 final products were thawed and cultured with both the 20-ml and the 2-ml aliquot. one of these pbpc products had the positive culture result with the 20-ml retested samples. nevertheless, the same pbpc product had the negative result with the 2-ml post-thaw pbpc sample and the 20-ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the 2-ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) (10 surfaces, 1 air), hôpital provincial g en eral de r ef erence (hpgrk) (24 surfaces, 2 air) and the national blood transfusion centre (cnts) (20 surfaces, 2 air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates (23.7 cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd30, kikkoman), expressed as relative light units (rlu) per 100 cm². air was sampled by active sampling (500 liter; spinair, iul) on cled and macconkey medium. in parallel, particles > 0.5 lm and > 5 lm were counted using a particle counter (14,15 liter; metone 227a). culture media were incubated for 48 h at 35°c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was 27 (6-60) cfu/rodac, 69 (6-103) cfu/rodac, 82 (3-132) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes (43 and 60 cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator (132 cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per 100 cm² was 2.906 (778-26.497) rlu at hpkll, ) rlu at cnts and 35.235 (1.754-348 .052) at hpgrk. atp results and total viable count were not correlated (n = 49, p = 0.52). median (range) bacterial count in the air was 258 (210-375) cfu/500l for all sites together. there was no correlation found between total bacterial count and particles > 0.5 lm or > 5 lm (r = 0.7 and r = 0.6 respectively; p < 0.05; n = 5) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january 1st 2020. however, we already decided to voluntarily test all our blood products since january 2015. aims: in this study, we present our results of a 100% screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january 2015 to december 2018, a total of 386,307 allogenic blood donations from 69,956 individual german blood donors were screened in a minipool format of 96 samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from 4.8 ml plasma using the chemagen msm-i extractor (viral 5k, perkin elmer chemagen gmbh). the 95% lod of the assay was determined to 4.66 iu/ml (447 iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for 3 months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf1. results: in total, 274 hev rna positive donors were identified. of these, 274 hev rna-positive donors, 216 were nat-only positive donations (78.83%, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer (1.09%), 30 donors showed reactive igm and igg titers (10.95%), 25 donors already had isolated igg titers (9.12%). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely 62 donors showed elevated alt levels (22.63%), mostly within a double increase within the reference range (16.06%), only 6.57% of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > 1,000 iu/ml compared to the group with viral loads between 100 and 1000 iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype 3 in all cases. the month-dependent incidence ranges from 1:719 to 1:3,781 blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february 2016, and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within 1 to 2 weeks. however, in whole blood, zikv rna might present positive up to day 101 after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of 200 ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at 37°c for 3 days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at 37°c for 3 days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to 10 9 copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < 0.05) in type o rbcs and lower than those in plasma (p < 0.05) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting 20 residents from 2000 to 2014. during 14 august to 4 september 2018, another outbreak of 29 confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district (19 cases) and the other in cheung chau, an outlying island (10 cases). aims: we assessed the risk of dengue transmission from blood donors during the 2018 outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, 2013) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of 15-to 64-year-old patients in the 2018 outbreak and residents of the same age range in hong kong and wts district as at mid-2018 respectively (16-65 years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during 12 august to 8 september 2018 after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, 2009 ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in 778,000 (266,000-1,822,000) territory-wide for the 28-day study period but increased to one in 147,000 (50,000-345,000) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was 1 in 279,000 (118,000-752,000) territory-wide and 1 in 52,000 (21,000-188,000) in wts during the same period. the eufrat also predicted a territory-wide issue of 0.08 unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at 1 in 778,000 during the 2018 outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by 4 folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july 2017 to december 2017 at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version 21.0 (ibm). results: in our study population there were 445 (98.9%) males and 5 (1.1%) females. the mean age of recruited blood donors was 28.38 (sd ae 7.34), with a range of 18-55. younger donors were more common with a frequency of 18-27 year olds of 249 (55.3%). we found an overall hev igg prevalence of 17.5% and an hev igm prevalence of 9.1%. there were 10 (2.2%) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was 33.8 (sd ae 41.0) with a range of 4-554 iu/l. the serum alt levels were elevated (>45 iu/l) in 73 (16.3%) blood donors. there was significant correlation (p=<0.001) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are 20 million hev infections, 3 million acute hev cases and 56 thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively 4.0% in new zealand, 13.5% in britain, 20.6% in denmark, 16% in the united states and 27.0% in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from 5552 blood donors were collected from april 2017 to april 2008 at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were 20.05% (1113/5552), 0.76% (42/5552) and 0.04% (2/5552), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = 1.089, p < 0.001; igm or = 1.055, p < 0.001). donors who were zhuang minority (32.69%, 7.69%) showed higher anti-hev than those who were han ethnicity (19.89%, 0.70%), and the difference was statistically significant (igg or = 2.052, p = 0.023; igm or = 12.029, p < 0.001). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population (60-100% seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested 20 h after infection, washed and used for spiking of plasma. plasma units (n = 2, 290 ml) were spiked with infected cell suspension (10% v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b2 illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses (0 after addition of mb, 30, 60, 90 and 120 [standard] j/cm 2 ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of 4.2 (bag no. 1) and 4.5 (bag no. 2) log 10 tcid 50 /ml was achieved in the plasma units. in hold samples, a mcmv titer of 3.8 (bag no. 1 and bag no. 2) log 10 tcid 50 /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ 2.9 log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log 10 reduction factors ranged from 4.2 (bacillus cereus) to 6.1 (serratia liquefaciens) for the tested bacteria, and from 3.1 (emcv) to ≥ 4.9 (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below 0.8% until day 35 of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a-1,2-fucosyltransferase (fuct2), encoded by fut2 gene, determines the secretor status of an individual. about 80% of caucasian population have a functional copy of fut2 (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: 126 samples of semen, 68 from infertile men and 58 from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who 2010 criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners (76.9%) were significantly higher than those from fertile couples (21.6%) (p < 0.03) the results obtained by pcr in sperm cells correlated 100% with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut1 gene (h | h), mandatory linked with an active fut2 gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric231-pe and a 1:1 mixture of bric231-pe/bric231, ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from 29 blood donors with different abo phenotypes (o (5), a 1 (5), a 2 (5), b (5), a 1 b (5), a 2 b (4)) and 2 patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a 1:1 antibody mixture (bric231-pe/bric231) covering approx. half of the h-binding sites by unconjugated bric231. in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric231-pe. rbcs coated with bric231-pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric231-pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen 1,2 , x xu 1,2 , x hong 1,2 , k ma 1,2 , j he 1,2 , j chen 1,2 and f zhu 1,2 1 blood centre of zhejiang province 2 zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: 109 samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of 109 samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province (2018rc029). results: 69 samples in 80 blood donors showed increased expression of h antigen, of which 47 were identified as abo subgroups. there were 22 enhanced reactions in 29 hemopathic patients. however, 19 were finally confirmed as normal abo genotypes. no statistical significance (86.3% vs 75.9%, p > 0.05) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is 50 and 3 respectively in blood donors and hemopathic patients. extremely significant statistical differences (68.1% vs 13.6%, p < 0.005) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november 2018 to january 2019, 55 cases (0.6%) of abo discrepancies among 9,550 abo blood type tests performed using the 15-min reaction mode of ih-500 in chonbuk national university hospital blood bank were identified. we compared the test results of 15-min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a1 and abo genotyping. results: in the immediate reaction using different red cell reagents, 45 cases (81.8%) of discrepancies disappeared and 10 cases (18.2%) remained discrepancies. all abo discrepancies observed in the 15-min reaction were due to serum side causes, and one case (1.8%) was due to both of serum and red cells side cause. nonspecific response (28 cases, 50.9%), cold antibody (20 cases, 36.4%), rouleaux formation (3 cases, 5.5%), cis-ab (3 cases, 5.5%), and abo subtype (1 case, 1.8%) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the 15-min reaction. summary/conclusions: ih-500, an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the 2 years data from jan 2017 to dec 2018. the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel 2016 and spss (version 22). results: during study period a total of 1970 patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were 20.91%, 37.51%, 32.74%, 8.83% for a, b, o and ab respectively. the common neurological tumours found in our study were glioma (33.55%) followed by pituitary adenoma (20.05%), meningioma (18.58%), schwannoma (8.98%), cavernoma (2.54%), neuroma (2.23%) and space occupying lesions (14.06%). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in 47.73% o group patients as compared to other blood groups which was found statistically significant (p < 0.05). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen (38.2%) is more common as compared to o blood group(33.4%). background: rhd and rhce represent homologous genes in head-to-head position on chromosome 1 (chr1, p36.11). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band 3 and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, 2016). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, 1955) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad-3/brad-5/fog-1 (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric-69pe (ibgrl). results: a total of 146 samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr(21), r1r(20), r1r1(23), r2r(18), r0r(15), r1r2(27), r2r2 (22)). variant expression of rhd by different rhce phenotypes using brad-3-pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples (3) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad-5-pe and fog-1-pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c.48g>c, c.201a>g, c.203a>g of exon 1, exon 2 resp. and intron 2) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c.676c>g, exon 5) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon 5 is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh26 antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:-26, and the antibody specificity anti-rh26. most polyclonal anti-c contain anti-c and anti-rh26. previous studies have shown 2 of 10 monoclonal anti-c reagents are actually anti-rh26. these reagents will not detect the c antigen where the red cells are rh:-26. aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r 1 r) was inconsistent with the current donation phenotype c-(r 1 r 1 ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a 2 + reaction by tube with bio-rad seraclone â (2) [clone ms35] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk7300 using beckman coulter anti-c [clone 951] blood grouping reagent and tested positive (4) reaction on the immucor neo using immuclone â (1) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd*01/*01n.01 and rhce*01.15/*02 with a predicted phenotype of c+, c+ w , d+, e-, e+, rh:55 (locr+), rh:-26. a review of the donor's historical records indicated the donor tested as c+ on the pk7200, which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms33]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:-26 variant associated with the rhce*01.15 allele. reagents containing clones ms-33 and ms35 correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk7300 and associated anti-c [clone 951] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:-26 phenotype, which is in contrast to the previous report by faas et al, transfusion, 1997 where it was demonstrated that clone 951 reacted with c+ rh:-26 bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since 2017 at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of 6 monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in 32 samples: a) by using the partial d kit, 23 samples were typed: four samples were characterized as "partial d" (3 dfr, 1diii) and 19 as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type 1" three samples, "weak d type 4.0 or 4.3" one sample). b) the nine (9) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized 2 samples as "weak d type 1", one sample as "weak d type 3" and another as "weak d type 11". results are pending for 5 samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in 68,75% of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over 200 rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ 50 years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type 1, 2 or 3 are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type 1, 2 and 3. aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from 2013/01/01 to 2018/10/01 we analysed 119.845 samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs232, bs226) (biorad, dreieich, germany), swing maestro [lmh 59/20 (ldm3) + 175-2 and th-28 + rum-1 + ldm1] and ih-1000 [lmh 59/20 (ldm3) + 175-2] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from 119.845 samples 300 (0,25%) were swd. 71/300 (24%) were referred to rhd genotyping. 55/71 (77%) samples were weak d type 1, 2 or 3, while 16/71 (23%) were weak d type 14 and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types 1, 2 and 3. clearly negative serologic reactions were given in 27/29 samples with bs 226 and bs 232, in 30/33 samples with lmh 59/20 (ldm3) + 175-2 and in 18/39 samples with th-28 + rum-1 + ldm1. summary/conclusions: the prevalence of swd in this study is rather low (0,25%). after rhd genotyping 77% of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of 3243 randomly selected saudi donors, who donated blood between january and june 2018, were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k1 phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of 3243 saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in 93.7%, while k antigen was positive in 11.1%. among other studied rh antigens, e was the most common (98.1%) followed by c (78.2%), c (70.3%) and e(26.9%). dce/dce (34.2%) and dce/dce (5.3%) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent (11.9%) with almost 5 times higher frequency compared that reported in caucasians (2.0%). the rare phenotype dce/dce was found in 3 donors (0.09%), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a 58-year-old male patient with lung cancer without previous transfusions was admitted (06/2009) for surgery. his hemoglobin was 13.2 g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july 2017, the patient was admitted for colon cancer surgery with a hemoglobin of 11,0 g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge*01.-03. an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of 0.35% and the antibody was considered not to be clinically relevant. the mi was interpreted as following: 0-3% not significant; 3-5% inconclusive; >5% clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received 2 units of ge:-2,-3 prbcs without any transfusion reaction. one and a half year later (11/2018), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was 87 g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july 2017 indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following 16 days the patient received a total of 5 units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december 12 th and 16 th (12 days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of 0.8% und 1% respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from 1 st april, 2013 to 30 th september, 2015 (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a1 lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen (3-cell) and identification (11-cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected 104 (0.072%) abo discrepancies out of the total 144279 donor samples tested during the study period. out of these, 135043 (93.6%) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody (33/104; 31.73%), followed by weak anti-a antibody and weak subgroups of a (24/104 each; 23.07% each) and weak subgroups of b (5/104; 4.8%). the remaining 17.3% (18/104) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was 0.02% ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within 3 days after receiving a patient's sample. however, in some cases, retests could be performed after more than 3 days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih-500 and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih-500 (bio-rad, 1785 cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, 1 week after storage at 4°c and 1 month after storage at à20°c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih-500 and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, 15; anti-le a , 4; anti-di a , 4; anti-c+e, 3; anti-m, 3; anti-d, 2; anti-c, 1; anti-k, 1; anti-jk a , 1: anti-xg a , 1; unidentified antibody, 13; autoantibody, 2 cases. with regard to the changes in reactivity owing to storage, 26 (52%) samples (anti-e+c, 12; anti-m, 3; anti-di a , 3; anti-d, 2; anti-c+e, 2; anti-le a , 1; anti-c, 1: autoantibody, 1; unidentified antibody, 1) showed identical reactivities after 1 week and 1 month storage by both ih-500 and tube methods. however, 19 (38%) samples, comprising 12 unidentified antibodies, 3 anti-le a , 1 anti-c+e, 1 anti-e, 1 anti-e+c, and 1 autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after 1 month storage, while one sample with anti-xg a showed decreased reactivity only after 1 month storage. higher reactivities were observed in all samples detected using the ih-500 analyzer than manual tube methods (p < 0.005, wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih-500 and tube methods were the same in all storage states; however, reactivities were higher in ih-500 than in the tube method. twenty-six (52%) of 50 samples showed identical reactivities after 1 week refrigeration and 1 month freezing. nineteen (38%) samples showed decreased reactivities after storage; however, 12 (12/19, 63%) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than 3 days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel 1 , d huber-marcantonio 1 , n rufer 1 , g canellini 1 and c niederhauser 2 1 unit of transfusion medicine, interregional blood transfusion src, lausanne 2 laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of 3'753 dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (<14 days). results: a positive dat was found for igg and c3d in 421 out of 3'753 (11.2%) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, 244 (57%) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only 10.7% (26/244) of the cases. from the 161 patients transfused within the last 14 days and having a positive dat, 60 (14%) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms (244 versus 60 samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last 14-days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd38 monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin #16-02). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd38 antibodies. vox sanguinis (2019) 114 (suppl. 1), 5-240 methods: edta-anticoagulated whole blood samples coming from 30 patients in different stages of treatment with daratumumab and 5 with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd38 antibodies treatment. in these cases, 6 of 7 negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed 100% agreement with genotype id core xt results. focusing in the data obtained during the treatment, 8 negative antigens were type as positive in gel technology (12% of the tests). mdmulticard agreed with genotype in 100% of the analyzed antigens. as complementary data, 13 of 66 patient-treated samples had dat or ac positive and 59 showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd38-directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes 25 ll and 50 ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and 20 donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card (50 ll at 0.8%). further, sample dilutions were dispensed into the card (25 ll or 50 ll). subsequently, cards were incubated 15 min, 37°c (coombs technique) and 15 min, 18-25°c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = 80 titrations, titer ranged 0-256) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ 0.5 titer in the 98% of the cases). differences between dg gel coombs and neutral (saline technique) (n = 80 titrations, titer ranged 2-512) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at 37°c and igg antibodies. differences were > 1 titer in 35% of the comparisons and ≤ 1 titer in the rest of the cases (65%). comparing sample volumes of 25 ll and 50 ll in all cards (n = 160 titrations), higher titers were observed using 50 ll, as expected. differences were 1 titer in the 51% of the comparisons, <1 titer in 44% and > 1 titer in the 5% of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged 0-18 years, diagnosed with aiha, between april 2017-december 2018 (21 months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: 10 patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from 2.5 to 9 grams/dl. the initial presentation was severe anemia in 7 children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in 3. the trigger for haemolysis was infection in 4 children. rheumatological evaluation was performed for 5 children out of whom 2 were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for 4 and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in 9 out of 10 aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of 9 dat positive cases had igg & c3d positivity on monoclonal dat testing whereas rest 7 had only igg coating the red cells. dat titration was more than 1:300 in 4 patients; where only 1 of these 4 patients had both igg1 and igg3 coating and rest 3 had only igg1. alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a 4-6 weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in 2 and rituximab was used in 3. out of these children 5 children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a 64-year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and 5-fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day 1), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day 5), two units of rbcs (day 6) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day 24 were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at 1 and 5 mg/ml. results: dat was positive (anti-igg 2 + , anti-c3d 2 + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer 2) and enzymetreated (titer 8) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at 5 mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p-331 singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd38 used in the treatment of multiple myeloma and has been known to bind to cd38 on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october 2016 to december 2018 was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of 55 patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in 23 of the 55 patients while the use daratumumab was mentioned in the serology request form of the other 32 patients. the median tat for red cell antibody screen and identification is 212 min (range: 47-877) if information on daratumumab was provided prior to start of testing and 301 min (range: 133-1417) if information was not provided prior to testing. the median tat for routine testing is 198 min (range: 15-2245). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value 0.72634). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value 0.00694). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd38 monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd38 is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd38 on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd38 interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd38 tertiary structure, avoiding the binding and testing interference by the anti-cd38 daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since 2016, 32 patients were studied, from which 16 were transfused with 108 blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd38 and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd38 is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july 2014 and dec 2018 (54 months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of 22888 patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in 145 patients and autoantibody was detected in 53 patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha (58%) and 20% of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group (45% vs 34%, p < 0.05). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in 21 where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood (1.2% vs. 5%, p < 0.05). during the period of study we obtained 10 cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk7300, 250,599 donation samples were tested and 4,895 (1.95%) were found absc positive. antibodies to red cells were identified in 250 donations out of 4,895 (5.11%) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was 0.10%. the top 5 most frequent antibody specificities were: nonspecific cold antibodies (28.8%), anti-e (26.8%), anti-mi a (19.6%), anti-m (16.8%) and anti-le a (4.0%). a total of 225,124 donations were screened for atypical antibodies by ih-1000 and 2,275 (1.01%) were screened positive. among these, anti-red cell antibodies were identified in 1,239 samples (54.46%), which was significantly higher than those identified in pk7300 screened positive samples (p < 0.00001). the prevalence rate for atypical antibody as screened positive by ih-1000 and with confirmed red cell specificities was 0.55%, which was also significantly higher (p < 0.00001). the top 5 most frequent antibody specificities were: anti-mi a (55.3%), anti-m (21.1%), anti-le a (10.2%), anti-e (6.5%) and non-specific cold antibodies (3.6%). anti-fy b was detected in 7 cases, which would be missed detection by enzyme treated reagent cells on pk7300 system. summary/conclusions: the performance of the ih-1000 system using a 3-cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk7300 in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of 0.55% in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus 2019) reported a rbcalloimmunization prevalence of 11.4%, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a 18 years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from 1990 to 2018 thalassaemia patients, with a minimum follow up of 1 year and transfused with more than > 10 rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall 57 patients (38 females, 19 males) were included in our retrospective analysis: 54 patients were affected by thalassaemia major and 3 by thalassaemia intermedia. rbc alloimmunization prevalence was 12.3% (7 patients): 3 patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected (1 anti-h, 1 anti-cw, 4 anti-e, 1 kpa, 1 anti-jka, 1 anti-jkb, 1 anti-m and 1 anti-lua). in 2 out of 3 alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining 4 patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the 7 patients positive for alloantibodies, 6 were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: 12.3% vs 11.4%; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since 1998. recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april 2013. that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in 3642 patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in 759 patients, acute leukemia in 600, multiple myeloma in 390, chronic lymphatic leukemia in 319, chronic myeloid leukemia in 218, aplastic anemia in 147, hemophilia in 105, myelodysplastic syndrome in 73, and other hematological diseases in 1031. the frequency of detection of antibodies to antigen c (0.25% vs 0.06%) and to antigen e (0.46% vs 0.12%) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly (0.10% vs 0.06%, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since 1998, therefore the immunization index for these antigens remained unchanged and amounted to 0.10% vs 0.12% for anti-c antibodies; 0.36% vs 0.36%for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included 466 first time patients of hematology clinic in 2017-2018. typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in 84 (18.0%) patients. the total number of complex cases was 96. the double population of red blood cell was most often determined in antigens of the rhesus system (10.9% of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in 31 cases (60.8% of patients with the chimera for rhesus and kell antigens), cin 23 (45.1%), sin 15 (29.4%), e -5 (9.8%), cw -8 (15.7%), k -5 (9.8%). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in 0.6% of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients (1.1%) was due to a decrease in the production of antibodies -4 cases and the appearance of extra agglutinins -1 case. autoantibodies were detected in 3.9% of all patients, including 0.6% of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in 2.8% of patients, including specific anti-din 4 (0.86%), anti-dcin 2 (0.43%), anti-kin 1 (0.21%); antibodies of unidentified specificityin 2 (0.43%), polyspecificin 4 (0.86%). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of 3,49,497 crossmatches were performed over period of last four & half years, out of which 867 units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c3d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of 3,49,497 crossmatches, only 867 units were found to be incompatible (0.14%). the major cause for incompatibility found in patients was aiha-(32.87%). other causes of incompatibility were infections (27.44%), multiple transfusions (17.41%), trauma (11.23%), evan's syndrome (4.15%), rh negative mother (3.57%), sca (2.99%) & incompatibility due to dat positive packed red cells (0.34%).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb-1) was admitted to our hospital in january 2017 for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu4 / atg protocol (5 days of 250 mg iv. fludarabine, 4 days of busulfan 792 mg iv, 2 days of 300 mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of 2016, was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december 2016. results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and 0.2 m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a 75-year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in 2004, with unknown transfusion history. her obstetric history was g6p4a2. the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a 0 rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from 9.0 g/dl to 12.0 g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (>99% prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of 1.2%, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no 0 rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an 18-years-old female patient with scd presented to our hospital with hemoglobin level of 4 g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial 11 cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r1r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to 2.8 g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to 8 g/dl in 2 weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r1r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than 1% of most populations are known as low incidence antigens and those present on more than 90% are known has high incidence antigens. the mns blood group system consists of 49 antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are 35 low incidence and 10 high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns49) antigen. anti-jenu was first described in a thai patient with thalassemia where only 2 compatible units were found following screening of 3600 units. the jenu negative phenotype is a rare phenotype with an estimated frequency of 0.06%. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of 4 + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd47 is also expressed on red blood cells (rbcs). recently, many drugs targeting cd47 have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd47 targeted high affinity sirpa fusion protein alx148. methods: a 35-year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx148 clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past 8 months. she had no previous transfusion history during the past two years. after two infusions of alx148, two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and 37°c albumin phase, and 2 + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and 37°c albumin phase, but 2 + at ahg phase. antibody screening (2 cells) and identification (11 cells) all showed 3 + at ahg phase using gel cards. direct antiglobulin test was 4 + for anti-igg and 3 + for anti-c3d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to 7.8 g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between 11.1-12.0 g/dl but it decreased again to 9.2 g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx148, and antibody screening and dat decreased to 0-1+ reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to 13.6 g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd47 is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms15/seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons 4-11 was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk*01(28a,226a,303a,588g)/jk*02. to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk*01(28a,226a,303a,588g)/ jk*01 and jk*02/jk*02, respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk*01(28a,226a,303a,588g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within 3 days of transfusion if a patient has been transfused or pregnant in the preceding 3 months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this 3 day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a 60 y/o man who presented to the ed (hospital day 0, hd0) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (<3 days). methods: specimen #1 (s1) was sent on hd0 for type & abs (t&s) and crossmatch (xm) of 2 rbcs. abs and immediate spin xm were negative; there was no patient history. by hd9, he had 4 negative t&s specimens (hd0: s1; hd2: s2&3; hd6: s4) and had been transfused 4 rbcs (hd2: 2; hd5: 2) via electronic xm (exm). at 1730 hr on hd9, 2 rbcs were requested and could have been issued via exm since s4 was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s5 was requested and received at 1801 hr. results: s5 showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from 10.2 (14.0-17.5 g/dl)/30.1 (41.5-50.4%) on hd6 to 6.9/ 20.6 on hd9. during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd9, which he tolerated well with an increase of hemoglobin from 6.9 g/dl to 8.6 g/ dl. he did well post transfusion with stable h/h between 8.1/24.2.0 to 8.5/25.3. he was discharged on hd19. repeat abs on s4 was negative. of the 4 rbcs transfused before s5, one was e+ and four jk(a+). the family reported that he was injured 20 years prior and had been admitted to 3 hospitals, but was unaware of transfusion. hospital #1 (h1) reported admissions 20 years ago (2 rbcs transfused) and 4 years ago; all abs were negative. h2 admission was 5 years ago with positive abs and inconclusive workup. h3 admission 4 years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than 3 days from the specimen collection, likely a secondary immune response to sensitization from a transfusion 20 years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every 3 days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a 75 year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong (4+). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr1), a high frequency antigen expressed by the abcg2 gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and 16 exons and adjacent intronic sequence of the abcg2 gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive (2+) in iat test, while the self-control was negative. the antiserums reacted strongly (4+ in iat test in gel card) with the papain-treated cells, but kept the same reaction strength (2+) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was 2. in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg2 gene in the patient and her brother revealed one homozygous nonsense mutation in exon 4 (c.376c>t, p.gln126x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals (98% or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, 76 years old, o rhd+, previously transfused with 4 red blood cells concentrates in 2006, was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected (2+ in all the 3 screening cells). in order to identify the specificity of the antibody, a panel of 11 cells was tested; the result was considered inconclusive, due to positive reactions (2+) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel (2+ with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg1 and igg3) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from 80865 blood samples, four donors with jk (a-b-) were selected, at a frequency of 0.0049%. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs5-1g>a combining with heterozygote of 359g>a (gly120glu) and heterozygote of 896g>a (gly299glu) combining with heterozygote of 956c>t(thr319met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs5-1g>a, 896g>a and 956c>t were common mutations in the before reports, while 359g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk3. background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of 10 blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: 5,213 blood donors from age 19 to 54 were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of 10 blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce 0.3%, rhce*ce 65.0%, rhce*ce 28.8%, rhce*ce 5.9% -kel*k_kpb_jsb allele 100% -jk*a allele 49.1%, jk*b allele 50.8%, jk*b_null allele 0.1% -fy*a allele 92.8%, fy*b allele 7.2% -gypa*m allele 51.1%, gypa*n allele 48.9% -gypb*s allele 5.1%, gypb*s allele 94.8%, gypb*mur allele 0.1% -di*a allele 4.7%, di*b allele 95.3% -do*a allele 10.1%, do*b allele 89.9% -co*a allele 100% -yt*a allele 100% -lu*b allele 100% summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding 16 blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu (8/14), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c.697c>g, and rhce*c.733c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c.667g>t were tested by pcr-ssp. a total of 1111 probands including 800 individuals from syria, 147 from iran, 123 from the arabian peninsula and 41 from northern african countries were included. results: 2% of the donors were homozygous for the fy null (fy*-67t>c, fy*02n.01) mutation, 16.2% carried the heterozygous mutation. 0.4% of the syrian probands were heterozygous for the do*350c>t mutation (both, do*jo1 and do*jo2; jo(a+/ a-)) that is virtually unknown in caucasian donors. 0.8% of the syrian donors heterozygously carried the kel*02.06 allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel*02.06 carriers were identified. 1.8% of the syrian and 1.5% of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c.251c>g, intron 5 + 5 g mutation, inducing the s-u+ w phenotype. 3.6% of all and 29.3% of northern african donors were heterozygous for the rhce*c.733c>g substitution, 0.3% of the syrian donors carried rhce*c.697c>g (heterozygously) and 0.004% of all donors were heterozygous for rhce*667g>t. heterozygosity for vel deficiency (vel*-01) was detected in 21 individuals (2%; 16 of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp1*c.601delg (co*01n.06) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals (2 blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: 1/gel cards diaclon id abo/d (anti-a: clone a5, anti-b: clone g1/2, anti-a,b: clone es131, es15+ birma 1+ es4; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m297/628 = la-2; bio-rad); 2/tube techniques with: anti-a (birma 1; a-11h5, a1 s.pa1m095, c.9113d10), anti-b (lb-2, b-6f9, c.9621a8). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of +7.21-kb site of abo gene to cover sequences ranging from the end of intron 1 to 3 0 utr of the abo gene. additionally sequence of exon 1 of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals (2 blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma 1 (1+ to 2+), a-11h5 (1+ to 2+) and c.9113d10 (weak+ to 1+); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a1 antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of 1061delc typical for abo*a2 alleles). in all these individuals abo sequencing of 7.21-kb fragment confirmed the heterozygous genotype with 7 polymorphisms characteristic for abo*b.01 allele (297a>g; 526c>g; 657c>t; 703g>a; 796c>a; 803g>c; 930g>a) and detected a novel abo*a allele sequence with duplication-based insertion of 21 bp after 564 position (abo*a c.dup543_563; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon 188 (p.dup_182_188qdvsmrr), with synonymous change of the repeated codon 182 (cgc>cgg) and 188 (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup543_563 allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a1.01 allele and the same abo*a c.dup543_563 allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in 1990 of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons 1-7 (including 50 base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a1 in plasma. abo genotyping gave the genotype abo*a1.01/o2.01 usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately 85%) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately 15% of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a1.01-like allele except for a novel mutation located in intron 5, c.239+4g. the o 2 allele had an additional snp, c.689g>a, consistent with the abo*o2.03 allele variant summary/conclusions: a previously unreported variant, c.239+4a>g, likely effecting the 5 0 -donor splice site of intron 5 was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. 374+4a>g mutation is located in the 5 0 -end of intron 6 and is predicted to cause partial skipping of exon 6. strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately 2% positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past 3 years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a2, b, o1 and o2 alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon 6, intron 6 and exon 7 were amplified using a forward primer targeting the 261g nucleotide (to exclude o1 alleles amplification) in combination with either b, a2 or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of 16 samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in 8 out of 12 samples with suspected a subgroup alleles, the c.804insg insertion was detected corresponding to the abo*ael.01 allele. the abo*aw31.02-05 variant, a hybrid a1-o1v allele, was found in 2 cases. in 1 case we found the c.722g>c change, previously reported associated with weak a antigen expression. finally, a novel c.961c>g change was detected in an a2 allele. b(a) or cis-ab suspected alleles: the abo*b(a)04 variant carrying the c.640a>g change was found in 1 of 3 samples with bo1 genotype but a weak antigen expression. in the remaining 2 cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b01.02 allele carrying the nucleotidic change c.926a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon 7 of the abo gene, in 14 out of 16 samples, including 2 novel alleles. chimerism was suspected in 2 cases of a antigen expression in samples with b1o1 genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but 8% of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a 20-year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih-500; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a 3 b 3 phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a102/b101/o01. however, in buccal swabs, the patient showed a102/o01 and his brother showed b101/o01. other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d8s1179, d21s11, d7s820, csf1po, th01, d13s317, d16s539, d19s433, d18s51, d5s818, and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case #1 is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.29-5t>g in heterozygosity on an otherwise common a1 allele, and in trans an abo*b.01 allele. case #2 is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c.29-5t>g in heterozygosity on an a background, and in trans an abo*o.01.01 allele. given that this variant is located near the intron 1 splice acceptor site, abo*29-5g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case #3 is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c.467c>t (pro156leu) and c.709t>a (tyr237asn), both in heterozygosity on an otherwise common a1 allele, with an abo*o.01.01 allele in trans. thus, the data establish an association of allele abo*467t,709a with an aweak-like phenotype. case #4 is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c.479c>g (pro160arg) in heterozygosity on an a2 background, and in trans an abo*o.01 allele. an interpretation of the data is that variant c.479c>g weakens the activity of the a2 transferase, with allele abo*a2(479g) encoding the aweak-like phenotype detected by serology. case #5 is a 9 year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.803g>c (gly268ala) in heterozygosity on an a2 background, and in trans an abo*o.01.02 allele. the serology and molecular results suggest that allele abo*a2(803c) encodes a cisab weak phenotype. case #6 is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c.739g>a (glu247lys) and c.871g>a (asp291asn), both in heterozygosity, in trans, and on a1 backgrounds. variant c.871g>a by itself constitutes allele abo*a3.01. the phenotype encoded by abo*739a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case #1 is a 35 year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a1/abo*o1. sequencing detected variant c.107insg (val36gly>fs56ter) in heterozygosity on an otherwise common a1 allele, and in trans an abo*o.01.01 allele. it is unclear how the early truncation of the a1 transferase encoded by allele abo*107insg still allows for some residual enzyme activity, as suggested by the reverse a type. case #2 is a recently-transfused 72 year-old black patient with an unresolved abo type. sequencing detected variant c.297a>g (silent) in homozygosity and variant c.1049c>t (ala350val) in heterozygosity, both on an o1 background, with an abo*b.01 allele in trans. although variants c.297a>g and c.1049c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o1(297g,1049t) allele. case #3 is a 40 year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon 7 detected variant c.436c>t (arg146cys) in heterozygosity on an abo*b allele background, and in trans an abo*o.01.01 allele. the phenotype encoded by allele abo*b(436t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case #4 is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a 2+ mixed field (mf) in tube. reverse type on a1 cells 1+ in gel, 0/1+ in tube. sequencing of genomic dna and cloned pcr products covering exons 6-7 detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*o.01.09 allele. case #5 is the newborn baby of case #4, with a forward type a 1+ mf in gel, a 3+ mf in tube. sequencing of the baby's dna detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*b.01 allele. from these results it is inferred that the phenotype encoded by allele abo*784c is a3-like. case #6 is an 18 year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons 5-6 and 6-7 detected variant c.979c>t (gln327ter) in heterozygosity, and in trans an abo*o.01.02 allele. the truncation of the a1 transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo*979t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. variants reported to date in the intron 1 enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut1 could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v5.1 170221 of red cell immunogenetics and blood group terminology working group of the isbt, 55 fut1 alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut1 alleles for the 19 chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut1 was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut1 sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut1 homozygous mutations were found in the 12 individuals and fut1 heterozygous changes were existed in 7 individuals after bidirectionally sequencing. .05%, 7.89%, 5.26%, 5.26%, 5.26%, 2.63%, 2.63%, 2.63% respectively in the 19 individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut1*01n.06(c.551_552delag), fut1*01w.09 (c.658c>t) and fut1*01w.01 (c.293c>t) were abolished in vitro assay, while fut1 mrna levels of them had no effect compared with wild type. summary/conclusions: the fut1 mutations in the para-bombay individuals were various. the most common fut1 allele in the chinese individuals with para-bombay phenotype was fut1*01n.06(c.551_552delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a1 and a2. later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a1, and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons 6 and 7 of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron 1 enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron 1 enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o1v/a 1 . dna sequence analysis showed result abo*a01 (28+5859a), abo*o.01.02. the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a01(28+5859a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d175-2, immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately 85% of o d+c+ cells. the patient's genotype was identified as abo*o.01/*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a1/*o1 genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a1 homozygote result. sanger sequencing of abo exons 6 and 7 also gave anomalous reactions: no abo*a allele was detected. homozygosity for c.261delg was observed as well as heterozygosity for c768c/a. this result therefore suggests the patient's genotype is abo*o.01/*o.01.26. next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr 3 0 -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing 4 copies of the enhancer region were present. presence of a single copy of the 43-bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, 9-30% of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd*01el.01 (rhd*1227a) is most prevalent (>99%) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from 2017 to 2018, the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th-28/ms-26, igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of 129 serologically apparent d-chinese patients (female, n = 128; male, n = 1) with alloanti-d were identified. different titers of alloanti-d from 1:2 to 1:4096 (≤1:16, n = 60; >1:16, n = 69) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = 11; anti-d mixed anti-e, n = 5). serological rhd typing confirmed the serologically apparent d-phenotype. rhd*01n.01/01n.01 (homozygous rhd gene deletion) genotype was identified in majority of them (123/129, 95.3%) by rhd zygosity analysis, while rhd*01n.03/ 01n.01 genotype (n = 5) and rhd*01n.05/01n.01 genotype (n = 1) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average 25% frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, 2005; legler, trans. med., 2001; richard, transfusion, 2007) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all 10 rhd exons were sequenced. the sequencing data revealed the mutation c.148+2delt in the splice donor site of exon 1. to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns4) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c.148+2delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing 9 monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor 1 were predicted to be rh:-1,-2,-3,4,5. however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position 1151 which is the second last nucleotide of exon 8 and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon 8 was identified. the mutation c.1151c>g leads to the amino acid substitution t384r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., 2011) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd*1151g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor 2. further serological determination of the rhd antigen with 9 different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c.526g>c in exon 4. this mutation causes a p.a176p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c.1151c>g and c.526g>c harbouring an amino acid substitution within a transmembrane segment. the c.1151c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c.1151c>g suggests that the t384r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c.526g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id1 is a d-negative donor self-declared as african descent. sample #id2 is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id1 was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id1 were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id2 was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional (3d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id1, a single nucleotide missense change, i.e. c.1151c>g in exon 8, was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position 384 (p.thr384arg) of the rhd protein. analysis in the 3d model clearly suggests a dramatic impact of the p.thr384arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id1. in sample #id2, the single c.325a>g transition was found in exon 2 leading to a threonine-to-alanine substitution at amino acid position 109 (p.thr109ala). amino acid 109 is located in rhd protein extracellular loop 2, and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id2. summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type 4 and diva cluster. in africans, the 16 most frequent were typically associated with alleles of the weak d type 4 (including dol and rhdpsi), diva and dau clusters with f223v occurring in > 10% of alleles; in addition the key mutations of weak d type 1 and dii and two inactivating mutations (c.1056_1057inst and c.1060delg) not reported in rhb were among the first 20 polymorphisms. in east asians, rhd(1227g>a) at 0.8% was most frequent, followed by dfv, weak d type 33, dbo-3, key mutations of diva and weak d type 4 cluster as well as rhce-like substitutions and the mutations of weak d type 25, type 15, type 66, rhd(a85v), dvl-1, weak d 119 and rhd(n135s). weak d type 151 and rhd(t148r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type 45 and type 33 in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r 0 haplotype and 1 probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r 1 r 1 , r 2 r 2 , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v3.1 cycle sequencing kit. sequence data was aligned to rhd_ng_007494.1. rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on 201 rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using 2 different anti-d igm clones (clone 1, dvi+: ldm1+esd1m; clone 2, dvi-: rum-1, th28) and 2 different anti-d igg clones (clone 1: ms26; clone 2: d415 1e4). all donors with d-negative results (n = 201, divided into 153 subjects with rhc+, 45 with rhe+ and 3 with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further 101 donors, studied prospectively. in 92.5% of donors (n = 186), the molecular analyses showed the complete deletion of the rhd locus, while in 15 cases (7.5%) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these 15 donors revealed the presence of 9 negative and 4 weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd*01n.82 (2 cases), rhd*01n.83 (1), rhd*01n.80 (1), rhd*01n.61 (1) , rhd*01n.05 (3), rhd*01el.08 (1), rhd*01el.18 (1), rhd*01el.17 (1), rhd*01w.54 (1) . moreover we found a donor with a lack of signal encompassing exons 1-5 of the rhd sequence (bioarray rhd beadchip), while 2 additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about 1 in 50 cases (4 subjects out of 201), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of 43960 serologically typed donors, 85.5% were d+, 14.0% d-and 0.5% weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in 22 blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: 62% weak d type 1, 28% weak d type 3, one donor was typed as weak d type 14 and another one as weak d type 11. these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from 2+ to 4+ with iat, except for the weak d type 11 with the score of <1+ which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was 19%. one of the weak d type 1 donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type 14, as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a 14 year-old young woman with parameters of hemolytic anemia: low hemoglobin level (10 g/dl), reticulocytosis (14%), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron 4 and the 3 0 untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c.920c>t mutation in rhag exon 6 in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c.920c>t mutation is responsible for the p.ser307phe amino acid substitution predicted to be in the 10 th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c.920c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of 15692 individual blood donors within a 15 month period, 1688 (10.76%) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from 112 out of 151 (8.95%) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron 4, exon 7 and exon 10 by a multiplex pcr-ssp method. the reaction was conducted in a final volume of 20 ll with primers that were applied as described by f. wagner et al. (bmc genetics, 2001 ) except antisense primer for exon 10 and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a 4% agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of 112 d-individuals analyzed, 101 were ddccee, 8 ddccee, 2 ddccee and one had a ddccee phenotype. molecular analysis showed that 104 (92.86%) were negative for all four rhd dna regions. among the other 8 samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron 4, exon 7 and exon 10, three for rhd promoter and exon 10, and two for exon 10 alone. further genotyping revealed five hybrid rhd-ce-d alleles [3 rhd-ce(2-9)-d and 2 rhd-ce(3-7)-d], one allele represented the del(m295i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than 100 weak d types have been described to date. transfusion recipients with weak d type 1, 2, or 3 are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is 5 anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band 3, as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, 4 haplotypes containing the mcc b and sl2 polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr1 polymorphisms and haplotypes, especially those containing mcc b and sl2 snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr1 polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do*01 and do*02 backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a 42 year old woman from the northeast brazil with a history of 4 pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the 3 exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology 2 diagnostic immunohematology 3 experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises 360 blood group antigens. most antigens (322) belong to one of the 36 blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of 38 antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in 1967.~91% of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in 96% of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a 3-cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b4galnt2 gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad 3-cells panel, containing donor 626521 with high expression of sd a . additionally, 200 pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b4galtnt2 exon 1-11. results: sequencing of b4galtnt2 revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in 6 controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon 10, c.1396t>c (enst00000300404.2, rs7224888) changes a cysteine to arginine at position 466 of the protein. the second mutation in exon 11 c.1590a>g (rs16946912) does not change an amino acid. both snps have a maf of 0.11 and therefore we expect that 2.1% of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b4galtnt2 gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon 10 causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~4% sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b4galnt2 and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by 5ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl8 was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = 0.016). similar result was also observed in rbc chemokine scavenging of ccl2 (p = 0.038). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = 0.021). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp1pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a 66-year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp1pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was 12.9 g/dl. 800 ml of blood was withdrawn through a radial arterial catheter in two 400 ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of 6% hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged 48 h later with a hemoglobin level of 12.3 g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp1pk and sequencing of the a4galt gene, which were conducted according to the protocols by koda et al.(transfusion. 2002) . the proband and her brother were homozygous for c.1029dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[140a], gypb*s_null(ivs5 + 5t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c.140c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about 0.06% and in thais with 0.04%. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom*12 allele. the lack of the serf antigen, serf(à) on red cells is caused by a single nucleotide polymorphism, c.647c>t in exon 5 of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(à) and a serf(à) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom*12 allele among blood donors. aims: this study aimed to identify the crom*12 allele among thai blood donors leading to predicted serf(+) and serf(à) phenotypes. methods: dna samples obtained from 1,512 central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom*12(+) and crom*12(à) among 1,512 central thais were 0.992 (3,001/3,024) and 0.008 (23/3,024), respectively. the homozygous of crom*12(à/à) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen 100 samples together with 23 heterozygous crom*12(+/à) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom*12(+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than 25 years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o1, o2, a2 and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, 31 from patients and 25 from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd1227g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene 1227 g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd 1227g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd 1227g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to 5 0 ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd 1227g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon 9 sequencing was performed to determine rhd 1227g>a genotype. results: a total of 84 samples were genotyped for rhd 1227g>a by pcr with t mshift primers. 32 samples were typed as 1227a+/g-, 22 samples were typed as 1227a-/g+, 6 samples were typed as 1227a+/g+ and 24 samples were typed as 1227a-/g-. two samples typed as 1227a+/g+ by pcr-ssp but 1227a+/g-by pcr with t m -shift primers were confirmed as 1227a+/g-by rhd exon 9 sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd 1227g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the 115 dna samples, 75 (65.2%) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon 8, which is functionally not relevant as reported before, was found in 28 samples, including 11 and 17 samples carrying respectively rhd-ce(8)-d and rhce-d(8)-ce (two homozygous each). except two samples that require additional studies (1.7%), rhd zygosity was resolved successfully: 60 (n = 2 rhd gene copies; 52.2%), 49 (1; 42.6%) and 4 (0; 3.5%). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples (4/115, 3.5%) carrying rhd*dau5, rhd*dv.2, a rhd*diiia-like allele, and a novel rhd*d-ce(7:g329h-y330s-n331i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd*03n.01 and rhd*diiic, were also found in trans with a normal rhd*01 allele. in rhce, c/c genotype could be resolved. the rhce*ce16 (rhce*ce (48c)-d(9)-ce) allele, which is commonly cis-associated with rhd*ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = 19) vs. non-bleeding (n = 15) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd63), p-selectin (cd62p), activated gpiib/iiia (pac-1) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd42b). normal healthy reference levels were available. results: the platelet count in bleeding (72 9 10 9 /l) and non-bleeding (68 9 10 9 /l) patients was comparable (p = 0,66). bleeding patients had a higher bat score compared to non-bleeding patients (10 vs. 2, p < 0,01). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) (86,16% and 4,96mfi) were higher, compared to bleeding patients (64,08% and 2,44mfi, both p < 0,05), and the proportion of ps+cps correlated negatively with bat score (r 2 =0,26, p < 0,01). cd63 + cp was higher in non-bleeding (97,25% and 10,54mfi) compared to both bleeding patients (94,88% and 6,89mfi) and significantly higher than the reference level (88,44% and 5,36mfi, both p < 0,05). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist (134,12 mfi unstimulated vs 32,38 mfi reference, p < 0,01). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa-1, hpa-2, hpa-3, hpa-5 and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa-1a, anti-hpa-2b, anti-hpa-3a, anti-hpa-5a samples were used to validate the specificities of the luminex assay. the anti-hpa-1a, anti-hpa-3a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to 1/1024). results: 44 samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa-1a, hpa-2b, hpa-3a, or hpa-5a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa-1a, and anti-hpa-3a were 1:512 and 1:64, respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of 44 samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of 358 individuals with normal abo groups (101 group a, 100 group b and 101 group ab individuals, and 56 group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg1 anti-b antibody (9431pe bgrl1). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v5.01. the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than 0.05 were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about 66.22% of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: 40 cases were found with antibody positive. among them, 6 cases (15%) were only anti-hla-i positive, 4 cases (10%) were only anti-hpa positive, 30 cases (75%) were both anti-hla-i and anti-hpa positive. 8 cases were found without anti-hla-i or anti-hpa. among the 34 cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were 73.5%, 61.2%, 50%, 44.1%, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > 0.05). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of 303 blood donors and 302 hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v 2 test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr3b*01 (hna-1a), fcgr3b*02 (hna-1bd), and fcgr3b*03 (hna-1bc) alleles were 0.384, 0.584 and 0.032; for the slc44a2*1 (hna-3a) and slc44a2*2 (hna-3b) alleles, 0.804 and 0.196; for the itgam*1 (hna-4a) and itgam*2(hna-4b) alleles, 0.898 and 0.102; for the itgal*1 (hna-5a) and itgal*2 (hna-5b) alleles, 0.708 and 0.292, respectively. in hematological patients, the gene frequencies for hna-1a/1bd/bc, -3a/3b, -4a/4b, and -5a/5b were 0.376/0.588/0.036, 0.795/0.205, 0.887/0.113, and 0.699/0.301, respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna -1, -3-5 for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = 605). the predicted risk of hna-1, -3, -4, -5 incompatibilities in this cohort were 34.9%, 26.9%, 19%, and 32.9%, respectively. the possible risk of hna-1a, -1bd, and -1bc alloimmunization were 0.233, 0.143, and 0.064, respectively; of hna-3a and -3b alloimmunization, 0.037 and 0.231; of hna-4a and -4b alloimmunization, 0.01 and 0.163; of hna-5a and -5b alloimmunization, 0.080 and 0.250, respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons (5, 7 or 10) of the rhd gene. methods: 2004 samples were tested from 01/12/2017 to 01/12/2018 using free dna fetal kit â rhd. 44 samples (2,2%) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce(4-7)-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. 65% of the identified variants are rhd negative alleles including alleles leading to partial rh2 antigen expression. unexpected alleles are found such as weak d type 1, 2, 5 or 11. these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on 20 cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed 16 of 20 (80%) pregnant women were rhd*weak d type 1 and not at risk for anti-d. rhd*weak d type 3 were typed in 3 cases (15%) and 1 case was rhd*weak partial 4.0 and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that 95% of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may 2018, uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of 3 ml. all material used passed pre-acceptance serological testing; samples were dispatched to 298 participants in 17 countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of 1.1 ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the 36 participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed 2 + reactions vs. two reagents by tube, cf. 4 + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of 3.7 ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with 4/17 anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all 16 respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa-1a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from 133 plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = 29), s/s (n = 13), hpa-1a (n = 49), hpa-3 (n = 8), hpa-5 (n = 14), hpa-15 (n = 20) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of 100-16,000 reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from 0,01% to 26,4% (one case was for advanced pregnancy -38 week of gestation). in 19/133 tested samples false positive results were detected at the level of 1 or 2 unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa-1a, -3a/b, -5a/b, -15a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh1) and anti-c (rh4) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih-500 system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih-500 system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from 29 samples containing anti-d and 20 containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih-500 system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < 0.0001, r = 0.79 and p < 0.0001, r = 0.82 respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < 0.0001) but gel scores have a better correlation coefficient than tube scores (r = 0.78 versus 0.55). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were 75 and 35, corresponding respectively to 5 ui/ml (250 uchp/ml) of anti-d and 7.5 ui/ml (500 uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih-500 system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from 250 pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january 2008 to december 2016. we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ 1:8 for anti kell antibodies and ≥ 1:32 for other specificities. results: out of 250 women, 143 (57.2%) displayed clinically significant antibodies, 92 (36.8%) clinically insignificant antibodies and 15 (6%) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d (16.8%) also in combination with other rh antibodies (8.8%), while anti-k accounted for 10%, anti-e for 10% and antibodies against high-incidence antigens for 2.4%. anti-m and anti-le a antibodies were also found (16.8% and 8% respectively) but they were not clinically significant. among 143 women with clinically relevant antibodies, 37 showed a critic antibody title and they underwent gynecological and obstetric monitoring. 21 fetuses resulted affected by hdfn, displaying anti-d in 16 cases and anti-kell in 5. 11 fetuses with severe hdfn (anti-d in 7 and anti-kell in 4) required iuts, 2 were treated with et, 8 received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from 0,4% to 2,7% worldwide. there are over 400 erythrocyte surface antigens, of which more than 43 have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in 2016 and 2017. methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during 2 years, from january 2016 to december 2017. in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, 5982 pregnant women were attended in hospital de braga. the laboratory registered 100 positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was 1,7%. anti-d was the most common antibody found (58,5%). anti-d prophylaxis given during pregnancy was responsible for 51 of 62 cases and maternal antibody titer levels did not exceed 8 among these cases. the prevalence of non-rhd immunization was 33%. anti-e (9,4%) was the most frequent alloantibody other than anti-d followed by anti-m (5,7%) and anti-c (4,7%). multiple maternal antibodies were found in 5 pregnant women. four women had 2 types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had 3 types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in 45% of them and phototherapy was given in 14%. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was 1,7%. the erythrocyte antibody screening showed that anti-d was the most common antibody found (58,5%) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was 33%. the other most frequent alloantibodies were anti-e (9,4%), anti-m (5,7%) and anti-c (4,7%). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may 2018, the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih-500 system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard (01/572) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the 2-months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of 2 have lower values. the highest differences (more than 2 to 3 dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel1) and anti-m (mns1) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih-500 system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a 1-day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was 13 mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes (15.2%), ldh (1162 iu/l) and g6pd (25.3 u/ghb); dat (+/-), iat (-), anemia (hb 8.7 g/dl, hct 28%), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs (3 + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with 1:1024 and 1:2048. the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to 14.6, bilirubin level was within normal range, she was discharged. another 5-days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was 15 mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes (5.2%), dat (+/à), iat (à), hb 12. background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in:1,-2 phenotype (in(a+b-)) are observed with a frequency of < 0.1% in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon 2 of cd44. results: in a 27-year-old pregnant (para 1) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of 1:2 was detected by iat (negative with papain-treated cells) at gestational week (gw) 32 and 36. the mma, performed in duplicate on samples taken at these dates, showed a mi of 0.5%/4.8% and 7.7%/6.3% respectively. the mi was interpreted as follows: 0-3% not relevant; 3-5% inconclusive; >5% clinical significant. the patient's parents were typed heterozygous, in:1,2 whereas her husband was homozygous, in:-1,2. due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw 41 without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw 32, was inconclusive whereas the second mma, performed in gw 36, indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included 20 immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period 2013 and 2018. results: the "critical titre" in our study was 8, meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only 2 newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than 4. moderate hemolytic disease of fetus and newborn were caused between the titre values 8-32. the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, 16 weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons 4, 5 and 10. rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all 3 exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at 3 nucleotide positions (c.1154, c.1193, c.1227) located in rhd exon 9 suggesting exon 9 was either deleted or rhce-replaced. paternal and cord gdna sequencing detected 4 out of 6 snps (c.186g>t, c.410c>t, c.455a>c, c.602c>g) associated with diiia phenotype plus 2 additional snps (c.604g>a, c.733g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from 5.3 iu/ml (16 weeks gestation) to 166 iu/ml (33 weeks gestation). the fetus required 3 intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon 9 deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd109 is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of 170 kda. in addition to being expressed on human plts, cd109 is expressed on activated t-cells, endothelial cells, cd34 + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa-15a and -15b are 0.505 and 0.495, respectively. based on these data, the risk of alloimmunization against hpa-15 alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa-15 alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa 15b alloantibodies by maipa and icfa. methods: a 24-year-old mother, gravida 2/para 0. the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy 33 weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy 24 weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to 4.7 9 10 10 /l, wbc count dropped to 2.96 9 10 10 /l, including neutrophil 37%, lymphocyte 16%, mononuclear 43%, eosinophil 2%, basophil 2%, red blood cells were normal, hb was 111 g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa-1a/a, -2a/a, -3a/a, -4a/a. -5a/a, 6a/a, 7a/a, 15a/b, naka (+) and the maternal was hpa-1a/a, -2a/b, -3a/a, -4a/a. -5a/a, 6a/a, 7a/ a, 15a/a, naka (+). the paternal genotype was hpa-1a/a, 2a/b, 3a/a, 4a/a, 5a/a, 6a/a, 7a/a, 15a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa-15aa and -15bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa-15a/a (donors 1), hpa-15a/b (donors 2) and hpa-15b/b (donors 3) donors by maipa. the mother's serum showed no reactivity against 15a/a plts, weak positive reactivity against 15a/b plts (od values 0.28), but strong reactivity against 15b/b plts (od values 0.38).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa-15genotyped donors (anti-hpa-15b average value 9.8). summary/conclusions: in this study, we found anti-hpa-15b in a case of fnait (patient hpa-15aa, blood group o) using the maipa technique. we were able to detect the presence of hpa-15b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in 1:10000 live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = 40). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, 2014 ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at 4 weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. 19%) are deceased due to organ rejection, and 8/36 patients (22%) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of 8-methoxypsoralen (8-mop) and irradiation with uv-a light. however, the addition of 8-mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with 8-mop+uv-a compared to uv-c treatment without additional 8-mop. methods: we used an in vitro 72 h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with 0,2 lg/ ml 8-mop plus 2 j/cm 2 uv-a treated cells and 2 j/cm 2 (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and 7-aad flow cytometry standings. results: the apoptosis analysis of cd3 cd4 t-helper cells, cd3 cd8 cytotoxic tcells, cd19 b-cells, cd14 monocytes, cd3 neg cd56 nk-cells and cd3 cd56 nkt cells revealed no statistical differences in almost all of these cell types after treatment with 8-mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after 72 h were similar in both treatment groups. summary/conclusions: the addition of 8-mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the 8-mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of 8-mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with 8-mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with 16 bedded micu and received tpe therapy between 1 june, 2016 and 31 december 2018. all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln9000 apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone 269 tpe procedures. among them, thirty were female patients (43%). the median age 45 (13-75) years. guillain-barre syndrome (gbs) was the most common indication (72%) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were 48 (18%) episodes of patient related complications during the tpe treatments. in 8 (3%) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts 13 levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of 9 years (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in 46 patients with diagnosis of maha (27-ahus, 16 -ttp, 1 each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was 19.94 ae 19.58 years with m:f as 1.5:1. number of procedures per patient varied from 1 to 27. post pe recovery was observed within 10-14 days with statistically significant increase in mean platelet count from 40.05 ae 5.9 to 82.11 ae 12.10 9 10 9 /l (p = 0.000) and significant decline in mean lactate dehydrogenase level from 48.91 ae 34.73 to 10.98 ae 6.49 lkat/l (p = 0.000). there was also significant decline in mean percentage of schistocytes in peripheral smear from 5.44 ae 3.96% to 0.56 ae 0.89% (p = 0.000). the mean serum urea changed from 48.88 ae 24.56 to 24.50 ae 17.78 mmol/l and creatinine from 266.11 ae 142.59 to 173.09 ae 127.34 lmol/l (p = 0.000 and 0.001 respectively) with significant increase in urine output from 0.71 ae 0.53 to 1.06 ae 0.33 ml/kg/h (p = 0.000). adverse events were observed in 10 patients (21%), allergic reaction to replacement fluid (n = 6) being the commonest followed by hypotension (n = 2), rigors and chills (n = 2). overall survival rate at 6 months was 89%. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a 17 year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since 2-3 days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, 6 cycles of tpe were performed on daily basis. after 6 cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [1] for motor performance, patient was assessed on a disability scale (0 = healthy; 1 = minor symptoms; 2 = able to walk 5 meters without support; 3 = able to walk 5 meters with support; 4 = confined to bed or wheelchair; 5 = requiring assisted ventilation; 6 = dead).patient's motor performance was increased to scale 1(upper limb) and 2(lower limb) from scale 5, deep tendon reflexes were normal. visual function began to improve 1 week after the treatment. visual acuity was 6/6 after 4 weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of 10% has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july 2014 to july 2018. a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > 10% but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than 10%, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were 10.6%, 11% and 31%. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused 46 days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between 9-16 days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from 01/01/2017 to 31/12/2018 in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of 6 patients met the inclusion criteria described. however, 1 patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded 88 exchange procedures (42 pmet and 46 aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why 2 patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = 2), stroke (n = 1) and recurrent vaso-occlusive crisis with multiorgan failure (n = 2). for both procedures, target values were to obtain a pre-exchange hbs level ≤ 30% for stroke and cerebral vasculopathy and ≤ 50-60% for other indications. the median hbs level before pmet was 42,6% (31,3-59,1) and 38,4% (18, 9) before aet. we documented a higher hbs level prior to the next procedure in 11,4% of patients (n = 10). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: 1356,1 vs. aet: 1314,7 ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: 360 vs. aet: 60 min and pmet: 21 vs. aet: 24 weeks, respectively). annual rbc requirements per procedure were superior (median 2 vs. 4 units) and the overall costs related with aet were 2,2 times higher -18.180,93€ and 8.174,79€ aet and pmet, respectively (estimated cost per session aet: 790,48€ and pmet: 389,28€). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo2) of 87% on air. the patient did not show improvement in spo2 level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo2 of 97% on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july 2016 to august 2017 were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these 46 (43%) were done on the mcs plus (haemonetics corporation) and rest 61 (57%) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = 0.000). the systolic (p = 0.010) and diastolic (0.001) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < 0.001), the age of the patient (p < 0.001) and the pre-procedure ph (p = 0.002). procedurerelated complications occurred during 30 procedures of which 13 complications (12.15%) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of 16 years (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . tpe procedures were done on two different apheretic devices (cs 3000 plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of 356 tpe (range 1-22/patient with mean of 6.2 procedures) were performed for 55 pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total 44 patients, neuromyelitis optica (category ii) in 4 patients, rapid proliferative glomerulonephritis (category i), c3 glomerulopathy in 3 patients each and one patient of infective hemophagocytosis. the average age of patient population was 7.8 yrs (1.2-13 years) . the male:female ratio was 3:1 with an average weight of 25.5 kgs. adverse events were observed during 20 (5.61%) procedures. most commonly observed adverse events were allergic reaction to replacement fluid (1.4%) followed by hypotension (1.1%), line occlusion (0.8%), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each (0.28%).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of 160 registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = 80)they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ 50 g); and group ii (n = 80)they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion 60, ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected 1 h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = 0.000) in group i (mean ae standard deviation: 67.86 ae 8.07 g; range: 50.80-92.13 g) than group ii (60.92 ae 8.29 g; range: 40.86-86.76 g). the mean hb increment in group i patients (3.26 ae 0.83 g/dl) was significantly higher (p = 0.04) than the group ii patients (3.00 ae 0.76 g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = 0.000 in groups i and ii), age (p = 0.001 for group i; p = 0.032 for group ii), body surface area (bsa) (p = 0.002 for group i; p = 0.000 for group ii) and blood volume (p = 0.006 for group i; p = 0.000 in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = 0.000 in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of 50 g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under 50 years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between 20/03/2004 and 01/09/2015 was collected. for the current study, male transfusion recipients under 50 years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (<10 days storage) or 'old' (>24 up to 36 days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was 0.47 day shorter when comparing the 221 patients who died, to 1,623 patients who survived (ci: à1.41 to 0.48). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was 0.64 day longer when comparing the 27 patients who died, to 101 patients who survived (ci: à2.53 to 3.80). in the single-transfusion cohort, 1,280 patients received a fresh rbc transfusion from a male donor, 52 of whom died; 138 patients received a fresh transfusion from an ever-pregnant female donor, 9 of whom died. 193 patients received an old transfusion from a male donor, 7 of whom died; 16 patients received an old transfusion from an ever-pregnant female, 2 of whom died. the 3-years cumulative incidence of death among young male recipients was 4.7% (confidence interval (ci): 3.6% to 6.2%) after a fresh transfusion from a male donor and 4.8% (ci: 2.2% to 10.4%) after a fresh transfusion from an ever-pregnant female donor. the 3-years cumulative incidence of death was 7.2% (ci: 3.8% to 13.6%) after an old transfusion from a male donor and 15.4% (ci: 4.1% to 48.8%) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at 3 years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from 50 to 80%. in our previous investigation, we found that premedication rate was 92.5% in the outpatients in 2017, which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, 2017 was retrospectively surveyed. evidence-based clinical practice was initiated since january, 2018. clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, 2018. the incidences of atrs and premedication rates in 2017 and 2018 were compared using chi-square test. a p value less than 0.05 was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, 2017 to september, 2018, a total of 5,018 blood units were transfused in the outpatients with 2,453 transfusion events. of these, 25 cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was 92.5% in 2017, and was significantly decreased to 77.9% in 2018 (p < 0.001). it was reported that the incidences of atrs in 2017 and 2018 were 0.48% and 0.52% per unit, respectively. there was no remarkable difference between the incidence of atrs in 2017 and 2018 (p = 0.642). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of 19 granulocyte transfusions in 15 hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) 5-10 lg/kg and tablet dexamethasone 8 mg, 10-12 h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within 30 days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of 1 9 10 10 per unit was fulfilled in 90% of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < 500/ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least 48 h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within 24 h) increased significantly (median value: 350/ll) as compared to baseline levels (median value: 40/ll) (p < 0.05). infection related mortality was observed in only 20% (3 out of 15) of patients. patients became afebrile within 2-4 days and culture negative within 3-6 days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: 1.5-3.0 9 10 8 cells/kg and high dose: >3.0 9 10 8 cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in 2016, we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for 2017. results are compared to the pilot audit and summarized below. methods: we collected data on 3 performance indicators from 7 acute public care hospitals for 4 weeks each in march and august 2017 (the pilot audit covered 2 weeks in 2016). the performance indicators were: 1). percentage compliance to documentation of red blood cell transfusion indications 2). percentage of patients screened for pre-operative anaemia, 14 to 45 days before surgery 3). peri-operative transfusion rates (3 days before to 3 days after surgery) for 6 commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator 3) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in 2012. results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator 1), 2 hospitals had a compliance of 57-61%, the remaining 5 had a compliance of 90-100%. all 7 hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from 75% in 2016 to 100% in a hospital that had newly mandated electronic ordering. for indicator 2), results ranged from 30% to 92%. 2 hospitals made notable improvements when compared to 2016, achieving 83% and 88% respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least 2 weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator 3), mean number of transfused units for each surgery ranged from 1.5 to 2.8 units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at 46% and lowest for tkr at 7%. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator 3) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november 2017 and march 2018. the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha 3 months after an allogeneic stem cell transplant, who has received multiple transfusions in the last 2 weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently 48 g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of 104 g/l and returns to clinic at a 2-week interval with symptoms of fatigue. he is actively haemolysing and commenced on 1 mg/kg prednisolone. results: there was a 42% (58/137) response rate by trusts. faced with a 4-6 h delay for allo-adsorption studies, 68% (38/56) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, 7% (4/56) would transfuse with o rh d negative red cells and 25% (14/56) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. 2017 british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the 2017 serious hazards of transfusion (shot) report, the most serious and fatal of 95 cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of 38 g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < 60 g/l after an hb fall of at least 40 g/l in the previous 2 weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits (2008) (2009) (2010) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between 14 th and 27 th may 2018 excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines (2008) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to 2 or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: 105 episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of 241 rbc units to 103 patients and the discard of 2 rbcs (due to incorrect transport). of the 105 episodes, 78 episodes (74%) involved an eventual switch to group-specific rbcs (range of emergency units, 1-13 units). the main requester was the emergency department (53%). the most common clinical indication for transfusion was acute gastrointestinal bleeding (49%). of the 105 episodes, 28 episodes (27%) did not meet the guidelines for emergency use because > 2 units of emergency uncrossmatched o rhd-neg rbcs were issued. 2 episodes (2%) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. 30 episodes (29%) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > 1 h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding (6%), receipt of an unsuitable pretransfusion sample requiring sample recollection (5%), delay in pre-transfusion sample processing (4%), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding (10%). only one patient was investigated for potential transfusion-related adverse outcome (1%) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, 105 episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with 241 rbcs issued and 2 rbcs discarded. a significant proportion of episodes (29%) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within 1 h before transfusion, 1 h after and 14-24 h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap-6) was assessed by flow cytometry by measuring p-selectin and pac-1 expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of 22 patients. the mean platelet count before transfusion was 8 9 10 9 /l (range 2-34 9 10 9 /l). 1 h cci was 11 9 10 9 /l and 14-24 h cci was 6 9 10 9 /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at 1 h after and 14-24 h after transfusion compared to before transfusion (p < 0.05). pac-1 expression after stimulation with adp was significantly higher at 14-24 h after transfusion (p < 0.001), but not at 1 h after transfusion. in rotem, clot amplitude at 10 and 20 min (a10 and a20) as well as maximum clot firmness (mcf) improved after transfusion (p < 0.05). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s =0.53 and 0.45 respectively, p < 0.001). absolute platelet count was also significantly correlated with mcf (r s =0.61, p < 0.001), where 84% of patients with a platelet count of more than 20 9 10 9 /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than 20 9 10 9 /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule-1 [soluble vcam-1]) in patients with scd in their non-crisis, "steady state." methods: patients, at least 10 years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: 15, hbsb thalassemia: 10) were enrolled (females: 15 [60%]), with a median age of 23 years (iqr: 11). following 6 months of hu, median values for wbc count (9.02 9 10 9 /l vs. 7.22 9 10 9 /l, p = 0.007) and d-dimer (1243.8 ng/ml vs. 830.2 ng/ml, p = 0.028) were significantly lower than baseline values, while the mean corpuscular volume (76.0 fl vs. 88.0 fl, p = 0.001) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin (9.1 g/ dl vs. 9.6 g/dl, p = 0.72), platelet count (250 10 9 /l vs. 196.5 10 9 /l, p = 0.71), lactate dehydrogenase (744 u/l vs. 567.5 u/l, p = 0.23) or soluble vcam-1 (567.8 ng/ ml vs. 526.4 ng/ml, p = 0.33) following 6 months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included 478 confirmed breast cancer patients planned for elective breast surgeries from january 2012 to december 2017. patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor 2 (her -2) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤40 years] and older group (>40 years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < 0.05 was considered statistically significant. results: of the total 478 patients most underwent wide local excision of the breast and modified radical mastectomy. a total of 16 patients received 71 units of blood and blood components in all categories of surgeries. only 103 were younger women (≤40 years) with mean age of 31 years. non-transfused patients were significantly more than transfused ones (p < 0.05). frequency of blood transfusion was more in young patients (4.9%). seven (22.6%) of the total 31 stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy (8.8%). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in 2009 and fully revised in 2016 by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a 700-bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in 2016. it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ 7 g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ 65 years, age ≤ 6 months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep 2016 to feb 2018 were analyzed. results: the number of transfusion-indications analyzed was 16138 for rbcs, 11158 for plts and 6024 for ffps. the most common indications for transfusion were chronic disease for rbcs (7977/16138, 49.4%), bleeding prevention for plts (5726/11158, 51.3%) and 'other' for ffp (2180/6024, 36.2%). 'hb ≤ 7 g/dl' was the most frequent sub-indication of chronic disease (3570/7977, 44.8%), and hematologic disease was the most frequent sub-indication of bleeding prevention (3432/ 5726, 59.3%). many clinicians entered transfusion indication as 'other': rbcs (2866/ 16138, 17.5%), plts (856/11158, 7.7%) and ffp (2180/6024, 36.2%). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; 82.9% of rbcs and 54% of plts. of the indications entered as 'other' in ffp, 80.3% were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep 2016. transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged 1-18 years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and 1 h post-transfusion, the expression of cd62p on platelet was determined by flow cytometry method. results: there were 102 subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd62p for nonleukodepleted and leukodepleted groups were 26.2% and 27.7%, and the mean increase of post-transfusion platelet cd62p for non-leukodepleted was 10.1% and the mean decrease of leukodepleted groups was 3.3%. it was shown the increase of post-transfusion platelet cd62p for non-leukodepleted group, and it was significantly (p < 0.05) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd62p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of 158 patients hospitalized in the period january -may 2018 in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < 12 g/dl and males < 13 g/dl), from the 158 patients included in the study, 61 (39%) were anaemic. from 117 males in the study, 40 (34%) of them were anaemic based on hb lab values, whereas from 41 women in the study anaemic were found to be 21 (51%) of them. from the 61 anaemic patients in the study, 35 (57.4%) of them with mild anaemia, 23 (37.7%) with moderate anaemia and 3 (4.9%) with severe anaemia. in the total of anaemic female 38.1% are under 65, while 61.9% are over/or 65 years old. in the total of anaemic males, 35% are under 65, while 65% are over/or 65 years old. it is noticed that most of them are with normochromic normocytic 67.2%, normocytic hypochromic anaemia 16.4%, hyperchromic microcytic anaemia 8.2%, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively 1.6% and microcytic normochromic anaemia 4.9%. the average value of preoperative hb decreased from 12.8 g/dl before surgery to 10.3 g/dl after surgery, so there is a decrease of approximately 2.5 g/dl of hb value. in our 158 patients, 48% (76) were transfused and the remaining 52% (82) were not transfused. from 76 transfused patients 41 (54%) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, 2003 to december, 2017, the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january 2008 to december 2009, the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of 988 patients were performed with unexpected antibody identification tests. of 1896 patients who underwent more than 2 screening tests, 25 (1.3%) were positive. 1871 were consistently unexpected antibody negative. the patients with solid tumors (n = 375, 56.3%) and those with hematologic diseases (n = 112, 16.8%) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = 0.0002). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group (65/988, 6.6%) than in the negative group (77/1871, 4.1%) (p = 0.006). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group (28/1871, 1.5%) than in the positive group (5/988, 0.5%) (p = 0.019). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in 2018 there were three female patients in the clinics of the centre for observation, age between 29 and 51 years (me = 39) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)-2, acute myeloid leukemia (aml)-1. individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac3/4 were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd41a. the density of fixed paig, pac was © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose 0,4 g/kg per day, for 3 days. one patient with aml received ivig-iggam therapy in the standard dose 5,0 ml/kg/day for 3. results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa-1 mfi-paigg reduced from 926 to 65; while the patient with aml: paiga reduced from 5506 to 187, and paigm from 1971 to 302, pac3 from 691 to 31, pac4 from 404 to 103. the patient with aa-2 over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the 3 patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells (60% vs 78%) and fresh frozen plasma (36% vs 84% p<o.oo1). more patients in the study group received platelets (72% vs 60%) and cryoprecipitate (50% vs 36%). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a 33-years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a 33-year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of 3 years at dubai thalassemia center. she was admitted to the hospital with 5 days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of 7.8 g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was 8.5 g/dl with a positive dat with both igg+4 and c3+4, patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin 5.6 g/dl, mcv 85.0, platelets count 216000, crp 3.6, retic count 2.18, blood culture no growth, total bilirubin 1.5, malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg 57.1 and igm 21.3, she was started on azithromycin for 5 days and received 2 units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to 9.3 g/dl. she did not show any improvement in her condition over the following 2 weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for 10 days. screening for collagen disease were all negative apart from high esr of 130 mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was 11.8 g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to 85 mm/hr and hemoglobin 10.2 g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of 1.2 g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first 60 min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january 1, 2011 to january 1, 2013 in 61 dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within 60 min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within 120 and within 180 min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of 1216 women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in 780 women (64.1%). in this cohort, seven maternal deaths (0.6%), 62 hysterectomies (5.1%) and 159 arterial embolizations (13.1%) were observed. plasma transfusion within 60 min in 114 women was matched on time-dependent propensity scores with 114 women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, 21.2% versus 19.9%, adjusted odds ratio 1.09 (95% confidence interval 0.57-2.08). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within 60 min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included 60 patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november 2017-july 2018. patients were previously taken asa at a dose of 100 mg per day, but therapy was discontinued 5 days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi 790-1410 au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (<790 au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately 30% of carrier females have factor ix clotting activity lower than 40% and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: 45 year old woman, with no history of abnormal haemorrhage, healthy, up to 2004 when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of 20% fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in 2008, after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since 2009. in 2012, she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with 2000 iu recombinant fix (rfix) daily, for four days, and up to 48 h after discharge maintaining basal levels of fix of 60% and with no major complications reported. in 2013, the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of 4000 iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of 3000 iu rfix are administered from day 1 (d1) and up to d7 maintaining fix levels around 60-80%. the patient is operated at d8 under the cover of 2000 iu of rfix, twice per day, and up to d14 after surgery reaching fix levels of around 80-110%. at d15 the dose was diminished to 2000 iu of rfix daily and at d17 she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february 2018 to january 2019. the study was approved from institutional review board. pack red cells (prc) transfusion in case of < 7 g/dl hemoglobin (hb) with cardiac history and < 7 g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < 50 9 10 9 /l, sepsis with bleeding and < 100 9 10 9 /l, patients on active chemotherapy without bleeding but platelet count < 10 9 10 9 /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version 23.0 was used for descriptive and inferential statistics. results: a total of 250 transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data 194(78%). out of 250, 134 (54%), 100(40%) and 16(6%) prcs, platelets and ffp were transfused respectively. out of 100 platelets, 10(10%) were platelet apheresis. 80/134 (60%) prcs and 22/90 (24%) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than 1 unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = 0.121). thirty six (45%) of transfusions were done with more than 1 units of prcs in non genuine group. the mean hb level of patients received 1 unit and more than 1 units prcs was statistically same (p = 0.868) and it was found out that there was same increment in hb in both the groups post transfusion. (p = 0.867). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = 4) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period (1 h) sheep were bled~40% of their estimated blood volume (estimated at 65 ml/kg) to a mean arterial blood pressure (map) of 30-40 mmhg. sheep were then resuscitated with either plasmalyte â (up to 20 ml/kg/ hr; n = 3) or transfused with sheep red cells (n = 1) to achieve a map > 65 mmhg. sheep were euthanised 4 h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of 931.7 ae 135.3 ml blood which combined with iatrogenic blood loss (~300 ml) corresponded to an average 40.2 ae 2.4% blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = 30-40 mmhg, lactate > 4 mm, svo 2 < 60%). across all four sheep the nadir map averaged 40.5 ae 13.1 mmhg, lactate peaked at 3.9 ae 1 mmol/ l, and nadir svo 2 was 41.3 ae 17.9%. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve 100% incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p-515 bilirubin were recorded within the 28-day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a 15-min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c3d. the rh group and non-rh group had 11 and 10 cases performed manually, and results were 2+ or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in 31/44 (70%) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in 2017, out of 7,276 notified atrs, 113 (1.5%) and 6 (0.1%) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january 1 st 2010 to june 30 th 2018. each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, 9,544 atr were reported, of which 29 included unconsciousness and/or convulsions (0.3%). of these 29 patients, 13 were females (44.8%) and 16 males (55.2%). unconsciousness alone was frequently observed (21 reports, 72.4%). convulsions were notified in 8 reports (27.6%) and were associated with unconsciousness in 2 of them. the diagnosis of seizure, with no other clinical signs, was established in 2 cases (6.9%). unconsciousness and/or convulsions were present in 8 allergic reactions (27.6%), 4 cases of transfusion-associated circulating overload (13.8%), 3 cases of suspected transfusion-transmitted bacterial infections and 2 hypertensive reactions. in allergic atrs, unconsciousness was notified in 7 cases and unconsciousness associated with convulsions in one. twelve atrs were severe (41.4%), 10 were life-threatening (34.5%) and in 4 cases, they resulted in the death of the recipient (13.8%). of the 8 allergic atrs, 4 were severe and 4 life-threatening. red blood cell concentrate was involved in 15 atrs (51.7%) and platelet concentrate in 9 (31.1%), including 5 cases with apheresis platelet concentrate and 4 cases with pooled platelet concentrate. fresh frozen plasma was involved in 5 atrs (17.2%). nevertheless, the imputability of the blood product was excluded or unlikely in 11 atrs (37.9%). in the 3 suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in 7 and 9 atrs respectively, but was certain in only 2 atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr (26 ars, 89.7%). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr 3.0, 95% ci 1.5-5.7, p = 0.001). this effect was most pronounced in the td-rs subgroup (hr 6.0, 95% ci 2.2-16.2, p < 0.001). similarly, elevated lpi levels were associated with inferior pfs (hr 3.4, 95% ci 1.9-6.3, p < 0.001) for the whole study population and the td-rs subgroup (hr 8.2, 95% ci 3.4-21.0, p < 0.001). in total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 5 patients desferrioxamine). lpi levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = 4 of each) was cultured with rpmi media (37°c, 5% co 2 ) alone or with the addition of lps (1-100 lg/ml; derived from escherichia coli 055: b5). the inflammatory response was assessed after 2 h (h), 6 h, 12 h, 24 h, 36 h and 48 h. supernatant was harvested at each time point and stored at à80°c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il-1b, il-6, il-8 and il-10). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il-1b and il-10 but not il-6 or il-8. il-1b production was significantly increased following stimulation of 100 lg/ml lps for 6 h (p = 0.043) and declined following 36 h incubation. release of il-10 was significant 12 h post-lps stimulation with 100 lg/ml (p = 0.030) and reached a maximum at 24 h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps (1 lg/ml), although the incubation times differed. il-1b was significantly increased following 2 h incubation (p = 0.002), with increasing levels observed up to 24 h post-lps stimulation. il-8 production was evident from 6 h and reached significance at 24 h post-lps stimulation (p = 0.009). il-10 was significantly increased following stimulation of 5 lg/ml lps for 6 hr (p = 0.043) with lower concentrations of lps resulting in il-10 production at 24 h (p = 0.008). release of il-6 was significant after 6 h of 50 lg/ml lps stimulation (p = 0.046), with lower concentrations of lps resulting in il-6 production at 24 h (p = 0.015). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. 1. rhdig inappropriately administered (unnecessary exposure) (n = 17, 40%) administered to: -rhd positive woman (n = 9) -rhd negative mother with rhd negative neonate (n = 5) -woman with immune anti d (n = 1) -administered in error (instead of other ig) (n = 2) 2rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = 17, 40%) -omitted (n = 14) -delayed (n = 1) -inadequate dose (n = 2) 3administration without correct patient identification (n = 6, 14%) 4storage & handling (n = 3, 7%) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo 2 agencia gallega de sangre, organos y tejidos, galicia 3 banco de sangre y tejidos de cantabria, cantabria 4 banco de sangre de la rioja, la rioja 5 banco de sangre y tejidos de navarra, navarra 6 banco de sangre y tejidos de arag on, aragon 7 fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon 8 fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain 9 terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in 17 autonomous regions, the hemovigilance system is structured in three levels: (1) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to (2) the regional hemovigilance coordinator, who communicates all the region's data to the (3) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in 2008. the mirasol prt system for platelets and plasma was introduced in 2010 and is currently being used in 10 of the 17 spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from 2012 to 2017 as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between 2012, when 4,196 mirasol treated blood products were issued to hospitals and 2017 with 56,229 mirasol products issued. due to low number of transfusions of mirasol-treated blood components in 2012 and 2013, notification rates began to be analyzed in 2014, showing ae rates of 0.2%, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around 0.23). rate of ae after transfusion of m-p is fluctuant between 0.06 and 0.17. this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around 80%) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > 2 reactions in 2016 and 2017 of respectively 0.18 and 0.07 no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period (2012) (2013) (2014) (2015) (2016) (2017) . at the national level, nine cases of bacterial transfusion transmission (with g&i > 2) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > 2 is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error (75%) with the lowest frequency in equipment failure (10%), compared to 21% and 29%, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes (25%) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in 2007-2017 were 80 in 8,385,448 blood units (1/104,818) issued for transfusion. these resulted in 27 serious reactions (34%) (1 fatal, 11 life-threatening) . another 21 (26%) were related with ibct that did not cause a reaction. near misses (component not transfused) were 24 (40%) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of 24 months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: 42,403 blood components were issued during the study period, while transfusion feedback forms for 37,816 components (89.1%) were received in the transfusion medicine department. delay in starting the transfusion (more than 30 min after issue) was observed in 2965 transfusion events (6.9%). the component transfusion time exceeded the permissible limits for 930 component (2.1%).the overall total permissible time for completion of components exceeded permissible limit in 2217 (5.2%) of transfusion events. the pediatric ward (23.9%), icu and ot complex (25.4%) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the 2965 delayed transfusions after issue, 2120 (71.5%) were during the routine hours i.e. between 7 am to 7 pm and 845 (28.5%) were in the non routine hours i.e. between 7 pm to 7 am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for 2013-2016 in comparison to previous years of surveillance, [2006] [2007] [2008] [2009] [2010] [2011] [2012] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for 2013-2016 89 sets of annual aggregated data from 25 countries covering 85,521,393 bcs issued were analyzed. all ars totalled 76,907 and infectious ars amounted to 285 (0.4%). the overall incidence of the infectious ars was 0.33/ 100,000 units of bcs issued. bacterial infections were the most frequent (188, 66%), next viral (84, 29.5%) and then parasitic (13, 4.5%). serious were 46% and there were 11 fatalities (3.9%, incidence 0.013/100,000). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the 84 viral sttis included 17 hbv (20%), 10 hcv (12%) and 2 hiv (2.5%). the 55 recorded as "other" (65.5%) including 24 cases of hev, one case of parvovirus b19, one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were 15 (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for 2006-2012 shows a consistent overall incidence in total sttis (0.33 vs 0.4/100,000). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in 2013-2016, p < 0.001) and an almost doubled rate of parasitic infections (p < 0.001). compared to the earlier period, there were many fewer hbv infections (17 vs 160) and many more hev. a similar reduction in the rate of hcv and hiv was observed in 2013-2016 in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january 2015-december 2018), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was 72 months (interquantile range-iqr 108). the median for the numbers of individual transfusions in children in a year was 12 (iqr 17). the median for the numbers of blood components individually transfused to patients was 18 (iqr 24). patients, anaphylactic transfusion reactions in 4 patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of 2.7 per 100000 units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of 11 and in 100000 units and 26 in 100000 units, respectively. while the incidence of transfusion reactions in children was found 0.62% in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of 2.7 per 100000 units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria 6 hospital general universitario, ciudad real, spain 7 hospital nostra senior de meritxell, andorra, andorra 8 banco sangre y tejidos, santander 9 banc de sang i teixits, barcelona, spain 10 fundaci on hematol ogica colombia, bogot a, colombia 11 centro regional de transfusi on de almer ıa, almeria 12 complejo hospitalario de navarra, pamplona 13 fundaci on banc de sang i teixits illes balears, palma de mallorca 14 hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in 2018 fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to 3 patients. two patients of 55 years of age diagnosed of acute myeloblastic leukemia (case 1 and 2) and chronic lymphatic leukemia (case 3). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors 1 month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all 3 cases, the story was judged as sufficient for analysis. the majority of reviewers (93%) diagnosed a chain of errors. there was agreement of 95% with respect to the initial process affected. the initial error was communication (42%), monitoring (45%) and compliance (62%), in cases 1, 2, and 3. 2-3 human errors were detected per case (average: 2.2, 2.7 and 2.4 errors respectively) and 2-3 latent errors per case (average: 1.8, 2.7 and 2.1, respectively). the latent errors most punctuated were: failures in the quality of the protocols (24%), in the transfer of important knowledge (22%), in the available technology (21%) and in the information to the patient (10%). all the participants contributed feasible measures of improvement according to root causes: 1) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, 2) train staff in knowledge important for safety, 3) communicate with computer application providers to improve the effectiveness and visibility of the alerts and 4) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from 2007 to 2017, reports of taco increased from 6 to 92; deaths from 1 to 7, major morbidity from 3 to 20. known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, <3 years, >60 years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last 3 years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case 1: a patient in their 50s weighing 67 kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb 37 g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of 100 g/l. -case 2: a patient in their 50s presented with a 4-week history of weakness and dizziness and had felt unwell for 6 months. the hb was 34 g/l, ferritin 26 lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of 51 g/l. a third unit was transfused over 125 min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case 3: a patient in their 90s with severe megaloblastic anaemia, hb 41 g/l and peripheral oedema developed taco after transfusion of 3 units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b12 deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b12 deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since 2015. results: total of 121,076 different blood components were applied in the period between 2015-2018. department for quality assurance and control has received 4 serious adverse reactions, 1 serious adverse event, 157 reports of transfusion reactions, of which 11 (7%) were inadequately filled, in the same period. from the above, 54 (34.39%) were transfusion reactions to erythrocyte blood components which were applied, 86 (54.77%) to platelet components and 17 (10.82%) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions (88 or 56.05%), followed by allergic reactions (50 or 31.84%). two transfusion reactions (1.27%) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was 0.004% (5 of 121,076) and 0.12% were reported transfusion reactions (157 of 121,076). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > 70 years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of 93 patients were analyzed (48 women, 45 men), with a median age of 86 years (70-97). in total, 189 ch were transfused. 70 patients received 2 ch, 3 patients 3 ch, 10 patients received 1 ch. 10 patients were transfused at two different times. the median hb prior to transfusion was 7.6 g/dl. in the patients who received 2 ch was 7.5 g/dl, those who received 3 ch: 6 g/dl and those who received 1 ch: 7.75 g/dl. the infusion time could be estimated in 84% of the patients. in those who received 2 ch was 223.62 min; 249.33 min in those who received 3 ch and 109.75 min in those who received 1 ch. 13 patients (14% of the total) suffered some type of adverse effect related to the transfusion. in 9 patients there was an increase in posterior ta, in 2 an increase in hr, in 1 an episode of hypotension and in another one episode of acute respiratory failure. 70% of those who had an adverse effect were older than 85 years. patients with aht after transfusion, 75% received 2 ch and the remainder 1 ch. among their background, 66% had a history of ischemic heart disease. 33% also had a positive balance. the average previous bp was 137/65 mmhg and the subsequent one was 182/72 mmhg. 55% of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. 2 ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > 85 years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse 2ch than 1ch. -we have appreciated that in those patients receiving 3ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february 2018 to december 2018. the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version 23.0. results: a total of 108 ida patients were analyzed in which 74(69%) were females and 34(31%) were males. the most common symptom in females and males was fatigue followed by body aches in females 12(16%) and pallor in males 10(29%). menorrhagia was present in 24(44%) of females of reproductive age. surgical history was present in 12(16%) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was 8.0 ae 2.2, 60.5 ae 9.6, and 20 ae 8.3 while in males it was 7.8 ae 2.3, 63 ae 14.3 and 18.4 ae 6.1 respectively. sixty two (84%) females were advised oral and i/v iron and 12 (16%) received transfusion. however, in males 8(24%) received transfusion and 26 (76%) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of 3 months follow up in males and females was same as that the transfusion (p > 0.05). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd34 + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo (100 ng/ml). after 12, 14 and 18 days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd41a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day 0), cultured but unselected samples (day 7), and cultured, selected samples (day 12, 14 and 18) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of 65140 genes were detected, of which 16612 showed up-regulation and 48528 down-regulation. among these genes, 8 differentially expressed genes (degs) (fold change ≥ 10; false discovery rate < 0.05) were selected were further validated with rq-pcr, including gabre, cdhr1, wasf3, pkhd1l1, thbs1, pf4v1, lrrc32 and lgals12. the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf4v1 mrna expression level was highest at day 14, lgals12 was highest at day 18. summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: 7-8 g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between 2011-2018. patients with hb level measurement within 90 days pre-surgery were included. receiving > 1 blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, 2591 patients underwent hip or knee surgery; 231 were excluded from the analysis due to receiving > 1 blood unit. hb measurement within 90 days pre-surgery was available for 1202 patients. hip or knee surgery was performed in 588 (49%) and 614 (51%) patients, respectively. women comprised 60% (n = 356) of patients who underwent hip surgery. in the hip-surgery group, 14.5% of patients required bt, with this need being slightly higher among women (31.5% vs. 26.2%; p-value=ns). only 50 (8%) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it (11.93 g/dl versus 12.8 g/dl for women and 12.3 g/dl vs. 13.8 g/dl for men; p-value < 0.001). hospitalization was longer in transfused patients compared to non-transfused ones (mean 7.52 vs. 6.91 days, p-value = 0.018) and in patients with a low hb level (female < 12, male < 13.5) than in those with a high hb level, irrespective of receiving bt (p-values < 0.00045). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < 0.05). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for 1 blood unit was 0.43 in the hb 11 g/dl group and 0.15 in hb 13 g/ dl group (35%>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd36, known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd36 deficiency was cd36 not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd36 deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd36 is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd34 + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd36 monoclonal antibody on cd34 + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd36 monoclonal antibody on proliferation and differentiation of human cd34 + hematopoietic stem (progenitor) cells in vitro. methods: choose 3 healthy full-term maternal women without various obstetric complications, take cord blood 20 ml. after density gradient centrifugation of ficoll cell separation solution, cd34 + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for 2-3 generations. mtt was used to examine the effect of anti-cd36 monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd34 + hematopoietic stem (progenitor) cells. the effect of anti-cd36 monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after 10-14 days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd34 + were sorted by flow cytometry, and about 0.44% of cd34 + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd36 monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value (0.9 ae 0.15) of anti-cd36 monoclonal antibody group (2 mg/ml) was decreased than normal group (1.05 ae 0.12) (p < 0.05), and the od value (0.81 ae 0.11) was significantly decreased at the cd36 monoclonal antibody concentration of 32 mg/ml (p < 0.01). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > 0.05). in the annexin v flow detection, the apoptotic rate of anti-cd36 monoclonal antibody group (2 mg/ml) was statistically increased than the normal group (p < 0.05). anti-cd36 monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g1 phase cells increased, and g1/s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was 169 ae 12, the number of cfu-e/bfu-e clones in the control group was 172 ae 12, and the number of cfu-e/bfu-e clones in the anti-cd36 monoclonal antibody culture group was 85 ae 6. the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd36 monoclonal antibody culture group was significantly lower than that of the other groups (p < 0.05). summary/conclusions: anti-cd36 monoclonal antibody can reduce the proliferation of human cd34 + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. 11 (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of 159 autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd 34+ cells concentration in blood higher than 20/ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. 11 or cobe spectra v. 6, v. 7, terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in 56 transplanted patients were assessed. results: standard collections were performed in 52 patients. the yield of cd 34+ cells was high, and no significant differences were found between the numbers of cd 34+ cells prepared from spectra optia 8,6 (1,3-41) 9 10 6 and cobe spectra 10,9 (1, 6 ) 9 10 6 /kg b. w. (a = 0,05; pval 0,619). the dependence of cd 34+ cell yield on the precollection concentration of cd 34+ cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = 0,95, and cobe spectra r = 0,93. lvl collections were performed in 107 of patients, and there were no significant differences between the numbers of cd 34+ cells prepared by cmnc spectra optia 10,9 (2-61,2)9 10 6 and cobe spectra 9,3 (2,4-86) 9 10 6 /kg b.w. (a = 0,05; pval 0,35). the relations between the precollection cd 34+ cells concentration in blood and the numbers of cd 34+ cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = 0,93, and cobe spectra r = 0,78, respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia (45%) than in cobe spectra (57%). a group of 12 patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra 11 days, while in platelets from cmnc 14 days, and from cobe spectra 12 days, respectively. the number of 44 patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with 11 and 13 days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd 34+ cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd34 + cells/kg. this number depends on the pre-apheresis cd34 + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in 1 day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd34 number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in 1 day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in 1 day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd34 + cells collected/(pre-apheresis cd34 + number*processed volume)*100%. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd34 + number, patient weight and the requested number of cd34 + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in 1 day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = 64% (n = 348). a ce of 40% was chosen as the cut-off for the cd34 calculation tool. using the cd34 calculation tool: allogeneic donors (n = 61): mean ce = 61%, mean blood volume processed = 3.3 9 tbv, mean time: 253 min, 37 donors were finished in 1 day collection (61%) autologous patients (n = 205): mean ce = 65%, mean blood volume processed = 2.4 9 tbv, mean time = 218 min, 173 patients were finished in 1 day (83%). the calculation failed in only 1 case (0.4%). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd34 calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from 2001 till 2019. all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was 2 9 10 6 /kg cd34 + cells and 2 9 10 8 /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c53000, cobe spectra and spectra optia using conventional-volume apheresis processing the 2-2.5 total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after 4 to 5 days of g-csf subcutaneous administration at a dose of 10 lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were 159 apheresis procedures performed in 106 healthy sibling donors. there were 75 males and 31 females, aged 20-55. one to two apheresis procedures were required to collect adequate graft. the single procedure usually took 3-4 h and the volume of collected stem cells was 50-220 ml. the needed number of mnc and cd34 + cells was successfully collected by 1.5 apheresis. there were 29 abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia -57 patients (53.7%), acute lymphoblastic leukemia -14 patients (13.2%), chronic myeloid leukemia -9 patients (8.5%), myeloproliferative disorders -8 patients (7.5%), myelofibrosis -5 patients (4.7%), severe aplastic anemia -5 patient (4.7%), non-hodgkin lymphoma -3 patient (2.8%), multiple myeloma -3 patient (2.8%), chronic lymphoblastic leukemia -1 patients (0.9), hodgkin disease -1 patient (0.9%). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from 400 ml whole blood collected from 32 healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of 35 days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw 125,000, 98% of hydrolysis) using electrospinning technology (nanospider tm 1ws500u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of 700-900 x10 6 plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from 10% pva solution using water: ethanol (8:2) solvent system. materials with proteins were electrospun from solution containing 10% of pva and 10% of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: 340 ae 120 nm for pristine pva fibers and 350 ae 148 nm for pva with incorporated proteins (pva/pl). pva/pl layers contain 5-10 mg of protein per gram of pva. 60% of the proteins are released during the first day (burst release) followed by a gradual release of up to 2 weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv18-01-00332. background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) 1, 2 and 3 which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp-4, comprises less than 1% of the total defensins in neutrophils and has a distinct sequence from hnp1-3. the other two, human defensin 5 and 6, are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp 1-3) are 3.4 kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at 22°c by leukoflex lst-1 filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph 7.4, without mgcl2 and cacl2, containing 5 mm edta and 2.5% sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs 1x at a concentration of 1 9 10 7 cells/ml. for degranulation, cells were stimulated with 100 nm of formylmethionyl-leucyl-phenylalanine (fmlp) for 5 min followed by stimulation with 10 lm of cytochalasin b for 5 min. supernatant was collected by centrifugation at 200 9 g for 6 min. supernatant was incubated with mouse monoclonal antibody to hnp 1-3 and purification of hnp 1-3 was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the 3.4 kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst-1 filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing 5-aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct4, sox2 and nanog were performed after 24 and 72 h and 1 week results: the results indicated that after 24 h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a with extract expressed oct4 with weak intensity after 24 h. no expression of oct4, sox2 and nanog were observed in other groups at the same time. after 72 h, oct4, sox2 and nanog were over expressed in the cells that were treated with 5-aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct4 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct4 and sox2 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase (5-aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than 150 nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon 1, 5, 10 and 1227a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of 1227 g>a and rhd exon 1, 5, 10. samples with rhd gene deletion homozygous/heterozygous, 1227 g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd1-rhce(2-9)-rhd10 homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were 1 in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon 1 (87°c) to internal control (77°c) were 2.33 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.09-2.21 in alleles of rhd gene deletion/normal rhd, 2.53-2.66 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 10 (81°c) to internal control (77°c) were 3.35 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 4.29-4.39 in alleles of rhd gene deletion/normal rhd, 4.67-6.10 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 5 (81°c) to internal control (77°c) were 3.64 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.49 in alleles of rhd gene deletion/rhd1-rhce(2-9)-rhd10 3.47-3.59 in alleles of rhd gene deletion/normal rhd, 3.97-4.77 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types 1, 2 and 3 not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total 583 dna samples were tested in 87 pools. fifteen (15) pools (181 samples) gave rhd deletion genotype and seventy two (72) pools (402 samples) resulted to the presence of rhd gene. the 72 positive pools were also analyzed individually. the genotype results obtained were: rhd exon 1 no amplification (1), rhd exon 6 and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, 100% accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: 1 sample rhd*93-94inst (del), 1 sample rhd*ivs3+1g (del), 9 samples rhd*weak d type 11 (partial d), 1 sample rhd*weak d type 5 (weak d), 1 sample rhd*weak d type 61 (weak d) and not described: 1 sample rhd*del 1-5 (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to 20 pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type 1, 2, and 3 which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~12-kilobase duplication event, including exon 3, was predominantly identified in 58.3% d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the 70 rhd variants included in this study, 49 samples (70%) showed presence of indian specific allele i.e. exon 3 duplication. seventeen rhd variants samples showed presence of both exon 5 and 10. qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r 1 r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. -128 non-invasive fetal kel1 genotypes from allo-immunized anti-kel1 women were done (34 positive confirmed fetuses, 6 undetermined, 16 positive non-confirmed, 22 negative non-confirmed and 50 negative confirmed). -190 non-invasive fetal rhc genotypes from allo-immunized anti-rh4 women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh3 women were done (66 positive foetuses, 1 undetermined for 21,5% of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła 1 , m krzemienowska 2 , m pelc-kłopotowska 2 , m jurkowska 3 , m debska 4 , m uhrynowska 2 and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since 2016 but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type 1, 2 and 3. aims: summary of a 3-year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of 366 pregnant women determined with standard serology as rhd-negative (in 8-38 week of gestation) was examined for the presence of exons 5 and 7 of rhd and ccr5 by realtime pcr using lc480ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in 123 cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr5 indicated a maternal d variant (d ct ccr5-rhd >2); the genetic follow-up of six of them identified: rhd*01w.2 in 4 cases, rhd*01w.1 and rhd*15. in 235 cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining 67% of mothers. in 1.9% cases with maternal d variant rhd nipt was not possible. however, in 5/6 such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type 1 and 2 in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh23 (d w ), as these are almost exclusive to african populations. rh23 is encoded by several types of rhd*dv as well as by dau-5. anti-rh23 is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida 4, para 1, causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at 37°c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh23 in addition to anti-fy 5 , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd*10.05 which encodes rh23. the newborn's phenotype was a r0r k-, fy(a-b+) and most likely rh23-and jk (a+b+), considering both maternal and paternal (a r0r, k-k+, fy(a-b+), jk(a+b-), rh23-) predicted phenotypes. the neonatal serum contained maternal anti-a1, anti-rh23 and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy 5 during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh23, derived from several rh23 + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from 22% in the patients younger than 60 years to 58% in the patients older than 80 years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with 59%. only 48,5% achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene 17%-33% patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above 200 mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a 28y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at 30y, a sister with similar profile age 26y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin 75 mg od, ticagrelor 90 mg bd, rosuvastatin 40 mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux5a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column 5a20 (evaflux, kawasumi, japan) as the second step. a total of 1.5x plasma volume(4470 ml) was processed. the patient was given continuous calcium infusion. the flow rate of 50 ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by 60%. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from 2010. hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: 101, 95, 111 in the last 3 years. delayed transfusion in relation to mh was reported for 54 cases 2016-2018 contributing to death in 6 patients 7%) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in 31/71 (43.7%): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, 2 units); 14% of women were transfused with fresh frozen plasma (median, 2 units) and 2% with platelets. mean pre-and post-transfusion hemoglobin levels were 6.7 g/dl and 9.0 g/dl, respectively, representing an increment of 1.0 g/dl per rbc unit transfused (1.09 g/dl in soweto and 0.83 g/dl in durban). indications for transfusion included obstetric hemorrhage (31%), chronic anemia (29%), surgery or anesthesia (13%), other (9%) and not specified (17%). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ 26 weeks (odds ratio = 7.07, 95% confidence interval 3.52-14.24). surgical blood loss was a common indication in trimester 1 (21%) that declined to 7% then 1% in trimesters 2 and 3. summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period 2016-2018. results: there were evaluated 1,281 688 of blood products administrations in 117,414 patients in the cr during defined three years period. announced 1,456 (0,11%) transfusion reactions including 50 severe transfusion reactions (20 9 adjudged with grade 3). the most frequent types of severe transfusion reactions: anaphylaxis 20, trali 7x, taco 5x, hcv 4x, hbc 2x, bacterial infection 1x, delayed hemolysis 1x. transfusion reactions incidence according to administered bp: red blood cells products: 882,017 administrations, 883 transfusion reactions (fnhtr 386x, allergy 227x, circulatory overload 66x, anaphylaxis 6x, trali 4x, hbv 2x, hcv 1x) platelets: 88,438 thrombocyte administrations, including 178 transfusion reactions (allergy 129x, fnhtr 30x, anaphylaxis 5x, circulatory overload 8x, delayed immune hemolysis 3x, acute circulatory overload 8x. granulocytes: 203 administrations, 2 transfusion reactions plasma: 293,801 administrations, 359 reactions reported (allergy 278, fnhr 25, circulatory overload 14, anaphylaxis 17x, trali 4x, hbv 3x, ards 1x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: 1. plasma for clinical uses from male blood donors, 2. prestorage of leucocyte reduced blood products, 3. production of platelets in additive solutions, 4. implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of 5 they were mainly associated with deviation in processing (22.4%) and attributed to equipment failure and materials (71%) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < .001) annual rate of total aes by 28% (95% confidence interval 26-30) ) 10% fibrinogen-depleted phpl or (3) 10% fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene 2.1 st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd73+/90+/105+ and cd14-/19-/34-/45-/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) 5%) were male and 31(36.5%) female. mean age of abo matched and mismatched groups were (6.99 ae 4.6) years. most common indications for transplant included beta thalassemia major 53(60.2%), aplastic anemia in 25(28.4) and pure red cell aplasia 2(2.35%). source of stem cell was bone marrow in 45 and peripheral blood 40 patients abo matched while abo mismatched group comprised of 28(32.9%) patients with further subdivision into major (n = 13), minor (n = 13) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = 401) and (n = 1837) comparably(n = 102) and (n = 480) in mismatched group. primary and secondary graft failure in matched group was 8.77% and 12.28% patients while in mismatched group graft failure was observed in 4(14.28%) patients respectively. positive dct in abo matched group in 1(1.75%) patient, whereas 2(7.14%) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia 1 , c sabia 1 , f cavalca 1 , g nicoletti 2 and c napoli 1 in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr4 and dr16 in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested 5 sera pre-treated with fbs from non-sensitized patients that showed the dr4/dr16 pattern. in particular, 5 ll of fbs was added to 95 ll of patient serum; incubated for 30 min at 37°c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from 5 patients with documented dsa including dr4/dr16 and 5 patients without hla antibodies. results: dr4/dr16 non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr4 and dr16 dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre 2 transfusion medicine, national blood transfusion center 3 transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from 17,992 healthy donors from which group a (36,8%), group o (41,7%), b (16,2%) and group ab (5,3%) resulted. the study was conducted in 3727 patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period 2011-2017 results: among the 3727 patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a (39.7%), followed by 0 (39.5%), b (15.2%) and ab (5.4%). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a (42%), in gastric cancer a (43%), colorectal cancer a (39, 6%), breast cancer a (41%), cervical cancer a (43%) and ovarian cancer a (42%) versus a (36.8%) in the control group. a high incidence of blood b is seen in multiple myeloma b (22%) and cervical cancer b (20%) versus b (16.2%) in the control group. blood group ab has a high incidence in malignant lymphoma ab (10%) versus (5.3%) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types 1, 2 and 3, are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a 12-year-old patient typed as weak d type 3, receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma (2+ by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type 3. rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type 3. mma showed the anti-d was clinically significant (>5%). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type 3 allele. weak d type 3 patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band 3/diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: 1.1. predictprotein (https://predictprotein.org); 1.2. spider2 (http://sparks-lab.org/server/spider2/); 1.3. dsc (discrimination of protein secondary structure class): using an in-house implementation; 1.4. jpred4 (http://www.compbio.dundee.ac.uk/jpred4/); 1.5. raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: 2.1. robetta (http://robetta.bakerlab.org/submit. jsp); 2.2. falcon (http://protein.ict.ac.cn/treethreader/); 2.3. itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: 3.1. verify3d (http://servicesn.mbi.ucla.edu/verify3d/); 3.2. prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: 4.1. gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); 4.2. gramm (http://va kser.compbio.ku.edu/main/resources_gramm1.03.php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = 10) carrying fetuses with known genotype was loaded into 3% agarose gel casette (3% df marker q3, sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from 45 min to 2 h 30 min of electrophoresis. results for real-time pcr detection of fetal rhd, kel*1 and ccr5 (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr5) was significantly lower in dna samples tested after gel selection (from 8.4 to 25.9geq/pcr) versus the level obtained from non-processed plasma dna (from 130 to 133geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from 1,5 to 6.6geq/pcr versus 10.8-11.2geq/pcr for non-processed plasma dna. results for kel*1 detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel*1-negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel*1 positive amplification was observed. however, kel*1 detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel*1-negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel*1 positive genotype was obtained. the total dna level in samples from k-negative women was from 18.3 to 18.7geq ccr5/pcr after gel selection versus from 52 to 746geq ccr5/ pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh1 quantification assay using ih-500 (bio-rad â ): promising results for monitoring rh:-1 pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh1 immunoglobulins since 1970 complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh1 microtitration. it is a column agglutination technology using red blood cells rh: 1, -2, -3,4,5 (r0r) . it permits to quantify low levels of anti-rh1 in comparison to a range of an anti-rh1 standard. performed since 1999 at the cnrhp and automated on evo clinical base tecan in 2008 (dilutions and distribution), anti-rh1 microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih-500 system from bio-rad â . methods: on ih-500, the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r0r, anti-rh1 standard). the performances of the method were evaluated using three internal quality control (icq) (2 cnrhp home-made at 2 and 12 ng/ml and 1 bio-rad â at 12 ng/ml) on papainized r0r (plc) and native r0r (nlc). a comparison of results from patient sera ranging from 1.5 to 48 ng/ml was done between ih-500 and evo clinical base tecan. results: the results of the 3 qci are similar between the different reagents used. there is no significant difference between the 2 types of red blood cells except for the limit of detection: 1.5 ng/ml in plc -6 ng/ml in nlc. for the 3 qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between 0 and 15%, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on 50 samples in plc and 44 samples in nlc was satisfactory (deming plc: r2 = 0.80 y = 0.89x + 0.78 -nlc: r2 = 0.86 y = 1.08x-0.25). summary/conclusions: the anti-rh1 microtitration on the ih-500 offers similar performances to the method conducted at the cnrhp. the ih-500 allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh1. this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab0i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about 1.5-2% of cases haemolytic disease demands transfusion support. aims: analysis hdfn from 2011 to 2018. methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group 0 rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (<5 days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit 70 + 5%, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit 40% -60% and irradiated. the ec used in post-natal transfusions is usually divided into rates of 70 ml, hematocrit 55 ae 5%. results: in last 7 years, we calculated 59 neonates with hdfn (28 males and 31 females): 31 with rhdi hdfn, 19 with ab0i hdfn and 9 with hdfn due to incompatibility for other red blood cell antigens. we have produced 81 iut: 48 for our hospitalized patients and 33 for patients located in other hospitals. 25 of these 48 patients, who received iut, were immunized: 19 showed anti-d antibody and 6 antibodies different from anti-a and anti-b. 7, of the 31 infants with rhdi hdfn, were transfused in utero. 4 neonates on 59 (6.8%) have performed et: 1 with ab0i hdfn and 3 with rhdi hdfn; the latter had also been transfused in utero. 27 neonates on 59 were transfused after birth: 18 with rhdi hdfn, 3 with ab0i hdfn and 6 with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous (52.7% of the total) and are those who have requested the highest blood supply both in the antenatal period (39.6%) that postnatal (9.6% performs et, 66.6% performs postnatal transfusions). neonates with aboi hdfn are 32.3%: nobody has received iut, only one has been subjected to et, and 11% has transfused after birth. patients with hdfn due to other antigens are 15%, have undergone iut 12.5% and were transfused after birth 22.2%. background: according to british guidelines on neonatal transfusion, since 2012 we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ 30 weeks and/or a birthweight lower than 1500 g, until 4 months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab0 direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab0/rh confirmation. blood transfusions were performed with 0 negative kell negative (0 cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than 5 days. ec were subdivided in 70 ml aliquots with a hematocrit of 55 ae 5%. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with 72 h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october 2012 to the end of 2018, 694 premature newborns received 2328 ec transfusions within their first 4 months of life. in 51% of cases (n = 1186), transfusion requirement was comprised within the 'small volume transfusions'. another 44% of cases (n = 1035), requiring further ec administration, was requested of a blood sample for t&s determination and 5% (n = 107) for a cross-match test. in 79.4% of newborns (n = 551), being transfused within the " small volume transfusions", blood requirement of 1401 ec was fulfilled by the initial blood test (1102 blood samples). 13.3% of newborns (n = 92) received more than 5 transfusions (6-21; median = 8) accounting for 820 ec released and for this group 381 blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, 381 blood tests were performed to sustain 643 infants transfused with 2221 units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of 75% of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first 4 months of life. background: glucose-6-phosphate dehydrogenase deficiency (g6pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g6pd 202 genotype is the most common g6pd genotype in sub saharan africa (ssa). some studies have linked blood from g6pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g6pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g6pdd 202 genotype among donors in two regions in uganda. it also described the effect of g6pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: 3,255 blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g6pd 202, haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g6pd 202 and co-inheritance with a-thalassaemia (n = 2,546) and haemoglobin s (n = 2,642) on the haematological quality of blood packs. a subset of 142 donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g6pdd. results: based on g6pd 202 genotyping, 10.3% (n = 274) of the blood samples used in the trial were deficient for g6pd enzyme while 5.3% (n = 142) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = 0.010) and whole blood (p = 0.009) donations of heterozygous g6pd 202 genotype. co-inheritance of g6pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was 83.3% sensitive and 0.8% specific for detecting donor blood packs with g6pdd. summary/conclusions: the prevalence of g6pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g6pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in 39 hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after 21 days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < 1000) and cdc-xm became negative. cdcxm labeled positive at ≥ 10%. t-cell fcmx was considered positive above 42 mfi and b-cell fcmx was considered positive above 120 mfi. lmxm was considered positive above 500 mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in 8 patients (20.5%). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the 39 cases initially screened 35 showed dsa positivity in sab. desensitization was done in those 35 cases only. in our study, sab was positive for class ii alone in 13(37%) while in remaining 22 (63%) cases it was positive for both class 1 and class ii. the number of pre transplant tpe procedures required was 6.7 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) . the mean number of post-transplant tpe sessions required was 0.7 (range, 1-6). during pretransplant and post transplant tpe procedures, five (14.3%) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean-8.5, median-7 days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx 17 months with the longest duration of follow up of 36 months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < 22%, we custom prime the tubing set with 5% albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient #1 is a 56-year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every 4 weeks, starting in 2012. the patient has completed 84 procedures with 3-4 units of washed red cells transfused to achieve a target hct goal of 45 to 57%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #2 was a 25-year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent 9 prx procedures with 2-3 units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient #3 is a 33-year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone 38 prx procedures with 3-5 red cell units transfused to achieve a hct goal of 30%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #4 is a 48-year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with 4-5 red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the 2016 american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since 2011. aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days 1, 2, 3, 5, 7, 9 along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos 7 aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of 63 variables was defined as possible confounders by a panel of experts. after discussion, 9 global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and 7 cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: 1018 patients were extracted from databases and further analyzed. the mean age of this group was 65,9 years (sd +/-14,1 years) and 67.3% of them were male. the mean duration of surgery was 256 min (sd +/-88,3 min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < 0.001). in 2011, the mean use was 2,34 units per operation (sd +/-2,277), which changed to 1,38 units (sd +/-2,158) in 2014. three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over 4 years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < 0.001). in the specific group of patients undergoing cardiac surgery with cpb (n = 640), the use of rbc was also significantly reduced (p < 0.001). in 2011, the mean use was 3,06 units per operation (sd +/-0,448) and this changed to 1,94 units (sd +/-0,167) in 2014. after correction for the 3 cpb variables that notably changed over the 4 years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over 4 years still remained statistically significant (p < 0.001). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between 2011 and 2014. this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from 14 adult pid and 13 adult sid patients from medical records and pathology reports, for their last 12 months of ivig and their first 12 months of scig. the starting and maintenance dose was 0.4 g/kg/month for ivig, transitioning immediately to 0.1 g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of 62.9 years versus 43.4 years in pid patients (p = 0.007). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = 0.041). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. 55% of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig (9.3 g/l) compared to ivig (8.4 g/l), and fewer infections on scig than ivig (mean annual infection rate of 1.64 vs 2.14 respectively). sid patients had higher mean serum igg trough levels on scig (8.4 g/l) than ivig (7.1 g/l) (p = 0.009) but experienced more infections while on scig versus ivig (mean annual infection rate of 2.15 vs 1.62 respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd62p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd62p expression. aims: to analyze the increase of platelet cd62p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante 2', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june 2014 to august 2018. the medical helicopter (ec 145t2) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and 35 days after collection, were performed. the statistical analysis used spss 23.0 version (significance p < 0,05). results: 138 prbc samples were evaluated, 48,5% (67) from case group and 51,5% (71) from control group. analyses were tested day 1 and day 35 after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered 35 prbc transfusion to 28 patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a 35-day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in 2010 the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of 11 deaths and 83 instances of harm due to delay over a 4-year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of 18 adult patients over a period of 7 months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all 18 cases. results: the abg analysis of all 18 patients showed decrease in the ph, increase in pco 2 , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the 18 cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of 18 cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in 4 cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in 2 cases. platelets were transfused in 5 patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of 40 adult surgical oncology patients who received massive transfusions and was conducted for a period of 7 months. every case of a massive transfusion was studied under the following headings (1) patient's details (2) patients base-line laboratory tests (3) resuscitation with transfusion (4) intra-operative laboratory tests (5) thromboelastography (teg) (6) post-operative complications (7) duration of stay in the hospital (8) 30 day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by 2 nd or 3 rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of 13 (32.5%) patients had ssi, 14 (35%) had surgical complications and 22 (55%) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, 38 (95%) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of 725 outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december 2017. laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted 15 ae 17.52%, but 49.72% of patients had a ttr less than 60%. patients were at high risk of thrombosis in 6.15% of time (inr < 1.5) and high risk of bleeding in 2.2% of time (inr > 4.5). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated 1.9 million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo 2 ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between 2005 and 2015 in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = 505) with two similarly exposed non-alloimmunized control recipients (n = 1010) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > 30 ml/min/1.73 m 2 , 'moderate renal failure' i.e. gfr ≥ 10-30 ml/min/1.73 m 2 during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < 10 ml/min/1.73 m 2 and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among 441 (87.3%) cases versus 838 (83.0%) controls; moderate renal failure among 24 (4.8%) cases versus 54 (5.3%) controls; and severe renal failure among 40 (7.9%) cases versus 108 (11.7%) controls. among the latter, 30 cases and 97 controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr 0.82, 95% ci 0.67-1.01 and adjusted rr 0.81, 95% ci 0.58-1.11, respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr 0.48, 95% ci 0.39-0.59) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir2dl1, kir2dl2/kir2dl3, kir3dl1, kir3dl2 and hla-a, -b, -c, -drb1, -dqb1 genotyping. 58 pairs of hla 10/10 identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c1/c1 homozygote group (48 cases), c1/c2 heterozygote group (8 cases) and c2/c2 homozygote group (2 cases). according to the expression of 3dl1, 53 cases were 3dl1 positive and 5 cases were 3dl1 negative. there were 5 cases of bw4/bw4, 24 cases of bw4/bw6 and 24 cases of bw6/bw6 in the 3dl1 positive samples. according to the expression of a3/a11, they were divided into three groups: a3/a11 negative group (28 cases), a3/a11 heterozygous group (28 cases) and a11/ a11 homozygote (2 cases). according to kir genotyping, kir haploidentical group (38 cases) and kir haploid mismatched group (20 cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of 58 cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v5.01. results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir3dl1 was positive. it was conducive to neutrophil and platelet remodeling when bw4/bw6 and a3/a11 was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to 5-7 days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = 3), prepared in 30% plasma/70% ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for 7 days (d) and sampled on d2, d5 and d7. cryopreserved sheep pcs (n = 3), prepared by the addition of 5-6% dimethyl sulfoxide, were stored at à80°c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), anti-inflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), 12-hete and 15-hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < 0.05 using paired t-test. results: in rt stored sheep pc supernatant il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete were detected at d2, d5 and d7. storage duration significantly increased accumulation of ip-10 at d5 (253.4 ae 266.7 pg/ml compared to 569.7 ae 272.7 pg/ml, p = 0.008) and further increased at d7, and il-8 at d7 (3477 ae 937.4 pg/ml compared to 5092 ae 521.1 pg/ml, p = 0.0460). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete than rt stored d5 pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il-8 with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than 10 cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = 5), prepared according to standard human procedures with minor modifications, were stored (2-6°c, 42 days (d) ) and sampled at d2 and d42. supernatant was prepared by double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), antiinflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), 12-hydroxyeicosatetraenoic acid (hete) and 15-hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < 0.05). results are mean ae standard deviation. results: at day 2, aa (75,142 ae 47,205 pg/ml), 12-hete (849.1 ae 235.6 pg/ml), 15-hete (87.6 ae 32.7 pg/ml) and il-1b (144.7 ae 198.9 pg/ml) were detectable in sheep prbcs supernatant. at day 42, storage duration significantly increased concentrations of aa (136,254 ae 60,433 pg/ml, p = 0.0425) and 15-hete (380.9 ae 116.3 pg/ml, p = 0.0022) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as 1 in 2,500 transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within 28 days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for 50 years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from 16% to 0.2%, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately 0.01 deaths per 1000. blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since 2007. since january 2015, rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = 93) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january 2015-december 2018, 45 reports were received; 43 reports were validated, with 2 reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in 2017 and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, 2019, depending on their iron parameters as having either: -idaserum ferritin < 30 mg/l -chronic inflammation with idaserum ferritin 30-100 mg/l with transferrin % of < 20%/crp > 5 mg/l -anaemia of chronic inflammationserum ferritin > 100 with transferrin % of < 20%/crp > 5 mg/l patients were considered eligible for iv iron if the following criteria were met:1. an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short 2. the anaemia pro-forma was completed 3. hb was ≤ 120 g/l 4. they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, 20 days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included 80 patients. 48 patients were classified as having ida and 32 patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of 96 g/l (55-120), a mean mcv of 77.4 fl and a mean serum ferritin of 16 lg/l. those with chronic inflammation with ida had a mean hb of 106 g/l (77-120), a mean mcv of 87.9 and a mean serum ferritin of 57 lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was 119 g/l (100-149) with an average increment of 23 g/l and in the group with chronic inflammation with ida the average post iv iron hb was 117 g/l (85-129) with an average increment of 11 g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb 20 days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january 2012-august 2018, which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were 154 products dispensed to 53 patients. the recipients were male patients diagnosed with prostate cancer with a mean age of 74 years. there were 3 doses (a complete course) administered to 50/53 (94%) recipients and a partial course (1-2 doses) administered to 3/50 (6%) recipients, for a total of 154 products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were 5 adverse events described in provider notes; 2 of them necessitating transfer to the emergency department, and 1 requiring hospital admission. of the 154 infusions, 6 infusions were documented in a chemotherapy note rather than a transfusion note (4%), and 59 (38%) were documented as both a transfusion and a chemotherapy administration. there were 6 additional deviations from the blood product administration policy: 2 cases where the consent check was not performed, 1 case where the product was infused with ringer's lactate rather than normal saline, and 3 cases where the 2-person 3-way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref 10310) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till 2017, at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ 20,000/ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = 31) and cmnc (n = 88) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in 134 consecutive cmnc procedures, including 88 autologous and 46 allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd34 + count ≥20/ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency-2 (ce-2) was defined as the total cd34 + amount in the collection bag divided by the amount of cd34 + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time (333 ae 59 and 289 ae 73 min, respectively; p = 0.001), the total blood volume processed (3.4 ae 0.8 and 2.4 ae 0.8, respectively; p = 0.001) and the final volume in the collection bag (337 ae 77 and 291 ae 84 ml; p = 0.001). the mean ce-2 in autologous versus allogeneic donors was 49 ae 24 and 66 ae 22, respectively (p = 0.003). using cmnc, the collection was effective in 94% of allogeneic and 63% of autologous donors. in autologous donors, a significantly lower collection bag volume (341 ae 84 and 396 ae 132, respectively; p < 0.01) and increased total wbc in the collection bag (263 ae 119 versus 149 ae 36, respectively; p < 0.000) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd34 + count following g-csf therapy; 7 of them achieved a cd34 count ≥20 and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd34 + count ≥ 20/ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd34 + cell counts are obtained with both methods. . tbv processed ranged from 1-4.8 tbv with mean of 2.6, average was 2.65 tbv for females and 2.74 tbv for males mean pre-apheresis cd34 + count was 92.89 cells/ll (range 33.14-152.64). mean postapheresis cd34 + count was 1402.3 cells/ll (559.02-2245.5). mean cd34 + cells x10 6 / kg recipients body weight was 7.5 (range: 2.77-12.33). our target yield was ≥3 9 10 6 cd34 + cells/kg body weight of the recipient and in only 2/34 (6%) cases, the yield was <3. 18/34 (52.9%) procedures were lvl and 16/34 (47.1%) were svl. summary/conclusions: most of our pbsc were done for haematological indications (85.3%) and the target dose was 3 9 10 6 cells/ll in single leukapheresis. in 32 cases (94%), target yield was achieved, only 2 cases had <3 but >2 yield. in our study donors <5 years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd34 + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd34 yield as high wbc count did not convert into high cd34 yield or vice versa. high prepheresis cd34 + count gave higher postpheresis cd34 + count. large volume leukapheresis (lvl), >3 tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd34 + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd34 + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid 1980s and early 1990s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our 18 year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine 11 hla loci (hla-a, -b, -c, drb1/3/ 4/5, dqb1, dpb1, dpa1, dqa1) in potential bone marrow donors from poland. the research included 18,500 potential bone marrow donors registered between 2017 and 2018. a novelty of this paper was that the amplification of all 11 hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the 11 hla loci (hla-a, -b, -c, drb1/3/4/5, dqb1, dpb1, dpa1, dqa1) of potential bone marrow donors was made by using the alltype tm ngs 11-loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of 18,500 donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c*12: 143, c*12: 30, c*05: 37, c*07: 151, drb1*11: 28, c*14: 04, b*51: 22, c*15: 13, dqb1*03: 12, drb1*14: 87, drb1*11:69. summary/conclusions: 1. new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. 2. the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. 3. the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn5 dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version 1.2.0(one lambda inc.)with the default setting. 94 cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: 94 samples were successfully tested with two methods and the coincidence rate between two sequencing methods was 100%. with the next-generation sequencing method, the probability of ambiguous results among 94 samples in our study is 1.06%(1/94) for hla-a, 5.31% (5/94) for hla-b and 0% (0/94)for hla-c. however, the probability of ambiguous results with the sanger sequencing method is 96.8% for hla-a, 95.7% for hla-b, 100% for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques.